Groove is the experience of wanting to move when hearing music, such as snapping fingers or tapping feet. This is a central aspect of much music, in particular of music intended for dancing. While previous research has found considerable consistency in ratings of groove across individuals, it remains unclear how groove is induced, that is, what are the physical properties of the acoustic signal that differ between more and less groove-inducing versions. Here, we examined this issue with a performance experiment, in which four musicians performed six simple and six complex melodies in two conditions with the intention of minimizing and maximizing groove. Analyses of rhythmical and temporal properties from the performances demonstrated some general effects. For example, more groove was associated with more notes on faster metrical levels and syncopation, and less groove was associated with deadpan timing and destruction of the regular pulse. We did not observe that deviations from the metrical grid  were a predictor of groove. A listener experiment confirmed that the musicians' manipulations had the intended effects on the experience of groove. A Brunswikian lens model was applied, which estimates the performer-perceiver communication across the two experiments. It showed that the communication achievement for simple melodies was 0.62, and that the matching of performers' and listeners' use of nine rhythmical cues was 0.83. For complex melodies with an already high level of groove, the corresponding values were 0.39 and 0.34, showing that it was much more difficult to “take out” groove from musical structures designed to induce groove. Groove is a central aspect of music appreciation, closely connected to functional uses of music such as dance, drill, and ritual. Given that these behaviors involve synchronization and coordination, which relies on a common temporal reference, it seems likely that the temporal properties of this signal and its representation might be crucial to the understanding of groove  result in an expressive variability of the heard durations against a steady and isochronous metrical grid, and in that sense, it can also be considered as an interaction between the physical and perceptual properties of the rhythm. It consists of slightly anticipated or delayed singular sound events (unsystematic MT), repetitive patterns of such deviations (systematic MT), or local changes in the tempo. Typical MT deviations are smaller than what is comprehensibly conveyed by notation, i.e., shorter than the durations that can be considered to be actual metrical subdivisions (<100 ms). MT has now repeatedly been associated with less groove  and their musical notation is found in Appendix.Four professional male musicians were paid the equivalent of $20 per hour to perform 12 monophonic melodies in various conditions. They were 24–44 years old, could play several instruments, and had a broad experience of different musical styles. The musicians were given recordings of 12 melodies a few days before the recording session. These were deadpan performances without timing or loudness variability, rendered in the sequencer software Cubase 5.0 . Melodies which were thought to be unfamiliar to participants were selected, in order to minimize possible extra-musical associations. Six of them (1–6) have a simple rhythmic structure similar to child songs like “Twinkle, twinkle little star” and were composed for this study, because it was difficult to find such melodies that were unknown to the participants. The other six (7–12) are more complex and were adapted from commercially available recordings of jazz and rock style music. The duration of the songs was 16–25 s . The musician had to play the melody in synchrony with the beat, which allowed analyzes of its rhythmical properties in relation to the beat and its underlying metrical grid. The musicians played a Yamaha DX7s keyboard connected to a PC via a PreSonus Audiobox USB MIDI interface.The production session began with the musician recording all 12 melodies in a deadpan version similar to the ones they had listened to. All musicians reproduced the melodies accurately, with the same pitches and canonical note values. After a short break, the musician was asked to perform each melody in two versions, one with the intention of maximizing groove and the other with the intention to minimize groove.The constraints for the performance were that (1) the melody in the original version had to be recognizable and (2) the same pitches as in the original version had to be used in the same order. Thus, each note could be manipulated in four ways, namely its timing, duration, loudness, and whether its note value was split into more notes with the same pitch or merged with adjacent notes. This was then repeated for all melodies in the orders 1, 7, 2, 8, 3, 9, 4, 10, 5, 11, 6, 12 for two of the musicians, and 12, 6, 11, 5, 10, 4, 9, 3, 8, 2, 7, 1 for the two other musicians. For each melody, the order of the groove conditions was randomized by flipping a coin. The musician was allowed to re-do each condition until he was satisfied with the performance. In total each musician produced 36 performances, 12 deadpan, 12 with groove, and 12 without groove.The independent variables were the type of melody (simple or complex) and groove intention (whether it was performed to minimize or maximize groove). We also include six levels of melody and four levels of performer so as to sample the populations of melodies and performers. The purpose of this was to generalize to these populations, based on the assumption that general tendencies emerge across instances. This constitutes a mixed experimental design with three within-participants (2 melody type × 2 groove intention × 6 melodies) and 1 between-participants (4 musicians) variables, which equals a total of 96 experimental conditions. Although each musician produced 12 deadpan performances, these were not included in the statistical analysis because they were quite accurate and the small performance variability in them could be attributed to unintentional error. Rather, the 12 sequencer-generated versions were used for comparisons of means in the following analyzes, totaling 108 performances.A number of parameters were calculated from the MIDI data, obtained through the List view in Cubase. This representation of the performance shows the pitch, onset- and offset time, and duration for each sound event. Event density was simply the number of events. Pitch was used to identify the tones in the melody. For each sound event, timing is represented as the position in the measure, defined by the time signature, the position within the beat (1–4), and the number of ticks. This information was used to describe the rhythmic properties of the performance as well as deviations from the canonical metrical structure, i.e., from an idealized deadpan performance. The List view denotes how late a sound event is from the prior 16th note position, rather than how early or late it is from the closest position. Events occurring on 8th note positions were therefore correctly assigned to the closest metrical level by the spreadsheet formula =IF=3;1;0), where POS is the position between 1 and 4 within each beat/quarter note. The time resolution in terms of the number of ticks per beat can be specified in the MIDI protocol, and was in this case set to 480. As the beat constituted quarter notes, the duration of a 16th note is 120 ticks.Events occurring on 16th note positions were similarly detected by =IF=2; POS+IF=4)=TRUE;1;0). These formulae simply add one 16th note to the prior position if the event is delayed more than one half 16th note (60 ticks). The same formulae were applied to offset positions and note durations, which means that the detected note values were defined by the actual values of performed events, and were unrelated to the notation, which was rather based on the deadpan versions.The number of ticks that differ from the closest subdivision of 60 ticks constitute the deviation from the closest 16th note, defined as 60 − abs(60 − TICKS), and this unsigned asynchrony from the metrical grid constituted our measure of MT. Because time is defined as a fraction of the beat in the MIDI protocol, one tick corresponds to 60,000/120/480 = 1.04167 ms when the beat tempo is 120 BPM, and 0.83334 ms for 150 BPM. Time deviations are perceptually proportional to the beat however, so the ticks are in fact more relevant than the absolute time, and were used in the subsequent analyzes.In sum, each note event was assigned a value of 0 or 1 depending on whether it occurred on the relevant metric positions (both 8th and 16th positions), and the parameter used in the following analyzes was the proportion of such events in the performance. The MT parameter was the absolute time difference from the closest 16th note position expressed as a proportion of a 32nd note (TICKS/60). These calculations were also done for event offsets and event durations. The means of these nine values were computed across all events in each performance. Thus, each performance could be characterized by 3 × 3 = 9 parameters plus the number of events. These parameters reflect the amount of syncopation by the fact that syncopation yields more events on fast note values in the same melody.In addition, we computed a pure measure of syncopation based on a previously published algorithm  and format it according to the requirements of the external.The algorithm was originally implemented in the programming language C as a Max/MSPN = 48) between groove intention and syncopation were 0.30 (p < 0.05) for simple melodies and 0.27 (n.s.) for complex ones.Table A shows that both the number of notes and the syncopation measure are greater in the maximum groove condition. That syncopation is greatest for the deadpan version is because melody 12 has an extremely high rating . All performances featured the same simultaneous kick and hi-hat beat that the musicians heard, in order to provide an unambiguous metric reference and the same conditions that the musicians had. Listener ratings were obtained by a custom made software that played the music examples through the computer sound card and collected the responses. Each listener rated 48 performances from two musicians plus the 12 deadpan versions.In order to minimize possible response bias, the main purpose of the experiment was not disclosed to the listeners, who were instead told that the study was about rhythm perception generally. This was the main reason why the rating scales also included how much they liked the music and how familiar it was to them. In response to the global question “How well do the following terms describe your experience of the music?” and the terms “bekant” (familiar), “bra” (likeable), and “rörelseskapande” (movement inducing) participants entered ratings ranging from 0 to indicate “not at all” up to 10 to indicate “entirely.” Movement inducing was defined in the instructions as “the sensation of wanting to move some part of your body in relation to some aspect of the music.” The ratings were entered through a horizontal slider on the computer screen that could be moved with arrow keys or the mouse, and whose initial default position was set to 5.Each participant made the ratings individually, seated in front of a computer in a quiet room and listening through headphones. The participant read a paper with instructions, including the definitions of each rating scale given above, and was asked if the task and terminology was clear. After signing the informed consent form, the participant underwent a training session in which three music examples were rated, different from the ones in the experiment proper, set the volume to a comfortable level, and became familiarized with the program and the procedure. During the experiment proper, which comprised 60 (2 musicians × 2 groove intention + 1 deadpan × 12 melodies) music examples, the participant was left alone in the room to avoid distraction. Upon completion of the experiment the participants were asked about their experience including how difficult it was, if they were tired, and if they felt comfortable with their performance. The entire session took 30–40 min. During the session participants controlled the pace by clicking a button to proceed to the next example. The sliders were blocked while the music was playing, to force them to listen to the whole stimulus before rating.The independent variables were the same as in Experiment 1, but the DV was the rating of perceived groove. It would have been too demanding to rate all 108 performances, so they were divided such that one listener groups rated the deadpan performances and all performances of two musicians, and the other did the same but for the other two musicians. This constitutes a mixed experimental design with 3 within participants variables (2 melody type × 2 groove intention × 6 melodies × 2 musicians (= 48 conditions) and 1 between participants variable (2 listener group), which adds up to 48 × 2 = 96 conditions. In addition to this were the 12 deadpan performances included as a comparison. The conditions were given in five blocks, starting with the deadpan versions, and then two blocks for each musician balanced according to ABBA. Within each block were the melodies were presented in the same random order, but with groove intention randomly varied, which was balanced by the opposite intention in the other block for the same musician. For example, if the melodies are numbered 1 through 12 for simplicity, they occurred in the second block in the order −9, +11, −1, +7, +6, −12, +10, −5, −8, +4, +3, −2, where—denotes minimum groove and + maximum groove. Consequently, they appear in the fifth block (the 2nd A in ABBA) with reversed groove intention, that is, +9, −11, +1, −7, −6, +12, −10, +5, +8, −4, −3, +2. The order in each B block was the same. The purpose of this presentation order was to dilute order effects across both melodies and musicians while at the same time balancing out possible large-scale effects such as fatigue.F = 47.06, p < 0.000001], melody type , and melody , but not of musician or listener group . There were significant 2-way interactions for groove intention × melody type , melody × melody type , and musician × listener group . Since neither melody nor musician number relate to any meaningful dimension, all effects including any of these variables are ignored in the following analysis. They include four significant and six non-significant three-way interactions and four non-significant four-way interactions. Melody and musician number were included in the ANOVA model because possible effects would then be accounted for and hence reduce the amount of unexplained variance. Figure The interviews indicated that all participants understood the instructions, felt comfortable with the task, and did not find it difficult or tiring. The whole design was subjected to a mixed model 2 groove intention × 2 melody type × 6 melodies × 2 musician × 2 listener group five-way ANOVA. There were main effects of groove intention [The so-called lens model is suitable for analyzing communication processes such as the present one, in which an intended message may be conveyed through multiple cues that may be combined to interpret the message by the receiver  and receivers (Rr), respectively, and an un-modeled component that consists of the correlation between the residuals in the regression models C and the residual variation of the models  expresses the first-order correlation between the sender and receiver as a function of a matching factor G, the multiple correlations of the regression models for senders (Rar) then equals the simple  correlation between groove intention and groove ratings. The lens model factors are summarized in Table G is calculated as the correlation between the predicted values for the sender and receiver regression models, and C is likewise the correlation between the residual values for the sender and receiver regression models. With proper correction for the unexplained variance in the second term of Equation 1, the achievement  a common reliance on cues not included in the regression models, (b) chance agreement between random model errors, (c) cue interactions common to both models, or (d) nonlinear cue function forms common to both models Cooksey, . That C The differences between simple and complex melodies seen in Figure s and rr values in Table The main purpose of the present study was to investigate what musicians do to increase and decrease groove and validate to what extent these strategies are effective, according to listeners' ratings. The results demonstrates that groove is conveyed by simple means, including syncopation. For the simple melodies, the musicians' intention explained 56 percent of the variance in the performance parameters, which in turn explained 47 percent of the variance in the listeners' ratings .Groove is a treasured property of much music, such as jazz, reggae, samba, salsa, and many more styles. Within these idioms, music groups and even individual musicians are known to produce more or less groove . A closer examination of the relation between syncopation and groove may be informative of cognitive mechanisms related to the perception of complex rhythmic structures.Conceived and designed the experiment: Guy Madison. Performed the experiment: Guy Madison. Analyzed the data: Guy Madison, George Sioros. Contributed analysis tools: George Sioros. Wrote the paper: Guy Madison, George Sioros.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.


