Multivariate analysis using the combined data from all radiotracers clearly separated the different groups along the first and second principal components according to increased PIB retention/decreased FDG uptake (component 1) and increased DED binding (component 2). Presymptomatic ADAD mutation carriers showed significantly higher PIB retention than non-carriers in all brain regions except the hippocampus. DED binding was highest in presymptomatic ADAD mutation carriers. This suggests that non-fibrillar Aβ or early stage plaque depostion might interact with inflammatory responses indicating astrocytosis as an early contributory driving force in AD pathology. The novelty of this finding will be investigated in longitudinal follow-up studies.Studying autosomal dominant Alzheimer’s disease (ADAD), caused by gene mutations yielding nearly complete penetrance and a distinct age of symptom onset, allows investigation of presymptomatic pathological processes that can identify a therapeutic window for disease-modifying therapies. Astrocyte activation may occur in presymptomatic Alzheimer’s disease (AD) because reactive astrocytes surround β-amyloid (Aβ) plaques in autopsy brain tissue. Positron emission tomography was performed to investigate fibrillar Aβ, astrocytosis and cerebral glucose metabolism with the radiotracers  Alzheimer’s disease (AD) is a progressive brain disorder with gradually occurring cognitive decline. The time course of the underlying pathological changes remains largely veiled. Increasing evidence argues that these changes start decades before the onset of clinical symptoms. The order and magnitude of these processes are hitherto not well understood. The typical histopathology of AD includes the presence of β-amyloid (Aβ) plaques, neurofibrillary tangles, neuronal cell dysfunction and loss, and the activation of glial cells. It has been hypothesized that the accumulation of Aβ plaques plays a causative role in the disease development24PSEN1), presenilin 2 (PSEN2), or amyloid precursor protein (APP) genesAD is sporadic (sAD) in the great majority of cases, but 1–5% of Alzheimer patients suffer from the autosomal dominant form of the disease (ADAD), which is caused by mutations in the presenilin 1  binds to monoamine oxidase B (MAO-B) on the outer mitochondrial membrane in astrocytes; increased DED binding is thought to reflect reactive astrocytosis1511C-DED PET study of sAD patients, we found evidence for early astrocytosis in 11C-PIB positive patients with mild cognitive impairment (MCI PIB+)The PET tracer This cross-sectional study reports the baseline results from a large, ongoing, longitudinal study aiming to examine the temporal and regional relationships between astrocytosis, Aβ deposition, and glucose metabolism as a measure of neurodegeneration in ADAD and sAD. Here we demonstrate for the first time the presence of significant astrocytosis decades before the occurrence of clinical symptoms.The demographic data for the subjects are presented in All raw test scores were transformed into z-scores . Z-score2(X)), and its cross-validated predictability, Q2(X), was 0.61 The principal component analysis PCA model accounted for 67% of the variance of the original data .The sAD and MCI PIB+ patients had increased PIB retention compared to ADAD non-carriers with effect sizes r > 0.80 (p < 0.001) in all ROIs, except the hippocampus which did not show a significant difference. Both sAD and MCI PIB+ groups showed significantly increased PIB retention compared to presymptomatic ADAD mutation carriers in all cortical regions, most pronounced in parieto-temporal cortex , but differences were not significant in subcortical regions. Presymptomatic mutation carriers had higher PIB than ADAD non-carriers in all ROIs , except for the hippocampus.Comparisons of four groups  using Kruskal-Wallis tests in each of 11 ROIs (except for the cerebellum), showed a trend toward statistical significance for the anterior cingulate cortex, the thalamus and the hippocampus (p = 0.059–0.069) . The higThe pattern of cerebral glucose metabolism as measured by FDG uptake was significantly different between the groups ; the KruThe statistical parametric mapping (SPM) results generally demonstrated ongoing progressive pathology in ADAD mutation carriers as the expected age at symptom onset approached , consistin vivo pathology, while cerebral glucose hypometabolism is a downstream marker that monitors the course of neurodegenerationThe rapid development of molecular imaging has provided powerful tools for the detection of AD pathology at an early stage of the disease. These tools, in combination with the currently available biomarkers, provide an unprecedented opportunity to define a therapeutic window for use in the development of potential preventive/disease-modifying therapies. For example, high retention of specific Aβ PET tracers such as Pittsburgh compound-B in patients with mild cognitive impairment seems to predict a high risk of developing Alzheimer’s disease18212223The amyloid cascade hypothesis, the focus of AD research for decades8278postmortem AD brain tissue using 3H-DED autoradiography and glial fibrillary acidic protein (GFAP) immunohistochemistry3132postmortem GFAP reactive astrocytes and both in vivo11C-PIB and in vitro3H-PIB binding. However, no correlation was found between postmortem3H-DED and in vivo11C-PIB3H-DED reactive astrocytes and fibrillar Aβ in AD autopsy brains, using regional and laminar distribution analysesin vivo PET imaging findings such as a lack of correlation between plaque deposition, metabolic dysfunction, atrophy, and clinical outcome (e.g. cognitive dysfunction) in AD patientsThe chronological order and relative causality of astrocyte activation, fibrillar Aβ deposition and glucose hypometabolism, have not been established, yet. Interestingly, it has been shown in cultured rat astrocytes that Aβ induces MAO-B expressionin vitro studies on reactive glia, less is known about their function in vivo. PET imaging of astrocytosis in mild cognitive impairment and sAD patients using 11C-DED has demonstrated elevated astrocytosis in MCI PIB+ patients , suggesting that astrocytosis is an early event in sAD patients11C-DED binding correlated with decreased gray matter density in the parahippocampus of MCI PIB+ patientsin vivo studies using 11C-DED in ADAD have been reported to date. Moreover, in vivo studies on astrocytosis are required to investigate the different roles of the reactive astrocytes across different disease stages, especially with regard to neuroprotection vs. neurotoxicity. Furthermore, microglia and astrocytes show differential relationships to Aβ pathology and seem to play different roles in inflammation in AD14In vivo PET studies using tracers for the translocator protein (TSPO) as a marker for microglial activation, showed discordant results with both increased tracer uptake in AD patients as compared to healthy controls and no difference between patients and controls373839While there are several postmortem and in vivo studies suggesting a general lack of correlation between reactive astrocytes and fibrillar Aβ. The observed early astrocytosis might have been caused by Aβ oligomers or other pathological features such as intraneuronal hyperphosphorylated tau protein and adds support to the idea of glial activation as independent of other pathological substrates in AD.In this study, the finding of early astrocytosis roughly coinciding temporally but not necessarily spatially with fibrillar Aβ deposition is consistent with evidence from The finding of early astrocytosis in the AD continuum is strengthened by recent findings of YKL-40, a potential astrocyte-derived biomarker measured in CSF, being strongly related to Aβ in AD patients and with tau pathology as a marker for neurodegeneration, most pronounced at early pre-dementia stages of AD4243444311C-DED binding in addition to PIB and FDG examinations. The PCA model showed a clear separation pattern between the different groups using the three different PET tracers in 24 brain regions allowed. Interestingly, the direction of the measured signal for the PET biomarkers PIB and FDG (which indicated worsening of amyloid deposition and glucose hypometabolism with time) differed from that for DED (which indicated decreased astrocytosis). This observation clearly reveals an opposite direction in time course between PIB and DED although these PET tracers show similar early presymptomatic presence but change differently in disease progression.We also compared cases of ADAD and sAD in this study, since a common neuropathological pathway has been suggestedin vitro at autopsyin vivo using PET DED imagingThe highest level of DED binding was observed in presymptomatic ADAD mutation carriers, while DED binding was low in symptomatic ADAD mutation carriers. Interestingly, earlier studies in healthy subjects have reported an age-related increase in MAO-B activity in healthy human brains, as investigated PSEN1 mutation carriers, suggesting that the thalamus may also be involved in early disease mechanisms in ADAD patients. Subtle cognitive dysfunction in ADAD patients has been detected relatively close to the onset of symptoms (from about five years EYO)The increase in PIB retention occurred early in presymptomatic ADAD carriers, predominantly in the anterior and posterior cinguli and the basal ganglia. This pattern of early changes in the subcortical brain regions is in agreement with recent ADAD studiesTo conclude, we have demonstrated that measures of astrocytosis in ADAD mutation carriers can be observed decades before symptom onset, possibly coinciding with early fibrillar Aβ plaque deposition, both of which are followed later on by impaired glucose metabolism. Multivariate analysis of the PET results clearly separated the subject groups in a different order for the PIB/FDG tracers compared to the DED tracer. The analysis also suggested that DED might not yield equally high levels of sensitivity or specificity as PIB/FDG. It appears, however, that astrocytosis is initiated very early on, possibly before or at a similarly early stage as fibrillar Aβ deposition; this supports the notion that non-fibrillar forms of amyloid might cause inflammatory responses but also supports the possibility that astrocytosis is an important early contributory driving force in AD pathology. The novelty and importance of these findings will be confirmed in ongoing longitudinal studies.PSEN1 carrier was excluded for incomplete penetrance of the mutation. Eleven MCI patients, seven sAD patients, and 21 ADAD family members were eligible for inclusion , Swedish APP (APPswe), and presenilin 1 (PSEN1) (p.H163Y) mutations. Among the 21 family members, nine were carrying an ADAD mutation: two carried the APParc, two the APPswe, and five the PSEN1 mutations. The presence of the mutations in the subjects was confirmed by sequencing.The ADAD families are part of an ongoing longitudinal clinical study at the Karolinska Institute and were recruited without reference to their mutation status. The family members are regularly followed up clinically and examined using neuropsychological assessment, MRI, collection of CSF, and blood testsAPParc mutation carriers were not included in the analyses because, according to our previously published results, symptomatic carriers of this mutation do not show PIB PET retention, while exhibiting other clinical and biomarker features comparable with other mutation carriersAll participants underwent complete clinical examination, DED-, PIB-, and FDG-PET, MRI, and neuropsychological testing. The PIB PET data from the two All participants underwent routine clinical neuropsychological testing, which involved tests for global cognitive function, language, visuospatial function, episodic memory, attention, and executive ability, typically within six months of the PET examinations. Test scores were converted into z-scores in comparison with a reference group of healthy elderly people from the