Our objective was to find out if there are antibodies binding to homocitrulline-containing type I and II collagen carboxyterminal telopeptides in sera of patients with rheumatoid arthritis (RA), and if these antibodies cross-react with citrulline and homocitrulline in the same peptide sequence.A total of 72 RA and 72 control sera were analyzed for binding using enzyme-linked immunosorbent assay to citrulline- or homocitrulline-containing type I and II collagen carboxyterminal telopeptides, as well as to cyclic citrullinated peptide (CCP) and to mutated citrullinated vimentin (MCV). Specificities of the antibodies were tested using inhibition-ELISA.Of the RA sera, 39 (54%) and 41 (57%) were positive for binding to CCP and MCV, respectively. Further, 34 (47%) and 30 (42%) of the patients had specific antibodies binding to and being inhibited by citrulline-containing type I collagen telopeptides and by citrulline-containing type II collagen carboxyterminal telopeptides, respectively. The corresponding figures regarding homocitrulline-containing type I and homocitrulline-containing type II collagen telopeptides were 16 (22%) and 14 (19%). Most of the patients, who were seropositive for citrullinated peptides, showed binding in multiple assays. A total of 10 (14%) RA patients were positive for all the tested peptide pairs, while 28 (39%) of them had antibodies that contained overlapping specifities between citrulline and homocitrulline in the same peptide sequence.Antibodies to both citrulline and homocitrulline containing type I and II collagen telopeptides can be found in sera of RA patients. These antibodies are not constant from one RA patient to another, but contain separate or overlapping specificities within the same peptide sequence varying between individuals. Our results suggest some relationship between citrulline and homocitrulline-recognizing antibodies, since homocitrulline antibodies exist mainly in individuals seropositive to anti-CCP and anti-MCV.The online version of this article (doi:10.1186/s13075-014-0515-z) contains supplementary material, which is available to authorized users. The search for citrullinated antigens in patients with rheumatoid arthritis (RA) has been in focus during recent years. The identification of the original immunogen for their production could be the key to the prevention of the immune response and the consequent cascade of the inflammatory process leading to tissue damage and loss of function. Antibodies against citrullinated proteins are a frequent finding in RA patients and may precede the onset of clinical symptoms by several years -3. We haLately, also, the contingent effect of carbamylation on the pathogenetic process of RA has become of interest. Carbamylation through myeloperoxidase is part of a natural defense mechanism and takes place in inflammatory conditions (reviewed in ), and haAltough ACPAs arise in RA patients years before the onset of clinical disease, the origin of these antibodies is not known. The RA patients’ antibody repertoire has been reported to go through epitope spreading between citrullinated antigens ,14,15. TThe study was conducted according to the requirements of the Declaration of Helsinki and was approved by the ethics committee of the Central Hospital of Jyväskylä. All patients gave verbal informed consent before inclusion in the study. In the 1980s, when the study was designed and conducted, no Good Clinical Practice instructions existed. Verbal consent in clinical studies was common praxis. The study design was accepted by the ethics committee of the hospital.et al. [A total of 72 RA patients diagnosed in the years 1987 and 1988 in the Jyväskylä Central Hospital according to the Ropes criteria (Ropes 1959), whose sera were available were included in the present study [et al. . Sera frAll sera were tested by enzyme-linked immunosorbent assay (ELISA) for binding of immunoglobulin G (IgG) to citrullinated and homocitrullinated type I and II collagen carboxyterminal telopeptides under standard conditions, and the binding was inhibited by addition of soluble antigens  to a finAnti-CCP (Immunoscan RA (Mark 2) from Euro Diagnostica, Malmö, Sweden) and anti-MCV  assays were performed according to the manufacturers’ instructions. All control and patient sera were tested in duplicate.P <0.01.All 72 control sera included were negative for anti-CCP (<25 U/ml) and anti-MCV (<20 U/ml). Logistic regression analysis was used for evaluation of the association of each test or test type (citrulline or homocitrulline tests) with bone erosions in RA patients . Differences were considered significant at Patients with RA were found to have antibodies that bind to citrulline and homocitrulline-containing sequences related to type I and II collagen telopeptides Figures  and 2. TWe measured specific antibodies to citrullinated type I collagen-related peptides using inhibition ELISA, where similar peptides were used both for binding the serum antibodies and in smaller, soluble form also to inhibit the binding. We found CitI specific antibodies in 34 (47%) of the 72 patient sera, while 16 (22%), 30 (42%) and 14 (19%) of the sera contained antibodies specific for HcitI, CitII and HcitII sequences, respectively. Furthermore, a total of 39 (54%) and 41 (57%) of the patient sera bound the respective antigens in direct ELISA for anti-CCP and anti-MCV. Total ELISA data can be found in Additional file Most of the sera that bound citrullinated peptides were reactive in multiple assays. A total of 10 (14%) sera contained specific antibodies to all four peptide pairs. Moreover, 28 (39%) of the patients had antibodies with overlapping specificities, so that the binding could be inhibited by peptides with different ureido group-containing amino acids. Of these, 16 (22%) sera showed such binding to citrulline-containing peptide that could be inhibited by homocitrulline-containing peptide. For another 16 (22%) sera the binding to homocitrulline-containing peptide could be inhibited by the corresponding citrulline-containing peptide. In only four (6%) of the cases were the antibodies cross-reactive and could be inhibited simultaneously in both directions.We could analyze sera taken at two different time points from 19 patients (data not shown). There was only slight variation in the titers of antibodies to specific collagen telopeptide sequences between the two samples. We also preliminarily tested sera from 38 hemodialysis patients for binding to citrulline- and homocitrulline-containing peptides to exclude a possible effect of high serum urea levels and found the results comparable to those of healthy controls.P = 0.043), CitII  and HcitII  reached close to statistically significant association with erosive RA, whereas for antibodies against HcitI  the association did not reach statistical significance. When the presence of joint erosions was checked against the presence of antibodies to either CitI or CitII in patient sera, the OR was 3.6 (P = 0.012), comparable to that of the combined homocitrulline tests; if a patient serum contained either HcitI or HcitII antibodies, the OR for erosions was 3.7 (P = 0.037).The association of the results of each inhibition-ELISA to erosive disease was assessed using logistic regression analysis Table . MCV hadWe have previously obtained results in rabbit experiments indicating that antibodies against citrullinated antigens can also be induced with homocitrulline-containing immunogens  and thatThe antibodies binding to citrullinated and carbamylated proteins are induced even years prior to clinical RA onset ,22. The We found that RA patients had specific antibodies to the CitI, CitII, HcitI and HcitII antigens. The antibodies binding to citrulline- or homocitrulline-containing telopeptides could be detected separately or simultaneously in the same serum. Evidently the antibodies present in sera of RA patients were able to discern between citrulline and homocitrulline in the same peptide-sequence, although there were also some overlapping specificities. The overlapping specificities were shown as the sera were able to bind to a citrullinated antigen, but could be inhibited by a soluble homocitrullinated peptide or vice versa. This phenomenon was usually detected only in one direction in a particular serum sample, but in two of the sera there was a cross-reaction in both directions  and no definite conclusions about long term variation between antibody titer can be drawn. In any case, it is worth noticing that the level of antibodies against homocitrulline-containing antigens in the 38 hemodialysis patient sera was comparable with that of healthy controls (data not shown).et al. [Shi and others have previously found 45% of RA patients to be positive for IgG antibodies that recognize carbamylated protein . The antet al. .The structural homology of two amino acids is a challenge for the immune system and a predisposing factor for development of overlapping specificities. In our study, the homocitrulline-containing sequences have no match in the human body, but the results show us properties of the binding antibodies. It is most probable that the level of cross-reacting antibodies is dependent on sequence homology of the neighboring amino acids in the immunogen and by analyzing with unnatural sequences it is not possible to directly identify the real target of these antibodies.Our study does not reveal the original immunogen for anti-citrulline or anti-homocitrulline antibodies. The homocitrulline-containing type I and II collagen carboxyterminal telopeptides used in this study cannot be original immunogens, because the sequences we used do not exist in the body. However, the fact that homocitrulline-binding antibodies mainly exist in individuals showing binding in anti-CCP and anti-MCV assays suggests that some relationship between anti-homocitrulline and anti-citrulline antibodies exist.At least one situation is known where citrullination and carbamylation are present together. NETs contain both hypercitrullinated histones and myeloperoxidase. In addition, in smokers the thiocynate released from tobacco smoke favors carbamylation. Thus, NETs in the oral cavity or in synovial tissue may be the place for the coexistence citrullination and carbamylation where the antibodies with overlapping specificities could also have a role in the development of chronic inflammation.Antibodies to both citrulline- and homocitrulline-containing sequences related to type I and II collagen telopeptides can be found in sera of RA patients. These antibodies are not constant from one RA patient to another, but contain separate or overlapping specificities within the same peptide sequence varying between individuals. Our results suggest a relationship between anti-citrulline and anti-homocitrulline antibodies, since anti-homocitrulline antibodies were found to exist mainly in individuals seropositive to anti-CCP and anti-MCV.


