Crystal polymorphism in fragment-based lead discovery of ligands of the catalytic domain of UGGT, the glycoprotein folding quality control checkpoint

None of the current data processing pipelines for X-ray crystallography fragment-based lead discovery (FBLD) consults all the information available when deciding on the lattice and symmetry (i.e., the polymorph) of each soaked crystal. Often, X-ray crystallography FBLD pipelines either choose the polymorph based on cell volume and point-group symmetry of the X-ray diffraction data or leave polymorph attribution to manual intervention on the part of the user. Thus, when the FBLD crystals belong to more than one crystal polymorph, the discovery pipeline can be plagued by space group ambiguity, especially if the polymorphs at hand are variations of the same lattice and, therefore, difficult to tell apart from their morphology and/or their apparent crystal lattices and point groups. In the course of a fragment-based lead discovery effort aimed at finding ligands of the catalytic domain of UDP–glucose glycoprotein glucosyltransferase (UGGT), we encountered a mixture of trigonal crystals and pseudotrigonal triclinic crystals—with the two lattices closely related. In order to resolve that polymorphism ambiguity, we have written and described here a series of Unix shell scripts called CoALLA (crystal polymorph and ligand likelihood-based assignment). The CoALLA scripts are written in Unix shell and use autoPROC for data processing, CCP4-Dimple/REFMAC5 and BUSTER for refinement, and RHOFIT for ligand docking. The choice of the polymorph is effected by carrying out (in each of the known polymorphs) the tasks of diffraction data indexing, integration, scaling, and structural refinement. The most likely polymorph is then chosen as the one with the best structure refinement Rfree statistic. The CoALLA scripts further implement a likelihood-based ligand assignment strategy, starting with macromolecular refinement and automated water addition, followed by removal of the water molecules that appear to be fitting ligand density, and a final round of refinement after random perturbation of the refined macromolecular model, in order to obtain unbiased difference density maps for automated ligand placement. We illustrate the use of CoALLA to discriminate between H3 and P1 crystals used for an FBLD effort to find fragments binding to the catalytic domain of Chaetomium thermophilum UGGT

Mutations in KCNK4 that Affect Gating Cause a Recognizable Neurodevelopmental Syndrome

Aberrant activation or inhibition of potassium (K+) currents across the plasma membrane of cells has been causally linked to altered neurotransmission, cardiac arrhythmias, endocrine dysfunction, and (more rarely) perturbed developmental processes. The K+ channel subfamily K member 4 (KCNK4), also known as TRAAK (TWIK-related arachidonic acid-stimulated K+ channel), belongs to the mechano-gated ion channels of the TRAAK/TREK subfamily of two-pore-domain (K2P) K+ channels. While K2P channels are well known to contribute to the resting membrane potential and cellular excitability, their involvement in pathophysiological processes remains largely uncharacterized. We report that de novo missense mutations in KCNK4 cause a recognizable syndrome with a distinctive facial gestalt, for which we propose the acronym FHEIG (facial dysmorphism, hypertrichosis, epilepsy, intellectual disability/developmental delay, and gingival overgrowth). Patch-clamp analyses documented a significant gain of function of the identified KCNK4 channel mutants basally and impaired sensitivity to mechanical stimulation and arachidonic acid. Co-expression experiments indicated a dominant behavior of the disease-causing mutations. Molecular dynamics simulations consistently indicated that mutations favor sealing of the lateral intramembrane fenestration that has been proposed to negatively control K+ flow by allowing lipid access to the central cavity of the channel. Overall, our findings illustrate the pleiotropic effect of dysregulated KCNK4 function and provide support to the hypothesis of a gating mechanism based on the lateral fenestrations of K2P channels

Cystic echinococcosis in wild boars (Sus scrofa) from southern Italy: Epidemiological survey and molecular characterization.

Cystic Echinococcosis (CE) caused by Echinococcus granulosus sensu lato (s.l.) is one of the most important parasitic zoonotic diseases in the world and it represents an important public health and socio-economic concern. In the Mediterranean basin, CE is widespread and it is endemic in Italy, with major prevalence in southern areas. Several studies have investigated CE in domestic pigs, however, such data in wild boars are scant. In the last decades the wild boar population in Italy has increased and this ungulate could play an important role in the spreading ofCEinthewild.Here wereporton theprevalenceandfertility rateofhydatid cystsinwildboarsthat were shot during two hunting seasons (2016–2017) in the Campania region of southern Italy. For each animal, a detailed inspection of the carcass and organs (lungs, liver and spleen) was performed and when cysts were found, their number, morphology and fertility were determined by visual and microscopic examination. Cysts were classified morphologically as fertile, sterile, caseous and calcified. Protoscoleces and germinal layers were collected from individual cysts and DNA was extracted to identify different strains/genotypes of E. granulosus s.l. Outofatotalof2108wildboars93(4.4%)werefoundpositiveforCE.Infectedanimalswere45malesand48 females, aged between 1 and 8 years. The average number of cysts per wild boar was 1.3 (min 1 - max 13). The total number of cysts collected was 123, of which 118 (95.9%) in the liver, 4 (3.3%) in the lungs and 1 (0.8%) in the spleen. Of all analyzed cysts, 70 (56.9%) were fertile and 53 (43.1%) sterile/acephalous. The presence of fertile cysts in 19.4% of CE-positive animals is noteworthy. Overall, molecular diagnosis showed 19 wild boars infected with the pig strain (G7)

