Peripheral nerve injury impairs motor and sensory function in humans, and its functional recovery largely depends on the axonal outgrowth required for the accurate reinnervation of appropriate targets. To better understand how motor and sensory nerve fibres select their terminal pathways, an unbiased cDNA microarray analysis was conducted to examine differential gene expression patterns in peripheral efferent and afferent fibres at different developmental stages in mice. Gene ontology (GO) and Kyoto Enrichment of Genes and Genomes (KEGG) analyses revealed common and distinct features of enrichment for differentially expressed genes during motor and sensory nerve fibre development. Ingenuity Pathway Analysis (IPA) further indicated that the key differentially expressed genes were associated with trans-synaptic neurexin-neuroligin signalling components and a variety of gamma-aminobutyric acid (GABA) receptors. The aim of this study was to generate a framework of gene networks regulated during motor and sensory neuron differentiation/maturation. These data may provide new clues regarding the underlying cellular and molecular mechanisms that determine the intrinsic capacity of neurons to regenerate after peripheral nerve injury. Our findings may thus facilitate further development of a potential intervention to manipulate the therapeutic efficiency of peripheral nerve repair in the clinic. However, numerous studies have indicated that this endogenous regenerative capacity is still limited since functional recovery is usually poor in the context of complete axonal transection with relatively large gap distances5. Peripheral nerve injury has a deleterious impact on daily life, as it significantly impairs motor and sensory function in patients. Therefore, a better understanding of how efferent and afferent nerve fibres select their terminal pathways, and further, how proximal axonal sprouts reach and reinnervate their topographically appropriate targets6, is of fundamental interest to both clinical and basic neuroscientists9.Injured axons in the adult mammalian peripheral nervous system (PNS), unlike those in the central nervous system (CNS), were considered to have the intrinsic ability to initiate regeneration in the appropriate context11. A central issue in neural regeneration is the manner in which various neurons reestablish their distinct and predictable phenotypes by forming functionally appropriate synaptic connections10. Previous studies have indicated that regenerating motor axons are guided through the motor pathways to reestablish nerve-muscle contact in a process termed ‘preferential motor reinnervation’13. However, the molecular mechanism underlying this phenomenon is not yet fully understood15.Generally, functional recovery after peripheral nerve injury largely depends on the accuracy of axonal pathfinding and the efficiency of reinnervation of distal target organs17. Efferent nerve fibres are axons originating from motor neurons, while afferent nerve fibres are processes extending from sensory neurons. In the spinal cord, motor neuron cell bodies are present in the ventral horn of the grey matter, and receive inputs from interneurons transmitting signals via sensory integration feedback18. Efferent nerve fibres conduct signals away from the CNS to their target peripheral organs, and form neuromuscular junctions with innervated muscles. Unlike motor neuron somas, sensory neuron somas are located in the DRGs of the PNS. Axonal processes from groups of afferent neuron somas extend in two opposite directions: the peripheral branch runs into the spinal nerve to innervate the peripheral target organs, while the central branch runs to the dorsal horn of the spinal cord, ultimately conveying sensory information from the PNS to the CNS19. During the development of selective sensory-motor circuits, fundamental biological processes, such as axon guidance, selective target reinnervation, and synaptic plasticity in neural networks, must be regulated properly to establish the appropriate neural circuits20.The sciatic nerve is the largest single nerve in humans. The rodent sciatic nerve injury model is the most commonly used model for studying peripheral nerve regeneration. The spinal segments associated with the sciatic nerve correspond to the lumbar dorsal root ganglia (L4-L6 DRGs)21. The aim of this study was to provide a framework for the integrated analysis of gene regulatory networks during peripheral motor and sensory neuronal differentiation/maturation. We also aimed to further elucidate the biological processes and molecular mechanisms underlying the activation of the intrinsic neuronal regenerative capacity after peripheral nerve injury. These data provide new insights for the development of potential interventions designed to manipulate the therapeutic efficiency of peripheral nerve repair in the clinic.In the present study, an unbiased cDNA microarray analysis was performed to examine differential gene expression patterns in both efferent and afferent nerve fibres at different developmental stages in mice. The detection window was from the postnatal day 0 (P0) to 5 weeks (5 W), as the de novo axonal sprouting and synaptic formation and remodelling during the regenerative processes is analogous with those in the early developmental stagesWe performed cDNA microarray analysis using RNA samples extracted from efferent and afferent nerve fibres originating from the L4–L6 segments of the spinal cord. The dissection range is illustrated using circles in the spinal cord diagram Fig. . At diffThe sample correlation matrices indicated that all the 24 samples (8 groups in triplicate) could be approximately clustered into four sets corresponding to the four experimental time points Fig. . AdditioThe histograms in Fig. Figure The temporal expression profiles of the significantly differentially expressed genes in efferent and afferent nerve fibres were visualized using Short Time-series Expression Miner (STEM) software. A comparison of the five distinct expression patterns of the genes differentially expressed between the efferent and afferent nerve fibres is presented in Fig. th of the differentially expressed genes in the efferent and afferent nerve fibres were first up-regulated and then down-regulated, while a little less than 1/3rd of the differentially expressed genes were first down-regulated and then up-regulated at all developmental stages. The gene expression patterns in the motor and sensory nerve fibres during development are shown in Fig. Hierarchical clustering analysis was performed on differentially expressed genes in efferent and afferent nerve fibres at each experimental time point. The results were visualized using heat map images. The heat maps indicated that nearly 1/4In order to analyse the patterns of differential gene expression in the efferent and afferent nerve fibres during the different developmental stages, the GO analysis database integrated with DAVID’s toolkit was used to identify the most significant differentially expressed genes associated with neuronal development and outgrowth. There genes were categorised based on the following defined terms: Biological Processes, Cellular Components, and Molecular Functions Fig. .Figure 4The enriched subcategories under Biological Processes in the efferent and afferent nerve fibres during development are presented in Fig. In the Cellular Components category, the GO analysis indicated that the excitatory synapse and inhibitory synapse subcategories had no statistically significant differences between the efferent and afferent nerve fibres at P0, although they had significant differential expression starting at 1 W. In addition, the differential expression patterns were similar to those in the presynaptic and postsynaptic membrane subcategories Fig. .The enrichment of Molecular Function in the different developmental stages is summarized in Fig. The GO analysis provided overall insight into the cellular and molecular regulation of motor and sensory neuronal cell fate determination. Kyoto Enrichment of Genes and Genomes (KEGG) analysis was further performed to identify critical signalling pathways during motor and sensory neuronal differentiation/maturation Fig. . It is nThe Z-scores calculated from the average expression profiles of the differentially expressed genes involved in the six most critical signalling pathways during development are listed in Fig. Although the differential expression signatures in the efferent and afferent nerve fibres during development had common characteristics, there were demonstrable differences between the two types of nerve fibres. Such differences were observed in the glutamatergic and dopaminergic synapse pathways. For instance, the significant differential expression of proteins in glutamatergic synapses was first detected at 1 W in efferent nerve fibres. In contrast, this distinctive change in expression was first observed at 3 W in the afferent nerve fibres. The significant differential expression of proteins at dopaminergic synapses was first detected at 1 W in efferent nerve fibres, and at 5 W in the afferent nerve fibres Fig. .Gabra1, Gabrb2, and Slit2 were differentially expressed between efferent and afferent nerve fibres as early as P0, and that multiple types of GABA receptors (GABARs) were consistently differentially expressed at later developmental stages. For instance, Gabra1, Gabra2, and Gabrb2 were found to be differentially expressed at 1 W; Gabra3 and Gabrb1 were still differentially expressed at 3 W; and Gabra2 and Gabra3 were consistently differentially expressed between the efferent and afferent nerve fibres during development. Intriguingly, the neurexin family members Nrxn1 and Nrxn2 were significantly differentially expressed between the efferent and afferent nerve fibres at 1 W, and Nrxn1 was differentially expressed even at later developmental stages. Nlgn1, which encodes neuroligin 1, the post-synaptic binding partner of neurexin, was differentially expressed between the efferent and afferent nerve fibres at 3 W and 5 W. This suggests that the developmental signature of the neuroligins was different from that of the neurexins during efferent and afferent nerve fibre development.To further identify key genes differentially expressed between the efferent and afferent nerve fibres at each developmental time point, gene network diagrams were created using the IPA database Fig. . We founGabrg2, Gabrb2, and Nrxn1 were dramatically downregulated at 1 W when compared with P0. This downregulation was sustained thereafter. Notably, a larger number of GABA receptors, such as Gabra1, Gabra4, and Gabra5, were further downregulated during motor nerve fibre development from 3 W to 5 W. Nlgn1 was involved in motor nerve fibre development from 3 W to 5 W. In comparison, the slit guidance ligand (SLIT) family members Slit2 and Slit3 were downregulated in the extracellular space in sensory nerve fibres at 1 W when compared with P0. These mRNAs interacted with roundabout homologue 1 (ROBO1), which was downregulated in the plasma membrane. In addition, GABAR family members, such as Gabra3, Gabrb2, and Gabrg3, were down-regulated in sensory nerve fibres at 1 W when compared with P0. However, changes in the expression levels of these proteins in sensory nerve fibres were not as obvious as those observed in motor nerve fibres during development. For instance, the Gabrb2 was dramatically downregulated together with Nrxn3 at 3 W and 5 W, while Gabra3 was only upregulated at 5 W. Furthermore, Nrxn2 and Nrxn3, but not Nrxn1, were downregulated together with Nlgn3, which is involved in sensory nerve fibre development.The regulatory networks for the genes differentially expressed during motor and sensory nerve fibre development were further analysed using the IPA