Reelin, a multifunctional extracellular protein that is important for mammalian brain development and function, is secreted by different cell types in the prenatal or postnatal brain. The spatiotemporal regulation of Reelin expression and distribution during development relates to its multifaceted function in the brain. Prenatally Reelin controls neuronal radial migration and proper positioning in cortical layers, whereas postnatally Reelin promotes neuronal maturation, synaptic formation and plasticity. The molecular mechanisms underlying the distinct biological functions of Reelin during and after brain development involve unique and overlapping signaling pathways that are activated following Reelin binding to its cell surface receptors. Distinct Reelin ligand isoforms, such as the full-length protein or fragments generated by proteolytic cleavage differentially affect the activity of downstream signaling pathways. In this review, we discuss recent advances in our understanding of the signaling transduction pathways activated by Reelin that regulate different aspects of brain development and function. A core signaling machinery, including ApoER2/VLDLR receptors, Src/Fyn kinases, and the adaptor protein Dab1, participates in all known aspects of Reelin biology. However, distinct downstream mechanisms, such as the Crk/Rap1 pathway and cell adhesion molecules, play crucial roles in the control of neuronal migration, whereas the PI3K/Akt/mTOR pathway appears to be more important for dendrite and spine development. Finally, the NMDA receptor (NMDAR) and an unidentified receptor contribute to the activation of the MEK/Erk1/2 pathway leading to the upregulation of genes involved in synaptic plasticity and learning. This knowledge may provide new insight into neurodevelopmental or neurodegenerative disorders that are associated with Reelin dysfunction. Cleavage at this site releases a six amino acid carboxy-terminal peptide, reducing signaling activity and hindering dendrite development in the postnatal neocortex  and the very low-density lipoprotein receptor (VLDLR). These proteins belong to the low-density lipoprotein receptor (LDLR) family. They have partial functional redundancy and play an essential role in Reelin-mediated neuronal migration based on the observation that double knockout mice display a in vitro  is an intracellular adaptor protein that is essential for Reelin signal transduction. This protein binds the cytoplasmic tail of lipoprotein receptors, including ApoER2 and VLDLR (Trommsdorff et al., Crk/CrkL mutant mice display a reeler-like cortical phenotype (Park and Curran, Genetic studies demonstrated that Dab1, and thus Reelin signaling, is specifically required for a specific mode of radial migration termed somal or terminal translocation, but not for glial-guided locomotion (Franco et al., PAFAH1b1 gene that is responsible for human lissencephaly type I, may be particularly relevant to cortical development. Lis1 binding to phospho-Dab1 is Reelin-dependent, and genetic interaction between Dab1 and PAFAH1b1 demonstrates a functional relationship between these proteins (Assadi et al., PAFAH1b alpha subunits bind specifically to VLDLR, potentially promoting the interaction between Lis1 and Dab1 downstream of this receptor (Zhang et al., In addition to Crks and Rap1, biochemical studies identified several molecules that may be involved in Reelin-dependent neuronal migration. These include proteins that regulate cytoskeletal dynamics and cell motility, such as Lis1, Nckβ and N-WASP (Assadi et al., reeler mice. Dendritic defects are also apparent in immature hippocampal or cortical cultures isolated from mutant mice, but not in mature cultures (Niu et al., . Since Reelin treatment rescued these defects, these in vitro studies first demonstrated that Reelin directly promotes dendrite development. Following studies further demonstrated that Reelin enables initial dendritic outgrowth by promoting the extension of the Golgi apparatus into apical dendrites (Matsuki et al., in vitro in a manner that is dependent on SFK activity and Dab1 phosphorylation (Beffert et al., in vivo studies demonstrated that the Crk adaptor proteins are required for Reelin-induced Akt phosphorylation, placing the kinase functionally downstream of these adaptors (Park and Curran, Dendrite outgrowth is disrupted in homozygous Other molecules that have been implicated in Reelin-dependent dendrite outgrowth include the amyloid precursor protein (APP; Hoe et al., reeler mice exhibit altered hippocampal synaptic plasticity and multiple behavioral abnormalities, such as defects in executive function and contextual fear conditioning learning (Brigman et al., in vivo (Beffert et al., ++ influx through the NMDAR (Beffert et al., ++ influx, Erk1/2 signaling and CREB-dependent IEGs transcription (Telese et al., Heterozygous ++ signaling (Hellwig et al., Reelin gene in adult mice does not result in impaired synaptic plasticity. However, it renders the adult brain strikingly sensitive to amyloid-induced synaptic suppression, leading to profound learning disabilities (Lane-Donovan et al., In addition to its well-documented postsynaptic effects, Reelin also acts presynaptically, causing a rapid enhancement of spontaneous neurotransmitter release. This effect is due to the mobilization of VAMP7-containing synaptic vesicles, and requires canonical ApoER2/VLDLR receptors, PI3K and CaGHL wrote the first draft of the manuscript and made the figures. GD wrote and revised the manuscript.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.


The grant number is GR09/0021.In the Funding section, the second grant number from the funder Instituto de Salud Carlos III (Madrid, Spain;


Morbilivirus . The clal., 2011; Fazlalial., 2011). Expandal., 2011; Liu et al., 2011). Furtheal., 2011; Leuridaal., 2011). Vaccinal., 2011; Sheikh al., 2011). Many ial., 2011). Among al., 2011). Furtheal., 2011). Therefore, keeping in view the importance and significance of measles, this preliminary study was designed to develop an ELISA for detection of humoral immunity. For this purpose ELISA plates were coated with measles virus CAM-70 strain  which contain not less than 1000 CCID50 in 0.5 ml. Coating of plates was performed using carbonate buffer as described by van der Werff (2008). BrieflThe authors declare that they have no conflict of interest.


The fossil record of crocodylians and their relatives (pseudosuchians) reveals a rich evolutionary history, prompting questions about causes of long-term decline to their present-day low biodiversity. We analyse climatic drivers of subsampled pseudosuchian biodiversity over their 250 million year history, using a comprehensive new data set. Biodiversity and environmental changes correlate strongly, with long-term decline of terrestrial taxa driven by decreasing temperatures in northern temperate regions, and biodiversity decreases at lower latitudes matching patterns of increasing aridification. However, there is no relationship between temperature and biodiversity for marine pseudosuchians, with sea-level change and post-extinction opportunism demonstrated to be more important drivers. A ‘modern-type' latitudinal biodiversity gradient might have existed throughout pseudosuchian history, and range expansion towards the poles occurred during warm intervals. Although their fossil record suggests that current global warming might promote long-term increases in crocodylian biodiversity and geographic range, the 'balancing forces' of anthropogenic environmental degradation complicate future predictions. Crocodylians and their relatives have a rich evolutionary history. Here the authors show long-term decline of terrestrial crocodylians driven by decreasing temperatures but no relationship between temperature and biodiversity for marine crocodylians over their 250 million year history. Ecological models can predict the responses of extant species distributions to rising temperatures. However, only the fossil record provides empirical evidence of the long-term interactions between climate and biodiversityOngoing climate change, with projected global warming of 2.0–4.8°C over the next centurySarcosuchus, which reached around 12 m in length and weighed up to 8 metric tons68Pseudosuchia is a major reptile clade that includes all archosaurs closer to crocodylians than birds, and made its first fossil appearance nearly 250 million years ago, at the time of the crocodile–bird split8101112Here we examine the effect of climate on spatiotemporal patterns in pseudosuchian biodiversity over their 250 million year history, using a comprehensive fossil occurrence data set. Our study is the first to analyse climatic drivers of pseudosuchian biodiversity through the group's entire evolutionary history, applying rigorous quantitative approaches to ameliorate for uneven sampling across both time and space. Furthermore, this is the only comprehensive, temporally continuous fossil occurrence dataset for a major extant vertebrate group: equivalent datasets are not currently available for mammals, birds, squamates, teleosts, or other groups with evolutionary histories of similar durations.An uncorrected global census of pseudosuchian genera  shows anN=38, R2=0.221; Subsampled pseudosuchian biodiversity reached a peak during the earliest intervals of the group's history and shows a long-term pattern of gradual decline towards the present day . SubstanOnly the crocodylomorph clade survived the Triassic/Jurassic mass extinction (201 Myr ago)10162416Sampling of palaeotropical non-marine crocodylomorphs is limited throughout the Jurassic–Cretaceous  and 4a. 88The Cretaceous witnessed the non-marine radiations of notosuchians819931The effect of the K/Pg mass extinction on crocodylomorphs has previously been perceived as minor or non-existent192819283436353636δ18O palaeotemperature proxy17Relative changes in subsampled non-marine biodiversity in both North America and Europe track each other and the 7  during the early Paleogene greenhouse world 66–41 Myr ago). There is no evidence for transient biodiversity increases driven by the short-term Paleocene–Eocene Thermal Maximum (56 Myr ago), possibly because the timescale of species origination and phenotypic divergence that would allow speciation to be recognisable in the fossil record is longer than that of this rapid climatic event  includes a near-comprehensive dataset of all published pseudosuchian occurrences spanning the Middle Triassic through to the Pleistocene, a period of nearly 250 million years. Pseudosuchian body fossil occurrences that could be assigned to genera  were downloaded from this database , accessed via Fossilworks (http://fossilworks.org), on 23 February 2015.Following extensive work to ensure that occurrences and taxonomic opinions were consistent and up-to-date with the literatureGenera were used so as to incorporate specifically indeterminate material, enabling us to include more data points in our analyses, and also to avoid problems with using species as a unit for estimating palaeobiodiversityAlligator and Crocodylus, have been used as ‘wastebasket taxa' for indeterminate or non-referable fossil species. Therefore, we modified occurrences of Crocodylus and Alligator before analysis, as explained in the Extant genera, especially et al. (in review). Environmental assignment (marine versus non-marine) for post-Ypresian taxa was based primarily on facies data recorded in the PaleoDB and information presented in refs et al.Aktiogavialis, Piscogavialis and Siquisiquesuchus; these were omitted from the marine crocodylomorph data set of Martin et al.Tomistoma were probably marineT. schlegelii) are non-marine, or their environments are unknown; as such, here we treat Tomistoma as non-marine.The resultant pseudosuchian data set was subdivided into non-marine  stratigraphic time bins . Althougu, singleton taxa were defined based on occurrences within collections58In determining Good's Patterns in non-marine and marine taxa were analysed separately, to avoid problems with sampling from heterogeneous environments58http://www.scotese.com) to obtain reconstructed palaeogeographic positions for each occurrence.In addition to producing sub-sampled terrestrial and marine biodiversity curves through time, we also analysed the palaeolatitudinal distribution of terrestrial biodiversity. This was implemented through plots of subsampled regional biodiversity against the regional palaeolatitudinal centroid, as well as plots of subsampling curves within time bins . Plots oδ18O palaeotemperature proxies and sequence stratigraphic eustatic sea level estimates in two sets of analyses: (1) terrestrial pseudosuchian biodiversity in North America and Europe were each compared to the Zachos et al.et al.δ18O from the Prokoph et al.We compared subsampled genus biodiversities at a quorum of 0.4 to δ18O and sea level, with and without the application of first differencing.Although a contentious issue in crocodylomorph phylogeny, we follow the most recent placement of Thalattosuchia as a basal clade outside of Crocodyliformeset al.δ18O values of fish teeth from the Western Tethys. One potential problem with this method is that the fish teeth are from a variety of different species and genera, with Lécuyer et al.δ18O can occur. In addition, there might be differences between the isotopic fractionation that occurs between phosphate and water, and that which takes place in the fish teethδ18O curves of Prokoph et al.et al.δ18O dataset for deep sea palaeotemperatures of Zachos et al.et al.1716The similarity of these independent isotopic databases17Statistical comparison was made using time series approaches, specifically generalised least squares (GLS) regression incorporating a first-order autoregressive model .Supplementary Figures 1-7, Supplementary Tables 1-2, Supplementary Methods and Supplementary ReferencesNon-marine pseudosuchian dataMarine pseudosuchian dataTime intervals tablePERL script for SQS (provided by John Alroy)et al. (2008) palaeotemperature proxy isotope dataZachos et al. (2005) sea level dataMiller et al. (2008) palaeotemperature proxy isotope dataProkoph


