Several lines of evidence have shown a close correlation between increased H2S production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying compounds selectively and potently inhibiting the human H2S-synthesizing enzymes may therefore prove beneficial for pharmacological applications. Here, the human enzymes CBS, CSE and MST were expressed and purified from Escherichia coli, and thirty-one pyridine derivatives were synthesized and screened for their ability to bind and inhibit these enzymes. Using differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), circular dichroism spectropolarimetry (CD), and activity assays based on fluorimetric and colorimetric H2S detection, two compounds (C30 and C31) sharing structural similarities were found to weakly inhibit both CBS and CSE: 1 mM C30 inhibited these enzymes by approx. 50% and 40%, respectively, while 0.5 mM C31 accounted for CBS and CSE inhibition by approx. 40% and 60%, respectively. This work, while presenting a robust methodological platform for screening putative inhibitors of the human H2S-synthesizing enzymes, highlights the importance of employing complementary methodologies in compound screenings.Biosynthesis of hydrogen sulfide (H H2S is endogenously synthesized by specialized enzymes. Yet, it can also be produced by the gut microbiota as an intermediate or end-product of microbial metabolic pathways, or be released from persulfides and polysulfides, endogenously produced or derived from dietary intake (reviewed in3). The human enzymes that synthesize H2S are cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) from the transsulfuration branch of the methionine cycle, and 3-mercaptopyruvate sulfurtransferase (MST)4. The signalling function of H2S is mostly related to the ability of this molecule to modify target proteins by mediating the persulfidation of cysteine thiols or direct interaction with metal centers, particularly heme moieties (reviewed in6). Being potentially toxic, H2S is disposed via a mitochondrial sulfide oxidation pathway in many cell types7 and proposedly via haemoglobin in red blood cells8. The production and breakdown of H2S are kept under tight regulatory control at all levels, transcriptional, translational and post-translational, by complex mechanisms which also involve the other two ‘gasotransmitters’, as reported for CBS14. Indeed, the relevance of H2S homeostasis is attested by the still growing association between altered H2S metabolism and human pathologies, from cardiovascular and neurodegenerative diseases to cancer (reviewed in16). Decreased endogenous H2S production, due to CSE knock-out or pharmacological inhibition, leads to hypertension and early development of atherosclerosis (reviewed in17). H2S has also been implicated in neuronal disorders. Indeed, a correlation between elevated H2S levels and amyotrophic lateral sclerosis (ALS) in human patients was reported18, while CSE deficiency was found to mediate neurodegeneration in Huntington’s disease19. Furthermore, several lines of evidence have shown a close correlation between increased expression of CBS and CSE and different cancer types, namely colorectal, ovarian, breast, prostate and melanoma22, where increased H2S production was suggested to promote cellular proliferation and energy metabolism22, and contribute to drug resistance24. Accordingly, in the human colorectal cancer cell line HCT 116, shRNA-mediated silencing of CBS or its pharmacological inhibition by aminooxyacetic acid (AOAA) significantly impaired cellular proliferation and migration, and tumor xenograft growth22.Once merely regarded as a toxic substance for virtually all life forms, hydrogen sulfide , as well as several other pyridoxal 5′-phosphate (PLP)-dependent enzymes25. Propargylglycine, a well-studied inhibitor of human CSE, though displaying an IC50 in the tens of micromolar range, requires millimolar concentrations to fully inhibit the enzyme in vivo25, and only non-selective and weak substrate-like inhibitors have so far been reported for human MST, none of them suitable for biological studies26.Given this association between H27, or on cell lines that endogenously overexpress CBS29. Recently, benserazide was reported to inhibit CBS activity with good selectivity, to impair colon cancer cell proliferation in vitro, and tumor growth in vivo29. As for CSE, a screening has been reported, where a few compounds were observed to inhibit CSE, although they lacked specificity, as they also inhibited CBS30. Recently, Hanaoka and co-workers performed a compound screening against mouse MST and identified four pyrimidone derivatives with high inhibitory effect against purified MST and MST-overexpressing cells26. Despite the potential interest on these newly discovered compounds, there is still the need to discover selective high affinity inhibitors for all three H2S-synthesizing enzymes.Compound screening campaigns have been reported mostly for CBS, but also for CSE and MST. Regarding CBS, screening studies have either focused on direct inhibition of the purified recombinant protein31. Biophysical methodologies that allow to directly evaluate the interaction between a target protein and the screened compounds are often used as a first line of screening, particularly if it is unfeasible, due to technical and/or budgetary reasons, to employ an enzymatic activity assay in a high-throughput fashion (reviewed e.g. in32). First line screening methodologies include differential scanning fluorimetry , surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and fluorescence polarization. Once hit compounds are identified in the first line of screening, complementary methodologies, such as in vitro activity assays, isothermal calorimetry and X-ray crystallography, ensue to validate lead molecules and feed information into the pipeline for compound optimization. High-throughput H2S-detecting methods have thus far commonly employed the fluorescence assay based on the 7-azido-4-methylcoumarin (AzMC) dye. AzMC is selectively reduced by H2S to 7-amino-4-methylcoumarin with concomitant increase in fluorescence33. Despite its specificity, AzMC not only reacts with H2S slowly, but is also prone to chemical interference29. Other approaches have been recently reported based on non-commercially available H2S probes (e.g.34). The colorimetric methylene blue method is one of the most commonly used methods for H2S measurement. The method is based on the reaction of H2S with N,N-dimethyl-p-phenylenediamine (NNDPD), followed by iron chloride (FeCl3)-mediated formation of the methylene blue dye, which is detected by visible absorption spectroscopy35.High-throughput screening campaigns typically employ orthogonal methods that complement the information from each other and progressively narrow down the number of tested compounds from up to millions of compounds to a few lead molecules2S-synthesizing enzymes CBS, CSE and MST. Both SPR and DSF didn’t detect any strongly interacting compound. An activity-based screening using the H2S-detecting AzMC fluorescent probe indicated positive hits. However, a counter-screen with the colorimetric methylene blue method revealed direct interference of the tested compounds with AzMC, thus rebutting the finding of positive hits, and allowed the identification of two compounds weakly inhibiting CBS and CSE. The experimental setup herein presented offers a robust platform for future compound screenings targeting the three human H2S-synthesizing enzymes.Herein, we employed a combination of orthogonal biophysical and functional assays to screen a library of synthetic pyridine derivatives against the human HThirty-one pyridine derivatives Table  were syn2S-synthesizing human enzymes tCBS, CSE and MST, recombinantly expressed and purified from E. coli. For each target protein, the DSF assays were preliminarily optimized in terms of protein and dye concentration, resulting in the following conditions : tCBS , CSE  or MST ; final dye concentration: 1x. As shown in Fig. Tm1 = 45.7 °C (60%) and Tm2 = 49.8 °C (40%), yielding a weighted mean value Tm_‘Ave’ = 47.4 °C. To check whether tCBS was partly unfolded at the initial temperature of 20 °C, thermal unfolding was also monitored by Far-UV CD spectropolarimetry. As shown in Fig. Tm of 58.6 °C. DSF was employed to screen the effect of the pyridine derivatives at 200 μM concentration on the thermal denaturation profile of tCBS. Statistical validation of the assay was obtained by incubating tCBS with 200 μM AOAA as the negative control , to be compared with the non-incubated enzyme as positive control  , four compounds, namely C2, C14, C28 and C29, yielded |ΔTm_‘Ave’| ≥ 1.0 °C.The newly synthesized derivatives were assayed by two complementary biophysical techniques, namely DSF and SPR, for their ability to bind to the HTm at 70.4 °C. While precluding reliable estimates of apparent Tm values, the DSF thermal denaturation profiles still allow analysing relative changes upon compounds screening, particularly in terms of the high basal fluorescence measured at 20 °C and intensity of each transition. To quantitatively analyse the effect of the pyridine derivatives on CSE thermal denaturation, we established two parameters based on fluorescence ratios along the thermal denaturation profile , to be compared with the non-incubated enzyme as positive control   and Tm2 = 56.2 °C (40%) by DSF yielding Tm_‘Ave’ = 48.2 °C, to be compared with Tm1 = 45.0 °C (60%) and Tm2 = 58.8 °C (40%) by CD spectropolarimetry yielding Tm_ave = 50.5 °C. Possible interactions of the pyridine derivatives with MST were evaluated by DSF , to be compared with the non-incubated enzyme as positive control   displayed poor solubility, thus precluding an analysis of their interaction with the target proteins by SPR. Moreover, the results obtained for several compounds revealed solubility issues affecting differently the sensorgrams for the three enzymes  and in its presence  . However, all the sixteen compounds tested were found to interfere with the AzMc probe, as they markedly decreased the dye signal . This led us to quit with assays based on the AzMc fluorescent probe and screen the whole compound library on each target enzyme  by detecting H2S with the colorimetric methylene blue (MB) assay.The screening was complemented with activity measurements to test the inhibitory efficacy of the synthetic compounds towards the target enzymes. A