MPAs  frequently occur in neurodevelopmental disorders because both face and brain are derived from neuroectoderm in the first trimester. Conventionally, MPAs are measured by evaluation of external appearance. Using MRI can help overcome inherent observer bias, facilitate multi-centre data acquisition, and explore how MPAs relate to brain dysmorphology in the same individual. Optical MPAs exhibit a tightly synchronized trajectory through fetal, postnatal and adult life. As head size enlarges with age, inter-orbital distance increases, and is mostly completed before age 3 years. We hypothesized that optical MPAs might afford a retrospective ‘window’ to early neurodevelopment; specifically, inter-orbital distance increase may represent a biomarker for early brain dysmaturation in autism.We recruited 91 children aged 7–16; 36 with an autism spectrum disorder and 55 age- and gender-matched typically developing controls. All children had normal IQ. Inter-orbital distance was measured on T1-weighted MRI scans. This value was entered into a voxel-by-voxel linear regression analysis with grey matter segmented from a bimodal MRI data-set. Age and total brain tissue volume were entered as covariates.Intra-class coefficient for measurement of the inter-orbital distance was 0.95. Inter-orbital distance was significantly increased in the autism group . The autism group showed a significant relationship between inter-orbital distance grey matter volume of bilateral amygdalae extending to the unci and inferior temporal poles.Greater inter-orbital distance in the autism group compared with healthy controls is consistent with infant head size expansion in autism. Inter-orbital distance positively correlated with volume of medial temporal lobe structures, suggesting a link to “social brain” dysmorphology in the autism group. We suggest these data support the role of optical MPAs as a “fossil record” of early aberrant neurodevelopment, and potential biomarker for brain dysmaturation in autism. Minor physical anomalies (MPAs) occur more frequently in neurodevelopmental disorders such as autism Minor physical anomalies (MPAs) are neurodevelopmental markers which manifest as unusual morphological features of the face or physique Traditionally, MPAs have been evaluated by visual examination of the body using the Waldrop scale inter alia to genes acting early in development or prenatal environmental risk factors In the current study, we propose that optical MPAs can serve as a putative biomarker to provide an accessible ‘window’ onto early neurodevelopment. The rationale for this is simple but compelling. First, it is known that the causes of autism may be linked In the present study, we describe the use of MRI to carefully measure inter-orbital distance. Considering that MPAs are established in intra-uterine life, that head size expansion is accompanied by an increase in inter-orbital distance increase in infancy, and there is dramatic head size expansion early in autism, we hypothesize that, in older children with autism, this will be reflected in greater inter-orbital distance. In addition, we use voxel-based methods to determine brain regions where grey matter anatomy may have been subject to the same developmental pressure in autism.The total sample comprised 91 children from our database who had trimodality MRI data available for analysis . Thirty-six children had an independent clinical diagnosis of autism spectrum disorder , were aged 7–16 years, and attending mainstream schools. Verbal IQ was estimated on the verbal subset of the WISC. Thirty of these passed cut-off for autism on all three domains of the ADI-R (Autism Diagnostic Interview – Revised version (ADI-R) A, B, C domains © Western Psychological Press, 2003). We also included 6 children classified as having pervasive developmental disorder not otherwise specified on the ADI-R (5 missed cut-off on 1 domain by 1 point). Exclusion criteria included a clinical diagnosis of co-morbid psychiatric or medical condition such as epilepsy, history of head injury, or clinically detectible genetic syndrome associated with autism such as Fragile X syndrome. Fifty-five age- and gender-balanced healthy controls  from local mainstream schools had been screened for major psychiatric illness using the structured parental interview of the Cantonese Diagnostic Interview Schedule for Children – Fourth version (DISC-IV), All MRI scans were obtained on a GE Signa 1.5 Tesla system  in Queen Mary Hospital, Hong Kong.T1-weighted scans were used for measurement of MPAs since they give clear grey-white definition of soft tissue and muscle landmarks. The MRI scanner prioritized clinical scan image quality such that in the course of this research study the T1 acquisition sequences were of two types. These were evenly distributed across the patient and control groups so that two thirds of the subjects in each group were scanned with sequence 1 , and the rest of subjects with sequence 2 . Although the T1 protocol variation was not expected to affect our results, we took care to enter T1 protocol as a covariate in the analyses of optical parameters.Interleaved dual-echo fast-spin echo (FSE) PD/T2 scans were used for voxel based morphometry. We have previously described the use of this protocol for whole brain voxel based analysis Scanning time for each subject was approximately 20 minutes. Each scan was screened by a consultant radiologist to exclude clinically significant abnormalities. All MRI scans were then assigned numerical codes to ensure that analyses were performed blind to group status.http://www.fil.ion.ucl.ac.uk). A reverse transformation could then be applied to the image to adjust tilt. This re-aligned image was resliced to a new volume with isotropic 1×1×1 mm voxels.T1 images were scrutinized along the sagittal and coronal planes in order to observe for rotational tilt due to ‘roll’ or ‘pitch’ in each plane respectively. For each image, a transformation matrix quantifying the tilt angle was constructed so that realignment could be achieved using SPM2 software  was used for MPA manual measurements. The axial slice which displayed the largest diameter for both eyes was selected for measurements of the distance between the orbits. The inter-orbital distance was defined as the most medial point of the orbits located at the point of intersection between nasal bone and eyeball The MRIcro viewer software . For this method, the PD/T2 images were segmented into tissue maps representing grey matter, white matter and CSF and linearly normalized to standard space to create customized study-specific templates. Next, as our study examined grey matter only, the grey matter maps in raw space were normalized non-linearly to the study-specific template. Finally, the normalized and segmented PD/T2 images were modulated with their Jacobian determinants to reflect their original volumes in native space following Good's optimized method Images were preprocessed using SPM2 software. The preprocessing follows the optimized method We conducted voxel-wise linear regression of grey matter volume and inter-orbital distance in each group separately using Cambridge Brain Activation software (CAMBA). Modulated grey matter maps from the preprocessing step were used to assess the significance of three-dimensional cluster statistics by non-parametric permutation testing Regions found to correlate significantly with inter-orbital distance in each group were exported as separate masks. Each mask was applied to the modulated grey matter maps of both control and autism group, and individual grey matter volume within this volume-of-interest were extracted.Inter-class coefficient ICC for MPA measurements performed 1 week apart was 0.95. There was no significant group difference in age, verbal IQ or total brain volume. As shown in For the autism group only, in a voxel-wise linear regression analysis we found a significant positive correlation between inter-orbital distance and the grey matter volume of the bilateral amygdala, extending to unci and inferio-medial poles of the bilateral temporal lobes and left orbito-frontal lobe (p<0.001), see The major finding in our study is that children with autism spectrum had significantly larger inter-orbital distance than the typically developing control group. In the autism group, inter-orbital distance was significantly positively correlated with the volume of the bilateral amygdalae, extending to infero-medial temporal lobes and orbito-frontal lobe. These results, covarying for age and total intracranial volume were confirmed in a volume-of-interest correlation analysis in SPSS. The increase in inter-orbital distance in the autism group was independent of symptom ‘scores’ on the diagnostic algorithm of the ADI-R.Cohen has written extensively on malformations of the craniofacial regions The dominant direction of cranial base expansion is antero-posterior In the autism group, grey matter volume of the amygdalae  and inferior pole of superior temporal lobes) was positively correlated with inter-orbital distance. This was not observed in the typical control group. The inference is that in this group, the growth of midline bony and brain regions are tightly linked; that is, regions involved in the regulation of socialization, emotion and memory We previously examined regional brain volumes in these children and did not identify a between-group difference in amygdala volume in the autism group compared to typically developing controls We capitalized on voxel-based morphometry to explore the relationship between brain structure and MPAs for each individual. Thus far, just one other study has used MRI to examine the relationship between brain morphology and MPAs and they performed this in individuals diagnosed with schizophrenia In terms of possible casual mechanisms, convention holds that minor physical anomalies develop during the first trimester We acknowledge inherent limitations of the methodology concerning the choice of reference axis for performing MPA measurements. We had considered a range of choices for baseline axis, all of which leverage on reliable and easy identification by external inspection or from bony X-rays. For example the orbito-canthal plane is formed by joining the external angle of the eye (canthus) to the ear canal . Alternatively, the Frankfurt horizontal plane according to the Boas atlas is classically taken as including the base of the orbits and the external auditory meatus and parallel to the horizontal plane, a plane which is commonly adopted by anthropologists in archaeological excavations In summary we found significantly increased inter-orbital distance in children with autism which was highly correlated with larger amygdala size in intellectually able children with autism. Thus MPAs may indicate early disruption of brain development even in intellectually able. We note that other non-brain dysmorphologies merit evaluation too and are in the process of extending our range of measurements. Replication is awaited in other study populations but these novel findings suggest MPAs may in time provide a useful additional biomarker for autism.


