NGATHA genes (NGA) form a small subfamily within the large family of B3-domain transcription factors of Arabidopsis thaliana. NGA genes act redundantly to direct the development of the apical tissues of the gynoecium, the style, and the stigma. Previous studies indicate that NGA genes could exert this function at least partially by directing the synthesis of auxin at the distal end of the developing gynoecium through the upregulation of two different YUCCA genes, which encode flavin monooxygenases involved in auxin biosynthesis. We have compared three developing pistil transcriptome data sets from wildtype, nga quadruple mutants, and a 35S::NGA3 line. The differentially expressed genes showed a significant enrichment for auxin-related genes, supporting the idea of NGA genes as major regulators of auxin accumulation and distribution within the developing gynoecium. We have introduced reporter lines for several of these differentially expressed genes involved in synthesis, transport and response to auxin in NGA gain- and loss-of-function backgrounds. We present here a detailed map of the response of these reporters to NGA misregulation that could help to clarify the role of NGA in auxin-mediated gynoecium morphogenesis. Our data point to a very reduced auxin synthesis in the developing apical gynoecium of nga mutants, likely responsible for the lack of DR5rev::GFP reporter activity observed in these mutants. In addition, NGA altered activity affects the expression of protein kinases that regulate the cellular localization of auxin efflux regulators, and thus likely impact auxin transport. Finally, protein accumulation in pistils of several ARFs was differentially affected by nga mutations or NGA overexpression, suggesting that these accumulation patterns depend not only on auxin distribution but could be also regulated by transcriptional networks involving NGA factors.The four  The carpel is the female reproductive organ of the angiosperm flower and its most distinctive feature. Carpels typically occur at the center of the flower forming the gynoecium, most commonly fused into a single pistil (a syncarpic gynoecium) or less frequently as individual organs that collectively form an apocarpic gynoecium composed of several pistils. The gynoecium confers major advantages to flowering plants: provides protection for the ovules; enables pollen capture and pollen tube guidance and supports self- and inter-specific incompatibility; finally, after fertilization of the ovules, the gynoecium develops into a fruit, which protects the developing seeds and facilitates seed dispersal  mutants, affected in the AUXIN RESPONSE FACTOR 3 gene; the phenotypes of mutants defective in auxin transport such as pinoid (pid) or pin-formed1 (pin1); and on the effects of inhibiting polar auxin transport (PAT) in gynoecium morphology. According to this model, auxin would be produced in the apical end of the pistil and transported basipetally, creating a gradient along the apical-basal axis that would be translated into the differentiation of the different functional modules: high apical auxin levels would direct the differentiation of style and stigma, intermediate levels would specify the ovary, and low basal levels, the gynophore. The Nemhauser model has been very useful to frame the role of different players in Arabidopsis carpel development, but conclusive proof of the proposed auxin gradient has never been obtained. Actually, detailed descriptions of auxin accumulation throughout gynoecium development using a DR5rev::GFP reporter have shown that auxin maxima are formed in the apical domain, first as isolated foci and later as a continuous apical ring, while the proposed gradient cannot be observed , a gene encoding a flavin monooxygenase-like enzyme involved in auxin synthesis , nga2-2 (line SM.20993) nga3-3 (AMAZE En-1 line 6AAi79), and nga4-3 (AMAZE En-1 line 6AAB133i) alleles were used to generate nga quadruple mutants. Genotyping was performed as previously described . Libraries for sequencing were prepared from 2–4 μg total RNA using Illumina TruSeq RNA kits and sequenced with Illumina HiSeq2000. Quality control on the raw sequence data was done using FastQC (Babraham Bioinformatics). Reads were aligned to whole genome sequences from the TAIR10 A. thaliana database (www.arabidopsis.org) and analyzed using the CLC Genomics workbench (www.clcbio.com). RPKM (reads per kilobase per million) was considered as expression values. Two biological replicates for wildtype and three for nga and 35S::NGA3 were used for sequencing. After normalization, Bagerley's test and a FDR correction were used for statistical analysis of samples. Genes with a corrected FDR p-value < 0.05 and with a fold change >1.4 or <-1.4 were selected for gene ontology analysis with the agriGO toolkit . GFP was excited using a 488 nm line of an argon ion laser. GFP emission spectra were collected between 500–550 nm and plastid autofluorescence was collected between 601 and 790 nm.For the micro-application experiment, 80 mg of indolacetic acid (IAA)  were dissolved in 2 mL of ethanol. IAA, or ethanol for mock treatment, was added to 10 gr of lanolin containing 2.5% liquid paraffin. The lanolin paste was applied to the apical end of stage 8–10 gynoecia using plastic pipette tips under a dissecting microscope, resulting in apical parts completely covered by lanolin paste. The gynoecia were observed after 2 weeks and photographed under a dissecting scope.nga mutants and plants overexpressing NGA3  encodes an enzyme that catalyzes the conversion of IAM to IAA and it has been proposed to contribute to auxin synthesis through an alternative pathway to the TAA1/YUC route . A limited partial rescue of the nga phenotypes was observed, with restored apical closure of the gynoecium, but no development of style or stigma typical cells, suggesting that the lack of auxin accumulation in this domain was probably not the only factor causing the nga phenotypes  and at the valve margins  do not show phenotypic defects  are reduced, but not absent, from nga mutant apical gynoecia. Interestingly, NGA3 constitutive expression appears to induce the expanded expression at valve margins of ARF1, ARF11, and ARF18, similarly to what was observed for TAA1 expression, suggesting that they might be responsive to local auxin synthesis putatively mediated by TAA1. It has been described that 35S::NGA3 fruits have enlarged dehiscence zones and thus, this expanded expression domains could be also due to an indirect effect of NGA3 overexpression on fruit morphology. Because no function has been assigned yet to ARF1, ARF11, or ARF18 in gynoecium development, it remains to be studied whether ARF regulation may mediate NGA functions in this process.Finally, protein accumulation patterns for several ARFs expressed through gynoecium development have been described in wildtype, YUC family, as well as TAA1 and AMI1 were not expressed in this domain in nga quadruple mutants, but only showed moderately increased expression in 35S::NGA3 lines. Accordingly, DR5rev::GFP showed no activity in nga mutants but no significant differences in 35S::NGA3 pistils when compared to wildtype. It is thus tempting to speculate that NGA could only direct auxin synthesis in the presence of other factors, for which SHI/STY family members are strong candidates. In addition, NGA altered activity affected the expression of PID and WAG2, regulators of PIN subcellular localization, and thus likely had an impact on auxin transport in parallel to the effect on auxin synthesis. Finally, protein accumulation in pistils of several ARFs was differentially affected by nga mutations or NGA overexpression, suggesting that these accumulation patterns depend not only on auxin distribution but could be also regulated by transcriptional networks involving NGA factors. Again, NGA3 constitutive expression did not result in wide activation of ARF expression in the gynoecium, reinforcing the idea of NGA requiring additional factors to exert their regulatory functions.In summary, our work shows that NGA factors impact on auxin signaling pathways at multiple levels throughout pistil development. First, and more importantly, NGA factors appear to be essential, but not sufficient for auxin synthesis in the apical developing gynoecium, since several members of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Radiation and drug resistance are significant challenges in the treatment of locally advanced, recurrent and metastatic breast cancer that contribute to mortality. Clinically, radiotherapy requires oxygen to generate cytotoxic free radicals that cause DNA damage and allow that damage to become fixed in the genome rather than repaired. However, approximately 40% of all breast cancers have hypoxic tumor microenvironments that render cancer cells significantly more resistant to irradiation. Hypoxic stimuli trigger changes in the cell death/survival pathway that lead to increased cellular radiation resistance. As a result, the development of noninvasive strategies to assess tumor hypoxia in breast cancer has recently received considerable attention. Exosomes are secreted nanovesicles that have roles in paracrine signaling during breast tumor progression, including tumor-stromal interactions, activation of proliferative pathways and immunosuppression. The recent development of protocols to isolate and purify exosomes, as well as advances in mass spectrometry-based proteomics have facilitated the comprehensive analysis of exosome content and function. Using these tools, studies have demonstrated that the proteome profiles of tumor-derived exosomes are indicative of the oxygenation status of patient tumors. They have also demonstrated that exosome signaling pathways are potentially targetable drivers of hypoxia-dependent intercellular signaling during tumorigenesis. This article provides an overview of how proteomic tools can be effectively used to characterize exosomes and elucidate fundamental signaling pathways and survival mechanisms underlying hypoxia-mediated radiation resistance in breast cancer. In the United States, breast cancer is the most common non-skin cancer and the second leading cause of cancer-related death in women. It is estimated that women have a 12% risk of developing breast cancer in their lifetime. Although targeted therapies such as the use of Trastuzumab have been shown to improve survival in the adjuvant treatment of HER-positive breast cancer [It has been shown that 40% of all breast cancers and 50% of locally advanced breast cancers contain hypoxic regions where the effectiveness of radiation and chemotherapy is reduced . HypoxicThere has been recent interest in the role of exosomes in mediating the cross-talk between various cell types ,6,7,8,9.in vitro using a murine model, the results suggest several important functional implications of the role of exosomes in the hypoxic tumor microenvironment. In vitro data suggest an association among hypoxia, exosome-mediated signaling and invasive tumor phenotypes [et al. have shown that breast cancer cells grown under hypoxic conditions release greater numbers of exosomes than cells grown under normoxia [et al. have demonstrated that the proteome and mRNA profiles of exosomes closely reflect the oxygenation status of donor glioma cells and patient tumors, and that exosomal signaling is a potentially targetable inducer of hypoxia-dependent intercellular signaling during tumor development [The hypoxia-induced release of exosomes from cancer cells has been hypothesized to cause the malignant transformation of normal recipient cells, which results in malignant cell proliferation and migration ,10,21 asenotypes ,11,24, aelopment ,26. An iA better understanding of the role for exosome signaling in cancer cell communication may allow for the development of novel strategies to overcome the therapeutic challenges related to tumor hypoxia. New deep coverage proteomic approaches that involve mass spectrometry instrumentation