By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development.A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of  Age-related differences in clinical susceptibility to infection have been extensively documented, with diminished protective responses and enhanced susceptibility observed in pre-term and term infants, as well as in older adults when compared to young adults –5. This Differences in innate immune responses are thought to contribute to the overall susceptibility observed in neonates and older adults ,6. NeonaThe approaches used to identify age-dependent differences that lead to an increased risk to suffer from infection at the extreme ends of life have been largely balkanized and focused on a few particular components, the choice of which appears to depend on the expertise of a given group of investigators. What has been missing is an unbiased yet comprehensive interrogation of the events that occur in the very young and the very old following recognition of an infectious threat. In addition to our deficiency in knowledge of age-dependent differences in the immune system, little is known about the molecular mechanisms responsible for these differences. Reduced activation of transcription factors such as interferon response factor 3 (IRF3), defects in nucleosome remodeling, and differences in the expression of pattern recognition receptors and signaling molecules (e.g. MyD88) are among the mechanisms that have been proposed to be responsible for the diminished innate immune responses observed in neonates ,14–16.Age-dependent differences in hematopoietic stem cells and in the development of hematopoietic lineages have also been observed, providing one possible explanation for the immune response differences –19. Accoex vivo infectious exposure of defined cell populations, along with the identification of differences in constitutive gene expression in these populations, would be of considerable value.DNA microarrays were previously used to obtain genome-scale insight into age-dependent differences in gene expression following infectious exposure . More reListeria monocytogenes (Lm) infection in cord blood monocytes and in peripheral blood monocytes from young and older adults. LPS provides an example of a well-defined innate immune stimulator; Lm causes suffering and dying in the very old and the very young, while most young adults rarely even display symptoms if infected [In this study, RNA-seq was used to compare the gene expression responses to LPS stimulation or infected . Our datinfected . In contThis study was specifically approved by the Research Ethics Board of the University of British Columbia (Protocol H13-00347). Informed written consent from all enrolled adult participants or the next of kin, care givers, or guardians on the behalf of the cord blood participants involved in our study was obtained for all study participants. Animal research for this study was specifically approved by the UCLA Chancellor’s Animal Research Committee (Protocol 1999-073-53E).6 cells/well. Monocytes were immediately stimulated with LPS (10 ng/ml) (InvivoGen tlrl-eblps) for 0, 1, and 6 hrs, or were infected with Lm at MOI = 5 for 0, 2, and 6 hrs. These time points were selected on the basis of pilot experiments, which showed that they capture the first and second major waves of gene activation in response to LPS stimulation and Lm infection. Wild-type (WT) Lm strain 10403s was provided by Dr. D. Portnoy  and grown as described [Lm experiment, uninfected cells (referred to as 0-hr time point) were collected after culturing without Lm for 2 hrs; in contrast, the unstimulated cells in the LPS experiment were collected immediately after isolation.Samples of cord blood from healthy, full-term elective Caesarean sections without labor and samples of healthy young adult (ages 19–45) and older adult (aged 65 and older) peripheral blood were collected directly into sodium heparin-containing vacutainers (BD Biosciences). Mononuclear cells were isolated by density gradient centrifugation within two hours of blood collection to avoid alterations of cell properties . Positivescribed . For the-/-, or IFNAR-/- mice as described [Mouse macrophages were prepared from the bone marrow of 6-week-old C57BL/6, IRF3escribed ,25, and Human monocyte RNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Strand-specific libraries were prepared using 120 ng RNA input according to the “deoxyuridine triphosphate (dUTP)” method . Mouse mhttp://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). Coexpressed gene classes were evaluated with Cluster3 by applying k-means clustering to mean-centered log2(RPKM) expression values [http://blast.ncbi.nlm.nih.gov/Blast.cgi). Pscan was used to detect DNA motifs overrepresented in each class between nucleotides -450 and +50 relative to the transcription start site [All bioinformatic analyses were conducted using the Galaxy platform . Reads wn values . Statistn values . Mouse oart site .Lm. For the LPS experiments, samples were collected 0, 1, and 6 hrs post-stimulation. For the Lm experiments, samples were collected 0, 2, and 6 hrs post-infection. After mRNA isolation and cDNA library preparation, RNA-seq was performed. The number of mapped reads ranged from 3.4 x 106 to 1.3 x 107 per sample.An attractive starting point toward a full understanding of age-related differences in immune responses is to employ RNA-seq to carefully examine mRNA transcript levels following stimulation or infection of defined cell types. Toward this goal, peripheral blood monocytes were obtained from healthy young adults and healthy older adults. In addition, neonatal monocytes were obtained from umbilical cord blood samples; cord blood is known to consist almost exclusively of neonatal cells . MonocytAn examination of the data sets from the LPS experiment identified 1147 annotated RefSeq genes that were induced by at least five-fold at the 1- or 6-hr time point (relative to the unstimulated sample) in at least one sample from any age group, and that exhibited a transcript level exceeding four RPKM following induction. To examine the relationship between the different time points and age groups in the response to LPS, hierarchical clustering was performed with these 1147 genes . This anSmall age-related differences were also observed with the 6-hr time-point data, in that, with only one exception , each age group clustered separately from the others. In contrast, the nine 1-hr time-point samples correlated closely, with no apparent age-related differences. The Pearson correlation values  used for the hierarchical clustering are shown in Lm data sets identified 865 annotated RefSeq genes that were induced by at least five-fold at the 2-hr or 6-hr time point in at least one sample, and that exhibited a transcript level exceeding four RPKM following induction. The hierarchical clustering results and the Pearson correlation values revealed even stronger correlations between age groups at each time point than were observed with the LPS data  was identified that showed substantial age-related differences . Genes iLm-induced genes also revealed extensive similarities among the three age groups  that also exhibited differential expression between neonates and young adults at a high level of statistical significance (p<0.01). Only 118 of the 1147 LPS-induced genes met these criteria.The 118 genes . 14 of the 16 genes also exhibited reduced expression in Ifnar—/- macrophages (column 10). IRF3- and/or IFNAR-dependence was also observed for most Group V genes for which mouse orthologs were both expressed and induced in mouse macrophages  rather than a reduction in IFNAR signaling .Lm infection were generally found to exhibit IRF3- and/or IFNAR-dependence  exhibited transcript levels that were significantly different (p<0.01) in unstimulated cells from young adults in comparison to older adults (Lm experiment (data not shown), but the number of genes showing differential expression was lower, probably because the unstimulated cells for the Lm experiment were cultured for 2 hrs prior to collection, whereas the unstimulated cells in the LPS experiment were collected without culturing.To evaluate gene expression differences between young adults and older adults, we first used the strategy described above to identify differentially induced genes. This analysis revealed smaller differences in transcriptional induction than were observed when comparing the neonatal and young adult profiles (data not shown), suggesting that the pathways involved in the responses to LPS and r adults . For theImportantly, although relatively large differences in expression between young adults and older adults were observed in the unstimulated cells, the magnitudes of the differences were generally lower after stimulation. This is apparent in ex vivo setting, with subsequent experiments focusing on how these intrinsic differences contribute to clinical observations in the far more complex in vivo setting.The diminished capacity of human neonates and older adults to mount an immune response to infectious agents has been well documented ,2. HowevLm infection. The improved dynamic range of the RNA-seq method in comparison to microarray methods [In this study, RNA-seq was used to examine the intrinsic response of blood monocytes to LPS stimulation and  methods  led to tLm. In the previous study, a large number of IRF3- and Type 1 IFN-dependent genes were found to be expressed at reduced levels in neonates. The reduced expression of these genes was attributed to reduced IRF3 activity because the neonatal and adult cells responded similarly to direct stimulation with IFNβ. Reduced IRF3 activity would lead to a broad reduction in the expression of IFN-dependent genes because IRF3 is critical for the initial induction of IFNB transcription in LPS-stimulated cells.Our results strengthen previous evidence that reduced IRF3 activity makes a major contribution to the deficient innate responses of neonates to infectious stimuli . The prein vitro DNA-binding activity was similarly induced [Lm-infected cells. LPS and Lm activate IRF3 via different signaling pathways–the TRIF pathway for LPS and the STING pathway for Lm [Interestingly, the previous study found that IRF3 translocated to the nucleus similarly in neonatal and adult cells, and its  induced . However induced . An addiy for Lm ,35–suggeIn addition to elucidating the specific mechanism, it will be important to understand why this difference exists between neonatal and adult monocytes. The simplest model is that neonatal monocytes are fundamentally different from adult monocytes and represent a developmentally distinct monocyte subtype. However, this model predicts that prominent gene expression differences would be observed prior to stimulation. The differentially expressed genes would be expected to include cell-surface markers that define different myeloid cell populations and genes that might help regulate IFN responses. Surprisingly, the expression profiles of the unstimulated monocytes from neonates and adults were remarkably similar (data not shown), with no large differences suggesting that they represent different myeloid subtypes, and no differences that would be predictive of the differential induction of IRF3-dependent genes.One possible explanation for this apparent paradox is that the differences between neonatal and adult monocytes are due to the differential expression of micro-RNAs or long noncoding RNAs, which were not examined in this analysis. However, the differential expression or processing of non-coding RNAs would be expected to require the differential expression of transcription factors that regulate the non-coding RNA genes, or the differential expression of processing enzymes; these protein-coding genes would have been included in our analysis. Differences in alternative pre-mRNA splicing also were not examined in our analysis. Once again, differential splicing would be expected to require the differential expression of genes encoding splicing factors. A more likely possibility is that the pronounced difference in the induction of IRF3-dependent genes is regulated by genes whose expression levels vary by only a small and statistically insignificant amount.in vitro [Because the RNA-seq profiles failed to provide evidence that the neonatal and adult cells represent developmentally distinct monocyte subtypes, the neonatal-adult differences may instead be due to environmental differences that act on the fully differentiated cells to influence their capacity to induce IRF3 activity. Such a mechanism would need to influence IRF3’s capacity for induction for a prolonged time period, because the IRF3 difference has been observed in dendritic cells differentiated for several days in vitro . This enTo summarize, the results of this study will help guide future efforts to understand the mechanisms responsible for the immune deficiencies observed in neonates and older adults. The results suggest that the intrinsic properties of blood monocytes are remarkably stable throughout life and vary to only a limited extent. The reduced capacity of neonatal monocytes to activate IRF3-dependent genes could play an important role in the deficient response of neonates to many microbial pathogens. Furthermore, the low-level inflammation that is readily apparent in monocytes from older adults could also influence anti-microbial responses. RNA-seq studies to quantitatively characterize intrinsic age-related differences in other innate and adaptive immune cell types should provide additional insights and should ultimately suggest strategies to enhance immune responses in deficient populations.S1 FigAn expanded version of (TIF)Click here for additional data file.S2 FigLm-induced genes that are differentially expressed in cord blood and young adult monocytes. RefSeq IDs and gene names are shown for human genes and their mouse orthologs.An expanded version of (TIF)Click here for additional data file.S3 FigAn expanded version of (TIF)Click here for additional data file.