database. The gene network diagram suggests that the differentially expressed genes were in highly correlated clusters in well-regulated networks during development Fig. . During Mpz, Slit2, Unc13c, Ncam1, Nrxn1, Nlgn1, Gabrb2, Gabra1, and Npy. The heat map in Fig. Slit2 mRNA expression in afferent nerve fibres was significantly up-regulated when compared with that in efferent nerve fibres at P0, although this trend was reversed thereafter. Nrxn1 mRNA expression in afferent nerve fibres was significantly up-regulated when compared with that in efferent nerve fibres at P0. Interestingly, this differential expression pattern was even more obvious than that for Slit2 because Nrxn1 mRNA expression levels were decreased during development in efferent nerve fibres. Coincidentally, the expression patterns of Nlgn1, Gabrb2, and Gabra1 mRNA were similar to that of Nrxn1. These similarities indicated that the above genes may have essential roles in motor and sensory nerve fibre development. The RT-qPCR results were consistent with the data from the microarray analysis.Several genes were considered representative regulatory genes. These genes were selected from the microarray analysis data for RT-qPCR validation. They included 23. When injured neurons have achieved homoeostasis after trauma-induced disruptions, the intrinsic neuronal regenerative capacity is activated to prompt the proximal stump of the nerve trunk to initiate the regenerative programme. The key neuronal element responsible for axonal regeneration is the growth cone, which is a specialized structure in both motor and sensory neurons25. Generally, growth cone navigation for achieving synaptic rearrangement exhibits a molecular correlation with neuronal differentiation/maturation26, indicating that the regenerative procedure may be fundamentally similar to the process of axon sprouting in the developing neural system2.Under normal physiological circumstances, neurons maintain proper functioning condition by transporting subcellular components to growth cone terminals via axoplasmic flow. However, when the continuity of axons is completely disrupted, the distal stump of the nerve trunk initiates a process known as Wallerian degeneration27. However, other evidences have indicated that it is the artificial culture condition that altered the gene expression patterns, and resulted in the distinct phenotypic characteristics of differentially expressed genes28. In addition, isobaric tags for relative and absolute quantitation-based analysis of total protein levels in different types of nerves in a quantitative proteomics approach revealed that different motor and sensory nerves have distinct protein expression profiles in naive rats29. In the in vivo regeneration model, the growth factor expression profiles between denervated motor and sensory nerves had obvious differences, further indicating that Schwann cells express distinct motor and sensory phenotypes that specifically benefit motor and sensory axonal regeneration, respectively30.Peripheral motor and sensory nerves have distinct functions that contribute to the maintenance of normal physiological activities, especially their unique abilities to accurately and selectively reinnervate terminal nerve pathways. Recent studies have demonstrated that Schwann cells obtained from different anatomical contexts can express distinct motor and sensory phenotypes in cell culture systems32, which may also play important roles in peripheral neural development and regeneration. We will further discuss these phenomena with respect to the involvement of Schwann cells as well.In order to decipher the cellular and molecular events during motor and sensory neuron development, microarray analysis was conducted to identify differential gene expression patterns in both efferent and afferent nerve fibres during different developmental processes. Considering the genders of patients who have peripheral nerve injury in the clinic, both sexes of the C57BL/6 mice were used equally across the analyses. In this study, the efferent nerve fibres mainly comprise axons of motor neurons whose cell bodies are located in the ventral horn of the spinal cord, while, the afferent nerve fibres mainly comprise the central branches of axons extending from DRG neurons that are biochemically and anatomically comparable to the efferent nerve fibres. In the peripheral nerve fibres, myelinating Schwann cells inevitably remain in the microarray samples collected using conventional dissecting methods33. The SLIT proteins are best known for their classic roles in mediating chemorepulsion through the ROBO receptors during axon guidance34. However, accumulating evidence indicates that the functional repertoire of SLIT proteins and ROBO also plays essential roles in neurogenesis, stem cell regulation, and organ development35. It has been reported that SLIT2 contributes to branching/arborisation of sensory trigeminal axons in the developing mammalian CNS36. Intriguingly, several groups have reported that SLIT1 is only expressed in peripheral neurons, while other SLIT family members, such as SLIT2 and its receptors ROBO1 and ROBO2 are also expressed in Schwann cells37, and SLIT2 functions as a repellent in cultured Schwann cells via Ras homologue gene family member A-myosin signalling pathway38. In this study, Slit2 mRNA expression was found to be higher in sensory nerve fibres than in motor nerve fibres at the P0 developmental stage. This is consistent with previous reports describing Slit expression patterns in the embryonic spinal cord. Intriguingly, this developmental characteristic changes after 1 W. The Slit2 mRNA expression level was dramatically up-regulated in motor nerve fibres from 1 W to 5 W, so that its expression in motor nerve fibres was significantly higher than that in sensory nerve fibres at the 3 W and 5 W developmental stages. The differential expression patterns of SLIT proteins in efferent and afferent nerve fibres led to our speculation regarding the specific regulatory role of SLIT/ROBO signalling in motor and sensory neuronal differentiation/maturation across different developmental stages in mice.Summarizing the GO analysis results, genes involved in axon guidance, cell differentiation, and GABAA receptor activity subcategories are significantly differentially expressed at all the four time points examined, indicating that the fundamental molecular and cellular mechanisms underlying the regulation of motor and sensory nerve differentiation/maturation may be biologically distinct. Commissural axons grow along stereotyped dorsoventral trajectories in the early developing spinal cord. The projection patterns are encoded by the spatiotemporal distributions of axon guidance cues, specifically netrin-1/ DCC signalling for ventral attraction and SLIT/ROBO signalling for dorsal repulsion39. Recent studies have indicated that glutamate plays an essential role in sensory input transduction, especially in the nociceptive afferent signalling pathways. Peripheral neuropathic pain and the involvement of inflammatory processes could be attenuated by pharmacologic manipulation of the glutamatergic system40. Neurexins constitute a family of polymorphic cell-surface proteins with hundreds of alternatively spliced isoforms that are essential to the synapse architecture. Recent studies have indicated that distinct spliced isoforms of neurexins are selectively expressed in particular neurons, implying that each type of neuron may have a unique neurexin expression pattern41. Notably, α-neurexins are not strictly limited in their function as synaptic terminal-specific proteins but are also involved in axon-Schwann cell and perineurial fibroblast interactions42. In addition, contactin-associated protein (CASPR)/paranodin, which is a vertebrate homolog of neurexin IV, has been reported to localize at the paranodal region of the Ranvier nodes; CASPR/paranodin links neuronal membrane components with the axonal cytoskeletal network and Schwann cells43. In this study, neurexin 1 was found to be significantly differentially expressed between efferent and afferent nerve fibres in the postnatal developmental stages from 1 W to 5 W, suggesting that neurexin 1 may serve as a central hub gene involved in modulating postnatal sensory neuron development. It is noteworthy that neurexin 1 is predominantly expressed both in motor and sensory axons at P0, implying that neurexin 1 plays fundamental roles in embryonic neural development. Neuroligins are thought to be necessary for maintaining synapse integrity, and function through recruitment of GABARs and ionotropic glutamate receptors. The microarray results indicated that neuroligin 1 mRNA expression level gradually decreased as motor neuron development proceeded, and that the significant differential expression between motor and sensory neurons was only detected at relatively mature stages . These data suggested that neuroligin 1 may follow a different modulating mechanism than its trans-synaptic partner neurexin 1 in regulating motor and sensory neuron development.Trans-synaptic interactions involving neuroligin-neurexin complexes are also implicated in the organization of excitatory glutamatergic and inhibitory GABAergic synapses, and play fundamental roles in the regulation of synaptic cell adhesion44. Moreover, recent evidence has confirmed that GABAergic inputs occur in the mouse embryonic spinal cord as early as at the embryonic age of 12.5 days, implying that GABAergic functions may be essential at very early developmental stages in mice45. The GABA-induced activities are mediated via its receptors, chloride-permeable ionotropic GABAA and GABAC receptors, as well as G-protein-coupled metabotropic GABAB receptors46. Interestingly, accumulating evidence demonstrates that the myelin-producing Schwann cells also express GABAA and GABAB receptors during neural development and regeneration, and that GABAA and GABAB receptors cross-interact with neuroactive steroids48 via activation of Src and phospho-focal adhesion kinase signalling49. The GABAB receptors are involved in myelination in Schwann cells but not in their proliferation50, and GABAB receptors are fundamental in regulating Schwann cell maturation and small nociceptive C-fibres52. The microarray data in this study indicated that Gabra1 and Gabrb2 levels were dramatically decreased in motor and sensory nerve fibres after 1 W. This was especially obvious in motor neuron development, consistent with previous reports that GABAergic activity contributes to the early stages of CNS development. Moreover, Gabra1 and Gabrb2 were significantly differentially expressed only at relatively early developmental time points (P0 and 1 W). To complicate matters more, other GABAR subtypes have been shown to be implicated in neuronal differentiation/maturation events in the following developmental windows (3 W and 5 W), further indicating that GABA and its various receptors have substantial roles in determining motor and sensory neuronal cell fate specification across development in mice.The principal inhibitory neurotransmitter, GABA, is broadly distributed in both CNS and PNS. Accumulating evidence indicates that the formation of GABAergic synapses is crucial for the formation and maintenance of stereotypic neural networks during CNS developmentThe current findings reveal fascinating aspects of differential gene expression signatures between peripheral efferent and afferent nerve fibres at different developmental stages in mice. The precise and balanced trans-synaptic cell adhesion system is crucial for the establishment of functioning neuronal circuits. Thus, accurate reinnervation of motor and sensory original targets requires appropriate synaptic plasticity, which is needed to fine-tune neural development and regeneration. Generally, the