with high sensitivity, resolution and mass accuracy, and more stringent exosome isolation and purification strategies will yield valuable insight into the identification and function of exosomes that are uniquely released under hypoxic conditions and contribute to radiation resistance in breast cancer. In this paper we will describe the role of hypoxia in breast carcinogenesis, basic exosome biology, and the potential for manipulating exosome-mediated intercellular signaling to increase the efficacy of breast cancer radiotherapy.Approximately 40% of all breast cancers contain hypoxic microenvironments where chemotherapy and radiation are less effective . In normHypoxia is a main force that drives cancer cells to adopt a more radiation resistant and invasive phenotype . ChronicThe ability of molecular oxygen to influence the biological effect of ionizing radiation is known as the oxygen effect . Hypoxicet al. demonstrated that the exposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes in the conditioned media from these cells, as determined by Western blot detection of the exosomal protein CD63 [In breast cancer cells, hypoxia has been shown to promote the release of exosomes, which is partly mediated by HIF-1α . In theiein CD63 . FurtherInterestingly, we have identified a novel non-HIF-1α-mediated response to hypoxia in breast cancer cells and high-grade or recurrent breast tumors . Our resSeveral mechanisms of radiation resistance in tumor cells under hypoxic conditions involve the function of biological molecules including oxygen, hydrogen, miR-210, HIF-1 and N-myc downstream-regulated gene 2 (NDRG2) ,42,43,44It is well established that hypoxia is directly linked to the poor prognosis of breast tumors. In addition, it has been shown that hypoxia leads to the increased production of exosomes ,23. TherExosomes are microvesicles that are known to alter the cellular phenotype of target cells and play an essential role in the growth and/or metastasis of breast cancer ,47. TheyIn addition to having a characteristic morphology, exosomes have unique protein and lipid compositions . BecauseIn vitro, exosomes that carry the HER2 receptor on their surface act as molecular “sponges”, binding and sequestering the monoclonal anti-HER2 antibody, Herceptin®, and reducing the amount of the antibody that is available to target the actual cancer cells for anti-cancer therapy [Exosome biogenesis begins with the internalization of plasma membrane receptors that are targeted to the endosome. This inward budding of the endosomal membrane results in the formation of intraluminal vesicles (ILVs), which collect in the mature or late endosome, now termed multivesicular bodies (MVBs). MVBs can then bud and fuse with the cell membrane to be released as exosomes into the extracellular environment. The multi-protein complexes that are involved in this process include the various endosomal sorting complex required for transport (ESCRT) proteins that are involved with internalized protein recognition and the invagination of the endosomal membrane. The energy that is required for the concomitant dissociation of the ESCRT machinery and scission of the ILV from the endosomal membrane is provided by the multimeric protein complex known as the VPS4 complex. Studies from our laboratory using the HER2 over-expressing breast cancer cell line SKBR3 have demonstrated that hypoxia or reactive oxygen species (ROS)-induced VPS4 dysregulation leads to the accumulation of the ESCRT machinery in exosomes, as well as an increase in proteins that are associated with exosome biogenesis and trans-Golgi network receptor recycling ,54,55 F. Further therapy ,57. It h13C or 15N arginine and lysine. These heavy-labeled exosomal proteins could be easily separated from any normal and “light”-labeled lysine and arginine-containing proteins that are derived from fetal bovine serum. Therefore, one of the long term goals of our approach is to generate a series of SILAC-labeled tumor-derived internal standards to monitor the secretion of tumor exosomes in various biological fluids derived from cancer patients. SILAC-based quantitative proteomics is discussed in further detail in Exosomes can be isolated from a variety of biological matrices. Ultracentrifugation and sucrose density gradient centrifugation are commonly accepted methods of isolating exosomes . AlthougVarious strategies, such as immunohistochemistry and functional or molecular imaging, have been adopted to assess tumor hypoxia in various cancer types. However, a significant unmet need resides in the development of strategies to overcome the radiation resistance of tumor cells. The results from clinical studies have shown that cancer treatment strategies that are targeted toward hypoxia-mediated pathways necessitate a comprehensive analysis of the complex network of intercellular communication that influences the tumor microenvironment .Under hypoxic conditions, cancer cells secrete exosomes that modulate their local and distant environment to facilitate tumor angiogenesis and metastasis . Exosomeet al. determined that in melanoma cisplatin resistance is associated with the exosomal trapping and export of cisplatin, which contributes to multi-drug resistance [Proteins associated with immune evasion, cell proliferation, cell invasion, metastasis, and angiogenic factors have all been identified in secreted exosomes, illustrating their potential role in tumor progression, as well as in mediating chemoresistance ,66,67,68sistance . Furthersistance . For thesistance . One sucImproved exosome purification strategies coupled with improved mass spectrometry-based proteomic tools have significantly enhanced the analysis of the molecular composition of exosomes. Advances in mass spectrometry instrumentation that have resulted in increased sensitivity and mass accuracy have enabled significant improvements in the depth of exosomal proteome coverage. Proteomic analysis has revealed that exosomes contain a common set of membrane and cytosolic proteins, and they also contain distinct subsets of proteins that could be associated with cell type-specific functions.in vitro sources and biological fluids has been provided by Simpson et al. [Exosomes that are derived from several types of tumor cells  or severe (0.1% O2) hypoxia, King et al. provided evidence for the importance of understanding the hypoxic tumor phenotype that is characterized by the increased release of exosomes by hypoxic cancer cells into their microenvironment to promote their own survival and invasion [Exosomes are unique entities for biomarker analysis that have the potential to provide novel targets for therapeutic intervention. Using breast cancer cell lines that were cultured under moderate  combined with mass spectrometry is a strategy that can permit the quantitative proteomic analysis of cell culture-derived exosomes. SILAC is based on the metabolic incorporation of an isotopically “light” or “heavy” form of amino acids into proteins, the mass spectrometry analysis of which results in quantitative information regarding protein relative abundance . Using tThe majority of the mass spectrometry-based proteomic analyses that have been described here followed a canonical “shotgun” workflow  whereby In typical MRM experiments, specific precursor ions representing peptides of interest are mass selected and fragmented, and the signals for only a few predefined fragment ions for each peptide of interest are monitored. This enables highly specific and quantitative multiplexed proteomic assays to be conducted. MRM-based proteomic assays have significant potential in the analysis of the exosome proteome in the context of biomarker development.The “shotgun” or discovery-based proteomic analysis described above permits the objective identification of candidate exosomal proteins as functional biomarkers that are associated with the hypoxic microenvironments of breast tumors. Exosomal proteins, such as plasma membrane proteins, cell surface molecules, cytokines, and proteins that are localized to the cytoplasm, with significant changes in relative abundance that correlate with various disease states could be identified as significant functional biomarkers. We have a developed SILAC-based proteomics quantification software tool, termed IsoQuant, that provides users with a convenient quantification framework to calculate peptide and protein relative abundance ratios . For SILOur preliminary studies have indicated that hypoxia or the decreased expression of the MVB-associated protein VPS4B in SKBR3 breast cancer cells can cause an increase in the relative abundance of exosome protein cargo in cultured cells. Several mass spectrometry-based quantitative proteomic methods other than metabolic stable isotope labeling have been developed. Some of these methods include label-free quantification, whereby protein abundance changes are determined by measuring mass spectral peak intensities or extracted ion chromatogram peak areas and spectral counts , and isoStatistical and bioinformatic analyses can be used in exosomal proteomic profiling to predict the protein interaction networks and pathways that are involved in cellular phenotype changes in response to various physiological conditions, including increased hypoxia in the breast tumor microenvironment. Results from our preliminary studies indicated the activation of ERK2 and glycogen synthase kinase-3 α and ß (GSK3α/ß) in exosomes that were isolated from SKBR3 cells with decreased levels of VPS4B expression . These rWe also conducted a Kyoto Encyclopedia of Genes and Genomes (KEGG) protein interaction network analysis of the exosomal proteome of SKBR3 cells that were cultured under hypoxic conditions, and we determined that the ErbB2/HER2 signaling pathway is highly activated. Furthermore, we found that EGF preferentially promoted the assembly of filamentous actin (F-actin) and the formation of filopodia in SKBR3 cells with decreased VPS4B expression. The EGFR signaling pathway plays a critical role in the regulation of cell proliferation, survival and differentiation in breast cancer ,106,107.The proteomic profiling of exosomes that are released from breast cancer cells that are cultured under hypoxic conditions can yield valuable insight into the signaling pathways that are activated under these conditions. Signaling pathway and network analysis permit the visualization of large-scale proteomic datasets to enable the prioritization of functional protein targets to pursue for targeted analyses.In the past few years, mass spectrometry-based proteomics methods have contributed significantly to the elucidation of the molecular composition of exosomes. It has been shown that the proteome profiles of tumor-derived exosomes are associated with the oxygenation status of patient tumors and that exosome signaling pathways are potentially targetable drivers of hypoxia-dependent intercellular signaling during tumorigenesis. Given that hypoxia is known to contribute to the radiation resistance of cancer cells and approximately half of locally advanced breast cancers have hypoxic regions where chemotherapy and radiation are less effective, the use of proteomic applications to characterize hypoxia-induced radiation resistance in breast cancer could have significant clinical utility.This article has provided an overview of how proteomic tools can be effectively used to characterize the role of exosomes in hypoxia-mediated radiation resistance in breast cancer. The identification of an exosomal proteomic “signature” could potentially be used to enhance the efficacy of radiation cancer treatment strategies that are rendered ineffective due to hypoxic tumor microenvironments. Further advances in mass spectrometry-based proteomic technologies and the continued development of selective and efficient exosome isolation and enrichment strategies will be of great importance when elucidating the fundamental mechanisms that underlie radiation resistance in breast cancer.


