Analysis of oxidised and glycated aminophospholipids: complete structural characterisation by C30 liquid chromatography-high resolution tandem mass spectrometry

The aminophospholipids (APL), phosphatidylethanolamine (PE) and phosphatidylserine (PS) are widely present in cell membranes and lipoproteins. Glucose and reactive oxygen species (ROS), such as the hydroxyl radical (•OH), can react with APL leading to an array of oxidised, glycated and glycoxidised derivatives. Modified APL have been implicated in inflammatory diseases and diabetes, and were identified as signalling molecules regulating cell death. However, the biological relevance of these molecules has not been completely established, since they are present in very low amounts, and new sensitive methodologies are needed to detect them in biological systems. Few studies have focused on the characterisation of APL modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS), mainly using C5 or C18 reversed phase (RP) columns. In the present study, we propose a new analytical approach for the characterisation of complex mixtures of oxidised, glycated and glycoxidised PE and PS. This LC approach was based on a reversed-phase C30 column combined with high-resolution MS, and higher energy C-trap dissociation (HCD) MS/MS. C30 RP-LC separated short and long fatty acyl oxidation products, along with glycoxidised APL bearing oxidative modifications on the glucose moiety and the fatty acyl chains. Functional isomers (e.g. hydroxy-hydroperoxy-APL and tri-hydroxy-APL) and positional isomers (e.g. 9-hydroxy-APL and 13-hydroxy-APL) were also discriminated by the method. HCD fragmentation patterns allowed unequivocal structural characterisation of the modified APL, and are translatable into targeted MS/MS fingerprinting of the modified derivatives in biological samples.publishe

Evaluation of air oxidized PAPC: A multi laboratory study by LC-MS/MS

Oxidized LDL (oxLDL) has been shown to play a crucial role in the onset and development of cardiovascular disorders. The study of oxLDL, as an initiator of inflammatory cascades, led to the discovery of a variety of oxidized phospholipids (oxPLs) responsible for pro-inflammatory actions. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is frequently used by the scientific community as a representative oxPL mixture to study the biological effects of oxidized lipids, due to the high abundance of PAPC in human tissues and the biological activities of oxidized arachidonic acids derivatives. Most studies focusing on oxPAPC effects rely on in-house prepared mixtures of oxidized species obtained by exposing PAPC to air oxidation. Here, we described a multi-laboratory evaluation of the compounds in oxPAPC by LC-MS/MS, focusing on the identification and relative quantification of the lipid peroxidation products (LPPs) formed. PAPC was air-oxidized in four laboratories using the same protocol for 0, 48, and 72 h. It was possible to identify 55 different LPPs with unique elemental composition and characterize different structural isomeric species within these. The study showed good intra-sample reproducibility and similar qualitative patterns of oxidation, as the most abundant LPPs were essentially the same between the four laboratories. However, there were substantial differences in the extent of oxidation, i.e. the amount of LPPs relative to unmodified PAPC, at specific time points. This shows the importance of characterizing air-oxidized PAPC preparations before using them for testing biological effects of oxidized lipids, and may explain some variability of effects reported in the literature

Dose-Dependent Impairment of the Immune Response to the Moderna-1273 mRNA Vaccine by Mycophenolate Mofetil in Patients with Rheumatic and Autoimmune Liver Diseases

