In parallel, primary neuronal cultures are adapted to study axonal transport but they lack the physiological organization of neuronal networks, which in turn may bias observations. We previously developed a microfluidic platform to reconstruct a physiologically-relevant and functional corticostriatal network in vitro that is compatible with high-resolution videorecording of axonal trafficking. Here, using this system we report progressive changes in axonal transport kinetics of both dense core vesicles and mitochondria that correlate with network development and maturation. Interestingly, axonal flow of both types of organelles change in opposite directions, with rates increasing for vesicles and decreasing for mitochondria. Overall, our observations highlight the need for a better spatiotemporal control for the study of intracellular dynamics in order to avoid misinterpretations and improve reproducibility.Studying intracellular dynamics in neurons is crucial to better understand how brain circuits communicate and adapt to environmental changes. In neurons, axonal secretory vesicles underlie various functions from growth during development to plasticity in the mature brain. Similarly, transport of mitochondria, the power plant of the cell, regulates both axonal development and synaptic homeostasis. However, because of their submicrometric size and rapid velocities, studying the kinetics of these organelles in projecting axons  The motile nature and submicrometric size of cellular organelles make their study extremely difficult in vivo because it requires technology with high spatial and temporal resolution that have yet to be developed. This is particularly true for axonal trafficking of dense core vesicles (DCV) that transports key elements for neuronal growth and transmission. In fact these organelles that are only few hundreds nanometers in size can travel at several micrometers per seconds1, which make them extremely difficult to track. Similarly, the relatively small size and highly dynamic nature of mitochondria renders their observation equally challenging in vivo and requires high-resolution and high-frequency image acquisitions2. Consequently, the exact molecular events controlling subcellular rearrangements and intracellular trafficking in axons and in dendrites within neuronal networks are not fully understood.Understanding mechanisms that drive the establishment, maturation, function and dysfunction of neuronal networks on a subcellular level requires microscopic approaches that are often technically challenging in the in vivo and in vitro analyses and that would allow systematic and reproducible analyses of intracellular dynamics.One way to overcome these limitations is to use primary cultures of neurons that are extracted from embryonic brain and seeded in a dish. However without a proper control of neurite outgrowth and directionality, neurons often make random, nonspecific, multidirectional and uncontrolled synaptic contacts that may jeopardize the validity of observations. The difficulty to recapitulate the complexity of brain networks composed of multiple neuronal identities complicates the assessment of microscopic events at homo- or heterotopic synapses. In addition, intracellular dynamics are often studied at a unique time point within a given culture, although intracellular dynamics may vary between developing and matured neurons, but also from one culture condition to another. This lack of rigorous temporal identification may therefore affect the dynamicity of organelles and may lead to discrepancies between studies. Therefore, there is a crucial need to develop culture systems that could bridge the gap between in vitro microfluidic system for recording intracellular dynamics with spatial and temporal control by reconstituting a compartmentalized, oriented and functional neuronal network3. Space compartmentalization was achieved using a 3-chamber microfluidic design allowing the separation of the different components of neuron’s architecture 4. Time compartmentalization was achieved by determining the different stages of neuronal network development using selective markers of neurite outgrowth, synapse formation and transmission, as well as neuronal activity. Because of the standardized architecture and specific physical and chemical constraints of the microfluidic platform, neuronal networks develop with specific kinetics that are similar through different devices. In this configuration, network development can be synchronized between different conditions, thus facilitating systematic analyses and reproducibility7. Using these spatiotemporal features, we cross-compared axonal trafficking of two motile organelles, dense core vesicles and mitochondria, throughout network maturation. We found marked changes in the dynamicity of axonal trafficking for both organelles that correlated with the progressive maturation of the network. Interestingly, trafficking kinetics of vesicles and mitochondria evolved in opposite directions, as demonstrated by the progressive acceleration and densification of anterograde vesicles compared to the dramatic reduction in motile mitochondria in mature axons.We recently reported an 3. This system uses a silicon polymer-based microfluidic device composed of two fluidically-isolated neuronal chambers that are connected via a set of thin microchannels through which neurites can grow and contact each other in an intermediate synaptic compartment. We previously determined optimal conditions using physical and chemical constraints  so that neuronal cultures between distinct devices are standardized and can be compared3. Using primary neuron cultures, we reconstituted a corticostriatal network in which cortical neurons project to striatal target neurons through oriented axodendritic connections  can be systematically identified at each stage of the culture, which allows to establish a temporal map of intracellular dynamics. We therefore took advantage of this system to cross-compare the trafficking kinetics of axonal DCVs and mitochondria at different stages of network maturation .We have recently developed a microfluidic-based approach that enables the reconstruction of a time- and space-controlled neuronal network compatible with fast spinning confocal videomicroscopyons Fig. . Because9. Mitochondrial transport was analyzed using cytochrome c oxidase subunit VIII fused to the fluorescent DsRed2 protein (Mito-DsRed2), a widely used reporter of mitochondria trafficking10. Videorecording was performed at high frequency and high resolution using spinning-disk confocal videomicroscopy. To ensure that organelle trafficking was recorded in axons only, the field of acquisition was placed over the most distal section of the 500 µm-long channels , retrograde (synapse to soma), pausing  and static (no movement). The velocity and number of motile units were used to evaluate the net directional flux  and the linear flow rate .To study the dynamics of DCVs, we used lentiviral expression of Brain-Derived Neurotrophic Factor fused to the fluorescent mCherry protein (BDNF-mCh) in cortical neurons. BDNF is a prototypical cargo of DCVs endogenously expressed in cortical neurons and transported in axons to promote the survival of striatal neurons3. Corticostriatal transmission was determined using the glutamate sensor iGluSnFR12 that selectively measures glutamate release from cortical axons since cortical projecting neurons are glutamatergic while striatal neurons are GABAergic14  and postsynaptic density protein-95 (PSD95) . The number of iGluSnFR-positive spots then progressively increased until reaching a steady state at DIV 14, indicating that the network was fully functional after 2 weeks in culture , mid (DIV 10) and late (DIV 14 and DIV 21) Fig. . VelocitWe then studied mitochondrial trafficking in cortical axons from early to late stages of development using the Mito-DsRed2 mitochondrial marker Fig. . We obse16. To do so, cortical neurons were transiently incubated with the dye at DIV 4 (early immature) or at DIV 14 (late mature) and mitochondria axonal trafficking