The 2013 International League Against Epilepsy (ILAE) classification segregates HS into typical (type 1) and atypical (type 2 and 3) groups, based on the histological patterns of subfield neuronal loss and gliosis. In addition, granule cell reorganization and alterations of interneuronal populations, neuropeptide fibre networks and mossy fibre sprouting are distinctive features of HS associated with epilepsies; they can be useful diagnostic aids to discriminate from other causes of HS, as well as highlighting potential mechanisms of hippocampal epileptogenesis. The cause of HS remains elusive and may be multifactorial; the contribution of febrile seizures, genetic susceptibility, inflammatory and neurodevelopmental factors are discussed. Post-mortem based research in HS, as an addition to studies on surgical samples, has the added advantage of enabling the study of the wider network changes associated with HS, the long-term effects of epilepsy on the pathology and associated comorbidities. It is likely that HS is heterogeneous in aspects of its cause, epileptogenetic mechanisms, network alterations and response to medical and surgical treatments. Future neuropathological studies will contribute to better recognition and understanding of these clinical and patho-aetiological subtypes of HS.Hippocampal sclerosis (HS) is a common pathology encountered in mesial temporal lobe epilepsy (MTLE) as well as other epilepsy syndromes and in both surgical and  A recent consensus classification system, validated through the neuropathology taskforce of the International League Against Epilepsy (ILAE) D,E), and scattered hypertrophic neurones (particularly in CA4)  with cytoplasmic distention by microtubules and neurofilaments and increased dendritic complexity vs. complete subfield damage in resected specimens is, as yet, uncertain The histological hallmarks of HS have long been recognized since the earliest PM and surgical series 1Although there is no agreed definition for GCD in HS .Axonal sprouting, a common process in the developing brain, is revived in adult tissues in response to seizures. Such plasticity may represent primarily a reparative response to hippocampal neuronal loss, but may ultimately be mal-adaptive and pro-epileptogenic 2The extent of mossy fibre sprouting can vary between HS cases Figure A–D. The Although the ILAE classification of HS is based on patterns of principal pyramidal cell loss, stereotypical alterations to hippocampal interneuronal cell types also accompany this process. This has been studied more extensively in experimental models, where functional alterations in relation to cellular, network changes and epileptogenesis, can be directly correlated with histological changes to interneuronal groups C), abnormal dendritic projections with altered distribution of spines 2C). Increased complexity of parvalbumin-positive chandelier cells and terminals in the pyramidal cell layer and the dentate gyrus has also been noted The predominant qualitative and quantitative change reported in human HS studies include loss of protein expression or reduction in interneuronal number 3NPY fibre networks is the best studied and argued to ‘define’ the epileptogenic hippocampus Neuropeptides colocalize with inhibitory neurones, are stored in large dense vesicles and when released, have longer half-lives than neurotransmitters, thus modulating neuronal or network activity over longer periods somatostatin-expressing neurones in HS, similar to NPY, have demonstrated a reduction in cell number and radial sprouting of fibres in the dentate gyrus Galanin is a small peptide of 29 amino acids with high concentrations in the adjacent amygdala Substance P has been shown to have pro-epileptic effects with increased expression shown in status epilepticus Studies of A neurotransmitter receptor expression, distribution and assembly has also been reported in HS; these mediate fast postsynaptic inhibition Abnormalities in the regulation of synaptic neurotransmission in HS can occur at any level from protein synthesis, release, to receptor distribution and assembly and subsequent signalling cascades Gliosis is a striking component of HS with chronic, fibrillary gliosis in CA1 and a radial gliosis in the dentate gyrus F).Over the last decade there has been a trend away from a ‘neurocentric’ focus with more directed investigations addressing the participation of astrocytes in the causation of seizures 4G).Abnormalities of the vasculature have been reported in HS, with proliferation of micro-vessels 4SCN1A gene The aetiology of HS is still controversial and is likely to be multifactorial. It is widely considered an acquired pathology. Alfred Meyer's seminal hypothesis from the 1950s proposed that an initiating event, injury or insult the initial precipitating injury’ (IPI), particularly prolonged febrile convulsions early in life, primed the immature hippocampus for the subsequent development of HS 2+, resulting in osmolytic stress and cellular free-radical production, culminating in necrosis of neurones Necrotic or apoptotic neurones are only very occasionally seen in surgical specimens of HS as evidence of ongoing neuronal death. It is generally regarded that seizure-induced neuronal injury and subsequent loss results from excitotoxic, glutamatergic neurotransmission, excessive Na+ and CaIn HS, neuronophagia or focal infiltrates of microglia are occasional findings. There is evidence to support activation of both the innate and adaptive immune system, for example IL-1β and IL-1 receptor upregulation, has been noted in astrocytes, microglia and neurones in HS, and intercellular adhesion molecule 1 (ICAM-1) and kallikrein expression in glia Hippocampal sclerosis can be observed in association with generalized or focal malformations of cortical development (MCD) as a dual pathology Although the bulk of neuropathology research in the past decades has been carried out on surgical tissue with its obvious advantages optimally preserved tissues, homogeneous clinical cohorts and access to up-to-date electroencephalography (EEG) and neuroimaging studies we should not neglect the ongoing contribution that autopsy samples can provide in the study of HS in epilepsy. Primarily, PM tissues enable comparisons of HS occurring in epilepsy syndromes other than TLE (also known as secondary HS The cellular and pathological basis of structural changes in extrahippocampal regions associated with HS typically manifests as neuronal loss and gliosis with few studies exploring the contribution of interneuronal populations Although, benign forms of TLE and HS exist with infrequent and well controlled seizures In summary, there is accumulating evidence that HS in epilepsy is likely to be heterogenous in many aspects, including its aetiology, genetics, epigenetics, networks involved, patterns of neuronal loss as well as responses to drugs and surgical treatments


S100A family of genes at least 2 weeks after sepsis, they do not express the cytokines observed in whole brain homogenates. Our results indicate that in a naturalistic model of infection, sepsis results in long-term neuroinflammation, and that this sustained inflammation is likely due to interactions among multiple cell types, including resident microglia and peripherally derived myeloid cells.Survivors of sepsis often experience long-term cognitive and functional decline. Previous studies utilizing lipopolysaccharide injection and cecal ligation and puncture in rodent models of sepsis have demonstrated changes in depressive-like behavior and learning and memory after sepsis, as well as evidence of myeloid inflammation and cytokine expression in the brain, but the long-term course of neuroinflammation after sepsis remains unclear. Here, we utilize cecal ligation and puncture with greater than 80% survival as a model of sepsis. We found that sepsis survivor mice demonstrate deficits in extinction of conditioned fear, but no acquisition of fear conditioning, nearly two months after sepsis. These cognitive changes occur in the absence of neuronal loss or changes in synaptic density in the hippocampus. Sepsis also resulted in infiltration of monocytes and neutrophils into the CNS at least two weeks after sepsis in a CCR2 independent manner. Cellular inflammation is accompanied by long-term expression of pro-inflammatory cytokine and chemokine genes, including TNFα and CCR2 ligands, in whole brain homogenates. Gene expression analysis of microglia revealed that while microglia do express anti-microbial genes and damage-associated molecular pattern molecules of the  Sepsis, a syndrome of infection and systemic inflammatory response, is a leading cause of mortality in hospitalized patients . As careThe central nervous system response to sepsis has been extensively investigated using systemic administration of high-dose lipopolysaccharide (LPS), as a model of sepsis. This model produces both deficits in learning and memory and increased depressive behavior for at least 30 days following the systemic inflammatory insult ,10. AcutCecal ligation and puncture (CLP) is a reproducible, titratable, naturalistic model of abdominal sepsis in which intra-abdominal infection from controlled bowel perforation leads to systemic inflammation . Severe Here, we hypothesize that sepsis results in persistent neuroinflammation over weeks to months. Using a form of CLP with high (>80%) survival, we have found that CLP results in persistent deficits in extinction learning 50 days after CLP. These changes occurred in the absence of overt increases in cell death or synaptic structure in the hippocampus. CLP did, however, result in long lasting vasculopathy, elevation in brain cytokines, and CNS myeloid inflammation. While microglia do not appear to be the source of these cytokines, transcriptome analysis does indicate that CLP results in distinct changes to microglial gene expression. These results highlight the importance of chronic neuroinflammation after sepsis even in the absence of ongoing overt neuronal death. The neuroinflammatory response after polymicrobial sepsis likely depends on interactions between multiple immunologically active cell types, and is not a simple function of microglial activation alone.