The dynamic switch mechanism that leads to activation of LRRK2 is embedded in the DFGψ motif in the kinase domain.

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson's disease (PD). Although the precise mechanisms that control LRRK2 regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a "brake" that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2

Meson retardation in deuteron electrodisintegration

The effect of meson retardation in $NN$-interaction and exchange currents on deuteron electrodisintegration is studied in a coupled channel approach including $NN$-, $N \Delta$- and $\pi d$-channels. It is shown that the influence of retardation depends on the energy regime: Whereas below $\pi$-threshold calculations with static and retarded operators yield almost identical results, they differ significantly in the $\Delta$-region. Especially, the longitudinal and the longitudinal-transverse interference structure functions are strongly affected.Comment: 6 pages, 6 figure

Double polarization experiments at intermediate energy

At modern electron accelerators with highly polarized, intense, high duty factor beams double polarization coincidence experiments became feasible with good statistical accuracy. The strong potential towards the precise determination of small nucleon structure quantities is illustrated by two recent examples from MAMI. The measurement of $G_E^n$ in the quasifree reaction $D(\vec e, e'\vec n)p$ lead to a new parametrization of $G_E^n$ which is significantly above the previously preferred one from elastic $e-D$ scattering. A $p(\vec e, e'\vec p)\pi^0$ experiment at the energy of the $\Delta$ resonance yields preliminary results for the longitudinal quadrupole mixing. Both experimental errors and model uncertainties are complementary to unpolarized measurements.Comment: 8 pages, 6 figures, plenary talk given at PANIC'9

Fifteen minute consultation: Managing neonatal and childhood herpes encephalitis

Herpes simplex encephalitis (HSE) is the most common single cause of viral encephalitis in infants and children. Treated or untreated, it can be associated with considerable morbidity and mortality, and its presentation is usually insidious and non-specific. Prompt and careful investigation is important in order to establish the diagnosis so that treatment can be optimised. We address some common questions arising when diagnosing and treating presumed HSE throughout childhood

Differential binding studies applying functional protein microarrays and surface plasmon resonance

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays

What doesn't kill you makes you stranger: Dipeptidyl peptidase-4 (CD26) proteolysis differentially modulates the activity of many peptide hormones and cytokines generating novel cryptic bioactive ligands

Dipeptidyl peptidase 4 (DPP4) is an exopeptidase found either on cell surfaces where it is highly regulated in terms of its expression and surface availability (CD26) or in a free/circulating soluble constitutively available and intrinsically active form. It is responsible for proteolytic cleavage of many peptide substrates. In this review we discuss the idea that DPP4-cleaved peptides are not necessarily inactivated, but rather can possess either a modified receptor selectivity, modified bioactivity, new antagonistic activity, or even a novel activity relative to the intact parent ligand. We examine in detail five different major DPP4 substrates: glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), peptide tyrosine-tyrosine (PYY), and neuropeptide Y (NPY), and stromal derived factor 1 (SDF-1 aka CXCL12). We note that discussion of the cleaved forms of these five peptides are underrepresented in the research literature, and are both poorly investigated and poorly understood, representing a serious research literature gap. We believe they are understudied and misinterpreted as inactive due to several factors. This includes lack of accurate and specific quantification methods, sample collection techniques that are inherently inaccurate and inappropriate, and a general perception that DPP4 cleavage inactivates its ligand substrates. Increasing evidence points towards many DPP4-cleaved ligands having their own bioactivity. For example, GLP-1 can work through a different receptor than GLP-1R, DPP4-cleaved GIP can function as a GIP receptor antagonist at high doses, and DPP4-cleaved PYY, NPY, and CXCL12 can have different receptor selectivity, or can bind novel, previously unrecognized receptors to their intact ligands, resulting in altered signaling and functionality. We believe that more rigorous research in this area could lead to a better understanding of DPP4’s role and the biological importance of the generation of novel cryptic ligands. This will also significantly impact our understanding of the clinical effects and side effects of DPP4-inhibitors as a class of anti-diabetic drugs that potentially have an expanding clinical relevance. This will be specifically relevant in targeting DPP4 substrate ligands involved in a variety of other major clinical acute and chronic injury/disease areas including inflammation, immunology, cardiology, stroke, musculoskeletal disease and injury, as well as cancer biology and tissue maintenance in aging

