Pathogen infection triggers complex molecular perturbations within host cells that results in either resistance or susceptibility. Protein acetylation is an emerging biochemical modification that appears to play central roles during host–pathogen interactions. To date, research in this area has focused on two main themes linking protein acetylation to plant immune signaling. Firstly, it has been established that proper gene expression during defense responses requires modulation of histone acetylation within target gene promoter regions. Second, some pathogens can deliver effector molecules that encode acetyltransferases directly within the host cell to modify acetylation of specific host proteins. Collectively these findings suggest that the acetylation level for a range of host proteins may be modulated to alter the outcome of pathogen infection. This review will focus on summarizing our current understanding of the roles of protein acetylation in plant defense and highlight the utility of proteomics approaches to uncover the complete repertoire of acetylation changes triggered by pathogen infection. Protein lysine acetylation is a reversible covalent modification that was first discovered on histones more than 50 years ago . In geneWhile protein acetylation was originally discovered on histones specifically it has long been known that non-histone proteins are also acetylated . InitialFigure 1. Specifically, total proteins are extracted from cells or tissues, then the total proteins are digested to peptides, and the acetylated peptides are enriched using the anti-acetyllysine antibodies. Following enrichment the acetylated peptides are detected and quantified by using LC–MS/MS methods. Using this methodology, 100s and 1000s of acetylated sites have been identified in plants and non-plant eukaryotic systems, respectively. Organisms that have been utilized for either global or organellar acetylome profiling include Arabidopsis  have been shown to acetylate both histone and non-histone proteins . Lysine etylases .Figure 2). Studies have found that the expression level of HAT genes is induced by treatment with hormones as well as pathogen infection , a member of the plant-specific HD2 subfamily of HDACs in rice, is increased in the compatible reaction and decreased in the incompatible reaction after infection by the fungal pathogen Magnaporthe oryzae (HDT701 in transgenic rice leads to decreased levels of histone H4 acetylation and enhanced susceptibility to the rice pathogens M. oryzae and Xanthomonas oryzae pv. oryzae (Xoo). In contrast, silencing of HDT701 in transgenic rice causes elevated levels of histone H4 acetylation and elevated transcription of PRR and defense-related genes, increased generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, as well as enhanced resistance to both M. oryzae and Xoo.In line with these observations, a direct role for HDACs in modulating histone acetylation of defense genes and thereby plant resistance has been shown. The transcription level of Histone Deacetylase701  reduces the histone acetylation level in the coding region of several plant defense genes, including PLANT DEFENSIN1.2 (PDF1.2), WRKY33 and OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 (ORA59), which results in the repression of these genes and leads to suppression of plant defense . One example of such an interaction is from the type III secreted effector, HopZ1a, which is secreted by Pseudomonas syringae and functions as an acetyltransferase. HopZ1 is able to self-acetylate and acetylate tubulin, which results in the destruction of plant microtubule networks, inhibits protein secretion and ultimately suppresses cell-wall mediated defense (Ralstonia solanacearum, also has acetyltransferase activity. PopP2 directly acetylates the C-terminal WRKY transcription factor domain of the NLR RRS1 as well as WRKY transcription factors. Acetylation of RRS1 or WRKYs abolishes DNA binding activity. In the case of RRS1 acetylation, RPS4 dependent immunity is activated. Conversely, acetylation of WRKY transcription factors results in immune suppression (P. syringae, have been found to acetylate multiple members of the RPM1 immune complex and depress plant immunity response (While the majority of research has investigated the role of histone acetylation in defense there are several studies that have established a role for non-histone protein acetylation in plant immunity. For instance, pathogen produced type III effectors can acetylate selected non-histone proteins in host cells, thereby triggering plant immunity response ( defense . A simil defense . Additiopression . Finallyresponse .Lysine acetylation has emerged as a major post-translational modification impacting a diverse array of cellular processes. In the context of plant defense signaling it is well established that modulation of histone acetylation levels of defense genes is critical for appropriate immune responses. There is also mounting evidence that acetylation of non-histone proteins impacts plant immunity. However, to date non-histone protein acetylation of plant proteins has only been shown to result from pathogen effectors that encode acetyltransferase enzymes. This raises the question of whether plant HATs or HDACs directly modulate acetylation status of non-histone proteins during pathogen infection. The recent development of robust proteomic methodologies to globally identify and quantify lysine acetylation enables us to now identify proteome-wide changes in acetylation triggered during immune responses. Detailed follow-up studies of specific alterations in acetylation triggered by pathogen infection should shed light on whether plants directly modulate non-histone protein acetylation during defense signaling.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neurologic outcomes following pediatric cardiac arrest are consistently poor. Early initiation of cardiopulmonary resuscitation has been shown to have positive effects on both survival to hospital discharge, and improved neurological outcomes after cardiac arrest. Additionally, the use of therapeutic hypothermia may improve survival in pediatric cardiac arrest patients admitted to the intensive care unit. We report a child with congenital hypertrophic obstructive cardiomyopathy and an out-of-hospital cardiac arrest, in whom the early initiation of effective prolonged cardiopulmonary resuscitation and subsequent administration of therapeutic hypothermia contributed to a positive outcome with no gross neurologic sequelae. Continuing efforts should be made to promote and employ high-quality cardiopulmonary resuscitation, which likely contributed to the positive outcome of this case. Further research will be necessary to develop and solidify national guidelines for the implementation of therapeutic hypothermia in selected subpopulations of children with OHCA. Epidemiologic studies regarding the outcome of out-of-hospital cardiac arrest (OHCA) in children show heterogeneous results. OHCA in Netherlands resulted in 24% 12/51) survival to hospital discharge; however, larger studies in the United States, Canada, and Japan revealed overall survival rates ranging from 6.4% (40/621) to 8.5% (700/8240) 2/51 surv. While rWe describe a 12-year-old female with a known history of hypertrophic obstructive cardiomyopathy (HOCM) who responded to therapeutic hypothermia following prolonged CPR for sudden cardiac arrest.A 12-year-old Caucasian female with a known history of HOCM  and Wolff-Parkinson-White syndrome (WPW) without supraventricular tachycardia collapsed while waiting at the school bus stop. She was unresponsive and pulseless, raising concern for cardiac arrest. Her father, a trained emergency medical technician (EMT) who witnessed the event, immediately performed CPR in the field for an estimated 30 minutes until emergency medical personnel arrived. Cardiac rhythm analysis revealed ventricular fibrillation. Manual external defibrillation (DC cardioversion) was utilized twice during transport to a local hospital, the second of which returned her to a sinus rhythm. She remained unconscious and was hemodynamically unstable, requiring endotracheal intubation, multiple normal saline intravenous boluses, and pressor therapy. She was stabilized and subsequently transferred to the PICU in our tertiary care children's hospital for further evaluation and treatment.The patient's EKG upon admission showed a QTc of 508 milliseconds, which was prolonged from her baseline of 458 milliseconds. Due to her witnessed collapse and prompted initiation of CPR, a therapeutic hypothermia protocol was initiated with a 48-hour cooling regimen. Goal temperatures were between 32.5 and 33 degrees Celsius during cooling. The patient was maintained on antiepileptic therapy for seizure prophylaxis, as well as fentanyl and midazolam drips for appropriate sedation while undergoing therapeutic hypothermia. An EEG performed within 24 hours of admission showed “intermittent diffuse background slowing with no epileptiform discharges, focal features, or asymmetries.” The patient was rewarmed at hour 48 at a rate of 1 degree Celsius every six hours over the course of the ensuing 48 hours, ultimately accomplishing euthermia between 36.5 and 37.5 degrees Celsius. Extubation trials failed on hospital day 5 due to delirium and shallow work of breathing; following CPAP trials on hospital day 10, successful extubation occurred with no gross neurologic, renal, or hepatic insults noted on clinical and laboratory evaluation. An MRI  of the brain at day 10 revealed no structural abnormalities or evidence of gross ischemia. An adenosine challenge was also conducted in the electrophysiology laboratory, which revealed that the cardiac preexcitation was secondary to a fasciculoventricular pathway not capable of participating in tachycardia. Therefore, it was determined that this accessory pathway did not contribute to tachycardia leading to cardiac arrest in this instance. An automated implantable cardioverter defibrillator was placed on hospital day 15. The patient was discharged on hospital day 17 with no neurological sequelae to date. Follow-up EKGs have demonstrated a return to her baseline QTc of 460 milliseconds. She has experienced no recurrent episodes of torsades de pointes, ventricular tachycardia, or ventricular fibrillation.This case correlates with existing studies that have demonstrated a survival benefit of early initiation of CPR . It is tOver the past half century, therapeutic hypothermia has been increasingly used as an adjunctive component of resuscitation in a variety of critically ill pediatric populations with mixed results. A multicenter, multinational randomized control trial comparing the efficacy of therapeutic hypothermia to normothermia in pediatric traumatic brain injury (TBI) patients demonstrated no mortality benefit associated with the use of hypothermia . DespiteMuch of the current peer-reviewed evidence for the utilization of therapeutic hypothermia in young children is based on patients who suffered cardiac arrest secondary to a respiratory etiology (most commonly drowning or choking) as opposed to those of cardiac origin . LikewisThis case is an illustration of the utility of both high-quality CPR in the field and therapeutic cooling in the PICU in a child with OHCA and accompanying congenital heart disease. Continuing efforts should be made to promote and employ high-quality CPR in the general population, which likely contributed to the positive outcome in this instance. Further research is necessary to develop guidelines for routine utilization of therapeutic hypothermia in selective groups of children with OHCA like those with underlying cardiac etiology.