Etoposide derives from podophyllotoxin, a toxin found in the American Mayapple. It was first synthesized in 1966 and approved for cancer therapy in 1983 by the U.S. Food and Drug Administration . Starti DNA topoisomerases are essential enzymes that regulate the topological state of the genetic material by introducing transient breaks in the DNA molecule. They are involved in fundamental biological processes such as DNA replication, transcription, DNA repair and chromatin remodeling. The unwinding and rewinding of the double helix, the protein movement along DNA and the coiling of DNA in higher-order structures lead to topological entanglements that are resolved by topoisomerases by enabling topological transformation through two transesterification reactions. A tyrosine in the active site of the enzyme initiates the first transesterification and forms a covalent adduct with the phosphate in the backbone of the DNA molecule, thus generating a transient interruption of the double helix through which topological transformations can occur. The second transesterification reseals the DNA break (re-ligation) and regenerates the free tyrosine. Based on their structure and mechanism of action, topoisomerases are grouped into type I (TopoI) and type II (TopoII) enzymes. TopoI enzymes carry out strand passage through a single-stranded break, while TopoII activities involve the production of a double-strand break . TopoIIThe covalent topoisomerase-cleaved DNA complex, referred to as cleavable or cleavage complex, is a short-live intermediate in this reaction. However, it can be stabilized by several compounds leading to the production of high levels of protein-associated breaks in the genome that are extremely toxic for the cell. Etoposide poisons the TopoII cleavage complexes (TopoIIcc) and inhibits the second step of the reaction (i.e. DNA re-ligation). The recent high-resolution of the ternary complex between TopoII, DNA and etoposide has revealed the elements that are crucial to the stabilization of the cleavable complex. Interactions with specific amino acids of the enzyme are critical for etoposide to enter the TopoII-DNA complex. The active role played by TopoII in promoting and stabilizing the ternary complex is consistent with the notion that the drug by itself displays low-affinity toward free DNA and is a poor DNA intercalator . TopoIIThe issue of isoform specificity has potential clinical implications. For instance, since TopoIIα is not expressed appreciably in quiescent cells, etoposide targeting of TopoIIβ in differentiated tissues, such as cardiac cells, could account for much of the off-target toxicity of the drug . TopoIIMLL) gene on chromosome 11q23 and several partner genes. This translocation appears to be linked to the trapping of a TopoIIcc in the MLL gene . Etoposide-induced t-AML is frequently associated with balanced translocations between the mixed lineage leukemia . In). InMLL)Azarova and colleagues  used sk (MDM2) gene encoding an ubiquitin ligase involved in proteasome-mediated protein degradation. Interestingly, a single nucleotide polymorphism (SNP) SNP309 (T/G) in the gene promoter produces MDM2 upregulation, and results in increased susceptibility to cancer and decreased response to radiation therapy and DNA-damaging drugs including TopoII poisons. Indeed cancer cell lines homozygous for SNP309 are selectively resistant (10-fold) to etoposide, mitoxantrone, amsacrine, and ellipticine. This effect arises from the ability of MDM2 protein to bind and target TopoII for degradation. Knockdown of MDM2 by RNAi stabilizes TopoII and decreases the resistance to TopoII-targeting drugs. Given the frequency of SNP309 in the general population (40 % of T/G heterozygosity and 12 % of G/G homozygosity), this polymorphism may represent a relatively common determinant of drug sensitivity with important implications for personalized cancer chemotherapy .The prolonged presence of TopoII-cleavage complexes and their associated single and double strand DNA breaks interfere with a number of DNA transactions, such as DNA replication and transcription, and must be efficiently removed by the cell. The first step in this process is the displacement of the protein cap from 5'-end of the break in order to make it accessible to DNA repair machineries. Schizosaccharomyces pombe the nuclease activity of the Mre11/Rad50/ Nbs1 (MRN) complex, which is essential for repairing DSBs, removes TopoII from the DNA . This pal., 2009; Yu et aal., 2009), which al., 2009). Howeveels, 2010). These Another mechanism has been discovered by Zhang and colleagues  and inv'-tyrosine phosphodiesterase has been identified for the excision of TopoII-DNA adducts . TTRAP al., 2006)), it haal., 2006; Gao et al., 2006). Notablal., 2006) and raiLIG4-/-) and of the Ku70 subunit of Ku heterodimer (KU70-/-) in chicken DT40 cells, which completely impairs NHEJ, makes the cells extremely sensitive to etoposide. In contrast, DNA repair and recombination protein RAD54-/- knock out cells (defective in HR) are much less hypersensitive. Collectively these results provide evidence that NHEJ is the predominant pathway for the repair of etoposide-induced TopoII-mediated DNA damage . Prolifal., 2003). In agral., 2006). Moreoval., 2006) have real., 2006). Finallal., 2006), reinfoDNA damage induces an evolutionary conserved network of sensors, mediators and effectors, called DNA damage response (DDR), that sense the damage and spread the signal throughout the cell by a signal amplification cascade. As a consequence cell cycle progression is slowed-down or arrested to prevent the duplication and transmission of damaged DNA to daughter cells and to coordinate DNA repair pathways . Single-stranded DNA and DSBs are sensed by specialized complexes that recruit to the lesions and activate two large protein kinases, ataxia telangiectasia and Rad3-related (ATR) and ataxia-telangiectasia mutated (ATM), resulting in the local phosphorylation of the histone variant H2AX . PhosphLong stretches of single-stranded DNA derived by replication stress, are bound by the single-stranded DNA-binding protein replication protein A (RPA) and represent the signal for ATR recruitment. Therefore, while DSBs primarily activate ATM, processing of DSBs by specific endonucleases in S and G2 phases can produce extended single-stranded DNA regions, resulting in ATR activation indicating that both ATM and ATR kinases may be engaged at the same lesion. The local increase in the activity of these kinases causes the activation of two additional checkpoint kinases, Chk2 and Chk1 respectively, that diffuse throughout the nucleus and give raise to signaling pathways that converge on key decision-making factors, such as p53 and the cell-division cycle 25 (CDC25) phosphatases. DDR activation triggers a deep reorganization of nuclear functional compartments with the formation of discrete nuclear foci to which damaged DNA, DNA repair enzymes and auxiliary factors are recruited  patients, result in hypersensitivity to etoposide . In parThe effect on the subcellular distribution of proteins is not limited to the replication compartments. Indeed, using spatial proteomics, a mass spectrometry-based approach for measuring the subcellular distribution of the proteome, Lamond group has recently observed that etoposide treatment of HCT116 cells results in the dissociation of the proteasome from inhibitory proteins in the cytoplasm and its re-location in the nucleus with the ensuing association with proteasome activators .Nuclear functions such as DNA replication and transcription are carried out within a highly organized three-dimensional environment, which starts with the packaging of DNA and associated proteins into chromatin. Post-translational histone modifications (PTMs) by affecting the higher-order chromatin structure directly influence the gene function, either activating or inhibiting transcription depending on the type of PTM, the residue which is modified and the distribution of modified histones along the gene . Among in vivo  is significantly decreased when cells are subjected to pre-treatment with the HDACs inhibitor (HDACi) trichostatin A (TSA). Along this line it is worth noticing that HDACi also induce sensitization to etoposide of multidrug-resistant cancer cells  and histone acetyltransferases (HATs) that also modulate the acetylation/deacetylation status of non-histone proteins, including transcription factors and co-regulators , and Toal., 2000). These al., 2008; Hajji eal., 2008). This eal., 2008). AlthouThe melanoma antigen 1 (MageA1) is one of the first identified tumor-specific antigens . EmergiChromatin organization and chromosome territories may be actually targets of etoposide treatment. In this regard it is worth noticing that repositioning of TopoIIcc could account for the relocation of the ETO/MTG8/ RUNX1T1 gene (located on chromosome 