Safirinium dyes as well as their reactive N-hydroxysuccinimide (NHS) esters bearing diverse substituents were synthesized and evaluated experimentally in terms of their lipophilicity by means of reverse-phase and immobilized artificial membrane high-performance liquid chromatography. Subsequently, the selected compounds were employed as novel cellular imaging agents for staining Gram-positive and Gram-negative bacteria, human kidney cell line, as well as human skin tissue. The analyzed dyes allowed for visualization of cellular structures such as mitochondria, endoplasmic reticulum, and cellular nuclei. They proved to be useful in fluorescent staining of stratum corneum, especially in the aspect of xenobiotic exposure and its penetration into the skin. The best results were obtained with the use of moderately lipophilic NHS esters of Safirinium Q. The development of Safirinium dyes is a promising alternative for commercially available dyes since the reported molecules have low molecular masses and exhibit efficient staining and remarkable water solubility. Moreover, they are relatively simple and low-cost in synthesis.Low-molecular synthetic fluorophores are convenient tools in bioimaging applications. Several derivatives of  Despite the widespread use of small organic fluorophores, the number of molecular scaffolds having tunable emission wavelengths and adjustable lipophilicity is limited12. The rational design of molecular probes is still a challenging task due the complexity of the processes underlying photophysical phenomena. Thus, the development of novel fluorescent molecules able to stain biological structures is an issue of major importance13.Fluorescent sensors and probes are of great interest due to their versatile applicability in chemical, environmental, and biological sciencesSafirinium P or Q, respectively16. The developed dyes display high fluorescence quantum yields, large Stokes shifts, as well as distinct absorption and emission spectra. Moreover, these compounds demonstrate remarkable water solubility with no dependence of optical properties on pH, and lack of toxicity. The carboxyl group present within the structure of the dye can be easily converted into N-hydroxysuccinimide (NHS) ester form, which is reactive toward nucleophiles. This approach has been successfully applied for fluorescent staining of Bacillus subtilis spores as well as labeling of lysine-containing peptides and proteinogenic amino acids18. In addition, the conjugation of Safirinium core to fluoroquinolone antibiotic or triterpenoic acid resulted in the development of new hybrid dual-acting antibacterial agents with enhanced penetration into the bacterial cells21 or cytotoxic agents targeting endoplasmic reticulum22, respectively. Safirinium compounds were also used as quaternary ammonium ionization tags (ITs) that enable peptides identification as a result of the presence of a permanent positive charge localized on the nitrogen atom within the triazolinium moiety18.Recently we have reported synthesis and optical properties of novel low molecular weight UV-fluorescent dyes bearing fused pyridino- or quinolino-triazolinium systems named Safirinium derivatives prompted us to exploit their potential usage in staining of biological structures by means of fluorescence microscopy. Lipophilicity and (phospho)lipophilicity parameters for the novel and previously reported fluorescent Safirnium-type labeling tags were assessed experimentally with chromatographic methods and compared with these assessed for commercially available dyes, namely fluorescein and sulforhodamine B. The most promising compounds were tested for their applicability in visualization of bacteria, human kidney cells, and skin tissue.The advantageous features of Safirinium chromophore chemical structure and selection of fluorescent probes that can efficiently stain biological structures.The presented work is focused on the targeted modification of Safirinium P (2a–h) and Q (5a–r) dyes bearing diverse alkyl substituents within the triazolium and quinoline moieties were designed and synthesized to attain the structures with optimal staining properties (Scheme a]pyridin-2-ium-8-carboxylate (2a–h) and 1,2-dihydro-triazoloquinolin-2-ium-4-carboxylate (5a–r) were obtained in the fluorogenic tandem Mannich—electrophilic amination reactions16 with use of two types of profluorophores, i.e. 4,6-dimethylisoxazolopyridin-3(1H)-one 1 and isoxazoloquinolin-3(1H)-ones 4a–g, respectively. Various secondary amines were utilized in order to modify lipophilicity of the final products. The obtained zwitterionic dyes were converted into hydrochlorides and subsequently reacted in anhydrous dimethylformamide (DMF) with N-hydroxysuccinimide (NHS) in the presence of N,N’-diisopropylcarbodiimide (DIC) as a coupling agent to yield a series of amine-reactive Safirinium P  and Q  esters. The structures of the products were confirmed with MS, IR, as well as NMR data , and slow-stirring methods are recommended for lipophilicity determination25. Currently, among these methods, the reversed phase high-performance liquid chromatography (RP-HPLC) is currently the most popular tool for lipophilicity assessment26. RP-HPLC provides reproducible results in the course of relatively short analysis, combining full automation and reduction of organic solvents expenditure compared to the traditional shake-flask procedure27. Moreover, the RP-HPLC approach can be augmented with application of biosimilar stationary phases, such as immobilized artificial membrane (IAM), that are commercially available nowadays. The chromatographically determined lipophilicity and (phospho)lipophilicity indices for the investigated dyes are summarized in Table n-octyl and n-decyl, substituents on the quaternary nitrogen atom  proved the highest values of (phospho)lipophilicity. Contrary, molecules 2a–c, f–h and 5a,f exhibit distinct hydrophilic character, as indicated by low logkw values. Consequently, the latter compounds showed marginal affinity to phospholipids and migrated with the front of the mobile phase in IAM-HPLC. All these derivatives have a carboxyl group, which ionization can lead to the reduction of affinity to phospholipids. Generally, a molecule should have optimal lipophilicity values and affinity for phospholipids to effectively penetrate the biological membranes. CHIIAM and logkw indices of the tested substances and two reference dyes, fluorescein  and sulforhodamine B  are plotted in Fig. Lipophilicity of a molecule should be considered when developing new dyes since it is a crucial determinant of the ability to penetrate the lipid bilayer of cellular membranes. Generally, lipophilicity can be assessed using theoretical and experimental approaches. Nevertheless, several studies indicated that calculation methods may lead to unsatisfactory results2 and 5 as well as their NHS esters 3 and 6 were photostable—no changes were observed in the MS spectra of the water solutions of the dyes and only a minor decrease in the intensity of fluorescence was noted after 24 h irradiation with UV lamp . Moreover, dyes 2 and 5 were stable in a broad range of pH (1–10), while their N-hydroxysuccinimide derivatives 3 and 6 proved stable under acidic, neutral, and slightly basic conditions. As expected, in alkaline pH, the reactive esters tend to be susceptible to hydrolysis and convert back into the free carboxylic acid forms 2 and 5, respectively, as evidenced by MS analysis (data not shown).Further experiments revealed that the synthesized dyes of type Safirinum derivatives: 2a,d, 3d, 5c,l and 6q for one-step non-specific labeling of Gram-negative E. coli and Gram-positive B. mycoides bacteria has been tested. In the experiment, bacteria were simply incubated 105 min in phosphate buffered solution (PBS) with the appropriate dyes at a concentration of 2.5 mg/mL and, after washing out the non-bound compounds, analyzed by fluorescence microscopy. The compounds 2a,d and 5c stained both bacteria not-efficiently producing a weak fluorescence signal merging with background fluorescence for image acquisition times greater than 1 s  at a concentration of 2.5 mg/mL with respect to eukaryotic cells. The Safirinium derivatives were examined for potential application in visualization of cellular organelles by means of confocal microscopy. In this experiment, HEK293 cells we used and the slides were prepared in accordance with the described procedure. On the basis of the obtained results, it was observed that compounds 2a,d and 5c produced weak fluorescence signals . In the case of the dyes 5l and 6q the fluorescence signals of the stained cells were effective and quick fading of the dyes were not evidenced. The staining effects with all of the dyes tested on non-permeabilized and permeabilized cells were almost identical, indicating that these compounds do not require an increased membrane permeability to stain effectively. Next, we decided to elucidate the exact subcellular localization of the 5l and 6q