The endocytic pathway is essential for processes that define how cells interact with their environment, including receptor signalling, cell adhesion and migration, pathogen entry, membrane protein turnover and nutrient uptake. The spatial organisation of endocytic trafficking requires motor proteins that tether membranes or transport them along the actin and microtubule cytoskeletons. Microtubules, actin filaments and motor proteins also provide force to deform and assist in the scission of membranes, thereby facilitating endosomal sorting and the generation of transport intermediates. Several2Microtubules support intracellular transport in a manner that is dependent on their intrinsic polarity. They possess a dynamic plus end that, in most cells, grows towards the periphery and a minus end that is anchored in the microtubule organising centre, normally located near the nucleus in non-dividing cells. Microtubules support both short- and long-range movements, using two types of motors: kinesins, with a few exceptions, move towards plus ends Dynein is a large (1–2 MDa) motor complex . A specific isoform of myosin VI  assists the transfer of nascent endocytic vesicles away from the actin-rich cell periphery in preparation for their delivery to the microtubule transport system One protein that may regulate the switch from actin- to microtubule-based movement of endocytic compartments is Huntingtin (Htt), in which expansion of poly-glutamine repeats leads to Huntington's disease 5It is well established that all types of endosome move actively along microtubules. This is particularly clear in neurons, but extensive motility is also seen in cultured cells such as HeLas. High frame rate imaging and automated particle tracking has recently provided insight into these dynamics.−1, and peak observed speeds often of 4–6  μm s−1Rab5-positive early endosomes move with an inward bias runs), interspersed with somewhat longer periods (rests/pauses) of non-directed (i.e. diffusive) motion, and with occasional reversals run/rest behaviour, but runs often terminate when endosomes contact ER tubules Long-range movements occur in short bursts of directed motility  may be important in some circumstances The inward movement and localisation of all endocytic compartments is determined largely by dynein, since inhibition of dynein causes scattering of early endosomes, late endosomes and lysosomes and disrupts endosome motility Glued to CLIP-170 Glued dynactin subunit can also bind to microtubules directly via its CAP-Gly and basic regions It has been proposed that dynein-dependent movement is initiated by a cascade of protein interactions located at the plus ends of microtubules that ‘capture’ the endocytic vesicle and then activate motility. Several proteins (termed +TIP proteins) including EB1, EB3 and CLIP-170 accumulate at the plus ends of growing microtubules where they regulate microtubule dynamics and interactions with the cell cortex Ustilago maydis and Aspergillus nidulans, where dynein supports the retrograde movement of early endosomes away from the hyphal tip, whilst kinesin-3 moves them to the tip However, whilst the dynactin-mediated microtubule ‘capture’ pathway may be important for activating the transport of endosomes and other organelles from microtubule plus ends in the distal regions of neurons A. nidulansA. nidulans and U. maydis links the dynein-dynactin complex to early endosomes Endocytic compartments use a range of mechanisms to engage this multisubunit motor . SurprisGluedDynein/dynactin are linked to late endosomes/lysosomes via Rab7-interacting lysosomal protein (RILP) and Oxysterol-binding protein-related protein-1L (ORP1L), which bind Rab7 to generate a membrane-bound tripartite complex Further routes for recruiting dynein to lysosomes have been identified recently. In Zebrafish neurons, the single LIC interacts with c-Jun N-terminal kinase interacting protein 3 (JIP3), to mediate the retrograde transport of lysosomes 5.2DrosophilaU. maydisA. nidulansBoth kinesin-1 and -2 move early endosomes in vitro in vitro Plus end-directed transport of late endosomes and lysosomes is driven by several kinesins. Hence, whilst disruption of the kinesin-2 KIF3A motor subunit induces the clustering of both late endosomes and lysosomes 5.3Endosomes moving along microtubules do on occasion switch directions, suggesting that at least a portion carry both plus- and minus-end-directed microtubule motors. Switching is fairly rare for early endosomes Dictyostelium and in HeLa cells Dictyostelium at least, several, weaker, dynein motor units balance each kinesin U. maydis, though here dynein appears to be the stronger motor Although the mechanism by which bidirectional transport can occur Such behaviour suggests that some form of coordinate regulation is in play. Htt is implicated in controlling the direction of movement of neuronal endosomes, since Htt phosphorylation by Akt stimulates plus-end-directed movement, while retrograde movement predominates in the presence of dephosphorylated Htt 6Dynein is important for sorting recycling cargo such as Tf away from degradative cargo such as EGF C. elegans gut epithelium Dynein is required not only for the generation of recycling intermediates but also for maintaining the position of the recycling endosome itself. This is achieved at least in part by the binding of LICs to the Rab11 effector FIP3 The generation of tubular sorting intermediates at the endosome is also assisted by actin In addition to the role of actin polymerisation in promoting membrane deformation, separation of membrane domains and carrier biogenesis, actin motors are also prominent at the endosome. Myosin VI acts with LMTK2 (lemur tyrosine kinase 2) to help TfR exit the sorting endosome and reach the Rab11-positive recycling endosome In summary, microtubules and their associated motor proteins function in concert with actin assembly and myosins to promote membrane tubulation and endosomal maturation. The mechanical forces exerted by these components may ultimately combine to induce membrane tension at critical points in the process in order to generate, elongate and break membrane tubules. Clearly, however, much work is required to clarify how these molecular events are ordered.7Once cargo has been transported through the recycling endosome and has reached the cell periphery, transport to the plasma membrane is facilitated by the action of the actin cytoskeleton and its associated motors, principally myosin Vb. Type V myosins are widely implicated in carrier transit to the cell surface. Consistent with this, myosin V isoforms bind multiple Rabs involved in exocytotic processes, including Rab3, Rab27A, Rab8 and Rab10 (see null mice display accelerated delivery of fluid-phase content to acidified endosomes It remains unclear how such short-range motility contributes to the transport of cargo over large distances 8The cytoskeleton and motors are essential for maintaining the spatial distribution of endocytic compartments within cells, and act at crucial points to facilitate transport steps such as membrane remodelling, carrier formation and cargo sorting. Paradoxically, experimental conditions that disrupt the positioning and motility of membranes profoundly are often of only minor consequence for the overall efficiency of endocytic functions such as receptor recycling or lysosomal delivery, at least in cultured cell models. This suggests that other transport steps are rate limiting in these cells, where distances between compartments are generally short, and indeed are often reduced by interventions such as microtubule motor disruption. In physiological systems, however, the movement and spatial distribution of endosomes is likely to be finely tuned and critical for cellular function. This is most clearly the case in highly polarised cells such as neurons, where the movement of endosomes and/or lysosomes is essential for processes such as dendritic branching


Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs. Advances in stem cell therapy for treating cardiovascular diseases have been hampered by the complexity to understand the modality by which stem cells are committed to the cardiovascular lineage once injected and what molecular mechanisms underlie stem cell regenerative potential. In combination with the transdifferentiation ability toward the cardiac lineage, stem cell candidates should have also additional attributes such as high achievable yield, reproducible in vitro expansion, and immunotolerance. Among those adult stem populations under clinical investigation, mesenchymal stem cells (MSCs) have been reported to show all the above-mentioned features –4. PartiADMSCs are routinely expanded in platelet lysate (PL), a hemocomponent enriched with growth factors and cytokines already employed as a substitute for foetal bovine serum (FBS) in cell culture and for treating those medical applications where repair, regeneration, and neoangiogenesis are desirable –10. InteWe have already described both the suitability of the GMP-compliant PL to enhance the biological properties of a novel tissue source of human ADMSCs derived from the mediastinal depots  and theiIn this brief report, we show for the first time the ability of a GMP-compliant PL  to potentially promote a permissive state of cardiovascular commitment in human mediastinal ADMSCs and to cause epigenetic modifications mainly based on acetylation of histones 3 and 4.2. Nonadherent cells were removed after 3 days. Cell count and viability were evaluated by Trypan Blue . ADMSC cultures were characterized according to the criteria of the International Society for Cellular Therapy (ISCT) . Negative controls consisted of the secondary antibody alone  added at room temperature for 2 hours. Images were acquired by fluorescent microscope . Nuclei were counterstained by DAPI .


