Experimental investigations revealed that the inhibition effectiveness of the investigated inhibitors (INHs) by the Tafel polarization method followed the order: MA-1740 (96.9%) > MA-1615 (95.6%), demonstrating higher efficiency than inhibitors of similar structure reported in the literature. The investigated bithiophene derivatives exhibit mixed-type corrosion inhibition characteristics by blocking the active sites on the surface of C-steel. EIS study revealed that the INHs behave as interface-type corrosion inhibitors. UV–Visible spectrometric measurements confirmed a complex formation between the Fe Despite the large number of ongoing research projects that aimed to design new corrosion resistant materials to cater for different types of industrial applications, C-steel is still the black horse among other metals due to its versatility, cost effectiveness, and excellent mechanical properties. However, one main drawback of using it in industries is that it easily corrodes when exposed to aggressive acids such as hydrochloric acid, which is widely used in oil and gas operations such as pickling, cleaning, and oil-well acidizing3. Nowadays, investigating the inhibitory performance of C-steel corrosion in such acid medium has received a considerable attention in academia and across the industrial domain. Over the last three decades, there have been some studies devoted to the development of efficient and cost-effective corrosion inhibitors. One of the most promising class of inhibitors are those organic compounds with hetero-atoms such as nitrogen, oxygen, sulfur, and phosphorus, which have the ability to adsorb on a metallic surface, and thereby protecting the surface from corrosion attack4. Bi- and oligochalcophenes play an important role as advanced materials, which have been applied into numerous fields and applications. These new class of organic compounds can be used as organic semiconductors and radioactive materials due to their luminescent properties5. Thiophene-containing cationic compounds are important synthetic precursor for biologically active molecules8. These types of compounds have significant use in solvatochromic, photosensitizing, and photovoltaic cells applications12 The idea depends on the presence of donor–(π-spacer)–acceptor in conjugation with each other consequently14. These compounds bear cyano group/or cationic amidine group which act as electron withdrawing acceptor, and electron donating group representing in thiophene ring. Owing to growing concerns on the toxic effects of such corrosion inhibitors, ongoing studies are conducted to develop new compounds which have safe impact on environment. The corrosion inhibition performance of bichalcophene compounds have been previously investigated in literature for C-steel alloys. The maximum %IE results EIS corrosion measurements are summarized in Table 17. The readability and accessibility of certain compounds that occur naturally in our local environment is a major advantage in harnessing their potentials as potential eco-friendly corrosion inhibitors. We aim in this study to advance our work by introducing two novel compounds and bench their corrosion inhibition potency against other bichalcophene compounds that previously reported in literature. This study encompasses synthesis work, experimental studies, surface analysis.Corrosion is commonly defined as the degradation of materials as a result of interaction with the surrounding environment. It occurs to metallic materials when exposed to corrosive environments, causing destructive industrial problems that result in a huge economic loss for many countries around the world. Not only does corrosion cause huge material loss, but also compromise human safety1H-NMR, 19F-NMR, and Mass spectra of inhibitors see the −, so we assume that this counter ion has little effect in the prevailing acid environment of 1.0 M HCl compared to the cationic part.The structure and molecular formula of studied cationic aryl bithiophene/terthiophene derivatives MA-1615, and MA-1740 are given in Table 2 was prepared starting from 4--2-fluorobenzonitrile (1)21, on treatment with NBS in DMF adopting the reported methodology21. Bromobithiophene compound 2 was obtained as a yellow solid in 87% yield, m.p. 170–171 °C (EtOH). Rf = 0.72, petroleum ether-EtOAc (8:2). IR (KBr) ν/cm−1; 3088 , 2230 , 1615, 1555, 1520 . 1H-NMR (DMSO-d6); δ 7.24–7.27 , 7.42 , 7.66 , 7.81 , 7.87–7.94 ppm . MS (EI) m/e (rel.int.); 363, 365 . Anal. Calcd. for: C15H7BrFNS2 (364.25): C, 49.46; H, 1.94; N, 3.85 Found: C, 49.23; H, 2.05; N, 3.77%.Bromobithiophene derivative 3 was prepared adopting a Stille coupling conditions between 4--2-fluorobenzonitrile (2), and 2-tributyltin thiophene adopting the reported methodology21. Compound 3 was obtained as an orange solid in 54% yield, m.p. 211–212 °C (DMF/EtOH). Rf = 0.63, petroleum ether-EtOAc (8:2). IR (KBr) ν/cm−1; 3067 , 2228 , 1612, 1556, 1513 . 1H-NMR (DMSO-d6); δ 7.10–7.12  7.32 , 7.37 , 7.39 , 7.46 , 7.55–7.57 , 7.67 , 7.83 , 7.88–7.95 ppm . MS (EI) m/e (rel.int.); 367 . Anal. Calcd. for C19H10FNS3 (367.48): C, 62.10; H, 2.74; N, 3.81 Found: C, 61.93; H, 2.92; N, 3.76%.Terthienyl compound 4 was prepared by treatment of terthienyl-fluorobenzonitrile compound 3 with lithium bis-trimethylsilylamide adopting reported methodology21. Compound 4 was obtained as a reddish-brown solid in 63% yield, m.p. 292–293 °C. IR (KBr) ν/cm−1; 3389 , 3063 , 1655, 1618, 1525 . 1H-NMR (DMSO-d6); δ 7.09–7.12 , 7.30–7.46 , 7.56 , 7.64–7.72 , 7.80–7.86 , 9.38 , 9.49 ppm . 19F-NMR (DMSO-d6); − δ112.88 ppm . MS (EI) m/e (rel.int.); 384 , 385 , 367 . Anal. Calcd. for C19H13FN2S3-1.0HCl (420.97): C, 54.21; H, 3.35; N, 6.65 Found: C, 54.37; H, 3.42; N, 6.53%.Terthienylbenzamidine compound 2)  and Cu(I)CN  in dry DMF (25 mL) was refluxed at 120–130 °C for 48 h. The reaction mixture was poured onto water/ammonia and extracted with ethyl acetate. The extract was washed with water and brine, dried over Na2SO4, then recrystallized from ethanol/EtOAc to afford the dicarbonitrile compound 5 as a yellow solid in 52% yield, m.p. 221–222.5 °C. Rf = 0.62, petroleum ether-EtOAc (8:2). IR (KBr) ν/cm−1; 3090 , 2212 , 1614, 1551, 1489 . 1H-NMR (DMSO-d6); δ 7.57 , 7.67 , 7.71 , 7.89 , 7.93–7.96 , 7.98 ppm . MS (EI) m/e (rel.int.); 310 . Anal. Calcd. for: C16H7FN2S2 (310.37): C, 61.92; H, 2.27; N, 9.03 Found: C, 62.05; H, 2.38; N, 8.83%.A mixture of 4--2-fluorobenzonitrile  ν/cm−1; 3342, 3187 , 3087 , 1663, 1619, 1523 . 1H-NMR (DMSO-d6); δ 7.65 , 7.66 , 7.73–7.75 , 7.89 , 8.13 , 7.91 , 9.22 , 9.45 , 9.51 , 9.54 ppm . 19F-NMR (DMSO-d6); − δ112.76 ppm . MS (EI) m/e (rel.int.); 344 , 327 , 310 . Anal. Calcd. for C16H13FN4S2-2.0HCl (417.35): C, 46.05; H, 3.62; N, 13.42 Found: C, 45.78; H, 3.78; N, 13.17%.Bithiophene diamidine compound 6 M) to 500 µM (5 × 104 M). Volta lab 80 (Tacussel-Radiometer PGZ-402) was used for electrochemical tests {Potentiodynamic polarization measurements (PP) and Electrochemical impedance spectroscopy (EIS)}. Electrochemical tests were executed by the typical 250 ml sealed electrochemical glass cell containing a finely cut API 5L X70 grade C-steel as working specimen, with an exposed surface area for 1 cm222. Platinum metallic sheet was mounted as the auxiliary electrode, and saturated calomel electrode (SCE) was utilized as the reference electrode. The surface of the working electrode was polished to mirror finish with various grades of emery papers (from 320 to 2000), degreased by acetone, cleaned by demineralized water and finally dried at room temperature before starting the tests. The reference electrode was placed close to the working electrode surface using Luggin–Haper capillary to nullify the iR potential drop between both the electrodes ends. EIS measurements were conducted via AC signal (10 mV) peak to peak, in potentiostatic mode at open-circuit steady state potentials, in the frequency extent between 100 kHz and 20 mHz23. Potentiodynamic polarization (PP) tests were executed using the scan rate at 1 mV s−1 and sweeping the electrode potential ± 300 mV relative to the steady state potential24. To ensure the consistency and accuracy of the results, all tests had been repeated three times.Corrosion tests were conducted on API 5L X70 grade C-steel specimen with the following elemental structure (wt. %): C, 0.026; Mn, 1.51; Si, 0.10; S, 0.02; N, 0.27; Ni, 0.16; Al, 0.35; Cr, 0.27; Cu, 0.28; Nb, 0.93; Ti, 0.11; and remainder Fe. The corrosive electrolyte of 1 M HCl was freshly prepared by dilution of analytical grade HCl with a concentration of 37% using bi-distilled water. The concentration of MA-1615 and MA-1740 inhibitors in corrosive solution ranged from 1 , degreased with acetone, cleaned by demineralized water and finally dried at room temperature before starting the weight loss experiments. After weighing accurately, the specimens were immersed in 250 ml beaker, which contained 150 ml aggressive solution with and without addition of different concentrations of the tested inhibitors. After the required immersion time (48 h), the test specimens were removed, washed with double distilled water, dried by a jet of air and finally weighed. The surface coverage area (θ) for the different concentrations of the investigated inhibitors in 1 M HCl “aggressive solution” and the inhibition efficiency from weight loss, ηw (%) were calculated by the following Eq.  in (mg cm−2 h−1) was calculated by the following Eq.  of inhibitor solution, and the corrosive solution containing (500 µM) of investigated inhibitor after immersion of C-steel sample at 30 °C for 28 h. The spectra were measured using a PG instruments T80+ spectrometer.25.Morphological investigation was carried out by Nanosurf Flex AFM for API X70 type C-steel samples, with dimensions 10 × 10 × 2 mm. The API 5L X70 steel surface was pretreated in the identical way of the working electrode and immersed for 24 h in 1 M HCl solution without and with 0.001 M of MA-1740 at 298 ± 1 K. Samples were collected, and then dried for analysis with AFM4  as a base peak. Structure of the newly synthesized terthienybenzamidine derivative 4 was assigned based on its elemental and spectral analyses. IR spectra of compound 4 indicated the disappearance of nitrile group and displayed new peaks corresponding for N–H stretching vibrations at ν′ 3460, 3389 cm−1. 1H-NMR spectrum of the monocationic compound 4 gave two singlet signals at δ 9.38  and 9.49  of the cationic amidine group, plus five signals integrated for ten protons of terthienyl moiety and 1,3,4-trisubstituted benzene ring. 19F-NMR displayed a singlet signal at − δ112.88 ppm  of fluorine atom. Mass spectrum of terthienyl fluorobenzamidine derivative 4 gave a m/z peak at 384 of its molecular ion peaks (M+), and m/z peak at 385 (M+ + 1) as a base peak, along with a fragment with m/z peak at 367 due to loss of a molecule of ammonia.Structure of the newly synthesized terthienylbenzonitrile derivative 6  with Cu(1)CN in DMF at 120–130 °C to afford the dicarbonitrile derivative 5. Dicarbonitrile derivative 5 was allowed to react with LiN(TMS)2, followed by hydrolysis with hydrogen chloride. The resultant precipitate was neutralized with 1 N NaOH to furnish the corresponding free base of diamidine 6. The diamidine hydrochloride salt 6 was prepared by treatment of its free base of diamidine with hydrogen chloride in ethanol.The preparation of the new bithiophene diamidine 6 Fig.  begins w5 was assigned based on its elemental and spectral analyses. IR spectra of the bithiophene dinitrile 5 indicated the presence of nitrile group with stretching vibrations at 2212 cm−1. 