Coincident parallel fibre (PF) and climbing fibre (CF) input to cerebellar Purkinje cells (PCs) induces long-term depression (LTD) of PF synapses. PF LTD is widely considered as a synaptic mechanism of cerebellar learning and pattern recognition ,2]. It hWe have previously studied the effect of non-specific synaptic plasticity (NSSP) on pattern recognition in an artificial neural network. In the absence of noise in the input patterns, introducing NSSP resulted in a decreased pattern recognition performance, similar to the loss of performance reported for unsupervised learning based on Oja’s rule . HoweverThe present study investigates the effect of non-specific LTD on pattern recognition in a PC model with a realistic distribution of PF synapses and one synapse / spine. We placed 14,740 spines along the thin dendrites and distributed them uniformly, but with random orientation. Following the experimental evidence we modeled non-specific LTD as a Gaussian function of distance. We presented stored (noisy) and novel patterns to the PC model and determined the pattern recognition performance by measuring signal-to-noise (s/n) ratios for the difference in pauses in PC spiking caused by the presentations of input patterns; as shown previously , pauses In the absence of local spatial noise the pattern recognition performance deteriorated in PC models with non-specific LTD. For non-specific LTD with standard deviations of ≥ 1.25µm, the s/n ratio approached zero and pattern recognition became impossible.Introducing local spatial noise in the patterns also decreased the pattern recognition performance, both in the presence and absence of non-specific LTD. However, when the amount of noise in the stored patterns was ≥ 30%, PC models with non-specific LTD outperformed models where LTD was specific to active PF synapses. This improvement of pattern recognition by non-specific LTD was particularly pronounced when the spatial distribution of noise in the input patterns matched the spatial spread of the non-specific synaptic plasticity. In simulations where the extent of the spread of the noise exceeded the spread of the non-specific LTD, the beneficial effect of the non-specific plasticity disappeared in a graded manner with an increasing spread of noise.Our simulation results predict that a possible computational function for non-specific LTD at PF – PC cell synapses is to provide robustness against local spatial noise.


Schistosoma haematobium may increase the risk of HIV infection in young women. Therefore, the treatment of Schistosoma haematobium with praziquantel could be a potential strategy for reducing HIV infection. Here we assess the potential cost-effectiveness of praziquantel as a novel intervention strategy against HIV infection.Epidemiological data from Zimbabwe suggests that genital infection with We developed a mathematical model of female genital schistosomiasis (FGS) and HIV infections in Zimbabwe that we fitted to cross-sectional data of FGS and HIV prevalence of 1999. We validated our epidemic projections using antenatal clinic data on HIV prevalence. We simulated annual praziquantel administration to school-age children. We then used these model predictions to perform a cost-effectiveness analysis of annual administration of praziquantel as a potential measure to reduce the burden of HIV in sub-Saharan Africa.We showed that for a variation of efficacy between 30–70% of mass praziquantel administration for reducing the enhanced risk of HIV transmission per sexual act due to FGS, annual administration of praziquantel to school-age children in Zimbabwe could result in net savings of US$16–101 million compared with no mass treatment of schistosomiasis over a ten-year period. For a variation in efficacy between 30–70% of mass praziquantel administration for reducing the acquisition of FGS, annual administration of praziquantel to school-age children could result in net savings of US$36−92 million over a ten-year period.In addition to reducing schistosomiasis burden, mass praziquantel administration may be a highly cost-effective way of reducing HIV infections in sub-Saharan Africa. Program costs per case of HIV averted are similar to, and under some conditions much better than, other interventions that are currently implemented in Africa to reduce HIV transmission. As a cost-saving strategy, mass praziquantel administration should be prioritized over other less cost-effective public health interventions. chistosoma haematobium increases the risk of becoming infected with HIV among women in sub-Saharan Africa. Praziquantel is an oral, nontoxic, inexpensive medication recommended for treatment of schistosomiasis, which might be able to prevent the development of genital schistosomiasis. We constructed a mathematical model of female genital schistosomiasis and HIV infections, which we calibrated using epidemiological data from Zimbabwe. We used this model to investigate the potential cost-effectiveness of mass drug administration with praziquantel as an intervention strategy for reducing HIV transmission in sub-Saharan Africa. We showed that mass drug administration with praziquantel may be a timely, innovative, and cost-saving intervention strategy for HIV prevention in sub-Saharan Africa. As a cost-saving strategy, mass drug administration with praziquantel should be prioritized over other less cost-effective public health interventions. Our findings indicate the possible benefit of scaling up schistosomiasis control efforts in sub-Saharan Africa, and especially in areas were Schistosoma haematobium and HIV are highly prevalent.Evidence from epidemiological and clinical studies supports the hypothesis that genital infection with S Schistosoma haematobiumSub-Saharan Africa continues to bear a disproportionate share of the global HIV burden There are three lines of evidence that indicate to an association between FGS and an elevated risk of HIV infection. Firstly, there is a strong statistical association between FGS and HIV transmission. Several cross-sectional epidemiological studies have reported that in sub-Saharan Africa, the region most heavily affected by the HIV/AIDS pandemic, women with FGS have a three- to four-fold increased odds of having HIV compared to women without FGS S. haematobium-endemic areas.Praziquantel is a highly effective anti-schistosomal chemotherapy agent against schistosomal morbidity that may be able to prevent FGS and the clinical manifestations associated with enhanced HIV susceptibility To estimate the potential cost-effectiveness of preventing HIV infection in sub-Sahara Africa through mass treatment of schistosomiasis, we constructed a mathematical model for genital schistosomiasis and HIV infections in the adult population  from which we quantify FGS prevalence in the HIV model. The compartmental diagram in Initially, we modeled a Zimbabwean population with no praziquantel treatment. We divided the population into males and females, and high and low sexual activity risk groups defined according to rate of sexual partner change. The state variable of the model are given by We used Bayesian Markov Chain Monte Carlo (MCMC) Praziquantel is a highly effective anti-schistosomal therapy agent against schistosomal morbidity To estimate the funding required to implement the alternative strategies of praziquantel treatment, we used the WHO dose for praziquantel (2.5 tablets of 600 mg per child per year) We calculated the potential cost-effectiveness of mass praziquantel administration to school-age children for reducing HIV transmission. The status quo was to be no mass treatment of schistosomiasis, as is currently the case in Zimbabwe. We measured the effectiveness of the intervention in terms of the number of HIV cases averted during a baseline intervention period of 10 years. We measured cost-effectiveness in terms of program costs per HIV case averted and averted medical care costs over the ten-year intervention period as the base line duration, which was also varied to assess the impact of the duration of intervention on its cost-effectiveness.To identify the contribution of each model parameter to the variability of the number of HIV cases averted, we calculated the partial rank correlation coefficients (PRCCs) To fit our dynamic model to the epidemiological HIV/FGS data from rural Zimbabwe We used the estimated mean values of the epidemiological parameters of the dynamic model input to calculate the incidence of HIV and FGS from 2000 to 2009. We considered two scenarios for the mechanism through which annual praziquantel administration for may reduce HIV transmission. In the first scenario, we assumed that for women who received praziquantel during their childhood, treatment reduces their enhanced risk of HIV transmission per sex act. In this scenario, the model predicted that, for an efficacy of 30%, annual administration of a single dose of praziquantel to all school-age children could avert 21,120  cases of HIV at a program cost of US$259.31 per HIV case averted over a ten-year period . When adFor the first scenario of the mechanistic basis of praziquantel in which the elevated risk of HIV acquisition is mitigated, variation in the number of HIV cases averted was primarily driven by nerships . For thenerships , highligS. haematobiumOur analysis demonstrated that mass administration of praziquantel could possibly be a cost-saving intervention for preventing HIV infection in Zimbabwe. For a wide range of efficacies, annual treatment of school-age children between 5 and 14 years could result in saving between US$15.8 and US$101.4 million medical costs over a ten-year period for a program cost of US$51.68–259.31 per HIV case averted. We found that even the indirect benefits of reducing HIV transmission alone may be sufficient to make mass praziquantel administration a cost-saving intervention. As a cost-saving intervention, mass praziquantel administration should be prioritized over other less cost-effectiveness public health interventions. Given the additional health benefits of reducing schistosomiasis morbidity that were not considered, because our model does not explicitly capture the complex age-structure dynamics of Cost-savings arise at the interface of the low cost of praziquantel, its high efficacy, and elevated risk of HIV among FGS-infected women. Mass treatment of schistosomiasis also compares favorably to other biomedical interventions against HIV transmission. Treatment of sexually transmitted diseases is estimated to cost between $304 and $514 per HIV case averted Our analyses indicate the potential for mass treatment of schistosomiasis as an innovative and cost-saving public health tool for preventing HIV infections in sub-Saharan Africa. Given that epidemiological and clinical data on FGS dynamics are scarce, our model does not account for the potential natural acquisition of partial immunity to FGS, with age, among adult women Praziquantel prevents long-term schistosomiasis induced morbidity by killing egg-laying schistosomes in the host Epidemiological studies have shown that women infected with genital schistosomiasis have a three- to four-fold increased odds of having HIV compared to women without genital schistosomiasis Our results suggest that mass treatment of schistosomiasis in sub-Saharan Africa would not only have direct health benefits of reducing schistosomiasis infections, it may also avert cases of HIV and reduce the cost burden on the Zimbabwean medical system. By considering the impact of this neglected disease on HIV transmission, public health efforts can be expanded to include a broader set of cost-effective control strategies.Protocol S1Mathematical details and model parameters.(DOC)Click here for additional data file.