Karolinska University Hospital HuddingeThe PET investigations were performed at Uppsala PET center on ECAT EXACT HR+ (Siemens/CTI) and GE discovery ST PET/CT  scanners. The PET scans for all three radiotracers were commonly performed in the order PIB, DED, and FDG in each subject on the same day with 2–3 hours between tracer injections. For a few subjects, and due to tracer synthesis failures, two scans were within four weeks of the others. The orbito-meatal line was used to center the heads of the participants. The emission scans for the DED investigation consisted of 19 time frames  with a total duration of 60 min; the emission scans for the PIB investigations consisted of 24 frames  over 60 min. A late 40–60 min PIB sum image was created and used for subsequent image analysis. For each FDG emission scan, seven frames  were acquired over 45 min. A late 30–45 min FDG sum image was created and used for subsequent analysis. Patients were required to fast for 4 h preceding the FDG scan, which was performed in a quiet room with dimmed light and eyes closed. The mean injected doses for each tracer were DED: 209 ± 57 MBq, PIB: 217 ± 74 MBq, and FDG: 232 ± 45 MBq. All emission data were acquired in 3D mode and reconstructed with filtered back-projection using a 4 mm Hanning filter, resulting in a transaxial spatial resolution of 5 mm in the field of view. The matrix included 128 × 128 pixels, and a 2.5 zoom factor was used. The reconstructed frames were re-aligned to correct for patient motion during each PET scan. The PET protocol followed the protocol established in our previous publicationAll patients and ADAD family members underwent structural T1 MPRAGE MRI scanning using a 3T (Siemens Trio) scanner at the Karolinska University Hospital Huddinge, Stockholm, on average within five months of the PET examinations.All images were processed and analysed using a probabilistic atlas approach, as described previouslyAll T1 MR reference images were then segmented into gray and white matter tissue classes using SPM8 Raw co-registered and re-sliced FDG (Bq/ml) and PIB (Bq/ml) PET data for each patient were sampled using the same individual digital atlases. Using this method, mean FDG uptake and PIB retention values were measured for each atlas region, as described previouslyk3 in the reference region for irreversible binding with a fixed correction factor of 0.01, which was the minimum value for correction still leading to linearization in the model. This graphical reference Patlak model resulted in two measurements: the intercept  and the slope (kI = net DED binding to MAO-B). Since our main interest was to evaluate DED binding we used the slope value in subsequent analyses.The PET data for each participant were analysed using the individual brain atlases generated in the steps described above. Regional parametric data were generated from dynamic DED data from 20 to 60 minutes. No arterial blood samples were available as an input function for the DED modelling because of the clinical character of the study. Instead, a modified Patlak reference tissue model was used for kinetic analysis, according to earlier study methods17a priori information about groups for the analysis. Statistically, PCA reduces the dimensionality and complexity of the data by finding lines and planes in the n-dimensional space (n = number of variables in the model) that approximates the data in the best way possible in the least squares sense. This gives us the opportunity to get an overview of the data to observe group belonging, trends and outliers. It is also possible to view relationships between the observations and the variables. One of the advantages of multivariate methods like PCA is that it can handle many more variables than observationsPrincipal component analysis (PCA)The PCA model was created to include all individuals: sAD, MCI PIB+ patients, MCI PIB− patients, ADAD mutation non-carriers, presymptomatic ADAD mutation carriers, and symptomatic ADAD mutation carriers. In total 72 variables  were included for each subject in the PCA analysis. The pre-processing steps mean-centering and unit variance scaling were performed. Mean-centering improves the interpretability of the data by subtracting the variable average, which repositions the data set around the origin. Large variance variables are more likely to be expressed in modelling than low variance variables. Consequently, unit variance scaling was selected to scale the data appropriately. This scaling method calculates the standard deviation of each variable. The inverse standard deviation is used as a scaling weight for each PET measurement. The results from the PCA were visualized by plotting the first two components in a scatter plot. Each point in the scatter plot represents one individual subject. Loading plots were also created to illustrate how the PET variables included in the model influenced the observed pattern in the scatter plot along each component (only the 25 most important variables are shown).n ≥ 5 being compared were: presymptomatic ADAD mutation carriers, MCI PIB+, sporadic AD (sAD) and ADAD mutation non-carriers. The comparisons were performed for 11 bilateral ROIs: frontal, parietal, temporal, occipital, anterior and posterior cingulate cortices, caudate nucleus, putamen, thalamus, hippocampus and cerebellum. Significance level was set at p < 0.05. Size effects (r) of Mann-Whitney comparisons were calculated using r = z/(√N), where z is the Mann-Whitney z and N the sum of individuals from the two groups being compared. Due to the small sample sizes of the MCI PIB− and symptomatic ADAD mutation carrier groups, individual z-score values were obtained instead with reference to the ADAD non-carrier group; z-score values were considered abnormal at |z| > 1.645.For each PET tracer, regional mean uptake values were compared between groups using two-tailed non-parametric Kruskal-Wallis tests, followed by post-hoc Mann-Whitney U tests in SPSS software. Non-parametric tests were applied due to the small sample sizes. The groups with −1) images in standard MNI space for each participant. After spatial normalization, each individual PET image from each ADAD mutation carrier was compared separately, using a two-sample t-test (SPM), with images from a group of five non-carriers (controls) who were the most proximal in age to the mutation carrier investigated. An explicit binary gray matter mask was used so that only voxels within the mask were compared in the SPM analysis. All SPM results for each tracer were analysed at a p value of <0.001 (uncorrected for multiple comparisons).The PET data  from each participant, which had been realigned to the DED image in native space, were spatially normalized using non-linear transformation from the segmentation of the T1 MRI data. This resulted in PIB and FDG ratio (/pons) images and DED slope .


Transcription control insights using blood ChIP sequencing compendium. in vivo. Although this technology has led to many important discoveries, the rapidly increasing number of publicly available ChIP-sequencing datasets still remains a largely unexplored resource. Using a compendium of 144 publicly available murine ChIP-sequencing datasets in blood, we show that systematic bioinformatic analysis can unravel diverse aspects of transcription regulation; from genome-wide binding preferences, finding regulatory partners and assembling regulatory complexes, to identifying novel functions of transcription factors and investigating transcription dynamics during development.Transcription factors play a key role in the development of a disease. ChIP-sequencing has become a preferred technique to investigate genome-wide binding patterns of transcription factors  Moreover, dysregulation of transcription has been widely proven to be a major contributor to human pathologies, with the recent development of small molecule drugs targeting protein interactions between transcriptional regulators generating much excitement.cis-regulatory DNA elements and trans-acting transcription factors (TFs) representing the fundamental basis of transcriptional control, the delineation of comprehensive collection of regulatory sequences together with knowledge of the TFs bound to them will be essential to gain global insights into transcriptional control mechanisms. Over the past 10 years, chromatin immunoprecipitation (ChIP) followed by microarray (ChIP-chip) or sequencing (ChIP-Seq) have become the most widely used approaches for genome wide identification and characterization of in vivo protein–DNA interactions. Due to the rapid drop in the cost of high throughput sequencing, ChIP sequencing has become the method of choice for the generation of high resolution maps of genome-wide protein–DNA interactions in mammalian systems.3With the interaction between 10 and only a few larger studies reporting 10 or more factors12 or a single factor across multiple cell types.13 We have previously shown14 that unlike gene expression data, ChIP-Seq datasets produced by different laboratories can be readily integrated. This analysis revealed that genome wide transcription factor binding profiles are largely governed by cellular context. We recently reported a TF ChIP-Seq compendium containing 144 publicly available studies pertaining to the mouse blood system.15 Using this dataset, here we show how concerted bioinformatic analysis of such a high quality hand-curated compendium can reveal previously unknown aspects of transcriptional control. This includes identification of those TF-bound sites most likely to be functional, prediction of TF interactions and multicomponent complexes, specific functionality of individual TFs and the dynamics of transcriptional regulation during differentiation and development.To gain a holistic view of transcriptional control during development, it is essential to generate genome scale maps of key transcription factors across multiple cell types. However, generating such genome-scale maps in many different cell types remains a daunting task for individual research groups due to limited human and financial resources. Moreover, each individual TF requires careful validation of antibody reagents, which limits the potential throughput of large-scale initiatives. Indeed, bespoke protocols are often developed by individual groups with specialist expertise, so that published ChIP-Seq studies commonly report binding maps for less than a handful of TFs15 to obtain 270 261 regulatory regions with at least one factor binding. We classified peaks into two groups: promoter and enhancer peaks by defining the peaks within 1 kb of TSS as promoter peaks. 