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Phenotyping and genotyping for the dihydropyrimidine dehydrogenase test in Italy: a precise diagnostic strategy to detect rare variants and improve drug administration
No full textIntroduction: Dihydropyrimidine dehydrogenase (DPD) is the enzyme implicated in the catabolism of the fluoropyrimidines (FP), a class of chemotherapeutics used to treat many cancers. The DPYD gene is known to have a huge number of variants potentially associated with DPD deficiency. Therefore, phenotypic and genotypic characterization of DPD is fundamental for cancer patients before undergoing treatment with FP. The AIOM (Italian Association of Medical Oncology) requires genetic analysis, with the purpose to identify a panel of 5 single nucleotide polymorphisms associated with 5-fluorouracil (5-FU)-induced toxicity, while the biochemical test, which measures uracil levels to predict the residual activity of DPD, is only recommended and not mandatory. Methods: Single nucleotide polymorphisms were analyzed by real-time polymerase chain reaction (PCR) from peripheral blood. Uracil and dihydrouracil were quantified using an ultra-performance liquid chromatography/tandem mass spectrometry system in plasma obtained from patients before 5-FU treatment. Sanger sequencing was performed for the DPYD gene. Results: Here, we describe the case of a 70-year-old Caucasian male patient with pancreatic cancer for whom real-time PCR highlighted a heterozygous pathogenic variant c.1905 + 1G > A associated with a 50 % reduction of DPD enzymatic activity. This finding was not confirmed by the biochemical test which revealed a complete absence of DPD activity. By performing Sanger sequencing, we highlighted the concomitant presence of a likely pathogenic variant c.2622 + 1G > A, not compatible with the administration of 5-FU. Conclusion: Thus far, guidelines provide a limited panel for the identification of pathogenic or likely pathogenic variants of the DPYD gene, therefore we encourage the mandatory use of biochemical tests as a precise diagnostic strategy to detect rare variants and improve drug administration
Expression of Recombinant Proteins for the Development of a c-ELISA Kit for the Serological Diagnosis of African Horse Sickness
No full textAfrican horse sickness (AHS) is a devastating arboviral disease of equids with mortality rates
reaching 95% in susceptible horses. Reliable serological diagnostic tools are essential for
surveillance, outbreak management, and facilitating international equine trade. This study aimed to
develop a competitive enzyme-linked immunosorbent assay (c-ELISA) using a recombinant VP7
protein for the serological diagnosis of African horse sickness virus (AHSV). The VP7 core protein
was selected as the diagnostic antigen due to its high conservation across all nine AHSV serotypes
and group-specific antigenic properties.
Two variants of recombinant VP7 were evaluated: wild-type VP7 (WT-VP7) and a solubilityenhanced variant with a leucine to arginine substitution at position 345 (VP7-L345R). Both were
expressed using a baculovirus expression system in Sf9 insect cells. A comprehensive optimization
study identified extremely low multiplicity of infection (MOI 0.001) combined with extended culture
periods (120 hours post-infection) as optimal for VP7-L345R expression, yielding approximately 10
mg of purified protein from 0.75 L culture at 1.5 × 10^6 cells/mL. The recombinant VP7-L345R
demonstrated enhanced solubility with substantial secretion into culture supernatant, facilitating
simplified purification via immobilized metal affinity chromatography, while preserving essential
antigenic epitopes.
A panel of five monoclonal antibodies (Mabs) against VP7 was characterized through
immunoblotting and indirect ELISA. All recognized the recombinant VP7-L345R, confirming that
the solubility-enhancing mutation did not compromise critical antigenic determinants. Systematic
evaluation in a competitive format identified Mab 5D4F7 as optimal for c-ELISA development due
to its superior dynamic range, discriminatory capacity, and reproducibility. The finalized assay
utilized microplates coated with 1 μg/mL of purified VP7-L345R and Mab 5D4F7 at a 1:60,000
dilution.
This study has successfully produced and characterized the key reagents necessary for developing a
VP7-based c-ELISA for AHSV diagnosis. The solubility-enhanced VP7-L345R antigen, combined
with characterized monoclonal antibodies, provides a robust foundation for serological detection of
AHSV antibodies. These optimized reagents address an important need for enhanced surveillance
capabilities in the face of changing global patterns of vector distribution and equine movement.