1H-NMR spectrum of compound 5 displayed four doublet signals (one proton each) of bithiophene moiety at δ 7.57, 7.67, 7.89, 7.98 ppm, plus two signals integrated for three protons of 1,3,4-trisubstituted benzene moiety. Mass spectrum of compound 5 gave a molecular ion peak m/z at 310 (M+) as a base peak. The newly synthesized bithienylbenzamidine derivative 6 was assigned based on its elemental and spectral analyses. IR spectra of the diamidine 6 indicated the disappearance of the carbonitrile group and displayed new peaks corresponding for N–H stretching vibrations at ν′ 3342, 3187 cm−1. 1H-NMR spectrum of compound 6 gave four singlet signals at δ 9.22 (2H), 9.45 (2H), 9.51 (2H), 9.54 (2H) characteristic for the cationic diamidine groups, four doublet signals at δ 7.65 (1H), 7.66 (1H), 7.89 (1H), 8.13 (1H) of bithiophene-H′s, plus two signals integrated for three protons of 1,3,4-trisubstituted benzene moiety. 19F-NMR displayed a singlet signal at − δ112.76 ppm  of fluorine atom. Mass spectrum of bithiophene diamidine compound 6 gave a m/z peak at 344 of its molecular ion peak (M+), and m/z peak at 327 (M+- NH3) as a base peak, along with a fragment with m/z peak at 310 due to loss of second molecule of ammonia  of the inhibitors. As shown in Fig. w% values of MA-1156 and MA-1740 are 93.57% and 94.16%, respectively. The inhibition behavior of the cationic aryl bithiophene/terthiophene derivatives can be explained by the adsorption of their molecules on the surface of carbon steel. According to Hammet substituent coefficient, MA-1740 has one more substituent –NH2 group that has negative value of σ (− 0.22) this indicates that capacity of MA-1740 to adsorb on metal surface is higher than MA-1615 as a result of increase in the electron density on the adsorption sites26.Weight loss is a generally used technique for estimating the corrosion inhibition efficiency of inhibitors, and it has high reliability because the average corrosion rate can be determined over time. The corrosion behavior of API 5L X70 steel in 1 M HCl without and with different doses of MA-1740 and MA-1615 was studied by weight loss method. Figure icorr) for the API 5L X70 grade C-steel sample in the acidic medium shows a decreasing trend. This behaviour demonstrates the ability of the cationic aryl bithiophene/terthiophene derivatives to increase in the inhibition efficiencies and to reduce the corrosion rate caused via acidic solution. Looking at the PP plots showed that the anodic and cathodic lines, it is obvious that the curves tend to decline, which is an indication of the adsorption of cationic aryl bithiophene/terthiophene derivatives on the C-steel surface lower cathodic hydrogen evolution and anodic dissolution of Fe ions. Additionally, the difference in the values of Tafel slopes (βa and βc) with different doses of cationic aryl bithiophene/terthiophene derivatives was small, indicating that the cationic aryl bithiophene/terthiophene derivatives hindered the corrosion of API 5L X70 type C-steel via inhibiting both anodic and cathodic reactions, without changing the corrosion process mechanism28. Tafel polarization was carried out at different temperature for the dicationic bithiophene inhibitor MA-1740 at optimum concentration of study as in Fig. ηp) can be calculated by the following equation30:Ecorr) to change to more positive and negative potentials compared with the blank solution. The Ecorr values were significantly less than 85 mV/SCE, indicating that the studied cationic aryl bithiophene/terthiophene derivatives were mixed-type inhibitors33. In addition, the inhibitory activity of cationic aryl bithiophene/terthiophene improved by the presence of lone pairs of electrons and thiophene moiety in these compounds, as well as number of cationic amidine groups. Interestingly, addition of second cationic amidine group improvement outweighs the addition of third thiophene group and this may be attributed to solubility effect of the cationic group. For that reason, cationic aryl bithiophene/terthiophene derivatives can be ordered according to their efficiency as anti-corrosion of API 5L X70-type C-steel in HCl solution as follows: MA-1740 > MA-1615.Tafel plots of immersed API 5L X70 grade C-steel electrode in 1 M HCl without and with different doses of MA-1740 at 25 °C is represented in Fig. 34. The corrosion behaviour of API 5L X70-type C-steel in HCl solution without and with different doses of the cationic aryl bithiophene/terthiophene derivatives was demonstrated by impedance tests. Figures 36. In addition, the Bode curves for the studied inhibitors in Fig. 37.EIS is a widely utilized tool in corrosion inhibition studies of metalsRs), charge transfer resistance (Rct), and the constant phase element (CPE). Cdl is the double-layer capacitor. The CPE impedance (ZCPE) is calculated by the following equations40:j is the imaginary root (j2 = − 1), Y0 represents the modules of CPE, ω represents the angular frequency, n represents the deviation index in terms of a phase shift, 2 fmax represents the frequency at the maximum value of the imaginary component of EIS spectra. The values of 41:ct values is higher when the dose of cationic bithiophene/terthiophene additives increases in test solutions. While we notice drop in the values of Cdl with uplifting the concentration of inhibitors in acid medium. This action can be correlated to the adsorption of studied cationic bithiophene/terthiophene derivatives on C-steel/electrolyte interface that blocks corrosion reactions on C-steel surface and protect the metal from further acid attack42. The data in Table 43.The equivalent circuit for data fitting experiments was presented in Fig. 45. The EIS results revealed that the value of Rct increases gradually with the increase in concentration of the synthesized inhibitors and this indicates an increase in the corrosion inhibition efficiency, which agrees with the potentiodynamic polarization results obtained.In view of the results presented in Table 2) is listed in Table Regression coefficients  and slope of these linear curves were found close to unity, indicating that the adsorption of these cationic aryl bithiophene/terthiophene derivatives on the surface of API 5L X70 type C-steel in 1 M HCl solution follow the Langmuir isotherm. The Gibbs standard free energy 48:R is the gas constant, T represents the absolute temperature, and Kads and Kads indicate the strong adsorption ability of these studied cationic aryl bithiophene/terthiophene derivatives on the surface of API 5L X70 type C-steel in 1 M HCl solution, and then enhanced protection against corrosion. From the results in Table G°ads values of the studied inhibitors ranged from − 33.88 to − 36.13 kJ mol−1, so it is the adsorption process occurs through both physical and chemical adsorption type of interactions51.The inhibition potential of corrosion inhibitor depends on the adsorption behaviour on C-steel surface. To investigate the type adsorption reaction of the cationic aryl bithiophene/terthiophene derivatives on API 5L X70-type C-steel surface, the adsorption isotherms must be investigated. Langmuir adsorption isotherm showed the best fit for the tested cationic aryl bithiophene/terthiophene derivatives that is defined as follows54. In our present work, in order to confirm the possibility of a complex formation between the C-steel electrode and the inhibitor molecules, UV–Vis spectrometric measurements were recorded for (500 µM) of the inhibitor, (500 µM) HCl solution containing (500 µM) of inhibitor after C-steel immersion for 28 h at 30 °C  HCl solution57.According to the literature reports, the influence of corrosion inhibition on metals in the test media in the presence of an inhibitor may be attributed to the complex formation between a metal and an inhibitor °C Fig. a,b. The  °C Fig. a,b show  °C Fig. a,b displRa) obtained for the polished C-steel surface was 38.2 nm and the average surface roughness (Ra) of C-steel surface in 1 M HCl was 467.1 nm while the Ra value in the presence of 0.001 M of MA-1740 was 103.7 nm. These surface roughness values proved that the inhibitor MA-1740 molecules protect the surface of C-steel, most probably by forming a protective film from inhibitor molecules on API 5L X70 C-steel surface59.Atomic force microscope (AFM) analysis is a powerful surface analysis technique to assess the morphological surface changes for the used substrate prior to and after the incorporation of corrosion inhibitors in test solutions. Figure 3. The adsorption of these investigated compounds, on the other hand, can occur chemically onto the steel surface through the high electron density cloud of the π-electrons aromatic cycles that possess S heteroatoms, and the imine group (–C=N–) that facilitate the extension of the double bond conjugation on the whole structure enhancing the electron distribution and a more planar conformation on the substrate surface60. The chemical adsorption is also feasible via the coordinating bonds that can be formed between the lone-paint of electron on the heteroatoms (S) of thiophene rings and the vacant d-orbitals of Fe atoms in API 5L X70 type C-steel surface62. The variation between the two inhibitor molecules MA-1740, and MA-1615 in terms of their chemical structure feature was responsible for their different corrosion inhibition performance, as reported throughout all this study. Examining these structural deviations, the MA-1740 molecule possessed two amidine groups and two thiophene rings, and MA-1615 molecule possessed three thiophene rings and one amidine group. Accordingly, these cationic aryl bithiophene/terthiophene derivatives can build up a protective film on the surface of API 5L X70 type C-steel via physical and chemical reactions, protecting the X70 C-steel from further dissolution.The cationic aryl bithiophene/terthiophene derivatives showed outstanding corrosion inhibitive properties which is attributed to the adsorption of these cationic aryl bithiophene/terthiophene derivatives on API 5L X70 type C-steel surface in 1 M HCl through either physisorption or chemisorption type of interaction as presented in Fig. Two novel cationic aryl bithiophene/terthiophene derivatives MA-1615, MA-1740 were synthesized, characterized by spectral means.The new aryl bithiophene/terthiophene derivatives were investigated as corrosion inhibitors in HCl for API 5L X70 C-steel using electrochemical techniques, which showed higher corrosion inhibition action than similar compounds in structure in literatureThe adsorption of the two investigated cationic inhibitors on API 5L X70 C-steel surface followed Langmuir isotherm and blocking the corrosion reaction sites. The negative sign of Rct and IE % and a decline in the value of Cdl.Tafel curves indicated that the two investigated cationic inhibitors impede the cathodic and anodic reactions simultaneously, and therefore considered as mixed-type inhibitors. EIS measurements indicate that the increase in the inhibitor′s concentration is accompanied with a rise in the values of both AFM pictures confirmed the presence of protective layer on API 5L X70 C-steel surface via the adsorption of the two investigated cationic bithiophene/terthiophene inhibitors. This protective layer protects API 5L X70 steel surface from further acid attack.The presence of dicationic group in MA-1740 has more structural effect on increasing the corrosion inhibition than introducing third thiophene ring.Supplementary Information.


