Development of new RT-PCR assays for the specific detection of BA.2.86 SARS-CoV-2 and its descendent sublineages.

The SARS-CoV-2 BA.2.86 variant, also known as Pirola, has acquired over 30 amino acid changes in the Spike protein, evolving into >150 sublineages within ten months of its emergence.

EU surveys insights: analytical tools, future directions, and the essential requirement for reference materials in wastewater monitoring of SARS-CoV-2, antimicrobial resistance and beyond.

Background: Wastewater surveillance (WWS) acts as a vigilant sentinel system for communities, analysing sewage to protect public health by detecting outbreaks and monitoring trends in pathogens and contaminants. To achieve a thorough comprehension of present and upcoming practices and to identify challenges and opportunities for standardisation and improvement in WWS methodologies, two EU surveys were conducted targeting over 750 WWS laboratories across Europe and other regions. The first survey explored a diverse range of activities currently undertaken or planned by laboratories.

Detection and Quantification of Botanical Impurities in Commercial Oregano () Using Metabarcoding and Digital PCR.

DNA technology for food authentication is already well established, and with the advent of Next Generation Sequencing (NGS) and, more specifically, metabarcoding, compositional analysis of food at the molecular level has rapidly gained popularity. This has led to several reports in the media about the presence of foreign, non-declared species in several food commodities. As herbs and spices are attractive targets for fraudulent manipulation, a combination of digital PCR and metabarcoding by NGS was employed to check the purity of 285 oregano samples taken from the European market.

DNA Accounting: Tallying Genomes to Detect Adulterated Saffron.

The EU General Food Law not only aims at ensuring food safety but also to 'prevent fraudulent or deceptive practices; the adulteration of food; and any other practices which may mislead the consumer'. Especially the partial or complete, deliberate, and intentional substitution of valuable ingredients (e.g.

A roadmap for the generation of benchmarking resources for antimicrobial resistance detection using next generation sequencing.

Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time.

Nuclear DNA barcodes for cod identification in mildly-treated and processed food products.

Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group.

The challenges of designing a benchmark strategy for bioinformatics pipelines in the identification of antimicrobial resistance determinants using next generation sequencing technologies.

Next-Generation Sequencing (NGS) technologies are expected to play a crucial role in the surveillance of infectious diseases, with their unprecedented capabilities for the characterisation of genetic information underlying the virulence and antimicrobial resistance (AMR) properties of microorganisms.  In the implementation of any novel technology for regulatory purposes, important considerations such as harmonisation, validation and quality assurance need to be addressed.  NGS technologies pose unique challenges in these regards, in part due to their reliance on bioinformatics for the processing and proper interpretation of the data produced.

Novel nuclear barcode regions for the identification of flatfish species.

The development of an efficient seafood traceability framework is crucial for the management of sustainable fisheries and the monitoring of potential substitution fraud across the food chain. Recent studies have shown the potential of DNA barcoding methods in this framework, with most of the efforts focusing on using mitochondrial targets such as the and genes. In this article, we show the identification of novel targets in the nuclear genome, and their associated primers, to be used for the efficient identification of flatfishes of the family.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B feed additive.

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive.

Towards Plant Species Identification in Complex Samples: A Bioinformatics Pipeline for the Identification of Novel Nuclear Barcode Candidates.

Monitoring of the food chain to fight fraud and protect consumer health relies on the availability of methods to correctly identify the species present in samples, for which DNA barcoding is a promising candidate. The nuclear genome is a rich potential source of barcode targets, but has been relatively unexploited until now. Here, we show the development and use of a bioinformatics pipeline that processes available genome sequences to automatically screen large numbers of input candidates, identifies novel nuclear barcode targets and designs associated primer pairs, according to a specific set of requirements.

Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF.

Human amnion-derived cells: prospects for the treatment of lung diseases.

Lung diseases represent a significant burden of morbidity and mortality worldwide. Current therapies have not proven adequate in the long term and are often associated with significant side effects. There has been recent interest in the regenerative/reparative potential of cell-based therapies, including cells derived from the placental tissues.

Cystic fibrosis newborn screening: distribution of blood immunoreactive trypsinogen concentrations in hypertrypsinemic neonates.

The IRT screening test for the use in diagnosing newborns with CF has a high sensitivity but is not very specific resulting in a large number of screened positive infants found to have a normal sweat test. The aim of this study was to analyze the differences in b-IRT levels among different groups of newborns positive to NBS.Population data included all b-IRT positive (>99th centile) neonates born in Lombardia from 2000 to 2007.