The cerebrospinal fluid (CSF) biomarkers amyloid-β (Aβ), tau and phosphorylated tau (p-tau181) are now used for the diagnosis of Alzheimer’s disease (AD). Aβ40 is the most abundant Aβ peptide isoform in the CSF, and the Aβ 42/40 ratio has been proposed to better reflect brain amyloid production. However, its additional value in the clinical setting remains uncertain.A total of 367 subjects with cognitive disorders who underwent a lumbar puncture were prospectively included at three French memory centers . The frequency of positive, negative and indeterminate CSF profiles were assessed by various methods, and their adequacies with the diagnosis of clinicians were tested using net reclassification improvement (NRI) analyses.P = 0.03), but it failed to improve the classification of subjects . In contrast, the use of Aβ 42/40 ratio instead of Aβ42 levels alone in patients with a discrepancy between p-tau181 and Aβ42 led to a reduction by half of the number of indeterminate profiles  and was further in agreement with clinician diagnosis .On the basis of local optimum cutoffs for Aβ42 and p-tau181, 22% of the explored patients had indeterminate CSF profiles. The systematic use of Aβ 42/40 ratio instead of Aβ42 levels alone decreased the number of indeterminate profiles  contains supplementary material, which is available to authorized users. Dementia is a growing public health concern in aging populations. The number of people affected worldwide is set to reach 66 million by 2030 . AlzheimThe use of biomarkers has strengthened the link between clinical dementia and AD pathophysiology . CurrentAβ40 is the most abundant Aβ peptide isoform in the CSF. Compared with CSF Aβ42 levels alone, Aβ 42/40 ratio is suggested to account for the constitutive interindividual differences in total CSF Aβ peptide load between high- and low-amyloid-producing individuals . The Aβ According to national recommendations, CSF analyses are routinely used during testing of patients with cognitive disorders in French memory centers ,17. PatiAll patients had a thorough examination, including clinical and neuropsychological evaluations, biological measurements and brain imaging. Patients with AD were included according to the criteria for probable AD as defined by the National Institute on Aging-Alzheimer’s Association . On the This research study was approved by the local ethics committees of each hospital  according to the manufacturer’s instructions. Concentrations of total tau greater than 1,200 pg/ml were well above the detection limit and thus were not recalculated after dilution because of the constraints in the procedure. The biological teams involved in CSF analyses were blinded to the clinical diagnoses.Lumbar punctures were performed on fasting patients within 1 month following their clinical diagnosis, usually between 9:00 am and 12:00 reported , all thrreported . Each CSCSF cutoffs used in the analyses were determined on the basis of the population included in the present study. We computed both overall and local cutoff receiver operating characteristic (ROC) curves using STATA software . ROC curves were built for each biomarker by plotting sensitivity and 1 − specificity to discriminate AD from non-AD patients. Optimum cutoff values were determined using two methods: the highest Youden index  and lowest distance between ROC plot and point (0.1). We checked that both approaches led to similar findings, and the presented results correspond to the highest Youden index. STATA code used for the determination of optimum cutoff is detailed in the Additional file CSF results were defined as negative  or positive . An indeterminate CSF profile was defined by the association of one positive biomarker and one negative biomarker.We chose to evaluate tau pathology based on CSF p-tau181 levels alone because p-tau181 and total tau levels were highly correlated in our sample (Pearson’s correlation coefficient = 0.87). Furthermore, p-tau181 has previously been reported to be more discriminant than total tau . Analyse2 test for categorical variables.The subjects’ characteristics are presented overall, by diagnosis (AD vs. non-AD) and by CSF collection centers. The various groups  were compared using analysis of variance for continuous variables and χWe computed ROC curves to evaluate the capacity of each CSF biomarker to discriminate AD from non-AD subjects. Analyses were performed in the overall study population and then stratified by centers. Optimum cutoffs for each biomarker were defined using the highest Youden index to discriminate in the best possible manner AD from non-AD subjects, thus maximizing sensitivity and specificity of the analyses based on ROC curve analyses. Then we used local optimum cutoffs for further analysis because intersite differences were present.Method 1: interpretation based on CSF p-tau181 and on CSF Aβ42Method 2: interpretation based on CSF p-tau181 and on Aβ 42/40 ratioMethod 3: a decisional algorithm based, in a first step, on CSF p-tau181 and on CSF Aβ42. Then, in case of discrepancy between p-tau181 and Aβ42, we used, in a second step, the Aβ 42/40 ratio in place of Aβ1–42To evaluate the interest of ratio Aβ 42/40 versus CSF Aβ42 alone, we compared three methods to interpret CSF results:For all three methods, we first determined the percentage of AD, non-AD and indeterminate biological CSF profiles. Proportions of indeterminate profiles according to the methods were compared using the McNemar test. Then we used a net reclassification improvement (NRI) method to compare method 2 to method 1, and method 3 to method 1, among AD patients and non-AD patients . Briefly, NRI is a statistical tool used to assess improvement in model performance offered by a new method of classification compared with a reference method ,24. The P (up | event) ‐ P (down | event) + P (down | non ‐ event) ‐ P (up | non ‐ event) [NRI = ‐ event) P-value [The null hypothesis for NRI = 0 was tested using Z-statistics following McNemar asymptotic test for correlated proportions . We provP-value .P-values were two-tailed, and P ≤ 0.05 was considered statistically significant. Statistical analyses were performed using STATA version 10 and SAS version 9.2 .All resulting P = 0.06), but this relationship was attenuated after adjustment for age and sex (P = 0.19).In this study, we enrolled a total of 367 participants who received a lumbar puncture between 1 September 2012 and 1 September 2013. The study was conducted at three study centers . The subjects’ characteristics are summarized in Table P = 0.39).The percentages of patients with AD across centers ranged from 31% to 59% Table . There wThe results of ROC curve analysis comparing AD with non-AD patients and optimum cutoffs are presented in Table 1–42 and CSF p-tau181 levels (non-AD profile or AD-type profile), and 83 participants (23%) had indeterminate CSF results.Figure P = 0.03 compared with method 1). (3) Use of CSF p-tau181 and CSF Aβ42, and then Aβ 42/40 ratio in case of discrepancy (method 3), attenuated the number of indeterminate results to 38 patients .Figure P = 0.64). By contrast, the use of Aβ 42/40 ratio only in patients with discordant results between p-tau181 and Aβ42 (method 3) improved the classification of patients, with a NRI at 10.5%  compared with method 1.Figure The Aβ 42/40 ratio was the only biomarker that was consistent between centers in AD and non-AD patients. We may therefore hypothesize that this ratio is less sensitive to preanalytical and analytical conditions.P = 0.64).The systematic use of Aβ 42/40 ratio instead of Aβ42 decreased the number of indeterminate CSF profiles but failed to improve the classification of patients based on clinical diagnosis .A strategy based on the use of Aβ 42/40 ratio instead of Aβ42 in patients with initially discrepant results between p-tau181 and Aβ42 led to a reduction of the number of indeterminate CSF profiles (10% vs. 22%) and improved the classification of patients (NRI = 10.5%, In this clinically based, multicenter study, we evaluated the systematic assessment of CSF Aβ40 as part of a battery of CSF biomarkers measured in patients with cognitive disorders. We made the following observations:Consequently, the assessment of Aβ 42/40 ratio could be helpful and can be recommended as a second step when the initial CSF profile remains indeterminate.Researchers in several previous studies have investigated the use of Aβ 42/40 ratio to differentiate patients with AD from patients with other cognitive disorders, and the reported results are heterogeneous. In a previous study, researchers found that Aβ 42/40 ratio outperformed Aβ42 alone in differentiating AD from non-AD patients, but the diagnostic performance of the combination of CSF tau and CSF Aβ42 was not improved by the use of Aβ 42/40 ratio instead of Aβ42 alone . In anotOur findings confirm and strengthen the results of a recent study in which researchers reported that the use of the Aβ 42/40 ratio may contribute to decreases in the proportion of indeterminate CSF profiles in the clinical setting .The proportion of patients with AD varied greatly at the three centers, reflecting the differences in recruitment and practices of memory clinics. Despite significant intercenter differences in reporting CSF Aβ40 and CSF Aβ42 levels, the mean Aβ 42/40 ratios were comparable across the three centers, ranging from 0.044 to 0.049 in patients with AD. We therefore hypothesize that the use of the CSF amyloid ratio could contribute to decreased preanalytical and analytical sources of variability among centers. Interestingly, we found that optimal cutoff for the ratio in the overall study population was equal to 0.055, which was comparable to the 0.057 cutoff recently reported by another team in a monocentric study .Very few data are available concerning indeterminate profiles of CSF biomarkers. In our study population, on the basis of routine clinical practice, this situation was not rare, being observed in 22% of the patients even after optimization procedures using local optimum cutoffs. We have shown that Aβ 42/40 ratio instead of Aβ42 leads to a clear biological conclusion in more than 50% of these indeterminate cases. Reclassification analyses also showed that this approach is more congruent with the diagnosis by clinicians.This study has several