Coalescence of the sites of cowpea mosaic virus RNA replication into a cytopathic structure

Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this proces

Regulated Membrane Localization of Tiam1, Mediated by the NH2-terminal Pleckstrin Homology Domain, Is Required for Rac-dependent Membrane Ruffling and C-Jun NH2-terminal Kinase Activation

Rho-like GTPases, including Cdc42, Rac, and Rho, regulate signaling pathways that control actin cytoskeletal structures and transcriptional activation. The Tiam1 gene encodes an activator of Rac1, and similarly to constitutively activated (V12)Rac1, overexpression of Tiam1 in fibroblasts induces the formation of membrane ruffles. Tiam1 contains a Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, hallmarks for activators of Rho-like GTPases. Unique for Tiam1 are an additional PH domain and a Discs-large homology region in the NH2-terminal part of the protein. Here we show that both in fibroblasts and COS cells, membrane localization of Tiam1 is required for the induction of membrane ruffling. A detailed mutational analysis, in combination with confocal laser scanning microscopy and immunoelectron microscopy, demonstrates that the NH2-terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH2-terminal PH domain of Tiam1 can be functionally replaced by the myristoylated membrane localization domain of c-Src, indicating that the primary function of this PH domain is to localize the protein at the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor stimulation induces membrane translocation of Tiam1. Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK). This Rac-dependent stimulation of JNK also requires membrane association of Tiam1. We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways

Lokomat guided gait in hemiparetic stroke patients:the effects of training parameters on muscle activity and temporal symmetry

Purpose: The Lokomat is a commercially available robotic gait trainer, applied for gait rehabilitation in post-stroke hemiparetic patients. Selective and well-dosed clinical use of the Lokomat training parameters, i.e. guidance, speed and bodyweight support, requires a good understanding of how these parameters affect the neuromuscular control of post-stroke hemiparetic gait. Materials and methods: Ten stroke patients (unilateral paresis, 7 females, 64.5 ± 6.4 years, >3months post-stroke, FAC scores 2–4)) walked in the Lokomat under varying parameter settings: 50% or 100% guidance, 0.28 or 0.56m/s, 0% or 50% bodyweight support. Electromyography was recorded bilaterally from Gluteus Medius, Biceps Femoris, Vastus Lateralis, Medial Gastrocnemius, and Tibialis Anterior. Pressure sensors placed under the feet were used to determine the level of temporal gait symmetry. Results: Varying guidance and bodyweight support had little effect on muscle activity, but increasing treadmill speed led to increased activity in both the affected (Biceps Femoris, Medial Gastrocnemius, Tibialis Anterior) and unaffected leg (all muscles). The level of temporal symmetry was unaffected by the parameter settings. Conclusions: The Lokomat training parameters are generally ineffective in shaping short term muscle activity and step symmetry patients with hemiparetic stroke, as speed is the only parameter that significantly affects muscular amplitude. Trial Registration: d.n.a.IMPLICATIONS FOR REHABILITATIONThe Lokomat is a commercially available gait trainer that can be used for gait rehabilitation in post-stroke hemiparetic patients.This study shows that muscle amplitude is generally low during Lokomat guided walking, and that treadmill Speed is the main training parameter to influence muscular output in stroke patients during Lokomat walking.Varying Guidance and Bodyweight Support within a clinical relevant range barely affected muscle activity, and temporal step symmetry was unaffected by variation in any of the training parameters.Based on the findings it is advised to increase speed as early as possible during Lokomat therapy, or use other means (e.g. feedback or instructions) to stimulate active involvement of patients during training. The Lokomat is a commercially available gait trainer that can be used for gait rehabilitation in post-stroke hemiparetic patients. This study shows that muscle amplitude is generally low during Lokomat guided walking, and that treadmill Speed is the main training parameter to influence muscular output in stroke patients during Lokomat walking. Varying Guidance and Bodyweight Support within a clinical relevant range barely affected muscle activity, and temporal step symmetry was unaffected by variation in any of the training parameters. Based on the findings it is advised to increase speed as early as possible during Lokomat therapy, or use other means (e.g. feedback or instructions) to stimulate active involvement of patients during training.</p

Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis

Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis

An energy vision for a planet under pressure

Worldwide, global energy systems face an array of challenges, from access for the poor to reliability and security. Meanwhile, the provision of energy creates local human and ecological health impacts as well as dangerous global climate change. Addressing these issues simultaneously will require a fundamental transformation of the energy system. Recent assessments show that such a transformation is achievable in technological and economic terms, but it will present formidable supply- and demand-side challenges as well as problems of governance, transparency and reliability across scales. This policy brief presents a long-term vision for the energy system and describes the elements required for the transition towards this vision. To succeed, this transformation must integrate several key components, including a focus on high levels of energy efficiency and the scale up of investments in technology deployment as well as research, development and demonstration (RD&D)

Справа Івана Дзюби

У статті автор, використовуючи документи Галузевого державного архіву СБ України, досліджує постать видатного літературознавця, громадського діяча Івана Дзюби у контексті боротьби співробітників органів держбезпеки УРСР з «українським буржуазним націоналізмом».В статье автор, используя документы Отраслевого государственного архива СБ Украины, исследует личность выдающегося литературоведа, общественного деятеля Ивана Дзюбы в контексте борьбы сотрудников органов госбезопасности УССР с «украинским буржуазным национализмом».Using the documents of State branch archive of State Security of Ukraine, the author investigates the personality of Ivan Dzyuba during the struggle of KGB of the UkSSR against the «Ukrainian bourgeois nationalism»

Nanomedicines: An approach to treat placental insufficiency and the current challenges

INTRODUCTION: Preeclampsia and fetal growth restriction are common pregnancy complications that significantly impact perinatal health and offspring development later in life. The origin of these complex syndromes overlap in placental insufficiency. Progress in developing treatments for maternal, placental or fetal health is mainly limited by the risk of maternal and fetal toxicity. Nanomedicines are a promising approach to safely treat pregnancy complications since they can regulate drug interaction with the placenta to enhance efficacy of the treatment while minimizing exposure of the fetus. METHODS: This narrative review discusses the current developments and challenges of nanomedicines during pregnancy with a focus on preclinical models of placenta insufficiency syndromes. Firstly, we outline the safety requirements and potential therapeutic maternal and placental targets. Secondly, we review the prenatal therapeutic effects of the nanomedicines that have been tested in experimental models of placental insufficiency syndromes. RESULTS: The majority of liposomes and polymeric drug delivery system show promising results regarding the prevention of trans-placental passage nanomedicines in uncomplicated and complicated pregnancies. The others two studied classes, quantum dots and silicon nanoparticles, have been investigated to a limited extent in placental insufficiency syndromes. Characteristics of the nanoparticles such as charge, size, and timing of administration have been shown to influence the trans-placental passage. The few available preclinical therapeutic studies on placental insufficiency syndromes predominantly show beneficial effects of nanomedicines on both maternal and fetal health, but demonstrate contradicting results on placental health. Interpretation of results in this field is complicated by the fact that results are influenced by the choice of animal species and model, gestational age, placental maturity and integrity, and nanoparticle administration route. CONCLUSION: Nanomedicines form a promising therapeutic approach during (complicated) pregnancies mainly by reducing fetal toxicity and regulating drug interaction with the placenta. Different nanomedicines have been proven to effectively prevent trans-placental passage of encapsulated agents. This can be expected to dramatically reduce risks for fetal adverse effects. Furthermore, a number of these nanomedicines positively impacted maternal and fetal health in animal models for placental insufficiency. Demonstrating that effective drug concentrations can be reached in the target tissue. While these first animal studies are encouraging, more research is needed to better understand the influence of the pathophysiology of this multi-factorial disease before implementation in clinical practice can be considered. Therefore, extensive evaluation of safety and efficacy of these targeted nanoparticles is needed within multiple animal, in vitro, and/or ex vivo models. This may be complemented by diagnostic tools to assess the disease status to identify the best time to initiate treatment. Together these investigations should contribute to building confidence in the safety of nanomedicines for treating mother and child, as safety has, understandably, the highest priority in this sensitive patient groups