Fine characterization of the recurrent c.1584+18672A>G deep-intronic mutation in the cystic fibrosis transmembrane conductance regulator gene.

Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings.

Amniotic mesenchymal stem cells: a new source for hepatocyte-like cells and induction of CFTR expression by coculture with cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs) were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic) lineages.

A wide methodological approach to identify a large duplication in CFTR gene in a CF patient uncharacterised by sequencing analysis.

Background: PCR-based diagnostic procedures are not able to characterise 6% of CF alleles. Recently, the application of array-CGH and of CFTR mRNA analysis has allowed the identification of new copy number mutations and splicing defects, that account for 2% and 13% of CF alleles, respectively, in the Italian population.

Methods: Here, we report the characterisation of a large duplication in CFTR gene through different methods: MLPA assay, RT-PCR and high-resolution array-CGH.

Use of parthenogenetic activation of human oocytes as an experimental model for evaluation of polar body based PGD assay performance.

Purpose: To develop an experimental model to assess the feasibility of polar body preimplantation genetic diagnosis without requiring oocyte fertilization.

Methods: First polar body was removed from donated oocytes and second polar body was biopsied after parthenogenetic activation. Molecular analysis on both polar bodies involved a fluorescent multiplex polymerase chain reaction of short-tandem repeat markers, closely linked to genes of interest.

A novel donor splice site characterized by CFTR mRNA analysis induces a new pseudo-exon in CF patients.

Background: The CFTR gene is tightly regulated and differentially expressed in many mucosal epithelial cell types. There is evidence of an increasing number of genomic variations in the intronic regions influencing mRNA splicing, and also the level of normal CFTR transcript.

Methods: In the present study, we investigate the molecular defect by RT-PCR analyzing the mRNA of 25 cystic fibrosis (CF) patients in whom only one or no CF allele had been identified after DNA analysis (of all the exons of the CFTR gene).

Borderline sweat test: Utility and limits of genetic analysis for the diagnosis of cystic fibrosis.

Objective: The sweat test remains the gold standard for the diagnosis of Cystic Fibrosis (CF) even despite the availability of molecular analysis of Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). We investigated the relationship between CFTR mutation analysis and sweat chloride concentration in a cohort of subjects with borderline sweat test values, in order to identify misdiagnosis of CF.

Design And Methods: In the period between March 2006 and February 2008 we performed 773 sweat tests in individuals referred for suspect CF.

Effects of metabolic genotypes on intermediary biomarkers in subjects exposed to PAHS: results from the EXPAH study.

Data from the EXPAH project on PAH exposure and intermediary biomarkers were analyzed with respect to individual genotypes at seven metabolic gene loci. The GSTM1 null allele was associated with significantly higher levels of two biomarkers, malondialdehyde-2'-deoxyguanosine and benzo[a]pyrene DNA adducts in the total population from three Central and Eastern European countries. The CYP1B1 Leu/Val variant demonstrated effects on both markers of oxidative DNA damage in opposite directions, producing a higher level of M(1)dG with a trend from wild type (Leu/Leu) to heterozygotes to homozygous (Val/Val) variants, whereas the effects of these variants were reversed for 8-oxodG.

Meta- and pooled analyses of the cytochrome P-450 1B1 Val432Leu polymorphism and breast cancer: a HuGE-GSEC review.

The association between the cytochrome P-450 1B1 (CYP1B1) Val432Leu polymorphism and breast cancer was assessed through a meta-analysis of all published case-control studies and a pooled analysis of both published and unpublished case-control studies from the Genetic Susceptibility to Environmental Carcinogens (GSEC) database ( www.upci.upmc.

A common CYP1B1 polymorphism is associated with 2-OHE1/16-OHE1 urinary estrone ratio.

Cytochrome P450 (CYP) is a multigene family of enzymes involved in important life functions; some of these genes are inducible and are implicated in the oxidative metabolic activation and detoxification of many endogenous and exogenous compounds. CYP1B1 codes for an enzyme that catalyses the production of a 2- and 4-hydroxyl group in estrone and estradiol, while CYP1A1 catalyzes the 2-hydroxylation of estradiol in endometrium. The two genes were evaluated in a cohort of 150 subjects: African-American women had significantly lower 2-hydroxyl estrone/16-hydroxyl estrone (2-OHE1/16-OHE1) urinary metabolite ratios than Caucasian women (2.