Uveal melanoma (UM) is the most common adult eye cancer. UM originates in the iris, ciliary body or choroid (collectively known as the uvea), in the middle layer of the eye. This first or primary UM is treated by targeting cancer cells using ocular radiation implants or by surgical removal of the eye. However, when UM spreads to the liver and other parts of the body, patients have a poor survival prognosis. Unfortunately, there are no effective treatment options for UM that has spread. Our aim is to help identify effective treatments for UM. In our study, we identified that the drug ACY-1215 prevents the growth of cells derived from UM in the eye and a UM that spread to the liver. Our pre-clinical study uncovered a potential treatment approach for advanced UM.p = 0.0001) dose-dependent reduction in surviving clones of the primary ocular UM cells, Mel270, was observed upon treatment with increasing doses of ACY-1215. Treatment of OMM2.5 MUM cells with ACY-1215 resulted in a significant (p = 0.0001), dose-dependent reduction in cell survival and proliferation in vitro, and in vivo attenuation of primary OMM2.5 xenografts in zebrafish larvae. Furthermore, flow cytometry revealed that ACY-1215 significantly arrested the OMM2.5 cell cycle in S phase (p = 0.0001) following 24 h of treatment, and significant apoptosis was triggered in a time- and dose-dependent manner (p < 0.0001). Additionally, ACY-1215 treatment resulted in a significant reduction in OMM2.5 p-ERK expression levels. Through proteome profiling, the attenuation of the microphthalmia-associated transcription factor (MITF) signaling pathway was linked to the observed anti-cancer effects of ACY-1215. In agreement, pharmacological inhibition of MITF signaling with ML329 significantly reduced OMM2.5 cell survival and viability in vitro (p = 0.0001) and reduced OMM2.5 cells in vivo (p = 0.0006). Our findings provide evidence that ACY-1215 and ML329 are efficacious against growth and survival of OMM2.5 MUM cells.Metastatic uveal melanoma (MUM) is characterized by poor patient survival. Unfortunately, current treatment options demonstrate limited benefits. In this study, we evaluate the efficacy of ACY-1215, a histone deacetylase inhibitor (HDACi), to attenuate growth of primary ocular UM cell lines and, in particular, a liver MUM cell line in vitro and in vivo, and elucidate the underlying molecular mechanisms. A significant ( Uveal melanoma (UM) is the most common adult intraocular cancer, afflicting approximately 4.3 per million people worldwide . AlthougHistone deacetylase inhibitors (HDACi) have garnered widespread interest in the past two decades as anti-cancer agents ,19,20,21In relation to selective HDAC inhibition, histone deacetylase 6 inhibitors (HDAC6i) have shown promise as anti-cancer agents in pre-clinical studies and are currently under clinical trial investigations as a monotherapy or combinatorial therapy for lymphoproliferative disease, hematologic malignancies and solid tumors ,28,29,30Here, we investigated the efficacy of ACY-1215 in inhibiting growth of a primary ocular UM cell line and then focused on ACY-1215 effects on the viability and growth of the OMM2.5 MUM cell line and the underlying molecular mechanisms. ACY-1215 significantly attenuated growth of the MUM cell line, OMM2.5, and this effect correlated with reduced levels of microphthalmia-associated transcription factor (MITF).p = 0.0001) decreases at 5, 10, 20 and 50 µM concentration of ACY-1215, respectively, compared to vehicle control (0.5% DMSO) (p = 0.0001) decreases when treated at 5, 10, 20 and 50 µM, respectively, compared to vehicle control (p = 0.095) or OMM2.5  cells  for 96 h. We observed a significant, dose-dependent reduction in OMM2.5 cell viability when treated at 20 µM  and 50 µM  ACY-1215, compared to vehicle control (p = 0.0001) reduction in OMM2.5 cell viability was observed when treated with 15% H2O2. There was no significant difference in cell viability observed when treated with 0.5, 1, 5 and 10 µM ACY-1215. From the MTT assay, the IC50 for ACY-1215 in OMM2.5 cells was calculated to be 6.51 µM  were selected to determine their efficacy in reducing long-term proliferation of human UM cell lines derived from primary ocular UM (Mel285 and Mel270) and a metastatic liver (OMM2.5) UM ,36,37. C5% DMSO) B,C. Simi control B,D. Pati4) cells B–D. AddiTT assay E,F. OMM2 control E. Simila 6.51 µM F. As ourp < 0.0001) with 20 µM ACY-1215 treatment compared to vehicle controls  and 24  hours post treatment (hpt) with 20 µM ACY-1215 compared to 0.5% DMSO-treated OMM2.5 cells, confirming the inhibitory effects of ACY-1215 , Cox proportional hazards models and Kaplan–Meier survival curves were generated. Kaplan–Meier survival curves were generated with a cut-off of 50% to demarcate a high or low HDAC6 expression, and the Log-rank test was used to compare survival probability between the groups. Interestingly, high HDAC6 transcript expression was significantly associated with better overall survival, but not with progression-free survival   C.50 of ACY-1215 in an enzymatic-based assay is 4.7 nM, at which ACY-1215 acts as a highly potent and selective HDAC6 inhibitor ..50 valuer assay) A.p = 0.005) when treated with 0.05 µM ML329 treatment compared to 0.5% DMSO (p = 0.0001) decreases at 0.1 to 50 µM concentration of ML329, compared to vehicle controls (p = 0.0001) in the number of surviving clones, compared to 0.5% DMSO. Given that MITF was found to play a role in OMM2.5 cell survival, correlations between MITF expression and UM patient OS/PFS was investigated. Curiously, high or low MITF expression levels were not significantly associated with better OS nor PFS , as shown by the Kaplan–Meier survival curves  reduction in OMM2.5 primary xenograft cell fluorescence was detected after normalization, following treatment with 1.25 µM ML329 compared to vehicle controls  . Althoug50 value . As befocontrols A,C. ExpeMetastatic UM (MUM) is a poor prognosis cancer, lacking effective treatment options. Our study has provided evidence that small molecule drugs ACY-1215 and ML329 are efficacious in conferring anti-cancer effects in a MUM cell line, both in vitro and in vivo. To the best of our knowledge, this is the first study to provide evidence linking the HDACi ACY-1215 and MITF in OMM2.5 MUM cells.Three commercially available, first-generation HDAC6i were screened in UM and MUM cell lines, and ACY-1215 was selected for follow-up studies. ACY-1215, either as a monotherapy or in combination with other drugs, is presently in clinical trials for several cancers ,49. We o50 value for ACY-1215 in enzymatic assays is 4.7 nM. In OMM2.5 cells, we do not observe a significant reduction in cell survival and viability at ACY-1215 treatment concentrations of less than 5 μM; therefore, we postulate that using lower concentrations of ACY-1215 that are within the selective range for HDAC6 inhibition will not offer the desired treatment benefits in this cell line. Importantly, HDAC6 was indeed inhibited by ACY-1215 at the concentration we used, as its substrate, acetylated α-tubulin, was significantly upregulated. Furthermore, from our proteomics data we also identified proteins involved in microtubule polymerization and regulation of microtubule polymerization or depolymerization to be significantly altered [However, pure HDAC6 inhibition mediated effects must be inferred with caution, as higher doses of ACY-1215 result in non-selective inhibition, and the observed beneficial effects are mediated by additional targets ,56. In a altered . IrrespeProteome profiling of ACY-1215-treated OMM2.5 cells was key in deducing potential mechanisms of action. We discovered that the MITF signaling pathway and associated factors were significantly downregulated upon treatment with 20 μM ACY-1215, given our treatment regime. Tying in with the concentration of ACY-1215 used, our findings are in line with another study, whereby it was reported that treatment of melanoma and clear cell sarcoma cells with different pan-HDAC inhibitors resulted in reduced MITF expression in vitro and in vivo in a mouse melanoma xenograft model .2 phases, suppressed cell migration and invasion in vitro and tumor formation in vivo; an opposite effect was observed when MITF was overexpressed [The role of MITF has been extensively studied in cutaneous melanoma ,61,62. Mxpressed . In the xpressed . In our 2/M phase and an increase in apoptosis [0/G1 phase and a time-/dose-dependent proapoptotic effects of ACY-1215 uncovered in lymphoma cell lines [GNAQ/GNA11 mutations are associated with constitutive activation of the MAPK/ERK signaling pathway, although heterogeneity in MAPK/ERK signaling has been observed across UM samples with GNAQ/GNA11 mutations [GNAQ, which is known to result in constitutively active MAPK/ERK signaling in UM [Q209 mutations; hence, these cell lines represent a large cohort of UM tumors [Significantly, several studies have independently shown that ACY-1215 regulates cell cycle and cell death mechanisms in various cancers. In HCT-116 and HT29 colorectal cancer cells, a reduction in cell proliferation and viability was noted in a time- and dose-dependent manner, and apoptosis was also observed at non-selective ACY-1215 concentrations ,77. Intepoptosis . Similarll lines . More rell lines . Collectll lines ,82. Furtll lines ,84,85. Ill lines . In A375ll lines . Interesll lines . Howeverll lines . This inutations ,91,92,93ng in UM . ReporteM tumors ,96.HDAC6 expression and OS probability, highlighting a possible involvement of HDAC6 in UM prognosis. Moreover, HDAC2 and SIRT2 expression correlated with OS, while HDAC4 expression showed correlations to PFS. HDAC 1 and 3 expression was not correlated with either OS or PFS, even though HDAC 1, 2, 3, 4 and Sirtuin 2 (SIRT2) expression was detected in UM eye samples [Going forward, it will be worthwhile to evaluate potential synergistic effects between ACY-1215 and Dacarbazine or MITF/MEK inhibitors in MUM cells, given that many studies are reporting increased benefits with combinatorial treatment regimes. Although promising, the role of ACY-1215 and ML329 needs to be thoroughly investigated in UM and MUM patient samples. Currently, there is no clear evidence linking either HDAC6/Class I/II HDAC or MITF in MUM prognosis. Immunohistochemistry-based expression analysis of 16 primary UM samples detected variable low levels of HDAC6 expression, with no correlation between HDAC6 expression levels and UM in this limited sample size . Based o samples . A limit samples ,98. Comp samples . Taken tThis study highlights that ACY-1215 regulates the survival of primary and metastatic UM cell lines and provides evidence that this involves the regulation of MITF in OMM2.5 cells. Our data suggest that ACY-1215- and ML329-related signaling pathways offer novel options for identifying therapeutic targets for the treatment of MUM, which needs to be considered and further evaluated.2.Mel270, Mel285 and OMM2.5 cells  were cultured with complete media containing RPMI 1640 with GlutaMAX™ Supplement  + 10% fetal bovine serum  + 2% penicillin–streptomycin (PEST) in T175 flasks for no more than 20 passages . ARPE-193 cells/mL  and allowed to adhere overnight. Initial drug screens were performed with Mel285 cells seeded at 1.5 × 103 cells/mL, and Mel270/OMM2.5 cells were seeded at 9 × 103 cells/mL. The following day, cells were treated with either 0.5% DMSO (vehicle control) or 20 µM Dacarbazine  or HDACi , ACY-1215 (SelleckChem) and Tubacin (Sigma-Aldrich)) at increasing doses, ranging from 1 µM, 5 µM, 10 µM, 20 µM and 50 µM, prepared in complete media. MITF inhibitor ML329  was tested at increasing concentrations, ranging from 0.05 µM, 0.1 µM, 0.25 µM, 0.5 µM, 1 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM, 20 µM and 50 µM. All drugs were dissolved in DMSO to prepare stock solutions. Cells were treated with 2 mL of desired drug solution per well in duplicate and incubated at 37 °C with 5% CO2 for 96 h. Drug solutions were removed, and wells were washed twice with 1 × phosphate-buffered saline . Fresh complete media were added to the plates, and