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Summer School in Pharmaceutical Analysis

Introduction. The ‘omics sciences are currently in development offering a new and combined perspective of cellular and organismal environment. Among these, genomics and proteomics are, probably, the most developed and already provided a large amount of data. On the contrary, lipidomics is still an emerging field with ongoing development of analytical and bioinformatics methods necessary to address the structural and physiochemical diversity of lipids [1,2]. The importance to provide a strong methodological approach paired to a rigorous data interpretation is explained by the recent discovery of the lipids’ key role in many biological processes. Indeed, they act not only as passive structural components or energy depots but also as second messengers in signal transduction, regulators of inter-cellular interactions and of surface charge. Again, most of lipids are involved in many diseases such as diabetes, atherosclerosis, cancer, neurodegenerative and dermatological ones [2]. Mass spectrometry (MS) technique, thanks to the recent significant advances, is the most suitable analytical method in many of ‘omics sciences, including lipidomics, making easier their integration [3]. Nevertheless, mainly for lipidomics, careful optimization of protocols is needed to reach the complete coverage of all lipid classes (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides) with an acceptable level of resolution.

The aim of my research is to integrate lipidomics analyses with quantitative proteomics methodologies for a more complete, multi-omics vision of intra- and extra-cellular processes in several biological conditions (physiological status, drug exposure, pathological conditions). In particular, during my PhD I applied proteomics and/or lipidomics approaches to investigate: i) effects of carnosine on nude mice skin to prevent UV-A damage; ii) Optimization of a reliable, reproducible, and fast methodology based on high resolution MS for the characterization and relative quantification of fatty acid esters of hydroxy fatty acids (FAHFAs) in human white adipose tissue (WAT) of obese patients; iii) Evaluation of molecular effects of low-molecular-weight hyaluronic acid in human dermal fibroblasts through advanced quantitative proteomics and semi-quantitative lipidomics; iv) Analysis of lipidome and proteome profile changing induced by Y-oryzanol prevention treatment in obese rats.

Study of carnosine’s effect on nude mice skin to prevent UV-A damage. The skin is an important barrier against external attacks from bacteria, radicals, or radiations. UV-A radiations (315-400 nm) cause significant impairment of this barrier, inducing inflammation, oxidative stress, and wrinkle formation, thereby promoting photoaging [4,5]. Previous studies reported that carnosine, a potent antioxidant, and carbonyl scavenger agent, may prevent photoaging features in the skin of hairless mice exposed to UV-A radiations. Here a quantitative proteomic approach in high-resolution MS (HRMS, Orbitrap Fusion™ Tribrid™ Mass Spectrometer -Thermo Scientific) was used to analyze the changes evoked by carnosine in the skin proteome of hairless mice exposed to UV-A. This approach allowed to quantify more than 2480 proteins, among them consistent differences were observed for 89 proteins in UV-A exposed vs control unexposed skins, and 252 proteins in UV-A-exposed skin preventively treated by carnosine (UVAC) vs UV-A. Several functional pathways were altered in the skin of UV-A exposed hairless mice, including the integrin-linked kinase, calcium signaling, fibrogenesis, cell migration and filament formation, mitochondrial function and metabolism. On the contrary, pre-treatment with carnosine prevented from UV-A induced proteome alterations. In conclusion, this study emphasizes the potency of a proteomics approach to identify the consequences of UV-A radiations in the skin, and points out the capacity of carnosine to prevent the alterations of skin proteome evoked by UV-A.

