A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA edit via FRET in a binary system

Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, in vitro and in cellulo. A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C → U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization. Herein we introduce a binary probe system based on fluorescence resonance energy transfer (FRET) that distinguishes three possible states i.e. (i) absence of target, (ii) presence of edited (matched) and (iii) unedited (single base mismatched) target. To address the shortcomings of read-out via FRET, we designed donor probes that avoid bleed through into the acceptor channel and nevertheless provide a high intensity of FRET signaling. We show the combined use of thiazole orange (TO) and an oxazolopyridine analogue (JO), linked as base surrogates in modified PNA FIT-probes that serve as FRET donor for a second, near-infrared (NIR)-labeled strand. In absence of target, donor emission is low and FRET cannot occur in lieu of the lacking co-alignment of probes. Hybridization of the TO/JO-PNA FIT-probe with the (unedited RNA) target leads to high brightness of emission at 540 nm. Co-alignment of the NIR-acceptor strand ensues from recognition of edited RNA inducing emission at 690 nm. We show imaging of mRNA in fixed and live cells and discuss the homogeneous detection and intracellular imaging of a single nucleotide mRNA edit used by nature to post-transcriptionally modify the function of the Glycine Receptor (GlyR)

Sequestration of Martian CO2 by mineral carbonation

Carbonation is the water-mediated replacement of silicate minerals, such as olivine, by carbonate, and is commonplace in the Earth’s crust. This reaction can remove significant quantities of CO2 from the atmosphere and store it over geological timescales. Here we present the first direct evidence for CO2 sequestration and storage on Mars by mineral carbonation. Electron beam imaging and analysis show that olivine and a plagioclase feldspar-rich mesostasis in the Lafayette meteorite have been replaced by carbonate. The susceptibility of olivine to replacement was enhanced by the presence of smectite veins along which CO2-rich fluids gained access to grain interiors. Lafayette was partially carbonated during the Amazonian, when liquid water was available intermittently and atmospheric CO2 concentrations were close to their present-day values. Earlier in Mars’ history, when the planet had a much thicker atmosphere and an active hydrosphere, carbonation is likely to have been an effective mechanism for sequestration of CO2

Congenital Hypogonadotropic Hypogonadism Due to GNRH Receptor Mutations in Three Brothers Reveal Sites Affecting Conformation and Coupling

Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor (GNRHR1) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gαq/11 signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote

Optotaxis: Caged Lysophosphatidic Acid Enables Optical Control of a Chemotactic Gradient.

Lysophosphatidic acid (LPA) is a serum-borne lipid mediator that binds to a variety of different G protein-coupled receptors to trigger an exceptionally wide range of biological effects, including cell survival and differentiation, cancer cell migration, and embryonic development. Here we synthesized caged LPA (cgLPA), a "photolysable" coumarin-masked derivative of LPA. We demonstrate that illumination of cgLPA with 405 nm light liberates bioactive LPA on a subsecond scale to evoke Ca(2+) signaling, Rho activation, and cytoskeletal contraction. In addition, we developed an "optotaxis" assay to attract melanoma cells through a stable chemotactic gradient by repeated liberation of LPA through local photolysis of extracellular cgLPA. We expect that this method of light-controlled chemotaxis will be generally applicable to a large variety of small molecules that drive cellular migration or other responses

Effectiveness of Extrafine Single Inhaler Triple Therapy in Chronic Obstructive Pulmonary Disease (COPD) in Germany – The TriOptimize Study

