Filamentous β-amyloid aggregates are crucial for the pathology of Alzheimer’s disease. Despite the tremendous biomedical importance, the molecular pathway of growth propagation is not completely ...understood and remains challenging to investigate by simulations due to the long time scales involved. Here, we apply extensive all-atom molecular dynamics simulations in explicit water to obtain free energy profiles and kinetic information from position-dependent diffusion profiles for three different Aβ9–40-growth processes: fibril elongation by single monomers at the structurally unequal filament tips and association of larger filament fragments. Our approach provides insight into the molecular steps of the kinetic pathway and allows close agreement with experimental binding free energies and macroscopic growth rates. Water plays a decisive role, and solvent entropy is identified as the main driving force for assembly. Fibril growth is disfavored energetically due to cancellation of direct peptide–peptide interactions and solvation effects. The kinetics of growth is consistent with the characteristic dock/lock mechanism, and docking is at least 2 orders of magnitude faster. During initial docking, interactions are mediated by transient non-native hydrogen bonds, which efficiently catch the incoming monomer or fragment already at separations of about 3 nm. In subsequent locking, the dynamics is much slower due to formation of kinetically trapped conformations caused by long-lived non-native hydrogen bonds. Fibril growth additionally requires collective motion of water molecules to create a dry binding interface. Fibril growth is further retarded due to reduced mobility of the involved hydration water, evident from a 2-fold reduction of the diffusion coefficient.
of the
species complex is an important hemibiotrophic pathogen of vegetables and fruits in temperate regions worldwide. In apple, it is one of the primary species responsible for bitter rot disease. ...Understanding the disease cycle is complicated because many broadleaf plants can be hosts of
By detecting and quantifying rain-splashed
species complex conidia in more than 500 samples from heavily bitter-rot-infected apple orchards and nearby forested woodlots over two summers, we show that conidial quantities were higher in the woodlots than in the orchards. Testing of more than 1,000 surface-disinfected leaves of apple and 24 different forest plant species showed that overall
was an abundant leaf endophyte, with high variation in leaf colonization area. Endophytic isolates from leaves were pathogenic on apples, and multilocus sequence analysis showed 100% identity between most isolates from leaves and diseased fruits. Apple leaves endophytically infected with
were present in a conventionally managed orchard and abundant in an untreated orchard. These lines of evidence, in the context of previously published research, lead us to hypothesize that the main ecological role of
is that of a leaf endophyte, which we present as a generalized
infection cycle that provides an updated framework for its integrated management in agricultural systems.
Summary
The most common forms of acquired epilepsies arise following acute brain insults such as traumatic brain injury, stroke, or central nervous system infections. Treatment is effective for only ...60%‐70% of patients and remains symptomatic despite decades of effort to develop epilepsy prevention therapies. Recent preclinical efforts are focused on likely primary drivers of epileptogenesis, namely inflammation, neuron loss, plasticity, and circuit reorganization. This review suggests a path to identify neuronal and molecular targets for clinical testing of specific hypotheses about epileptogenesis and its prevention or modification. Acquired human epilepsies with different etiologies share some features with animal models. We identify these commonalities and discuss their relevance to the development of successful epilepsy prevention or disease modification strategies. Risk factors for developing epilepsy that appear common to multiple acute injury etiologies include intracranial bleeding, disruption of the blood‐brain barrier, more severe injury, and early seizures within 1 week of injury. In diverse human epilepsies and animal models, seizures appear to propagate within a limbic or thalamocortical/corticocortical network. Common histopathologic features of epilepsy of diverse and mostly focal origin are microglial activation and astrogliosis, heterotopic neurons in the white matter, loss of neurons, and the presence of inflammatory cellular infiltrates. Astrocytes exhibit smaller K+ conductances and lose gap junction coupling in many animal models as well as in sclerotic hippocampi from temporal lobe epilepsy patients. There is increasing evidence that epilepsy can be prevented or aborted in preclinical animal models of acquired epilepsy by interfering with processes that appear common to multiple acute injury etiologies, for example, in post–status epilepticus models of focal epilepsy by transient treatment with a trkB/PLCγ1 inhibitor, isoflurane, or HMGB1 antibodies and by topical administration of adenosine, in the cortical fluid percussion injury model by focal cooling, and in the albumin posttraumatic epilepsy model by losartan. Preclinical studies further highlight the roles of mTOR1 pathways, JAK‐STAT3, IL‐1R/TLR4 signaling, and other inflammatory pathways in the genesis or modulation of epilepsy after brain injury. The wealth of commonalities, diversity of molecular targets identified preclinically, and likely multidimensional nature of epileptogenesis argue for a combinatorial strategy in prevention therapy. Going forward, the identification of impending epilepsy biomarkers to allow better patient selection, together with better alignment with multisite preclinical trials in animal models, should guide the clinical testing of new hypotheses for epileptogenesis and its prevention.
