Nanodomain cAMP signalling in cardiac pathophysiology: potential for developing targeted therapeutic interventions.

3', 5'-cyclic adenosine monophosphate (cAMP) mediates the effects of sympathetic stimulation on the rate and strength of cardiac contraction. Beyond this pivotal role, in cardiac myocytes cAMP also orchestrates a diverse array of reactions to various stimuli. To ensure specificity of response, the cAMP signaling pathway is intricately organized into multiple, spatially confined, subcellular domains, each governing a distinct cellular function. In this review, we describe the molecular components of the cAMP signalling pathway, how they organized are inside the intracellular space and how they achieve exquisite regulation of signalling within nanometer-size domains. We delineate the key experimental findings that lead to the current model of compartmentalised cAMP signaling and we offer an overview of our present understanding of how cAMP nanodomains are structured and regulated within cardiac myocytes. Furthermore, we discuss how compartmentalized cAMP signaling is affected in cardiac disease and consider the potential therapeutic opportunities arising from understanding such organization. By exploiting the nuances of compartmentalized cAMP signaling, novel and more effective therapeutic strategies for managing cardiac conditions may emerge. Finally, we highlight the unresolved questions and hurdles that must be addressed to translate these insights into interventions that may benefit patients.

Recurring EPHB1 mutations in human cancers alter receptor signalling and compartmentalisation of colorectal cancer cells.

BACKGROUND: Ephrin (EPH) receptors have been implicated in tumorigenesis and metastasis, but the functional understanding of mutations observed in human cancers is limited. We previously demonstrated reduced cell compartmentalisation for somatic EPHB1 mutations found in metastatic colorectal cancer cases. We therefore integrated pan-cancer and pan-EPH mutational data to prioritise recurrent EPHB1 mutations for functional studies to understand their contribution to cancer development and metastasis. METHODS: Here, 79,151 somatic mutations in 9,898 samples of 33 different tumour types were analysed with a bioinformatic pipeline to find 3D-mutated cluster pairs and hotspot mutations in EPH receptors. From these, 15 recurring EPHB1 mutations were stably expressed in colorectal cancer followed by confocal microscopy based in vitro compartmentalisation assays and phospho-proteome analysis. RESULTS: The 3D-protein structure-based bioinformatics analysis resulted in 63% EPHB1 mutants with compartmentalisation phenotypes vs 43% for hotspot mutations. Whereas the ligand-binding domain mutations C61Y, R90C, and R170W, the fibronectin domain mutation R351L, and the kinase domain mutation D762N displayed reduced to strongly compromised cell compartmentalisation, the kinase domain mutations R743W and G821R enhanced this phenotype. While mutants with reduced compartmentalisation also had reduced ligand induced receptor phosphorylation, the enhanced compartmentalisation was not linked to receptor phosphorylation level. Phosphoproteome mapping pinpointed the PI3K pathway and PIK3C2B phosphorylation in cells harbouring mutants with reduced compartmentalisation. CONCLUSIONS: This is the first integrative study of pan-cancer EPH receptor mutations followed by in vitro validation, a robust way to identify cancer-causing mutations, uncovering EPHB1 mutation phenotypes and demonstrating the utility of protein structure-based mutation analysis in characterization of novel cancer genes. Video Abstract.

A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterisation in different cell types.

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.

Mitochondrial protein import as a quality control sensor.

Mitochondria are organelles involved in various functions related to cellular metabolism and homoeostasis. Though mitochondria contain own genome, their nuclear counterparts encode most of the different mitochondrial proteins. These are synthesised as precursors in the cytosol and have to be delivered into the mitochondria. These organelles hence have elaborate machineries for the import of precursor proteins from cytosol. The protein import machineries present in both mitochondrial membrane and aqueous compartments show great variability in pre-protein recognition, translocation and sorting across or into it. Mitochondrial protein import machineries also interact transiently with other protein complexes of the respiratory chain or those involved in the maintenance of membrane architecture. Hence mitochondrial protein translocation is an indispensable part of the regulatory network that maintains protein biogenesis, bioenergetics, membrane dynamics and quality control of the organelle. Various stress conditions and diseases that are associated with mitochondrial import defects lead to changes in cellular transcriptomic and proteomic profiles. Dysfunction in mitochondrial protein import also causes over-accumulation of precursor proteins and their aggregation in the cytosol. Multiple pathways may be activated for buffering these harmful consequences. Here, we present a comprehensive picture of import machinery and its role in cellular quality control in response to defective mitochondrial import. We also discuss the pathological consequences of dysfunctional mitochondrial protein import in neurodegeneration and cancer.

