Two dipolar biphenyls show significant 180° orientational disorder resulting in bipolar as-grown crystals. An added symmetrical biphenyl interferes with polarity formation and inverts the initial bipolar state. 2 or CN) are predominantly present at crystal surfaces. However, the stochastic process of polarity formation can be influenced by adding a symmetrical biphenyl to a growing system. For this case, Monte Carlo simulations predict an inverted net polarity compared with the growth of pure BNBP and BCNBP. SPEM results clearly demonstrate that 4,4′-dibromobiphenyl (DBBP) can invert the polarity for both crystals. Phenomena reported in this paper belong to the most striking processes seen for molecular crystals, demonstrated by a stochastic process giving rise to symmetry breaking. We encounter here further examples supporting the general thesis that monodomain polar molecular crystals for fundamental reasons cannot exist.180° orientational disorder of molecular building blocks can lead to a peculiar spatial distribution of polar properties in molecular crystals. Here we present two examples [4-bromo-4′-nitrobiphenyl (BNBP) and 4-bromo-4′-cyanobiphenyl (BCNBP)] which develop into a bipolar final growth state. This means orientational disorder taking place at the crystal/nutrient interface produces domains of opposite average polarity for as-grown crystals. The spatial inhomogeneous distribution of polarity was investigated by scanning pyroelectric microscopy (SPEM), phase-sensitive second harmonic microscopy (PS-SHM) and selected volume X-ray diffraction (SVXD). As a result, the acceptor groups (NO In particular, the topological packing and possible strong interaction motifs are the base to assign a possible mechanism of disorder which during growth is producing sectors showing altered polar properties. It is important to notice that the orientational disorder we are addressing here exclusively emerges from the attachment of incoming molecules at the crystal/nutrient interface. Once in the bulk, elongated prolate top molecules will not undergo reversal at temperatures far below melting. The analysis presented attempts to understand polarity evolution in 4-bromo-4′-nitrobiphenyl (BNBP) and 4-bromo-4′-cyanobiphenyl (BCNBP) crystals in the frame of a general theory describing stochastic polarity formation due to 180° orientational disorder of dipolar molecules , the topological packing of antiparallel dipoles will develop growth-induced faults, which are kinetically stabilized in the bulk. When starting from a polar seed the crystal may perform a reversal transition x are investigated: The symmetrical molecules undergoing similar synthon interactions induce an inversion of the bipolar state, i.e. the bipolar state changes the polarization direction in corresponding domains. The systems BNBP and BCNBP are both showing (i) a bipolar state as pure crystals, and (ii) they undergo polarity inversion of the bipolar state when e.g. 4,4′-dibromobiphenyl (DBBP) is added to the solution of growth.Recently we have been able to demonstrate experimentally this kind of reversal transition  Isothermal evaporation of solvent , (ii) lowering the temperature of the solutions , and (iii) sublimation in evacuated ampoules (150°C) have all produced bipyramidal prisms. In (ii) rhombic plates were obtained using chloroform and toluene. There is no evidence for polymorphic forms. Melting occurred in the range 175–177°C. All crystals were transparent and colourless. The bipyramidal form expresses faces such as: (110),  isothermal evaporation of solvent  was applied. BCNBP also develops bipyramidal transparent prisms showing the following faces: (2) of a thickness of 20–40 µm were obtained.For second harmonic microscopy, thin melt grown crystals of BNBP, BCNBP were produced. For this, powder was placed in between two glass plates heated above melting. By slow cooling, large single crystalline areas  was added to growth solutions of butan-2-one. For comparison and the exclusion of solvents effects, some experiments were also performed in evacuated ampoules.2.3.X-AREA and X-RED32 software; Stoe & Cie, 2002Kα radiation (λ = 0.71073 Å). The intensities were collected in the ω scan mode (scan width ω = 1°) and data reduction was achieved by means of the X-RED32 program.Prior to X-ray experiments, all areas of BNBP and BCNBP crystals were visualized by scanning the polarization distribution on the faces of the crystal. The native crystals were glued on a glass fibre with a two-component epoxy resin. Reflections were measured at room temperature using a Stoe Image Plate 2 Diffractometer System (3) into a beam of diameter 0.5 mm.At first, full crystals were exposed in order to obtain the metric and structural models. Then, selected volume X-ray diffraction (SVXD) was performed by centring the volumes of interest .2.4.et al., 2000dT, the induced current in the capacitor used for measurements is proportional to the absolute value of the polarization P. BNBP and BCNBP bipyramidal crystals were polished parallel to the -plane and placed into a capacitor (b perpendicular to the electrodes). Additionally, the -plane of a BNBP prism was polished. A modulated laser  reduced to a spot size of less than 10 µm locally heated the surface of the cut plane. The thermally induced current amplified by a Keithley 428 was measured by a lock-in amplifier (Stanford Research SR830).Scanning pyroelectric microscopy (SPEM) allows us to map the spatial polarization distribution of molecular crystals showing a pyroelectric effect  coupled to a dynamic photon counting camera  was used in transmission mode. The Leica microscope was additionally joined to a 3CCD colour video camera (DXC-950P Sony). Objectives of 5× and 10× magnifications (LMPLFL Olympus) were used. Single crystals were mounted on glass slides.For SHG measurements we used a Q-switched Nd:YAG laser  providing a repetition rate of 10 Hz with a pulse width of 20–25 ns and a pulse energy of ∼ 25 mJ. This laser generated a fundamental beam at 1064 nm with a pulse intensity of 10 MW cm3.3.1.b-axis of the monoclinic cell. Typical synthon interactions —NO2⋯Br—, —CN⋯Br— give a topological packing into infinite antiparallel chains. A head-to-tail disorder of NO2/Br or CN/Br terminal groups is observed for one chain at least, the carbon skeleton of the biphenyl moiety being undistinguishable. The structures seem to be centrosymmetric but refinements in such a space group did not succeed in stabilizing acceptable models. The best refinements were obtained in the polar space group P21 with four independent molecules in the asymmetric unit cell, only one of them showing a head-to-tail 180° disorder  In BNBP, Flack parameters of individual domains (here right and left parts) are found to be 0.06 (4) and 0.10 (4), respectively. The absolute structure is consequently satisfactorily determined. Refined molecule populations for the asymmetric unit result in a 1.584 (4) bromine group toward the +b direction, 2.416 (4) toward −b for the right part, and a 2.361 (6) bromine group toward +b direction, 1.639 (6) toward −b for the left part. (ii) In BCNBP Flack parameters of each domain, i.e. top and bottom, values probing here again the monopolar character of the selected volumes are respectively refined to 0.085 (25) and 0.061 (19). For the top-volume, the refined molecule populations of the bromine group in the asymmetric unit give a population of 2.293 (4) toward the +b direction and 1.707 (4) toward the −b direction. In the case of the bottom-volume investigated, we find a 1.539 (3) bromine toward the +b direction, 2.461 (3) toward the −b direction.A Flack parameter around 0.5 is obviously not adequate to assess the relationship between polarity, morphology and structure. In order to verify the structural model constituted by only one disordered molecule and to clarify the polarity/morphology relationship, the SVXD method was applied to investigate separately the structures of two selected domains on each compound. Due to geometrical constraints to keep the same diffracting volume in the focused beam, the diffraction data are tarnished by some weaknesses , 2, CN) are pointing in a predominant way from inside the crystal to outside, i.e. towards the growing interface.With all these results the relationship between the bipolar crystal morphology and the absolute structure can be established: In both crystal systems the acceptor groups .As mentioned during the introduction both a centric and a polar seed can end up in a bipolar state, in other words, can represent 180° non-classical twins. For this we have investigated the spatial variation of the Flack parameter (see b-axis. The only likely space group is P21 (2). In case the reader accepts that in BNBP and BCNBP orientational disorder is a grown-in phenomenon, we are at the point of raising a basic question: What is the symmetry of the seed? Following the most recent results , i.e. polar seed followed by a reversal transition .Summarizing X-ray data and physical measurements, we can assign a polar structure to sectors involving the 3.2.et al., 2010a) and (ii) SHG results (b)–(d). Because of intergrown parts, the analysis is restricted on the parts that are marked (white broken lines).Preliminary to the analysis by SPEM we performed edge-defined thin film melt growth for both BNBP and BCNBP. A further goal was to compare solution, melt and vapour growth results because of the known effects of solvents  let us localize the seed region, because of the appearance of two domains featuring opposite polarities. These PS-SHM experiments allow us only to conclude that (i) significant 180° orientational disorder is present, (ii) the disorder varies in space, (iii) a bipolar growth state is obtained and (iv) the phenomenon is also present at high temperature, i.e. for growth upon a supercooled melt. No further distinction on the possible polar state of the seed can be made here.By turning the crystal at a fixed polarization of the laser, the direction of maximum response was found. At 90° to this orientation no signal appeared, meaning that the β4.P is mainly emerging from a sum over the polarity of individual molecules (modified by the effect of their surrounding molecular field), P is proportional to the sum of weighted SOF values: P = ε(SOF(Br1)α1 + SOF(Br2)α2 + SOF(Br3)α3 + SOF(Br4)α4), where αi  are factors taking into account (i) the inclination of μmol with respect to axis 2, and (ii) the modification of μmol by its surrounding, ε being a conversion factor. Depending on the degree of orientational disorder, the spatial response Px,y,z) and P = 0 (centric structure).SPEM measuring the local pyroelectric response of a material provides a map of the polarization distribution in BNBP and BCNBP crystals. Because b,c)-plane of two polished crystals we find two parts of obviously opposite polarity . This was obtained by measuring the direction of the discharge current. Assuming a negative effective pyroelectric coefficient, on average NO2 groups are oriented towards the nutrient. The spatial variation becomes evident (right side) when changing the frequency of the modulated heat source: At lower frequency  a different degree of net polarization and distribution is obtained compared with 1025 Hz . At low frequency the penetration depth is larger than at high values -plane revealed a similar behaviour. From these measurements we can conclude: BNBP as a seed  than the other. However, there are additional defects, visible in optical microscopy. The nitro-groups are preferentially pointing towards the growing interface (as confirmed by X-ray).Measuring a second crystal prepared for scanning the , representing a polar structure with nearly full parallel alignment of dipoles  groups are preferably covering the surfaces in the ±b directions of the bipolar object, stochastic theory of polarity formation  groups preferably cover the polar faces Fig. 7. So, mosA–π–D)x1 −  (D–π–D)x predict  revealed clearly an inverted situation in both cases  interactions can be considered as strong