8) in physical proximity to the AML1 gene (on chromosome 21) observed in cycling cells upon etoposide treatment. Rubtsov and colleagues  have sucis acting non-coding RNA. This fascinating finding concerns the dormant allele of UBE3A gene whose mutation causes the neurodevelopmental disorder known as Angelman syndrome. In neuron, the paternal allele of the ubiquitin protein ligase E3A (UBE3A) is intact but epigenetically silenced by an imprinted antisense RNA. Etoposide treatment un-silences the paternal allele reducing the transcription level of the cis acting antisense RNA. This observation, besides suggesting a potential therapy for patient bearing mutations in the UBE3A maternal allele, sheds a new light on additional molecular mechanisms and therapeutic potential of TopoII poisons.Another connection between etoposide, chromatin organization and gene expression has been recently reported by Huang and coworkers . These A further mechanism through which etoposide can down-regulate transcription of specific genes, has been unveiled by Takami and colleagues  that, bEtoposide can affect transcription also through the bridging integrator-1 (BIN1) protein originally identified as a c-MYC-interacting pro-apoptotic tumor suppressor. Several splice variants of BIN1 harbor a MYC-binding domain (MBD), thus retaining the ability to physically interact with and hence to inhibit the oncogenic functions of c-MYC. Krainer group  showed 2+/Mn2+ dependent 1G (PPM1G), a nuclear member of the PP2C family of Ser/Thr phosphatases  of human genes . A protal., 2012). In parIn line with the results of large-scale proteomic analysis .It is known that high etoposide concentrations can trigger caspase-mediated apoptosis which mainly occurs through the cytocrome c/caspase 9 pathway . After There are also evidences of the involvement of a Fas ligand (FasL) pathway in etoposide-mediated apoptosis . EtoposAs stated above etoposide treatment induces the formation of DSBs also in non-replicating cells thus perturbing gene transcription. This has been proven in resting human T lymphocytes where etoposide treatment activates DDR and induces phosphorylation of ATM and of its substrates, H2AX and p53. The final effect is the activation of the pro-apoptotic PUMA (p53 upregulated modulator of apoptosis) protein and of caspases, leading to apoptotic cell death. Intriguingly, in resting cells inhibition of ATM activity with Ku55933 blocks DDR and apoptosis by preventing PUMA expression and caspase activation, while the same protocol increases the cytotoxic effects of etoposide on proliferating tumor cells . This oThe tumor suppressor p53 plays an important role in etoposide-induced apoptosis and is finely regulated by the drug . It hasAutophagy is a catabolic pathway that helps cells to degrade unnecessary or misfolded components and to recycle nutrients during metabolic stress. Although autophagy can be seen as a cellular pro-survival mechanism, it can also promote non-apoptotic programmed cell death . Th). Th52]), that is controlled by the activity of hVps34 kinase and its interacting proteins beclin-1 and p150/Vps35 to form the so called PI-3K (phosphatidylinositide 3-kinase) complex on the membrane of the preautophagosome; enlongation, in which the preautophagosome membrane grows and fuses its extremities thanks to the action of the atg5-atg12-atg16 protein complex. Once the autophagosome is correctly assembled it maturates by fusing with a lysosome giving raise to an autophago-lysosome ; nucleaal., 2008). Macroaal., 2008). Microautophagy is a process involved in the degradation of entire regions of cytosol directly performed by lysosomes through membrane invagination or evagination and without the formation of autophagolysosomes . FinallEtoposide is a well-known trigger of apoptotic pathways but recent findings suggest its involvement in autophagic pathways as well. It is still unclear, however, if the induction of autophagic processes by etoposide causes cell death or plays a pro-survival function. In this regard it is worth noticing that Katayama and colleagues have shown that etoposide induces an autophagy-dependent production of ATP in multiple glioma cells that actually protects cells and may contribute to drug resistance . This iHowever, a study aimed at dissecting the mechanism by which etoposide induces cell death, showed that etoposide treatment of Hep3B hepatoma cells causes not only apoptotic but also autophagic phenotypes. Autophagy inhibition by 3-MA and caspase inhibition by zVAD-fmk effectively decreased autophagic and apoptotic phenotypes, respectively. However, both drugs when used separately only partially prevented cell death indicating that etoposide concomitantly induces autophagic cell death and apoptosis in Hep3B cells . We apologize to colleagues whose work could only be cited indirectly. This work has been supported by grants from the Associazione Italiana per la Ricerca sul Cancro AIRC, the PNR-CNR Aging Program 2012-2014 CNR-MIUR, the Flagship project Epigen CNR-MIUR, the MbMM project 2014-2015 Accordo Quadro Regione Lombardia-CNR to GB. FZ is a student of the PhD program in Genetics, Molecular and Cellular Biology of the University of Pavia and is supported by a fellowship of the MbMM project.


Considering the second-order spectral correlation function of SASE-FEL radiation allows a real-time observation of the photon pulse duration during spectra acquisition. For photon diagnostics at free-electron lasers (FELs), the determination of the photon pulse duration is an important challenge and a complex task. This is especially true for SASE FELs with strongly fluctuating pulse parameters. However, most techniques require an extensive experimental setup, data acquisition and evaluation time, limiting the usability in all-day operation. In contrast, the presented work uses an existing approach based on the analysis of statistical properties of measured SASE FEL spectra and implements it as a software tool, integrated in FLASH’s data acquisition system. This allows the calculation of the average pulse durations from a set of measured spectral distributions with only seconds of delay, whenever high-resolution spectra are recorded. For experiments of this type and for the correct interpretation of the obtained data, the precise knowledge of the pulse duration is essential. Furthermore, many experiments require even a control of these ultra-short pulses during run time. However, at FELs, like the XUV/soft X-ray laser FLASH in Hamburg  or X-ray light sources such as free-electron lasers (FELs) provide ultra-fast pulse durations ranging from a few to hundreds of femtoseconds, a spectral peak brightness which exceeds those of third-generation light sources like storage-ring-based synchrotrons or high-order harmonics of intense laser femtosecond pulses  can be interpreted as spectral coherence which yields a relation to the photon pulse duration  by altering the electron bunch charge over the range 0.2–0.5 nC, while leaving other machine parameters constant. The electron bunch duration was measured using the TDS as described above. The corresponding photon pulse durations calculated by the second-order correlation analysis were significantly shorter than values measured so far (23%). However, recently published results from another SASE FEL  was demonstrated. We have also shown the monitoring of changes in the photon pulse duration while accelerator parameters are changed (e.g. bunch charge). The presented method provides a valuable tool for future investigations of the dependency of different machine operation settings on the pulse duration. While the method works well, there might be limits: in particular, it is unclear how far this parameter range can be extended. Defining these boundary conditions in which the spectral correlation analysis method works reliably and where the assumptions begin to fail is the subject of future work.The second-order correlation analysis of XUV spectra (Lutman