7.5% of the total peaks belonged to promoters and all non-promoter peaks were classified as putative enhancers. The hierarchical clustering of enhancers clustered them according to the cell type (14We collected genome-wide binding patterns (peaks) of 144 publicly available murine ChIP-sequencing datasets for 53 transcription factors in 15 major blood lineages and leukemiaell type  irrespecet al., 2009).The promoter regions did not show a strong cell type specific clustering but clustered into two major clusters . ClusterAs Pu.1 peaks in both promoters as well as enhancers cluster according to the factor rather than the cell type, we characterized them in more detail. The 5-way Venn diagram of Pu.1 in promoter regions showed a high overlap of binding sites with about 50% of peaks present in all cell types  whereas 8 To check if TFs have distinct interacting partners in promoter and enhancer regions, we calculated cis-regulatory motif enrichment in promoter and enhancer regions separately for each factor using HOMER software. The sequence motif of the transcription factor chipped was enriched in both promoters and enhancers in most samples. Most samples also exhibited promoter-specific and enhancer-specific sequence motifs  motif is enriched only for Pu.1 enhancers in B cells while MafA (macrophage activating factor) motif is only enriched in Pu.1 enhancers in macrophages.It is well established that transcription factors bind to different interacting partners in a cell type specific manner to drive gene expression.Taken together, the data support the suggestion that tissue-specificity is a common feature of enhancers rather than promoters.p-value: 6.6 × 10–18), ‘hematopoiesis’  and ‘blood vessel development’  demonstrating that hematopoietic regulatory genes have more binding sites in their gene loci. In an individual ChIP-sequencing experiment, most gene loci are associated with only one peak with an average of 1.8 peaks per gene. Genes with more than 5 peaks in their gene locus were enriched for hematopoietic functions. Transcription factor gene loci have an average of 2.5 peaks per gene, in agreement with previously reported suggestions that TF gene loci have a higher number of regulatory elements than average. This difference is statistically significant even after correcting for the gene length .We mapped peaks across 15 blood lineages to their nearest genes resulting in an average of 13.5 peaks per gene. The 19 869 unique gene loci were associated with peaks ranging from a single peak to over 200 peaks. The 726 genes with more than 50 peaks in their gene loci are enriched for functional categories ‘transcription regulation’  for all samples (y axis) highlighted in yellow. The enriched motifs are useful in three ways. Firstly, they validate the chipped TF e.g. the Cepb motif is enriched in the two samples CebpA and CebpB  can accurately predict the presence of CpG islands in promoters suggesting that they are structural elements critical for CpG island function. In line with this, transcription factors such as the three ETS factors Erg, Fli1 and Pu.1 were enriched for CpG rich regions. Interestingly, peaks of components of the Ldb1 complex  occurred significantly less often than expected by chance in CpG rich regions .Physical interaction of TFs is an important aspect in determining tissue specific gene expression, and cooperative binding to DNA may be subject to spatial constraints. For each TF pair, we mapped the sequence motifs to peaks bound by both TFs and calculated the distance between two motifs. We selected motif pairs displaying a specific distance preference in at least two independent ChIP-seq experiments. Importantly, this analysis recovered previously known spacing of 8–10 bps between GATA and E-box motifs involved in binding of Gata1/Scl/E2A/Lmo2 multiprotein complexes.et al., 2008).32 The second pattern, GATA and CTG or GTC, had a 9 bp gap mapping to GATA and a half Ebox binding as a part of the Ldb1 complex. The final pattern, Gfi1b and (A/T)GC, had a 2 bp gap . We found 3 binding distance preferences; the first pattern, GATA and GAT, had a 3/4 bp gap consistent with Gata factors binding as homo-dimers validated by the crystal structure ’, ‘immune system development ’, ‘B cell receptor signalling pathway ’ are enriched in genes near Pu.1 peaks in HPC7 and B cells and not in macrophages while macrophage specific functional categories such as ‘endocytosis ’ and ‘inflammatory response ’ are over-represented in genes near Pu.1 peaks in HPC7 and macrophages and not in B cells. This is a strong indicator of lineage priming in the progenitor cells and therefore provides global confirmation for one of the most hotly debated topics in stem cell biology.TFs are major determinants of cell fate and lineage choice. However, most lineage determining TFs are expressed across multiple lineages, suggesting that combinatorial interactions are critical in determining cell type specificity. By merging datasets from different studies, the TF ChIP-seq compendium serves as an excellent resource in the study of genome wide binding patterns of the same TF in multiple cell types. Grouping the genome wide binding patterns of Pu.1 in haematopoietic progenitor cells (HPCs) along with two mature cell types (macrophages and B cells) highlights that cell type specific, as well as ubiquitous binding events are present in both promoters and enhancers with ubiquitous binding events being more common in promoters. T and B cells specific functional categories such as ‘lymphocyte activation  or under-represented (blue) compared to 100 randomizations. Using HOMER8 and based on gene context or repeat elements, peaks were sorted into 9 categories: 3′ UTR, 5′ UTR, exons, introns, intergenic regions, promoters, LINE, SINE and LTR. CpG islands were downloaded from UCSC. A list of transcription factors in mouse was downloaded from RIKEN.29 To find distance preferences between pairs of TFs, the sequences for peaks bound by both transcription factors were obtained using UCSC Galaxy and the binding locations of each sequence motif were determined using TFSBsearch.30cis-Regulatory sequence motifs were downloaded from the JASPAR library26 and the motifs were searched in peaks using TFSBsearch;30 over-representation was calculated with respect to 100 random sequence sets of the same number and lengths of real peak sequences. Functional enrichment was calculated using DAVID.31 Most analysis was done using Perl, MATLAB and R scripts.The Genome-wide binding patterns of 53 transcription factors in 15 major blood lineages and leukaemia were obtained from The advent of next generation sequencing technologies has led to a dramatic shift in modern biological research, where bioinformatic processing and interpretation of large-scale datasets are rapidly replacing data generation as the major bottleneck. Moreover, bioinformatic analysis of genome-scale datasets is often restricted to the particular context of the paper that first reported them, even though the raw data are made publicly available in online repositories. Consequently, a whole potential treasure trove of biological insights remains essentially unexplored.To ameliorate this situation, progress on two fronts will be vital. Firstly, significant efforts need to be invested into the generation of data integration platforms that facilitate cross-referencing between the multiple independent studies. Secondly, bioinformatic analysis strategies need to be developed to facilitate extraction of novel biological hypotheses from integrated genome-scale resources.In this paper, we have addressed the latter issue and provided seven examples of bioinformatic analysis that together have allowed us to develop a number of new hypotheses on transcriptional control mechanisms with the potential to transform our understanding of blood cell development. Importantly, both the procedures outlined as well as the take-home messages learned should be readily transferable to the exploitation of ChIP-Seq datasets in other cellular systems, and thus have the potential to significantly advance our understanding of a wide range of both normal and pathological cellular processes.


Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure. Although researchers have made significant progresses in elucidating the pathophysiology of this complex syndrome over the years, the absence of a universal detail disease mechanism up until now has led to a series of practical problems for a definitive treatment. This study aimed to predict some genes or pathways associated with sepsis-related ARDS based on a public microarray dataset and to further explore the molecular mechanism of ARDS.CCNB1), cyclin B2 (CCNB2) and topoisomerase II alpha (TOP2A). A total of seven transcription factors including forkhead box protein M1 (FOXM1) and 30 target genes were revealed in the transcription factor-target gene regulation network. Furthermore, co-cited genes including CCNB2-CCNB1 were revealed in literature mining for the relations ARDS related genes.A total of 122 up-regulated DEGs and 91 down-regulated differentially expressed genes (DEGs) were obtained. The up- and down-regulated DEGs were mainly involved in functions like mitotic cell cycle and pathway like cell cycle. Protein–protein interaction network of ARDS analysis revealed 20 hub genes including cyclin B1 (CCNB1, CCNB2 and TOP2A, as well as transcription factors like FOXM1 might be used as the novel gene therapy targets for sepsis related ARDS.Pathways like mitotic cell cycle were closed related with the development of ARDS. Genes including  Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure . Its devPrevious study has indicated that ARDS has a close relation with severe sepsis , but litBAD) gene is a candidate gene associated with the development of ARDS. Besides, variants in more than 30 genes have been associated with ARDS  were revealed in the present regulatory network  (Table P = 0.0035)  was originally discovered as a soluble protein that altered migratory cell behavior and bound to hyaluronan [The transcription factor-target gene regulation network analysis in this study revealed seven transcription factors including tability . In thisaluronan . HMMR isaluronan  have sugDespite of the results obtained above, there were some limitations in this study. Firstly, no verification experiments based on cells or tissues or joint analysis of expression profile data were performed to confirm our results, besides, the sample size was small. Secondly, due to the data themselves, there was no correction for multiple comparisons in the DEGs identification. Thirdly, the mRNA expression profile used in this study was extracted from blood leukocytes, and another important cells in ARDS, such as epithelial and endothelial cells of the lung had not been studied. Therefore, more investigations related to another cells in ARDS with experimental verification and diverse samples are needed in the further study.CCNB1, CCNB2 and TOP2A, as well as transcription factors FOXM1 may be used as the novel gene therapy targets for ARDS.In conclusion, the pathways like mitotic cell cycle were closed related with the development of sepsis related ARDS. Genes including


The nosological assignment of congenital ocular motor apraxia type Cogan (COMA) is still controversial. While regarded as a distinct entity by some authorities including the Online Mendelian Inheritance in Man catalog of genetic disorders, others consider COMA merely a clinical symptom.We performed a retrospective multicenter data collection study with re-evaluation of clinical and neuroimaging data of 21 previously unreported patients  diagnosed as having COMA.n = 6) or intellectual disability (n = 4). Re-evaluation of MRI data sets revealed a hitherto unrecognized molar tooth sign diagnostic for Joubert syndrome in 11 patients, neuroimaging features of Poretti-Boltshauser syndrome in one case and cerebral malformation suspicious of a tubulinopathy in another subject. In the remainder, MRI showed vermian hypo-/dysplasia in 4 and no abnormalities in another 4 patients. There was a strong trend to more severe cognitive impairment in patients with Joubert syndrome compared to those with inconclusive MRI, but otherwise no significant difference in clinical phenotypes between these two groups.Ocular motor apraxia (OMA) was recognized during the first year of life and confined to horizontal pursuit in all patients. OMA attenuated over the years in most cases, regressed completely in two siblings, and persisted unimproved in one individual. Accompanying clinical features included early onset ataxia in most patients and cognitive impairment with learning disability  contains supplementary material, which is available to authorized users. The term congenital ocular motor apraxia (COMA) was introduced by Cogan in 1952 when he described a particular disorder of voluntary horizontal gaze in four children [During subsequent decades a wide range of disorders associated with ocular motor apraxia (OMA) was recognized . HoweverOn the other hand, early-onset OMA is a frequent symptom of Joubert syndrome (JBTS), accompanied by infantile muscular hypotonia, early-onset ataxia, neonatal breathing abnormalities with episodic tachypnea and apnea, as well as developmental delay/intellectual disability –12. ThusMRI in COMA was reported to be either normal or show a vermian