Besides their neuronal support functions, astrocytes are active partners in neuronal information processing. The typical territorial structure of astrocytes (the volume of neuropil occupied by a single astrocyte) is pivotal for many aspects of glia–neuron interactions.Individual astrocyte territorial volumes are measured by Golgi impregnation, and astrocyte densities are determined by S100β immunolabeling. These data are compared with results from conventionally applied methods such as dye filling and determination of the density of astrocyte networks by biocytin loading. Finally, we implemented our new approach to investigate age-related changes in astrocyte territories in the cortex and hippocampus of 5- and 21-month-old mice.The data obtained by our simplified approach based on Golgi impregnation were compared to previously published dye filling experiments, and yielded remarkably comparable results regarding astrocyte territorial volumes. Moreover, we found that almost all coupled astrocytes (as indicated by biocytin loading) were immunopositive for S100β. A first application of this new experimental approach gives insight in age-dependent changes in astrocyte territorial volumes. They increased with age, while cell densities remained stable. In 5-month-old mice, the overlap factor was close to 1, revealing little or no interdigitation of astrocyte territories. However, in 21-month-old mice, the overlap factor was more than 2, suggesting that processes of adjacent astrocytes interdigitate.Here we verified the usability of a simple, versatile method for assessing astrocyte territories and the overlap factor between adjacent territories. Second, we found that there is an age-related increase in territorial volumes of astrocytes that leads to loss of the strict organization in non-overlapping territories. Future studies should elucidate the physiological relevance of this adaptive reaction of astrocytes in the aging brain and the methods presented in this study might be a powerful tool to do so. Protoplasmic astrocytes are the primary glial cell subpopulation in the brain gray matter. Besides maintaining topographic relationships and structural integrity, these cells contribute to ion and transmitter homeostasis This raises the question of how the spatial arrangement of astrocytes is organized with respect to their neighbors. If every astrocyte occupies a defined volume of the neuropil, does this mean that no other astrocyte invades the same volume, or is there an overlap between the territories occupied by neighboring astrocytes? An overlap would ensure support of a given neural tissue compartment even when an astrocyte becomes dysfunctional. However, astrocytes avoid intense interdigitation with processes from neighboring cells by “tiling” – a process that results in territorial volume overlap of only 4–6% between adjacent astrocytes in adult mouse hippocampus Unfortunately, the data are sparse and were obtained from different brain regions, by different methods, from animals of different age, and even from different species, making comparisons difficult. The sparseness of the data is unsurprising, as intracellular dye filling of neighboring astrocytes and morphometric electron microscopy studies are sophisticated, time-consuming methods.To obtain information on the spatial arrangement of astrocytes in different brain regions requires reliable data on the number of astrocytes and the volume they access. Ogata and Kosaka We devised a simple, versatile method to assess the overlap between adjacent astrocyte territories. Specifically, the average volume accessed by a single astrocyte is measured on stacks of confocal images of Golgi-labeled astrocytes and multiplied by the number of astrocytes in a larger unit volume, counted on histological sections immunolabeled for an astrocyte-specific protein, S100β. This “total astrocyte-accessed volume” is divided by the unit brain volume. In the case of astrocyte tiling, the resulting overlap factor should be close to 1. Values higher than 1 would indicate sharing of territories between adjacent astrocytes, and values below 1 would indicate incomplete penetration of the whole neuropil by astrocyte processes.Using this method, we compared the organization of astrocyte territories in the hippocampus and cerebral cortex of mice at 5 months and 21 months of age. These territories might differ in the brains of adult and old mice, because astrocytes are thought to become hypertrophic with advancing age and because neurons in these two regions appear to be especially prone to age-related degeneration Female and male C57Bl6, 129Ola and 129Sv mice were used in the study. All experiments were performed in accordance with the European Communities Council Directive 86/609/EEC and German guidelines for welfare of experimental animals (Tierschutzgesetz) and were approved by the local authorities .All mice were perfused with 4% phosphate-buffered paraformaldehyde and decapitated, and the brains were removed. From each mouse, one hemisphere was processed with the Golgi method as described The other brain hemisphere of each mouse was postfixed in 4% paraformaldehyde, equilibrated with 30% phosphate-buffered sucrose, and cut into 30-µm-thick coronal frozen sections with a freezing microtome. For immunofluorescence labeling of the astroglial marker S100β, free-floating sections were incubated overnight with a rabbit anti-S100β antibody . The tissue was then incubated with carbocyanine (Cy) 2–conjugated donkey anti-rabbit IgG  for 1 h. Next, the remaining free binding sites of Cy2-anti-rabbit IgG were blocked with 50% rabbit antiserum for 3 h. The sections were then incubated overnight with digoxigenylated rabbit anti-GFAP antibodies (10 µg/ml) as described In control experiments, omission of primary antibodies resulted in the expected absence of cellular labeling. In addition, Cy3-anti-GFAP antibodies produced the same staining pattern as digoxigenylated anti-GFAP antibodies successively revealed by Cy3-anti-digoxin (data not shown).We focused on two brain regions: the neocortex  and the CA3 area (stratum moleculare) of the hippocampus. The volume accessed by a single astrocyte  was defined as the space over which the astrocyte processes extend, including the finest elaborations of the main branches. To calculate this volume, three-dimensional z-stacks from Golgi-impregnated brain slices were obtained with a Zeiss confocal laser-scanning microscope 510 Meta in reflection mode. Single cells were split into 1-µm-thick optical slices . The area of each cross-section was measured with Zeiss Image Analyzer Software. With this technique, multiple two-dimensional areas per slice could be calculated up to the cell's three-dimensional volume. Alternatively, the area of maximal extension of each astrocyte was recorded on maximum projections of an astrocyte. This area was treated as circular and used to calculate the volume of a theoretical “astrocyte globe” . FinallyTo calculate this factor, defined as the degree of tissue shrinkage after embedding, the thickness of the 20 embedded slices (included preparations from adult and aged mice) was measured and normalized to the thickness of the original slices (150 µm). Subtraction from 100% results in shrinkage of about 13.8%. Assuming that all structures shrink to about the same degree, we included this factor in our calculations, which led to slightly increased astrocyte volumes. Potential shrinkage from postfixation was not determined and might lead to a slight underestimation of the volume of astrocyte territories. Shrinkage associated with embedding was not considered for the immunohistochemical staining used to determine the astrocyte cell number, as we could count all astrocytes in the 30-µm-thick slice (frozen sections).To assess the complexity of the process architecture, we counted the branchings of all main processes up to 15 µm from the soma center of each astrocyte.3 as the counted number of astrocytes/[area where astrocytes were counted (mm2) x slice thickness (mm)].Immunohistochemically stained sections were analyzed for the number of cells expressing the calcium-binding protein S100β, a reliable marker of astrocytes in mammalian brain Next, we calculated the overlap factor for multiple astrocytes within the observed volume of brain tissue by multiplying the mean astrocyte territorial volume by the number of cells (estimated by counting S100β-positive cells), which showed us the whole three-dimensional space occupied by all astrocytes summed up. This number, divided by the volume of the measured brain tissue, results in the overlap factor, which indicates the extent to which cell processes of individual astrocytes overlap with each other.We applied Golgi-impregnation on murine brain slices and used a confocal microscope in reflection mode to examine the cells . The GolAdditionally, we compared the results of the present study with data from earlier dye filling experiments performed on adult mice ; Table 13 in the neocortex and 38,361±3174 µm3 in the hippocampus of adult mice (3) than in adult mice. Territorial volumes in hippocampal astrocytes were also more than twofold larger in old mice   . Thus, a174 µm3) .3 did not change with age in cortical or hippocampal slices   .Finally, to estimate the extent of interdigitation between processes of neighboring astrocytes, we calculated the overlap factor multplying the absolute number of astrocytes by the average astrocyte territorial volume and divided the result by the volume of the brain tissue studied. In adult mice, the resulting overlap factor was 0.47 in the cortex and 0.86 in the hippocampus . In old In this study, we established a fast, reliable method to quantitatively assess the morphology and spatial arrangement of astrocytes in different brain regions. Using this method, we showed that aging correlates with remarkable increases in astrocyte cell volume and in the overlap of astrocyte territories. The tiling of astrocyte territories led to a negligible overlap of individual astrocyte territories in adult brain, whereas both the size and overlap of the territories increased in old brains. This increase may be required to compensate for increased metabolic demands and decreasing functional capabilities of aged astrocytes.Previous work in this field focused on analyzing defined groups of astrocytes filled with different fluorescent dyes Our approach, based on Golgi staining and S100β immunohistochemistry, will make it possible to acquire comprehensive data sets from astrocytes largely independent of the brain region and of the heterogeneity of the astrocyte population since we found that data from dye filling experiments on astrocytes of adult mice Nonetheless, for both brain regions studied the resulting overlap factors in our study are significantly lower than 1.0 as had been assessed by earlier dye-filling experiments 333 (present study), to 43,400 µm33Considerable work has been done to characterize the spatial organization of protoplasmic astrocytes in brain gray matter. For example, the reported values of the average volume occupied by a hippocampal astrocyte, varies from 16,400 µmWe largely confirmed these data, as the overlap factor we calculated that was less than, but close to 1 in adult mice, both in the hippocampus and in the neocortex. Importantly, we found that age is a primary determinant of the mean astrocyte territorial volume in both regions. The territory infiltrated by the fine processes of an average astrocyte more than doubled between 5 and 21 months. Yet, the astrocytes showed few signs of reactive gliosis, such as a slight increase in the extension of their main processes (indicated by the increased size of their territories) and their enhanced branching. These findings do not necessarily contradict earlier studies consistently demonstrating an age-related upregulation of GFAP in astrocytes in general, and, specifically, in the hippocampus Since astrocytes are strictly organized into a network of non-overlapping territories in adult mice, it is tempting to speculate that this is the morphological equivalent of the functional units that glial cells build with the neuronal structures they enwrap It is less easy to explain the increase in the astrocyte territorial volume and, consequently, in the astrocyte overlap in old mice. Reactive astrocytes in murine epileptic brain show similar structural changes, including a 10-fold increase in the overlap factor 2O2It is not known whether the reorganization of astrocyte territories in epilepsy is the reason for or a consequence of neuronal hyperexcitability. In traumatic brain injury, both the astrocyte territories and the limited overlap of neighboring astrocyte territories are unchanged Taken together, we present a versatile, simple method to assess the volume of astrocyte territories and to determine astrocyte cell densities in various brain regions. First results obtained with this approach underline the importance to analyze animals of exactly known age if one wants to obtain information about whether and how, in addition to age, pathophysiological mechanisms affect the size and overlap of astrocyte territories.