strengths, including its large size, its multicentric and prospective design, and the use of a common CSF polypropylene tubing at each of the three centers to standardize CSF evaluation. In addition, we used a NRI method that compares different strategies of biomarker analyses and is more precise than traditional analyses based on ROC curves. The main limitation of the results is the lack of neuropathological validations. Also, clinicians were not blinded to CSF results prior to clinical diagnosis, which may generate a circular reasoning bias in our findings. The absence of a centralized measurement for CSF biomarkers is another limitation, owing to the persistence of an intersite variability despite the use of a common tubing to collect CSF. Furthermore, the non-AD group was heterogeneous and included patients with cognitive disorders, of both psychiatric and neurologic origins. However, our study was aimed at reflecting the standard practice at memory clinics. The wide variety of patients with a number of diseases reflects the broad spectrum of cognitive complaints referred to memory centers. Finally, this study excluded patients with MCI. Inclusion of these patients in future studies will help to determine the links between the amyloid ratio and the rate of conversion from MCI to AD.Using a large prospective multicenter cohort of patients with cognitive disorders, we did not find an added value for the systematic assessment of the CSF Aβ 42/40 ratio. However, in cases of discrepancy between CSF p-tau and CSF Aβ42, the use of Aβ 42/40 ratio allowed reaching a biological conclusion in more than 50% of indeterminate results and improved the biological congruence with clinical diagnoses.


MCPH1  and ASPM  genes cause primary microcephaly. Both are centrosomal associated proteins involved in mitosis. Microcephalin plays an important role in DNA damage response and ASPM is required for correct division of proliferative neuro-epithelial cells of the developing brain. Reduced MCPH1 mRNA expression and ASPM mRNA over-expression have been implicated in the development of human carcinomas. Epithelial ovarian cancer (EOC) is characterised by highly aneuploid tumours. Previously we have reported low Microcephalin and high ASPM protein levels and associations with clinico-pathological parameters in malignant cells from ascitic fluids. To confirm these previous findings on a larger scale Microcephalin and ASPM expression levels and localisations were evaluated by immunohistochemistry in two cohorts; a training set of 25 samples and a validation set of 322 EOC tissue samples. Results were correlated to the associated histopathological data. In normal ovarian tissues the Microcephalin nuclear staining pattern was consistently strong. In the cancer tissues, we identified low nuclear Microcephalin expression in high grade and advanced stage tumours (p<0.0001 and p = 0.0438 respectively). ASPM had moderate to high nuclear and low to moderate cytoplasmic expression in normal tissue. Cytoplasmic ASPM expression decreased with tumour grade and stage in the serous subtype of EOC (p = 0.023 and p = 0.011 respectively). Cytoplasmic ASPM increased with tumour stage in the endometrioid subtype (p = 0.023). Increasing tumour invasiveness (T3) and lymph node involvement (N1) also correlated with a decrease in cytoplasmic ASPM in EOC (p = 0.02 and p = 0.04 respectively). We have validated previous findings of deregulated expression of Microcephalin and ASPM in EOC by confirming associations for low nuclear Microcephalin levels and high cytoplasmic ASPM levels in a larger scale tumour tissue study. Microcephalin and ASPM may prove useful biomarkers in EOC.Mutations in the  Approximately 225,000 new cases of ovarian cancer were diagnosed in 2008 worldwide, comprising 4% of all female cancers MCPH1 and MCPH5 genes encode Microcephalin and the abnormal spindle-like microcephaly-associated protein (ASPM) respectively MCPH1 and MCPH5 are two of ten microcephaly genes identified, which are implicated in autosomal recessive primary microcephaly (MCPH) N-terminally located and two in the C-terminus BRCT-repeat inhibitor of hTERT expression), which was initially identified as a transcriptional repressor of human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase N-terminal microtubule binding domain C-terminus consisting of a single Armadillo like sequence and a region involved in cytokinesis The et al. reported that MCPH1 copy number was decreased in 40% (35/87) of advanced EOC and in 72% (39/54) of breast cancer cases MCPH1 mRNA was decreased in 63% (19/30) of EOCs MCPH1 by deletion, promoter methylation and mutation was identified in an oral squamous cell cancer study Several lines of evidence indicate that Microcephalin and ASPM play a role in carcinogenesis. At the DNA level, Rai ASPM mRNA levels were increased in tumour and transformed human cells ASPM mRNA and protein levels were also identified in glioblastoma multiforme (GBM), where they were associated with increasing tumour grade ASPM mRNA upregulation was identified in 66% (162/247) of hepatocellular carcinomas, an observation associated with increased invasion, high stage and early tumour recurrence In contrast, Our recent EOC study determined a correlation between Microcephalin and ASPM levels with tumour grade and survival in cell lines and in primary cultures of malignant cells derived from ovarian ascites samples Appropriate ethical approval was obtained from the Local Research Ethics Committee of the Leeds Teaching Hospitals NHS Trust, Leeds, UK, (REC reference 09/H1306/96). All participants provided written informed consent and all data were analysed anonymously.A cohort of 25 tumour archival, formalin-fixed, paraffin-embedded (FFPE) blocks were obtained from the histopathology department of St James's University Hospital, Leeds and used to create the training set. The sample set in this cohort represented four major (most frequently encountered) EOC subtypes  and were predominately grade 3 tumours. All the blocks in this cohort were then combined into one in-house tissue microarray (TMA). A normal cohort consisted of 16 ovarian tissue samples taken from either normal ovary or from normal tissue adjacent to tumour plus 4 normal fallopian tube and 4 normal endometrium samples were also available.Another independent cohort of 322 EOC samples was obtained from Tissue Array Networks and was designated the validation set. The validation set consisted of 294 different samples of five major EOC subtypes , plus 8 normal ovarian tissues taken from normal ovary and 20 from normal tissue adjacent to tumour. The 322 cases were represented in 616 cores, with double cores for cancer cases and a single core for normal tissue. Tissue cores were 0.6 mm in diameter with a thickness of 5 µm. Clinical data  staging and TNM staging et alAn in-house TMA was constructed from 25 EOC samples at the Leeds Institute of Cancer and Pathology facility following a protocol described by Ellis 2O2 in methanol. Antigen retrieval was carried out using Vector antigen retrieval buffer (Vector Laboratories Ltd) in a pressure cooker for 4 minutes for Microcephalin and 2 minutes for ASPM. Sections were blocked with 1∶10 vector casein solution (Vector Laboratories) to reduce non-specific staining. On the TMA sections the rabbit anti-Microcephalin antibody ab2612 (Abcam) was used at 1/300 and the rabbit anti-N-terminal ASPM antibody 216.1 To optimise the Microcephalin and ASPM staining protocol, the in house EOC TMA sections were stained using a range of antibody dilutions and antigen retrieval methods. Microcephalin and ASPM were stained separately under the same conditions except for the antibody retrieval and dilution. Slides were deparaffinised and rehydrated using xylene and ethanol series. To block hydrogen peroxidase activity, slides were soaked in 3% HIn order to determine the staining specificity of the Microcephalin antibody on FFPE samples, an immunizing peptide blocking experiment was performed on EOC tissue sections and on the 25 core in-house TMA. The antibody was neutralized by pre-absorption with the BRIT1 peptide ab13737 (Abcam). The antibody was diluted to its predetermined working concentration (described above) and incubated with a tenfold excess of peptide overnight at 4°C before application to the tissue. Tissue sections with neutralized antibody were compared with corresponding tissue sections stained with antibody alone.C-terminus of ASPM. Similar staining results were obtained with both antibodies with only slight differences between cytoplasmic staining levels.We had already determined the antibody specificity of the ASPM antibody 216.1 in previous work by peptide blocking immunofluorescence experiments MCPH1 and ASPM were knocked down in the U-2 OS cell line  by siRNA as previously described For further validation of the specificity and sensitivity of the antibodies and to control for the effects of formalin fixation and antigen retrieval, All the TMA sections were scanned using the Aperio Scan Scope slide scanner (Aperio Technologies) at 40x magnification, using morphometry tools in Imagescope software (Aperio). For both Microcephalin and ASPM the replicate cores for each sample were scored for staining intensities of nuclear and cytoplasmic staining  as well as the proportion of cells with positive nuclear or cytoplasmic staining within the cores (proportion score). To analyse the Microcephalin staining the semi-quantitative Allred (quick) 8-unit system was used P≤0.05 values was considered statistically significant.The non-parametric Kruskall-Wallis (K-W) and Wilcoxon-Mann-Whitney (W-M-W) test were used to identify the association between Microcephalin and ASPM with clinical variables . All statistical tests were two-sided, and a The Microcephalin and ASPM antibodies were initially optimized for use on paraffin-embedded TMA sections. Immunohistochemistry with these antibodies demonstrated that Microcephalin and ASPM were localized in the nucleus and in the cytoplasm of both the normal  and EOC The staining pattern for ASPM antibody 279.3 performed in parallel to the staining with antibody 216.1 revealed that cytoplasmic staining was similar for both antibodies. However, 216.1 showed a more pronounced nuclear staining pattern than 279.3 and consequently this