Marco Pallecchi

Peptides are increasingly employed as active ingredients in both therapeutic and cosmeceutical applications due to their high biological specificity, favorable safety profiles, and expanding market relevance. In topical formulations, peptide activity is typically confined to the skin, where widespread dermal proteases may significantly affect their stability and efficacy. Despite the growing use of bioactive peptides in dermatological and cosmeceutical products, robust analytical methodologies for assessing their susceptibility to dermal enzymatic degradation remain limited. In this study, a two‐dimensional HPLC–MS/MS (2D‐HPLC–MS/MS) method based on ion trap detection was developed and validated for the quantitative evaluation of peptide stability in human skin homogenate (HSH). The analytical setup integrates online cleanup with chromatographic separation on a silica‐based pentafluorophenyl (PFP) column, enabling reliable analysis of peptides with different polarity profiles within a single workflow. The system enables direct injection of sample solutions with relatively high organic solvent content, allowing limited sample dilution and preservation of analytical sensitivity. A key feature of the method is the use of isomeric peptide analogues as internal standards, monitored under identical MS/MS conditions as their corresponding analytes. This approach provides effective correction for ionization and fragmentation variability while offering a practical alternative to stable isotope‐labelled standards. The ion trap mass analyzer ensured controlled and reproducible fragmentation behavior, supporting robust quantitative performance. The developed method demonstrated satisfactory linearity, sensitivity, precision, accuracy, and minimal matrix effects. Application to stability studies of three peptides with distinct polarity profiles confirmed its suitability for monitoring degradation kinetics in HSH. Overall, this 2D‐HPLC–MS/MS strategy provides a versatile analytical platform for dermal peptide stability assessment and supports early‐stage screening and preclinical studies of bioactive peptides intended for topical applications.

Background/Objectives: Age-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly and is characterized by a multifactorial etiology. Emerging evidence points to the potential involvement of the gut–retina axis in AMD pathogenesis, prompting exploration into novel therapeutic strategies. This study aims to investigate the effects of some micronutrients (such as lutein and zeaxanthin) and saffron (as a supplement)—known for their anti-inflammatory properties—on ophthalmological and microbial parameters in neovascular AMD (nAMD) patients. Methods: Thirty naive nAMD patients were randomized to receive daily micronutrient supplementation alongside anti-VEGF (vascular endothelial growth factor) therapy, or anti-VEGF treatment alone, over a 6-month period, with comparisons made to a healthy control (HC) group (N = 15). Ophthalmological assessments, biochemical measurements, and stool samples were obtained before and after treatment. Gut microbiota (GM) characterization was performed using 16S rRNA sequencing, while short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), and long-chain fatty acids (LCFAs) were analyzed with a gas chromatography–mass spectrometry protocol. Results: Compared to HC, nAMD patients exhibited reduced GM alpha diversity, altered taxonomic composition, and decreased total SCFA levels, in addition to elevated levels of proinflammatory octanoic and nonanoic acids. Micronutrient supplementation was associated with improved visual acuity relative to the group treated with anti-VEGF alone, along with a decrease in the total amount of MCFAs, which are metabolites known to have adverse ocular effects. Conclusions: In conclusion, despite certain limitations—such as the limited sample size and the low taxonomic resolution of 16S rRNA sequencing—this study highlights compositional and functional imbalances in the GM of nAMD patients and demonstrates that micronutrient supplementation may help restore the gut–retina axis. These findings suggest the therapeutic potential of micronutrients in enhancing ocular outcomes for nAMD patients, underscoring the complex interaction between GM and ocular health.