Functional brain imaging studies have improved our knowledge of the neural localization of language functions and the functional reorganization after a lesion. However, the neural correlates of agrammatic symptoms in aphasia remain largely unknown. The present fMRI study examined the neural correlates of morpho-syntactic encoding and agrammatic errors in continuous language production by combining three approaches. First, the neural mechanisms underlying natural morpho-syntactic processing in a picture description task were analyzed in 15 healthy speakers. Second, agrammatic-like speech behavior was induced in the same group of healthy speakers to study the underlying functional processes by limiting the utterance length. In a third approach, five agrammatic participants performed the picture description task to gain insights in the neural correlates of agrammatism and the functional reorganization of language processing after stroke. In all approaches, utterances were analyzed for syntactic completeness, complexity, and morphology. Event-related data analysis was conducted by defining every clause-like unit (CLU) as an event with its onset-time and duration. Agrammatic and correct CLUs were contrasted. Due to the small sample size as well as heterogeneous lesion sizes and sites with lesion foci in the insula lobe, inferior frontal, superior temporal and inferior parietal areas the activation patterns in the agrammatic speakers were analyzed on a single subject level. In the group of healthy speakers, posterior temporal and inferior parietal areas were associated with greater morpho-syntactic demands in complete and complex CLUs. The intentional manipulation of morpho-syntactic structures and the omission of function words were associated with additional inferior frontal activation. Overall, the results revealed that the investigation of the neural correlates of agrammatic language production can be reasonably conducted with an overt language production paradigm. For each utterance the relevant concepts and their associated lemmas in the mental lexicon are retrieved. Besides the word, its morpho-syntactic features such as number, tense, grammatical gender and word class as well as the syntactic frame defining its possible structural environment are activated. In a morpho-syntactic encoding process the lexical items with their specific constraints are combined into a syntactic structure and an utterance is generated  many neuroimaging studies have shown correlates of single aspects of morpho-syntactic processing in the neurologically normal brain. Morpho-syntactic processes on word-level  have often been localized in the left pars opercularis and pars triangularis of the IFG in overt production and covert tasks ] is associated with reduced effects in the well-known predominantly left hemisphere network reported for syntax processing including pars triangularis and pars opercularis of the IFG, the Rolandic operculum and posterior temporal regions. This is applicable for the production as well as the planning of utterances.- Morphological errors in the healthy speakers are correlated with activation in the left pars triangularis and pars opercularis of the IFG and in the left MFG as well as in in additional brain areas subserving monitoring processes.- In the agrammatic speakers, effects for completeness, complexity and morphology show in similar left hemisphere activation networks as the respective effects in the healthy speakers with activation in preserved and perilesional tissue.- Agrammatic language production goes along with stronger effects in the areas described above when compared with unimpaired language production and language production in the healthy speakers.- Agrammatic language production is associated with enhanced right hemisphere activations when compared with unimpaired language production and language production in the healthy speakers.Participants gave their written informed consent before participating in the study, that was approved by the ethics committee of the Medical Faculty RWTH Aachen University (reference no. EK 040/07).Twenty subjects with no history of neurological, psychiatric or speech and language disorders participated in the study. All were native German speakers and presented with normal or corrected-to-normal vision. Five subjects had to be excluded from the study due to technical problems during fMRI image acquisition. The final experimental group consisted of 6 female and 9 male participants in the age range of 23 to 67 years (mean 43.53). Handedness was assessed by the Edinburgh Handedness Inventory , showing agrammatic speech production and being able to perform the picture description task using utterances above single-word level, participated in the study. To avoid movement artifacts, participants with severe apraxia of speech were not included. Further exclusion criteria were a history of psychiatric disease, premorbid language disorders or any contraindication for fMRI. The age ranged from 21 to 48 years (mean 39.2). All aphasic speakers presented with chronic moderate to mild Broca's aphasia [30–51 months post onset (mean 39.4)] secondary to a left hemisphere cerebral vascular accident affecting different brain regions with lesion foci in the insula lobe, inferior frontal, superior temporal and inferior parietal areas. The lesion overlap in Figure  woman, sIn experiments 1 and 2, spontaneous language was elicited using 9 black and white line drawings showing complex scenes like a market Figure . EarlierDuring fMRI the stimuli were presented via goggles  using Presentation 11.0 software . Each picture appeared in randomized order for 3 min, in which the participants described in detail what was happening in the picture. All pictures were followed by a blank screen for 55 s and subsequently a fixation cross for 5 s, to focus the participants' attention on the upcoming picture. During scanning verbal output was recorded using the headphone's microphone and Audacity 2.0.1, an open source program for recording and editing sound.Max. 3 Wörter (three words at most) was shown 5 s before the 1st, 3rd, 5th, 7th, and 9th picture appeared (3W-condition). Five seconds before the 2nd, 4th, 6th, and 8th picture the instruction on the screen was Natürlich beschreiben . Before scanning both conditions were practiced with one additional picture with the healthy participants. Feedback was given on the way of description to avoid deictic descriptions and labeling. Furthermore, it was made sure that the participant was able to accomplish the descriptions in the 3W-condition properly.There is a relation between utterance length and syntactic structure  using a Philips SENSE head coil eight Elements. For each participant 535 functional scans were acquired in 42 sagittal slices of 3.75 mm thickness without interslice gap covering the whole brain using a fast-field-echo gradient EPI sequence, a repetition time (TR) of 4000 ms, echotime of 30 ms, a flip angle (FA) of 90° and a 240 mm field of view (FOV). The in-plane resolution was 3.75 × 3.75 mm. The structural T1-weighted MP-RAGE images were taken with a 3D FFE sequence .Speech recordings were transcribed using ASPA , a CLU was classified as simple or complex, depending on whether or not it was part of a compound sentence. Beyond that each CLU was marked as complete or incomplete , a CLU was a simple sentence that was either complete or incomplete caused by a missing verb or by a missing obligatory argument like an object. Simple complete CLUs were further divided into morphologically correct or impaired CLUs, caused by at least one missing or wrong function word and/or inflectional element.To capture the conceptual and morpho-syntactic planning of the CLUs .On morphological level, morphological correct and impaired CLUs were distinguished. A CLU with an omission or substitution of a closed class word  was classified in one category. When the error concerned the inflection of an open class word, the CLU was further assigned to a second morphological category. Because one CLU can contain morphological errors concerning open and closed class words at the same time, a mixed category was included. Morphological errors that were corrected were nonetheless assigned to one of the morphological categories.The data were analyzed using SPM8  running on MATLAB 7.13 R2011b  in combination with the SPM Anatomy Toolbox  level, only conditions with at least 15 events were considered for statistical analysis  level. Results on completeness, complexity, and morpho-syntactic planning were calculated in a second level analysis enclosing the following categories: complex complete CLUs, simple complete CLUs, pauses/interjections between CLUs in the NAT-condition as well as simple complete CLUs, simple incomplete CLUs with a missing verb, pauses/interjections between CLUs in the 3W-condition. The second level analysis to calculate results on the morphological processing contained the categories: simple complete CLUs with morphological errors, pauses/interjections between CLUs in the 3W-condition as well as simple complete CLUs and pauses/interjections between CLUs in the NAT-condition.P < 0.05, a cluster extent of 14 contiguous resampled voxels was indicated as necessary in the control group assuming an individual voxel Type I error of P < 0.001.Specific effects were tested as linear t-contrasts. The calculated contrasts are listed in Table The aphasic participants' data were analyzed on single subject level in order to avoid problems associated with grouping data from patients with heterogeneous lesion sizes and locations and with small sample sizes .Specific effects were tested as linear t-contrasts  were extracted. SpmT-values indicate the intensity of activation at the given voxel. In the correspondent aphasic speakers' t-contrasts the spmT-values were extracted at the equivalents of the groups' local maxima and compared to the healthy speakers' maxima with a one-sample t-test.To find out if the brain regions showing a significant BOLD effect in the healthy speakers were involved in the processing of the same morpho-syntactic aspects in the aphasic speakers, the spmT-values at the healthy speakers' local maxima (voxels with activation peaks in the