was analyzed  with global neuronal activity . By recording axonal trafficking dynamics of two distinct cargoes (DCVs and mitochondria) at different developmental stages of the network, we found opposite changes in their dynamicity that we could directly link with the progressive maturation of the neuronal circuit.In this study, we used a microfluidic platform that allows the In vivo, brain structures such as the cortex and the striatum are physically separated. They connect over long distances via oriented, unidirectional axodendritic contacts. Microfluidic devices are unique tools to isolate and compartmentalize cells in vitro. By using an optimized version of the previous generation of compartmentalized microfluidics we reconstructed an in vitro corticostriatal network compatible with subcellular investigations4. In this system, cortical neurons specifically project their axons onto striatal neurons through a series of individual microchannels while cell bodies remain in their respective seeding chambers. This facilitates the study of specific intracellular events in fluidically isolated cell compartments . Furthermore, the presence of an intermediate chamber connecting axonal and dendritic channels further isolates synaptic contacts which allows the study of molecular dynamics in pre- and post-synaptic elements of the synapse.in vitro, such as cortico-cortical, cortico-hippocampal, hippocampo-hippocampal networks or even neuron-muscle connections19. In addition, this system can be used in normal and pathological conditions using neurons from transgenic mouse models3 or induced stem cells from human patients, to study cellular and molecular alterations underlying network dysfunctions.Microfluidics are highly versatile in nature so that they can be used with virtually any types of cells, including neuronal and non-neuronal cells, as long as their primary culture is made possible. Consequently many types of physiological neural networks can be reconstituted 15 or iGluSnFR to quantify excitatory transmission (a readout of synapse maturation)12. A wide variety of other biological analyses can be envisaged such as nuclear gene expression using luciferase reporter gene20, axonal endoplasmic reticulum dynamics using ER-GCaMP-15021, activity-dependent cytoskeleton remodeling using live actin imaging22, and exocytosis using pHluorin-tagged reporters23.Because microfluidic systems are compatible with high-resolution microscopy they allow the simultaneous analysis of multiple subcellular events, including intracellular transport, synapse dynamics, transmission, neuronal activity and global network synchrony Fig. . In this9. This is particularly exemplified in corticostriatal disorders such as Huntington’s disease in which BDNF axonal transport is deeply affected24. In addition, both BDNF and mitochondria axonal trafficking have been shown to be critical for the development and branching of cortical projecting neurons26. The combined analyses of BDNF and mitochondria axonal transport using our time-controlled microfluidic device revealed opposite regulatory changes during the development of the system. These observations could be used to further investigate in vivo the exact contributions of both cellular event in the growth and branching of cortical axons.The corticostriatal neuronal circuit is particularly relevant for the study of BDNF trafficking because the trophic support of striatal neurons depends on cortical axonsin vivo study reporting decreased motility of mitochondria in adult brains compared to developing neuronal circuits27, which supports the physiological integrity of our reconstituted network. Interestingly, the number of motile mitochondria dramatically decreases in the late, mature stages of network maturation whereas the total number of axonal mitochondria remains stable over time. This 2-fold reduction in motility leads to an increase of stationary mitochondria in the axon over time. In contrast, transport kinetics of DCVs demonstrated increased dynamics upon network maturation. These opposite changes therefore suggest distinct regulatory mechanisms and molecular actors which may be selective for each organelle. In the case of mitochondria trafficking, increased electrical activity and Na+ channel activation have been shown to slow axonal transport and to increase the number of stationary mitochondria28. This activity-dependent regulation of mitochondrial transport in axons may be mediated by the direct coupling between kinesin and syntaphilin29. Immobilization of mitochondria via syntaphilin overexpression has been associated with increased axonal branching and synaptic formation in the distal axon26. In contrast, activity-dependent increase in vesicular trafficking has been associated with microtubule polyglutamylation30, kinesin phosphorylation31 or motor redistribution32, although these mechanisms have not been studied specifically in axons and remained to be elucidated. These distinct molecular mechanisms may therefore underlie the opposite changes in mitochondria and vesicular motility during axonal maturation and synapse formation.Using the microfluidic platform, we observed that the axonal trafficking of secretory vesicles and mitochondria progressively evolve in opposite directions during network maturation. Our observation of decreased mitochondrial dynamicity agrees with a recent 34. The 1–3 μm/s velocity range recorded in the early, immature stage of network culture is much more comparable to what was found in these studies. Interestingly, changes in vesicular dynamicity are not restricted to velocities. Indeed, the number and directionality of vesicles along the axon both drastically increased throughout network maturation. As a result, the directional flux of axonal vesicles becomes progressively more anterograde toward presynaptic sites which correlates well with the increasing need in synaptic vesicles and secretory proteins at mature synapses17. Interestingly, the species used for neuronal culture is also critical for transport kinetics. We previously reported axonal trafficking of DCVs and mitochondria at similar time points using mouse cortical neurons that showed slight differences with the present study using rat cortical neurons3. While BDNF axonal trafficking remained quite similar between both studies , mitochondria axonal transport showed marked differences, especially on the number of motile units (2-fold increase in rat cultures) and linear flow rate (3-fold increase). These observations show that the stage of neuronal maturity as well as the species used for neuronal cultures are important factors of variability that should be taken into account for the reproducibility of observations between studies.In the late, mature stage of the network we observed vesicular velocities ranging from 3 to 7 μm/s, which is much higher than velocities traditionally reported in the literatureTaken together, our results demonstrate that studying intracellular dynamics in organized and controlled co-cultures, instead of mixed, free neuronal cultures, is crucial to understand molecular mechanisms that occur in mature circuits. These mechanisms are likely to be different for developing versus mature neurons, and will vary depending on the cargo of interest. Furthermore our data reveals the importance of choosing the correct time window for observing biological mechanisms depending on the scientific question in order to improve the reproducibility of studies.3. Microchambers were coated with poly-D-lysin (0.1 mg/ml) in the upper and synaptic chambers, and with a mix of poly-D-lysin (0.1 mg/ml) + laminin (10 µg/ml) in the lower chamber overnight at 4 °C, followed by careful washing with growing medium . Primary cortical and striatal neurons were prepared as previously described35. Briefly, cortex and ganglionic eminences were dissected from e17.5 rat embryos, digested with a papain and cysteine solution followed by two incubations with trypsin inhibitor solutions, and gentle mechanic dissociation. Dissociated cortical and striatal neurons were re-suspended in growing medium