Objectives: In systemic sclerosis (SSc), gastrointestinal involvement is one of the earliest events. We compared the gut microbiota (GM), its short-chain fatty acids (SCFAs) and host-derived free fatty acids (FFAs) in patients with very early diagnosis of SSc (VEDOSS) and definite SSc. Methods: Stool samples of 26 patients with SSc, 18 patients with VEDOSS and 20 healthy controls (HC) were collected. The GM was assessed through 16S rRNA sequencing, while SCFAs and FFAs were assessed by gas chromatography-mass spectrometry. Results: In patients with VEDOSS, an increase in Bacteroidales and Oscillospirales orders and a decrease in Bacilli class, Blautia, Romboutsia, Streptococcus and Turicibacter genera was detected in comparison with HC. In patients with SSc, an elevated number of Acidaminococcaceae and Sutterellaceae families, along with a decrease of the Peptostreptococcaceae family and Anaerostipes, Blautia, Romboutsia and Turicibacter genera was found in comparison with HC. Patients with SSc and VEDOSS had a significantly lower butyrate and higher acetate with respect to HC. In VEDOSS, an increase in Oscillospiraceae family and Anaerostipes genus, and a decrease in Alphaproteobacteria class, and Lactobacillales order was identified with respect to SSc. Moreover, patients with VEDOSS exhibited higher acetate and lower valerate compared with definite SSc. Conclusion: A GM dysbiosis with depletion of beneficial anti-inflammatory bacteria (especially butyrate-producing) and a significant decrease in faecal butyrate was identified in patients with VEDOSS. This early GM imbalance may foster the growth of inflammatory microbes, worsening intestinal dysbiosis and inflammation in early SSc stages. The potential butyrate administration in early disease phases might be considered as a novel therapeutic approach to mitigate gastrointestinal discomfort and progression preserving patient’s quality of life.

Oleuropein (OLE), a phenolic compound particularly abundant in the olive leaves, has been reported to have beneficial activities against colorectal cancer (CRC). In vitro studies suggested that these latter could be due to a modulation of the intestinal microbiota. Aiming to evaluate if OLE could affect the intestinal microbiota and the plasma metabolome, an antioxidant oleuropein-rich leaf extract (ORLE) was administered for one week to PIRC rats (F344/NTac-Apc^am1137), a genetic model mimicking CRC. ORLE treatment significantly modulated the gut microbiota composition. Plasma metabolomic profiles revealed a significant predictive ability for amino acids, medium-chain fatty acids, and aldehydes. Pathway analysis revealed a significant decrease in phosphatidylcholine accumulation (LogFC = −1.67) in PIRC rats. These results suggest a significant effect of ORLE administration on faecal microbiota profiles and plasma metabolomes, thereby offering new omics-based insights into its protective role in CRC progression.

Metformin is the most prescribed glucose-lowering drug worldwide; globally, over 100 million patients are prescribed this drug annually. Some different action mechanisms have been proposed for this drug, but, surprisingly, no metabolite of metformin has ever been described. It was considered interesting to investigate the possible reaction of metformin with glucose following the Maillard reaction pattern. The reaction was first performed in in vitro conditions, showing the formation of two adducts that originated by the condensation of the two molecular species with the losses of one or two water molecules. Their structures were investigated by liquid chromatography coupled with mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS) and accurate mass measurements (HRMS). The species originated via the reaction of glucose and metformin and were called metformose and dehydrometformose, and some structural hypotheses were conducted. It is worth to emphasize that they were detected in urine samples from a diabetic patient treated with metformin and consequently they must be considered metabolites of the drug, which has never been identified before now. The glucose-related substructure of these compounds could reflect an improved transfer across cell membranes and, consequently, new hypotheses could be made about the biological targets of metformin.

The various forms of interactions that microorganisms engage in within fermented foods result in distinct metabolic product patterns. Fermentation products often feature both yeasts and bacteria, each possessing characteristics that can enhance the overall quality of the food, thus benefiting consumers. Kefir, a fermented milk originating from grains containing a unique and intricate blend of bacteria and yeasts living in a symbiotic relationship, is a valuable model for studying the evolution of the interactions between yeasts and bacteria. Targeted metagenomics was applied to investigate the microbiome of a batch of traditional Romanian kefir and further examine the growth and metabolic properties of the dominant yeast and bacterial strains isolated from this batch. In contrast to yeast, which is either unaffected or harmed by the presence of bacteria, our study revealed that a specific strain of Lactobacillus (L. rhamnosus) derived from the kefir batch benefited from the presence of Saccharomyces cerevisiae. The analysis of short-chain fatty acids (SCFAs) produced by in vitro cultures of these two chosen strains indicated significant changes in SCFA levels compared to single cultures. The dynamic interactions described in this and other studies on kefir emphasize the importance of a more profound comprehension of the ecological mechanisms governing interactions between yeast, bacterial, and mammalian cells.