Since the reprogramming of adult human terminally differentiated somatic cells into induced pluripotent stem cells (hiPSCs) became a reality in 2007, only eight years have passed. Yet over this relatively short period, myriad experiments have revolutionized previous stem cell dogmata. The tremendous promise of hiPSC technology for regenerative medicine has fuelled rising expectations from both the public and scientific communities alike. In order to effectively harness hiPSCs to uncover fundamental mechanisms of disease, it is imperative to first understand the developmental neurobiology underpinning their lineage restriction choices in order to predictably manipulate cell fate to desired derivatives. Significant progress in developmental biology provides an invaluable resource for rationalising directed differentiation of hiPSCs to cellular derivatives of the nervous system. In this paper we begin by reviewing core developmental concepts underlying neural induction in order to provide context for how such insights have guided reductionist in vitro models of neural conversion from hiPSCs. We then discuss early factors relevant in neural patterning, again drawing upon crucial knowledge gained from developmental neurobiological studies. We conclude by discussing open questions relating to these concepts and how their resolution might serve to strengthen the promise of pluripotent stem cells in regenerative medicine.  The process of neurodevelopment is spatiotemporally regulated and necessitates sequential, progressive restrictions in cell fate. Although some interspecies differences in both cytoarchitecture and molecular machinery do exist between mouse and man, rodent models have illuminated key underlying mechanisms of lineage restriction to a variety of cell types. These insights have provided invaluable guidance for the predictable manipulation of human pluripotent stem cells (hPSCs) into myriad cell fates. From the point of fertilisation of the secondary oocyte, cells commence asymmetric division and sequentially give rise to the 2-, 4-, and then 8-cell stage blastomere, which subsequently develops into the blastocyst . Oct3/4  neural induction specifies a region of the embryonic ectoderm to form the neural plate , and dorsoventral (D-V) axes  and subsequently terminal differentiation into diverse postmitotic neuronal subtypes . Such in interspecies suggests evolutionary conservation of underlying mechanisms. The notion of inductive signals orchestrating the process of neural induction has become widely accepted. Accumulating evidence suggests a spatiotemporal interdependence of several signalling pathways in neural induction, which somewhat challenges the concept of organiser tissue. The molecular pathways underlying neural induction remained elusive until the 1990s, when Xenopus studies first reported that transient dissociation of gastrula-stage animal caps into single cells resulted in neural fate acquisition and that misexpression of a dominant-negative Activin receptor, since being discovered to inhibit multiple transforming growth factor (TGFβ-) related factors, ectopically generated neural tissue at the expense of mesoderm specification. These studies suggest that neural induction may occur through a “de-repression” strategy . The first mechanistic insights into neural induction originate from seminal experiments by Spemann and Mangold in the early part of the twentieth century. In these studies, dorsal mesoderm was transplanted into the ventral embryo and generated a secondary host-derived neural tube. The graft itself was found to contribute to secondary mesodermal structures including the notochord, while the neural tissue was host-derived. The ability of the dorsal blastopore lip to reprogram surrounding tissues when transplanted ectopically justifies its designation as “organiser tissue.” Equivalent organiser regions in other vertebrates were subsequently discovered by the elegant work of Waddington in the 1930s, including “Hensen's node” in birds and mammals . Organisβ signalling is relatively well understood: ligand binding causes receptor dimerization and initiates a signal transduction pathway and activates a family of cytoplasmic proteins, the Smads, by phosphorylation. Eight Smad proteins are encoded in the human genome, although only five of these  act as substrates for the TGF receptor family; these are commonly referred to as “receptor-regulated Smads,” or just “RSmads.” Broadly, the TGF-β signalling superfamily encompasses both the Activin/Nodal and bone morphogenetic protein (BMP) signalling pathways [The molecular machinery of TGF-pathways . The subIn the early 1990s Noggin, Follistatin, and Chordin were identified as genes encoding proteins with neuralizing activity that were expressed in organiser tissue. These proteins are inhibitors of BMP signalling, with a particular bias towards antagonising BMP4, an inhibitor of neural fate. BMP4 is expressed widely at the onset of gastrulation  but is sThese facts, taken together, allow a simple molecular pathway for neural induction to be considered: the extraembryonic ectoderm produces BMPs to promote epidermal differentiation, while neural inducing regions (organiser tissues) antagonize BMPs to permit neural induction Figures . This caβ superfamily, including Nodal, is also well established [A significant majority of studies have focused on the role of BMP inhibition in neural induction during vertebrate development. However, the importance of other members of the TGF-ablished . Nodal aablished , while Nablished . A role ablished –15 and cablished . Nodal iablished , which pablished –16, 18, FGFs are a diverse collection of secreted diffusible glycoproteins that act by binding with differential affinity to four classes of extracellular receptor (FGFR 1–4). The precise role of FGF signalling in neural induction remains controversial, but studies collectively suggest an early function to promote competence for neural conversion and later functions in transcriptional antagonism of BMP. Another important member of the FGF family, FGF8, is expressed in the mouse embryo in the extraembryonic ectoderm and the epiblast before and during gastrulation Figures . FGF8 acβ-catenin pathway (to promote proliferation), or the noncanonical JNK pathway  in an FGF2-dependent manner [WNTs are secreted glycoproteins responsible for establishment of the dorsoventral axis of the embryo, a direct consequence of which is the acquisition of neural identity. Administration of mRNA encoding WNTs (or their effectors) into the animal hemisphere of one-cell embryos by injection generates ectopic neural tissue. WNT signalling is itself activated by BMP4 and implicated in a Nodal positive feedback loop  Figure . The AVEt manner .These findings collectively suggest that neuroectoderm specification is likely more complex than the “default” (BMP4 inhibition) or “organiser”  models might suggest. The effects of each relevant signalling pathway are temporally regulated and determined by developmental context, justifying their systematic investigation  in the neural conversion of hPSCs .Once specified, the neuroectoderm is subsequently regionalized along the R-C axis of the embryonic body Figures . OrganisThe precise timing and mechanisms of neuraxial patterning remain unresolved. A popular model is that neural induction initially specifies rostral precursors, upon which caudalising signals subsequently respecify positional identity in a progressive and stereotyped manner to establish subdivisions of the posterior neuraxis. Some of the signalling pathways implicated in neural induction also appear to play key roles in early R-C and D-V patterning at later stages ; they esEvidence from animal studies suggests that spatially and functionally distinct cell populations organise development of head and trunk structures . The hea+/− and Smad 3−/− mutant embryos exhibit a miniaturized head-like structure [Studies using a range of approaches have shown that AVE is necessary for normal forebrain development with Nodal signalling being critical in this process . Collecttructure . In zebrtructure . Howevertructure , 26.β superfamily) plays an important role in Hox8–Hox10 gene expression at thoracic and lumbar neural tube regions [A FGF signalling gradient operates along the R-C axis to induce the expression of paralogous Hox genes in the neural tube. Hox genes located at one end of the cluster (3′ end) are expressed more rostrally in response to low levels of FGF; conversely genes at the opposite end (5′ end) are expressed caudally in response to high levels of FGF . Differe regions . Drosophila gene hedgehog, which encode secreted signalling proteins. Sonic hedgehog (SHH) transpired as the ventrally secreted morphogen conferring D-V neural tube polarity  are ventrally located, while the cerebral cortex is dorsally positioned. R-C and D-V patterning is carefully integrated in a highly stereotyped manner. Broadly, ventral regional specification requires activation of both the Nodal and Sonic hedgehog pathways with antagonism of BMP signalling. Over and beyond its role in R-C patterning, RA is required for intermediate zone specification within the D-V axis. Likewise, FGF also plays important roles in ventral domain specification. The major contributors to D-V axis formation are BMPs and WNTs dorsally, and Sonic hedgehog ventrally . Distincpolarity . It is npolarity . Its funpolarity .How is positional identity imposed on precursor cells? Several studies have implicated a group of factors, predominantly the homeodomain (HD) and basic helix-loop-helix (bHLH) transcription factors, as crucial regulators here. These are expressed in strictly organised arrays along the D-V axis of the neural tube. Individual proteins are designated as classes I or II by their response to SHH signalling. Class I proteins are repressed by SHH, thus defining their ventral limit of expression, while class II protein expression is induced by SHH and defines dorsal expression boundaries. Specifically in the context of spinal cord development, such cross-repressive interactions allow the establishment of five distinct ventral precursor domains, which in turn permit the specification of distinct neuronal subtypes. Gain- and loss-of-function experiments have further supported this putative mechanism across different species, where ectopic expression of HD proteins predictably changed the regional allocation of individual neuronal subtypes within the neural tube , 42. A sSHH signalling does not appear to contribute to patterning in the dorsal neural tube. However, BMPs have similar and complementary roles in dorsal patterning of the neural tube and telencephalon . These sThese aforementioned developmental studies provide a conceptual framework to rationalise both neural induction strategies and bespoke programmes of morphogenetic cues for the directed differentiation of hPSCs to clinically relevant and region specific neurons (summarized in  selective Rho-associated kinase (ROCK) inhibitor permits