Key components in this response include enzymes involved in the biosynthesis of deoxymugineic acid , the deoxymugineic acid efflux transporter (TOM1), the Fe(III)-deoxymugineic acid transporter (OsYSL15), and Fe2+ transporters . In whole roots, these proteins are expressed in a coordinated manner with strong transcriptional induction in response to Fe deficiency. Radial transport of Fe to xylem and phloem is also mediated by the mugineic acid family phytosiderophores, as well as other chelators and their transporters, including Fe(II)-nicotianamine transporter (OsYSL2), phenolics efflux transporters (PEZ1 and PEZ2), and citrate efflux transporter (OsFRDL1). Among these, OsYSL2 is strongly induced under conditions of Fe deficiency. Both transcriptional induction and potential feedback repression mediate the expressional regulation of the genes involved in Fe uptake and translocation in response to Fe deficiency. The transcription factors IDEF1, IDEF2, and OsIRO2 are responsible for transcriptional induction, whereas the ubiquitin ligases OsHRZ1 and OsHRZ2, as well as the transcription factors OsIRO3 and OsbHLH133, are thought to mediate negative regulation. Furthermore, IDEF1 and OsHRZs bind Fe and other metals, and are therefore candidate Fe sensors. The interacting functions of these regulators are thought to fine tune the expression of proteins involved in Fe uptake and translocation.Iron (Fe) is an essential element for most living organisms. To acquire sparingly soluble Fe from the rhizosphere, rice roots rely on two Fe acquisition pathways. The first of these pathways involves Fe(III) chelators specific to graminaceous plants, the mugineic acid family phytosiderophores, and the second involves absorption of FeThe online version of this article (doi:10.1186/s12284-014-0027-0) contains supplementary material, which is available to authorized users. Among u et al. ). The tr2+ uptake is coupled to ferric-chelate reductase activity on the root surface, which is strongly induced under conditions of Fe deficiency . Howevearschner ), thus i2+ uptake, although this effect may be limited as the PEZ2 transcript shows little induction under conditions of Fe deficiency . To avoUnder Fe-deficient conditions, the enzymes and transporters responsible for Fe uptake are induced not only in the epidermis/exodermis, but also in the cortex and vascular bundle Table  . The Ose et al. ; Ishimare et al. ). The NAe et al. ) are thoOsFRDL1 is specifically expressed in root pericycle cells, with no apparent induction under conditions of Fe deficiency (Table Citrate is an Fe(III) chelator and is thought to play a dominant role in xylem Fe transport in non-graminaceous plants . In ricPEZ1 is expressed specifically in the vascular bundle, while PEZ2 is expressed in the epidermis/exodermis, cortex, and vascular bundle . Both PArabidopsis thaliana (AtFPN1/AtIREG1) is a likely candidate  for theOsVIT1, OsVIT2, and MIT expression are repressed under conditions of Fe deficiency . The miAs reviewed above, the levels of expression of numerous rice genes involved in Fe uptake and translocation are strongly induced under conditions of Fe deficiency at the transcriptional level. Highly conserved temporal and spatial patterns of expression are observed, especially with regard to genes involved in DMA-based Fe uptake . IDE1 ai et al. , 2004]))cis-actii et al. , 2009];;cis-actii et al. ). IDEF1 on Table . At latei et al. , [2009],i et al. ). IDEF2 o et al. ), whereai et al. , 2013]))cis-actii et al. , [2009],i et al. ; Ogo et i et al. ; Itai eti et al. ). Both Ig et al. ).OsIRO2 itself is positively regulated by IDEF1, forming a transcriptional cascade enhancing the expression of genes involved in Fe(III)-DMA uptake and translocation . Howeveg et al. ).in silico prediction of cis-sequences over-represented in Fe deficiency-induced gene promoters revealed that the IDEF1-, IDEF2-, and OsIRO2-binding sequences are enriched within the 500-bp promoter regions of Fe deficiency-induced genes  and AGCTAGCT (designated DCEp1 for putative downstream core element 1), as well as common regulatory sequences, such as TATA-box and upstream TFIIB-recognition elements near the transcription start sites of Fe deficiency-inducible genes . The eni et al. ). Regulavs. other metals.IDEF1 binds ferrous Fe and other divalent metals, such as zinc (Zn) and nichel, via its histidine-asparagine repeats and proline-rich regions . In ricOsHRZ1 and OsHRZ2 knockdown show substantial tolerance to low Fe availability, and accumulate large amounts of Fe in shoots and seeds irrespective of Fe nutritional conditions. These knockdown lines also show enhanced expression of Fe deficiency-inducible genes involved in Fe uptake and/or utilization even under Fe-sufficient conditions, but this enhancement is much less obvious under prolonged Fe deficiency  interacts indirectly with the OsIRO3 homolog POPEYE (PYE) by binding another subset of basic helix-loop-helix transcription factors  from thcy Table . OsHRZ1 y Figure . In Arabg et al. ). BTS ang et al. ). BTS kng et al. ).OsHORZ1 knockdown show increased susceptibility to Fe deficiency and repression of Fe deficiency-inducible genes . OsHORZi et al. ). Thus, OsNAS1, OsNAS2, OsYSL15, and OsIRT1 under both Fe sufficiency and deficiency, and this enhancement may be mediated by the OsIRO2 pathway . Knockoi et al. ). GibberTakasaki ; Yoshii Takasaki ). A jasmTakasaki ) has recg et al. ). OsRMC In Strategy-I plants, systemic Fe signal derived from shoots is thought to determine whether Fe deficiency response in roots takes place (Giehl et al. ; García 2+ uptake. Research has also elucidated the function of DMA in internal Fe translocation, in addition to its known role in Fe uptake. The differential spatial expression of genes responsible for Fe uptake and translocation in response to Fe deficiency is highly coordinated and is well correlated with their physiological functions. In addition to positive regulation of these genes at the transcriptional level, negative regulation at both the transcriptional and translational levels may also function in fine tuning the Fe deficiency response. Although the Fe-sensing mechanism remains unclear, further characterization of IDEF1 and OsHRZs may in fact consolidate their respective roles as Fe sensors. Despite these advances in our understanding, a potentially large proportion of the molecules involved in intercellular and subcellular Fe movement have not yet been identified. Further investigation into these issues will help to develop novel tools for producing Fe-efficient and Fe-fortified crops with increased versatility.Rice responds to low Fe availability by inducing enzymes, transporters, and regulators that participate in Fe uptake from the rhizosphere and Fe translocation within the plant body. Recent studies demonstrated that rice utilizes both classical DMA-based Fe uptake and direct FeTK and RNI analyzed the microarray data. TK wrote the manuscript with critical revision by RNI and NKN. All of the authors read and approved the manuscript.


