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Deforestation and forest degradation in the tropics may substantially alter soil N-oxide emissions. It is particularly relevant to accurately quantify those changes to properly account for them in a REDD+ climate change mitigation scheme that provides financial incentives to reduce the emissions. With this study we provide updated land use (LU)-based emission rates (104 studies, 392 N2O and 111 NO case studies), we determine the trend and magnitude of flux changes with land-use change (LUC) using a meta-analysis approach (44 studies, 135 N2O and 37 NO cases) and evaluate biophysical drivers of N2O and NO emissions and emission changes for the tropics. The average N2O and NO emissions in intact upland tropical forest amounted to 2.0 ± 0.2 (n = 90) and 1.7 ± 0.5 (n = 36) kg N ha−1 yr−1, respectively. In agricultural soils annual N2O emissions were exponentially related to N fertilization rates and average water-filled pore space (WFPS) whereas in non-agricultural sites a Gaussian response to WFPS fit better with the observed NO and N2O emissions. The sum of soil N2O and NO fluxes and the ratio of N2O to NO increased exponentially and significantly with increasing nitrogen availability (expressed as NO3− / [NO3−+NH4+]) and WFPS, respectively; following the conceptual Hole-In-the-Pipe model. Nitrous and nitric oxide fluxes did not increase significantly overall as a result of LUC (Hedges's d of 0.11 ± 0.11 and 0.16 ± 0.19, respectively), however individual LUC trajectories or practices did. Nitrous oxide fluxes increased significantly after intact upland forest conversion to croplands (Hedges's d = 0.78 ± 0.24) and NO increased significantly following the conversion of low forest cover (secondary forest younger than 30 years, woodlands, shrublands) (Hedges's d of 0.44 ± 0.13). Forest conversion to fertilized systems significantly and highly raised both N2O and NO emission rates (Hedges's d of 1.03 ± 0.23 and 0.52 ± 0.09, respectively). Changes in nitrogen availability and WFPS were the main factors explaining changes in N2O emissions following LUC, therefore it is important that experimental designs monitor their spatio-temporal variation. Gaps in the literature on N oxide fluxes included geographical gaps (Africa, Oceania) and LU gaps (degraded forest, wetland (notably peat) forest, oil palm plantation and soy cultivation).

Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids., (© 2024. The Author(s).)