The purpose of this study was to evaluate the efficacy and safety of the Moderna-1273 mRNA vaccine for SARS-CoV-2 in patients with immune-mediated diseases under different treatments. Anti-trimeric spike protein antibodies were tested in 287 patients with rheumatic or autoimmune diseases (10% receiving mycophenolate mofetil, 15% low-dose glucocorticoids, 21% methotrexate, and 58% biologic/targeted synthetic drugs) at baseline and in 219 (76%) 4 weeks after the second Moderna-1273 mRNA vaccine dose. Family members or caretakers were enrolled as the controls. The neutralizing serum activity against SARS-CoV-2-G614, alpha, and beta variants in vitro and the cytotoxic T cell response to SARS-CoV-2 peptides were determined in a subgroup of patients and controls. Anti-SARS-CoV-2 antibody development, i.e., seroconversion, was observed in 69% of the mycophenolate-treated patients compared to 100% of both the patients taking other treatments and the controls (p < 0.0001). A dose-dependent impairment of the humoral response was observed in the mycophenolate-treated patients. A daily dose of >1 g at vaccination was a significant risk factor for non-seroconversion (ROC AUC 0.89, 95% CI 0.80-98, p < 0.0001). Moreover, in the seroconverted patients, a daily dose of >1 g of mycophenolate was associated with significantly lower anti-SARS-CoV-2 antibody titers, showing slightly reduced neutralizing serum activity but a comparable cytotoxic response compared to other immunosuppressants. In non-seroconverted patients treated with mycophenolate at a daily dose of >1 g, the cytotoxic activity elicited by viral peptides was also impaired. Mycophenolate treatment affects the Moderna-1273 mRNA vaccine immunogenicity in a dose-dependent manner, independent of rheumatological disease

Mapping Unsaturation in Human Plasma Lipids by Data-Independent Ozone-Induced Dissociation

Over 1500 different lipids have been reported in human plasma at the sum composition level. Yet the number of unique lipids present is surely higher, once isomeric contributions from double bond location(s) and fatty acyl regiochemistry are considered. In order to resolve this ambiguity, herein, we describe the incorporation of ozone-induced dissociation (OzID) into data-independent shotgun lipidomics workflows on a high-resolution hybrid quadrupole-Orbitrap platform. In this configuration, [M + Na]+ ions generated by electrospray ionization of a plasma lipid extract were transmitted through the quadrupole in 1 Da segments. Reaction of mass-selected lipid ions with ozone in the octopole collision cell yielded diagnostic ions for each double bond position. The increased ozone concentration in this region significantly improved ozonolysis efficiency compared with prior implementations on linear ion-trap devices. This advancement translates into increased lipidome coverage and improvements in duty cycle for data-independent MS/MS analysis using shotgun workflows. Grouping all precursor ions with a common OzID neutral loss enables straightforward classification of the lipidome by unsaturation position (with respect to the methyl terminus). Two-dimensional maps obtained from this analysis provide a powerful visualization of structurally related lipids and lipid isomer families within plasma. Global profiling of lipid unsaturation in plasma extracts reveals that most unsaturated lipids are present as isomeric mixtures. These new insights provide a unique picture of underlying metabolism that could in the future provide novel indicators of health and disease

New combinatorial strategies to improve the PARP inhibitors efficacy in the urothelial bladder Cancer treatment

Abstract Background Novel therapeutic strategies are urgently needed for the treatment of metastatic Urothelial Bladder Cancer. DNA damaging repair (DDR) targeting has been introduced in cinical trials for bladder cancer patients that carry alterations in homologous DNA repair genes, letting to envisage susceptibility to the Poly (adenosine diphosphate [ADP]) ribose polymerase (PARP) inhibitors. Main body PARP inhibition, by amplifying the DNA damage, augments the mutational burden and promotes the immune priming of the tumor by increasing the neoantigen exposure and determining upregulation of programmed death ligand 1 (PD-L1) expression. Thus, the combination of PARP-inhibition and the PD/PD-L1 targeting may represent a compelling strategy to treat bladder cancer and has been introduced in recent clinical trials. The targeting of DDR has been also used in combination with epigenetic drugs able to modulate the expression of genes involved in DDR, and also able to act as immunomodulator agents suggesting their use in combination with immune-checkpoint inhibitors. Conclusion In conclusion, it may be envisaged the combination of three classes of drugs to treat bladder cancer, by targeting the DDR process in a tumor context of DDR defect, together with epigenetic agents and immune-checkpoint inhibitors, whose association may amplify the effects and reduce the doses and the toxicity of each single drug

High Biodiversity Arises from the Analyses of Morphometric, Biochemical and Genetic Data in Ancient Olive Trees of South of Italy