Immunoendocrinology is an important field that studies the connection between the immune and endocrine systems. Hormones, including molecules like vitamin D and metabolic components, and neurotransmitters are compounds produced by different cell types that are capable of regulating the cross talk among cells from different tissues. During the last years, it became evident that hormones and neurotransmitters are specific modulators of cells of the immune system by fine-tuning their activation and key functions.This special issue covers the most recent research elucidating how hormones are able to regulate the recruitment of immune cells to an inflammatory site as well as to explain the hormonal impact on the intracellular signaling cascades that follow after tissue injury. It will cover the mechanisms by which hormones can influence inflammatory process.Diabetes mellitus is the most common cause of end-stage renal disease and chronic low-grade inflammation is an important factor in the pathogenesis of diabetic complication. Mycophenolate mofetil (MMF) has an anti-inflammatory effect, inhibiting lymphocyte proliferation. J.-W. Seo et al. study the effect of MMF on diabetic nephropathy and investigate its action mechanisms in type 2 diabetic mouse models. They demonstrated that MMF treatment attenuates diabetic nephropathy by decreasing CD4+ T cell infiltration and its related cytokines and chemokines. This anti-inflammatory effect of MMF attenuates podocyte apoptosis independent of glucose control.MicroRNAs are small noncoding RNA molecules that regulate gene expression in all cell types, which are involved in a wide range of biological processes, exerting functional effects at cell, tissue, and organ levels. G. Ventriglia et al. summarize the most recent data on the potential involvement of microRNAs in autoimmune diabetes and indicate that miRNAs represent major players in the dialogue between beta cells and immune system, regulating several aspects of their function and survival. Trypanosoma cruzi induces a hormonal systemic deregulation. A. F. Nardy et al. illustrated how the sphingosine-1-phosphate (S1P) system regulates both thymic and lymph node T cell egress which is essential for producing and maintaining the recycling T cell repertoire. The presence of this T cell receptor (TCR) bearing cells in the periphery may have potential implications for disease outcome. The authors discuss the possibility that the early egress of undifferentiated CD4−CD8− T cells plays a role in the immunopathologic events in Chagas disease by altering adaptive immune responses.Infection with the protozoan parasiteThe current evidence of the role of liver dendritic cells (DC) in the progression from nonalcoholic fatty liver disease (NAFLD) to fibrosis is reviewed by P. Almeda-Valdes et al. Link between lipid metabolism and DC function suggests that immunogenic DC are associated with liver lipid storage, which might represent a possible pathophysiological mechanism in NAFLD.β suppresses melatonin secretion and its action seems to be targeted on the reduction of pineal gland arylalkylamine-N-acetyltransferase (AA-NAT) protein expression.Interactions between the pineal gland and the immune system have been studied ex vivo. A. P. Herman et al. provide evidence on how interleukin- (IL-) 1Varicocele is the most common cause of infertility in men and the development of varicocele-related testis damage may be caused by disruption of homeostasis between cell proliferation and cell death. In addition, apoptosis is a physiological process by which a sequence of intracellular events results in the programmed elimination of a cell from its environment. Specifically, alterations in the apoptosis of germ cells may be crucial in varicocele-related human infertility and, as a direct consequence, targeting apoptosis may represent an alternative and rational therapeutic strategy in the treatment of varicocele complications. L. Minutoli et al. studied the effect of Polydeoxyribonucleotide (PDRN), an agonist of adenosine A2A receptor, on testicular Neuronal Apoptosis Inhibitory Protein (NAIP) and survivin expression in an experimental model of varicocele. They found that PDRN may represent a rational therapeutic option for accelerating recovery from depressed testicular function through a strategic modulation of apoptosis in experimental varicocele.


Drosophila have shown a paucity of male-biased genes  on the X chromosome. Dosage compensation (DC) is a regulatory mechanism of gene expression triggered in males that hypertranscribes the X-linked genes to the level of transcription in females. There are currently two different hypotheses about the effects of DC on the distribution of male-biased genes: (1) it might limit male-expression level, or (2) it might interfere with the male upregulation of gene expression. Here, we used previously published gene expression datasets to reevaluate both hypotheses and introduce a mutually exclusive prediction that helped us to reject the hypothesis that the paucity of male-biased genes in the X chromosome is due to a limit in the male-expression level. Our analysis also uncovers unanticipated details about how DC interferes with the genomic distribution of both, male-biased and female-biased genes. We suggest that DC actually interferes with female downregulation of gene expression and not male upregulation, as previously suggested.Several studies in  Drosophila (fruit flies), females carry two X chromosomes (XX) while males carry one X chromosome and one Y chromosome (XY). Because the Y chromosome is highly degenerated and nearly devoid of genes . These four gene expression profiles were obtained using microarrays and the expression level measured as hybridization signal intensity. The DCC binding regions were previously identified from high-resolution ChIP-chip mapping data in MSL3 mutant male embryos. According to the original study, a gene was classified as “bound” if it overlaps with a DCC binding region and as “unbound” otherwise .Data manipulation and statistical analysis were performed in R , supporting the DC interference hypothesis  is significantly lower for X-linked genes compared to autosomal genes 3-7. However, mutations in these genes do not upregulate X-linked genes or even cause a generalized misregulation of the X-linked genes. Therefore, to our knowledge, there is no evidence to suggest that these proteins impede upregulation of X-linked genes in males . This observation is incompatible with the hypothesis that DC and other chromatin remodeling proteins impede the upregulation of gene expression (Vicoso and Charlesworth Drosophila (Chintapalli et al. Chromatin modifications associated with DC and open chromatin structure still persist in females (Jin et al.


The aim of this study was to quantify potential differences in count, frequency and pattern of high-intensity transient signals (HITS) during transapical transcatheter aortic valve implantation (TA-TAVI), by comparing the Symetis Acurate TA (SA) with the balloon-expandable Edwards Sapien XT (ES) system.Recently, the Symetis Acurate TA revalving system has been introduced for TA-TAVI. The Symetis Acurate TA aortic bioprosthesis is self-expanding and is deployed by a specific two-step implantation technique. Whether this novel method increases the load of intraprocedural emboli, detected by transcranial Doppler ultrasound (TCD) as HITS, or not is not clear.Twenty-two patients  displayed continuous TCD signals of good quality throughout the entire TA-TAVI procedure and were included in the final analysis. Data are presented as median with interquartile ranges.No significant differences were detected in total procedural or interval-related HITS load . With both devices, HITS peaked during prosthesis deployment (PD), whereas significantly fewer HITS occurred during instrumentation  or post-implantation PI . PD-associated HITS amounted to almost half of the total HITS load. One patient suffered new disabling stroke at 30 days. Thirty-day mortality amounted to 13.6% (3 of 22 patients).Simplified transapical delivery using the self-expanding SA device does not increase HITS, despite of a two-step deployment technique with more interactions with the native aortic valve, when compared to the balloon-expandable ES valve. The similarity in HITS count, frequency and pattern with the two systems suggests a common mechanism for the release of cerebral microemboli. In selected high-risk patients with severe aortic valve stenosis, early and late results of the PARTNER trial allow for a direct comparison of event rates of stroke and transient ischemic attack between transcatheter aortic valve implantation (TAVI) and surgical aortic valve replacement (SAVR) The passage of stiff guidewires, catheters and the delivery system through the aortic arch  and through the degenerative aortic valve as well as, but also balloon aortic valvuloplasty and prosthesis deployment may detach atheromatous or calcified debris, and may cause cerebral microembolization with occasionally devastating complications Rapid innovation of self-expanding TAVI devices As one of these innovations, the self-expanding Symetis ACURATE TA system has been designed for the transapical access and has received CE mark approval at the end of 2011. The device combines various new prosthesis- and implantation-related features designed to facilitate precise and reproducible deployment. The prosthesis is delivered by a two-step deployment technique, allowing tactile feedback control by interaction of the Acurate frame with the native aortic valve. This technique increases deployment accuracy, but might also increase friction and hence, risk of cerebral thromboembolism.The aim of the current study was to investigate the incidence, rate and temporal pattern of HITS during transapical TAVI (TA-TAVI), using either the new Symetis ACURATE (SA) device or the conventional Edwards SAPIEN (ES) prosthesis and implantation technique.Consecutive patients undergoing TA-TAVI have been analyzed for the purpose of this study. The patients are a subset of our institutional prospective single-center registry initiated in July 2007. Selection of the SA or ES device was non-randomized and guided by patient and device parameters. All patients had given written informed consent to undergo TA-TAVI under general anesthesia with invasive and noninvasive monitoring, and for the scientific use of their anonymized data. Approval for retrospective study was given by the Cantonal Ethics Committee Bern, and granted by the Institutional Research Directorate. The corresponding author takes full responsibility for the integrity of the data. All authors have read the manuscript and agree with the present version.2; aortic valve mean pressure gradient >40 mmHg on echocardiography), age >80 years, and/or a logistic EuroScore ≥15%, or age >70 years with appreciated high or prohibitive risk of morbidity or/and mortality for SAVR. Selection of the SA or ES device occurred at the clinical discretion of the institutional heart team based on aortic root anatomy, distribution of valvular calcification and technical characteristics of the prostheses.During the period from March 2012 to February 2013, thirty-one patients underwent TA-TAVI at our institution. Sixteen patients were treated with the SA, and 15 patients with the ES system. Patients were considered for a TA-TAVI procedure based on clinical, echocardiography and computed tomography findings by the institutional heart