B-cell lymphoma in children accounts for about 10% of all pediatric malignancies. Chemotherapy has been very successful leading to an over-all 5-year survival between 80 and 90% depending on lymphoma type and extent of disease. Therapeutic toxicity remains high calling for better targeted and thus less toxic therapies. Therapeutic antibodies have become a standard element of B-cell lymphoma therapy in adults. Clinical experience in pediatric lymphoma patients is still very limited. This review outlines the rationale for antibody treatment of B-cell lymphomas in children and describes potential target structures on B-cell lymphoma cells. It summarizes the clinical experience of antibody therapy of B-cell lymphoma in children and gives an outlook on new developments and challenges for antibody therapy of pediatric B-cell lymphoma. Pediatric lymphomas account for 10% of all pediatric malignancies  or restricted to B-cell subpopulations . Possible side effects of lymphoma antibody therapy are therefore a direct consequence of target protein expression by normal B-cells. B-cell lymphoma surface molecules that at least partly fulfill the above mentioned requirements for antibody targets are CD19, CD20, CD21, CD22, and CD30 (see Table 9 or restin vitro and in vivo (mouse models) activity against CD19-expressing lymphomas  and functions as an immune inhibitory receptor on B-cells . In adults, Rituximab has been applied successfully to treat B-cell lymphomas  is a third way to increase clinical efficacy. Brentuximab vedotin is a good example for this concept  (Ansell et al., Finally, combinational antibody therapy, e.g., combining antibodies against two or more lymphoma antigens (Alinari et al., Experience with use of therapeutic antibodies in adult lymphoma patients is rapidly expanding. The successes of antibodies such as Rituximab and Ipilimumab have sparked the development of biosimilars Carey, , and of National and international registries of all pediatric B-cell lymphoma patients receiving antibody-based therapies would be a first step to increase available treatment data. In addition, antibody-therapy must be included and evaluated in future national and international cooperative B-cell lymphoma therapy trials. It is encouraging that currently at last six studies evaluating Rituximab in pediatric B-cell lymphoma treatment are recruiting patients (see Table In vitro screening of primary lymphoma cells for binding to therapeutic antibodies and subsequent cytotoxicity can be a first step to choose therapeutic antibodies for individual patients. Another way to assess antibody efficacy may be in vivo testing in animal models, such as immunodeficient mice harboring a human immune system (Traggiai et al., However, if clinical testing cannot be done in a timely fashion, other options must be explored. 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.


There is no consensus on how to investigate men with negative transrectal ultrasound guided prostate biopsy (TRUS-B) but ongoing suspicion of cancer. Three strategies used are transperineal (TP-B), transrectal saturation (TS-B) and MRI-guided biopsy (MRI-B). We compared cancer yields of these strategies.Papers were identified by search of Pubmed, Embase and Ovid Medline. Included studies investigated biopsy diagnostic yield in men with at least one negative TRUS-B and ongoing suspicion of prostate cancer. Data including age, PSA, number of previous biopsy episodes, number of cores at re-biopsy, cancer yield, and Gleason score of detected cancers were extracted. Meta-regression analyses were used to analyse the data.Forty-six studies were included; 12 of TS-B, 14 of TP-B, and 20 of MRI-B, representing 4,657 patients. Mean patient age, PSA and number of previous biopsy episodes were similar between the strategies. The mean number of biopsy cores obtained by TP-B and TS-B were greater than MRI-B. Cancer detection rates were 30·0%, 36·8%, and 37·6% for TS-B, TP-B, and MRI-B respectively. Meta-regression analysis showed that MRI-B had significantly higher cancer detection than TS-B. There were no significant differences however between MRI-B and TP-B, or TP-B and TS-B. In a sensitivity analysis incorporating number of previous biopsy episodes (36 studies) the difference between MRI-B and TP-B was not maintained resulting in no significant difference in cancer detection between the groups. There were no significant differences in median Gleason scores detected comparing the three strategies.In the re-biopsy setting, it is unclear which strategy offers the highest cancer detection rate. MRI-B may potentially detect more prostate cancers than other modalities and can achieve this with fewer biopsy cores. However, well–designed prospective studies with standardised outcome measures are needed to accurately compare modalities and define an optimum re-biopsy approach. Prostate cancer is the most common male cancer A number of different techniques for prostate re-biopsy have been developed and tested in an attempt to improve cancer detection rates following an initial negative biopsy. These include saturation biopsy approaches  or image guided  biopsies. In the initial biopsy setting it has been shown that the use of saturation biopsy techniques, which obtain greater number of prostate cores, either by transrectal or transperineal routes have no advantage over standard TRUS-B with respect to cancer detection rate What remains unclear from the existing literature is which repeat biopsy strategy offers the highest cancer detection rate in patients with a negative TRUS-B but ongoing suspicion of prostate cancer A literature search of Pubmed, Embase and Ovid Medline databases was performed using the search terms ‘prostate’ and ‘biopsy’. Results were limited to the English language published since 1st January 1995 and up to January 2012. Three re-biopsy strategies were compared in this study: TP-B; multiple needle core prostate biopsy obtained through the perineum with or without the use of a brachytherapy grid template under transrectal ultrasound (TRUS) guidance; TS-B; multiple needle core prostate biopsy obtained transrectally under TRUS guidance and MRI-B; multiple needle core prostate biopsy obtained after the use of MRI to identify areas of the prostate suspicious for the presence of cancer. We did not distinguish between the various functional imaging modalities or biopsy approaches used in MRI-B for simplicity of analysis due to the diversity of techniques described in the current literature. The inclusion criteria for this study were papers investigating biopsy diagnostic yield in men who had one or more previous negative TRUS-B, who were undergoing one of the three repeat biopsy strategies outlined above for ongoing suspicion of prostate cancer, defined as elevated or rising PSA, abnormal digital rectal examination (DRE) of the prostate and/or previous high grade prostatic intraepithelial neoplasia (HGPIN) or atypical small acinar proliferation (ASAP) on previous prostate biopsy. Studies of men with known prostate cancer were excluded. In papers where both initial and repeat biopsies were being studied, only data relating to patients undergoing the repeat biopsy procedure were analysed.The following data were extracted from each paper: first author, year of publication, study size, mean patient age, mean PSA at repeat biopsy, mean number of previous biopsy episodes, mean number of biopsy cores taken at repeat biopsy, cancer detection rate, and Gleason scores of detected cancers. Statistical analysis was performed by RCH and RAP. For each strategy, weighted summary statistics, which weight according to the sample size of each individual study, were calculated for mean patient age, mean PSA, mean number of previous biopsy episodes, and mean number of cores obtained by the repeat biopsy strategy. We were not able to weight by the inverse study variance for these variables because estimates of within-study variability were unavailable. For the primary outcome (cancer detection rate) however an estimate of the variance could be extracted using the formula for the variance of a proportion, and so this variable was weighted by the inverse variances. A meta-regression analysis compared the overall prostate cancer detection rate (percentage of patients diagnosed with prostate cancer) between the three re-biopsy strategies having adjusted for differences in mean patient age and PSA between the strategy cohorts. In order to account for heterogeneity between studies, a random-effects meta-regression analysis was conducted using inverse-variance weights. The amount of residual heterogeneity between studies was assessed using a Q-test, based on the DerSimonian-Laird estimator. Publication bias was assessed using a funnel plot of the inverse sample variance against model residuals  and a raThe literature search yielded 1,943 papers, which were then individually screened for this study. 1,884 papers were excluded, as they did not meet the inclusion criteria. Forty-nine papers met the inclusion criteria for this study, however three of these were subsequently excluded. In two of these papers data on mean age and mean PSA was not available, Baseline characteristics of the studies analysed are shown in The weighted summary values for mean patient age, mean PSA at the time of re-biopsy, mean number of previous biopsy episodes, and mean number of prostate cores obtained at re-biopsy for each strategy are displayed in Forty-six studies comprising 14 TP-B, 12 TS-B, and 20 MRI-B papers were analysed. In the meta-regression analysis MRI-B had a significantly higher cancer detection rate than TS-B  . HoweverA sensitivity analysis was next performed, adjusting for the mean number of biopsy episodes prior to the re-biopsy strategy . Ten stuTwenty-eight studies (61%) had median Gleason score available for this analysis. Comparison of median Gleason scores by Fisher’s exact test did not reveal any significant differences between the strategies (p = 0·90). As only 28 studies had complete data available for analysis of Gleason score we interpret these results with caution. Nevertheless, we were unable to find any evidence of a difference in the reported clinical significance (as defined by Gleason sum) of the detected cancers between strategies.In this study we addressed the question of which repeat biopsy strategy is most effective at diagnosing prostate cancer in men with initial negative TRUS-B and ongoing suspicion of prostate cancer. There is currently no published consensus on which re-biopsy approach should be used in this group of men and the decision as to which strategy to use is largely based on institutional practice and the availability of a particular technology. A key observation was the large heterogeneity across the studies included in the analysis with regards the patient demographics, biopsies taken, previous biopsy episodes, and outcome reporting. Our conclusions were therefore based on analyses following correction for these variables as much as possible. In the initial meta-regression analysis our results demonstrated that MRI-B detected significantly more cancer than TS-B, but there were no significant differences between either MRI-B and TP-B, or TP-B and TS-B. In a subset analysis correcting for number of prior biopsy episodes however, this difference was not maintained. This was almost certainly due to a loss of statistical power resulting from the reduced number of studies available for inclusion in the sensitivity analysis as when the 36 studies from the sensitivity analysis were re-analysed using the initial meta-regression model, there were no significant differences between the three re-biopsy strategies. Additionally, the weighted summary statistics  demonstrIn this study TP-B and MRI-B had very similar cancer detection rates but the latter achieved this with fewer biopsy cores. It could therefore be said that MRI-B offers higher cancer yield per biopsy core than the other strategies. These results are consistent with the recent systematic analysis by Moore It is well recognised that many detected cancers will be indolent and not require active intervention The use of MRI for targeted prostate biopsy is a rapidly evolving field and recent data suggests an improving ability to better detect clinically significant cancers after initial negative investigation This study has a number of inherent limitations. Data was extracted from published manuscripts, rather than from original patient data, so a degree of reporting bias is inevitable. We have acknowledged that there is heterogeneity of data across the studies analysed despite efforts to standardise the data included in the meta-regression analysis. We were unable to formally compare the weighted summary statistics with statistical methodology as data relating to within-study variability was not available from the published manuscripts. We did not differentiate between the various techniques of MRI-B, and so our findings with respect to this strategy may not be applicable to each of the individual techniques described in the literature. Finally, this study was not able to accurately assess and compare the complication rates or costs of each modality as these factors were often not well recorded in the papers reviewed. An evaluation of these aspects in a prospective study will be crucial in determining the health economic cost benefits of each modality.This study has compared the cancer detection rates of three re-biopsy strategies in men with initial negative biopsies and ongoing suspicion of prostate cancer. The main meta-regression analysis demonstrated that MRI-B had a significantly higher cancer detection rate than TS-B, but this result was not maintained after adjustment for the mean number of prior biopsy episodes. Notably MRI-B required the fewest biopsy cores to achieve the greatest cancer detection rate. We observed considerable heterogeneity in the studies in the literature. There is an urgent need for prospective national and international audit and a common reporting format to improve the quality of data for accurate comparison of biopsy strategies and to investigate the complication rates associated with each strategy. Quality of life and health economic analyses should also be incorporated to better inform clinical decision on the most efficient and cost effective re-biopsy strategy.Figure S1Details of the literature search strategy and results are displayed in the PRISMA flow diagram.(DOC)Click here for additional data file.Figure S2The PRISMA checklist, outlining the accepted standard for meta-analysis.(PDF)Click here for additional data file.