physiological and molecular mechanisms that modulate peripheral nerve regeneration are most intensively studied in the nerve injury model. These data serve as valuable resources, and provide a new perspective regarding the possible cellular and molecular mechanisms underlying motor and sensory development. Further in-depth studies are needed to identify the hub genes and key molecular events that regulate peripheral motor and sensory neuronal cell fate determination.C57BL/6 mice at different developmental stages  were supplied by the Experimental Animal Center of Nantong University. All animal protocols were approved by the Committee of Nantong University, and were performed in accordance with the guidelines of the Administration Committee of Experimental Animals, Jiangsu Province, China.2 by inhalation. The lumbar vertebral canal was opened, and the spinal cord was exposed. After the spinal arachnoid was carefully peeled off under a dissecting microscope, the efferent and afferent nerve fibres arising from spinal cord segments L4–L6 were dissected and separately collected into 8 tubes containing ice-cold D-Hank’s solution  corresponding to the 8 groups (four time points and two types of nerve fibres). Tissues were then cut into small pieces (no larger than approximately 1 mm3) and transferred into liquid nitrogen, where they were stored until further processing.The animals  were sacrificed using an overdose of COSamples were electronically homogenized on ice and then centrifuged at 13,200 rpm for 15 minutes at 4 °C. The resulting supernatant was collected and used in the following steps. Total RNA was isolated using mirVana RNA Isolation Kit  according to the product manual. After the total RNA was purified using the RNeasy Mini Kit , its quality was determined based on RNA integrity number (RIN) obtained using a bioanalyzer 2100 system . Samples with RIN values ≥7 were used for further procedures.The cDNA was labelled using Cyanine-3-CTP Quick Amp Labeling Kit . Agilent SurePrint G3 Mouse GE V2.0  microarrays (Agilent Technologies) were hybridized using the Agilent Gene Expression Hybridization Kit . The arrays were then scanned using an Agilent Scanner G2505C (Agilent Technologies). Agilent feature-extraction software (version 10.7.1.1) was used to analyse the microarray images and obtain raw data. Further, GeneSpring GX software (version 12.5) was used to complete the basic analysis. The raw data were normalized using the quantile algorithm. The R software (version 2.13.0) was used for deeper analysis of the microarray data. The limma (linear regression model) package was used to statistically analyse differentially expressed genes.p value < 0.05 and a mean expression fold change greater than 2 were considered statistically significant.The microarray analysis was performed by Shanghai OEBiotech Technology Co., Ltd. . For each group, data from three independent experiments were assessed to ensure reproducibility. All data  was Minimum Information About a Microarray Experiment-compliant, and has been deposited to the National Center for Biotechnology Information database (accession number: GSE113820). Genes and pathways with 17. Hierarchical clustering analysis was performed using Multi Experiment Viewer software (version 4.9). In addition, the STEM software (version 1.3.11) was used to visualize the enrichment analysis of the temporal expression profiles of differentially expressed genes.We performed PCA using the “Population PCA” tool on the Harvard Medical School webpage. The Z-scores (standard scores) were calculated and hierarchical clustering was performed based on log2-transformed mean-centred datasets, as described previously53, and the expression profiles for each GO subcategory were calculated as previously described23.The differentially expressed genes were classified using the following GO categories: Biological Processes, Cellular Components, and Molecular Functions. Database for Annotation, Visualization, and Integrated Discovery (DAVID) bioinformatic resources were used for further analysis of GO category enrichment23.The KEGG bioinformatics database was integrated with DAVID tools, and used to systematically screen differentially expressed genes, essentially as previously describedwww.ingenuity.com; Redwood City, CA, USA) is an online software package used to identify canonical pathways and gene networks, and to further categorise specific physiological processes. The Ingenuity Pathway Knowledge Base was used for deep analysis of the global molecular network, and revealed interactions among the differentially expressed genes.The IPA ; PCR was performed using SYBR Green Premix Ex Taq (Takara) on the StepOne Real-Time PCR System (Applied Biosystems) in triplicate for each sample. The relative mRNA level was calculated using the comparative 2−ΔΔCt method . The sequences of the primer pairs are listed in Table We performed RT-qPCR analysis of the mRNA expression levels of the differentially expressed genes identified in the microarray analysis, including p values <0.05 were considered statistically significant.All quantitative data are presented as mean ± standard deviation. The data were analysed using SPSS 11.5 software . Statistical analysis was performed using one-way analysis of variance, followed by Scheffe’s post hoc t-tests; Dataset 1Dataset 2Dataset 3


Arc to index neuronal activation in response to terpinene re-exposure, stressed cage-mate induced associative learning was measured. Companion rats showed increased neuronal activity in the accessory olfactory bulb, while terpinene + shock-conditioned rats showed increased activity in the main olfactory bulb. Both groups had enhanced activity in the anterior basolateral amygdala and central amygdala. To test involvement of pheromone mediation, in the 2nd paradigm, we paired terpinene with soiled bedding from odor + shock rats or a rat alarm pheromone. Both conditioning increased rats’ freezing to terpinene. Blocking NMDA receptors in the basolateral amygdala prevented odor-specific learning suggesting shock and pheromone-paired pathways converge in the amygdala. An alarm pheromone thus enables cue-specific learning as well as signalling danger.Alarm pheromones alert conspecifics to the presence of danger. Can pheromone communication aid in learning specific cues? Such facilitation has an evident evolutionary advantage. We use two associative learning paradigms to test this hypothesis. The first is stressed cage mate-induced conditioning. One pair-housed adult rat received 4 pairings of terpinene + shock over 30 min. Ten minutes after return to the home cage, its companion rat was removed and exposed to terpinene. Single-housed controls were exposed to either terpinene or shock only. Companion rats showed terpinene-specific freezing, which was prevented by β-adrenoceptor blockade. Using  These molecules, even in minute amounts, can be detected by the vomeronasal system and main olfactory system of another member of the same species (conspecific) and evoke anxiety or fear reactions in the conspecifics6. A rat alarm pheromone released in response to perianal stimulation and footshock has recently been identified2. The release of this pheromone is associated with increases in anxiety behaviors7, hyperthermia to stress events8, and increased auditory startle responses9 in receiving rats. Thus, alarm pheromones can prepare conspecifics for potential danger and thereby aid in the communication of fear to conspecifics without their direct exposure to an inherently stressful situation. But is cue specific fear learning engaged by an alarm pheromone?Fear learning is evolutionarily essential for species’ survival, without which dangerous situations may lead to not only the death of individuals but eventual extinction of the species. Fortunately, one does not have to directly experience danger in order to acquire an adaptive fear; there are different means for the communication of fear. Humans explore their environment crucially through vision and communicate extensively through languages. Humans can learn fear through the observations of others’ experiences. Similarly, just hearing a frightening recollection can lead to avoidance of a dangerous situation in the future. Other animals, such as rodents, rely heavily on their sense of smell to navigate their environment and to communicate. An alarm pheromone is a type of volatile pheromone that is released by rodents when they are stressedThe nature of fear learning is essentially associative. A sight, a sound or a smell associated with a harmful stimulus, can later trigger the recall of the fearful event. This is commonly modeled in rodents by classical conditioning, in which a harmful stimulus  is presented concurrently with a neutral stimulus . Animals develop fearful or defensive responses to the neutral stimulus. Can such classically conditioned fear be transferred to another conspecific in the absence of an external aversive stimulus through pheromone communication? If so, does pheromone-mediated memory utilize distinct neural circuitry from that which mediates classically conditioned fear?2 produced similar odor fear conditioning in naïve odor-exposed rats. To determine whether these two differentially acquired memories  utilize distinct neural circuitries, immediate early genes Arc and Homer1a (H1a) were used to map cells activated by the conditioned odor and a novel control odor across several regions of the brain. We included brain structures specialized for specific odor processing such as the main olfactory bulb (MOB), olfactory tubercle (OT), and piriform cortex (PC)11; for pheromone processing such as accessory olfactory bulb (AOB), and the medial amygdala (MeA)12; and for fear memory formation such as lateral amygdala (LA), basolateral amygdala (BLA) and central amygdala (CeA)16. Our results reveal converging pathways in the common fear circuitry of the amygdala for both types of learning.We developed a pheromone fear communication model in adult rats by housing naïve non-stressed rats (companions) with odor/shock-conditioned rats. The companion rats developed conditioned freezing to an odor that they were exposed to shortly after housing with the odor/shock-conditioned rats. Replacing the odor/shock conditioned rats with their soiled bedding or with the alarm pheromone molecules 4-methylpentanal plus hexanaln = 110 total) were assigned randomly to groups. Rats were housed in polycarbonate cages on a 12 h light/dark cycle, given ad libitum access to food and water, with all behavioural manipulation completed during the light phase of the light cycle. Odor + shock conditioned rats (O+/S+) were housed with same sex companion rats one week before the experiments and for the duration of the experiments, while other groups were housed alone. All procedures were approved by the Memorial University Institutional Animal Care Committee and carried out in compliance with the guidelines of the Canadian Council on Animal Care.Sprague Dawley rats  of both sexes  and octanol (2.67%). These odorants were chosen as they are neither innately appetitive nor aversive to adult rats, and the concentrations were chosen so that the odors would emit a vapor-phase partial pressure of 1 PascalApparatus: All behavioural training and testing was completed with a custom-made olfactometer for air and odorant delivery attached to the shock chamber: a plexiglass chamber that sits on top of an electrified grid, connected to a shock generator/scrambler (Muromachi Kikai Model SGS-003DX). Polyvinyl carbonate bottles were used for each odor and connected to the olfactometer by C-flex tubing pinched shut when not in use. Evacuation tubing with a fan was attached to the top lid of the shock chamber to promote odor removal.via the evacuation tubing. The fourth day