antibody was used to stain the EOC validation set. Results for staining patterns of representative cores and a summary of the staining results are shown in To further confirm the validity of the antibodies to stain paraffin-embedded tissues, the antibodies were tested on paraffin-embedded U-2 OS cells with and without siRNA knockdown for Microcephalin or ASPM. Knockdown cells for both genes showed very weak staining relative to the control siRNA .p<0.0001) (p = 0.0438) (p = 0.0581). No significant changes in Microcephalin expression were identified between different pathological subtypes (p = 0.486)  of normal ovarian epithelial cells demonstrated positive Microcephalin staining while this was slightly lower (70–80%) in the endometrium and fallopian tube samples . Initial<0.0001) . Microce 0.0438) . Patient 0.0438) , but the= 0.486) . However= 0.486) . Figure The normal ovarian epithelium, endometrium and fallopian tube showed variable ASPM expression, with moderate to high nuclear expression and low to moderate cytoplasmic ASPM staining . The ASPIn the validation set nuclear and cytoplasmic staining was also observed. The staining patterns differed between tumour samples and examples of low and high ASPM expression are shown in p = 0.0438) (p = 0.0032)  . Similar 0.0032) . Microce 0.0032) .For ASPM, an association between cytoplasmic staining levels and tumour invasiveness was identified. High cytoplasmic ASPM levels were found at T1, which decreased significantly with T2 and T3 (p = 0.0198) . High cyEOC is frequently first diagnosed at an advanced stage due to the lack of symptoms and reliable early detection methods. Consequently the identification of diagnostic and prognostic biomarkers for EOC is of major clinical importance. In this study we investigated the expression of two MCPH proteins, Microcephalin and ASPM, in EOC tumour tissue samples. EOC displays a high degree of aneuploidy. DNA damage response proteins are a vital defence against genomic instability and defects in these proteins may lead to cancer. Microcephalin has a known role in DNA repair and ASPM for spindle function during mitosis. The purpose of this study was to determine whether Microcephalin and ASPM expression are associated with clinicopathological parameters in patients with EOC. Both proteins had been shown to be deregulated in other cancers in a manner that was associated with tumour progression p<0.0001 and p = 0.0438 respectively). These findings match our previous study on ovarian ascites samples that indicated that Microcephalin expression was reduced in cell cultures derived from ascites of EOC patients with advanced tumours MCPH1 DNA copy number in 72% (39/54) of breast cancers Recently we reported an association of Microcephalin and ASPM levels in malignant cells derived from ascitic fluids from EOC patients with various clinic-pathological parameters MCPH1 deletion mutations in the C-terminal BRCT domains that have previously been shown to result in Microcephalin moving from a nuclear to cytoplasmic localization similar to BRCA1MCPH1 splice variants reported mutation or deletion of nuclear localization signals within the MCPH1 gene resulted in a localization change from nuclear to cytoplasmic p = 0.0051). However due to non-specific background cytoplasmic staining observed in the blocking peptide experiment, cytoplasmic expression was not included here.Previously, we identified a correlation between the abnormal localization of Microcephalin with tumour grade in primary cultures of malignant cells derived from ascitic fluids from patients with EOC. In these cells, cytoplasmic Microcephalin increased with tumour grade. We suggested that might be because of Our findings of reduced expression of the DNA repair protein Microcephalin in high grade tumours but few low grade tumours is consistent with the molecular characteristics of these different tumour types. Loss of DNA repair function e.g. BRCA genes leading to chromosomal instability and a complex genome is common in high grade cancers. In contrast to low grade tumours which do not tend to display chromosomal instability and BRCA mutations.Similarly to Microcephalin expression, in this study we recapitulated our recent findings on cytoplasmic ASPM levels and tumour grade. In primary cultures of malignant cells derived from ovarian ascites samples we had determined that cytoplasmic ASPM levels decreased with tumour grade. In this study we were able to verify this result in the serous subtype in the large-scale validation set of EOC tumour samples. In addition we were able to determine associations of cytoplasmic ASPM levels with disease stage, where high cytoplasmic ASPM levels were predominantly found in low stages (1 and 2) in the serous subtype and an increase of cytoplasmic ASPM levels with stage in the endometrioid subtype. It has been suggested that the different subtypes of EOC follow different molecular pathways and this may be represented in our results. Cytoplasmic ASPM levels were also found to be associated with tumour invasiveness with highest cytoplasmic ASPM levels found only when the tumour remained confined to the ovaries (T1). This may indicate that high levels of cytoplasmic ASPM are important for tumourigenesis but not for tumour progression.Although cytoplasmic and nuclear locations of ASPM in other tumour types have been noted in the literature Interestingly, our findings of different cytoplasmic ASPM expression levels and associations with tumour stage among two subtypes reflect recent findings in the literature. For example, Köbel et al, 2008 In summary, in this study we investigated the potential use of Microcephalin and ASPM expression in clinical practice in EOC in a large scale study. We conclude that deregulation of Microcephalin and ASPM expression is significantly associated with tumourigenesis. The results from this study warrant the further investigation of Microcephalin and ASPM as potential biomarkers in EOC.Figure S1Characterization of the anti-Microcephalin antibody. Immunohistochemical staining of Microcephalin in the absence (A and C) and presence (B and D) of the peptide the antibody was raised against illustrating, low non-specific cytoplasmic background staining. A and B are 40x magnification, C and D are 10x magnification.(TIF)Click here for additional data file.Figure S2Comparison of two different ASPM antibodies. The staining patterns of the N-terminal 216.1 and C-terminal 279.3 ASPM antibodies 216.1 (A and C) and 279.3 (B and D) revealed identical cytoplasmic and nuclear localization and relative protein expression levels. A and B TMA core obtained for the same patient sample showing low level ASPM expression. C and D TMA core obtained for the same patient showing high ASPM expression. All images are 6.2x magnification.(TIF)Click here for additional data file.Figure S3Conformation of antibody specificity by siRNA knockdown in U-2 0S cells. Immunohistochemistry analysis of paraffin embedded U-2 0S cells after siRNA knockdown stained with anti Microcephalin (A and B) or ASPM (C and D) antibodies respectively. Cells transfected with scrambled control siRNA showed high Microcephalin (A) and ASPM (C) expression respectively. Cells transfected with MCPH1 (B) or ASPM (D) siRNA showed low expression. All images are x40 magnification.(TIF)Click here for additional data file.Table S1Analysis of the staining intensities of two different ASPM antibodies 216.1 and 279.3 performed on the training set TMA.(DOCX)Click here for additional data file.


The colony stimulating factor granulocyte (G-CSF) is a glycoprotein capable of promoting the survival, proliferation and differentiation of hematopoietic cells. Studies demonstrate the cytoprotective action of G-CSF against renal injury by ischemia/reperfusion injury in murine models. But the literature is still controversial in relation to the risk of worsening renal function after useG-CSF, which motivated the study to elucidate possible interference on the renal function of the treated animals.Evaluate the renal function of rats after treatment with G-CSF at different doses of the drug.Male Wistar rats (n = 18), 200g approx. (CEUA050/2013), divided into 3 groups  : control group (C) 5% glucose solution (solvent) groups treated with G-CSF at a dose of 10 (G10) and 50 (G50) mg / kg / per 5 days. After treatment, the rats were placed in metabolic cages for urine collection and obtaining urine volume. Values were obtained from creatinine, proteinuria, urea and number of circulating leukocytes. The results were expressed as mean ± SEM. The averages ofvalues between groups were calculated using one - way ANOVA followed by post hoc Fisher for comparison between differentgroups.A significant increase in the number of circulating leukocytes in animals treated with G- CSF . There was not a significant increase in urine volume after 24 hours treatment with G-CSF. There was no significant difference between the values of creatinine clearance, proteinuria and Urea, among groups C, G10 and G50.There was no impairment of renal function in animals treated at doses of 10 and 50 mg / kg / per 5 days.Financial support: FAPES


Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent a continuum of devastating neurodegenerative diseases, characterized by transactive response DNA-binding protein of 43 kDa (TDP-43) aggregates accumulation throughout the nervous system. Despite rapidly emerging evidence suggesting the hypothesis of ‘prion-like propagation’ of TDP-43 positive inclusion in the regional spread of ALS symptoms, whether and how TDP-43 aggregates spread between cells is not clear. Herein, we established a cerebrospinal fluid (CSF)-cultured cell model to dissect mechanisms governing TDP-43 aggregates formation and propagation. Remarkably, intracellular TDP-43 mislocalization and aggregates were induced in the human glioma U251 cells following exposure to ALS-FTD-CSF but not ALS-CSF and normal control (NC) -CSF for 21 days. The exosomes derived from ALS-FTD-CSF were enriched in TDP-43 C-terminal fragments (CTFs). Incubation of ALS-FTD-CSF induced the increase of mislocated TDP-43 positive exosomes in U251 cells. We further demonstrated that exposure to ALS-FTD-CSF induced the generations of tunneling nanotubes (TNTs)-like structure and exosomes at different stages, which mediated the propagation of TDP-43 aggregates in the cultured U251 cells. Moreover, immunoblotting analyses revealed that abnormal activations of apoptosis and autophagy were induced in U251 cells, following incubation of ALS-CSF and ALS-FTD-CSF. Taken together, our data provide direct evidence that ALS-FTD-CSF has prion-like transmissible properties. TNTs-like structure and exosomes supply the routes for the transfer of TDP-43 aggregates, and selective inhibition of their over-generations may interrupt the progression of TDP-43 proteinopathy. Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, in which loss of motor neurons leads to progressive weakness of the voluntary muscles. Frontotemporal dementia (FTD) is a form of dementia clinically characterized by behavioral dysfunction and changes in personal and social conduct. Pathological studies of ALS and FTD reveal significant neuropathological overlap, suggesting that they represent different manifestations among a spectrum of phenotypes of the similar underlying pathogenesis -4. The fA breakthrough in understanding sporadic neurodegenerative disease progression was the discovery of ‘prion-like propagation’ of protein aggregates a couple of years ago , in whicThe present study aimed to substantiate the hypothesis of prion-like propagation of TDP-43 aggregates, and further to dissect the potential mechanism underlying the intercellular transfer. We find that ALS-FTD-CSF incubation with U251 cells generates TDP-43 mislocalization, and the cell-to-cell transmission of TDP-43 aggregates is mediated via exosomes and TNTs-like structure. Furthermore, incubation of ALS-CSF and ALS-FTD-CSF with U251 causes toxic to the cells.In the past 10 years, an increasing list of neurodegenerative diseases have been shown to manifest self-perpetuating seeded aggregation and spreading phenomena, the ‘prion-like’ pathological process. But there is no evidence to date that misfolded protein aggregates can spread between individual organisms to cause an acquired disease. To test whether TDP-43 aggregates can propagate via body fluid of patients, we established a CSF cell culture model using U251 cells. U251 cells were incubated for 0, 1, 3, 5, 7, 10, 14, 18, 21, and 28 day in DMEM containing 30% v/v of CSF. After exposure to CSF for 21 days, U251 cells showed a reduced rate of growth and diverse morphological changes in ALS-CSF and ALS-FTD-CSF treated groups compared with the controls Figure . We nextTo further test whether treatment of ALS-CSF and ALS-FTD-CSF can induce the seeding of TDP-43 proteinopathy in cultured U251 cells, we performed immunocytochemistry staining for TDP-43 positive aggregates. When cells were incubated for 21 days, surprisingly, the formation of the intracellular TDP-43 aggregates and the mislocalization of TDP-43 from the nucleus to the cytoplasm were observed in the U251 cells of ALS-FTD-CSF group, but not in the ALS-CSF and NC-CSF groups, as shown in Figure Release of exosomes, small vesicles of endocytic origin, from a variety of different neuronal cell lines has been described , and theFrom others , 21, theTo test the hypothesis, we first investigated whether flotillin-1, a lipid raft marker, is increased in U251 cells following incubation with ALS-FTD-CSF. Immunoblotting analysis showed the expression level of flotillin-1 significantly higher in the cells following ALS-FTD-CSF incubation than NC-CSF for two weeks Figure , 3C. NexIntracellular aggregates are considered toxic to cells as they inducing apoptosis, autophagy, and other disturbances in internal environment . In ordeOur above-mentioned results showed that TDP-43 CTFs expression levels were increased in the exosomes derived from ALS-FTD-CSF, and earlier studies from others -27 demanTDP-43 proteinopathies represent a novel class of neurodegenerative disorders akin to α-synucleinopathies and tauopathies. A broad clinic-pathological spectrum of a single disorder shares similar disease mechanisms linked to TDP-43 proteinopathies, such as ALS and FTD , 29. NotAccording to the clinical symptoms, the spread mechanism of ALS and FTD lesion is explained by propagation, and classified as contiguous type and non-contiguous type . It is sin vitro, characterized by decreased growth of cell, cytoskeletal abnormal and transmission of TDP-43 aggregates from ALS-FTD-CSF to the cultured cells. Previous research revealed that activated astrocytes become capable of damaging healthy neighboring motor neurons, and astrocytes start producing a host of toxic molecules . After clearing the lysate by centrifugation, samples were heated in loading buffer, and equal amounts of protein were loaded and separated by SDS-PAGE. After transfer to nitrocellulose membranes, blots were blocked with 10% nonfat dry milk in TBST  for 30 min, and then incubated with primary antibodies overnight at 4°C. After washing 3 times in TBST, the membrane was incubated with anti-rabbit IgG  or anti-mouse IgG  for 1 h. Membranes were washed three times and proteins were visualized after ECL  treatment. The primary antibodies used were rabbit polyclonal anti-TDP-43 antibody , rabbit polyclonal anti-flotillin-1 antibody , mouse monoclonal anti-GAPDH antibody , rabbit polyclonal anti-caspase-3 antibody , mouse monoclonal anti-p53 antibody , rabbit polyclonal anti-Bcl-2 antibody , rabbit polyclonal anti-LC3 antibody , mouse monoclonal anti-Beclin-1 antibody  and rabbit polyclonal anti-GFP antibody .Cells were collected from the plates and centrifugation at 500 × p-value lower than 0.05.All statistical analyses were performed using SPSS statistical software package . Data were shown as mean±SD. Multiple comparisons were tested with ANOVA and Bonferroni procedure. All tests were considered significant at


Pythium insidiosum iron acquisition mechanisms are unknown. We previously showed that the iron chelator deferasirox had weak activity in vitro and in rabbits with experimental pythiosis. Here we show that deferasirox causes damage to P. insidiosum hyphae in vitro, but that activity is diminished in the presence of exogenous iron. The tissue activity of the proinflammatory enzyme adenosine deaminase and the histological pattern observed in pythiosis lesions of rabbits treated with deferasirox were similar to the ones in animals treated with immunotherapy. Pythium insidiosum often cause cutaneous, gastrointestinal, ocular or systemic disease in humans and animals. Despite significant advances in diagnosis and treatment, pythiosis remains a difficult-to-treat infection, characterized by necrotizing eosinophilic and granulomatous inflammation and frequent lack of response to antifungal therapy [Infections by the oomycete  therapy , 2.It is known that the purinergic system participates in immune system modulation during infections, including in pythiosis . In partP. insidiosum [P. insidiosum has elicited interest in the role of iron in pythiosis [Iron is essential for both the pathogen and the host, and not only iron deficiency but iron overload can significantly impair immune function by altering T and B cell proliferation . The presidiosum  and the ythiosis .in vitro against P. insidiosum strains. Similarly, subcutaneous lesions were only slightly decreased following deferasirox treatment when compared to placebo-treated animals [P. insidiosum growth and to explore the immunomodulatory properties of deferasirox in pythiosis lesions, we evaluated the activity of deferasirox in vitro and in lesions of rabbits treated with the iron chelator and/or immunotherapy.The iron chelator deferasirox has shown potential as a therapeutic intervention for pathogenic fungi , 11. The animals . To bettH-tetrazolium-5-carboxanilide (XTT) colorimetric assay. Triplicate Eppendorf tubes containing 103P. insidiosum zoospores (strain CBS 101555) diluted in 1 mL RPMI 1640 broth and deferasirox at the MIC concentration (50 μg/ml) [We determined the time course of deferasirox-induced hyphal damage with the use of the 2, 3-bis(2-methoxy-4-nitro-5-sulfophenyl)-20 μg/ml) , iron (00 μg/ml) . The optP. insidiosum hyphae, we performed viability staining with bis- trimethine oxonol (DiBAC) . P. insidiosum zoospores were incubated in Eppendorf tubes with RPMI 1640 plus 0.15% (wt/vol) Junlon  at 37°C with shaking for 4 h. Tubes were centrifuged at 4,000 g to remove the media, and germlings were resuspended in RPMI containing deferasirox at the MIC concentration, 0.125% iron or both. Drug-free medium served as negative control. After 6 h incubation at 37°C with shaking, DiBAC staining was performed as previously described [To determine whether deferasirox causes damage to P. insidiosum infected animals were treated by oral gavage with 15 mg/kg/day deferasirox, subcutaneous immunotherapy  at 14 d intervals, a combination of both, or placebo, with treatments starting 25 d post-infection [Rabbit pythiosis lesions used in the current study for histopathology and ADA activity assessment were obtained from a previous study in which groups of five nfection . At day Lesion samples were fixed in formalin, embedded in paraffin and sections stained with hematoxylin and eosin (H&E) to evaluate the inflammatory cell infiltrate and Grocott’s methenamine silver nitrate (GMS) to evaluate hyphal morphology . Two lesLesion slices were washed and homogenized in  50 mM phosphate buffer solution (pH 7) on ice using a tissue homogenizer. The homogenate was centrifuged at 10,000 X g for 60 min and clear upper supernatant fluids were used for protein and ADA activity measurements. Protein was determined by the method of Lowry et al. , and ADAThe variables were compared among groups by one-way analysis of variance and Tukey’s test (α = 0.05). Furthermore, a Pearson’s correlation analysis was performed to evaluate the relationship between mean lesion areas and enzymatic activities.P. insidiosum zoospores in RPMI 1640 medium reached a peak of growth at 16 h of incubation. At the MIC concentration, the iron chelator deferasirox fully inhibited P. insidiosum growth, and this inhibition was partially abrogated by the concomitant addition of 0.125% FeCl3 (P. insidiosum supplemented with iron was lesser than the observed in the control medium (RPMI only).5% FeCl3 . InteresP. insidiosum germlings treated with deferasirox at the MIC concentration Click here for additional data file.