clusters of activity) of the t-contrasts corrected at cluster level  the average number of CLUs per picture reduced to 24.4. The aphasic speakers' performance is shown in Table P = 0.001; 3W < NAT: P = 0.001) and in the speech rate (words per minute) . The complexity was lowest in 3W and highest in NAT with significant differences between the three groups . The aphasic speakers showed a significantly lower percentage of words than the healthy speakers in both conditions  as well as a lower type token ratio . The percentage of open class words was significantly higher in 3W than in NAT and in the aphasic speakers .The statistical analysis of differences between the aphasic speakers' and the healthy participants' speech production in the 3W and NAT-condition revealed significantly lower values in the aphasic speakers' group (AS) and in 3W as in NAT for completeness  revealed an effect in the left pMTG extending to the angular gyrus (AG) in the inferior parietal cortex. In addition, a homologous cluster with a maximum in the pMTG showed that was smaller in extent though. Further activation clusters were found in the left hemisphere in the precuneus, thalamus, the paracentral lobule, and the inferior parietal lobule (IPL). In the right hemisphere effects showed in the anterior cingulate cortex, the middle occipital gyrus and the right MFG. See Figure There was no BOLD effect found for the reverse contrast contrasting incomplete with complete simple CLUs.An effect for complexity, i.e., the contrast of complex with simple CLUs  revealed deviant activation strength at at least four maxima in all aphasic speakers except A4 . In A1 and A3 deviant spmT-values were significantly lower, in A2 and A4 either significantly higher or lower than the healthy speakers' values. All maxima were affected. The spmT-value at the maximum in the IPL was significantly lower in two and significantly higher in the other two aphasic speakers. A4 had a lesion at six of the nine maxima. See Table The comparison of the spmT-values (indicating intensity and significance of activity) of the individual aphasic speakers with the spmT-values of the healthy group at the nine local maxima  for completeness reported in experiment 1  in the group of healthy speakers and the aphasic speakers, the spmT-values of this contrast were not compared.Results of aphasic speakers' fMRI data for the contrast of simple complete vs. simple incomplete CLUs with a missing verb are graphically presented in Figure An increased activation pattern for incomplete utterances with a missing verb was found in left and right frontal and in right temporal areas in A2. In A1 and A2 effects were found in left and right parietal regions. Effects in the cingulate cortex showed in A1, A2, A4, and A5, in the precuneus in A1 and A2. Overall, smaller differences in activation patterns were found in A3, A4, and A5.Only A1 showed a sufficient amount of CLUs that were complex and complete Table  to analyThe reverse contrast resulted in an effect for simple sentences in the left and right frontal cortex, the left temporal and occipital cortex and the precuneus.Figure When compared with morphologically correct CLUs, BOLD effects for morphological errors were found in frontal and temporal regions in A3 (LH) and A5 , who both primarily presented with substitutions of function words. Left hemispheric parietal activation appeared in A5. Additional clusters included the cingulate cortex in A2 (only omissions of function words) and A5 and the precuneus in A3 and A5. There was no BOLD effect found for A1 and A4 in this contrast, who both showed CLUs with omissions as well as substitutions of closed class words. A3 showed distinctly more effects for the morphological errors than in the reverse contrast for the correct CLUs.In the present fMRI study, the neural correlates of syntactic completeness, complexity and the production of function words in normal and disordered grammatical processing in continuous language production were studied with a new paradigm. In experiment 1, the neural mechanisms underlying natural morpho-syntactic processing in a picture description task were analyzed in 15 healthy speakers. In the same group, agrammatic-like speech behavior was induced to study the underlying functional processes. In experiment 2, five agrammatic participants performed the picture description task to gain insights in the neural correlates of agrammatism and the functional reorganization of language processing after stroke. In both experiments, agrammatic language production was contrasted with unimpaired language production. The main results shall now be discussed in detail.In experiment 1, non-brain-damaged participants described pictures with natural spontaneous speech on the one hand and with utterances with a reduced morpho-syntax (at most three words per utterance) on the other hand. By contrasting corresponding categories we were able to reveal those brain regions underlying three aspects of spontaneous speech production that are often impaired in agrammatism: syntactic completeness, complexity and the correct use of function words. All aspects were associated with activation in the left pMTG and the IPL. Complex and complete CLUs were associated with an additional effect in the left AG and SMG. The omission of function words was linked to an additional bilateral frontal effect in the pars triangularis and pars opercularis of the IFG, the Rolandic operculum and the MFG. These findings are in line with earlier studies on morpho-syntactic processing and give further insights into the neural mechanisms underlying spontaneous language production.Studies on sentence production reported stronger activation for syntactically more elaborated conditions, e.g., complete vs. incomplete CLUs . For this reason the intensity of activity at the healthy speakers' activation peaks  and the intensity of activity  in the aphasic speakers at the equivalent voxels of the groups' local maxima were compared. In every agrammatic speaker (except A4) and each contrast, at least four equivalents of the groups' local maxima showed deviant activation strength which was significantly increased at some and decreased at other maxima. Regarding the maxima that showed divergences the five aphasic speakers did not present with consistent activation patterns. One maximum could be associated with increased activation in the one and with decreased activation in the other aphasic speaker. Critically one can note that the healthy speakers' categories varied from the aphasic speakers' categories and thus the contrasts were not comparable. This is, to a certain amount, true for the 3W-categories used in the completeness contrast that were compared with the aphasic speakers' categories not underlying any limitations in utterance length. However, in both groups the contrasted categories differed only in one feature: the verb that was produced or not. In the healthy and the aphasic speakers the fMRI results for this contrast thus revealed the areas associated with completeness (verb production) in sentence generation. In the second comparison (complexity), the two contrasted categories contained only CLUs of the NAT-condition, where speakers described the pictures without a target utterance length just like the aphasic speakers did. Still the comparison of A1's spmT-values with that of the healthy speakers' revealed increased and decreased deviations at several maxima. In sum, the aphasic speakers showed individual activation patterns at the local maxima of the healthy speakers with deviant activation strength at atleast four maxima per contrast.This analysis suggests that the basis of morpho-syntactic processing in the lesioned brain varies from that in healthy speakers. To get further insight into the individual activation patterns, we contrasted brain activation correlated with agrammatic CLUs with activation of undisturbed speech production in the aphasic speakers. As in experiment 1, the three aspects of spontaneous speech production often impaired in agrammatism  were analyzed.In experiment 1, incomplete CLUs with a missing verb were associated with decreased activation. In experiment 2, two aphasic participants (A3 and A5) showed a similar activation pattern but with additional activation in areas not specifically involved in language processing. In two aphasic speakers (A1 and A2) incompleteness was associated with increased as well as decreased activation. In A4 differences in both directions did not show in language-specific areas. Only A1 produced enough complex complete CLUs to analyze the effects in activation between complex (with embedding) and simple CLUs. The production of simple sentence constructions was associated with decreased activation when compared with complex sentences in the group of healthy speakers. A1 showed the same pattern but with additional increased activation. The category morphological errors concerning open class words embraced different symptoms in the individual aphasic speakers: While A1 and A4 produced omissions and substitutions of function words, A2 showed primarily omissions  and A3 and A5 predominantly substitutions of function words. Omissions of function words were associated with increased activation in the healthy speakers but with primarily decreased activation in the aphasic speakers as was also found for the combination of omissions and substitutions. Substitutions however, were correlated with primarily increased activation in language specific areas. Thus, neither for the different symptoms nor for the individual aphasic speakers stringent activation patterns (decreased and/or increased) were found. In the literature aphasic errors have mostly been associated with an enhancement in activation in right and left hemisphere regions subserving language and monitoring processes  that has, however, been reported to be involved in syntactic or general language processing, monitoring and attention processes , causing the errors. These issues could be further investigated with repetitive transcranial magnetic stimulation (rTMS) or transcranial direct current stimulation (tDCS) applied to the identified areas (Devlin and Watkins, 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.