consisted of a 30 min behavioural testing session in the same conditioning chamber. Medical air was delivered in the first half of the session and an odorant was delivered in the second half. Rats were tested with terpinene and octanol (a control odor) on the same day, and the order of the odorant testing was randomized and counter-balanced. The percentage of freezing time in response to the terpinene and octanol exposure was measured.Odor conditioning and testing: All rats were habituated to the shock chamber for one 30 min session each on two consecutive days with clean air pumped through the shock chamber. On the third day, rats were trained individually with four separate exposures to either odor, shock, or odor and shock, depending on their respective groups at 5, 15, 20, and 30 min during a 30 min training session. Odorant (terpinene) was delivered for 1 min at each time point. Shock was delivered at the last sec of the odor delivery (0.5 mA for 1 sec). Between each experiment, the shock chamber and grids were thoroughly cleaned with 70% ethanol and clean paper towels. There was a 15 min interval between the chamber cleaning and next experiment, where residual smell and ethanol were removed −/S+, rats were housed alone and received shock only, no odor during the experiment; (2) O+/S−, rats were housed alone and received terpinene odor but no shock during the training; (3) O+/S+, rats received both terpinene odor and shock; and (4) O+/Comp (companion), rats were housed with O+/S+ rats and exposed to terpinene only during the training. O+/S+ rats were returned to the cages with O+/Comp rats immediately following the odor/shock conditioning. Ten minutes later, O+/Comp rats were subjected to the odor only conditioning. A subset of the O+/Comp rats in this group received saline  during the habituation and 40 min before the training; (5) O+/Comp + Prop, O+/Comp rats received saline during the habituation and propranolol  40 min before the training. (6) O−/Comp, companion rats were housed with the O+/S+ rats but not exposed to the conditioned odor.Six groups were examined: (1) OArc and H1a mRNA visualization on the 4th day. Animals were given a final odor exposure in lieu of behavioural testing. Rats were placed in a sealed plexiglass container ventilated with a continuous flow of charcoal-filtered air for 1.5 hrs. Rats were then given a 5 min exposure to octanol, another 20 min of charcoal-filtered air, then a 5 min exposure to the conditioned odor, terpinene, followed by immediate isoflurane anaesthesia and decapitation. Brains were collected and flash frozen in 2-methylbutane immersed in an ethanol/dry ice slurry and kept at −80 °C.Neural circuit mapping: A separate cohort trained identically to groups 1–4 as described above underwent tissue collection for in situ hybridization (FISH): Brains were trimmed so that the cerebellum was discarded, and only the right hemisphere was analyzed. The right hemispheres of rats from each behavioural group were arranged so that the rostral end of their olfactory bulbs touched a razor blade to align them at the same rostral-caudal level. Brains were then arranged in a custom-made plastic box filled with OCT medium at −20 °C in a cryostat and frozen in a block. Coronal sections of 20 μm were collected on 2% 3-aminopropyltriethoxysilane treated slides. Five representative slides over the rostral-caudal range of each of the MOB, AOB, aPC/tubercle, and pPC/amygdala were chosen for FISH and stored at −20 °C.Fluorescence 18. Briefly, full length Arc riboprobes conjugated to digoxigenin and H1a riboprobes conjugated to fluorescein were obtained using commercial transcription kits (Maxiscript) and RNA labeling mixes (Roche). Riboprobes were purified using RNA mini quickspin columns (Roche) and verified via agarose gel.The double FISH protocol was established previouslyArc and H1a probes. Slides were hybridized overnight in a 56 °C oven. All steps until this point were performed in the absence of RNAse. Slides were washed in a series of sodium citrate solutions followed by cleavage of any remaining single-stranded RNA using RNAse A. Endogenous peroxidases were quenched with H2O2 and slides blocked with 5% sheep serum (Sigma-Aldrich). Arc riboprobe was detected with anti-digoxigenin-POD (Roche) and a TSA cyanine-3 substrate kit (Perkin Elmer). Following Arc detection slides were dipped in 2% H2O2 solution to quench any residual HRP activity. H1a riboprobe was detected with anti-fluorescein-POD (Roche) and a TSA Fluorescein Tyramide substrate kit (Perkin Elmer). Nuclei were counterstained with DAPI (Sigma-Aldrich). Slides were coverslipped with Vectashield antifade medium (Vector Laboratories), sealed with clear nail polish, and kept at 4 °C before confocal microscopy scanning.Slides were thawed for 30 minutes at room temperature, fixed in 4% paraformaldehyde at 4 °C, bathed in acetic anhydride and acetone/methanol (Fisher Scientific), and treated with pre-hybridization buffer and hybridization buffer (Sigma-Aldrich) containing Image Acquisition and Analysis: All slides were scanned in a Fluoview FV1000 confocal microscope (Olympus). All images were taken at 20X magnification. The photomultiplier tube assignments, confocal aperture size, and contrast remained constant for each slide. The z-stacks  were taken throughout the thickness of the section and were acquired from 3–4 slides for each animal.The mitral cell layer was analyzed in the olfactory bulbs, including the dorsolateral and ventral medial regions in the MOB. Layer II was analyzed in the PC, and the dense cell layer was analyzed in the OT. Images were analyzed from the center of each of the amygdala subdivisions.H1a+ and Arc+ cells, counting was done manually by checking 20% of the mid-range of the stack that comprised each cell. Average cell counts of Arc+ cells were divided by the average cell counts of H1a+ cells to compute a ratio of cells active to the conditioned odor versus cells active to the control odor for each animal.ImageJ software was used for counting cells in the scanned images. In all areas except the OBs total cell counting was done automatically for the DAPI stained nuclei; images were cropped to include only the area of analysis, transformed to binary images (black and white), and cells were counted using the “Analyze Particles” function in ImageJ. For the +/S− (terpinene odor only); (2) Ph-T (pheromone paired with terpinene). Rats were housed alone and exposed to the clean bedding with a piece of filter paper soaked with 0.75 mL 4-methylpentanal (1.3 × 10−6 M) and hexanal (8.7 × 10−6 M) binary mixture (dissolved in purified water)2 on top of the bedding and received terpinene as the conditioned odor. (3) SB-T (soiled-bedding conditioned with terpinene); (4) SB-Oc (soiled-bedding conditioned with octanol). Rats were housed alone and exposed to the soiled bedding. A donor rat was shocked to release pheromone in the shock chamber (4 shocks during 30 min). The soiled bedding was woodchip bedding placed underneath the shock chamber during the donor rat shock and was subsequently left untouched for the conditioning of the SB rat.Behavioral study: Four groups were included: (1) OHabituation, odor delivery during the training, and testing were carried out in the same manner as in Experiment 1, except testing lasted 10 min , instead of 30 min. Additionally, Experiment 1 and 2 were carried out in two different rooms with different experimenters.To study the role of NMDA receptors in the basolateral amygdala, a separate cohort underwent cannular implantations. Cannular surgeries were performed 1 week before the behavioral experiments. During surgeries, rats were anesthetized with isoflurane gas and secured in a stereotaxic apparatus. Two holes were drilled 2.5 mm posterior, and 4.9 mm bilateral relative to bregma for the BLA. Guide cannulae were inserted 7.8 mm ventral to the skull surface. Guide cannulae were secured by dental cement to two skull screws. The skin was sutured and the rats were returned to their cages for recovery.+/S+ and pheromone molecule conditioned (O+/Ph) rats were infused with either saline or D-APV  bilaterally into the BLAs 30 min before the conditioning experiments. Infusion tubing and cannular attachment were performed during habituation for animals to become acclimated to the attachment of the infusion tubing.Opost-hoc Bonferroni tests were used to compare different groups in Figs OriginPro 9.0 was used to analyze the datasets. One-way ANOVAs plus +/Comp) of the O+/S+ conditioned rats were able to form cue-specific fear memory when subsequently exposed to the conditioned odor terpinene  before the training. Additionally, we tested where the CS-UCS association occurs by including a group of companion rats that were not subsequently exposed to the conditioned odor (O−/Comp). The absence of learning in the O−/Comp group would suggest that any residual terpinene smell on the O+/S+ rat is not sufficient to induce associative learning in the O+/Comp rat during the social interaction, although it does not exclude any priming effects of social interaction on subsequent odor conditioning.In the first experiment, we tested whether the companion rats  and O+/S− , and between the O+/S− and O+/Comp . Pre-training infusion of propranolol (O+/Comp + Prop) prevented the formation of odor-specific memory . The companion rats without terpinene exposure (O−/Comp) spent significantly less time freezing in terpinene  than the O+/S+ rats . No significant difference was observed in animals exposed to the control odor octanol 19. This technique utilizes the immediate-early genes H1A and Arc to visualize cells that are active to two temporally distinct events. H1A is expressed in the nucleus approximately thirty minutes following a stimulus, while Arc appears in the nucleus approximately five minutes after stimulus presentation18. Four groups were used for this experiment: O−/S+, O+/S−, O+/S+ and O+/Comp. Twenty-four hours following odor conditioning, rats were exposed to octanol for 5 min, clean air for 20 min, terpinene for 5 min, and then immediately sacrificed. Cells expressing H1A were those activated by the control odor octanol, while cells expressing Arc were those activated by the conditioned odor terpinene  as a way of normalizing the activation profiles in each region. This within-tissue control protocol reduces variation from intrinsic variability in individual animal response levels to odor input and from variability related to differences in tissue processing. This approach enhanced the signal to noise ratio and resulted in the use of fewer animals for statistical comparisons . Significant differences among groups were observed in the dorsal lateral MOB  than O−/S+ , and O+/S− rats  compared to O−/S+ , and O+/S− rats , and O+/S− rats , significantly different from O+/S+  compared to O−/S+ , and O+/S− rats , and O+/S− rats . Another group of rats were exposed to the soiled bedding (SB) from the donor shocked rats, but were never physically in contact with the donor rats. A subgroup of the SB rats were conditioned with terpinene (SB-T) while another subgroup was conditioned with octanol (SB-Oc). All rats were tested for freezing in the presence of terpinene and octanol separately . The SB-T group also showed more freezing  compared to the O+/S− group  or pheromone conditioned training  and tested freezing behavior 24 hr later. Both forms of learning were prevented by the D-APV infusions. In O+/S+ rats, the D-APV infused group  showed significantly less freezing than the saline infused control group  also spent significantly less time freezing than the saline infused group  as that observed in pups22 remains to be examined.Rat pups have also been shown to acquire an odor aversion when a novel odor is paired with their mother’s fear+/S+) is correlated