Ependymal cells (ECs) form a barrier responsible for selective movement of fluids and molecules between the cerebrospinal fluid and the central nervous system. Here, we demonstrate that metabolic and barrier functions in ECs decline significantly during aging in mice. The longevity of these functions in part requires the expression of the myristoylated alanine-rich protein kinase C substrate (MARCKS). Both the expression levels and subcellular localization of MARCKS in ECs are markedly transformed during aging. Conditional deletion of MARCKS in ECs induces intracellular accumulation of mucins, elevated oxidative stress, and lipid droplet buildup. These alterations are concomitant with precocious disruption of ependymal barrier function, which results in the elevation of reactive astrocytes, microglia, and macrophages in the interstitial brain tissue of young mutant mice. Interestingly, similar alterations are observed during normal aging in ECs and the forebrain interstitium. Our findings constitute a conceptually new paradigm in the potential role of ECs in the initiation of various conditions and diseases in the aging brain. They are highly polarized with adherens junctions at their apical interface allowing them to form a highly selective barrier between the interstitial tissue and cerebrospinal fluid (CSF) in the ventricles  is critical for previously unappreciated biological functions in ECs related to their aging. Selective deletion of MARCKS in ependyma results in precocious emergence of biomarkers for aging, both in ECs and in the brain interstitium. MARCKS is a known regulator of cell polarity, cytoskeletal signaling, cell migration, and embryonic brain development  was confirmed using antibody labeling, which additionally revealed that its subcellular localization mirrored the distribution of MARCKS in 2M ECs, enriched at the apical surface   mouse brains revealed MARCKS loses its apical clustering and becomes less polarized in the cytosol, whereas p-MARCKS is sparsely distributed Fig. and S5. ines Fig.. Thus, Mζ in ECs, we focused on determining how the phosphorylation state of MARCKS may influence its localization and function. In lung epithelial cells, phosphorylation of MARCKS is known to regulate its association with filamentous actin (F-actin) near the plasma membrane. MARCKS dissociates from actin networks and the plasma membrane upon phosphorylation by PKC -bisphosphate P₂) in neurons  and by performing a FITC–dextran (FD4) assay in wholemounts using Ussing chambers Fig.. TER, a Finally, the impact of precocious aging of MARCKS-cKO ECs on potential gliosis and neuroimmune responses in interstitial brain tissue was assessed. First, effects of normal aging on the integrity of the ependymal layer in the forebrain were documented in wholemount preparations of the ventricular walls in young and old brains. Imaging clearly illuminated scarring of ECs accompanied by astrocyte infiltration into the scarred regions . These pet al., et al., et al., et al., MARCKS-dependent mechanisms used as a model in our study likely represent one of many that can influence aging and maintenance of ECs. ECs have been credited with multiple functional attributes which are possibly influenced by MARCKS-dependent cellular and molecular mechanisms. For example, a unique population of ependyma in the choroid plexus is responsible for the production of CSF and various transport proteins  may be indicative of a potential role for MARCKS on biomechanical properties of stroke in EC cilia. Indeed, the apical clustering of MARCKS near the base of EC cilia is suggestive of potential interaction with molecular motors that facilitate the beating of motile cilia. Ependyma in the young adult brain have been described to be heterogeneous based on their multiciliated or biciliated morphology  was originally identified in intestinal goblet cells and gained attention for its selective expression in the lung epithelium and its role in mucus secretion during airway hyper-responsiveness (Nakanishi et al., et al., et al., et al., et al., et al., Ependyma have been shown to accumulate lipids in the aged mouse brain (Bouab et al., We also found elevation of 4-HNE and nitrotyrosine in ECs of MARCKS-cKO mice similar to aged wild-type brains. 4-HNE is an α,β-unsaturated hydroxyalkenal that is produced by lipid peroxidation and tyrosine nitration. It is upregulated in instances of elevated reactive nitrogen species and, together with nitrotyrosine, is routinely used as a biomarker for oxidative stress. Interestingly, elevated intracellular oxidative stress has been demonstrated to lead to induction of lipid droplet formation and fatty acid accumulation in several cell types (Kuramoto et al., et al., et al., Our findings have revealed that selective disruption of barrier functions in ECs can age the interstitial brain tissue Fig.. This paet al., et al., However, ependyma-infiltrated astrocytes may contribute toward elevated oxidative damage in periventricular tissues. For example, it was recently demonstrated that age-associated increase in mitochondrial metabolism in astrocytes produces detrimental reactive oxygen and nitrogen species (Jiang & Cadenas, in vivo model for assaying the role of ECs in maintenance of brain homeostasis under various disease states and environmental stimuli with potential relevance to human aging.In summary, our study demonstrates for the first time that MARCKS is required for maintaining the ependymal barrier integrity and sustaining forebrain homeostasis during normal aging. In addition, we have identified that mucins are expressed by, and may be secreted from, ependymal cells into the brain interstitium in an age- and MARCKS-dependent manner. These functions are correlated with lipid droplet formation in ependyma raising the possibility that MARCKS-dependent mucin expression and distribution in the brain parenchyma may be related to lipid metabolism in aging ECs. Thus, conditional MARCKS deletion in ependyma together with the apparent precocious aging of ECs provides a highly suitable Experimental Procedures are available in Supporting information.


Escherichia coli and related enteric species is cytoplasmic accumulation of phosphorylated sugars such as glucose-6-phosphate or the non-metabolizable analog α-methylglucoside-6-phosphate. This “glucose-phosphate stress” triggers a dedicated stress response in γ-proteobacteria including several enteric pathogens. The major effector of this stress response is a small RNA (sRNA), SgrS. In E. coli and Salmonella, SgrS regulates numerous mRNA targets via base pairing interactions that result in alterations in mRNA translation and stability. Regulation of target mRNAs allows cells to reduce import of additional sugars and increase sugar efflux. SgrS is an unusual sRNA in that it also encodes a small protein, SgrT, which inhibits activity of the major glucose transporter. The two functions of SgrS, base pairing and production of SgrT, reduce accumulation of phosphorylated sugars and thereby relieve stress and promote growth. Examination of SgrS homologs in many enteric species suggests that SgrS has evolved to regulate distinct targets in different organisms. For example, in Salmonella, SgrS base pairs with sopD mRNA and represses production of the encoded effector protein, suggesting that SgrS may have a specific role in the pathogenesis of some γ-proteobacteria. In this review, we outline molecular mechanisms involved in SgrS regulation of its target mRNAs. We also discuss the response to glucose-phosphate stress in terms of its impact on metabolism, growth physiology, and pathogenesis.Bacteria adapt to ever-changing habitats through specific responses to internal and external stimuli that result in changes in gene regulation and metabolism. One internal metabolic cue affecting such changes in  Over the last decade, small RNAs have emerged from relative obscurity to take their places as central regulators of gene expression in organisms from all three domains of life. While hundreds of small RNAs in dozens of bacterial genomes have been identified by computational or experimental methods, the functions of the vast majority of these remain a mystery. Nevertheless, we have learned a great deal about a small number of “model” bacterial sRNAs, including how their production is regulated, what targets they in turn regulate and the physiological outcomes of sRNA-mediated regulation. In this review, we first provide a brief overview of features of regulatory sRNAs that act on mRNAs through base pairing interactions. We then focus on one well-characterized sRNA, SgrS (sugar transport related sRNA) and describe its activities on target mRNAs and how these activities regulate bacterial metabolism and pathogenesis.Several mechanistic classes of sRNAs have been identified in diverse bacterial species. Many characterized sRNAs act by base pairing with mRNA targets to control mRNA stability and translation. Such sRNAs are often between 50 and 300 nucleotides in length and require an RNA chaperone, Hfq, for their stability and regulatory effects on target mRNAs  Hfq-binding sRNA  and this is independent of manX regulation , a glutamate-pyruvate aminotransferase  is highly conserved, suggesting that SgrR regulates sgrS expression in all these organisms. All identified SgrS homologs contain a Rho-independent terminator and most possess an additional stem-loop structure upstream of the terminator; these two structures are important for Hfq binding to SgrS   was not sufficient to allow growth rescue during glucose-phosphate stress conditions. This is in part due to very low levels of SgrT produced from the native sgrS allele in E. coli -1 and SPI-2 (Brumell et al., sopD by SgrS involves base pairing interactions between the conserved region of SgrS and the early coding sequence of sopD mRNA (Figure sopD mRNA degradation (Papenfort et al., Salmonella encodes a second SopD protein, SopD2, which shares 42% identity with SopD and likely arose from a duplication (Jiang et al., sopD2 base pairing interaction differs from SgrS-sopD at only a single position, a wobble G:U base pair instead of the G:C base pair. Remarkably, this interaction that differs by only a single hydrogen bond prevents regulation of sopD2 by SgrS (Papenfort et al., Although SgrS is conserved among enteric bacteria, divergence in primary sequence has resulted in species-specific target regulons, exemplified by the finding that A Figure ; the intsopD regulation by SgrS is not yet clear, the inclusion of sopD in the Salmonella SgrS regulon illustrates plasticity in the evolution of sRNA regulons. The presence of sgrR-sgrS-sgrT in the same genomic context in pathogenic and non-pathogenic γ-proteobacteria (Horler and Vanderpool, Salmonella. Studies of other SgrS homologs in pathogenic and non-pathogenic enteric bacteria will surely shed light on the breadth of regulatory activities of this fascinating dual-function sRNA.While the biological significance of 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.


Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and apoplastic metabolic pathways, in tissue differentiation and organ development.Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell-wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H In Arabidopsis thaliana 10 CuAO genes are present, among which only eight encode for putative functional CuAOs AtCuAOε1  and AtCuAOε2  are consecutive fragments of a copy of AtCuAOδ gene. Phylogenetic analysis evidenced that plant CuAOs form three clades (I-III), clade I being composed of three subgroups (Ia-Ic) and clade II of two  putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and thermospermine (Therm-Spm) are involved in several physiological processes, such as cell proliferation, differentiation and defense responses  are localized in the apoplast  and the other members of clade III in peroxisomes , AtCuAOγ1 (clade IIa) as well as Nicotiana tabacum CuAO1 (NtDAO1), oxidize mainly Put, Cad, and Spd , which shows preference for N-methyl-Put and is involved in nicotine biosynthesis, although all three proteins are clustered together in clade III  was shown to produce Nor-Spd from Therm-Spm , which are localized intracellularly and show an -AtPAO5, 2O2; hydroxyl radical, •OH; singlet oxygen, 1O2) and nitric oxide (NO) in orchestrating developmental processes, as well as in being involved in signaling of both local and systemic defense responses in plants. The apoplast is a major “hub” for these chemical species. Their accumulation in large amounts and the complexity of the regulatory mechanisms involved in their biosynthesis reflect the peculiar role of this compartment in physiological events that depend on temporarily regulated and spatially restricted ROS and NO signatures , transition metal ions and H2O2 -mediated NO production  distribution  in plants , which is clustered together with AtCuAOδ in clade IIb, was attributed to the implication of PA metabolism in physiological processes taking place during fruit ripening , are controlled by Therm-Spm and Spm  and an AtPAO5-like (GhPAO4) PAO may play a crucial role in the generation and differentiation of embryogenic callus during somatic embryogenesis  have a role in shoot regeneration from root cultures (Lim et al., Some studies suggest that in CuAO and PAO gene families regarding catalytic activity, subcellular localization, expression pattern and physiological roles of the encoded proteins. Indeed, important links to developmental and stress-related events are emerging for CuAOs and PAOs through ROS/NO production and regulation of specific PA levels.Numerous recent studies have evidenced an extraordinary complexity in All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Although their causative mechanisms are quite diverse, these diseases share the common feature of an uncontrolled and progressive accumulation of fibrous tissue macromolecules in affected organs leading to their dysfunction and ultimate failure. The pathogenesis of fibrotic diseases is complex and despite extensive investigation has remained elusive. Numerous studies have identified myofibroblasts as the cells responsible for the establishment and progression of the fibrotic process. Tissue myofibroblasts in fibrotic diseases originate from several sources including quiescent tissue fibroblasts, circulating CD34+ fibrocytes, and the phenotypic conversion of various cell types including epithelial and endothelial cells into activated myofibroblasts. However, the role of the phenotypic transition of endothelial cells into mesenchymal cells  in the pathogenesis of fibrotic disorders has not been fully elucidated. Here, we review the evidence supporting EndoMT’s contribution to human fibrotic disease pathogenesis.Fibrotic diseases encompass a wide spectrum of clinical entities including systemic fibrotic diseases such as systemic sclerosis, sclerodermatous graft  The resulting fibrosis disrupts the normal architecture of the affected organs, leading to their progressive dysfunction and eventually to their functional failure ,2,3. Thee (GVHD) , and IgG disease ,11, as w disease ,13, and  disease , pulmona disease , liver [ disease , and kid disease . Further disease , asbesto disease , or aris disease , and oth disease  and Chag disease . The mosFibrotic disorders collectively affect a very large number of individuals, and owing to the current lack of effective therapeutic approaches, result in very high morbidity and mortality rates. Indeed, it has been estimated that the mortality caused by fibrotic diseases in the Western developed countries is as high as 45% of the total mortality . AlthougGiven the wide variety of affected organs, the chronic nature of the fibrotic processes, the lack of effective antifibrotic therapies, and the large number of individuals suffering their devastating effects, fibrotic diseases pose a serious public health problem representing an enormous challenge to health services worldwide and causing a serious economic burden to society. Despite the remarkable detrimental consequences of the fibrotic disorders for human health, there are very few approved therapeutic agents for these conditions. However, the recent elucidation of important alterations in crucial regulatory pathways involved in the fibrotic process and the identification of the central role of activated myofibroblasts in the production and abnormal deposition of ECM in this process provide a sound basis for the search and eventual identification of novel and effective means of therapy for these devastating diseases ,28,29.in vivo, myofibroblasts induce changes in the biomechanical properties of the affected tissues causing a progressive increase in tissue stiffness, a newly recognized extremely potent profibrotic stimulus [Numerous recent studies have demonstrated that myofibroblasts are the cells ultimately responsible for the severe fibrotic process in the fibrotic disorders ,31,32,33stimulus ,37.et al. [Given the crucial role of myofibroblasts in the pathogenesis of systemic and organ-specific fibrotic disorders including SSc and Idiopathic Pulmonary Fibrosis (IPF), there has been extensive investigation aimed at the precise identification of their cellular origin ,39,40,41et al. . Indeed,et al. ,70. HoweIn the following sections, we will review the available evidence that supports a role for EndoMT in the pathogenesis of various human fibrotic disorders. First, we will review studies of the molecular mechanisms of EndoMT emphasizing various pathways known to participate in the pathogenesis of tissue fibrosis, then, we will briefly discuss the occurrence of EndoMT in various experimentally-induced animal models of tissue fibrosis, and finally, we will review the occurrence of EndoMT in cultured human EC and in tissues from patients with various human fibrotic disorders.In contrast with the extensive studies on the molecular events and pathways involved in EMT , the mecThe TGF-β family of proteins comprises several pleiotropic growth factors that play crucial roles in numerous physiological processes including embryogenesis, cellular development and differentiation, immunologic system development, inflammatory response functions, and wound repair ,72. Owinin vitro; (2) TGF-β induction of EndoMT was associated with a strong upregulation in the expression of the transcriptional repressor Snail1 indicating that Snail1 is directly involved in TGF-β-induced α-SMA expression; and (3) induction of α-SMA expression involved the c-Abl kinase and protein kinase C-δ (PKC-δ), as specific inhibition of their kinase activities with imatinib mesylate and rottlerin, respectively, or by knockdown of their corresponding transcripts with specific siRNA abrogated the marked increase in TGF-β induced α-SMA and Snail1 expression and protein levels. These observations confirmed the potent induction of EndoMT by TGF-β1 in vitro [in vivo studies. The study of Cooley et al. demonstrated that a TGF-β-neutralizing antibody decreased EndoMT and resulted in a profound reduction in neo-intima formation in a mouse model of interpositional vein grafts [et al. generated heterozygous mice with endothelium-specific knock-out of the TGF-receptor II (TβRII) gene and demonstrated that the resulting partial TβRII deletion abrogated EndoMT and reduced tubulointerstitial kidney fibrosis in two murine models of renal fibrosis [Given the crucial role that the TGF-β family of profibrotic growth factors plays in the development of tissue fibrosis and in the pathogenesis of numerous fibrotic diseases, several studies have demonstrated that TGF-β is involved in the generation of myofibroblasts through EndoMT ,82,83,84n grafts . In anotfibrosis . The rolfibrosis .The morphogens Notch and Hedgehog play crucial roles during embryonic development. Although in the adult these pathways are very highly regulated, their aberrant activation may lead to various pathological consequences, including the development of fibrotic diseases ,88,89. TRecently, the Sonic Hedgehog (SHh) pathway, another important morphogen, has also been shown to participate in the pathogenesis of various fibrotic disorders ,96,97,98The Wnt proteins comprise a multigene family of secreted glycoproteins that play crucial roles during embryogenesis signaling through canonical and non-canonical pathways ,100. NumCav1 knock out mice (Cav1−/−) showed that these mice exhibited extensive skin and lung fibrosis [Cav1−/− mice [Cav1−/−mice in vivo and suggested that CAV1 deficiency participates in the development and progression of tissue fibrosis and fibrotic diseases through the establishment of EndoMT.CAV1, the main protein component of caveolae plays an important role in the internalization, trafficking and degradation of TGF-β receptors and, therefore, is involved in the regulation of TGF-β signaling and TGF-β-mediated fibrotic responses ,109,110.fibrosis . Subsequfibrosis ,112,113,fibrosis . These s−/− mice . The resThe important role of ET-1, the most potent endogenous vasoconstrictor polypeptide identified to date, in the regulation of multiple vascular functions and its participation in various human diseases including Primary and Secondary Pulmonary Arterial Hypertension (PAH) has been well recognized. However, numerous recent studies have shown that ET-1 also displays strong profibrotic effects and it is involved in various aspects of the fibrotic process ,117. Recin vitro and in vivo [Numerous recent studies have demonstrated an important role of hypoxia in the development or progression of various fibrotic diseases including SSc, kidney, and cardiac fibrosis ,123,124. in vivo . However in vivo  and anot in vivo .One novel pathway that has been recently recognized as a potentially important participant in the pathogenesis of fibrotic processes through reciprocal interactions with TGF-β involves reactive oxygen species (ROS) ,131. AltAlthough there are multiple sources of intracellular ROS, extensive studies have shown that most ROS production derives from the activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system. NOX4 is one of seven NADPH isoforms, however, unlike other members of the NOX family, it is constitutively active and the regulation of its effects occurs mainly at the expression level. The role of NOX4 as an important downstream mediator of TGF-β-induced myofibroblast generation, its contribution to the potent TGF-β profibrotic effects, and its participation in the pathogenesis of tissue fibrosis in various fibrotic disorders such as IPF and liver fibrosis have been recently demonstrated ,143,144.MicroRNAs (miRNAs) are small non-coding RNA that modulate the expression of a large number of protein coding genes at the post-transcriptional level . Recent et al. [The pioneering studies of Zeisberg et al.  clearly et al. . Anotheret al. . Other set al. ,118. Howet al. .et al. [et al. [et al. [Li and Bertram  recentlyet al.  examinedet al. . In anot [et al.  employed [et al. . These s [et al.  employin [et al. ,167.et al. [in vivo in this animal model.The role of EndoMT in experimentally induced pulmonary fibrosis and pulmonary arterial hypertension was examined in several recent studies. The pioneering studies of Hashimoto et al.  evaluateet al. . The resin vitro with EC isolated from affected tissues. The study of Zeisberg et al. [in vitro studies with isolated human coronary artery EC. These studies showed that, following TGF-β1 treatment, the cells became spindly shaped and lost their EC molecular markers and acquired the ability to produce various fibroblast-specific macromolecules including vimentin and type I procollagen. Several other studies have examined the occurrence of EndoMT in cultured human EC of various types. A study by Kitao et al. [in vitro in cultured human dermal microvascular EC and demonstrated the induction of a mesenchymal cell phenotype by TGF-β1 with a change to a spindle cell morphology in monolayer culture and initiation of expression of α-SMA and COL1A. In another study Rieder et al. described the phenotypic conversion of human microvascular intestinal EC into mesenchymal cells following exposure to a combination of proinflammatory cytokines  in vitro and suggested that intestinal EC exposed to an inflammatory environment may participate in the intestinal fibrotic process which accompanies intestinal inflammatory diseases in vitro [et al. examined the effects of interferons α and γ on human dermal microvascular EC and demonstrated that interferon γ induced morphological and molecular/gene expression changes in these cells consistent with their transdifferentiation into profibrotic myofibroblasts [in vitro [In contrast with the extensive evidence from animal models demonstrating the occurrence of EndoMT in the development of experimentally-induced tissue fibrosis there are only few studies that have provided evidence indicative of the occurrence of EndoMT in human fibrotic disorders. Most studies have been performed g et al.  describio et al.  examinedin vitro . Chrobakroblasts . Recentlin vitro . The resin vitro . The samin vitro .in vitro following exposure to TGF-β1 or to other cytokines or inducing agents. In contrast, there are few studies that have demonstrated the occurrence of EndoMT directly in vivo, although some recent publications have described results demonstrating the presence of EC expressing simultaneously EC molecular markers such as, for example, CD31/PECAM-1 or von Willebrand Factor and proteins such as α-SMA, or type I and III interstitial collagens that are specific for myofibroblastic mesenchymal cells. Most of these studies identified cells co-expressing both types of molecular markers in the affected tissues employing immunofluorescence microscopy studies with confocal microscopy. These studies are summarized in All the studies described in the previous section demonstrated that EC isolated from fibrotic tissues from various human fibrotic disorders were capable of undergoing EndoMT et al. [et al. [et al. [Despite the clinical importance and the demonstration of morphological and molecular alterations indicative of EndoMT during experimentally-induced pulmonary fibrosis , there aet al.  were amoet al.  and the et al. . In the  [et al.  employed [et al. , assesseWe recently performed a study to examine the possibility that EndoMT is involved in the fibrotic process of SSc-associated pulmonary fibrosis . ImmunohTissue fibrotic reactions represent the most serious complications of radiation therapy and intense investigation is being conducted to understand the intimate mechanisms involved in the devastating organ fibrosis and dysfunction induced by radiation therapy. Recent studies have examined the potential role of EndoMT in the severe radiation-induced fibrotic process. One study examined the role of EndoMT in the development of radiation-induced pulmonary fibrosis . Using iet al. [et al. [in vivo during the development of diabetic kidney disease-associated kidney fibrosis [Several studies have described evidence supporting the occurrence of EndoMT in human tissues from patients with cardiac fibrosis ,171 and et al.  examinedfibrosis .The possibility that EndoMT of portal vein endothelium via TGF-β/Smad activation may be involved in portal venopathy was examined recently . The resin vitro studies with human EC of various tissue origins, experimental animal models of tissue fibrosis, and analysis of tissues from patients with various fibrotic diseases certainly indicate that EndoMT plays an important role in the pathogenesis of these common and often fatal disorders. The extensive literature published about the participation of EndoMT in the fibrotic process and the demonstration of its occurrence in affected tissues from patients with SSc-associated pulmonary fibrosis and PAH, vein graft fibrosis, intestinal fibrosis, and radiation-induced organ fibrosis indicate that such a role should no longer be considered controversial but it is a reality, as discussed recently [The results of the numerous studies reviewed here including recently ,176. A c