When we think about cancer, the link to development might not immediately spring to mind. Yet, many foundational concepts in cancer biology trace their roots back to developmental processes. Several defining traits of cancer were indeed initially observed and studied within developing embryos. As our comprehension of embryonic mechanisms deepens, it not only illuminates how and why cancer cells hijack these processes but also spearheads the emergence of innovative technologies for modeling and comprehending tumor biology. Among these technologies are stem cell-based models, made feasible through our grasp of fundamental mechanisms related to embryonic development. The intersection between cancer and stem cell research is evolving into a tangible synergy that extends beyond the concepts of cancer stem cells and cell-of-origin, offering novel tools to unravel the mechanisms of cancer initiation and progression., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Charcot-Marie-Tooth disease is the most common inherited disorder of the PNS. CMT1A accounts for 40-50% of all cases and is caused by a duplication of the PMP22 gene on chromosome 17, leading to dysmyelination in the PNS. Patient-derived models to study such myelination defects are lacking as the in vitro generation of human myelinating Schwann cells has proved to be particularly challenging. Here, we present an induced pluripotent stem cell-derived organoid culture, containing various cell types of the PNS, including myelinating human Schwann cells, which mimics the human PNS. Single-cell analysis confirmed the PNS-like cellular composition and provides insight into the developmental trajectory. We used this organoid model to study disease signatures of CMT1A, revealing early ultrastructural myelin alterations, including increased myelin periodic line distance and hypermyelination of small axons. Furthermore, we observed the presence of onion-bulb-like formations in a later developmental stage. These hallmarks were not present in the CMT1A-corrected isogenic line or in a CMT2A iPSC line, supporting the notion that these alterations are specific to CMT1A. Downregulation of PMP22 expression using short-hairpin RNAs or a combinatorial drug consisting of baclofen, naltrexone hydrochloride and D-sorbitol was able to ameliorate the myelin defects in CMT1A-organoids. In summary, this self-organizing organoid model can capture biologically meaningful features of the disease and capture the physiological complexity, forms an excellent model for studying demyelinating diseases and supports the therapeutic approach of reducing PMP22 expression., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Objective: We performed a 1-year evaluation of a novel strategy of simultaneously analyzing single nucleotide variants (SNVs), copy number variants (CNVs) and copy-number-neutral Absence-of-Heterozygosity from Whole Exome Sequencing (WES) data for prenatal diagnosis of fetuses with ultrasound (US) anomalies and a non-causative QF-PCR result., Methods: After invasive diagnostics, whole exome parent-offspring trio-sequencing with exome-wide CNV analysis was performed in pregnancies with fetal US anomalies and a non-causative QF-PCR result (WES-CNV). On request, additional SNV-analysis, restricted to (the) requested gene panel(s) only (with the option of whole exome SNV-analysis afterward) was performed simultaneously (WES-CNV/SNV) or as rapid SNV-re-analysis, following a normal CNV analysis., Results: In total, 415 prenatal samples were included. Following a non-causative QF-PCR result, WES-CNV analysis was initially requested for 74.3% of the chorionic villus (CV) samples and 45% of the amniotic fluid (AF) samples. In case WES-CNV analysis did not reveal a causative aberration, SNV-re-analysis was requested in 41.7% of the CV samples and 17.5% of the AF samples. All initial analyses could be finished within 2 weeks after sampling. For SNV-re-analysis during pregnancy, turn-around-times (TATs) varied between one and 8 days., Conclusion: We show a highly efficient all-in-one WES-based strategy, with short TATs, and the option of rapid SNV-re-analysis after a normal CNV result., (© 2023 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Advancements in lab-on-a-chip technologies have revolutionized the single-cell analysis field. However, an accessible platform for in-depth screening and specific retrieval of single cells, which moreover enables studying diverse cell types and performing various downstream analyses, is still lacking. As a solution, FLUIDOT is introduced, a versatile microfluidic platform incorporating customizable microwells, optical tweezers and an interchangeable cell-retrieval system. Thanks to its smart microfluidic design, FLUIDOT is straightforward to fabricate and operate, rendering the technology widely accessible. The performance of FLUIDOT is validated and its versatility is subsequently demonstrated in two applications. First, drug tolerance in yeast cells is studied, resulting in the discovery of two treatment-tolerant populations. Second, B cells from convalescent COVID-19 patients are screened, leading to the discovery of highly affine, in vitro neutralizing monoclonal antibodies against SARS-CoV-2. Owing to its performance, flexibility, and accessibility, it is foreseen that FLUIDOT will enable phenotypic and genotypic analysis of diverse cell samples and thus elucidate unexplored biological questions., (© 2023 Wiley-VCH GmbH.)

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field., Competing Interests: Declaration of interests S.W.S. is on the scientific advisory council of the Lewy Body Dementia Association and the MSA Coalition. S.W.S. is an editorial board member for the Journal of Parkinson Disease and JAMA Neurology. S.W.S. received research support from Cerevel Therapeutics. M.K. serves on the scientific advisory boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, and Alector and is a consultant to Modulo Bio and Recursion Therapeutics. Participation by researchers from Data Tecnica International, LLC in this project was part of a competitive contract awarded to Data Tecnica International, LLC by the National Institutes of Health to support open science research. M.A.N. also currently serves on the scientific advisory board for Clover Therapeutics and is an advisor to Neuron23 Inc. E.A. is founder, shareholder, and scientific advisor of Cholestenix, Ltd., (Published by Elsevier Inc.)

Genetic pain loss includes congenital insensitivity to pain (CIP), hereditary sensory neuropathies and, if autonomic nerves are involved, hereditary sensory and autonomic neuropathy (HSAN). This heterogeneous group of disorders highlights the essential role of nociception in protecting against tissue damage. Patients with genetic pain loss have recurrent injuries, burns and poorly healing wounds as disease hallmarks. CIP and HSAN are caused by pathogenic genetic variants in >20 genes that lead to developmental defects, neurodegeneration or altered neuronal excitability of peripheral damage-sensing neurons. These genetic variants lead to hyperactivity of sodium channels, disturbed haem metabolism, altered clathrin-mediated transport and impaired gene regulatory mechanisms affecting epigenetic marks, long non-coding RNAs and repetitive elements. Therapies for pain loss disorders are mainly symptomatic but the first targeted therapies are being tested. Conversely, chronic pain remains one of the greatest unresolved medical challenges, and the genes and mechanisms associated with pain loss offer new targets for analgesics. Given the progress that has been made, the coming years are promising both in terms of targeted treatments for pain loss disorders and the development of innovative pain medicines based on knowledge of these genetic diseases., (© 2022. Springer Nature Limited.)