fluorescence-based assay using the H2S, each derivative was tested in preliminary control assays using 3 mM GYY4137 as the H2S source . As shown in Supplementary Table 2S. The MB method was therefore employed to evaluate the effect of each compound on the H2S-producing activity of tCBS, CSE and MST  is endogenously produced to accomplish the regulation of numerous physiological processes, ranging from neoangiogenesis37, vasorelaxation and blood pressure38 to cardioprotective39, antinflammatory40 and antioxidant41 effects. Altered H2S metabolism is associated with multiple human pathologies, such as cardiovascular17 and inflammatory42 disorders, neurodegeneration19 and cancer43. Therefore, the development of compounds targeting the three H2S-synthesizing enzymes, namely CBS, CSE and MST, may prove beneficial for future therapeutic strategies, as posited for CBS28. To date, only unspecific or relatively weak inhibitors have been reported for CBS. While AOAA displays a half-maximal inhibitory concentration (IC50) of approximately 3 μM for human recombinant CBS, it also inhibits human CSE with even higher potency, as well as other PLP-dependent enzymes25. Whereas benserazide appears more specific towards CBS, it exhibits lower potency29. The present work attempted to identify new CBS, CSE and MST inhibitor scaffolds, using combined biophysical and biochemical approaches. The tested compounds were assembled in a composite library of 31 pyridine derivatives.Hydrogen sulfide (H45). The usage of both methodologies thus affords a more robust approach46.A combination of orthogonal biophysical and biochemical methods was applied to provide a robust and effective platform to identify putative ligands. Despite the fact that both DSF and SPR are able to detect protein-ligand interactions, though through different mechanisms, frequently in compound screenings the lists of hits identified by either method are only partially overlapping. Both methodologies are typically employed due to the ease of setting up such assays, and to the relatively low amounts of protein required  were found to mildly affect tCBS thermal stability (|ΔTm_‘Ave’| ≥ 1.0 °C), though with |Z-score| < 3.0  and were best fitted with two apparent ues Fig. . The disSimilarly to tCBS, CSE showed high basal fluorescence intensity at resting temperature, possibly related to the presence of the PLP cofactor. Indeed, the CD-monitored thermal denaturation profile revealed CSE to be remarkably stable, ruling out protein instability at resting temperature Fig. . Despite47 and other transulfurases, like rhodanese48. From the four analysed parameters , compounds putatively interacting with MST were identified based on a combination of defined criteria related to Tm shifts and corresponding Z-score values. According to these criteria, only two compounds (C2 and C30) were found to cause shifts in both Tm1 and Tm2 with |Z-score| ≥ 3.0, and notably C30 caused the greatest ΔTm_‘Ave’ (−1.4 °C) among all tested compounds  consistent with an excellent assay, while Ratio B exhibited a slightly lower Z’-factor (+0.31), yet consistent with a good assay (Supplementary Table Tm2) were found to be characterized by Z’-factors  consistent with an excellent assay (Supplementary Table Tm2 exhibited a slightly positive Z’-factor (+0.08), unlike Tm1 and Tm_‘Ave’  . The disadvantages of using a fluorescence-based technique involving an added dye are overcome by SPR, which is a label-free methodology. Herein a CM5 chip was employed to immobilize all target proteins, based on covalent linkage to the carboxymethylated surface of exposed lysine residues distributed through the protein surface, thus offering different possible binding orientations. Pyridine derivatives were screened at four concentrations, from 25 to 200 μM, which already proved to be above the desired solubility for 15 compounds . Despite this limitation and the current lack of adequate controls to statistically validate the use of SPR for high-throughput screening, we observed interactions for a limited number of compounds, based on the proportionality between steady-state response units and compound concentration: 15% of the compounds in the library for tCBS, ~6% for CSE and ~10% for MST , were employed for the first time for the three human H2S-synthesizing enzymes CBS, CSE and MST altogether. Testing a library of 31 pyridine derivatives, a relatively low overlap was observed between DSF and SPR outputs in terms of compounds possibly interacting with the target enzymes. The possibility of including functional approaches such as enzymatic inhibition assays in screening campaigns increases the robustness of the resulting hit compound identification. Herein, only two out of the 31 tested compounds, namely C30 and C31, displayed a weak inhibitory activity towards both tCBS and CSE. Notably, compound C31 was also identified by DSF as a hit compound for CSE and likely interactor of the same protein by SPR.Herein, orthogonal biophysical screening methods, based both on protein-compound interaction (DSF and SPR) and on enzymatic inhibition  to the implementation of more functional assays (enzymatic activity). From this perspective, we hope that the experimental setup herein presented, that integrates analysis of protein-ligand interactions by both DSF and SPR, and protein thermal denaturation investigation by Far-UV CD, with activity assays based on fluorimetric and colorimetric HAll chemicals were purchased from Sigma, except GYY4137 (from CAYMAN), the Protein Thermal Shift Dye Kit™ (from Applied Biosystems) and the SPR Amine Coupling Kit, type 2 .56. Compound purity was assessed by high-performance liquid chromatography in a Waters Alliance system coupled to a Waters 2485 UV/Vis detector.Herein, a composite library of thirty-one pyridine derivatives was assembled Table , based oTo the mixture of 0.3 g (1 mmol) 2,6-dimethyl-1,4-dihdropyridine-3,4,5-tricarboxylic acid 3,5-diethyl ester in 5 ml of dimethylformamide, 0.37 g (2 mmol) potassium carbonate and 0.19 g (1 mmol) iodoacetamide were added. The reaction mixture was heated at 60 °C for 3 h. Afterwards, the reaction mixture was evaporated, and the residue was dissolved in water and extracted with dichloromethane. The dichloromethane layer was dried over magnesium sulfate, filtered and evaporated. Residue was crystallized with diethyl ether and 0.2 g (56.5%) of compound C16 was obtained.1H-NMR : 8.98 , 7.36 , 6.85 , 4.71 , 4.32 , 4.16–4.02 , 2.23 , 1.19 ppm .Compound C16: −. Mp. = 183–184 °C.m/z = 353[M-H]16H22N2O7: C, 54.23; H, 6.26; N, 7.91. Found: C, 54.25; H, 6.22; N, 7.80.Calculated for CTo the mixture of 1 g (3.3 mmol) 2,6-dimethyl-1,4-dihdropyridine-3,4,5-tricarboxylic acid in 35 ml acetone, 1.36 g (9.9 mmol) potassium carbonate was added. The obtained mixture was stirred and refluxed, and 0.56 g (3.3 mmol) of bromo-acetic acid ethyl ester was added. Then, the reaction mixture was refluxed for 4 h, cooled to room temperature and the formed potassium bromide was filtered off. The filtrate was evaporated, dissolved in 15 ml of ethanol and left for 12 h in the refrigerator for crystallization. After filtration and crystallization of the obtained precipitate from benzene, 0.78 g (61%) of compound C17 was obtained.1H-NMR : 5.84 , 4.98 , 4.53 , 4.25–4.12 , 2.32 , 1.29–1.21 ppm .Compound C17: −. Mp. = 110–111 °C.m/z = 382[M-H]18H25NO8: C, 56.39; H, 6.57; N, 3.65. Found: C, 56.54; H, 6.55: N, 3.52.Calculated for CCompound C18 (58% yield) was prepared similarly to C17.1H-NMR : 7.86–7.42 , 6.19 , 5.23 , 5.05 , 4.26–4.14 , 2.30 , 1.27 ppm .Compound C18: −. Mp. = 126–128 °C.m/z = 414[M-H]22H25NO7: C, 63.61; H, 6.7; N, 3.33. Found: C, 63.70; H, 6.03; N, 3.32.Calculated for CCompound C19 (57% yield) was prepared similarly to C17.1H-NMR : 8.96 , 7.87 , 7.03 , 5.26 , 4.84 , 4.16–4.03 , 3.84 , 2.24 , 1.19 ppm .Compound C19: −. Mp. = 155–156 °C.m/z = 444[M-H]23H27NO8: C, 62.01; H, 6.11; N, 3.14. Found: C, 61.72; H, 6.02; N, 3.04.Calculated for CSodium hydroxide  in 3 ml of water was added to a solution of 2 g (6 mmol) ester C26 in 10 ml of ethanol and mixed for 40 min. Then, the reaction mixture was evaporated and the residue dissolved in water. The water solution was acidified to pH = 5 with hydrochloric acid, and the obtained precipitate was filtered and crystallized from ethanol. 1.06 g (58%) of compound C25 was obtained.1H-NMR : 11.79 , 9.10 , 4.52 , 4.13–4.00 , 2.35 , 2.22 , 2.16 , 1.18 , 1.02 , 1.00 ppm .Compound C25: +. Mp. = 165–170 °C.m/z = 308[M + H]16H21NO5. H2O: C, 59.07; H, 7.13; N, 4.30. Found: C, 59.58; H, 7.07; N, 4.26.Calculated for CTo 13 g (0.1 mol) of ethylacetoacetate in 0.2 ml of piperidine, 0.2 ml acetic acid and 20.4 ml (0.1 mol) of a 50% solution of ethyl glyoxalate in toluene was added. The obtained solution was mixed for 48 h at room temperature. Then, 13.9 g (0.1 mol) of dimedone enamine in 30 ml of ethanol was added to the reaction mixture and refluxed for 1 h. After cooling, the mixture began to form precipitates which were filtered and crystallized from ethanol and as a result 10 g (30%) of compound C26 was obtained.1H-NMR : 5.95 , 4.85 , 4.23–4.01 , 2.33 , 2.30 , 2.27 , 1.18 , 1.11 , 1.09 ppm .Compound C26: +. Mp. = 194–196 °C.m/z = 336[M + H]18H25NO5: C, 64.46; H, 7.51; N, 4.18. Found: C, 64.41; H, 7.41; N, 4.12.Calculated for CA mixture of 0.7 g (5 mmol) of 3-amino-5,5-dimethyl-cyclohex-2-enone, 0.56 g (5 mmol) of 2-thiophene carboxaldehyde and 0.95 g (5 mmol) of 3-oxo-butyric acid ethoxycarbonylmethyl ester in 10 ml of ethanol was stirred at room temperature for 3 days. The reaction mixture was cooled to −10 °C and the product crystallization was observed. The crude product was recrystallized from ethanol and 0.85 g (42%) of C27 was obtained.1H-NMR : 9.36 , 7.17 , 6.81 , 6.72 , 5.20 , 4.65 , 4.10 , 2.37 , 2.27 , 2.15 , 1.17 , 1.02 , 0.93 ppm .Compound C27: +. Mp. = 188–190 °C.m/z = 404[M + H]21H25NO5S: C, 62.51; H, 6.25; N, 3.47. Found: C, 62.41; H, 6.34; N, 3.34.Calculated for CTo a hot solution of 0.3 g (0.74 mmol) of methylester C27 in 7 ml of ethanol, a solution containing 0.033 g (0.83 mmol) of sodium hydroxide in 0.8 ml of water was added and boiled for 3 minutes. The solvent was evaporated. The obtained precipitates of crude product were suspended in diethyl ether. After filtration and washing with diethyl ether, 0.26 g (90%) of sodium salt C28 was obtained.1H-NMR : 7.11 , 6.79–6.76 , 5.21 , 4.15 , 2.35 , 2.27 , 2.15 , 1.01 , 0.94 ppm .Compound C28: −. Mp. = 195–196 °C.m/z = 374[M-Na + H]19H20NNaO5S·2,5H2O: C, 51.57; H, 5.65; N, 3.16. Found: C, 51.24; H, 5.10; N, 3.03.Calculated for Cs-adenosyl-l-methionine-binding regulatory domain, was expressed and purified as described in12. Human CSE and MST were expressed and purified as described in Sun et al.30 and in Yadav et al.47 respectively.A truncated version of human CBS (tCBS), lacking the 57. Using 96-to-1536-well plates in an RT-PCR instrument, protein denaturation is monitored as a function of temperature increase by making use of a fluorescent dye that emits light upon binding to the buried hydrophobic amino acid residues that become exposed as the protein unfolds. Typically, thermal denaturation profiles are sigmoidal and the protein melting temperature (Tm) is estimated from the inflexion point. Ligand binding is evaluated from the ability of a given compound to either stabilize or destabilize the target protein, which is reflected in an increase or decrease in the Tm. Here, DSF measurements were carried out in 384-well plates in an Applied Biosystems QuantStudio™ 7 Flex Real-Time PCR. Prior to testing the compounds, the assay was optimized for each target protein in terms of concentration of the proteins (1 to 6 μM) and the fluorescent dye . Once conditions were optimized (see Results), the compounds were assayed by mixing in each well 14 µl of tCBS , CSE , or MST  in Buffer A phosphine [TCEP], pH 7.2), with 2 µl of compound solution . The microplate was incubated at 4 °C for 2 hours to favour possible interactions with the target proteins. Prior to the measurement, the fluorescent dye was pre-diluted in Buffer A to a 5x working concentration, and 4 µl of the dye solution were dispensed into each well. The microplate was sealed with optical adhesive covers (Applied Biosystems) and centrifuged for 30 s at 500 × g. Using the QuantStudio™ 7 Flex Real-Time PCR System, the microplates were incubated at increasing temperatures, from 20 °C to 90 °C with increments of 1 °C/min. Protein unfolding was monitored with the ROX filter (excitation wavelength = 580 ± 10 nm and emission wavelength = 623 ± 14 nm). Data from triplicate curves were averaged and, for tCBS and MST, the melting temperatures were determined by fitting the data to biphasic sigmoidal curves . For statistical validation of the DSF assays towards these protein targets, control assays were performed employing 200 μM AOAA as negative control both for tCBS and CSE, and 2 mM 3-mercaptopyruvate for MST, with the compound-free enzymes being used as positive controls.DSF allows to gain information on ligand binding to a target protein from the observed changes in the protein thermal denaturation profileFar-UV circular dichroism (CD) thermal denaturation curves were recorded in a Jasco J-815 spectropolarimeter equipped with a Jasco CDF-426S Peltier temperature controller, in a 0.1 cm path length quartz cuvette. CSE was diluted to 0.15 mg/mL in 50 mM NaPi buffer, 300 mM NaCl, 0.5 mM TCEP, 10% glycerol, pH 7.5, while tCBS and MST were diluted to 0.2 mg/mL in 10 mM Hepes buffer, 150 mM NaCl, 1 mM TCEP, pH 7.2. CD spectropolarimeter experimental setup: λ = 222 nm; temperature range, 20–90 °C; slope, 1 °C/min; data pitch, 0.5 min; data integration time, 1 s; bandwidth, 1 nm; nitrogen flow, 4 L/min. Thermal denaturation curves were analyzed with Graphpad (Prism), fitting the data to a monophasic (tCBS and CSE) or biphasic (MST) sigmoidal curve.Putative interactions of the tested compounds with tCBS, CSE, and MST were investigated by surface plasmon resonance (SPR). Assays were carried out in a Biacore 4000 instrument  at 25 °C. First, a pH scouting was performed on a CM5 chip for immobilization optimization using the following buffers: 10 mM sodium acetate pH 5.0, 5.5 and 5.8, 10 mM Bis-Tris pH 6.0 and 6.5, 10 mM sodium phosphate pH 7.0, 10 mM HEPES pH 7.5, or 10 mM Tris-HCl pH 7.5.N-hydroxysuccinimide for 1.5 min. Proteins were coupled to the surface with a 5 to 10 min injection time at a flow rate of 10 µL min−1 in order to reach 5000 to 10000 response units (RU). The remaining activated carboxymethylated groups were blocked with a 5 min injection of 1 M ethanolamine pH 8.5.tCBS, CSE and MST were diluted to 5 µg/mL (tCBS) or 10 µg/mL (CSE and MST) in their corresponding immobilization buffer  and immobilized onto CM5 sensor chips using the standard amine coupling procedure. HBS-N, which consisted of 10 mM Hepes pH 7.4 and 150 mM NaCl, was used as the background buffer. Prior to immobilization, the carboxymethylated surface of the chip was activated with 20 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 5 mM 2, pH 7.2.) and injected at four different concentrations using 2-fold dilutions series, with the highest concentration tested being 200 µM. Interactions were qualitatively assessed from the obtained plots of steady-state analyte binding levels against the concentration, making use of the provided Biacore 4000 evaluation software .Compounds  were directly diluted in running buffer  TWEEN-20, 5 mM MgCl2S production activity assays were adapted from Thorson et al.33. Assays were carried out in 96-well black plates, using the H2S-selective fluorescent probe AzMC and a FLUOstar Optima BMG Labtech plate reader. The reaction mixture, in 200 mM Tris-HCl pH 8.0, contained 1.12 µg recombinant human tCBS per well (100 nM), 0.5 mM homocysteine, 50 µM PLP, and 50 µM AzMc (diluted from a 49.7 mM stock in DMSO). Compounds dissolved in DMSO were added to each well to yield a final concentration ranging from 15.6 µM to 1 mM (5% DMSO) in a total assay volume of 250 µl. The plate was incubated at 37 °C for 10 min and the reaction was then triggered by adding the 10 mM l-cysteine. The increase in the probe fluorescence  was monitored over 1 hour at 37 °C. The reader was set up to automatically shake the plate for 5 seconds prior to each data acquisition. Data were analysed using Excel and activity was calculated from the initial slope of the fluorescence increase after l-cysteine addition. Control experiments to evaluate a possible interference of the tested compounds with the probe were done by replacing tCBS with the H2S donor GYY4137 .Fluorimetric H2S, was adapted to 96-well plates, as reported by Druzhyna et al. for CBS29, and improved in order to i) avoid H2S escape from the reaction mixture and headspace before trapping sulfide with zinc acetate, and ii) be extended to the other two H2S-synthesizing enzymes (CSE and MST). Prior to the enzymatic inhibition assays, control absorbance measurements were performed in 96-well plates: at λ = 600 nm to evaluate the compounds solubility under the assays conditions, and at λ = 690 nm to evaluate their interference with the methylene blue read outs (and employ a correction factor whenever needed).The colorimetric methylene blue assay, commonly used to detect H2S production assays, 110 µL of reaction mixture contained 0.5 mM or 1 mM of tested compound, tCBS (10 µg/well) or CSE (30 µg/well) in 50 mM Tris-HCl pH 8.0, and 5 µM PLP. In the case of tCBS, 2 mM homocysteine was also added. After keeping the plate on ice for 30 min, the reactions by tCBS and CSE were respectively triggered by adding either l-cysteine  alone or plus homocysteine , The plate was sealed with vinyl adhesive films and incubated for 2 h at 37 °C. Regarding MST, a 110 µL-reaction mixture contained 0.5 mM or 1 mM of each compound, 30 µg/well MST in 50 mM Tris-HCl pH 8.0, and 10 mM dithiothreitol. After keeping the plate on ice for 30 min, the reaction was triggered by adding 1.5 mM sodium 3-mercaptopyruvate. The plate was sealed with vinyl adhesive films and incubated for 1 h at 37 °C. After the above mentioned incubation times, the CBS, CSE and MST reaction plates were kept on ice for 15 min. Then, 110 µl of 4% zinc acetate were dispensed to each well by punching a hole through the strip with a gas-tight Hamilton syringe. After 15-min incubation on ice, the films were removed and 15 µl of 60 mM NNDPD  and 15 µl of 90 mM FeCl3  were dispensed to each well . The plate was incubated for 10 min at room temperature in the dark and the absorbance measured afterwards at 690 nm. Data were acquired using the plate reader Thermo Scientific Appliskan Multimode.For CBS- and CSE-catalyzed H49. Data were reported in the tables with their corresponding Z-score and coefficient of variation (CV). A |Z-score| threshold value of 3 was assumed to evaluate the effect of the screened compounds. All calculations were performed using Excel (Microsoft).The Z’-factor was evaluated for each assay as described inSupplementary information


ND) was calculated to estimate mGluR1 availability, and partial volume correction was applied to the BPND values. Mini Mental State Examination (MMSE) scores were also obtained (22.0 ± 4.8). No significant difference was observed in BPND between the AD and control groups in the anterior lobe (p = .30), posterior lobe (p = .95), vermis (p = .96), frontal cortex (p = .61), parietal cortex (p = .59), or temporal cortex (p = .27). No significant correlation was observed between BPND and MMSE scores in the anterior lobe (p = .59), posterior lobe (p = .35), vermis (p = .92), frontal cortex (p = .78), parietal cortex (p = .83), or temporal cortex (p = .82). In conclusions, this study suggests that mGluR1 availability is unchanged in the relatively early stage of AD. However, because regional mGluR1 availability may change with the progression of AD, further longitudinal follow-up is necessary.Imaging of type 1 metabotropic glutamate receptor (mGluR1) has recently become possible using positron emission tomography (PET). To date, little evidence exists on the role of mGluR1 in the pathophysiology of Alzheimer's disease (AD). We aimed to examine mGluR1 availability in patients with AD. Ten patients with AD (78.9 ± 5.9 years) and 12 age-matched volunteers (74.6 ± 2.6 years) underwent PET using an mGluR1 radiotracer. All patients were anti-dementia drug-naive. Volumes-of-interest were placed on the anterior and posterior lobes and vermis in the cerebellum and frontal, parietal, and temporal cortices. The binding potential (BP •Metabotropic glutamate receptors (mGluR) can be affected in AD.•Little evidence exists on the role of type 1 mGluR (mGluR1) in AD.•We examined mGluR1 availability in patients with AD.