dyes. Hence, in order to label the organelle the following fluorescent dyes have been utilized: MitoTracker Green dye to stain mitochondria, DiOC6 to stain mainly endoplasmatic reticulum and DAPI to stain cellular nuclei. It was found that the novel fluorescent dyes feature intracellular accumulation sites. Thus, compound 5l targets mainly mitochondria and endoplasmic reticulum of cells, whilst the 6q dye localizes primarily in cellular nuclei  and hydrogen bond donor (HBD) is no more than 10 and 5, respectively30. An optimal dye should feature affinity to the skin structures, i.e. stratum corneum, which is built of corneocyte cells (composed of proteins—mainly keratin) and lipids of the lipid bilayer surrounding corneocytes . The most commonly used fluorescent dyes in the above context are fluorescein and rhodamine derivatives.In the case of human skin imaging, the potential dyes must have the ability to diffuse deeply into the skin layer. The literature reports show that the penetration of the 2b,d and 3d) and 9 derivatives of quinoline  were used in skin staining experiments to select the compounds exhibiting desirable properties and the best staining effects. Derivatives with free carboxyl groups  as well as compounds with reactive carbonyl  groups were utilized to verify the influence of structural modifications on cell structure visualization.In the next step, 3 pyridine-based dyes —a siloxane with a cyclic structure.2b,d), the long-lasting staining of the control and the test samples was not attainable due to the quick fading of the dyes (after approx. 30 s). It was concluded that this effect could result from evaporation of the solvent from the surface of the formulations in which the fluorescent compounds were dissolved. In contrast, the quinoline derivatives  retained longer fluorescent properties, although they did not prove effective ability to penetrate stratum corneum structures, staining mainly the surface of the skin  surrounding the corneocytes was observed. The obtained result is consistent with the mechanism of the skin barrier crossing described in the scientific literature, which assumes that ionized compounds feature much lower permeation coefficients than non-ionized compounds. Thus, proteins such as keratin present in corneocytes are ionized under physiological conditions presenting both positively and negatively charged fragments. For example, acidic proteins feature negatively charged amino acid side chains incorporating aspartic or glutamic acids, while basic proteins prove positively charged side chains build from lysine, arginine or histidine31. On the contrary, lipids bear negative charges, therefore it is generally assumed that the skin can act as a negatively charged membrane32. Consequently, electrostatic interactions may occur between the structural elements of the stratum corneum and ionized xenobiotics. The above results in repulsion of molecules , which makes overcoming the skin barrier difficult or impossible. Thus, on the basis of the obtained results, it can be concluded that the negative charges located on the carboxylate groups (COO-) in the fluorescent dyes 2b,d and 5p,q condition poor or prevent penetration into the structures of stratum corneum. Consequently, positive charges on nitrogen atoms are sterically hindered making the COO- groups exposed to electrostatic interactions with the negatively charged lipids of the lipid bilayer. This observations can be further supported by retention parameters obtained in the HPLC experiments. Hence, albeit logkw values of the examined dyes 2b,d and 5p,q  are comparable the corresponding parameters assessed for the reference fluorescein and sulforhodamine B , the latter proved considerable higher affinities to phospholipids in CHIIAM chromatography than the tested Safirinium dyes with (phospho)lipophilicity indices of 23.80 and 27.80 vs ND, 2.50, 6.60 and 16.80, respectively  and triazoloquinolinium-carboxylate (6p) in terms of their utilization in imaging the stratum corneum structures. These two compounds were selected, taking into account their HPLC lipophilicity indices of 1.71 and 1.77, respectively, that correspond to the lipophilicity parameters assessed for the reference fluorescein (2.55) and sulforhodamine B (1.52). The conducted experiments has shown that compound 6p of quinoline system and ethyl substituents within the triazolinium ring displays favorable fluorescent and diffusion properties to the skin in relation to the compound 3d based on dibutyl-triazolopyridinium system  than ester 3d (37.10), fluorescein (23.80) and sulforhodamine B (27.80). It should be pointed out that the dye 5p featuring free carboxyl group (logkw 1.82 and CHIIAM = 6.60), despite having analogical physicochemical parameters to ester 6p (logkw 1.77 and CHIIAM = 6.80), does not stain corneocytes within stratum corneum structures. Moreover, it was noted that application of the model liquid substance D4 to the skin increases of fluorescence intensity, which can be related to the solubility of the applied lipophilic dye (6p) in the model lipophilic substance . In conclusion, it was found that the reactive carbonyl moiety within the dye structure is crucial for visualization of corneocytes since it enables diffusion of the fluorescent dye into the stratum corneum.The next stage covered the assessment of escein 2.5 and sulstratum corneum structures has been evaluated in relation to alkyl substitution pattern that condition lipophilicity of the N-hydroxysuccinimide esters of Safirinium Q dyes. Hence, compounds 6b, 6c, 6p and 6q of logkw and CHI-IAM parameters in the ranges 1.77 to 2.64 and 2.45 to 32.10, respectively, have been investigated .The visualization efficiency of Safirinium Q chromophore two novel and one known N-hydroxysuccinimide esters useful for visualization of stratum corneum structures were selected:a]quinolin-2-ium chloride (6c);2,2-dibutyl-4-carbonyl)-1,2-dihydro-triazoloquinolin-2-ium chloride (6p);2,2-diethyl-4-carbonyl)-8-methyl-1,2-dihydro-triazoloquinolin-2-ium chloride (6q).2,2-dibutyl-4-carbonyl)-8-methyl-1,2-dihydro-triazolopyridin-2-ium-8-carboxylate (2d) and dihydro-triazoloquinolin-2-ium-4-carboxylates  were obtained from the corresponding isoxazolones  dissolved in methanol (10 mL). Dibutylamine  and formaldehyde 35 wt. % aqueous solution  were added to the solution and the resulting mixture was stirred at room temperature for 16 h. The progress of the reaction was monitored by LC–MS. The reaction mixture was evaporated under reduced pressure and subsequently, the crude product was washed with acetone (2 × 3 mL).2,2-Dibutyl analogues of dihydro-triazolopyridin-3(1H)-one 1 (0.312 g). Yield: 83% (0.481 g); m.p. 139–141 °C; 1H NMR : δ = 0.99 , 1.35–1.47 , 1.74–1.84 , 2.20 , 2.30 , 3.45–3.62 , 5.72 , 5.99 ; 13C NMR : δ = 12.6, 16.9, 18.4, 19.2, 24.1, 66.2, 72.9, 110.1, 118.0, 139.1, 146.2, 155.6, 170.2; IR (KBr): 3394, 3001, 2962, 2983, 2871, 1668, 1634, 1619, 1560, 1529, 1467, 1374, 1356, 1188, 801, 606 cm−1; MS (ESI) m/z: 306 [M + 1]+.Obtained from 4,6-dimethylisoxazoloquinolin-3(1H)-one 4a (0.354 g). Yield: 98% (0.610 g); m.p. 176–179 °C; 1H NMR : δ = 0.98 , 1.37–1.49 , 1.76–1.87 , 3.60–3.77 , 5.84 , 7.18 , 7.32 , 7.66 , 7.74 , 8.18 ; 13C NMR : δ = 12.6, 19.2, 24.2, 66.1, 71.8, 113.2, 121.0, 121.9, 123.4, 129.9, 132.4, 134.3, 140.9, 154.9, 167.8; IR (KBr): 3401, 2962, 2934, 2875, 1612, 1588, 1569, 1540, 1457, 1365, 1348, 1297, 1216, 809, 762, 594, 528 cm-1; MS (ESI) m/z: 328 [M + 1]+.Obtained from isoxazoloquinolin-3(1H)-one 4f. (0.380 g). Yield: 91% (0.590 g); m.p. 230–235 °C; 1H NMR : δ = 1.02 , 1.45–1.50 , 1.80–1.89 , 2.56 , 3.67–3.79 , 5.88 , 7.11 , 7.28 , 7.79 , 8.71 ; 13C NMR : δ = 12.6, 19.2, 21.0, 24.2, 66.3, 71.8, 111.9, 114.0, 118.1, 125.4, 130.9, 135.7, 146.8, 147.1, 154.1, 163.5; IR (KBr): 3422, 3003, 2962, 2978, 2897, 2874, 1721, 1616, 1555, 1221, 1199, 1182, 1159, 1119, 816, 736 cm-1; MS (ESI) m/z: 342 [M + 1]+.Obtained from 7-methylisoxazolopyridin-2-ium (3d) and 4-carbonyl)-1,2-dihydro-triazoloquinolin-2-ium  chlorides were obtained according to literature procedures reported earlier by our research group14 with only minor modifications. The compounds 2d and 5c,q (0.35 mmol) obtained in zwitterionic forms were dissolved in methanol and acified with methanolic HCl solution to get hydrochlorides 2d and 5c,q. The solvent was evaporated under reduced pressure, then the resulted solids were dissolved in anhydrous DMF (7 mL). Subsequently, NHS  and (DIC)  were added and the reaction mixtures were stirred for 2–4 h at room temperature. The progress of the reaction was monitored by LC–MS. After the completion of the reaction, the solvent was evaporated and the crude product was isolated. Compound 2d was dissolved in water (5 mL) and filtered, then the filtrate was evaporated. The resulted solid was washed with acetone (3 × 2 mL) and dryied under vacuum. The compounds 5c,q were washed with acetone (3 × 2 mL) and dried, then purified by crystallization from methanol/aceton mixture .Dibutyl