hypoplasia, preferentially of the inferior portion of the vermis , 14. A rIn contrast, a highly characteristic neuroimaging feature of JBTS in axial MRI of the brainstem was recognized in 1997 and designated the “molar tooth sign” (MTS) . The “moOur study presented here originally aimed at identification of the gene associated with COMA. However, re-evaluation of clinical and neuroimaging data of patients recruited with the diagnosis of COMA indicated that delineation of COMA versus JBTS and clarification of the nosological relationship between these two conditions are mandatory and a prerequisite for genetic investigations in COMA.This study was approved by the ethics committee of the Faculty of Medicine, University of Göttingen (file no. 19/5/14). Written informed consent was obtained from all families.n = 4) and (ii) using an email based acquisition of rare neurological disorders in childhood  [n = 16) and Switzerland (n = 1).Patients diagnosed as having COMA were collected (i) from the cohort of patients of the Department of Pediatric Neurology, University Medical Center Göttingen ( ESNEK”)  from pedInclusion criteria for the definite COMA cohort comprised written informed consent of the parents or the patient or both, early-onset OMA (diagnostic recognition within the first year of life), and availability of an MRI in technical quality adequate for assessment of especially brainstem and cerebellum. We did not include patients with the MTS already recognized on MRI, thus with a preexisting diagnosis of JBTS.Demographic data, neurological features and information about the developmental stage were compiled by review of the clinical histories and by clinical-neurological follow-up investigations. Information about ophthalmological features was collected from neuro-ophthalmological or pediatric-neurological reports. Cognitive function was assessed using standardized neuropsychological tests whenever possible. Otherwise, the stage of mental developmental was appraised from the patient’s history, clinical examination, and reports from nursery or school. Additional data were collected in an interview with the parents conducted by telephone using a standardized questionnaire  . All avand birthday, neuroophthalmological features not consistent with OMA). In additional three patients cranial MRI was not available in sufficient technical quality. One patient already had a molecular genetic diagnosis of JBTS. Thus 21 patients were included in this study . Parental consanguinity was reported in one patient (#7). Table A total of 28 patients were recruited via email based acquisition and from our own department. Among these, critical reappraisal of disease course and clinical features revealed that phenotypes were not consistent with COMA in three cases , nystagmus (n = 5), head tilting (n = 3), and strabism (n = 2), with ages at onset ranging from 4 weeks to 6 months.In all patients OMA was recognized by attending physicians during the child´s first year of life. Most children (15 of 21 cases) presented with visual problems already in the first months of life, before OMA was definitely diagnosed, occasionally giving rise to the concern of severe visual impairment or even blindness. Initial visual symptoms included lack of fixation and visual pursuit  at age 4 and 5 years. Additional oculomotor features comprised nystagmus in three patients and Duane syndrome in two siblings (#8 and #9).n = 7) or intellectual disability .In most patients, early-onset cerebellar ataxia together with muscular hypotonia constituted further clinical hallmarks with gradual amelioration during the first decade, while general clumsiness persisted. Motor development was delayed in most cases, unsupported walking was achieved at ages ranging from 14 months to four years (mean 25 months). All subjects showed gradual ongoing improvement of motor skills. Development of both, language perception and expressive speech was delayed as well in all but two cases. An impairment of cognitive development was observed in 11 cases comprising learning disability  had retinal coloboma, neonatal breathing dysregulation, and persistently elevated liver enzymes. These features are well-known in JBTS with liver involvement due to utations , but hadP1L gene . No otheIn our cohort of 21 patients with clinical phenotypes consistent with COMA a reappraisal of MR images primarily judged to be normal revealed a MTS in 11 patients. Thus the majority of patients clinically diagnosed to have COMA can in fact be assigned to JBTS. All these MR images had previously been analyzed by board-certified radiologists or neuroradiologists, in some cases with explicit question whether there is a MTS, and had been rated as normal.Several factors contribute to the difficulties in recognition of the MTS in our cohort: The molar tooth is a characteristic, but subtle MRI sign which is easily missed. It was recognized as a typical hallmark of JBTS in neuroimaging only in 1997, 28 years after first description of JBTS as a clinical syndrome . MRI wasIn two additional patients of our cohort re-evaluation of MRI revealed otherwise specific neuroimaging features. An 18-year-old man (#7) with clinical phenotype consistent with COMA had clear MRI characteristics of Poretti-Boltshauser syndrome, and a 10-year-old boy showed neuroimaging features pointing to a tubulinopathy. Appropriate molecular genetic investigations are under way in these two cases. In the remaining 8 patients with clinical phenotype consistent with COMA MRI revealed no conclusive findings.These results strongly underline that a brain MRI of a child with COMA needs to be reviewed by a neuroradiologist or neurologist with experience in pediatric posterior fossa diseases, particularly malformations. As has been shown for brain MRI in children with cerebral palsy the accuracy of neuroimaging reports clearly depends on the expertise of those reporting . An expeAmong the four patients originally described by Cogan in 1952, no other neurologic symptoms were present in two, while one had convulsions shortly after birth without neurologic sequelae and one had a persistent extrapyramidal disorder assigned to intrauterine carbon monoxide poisoning . It is oIt has been doubted “whether Cogan-type oculomotor apraxia can exist as an isolated entity” , and a cATM (AT), aprataxin (AOA1) and senataxin (AOA2) are involved in mechanisms of DNA repair.In contrast to this spectrum of conditions related to COMA, acquired OMA especially occurs in a range of different genetic disorders comprising ataxia teleangiectasia (AT), ataxia with ocular motor apraxia 1 (AOA1), and ataxia with ocular motor apraxia 2 (AOA2). The associated genes Our results indicate that a large subgroup of patients diagnosed with “benign” or “idiopathic” COMA in fact have JBTS, as the MTS was missed on MRI – which may easily happen. Therefore meticulous analysis of the MRI in patients with COMA is advisable. Furthermore we found no consistent differences in clinical phenotypes  between patients with the MTS and those with “benign” or “idiopathic” COMA, apart from one case with Poretti-Boltshauser syndrome and one patient with clinical features pointing to JBTS which were however not previously recognized in their diagnostic value in these patients.In the future, delineation of new clinical and genetic entities associated with early-onset OMA will possibly allow for assignment of hitherto ill-defined cases of “idiopathic” COMA to a specific diagnosis. Additional genetic studies using whole exome sequencing are needed to clarify whether a subset of cases with early-onset OMA without the MTS on MRI frames a nosological entity with a distinct genetic basis and autosomal recessive inheritance, possibly constituting a further part of the clinical and genetic spectrum of ciliopathies.AOA1, ataxia with ocular motor apraxia 1; AOA2, ataxia with ocular motor apraxia 2; AT, ataxia teleangiectasia (AT); COMA, congenital ocular motor apraxia; JBTS, Joubert syndrome; MTS, molar tooth sign; OMA, ocular motor apraxia


The anatomy of cerebral cortex is characterized by two genetically independent variables, cortical thickness and cortical surface area, that jointly determine cortical volume. It remains unclear how cortical anatomy might influence neural response properties and whether such influences would have behavioral consequences. Here, we report that thickness and surface area of human early visual cortices exert opposite influences on neural population tuning with behavioral consequences for perceptual acuity. We found that visual cortical thickness correlated negatively with the sharpness of neural population tuning and the accuracy of perceptual discrimination at different visual field positions. In contrast, visual cortical surface area correlated positively with neural population tuning sharpness and perceptual discrimination accuracy. Our findings reveal a central role for neural population tuning in linking visual cortical anatomy to visual perception and suggest that a perceptually advantageous visual cortex is a thinned one with an enlarged surface area. •Variability in cortical thickness and surface area has opposite functional impacts•Smaller human visual cortical thickness links to high neural and perceptual acuity•Larger human visual cortical surface area links to high neural and perceptual acuity Song et al. showed that large brains are not necessarily advantageous. Instead, the two dimensions, thickness and surface area, of human brain have opposite impacts on visual perception. A perceptually advantageous brain is a thinned one with enlarged surface area. The cerebral cortex is a neural sheet composed vertically of ontogenetic cortical columns and horizontally of laminar layers . The surThis substantial variability in cortical anatomy has attracted great interest in the study of its behavioral consequences, and recent progress has been made in identifying correlations between higher performance on a variety of behavioral tasks and larger local cortical volume in task-relevant cortical regions . HoweverTo test our two hypotheses, we therefore investigated whether cortical thickness and cortical surface area, which both contribute to cortical volume, had similar or different functional impacts for neural response properties and human behavioral performance. In human cerebral cortex, the neural response properties of many cortical regions are hard to characterize using noninvasive neuroimaging techniques. Due to the limited spatial resolution of fMRI signals, different neurons within a single fMRI voxel tend to exhibit heterogeneous response properties that often render the voxel-level characterization of neural responses qualitatively different from the response properties of single neurons. An exception to this limitation is early retinotopic visual cortices. Neurons in early visual cortices respond to visual field position in an orderly fashion, where cortically adjacent neurons are tuned to spatially adjacent visual field positions . This reUsing human early visual cortices (V1 and V2) as a model system, we investigated how visual cortical thickness and visual cortical surface area influenced neural population tuning for visual field position, and whether such influences had behavioral consequences on perceptual discrimination for visual field position. Since visual cortical thickness captures the differences between visual field positions in the cortical architecture at corresponding visual cortical locations, we studied how thickness at different visual cortical locations related to the width of neural population tuning and the0 of perceptual discrimination for corresponding visual field positions. On the other hand, since visual cortical surface area reflects the differences between individuals in the proportion of cortex devoted to early visual processing, we studied how surface area of early visual cortices influenced the position tuning width and position discrimination threshold across the visual field in