Developmental validation of a multiplex qPCR assay for simultaneous quantification of nuclear and mitochondrial DNA

Quantification of human DNA is key in forensic genetics. A more accurate estimate of the amount of DNA is essential for planning and optimising genotyping assays, as is evaluating the presence of PCR inhibitory substances and DNA degradation status. Multiplex qPCR assays are helpful in forensics because they can quantify different targets simultaneously, thus saving valuable samples, time, and labour. The aim of this study was to highlight the challenges in the developmental validation of a multiplex real-time PCR assay and the drawbacks encountered in translating a previously described and validated assay (SD quants) to a different technology by modifying the dye probes and reagent mix to be used in a different instrument. We developed a TaqMan probebased multiplex qPCR using reagents and fluorescent probes adapted for the Rotor-Gene 6000 instrument (QIAGEN, Hilden, Germany). The initial assay combined two mitochondrial DNA (mtDNA) and two nuclear DNA (nDNA) targets, with amplification products of different sizes (mtDNA = 69 and 143 bp; nDNA = 71 and 181 bp), to estimate the DNA degradation status and an internal positive control (IPC) to detect potential inhibitors. During the initial testing of the assay, we observed an interaction between the 69 bp mtDNA target and the 71 bp nDNA target probe, and experiments were conducted to resolve this issue without success. We removed the small nDNA target (71 bp) and changed from a 5-plex to a 4-plex qPCR assay (qMIND). The final tetraplex assay was tested on 105 forensic samples and/or small amounts of degraded DNA, such as bones, teeth, fingernails, formalin-fixed paraffin-embedded tissues (FFPE), and hair shaft samples. The quantification results were compared with data acquired from the same samples using another commercially available quantification system commonly used in forensic laboratories. In addition, the short tandem repeat (STR) profiles were investigated to determine their correlation with the quantitative values obtained. Overall, the qPCR assay was robust and reliable for DNA quantification in samples commonly used in forensic practice

Molecularly imprinted polymers (MIPs) for SARS-CoV-2 omicron variant inhibition: An alternative approach to address the challenge of emerging zoonoses

Emerging zoonoses pose significant public health risks and necessitate rapid and effective treatment responses. This study enhances the technology for preparing Molecularly Imprinted Polymers (MIPs), which function as synthetic nanoparticles targeting SARS-CoV-2 receptor-binding domain (RBD), specifically the Omicron variant, thereby inhibiting its function. This study builds on previous findings by introducing precise adjustments in the formulation and process conditions to enhance particle stability and ensure better control over size and distribution, thereby overcoming the issues identified in earlier research. Following docking studies, imprinted nanoparticles were synthesized via inverse microemulsion polymerization and characterized in terms of size, morphology and surface charge. The selective recognition properties and ability of MIPs to obstruct the interaction between ACE2 and the RBD of SARS-CoV-2 were assessed in vitro, using Non-Imprinted Polymers (NIPs) as controls, and rebinding studies were conducted utilizing a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). The synthesized nanoparticles exhibited uniform dispersion and had a consistent diameter within the nanoscale range. MIPs demonstrated significant recognition properties and exhibited a concentrationdependent ability to reduce RBD binding to ACE2 without cytotoxic or sensitizing effects. MIPs-based platforms offer a promising alternative to natural antibodies for treating SARS-CoV-2 infections, therefore representing a versatile platform for managing emerging zoonoses

Second-Line Medications for Women Aged 10 to 50 Years With Idiopathic Generalized Epilepsy