cells were allowed to grow for an additional 10 days at 37 °C with 5% CO2. Clones were washed twice and fixed with 4% paraformaldehyde/formaldehyde for 10 min at room temperature (RT). Clones were stained with 0.5% crystal violet solution  for 10 min–2 h at RT, shaken, washed and dried (once desired staining was achieved). Plates were imaged using the GelCount™ system  and analyzed using the ColonyCountJ Plugin  in ImageJ v1.53e [p value of < 0.05 was considered statistically significant. Experiments were performed in triplicates/quadruplicates.All three cell lines were seeded into 6-well plates at 1.5 × 10J v1.53e . Statist2O2 or 0.5, 1, 5, 10, 20 and 50 µM ACY-1215 (SelleckChem), prepared in complete media for 96 h. Drug solution was removed, MTT -2,5-diphenyltetrazolium bromide) dye and serum-free media were added in a 1:10 ratio to each well and incubated in the dark for 2.5 h at 37 °C. Following this, 100% DMSO (1:1 ratio) was added to each well, and absorbance was measured at 570 nm using a SpectraMax® M2 microplate reader . One-way ANOVA with Dunnett’s Test for Multiple Comparisons statistical analysis IC50 value for ACY-1215 was calculated using GraphPad Prism v8.00 .Effect of ACY-1215 on OMM2.5 cell viability was determined using 3--2,5-diphenyltetrazolium bromide) MTT assay, as described previously . BrieflyTg(fli1a:EGFP) background were raised in embryo media containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM MgCl2, 0.33 mM CaCl2 and 0.003% phenylthiourea (PTU), in a Petri dish at 28.5 °C incubator. Adult Tg(fli1a:EGFP) zebrafish were maintained in a 14 h light/10 h dark cycle in a recirculating water system at 28 °C. OMM2.5 cells were prepared for transplantation, as described in a previously published report [TM, Tritech Research; Los Angeles, CA, USA) into the perivitelline space of 2-day-old Tg(fli1a:EGFP) zebrafish larvae under anesthesia . Larvae were imaged using a fluorescent microscope  for red fluorescence and placed individually into 48-well plates. Only larvae with tumor cells correctly implanted in the perivitelline space were included in the study. Larvae (0 days post treatment (dpt) were treated with either 0.5% DMSO, 20 µM ACY-1215 , 20 µM Dacarbazine  or 1.25 µM ML329 for 3 days at 35 °C and imaged at both perivitelline space and caudal vein plexus post treatment (25–32 pooled larvae were used per treatment group). Differences in transplanted OMM2.5 Dil-labeled cells’ primary fluorescence between 0 dpt and 3 dpt were measured, normalized and calculated using ImageJ. Before drug treatment, toxicity assays were performed with either 0.5% DMSO, ACY-1215, Dacarbazine or ML329 (ranges from 1–100 µM). A total of 8 larvae (4 larvae/well) per treatment group were exposed to the desired concentration of drug solutions for 3 days in 24/48-well plates at 35 °C and imaged at 3 dpt. One-way ANOVA with Dunnett’s Test for Multiple Comparisons or Student’s T test statistical analyses were performed using GraphPad Prism v7.00 .Zebrafish rearing and husbandry were performed in accordance with ethical regulations of the Linköping Animal Research Ethics Committee. Only larval, and not animal forms, of zebrafish were used in the study Zebrafish embryos/larvae from d report . OMM2.5 6 cells per well and drug treated for 4 or 24 h with 0.5% DMSO or 20 µM ACY-1215, in duplicate (N = 4). Protein was isolated using PreOmics iST for protein/proteomics preparation kit , according to manufacturer’s protocol. Mass spectrometry and bioinformatic analysis of samples were performed as described previously [Homo sapiens database (2020_05) containing 75,074 entries [p value of ≤ 0.05) was performed using ClueGo (v2.5.8) [Homo sapiens (9606) marker set. GO: Biological Process functional pathway databases, consisting of 18,058 genes, were used. GO tree levels  and GO term restriction  were set, and terms were grouped using a Kappa Score Threshold of 0.4. The classification was performed by two-sided hypergeometric enrichment test, and its probability value was corrected by the Benjamini–Hochberg method .OMM2.5 cells were seeded at a density of 1 × 10eviously . Slight  entries . Pathway(v2.5.8)  and Clue(v2.5.8)  plugins (v2.5.8) , with th6 or 4 × 105, and immunoblotting was performed as described [Protein was isolated from Mel270, Mel285, OMM2.5 and ARPE19 cells at a cell density of 1 × 10escribed . For val2. Cells were trypsinized and filtered through 50 µm CellTrics filter. Live cells were labeled sequentially with YO-PRO™-1 Iodide  for 15 min and Propidium Iodide  for 3 min, in the dark, at RT, to analyze apoptotic events. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol at 4 °C. After being fixed, cells were labeled with 1.25 µL of 1 mg/mL PI stock and co-treated with 2.5 µL of 10 mg/mL RNase A enzyme (ThermoFisher Scientific) for 30 min at RT, in the dark. All samples were run on a BD AccuriTM C6 Flow Cytometer , and up to 50,000 events were recorded per sample (N = 3–4). YO-PRO™-1 Iodide and PI were excited using a 488 nm laser, and its fluorescence was collected using FL-1 channel (B530/30 band pass filter) and FL-3 channel (B675LP band pass filter), respectively. For cell cycle analysis, PI was excited using a 488 nm laser and its fluorescence collected using FL-2 channel (575/25 band pass filter). The collected samples were gated based on controls (DMSO/Etoposide) and preliminarily analyzed using CFlow Plus Software . Reanalysis was performed using FCS ExpressTM De Novo (Research Edition) v6 software. The instrument was calibrated with manufacturer’s specifications prior to use. Two-way ANOVA followed by Tukey’s Multiple Comparison test or Dunnett’s Test for Multiple Comparisons statistical analyses were performed using GraphPad Prism v7.00 . Detailed information on flow cytometry experiments and analysis performed are provided in A total of 300,000 OMM2.5 cells were seeded and treated with 0.5% DMSO, 50 µM Etoposide , 10, 20 and 50 µM ACY-1215 or 20 µM Dacarbazine, in duplicate (N = 3–4) for 4, 24 and 96 h at 37 °C with 5% COSurvival analyses were performed with package “survminer”, R v3.5.0 . Gene expression and clinical data from 80 primary UM included in The Cancer Genome Atlas (TCGA) were collected from the cBioPortal. RNA-seq data were downloaded in Fragments Per Kilobase of exon per million fragments Mapped (FPKM) and then converted to log2 scale. The associations between gene expression and prognosis were assessed by Cox proportional hazard regression models. Progression-Free Survival (PFS) and Overall Survival (OS) were used as end points. For categorization of the gene expression into “High” and “Low” categories, median values were used as cut-off. Survival probabilities were plotted on a Kaplan–Meier curve, and a Log-rank test was used to compare the two groups. Progression-free survival is defined as time until metastatic recurrence. Overall survival is defined as death by any cause.This research provides evidence that ACY-1215 and ML329 should be further investigated to establish their potential as treatment option(s) for MUM. Specifically, this study proves the efficacy of ACY-1215 as an anti-cancer agent for MUM cells, OMM2.5, in vitro and in vivo. We have additionally elucidated that ACY-1215 treatment reduces MITF expression in OMM2.5 cells.


Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn2+, Co2+, and Fe2+ increased the alginate degrading activity, but it was inhibited in the presence of Zn2+ and Cu2+. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium,  Alginate is the most abundant polysaccharide located in the matrix and in the cell wall of brown seaweed. It is composed of α-L-guluronate (G) and β-D-mannuronate (M) as the major monomeric units . These uAscophyllum nodosum and 25 to 44% for Laminaria digitata 9.5 kDa) , and AlgStreptomyces sp. AVP053U2 database, with 99% confidence (Proteinpilot v4.5). The peptide sequences of length 12–26 amino were converted to DNA sequence by using a codon usage table for the G + C rich Streptomyces [Streptomyces luridiscabiei using Geneious software. After obtaining the full alginate lyase gene sequences, the complete amino acid sequences were found by using the translation tool ExPASy  to convert DNA to protein. The result showed that the AlyDS44 alginate lyase gene was composed of 780 bp , encoding 260 amino acid residues. Each of the 10 peptides sequenced had 100% identity with the genetic sequence.The purified AlyDS44 band was subjected to nanospray qTOF mass spectrometry  analysis, and 10 peptides were matched against the ptomyces . The algVibrio spp. and Streptomyces sp. ALG5, which have different substrate specificity, were compared with the identified amino acid sequences of the alginate lyase from strain DS44 to reveal the regions of similar sequences. The multiple sequence alignment of the catalytic domain of AlyDS44  families, namely the PL5, PL6, PL7, PL14, PL15, PL17, PL18, PL31, PL32, PL34, PL36, and PL 39 families, based on the similarity of protein sequences and structural features ,25,26. T AlyDS44  indicate regions . Otherwiubstrate ,39. The p. ALG-5 , which dThe negative-ion electrospray ionization mass spectra of hydrolysis products of alginate by AlyDS44 is shown in After degrading for 24 h at 37 °C, the main AlyDS44 degradation products were determined as disaccharides, trisaccharides, and tetrasaccharides. The results verified that AlyDS44 alginate lyases cleaved the glycosidic bonds in alginate by β-elimination reaction and also indicated that this enzyme is an endo-type alginate lyase ,32.The maximum activity of AlyDS44 was obtained at pH 8.5 in 0.02 M Tris-HCl buffer a. GreateMicrobulbifer sp. ALW1, Marinimicrobium sp. H1, and Shewannella sp. YHI [Sphingomonas sp. MJ-3 and Microbulbifer sp. 6532A, which have an optimal temperature of 50 °C [There are significant differences in terms of the optimal temperatures of alginate lyases . The opt sp. YHI ,32,40, bof 50 °C .Vibrio harveyi AL-28 [AlyDS44 alginate lyase was active in the presence of a wide concentration range of NaCl (0.2–1 M) and showed a highest activity of 125% in 0.6 M NaCl. Interestingly, AlyDS44 could maintain a high activity at a concentration of 1 M NaCl, which was similar to AlyAL-28 lyase extracted from yi AL-28 . Therefoyi AL-28 ,32,43. T2+, Co2+, and Fe2+ could enhance the alginate degradation activity of AlyDS44. These results were similar to reports of M3 lyase and alginate lyase from Streptomyces spp. [Isoptericola halotolerans CGMCC 5336 [2+ and Co2+, with increases of 242% and 219% relative activity, respectively. Ca2+ and Mg2+ had no effect on the enzyme activities [2+ and Cu2+, while Fe3+ showed a slight inhibitory effect, as observed for other alginate lyases [As shown in ces spp.  and the MCC 5336 . Surpristivities . In conte lyases ,38,46. MMicrobulbifer sp. ALW1 [Vibrio sp. NJ-04 [Marinimicrobium sp. H1 [AlyDS44 exhibited the highest activity toward sodium alginate . Furthersp. ALW1 , marine p. NJ-04 , and Marm sp. H1 . The resFor the control samples, 0.5% of sodium alginate, PolyM, and PolyG in 0.02 M Tris-HCl buffer were used. The degradation ability of AlyDS44 is shown in After 24 h incubation at 37 °C, degradation products that had smaller sizes, of 18.7 KDa (15.2%), 6.6 KDa (15.3%), and 3.4 KDa (35%), were detected. These results indicate that AlyDS44 worked effectively on the sodium alginate substrate and degraded it into oligosaccharides of approximately 30-times-lower molecular weight. In comparison to sodium alginate, PolyM and PolyG had lower initial molecular weights of 17.7 KDa (79%) and 14 KDa (80%), respectively. After the commencement of incubation (1 h), their chains were cut into smaller sizes of 15.8 KDa (65.5%) for PolyM and 10.9 KDa (65.8%) for PolyG. They were subsequently broken down to MW sizes of 9.8 KDa (54.7%) for PolyM and 3.1 KDa (51%) for PolyG after 24 h incubation. Moreover, the smallest molecular weight of degradation products detected from the reaction between AlyDS44 and PolyG substrate was 0.5 KDa, which accounted for 34.5% of the total. Therefore, it was concluded that AlyDS44 possesses strong activity against alginate substrate and could