Chronic endometritis (CE) is an unusual endometrial inflammation characterized by stromal plasmacyte infiltration. CE is easily missed due to its subtle symptoms and demanding histopathological examinations. Although the immunohistochemistry for the plasmacyte marker CD138 has facilitated the detection of endometrial stromal plasmacytes, the accuracy and biases of this method for CE diagnosis remain poorly understood. The aim of this study was to investigate the inter- and intra-observer variability in the immunohistochemical detection of stromal plasmacytes in the human endometrium. A total of 80 CE and 20 non-pathological archival hematoxylin-stained endometrial preparations with or without immunostaining for CD138 were evaluated independently by two experienced observers and two inexperienced observers. Endometrial stromal plasmacytes in unit areas were counted in the hematoxylin-stained and CD138-immunostained preparations. Each preparation was subdivided into 11 categories by every five plasmacyte counts. The second evaluation was performed four weeks after the first evaluation. The immunohistochemical detection method was superior to conventional histopathological evaluation in both the inter- and intra-observer agreement, irrespective of the experience level of the observers. The linear weighted κ coefficient for intra-observer agreement was higher in the experienced observers than in the inexperienced observers. The inter-observer agreement among the four observers by the immunohistochemical detection method was similarly good between the first and second evaluation. There was no significant inter- or intra-observer variability in the paired comparison of the individual samples. These findings validate the use of immunohistochemistry for CD138 as an accurate and less biased diagnostic tool for CE. Chronic endometritis (CE) is an inflammatory condition characterized by endometrial stromal plasmacyte infiltration which are unusual leukocyte populations in this mucosal tissue. CE is easily missed in gynecological practice, as it is typically asymptomatic or oligosymptomatic with mild manifestations of leukorrhea, pelvic discomfort or slight vaginal bleeding. Even symptomatic CE may be overlooked since endometrial sampling and histopathological detection of plasmacytes are required for diagnosis. In addition, endometrial stromal plasmacytes are difficult to detect using conventional tissue staining techniques .in vitro fertilization-embryo transfer, unexplained infertility and unexplained recurrent miscarriages  and labeled streptavidin-biotin method . Of them, 80 specimens were diagnosed with CE by two experienced observers of human endometrial specimens, whereas the remaining 20 specimens were diagnosed as non-pathological .Under a light microscope , the preparations were evaluated independently by two experienced observers and two inexperienced undergraduate medical trainees unconnected with the study. Instruction was given to the inexperienced observers on the basic endometrial morphology, cell components and appearance of plasmacytes. In the hematoxylin-stained preparations, endometrial stromal plasmacytes were counted in 20 non-overlapping stromal areas according to the nuclear chromatin rearrangement which appeared as clock-face or spoke-wheel patterns. In the CD138-immunostained preparations, the number of immunoreactive cells was counted in the same number of stromal areas. The observers were blinded to each other’s findings until every observer had finished their evaluations. Each preparation was subdivided into 11 categories by every five cell counts. The second evaluation was performed 4 weeks after the first.The inter-observer or intra-observer variability was calculated using weighted and Fleiss’ κ statistics. The multiple comparisons between the datasets were performed with Tukey’s test. The intra-observer variability for an individual was compared using the Wilcoxon signed-rank test. P<0.05 was considered to indicate a statistically significant difference.+ stromal plasmacytes in all preparations using the immunohistochemical method. The duration required for microscopic evaluation per preparation in the immunohistochemical method was similar (P>0.66) between the experienced  and inexperienced observers .Under the conventional histopathological method, the inexperienced observers (3 and 4) failed to complete the full evaluation of all 100 preparations within one week. By contrast, they were able to identify and enumerate endometrial CD138In the 11-tier evaluation system, the linear weighted κ coefficient for inter-observer agreement was higher in the immunohistochemical method (0.968–0.976) than in the conventional histopathological method (0.687–0.771), irrespective of the experience level of the observers  and III.The immunohistochemical method showed good intra-observer agreement (linear weighted κ coefficient 0.933–0.968) for the experienced and inexperienced observers  and IV, In the present study, favorable inter- and intra-observer agreements for the detection of endometrial stromal plasmacytes using immunohistochemistry for CD138 were demonstrated. The inexperienced observers who failed to complete the full evaluation using the conventional histopathological method were able to detect and count plasmacytes easily by the immunohistochemical method, confirming the superiority of this approach for objectivity and reproducibility in the diagnosis of CE.By contrast, the conventional histopathological method showed a certain degree of intra-observer variability. Bayer-Garner and Korourian reported that the diagnosis rate of CE by the conventional histopathological method 15%) was significantly lower than that by the immunohistochemical method (42%), in endometrial curettage samples 5% was si.One major concern in the sole immunohistochemical detection of CE is that the representative clones of anti-human CD138 antibodies detect not only stromal plasmacytes but also endometrial glandular/surface epithelial cells which constitutively express this proteoglycan. Although CD138 immunostaining in endometrial epithelial cells is weaker than in the stromal plasmacytes in the CE samples , the conIn conclusion, immunohistochemistry for CD138 is a convenient and reliable tool for the detection of endometrial stromal plasmacytes in CE. Wider use of this methodology is expected to improve diagnostic accuracy, simplify testing and reduce examiner bias.