short-range contacts. Reversal at the surface involves —Br⋯Br— (D⋯D) and —NO2⋯O2N— or —CN⋯CN— (A⋯A) contacts, where —Br⋯Br— may show a local energy minimum and —A⋯A— is destabilizing. Consequently, we have (i) EDD − EAA ≠ 0, and most likely (ii) EAA > EDD. The first condition ensures that polarity can evolve, the second is giving preference for A-groups at the surface. The way in which BNBP and BCNBP crystals develop into a bipolar state may allow us to distinguish between a (i) centric and a (ii) polar seed: The rather large transition zone of BNBP x1 −  (D–π–D)x, i.e. the distribution coefficient is low. Here we identify a lack of understanding, because MC simulations for model systems also predicted a low distribution coefficient, but obviously larger than found experimentally.Solution and vapour grown BNBP and BCNBP crystals can express a regular and symmetrical morphology featuring flat faces, despite representing inside a twinned, BP Fig. 6 may be dThe present results end up in a first step analysis. From here, deeper crystallography  may investigate volume parts of DNBP or DCNBP crystals being well characterized by SPEM, with the aim of disclosing more about the peculiar disorder of such types of disordered crystalline states.10.1107/S2052252516006709/bi5055sup1.cifCrystal structure: contains datablock(s) BNBP_left, BNBP_right, BCNBP_bottom, BCNBP_top. DOI: 10.1107/S2052252516006709/bi5055BNBP_leftsup2.hklStructure factors: contains datablock(s) shelxl. DOI: 10.1107/S2052252516006709/bi5055BNBP_rightsup3.hklStructure factors: contains datablock(s) shelxl. DOI: 10.1107/S2052252516006709/bi5055BCNBP_bottomsup4.hklStructure factors: contains datablock(s) shelxl. DOI: 10.1107/S2052252516006709/bi5055BCNBP_topsup5.hklStructure factors: contains datablock(s) shelxl. DOI: Click here for additional data file.10.1107/S2052252516006709/bi5055BNBPsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2052252516006709/bi5055BCNBPsup7.cmlSupporting information file. DOI: 1475349, 1475350, 1475351, 1475352CCDC references:


N = 147) using neuroimaging, magnetencephalography and psychoacoustics. All disorder subgroups exhibited an oversized left planum temporale and an abnormal interhemispheric asynchrony (10–40 ms) of the primary auditory evoked P1-response. Considering right auditory cortex morphology, bilateral P1 source waveform shapes, and auditory performance, the three disorder subgroups could be reliably differentiated with outstanding accuracies of 89–98%. We therefore for the first time provide differential biomarkers for a brain-based diagnosis of dyslexia, ADHD, and ADD. The method allowed not only allowed for clear discrimination between two subtypes of attentional disorders (ADHD and ADD), a topic controversially discussed for decades in the scientific community, but also revealed the potential for objectively identifying comorbid cases. Noteworthy, in children playing a musical instrument, after three and a half years of training the observed interhemispheric asynchronies were reduced by about 2/3, thus suggesting a strong beneficial influence of music experience on brain development. These findings might have far-reaching implications for both research and practice and enable a profound understanding of the brain-related etiology, diagnosis, and musically based therapy of common auditory-related developmental disorders and learning disabilities.Dyslexia, attention deficit hyperactivity disorder (ADHD), and attention deficit disorder (ADD) show distinct clinical profiles that may include auditory and language-related impairments. Currently, an objective brain-based diagnosis of these developmental disorders is still unavailable. We investigated the neuro-auditory systems of dyslexic, ADHD, ADD, and age-matched control children ( The auditory cortex (AC) is very widely connected with different brain regions, including subcortical, prefrontal and parietal areas, where attentional and default networks are hosted. In view of the strong interdependence of such integrated networks D belong to the most common developmental disorders in childhood and adolescence with a worldwide prevalence of each about 5–10% D is merely diagnosed on the basis of observable behavior, which may lead to considerable variability among informants, cultures, and countries , subjects with ADHD , subjects with ADD , and normal controls .We measured the neuro-auditory profiles of 147 children in a cross-sectional design by means of structural MRI, MEG, and psychoacoustic tests. Participants belonged to one of four groups, namely dyslexics  = 0.26, n.s.) and self-indicated handedness  = 3.0, p = 0.032) may be due to a relatively higher sustained attention, which is not only impaired in ADHD and ADD, but may also be affected to some extent in dyslexia  or F 98.8 (ADD) according to the International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10) were included in the study. However, accuracies of behavioral classification scales lie between 47 and 79%, which clearly affects their clinical validity ; moreover, only controls with DISYPS-values outside the critical ranges were included (N = 30). All discriminant analyses were calculated as well for the original as for the more strictly defined revalidated sample.In order to improve diagnostic accuracies for our sample, the original classifications were re-validated on the basis of two criteria. Informal interviews were conducted with the responsible experts to clarify if potential comorbidities had arisen after the initial diagnoses. In addition, parents had to fill out the “Parents assessment sheet for hyperactive disorder” (FBB-HKS), which is part of the “Diagnostic System for Psychiatric Disorders in Children and Adolescents” . According to the selection criteria for our original sample, where such cases were excluded in advance as far as possible , this number is lower than in the normal population ” supported by the German Federal Ministry of Education and Research].SD = 0.45). The long-term sample included 79 control children without developmental disorders  who also participated in a larger comprehensive longitudinal study on potential benefits of musical training on brain and behavior, starting in 2009. In that study, as yet the 3rd measurement timepoint has been completed and a 4th and 5th follow-up measurement will be carried out until 2019. The cross-sectional data of this study basically correspond to the 2nd measurement timepoint. The age of the longitudinal control children was 8.5 ± 0.7 years at MTP1 and 12.2 ± 0.8 years at MTP2. The children of the control group (musicians and non-musicians) and the ADHD and ADD groups (non-musicians only) were also included in a first longitudinal comparison addressing the development of the auditory system over a shorter time span of about 1 year  was used as a classification criterion. The IMP was defined as the product of the number of years of formal music education and the amount of hours per week spent practicing a musical instrument. Similarly to the cross-sectional sample, the longitudinal sub-sample was divided into (a) “control group” vs. “pooled disorder group”  and (b) according to the cumulative amount of musical practice at the 2nd measurement timepoint  into “musicians” (IMP > 4) vs. “non-musicians” (IMP ≤ 4). The control group comprised 79 participants  and the disorder group comprised 30 participants . Noteworthy, such children were very rare (less than 5%) and had to be recruited all over Germany and Switzerland over almost 1 year.For the assessment of musical expertise an index of cumulative musical practice  the MRI or MEG data were not of sufficient quality for further processing. Accordingly, these children were only included in the analyses of psychoacoustic data and parts of the neurological analyses.The local research ethics committee of the Medical Faculty of the Ruprecht Karls University Heidelberg approved all experimental procedures in accordance to the Helsinki declaration. Parents provided informed consent in written form and subjects informed assent.y = 0 , repetition time (TR) = 1930 ms, echo time (TE) = 3.47 ms, flip angle 15°) was performed to investigate the anatomy of AC. A standardized individual approach of three-dimensional gray matter surface reconstruction of auditory subareas  was applied to account for individual morphology and gyrification patterns  it was not necessary to acquire EOGs. Then subjects were led to the magnetically shielded room, where the MEG measurements took place. They were requested to sit relaxed under the dewar of the MEG system. Foam ear pieces (Ethymotic ER3) were connected via 90 cm plastic tubes (diameter 3 mm) to small shielded transducers that were fixed in boxes next to the subject's chair. At the beginning of the MEG recordings the head position inside the dewar was determined. The loudness of the stimulation was adjusted to an equal subjective loudness (50 dB nSL) as determined by a Brüel and Kjaer artificial ear (type 4152). Stimuli were presented binaurally.Children were instructed to listen passively to the presented sounds over a measurement-duration of about 15 min. They were allowed to watch a silent movie in order to keep them seated and calm and to reduce potential artifacts. The stimuli were presented in pseudo-randomized order and consisted of seven different instrumental sounds  and four synthetically generated harmonic complex tones, each with a duration of 500 ms and a pseudo-randomized interstimulus interval between 400 and 500 ms. Each of the 11 sounds was presented 100 times, while auditory evoked fields were recorded. This high repetition rate guaranteed a good signal-to-noise-ratio as a necessary predisposition for robust source modeling and reliable source waveform analyses.The auditory evoked fields were recorded with a bandpass filter of 0.00 (DC)−330 Hz and a sampling rate of 1000 Hz. Prior to averaging, data were automatically inspected to exclude external artifacts by using the BESA Research Event-Related Fields (ERF) module. By applying the automatic Artifact Scan tool, on average about 3–7 noisy (bad) channels were excluded and about 10% of all epochs exceeding a gradient of 600 fT/cm×s and amplitudes either exceeding 3000 fT/cm or falling below 100 fT/cm were rejected from further analysis. Thereby, the major part of endogenous artifacts, like eye blinks, eye movements, cardiac activity, face movements, and muscle tensions could be accounted for. Due to the extremely high signal-noise-ratio (noise reduction factor of 33.2 when averaging over 1100 epochs) and systematic pseudo-randomization of the interstimulus intervals (see above), the influence of potential remaining artifacts was negligible. The efficiency of the procedure has also been demonstrated in a pilot study, where two additional EOG electrodes and one ECG electrode were used during the recordings and where continuous data were subsequently corrected manually for vertical and horizontal eye movements and ECG artifacts  designed for high-quality auditory testing. These headphones provide a passive attenuation of about 30 dB within the frequency range of the used stimuli. Stimulus intensity was adjusted to 65 dB SPL using a Brüel and Kjaer artificial ear, type 4152. The order of the behavioral tasks and neurological measurements was counterbalanced to avoid sequence effects. The total measurement time of the behavioral tests was about 3 h including three to four breaks (each about 10–15 min) between the different tasks.In order to test basic sound discrimination abilities, the “Dinosaur” threshold estimation program  or formant transitions . Participants have to decide, whether the items sound equal or different.Verbal repetition: Subjects have to repeat the word or non-word pairs directly after presentation.Analysis of consonant clusters with voiced and voiceless stop sounds at the initial position of a word: Subjects have to tell, which is the first and which the second phoneme of the presented word.T-values) are available for children of grade 1, 2, 3, and 