and plated at a final density of ~7000 cells/mm2. Cortical neurons were plated on the upper chamber after addition of growing medium in the synaptic chamber to balance compartmental pressure. Striatal neurons were then added in the lower chamber. Neurons were left in the incubator for at least 3 hours before all compartments were gently filled with growing medium. Fluorescent markers and cargoes were expressed in neurons using lentiviruses (LV) or adeno-associated viruses (AAV) infections at DIV 2, or were electroporated with plasmids using Amaxa Nucleofactor (Lonza) before plating. Cultures in the microfluidics were visually inspected before acquisitions. Low-quality cultures (i.e. containing dead/clustered cells or with cells invading the synaptic chamber) were removed. All experimental procedures were performed in an authorized establishment  in strict accordance with the recommendations of the European Community (86/609/EEC) and the French National Committee (2010/63) for care and use of laboratory animals.Microfluidic chambers were fabricated and prepared as previously described36 and pDsRed2-Mito (plasmid #632421 from Clontech).The following constructs, LVs and AAVs were used: LV.PGK.GFP (pRRLSIN.cPPT.PGK-GFP.WPRE plasmid #12252 from Addgene), LV.CMV.mCherry (pLV-mCherry plasmid #36084 from Addgene), LV.PGK.iGluSnFR (pCMV(MinDis).iGluSnFR plasmid #41732 from Addgene cloned into pRRLSIN.cPPT.PGK-GFP.WPRE plasmid #12252 by replacing GFP with iGluSnFR), AAV5.SYN.GCaMP6f (#AV-5-PV2822 from U Penn Vector Core facility), LV.PGK.BDNF-mChCulture medium of cortical neurons was complemented at DIV 3 or DIV 13 with 100 nM Mitotracker-Red CMXRos  for 45 min and then washed twice with fresh NB-B27. Videorecordings were performed 24 h later at DIV 4 or DIV 14, respectively.+ 1024, Princeton Instrument) and maintained at 37 °C and 5% CO2. Images were taken at 5 Hz for 30 s for BDNF-mCh trafficking using a ×63 oil-immersion objective (1.46 NA), at 1 Hz for 5 min for Mito-DsRed2 using a ×63 oil-immersion objective (1.46 NA), and at 5 Hz for 30 s for GCaMP6f using a ×20 objective (0.8 NA). iGluSnFr was imaged live using z-stack acquisitions with a ×63 oil-immersion objective (1.46 NA) in the synaptic chamber. Fixed immunostaining images of SYP/PSD95 were obtained using z-stack acquisitions with a ×63 oil-immersion objective (1.4 NA) using an inverted confocal microscope  coupled to an Airyscan detector to improve signal-to-noise ratio and spatial resolution.Live-cell videorecordings were performed using an inverted microscope  coupled to a spinning-disk confocal system  connected to wide-field electron-multiplying CCD camera (ProEMNeurons were fixed by filling the microchambers with PFA/Sucrose (4%/4% in PBS) for 20 min at room temperature (RT) followed by PBS washes and were incubated for 1 h at RT with a blocking solution . The synaptic compartment was then incubated with Synaptophysin  and PSD95  primary antibodies overnight at 4 °C and appropriate fluorescent secondary antibodies were incubated for 1 h at RT. The immunofluorescence was maintained in PBS for a maximum of one week in the dark at 4 °C before confocal acquisitions. SYP/PSD95 colocalization analyses were performed using ImageJ. Airyscan images were first thresholded to remove non-specific signal and an area of interest of at least 100 μm in length was defined around neurites. The number of synaptophysin spots overlapping, juxtaposed or separated by no more than 2 pixels (130 nm) to PSD95 spots were counted manually. 3 regions of interest per chamber were randomly selected in at least 6 microchambers from 3 independent cultures (n = number of fields).2 2 mM, glucose 10 mM, glycine 200 µM, strychnine 1 µM). iGluSnFR images were filtered by size using the Bandpass Filter plugin in ImageJ and were then binarized to remove non-specific signals. A region of interest of at least 50 μm in length was defined around neurites using transillumination. The number of spots was then counted using the Analyze Particle plugin. 4 fields per chamber from at least 6 microchambers from 3 independent cultures were analyzed (n = number of fields).Live acquisition of iGluSnFR fluorescence was performed after stimulating cortical neurons for 5 min at 37 °C using chemical stimulation  from 9 microchambers prepared from 3 independent cultures (n = neurons unless otherwise stated).Single cell analysis of calcium fluorescence was performed using Matlab 2014b. Cell bodies detection was achieved manually and ΔF/F traces were calculated using FluoroSNNAP software10 with a length of 100 µm (x-axis) and a total time of 30 s (BDNF-mCh) or 5 min (mito-DsRed2) (y-axis) to extract the following kinetics parameters:Live-cell video-acquisitions of BDNF-mCh and Mito-DsRed2 were analyzed using kymographs generated using KymoToolBox plugin for ImageJp < 0.05.Statistical analyses were performed using Prism5 (GraphPad Software). The effect of time on synaptic formation (SYP/PSD95), synaptic transmission (iGluSnFR), network connectivity (GCaMP6f) and trafficking kinetics  was analyzed using one-way ANOVA followed by a Tukey post hoc test. Correlations studies were also analyzed using one-way ANOVA followed by a Tukey post hoc test. Early and late effects were compared using unpaired two-tailed Student’s t-test. The distributions of motile vesicles (BDNF-mCh) were analyzed using a Pearson’s chi-square test. Results are expressed as mean ± SEM. The criterion for statistical significance was set at Supplementary InformationMovie S1Movie S2


TP53 gene encoded protein p53 plays a critical role in tumor suppression, while its germline mutation causes Li-Fraumeni syndrome (LFS), an autosomal dominant cancer predisposition disorder [TP53 R337H that gives rise to a variant of LFS is highly prevalent in southern and southeastern Brazil with an estimated mutation carrier frequency of ~0.3%, translating to hundreds of thousands of individuals [TP53 R337H has been associated with an increased risk of pediatric adrenocortical carcinoma relative to other cancers typically associated with LFS. The majority of LFS mutations occur within the DNA binding domain of p53 that compromises the transactivation of its target genes, but the R337H mutation is located in the oligomerization domain of the carboxy terminus with grossly preserved p53 transcriptional activity by in vitro studies [in vitro assays [in vivo, we recently reported the generation of a mouse model with knockin of the p53 R334H mutation (human TP53 R337H homolog) and examined how it affects tumorigenesis as well as the oligomerization and activity of p53 in response to DNA damage under physiological conditions [The disorder . LFS is ividuals . Althoug studies . Howevero assays –4. Givenp53 R334H mutation (p53334H/H) developed normally and did not show any significant difference in terms of either cancer incidence or life span compared with wild-type mice, consistent with the low cancer penetrance observed in humans with the TP53 R337H mutation [p53334H/H mice with more malignant histopathologic features. In parallel, there was evidence of increased DNA damage and decreased transactivation of p53 target genes in the DEN-treated liver tissue of p53334H/H mice compared with that of wild-type mice. Mechanistically, p53334H/H mouse liver tissue showed decreased levels of dimers and tetramers but increased monomers of p53 after DEN treatment, constituting the first in vivo demonstration of its diminished oligomerization capacity consistent with the observed decrease in its transcriptional activity (Figure Mice that are homozygous for the y Figure .in vivo. Because patients with the TP53 R337H mutation have also been reported to have decreased plasma ascorbic acid levels [in vivo and affect tumorigenesis. Vitamin C deficiency is also common during pregnancy which could further potentiate the development of pediatric adrenocortical carcinoma in these genetically predisposed patients [p53 R334H mouse model, some of these questions could be addressed in the laboratory and may lead to insights for follow up by clinical investigation with the goal of improving the management of this large population of patients with a unique p53 mutation.Structural studies have suggested that arginine 337 within the α-helix motif of human p53 forms a salt bridge with aspartate 352 of another p53 molecule, helping to stabilize p53 homodimers that in turn dimerize to form tetramers . The subd levels , it is tpatients . Finallypatients , the inc