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Characterization and relative quantification of fatty acid esters of hydroxy fatty acids (FAHFAs) in human white adipose tissue (WAT) of obese patients. Elevated levels of circulating fatty acids (FA) are generally associated with diabetes and obesity. However, certain FA (dietary omega-3 fatty acids, endogenous palmitoleate etc.) have positive metabolic effects partially reverting these conditions. Similarly, a new class of endogenous lipids called fatty acid esters of hydroxy fatty acids (FAHFAs), present in adipose tissue, kidney, pancreas, and serum, have shown analogous biological activities. Indeed, studies on insulin-resistant individuals which have lower palmitic acid esters of hydroxystearic acid (PAHSA) concentrations in serum and adipose tissue, suggest that FAHFAs deficiency may contribute to metabolic diseases [6]. Furthermore, FAHFAs, particularly 9-PAHSA and 5-PAHSA, have shown anti-inflammatory and immunomodulatory effects [7]. Increasing evidence on the physiological roles of FAHFAs motivates a more extensive characterization of these lipids as possible biomarkers and therapeutic targets for pathological conditions such as diabetes or obesity. Nevertheless, the low concentration in human tissues paired to the large structure heterogeneity of this lipids class challenges current analytical methods for their accurate identification and quantification. Moreover, the major amount of FAHFAs in cells is incorporated into triacylglycerols (TGs) by esterification to the glycerol backbone, requiring lipolysis (i.e., TGs hydrolysis) to quantify free FAHFAs [8]. Considering the current analytical limitations, our principal goal was to optimize a reliable, reproducible, and fast methodology based on high resolution MS for the characterization and relative quantification of FAHFAs in human white adipose tissue (WAT) of obese patients. To facilitate the reliable identification of FAHFAs in a complex human matrix, first optimization of sample processing and instrumental parameters were performed using individual and pooled mixtures of FAHFAs standards (n=15). Then, the procedures were applied to the real samples i.e., human white adipose tissue (WAT, n=169) corresponded to abdominal visceral (VAT) and subcutaneous (SAT) fat depots of obese insulin sensitive patients (IS, n=34; BMI > 40kg/m²), obese insulin resistant patients (IR, n=47; BMI > 40kg/m²) and lean subjects used as controls (L, n=5; BMI ‹25kg/m²). Briefly, since TGs are the major reservoir of FAHFAs, at first, we identified the optimum conditions that would hydrolyze esterified FAHFAs from TGs without degradation. Due to low physiological abundance of FAHFAs in human tissue, amino-propyl–NH2 (Strata NH2) solid phase extraction (SPE) enrichment was performed to separate FAHFAs from FFA released by TG hydrolysis. Finally, considering the low abundance of endogenous FAHFAs in adipose tissue samples and the expected large amounts of FFAs released from hydrolyzed TGs, a C18- reverse phase (C18-RP) SPE allowed the separation of these two lipid classes. Instrumental analyses were conducted on high-resolution MS (HRMS) (UHPLC/Q-Exactive Plus™, Thermo Scientific) applying a parallel reaction monitoring (PRM) method. 61 FAHFAs were manually identified, 32 out of those considered for the final analyses being present in > 70% of the samples and at least in two groups. 16 of 32 showed a significant alteration between groups (p value < 0.05; fold change >1.5) supporting a different response based not only on the metabolic status (lean vs obese) but also on the type of adipose tissue (SAT vs VAT). To conclude, considering the novelty and complexity of FAHFAs research (so far mainly conducted in vivo), this work aimed for the first time to set up a reliable and reproducible protocol to study them in human adipose tissue giving new perspective for the treatment of metabolic diseases.

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Evaluation of molecular effects of low-molecular-weight hyaluronic acid in human dermal fibroblasts through advanced quantitative proteomics and semi-quantitative lipidomics. Hyaluronic acid (HA) is a glycosaminoglycan physiologically synthesized by several human cell types, but it is also a very common ingredient of commercial products, from pharmaceuticals to cosmetics due to its widespread distribution in humans and its diversified physiochemical proprieties including biocompatibility, biodegradability, mucoadhesivity, viscoelasticity and hygroscopicity [8]. Despite its extended use and preliminary evidence showing even also opposite activities to the native form, the precise intra- and extra-cellular effects of HA at low-molecular-weight (LWM-HA) are currently unclear [9]. At this regard, our first aim was to identify in-depth and quantify proteome’s changes in normal human dermal fibroblasts after 24 hours treatment with 0.125, 0.25 and 0.50 % LMW-HA (20-50 kDa) respectively vs controls. To do this, a label-free quantitative proteomic approach based on high-resolution mass spectrometry was used (Dionex Ultimate 3000 nano-LC system -Sunnyvale CA, USA- connected to Orbitrap Fusion™ Tribrid™ Mass Spectrometer -Thermo Scientific, Bremen, Germany). Overall, 2328 proteins were identified of which 39 significantly altered by 0.125 %, 149 by 0.25 % and 496 by 0.50 % LMW-HA. Protein networking studies indicated that the biological effects involve the enhancement of intracellular activity at all concentrations, as well as the extracellular matrix reorganization, proteoglycans and collagen biosynthesis. Moreover, the cell’s wellness was confirmed, although mild inflammatory and immune responses were induced at the highest concentration. Influenced by the treatment also lipids metabolism related proteins, also in this case with 0.50% LMW-HA [10]. Then, the LWM-HA effects were investigated from the lipidome point of view. 1380 lipids unique by structure were detected by HRMS (UHPLC/Q-Exactive Plus™, Thermo Scientific) of which 477 lipids were filtered out by the data manual curation not matching the identification criteria (presence of class characteristics fragments, retention time, fragmentation pattern products at MS2 level) leaving 903 lipids (n=384 if considered at bulk level). Triacylglycerols (TGs), Ceramides (Cer) and Phosphatidylcholines (PCs) are the most represented classes (45.2%, 12.1% and 11.3% respectively). 563 out of 903 features significantly altered vs untreated control group based on One-way Anova test (adjusted p value ‹0.05, Fisher’s test) showing a clear cluster separation between 0.50% LMW-HA group and the remainders suggesting a cellular effect related strictly to this concentration as previously saw with the proteomics analyses. Ceramides and hexosy-1-ceramides, among the most important lipids classes from the biological and functional point of view both at the stratum corneum and deeper fibroblasts layer, resulted also among those most significantly affected by LMW-HA 0.50%. Although separated proteomics and lipidomics analyses allowed us to understand the cellular LWM-HA effects from different point of views, combining them provided an even further comprehension about its impact. At this regard, as final goal of this project the significant altered proteins (n= 495) and lipids through the 0.50 % LMW-HA treatment were integrated with Ingenuity Pathways Analysis software (IPA) resulting in 25 networks including 26 lipids belonging to different classes and biological functions such as mitochondrial activity, cell signaling, metabolism, and energy storage. The more complete comprehension of intra- and extra-cellular effects of LMW-HA here provided by an advanced analytical approach and integratomics analyses will be useful to further exploit its features and improve current formulations.