The allelic architecture of complex traits is likely to be underpinned by a combination of multiple common frequency and rare variants. Targeted genotyping arrays and next-generation sequencing technologies at the whole-genome sequencing (WGS) and whole-exome scales (WES) are increasingly employed to access sequence variation across the full minor allele frequency (MAF) spectrum. Different study design strategies that make use of diverse technologies, imputation and sample selection approaches are an active target of development and evaluation efforts. Initial insights into the contribution of rare variants in common diseases and medically relevant quantitative traits point to low-frequency and rare alleles acting either independently or in aggregate and in several cases alongside common variants. Studies conducted in population isolates have been successful in detecting rare variant associations with complex phenotypes. Statistical methodologies that enable the joint analysis of rare variants across regions of the genome continue to evolve with current efforts focusing on incorporating information such as functional annotation, and on the meta-analysis of these burden tests. In addition, population stratification, defining genome-wide statistical significance thresholds and the design of appropriate replication experiments constitute important considerations for the powerful analysis and interpretation of rare variant association studies. Progress in addressing these emerging challenges and the accrual of sufficiently large data sets are poised to help the field of complex trait genetics enter a promising era of discovery. GWASs are designed to provide a survey of common variation [minor allele frequency (MAF) > 0.05], therefore examining only a portion of the genomic landscape of complex traits. Low-frequency (MAF 0.01–0.05) and rare (MAF < 0.01) variation has thus far been more challenging to access. Early studies on data from deep sequencing of small numbers of loci and more recently larger-scale studies (e.g. 1000 Genomes Project) demonstrate that rare variants constitute the majority of polymorphic sites in human populations (The genetic architecture of complex traits has not been fully elucidated yet. Following the advent of genome-wide association studies (GWASs) and large-scale consortial meta-analyses of GWASs, several thousands of variants have been robustly associated with complex phenotypes of medical relevance (y MAF 0.0–0.05 andulations .http://genome.sph.umich.edu/wiki/Exome_Chip_Design), metabochip  risk of sick sinus syndrome  levels revealed an aggregation of rare alleles in individuals with low HDL-C compared with those with high HDL-C . Resequeease HDL . Recentl to date . An aggr to date  and with to date . The fir to date .The study of rare variation can be empowered by focusing on population isolates ,32. IsolAPOC3 gene with HDL-C and triglycerides levels was first detected in the Amish founder population and later confirmed in a Greek population isolate from Crete. R19X appears to have drifted up in frequency independently in the two population isolates  –36. Assoisolates ,38.www.uk10k.org) has undertaken high-depth WES of 6000 and low-depth WGS of 4000 well-phenotyped individuals primarily from the UK. The imputation of low-frequency and rare variants can be challenging compared with common alleles, and the availability of very large-scale reference panels can improve imputation performance. It is envisaged that large-scale WGS efforts will join forces to generate an overarching reference panel to enable efficient and widespread use of the generated data.Large-scale collaborative efforts aiming to help understand the full spectrum of sequence variation serve as valuable resources for the scientific community. The 1000 Genomes Project  has helpStatistical genetics considerations of rare variant association analysis have been the focus of intensive method development over the last few years. The single-point analysis of rare variants is under-powered, because not enough copies of the rare variant allele are observed in sample sizes typically available to date. Instead of examining the association of each rare variant in isolation, multivariate methods that combine information across multiple variant sites within a gene or other functional genomic region are a viable alternative strategy Fig. . A plethCollapsing methods aggregate information across multiple variants within a region of interest into a single quantity, which is then used to test for trait association with an accumulation of rare minor alleles. Collapsing methods vary in the way they collapse the variants and in the chosen statistical test ,47, for Genotype uncertainty metrics for imputed genetic variants or for sequencing-derived variants could be incorporated as weights in different statistical tests, instead of filtering out variants with low imputation or quality scores, to increase association power as shown by . MoreoveP = 5 × 10−8 based on the number of independent common-frequency variants across the genome calculated based on the European population data from the HapMap Project  approach is that apart from calculating asymptotic P-values, it also evaluates significance in an empirical and numerically stable way via an adaptive Monte-Carlo simulation scheme. Another unique feature of Liu et al.'s approach is its ability to conduct conditional meta-analysis of gene-level tests. Lumley et al., Lee et al. and Liu et al. (2013) show in simulation studies that their proposed approaches are as efficient as an analysis that pools individual-level data together. The evaluation of different meta-analysis approaches of rare variant tests is an active field of study.Liu et al  and Tanget al  suggest et al ,47, variet al  and SKATet al . Their mPopulation stratification at rare variants is an important consideration for next-generation association studies. Rare variants show increased population specificity . Based oStrategies for replication of associations discovered in low-frequency and rare variants depend on the allelic architecture of the associated locus. For example, if a single low-frequency or rare variant is driving the signal, replication can be sought by genotyping the implicated variant in an independent sample set e.g. . For assThe emerging generation of studies in search of low-frequency and rare variants affecting complex traits will require robust strategies to ensure high power in the context of an appropriate statistical framework. It is anticipated that sequence-based meta-analysis across diverse populations, including populations of African descent, will empower novel locus discovery, and that accruing the necessary sample sizes is likely to be a key determinant of success. Initial insights into the contribution of rare variation indicate a firm role in complex trait aetiology and suggest a combination of potential allelic architectures underpinning biological phenotypes. Their powerful detection will require tailored study design and analysis approaches.Wellcome Trust (098051). K.P. is funded by Arthritis Research UK (19542). Funding to pay the Open Access publication charges for this article was provided by The Wellcome Trust (098051).K.P., I.T. and E.Z. are funded by the


Patients undergoing hemodialysis treatment have a six-fold increased risk for stroke relative to the general population. However, the effect of hemodialysis on cerebral blood flow is poorly studied and confounding factors like blood pressure and ultrafiltration as well as temperature changes have rarely been accounted for. The aim of our study was to use state-of-the-art technology to evaluate the effect of a single dialysis session on cerebral perfusion as well as on vascular stiffness.Chronic hemodialysis patients  were recruited. Cerebral blood flow and arterial pulse wave velocity were measured before and immediately after a hemodialysis session. To exclude effects of volume changes we kept ultrafiltration to a minimum, allowing no change in body weight. Isothermic conditions were maintained by using the GENIUS single-pass batch-dialysis system with a high-flux polysulfone dialyser. Cerebral blood flow was measured by contrast-enhanced computed tomography. Pulse wave velocity was measured using the SphygmoCor  device by a single operator.This study shows for the first time that isovolemic, isothermic hemodialysis neither affected blood pressure or heart rate, nor total or regional cerebral perfusion. There was also no change in pulse wave velocity.Mechanisms other than the dialysis procedure itself might be causative for the high incidence of ischemic strokes in this patient population. Moreover, the sole removal of uremic toxins does not lead to short-term effects on vascular stiffness, underlying the importance of volume control in this patient population. The high prevalence of cardiovascular disease in chronic kidney disease (CKD) stage 5 D, i.e. dialysis patients has been known for decades The study protocol was approved by the Hannover Medical School Ethics Committee (protocol # 3952). Stable adults undergoing chronic hemodialysis treatment were studied after written informed consent was obtained from the patients.Participants included were undergoing chronic hemodialysis for at least one year with a regimen of at three times per week. None of the patients suffered from instable coronary artery disease or had signs of severe heart failure (>NYHA II). The patients had no signs of infections based on c-reactive protein (CRP) and clinical examination. Exclusion criteria were participation in other studies during the previous three month, allergy against contrast agents or iodine, hyperthyroidism or unclear dysfunction of thyroid gland, premenopausal women, history of radiation of the cranium, residual diuresis (≥500 ml/day), history of stroke within six weeks prior to study participation, taking nutrition supplements containing L-arginine, and any further conditions at the discretion of the treating physician.The study participation took place on the patient’s midweek dialysis day. After measurement of the predialysis body weight, heart rate, blood pressure and body temperature the dialysis needles  were placed as usual in the dialysis fistula and a blood gas analysis (BGA) including determining sodium and potassium was performed.Afterwards the patient was taken to the computed tomography (CT) scan in supine and resting position, to rule out influences by altered hemodynamics. The initial CT-scan was performed as described below and the patient was taken back to the dialyis unit. The four hour lasting dialysis session was performed using the Genius®  single-pass batch-dialysis system with a high-flux polysulfone dialyser FX 60 . The dialysis solution was composed according to the patient’s requirements. The blood flow and the countercurrent dialysate flow ranged between 200 and 250 ml/min, there was no net ultrafiltration. After 4 hours the patients underwent a second pulse wave analysis and subsequently a second CT.The method used was described previously and adapted due to modified CT hardware Carotid-aortic PWV was determined using a validated system , which employs high-fidelity applanation tonometry by a pencil-type probe for non-invasive registration of peripheral arterial pressure waves. Pulse waveforms of the common carotid artery and the femoral artery were obtained sequentially and PWV was calculated as the distance between the suprasternal notch and the femoral artery recording site minus the distance between the suprasternal notch and the carotid artery recording site, divided by the time interval between the feet of the flow waves. The device uses the foot-to-foot methods as described previously Body temperature was measured with an infrared tympanic thermometer  with an accuracy of +/−0.1°C.2 or pH changed significantly Click here for additional data file.


Hysteresis of the pressure-volume loop is a measure for the additional energy that is required during inspiration to recruit and inflate additional alveoli. The hysteresis area is usually constructed using data from a low-flow inflation/deflation maneuver; that is, from a quasi-static situation that the lung never sees during ongoing ventilation. However, during the dynamic conditions of mechanical ventilation the hysteresis area is biased by resistive pressure portions. Therefore we uncoupled flow and volume by linearizing expiratory flow (flow-controlled expiration). This enabled calculation of compliance separately for inspiration and expiration. We hypothesized that the volume-dependent intratidal compliance profiles differ between inspiration and expiration, describing a dynamic hysteresis behavior.2O. The flow-controlled expiration was realized by a computer-controlled expiratory resistance which was adjusted in a fashion that expiratory flow was strongly limited in the beginning and continuously facilitated towards the end of expiration. Using the gliding-SLICE method [In five Swedish Landrace Hybrid pigs weighing 26 ± 2 kg the lungs were ventilated in the volume-controlled mode. PEEP was set to 0, 6, 12 and 15 cmHIntratidal compliance profiles of inspiration and expiration differed strongly in mean value and slope at low PEEP. With increasing PEEP the inspiratory compliance profile approximated closer to the expiratory compliance profile. This was accompanied by a decreased relative hysteresis area by 26%.Flow-controlled expiration allows for calculation of respiratory system compliance separately for inspiration and expiration. This compliance displays the hysteresis behavior of the respiratory system during uninterrupted ventilation. Such analysis, which is similar to the time-honored quasi-static hysteresis area analysis, could be helpful for finding an optimal PEEP.


