Vesicle-based cell-free synthesis of short and long unspecific peroxygenases

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal enzymes that catalyze the oxyfunctionalization of non-activated hydrocarbons, making them valuable biocatalysts. Despite the increasing interest in UPOs that has led to the identification of thousands of putative UPO genes, only a few of these have been successfully expressed and characterized. There is currently no universal expression system in place to explore their full potential. Cell-free protein synthesis has proven to be a sophisticated technique for the synthesis of difficult-to-express proteins. In this work, we aimed to establish an insect-based cell-free protein synthesis (CFPS) platform to produce UPOs. CFPS relies on translationally active cell lysates rather than living cells. The system parameters can thus be directly manipulated without having to account for cell viability, thereby making it highly adaptable. The insect-based lysate contains translocationally active, ER-derived vesicles, called microsomes. These microsomes have been shown to allow efficient translocation of proteins into their lumen, promoting post-translational modifications such as disulfide bridge formation and N-glycosylations. In this study the ability of a redox optimized, vesicle-based, eukaryotic CFPS system to synthesize functional UPOs was explored. The influence of different reaction parameters as well as the influence of translocation on enzyme activity was evaluated for a short UPO from Marasmius rotula and a long UPO from Agrocybe aegerita. The capability of the CFPS system described here was demonstrated by the successful synthesis of a novel UPO from Podospora anserina, thus qualifying CFPS as a promising tool for the identification and evaluation of novel UPOs and variants thereof

Design and Synthesis of CNS-targeted Flavones and Analogues with Neuroprotective Potential Against H2O2- and Aβ1-42-Induced Toxicity in SH-SY5Y Human Neuroblastoma Cells

With the lack of available drugs able to prevent the progression of Alzheimer’s disease (AD), the discovery of new neuroprotective treatments able to rescue neurons from cell injury is presently a matter of extreme importance and urgency. Here, we were inspired by the widely reported potential of natural flavonoids to build a library of novel flavones, chromen-4-ones and their C-glucosyl derivatives, and to explore their ability as neuroprotective agents with suitable pharmacokinetic profiles. All compounds were firstly evaluated in a parallel artificial membrane permeability assay (PAMPA) to assess their effective permeability across biological membranes, namely the blood-brain barrier (BBB). With this test, we aimed not only at assessing if our candidates would be well-distributed, but also at rationalizing the influence of the sugar moiety on the physicochemical properties. To complement our analysis, logD7.4 was determined. From all screened compounds, the p-morpholinyl flavones stood out for their ability to fully rescue SH-SY5Y human neuroblastoma cells against both H2O2- and Aβ1-42-induced cell death. Cholinesterase inhibition was also evaluated, and modest inhibitory activities were found. This work highlights the potential of C-glucosylflavones as neuroprotective agents, and presents the p-morpholinyl C-glucosylflavone 37, which did not show any cytotoxicity towards HepG2 and Caco-2 cells at 100 μM, as a new lead structure for further development against AD.Fundação para a Ciência e a Tecnologia-UID/Multi/0612/2019Unión Europea-D3i4AD), FP7-PEOPLE-2013-IAPP, GA 61234

Effect of ritonavir on the pharmacokinetics of the benzimidazoles albendazole and mebendazole: an interaction study in healthy volunteers

Background: Benzimidazoles are often used concomitantly with protease inhibitors in patients with helminthic disease and HIV infection. Low bioavailability and extensive first-pass metabolism make benzimidazoles prone to pharmacokinetic drug interactions. The aim of the present study was to investigate potential drug interactions between the benzimidazoles albendazole and mebendazole and the potent CYP3A4 inhibitor ritonavir. Methods: Sixteen healthy volunteers were administered a single oral dose of 1,000mg mebendazole or 400mg albendazole (2 × n = 8). AUC, Cmax, and t1/2 of mebendazole, albendazole, and albendazole sulfoxide were studied in absence and after short-term (2 doses) and long-term (8days) treatment with ritonavir 200mg bid. Results: Pharmacokinetic parameters of albendazole and mebendazole were not changed by short-term administration of ritonavir. However, long-term administration of ritonavir resulted in significant changes in albendazole and mebendazole disposition, with a significant decrease in AUC0-24 (27 and 43% of baseline for albendazole and mebendazole, respectively) and Cmax (26 and 41% of baseline, respectively). Conclusion: The AUC0-24 of benzimidazoles decreased after long-term use of ritonavir, while no changes in pharmacokinetic profiles were observed under short-term administration. These findings might help to optimize benzimidazole efficacy when used in combination with protease inhibitor

Morphology and reproduction in the Hapalocarcinus marsupialis Stimpson, 1859 species complex (Decapoda: Brachyura: Cryptochiridae)