Keep your friends close: Host compartmentalisation of microbial communities facilitates decoupling from effects of habitat fragmentation.

Root-associated fungal communities modify the climatic niches and even the competitive ability of their hosts, yet how the different components of the root microbiome are modified by habitat loss remains a key knowledge gap. Using principles of landscape ecology, we tested how free-living versus host-associated microbes differ in their response to landscape heterogeneity. Further, we explore how compartmentalisation of microbes into specialised root structures filters for key fungal symbionts. Our study demonstrates that free-living fungal community structure correlates with landscape heterogeneity, but that host-associated fungal communities depart from these patterns. Specifically, biotic filtering in roots, especially via compartmentalisation within specialised root structures, decouples the biogeographic patterns of host-associated fungal communities from the soil community. In this way, even as habitat loss and fragmentation threaten fungal diversity in the soils, plant hosts exert biotic controls to ensure associations with critical mutualists, helping to preserve the root mycobiome.

Myocardial subcellular glycogen distribution and sarcoplasmic reticulum Ca2+ handling: effects of ischaemia, reperfusion and ischaemic preconditioning.

Ischaemic preconditioning (IPC) protects against myocardial ischaemia-reperfusion injury. The metabolic and ionic effects of IPC remain to be clarified in detail. We aimed to investigate the effect of IPC (2 times 5 min ischaemia) on the subcellular distribution of glycogen and Ca2+-uptake and leakiness by the sarcoplasmic reticulum (SR) in response to ischaemia-reperfusion in cardiomyocytes of isolated perfused rat hearts (Wistar rats, 335 ± 25 g). As estimated by quantitative transmission electron microscopy, the pre-ischaemic contribution [%, mean (95% CI)] of three sub-fractions of glycogen relative to total glycogen was 50 (39:61) as subsarcolemmal, 41 (31:50) as intermyofibrillar, and 9 (5:13) as intramyofibrillar glycogen. After 25 min of ischaemia, the relative contribution (%) of subsarcolemmal glycogen decreased to 39 (32:47) in control hearts (Con) and to 38 (31:45) in IPC. After 15 min reperfusion the contribution of subsarcolemmal glycogen was restored to pre-ischaemic levels in IPC hearts, but not in Con hearts. IPC increased the left ventricular developed pressure following ischaemia-reperfusion compared with Con. In saponin-skinned cardiomyocyte bundles, ischaemia reduced the SR Ca2+-uptake rate, with no effect of IPC. However, IPC reduced a SR Ca2+-leakage at pre-ischaemia, after ischaemia and during reperfusion. In conclusion, subsarcolemmal glycogen was preferentially utilised during sustained myocardial ischaemia. IPC improved left ventricular function reflecting reduced ischaemia-reperfusion injury, mediated a re-distribution of glycogen towards a preferential storage within the subsarcolemmal space during reperfusion, and lowered SR Ca2+-leakage. Under the present conditions, we found no temporal associations between alterations in glycogen localisation and SR Ca2+ kinetics.

Sitting in the driver's seat: Manipulation of mammalian cell Rab GTPase functions by apicomplexan parasites.

Many intracellular microbial pathogens subvert, disrupt or otherwise modulate host membrane trafficking pathways to establish a successful infection. Among them, bacteria that are trapped in a phagosome during mammalian cell invasion, disengage the programmed degradation process by altering the identity of their replicative niche through the exclusion or recruitment of specific Rab GTPases to their vacuole. Many viruses co-opt essential cellular trafficking pathways to perform key steps in their lifecycles. Among protozoan parasites, Apicomplexa are obligate intracellular microbes that invade mammalian cells by creating a unique, nonfusogenic membrane-bound compartment that protects the parasites straightaway from lysosomal degradation. Recent compelling evidence demonstrates that apicomplexan parasites are master manipulators of mammalian Rab GTPase proteins, and benefit or antagonise Rab functions for development within host cells. This review covers the exploitation of mammalian Rab proteins and vesicles by Apicomplexa, focusing on Toxoplasma, Neospora, Plasmodium and Theileria parasites.