deriatives of 8-carbonyl)-5,7-dimethyl-2,3-dihydro-triazolo+.Obtained from compound 5c (0.115 g). Yield: 43% (0.069 g); m.p. 168–169 °C; 1H NMR : δ = 0.93 , 1.34–1.48 , 1.70–1.83 , 2.93 , 3.68–3.96 , 5.94 , 7.26 , 7.43 , 7.92 , 8.12 , 9.11 ; IR (KBr): 3341, 2967, 2935, 2876, 1774, 1738, 1718, 1619, 1572, 1459, 1362, 1246, 1202, 1168, 1138, 1093, 1070, 996, 765, 665 cm-1; MS (ESI) m/z: 425 [M + 1]+.Obtained from compound 5q (0.120 g). Yield: 96% (0.161 g); m.p. 199–202 °C; 1H NMR : δ = 0.93 , 1.34–1.38 , 1.70–1.81 , 2.60 , 2.92 , 3.62–3.74 , 5.93 , 7.13 , 7.28 , 8.00 , 9.05 ; IR (KBr): 3341, 3023, 2966, 2935, 2876, 1775, 1740, 1618, 1562, 1198, 1096, 1066, 920, 816, 765, 644 cm-1; MS (ESI) m/z: 439 [M]+.Obtained from compound The determinations of lipophilicity and affinity to phospholipids were performed using chromatographic gradient approaches. Each chromatographic experiment was carried out using the Prominence-1 LC-2030C 3D HPLC system  controlled by LabSolution system . Detection of solutes was performed at 200 nm using DAD detector.Organic solvents, i.e. acetonitrile and methanol (HPLC grade for liquid chromatography), sodium phosphate dibasic dehydrate, and sodium phosphate monobasic monohydrate were purchased from Sigma-Aldrich . Ultrapure water used for buffer mobile phase preparation was obtained with Millipore Direct-Q 3 UV Water Purification System .18 4.6 × 150 HPLC column with a linear gradient 25–98% phase B (where phase A was water and phase B was methanol) with a flow rate of 1 mL/min. The temperature of the chromatographic column was controlled and set to 30.0 °C. Two gradient runs of diverse gradient time (tG corresponds to 15 min. and 30 min.) were performed. The retention times (tR) of the investigated solutes are listed in Table A. These data were used as input values for logkw calculation (i.e. the logk retention factor extrapolated to 0% organic modifier) using DryLab 6.0 software , according to the theory proposed by Snyder and co-workers. The dwell volume of the HPLC system was 0.780 mL, whereas the measured dead time for the utilized HPLC columns was 1.401 min.The RP-HPLC analyses were performed on Knauer 100–5 C35. The chromatographic analyses were performed in duplicate.The IAM-HPLC analyses were carried on IAM.PC.DD2 column  with a linear gradient 0–85% phase B (where phase A was 10 mM phosphoric buffer at pH 7.4 and phase B was acetonitrile) at a flow rate of 1.5 mL/min and constant temperature of 30.0 °C. The standards of octanonophenone, butyrophenone, and acetanilidine were provided by Alfa Aesar ; acetaminophen, theophylline, benzimidazole, acetophenone and indole were purchased from Sigma-Aldrich ; heptanophenone, hexanophenone, valerophenone, propiophenone, acetophenone were acquired from Acros Organic . The reference substances were utilized to determine CHI indices of the investigated dyes according to the protocol proposed by Valkó and co-workersE. coli BL21DE3 (Merck), E. coli BL21DE3 encoding pSFOXB20-daGFP plasmid (Oxford Genetics) for constitutive production of GFP (Green Fluorescent Protein) and Bacillus mycoides (ATCC 6462). E. coli and B. mycoides were cultivated overnight with agitation at 37 and 30 °C, respectively, in Luria broth (LB) supplemented with the kanamycin (20 μg/mL) for BL21DE3/pSFOXB20-daGFP strain. Overnight cultures were suspended in phosphate-buffered saline (PBS) to OD600 = 0.5. The cells were incubated at room temperature in 70% isopropanol for 15 min to prepare bacteria with a permeabilizded membrane. Then the permeabilized cells were washed with PBS and finally suspended in PBS buffer to OD600 = 0.5. B. mycoides aggregates were induced by incubation of bacteria at room temperature without shaking for 4 h in PBS. For fluorescence microscopy, bacteria suspended in PBS were incubated with 2a,d, 3d, 5c,l and 6q dyes at a final concentration of 2.5 mg/mL for 105 min at room temperature in the dark. Then, the non-bound dyes were removed by washing with PBS buffer. The E. coli and B. mycoides as permeabilized, non-permeabilized or aggregated cells were analyzed in suspension as a drop loaded on a slide glass using the Olympus IX73 inverted fluorescence microscope equipped with the UCPlanFL N 20x/0.70, a Olympus U-HGLGPS fluorescence light source and a Hamamatsu Orcaflesh 2.8 CMOS digital camera. For blue and green fluorescence the following filter sets were used: DAPI HC BP, GFP-1828A and TRITC-B-000 , respectively. Each type of experiment was repeated 3 times.The following bacterial strains were used: 2.Human embryonic kidney cell line (HEK-293) from American Type Culture Collection  was used in this study. The cells were maintained in cell media consisting of Dulbecco’s modified Eagles medium (DMEM)  with 10% fetal bovine serum (FBS), , 1% penicillin/streptomycin , and 1% L-glutamine . HEK-293 cells were cultured in an incubator at 37 °C and 10% CO2a,d, 3d, 5c,l and 6q dyes at final concentration of 2.5 mg/mL diluted with PBS × 1. Nuclei were stained in a 0.25 μg/mL 4′,6-diamino-2-phenylindole (DAPI) solution. MitoTracker Green at concentration of 500 nM was used to stain the mitochondria. 3,3′-Dihexyloxacarbocyanine iodide (DiOC6) was used for the staining of a cell's membranes including endoplasmic reticulum and vesicle membranes, at final concentration of 5 µM.In this study the cells were grown in slides placed in a 35 mm dish. After 24 h incubation the cells were washed with PBS × 1 and fixed by adding 1 mL per dish of a 4% paraformaldehyde solution for 15 min at room temperature. To prepare cells with a permeabilized membranes, the cells were incubated at room temperature in 0.2% PBS-Triton × 1100 for 10 min. After washing twice with PBS × 1, the cells were incubated for 105 min at room temperature in the dark with The slides were observed using the confocal microscope (Zeiss LSM 800) at magnifications of 63×, applying appropriate filters and wavelengths.2b and 5b was examined in human embryonic kidney 293 cells (HEK 293) using the 3--2,5-diphenyltetrazolium bromide (MTT) cell viability assay. No reducing trends to the cell viability or changes in cell morphology were observed (data not shown). All the tested compounds were found to be non-toxic up to the concentration of 20 µM.The cytotoxicity of the prepared compounds 2). All measurements of skin samples demonstrated values above the limit of 2 kΩ/cm2 (average 5 kΩ/cm2), indicating the integrity of the skin38. After placing the fragment in a chamber, 10 µL of siloxane D4 was applied to the skin surface of the test sample (control sample—the same procedure but without application). After 24 h of incubation, the remaining siloxane was removed from the sample surface using successively: filter paper strips, a hexane-soaked swab, and by removing the top three layers of stratum corneum by tape-stripping technique with special adhesive tapes . The epidermis layer was then isolated from dermis using the heat-separation technique . Then, epidermis was washed with distilled water and cut into smaller pieces. Samples were next transferred to a microscope slide with the stratum corneum on top and dried for 2 h.Human cadaver skin was used, obtained during necropsy. Skin section  from the abdominal region of males and females in the age group of 35–50 years was collected 48 h hours after the time of death. The study has been approved by the Independent Bioethics Commission for Research at the Medical University of Gdańsk (no NKBBN/309/2013 and no NKBBN/449/2020). After separation of subcutaneous tissue, skin was washed with purified water and 0.9% w/v NaCl solution, dried and fragmented into smaller pieces, protected with aluminium foil and stored in a freezer at −20 °C, labelled with the batch number (donor). Prior to examination, a section of skin was thawed. Then, skin sample integrity was examined by the Trans-Epithelial Electrical Resistance technique (TEER). Millicell ERS-2  data bridge connected to two stainless steel electrodes was used; ER was expressed in kΩ for the surface area of the exposed skin .We declare that the Microscopic images were recorded using a Nikon Eclipse 50 fluorescence microscope with a mercury lamp. 10 µL of the appropriate dye was topically applied to epidermal sample, omitting the pore areas left by the hair follicles. The sample was placed in a chamber consisting of two Petri dishes, lined with a piece of lignin, previously moistened with purified water. The incubation time at 32 °C ± 1 °C in a thermostat was 10 min. In the next step, the residual dye was gently removed from the sample surface with a piece of lignin. Next, samples were examined using the fluorescence microscope at magnifications of 4, 10, 40, and 100 x, applying appropriate filters and wavelengths. A series of images were taken at different depths of the sample (changing the position of the table by 0.5–1.0 µm average) with utilization of automatic z-axis drive. Image registration and processing was performed using "NIS Elements AR3.2" software.Supplementary Information.