with enhanced activation of the MOB and BLA during memory recall. The MOB has been shown to be a critical site for odor associations in multiple learning models29. The BLA is essential for shock-mediated conditioning including contextual fear conditioning30 and odor conditioning22. Hebbian plasticity requires coincident inputs of both CS and UCS onto common postsynaptic cells31. While the BLA receives olfactory inputs and somatosensory inputs such as those induced by electrical shock33, whether MOB neurons receive direct somatosensory inputs is unknown. Alternatively, locus coeruleus (LC) neurons release NE following shock35. The MOB36 and BLA38 receive extensive LC projections. Odor and NE inputs could converge on both MOB and BLA neurons to initiate plasticity mechanisms. It has been shown that both Hebbian plasticity and neuromodulation by NE are required for tone-shock fear conditioning in the amygdala39. MOB plasticity could reinforce BLA potentiation either through its direct projections to the BLA, or via the PC or the MeA projections42, as suggested by our retrograde tracing data. In fact, both the PC and BLA exhibit potentiated odor responses following fear learning in another study43. In associative odor reward learning, PC neurons exhibit more reliable activation to the conditioned odor, although the absolute number of activated cells does not increase45. This may occur during aversive odor shock conditioning as well.Freezing here in the classically conditioned rats (O47, with the MOB system being responsible for volatile odorant detection and the AOB system detecting pheromones, recent evidence has revealed that the two systems have considerable overlap in terms of chemosignal detection and the behavioural effects they mediate49. A subset of vomeronasal neurons express odorant receptors and project to the AOB50. The AOB system can thus detect both odorants and pheromones51. We suggest potentiation of AOB neurons is linked to pheromone stimulation acting as a UCS in the associative learning of the CS odor with both signals converging on and potentiating common neurons in the AOB. Another possibility is that amygdala cortifugal input shaped the potentiated responses in the AOB despite an absence of greater activation in the back-projecting cortical amygdala nucleus52. The ability of terpinine exposure following a 10 min exposure to the O+/S+ rat to promote equally strong specific cue odor learning in the companion rat is somewhat surprising, given the long-standing evidence that CS must precede or be contiguous with the US in associative learning. Companion rats were able to associate the pheromone released from a stressed rat with a subsequent odor cue in the absence of the stressed rats, however an association is not formed without the subsequent odor cue (Fig. 7 could prime pheromone-activated neurons for later association with odor activation. The odor stimulus would then subsequently become sufficient to drive AOB neurons in the absence of the pheromone stimulus. Our data showing how β-adrenoceptor blockade prevents pheromone learning is consistent with the involvement of NE. However, our data do not exclude the possibility that other priming effects may occur during social interaction between the stressed and companion rats such as residual odor smell on the stressed rats or ultrasonic communication.It is intriguing that pheromone-conditioned learning in companion rats leads to enhanced AOB activation to the conditioned odor upon memory recall. Although initially regarded as functionally independent systems16. Elevated CeA activation was observed in both types of learning in our study. Our results regarding the classical and pheromone conditioning pathways are summarized in Fig. Similar to classically conditioned fear, stressed cage mate-conditioned fear activates the BLA during memory recall. Abolishing plasticity in the BLA by NMDA receptor blockade prevents both classical and pheromone-conditioned fear memory formation. From the BLA, information flows to the medial portion of the CeA which sends output to the periaqueductal grey and hypothalamus to mediate freezing and autonomic responses associated with fearFunctionally speaking, it is advantageous for an animal to associate environmental cues with a potential danger even when he navigates away from the source of the alarm pheromone, as the source of the alarm pheromone, such as a stressed or injured rat, may have parted from the actual site of the danger. This is mimicked in our behavioral paradigm when the companion rats received the pheromone in the home cage from shock-treated rats that had already parted from the shock chamber.In summary, we now report that alarm pheromone released from a stressed rat can serve as a UCS and produce associative learning in a receiver rat. Whether additional input from the stressed rat primes or amplifies these effects remains to be clarified, but the pheromone alone experiments demonstrate that this input suffices to produce conditioning. Unlike classical odor conditioning that leaves a memory trace in the MOB, pheromone conditioning potentiates AOB activation and the AOB appears to mediate the specific odor cue association as well. However, the two forms of learning activate common fear pathways in the amygdala. BLA plasticity is critical for both classical and pheromone conditioned learning. Our study sheds light on how animals communicate with each other in nature and how they may avoid danger through pheromone-mediated associative learning.Supplementary Figures


Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus. A potentially novel ampelovirus was also detected by a complimentary Illumina sequencing approach. The full-length genome sequence of yam chlorotic necrosis virus was determined using Sanger sequencing, which enabled determination of the coverage and sequencing accuracy of the MinION technology. Whereas the total mean sequencing error rate of yam chlorotic necrosis virus-related MinION reads was 11.25%, we show that the consensus sequence obtained either by de novo assembly or after mapping the MinION reads on the virus genomic sequence was >99.8% identical with the Sanger-derived reference sequence. From the perspective of potential plant disease diagnostic applications of MinION sequencing, these degrees of sequencing accuracy demonstrate that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length positive-sense single-strand polyadenylated RNA plant virus genomes.We here assessed the capability of the MinION sequencing approach to detect and characterize viruses infecting a water yam plant. This sequencing platform consistently revealed the presence of several plant virus species, including  These discoveries have strengthened our understanding of the ecological roles and impacts of viral communities, indicating that viruses are likely essential components of ecosystems as diverse as the human gut6 and the oceans8. For example, metagenomics approaches that consider the spatial arrangements of plant samples, and the precise environmental contexts of individual sampling sites have revealed the impact of agriculture on the distribution and prevalence of plant viruses at the ecosystem scale9. While the usefulness of viral metagenomics for diagnostics and viral surveillance is still debated13, these approaches have proven effective for the discovery of unknown etiological agents15. Specifically, in plant virology, viral metagenomics approaches focusing on the analysis of virus-derived small interfering RNAs (siRNA) has gained in popularity for the detection of both known and previously uncharacterized plant viruses within infected plants17.Metagenomic approaches have enabled the discovery of hundreds previously unknown virus species18. Although viral metagenomics approaches have both expanded our view of global viral biodiversity, and paved the way towards a better understanding of viral ecology and evolution, several methodological biases undermine the effective analysis and taxonomic assignment of the sequence reads and contigs that have been generated using these approaches20.Viral metagenomics methodologies, which until now have been primarily based on high-throughput second generation sequencing technologies , have generated billions of ~150–300 nucleotides (nt) or of 21–24 nt small RNA (sRNA) reads and, depending on the sequencing coverage, have been successful for detecting and characterizing low-frequency genetic variants within viral populations21. Whenever a known related reference sequence is unavailable to guide the mapping of short reads, the reads must be assembled de novo to obtain either full genome sequences, or large genomic sequence “contigs”. These de novo assembled sequences are in most cases likely to be chimaeras of reads from different viral variants23: a factor that should exclude these sequences from being used to make phylogenetic and demographic inferences. In addition, differentiation between exogenous and endogenous virus sequences remains difficult to achieve based on de novo assembled short read contigs because the genomic environment of these contigs is usually missing.One of the most important of these methodological issues relates to the production and analysis of short reads24. This technical problem is compounded by the dearth of viral taxa that are represented in public nucleotide sequence databases such as GenBank. A consequence of this is that, up to 70% or more of the reads that are generated by some environmental viral metagenomic studies, end up being labeled as “dark matter” because they have no detectable homology to sequences within the public nucleotide sequence databases25.A second methodological issue with metagenomic approaches that employ second generation sequencing methods relates to the difficulty of creating scaffolds by chaining reads and contigs together. Failure to build large enough scaffolds or contigs can reduce the proportions of reads that can be reliably identified as being related to known virus species using alignment-based approaches such as BLASTde novo from short sequence reads. These techniques could therefore avoid the problems mentioned above26. Among the third-generation sequencing techniques is that implemented in the Oxford Nanopore MinION. The MinION has recently proved effective for delivering, in real time, average reads lengths of >15 kilobases29. While the per nucleotide error rate of the MinION long reads is frequently in excess of 20% – substantially higher than that of the shorter Illumina reads30 – the high degrees of genome coverage that are achievable together with post-sequencing data analysis can still yield consensus sequences that are >99% accurate31 and can be effective for identifying recombinant viral variants32. The MinION has been used to detect and determine the full (or nearly full) genome sequences of a range of animal and human viruses, including ebola33, dengue34, zika35, influenza31, cowpox36, and Ross River virus37. The only published applications of the MinION to sequencing plant virus genomes involved the detection of maize streak virus, maize yellow mosaic virus and maize totivirus in maize plants38 and plum pox virus in plum plants39.Third generation sequencing techniques that are capable of generating much longer reads from individual RNA or DNA molecules promise to eliminate the need to assemble contigs Badnavirus genus), yam mild mosaic virus  and yam chlorotic necrosis virus . The full-length genome sequence of YCNV was determined using a Sanger sequencing approach to enable the comparative quantification of the degrees of sequencing coverage and accuracy using the Illumina and MinION platforms. We show that consensus sequences of YCNV whether obtained by de novo assembly or by mapping of the MinION reads to a reference