survival of dissociated hPSCs, thus allowing systematic manipulations to cell fate after dissociation [“Default” neural conversion from hPSCs to forebrain neuronal subtypes has been demonstrated in a variety of systems including chemically defined suspension culture, not requiring extrinsic signals, as well as in an adherent culture method , 44, 45.ociation . Such deociation .Prior to terminal differentiation, if specified dorsal telencephalic precursors are exposed to SHH and/or a WNT antagonist, they are ventralised to generate subpallial derivatives . Upon terminal differentiation, these ventralised telencephalic cultures give rise to GABAergic projection neurons and interneurons. Clinically relevant cell types originate from the LGE  and the MGE . Further sophistication can be added to the aforementioned directed differentiation strategies by carefully regulating SHH and WNT pathways . For example, a low concentration of SHH alone permits the specification of both LGE and MGE derivatives, whereas if a WNT antagonist is added to SHH, the more ventral MGE  neurons are preferentially specified at the expense of LGE  neurons. Some elegant and ontogeny recapitulating strategies have been defined for the generation of authentic DARPP32 expressing medium spiny projection neurons , 47.Differentiating hPSCs into midbrain dopaminergic neurons has maintained great enthusiasm likely owing to their potential to understand and treat Parkinson's disease. Although dopaminergic neurons exist throughout the nervous system, there is a region-specific functional heterogeneity that has been experimentally demonstrated by performing anisotopic implantation experiments . Midbrai directed differentiation of hPSCs in regenerative medicine [β inhibitors such as CHIR99021 used here for WNT pathway activation do have off target effects  [β inhibitor CHIR99021 [β inhibition and the identification of additional key regulatory pathways would be of great importance to establish.Against this background, initial approaches to midbrain differentiation were based on FGF8 for R-C patterning to the region of the midbrain, and SHH for ventralization into dopaminergic neurons, although the yields were low (approx. 30%) using such strategies. Furthermore, subsequent studies have raised the possibility that PA6 and SHH/FGF8-based approaches alone are not sufficient to generate authentic midbrain dopaminergic neurons , 51. Themedicine . A furthmedicine . Althoughan WNT) . AdditioHIR99021 , 54. In Broadly, evolutionary pathways appear to be more conserved in caudal (primitive) regions of the CNS such as the hindbrain. The hindbrain can be divided into rostral and caudal portions, which are separated by rhombomere 4 (r4). Neurons derived from rostral regions project to and innervate myriad brain regions, whereas the caudal portion, located in the myelencephalon, gives rise mainly to descending spinal projections. The brain innervating central serotonergic neurons, originating from r2-3 of the rostral raphe, contribute to higher order brain functions and are implicated in a range of psychiatric disorders. By using EGF and FGF2 in the maintenance media, so-called “long-term self-renewing rosette-type” hPSC-derived neural precursors can be expanded which exhibit a ventral anterior hindbrain-like expression profile after prolonged culture . These pThere are few reports of cerebellar differentiation with demonstration of electrophysiologically mature and functional Purkinje- and granule-cell specification , 58. A rThe generation of functional spinal cord derivatives, including motor neurons, has been achieved from hPSCs through a variety of approaches using insights from developmental biology , 60–62. Treating cultures with RA typically results in a cervical or brachial positional identity , 65. MorNeural crest cells are highly migratory and give rise to myriad differentiated cell types including (i) sensory and autonomic neurons and Schwann cells, (ii) chromaffin cells in the adrenal medulla, (iii) melanocytes, and (iv) cranial skeletal and connective tissue components. The fate of the neural crest cells is largely determined by where they migrate to/settle. From an hPSC perspective, striking phenotypic consequences have been demonstrated based on plating density, and this provides a strategy to generate neural crest derivatives. A high plating density favours PAX6 expressing central nervous system precursors while low plating density specified neural crest-like differentiation . Using vThe unrivalled complexity of the mammalian central nervous system is enabled by a series of progressive and sequential events during embryogenesis. The degree of interconnectedness within the central neuraxis is somewhat surprising given its impressively precise organisation into discrete regions. Evolutionary conservation of developmental processes underlying the organisation of such discrete neural regions becomes increasingly less applicable to more rostral  components, like the forebrain. The hPSC platform is emerging as an important reductionist in vitro system to interrogate aspects of human development, which have remained experimentally inaccessible until now. postmitotically, which has been reported [Current approaches towards such directed differentiation of hPSCs often fail to capture the dynamic and overlapping nature of neurodevelopmental processes. For instance, neural induction and patterning are often conceptualised as mechanistically distinct processes. However, a bias towards different regional fates will likely be determined by the neural conversion paradigm employed. Similarly current differentiation strategies do not yet fully acknowledge or exploit the ability to influence cell (subtype) fate decisionsreported –71. As sDevelopmental principles are a crucial resource for defining ontogeny recapitulating directed differentiation protocols for hPSCs . In addiFinally, the hPSC field stands to benefit from defining multiple directed differentiation protocols that employ closely aligned methods for neural conversion and similar protocol durations. This may then permit more meaningful comparison between region-specific neurons, without the potentially confounding issue of differential cellular maturation. Indeed such an approach was recently utilised to show region-specific phenotypes using iPSCs derived from patients with Alzheimer's disease and motor neuron disease . Taken t


Recently, a new paradigm for the development of heart failure with preserved ejection fraction (HFpEF) has been proposed, which identifies a systemic pro-inflammatory state induced by comorbidities as the origin of microvascular endothelial cell inflammation and subsequent concentric cardiac remodeling and dysfunction. This review further discusses the pivotal role of the inflamed endothelium in the pathogenesis of HFpEF-specific cardiac remodeling. The potential importance of reciprocal interactions of the endothelium with cardiac fibroblasts and cardiomyocytes and with the cardiac neurohumoral response in this cardiac remodeling process is outlined. Epidemiological studies demonstrate that 50 % of all heart failure patients suffer from heart failure with preserved ejection fraction (HFpEF), which—in contrast to heart failure with reduced ejection fraction (HFREF)—cannot be adequately treated with current available therapeutical strategies . DiabeteThis review gives a brief overview on how systemic inflammation induced by comorbidities influences endothelial cell behavior and signaling and how this affects the interaction of the endothelium with cardiomyocytes and cardiac fibroblasts, and the cardiac neurohumoral response, and subsequent cardiac remodeling. On the other hand, this review points out that those cells in turn influence the inflammation or directly the endothelium, aggravating the initial inflammatory process Fig. . The impThe endothelium, the physical barrier between the blood and vascular wall, plays a pivotal role in cardiovascular homeostasis by regulating vasomotor tone, vascular permeability, and cardiac function . The rel2•) rather than NO [2• production and subsequent formation of peroxynitrite (ONOO–), which enhances eNOS uncoupling via reducing the affinity of eNOS for tetrabiopterin and via limiting the de novo synthesis of tetrabiopterin [The HFpEF-associated comorbidities lead to endothelial dysfunction, a condition characterized by impaired endothelium-dependent vasodilatation and “endothelial activation,” referring to a state in which the endothelium loses its physiological properties and shifts towards a pro-inflammatory, pro-coagulatory, and vasoconstrictor state . Particu than NO , 21. Hyp than NO  leads toiopterin .The cardiac endothelium comprises the endothelial cells of the coronary microvasculature, of the endocardium, and of the intramyocardial capillaries. Cardiomyocytes lie within the coronary microvascular network maximum 3 μm from endothelial cells. This specific anatomical arrangement allows not only adequate blood supply but also facilitates the bidirectional communication among those cells –26. The Endothelial dysfunction/activation may contribute to cardiac fibrosis by different means. Murdoch et al.  recentlyBesides immune cells, also fibrocytes, circulating monocyte-derived cells with tissue remodeling properties of fibroblasts , which aSecondly, endothelial dysfunction contributes to cardiac fibrosis via the reduced bioavailability of NO, known to exert direct anti-fibrotic effects involving the cyclic guanosine monophosphate (cGMP) pathway , 42. VetThirdly, NO deprivation , 48 leadIn summary, these findings suggest that endothelial dysfunction/activation and downstream pathways including not only inflammation but also far less appreciated EndMT, are important mechanisms contributing to cardiac fibrosis and the development of HFpEF . CardiacThe endocardial endothelial cells and the endothelial cells of intramyocardial capillaries regulate the contractile state of cardiomyocytes via autocrine and paracrine signaling involving NO and endothelin-1. Furthermore, co-culture of cardiac endothelial cells with cardiomyocytes protects those cardiomyocytes from hydrogen peroxide-induced apoptosis through neuregulin-erbB4 signaling . This in2+/calmodulin-dependent protein kinase II [2+/calmodulin-dependent protein kinase II and its involvement in NO synthesis as a result of Ca2+-dependent activation of eNOS [2+/calmodulin-dependent protein kinase II may further contribute to the relaxation of titin via triggering NO release. Due to the potential risk of arrhythmic death, the induction of protein kinase A via ß adrenergic stimulation is excluded as therapeutical option. Blocking the breakdown of the downstream NO target cGMP via sildenafil has not been successful in HFpEF patients despite promising experimental studies (see infra). In contrast, favorable effects in improving diastolic dysfunction in patients with HFpEF have recently been found by the use of the neprilysin inhibitor (LCZ696), which inhibits the breakdown of natriuretic peptides. Neprilysin activity is induced in obese patients [Particularly, the contribution of cardiomyocyte stiffness in diastolic LV stiffness and HFpEF is established. Besides intracellular calcium dysregulation, involving dysfunction of the sarcoplasmic reticular adenosine triphosphate (ATP)-driven pump (SERCA), phospholamban (PLB), and/or ryanodine receptor (RYR) 2, cardiomyocyte stiffness is mainly regulated by the giant sarcomeric protein titin . Titin sinase II  phosphorinase II . Proteininase II  and G [7inase II , 74•• ph of eNOS , it is tpatients . The finpatients  may explpatients .In summary, the importance of NO bioavailability and oxidative stress in titin relaxation (of the N2B segment) •• as welAs it has already been broadly outlined before, HFpEF comorbidities provoke a systemic inflammatory state, which severely affects the coronary microvascular endothelium , 79. TheCardiovascular autonomic neuropathy in diabetes mellitus is associated with LV diastolic and systolic dysfunction, LV hypertrophy, and higher LV mass index . FindingHFpEF is a complex disorder caused by multifactorial stresses secondary to comorbidities. This review highlights the importance of endothelial dysfunction or deterioration at the early onset of the pathogenesis of HFpEF and stresses the subsequent complexity of cardiac remodeling due to bidirectional interactions between endothelial cells, cardiomyocytes, fibroblasts, and cardiac neurohumoral activation. It further strengthens the need for an early diagnosis of endothelial dysfunction •• enabli