degrade PolyG more effectively than PolyM.Durvillaea potatorum (bull kelp), which belongs to the brown seaweed group. Bull kelp was collected at Rivoli Bay , Beachport, South Australia and was used as a carbon source for the growth of actinobacteria and as a substrate for producing alginate lyase.The seaweed used in this study was Streptomyces strain DS44 grown on HPDA plates for at least 7 days were inoculated into 50 mL of IM22 inoculum medium  in 250 mL Erlenmeyer flasks and then cultured at 27 °C, with shaking at 150 rpm. After three days, the inoculum was transferred to 50 mL production medium in 250 mL Erlenmeyer flasks at 5% (v/v). The production medium was prepared by adding 2% bull kelp powder (prepared by grinding and sieving) to 5 g/L peptone, 1 g/L yeast extract, 1 g/L glucose, 20 g/L NaCl, 2 g/L K2HPO4, 0.2 g/L MgSO4.7 H2O, and 5 g/L NH4Cl. The medium was sterilized by autoclaving at 121 °C for 15 min. After inoculation, the flasks were incubated at 27 °C on a rotary shaker at 150 rpm for 7 days.Two loopfuls of spore mass of isolated Total genomic DNA of strain DS44 was isolated using a modified CTAB (hexadecyl trimethyl ammonium bromide) protocol based on the method described by Doyle and Doyle . DNA quag for 20 min at 4 °C. The protein was then precipitated from the supernatant by stepwise ammonium sulfate addition, and the crude enzyme was collected at 60% saturation overnight at 4 °C. The solution was centrifuged at 10,000× g at 4 °C for 15 min. The precipitate was collected and dissolved in 20 mM Tris-HCl buffer. It was dialyzed by using dialysis tubing  in 1 L of the same buffer at 4 °C for desalting. The buffer was changed every two hours for 6 h. After dialysis, the supernatant was used as a partially purified enzyme for testing enzyme activity.The broth cultures were centrifuged at 10,000× TM column, 5/50 GL, GE Healthcare/Cytiva, Marlborough, MA, USA) attached to a FPLC . The column had been previously equilibrated with buffer A (20 mM Tris-HCl pH 8.0). The pump was run at a flow rate of 1 mL/min. The enzyme was eluted with a linear gradient of 0–1 M NaCl in buffer B . The fractions obtained from anion exchange chromatography were evaluated for enzyme activity and protein concentration using a Pierce BCA Protein Assay Kit . The fractions possessing the highest specific enzyme activity were pooled and kept in the freezer at −20 °C before loading onto a SuperdexTM 75 column . The concentrated pooled fraction (1 mL) was loaded onto the column and elution was performed with 20 mM Tris HCl (pH 8.0) buffer containing 0.1 M NaCl at a flow rate of 0.5 mL/min. The fractions (1 mL) obtained from the size exclusion column were evaluated for their enzyme activity and protein concentration. The fractions with a high specific enzyme activity were stored at −20 °C and subsequently used for characterization studies.The concentrated enzyme was applied to an anion exchange column  reagent . A reactThe relative activity was calculated as the percentage ratio of activity at a given pH/temperature to the activity at optimum pH/temperature.Statistical analysis: all results are based on an average of three experiments. The results were analyzed statistically by one-way ANOVA in SPSS.TM TGX Stain-FreeTM Precast Gel  was used for running SDS-PAGE. Reference proteins (5 uL) of Precision Plus Protein Unstained Standards , which are a mixture of ten Strep-tagged, recombinant proteins (10–250 kD), including three reference bands , were used as molecular mass markers. Next, 5 µL of 4× loading buffer was added to 15µL of sample, then the mixture was boiled at 95 °C for two minutes, centrifuged briefly and loaded onto a 4–20% stain free gel . The gel was run at 300 V for approximately 20 min using a criterion tank (Bio-Rad) containing 1× running buffer. After electrophoresis, the gel was placed on a Stain Free Tray and the gel was imaged using automated exposure by a Gel Doc EZ imager  to visualize protein bands.The apparent molecular weight of AlyDS44 was determined by SDS-Polyacrylamide Gel Electrophoresis. A 4–20% Criterionwww.sciex.com, accessed on 6 October 2021) using protein mode against the Streptomyces amino acid database (UniProt).The AlyDS44 band was excised from the gel, washed, reduced/alkylated, and digested with Trypsin, as described previously . AlginatVibrio sp. QY101 (Genbank: AAP45155.1), AlyVOA from Vibrio sp. O2 (Genbank: ABB36771.1), A9mT from Vibrio sp. A9m (Genbank: BAH79132.1), AlgNJU-03 from Vibrio sp. NJU-03 (ASA33933.1), and ALG5 from Streptomyces sp. ALG5 (Genbank: ABS59291.1) were used for multiple sequence alignment with AlyDS44.The matched peptide sequences were analyzed using Clustal Omega (EMBL-EBI) to identify sequences with homology to reported alginate lyases. Amino acid sequences of alginate lyase AlyVI from Electrospray-ionization mass spectroscopy  was used to analyze the mass/charge ratio of the alginate lyase degradation products. Samples were run in negative ionization mode. A reaction containing 0.5% sodium alginate in 0.02 M Tris-HCl buffer (pH 8.0) was incubated with purified enzyme at 37 °C for 24 h. Samples were diluted in 1:50 methanol and then injected into electrospray. Mass calibration was performed via injection of sodium chloride. Mass range of the scans was from m/z 150 to 2000.The reaction solution containing purified enzyme (100 µL) and substrate  with a range of pH values  was incubated for 2 h at 37 °C. The enzyme activity was measured by the DNS assay.The purified enzyme was incubated with the substrate  in 0.02 M Tris-HCl buffer at 15 °C, 25 °C, 35 °C, 45 °C, 65 °C, and 75 °C for 2 h.The purified enzymes were incubated with the substrate  in 0.02 M Tris-HCl buffer at NaCl concentrations of 0.2 M, 0.4 M, 0.6 M, 0.8 M, and 1.0 M for 2 h at 37 °C.3, CoCl2, CuSO4, FeSO4, FeCl3, MgSO4, MnCl2, ZnCl2) was incubated for 2 h at 37 °C.The reaction mixture containing purified enzyme and substrate  in 0.02 M Tris-HCl buffer and 0.2 mL of 5 mM the appropriate salt , and polyguluronic (PolyG), were used. Each reaction contained 100 µL each of purified enzyme and 500 µL 0.5% of each substrate in 0.02 M Tris-HCl buffer (pH 8.0). They were incubated at 37 °C for 2 h and then enzyme activity was evaluated by DNS assay.g for 5 min and then 100 µL of sample was placed into an HPLC vial. A 50 µL sample was injected and run through the column with a flow rate of 1.0 mL/min. The peaks were detected using a refractive index (RI) detector.To identify alginate hydrolysis ability of AlyDS44, the molecular weight of hydrolysis products was determined by HPLC  with a GPC/SEC column: PL aquagel-OH Mixed-H 8 um 300 × 7.5 mm (6 kDa–10 MDa) and PL aquagel-OH 30 8 µm 300 × 7.5 mm (0.1–60 kDa). The mobile phase was 0.1 M sodium nitrate (isocratic). The standards and tested samples were dissolved in mobile phase. For preparing the sample, 100 µL of purified enzyme was incubated with 500 µL of substrate, including 0.5% sodium alginate, 0.5% PolyM, and 0.5% PolyG in 0.02 M Tris-HCl buffer (pH 8.0) at 37 °C. Then, 100 µL of sample was taken at 1 h and 24 h incubation and diluted with 100 µL of 0.1 M sodium nitrate. The sample was centrifuged at 19,000× 2+, Co2+, and Fe2+ and inhibited by Zn2+ and Cu2+. Finally, the amino acid sequence of DS44 showed more than 10% difference in amino acid residues from a closely related alginate lyase from Streptomyces sp. ALG-5, which demonstrates that AlyDS44 from Streptomyces luridiscabiei is a novel enzyme. To sum up, AlyDS44 is a candidate to be an industrial enzyme for the efficient production of alginate oligosaccharides.In this study, the purification and characterization of a novel alginate lyase AlyDS44 was accomplished. The successful purification of the alginate lyases was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and size exclusion chromatography. AlyDS44 has a molecular weight of 28.6 KDa. The alginate lyase gene was composed of 780 bp, encoding 260 amino acid residues. AlyDS44 could degrade both PolyM and PolyG, indicating that it is bifunctional alginate lyase; however, it preferentially degraded PolyG. This characteristic suggests that AlyDS44 has the potential to produce alginate oligosaccharides with lower degrees of polymerization. Moreover, based on the highly conserved regions of their amino acid sequences, this enzyme was classified to the PL7 family. AlyDS44 could hydrolyze sodium alginate to produce alginate disaccharides and trisaccharides as the main end products, indicating that it was an endo-type alginate lyase. Furthermore, this enzyme showed the highest activity at pH 8.5, when the degradation reaction was performed at 45 °C. AlyDS44 also exhibited an unchanged activity at high concentrations of NaCl, which led it to be grouped into salt-tolerant alginate lyases, isolated from marine bacteria. AlyDS44 was more active in the presence of Mn


Fridericia chica (Bignoniaceae) is a traditional medicinal plant. The aim of this research was to determine the protective effects of the hydroethanolic extract from the F. chica leaves (HEFc) against the cytotoxicity of zearalenone (α-ZEL) and β-ZEL on SH-SY5Y cells. Free radical scavenging activity of HEFc was evaluated using the DPPH method. The cytotoxicity of both zearalenone metabolites and HEFc was examined using MTT test, as was the cytoprotective effects of the HEFc on cells treated with these mycotoxins. The chemical composition of HEFc was determined using UPLC-QTOF-MS/MS. HEFc elicited good DPPH radical scavenging activity following a concentration-dependent relationship. Cells exposed to α-ZEL exhibited a viability ˂50% after 48 h of treatment (25 and 50 µM), while those exposed to β-ZEL showed viability ˂50% (100 µM) and ˂25% (25-100 µM) after 24 and 48 h of exposure, respectively. HEFc showed a significant increase in cell viability after exposure to α-ZEL (25 and 50 µM) and β-ZEL (6–100 µM) (p < 0.05). UPLC-QTOF-MS/MS analyses allowed the identification of 10 phytochemical components in the HEFc. In short, the hydroethanolic extract of F. chica grown in Colombian Caribbean can protect against the effects of mycotoxins and it is a valuable source of compounds with antioxidant properties. Fridericia chica (Bonpl.) L.G. Lohmann, Bignoniaceae, which is known as “limpia diente” and grows frequently in tropical regions in South America and Africa.Colombia is one of the countries in the world that is considered to be mega-diverse due to its various ecosystems . BioprosFridericia chica (synonym Arrabidaea chica) leaves are usually employed as a red dye, in fact, it has traditionally been used by Sinu artisans of the Colombian Caribbean to make the vueltiao hat [F. chica comes from anthocyanidins, a class of phenolic compounds with known antioxidant properties [F. chica, such as isoscutellarein, 6-hydroxyluteolin, hispidulin, scutellarein, luteolin, apigenin, and hispidulin [tiao hat . The redoperties , and extoperties , antiprooperties , wound hoperties , antispaoperties , photoproperties , and leioperties  activitispidulin , this laspidulin .Fusarium and infect mainly cereals and other plant products [Exposure to environmental toxins is key in the development of neuropathological diseases . Within products . The toxproducts . In addiproducts ,17. In fproducts  and granproducts .F. chica (HEFc) grown in northern Colombia to mitigate the effects of α-ZEL and β-ZEL in undifferentiated human neuroblastoma, as well as to characterize its constituents.The human neuroblastoma dopaminergic neuronal cell line SH-SY5Y has been used as an in vitro model for numerous neurotoxicity and cytoprotection experiments. The aim of this work was to investigate the potential of a hydroethanolic extract of 50 value was found to be 709 µg/mL.The results of the free radical scavenging activity of the HEFc are presented in 50 values for SH-SY5Y cells at 24 and 48 h were 61.2 µg/mL (45 to 83 µg/mL) and 53.8 µg/mL (24.0 to 116.6 µg/mL), respectively  for α-ZEL and 10.5 µM (CI95: 7.1 to 15.7 µM) for β-ZEL (The cytotoxicity of α-ZEL and β-ZEL in undifferentiated human neuroblastoma cells are displayed in or β-ZEL .p < 0.05) after the first 24 h of incubation. HEFc (16 µg/mL) significantly increased in cell proliferation in samples with low concentrations of α-ZEL  after treatment for 48 h. At the highest α-ZEL concentrations tested (25–50 µM), simultaneous co-exposure with the extract was able to significantly increase cell viability (p < 0.05) by 18–21%.The protective effects of HEFc against cytotoxicity in SH-SY5Y cells induced by α-ZEL and β-ZEL after 24 and 48 h exposures, are shown in The treatment with HEFc (16 µg/mL) in undifferentiated human neuroblastoma cells exposed for 24 h with β-ZEL only displayed some protection in viability (~20%) at the highest tested mycotoxin concentration (100 µM) tested. After prolonged exposure (48 h), HEFc was able to significantly recover cell viability at an extent similar to that observed after 24 h (~20%); however, the concentration range displaying the effect was greater (12.5–100 μM) .The results of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) analysis of the HEFc are shown in F. chica (HEFc) obtained from fallen leaves collected from trees grown in the Colombian Caribbean, the protective effects of the extract on zearalenone metabolites-induced citotoxicity in SH-SY5Y cells, and its chemical composition.This study evaluated the free radical scavenging activity of a hydroethanolic extract of F. chica is a plant widely known for its anti-inflammatory [50 of the extract was 709 µg/mL, a value much than that reported in extracts obtained from leaves of the same species from Brazil (IC50 of 13.5 µg/mL) [ammatory ,5, antibammatory ,21 healiammatory , and antammatory ,23. Howe5 µg/mL)  or Argen5 µg/mL) ,25.F. chica have been previously reported [50 found here after 24 and 48 h treatment (61.2 and 53.8 µg/mL), were moderately higher than the IC50 values (<30 µg/mL) suggested as a criteria to extract promising agents for anticancer drug development [50, 26.9 µg/mL), and Jurkat cells  [Although some biological properties of reported ,25; the elopment . Howeverls IC50, .9 µg/mL 9 µg/mL) .50 after 48 h of exposure. These results are similar to those obtained by other authors [50 values after 48 h of exposure. In the present study, it was found that a concentration of 17.9 µM (α-ZEL) and 10.5 µM (β-ZEL) was required to reach the IC50 values in SH-SY5Y cells, while other types of cell lines appear less sensitive  [Zearalenone metabolites at high concentrations produce an increase in cell death after the first 24 h of treatment, finally reaching values below the IC authors ,28. Howe1 cells) . It is k1 cells) . In this1 cells) .The brain is also a target for estrogens and phytoestrogens, such as ZEA or its metabolites, as they are known to cross the blood-brain barrier . AlthougThe characterization of HEFc showed that some of the tentatively identified compounds has been reported to have potential anti-osteoporotic, anti-inflammatory, antiplatelet, anticonvulsant, and anticancer ,34. FlavF. chica leaves exerts significant protective effects against mycotoxin-induced cytotoxicity in undifferen-tiated human neuroblastoma cells. This may be due to the presence of compounds with antioxidant properties present in the extract. These results may contribute to new approaches for treatment against the effects of mycotoxins.The present study demonstrates that hydroethanolic extract from the F. chica (Bonpl.) L.G. Lohmann, Bignoniaceae were collected between January and December 2018 in the municipality of Sincelejo (9°14′20″ N-75°25′17″ W), department of Sucre . The specimens were identified by Pedro Alvarez Perez in the Herbarium of the University of Sucre , and a voucher specimen was deposited and registered in this herbarium (004537). Leaves of F. chica were dried at room temperature. Subsequently, dried leaves were cut and powder (30 g) was mixed with  with 70% ethanol in water for 4 h at 200 rpm using an automated Shaker. The extract was filtered and the solvent evaporated in an oven at 70 °C under a hood. The extract was a black reddish solid, and was stored at −20 °C until analysis.The fallen leaves of DPPH radical scavenging assay was determined using the kit from Bioquochem . Initially, a stock solution at 100,000 µg/mL of the hydroethanolic extract from the HEFc with MilliQ water was prepared. All extract samples were tested in duplicate at seven different concentrations , then, sample extracts (20 μL) were reacted with 200 μL of the DPPH solution in the dark at room temperature. The absorbance at 517 nm was performed in the Varioskan™ LUX Multimode Microplate Reader . The percentage of inhibition of the radical DPPH•+ for each standard point was obtained with the following formula:R standard curve was used to determine TEAC  of the tested concentrations of extracts. The calculation was performed using the following formula:A Trolox® CRL-2266™) were maintained following the methods previously reported [6 cells/plate) for 24 h. Briefly, the cells were exposed to 100 μL of medium containing different concentrations of α-ZEL (0.4 to 50 μM), β-ZEL (0.4 to 100 μM) and HEFc (4 to 1000 µg/mL) individually for 24 and 48 h. For all cases, the viability was examined using MTT test (50 using Prism 6.0 (GraphPad Software Inc.).SH-SY5Y NB cells  for 24 h at 37 °C in a 5% CO2 atmosphere and these were exposed to two independent treatment combinations. The first consisted of a mixture of HEFc (16 µg/mL) with eigth concentrations of α-ZEL , for the second, the combination of HEFc (16 µg/mL) and nine concentrations of ZEN metabolites  for 24 and 48 h were tested. The plates were incubated for 24 and 48 h at 37 °C, in a 5% CO2 atmosphere and the viability was examined by MTT assay . The supernatants were transferred to auto sampler vials. Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) was utilized to separate and characterize the components. Chromatographic separation was performed using an Agilent 1290 UPLC system  employing a YMC Carotenoid column   with a mobile phase flow rate of 0.3 mL/min, where the mobile phase A and B were 0.1% HCOOH acid in ddH2O and acetonitrile, respectively. Starting conditions were 95:5 A: B, held for 1 min, followed by a linear gradient to 5:95 at 12 min, with a hold to 15 min. Column re-equilibration was accomplished by returning to 95:5 A:B at 16 min and holding until 21 min. The mass analysis was acquired with an Agilent 6545 Q-TOF MS  with ESI capillary voltage +3.5 kV, N2 gas temperature 320 °C, drying gas flow rate 8.0 L/min, nebulizer gas pressure 35 psig, fragmentor voltage 135 V, skimmer 65 V, and OCT RF 750 V. Mass spectral data were gathered in profile mode. Mass accuracy was enhanced by infusing Agilent Reference Mass Correction Solution (G1969-85001). The instrument was operated from 100 to 1200 m/z at a scan rate of 2 spectra/s. MS data scans were obtained using Agilent MassHunter Acquisition software (v. B.06). MS/MS was achieved in a data-dependent acquisition mode on composite samples. Peak deconvolution and integration was executed using Agilent ProFinder (v. B.06). Peak annotations were carried out with METLIN (metlin.scripps.edu) metabolite databases, with a mass error around 1 ppm. Identifications were supported by MS/MS spectra comparisons.The HEFc was diluted 1/10 in 50% acetonitrile mixed with 0.1% HCOOH. Samples were sonicated for 5 min and then centrifuged at 16,000 50 values of HEFc and ZEN metabolites were analyzed using a non-linear sigmoid curve fit. Statistical comparison was performed using one-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test. In addition, multiple Student t-test was conducted to investigate different mean cell viabillity between mixture (extract and metabolite) and metabolite alone. Results were considered significant at p < 0.05.The data are presented as mean±SEM and analyzed statistically by GraphPad Prism 8.0 . The IC


Primary open-angle glaucoma is established by the disruption of trabecular meshwork (TM) function. The disruption leads to increased resistance to the aqueous humor (AH), generated by the non-pigmented ciliary epithelium (NPCE). Extracellular vesicles (EVs) participate in the communication between the NPCE and the TM tissue in the ocular drainage system. The potential use of NPCE-derived EVs to deliver siRNA to TM cells has scarcely been explored. NPCE-derived EVs were isolated and loaded with anti-fibrotic (SMAD7) siRNA. EV’s structural integrity and siRNA loading efficiency were estimated via electron microscopy and fluorescence. Engineered EVs were added to pre-cultured TM cells and qRT-PCR was used to verify the transfer of selected siRNA to the cells. Western blot analysis was used to evaluate the qualitative effects on Wnt-TGFβ2 proteins’ expression. EVs loaded with exogenous siRNA achieved a 53% mRNA knockdown of SMAD7 in TM cells, resulting in a significant elevation in the levels of β-Catenin, pGSK3β, N-Cadherin, K-Cadherin, and TGFβ2 proteins in TM cells. NPCE-derived EVs can be used for efficient siRNA molecule delivery into TM cells, which may prove to be beneficial as a therapeutic target to lower intraocular pressure (IOP). Primary open-angle glaucoma (POAG), the predominant form of glaucoma ,2, is a In-vivo studies on fibronectin matrix remodeling showed that cadherin-mediated cell-cell contact modifies tissue tension and integrin-mediated cell-matrix interactions . TM cellThe cytokine TGFβ2 is an immunosuppressive factor found in elevated levels in glaucomatous patients  and may Electroporation applies an electrical current to the phospholipid bilayer of the exosomes, leading to the formation of temporary pores. Although electroporation leads to beneficial loading of siRNA over chemical transfection, it might lead to exosome instability, reducing the loading capacity. To ensure that the tested parameters for achieving maximal siRNA loading into the EVs did not lead to the structural instability of the NPCE-derived vesicles, we used TRPS technology and Cryo-TEM analysis. 12/mL concentration of NPCE EVs following the detailed conditions for electroporation of condition 1 were closest to the values of the non-electroporated EVs’ control group (with a size of 76.67 ± 11.06 nm and concentration of 1.38 × 1012/mL). For the other three groups (Conditions 2–4), a trend of decreases in EVs’ size and concentration were observed compared to the control group. Values are the mean of three measurements for each condition. Characterization of NPCE EVs was previously done using exosomes markers: CD81, Alix, and TSG101, as was determined by and Exo-Check analysis and Western blot analysis [The size and concentration of EVs following four different electroporation protocols as well as untreated EVs as control were measured using Tunable Resistive Pulse Sensing (TRPS) technology. Our analysis indicated that the most suitable conditions for electroporation of EVs are 400 v, 125 µF, and one electric pulse  to electroporated TM cells (3.146 ± 1.315) indicated that the electric pulse itself contributed to gene activation. Electroporation of non-targeting siRNA to TM cells did not cause a change in SMAD7 mRNA levels compared to untreated TM cells. Data were normalized to the average mRNA level of GAPDH .6 TM cells. A significant decrease (*** p < 0.001) in the mRNA levels of TM cells incubated with NPCE EVs loaded with siRNA against SMAD7 (p < 0.05) in mRNA levels of treated TM cells with NPCE EVs compared to untreated ones was seen for the target gene SMAD7 (p < 0.05) in mRNA levels of treated TM cells with NPCE EVs compared to untreated ones, was seen for the target gene SMAD7  in p-GSK3β (p < 0.001) (p < 0.001) in N-Cadherin (p < 0.05). In addition, in TM cells incubated with NPCE EVs loaded with SMAD7 siRNA, a significant increase (*** p < 0.001) in pGSK3β levels was found compared to untreated TM cells  was determined A. A sign p-GSK3β C and β-C< 0.001) D expressCadherin G, β-CateCadherin D, p-GSK3Cadherin C, GSK3β Cadherin B, and TGCadherin E levels Cadherin F in treaTM cells C. No difDespite the available treatments for POAG disease, glaucoma is still the primary cause for irreversible blindness worldwide with no preventive treatment . Recentlp < 0.001) silencing of the SMAD7 gene in TM