Chlorogenic acids, the esters of caffeic and quinic acids, are the main phenolic acids detected in Acmella oleracea extracts and have gained increasing interest in recent years due to their important biological activities. Given their structural similarity and instability, the correct analysis and identification of these compounds in plants is challenging. This study aimed to propose a simple and rapid determination of the A. oleracea caffeoylquinic isomers, applying an HPLC-MS/MS method supported by a mathematical algorithm (Linear Equation of Deconvolution Analysis (LEDA)). The three mono- and the three di-caffeoylquinic acids in roots of Acmella plants were studied by an ion trap MS analyzer. A separation by a conventional chromatographic method was firstly performed and an MS/MS characterization by energetic dimension of collision-induced dissociation mechanism was carried out. The analyses were then replicated using a short HPLC column and a fast elution gradient (ten minutes). Each acquired MS/MS data were processed by LEDA algorithm which allowed to assign a relative abundance in the reference ion signal to each isomer present. Quantitative results showed no significant differences between the two chromatographic systems proposed, proving that the use of LEDA algorithm allowed the distinction of the six isomers in a quarter of the time.

The composition of the gut microbiota (GM) undergoes significant changes during pregnancy, influenced by metabolic status, energy homeostasis, fat storage, and hormonal and immunological modifications. Moreover, dysbiosis during pregnancy has been associated with preterm birth, which is influenced by factors such as cervical shortening, infection, inflammation, and oxidative stress. However, dysbiosis also affects the levels of lipopolysaccharide-binding protein (LBP), short-chain fatty acids (SCFAs), and free fatty acids (FFA) in other tissues and the bloodstream. In this study, we investigated the plasmatic levels of some pro-inflammatory cytokines, such as matrix metalloproteinases-8 (MMP-8), interleukin-8 (IL-8), heat shock protein 70 (Hsp70), and microbial markers in pregnant women with a short cervix (≤25 mm) compared to those with normal cervical length (>25 mm). We examined the differences in the concentration of these markers between the two groups, also assessing the impact of gestational diabetes mellitus. Understanding the relationship between GM dysbiosis, inflammatory mediators, and cervical changes during pregnancy may contribute to the identification of potential biomarkers and therapeutic targets for the prevention and management of adverse pregnancy outcomes, including preterm birth.

The tandem mass spectrometry (MS/MS) approach employing an ion trap mass analyzer (IT) was evaluated in isomers recognition. The proposed approach consists of sole, simple, and rapid liquid chromatographic separation (HPLC) without requiring resolution between the analytes. Then, the MS/MS properties were optimized to solve the signal assignment using post-processing data elaboration (LEDA). The IT-MS/MS experiment uses the same site, helium as collision gas, and different time steps to modify the applied conditions on the studied ions. Nevertheless, helium cannot ensure the quick energization of the precursor ion due to its small cross-section. Then, different combinations between excitation amplitude (ExA) and excitation time (ExT) were tested to achieve the activation of the fragmentation channels and the formation of the MS/MS spectrum. Usually, the IT-MS/MS acquisition cycle is longer for other multistage instruments, decreasing the frequency of sample data collection and influencing the chromatographic profile. To solve these problems, two time segments were set up, and the elution conditions were optimized with a compromise between peaks distinction and run time reduction. The developed HPLC-MS/MS method was checked and applied to analyze a series of human plasma samples spiked with an equimolar mixture of pair of isomers.

Persistent pain can be managed with opioids, but their use is limited by the onset of tolerance. Ultramicronized N-palmitoylethanolamine (PEA) in vivo delays morphine tolerance with mechanisms that are still unclear. Since glial cells are involved in opioid tolerance and mast cells (MCs) are pivotal targets of PEA, we hypothesized that a potential mechanism by which PEA delays opioid tolerance might depend on the control of the crosstalk between these cells. Morphine treatment (30 μM, 30 min) significantly increased MC degranulation of RBL-2H3 cells, which was prevented by pre-treatment with PEA (100 μM, 18 h), as evaluated by β-hexosaminidase assay and histamine quantification. The impact of RBL-2H3 secretome on glial cells was studied. Six-hour incubation of astrocytes with control RBL-2H3-conditioned medium, and even more so co-incubation with morphine, enhanced CCL2, IL-1β, IL-6, Serpina3n, EAAT2 and GFAP mRNA levels. The response was significantly prevented by the secretome from PEA pre-treated RBL-2H3, except for GFAP, which was further upregulated, suggesting a selective modulation of glial signaling. In conclusion, ultramicronized PEA down-modulated both morphine-induced MC degranulation and the expression of inflammatory and pain-related genes from astrocytes challenged with RBL-2H3 medium, suggesting that PEA may delay morphine tolerance, regulating MC-astrocyte crosstalk.