Further validation studies are required to establish the full diagnostic performance of this assay under
field conditions
New analytical challenges in characterization of antibody-drug conjugates
No full textSince 1913, when Paul Ehrlich introduced the concept of “selective toxicity”, new strategies to obtain quality control, quantification of the drug were mandatory. For this reason, the introduction of antibody drug conjugates (ADC) has shown many critical points, being these systems made of small- and high-molecular weight compounds. Being ADCs a novelty for showing therapeutic action, they were subjected to a grow up in studies. There are in literature several approaches to follow ADCs for in vivo and in vitro studies because type of conjugation, release of the drug, and body-distribution are characteristic for each of them, resulting in many critical steps. Liquid chromatography (LC) and capillary electrophoresis (CE) are the most common analytical technique used in order to obtain the aim. For the characterization of ADCs, there are several chromatographic techniques. HPLC finds application in both small and large molecules, related to the availability of various modes of separation (reversed-phase, size exclusion, ion exchange, mixed-mode etc.). Capillary electrophoresis (CE) finds its main application in large molecule therapeutics, where its electrophoretic separation mechanism offers a distinct, and often superior, separation of macromolecules compared to classic chromatographic techniques. Several modes of CE, including capillary electrophoresis sodium dodecyl sulphate (CE-SDS), capillary zone electrophoresis (CZE), and capillary isoelectric focusing (CIEF) or imaged capillary isoelectric focusing (iCIEF) are commonly utilized in characterization of the critical quality attributes (CQAs) of monoclonal antibody drugs such as charge variants, size variants, and positional isomers/purity etc. In this review, analytical methods for physicochemical characterization of ADCs will be reported and analysed in order to highlight as the chromatographic procedures allow obtaining a complete ADCs evaluation and characterization
GWAS Study Applied to Phenotypically Slow Growth Strains of Listeria monocytogenes Workflow and Outcome
No full textListeria monocytogenes (Lm) is a serious public health foodborne pathogen cause of listeriosis, usually in elderly, pregnant and immunocompromised people, linked to consumption of contaminated food, especially ready-to-eat (RTE) products. Different protocols can be used to detect Lm, and ISO11290-1:2017 is the reference method in Europe. Through molecular techniques such as whole genome sequencing (WGS) it is possible to discriminate between Lm strains, which are unequally distributed between clinical cases, food or food related environments, probably also due to enrichment step bias towards some Lm serogroup (IIa) compared to IVb. In the present work a set of Lm strains, detected in clinical cases and food, was investigated to define Lm strains growth ability after incubation in Half Fraser broth, and Genome Wide Association Studies (GWAS) applied to correlate the growth phenotype traits to presence of relevant genes. GWAS enabled the identification of a more relevant cassette of genes associated to a holin region of bacteriophage A118 and the determination of the distribution of relevant genes, highlighted from GWAS analysis within a population of Lm IVb and IIa
Diritto internazionale e sostenibilità: l'impatto delle tecnologie digitali sul diritto a un ambiente salubre.
No full textil presente lavoro di tesi ha ad oggetto il rapporto – talvolta sinergico, talvolta conflittuale - tra l’impiego delle tecnologie digitali e il godimento del diritto umano a un ambiente salubre, recentemente riconosciuto dal consiglio dei diritti umani e dall'assemblea generale delle nazioni unite. in questo senso, anche se in anni recenti si è assistito ad un ottimismo incondizionato da parte delle organizzazioni internazionali e dagli stati verso le tecnologie digitali (e in generale verso il progresso tecnologico), quali “panacea” delle questioni ambientali che caratterizzano la nostra epoca, tale fiducia oggi viene messa in crisi dalle evidenti problematiche ambientali che sorgono dall’impiego di tali tecnologie: si pensi, ad esempio, al consumo di energia elettrica (ed alle conseguenti emissioni di gas serra), alla produzione di rifiuti elettronici o all'estrazione di terre rare necessarie per assemblare tali dispositivi. ciò trova un riflesso anche nel diritto internazionale dell’ambiente e nel diritto internazionale dei diritti umani, che nel corso degli anni hanno dato vita (non senza criticità) a vari strumenti a promozione della sostenibilità ambientale di natura eterogenea. suddetti strumenti si trovano oggi ad essere “votati” anche alla disciplina delle tecnologie digitali (traino della c.d. Quarta Rivoluzione Industriale), le quali peraltro sono state motivo del concepimento di inediti atti normativi di respiro internazionale
Exploring the Antioxidant Roles of Cysteine and Selenocysteine in Cellular Aging and Redox Regulation
No full textAging is a complex, universal biological process characterized by the progressive and irreversible decline of physiological functions across multiple organ systems. This deterioration is primarily driven by cumulative cellular damage arising from both intrinsic and extrinsic stressors. The free radical theory of aging, first proposed by Denham Harman in 1956, highlights the role of reactive oxygen species (ROS), byproducts of normal metabolism, in driving oxidative stress and age-related degeneration. Emerging evidence emphasizes the importance of redox imbalance in the onset of neurodegenerative diseases and aging. Among the critical cellular defenses against oxidative stress are sulfur-containing amino acids, namely cysteine (Cys) and selenocysteine (Sec). Cysteine serves as a precursor for glutathione (GSH), a central intracellular antioxidant, while selenocysteine is incorporated into key antioxidant enzymes such as glutathione peroxidases (GPx) and thioredoxin reductases (TrxR). These molecules play pivotal roles in neutralizing ROS and maintaining redox homeostasis. This review aims to provide an updated and critical overview of the role of thiol-containing amino acids, specifically cysteine and selenocysteine, in the regulation of redox homeostasis during aging
Finanziare le politiche europee attraverso il debito comune UE: riflessioni sul dilemma dell’emergenza e sulla conciliabilità dell’istituto con i principi di bilancio e di stabilità finanziaria sovranazionali
No full textNel contesto attuale segnato da crisi sistemiche e urgenze ricorrenti, l’emissione di debito comune da parte dell’Unione europea si configura come uno strumento sempre più utilizzato per attuare politiche pubbliche. Tuttavia, sorgono interrogativi sulla compatibilità di tale strumento con il diritto primario dell’UE, specie al di fuori di scenari emergenziali. Il contributo esplora questa tematica attraverso cinque questioni giuridiche connesse agli artt. 122, 310, 311 e 123-125 TFUE, con particolare attenzione al caso studio del programma NextGenerationEU. L’analisi si concentra sul fondamento giuridico per l’assistenza finanziaria, sulla base giuridica per l’indebitamento, sulla coerenza con i principi di bilancio dell’Unione, sulla ripartizione della responsabilità tra Stati e istituzioni europee, e sulla compatibilità con le regole di stabilità finanziaria. L’elaborato evidenzia i margini di flessibilità e i vincoli strutturali insiti nei Trattati