Mutations impairing GSK3-mediated MAF phosphorylation cause cataract, deafness, intellectual disability, seizures, and a down syndrome-like facies

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing developmen

Can whole body vibration exercises promote improvement on quality of life and on chronic pain level of metabolic syndrome patients? A pseudorandomized crossover study

Quality of life (QoL) is one of the most important health outcome concepts expressed subjectively. Chronic pain (CP) is an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Taking into account the poor QoL and the CP already described in metabolic syndrome (MSy) individuals, this study aimed to evaluate the effects of whole body vibration exercises (WBVE) on these parameters in this population. Thirty-three MSy patients were divided in subgroups A [whole body vibration exercise group (WBVeG), n = 17, 15 females/2 males, 61.1 ± 8.4 yr] and B (control group, n = 16, 14 females/2 males, 58.2 ± 9.1 yr). Subgroup A performed 10 sessions (2 times/wk) of WBVE (18 min/session, with a frequency from 5 up to 14 Hz and a peak-to-peak displacement of 2.5, 5.0, and 7.5 mm) on a side-alternating vibrating platform (VP). Subgroup B did the same protocol, but the VP was turned off. The individuals answered the World Health Organization Quality of Life bref (WHOQoL-bref) questionnaire before the first and after the 10th session. The chronic pain level (CPL) was measured by a numeric rating scale (0–10) before and at the end of each session. Significant improvements were found in physical health ( P = 0.05) and psychological health ( P = 0.04) domains of WHOQoL-bref in WBVeG. A significant acute reduction of the CPL was found in the WBVeG after the protocol, considering the first session and at the last session. WBVE marginally improved physical health and psychological health and decrease the CPL in acute interventions. NEW & NOTEWORTHY Metabolic syndrome patients experience poor quality of life, frequently associated with lack of exercise and bad dietary habits. Additionally, factors such as obesity, neuromusculoskeletal impairment, and peripheral endothelial dysfunction result in a chronic pain level. Whole body vibration exercise might represent a suitable physical therapy, since it is easy to perform, low cost, safe, and capable of promoting an improvement of quality of life and reducing chronic pain level during acute interventions in metabolic syndrome individuals

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, UDP-glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 “WY” conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 μM binding affinity for CtUGGTGT24 in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 μM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors

Measurements of Higgs boson production and couplings in diboson final states with the ATLAS detector at the LHC

Measurements are presented of production properties and couplings of the recently discovered Higgs boson using the decays into boson pairs, H →γ γ, H → Z Z∗ →4l and H →W W∗ →lνlν. The results are based on the complete pp collision data sample recorded by the ATLAS experiment at the CERN Large Hadron Collider at centre-of-mass energies of √s = 7 TeV and √s = 8 TeV, corresponding to an integrated luminosity of about 25 fb−1. Evidence for Higgs boson production through vector-boson fusion is reported. Results of combined fits probing Higgs boson couplings to fermions and bosons, as well as anomalous contributions to loop-induced production and decay modes, are presented. All measurements are consistent with expectations for the Standard Model Higgs boson

Standalone vertex finding in the ATLAS muon spectrometer

A dedicated reconstruction algorithm to find decay vertices in the ATLAS muon spectrometer is presented. The algorithm searches the region just upstream of or inside the muon spectrometer volume for multi-particle vertices that originate from the decay of particles with long decay paths. The performance of the algorithm is evaluated using both a sample of simulated Higgs boson events, in which the Higgs boson decays to long-lived neutral particles that in turn decay to bbar b final states, and pp collision data at √s = 7 TeV collected with the ATLAS detector at the LHC during 2011

Hunt for new phenomena using large jet multiplicities and missing transverse momentum with ATLAS in 4.7 fb−1 of √s=7 TeV proton-proton collisions

Results are presented of a search for new particles decaying to large numbers of jets in association with missing transverse momentum, using 4.7 fb−1 of pp collision data at s√=7TeV collected by the ATLAS experiment at the Large Hadron Collider in 2011. The event selection requires missing transverse momentum, no isolated electrons or muons, and from ≥6 to ≥9 jets. No evidence is found for physics beyond the Standard Model. The results are interpreted in the context of a MSUGRA/CMSSM supersymmetric model, where, for large universal scalar mass m 0, gluino masses smaller than 840 GeV are excluded at the 95% confidence level, extending previously published limits. Within a simplified model containing only a gluino octet and a neutralino, gluino masses smaller than 870 GeV are similarly excluded for neutralino masses below 100 GeV