Antibodies (Abs) are among the most important class of biologicals, showcasing a high therapeutic and diagnostic value. In the global therapeutic Ab market, fully-human monoclonal Abs (FH-mAbs) are flourishing thanks to their low immunogenicity and high specificity. The rapidly emerging field of single-cell technologies has paved the way to efficiently discover mAbs by facilitating a fast screening of the antigen (Ag)-specificity and functionality of Abs expressed by B cells. This review summarizes the principles and challenges of the four key concepts to discover mAbs using these technologies, being confinement of single cells using either droplet microfluidics or microstructure arrays, identification of the cells of interest, retrieval of those cells and single-cell sequence determination required for mAb production. This review reveals the enormous potential for mix-and-matching of the above-mentioned strategies, which is illustrated by the plethora of established, highly integrated devices. Lastly, an outlook is given on the many opportunities and challenges that still lie ahead to fully exploit miniaturized single-cell technologies for mAb discovery.

Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C-terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient-derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V-containing proteins. We identified multiple IxI/V-bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V-binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein-protein interactions., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Alphabaculoviruses ( Baculoviridae ) are pathogenic DNA viruses of Lepidoptera that have applications as the basis for biological insecticides and expression vectors in biotechnological processes. These viruses have a characteristic physical structure that facilitates the transmission of groups of genomes. We demonstrate that coinfection of a susceptible insect by two different alphabaculovirus species results in the production of mixed-virus occlusion bodies containing the parental viruses. This occurred between closely related and phylogenetically more distant alphabaculoviruses. Approximately half the virions present in proteinaceous viral occlusion bodies produced following coinfection of insects with a mixture of two alphabaculoviruses contained both viruses, indicating that the viruses coinfected and replicated in a single cell and were coenveloped within the same virion. This observation was confirmed by endpoint dilution assay. Moreover, both viruses persisted in the mixed-virus population by coinfection of insects during several rounds of insect-to-insect transmission. Coinfection by viruses that differed in genome size had unexpected results on the length of viral nucleocapsids, which differed from those of both parental viruses. These results have unique implications for the development of alphabaculoviruses as biological control agents of insect pests. IMPORTANCE Alphabaculoviruses are used as biological insecticides and expression vectors in biotechnology and medical applications. We demonstrate that in caterpillars infected with particular mixtures of viruses, the genomes of different baculovirus species can be enveloped together within individual virions and occluded within proteinaceous occlusion bodies. This results in the transmission of mixed-virus populations to the caterpillar stages of moth species. Once established, mixed-virus populations persist by coinfection of insect cells during several rounds of insect-to-insect transmission. Mixed-virus production technology opens the way to the development of custom-designed insecticides for control of different combinations of caterpillar pest species., (Copyright © 2021 American Society for Microbiology.)

Mauritia flexuosa palm swamp, the prevailing Peruvian Amazon peatland ecosystem, is extensively threatened by degradation. The unsustainable practice of cutting whole palms for fruit extraction modifies forest's structure and composition and eventually alters peat-derived greenhouse gas (GHG) emissions. We evaluated the spatiotemporal variability of soil N 2 O and CH 4 fluxes and environmental controls along a palm swamp degradation gradient formed by one undegraded site (Intact), one moderately degraded site (mDeg) and one heavily degraded site (hDeg). Microscale variability differentiated hummocks supporting live or cut palms from surrounding hollows. Macroscale analysis considered structural changes in vegetation and soil microtopography as impacted by degradation. Variables were monitored monthly over 3 years to evaluate intra- and inter-annual variability. Degradation induced microscale changes in N 2 O and CH 4 emission trends and controls. Site-scale average annual CH 4 emissions were similar along the degradation gradient (225.6 ± 50.7, 160.5 ± 65.9 and 169.4 ± 20.7 kg C ha -1  year -1 at the Intact, mDeg and hDeg sites, respectively). Site-scale average annual N 2 O emissions (kg N ha -1  year -1 ) were lower at the mDeg site (0.5 ± 0.1) than at the Intact (1.3 ± 0.6) and hDeg sites (1.1 ± 0.4), but the difference seemed linked to heterogeneous fluctuations in soil water-filled pore space (WFPS) along the forest complex rather than to degradation. Monthly and annual emissions were mainly controlled by variations in WFPS, water table level (WT) and net nitrification for N 2 O; WT, air temperature and net nitrification for CH 4 . Site-scale N 2 O emissions remained steady over years, whereas CH 4 emissions rose exponentially with increased precipitation. While the minor impact of degradation on palm swamp peatland N 2 O and CH 4 fluxes should be tested elsewhere, the evidenced large and variable CH 4 emissions and significant N 2 O emissions call for improved modeling of GHG dynamics in tropical peatlands to test their response to climate changes., (© 2020 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