-/- mice were of the strain B6.129S4-Ccr2tm1Ifc/J.Wild-type mice used in these experiments were male C57BL/6 mice obtained from Jackson Laboratory and used at 8–10 weeks of age. CCR2All procedures involving animals were undertaken in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. The study protocol was approved by the University Committee on the Use and Care of Animals of the University of Michigan (protocol #PRO00004786).2 inhalation in this circumstance. All deaths occurred within 5 days of CLP, were attributed to the procedure, and were considered expected.Cecal ligation and puncture was performed as previously described . Animal Mice were tested in one of four chambers equipped with a stainless steel floor grid designed for mice (Med Associates). The chambers were arranged in a 2 x 2 grid in an isolated room separate from the room where mice were housed. Each chamber was equipped with an overhead camera, with video signals sent to Actimetrics FreezeFrame software. Foot shock generation and auditory tones were controlled by the same software. Freezing was quantified by the Actimetrics FreezeFrame software based on frame-by-frame analysis of the video signals, digitized at 4 Hz. Testing was performed at the same time each day.The conditioned context was defined by placing 95% ethanol in a pan below each grid, lighting the room with white light, and auditory stimulation with white noise. Mice were placed individually in each chamber, and four mice were tested at a time. During conditioning, mice were placed in the chamber 3 minutes prior to the onset of a 30 second tone. At the end of the tone, mice received the unconditioned stimulus (2 second 0.5 mA foot shock). Mice remained in the chamber for an additional 30 seconds and were then removed and returned to their home cages. Conditioning was performed once daily for a total of 3 sessions on consecutive days. On the fourth day, generalization of the freezing response and the conditioned response to tone were tested by placing mice in novel context, defined by placing 1% acetic acid in the pan below each grid, lighting the room with red light, and removing the white noise. Response to the novel context was measured by freezing during 3 minutes prior to the tone being played. The tone was played 3 times at 30 second intervals, and freezing in response to tone was averaged over the interval after each tone. On the fifth day, contextual fear conditioning was measured by freezing when mice were placed back into the conditioned context for 3 minutes.Experiments were carried out in the same manner as contextual fear conditioning, with the exception that a 30 second trace interval was introduced between the tone and unconditioned stimulus.Extinction was measured starting on the eighth day after the initiation of fear conditioning. Mice were placed back in the conditioned context for 30 minutes without any additional stimuli. Freezing was quantified over the entire session. Extinction continued for four consecutive days.2 inhalation, and trascardially perfused with ice-cold phosphate buffered saline (PBS). The brain was then removed and immediately frozen in liquid nitrogen, and stored at -80°C. Whole brain mRNA was prepared by homogenization in Trizol (Invitrogen), followed by centrifugation to remove fatty debris, extraction in chloroform, and isopropanol precipitation as per the manufacturer’s instructions.Mice were deeply anesthetized by COReal-time RT-PCR was performed on an ABI Prism 7000 thermocycler  and analyzed as previously described . Gene-spSingle cell suspensions were prepared as previously described . Brieflymid/CD11b+/CD64+ cells, and monocytes as CD45hi/CD11b+/Ly6C+/Ly6G- cells. Cells were sorted directly into Trizol and stored at -80C until extraction by chloroform and isopropanol precipitation. RNA concentration was quantified by spectrophotometry  and quality was assessed by chip electrophoresis . Preparation of cDNA, probe labeling, and hybridization to Affymetrix Mouse Genome 430 2.0 arrays was performed by the University of Michigan Microarray Core Facility. Expression values were derived using robust multiarray averages in the Bioconductor suite implemented in the R statistical environment . Mi. Mi40]. Given that fear conditioning is a robust form of hippocampal dependent learning, we increased the difficulty of the task by introducing a trace interval between the conditioned and unconditioned stimuli  42]. Th. Th42]. p < 1 x 10−5 for effect of day). However, post CLP animals froze more than sham-operated or untreated mice (p = 0.038 for effect of group), indicating a defect in extinction of conditioned fear.In order to further interrogate whether CLP results in deficits in behaviors which require coordination between multiple brain regions, and are not primarily dependent on the hippocampus, we studied extinction of contextual fear conditioning in the animals which had undergone contextual trace fear conditioning . All groHigh dose systemic LPS administration causes death of neurons in the hippocampus  We examiMetabolic insults and inflammation may also lead to derangement of synaptic structure without resulting in irreversible cell death, and CLP has been reported to result in long lasting decreases in dentritic spine density . We examWhile CLP does not result in significant structural changes in the hippocampus, we did note histologic evidence of an altered brain environment. CLP resulted in perivascular deposition of IgG, a marker of increased blood-brain barrier permeability  14 days CLP was also associated with increased immunoreactivity for the complement component C1q, which may be blood borne and also produced within the CNS . Immunorhi/CD11b+ myeloid cells 14 days after CLP when compared to sham operated controls  in pooled brain homogenates  of unoperated, sham, and CLP mice 14 days after operation. Gene expression was then examined via microarray analysis. Genes were considered differentially expressed if their expression differed at least 2-fold, and the robust multiarray average (RMA) expression value of at least one gene in the pair was 24.Although amoeboid morphology is often taken as a surrogate for microglial activation , we hypoBy this criterion, 97 genes were differentially regulated among CLP survivors and unoperated age-matched controls, and 86 genes were differentially regulated among CLP survivors and sham operated controls.Fold change in gene expression for genes that were differentially expressed in CLP survivors compared to both sham and unoperated controls are shown . These iCognitive decline and affective disorders are significant clinical problems contributing to functional disability in sepsis survivors. Numerous studies examine the acute neuroinflammatory response to sepsis, modeled either by CLP or high dose LPS in rodent models. Among studies which have examined the long-lasting CNS effects of sepsis survivorship in animal models, most have demonstrated long-lasting behavioral deficits and neuroinflammation using either high-dose LPS or CLP with high mortality, models which likely have limited fidelity to the physiology of patients with high rates of sepsis survival as indicated in epidemiologic studies.Here, we employed CLP with antibiotic administration to achieve a model of sepsis with approximately 80% survival, similar to that observed in epidemiologic studies of sepsis survivors. While sepsis survivor animals did not demonstrate deficits in hippocampal dependent fear conditioning, survivors of this milder insult nonetheless had deficits in extinction learning. Extinction learning has been proposed as a rodent model of post traumatic stress disorder (PTSD), which has been reported in 25–35% of sepsis survivors ,53. Exti+ cells diminished. This decoupling of chemokine expression and cellular infiltration suggests that these molecules may play a role beyond leukocyte chemoattraction to the brain. Indeed, we found that although CCR2 deficiency results in reduced trafficking of monocytes from the bone marrow to the periphery, as has been previously described, CCR2 expression was not necessary for CNS recruitment of Ly6Chi monocytes. While CCL2 is elevated and monocytes are recruited to the brain acutely following systemic LPS injection and contribute to a pro-inflammatory environment in that model [Listeria encephalitis, a disease caused by an intracellular pathogen with tropism to the CNS [We found that sepsis survivor mice continue to experience significant neuroinflammation for weeks to months after CLP. This neuroinflammation does not appear to correlate to diffuse neuronal death or loss of synaptic density. Furthermore, we observed that brain levels of CCL2 and CCL12, ligands for CCR2, continue to increase even as the proportion of CNS CCR2at model . In cont the CNS . Importaex vivo [in vivo. Under conditions of dopamine excess after methamphetamine administration, TNFα decreases dopamine levels by increasing reuptake [While cellular inflammation and infiltration likely do play important roles in brain injury and recovery after sepsis, the co-incidence of elevated cytokine and chemokine levels with a deficit in extinction learning raise the possibility that soluble inflammatory mediators affect neuronal function directly after sepsis. Certainly, cytokine signaling plays a role in a global inflammatory response and alters neurotrophic factors and other important signals in the brain environment . Howeverex vivo . Conversex vivo  and increx vivo  in vivo.reuptake . It is lreuptake . StudiesStudies examining the acute effects of LPS in the hours to days following administration suggest that microglia are dynamic producers of cytokines and regulators of the inflammatory environment ,48. ChroThe difference between these observations and the more typical amoeboid activation observed after LPS may be a result of altered kinetics of exposure to pro- and anti-inflammatory stimuli after CLP compared to a single high-dose administration of LPS. True infection exposes the immune system to varying levels of pathogen and damage associated molecular patterns (PAMPs and DAMPs) over hours to days. While more exposure to LPS may simply result in potentiation of the immune response , it is eIn addition to myeloid cell infiltration and microglial activation, we observed evidence of IgG deposition in a patchy vascular pattern 14 days after CLP. Both LPS and CLP may reduce the integrity of the blood-brain barrier and injure cerebrovascular endothelium in the acute setting . While ain vitro to be induced by TNFα in immune and endothelial cells [The chronicity of neuroinflammation, reflected in microvascular injury, myeloid cell infiltration, and cytokine expression, after sepsis suggests a reprogramming of the innate immune response. We speculate that a paracrine and autocrine signaling among multiple immunologically active cell types, including CNS resident microglia, bone marrow derived monocytes and neutrophils, and endothelium result in chronic neuroinflammation. This model is suggested by long-lasting elevation in cytokines even after the period of acute illness has passed. It is notable that both CCL2 and CXCL10, which are increased 50 days after CLP, have been shown al cells ,74 and tal cells . Conversal cells  and alsoal cells . TNFα sial cells . Further