Shigella and ETEC vaccine development. The workshop featured four short presentations which highlighted how genomic applications can be used to assist in the identification of genetic patterns related to the virulence of disease, or host genetic factors that could contribute to immunity or successful vaccine responses. Following the presentations, there was an open debate with workshop attendees to discuss the best ways to utilise such genomic studies, to improve or accelerate the process of both Shigella and ETEC vaccine development. The workshop concluded by making specific recommendations on how genomic research methods could be strengthened and harmonised within the ETEC and Shigella research communities.Thanks to the modern sequencing era, the extent to which infectious disease imposes selective pressures on the worldwide human population is being revealed. This is aiding our understanding of the underlying immunological and host mechanistic defenses against these pathogens, as well as potentially assisting in the development of vaccines and therapeutics to control them. As a consequence, the workshop “How genomics can be used to understand host susceptibility to enteric infection, aiding in the development of vaccines and immunotherapeutic interventions” at the VASE 2018 meeting, aimed to discuss how genomics and related tools could be used to assist  For instance, whole genome sequencing (WGS)/next-generation sequencing (NGS) can be used to provide an insight into genomic diversity and evolution of different microbial species When considering the use of genomics, related genomic technologies and methods should also be considered. Examples include: transcriptomic analysis of host-pathogen interactions upon infection or vaccination; proteomic analysis to study protein-protein interactions within the pathogen or between the host and pathogen; pathogen and host rapid phenotyping; immunoproteomic analysis; and data mining of various genomic or protein databases Shigella and ETEC vaccines. The workshop began with four short presentations that highlighted how genomics and related tools can successfully be used to aid the identification of genetic patterns related to the virulence of disease, or host genetic factors that could contribute to immunity or successful vaccine responses. The presentations were then followed by an open workshop debate on the best ways to utilise such genomic based studies to improve ETEC and Shigella vaccine development. The workshop concluded by making specific recommendations on how genomic research methods could be strengthened and harmonised in the ETEC and Shigella vaccine development fields.The purpose of this VASE 2018 workshop was to discuss how genomics and related applications could be applied to facilitate the development of 22.1Lynda Mottram  described how she has used Genome-wide Association study (GWAS) data to identify potential human genetic biomarkers of severe ETEC infection susceptibility/possible ETEC vaccine efficiency a), could be a human intestinal binding receptor of ETEC colonization factor I (CFA/I), and related colonisation factor (CF) fimbriae a phenotyped Bangladeshi children are more susceptible to symptomatic than asymptomatic ETEC CFA/I infection a phenotype (caused by homozygous fucosyltransferase 2 [FUT2] single nucleotide polymorphisms [SNPs]) has also been previously associated with susceptibility to other enteric infections, as well as lower immunoglobulin A (IgA) antibody responses to rotavirus vaccination FUT2 SNP could also be used as a human genetic biomarker of susceptibility to severe ETEC CFA/I and related CFs diarrhoeal disease/and or vaccine efficiency associated with ETEC vaccines containing CFA/I.There is evidence to suggest that the human small intestinal glycan antigen Lewis a  found in 2,504 human genetic sequences of 26 different human populations worldwide Dr Mottram initially searched for the frequency of known FUT2 genetic mutations  identified in the GWAS studied individuals, was downloaded FUT2 SNPs, a further analysis of The 1000 Genomes Project FUT2 genetic variation dataset was performed using the Ensembl Variation Effect Predictor (VEP) tool FUT2 single nucleotide SNPs candidates, that were present in the Bangladeshi population Consequently, a variant calling file (VCF) from The 1000 Genomes Project server that contained all the FUT2 non-synonymous SNPs in Lea phenotyped Bangladeshi children, who had been previously clinically monitored for ETEC infection during the first two years of life were then assessed FUT2 SNPs; rs200157007-TT and rs601338-AA, that were strongly associated with symptomatic ETEC infection and the Lea phenotype in the Bangladeshi children Using RT-PCR SNP genotyping methods, the frequency of these three a phenotyped Bangladeshi children could be found. Subsequently, further clinical studies in other ETEC endemic areas, as well as further in-vivo analysis are needed to evaluate if these FUT2 SNPs could be used as host biomarkers of ETEC CFA/I (and related CFA/I CFs) infection susceptibility, or vaccine efficiency of ETEC vaccine candidates containing the CFA/I antigen.However, potentially due to the limited number of samples from which human gDNA could be successfully extracted from, only a trend but not a statistical relationship with rs200157007-TT and rs601338-AA SNPs, symptomatic ETEC expressing CFA/I or related CF infection, and the Le2.2James Fleckenstein  explained how his team is complementing their host-pathogen interaction studies with genomics and related “omics” tools, to identify and characterise noncanonical ETEC vaccine candidates that are associated ETEC pathogenesis; i.e. novel ETEC virulence factors associated with ETEC adhesion, intestinal colonisation and toxin delivery during severe ETEC infection.phoA transposon-based mutagenesis study using the fully sequenced human challenge ETEC H10407 strain, to identify secreted or surface-expressed antigens. This study enabled the identification of a number of novel ETEC plasmid-encoded virulence loci, including the eatA autotransporter and the etpBAC two-partner secretion system, that were expressed in the virulent ETEC H10407 strain, but not in the fully sequenced E. coli K-12 strain Initially, Dr Fleckenstein’s group performed a TnShigella flexneriin-vitro enteroid  model By combining genomic database mining and immunoproteomics, EatA has been found to be a member of the serine protease autotransporter family, as well as being fairly conserved among a geographically and phylogenetically diverse group of ETEC strains Likewise, EtpA has found to be a large glycosylated exoprotein, secreted via the etpBAC two-partner secretion system (TPS) +/+ wild-type intestinal cell line and blood group A knockout HT-29A−/− cells (derived by CRISPR-Cas9 engineering to eliminate the gene encoding blood group A glycotransferase) Using a combination of human glycan arrays, biolayer inferometry, noncanonical amino acid labelling and hemagglutination studies, EtpA has been identified as a dominant ETEC blood group A–specific lectin/hemagglutinin Such molecular and genomic characterisation of EtpA has enabled further collaborations with Dr Chakraborty see  to show 2.3In the third presentation, Subhra Chakraborty  described how genomics is being used to evaluate human host responses to ETEC during CHIM studies.5 or 106 colony-forming unit (CFU) of ETEC strain H10407 (LT+ ST+ CFA/I+ and O78+). Following the ETEC H10407 challenge, 29 out of the 30 volunteers were found to shed ETEC bacteria. However, the rates of moderate to severe diarrhoea (MSD) compared to asymptomatic ETEC carriage varied between the volunteers of the same dosing cohorts A total of 30 naive ETEC