A number's game: Towards a solution to the policing of ringfenced COVID Hotspots.

ISSUED ADDRESSED: COVID-19 has highlighted the disruptive, cross-sectorial effects a sudden-onset pandemic has on a globally interconnected world. The control of community transmission requires the identification and isolation of hotspots, commonly achieved through blanket measures or through ring-fencing. A simpler and more readily policeable, and geographically more flexible system is needed that allows both law enforcement and the public to detect people moving outside the ringfenced areas. METHODS: A narrative examination of the border closure between New South Wales and Victoria in June to September 2020. RESULTS: Enforcement of people's movement in and out of ring-fenced areas relies on voluntary, ethical compliance coupled with legal prosecution of violators. Despite extensive community health promotion for COVID-safe behaviour, the events of 2020 showed multiple, flagrant violations which were caught during random spot checks, as well as at the Murray River, a hard border set up along readily controllable and patrollable features (bridges). CONCLUSIONS: Given that most medium and long-distance movement in Australia is vehicular-based, this paper advocates for the introduction of European-style local government area based car registration which makes 'out of bounds' vehicle traffic readily recognisable by their number plates. SO WHAT?: Public health promotion, coupled with convenient and ubiquitous observation and enforcement tool, is likely to moderate community behaviour and ensure increased compliance with the directives of health authorities and associated promotion of COVID-safe behaviour.

Heterogeneity in subcellular muscle glycogen utilisation during exercise impacts endurance capacity in men.

KEY POINTS: When muscle biopsies first began to be used routinely in research on exercise physiology five decades ago, it soon become clear that the muscle content of glycogen is an important determinant of exercise performance. Glycogen particles are stored in distinct pools within the muscles, but the role of each pool during exercise and how this is affected by diet is unknown. Here, the effects of diet and exercise on these pools, as well as their relation to endurance during prolonged cycling were examined. We demonstrate here that an improved endurance capacity with high carbohydrate loading is associated with a temporal shift in the utilisation of the distinct stores of glycogen pools and is closely linked to the content of the glycogen pool closest to actin and myosin (intramyofibrillar glycogen). These findings highlight the functional importance of distinguishing between different subcellular microcompartments of glycogen in individual muscle fibres. ABSTRACT: In muscle cells, glycogen is stored in three distinct subcellular pools: between or within myofibrils (inter- and intramyofibrillar glycogen, respectively) or beneath the sarcolemma (subsarcolemmal glycogen) and these pools may well have different functions. Here, we investigated the effect of diet and exercise on the content of these distinct pools and their relation to endurance capacity in type 1 and 2 muscle fibres. Following consumption of three different diets (normal, mixed diet = MIX, high in carbohydrate = HIGH, or low in carbohydrate = LOW) for 72 h, 11 men cycled at 75% of V̇O2 max until exhaustion. The volumetric content of the glycogen pools in muscle biopsies obtained before, during, and after exercise were quantified by transmission electron micrographs. The mean (SD) time to exhaustion was 150 (30), 112 (22), and 69 (18) minutes in the HIGH, MIX and LOW trials, respectively (P < 0.001). As shown by multiple regression analyses, the intramyofibrillar glycogen content in type 1 fibres, particularly after 60 min of exercise, correlated most strongly with time to exhaustion. In the HIGH trial, intramyofibrillar glycogen was spared during the initial 60 min of exercise, which was associated with levels and utilisation of subsarcolemmal glycogen above normal. In all trials, utilisation of subsarcolemmal and intramyofibrillar glycogen was more pronounced than that of intermyofibrillar glycogen in relative terms. In conclusion, the muscle pool of intramyofibrillar glycogen appears to be the most important for endurance capacity in humans. In addition, a local abundance of subsarcolemmal glycogen reduces the utilisation of intramyofibrillar glycogen during exercise.

Implementation of OIE international standards: challenges and opportunities for monitoring.