For entry and infection viruses have developed numerous strategies to subjugate indispensable cellular factors and functions. Host cell lipids and cellular lipid synthesis machinery are no exception. Not only do viruses exploit existing lipid signalling and modifications for virus entry and trafficking, they also reprogram lipid synthesis, metabolism, and compartmentalization for assembly and egress. Here we review these various concepts and highlight recent progress in understanding viral interactions with host cell lipids during entry and assembly. The biological functions of lipids range from membrane formation to energy storage and signalling , glycophosphatidylinositol (GPI)-anchored proteins, and transmembrane proteins can cluster into discrete domains, called lipid rafts  or glycosphingolipids . Upon specific binding to ganglioside GM1, SV40 reduces GM1’s diffusion rate. This stabilized SV40-GM1 complex then recruits cholesterol to generate a lipid raft. The interaction induces actin-dependent immobilization of the virus-ganglioside complex, followed by virus-induced invagination of the plasma membrane. An elegant study by Ewers et al., et al., et al., Low-density lipoprotein receptors, also known as cholesterol receptors, are used by several members of the Flaviviridae family, including hepatitis C virus (HCV)  and Niemann-Pick C1-like 1 (NPC1L1) are important entry factors for filovirus . Entry of VSV has been suggested to require the interaction between the viral glycoprotein G and the negatively charged phospholipid phosphatidylserine (PS) on the cell surface  is the least abundant phospholipid in the cell membrane, but it is also one of the most versatile signalling molecules in cells and plays a central role in endosome trafficking and maturation. Differential phosphorylation of PI, regulated by specific PI kinases and phosphatases, results in the formation of different PI phosphate (PIP) species  signalling pathway is one of the most important PI-mediated signalling cascades activated during virus entry. Activation of PI3K and subsequent generation of PIP3 serves as docking platform for proteins carrying lipid-binding domains, including Akt, the main effector of PI3K signalling  provide a scaffold for the viral replication machinery, serve to concentrate the viral and cellular factors needed for assembly of new virions, and provide a protective environment for avoidance of cellular innate immune responses. Examples include the large dsDNA poxviruses which transiently recruit endoplasmic reticulum (ER)-derived cisternae around viral RCs , for instance, recruits PI4K-IIIα to virus replication sites to increase local levels of PI(4)P  for assembly of infectious viral particles  cycle or by manipulating the carbon source used by the cells to generate energy and macromolecules, several viruses take control of central energy metabolism to promote synthesis of cholesterol and fatty acids. This phenomenon has been described for two large enveloped viruses: HCMV  or endosomes  (Ichihashi and Oie, et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Since its inception, viral apoptotic mimicry has proven to be a widespread lipid-mediated entry mechanism used by several enveloped viruses including: pichinde, cytomegalo, lassa fever, lenti, dengue, ebola and Marburg viruses (Callahan The fundamental role of lipids in virus biology and infection is becoming increasingly clear. New technologies, such metabolomics, and advances in mass spectrometry-based lipidomics are allowing for systematic characterization of the alterations in host lipid metabolism, as well as cellular and viral lipid profiles induced by viral infection.As highlighted in this review, lipids serve to orchestrate different stages of viral replication, ranging from entry to spread. Viruses take control of lipid-mediated signalling to co-ordinate viral entry and intracellular trafficking. Later during infection, they actively modify intracellular membrane for replication, re-direct lipid metabolism to produce sufficient membrane for the assembly of new particles, and modify cell membrane lipid content to ensure infectivity of those virions.Systematic characterization of how viruses take control of and alter lipid metabolism is now needed to unravel the common strategies used by these different viruses. Such studies may serve for the identification of infection biomarkers; and together with the development of therapeutics targeting subsets of lipid synthesis enzymes it may be possible to identify novel broad-spectrum therapeutic agents that target virus modified lipid metabolism. Overall, a deeper understanding of how viruses manipulate the host cells lipid program will serve to further our understanding of the cellular mechanisms that govern lipid modification, compartmentalization and metabolism.


The Editorial on the Research TopicImmunogenic Cell Death in Cancer: From Benchside Research to Bedside RealityGarg et al.; .Immunogenic cell death (ICD) has emerged as a cornerstone of therapy-induced antitumor immunity –3. ICD i et al.; ]. The imDerer et al. (Udo S. Gaipl lab). It is noteworthy that ICD can also be achieved by various “smart” combinatorial strategies – an important point for clinically applied non-ICD inducers, discussed in details by Bezu et al. (Guido Kroemer lab).Most potent ICD inducers, characterized so far, elicit danger signaling through oxidative-endoplasmic reticulum stress . SeveralFucikova et al. (Radek Spisek lab). The promising results generated by systemically administered ICD inducers have also paved way for application of ICD-based dendritic cell (DC) vaccines  and lymphoma by Zappasodi et al. (Massimo Di Nicola lab). In the latter case, it is clear that the field is moving toward chimeric antigen receptor (CAR)-T cell’s application, and it will be interesting to see its combination with ICD in near future.Several lines of experimental evidence have established the validity of ICD. However, the overreliance on usage of prophylactic vaccination in transplantable (heterotopic) tumor models has attracted some criticism . While tvaccines . This imKersten et al. (Karin E. de Visser lab). The strategies for targeting epigenetic processes to improve immunotherapy are further discussed by Wachowska et al. (Jakub Golab lab).Nevertheless, the insurmountable complexity of cancer makes it inevitable that in certain contexts, ICD may fail. This failure may stem from various factors, e.g., tumor heterogeneity , MHC-levGarg et al.). This classification paper brings together >50 authors from the fields of ICD and DAMPs, and tries to reach a comprehensive accord on various terminologies related to DAMPs/ICD, the historical background of these concepts, ICD classification system (Type I vs. Type II inducers), and the relevant preclinical/clinical criteria crucial for the field(s) . We hope that this consensus paper will be a useful literature resource for various researchers/clinicians. These contributions, while summarizing the status quo, have also exposed a set of major questions and challenges that still need to be addressed.We believe that the valuable contributions of key researchers/clinicians toward this research topic/special edition have largely fulfilled its primary aim, i.e., to foster a critical discussion on experimental and clinical relevance of ICD. In fact, to further summarize and organize the fields of ICD and DAMPs, we have produced a multi-author consensus paper within this research topic that attempts to classify DAMPs and ICD inducers with an eye on translational potential of ICD  apoptosis-associated immunosuppressive processes?Does the role of ROS in ICD extend beyond a proximal stressor? e.g., ROS-elicited oxidation-associated molecular patterns/OAMPs have been shown to mediate immunogenic potential  cancer mice models  clinical trial.Can ICD withstand the operational/regulatory (GLP/GMP/GCP) hurdles associated with anticancer vaccines-production? [indications for which are emerging (emerging ]Characterizing ICD-resistance mechanisms in the clinic.Characterizing reliable ICD-biomarker(s) detectable in patient tumor/sera-samples.Investigating ICD as a source of robust prognostic/predictive/mechanistic biomarkers [a point investigated recently in some studies  is incontrovertibly valid; but, owing to the incomprehensible complexity of cancer, the “specifics of ICD”  will always remain open to amenability and variations. We envisage that overtime various “variants” of ICD may emerge that differ from each other in a manner dependent upon, the type of anticancer therapy, cancer cell death pathways, cancer-types, tumor antigen make-up, the ADG wrote the manuscript. PA provided senior supervision and critically revised the manuscript.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.


Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by the expansion of a polyglutamine stretch within the huntingtin protein (HTT). The neurological symptoms, that involve motor, cognitive and psychiatric disturbances, are caused by neurodegeneration that is particularly widespread in the basal ganglia and cereberal cortex. HTT is ubiquitously expressed and in recent years it has become apparent that HD patients experience a wide array of peripheral organ dysfunction including severe metabolic phenotype, weight loss, HD-related cardiomyopathy and skeletal muscle wasting. Although skeletal muscles pathology became a hallmark of HD, the mechanisms underlying muscular atrophy in this disorder are unknown. Skeletal muscles account for approximately 40% of body mass and are highly adaptive to physiological and pathological conditions that may result in muscle hypertrophy  or atrophy . The atrophy is caused by degeneration of myofibers and their replacement by fibrotic tissue is the major pathological feature in many genetic muscle disorders. Under normal physiological conditions the muscle function is orchestrated by a network of intrinsic hypertrophic and atrophic signals linked to the functional properties of the motor units that are likely to be imbalanced in HD. In this article, we highlight the emerging field of research with particular focus on the recent studies of the skeletal muscle pathology and the identification of new disease-modifying treatments. HTT) gene which are normally observed in healthy objects. Neurodegeneration, particularly widespread in the striatal nuclei, basal ganglia and cereberal cortex, is a source of neurological symptoms that involve motor, cognitive and psychiatric disturbances  is neurodegenerative disorder caused by the expansion of polyglutamine stretch within the huntingtin protein (HTT)  developed signs of a slowly progressing myopathy with elevated creatine kinase levels many years before first signs of chorea were detected. Muscle biopsy revealed a mild myopathy with mitochondrial pathology including a complex IV deficiency  could be considered as an additional mechanism of HD pathology  transcripts were significantly reduced and defects in mRNA processing were detected (Waters et al., Transcriptional deregulation is a typical feature of HD pathology in the brain (Luthi-Carter et al., To better understand a mechanism underlying muscle wasting in the R6/2 mouse model, key pathways governing protein metabolism, apoptosis and autophagy were examined. R6/2 mice exhibited increased adiposity and elevated energy expenditure without altered food intake. A total protein synthesis was unexpectedly increased in the gastrocnemius muscle by 19%, which was associated with over-activation of rapamycin mTOR signaling (She et al., HTT resulted in increased PGC-1α expression in HD myoblast, while PGC-1α rescue led to increased expression of markers for oxidative muscle fibers and reversal of blunted response for GPA in HD mice (Chaturvedi et al., c oxidase by 15%. Complex I–dependent respiration of HD mitochondria showed more sensitivity to inhibition by Ca2+ than in wild-type mitochondria (Gizatullina et al., Since mitochondrial dysfunction might play a crucial role in HD pathology (Quintanilla and Johnson, As it has been mentioned in the previous paragraph, enhancing PGC-1α activity might be a good strategy to improve skeletal muscles function in HD. Indeed, pharmacologic treatment with the pan-PPAR agonist bezafibrate restored the PGC-1α, PPARs and downstream genes to wild type levels. It also prevented conversion of type I oxidative to type II glycolytic muscle fibers as well as increased muscle mitochondria numbers. Finally, bezafibrate rescued lipid accumulation and apparent vacuolization of brown adipose tissue in the HD mice (Johri et al., in vivo (Fujimoto et al., The other strategy to improve muscle function in HD is based on the heat shock machinery modulation that could suppress mHTT aggregation (Labbadia and Morimoto, Recent studies also showed that HDAC4 function in the cytoplasm (Mielcarek et al., HD is a complex disease that has a peripheral component to its pathophysiology. Transcriptional changes in the HD skeletal muscles were comparable to those observed in the different brain regions and skeletal muscle wasting/atrophy is likely to be an important portion of HD pathogenesis. Some of the molecular and physiological changes in HD muscles can be detected, even in the pre-symptomatic HD individuals. On the molecular level, mitochondrial dysfunctions, PPAR alpha signaling and HSF1 activation were identified as major players in the muscle HD-related pathology. The major pathological pathways identified in skeletal muscles have been summarized in Figure Conflict of Interest Statement: 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.