cells  cell lines [g. Two hundred milliliters of supernatant were collected and transferred to a 200 mL medium: containing 2 mM L-glutamine, 0.1 mg/mL streptomycin, and 100 units/mL of penicillin. EV-depleted serum was used along with all experiments.Cells were cultured according to previously published conditions . Brieflyll lines  were useTo examine whether the attenuation of the Wnt-TGFβ2 signaling pathway in TM cells may be established, NPCE EVs were loaded with SMAD7 siRNA, using a Gene Pulser II System  based on Alvarez-Erviti L., and Momen-Heravi, F. publications ,65. NPCEg for 10 min, 2000× g for 10 min, and 10,000× g for 30 min. NPCE EVs were then pelleted at 100,000× g for 70 min and washed with PBS 0.1M, pH = 7.2, and recovered at 100,000× g for 70 min. The pelleted EVs were suspended in PBS 0.1M, pH = 7.2, and stored at −80 °C until use.EVs isolation was achieved by a series of centrifugations at 4 °C . Briefly13 beads/mL  were used to calibrate size and concentration, following the manufacturer’s instructions. Samples were diluted 1000-fold with PBS 0.1 M buffer, pH 7.2, and measured up to 10 min. The movement of the particle through the membrane is identified as a change in the ionic stream causing current changes. The signal power is proportional to the particle size. According to the number of particles and their velocity at a specific time, the qNano determines EV’s sizes and concentration.To determine the optimal EVs concentration for electroporation, and to exclude alternations in EVs size or amount following the electric pulse, EVs at two different concentrations (0.25 µg/µL and 0.5 µg/µL), chosen based on literature, were mixed with non-targeting siRNA (1nmole/mL) and electroporated with one or two electric pulses. Samples containing EVs without an electric pulse were used as a negative control. To eliminate the excess siRNA, the EVs were treated with 1 unit of RNAse H. Before TRPS analysis, EVs samples were passed through 0.22 µm filters to eliminate contaminating debris. Then, NPCE EVs’ size and concentration were determined by the qNano  instrument, using the Tunable Resistive Pulse Sensing (TRPS) technology with an NP150 membrane (85–300 nm) . The app7 particles/2.5 μL PBS 0.1M, pH = 7.2, were directly adsorbed onto glow-discharged carbon grids with holes blotted at 95% humidity and plunged into liquid ethane. Samples were imaged at liquid nitrogen temperature using a Tecnai 12 TWIN TEM transmission Cryo-electron microscope, operating at 200 kV . A minimum of 50 digital images was taken for each group, using a cooled slow-scan CCD camera . Normal or damaged-shaped EVs were evaluated as a percentage of the total particle population in each image.Evaluation of NPCE EVs membrane integrity following electroporation was conducted using Cryo-transmission electron microscopy (TEM) analysis. Untreated EVs, and four conditions of electroporated EVs in one of two concentrations (0.5 µg/µL vs. 0.25 µg/µL), and a different number of pulses (1 vs. 2), were examined. From each condition, EVs in a concentration of 10g for 70 min at 4 °C to remove the unincorporated dye [9 EVs/mL).Purified NPCE-derived EVs were labeled with 2 mM of DiD; a lipophilic fluorescent dye  at concentration of 2.5 µL/mL PBS 0.1M, pH = 7.2, for confocal microscopy. EVs were incubated for 10 min with the DiD solution at room temperature to ensure uniform labeling. After staining, labeled EVs were suspended in PBS  and pelleted by ultracentrifugation at 100,000× ated dye . The fing at 4 °C to remove unbound siRNA and RNAse H residues, as will be described later in more detail. Pellets were re-suspended in 200 µL PBS, introduced to 96 wells plate, and siRNA fluorescence was determined using a fluorescence infinite m200 (TECAN) device reader (Infinite 200) to calculate the percentage of encapsulation compared to a sample containing EVs without the electric pulse used as a negative control.The encapsulation efficacy of siRNA in EVs was evaluated as follow; 10 μg of NPCE EVs were mixed with 10μg of siGLO green transfection indicator  in a total volume of 400 µL holding 10µg EVs in PBS 0.1 M, 10 µg siRNA, 50 mM Trehalose and 1 mM EDTA). Samples were electroporated in 0.4 cm cuvettes with aluminum electrodes using a Gene Pulser II System . Samples were diluted 10-fold with PBS 0.1 M and centrifuged for 70 min at 100,000× 9/mL) were mixed with 5 µL of Fluromount-G mounting media , and a drop of 8 µL was transferred to each coverslip. Detection of siGLO and EVs membrane was performed using an FV1000-IX81 confocal microscope  equipped with a 60x objective. Ten pictures from 3 different slides for each treatment were captured and analyzed by MacBiophotonics Image J software (Version 1.53k14).The percentage of encapsulated siRNAs into NPCE EVs following electroporation was assessed by confocal microscopy. NPCE electroporated EVs loaded with 10 μg siGLO transfection indicator and non-electroporated EVs were labeled with DiD at a final concentration of 5 mg/mL PBS for membrane staining. A total of 4 µL of labeled EVs  to 0.8 × 106 TM cells by electroporation, using a Gene Pulser II System . Suitable conditions of 170 v, 2500 µF, 85–140msec, and one pulse were used for electroporation. Post-electroporation, 300 µL of TM cells loaded with SMAD7 siRNA were transferred at once to 6-well sterile plates. Each well-contained 950 µL of 20% DMEM, pre-heated medium (37 °C) was added to reach a final concentration of 500 nM siRNA/1.25 mL medium/well. Twenty-four hours later, the growth medium was replaced with fresh medium contains 20% (vol/vol) FBS. To reach maximal effect, 100% confluent TM cells were harvested 48 h post-incubation. mRNA levels were determined by qRT-PCR analysis to estimate whether the attenuation of SMAD7 gene expression was achieved. Untreated TM cells, TM cells + non-targeting siRNA, electroporated TM cells without siRNA, and three different sequences of siRNA target gene were examined.Prior to electroporation of NPCE EVs with siRNA, a validation of the effectiveness of loading siRNA sequence for targeting SMAD7 in TM cells was done based on work done by Wei Xue et al. . Briefly9 EVs/mL PBS 0.1M, pH = 7.2) and 10 μg siRNA (1:1 w/w). EVs were suspended in PBS 0.1M, pH = 7.2 with a supplement of 50 mM Trehalose  and 1 mM EDTA (Sigma-Aldrich). Pre-electroporation tubes containing EVs and siRNA were incubated for 10 min on ice. Suitable conditions of 400 v, 125 µF, 10–15 msec, and one pulse were used for electroporation. After electroporation, all cuvettes were incubated on ice for 30 min. A total of 1 unit of RNAse H was added to each treatment for 5 min at 37 °C to eliminate aggregates formation in the solution. After 20 min, the activity of RNAse H was neutralized by 1 µL of 0.5 M EDTA/400 µL sample. Then, solutions of electroporated or non-electroporated EVs were diluted with PBS 0.1 M, pH = 7.2 to a final volume of 30 mL and were ultra-centrifuged using Beckman Coulter ultracentrifugation rotor SW28, for 70 min, at 4 °C and 100,000× g to exclude siRNA or EVs debris and RNase H residues. Pellets were suspended in 100 µLPBS 0.1 M + 1% w/v BSA and were added to TM cells by treatments. Untreated TM cells, TM cells + NPCE EVs (non-electroporated) and TM cells + NPCE EVs loaded with non-targeting siRNA  were used as control in all experiments.NPCE-derived EVs were suspended in 0.4 cm cuvettes with aluminum electrodes using a Gene Pulser II System . Human SMAD7 , Stealth siRNA, 20 nmole, Invitrogen, Hercules CA, USA) was examined. For every electroporation, the sample volume was fixed at 400 μL, containing 10 μg EVs  in 6-well plates. Twenty-four hours post-electroporation, cell viability was assessed by Trypan Blue (Sigma-Aldrich) uptake. All samples were assayed in triplicates, and the viability of each treatment was compared to non-electroporated TM cells.Trypan Blue protocol  was used6 cells in 1 mL/well) to reach a confluence of 2 × 106 cells on the day of the experiment. Twenty-four hours later, the growth medium was replaced with fresh EV-depleted medium. 100% confluent TM cells were exposed for an additional 24 h to electroporated NPCE EVs with SMAD7 siRNA to examine the effects of engineered EVs on the signal transfer in TM cells. TM cells incubated with electroporated EVs or EVs without the electric pulse and TM alone were used as controls.TM cells were seeded in 6-well sterile plates  according to the manufacturer’s instructions. RNA quality and quantity were assessed at 260 nm using a NanoDrop2000 Spectrophotometer . Equal amounts (1 µg) of RNA were reverse transcribed in triplicates using Quanta bio qScript cDNA Synthesis kit . Changes in mRNA levels of SMAD7, TGFβ2, GSK-3β, and β-Catenin genes were determined by qRT-PCR with an Applied Biosystems Real-Time PCR QuantStudio™ 1 System (Version 1.5.1), using Syber Green Master Mix . The human GAPDH gene was used as endogenous control. The mRNA levels were calculated using QuantStudio Design & Analysis Software . All primers were purchased from IDT , and their sequences are detailed in 2, 1% Triton-X100, and 10 μL of protease phosphatase inhibitors per 1mL buffer. After incubation on ice for 45 min, the lysates were sonicated for 15 min (40% Amplitude without pulses and), centrifuged at 12,000× g at 4 °C, and the supernatant was collected. Supernatants were analyzed for protein concentration using the Bradford assay. Absorbance was recorded on a microplate reader at 595 nm .Following incubation of TM cells with electroporated or non-electroporated NPCE-derived EVs with siRNAs and TM with no treatment as control, TM cells were lysed as established in our earlier works . BrieflyFor the evaluation of the expression, levels of Wnt-TGFβ2 signaling pathway proteins, 20 μg of protein extracted from TM cells for each sample were mixed with Laemmli buffer  containing 0.1% β-mercaptoethanol, boiled for 5 min at 95 °C and separated on a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane, blocked in 5% BSA with 0.5% Tween 20, then probed overnight with primary antibodies to detect and analyze the expression levels of pGSK-3β , GSK-3β , β-Catenin , TGF-β2 , K-Cadherin  and N-Cadherin . Membranes were incubated for 1hr at room temperature with anti-mouse secondary antibodies  against GSK-3β, N-Cadherin, and TGF-β2 or anti-rabbit secondary antibodies  against pGSK-3β, β-Catenin, and K-Cadherin. β-Actin levels were determined using anti-β-Actin . Immune complexes were detected with chemiluminescence reagent , followed by exposure to Kodak X-ray film . Semi-quantitative analysis was carried out for all Western blot experiments using a computerized image analysis system, MacBiophotonics ImageJ software (Version 1.53k14).p < 0.05.Data are presented as mean ± standard deviation. Statistical evaluation of one-way ANOVA was performed with GraphPad Prism version 5 software . Differences between groups were tested using Tukey’s test. Two-way ANOVA and Bonferroni multiple comparisons were used to determine a statistical difference in the siRNA fluorescence measurements. Student t-test was performed to determine changes in the cell viability comparison. All tests were considered significant at * NPCE-derived EVs, electroporated with SMAD7 siRNA were able to modify TM cells Wnt-TGFβ2 signaling pathway in vitro. These results may be beneficial as a potential approach for medical intervention to treat POAG patients. NPCE-derived EVs, electroporated with SMAD7 siRNA were able to modify TM cells Wnt-TGFβ2 signaling pathway in vitro. These results, aimed at intervening in the AH drainage resistance—the main cause of intraocular pressure. In the present study, we demonstrated a specific delivery of siRNA against SMAD7 gene into TM cells through targeted NPCE EVs that modulated genes and proteins expression related to WNT-TGFβ2 signaling pathway pressure increase, offer a new way of future intervention that requires further in-vivo research in the TM-SC pathway. Charging exosomes with specific siRNAs can be used as local biological therapy subject to the development of an effective form of administration, in addition to or as an alternative to existing therapy.


