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Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity. RASTMIN delivers ~1–2 nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures.Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale. Recently, the method called MINFLUX has achieved a ~10-fold improvement in spatial resolution over previously developed techniques, reaching molecular resolution with moderate photon counts. However, the technical complexity of this technique has hindered its widespread application. The new technique called RASTMIN provides equivalent resolution to MINFLUX while it can be implemented in standard scanning (confocal) microscopes.

Recent advances in fluorescence nanoscopy have pushed resolution to the 1–10 nm range, enabling the direct visualization of individual molecules even in crowded biological environments. Achieving this level of precision requires rigorous sample drift control. Techniques such as MINFLUX and RASTMIN, which rely on keeping the sample fixed within an excitation pattern, demand active drift correction to achieve their theoretical nanometer-scale resolution limits. Here, we present an active stabilization system for super-resolution microscopy that delivers sub-nm precision for hours. Featuring a simple optical design, the system can be added as a separate module to any fluorescence microscope. We also provide an open-source control software including a user-friendly graphical interface readily adaptable to different setups. We demonstrate the adaptability and performance of the stabilization system with p-MINFLUX and RASTMIN measurements performed in two different setups, reaching the theoretical Cramér-Rao Bound and resolving ~10 nm distances within DNA origami structures.

Niclosamide (NICLO) is a recognized antiparasitic drug being repositioned for Helicobacter pylori. The present work aimed to formulate NICLO nanocrystals (NICLO-NCRs) to produce a higher dissolution rate of the active ingredient and to incorporate these nanosystems into a floating solid dosage form to release them into the stomach slowly. For this purpose, NICLO-NCRs were produced by wet-milling and included in a floating Gelucire l3D printed tablet by semi-solid extrusion, applying the Melting solidification printing process (MESO-PP) methodology. The results obtained in TGA, DSC, XRD and FT-IR analysis showed no physicochemical interactions or modifications in the crystallinity of NICLO-NCR after inclusion in Gelucire 50/13 ink. This method allowed the incorporation of NICLO-NCRs in a concentration of up to 25% w/w. It achieved a controlled release of NCRs in a simulated gastric medium. Moreover, the presence of NICLO-NCRs after redispersion of the printlets was observed by STEM. Additionally, no effects on the cell viability of the NCRs were demonstrated in the GES-1 cell line. Finally, gastroretention was demonstrated for 180 min in dogs. These findings show the potential of the MESO-PP technique in obtaining slow-release gastro-retentive oral solid dosage forms loaded with nanocrystals of a poorly soluble drug, an ideal system for treating gastric pathologies such as H. pylori.

Ca²⁺ release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP₃Rs) plays a relevant role in numerous physiological processes. IP₃R-mediated Ca²⁺ signals involve Ca²⁺-induced Ca²⁺-release (CICR) whereby Ca²⁺ release through one open IP₃R induces the opening of other channels. IP₃Rs are apparently organized in clusters. The signals can remain localized (i.e., Ca²⁺ puffs) if CICR is limited to one cluster or become waves that propagate between clusters. Ca²⁺ puffs are the building blocks of Ca²⁺ waves. Thus, there is great interest in determining puff properties, especially in view of the current controversy on the spatial distribution of activatable IP₃Rs. Ca²⁺ puffs have been observed in intact cells with optical techniques proving that they are intrinsically Ca²⁺ dyes, slow exogenous buffers (e.g., EGTA) to disrupt inter-cluster CICR and UV-photolyzable caged IP3. Single-wavelength dyes increase their fluorescence upon calcium binding producing images that are strongly dependent on their kinetic, transport and photophysical properties. Determining the artifacts that the imaging setting introduces is particularly relevant when trying to analyze the smallest Ca²⁺ signals. In this paper we introduce a method to estimate the expected signal-to-noise ratio of Ca²⁺ imaging experiments that use single-wavelength dyes. The method is based on the Number and rightness technique. It involves the performance of a series of experiments and their subsequent analysis in terms of a fluorescence fluctuation model with which the model parameters are quantified. Using the model, the expected signal-to-noise ratio is then computed. Equivalence classes between different experimental conditions that produce images with similar signal-to-noise ratios can then be established. The method may also be used to estimate the smallest signals that can reliably be observed with each setting.