general.For a group of 20 healthy participants, we used structural MRI, high-resolution fMRI, and visual psychophysics to measure anatomy of early visual cortices (V1 and V2), population tuning of visual cortical neurons, and performance in perceptual discrimination, respectively. The psychophysical experiments assessing perceptual discrimination were conducted outside the scanner, while the neuroimaging experiments assessing visual cortical anatomy and neural population tuning were performed inside the scanner. During data analysis, we explored the relationships among these independent measures reflecting cortical anatomy, neural response properties, and behavioral performance, respectively.Delineation of early visual cortices (V1 and V2) used the standard method of retinotopic mapping . The mapAfter the delineation of early visual cortices, we measured visual cortical thickness and visual cortical surface area by applying the surface-based analysis on the structural MRI data collected using the standard T1-weighted MRI sequence. In the analysis, the structural MRI data were segmented into different cortical tissues, from which the 3D cortical surface was reconstructed in a smooth triangle-mesh model, with each vertex of this mesh representing a single cortical location distinguishable by MRI . Based oOur MRI-based measure of visual cortical anatomy was also potentially vulnerable to any confounding influences of data acquisition sequence. Specifically, while the T1-weighted MRI sequence we employed is a widely used standard protocol, the signal in fact represents a combination of magnetic-field-specific and biological-tissue-specific components. As a result, the T1-weighted MRI images had inhomogenous intensity and low tissue contrast that could potentially lead to bias in the segmentation of cortical tissues. To address this limitation in quality of the standard T1-weighted MRI sequence, in control experiments, we collected the structural MRI data using a state-of-art quantitative-T1 MRI sequence, at both a high resolution (0.8 millimeter [mm] isotropic voxels) and a standard resolution (1 mm isotropic voxels). Through the detection of multiple parametric signals, the quantitative-T1 MRI sequence factored out the magnetic-field-specific component and directly reflected the physical property of the underlying biological tissue . As such2 to 3,328 mm2 and V2, 1,611 mm2 to 2,936 mm2) that was correlated between V1 and V2  and did not correlate with the measure of visual cortical surface area . Therefore, we concluded that the retinotopically delineated visual cortical surface area captured true anatomical variability.From our MRI-based measure of visual cortical anatomy, we studied variability in visual cortical surface area and visual cortical thickness. Consistent with previous reports , the retntricity B. Given Compared to the large degree of interindividual variability in visual cortical surface area, the average thickness of early visual cortices varied across participants to a much smaller degree from 2 mm to 2.5 mm. Nevertheless, within individual participants, visual cortical thickness varied across different visual cortical locations from 1 mm to 4 mm following a Gaussian distribution . In addiWhile the structural MRI data allowed a noninvasive, in vivo measure of visual cortical anatomy, the measure was, at the same time, limited by its indirect nature. In contrast, a direct measure of cortical anatomy  is possible from postmortem histology. Therefore, we further addressed the reliability of our in vivo MRI measure by comparing it with an in vitro histology measure derived from postmortem human brain. Conventional analysis of histology data employs a slice-based approach that is constrained by the slice orientation and has a limited sampling coverage. For example, the slice-based measure of cortical thickness is only valid for histology slices orthogonal to the cortical surface. To overcome this limitation, we developed a surface-based approach and applied it to a data set of high-resolution (40 μm isotropic pixel) whole-brain  histology images , taken consecutively every 300 μm along the dorsoventral axis of a postmortem human brain. In the analysis, we manually segmented each histology image into different cortical tissues, from which we reconstructed the 3D cortical surface and acquired the surface-based measure of visual cortical anatomy  and from sulci to gyri , where the slope of this eccentricity-dependent increase varied across participants over 2-fold. In addition to this eccentricity-dependent variability , we founThe measure of perceptual discrimination for visual field position was based on the standard staircase procedure with a two-alternative forced-choice task. In the experiment, the visual field position difference between two horizontally offset stimuli was varied in a 2-up-1-down staircase to assess the minimum position difference that participants could discriminate. To improve the reliability of this perceptual discrimination measure, we conducted two separate experiments, employing respectively, a spatial forced-choice task where participants discriminated the position difference between two concurrently presented stimuli, and a temporal forced-choice paradigm where participants discriminated the position difference between two sequentially presented stimuli. We found that the measure of the position discrimination threshold from these two different experiments was correlated across participants , suggesting that the measure reflected a robust, trait-like perceptual variability  and six polar angles . Such a distributed coverage of the visual field allowed a comprehensive characterization of intraindividual perceptual variability. Specifically, by distributing the 13 visual field positions along both the axis of eccentricity and the axis of polar angle, we separately assessed the eccentricity-dependent and the eccentricity-independent component of intraindividual perceptual variability that reflected respectively, how the position discrimination threshold changed along visual field eccentricity and varied across different visual field positions at the same eccentricity . Across a total of 20 participants and six visual field positions at 4.7 degree eccentricity, we plotted the position tuning width of V1 neural populations and position discrimination threshold of human participants, first against each other to address the behavioral significance of neural population tuning A and theWe found that the position tuning width of V1 neural populations correlated positively with the position discrimination threshold of our participants  suggested that thickness and surface area of human early visual cortices (V1 and V2) had opposite impacts on neural population tuning that in turn affected perceptual discrimination. To study whether these observations were specific to certain eccentricity or were generalizable across the visual field, we conducted further analyses to address the impacts that visual cortical anatomy had on eccentricity-dependent variability in neural population tuning width and perceptual discrimination threshold. Specifically, by fitting the position tuning width and position discrimination threshold as a function of visual field eccentricity, we explored how visual cortical anatomy might relate to the slope and the intercept of the fit. A relationship with the slope of the fit would indicate that the position tuning width and position discrimination threshold got more dependent on visual cortical anatomy as one approached the peripheral visual field, whereas a relationship with the intercept of the fit would indicate an increased dependence toward the central visual field.Across the group of 20 participants and the retinotopically delineated coverage of the visual field (0.25–7.2 degree eccentricity), we plotted the position tuning width at individual V1 locations (vertices) against visual field eccentricities these locations responded to and V1 surface area of the participants. The data were binned into a data grid where individual data points represented the position tuning width averaged over V1 locations (vertices) that were from the same participant and responded to similar eccentricities A. This 3Thus, the functional impacts of V1 surface area on neural population tuning width, which we observed at 4.7 degree eccentricity, were generalizable across the visual field. To study the functional impacts of V1 thickness, we applied a similar analytic approach. Across the group of 20 participants and the retinotopically delineated coverage of visual field (0.25−7.2 degree eccentricity), we plotted the position tuning width at individual V1 locations (vertices) against visual field eccentricities that these locations responded to and V1 thickness at these locations. The data were binned into a data grid where individual data points represented the position tuning width averaged over V1 locations (vertices) that were similar in thickness and responded to similar eccentricities A. ThrougThese analyses suggested that, across the visual field, neural population tuning in V1 for a specific visual field position was affected jointly by thickness at corresponding V1 locations and surface area of V1. We further investigated whether such influences on neural population tuning would have behavioral consequences on perceptual discrimination. The threshold of perceptual discrimination, measured at 13 nonoverlapping visual field positions covering three eccentricities  and six polar angles , was projected onto V1 to generate a cortical map of perceptual acuity. This cortical projection allowed us to relate perceptual acuity at different visual field positions with V1 anatomy at corresponding cortical locations, using analyses similar to the ones on neural population tuning width.On a vertex-by-vertex level, we plotted the position discrimination threshold at individual V1 locations (vertices) against visual field eccentricities that these locations responded to and V1 thickness at these locations or V1 surface area of the participants A. The daTogether, these analyses suggested that our observations at 4.7 degree eccentricity, where thickness and surface area of V1 exerted opposite influences on the position tuning width of V1 neural populations with behavioral consequences on the position discrimination threshold of human participants, were generalizable across the visual field. To investigate whether such generalization was also observable in V2, we plotted the position tuning width  and posiIt is intuitive to assume that a larger cortical volume has some behavioral advantage. Indeed, within species, a positive correlation is usually observed between performance on behavioral tasks and local cortical volume in task-relevant cortical regions . HoweverWe used early visual cortices as a model system, since the orderly representation of visual field position in early visual cortices allowed fMRI-based, noninvasive characterization of neural population tuning . UtiliziOur findings suggested a larger visual cortical volume is not in itself advantageous for visual perception. Instead, a perceptually advantageous cortical design may involve a thinned visual cortex with an enlarged surface area. This is consistent with the developmental trend that sensory experience drives the expansion of sensory cortical maps, but thinning of sensory cortex . MoreoveSpecifically, thickening of visual cortex is likely to burden intracortical processing, as the axons and the dendrites of interlaminar connections would need to double and quadruple in diameter to improve interlaminar conduction speed in order to maintain the same interlaminar processing time (delay) . Due to Limited by the current resolution of noninvasive neuroimaging techniques, an empirical assessment of intracortical processing in human participants is not easy. Nevertheless, it might be of interest for future studies to explore whether a visual cortical model that incorporated these hypothetical changes in intracortical processing could reproduce our empirically observed correlations between visual cortical anatomy and neural population tuning. Regardless of the underlying mechanisms, our findings revealed that the population tuning properties of visual cortical neurons play an important role in linking visual cortical anatomy to visual perception. We showed that it is not cortical volume per se, but rather the associated neural response properties that mediate the influences of cortical anatomy on behavioral performance. This raises concerns for the classical approach taken in studying the anatomical basis of behavioral performance, where one simply searches for cortical regions whose local volume correlates positively with behavioral performance . By demoIn a group of 20 healthy human adults, we studied the relationships among the anatomy of early visual cortices (V1 and V2), the width of neural population tuning, and the threshold of perceptual discrimination, measured respectively using structural imaging, fMRI, and visual psychophysics. First, we acquired the measure of visual cortical anatomy by applying the surface-based analysis to early visual cortices delineated on the structural imaging data. To improve the reliability of the measure, we used different experimental paradigms, where we delineated early visual cortices retinotopically according to the phase-encoded map , retinot


