In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF

Aflatoxin M1 in cow, sheep, and donkey milk produced in Sicily, Southern Italy

Samples (n = 485) of raw (n = 394) or heat-treated (n = 91) milk of three different species (cow, n = 170; sheep, n = 133; donkey, n = 84), collected 2013–2016 in Western Sicily (Southern Italy), were analyzed for aflatoxin M1 (AFM1) by enzyme-linked immunosorbent assay (ELISA). Positive ELISA results were further analyzed by HPLC with fluorescence detection. Both methods had a detection limit for AFM1 in milk of 7 ng kg−1. ELISA yielded 12.9 and 5% positives in cows and sheep milk, respectively, all samples of donkey milk were negative. Levels of AFM1 were in most cases at 0.007–< 0.05 μg kg−1, only two samples (sheep milk) slightly exceeded the European Union maximum level of 0.05 μg kg−1. Only 6% of the samples were positive for AFM1 in a concentration range of 0.008–0.15 μg kg−1. Only milk samples collected directly from farms were positive. Overall, the levels were much lower than previously reported for Southern Italy cow and sheep milk samples purchased in retail stores. The results of this work indicate a continuous improvement of the feeding techniques on dairy farms of Southern Italy, which is essential to ensure consumers’ protection

Quantitative Evaluation of <i>CFTR</i> Gene Expression: A Comparison between Relative Quantification by Real-Time PCR and Absolute Quantification by Droplet Digital PCR

In the precision medicine era of cystic fibrosis (CF), therapeutic interventions, by the so-called modulators, target the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The levels of targetable CFTR proteins are a main variable in the success of patient-specific therapy. In turn, the CFTR protein level depends, at least in part, on the level of CFTR mRNA. Many mechanisms can modulate the CFTR mRNA level, for example, transcriptional rate, stability of the mRNA, epigenetics, and pathogenic variants that can affect mRNA production and degradation. Independently from the causes of variable CFTR mRNA levels, their exact quantitative assessment is of great importance in CF. Methods with high analytical sensitivity, precision, and accuracy are mandatory for the quantitative evaluation aimed at the amelioration of the diagnostic, prognostic, and therapeutic aspects. This paper compares, for the first time, two CFTR gene expression quantification methods: a well-established method for the relative quantification of CFTR mRNA using a real-time PCR and an innovative method for its absolute quantification using a droplet digital PCR. No comprehensive methods for absolute CFTR quantification via droplet digital PCR have been published so far. The accurate quantification of CFTR expression at the mRNA level is a critical step for the personalized therapeutic approaches of CF

3,5-DIIODO-L-THYRONINE-INDUCED MODIFICATION IN PITUITARY-THYROID AXIS IN RATS FED HIGH-FAT DIET. A PRELIMINARY REPORT

Experimental observations highlight that the administration of 3,5-diiodo-L-thyronine (T2) may decrease the body weight and the plasma levels of cholesterol and triglycerides and may prevent the onset of hepatic steatosis in rats fed diets rich in lipids (HFD). On the basis of these findings we have carried out some in vivo studies to evaluate the effects of increased levels of T2 on pituitary thyroid axis function in HFD rats. Fifteeen Wistar male rats were divided in 3 groups. The first group (N) was fed with a standard diet. The second group (G) was fed with a diet high in fat (HDF), while the third group (GT2) was additionally administered intraperitoneally with T2 (70 ug/100g body weight) for 3 days a week up to 4 week. Blood samples from animals were collected and stored at -20°C until 3rd generation and TSH, T3, T4, ACTH, triglycerides, cholesterol, glucose ALT, AST, Alkaline Phosphatase were assayed. Furthermore, rat liver from rats underwent histological examination to assess the degree of steatosis. The administration of T2 (70 ug/100 gr body weight 3 times a week up to 4 weeks suppressed TSH secretion in HDF rats. Unlike observed in the liver of rats from group N and group GT2, the histological examination of the liver from G group rats showed the presence of hepatic steatosis. These preliminary data highlight that the administration of 70 ug/100 b.w. of T2 inhibits TSH secretion and prevent the onset of hepatic steatosis in HFD rats

New Macrocyclic Amines Showing Activity as HIV Entry Inhibitors Against Wild Type and Multi-Drug Resistant Viruses †

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Effects of contaminants detected in animal biological indicators of Sicily on the zebrafish's genome

The research aimed to obtain data on the presence of toxic substances in dairy surrounding the Sicilian risk areas and to analyse the in vivo effects of these toxic agents in embryos and larvae of zebrafish. We examined 368 milk samples from cattle and sheep farms (50 for dioxins and 318 for heavy metals detection, respectively). The analysis of heavy metals has provided the development of an ICP-MS method. The dioxins levels were assessed by a HRGC/HRMS method. The results on milk samples revealed a significantly greater presence of dioxins in sheep milk, with average values near the limit of the Reg. EC 1259/2011. Ten samples that came from farms close to Bellolampo dump have detected concentrations greater than EC Reg. 1259/2011 limits. Only six samples of sheep milk have detected Pb concentrations over the LOD but below the limits imposed by the EC Reg. 1881/2006. The highest concentrations of dioxin detected in milk samples studied lead to a decrease in the survival of the larvae and malformations of the skeletal and circulatory systems. Furthermore, an over-expression of genes involved in phenomena of environmental stress such as hsp70, gadd45b, ATF3 CYP1A was found. The data obtained in this study confirm the close relationship between environmental factors and health effects of the population

L1077P CFTR pathogenic variant function rescue by Elexacaftor–Tezacaftor–Ivacaftor in cystic fibrosis patient-derived air–liquid interface (ALI) cultures and organoids: in vitro guided personalized therapy of non-F508del patients

Abstract Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro, which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials

Refinement of the clinical and mutational spectrum of UBE2A deficiency syndrome

UBE2A deficiency, that is, intellectual disability (ID) Nascimento type (MIM 300860), is an X-linked syndrome characterized by developmental delay, moderate to severe ID, seizures, dysmorphisms, skin anomalies, and urogenital malformations. Forty affected subjects have been reported thus far, with 31 cases having intragenic UBE2A variants. Here, we report on additional eight affected subjects from seven unrelated families who were found to be hemizygous for previously unreported UBE2A missense variants (p.Glu62Lys, p.Arg95Cys, p.Thr99Ala, and p.Arg135Trp) or small in-frame deletions (p.Val81_Ala83del, and p.Asp101del). A wide phenotypic spectrum was documented in these subjects, ranging from moderate ID associated with mild dysmorphisms to severe features including congenital heart defects (CHD), severe cognitive impairment, and pineal gland tumors. Four variants affected residues (Glu62, Arg95, Thr99 and Asp101) that contribute to stabilizing the structure of the E3 binding domain. The three-residue in-frame deletion, p.Val81_Ala83del, resulted from aberrant processing of the transcript. This variant and p.Arg135Trp mapped to regions of the protein located far from the E3 binding region, and caused variably accelerated protein degradation. By reviewing available clinical information, we revise the clinical and molecular profile of the disorder and document genotype-phenotype correlations. Pineal gland cysts/tumors, CHD and hypogammaglobulinemia emerge as recurrent features