A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 -TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia.The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via  How didractions . It was ractions , 6.In this paper we give a brief overview of the modulatory role of receptor heteromers in the pain pathways in the CNS and in peripheral nociceptors. They seem to be integrators of nociceptive and antinociceptive wiring (WT) and volume transmission (VT) signals with a major role for the opioid receptor heteromers. Their relevance in the mechanisms for the antinociceptive actions of acupuncture will be discussed.δ) afferents that mediate well-localized fast pain. The second class of nociceptor includes small diameter unmyelinated “C” fibers that mediate poorly localized, slow pain [δ nociceptors terminate most superficially, synapsing upon large projection neurons in lamina I and interneurons located in outer lamina II. Thus, primary afferent nociceptors convey noxious information to projection neurons within the dorsal horn of the spinal cord . One major class includes medium diameter myelinated  mainly operate via VT [In these pain circuits we will describe how opioid receptor containing heteromers may play a role in the modulation of pain transmission, offering novel targets for antinociceptive drugs. They may be involved in conveying the antinociceptive actions of acupuncture  since ine via VT –27 and lAs mentioned in the introduction, the concept of intramembrane receptor-receptor interactions in receptor heteromers was born in the analysis of neuropeptide-monoamine receptor-receptor interactions in 1980-81. The concept of G protein-coupled receptor (GPCR) heteromers was later confirmed in 1998-1999 by studies reporting that two non-functional GPCR monomers, gamma amino butyric acid (GABA)B1 and GABAB2 receptors, can assemble in a signalling heterodimer, the GABAB receptor at the cell surface , 29. 3+) or terbium (Tb3+) cryptate, as a donor molecule and a compatible acceptor chromophore, such as alexafluor 647 or allophycocyanin [Different resonance energy transfer methods have been used to study the existence of GPCR heteromers –33. Fluococyanin .Renilla luciferase-GFP2/YFP pair is used in the tagging of the two receptors, in the presence of the substrate h-coelenterazine or coelenterazine-400 on which Renilla luciferase acts to produce through oxidation a bioluminescent signal. Then the energy transfer between the generated luminescence and YFP or GFP2 occurs when the distance between these proteins is less than 10 nm leading to a fluorescence emission from YFP or GFP2 [Principle of the detection of GPCR heterodimerization using the bioluminescence resonance energy transfer BRET method is similar to the FRET method except a  or GFP2 .On the other hand, the principle of bimolecular fluorescence complementation (BiFC) methods is based on the complementation of the N-terminal and C-terminal fragment of a fluorescent protein  . After iA drawback of these methods is the fact that they involve the ectopic expression and/or overexpression of the fusion receptors, thereby, sometimes promoting the formation of artefacts. It is therefore advisable, whenever possible, to consider the physiological expression levels of the receptor pairs under study.ex vivo [However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems (mainly by BRET and FRET methodologies), the existence of such heteromers in the CNS and other tissues remains largely unknown, mostly because of the lack of appropriate methodology. Recently, a well-characterized in situ proximity ligation assay (in situ PLA) has been adapted to confirm the existence of GPCR heteromers in brain slices ex vivo –39.In situ PLA is based on a pair of antibodies that can bind to target proteins and to which oligonucleotides have been attached. When the so-called proximity probes recognize a target, for example, the receptor heteromer, the attached oligonucleotides are brought into a sufficiently close spatial proximity to allow them to join followed by ligation of the two linear oligonucleotides into a circular DNA molecule. This newly formed DNA circle strand can serve as a template for rolling circle amplification (RCA), resulting in a long single-stranded rolling circle product (RCP) attached to one of the proximity probes. Since the RCP is linked to the proximity probe, it is attached at the site where the proximity probe bound, which means that it can be used to reveal the location of the receptor complex , 41. TheIn the beginning, allosteric mechanisms were only discussed in terms of intramolecular interactions within a receptor between orthosteric and allosteric sites. This was the classic pharmacology , left.Now we have moved into a novel pharmacology, where intermolecular receptor-receptor interactions can occur and results in novel receptor recognition, pharmacology and signaling , right. β-arrestin or to calmodulin [Allosteric mechanism causes a marked rise of the repertoire of GPCR recognition, pharmacology, trafficking and signaling of the participating protomers. This is achieved through changes in recognition, G protein selectivity, and signaling cascades with among others switching from G proteins to lmodulin , 45. Itslmodulin , 37, 46.The term moonlighting protein is used to describe multifunctional proteins in which several functions can be found in a single strand of amino acids unrelated to splicing, posttranslational changes, and so forth , 48. In We are interested in the receptor interface since it can be a target for novel drugs by their ability to block or mimic the allosteric receptor-receptor interactions , 49–51. Based on a mathematical approach developed by Dr. Tarakanov, we have deduced, based on 48 pairs of receptors that form or not form heterodimers, a set of triplet amino acid homologies that may be critically involved in receptor-receptor interactions . We callThe pro-triplet theory has recently became validated , 62 undeβ-endorphin neurons in case of MOR and DOR and of the dynorphin neurons in case of KOR  with reductions of changes in gene expression and reduction of morphine action [An interesting concept was introduced by Rozenfeld et al. in 2007  to show e action . β-arrestin2 from the heteroreceptor complex and return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. It may also be associated with a certain disruption of MOR-DOR heteromers into MOR homomers increasing the MOR homomer/MOR-DOR ratio . Treatment with DOR antagonists reduces the β-arrestin2 coupling in the MOR-DOR heteromer and enhances MOR binding, signaling and morphine-induced antinociception; which may enhance MOR-DOR disruption. It will be of substantial interest to study if acupuncture could favor the formation of MOR homomers versus MOR-DOR heteromers during chronic morphine treatment.All the data support a role of this heteromer in morphine tolerance and dependence, since this heteromer develops a reduced G protein-coupling with signaling mainly operating via The field of MOR-KOR heteromers is also exciting. Formation of MOR-KOR heterodimer is gender-dependent and mediates female-specific opioid analgesia  Figure . Spinal The evidence also shows that the contribution of dynorphin/KOR (part of MOR-KOR heterodimer) to spinal morphine antinociception is dependent on the stage of the ovarian cycle , 75. SpiFurther work has indicated that spinal synthesis of estrogen and concomitant signaling by membrane ER regulate spinal MOR-KOR heterodimerization and female-specific spinal morphine antinociception , 77. TheBiochemical and behavioral experiments suggest that ERs work in a cooperative manner as part of a macromolecular complex to increase KOR/MOR expression. Estradiol (E2)  triggers the formation of a signaling complex that contains multiple ERs and enhances heterodimerization of KOR and MOR. Transcriptional effects of progesterone (P4) are essential either for the formation of the ERs signaling complex and/or the heterodimerization of KOR with MOR , 77. In our view the formation of receptor mosaics of activated membrane ER complexes and MOR and/or KOR may take place markedly enhancing the formation of MOR-KOR heteromers in the plasma membrane through allosteric changes in MOR and/or KOR. Possibly, progesterone may contribute through transcriptional increases of opioid receptor interacting proteins that may be essential for the MOR-KOR heteromer formation from the ERs-MOR and/or ERs-KOR heteromer complexes. In summary, the gender- and ovarian steroid-dependent recruitment of spinal cord MOR-KOR heterodimers would provide a way to influence the balance between antinociceptive and pronociceptive functions of the spinal dynorphin/KOR opioid system. Impaired formation of MOR-KOR heteromers could be a biological determinant of various types of chronic pain states that are substantially more common in women than men . The nociceptin receptor or opioid receptor-like receptor 1 (ORL1) belong to the class of Gi/o-linked receptors , 79 and Upon dimerization with opioid receptors, ORL1 regulation of N-type calcium channels is altered. ORL1 can function as a molecular link that allows MORs to trigger N-type calcium channel internalization . Thus, MThese results are of high interest since opioid and opioid-like receptors play a key role in controlling pain signaling in primary afferent terminals in the dorsal horn by two primary mechanisms , 82. Theδ nociceptors release a variety of neurotransmitters, activating output neurons in lamina I of the dorsal horn forming part of the pain pathways from the dorsal horn. One important location of the opioid-ORL1 heteromers may therefore be at the central terminals of these nociceptors to inhibit release of transmitters from them involving increased internalization of the N-type Ca2+ channels as well as activation of inwardly rectifying K+ channels. Again it would be of high interest to explore how acupuncture would modulate the formation of these receptor heteromeric complexes in the dorsal horn.Looking at the primary afferents to the dorsal horn clearly activated C and AEarly work indicates that agonists acting at the alpha-2A adrenergic receptor subtype  and opioid receptors have analgesic properties and act synergistically when co-administered in the spinal cord. The alpha-2AAR subtype is the primary mediator of alpha2 adrenergic spinal analgesia and is necessary for analgesic synergy with opioids and feedback inhibition of capsaicin-induced hyperalgesia , 84. OthThe question is if alpha-2AAR-MOR heteromers can participate in these synergistic actions? The major origin of NA innervation of the dorsal horn by the descending bulbo-spinal NA systems , 12 is tImmunoreactivity for both alpha-2AAR and MOR is observed in the superficial layers of the dorsal horn of the spinal cord. The primary localization of the alpha-2AAR in the rat spinal cord is on the terminals of capsaicin-sensitive, SP-containing primary afferent fibers (colocation with MOR IR). Thus, alpha-2AAR-MOR heteromers may exist on these terminals.The role of the receptor-receptor interactions in the alpha-2AAR and MOR heteromers was found to be an unexpected one . There eIt follows that synergy of alpha-2AAR agonist and morphine in antinociception cannot be explained by receptor-receptor interactions in the alpha-2AAR-MOR heteromers. In this case, interactions at the level of signaling pathways and ion channels controlled by the corresponding homomers may be involved as well as a location in different nerve cells of the neuronal network synergizing in favoring an output pathway leading to antinociceptive effects.Synergistic interactions also exist between cannabinoid and opioid analgesia . The canCB1s are present at many locations in the pain networks namely in peripheral terminals of primary sensory neurons, at synapses in the spinal cord, and in pain circuits of the brain. At spinal synapses, CB1 could be on nerve terminals of afferent neurons, on interneurons, and/or on terminals of pathways originating in supraspinal regions . 