Excess alcohol consumption is a potential risk factor for cardiovascular diseases and is linked to accelerated aging. Drug discovery to reduce toxic cellular events of alcohol is required. Here, we investigated the effects of ethanol on human umbilical vein endothelial cells (HUVECs) and explored if doxycycline attenuates ethanol-mediated molecular events in endothelial cells. Initially, a drug screening using a panel of 170 drugs was performed, and doxycycline was selected for further experiments. HUVECs were treated with different concentrations (300 mM and 400 mM) of ethanol with or without doxycycline (10 µg/mL). Telomere length was quantified as telomere to single-copy gene (T/S) ratio. Telomere length and the mRNA expression were quantified by qRT-PCR, and protein level was analyzed by Western blot (WB). Ethanol treatment accelerated cellular aging, and doxycycline treatment recovered telomere length. Pathway analysis showed that doxycycline inhibited mTOR and NFκ-B activation. Doxycycline restored the expression of aging-associated proteins, including lamin b1 and DNA repair proteins KU70 and KU80. Doxycycline reduced senescence and senescence-associated secretory phenotype (SASP) in ethanol-treated HUVECs. In conclusion, we report that ethanol-induced inflammation and aging in HUVECs were ameliorated by doxycycline. Aging is strongly associated with cardiovascular diseases. With increasing age, the risk of multiple cardiovascular diseases increases. The senescence of vascular cells is associated with age-related cardiovascular diseases ,2,3. SenThe senescence and dysfunction of vascular endothelial cells play a vital role in vascular aging, which results in the initiation, progress, and advancement of cardiovascular diseases . The senDoxycycline is a broad-spectrum antibiotic belonging to the tetracycline class, and it has an anti-inflammatory effect. It has been reported to inhibit the activation of NF-κB ,13. BothThis is the first report showing that ethanol accelerated cellular aging and senescence in HUVECs. Doxycycline treatment reduced ethanol-enhanced aging and cellular senescence by inhibiting NFκ-B activation and the mTOR pathway.2. Cells were seeded in the T75 cell culture flask after thawing. When the cells were 90% confluence, they were incubated with trypsin for 4 min at 37 °C, and then cells were seeded in 10 cm cell culture plate, 5000 cells/cm2. For experiments, the endothelial cells were used at passage 7 and were seeded in 6-well plates. After 24 h, the medium was changed, and the new medium containing either ethanol (300 mM or 400 mM), doxycycline (10 µg/mL), or 400 mM ethanol combined with doxycycline (10 µg/mL) was added to the culture; the medium was changed after two days. Doxycycline was purchased from Sigma-Aldrich (D1822).HUVECs were obtained from Promocell  and maintained in the medium consisting of endothelial cell medium  supplemented with endothelial growth factors  at 37 °C in a 95% humidified atmosphere containing 5% COIn the first experiment, the endothelial cells were treated with an endothelial cell medium containing either 300 mM or 400 mM ethanol. In the second experiment, the endothelial cell medium was supplemented with either 400 mM ethanol or 400 mM ethanol combined with doxycycline (10 µg/mL). The endothelial cell medium alone was used for the control group. The medium was changed after two days. After four days of treatment, DNA was extracted from cell pellets using innuPREP DNA Mini Kit . For telomere length measurement, 1 ng DNA was utilized. For qPCR the primers  telomere2). The medium was changed the next day with a new medium alone (for control) or medium containing ethanol , doxycycline  and combination of ethanol (400 mM) and doxycycline (10 µM). The medium was changed after two days. The readout of the cell survival was performed using luminescence-based CTG assay  according to the manufacturer’s instructions, except that the reaction agent was diluted in 1:1 with PBS. The CTG assay was performed immediately after changing the medium on day 0 and on day 1, day 2, and day 4.For the CTG assay, the cells were seeded in 96-well plate , or 400 mM ethanol combined with doxycycline (10 µg/mL). For the control group, the endothelial cells were treated with endothelial cell medium alone. After 24 h of treatment, the total protein was extracted using RIPA Buffer, then determined calorimetrically using the DC Protein Assay Kit , according to the manufacturer’s instructions, and measured with the Paradigm micro-plate reader. Total protein  was loaded on 12% sodium dodecyl sulfate-polyacrylamide gel and run at 60 volt for 20 min, then continued with 110 Volt for 30–60 min, which was further transferred onto a polyvinylidene difluoride membrane at 250 mA for 120 min. The non-specific binding was blocked with 5% skim milk (0.05% TBST) for 1 h. The membranes were incubated with primary antibodies (as reported in For quantitative PCR (qPCR), total RNA was extracted from endothelial cells treated with either endothelial cell medium alone (control group) or endothelial cell medium containing either 400 mM ethanol or 400 mM ethanol combined with doxycycline (10 µg/mL) for 24 h. The Nucleo Spin RNA kit  was utilized to extract total RNA following the manufacturer’s instructions. RNA (1.2 µg) was utilized to reverse transcribe with M-MLV Reverse Transcriptase kit , Random Hexamer Primers , and RiboLock RNase Inhibitor . qPCR was performed with AceQ SYBR qPCR Master Mix  on Bio-Rad with an initial denaturation of 95 °C for 8 min, followed by 45 cycles of 95 °C for 15 s, 58.9 °C for 30 s, and 72 °C for 30 s, followed by melting curve. The relative mRNA expressions were calculated after normalizing them to β-actin expression. The primer sequences are listed in HUVECs were seeded in a 6-well plate. Once the cells were more than 95% confluence, a 10 μL sterile pipette tip was used to make a scratch on the monolayer of endothelial cells. After the scratch, the endothelial cells were washed 3 times with PBS, and a new endothelial cell medium containing either 400 mM ethanol or 400 mM ethanol combined with doxycycline (10 µg/mL) was added to the culture. For the control group, the endothelial cells were treated with endothelial cell medium alone. At 0 h, 6 h, 12 h, and 24 h following the scratch assay, images of cell migration in three randomly selected fields were taken for each well using an optical microscope. Finally, the area of the wound was measured by Image J. All experiments were performed with three biological triplicates, and each biological replicate had three technical triplicates.β-Gal staining was detected using a Senescence Cells Histochemical Staining Kit , following manufacturer instructions. The endothelial cells were treated with endothelial cell medium alone (control group) or endothelial cell medium supplemented with either 400 mM ethanol or 400 mM ethanol combined with doxycycline (10 µg/mL). The medium was changed after two days. After 4 days, the cells were fixed in the fixation buffer provided with the Senescence Cells Histochemical Staining Kit. The fixed samples were stained with fresh solution for SA-beta-galactosidase activity at 37 °C for 7 h, followed by aspiration with the staining solution. After that, the cells were overlaid with a 70% glycerol solution and stored at 4 °C. The images were taken with an optical microscope. Finally, the number of stained cells was counted using Image J. The experiment was performed with three biological triplicates.2). The medium was changed the next day with a new medium alone (for control) or a medium containing ethanol (400 mM), doxycycline (10 µM), and a combination of ethanol (400 mM) and doxycycline (10 µM). After two hours of incubation, immunofluorescence staining was performed. The cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton™ X-100 for 10 min, and blocked with 5% BSA for 1 h at RT. The cells were incubated overnight at 4 °C with primary antibody 8-OHDG . The next day, the cells were washed three times and then labeled with Secondary antibody  for 60 min at RT. Nuclei were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) for 10 min. The images were captured at 20× magnification.The cells were seeded in a 96-well plate  of ethanol on the telomere length of endothelial cells. The endothelial cells were treated with an endothelial cell medium containing either 300 mM or 400 mM ethanol. The endothelial cell medium without ethanol was used for the control group. Telomere length was quantified as T/S. The telomere length was shortened by more than 50% in ethanol-treated HUVECs as compared to that in the untreated control  assay. The cells were treated with different concentrations of ethanol, doxycycline, and combined ethanol (400 mM) and doxycycline (10 µM). The medium was changed after two days. The CTG assay was performed on day 0, day 1, day 2, and day 4. On day 0, no difference was observed between the groups  = 49.26 ± 9.34%, ethanol (300 mM) = 48.05 ± 8.93%, ethanol (400 mM) = 50.22 ± 15.67%, ethanol (600 mM) = 51.76 ± 