genome were >99.8% identical to the Sanger-derived sequence of this virus, which indicates that the MinION should in a near future reliably enhance research on, and the monitoring of, plant viruses.Here, we aimed at using the MinION for detecting and characterizing several plant viruses infecting a single yam plant. This sequencing platform proved efficient for detecting Dioscorea bacilliform virus  infected by YMMV and YCNV was collected in Kerala (India), in 2010 and maintained in vivo at the DSMZ Plant Virus Department (PV-1066).A water yam plant  molecules, including small 21–24 nucleotides (nt) interfering RNAs, were extracted from symptomatic leaves of the water yam sample using a RNAzolRT kit  following the manufacturers protocol . The small RNA fraction was quantified in a Qubit fluorimeter using a Qubit RNA HS Assay Kit  and checked for quality in a bioanalyser 2100 (Agilent). RNA passing the quality check was used for library preparation and subjected to high-throughput sequencing on an Illumina “Hi-Seq 2000” instrument using the services of a commercial company .de novo assembled sequences (using the CAP3 sequence assembly program40) from publicly available D. alata expressed sequence tag (EST) resources  was performed using BLASTn and BLASTx searches against the GenBank nucleotide collection (nt/nr) sequence databases implemented in the software KoriBlast 3.1  with a maximum e-value threshold of 10−3.A systematic search of viral sequences in 41 with default parameters) of the consensus sequence of YCNV obtained using the sRNA Illumina approach, one 2306 nt long sequence with a high degree of similarity (80.7%) to YCNV recovered from D. alata EST sequences obtained from a plant grown in Nigeria42, and sequences of known macluravirus species (Chinese yam necrotic mosaic virus (CYNMV) and yam chlorotic necrotic mosaic virus (YCNMV)). RT-PCR reactions were performed using the Qiagen OneStep RT-PCR Kit . The 25 μL RT-PCR reaction mix consisted of 1 μL of eluted RNA, 14 μL of RNAse-free water, 5 μL of RT-PCR buffer (5X), 1 μL of dNTP mix (10 mM), 1.5 μL of each primer (10 μM) and 1 μL of RT-PCR enzyme mix. The RT-PCR program was as follows: 50 °C for 30 min, 95 °C for 15 min, 35 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 or 2 min with a final 72 °C extension for 10 min. PCR products were analyzed by electrophoresis on a 1.2% agarose gel in TAE buffer stained with ethidium bromide and visualized under UV light. Since the extreme terminal ends were not covered, the exact termini were analyzed by 5′- and 3′-RACE. To amplify and sequence the 5′ region of YCNV genome, cDNAs were produced with the virus specific primer YamMac31R  as described by the manufacturer. A set of 30 overlapping primers (Supplementary Table One-hundred mg of yam leaves were ground with a mortar and pestle in liquid nitrogen and 450 µL of RLC buffer (Qiagen) with 0.1% of ß-mercaptoethanol (Sigma Aldrich) were added in a 1.5 ml tube, which was then vortexed twice and kept for five minutes at room temperature. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen). A concentration of 25 ng/μl of total RNA was quantified by the Qubit fluorometer (Life technologies). The MinION sequencing library was prepared using the SQK-PCS108 cDNA-PCR kit (Oxford Nanopore Technologies Ltd). Briefly, cDNA was first generated using RT and the strand-switching method  using a kmer-based approach (k = 8). Specifically, for each read  identified as having detectable homology to the reference sequence, the coordinates of all perfectly matching 8 nt long query sequence fragments were identified on the reference sequence. All perfectly matching 8 nt fragments that were within 20 nucleotides of at least one other perfectly matching 8 nt long fragment on either the query or reference sequences were flagged as being accurately mapped and each of the nucleotides within these fragments was identified as being accurately sequenced . When 8mer fragments of the query sequence that contained nucleotides that were not denoted as matches in this initial scan (i.e. those nucleotides that were not within any 8 mer fragment that was accurately mapped), were bounded by two accurately mapped 8 mer fragments, the portion of the reference sequence between the mapping coordinates of these accurately mapped fragments were pairwise aligned  with the unmapped query sequence nucleotides. Matching nucleotide positions, mismatched nucleotide positions and the numbers and lengths of insertions and deletions in the query sequence fragment were then enumerated from these pairwise alignments. The 3′ and 5′ unmapped fragments of the query sequences were not used to enumerate numbers of matching nucleotides, mismatching nucleotides, insertions and deletions.Illumina and MinION reads and contigs were compared to sequences in the GenBank database using DIAMOND47 and de novo assemblies were generated using SPAdes 3.6.2 software48 and CAP340. MinION reads de novo assembly was done using Canu v1.649 with the suggested parameters for Nanopore R9 1D sequencing (-nanopore-raw overlapper = mhap utgReAlign = true) using reads >1 kb (minReadLength = 1000) and a genome size of 20 kb (genomeSize = 20 k).Illumina reads cleanups and corrections were performed using the CutAdapt version 1.8.1 program50 using the end-to-end and very sensitive option. All reads mapped by Bowtie2 were kept in the analysis. Resulting bam alignment files were then processed with samtools 1.351 to produce mpileup files. Bases with a phred score lower than 13 were filtered out. The resulting mpileup files were then parsed with custom python and R scripts to extract positions and coverages , while the similarity was computed using the fifth column of the file, which displayed the numbers of matches, mismatches and indels at each position in the original bam files. Similarity was reported as percentage of match over the coverage. Means of similarity across 100 nt long sliding windows (step size: one nucleotide), were used for clarity of display. Dataset and scripts are available in a github repository at the address: https://github.com/loire/roumagnac2018_figs. Consensus sequences of the YCNV genome were obtained from mapped sRNA Illumina reads, mapped MinION reads, and jointly mapped sRNA Illumina plus MinION reads. Pairwise identity analyses of the reference YCNV Sanger sequence and de novo Minion contigs, mapped sRNA Illumina reads-, mapped MinION reads-, and mapped sRNA Illumina/MinION reads- consensus sequences were carried out using the MUSCLE-based pairwise alignment option implemented in SDT v1.252.Both sets of reads were independently and jointly mapped to the complete Sanger YCNV genome with Bowtie2 v2.2.941. Maximum-likelihood phylogenetic trees were inferred using PhyML 3.153 implemented in MEGA version 6.0654 with the nucleotide substitution model GTR + G + I selected as best-fit models by jModelTest55. Branch supports were tested using 1000 bootstrap replicates.All available macluravirus full genomes were downloaded from GenBank and aligned using MUSCLE−4 < DIAMOND e-values < 1.7 × 10−34) were related to DBV, 17 to pestiviruses , 5 to bymoviruses  and 2 to begomoviruses . Interestingly, DBV is reportedly the most prevalent virus in yam56 and transcriptionally active endogenous geminiviral (EGV) sequences have also been identified integrated within the genomes of many yam species57. The two begomovirus MinION reads recovered in this study share 67% and 89% identities with previously identified EGVs in D. alata. Five reads were assigned to the Bymovirus genus: a sister genus of the Macluravirus genus in the Potyviridae family. These reads may have been misassigned due to the potentially high rate of MinION sequencing errors and may actually be macluravirus-related reads.With the intention of comparing the potential utility of Illumina and MinION sequencing platforms within a virus discovery context, we attempted to determine the complement of viruses present within a single yam plant using a Nanopore MinION device. In total, 2,036,598 reads were generated of which 41,487 (2%) were identified as likely being virus-derived using DIAMOND searches .A total of 15,365,074 small RNA - including small interfering RNA - raw Illumina sequence reads were generated from the water yam sample. In agreement with the analysis of the MinION reads, 56 contigs >100 nt long that were obtained by es Table . OverallDe novo assembly of MinION reads yielded two large contigs. BLASTn comparisons between these two contigs and all sequences in GenBank indicated that the highest similarity scores were detected with YMMV  for contig #1 (8791 bp) and YCNV  for contig #2 (7277 bp).59. Like the other known annotated macluravirus genomes, this virus lacks a P1 proteinase, and is likely to use its HC-Pro and NIa proteinases for polyprotein cleavage. The HC-Pro self-cleavage site between HC-Pro and P3 is likely the FVG/V site located at aa positions 259 to 262 of the polyprotein.A 8263-nt full length genome sequence of YCNV isolate Kerala (accession number MH341583) was further assembled and annotated, confirming that it possesses a typical macluravirus genome organization Fig. , includi60, five putative NIa proteinase cleavage sites were identified that have the canonical dipeptide Q(E)/M(A) with a conserved L residue at the −2 position from the cleavage site. These potential cleavage sites were located at positions 569 , 630 , 1289 , 1369 , and 2340  of the polyprotein  is present, followed by a short PIPO region of 59 aa, ending at a stop codon at positions 1538–1540 of the genome.Based on the alignment of the 2628 aa long polyprotein with artichoke latent virus (ArLV) Chinese yam necrotic mosaic virus (CYNMV) and YCNV isolate YCNV-YJish from Chinaein Fig. . Two add62. The virus sharing the highest genome-wide pairwise identity with YCNV isolate Kerala (81.9%) is YCNV isolate YJish  that covered 87.5% of the full-length YCNV genome and shared 99.889% pairwise identity with the reference Sanger sequence of this genome  sequences: which would have presumably been plant or plant-associated bacterial/fungal derived polyadenylated mRNA transcripts  tract (e.g. ampeloviruses) could potentially go undetected using the MinION protocol that was applied here. This bias would probably not have been an issue if viral genomes were randomly amplified. Interestingly, a recent study has shown that MinION enables rapid detection of enteric viruses following the ligation of sequencing adapters to randomly pre-amplified viral genomes63. Specifically, viral metagenomics approaches based on random amplification of virion-associated nucleic acids (VANA) purified from virus-like particles have proven effective in the discovery of novel RNA and DNA plant viruses65, which paves the way towards sequencing all types of plant viruses using the MinION technology. In addition, MinION 1D2 libraries should be used because they offer increased accuracy advantages relative to the existing 1D approach. However, PCR-free MinION detection of plant viruses may be problematic because of how technically difficult and time-consuming it is to purify large-enough quantities of plant virus DNA/RNA from plant tissue samples.Overall, the present study shows that the MinION is an efficient means of detecting and characterizing positive sense ssRNA viral genomes that have 3′ poly(A) tracts. It also indicates that the Potyviridae genera (nt sequence identity less than 76% either in the coat protein gene or over the whole genome), suggesting that the water yam plant used in this study is infected by YCNV. While the genome coverage of the mapped Illumina reads was better than that of the mapped MinION reads, the consensus YCNV sequences generated by both approaches shared more than 99.83% identity with the YCNV reference Sanger sequence  