Neurobeachin (Nbea) have been generated independently. Although both models have similar phenotypes, one striking difference is the dwarf phenotype observed in the heterozygous configuration of the GH240B model that is generated by the serendipitous insertion of a promoterless human growth hormone (hGH) genomic fragment in the Nbea gene. In order to elucidate this discrepancy, the dwarfism present in this Nbea mouse model was investigated in detail. The growth deficiency in Nbea+/− mice coincided with an increased percentage of fat mass and a decrease in bone mineral density. Low but detectable levels of hGH were detected in the pituitary and hypothalamus of Nbea+/− mice but not in liver, hippocampus nor in serum. As a consequence, several members of the mouse growth hormone (mGH) signaling cascade showed altered mRNA levels, including a reduction in growth hormone-releasing hormone mRNA in the hypothalamus. Moreover, somatotrope cells were less numerous in the pituitary of Nbea+/− mice and both contained and secreted significantly less mGH resulting in reduced levels of circulating insulin-like growth factor 1. These findings demonstrate that the random integration of the hGH transgene in this mouse model has not only inactivated Nbea but has also resulted in the tissue-specific expression of hGH causing a negative feedback loop, mGH hyposecretion and dwarfism.Two knockout mouse models for the autism candidate gene  NEUROBEACHIN (NBEA) has previously been identified as a candidate gene for autism spectrum disorder (ASD) based on associations and linkage studies and patients with monoallelic inactivation of NBEA Nbea heterozygous and knockout mouse models have been used. Independently, two models have been generated in which the Nbea gene is disrupted growth hormone (hGH) genomic fragment. In the GH240B transgenic line this insertion occurred in intron 1 of Nbea leading to a genuine null allele GH minigene was examined and could not be detected. The second Nbea mouse model was generated by means of the gene-trap vector system (further referred to as the gene-trap mouse) −/−Nbea mice +/−Nbea mice also revealed abnormal morphology of dense-core granules in blood platelets, similar to the first described patient with NBEA haploinsufficiency. Furthermore, a decrease in several actin-associated peptides was observed. In addition, these studies in blood platelets confirmed the important role of NBEA as an A-kinase anchoring protein, affecting phosphorylation levels of several PKA substrates A and glutamate receptors To further investigate the function of NBEA, Nbea−+/ mice. This growth deficiency is only present in the GH240B Nbea−+/ mouse model Nbea−+/ mice have a normal stature accompanied by a higher body weight due to an increased adipose tissue mass One striking difference between both mouse models is the presence of a dwarf phenotype in Nbea−+/ mice was investigated in detail. A growth curve was generated and the expression of genes in the GH pathway was measured at mRNA and protein level. Alterations in body composition were detected via dual-energy X-ray absorptiometry (DEXA). Pituitary cell numbers were determined and pituitary size and the ultrastructure of somatotrope cells were examined. The responsiveness of somatotrope cells to secretagogues was analyzed in ex vivo cultures of anterior pituitaries. Finally, hGH expression, which was previously found to be undetectable, was re-examined in serum, pituitary, hypothalamus, hippocampus and liver.In order to elucidate this discrepancy, the dwarf phenotype of GH240B ad libitum. All experiments were performed with 12 week old male mice unless otherwise stated.The GH240B transgenic line All experiments were approved by the ethical research committee of KU Leuven in accordance with the declaration of Helsinki (project number P182/2011).Nbea+/+ mice and 17 male Nbea−+/ mice were included.Mice were weighed weekly starting at postnatal day 2 until mice reached adulthood (12 weeks of age). Afterwards, mice were weighed once a month until they were 1 year of age. In total 10 male Mice were anesthetized by intraperitoneal injection with 10 mg/kg Xylazine  and 100 mg/kg Ketamine . The body composition of anesthetized mice (n = 5/genotype) was measured by DEXA  using ultra-high resolution  and software version 1.45.RNA of the anterior pituitary, hypothalamus and liver was extracted with the Nucleospin RNA XS or L kit  according to the manufacturer's protocol. Per experiment, an equal amount of RNA was reverse transcribed to cDNA using the iScript cDNA synthesis kit . Real-time qRT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad). 40 cycles of annealing/extension for 1 min at 60°C were carried out with the MyiQ single color real-time PCR detection system (Bio-Rad). The relative amount of mRNA expression was calculated using the method of Livak with normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as reference gene Murine (m)GH levels were quantified using the Rat/Mouse Growth hormone ELISA  according to the supplier's protocol. The content of human (h)GH was calculated by means of the HGH human direct ELISA kit  according to the manufacturer's instructions. The plasma samples measured were derived from blood taken at 3 different time points to cover the peak of GH release in the mouse, namely 09:30 h, 10:00 h and 10:30 h Mice were anesthetized with an intraperitoneal injection of 50 mg/kg Nembutal . After gravitational perfusion of the mice with Karnovsky's fixative , pituitary glands were isolated and additionally fixed for 2 hours at room temperature, followed by an overnight immersion at 4°C in Karnovsky's fixative. After the samples were washed and postfixed with 1% OsO4, they were dehydrated through a series of increasing ethanol concentrations and embedded in epoxy resin. Ultrathin sections (70 nm) were collected on formvar-coated copper grids and stained with lead citrate and uranyl acetate.TEM of adenopituitary tissue sections was performed at 80 kV using a JEM1400 transmission electron microscope . All micrographs were acquired on a SIS Quemesa camera with the same spatial resolution. One pixel corresponds to 12.44 nm. Somatotropes were identified based on their morphological description as defined in 2 asphyxiation followed by immediate decapitation. The neural and intermediate lobes of the pituitary were discarded using a stereomicroscope. The anterior lobe (anterior pituitary) was isolated and dissociated into individual cells with trypsin. Cells were resuspended in serum-free defined medium  and spun down on Cytospin slides . Cells were permeabilized with 0.5% saponin  after fixation in 4% paraformaldehyde, and were incubated overnight with antibodies against murine GH , prolactin (PRL) , adrenocorticotropic hormone (ACTH)  and α-glycoprotein subunit (αGSU) . Hormone antisera were obtained from Dr. A. F. Parlow . Subsequently, cells were incubated for 1 h with fluorescently labeled secondary antibodies  and 6-diamidino-2-phenylindole (DAPI). Images were captured using an Olympus IX81 microscope. Cells were counted using ImageJ  in at least 5 random cytospin fields  and the proportions of immunopositive cells on the total anterior pituitary cells were determined. To take into account the smaller size of the anterior pituitary and the correspondingly lower number of total anterior pituitary cells  in the +/−Nbea GH240B mice, we calculated the decrease in absolute cell number of the different cell types .This experiment was performed as described before Primary cultures were generated based on the protocol described Anterior pituitary, hypothalamus and liver were homogenized in RIPA buffer containing a protease and phosphatase inhibitor cocktail. Anterior pituitaries and hypothalami of littermates were pooled and homogenization was performed manually using a pellet pestle for 1.5 ml tubes. Liver tissue was homogenized with a motor-driven Potter-Elvehjem tissue grinder . The protein concentration of the samples was quantified with a BCA protein assay . 25 µg of lysate was loaded on a 10% Bis-Tris gel . Proteins were transferred to Protran Nitrocellulose membrane  and incubated with antibodies against human GH (hGH)  and actin , used for normalization. Afterwards, membranes were incubated with HRP-conjugated secondary antibody  and proteins were visualized with western blotting ECL detection reagent.Total pituitaries were dissected, followed by an overnight fixation in 4% formaldehyde. After rinsing in 70% ethanol, pituitaries were embedded in paraffin according to standard procedures. Tissue blocks were cut in 5 µm sections with subsequent removal of paraffin with Histo-Clear  and dehydration through serial ethanol solutions. Preceding a 30 min block in 10% goat serum in PBS with 2% BSA, sections were incubated for 20 min in target retrieval solution, pH 6 (Dako). Sections were incubated for 1.5 h with mouse anti-hGH antibody  and subsequently for 1 h with Alexa 488–labeled anti-mouse antibody  before mounting in ProLong Gold Antifade Reagent (Dako) containing DAPI. Immunofluorescence was visualized by Axiovision software  with the Axio Imager Z1 microscope (Zeiss).t test, analysis of variance (ANOVA) or repeated measures (RM)-ANOVA procedures with Tukey test post hoc comparison using Statistica version 9.0 . All statistical tests were performed with 0.05 as the α level of significance .Data are presented as mean with standard error of the mean (SEM). Significance of differences between mean values were analyzed using (where appropriate) two-tailed Nbea−+/ GH240B mice, their body weight was measured from postnatal day 2 until mice were 1 year old  = 17.3, P<0.001). This dwarf phenotype was apparent after 4 weeks and became statistically significant starting 7 weeks after birth and persisted into adulthood. As mice became older, Nbea−+/ mice showed an increased weight gain in comparison to Nbea+/+ mice  = 8.4, P<0.001) , despite similar weight (To characterize the growth retardation of year old . The bodP<0.001) . At 1 yer weight .Nbea−+/ mice. Therefore, the body composition of adult mice was determined by DEXA. Although the total tissue mass was significantly lower in +/−Nbea mice than in Nbea+/+ mice     , Nbea+/P<0.001) . In addiP<0.001) .Nbea−+/ mice in combination with the altered body composition suggested abnormalities in the GH signaling cascade. To assess alterations in the GH/IGF-1 axis, mRNA expression of the relevant genes was analyzed in anterior pituitaries, hypothalami and livers from adult mice (Gh-releasing hormone (Ghrh) expression was detected  (Ghrelin was too low