Downexpression of miR-200c-3p Contributes to Achalasia Disease by Targeting the PRKG1 Gene

Achalasia is an esophageal smooth muscle motility disorder with unknown pathogenesis. Taking into account our previous results on the downexpression of miR-200c-3p in tissues of patients with achalasia correlated with an increased expression of PRKG1, SULF1, and SYDE1 genes, our aim was to explore the unknown biological interaction between these genes and human miR-200c-3p and if this relation could unravel their functional role in the etiology of achalasia. To search for putative miR-200c-3p binding sites in the 3′-UTR of PRKG1, SULF1 and SYDE1, a bioinformatics tool was used. To test whether PRKG1, SULF1, and SYDE1 are targeted by miR-200c-3p, a dual-luciferase reporter assay and quantitative PCR on HEK293 and fibroblast cell lines were performed. To explore the biological correlation between PRKG1 and miR-200c-3p, an immunoblot analysis was carried out. The overexpression of miR-200c-3p reduced the luciferase activity in cells transfected with a luciferase reporter containing a fragment of the 3′-UTR regions of PRKG1, SULF1, and SYDE1 which included the miR-200c-3p seed sequence. The deletion of the miR-200c-3p seed sequence from the 3′-UTR fragments abrogated this reduction. A negative correlation between miR-200c-3p and PRKG1, SULF1, and SYDE1 expression levels was observed. Finally, a reduction of the endogenous level of PRKG1 in cells overexpressing miR-200c-3p was detected. Our study provides, for the first time, functional evidence about the PRKG1 gene as a direct target and SULF1 and SYDE1 as potential indirect substrates of miR-200c-3p and suggests the involvement of NO/cGMP/PKG signaling in the pathogenesis of achalasia.     

Blocking Muscarinic Receptor 3 Attenuates Tumor Growth and Decreases Immunosuppressive and Cholinergic Markers in an Orthotopic Mouse Model of Colorectal Cancer

Tumor cells have evolved to express immunosuppressive molecules allowing their evasion from the host’s immune system. These molecules include programmed death ligands 1 and 2 (PD-L1 and PD-L2). Cancer cells can also produce acetylcholine (ACh), which plays a role in tumor development. Moreover, tumor innervation can stimulate vascularization leading to tumor growth and metastasis. The effects of atropine and muscarinic receptor 3 (M3R) blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), on cancer growth and spread were evaluated in vitro using murine colon cancer cell line, CT-26, and in vivo in an orthotopic mouse model of colorectal cancer. In the in vitro model, atropine and 4-DAMP significantly inhibited CT-26 cell proliferation in a dose dependent manner and induced apoptosis. Atropine attenuated immunosuppressive markers and M3R via inhibition of EGFR/AKT/ERK signaling pathways. However, 4-DAMP showed no effect on the expression of PD-L1, PD-L2, and choline acetyltransferase (ChAT) on CT-26 cells but attenuated M3R by suppressing the phosphorylation of AKT and ERK. Blocking of M3R in vivo decreased tumor growth and expression of immunosuppressive, cholinergic, and angiogenic markers through inhibition of AKT and ERK, leading to an improved immune response against cancer. The expression of immunosuppressive and cholinergic markers may hold potential in determining prognosis and treatment regimens for colorectal cancer patients. This study’s results demonstrate that blocking M3R has pronounced antitumor effects via several mechanisms, including inhibition of immunosuppressive molecules, enhancement of antitumor immune response, and suppression of tumor angiogenesis via suppression of the AKT/ERK signaling pathway. These findings suggest a crosstalk between the cholinergic and immune systems during cancer development. In addition, the cholinergic system influences cancer evasion from the host’s immunity.     

Lipidomic Approaches to Study HDL Metabolism in Patients with Central Obesity Diagnosed with Metabolic Syndrome

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated “omics” approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the “lysophosphatidylcholines to phosphatidylcholines” and “cholesteryl ester to free cholesterol” ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated “omics” approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.     