4.Each subtest may be evaluated separately; a total score is obtained by summing up all correct answers (maximum: 62). Normative data , “Musical expertise” , and “Hemisphere” . For the MRI-data “HG volume,” “PT volume,” and “HG/PT-ratio” and for the MEG data “P1 latency,” “P1 width,” and “P1 amplitude” were considered as dependent variables. Moreover, measures of functional lateralization were introduced and considered in corresponding two-way ANOVAs. These comprised “Absolute P1 asynchrony” and the relative asymmetry values “ΔL,” “ΔW,” and “ΔA.”Likewise, performance in each of the psychoacoustic tests was analyzed in two-way ANOVAs with the dependent variables “Difference limens for frequency,” “intensity,” “onset ramp,” “duration,” “meter,” “rhythm,” “melody,” as well as “Subjective pitch perception.”post-hoc comparisons between controls, dyslexics, ADHD-children, and ADD-children were performed with the Tukey-Test (including a Bonferroni correction for multiple comparisons). Otherwise, the Tamhane Test was used. In case of significant interactions, the mean values of interest were compared with the Tukey-HSD.In case of a significant main effect “Disorder” and homogeneous error variances (as indicated by the Levene-Test), For the supplementary analysis of longitudinal data on the effects of musical practice a 2-way ANOVA was performed on the independent variables “Disorder”  and “Musical expertise”  and the dependent variable “Absolute P1 asynchrony.”In addition, discriminant analyses were performed to investigate how well different neural parameters segregate (a) the control group from the pooled disorder group and (b) the three disorder subgroups . The considered predictor variables were (1) MRI-based morphology of AC: “HG/PT ratio right,” “HG/PT ratio left”; (2) MEG-based functional lateralization of AC: “Absolute P1-asynchrony,” “ΔL,” “ΔW,” “ΔA”; (3) Behavioral measures: (a) Auditory discrimination of frequency, intensity, onset-ramp, tone duration (Dino Test) and meter (Metric Test); (b) Musicality (rhythmic and melodic scores of IMMA); (c) Subjective pitch (AAT-score).correct positive/; the specificity “SPEC” indicates the capacity to correctly identify non-deficit cases = Ncorrect negative/. The hit rate indicates the proportion of correctly classified cases in general = (Ncorrect positive + Ncorrect negative)/Nall cases.Sensitivity, Specificity, and Hit rate. The sensitivity “SEN” is defined as the capacity to correctly identify cases with deficit = NAll statistical analyses were carried out with the software package IBM SPSS Statistics Version 21.0.0.0.2) please refer to Table Using standard segmentation techniques . We focused on the first positive response complex (P1), peaking around 60–110 ms after tone onset and being the most prominent component in children at the elementary school age.F = 11.2, p = 1.3 × 10−6, part. η2 = 0.19). As evident from Table p = 9.4 × 10−6), ADHD (p = 1.7 × 10−5), and ADD (p = 0.0001). While controls showed fairly symmetric P1 sources in left and right HG, all disorder subgroups were characterized by atypical locations on left PT. The result is graphically illustrated in Figure All three disorder subgroups showed aberrant mean P1 source locations  please refer to Table Responses to acoustic stimulation were generally faster on the right side , but varied substantially across groups. Controls showed well-balanced bilateral responses with an average absolute P1-asynchrony (peak latency difference) of 3.7 ± 1.6 ms. Intriguingly, such asynchronies were about five times larger in the disorder subgroups ; for statistical significance values and effect sizes  to functional asynchrony, both measures were found to be negatively correlated , amplitude (ΔA), and width (ΔW) and compared across groups  and more complex auditory pattern recognition, such as meter, rhythm, melody, and pitch perception, were tested . As compared to the control group, dyslexics performed significantly poorer in almost all categories. Moreover, their subjective pitch perception was dominated by the aspect of spectral timbre. Children with ADHD were characterized only by lower scores in melodic and rhythm processing, whereas children with ADD did not show any auditory impairment at all Figure .F = 47.3, p = 4.5 × 10−10, part. η2 = 0.31). The extent of this long-term synchronization was directly correlated to the amount of musical practice between MTP1 and MTP2 ; for more details please see Figure F = 19.1, p = 2.9 × 10−5, part. η2 = 0.15.Due to our previous findings that musical training leaves the gross morphological structures of AC unchanged, but increases the neural efficiency of AC functions  and the functional synchronization between the right and left hemisphere (“Absolute P1-asynchrony”) segregate the control group from the pooled disorder group. The established discriminant function allowed for a correct assignment of 84.4% of all cases Table .In an attempt to discern the different types of developmental disorders, as a second step pair-wise discriminant analyses were performed on those neurological and behavioral measures that showed reliable differences between disorder subgroups  and specificity values signifying the correct identification of controls  were obtained  and to a lesser extent in right AC  may therefore be due to genetic factors, a lack of neural pruning, or both. We speculate that these morphological anomalies may hinder the build-up of reliable interconnections between bilaterally homotopic regions via the corpus callosum D treatments with methylphenidate and related stimulants, which are controversially discussed in the public, only positive side-effects are to be expected from musical training (Schellenberg, As a future perspective, a cross-validation of our findings may lead to an even more generalized view of the presented inferences. This would have far-reaching implications for research and practice and provide a deeper understanding of the etiology, diagnosis, and musically based therapy of common auditory-related developmental disorders.BS, CG, PS, and ASP substantially contributed to the conception and design of the work, BS, CG, DE, NG, PS, and ASP were involved in the acquisition of the data, BS, CG, VB, JB, DE, MBL, MW, MBR, AS, RP, SS, PS, and ASP in the anaylsis and interpretation of the data. BS, CG, PS, and ASP drafted the work, VB, DE, JB, NG, MBL, MW, MBR, AS, SS, and RP revised it critically for important intellectual content. All authors agree to be accountable for the content of the work and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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 identification of early, preferably presymptomatic, biomarkers and true etiologic factors for Alzheimer’s disease (AD) is the first step toward establishing effective primary and secondary prevention programs. Consequently, the search for a relatively inexpensive and harmless biomarker for AD continues. Despite intensive research worldwide, to date there is no definitive plasma or blood biomarker indicating high or low risk of conversion to AD.Magnetic resonance imaging and β-amyloid (Aβ) levels in three blood compartments  were measured in 96 subjects . Pearson correlations were completed between 113 regions of interest (ROIs)  and Aβ levels. Pearson correlation analyses adjusted for the covariates age, sex, apolipoprotein E (ApoE), education and creatinine levels showed neuroimaging ROIs were associated with Aβ levels. Two statistical methods were applied to study the major relationships identified: (1) Pearson correlation with phenotype added as a covariate and (2) a meta-analysis stratified by phenotype. Neuroimaging data and plasma Aβ measurements were taken from 630 Alzheimer’s Disease Neuroimaging Initiative (ADNI) subjects to be compared with our results.P = 0.0007) after adjustment for the covariates age, gender and education, ApoE and creatinine levels. The correlation remained almost the same  if phenotype is also added as a covariate. The association between both measurements was independent of cognitive status. The left hemisphere entorhinal cortex also correlated with Aβ(1–40) cell-bound fraction. AB128 and ADNI plasma Aβ measurements were not related to any brain morphometric measurement.The left hippocampus was the brain region most correlated with Aβ(1–40) bound to blood cell pellets  = −0.37, Association of cell-bound Aβ(1–40) in blood with left hippocampal volume was much stronger than previously observed in Aβ plasma fractions. If confirmed, this observation will require careful interpretation and must be taken into account for blood amyloid-based biomarker development. Alzheimer’s disease (AD) is the most frequent cause of dementia in Western societies. Neuropsychological evaluation remains the most useful tool for the diagnosis of AD and mild cognitive impairment (MCI) . HoweverAlthough Aβ levels in plasma have been widely investigated as a potential biomarker for AD evaluation, the development of a blood-based test to diagnose AD has remained elusive. Therefore, no definitive plasma or blood biomarker that can be used to indicate high or low risk of conversion to AD has been confirmed to date . ImportaOur group is actively involved in the development of novel sandwich ELISA colorimetric tests for detection of Aβ using whole blood instead of plasma alone ,13]. In. In13]. In this work, we aim to establish a relationship between blood Aβ levels obtained using these novel ELISA techniques and brain morphometry measured using MRI in healthy and cognitively impaired individuals. We postulated that if the blood Aβ load is associated with AD, those biomarker levels could be related to brain regions of interest (ROIs) previously defined for AD. Therefore we undertook a data exploration to identify blood Aβ measurement methods which correlated with brain volume.The study included 96 participants divided into three clinical groups based on their cognitive status, comprising 33 patients with amnesic MCI, 14 patients with AD and 49 HCs. AD, HC and MCI criteria used to recruit subjects in this study are described in our earlier work . BrieflySubject demographic characteristics are listed in Table Blood samples from each participant were drawn on the morning after an overnight fast and were collected in polypropylene vials with ethylenediaminetetraacetic acid and a protease inhibitor cocktail . The samples were immediately cooled to 4°C until processing, which occurred within 24 hours after collection. Blood samples were centrifuged, and both the plasma and the cellular pellet (CP) were divided into aliquots and stored in polypropylene tubes at −80°C until analyzed. The material was not thawed or refrozen at any time. All samples were analyzed in triplicate in the same run for each of the three blood fractions using two specific sandwich ELISA kits, ABtest 40 and ABtest 42 , as described elsewhere . MR. MR26]. ln was calculated as i runs over individuals and 〈Aβ〉 is the median value for the fraction. This new quantity characterized the amyloid fraction distributions in plasma for the present study.All the statistical procedures were carried out using R software . Becauseln values with age, gender and ApoE, education and creatinine levels [Pearson partial correlations were calculated between the MRI measurements for 45 ROIs given in the Freesurfer subcortical atlas and different Aβe levels -30] as  as ln valn values and cortical volumes and cortical thickness average for 68 ROIs in the Freesurfer cortical atlas. There was no correction for multiple comparisons in any case.Pearson partial correlations adjusted for the covariates age, gender and ApoE, education and creatinine levels were also calculated between Aβln values and also between the 68 Freesurfer cortical atlas ROI volumes and thickness average values and Aβln values. No correction for multiple comparisons was done.The whole procedure was repeated for both data sets, AB128 and ADNI, taking age, gender and ApoE, education and creatinine levels and phenotype as covariates. Partial correlations