Conformational dynamics in microRNAs : the example of miR-34a targeting Sirt1 mRNA

In biology, regulatory mechanisms are essential to achieve complex tasks, as virtually every process can be positively or negatively modulated in its outcome, upon different cues. In humans, microRNAs (miRNAs) constitute a fundamental layer of post-transcriptional gene expression regulation. This class of molecules finely tune protein expression, by downregulating messenger RNAs (mRNA) levels and their translation. The mechanism by which miRNAs find and act upon their targets primarily relies on their nucleotide sequence, relative to the corresponding binding site on the mRNA. The development of an exhaustive miRNA–mRNA interactome is particularly attractive because of the profound implication for basic biology as well as for diagnostics and therapeutics in human health. However, computational prediction of target sites and associated downregulation levels, using the limited sequence determinants available, is still an outstanding challenge in the field. In this thesis, we bring forward the hypothesis that modeling of miRNA–mRNA pairs might benefit from considering the inherent structural flexibility of these complexes, at the molecular level. In the introductory chapter, we present the structural features of RNAs with a focus on their conformational dynamics and NMR spectroscopy as a tool to investigate these motions. The molecular details of miRNA biogenesis and function are later introduced to contextualize the results of Paper I. Finally, the challenges associated with RNA sample preparation are discussed in light of the work presented in Paper II. In Paper I, we show that a miRNA–mRNA pair involved in a cancer-regulating pathway exploits its flexibility to toggle between lower and higher target repression states. This study shows that suboptimal structures of a given miRNA–mRNA pair, that are overlooked by computational prediction and that often elude experimental detection, can be functionally relevant and are essential to draw a mechanistic picture of miRNA function. The methods used in Paper I for RNA sample preparation and molecular simulation are described in Paper II and II, respectively. While these methods were essential to achieve the results of Paper I, they also find widespread application in the RNA field

Unsupervised estimation of signal-dependent CCD camera noise

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Vitamin D status and cholecalciferol supplementation in chronic kidney disease patients: An Italian cohort report

This study investigated the factors associated with hypovitaminosis D, in a cohort of 405 prevalent patients with chronic kidney disease (CKD) stages 2–4, living in Italy and followed-up in tertiary care. The effect of cholecalciferol 10,000 IU once-a-week for 12 months was evaluated in a subgroup of 100 consecutive patients with hypovitaminosis D. Vitamin D deficiency was observed in 269 patients (66.4%) whereas vitamin D insufficiency was found in 67 patients (16.5%). In diabetic patients, 25-hydroxyvitamin D deficiency was detected in 80% of cases. In patients older than 65 years, the prevalence of hypovitaminosis D was 89%. In the univariate analysis, 25-hydroxyvitamin D was negatively related to age, parathyroid hormone (PTH), proteinuria, and Charlson index, while a positive relationship has emerged with hemoglobin level. On multiple regression analysis, only age and PTH levels were independently associated with 25-hydroxyvitamin D levels. No relationship emerged between vitamin D deficiency and renal function. Serum levels of 25-hydroxyvitamin D or prevalence of hypovitaminosis D did not differ between patients on a free-choice diet and on a renal diet, including low-protein, low-phosphorus regimens. Twelve-month oral cholecalciferol administration increased 25-hydroxyvitamin D and reduced PTH serum levels. In summary, hypovitaminosis D is very prevalent in CKD patients (83%) in Italy, and it is similar to other locations. PTH serum levels and age, but not renal function, are the major correlates of hypovitaminosis D. Implementation of renal diets is not associated with higher risk of vitamin D depletion. Oral cholecalciferol administration increased 25-hydroxyvitamin D and mildly reduced PTH serum levels. Oral cholecalciferol supplementation should be recommended as a regular practice in CKD patients, also when serum 25-hydroxyvitamin D determination is not available or feasible

Fate of soil organic carbon and polycyclic aromatic hydrocarbons in a vineyard soil treated with biochar

The effect of biochar addition on the levels of black carbon (BC) and polcyclic aromatic hydrocarbons (PAHs) in a vineyard soil in central Italy was investigated within a two year period. Hydropyrolysis (HyPy) was used to determine the contents of BC (BCHyPy) in the amended and control soils while the hydrocarbon composition of the semi-labile (non-BCHyPy) fraction released by HyPy was determined by gas chromatography-mass spectrometry, together with the solvent-extractable PAHs. The concentrations of these three polycyclic aromatic carbon reservoirs, changed and impacted differently on the soil organic carbon over the period of the trial. The addition of biochar (33 ton dry biochar ha-1) gave rise to a sharp increase in soil organic carbon which could be accounted for by an increase of BCHyPy. Over time, the concentration of BCHyPy decreased significantly from 36 to 23 mg g-1, and as a carbon percentage from 79% to 61%. No clear time trends were observed for the non-BCHyPy PAHs varying from 39 to 34 µg g-1 in treated soils, not significantly different from control soils. However, the concentrations of extractable PAHs increased markedly in the amended soils, and decreased with time from 153 to 78 ng g-1 remaining always higher than those in untreated soil. The extent of the BCHyPy loss was more compatible with physical rather than chemical processes

Retrieving soil moisture in rainfed and irrigated fields using Sentinel-2 observations and a modified OPTRAM approach