•mGluR1 availability was unchanged in the relatively early stage of AD. In concin vivo studies focusing on mGluR1 in the cerebellum, very few in vivo studies have investigated the role of mGluR1 in the cerebrum, probably because its expression in the cerebrum is relatively poor. However, because metabotropic glutamate receptors can be involved in the regulation of neuronal excitability and synaptic plasticity, cerebral mGluR1 playing a critical role in the development and protection of neurons in some neurological and psychiatric disorders is unsurprising , and found that mGluR1 expression in the cerebrum can decrease with progression of the pathological stages of AD pyrimidin-4-yl]-1,3-thiazol-2-yl] -4-11C-methoxy-N-methylbenzamide (11C-ITMM) and PET.Compared to the number of rprising . Ostapchrprising . In contes of AD . This di22.111C-ITMM PET and magnetic resonance imaging (MRI) scans. The participants comprised 10 patients  who visited a neurologist complaining of the development of memory loss, and 12 age-matched volunteers . All patients underwent 18F-fluorodeoxyglucose (18F-FDG) PET, which provides an index of regional glucose metabolism, and exhibited a pattern characteristic of AD; 18F-FDG uptake decreased in the posterior cingulate, precuneus, and/or temporoparietal cortices . The subsequent 11C-Pittsburgh compound B (11C-PiB) PET yielded positive findings for Aβ accumulation in all patients . All patients were anti-dementia drug-naive at the time of 11C-ITMM PET, and the Mini Mental State Examination (MMSE) and Clinical Dementia Rating scale (CDR) scores of the patients were 22.0 ± 4.8 and 0.8 ± 0.3 (0.5 or 1), respectively  and 0, respectively (This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Tokyo Metropolitan Institute of Gerontology (H26-81). Written informed consent was obtained from all participants who underwent ectively . Althougcriteria  in the rectively . Data ar2.211C-ITMM, was synthesized as described previously . For the control group, the corresponding values were 542.7 ± 67.1 MBq and 84.4 ± 32.1 GBq/μmol, respectively. The frame arrangements for the dynamic PET data were 20 s × 3 frames, 30 s × 3 frames, 60 s × 5 frames, 150 s × 5 frames, and 300 s × 10 frames. Data were reconstructed after correction for decay, attenuation, and scatter.The radioligand, eviously , and wasThe MRI scanning was performed using the Discovery MR750w 3.0 T scanner  in three-dimensional mode . The PET and MRI data were processed using the FMRIB Software Library version 5.0.4 .2.3The cerebellar subregions, three cortical lobes  and white matter were defined as volumes-of-interest (VOIs). The VOIs for the anterior and posterior lobes and vermis in the cerebellum were selected from the probabilistic cerebellar atlas included in the FSL. The VOI for the whole cerebellum was created by combining the VOIs of the cerebellar subregions. The VOIs for the three cortices were selected from the Montreal Neurological Institute (MNI) structural atlas included in the FSL. The VOI for the white matter was created in the MNI space by combining the VOIs of the superior corona radiata, superior longitudinal fasciculus, splenium of the corpus callosum, and pons to include sufficient volume for the subsequent kinetic analysis. The VOIs of the superior corona radiata, superior longitudinal fasciculus, and splenium of the corpus callosum were sampled from the JHU DTI-based white matter atlas included in the FSL, and the pons VOI was manually drawn in the MNI space. These VOIs in the MNI space were then transformed into native space and used for the subsequent kinetic analysis.ND) was then calculated to estimate mGluR1 availability. Additionally, BPND maps were generated in native space using the PMOD PXMOD version 3.4 and the SRTM2 . First, the time-activity curves were fitted using the Simplified Reference Tissue Model (SRTM) to provide an estimation of k2′, the rate constant for the transfer of the radioligand from the reference region to the plasma . We usedhe SRTM2 .2.4ND values because of a partial volume effect. To overcome this problem, a two-compartmental partial volume correction was applied to the BPND values of all of the participants . Statistical significance was set at p < .05.The differences in variables between the AD and control groups were tested using a two-tailed t-test. The relationships between BP311C-ITMM PET are summarized in 18F-FDG and 11C-PiB images showed the pattern characteristic of AD.The characteristics of all participants at the time of ND values in each region are shown in ND values were 5.13 ± 0.38, 4.57 ± 0.43, 5.24 ± 0.40, 5.11 ± 0.42, 2.24 ± 0.31, 2.07 ± 0.47, and 2.36 ± 0.34 in the whole cerebellum, anterior lobe, posterior lobe, vermis, frontal cortex, parietal cortex, and temporal cortex, respectively (mean ± SD). For the control group, the corresponding values were 5.15 ± 0.42, 4.77 ± 0.46, 5.23 ± 0.42, 5.10 ± 0.40, 2.31 ± 0.31, 2.16 ± 0.21, and 2.19 ± 0.34, respectively. No significant difference was observed between the AD and control groups in the whole cerebellum (p = .91), anterior lobe (p = .30), posterior lobe (p = .95), vermis (p = .96), frontal cortex (p = .61), parietal cortex (p = .59), or temporal cortex (p = .27).Plots of the corrected BPND maps were averaged and visually inspected to identify any regional differences in the cortical BPND values between the AD , anterior lobe , posterior lobe , vermis , frontal cortex , parietal cortex , or temporal cortex .No significant correlation was observed between the corrected BP4ND values. Although we showed results only from the corrected BPND values, regardless of partial volume correction , we found no difference in mGluR1 availability between the AD and control groups across the brain, and no correlation between mGluR1 availability and MMSE scores.The objectives of this study were to assess whether mGluR1 availability differs between the AD and control groups and whether mGluR1 availability and MMSE scores are correlated in patients with AD. Because patients with AD and elderly volunteers are susceptible to a partial volume effect, we applied partial volume correction to the BPMetabotropic glutamate receptors are likely to be involved in regulating excitatory neurotransmission and synaptic plasticity, which are affected in AD . Eight s18F-FDG, 11C-PiB and voxel-based morphometry analyses (see a supplementary file). Therefore, one cannot deny the possibility that regional mGluR1 availability may change with the progression of the clinical stages of AD. Further follow-up of the AD patients in this study will be needed to elucidate this issue.The present study had some limitations. The number of patients with AD was relatively small, and the patients were predominantly distributed in the early stage of the AD spectrum based on the clinical course of each patient and results from


The Epstein-Barr virus (EBV) oncoproteins latent membrane protein 1 (LMP1) and LMP2A constitutively activate multiple signaling pathways, and both have been shown to interact with cellular ubiquitin ligases and affect cellular ubiquitination. To detect the LMP1- and LMP2A-mediated effects on the global cellular proteome, epithelial cell lines expressing LMP1 or LMP2A were analyzed using label-free quantitative proteomics. To identify proteins whose ubiquitination is affected by the viral proteins, the cells were cultured in the presence and absence of deubiquitinase (DUB) and proteasome inhibitors. More than 7,700 proteins were identified with high confidence and considerably more proteins showed significant differences in expression in the presence of inhibitors. Few of the differentially expressed proteins with or without inhibitors were common between LMP1 and LMP2A, confirming that the viral proteins induce unique changes in cell expression and function. However, ingenuity pathway analysis (IPA) of the data indicated that LMP1 and LMP2A modulate many of the same cellular regulatory pathways, including cell death and survival, cell movement, and actin filament dynamics. In addition, various proteasome subunits, ubiquitin-specific peptidases and conjugating enzymes, vesicle trafficking proteins, and NF-κB and mitogen-activated protein kinase signaling proteins were affected by LMP1 or LMP2A. These findings suggest that LMP1 and LMP2A may commonly target critical cell pathways through effects on distinct genes, with many cellular proteins modified by ubiquitination and/or degradation. The Epstein-Barr virus proteins latent membrane protein 1 and 2 have potent effects on cell growth and signaling. Both proteins bind to specific ubiquitin ligases and likely modulate the cellular proteome through ubiquitin-mediated effects on stability and intracellular location. In this study, a comprehensive proteomic analysis of the effects of LMP1 and LMP2A revealed that both proteins affected proteasome subunits, ubiquitin-specific conjugases and peptidases, and vesical trafficking proteins. The data suggest that the effects of these proteins on the abundance and ubiquitination of cellular proteins are in part responsible for their effects on cell growth regulation. The EBVormation . LMP1 isormation . Recent ormation , 4. In crmation 46. Howevemation 468.LMP2A is not essential for B-cell immortalization; however, it affects cell signaling and blocks signal transduction from the B-cell receptor (BCR) –12. The –15–18–Both LMP1 and LMP2A interact with cellular ubiquitin ligases that can affect both the levels and locations of cellular proteins. LMP1 is considered a constitutive member of the tumor necrosis factor receptor family and interacts with the same factors, TRAF1, -2, -3, -5, and -6, TRADD, and A20, most of which have ubiquitin ligase activity through a RING finger domain –26. LMP2–N-ethylmaleimide and MG132, respectively. The use of deubiquitinase inhibitors would stabilize proteins whose ubiquitination is induced by the viral proteins, while the proteasome inhibitors would block their degradation. This approach would tentatively identify proteins that would otherwise be undetectable due to virus-mediated effects on ubiquitination and protein turnover. The ubiquitinated proteins include proteins whose levels are affected by the viral proteins through protein degradation but also would include proteins whose ubiquitination is induced by LMP1 or LMP2A to modulate cellular location or activity. The effects of these viral proteins on the proteome could have considerable