2+ channels and activation of inwardly rectifying K+ channels in afferent terminals and dorsal horn neurons as discussed for the alpha-2AAR and MORs.A strong colocalization of CB1 and MOR has been observed in lamina II interneurons . The CB1Rios et al. in 2006 demonstrated that CB1 forms heteromers with opioid receptors . A BRET Simultaneous activation of MOR and CB1 leads to a significant attenuation of the increase in MAPK phosphorylation response seen upon activation of the individual protomers. Thus, upon agonist coactivation antagonistic receptor-receptor interactions develop in the CB1-MOR heteromers as observed for the alpha-2AAR-MOR heteromers. However, when the CB1 protomer alone is activated in the CB1-MOR heteromer there is a marked increase in signaling compared with the agonist activation of CB1 monomer/homomer.Similar results were obtained on neurite outgrowth in Neuro-2A cells expressing MOR and CB1 's. Agonist-induced neurite outgrowth in Neuro-2A cells treated with a combination of 100 nM DAMGO or morphine, and 100 nM CB1 agonist HU-210 is markedly reduced upon coactivation while increased with single agonist activation. Upon coactivation a substantial cross-inhibition of the phosphorylation of Src and STAT3 is observed .Antagonistic allosteric interactions in CB1-MOR heteromers may underlie the attenuation of the Src-STAT3 pathway signaling which could be one of the mechanisms leading to reduction of neurite outgrowth. MOR-CB1 interactions will thus upon coactivation lead to cross-inhibition of neuritogenesis involving inhibition of the Src-STAT3 pathway. Such a phenomenon may be of substantial importance. Thus, it may lead to counteraction of the plasticity changes seen in discrete pain networks leading to chronic pain . If so, There exist indications for the existence also of a CB1-D2R heteromer in which A2AR may participate , 102 in There may also exist D4R-MOR heteromers in the nucleus accumbens and dorsal striatum of relevance to the treatment of addiction . Thus, DIn 1983 we discovered neuronal gastrin releasing peptide (GRP) IR in the rat CNS . In the Sun and Chen discovered that GRPR mediates the itch sensation in the spinal cord which brought GRP transmission into the spotlight . In 2011β/IP3-dependent Ca2+ signaling pathway)  . They shThe data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D-GRPR heterodimerization. The evidence demonstrates that the C-Terminus of the MOR1D is critical for the MOR1D-GRPR heterodimer formation. The difference between MOR1 and MOR1D isoforms lies in a motif consisting of seven amino acids (RNEEPSS) located in the C-terminus of the MOR1D.in vivo. It specifically blocked morphine induced scratches while leaving GRP induced scratches intact, morphine induced analgesia unaltered and a reduction in the coimmunoprecipitation of MOR1D-GRPR levels.To test the spinal functions of the heteromer, a Tat-fusion peptide (Tat-MOR1D CT) was synthesized. The Tat-motif (YGRKKRRQRRR) belong to a trans-activating domain of a HIV protein that can permeate the cell membrane allowing, after intrathecally injection into the spinal cord, the introduction of the fused MORD1 C-terminal (RNEEPSS motif) into the cells. Introduction of the Tat-MOR1D CT permits its competition with MOR1D for physical contacts with GRPR β3 and IP3R3, two downstream effectors of GRPR, specifically blocked morphine induced scratches but not morphine induced analgesia [New insights into opioid-induced itch prevention was in this way obtained. They also demonstrated that molecular and pharmacologic inhibition of PLC-nalgesia . Based o2+ permeability and the capsaicin activation of this channel kills the cells.Of high interest are the peripheral nociceptors and their Transient receptor potential cation channel, subfamily A, member 1 (TRPA1)-Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) heteromeric complexes. TRPV1, one heat nociceptor, is the most famous one, since it represents the capsaicin or vanilloid receptor, activated by ingredients in “hot” chili peppers . TRPA1 iBy means of acceptor bleaching FRET, a direct interaction between TRPA1 and TRPV1 on the plasma membrane was observed . The incMustard oil activates single channel currents in TRPA1 and TRPA1-TRPV1 expressing CHO cells. Vigorous activation of channels in TRPA1 and TRPA1-TRPV1 expressing cells was observed but not in untransfected CHO cells with multiple conductance states, performed in cell-attached configuration in voltage clamp mode . However2+ and/or directly, involving allosteric receptor-receptor interactions between these receptor channels within a heteromeric complex.These results support the hypothesis that TRPV1 and TRPA1 in nociceptors may form a heteromeric receptor ion channel complex and that TRPV1 can influence intrinsic characteristics of the TRPA1 channel also independent of intracellular calcium , 115. TrThe demonstrated pharmacological cross-desensitization between capsaicin and mustard oil responses can involve desensitization of TRPA1 and TRPV1 ion channel activities in their heteromeric complexes which may contribute to inhibition of nociceptor signaling leading to antihyperalgesia and antinociception , 115. OnThere is also a role of ionotropic cannabinoid receptors (ICR) in peripheral antinociception and antihyperalgesia. The known ICRs are members of the family of transient receptor potential channels (TRP) and remarkably include TRPV1, TRPV2, TRPV4, TRPM8, and TRPA1 (see above). The majority of ICRs are expressed in nociceptive sensory neurons, which can detect and respond to noxious mechanical, thermal and chemical stimuli. Nevertheless, the cannabinoids produce a profound antihyperalgesia and the mechanism has not yet been established . 2+ accumulation. As a result, cannabinoid-gated responses might not reach the threshold levels required to excite nociceptors. Moreover, slow depolarization of nociceptor membrane potentials might lead to inactivation of voltage gated channels that, in turn, inhibits the generation of action potentials.One possible hypothesis addressing this issue is that partial activation of ICRs does not necessarily generate excitation  of nociceptors. From this perspective, it is interesting that cannabinoids are not full agonists for TRP channels. Indeed, cannabinoids typically evoke a slow generation of small inward currents and CaTo understand how activation of ICRs leads to inhibition of nociceptors, molecular mechanisms of desensitization of TRP channels by ICR-activating cannabinoids have been studied . The resBased on the existence of TRPA1-TRPV1 heteromeric complexes we may also hypothesize the following mechanism: Ionotropic cannabinoids can activate antagonistic allosteric channel-channel interactions in such types of heteromeric complexes. This allosteric mechanism especially upon coactivation of the two TRP channels by selective ionotropic cannabinoids may produce cross-desensitization of the two nociceptor channels leading to antinociception and antihyperalgesia. The ionotropic cannabinoids may best be regarded as negative allosteric modulators of TRPA1-TRPV1 heteromeric complexes and other types of TRP heteromeric complexes. It is presently unknown to which extent metabotropic CB1 and CB2 may participate in the modulation of the peripheral nociceptors and in the mediation of the actions of the ionotropic cannabinoids. A dynamic interplay between CB1/CB2 and TRP channels as to heteromerization is however, an interesting possibility.Other mechanisms for acupuncture induced analgesia likely also exist since analgesic effects also develop in distant parts of the body. There exists a mechanism called diffuse noxious inhibitory control (DNIC) which results in reduction of pain from the experimental noxious stimulus when a nociceptive stimulus is applied to a region remote to the test area , 117. DNIt should also be noted that sensory neuropeptides like substance P may be released from nociceptors upon acupuncture and both acupuncture and sensory neuropeptides increase cutaneous blood flow . It is o Understanding the role of MOR-DOR, MOR-KOR, alpha-2AAR-MOR, and CB1-MOR heteromers in key pain circuits in the CNS. Of special interest will be to outline the role of opioid receptor heteromers as a target for the treatment of pain including acupuncture and the role of MOR1D-GRPR heteromer in itch and as a target for the anti-pruritus actions of acupuncture.  Understanding the role of TRPA1-TRPV1 heteromeric ion channel complexes in nociceptor function and their role in the antinociceptive and antihyperalgesic actions of cannabinoids. Characterization of the receptor interfaces in distinct opioid receptor and TRPA1-TRPV1 heteromers and putative ERs-MOR-KOR heteromeric complexes. The receptor interface is a novel target enabling modulation of the allosteric receptor-receptor interactions. Understanding the pharmacology of the above receptor heteromers. A major targets for the therapeutic effects of antinociceptive drugs which may mediate side effects. Understanding the link of potential changes in distinct opioid receptor and TRPA1-TRPV1 heteromer structure and function to plasticity changes in the pain pathways in chronic pain syndromes. Discovery of novel key receptor heteromers is still to come. Finally, discrete heteromers may also be targets for chemical ingredients mediating the medicinal properties and the side-effects of plants that is, herbal medicine.The role of the receptor heteromers in pain modulation may be studied along the following lines. The usage of terms in medicine often varies widely. For this reason, it is convenient and helpful to authors and readers if words can be used with an agreement in their technical meaning. The definition provided in this Glossary are intended to be specific andexplanatory and to serve as a useful framework, not as a constraint on feature development for members who work in the field of pain or are interested in this review article topics .Acupuncture. Acupuncture is a procedure involving the stimulation or inhibition at ananatomical location on or in the skin by a variety of techniques. A number of effects on painphysiology have been identified, the most important being the activation of the endogenousopioid system and the spinal modulation of pain signaling through activation of touch fibers (Aβ fibers).Analgesia. Absence of pain in response to stimulation that would normally be painful. The stimulus is defined by its usual subjective effects.Hyperalgesia. An increased response to a stimulus that in normally painful. Hyperalgesia reflects increased pain on supra-threshold stimulation. For pain evoked by stimuli that usuallyare not painful, the term allodynia is preferred, while hyperalgesia is more appropriately usedfor cases with an increased response at a normal threshold, or at an increased threshold, such asin patients with neuropathy. It should also be recognized that with allodynia the stimulus andthe response are in different modes, whereas with hyperalgesia they are in the same mode.Meridian. In the Chinese medicine acupuncture means each of a set of pathways in thebody along which vital energy is said to flow. There are twelve such pathways associated with specific organs.Nociception. Nociception is the sensory component of pain. It encompasses the peripheral and the central neuronal events following the transduction of damaging mechanical, chemicalor thermal stimulation of sensory neurons (nociceptors).Nociceptor. A receptor preferentially sensitive to a noxious stimulus or to a stimulus thatwould become noxious if prolonged. Often called a pain receptor.Noxious Stimulus. A noxious stimulus is one that is damaging to normal tissue.


