High Glucose Exposure Impairs L-Cell Differentiation in Intestinal Organoids: Molecular Mechanisms and Clinical Implications

Intestinal organoids are used to analyze the differentiation of enteroendocrine cells (EECs) and to manipulate their density for treating type 2 diabetes. EEC differentiation is a continuous process tightly regulated in the gut by a complex regulatory network. However, the effect of chronic hyperglycemia, in the modulation of regulatory networks controlling identity and differentiation of EECs, has not been analyzed. This study aimed to investigate the effect of glucotoxicity on EEC differentiation in small intestinal organoid platforms. Mouse intestinal organoids were cultured in the presence/absence of high glucose concentrations (35 mM) for 48 h to mimic glucotoxicity. Chronic hyperglycemia impaired the expression of markers related to the differentiation of EEC progenitors (Ngn3) and L-cells (NeuroD1), and it also reduced the expression of Gcg and GLP-1 positive cell number. In addition, the expression of intestinal stem cell markers was reduced in organoids exposed to high glucose concentrations. Our data indicate that glucotoxicity impairs L-cell differentiation, which could be associated with decreased intestinal stem cell proliferative capacity. This study provides the identification of new targets involved in new molecular signaling mechanisms impaired by glucotoxicity that could be a useful tool for the treatment of type 2 diabetes.     

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5′UTR and 3′UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband’s group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3′UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.     

Engineering of Janus-Like Dendrimers with Peptides Derived from Glycoproteins of Herpes Simplex Virus Type 1: Toward a Versatile and Novel Antiviral Platform

Novel antiviral nanotherapeutics, which may inactivate the virus and block it from entering host cells, represent an important challenge to face viral global health emergencies around the world. Using a combination of bioorthogonal copper-catalyzed 1,3-dipolar alkyne/azide cycloaddition (CuAAC) and photoinitiated thiol–ene coupling, monofunctional and bifunctional peptidodendrimer conjugates were obtained. The conjugates are biocompatible and demonstrate no toxicity to cells at biologically relevant concentrations. Furthermore, the orthogonal addition of multiple copies of two different antiviral peptides on the surface of a single dendrimer allowed the resulting bioconjugates to inhibit Herpes simplex virus type 1 at both the early and the late stages of the infection process. The presented work builds on further improving this attractive design to obtain a new class of therapeutics.     

Reduction of immunoreactivity of bovine beta-lactoglobulin upon combined physical and proteolytic treatment

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.     

Near-infrared spectroscopy to assist authentication and labeling of Asiago d’allevo cheese

This study investigated the possibility of using near-infrared spectroscopy (NIRS) for the authentication of Asiago d’allevo, a protected designation of origin cheese from northern Italy. Latent variable models applied on spectral data were developed and used to estimate several chemical properties and to classify the available dataset according to the location and management of the cheesemaking factory (lowland and alpine), the ripening age (6, 12, 18 and 36 months), the altitude of milk production (low, medium, medium–high and high), and the period of the year of the cheese production (May, July and September). The variable importance in projection index was used to identify the most informative spectral regions for discrimination. Results showed that NIR spectra can be used both to accurately estimate several chemical properties and to classify the samples according to the different experimental conditions under investigation with the same discrimination capacity provided by traditional chemical analysis.     

A 3D moisture-stress FEM analysis for time dependent problems in timber structures

This paper presents a 3D moisture-stress numerical analysis for timber structures under variable humidity and load conditions. An orthotropic viscoelastic-mechanosorptive material model is specialized on the basis of previous models. Both the constitutive model and the equations needed to describe the moisture flow across the structure are implemented into user subroutines of the Abaqus finite element code and a coupled moisture-stress analysis is performed for several types of mechanical loads and moisture changes. The presented computational approach is validated by analyzing some wood tests described in the literature and comparing the computational results with the reported experimental data.     

Experimental validation of the discrete-time MCS adaptive strategy

This paper is concerned with the implementation and experimental validation of a discrete-time model reference adaptive control strategy, known as Minimal Control Synthesis (MCS) algorithm. After discussing the proof of stability of the algorithm when applied to discretized models of continuous-time plants, the problem of controlling a highly nonlinear electro-mechanical device is taken as a representative case of study. It is shown that the discrete-time MCS is an effective strategy to solve the problem while guaranteeing robustness to unmodeled nonlinear dynamics over a wide range of test manoeuvres.     

Analytical and graphical design of lead–lag compensators

In this article an approach based on inversion formulae is used for the design of lead–lag compensators which satisfy frequency domain specifications on phase margin, gain margin and phase (or gain) crossover frequency. An analytical and graphical procedure for the compensator design on the Nyquist and Nichols planes is presented with some numerical examples.     

Stratigraphic analysis of intercalated graphite electrodes in aqueous inorganic acid solutions

A detailed stratigraphic investigation of the intercalation mechanism when graphite electrodes are immersed inside diluted perchloric(HClO₄)and sulfuric(H₂SO₄)electrolytes is obtained by comparing results when graphite crystals are simply immersed in the same acid solutions.By combining time-of-flight secondary ion mass spectrometry(ToF-SIMS)and in-situ atomic force microscopy(AFM),we provide a picture of the chemical species involved in the intercalation reaction.The depth intensity profile of the ion signals along the electrode crystal clearly shows a more complex mechanism for the intercalation process,where the local morphology of the basal plane plays a crucial role.Solvated anions are mostly located within the first tens of nanometers of graphite,but electrolytes also diffuse inside the buried layers for hundreds of nanometers,the latter process is also aided by the presence of mesoscopic crystal defects.Residual material from the electrolyte solution was found localized in well-defined circular spots,which represent preferential interaction areas.Interestingly,blister-like micro-structures similar to those observed on the highly oriented pyrolytic graphite(HOPG)surface were found in the buried layers,confirming the equivalence of the chemical condition on the graphite surface and in the underneath layers.