In disease diagnostics, single- and multiplex nucleic acid (NA) detection, with the potential to discriminate mutated strands, is of paramount importance. Current techniques that rely on target amplification or protein-enzyme based signal amplification are highly relevant, yet still plagued by diverse drawbacks including erroneous target amplification, and the limited stability of protein enzymes. As a solution, we present a multicomponent nucleic acid enzymes (MNAzymes)-based system for singleplex and multiplex detection of NA targets in microwells down to femtomolar (fM) concentrations, without the need for any target amplification or protein enzymes, while operating at room temperature and with single base-pair resolution. After successful validation of the MNAzymes in solution, their performance was further verified on beads in bulk and in femtoliter-sized microwells. The latter is not only a highly simplified system compared to previous microwell-based bioassays but, with the detection limit of 180 fM, it is to-date the most sensitive NAzyme-mediated, bead-based approach, that does not rely on target amplification or any additional signal amplification strategies. Furthermore, we demonstrated, for the first time, multiplexed target detection in microwells, both from buffer and nasopharyngeal swab samples, and presented superior single base-pair resolution of this assay. Because of the design flexibility of MNAzymes and direct demonstration in swab samples, this system holds great promise for multiplexed detection in other clinically relevant matrices without the need for any additional NA or protein components. Moreover, these findings open up the potential for the development of next-generation, protein-free diagnostic tools, including digital assays with single-molecule resolution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Axonal transport is a highly complex process essential for sustaining proper neuronal functioning. Disturbances can result in an altered neuronal homeostasis, aggregation of cargoes, and ultimately a dying-back degeneration of neurons. The impact of dysfunction in axonal transport is shown by genetic defects in key proteins causing a broad spectrum of neurodegenerative diseases, including inherited peripheral neuropathies. In this review, we provide an overview of the cytoskeletal components, molecular motors and adaptor proteins involved in axonal transport mechanisms and their implication in neuronal functioning. In addition, we discuss the involvement of axonal transport dysfunction in neurodegenerative diseases with a particular focus on inherited peripheral neuropathies. Lastly, we address some recent scientific advances most notably in therapeutic strategies employed in the area of axonal transport, patient-derived iPSC models, in vivo animal models, antisense-oligonucleotide treatments, and novel chemical compounds.

Purpose: Characterization of virosomes, in late stage preclinical development as vaccines for Respiratory Syncytial Virus (RSV), with a membrane-incorporated synthetic monophosphoryl lipid A, 3D-PHAD® adjuvant., Methods: Virosomes were initially formed by contacting a lipid film containing 3D-PHAD® with viral membranes solubilized with the short chain phospholipid DCPC, followed by dialysis, later by adding solubilized 3D-PHAD to viral membranes, or to preformed virosomes from DMSO., Results: Virosomes formed from lipid films contained the membrane glycoproteins G and F, at similar F to G ratios but lower concentrations than in virus, and the added lipids, but only a fraction of the 3D-PHAD®. By single particle tracking (SPT), the virosome size distribution resembled that seen by cryo-electron microscopy, but dynamic light scattering showed much larger particles. These differences were caused by small virosome aggregates. Measured by SPT, virosomes were stable for 300 days. 3DPHAD ® incorporation in virosomes could be enhanced by providing the adjuvant from DCPC solubilized stock, but also by adding DMSO dissolved adjuvant to pre-formed virosomes. Virosomes with 0.1 mg/mg of 3D-PHAD®/viral protein from DMSO induced antibody titers similar to those by virosomes containing 0.2 mg/mg of DCPC-solubilized 3D-PHAD®., Conclusions: Stable 3D-PHAD® adjuvanted RSV virosomes can be formulated.

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch's postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.