Adeno associated vectors (AAV) have shown considerable promise to treat various genetic disorders in both preclinical and clinical settings mainly because of its safety profile. However, efficient use of AAV to deliver genes in immune-competent sites like muscles and liver requires very high doses which are associated with concomitant cellular immune response against the viral capsids leading to destruction of the transduced cells. Coupled with that, there are enough evidences that at high doses, AAV particles are subjected to increased cellular phosphorylation/uniquitination leading to proteasome mediated degradation and loss of the viral particles. The presence of preexisting immunity against AAV further adds on to the problem which is acting as a major roadblock to efficiently use it as a gene therapy vector in the clinics. To overcome this, rational bioengineering of AAV capsid becomes a prime tool by which specific amino acid residue(s) can be suitably modified/replaced by compatible residue(s) to create vectors having lower host immune response and higher intracellular trafficking rate. This article reviews the various aspects of rationally designing AAV capsids like by site-directed mutagenesis, directed evolution and combinatorial libraries which can create vectors having not only immune evasive property but also enhanced gene expression and transduction capability. One or more combinations of these strategies have strong potential to create novel vectors which will have suitable clinical efficiency even at a low dose. Despite the reported success it is becoming increasingly clear that humoral and cell mediated immune response against the vector is a major impending factor towards the efficacy of gene therapy [Adeno-associated virus (AAV) is a non-pathogenic parvovirus that has been widely used as a vector of choice for gene therapy. Although the virus has been detected in many different tissues of several animal species  it has n therapy . Preexis therapy ,11.AAV has been used in several clinical trials for both inherited and non-inherited diseases with considerable success Table . In the In contrast to these clinical studies, which targeted immune privileged sites, AAV has limited success when it came to treat monogenic diseases like haemophila B and lipoprotein lipase (LPL) deficiency following intravenous, intrahepatic or intramuscular administration Table . For exaet al., tried to transiently suppress the immune system to inhibit AAV capsid specific T cell response against transduced hepatocytes expressing F.IX transgene in rhesus macaques [One of the major barriers to successful gene delivery with AAV vectors is the humoral immunity to wild type vectors. Humans are natural carriers of AAV genome. Neutralizing antibodies (NAb) to AAV AAV1 and 2) in humans was first reported in the early 60s and 70s ,19. Rece and 2 inmacaques . But no To summarize, immune-suppression can be advantageous as it represses the body’s immune response long enough for the AAV capsid proteins to be not recognized by our defense mechanism thereby preventing NAb formation and allowing readministration of the vector. However this strategy will not be useful to circumvent preexisting NAb against AAV capsid. Thus, alternate strategies like rational capsid modifications must be looked into to evade these neutralizing antibodies.invitro. Extending this finding invivo the authors were able to elucidate a negative effect of tyrosine phosphorylation on viral intracellular trafficking and transgene expression [invitro (~10 fold) and invivo  transduction efficiency of the novel vectors.The ubiquitin–proteasome pathway has been shown to play an essential role in AAV intracellular trafficking ,27 and tpression . Thus baFollowing this finding, tyrosine mutant AAV vectors were used in other target sites like retina , skeletaApart from tyrosines, serines (S), threonines(T) and lysines(K) are also potential sites for phosphorylation and or ubiquitination on the AAV capsids and traditionally mutating them could augment AAV transduction efficiency. It has been shown earlier that targeted inhibition of the serine/threonine kinase phosphorylation of a cellular protein FK506-binding protein (FKBP52), improved AAV mediated gene transfer by 30-fold compared to ~5 fold increase seen with inhibition of tyrosine kinases alone . It is aGoing by this logic, several studies by our laboratory at Christian Medical College, Vellore, India, had generated S/T/K mutants on AAV1, 2, 5 and 8 which has proven to be more efficient in hepatic gene delivery as compared to their wild type counterparts -40. S/T Tenney et al., [In summary, rational point mutations on AAV capsids have shown considerable promise and this field is still wide open to explore especially since we have access to the 3 dimensional (3D) structures of several clinically important AAV serotypes -49. Usin et al.,  could deet al., [et al., [et al., in 1999 [et al., [et al., described a rationally designed chimeric AAV2.5 [et al., [Another approach to create novel AAV variant is to insert known ligands into the AAV capsid thereby allowing retargeting to specific cell types to which the WT vectors have a low affinity . By thiset al.,  where th[et al.,  demonstr in 1999 . In this in 1999 . However[et al.,  generate[et al., . Two new[et al., -59. Bowl[et al.,  describe[et al.,  as well [et al., .in vitro evolution strategy was first described by Grimm et al.,, in 2008 [et al., in 2008 [et al., [in vivo at serum levels which were much higher than what is required to neutralize the wild type vector. Additionally, both the mutants elucidated increased gene expression when compared to the wild type. Using DNA shuffling in an in vitro model of human ciliated airway epithelium, Li et al., [et al., [invivo. In this study they enriched for an AAV variant which showed widespread delivery to the outer retina and reverses the disease phenotypes of X-linked retinoschisis and Leber’s congenital amaurosis in murine models. This vector also efficiently transduced primate photoreceptors from the vitreous.Chimeric capsids based on  in 2008 . In this in 2008  created [et al.,  utilized et al.,  were abl[et al.,  successfet al., [et al., [et al., [et al., [We have seen in clinical trials using AAV that in targeting immune competent sites like liver and muscles, preexisting humoral immunity acts as an impediment to long term gene expression. Thus it necessitates basic knowledge of immunogenic epitopes on AAV capsids to rationally design AAV variants that can evade this immune response. Peptide scanning to map neutralizing epitopes for antibodies against AAV capsid opens up another rational approach to bioengineer AAV capsids. In one such early study Moskalenko et al.,  identifi[et al.,  where th[et al., . These A[et al., . In late[et al.,  which le[et al.,  created [et al.,  and Hutt[et al.,  demonstrAlthough AAV has gained immense popularity as a gene therapy vehicle to treat several genetic disorders, there is still a persistent need to further improve on the vector capsid design and engineering which can bypass the problem of neutralization by preexisting antibodies as well as T-cell mediated immune clearance. Over the past decade, many technologies have been used to make the AAV capsids less immunogenic and more efficient. For example, coating of AAV particles by inert polymers like polyethylene glycol (PEG) has been shown to modestly decrease its immunological properties. Site directed mutagenesis of amino acid residues (S/T/K/Y) on AAV capsids based on their phosphorylation status and presence on B- cell epitope has created novel vectors with reduced antibody response as well as high transgene expression. Rationally creating point mutations does not seem to interfere with their overall safety profile or packaging efficiency when compared to wild type vectors. Thus, it enables us to achieve high gene expression at a low vector dose which will further reduce the chance of eliciting immune response against the viral particles. Also, inserting known peptides at specific sites on AAV capsids can alter the natural tropism of AAV which is extremely helpful for targeted gene delivery at specific organs. Finally directed evolution of AAV can create novel chimeric vectors which can also have reduced neutralizing antibody response along with high target site specificity.An alternate strategy that can be employed in quest of a ‘stealth’ vector is to isolate/screen novel AAV’s from human sources . This st