Determining the body fluids where secreted proteins can be secreted into is important for protein function annotation and disease biomarker discovery. In this study, we developed a network-based method to predict which kind of body fluids human proteins can be secreted into. For a newly constructed benchmark dataset that consists of 529 human-secreted proteins, the prediction accuracy for the most possible body fluid location predicted by our method via the jackknife test was 79.02%, significantly higher than the success rate by a random guess (29.36%). The likelihood that the predicted body fluids of the first four orders contain all the true body fluids where the proteins can be secreted into is 62.94%. Our method was further demonstrated with two independent datasets: one contains 57 proteins that can be secreted into blood; while the other contains 61 proteins that can be secreted into plasma/serum and were possible biomarkers associated with various cancers. For the 57 proteins in first dataset, 55 were correctly predicted as blood-secrete proteins. For the 61 proteins in the second dataset, 58 were predicted to be most possible in plasma/serum. These encouraging results indicate that the network-based prediction method is quite promising. It is anticipated that the method will benefit the relevant areas for both basic research and drug development. Protein secretion is a universal biological process occurring in all organisms. Secreted proteins such as hormones, digestive enzymes, neurotransmitters as well as antibodies, play vital regulatory roles in various biological activities such as reproduction, digestion, nerve conduction and immunization However, how to realize the identification is still a big challenge even having the advanced proteomics technologies because there are a large amount of proteins with a variety of modifications in body fluids According to a recent comprehensive review (1) Open the web-page at http://www.uniprot.org/ (Release 2011_05). (2) Click the button “Fields”, followed by selecting “Subcellular location” for (3) Click Add & Search. Thus we collected a total of 1,019 experiment-validated human secreted proteins. Subsequently, these proteins were mapped to 11 different kinds of body fluids contained in the human body fluid database “Sys-BodyFluid” http://lifecenter.sgst.cn/bodyfluid/), where the body fluid proteome data was manually collected from 50 peer-review publications. Finally, a total of 682 human proteins have been obtained that can be secreted into the aforementioned body fluids.The human secreted proteins were retrieved from UniProt http://string.embl.de/), which is a database dedicated to both physical and functional interactions. Information derived from 3 kinds of sources  was integrated into several PPI networks. As done by previous investigators in using the intuitive graphic representation to deal with complicated biological systems, such as enzyme-catalyzed system The human protein-protein interaction (PPI) networks were retrieved from STRING Of the 682 human secreted proteins, we have found that 153 proteins have no PPI information nor interact with any of the other secreted proteins, while 529 proteins interact with at least one of the other proteins in the human PPI network from STRING. Thus, we obtained a working PPI network that consists of 529 nodes (proteins) and 27,176 interaction units. Such 529 human secreted proteins in the newly constructed PPI network were used as the training dataset for developing the current network-based method.The distribution of the 529 human secreted proteins classified according to the 11 different types of body fluids is shown in Table S1).Two testing datasets were used in this study. The first one contains 57 blood-secreted proteins, which was obtained as follows. First of all, 305 blood-secreted proteins were retrieved from the positive dataset in Table S2) for the second testing dataset.The second testing dataset contains 61 proteins as obtained as follows. From Many interacting proteins must co-occur in the same location to participate in the biological processes Given a query protein, the higher interaction confidence score between it and its interacting counterpart, the more likely they are to be secreted into the same body fluid. Also, the more its interacting proteins in a certain body fluid, the more likely it is to be secreted into such body fluid n proteins in the PPI network as n proteins in the PPI network can be described asj-th body fluid j-th body fluid j-th body fluid First, let us denote the orth cf. Table 1.rd body fluid  will have the maximum likelihood (cf. th body fluid (Plasma/Serum) will have the second maximum likelihood, and that th body fluid (Urine) will have the third maximum likelihood. And so forth. The predicted results thus obtained are called the 1st order predicted body fluid, the 2nd order predicted body fluid, the 3rd order predicted body fluid, and so forth.Since many secreted proteins can be secreted into more than one body fluid, our method is dedicated to provide flexible information by predicting possible body fluids for secreted proteins, rather than the most likely body fluid. To realize this, let us sort the 11 elements of each row in Eq.4 according to descending order. By doing so, we obtain a hood cf. Table 1,(1) For the independent dataset test, although all the proteins used to test the predictor are outside the training dataset used to train it so as to exclude the “memory” effect or bias, the way of how to select the independent proteins to test the predictor could be quite arbitrary unless the number of independent proteins is sufficiently large. This kind of arbitrariness might result in completely different conclusions. For instance, a predictor achieving a higher success rate than the other predictor for a given independent testing dataset might fail to keep so when tested by another independent testing dataset (2) For the subsampling test, the concrete procedure usually used in literatures is the 5-fold, 7-fold or 10-fold cross-validation. The problem with this kind of subsampling test is that the number of possible selections in dividing a benchmark dataset is an astronomical figure even for a very simple dataset, as demonstrated by Eqs.28–30 in (3) In the jackknife test, all the proteins in the benchmark dataset will be singled out one-by-one and tested by the predictor trained by the remaining protein samples. During the process of jackknifing, both the training dataset and testing dataset are actually open, and each protein sample will be in turn moved between the two. The jackknife test can exclude the “memory” effect. Also, the arbitrariness problem as mentioned above for the independent dataset test and subsampling test can be avoided because the outcome obtained by the jackknife cross-validation is always unique for a given benchmark dataset. Accordingly, the jackknife test has been increasingly and widely used by those investigators with strong math background to examine the quality of various predictors  prediction accuracy is 79.02%, indicating that the 1st-order predicted body fluid for the secreted proteins is believable. The 11th-order (least likely) prediction accuracy is 6.99%, indicting that the likelihood that the query protein is secreted into the 11th-order predicted body fluid is very low and such predicted body fluid can be ignored.In this study, the network-based method was applied to the 529 human secreted proteins to predict the body fluids where they were secreted. All the 11 order jackknife cross-validation accuracies are shown in k (cf. Eq.10) was set to be 4 ([3.23]+1), i.e., we consider the first 4 order predicted body fluids from the 11 order prediction. The likelihood that the first 4 order predicted body fluids contain all the true body fluids that the proteins can be secreted into is 0.6294 according to Eq.10, indicating that the first 4 order predicted body fluids should be paid more attention to than others in the 11 predicted body fluids.The average number of body fluids that each secreted protein in the PPI network can be secreted into is 3.23 according to Eq.9. Hence, a random guess of body fluid for the secreted proteins will have a 29.36% (3.23/11) success rate, lower than the first 5 order prediction accuracies. The parameter Many important biological activities are mediated by proteins interactions. The interacting proteins should co-occur spatially and temporally to intact with each other Table S1). The 11 order prediction accuracies for the 57 blood-secreted proteins by the network-based method are listed in st prediction accuracy is 96.49%, i.e., 55 of 57 proteins were predicted to be secreted into plasma/serum in the 1st prediction. And the 2nd prediction accuracy is 3.51%, and all the other accuracies are 0. In other words, the first 2 predictions cover the secreted locations of all the 57 blood-secreted proteins. Apparently, the results indicate a good performance of the network-based method for secreted proteins in blood. Except the proteins in the training dataset and the 57 blood-secreted proteins, few secreted proteins in other body fluids have been found in the present researches. Therefore our method was evaluated on the blood-secreted proteins.Now, let us demonstrate the prediction method on an independent testing dataset that contains 57 blood-secreted proteins .The 61 possible marker proteins listed in Based on the quite promising results obtained through this study, we can now propos a way to discover disease biomarker in body fluids. After screening the proteins showing abnormal expression levels in various diseases and identifying their subcellular locations As is discussed above, the predicted body fluids of the first 4 orders can be regarded as the candidate locations of the secreted proteins. Biologists can focus on these body fluid candidates, which can save a lot of time and labor so as to accelerate the research progress. The predicted body fluids with the last 5 or 6 orders might be excluded for consideration owing to their low accuracies.Considering the effectiveness of the network-based method for human secreted protein, it is possible to apply the current method to predict the locations of secreted proteins in other species. The PPI network can be collected from numerous sources including STRING The performance of the network-based method can be further improved via the following two avenues. The first one is to collect the PPI data of high quality to exclude the false positive interaction, which was expected to improve the prediction accuracies. The second way is to collect as much PPI data as possible for constructing the PPI network, which was expected to make the method cover as many secreted proteins as possible.In this study, a multi-target model was developed for assigning the human secreted proteins to the body fluid categories based on the PPI network. Since it is the first computational method to annotate the body fluids where human protein can be secreted into, it is anticipated that the method will benefit the relevant experimental researches and stimulate a series of follow-up investigations into this emerging and challenging area.Table S1The 57 blood-secreted proteins used to test the network-based method.(DOC)Click here for additional data file.Table S2The 61 abnormally expressed proteins (possible biomarkers) used to test the network-based method that were involved with various cancers.(DOC)Click here for additional data file.