H10407 subjects were enrolled into a CHIM study, in an inpatient unit at the Centre for Immunization Research, John Hopkins University. These volunteers were randomly assigned to one of two dosing groups; 10P = 0.02) in the asymptomatic subjects Dr Chakraborty pre-screened the volunteers for pre-existing ETEC antibody titres before ETEC H10407 challenge. This was performed using antibodies in lymphocyte supernatants (ALS), sera and faecal ELISA assays, and included; lipopolysaccharide (LPS) IgA and IgG, LTB IgA and IgG, and CFA/I IgA and IgG analysis. Dr Chakraborty compared subjects who subsequently developed asymptomatic (n = 24) verses MSD (n = 6) subjects, and observed only significantly higher pre-challenge LTB IgG antibody sera titres  versus asymptomatic (n = 6) at the baseline time point Tubb2A, Tubb2b, Tubb3 and Tubb4B) which have previously been associated with E. coli pathogenesis, to be up-regulated in the asymptomatic (resilient to infection) dataset. In comparison, C4BPA an inhibitor gene associated with the classical complement pathway was down-regulated in the asymptomatic (resilient to infection) dataset This microarray analysis identified 29 differentially expressed gene probes that were potentially associated with resilience to severe ETEC infection. Of these 29 identified probes, gene probe sets associated with major histocompatibility complex (MHC) protein binding and MHC class I protein binding molecules were significantly up-regulated in the asymptomatic dataset. The analysis also identified four tubulin genes  gene sequencing gut microbiome analysis performed on stool specimens from a subset of these ETEC H10407 infected individuals; MSD (n = 5) and asymptomatic (n = 6) individuals. The aim of this analysis was to evaluate if pre-infection microbiota could be used to predict the onset of severe diarrheal ETEC disease Escherichia as well as Bacteroides dorei, Prevotella species, Alistipes onderdonkii, Bacteroides species (ovatus), and Blautia species. In contrast, the faecal microbiota of the asymptomatic carriers  were enriched with normalised 16S rRNA gene sequences including Sutterella species, Prevotella copri, and Bacteroides vulgatusResults from the 16S rRNA analysis revealed the MSD individuals  had a higher concentration of faecal 2.4E. coli (STEC) and F4+ fimbriae ETEC strains. The susceptibility of pigs to F18+ and F4+ E. coli is determined by the presence of F18 and F4 specific host receptors in the brush boarder of pig’s small intestine. Therefore, understanding the genetic composition of these F18 and F4 host receptors is useful for animal health and subsequent breeding management.In the final presentation, Eric Cox  described the use of genomics and related genomic tools to examine the genetic susceptibility of pigs to F18+ fimbriae ETEC/shigella toxin-producing E. coli intestinal binding receptor had been previously thought to be only encoded by FUT1, a host gene that encodes for α fucosyltransferase blood group AO antigens on small intestinal type 2 glycan chains in pigs. Meijerink et al. FUT1 SNP at nucleotide position bp307 (G=>A transition) of the FUT1 open reading farm (ORF) was closely linked to susceptibility to F18+ E. coli infection, and thus genetic variations in this FUT1 M307 SNP can be used as a specific genetic marker for selecting and breeding pigs which are resistant to F18+ E. coli infections The functional pig F18+ E. coli infection susceptibility E. coli binding to pig A and O blood groups Conversely, Prof. Cox’s group has discovered that F18+ fimbriae attach to the small intestine of young piglets by binding to mucosal type 1 core glycans chains that express A/O blood group determinants, with this host-pathogen interaction directly correlating with F18+ E. coli infection might not be an absolute correlate with the FUT1 M307 related SNP. Prof. Cox’s group is currently analysing data to suggest the genetic regulation of FUT2 (a gene closely related to FUT1), might also be controlling blood group A and O blood expression in the pigs small intestine, and thus piglet susceptibility to F18+ E. coli infection. Interestingly, the expression of AO antigens in the pig’s small intestine is also directly correlate with age, as Prof. Cox’s group has noticed that new-borns are always resistant to F18+ E. coli infection independent of the FUT1 M307 SNP, and that susceptibility to F18+ E. coli infection becomes highest just after weaning at 6–8 weeks old Recent genetic studies have further identified that piglet susceptibility to F18+ Prof. Cox also described the current work to genetically define the F4+ ETEC fimbriae binding receptor in the porcine small intestine. There are three antigenic variants of F4+ ETEC fimbriae , with each antigenic variant showing a different binding pattern to brush border membrane proteins of small intestinal enterocytes. Only a small number of piglets completely lack an intestinal receptor to F4+ ETEC fimbriae, and are therefore resistant to F4+ bacteria and subsequent diarrhoea caused by F4+ ETEC strains. In Belgium however, most Flemish farmed pigs express F4ab or F4ac intestinal receptors, so Flemish pigs are subsequently more susceptible to F4ab+ or F4ac+ associated ETEC infections TRFC), mucin 4 (MUC4), mucin 13 (MUC13), mucin 20 (MUC20), solute carrier family 12 member 8 (SLC12A8), myosin light chain kinase (MYLK), karyopherin alpha 1 (KPAN1), beclin-1 associated RUN domain containing protein (KIAA0226), lactosylceramide 1,3-N-acetyl-β-D-glucosaminyl transferase (B3GNT5) loci, but none of the polymorphisms were found to be causative. Therefore, the Gent University researchers conducted a GWAS study using 120 precisely F4ab/ac receptor phenotyped Flemish pigs (i.e. 52 F4ab+/Fac+ E. coli resistant and 68 F4ab+/Fac+ E. coli susceptible pigs) from 5 different Belgium farms, based on MUC4 and MUC13 polymorphism and in vitro villous adhesion of F4 = and F4ac+ E. coli. DNA was isolated from the blood samples of these phenotyped pigs and then sequenced using the Porcine SNP60 BeadChip (Illumina) microarray Several studies located the F4ab/acR locus(loci) on chromosome 13 and several linkage studies proposed different loci as candidate receptor loci such as the transferrin, the transferrin receptor . Unfortunately, these genetic regions lacks annotated genes, and contain a sequence gap based on the sequence of the porcine GenomeBuild 10.2. Subsequently, it can only be currently hypothesise that a porcine orphan gene or trans-acting element in the candidate region determines F4ab/F4ac ETEC susceptibility in pigs This GWAS study revealed that pig F4ab/ac ETEC susceptibility, is instead likely highly associated with two SNPs and genetic regions adjacent to 3Shigella vaccine development.Following the presentations, the workshop moved to an open discussion with attendees on how genomic and related genomic tools could be best utilised to accelerate the current process of ETEC and To potentially aid the development of new vaccines, a good part of the workshop discussion centred on the use of genomics during molecular epidemiology studies. The assuming advantages of this is that WGS or related high throughput genomic platforms could be used to characterise the genomic diversity of the pathogen, as well as take into account the genetic/immunological variability’s of the host. Such information could be used to critically define host-pathogen dynamics during infection, and thus define correlates of protection.For this to happen, some workshop attendees suggested such studies would have to be large-scale multi country epidemiological studies , where strong population data and phenotypic information was collected on both the pathogen and the host. To aid the subsequent identification of any genomic host biomarker, associated with infection susceptibility, these epidemiological studies would also importantly need to record the immune response elicited in each infected individual. Another alternative option to using large-scale epidemiological studies could be to use CHIM studies to define host-pathogen dynamics during infection in a smaller