Enterocytes are unique cells governing an array of processes. They are covered by the gut glycocalyx, which is an extraneous carbohydrate-rich coat and an integral part of the plasma membrane. The intestinal glycocalyx and secreted mucins constitute a glycosylated milieu which has a number of physiological and protective functions. One of the important functions of the glycocalyx is its barrier function against microbial adherence to different membrane glycolipids. Thus, the glycocalyx is an important part of the mucosal immune system in newborns. The aim of our study was to identify the carbohydrates in the small bowel glycocalyx of Balb/c mice at different ages. We used plant lectins with different sugar specificities. Fluorescein-labelled lectins binding different carbohydrate moieties were detected using confocal laser scanning microscopy. Biotinilated lectins were used for transmission electron microscopy observations of the constituents of the gut glycocalyx at different periods of postnatal development in mice. Different carbohydrate moieties that were identified in the murine intestinal glycocalyx followed different distribution patterns and characteristics. Carbohydrates present on the mucus surface depended on tissue localization, cell type and stage of development. The distribution and mucins glycosylation could be of interest in analysing the response of the mucosal barrier to intestinal pathogens causing infection or inflammation. Enterocytes are unique cells governing an array of processes. They are covered by a specific coat of carbohydrates responsible for gut colonization – the gut glycocalyx. It plays an important role as a barrier against the attachment of different micro-organisms like bacteria, viruses and protozoa. Thus, the glycocalyx is an integral part of the mucosal immune system in newborns. The distribution of carbohydrates on the luminal surface along the intestine not only depends on the part of the organ, but is also age related.Plant lectins (often named as agglutinins) can bind reversibly to specific monosaccharaides or oligosaccharides. It is considered that the biological function of lectins is to provide defence against different kinds of plant-eating organisms. They may also serve as a depot of nitrogen or as specific recognition factors. At the same time, lectins may interact with glycoconjugates of other organisms. Many stuN-acetyl-D-glucosamine of epithelial cells and M cells.[N-acetyl-D-glucosamine serves as a building block for many oligosaccharides present in maternal milk. It can also facilitate the colonization of the gut by Bifidobacterium species in infants. The glycoconjugates composition of intestinal epithelial cells depends not only on the gut microbial flora, but also on its interaction with dietary constituents altering the mucosal architecture and the activity of endocrine cells.[Wheat germ agglutinin (WGA) is a nontoxic lectin which is derived from wheat flour and is highly resistant against gastric digestion. WGA binds specifically to  M cells. It has b M cells. At the sne cells.,12α-d-mannosyl and α-d-glucosyl residues. It can interact both with intestinal epithelial cells and with macrophages, stimulating their production of NO and proinflammatory cytokines.[Concanavalin A (Con A) is a lectin from Jack bean. It binds specifically glycoproteins and glycolipids containing ytokines.The aim of the present study was to trace age-dependent changes in carbohydrates in the small bowel glycocalyx of Balb/c mice.Balb/c murine intestinal explants from animals of different ages, starting from days 5, 10, 20 and 30, were used. To prepare samples for transmission electron microscopy (Opton EM 109), intestinal explants were blocked with bovine serum albumin (BSA), using a standard pre-embedding protocol, and incubated with biotinilated WGA  or with biotinilated Con A  overnight. Then, streptavidin colloidal gold solution  was added overnight. Small sections of duodenum, jejunum or ileum were embedded in resin (Durcopan) and processed for ultra-thin sectioning. After contrasting, grids were observed under transmission electron microscope at different magnifications. Fluorescein-labelled lectins binding different carbohydrate moieties were detected using fluorescent microscopy or confocal laser scanning microscopy (CLSM). We performed cryo-sectioning of different gut parts from animals at all ages. Ultra-thin cryo sections were fixed in 4% formaldehyde solution, blocked with BSA and then stained with fluorescein-labelled WGA (25 μg/mL) or with fluorescein-labelled Con A (25 μg/mL) overnight at 4 °C. After waAll animal procedures were carried out in accordance with the guidelines of the Animal Ethics Committee at IEMPAM-BAS.The differences in the distribution patterns of sugar residues in the gut lumen could be one explanation for the site-specificity of certain pathogens. GlycoconWGA specifically binds N-acetyl-β-glucosamine oligomers. We detected that colloidal-gold staining of WGA was abundant on the surface of whole villi covering the duodenum, jejunum and ileum of murine small intestine a). Age-rCLSM and fluorescent microscopy  observatTaken together, these results show that the reaction of mannose with Con A was detected from days 10 on the surface of murine enterocytes, whereas N-acetyl-β-glucosamine was constantly present during all days of the examination in all parts of the murine gut. As a next step in our studies, the distribution patterns of fucose, galactose and sialic acid on murine enterocytes are currently under examination.The present study showed that there are differences in lectins binding during the first 30 days of murine life, suggesting a possibility for various micro-organisms to colonize the developing gut. Different carbohydrate moieties were identified in the glycocalyx of murine small intestine. They followed different distribution patterns and characteristics. Since the carbohydrates present on the mucous surface depend on the tissue localization, cell type and the stage of development, the distribution and mucins glycosylation could be of interest in analysing the response of the mucosal barrier to intestinal pathogens causing infection or inflammation. No potential conflict of interest was reported by the authors.


Human T-cell lymphotropic Virus Type I (HTLV-1) is a retrovirus that generates an inflammatory response that can trigger a neurological disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) which appears in 5-10% of infected people around the world. This inflammatory process is due the action of anti-Tax CD8+ lymphocytes that employ cytokines in order to stop the predominant infiltration of infected CD4+ T lymphocytes. IL8 is a pro-inflammatory cytokine that induces the activation and migration of neutrophils towards the sites of infection and it is continuously expressed in HTLV-1 infected cells. The SNP rs4073, located in the promoter of IL-8 gene, has been found associated to some inflammatory diseases and we hypothesized that it could be also a risk factor for the HAM/TSP development. The aim of our study was to evaluate the association of IL8 gene polymorphism SNP rs4073 and the presence of HAM/TSP. The study includes 243 infected subjects between asymptomatic (AC) and HAM/TSP patients. The genotyping of the polymorphisms was performed using the tetra-primer ARMS-PCR (Amplification Refractory Mutation System) method, 37 ancestry informative markers (AIMs) were used to correct by ethnicity and to avoid false positive results due to population stratification effect. The statistical analysis was conducted using the chi-square and Kruskal-Wallis test for possible differences among the groups. Linear and logistic regression analysis was performed using age, gender and ethnicity. The results show that none of the SNP rs4073 genotypes achieved a significant difference between AC and HAM/TSP, neither significant difference was found with proviral load (p>0.05). Our results suggest that the increase of the inflammatory response in HAM/TSP patients may not be caused by the SNP rs4073 in the promoter site of IL8, but the over expression of these gen could be possibly manage by another SNP or any other mutation in the enhancer region.


Primary percutaneous coronary intervention (PCI) has unequivocally proven to be the optimal reperfusion treatment strategy in patients with ST-segment elevation myocardial infarction, whereby timing is simply an issue of the sooner the better. In recent years, it has additionally become apparent that an early invasive approach is also beneficial in patients with non-ST-segment elevation acute coronary syndromes (NSTE-ACS). The definition of early in this subset of patients is, however, ambiguous. Based on risk stratification parameters, high-risk patients (i.e. GRACE score > 140) are preferably subjected to invasive diagnosis and potential PCI within 24 h whereas in low-intermediate risk patients, urgency of coronary angiography is of lesser importance and advocated to occur within a timeframe of 72 h after the initial diagnosis. These guidelines dictate the timeframe in which the invasive approach should be employed at the latest but do not indicate whether an immediate treatment may yield an even better outcome [the Netherlands Heart Journal, Oosterwerff et al. report on the long-term follow-up data of the OPTIMA trial exploring the effects of an immediate vs. deferred PCI in NSTE-ACS patients with coronary anatomy amenable for percutaneous intervention [In this issue of rvention . Even thrvention . Whereasrvention .The debate pertaining to the optimal timing of intervention in patients presenting with NSTE-ACS will therefore continue as results from ongoing trials as the OPTIMA 2 and TRANSIENT are eagerly awaited . In the None.