functional outcomes in multiple aspects of cellular function, including signaling, endocytosis, and vesicle trafficking.To identify proteins and pathways directly and indirectly affected by LMP1 and LMP2A through effects on protein levels, the individual cellular proteomes of cells expressing LMP1 or LMP2A were determined in the absence or presence of deubiquitinase and proteasome inhibitors P < 0.05) in comparison to the pBabe-containing control cells, with an almost equal distribution of increased and decreased expression. In the LMP2A-expressing cells, 433 proteins (P < 0.05) were changed in comparison to the control cells; however, a considerably greater number of proteins were decreased relative to control cells, with 308 decreased and 125 increased. These data suggest that both LMP1 and LMP2A affect the cellular proteome through proteasome-mediated effects on levels of specific proteins. All P values and fold changes for each comparison are presented in Equal amounts of cell lysates from MCF10 epithelial cells expressing LMP1, LMP2A, or pBabe empty vector control, incubated for 6 h with or without inhibitors, were fractionated by basic reverse-phase high-performance liquid chromatography (RP-HPLC) and were analyzed using LC-tandem mass spectrometry (LC-MS/MS). Two biological replicates with two technical replicates of each were analyzed using MaxQuant software . A total10.1128/mBio.00959-18.1TABLE S1 P values, fold changes, and gene names are provided for t test comparisons between LMP1 and pBabe without inhibitors, LMP2A and pBabe without inhibitors, LMP1 and pBabe with inhibitors, and LMP2A and pBabe with inhibitors. The results for each t test are presented on a separate tab. Download TABLE S1, PDF file, 1.9 MB.Proteomics data set. Copyright © 2018 DeKroon et al.2018DeKroon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Comparison of the individual, significantly different proteins indicated little overlap between groups with and without inhibitors for either LMP1 (2.4%) or LMP2A (0.8%) ; Table 2P values on the protein ubiquitination pathway in LMP1 with and without inhibitors and in LMP2A with inhibitors compared to the pBabe controls. Canonical pathways involved in EIF2 signaling, regulation of eIF4 and p70s6K signaling, actin cytoskeleton signaling, regulation of actin-based motility by Rho, integrin, FAK, PI3K/Akt, ERK/MAPK, and p53 signaling were all differentially regulated. Although not all of these pathways had a significant number of proteins associated with them to indicate activation or deactivation, the results did indicate that there were a number of significantly different proteins for these associated functions. Multiple pathways involved in vesicle formation and endocytosis, including Clathrin- and Caveolar-mediated endocytosis, were also differentially regulated , LMP2A-noInh (LMP2A versus pBabe without inhibitors), LMP1+Inh (LMP1 versus pBabe with inhibitors), and LMP2A+Inh (LMP2A versus pBabe with inhibitors), with specific attention on the protein ubiquitination pathway, vesicle formation and trafficking, transcription and translation, cytoskeleton cell movement and cell junctions, and pathways previously described for LMP1 and LMP2A , and S5.10.1128/mBio.00959-18.2TABLE S2 P value and fold change data for all proteins associated with vesicle formation and trafficking pathways in the IPA analysis are shown. In each pathway, the significant data for each comparison  are presented, with significant P values highlighted following the convention used for IPA. Green cells indicate significantly downregulated levels versus pBabe, and red cells indicate significantly upregulated levels versus pBabe. For each protein (or gene) that was significant in one comparison, data for other comparisons are also presented, unless they were from the manual curation. Download TABLE S2, PDF file, 0.11 MB.IPA data: vesicle formation and trafficking. The Copyright © 2018 DeKroon et al.2018DeKroon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00959-18.3TABLE S3 P value and fold change data for all proteins associated with transcription and translation pathways in the IPA analysis are shown. In each pathway, the significant data for each comparison  are presented, with significant P values highlighted following the convention used by IPA. Green cells indicate significantly downregulated levels versus pBabe, and red cells indicate significantly upregulated levels versus pBabe. For each protein (or gene) that was significant in one comparison, the data from other comparisons are also presented. Download TABLE S3, PDF file, 0.24 MB.IPA data: transcription and translation. The Copyright © 2018 DeKroon et al.2018DeKroon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00959-18.4TABLE S4 P value and fold change data for all proteins associated with cytoskeleton, cell movement, or cell junction pathways in the IPA analysis are shown. In each pathway, the significant data for each comparison  are presented, with significant P values highlighted following the convention used by IPA. Green cells indicate significantly downregulated levels versus pBabe, and red cells indicate significantly upregulated levels versus pBabe. For each protein (or gene) that was significant in one comparison, the data in other comparisons are also presented. Download TABLE S4, PDF file, 0.16 MB.IPA data: cytoskeleton, cell movement, and cell junctions. The Copyright © 2018 DeKroon et al.2018DeKroon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00959-18.5TABLE S5 P value and fold change data for all proteins associated with known LMP1- or LMP2A-associated pathways in the IPA analysis are shown. In each pathway, the significant data for each comparison  are presented, with significant P values highlighted following the convention used by IPA. Green cells indicate significantly downregulated levels versus pBabe, and red cells indicate significantly upregulated levels versus pBabe. For each protein (or gene) significant in one comparison, the data in other comparisons are also presented. Download TABLE S5, PDF file, 0.25 MB.IPA data: other or known LMP1-LMP2A pathways. The Copyright © 2018 DeKroon et al.2018DeKroon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 1.92 × 10−5)  , some of × 10−5)  identifi × 10−5)  30). In. InP = 1 × 10−5) , 31, 32.P = 2.35 × 10−6)  . Some of × 10−6)  also add × 10−6) , PSMG1 ( × 10−6) , and UFMP = 4.26 × 10−5)  . This gr × 10−5)  added th × 10−5) , and PIA × 10−5) . LMP2A h × 10−5) . These pP = 0.0126; Z score,1.987) and “Autophagy of cells”  categories in the Disease and Biofunctions group (P = 0.00019), which is a metastasis suppressor that inhibits stress-induced autophagy in cancer cells (P = 0.048) which when downregulated enhances autophagy and endoplasmic reticulum (ER) stress-induced apoptosis (P = 0.015), which mediates autophagy-dependent protein degradation of hMSH4, a MutS protein involved in regulating DNA double-strand break repair in homologous recombination , ARPC4, and Flottilin-2 (FLOT2), had significantly different expression in LMP1-expressing cells than in the pBabe control. However, the IPA pathways associated with these proteins did not contain a significant number of proteins . In addition, manual curation found a number of other proteins with functions in vesicle formation control , includiP = 0.0154) and Fusion of late endosomes (P = 0.0176) categories, both of which only contained Rab7a from our data set, which was downregulated 1.678-fold (P = 0.04)  . Rab7A i = 0.04) . Rab7a w = 0.04) . Other d = 0.04) . Manual  = 0.04)  46, 47), 47P = 0 = 0.04) .P = 0.00219), Endocytosis (P = 2.29 × 10−4), Macropinocytosis (P = 0.01), Viral entry via endocytic pathway (P = 0.0437), and Caveolar-mediated endocytosis signaling (P = 0.0497) (P = 0.039), which is essential for phagocytosis and migration (P = 0.037), which contains a novel ATG16L1-binding motif and promotes local activation of LC3 . It has been demonstrated to bind ATG16L1 and mediate autophagy during Staphylococcus aureus infection (P = 0.034) (P = 0.028). WDR6 is a negative regulator of amino acid starvation-induced autophagy  . The Aut7 . The A 0.0497) . Manual igration , TMEM59 = 0.034)  and WDR6utophagy .Similarly, the Endocytosis pathway included 24 proteins in total, with 11 proteins upregulated and 13 downregulated. Upregulated proteins included ATP6V0D1, CLIC3, Ezrin (EZR), and RAC2, while downregulated proteins included Rab7a, SNX3, ATG2A, and AP1S1. Ezrin acts as a cross-linker between membranes and the cytoskeleton and is important for endocytosis, phagocytosis, and vesicle trafficking, enhancing cell motility and invasiveness. It is known that Ezrin phosphorylation is induced by LMP1-enhanced cell motility . Manual P = 2.63 × 10−4), Fusion of late endosomes (P = 0.0095), and Endocytosis by tumor cell lines (P = 0.013). The Endocytosis group contained Annexin-6 (ANXA6), which has been suggested to mediate the aggregation and fusion of endosomes during exocytosis in epithelial layers . Ot. OtP = 2l layers . CHMP3 il layers . Manual l layers , 62. EXOP = 4.65 × 10−3), tRNA charging (P = 1.18 × 10−2), translation of protein (P = 2.52 × 10−4), translation of mRNA (P = 7.45 × 10−5), expression of protein (P = 4.69 × 10−4), and processing of RNA (P = 9.51 × 10−3). In EIF2 signaling EIF2AK2, EIF3L, EIF4A1, and RPL12 were downregulated while EIF2B4 was upregulated. EIF2AK2, EIF2B4, and EIF3L were also included in the Translation of mRNA, Translation of protein, and Expression of protein pathways. All other proteins associated with these pathways are listed in P = 3 × 10−4). IPA found other pathways with LMP2A-noInh, but each of these had only one protein listed, ribosomal protein S25 (RPS25) in EIF2 signaling and eIF4-p70S6K signaling and glycyl-tRNA-synthetase (GARS) in tRNA charging.IPA also revealed a number of overlapping pathways containing significant numbers of proteins with functions in transcription and translation . For LMPWith the addition of inhibitors, a greater number of transcription and translation pathways had significant scores with IPA. In addition, there were a greater number of differentially expressed proteins associated with these pathways, most of which were downregulated . For insP = 6.27 × 10−3), in which ARPC4, CAPN1, and RAP1B were downregulated while CAPNS1 and vinculin (VCL) were upregulated. Other pathways had proteins listed for LMP1-noInhs