Antirrhinum majus, the TCP transcription factor CYCLOIDEA is the molecular key regulator driving the formation of floral monosymmetry. Although most Brassicaceae form a polysymmetric corolla, six genera develop monosymmetric flowers with two petal pairs of unequal size. In the monosymmetric crucifer Iberis amara, formation of the different petal pairs coincides with a stronger expression of the CYC-homolog IaTCP1 in the small, adaxial petals.In the co-evolution between insects and plants, the establishment of floral monosymmetry was an important step in angiosperm development as it facilitated the interaction with insect pollinators and, by that, likely enhanced angiosperm diversification. In Iberis amara. About 9 Gb of sequence data was generated, processed and re-assembled into 18,139 likely Iberis unigenes, from which 15,983 showed high sequence homology to Arabidopsis proteins. The transcriptome gives detailed insight into the molecular mechanisms governing late petal development. In addition, it was used as a scaffold to detect genes differentially expressed between the small, adaxial and the large, abaxial petals in order to understand the molecular mechanisms driving unequal petal growth. Far more genes are expressed in adaxial compared to abaxial petals implying that IaTCP1 activates more genes than it represses. Amongst all genes upregulated in adaxial petals, a significantly enhanced proportion is associated with cell wall modification and cell-cell signalling processes. Furthermore, microarrays were used to detect and compare quantitative differences in TCP target genes in transgenic Arabidopsis plants ectopically expressing different TCP transcription factors.In this study, RNA-Seq was employed to reconstruct the petal transcriptome of the non-model species Iberis amara, is likely driven by the enhanced activity of IaTCP1 in adaxial petals.The increased occurrences of genes implicated in cell wall modification and signalling implies that unequal petal growth is achieved through an earlier stop of the cell proliferation phase in the small, adaxial petals, followed by the onset of cell expansion. This process, which forms the monosymmetric corolla of The online version of this article (doi:10.1186/s12870-014-0285-4) contains supplementary material, which is available to authorized users. In the co-evolution between plants and insects, the development of floral monosymmetry was an important step, facilitating angiosperm speciation as a response to the adaptation to the visual senses of insect pollinators . MonosymTCP transcription factor CYCLOIDEA (CYC) as the molecular key regulator of monosymmetry development in Antirrhinum  was used as a housekeeping gene for normalization.Specificity of each PCR was assessed by gel analysis and examination of the melt curve, generated after each reaction. The mean normalized expression (MNE) for each sample  is baseIberis flowers on three biological replicates  adaxial and abaxial petals of stage A1 Iberis flowers, which were dissected into the upper petal blade and the lower petal claw  was set to 1. An expression increase in adaxial petals of 1.5-times or above in at least two biological replicates was considered as a differential expression and consequently as a further corroboration of RNA-Seq data.IaTCP1, TCP1 and CYC, respectively, under the control of the CaMV35S-promoter [Arabidopsis Genome Arrays  and statistical data analysis were carried out at the Integrated Functional Genomic service unit from the University of Münster (Germany). Only genes showing a minimal expression fold change of two and a p-value ≤0.05 were considered as being differentially expressed. Microarray expression data were determined in comparison to transgenic plants harbouring the empty vector. Data were corroborated for selected genes by reverse transcriptase PCR performed as in [Ran3 GTPase (At5g55190). Primer sequences are given in Additional file Two technical samples of total RNA were prepared from each transgenic T2 Line expressing promoter  or the eed as in   and examined with a digital scanning electron microscope (Zeiss Auriga). Stage A1 and A2 petals pictures were taken with a binocular with integrated camera .The sequence datasets supporting the results of this article are available from the Short Read Archive (SRA) database at NCBI under the accession SRP048782. Assembled contigs are provided in FASTA format in Additional file


Muscle stem cells, termed satellite cells, are crucial for skeletal muscle growth and regeneration. In healthy adult muscle, satellite cells are quiescent but poised for activation. During muscle regeneration, activated satellite cells transiently re-enter the cell cycle to proliferate and subsequently exit the cell cycle to differentiate or self-renew. Recent studies have demonstrated that satellite cells are heterogeneous and that subpopulations of satellite stem cells are able to perform asymmetric divisions to generate myogenic progenitors or symmetric divisions to expand the satellite cell pool. Thus, a complex balance between extrinsic cues and intrinsic regulatory mechanisms is needed to tightly control satellite cell cycle progression and cell fate determination. Defects in satellite cell regulation or in their niche, as observed in degenerative conditions such as aging, can impair muscle regeneration. Here, we review recent discoveries of the intrinsic and extrinsic factors that regulate satellite cell behaviour in regenerating and degenerating muscles. Summary: This Review discusses how satellite stem cell behaviour is regulated during regeneration and degeneration by a complex balance between extrinsic cues and intrinsic regulatory mechanisms. Satelli muscle) . In rest muscle) . Thereaf muscle) . Satelli muscle) .Fig. 1.Following muscle injury, activated satellite cells give rise to the myogenic progenitors needed to form new myofibers, but they also need to self-renew to maintain the satellite cell pool. Repeated injury experiments have shown that satellite cell numbers remain constant even after multiple traumas . The abiIn recent years, many intrinsic and extrinsic factors governing satellite cell functions have been discovered. Together, these studies demonstrate that the fine-tuned regulation of cell cycle repressors and activators is crucial to coordinate the different states of satellite cells. In this Review, we analyze the intrinsic mechanisms that control satellite cell cycle progression throughout the different stages of adult myogenesis. We also describe how intrinsic mechanisms and extrinsic signals from the satellite cell niche cooperate to influence satellite cell fate decisions, in particular self-renewal versus myogenic commitment. Finally, we discuss the intrinsic defects of aged satellite cells and describe how these deficits modulate the ability of satellite cells to appropriately regulate the cell cycle and cell fate decisions, which ultimately affects their regenerative capacity.MyoD prenatally  and committed satellite cell (YFP+) subpopulations, with the former resulting in long-term engraftment into the transplanted muscle while the latter leading to differentiation and fusion to the host myofibers  as well as a subpopulation of satellite stem cells (i.e. cells that are able to self-renew and maintain the satellite cell pool). However, whether the satellite stem cell populations identified with the various reporter mouse models represent the same or different subsets of satellite stem cells remains to be determined.Muscle regeneration is characterized by different myogenic stages, namely: activation, proliferation, differentiation, and self-renewal/return to quiescence. Careful regulation of the cell cycle is essential to ensure appropriate progression through these various overlapping states. The following sections describe the intrinsic mechanisms and extrinsic signals that regulate the satellite cell cycle.0 state  and 1C , the retinoblastoma tumor suppressor protein , regulator of G-protein signalling 2 and 5 , peripheral myelin protein 22 (Pmp22), and the negative regulator of fibroblast growth factor (FGF) signalling sprouty 1 (Spry1). These quiescence genes act in concert to prevent the precocious activation of quiescent satellite cells, and it was shown that the conditional knockout of p27Kip1 or Rb protein results in aberrant satellite cell activation and proliferation . The overall requirement of miRNAs in satellite cell quiescence is exemplified by their precocious activation after deletion of Dicer, the enzyme involved in pre-miRNA hairpin cleavage . Althougle entry . Additiommitment .0 quiescent and activated cell states, termed GAlert  at the site of injury could have a systemic effect and activate mTOR signalling in distant quiescent satellite cells, thereby driving their entry into the GAlert state , p19Arf (Cdkn2a) and p57  or to return to quiescence, two processes that involve cell cycle exit . This ce and p57 .Spry1 null mice show an impaired ability to return to quiescence, resulting in a decreased satellite cell pool after muscle injury  cell, whereas the committed daughter (Myf5-positive) cell inherits the Notch ligand Delta1 . Accordi of Myf5 . However of Myf5 . During f5 (Myf5 .+ MyoD− reserve cell and one Pax7− MyoD+ committed myogenic cell (+ Myog− and one Pax7− Myog+ daughter cell (Lo) perform random DNA segregation, those expressing higher levels of Pax7 (Pax7Hi) mostly perform asymmetric DNA segregation during cell division  the unequal distribution of polarity proteins and cell fate determinants; and (2) mitotic spindle orientation. How these processes are regulated in satellite cells remains largely elusive, although recent studies highlight a role for members of the Partitioning-defective protein (PAR) family, which are master regulators of cell polarity establishment in many different stem cell types . In muscThe analysis of different adult stem cell populations, such as epithelial stem cells, neural stem cells and haematopoietic stem cells, has revealed that in all these tissues the stem cell niche provides essential cues that influence cell fate decisions . In skelA number of proteins located in the satellite cell niche have been shown to affect the orientation of division and, hence, cell fate choice. For example, collagen VI present in the ECM reduces myogenic commitment and promotes the self-renewal of activated satellite cells , and itsin vitro on symmetric or asymmetric micropatterns coated with fibronectin and fibrinogen  and increases H4K16 acetylation, ultimately leading to the activation of muscle gene transcription , for example, is one factor that is overexpressed in old muscle and that induces phosphorylation and activation of Smad3 in aged satellite cells . Smad3 aSerum from aged mice also contains a higher concentration of Wnt ligands . CanonicINK4a, an alternative spliced variant of p19Arf (Consistent with the inability of aged satellite cells to maintain quiescence it was demonstrated in geriatric mice (30 months old) that a proportion of satellite cells displays a loss of reversible quiescence (pre-senescence) . Under pf p19Arf , is at lf p19Arf . This geSatellite cells are the protagonists of muscle regeneration. To appropriately fulfil their functions, they must maintain a dynamic balance between their different cell states, namely quiescence, commitment, differentiation and self-renewal. As we have highlighted above, many intrinsic mechanisms are required to regulate the cell cycle and cell fate determination in satellite cells, and dysregulation of these mechanisms results in the loss of regeneration in degenerative conditions, including aging. In particular, aged satellite cells lose their ability to maintain quiescence (through increased cycling or senescence) and, once activated, their cell fate is forced toward commitment to the myogenic lineage in lieu of self-renewal. Whether these changes are caused by modifications in the satellite cell niche or by intrinsic satellite cell defects remains a matter of debate. Some dysfunctions in aged satellite cells can be rescued by a young environment, whereas others cannot, suggesting that both intrinsic and extrinsic deficiencies are present simultaneously.Many recent studies have shown that, regardless of their origin, these deficits can be overcome by pharmacological inhibitors. These discoveries could represent a cornerstone in the treatment of pathologies such as sarcopenia, an irreversible loss of muscle mass induced by aging that causes serious health issues and currently has no therapy. It is likely that intrinsic perturbations in satellite cell regulatory mechanisms are present in other pathologies, including muscular dystrophies, and further studies are needed to explore these possibilities and to aid our understanding of muscle development, regeneration and degeneration.