The standard of care for smouldering multiple myeloma is observation. We did the QuiRedex study to compare early treatment with lenalidomide plus dexamethasone with observation in patients with high-risk smouldering multiple myeloma. Here we report the long-term follow-up results of the trial. We did this open-label, randomised, controlled phase 3 study at 19 centres in Spain and three centres in Portugal. Patients aged 18 years or older with high-risk smouldering multiple myeloma were randomly assigned (1:1), via a computerised random number generator, to receive either early treatment with lenalidomide plus dexamethasone or observation, with dynamic balancing to maintain treatment balance within the two groups. Randomisation was stratified by time from diagnosis of smouldering multiple myeloma to study enrolment (≤6 months vs >6 months). Patients in the treatment group received nine 4-week induction cycles (lenalidomide 25 mg per day on days 1–21, plus dexamethasone 20 mg per day on days–1–4 and days 12–15), followed by maintenance therapy (lenalidomide 10 mg per day on days 1–21 of each 28-day cycle) up to 2 years. Group allocation was not masked from study investigators or patients. The primary endpoint was time from randomisation to progression to symptomatic myeloma. The primary analysis was based on the per-protocol population, restricted to patients who fulfilled the protocol in terms of eligibility. Safety assessments were based on the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT00480363. Between Nov 8, 2007, and June 9, 2010, 125 patients were enrolled and underwent randomisation. 119 patients comprised the per-protocol population and were randomly assigned to receive either lenalidomide plus dexamethasone (n=57) or observation (n=62). The cutoff date for this update was June 30, 2015. Median follow-up for surviving patients was 75 months (IQR 67–85). Lenalidomide plus dexamethasone continued to provide a benefit on time to progression compared with observation (median time to progression not reached [95% CI 47 months–not reached] vs 23 months [16–31]; hazard ratio [HR] 0·24 [95% CI 0·14–0·41]; p<0·0001). Progression to multiple myeloma occurred in 53 (86%) of 62 patients in the observation group compared with 22 (39%) of 57 patients in the treatment group. At data cutoff, ten (18%) patients had died in the treatment group and 22 (36%) patients had died in the observation group; median overall survival from the time of study entry had not been reached in either group (95% CI 65 months–not reached vs 53 months–not reached; HR 0·43 [95% CI 0·21–0·92], p=0·024). Survival in patients who had received subsequent treatments at the time of progression to active disease did not differ between groups (HR 1·34 [95% CI 0·54–3·30]; p=0·50). The most frequently reported grade 3 adverse events in patients given lenalidomide plus dexamethasone were infection (four [6%]), asthenia (four [6%]), neutropenia (three [5%]), and skin rash (two [3%]); these events all occurred during induction therapy. No grade 4 adverse events occurred, but one (2%) patient in the lenalidomide plus dexamethasone group died from a respiratory infection during induction therapy The frequency of second primary malignancies was higher in patients in the treatment group than in those in the observation group (six [10%] of 62 patients vs one [2%] of 63 patients), but the cumulative risk of development did not differ significantly between the groups (p=0·070). This study is, to our knowledge, the first randomised trial in which early treatment has been assessed in selected patients with high-risk smouldering multiple myeloma. Positive results from ongoing trials would support the use of early treatment for patients with high-risk disease in the near future. Pethema (Spanish Program for the Treatment of Hematologic Diseases).