What cellular and network properties allow reliable neuronal rhythm generation or firing that can be started and stopped by brief synaptic inputs? We investigate rhythmic activity in an electrically-coupled population of brainstem neurons driving swimming locomotion in young frog tadpoles, and how activity is switched on and off by brief sensory stimulation. We build a computational model of 30 electrically-coupled conditional pacemaker neurons on one side of the tadpole hindbrain and spinal cord. Based on experimental estimates for neuron properties, population sizes, synapse strengths and connections, we show that: long-lasting, mutual, glutamatergic excitation between the neurons allows the network to sustain rhythmic pacemaker firing at swimming frequencies following brief synaptic excitation; activity persists but rhythm breaks down without electrical coupling; NMDA voltage-dependency doubles the range of synaptic feedback strengths generating sustained rhythm. The network can be switched on and off at short latency by brief synaptic excitation and inhibition. We demonstrate that a population of generic Hodgkin-Huxley type neurons coupled by glutamatergic excitatory feedback can generate sustained asynchronous firing switched on and off synaptically. We conclude that networks of neurons with NMDAR mediated feedback excitation can generate self-sustained activity following brief synaptic excitation. The frequency of activity is limited by the kinetics of the neuron membrane channels and can be stopped by brief inhibitory input. Network activity can be rhythmic at lower frequencies if the neurons are electrically coupled. Our key finding is that excitatory synaptic feedback within a population of neurons can produce switchable, stable, sustained firing without synaptic inhibition. Rhythmic actions like chewing, scratching and walking need to be switched on and off. Once started, rhythms are generated by networks of neurons in the brain and spinal cord which drive muscles. We use computer models of the swimming network in young frog tadpoles to ask how electrically-coupled brainstem neurons with feedback excitation generate rhythmic activity and how this can be switched on and off by sensory stimuli. Young tadpoles swim for several seconds when touched and stop when they contact a solid object. Swimming rhythms can be generated by minimal populations of ~30 reticulospinal neurons on each side of the brainstem. The sensory pathways providing excitatory start and inhibitory stop signals are known. In our model network, based closely on biological evidence, brief synaptic excitation can activate a stable swimming rhythm sustained by mutual excitation among the reticulospinal neurons; swimming can be stopped by brief inhibition. Model networks of more generic neurons with mutual excitation can produce switchable, sustained non-rhythmic firing. We conclude that mutual excitation, providing positive feedback, can enable stable rhythmic or non-rhythmic firing in small neuron populations that can be rapidly turned on and off by sensory inputs. Many rhythmic motor patterns are generated within the nervous system by networks of neurons –6. The mXenopus tadpole, responds to brief touch stimulation with several seconds of swimming which stops when it contacts a solid object. The sensory pathways controlling swimming and the specific populations of CNS neurons involved in generating the swimming rhythm have been defined in detail anatomically and physiologically  o = 0.5 mM. In simulations of the network in zero extracellular Mg2+, the vdepMg2+(V) term was set to 1. Feedback synaptic connections were made with a fixed probability of 0.2 between dINs.A custom Python toolbox ('morphforge’) was used to perform simulations of small networks . A dIN nf A and B. AMPAR af A and B. A scali2 = 0.1).In experiments to generalise conclusions from our models of tadpole neurons we used a population of 30 simpler, single-compartment Hodgkin-Huxley neurons with direct soma to soma electrical connections. These neurons had leak, sodium and potassium currents ,30. NMDA1, t2…tn}. The time of the kth spike, tk was generated from a normal distribution tk = N, where μk and σ k are the means and standard deviations of the kth spike  to head-skin stimuli  were useth spike . The valeach dIN . A set oeach dIN .in situ step current injections but they fire repetitively at low frequencies (once per cycle) during swimming. Modelling the dIN population has already shown how the single spiking behaviour could be a result of their electrical coupling, rather than their individual membrane properties  was increased, either by adding more IPSPs or increasing the interval between them  parameters . As previn vivo about the mechanisms controlling action potential firing in neurons driving motor responses. To generalise our conclusions we have also studied a simple network of generic neurons with mutual synaptic excitation.Persistent activity occurs in motor systems controlling the position of eyes and limbs and also in areas of cerebral cortex involved in short-term memory and in many sub-cortical brain regions . The detFor the network controlling fictive swimming in hatchling tadpoles, we have demonstrated central roles in sustaining activity for mechanisms identified experimentally. These are: voltage-gated membrane channels; feedback glutamate excitation; and voltage-gated magnesium block of NMDA channels ,18,42. WCellular pacemaker mechanisms and network mechanisms involving Post Inhibitory Rebound (PIR) firing have been proposed to complement each other in producing rhythms in many networks, from molluscs to mammals, –52. ThisOne of the main conclusions of this modelling study is to confirm the hypothesis that a population of neurons with mutual glutamatergic synapses can generate self-sustaining pacemaker-like activity by activating their own NMDARs ,19. DuriThe NMDAR-mediated feedback mechanism underlying rhythm generation in the tadpole CNS will work in other populations of neurons with much higher intrinsic maximum firing frequencies, for example classical Hodgkin-Huxley-type neurons. Our simple models of these networks showed that at the higher frequencies, electrical coupling had little effect and, as a result, these populations generated unsynchronised firing activity. Although non-rhythmic, such small populations with feedback excitation would be particularly suited to systems where activity needed to be switched on and off quickly by synaptic input.Using the tadpole as a case study we have produced a model, based closely on experimental results, of a specific small CNS neuron population with mutual NMDAr mediated excitation, which is capable of reproducing key experimental results. However, it is an oversimplification. For example, our model dIN network could fire rhythmically in the frequency range of tadpole swimming during physiologically realistic levels of excitation, but was also stable when firing faster at higher levels of excitation (~60 Hz). The model network could also fire rhythmically in the absence of NMDA channel voltage dependency  and this does not occur experimentally . During Although this work has focused on electrically-coupled neurons on a single side of the CNS, the tadpole has bilateral populations of neurons which form connections with other neuron populations to produce rhythmic, antiphasic firing on each side of the body during swimming ,15. Mid-Many real neuronal networks generate rhythmic or continuous activity controlled by synaptic input. Our model of a population of tadpole hindbrain neurons sustains rhythmic firing through positive feedback onto itself. In many domains, such positive feedback risks uncontrolled exponential growth and instability. However, neuronal circuits contain many saturating non-linearities which constrain firing activity and permit the use of positive feedback. All neurons have a limited upper firing frequency, which in the tadpole dINs is particularly low. Additionally, the saturation of the NMDARs at feedback synapses, inactivation of sodium conductances, and the activation of non-inactivating potassium conductances will help to prevent run-away depolarisation and firing even without synaptic inhibition. We have explored major features of the pacemaker mechanisms in the neuronal population driving tadpole swimming and have shown that similar mechanisms could also lead to stable firing at higher frequencies and in generic neuron populations. In both cases the voltage dependence of NMDARs can facilitate strong drive to neurons without causing them to lock up. We have built a model that reproduces many experimental findings although the specific channel currents which limit firing frequencies in tadpoles remain to be clarified experimentally. We have demonstrated that electrical coupling plays a crucial role in synchronizing pacemaker population firing so that, over lower frequency ranges, stable rhythmic activity is generated. Finally, we have demonstrated that small, biologically-realistic populations of neurons with mutual NMDAR mediated excitation are able intrinsically to sustain stable firing which can be switched on and off via conventional excitatory and inhibitory synaptic input pathways. Small neuron populations with these characteristics seem ideal for activating motoneurons to control posture or eye position and intermittent rhythmic actions like locomotion.S1 Paper Simulations(ZIP)Click here for additional data file.