team consisting of interventional cardiologists and cardiac surgeons. Selection criteria for TA-TAVI were severe symptomatic aortic stenosis  or an Edwards  was selected. In all patients TA-TAVI included echocardiography-guided anterolateral mini-thoracotomy, access site preparation, insertion of the apical introducer and balloon aortic valvuloplasty of the calcified native aortic valve during rapid ventricular pacing (RVP). This was followed by angiography-guided delivery of the TA-TAVI device. The balloon-expandable ES was deployed by a single implantation step during RVP. The self-expanding SA device was deployed by a specific two-step implantation technique. The first step consisted of partial release with unsheathing of the stabilization arches and the upper crown, to obtain initial valve positioning with optimal axial alignment. After commissural alignment under gentle retrograde pull, the upper crown was reversibly brought in touch with the calcified leaflets of the native aortic valve until tactile feedback could be felt. In a second step, final placement was achieved with full prosthesis deployment during stable tactile feedback. RVP could be initiated at the surgeon’s preference but was not mandatory.All TA-TAVI procedures were performed in general endotracheal anesthesia using propofol and remi-fentanil. Continuous anesthesia monitoring consisted of ECG, pulse oximetry, capnography, invasive arterial and central venous pressure, transesophageal echocardiography, processed EEG (BIS) and bilateral transcranial Doppler sonography. Postoperatively, patients were either extubated in the angiography suite, or remained intubated until circulatory and respiratory stabilization. Thereafter, all patients were transferred to the intensive care unit.Intraprocedural anticoagulation consisted of IV bolus administration of heparin (70–100 IU/kg) with the goal to achieve an activating clotting time of >250 s throughout the implantation procedure. The oral antiplatelet regimen was started with acetylsalicylic acid (100 mg/d) and a loading dose of 300 mg clopidogrel, which was administered six hours after apical access closure. Patients were discharged with a prescription of acetylsalicylic acid 100 mg/day indefinitely and clopidogrel 75 mg/day for 3–6 months. If oral anticoagulation was indicated, warfarin was combined with either acetylsalicylic acid or clopidogrel.3, Model# PMD 150; Spencer Technologies, Seattle, WA, USA) to provide an optimal signal-to-noise ratio. Sample volume was set to 3 mm for all recordings. Using the 33 gate Power M-mode function for rapid vessel localization, characteristic blood flow velocity spectra were detected with high signal quality by insonation of the MCA at a depth between 46 and 58 mm. Automated HITS monitoring used the multi-depth embolic detection mode with artefact rejection. A post-procedural analysis of all TCD recordings was conducted by one of the authors (GE) in order to confirm the quantity and quality of microembolic signals.Transcranial Doppler ultrasonography and HITS detection were performed as described previously by our group As the primary endpoint of the analysis, the cumulative count of HITS and the specific HITS load related to the following predefined procedural intervals were analyzed:Instrumentation  prior to and until balloon aortic valvuloplasty of the native aortic valve, (IN)Balloon aortic valvuloplasty of the native aortic valve, (BAV)Prosthesis deployment (PD) andPost-implantation period including any post-dilatation maneuvers (PI).The duration of each period was documented and also validated using the interventional protocols. Phase-specific HITS rate was defined as the ratio of HITS count per specific interval duration .GE) prior to the procedure and after TA-TAVI between post-procedural days 1 and 6 by performing a gross neurological status and by confusion assessment using the CAM-ICU questionnaire All patients were examined  for continuous measures. HITS counts were not normally distributed, and were thus compared between different procedural steps using Friedman Repeated Measures ANOVA on ranks; for comparison between the two prosthesis types, Mann-Whitney rank sum test was used. p<0.05 was considered significant. Analyses were performed using SigmaPlot for Windows, Version 10.0 and SigmaStat for Windows, Version 3.0 .Of 31 patients undergoing TA-TAVI during the observation period, 22 patients (71%) displayed MCA spectra of good quality throughout the entire procedure, and were included in the final analysis. SA and ES implantations were equally distributed in each study arm (n = 11).In the remaining 9 patients, TCD monitoring was not performed either due to logistic reasons (4/31) or due to absence of an appropriate temporal bone window (3/31), or data acquisition was terminated prematurely after loss of the MCA signal during the procedure (2/31).Baseline characteristics and procedural data of the study population are summarized in In the SA group, the majority of the patients received size M prostheses (73%). In group ES, sizes 26 mm and 29 mm were implanted in 90% of the cases. Immediate implantation and device success was 100%. Delirium occurred in 7/22 (32%) patients, and disabling stroke in 1/22 (4.5%). The affected patient suffered an ischemic stroke on the day of TAVI as a consequence of severe carotid artery stenosis). Hemispheric HITS count and total HITS occurrence in this patient, however, were not different from the cohort’s . All-cause 30-day mortality was 3/22 (13.6%). Intra- and post-procedural data as well as adverse outcomes were not significantly different between SA and ES groups .Total intraprocedural cumulative HITS count did not significantly differ between devices , nor differed the interval-specific HITS load between SA und ES . This reTotal as well as interval-specific HITS load showed symmetrical distribution to left and right brain hemisphere, and hence, similar time patterns. The devices did not significantly differ from each other when interval-specific HITS load was related to interval duration  .In contrast, the procedural phase itself had a much larger impact: comparisons between procedural intervals demonstrated clearly that valvuloplasty (BAV) and deployment (PD) both had high and very similar phase-specific HITS rates, whereas instrumentation (IN) and post-implantation (PI) had a comparably low HITS rate in common .This study investigated and compared count, frequency and intraprocedural pattern of HITS during TA-TAVI with either the self-expanding Symetis ACURATE TA revalving system or the balloon-expandable Edwards Lifesciences SAPIEN XT setup.The main findings are, first, that both transapical devices release a similar total and interval-specific HITS load during and immediately after implantation. Second, deployment of the prosthesis is by far the period during which most HITS are generated. Independent of the device, balloon aortic valvuloplasty and valve deployment are associated with high and very similar HITS rates, although their duration and absolute HITS count differ.We observed no difference in procedural and clinical outcomes  between SA and ES, although these were not endpoints in our small series.Reduction of the microembolic load generated by TA-TAVI is an essential contribution to improving patient outcomes. New TA-TAVI devices may offer better deployment accuracy or ease of handling, but very little is known about the impact of these changes on cerebral microembolic release. The SA system features single-operator tactile feedback-guided deployment, but by this, may intensify device-calcification interaction prior to final implantation In fact, no significant difference was found between the devices. SA produced a HITS rate and pattern very similar to that of the ES prosthesis. Several reasons may explain these results: user experience with the SA device is shorter than with ES technology, which is implanted already for 7 years in our institution. Thus, an operator-dependent increase in embolic load might still be produced by SA. A controlled prospective study with adequate power and at comparable points in the teamś learning curves may be necessary to observe true device-related differences. In addition, as we did not systematically perform non-contrast enhanced CT scan images, we are not able to investigate the association of overall aortic root calcification and its impact on HITS. However, at this point in time there is no clear evidence for an association between calcification amount or distribution and a higher rate of HITS during TA-TAVI, but will be definitely addressed in future studies. A further limitation of the study is related to the method of HITS detection. HITS were detected automatically using the automated multi-depth embolic detection software with artifact rejection. In this regard HITS detection has been performed according to the manufacturer’s specifications and current standards published in literature on this area. Nevertheless, our equipment was not able to differentiate between gaseous and particulate emboli. Therefore, the results do not allow to make statements on the quality of HITS. In this regard, it is possible that SA and ES exhibited different counts of particulate or gaseous HITS.In view of our sample size we did not expect to see major differences because of the relatively low event rate of stroke or mortality. Although type II error due to low power (β = 0.278) is a clear limitation of the present series, our data appear useful for planning controlled comparative trials in the future. Second, the clinical outcome of patients treated with SA or ES was similar, therefore it is not surprising that the total HITS count is comparable. Apparently, neither the design improvements nor the seemingly advantageous two-step implantation of the SA device influence the mechanism of microemboli generation during TA-TAVI.Today, there is still a very limited number of TCD studies, although the number of TA-TAVI procedures increases rapidly. None so far provided information about HITS occurrence with the SA device operator-independent, intervention-related HITS generation during TA-TAVI. The procedural phase appears to be the leading determinant of the rate of microemboli release to the brain.Matching the present results to those of a previous study from our group allows to compare HITS patterns, owing to an identical definition of procedural phases We conclude that balloon valvuloplasty and valve deployment are still the major contributors of intraprocedural HITS generation, most likely due to mechanical friction of the introducer system and/or the device itself within the aortic root. In this regard, the novel Symetis ACURATE TA system showed at least no inferiority to transapically implanted Edwards SAPIEN XT valves.The results of this study indicate that the device-specific implantation technique and stent configuration of the Symetis ACURATE TA prosthesis are not associated with a substantial increase or decrease of cerebral microembolism. The similarity in HITS patterns of a variety of devices supports the hypothesis of a common pathomechanism for cerebral microembolism in TA-TAVI. Most probably this is related to hardware-tissue friction in the aortic root during balloon valvuloplasty and valve deployment. The evolution of next-generation TA-TAVI prostheses should also focus on improved deployment techniques which are less traumatic when performed in degenerated and calcified surfaces of the aortic root.


Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of “Methylation-Insensitive Primers” (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies. This difference was much more evident in brain and muscle tissues: all the cytosines (except the 1149) resulted significantly hypomethylated when the methylation assay is performed using MethPrimers vs. MIPs in the most methylated condition . It is therefore evident that in experimental conditions in which DNA methylation is high MethPrimers show low power of detection compared to MIPs, eventually failing to evidence differences between differential methylation in two samples.12 cells  and in m12 cells . When mythylated  vs. hypothylated  experimePSEN1 in SK-N-BE cells grown in hypermethylating (S-adenosylmethionine supplemented) or hypomethylating (B vitamin deficiency) conditions  between amplification efficiency and DNA methylation level. This result indicates that the more DNA is non-CpG methylated, the less MethPrimers are able to bind to the bisulfite-modified product indicating that these primers negatively select the high methylation (poorly modified) DNA fraction in the sample. As expected, MethPrimers failed to amplify untreated PCR products whereas MIPs show similar amplification efficiency for both treated and untreated PCR fragments.In order to confirm these observations by an independent approach, we also compared, by real-time PCR on bisulphite-modified genomic DNA, the amplification efficiency of each primer set respect to differential methylation statuses . Fig. 2Amyogenin were already published  x100). Then, we calculated the average methylation % over the replicated cell culture experiments or over the 4 tissue samples for each experimental condition. Raw data related to DNA methylation results are shown in DNA was extracted from cells and tissues by classical phenol-chloroform method . BisulphGenBank accession numbers, primer names, sequence and position, expected products and annealing temperatures of the Methylation Insensitive Primers (MIPs) used for bisulphite analysis were already published , 10. Seqmyogenin and PSEN1 promoter, obtained from genomic DNA as template with the same MIPs primers used for bisulphite PCR; the same purified PCR products where methylated in vitro with SssI methylase (New England Biochemistry), that methylates only cytosines in CpG dinucleotides, and were used as positive controls. We adopted all the possible cautions and controls to be sure that no methodological troubles could bias our analysis. In particular: 1) DNA samples to be compared were purified in parallel and modified in the same bisulfite assay; 2) amplifications with MIPs and with MethPrimers were performed on two aliquots of the same bisulfite-modified sample; 3) PCR products obtained by both MIPs and MethPrimers were always cloned in the same assay; 4) positive and negative controls were always used in each bisulfite assay; 5) clones were sequenced using two different instruments (the in-lab Applied Biosystems instrument and in service by Primm).We also used different bisulphite modification assays as random control in samples characterized by low and high (CpG and non-CpG) methylation to ensure that cytosine conversion was complete. In particular, standard bisulphite procedures  with modmyogenin as reference gene when assessing human PSEN1 amplification efficiency and exogenous human PSEN1 when assessing mouse myogenin amplification efficiency. To this end, standard amount (50 ng) of mouse myogenin or human PSEN1 PCR product were respectively added to each sample before splitting it in the tubes for the amplification. We preliminary verified that primers used to amplify mouse myogenin were not able to amplify myogenin in human samples and that those used to amplify human PSEN1 were not able to amplify PSEN1 in mouse samples. Additionally, bisulphite-treated and bisulphite-untreated PCR products were amplified as controls; as expected, MethPrimers failed to amplify untreated PCR products.SybrGreen PCR real-time assay was performed to assess the efficiency of amplification of MIPs and MethPrimers in samples with differential DNA methylation levels. 1 μg of DNA was used in each real-time reaction, performed in triplicate as previously described . Data arPSEN1 promoter: i) PvuII has one recognition site at the cytosine 862 of the sequence and is sensitive to CpT methylation; ii) EcoNI has one recognition site at the position 1204 and is methylation insensitive; iii) EcoRI has no recognition sites. The digested samples were then amplified as previously described by the same couple of MIPs primers used for bisulphite analysis. All the electrophoresis gels were analyzed using a computerized densitometer . Controls with heat-inactivated endonucleases and on non-methylated PCR products were performed. Specificity of digested samples was confirmed by molecular weight comparison with DNA molecular weight markers and by sequencing.DNA digestion and PCR reactions were performed as previously described . BrieflyStatview statistical software was used to calculate any significant difference reported in this paper. Histograms show the mean value ± s.d. Asterisks in figures evidence the statistically significant differences; differences lacking of remarks are to be considered non-significant.Analysis on methylation data was performed using non-parametric tests since the experimental method we applied  results in percent values (methylation %) for many cytosines  in each sample. Mann-Wittney test was used to calculate inter-sample differences (i.e. when comparing HM and LM samples); Wilcoxon test was used to calculate intra-sample differences (i.e. when comparing MIPs and MethPrimers data).One-way ANOVA was computed and Bonferroni post-test was used to evaluate any significant difference in the Real-Time PCR assays. To assess the correlation between quantitative variables, we computed the linear correlation coefficient r (Pearson’s) with the corresponding significance level.Work on human subjects: post-mortem brains used in the present study were obtained from the Institute of Neuropathology and Brain Bank (HUB-ICO-IDIBELL Biobank) following the guidelines of the Declaration of Helsinki, and according to the Spanish and Catalonian Autonomous regulations on this matter, and the approval of the local Ethics Committee of the Bellvitge University Hospital. Work on vertebrate animals: embryonic mouse tissues used in the present study were collected in the Dept. of Surgery “P. Valdoni”, Sapienza University of Rome after Institutional and National approval according to the EU laws.S1 Figmyogenin (up) and human PSEN1 (down) 5’-flanking regions. Numbers on the left of the DNA sequences indicates the base-number as prorated in the GeneBank sequences. MethylC-Insensitive Primers (MIPs) are indicated in bold-blue and MethPrimers are indicated in bold/underline. b) Characteristics of the oligonucleotides used as primers to investigate mouse myogenin and human PSEN1 methylation. The position of each primer is indicated below the sequence.a) Schematic representation of the investigated region in mouse (PDF)Click here for additional data file.S2 Figmyogenin promoter. Light grey columns represent the result obtained by using MIPs, whereas dark grey columns represent the results obtained by using MethPrimers. a) C2C12 in 10% FCS (high methylation); b) C2C12 in 1% FCS (low methylation); c) Embryonic brain (high methylation); d) Embryonic muscle (low methylation). Detection of non-CpG methylation is clearly defective when MethPrimers are used, particularly in high methylation conditions.Histograms show the methylation percent measured for each (both CpG and non-CpG) cytosine in the investigated region of the mouse (PDF)Click here for additional data file.S3 FigPSEN1 promoter. Light grey columns represent the result obtained by using MIPs, whereas dark grey columns represent the results obtained by using MethPrimers. a) SK-N-BE + SAM (high methylation); b) SK-N-BE in B vitamin deficient medium (low methylation); c) Cortical brain tissue from control subjects (high methylation); d) Cortical brain tissue from Alzheimer’s Disease subjects (low methylation). As for myogenin, it is evident, also in PSEN1 promoter, that detection of non-CpG methylation is defective when MethPrimers are used, particularly in high methylation conditions.Histograms show the methylation percent measured for each (both CpG and non-CpG) cytosine in the investigated region of the human (PDF)Click here for additional data file.S4 FigPSEN1 promoter in high methylated (HM) and low methylated (LM) samples. PvuII is inhibited when the target sequence is methylated on the CpT moiety, as schematized in the table. PCR after incubation with the enzyme shows that HM sample and (at lower level) LM sample are incompletely cut, indicating the presence of non-CpG methylation. Use of methylation-insensitive endonuclease (EcoNI) and of unmethylated controls (PCR products) demonstrate that the DNA is not resistant for other intrinsic factors and that PvuII is able at cutting when the target sequence is unmethylated. MM: Molecular weight marker; PvuII Inact.: heat inactivated PvuII.Non-CpG methylation was confirmed in SK-N-BE cells by methylation-sensitive endonuclease assay on (PDF)Click here for additional data file.S5 FigRepresentation of the raw methylation data for each gene/experimental condition/sample/sequenced clone. Black boxes represent methylated cytosines, white boxes represent unmethylated cytosines, as described in the graphic legend.(PDF)Click here for additional data file.