but their P values were not significant. In addition, for LMP2A-noInh there were no pathways with statistically significant numbers of proteins in these functional categories. With the addition of inhibitors to LMP2A-expressing cells, only the Rac signaling (P = 1.32 × 10−2) and Binding of filaments (P = 4.51 × 10−3) pathways contained significant numbers of proteins. In Rac signaling, six proteins were downregulated  and one was upregulated (NF-κB1), while in Binding of filaments all four proteins were downregulated . It is intriguing that LMP2A would affect the expression of NF-κB1, a major component of the canonical NF-κB pathway that is activated by LMP1. This provides another mechanism for synergy between LMP1 and LMP2A.With the addition of inhibitors, the number of cytoskeleton, cell movement, and cell junction pathways containing significant numbers of proteins identified via IPA increased dramatically in LMP1-expressing cells . WithoutP = 3.67 × 10−5), Actin cytoskeleton signaling (P = 9.58 × 10−6), Epithelial adherens junction signaling (P = 4.98 × 10−5), Sertoli cell-Sertoli cell junction signaling (P = 3.15 × 10−6), Tight junction signaling (P = 6.25 × 10−4), Rac signaling (P = 2.6 × 10−2), RhoA signaling (P = 6.52 × 10−3), Leucocyte extravasation signaling (P = 4.52 × 10−4), and ILK signaling (P = 5.18 × 10−3).In contrast, with LMP1+Inh cells the number of pathways IPA found with statistically significant numbers of proteins increased to 13 . These pP = 1.9 × 10−2), although only 3 proteins were listed. These were SERPINB5 and Stratifin (SFN), which were upregulated, and PCNA, which was downregulated. For LMP1+Inh, p53 signaling contained PERP (TP53 apoptosis effector) was downregulated 7.4-fold and TP53IP3 was upregulated 3.2-fold. LMP1+Inh also contained a significant number of proteins in the CDC42 signaling pathway (P = 1.67 × 10−2), with 8 of 10 proteins being upregulated. Some of these included IQGAP3, HLA-B, FDG1, and ARPC1A.A number of functional pathways previously described for LMP1 and LMP2A were also implicated by IPA . In LMP1P = 1.33 × 10−3), PI3K/AKT signaling (P = 3.26 × 10−3), and PTEN signaling (P = 8.34 × 10−3) pathways contained significant numbers of proteins. In the mTOR signaling pathway, 9 of 11 proteins were downregulated and this included a number of EIF3 and EIF4 proteins. In the PI3K/AKT signaling pathway, 6 of 8 proteins were downregulated and also included INPP5K, MAP2K2, and PPP2R2A. For the PTEN signaling pathway, in which 5 of 7 proteins were downregulated, INPP5K, MAP2K2, and RPS6KB1 were also included , and manual curation found NF-κB-activating protein (NKAP) (P = 0.0379) was significantly upregulated in LMP2A+Inh compared to its pBabe control. In addition, IKBKG was upregulated 1.742-fold in LMP1 with inhibitors (P = 5.03 × 10−3), while TANK-binding kinase (TBK1) was downregulated 1.305-fold (P = 0.0425)  . These dP = 0.03), in contrast to the MS/MS data which indicated greater variance and were not significantly different (P < 0.05) . Western transfers also suggested that Rab11-FIP1 was significantly increased by LMP1 in the presence of inhibitors, in agreement with the MS/MS data. Interestingly, Rab11 was also shown to be increased based on immunoblotting, with significant variability   and C. Wiability  and E.This is the first analysis of the effects of the EBV proteins LMP1 and LMP2A on the cellular proteome. As both proteins bind ubiquitin ligases, to identify proteins potentially regulated through this mechanism, the proteomes were also assessed in the presence of inhibitors of both ubiquitination and proteasome-mediated degradation. Not surprisingly, this analysis identified considerably more proteins in the presence of inhibitors that are apparently regulated by LMP1 or LMP2A through effects on ubiquitination or proteasomal processing. Inhibition did not necessarily increase the abundance of proteins, and considerable numbers of the affected proteins decreased in the presence of inhibitors. It is possible that these proteins are regulated at some level by LMP1 or LMP2A through effects on negative regulators of expression which are not degraded with proteasomal inhibition. Additionally, the inability to deubiquitinate proteins could also result in their degradation through standard lysosomal mechanisms.in vivo. However, these data identified many proteins that are likely affected by LMP1 and LMP2A through ubiquitination and proteasomal turnover. Interestingly, multiple specific proteasome components were affected, and the effects of the viral proteins on these components may contribute to proteasome function and specificity.However, the differences in the levels of expression suggest that LMP1 and LMP2A modulate the ubiquitination of these proteins. Both proteins also affected multiple ubiquitin-conjugating enzymes and peptidases, which could affect the specificity for ubiquitin targeting. It is known that LMP1 activates NF-κB through effects on the function of the canonical NF-κB inhibitor IκBα, and the data presented here indicate that it is highly ubiquitinated in the presence of LMP1 . AdditioThe effects of LMP1 and LMP2A on cellular expression have previously largely been identified by changes in transcription. Although most of these data have been determined in EBV-infected lymphocytes, comparison of these data to existing transcriptional data identified several genes in common. Approximately 39% of genes modulated by LMP2A without inhibitors have been shown to be affected transcriptionally, while 29% with inhibitors were also identified transcriptionally , 66. SimIt is interesting to overlay the effects of both LMP1 and LMP2A on the pathways most clearly modulated. Evaluation of the effects on pathways involved in vesicle trafficking  showed tIn these same MCF10A cells, we previously showed that LMP2A activates autophagy to prolong survival during the differentiation process of luminal formation . In contThe importance of virus-mediated effects on the ubiquitin system was clearly revealed in initial studies demonstrating that growth-transforming viral proteins such as E6 induced growth through specific effects and interactions with ubiquitination components, such as E6AP. The data presented here revealed that the EBV oncogenes also likely affect the abundance of many proteins through effects on ubiquitination. Additionally, these proteomics data suggest altered regulation of both endosomal vesicle trafficking and autophagosome-to-lysosome flux with the expression of LMP1 or LMP2A. The ability to affect vesicle formation and trafficking would also contribute to the regulation of many signaling complexes, such as those that are associated with endocytosed receptors or occur at the vesicle membrane. It will be important to further dissect how these virus-mediated effects on protein ubiquitination alter specific protein localization.2. Each treatment/experimental group was plated in 6 replicates in 15 cm dishes. Treatment groups consisted of pBabe-, LMP1- or LMP2A-expressing cells untreated or incubated for 6 h with the DUB and proteasome inhibitors N-ethylmaleimide  and MG-132 , respectively.MCF10A breast epithelial cells expressing LMP1, LMP2A, or pBabe vector  were culg for 15 min at 4°C. Protein concentrations were estimated using a colorimetric protein assay . For each treatment group, 5 mg of protein was reduced with 5 mM dithiothreitol (DTT) for 45 min at room temperature, followed by 10 mM N-ethylmaleimide for 30 min while protected from light. Samples were digested with 5 µg LysC  for 4 h at 37°C, then diluted to 2 M urea  by using 50 mM Tris (pH 7.5) and digested with 100 µg sequencing-grade trypsin  overnight at 37°C.Cells were lysed at 4°C in denaturing lysis buffer  with the addition of protease inhibitors  and DUB and proteasome inhibitors when applicable. The resulting lysates were centrifuged at 13,000 × 18 Sep-Pak Vac RC cartridge (Waters) according to the manufacturer’s instructions.Following digestion, samples were acidified with formic acid (FA) to a final concentration of 0.5% and consequently desalted using a 500-mg C18 column (Waters).To increase the depth of coverage and quantitation of these lysates, the trypsin-digested desalted samples were fractionated using strong cation exchange (SCX) chromatography, collecting 32 fractions in each run. These fractions were then recombined noncontiguously into 8 final fractions per sample, such that each resulting fraction contained a less complex mixture of peptides with a maximum range of charges. Each fraction was subsequently analyzed by nano-liquid chromatography (nano-LC) run in-line with MS/MS identification of peptides. Samples were fractionated by basic RPLC on an XBridge 4.6- by 250-mm CSamples were resuspended in 0.1% formic acid. Identification and quantitation of proteins were performed via RPLC-MS/MS on a two-dimensional nano-LC Ultra system coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Separation of the tryptic peptides was achieved with a 70-min linear gradient of 2 to 30% buffer B in buffer A at 200 nl/min, where buffer A was an aqueous solution of 0.1% formic acid and buffer B was a solution of 0.1% formic acid in acetonitrile (ACN).m/z and a retention time-aligned extracted ion chromatogram for each peptide. All 4 replicates  of each sample group were included in the LFQ experimental design, with protein-level quantitation and normalization performed using unique and razor peptide features. Statistical analysis was performed using Perseus (version 1.4.1.3), and the differences in expression profiles were further analyzed using Ingenuity Pathway Analysis software.Mass spectra were processed, and peptide identification was performed by using the Andromeda search engine found in MaxQuant software version 2.2.1. Peptides were identified using a peptide false-discovery rate (FDR) of 0.05 and a protein FDR of 0.01 with at least 2 unique peptides. Label-free quantitation (LFQ) was calculated by utilizing the measured area under the curve (AUC) of