Bitter rot is a major disease of apple fruit in warm and humid regions. It is caused by various species in the Colletotrichum gloeosporioides and C. acutatum species complexes, of which C. fioriniae ...of the C. acutatum species complex is most common in the Mid-Atlantic region of the United States. While bitter rot management begins with good cultural practices, fungicides are generally used for consistent control. Fungicides should be applied before or during infection periods, but the timing of infection is unclear due to the hemibiotrophic lifestyle of the causal species. To determine when infection periods occur, we quantified C. fioriniae spore dispersal throughout three growing seasons and compared the temporal susceptibility of apples in two seasons of field trials. Spores were detected in rainwater from bud break to leaf drop, with the highest spore quantities in the summer and early fall correlating with optimal temperatures for C. fioriniae. Late-season-inoculated fruit had more bitter rot than early-season-inoculated fruit, but this was also positively correlated with periods of optimal temperatures and moisture for infection. In the context of previous experiments, these results suggest that infection periods are primarily determined by temperature and moisture rather than apple fruit phenology. Based on the relative numbers of spores, biotrophic and necrotrophic infections, only a tiny proportion of spores establish viable biotrophic infections, but a relatively high proportion of biotrophic infections switch to necrotrophy. We suggest bitter rot management should focus on preventing initial biotrophic infections by protecting apples during weather conditions that favor infection.
Tay-Sachs disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Here, we describe an adeno-associated virus (AAV) gene therapy expanded-access trial in ...two patients with infantile TSD (IND 18225) with safety as the primary endpoint and no secondary endpoints. Patient TSD-001 was treated at 30 months with an equimolar mix of AAVrh8-HEXA and AAVrh8-HEXB administered intrathecally (i.t.), with 75% of the total dose (1 × 10
vector genomes (vg)) in the cisterna magna and 25% at the thoracolumbar junction. Patient TSD-002 was treated at 7 months by combined bilateral thalamic (1.5 × 10
vg per thalamus) and i.t. infusion (3.9 × 10
vg). Both patients were immunosuppressed. Injection procedures were well tolerated, with no vector-related adverse events (AEs) to date. Cerebrospinal fluid (CSF) HexA activity increased from baseline and remained stable in both patients. TSD-002 showed disease stabilization by 3 months after injection with ongoing myelination, a temporary deviation from the natural history of infantile TSD, but disease progression was evident at 6 months after treatment. TSD-001 remains seizure-free at 5 years of age on the same anticonvulsant therapy as before therapy. TSD-002 developed anticonvulsant-responsive seizures at 2 years of age. This study provides early safety and proof-of-concept data in humans for treatment of patients with TSD by AAV gene therapy.
Apple bitter rot caused by Colletotrichum species is a growing problem worldwide. Colletotrichum spp. are economically important but taxonomically un-resolved. Identification of Colletotrichum spp. ...is critical due to potential species-level differences in pathogenicity-related characteristics. A 400-isolate collection from New York apple orchards were morphologically assorted to two groups, C. acutatum species complex (CASC) and C. gloeosporioides species complex (CGSC). A sub-sample of 44 representative isolates, spanning the geographical distribution and apple varieties, were assigned to species based on multi-locus phylogenetic analyses of nrITS, GAPDH and TUB2 for CASC, and ITS, GAPDH, CAL, ACT, TUB2, APN2, ApMat and GS genes for CGSC. The dominant species was C. fioriniae, followed by C. chrysophilum and a novel species, C. noveboracense, described in this study. This study represents the first report of C. chrysophilum and C. noveboracense as pathogens of apple. We assessed the enzyme activity and fungicide sensitivity for isolates identified in New York. All isolates showed amylolytic, cellulolytic and lipolytic, but not proteolytic activity. C. chrysophilum showed the highest cellulase and the lowest lipase activity, while C. noveboracense had the highest amylase activity. Fungicide assays showed that C. fioriniae was sensitive to benzovindiflupyr and thiabendazole, while C. chrysophilum and C. noveboracense were sensitive to fludioxonil, pyraclostrobin and difenoconazole. All species were pathogenic on apple fruit with varying lesion sizes. Our findings of differing pathogenicity-related characteristics among the three species demonstrate the importance of accurate species identification for any downstream investigations of Colletotrichum spp. in major apple growing regions.
Secondary nucleation pathways in which existing amyloid fibrils catalyze the formation of new aggregates and neurotoxic oligomers are of immediate importance for the onset and progression of ...Alzheimer’s disease. Here, we apply extensive all-atom molecular dynamics simulations in explicit water to study surface-activated secondary nucleation pathways at the extended lateral β-sheet surface of a preformed Aβ9–40 filament. Calculation of free-energy profiles allows us to determine binding free energies and conformational intermediates for nucleation complexes consisting of 1–4 Aβ peptides. In addition, we combine the free-energy profiles with position-dependent diffusion profiles to extract complementary kinetic information and macroscopic growth rates. Single monomers bind to the β-sheet surface in a disordered, hydrophobically collapsed conformation, whereas dimers and larger oligomers can retain a cross-β conformation resembling a more ordered fibril structure. The association processes during secondary nucleation follow a dock/lock mechanism consisting of a fast initial encounter phase (docking) and a slow structural rearrangement phase (locking). The major driving forces for surface-activated secondary nucleation are the release of a large number of hydration water molecules and the formation of hydrophobic interface contacts, the latter being in contrast to the elongation process at filament tips, which is dominated by the formation of stable and highly specific interface hydrogen bonds. The calculated binding free energies and the association rates for the attachment of Aβ monomers and oligomers to the extended lateral β-sheet surface of the filament seed are higher compared to those for elongation at the filament tips, indicating that secondary nucleation pathways can become important once a critical concentration of filaments has formed.