Due to the practicalities of recruitment, randomisation of participants in surgical trials may need to take place prior to the day of surgery. However, substantial post-randomisation delays in receiving surgery due to limited capacity may then occur. This can create a tension between timing the follow-up from randomisation  and surgery . It is unclear what impact alternative follow-up timings have upon the outcome.TOPKAT compared the clinical and cost effectiveness of total or partial knee replacements for medial compartment osteoarthritis. An additional assessment at 1 year post surgery of the primary outcome measure, Oxford Knee Score (OKS) was administered to patients who had a waiting time following randomisation of greater than 12 weeks as well as baseline and post-randomisation follow-up. Mean difference (MD) between treatments ) was calculated using 1 year post-surgery and post-randomisation data.Waiting times for surgery (0-407 days) resulted in a number of participants (n=134 of 531 recruited) receiving their 1 year post randomisation follow up questionnaire at a time point much earlier than 1 year post surgery. However, the mean difference in OKS was very similar for post randomisation ) and surgery ) analyses.Results of the 1 year post-randomisation and surgery follow-ups were very similar. Further assessment in other trials is required to explore the generalisability of this finding. Timing of follow up needs to be carefully chosen to ensure interpretable results.


The expression of functional B cell antigen receptors (BCRs) is essential for development, survival and activation of B cells. During early developmental stages, immature B cells exclusively express IgM. Cells that pass the immature B cell stage and leave the bone marrow coexpress BCRs of IgM and IgD isotypes, which differ in the usage of the heavy chain (HC) μ versus δ that are generated by alternative poly-adenylation and splicing. It is currently unclear how the prevalent expression of the μHC over δHC at the early stages of B-cell development is reverted in mature B cells, where IgD becomes the dominant antigen receptor. The reason for this tightly regulated expression and the individual function of IgD versus IgM are poorly understood. Another remarkable difference is that μHC can be found both as membrane-associated BCR and soluble antibody, whereas IgD antibodies are barely detectable in the serum and δHC is predominantly part of membrane-associated IgD BCR.in vitro [in vivo by selective expression or deletion of either μHC or δHC in transgenic animal models. This showed that, while IgM and IgD may largely substitute for each other, the development of innate-like B cells was impaired in IgM knockout mice whereas IgD knockout B cells show defects in affinity maturation [This points to a cell-associated signaling specific function of IgD, which is supported by experiments suggesting that activation IgD as compared with IgM BCR leads to specific kinetics of phosphorylation of intracellular proteins in vitro . The rolturation –3.Continuous exposure to soluble neo-self-antigen in transgenic mice expressing hen-egg-lysozyme (HEL) and the cognate BCR (IgHEL) manifests with downregulation of surface IgM while IgD expression is unaffected . Periphein vitro reconstitution system, model BCRs including the IgHEL were investigated as IgM and IgD receptors bearing the same antigen specificity. Surprisingly, the tested BCRs responded to treatment with low-valence antigens, such as soluble HEL, only when expressed as IgM but not when expressed as IgD BCR. Treatment with multivalent antigens however resulted in comparable activation of all receptors [Using an eceptors .These data suggested that anergic B cells might not respond to the treatment with soluble monovalent antigens and maintain IgD expression on B cells simply because IgD requires polyvalent antigen for stimulation. Testing this hypothesis on splenic cells revealed that anergic B cells from IgHEL transgenic mice are fully responsive to polyvalent antigen. Characterization of the molecular mechanism in more detail identified the hinge region in the heavy chain of IgD as the essential element for the distinctive IgD function. It seems that the hinge region allows the two arms of IgD to act as pincers that promote binding of low-valence antigen by one IgD, thereby preventing BCR-BCR interaction.Together, it is tempting to speculate that anergy is a regular step of normal B cell development towards mature B cells and that soluble self-antigens are involved in the generation of mature B cells. Moreover, the increased expression of IgD provides mature B cells with an antigen receptor, which is optimized for activation by multimeric immune complexes and for efficient recruitment into T cell-dependent immune responses. Intriguingly, an additional level of regulation emerges as monovalent antigens may interfere with polyvalent antigens for IgD binding. In fact, soluble HEL prevents the activation of IgHEL splenic cells expressing IgD BCR by multimeric HEL. Thus, it is conceivable that soluble self-antigens, while contributing to the maturation of B cells, block mature B cell activation by interfering with immune complexes containing self-antigen. It seems that the balance between soluble and multimeric antigen in immune complexes is an important parameter for mature B cell activation. This balance might be shifted under conditions of chronic inflammation or infection where immune complexes containing self-antigens may be increased thereby leading to chronic B cells activation and eventually autoimmune diseases or continuous proliferation. This scenario points to the potential use of soluble auto-antigens to control autoimmune diseases or lymphoproliferative disorders if the abnormal cells express IgD. On the other hand, the ratio of soluble versus complex antigen might be a key parameter for the design of protective immunization and vaccination as IgD expression is optimal for recruitment into T cell-dependent immune responses, which include the generation affinity-matured memory cells. Since IgG-type BCRs expressed on memory B cells also contain a hinge region similar to IgD, it is also conceivable that memory B cell responses are also regulated by the ratio of low-valence to multi-valence antigen.The emerging scenario suggests that the expression of IgD raises the activation threshold, renders cells inducible selectively by complex antigen and directs the cells towards memory responses, while the control by low-valence antigens contributes to B cell maturation and tolerance. On the other hand, the high sensitivity of IgM BCR may be important for stringent selection of early immature B cells and may also confer transformed cells with a receptor isotype that efficiently reacts to multiple stimuli including low-valence antigen.


The nanoscale chip-integrated all-optical logic parity checker is an essential core component for optical computing systems and ultrahigh-speed ultrawide-band information processing chips. Unfortunately, little experimental progress has been made in development of these devices to date because of material bottleneck limitations and a lack of effective realization mechanisms. Here, we report a simple and efficient strategy for direct realization of nanoscale chip-integrated all-optical logic parity checkers in integrated plasmonic circuits in the optical communication range. The proposed parity checker consists of two-level cascaded exclusive-OR (XOR) logic gates that are realized based on the linear interference of surface plasmon polaritons propagating in the plasmonic waveguides. The parity of the number of logic 1s in the incident four-bit logic signals is determined, and the output signal is given the logic state 0 for even parity (and 1 for odd parity). Compared with previous reports, the overall device feature size is reduced by more than two orders of magnitude, while ultralow energy consumption is maintained. This work raises the possibility of realization of large-scale integrated information processing chips based on integrated plasmonic circuits, and also provides a way to overcome the intrinsic limitations of serious surface plasmon polariton losses for on-chip integration applications. An all-optical logic parity checker has the function of determining the parity of the number of logic 1 signals propagating in optical computing systems and thus determining whether the logic signals are correctly transmitted, i.e., the logic signals are correct for an even number of logic 1s, while an error exists in the logic signals for an odd number of logic 1s234672, which is four orders of magnitude lower than that of previously reported devices based on semiconductor optical amplifiers and third-order nonlinear optical materials2345678Here, we report a simple and effective strategy for direct realization of a nanoscale chip-integrated all-optical logic parity checker with ultracompact feature size, ultralow energy consumption, and high contrast ratio in an integrated plasmonic circuit. The all-optical logic parity checker consists of two-level cascaded exclusive-OR (XOR) logic gates, which are realized on the basis of the linear interference of surface plasmon polaritons (SPPs) propagating in U-shaped plasmonic waveguides. When compared with conventional plasmonic slot waveguides, the distinctive features of the U-shaped plasmonic waveguides lie in their strong light-field localization, relatively low propagation losses, high transmission around sharp bends, and high tolerance to structural imperfections11et al.et al.The U-shaped plasmonic waveguide structure is shown schematically in et al. showed that the intensity contrast ratio between the output logic values of 1 and 0 was calculated from 10·log(P1/P0), where P1 and P0 are the signal intensities of the logic 1 and logic 0, respectivelyThe all-optical logic XOR gate has an asymmetric Y-shaped configuration, as shown in The sample all-optical logic parity checker consists of two-level cascaded XOR logic gates that were formed using U-shaped plasmonic waveguides, as shown in 2 in our experiment, and this represents a reduction of four orders of magnitude when compared with the corresponding values from previous reports12345678et al. noted that the ultralow incident signal light intensity of only 100 kW/cm2 is comparable with that of efficient ultralow-power all-optical switching devices based on silicon photonic crystal nanocavitieset al.Because of the strong field confinement effect of U-shaped plasmonic waveguides, the minimum feature size of the sample all-optical parity checker was only 15 μm, which is more than two orders of magnitude smaller than the previously reported results12345678The output logic state of the sample all-optical parity checker described above not only reflects the parity of the logic 1 in the incident four-bit logic signals, but can also be used as the parity bit for the incident logic signals. The sample all-optical parity checker can therefore also function as an all-optical parity generator for the incident four-bit logic signals. We can add a parity bit, i.e., the output logic state of the all-optical parity