Morphometric, biochemical and genetic analyses were conducted on Olea europaea L. of Campania, an area of Southern Italy highly suited to the cultivation of olive trees and the production of extra virgin olive oil (EVOO). We aimed to characterize the distribution of morphological, biochemical and genetic diversity in this area and to develop a practical tool to aid traceability of oils. Phenotypes were characterized using morphometric data of drupes and leaves; biochemical and genetic diversity were assessed on the basis of the fatty acid composition of the EVOOs and with microsatellite markers, respectively. We provide an open-source tool as a novel R package titled ‘OliveR’, useful in performing multivariate data analysis using a point and click interactive approach. These analyses highlight a clear correlation among the morphological, biochemical and genetic profiles of samples with four collection sites, and confirm that Southern Italy represents a wide reservoir of phenotypic and genetic variability

New Site of Modification of 23S rRNA Associated with Clarithromycin Resistance of Helicobacter pylori Clinical Isolates

Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 μg/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an HhaI restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates

Computational and Experimental Drug Repurposing of FDA-Approved Compounds Targeting the Cannabinoid Receptor CB1

The cannabinoid receptor 1 (CB1R) plays a pivotal role in regulating various physiopathological processes, thus positioning itself as a promising and sought-after therapeutic target. However, the search for specific and effective CB1R ligands has been challenging, prompting the exploration of drug repurposing (DR) strategies. In this study, we present an innovative DR approach that combines computational screening and experimental validation to identify potential Food and Drug Administration (FDA)-approved compounds that can interact with the CB1R. Initially, a large-scale virtual screening was conducted using molecular docking simulations, where a library of FDA-approved drugs was screened against the CB1R's three-dimensional structures. This in silico analysis allowed us to prioritize compounds based on their binding affinity through two different filters. Subsequently, the shortlisted compounds were subjected to in vitro assays using cellular and biochemical models to validate their interaction with the CB1R and determine their functional impact. Our results reveal FDA-approved compounds that exhibit promising interactions with the CB1R. These findings open up exciting opportunities for DR in various disorders where CB1R signaling is implicated. In conclusion, our integrated computational and experimental approach demonstrates the feasibility of DR for discovering CB1R modulators from existing FDA-approved compounds. By leveraging the wealth of existing pharmacological data, this strategy accelerates the identification of potential therapeutics while reducing development costs and timelines. The findings from this study hold the potential to advance novel treatments for a range of CB1R -associated diseases, presenting a significant step forward in drug discovery research

Therapeutic Efficacy of a Coriolus versicolor-Based Vaginal Gel in Women with Cervical Uterine High-Risk HPV Infection. A Retrospective Observational Study

Introduction A Coriolus versicolor-based vaginal gel is available for treating women with cervical uterine high-risk human papillomavirus (HPV) infection through re-epithelizing and re-balancing microbiota actions. Methods A longitudinal retrospective observational study was performed to evaluate efficacy and safety of the gel. Women treated with Coriolus versicolor-based vaginal gel were compared with women not treated with the gel. Both groups were monitored for HPV infection by an HPV DNA test, Pap smear (cytology) and colposcopy at baseline and after 6 months. Results Overall, 183 high-risk HPV positive women were enrolled (97 treated and 86 controls). After 6 months, the HPV DNA test became negative in 67.0% versus 37.2% of treated and controls, respectively (p < 0.0001). Furthermore, 76.1% versus 40.8% registered a colposcopy improvement (p = 0.0005) and 60.4% versus 40.8% showed a remission (p = 0.05), for treated versus controls, respectively. Regarding to cytology, 78.5% of treated versus 37.7% of controls registered an improvement, while 70.8% of treated versus 34.8% of controls had a remission (p < 0.0001 for both variables). At multivariate analyses adjusted for age, smoking habit, and use of estroprogestinic pill, compared to controls, women treated with the gel showed a significantly higher likelihood to experience the clearence of HPV DNA (OR 4.81; 95% 2.43-9.53), and remission at colposcopy (OR 2.30; 95% 1.00-5.31), and cytology (OR 5.13; 95% 2.40-10.96) at 6 months. No adverse event was reported during the follow-up. Conclusions The use of a Coriolus versicolor-based vaginal gel in high-risk HPV patients is safe and effective based on all examined tests