Symbiotic relationships contribute considerably to the high biodiversity found on coral reefs. Coral-dwelling gall crabs (Cryptochiridae) represent a prime example of coral-associated invertebrates that exhibit obligate relationships with their host. The induction of a skeletal modification in the coral, used as a dwelling by the crab, is the most remarkable aspect of this close association. Here we examined Hapalocarcinus marsupialisStimpson, 1859, collected from Pocillopora and Stylophora corals in the Saudi Arabian Red Sea. Specimens were DNA barcoded, and five distinct clades were revealed, providing further evidence for the hypothesis that H. marsupialis is a species complex. Divergence (COI) between the clades ranged from 3.2 to 15.7%. The putative species were tested for differences in morphology and reproduction. Crabs were examined regarding morphometric characters (carapace length and width, pleon (abdomen), chelae, and growth patterns) and reproductive traits. The data were pooled and analysed by host genus and putative species, which revealed significant differences for most of the measured variables in female crabs. Specimens retrieved from Pocillopora were significantly larger (up to 49 %) and had higher fecundity than those inhabiting Stylophora. For reproductive output (RO) no differences at species- or host-genus level were detected. The average RO of ~70% over all specimens is high compared to other brachyurans, supporting the assumption that symbiotic brachyurans invest more energy in reproduction than their free-living counterparts. Discrepancies with published data on growth and reproduction of Hapalocarcinus are discussed. Our results demonstrate the usefulness of morphometric traits and fecundity in separating the clades in the H. marsupialis complex, and prepare the ground for further morphometric studies on the genus and other symbiotic brachyurans. Moreover, it highlights the need to check for the presence of cryptic species when studying aspects of the biology of a species

Entire functions with Julia sets of positive measure

Let f be a transcendental entire function for which the set of critical and asymptotic values is bounded. The Denjoy-Carleman-Ahlfors theorem implies that if the set of all z for which |f(z)|>R has N components for some R>0, then the order of f is at least N/2. More precisely, we have log log M(r,f) > (N/2) log r - O(1), where M(r,f) denotes the maximum modulus of f. We show that if f does not grow much faster than this, then the escaping set and the Julia set of f have positive Lebesgue measure. However, as soon as the order of f exceeds N/2, this need not be true. The proof requires a sharpened form of an estimate of Tsuji related to the Denjoy-Carleman-Ahlfors theorem.Comment: 17 page

Electrostatic boundary value problems in the Schwarzschild background

The electrostatic potential of any test charge distribution in Schwarzschild space with boundary values is derived. We calculate the Green's function, generalize the second Green's identity for p-forms and find the general solution. Boundary value problems are solved. With a multipole expansion the asymptotic property for the field of any charge distribution is derived. It is shown that one produces a Reissner--Nordstrom black hole if one lowers a test charge distribution slowly toward the horizon. The symmetry of the distribution is not important. All the multipole moments fade away except the monopole. A calculation of the gravitationally induced electrostatic self-force on a pointlike test charge distribution held stationary outside the black hole is presented.Comment: 18 pages, no figures, uses iopart.st

Exploring the genetics of irritable bowel syndrome: A GWA study in the general population and replication in multinational case-control cohorts

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11\u2005326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31 710(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions

A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models

© 2023 The Author(s). Published by Oxford University Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.Peer reviewe

Optimal follow-up after acute pulmonary embolism: a position paper of the European Society of Cardiology Working Group on Pulmonary Circulation and Right Ventricular Function, in collaboration with the European Society of Cardiology Working Group on Atherosclerosis and Vascular Biology, endorsed by the European Respiratory Society

This position paper provides a comprehensive guide for optimal follow-up of patients with acute pulmonary embolism (PE), covering multiple relevant aspects of patient counselling. It serves as a practical guide to treating patients with acute PE complementary to the formal 2019 European Society of Cardiology guidelines developed with the European Respiratory Society. We propose a holistic approach considering the whole spectrum of serious adverse events that patients with acute PE may encounter on the short and long run. We underline the relevance of assessment of modifiable risk factors for bleeding, of acquired thrombophilia and limited cancer screening (unprovoked PE) as well as a dedicated surveillance for the potential development of chronic thromboembolic pulmonary hypertension as part of routine practice; routine testing for genetic thrombophilia should be avoided. We advocate the use of outcome measures for functional outcome and quality of life to quantify the impact of the PE diagnosis and identify patients with the post-PE syndrome early. Counselling patients on maintaining a healthy lifestyle mitigates the risk of the post-PE syndrome and improves cardiovascular prognosis. Therefore, we consider it important to discuss when and how to resume sporting activities soon after diagnosing PE. Additional patient-relevant topics that require Focused counselling are travel and birth control