Many biological materials are composite structures, interpenetrating networks of different types of fibers. The composite nature of such networks leads to superior mechanical properties, but the origin of this mechanical synergism is still poorly understood. Here we study soft composite networks, made by mixing two self-assembling fiber-forming components. We find that the elastic moduli of the composite networks significantly exceed the sum of the moduli of the two individual networks. This mechanical enhancement is in agreement with recent simulations, where it was attributed to a suppression of non-affine deformation modes in the most rigid fiber network due to the reaction forces in the softer network. The increase in affinity also causes a loss of strain hardening and an increase in the critical stress and stain at which the network fails.

The Glossinavirus (Glossina pallidipes salivary gland hypertrophy virus (GpSGHV)) is a rod-shaped enveloped insect virus containing a 190,032 bp-long, circular dsDNA genome. The virus is pathogenic for the tsetse fly Glossina pallidipes and has been associated with the collapse of selected mass-reared colonies. Maintenance of productive fly colonies is critical to tsetse and trypanosomiasis eradication in sub-Saharan Africa using the Sterile Insect Technique. Proteomics, an approach to define the expressed protein complement of a genome, was used to further our understanding of the protein composition, morphology, morphogenesis and pathology of GpSGHV. Additionally, this approach provides potential targets for novel and sustainable molecular-based antiviral strategies to control viral infections in tsetse colonies. To achieve this goal, identification of key protein partners involved in virus transmission is required. In this review, we integrate the available data on GpSGHV proteomics to assess the impact of viral infections on host metabolism and to understand the contributions of such perturbations to viral pathogenesis. The relevance of the proteome findings to tsetse and trypanosomiasis management in sub-Sahara Africa is also considered., (Copyright © 2012 International Atomic Energy Agency. Published by Elsevier Inc. All rights reserved.)

In animal cells a double-membrane-bound structure, the autophagosome, encloses a portion of the cytoplasm. The encapsulated material becomes digested after fusion of the autophagosome with a vesicle containing lytic enzymes. The autophagosome is then termed autolysosome. In intact plants, structures similar to animal autophagosomes/autolysosomes have been found only in a few types of cells. Additionally, some early papers indicated that plastids can function similar to autophagosomes/autolysosomes. Here, we report that plastids in Dendrobium cv. Lucky Duan petals produced an endocytosis-like invagination of the two outer membranes. The opening between the invagination space and the cytoplasm was almost isodiametric, less than 0.2 μm in diameter. The volume of the space formed by the invagination had a maximum of about half of the total plastid volume. Staining of the invagination lumen for acid phosphatase, a marker of organelles showing autophagic activity, was positive. Membranes and numerous ribosomes were observed inside the lumen of the invagination. The structure of the material inside the lumen varied from that of the cytoplasm to uniform electron-translucent, indicating that the enclosed cytoplasmic material became completely digested. No support was found for the idea that the material engulfed by the plastid or the whole plastid became transferred to a vacuole. Taken together, the data suggested the hypothesis that plastids in Dendrobium petal mesophyll cells can function in a way similar to both autophagosomes and autolysosomes in animal cells.

Dyslexia is the most common childhood learning disorder and it is a significantly heritable trait. At least nine chromosomal loci have been linked to dyslexia, and additional susceptibility loci on other chromosomes have been suggested. Within two of these loci, DYX1C1 (15q21) and ROBO1 (3p12) have recently been proposed as dyslexia candidate genes through the molecular analysis of translocation breakpoints in dyslexic individuals carrying balanced chromosomal translocations. Moreover, genetic association studies have indicated a cluster of five dyslexia candidate genes in another linkage region on chromosome 6p22, although there is currently no consensus about which of these five genes contributes to the genetic susceptibility for dyslexia. In this article, we report the identification of four new dyslexia candidate genes (PCNT, DIP2A, S100B, and PRMT2) on chromosome region 21q22.3 by FISH and SNP microarray analyses of a very small deletion in this region, which cosegregates with dyslexia in a father and his three sons., (2008 Wiley-Liss, Inc.)