were calculated between the 45 Freesurfer subcortical atlas ROI volumes and Aβln CP Aβ(1–40) values and more correlated brain ROIs was conducted. The subjects were divided into phenotype groups , and each group was analyzed separately. Linear regressions were performed between Aβln CP Aβ(1–40) and left hippocampal volume in each case.An additional analysis was performed to determine the association between Aβln values for each amyloid fraction and MMSE score.Finally, Pearson partial correlations adjusted for the covariates age, gender and ApoE, education and creatinine levels were also calculated between Aβln compartments with age, gender and ApoE, education and creatinine levels were taken as covariates  fraction , as shown in Table ln fraction that remained significant when associated with left hippocampal volume was again the Aβln CP Aβ(1–40) fraction.Partial Pearson correlations were calculated for 45 ROIs contained in the Freesurfer subcortical atlas, and Aβln values and 68 ROI volumes from the Freesurfer cortical atlas and thickness average segmented in Freesurfer  and left hemisphere entorhinal thickness average  emerged as the most significant cortical measurements for Aβln CP Aβ(1–40).The same procedure was repeated between the plasma Aβln CP Aβ(1–40) fraction  for subcortical volumes, as shown in Table P =0.008) was the most significant of the cortical volumes  and thickness average  for Aβln CP Aβ(1–40).Pearson correlations were repeated, but this time also including phenotype as a covariate  adjusted for the covariates age, gender, ApoE, education and creatinine levels, and no association was found. In addition, no association was obtained when the analysis was adjusted for the covariates age, gender, ApoE, education, creatinine levels and diagnostic category. These results are fully compatible with our findings in TP. The partial correlations between Aβln values and MMSE scores showed no correlation between both magnitudes.Stratification analyses by phenotypic groups suggested that each diagnostic group had a similar correlation with left hippocampal volume in terms of effect size and significance. In other words, the observed correlations cannot be attributed to any specific cognitive subgroup, as shown in Figure The major finding of this study is that cell-bound Aβ was correlated with left hippocampal volume, a major area of AD pathology. Therefore, even when cell-bound Aβ levels do not distinguish HC, MCI and AD satisfactorily , they arln CP Aβ(1–40) level shown in Figure ln CP Aβ(1–40) level.On the other hand, the relationship we found seems to be independent of subject phenotype. Indeed, the similarity between the correlations with and without phenotype as covariates points out that the relationship to hippocampal volume is purely physiological. The same conclusion can be extrapolated from the slopes of linear regressions between left hippocampal volume and Aβln CP Aβ–40 levelad hoc analysis showed that blood CP Aβ(1–40) level could be used as a suggestive proxy for hippocampal volume and thus could be a useful screen for AD, even if it must be used with other biomarkers. Further studies are necessary to validate this working hypothesis.The unexpected association between CP Aβ(1–40) level and left hippocampal volume was examined exhaustively and, after a statistical analysis, proved to be consistent enough to be reported. This newly established relationship deserves further research. Even when replications were needed, the The major limitation of this study is the sample size. The study in fact involved a modest number of subjects. Only independent replications will be able to help clarify whether the observed associations and statistical significance are related to true findings or to random statistical oscillations. If we can finally confirm this observation, our findings may open new avenues to developing an AD biomarker related to the intrinsic properties of Aβ bound to blood cells. This finding might also have several physiological and pathological implications beyond the scope of this study. Another important limitation of the study is that no correction for multiple comparisons was done. Therefore, further studies are necessary to clarify the effect and function of Aβ peptides bound to different blood cells.The cell-bound Aβ(1–40) fraction was found to correlate with volume in the left hippocampus, a major site of AD pathology. However, other plasma Aβ fractions did not correlate with hippocampal volume. This suggests a different behavior, biochemically, for the traditionally measured Aβ(1–40) and Aβ(1–42) biomarkers and the CP Aβ(1–40) fraction. This newly established relationship deserves further research. Blood CP Aβ(1–40) may prove to be a useful screening test for AD, even as part of a composite biomarker.AD: Alzheimer’s diseaseADNI: Alzheimer’s Disease Neuroimaging InitiativeApoE: Apolipoprotein EAβ: Amyloid-βCDR: Clinical Dementia RatingCP: Cellular pelletCSF: Cerebrospinal fluidDA: Directly accessibleELISA: Enzyme-linked immunosorbent assayHC: Healthy controlMCI: Mild cognitive impairmentMMSE: Mini Mental State ExaminationMRI: Magnetic resonance imagingROI: Region of interestRP: Recovered from plasmaTP: Total in plasmaAR is a shareholder of Neopharm Obesity and Oxigene. PP, ISJ, VPG and MS are employees of Araclon Biotech Ltd. MS and ISJ are shareholders of Araclon Biotech Ltd. MBo is a consultant to Novartis and Esteve Pharmaceuticals; she is supported in part by FIS/EC 11-358 and FIS/P 10-00945 funds from Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain (AATM/390-6-2009), Generalitat de Catalunya ; and she is a member of Advisory Boards of Grifols, Lilly, Elan, Nutricia, Genentech and Roche. OSG, SV, IH, AL, MBu, MI, MAT, JG and LT have nothing to disclose. This work was funded by Araclon Biotech And Fundació ACE Memory Clinic and was also supported by the Spanish Ministry of Health through Instituto de Salud Carlos III (Madrid) (FISS PI10/00954) and by Agència d’Avaluació de Tecnologia i Recerca Mèdiques, Departament de Salut de la Generalitat de Catalunya (grant 390). Fundació ACE Memory Clinic is a CIBERNED-associated site.MS, MBo, LT, ISJ, PP, AR and OSG conceived of and designed the experiments. PP, ISJ, VPG, MAT, JG, AL, MBu, MI and IH performed the experiments. OS, SV, AR, PP, MBo, LT and MS analyzed the data. PP, VPG, SV, OS and AR contributed reagents/materials/analysis tools. OS, AR, PP, MS, MBo and LT wrote the manuscript. All authors read and approved the final manuscript.Excel spreadsheet with partial correlations adjusted for the covariates age, ApoE, gender, education and creatinine levels. (a) Partial Pearson correlations between amyloid-β fractions and volume Freesurfer subcortical ROIs. (b) Partial Pearson correlations between amyloid-β fractions and volume of Freesurfer cortical ROIs. (c) Partial Pearson correlations between amyloid-β fractions and cortical thickness of Freesurfer cortical ROIs.Click here for fileExcel spreadsheet with partial correlations adjusted for the covariates age, ApoE, gender, education, creatinine levels and phenotype. (a) Partial Pearson correlations between amyloid-β fractions and volume Freesurfer subcortical ROIs. (b) Partial Pearson correlations between amyloid-β fractions and volume of Freesurfer cortical ROIs. (c) Partial Pearson correlations between amyloid-β fractions and cortical thickness of Freesurfer cortical ROIs.Click here for file


TNF) –308G>A and nitric oxide synthase 3 (NOS3) +894G>T polymorphisms and migraine. We performed a meta-analysis of case-control studies to evaluate whether the TNF –308G>A and NOS3 +894G>T polymorphisms confer genetic susceptibility to migraine.Conflicting data have been reported on the association between tumor necrosis factor  and 95% confidence intervals (95% CI) assuming allele contrast, dominant model, recessive model, and co-dominant model as pooled effect estimates.We performed an updated meta-analysis for TNF –308G>A and six studies in 1055 migraineurs and 877 controls for NOS3 +894G>T were included in the analysis. Neither indicated overall associations between gene polymorphisms and migraine risk. Subgroup analyses suggested that the “A” allele of the TNF –308G>A variant increases the risk of migraine among non-Caucasians . The risk of migraine with aura (MA) was increased among both Caucasians and non-Caucasians. Subgroup analyses suggested that the “T” allele of the NOS3 +894G>T variant increases the risk of migraine among non-Caucasians .Eleven studies in 6682 migraineurs and 22591 controls for TNF –308G>A polymorphism may act as a genetic susceptibility factor for migraine among non-Caucasians and that the NOS3 +894G>T polymorphism may modulate the risk of migraine among non-Caucasians.Our findings appear to support the hypothesis that the  Migraine is characterized by recurrent, moderate to severe, throbbing headache attacks, associated with photophobia, phonophobia, nausea, and vomiting. Migraine is remarkably common, affecting approximately 11% of the adult population around the world . This coThe etiology of migraine is complex, involving both multiple genetic and environmental factors . It is nSterile meningeal inflammation is a key mechanism that may underlie the sustained activation and sensitization of perivascular meningeal nociceptors. Cytokines and nitric oxide (NO) have been implicated in the pathogenesis of migraine in both animal and human studies .Tumor necrosis factor-alpha (TNF-α) is an important cytokine that can promote powerful hyperalgesia by causing prostanoid release, increasing nerve growth factor (NGF) and bradykinin receptor expression, or by modulation of activity within sympathetic fibers. Several studies have indicated changes in serum , plasma NO is a potent endogenous vasodilator, which is a key molecule affecting the pain associated with migraine , as it wl-arginine and molecular oxygen by the NO synthase (NOS) family. In endothelial cells, NO is synthesized by the endothelial NOS (eNOS). Deregulation of eNOS influences the plasma level of NO, which has an anti-inflammatory effect. The haplotype of the NOS3 gene is associated with variability in endogenous NO formation  OR “migraine disorders” [All Fields] OR “migraine” [All Fields] OR “migraine disorder” [All Fields] OR “headache disorders” [MeSH Terms] OR “headache disorders” [All Fields] OR “headache” [All Fields] OR “headache disorder” [All Fields])) OR “migraine with aura” [MeSH Terms] OR “migraine with aura” [All Fields] OR “migraine without aura” [MeSH Terms] OR “migraine without aura” [All Fields]) AND  AND  OR “variant” [All Fields] OR “variants” [All Fields] OR “rs1800629” OR “TNF –308G>A”) AND “humans” [MeSH Terms]. In addition, the reference lists of selected papers and potentially relevant studies in the previous meta-analyses were also screened to further identify potentially relevant papers through reference association. The last search was updated in July 2014. The selection process is shown as a flow chart in Two of the authors (MC and WT) searched the PubMed, EMBASE, Science Citation Index (SCI), and Cochrane Library electronic databases for tumor necrosis factor and endothelial nitric oxide synthase  AND “headache” OR “headache disorder” OR “migraine” OR “migraine disorder” OR “migraine with aura” OR “migraine without aura” AND “gene” OR “polymorphism” OR “genetic variation” OR “polymorphisms” OR “rs1800629” OR TNF –308G>A or NOS3 +894G>T polymorphism and migraine risk based on a case-control or cohort design, and (ii) that contained information on the sample sizes, distribution of genotypes, and allele frequencies for the polymorphisms investigated among migraineurs and non-migraineurs allowing for calculation of crude risks for migraine. In publications with overlapping cases and/or controls, the most recent or largest population was chosen.The meta-analysis included only articles (i) that evaluated the association of the In the first step of the analysis, two of the investigators (MC and WT) independently identified all studies not meeting