Transporters comprise the largest family of membrane proteins in human organism, including members of solute carrier transporter and ATP-binding cassette transporter families. They play pivotal roles in the absorption, distribution and excretion of xenobiotic and endogenous molecules. Transporters are widely expressed in various human tissues and are routinely evaluated during the process of drug development and approval. Over the past decade, increasing evidence shows that drug transporters are important in both normal physiology and disease. Currently, transporters are utilized as therapeutic targets to treat numerous diseases such as diabetes, major depression, hypertension and constipation. Despite the steady growth of the field of transporter biology, more than half of the members in transporter superfamily have little information available about their endogenous substrate(s) or physiological functions. This review outlines current research methods in transporter studies, and summarizes the drug-transporter interactions including drug-drug and drug-endogenous substrate interactions. In the end, we also discuss the therapeutic perspective of transporters based on their physiological and pathophysiological roles. Movement of many drugs and endogenous molecules across the cell membrane is governed by protein transporters. In general, membrane transporters are divided into two major superfamilies—the ATP binding cassette (ABC) family and the solute carrier (SLC) family. To date, more than 400 transporters have been annotated in the human genome. The ABC transporters are primary active transporters that utilize the energy from ATP hydrolysis to transport substrates across the membrane. SLCs can be either facilitative transporters, which transport their substrates down the gradient across the membrane, or secondary active transporters, which transport their substrates against the gradient across the membrane by coupling a downhill transport of another substrate. Major drug transporters are listed in Table The biological function of transporters has been investigated using multiple approaches. Among them, metabolomic studies have been quite valuable for substrate identification of transporters systemically, which identify the alterations of many metabolites or their substrates directly. Alternatively, the uptake of substrate candidates can be evaluated by radio-tracer or radio-free LC/MS quantification. This method was used to identify thiamine and ergothioneine as the physiological substrates of OCT1 (SLC22A1) and OCTN1 (SLC22A4) respectively . Clinical genomics also reveal involvement of transporters in bioconversion and toxicity of drugs. One of the most compelling examples of the critical role of transporters in drug toxicity stems from a GWAS encompassing 85 patients with myopathy and 90 normal control individuals  that has been shown to be involved in the hepatic uptake of statins. GWASs have also associated SLCO1B1 and SLCO1B3 variants with bilirubin levels and unconjugated hyperbilirubinemia  cells. The colon carcinoma cell line Caco-2 are especially useful for the study of intestinal transporters, since these cells exhibit morphological and functional similarity with the enterocytes lining the small intestine  of a membrane transporter frequently begins with an o Bröer, . If combThe primary goal of transporter assays is to determine the kinetics of substrate transport. For the primary active transporter, like the ABC transporter, substrate translocation across the cell membrane is directly correlated to ATP hydrolysis. Thus ATPase assays that measure the inorganic phosphate release using colorimetric measurement can be utilized to assess active transporter kinetics , encoded by SLC46A1, incorporates folate by coupling a proton co-transport down the proton gradient at pH 5.5, however, this transport activity is absent at neutral pH , TALEN and CRISPR/Cas-based genomic engineering provides powerful and convenient tools to generate transgenic or knockout animals for a gene of interest . Potential drug-drug interactions (DDIs) may cause severe, adverse reactions in patients due to alteration in ADME by transporters. Most DDIs via transporters involve, but are not restricted to, the main transporters in drug ADME: P-gp, BCRP, OATP1B1/1B3, OAT1/3, OCT1 and OCT2  to exogenous or endogenous stimuli. Altered drug ADME during inflammation has long been primarily ascribed to changes of enzymes in drug metabolism. An example of this is decreased expression of P450, the main drug metabolizing enzyme in liver, following inflammation. Moreover, recent studies have also shown that the expression and activity of numerous transporters are also changed in inflammatory condition. In experimental cell and animal models, inflammatory conditions induced down regulation of MDRs, MRPs and OATPs. Consequently, the ADME of drugs which are substrates of these transporters would be broadly affected  due to cholestasis. The transporter BSEP plays an important role in the disposition and exportation of bile salts from liver. BSEP inhibition by drugs can cause accumulation of bile salts in liver, thus leading to cholestasis. There is a strong correlation between inhibition of the Oct1 KO mice provide a model to investigate the function of Oct1 systemically. Thiamine is converted to thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) in cytosol, which serves as the essential co-enzyme for energy generation and glucose metabolism. Due to reduced thiamine uptake in liver, thiamine-related changes were observed in the serum of Oct1 KO mice. These included reduced levels of TMP and TPP and higher levels of pyruvate and alpha-ketoglutarate, which are the substrates of pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (OGDH)  and OCT1 might play a critical role in the uptake of serotonin in liver , the homologue of mammal SLC13A5, confers extended lifespan in a caloric restriction-like manner (Rogina et al., Slc13a5 in mice results in reduction in the liver uptake of citrate from circulation accompanied by an increase in mitochondrial biogenesis, lipid oxidation, energy expenditure and decrease in hepatic de novo lipogenesis. The loss of Slc13a5 also protects mice from high-fat diet induced obesity and insulin resistance (Birkenfeld et al., Drosophila INDY, leaving mitochondria citrate transporter activity unchanged (Sun et al., Vibrio cholera at 3.20 Å. This provides the structural basis for understanding the substrate binding properties as well as conformational change upon substrate binding (Mulligan et al., In 2+-insulin hexamers in the secretory granules of the pancreas, which releases the physiologically active form of insulin in the serum driven by a pH change (Xu et al., 2+ transport activity than the Trp325 ZnT8 (Nicolson et al., Zinc transporters are reported to be involved in the pathophysiology of cancer, cardiovascular diseases and Alzheimer’s disease making them possible therapeutic targets for treatment of these diseases (Prasad et al., The exact relationship between ZnT8 activity and diabetes risk, however, remains controversial. Zn ions have several effects on the regulation of insulin production and clearance. Co-secretion of Zn ions with insulin from beta cells inhibits insulin production in an autocrine and paracrine fashion through the K-ATP channel (Bancila et al., Intriguingly, a recent GWAS reported that 12, rare truncating mutations in ZnT8 were surprisingly protective against developing diabetes and, collectively, explained a 65% reduction in diabetes risk (Flannick et al., Transporter biology is a rapidly changing field in pharmaceutical research. Identifying the function of transporters in physiological and pathological conditions is garnering significant attention for drug development. Many drugs, nutrients and metabolites are moved by transporters not only through the plasma membrane but also among subcellular organelles like vesicles, ER, lysosome and mitochondria. Transporters remain one of the most underestimated fields for drug targets. Within the hundreds of annotated transporters in human genome, more than half are orphan transporters with unknown substrates or function. The nature of these transporters, including their expression and localization in organs and tissues, subcellular localization, endogenous substrates, largely remains elusive. As discussed in this review, transporters can be of significant therapeutic potential in the treatment of various diseases. A comprehensive dissection of the physiological roles of the transporter could be a key step to excavate this “gold mine”. Our growing knowledge of transporter biology will greatly accelerate the discovery of novel drug targets and development of first-in-class therapies.