10.25%, ethanol (800 mM) = 54.26 ± 9.97%, doxycycline (1 µM) = 48.32 ± 11.54%, doxycycline (10 µM) = 46.72 ± 10.73%, doxycycline (50 µM) = 46.71 ± 12.83%, doxycycline (100 µM) = 48.30 ± 11.91%, ethanol + doxycycline = 43.12 ± 9.23%, p < 0.001, ** p < 0.01 p < 0.05, p < 0.001, p < 0.01, * p < 0.05, p < 0.01, * p < 0.05, p-4EBP1, but it did not affect its relative protein expression in ethanol-treated HUVECs  in endothelial cell medium. The control group was treated with endothelial cell medium alone. The medium was changed after two days. On day 4, we collected the cell pellets and extracted DNA. Telomere length quantification showed that doxycycline treatment ameliorated ethanol-accelerated telomere shortening in HUVECs .To investigate ethanol-induced oxidative stress, we performed immunofluorescence staining for oxidative stress marker 8-OHDG. HUVECs were treated with ethanol (400 mM), doxycycline (10 µM), and a combination of ethanol (400 mM) and doxycycline (10 µM) for 2 h. The endothelial cell medium alone was used for control. Ethanol increased oxidative stress, which was ameliorated by doxycycline (Control = 0.58 ± 0.53, Ethanol = 90.52 ± 2.65, doxycycline = 3.84 ± 4.94, ethanol ± doxycycline = 12.37 ± 15.51, p < 0.001, ** p < 0.01, * p < 0.05 p < 0.01, * p < 0.05, p < 0.01, * p < 0.05, NFκ-B is known to play an important role in inflammation, aging and cellular senescence ,15. Ethap < 0.05, p < 0.01, * p < 0.05, p < 0.001, ** p < 0.01 * p < 0.05, p < 0.001, p < 0.01, p < 0.001, ** p < 0.01, * p < 0.05, Doxycycline restored the relative protein expression of DNA repair proteins KU70 (Control = 100 ± 10.12, ethanol = 80.38 ± 2.71, doxycycline = 91.53 ± 0.17, ethanol + doxycycline = 99.31 ± 9.67, n = 3, * p < 0.001, It has already been known that increased inflammation and telomere shortening lead to cellular senescence ,4,14. Top < 0.01, * p < 0.05 p < 0.001, p < 0.001, * p < 0.05, p < 0.001, * p < 0.05, p < 0.001, ** p < 0.01, p < 0.001, ** p < 0.01, * p < 0.05 p < 0.01, As doxycycline inhibited ethanol-induced senescence in HUVECs, we further investigated if doxycycline also reduces SASP in HUVECs exposed to ethanol. The ethanol-treated endothelial cells showed increased relative mRNA expression of pro-inflammatory molecules IL-1β, IL-8, MCP-1, E-selectin, ICAM-1, and VCAM-1 A–F. Doxyp < 0.05, p < 0.01, * p < 0.05 p < 0.001, ** p < 0.01 p < 0.001, ** p < 0.01 p < 0.001, p < 0.001, ** p < 0.01, * p < 0.05, p < 0.01, p < 0.001, ** p < 0.01, * p < 0.05 p < 0.01, The senescent cells release MMPs ,15, whicIn this study, we report that ethanol accelerated molecular aging caused cellular senescence and induced endothelial SASP and dysfunction. Doxycycline dampened ethanol-mediated inflammation and aging in endothelial cells and attenuated cellular dysfunction. Alcohol abuse is one of the potential risk factors associated with cardiovascular diseases. Studies suggest alcohol abuse causes aging ,11, but We treated HUVECs with two different concentrations of ethanol. Ethanol accelerated aging in HUVECs . The telDoxycycline did not affect the relative mRNA expression of aging markers P16 and P21 E,F, but The migration of endothelial cells is an important process for angiogenesis and repair/healing of damaged tissue. Previous studies have reported that the senescent cells show retarded migration and are not efficient in the repair/healing of damaged tissue ,25. EthaThe senescent cells have been shown to release MMPs ,15, and Telomere shortening is associated with cardiovascular diseases, but this association does not state that telomere shortening has a causal link to cardiovascular diseases. It is possible that the risk factors that lead to cardiovascular diseases also caused telomere shortening, and the factors that reduced vascular aging also inhibited telomere shortening. Alcohol is a potential risk factor for cardiovascular diseases, and in the present study, we showed that ethanol induced telomere shortening and premature senescence. Doxycycline reduced ethanol-induced inflammaging in HUVECs, possibly by inhibiting activation of the mTOR and NF-kB pathways.


Vitamin K, especially its K2 form, is considered to be a protective factor against developing vascular changes and bone lesions that are common complications in kidney transplant (KTx) recipients. There is a growing number of studies showing that KTx patients are at risk of vitamin K deficiency. The aim of this study was to evaluate the intake of vitamin K1 and K2 in the diet of patients in the late period after KTx. During a routine visit at one outpatient transplantation clinic in Central Europe, a diet survey questionnaire was filled in by 151 clinically stable KTx recipients and compared with medical history, anthropometric measurements and laboratory tests. Mean vitamin K1 intake was 120.9 ± 49 μg/day and vitamin K2  intake 28.69 ± 11.36 μg/day, including: MK-4: 25.9 ± 9.9 μg/day; MK-5: 0.1 ± 0.2 μg/day; MK-6: 0.2 ± 0.4 μg/day; MK-7: 0.2 ± 0.23 μg/day; MK-8: 1 ± 1.9 μg/day; MK-9: 0.9 ± 2.3 μg/day; and MK-10: 0.2 ± 0.5 μg/day. Our study showed that KTx recipients’ diets contained adequate amounts of vitamin K1, whereas the intake of vitamin K2 seemed insufficient. Cardiovascular (CV) diseases and bone disorders  are the most common complications in kidney transplant (KTx) recipients ,2,3,4. V2), as well as patients following definite diets , were not eligible for the study.Adult patients > 12 months after KTx and under the care of one transplant outpatient clinic in central Poland were considered eligible for the study. Patients using dietary supplements (including any form of vitamin K), with currently diagnosed cancer, symptoms of inflammation (acute or chronic), liver disorders, severe heart failure or advanced graft disfunction (estimated glomerular filtration rate (eGFR) < 15 mL/min/1.73 mI.A complete menu submitted by the patient from the three consecutive working days immediately prior to the visit. The questions concerned the intake and portion sizes of three main meals and any snacks.II.Product groups to accurately determine the food consumed and to obtain information on vitamin K1 and K2 content. The diary listed eight product groups: 1: meats, eggs, fish; 2: dairy products; 3: bread, cereals, pasta; 4: fats; 5: vegetables; 6: fruits; 7: nuts, seeds; 8: sweets.During a routine outpatient transplantation visit, the patients, in the presence of a clinical dietitian, completed a food-frequency questionnaire (FFQ) , a meal Using the information from both parts of the diary, a summary of the nutrient composition (including PK and MK) of the consumed food was made using the USDA Food Composition Databases . The MK On the same day, the patient’s anthropometric measurements were taken. Height measurement was assessed in the Frankfurt position using a height gauge attached to the medical balance with accuracy of 0.5 cm. Body weight and body mass index (BMI) were determined using a body composition analyzer . Data about medications (including immunosuppressive drugs) and comorbidities such as diabetes mellitus (DM), bone fractures and CV diseases  were obtained from the patients’ medical histories and analysis of their medical records. The graft function was determined by eGFR according to the CKD-EPI (Chronic Kidney Disease–Epidemiology Collaboration) formula on the basis of serum creatinine concentration assessed during the same visit in routine laboratory tests. The results of serum lipid concentration—total cholesterol (TC), HDL cholesterol and triglycerides (TG)—assessed within the previous 6 months were also taken from the medical records. The LDL cholesterol fraction was calculated using the Friedewald formula . TC and All patients gave informed consent to participate in the study. The study was approved by the Bioethics Committee of the Polish Mother’s Memorial Hospital Research Institute (Decision No 77/2018).p < 0.05. Analyses were performed in the R environment using the following packages: data.table, rstatix, tidyverse, ggpubr, Hmisc, corrplot, PerformanceAnalytics, scales, and gtable.For statistical analysis, the mean, standard deviation, median, minimum, maximum and quartile range were used to analyze the data on dietary intake and food composition. Examination of the distribution of variables was performed using the Shapiro–Wilk test. The nonparametric Mann–Whitney–Wilcoxon test of one sample and for two independent variables and the Kruskal–Wallis test for three or more independent variables were used to compare the data. The nonparametric Wilcoxon test was used as a post-hoc test for significant comparisons with the Kruskal–Wallis test. Spearman’s test was used to observe the correlation of nonparametric variables. Statistical significance was taken as 2, meaning that ultimately 151 patients, 91 men and 60 women, were eligible for the analysis. The mean age of the KTx respondents was 54.4 ± 12.9 years, with mean 9.7 ± 5.6 years after KTx and with the mean eGFR 53.02 ± 18.7 mL/min/1.73 m2. Eight patients had eGFR ≥ 90 mL/min/1.73 m2; 39 recipients 60–89.99 mL/min/1.73 m2; most