Aging is associated with changes in the motor system that, over time, can lead to functional impairments and contribute negatively to the ability to recover after brain damage. Unfortunately, there are still many questions surrounding the physiological mechanisms underlying these impairments. We examined cortico-spinal excitability and plasticity in a young cohort (age range: 19–31) and an elderly cohort (age range: 47–73) of healthy right-handed individuals using navigated transcranial magnetic stimulation (nTMS). Subjects were evaluated with a combination of physiological  and behavioral  measures at baseline and following one session of low-frequency (1 Hz) navigated repetitive TMS (rTMS) to the right (non-dominant) hemisphere. In the young cohort, the inhibitory effect of 1 Hz rTMS was significantly in the right hemisphere and a significant facilitatory effect was noted in the unstimulated hemisphere. Conversely, in the elderly cohort, we report only a trend toward a facilitatory effect in the unstimulated hemisphere, suggesting reduced cortical plasticity and interhemispheric communication. To this effect, we show that significant differences in hemispheric cortico-spinal excitability were present in the elderly cohort at baseline, with significantly reduced cortico-spinal excitability in the right hemisphere as compared to the left hemisphere. A correlation analysis revealed no significant relationship between cortical thickness of the selected region of interest (ROI) and MEPs in either young or old subjects prior to and following rTMS. When combined with our preliminary results, further research into this topic could lead to the development of neurophysiological markers pertinent to the diagnosis, prognosis, and treatment of neurological diseases characterized by monohemispheric damage and lateralized motor deficits. Age-associated decline in muscle strength and motor performance has been well documented  paradigms to explore interhemispheric communication  is a tool well suited to aiding in the determination of whether older-adult ipsilateral activation is due to inhibitory signal disruption or active task recruitment. Suppression of motor evoked potentials (MEP) induced by suprathreshold magnetic stimulation has been demonstrated following slow frequency (1 Hz or less) rTMS applied for 5 min or more over the motor cortex . The non-dominant cortex was chosen because the majority of studies demonstrating a difference in ipsilateral motor cortical activation between young and old have made this determination using dominant motor activity  and eight were placed in the old cohort  equipped with a Nexstim 59 mm mean winding diameter figure-of-eight TMS coil and connected to a magnetic stimulator  delivering biphasic pulses. Single and rTMS were both delivered using biphasic pulses delivered through the same coil. During stimulation, surface electromyography (EMG) was recorded and monitored continuously online  using pre-gelled, disposable Ag/AgCl electrodes (10 mm diameter). Active electrodes were attached to the skin overlying the first dorsal interosseus (FDI) muscle and reference electrodes were placed over the metacarpophalangeal joints. The EMG signals were filtered (8-500 Hz), amplified, displayed, and stored for off-line analysis. The TMS system delivered trigger pulses that synchronized the TMS and EMG systems.During TMS measurements, the subjects sat in a comfortable recliner and held their hands supine on their laps. The subjects remained silent during the study to avoid speech-induced modulation of cortical excitability. The subjects were also monitored for drowsiness and asked to keep their eyes open throughout the experiment. Relaxation of the measured muscle was controlled by continuous visual and audio EMG monitoring.Prior to TMS, all subjects underwent a high-resolution T1-weighted structural MRI scan. Imaging data were fed to the navigation software  for automatic 3D brain reconstruction that was used to guide navigation and deliver TMS over M1 (“hot spot”). In each session, the motor cortical output was mapped carefully for the optimal representation of the FDI muscle on both hemispheres Figure .We determined the optimal scalp positions and lowest stimulation intensity capable of eliciting MEPs in the contralateral FDI muscle. According to the recommendations of the International Federation for Clinical Neurophysiology, motor threshold (MT) was defined as the lowest stimulator output intensity that produced at least five MEPs out of 10 consecutive stimuli of at least 50 μ V peak-to-peak amplitude  (short duration of EMG suppression following MEP elicitation) was obtained under resting conditions during a submaximal isometric finger and thumb contraction at intensity of approximately 20% of maximal voluntary contraction (MVC) strength. Specific details on these procedures are provided in previous study , and a suprathreshold test stimulus (TS: 120% of rMT) with two different inter stimulus intervals (ISI: 3 and 12 ms). Ten TS evoked responses were collected for each CS intensity and each ISI condition. Non-conditioned (control) MEP response was determined from ten stimuli at the TS intensity (120% of MT)  were delivered to the right FDI hot spot at an intensity of 120% of rMT to determine baseline MEP amplitude and latency.All subjects then received a train of 1600 stimuli of 1 Hz rTMS at 90% rMT to the right (non-dominant) M1. rTMS was delivered over the optimal site for induction of MEPs in the left FDI muscle. rTMS in the 1 Hz range has previously been shown (in most subjects) to suppress excitability of the motor cortex in the targeted hemisphere and increase excitability in the contralateral motor cortex  was measured according to a previously described protocol that exhibits good validity and test-retest reliability .Subjects were instructed to press the space bar on a common keyboard with their index finger as fast as possible in response to an on-screen stimulus. The time between stimulus onset and response was recorded. Two trials of 20 stimuli each were presented to each participant prior to and after rTMS.http://surfer.nmr.mgh.harvard.edu), which is documented and freely available for download online (http://surfer.nmr.mgh.harvard.edu/). The technical details of these procedures are described in prior publications . Cortico-spinal excitability was assessed by measuring peak-to-peak amplitude of MEPs in the contralateral FDI muscle in response to single TMS pulses. To minimize the variability of TMS-induced single-pulse responses, the largest and smallest MEP amplitude responses were excluded from analysis—no additional single-pulse MEP responses were excluded. Behavioral measures, for the respective hand both before and after rTMS, were averaged across all trials performed by each participant. Additionally, we computed the percent change (%Δ) in MEP amplitude pre- to post-rTMS. As young and elderly populations are independent and a normal distribution cannot be assumed, interhemispheric and pre/post-TMS measures were compared using the nonparametric Wilcoxon signed rank test.Cortico-spinal excitability was assessed by measuring peak-to-peak amplitude of MEPs in the contralateral FDI muscle in response to single TMS pulses. MEP amplitude and SP duration were determined as the mean response of 10 trials in each subject. The percent change in MEP (MEP %Δ) as a result of paired-pulse relative to the non-conditioned response were computed and used in the subsequent analyses. We also computed a MEP defined as (MEP %Δ after rTMS/MEP %Δ before rTMS) for each ISI and each hemisphere, which we used in correlation analysis.p < 0.05. All correlations were performed using Spearman's rank correlation. All statistical analyses were performed using MatLab (version 7.4.0).To compare the distribution of the ages of subjects between the young and elderly groups, we used a two-sample Kolmogorov-Smirnov goodness-of-fit hypothesis test. To compare groups on the binary classification of gender, we used Fisher's exact test. A Mann-Whitney U-test was used to compare between the younger and the older groups for all additional measures. All statistical tests were two-tailed, with statistical significance defined at There were no differences in gender, EHI handedness scores, or MMSE scores between the groups Table .r = −0.154, p = 0.672; after rTMS: r = −0.219, p = 0.544) and old , thus suggesting that there are no differences in cortical thickness confounding the results presented . Moreover, the amplitude of MEP elicited from the right hemisphere was significantly reduced in older subjects  than younger subjects in the non-dominant (right) hemisphere  or right hemisphere  and right hemispheres  and intracortical facilitation (ICF). MEP %Δ pre-rTMS for 3 ms ISI (SICI) was not significant between the young and old cohorts in the left  and right hands . With regards to the 9-hole peg task, there was only a significant difference at baseline (p = 0.043) between the two groups  and finger tapping tasks only Tables , 4. RTs p = 0.037). Conversely, MEPs from the right (stimulated) M1 decreased significantly (p = 0.010) in the young subjects. The effect of rTMS was not significant in either the left or right hemisphere in the old group ; although the pattern of left M1 MEP amplitude increase/right M1 MEP amplitude decrease pattern was present  or right hemisphere or right hemisphere  was not significant between the young and old cohorts in the left  rTMS . As at baseline, this measure showed a significant difference between groups - and after rTMS (p = 0.006).Neither the young nor old cohorts showed any significant changes from baseline with regards to RT with the right (non-stimulated hemisphere) hand (Young: p = 0.008). No effects were found for either hand in either cohort for the finger tapping or 9-Hole Peg tasks.For the grip strength task, there was a significant increases in the right hand of the elderly cohort following stimulation . However, in the left hemisphere, we found a weakly negative, non-significant association  and right  hemispheres . We also observed that older adults had a reduced SP compared to young cohort group.Additionally, we found reduced motor cortex excitability, reduced cortical inhibition, and minimal task-related modulation of corticospinal responses in the non-dominant hand of older-adults as compared to younger subjects. In contrast, we found increased task-dependent modulation of the corticospinal pathway in the dominant hand of older adults. These differences in the corticospinal control in the right and left hands of older adults may be reflective of neural changes brought about by many years of one-hand preference during skilled motor tasks.We also observed increased SICI and a longer SP in older adults. This indicates increased GABAergic inhibition with age under resting conditions. Additionally, we found a decreased ICF in older adults. This could be the result of the inhibitory increase or a wholly separate facilitatory (glutamatergic) decrease.Behaviorally, there were significant increases for both the young and old groups in the RT task in the left  hand following rTMS. This result is consistent with the neurophysiologic findings of a greater inter-hemispheric impact of 1 Hz rTMS .Behaviorally, there were significant increases for both the young and old groups in the RT task in the left  hand following rTMS. This result is consistent with the neurophysiologic findings of a greater inter-hemispheric impact of 1 Hz rTMS. It is worth reiterating that our relatively small sample and unbalanced group sizes likely influenced our results. However, we hope these findings inspire additional and larger research projects within this field.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.


Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) co-infect five million people worldwide. HIV-HCV co-infection represents the major cause of liver related morbidity among the HIV infected patients. The current HCV standard of care consisting of pegylated α-interferon and ribavirin has variable efficacy and considerable side-effects. A major step towards a more efficient and better tolerated therapy has been done by the recent approval of the first direct acting antivirals (DAAs) to enter the clinic. Moreover, there are numerous DAAs which are advanced in clinical trials targeting different steps in the viral life cycle. Due to the variety and efficacy of HCV drugs in the clinic or in the pipeline, there is hope for an interferon free treatment. However, despite the increased efficacy of DAAs with or without interferon and ribavirin, viral resistance still represents an issue. Thus, phenotyping resistance “in vitro” should help the clinician to better personalize the HCV treatment. HCV drug resistance phenotyping has become possible due to the HCV cell culture system.HCV cell culture (HCVcc) system relies on the JFH-1 strain of genotype 2a which is able to replicate in cell culture without adaptive mutations. HCVcc system allows the analysis of every step in the viral life cycle: replication, secretion and infectivity. Clinically relevant chimeric viruses are available covering different HCV genotypes. This allows the “in vitro” evaluation of the viral fitness starting from patient derived viral sequences. We are presenting the complementarity between drug resistance genotyping and HCVcc system in understanding the mechanisms of HCV drug resistance. The use of a drug resistance phenotyping assay for clinical purpose will be discussed.