Objectives: To evaluate the effects of folic acid supplementation on isolated oral cleft recurrence and fetal growth. Patients and Methods: The study included 2,508 women who were at-risk for oral cleft recurrence and randomized into two folic acid supplementation groups: 0.4 and 4 mg per day before pregnancy and throughout the first trimester. The infant outcome data were based on 234 live births. In addition to oral cleft recurrence, several secondary outcomes were compared between the two folic acid groups. Cleft recurrence rates were also compared to historic recurrence rates. Results: The oral cleft recurrence rates were 2.9% and 2.5% in the 0.4 and 4 mg groups, respectively. The recurrence rates in the two folic acid groups both separately and combined were significantly different from the 6.3% historic recurrence rate post the folic acid fortification program for this population (p = 0.0009 when combining the two folic acid groups). The rate of cleft lip with palate recurrence was 2.9% in the 0.4 mg group and 0.8% in the 4 mg group. There were no elevated fetal growth complications in the 4 mg group compared to the 0.4 mg group. Conclusions: The study is the first double-blinded randomized clinical trial (RCT) to study the effect of high dosage folic acid supplementation on isolated oral cleft recurrence. The recurrence rates were similar between the two folic acid groups. However, the results are suggestive of a decrease in oral cleft recurrence compared to the historic recurrence rate. A RCT is still needed to identify the effect of folic acid on oral cleft recurrence given these suggestive results and the supportive results from previous interventional and observational studies, and the study offers suggestions for such future studies. The results also suggest that high dosage folic acid does not compromise fetal growth. Oral clefts are among the most common birth defects worldwide, with incidence ranging from 1/200 births in Amerindian populations to about ½,500 births in African populations and an intermediate incidence for European populations at about 1/700 births . Oral clThe etiology of oral clefts is complex involving genetic and environmental factors with both independent and interactive effects. Although much of the genetic etiology, particularly functional and developmental pathways, remain unknown, several genes/loci have been implicated in oral clefts ,7,8,9, wThere has been a wide interest in evaluating the role of maternal nutrition particularly the consumption of folate rich diets and use of folic acid supplements in oral cleft recurrence and occurrence. A detailed review of this literature is presented elsewhere . Severali.e., mother is affected or has had a previous child with oral clefts) is higher by about 40 times than overall prevalence in the general population . Several studies have evaluated the effects of folic acid supplementation, particularly high versus low doses on oral cleft recurrence. Most of these studies found significant protective effects of high doses—on average a decrease of 50% in recurrence . Howeveeview in , the eviversus low-dose folic acid supplementation on oral cleft recurrence among children of Brazilian women [The Oral Cleft Prevention Program (OCPP) was as a double blinded RCT to assess the effect of high-dose an women . We descThis study aimed at testing the effect of 4 mg folic acid taken daily before pregnancy throughout the first trimester by women who were themselves born with a cleft or had had a prior child with a cleft on recurrence of clefting in a subsequent child compared to taking the currently recommended 0.4 mg dose . A placeetc.). The folic acid pills were specifically manufactured for the study by ATIVUS Pharmaceutical Industries in Brazil. The manufacturer conducted quality control tests for each batch of pills including identification test of the raw folic acid material used in the pills (prior to production), assay tests for the folic acid dosage in the pills, content uniformity tests (to ensure that the dosage is consistent across pills), and dissolution tests to ensure that the pills dissolve adequately in a standard time.The study had a double-blinded randomized design, where both investigators and participants were blinded to the study group assignment . ParticiParticipants who had a delay in menstruation of 14 days or more at baseline underwent a pregnancy test before randomization; only those with a negative test were randomized [Participants confirmed to be pregnant were advised by the study staff to stop taking the study pills at the end of the first trimester of pregnancy . There wAfter delivery, all live births were followed in-person by the study staff to measure oral cleft status. Mothers also completed an interview with the study staff after delivery either in-person or over the phone to obtain data on perinatal and infant health outcomes . All intThe OCPP aimed at observing 1,582 births (791 births in each folic acid group) in order to achieve 80% power for a 50% reduction in a baseline rate of 5% recurrence. The study assumed a birth rate of 0.0957 births per participant-year, which implied that about 16,531 participant-years were required to observe the 1,582 births.All potential participants were screened for eligibility using the same data forms and inclusion criteria across all sites by trained and qualified study staff before they were enrolled . After cThe study was conducted at six craniofacial clinics in Brazil (see detailed list in online Appendix). The clinics were selected based on their long experience in providing care to patients with oral clefts and most of them are considered referral centers for oral cleft care. Participants were identified from the population of patients served by these clinics. Enrollment was first initiated in 2004 using an outreach model for the patient pool served by the first study clinic. In that model, the study staff conducted meetings with the study participants in their local communities during which data were collected and study pills provided. In 2005, a clinic-based recruitment model was initiated at two new sites, where participants were recruited and interviewed at the study clinic. The clinic-based model collected data from participants over phone interviews and mostly delivered study pills via express mail; in-person follow-ups were completed with the participant every 6 months when possible. In 2007, the first site switched from the outreach model to a clinic-based model, and two new sites were added under the clinic-based model. A sixth site following the clinic-based model was added in 2008.The study participants were women 16–45 years of age who have nonsyndromic/isolated oral clefts or had had at least one natural child of any age with nonsyndromic/isolated oral clefts who and who had received care at the study clinics . The stuSyndromic/non-isolated cleft status including cases with recognized syndromes, cases with a chromosome abnormality, cases with one or more other major structural anomaly, cases with cognitive delay , or cases exposed to phenytoin or valproic acid in utero.Any first degree relative  who has cleft palate only .The woman or her husband/partner was sterilized .Using intrauterine devices or injectible contraceptives (added in 2008).Using anti-epileptic drugs .Using drugs that contain benzodiazepines .Women who were pregnant at screening.Women who were planning to move outside of the catchment area of the study within the next year.12 deficiency as determined from testing participants’ blood samples in the study before supplementation (B12 level below 174 pg/mL or 134.328 pmol/L), which may be masked by folic acid.Women who had BWomen who were allergic to folic acid.Participants who met any of the following criteria at the time of screening were excluded from participation :Syndrom12 after randomization and a hematologist recommended that the woman be fully withdrawn from the study (no participants met this criterion), and taking epileptic drugs unless the participant is pregnant. B12 levels were assessed every 4 months between 2004 and 2006. After that, B12 levels were assessed once every 12 months.Participants who did not meet these criteria at enrollment because these were missed during the initial screening or the subject met these criteria after enrollment but were later found to meet the following exclusion criteria were discontinued from participation: definite sterilization of the woman and/or husband/partner, detecting BThe primary outcome of the study was the recurrence of isolated oral clefts, defined as the birth of a child with an oral cleft to the study women during their participation . This isThe study evaluated several secondary outcomes. The cleft recurrence rates in the OCPP were compared to historic recurrence rates. We calculated these rates by surveying women who are themselves affected or have had children affected with isolated cleft lip with/without cleft palate who were obtaining care from the same clinics involved in the OCPP but who did not enroll in the OCPP for reasons unrelated to cleft recurrence risk . Only cases with cleft lip with/without cleft palate were included in the historic recurrence survey; cases with cleft palate only were excluded. The study staff interviewed these women in 2011–2012 for their complete pregnancy history. We calculated the historic recurrence rates for the period after the initiation of the folic acid fortification program (after 2004) since the OCPP infants were born during this period and fortification may affect oral cleft recurrence. For affected women, the recurrence rate was defined as the proportion of affected children among all live births. For unaffected women with an affected child, the recurrence rate was defined as the proportion of affected children among all live births after the affected child.The severity of oral clefts, birth weight, gestational age, and preeclampsia were also evaluated as secondary outcomes. In addition, we report here a comparison of head circumference, length at birth, and Apgar scores between the study births in the two folic acid groups.t-test for continuous outcomes. A Wilcoxon rank-sum (or Mann-Whitney) test was also used for the continuous outcomes since they did not meet normality tests (except for head circumference). Fisher exact tests and student’s t-tests were also used to compare the baseline characteristics between the two folic acid groups. We compared the OCPP recurrence rates to the historic rates using a one-sample z-test for proportions.Outcomes were compared between the two doses of folic acid to which the women were assigned randomly at enrollment. Data for all women and infants were analyzed in the treatment arm to which the women were randomized, even when the women did not comply fully with the assigned treatment regimen as described above. We recognize that the sample size does not provide adequate power to identify moderate effects for small frequency outcomes when analyzing the live birth sample. Given the randomized design of the study, we compared the outcomes between the two folic acid groups using two-group comparison tests including a Fisher’s exact test for binary outcomes and a Student’s At the beginning of the study, there was no limit on how long a woman may participate. Participants were allowed to enroll again in the study in the same treatment group after their pregnancy if they wished to do so and if they still met all the eligibility criteria. Nine women participated a second time and had live births. In 2008, participation time was limited to three years. The study participants participated in the study until their delivery