The T-box family transcription factor Brachyury is overexpressed in a variety of human carcinomas, including lung, breast, colon, ovarian and prostate. Brachyury has been shown to promote epithelial to mesenchymal transition (EMT) in tumor cells, a critical step in the path to metastasis. An HLA-A2 epitope of Brachyury has been shown to expand human T cells that are capable of lysing Brachyury-expressing tumor cells in an HLA-dependent manner. A phase I clinical trial is ongoing at the NCI using a recombinant yeast Brachyury vaccine. We have previously demonstrated that agonist epitopes of tumor-associated antigens are more effective than native epitopes at activating antigen-specific T cell responses. The current study sought to identify an agonist of the Brachyury HLA-A2 epitope in order to increase T cell activation and tumor lysis.A novel agonist epitope of Brachyury was generated by residue substitution of the native epitope. Characterization of this epitope as an agonist included; comparison of HLA-A2 binding affinity and stability, interferon-γ production by epitope-specific T cell lines, FACS analysis of activation markers, as well as Brachyury and HLA-A2 specific lysis of tumor cells. The presence of Brachyury-specific T cells in cancer patients that recognize the agonist peptide was determined by ELISPOT.The agonist epitope was shown to bind HLA-A2 with higher avidity and stability than the native. PBMC from colon and ovarian cancer patients reacted to the agonist peptide in an ELISPOT assay. Tetramer staining revealed Brachyury agonist-specific T cells in the PBMC of a prostate cancer patient after in vitro stimulation with the agonist peptide. T cell lines generated from both the native and agonist epitopes produced higher levels of interferon-γ in response to stimulation with the agonist epitope, and they also expressed higher levels of intracellular Ki-67 and perforin. The agonist-specific T cell line lysed a variety of Brachyury-expressing tumor cells more efficiently.An agonist epitope for Brachyury has been identified that, as compared to the native epitope, increased T cell activation and cytotoxic activity against tumor cells expressing native Brachyury. A T cell line generated with the agonist peptide displayed increased activation  and cytotoxic activity upon subsequent stimulation with the peptide. This study supports the use of the Brachyury agonist epitope as a cancer vaccine strategy.


Adoptive cell transfer (ACT) of tumor specific T lymphocytes, after host immunodepletion, was shown to mediate objective cancer regression of metastatic melanoma. Effective T cell activation depends, among other factors, on the functional avidity of the peptide-MHC complex (pMHC) to the T cell receptor (TCR), i.e. on the affinity and the number of pMHC-TCR contacts. Since the T cell repertoire is controlled by negative and positive selection in the thymus, naturally occurring TCRs have mostly low affinities, in the range of 1-100µM. Moreover, unlike antibodies whose affinities improve over time by somatic hypermutation (SHM), TCR do not undergo SHM. In our research we developed a system that can increase the affinity of a TCR to its ligand by subjecting TCR genes to somatic hypermutation, directed by the mutator enzyme Activation Induced cytidine Deaminase (AID). Affinity maturation reactions are performed ex-vivo in easy transfectable cells and affinity maturated TCRs are selected by tetramer staining followed by FACS sorting. The affinity maturation system is designed to be modular so the maturation can be done in several cycles in order to optimize TCR affinity. The affinity maturated TCRs are used to create anti-tumor reactive T cells by means of gene transfer into naïve lymphocytes or anti-viral CTL. These transformed T cells are then functionally tested in-vitro and in-vivo with murine melanoma models. Using this system we are trying to define the parameters that govern the changes in affinity as well as the biological consequences of these changes. Such systems can potentially be used to augment T cell responses to low immunogenic Tumor Associated Antigen (TAA) peptides, for the treatment of cancer.


MicroRNAs  are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls.We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs .Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202.MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use. Breast cancer (BC) is one of the leading causes of cancer death among women worldwide MicroRNAs  are a novel class of endogenous, non-coding, single-stranded RNAs, first described in 1993 by Lee et al. in C. elegans MiRNA loci are statistically over-represented at fragile genomic regions that are commonly amplified or deleted in human cancers, implying a connection of miRNAs with cancer initiation and progression The finding of a decrease of miR-15a and miR-16-1 in patients with chronic lymphocytic leukaemia was one of the first direct links between regulative miRNAs and cancer Aim of the present study was to analyze the miRNA expression patterns in whole blood of patients with early stage BC in comparison to healthy controls using a miRNA microarray chip.All patients of this study participated in a prospective case control study for the molecular detection of breast cancer (MODE-B Study). Patients presenting at our specialized breast cancer unit with suspect breast lesions are routinely asked to participate in this still ongoing MODE-B Study. The presented data are results from the monocentric miRNA pilot-study.The MODE-B Study was approved by the Ethics Committee of the Medical Faculty of the Friedrich-Alexander University Erlangen-Nuremberg (reference number 3937). Written informed consent was obtained from every patient and control individual before blood was taken.Venous blood samples (non-fasting) (2.7 mL per patient) were collected from cases and controls in EDTA blood tubes  containing 1.6 mg EDTA as anticoagulant and stored at −20°C until further processing. We selected the first 48 consecutive early stage BC patients suitable for this prospectively planed miRNA-biomarker-study.This study comprises two case control studies, a discovery study  and a validation study (RT-qPCR of selected miRNAs). Patients were included in the specialized breast unit. They either referred themselves because of a newly palpable breast lesion or were referred to the breast unit by their physicians because of suspicious lesions for further breast diagnostics and biopsy.The independent RT-qPCR validation cohort consisted of consecutive early stage BC patients diagnosed within the German BC screening program. These differences in patients' recruitment between the microarray study cohort and the RT-qPCR validation cohort resulted inevitably in a lower risk profile of patients in the validation cohort , which wTotal RNA extraction was performed as published previously The quality and quantity of the RNA was evaluated by 260/280 ratio using NanoDrop spectrophotometry (NanoDrop ND-1000) and Agilent 2100 Bioanalyzer .Each array of the Geniom® Biochip contains 11 replicates of 1100 miRNAs and miRNA star sequences as annotated in the Sanger database miRBase15.0 The approximate normal distribution of the measured data was verified by Shapiro–Wilk test with a median P-value of 5.9E-20. MiRNAs with different expression levels between BC patients and controls were identified by unpaired two-tailed parametric t-test. P-values obtained for each individual miRNA were adjusted for multiple testing by Benjamini–Hochberg adjustment CT) method. The average CT value of the control miR-16 for every sample was subtracted from the CT value for each respective mature miRNA reaction, resulting in the ΔCT value. The fold changes in miRNAs were calculated by the equation 2−ΔΔCt where the comparative cycle threshold (ΔΔCT) is defined as the difference between ΔCT (Cancer) minus ΔCT (Control) as described previously Two miRNA  which were differentially expressed in the microarray assays were analyzed in an independent validation cohort by RT-qPCR. Among the most deregulated miRNAs from our microarray experiments we selected miRNAs for PCR-validation with purchasable and well established primers to reduce sources of error. The miRNA isolation and RNA quantification of the independent validation cohort was identical to the microarray analysis. TaqMan miRNA assays from ABI  were purchased for miR-202 and miR-718. First, each miRNA was specifically reverse transcribed according to manufactures protocol using TaqMan miRNA RT-Kit with stem-loop RT-primer and ABI7300 (ABI). Second, each sample was analyzed in duplicates for each specific miRNA using a RT-primer with universal master mix II (without Uracil-N-glycosylase) on the Applied Biosystems 7300 Sequence Detection System according to manufactures protocol. The cycle thresholds (Ct) for BC patients and their age-matched healthy controls were calculated and normalized to miR-16 , which was found in the literature as the most widely-used endogenous control miRNA for RT-qPCR. Each analysis also contained inter- and intra-assay replicates. High Ct-values indicated low miRNA quantity and vice versa. The expression levels of miRNAs in BC patients relative to their age-matched healthy controls were calculated using the comparative cycle threshold  and the consecutive patient recruitment.We used miRNA microarray technology to analyze the miRNA expression profiles of early stage BC patients compared to healthy controls from frozen EDTA-whole-blood and validated the results with RT-qPCR in an independent early stage BC cohort. Research of circulating miRNAs as blood based biomarkers is still in its infancy. However, this study as well as other recent studies indicate that miRNA-analyses have diagnostic and prognostic potential and could improve early stage BC detection in the future. There are several possible applications of miRNA profiling conceivable in the future. Firstly, miRNA profiles could help to reduce unnecessary breast biopsies if miRNA sets could be identified which reliably identify BC free individuals. Secondly, miRNA profiling could be used as a pre-screening method for example by general practitioners to identify women with an urgent need for breast diagnostics. Thirdly, in younger patients with dense breast tissue a future miRNA-based BC screening could possibly provide better sensitivity and specificity than the mammography even without radiation exposure.Our study detected several significant deregulated miRNAs in frozen whole blood of early stage BC patients, which should be analyzed further with regard to their function in breast cancer development and progression. Moreover, large prospective clinical studies are clearly warranted to confirm our preliminary results and further explore the existing potential of circulating miRNAs in serum, plasma or whole blood as diagnostic and therapeutic BC biomarkers.Figure S1Classification plot of microarray signatures. This is a classification plot demonstrating that a multimarker signature increases test accuracy, specificity and sensitivity depending upon the number of miRNAs that compose the diagnostic signature.(TIF)Click here for additional data file.Table S1MiRNAs differentially expressed in different subgroups.(DOC)Click here for additional data file.