checker sample, to the last part of the incident logic signal to form new five-bit logic signals where the lowest digit is used as the parity bit. The strategy proposed here can also easily be extended to realize the functionality of an all-optical checker and generator to be applied to multi-bit complex logic signals by simply increasing the number of XOR gates in the first and second levels. This work therefore not only paves the way towards the construction of ultrahigh-speed and ultrawide-band information processing chips and optical computing systems based on plasmonic nanostructures, but also offers a way to overcome the intrinsic obstacle of the SPPs for ultralarge-scale on-chip integration applications. These results are completely different to the results obtained in ref. et al. noted that a high quality factor of more than 4000 can be obtained in a plasmonic coupled resonator optical waveguide, and this indicates that plasmonic coupled resonator optical waveguides are excellent candidates for construction of the delay line unit for the data streamet al. noted that plasmonic coupled resonator optical waveguides could also be constructed using coupled plasmonic coplanar nanocavities formed by several defect units in a one-dimensional plasmonic crystal consisting of an array of periodic nanogratings etched at the center of a plasmonic waveguideet al. also stated that this type of plasmonic coupled resonator optical waveguide was highly suitable for integration into a plasmonic platform with a waveguiding geometryIn an actual data stream, the bit signals appear sequentially. Wood In summary, we have directly realized a nanoscale chip-integrated all-optical logic parity checker in integrated plasmonic circuits. An overall minimum feature size of 15 μm, ultralow energy consumption, and an ultrahigh intensity contrast ratio between the output logic states of 1 and 0 of 30 dB were realized simultaneously. This work not only opens up the possibility of realization of large-scale integrated information processing chips based on integrated plasmonic circuits, but also offers a way to overcome the intrinsic limitation of the serious losses of SPPs for on-chip integration applications.2. A focused ion-beam etching system  was used to prepare the U-shaped plasmonic waveguides, the XOR gates, and sample parity checkers.300-nm-thick gold films were fabricated using a laser molecular beam epitaxy (LMBE) system . We used the output beam (at an operating wavelength of 248 nm with a pulse repetition rate of 5 Hz) from an excimer laser system  as the excitation light source. The beam was focused on a gold target mounted on a rotating holder that was located 15 mm from the silicon dioxide substrates. The typical energy density of the excitation laser was approximately 450 mJ/cmIn the experiments, a nano-spectroscopy measurement system was used to measure both the waveguiding properties of the plasmonic waveguides and the logic performance of the sample all-optical logic parity checker. The input-coupling grating was normally illuminated from the rear using a home-made fiber laser system. The optically-thick gold film can prevent the direct transmission of the incident laser beam. The incident signal laser beam was focused into a 30-μm-diameter spot. The center of the focused laser beam was located at the midpoint of a line segment formed by input-coupling ports A and D of the sample all-optical logic parity checker, and at the midpoint of a line segment formed by input-coupling ports A and B of the sample all-optical logic XOR gate. This guarantees that all input-coupling ports can be excited uniformly, i.e., the incident signal intensities would have almost identical values for all input-coupling ports. The line width of the laser spectrum curve was less than 1.5 nm, thus ensuring that only the desired quasi-monochromatic SPPs can be excited by the input-coupling gratings. The guided SPP mode was scattered by a decoupling grating etched into the output port of the plasmonic waveguide. The scattered light was then collected using a long working distance objective , and imaged using a CCD imager. The intensity of the scattered light at the decoupling port was obtained directly from the CCD image, and no normalization was required.How to cite this article: Wang, F. et al. Nanoscale on-chip all-optical logic parity checker in integrated plasmonic circuits in optical communication range. Sci. Rep.6, 24433; doi: 10.1038/srep24433 (2016).


This report investigates the influence of surface chemistry (or wettability) on the evaporation performance of free-standing double-layered thin film on the surface of water. Such newly developed evaporation system is composed of top plasmonic light-to-heat conversion layer and bottom porous supporting layer. Under solar light illumination, the induced plasmonic heat will be localized within the film. By modulating the wettability of such evaporation system through the control of surface chemistry, the evaporation rates are differentiated between hydrophilized and hydrophobized anodic aluminum oxide membrane-based double layered thin films. Additionally, this work demonstrated that the evaporation rate mainly depends on the wettability of bottom supporting layer rather than that of top light-to-heat conversion layer. The findings in this study not only elucidate the role of surface chemistry of each layer of such double-layered evaporation system, but also provide additional design guidelines for such localized evaporation system in applications including desalination, distillation and power generation. This paper studies the impact of surface chemistry on the evaporation performance of the localized solar-driven evaporation system using anodic aluminum oxide (AAO)-based gold nanoparticle (AuNP) films. Through surface group functionalization and modulation, the surface chemistry of the top light-to-heat conversion layer and the bottom supporting layer were tailored to study the effect . The stuhow and to what extent the surface chemistry of both the top light-to-heat conversion layer and the bottom supporting layer in the floating double-layered geometry controls the speed of water evaporation.Evaporation is a critical phase change phenomenon in many industrial processes, such as in desalination, distillation, and power generation12455781011121314157559In this study, nanoporous AAO membrane was used as the bottom supporting layer to provide mechanical stability for the top AuNP film layer. AAO membrane, possessing superior transport and the tunability in selectivity, has drawn considerable attention in a wide range of chemical and biological applications, especially in chemical separation, catalysis, nanotemplating and biosensor19202111/ml) was filtered by a nanoporous AAO membrane (Whatman®6809-6002 Anodisc: pores of 200-nm diameter on bottom surface and pores of 20-nm diameter on top surface) through a vacuum filtration process and a shiny dark-red AuNP film was generated , while other parameters of the setup were kept the same. As shown in Further investigation on AANF’s evaporation performance under strong light intensity was carried out by employing a Fresnel lens  to focus the Xenon light upon the surface of AANF  to quantify the difference in evaporation rate between hydrophilic and hydrophobic AAO-based AANFs under specific incident light intensity  and RAANF(B) are the evaporation rates of hydrophilic and hydrophobic AAO-based AANFs, respectively; RHLA and RHBA are the evaporation rates of hydrophilic and hydrophobic AAO membrane, respectively; Rr is the difference between relative evaporation rates. The calculated ΔRr under different incident light intensity is shown in Rr = 0.39, intensity = ~3.2 kW/m2; ΔRr = 2.23, intensity = ~14.3 kW/m2), which shows that plasmonic nanoparticle film can differentiate the evaporation rates of hydrophilic and hydrophobic AAO during solar light driven evaporation and the difference can be amplified when the incident light intensity increases. Due to the relatively large fluctuation in evaporation during the first minute as the evaporation stabilizes, only evaporation rates between 1 and 12 minutes are shown in where With the flexibility of controlling the chain length of alkyl thiols, we also tailored the organic tether group attached to the AuNP film surface by using thiols with different carbon chain lengths and studied the evaporation performance of HBN-HLA. Different thiols could potentially change the optical absorption performance of the AuNPs and resulted in the difference in heat localization and evaporationTo help understand the AANF evaporation performance, we further examined the morphologies of nano-porous AAO membranes by SEM and proposed an evaporation mechanism of AAO-based film in The evaporation performance of AAO membranes covered with AuNP films, however, was different. When AANF is exposed to simulated solar light radiation, the surface temperature of AuNP film will be increased due to the highly efficient light-to-heat conversion2) illumination, small bubbles formed and merged under hydrophobic AAO-based AANFs and the cycle of bubble formation, growth and departure were in the time scale of several minutes. In contrast with weak light, under strong light (~14.3 kW/m2) illumination, huge bubbles (~one or two centimeters in diameter) formed and the cycle length of bubble formation, growth and departure was reduced to the time scale of ~10 s of seconds. These bubbles formed under hydrophobic AAO-based AANFs can act as insulating layer, which might leads to smaller temperature rise at the water surface and lower evaporation rate than those of hydrophilic AAO-based AANFs . The IR camera was calibrated using a calibrated thermocouple  with the heating of AANF floating on the surface of water using an electronic heater. The measurement uncertainty of the calibrated IR camera is ~1.0 °C in the temperature range we studied.4) was prepared by dissolving tetrachloroauric acid  in water. 50 ml of the HAuCl4 aqueous solution was boiled in a 100-ml round-bottom flask with a condenser attached and the solution was intensively stirred during heating. After adding 2.5 ml of 38.8 mM sodium citrate to the boiling solution, the mixture was boiled for 15 minutes and stirred for additional 1 hour until the solution cooled down.Solution of 38.8 mM trisodium citrate dihydrate (99% Aladin) was made by directly dissolving sodium citrate powder in DI water . Aqueous solution of 1 mM tetrachloroauric acid  AuNPs were synthesized by stepwise NHThe as-prepared 100-nm colloidal gold nanoparticle solution was placed in an undisturbed container to allow for the nature precipitation. The 100-nm particles precipitated at the bottom after two weeks. We collected the precipitates and re-dispersed them in DI water. Concentrated colloidal gold nanoparticle solutions were stored at room temperature for future use. All the experiments in this report used such concentrated 100-nm colloidal AuNP solutions.How to cite this article: Yu, S. et al. The impact of surface chemistry on the performance of localized solar-driven evaporation system. Sci. Rep. 5, 13600; doi: 10.1038/srep13600 (2015).