The World Organisation for Animal Health (OIE) is a major actor in international cooperation to improve animal health and welfare throughout the world. The OIE sets international standards to support Member Countries in their efforts to prevent and control animal diseases, strengthen Veterinary Services and Aquatic Animal Health Services, and facilitate safe international trade. Member Countries face many challenges in the implementation of OIE standards. Poor governance and a lack of resources and technical capacity are often major constraints. Trade concerns raised at the World Trade Organization (WTO) can also be a signal that countries are experiencing difficulties in implementing international standards. In May 2018, the World Assembly of OIE Delegates adopted a resolution recommending the establishment of an observatory to monitor the implementation of OIE standards. This monitoring mechanism will help the OIE to improve its international standard-setting process and identify the capacity-building needs of Member Countries. Monitoring implementation will be challenging as the OIE does not prescribe a specific procedure for implementing OIE standards. World Organisation for Animal Health Member Countries use a range of approaches to implement OIE standards, because of differences in animal health situations, legal frameworks and procedures, trade profiles, and acceptable levels of risk. Given this complexity, this article proposes a 'cross-over' approach to monitoring implementation that would require the collection of information from various trusted sources, such as the World Animal Health Information System (WAHIS), the OIE Performance of Veterinary Services (PVS) Pathway mission reports and the WTO Sanitary and Phytosanitary Information Management System database. This approach aims to document what is currently happening and to identify potential patterns in Member Country practices when implementing OIE standards.

L'Organisation mondiale de la santé animale (OIE) est un acteur majeur de la coopération internationale oeuvrant à l'amélioration de la santé et du bien-être animal dans le monde. L'OIE élabore des normes internationales visant à soutenir les efforts déployés par ses Membres pour prévenir et lutter contre les maladies animales, renforcer les Services vétérinaires et les Services chargés de la santé des animaux aquatiques, et faciliter un commerce international sûr. Les Membres rencontrent de nombreuses difficultés lors de la mise en oeuvre des normes de l'OIE. Une mauvaise gouvernance et l'insuffisance des ressources et des capacités techniques constituent souvent des contraintes majeures. Les préoccupations commerciales soulevées au sein de l'Organisation mondiale du commerce (OMC) sont également révélatrices des difficultés rencontrées par les pays pour mettre en oeuvre les normes internationales. En mai 2018, l'Assemblée mondiale des Délégués de l'OIE a adopté une résolution recommandant la création d'un observatoire destiné à assurer le suivi de la mise en oeuvre des normes de l'OIE. Grâce à ce mécanisme de suivi, l'OIE sera à même d'améliorer le processus d'élaboration de ses normes internationales et d'identifier les besoins de ses Membres en matière de renforcement des capacités. Le suivi de la mise en oeuvre ne sera pas une tâche facile, dans la mesure où l'OIE ne prescrit pas de procédure spécifique pour appliquer ses normes. Les Membres de l'OIE recourent à diverses approches pour mettre en oeuvre les normes de l'OIE, dictées par des différences dans les situations zoosanitaires, les cadres et procédures juridiques, les profils commerciaux et les niveaux de risque considérés comme acceptables. Face à une telle complexité, les auteurs proposent une méthode transversale de suivi de la mise en oeuvre, qui passe par la collecte d'informations auprès de diverses sources fiables telles que le Système mondial d'information sanitaire de l'OIE (WAHIS), les rapports de mission du Processus de l'OIE pour évaluer la Performance des Services vétérinaires (Processus PVS) et la base de données de l'OMC de gestion des renseignements sanitaires et phytosanitaires. Cette approche vise à documenter ce qui se fait actuellement et à identifier d'éventuelles tendances dans les pratiques des Membres en matière de mise en oeuvre des normes de l'OIE.