The aim of this study was to compare the recovery of long-term facial nerve function between patients who received sural grafts and those who underwent hypoglossal-facial anastomosis techniques following translabyrinthine vestibular schwannoma surgery. This study included 25 patients with vestibular schwannomas treated with translabyrinthine tumor removal. All patients had large tumors with a mean tumor size of 3.12 cm. Of these patients, six had progressive tumor enlargement symptoms and had been treated previously with stereotactic irradiation. Preoperatively, all patients had normal facial functions, and total tumor removal with a translabyrinthine approach was achieved in all cases. During surgery, the facial nerve was interrupted in all 25 patients. Two types of facial reanimation were performed. Sural grafts were placed in 13 patients and hypoglossal-facial (VII–XII) anastomosis was performed in the other 12. Facial nerve function and surgical outcomes were observed upon discharge, in the short term (one year following surgery), and in the long term (three years following surgery). Total facial paresis was observed in all patients upon discharge. In the sural graft group, House-Brackmann grade III facial function was achieved in four patients upon short-term evaluation and in ten upon long-term evaluation, while House-Brackmann grade IV facial function was achieved in nine patients upon short-term evaluation and three in the long term. In the VII-XII anastomosis group, House-Brackmann grade III facial function was achieved in two patients in the short term and eight in the long term, and House-Brackmann grade IV facial function was achieved in ten patients in the short term and four in the long term. There was a statistically significant difference in the facial recovery results between the short- and long-term follow-up periods. The sural graft group exhibited a marked improvement in results compared with the VII–XII anastomosis group, but no statistically significant difference in facial function was observed between the two facial reanimation groups at either the short- or long-term follow-up. In the sural graft group, synkinesia, noted in three patients, was the most frequently observed complication. Claudication was common upon discharge (four patients), but diminished during follow-up. Disarticulation was the most common complication in the VII–XII anastomosis group (five patients); numbness of the tongue was the second most common complication (four patients). None of the patients developed dysphagia. Facial reanimation is an effective procedure for the surgical rehabilitation of static and dynamic facial nerve functions. Significant improvement in facial nerve function may occur more than three years after surgery. Despite morbidities such as synkinesia, the sural graft technique demonstrates greater improvements in facial nerve function than VII–XII anastomosis in the short and long term following surgery, but this conclusion requires confirmation by larger studies with a greater number of patients. Vestibular schwannoma surgery is currently performed with low morbidity and mortality rates due to improvements in diagnostic capabilities and advances in perioperative monitoring and microsurgical techniques . IntraopThe aim of the current study was to compare the short-and long-term facial recovery results and surgical outcomes of patients who had undergone sural grafting and VII–XII anastomosis for the reanimation of interrupted facial nerves during the removal of translabyrinthine vestibular schwannomas.All vestibular schwannoma surgeries performed by surgeons at Xinhua Hospital  between 2003 and 2006 were screened for the following selection criteria: i) translabyrinthine total tumor removal; ii) normal preoperative facial nerve function (House-Brackmann grade); iii) interruption of the facial nerve during surgery and immediate reanimation by sural graft or VII–XII anastomosis; and iv) data available at all postoperative points in time . These criteria were met in 25 cases. These patients became the subjects of this study. Written informed consent was obtained from patents and their families. The study was approved by the Ethics Committee of Xinhua Hospital.The characteristics of the patients in the two groups are shown in The patients were divided into two groups, odd and even according to the last number of their ID. Odd patients received sural graft facial reanimation and even patients received VII–XII anastomosis. The four patients in whom it was not possible to identify the proximal stumps of the facial nerves were placed in the even group.Facial function and surgical data were obtained from Xinhua Hospital records. Patients who underwent vestibular schwannoma removal were routinely followed up for facial function and surgical outcome by interview approximately one and three years following surgery. All data were accessible for the purpose of this study.During the follow-up period, any complications developed by the facial reanimation patients, such as synkinesia, claudication, disarticulation, numbness of the tongue and dysphagia, were assessed.Statistical comparisons were performed using parametric and non-parametric tests, as appropriate. Assessments were performed using two-tailed probabilities. P<0.05 was considered to indicate a statistically significant difference. Statements showing no difference between groups indicate that a statistical test was performed and failed to reject the null hypothesis.House-Brackmann facial grade VI function was observed in all 25 patients upon discharge. Significant recovery of facial nerve function was observed at both postoperative time intervals: one year (short term) and three years (long term). Facial nerve grades for the two groups at the short- and long-term intervals are shown in A comparison of the facial recovery results of the techniques revealed that, upon short-term follow-up, House-Brackmann grade III facial function was achieved in four (30.8%) and two (16.7%) cases in the sural graft and VII–XII anastomosis groups, respectively. Upon long-term follow-up, House-Brackmann grade III facial function was achieved in ten (76.9%) and eight (66.7%) cases in the sural graft and VII–XII anastomosis groups, respectively. It appeared that facial nerve function recovery was improved in the sural graft group compared with the VII–XII anastomosis group in both the short- and long-term follow-up, but no statistically significant difference was observed between the groups .Some special outcomes following facial animation were observed during postoperative follow-up. In the sural graft group, claudication was common upon discharge (four patients), but diminished over time. Synkinesia, the most common cause of complaint, was observed in three patients. In the VII–XII anastomosis group, disarticulation was the most common complication, observed in five patients. Numbness of the tongue was the second most common complication, observed in four patients. None of the patients developed dysphagia and synkinesia.In this type of large tumor, total tumor removal is achieved exclusively through the translabyrinthine approach ,9. The tThe incidence of facial nerve interruption has been reduced significantly due to improvements in surgical techniques and perioperative monitoring. It has been reported that automatic facial nerve preservation is achieved in >90% of large vestibular schwannoma patients. However, a certain degree of facial interruption is inevitable, particularly in patients with large or recurrent tumors .Various techniques may be used for facial reanimation depending on the characteristic of the proximal stump of the facial nerve and the preference of the surgeon . Sural gThis study compared patients who underwent surgery during the same period by surgeons with the same level of experience. The mean patient age was 46.69 years in the sural graft group and 47.00 years in the VII–XII anastomosis group. No statistical difference in age was detected between the groups. Tumor size is the most important factor affecting facial function following surgery. The mean tumor size was 3.15 cm in the sural graft group and 3.08 cm in the VII–XII anastomosis group. No statistically significant difference was detected between the groups with regard to tumor size.We compared the short- and long-term outcomes of facial reanimation. In the sural graft group, House-Brackmann grade III facial function was achieved in four patients in the short term and in ten patients in the long term. House-Brackmann grade IV facial function was achieved in nine patients in the short term and in three patients in the long term. In the VII–XII anastomosis group, House-Brackmann grade III facial function was achieved in two patients in the short term and eight patients in the long term. House-Brackmann grade IV facial function was achieved in ten patients in the short term and in four patients in the long term. There was a statistically significant difference between the facial recovery results across the short- and long-term follow-up periods. The outcomes of facial reanimation were acceptable following sural grafting and VII–XII anastomosis.We compared the long-term outcomes of facial recovery following the different surgical facial reanimation techniques. Sural grafting appeared to produce superior facial recovery compared with VII–XII anastomosis in the short term (House-Brackmann grade III 30.8 vs. 16.7%) and in the long term (House-Brackmann grade III 76.9 vs. 66.7%). However, no statistically significant difference was observed between the surgical groups. Selecting the appropriate surgical technique for facial reanimation requires careful consideration of the complications and benefits of all available surgeries. A study sampling a greater number of patients may provide further insight into the value of various surgical procedures.Following surgery, synkinesia developed in 23.1% (3/13) of sural graft cases in the present study. No significant synkinesia was observed in the VII–XII anastomosis group. This observation is explained by the fact that the healing of two nerves led to a greater incidence of incorrect regeneration by the facial nerve fibers .Loss of function of the hypoglossal nerve led to numbness of the tongue and disarticulation, which was occasionally accompanied by severe facial paresis. Following surgery, rehabilitation training of the hypoglossal and facial nerves may improve functional recovery.In conclusion, facial reanimation remains an effective procedure for the surgical rehabilitation of static and dynamic facial nerve functions. A significant improvement in facial nerve function may occur more than three years following surgery. Despite such morbidities as synkinesia, the sural graft technique is accompanied by a greater improvement in facial nerve function compared with VII–XII anastomosis in both the short- and long-term evaluations, but this requires confirmation by larger studies.