number of individuals, in a controlled environment Shigella scientific communities to harmonise which infection time points are used to collect data related to immunological and genomic analysis. Furthermore, to ensure that sample collection does not affect the related genomics results obtained, it might also be important to standardise what human samples are collected, and how they are collected and stored at both the epidemiological field site or in the clinic.However, the workshop discussion emphasized that for genomics analysis to be used efficiently in such molecular epidemiological or CHIM studies, it would be important for both the ETEC and Shigella reference strains that represent their worldwide geographical, temporal and phenotypic diversity. It is well recognised the role played by the erstwhile WHO reference laboratories, NIH, CDC, USDA as well those such as the Sanger Institute in generating genomic databases of Shigella and ETEC stains, who make reference strains/genomic sequences available to the scientific community. However, perhaps it could be advantageous to the ETEC and Shigella scientific communities to work with a harmonised set of genomically annotated ETEC and Shigella reference strains, as well as their related genomic sequences. Such a harmonised, defined and fully sequenced strain collection could be used in all the associated ETEC and Shigella vaccine developmental studies performed by different laboratories worldwide.Likewise, other workshop attendees suggested that WGS/NGS can be used to fully annotate a group of ETEC or in-silico analysis of multiple whole genome sequences are used to identify highly conserved antigen in pathogenic strains but not commensal strains was also mentioned as a successful method of vaccine discoveryThe workshop speaker’s sessions highlighted how high throughput genomic screening for virulence factors could offer potential for the rational development of new vaccine candidates, which could stand-alone or complement the vaccines currently in development. Workshop attendees highlighted use of proteomics in-vivo vaccine developmental studies. Animal studies are considered to be very important to evaluate vaccine efficacy. However, some animal models never perfectly correlate with the response in humans to ETEC or Shigella infection. Subsequently, as well as using gene edited bacterial strains to characterise vaccine antigen candidate expression and function, it was discussed that genomics analysis could also be used to define specific host-pathogen interactions during infection. Perhaps also, genomics analysis could also be used in animal studies to assess for the presence of a specific genetic biomarker that predict the mucosal efficiency or toxicity of a vaccine, or even how effective a vaccine would be in humans.Similarly, the group discussed the advantages of genomic analysis during preclinical Others suggested the use of high throughput screens using large scale sero-epidemiological studies in-vitro and in-vivo modelling, a vaccine next goes into clinical trial. Here the group discussed if genomics could also be used to improve the process by identifying and testing for biomarkers that alert researcher to toxicity or efficiency issues early in the clinical trial process. Moreover, using GWAS studies, it may also possible to identify the most genetically susceptible populations for a particular disease and thereby reduce the sample size needed for an effective trial, which could also reduce the cost of such clinical trials. However, whilst the group felt this was important, it was considered that further research is still necessary to identify such defined host biomarker(s) in the ETEC and Shigella fields.Following epidemiological studies, searches for antigens, 4Shigella research communities, and the harmonisation and standardisation of efforts to facilitate exchange of genomic sequencing, information and materials. The specific recommendations proposed from this workshop are:1.Genomics and related genomic applications can be used to complement classical vaccinology approaches2.To effectively use genomics and related technologies in ETEC and Shigella vaccine development, immunological and genomic sample collection time points should be standardised and harmonized amongst different laboratories3.The types of sample  collected from the host for genomic related analysis should be standardised, to avoid genomic skewing of data4.Shigella scientific communities, genomic collection and storage standard operating procedures (SOPs) could be established for use amongst the different ETEC and Shigella research groupsTo standardise genomic testing in the ETEC and 5.Shigella reference strains for use in CHIM, vaccine antigen discovery, and host-pathogen interaction studiesEstablish a harmonised global set of genomically annotated ETEC and 6.Shigella pathogenesis studies.Use genomics to aid the development of more genetically defined humanised models for use in ETEC and The workshop emphasised the importance of scientific interaction among members of the ETEC and


Fusion transcripts can contribute to diversity of molecular networks in the human cortex. In this study, we explored the occurrence of fusion transcripts in normal human cortex along with single neurons and astrocytes. We identified 1305 non-redundant fusion events from 388 transcriptomes representing 59 human cortices and 329 single cells. Our results indicate while the majority of fusion transcripts in human cortex are intra-chromosomal (85%), events found in single neurons and astrocytes were primarily inter-chromosomal (80%). The number of fusions in single neurons was significantly higher than that in single astrocytes (p < 0.05), indicating fusion as a possible contributor towards transcriptome diversity in neuronal cells. The identified fusions were largely private and 4 specific recurring events were found both in cortex and in single neurons but not in astrocytes. We found a significant increase in the number of fusion transcripts in human brain with increasing age both in single cells and whole cortex . This is likely one of the many possible contributors for the inherent plasticity of the adult brain. The fusion transcripts in fetal brain were enriched for genes for long-term depression; while those in adult brain involved genes enriched for long-term potentiation pathways. Our findings demonstrate fusion transcripts are naturally occurring phenomenon spanning across the health-disease continuum, and likely contribute to the diverse molecular network of human brain. Earlier studies have reported wide variety of DNA level diversity in neuron rich regions of normal human brain - ranging from whole chromosomes3, large-scale retro transpositions5, and copy number variations at the single neuron level6. We have also shown the diversity in the non-coding RNA of human brain attributed through the RNA editing mechanisms in miRNAs and their possible role in biological outcome7. In this study, we embarked on investigation of fusion transcripts in human brain - another possible mechanism by which the transcriptome can contribute to the diversity of complex systems.Biological systems utilize their built-in flexibility to respond to unknown situations that challenge their ‘fitness’ to adapt and respond. This flexibility usually increases with increased complexity and diversity of more recently evolved species. Human biology, in particular of the human brain, is one of the most diverse natural systems. At a molecular level, this flexibility is created and maintained by contributions from all layers of information - namely, DNA, RNA and Proteins, usually following an increasing order of diversity. We have earlier reported the extent of DNA level diversity and its possible role due to somatic single nucleotide variations in normal human brain9. These fusions may arise at the genomic level by various structural rearrangements like deletion, duplication, inversion or translocation10 as well as at transcriptomic level by RNA-polymerase read-through12, cis- or trans-splicing13. Thus, investigation