is YCNV isolate YCNV-YJish Fig. . This de20. First, long reads would solve the assembly chimaera problems that have plagued attempts to use short-read sequencing platforms such as Illumina for sequencing genomes directly from non-clonal viral populations. Secondly, long-read sequencing platforms like MinION will yield smaller numbers of unassigned reads (i.e. will reduce the amount of dark matter) by increasing the efficiency with which reads can be assembled into contigs. While the sizes of these contigs will be larger, the probability that highly variable genes (such as silencing suppressor or movement protein encoding genes) will be accurately linked within contigs to more conserved genes  will be increased.Besides this promise, the potential utility of the MinION platform for generating sequencing reads in excess of 10 Kb could have a major impact on viral metagenomics studies. Reads of this length would encompass the genomes of most viruses with RNA or ssDNA genomes. This would correct two of the most important biases that arise during metagenomics studiesde novo assembly of MinION reads, which might still result in chimeric consensus sequences that mix reads originating from different viral variants. Consequently, there are several areas in which improvement is still needed before sequences generated by this technology can be used reliably for downstream analyses such as to test for evidence of genetic recombination, adaptive evolution, or resistance breaking mutations.However, the per-read error rate of the MinION remains very high (most often exceeding 10%) and the total numbers of sequenced nucleotides are lower than Illumina and other shorter reads platforms. Also, it remains to be determined whether the MinION platform is capable of differentiating between exogenous and endogenous viral sequences, between the different components of segmented virus genomes or between viral variants. These limitations could hinder reliable 66 and the mobile real-time surveillance of Zika viruses in Brazil67. While field-monitoring of plant viral diseases is a still-missing first step towards identifying and controlling the emergence of new plant diseases, widespread use of highly portable sequencing platforms like the MinION is likely to revolutionize plant disease diagnostics and is already poised to enable the implementation of reliable epidemiosurveillance networks.Nevertheless, successfully demonstrating that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length plant virus genomes is an important first step towards the application of highly portable sequencing platforms like the MinION to field-based plant virus diagnostics such as has already been attempted for the identification of closely-related plant speciesSupplementary Material


Demographic data were recorded on admission: age 39 ± 19, Glasgow Coma Score 5 (3–8). Neutrophil Gelatinase-Associated Lipocalin and inflammatory mediators were measured in plasma on admission and after 24, 48 and 72 hours; urine were collected for immunoelectrophoresis having healthy volunteers as controls. Human renal proximal tubular epithelial cells were stimulated with patients or controls plasma. Adhesion of freshly isolated human neutrophils and trans-epithelial electrical resistance were assessed; cell viability (XTT assay), apoptosis (TUNEL staining), Neutrophil Gelatinase-Associated Lipocalin and Megalin expression  were measured. All patients with normal serum creatinine showed increased plasmatic Neutrophil Gelatinase-Associated Lipocalin and increased urinary Retinol Binding Protein and α1-microglobulin. Neutrophil Gelatinase-Associated Lipocalin was significantly correlated with both inflammatory mediators and markers of tubular damage. Patient’ plasma incubated with tubular cells significantly increased adhesion of neutrophils, reduced trans-epithelial electrical resistance, exerted a cytotoxic effect and triggered apoptosis and down-regulated the endocytic receptor Megalin compared to control. Plasma of brain injured patients with increased markers of subclinical acute kidney induced a pro-inflammatory phenotype, cellular dysfunction and apoptotic death in tubular epithelial cells.Acute kidney injury following traumatic brain injury is associated with poor outcome. We investigated  Recent studies reported an incidence of AKI equal to 8–20% in TBI patients6 using serum creatinine increase and urine output decrease as criteria for AKI. However it has been reported that 15–20% of patients who do not fulfill these criteria are likely to have acute tubular damage, a condition recently defined as “subclinical AKI”7.In patients with traumatic brain injury (TBI), outcome is highly influenced by non-neurological complications8, is mediated by the release of both pro- and anti- inflammatory cytokines and chemokines12. Interleukin-6 (IL-6), up-regulated in human serum and cerebrospinal fluid following TBI15, is also expressed in renal tubular epithelial cells during AKI, promoting neutrophil infiltration and exacerbating renal injury16. Consequently, systemic inflammation occurring after TBI has been proposed as a possible mechanism of AKI causing functional alterations and apoptosis of tubular epithelial cells similarly to the sepsis-associated AKI. Nevertheless in brain injured patients cellular mechanisms of subclinical AKI have not been fully elucidated.In severe brain injured patients, a systemic inflammatory reaction, triggered by the primary injury and associated with secondary insultsin vitro study, we exposed human kidney-derived tubular epithelial cells to plasma of TBI patients with evidence of subclinical AKI.We hypothesized that following TBI, tubular cells represent a target of the inflammatory reaction leading to a consequent cellular dysfunction. To test this hypothesis, we performed a prospective observational clinical study to identify severe TBI patients with subclinical AKI and increased inflammatory reaction. In the The Institutional Review Board  approved this prospective observational study. If the patients were incompetent, they were included into the study and informed consent was delayed. The family was informed of the study . Written informed consent for using collected data was hence obtained from the patient (if competent) or from the family (in case of death or if the patient remained incompetent). All methods were performed in accordance with the relevant guidelines and regulations.17, during 18 months from September 2011 up to March 2013.All patients with severe TBI consecutively admitted to the NeuroIntensive Care Unit at the Trauma Center of the “Azienda Ospedaliera Univerisitaria Città della Salute e della Scienza di Torino” were included as part of a European network for high resolution data collection after TBI, the Brain Monitoring with Information TechnologyInclusion criteria were age older than 18 years and severe traumatic brain injury (Glasgow Coma Score lower than 9). The severity of brain injury defined as GCS < 9 was confirmed after patient stabilization in ICU.18) including diabetes; patients with extracranial injuries causing severe hemorrhagic shock and requiring transfusion on admission; immunosuppression, pregnancy, lack of consent.Exclusion criteria were GCS = 3, history of known chronic kidney disease (eGFR < 60 ml/min: Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula19. All patients were sedated, intubated and mechanically ventilated. Glasgow Coma Scale (GCS), Injury Severity Score (ISS) for the assessment of multiple injuries, Acute Physiology and Chronic Health Evaluation score (Apache II) to quantify the severity of illness and Marshall scale to classify head CT scan lesions were recorded on admission.Patients were managed according to the Brain Trauma Foundation Guidelines20, haemoglobin, white blood cell count.During the ICU stay, patients were continuously monitored to detect episodes of secondary insults (i.e. hypotension). Acute kidney injury (AKI) was assessed using the RIFLE criteria: serum creatinine (SCr) and 24 hrs urine outputBlood samples for analysis of Neutrophil Gelatinase-Associated Lipocalin (NGAL) and cytokine levels were collected at the time of inclusion (T0), at T24, T48 and T72 h. All plasma samples were centrifuged and then frozen at −80 °C until analyzed.For the quantitative measurement of NGAL concentration , the Triage® NGAL test was used, a fluorescence-based immunoassay used in conjunction with the Triage Meter , a fluorescence spectrometer.The Bioplex assay for 27 cytokines was carried out in 96-well microplates using the Bio-Plex Pro Human Cytokine 27-plex Assay kit  at the Bioclarma - Research and Molecular Diagnostics, Torino, Italy. Plasma samples were diluted at 1:4 and run in duplicate.Urine immunoelectrophoresis: Urine samples were collected at T48 for the evaluation of proteinuria; due to the emergency admission it was not possible to perform this analysis on fresh urine samples at T0 and on T48 were available in 10 patients. Determination of proteinuria was performed by the Hydragel 5 Proteinurie KIT . Electrophoresis on neutral buffered SDS-agarose gels separated the urinary proteins by molecular weight and distinguished the proteins of tubular ) and glomerular (>70 kDa) origin. The resulting electrophoretic patterns, stained with acid violet, were visually compared to molecular weight protein markers in the reference track to identify the individual proteins and thus classify proteinuria. Pooled samples for age- and gender-matched healthy volunteers served as controls.22.PTEC were obtained from normal cortex fragments of surgically removed kidneys. Immortalized tubular cell line is generated by infection with a hybrid Adeno5/SV40 virus as previously described2. PTEC were stimulated with 10% of patients’ plasma. Plasma from healthy volunteers were used as control. Optimal plasma percentage was determined on the basis of previous studies conducted in our laboratory23. In all the parts of the in vitro study PTEC monolayers were incubated with 10% TBI patients’ or healthy volunteers’ plasma.Cell cultures were maintained in RPMI  containing 10% of fetal bovine serum (Gibco BRL Life Technologies Inc.) at 37 °C in humidified air with 5% CO24.Neutrophils were isolated from healthy adult volunteer’s blood sample according to published methods2. After 30 minutes, non-adherent cells were removed and the monolayers were washed several times with 1X PBS. At this point, adhered PMN were counted using a fluorescence microscope.Whole blood added with EDTA was collected, layered over a density gelatin gradient and centrifuged. The neutrophil layer was aspirated and residual erythrocytes lysed. Cells were then washed, counted and re-suspended in buffer to obtain the desired concentration. Neutrophils were labeled with PKH26 red fluorescent cell linker kit . Confluent monolayers of PTEC in 48-well plates were previously incubated with 10% of TBI or healthy plasma for 8 hours. PKH26-labeled PMN were added to the monolayers and the plates were incubated at 37 °C with 5% CO23.Cell polarity was assessed by Trans-Epithelial Electrical Resistance (TEER) and by expression of proteins typical of fully differentiated PTEC such as the endocytic receptor megalin® collagen-coated polycarbonate membranes  and allowed to reach confluence before the addition of 10%of TBI patients’ plasma or control healthy plasma incubated for 12 h. An epithelial volt-ohm meter  was used to determine TEER values. All measurements were performed in triplicate for each well, normalized for the area of the membrane used in the experimental procedure and expressed as ohm/cm2.PTEC were plated on TranswellsTotal RNA was extracted using the RNeasy Mini Kit .RNA yield and quality was determined using a NanoDrop Spectrophotometer . Complementary cDNA was generated by