Multiple sclerosis (MS) damages central white matter pathways which has considerable impact on disease-related disability. To identify disease-related alterations in anatomical connectivity, 34 patients (19 with relapsing remitting MS (RR-MS), 15 with secondary progressive MS (SP-MS) and 20 healthy subjects underwent diffusion magnetic resonance imaging (dMRI) of the brain. Based on the dMRI, anatomical connectivity mapping (ACM) yielded a voxel-based metric reflecting the connectivity shared between each individual voxel and all other brain voxels. To avoid biases caused by inter-individual brain-shape differences, they were estimated in a spatially normalized space. Voxel-based statistical analyses using ACM were compared with analyses based on the localized microstructural indices of fractional anisotropy (FA). In both RR-MS and SP-MS patients, considerable portions of the motor-related white matter revealed decreases in ACM and FA when compared with healthy subjects. Patients with SP-MS exhibited reduced ACM values relative to RR-MS in the motor-related tracts, whereas there were no consistent decreases in FA between SP-MS and RR-MS patients. Regional ACM statistics exhibited moderate correlation with clinical disability as reflected by the expanded disability status scale (EDSS). The correlation between these statistics and EDSS was either similar to or stronger than the correlation between FA statistics and the EDSS. Together, the results reveal an improved relationship between ACM, the clinical phenotype, and impairment. This highlights the potential of the ACM connectivity indices to be used as a marker which can identify disease related-alterations due to MS which may not be seen using localized microstructural indices. Multiple sclerosis (MS) is a disease with an inflammatory, demyelinating and neurodegenerative disease course. It diffusely affects the structural and functional integrity of white matter (WM) tracts, contributing to disease-related disability. Regional structural abnormalities can be readily detected with magnetic resonance imaging (MRI) which explains the pivotal role of conventional MRI techniques for early diagnosis of MS Diffusion magnetic resonance imaging (dMRI) is an imaging modality sensitive to the mobility of water molecules. It is sensitive to disease-induced changes in microstructure because the diffusion process of water is determined by the microstructural boundaries of the local brain tissue environment. To characterize the diffusion properties within a voxel and to estimate the most probable axonal fiber direction, diffusion tensor imaging (DTI) can be used While DTI indices reflect localized microstructural properties of brain tissue, other indices such as anatomical connectivity mapping (ACM) are able to give a more complex description of the health state of the WM network. ACM does this by estimating a connectivity index reflecting the strength of connectivity a voxel has with the rest of the brain. Since MS is a very heterogeneous disease and each voxel of the ACM may be sensitive to microstructural damage at a distant site, we believed it may be able to supply disease contrast not seen with localized DTI indices. The voxel-specific whole-brain anatomical connectivity indices of ACM are estimated by initiating probabilistic streamlines from all voxels within a whole-brain mask and counting the number of streamlines passing through each voxel within the mask. The ACM approach was first proposed by Here we apply ACM to study the disruption of anatomical connectivity in relapsing remitting MS (RR-MS) and secondary progressive MS (SP-MS). Unlike previous works we minimized the influence of inter-subject anatomical differences by estimating ACM in a common stereotactic space. Given the diffuse pathology of MS, we hypothesized ACM to reveal widespread reductions in motor-related WM compared with healthy subjects and that these reductions will differ depending on the clinical form of MS. The principal reasoning behind the motor-related hypotheses is that MS patients are expected to experience motor disabilities. The motor-related reduction should be apparent when comparing patients to healthy subjects and RR-MS patients to SP-MS patients. The between MS group connectivity reduction is hypothesized since the SP-MS patients of our study exhibit an increased motor disability compared to RR-MS patients, as measured by a higher extended disability status scale (EDSS) score Thirty-four patients suffering from MS fulfilling the revised McDonald criteria The study was approved by the Scientific Ethical Committee of the municipalities of Copenhagen and Frederiksberg (protocol no. KF01 – 131/03 with addendum) and all subjects gave written informed consent.3 isotopic resolution. Also acquired was a three-dimensional T2-weighted image (T2) with a TR of 3000 ms, TE of 354 ms, FLA = 180° and a matrix of 196×256×192, resulting in a 1.1×1.1×1.1 mm3 isotropic resolution and a three-dimensional T2-weighted fluid attenuation inversion recovery (FLAIR) image using the sequence parameters, TR = 6000 ms, TE = 353 ms, FLA = 180° and a matrix of 220×256×192 (1.1×1.1×1.1 mm3 resolution).All MRI data were acquired on a Siemens TRIO 3 tesla scanner using an eight-channel surface head-coil . The structural MRI protocol included a three-dimensional T1-weighted image using a magnetization prepared rapid gradient echo (MPRAGE) sequence with a repetition time (TR) of 11400 ms, echo time (TE) of 2.32 ms, flip angle (FLA) of 9° and a matrix of 182×218×182, resulting in a 1 mm3 isotropic voxel resolution. For each subject, ten non-diffusion weighted images (b0) and 61 diffusion weighted images (DWIs) were acquired using a b-factor of 1200 s/mm2. In addition, a field map was obtained used a double gradient echo sequence  resulting in a voxel resolution of 2×2×3 mm3.Diffusion MRI (dMRI) was acquired using a twice-refocused spin echo sequence http://www.fil.ion.ucl.ac.uk/spm/) The DWIs were simultaneously corrected for eddy currents and motion by co-registering them with the first of the ten b = 0 images using a 12 parameter affine transformation Based on T2, FLAIR and MPRAGE modalities WM lesions were segmented using an automated segmentation method described by http://cmic.cs.ucl.ac.uk/camino/) 2. To determine the number of fibers in a voxel, we used the classification approach proposed by The multi-tensor model by http://www.fmrib.ox.ac.uk/fsl/) 3 isotropic resolutions matching the FMRIB58_FA atlas.To ensure ACMs with cross-subject comparability, it is important to prevent cross-subject brain shape variability from biasing the connectivity values of the ACM. To avoid this, the tensor models were spatially normalized into a common space The values of an ACM reflect the connectivity/dis-connectivity that each individual voxel has with the rest of the brain. The ACM is estimated by repeating probabilistic tractography for every seed voxel of the brain and counting the number of probabilistic streamlines passing through each individual voxel The brain mask was derived from the FMRIB58_FA atlas by identifying voxels with FA≤0 as background and the rest as voxels belonging to the brain mask. This definition is inherited directly from the FMRIB58_FA atlas documentation. This whole-brain mask is used both as a seed mask and as a streamline stopping criteria. We used the PICo probabilistic tractography method by To minimize the uncertainty associated with voxel-wise connectivity estimates of an ACM it is important to choose a sufficiently large number of streamlines. We therefore estimated the average uncertainty of each ACM voxel as a function of the number of streamlines in five healthy subjects. Individual subject ACMs were estimated five times for a fixed number of streamlines  and the voxel-wise coefficient of variation (CV) was calculated The estimation of a lesion probability map was done by deforming and resampling all lesion masks towards the common atlas space (FMRIB58_FA), using the spatial normalization previously described. This was followed by a voxel-wise summation of the spatially normalized lesion masks, each voxel quantifying the number of observed lesions divided by the total number of subjects.Regarding the voxel-based measures of whole-brain connectivity, we hypothesized that both, the group of RR-MS and SP-MS patients, would show increased connectivity relative to a group of healthy subjects. In addition, we expected that SP-MS patients (with a higher range of EDSS scores) would display reduced connectivity relative to RR-MS patients (with a lower range of EDSS scores). These hypotheses, commonly referred to as alternative hypotheses, were examined by testing the null hypotheses which assumes no differences between groups. The hypotheses were investigated using a general linear model (GLM), including group, age, gender and a voxel-based atrophy measure as independent covariates while using the ACM values as the dependent variable. The purpose of the voxel-based atrophy covariate is to adjust for potentially confounding effects that tissue atrophy may have on the spatially normalised ACM and FA. For instance, atrophy in the form of enlarged ventricles might introduce consistent errors in the spatial normalization process Prior to fitting voxel-wise GLMs, the ACM data were smoothed using a Gaussian kernel with a full-width half-maximum value of 4 mm. After fitting the GLMs, the GLM parameters were examined using one-tailed two sample t-tests with the assumption of unequal variance, testing for voxel-wise significant differences between the group parameters of the GLM. The same voxel-based GLM analyses were repeated using FA. The additional FA-based analyses enabled us to compare the outcome of analyses based on a measure reflecting localized microstructural tissue properties (FA) with analyses based on a measure reflecting