ZnS hierarchical nanospheres (HNs) were prepared via a solution-based route and their sensing performances were manipulated by Mn2+ doping. The responses of the sensors based on ZnS HNs towards 8 explosives generally increase firstly and then decrease with the increase of the doped Mn2+ concentration, reaching the climate at 5% Mn2+. Furthermore, the sensory array based on ZnS HNs with different doping levels achieved the sensitive and discriminative detection of 6 analytes relevant to IEDs and 2 military explosives in less than 5 s at room temperature. Importantly, the superior sensing performances make ZnS HNs material interesting in the field of chemiresistive sensors, and this simple method could be a very promising strategy to put the sensors based on thin-films of one-dimensional (1D) nanostructures into practical IEDs detection.A simple method combing Mn However, in order to avoid the W18O49 NWs forming severe aggregation and to obtain well-dispersed NWs, further surface modification is needed, which requires a large amount of organic solvents, such as chloroform and oleylamine. Three-dimensional (3D) nanostructures containing ordered NWs are promising candidates for building thin-film sensors due to the efficient exposure of NWs and the excellent charge transport property9Chemiresistive sensors based on 1D nanostructures, such as nanotubes and nanowires (NWs), have attracted a great deal of attention because of their exquisite sensitivity and fast response to the surrounding environment12354via ion chromatography114282+ was often doped into the ZnS host to manipulate the optical properties, such as light-emitting and fluorescence322+ doped ZnS342+ doping in ZnS could also influence the electronic properties, for example, the charge carrier density, and thus the gas sensing performance. As a result, a systematical study by employing Mn2+ as the doping element in 3D ZnS hierarchical structure would not only help to promote the explosive vapor sensing performance, but also help to understand the chemiresistive sensing mechanisms of ZnS.More recently, improvised explosive devices (IEDs) are extensively used in terrorist attacks, such as those in Pune, India 2010), Oslo, Norway 2011), Boston, USA 2013) and Abuja, Nigeria 2014), owing to the readily available, low cost and legally purchased components, Boston,12 and Abuj1113, owing t5151617181910, Oslo,13via the solution-based route and the resulting sensing performances were manipulated by Mn2+ doping. Based on it, ZnS HNs with different Mn2+ doping concentrations were fabricated into a sensory array, achieving the rapid, sensitive and recognizable detection towards vapors of the analytes relevant to IEDs, such as urea, urea fertilizer (UF), black powder (BP), PP, SP, and PN, as well as trinitrotoluene (TNT) and 2, 4-Dinitrotoluene (DNT) at room temperature.In this paper, ZnS HNs containing ordered NWs vertically aligned on the surface were prepared 2+:ZnS HNs were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The SEM images with low magnification  characterization was conducted  and 6 improvised explosive vapors  saturated at room temperature were used as the testing vapors, respectively. It can be easily observed that the responses of the Mn2+:ZnS HNs-based sensor are significantly enhanced compared with those of the pure ZnS HNs-based sensor towards a certain explosive vapor  in general. This changing trend of the response values with the increase of the doped Mn2+ clearly indicates that Mn2+ concentration has an obvious influence on the sensing performance of ZnS HNs and Mn2+ doping is an effective strategy to manipulate the response of a sensor based on ZnS HNs.To systematically investigate the gas sensing performance of the sensors based on pure ZnS and Mnve vapor . It is a2+ (corresponding to 5% Mn2+ in the precursor), the response values to TNT, DNT, SP, BP, UF, PP, and PN are greatly improved to 135.7, 232.1, −51.3, 95.2, 58.5, 46.1 and 35.7%, respectively. Although the response values decrease with the further increase of doped Mn2+, the response values of the sensor based on the ZnS HNs with 10% Mn2+ to the analytes (except urea) are larger than those of the sensor based on pure ZnS HNs. This result also indicates that the doping of Mn2+ can effectively improve the gas sensing performance of ZnS HNs.It is known that with the help of a sensory array, the discrimination towards different target gases could be realized. Thus, all the four sensors in the present study were organized into a sensory array . Figure This sensory array also shows fast response and recovery characteristics towards all the saturated explosive vapors at room temperature . It is s2+:ZnS HNs-based sensors were further confirmed by the comparison with other recently reported chemiresistor or Schottky sensors towards explosive vapors  were further analyzed using a principal component analysis (PCA) method. It is shown that the clusters aggregate separately and are far from each other , which i2+ is incorporated into ZnS, there are Mn-S bonds appeared in Mn2+:ZnS host. Compared with that of Zn-S (2.341 Å), the bond length of Mn-S (2.431 Å) is longer2+:ZnS NWs 2∙2H2O), L-cysteine (C3H7NO2S), manganese acetate (Mn(CH3COOH)2∙4H2O), ethanol amine, urea, potassium permanganate (PP), potassium nitrate (PN), and sulphur powder (SP) are analytical grade. 2, 4-Dinitrotoluene (DNT) was purchased from Sigma-Aldrich. Trinitrotoluene (TNT) was obtained from the National Security Department of China and recrystallized with ethanol before use. Urea fertilizer (UF) was purchased from shopping store. Black powder (BP) was self-made by blending SP, PN and graphite powder with the molar ratio of 1:2:3.Zinc acetate (Zn(CH3COOH)2∙2H2O, 4 mmol C3H7NO2S and a different given amount  of Mn(CH3COOH)2∙4H2O were placed in each beaker. Second, 24 mL of deionized water was added into each of the above beaker and the mixture was dispersed to form a homogeneous solution at room temperature by constant stirring for 30 min. Third, 16 mL of ethanol amine was added to the above solution and was continually stirred for 10 min. Then the resulting mixture was transferred into a Teflon-lined stainless autoclave of 60 mL capacity and maintained at 160 °C for 2 h. The system was then cooled to ambient temperature naturally. After that, the precipitation was washed with ethanol and distilled water in sequence and repeated 3 times, finally dried in oven overnight at 40 oC to get pure ZnS and Mn2+:ZnS HNs.First, four beakers were used and 2 mmol Zn. Field-emission scanning electron microscope  and transmission electron microscope  were used to characterize the morphology of the samples. Energy disperse spectroscopy (EDS) was used to quantitatively evaluate the composition of the impurity element in the Mn2+:ZnS HNs. The content of Mn element in ZnS HNs was defined as the atomic ratio of Mn/(Mn+Zn) measured.X-ray diffraction (XRD) measurement was conducted using powder XRD  saturated explosive vapor was obtained by putting solid explosive powder at the bottom of a conical flask (250 mL) and sealed for 48 h. For gas sensing test, the sensor was inserted into the saturated vapor of an explosive vapor (a = (Rg − Ra)/Ra × 100%, where Ra and Rg are the electrical resistances of the sensor in air and in explosive vapor. The response time is defined as the period in which the sensor resistance reaches 90% of the response value upon exposure to the explosive vapor, while the recovery time is defined as the period in which the sensor resistance changes to 10% of the response value after the explosive vapor is removed.The Mntability . The roove vapor . After tHow to cite this article: Zhou, C. et al. Ultrasensitive, Real-time and Discriminative Detection of Improvised Explosives by Chemiresistive Thin-film Sensory Array of Mn2+ Tailored Hierarchical ZnS. Sci. Rep. 6, 25588; doi: 10.1038/srep25588 (2016).


Amyloid β-protein (Aβ) containing amyloid plaques and abnormal phosphorylated τ-protein containing neurofibrillary tangles (NFTs) are hallmark lesions of Alzheimer's disease. Both Aβ plaques and NFTs show hierarchical patterns in which the areas of the brain are subsequently affected by Aβ plaques and NFTs, respectively  and GE-Healthcare (UK), a speaker honorarium from GE-Healthcare (UK) and collaborated with Novartis Pharma Basel (Switzerland).


Recombinant human Granulocyte-Colony Stimulating Factor (rhG-CSF) supplementation seems to be a promising innovative therapy in reproductive medicine, used in case of recurrent miscarriage, embryo implantation failure or thin endometrium, although its mechanisms of action remain unknown. Our aim was to identify possible endometrial pathways influenced by rhG-CSF.ex-vivo culture. Middle luteal phase endometrial biopsies were cultured on collagen sponge with or without rhG-CSF supplementation during 3 consecutive days. Variations of endometrial mRNA expression for the selected targets were studied by RT-PCR.Hypothetical molecular interactions regulated by G-CSF were designed through a previous large scale endometrial microarray study. The variation of endometrial expression of selected target genes was confirmed in control and infertile patients. G-CSF supplementation influence on these targets was tested on an endometrial At the highest dose of rhG-CSF stimulation, the mRNA expression of these selected target genes was significantly increased if compared with their expression without addition of rhG-CSF. The selected targets were G-CSF Receptor (G-CSFR), Integrin alpha-V/beta-3 (ITGB3) implicated in cell migration and embryo implantation, Plasminogen Activator Urokinase Receptor (PLAUR) described as interacting with integrins and implicated in cell migration, Thymidine Phosphorylase (TYMP) implicated in local angiogenesis, CD40 and its ligand CD40L involved in cell proliferation control.ex-vivo model should be tested in-vivo. The strict indications or counter indication of rhG-CSF supplementation in reproductive field are not yet established, while the safety of its administration in early pregnancy on early embryogenesis still needs to be demonstrated. Nevertheless, rhG-CSF appears as a promising therapy in some difficult and unsolved cases of reproductive failure. Indications of pre-conceptual rhG-CSF supplementation may derive from a diagnosed lack of endometrial expression of some target genes.RhG-CSF seems able to influence endometrial expressions crucial for implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. These variations observed in an  Recombinant Human Granulocyte-Colony Stimulating Factor (rhG-CSF) is