Importance: Women with idiopathic generalized epilepsy (IGE) face challenges in treatment due to limited options that are both effective and safe. Objective: To evaluate the effectiveness and safety of substitution monotherapy vs add-on therapy as second-line options for women who might become pregnant with IGE after failure of first-line antiseizure medications (ASMs) other than valproic acid. Design, Setting, and Participants: Multicenter retrospective comparative effectiveness cohort study at 18 primary, secondary, and tertiary adult and children epilepsy centers across 4 countries, analyzing data from 1995 to 2023. Participants were women aged 10 to 50 years diagnosed with IGE who were prescribed a second line of ASM. Main Outcomes and Measures: Treatment failure (TF), defined as the replacement or addition of a second ASM due to ineffectiveness, was compared between patients receiving ASM add-on or substitution monotherapy using inverse probability of treatment weighting (IPTW)-adjusted Cox proportional hazards regression. Exploratory analyses were also conducted to assess the effectiveness of individual ASMs and various ASM combinations. Results: This study included 249 women with a median (IQR) age of 18.0 (15.5-22.0) years. Among them, 146 (58.6%) received an add-on regimen, and 103 (41.4%) received substitution monotherapy. During follow-up, TF occurred in 48 patients (32.9%) receiving add-on therapy and 36 (35.0%) using substitution monotherapy, with no significant differences between groups (IPTW-adjusted hazard ratio [HR], 0.89; 95% CI, 0.53-1.51; P =.69). ASM discontinuation due to ineffectiveness or adverse effects occurred in 36 patients (24.7%) receiving add-on therapy and 29 (28.2%) receiving substitution monotherapy, showing no significant differences (IPTW-adjusted HR, 0.97; 95% CI, 0.57-1.65; P =.92). Rates of ASM discontinuation due to adverse effects only were low in both groups, occurring in 13 patients (9.0%) receiving add-on therapy and 9 (8.7%) receiving a substitution monotherapy. Among add-on regimens other than valproic acid, the combination of levetiracetam and lamotrigine demonstrated a lower risk of TF compared with other combinations with levetiracetam plus other ASM (adjusted HR, 2.41; 95% CI, 1.12-5.17; P =.02) and lamotrigine plus other ASM (adjusted HR, 4.03; 95% CI, 1.73-9.39; P =.001). However, valproic acid remained the most effective second-line ASM when considering individual agents. Conclusions and relevance: In this comparative effectiveness study of second-line treatment strategies for women with IGE, no significant differences were observed between substitution monotherapy and add-on therapy

Olive Leaf Extracts With High, Medium, or Low Bioactive Compounds Content Differentially Modulate Alzheimer's Disease via Redox Biology

Alzheimer's disease (AD) involves β-amyloid plaques and tau hyperphosphorylation, driven by oxidative stress and neuroinflammation. Cyclooxygenase-2 (COX-2) and acetylcholinesterase (AChE) activities exacerbate AD pathology. Olive leaf (OL) extracts, rich in bioactive compounds, offer potential therapeutic benefits. This study aimed to assess the anti-inflammatory, anti-cholinergic, and antioxidant effects of three OL extracts (low, mid, and high bioactive content) in vitro and their protective effects against AD-related proteinopathies in Caenorhabditis elegans models. OL extracts were characterized for phenolic composition, AChE and COX-2 inhibition, as well as antioxidant capacity. Their effects on intracellular and mitochondrial reactive oxygen species (ROS) were tested in C. elegans models expressing human Aβ and tau proteins. Gene expression analyses examined transcription factors (DAF-16, skinhead [SKN]-1) and their targets (superoxide dismutase [SOD]-2, SOD-3, GST-4, and heat shock protein [HSP]-16.2). High-OL extract demonstrated superior AChE and COX-2 inhibition and antioxidant capacity. Low- and high-OL extracts reduced Aβ aggregation, ROS levels, and proteotoxicity via SKN-1/NRF-2 and DAF-16/FOXO pathways, whereas mid-OL showed moderate effects through proteostasis modulation. In tau models, low- and high-OL extracts mitigated mitochondrial ROS levels via SOD-2 but had limited effects on intracellular ROS levels. High-OL extract also increased GST-4 levels, whereas low and mid extracts enhanced GST-4 levels. OL extracts protect against AD-related proteinopathies by modulating oxidative stress, inflammation, and proteostasis. High-OL extract showed the most promise for nutraceutical development due to its robust phenolic profile and activation of key antioxidant pathways. Further research is needed to confirm long-term efficacy

Psychosocial Interventions in the Rehabilitation and the Management of Psychosis and Schizophrenia: A Systematic Review on Digitally-Delivered Interventions