High-resolution mass spectrometry, such as Fourier-transform mass spectrometry (FTMS), is capable of resolving Type-II overlap depending on mass resolving power. In this work, we evaluated FTMS quantification accuracy of lipid species affected by Type-II overlap. Spike experiments with lipid species pairs of various lipid classes were analyzed by flow injection analysis-FTMS. Accuracy of quantification was evaluated without and with Type-II correction (using relative isotope abundance) as well as utilizing the first isotopic peak (M+1). Isobaric peaks, which were sufficiently resolved, were most accurate without Type-II correction. In cases of partially resolved peaks, we observed peak interference causing distortions in mass and intensity, which is a well-described phenomenon in FTMS. Concentrations of respective species were more accurate when calculated from M+1. Moreover, some minor species, affected by considerable Type-II overlap, could only be quantified by M+1. Unexpectedly, even completely unresolved peaks were substantially overcorrected by Type-II correction because of peak interference. The described method was validated including intraday and interday precisions for human serum and fibroblast samples. Taken together, our results show that accurate quantification of lipid species by FTMS requires resolution-depended data analysis.Lipidomics data require consideration of ions with near-identical masses, which comprises among others the Type-II isotopic overlap. This overlap occurs in series of lipid species differing only by number of double bonds (DBs) mainly because of the natural abundance of  The overlap of the monoisotopic peak (MDB=i+0) with the second isotopic peak of a species of the same lipid class and number of C-atoms but with one additional DB (MDB=i+1+2) is termed Type-II isotopic effect  , 12, 13.In this work, we evaluated the accuracy of quantification of lipid species affected by Type-II overlap by flow injection analysis (FIA)-FTMS. Concentration derived from three different data processing strategies, i.e., without and with Type-II correction and application of M+1 were evaluated. Finally, a workflow depending on mass resolving power was proposed to minimize peak interference effects as far as possible.Chloroform and 2-propanol were purchased from Roth  and methanol from Merck . All solvents were HPLC grade. Ammonium formate and cholesteryl ester (CE) standards were purchased from Sigma-Aldrich . Triglyceride (TG) and diglyceride (DG) standards were purchased from Larodan  and dissolved in 2,2,4-trimethylpentane/2-propanol (3:1 v/v). Phosphatidylcholine (PC), ceramide (Cer), sphingomyelin (SM), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE) standards were purchased from Avanti Polar Lipids  and dissolved in chloroform.2 in a humidified incubator at 37°C as described previously , LPC 18:0 (0.18 pmol/μl), DG 36:0 (0.18 pmol/μl), CE 18:0 (0.18 pmol/μl), SM 36:1;2 (0.18 pmol/μl), Cer 42:1;2 (0.018 pmol/μl), and TG 54:0 (0.18 pmol/μl). The mixture “M1” was composed of the corresponding species of the same lipid class with exactly one additional DB: LPE 18:1 (0.018 pmol/μl), LPC 18:1 (0.18 pmol/μl), DG 36:1 (0.18 pmol/μl), CE 18:1 (0.18 pmol/μl), SM 36:2;2 (0.18 pmol/μl), Cer 42:2;2 (0.018 pmol/μl), and TG 54:1 (0.18 pmol/μl). A volume of 25 μl of both mixtures subjected to lipid extraction, corresponding to 0.018 pmol/μl (0.1 nmol added to lipid extraction) LPE and Cer, and 0.18 pmol/μl (1 nmol added to lipid extraction) LPC, DG, CE, SM, and TG was defined as ratio M1/M0 = 1:1. Samples were analyzed with a constant amount of the M0 mixture and variable amounts of the M1 mixture. Concentrations of the stock solutions were adjusted by FTMS quantification using the respective internal standards. The internal standard mixture used for quantification was composed of CE 17:0 (0.7 pmol/μl), Cer 32:1;2 (0.005 pmol/μl), DG 28:0 (0.11 pmol/μl), LPC 13:0 (0.15 pmol/μl), LPE 13:0 (0.011 pmol/μl), PC 28:0 (0.23 pmol/μl), SM 30:1;2 (0.007 pmol/μl), and TG 51:0 (0.26 pmol/μl). The concentration of the infusate was calculated by dividing the amount added to the extraction in nmol by a factor of 5.598 derived from dilution during sample preparation.Samples were spiked with internal standards before lipid extraction (solvent of standards was removed by vacuum centrifugation). A serum amount of 10 μl, cell homogenate containing 100 μg of protein, or tissue homogenates containing a wet weight of 2 mg were subjected to extraction. The samples were extracted according to the procedure described by Bligh and Dyer  with a tm/z range 500–1,000 for 1 min. Negative ion mode FTMS data were recorded in m/z range 400–650 for LPE ([M–H]−) and LPC ([M+HCOO]−) and m/z range 520–960 for Cer, SM, and PC quantification (as [M+HCOO]−). All experiments used a maximum injection time of 200 ms, an automated gain control (AGC) of 1 × 106, three microscans, and a target resolution of 140,000 (at m/z 200). Changes in resolution and AGC are indicated in the corresponding figures.Lipid quantification was performed by direct flow injection on a hybrid quadrupole-Orbitrap mass spectrometer QExactive  equipped with a heated electrospray ionization source and a standard-sized Orbitrap. The ion source was operated using the following settings: spray voltage of 3.5 kV, S-lens RF level 50, capillary temperature of 250°C, aux gas heater temperature of 100°C, and settings of 15 for sheath gas and 5 for aux gas. Enhanced Fourier-transform was applied for signal processing . All datm/z 470–1,030 using profile mode, a max injection time of 100 ms, AGC of 1 × 105, three microscans, and a target resolution setting of 480,000 and 120,000. Negative ion mode was performed in a second injection in scan range m/z 360–675 and m/z 530–1,080 for 2 min each using the parameters as described before.Samples were infused with the robotic nanoflow ion source TriVersa NanoMate  connected to an Orbitrap Fusion Tribrid equipped with a high-field Orbitrap analyzer  as described previously . Full scm/z-tolerance set to ±0.0045 for intensity picking. The extracted data were exported to Microsoft Excel 2016 and further processed by self-programmed Macros. For the isotope Type-II effect  at m/z 200]. In Orbitrap mass analyzers, mass resolution R is inversely proportional to the square root of m/z  led to a substantial overcorrection , SM 36:2;2 (m/z 775.5971) and to a lesser extent even for TG 54:0 (m/z 908.8641) but not for LPC 18:0 (m/z 568.3620)  .Fig. 4Lo6 to 5 × 104 did not affect the quantification for all compared correction algorithms . Here, concentrations calculated after Type-II correction were more accurate at lower mass resolution arguing for a reduction of interference effects at lower resolutions , sufficient peak resolution and therefore no peak interference were observed for species at lower m/z (LPE 18:0 at m/z 478 and LPC 18:0 at m/z 568). More accurate M+1-derived concentrations were obtained for DG 36:0 at m/z 643; CE 18:0 at m/z 671; Cer 42:1;2 at m/z 695; SM 36:1;2 at m/z 776. For the Type-II overlap with mass difference of 8.94 mDa, a separation of equal abundant peaks by exactly 1× FWHM (Gaussian peak shape model) is achieved at approximately m/z 679. Based on our findings, peak interference should be considered starting at m/z 600 corresponding to a calculated peak resolution of about 0.8× FWHM (m/z (m/z 670 and species at higher m/z (m/z 200). This corresponds to a theoretical peak separation of less than 0.25× FWHM. Adapted to a resolution setting of 140,000, the intensities of the isobaric ions should be additive for species of above m/z 1,700. However, further investigations are necessary to evaluate the range toward 0.25× FWHM peak separation. Importantly, beside the theoretical peak separation, the ratio of the isobaric ions needs consideration, which is in our workflow reflected by the peak configurations  compared to Mi+1+2. Therefore, M+1 quantification provides usually more accurate concentrations for such peaks. However, M+1 peak intensity is substantially lower compared with the monoisotopic peak compromising the sensitivity and thus analytical performance for minor species. Practically, we observed almost no deviations between uncorrected and M+1 concentrations for lipid species in plasma and fibroblast samples (m/z 200).Considering accurate quantification, we recommend the calculation of both uncorrected and M+1-concentrations, in particular for respective e occurs . Compari samples  and S2.  samples A. Howevem/z 200), which can be achieved either by advanced instrumentation  . Further isobaric interferences may derive from other adduction ions like sodium requiring additional correction . Accuratve.org/) .The data supporting this study are available in the article, the This article contains The authors declare that they have no conflicts of interest with the contents of this article.