Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for ...DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1T112A), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1Δ109–116), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.
Abstract
Group-living animals often experience within-group competition for resources like shelter and space, as well as for social status. Because of this conflict, residents may aggressively resist ...joining attempts by new members. Here, we asked whether different forms of competition mediate this response, specifically competition over 1) shelter, 2) spatial position within groups, and 3) social or sexual roles. We performed experiments on wild groups of Neolamprologus multifasciatus cichlids in Lake Tanganyika, either increasing or decreasing the number of shelters (empty snail shells) within their territories. We predicted that increases in resource abundance would reduce conflict and lower the aggression of residents toward presented conspecifics, while decreases in resources would increase aggression. We explored the effects of social conflict and spatial arrangement by introducing same or opposite sex conspecifics, at greater or lesser distances from resident subterritories. We found that changing the abundance of shells had no detectable effect on the responses of residents to presented conspecifics. Rather, aggression was strongly sex-dependent, with male residents almost exclusively aggressing presented males, and female residents almost exclusively aggressing presented females. For females, this aggression was influenced by the spatial distances between the presented conspecific and the resident female subterritory, with aggression scaling with proximity. In contrast, presentation distance did not influence resident males, which were aggressive to all presented males regardless of location. Overall, our results show that group residents respond to presented conspecifics differently depending on the type of competitive threat these potential joiners pose.
Group living is associated with numerous challenges, including how to share limited space and resources among group members, and how to divide social roles. These challenges generate conflict among group members and outsiders. We find that changing within-group resource abundance does not affect responses, but that aggression is highest when residents and outsiders are of the same sex, and for females, when the outsider is in close proximity to her subterritory.
The Linac Coherent Light Source free electron laser is a source of high brightness x rays, 2×10(11) photons in a ∼5 fs pulse, that can be focused to produce double core vacancies through rapid ...sequential ionization. This enables double core vacancy Auger electron spectroscopy, an entirely new way to study femtosecond chemical dynamics with Auger electrons that probe the local valence structure of molecules near a specific atomic core. Using 1.1 keV photons for sequential x-ray ionization of impulsively aligned molecular nitrogen, we observed a rich single-site double core vacancy Auger electron spectrum near 413 eV, in good agreement with ab initio calculations, and we measured the corresponding Auger electron angle dependence in the molecular frame.
A new process to produce back-injected self-reinforced composites (SRCs) is presented. In contrast to other investigations on back-injection of SRCs, a process is presented which allows compacting ...and back injection of SRCs in one step where the SRCs are partly consolidated only via melt pressure inside the cavity. The mechanical properties of SRCs depend to a large extent on the process parameters of temperature and pressure during manufacture. These parameters are not yet known for back-injected areas. Sensors inside of the cavity measure the influences on the temperature and pressure conditions in the cavity. Initial studies on adhesion were carried out and analysed. For this purpose, shear tests of the back-injected component were carried out and a maximum shear strength of 5.81 MPa was determined for the materials used here. The investigations also show a dependence on the Distance from the Gate (D
) and the Mass temperature (T
). First microscopic examinations show good bonding between the SRC and the injection molded part, with no voids or air pockets in the boundary layer. It can also be seen that successful consolidation takes place in the area of the back injection.
The mechanical properties of self-reinforced composites (SRCs) produced in a hot compaction process significantly depend on the process parameters. Only a little deviation of the process temperature ...or pressure causes the component to act differently under mechanical load. This is a chance and a challenge at the same time, since this process is difficult to handle but by properly controlling the process parameters, the mechanical properties can be adjusted, even locally for one component. In this research SRC are manufactured in a hot compaction process. A correlation between process parameters and density is found. Density increased from 0,8 to 0,91 g/cm³ by increasing temperature and pressure in the hot compaction process. The different thermal properties in the direction of orientation (IP) and transverse to orientation (TP) are measured with a laser flash device. It was found that, due to a change in density and molecular orientation, diffusivity and conductivity are influenced in different degrees in IP and TP directions. For interpretation of thermal measurement results, microstructures are analysed with a confocal laser scanning microscope after preparing the specimen with a permanganate etching. A schematic model of conductive path is worked out and discussed. With measurement data the anisotropy of IP and TP diffusivity is calculated, and a model is built to describe relative density related to anisotropy. The highest anisotropy between IP and TP diffusivity was calculated with a ratio of 6 at a relative density of approximately 0,82 g/cm³. Since mechanical properties in correlation to process parameters have already been investigated, results of this investigation, in combination with previous research on mechanical properties, will enable the development of a non-destructive testing method for SRCs by measuring the thermal diffusivity.