A 60-year-old man with a history of implantation of two bare-metal stents 2 years prior presented to the emergency department with new-onset chest pain. He has been regularly taking angiotensin-converting enzyme inhibitors, beta blockers and aspirin. Aspirin was suspended for 10 days prior to the current hospitalization in order to perform surgery to remove a kidney tumor. He underwent coronary angiography, which revealed a right coronary artery with a distal intraluminal defect within the stents, suggesting thrombus. Intravascular ultrasound demonstrated a severe malapposition and underexpansion throughout the entire length of the stents containing thrombus. In this case, the mechanisms of very late stent thrombosis after bare-metal stent most likely were a combination of mechanical factors (severe stent undersizing during the index procedure) and pharmacological factors (aspirin discontinuation). Endothelization and healing at the site of stent implantation in patients with STEMI may be delayed substantially. Furthermore, although there is no difference in the frequency of stent thrombosis with both stents types, the relatively high very late stent thrombosis rate recently documented with BMS is notable and refuses the prior conception that, at least in STEMI, very late stent thrombosis is a rare phenomenon. In addition, data and imaging documenting the mechanism of very late stent thrombosis in STEMI patients is rare. The aim of this case was to report the potential mechanisms of very late stent thrombosis after BMS by intravascular ultrasound (IVUS).Primary percutaneous coronary intervention with stent implantation has emerged as the treatment of choice for most patients with evolving ST-segment elevation myocardial infarction (STEMI). Nevertheless, emerging data suggest that risk of stent thrombosis in STEMI is relatively increased in patients without STEMIA 60-year-old man with a history of implantation of two BMS (2.25x28mm and 2.5x28mm) for an inferior STEMI  2 years . The risk of stent thrombosis in the STEMI population is increased threefold compared with non-STEMI patients, and occurs with similar frequency in BMS and DES. The higher rate of stent thrombosis in patients with STEMI may be related to the underlying plaque composition responsible for the condition. Stent strut penetration of an underlying necrotic core may augment inflammation, platelet activation, and fibrin deposition and inhibit neointimal growth, resulting in uncovered stent struts. Furthermore, the incidence of positive arterial remodeling and late acquired incomplete stent apposition is higher in patients with acute coronary syndromes, especially in those with DES. Finally, infarct-related artery vasoconstriction, reduced epicardial flow, and thrombus may lead to stent undersizing, as likely occurred in the current case at the time of STEMI treatment, when a 4.5mm vessel was treated with a 2.5mm stent. Over time, when flow was reestablished, thrombus dissolved, and the vessel increased in size, which developed malapposition and underexpansion of the stent. In this case, the mechanisms of very late stent thrombosis after BMS most likely were a combination of mechanical factors (severe stent undersizing during the index procedure with or without superimposed positive remodeling and infarct-related thrombus dissolution during the 2 year follow-up period) and pharmacological factors (aspirin discontinuation).Although it is a relatively low-frequency event, stent thrombosis is associated with high risk of myocardial infarction and deathThis case reports the mechanisms of very late stent thrombosis 2 year after bare-metal stent implantation. Intravascular ultrasound plays an important role in identifying mechanical factors related to stent thrombosis as well as guiding its therapy accordingly.


Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na+/H+ antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway.Comparative genome analysis revealed seven uncharacterized genes,  Streptomyces venezuelae strain ATCC 10712 (p-aminobenzoic acid (PABA) that is required for folic acid biosynthesis (Chloramphenicol (CHL) is a bacteriostatic antibiotic that inhibits protein synthesis by binding to the 50S subunit of the bacterial ribosome. It is active against both Gram-negative and Gram-positive bacteria, including many multiply drug-resistant strains. CHL is produced by several Gram-positive soil actinomycetes, but its biosynthesis has been analyzed mostly in ynthesis .S. venezuelae resulted in the identification of 12 genes with a proven or likely role in CHL production  or DNA agar medium  or in groups (sven0909 to sven0912 and sven0913–sven0915) with a hygromycin (HYG) resistance cassette amplified from pIJ10700 using the primer pairs listed in Table S1 in the supplemental material, as described by Gust et al.  were reeviously .sven0913, sven0914, and sven0915 deletion mutants, PCR products were generated by high-fidelity PCR using the primers listed in Table S1 in the supplemental material. For the complementation of Δsven0913 and Δsven0915, the PCR products extended from the beginning of the upstream intergenic region to the stop codon of each gene. The fragments were cloned into pRT802  containing the driver plasmid pR9406 and the nonmethylating E. coli strain ET12567, according to standard procedures  was generated by high-fidelity PCR using the primers listed in Table S1 in the supplemental material. This fragment was cloned into the integrative vector pIJ10257 , as described previously  fitted to an Agilent 1100 HPLC system with a diode array detector and analyzed using a method modified from He et al. (Chloramphenicol production was quantified by high-performance liquid chromatography (HPLC). The culture supernatants were filtered through VectaSpin Micro polysulfone 0.2-mm columns , injected onto a Spherisorb 5-mm ODS2 4.6 by 250 mm CS. venezuelae and subsequent DNA microarray analysis were carried out as described previously  and homogenized using a FastPrep instrument (BIO 101). Two pulses of 30 s of intensity 6.0 were applied, with cooling down for 1 min on ice between pulses. The supernatants were centrifuged for 10 min at 13,000 rpm and then treated according to the instructions given in the RNeasy kit . The RNA samples were treated with DNase I (Invitrogen) until they were free of DNA contamination. The RNA was quantified, and equal amounts from each sample were converted to cDNA, according to the manufacturer's instructions . The oligonucleotide pairs listed in Table S1 in the supplemental material were used to amplify the genes representing each of the putative operons within the CHL biosynthetic cluster, as well as the left and right flanking genes . Amplification was also attempted using the same oligonucleotide pairs on RNA samples that had not been treated with reverse transcriptase to confirm a lack of DNA contamination.eviously . RNA waseviously  from 2.5Streptomyces sp. strain OH-4156  adjacent to the previously identified CHL biosynthetic gene cluster (sven0916–sven0928) that were absent in the nonproducing strain  with 44% amino acid sequence identity to StrR, the pathway-specific activator of the streptomycin biosynthetic gene cluster in Streptomyces griseus, a phosphopantetheinyl transferase (PPTase) (sven0914), and a Na+/H+ antiporter (sven0913). In contrast, the expression patterns of sven0909–sven0912, all encoding hypothetical proteins, were different from those of the known CHL biosynthetic genes, suggesting that they may not be part of the CHL cluster  and sven0913–sven0915 (Δsven0913–Δsven0915) in E. coli, and the mutated cosmids were transferred to S. coelicolor M1152 by conjugation, yielding M1583 (Δsven0909–Δsven0912) and M1584 (Δsven0913–Δsven0915), respectively. The integration of each cosmid was confirmed by PCR. The two strains, together with S. coelicolor M1152 carrying pAH91 (M1581), were grown in GYM liquid medium , the putative transcriptional activator, abolished antibiotic activity (sven0914 (to give M1586), coding for a putative PPTase, or sven0915 (yielding M1587), encoding a putative Na+/H+ antiporter, decreased the level of CHL production to around 40% of that observed with the wild-type gene cluster. Presumably, the roles that Sven0914 and Sven0915 play in CHL biosynthesis can be at least partially substituted by proteins encoded elsewhere in the S. coelicolor genome.To investigate the individual roles of activity  and CHL activity . In contsven0913 and sven0915) or from the constitutive ermE* promoter (sven0914). In each case, CHL production was restored to the level observed with the wild-type gene cluster , consistent with the proposed role of sven0913 as a cluster-situated transcriptional activator of the CHL biosynthetic gene cluster.As mentioned previously, we were unable to detect chloramphenicol production in nditions . Since opIJ10257  under thoduction . The consven0913 plays a role in activating transcription of the CHL biosynthetic gene cluster, real-time PCR (RT-PCR) analysis was performed on the following strains: wild-type S. venezuelae, M1582 (S. venezuelae with constitutive expression of sven0913), M1581 (S. coelicolor M1152 carrying pAH91), and M1585 (M1581 with sven0913 deleted). RNA was extracted from each of the cultures and RT-PCR conducted using primers amplifying the transcripts from several regions of the CHL gene cluster as well as from flanking genes . While the constitutive expression of sven0913 in S. venezuelae resulted in elevated levels of expression of the genes predicted to lie within the CHL gene cluster, it had no effect on the flanking genes sven0912 and sven0930 (sven0913 in S. coelicolor (M1585) reduced the levels of expression of the predicted CHL biosynthetic genes but had no effect on those of the flanking genes. These results confirm that Sven0913 is a previously unidentified transcriptional activator that plays a crucial role in the regulation of CHL biosynthesis.To confirm that sven0930 . ConversS. venezuelae (GenBank accession no. FR845719) had identified an additional previously undetected gene in the CHL biosynthetic gene cluster, sven0925, whose 108-amino-acid product failed to show clear similarity to the proteins of known function in the NCBI database. A more recent BLASTp search revealed a large number of homologues present in the genome sequences, some of which were annotated as acyl carrier proteins (ACPs). Based on this observation, Sven0925 was subjected to analysis using the Phyre2 protein structure prediction server (http://www.sbg.bio.ic.ac.uk/phyre2). The program predicts with 97% to 98% confidence that Sven0925 is a homologue of structurally characterized ACPs from a variety of bacterial species; for example, 77 residues of Sven0925 (71% of its sequence) were modeled with 98.0% confidence to the structure of the holo-acyl carrier protein ne2163 from Nitrosomonas europaea . Moreover, Sven0925 possesses a conserved serine at position 50 that is predicted to be the catalytic residue needed for the addition of the 4′-phosphopantetheine moiety required for the activity of the holo-ACP . Together with the expression profile of