Introduction: Bleeding occurs frequently in liver surgery. Unbalance between tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) concentrations might increase bleeding. Our aim was to analyze perioperative fibrinolytic changes during liver surgery.Materials and Methods: We evaluated 15 patients for inclusion into a prospective pilot study of liver surgery. We assessed fibrinolysis by plasma PAI-1 and t-PA: before surgery (T1), before Pringle maneuver , at the end of surgery (T3) and 24 h postoperatively (T4), and registered demographic and laboratory data, extent and duration of surgery, hemodynamic parameters, blood loss, and transfused volumes of blood products. Data presented as mean ± SD. Significance at P < 0.05.Results: After exclusion of six patients only undergoing biopsies, we included six women and three men aged 49.1 ± 19.6 years; two patients with liver metastases of colorectal cancer and hepatocellular carcinoma, respectively, two with focal nodular hyperplasia, two with hepatic hemangioma, and one with angiomyolipoma. Six patients underwent PM. PAI-1 plasma concentration (n = 9) rose from 6.25 ± 2.25 at T1 through 17.30 ± 14.59 ng/ml at T2 and 28.74 ± 20.4 (p = 0.007) and 22.5 ± 16.0 ng/ml (p = 0.04), respectively, at T3 and T4. Correspondingly, t-PA plasma concentration (n = 9) increased from 4.76 ± 3.08 ng/ml at T1 through 8.00 ± 5.10 ng/ml (p = 0.012) at T2 and decreased to 4.25 ± 2.29 ng/ml and 3.04 ± 3.09 at T3 and T4, respectively. Plasma t-PA level at T2 was significantly different from those at T1, T3, and T4 (p < 0.004). In PM patients, t-PA levels increased from T1, peaked at T2 (p = 0.001), and subsequently decreased at T3 and T4 (p = 0.011 and p = 0.037), respectively. Mean blood loss was 1,377.7 ± 1,062.8 ml; seven patients received blood products. Patients with higher PAI-1 levels at T3 received more fresh frozen plasma  and red blood cells .Conclusions: During liver surgery, fibrinolysis increased, as evidenced by rises in plasma PAI-1and t-PA, especially after start of surgery and following PM. Transfused volumes of blood products correlated with higher plasma concentrations of PAI-1. Confirming this tendency requires a larger cohort of patients. Liver resection is a commonly used procedure for treatment of both benign and malignant hepatic diseases. Although, both anesthetic and surgical techniques have progressed during the last few years, resulting in lower perioperative morbidity, serious complications still occur .The liver is involved in synthesis and clearance of proteins of the fibrinolytic system, which is responsible for the degradation of thrombi in the vasculature –4. FibriTo reduce bleeding during liver resection, the surgeon often uses the so-called Pringle maneuver (PM), which consists in clamping the hepatoduodenal ligament, thereby interrupting blood flow through the hepatic artery and portal vein . In the Hepatic bleeding might occur, not only following activation of fibrinolysis, but also secondary to reduced production or rapid consumption of coagulation factors. Hence, limited reserves of coagulation proteins might play a decisive role for the outcome when bleeding occurs in patients with liver diseases , 11, 12.In the present prospective pilot study, we speculate, whether increased fibrinolysis develops secondary to low liver clearance of tissue plasminogen activator (t-PA) in combination with protein C inactivation of PAI-1 . These aFifteen subsequent patients, who were scheduled for liver resection surgery, were included in a prospective pilot observational study in Pauls Stradins Clinical University Hospital in Riga, Latvia. The Medical Research Ethics Committee approved the study protocol and the informed consent form .We defined the following exclusion criteria: patients < 18 years of age, tumor considered as being radically inoperable or surgery limited to tumor biopsy only (5 patients) or cholecystectomy (1 patient). Therefore, after further analysis, we excluded six patients, as depicted in Figure All patients underwent standard general anesthesia including endotracheal intubation. We induced anesthesia by injection of midazolam 2.5 mg , fentanyl  0.01 mg/kg, atracurium  0.5 mg/kg and propofol  1.5–2 mg/kg intravenously.Anesthesia was maintained with inhalation of sevoflurane  at 0.8–1.2 MAC, combined with continuous infusion of fentanyl  0.01–0.03 mg/kg/min. Muscle relaxation was obtained with atracurium 0.25 mg/kg per h. All patients received tranexamic acid (TXA)  10 mg/kg before skin incision. We also provided postoperative epidural analgesia with bupivacaine  0.5% solution 0.05–0.08 mg/kg at the end of surgery.2O to minimize bleeding. If concomitantly, tachycardia (heart rate > 100/min), hypotension (mean blood pressure < 65 mmH2O), or oliguria (< 0.5 ml/kg/h) should occur, we administered vasoactive agents, for instance, norepinephrine or dopamine at doses of 0.05–0.2 μg/kg/min and 3–7 μg/kg/min, respectively. Patients, who had blood losses of >10 ml/kg and/or demonstrated hemodynamic instability and signs of severe hypovolemia, including hemoglobin levels < 9 g/L, received blood transfusions.On purpose, we accepted relative hypovolemia during surgery, as assessed by keeping the central venous pressure (CVP) between 4 and 8 cmHWe collected blood from a peripheral vein into heparin tubes that we cool centrifuged  for 5 min at 3,000 × g. The plasma supernatant was removed from the spun samples and frozen to −70°C until the time of analysis of biomarkers of coagulation and fibrinolysis.We analyzed fibrinolytic activity at four data collection points by means of the markers PAI-1 and t-PA. Firstly, we determined baseline fibrinolytic activity before surgery (T1). Secondly, we assessed PAI-1 and t-PA plasma concentrations during surgery, before initiation of the Pringle maneuver (T2), in order to evaluate the influence of surgically created tissue damage. Thirdly, we determined the markers at the end of surgery (T3) to assess the impact of liver hypoperfusion on fibrinolytic activity, and fourthly, 24 h after surgery (T4), to detect postoperative fibrinolytic activity, which commonly stays enhanced a few days after liver resection .actibind, an ELISA method. Plasma concentrations of PAI-1 and t-PA are normally < 20 ng/ml and < 10 ng/ml, respectively.PAI-1 and t-PA  were analyzed with In addition to PAI-1 and t-PA, we determined the fibrinogen plasma concentration in citrated plasma  preoperatively and analyzed prothrombin (PT) with a PT complex assay . We performed all the coagulation tests by means of Sysmex® CA-1500 . Hemoglobin (Hb), hematocrit (Hct), platelets, white and red blood cell counts were analyzed by means of a Beckman Coulter LH 750 Hematology Analyzer.post-hoc test for all pairwise multiple comparisons of PAI-1 and t-PA vs. time (T1-T4). We used linear regression (Pearson's correlation coefficient) to analyze the relationships between demographic and surgical data as well as PAI-1 and t-PA vs. bleeding volumes. Furthermore, we used Chi-square test to analyze categorical data. We defined a p < 0.05 as a statistically significant difference and correspondingly, p > 0.05 as not significant (NS). We also calculated sample sizes with comparisons of two means or paired sample t-test by using PAI-1 and t-PA data provided α ≤ 0.05 and β ≤ 0.2.Data were analyzed with SPSS  and SigmaPlot , as appropriate. Continuous variables were presented as mean ± standard deviation (SD) or as median and interquartile range; categorical variables as percentages (%). We checked the data for normal distribution with Kolmogorov-Smirnov and Shapiro Wilks tests and used ANOVA for repeated measurements followed by Student-Newman-Keuls Totally 15 consecutive patients underwent liver resection during a 6 months period whereof six patients met exclusion criteria. Table 2O, ranging from 2.5 to 8 cm H2O during surgery indicating relative hypovolemia. Mean systolic blood pressure was 115.2 ± 7.5 mmHg and mean heart rate was 72 ± 17 beats/min.The extent of liver resection was from one to four segments with an average duration of surgery of 339.4 ± 230.5 min (min. 190–max. 910 min). The Pringle maneuver lasted from 20 to 25 min (mean 20.5 min). Mean CVP was 4.9 ± 1.9 cm HFive patients had significant bleeding during surgery, each of them lost more than 1,000 ml of blood. Average blood loss was 1,378 ± 1,063 ml. Seven patients received transfusions of packed red blood cells and fresh frozen plasma, whereas only two of them required fibrinogen substitution with cryoprecipitate  30 ± 2.9 s, prothrombin time (PT) 104 ± 14%, International Normalized Ratio (INR) 0.9 ± 0.07 and fibrinogen plasma concentration 3.6 ± 0.18 g/L.Before surgery, the patients had the following mean blood cell counts: white blood cells 7.30 ± 1.98 × 10n = 9) increased from 6.25 ± 2.24 ng/ml at T1 through 17.30 ± 14.59 ng/ml during surgery before start of the Pringle maneuver at T2 and reached its peak concentration of 28.74 ± 20.41 ng/ml (p = 0.007) at T3. At T4, PAI-1 plasma level decreased to 22.5 ± 16.2 ng/ml, but still remained elevated in comparison with T1 (p = 0.04).As shown in Figure n = 9) rose from 4.76 ± 3.08 at T1 to 8.00 ± 5.1 at T2, and decreased to 4.25 ± 2.29 ng/ml and 3.04 ± 3.1 at T3 and T4, respectively. Thus, the t-PA level at T2 differed significantly from those determined at T1, T3, and T4 (p < 0.004).Correspondingly, as depicted in Figure We used the intragroup changes in PAI-1 concentrations between T1 and T3, and the differences in t-PA level between T2 and T4 to calculate sample sizes of a future prospective study. Based on mean clinical relevant intragroup differences of PAI-1 and t-PA of 22.5 and 5.00 ng/ml, respectively, we estimated sample sizes of 9 and 13 patients for each variable, respectively. Concerning patients undergoing Pringle maneuver, the least sample size required for obtaining significant intragroup differences in PAI between T1 and both T3 and T4, was 10 patients. Between those who underwent Pringle maneuver at T3 vs. those who did not, mean difference in PAI-1 was 14 ng/ml. Given standard deviations of 19.00 ng/ml and 23.5 ng/ml, respectively, would require 60 and 30 patients in the Pringle and the non-Pringle groups.r = 0.9; p = 0.002) and fresh frozen plasma . We did not find corresponding relationships between t-PA and transfused volumes of blood products.Statistically significant correlations were observed at T3 between PAI-1 plasma levels and transfused volumes of fresh frozen plasma and red blood cells. As depicted in Figures Plasma concentrations of PAI-1 increased gradually with the extent of liver resections, albeit without reaching statistical significant difference. At the end of surgery (T3), mean PAI-1 plasma concentration was 13.6 ± 19 ng/ml in patients subjected to