Abstract Surface soil water content plays an important role in driving the exchange of latent and sensible heat between the atmosphere and land surface through transpiration and evaporation processes, regulating key physiological processes affecting plants growth. Given the high impact of water scarcity on yields, and of irrigated agriculture on the overall withdrawal rate of freshwater, it is important to define models that help to improve water resources management for agricultural purposes, and to optimize rainfed crop yield. Recent advances in satellite-based remote sensing have led to valuable solutions to estimate soil water content based on microwave or optical/thermal-infrared data. This study aims at improving soil water content estimation at high spatial and temporal resolution, by means of the Optical Trapezoid Model (OPTRAM) driven by Copernicus Sentinel-2 data. Two different model variations were considered, based on linear and nonlinear parameters constraints, and validated against in situ soil water content measurements made with time domain reflectometry (TDR) on irrigated maize in central Italy and on rainfed maize and pasture in northern Italy. For the first site the non-linear model shows a better correlation between measured and estimated soil water content values (r = 0.80) compared to the linear model (r = 0.73). In both cases the modeled soil moisture tends to overestimate the measured values at medium to high water content level, while both models underestimate soil moisture at low water content level. Estimated versus measured normalized surface soil water for rainfed pasture plots from nonlinear OPTRAM parametrized based on irrigated maize parameterization (SIM1), and site-specific parametrization for rainfed pasture (SIM2), indicate that both models (SIM1 and SIM2) are comparable for rotational grazing pasture (RMSEsim1 = 0.0581 vs. RMSEsim2 = 0.0485 cm3 cm-3) and the continuous grazing pasture (RMSEsim1 = 0.0485 vs. RMSEsim2 = 0.0602 cm3 cm-3), while for the rainfed maize plots SIM1 shows lower RMSE (average for all plots RMSE = 0.0542 cm3 cm-3) compared to the site-specific calibration model (SIM2 – average for all plots RMSE = 0.0645 cm3 cm-3). Finally, OPTRAM estimations are close to in situ measurement values while Surface Soil Moisture at 1 km (SSM1 km) tends to underestimate the measurements during maize crop growing season. Soil moisture retrieval from high-resolution Sentinel-2 optical images allows water stress conditions to be effectively mapped, supporting decision making in irrigation scheduling and other crop management

Clinical Features, Cardiovascular Risk Profile, and Therapeutic Trajectories of Patients with Type 2 Diabetes Candidate for Oral Semaglutide Therapy in the Italian Specialist Care

Introduction: This study aimed to address therapeutic inertia in the management of type 2 diabetes (T2D) by investigating the potential of early treatment with oral semaglutide. Methods: A cross-sectional survey was conducted between October 2021 and April 2022 among specialists treating individuals with T2D. A scientific committee designed a data collection form covering demographics, cardiovascular risk, glucose control metrics, ongoing therapies, and physician judgments on treatment appropriateness. Participants completed anonymous patient questionnaires reflecting routine clinical encounters. The preferred therapeutic regimen for each patient was also identified. Results: The analysis was conducted on 4449 patients initiating oral semaglutide. The population had a relatively short disease duration (42%  60% of patients, and more often than sitagliptin or empagliflozin. Conclusion: The study supports the potential of early implementation of oral semaglutide as a strategy to overcome therapeutic inertia and enhance T2D management

One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is in vitro transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA

Dynamics of the Intrinsically Disordered C-Terminal Domain of the Nipah Virus Nucleoprotein and Interaction with the X Domain of the Phosphoprotein as Unveiled by NMR Spectroscopy

International audienceno abstrac

Determination of polycyclic aromatic hydrocarbons (PAHs) in an agricultural soil treated with biochar

The unavoidable presence of hazardous PAHs in biochar is a matter of concern due to possible health and ecological risks associated to its application in soils. Besides the direct effect of increased environmental PAH concentrations resulting from its use, a variety of indirect and ancillary factors may affect the behavior of biochar amended soils as a sink or source of PAHs. Understanding the impact of biochar on soil quality needs a considerable effort from laboratory and field studies. This contribution reports on the levels of PAHs in a cultivated soil one year after the addition of biochar. Biochar, deriving from slow pyrolysis of pruning orchard, was superficially distributed in the inter-row space of a vineyard at a rate of 22 t ha-1 in 2009 following a randomized block layout with 5 replicates and incorporated into the soil with a chisel plow tiller in the 0-30 cm depth. Soil is a sandy clay loam with sub-acid pH (5.4). Two soil sampling campaigns were made in August and December 2010. An analytical procedure targeted to the determination of PAHs in both biochar and soil matrices was recently developed [1]. Briefly, samples were spiked with surrogate PAHs, then PAHs were soxhlet extracted for 36 hours with an acetone/cyclohexane 1:1 mixture, purified by silica gel solid phase extraction (SPE) and analysed by GC-MS(SIM). The method was validated with the certified reference soil CRM (ERM-CC013a). All the US EPA PAHs were detected in the utilised biochar and summed up to 3.5 µg g-1, with naphthalene as the most abundant species followed by phenanthrene. The untreated soil samples exhibited total PAH concentrations two orders of magnitude lower than that of biochar and with a different distribution pattern characterised by higher abundance of phenanthrene and fluorene. After almost one year following biochar application, the total mean concentration values of PAHs in amended soils resulted higher than those of the untreated soils, both in August (52 vs. 31 ng g-1) and December (35 vs. 27 ng g-1). However, the differences were not statistically significant due to the high dispersion of PAH values between samples withdrawn from the same parcel (n = 5). The lower concentrations observed in winter for the treated soils suggest a seasonal variability superimposed to sampling heterogeneity. These preliminary results suggest that the soil contamination by PAHs following biochar application is not significant at the application rates currently recommended in agriculture (20–60 t ha-1) and the PAH load typically found in biochar from slow pyrolysis [1]. The long term persistence of PAHs and their potential to be bioaccumulated is under investigation. [1] D. Fabbri, A.G. Rombolà, C. Torri, K. A. Spokas, J.Anal.Applied Pyrol., 2012), http://dx.doi.org/10.1016/j.jaap.2012.10.003