The first time-resolved x-ray/optical pump-probe experiments at the SLAC Linac Coherent Light Source (LCLS) used a combination of feedback methods and post-analysis binning techniques to synchronize ...an ultrafast optical laser to the linac-based x-ray laser. Transient molecular nitrogen alignment revival features were resolved in time-dependent x-ray-induced fragmentation spectra. These alignment features were used to find the temporal overlap of the pump and probe pulses. The strong-field dissociation of x-ray generated quasi-bound molecular dications was used to establish the residual timing jitter. This analysis shows that the relative arrival time of the Ti:Sapphire laser and the x-ray pulses had a distribution with a standard deviation of approximately 120 fs. The largest contribution to the jitter noise spectrum was the locking of the laser oscillator to the reference RF of the accelerator, which suggests that simple technical improvements could reduce the jitter to better than 50 fs.
To examine the real-world performance of a support vector machine learning software (RetinaLyze) in order to identify the possible presence of diabetic retinopathy (DR) in patients with diabetes via ...software implementation in clinical practice.
1001 eyes from 1001 patients-one eye per patient-participating in the Danish National Screening Programme were included. Three independent ophthalmologists graded all eyes according to the International Clinical Diabetic Retinopathy Disease Severity Scale with the exact level of disease being determined by majority decision. The software detected DR and no DR and was compared to the ophthalmologists' gradings.
At a clinical chosen threshold, the software showed a sensitivity, specificity, positive predictive value and negative predictive value of 84.9% (95% CI: 81.8-87.9), 89.9% (95% CI: 86.8-92.7), 92.1% (95% CI: 89.7-94.4), and 81.0% (95% CI: 77.2-84.7), respectively, when compared to human grading. The results from the routine screening were 87.0% (95% CI: 84.2-89.7), 85.3% (95% CI: 81.8-88.6), 89.2% (95% CI: 86.3-91.7), and 82.5% (95% CI: 78.5-86.0), respectively. AUC was 93.4%. The reference graders Conger's Exact Kappa was 0.827.
The software performed similarly to routine grading with overlapping confidence intervals, indicating comparable performance between the two groups. The intergrader agreement was satisfactory. However, evaluating the updated software alongside updated clinical procedures is crucial. It is therefore recommended that further clinical testing before implementation of the software as a decision support tool is conducted.
We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall ...biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and in silico analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an in vivo model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections.
The objectives of this study were to quantify residual interfraction displacement of seminal vesicles (SV) and investigate the efficacy of rotation correction on SV displacement in marker-based ...prostate image-guided radiotherapy (IGRT). We also determined the effect of marker registration on the measured SV displacement and its impact on margin design.
SV displacement was determined relative to marker registration by using 296 cone beam computed tomography scans of 13 prostate cancer patients with implanted markers. SV were individually registered in the transverse plane, based on gray-value information. The target registration error (TRE) for the SV due to marker registration inaccuracies was estimated. Correlations between prostate gland rotations and SV displacement and between individual SV displacements were determined.
The SV registration success rate was 99%. Displacement amounts of both SVs were comparable. Systematic and random residual SV displacements were 1.6 mm and 2.0 mm in the left-right direction, respectively, and 2.8 mm and 3.1 mm in the anteroposterior (AP) direction, respectively. Rotation correction did not reduce residual SV displacement. Prostate gland rotation around the left-right axis correlated with SV AP displacement (R(2) = 42%); a correlation existed between both SVs for AP displacement (R(2) = 62%); considerable correlation existed between random errors of SV displacement and TRE (R(2) = 34%).
Considerable residual SV displacement exists in marker-based IGRT. Rotation correction barely reduced SV displacement, rather, a larger SV displacement was shown relative to the prostate gland that was not captured by the marker position. Marker registration error partly explains SV displacement when correcting for rotations. Correcting for rotations, therefore, is not advisable when SV are part of the target volume. Margin design for SVs should take these uncertainties into account.
•Phosphoethanolamine methyltransferases (PMT) are critical for the growth and development of nematodes.•Analysis of PMT1 from the pine wilt nematode (Bursaphelenchus xylophilus) confirms biochemical ...function.•The crystal structure of BxPMT1 reveals how the enzyme functions to methylate substrates.•Site-directed mutagenesis identifies key residues on two surface loops as critical for phosphobase methylation..
In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the β1d-α6 loop (W123F, M126I, and Y127F) and β1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.
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