Background Physical fitness is an important marker of current and future health status, yet the association between physical fitness and indicators of mental health in youth has not been systematically reviewed and meta-analyzed. Objective The aim of this work was to systematically review and meta-analyze the association between physical fitness components (i.e. cardiorespiratory fitness, muscular fitness, speed-agility, flexibility and fitness composite) and mental health indicators (i.e. psychological well-being and psychological ill-being) in preschoolers, children and adolescents. Design Systematic review and meta-analysis. Data Sources Systematic searches were conducted in PubMed, Web of Science and Scopus from database inception to May 2020. Eligibility Criteria Studies (cross-sectional, longitudinal and intervention designs) were included if they measured at least one physical fitness component and one mental health indicator in healthy youth (2–18 years). Results A total of 58 unique studies (52 cross-sectional, 4 longitudinal and 4 intervention studies) met all eligibility criteria and were included. There was a significant positive overall association between physical fitness and mental health in children and adolescents (pooled r  = 0.206, p  < 0.001). We found suggestive evidence of moderation by age group, fitness components and socioeconomic status (all p  < 0.08). No relevant studies focusing on preschoolers were identified. Evidence based on longitudinal and intervention studies was limited. Conclusion We observed a small to medium sized positive association between physical fitness and overall mental health in youth. However, as the majority of studies were cross-sectional, additional longitudinal and intervention studies are needed to provide evidence of causation. Trial Registration PROSPERO registration number CRD42017080005.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high‐throughput screening variant of the Mammalian Membrane Two‐Hybrid (MaMTH‐HTS) to map the protein–protein interactions of wild‐type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient‐specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient‐derived intestinal organoids has a significant effect on CFTR functional expression. SYNOPSIS A new MaMTH‐HTS platform is used with a Human ORFeome library to map the protein‐protein interactions of full‐length wildtype and F508del CFTR. Functional validations in multiple disease models uncovered proteins with potential roles in CFTR function and cystic fibrosis. MaMTH‐HTS identifies 447 interactors of wildtype and F508del CFTR. The CFTR interactomes are evaluated using traditional MaMTH and a fluorescence‐based assay is performed to monitor the effect of transiently expressed interactors on CFTR channel activity. siRNA‐mediated knockdown of candidate proteins reveals 19 interactors whose down‐regulation led to increased F508del CFTR trafficking and complex glycosylation. One candidate protein, Fibrinogen Like 2 (FGL2) has a significant effect on CFTR functional expression, as demonstrated in patient‐derived intestinal organoids. Graphical Abstract A new MaMTH‐HTS platform is used with a Human ORFeome library to map the protein‐protein interactions of full‐length wildtype and F508del CFTR. Functional validations in multiple disease models uncovered proteins with potential roles in CFTR function and cystic fibrosis.

Lipid oxidation is the principal process of edible oil degradation. Therefore, the objective was to determine the antioxidant effect of hop and oregano essential oils in sunflower oil under high temperatures, in order to replace the synthetic antioxidants. The sunflower oil was enriched with oregano and hop essential oils (0.02% w/w). These treatments were compared with sunflower oil with the addition of BHT (0.02% w/w) and another sample of sunflower oil (without additives). All treatments were exposed to 150 °C for 8 h. Samples were collected at 0, 2, 4, 6, and 8 h. Peroxide value (PV), conjugated dienes, oxidation, and essential oils volatile compounds were measured. Oregano and hop essential oil increased the oxidative stability of the sunflower oil under 150 °C. The PV of the control was 44.1 meq O 2 /kg at 8 h, while sunflower oil with the addition of hop and oregano essential oils were 14.4 meq O 2 /kg and 8.5 meq O 2 /kg at 8 h, respectively. Origanum vulgare essential oil had the best antioxidant effect, obtaining 80.7% less PV than the control, while Humulus lupulus showed 67.4% less PV than the control. In general, in comparison to the sunflower oil without additives, lower values of hexanal and 2-heptenal were observed in the samples enriched with essential oils. The principal components of these essential oils were found in the heated samples. Terpinen-4-ol (oregano) and β-myrcene (hop) were present at 4 h of the heat treatment. Thus, these terpenes appeared to be responsible for maintaining the antioxidant protection of sunflower oil throughout the heat treatment.

In view of the rising relevance of emerging pollutants in the environment, this work studies the photodegradation of three antibiotics, evaluating the effects of the pH of the medium and the concentration of dissolved organic matter. Simulated light (with a spectrum similar to that of natural sunlight) was applied to the antibiotics Ciprofloxacin (Cip), Clarithromycin (Cla) and Trimethoprim (Tri), at three different pH, and in the presence of different concentrations of humic acids. The sensitivity to light followed the sequence: Cip > Cla > Tri, which was inverse for the half-life (Tri > Cla > Cip). As the pH increased, the half-life generally decreased, except for Cla. Regarding the kinetic constant k, in the case of Cip and Tri it increased with the rise of pH, while decreased for Cla. The results corresponding to total organic carbon (TOC) indicate that the complete mineralization of the antibiotics was not achieved. The effect of humic acids was not marked, slightly increasing the degradation of Cip, and slightly decreasing it for Tri, while no effect was detected for Cla. These results may be relevant in terms of understanding the evolution of these antibiotics, especially when they reach different environmental compartments and receive sunlight radiation.