any of the pre-specified criteria by screening the titles and abstracts, and these studies were excluded. In the second step, the same investigators independently evaluated the full-text publications of the remaining studies. Studies were excluded if they did not meet all of the criteria. Any discrepancies were adjudicated by a third reviewer (ZD).Studies were excluded if one of the following existed: (1) studies with insufficient genotyping data of the participants; (2) case-only studies; (3) review articles; (4) animal studies; (5) editorials; (6) abstracts; (7) studies that reported associations between gene polymorphisms and recovery from a migraine attack; (8) studies that reported associations between gene polymorphisms and response to any therapy; (9) studies that deviated from Hardy—Weinberg equilibrium.Two of the investigators (MC and WT) independently reviewed, extracted, and entered data from the studies in a standardized data extraction form. Discrepancies were resolved by consensus. The items recorded were authors’ names, year of publication, ethnicity of subjects, study design, genotyping method, migraine status , diagnostic criterion of cases, gender of individuals in the study population, study size, allele and genotype frequencies, and additional potentially relevant information. We attempted to collect genetic information for all migraineurs and migraineurs with and without aura separately as well as for the whole study population and for females and males separately. If not presented in the article, allele and genotype frequencies were calculated when possible. For studies that did not allow extraction of all relevant information, including genotype and allele frequencies, we e-mailed the corresponding authors to obtain the missing information. Authors not responding within 1 week were sent up to two reminders by e-mail.http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp). The quality of each study was assessed and awarded stars based on indicators of quality, including selection of study population, comparability, and exposure assessment independently. The quality score ranged from 0 (worst) to 10 points (best). Any discrepancies were adjudicated by a third reviewer (LH).The quality of studies was assessed by the same two reviewers (MC and WT) independently using a modified 10-point Newcastle—Ottawa Scale (NOS) , dominant model , recessive model , and allele contrast . Heterogeneity across studies was detected by the χ2 test-based Q-statistic (P < 0.10 was deemed as evidence of heterogeneity) as well as I2 statistic, which takes values between 0% and 100%  [All analyses were performed using STATA version 12.0 . Unless otherwise stated, P < 0.05 was taken to indicate statistical significance. Hardy—Weinberg equilibrium (HWE) for each study was determined using the goodness-of-fit geneity) . When thgeneity)  was applgeneity)  was perfTNF –308G>A [NOS3 +894G>T [TNF –308G>A and NOS3 +894G>T [TNF –308G>A, we excluded one abstract [NOS3 +894G>T, one was excluded because no information about the distribution of genotypes was provided [TNF –308G>A and NOS3 +894G>T [TNF –308G>A was available but not that for NOS3 +894G>T. MC and WT independently read the articles, and 11 studies were finally included for TNF –308G>A and 6 studies were included for NOS3 +894G>T. Kappa statistic for agreement of TNF –308G>A between the two reviewers was 0.9, and that for NOS3 +894G>T was 0.86. Any discrepancies were adjudicated by a third reviewer (ZD). –308G>A , 44–46,  +894G>T , 47–52,  +894G>T . After fabstract . Anotherabstract . Among t +894G>T , informaTNF –308G>A, 7 [NOS3 +894G>T, six reported genotype and allele frequencies for all participants and for overall migraine as well as those stratified by aura [Among the 11 papers focusing on 08G>A, 7 , 37, 44 08G>A, 7 , 45. Aft08G>A, 7 . Among t by aura –51, 53,  by aura , 47. We TNF –308G>A polymorphisms [NOS3 +894G>T polymorphisms [The detailed characteristics of each study included in the meta-analysis are presented in orphisms , 44, 45 orphisms , 55, 56.TNF –308G>A, all studies were of a case-control design [NOS3 +894G>T, all studies were of a case-control design, and all studies were performed in adults. Three studies were performed in Caucasians [For l design , 44–46 el design . One stul design , and thel design , 45, 46.l design , 36, 46 l design , 34, 45.ucasians , 53, 56 ucasians , 51, 55.TNF –308G>A and NOS3 +894G>T among migraineurs and controls are listed in The distributions of genotypes and alleles for TNF –308G>A and NOS3 +894G>T polymorphisms, respectively.Tables TNF –308G>A with 6682 genotyped migraine cases and 22591 controls, no significant association between TNF –308G>A polymorphism and migraine risk was observed under any of the genetic models. Strikingly, the meta-analysis provided an OR of 1.74  under A vs. G, an OR of 1.82  under AA+GA vs. GG, and an OR of 1.78  under GA vs. GG among non-Caucasians, while none of the contrast models showed a significant association in Caucasian populations , as shown in By pooling the six selected studies with 1055 genotyped migraine cases and 877 controls for TNF –308G>A polymorphism and MA . Nevertheless, the reverse effects were observed in studies of the TNF –308G>A polymorphism and migraine without aura (MO). Using Galbraith plots, we identified individual studies as important sources of heterogeneity [There was no significant heterogeneity among studies of ogeneity , 51, as ogeneity , 30, 31.TNF –308G>A, P = 0.707 and 0.508, respectively, for NOS3 +894G>T)  Fig  and 6B.The overall results of this meta-analysis did not indicate associations between any of the polymorphisms investigated and migraine. However, the associations differed by ethnicity and clinical phenotype.TNF –308G>A polymorphism, stratified analysis by ethnicity suggested an increased risk of migraine among non-Caucasians. When stratifying the data by type of migraine, we observed an increased risk of MA among both non-Caucasians and Caucasians, but especially among non-Caucasians.Among studies investigating the NOS3 +894G>T variant increased the risk of migraine among non-Caucasians, which was driven by associations for MA. Assuming an allele T frequency of 10.64% among controls and using the TT vs. GT+GG genetic model, this study had about 84.3% power at a significance level of α = 0.05 to detect an effect size of 1.84 among non-Caucasians [Subgroup analyses suggested that the T allele of ucasians .NOS3 +894G>T genotype GT have an increased risk of allodynia [“Neurogenic inflammation” around the dural trigeminal afferents is known to play an important role in the generation of migraine attacks  and to cllodynia .TNF promoter has been shown to be associated with a high level of TNF-α expression [HLA alleles, which have high linkage disequilibrium with TNF. Individuals carrying the “A” allele as part of the HLA Al, B8, DR3 haplotypes, which are population-specific within the human MHC region [The levels of TNF-α and NO are under genetic control. An “A” at position –308 in the pression , 62. A fC region , may havC region , in whicTNF –308G>A and NOS3 +894G>T polymorphisms with predisposition to migraine have been examined in a number of studies, which reported inconsistent results. Due to the failure of independent association studies in identifying a role for the TNF –308G>A polymorphism in migraine, two meta-analyses were performed to reach definitive and reliable conclusions. Gu et al. [TNF –308G>A polymorphism with migraine based on six studies in Asian populations (985 cases and 958 controls); their results indicated that the TNF –308G>A polymorphism was associated with migraine risk in Asians. Subgroup analysis suggested that there was a statistically significant result for MA but not for MO. Another meta-analysis conducted by Schuerks et al. [TNF –308G>A polymorphism indicated no overall association with migraine. Subgroup analyses suggested that the “A” allele of the TNF –308G>A variant increases the risk of migraine among non-Caucasian populations, which was driven by associations for MO. In our study, subgroup analyses suggested that the risk of migraine with aura (MA) was increased among both Caucasians and non-Caucasians. Performance of a genome-wide association study (GWAS) enables identification of genetic variants in many multi-pathogenic diseases. To date, neither the TNF –308G>A nor the NOS3 +894G>T polymorphism has been included in GWAS.The associations of the u et al.  performes et al.  based onTNF –308G>A or NOS3 +894G>T polymorphism and migraine risk. There are two possible explanations for the observed overall lack of associations. Either, there may be really be no association and the positive results in some studies may have occurred by chance or due to certain study characteristics, or the pattern of association may be more complex and involve additional factors. In this context, considerations such as the heterogeneity among studies, different sources of controls, and variable sample sizes among the studies must be addressed.Despite their plausible associations with migraine, this meta-analysis revealed no general associations between the TNF –308G>A polymorphism with migraine and MO, which may have been due to differences between subgroups. Furthermore, assuming that the frequency of the “A” allele is 2.74% among controls and using the A vs. G genetic model, this study had only about 61.5% power at a significance level of = 0.05 to detect an effect size of 1.74 among non-Caucasians [NOS3 +894G>T, not all information was available, which would have been valuable to draw clear associations with migraine among populations of different ethnicities.Specifically, there were moderate to high degrees of heterogeneity among the studies investigating the association of the ucasians . To achiInherent bias, such as sampling bias, selection bias, publication bias, and within-study bias, cannot be avoided in a meta-analysis of observational studies. The following biases must be taken into consideration when interpreting the results of the present study. First, all of the studies included in the meta-analysis were published in English, so selection bias may have occurred. Second, we did not include fugitive literature, including academic dissertations and conference papers, so there may have been bias in provision of data. Third, there was a lack of unified sources of controls. The controls were selected from three sources: hospital-based, healthy population, and family members of the migraineurs, so selection bias may have occurred especially among the studies with hospital-based controls. Fourth, there was heterogeneity across the studies, which may have masked significance in the present study. Another limitation of our study was that we did not analyze the gene-to-gene or gene-environment interactions that may contribute to modification of migraine risk due to a lack of data among the existing publications eligible for this meta-analysis.TNF –308G>A polymorphism in the TNF region may represent a risk for migraine among non-Caucasians and MA among both Caucasians and non-Caucasians. The NOS3 +894G>T polymorphism of eNOS may represent a risk for migraine among non-Caucasians. However, given the heterogeneity among study designs, the results of this meta-analysis should be interpreted with caution. Further cohort studies based on pedigree are warranted to evaluate population-specific effects, including population stratification.In summary, the general results of our meta-analysis do not support a strong overall effect, but provide statistical evidence that the S1 Checklist(DOCX)Click here for additional data file.S2 Checklist(DOC)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 FigTNF –308G>A. B: S1B Fig Galbraith plot for NOS3 +894G>T.A: S1A Fig Galbraith plot for (EPS)Click here for additional data file.S1 TableMA: migraine with aura; MO: migraine without aura(DOCX)Click here for additional data file.S2 TableMA: migraine with aura; MO: migraine without aura(DOCX)Click here for additional data file.