of the recipients (n = 86) 30–59.99 mL/min/1.73 m2; and 18 recipients 15–29.99 mL/min/1.73 m2. The mean BMI was 26.4 ± 0.6 kg/m2 (18.7–40.8). The patients’ medical statuses were as follows: six patients were after second KTx; all patients had received a kidney from a deceased donor; six patients had a history of CV events, four with myocardial infarction and two with stroke after KTx; 36 recipients had DM, of whom 30 had been diagnosed with post-transplant diabetes (PTDM); 54 KTx patients had bone fractures in their medical history, of which 11 fractures occurred after KTx. Regarding medications, 85 patients were being treated with statins: 70 with atorvastatin, one with lovastatin, eight with rosuvastatin, and six with simvastatin. The mean serum TC was 202.6 ± 43.7 mg/dL, LDL 119.6 ± 34.9 mg/dL, HDL 56.8 ± 13.7 mg/dL, and TG 152.1 ± 70.8 mg/dL. All the patients were treated with antihypertensive medication. Moreover, seven patients were taking vitamin K antagonist (VKA).A total of 154 KTx patients completed the survey. Three patients were excluded because their eGFR was below 15 mL/min/1.73 mBased on the dietary intake analysis, the mean daily intake of vitamin K1 was 120.9 ± 49 μg/day (33.3–436.2 μg/day), whereas that of vitamin K2 was 28.69 ± 11.36 μg/day (2.73–72.21). The intake of particular isoforms of MK are presented in 2.There was no significant difference in vitamin K1 and K2 content in the diets of patients with respect to their graft function , except ± 10.9 vs. 26.2 ± 11.3 μg/day; p = 0.0037), in particular MK-4 . However, no significant differences were observed in association with TG level (p = 0.02), particularly its isoformsMK-4, MK-7 and MK-8, and TC level. A negative association was foundbetween vitamin K2  and MK-4  intake and HDL level. There were no significant correlations between the intake of vitamins K1 and K2 and the serum LDL fraction or TG. All correlations between vitamins K daily intakes and lipid levels are presented in There was a significantly higher dietary intake of vitamin K2 in patients with a normal TC level than with hypercholesterolemia , including MK-4  and MK-10 . On the other hand, patients who did not develop bone fractures consumed more MK-5 . There was also no relationship observed between the intake of vitamins K1 and K2 in patients with CV episodes; however, this group was very small. Similarly, no significant difference was found in the dietary intake of vitamin K between patients treated and those untreated with VKA, however again, the size of the group taking VKA was very small (n = 7). There were no significant differences in vitamin K1 and K2 intake between patients with and without DM. Detailed data are presented in KTx patients with higher BMIs consumed more vitamin K2  recommends an Adequate Intake (AI) of 1 µg/kg/day of PK for all age groups except the neonatal period . EFSA reBeulens et al., over a 10-year follow-up of Dutch men and women, showed a daily vitamin K1 intake of 200 ± 98 µg and a daily K2 intake of 31 ± 7 µg . VitaminBeulens et al. also showed that higher dietary intake of both forms of vitamin K may reduce the risk of DM in the healthy population . The proMultiple studies conducted in the general population show that a high intake of vitamin K1 and K2 has a protective effect on the skeletal system ,49,50,51The daily intake of vitamin K1 in KTx patients living in Poland is adequate, but their diet appears to be poor in vitamin K2. Further studies are needed in order to evaluate proper recommendations for the daily vitamin K1 and K2 intake of KTx recipients, as well as the necessity of its possible supplementation.


The HDL proteome has been widely recognized as an important mediator of HDL function. While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. Here, we compared three of the most common methods for HDL isolation, namely immunoaffinity (IA), density gradient ultracentrifugation (UC), and dextran-sulfate precipitation (DS), in terms of their effects on the HDL proteome and associated functionalities. We used state-of-the-art mass spectrometry to identify 171 proteins across all three isolation methods. IA-HDL contained higher levels of paraoxonase 1, apoB, clusterin, vitronectin, and fibronectin, while UC-HDL had higher levels of apoA2, apoC3, and α-1-antytrypsin. DS-HDL was enriched with apoA4 and complement proteins, while the apoA2 content was very low. Importantly, size-exclusion chromatography analysis showed that IA-HDL isolates contained subspecies in the size range above 12 nm, which were entirely absent in UC-HDL and DS-HDL isolates. Analysis of these subspecies indicated that they primarily consisted of apoA1, IGκC, apoC1, and clusterin. Functional analysis revealed that paraoxonase 1 activity was almost completely lost in IA-HDL, despite high paraoxonase content. We observed that the elution conditions, using 3M thiocyanate, during IA resulted in an almost complete loss of paraoxonase 1 activity. Notably, the cholesterol efflux capacity of UC-HDL and DS-HDL was significantly higher compared to IA-HDL. Together, our data clearly demonstrate that the isolation procedure has a substantial impact on the composition, subclass distribution, and functionality of HDL. In summary, our data show that the isolation procedure has a significant impact on the composition, subclass distribution and functionality of HDL. Our data can be helpful in the comparison, replication and analysis of proteomic datasets of HDL.  Three independent serum pools of six participants each, matched for age and sex, were prepared from the sera of the 18 subjects see . All subg) for 6 h in a 90Ti fixed angle rotor . After centrifugation, the HDL-containing band was collected, desalted via PD10 columns  and either immediately used for experiments or stored with 5% sucrose at −70°C  to 1.24 g/ml, and a two-step density gradient was generated in centrifuge tubes  by layering 3 ml density-adjusted plasma (1.24 g/ml) underneath a KBr-density solution (1.063 g/ml) as described . Tubes wat −70°C .HDL subspecies were isolated from human sera of healthy volunteers with modifications as described , 16. SerWe used a commercial available kit from Cell Biolabs (Nr.: STA-607). The isolation was performed according to the manufacturer’s instructions.2 and 0.49 mM MgCl2, pH 7.4 as running buffer . HDL samples (0.5 mg protein) were loaded with a 0.25 ml loop and were separated with a constant flow of 0.5 ml/min. For HDL2/3 separations, 0.25 ml fraction was collected, and fractions pooled as indicated in NGC QUEST FPLC System  equipped with a Superdex 200 Increase 50/300 column  was used with DPBS containing 0.9 mM CaClFor native gel electrophoresis, isolated HDL (5–15 μg protein per lane) was separated by native gel electrophoresis on 4%–16% gels . Gels were run at constant voltage of 150 V for 120 min. As a high molecular weight marker , containing bovine serum albumin (7.1 nm), lactate dehydrogenase (8.2 nm), B-phycoerythrin , and apoferritin band 2 (18.0 nm) was used to estimate the size of HDL. Afterwards, gels were either stained with a freshly prepared solution of Coomassie Brilliant Blue G-250 overnight (ThermoFisher) or used for blotting. Gels were transferred to polyvinylidene difluoride membranes with an iBlot 2 Dry Blotting System at 100V for 7 min at RT. Membranes were probed blocked with 5 % milk in PBS for 1 hour and incubated with the following primary antibodies diluted in 5 % milk in PBS overnight at 4°C. The list of antibodies used can be found in the supporting information .Membranes were washed carefully for at least three times with wash buffer and incubated with secondary HRP-conjugated antibodies  for 2 h at RT. Membranes were carefully washed and developed using Clarity ECL western reagents . Detection was performed on a Chemidoc Touch imaging system .HDL (5–10 μg) was solubilized in 100 mM ammonium bicarbonate in the presence of 0.2% sodium deoxycholate (Sigma-Aldrich), reduced with 5 mM dithiothreitol (Bio-Rad) and alkylated with iodoacetamide (Bio-Rad). Proteins were digested with trypsin from Promega  for 4 h at 37°C. A second trypsin aliquot was added to the samples  and incubated overnight at 37°C . Digestim/z 200) with AGC target set to 4 × 105, m/z range of 350–1550, and maximum injection time of 50 ms. MS2 resolution was set at 30,000 (at m/z 200) with AGC target of 5 × 104 and maximum injection time of 54 ms. For targeted analysis (DIA), peptides were quantified using Orbitrap resolution of 30,000 (at m/z 200) with AGC target of 5 × 105, precursor m/z range of 400–900, scan range of product ions between m/z 100 and 1000, maximum injection time of 54 ms, and isolation windows of 25 m/z with 0.5 m/z margins.Digested HDL proteins (50 ng) were loaded onto a trap column  and eluted onto a C18 column . Peptides were analyzed using an Easy-nLC 1200 UHPLC system (Thermo Scientific) coupled to an Orbitrap Fusion Lumos (Thermo Scientific) equipped with a nanospray FlexNG ion source (Thermo Scientific) in a 44 min gradient and normalized collision energy of 30 for HCD