Protein translation is a fundamental biochemical process and the regulation of this process in response to a variety of changes has been demonstrated to play a key role in cellular functional activity. Recently, the translational control of oncogenes is implicated in many cancers .in vitro and in vivo. In this study, we developed novel computational tools, specifically be tailored to study high throughput ribosome footprint data (Ribo-seq) [We recently reported a translation initiation factor eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of drug silvestrol . Inhibitibo-seq) , to iden4 motif associated with RNA G-quadruplex (GQ) structures s Figure . Our nov


Persistent neural activity has been the focus of neuroscientific research since it was first associated with complex cognitive behaviours. In particular, persistent firing has long been thought to be the neural mechanisms underlying short-term memory encoding and storage . This acPersistent activity elicited in a recurrent network comprising strong excitation in the local circuit has been extensively described in the literature (for a review see ). Howeve2+ currents governing the inward flow of calcium ions in the cell membrane. The intracellular calcium concentration mediates the opening of calcium-activated non-specific (CAN) ion channels [ms 200pA current injection, the neuron is capable of eliciting and maintaining persistent activity with a firing rate of 6Hz for long delay periods (~30s). Moreover, this behaviour is in accord with that displayed in neural recordings of hippocampal slice preparations [20-40Hz and inter-burst frequencies of 3Hz. These results hint towards a possible mechanism for the generation of memory-related oscillatory activity in the hippocampus.Building on previous findings , we modechannels , which carations . Connect


Reelin, a glycoprotein expressed by Cajal-Retzius neurons throughout the marginal layer of developing neocortex, has been extensively shown to play an important role during brain development, guiding neuronal migration and detachment from radial glia. During the adult life, however, many studies have associated Reelin expression to enhanced neuronal plasticity. Although its mechanism of action in the adult brain remains mostly unknown, Reelin is expressed mainly by a subset of mature interneurons. Here, we confirm the described phenotype of this subpopulation in the adult neocortex. We show that these mature interneurons, although being in close proximity, lack polysialylated neural cell adhesion molecule (PSA-NCAM) expression, a molecule expressed by a subpopulation of mature interneurons, related to brain development and involved in neuronal plasticity of the adult brain as well. However, in the layer II of Piriform cortex there is a high density of cells expressing Reelin whose neurochemical phenotype and connectivity has not been described before. Interestingly, in close proximity to these Reelin expressing cells there is a numerous subpopulation of immature neurons expressing PSA-NCAM and doublecortin (DCX) in this layer of the Piriform cortex. Here, we show that Reelin cells express the neuronal marker Neuronal Nuclei (NeuN), but however the majority of neurons lack markers of mature excitatory or inhibitory neurons. A detail analysis of its morphology indicates these that some of these cells might correspond to semilunar neurons. Interestingly, we found that the majority of these cells express T-box brain 1 (TBR-1) a transcription factor found not only in post-mitotic neurons that differentiate to glutamatergic excitatory neurons but also in Cajal-Retzius cells. We suggest that the function of these Reelin expressing cells might be similar to that of the Cajal-Retzius cells during development, having a role in the maintenance of the immature phenotype of the PSA-NCAM/DCX neurons through its receptors apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR) in the Piriform cortex layer II during adulthood. Reelin is a secreted extracellular matrix glycoprotein, identified 20 years ago by the study of the “reeler” mouse  and free access to food and water. All animal experimentation was conducted in accordance with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and was approved by the Committee on Bioethics of the Universitat de Valencia and by the Animal Ethical Committee of Southern Finland.At the age of 4 months old all the animals were deeply anesthetized with sodium pentobarbital (200 mg/Kg). Animals were perfused transcardially; first briefly with saline and then with 4% paraformaldehyde in sodium phosphate buffer 0.1 M, pH 7.4 (PB). The brains were extracted, postfixed for 30 min at 4°C and stored in PB 0.1 M and sodium azide 0.05%. Then, brains were cut in 60 μm thick sections using a vibratome . That same procedure was followed with three CD-1 mice pups at 6 days of age after birth (P6).Six mice were injected with adeno-associated viruses in two different regions of the right hemisphere. First, we injected 1 μL of AAV6-CAG-GFP in the olfactory bulb  targeting the mitral cell layer. We used this virus as an anterograde tracer, since it allow us to follow the projection of these neurons to the Piriform cortex. Those same animals were also injected in the Piriform cortex of the right hemisphere  with 1 μL of AAV5-CMV-mCherry targeting its layer II, to study the morphology of those cells expressing Reelin.Free-floating sections were processed as it follows: after washing with phosphate saline buffer (PBS), non-specific bindings were blocked by 10% normal donkey serum , 0.2% Triton-X100 (Sigma) in PBS for 1 h. Sections were then incubated for 48 h at 4°C with different primary antibody cocktails diluted in PBS—0.2% Triton-X100 . At least three different coronal sections were analyzed per animal between +0.5 and −2.0 mm Antero-Posterior relative to Bregma. From each section 20 cells were analyzed in the Piriform cortex, while in the neocortex all Reelin expressing cells of each stack where analyzed to obtain the percentages describing the neurochemical phenotype, expressed over the total of Reelin expressing cells.To analyze the animals injected with different adeno-associated viruses, we first obtained single confocal planes at each injection site to confirm the correct location of each injection. Then we obtained z-stacks in the Piriform cortex to study the dendritic arbor of dendrites expressing mCherry and axons expressing green fluorescent protein (GFP).In the mouse neocortex, Reelin-expressing cells were found scattered in all layers, although with a higher density in the Layer I Figure . When anWe then analyzed whether these Reelin expressing interneurons would also express the PSA-NCAM. We found that the vast majority of Reelin expressing interneurons do not express PSA-NCAM (4.1 ± 2.5%), although in very few cases we found a very weak PSA-NCAM expression in Reelin expressing interneurons Figure .In the Piriform cortex, Reelin-expressing cells were densely located in the layer II, being scarce in the layers I and III Figure . We founThen, we analyzed the expression of the glial fibrillary acidic protein (GFAP). We found out that none of these Reelin expressing cells expressed GFAP, therefore we discarded the possibility that these neurons would correspond to glial cells Figure .We also analyzed the expression of immature neuronal markers. We found that none of the Reelin expressing cells were immunoreactive for PSA-NCAM, the A3 subunit of the CNGA-3 ion channel Figures  or DCX .However, we found that these Reelin expressing cells in the Piriform cortex express the Neuronal Nuclei protein (NeuN) even if in some cells this expression is faint Figure .We analyzed the connectivity of these neurons by transfecting mCherry under a generic promoter (CMV) to the Piriform cortex combined with Reelin immunohistochemistry Figure . We obseWe analyzed the expression of the neuron-specific transcription factor T-box brain 1 (TBR-1) as it is expressed by neurons of pallial origin and by Cajal-Retzius neurons during brain development . We confirm that Cajal-Retzius cells in the marginal zone of the neocortex express both Reelin and TBR-1 Figure . Interespseudo-radial glia that expresses PSA-NCAM  and very low density lipoprotein receptor (VLDLR), on the surface of PSA-NCAM expressing neurons in the layer II Figures . These rIn the present study, we describe in detail the neurochemical phenotype of Reelin expressing cells in the neocortex and the Piriform cortex in the adult mouse brain. In the neocortex we confirm a widespread distribution of Reelin positive cells among all layers of the neocortex, confirming previous results based in the study of Reelin expression in the adult rodent brain (Alcántara et al., Reelin is expressed widely during development but is restricted to a subpopulation of neurons in the adult brain (Pesold et al., Similar to Reelin, PSA-NCAM is expressed by inhibitory neurons in the adult neocortex and it has been suggested to play an important role in adult brain plasticity as well (Nacher et al., In the present study, we also analyze the nature of Reelin expressing cells in the Piriform cortex, which has never been studied in detail before. We found Reelin positive cells scattered in the layers I and III and, as previously described, a large density of these cells packed in the layer II (Alcántara et al., Since the neurons in the layer II lack expression of GAD67 and the large number of cells present there, it was previously assumed these neurons would correspond to excitatory neurons. However, the neurochemical phenotype of these neurons has not ever been investigated. We found that Reelin expressing neurons of the layer II displayed indeed a divergent neurochemical phenotype: most of the Reelin expressing neurons of the layer II expressed neither GAD67 nor GluR2/3. Only few of these neurons express GluR2/3; a marker of excitatory neurons (Leranth et al., To explore further the phenotype of these Reelin expressing cells we discarded the possibility that these neurons would correspond to glia, and in fact our results showed that there was no colocalization with GFAP. Then we considered whether Reelin expressing cells might correspond to the immature neurons described in the layer II of paleocortex (Nacher et al., The presence of cortical guide scaffolds expressing PSA-NCAM resembling radial glia intuitively suggests a pathway from subventricular areas to the Piriform cortex as a diversion from the elbow of the rostral migratory stream as suggested previously (Shapiro et al., Considering these results, we decided to study the expression of the neuron-specific transcription factor TBR-1, a molecule found on pallium-derived neurons committed to differentiate into excitatory neurons and in Cajal-Retzius cells (Hevner et al., Altogether our results indicate that Reelin is expressed in the Piriform cortex layer II by different subpopulations of neurons including, but not limited to, semilunar glutamatergic neurons and interneurons. These results also suggest that Reelin might be involved through ApoER2 and VLDL in the maturation stage of the subpopulation of immature neurons located in the Piriform cortex layer II. Future experiments controlling the secretion of Reelin will be needed to understand yet the physiological role of Reelin in the adult brain.RG designed the study; RG, HC and LR-E performed the experiments; RG and HC analyzed the data; RG, EC and JN wrote the manuscript.This study was supported by ERC Grant No 322742—iPLASTICITY, Sigrid Juselius foundation and Academy of Finland Grant #257486. The Spanish ministry of economy and competitiveness: BFU2012-32512 and MICINN-PIM2010ERN-00577/NEUCONNECT and Generalitat Valenciana: PROMETEO2013/069.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.