Rice yield and quality are adversely affected by high temperatures, and these effects are more pronounced at the ‘milky stage’ of the rice grain ripening phase. Identifying the functional proteins involved in the response of rice to high temperature stress may provide the basis for improving heat tolerance in rice. In the present study, a comparative proteomic analysis of paired, genetically similar heat-tolerant and heat-sensitive rice lines was conducted. Two-dimensional electrophoresis (2-DE) revealed a total of 27 differentially expressed proteins in rice grains, predominantly from the heat-tolerant lines. The protein profiles clearly indicated variations in protein expression between the heat-tolerant and heat-sensitive rice lines. Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis revealed that 25 of the 27 differentially displayed proteins were homologous to known functional proteins. These homologous proteins were involved in biosynthesis, energy metabolism, oxidation, heat shock metabolism, and the regulation of transcription. Seventeen of the 25 genes encoding the differentially displayed proteins were mapped to rice chromosomes according to the co-segregating conditions between the simple sequence repeat (SSR) markers and the target genes in recombinant inbred lines (RILs). The proteins identified in the present study provide a basis to elucidate further the molecular mechanisms underlying the adaptation of rice to high temperature stress. Climate change and abiotic stress affect agriculture and crop production adversely. Among the various climatic factors affecting agriculture, temperature is one of the most important because higher temperatures adversely affect plant growth and yield  and mass spectrometry (MS) analyses provide an effective approach to investigate the molecular response of plants to stress. A previous study reported that >70 proteins in rice grains were differentially expressed when plants were exposed to high temperatures of 30–35 °C during the full-length grain-filling stage , originating from Xieqingzao B/N22//Xieqingzao B, were bred  markers and RIL populations.Two rice lines, heat-tolerant XN0437T and heat-sensitive XN0437S, were used as the plant materials in this study. They were inbred in a previous project, and showed significant differences in grain weight when exposed to high temperature during the ﬁrst half of the ripening period. These lines had a polymorphism rate of only 1.80% in the genome when the 887 SSR markers were used to detect the genetic polymorphism  at at least at one time point between the heat-tolerant and heat-sensitive lines. The experimental molecular weight and isoelectric point (pI) of a protein were estimated using its position on the 2-D gel.Image analysis of the silver-stained 2-DE gels was carried out using Image Master 2D platinum software version 5.0 . Each gel was analysed for spot detection, background subtraction, and protein spot OD intensity quantification. The gel image showing the highest number of spots and the best protein pattern was selected as the reference gel. All spot comparisons and analyses were coordinated using the reference gel, and its spots were then matched across all gels. The value of each spot was normalized as a percentage of the total volume in all of the spots present in the gel to correct the variability due to the quantitative variations in the intensity of protein spots. The heat-responsive proteins were detected based on the criteria that (i) the differential spots were reproduced on three biological replicates; (ii) spot volume variations changed at least 2-fold at at least one time point between the treatment and control; and (iii) spot volume variations changed  4700 proteomics analyser. The identities of the proteins were determined using MASCOT  software with the following optimized parameters: peptide mass tolerance of 100 ppm, a maximum of one missed tryptic cleavage, and allowing for iodoacetamide modiﬁcations including the oxidation of methionine. The peptide search was limited first to the taxonomy of rice. If no positive results were obtained, the search was performed again using green plants as the taxonomy. If peptides were matched to multiple members of a protein family, or if a protein appeared under different names and accession numbers, the entry with the highest score was selected. Identified proteins were grouped into individual categories according to their documented functions in MASCOT databases.To investigate the corresponding mRNA expression patterns of the proteins differentially expressed during high temperature stress, eight genes were selected and their expression patterns were monitored further. Total RNA was isolated from 0.5g of caryopsis using the Column Plant RNAout2.0 Kit  following the manufacturer’s instructions. Double-stranded cDNA synthesis and the cDNA concentrations of all samples were normalized against the expression of the ACT1 gene (GenBank ID: AK100267)  and transformed into DH5α-competent cells  following the manufacturer’s instructions. The positive clones verified by PCR were sequenced at the China branch of Invitrogen to confirm the identity of the products.The RILs inbred in a previous study  version 14.10 , and a two-tailed Student’s Supplementary Fig. S1 available at JXB online), but also the grain weight and net photosynthetic rate were decreased in both heat-tolerant (XN0437T) and heat-sensitive (XN0437S) rice lines after continued exposure to high temperatures . Compared with the heat-tolerant rice line, the net photosynthetic rate of the heat-sensitive rice line declined significantly (P < 0.05) after treatment with high temperature (38 °C) for 1 d. Several photosynthetic enzymes, such as Rubisco, undergo degradation in response to higher temperatures (>35 °C), which affects the photosynthetic rate  between heat-sensitive and heat-tolerant rice lines at at least at one time point after high temperature stress. The differentially displayed proteins (with spot volume variations between treated and control samples from heat-tolerant and heat-sensitive rice lines) that could be distinguished visually are shown in Based on the criteria used here for protein spot detection, 27 differentially expressed proteins were detected and their locations in 2-DE profiles are shown in in vivo or in vitro protein degradation when the experimental molecular weight is lower, and post-translational modifications such as glycosylation when higher than expected experimental molecular weights were observed  out of the 27 detected proteins could not be identified conclusively, which could be attributed to low protein spot volume and/or low quality spectra. Several of the identified proteins showed discrepancies between their experimental and expected molecular weight, consistent with previous reports , 16 proteins , and 20 proteins  were significantly up- and down-regulated in the heat-tolerant and heat-sensitive rice lines, respectively . These rOf the 10 proteins involved in biosynthesis , six  and 17.9kDa heat shock protein (OsUP2), were up-regulated in both heat-tolerant and heat-sensitive rice lines . HoweverThe protein involved in transcriptional regulation, the basic/helix–loop–helix (bHLH) transcription factor (OsUP9), was up-regulated in both heat-tolerant and heat-sensitive rice lines during high temperature stress.A comparison of the expression profiles of the differentially expressed proteins involved in energy metabolism, oxidation, and heat shock between heat-tolerant and heat-sensitive rice lines showed that the expression of most proteins was triggered earlier in heat-tolerant rice lines compared with heat-sensitive plants. It is possible that the self-conserving metabolism was triggered earlier in heat-tolerant rice lines than in heat-sensitive lines after exposure to high temperature conditions.l-ascorbate peroxidase (OsUP3), ADP-glucose pyrophosphorylase large subunit (OsDP3), thioredoxin type H (OsUP6), orthophosphate dikinase (OsDP4), and 18.1kDa heat shock protein (OsUP1)] were selected based on their possible functions and expression patterns in 2-DE analysis. The dbEST IDs in the NCBI database (www.ncbi.nlm.nih.gov/dbEST) and the PCR primers used to assess the eight representative genes are shown in Supplementary Table S1 at JXB online.Real-time qRT–PCR was used to monitor the expression patterns of the genes encoding the differentially expressed proteins at the transcriptional level. Eight representative genes  were identified that could regulate ROS levels. The expression levels of these proteins were significantly higher in both heat-tolerant and heat-sensitive rice lines during the process of adaptation to high temperature stress. l-Ascorbate peroxidase, putative malate dehydrogenase, and cytochrome c peroxidase were all up-regulated to a significantly greater extent in heat-sensitive rice lines compared with heat-tolerant rice lines after 3 d of high temperature stress (Reactive oxygen species (ROS) can be either beneficial or harmful to cells and tissues. At physiologically low levels, ROS function as ‘redox messengers’ in intracellular signalling and regulation, whereas excess ROS induce the oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death. Oxidative bursts induced by environmental stress trigger disturbances in the cellular redox balance, which are highly toxic. Plants have evolved complex regulatory mechanisms to prevent cellular injury by regulating the steady-state levels of ROS . The pere stress . In contThioredoxins (Trxs) are a multigene family of proteins that play a critical role in redox balance regulation via thiol–disulphide exchange reactions (Heat shock proteins (Hsps) are molecular chaperones that regulate the folding and accumulation of proteins as well as their localization and degradation in all plant and animal species . The sigl-ascorbate peroxidase (OsUP3), malate dehydrogenase (OsUP4), copper/zinc superoxide dismutase (OsUP7), and cytochrome c peroxidase (OsUP10) are all up-regulated. The signal of this redox imbalance is transduced to the Calvin cycle , and ADP-glucose pyrophosphorylase  is down-regulated. This affects