fragmentation. For untargeted analysis (DDA), peptides were analyzed using MS1 resolution of 120,000  as described (MaxQuant software (version 2.0.1.0) was used to search raw shotgun MS spectra against the human proteome . The criteria for protein detection and quantification included at least two peptides (at least one of them unique), with methionine oxidation and protein escribed . For DIA2+-dependent arylesterase activity was determined with a photometric assay using phenylacetate as previously described. HDL (0.5 μg protein) was added to 200 μl buffer containing 100 mmol/L Tris, 2 mmol/L CaCl2 (pH 8.0), and 1 mmol/L phenylacetate to a 96-well quartz glass plate . The rate of hydrolysis of phenylacetate was monitored by the increase of absorbance at 270 nm, and readings were taken every 15 s at room temperature to generate a kinetic plot. The slope from the kinetic chart was used to determine the increase in fluorescence per minute. Enzymatic activity was calculated with the Beer-Lambert Law from the molar extinction coefficient of 1310 mol1∗L−1∗cm−1 for phenylacetate.Ca3H]-cholesterol efflux was determined by incubating cells for 3 h with 50 μg protein/ml HDL. Cholesterol efflux was expressed as radioactivity in the cell culture supernatant relative to total radioactivity (in the cell culture supernatant and cells) of three independent experiments respectively, measured in duplicates. All steps were performed in the presence of 2 μg/ml of an acyl coenzyme A cholesterol acyltransferase inhibitor (Sandoz 58-035).J774.2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) in the presence of 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were plated on 48-well plates , cultured for 24 h, and loaded with 0.5 μCi/ml radiolabeled [3H]-cholesterol in DMEM supplemented with 2% fetal bovine serum and 1% penicillin/streptomycin in the presence or absence of 0.3 mM 8-(4-chlorophenylthio)-cyclic adenosine monophosphate overnight to induce the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1). After labeling, cells were rinsed with serum-free DMEM containing 1% penicillin/streptomycin and equilibrated with serum-free DMEM containing penicillin/streptomycin and 2 mg/ml bovine serum albumin for 2 h. Subsequently [96W20idf chips  were incubated with 10 mM L-cysteine for 10 min at RT, washed twice with PBS, followed by a coating with 1% gelatine for 30 min at 37°C. The human umbilical vein cell line (Ea.hy926) was maintained in DMEM containing 10% FBS, 1% HAT and 1% Penstrept, seeded at 30.000 per well, and grown to full confluence for two days. Cells were serum-starved prior to experiments and baseline recorded until stable, which was routinely achieved after 1–2 h. Afterward, 100 μg/ml HDL was added, and impedance monitored over a period of 20 h. For quantification, we used the impedance recorded at 4000 Hz at the 10 h time point.t test. Differences between three groups (IA vs. UC vs. DS) were analyzed with one-way ANOVA followed by Bonferroni’s multiple comparison test. Significance was accepted at ∗P < 0.05 and ∗∗P < 0.01. Statistical analyses were performed with GraphPad Prism, Version 6, and SPSS, Version 26. Principal component analysis was performed on Perseus (v.2.0.3.1) with imputation of missing values .Differences between two groups . Interestingly, we found that HDL isolated by IA contained a fraction larger than 12 nm, which was almost entirely absent with the other isolation methods. The distribution between the HDLn DS-HDL . To furtn DS-HDL .2/3 is located in the size range between 7.5 and 12 nm , IGκC (13%), clusterin (10%), apoC1 (9%), and apoC2 (4%) together with a small amount of apoB (0.3%) . The frand 12 nm .Table 3P2/3. The data from HDL2/3 suggests an increase in apoA2 and lower levels of apoA1 in UC-HDL , apoA4 (-73%), α-1-antytrypsin (-71%), apoB (-63%), serotransferrin (-87%), fetuin A (-90%), platelet basic protein (-99%), apo(a) (-88%), afamin (-65%), plasminogen (-90%), and a series of complement proteins (-60%-80%). After SEC separation, the majority of these proteins were either identified in the SEC fraction above 12 nm , apoB, platelet basic protein, plasminogen) or in the SEC fraction below 7.5 nm   , Table 52/3 between ∼7.5 and 12 nm. As expected, the highest abundance of apoA1 was observed throughout the HDL2/3 size range, while lower levels were visible at 5–7.5 nm indicating the presence of pre-β HDL in the isolates (2/3 in IA-HDL and UC-HDL, while lower levels of apoA1 were observed in the smaller HDL3 fraction in DS HDL. This result is consistent with proteomics data (2/3 (2/3 as well as in smaller particles around ∼6.5 nm. α-1-antitrypsin was present in HDL over the entire size range of HDL, with a significant accumulation at around 6.5 nm. Consistent with this observation, data from purification of total HDL into HDL2/3 showed a 70% reduction in α-1-antrypsin content (2/3. Haptoglobin-related protein was detected in HDL from all three isolation methods in a size range of ∼10–13 nm. ApoA-I, apoL1, and haptoglobin-related protein together form the lipid-rich trypanosome lytic factor 1 (2. According to the proteomic data, albumin content was highest in UC-HDL, with the majority detected in the SEC traction below 7.5 nm smaller pre-β-HDL size fraction (2/3. Of note, DS-HDL also contained serum amyloid A in the size fraction below 7.5 nm. Significant amounts of plasminogen were found only in DS-HDL in the size range of HDL3, while only small amounts were present in IA-HDL (2/3 were highest in UC-HDL (To further investigate the distribution of the identified proteins over the entire HDL size range, we performed native gel electrophoresis followed by Western blotting and antibody detection. The dashed line in isolates . ApoA1 dics data  and SEC ics data . Other aics data , 22. Anaics data . This frata (2/3 . Two oth content . This refactor 1 , which ifactor 1 . Westernfraction . Serum an IA-HDL . Westernn UC-HDL . In contn UC-HDL  and S2. n UC-HDL . Since t2/3 might be involved in the reduction of cholesterol efflux capability. Interestingly, DS-HDL was most potent in promoting cholesterol efflux. Our data suggest an increased content of apoA1 together with an increased content of larger HDL2 particles as the prime causes. Paraoxonase 1 is an atheroprotective enzyme that is mainly bound to HDL in the circulation (Next, we investigated whether the compositional differences observed in isolated HDL are linked to altered functionality. Cholesterol efflux was measured with the widely used system using cyclic adenosine monophosphate–treated J774 macrophages . Within culation , 28, 29.culation B. Importculation . We suspculation D. The reculation . We furtculation C. We alsculation D.Fig. 4FWhile a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. In this study, we systematically compared the composition and function of HDL particles isolated by common methods such as IA, UC, and DS. Especially IA chromatography with apoA1-specific antibodies has emerged as an alternative method for isolating HDL because UC alters the composition of lipoproteins , 31. In In the present study, we observed that IA-HDL contained subtypes that were completely absent in UC-HDL and DS-HDL and that differed in composition from the major forms of HDL. This is consistent with previous reports showing that apoA1 can form HDL subclasses with higher size ranges , 33. FurAn interesting observation of the present study was that IA-HDL was less potent in promoting cholesterol efflux and almost completely lost its paraoxonase activity. Remarkably, the prime mediator of cholesterol efflux, apoA1, was more abundant in IA-HDL than in UC-HDL. Therefore, other factors like changes in the subclass distribution and in the lipidation status might be responsible for the impaired cholesterol efflux capacity. For ABCA1-mediated efflux, the size and lipidation status of the cholesterol acceptor is of critical importance, and small and lipid poor forms of HDL are more effective acceptors . We did A further important observation of our study was that DS precipitation yielded HDL distinct of UC-HDL and IA-HDL. This is line with a recent report demonstrating that precipitations reagents can have a significant impact on the compositions and size of isolated HDL . DS does2/3 size range (SEC has been used as an alternative approach for the isolation of HDL , 37. Whize range , 40. Theze range , 40 and ze range . When usIn conclusion, our data are the first to provide an in depth assessment of proteomic features of HDL isolated by UC, IA purification, and DS. We have demonstrated that the use of different isolation methods resulted in the isolation of HDL that was compositionally and functionally distinct. This is of particular importance as especially the use of IA purification gains widespread use for HDL isolation from clinical cohorts , 44, 45.The data supporting this study are available in the article, the supplemental data, or available from the corresponding author upon reasonable request.This article contains The authors declare that they have no conflict of interest.