if they became pregnant or until they had completed three years of participation without becoming pregnant. After delivery, the participants were allowed to participate again if they had not completed a total of three years of participation in the study. We first report the analyses only for first-enrollments and first pregnancies. In additional analyses of cleft recurrence, we add women who participated a second time.Between January 2004 and May 2009, a total of 3,821 women were screened for eligibility into the study. Of these, 851 (22.3%) met one or more of the study exclusion criteria. Of the 2,970 eligible women, 2,508 women gave consent and were randomized into the study treatment and control groups; the 462 who were not randomized did not consent to participate in the study. Of the randomized women, 273 became pregnant during their first participation in the study. Of these pregnancies, there were 269 verifiable pregnancy outcomes ; 234 were live births, 33 were miscarriages, and two were still births. Of these live births, 225 live births occurred in families where the previous affected child or affected mother had cleft lip with/without cleft palate . Of the randomized women, 913 chose to discontinue their participation in the study before pregnancy, and five pregnant women were lost to follow up. On average, these women withdraw 20 months after randomization (ranging from 0 to 57). In 2009, the DSMB overseeing the study with the funding institute (NIDCR) recommended that all new recruitment be halted and enrollment of non-pregnant women participating in the study be ended due to the lower than anticipated enrollment and pregnancy rates, which implied that the sample of infants needed to achieve 80% power could not be reached over the planned project period. Therefore, the enrollment of all non-pregnant participants was terminated at all sites after that. All pregnant women were followed according to the protocol and their babies examined in person by a trained study staff while maintaining the blinding of the subject and study staff to the folic acid group assignment.p = 0.59 based on a one-sided Fisher’s exact test). The total recurrence rate combining the two folic acid groups was 2.7% (six affected out of 225 infants). One of the live births in the 4 mg group was diagnosed with Van-der-Woude syndrome (VWS), which is the most common syndromic form of oral clefting with a dominant genetic inheritance model [ce model  and aboup = 0.44). All three cleft cases in the 0.4 mg group were cleft lip with palate compared to one case in the 4 mg group; the rates of cleft lip with palate rates were 2.9% versus 0.8% in the 0.4 and 4 mg groups, respectively. Adding the births from families where the previously affected child or affected mother had cleft palate only or including the babies of mothers who participated a second time  has virtually no effect on the estimates of oral cleft recurrence as only a few cases are added as shown in Supplementary online VWS was not diagnosed when the mother of this case was screened for eligibility before enrollment and should have been excluded. Excluding the VWS case results in an isolated oral cleft recurrence rate of 1.6%  and compared them to the observed rates in p = 0.0009 when comparing the post-fortification expected recurrence rate to the observed recurrence rate in the combined group). We compared the OCPP rates first to the recurrence rate in the total historic group, and then to the recurrence rate in children exposed to the fortification program during the first trimester of pregnancy in order to account for any change in recurrence with fortification since women were randomized into the OCPP after the beginning of the fortification. The overall recurrence rate was 6.65% for having a prior affected sibling and 6.98% for affected mothers; these rates were fairly similar when restricting the sample to children exposed to the fortification program (6.47% for a prior affected sibling and 6.0% for affected mothers). Based on these historic recurrence rates and the proportions of affected women versus 3.7% in the 4 and 0.4 mg groups, respectively).p < 0.0001). Similarly, post-supplementation mean RBC folate levels were about 716 and 793 ng/mL in the 0.4 and 4.0 mg groups . Similar to serum folate, the post-supplementation mean RBC folate level was significantly higher in the 4 mg than 0.4 mg group (p = 0.0021).Based on the pill counts, median compliance was about 74% in both groups. The changes in blood folate levels also suggest good compliance with the study interventions. Based on a subgroup of participants with reviewed laboratory tests conducted at the laboratory at the study site at the Hospital de Clínicas de Porto Alegre for participants enrolled in the clinic-based model , mean post-supplementation serum folate levels were about 13.0 and 14.3 ng/mL in the 0.4 and 4 mg groups, respectively, representing an increase by about 1.7 and 3.1 ng/mL compared to the baseline levels (about 15% and 28%). The post-supplementation mean serum folate level was significantly higher in the 4 mg than 0.4 mg group  based on a two-sided chi-square test and 5% type-1 error. Therefore at the current sample size, the study does not have statistical power to detect a smaller difference (<6 percentage-points) in recurrence rates between the two folic acid groups; a larger sample is needed to achieve acceptable power. In contrast, there was a decline in recurrence in both OCPP groups separate and combined compared to historic control groups. The study has reasonable power (65%) to detect the observed difference in recurrence in the combined sample of 0.4 and 4 mg groups compared to the post-fortification historic recurrence rate reported in The study observes no difference in recurrence rates between the 4 mg and 0.4 mg groups. The small sample limits our ability to draw formal statistical inference on the effect of high dosage folic acid (4 mg) relative to low dosage (0.4 mg) on oral cleft recurrence based on comparing the recurrence rates between these two groups. With an observed recurrence rate of 2.9% in the 0.4 mg group, the current sample size provides 37% power to detect a 100% decrease—the maximum possible effect size —in recurrence in the 4 mg group based on a one-sided test  and a 5% type-1 error. However, at this sample size, the study has 80% power to detect a 6 percentage-point difference in recurrence between the two groups (The decrease in recurrence in the 0.4 mg group compared to the historic rate (similar to the 4 mg group) suggests that this dose may be effective in reducing cleft recurrence risk. However, more research is needed to test this hypothesis and quantify the effect. The relatively similar recurrence rates between the two folic acid groups may be due to the potential ineffectiveness of the higher dose in further reducing recurrence relative to the lower dose. This may also be complicated by the participants’ use of prenatal vitamins and folic acid supplements on their own as well as their dietary folate consumption. About 40% of the participants (based on data for 207 live births) reported using multivitamins and/or folic acid supplements other than those supplied by the study during the month before pregnancy and/or during the first trimester (37% in the 0.4 mg and 43% in the 4.0 mg). Even though these additional folate sources would elevate the folate levels in both the 0.4 and 4 mg folic acid groups, they may result in an average folate intake in the lower dose group that is markedly higher than the 0.4 mg per day.One limitation of the study is introducing some changes in recruitment strategies (such as limiting length of participation to 3 years) and inclusion/exclusion criteria  while the study was ongoing. Another limitation is the potential over-estimation of population recurrence risk based on the historic control groups since these were identified through craniofacial clinics providing care to patients with oral clefts. Therefore, women who have experienced a recurrence may have been overrepresented. This highlights the need to calculate these rates using population-based registries; however, these resources are relatively underdeveloped in Brazil and are currently not available for such efforts. These limitations should be considered when evaluating the generalizability of the study results.The study is the first to shed some light using a double-blinded randomized design on effects of high dosage folic acid on fetal development. The results suggest that high dosage folic acid does not compromise fetal growth or increase perinatal risks. These findings are consistent with previous observational study results suggesting positive effects of prenatal folic acid supplementation on development ,17, and


To date, genome-wide association studies have yielded discoveries of common variants that partly explain familial aggregation of diseases and traits. Researchers are now turning their attention to less common variants because the price of sequencing has dropped drastically. However, because sequencing of the whole genome in large samples is costly, great care must be taken to prioritize which samples and which genomic regions are selected for sequencing. We are interested in identifying genomic regions for deep sequencing using large multiplex families collected as part of earlier linkage studies. We incorporate linkage analysis into our search for Q1-associated alleles. Overall, we found that power was low for both whole-exome and linkage-guided sequencing analysis. By restricting sequencing to regions with high LOD peaks, we found fewer associated single-nucleotide polymorphisms than by using whole-exome sequencing. However, incorporating linkage analysis enabled us to detect more than half of the associated susceptibility loci (52%) that would have been identified by whole-exome sequencing while examining only 2.5% of the exome. This result suggests that incorporating linkage results from large multiplex families might greatly increase the efficiency of sequencing to detect trait-associated alleles in complex disease. Linkage studies have fallen out of favor in recent years as genome-wide association has become the new paradigm for gene discovery. However, genome-wide association itself is perhaps reaching its limit, because the price of sequencing has decreased and is likely to drop much further. At this point, the cost of whole-genome sequencing is still high enough that great care must be taken to select which samples or genomic regions to sequence. Much of this sequencing will not include newly collected samples but will use samples from existing studies, either of the case-control or pedigree variety. We are interested in the potential of large multiplex families , obtained as part of linkage studies, to guide subsequent sequencing efforts. This analysis could be done either by identifying highly informative individuals to sequence, by directing the analysis to gain greater power, or by prioritizing certain regions for deep sequencing rather than taking a genome-wide approach.In this paper, we explore the utility of linkage analysis of large pedigrees to prioritize certain genomic areas for sequencing. This method can be viewed as an extreme case of guiding an analysis for greater power . Of courIn this study, we compute the variance component logarithm of odds (LOD) scores for Q1 and Q4 for all 200 replicates provided in the Genetic Analysis Workshop 17 (GAW17) data set. The median heritability for the 200 simulation replicates is 58% for Q1 and 63% for Q4. We then examine the power of the 17 truly associated Q1 SNPs by controlling the type I error inferred