whole-brain connectivity (ACM).The corticopontine tracts and superior longitudinal fascicles of the left and right cerebral hemispheres were defined as tracts of interests. We chose the corticopontine tracts because they contain the major descending corticospinal and corticonuclear motor fibers and therefore, are critical to motor function 2We expected that regional reduced anatomical connectivity in the motor-related tracts might be closely related to inter-individual variations in clinical disability. The analyses were conducted by estimating a regional median ACM statistic within the aforementioned ROIs, which were correlated with the EDSS scores using Pearson's correlation coefficient. For the sake of comparison, the linear correlation was also calculated for the median FA within the ROIs. These analyses were supplemented by GLM analyses using models adjusted for confounding covariates. We fit the GLM using EDSS as the outcome measure, and using the median statistic, age and gender as explanatory covariates. The fitting correlation of the GLM and its coefficient of determination rWhite matter lesion load is often used as an imaging marker in MS treatment studies ACM estimation using probabilistic tractography with different numbers of streamlines per voxel revealed a decreasing SNR benefit as the number of streamlines increases . SNR becUsing the voxel-based GLM it was found that patients with RR-MS and SP-MS had widespread ACM reductions in all motor-related WM compared to healthy subjects as shown in Patients with RR-MS and SP-MS also demonstrated widespread decreases in FA , resultiIn summary, these results show that the pathology changes of both RR-MS and SP-MS are associated with widespread motor-related decreases in ACM and FA. They further suggest that based on summary statistics, FA and ACM appear equally informative about the clinical phenotypes.Comparing the findings of We found the median ACM statistics in the motor-related WM to be decreased in patients with SP-MS relative to patients with RR-MS (p = 0.013, two-sample t-test; The difference between the FA and ACM analysis results is likely because the ACM indices are increasingly global in nature as opposed to FA. We believe this property in part overcomes analysis difficulties caused due to different MS subjects with similar symptoms but having spatially heterogeneous disease pathologies. The effect of such heterogeneous pathology pattern is observed by the non-significant and widespread differences between the RR-MS and SP-MS lesion maps in The region-specific median ACM statistics within the motor-related WM tracts showed significant correlations with the individual EDSS scores . Correla2 = 0.35.For completeness and in comparison with Based on the connectivity information of ACM, we found widely reduced anatomical connectivity in the motor-related WM of RR-MS and SP-MS patients compared to healthy subjects. SP-MS patients showed voxel-wise reduced ACM values relative to RR-MS while no consistent decreases were found using FA. Accordingly, the motor-related disease symptoms of the two MS groups are significantly different; however the difference is only measurable using ACM. Regional ACM summary statistics correlated moderately with clinical disability as reflected by the EDSS score and showed correlation similar to or higher than FA summary statistics.Voxel-based analyses and region specific statistics provided converging evidence for a reduction in the cerebral WM for the ACM of MS patients, compared to a group of healthy subjects. This is in contrast with a previous study where ACM has been investigated in patients with RR-MS Several pathological processes may have contributed to reducing the ACM in patients with MS. Axonal damage, Wallerian degeneration, local inflammation and demyelination, all contribute to changing the ACM values, since these pathologies may affect the diffusion process, leading to changes in the principal directional uncertainty of the fODF, propagating through to the ACM. The decreased ACM of MS patients widely summarizes these pathologies, since an ACM voxel obtains its value based on streamlines that will have passed through areas of the WM tract with potentially different underlying disease pathology. Consequently an ACM not only reflect the localized tissue pathology as with DTI indices. It also reflects the accumulated effect that pathologies have on estimating a structural WM connection from a given voxel. As may be expected the results show decreased connectivity in patients with more severe symptoms.In addition to voxel-wise ACM reductions, MS patients also exhibited decreases in FA, suggesting that both ACM and FA are sensitive to measuring MS-related changes, although they have different interpretation. As with ACM values, FA changes can be a function of axonal damage, Wallerian degeneration, local inflammation and demyelination. However, the interpretations of the measurements are quite different. In particular the values of an ACM measure a spatially dependent whole-brain effect derived from the trajectories of streamlines passing through multiple voxels. In distinction, FA measures local microstructural tissue changes, which may to some degree also reflect distant tissue damage. While there may be some correlation between ACM and FA, they are by design sensitive to very different aspects of the dMRI measurements. The FA decreases observed here agree with the findings of We believe the difference between ACM and FA explains why group-wise comparisons based on ACM resulted in significant differences between SP-MS and RR-MS patients, while we were unable to find differences using FA. Additionally, the median ACM statistic of the entire motor-related WM ROI was significantly lower in patients with SP-MS than in patients with RR-MS. Together; these findings highlight the potential use of ACM to capture alterations in the connectivity pattern which are characteristic to the different clinical forms of MS.The significant voxel-based ACM decreases found was hypothesized prior to undertaking the study. The strongly localized ACM reductions in patients with SP-MS are plausible as the patient group with RR-MS had less motor impairment as reflected by their EDSS scores. The ACM decreases of SP-MS found in the superior longitudinal fasciculus were hypothesized to be motor-related but could also be related to differences in distinct cognitive functions between both groups. Since we did not expose patients to cognitive tests, we cannot comment on this possibility.Supplementing these findings, the differences in lesion probability maps between the two phenotypes were found to be non-significant. This is consistent with the finding of very few phenotype-related FA-related differences but in stark contrast with ACM. The ACM-related differences suggest it is indeed able to capture/quantify system specific spatially complex pathologies; in a way not observable using FA or lesion probability maps.In our patient cohort, region-specific median ACM statistics calculated within the WM ROIs correlated with MS disability as indicated by the EDSS score. Negative correlations were found between the median ACM statistics and individual EDSS scores. In contrast to our results, the study by Correlation analyses did not reveal any significant correlations between the median FA statistics and individual EDSS. However, when applying a GLM that adjusted for between-subject differences of age and gender, we found a linear relationship with EDSS for both the median ACM and median FA. The explanatory power of the linear relation was generally stronger for using the ACM than it was using FA. The median ACM in the right posterior corona radiata, right superior longitudinal fasciculus correlated more strongly with the EDSS than the corresponding median FA. Our results confirm that ACM is at least as good a marker of MS-related disability as FA. Since ACM and FA provide complementary information, it is possible that a combined measure that integrates the structural information provided by FA and ACM might be most sensitive to disability and disease progression. This possibility remains to be addressed in depth in future studies.The findings suggest that ACM may be used to relate regional anatomic disconnection with disease-related disabilities. A similar finding was made in a study involving amyotrophic lateral sclerosis patients, in which a tract-specific probabilistic index of the corticospinal tract was found to achieve better disease score correlation than FA When ACM is applied to patients with MS, probabilistic tractography needs to measure isotropic diffusion components in the affected brain tissue due to inflammation, demyelination, gliosis, and axonal injury. We combined the multi-tensor model with the Bingham distribution The multi-tensor model is one of numerous models that could be used for reconstructing local fiber configurations of WM. In the context of ACM, We introduced a method for obtaining a spatially normalized ACM, suited for group studies to account for the integrated pathology occurring along WM tracts of MS patients. Unlike previous work, this way of estimating ACM ensured that the connectivity estimates of ACM become minimally dependent on the individuals brain shape and size variation. We successfully applied the ACM, to detect consistent differences in whole-brain anatomical connectivity between two clinical sub-groups of MS. We were able to relate alterations in the ACM with disease-related disability in motor-related WM and showed that ACM provides complementary information to conventional DTI indices such as FA. However further large scale studies are warranted to explore the potential of ACM to map the magnitude and spatial distribution of anatomical disconnection in brain diseases and relate these connectivity changes with disease-related disabilities.