Listeria monocytogenes and highlights recent discoveries on the molecular mechanisms evolved by this intracellular bacterium to subvert IFN responses.Interferons (IFNs) are secreted proteins of the cytokine family that regulate innate and adaptive immune responses to infection. Although the importance of IFNs in the antiviral response has long been appreciated, their role in bacterial infections is more complex and is currently a major focus of investigation. This review summarizes our current knowledge of the role of these cytokines in host defense against the bacterial pathogen  Listeria monocytogenes is a pathogenic Gram-positive bacillus responsible for a foodborne disease in humans and animals called listeriosis , activation of pathogen-recognition receptors (PRRs) and subsequent production of pro-inflammatory cytokines  production during infection. Type I IFN production by infected cells can be controlled by Listeria multidrug efflux pumps MdrM and MdrT, via the secretion of the second messenger cyclic-di-AMP , Salmonella typhimurium and some viruses  is a paradigm for this, being an important mediator of innate and adaptive immune responses with a key role in clearance of viral and bacterial pathogens and in tumor control. IFN-γ was first described as an antiviral protein , Brucella abortus, Chlamydia muridarum, Francisella novicida, Salmonella enterica, Staphylococcus aureus, and Yersinia pestis , which encode proteins involved in a broad range of cellular functions (reviewed in MacMicking, Listeria infection has mostly been studied in the context of the IFN-γ pathway.The beneficial or detrimental effects of IFNs on Listeria cytoinvasion (Myers et al., Listeria infection by coordinating a potent oxidative and vesicular trafficking program (Kim et al., L. monocytogenes (Sonoda et al., L. monocytogenes (Cole et al., Listeria within granulomatous structures, thus avoiding massive T cell activation (Popov et al., The antilisterial activity of IFN-γ in phagocytic cells involves induction of oxidative and nitrosative defences, via increased expression of enzymes that control production of reactive oxygen and nitrogen species, such as NOX2/CYBB, DUOX2, and iNOS/NOS2 (MacMicking, Listeria infection is less documented. Zwaferink et al. have observed that upregulation of iNOS/NOS2 by IFN-β promotes necrotic death of macrophages (Zwaferink et al., Listeria infection of epithelia is not understood.The function of type I IFN-associated ISGs in L. monocytogenes (Archambaud et al., Of interest, a subset of ISGs is amongst the most induced genes in the intestinal tissue of gnotobiotic humanized mice infected orally with Listeria has evolved several mechanisms to avoid immune detection and evade IFN responses. It has been demonstrated that deacetylation of Listeria peptidoglycan by the deacetylase PgdA confers resistance to host lysozyme, thus preventing release of MAMPs, such as DNA, RNA and lipopeptides, that trigger IFN-β production (Boneca et al., Listeria pgdA mutants are rapidly killed in murine macrophages, which produce lysozyme, and induce a strong secretion of IFN-β compared to wild-type Listeria. The role of PgdA is not limited to the control of type I-IFN production as a pgdA mutant hyperinduces pro-inflammatory cytokines as well. Modification of peptidoglycan by PgdA is an extremely efficient mechanism of immune escape used by Listeria, which correlates with its critical role in virulence.Listeria has evolved a sophisticated strategy to modulate, either negatively or positively, the expression of ISGs in epithelial cells, by targeting a chromatin-repressive complex, BAHD1 (Bierne et al., Listeria infection promotes, albeit via an unknown mechanism, the targeting of BAHD1 at the promoter of a set of ISGs, thereby downregulating type I- and type III-IFN responses. On the other hand, Listeria can produce a nucleomodulin, LntA, which when secreted by intracellular bacteria, enters the nucleus of infected cells, binds BAHD1 and inhibits its function (Lebreton et al., Remarkably, Listeria-host interactions. Yet, our understanding of the immune response to Listeria, and more specifically the role IFNs and of their downstream effectors, is far from complete and often relies on studies performed in cultured cells or in mice. However, murine and human listeriosis differ in many aspects (Lecuit, Listeria in epithelial cells, is not functional for Listeria uptake in the mouse. Thus, the route of entry of Listeria is not strictly the same in mice and humans. Moreover, ISGs induced in response to infection are not identical in mice and humans. Additionally, murine hepatocytes do not respond to type III-IFNs (Hermant et al., L. monocytogenes. Altogether, species-specific differences provide limits to the use of mouse models in characterizing IFN pathways engaged during Listeria infection in humans, especially in key epithelial organs such as the gut, liver and placenta. It will be important to perform future studies using adapted animal models, such as humanized mice permissive to oral infection or transgenic mice with human xenografts (Walters et al., Listeria infection depends on the route and time of infection (Pontiroli et al., Listeria infection in vitro, but the relevant ISGs and their cellular functions remain to be identified. Validation of ISGs identified in cultured cells in adequate in vivo models or deduced from analyses of patient samples, will be required to address the complex role of IFNs and bacterial subversion strategies and provide new insights into Listeria pathogenesis.We have an extensive knowledge of the molecular and cellular mechanisms involved in  Lecuit, . For insThe 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.


S-nitrosoglutathione (GSNO) indicates the involvement of these organelles in the sulfur metabolism. This could suggest the participation of a new family of molecules designated as reactive sulfur species (RSS) which will possibly provide new functions for peroxisomes.Peroxisomes are ubiquitous organelles with a notable oxidative metabolism. In plants, these subcellular compartments have been shown to be involved in the metabolism of reactive oxygen and nitrogen species (ROS and RNS), whose components, hydrogen peroxide and nitric oxide (NO), are important molecules involved in signaling processes. The presence of new elements in plant peroxisomes such as glutathione reductase (GR), sulfite oxidase (SO), glutathione (GSH), and ·−2), hydrogen peroxide (H2O2), and RNS  and GSNO which are characterized by a broad spectrum of physiological/pathological activities. Both these molecular families (ROS and RNS) include radical molecules containing an unpaired electron as well as non-radical molecules and can also have dual effects depending on their cellular concentration. Thus, H2O2 and NO at low concentrations can function as signal molecules in the cell or may cause damage to cell components when overproduced as a consequence of adverse conditions  which does not reflect the complexity of their enzymatic composition  has been shown to be generated from L-cysteine by the pyridoxal-5′-phosphate-dependent enzyme. Thus, endogenous H2S can act as a neuromodulator in rat brain . Thus, high cellular GSH concentrations in an oxidative environment and the decomposition of S-nitrosothiols generate disulfide-S-oxides  is an essential mineral for plant growth and development  . This RNS is a highly oxidant compound capable of catalyzing the conversion of xanthine dehydrogenase to xanthine oxidase . SO catalyzes the conversion of sulfite to sulfate with the concomitant generation of H2O2  (Jiménez et al., In this context, the interactions of ROS, RNS and possibly RSS components in plant peroxisomes open up new challenges and a new area of research to determine the biochemical interactions and potential functions of these reactive species of oxygen, nitrogen and sulfur in peroxisomes, some of which play a very important role as signaling molecules in physiological and phyto-pathological processes (Yamasaki, 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.


Within this physiologic range of RBC turnover, a small fraction of hemoglobin (Hb) is released into plasma as free extracellular Hb. In humans, there is an efficient multicomponent system of Hb sequestration, oxidative neutralization and clearance. Haptoglobin (Hp) is the primary Hb-binding protein in human plasma, which attenuates the adverse biochemical and physiologic effects of extracellular Hb. The cellular receptor target of Hp is the monocyte/macrophage scavenger receptor, CD163. Following Hb-Hp binding to CD163, cellular internalization of the complex leads to globin and heme metabolism, which is followed by adaptive changes in antioxidant and iron metabolism pathways and macrophage phenotype polarization. When Hb is released from RBCs within the physiologic range of Hp, the potential deleterious effects of Hb are prevented. However, during hyper-hemolytic conditions or with chronic hemolysis, Hp is depleted and Hb readily distributes to tissues where it might be exposed to oxidative conditions. In such conditions, heme can be released from ferric Hb. The free heme can then accelerate tissue damage by promoting peroxidative reactions and activation of inflammatory cascades. Hemopexin (Hx) is another plasma glycoprotein able to bind heme with high affinity. Hx sequesters heme in an inert, non-toxic form and transports it to the liver for catabolism and excretion. In the present review we discuss the components of physiologic Hb/heme detoxification and their potential therapeutic application in a wide range of hemolytic conditions.Hemolysis, which occurs in many disease states, can trigger a diverse pathophysiologic cascade that is related to the specific biochemical activities of free Hb and its porphyrin component heme. Normal erythropoiesis and concomitant removal of senescent red blood cells (RBC) from the circulation occurs at rates of approximately 2 × 10 Hemolysis with release of free hemoglobin (Hb) and heme occurs in a wide range of disease states and clinical interventions, including genetic and acquired anemias such as sickle cell disease, burns, extracorporeal circulation and massive blood transfusion  state. The 32-kD Hb dimer readily passes through the glomerulus—if not bound by Hp—and is rapidly cleared by the kidneys , Hb exists as a tetrameric heme containing protein with a molecular weight of 64 kD. Following release from RBCs during intravascular hemolysis, extracellular Hb is in a dynamic tetramer ↔ dimer equilibrium  16-fold faster than tetrameric Hb (kox = 0.015 h−1)  Hb. While oxidized ferric (Fe3+) Hb usually remains below the level of detection in plasma, a large fraction of excreted Hb ultimately appears in the urine in the oxidized ferric state  may accumulate. The large Hb-Hp complex cannot be filtered by the kidney and in vitro translocation across endothelial monolayers is almost completely blocked  stabilizes Hb in a reduced state  oxidation state -6 effector pathway, increases expression of Hp in the liver and many parenchymal and non-parenchymal cells. In many species, Hp is one of the most extensively responding acute-phase plasma proteins. IL-6 has also been reported to enhance expression of CD163 on macrophages, suggesting that enhanced Hb sequestration and clearance capacity are general adaptive responses to infection and tissue injury . Additionally, NO synthase (NOS), may be uncoupled due to oxidation of the essential cofactor BH4, thus leading to the generation of O·−2 in place of NO. Accumulation of ONOO- may further contribute to the reduction of NOS activity and the disruption of the NOS dimer.Hx also appears to support vascular NO homeostasis as demonstrated by studies in heme-overloaded Hx-null mice and mouse models of hemolytic disorders  or lipid peroxides] occur in many disease states, particularly within the context of inflammatory reactions and during cycles of ischemia followed by reperfusion  oxo-ferryl Hb [Hb(Fe4+ = O)] generation, (2) ferric Hb [Hb(Fe3+)] generation, and (3) protein radical generation [·Hb(Fe4+ = O)]  during the peroxidative reaction sequence. However, Hp binding limits secondary reactions of these compounds with internal  and/or external substrates  or it can transfer to susceptible external molecules such as lipoproteins (“extrinsic reactions”). During the “intrinsic” free radical reactions within Hb, a defined peptide hotspot that is located at the α-globin/β-globin interface becomes the primary site where amino acid oxidation occurs. Among these amino acids is the highly susceptible β-chain Cys93, which can be oxidized to cysteic acid, as well as the α-chain Tyr42, which appears to be an important radical hub within Hb  Hb to low-affinity heme acceptors  Hb —the substrate for heme transfer—does not usually accumulate in plasma, even under severe hemolytic conditions  Hb-Hp to accumulate and to reach significant plasma concentrations. In a hemolysis model in guinea pigs, we detected up to 50% of ferric Hb-Hp 24 h after infusion of an Hp bolus for treatment of severe blood transfusion-associated hemolysis. Conceptually, the Hb-Hp complex in plasma may therefore be better compared with a polymerized Hb such as a hemoglobin-based oxygen carrier (HBOC). The intentionally slow clearance rates of HBOCs also allow these substances for significant accumulation of ferric species in vivo. Following infusion of most HBOCs there is no barrier against heme release and the oxidative damage that is associated with heme loss may be a significant component of vascular toxicity that accompanies administration of these therapeutic candidates . Hx sequesters heme in an oxidatively inert conformation in a complex with a 1:1 stoichiometry, until it is cleared by the liver. Heme transfer to more reactive biomolecules is therefore suppressed by Hx. Accordingly, heme-overloaded Hx-null mice show increased oxidative stress in blood vessels, endothelial activation, enhanced inflammation and decreased NO bioavailability  are therefore likely to modulate pathologies of the cardiovascular system such as atherosclerosis or typical vascular complications of hemolytic anemias. Oxidized lipoproteins are an established promoter of atherosclerosis and both Hb and heme can promote LDL oxidation (Balla et al., In humans, an Hp gene polymorphism exists that determines the three major phenotypes: 1-1, 2-1, and 2-2 (Levy et al., in vivo administration studies. These experimental Hp products resulted from industrial efforts to develop phenotype-specific therapeutic Hp products from pooled human plasma. Comparative investigations of two Hp products with predominant Hp 1-1 or Hp 2-2 composition found no significant differences in the intravascular sequestration or renal and vascular short term protection provided by dimeric and multimeric Hp phenotypes in a therapeutic setting of acute intravascular Hb exposure in guinea pigs and dogs (Lipiski et al., In the past, multiple functions of Hp, such as its anti-oxidative and CD163 adaptor functions, have been reported to be phenotype dependent (Asleh et al., Epidemiologic studies have also found controversial associations of Hp genotypes with the prevalence and clinical sequelae of atherosclerosis in the general population and in specific patient populations, particularly those with diabetes mellitus. However, more stratified analysis of several large observational studies now convincingly revealed that the Hp 2-2 genotype/phenotype is associated with increased relative risk of coronary artery disease of up to 10-fold in diabetic patients with a glycosylated HbA1c > 6.5 (Cahill et al., Thus, far, the pathophysiology underlying these associations is unknown. According to studies discussed above, the primary Hb detoxifying functions of Hp 1-1 and Hp 2-2 are comparable and can therefore not easily explain the epidemiologic differences. However, the biologic functions of Hp that have been investigated so far are more representative of the short term protective functions of Hp during acute hemolysis and may not fully reflect longer term antioxidant or immune modulatory functions of the protein that might be more relevant for chronic vascular disease development.Most epidemiologic studies of phenotype association with cardiovascular disease have been controlled for overall population heterogeneity and for established cardiovascular risk factors; however, these studies were not systematically controlled for differences in Hp plasma concentrations (Cahill et al., The controversial findings related to Hp phenotype-specific functions and associated disease state risks should certainly stimulate more extensive investigations. However, it now appears that, for therapeutic considerations, the Hp phenotype selection of a product will likely not be a primary determinant of clinical success (Lipiski et al., A genetic Hx polymorphism has been reported in populations of African ancestry (Kamboh et al., The only available data suggesting a strong association between plasma Hx level and disease risk come from studies evaluating the mouse model of Hx deficiency (Tolosano et al., Mouse models of sickle cell disease have a phenotype of chronic heme-driven endothelial activation and dysfunction that could be recovered by repeated Hx administration (Vinchi et al., Hemolysis has also been recognized to exacerbate some renal and vascular complications related to the transfusion of stored red blood cells. Retrospective clinical observation studies suggested a link between the storage duration of red blood cells and the incidence of cardiovascular complications such as myocardial infarction, stroke and death. One component of the so-called red blood cell storage lesion is an increased RBC fragility, which may lead to hemolysis post-transfusion. In a guinea pig transfusion model, increased fragility of older RBC could be linked to increased hemolysis in the post-transfusion period, appearance of free Hb in the circulation and Hb-triggered injury in the kidney and vasculature. All these pathologic changes could be prevented by Hp treatment at the time of old blood transfusion (Baek et al., Hemolysis with increased free Hb/heme levels and accompanying Hp/Hx depletion can be observed in some patients with severe sepsis. In a mouse sepsis model, Hx administration significantly reduced organ injury and mortality (Larsen et al., The rationale for the use of Hp and Hx as therapeutic agents is based on the idea that they act by scavenging circulating Hb and heme, with particular relevance to pathological conditions associated with hemolysis. Central to this concept is the notion that acute and chronic hemolytic conditions are characterized by depletion of the Hb and heme scavengers. Pre-clinical proof-of-concept animal models have demonstrated that Hp and Hx effectively attenuate Hb- and heme-induced vascular and renal pathologies. Based on these preliminary observations, it would appear rational to develop Hp and Hx for therapeutic use (Boretti et al., 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.