We aim to demonstrate the application of free-breathing prospectively gated carbon nanotube (CNT) micro-CT by evaluating a myocardial infarction model with a delayed contrast enhancement technique. Evaluation of murine cardiac models using micro-CT imaging has historically been limited by extreme imaging requirements. Newly-developed CNT-based x-ray sources offer precise temporal resolution, allowing elimination of physiological motion through prospective gating. Using free-breathing, cardiac-gated CNT micro-CT, a myocardial infarction model can be studied non-invasively and with high resolution. Myocardial infarction was induced in eight male C57BL/6 mice aged 8–12 weeks. The ischemia reperfusion model was achieved by surgically occluding the LAD artery for 30 minutes followed by 24 hours of reperfusion. Tail vein catheters were placed for contrast administration. Iohexol 300mgI/mL was administered followed by images obtained in diastole. Iodinated lipid blood pool contrast agent was then administered, followed with images at systole and diastole. Respiratory and cardiac signals were monitored externally and used to gate the scans of free-breathing subjects. Seven control animals were scanned using the same imaging protocol. After imaging, the heart was harvested, cut into 1mm slices and stained with TTC. Post-processing analysis was performed using ITK-Snap and MATLAB. All animals demonstrated obvious delayed contrast enhancement in the left ventricular wall following the Iohexol injection. The blood pool contrast agent revealed significant changes in cardiac function quantified by 3-D volume ejection fractions. All subjects demonstrated areas of myocardial infarct in the LAD distribution on both TTC staining and micro-CT imaging. The CNT micro-CT system aids straightforward, free-breathing, prospectively-gated 3-D murine cardiac imaging. Delayed contrast enhancement allows identification of infarcted myocardium after a myocardial ischemic event. We demonstrate the ability to consistently identify areas of myocardial infarct in mice and provide functional cardiac information using a delayed contrast enhancement technique. Yet there are significant limitations to this approach: 2-D ultrasound images cannot easily distinguish between nonfunctional infarct, ischemic, stunned, hibernating, and healthy myocardium, as these differences are largely functional rather than structural on the length scales visible with available in-vivo murine imaging modalities. The current gold standard for determining myocardial tissue viability in the clinical setting is magnetic resonance imaging (MRI) with delayed contrast enhancement, which combines the ease and high spatial resolution of structural imaging with a contrast administration protocol that illuminates necrotic myocardial tissue [The leading cause of mortality worldwide is cardiovascular disease. The World Health Organization’s most recent reports estimate that a total 17.3 million people died in 2008 as a result cardiovascular disease, or 30% of all global deaths, with 7.3 million attributed specifically to coronary heart disease [Delayed contrast imaging is made possible because the gadolinium-based vascular contrast agents used in MR imaging distribute themselves in the extracellular spaces of healthy myocardium soon after intravenous administration, while these agents are too large to be admitted into the normal myocardial cells. However, the cellular changes resulting from ischemia affect arterial flow rate and the permeability of the capillaries. Within necrotic and acute infarcted regions of myocardium, a loss of membrane integrity allows contrast agent molecules to leak from capillaries into these regions, and contrast is retained in these tissues past the typical wash-out time in uninjured extracellular spaces. The result is a characteristic delayed enhancement in necrotic regions of myocardium captured in MR images acquired during an appropriate time window during which gadolinium has leeched into infarcted tissue but has not yet leeched out again. This phenomenon has already been successfully demonstrated in murine MRI imaging studies . The samSurgically-induced ischemia and reperfusion of the LAD is also expected to significantly inhibit left ventricular myocardial function, decreasing the heart’s ability to contract and pump blood. Any change in cardiac output can be quantified by a comparison between the ejection fractions of subjects, which have undergone ischemia and reperfusion and the ejection fractions of control subjects. When blur-free micro computed tomography (micro-CT) images can be obtained from live murine subjects during diastolic and systolic cardiac phases, accurate ejection fractions can be calculated as a metric of left ventricular function. Thus, high quality cardiac micro-CT, when achieved, provides both structural and functional information about the murine heart following ischemia and reperfusion.In general, the ability to acquire high quality murine cardiac images using traditional micro-CT demands a high temporal resolution, which is challenging to achieve, since subjects experience rapid cardiac and respiration rates. Full scan times of 10–15ms would be necessary to complete a full, motion-free CT scan during a single systolic phase, but this is not possible with current technology due to gantry speed and x-ray tube flux limitations. Instead, physiological gating is necessary for in vivo cardiac imaging of mice, acquiring x-ray projection images only during the phase of interest over several successive cardiac cycles. Retrospective gating techniques allow for rapid scan times but involve several gantry rotations per scan and subsequently deliver a higher radiation dose to the subject .et al. and in small animal studies using micro-CT [Delayed iodine contrast enhancement of myocardial tissue in murine models has previously been demonstrated by Nahrendorf, micro-CT . Though micro-CT  and whicin-vivo micro-CT are overcome with the development of a micro-CT with carbon nanotube (CNT) cathode [Many of the difficulties of traditional  cathode . The dev cathode . By redu cathode , 11. X-r cathode . In the Here, we evaluate a murine model for acute myocardial infarction using the CNT micro-CT and a delayed contrast enhancement technique with an iodinated contrast agent administered in a bolus. The surgical model for ischemia and reperfusion was produced by surgical occlusion of the left anterior descending artery (LAD) with a suture. Ischemia was maintained for thirty minutes and was followed by twenty four hours of reperfusion prior to obtaining CT images. Two different iodine-based contrast media were administered to the subjects via tail vein catheter. Iohexol 300 mg/mL  was chosen to demonstrate the delayed contrast effect due to its rapid distribution and quick wash-out. Fenestra VC  , with diThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill (IACUC ID: 09-340.0). All surgeries were performed under sodium pentobarbital anesthesia, and all micro-CT imaging and contrast administration was performed under vaporized isoflurane anesthesia.Myocardial infarction was induced in eight C57BL/6 male mice aged 8–12 weeks (∼30g) (Jackson Laboratories) one day prior to the imaging study. The ischemia reperfusion model was achieved by surgically occluding the LAD artery for 30 minutes followed by 24 hours of reperfusion . In summTail vein catheters were placed for contrast administration immediately prior to imaging. During the imaging procedure, subjects were anesthetized using isoflurane vapor in medical grade oxygen . Animals breathed freely throughout the entire imaging procedure, preventing possible complications that could arise from intubation and forced ventilation . To faciThe CT imaging of this study was performed on a custom-built cone beam micro-CT system which consists of a field-emission x-ray source and a flat panel detector, both mounted on a step-and-shoot rotating gantry . The x-rAll CT images were acquired during the end exhalation phase of respiration and with a 0 or 55 msec delay following the R-wave, corresponding to the diastolic or systolic cardiac phases, respectively. The protocol, including contrast administration and image acquisition times, is displayed in 7 wild-type mice were also imaged in a similar protocol as controls. Physiologically-gated micro-CT images of these animals were obtained during the diastolic and systolic cardiac phases after administration of the iodinated lipid blood pool agent. These images were used as a baseline comparison for ejection fraction measurements.Images were acquired in a step-and-shoot protocol with 286 projections over a total of 200 degrees gantry rotation. Each x-ray projection exposure was 15 msec in duration with 50 kVp energy and a 3 mA cathode current. The system control software was written in Labview  and used to synchronize the flat-panel detector, operating on a 1 Hz fixed frame rate, and the x-ray source with the physiological trigger signals. The absolute minimum scan time of the system is 286 seconds to allow for gantry rotation and image readout. Synchronization requirements imposed by a physiologically-gated protocol increase the scan time to an approximate minimum of ten minutes per scan, although the exact scan duration is dependent upon individual cardiac and respiration rates.After acquisition, projections were preprocessed with a script in MATLAB  to eliminate defective detector pixels and lines and then reconstructed to a 3-D volume with commercial reconstruction software . As a part of 3D reconstruction, Hounsfield unit correction was performed to normalize to known attenuations of water and air. Scanner resolution after reconstruction was 77 μm isotropic.3.Using a partially-automatic segmentation algorithm based on volume-growing region competition snakes in the Dicom viewer ITK-SNAP , micro-CDia and VSys, as EF = (VDia − VSys)/VDia.CT estimates of LV volumes are commonly used in the clinic for calculation of ejection fraction . Using hA MATLAB program was written to measure infarcted tissue volumes