Lipocalin-2 is a sensitive and specific marker of bacterial iInfection in children

Abstract Introduction Bacterial infection is the leading cause of death in children globally. Clinical algorithms to identify children who are likely to benefit from antimicrobial treatment remain suboptimal. Biomarkers that accurately identify serious bacterial infection (SBI) could improve diagnosis and clinical management. Lipocalin 2 (LCN2) and neutrophil collagenase (MMP-8) are neutrophil-derived biomarkers associated with bacterial infection. Methods We evaluated LCN2 and MMP-8 as candidate biomarkers in 40 healthy controls and 151 febrile children categorised confirmed SBI, probable SBI, or viral infection. The diagnostic performance of LCN2 and MMP-8 to predict SBI was estimated by the area under the receiver operating characteristic curve (AUROC) and compared to the performance of C-reactive protein (CRP). Results Plasma LCN2 and MMP-8 concentration were predictive of SBI. The AUROC (95% CI) for LCN2, MMP8 and CRP to predict SBI was 0.88 (0.82-0.94); 0.80 (0.72-0.87) and 0.89 (0.84-0.94), respectively. The diagnostic performance of LCN2 in combination with CRP was significantly superior to either marker alone: AUROC 0.92 (95% CI: 0.88-0.96). Conclusion LCN2 is a sensitive and specific predictor of SBI in children which could be used to improve clinical management and antimicrobial stewardship. LCN2 should be further evaluated in prospective clinical studies

Extraction of electromagnetic neutron form factors through inclusive and exclusive polarized electron scattering on polarized 3He target

Inclusive 3He(e,e') and exclusive 3He(e,e'n) processes with polarized electrons and 3He have been theoretically analyzed and values for the magnetic and electric neutron form factors have been extracted. In both cases the form factor values agree well with the ones extracted from processes on the deuteron. Our results are based on Faddeev solutions, modern NN forces and partially on the incorporation of mesonic exchange currents.Comment: 28 pages, 29 Postscript figure

Effect of recent R_p and R_n measurements on extended Gari-Krumpelmann model fits to nucleon electromagnetic form factors

The Gari-Krumpelmann (GK) models of nucleon electromagnetic form factors, in which the rho, omega, and phi vector meson pole contributions evolve at high momentum transfer to conform to the predictions of perturbative QCD (pQCD), was recently extended to include the width of the rho meson by substituting the result of dispersion relations for the pole and the addition of rho' (1450) isovector vector meson pole. This extended model was shown to produce a good overall fit to all the available nucleon electromagnetic form factor (emff) data. Since then new polarization data shows that the electric to magnetic ratios R_p and R_n obtained are not consistent with the older G_{Ep} and G_{En} data in their range of momentum transfer. The model is further extended to include the omega' (1419) isoscalar vector meson pole. It is found that while this GKex cannot simultaneously fit the new R_p and the old G_{En} data, it can fit the new R_p and R_n well simultaneously. An excellent fit to all the remaining data is obtained when the inconsistent G_{Ep} and G_{En} is omitted. The model predictions are shown up to momentum transfer squared, Q^2, of 8 GeV^2/c^2.Comment: 14 pages, 8 figures, using RevTeX4; email correspondence to [email protected] ; minor typos corrected, figures added, conclusions extende

Extended Gari-Krumpelmann model fits to nucleon electromagnetic form factors

Nucleon electromagnetic form factor data (including recent data) is fitted with models that respect the confinement and asymptotic freedom properties of QCD. Gari-Krumpelmann (GK) type models, which include the major vector meson pole contributions and at high momentum transfer conform to the predictions of perturbative QCD, are combined with Hohler-Pietarinen (HP) models, which also include the width of the rho meson and the addition of higher mass vector meson exchanges, but do not evolve into the explicit form of PQCD at high momentum transfer. Different parameterizations of the GK model's hadronic form factors, the effect of including the width of the rho meson and the addition of the next (in mass) isospin 1 vector meson are considered. The quality of fit and the consistency of the parameters select three of the combined HP/GK type models. Projections are made to the higher momentum transfers which are relevant to electron-deuteron experiments. The projections vary little for the preferred models, removing much of the ambiguity in electron-nucleus scattering predictions.Comment: 18pp, 7 figures, using RevTeX with BoxedEPS macros; 1 new figure, minor textual changes; email correspondence to [email protected]