Engineering the Thermoelectrical Properties of PEDOT:PSS by Alkali Metal Ion Effect

Engineering the electrical properties of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)(PEDOT:PSS)holds great potential for various applications such a...     

Subspace identification analysis of RFX and T2R reversed-field pinches

Input–output datasets from two magnetic confinement fusion (MCF) experiments of the reversed-field pinch (RFP) type are examined. The RFP datasets, which are samples of the distributed magnetic field dynamics, are naturally divided into many smaller batches due to the pulsed-plasma operation of the experiments. The two RFP experiments considered are (i) EXTRAP T2R (T2R) with 64 inputs and 64 outputs and (ii) RFX-mod (RFX) with 192 inputs and 192 outputs. Both T2R and RFX are magnetohydrodynamically unstable and operates under magnetic feedback with optional dither injection. Using subspace system identification techniques and randomised cross-validation (CV) methods to minimise the generalisation error, state-space orders of the empirical systems are suggested. These system orders are compared to “stabilisation diagrams” commonly used in experimental modal analysis practice. The relation of the CV system order to the decay of the singular values from the subspace method is observed. Both (i) stable vacuum diffusion and (ii) unstable plasma response datasets are analysed. Apparent simulation and prediction errors are quantified for both cases using a deviation-accounted-for index. These results are purely data-driven. A simple approach towards exploitation of the subspace techniques for finite-element model refinement and data confrontation is presented.     

A spatial relation-based framework to perform visual information extraction

The Spatial Relation Query (SRQ) tool is a graphical software environment, supported by a SQL-like language, which enables users to perform information extraction driven by the visual appearance and the spatial arrangement of the information. The tool has been initially customised to work on specific application domains, like web pages and geospatial data. In this paper, we present the theoretical formalisation of the visual information extraction (VIE) task and accordingly the redesign of the SRQ tool, which is now a full-featured, general-purpose information extraction system. Moreover, we show a new application of the VIE framework to the analysis and visual information extraction from PDF files.     

Determination of pyrethroids in chicken egg samples: development and validation of a confirmatory analytical method by gas chromatography/mass spectrometry

The aim of this study was to develop and validate an efficient analytical method based on gas chromatography/tandem mass spectrometry (GC/MS/MS) for detection and quantification of six pyrethroids residues (Phenothrin, Permethrin, Cyfluthrin, Cypermethrin, Deltamethrin and Fenvalerate) in chicken eggs. The method was based on a preliminary liquid–liquid extraction of albumen‐free yolk samples, followed by a clean‐up by solid‐phase extraction. GC/MS/MS analyses were carried out in the selected reaction monitoring mode. Validation parameters such as specificity, detection capability, decision limit, precision, recovery, stability and ruggedness were determined, resulting in compliance with Decision 2002/657/EC. No complicated apparatus are required; moreover, low volumes of organic solvents and a nonintensive manual labour are required. These low costs and simple procedure, based on rapid and safe operations, may represent a useful tool in the routine analysis of pyrethroids pesticides, in the place of the currently used conventional techniques.     

Anisotropic molecular packing of soluble C60 fullerenes in hexagonal nanocrystals obtained by solvent vapor annealing

We describe the self-assembly of soluble, chemically modified fullerene [6,6]-phenyl C₆₁ butyric acid methyl ester (PCBM) into a new crystalline phase where the C₆₀ moieties are arranged in parallel layers. Minimum C₆₀ center-to-center distance is 10 Å within the layers, and up to 15 Å perpendicular to the layers. Highly anisotropic, mesoscopic hexagonal crystals of this material, with a lateral size of many microns and a thickness below 1 μm, are obtained from chloroform solution by solvent vapor annealing, and characterized by optical microscopy and X-ray diffraction. The crystalline structure is deduced combining experimental data with molecular modeling and ab initio calculations. The large difference in C₆₀–C₆₀ spacing indicates a high anisotropy in electrical and charge transport properties of this new phase.