To address these competitive requirements, the use of error-correcting codes is advised. Current barcoding systems are mostly built from short random error-correcting codes, a feature that strongly limits their multiplexing accuracy and experimental scalability. To overcome these problems on sequencing systems impaired by mismatch errors, the alternative use of binary BCH and pseudo-quaternary Hamming codes has been proposed. However, these codes either fail to provide a fine-scale with regard to size of barcodes (BCH) or have intrinsic poor error correcting abilities (Hamming). Here, the design of barcodes from shortened binary BCH codes and quaternary Low Density Parity Check (LDPC) codes is introduced. Simulation results show that although accurate barcoding systems of high multiplexing capacity can be obtained with any of these codes, using quaternary LDPC codes may be particularly advantageous due to the lower rates of read losses and undetected sample misidentification errors. Even at mismatch error rates of 10 Molecular barcoding provides the opportunity to multiplex next-generation sequencing  capacityN are first screened to ensure the satisfiability of chemistry constraints imposed by the target sequencing technology, e.g., barcodes designed for pyrosequencing platforms must avoid homopolymer regions. Candidate barcodes are then screened to ensure a minimum pairwise distance dmin that guarantees the unambiguous correction of dmin and the number M of legal barcodes must be accepted  was considered.Barcoding systems built from binary BCH, binary Golay, quaternary LDPC and BY barcodes were evaluated using a Quaternary Symmetric Channel (QSC) model . Under tollowing , 55, psN was limited to 25 bases. For each barcoding system of size N built with an error correcting code of size n, a wide range of error correction and multiplexing abilities were evaluated. This was accomplished by varying parameter t of binary BCH codes and parameter m of quaternary LDPC codes. For BCH barcodes, binary BCH codes of size n ∈ {15, 31, 63} and shortened versions of them were considered. For LDPC barcodes, quaternary LDPC codes of size n ≥ 16 were considered. LDPC codes of size n < 16 were disregarded due to difficulties in satisfying the mandatory LDPC sparse constraint. In addition, BY barcodes of size N ∈ {7, 8, 15} and Golay barcodes of size N = 12 were considered. For the sake of completeness, random barcodes reported in [N ∈ {8, 9, 10} and minimum edit (Levenshtein) distance dL ∈ {3, 5, 7} were also considered. Recalling that the Hamming distance is an upper bound of the edit distance, random barcodes were further screened to determine their minimum Hamming distance dH. Similar values were observed, i.e., dH ∈ {3, 5, 7} so that the correction of at least t ∈ {1, 2, 3} mismatch sequencing errors can be guaranteed.For practical purposes, orted in  of size M, their barcoding rate B, their probabilities pe of barcode identification errors and their probabilities pu of undetected multiplexation errors. For each N, M was defined as the maximum number of barcodes which were compatible with the given sequencing chemistry. Similarly, B was defined as the actual fraction of informative quads per barcode, i.e., B is close as possible to Barcoding systems were evaluated through their multiplexing capacity QSC type that generates mismatch sequencing errors with a probability ps. For a given set of sequencing chemistry constraints, the M and B accomplished by BCH and LDPC barcodes of size N will depend on the desired pe and pu for the given ps. With main focus on boosting experimental scalability without compromising multiplexation accuracy, BCH and LDPC barcodes of size N ≤ 25 able to fulfill the operational constraint M > ≥ 24 and pu ≤ 10−8 at ps = 10−2 were identified. For BCH barcodes, simulation results showed that the desired operational constraint could be only satisfied by shortening binary BCH codes of size n = 63. For LDPC barcodes, the desired operational constraint could be only satisfied by LDPC barcodes of size N ≥ 19.Let us consider an ideal sequencing channel of the N = 21 can be used to multiplex up to M = 86 samples with pe ≈ 10−5 and pu ≈ 10−8. By letting N to increase up to 25, one additional satisfactory configuration with pu ≈ 10−8 can be identified at N = 22 with M = 384 and pe ≈ 10−5. Note that for pu ≈ 0, pe essentially bounds the probability of read losses. Taking into account that the number of Illumina reads per flow cell currently ranges from 25 × 106 to 300 × 109, we may be interested in further pe reductions.As shown in pe ≈ 10−6, shortened BCH barcodes of size N at least 24 are required. BCH barcodes of size N = 24 can be used to multiplex up to M = 73 samples with pe ≈ 10−6 and pu ≈ 10−9. By letting N = 25, one additional satisfactory configuration with pu ≈ 10−9, M = 295 and pe ≈ 10−6 can be obtained. Details about the pe performance of BCH barcodes beyond ps = 0.01 are shown in Simulation results showed that to accomplish N = 19 can be used to multiplex up to M = 65 samples with pe ≈ 10−5 and pu ≈ 10−9. By letting N to increase up to 25, three additional satisfactory configurations with pu ≈ 10−9 can be identified at N = 21, 23, 24 with M = 210, 648, 1911 and pe ≈ 10−6. To further reduce pe in one order, LDPC barcodes of size N ≥ 23 are required. LDPC barcodes of size N = 23 can be used to multiplex up to 56 samples with pe ≈ 10−7 and pu ≈ 10−9. By letting N = 25, one additional satisfactory configuration with pu ≈ 10−9, M = 118 and pe ≈ 10−7 can be obtained. Details about the pe performance of LDPC barcodes beyond ps = 0.01 are shown in As shown in N = 7, 8 ) per iteration, being m = n − k the number of redundant symbols of the regular non-binary LDPC in GF(q) and j the number of non-zero entries per column of the LDPC matrix [q = 16 [N = 19 allowing the multiplexation of up to M = 65 samples using a quaternary regular  LDPC code with j = 3, the recovery of 12M identities in an Illumina MiSeq platform with a 177 MIPS processor using a maximum of 50 iterative decoding steps would take less than an hour.In this paper, LDPC barcodes demultiplexing was performed with the iterative decoding algorithm used for decoding non-binary LDPC codes for magnetic recording applications . Hence, C matrix , 61. Thi [q = 16 , is amen [q = 16 . For exanon-binary LDPC codes. Specifically, concatenated watermark codes [non-binary LDPC code and an inner sparse code, to which a watermark sequence has been added, could be used. Concatenated watermark codes rely on the ability of the inner decoder to transform insertion/deletion errors into mismatch errors and on the ability of the outer non-binary LDPC decoder to correct them. Regarding DNA barcoding applications of concatenated watermark codes, we expect samples identities are first mapped to hexadecimal strings, that these strings are LDPC encoded with a short hexadecimal LDPC code already optimized for transmissions over a quaternary symmetric channel, that hexadecimal LDPC codeword symbols are mapped to the quaternary sequencing layer with a non-linear quaternary sparse code and that resulting sparse quaternary sequences are finally perturbed with the addition of a well-known quaternary pseudorandom sequence defined as the pilot watermark signal. After this non-trivial processing of samples identities is performed, candidate barcode sequences able to deal with mismatch and indel sequencing errors could be obtained. We are currently putting together these pieces, looking forward to the design of concatenated watermark barcodes for the third generation Single-Molecule Real-Time (SMRT) long-read sequencing technology [Along this paper we have restricted our attention to sequencing errors of the mismatch type. Readers might reasonable argue that challenging sequencing errors are those dominated by insertions and deletions. We note, however, that these errors might be also tackled with short rk codes  built frchnology , for whichnology . We thuschnology  sequenciq = 4u and u ≥ 1. Let Γ be a bipartite graph with n left nodes called message nodes and m < n right nodes called check nodes. Let us map one-to-one the n message nodes of Γ to the n coordinates of q − ary codewords c = , ci ∈ {0, 1, 2, …,q−1}, i = 1,…,n. Provided the sum of neighboring positions for all check nodes among neighboring message nodes is zero in GF(q), Γ defines a q-ary linear code of size n able to carry k = n − m informative symbols. Thus, the code structure can be dissected into m component parity subcodes. In addition, if Γ is sparse, i.e., each message node is constrained by j < < m check nodes and each check node constraints v < < n message nodes, the code turns to be an LDPC code in GF(q). Finally, if n ⋅ j = m ⋅ v holds, a regular LDPC code is obtained.Let us start with a brief revision of LDPC codes in GF(q), ci and their noisy observations ri, i = 1, …, n. Rectangles are used to represent parity constraints over codeword symbols. Edges are put between codeword symbols and parity constraints. Legal LDPC codewords must fulfill the complete set of m parity constraints. On regular LDPC codes, any codeword symbol participates in exactly j ≥ 3 parity constraints. Rectangles are also used to represent probability functions pi = P(ri = a∣ci = b) modelling the transmission channel, i = 1…n. For a QSC model, pi = 1−p and a ≠ b holds. Iterative decoding provides estimates p and ri, i = 1,…,n , si ∈ {0, 1, 2, 3}, i = 1,…,k, by means of c = s ⋅ G.The construction of barcodes from quaternary LDPC codes is straightforward once Γ is given. Formally, Γ is described by the so-called parity check matrix D score to discriminate between prospective good and bad short quaternary LDPC codes, 20 sets of 50 random quaternary LDPC matrices were built from binary PEG templates with m rows, n columns and j = 3 non-zero entries per column. For each set, the best D-scoring code was selected and its barcoding performance assessed at ps = 10−2. The Spearman correlation coefficient S2 was then used to evaluate the correlation between D scores and the empirical log10pe accomplished by selected LDPC codes. Moderate negative associations were observed suggesting that the D score was indeed useful for the identification of prospective good short quaternary LDPC codes for barcoding purposes. For example, the observed correlation at ps = 10−2 for LDPC barcodes of size N = 24 carrying k = 5 informative quads was S2 = −0.52, p-value < 0.05. Practically, prospective good short quaternary LDPC codes were selected from sets of 1000 random instances. For each selected H, the corresponding generator matrix G with k rows and n columns was computed using the constraint G ⋅ Ht = 0. This was accomplished by means of an adaptation of I.V. Kozintsev software [H matrices in GF(q) using Gaussian elimination.To shed light into the