Cowpea mosaic virus (CPMV) moves from cell to cell by transporting virus particles via tubules formed through plasmodesmata by the movement protein (MP). On the surface of protoplasts, a fusion between the MP and the green fluorescent protein forms similar tubules and peripheral punctate spots. Here it was shown by time-lapse microscopy that tubules can grow out from a subset of these peripheral punctate spots, which are dynamic structures that seem anchored to the plasma membrane. Fluorescence resonance energy transfer experiments showed that MP subunits interacted within the tubule, where they were virtually immobile, confirming that tubules consist of a highly organized MP multimer. Fluorescence recovery after photobleaching experiments with protoplasts, transiently expressing fluorescent plasma membrane-associated proteins of different sizes, indicated that tubules made by CPMV MP do not interact directly with the surrounding plasma membrane. These experiments indicated an indirect interaction between the tubule and the surrounding plasma membrane, possibly via a host plasma membrane protein.

The movement protein (MP) of Cowpea mosaic virus (CPMV) forms tubules through plasmodesmata in infected plants thus enabling virus particles to move from cell to cell. Localization studies of mutant MPs fused to GFP in protoplasts and plants identified several functional domains within the MP that are involved in distinct steps during tubule formation. Coinoculation experiments and the observation that one of the C-terminal deletion mutants accumulated uniformly in the plasma membrane suggest that dimeric or multimeric MP is first targeted to the plasma membrane. At the plasma membrane the MP quickly accumulates in peripheral punctuate spots, from which tubule formation is initiated. One of the mutant MPs formed tubules containing virus particles on protoplasts, but could not support cell-to-cell movement in plants. The observations that this mutant MP accumulated to a higher level in the cell than wt MP and did not accumulate in the cell wall opposite infected cells suggest that breakdown or disassembly of tubules in neighbouring, uninfected cells is required for cell-to-cell movement.

Cowpea mosaic virus (CPMV) derivatives expressing movement protein (MP) green fluorescent protein (GFP) fusions (MP:GFP) were used to study the intracellular targeting and localization of the MP in cowpea protoplasts and plants. In protoplasts, a virus coding for a wild type MP:GFP (MPfGFP) induced the formation of fluorescent tubular structures, which shows that subcellular targeting and tubule formation are not affected by fusion of GFP to the C-terminus of the MP. In plants, MPfGFP infections were mostly confined to single epidermal cells and failed to achieve a systemic infection, probably because the fusion of GFP to the MP interfered with MP-virion interaction. MP:GFP mainly accumulated in fluorescent spots in the cell wall of epidermal cells of inoculated leaves, which may represent short tubular structures in modified plasmodesmata. At the cuticle-side of epidermal cells tubular structures were detected indicating that tubule formation in plants, as in protoplasts, does not require the presence of functional plasmodesmata. Furthermore, results were obtained which indicate that CPMV MP:GFP is able to traffic from cell-to-cell by itself. The possible significance of this finding is discussed.

SUMMARY Taxonomy: Cowpea mosaic virus (CPMV) is the type member of the Comoviridae and bears a strong resemblance to animal picornaviruses, both in gene organization and in the amino acid sequence of replication proteins. Little systematic work has been done to compare isolates of the virus from different parts of the world. Physical properties: Purified preparations of virus contain three centrifugal components; empty protein shells without RNA (T) and two nucleoprotein components (M and B), containing 24% and 34% RNA, respectively. The icosahedral particles have with a diameter of 28 nm, consist of 60 copies of two coat proteins, and are heat stable. Hosts: CPMV causes one of the most commonly reported virus diseases of cowpea (Vigna unguiculata), in which it produces chlorotic spots with diffuse borders in inoculated primary leaves. Trifoliate leaves develop a bright yellow or light green mosaic of increasing severity in younger leaves. The host range is rather limited, and few hosts are known outside the Leguminosae. The virus is transmitted by various beetles with biting mouthparts. Reported in Africa, the Philippines and Iran. Is apparently absent from North and South America. Useful website: http://mmtsb.scripps.edu/viper/1cpmv.html (structure); http://image.fs.uidaho.edu/vide/descr254.htm (general information).

Sorting can be an important mechanism for the transfer of information from one level of biological organization to another. Here we study the algorithm underlying worker sorting in Leptothorax ant colonies. Worker sorting is related to task allocation and therefore to the adaptive advantages associated with an efficient system for the division of labour in ant colonies. We considered four spatially explicit individual-based models founded on two-dimensional correlated random walk. Our aim was to establish whether sorting at the level of the worker population could occur with minimal assumptions about the behavioural algorithm of individual workers. The behaviour of an individual worker in the models could be summarized by the rule "move if you can, turn always". We assume that the turning angle of a worker is individually specific and negatively dependent on the magnitude of an internal parameter micro which could be regarded as a measure of individual experience or task specialization. All four models attained a level of worker sortedness that was compatible with results from experiments onLeptothorax ant colonies. We found that the presence of a sorting pivot, such as the nest wall or an attraction force towards the centre of the worker population, was crucial for sorting. We make a distinction between such pivots and templates and discuss the biological implications of their difference.

Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotiana benthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis.

The movement protein (MP) of cowpea mosaic virus (CPMV) forms tubules on infected protoplasts and through plasmodesmata in infected plants. In protoplasts the MP fused to GFP (MP-GFP) was shown to localize in peripheral punctate structures and in long tubular structures extending from the protoplast surface. Using cytoskeletal assembly inhibitors (latrunculin B and oryzalin) and an inhibitor of the secretory pathway (brefeldin A), targeting of the MP to the peripheral punctate structures was demonstrated not to be dependent on an intact cytoskeleton or functional secretion pathway. Furthermore it was shown that a disrupted cytoskeleton had no effect on tubule formation but that the addition of brefeldin A severely inhibited tubule formation. The results presented in this paper suggest a role for a plasma membrane host factor in tubule formation of plant viral MPs., ((c) 2002 Elsevier Science (USA).)

The competence of a Frankliniella occidentalis and a Thrips tabaci population to transmit Tomato spotted wilt virus (TSWV) was analysed. Adults of the F. occidentalis population transmitted this virus efficiently, whereas those of the thelytokous T. tabaci population failed to transmit. TSWV replicated in the midgut of the larvae of both populations after ingestion of virus; however, lower amounts accumulated in T. tabaci larvae than in F. occidentalis larvae. The virus was almost undetectable in T. tabaci adults, whereas high titres were readily detected in the F. occidentalis adults. The first infections in F. occidentalis larvae were detected by immunocytochemical studies in midgut epithelial and subsequently in midgut muscle cells, the ligaments, and finally in the salivary glands. The infections were weaker in the midgut epithelial and muscle cells of T. tabaci larvae, followed by an almost complete absence of any infection in the ligaments, and a complete absence in the salivary glands. Studies by electron microscopy revealed the budding of some virus particles from the basal membrane of midgut epithelial cells of F. occidentalis larvae into the extracellular space of the basal labyrinth. Enveloped virus particles were also seen in midgut muscle cells of F. occidentalis larvae. They were not discerned in epithelial and muscle cells of T. tabaci larvae and adults. This study showed that the rate of virus replication in the midgut and the extent of virus migration from the midgut to the visceral muscle cells and the salivary glands are probably crucial factors in the determination of vector competence.

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.

A model for the maturation of tomato spotted wilt virus (TSWV) particles is proposed, mainly based on results with a protoplast infection system, in which the chronology of different maturation events could be determined. By using specific monoclonal and polyclonal antisera in immunofluorescence and electron microscopy, the site of TSWV particle morphogenesis was determined to be the Golgi system. The viral glycoproteins G1 and G2 accumulate in the Golgi prior to a process of wrapping, by which the viral nucleocapsids obtain a double membrane. In a later stage of the maturation, these doubly enveloped particles fuse to each other and to the endoplasmic reticulum to form singly enveloped particles clustered in membranes. Similarities and differences between the maturation of animal-infecting (bunya)viruses and plant-infecting tospoviruses are discussed.

The jellyfish green fluorescent protein (GFP) coding sequence was used to replace the coat protein (CP) genes in a full-length cDNA clone of CPMV RNA-2. Transcripts of this construct were replicated in the presence of RNA-1 in cowpea protoplasts, and GFP expression could be readily detected by fluorescent microscopy. It was not possible to infect cowpea plants with these transcripts, but combined with a mutant RNA-2, in which the 48-kDa movement protein (MP) gene has been deleted infection did occur. With this tripartite virus (CPMV-TRI) green fluorescent spots were visible under UV light on the inoculated leaf after 3 days and a few days later on the higher leaves. These results show that the polyproteins encoded by RNA-2 do not possess an essential function in the virus infection cycle and that there is, contrary to what we have found so far for the proteins encoded by RNA-1, no need for a tight regulation of the amounts of MP and CPs produced in a cell. Subsequently, the GFP gene was introduced between the MP and CP genes of RNA-2 utilizing artificial proteolytic processing sites for the viral proteinase. This CPMV-GFP was highly infectious on cowpea plants and the green fluorescent spots that developed on the inoculated leaves were larger and brighter than those produced by CPMV-TRI described above. When cowpea plants were inoculated with CPMV RNA-1 and RNA-2 mutants containing the GFP gene but lacking the CP or MP genes, only single fluorescent epidermal cells were detected between 2 and 6 days postinoculation. This experiment clearly shows that both the capsid proteins and the MP are absolutely required for cell-to-cell movement.