Blood pressure homeostasis is maintained by several mechanisms regulating cardiac output, vascular resistances, and blood volume. At cellular levels, reactive oxygen species (ROS) signaling is involved in multiple molecular mechanisms controlling blood pressure. Among ROS producing systems, NADPH oxidases (NOXs), expressed in different cells of the cardiovascular system, are the most important enzymes clearly linked to the development of hypertension. NOXs exert a central role in cardiac mechanosensing, endothelium-dependent relaxation, and Angiotensin-II (Ang-II) redox signaling regulating vascular tone. The central role of NOXs in redox-dependent cardiovascular cell functions renders these enzymes a promising pharmacological target for the treatment of cardiovascular diseases, including hypertension. The aim of the present review is to focus on the physiological role of the cardiovascular NOX-generating ROS in the molecular and cellular mechanisms affecting blood pressure. Blood pressure is regulated by a dynamic equilibrium of different complex mechanisms Opie, . The maiNOX enzymes are membrane NADPH oxidases with the unique role of producing superoxide anions by one electron reduction of oxygen using NAD(P)H as electron donor  have been identified Lambeth,  Figure  and togeROS have been for long time considered as toxic byproducts of the chemical utilization of oxygen within the cells and oxidative stress has been linked to the pathogenesis of many disorders Sies, . It was Multiple physiological functions have been so far attributed to NOX enzymes. DUOX1 and 2 enzymes, first discovered in thyroid with a role in thyroid hormones synthesis , vascular smooth muscle cells (VSMCs), and adventitial fibroblasts.ECs express NOX1, NOX2, NOX4, and NOX5 , is involved in many vascular processes including vasoconstriction, fibrosis, hypertrophy, inflammation, and aging . For the role of NOX-dependent Ang II signaling in the endothelium see NOXs in the endothelium-dependent relaxation.Ang II induces activation of the enzymatic activity and increases expression of NOXs both in cultured VSMCs and intact arteries  generated by endothelial nitric oxide synthase (eNOS). The liposolubile NO diffuses across the membranes reaching VSMCs, where it increases cGMP levels by activating the soluble guanylate cyclase; the subsequent activation of cGMP-dependent kinases leads to a decrease of intracellular calcium levels and relaxation. Superoxide anions produced by NOXs react with NO to produce peroxynitrite  close to NOX2. Then, ryanodine receptors-2 activation leads to an increase of local cytosolic Ca2+ concentration and force development (Prosser et al., in vitro results in an increase of ROS levels correlated with the amplitude and the frequency of stretch (Prosser et al., in vivo during the normal cyclic stretching and shortening of cardiomyocytes at each heartbeat, where the Ca2+ spark can be dynamically modulated by ROS in dependence of pre-load and heart frequency.NOX-generating ROS contribute to the positive inotropic response to mechanical stretch in cardiomyocytes. Physiological stretch triggers a microtubule-mediated activation of NOX2 localized at t-tubule membranes. This mechanism referred by Prosser et al.  as X-ROS2+ concentration and myocardial contractility due to mechanical stretch, known as Anrep effect. This slow response follows within 1–2 min an increase of the afterload reaching a maximum after 10–15 min. In this case, NOX2-derived ROS mediates Ang II dependent ET-1 release. In cardiomyocytes, Ang II released by mechanical stretch (Sadoshima et al., +/H+ exchanger-1 (Akram et al., +, inhibition of Na+/Ca2+ exchanger, increase of cytosolic calcium concentration and contraction.NOX2 is also involved in the slow enhanced increase in intracellular Ca2+-ATPase, plasma membrane Ca2+ ATPase, L-Type Ca2+ channels and Nav are some examples of molecular target of ROS leading to modulation of intracellular Ca2+ levels linked to myocyte contractility (Sag et al., The role of NOXs in cardiomyocytes are not limited to mechanosensing. ROS produced by NOXs and by other sources such as mitochondria, are able to modulate different kinases phosphorylating proteins involved in calcium signaling; sarco/endoplasmic reticulum CaThe role of ROS in hypertension has been well documented (Lee and Griendling, Numerous studies using ROS scavengers or more specific NOX inhibitors, were aimed at evaluating the role of NOXs in the elevation of blood pressure in hypertensive animals (Lassègue et al., 2O2 could mediate compensatory relaxation acting as an endothelium-derived hyperpolarizing factor (Yada et al., NOX2 elevation is correlated with hypertension. Indeed, in transgenic mice with endothelial-specific overexpression of NOX2, Ang II causes a greater increase in ROS production and attenuated acetylcholine-induced vasorelaxation compared to wild-type (Murdoch et al., Human studies aimed at linking NOX dysfunction with hypertension, have shown that some polymorphisms in the gene encoding p22 phox and affecting enzymatic activity, are associated with hypertension (Zalba et al., Finally, another interesting aspect of NOX involvement in blood pressure homeostasis impairment is related to cigarette smoke, a risk factor of hypertension. Cigarette smoke condensate exposures have been correlated with ROS production, downregulation of enzymatic antioxidant cellular systems and cell toxicity (Russo et al., in vitro experiments. However, due to the low bio-availability, peptide inhibitors are not promising therapeutic tools.NOXs are now being considered as target of pharmacological intervention in patients with hypertension (Cai et al., A number of small molecule NOXs selective inhibitors have been developed. Among them there are NOX1 and NOX 4 selective inhibitors like GKT137831, GKT136901, and GKT901 (Takac et al., Multiple molecular mechanisms regulating blood pressure involve NOX signaling. Of great importance is the central role of NOXs in angiotensin signaling, in the availability of NO and in the cardiac mechanosensing. A general scheme of the main overall effects of NOX-mediated signaling in the cells of the cardiovascular system leading to blood pressure modulation are shown in Figure The current challenge in the NOX biology research field is represented by the better understanding of the mechanisms by which NOX isoforms exert their differential biological effects leading to the development of substances able to modulate specific redox-dependent cell functions.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.