of transcriptomic analysis using massively parallel sequencing strategies provides an opportunity to capture all fusion transcripts – irrespective of their genomic or transcriptomic origins15.Fusion transcripts have sequences from two or more genes, unlike conventionally spliced mRNA isoforms18. A few isolated studies have reported the existence of fusion RNAs within the normal human genome20.Fusion transcripts and their encoded products were earlier perceived to be an aberration with negative outcome – owing to their abundance in cancersIn this study, we elucidated the landscape of fusion transcripts in the frontal cortex of human brain. For a comprehensive understanding, we have explored the transcriptome at the tissue level (cortex) as well as at the level of single neurons and astrocytes. Further, we analysed age related changes in fusion transcript of human brain. To our knowledge this is the most comprehensive study of fusion transcripts in normal human brain.We analyzed 59 whole-transcriptome datasets comprising of 49 prefrontal cortices (public domain) and 10 frontal cortices (sequenced in-house). By analyzing ~4.2 billion sequencing reads we identified 88 fusion transcripts representing 38 non-redundant events  samples for experimental validation of the identified fusions. Based on the recurrence and supporting reads, 3 selected fusion events were validated using fusion specific PCR followed by Sanger sequencing. They include, (i) two recurring events, namely, KANSL1-ARL17A Fig.  also se along wiWe used single cell transcriptome data from human brain to resolve the tissue level data with increased resolution. For this we considered publically available transcriptomic libraries from 131 neurons and 62 astrocytes collected from 7 different healthy adult brains. Upon analyzing 500 million reads  fusions involving genes from two different chromosomes Fig.  – which Three recurring fusions were identified in single neurons which were also found to recur in cortex samples  number of fusions in neurons when compared to the astrocytes Fig. . SimultaTo ascertain any association between the fusion burden and aging brain we considered another data set encompassing brain transcriptomes from two distinct age groups. Towards this we analyzed a total of ~2 billion RNA sequencing reads from Dorso Lateral Frontal Cortices (DLFC) derived from 16 adult and 8 newborns together with 131 and 110 single neurons derived from 7 adult and 5 fetal brains respectively. We identified 63 unique fusions in 16 adult brains while 10 fusions in fetal brain. Similarly, 724 fusion events were observed in adult neurons and 302 in fetal neurons  in adult brain compared to the fetal brain, be it a cell or a tissue  Fig. . ConcurrTo know the functional significance of these genes that undergo fusion, we also performed their enrichment analysis using GSEA. Genes harboring fusion demonstrated a significant enrichment for the long-term potentiation pathway in adults while in fetal there was a significant representation for long-term depression pathway  Fig. .GDI2-FAM208B junction peptide which retained the reading frame from both of its parent genes.To test the translational ability of these fusion events we performed in-silico translation around the junction by translating them into six reading frames. These putative fusion peptides were first subjected to in-silico digestion with trypsin followed by MS identification. We used publically available MS/MS data from human cortex to map our digested peptides and identified 214 putative translated junctions. Among them 23% (49/214) were found in multiple proteomes  reported in at least 2 cell lines at a 40 kb resolution Fig. . This seBy comparing fusion breakpoints with the Hi-C interaction maps of three-dimensional chromosome conformation led us to detect nine fusion partners located in broad chromatin domains that are spatially proximal in normal cell nuclei Table . These eKANSL1-ARL17 fusion was one of the most recurring events comprising of first three exons from KANSL1 and last three exons from ARL17. It was identified in ~20% (12/59) of normal brain tissues (Supplementary Table KANSL1 and ARL17).To test the status of this particular fusion in disease condition we used a most malignant brain tumor i.e. Glioblastoma multiforme (GBM). By performing junction specific PCR, we validated the same event in 7 out of 9 (78%) GBM samples and further confirmed it by Sanger sequencing Fig. . The lar21. H1β duplication is the only subtype capable to produce KANSL1-ARL17 fusion. We confirmed H1β duplication in our in-house normal brain samples which were observed to have an allele frequency of 70%, (7/10) as it was detected in the case of GBM samples  in GBM while it was up-regulated (5 ×) to the same extent in normal brain when compared to its parent gene (KANSL1) Fig. . These r22. This is not only true for whole organisms but also true at the levels of tissues, organs and individual cells. Recent surge of next generation sequencing data has revealed that in the human brain this diversity is achieved and maintained not only by the inherited set of variations but also by the variability introduced de novo at the somatic level for both DNA and RNA25. Fusion transcripts are one of many possible ways to create and maintain the much needed diversity – especially in post-mitotic cells like human neurons. Fusion transcripts are now widely reported in the literature albeit with a bias towards their presence and function in diseases – especially for their oncogenic role in human cancers. However, these transcripts can have a big influence on the normal phenotypic outcome. It has been shown for fusion transcripts that the fusion event when created at the RNA level provide a growth advantage while the same event resulting from a DNA level translocation may lead to cancer13. The underlying explanation for this observation is likely due to the obligatory (and hence more) expression of the fusion RNA when it is formed at the DNA level. This suggests a functional continuum of the fusion transcripts, where the same events in moderation can provide an advantage while an excess is detrimental. The spectrum of fusion transcripts in normal human tissues and to what extent they are functionally relevant is an unanswered question. We embarked on this study in an attempt to address this issue focused to the normal human brain, particularly the cortex.The life span and ‘fitness’ of an organism is the sum of deleterious changes and counteracting repair and maintenance mechanisms that trigger in response to stimuliOur analysis showed the organization and distribution of the fusion transcripts are unique and distinct between the tissue and the single cells. At the tissue level we found more recurrence and majority of the events formed between two genes residing on the same chromosome. Notably, our analysis has identified KANSL1-ARL17 fusion as a recurring event in normal human cortex. Contrasting expression pattern between the KANSL1-ARL17 fusion and its parent gene, KANSL1 in GBM compared to the normal brain warrants further investigation, as it suggests their potential role in cancer.27 or possibly a mix of both. Further, a complete lack of redundancy in the single astrocytes was striking and cannot be explained. Further research on the same source of tissue and single cells might be able to shed light on these observations.On the other hand, the spectrum of fusion transcripts occurring from the single neurons and astrocytes show minimal or no recurrence and the vast majority formed by fusion of two transcripts coming from different chromosomes. Recurrence is an important factor to estimate their abundance. In our case, majority of our events were non-recurring between cells which might signify their low abundance or they may be false positive generated by template switching of reverse transcriptase during RT-PCR29. Genes harboring fusion demonstrated a significant enrichment for long-term potentiation (LTP) and long-term depression (LTD) pathways in adult and fetal brain respectively. Both of these pathways