Cumulative evidence has indicated that there is an important role for adult hippocampal neurogenesis in cognitive function. With the increasing prevalence of cognitive decline associated with neurodegenerative diseases among the ageing population, physical exercise, a potent enhancer of adult hippocampal neurogenesis, has emerged as a potential preventative strategy/treatment to reduce cognitive decline. Here we review the functional role of adult hippocampal neurogenesis in learning and memory, and how this form of structural plasticity is altered in neurodegenerative diseases known to involve cognitive impairment. We further discuss how physical exercise may contribute to cognitive improvement in the ageing brain by preserving adult neurogenesis, and review the recent approaches for measuring changes in neurogenesis in the live human brain. Given the overwhelming evidence showing adult neurogenesis in the mammalian brain –5 and itμm, two cell nuclei wide) from the SGZ to the granule cell layer (GCL), where they integrate into the existing circuitry [Newborn neurons are mainly produced from neural stem cells in two neurogenic zones of the adult brain: the subventricular zone (SVZ)/olfactory bulb (OB) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) . In the ircuitry . A dividircuitry   [Using exercise training as an upregulator for hippocampal neurogenesis, ann brain) . Furthern brain) . In spitNeurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) share the common characteristic of progressive loss of structure and/or function of neurons in the brain. Although neuronal degeneration predominantly affects specific neuronal populations  ergic neurons in HD, and cortical and hippocampal neurons in AD), all these neurodegenerative diseases are characterized by a more or less severe loss of certain cognitive functions including learning and memory. Concomitantly, several lines of evidence have shown that adult hippocampal neurogenesis might be altered in these neurodegenerative processes , 50.Physical activity has been repeatedly shown to improve cognition and prevent age-related cognitive decline in humans , particuHere, we review the functional role of adult neurogenesis in cognitive function and the emerging association between adult hippocampal neurogenesis and cognitive impairment in neurodegenerative diseases. We further discuss physical exercise-induced hippocampal neurogenesis and its relationship with cognitive improvement. Finally, we address the emerging techniques for measuring adult neurogenesis in live human brain.The functions of adult neurogenesis in the adult brain have been extensively investigated in the past decade. Numerous studies have suggested that neurogenesis in the DG may play an important role in hippocampal-dependent learning and memory –11, 56, Importantly, newly generated neurons have particular physiological properties that make them more susceptible to behavioral-dependent synaptic plasticity . Using rSince these newly generated neurons are linked to the functioning of the hippocampus, it is reasonable to speculate that they might play a role in mechanisms of hippocampal-dependent learning and memory. In agreement with this hypothesis, it has recently been shown that new neurons are indeed recruited into neuronal circuits involved in spatial learning and memory in the hippocampus . FurtherAlthough details of how newborn neurons modulate learning and memory are still unclear, recent findings have suggested that adult born neurons in the DG play a critical role in pattern separation, preventing memory interference from overlapping contexts , 65, 66.To test the hypothesis concerning the functional role of neurogenesis on pattern separation in the human brain, Dery and colleagues used the visual pattern separation task, a cognitive test that is believed to be neurogenesis-dependent and that uses some objects that are repeatedly presented across trials and some objects that are new but highly similar to previously presented ones. They observed a significant enhancement in performance on the visual pattern separation task together with lower depression scores in subjects who participated in exercise training a well-known enhancer of neurogenesis . This fiIn summary, it is currently believed that hippocampal new neurons are required for the separation of events based on their spatial and temporal characteristics (a process that preserves the uniqueness of a memory representation), as well as space representation, long-term memory retention, and flexible inferential memory expression .The contribution of altered adult hippocampal neurogenesis to the cognitive deficits that are characteristic of various neurodegenerative conditions such as AD, PD, and HD is still not fully elucidated. Nevertheless, since alterations in adult neurogenesis have been repeatedly shown in various animal models of these disorders  Table 1Table 1, γ-secretase, the enzyme responsible for cleaving the amyloid precursor protein (APP) into toxic amyloid-β (Aβ) peptides, the building blocks of senile plaques [AD is manifested by progressive cognitive deterioration, memory loss, behavioural changes, and eventually dementia. At the pathological level, AD is characterized by acetylcholine depletion, the accumulation of amyloid (or senile) plaques, and the formation of neurofibrillary tangles (NFT), which can lead to neuronal loss by apoptosis particularly in the cortex and hippocampus and severe brain atrophy . While t plaques .While the exact neurobiological mechanisms underlying the symptoms of AD are still unclear, severe neuronal loss in areas of the brain involved in learning and memory, such as the hippocampus and prefrontal cortex, is evident in the AD brain. Transgenic mouse models of AD show impairments in several hippocampal-dependent learning and memory tasks, such as spatial learning, object recognition, and contextual fear conditioning . AdditioβIII-tubulin) appears to be increased [β aggregates have been found to accumulate near neural precursor cells in the hippocampal DG [α may be important during cell proliferation [In human AD patients, the expression of several immature neuronal markers (doublecortin (DCX), polysialylated nerve cell adhesion molecule (PSA-NCAM), neurogenic differentiation factor (NeuroD), and ncreased , while tncreased  in the Dampal DG , 92 suggampal DG , whereasferation .α-synuclein-positive Lewy bodies and dystrophic Lewy neurites throughout the brain, which initially occur in the vagal nerve and OB and thereafter spread to other nuclei and cortical areas [PD is caused by death of dopaminergic neurons that project from the substantia nigra (SN) pars compacta to the striatum of the basal ganglia. PD is manifested by (1) severe motor symptoms characterized by a progressive impairment of movement control, akinesia, rigidity, and tremor; and (2) nonmotor symptoms such as cognitive decline, olfactory dysfunction, anxiety , and depal areas .The neurogenic regions of the adult brain are innervated by dopaminergic projections from the SN and the ventral tegmental area (VTA) –100; theThe animal models that have been most widely used in PD research are the unilateral 6-hydroxydopamine (6-OHDA) lesion rat model and the bilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion mouse model, which develop PD-like symptoms . Park an in vivo studies have also evaluated how hippocampal neurogenesis is altered by the expression of α-synuclein. Transgenic mice overexpressing human wild-type α-synuclein showed significantly fewer neurons both in the OB as well as in the DG of the hippocampus as compared to their control littermates, an effect that seems to result from a decrease in neuronal precursor survival [α-synuclein were shown to have impaired hippocampal neurogenesis due to a decrease in proliferation and survival of neural precursor cells [α-synuclein mouse model. Turning off the transgene expression did halt the progression of these symptoms, although no regression was observed [Severalsurvival , whereasor cells . In a diobserved .βIII-tubulin-positive cells in the DG of the hippocampus have also been found in postmortem tissue from PD patients, presumably as a consequence of dopaminergic denervation of these neurogenic regions [Finally, a decrease in the number of proliferating cell nuclear antigen (PCNA) positive cells (a marker of cell proliferation) in SVZ and a reduction in the number of nestin- and  regions , providi HD gene, which results in an expanded polyglutamine tract in the NH2-terminal of the protein huntingtin [HD is caused by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in thentingtin . In mostntingtin . In factntingtin –120.Mutant huntingtin is ubiquitously expressed throughout the organism. However, cell degeneration occurs mainly in the brain, particularly in the striatum and certain layers of the cortex , 121. Ne HD gene. These models primarily differ in the size of the expressed huntingtin fragment, the number of CAG repeats, the promoter driving the transgene, and consequently the expression of the mutant protein, as well as the background strain. As a consequence, each model exhibits unique phenotypes. Nevertheless, most demonstrate progressive neurological phenotypes  that mimic well the human condition [ HD gene (corresponding to approximately 3% of the entire gene) with 115 and 150 CAG repeats, respectively [ HD gene with 128 CAG repeats [Several rodent models are currently available to study the effects of the alteredondition –127. Theondition , 129 andondition –132 tranectively , and shoectively , 135. Inectively , a reducectively  and by 1ectively . In agreectively . This tr repeats  and repl repeats  while al repeats  and hipp repeats . In this repeats , once ag repeats  also fou repeats . Finally repeats .Although it is still unclear how the expression of mutant huntingtin gene might lead to a dysregulation of the neurogenic process, various mechanisms have been proposed to contribute to this disturbance . These iTaken together, these studies support the possible role of mutant huntingtin in disrupting the process of adult hippocampal neurogenesis in the HD brain. These neurogenic deficits can in turn contribute, at least in part, to the cognitive decline and depressive-like symptoms found in HD transgenic models. However, studies in postmortem human HD brains have shown no changes in hippocampal cell proliferation  and an aEven though tremendous advances have been made over the past few decades with regard to our understanding of the etiology of age-related neurodegenerative disorders, to date no effective treatments are available for individuals afflicted with these devastating neurodegenerative diseases. In recent years, physical exercise has emerged as the most effective, low-cost, and low-tech way for successful ageing, and therefore, it has the potential to represent a preventive or disease-slowing therapeutic strategy for age-related neurodegenerative diseases .In support of this hypothesis, a meta-analysis study has shown that 1 to 12 months of exercise in healthy adults brings behavioral benefits, including significant increases in memory, attention, processing speed, and executive function . MoreoveEvidence from animal studies has suggested that an enhancement in hippocampal neurogenesis may underlie the reported beneficial effects of exercise on cognitive function. Indeed, pioneer studies by van Praag and collaborators showed that physical running not only increased hippocampal neurogenesis , 43 but These beneficial effects of physical exercise on cognitive function suggest that exercise might indeed be used as a strategy to prevent cognitive decline in age-related neurodegenerative diseases. Physical exercise has been shown to prevent the age-induced decrease in hippocampal cell proliferation, neurogenesis , LTP, anThe exact unerlying mechanisms of how physical exercise promotes adult hippocampal neurogenesis is still unclear. Neurotrophins such as BDNF, insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) have been recognized as primary mediators of adult neurogenesis , 177–179Animal models of neurodegenerative diseases constitute valuable tools to unmask the underlying mechanisms by which exercise enhances adult neurogenesis, brain plasticity, and hence cognitive function in the diseased brain .