Schizophrenia patients were significantly less accurate than controls on both n-back tasks. No main effects or interactions with past SA on WM performance were observed. However, several fronto-parietal-thalamic regions showed an interaction between diagnostic group and past SA. These regions were significantly more active in controls with past SA compared to controls without past SA. Schizophrenia patients with or without past SA either showed no significant differences, or patients with past SA showed somewhat less activation compared to patients without past SA during WM. These results suggest robust effects of past SA on WM brain functioning in controls, but less impact of past SA in schizophrenia. This is consistent with previous literature indicating less impaired neurocognition in schizophrenia with SA.There has been little investigation of the effects of past substance abuse (SA) on working memory (WM) impairments in schizophrenia. This study examined the behavioral and neurobiological impact of past SA (6 months or longer abstinence period) on WM in schizophrenia. Thirty-seven schizophrenia patients (17 with past SA and 20 without) and 32 controls (12 with past SA and 20 without) completed two versions of a two-back WM task during fMRI scanning on separate days. Analyses focused on regions whose patterns of activation replicated across both  The lifetime prevalence rate of substance abuse (SA) among patients diagnosed with schizophrenia is approximately 50% . DespiteSeveral studies have shown a negative impact of past SA on WM brain functioning in non-psychosis samples. After 28 days of abstinence, adolescent marijuana users displayed aberrant brain activations evoked during WM in the dorsolateral prefrontal cortex (DLPFC), parietal cortex, and occipital regions compared to non-abusers . AlteredDespite the aforementioned neurobiological evidence for residual WM deficits associated with past SA in non-psychosis samples, relatively few research studies exist examining these effects in schizophrenia. The current literature has mixed results that are somewhat difficult to interpret . StartinAn interesting differential effect of past SA on neurocognitive functioning in schizophrenia patients compared to controls has been recently observed. Jockers-Scherübl and colleagues  found thThere are also fairly limited and mixed findings with regard to the effects of past SA on indices of brain structure and function in schizophrenia. To our knowledge, there have been no fMRI investigations of SA-related WM deficits in schizophrenia. Two social–cognitive fMRI studies indicated that schizophrenia patients with past SA had relatively preserved medial prefrontal cortex activity during processing of social emotion  and affeA review of the SA neuroimaging literature by Potvin and colleagues  suggesteAs described above, there is limited and mixed evidence regarding the impact of past SA on WM functioning in schizophrenia. Thus, the aim of this investigation was to do an initial examination of the impact of past SA on brain activation during WM performance in schizophrenia patients with and without past SA compared to healthy controls with and without past SA. To increase confidence in our findings through replication, participants completed one version of a two-back WM task during one scanning session and then completed a second version of a two-back WM task during a second scanning session on a different day. Given the inconsistent results in the prior literature, it is difficult to make specific predictions about the behavioral and functional impairments associated with 6 months or longer abstinence periods in the schizophrenia patients. However, the literature suggesting detrimental effects of past SA on WM performance and brain activation in non-psychosis samples leads us to hypothesize that schizophrenia patients with past SA will display the greatest degree of WM impairments in performance and brain activation in regions implicated in WM impairment, such as the DLPFC  and otheParticipants were recruited through the clinical core of the Conte Center for the Neuroscience of Mental Disorders (CCNMD) at Washington University in St. Louis. Exclusion criteria included: (a) any type of substance abuse/dependence present within the past 6 months; (b) the presence of any clinically unstable or severe medical disorder; (c) current or past head injury with documented neurological sequelae, and/or causing loss of consciousness; (d) meeting DSM-IV criteria for mental retardation; and (e) pregnancy, or any contraindication to MR. Controls were excluded if they had any lifetime history of, or first-order family member with, an Axis I psychotic disorder. Controls were also excluded if they endorsed any personal current or past mood or anxiety disorder other than specific phobias.n-back version A task. Thirty-one of the above schizophrenia participants (15 with past SA and 16 without) and 29 control participants (17 with past SA and 12 without) also completed the n-back version B task on a different scanning day. Fewer subjects completed the n-back version B task because they failed to return to the second scanning session on a different day. All participants provided written informed consent in accordance with Washington University Human Subjects Committee’s criteria. Schizophrenia diagnoses were based on a Structured Clinical Interview for DSM-IV-TR  and 32 controls (12 with past SA and 20 without) completed the SM-IV-TR  performeSM-IV-TR ] and NegSM-IV-TR ]. Based SM-IV-TR , we creaSM-IV-TR  was alson-back using either words or faces (unfamiliar). The two-back version was used because of previous literature indicating this level of WM processing elicits peak activation in schizophrenia patients compared to healthy controls  were acquired parallel to the anterior–posterior commissure plane.Functional scanning was performed on a 3-T Siemens Allegra head-dedicated system at the Research Imaging Center of the Mallinckrodt Institute of Radiology at the Washington University Medical School. First, a low resolution 3D sagittal T1-weighted MP-RAGE acquisition image was obtained . This MP-RAGE was then warped to Talairach space . Second,MR data were reconstructed into images, and normalized across runs by scaling whole-brain signal intensity to a fixed value (mode of 1000), and removing the linear slope on a voxel-by-voxel basis to counteract effects of drift. To correct for head motion, the images were aligned using six parameter rigid-body rotation and translation correction algorithms , 59. Then-back, we used a repeated measures design implementing a General Linear Model (GLM) executed in SPSSv19. These analyses included stimulus type (words vs. faces) as a within subject factor and diagnostic group (schizophrenia vs. controls) and past SA (no vs. yes) as between-subject factors, with mean WM accuracy as the dependent variable.To investigate the effects of past SA on WM accuracy performance in schizophrenia patients and controls on both versions A and B of the n-back tasks. Each GLM included regressors for linear trends within runs and baseline shifts between runs computed for every subject. BOLD responses to each block-type  were modeled as “boxcar” functions, and then convolved with a canonical hemodynamic response function to generate separate estimates for each condition. For clarity and brevity, only the main effects and interactions involving past SA  were considered when examining differences in activations during each WM task independently. Using an in-house software program (FIDL), estimated GLMs from each individual participant were then entered into whole-brain voxel-wise mixed effects analyses of variance (ANOVA) using stimulus type (words vs. faces) as the within subject factor, diagnostic group (control vs. schizophrenia) and past SA (no vs. yes) as between-subject factors, and subjects as a random factor.For the functional imaging data, GLMs were modeled within each participant to estimate voxel-wise magnitudes of task-related activity, with one GLM for each of the two n-back version A or the n-back version B. To do so, we thresholded the whole-brain statistical maps for the n-back version A and the n-back version B at Z > 2.5 and k = 13 based on Monte Carlo simulations, and created conjunction maps of regions showing the same effects across both tasks. Monte Carlo simulations were utilized to protect against inflated type 1 error . In addition, controls without past SA had parents with significantly higher levels of education compared to the parents of controls with past SA . Subsequently, age and highest parental education were entered into secondary behavioral and fMRI analyses as confounding variables. No significant differences emerged between the schizophrenia patients with and without past SA, with the exception of the schizophrenia patients with past SA having slightly higher disorganized symptomology . Past SA was not correlated with neither positive symptoms  nor negative symptoms  in the schizophrenia patients. Specific past SA characteristics for the subgroups of control and schizophrenia participants are presented in Table Overall, the schizophrenia patients and controls did not significantly differ in age, gender, ethnicity, handedness, or highest parental education. However, the mean number of years of education was higher in the control group, and the rate of smoking was higher for the schizophrenia group. With regard to subgroup differences  and n-back version B  was observed, such that schizophrenia patients were significantly less accurate than controls  or n-back version B , nor was there a significant diagnostic group by past SA interaction for either the n-back version A  or n-back version B .A significant main effect of diagnostic group for the n-back. We focused on regions that displayed significant main effects or interactions with group and/or past SA. First, as shown in Table n-back demonstrated a significant main effect of diagnostic group. When compared to the control participants, schizophrenia patients displayed significantly greater WM-related activation in the left superior temporal gyrus, left superior parietal lobule, left cerebellum tonsil and pyramis, and the right supramarginal gyrus. Second, there were a few regions that demonstrated a significant main effect of a past SA , most regions remained significantly different from controls when patients with past SA were added to the sample of patients without past SA. Such results suggest that a differential history of past SA in schizophrenia samples is not responsible for group differences from controls in functional brain activity during WM tasks.A number of regions showing interactions between diagnostic group and past SA in this study are those who have been similarly identified as showing group differences during WM and executive function tasks in prior studies . Thus, wThe goal of this initial investigation was to examine the effects of past SA in schizophrenia patients compared to healthy controls. This was accomplished by assessing both behavioral and neural activity that replicated across two versions of a two-back WM task in schizophrenia patients and controls with and without past SA. Although we had predicted that schizophrenia patients with past SA would demonstrate the highest level of WM impairment, our results indicated that past SA did not exacerbate behavioral or functional WM impairment in schizophrenia. An interesting differential effect of past SA was observed between controls and schizophrenia patients in WM-related brain functioning. Specifically, a number of significant WM-related brain activation differences were observed between controls with and without past SA, while only a few regions showed significantly different task-related activation between schizophrenia patients with and without past SA. These results remained significant after age and highest parental education were considered as cofounding variables. The results are discussed in more detail below.n-back. However, no significant differences in WM accuracy were observed in either controls or schizophrenia patients with and without past SA. These findings are consistent with other previous investigations observing equivalent or better neurocognitive performance in abstinent schizophrenia patients with past SA compared to patients without past SA  due to social skills involved in addiction maintenance (In summary, we found a differential impact of past SA on WM-related brain function in controls vs. schizophrenia patients. Controls with past SA displayed increased WM-related brain activation in several frontal-parietal-thalamic regions relative to controls without past SA. In contrast, schizophrenia patients with and without past SA only displayed differences in decreased activation in the middle frontal gyrus and inferior parietal lobule during only the ntenance , 67 and/ntenance . These pThe 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.