Torpedo californica, synucleins have been sequenced in all vertebrates, showing differences in the number of genes and splicing isoforms in different taxa. Although α-, β- and γ-synucleins share high homology in the N-terminal sequence, suggesting their evolution from a common ancestor, the three isoforms also differ in molecular characteristics, expression levels and tissue distribution. Moreover, their functions have yet to be fully understood. Great scientific interest on synucleins mainly derives from the involvement of α-synuclein in human neurodegenerative diseases, collectively named synucleinopathies, which involve the accumulation of amyloidogenic α-synuclein inclusions in neurons and glia cells. Studies on synucleinopathies can take advantage of the development of new vertebrate models other than mammals. Moreover, synuclein expression in non-mammalian vertebrates contribute to clarify the physiological role of these proteins in the evolutionary perspective. In this paper, gene expression levels of α-, β- and γ-synucleins have been analysed in the main organs of adult Xenopus laevis by qRT-PCR. Moreover, recombinant α-, β- and γ-synucleins were produced to test the specificity of commercial antibodies against α-synuclein used in Western blot and immunohistochemistry. Finally, the secondary structure of Xenopus synucleins was evaluated by circular dichroism analysis. Results indicate Xenopus as a good model for studying synucleinopathies, and provide a useful background for future studies on synuclein functions and their evolution in vertebrates.The α-, β- and γ-synucleins are small soluble proteins expressed in the nervous system of mammals and evolutionary conserved in vertebrates. After being discovered in the cartilaginous fish  Torpedo californica . P. P® MiniXenopus α-, β- and γ-syn isoforms were cloned BamHI-EcoRI in pGEX-2T to produce GST-fusion proteins. All plasmids were sequence-verified before transformation into E. coli BL21(DE3) cells that were grown in LB medium supplemented with ampicillin to OD600 0.5–0.6 when GST-syn expression was induced with 0.1 mM IPTG at 37 °C for 2–3 h. Cells were harvested and stored frozen at −80 °C until use. Cells were resuspended in lysis buffer  and sonicated to obtain a lysate that was clarified by centrifugation at 20,000× g for 20 min. All GST-syn isoforms were purified on GSH-Sepharose Fast Flow  according to the manufacturer’s instructions. To remove the GST tag, the purified fusion protein was treated with thrombin  for 2 h and repurified on GSH-Sepharose. Syn was recovered in the unbound and wash fractions. The purified protein was concentrated by ultrafiltration with Vivaspin10K filters (Sartorius). Protein content was measured with the microBCA assay and spectrophotometrically. CD spectra were recorded on a Jasco J-810 spectropolarimeter in the range 260–190 nm, with 0.1 cm cuvettes. All spectra are the average of at least 4 scans with buffer subtracted. For CD analyses, the proteins were exchanged in 10 mM potassium phosphate buffer pH 7, containing 50 mM Na2SO4 to remove chloride. Fluorescence spectra were recorded on a Fluoromax Jobin Yvon spectrofluorimeter at 20 °C with a 0.4 × 1 cm cuvette . Excitation was at 270 nm, emission spectra were collected between 280 and 500 nm, and the excitation and emission slit width was 5 nm.Coding sequences for α-, β- and γ-syns were obtained by PCR on an aliquot of the same cDNAs employed for qRT-PCR. Primer sequences are reported in w/v), 8 mM EDTA (v/v) and 50 mM dithiothreitol  [g for 20 min. The protein concentration was determined by the Bradford assay. For SDS-PAGE analysis, proteins were denatured by boiling in Laemmli Sample Buffer for 5 min. Then, 50 μg of protein was loaded in each lane and separated in 15% SDS-polyacrylamide gels according to Laemmli [Samples previously collected and constituted of tissues pooled from 6 animals were homogenized in a denaturing lysis buffer containing 30 mM Tris/HCl (pH 7.4), 1.5% sodium dodecyl sulphate   and prot Laemmli . After e Laemmli . Then me2O2. To avoid the non-specific antibody binding, sections were pre-incubated with normal horse serum  diluted 1:50 in PBST, containing 1% bovine serum albumin . Free-floating sections were then incubated for 5 days at 4 °C with ab27766 antibody  dilution 1:10,000. Sections were then incubated for 1 h at room temperature with biotinylated horse anti-mouse immunoglobulin (Vector Laboratories) and then incubated for 45 minutes at room temperature with avidin-biotin-peroxidase complex  diluted 1:2000 with PBST. The peroxidase activity was evidenced by a reaction with a solution containing 0.04% of 3.3-diaminobenzidine-tetrahydrochloride , 0.4% of nickel ammonium sulphate, and 0.003% of H2O2 in 0.5 M Tris-HCl buffer, pH 7.6 for 3 minutes at room temperature. Some sections were counterstained with the Nuclear Fast Red (Kernechtrot) solution  after the immunohistochemistry procedure. For control experiments, the primary antiserum was substituted with buffer or normal rabbit serum. None of the control sections showed positive immuno-staining. The stained sections were mounted on glass slides maintaining the seriality, dehydrated, cleared, and cover-slipped with Permount .Samples were fixed by immersion in PFA fixative , pH 7 at 4 °C for 24 h, then stored at 4 °C in 0.01 M phosphate buffer (PB) containing 15% of sucrose, embedded in PB containing 10% gelatin and frozen. Samples were frozen and cut on a cryostat  into 30-µm-thick coronal serial sections that were stored until use in 24-well plates containing cold 15% sucrose PB. Sections were enumerated to avoid misplacement, maintaining the seriality. Before immunohistochemical staining, the free-floating sections were treated with 0.01 M phosphate-buffered saline (PBS) containing 0.3% Triton X-100 (PBST) at 4 °C for 2 or 3 days to improve tissue permeability. Sections were pre-treated for 1 h at room temperature with PBST containing 0.1% sodium azide and 0.5% HMicrosoft Power Point and Corel Draw software and BioRender  were useXenopus laevis (The analysis of the sequence, secondary structure, expression and distribution of α-syn in s laevis  suggests