the energy-related product glyceraldehyde-3-phosphate (GAP), which limits the material available for starch synthesis. This causes the UDPase (OsDP5) and GBSS (OsDP8) to regulate the catalysis of glucose-1-phosphate (G-1-P) to starch in the cytoplasm and chloroplasts. The interchange between amylopectin and amylase is affected by the quantity of synthetic starch, and starch debranching enzyme is down-regulated. In response to high temperature stress, the glycolytic pathway (EMP) accelerates, as does the key enzyme d-glyceraldehyde-3-phosphate dehydrogenase (OsUP5) that catalyses conversion of triose phosphate to 1,3-diphosphoglyceric acid  in the EMP. This suggests that starch in plant cells is synthesized less and consumed more under high temperature conditions compared with normal.Based on the functions of the differentially expressed proteins and the expression patterns suggested by comparative proteomics and real-time PCR, a possible mechanism is proposed by which these proteins function in response to heat shock in the plant cell . InitialThe faster EMP under high temperature conditions results in increased production of pyruvate (Pyr). As a substrate signal of the tricarboxylic acid (TCA) cycle, high levels of Pyr accelerate the TCA cycle process, which increases the conversion of succinic-CoA (Su CoA) to succinic Acid (Su Acid) that is catalysed by the GDP (OsUP19). This suggests that GDI is up-regulated. Oxoglutarate is a substrate of glutelin synthesis in the TCA cycle, and is up-regulated when the TCA activity is increased. The up-regulation of oxoglutarate suggests that glutelin synthesis and glutamine synthetase (OsUP16) are up-regulated. The differentially expressed proteins regulate the above processes, supporting the present observation that the accumulation of starch in rice grains under high temperature stress was less than that under normal temperature conditions, while the glutelin accumulation of rice grains was accelerated under high temperature stress.Supplementary Table S2 at JXB online) were further compared with the proteomic results from 2-DE. The characteristics of 12 proteins  coincided with the results from 2-DE, but those of the other five proteins did not. Therefore, it is possible that these five proteins were in the same linkage groups as the co-segregating markers, but not at their exact locus. The genes mapped to rice chromosomes could provide useful information for further studies on fine genetic mapping and cloning the full-length genes of rice that regulate the heat tolerance trait.To date, only a small number of genes involved in the high temperature stress response of rice at the milky stage have been mapped to rice chromosomes due to the difficulty of selecting phenotypic traits of rice that are related to high temperature. A previous study identified the genetic locus of rice that is related to high temperature stress at the milky stage  locus numbers and PCR primer sequences of co-segregating markers of 17 mapped genes in rice that respond to high temperature stress at the early milky stage.


The aim of the present study was to investigate and compare the apical sealing ability of Resilon/Epiphany-filled root canals with those that were obturated with gutta-percha/AH-Plus endodontic sealer.A total of 60 extracted human single-rooted teeth were selected; 25 teeth for each test group and five for each control group. After conducting conventional endodontic treatment, the teeth were immersed in physiologic saline solution for thirty days, and subsequently sealed and stored in methylene blue dye solution for seven days. The teeth were sectioned to evaluate the linear apical leakage using a stereoscopic microscope. The data were statistically analyzed by non-parametric Kruskal-Wallis and Mann-Whitney U tests.The results showed significant differences between the two groups of endodontic sealers (P<0.001).Within the limitation of the present in vitro study, Resilon/Epiphany sealer had better apical sealing ability than gutta-percha/AH-Plus sealer. The main objectives of root canal treatment are to clean and shape the root canal, removing all organic material, and to seal the pulp chamber and canal. Success in endodontic treatment is predominantly determined by complete obturation of the canal system. Gutta-percha, which has been commonly used for this purpose, has universally been accepted as the gold standard for root canal filling materials. However, it fails to provide an adequate apical seal, as it lacks the ability to bond or adhere to both the root canal dentine and sealer 2]3]4][3][4]2][[4][3][4][3][4]2]. Thus, fA total of sixty newly extracted single-rooted human teeth were used in this study. All selected teeth were free from any open apices, cracks, and resorptive defects. Teeth were carefully cleaned with curettes to remove the soft-tissue remnants and stored in saline solution prior to instrumentation. The crowns of the teeth were sectioned at the cemento-enamel junction by using water-cooled diamond discs.The canal length was visually established by placing a size 15 K-type file  into each root canal until the tip of the file was visible at the tip of the apical foramen. The working length was established 1mm short of the apex. Cleaning and shaping were performed. The root canals were prepared with K-flexofile , with the crown-down technique until the working length was reached. Instruments were adjusted to the walls of the canals, followed by instruments in ascending order of diameter, to achieve an apical flare equal to that provided by the size 45 file. During the biomechanical preparation, the canal patency was maintained with a size 10 file and irrigation with 3% sodium hypochlorite (NaOCl) solution  was carried out. After preparation the canals were instrumented with size-10 and -15 files 2 mm beyond the foramen to standardize the foramina diameter. After using the last file, the roots were irrigated with physiologic saline solution and dried with absorbent paper points . The canals were irrigated between instrumentation with 17% ethylene diamine tetra-acetic acid (EDTA)  and 3% NaOCl. EDTA was used as the final rinse before root canal obturation, as required by the manufacturer of Epiphany to remove the smear layer. To remove the remaining NaOCl and EDTA solutions final irrigation was performed with 5 mL normal saline and dried with a paper point. This protocol was employed for all teeth to eliminate the introduction of an additional experimental variable. The debrided root canals were dried with multiple paper points and randomly divided into two experimental groups (25 for each group) and two control groups (five for each group).The teeth of group I were obturated using the lateral condensation technique using gutta-percha master point  dipped in AH sealer . Excess gutta-percha was removed and condensed with plugger 1 mm below the canal opening; the root canal opening was sealed with a ready-made temporary filling material Cavit . In group II before inserting the sealer, the self-etching primer (Epiphany primer) was introduced into the root canal with microbrush and after 30 seconds, the excess material was removed with absorbent paper points. Teeth were obturated using the lateral condensation technique, a pre-measured master point to fit the diameter and length of the root canal, coated with the sealer  was placed in the root canal and condensed with a finger spreader. The rest of the canal was filled with accessory points dipped in a small amount of sealer, which were polymerized for 40 seconds in the coronal portion. The excess points were removed with a hot instrument; the root canal opening was sealed with a ready-made temporary filling material (Cavit).The tooth roots in group III (positive control) were not obturated and were used to demonstrate dye leakage throughout the entire length of the canal. The coronal third was filled with Cavit. The root canals in group IV (control) were obturated with gutta-percha/AH-Plus. Finally, all the teeth were immersed in physiologic saline solution for 14 days. The solution was renewed each week.Apical leakage was estimated using a dye penetration test. The root surfaces were covered with two layers of nail varnish and sticky wax with the exception of the apical 1 mm, but the root surfaces of the group IV teeth were entirely coated with two layers of nail varnish and sticky wax to prevent possible leakage. The teeth were then placed into 2% methylene blue dye solution for seven days at 37ºC. After being removed from the dye solution, the teeth were washed with water and dried, and then the nail varnish and sticky wax were removed with a scalpel. The teeth were sectioned longitudinally in a bucco-lingual direction through the center of the root. Linear apical leakage was measured from the apex to the coronal extent of the methylene blue dye penetration. Maximum dye penetration was measured in millimeters in each half of each tooth from the apex to the most coronal part of the root canal to which the dye had penetrated usinga stereomicroscope at 25× . The results were collected, tabulated and statistically analyzed using non-parametric Kruskal-Wallis and Mann-Whitney U tests. The level of significance was fixed at P<0.05.Means and standard deviations for the degree of leakage of the experimental groups were measured . Dye penComplete obturation of the root canal system with impervious, biocompatible, and dimensionally stable filling-material is essential for successful root canal treatment. However, it has been reported that a complete seal of the root canal system is almost impossible with currently accepted materials and techniques using a combination of gutta-percha and root canal sealer . IdeallyLeakage studies on the sealing properties of endodontic materials are still important and relevant . DiffereOn the other hand, other studies using bacterial leakage tests as well as a dye penetration method, did not observe a difference between Resilon and gutta-percha 20]. Kan. Kan20].Within the limitation of the present study, Resilon/Epiphany sealer had better apical sealing ability than gutta-percha/AH-Plus sealer.