La Organización Mundial de Sanidad Animal (OIE) es uno de los actores principales de la cooperación internacional para mejorar la sanidad y el bienestar animales en todo el mundo. La OIE elabora normas internacionales destinadas a apoyar el esfuerzo de los Países Miembros por prevenir y combatir las enfermedades animales, fortalecer los Servicios Veterinarios y los Servicios de sanidad de los animales acuáticos y facilitar un comercio internacional seguro. Los Países Miembros afrontan numerosos desafíos a la hora de implementar las normas de la OIE. La insuficiencia de mecanismos de gobernanza y la falta de recursos y capacidad técnica constituyen a menudo obstáculos de gran calado. Las preocupaciones comerciales expresadas ante la Organización Mundial del Comercio (OMC) también pueden ser una señal de las dificultades que afrontan los países para implementar las normas internacionales. En mayo de 2018, la Asamblea Mundial de Delegados de la OIE aprobó una resolución en la que recomendaba la creación de un observatorio para el seguimiento de la implementación de las normas de la OIE. Este mecanismo ayudará a la OIE a mejorar su proceso de elaboración de normas internacionales y a identificar las necesidades de capacidades específicas de los Países Miembros. El seguimiento de la implementación no será fácil, pues la OIE no prescribe ningún procedimiento específico para poner en práctica sus normas. Los Países Miembros utilizan modalidades distintas debido a las diferencias en cuanto a situaciones zoosanitarias, ordenamiento y procedimientos jurídicos, perfiles comerciales y niveles de riesgo aceptables. Teniendo en cuenta esta compleja situación, los autores proponen aquí un enfoque «transversal¼ de seguimiento de la implementación de las normas, que requeriría reunir información de diversas fuentes fidedignas, como el Sistema Mundial de Información Zoosanitaria (WAHIS), los informes de las misiones de evaluación de las prestaciones de los Servicios Veterinarios (Proceso PVS) de la OIE y la base de datos del sistema de la OMC de gestión de información relativa a las medidas sanitarias y fitosanitarias. Tal enfoque tiene por objetivo documentar lo que está ocurriendo actualmente e identificar posibles patrones en las prácticas de los Países Miembros al implementar las normas de la OIE.

Multi-Compartment, Early Disruption of cGMP and cAMP Signalling in Cardiac Myocytes from the mdx Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is the most frequent and severe form of muscular dystrophy. The disease presents with progressive body-wide muscle deterioration and, with recent advances in respiratory care, cardiac involvement is an important cause of morbidity and mortality. DMD is caused by mutations in the dystrophin gene resulting in the absence of dystrophin and, consequently, disturbance of other proteins that form the dystrophin-associated protein complex (DAPC), including neuronal nitric oxide synthase (nNOS). The molecular mechanisms that link the absence of dystrophin with the alteration of cardiac function remain poorly understood but disruption of NO-cGMP signalling, mishandling of calcium and mitochondrial disturbances have been hypothesized to play a role. cGMP and cAMP are second messengers that are key in the regulation of cardiac myocyte function and disruption of cyclic nucleotide signalling leads to cardiomyopathy. cGMP and cAMP signals are compartmentalised and local regulation relies on the activity of phosphodiesterases (PDEs). Here, using genetically encoded FRET reporters targeted to distinct subcellular compartments of neonatal cardiac myocytes from the DMD mouse model mdx, we investigate whether lack of dystrophin disrupts local cyclic nucleotide signalling, thus potentially providing an early trigger for the development of cardiomyopathy. Our data show a significant alteration of both basal and stimulated cyclic nucleotide levels in all compartments investigated, as well as a complex reorganization of local PDE activities.

Dynamic Synthetic Cells Based on Liquid-Liquid Phase Separation.

Living cells have long been a source of inspiration for chemists. Their capacity of performing complex tasks relies on the spatiotemporal coordination of matter and energy fluxes. Recent years have witnessed growing interest in the bottom-up construction of cell-like models capable of reproducing aspects of such dynamic organisation. Liquid-liquid phase-separation (LLPS) processes in water are increasingly recognised as representing a viable compartmentalisation strategy through which to produce dynamic synthetic cells. Herein, we highlight examples of the dynamic properties of LLPS used to assemble synthetic cells, including their biocatalytic activity, reversible condensation and dissolution, growth and division, and recent directions towards the design of higher-order structures and behaviour.

Nuclear localisation of 53BP1 is regulated by phosphorylation of the nuclear localisation signal.

