Strengthening and sustainability of national immunization technical advisory groups (NITAGs) globally: Lessons and recommendations from the founding meeting of the global NITAG network.

National Immunization Technical Advisory Groups (NITAGs) provide independent, evidence-informed advice to assist their governments in immunization policy formation. However, many NITAGs face challenges in fulfilling their roles. Hence the many requests for formation of a network linking NITAGs together so they can learn from each other. To address this request, the Health Policy and Institutional Development (HPID) Center (a WHO Collaborating Center at the Agence de Médecine Préventive - AMP), in collaboration with WHO, organized a meeting in Veyrier-du-Lac, France, on 11 and 12 May 2016, to establish a Global NITAG Network (GNN). The meeting focused on two areas: the requirements for (a) the establishment of a global NITAG collaborative network; and (b) the global assessment/evaluation of the performance of NITAGs. 35 participants from 26 countries reviewed the proposed GNN framework documents and NITAG performance evaluation. Participants recommended that a GNN should be established, agreed on its governance, function, scope and a proposed work plan as well as setting a framework for NITAG evaluation.

A niche for adult neurogenesis in social behavior.

The structural and functional changes occurring into the brain is the hallmark of its tremendous capacity for dealing with the complexity that we are facing throughout life. It is also the hallmark of what neuroscientists refer as neuroplasticity. The continuous generation of cohorts of new neurons in some discrete regions of the adult brain, including the olfactory system, is a newly recognized form of neuroplasticity that has been recently the focus of neuroscience studies. Several lines of evidence indicate that this recruitment of newly-generated neurons is extremely sensitive to the overall neuronal activity of the host circuits. Therefore, adult neurogenesis represents, not only a constitutive replacement mechanism for lost neurons, but also a process supporting a capacity of neural plasticity in response to specific experience throughout life. The remarkable complexity of the social life offers a host of daily challenges that require a diversity of brain mechanism to make sense of the ever-changing social world. This review describes some recent findings which have begun to define reciprocal relationships between the production and integration of newborn neurons in the adult brain and social behavior. These studies demonstrate how this domain of research has the potential to address issues in the functional contribution of adult neurogenesis in the expression of some social traits as well in the role of some social contexts to finely regulate the production, survival and integration of adult newborn neurons.

Turning astrocytes from the rostral migratory stream into neurons: a role for the olfactory sensory organ.

Neurogenesis persists within a few restricted areas of the adult mammalian brain, giving rise to neurons that functionally integrate into preexisting circuits. One of these areas, the subventricular zone (SVZ), was believed, until recently, to be the unique source providing the adult olfactory bulb (OB) with newborn neurons. Because of the fact that neuroblasts derived in the SVZ migrate through the rostral migratory stream (RMS) en route to the OB, the existence of candidate neural stem cells within the RMS was long overlooked. Here, we confirm and considerably extend recent evidence for the existence of adult neural stem cells within the RMS, and go on to investigate their proliferative regulation. Specifically targeting RMS-astrocytes with lentiviral vectors encoding GFP, we demonstrate that glial cells in the RMS differentiate into both OB granule and periglomerular interneurons. In addition, ultrastructural analysis unambiguously reveals the astrocytic nature of stem cells in the adult RMS, and patch-clamp recordings demonstrate the functional integration of RMS-derived interneurons into OB circuitry. Proliferative regulation was investigated via two contrasting manipulations: exposure to an odor-enriched environment that enhances candidate stem cell proliferation in both the RMS and SVZ, and chemical lesion of the main olfactory epithelium that increases cell proliferation in the RMS only. New neurons in the adult OB can therefore arise from different neurogenic areas that can be separately regulated.

High-sensitivity analysis of specific peptides in complex samples by selected MS/MS ion monitoring and linear ion trap mass spectrometry: application to biological studies.

Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments in this field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios. They include highly specific identification of proteins present in low amounts, characterization and relative quantification of post-translational modifications such as phosphorylation and S-nitrosylation and species-specific peptide identification.

The how and why of adult neurogenesis.

Brain plasticity refers to the brain's ability to change structure and/or function during maturation, learning, environmental challenges, or disease. Multiple and dissociable plastic changes in the adult brain involve many different levels of organization, ranging from molecules to systems, with changes in neural elements occurring hand-in-hand with changes in supportive tissue elements, such as glia cells and blood vessels. There is now substantial evidence indicating that new functional neurons are constitutively generated from endogenous pools of neural stem cells in restricted areas of the mammalian brain, throughout life. So, in addition to all the other known structural changes, entire new neurons can be added to the existing network circuitry. This addition of newborn neurons provides the brain with another tool for tinkering with the morphology of its own functional circuitry. Although the ongoing neurogenesis and migration have been extensively documented in non-mammalian species, its characteristics in mammals have just been revealed and thus several questions remain yet unanswered. "Is adult neurogenesis an atavism, an empty-running leftover from evolution? What is adult neurogenesis good for and how does the brain 'know' that more neurons are needed? How is this functional demand translated into signals a precursor cell can detect? "[corrected].Adult neurogenesis may represent an adaptive response to challenges imposed by an environment and/or internal state of the animal. To ensure this function, the production, migration, and survival of newborn neurons must be tightly controlled. We attempt to address some of these questions here, using the olfactory bulb as a model system.