β plaques in the frontal cortex and hippocampus of TgCRND8 AD mice but also enhances their hippocampal-dependent learning in the Morris water maze [β deposition and tau phosphorylation as well as a decrease in APP phosphorylation and PS1 expression in the hippocampus [Several mouse models of AD have shown that running can promote neurogenesis and cognitive function in the AD brain. Short-term running is able to enhance cognitive function in aged Tg2576 mice . Long-teter maze . Similarter maze , while apocampus . HoweverOn the other hand, studies using the APOE-e4 transgenic mouse model have demonstrated the effect of running on restoring hippocampal plasticity and improving cognitive functions in this AD transgenic mouse model –195. AddEpidemiological studies have reported a reduced risk of developing dementia in elderlies with higher physical activity –201. NeuDespite the fact that there is abundant evidence suggesting that physical activity might be effective in reducing the risk of developing AD in humans, the exact mechanisms by which physical exercise reduces the risk of AD are still unknown. Animal studies have suggested that physical exercise might result in structural changes in the hippocampus that in turn may reduce the risk for AD future research linking the possible changes of the brain  with functional outcomes in AD patients or individuals with higher risk for AD will shed light on how physical exercise benefits these individuals.Several studies have shown that physical exercise can be beneficial in ameliorating some of the neuropathological and behavioural deficits characteristic of various PD rodent models –207. HowA few clinical studies have reported that physical exercise can improve motor function and cognitive performance in human PD patients , 210. ThThe use of voluntary physical exercise as a means to promote adult neurogenesis was initially tested in 5-week-old R6/2 HD mice . However bdnf gene expression that was specifically observed in R6/1 females but not in their male counterparts [ bdnf expression levels in R6/1 females versus males. Of note, it is reasonable to speculate that the inability of physical exercise to consistently upregulate bdnf gene expression and protein levels in the hippocampus of R6 mice [Nevertheless, other authors have found that exposure of R6/1 mice to physical exercise delayed the onset of rear-paw clasping and improved cognition in adulthood , while aterparts . Sex-spe R6 mice , 158 may R6 mice .Of note, it has also been reported that R6/1 HD mice show decreases in dendritic spine density and spine length both in striatal and cortical neurons . HoweverIn contrast to physical exercise, treatment of R6/1 mice with fluoxetine, a selective serotonin reuptake inhibitor (SSRI) antidepressant, was shown to abolish the impairment in adult hippocampal neurogenesis while also increasing cognitive performance  . These pThe first evidence for the occurrence of adult neurogenesis in the human brain came from a study by Eriksson and colleagues showing the presence of BrdU-positive cells in postmortem hippocampal and SVZ human tissue obtained from cancer patients that received BrdU injections in life for diagnostic purposes . However in vivo has emerged as an essential research area within the neurogenesis field. Within this scenario, the recent detection of adult neurogenesis in live human brains using magnetic resonance imaging (MRI) [ in vivo imaging method that has been employed to detect neurogenesis in humans consists in using proton nuclear magnetic resonance spectroscopy (1H-NMR). This technique uses the magnetic properties of protons to detect a specific biomarker of neural progenitor cells, N-acetylaspartate , in living tissue [ in vivo imaging methods as indirect ways to measure adult neurogenesis in the live human brain.The development of alternate methods that can be used to assess adult human neurogenesisng (MRI)  has provng (MRI) , based ong (MRI) , 173. Adng (MRI) , 218, thng (MRI) . An alteg tissue . Since tAnother indirect and noninvasive measure of adult neurogenesis in humans might be the measurement of peripheral biomarkers that correlate well with changes in adult neurogenesis. However, such peripheral biomarkers have not yet been clearly identified.As mentioned above, BDNF, IGF-1, and VEGF have been recognized as primary mediators of adult neurogenesis , 177–179Given this well-established relationship between various neurotrophins and adult hippocampal neurogenesis, it is reasonable to speculate that the peripheral levels of these trophic factors might be reliable biomarkers of adult hippocampal neurogenesis. However, the exact relationship between peripheral levels of neurotrophins and levels of hippocampal neurogenesis is still unclear. Rachman and colleagues have provided the first evidence that brain BDNF is the major contributor to the increase in plasma BDNF that is observed in response to exercise . Yau andIn agreement with the findings from animal studies, a dissociation between central and peripheral BDNF levels has also been shown in the clinical setting. Thus, an increase in the brain levels of BDNF was detected in blood samples from the internal jugular vein following 3 months of endurance training in healthy subjects, but no changes in peripheral BDNF levels were observed in these individuals . Indeed,IGF-1 is secreted primarily in the liver  and can A positive correlation between serum levels of IGF-1 and cognitive function has also been demonstrated in several clinical studies –237. ForVEGF, a 45 kDa heparin-binding homodimeric glycoprotein, is secreted by skeletal muscle and could be released into the circulation . Acute eVoss and colleagues have shown the first link between exercise-induced functional connectivity in the temporal cortex and changes in BDNF, IGF-1, and VEGF in healthy elderlies . They reIn conclusion, the relationship between exercise-induced changes in peripheral and central levels of neurotrophic factors has not yet been fully validated, and as such, it is still not feasible to use peripheral levels of neurotrophins as biomarkers for predicting changes in adult neurogenesis in human subjects. Further investigations will be needed to discern the interactions between hippocampal neurogenesis and peripheral and central changes in the levels of neurotrophic factors in animal models and humans, both in basal conditions and following different intervals of physical exercise.Several animal studies have provided evidence for a functional role of adult hippocampal neurogenesis in specific forms of hippocampal-dependent learning and memory. The multifactorial nature of adult neurogenesis implies that this complex process can be compromised by a variety of disease conditions and mounting evidence from rodent models over the last two decades suggests that alterations in the normal neurogenic capacity can either contribute to or be a consequence of a wide range of neurological disorders including AD, PD, and HD. Despite some inconsistencies in the literature, there seems to be an overall trend towards a decrease in neurogenesis with neurodegeneration. However, in some cases an upregulation of the endogenous neurogenic function has also been found, which may reflect an intrinsic attempt of the brain to regenerate itself and replace the neurons that are lost during the degenerative process. Furthermore, discrepancies between studies performed in animal models and postmortem human brains are also present in the literature and may reflect differences in the amount of progenitor cell proliferation present in the diseased human brains and the respective rodent models . in vivo imaging techniques for measurement of adult neurogenesis in the live human brain as well as the discovery of peripheral biomarkers that can be used to determine changes in hippocampal neurogenesis will certainly open new avenues to not only answer these questions but also to diagnose and follow the progression of cognitive decline in various neurodegenerative conditions as well as to measure the effectiveness of treatments aimed at manipulating adult hippocampal neurogenesis. The in vivo imaging techniques are promising and applications of these methods in clinical populations with neurodegenerative diseases merit future research to validate their reliability in clinical settings. On the other hand, with emerging knowledge about the functional significance of hippocampal neurogenesis in pattern separation of learning and memory formation, neurogenesis-dependent cognitive tasks  characteristic of various animal models of these disorders while also promoting neurogenesis. Further clinical studies are warranted to further elucidate the exact relationship between adult hippocampal neurogenesis and cognitive decline in various neurodegenerative diseases within the human population. The development and refinement of the currention task ) would bTo conclude, although the exact links between physical exercise, increase adult hippocampal neurogenesis and improved cognition are still unclear due to the current technical limitations, it is undisputable that exercise has a positive impact in the brain both during ageing and neurodegenerative processes that are associated with poor cognitive function including dementia. Therefore, physical exercise has now emerged as the most effective way to delay the aged-related cognitive decline associated with various neurodegenerative diseases. Finally, the development of new pharmacological cognitive enhancers that mimic the effects of physical exercise on the brain may also emerge as a new teherapeutic strategy to prevent cognitive decline in the ageing population.