used since late 1980's in haematological indications such as chemotherapy induced neutropenia In reproductive medicine, rhG-CSF supplementation seems to be one of the most promising innovative therapies. Indeed, in distinct countries, rhG-CSF supplementation, either systemic (subcutaneous administration) or local (intra uterine infusion), is evaluated in the context of some unexplained recurrent miscarriages, repeated embryo implantation failures or thin unresponsive endometrium. Two randomised studies using rhG-CSF supplementation on IVF stimulation protocols, in case of repeated miscarriages, suggest a higher live birth rate and fewer cases of pregnancy loss However, the mechanisms of action by which rhG-CSF would positively influence the embryo implantation are largely unknown. The objective of the present study was to identify the possible relevant pathways influenced by a local administration of rhG-CSF, involving target genes pre-selected from a previous large scale microarray 81 women were enrolled for this study. All patients were involved in Assisted Reproductive Technology (ART) programs and were less than 38 years of age. There was no statistically significant difference in age and BMI between the different groups. All patients were non-smoker. They all provided a written informed consent and this investigation was approved by our Institutional Review Board, Agence de la Biomedecine, under the trial registration number 2013-A00072-43.17 patients were fertile control women, involved in ART because of male infertility. All had delivered after either intra-uterine insemination or ICSI within the two first attempts. They were evaluated at least six months after their delivery and before a new attempt. The mean age in this group was 33 years (from 29 to 35 years old). The mean Body Mass Index (BMI) was 23.8 (from 21 to 27.9).3 during luteal phase. In this group, the mean age was 31.7 years (from 28 to 35) and the mean BMI was 22.6 (from 19 to 29).19 patients had repeated unexplained embryo implantation failure (IF) 15 patients had a history of unexplained recurrent miscarriages (RM) 30 other patients participated to our study. They were involved in a pre IVF cycle endometrial biopsy program . These endometrial biopsies were used for ex-vivo microhistocultures.Endometrial biopsies were programmed during a monitored natural cycle, 7 to 9 days after the ovulation surge (LH surge), during the hypothetical implantation window. These biopsies were performed with a standard Cornier pipelle , collecting material from superficial endometrial layers in order to minimise sampling variations.One part of the endometrial sample was formalin-fixed and paraffin-embedded for later histological study. We used a standard haematoxylin and eosin staining protocol on 3 µm thick sections for routine histological evaluation. Moreover, a histological endometrial dating, to classify patients as “in phase” or “out phase”, was performed according to the Noyes criteria Another part was collected in RPMI 1640 Glutamax medium for endometrial ex-vivo culture.A third and last part of the endometrial sample was transferred into an RNA stabilizer solution  for further RNA extraction.3) were taken from biopsies collected in RPMI 1640 Glutamax medium, during the 2 or 3 first hours after collection. We put these endometrial blocks on collagen sponge gels  which are placed into supplemented RPMI 1640 Glutamax culture medium. The medium supplementation was as follows: 15% heat-inactivated foetal calf serum, 1% penicillin and 1% streptomycin, non-essential amino acids, 1% sodium pyruvate , 50 nmol/l of estradiol and progesterone . Estradiol and progesterone supplementations were repeated daily. The cultures were maintained at 37°C in a 5% CO2 humid atmosphere.The endometrial microhistoculture model used in this study has been previously described by our team For rhG-CSF stimulation, the five culture conditions were: rhG-CSF at either 20, 100 or 200 ng/ml, or G-CSF blocking antibody (anti G-CSF) at 3 µg/ml . Recombinant protein or antibody was added to the culture medium every day during three consecutive days. After a three days incubation, the endometrial samples were collected in a RNA stabilizer solution  for RNA extraction and RT-qPCR, as described in the corresponding paragraph.To specifically target the rhG-CSF endometrial action, the same experiments were performed with Granulocyte-Macrophage-CSF (rhGM-CSF) at either 20, 100 or 200 ng/ml, or GM-CSF blocking antibody (anti GM-CSF) at 3 µg/ml .3) for rhG-CSF microhistocultures. Biopsies from 13 patients provided 39 samples (1 to 2 mm3) for control rhGM-CSF microhistocultures.The biopsies from 30 patients, involved in a pre IVF cycle endometrial biopsy program, were used for these ex-vivo microhistocultures. Biopsies from 17 patients provided 57 endometrial samples , as described, during the three days incubation period. Therefore, for the same endometrium, we could compare the target gene expressions under specific stimulation and EP condition.The total endometrial sample was homogenized with an Ultra Turrax T15 (IKA-WERKE) and the homogenate was then purified on Qiashredder columns . The total RNA was isolated with the Qiagen RNeasy mini kit  including the RNase-free DNAse set. Recombinant RNase inhibitor (10 units/µl of extracted RNA) was added to prevent RNA degradation. RNA quantity and quality were confirmed by the analysis with an Experion system and RNA Std Sens analysis kit . The RNA was then stored at −80°C. The RNA (1 µg) was first reverse-transcribed into cDNA using random primers and SuperScript III reverse transcriptase  following the manufacturer's instructions.Hypothetical pathways and molecular interactions putatively regulated by G-CSF were selected on the basis of gene expression deregulations according to the indications stemming from a previous large scale endometrial microarray study Specific primers for these targets genes and Ribosomal Protein L13A (RPL13A), as the reference gene, were constructed using the Universal Probe Library Assay Design Center (\.rocheapplied-science.com) and their sequences were searched against Gen-Bank sequences with the BLAST program to ensure the specificity of primers. Real-time PCR was carried out using a LightCycler 480 apparatus . Reactions were set up using the following final concentrations: 0.5 mM of sense and antisens primers, 1X 480 SYBER Green master mix and 4 ml of 1/20 diluted cDNA. Cycling conditions were as follows: denaturation (95°C for 5 min), amplification and quantitation  repeated 40 times, a melting curve program (65–95°C with a ramp rate of 2.2°C/s) and a cooling step to 4°C. In addition to the no-reverse transcription and no-template controls, an independently inter run calibrator (IRC) was included in each RT-PCR assay. This IRC was obtained from blast-cells. In each assay, an aliquot of the IRC cDNA was 20 times diluted and was submitted to the qPCR protocol as the unknown samples. PCR efficiencies for each quantified target and reference were calculated using known serial dilutions of each specific cDNA. Data were analysed using the LightCyclerH480 Software release 1.5.0. Each specific target transcription level was normalized to the geometric mean of the transcription levels of the reference gene, using the Advanced Relative Quantification of the LightCyclerH480 Software. Efficiency was controlled for each specific gene amplification.For each sample, the results are expressed in concentration ratio (target gene mRNA level/reference gene mRNA level). For microhistoculture samples, these ratios are compared between the stimulated culture condition and the EP basal condition, as described in the dedicated paragraph, each patient being her own control.The Wilcoxon test was used to compare target genes mRNA expressions in each patient group and in each culture condition. The Spearman test was used to search for a correlation between different gene expressions. The statistical assessments were performed using the Stat View software . The significance level was set up at p<0. 05.G-CSF receptor (G-CSFR) have been shown to be localised at the maternal foetal interface since the late 1980's Both the protein production and mRNA expression of Thymidine Phosphorylase (TYMP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, is a key angiogenesis promoting enzyme Integrin alpha-V/beta-3 (ITGB3), which is present in the endometrium Plasminogen Activator Urokinase Receptor (PLAUR), or uPAR (Urokinase Plasminogen Activator Receptor), is an essential actor in tissue remodelling through cell migration, proliferation and survival CD40 and its ligand CD40L are membrane molecules involved in the control of cellular proliferation via regulation of apoptotic mechanisms The mRNA expression variations for G-CSFR, TYMP, ITGB3 and PLAUR, observed under rhG-CSF stimulation, were found to be correlated, as shown in To specifically study the action of endometrial rhG-CSF on its selected target genes, we used the same ex-vivo experiments with rhGM-CSF supplementation during 3 consecutive days. This control supplementation did not induce any variation on mRNA expressions of G-CSFR, TYMP, ITGB3 or PLAUR, as observed with rhG-CSF adjunction.In all cases, adjunction of either anti G-CSF blocking antibody or anti GM-CSF blocking antibody did not show any variation in the target genes expression with the present ex vivo model. This may be due to existing redundant pathways or linked to the type of selected blocking antibody.After showing the difference of expression of some hypothetical endometrial targets in infertile patients, and after observing the variations of expression of these target genes under specific rhG-CSF stimulation ex vivo, this study illustrates the putative key role of G-CSF during the embryo implantation process. This cytokine seems able to modulate fundamental genes intervening in the local embryo adhesion, cell migration, tissue remodelling and angiogenesis, unavoidable for a successful implantation and further placentation.RhG-CSF appears actually as a promising innovative therapy in some difficult and unsolved cases of reproductive failure. However, strict indications of this supplementation in reproductive field are not established yet. Such expected endometrial actions may be tested after in vivo rhG-CSF supplementation and safety of such treatment still needs to be demonstrated on early stages of embryogenesis. Specific indications of pre conceptual rhG-CSF supplementation may also derive from diagnosed lack of endometrial expression of some of its target genes.