BACKGROUND INFORMATION: Repair of damaged DNA is essential for maintaining genomic stability. TP53-binding protein 1 (53BP1) plays an important role in repair of the DNA double-strand breaks. Nuclear localisation of 53BP1 depends on importin ß and nucleoporin 153, but the type and location of 53BP1 nuclear localisation signal (NLS) have yet to be determined. RESULTS: Here, we show that nuclear import of 53BP1 depends on two basic regions, namely 1667-KRK-1669 and 1681-KRGRK-1685, which are both needed for importin binding. Lysine 1667 is essential for interaction with importin and its substitution to arginine reduced nuclear localisation of 53BP1. Furthermore, we have found that CDK1-dependent phosphorylation of 53BP1 at S1678 impairs importin binding during mitosis. Phosphorylation-mimicking mutant S1678D showed reduced nuclear localisation, suggesting that phosphorylation of the NLS interferes with nuclear import of the 53BP1 CONCLUSIONS: We show that 53BP1 contains a classical bipartite NLS 1666-GKRKLITSEEERSPAKRGRKS-1686, which enables the importin-mediated nuclear transport of 53BP1. Additionally, we found that posttranslational modification within the NLS region can regulate 53BP1 nuclear import. SIGNIFICANCE: Our results indicate that integrity of the NLS is important for 53BP1 nuclear localisation. Precise mapping of the NLS will facilitate further studies on the effect of posttranslational modifications and somatic mutations on the nuclear localisation 53BP1 and DNA repair.

Perspective on architecture and assembly of membrane contact sites.

Membrane contact sites (MCS) are platforms of physical contact between different organelles. They are formed through interactions involving lipids and proteins, and function in processes such as calcium and lipid exchange, metabolism and organelle biogenesis. In this article, we discuss emerging questions regarding the architecture, organisation and assembly of MCS, such as: What is the contribution of different components to the interaction between organelles? How is the specific composition of different types of membrane contacts sites established and maintained? How are proteins and lipids spatially organised at MCS and how does that influence their function? How dynamic are MCS on the molecular and ultrastructural level? We highlight current state of research and point out experimental approaches that promise to contribute to a spatiomechanistic understanding of MCS functions.

Meeting after meeting: 20 years of discoveries by the members of the Exocytosis-Endocytosis Club.

Twenty years ago, a group of French cell biologists merged two scientific clubs with the aim of bringing together researchers in the fields of Endocytosis and Exocytosis. Founded in 1997, the first annual meeting of the Exocytosis Club was held in 1998. The Endocytosis Club held quarterly meetings from its founding in 1999. The first joint annual meeting of the Exocytosis-Endocytosis Club took place in Paris in April, 2001. What started as a modest gathering of enthusiastic scientists working in the field of cell trafficking has gone from strength to strength, rapidly becoming an unmissable yearly meeting, vividly demonstrating the high quality of science performed in our community and beyond. On the occasion of the 20th meeting of our club, we want to provide historic insight into the fields of exocytosis and endocytosis, and by extension, to subcellular trafficking, highlighting how French scientists have contributed to major advances in these fields. Today, the Exocytosis-Endocytosis Club represents a vibrant and friendly community that will hold its 20th meeting at the Presqu'Ile de Giens, near Toulon in the South of France, on May 11-13, 2017.

Development of a Minimal Photosystem for Hydrogen Production in Inorganic Chemical Cells.

Inorganic chemical cells (iCHELLs) are compartment structures consisting of polyoxometalates (POMs) and cations, offering structured and confined reaction spaces bounded by membranes. We have constructed a system capable of efficient anisotropic and hierarchical photo-induced electron transfer across the iCHELL membrane. Mimicking photosynthesis, our system uses proton gradients between the compartment and the bulk to drive efficient conversion of light into chemical energy, producing hydrogen upon irradiation. This illustrates the power of the iCHELL approach for catalysis, where the structure, compartmentalisation and variation in possible components could be utilised to approach a wide range of reactions.

Heterogeneous Pd catalysts as emulsifiers in Pickering emulsions for integrated multistep synthesis in flow chemistry.