to be conclusive. The expression levels of somatostatin (Sst), Sst receptors (Sstr) 4 and 5, and Gh receptor (Ghr) did not significantly differ between genotypes . In the anterior pituitary, a significant decrease in Gh mRNA expression was accompanied by a significant increase in Ghr and the most abundantly expressed somatostatin receptor Sstr2 mRNA expression  (Ghrh receptor (Ghrhr), Ghrelin receptor (Ghrlr) and Sstr5 were detected . One of the main consequences of peripheral GH signaling is the production of IGF-1 in the liver Igf-1 mRNA levels in the liver were determined, combined with the quantification of IGF-1 present in plasma. A significant decrease in Igf-1 mRNA in the liver was detected  which resulted in a significantly reduced level of IGF-1 in the plasma  (The observed dwarf phenotype of ult mice . In the = 0.008) . The exp= 0.002) . No sign= 0.001)  D and E.Nbea−+/ mice became apparent . Furthermore the cell composition of these anterior pituitaries was assessed and a 55%±3 and 55%±9 decrease in the absolute number of GH- and prolactin- (PRL) positive cells, respectively, was detected in the anterior pituitary of Nbea−+/ mice when compared to control mice   .To investigate any morphological abnormalities of the GH-producing cells, transmission electron microscopy was performed. However, the ultrastructure of the somatotrope cells and the large electron-dense granules containing GH appeared unaffected .Nbea−+/ mice secreted significantly less GH compared to Nbea+/+ mice  = 32.3, P<0.001 and post hoc comparison: basal medium: P<0.001 and stimulated medium: P<0.001)  = 21.0, P<0.001 and post hoc comparison: cell lysate: P = 0.003). Consistent with a reduced amount of GH per somatotrope cell in Nbea−+/ mice, the total GH content of 200,000 anterior pituitary cells, defined as the sum of GH in basal and stimulated medium and the cell lysate, was decreased in +/−Nbea mice with 71%±3 compared to the total GH content as measured in +/+Nbea mice , while the reduction of GH cells was only 55% (see above). We analyzed the effect on secretion by normalizing the absolute amount of GH in the medium for their total GH content and found no significant difference for genotype in basal nor in stimulated secretion  = 1.28, P = 0.27; condition: F  = 1135.9, P<0.0001; interaction genotype x condition: F  = 0.212, P = 0.65), indicating a normal responsiveness of GH secretion upon stimulation  . In addimulation . Taken tNbea mouse model used in this study was generated serendipitously by integration of a transgenic construct of hGH into intron 1 of the Nbea gene resulting in a Nbea null allele. Although the absence of hGH expression in the pituitary was previously shown by RT-PCR The GH was determined in different tissues . Although the hGH expression in the hypothalamus was also significantly higher compared to Nbea+/+ mice , the hGH expression was much lower in comparison to the anterior pituitary, albeit still clearly detectable. The liver of both Nbea−+/ and Nbea+/+ mice contained at best trace levels of hGH mRNA .Relative mRNA expression of h tissues . In the Nbea−+/ mice, but not in the hypothalamus and liver. As expected, no hGH could be detected in anterior pituitary, hypothalamus and liver of Nbea+/+ mice.To assess whether the mRNA levels correlated with protein levels, a western blot of hGH was performed on lysates of the same tissues . hGH proNbea−+/ mice , but at much smaller amounts than in the pituitary which is consistent with mRNA data (In order to determine the ratio of hGH relative to murine GH (mGH) in the anterior pituitary of  present , leadingRNA data . It is wTo find out whether hGH is more generally expressed in the brain, levels were also measured in lysates of the hippocampus since this region has the highest expression of NBEA. The values detected in both wild-type and heterozygous mice were below the detection limit of the ELISA .To analyze whether hGH is secreted under basal and stimulated conditions, the hGH levels were measured in the samples of the primary anterior pituitary cell cultures. Under basal conditions, 0.99±0.10 ng hGH was detected in the medium, just above the detection limit of the ELISA. After stimulation with IBMX and Forskolin, 3.3±0.3 ng hGH is secreted in the medium, which is 79 times less than mGH. The remaining level of hGH in the cell lysate was 3.5±0.1 ng hGH . The sigTo assess whether hGH could be detected in the circulation, blood samples were taken at 3 different time points, namely 09:30 h, 10:00 h and 10:30 h, encompassing the peak of GH release in the mouse Nbea−+/ GH240B mouse model generated by Su et alNbea−+/ GH240B mice have an increased percentage of fat mass concomitant with a decrease in lean mass and bone mineral density. This phenotype is the result of alterations in the GH/IGF-1 axis caused by hGH expressed from the transgene in the pituitary and hypothalamus of Nbea−+/ GH240B mice. GHRH levels in the hypothalamus are suppressed, resulting in reduced somatotroph stimulation, causing reduced cell numbers and mGH content.In this study we have delineated the dwarf phenotype in the GH was accidently inserted in intron 1 of Nbea, the absence of expression of hGH in the pituitary was verified by RT-PCR +/−Nbea mouse models regarding dwarfism prompted us to reevaluate the expression of hGH, especially since 5 different transgenic mouse models were generated with the same transgenic hGH construct and analysis of pituitary mRNA revealed hGH expression in 3 of these mouse models +/−Nbea GH240B mice, using qRT-PCR and ELISA. Most likely, the absence of HS I and II resulted in a minimal expression of hGH reflecting the basal functions of the proximal regulatory elements of the hGH gene present in the transgenic construct When the transgenic construct of hNbea−+/ GH240B mice is probably at the basis of the observed GH deficiency and subsequent dwarfism. Other transgenic mouse models with targeted expression of hGH in the hypothalamus also present dwarfism and similar alterations at the level of the pituitary to Nbea−+/ GH240B mice Ghrh mRNA in the hypothalamus is significantly reduced leading to a decrease in pituitary mGh mRNA and protein levels GH gene was inserted into the AlfpCre transgene leading to the presence of hGH protein in the hypothalamus and pituitary The presence of hGH in the hypothalamus of Nbea−+/ GH240B mice, the brain region with the highest expression of Nbea, indicating that expression of hGH is not directed by the Nbea promoter but promoter elements in the hGH transgene itself, only activated in specific tissues. This is also consistent with the fact that the two genes are in opposite directions The 500-fold lower levels of hGH in the pituitary compared to mGH and the undetectable levels in the blood preclude a significant effect of hGH on IGF-1 production in liver, while the low expression level of hGH in hypothalamus is apparently sufficient to locally activate the mGHR and suppress GHRH expression. A possible explanation can be that, since mGH is not expressed in hypothalamus and can only activate the mGHR in this tissue through an endocrine mechanism, the hGH/mGH ratio in hypothalamus will probably be many times higher than in pituitary. However, we did not determine this ratio in the hypothalamus. No hGH was detected in the hippocampus of Nbea−+/ GH240B mice, the total number of GH- and PRL-producing cells in the pituitary of mice with transgenic hGH expression in the hypothalamus is reduced whereas other cell types were unaffected leading to hypoplasia of the pituitary Ghrhr deficient little mice and the Pc1/3 knockout mice in which the maturation of proGHRH is blocked Ghrh promoter Similar to ex vivo cell cultures both under basal condition and upon stimulation with GHRH Nbea−+/ GH240B mice, Forskolin and IBMX were applied as secretagogues mimicking the rise in cAMP caused by GHRHr activation Nbea−+/ mice. The observed reduced presence of GH in the medium of cell cultures of Nbea−+/ mice, both in basal and stimulated conditions, is most likely caused by the reduced presence of somatotrope cells in the anterior pituitary of Nbea−+/ mice and the lower GH content per GH-producing cell. Since NBEA has been identified as a negative regulator of regulated secretion in vitroex vivo cell cultures derived from Nbea−+/ mice. A possible explanation for the similar ratios is that compensatory mechanisms in the somatotrope cell population of Nbea−+/ GH240B mice mask any ex vivo effect of Nbea haploinsufficiency on regulated secretion.The somatotrope cells present in the transgenic mice expressing hGH in the hypothalamus retain their functional capacity for GH secretion in Sstr4 and 5 were selected due to their predominant presence on neurons producing GHRH and Ghrelin Sstr2 and 5 are most highly expressed Sstr2 and 5 are also expressed in thyrotrophs Ghrh mRNA in the hypothalamus in combination with the increased mRNA level of Sstr2 in the anterior pituitary might be at the basis of the reduced mGh mRNA detected in the anterior pituitary of Nbea−+/ mice. Most likely, in an attempt to compensate for the decrease in mGH, the mRNA level of Ghr is elevated in the anterior pituitary of Nbea−+/ mice. The 85% reduction in mGh in anterior pituitaries of Nbea−+/ mice causes a 50% decrease in Igf-1 mRNA in the liver and a 66% decrease in IGF-1 protein detected in plasma. Reduced plasma IGF-1 levels correlate with the dwarf phenotype and increased fat percentage For the assessment of the mRNA profile of members of the GH cascade, only the most relevant SST receptors were included, although 5 different SST receptors are present both in pituitary and hypothalamus Nbea GH240B mouse model used in our experiments obfuscates the interpretation of body weight, although the body weight of 1-year-old Nbea−+/ mice exceeds the body weight of Nbea+/+ mice. Normal growth was reported for the Nbea−+/ gene-trap mice and they also have an increased ratio of adipose tissue resulting in a higher body weight Nbea haploinsufficiency rather than solely with the method of generating the Nbea mouse model. However, it cannot be excluded that the gene-trap insertion has affected the expression of other genes involved in adipogenesis.The dwarf phenotype in the An important issue arising from this study is whether the mouse model is still useful for studying NBEA function and its role in ASD. The two models are highly similar with the exception of growth deficiency. In addition, behavioral studies with the GH240B mouse model have shown abnormalities in sociability, and spatial learning and memory and show an increase in conditioned fear and in repetitive self-grooming behavior, making this a highly promising model for the study of ASD Another argument highlighting the usefulness of the GH240B model is that abnormalities in platelet morphology, which were first noticed in several ASD patients including one with NBEA haploinsufficiency, are also observed in platelets of this mouse model Nbea mouse models are mostly comparable. Taken together, this correlation between the two mouse models, combined with correlations with the ASD patient and cell lines, gives credence to all findings regarding NBEA function. However, to exclude the confounding effects of hGH we recommend using the gene-trap mouse models in the future to study the physiological function of NBEA and its role in the pathogenesis of ASD.This study has unveiled the molecular basis for the discrepancy between the two commonly used mouse models for NBEA deficiency. Despite the growth deficiency caused by recombinant hGH expression, the two Table S1Primers per gene used for quantitative reverse transcription PCR. The sequences of the primers are presented in a 5′ to 3′ orientation. Gh: growth hormone; Ghr: Gh receptor; Ghrh: Gh-releasing hormone; Ghrhr: Ghrh receptor; Sst: somatostatin; Sstr: Sst receptor; Ghrelinr: Ghrelin receptor; hGH: human GH; Igf-1: insulin-like growth factor 1; Gapdh: glyceraldehyde 3-phosphate dehydrogenase.(PDF)Click here for additional data file.