reverse transcription of 2 μg of high quality total RNA using the High Capacity cDNA Reverse Transcription Kit . The measurement of Megalin and NGAL mRNA levels was performed by SYBR green qRT-PCR analysis using the ABI Prism 7900 Sequence Detection System  and SYBR FAST . The following primers were used: Megalin forward 5′GCCGATGCATTTATCAAAAC3′, Megalin reverse 5′TCACATCCATCTTCATCTCC3′, NGAL forward 5′GGAAAAAGAAGTGTGACTACTG3′, NGAL reverse 5′GTAACTCTTAATGTTGCCCAG3′, GAPDH forward 5′ACAGTTGCCATGTAGACC3′, GAPDH reverse 5′TTTTTGGTTGAGCACAGG3′ . Relative quantification was performed using the standard curve method. The results for the target gene expression were normalized on GAPDH as the endogenous control, and the mean values of the vehicle were set as the calibrator.25.Cellular viability was studied by using the XTT-based colorimetric method , a sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 2, 3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. The viability of the cells stimulated with TBI plasma was compared to that of the cells stimulated with healthy control plasma. Cells were seeded into 96-well plates with 150 μl of medium RPMI and incubated with 10% of TBI and control plasma for 24 hours. Then, XTT solution was added and the optical density was measured at 490 nmIn Situ Apoptosis Fluorescein Detection Kit 25.PTEC cells were exposed to 10% of TBI patients’ plasma for 48 hours. In all groups studied, apoptosis was assessed by terminal de-oxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and by the detection of fragmented DNA using the ApopTag® Plus Continuous data are presented as mean and standard deviation or median and range. Correlation between plasmatic NGAL and cytokines or proteinuria was performed by linear regression analysis. Data normally distributed were analyzed by Student’s t-test, and data not normally distributed were analyzed using a Mann-Whitney U or Kruskal-Wallis test. Differences were considered significant when the probability value was <0.05. Statistical analysis was performed using SPSS-statistical software, version 21.0 .Twenty-three adult severe TBI patients  were enrolled in this study over a period of 30 months. Forty-three trauma patients were screened for eligibility see Fig. .Figure 126) those with the strongest correlation with markers of tubular damage were selected for the present analysis. Plasmatic NGAL and inflammatory mediator levels collected on T48 were significantly correlated: NGAL vs IL-6 , NGAL vs MCP-1  and NGAL vs MIP-1β  and that tubular injury is probably associated with the severity of the systemic inflammatory response. Indeed plasma NGAL and low molecular weight proteinuria were significantly increased compared to controls even if biomarkers currently used for diagnosis and risk of AKI (i.e. serum creatinine and estimated glomerular filtration rate (eGFR))33. The presence of low molecular weight proteinuria in TBI patients confirmed the putative presence of an ongoing subclinical AKI associated with tubular dysfunction. In future larger studies including patients with increased serum creatinine it would be interesting to investigate if there is a threshold level of NGAL corresponding to increased creatinine level.The increased plasmatic NGAL concentrations observed in the present study could be related to the inflammatory reaction occurring after TBI as well as to the development of AKI. The parallel increase of NGAL levels and proteinuria suggested the presence of a subclinical form of AKI. Indeed α-1microglobulin and RBP are low molecular weight proteins able to pass across the glomerular membrane into the Bowman’s capsule and then re-adsorbed by the proximal tubule through a mechanism involving the endocytic receptor megalinThe damage of tubular epithelial cells represents a hallmark of both ischemic and nephrotoxic AKI. Although several clinical studies reported an increased incidence of AKI in TBI patients, the mechanisms of renal damage have not been fully elucidated. Indeed, AKI develops even without severe episodes of hypoperfusion or exposure to nephrotoxins and tubular injury may be correlated to TBI-associated systemic inflammation and secondary organ damage.34. We previously found that plasma from patients with severe sepsis contains factors able to induce functional alterations of tubular and glomerular epithelial cells23. Part of inflammatory mediator data has been previously published from our group showing a significant correlation with secondary injury26. In the present study, we demonstrated that plasma from TBI patients vehicles molecules involved in the development of tubular dysfunction35. Moreover, it has been shown that IL-6 mediates a cytokine-dependent cell-mediated immune response that may exacerbate renal injury playing a critical role in the inflammatory response associated with AKI16. MCP-1 and MIP-1β are chemokines produced by leukocytes and endothelial cells during tissue injury: these molecules are involved in neutrophil infiltration attracting inflammatory cells from circulation into injured tissues36. Viedt and coworkers demonstrated that MCP-1 activates human tubular epithelial cells leading to a time- and dose-dependent increase of IL-6 and ICAM-1 secretion. Moreover, apart from its chemoattractant activity, MCP-1 may further increase the inflammatory response by inducing cytokine release and adhesion molecule expression in tubular epithelial cells37.On this basis, the mechanisms of TBI-associated renal dysfunction may be similar to those observed in experimental and clinical studies in patients with severe sepsis where AKI may be mainly ascribed to microvascular derangement as well as to functional alterations of tubular epithelial cells including loss of solute carrier expression, bioenergetic alterations due to an impairment of mitochondrial function and, ultimately, apoptosisin vitro study, PTEC were challenged with plasma of TBI patients in order to confirm the hypothesis of subclinical AKI generated by clinical observations. Plasma of TBI patients with subclinical AKI induced in PTEC neutrophil adhesion, altered permeability, neutrophil adhesion, reduced viability, enhanced apoptosis and increased expression of inflammatory molecules.In the 40 and up-regulated in response to inflammatory stimuli43. It has been shown that neutrophils are able to injury the epithelium through several mechanisms, such as regulating disassembly of tight junctions, mechanical force, and degradative effects of soluble mediators including elastases, matrix metalloproteinases, defensins and oxidants44. Different studies provided evidence that neutrophil transmigration through an epithelial monolayer is sufficient by itself to trigger apoptotic cell death45. However, tubular cell apoptosis in the course of systemic inflammation can be ascribed to an alteration of mitochondrial biogenesis and function as well as to the presence of pro-apoptotic circulating mediators able to interact with specific receptors located on tubular cells46. In this study, we found that after long term incubation with TBI plasma, tubular cells underwent apoptosis. The persistence of apoptotic tubular injury may lead to the possible lack of tissue regeneration leading to a maladaptive repair: on this basis, innflammation may have a relevant role in the extension phase of AKI and progression toward fibrosis and consequent development of chronic kidney disease46. Infiltrating inflammatory cells may release an additional load of harmful mediators able to perpetuate tubular injury.First, we found an increase in neutrophil adhesion to tubular epithelial cells. Adhesion is the initial stage of neutrophil trans-epithelial migration. Binding of neutrophils to the apical epithelium is known to be mediated by specific adhesion molecules such as ICAM1, expressed predominantly on the apical surface of epithelial cellsPolarity is a distinctive feature of epithelial cells mandatory to maintain two separate compartments with precise electrolyte concentrations. The mislocalization of specific solute carriers on tubular cell surface may lead to an increased influx of sodium and chloride in the distal tubule, thus leading to the triggering of tubulo-glomerular feedback with a consequent reduction of glomerular filtration rate. Moreover, the altered expression of other molecules usually located on the apical surface of PTEC may be responsible for typical alterations such as proteinuria observed in patients with sepsis and confirmed in our cohort of TBI patients.47. As we previously observed in septic patients with AKI, we found a significative decrease of megalin mRNA levels in PTECs incubated with TBI plasma.Megalin is a multiligand, endocytic receptor expressed in kidney proximal tubule luminal membranes and apical endocytic compartment that is responsible for normal tubular reabsorption of several filtered proteins, mediating the recovery of essential substances that would be otherwise lost in the urinein vitro study generates the hypothesis that subclinical AKI is underestimated in TBI patients using conventional biomarkers of renal function. This vulnerability of the kidney may evolve to clinical damage potentially leading to worse outcome.The presence of tubular cell dysfunction and apoptosis demonstrated in our 49. The presence of an unidentified and persistent tubular injury may therefore be clinically relevant in the setting of kidney transplantation. The tubular dysfunction associated with TBI may enhance kidney transplant damage due to ischemia-reperfusion injury and drug toxicity contributing to increase the incidence and duration of delayed graft function. On this basis, the use of functional and structural biomarkers of tubular damage in TBI patients should be encouraged.Furthermore, almost 10% of severe brain damaged patients may evolve to brain death and then may become potential organ donors26. We therefore tested the hypothesis that the inflammatory reaction triggered by the brain trauma could contribute to the development of tubular damage.In our patient population brain trauma was associated in more than 50% of the cases with extracranial injuries, but we did not include patients with severe hemorrhagic shock requiring transfusion to avoid that the severity of multiple trauma was the leading process starting inflammation and that a severe and prolonged hypothension was the main cause for tubular injury. Indeed it has been shown that in severe traumatic brain injured patients an inflammatory reaction is correlated both to the severity of primary injury and to the secondary insults occurring during the ICU stayWe performed an observational study in a small number of patients, indeed only a low percentage developed AKI according to KDIGO criteria. Further studies including large cohort of severe brain injured patients are required to further elucidate the mechanism of tubular injury following traumatic brain injury.The main limitations of the present study are related to the small number of patients and to the co-presence of extracranial injuries.To the best of our knowledge, we observed for the first time the consequence of TBI on renal function correlated to the inflammatory process associated with brain injury. TBI triggers a complex cascade of molecular and cellular events associated with systemic inflammation and secondary distal organ damage. Circulating pro-inflammatory cytokines and chemokines such as IL-6, MCP-1 and MIP-1ß are potentially responsible for a hidden form of AKI that may progress toward chronic kidney disease without manifest signs. These considerations are suggested both by the presence of increased levels of NGAL and low molecular weight proteinuria in the enrolled patients and by the evidence of tubular cell dysfunction and apoptosis induced in PTEC incubated with TBI plasma.