Within the "compartmentalised smart factory" approach of the ONE-FLOW project the implementation of different catalysts in "compartments" provided by Pickering emulsions and their application in continuous flow is targeted. We present here the development of heterogeneous Pd catalysts that are ready to be used in combination with biocatalysts for catalytic cascade synthesis of active pharmaceutical ingredients (APIs). In particular, we focus on the application of the catalytic systems for Suzuki-Miyaura cross-coupling reactions, which is the key step in the synthesis of the targeted APIs valsartan and sacubitril. An immobilised enzyme will accomplish the final product formation via hydrolysis. In order to create a large interfacial area for the catalytic reactions and to keep the reagents separated until required, the catalyst particles are used to stabilise Pickering emulsions of oil and water. A set of Ce-Sn-Pd oxides with the molecular formula Ce0.99-x Sn x Pd0.01O2-δ (x = 0-0.99) has been prepared utilising a simple single-step solution combustion method. The high applicability of the catalysts for different functional groups and their minimal leaching behaviour is demonstrated with various Suzuki-Miyaura cross-coupling reactions in batch as well as in continuous flow employing the so-called "plug & play reactor". Finally, we demonstrate the use of these particles as the sole emulsifier of oil-water emulsions for a range of oils.

The influence of nuclear compartmentalisation on stochastic dynamics of self-repressing gene expression.

Gene expression is an inherently noisy process. This noise is generally thought to be deleterious as precise internal regulation of biochemical reactions is essential for cell growth and survival. Self-repression of gene expression, which is the simplest form of a negative feedback loop, is commonly believed to be employed by cellular systems to decrease the stochastic fluctuations in gene expression. When there is some delay in autoregulation, it is also believed that this system can generate oscillations. In eukaryotic cells, mRNAs that are synthesised in the nucleus must be exported to the cytoplasm to function in protein synthesis, whereas proteins must be transported into the nucleus from the cytoplasm to regulate the expression levels of genes. Nuclear transport thus plays a critical role in eukaryotic gene expression and regulation. Some recent studies have suggested that nuclear retention of mRNAs can control noise in mRNA expression. However, the effect of nuclear transport on protein noise and its interplay with negative feedback regulation is not completely understood. In this paper, we systematically compare four different simple models of gene expression. By using simulations and applying the linear noise approximation to the corresponding chemical master equations, we investigate the influence of nuclear import and export on noise in gene expression in a negative autoregulatory feedback loop. We first present results consistent with the literature, i.e., that negative feedback can effectively buffer the variability in protein levels, and nuclear retention can decrease mRNA noise levels. Interestingly we find that when negative feedback is combined with nuclear retention, an amplification in gene expression noise can be observed and is dependant on nuclear translocation rates. Finally, we investigate the effect of nuclear compartmentalisation on the ability of self-repressing genes to exhibit stochastic oscillatory dynamics.

Intracanalicular Optic Nerve Swelling and Signal Change in Fulminant Untreated Idiopathic Intracranial Hypertension.

The role of the optic canal in the pathogenesis of papilloedema has been under scrutiny recently. Whether a larger canal precedes more severe papilloedema or is the result of bone remodelling from chronically raised pressure across a pressure gradient is not clear. The authors present the magnetic resonance imaging findings of a 29-year-old female with fulminant and untreated idiopathic intracranial hypertension. Imaging showed focal expansion and intrinsic signal changes of the intracanalicular optic nerve. The authors discuss the possibility of either fluid accumulation within the optic nerves from a water hammer effect across blocked optic canals resulting from the steep pressure gradient or opticomalacia (optic nerve softening) from chronic ischaemia.

Ribonucleoprotein bodies are phased in.

Intracellular compartments are necessary for the regulation of many biochemical processes that ensure cell survival, growth and proliferation. Compartmentalisation is commonly achieved in organelles with defined lipid membranes, such as mitochondria, endoplasmic reticulum or the Golgi apparatus. While these organelles are responsible for many localised biochemical processes, recent evidence points to another class of compartments that lack membrane boundaries. The structure and content of these bodies depend on their function and subcellular localisation, but they mainly incorporate proteins and RNA. Examples of these ribonucleoprotein bodies (RNPBs) include eukaryotic mRNA processing bodies (P-bodies) and stress granules (SGs). While most of these structures have been widely studied for their capacity to bind, store and process mRNAs under different conditions, their biological functions and physical properties are poorly understood. Recent intriguing data suggest that liquid-liquid phase separation (LLPS) represents an important mechanism seeding the formation and defining the function of RNPBs. In this review, we discuss how LLPS is transforming our ideas about the biological functions of SGs and P-bodies and their link to diseases.