Pressure waves distributed through the aorta by left ventricular contraction propagate at a rate known as the pulse wave velocity (PWV). PWV is a measure of aortic stiffness, and increased PWV has been correlated with increased coronary artery disease risk. PWV can be measured by velocity-encoded magnetic resonance (PC-MR), with velocity profiles collected at a minimum of two locations along the vessel. The commonly used simple linear regression method computes PWV from detected wavefronts in each profile and distance along the aorta. Recognizing the limitation imposed on wave front detection by simple maximum impulse by noise in the signal producing local maxima, we designed and tested a template-matching scheme to improve PWV estimation.In vivo CMR was performed on a 3T scanner  in perimenopausal women with a variable number of atherosclerosis risk factors but no evident atherosclerotic disease. The derivation cohort consisted of 12 scans (4 patients over 3 time points) and validation cohort consisted of 139 scans PC-MR was acquired in an oblique sagittal plane through the descending aorta with in-plane velocity encoding in the head-foot direction and prospective ECG triggering . Distance from the base of the aorta and velocity were collected along the centerline of the aorta. Wave detection was performed at each cross section by the following scheme. A generalized logistic function f = -Vmax+2Vmax/[1+exp{-a(t-c)}], c = tmax+ln(1/.99-1)/a, the center point, where Vmax is the maximum velocity, t is time, tmax is the time of the observed Vmax, and a was optimized to maximize the goodness of fit between the template and observed data between c and tmax. An example fit varying the control parameter, a, is shown in Figure The proposed method for estimation of pulse wave velocity yielded a 53% improvement in overall goodness of fit (p < 0.0001) when compared to estimations using maximum impulse detection Figure .A template-matching scheme provides more robust estimation of aortic PWV compared to maximum impulse detection. This approach warrants incorporation into longitudinal studies of cardiovascular risk and aortic pathophysiology to improve reliability of PWV as an outcome measure.NIH R01HL095563.


Lactobacillus rhamnosus CRL1505 to modulate the TLR3-mediated immune response in the respiratory tract and to increase the resistance to RSV infection. In addition, we review the role of interferon (IFN)-γ and interleukin (IL)-10 in the immunoregulatory effect of the CRL1505 strain that has been successfully used for reducing incidence and morbidity of viral airways infections in children.Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants and young children. Host immune response is implicated in both protective and immunopathological mechanisms during RSV infection. Activation of Toll-like receptor (TLR)-3 in innate immune cells by RSV can induce airway inflammation, protective immune response, and pulmonary immunopathology. A clear understanding of RSV–host interaction is important for the development of novel and effective therapeutic strategies. Several studies have centered on whether probiotic microorganisms with the capacity to stimulate the immune system (immunobiotics) might sufficiently stimulate the common mucosal immune system to improve defenses in the respiratory tract. In this regard, it was demonstrated that some orally administered immunobiotics do have the ability to stimulate respiratory immunity and increase resistance to viral infections. Moreover, during the last decade scientists have significantly advanced in the knowledge of the cellular and molecular mechanisms involved in the protective effect of immunobiotics in the respiratory tract. This review examines the most recent advances dealing with the use of immunobiotic bacteria to improve resistance against viral respiratory infections. More specifically, the article discuss the mechanisms involved in the capacity of the immunobiotic strain  Paramyxoviridae, and a highly contagious virus. Significant epidemiological studies have characterized RSV to be a relevant human pathogen that causes a major health burden worldwide .The first isolation of human respiratory syncytial virus (RSV) was performed in 1955 from a captive chimpanzee. The virus was quickly identified as a major respiratory pathogen in infants and children  might stimulate the common mucosal immune system to improve respiratory tract defenses. In this regard, it was demonstrated that some orally administered immunobiotics do have the ability to stimulate respiratory immunity and increase resistance to viral infections. During the last decade, scientists have significantly advanced in the knowledge of the cellular and molecular mechanisms involved in the protective effect of immunobiotics in the respiratory tract.Lactobacillus rhamnosus CRL1505 to beneficially modulate the immune response triggered by Toll-like receptor (TLR)-3 activation in the respiratory tract and to increase the resistance to RSV infection. In addition, we will discuss the role of interferon (IFN)-γ and interleukin (IL)-10 in the immunoregulatory effect of the CRL1505 strain that has been successfully used for reducing incidence and morbidity of viral airways infections in children . Recognition of viral PAMPs is achieved by cellular receptors known as pattern recognition receptors (PRRs) that are expressed in both respiratory epithelial cells and immune cells. PRRs sensors include the TLRs; C-type lectin receptors and; RNA-sensing RIG-I-like receptors (RLRs) including melanoma differentiation-associated protein 5 (MDA5) and, retinoic acid-inducible gene I (RIG-I) .Double-stranded RNA (dsRNA) is a replication intermediate of several virus that is able to sensitize innate immune system through TLR3. dsRNA is observed during most RNA virus replications like RSV. The important role of TLR3 in anti-viral immunity has been experimentally proved using TLR3 knockout mice and an artificial dsRNA, the synthetic dsRNA polyinosinic–polycytidylic acid [poly(I:C)]. TLR3-deficient mice have been found to have their anti-viral immune response impaired in challenge-experiments with dsRNA or poly(I:C) . Then, TRespiratory syncytial virus predominantly infects primary airway epithelial cells, but can also infect other structural airway and immune cells. Upon viral entry and activation of signaling complexes including TLR3 Figure A 6, 9),, 9,6, 9)Proliferation and activation of NK cells, as well as its anti-viral capacities are also important for the protection against RSV. An emerging trend born from multiple clinical studies of severely RSV-infected infants is a failure to generate a robust NK-cell response –13. In aIn addition, recent studies demonstrated an important role for macrophages in providing an immediate pro-inflammatory response , and proVirus elimination and the recovery from primary viral respiratory infection are primarily mediated by the adaptive immune response. Both cellular and humoral immune responses act directly to eliminate viral pathogens in the respiratory tract Figure .hiMHC-IIhi and plasmacytoid DCs (pDCs) that are CD11cintB220+. In addition, cDCs can be further divided into CD11b+CD103− cDCs (CD11b+ cDCs) and CD11b−CD103+ cDCs (CD103+ cDCs) (+ and CD103+) have essentially different locations within the lung tissue. CD11b+ cDCs are located in the parenchyma of the lung and they promote the recruitment of leukocytes through the production of pro-inflammatory chemokines. In contrast, CD103+ cDCs are located in the basal lamina and they are able to extend dendrites into the airway lumen, allowing them to sample potential foreign pathogens from the airway.The interaction of RSV with respiratory DCs results in activation and maturation of those cells, being both processes important in establishing virus-specific immunity. The quality and durability of the host immunity as well as the susceptibility to reinfection are significantly influenced by these early events during the initial immune response . Respira3+ cDCs) . Both po+ cDCs when compared with parenchymal CD103+ DCs populations. Whereas the number of CD11b+ cDCs increases, the total number of CD103+ cDC decreases and remained low during the course of the RSV infection  strains can exert their beneficial effect on the host through their immunomodulatory activity. These strains have been termed immunobiotics . AlthougThere are several lines of evidence that orally or nasally administered immunobiotics are capable of improving resistance against viral infections in the respiratory tract. Different aspects of respiratory anti-viral immunity can be beneficially modulated by immunobiotics, including the production of type I IFNs, the activity of NK cells, the generation of Th1 responses as well as the production of specific antibodies, and the regulation of inflammatory-mediated lung injury  cells. In addition, both IFN-γ and TNF-α were augmented in MLN cell cultures from mice receiving L. casei Shirota intranasally. These changes in MLN’s cytokine profile, induced by the immunobiotic treatment, explain the improvement of NK cells stimulation and the enhancement of the Th1 response  involved in the beneficial effect. Then, we evaluated the effect of the oral administration of L. rhamnosus CRL1505 on respiratory anti-viral immunity triggered by TLR3 activation. For this purpose, we used infant and adult BALB/c mice and the nasal administration poly(I:C) that is and artificial dsRNA analog and TLR3 ligand, to induce lung inflammation. This mice model allows us to imitate functional alterations and pro-inflammatory consequences of RNA viral infections in the lung. We showed that after nasal administration of poly(I:C) to BALB/c mice there was an increased inflammatory cell recruitment into the lung and production of pro-inflammatory cytokines, that were accompanied by a marked impairment of lung function  increases TLRs and transcription factors expression and induces the secretion of multiple cytokines and chemokines . IL-10 would be valuable for attenuating TLR3-mediated inflammatory damage in the lungs. Consequently, L. rhamnosus CRL1505 treatment could be used to beneficially modulate the balance between pro- and anti-inflammatory cytokines, allowing a reduction of lung tissue damage through an effective regulation of the inflammatory response.Host’s inflammatory response has to be tightly regulated during acute viral lung infection. A regulated inflammatory response enables pathogen elimination without the detrimental effects of inflammation on the delicate lung tissue where gas exchange