Systematic characterization of phosphorylation sites in NFATc2 by linear ion trap mass spectrometry.

Members of the nuclear factor of activated T cells (NFAT) family of transcription factors regulate transcription of genes involved in the function of many different cellular systems. Activity of NFAT proteins is regulated by a complex interplay of phosphorylation events that are still poorly understood. In this work, we take advantage of the high scanning speed of the linear ion trap to develop a method to make a systematic characterization of NFATc2 phosphorylation. The method is based on the simultaneous monitoring of all tryptic peptides that can be detected by MS and contain potential phosphorylation sites. By this approach, we detected six NFATc2 phosphorylation sites by c-Jun NH2-terminal kinase (JNK) in vitro in only one experiment; a further site was also identified by performing digestion in solution. Using this approach, we have also characterized five basal phosphorylation sites in NFATc2 protein expressed in HEK cell cultures. Two of these NFATc2 phosphorylation sites in vivo have not been described before. The simplicity and sensitivity of this technique, which can be applied to any potential modification, makes it particularly attractive for the systematic detection of post-translational modifications of specific target proteins both in vitro and in vivo.

Blockade of NFAT activation by the second calcineurin binding site.

Activation of NFAT transcription factors requires their dephosphorylation by the phosphatase calcineurin (CN). NFATs contain two CN binding motifs: PxIxIT and CnBP-B/CNBR2 (which we call LxVP). Here we carry out a detailed comparative analysis of the CN binding activity displayed by the PxIxIT and LxVP sites from different NFATs. Dose-response CN binding experiments with GST fusion proteins of NFATc1 and NFATc2 showed that NFATc1 binds CN in vitro more efficiently than does NFATc2. This difference in binding appears to be caused by the different CN binding potencies of the corresponding LxVP sites; thus while the LxVPc2 peptide fused to GST did not bind CN, GST-LxVPc1 bound it more efficiently than did GST-PxIxITc1 or GST-PxIxITc2. Furthermore, an NFATc2 chimera protein containing the LxVP motif from NFATc1 interacted with CN much more potently than did wild-type NFATc2. Free peptides spanning the LxVP motifs from NFATc1, c3 or c4 displaced CN from GST-NFATc1 and GST-NFATc2 more efficiently than any PxIxIT peptide. PxIxITc2 and LxVPc1 peptides were each able to cross-compete GST-LxVPc1-CN and GST-PxIxITc2-CN binding. In contrast with PxIxITc2, the LxVP peptide not only blocked CN-NFAT binding but also inhibited CN phosphatase activity in vitro. Furthermore, exogenous LxVPc1 blocked NFATc2 phosphorylation and nuclear translocation in vivo. These results suggest a model in which the different CN binding characteristics of the PxIxIT and LxVP sites enable different NFAT members to influence each others activities in cells where they are co-expressed.

c-Jun N-terminal kinase (JNK) positively regulates NFATc2 transactivation through phosphorylation within the N-terminal regulatory domain.

The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the transcription of cytokine genes and other genes involved in the regulation and function of the immune system. NFAT activity is regulated by the phosphatase calcineurin, which binds and dephosphorylates the NFAT N-terminal regulatory domain, a critical step required for nuclear translocation and transcriptional activity. Here we show that the mitogen-activated protein kinase (MAPK) JNK activates NFATc2-dependent transcription. Mass spectrometry revealed that JNK phosphorylates at least six residues within the NFATc2 regulatory domain in vitro. Transfection of cells with a chimeric construct encoding the GAL-4 DNA binding domain linked to wild-type NFATc2 showed that JNK stimulates the NFATc2 transactivation domain in activated Jurkat T lymphocytes, an effect that is inhibited by dominant-negative versions of JNK. Likewise, the mutation of the phosphorylation sites identified revealed that Thr(116) and Ser(170) are critical for the transactivation of NFATc2 by JNK. In addition, clustered mutation of the SP-conserved motifs of NFATc2 showed that SP1 and SP2, but not SP3, are also important for the inducible transactivation of NFATc2. Furthermore, mass spectrometry analysis of NFATc2-transfected cells indicated that the activation of the JNK pathway results in the in vivo phosphorylation of Thr(116). Our results indicate that, unlike other NFAT members, the transcriptional activity of NFATc2 is up-regulated by JNK. JNK-mediated phosphorylation of NFATs thus appears to play a differential physiological role among NFAT family members.

The linker region joining the catalytic and the regulatory domains of CnA is essential for binding to NFAT.

Calcineurin (CN) is an important regulator of developmental processes and in adults controls the immune response through its regulation of nuclear factor of activated T cells (NFAT). The physical interaction between CN and NFATs is an essential step in the activation of NFAT-dependent genes by calcium signals. Using deletional and substitutional analyses, we have identified a 13-amino acid region within CN that is essential for the interaction with NFAT and with two other CN-binding proteins, AKAP79 and Cabin-1. The interaction of CN with these proteins is selectively disrupted by substitution of specific amino acid residues within this region, indicating that NFAT and other CN-interacting proteins bind differentially to CN. This selectivity suggests that the region identified in CN could be a potential molecular target for immunosuppressive and other therapeutic interventions in diseases involving the CN/NFAT pathway.