from the association results with 218 unassociated SNPs for Q4 because the simulation model does not include any truly associated SNPs for Q4. This allows us to compare power and type I error rates for two sequencing strategies: (1) whole-exome sequencing followed by association tests on all SNPs detected from the whole exomes and (2) targeted sequencing of exomes under linkage peaks followed by family-based association tests using polymorphisms in these linked regions.For each of the 200 GAW17 simulation replicates, we used the 697 individuals from 8 families for linkage and association analyses. We did not split the large multiplex families into small families. We performed genome-wide variance components linkage analysis . We incoFor the association analysis, we computed residuals from a linear model that included Age, Sex, and Smoking status for traits Q1 and Q4 and used the residuals in subsequent association analyses. From the simulation model, we selected 17 SNPs that were truly associated with Q1. A set of 218 SNPs, including 201 SNPs that were not associated with Q1 and the true Q1 SNPs (17 SNPs), was tested for association with Q4. Each SNP was coded as 0, 1, or 2 with respect to the number of minor alleles and was used as a covariate in the RELPAL program in S.A.G.E. (version 6.0) [ikΥ is the trait value of individual i in pedigree k, ikX is the design vector for fixed effects for individual i , B is the coefficient vector of fixed effects, ikZ is the design vector for within-pedigree random effects, b is the coefficient vector for pedigree-specific covariates and polygenetic effects, and the ike are individual-specific random effects assumed to be independently and identically distributed [where tributed . SignifiWe evaluated the power for association using Q1 and type I error using Q4. Because we found unexplained genotype correlation across chromosomes, the unassociated trait Q4 was an appropriate choice to calculate the type I error. To address the problem of multiple testing, we applied two adjustments for significance thresholds using the Sidak correction:N is the number of statistical tests. First, an adjustment was based on the total number of SNPs  analyzed in the whole exome. The second adjustment was based on the number of SNPs under the 1.5-LOD support interval for regions with a LOD score greater than 3.3 in each of the 200 replicates. We applied the significance threshold for linkage signal at a LOD of 3.3 for a conservative genome-wide significance level [p-value threshold ranging from 0.05 to 2.81 × 10−6). P-values outside the LOD support area are set to 1 and are therefore never considered significant. That is, true Q1 risk alleles that are not under a support interval with a peak LOD score greater than 3.3 are not carried forward for association analysis in the linkage-guided strategy and thus are considered false negatives.where VEGFA and C4S4935 in VEGFC. After correcting for the number of SNPs tested in the genome-wide approach, we found that the power to detect both of these SNPs was greater than 99%. The power to detect the truly associated loci was greater than the nominal α level for only three of the remaining SNPs, all of them located in the FLT1 gene  for each replicate. The number of SNPs within 1.5-LOD support intervals from the most significant linkage peaks varied substantially by replicate. Significant linkage loci were observed at all but 12 of the Q1 replicates but only at 7 of the Q4 replicates. On average, 611.7 SNPs per replicate were under linkage peaks, which represent only about 2.5%  of the exome. The percentage of the genome included in the linkage peaks varied across replicates but was never larger than 7% of the whole exome. Therefore a great reduction in sequencing cost could be achieved by restricting sequencing to areas under linkage peaks. The linkage analysis of Q4 indicates that a much smaller percentage of the exome would be sequenced for unassociated traits, with only seven replicates requiring any sequencing at all, and that none of the regions overlapped in different replicates. The average proportion of the genome sequenced for unassociated traits if sequencing were restricted to linked regions would be 11.4/24,487, or 0.04%, which suggests a low false-positive rate.We examined the power for the true Q1 susceptibility loci using linkage results to guide our association analyses (Table Seven out of 43,600 SNPs showed significant association for Q4. However, among 218 unassociated SNPs only 10 were under the linkage region, and none of these were significant. This implies that the linkage-based sequencing produced zero false positives out of 43,600 tests.We examined 17 causal SNPs for Q1 and 218 unassociated SNPs for Q4. We then examined these SNPs using two sequencing paradigms: whole-exome and linkage-guided sequencing. Association results with the whole-exome sequencing approach with appropriate corrections accounting for multiple testing revealed that overall power to detect association with small effect sizes, regardless of SNP minor allele frequency, was quite low. Only two SNPs were detected with a power greater than 80%.For the second approach, we first performed genome-wide robust variance components linkage analyses for Q1 and Q4 using the supplied IBD sharing. Then, we identified SNPs linked to traits in each replicate, defined as being within a 1.5-LOD support interval of a LOD score greater than 3.3. Finally, we recomputed the power to detect each of the Q1 SNPs under a linkage-guided sequencing paradigm, using a less stringent multiple testing penalty that accounted only for SNPs falling under linkage peaks. Using the linkage results, we detected association with the two easily detected SNPs about 70% of the time. Comparing 90% with 70% of the power to detect only 2 of the 17 susceptibility loci might seem low, but it is important to keep in mind that power to detect the other Q1 loci is also low under a whole-exome paradigm. By using the linkage-guided approach to reduce the amount of sequencing, we found that restricting sequencing under the linkage peaks would have detected more than 52% of the loci found by whole-exome sequencing despite the fact that only 2.5% as much of the genome would have to be sequenced. This statistic seems better if we restrict our attention to two loci that could be detected with high power, where restricting sequencing under linkage peaks would have detected association approximately 70% of the time. This demonstrates that sequencing under linkage peaks can be an efficient strategy for examining large multiplex families in terms of the number of true associations obtained per base pair sequenced.Our method is only the first step in an evaluation of the utility of linkage information in association analysis. It would also be important to evaluate the difference between analyses of the full sample and analyses that sequenced only families that appeared to be linked. When we examined the significance of SNPs by family, it was clear that for most SNPs the evidence for association emerged from a single family or a small group of families. Performing pedigree-specific LOD score analysis may enable a further reduction in the number of base pairs to be sequenced without compromising the power to detect mutations associated with the traits of interest. One limitation of our study is that we did not account for population substructure in our current association analyses. Further analysis would be necessary to evaluate whether or not the substructure confounds the reported findings.The authors declare that there are no competing interests.SHC carried out all association analyses and power calculation, and participated in drafting the manuscript. CL carried out the linkage analyses and provided assistance with the power calculation, and participated in drafting the manuscript. JD conceived of the study, participated in the design of the study, and participated in drafting the manuscript. MWL conceived of the study, participated in its design and statistical analyses, and drafted the manuscript. GJ conceived of the study, participated in its design and statistical analyses, and helped to draft the manuscript. All authors read and approved the final manuscript.


Complete labia fusion is a rare clinical entity in post-menopausal women. The most common complications of this presentation are infections of the urinary tract and retention of urine in the vagina. We present the case of a post-menopausal woman with adnexal mass and abdominal pain due to fusion of the labia majora. To the best of our knowledge this is the first report in the literature of this complication.A 78-year-old Caucasian woman was admitted to our hospital due to abdominal pain and urination difficulty, along with fever and leucocytosis. On examination the labial majora were fused. Computed tomography of the abdomen revealed a cystic formation in the anatomical area of the right adnexa. Our patient had developed a pyosalpinx as a Sequela of labial fusion. At laparoscopy the right pyosalpinx was identified and resected, whereas the labia majora were reconstructed via dissection and separation.Labial fusion is a rare clinical entity in post-menopausal women and can have serious and unexpected complications. Though this presentation is rare, a clinical examination must be performed in detail in order to gain valuable information for an accurate diagnosis. Post-operational instruction must be given to patients in order to prevent the re-occurrence of the fusion and its complications. Labial fusion in adults is a rare clinical entity with only few described cases, in particular in post-menopausal women. The major symptom in those reported cases is urination anomalies and most commonly infections of the urinary tract. Pyosalpinx is most often a complication of pelvic inflammatory disease arising from a variety of causes. In post-menopausal women, pyosalpinx is commonly associated with primary fallopian tube malignancy. We report a case of a post-menopausal woman who developed a pyosalpinx following complete fusion of the labial majora.A 78-year-old Caucasian Greek post-menopausal woman was admitted to our hospital due to continuous pain in her lower abdomen along with fever. Both symptoms began one week prior to admission and had become progressively worse over the 24 hours before presentation when she experienced four vomiting episodes. Our patient reported a long-standing history of urinary incontinence and voiding difficulty.Her overall clinical examination did not reveal anything worrisome, although a gynecological examination was unfeasible due to complete labial majora fusion Figure . There wLaboratory blood analysis on admission revealed the following: leucocytosis (white blood cell (WBC) count 13.02 × 109/L) with neutrophilia (neutrophils 11.95 × 109/L) with a further rise on day two (WBC: 18.64 × 109/L) followed by a drop on day four (WBC: 12.82 × 109/L); an elevated C-reactive protein level (105 mg/L) with a further rise on day two (119 mg/L) followed by a drop on day four (59 mg/L); an elevated blood glucose level (165 mg/dL) followed by a drop on day two (142 mg/L) and then the level remained within normal limits throughout the rest of her hospital stay and finally an elevated lactate dehydrogenase level (286 U/L). Carcinoembryonic antigen (CEA), α-fetoprotein (AFP), β-human chorionic gonadotropin (β-HCG), cancer antigen 125 (CA 125), cancer antigen 19.9 (CA 19.9) and cancer antigen 15.3 (CA 15.3) levels were all normal. Serum urea, creatinine, sodium and potassium levels were within normal limits. A computed tomography (CT) scan of her abdomen on admission