sven0925, which conforms to that of the other CHL biosynthetic genes, we conclude that Sven0925 is indeed an ACP involved in CHL biosynthesis  suggested that sven0929 might be part of the CHL biosynthetic gene cluster. This inference was further supported by the microarray analysis  to the active holo form by covalent attachment of a coenzyme-A-derived phosphopantetheine group to a specific serine residue  lacks a sven0914 homologue, although all of the other genes required for CHL production are present and arranged in precisely the same manner as in S. venezuelae . A BLASTp search to find the possible homologues of Sven0914 in the heterologous host S. coelicolor identified Sco6673, with 54% amino acid sequence identity. A Pfam database search (sven0914 still results in some CHL production in the heterologous host (the Sco6673 and the Sco6673-like proteins of S. ambofaciens share 83% identity).Interestingly, not all natural products that require the activity of a PPTase for their synthesis contain the corresponding gene within their biosynthetic gene clusters, and instead, they utilize a PPTase encoded elsewhere in the genome. Consequently, it is not too surprising that the deletion of CC 39149   synthase and 4-amino-4-deoxychorismate synthase, were present in the cml gene cluster , sven0926 (cmlJ), sven0927 (cmlK), and sven0929 (cmlT), as well as sven0928 (cmlS), encoding a flavin-dependent halogenase. All five of these genes are clustered together and may well be contained in a single operon, consistent with a common function. Based on the similarity of CmlK to the adenylating enzymes PchD and DhbE, which utilize salicylic acid and 2,3-dihydroxybenzoic acid as substrates, respectively, Piraee, White, and Vining  mutants of S. venezuelae. BldM and WhiI are both orphan atypical response regulators that play a crucial role in morphological differentiation. Recent work has demonstrated that BldM and WhiI can form a heterodimer that activates transcription from so-called group II promoters, presumably integrating signals from two distinct developmental pathways  is directly activated by BldN at the onset of development (bldN on CHL gene transcription is presumably mediated through decreased levels of bldM transcription. Similarly, the transcription of whiI is activated from a single WhiG-dependent promoter (whiG on cml gene transcription.Surprisingly, microarray analyses revealed that the expression of the CHL biosynthetic gene cluster was markedly enhanced in pathways . Chromatelopment , 38, andpromoter , presuma


Intelligence is a fundamental ability that sets humans apart from other animal species. Despite its importance in defining human behaviour, the neural networks responsible for intelligence are not well understood. The dominant view from neuroimaging work suggests that intelligent performance on a range of tasks is underpinned by segregated interactions in a fronto-parietal network of brain regions. Here we asked whether fronto-parietal interactions associated with intelligence are ubiquitous, or emerge from more widespread associations in a task-free context. First we undertook an exploratory mapping of the existing literature on functional connectivity associated with intelligence. Next, to empirically test hypotheses derived from the exploratory mapping, we performed network analyses in a cohort of 317 unrelated participants from the Human Connectome Project. Our results revealed a novel contribution of across-network interactions between default-mode and fronto-parietal networks to individual differences in intelligence at rest. Specifically, we found that greater connectivity in the resting state was associated with higher intelligence scores. Our findings highlight the need to broaden the dominant fronto-parietal conceptualisation of intelligence to encompass more complex and context-specific network dynamics. Human intelligence can be broadly defined as the capacity to understand complex ideas, adapt effectively to the environment and engage in complex reasoning7101213Functional magnetic resonance imaging (fMRI) has been used to examine the relationship between individual differences in intelligence and brain activity during the engagement of cognitive abilities such as working memory1717Recent studies have investigated the relationship between individual intelligence scores and patterns of functional connectivity during resting state scans10141220212223Here we used convergent approaches to assess whether the P-FIT can be extended to task-free (resting state) contexts. We started by conducting an exploratory mapping of previous findings from studies that had investigated the relationship between resting state functional connectivity and measures of intelligence. Specifically, we mapped significant pairwise connections from four previous studies see  into a vResults from our mapping of studies  assessinCorrelations between lower resting state functional connectivity  and higher intelligence scores  involvedThough suggestive, the above results represent merely the overlap of findings from a small number of studies with varying sample sizes and regions of interest. Thus, to empirically test the hypothesis that resting state connectivity correlates of intelligence extend beyond the fronto-parietal network, we utilized resting state data from the Human Connectome Project. Specifically, we assessed positive and negative linear relationships between whole-brain resting state functional connectivity and intelligence scores of 317 unrelated participants .positive connectivity values were associated with higher intelligence scores (r = 0.38).Functional connectivity in a concentrated resting state network comprising regions of the fronto-parietal (yellow), default-mode (red), and cortex not associated with any specific network . No networks showed significant negative associations between intelligence and functional connectivity using these lower statistical thresholds.No associations were found between increased intelligence and decreased resting state functional connectivity. Considering the large sample size this is unlikely to be related to a lack of statistical power. Nevertheless, we performed the NBS again with two lower exploratory statistical thresholds Recent evidence suggests that the default-mode and frontal-parietal networks represent overarching systems of the brain, composed of several sub-networks that dynamically interact273032161734cooperation  between distinct brain regions comprising default-mode and fronto-parietal networks in the resting state are associated with higher intelligence scores. This pattern of connectivity-intelligence associations is consistent with other findings suggesting that higher global network efficiency is related to higher general intelligence measures373940Somewhat at odds with this functional dichotomy between fronto-parietal and default-mode network activity, our analyses suggest that greater How the brain self-reorganizes to achieve optimal configurations of functional networks across individuals with varying levels of intelligence is an open question. Recent neuroimaging work has suggested that transient cooperation between different neural systems, including fronto-parietal, cingulo-opercular and default-mode networks, is integral to complex cognitive tasks such as reasoning4239A strength of the current work is the use of a statistically robust network-based method to isolate brain-intelligence associations at rest. While sensitive to network-level associations between functional connectivity and intelligence, our approach may have overlooked edge-specific associations detected in previous work see . For exa10In summary, our study provides a novel characterization of large-scale networks that explain individual differences in intelligence in a state of rest. Our results suggest that intelligence is supported by activity within a diffuse neural system comprised of brain regions encompassing fronto-parietal and default-mode networks. Consistent with these findings, we propose the influential parieto-frontal intelligence theory (P-FIT) may need to be extended to address context-specific network interactions. The functional links between transitions from diffuse resting state dynamics and more segregated task dynamics and intelligence will be an important topic for ongoing research.® (Thomson Reuter) and Scopus® (Elsevier), and was last updated the 9th of December 2015. Corresponding authors were contacted and asked to provide additional details or whole-brain results if these were not included in the published papers. Our final sample included data from 207 healthy adult participants. In one case10For the explorative mapping of data on the relationship between intelligence scores and intrinsic neural activity we performed a manual literature search of English-language peer-reviewed fMRI studies linking measures of pairwise resting state functional connectivity with behavioural measures of intelligence in healthy human adults . The lit3) of the MNI sphere. In instances where several parcels were implicated from a single coordinate, only the parcel that overlapped the most was included. Edges were drawn between implicated parcels using their associated r-values from the original studies. Note that changing our criteria for an overlapping region, either by increasing or decreasing the voxel limit, or by increasing the sphere size to 10 mm, yielded very similar results to those reported below.Cortical regions resulting from the above mapping were transposed into a common functional brain parcellation comprising 333 cortical regions26age = 28.43 years, SDage = 3.77, rangeage = 22–36 years). Two relevant behavioural tasks were used as measures of general intelligence. The Penn’s Progressive Matrices (PMAT), a shortened version of Raven’s Progressive Matricesr = 0.38, p < 0.0001).We next conducted an analysis of data from a large, independent sample of healthy adult participants, to examine associations between intelligence and functional connectivity across the whole brain in a task-free context. The relationship between measures of intelligence and neural activity was assessed using high quality resting state fMRI data from the HCP3 voxels)5153101223Data consisted of whole brain echo-planar images (EPIs) with sub-second temporal resolution (time repetition of 720 ms) and high spatial resolution (2 mmhttps://sites.google.com/site/bctnet/comparison/nbs). The NBS is a powerful and sensitive statistical tool that controls for Type I error at the network-level. The use of NBS represents a distinct advantage in term of sensitivity over previous studies that corrected for multiple comparisons at the edge level10To assess the relationship between resting state functional connectivity and individual intelligence scores we used the network based statistic (NBS56http://immersive.erc.monash.edu.au/neuromarvl/) and in-house Matlab scripts.Figures were generated using BrainNet ViewerHow to cite this article: Hearne, L. J. et al. Functional brain networks related to individual differences in human intelligence at rest. Sci. Rep.6, 32328; doi: 10.1038/srep32328 (2016).