Analysis of lipidome and proteome profile changing induced by Y-oryzanol prevention treatment in obese rats. Grains are the most common staple food consumed worldwide and among them, rice (Oryza sativa) is the top one. It is very important so to consider its constituents, such as Y-oryzanol (Orz) that is present in the bran layer and comprises a mixture of ferulic acid esters and phytosterols. Orz has shown several biological activities and it’s often associated with fat and cholesterol-lowering, anti-inflammatory, anti-cancer, anti-diabetic effects and antioxidant ones by blocking lipid peroxidation [11,12]. It’s also recently become a great candidate in metabolic disorders like obesity, responsible of 13% of global deaths. The mechanisms under all these effects are still not completely clarified. Starting from observed proteomics changes lipids-related in heart samples (parallel project), our main goal was to describe and relatively quantify by HRMS (UHPLC/Q-Exactive™) the plasma lipidome changes in obese-induced rats (high-sugar [78%] and high-fat [22%] diet, n=16) w/wo preventive treatment with Orz (0.5% in the meal for 30 weeks) vs lean ones (n=16). The oxidized lipids profile could support the unmodified lipids analyses further than plasma proteomics ones to offer a more complete vision of obesity’s metabolism effects and Orz activity. Once spiked with Splash Lipidomix, (Avanti Polar Lipids, Alabaster, AL, USA) lipid extracts were obtained with liquid-liquid extraction using MTBE/MeOH/H2O (4:1.2:1, v/v + 0.1% BHT) method [13]. Lipid identification was based on LipidHunter vRC3_2 and Lipostar v1.3.2 software and the peak areas were manually integrated and curated using Skyline. Variation in lipid profile among groups was evaluated with MetaboAnalyst online software. 394 lipids unique by structure were identified mostly belonging to TGs followed to PCs. Once compared their concentrations among groups we saw, for TGs for example, a marked increase in the obese vs lean group but at the same time as Orz improved the conditions lowering their values respect to untreated obese as well as of CEs and ceramides as Cer(18:1/16:0), positively associated also with an increasing of cardiovascular risk [14]. Further changes were also observed in PLs classes with some PEs, LPEs, PIs lipid species upregulated in obese vs lean and decreased by Orz treatment. Opposite trend for some PCs, generally inversed correlated with obesity status. PC(18:0_22:5) for example, a marker used in obesity models [15], has shown a return to physiological-like conditions in the Orz treated group. As on-going steps, the analyses of oxidized lipids and plasma proteomics in to integrate the present results offering a more complete vision of Orz activity in obesity.

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Conclusions. To conclude, all these works demonstrate how omics sciences (proteomics and lipidomics in this case) through high-resolution mass spectrometry can provide a detail description of many biological processes triggered by different stimulus (drugs, environmental stress, metabolic status etc.). An even more complete vision is obtained by their integration (integratomics), an approach still scarcely applied in the omics field although it could offer a strong contribution in the field of personalized medicine connecting several expertise and perspectives.

References
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[4] Fisher GJ, Cutis 2005; 75:5-8
[5] Zhang M. Biosci Rep 2020, 40:7
[6] Yore MM, Cell. 2014;159(2):318‐332
[7] Kolar MJ, Anal. Chem. 2018, 90, 8
[8] Fallacara A. Polymers, 2018, 10
[9] Snetkov P, Polymers, 2020, 11;12(8):1800
[10] Radrezza S., JPBA 2020 Jun;185:113199
[11] H. Masuzaki, J Diabetes Investig 2019
[12] Minatel IO Int J Mol Sci. 2016;17(8):1107
[13] Matyash Vet, J Lipid Res. 2008
[14] R. Laaksonen, European Heart Journal, 2016
[15] Pickens, C.A., Sci Rep 2017

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