from the reconstructed 3-D image sets of the first Iohexol-enhanced CTs acquired for each subject. HU thresholds were defined in order to segment and measure the total volumes of infarct and of the LV myocardium. Infarct sizes are reported as a percentage of the total LV myocardium for best comparison with histology.After imaging, subjects were sacrificed and their hearts were cut into 1mm slices and stained with triphenyl tetrazolium chloride (TTC) to visualize uninjured and necrotic tissues. Slices were digitally photographed (both front and back) with an Olympus DP71 digital camera  with 200 ISO speed and 0.71 s exposure time. The resulting photographs were stored in jpeg format with dimensions of 1360 × 1024 pixels and 144 dpi.A MATLAB program was written to analyze each digitized histological slice to segment and measure the areas of myocardium with negative TTC stain uptake corresponding to infarcted tissue. The calculated infarct volume was reported as a percentage of total myocardium volume for each subject to eliminate the effects of tissue shrinkage, and a statistical comparison was made with the percent infarct volume obtained from CT images.All eight animals survived the surgery, and all survived the delayed enhancement imaging portion of the protocol. One animal with the most extensive damage to the myocardium did not survive to be imaged for the lipid blood pool agent imaging and ejection fraction measurements. High quality CT images were obtained for all surviving animals. During CT acquisition, the mean respiration rate of the ischemia reperfusion model mice was 108 ± 17 bpm and the mean cardiac rate was 420 ± 70 bpm. Obvious delayed contrast enhancement following Iohexol administration was seen in the LV wall in CT images for all subjects. The blood pool contrast agent revealed changes in cardiac function which were quantified by low ejection fractions. All subjects demonstrated areas of myocardial infarct in the LAD distribution in reconstructed CNT micro-CT images; these regions corresponded with the areas lacking TTC stain uptake in the histological results.Axial cross-sections of the micro-CT images for a representative subject are displayed in grayscale in CT numbers within regions of interest comprised of the blood pool, myocardium, and infarcted tissues were measured from the first two CT images acquired of each subject. They are plotted as HU over time in Delayed hyper-enhancement occurs in all imaging time-points following the administration of Iohexol but is strongest during the first image acquisition. While the CT numbers for blood and infarct are similar in many of the images (411 ± 56 HU and 431 ± 111 HU during the first image acquisition), the two are easily distinguishable within the context, since the infarct region is always imbedded within the myocardial wall. Both blood and infarct are clearly distinguishable from myocardium in all Iohexol-enhanced images (p < 0.0001) during the first of the two imaging time points. Because of the acquisition time for physiologically-gated micro-CT protocols (∼10–20 minutes), the initial wash-in portion of delayed enhancement could not be seen. However, the first CT image, acquired an average of 13 minutes after contrast injection, was optimal for visualizing hyper-enhancement of the infarcted region. If acquired within the appropriate time window, only one image is required to visualize the infarcted regions.A visual comparison was made between the delayed contrast micro-CT axial slices  and TTC-2 = 0.6897 to R2 = 0.8329.This relationship is quantified in In addition to the structural changes within the myocardium resulting from ischemia and reperfusion, micro-CT images provide information on heart function via ejection fractions, calculated from the images acquired during the diastolic and systolic cardiac phases . The aveThe results of CT image analysis and TTC-stained histological analysis confirm that the location, shape, and general size of the infarcted regions generally agree between modalities. While the average percent infarcted tissue from histological analysis is slightly greater than that derived by CT (32.2 ± 10.7% vs. 30.5 ± 7.8%), the difference may be partially explained by the non-isotropic spatial resolution of the images of the TTC-stained gross slices. Specifically, while reconstructed micro-CT images have a resolution of less than 80 microns in x, y, and z directions, photographing both the front and back of each 1mm TTC-stained slice results in an effective out-of-plane spatial resolution of only 500 microns. A possible result of this low out-of-plane resolution is an over-estimate of infarct volume reported by histology. Thinner slicing of harvested organs should improve accuracy but is practically challenging.While the volume of infarcted tissue can be measured by tissue staining, measurement of the cardiac function of ischemia-reperfusion animals can only be determined with in-vivo imaging. Micro-CT imaging facilitated accurate LV blood pool measurements during diastole and systole, so that cardiac function could be quantified by ejection fraction. The values of ejection fraction calculated for healthy subjects and for subjects following ischemia reperfusion were quantifiably different and statistically significant, demonstrating the use of CNT micro-CT for such functional measurements.The ability to identify infarcted tissue in the live animal is useful for longitudinal studies in mice. For example, infarct volume present immediately after surgery may increase or decrease through interventions. Without the ability to obtain high resolution images of infarcted tissue volume and distribution in vivo, the progress of an individual subject cannot be tracked, and many more mice would be required to evaluate an intervention statistically at intermediate time points. Despite the sensitive state of post-ischemic and reperfusion subjects, all study subjects survived 24 hours after surgery, and 7 of 8 subjects survived through the full imaging protocol. Given the noninvasive nature of the physiologically-gated micro-CT imaging protocol, longitudinal studies performed with the CNT micro-CT device are possible.As with all physiologically-gated micro-CT imaging, the utility of data from the resulting images is dependent upon the quality of the gating protocol. While prospective gating is robust to many deviations from true periodic physiological motion, errors in gating due to respiratory and cardiac instability can lead to heart and rib motion blur, which can result in incorrect automatic segmentation and infarct volume measurements. Improvements in animal handling and increasingly sensitive physiological monitoring will enhance overall image quality and the accuracy of measurements.Although prospective physiological gating and a free breathing protocol have many advantages, including minimized radiation dose and non-invasive handling, increased acquisition time results from the requirement of synchronizing both respiratory and cardiac motion for each x-ray projection. The ten to fifteen minute imaging time is on the same order of magnitude as the Iohexol contrast washout time, which poses a challenge in synchronizing imaging to the time-point of maximum iodine concentration in infarcted tissues. It would be optimal if the system scan time were reduced in order to allow acquisition of additional CT images prior to contrast washout. A reduction in total scan time could be achieved through a faster system gantry, one of the limitations in this prototype system. Alternatively, development of a contrast agent with a longer half-life with the correct properties to allow delayed contrast enhancement effects would have the same benefit as reducing scan time. Proposed improvements in the CNT micro-CT protocol, such as reducing the total number of projection images and applying a post-reconstruction bilateral filter to reduce Gaussian noise, could preserve image quality while simultaneously reducing scan time. Nonetheless, initial experimentation and the results of Nahrendorf et al  suggesteThe protocol of this study relied upon administration of two separate iodinated contrast agents; the long half-life lipid agent was administered only to guarantee sufficient contrast to perform LV volume measurements for ejection fraction. However, despite current hardware speed limitations, two CT image sets were able to be acquired during the half-life of Iohexol in the blood pool, and even images from the second acquisition exhibited sufficient contrast to measure LV blood pool volume. An amended procedure of sequential diastolic and systolic phase imaging immediately following Iohexol administration would shorten the study duration and eliminate the need for multiple contrast agents and resulting complications. Again, more rapid CT image acquisition would further facilitate the goal of single-contrast agent myocardial infarct imaging.The carbon nanotube micro-CT offers specific benefits over other imaging devices for in-vivo murine cardiac imaging. The short (15ms), high flux pulses produced by electronic triggering of the CNT cold cathode x-ray source allows a significant reduction in cardiac- and respiratory-motion blur. As this method of gated imaging does not require intubation and forced ventilation, it is minimally invasive and therefore appropriate for the most delicate of disease models . With CT images visualizing the delayed contrast effect, regions of myocardial infarct appear distinct from the surrounding uninjured tissue of the LV; these results compare in size, shape, and location to the regions of infarct indicated by TTC staining in histology. Furthermore, the pair of CT images obtained in diastole and systole allowed the measurement of ejection fractions, quantifying a significant decrease in heart function in subjects after ischemia and reperfusion. A delayed-enhancement contrast protocol, combined with the particular benefits of our novel CNT-cathode micro-CT and prospective respiratory and cardiac gating, provides a valuable new tool for the study of cardiac pathophysiology.