in conjunction provide neuronal synaptic plasticity that undergo age-related alterations33. Results from our analysis suggest aging leading to a substantial fusion load, which might affect the neuronal synaptic function. Evidence for their corresponding junction peptides further support their biological relevance. Later on we also identified the inter-chromosomal proximity between ENSG00000227733 and HYDIN genes from chromosome 1 and 16, respectively. Co-occurrences of chromatin interaction maps with these fusion breakpoints suggest spatial proximity can be one of the possible triggers.We found significantly higher number of fusions in neurons compared to the astrocytes. This observation, together with a considerably higher number of fusions in adult brain compared to the newborns – indicate a role of fusion transcripts in maintaining required diversity in neurons with restricted regenerative capabilities and age-dependent decline in turnover rate of the cellsIn summary, our genome-wide analysis establish fusion transcripts are naturally occurring phenomenon that span the health-disease continuum of the human brain.Frontal cortexes (Grey matter) were procured from post-mortem samples of road accident victims collected from NIMHANS Brain Bank, Bangalore, India. GBM samples were obtained from AIIMS, New Delhi, India. The samples were collected according to the Helsinki Declaration and the ethical review board of All India Institute of Medical Sciences, Delhi, India approved the project. Diagnosis and grading of tumor samples were done as per 2007 WHO classification. The details of the in-house samples used in the study are provided in Supplemental Table Total RNA was isolated by using miRvana miRNA isolation kit  as per the manufacturer’s instructions. RNA Libraries were prepared using Illumina’s TruSeq total RNA Sample preparation for 6 FCs and poly-A enriched protocol for rest of the 4 FCs following the manufacture’s protocol. Cluster generation and sequencing was done on Illumina HiSeq. 2000 using standard Illumina sequencing workflow. The in-house sequencing data is deposited at the sequence read archive (SRA ID: SRP045655).DNA isolation was done by using Omniprep Genomic DNA isolation kit  as per manufacturer’s protocol. Exome capture was done using Illumina TruSeq Exome capture kit. 100 base pair paired end sequencing was done using Illumina HiSeq. 2000 . The exome sequence data is also deposited at the sequence read archive (SRA ID: SRP045655).34 with the default parameters. Briefly, the quality filtered reads were aligned using Tophat (version 2.0.5) against transcriptome (UCSC hg19 annotations) and genome (hg19) both with 2 mismatches. Discordant reads were used to identify potential fusion candidates using its fusion-search module. Tophat-fusion-post was further used to filter out events supported by minimum 5 supporting reads. The software and detailed description is available at < https://ccb.jhu.edu/software/tophat/fusion_tutorial.shtml >. The same pipeline was used for both in-house and publicly available dataset for calling fusion transcripts.Raw data was checked for per base quality score and reads having 80% bases with phred quality score 30 and greater were only be considered for downstream analysis. Fusion transcripts were identified using the published pipelineTo determine if the origin of the detected fusions were genomic, we explored signatures for copy number variation around fusion breakpoint Genome-wide CNV calling was performed using two different platforms: exome sequencing and illumina’s 660 quad microarray for 4 out of 10 FC samples 36 with default parameters.Raw data from exome sequencing was checked for per base quality score and reads having 80% bases with phred quality score 30 and greater were carried forward for downstream analysis and rest were discarded. Filtered reads were aligned to the reference genome (hg19) using BWA (version 0.6.1)Isolation of the genomic DNA was done using a standard salting-out procedure to perform genome-wide genotyping by using the Infinium Human660W-quad BeadChip method . We used 200 ng of genomic DNA for each sample, in accordance with the manufacturer’s guidelines. The raw data files were processed by the GenomeStudio software package. To call CNVs, we used the PennCNV algorithm and applied a stringent criterion of at least 10 consecutive probes to show altered intensity to qualify as a CNV call. We used a threshold of 0.35 for the standard deviation for logR ratio of normalized intensity (LRR) and a threshold of 0.05 for the standard deviation for B allele frequency as explained in our earlier study.In order to confirm their genomic evidences for fusion events, we overlaid CNV coordinates obtained from above analysis with fusion breakpoints ( ± 500 Kb).http://weblogo.berkeley.edu/logo.cgi).Nucleotide sequences extracted from 50 bases up-stream (from 5’ gene) and down-stream (from 3’ gene) around the fusion breakpoint were tested for any sequence preference. This part of analysis was performed using an in-house Perl script along with an online tool WebLogo to calculate the frequency of each base around the junction. (37 (v0.5.8) and Bowtie38 (v2.2.6). All sequencing data was mapped to hg19 reference genome at a resolution of 40 kb. A di-tag was considered only if it recurred in ≥ 2 cell lines with MAPQ ≥ 30 for both mates with Z score > 1.96. In order to check if there is any association of chromosomal proximity with fusion transcripts, we overlaid the identified chromatin map with these fusion breakpoints.To determine the chromosome proximity we used Hi-C data from ENCODE phase 3 experiments for ten different cell lines as explained in Supplementary Table 39 and X!Tandem41 in integrated transcriptomic-proteomic pipeline EuGenoSuite42.To check the translational evidence for identified fusions, MS/MS Spectral datasets from human cortex were considered from PRIDE projects PXD000263, PXD004076, PXD004987, PXD005629 and PXD002775. These were downloaded from the PRIDE repository and were searched against the six frame translated database using OMSSA43.We performed in-silico trypsin digestion for our probable peptides with one miss-matched site. Further parameters involve 20 ppm precursor ion tolerance; 0.5 Da product ion tolerance; carbamidomethylation of cysteine as fixed modification; and oxidation of methionine and peptide N-terminal acetylation were used as variable modifications. Stringent FDR threshold of ≤ 1% was applied to the resulting PSMs and junction specific peptides were identified using a customized Perl script. Reading frames for these filtered hits were further confirmed by again mapping them with human proteome (RefSeq GRCh37.p13) using stand-alone BLAST tool45. We applied gene ontology enrichment analysis using KEGG pathways to generate gene clusters enriching similar biological processes. Gene cluster and their corresponding pathways with highest enrichment score and FDR q-value < 0.05 were only considered for literature mining.To understand the functional significance of genes harboring fusions, pathway analysis was performed using Gene Set Enrichment Analysis (GSEA)http://www.rproject.org) using rlog-transformed read count from whole transcriptome.Differential numbers of fusion transcripts between groups of samples were screened out using Wilcoxon rank-sum test. P < 0.05 was considered as statistically significant. Inter-individual differences between cells and tissues were quantified by using Principal Component Analyss and were performed in R platform (http://frodo.wi.mit.edu/primer3/) and their genomic locations were confirmed by UCSC’s In-silico PCR option. The list of primers used in this study is provided in Supplementary Table 46 and using B2M as the house-keeping gene.cDNA conversion of the extracted RNA (1 micro-gram) was done using High-Capacity cDNA Reverse transcription kit (Thermo Fischer Scientific) as per manufacturer’s protocol in a reaction volume of 20 µl. To target fusion junction we designed fusion specific primers by using Primer3 software (Supplementary figures.Supplementary dataset 1-10.