Z-component  coordinate of center-of-gravity (COG) were compared between two groups. Probabilistic tractography was performed to investigate diffusion indices of fibers between the striatum and frontal ROIs. COG Z-component coordinates of connectivity maps for OFC ROI were located in the more dorsal part of the VS in OCD patients compared to HC. Fractional anisotropy of fibers between the OFC and the striatum was higher in OCD patients compared to HC. Part of the pathophysiology of OCD might be understood by altered topography and structural connectivity of fibers between the OFC and the striatum.Fronto-striatal circuits are hypothesized to be involved in the pathophysiology of obsessive-compulsive disorder (OCD). Within this circuitry, ventral frontal regions project fibers to the ventral striatum (VS) and dorsal frontal regions to the dorsal striatum. Resting state fMRI research has shown higher functional connectivity between the orbitofrontal cortex (OFC) and the dorsal part of the VS in OCD patients compared to healthy controls (HC). Therefore, we hypothesized that in OCD the OFC predominantly project fibers to the more dorsal part of the VS, and that the structural connectivity between the OFC and VS is higher compared to HC. A total of 20 non-medicated OCD patients and 20 HC underwent diffusion-weighted imaging. Connectivity-based parcellation analyses were performed with the striatum as seed region and the OFC, dorsolateral prefrontal cortex, and dorsal anterior cingulate cortex as target regions. Obtained connectivity maps for each frontal region of interest (ROI) were normalized into standard space, and  Obsessive-compulsive disorder (OCD) is characterized by anxiety-provoking thoughts or images (obsessions) and repetitive behaviors that are performed to reduce anxiety (compulsions). Fronto-striatal circuits are hypothesized to be involved in the pathophysiology of OCD because both structural and functional neuroimaging research showed abnormalities in these circuits Within this circuitry, the ventral prefrontal cortex (vPFC) including orbitofrontal cortex (OFC) mainly projects fibers to the ventral striatum, while the dorsal frontal cortex such as dorsolateral prefrontal cortex (DLPFC) and dorsal anterior cingulate cortex (dACC) project to the dorsal striatum Our previous resting state fMRI research showed higher functional connectivity between the dorsal part of the ventral striatum and the OFC in OCD patients compared to HC while there was no significant change of functional connectivity between the ventral part of the ventral striatum and the OFC versus HC Additionally, we hypothesized that patients with OCD also show higher structural connectivity between the OFC and the ventral striatum compared to HC. Structural connectivity represents the anatomical white-matter connection between regions, which depends on degree of myelination and number of fibers, while functional connectivity refers to the synchrony between a continuous time series of brain activity . In geneThe subjects were 20 adult patients (9 men and 11 women) diagnosed with OCD (based on the DSM-IV criteria) and 20 healthy volunteers (6 men and 14 women) matched for age and handedness. Patients were recruited at the Kyoto Prefectural University of Medicine Hospital, Kyoto, Japan. All patients were primarily diagnosed using the Structured Clinical Interview for DSM-IV Axis I Disorders-Patient Edition (SCID) http://surfer.nmr.mgh.harvard.edu). High-resolution T1-weighted and diffusion-weighted images (DWIs) were obtained with a whole-body 3-Tesla MR system  with an eight-channel phased-array head coil. T1-weighted images were used to define anatomically correct regions of interests based on cortical and subcortical structures as described below. A combination of T1-weighted images and DWIs is therefore advantageous to delineate specific fibers within a neural circuitry. The scanning parameters of the T1-weighted three-dimensional magnetization-prepared rapid gradient-echo (3D-MPRAGE) sequences were as follows: flip angle, 10 degrees; acquisition matrix, 256×256×170; field of view, 25.6 cm; section thickness, 1.0 mm; voxel size, 1.0 mm×1.0 mm×1.0 mm; TR, 7.1 ms; and TE, 3.3 ms. The 3D-MPRAGE images were preprocessed using the FreeSurfer software package version 5.2.0  version 5.0.2 (http://www.fmrib.ox.ac.uk/fsl). All DWIs source data were corrected for eddy currents and head motion by registering each data point to the first b = 0 image with affine transformation. The FA, MD, AD, and RD maps were calculated using the DTIFIT program implemented in FSL. In order to perform connectivity-based parcellation and probabilistic tractography, probability distributions on 2 fiber directions were modeled at each voxel using FSL’s BedpostX program, based on a multifiber diffusion model Diffusion-weighted data were obtained using a single-shot spin-echo echo-planar sequence with 32 Stejskal–Tanner motion-probing gradient orientations. DWIs protocol were as follows: b = 1000 s/mmThe striatum ROIs were created with a combination of caudate, putamen, and nucleus accumbens that were extracted from the automated segmentation in FreeSurfer http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Cluster). In detail, we used following command line.Each subject’s b = 0 image was co-registered to a T1 image by applying the rigid transformation matrix, which was calculated by use of FSL’s FLIRT program cluster – in = <each connectivity map> – thresh = 0 – mm.Z-component  COG of spatially normalized connectivity maps were identified from above “cluster” command line. Then, they were compared between the two groups by analysis of covariance (ANCOVA) controlled for age and gender effects because both of them might affect white matter structure P<.05 .Here, – in, – thresh, and – mm arguments represent input file, threshold, and use millimeter (not voxel coordinates), respectively. COG was used as an index of fiber spatial distribution as it is used in previous connectivity-based parcellation studies P<.05 .The same 3 frontal ROIs were used as seed ROIs in probabilistic tractography. Each subject’s connectivity maps for each frontal ROI was thresholded at 25% of the maximum connectivity probabilities to exclude voxels with low connectivity probabilities. Thus, connectivity-based striatum ROIs were created and used as the target ROIs , Fig. S1Z-component coordinates of COG for each connectivity map. The statistical significance level was set at P<.05. Multiple comparisons were not corrected in this analysis. Data were analyzed using SPSS 21.0.In the patient group, multiple regression analyses were performed using each DWI index  as a dependent variable and the Y-BOCS, HDRS and HARS total scores as independent variables to examine whether abnormalities of the DWI indices in patients with OCD are state or trait markers. We also examined Spearman rank-order correlations between age of onset, duration of illness, and The demographic and clinical data are shown in Z-component coordinate of each connectivity map in two groups are shown in Z-component coordinates of the connectivity maps for the left OFC were significantly larger in patients with OCD than those of HC (P = .043). Patients with OCD also showed marginally significant larger Z-component coordinates of the connectivity maps for the right OFC (P = .056). That is, they were located in the more dorsal area of the striatum in patients compared to HC.Mean images of connectivity maps for each frontal ROI and examples of some subjects’ results are shown in MNI152 standard space  and eachP = .025). Patients with OCD also showed a trend for higher FA of fibers between the right OFC and the striatum compared to HC (P = .062). The results of all diffusion indices for fibers between the OFC and the striatum are shown in P = .043). Other results are shown in P = .091) of the left DLPFC-striatum fibers, for higher MD (P = .074) and higher AD (P = .061) of the right DLPFC-striatum fibers, and for higher FA of the right dACC-striatum fibers (P = .070) in OCD compared to HC.Mean image of tractography results and examples of some subjects’ results are shown in MNI152 standard space  and eachNone of the Y-BOCS, HDRS, and HARS total scores did predict DWI indices of fibers . Age of This is the first connectivity-based parcellation and probabilistic tractography study examining directly the fronto-striatal fiber topography and structural connectivity of non-medicated patients with OCD and matched HC. We found dorsally spreading projection of fibers between the OFC and the striatum in patients with OCD, as we hypothesized, and higher FAs of these fibers in patients with OCD compared to HC based on the connectivity-based parcellation and the probabilistic tractography analyses respectively.Previous DWI studies in OCD have shown abnormalities of white matter tracts, including the corpus callosum, anterior limb of the internal capsule, and cingulum Connectivity-based parcellation analyses showed dorsally spreading projection of fibers between the OFC and the striatum in patients with OCD, which is consistent with a finding of our previous resting state fMRI research that showed higher functional connectivity between the dorsal part of the ventral striatum and the OFC In addition, probabilistic tractography analyses showed the higher FAs of fibers between the OFC and the striatum. It can be affected by several factors such as degree of myelination, number of fibers, membrane permeability, and fiber orientation in each voxel. However, the left OFC-striatum fibers also showed lower RD that reflects water mobility perpendicular to the fiber axis. Myelination could decrease RD by increasing the barriers for water molecular diffusion perpendicular to the myelinated axons From both connectivity-based parcellation and probabilistic tractography results, we did not find any topographical and connectivity abnormalities in fibers between the DLPFC, dACC and the striatum except for trends for higher FA of the left DLPFC-striatum fibers, higher MD and higher AD of the right DLPFC-striatum fibers, and higher FA of the right dACC-striatum fibers in OCD compared to HC. These findings were only found in the unilateral hemisphere, though we did not have a hypothesis for these fibers’ abnormalities. Therefore, further studies will be necessary to confirm the involvement of white matter abnormalities in the fibers between the DLPFC, dACC and the striatum in patients with OCD.Multiple regression analyses showed that severity of OCD, of comorbid depression and anxiety did not predict DWI indices. A higher FA or lower RD in patients with OCD compared to HC probably represents a trait rather than a state marker. However, the results should be interpreted with extreme caution because of the small sample size of this study. This finding could be clarified by examining first-degree relatives of patients with OCD. Age of onset, but not disease duration showed a significant negative correlation with Z-component coordinates of COG for almost all connectivity maps. This finding hints to the hypothesis that abnormalities of fiber topography may be related to the specific pathophysiology of early-onset OCD, which could be examined by comparing early with late-onset-patients with OCD.Although the fiber topography and connectivity develop over time, it is unknown whether abnormalities of white matter structure are causally related to the etiology of OCD. FA values of white matter in healthy subjects increase during their childhood and adolescents Although all patients with OCD were non-medicated, medication could have affected the fronto-striatal fiber structure. We compared fiber topography and connectivity between two groups covarying not only age and gender but also the length of previous medication. The results remained unchanged see –S7. ThisOur findings from connectivity-based parcellation analysis might be related to clinical application. Dorsally spreading projection of fibers between the OFC and the striatum in patients with OCD might be particularly relevant to optimization of deep brain stimulation (DBS) settings since it targets the ventral striatum. DBS is the most promising treatment for refractory OCD patients although its mechanism is still unclear. Nucleus accumbens DBS seems to affect not only the nucleus accumbens itself but also the adjacent fibers because the active contact points of DBS effective in reducing anxiety and OCD symptoms were located near the internal capsule This study has several limitations. The first one is that we used the MNI305 atlas to transform original T1 images into standard space in the Freesurfer analysis. The atlas was not representative of our samples. This could be a limitation of the study although this method is validated and used as default setting in Freesurfer analysis In conclusion, our hypothesis was supported by the connectivity-based parcellation analysis. That is, the OFC predominantly project fibers to the more dorsal part of the striatum in patients with OCD compared to HC. This study also showed hyper structural connectivity of fibers between the OFC and the striatum. Part of the pathophysiology of OCD might be understood by altered topography and structural connectivity of fibers between the OFC and the striatum.Figure S1Randomly selected examples of the connectivity maps (left panel) and connectivity-based ROIs (right panel) for each frontal ROI  in the 3D-MPRAGE space. Yellow represent high probabilities of structural connection with each ROI, while red represents low probabilities. The ventral striatum had high probabilities of connection to the OFC, while the dorsal striatum had high probable connection with the DLPFC and dACC. Connectivity-based ROIs were created by thresholding connectivity maps at 25% and used as the target ROIs in following probabilistic tractography analysis.(JPG)Click here for additional data file.Figure S2Randomly selected examples of tractography results. The frontal (blue), striatum (red) regions, and delineated fibers (green) were shown in diffusion space rendered on each subject’s fractional anisotropy map. Fibers between the OFC, DLPFC and the striatum were shown in axial section, while fibers between the dACC and the striatum were shown in coronal section. Images are shown with and without the ROIs and fibers to clarify the locations. Left-right orientation is according to radiological convention.(JPG)Click here for additional data file.Table S1Diffusion indices of fibers between the DLPFC and the striatum.(DOC)Click here for additional data file.Table S2Diffusion indices of fibers between the dACC and the striatum.(DOC)Click here for additional data file.Table S3Results of multiple regression analyses predicting each DWI index from the Y-BOCS, HDRS and HARS total scores in patients with OCD .(DOC)Click here for additional data file.Table S4Z-component  coordinates of center-of-gravity (COG) for each connectivity map in the MNI152 standard space controlling for past medication effects.(DOC)Click here for additional data file.Table S5Diffusion indices of fibers between the OFC and the striatum controlling for past medication effects.(DOC)Click here for additional data file.Table S6Diffusion indices of fibers between the DLPFC and the striatum controlling for past medication effects.(DOC)Click here for additional data file.Table S7Diffusion indices of fibers between the dACC and the striatum controlling for past medication effects.(DOC)Click here for additional data file.