ability of the software  for the QSC model. Corrupted BCH barcodes were first deinterleaved, mapped to the binary domain using inverse mapping tables and decoded with the implementation of the Berlekamp-Massey decoding algorithm in [GF(2) were then mapped to GF(4) to recover original barcode sequences. A similar procedure was used to recover corrupted Golay, LDPC and BY barcodes. Golay barcodes were first deinterleaved, mapped to the binary domain and then decoded with the implementation of the arithmetic decoding algorithm in [ps of a base mismatch used as input to the QSC channel model. BY barcodes were first deinterleaved and then decoded as indicated in [Simulation experiments were performed to analyze the robustness of BCH, Golay, LDPC, BY and Random barcodes over the rithm in . Decodedrithm in . LDPC barithm in  and implrithm in . The LDPcated in . Finallycated in  with exppe of barcode identification error was then estimated by Montecarlo simulation. For this purpose, T = 100 random samples comprising C = 107 barcode sequences were used. Samples were obtained by performing sampling with replacement over the sets of valid barcode sequences obtained after the Barcrawl filtering stage. For each sample Si, the proportion pe,i of barcodes identification errors and sample variances i = 1, …, T. At the end, the pooled sample mean pe estimations and thus, 95% confidence intervals for the case pu of undetected multiplexation errors.The probability ere used . A base g10sepp¯e. A similM and the barcoding rate B are fundamental properties of any coding-based barcoding system. They follow from counting all candidate barcode sequences that are compatible with a predefined set of DNA manipulation constraints. For random barcodes of size N, 4N candidate barcode sequences must be first individually screened to remove those with undesirable composition patterns. Concerning their posterior use for error correction purposes, remaining sequences must be then globally analyzed to ensure a predefined minimum distance. Hence, for random barcodes, the estimation of M and B scales exponentially with N both in time and memory. On the other hand, the minimum distance is a built-in property of candidate barcode sequences when systematic error correcting codes are used and thus, only the presence of undesirable composition patterns must be controlled. For barcodes of size N built from linear codes of size n able to carry k informative symbols in GF(q), just qk sequences must be individually screened, k < < n. For modest k and q settings, e.g., k < 8 for q = 4, exact estimation of M and B can be accomplished. For larger k settings, a Montecarlo approach is required.The multiplexing capacity


Midichloriaceae has been described within the bacterial order Rickettsiales. It includes a variety of bacterial endosymbionts detected in different metazoan host species belonging to Placozoa, Cnidaria, Arthropoda and Vertebrata. Representatives of Midichloriaceae are also considered possible etiological agents of certain animal diseases. Midichloriaceae have been found also in protists like ciliates and amoebae. The present work describes a new bacterial endosymbiont, “Candidatus Fokinia solitaria”, retrieved from three different strains of a novel Paramecium species isolated from a wastewater treatment plant in Rio de Janeiro (Brazil). Symbionts were characterized through the full-cycle rRNA approach: SSU rRNA gene sequencing and fluorescence in situ hybridization (FISH) with three species-specific oligonucleotide probes. In electron micrographs, the tiny rod-shaped endosymbionts (1.2 x 0.25–0.35 μm in size) were not surrounded by a symbiontophorous vacuole and were located in the peripheral host cytoplasm, stratified in the host cortex in between the trichocysts or just below them. Frequently, they occurred inside autolysosomes. Phylogenetic analyses of Midichloriaceae apparently show different evolutionary pathways within the family. Some genera, such as “Ca. Midichloria” and “Ca. Lariskella”, have been retrieved frequently and independently in different hosts and environmental surveys. On the contrary, others, such as Lyticum, “Ca. Anadelfobacter”, “Ca. Defluviella” and the presently described “Ca. Fokinia solitaria”, have been found only occasionally and associated to specific host species. These last are the only representatives in their own branches thus far. Present data do not allow to infer whether these genera, which we named “stand-alone lineages”, are an indication of poorly sampled organisms, thus underrepresented in GenBank, or represent fast evolving, highly adapted evolutionary lineages.Recently, the family  Rickettsiales belongs to the Alphaproteobacteria and exclusively comprises obligate intracellular bacteria including causative agents for serious human diseases, such as Rickettsia rickettsii (Rocky Mountain spotted fever), and Rickettsia prowazekii (epidemic typhus) [Rickettsia, Anaplasma and Ehrlichia that stirred up the interest in this group. Later, the discovery of their close relationship to mitochondria fueled speculations on their phylogeny and evolution [Rickettsiaceae” or Rickettsia-like organisms (RLO) inhabiting non-haematophagous hosts opened further perspectives in this field, both from the evolutionary and ecological points of view [Rickettsiales will not only provide missing links needed to resolve the intricate evolutionary patterns within Rickettsiales per se and enlighten their role as partners in numerous symbiotic systems, but also broaden our knowledge of host-symbiont interaction and its development during evolution.The order  typhus) –3. For mvolution –6. Studi of view –10. StudRickettsiales comprises the families [Rickettsiaceae, with the genera Rickettsia, Orientia, Occidentia [Candidatus (Ca.) Megaira” [Ca. Cryptoprodotis” [Ca. Arcanobacter” [Ca. Trichorickettsia” and “Ca. Gigarickettsia” [Anaplasmataceae, with the genera Anaplasma, Wolbachia, Ehrlichia, Neorickettsia [Aegyptianella [Ca. Neoehrlichia” [Ca. Xenohaliotis” [Ca. Xenolissoclinum” [Ca. Midichloriaceae” (Midichloriaceae from now on). Recently, Midichloriaceae have been recognized as a clade or even a putative family by several authors [Holosporaceae, is presently debated. It fell at the base of Rickettsiales evolution in several phylogenetic trees based on SSU rRNA gene analyses  Rickcidentia , “CandidMegaira” , “Ca. Crrodotis” , “Ca. Arobacter” , “Ca. Trkettsia” ; ii) Anackettsia ,16, Aegytianella , “Ca. Nerlichia” , “Ca. Xealiotis” , and “Caoclinum” ; and iii authors ,21–25 an authors . The staes e.g. –30), and, andRickbacteria ,31–33. Wny e.g. ,26,30). ,30. RickRickettsiales show differences in their host range. Up to now, members of the Anaplasmataceae have been only detected in animals (Metazoa), thus suggesting a certain host specificity . The fatebrates , have betebrates . Rickett leeches ) and Holuclearia ); in Arcuclearia –39 and huclearia ; in SAR uclearia ,10,14 anuclearia ; in Excauclearia ,43. In pciliates ,45, cnidciliates ,47 and gciliates ,39, indiMidichloriaceae, with “Ca. Midichloria” as a type genus, revealed a striking biodiversity in the last years. Midichloriaceae as a whole show a wide host range. They can invade not only different arthropods including ticks, fleas, bed bugs, seed bugs and gadflies [Trichoplax adhaerens [Similarly, the recently described family gadflies ,48–51, bdhaerens  and cniddhaerens ,52. Assodhaerens ,54 and mdhaerens ,56, incldhaerens ,51,57, wdhaerens  and Cilidhaerens ,59,60. Idhaerens ,26) but Rickettsiaceae and Midichloriaceae, are found at various trophic levels of the food chain, suggesting the theoretical possibility of horizontal transfer of the bacteria from one host to another by trophic interaction. Though not yet proven for all Rickettsiales, recent findings support the idea of possible host shifts in some Rickettsiaceae [Midichloriaceae (i.e. “Ca. Defluviella procrastinata” in Paramecium nephridiatum and “Ca. Cyrtobacter zanobii” in Euplotes aediculatus) support the notion of the independent establishment of different symbiotic systems involving Midichloriaceae and ciliates during evolution [Rickettsiaceae and Midichloriaceae to terrestrial habitats by arthropods. Taking into account the frequent occurrence of Midichloriaceae in haematophagous ticks [Midichloriaceae towards vertebrates [Representative hosts of both, tsiaceae ,10. Datavolution ,60,61. Pus ticks ,23,62–66us ticks , it is otebrates ,56,57. TMidichloriaceae family. It was recently discovered in a Paramecium species collected from a wastewater treatment plant in Rio de Janeiro (Brazil). According to the taxonomic rules for uncultivable bacteria [Ca. Fokinia solitaria”. New insights into the evolutionary pattern of Rickettsia-Like Organisms are also discussed.In the present study we provide an ultrastructural, molecular and phylogenetic description of a novel bacterial endosymbiont representing a new solitary branch within the bacteria , we propParamecium strains Rio ETE_ALG 3VII, 3IX, 3X and 3XI were isolated from the wastewater treatment plant Estação de Tratamento de Esgoto Alegria  in February 2012. Sampling permission was provided by the State water and sewage company CEDAE . Monoclonal cultures (strains) were established and maintained at 22 ± 1°C in 0.25% Cerophyl-medium inoculated with Enterobacter aerogenes [The erogenes . Accordierogenes  and eukaerogenes  the hostParamecium cells were starved overnight in sterile Volvic water, washed again six times to minimize bacterial contamination and fixed in 70% ethanol. DNA was extracted applying the NucleoSpin® Plant DNA Extraction Kit , following the CTAB protocol for mycelium.Total DNA extraction was performed according to the following protocol: approximately 50 cells were washed by six successive transfers into sterile mineral water ; Alphaproteobacteria specific forward primer 16Sα_F19b 5'-CCTGGCTCAGAACGAACG-3' [For the molecular characterization of the endosymbiont, bacterial SSU rRNA genes were amplified with the GAACG-3'  and the GAACG-3'  using a GAACG-3'  at GATC m = 54.5°C), Fokinia_434 5'-ATTATCATCCCTACCAAAAGAG-3'  and Fokinia_1250 5'-ACCCTGTTGCAGCCTTCT-3' . Tm was determined by Eurofins GMBH  that synthetized the probes. FISH experiments using one of the species-specific probes in combination with the almost universal eubacterial probe EUB338 (either FITC- or Cy3-labeled [in silico determined using the TestProbe tool 3.0 (SILVA rRNA database project [10.6084/m9.figshare.2008524).In order to verify that the sequenced bacterial SSU rRNA gene amplicon derived from the endosymbiont, three species-specific probes were designed: Fokinia_198 5'-CTTGTAGTGACATTGCTGC-3'  were pe project ) and the project ) allowin project  and figsTM AF1  containing DAPI, and examined using the fluorescence microscope Nikon Eclipse Ti .FISH experiments were performed to