During the summer and fall of 2002, an epidemic (223 cases) and epizootic of West Nile virus infections occurred in Chicago. Retrospective spatial analysis demonstrated that age-adjusted human case rates were three times higher inside geographic areas with high early-season crow deaths than outside these areas. West Nile virus (WNV) activity was first identified in North America in New York City in 1999 (Corvus brachyrynchos) have preceded the detection of human infections. Tracking sightings of dead crows is advocated as an integral aspect of WNV activity surveillance systems . The first dead crow sighting recorded by the nonemergency hotline occurred on July 1; by October 19, a total of 3,837 dead crow sightings at distinct addresses had been reported through the hotline. These reports peaked during the week ending August 17 (week 33) .Spatial analysis was used to assess the relationship between the locations of dead crow sightings reported before the first human case of WNV infection was detected and the reported residence of human WNV case-patients. Spatial analysis was conducted by using ArcView Spatial Analyst version 1.1 in ArcView version 3.2, and maps were generated with ArcView version 8.2 . Dead crow sightings reported through the hotline from July 1 through August 11 were geocoded and mapped. Smoothed dead crow densities were generated by using kernel estimation to calculate density and a bandwidth radius of 1 mile. The kernel function was used to describe mathematically the geographic density of crow deaths by assigning a value to a point on the map; the value is a function of that point’s proximity to surrounding crow deaths (>65 years); effect modification and confounding were assessed; and the rates within and outside the high crow-mortality areas were compared by calculating a Mantel-Haenszel weighted incidence ratio with Greenland-Robins 95% confidence intervals (CI) in EpiInfo version 6.04 . Because WNV fever case-patients were often seen as outpatients and their inclusion could have introduced diagnostic access bias into the findings, the analysis was conducted a second time, with patients limited to those meeting the WNV meningoencephalitis case classification.Human cases of WNV infection were geocoded and mapped by using the case-patient’s reported street address of residence. Human case rates were calculated on the basis of the 2000 census population within and outside the high crow-mortality areas. To control for age, the data were stratified by age group  had address information sufficient for geocoding. Home address data were obtained for 219 (98%) of the 223 Chicago case-patients. The high crow-mortality area covered 124 square miles (54% of Chicago’s surface area), and 179 (82%) of the geocoded case-patients resided within it’s the city’s boundaries . The cruWe identified a spatial association between early-season crow deaths and residences of WNV-infected case-patients in Chicago. The findings indicate that a functioning dead-crow surveillance system could assist in prioritizing geographic areas for mosquito-control interventions, such as risk communication and larval and adult mosquito control. These interventions could occur before, or concurrently with, the identification of WNV-infected case patients in a given transmission season.The success of dead crow surveillance systems depends in part upon public participation, and the Chicago nonemergency hotline—a simple, well-recognized, and well-publicized means of collecting dead crow data—was an essential prerequisite to this analysis. One potential source of bias that accompanies these data, nonetheless, is the self-selection of persons who report crow deaths. Residents of certain neighborhoods might be more inclined to notify their local government upon seeing a dead bird than those in other neighborhoods.WNV-associated avian deaths have historically preceded human illness, and in Chicago, the epizootic peaked approximately 2 weeks before the human epidemic. Real-time analysis of incoming dead crow reports might be especially important because of the time lag between illness onset, confirmatory testing for WNV infection, report to the local health authority, and initiation of an epidemiologic case investigation to identify and adequately describe WNV-related human illness. In Chicago in 2002, the first case of WNV infection in a resident was reported on August 12, 13 days after the patient’s onset of illness.Whether a similar spatial association between early-season crow deaths and residences of WNV-infected case-patients will be evident in future seasons is unknown, as an estimated 81% of the Chicago-area crow population is thought to have died in 2002 (


A general paradigm to understand protein function is to look at properties of isolated well conserved domains, such as SH3 or PDZ domains. While common features of domain families are well understood, the role of subtle differences among members of these families is less clear. Here, molecular dynamics simulations indicate that the binding mechanism in PSD95-PDZ3 is critically regulated via interactions outside the canonical binding site, involving both the poorly conserved  Protein interactions play crucial roles in all biological processes. A common way of studying them is to focus on sub-parts of proteins, called domains, that mediate specific types of interactions. For instance, it is known that most PDZ domains mediate protein interactions by binding to the C-terminus of other proteins. Humans have more than 200 slightly different copies of these domains. At the level of the binding site, PDZ domains look quite similar. This is in apparent contradiction with their heterogeneous binding specificity. Using detailed molecular dynamics simulations in conjunction with statistical analysis, we predict that contacts outside of the canonical binding site play important roles in regulating protein interactions. Some of these contacts influence the overall dynamics of PDZ domains, providing an explanation for their allosteric effect. These interactions involve regions of the PDZ domains that are much less conserved, suggesting that they can help in differentiating selectivity in this large domain family. Molecular dynamics (MD) simulations indicate that residues upstream of the 4th C-terminal amino acid are crucial for binding. Specifically, lysines residues at position −4 and −7 in the CRIPT peptide are observed to dynamically interact with the Seminal X-ray crystallography experiments on the third PDZ domain of PSD95 in complex with the CRIPT C-terminal peptide indicated that peptide binding is realized through the last four residues (peptide positions 0 to −3), while the rest of the peptide is mostly disordered PDZ3 see  [1], [17These observations suggested that interactions beyond the canonical class-I motif are needed to achieve stable binding in native conditions , possibly with a major role of the Structural cluster analysis provides a quantitative classification of the non-canonical interactions  in the WT showed that the flexibility of the bound form is not affected by helix truncation . HoweverThese observations have important consequences for the interpretation of the entropic penalty upon binding to The important role of backbone dynamics is in contrast with recent NMR relaxation experiments which found a negligible contribution of the backbone compared to side chains flexibility Stabilization of the extra-domain helix is further mediated by a hydrophobic patch, formed by he  loop , it is fTo further investigate this hypothesis, we used PSIPRED The PSD95-PDZ3 To provide a dynamical picture of the process, we performed additional simulations of the DLG1-PDZ2:E6 complex see . Our calIn PDZ binding, the relatively limited information about peptide amino acids more distant from the C-terminus prevented a clear structural understanding of the effect and importance of these upstream side chains. Our work aims to fill this gap by providing calculations with both a canonical 5-mer CRIPT peptide as well as a longer 9-mer peptide in complex with PSD95-PDZ3. Three main results emerge from our work.First, we observe in our simulations that peptide binding is mediated by ionic interactions with the loop following the binding site, referred to here as the Second, we propose a mechanistic explanation for the microscopic origin of the binding entropic penalty in absence of the extra-domain helix of PSD95-PDZ3. In the apo form, the helix plays a crucial role in stabilizing both the carboxylate binding loop and the Third, analysis of 258 human PDZ domains as well as MD simulations of single-mutants allowed for the identification of an amino acid at the beginning of the Molecular dynamics simulations were performed using the GROMACS implementation is 2I0L  . The firfour-digits code. The first three digits describe the peptide-loop interactions, e.g. “110”. The last digit represents an id, encoding the peptide structural conformation . The latter was obtained by running a leader-based cluster-analysis on the ligand backbone  Distance between the 5-mer peptide side chain nitrogen of (TIF)Click here for additional data file.Figure S2CRIPT peptide affinity scheme for PSD95-PDZ3. (Top) Schematic representation of the domain-peptide complex. Experimental binding affinities from Ref. (TIF)Click here for additional data file.Figure S3Amino acid frequencies at position 328 in human PDZ domains. Contribution of paralog domains have been weighted as described in (TIF)Click here for additional data file.Figure S4Conservation of the (TIF)Click here for additional data file.Table S1Details of the simulations performed.(PDF)Click here for additional data file.Table S2Examples of PDZ structures in complex with ligands with unresolved residues between position −4 and −7.(XLSX)Click here for additional data file.Table S3Manually curated multiple sequence alignment of the 258 human PDZ domains used in this work.(XLSX)Click here for additional data file.Table S4Clusters of human PDZ paralogs.(XLSX)Click here for additional data file.


The antipsychotic drug risperidone has become increasingly popular as a treatment for children with various behavioral disorders, including autism. However, little is known about the long-term effects of early-life risperidone treatment on the brain and behavior. The frontal cortex is one of the primary targets of risperidone action in the brain, so the purpose of this study was to determine if early-life risperidone treatment altered neurotrophin expression in the prefrontal cortex during adulthood.Twenty-four rats  received daily injections from postnatal days 14-42. Rats were divided into two treatment groups (1.0 and 3.0 mg/kg risperidone) and a vehicle control. Brain tissue was collected on postnatal day 62. Prefrontal cortical tissue from the left hemisphere was examined for neurotrophin expression through the use of a rat neurotrophin RT-PCR array kit . Sections of prefrontal cortex were also used for immunohistochemistry.PCR results indicated an up-regulation of mRNA for the cytokine, Leukemia Inhibitory Factor (LIF), in the low and high dose treatment groups. Immunohistochemistry is being conducted to assess possible differences in LIF protein levels in the prefrontal cortex of the different treatment groups.This study indicates that early-life risperidone treatment has the potential to alter the expression of proteins involved in cell growth and differentiation with brain regions critical for cognitive control. The behavioral consequences of these cellular changes are an area that further research should address.