Graziella Eliza Ronsein

6‐Thioguanine (6‐TGua) is one of the thiopurines used as a cytostatic drug. When internalized by cells, 6‐TGua is metabolized through the purine salvage pathway and readily incorporated into DNA. Patients treated with these thiopurines are more prone to develop squamous cell carcinoma of the skin. The absorbance spectrum of 6‐TGua, in contrast to guanine (Gua) or any other canonical base, has a maximum absorbance at 342 nm. Therefore, 6‐TGua undergoes photoexcitation upon exposure to UVA radiation, with maximum absorption at 340 nm. In this study, the used approach unequivocally demonstrates the generation and quenching of ¹ O ₂ by 6‐TGua via the direct spectroscopic detection of ¹ O ₂ monomol light emission at 1270 nm. Chemiluminescence‐based methods were employed for the determination of the ¹ O ₂ generation quantum yield (Φ ¹ O ₂ ) and the total ¹ O ₂ quenching rate constant (k _t ). For the ¹ O ₂ quantum yield, we found values of 0.22 ± 0.03 for 6‐TGua and 0.12 ± 0.03 for 2′‐deoxy‐6‐thioguanosine (6‐TdGuo). These compounds presented k _t values of 1.5 × 10 ⁷ L mol ⁻¹ s ⁻¹ and 1.1 × 10 ⁷ L mol ⁻¹ s ⁻¹ , respectively. Through the comparison of these values with the ones obtained for 2′‐deoxyguanosine (dGuo) and its oxidation product 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo), we aim to provide new insights into the 6‐TGua‐containing DNA (DNA‐6‐TGua) reactivity towards ¹ O ₂ in a biological context.

Neutrophils are the innate immune system's first line of defense, and their storage organelles are essential to their function. The storage organelles are divided into 3 different granule types named azurophilic, specific, and gelatinase granules, besides a fourth component called secretory vesicles. The isolation of neutrophil's granules is challenging, and the existing procedures rely on large sample volumes, about 400 mL of peripheral blood, precluding the use of multiple biological and technical replicates. Therefore, the aim of this study was to develop a miniaturized isolation of neutrophil granules method, using biochemical assays, mass spectrometry–based proteomics and a machine learning approach to investigate the protein content of these organelles. Neutrophils were isolated from 40 mL of blood collected from 3 apparently healthy volunteers and disrupted using nitrogen cavitation; the organelles were fractionated with a discontinuous 3-layer Percoll density gradient. The method was proven successful and allowed for a reasonable separation and enrichment of neutrophil's storage organelles using a gradient approximately 37 times smaller than the methods described in the literature. Moreover, mass spectrometry–based proteomics identified 368 proteins in at least 3 of the 5 analyzed samples, and using a machine learning strategy aligned with markers from the literature, the localization of 50 proteins was predicted with confidence. When using markers determined within our dataset by a clusterization tool, the localization of 348 proteins was confidently determined. Importantly, this study was the first to investigate the proteome of neutrophil granules using technical and biological replicates, creating a reliable database for further studies.

The association between high-density lipoprotein (HDL) cholesterol and breast cancer (BC) remains controversial due to the high complexity of the HDL particle and its functionality. The HDL proteome was determined in newly diagnosed BC classified according to the molecular type [luminal A or B (LA or LB), HER2, and triple-negative (TN)] and clinical stage of the disease. Women (n = 141) aged between 18 and 80 years with BC, treatment-naïve, and healthy women [n = 103; control group (CT)], matched by age and body mass index, were included. Data-independent acquisition (DIA) proteomics was performed in isolated HDL (D = 1.063–1.21 g/mL). Results: Paraoxonase1, carnosine dipeptidase1, immunoglobulin mMu heavy chain constant region (IGHM), apoA-4, and transthyretin were reduced, and serum amyloid A2 and tetranectin were higher in BC compared to CT. In TNBC, apoA-1, apoA-2, apoC-2, and apoC-4 were reduced compared to LA, LB, and HER2, and apoA-4 compared to LA and HER2. ComplementC3, lambda immunoglobulin2/3, serpin3, IGHM, complement9, alpha2 lysine rich-glycoprotein1, and complement4B were higher in TNBC in comparison to all other types; complement factor B and vitamin D-binding protein were in contrast to LA and HER2, and plasminogen compared to LA and LB. In grouped stages III + IV, tetranectin and alpha2-macroglobulin were reduced, and haptoglobin-related protein; lecithin cholesterol acyltransferase, serum amyloid A1, and IGHM were increased compared to stages I + II. Conclusions: A differential proteomic profile of HDL in BC based on tumor molecular classification and the clinical stage of the disease may contribute to a better understanding of the association of HDL with BC pathophysiology, treatment, and outcomes.