detect the presence of endosymbiotic bacteria. At least 20 cells were washed three times in sterile Volvic and placed on SuperFrost Ultra Plus® slides . Cells were fixed with 2% paraformaldehyde (PFA), dehydrated in an ethanol gradient and air-dried. Fixed cells were covered with hybridization buffer  containiAlternatively, to reduce autofluorescence background signal, cells were incubated for 30 minutes at 4°C in 2% PFA in depression slides, transferred to microscope slides and incubated again for 30 minutes at 4°C. The surplus liquid was removed. One drop of ice-cold 70% methanol was added, immediately removed and the slides were transferred into a washing chamber filled with 2x PBS at room temperature. Hybridization was performed applying 10 ng/μL of each probe in the hybridization buffer containing optimal formamide concentration see . The sliAlphaproteobacteria-specific probe ALF1b , the Al-labeled ) and theCa. Fokinia solitaria” was aligned with the automatic aligner of the ARB software package version 5.2 [Midichloriaceae family, 17 members of the Anaplasmataceae and Rickettsiaceae and 10 sequences representing the outgroup. The alignment was optimized manually especially focusing on the predicted base pairing of the stem regions, referring to the SSU rRNA structure of E. coli provided by ARB. The aligned sequences were then trimmed at both ends to the length of the shortest one; gaps were treated as missing data. The resulting alignment . Then the cells were dehydrated in an ethanol gradient followed by ethanol/acetone (1:1), 100% acetone, and embedded in Epoxy embedding medium . The resin was polymerized according to the manufacturer’s protocol. The blocks were sectioned with a Leica EM UC6 Ultracut. Sections were stained with aqueous 1% uranyl acetate followed by 1% lead citrate.Ciliates were processed for electron microscopy as described elsewhere . BrieflyNegative staining was performed by first washing and starving the cells overnight in distilled water to decrease the abundance of food and environmental bacteria. Single cells were then squashed with the micropipette and the remaining were transferred onto grids covered with the supporting film. Staining was performed using aqueous 1% uranyl acetate. All samples were examined with a JEOL JEM-1400  electron microscope at 90 kV. The images were obtained with an inbuilt digital camera.Paramecium clone Rio ETE_ALG 3VII was submitted to the GenBank database (NCBI) under the accession number KM497527 .The sequence obtained from bacterial SSU rRNA gene of the endosymbiont of Paramecium strains isolated from the wastewater samples were submitted to morphological analyses. General morphological features like size, body shape and location of the cytoproct were typical for the Paramecium caudatum-aurelia clade. However, species identification proved to be equivocal, suggesting the possibility that we were dealing with a new species. A detailed description of the host including morphometric, ultrastructural, and molecular characterization will be provided in a separate publication.The Ca. Defluviella procrastinata”, symbiont of Paramecium nephridiatum. It is noteworthy that, compared to the other Midichloriaceae included in the analysis, the SSU rRNA gene sequences of both “Ca. Fokinia solitaria” and “Ca. Defluviella procrastinata” had four small insertions (2–13 nucleotides long) in the same positions . These insertions were paired two by two in the predicted rRNA structure, increasing the length of two stems in regions V1 and V2 respectively. Other two small insertions  were present in “Ca. Fokinia solitaria” only, which were predicted to increase the length of a third stem in region V3 of the rRNA molecule.Nearly full-length bacterial SSU rRNA gene sequences were obtained for the three strains Rio ETE_ALG 3VII, 3IX and 3X. The sequences were identical, hence, strain Rio ETE_ALG 3VII was used representatively for all three strains . NCBI Blastn results against nucleotide collection (nr/nt) showed the highest identity  with an uncultured bacterium from a lake in New York (accession number FJ437943) and “Paramecium strains. Overlapping signals of both probes indicated the presence of endosymbiotic bacteria belonging to Alphaproteobacteria in the cell cortex. Bacteria localized in digestive vacuoles (food bacteria) showed positive signals only with probe EUB338.In preliminary FISH experiments, positive signals with both probes (EUB338 and Alf1b) were observed in the cytoplasm of all m that should have guaranteed good specificity without formamide or with low formamide concentrations. Hybridization experiments with their target organism in a formamide range from 0 to 30%, confirmed the signal intensity was best between 0–15% formamide  of these host strains .After the choice of the GTR+I+G substitution model with jModelTest, the ML and BI trees were estimated . The monre e.g. ,60). Onl. OnlRickCa. Fokinia solitaria” from Paramecium strain Rio ETE_ALG 3VII affiliated to Midichloriaceae and was strongly associated  to “Ca. Defluviella procrastinata” endosymbiont of P. nephridiatum  monophyletic branch. One of the sequences derives from an uncultured bacterium of a freshwater lake in New York , the other one belongs to “Ca. Defluviella procrastinata”, an endosymbiotic bacterium inhabiting P. nephridiatum [Ca. Fokinia solitaria” and “Ca. Defluviella procrastinata”, supports this presumption and suggests that this feature could be a shared derived character of the two genera. The sequence FJ437943 does not share any of the insertions and therefore seems to retain the ancient condition. Nevertheless, as the identity values among the three sequences (84.9–88.5%) are far below the taxonomic threshold for discriminating bacterial genera , the thMidichloriaceae, such as genera “Ca. Midichloria”, “Ca. Lariskella” and “Ca. Bandiella”, have been observed in a great variety of host species and with a worldwide distribution [Midichloriaceae it is not clear, when and how many times the adaptation to terrestrial animals took place  on one hand, and fast evolving “stand-alone” symbionts, such as Lyticum, “Ca. Defluviella”, “Ca. Anadelfobacter” and “Ca. Fokinia”, on the other hand. To refer to the characteristic of “Ca. Fokinia”, represented by an isolated branch, and in accordance with the guidelines of the International Committee of Systematic Bacteriology [Ca. Fokinia solitaria” in honor to our appreciated colleague Professor Sergei I. Fokin, a prominent specialist in the study of bacterial symbionts of ciliates.Overall, the family eriology , we propMidichloriaceae-endosymbionts of ciliates are rod-shaped but differ significantly in their size, “Ca. Fokinia solitaria” being one of the smallest. There are also remarkable differences in the intracellular localization of the endosymbionts. “Ca. Fokinia solitaria” and “Ca. Cyrtobacter comes” [Lyticum species and “Ca. Anadelfobacter veles” reside in host vesicles [Ca. Fokinia solitaria” shows a defined distribution, stratified in a narrow layer in the host cortex. This area is known to be devoid of acid phosphatase (AcPase) activity, indicating the absence of lysosomes and autophagosomes [Ca. Fokinia solitaria”, permitting it to avoid the host defense mechanisms, especially because it is not surrounded by a protective symbiont-containing vacuole. The occurrence of “Ca. Fokinia solitaria” in autolysosomes in the inner parts of the cytoplasm seems to support this view. Autophagy is not only a process of degrading macromolecules or organelles to provide nutrition during starvation periods; it is also involved in other biological processes like development and differentiation, cell death as well as immune system and protection against pathogens [Ca. Fokinia solitaria” in one of the sampled Paramecium strains may be the result of xenophagy and implies that the endosymbiont is not necessary for the host species and is treated as a pathogen. On the other hand, several pathogens were found to be able to avoid, subvert or even utilize the hosts autophagic machinery for replication [All so far discovered r comes”  are not vesicles ,59. Onlyagosomes ,96. The athogens –99. In mathogens ) like viathogens , bacteriathogens –104 and athogens . Hence, lication ,107 and lication .Ca. Fokinia solitaria” gave no evidence for the existence of flagella . After vailable ,125, sugRickettsiales not associated with pathogenicity for vertebrates emerged recently [Midichloriaceae corresponding to six new genera have been described, or respectively molecularly characterized for the first time, in ciliate model organisms Paramecium and Euplotes, i.e. “Ca. Defluviella procrastinata”, “Ca. Cyrtobacter comes” and “Ca. C. zanobii”, “Ca. Anadelfobacter veles” [Lyticum sinuosum and L. flagellatum [Ca. Bandiella wodruffii” [Ca. Fokinia solitaria”. This high rate of new species descriptions indicates a general high abundance of different Midichloriaceae species in ciliates and, possibly, in protists. It seems very likely that more descriptions of new Midichloriaceae will follow providing us a better understanding of their phylogenetic relationships and host-endosymbiont interactions.A big diversity of recently . In almor veles” ,60, Lytigellatum , “Ca. Badruffii” , and thiCandidatus Fokinia solitaria” . Short rod-like bacterium (1.2 x 0.25–0.35 μm in size). Cytoplasmic endosymbiont of the ciliate Paramecium sp. strain Rio ETE_ALG 3VII . Basis of assignment: SSU rRNA gene sequence (accession number: KM497527) and positive match with the specific FISH oligonucleotide probes Fokinia_198 (5'-CTTGTAGTGACATTGCTGC-3'), Fokinia_434 (5'-ATTATCATCCCTACCAAAAGAG-3') and Fokinia_1250 (5'-ACCCTGTTGCAGCCTTCT-3'). Belongs to Midichloriaceae family in the order Rickettsiales (Alphaproteobacteria). Identified in Paramecium sp. strain Rio ETE_ALG 3VII isolated from a wastewater treatment plant in Rio de Janeiro (Brazil). Uncultured thus far.“S1 AlignmentMidichloriaceae family, 17 members of the Anaplasmataceae and Rickettsiaceae and 10 other Alphaproteobacteria representing the outgroup were aligned with “Ca. Fokinia solitaria” to perform phylogenetic analyses. The alignment was trimmed at both ends to reach the length of the shortest sequence on each side, the resulting 1,568 nucleotide columns are presented here.22 closely related sequences of the (S1_ALIGNMENT)Click here for additional data file.S1 TableThe parameters are given as total tree length (TL), reversible substitution rates (r(A<->C), r(A<->G), etc), stationary state frequencies of the four bases (pi(A), pi(C), etc), the shape of the gamma distribution of rate variation across sites , and the proportion of invariable sites (pinvar). The estimated sampling size (ESS) is shown as minimal (minESS) and average (avgESS) values. PSRF stands for potential scale reduction factor.(DOCX)Click here for additional data file.