A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.

The coding regions for cowpea mosaic virus (CPMV) capsid proteins VP37 and VP23 were introduced separately into a transient plant expression vector containing an enhanced CaMV 358 promoter. Significant expression of either capsid protein was observed only in protoplasts transfected simultaneously with both constructs. Immunosorbent electron microscopy revealed the presence of virus-like particles in extracts of these protoplasts. An extract of protoplasts transfected with both constructs together with RNA-1 was able to initiate a new infection, showing that the two capsid proteins of CPMV can form functional particles containing RNA-1 and that the 60-kDa capsid precursor is not essential for this process.

Cell-to-cell movement of cowpea mosaic virus particles in plants takes place with the help of tubules that penetrate presumably modified plasmodesmata. These tubules, which are built up by the virus-encoded 48-kDa movement protein (MP), are also formed on single protoplast cells. To determine whether the MP contains different functional domains, the effect of mutations in its coding region was studied. Mutations between amino acids 1 and 313 led to complete abolishment of the tubule-forming capacity, while a deletion in the C-terminal region resulted in tubules that could not take up virus particles. From these observations, it is concluded that the MP contains at least two distinct domains, one that is involved in tubule formation and that spans amino acids 1 and 313 and a second that is probably involved in the incorporation of virus particles in the tubule and that is located in the C terminus between amino acids 314 and 331.

The 33.6-kDa nonstructural (NSM) protein gene, located on the ambisense M RNA segment of tomato spotted wilt virus (TSWV), was cloned and expressed using the Escherichia coli pET-11t expression system. The protein thus produced was purified and used for the production of a polyclonal antiserum. Western immunoblot analyses of TSWV-infected Nicotiana rustica plants showed NSM synthesis only during a short period early in systemic infection. Although NSM was found associated with cytoplasmic nucleocapsid preparations, it was absent from purified virus particles. Analyses of subcellular fractions from young, systemically infected leaves showed the presence of NSM in fractions enriched for cell walls and cytoplasmic membranes, respectively. Furthermore, immunogold labeling of tissue sections of TSWV-infected N. rustica plants showed that this protein was found associated with nucleocapsid aggregates in the cytoplasm and in close association with plasmodesmata. The data obtained provide evidence that NSM represents the viral movement protein of TSWV, involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures.

The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection of plant hosts. Genome sequence comparisons of these plant RNA viruses have revealed that most of them are genetically related to animal cell-infecting counterparts; this led to the concept of "superfamilies". Comparison of genetic maps of representative plant and animal viruses belonging to the same superfamily (e.g. cowpea mosaic virus [CPMV] versus picornaviruses and tobacco mosaic virus versus alphaviruses) have revealed genes in the plant viral genomes that appear to be essential adaptations needed for successful invasion and spread through their plant hosts. The best studied example represents the "movement protein" gene that is actively involved in cell-to-cell spread of plant viruses, thereby playing a key role in virulence and pathogenesis. In this paper the host adaptations of a number of plant viruses will be discussed, with special emphasis on the cell-to-cell movement mechanism of comovirus CPMV.

On the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.

The accumulation of two proteins, the nucleocapsid (N) protein and a non-structural (NSs) protein both encoded by the S RNA of tomato spotted wilt virus (TSWV), was followed in larvae during development and in adults of Frankliniella occidentalis after ingesting the virus for short periods on infected plants. The amounts of both proteins increased, as shown by ELISA and Western blot analysis, within 2 days above the levels ingested, indicating multiplication of TSWV in these insects. Accumulation of these proteins and of virus particles was further confirmed by in situ immunolabelling of the salivary glands and other tissues of adult thrips. The accumulation of large amounts of N and NSs protein, the occurrence of several vesicles with virus particles in the salivary glands and the massive numbers of virus particles in the salivary gland ducts demonstrate that the salivary glands are a major site of TSWV replication. The occurrence of virus particles in the salivary vesicles is indicative of the involvement of the Golgi apparatus in the maturation of the virus particles and its transport to the salivary ducts.