Among people living with HIV/AIDS, illicit drug use is a risk for sub-optimal treatment outcomes. However, few studies have examined the relative contributions of different patterns of drug use on adherence to antiretroviral therapy (ART). We sought to estimate the effect of different types of illicit drug use on adherence in a setting of universal free HIV/AIDS treatment and care.Using data from ongoing prospective cohorts of HIV-positive illicit drug users linked to comprehensive pharmacy dispensation records in Vancouver, Canada, we examined factors associated with ≥95% prescription refill adherence using generalized estimating equations (GEE) logistic regression.Between 1996 and 2013, 692 ART-exposed individuals were followed for a median of 42.7 months (Interquartile Range: 29.1–71.7). In multivariable GEE analyses, heroin injection  as well as cocaine injection  were associated with lower likelihoods of optimal adherence. Methadone maintenance therapy  was associated with a greater likelihood of adherence.Periods of heroin and cocaine injection appeared to have the most deleterious impact upon antiretroviral adherence. The findings point to the need for improved access to treatment for heroin use disorder, particularly methadone, and highlight the need to identify strategies to support ART adherence among cocaine injectors. The development of antiretroviral therapy (ART) has led to substantial declines in HIV/AIDS-associated morbidity and mortality among many groups in many settings worldwide . AdherenIn many studies, active illicit drug use among individuals living with HIV/AIDS is associated with decreased access to HIV treatment, reduced medication adherence and increased mortality . AlthougData for these analyses were derived from the Vancouver Injection Drug Users Study (VIDUS) and AIDS Care Cohort to evaluate Exposure to Survival Services (ACCESS), two ongoing prospective observational cohorts of illicit drug users in Vancouver, Canada. Beginning in 1996, persons who had injected illicit drugs other than cannabinoids in the previous month were recruited into VIDUS. Since 2005, persons who used illicit drugs and were HIV-seropositive at baseline or during the study are subsequently followed in ACCESS. Both cohorts use identical recruitment and follow-up procedures to allow for combined analyses.Described in detail previously -10, the The present analyses considered all HIV-seropositive participants who had been dispensed at least one day of antiretroviral therapy (ART) between May 1996 and May 2013. The primary outcome of interest in this study was adherence to ART in the six month period prior to each study interview. As in previous studies , we estiExplanatory variables of interest included socio-demographic data: age (per 10 years older), gender , and Caucasian ancestry (yes vs. no). Variables related to drug use characteristics included: heroin injection (yes vs. no), cocaine injection (yes vs. no), amphetamine injection , crack cocaine smoking (yes vs. no), and enrollment in methadone maintenance therapy (yes vs. no). All drug use characteristics defined above were treated as time-updated based on questionnaire data pertaining to the six month period prior to each study interview ,13. We aAs a first step, we visually inspected trends in the proportion of all participants reporting heroin injecting, cocaine injecting, crack smoking and amphetamine injecting at each interview period. Next, we visually inspected changes in median adherence rate at each interview period over time.p-values are two-sided.Since serial measures for each subject  were available for many participants, we estimated the relationships between different socio-demographic, drug use and clinical factors and adherence using generalized estimating equations (GEE) with a logit-link function. As a first step, we conducted bivariable GEE analyses to determine which variables were statistically associated with adherence in unadjusted analyses. To adjust for potential confounding and identify factors that were associated with the outcome, all significant variables in the bivariable analyses were considered in the full multivariable model. With the drug use patterns being forced into the model, a backwards model selection procedure was used to identify the multivariate model with the best overall fit as indicated by the lowest quasilikelihood under the independence model criterion value. All statistical analyses were performed using the SAS software version 9.3 . All During the study period, 692 individuals were included in these analysis, among whom 213 (30.8%) were female, the median age at baseline was 42 years (Inter-quartile Range (IQR): 35–47) and the median follow-up duration was 42.7 months (IQR: 29.1–71.7). At baseline (Table The bivariable and multivariable GEE analyses of adherence to ART are shown in Table In this long-running community-recruited study of illicit drug users linked to comprehensive HIV clinical records in a setting of universal free HIV/AIDS treatment and care, we observed that periods of heroin and cocaine injection were independently and negatively associated with the likelihood of optimal adherence to ART. We did not observe a statistical relationship between periods of either amphetamine injection or crack cocaine smoking and the likelihood of optimal adherence to ART. Meanwhile, engagement in methadone maintenance therapy was independently and positively associated with optimal adherence to ART.Our finding of strong and independent links between heroin use, engagement in methadone maintenance and the likelihood of optimal ART adherence suggests that the expansion of evidence-based addiction treatment strategies, such as methadone, will likely improve retention in HIV/AIDS care among opioid-dependent illicit drug users -19. ImprIn our study, we did not observe a statistical relationship between amphetamine use and adherence to ART. Crystal methamphetamine use is particularly prevalent among some populations of people living with HIV/AIDS and persons at risk for HIV, especially men who have sex with men. -23 NumerAlthough we observed increasing levels of adherence over time, a substantial proportion of all participants displayed sub-optimal adherence to prescribed treatment at every interview period. Our model indicates that specific drug use patterns each had different relationships to antiretroviral adherence. For example, while periods of heroin injection were 23% less likely to be characterized by ≥95% adherence, there was no significant difference between levels of optimal adherence during periods of amphetamine injection. Unfortunately, our model does not offer specific insights into possible explanations for these divergent results. Thus, future research should investigate the possible behavioural-, psychologic-, social- and structural-level barriers and facilitators of adherence for different groups of illicit drug users in order to optimize substance abuse treatment and support ART adherence.There are some limitations to this study to note. Participant selection for this observational study was not random and the results of this study cannot be generalized to the wider population of HIV-positive illicit drug users. Regarding associations drawn from the results of the study, there is potential for unmeasured confounding. In an attempt to minimize the impact of confounding on the observed relationships we used multivariable modelling. Also, we have previously observed that at least 95% adherence to prescribed ART is strongly associated with viral suppression and survival ,16. NeweTo conclude, our study utilized data from a long-running community-recruited prospective cohort of HIV-seropositive illicit drug users in the setting of free and universal access to HIV care. Periods of injection heroin use and injection cocaine use were both independently and negatively associated with a lower likelihood of optimal adherence to ART while engagement in MMT was associated with higher levels of optimal adherence. Given the importance of addressing substance use in the community and the proven success of MMT at reducing the risk of drug-related harms, our findings support the need to spur efforts to improve access to treatment for problematic substance use among individuals living with HIV/AIDS.


Redox signaling has been widely reported to be involved in modulation of the cardiovascular system in both healthy and disease conditions. Redox imbalance such as oxidative stress contributes to the pathological process of many cardiovascular-related diseases such as diabetes, heart failure, and hypertension while also playing a negative role in the aging process –4. OveraIn addition to identification and description of central mechanisms mediated by redox signaling, understanding how sex differences also contribute to these physiological processes is becoming increasingly important. In this special issue, original research articles by F. Hao et al. and S. Dai et al. highlight that female sex hormones, such as estrogen, are critical to the operation of central neural pathways that modulate cardiovascular function through antioxidant and anti-inflammation mechanisms. The article by F. Hao et al. reports that estrogen replacement reduces oxidative stress in the rostral ventrolateral medulla (RVLM) as well as sympathetic outflow in ovariectomized (OVX) rats. These findings elucidate a potential antioxidant role of estrogen in the central nervous system. The second article by S. Dai et al. suggests that the chronic antihypertensive and anti-inflammatory effects of Compound 21 (a selective angiotensin type 2 receptor (AT2R) agonist) in DOCA/NaCl-induced female hypertensive rats may function through female sex hormones. As evidence, these beneficial effects of Compound 21 could be abolished by OVX. in vivo and in vitro exacerbates ROS production and neuronal apoptosis, indicating that HDAC6 might serve as a new antioxidant target in neurotrauma. In another report in this issue, Y. Ma and colleagues provided evidence that an active compound found in figs and mulberries, known as morin, abrogated immune cell migration as well as cytokine production and ROS attenuating inflammatory processes in a model of asthma. This study highlights a novel profile of morin as a potent antioxidant and anti-inflammatory agent for asthma. In addition, a paper by M. Zhao et al. investigated the potential redox signaling pathways involved in cardiomyocyte hypertrophy induced by palmitic acid, the most common saturated fatty acid found in animals, plants, and microorganisms. These investigators demonstrated that palmitic acid-induced cardiomyocyte hypertrophy is associated with activation of necroptosis markers such as receptor interacting protein kinases (RIPK) 1 and 3. Moreover, the authors observed crosstalk between ER stress and necroptosis induced by palmitic acid. They suggested that mammalian target of rapamycin (mTOR) signaling was a key factor mediating the palmitic acid-induced activation of necroptosis and cardiac hypertrophy. Finally, the article by T. Dong et al. provided a very interesting observation that renal denervation slowed the development of deep venous thrombosis (DVT). They utilized several different measures to show that the mechanisms related to DVT are related to oxidative stress, as antioxidants such as tempol  could achieve a protective effect similar to renal denervation. These findings extend our understanding of how neurons may affect the circulation via interactions with redox signaling.Several papers in this special issue also described new insights into the antioxidant therapeutic potential of various pharmacological and dietary compounds in spinal cord injury, asthma, and cardiomyopathy. M. Su and colleagues demonstrated a role for histone deacetylase-6 (HDAC6) in the protection of neurons after spinal cord injury and the resulting hypoxia-ischemia insult. The investigators demonstrate that inhibition of HDAC6The final topic of this special issue is related to exercise. It is well known that acute exercise can dramatically increase ROS production whereas long-term exercise training (ExT) plays an antioxidant and anti-inflammatory role –7. Two pIn summary, the articles presented in this special issue highlight the current advances in the research fields of redox biology, neural science, and exercise physiology. These articles enrich our understanding of how redox signaling interacts within the nervous system both at rest and during exercise. Moreover, the works highlight the therapeutic potential of antioxidants as effective treatments for various cardiovascular and neural diseases.