is produced. Therefore, an appropriate balance between pro-inflammatory and anti-inflammatory factors is crucial for an effective and safe response against RSV. In fact, it was described that excessive IL-10 production can induce a delayed virus clearance while exuberant production of TNF-α/IL-8/MCP-1 can lead to increased immunopathology . During injuries . MoreoveL. rhamnosus CRL1505-treated mice could be related to the increased activity lung DCs that are able to augment CD3+CD4+IFN-γ+ T cells numbers. In addition, we found increased levels of CD11bhigh and CD103+ DCs in lungs of L. rhamnosus CRL1505-treated mice after challenge with poly(I:C). Moreover, an improved MHC-II expression was found in both DCs populations when compared with controls. However, only CD103+ DCs showed higher production of IL-12 and IFN-γ in L. rhamnosus CRL1505-treated mice  challenge . The higted mice . In linehan IL-4 .L. rhamnosus CRL1505 could be useful as a prophylactic agent to control viral respiratory virus since this probiotic strain is a potent inducer of anti-viral cytokines. However, further research was needed to conclusively demonstrate the protective effect of the CRL1505 strain against real viral challenges. Therefore, we next examined whether oral administration of L. rhamnosus CRL1505 was able to reduce the susceptibility of infant mice to RSV infection. We demonstrated that oral administration of L. rhamnosus CRL1505 contributed to a significant decrease of RSV titers and lung tissue damage after the challenge with the respiratory pathogen  involved in the immunoprotective capacities of  changes . On the  CRL1505 . These s CRL1505 . Furthern of DCs  and macrn of DCs . Addition of DCs   or RSV challenges. On the other hand, priming with L. rhamnosus CRL1505 effectively improved levels of IFN-γ and IL-10 in the respiratory mucosa /RSV challenges. In addition to the CRL1505 strain, we also evaluated al cells , 82. Oury mucosa .L. rhamnosus CRL1506 had a significant effect on epithelial cells from the respiratory tract. It is known that type I IFNs increase the expression of genes that are involved in innate anti-viral defenses and the development of a strong Th1 response. Therefore, L. rhamnosus CRL1506, through the stimulation of anti-viral defenses in epithelial cells, could play a significant role in the improvement of innate and specific immune responses against respiratory viral infections . Both lactobacilli augmented IL-10 production in response to TLR3 activation, however, L. rhamnosus CRL1505 was more efficient than CRL1506 to increase the levels of this cytokine in the lung. Additionally, the markers of lung damage were lower in CRL1505-treated mice than in those receiving L. rhamnosus CRL1506  challenge . This finding also supports the idea that modulation of IL-10 is an effective way to improve the outcome of RSV disease. In addition, our results demonstrated that the nasal priming with immunobiotics is an interesting alternative to achieve the immunoprotective effect in the respiratory tract; since virus titers and lung alterations were significantly lower in mice nasally treated with L. rhamnosus CRL1505 than in those fed with the bacteria  and viable L. rhamnosus CRL1506 but not the heat-killed CRL1506 strain reduced lung damage by increasing IL-10 concentrations. These results suggest that the immunoregulatory effect of some probiotic bacteria can be changed after heat treatment. Therefore, not all heat-killed bacteria derived from immunobiotic will maintain their immunoregulatory capacities. This fact should be considered when selecting non-viable immunobiotic strains  administration of immunobiotic lactobacilli. Moreover, those treatments are able to increase the resistance to RSV challenge in both infant and adult hosts. We also showed that the anti-viral capacities of immunobiotic lactobacilli are strain dependent, as it has been reported for other probiotic effects. Comparative studies using two e origin , 80 alloe origin .L. rhamnosus CRL1505 or its cellular fractions as immunomodulators and anti-viral adjuvants in immunocompromised hosts.Our research also demonstrated that anti-viral respiratory defenses are beneficially modulated by heat-killed immunobiotics. This implies that non-viable immunobiotics could be an interesting alternative as mucosal adjuvants to improve respiratory defenses and protect against viral infections. The use of non-viable immunobiotics or their cellular fractions could have an important impact in the prevention of viral respiratory infections in immunocompromised hosts in which the use of live bacteria might be dangerous. In addition, heat-killed immunobiotic could have several technological advantages such as easier storage, and transportation and a longer product shelf-life. Therefore, an interesting topic for future research would be the evaluation of non-viable 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.


Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpes virus 8 (HHV-8) is one of the several carcinogenic viruses that infect humans. KSHV infection has been implicated in the development of Kaposi’s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman’s Disease. While KSHV infection is necessary for the development of KSHV associated malignancies, it is not sufficient to induce tumorigenesis. Evidently, other co-factors are essential for the progression of KSHV induced malignancies. One of the most important co-factors, necessary for the progression of KSHV induced tumors, is immune suppression that frequently arises during co-infection with HIV and also by other immune suppressants. In this mini-review, we discuss the roles of co-infection with HIV and other pathogens on KSHV infection and pathogenesis. Kaposi’s sarcoma-associated herpes virus (KSHV), the latest member of the human herpes virus family has been classified as a direct carcinogen by the International Agency for Research on Cancer  because in vivo and in vitro cell tropism but CD19+ B cells appear to be the primary target for long-term viral latency , a count that defines patient’s progression into AIDS. Following the introduction of combination antiretroviral therapies (cART), which are very effective in controlling HIV-1 replication and in restoring CD4+ T cell counts in HIV-1 co-infected patients, incidences of KS in HIV-1 infected populations have substantially declined but not quite eradicated  is a similar inflammatory condition arising as a result of severe systemic lytic reactivation of KSHV. It is associated with high systemic viral load and excessive cytokine production, notably, elevated levels of interleukin 6 (IL-6), and interleukin 10 (IL-10) . These fKaposi’s sarcoma-associated herpesvirus oncogenicity can be ascribed to its astounding capacity to encode several genes and miRNAs that target cell survival, apoptosis, and tumor suppressor pathways  is a small cationic peptide that can penetrate a variety of cells . It has While immune suppression associated with HIV infection appears to be one of the most important aspects of HIV’s capacity to induce tumors, reports suggest that it is probably not the only mechanism that favors KSHV driven oncogenesis . For exain vivo and in vitro  and endothelial cells (KSHV). Direct molecular interactions between KSHV and EBV can occur during chronic viral infections of B-lymphocytes, a natural latency target for both of these oncogenic viruses. While KSHV infection is a pre-requisite for PEL development and EBV infection is not, it is interesting to note that nearly 70% of PEL cell lines are co-infected with EBV. Both of these viruses may make the cellular microenvironment more hospitable for each other and direct or indirect interactions between them may influence pathogenesis of PELs  target crucial cellular pathways including apoptosis, cell-cycle control and immune-modulation pathways whose deregulation is important for long-term viral persistence of both KSHV and EBV . InteresIn addition to promoting EBV latency, KSHV infection can also enhance the infectivity of EBV indicating that EBV co-infection may confer survival advantages to KSHV. In KSHV infected B-lymphocytes, K-RTA has been shown to increase the infectivity of EBV through the upregulation of CD21 expression, a receptor for EBV entry .in vivo interactions between these two viruses, although not discovered yet, are likely to exist. Presence of HCMV has been detected in KS lesions and experimental evidences suggest that HCMV co-infection can induce KSHV lytic replication. Co-infection of HCMV in experimental systems is known to activate KSHV lytic replication cycle in a variety of cells including endothelial cells, fibroblasts, and keratinocytes  signaling (N-methyl transferases-EZH2 (enhancer of zeste homolog2) and SUV39H1  of a Gram-negative bacterium, P. gingivalis have been shown to enhance the infectivity of KSHV in nearby oral cells and also influence the expression of viral genes. These PAMPs of oral pathogens may upregulate cellular receptors for KSHV entry, increase production of reactive oxygen species (ROS), and modify intracellular signaling such as MAPK and NF-κB pathway, and consequently affect KSHV entry and/or gene expression , family of parasite encoded proteins that are expressed on the surface of IRBCs and their cognate host receptors expressed on the surface of endothelial cells. CD36 is the main host endothelial cell receptor that mediates the adherence of IRBCs . Additional epidemiological and experimental studies regarding interactions between KSHV and its frequently co-infecting microbes can impart better knowledge of underlying microbial synergy and help in defining new strategies for superior management of KSHV induced pathologies. It is becoming increasingly important to detect co-infecting pathogens and consider synergistic polymicrobial interactions while treating patients for a particular disease, as co-infecting pathogens can impact the overall outcome of the associated pathogenesis through a variety of mechanisms.Kaposi’s sarcoma-associated herpesvirus induced pathologies are multi-factorial diseases emerging from extremely complex interactions involving KSHV, countless host factors, and polymicrobial co-infections. While enormously complex interplay of multiple microbial interactions and how co-infected host responds to these interactions with regards to KSHV associated cancers is not well understood, it is clear that co-pathogenic infections have a potential to affect KSHV infection and pathogenesis (ST and SV wrote this review.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.