Background. The development of an effective human immunodeficiency virus (HIV) vaccine is a high global priority. Here, we report the safety, tolerability, and immunogenicity of a ...replication‐defective recombinant adenovirus serotype 5 (rAd5) vector HIV‐1 candidate vaccine. Methods. The vaccine is a mixture of 4 rAd5 vectors that express HIV‐1 subtype B Gag‐Pol fusion protein and envelope (Env) from subtypes A, B, and C. Healthy, uninfected adults were randomized to receive 1 intramuscular injection of placebo (n=6) or vaccine at dose levels of 109 (n=10), 1010 (n=10), or 1011 (n=10) particle units and were followed for 24 weeks to assess immunogenicity and safety. Results. The vaccine was well tolerated but was associated with more reactogenicity at the highest dose. At week 4, vaccine antigen–specific T cell responses were detected in 28 (93.3%) and 18 (60%) of 30 vaccine recipients for CD4+ and CD8+ T cells, respectively, by intracellular cytokine staining assay and in 22 (73%) of 30 vaccine recipients by enzyme‐linked immunospot assay. Env‐specific antibody responses were detected in 15 (50%) of 30 vaccine recipients by enzyme‐linked immunosorbant assay and in 28 (93.3%) of 30 vaccine recipients by immunoprecipitation followed by Western blotting. No neutralizing antibody was detected. Conclusions. A single injection induced HIV‐1 antigen–specific CD4+ T cell, CD8+ T cell, and antibody responses in the majority of vaccine recipients. This multiclade rAd5 HIV‐1 vaccine is now being evaluated in combination with a multiclade HIV‐1 DNA plasmid vaccine.
Apple growers in the Mid-Atlantic region of the United States have been reporting an increase in losses to bitter rot of apple and are requesting up-to-date management recommendations. Management is ...complicated by variations in apple cultivar susceptibility, temperature, rainfall, and biology of the Colletotrichum spp. that cause bitter rot. Over 500 apple fruit with bitter rot were obtained from 38 orchards across the Mid-Atlantic and the causal species were identified as Colletotrichum fioriniae and C. nymphaeae of the C. acutatum species complex and C. chrysophilum, C. noveboracense, C. siamense, C. fructicola, C. henanense, and C. gloeosporioides sensu stricto of the C. gloeosporioides species complex, the latter two being first reports. Species with faster in vitro growth rates at higher temperatures were more abundant in warmer regions of the Mid-Atlantic, while those with slower growth rates at higher temperatures were more abundant in cooler regions. Regional bloom dates are earlier and weather data show a gradual warming trend that likely influenced but was not necessarily the main cause of the recent increase in bitter rot in the region. A grower survey of apple cultivar susceptibility showed high variation, with the increase in acres planted to the highly susceptible cultivar Honeycrisp broadly corresponding to the increase in reports of bitter rot. These results form a basis for future studies on the biology and ecology of the Colletotrichum spp. responsible, and suggest that integrated bitter rot management must begin with selection of less-susceptible apple cultivars.
Galectin-9 (Gal-9) is a mammalian lectin secreted by endothelial cells that is highly expressed in rheumatoid arthritis synovial tissues and synovial fluid. Roles have been proposed for galectins in ...the regulation of inflammation and angiogenesis. Therefore, we examined the contribution of Gal-9 to angiogenesis and inflammation in arthritis.
To determine the role of Gal-9 in angiogenesis, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis, Matrigel tube formation, and mouse Matrigel plug angiogenesis assays. We also examined the role of signaling molecules in Gal-9-induced angiogenesis by using signaling inhibitors and small interfering RNA (siRNA). We performed monocyte (MN) migration assays in a modified Boyden chamber and assessed the arthritogenicity of Gal-9 by injecting Gal-9 into mouse knees.
Gal-9 significantly increased HMVEC migration, which was decreased by inhibitors of extracellular signal-regulating kinases 1/2 (Erk1/2), p38, Janus kinase (Jnk), and phosphatidylinositol 3-kinase. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. In mouse Matrigel plug assays, plugs containing Gal-9 induced significantly higher angiogenesis, which was attenuated by a Jnk inhibitor. Gal-9 also induced MN migration, and there was a marked increase in MN ingress when C57BL/6 mouse knees were injected with Gal-9 compared with the control, pointing to a proinflammatory role for Gal-9.
Gal-9 mediates angiogenesis, increases MN migration in vitro, and induces acute inflammatory arthritis in mice, suggesting a novel role for Gal-9 in angiogenesis, joint inflammation, and possibly other inflammatory diseases.
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