showed a cystic (7 × 4.5 cm) formation within the pelvis minor in the anatomical position of the right adnexa; the formation appeared to be tubular in shape most likely suggesting a hydrosalpinx or pyosalpinx.After assessing all available evidence we were led to believe that our patient had most likely developed a pyosalpinx as a complication of the labial majora fusion. Our patient was started on antibiotic treatment. Three days later she underwent a minor surgical procedure during which a small opening was made in the outer vulvar lips in order to assist urination. Following antibiotic treatment there was resolution of her symptoms and, as our patient was judged to be at high risk if placed under general anesthesia, a conservative treatment of the pyosalpinx was preferred. A week later our patient was discharged and an appointment for a follow-up visit was made in two weeks. During the scheduled appointment a decision would be made on whether to proceed to labial separation alone or combined abdominal surgical treatment of the pyosalpinx.On arrival for the follow-up visit her blood test results were normal except for her blood glucose (174 mg/dL) and lactate dehydrogenase (257 U/L) levels. Her CEA, AFP, CA 125, CA 19.9 and CA 15.3 levels were normal; β-HCG however was mildly elevated. A second CT scan of the abdomen confirmed persistence of the previous pyosalpinx finding. Furthermore, a 7 cm elongated cystic formation suggestive of a cystovaginal fistula was identified, as well as bilateral mild hydronephrosis as a result of the urinary bladder's over-distension.On day two of admission our patient underwent laparoscopic surgery during which the right pyosalpinx was identified and subsequently resected Figure , and in The pathology report from the tissue specimens retrieved from the operation showed acute and chronic inflammation in the fallopian tube mucosa and chronic inflammation in the skin of the labia majora. No signs of malignancy were found in either of the fallopian tubes or the labia majora.Pyosalpinx following labial fusion has not been reported previously in the literature and, to the best of our knowledge, this is the first case report of such Sequela. Labial fusion occurs mainly in the first years of life and can be congenital or acquired. The condition is rare in adults and only a few cases have been reported in the literature, predominantly in post-menopausal women. The pathophysiological mechanism is believed to be chronic inflammation and/or irritation of the vulvar skin arising from various conditions such as local inflammation , lichen Our patient had been previously diagnosed with labial fusion and although she recalled having been surgically treated, her condition reappeared. Recurrence of labial fusion has been previously described , and we These instructions in our view are essential for the maintenance of a good outcome. In our patient although there was no visible vulvar opening, it is likely that some amount of the urine that was accumulating inside the vagina causing urocolpos was constantly filtered through the fused labia, since our patient stated she was continuously wet.Chlamydia trachomatis, Neisseria gonorrhea, and microorganisms associated with bacterial vaginosis  In one of the follow-up visits, I was asked about my feelings regarding my problem. I mentioned that I found not being able to urinate properly distressing and I was embarrassed to be constantly wet. I was disappointed that the initial procedure some years ago failed and that I had not been instructed to be under regular follow-up, nor had I been given instructions to try and keep the vulvar introitus open. I was extremely pleased with the outcome of the second procedure. I am happy that I am able to completely empty my bladder and was surprised I recovered so quickly from the laparoscopic salpingectomy. I am very willing to do whatever I am instructed to prevent a recurrence of the fusion, and I agree to have my case shared with the medical community if it helps other women with a similar problem.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.GIT, SIP and GMM analyzed and interpreted the data from our patient regarding clinical presentation, and hematological and imaging data. GK and TS performed the first labial dissection and the operative laparoscopy. All authors contributed to writing the manuscript and approved the final version.


Pneumococcal meningitis (PM) is a severe and life-threatening disease that is associated with cognitive impairment including learning difficulties, cognitive slowness, short-term memory deficits and poor academic performance. There are limited data on cognitive outcomes following exposure to PM from Africa mainly due to lack of culturally appropriate tools. We report cognitive processes of exposed children as measured by auditory and visual event-related potentials.Sixty-five children  aged between 4-15 years with a history of PM and an age-matched control group of 93 children  were recruited from a well-demarcated study area in Kilifi. In the present study, both baseline to peak and peak-to-peak amplitude differences are reported.Children with a history of pneumococcal meningitis had significantly longer auditory P1 and P3a latencies and smaller P1 amplitudes compared to unexposed children. In the visual paradigm, children with PM seemingly lacked a novelty P3a component around 350 ms where control children had a maximum, and showed a lack of stimulus differentiation at Nc. Further, children with exposure to PM had smaller peak to peak amplitude (N2-P1) compared to unexposed children.The results suggest that children with a history of PM process novelty differently than do unexposed children, with slower latencies and reduced or absent components. This pattern suggests poorer auditory attention and/or cognitive slowness and poorer visual attention orienting, possibly due to disruption in the functions of the lateral prefrontal and superior temporal cortices. ERPs may be useful for assessment of the development of perceptual-cognitive functions in post brain-injury in African children by providing an alternate way of assessing cognitive development in patient groups for whom more typical standardized neuropsychological assessments are unavailable. Streptococcus pneumoniae . This occurred due to longer mean P1 latency (99 ± 22.5 ms vs 90 ± 11.0 ms) in children with exposure to PM compared to unexposed children  Figure . The intThe N2 latency had significant interaction effect of stimulus by diagnosis by age . The interaction effect of stimulus by diagnosis by age occurred due to a significantly faster N2 latency for novel stimuli for unexposed children at age 10-15 years compared to younger ages  but not for children exposed to PM . The N2 amplitude from baseline to peak did not differentiate between children with exposure to PM and unexposed children. However, the peak to peak amplitude (N2-P1) had main effects of age  and diagnosis . The main effect of age occurred due to decreasing amplitude differences with age. The main effect of diagnosis was due to significantly smaller amplitude differences for children exposed to PM compared to unexposed children  = 6.112, p = 0.015]. This occurred due to longer P3a latency (333 ± 29.7 ms vs 321 ± 15.1 ms) in children with exposure to PM compared to unexposed children   = 3.952, p = 0.026] and stimulus by electrode by diagnosis . The interaction of stimulus by diagnosis occurred due to larger amplitude associated with infrequent stimuli compared to frequent stimuli in unexposed children  but not in children with a history of PM [p = 0.386]. The interaction of stimulus by electrode by diagnosis was due to larger amplitudes associated with infrequent compared to frequent stimuli at Fz, Cz and Pz  for unexposed children but not for children with a history of PM .The present study used auditory and visual event related potentials to novelty in children exposed pneumococcal meningitis, to investigate whether the disease disrupted the neural processes underlying visual and auditory attentional orienting, and its possible effects on brain processing as a marker of cognitive function .Children exposed to PM had longer auditory P1 latencies than unexposed children. The P1 component has been thought to be an objective measure of cortical auditory function ,36 or prThe auditory N2 latency is influenced by attention and task difficulty ,44. It iThe P1-N2 peak to peak amplitudes to novel stimuli showed significant differences between children with exposure to PM and unexposed community children. This aimed at determining whether the amplitude of the N2 component was influenced by the diagnosis of the children independent of the P1 component. The smaller brain responses in children with exposure to PM suggests deficits in cognitive processes necessary to discriminate novel stimuli ,52.The P3a component reflects an involuntary attention switch from the actual focus of voluntary attention to the eliciting sound , and is The present study revealed absence of the visual P3a component in children exposed to PM. Some previous studies have found marked reduction and even absence of this component in patients with alcoholism ,64 and eGenerally, amplitudes are related to the strength of the neural association while the latencies represent the time to respond to the stimuli . The resPrevious research has reported potentially serious cognitive consequences in children with a history of pneumococcal meningitis ,12,42. TThe results of the auditory paradigm of study should be interpreted with caution since bacterial meningitis is known to cause subtle to profound hearing loss and the children were not fitted with hearing aids. Children with severe to profound hearing loss were excluded from the ERP paradigms to minimize obvious biases arising from sensory impairments. However, it is possible that subtle hearing loss and cortical blindness could have accounted for the differences in children with pneumococcal meningitis. The auditory N2 and visual P2 latencies are indices of processing speed and attention allocation and could have been affected by subtle impairments.Also, a lack of neuropsychological testing in this study meant that we could not compare the differences in function between children exposed to PM and those who were unexposed.Studies in Africa have shown a high HIV prevalence in children with meningitis . HoweverThe results of the passive ERP paradigm show that there is a difference between children exposed to pneumococcal meningitis and unexposed children. These exposed children generally have a slower brain processing as depicted by their longer ERP latencies of the various components. The results suggest impaired development of frontal cortex and/or its connecting pathways, possibly due to disruption of brain development by meningitis infection. This methodology may be useful for assessment of the development of perceptual-cognitive functions in African children providing an alternate way of assessing perceptual-cognitive development in patient groups for whom more typical standardized neuropsychological assessments are unavailable.The authors declare that they have no competing interests.MK, EOW, BGRN and CRJCN were responsible for the conception of the study, its design, data collection and critical review of the manuscript. HH and MK were involved in the recording and analysis of the event-related potentials. MdH, CRJCN and MK were involved in the evaluation and interpretation of results. MK wrote the initial draft of the manuscript and all other authors contributed substantially to the content. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/79/prepub