Background: Schizophrenia and psychotic disorders are disabling, complex and severe psychiatric conditions, which may pose a significant therapeutic challenge. Integrating current psychopharmacological treatment with psychosocial interventions demonstrated a higher efficacy in terms of prognosis. However, most schizophrenia or psychotic patients may have restricted or no access to evidence-based psychosocial interventions, mainly due to poor dissemination of specialized interventions or stigma. Therefore, we aim to systematically review all studies about the current evidence on the feasibility, acceptability, efficacy, effectiveness, and benefits of digitally-delivered psychoeducational and psychosocial interventions for individuals suffering from schizophrenia or psychotic disorders. Methods: A systematic literature review was conducted of the literature from 2000 to 2024 according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, by using PubMed-MEDLINE, Scopus and OVID databases and combining the search approach using both free text terms and Medical Subject Headings (MESH) headings for the topics “psychoeducation”, “psychosocial intervention” and “psychosis” and “schizophrenia”. Results: Out of a total of 3042 reviewed papers, 69 studies were included here. The interventions included web-based family and individual psychoeducation, integrated web-based therapy, social networking, peer and expert moderation, virtual reality-assisted and mobile-based psychosocial interventions. Results showed that digitally-delivered interventions have a positive effect in ensuring the continuity and maintenance of the effectiveness of psychosocial treatments, by providing personalized, flexible, and evidence-based interventions to patients with psychosis and/or schizophrenia. At the same time, the studies included demonstrated the acceptability and feasibility of this kind of intervention in clinical practice. Conclusions: Digital interventions have the potential to deliver non-stigmatizing, constantly available psychosocial and psychoeducational interventions in psychosis and schizophrenia by increasing access to mental health care and not costly interventions. However, further randomized controlled trials (RCTs) and observational studies should compare and evaluate the effectiveness and feasibility of web-based vs. face-to-face psychosocial interventions amongst schizophrenia and psychosis individuals

Monetization and the Fiscal Multiplier

We investigate the size of Italian fiscal multipliers over the period 1872–2006. By instrumenting total expenditure with defence expenditure, we measure the multiplier under different business and monetary policy regimes, also employing a time-varying parameters model to encompass structural changes. We explore the nexus between the multiplier and monetization using the treasury monetary base, a policy instrument used in Italy during the period, as a novel measure of monetization. Results suggest that when monetization and economic slackness occur jointly, the fiscal multiplier tends to be larger. Monetization does not significantly affect the multiplier during expansions

Commento Art. 158, TUIR

Commento all'art. 158, TUIR relativo al regime istitutivo ed applicativo della tonnage tax e del sistema di imposizione reddituale di vantaggio per le imprese marittime. Regime della rilevanza fiscale di plusvalenze e minusvalenze realizzate e le novità applicative introdotte dal D.Lgs. n. 192/202

Atypical vascular calcification with osseous and bone marrow metaplasia: post-mortem cases series with functional pathogenetic implications

Arterial vascular calcification is an ectopic mineralization similar to bone formation, acknowledged as an active process. Osseous metaplasia can be uncommonly observed inside vascular calcifications, mainly in the peripheral vessels, and rarely together with bone marrow cellularity, even if the incidence is underestimated. We hereby illustrate three cases, median age 82 years, female/male ratio 2/1, where osseous metaplasia with hematopoietic cells was incidentally observed within vascular calcifications: in one case in peripheral artery and in two cases in coronary arteries. Atherosclerosis and arterial hypertension were reported in the three cases. Final causes of death were pulmonary thromboembolism, myocardial infarction, and trauma due to car accident. Bone marrow metaplasia has important consequences for plaque stability, particularly in coronary arteries, because it could determine a risk per se of cardiovascular adverse events, but also a risk of adverse events in case of endovascular diagnostic or therapeutic procedures. Insight into the pathogenetic process and thus the possibility to assess the plaque rupture risk are of central importance for cardiovascular risk management

Dynamical behaviour of a two prey and one predator system with indirect effect and time delay

The effect of time delay on a population model consisting of two preys and one predator is investigated. The interactions within the system are primarily governed by a Holling type II functional response, which describes the relationship between the predation rate and the density of the first prey. Additionally, the model accounts for the indirect effects of predation, capturing the complex dynamics arising from these interactions. By incorporating time delay, the analysis explores how the temporal lag in responses affects the stability and behavior of the populations, such as reproduction, predation, or resource regeneration. This approach provides insights into the conditions under which populations coexist, oscillate, or collapse, offering a deeper understanding of predator–prey dynamics in ecosystems influenced by delayed feedback mechanisms. Conditions under which an equilibrium point loses stability and bifurcates into cyclic oscillations are established. Our study sheds light on the intricate relationship between time delay and the emergent dynamics in this ecological system. Finally, the validity of the theoretical results is verified through several numerical example