In the Republic in Congo, the national algorithm for the diagnosis of pulmonary tuberculosis (TB) relies on Ziehl-Neelsen (ZN) sputum smear microscopy, chest X-ray radiography (CXR) and clinical symptoms. Microscopy positive pulmonary TB (MPT+) is defined as symptoms of TB and a positive ZN smear. Microscopy negative pulmonary TB (MPT-) is defined as symptoms of TB, a negative ZN smear but CXR changes consistent with TB. The present cross-sectional study was designed to determine the prevalence of positive and negative MPT individuals among HIV positive and HIV negative individuals presenting to an ambulatory TB treatment center (CTA) in Brazzaville.All study participants underwent a physical examination, chest radiography and three ZN sputum smear examinations and HIV testing. Viral load and CD4 counts were determined for HIV positive individuals.775 individuals presented with symptoms of TB. 425 individuals accepted the voluntary HIV test. 133 (31.3%) were HIV positive (HIV+) and 292 (68.7%) were HIV negative (HIV-). Of the 292 HIV- individuals 167 (57%) were classified as positive MPT and 125 (43%) as negative MPT. Of the 133 HIV positive individuals 39 (29%) were classified as MPT + and 94 (71%) as MPT-.Our study shows that the prevalence of positive MPT individuals is lower among HIV positive individuals compared to HIV negative individuals in agreement to reports from other countries. The data suggest that a substantial number of HIV positive pulmonary TB cases are not detected by the national algorithm and highlight the need for new diagnostic tests in this population. There are approximately 34 million people currently living with HIV, one-third of these is estimated to also be infected with tuberculosis (TB) –3. AlthoIn sub-Saharan Africa, TB is often the first manifestation of HIV infection, and it is the leading cause of death among HIV infected patients –7. The cThe diagnosis of HIV-associated TB is often challenging and nucleic acid amplification tests, including the GeneXpert MTB/RIF assay and urinary LAM assays are known to improve the diagnosis of both smear-negative and extrapulmonary TB in HIV positive individuals .TB diagnosis worldwide currently heavily relies on sputum smear microscopy to detect acid-fast bacilli (AFB), as it is rapid, inexpensive, and highly specific for identifying active pulmonary tuberculosis (PTB) , 14. ThiThe study participants were recruited at the anti-tuberculosis center (CAT) of Brazzaville during the study period from February to June 2011. All consecutive TB suspects regardless of HIV status were eligible for the study. Informed consent was obtained from all study participants or their guardians. Ethical authorization was obtained from the Comité d’éthique Institutionnel de la Recherche en Santé (CERSSA) (No. 00000067/DGRST/CERSSA).Demographic data  and clinical data were recorded for all participants during a medical interview and a physical examination by a clinician. Chest radiography and sputum collection was performed according to the national diagnostic algorithm. Participants provided three sputum specimens over the course of 2 days. The first specimen was collected when the patient attended the CAT for the first time. A container was given to the participant for collecting an early morning sample at home the next day, and a third was collected again at the center when the patient brought back the early morning specimen. The samples were tested with the direct Ziehl-Neelsen light microscopy method following standard instructions on proper sputum submission. Sputum microscopy was performed at the onsite laboratory, with density of acid fast bacilli graded as 1, 2 or 3 + .Germany) and ImmunoComb II HIV 1 & 2 BiSpot . In case of discordance between these two tests, the enzyme-linked immunosorbent assay  was performed by the reference lab which is the Laboratoire National de Santé Publique.Blood samples were tested using two rapid tests, Determine HIV 1/2 test , following manufacturer’s instructions.Approximately 5 ml of blood was collected in an EDTA tube for counting T cells. The blood samples were processed immediately within 2 hours of collection, for determination of the absolute counts of CD4+, CD8+ cells by activated cell sorting (FACS) count . The determination of viral load was performed at the Laboratory of Molecular Biology at the Faculty of Health Sciences /University Marien Ngouabi, Brazzaville.At the first consultation, all patients suspected of TB were clinically assessed by a trained health professional. The assessment includes screening for clinical signs and symptoms such as current cough (if self-reported cough was > 2 weeks), fever, night sweats and weight loss. Sputum samples were collected and examined using Ziehl-Neelsen staining microscopy. A chest X-ray was performed and HIV testing was offered to all individuals.Pulmonary TB (PTB) positive microscopy: Patients with at least 2 positive sputum samples were classified as positive for pulmonary TB. These PTB positive patients were given the standard anti-TB treatment according to National guidelines.Pulmonary TB negative microscopy: Patients with at least 2 sputum samples negative for AFB and with symptoms of TB, a negative ZN smear but CXR changes consistent with TB were classified as negative for pulmonary TB. These PTB negative patients were treated with a full course of anti-TB chemotherapy if 15 days later the screening is positive.Patients without pulmonary TB: Individuals with negative sputum samples for AFB and no X-ray changes suggestive of TB.The demographic, clinical and laboratory results were entered into a database and analyzed using SPSS version 16 . The mean and standard deviation of the age, CD4 and CD8 T cells and viral load were calculated using student’s t-test for comparison. Logistic regression was used to evaluate the effect of age, gender and clinical parameters on HIV infection. Kruskal-Wallis test was used to compare the proportion of HIV infected patients with different levels of CD4+, CD8+ T cells and viral load between AFB positive and negative microscopy. Differences were considered statistically significant when the p value was ≤ 0.05.From February to June 2011, 775 patients attending the consultation service at the TB center of Brazzaville (CAT) were deemed suspected TB cases based on clinical examination  were classified as PTM positive and 125 (43%) as PTM negative. Of the 133 HIV + individuals 39 (29.3%) were classified as PTM positive and 94 (70.7%) as PTM negative  in patients on antiretroviral therapy with undiagnosed and those with untreated TB .Mycobacterium tuberculosis (Mtb) and rifampicin resistance [Many studies have confirmed the superior effectiveness of culture methods when compared to smear microscopy , 39, 40.sistance . GeneXpesistance –44. Recesistance , 46.The limit of the National algorithm for the diagnosis of TB infection is that no culture lab exists in the country. Moreover, with regard to high risk of HIV people to get TB infection, it would be very useful to test negative sputum samples with the WHO endorsed GeneXpert technology.In countries were TB and HIV are endemic, accurate and rapid diagnosis tools should be implemented. It is crucial to have access low-cost tools that are adapted to countries where resources are limited and to support health care providers with adequate training. This study showed that the current national treatment algorithm may adequately detect smear positive pulmonary TB in HIV- individuals.In the present study, a consistent number of patients with smear-negative microscopy and negative radiology were found; therefore it is possible that many patients with negative smear pulmonary TB did not have TB. In order to avoid over-TB treatment, it appears urgent to use locally specific diagnostic methods such as culture or GeneXpert. However, our data also suggest that additional diagnostic modalities are required to diagnose TB in HIV + individuals, especially those with smear-negative pulmonary tuberculosis.