Christian Gessner,1 Frederik Trinkmann,2,3 Sanaz Bahari Javan,4 Raimund Hövelmann,4 Valentina Bogoevska,4 George Georges,5 Elena Nudo,6 Carl-Peter Criée7 1Pneumologische Praxis Leipzig, Universitätsklinikum Leipzig, Institut für Klinische Immunologie, Leipzig, Germany; 2Pneumology and Critical Care Medicine, Thoraxklinik at University Hospital Heidelberg, Translational Lung Research Center Heidelberg (TLRC), Member of German Center for Lung Research (DZL), Heidelberg, Germany; 3Department of Biomedical Informatics (DBMI) at the Center for Preventive Medicine and Digital Health Baden-Württemberg (CPD-BW), University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany; 4Department of Medical Affairs, Chiesi GmbH, Hamburg, Germany; 5Corporate R&D, Chiesi USA Inc., Cary, NC, USA; 6Global Medical Affairs, Chiesi Farmaceutici S.p.A., Parma, Italy; 7Department of Sleep and Respiratory Medicine, Evangelical Hospital Goettingen-Weende, Bovenden, GermanyCorrespondence: Christian Gessner, Pneumologische Praxis Leipzig, Universitätsklinikum Leipzig, Institut für Klinische Immunologie, Tauchaer Straße 12, Leipzig, 04357, Germany, Tel +49 341 60 20 960, Email [email protected]: Real-word evidence on the effectiveness of switching from dual therapies or triple therapies (multiple inhalers) to extrafine single-inhaler triple therapy (efSITT), which consists of the inhaled corticosteroid (ICS) beclomethasone, the long-acting β2-agonist (LABA) formoterol and the long-acting muscarinic antagonist (LAMA) glycopyrronium, in patients with moderate-to-severe chronic obstructive pulmonary disease (COPD) is limited. The impact of switching to efSITT on health-related quality of life (HRQoL), COPD specific symptoms, lung function and treatment adherence were assessed in routine clinical care.Patients and Methods: Patients were recruited at 148 sites in Germany between 2017 and 2020 in this multicenter, non-interventional observational study. Demographics, clinical data and treatment history were collected at baseline. HRQoL (measured by COPD Assessment Test [CAT]), lung function and adherence (measured by Test of Adherence to Inhalers [TAI]) were assessed at baseline and after six months. Descriptive analyses were conducted by prior treatment and GOLD groups as well as for the overall population.Results: 55.1% of the 2623 included patients were male. Mean age was 65.8 years. 57.5% of the patients were previously treated with ICS+LABA+LAMA (multiple inhalers), 23.9% with ICS/LABA (single or two inhalers) and 18.6% with LAMA/LABA (single or two inhalers). After six months, largest mean improvements in the total CAT score were observed in the ICS/LABA (− 3.9) and LAMA/LABA (− 3.9) prior treatment groups as well as in patients in GOLD group B (− 2.9). In the overall population, the CAT items for cough, phlegm, and dyspnea decreased on average by − 0.4 points each. After six months, FEV1 increased by 2.0 percentage points in relation to predicted values. The percentages of measured sRtot and RV of predicted values decreased by 24.5 and 4.4 percentage points, respectively. The percentage of patients with good adherence increased from 67.8% to 76.5%.Conclusion: Treatment switch to efSITT resulted in an improvement of HRQoL, COPD specific symptoms, lung function parameters and adherence under real-world conditions.Keywords: COPD, extrafine single inhaler triple therapy, treatment adherence, CAT scor

Evidence for liquid liquid phase separation during the early stages of Mg struvite formation

In this work, we propose hydroxyapatite HA as a hard template to unlock the porosity of Fe N C catalyst materials. Using HA, a naturally occurring mineral that can be removed with nitric acid, in the synthesis generates a catalyst material with a unique porous network comprising abundant pores and interparticle cavities ranging from 10 to 3000 nm. Hard templating with HA alongside ZnCl2 as a micropore former results in a Fe N C catalyst based on naturally abundant peat with excellent oxygen reduction activity in alkaline conditions. A half wave potential of 0.87 V vs RHE and a peak power density of 1.06 W cm amp; 8722;2 were achieved in rotating ring disk electrode and anion exchange membrane fuel cell experiments, respectively, rivaling the performance of other state of the art platinum free catalysts presented in the literature. A combined approach of using renewable peat as a carbon source and HA as a hard template offers an environmentally friendly approach to high performance Fe N C catalysts with abundant porosit