Plastids rely on protein supply by their host cells. In plastids surrounded by two membranes (primary plastids) targeting of these proteins is facilitated by an N-terminal targeting signal, the transit peptide. In secondary plastids (surrounded by three or four membranes), transit peptide-like regions are an essential part of a bipartite topogenic signal sequence (BTS), and generally found adjacent to a N-terminally located signal peptide of the plastid pre-proteins. As in primary plastids, for which no wealth of functional information about transit peptide features exists, the transit peptide-like regions used for import into secondary ones show some common features only, which are also poorly characterized.i) pre-protein import is highly charge dependent. Positive net charge is necessary for transport across the plastid envelope, but not across the periplastid membrane. Acidic net charge perturbs pre-protein import within the ER. Moreover, we show that (ii) the mature domain of the pre-protein can provide intrinsic transit peptide functions.We modified the BTS (in the transit peptide-like region) of the plastid precursor fucoxanthin-chlorophyll a/c binding protein D (FcpD) fused to GFP as model substrate for the characterization of pre-protein import into the secondary plastids of diatoms. Thereby we show that (Our results indicate important characteristics of targeting signals of proteins imported into secondary plastids surrounded by four membranes. In addition, we show a self-targeting mechanism, in which the mature protein domain contributes to the transit peptide function. Thus, this phenomenon lowers the demand for pre-sequences evolved during the course of endosymbiosis. Primary plastids are organelles of endosymbiontic origin e.g. , 2. In in vivo experiments on apicomplexa and diatoms  can  can PhaePhaeodactylum tricornutum with respect to the transit peptide (TP) charge. We chose FcpD (NCBI accession number Z24768) as our model precursor, since its 24 amino acids (aa) long BTS displays all features of a typical diatom plastid pre-protein  between the sequence FAPA and GFP to spatially separate potential targeting information within the GFP from the BTS. Notably, neither the introduction of one nor of two subsequent spacers failed to block pre-protein import, because these precursors were still imported into the plastid (Table FAPA-[M(GGGGP)5]3GFP-; see Table M(GGGGP)5]3 spacer should restore proper GFP targeting into the plastid stroma. Therefore we substituted the third glycine in the spacer sequence with a lysine (FAPA-[M(GGKGP)-(GGGGP)4]3-GFP; see Table P. tricornutum and co-expressed this construct together with the saGFP11 fragment C-terminally fused to the FAPA-[M(GGKGP)-(GGGGP)4]3 construct. By the use of this technology we were therefore able to visualize solely that fraction of precursors which enter the plastid stroma (and not that part of the fusion protein that is stuck in the ER). As presented in Figure To analyze this charge dependence in more detail, and further discern the influence of the GFP reporter on protein import, we truncated the presequence of the FcpD BTS and deleted the entire charged region of the TP yielding the sequence "a Figure . In conta Figure . The infa Figure . In ordein vivo by light-microscopic approaches using fluorescently labelled proteins. However, the exact interpretation of the obtained results is often insufficient because of the resolution limits of microscopes. For instance, evaluation of successful pre-protein import into the four-membrane-bound plastid (apicoplast) of apicomplexan parasites such as Plasmodium falciparum is always an all-or-none decision, as one cannot distinguish between the sub-apicoplast compartments. In contrast, the cell size of the diatom P. tricornutum gives rise to the advantage that not only import into the plastid stroma can be investigated, but also transport across only some of the four surrounding envelope membranes can be tracked [P. tricornutum as a model system for studying pre-protein import into secondary plastids makes such studies more precise.The enslavement and reduction of once free-living cyanobacteria and phototrophic protists to what we today refer to as primary or secondary plastids was accompanied by subsequent cellular and genomic reductions. The plastid genome shrunk enormously by massive gene transfers [and by gene losses; e.g.  tracked ,15,17. TP. tricornutum relies on the presence of basic/positively charged aa residues [Like in land plant plastids, transit peptide-like function in residues ,25. Thisi) An impact of the reporter GFP on pre-protein import as already shown in Figure ii) import across the PPM can depend on discrete positively charged residues rather than on a global basic net charge of the TP, but not in either case  or within the GFP (or a combination of both).As outlined before, the TP of the FcpD BTS is compared to plant TP relatively short, and an influence of the mature part of FcpD on pre-protein import was taken into account. To investigate this in more detail, we trimmed the TP such that the four remaining residues of the transit peptide lack any charge. According to our and previous data [outlined in in vivo localization of a putative plastid protein by genetically fusing a potential BTS to GFP might be misleading.To demonstrate the influence of the GFP lysine in more detail, we introduced a spacer peptide between the truncated TP and wild type GFP  facilitated its own translocation, consequently reducing the degree of topogenic capacity necessary in the signal preceding the mature protein. This self-targeting in turn might have enhanced the successful expression of plastid genes targeted to the nucleus during evolution.(i) many factors of the translocation machineries are conserved in archaeplastida [(ii) some of them, as well as the putative import machinery embedded in the PPM, are present in complex plastids and the apicoplast, respectively [(iii) transit peptides are cross-species functional [e.g. Lastly, our results presumably can be generalized for the other plastid-harbouring organisms as well, since plastida ,29, (ii)ectively -33, and al e.g. , 37.EcoRI and 3' HindIII restriction sites. The presequences were linked via NcoI sites to GFP and YFP, respectively. DNA fragments were amplified using the PHUSION High-Fidelity DNA polymerase  or Biotools DNA polymerase . Amino acid substitutions were carried out by PCR-based mutagenesis using primers with respective sequences  of the self assembly GFP system [AluI restriction site of eGFP and 3' via HindIII. The plasmid used for this system, the induction procedure and the analysis are described elsewhere [ble gene of the plasmid pPhaT1 was substituted for the nat1 gene  conferring resistance to nourseothricin. All constructs were checked for fidelity as well as number and orientation of integrated spacers by sequencing.eGFP and eYFP constructs were generated with terminal 5' P system  was intrlsewhere . For cotPhaeodactylum tricornutum was carried out according to Apt et al. 1996 [Cultivation and biolisitc transformation of al. 1996 . Cotransal. 1996  for the GF carried out the experiments. MSS and UGM conceived the study. All authors analyzed the data and participated in writing, reading and approving the manuscript.Additional table 1: PCR primers used in this studyClick here for file