resection of one liver segment, and correspondingly 29 ± 19 ng/ml after resection of two segments, and 34.7 ± 32 ng/ml and 45 ng/ml after three and four liver segment resections, respectively.A similar tendency, was seen when analyzing the corresponding t-PA plasma concentrations. At the end of surgery (T3), t-PA was 3 ± 4.1 ng/ml in patients subjected to resection of a single liver segment, and 4.7 ± 1.3 ng/ml and 5.2 ± 3.5 ng/ml, respectively, in those who underwent two and three segment resections. Surprisingly, we found the lowest t-PA level of 2.9 ng/ml in the patient with four liver segment resections.n = 6) with those in whom the maneuver was not applied (n = 3). In those exposed to the Pringle maneuver, PAI-1 plasma concentration increased from 7.00 ± 2.37 ng/ml at T1 to 24.05 ± 19.16 ng/ml (NS) at T3, and further to 26.66 ± 18.17 ng/ml (NS) at T4. In subjects not exposed to the Pringle maneuver, PAI-1 increased significantly within group from 4.76 ± 1.05 ng/ml at T1 to 38.13 ± 23.46 ng/ml at T3 (p = 0.043), and decreased to 14 ± 7 ng/ml (NS) at T4.We also compared PAI-1 and t-PA plasma concentrations after surgery at T3 in patients subjected to the Pringle maneuver (p = 0.001), and subsequently decreased to 3.78 ± 2.30 at T3 (p = 0.011 vs. T2) and to 2.93 ± 3.14 ng/ml (p = 0.037 vs. T2) at T4. In patients not undergoing the Pringle maneuver, t-PA fell slightly from 5.56 ± 4.30 ng/ml at T1 to 5.20 ± 2.40 ng/ml at T3 and to 3.26 ± 3.68 ng/ml at T4 (NS). However, we found no intergroup differences in PAI-1 and tPA plasma concentrations depending on whether the Pringle maneuver was used or not.In patients subjected to the Pringle maneuver, plasma t-PA levels increased from 4.36 ± 2.68 ng/ml at T1 through 9.33 ± 4.53 ng/ml at T2  and anti-fibrinolytic (PAI-1) factors, as characterized by an increase in global fibrinolytic capacity and a reduction of clot lysis time, particularly in patients with liver cirrhosis . Liver sChild-Pugh score, which is widely used to assess the prognosis of patients with liver cirrhosis . We interpret these changes as secondary to activation of the fibrinolytic system in the presence of an insufficient inhibition. Increments in the concentration of t-PA were rather modest, as assessed by an elevation of 68% at T2 as compared with that at T1. Rapid increments in these marker concentrations at the start of surgery might be explained by endothelial and surgical tissue damages, as described by recent investigators .Our findings are consistent with those of Illig and co-authors, who studied primary fibrinolysis during supraceliac clamping of the aorta . The latAccording to recent studies, mean blood loss in otherwise healthy patients undergoing liver surgery is ~600–900 ml, which is less than in our patients. We found a blood loss ranging from 100 to 3,000 ml. This discrepancy might be due to comorbidities and a high age difference between the patients, combined with the fact that liver resection surgery in Latvia was implemented rather recently , 37.After analyzing the influence of the extent of tissue resection on fibrinolytic activity, we observed that in patients with more extensive liver resections, PAI-1 plasma concentrations increased before application of the Pringle maneuver and at the end of surgery, which might be explained by a larger surgical tissue trauma. Thus, apparently, the extent of liver surgery influenced t-PA plasma levels to a lesser degree. According to recent literature, fibrinolytic activity might increase before surgery in patients with chronic liver diseases, as documented by elevated t-PA plasma levels .Surprisingly, the patient with a four segment liver resection had a low t-PA plasma level at the end of surgery and 24 h postoperatively. Most likely, this is explained by a decrease in hepatic clearance and low t-PA plasma levels already before the surgery. Low plasma levels of t-PA at baseline could be due to high age and such co-morbidities as general atherosclerosis, arterial hypertension, type 1 diabetes and metabolic syndrome. These co-morbidities were frequently associated with reduced levels of t-PA and elevated PAI-1 concentrations . HoweverWe admit as a major limitation that the study is undersized to detect significant intragroup differences in t-PA and intergroup differences in both PAI-1 and t-PA between patients who underwent the Pringle maneuver and those who did not. The reason for this shortage is that we introduced systematic liver surgery rather recently. Moreover, because of limited experience both with anesthetic and surgical management of patients undergoing liver surgery we intentionally avoided to include a control group not given the prophylactic standard dose of tranexamic acid. This kind of surgery requires multidisciplinary treatment and application of demanding techniques, which in addition to patient factors explain both the small number of patients included, and the relatively high blood losses.Another limitation is the heterogenic group of patients studied. Some of them had different co-morbidities such as diabetes, primary arterial hypertension that might affect baseline fibrinolytic activity. We do not know to what degree fibrinolytic activation due to surgical trauma and blood loss occur in these patients in comparison with those without such cardiovascular co-morbidities.Child-Pugh scores, we should be cautious to extrapolate the results to a larger cohort of patients scheduled for liver surgery. In accordance with our sample size calculations, we expect that a future prospective multicenter study of at least 90 patients planned for liver surgery, which also includes the Pringle maneuver, might shed more light on fibrinolysis as a cause of perioperative bleeding.Unfortunatelly, we did not meassure changes in activated protein C plasma levels in parallel with changes in PAI-1 concentrations. Because of low sample sizes and no deaths in our pilot study, we were not able to analyze the impact of changes in plasma concentrations of PAI-1 on mortality or other measures of clinical outcome. However, we found corelations between PAI-1 level after surgery and transfused volumes of red blood cells and fresh frozen plasma.Considering the limited number of patients in this pilot study and their initial During liver resection surgery, fibrinolytic system activation increases as experienced by a significant release of PAI-1, in parallel with increments in t-PA, especially at the start of surgery. Application of the Pringle maneuver was associated with a trend toward more pronounced activation of fibrinolysis. Although, we found no associations between fibrinolytic markers and bleeding volumes, higher plasma concentrations of PAI-1 correlated highly with the volumes of transfused blood products. Further analysis including more patients is necessary to confirm the augmentation of fibrinolysis and its correlation with bleeding volumes during liver surgery.All authors have contributed equally to the design of the study, to analysis and interpretation of results, and drafting or revising the manuscript. AgO, JN, and AK conceived the study. AgO included patients into the study and got agreement of participation, as well as continued to collect the data. JN was responsible for administering anesthesia in a standardized manner to all the included patients and ArO and JG performed the surgery. IV, AK, and LV performed data analysis and interpreted the results. AgO and LB drafted and revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A homogeneous and dense microstructure was observed in all samples. With increasing Sm content, the crystal structure changed from tetragonal (x ≤ 0.06) to cubic (x = 0.07) and unit cell volume (V0) decreased continuously, which was mainly due to the substitution of Ba2+ ions by smaller Sm3+ ions in the perovskite lattice. Electron paramagnetic resonance investigation revealed that Mn ions were reduced from high valence to low valence under the role of Sm3+ donor, and only Mn2+ ions were observed at x = 0.07. The Curie temperature (Tc) moved to lower values, from 105.5 down to 20.4 °C, and the x = 0.07 sample satisfied Y5V specification with high permittivity  and low loss (tan δ < 0.03).The structure, valence state, and dielectric properties of (Ba Tsur eta/Ti < 1  have als−1 at room temperature, are shown in x ≤ 0.06, six Raman bands at 245, 304, 515, 722, 785, and 840 cm−1 were observed, and the 245, 304, 515, and 722 cm−1 bands corresponded to tetragonal BaTiO3 [−1, which was assigned to B1+E(LO+TO) [x = 0.07, suggesting a phase transition from tetragonal to cubic, which was in good accordance with the XRD results.Together with the XRD studies, we employed Raman spectroscopy measurements to investigate the structure and phase transformation. Raman spectra of BSTM ceramics, excited by a 532 nm laser in the range of 100–900 cml BaTiO3 ,28. The E(LO+TO) . This ba−1 was observed. We propose that doping with Sm3+ ions will induce the valence change of Mn ions from high valence to low valence for keeping charge neutrality. Since Mn2+ (0.67 Å) has a larger ionic radius than that of Mn3+ (0.58 Å) and Mn4+ (0.53 Å), the Mn–O bonds have higher covalency, and 722 cm−1 bands shift toward a higher frequency. A similar behavior was also found in Nd/Mn co-doped BaTiO3 [A noticeable shift in position for the band at 722–730 cmd BaTiO3 . −1 bands, which were not observed in BaTiO3 [−1 band was also observed in Ba(Ti1−xZrx)O3 [0.4Ba0.6Ti0.6RE0.4O3  [1g octahedral breathing mode. In order to investigate the origin of the 785 cm−1 band, the dielectric permittivity (ε′) of representative samples with x = 0.04 and 0.07 as a function of temperature at various frequencies (10–1 × 105 Hz) is shown in Tc) of BS4TM and BS7TM remained unchanged as the frequency increased. The result shows that there was no frequency dispersion in BSTM ceramics, which indicated that BSTM ceramics were not relaxor ferroelectrics, but normal ferroelectrics. Pokorny et al. [1g octahedral breathing mode was inactive in BaTiO3 since this mode is symmetrical. However, for BaTiO3-based ceramics with two or more B-site species, the A1g band became Raman-active. The presence of different ions in the center of the BO6 octahedra created asymmetry in the breathing-like mode, and the width and intensity of this band was largely temperature-independent in the vicinity of the cubic-to-tetragonal phase transition [x = 0.04 and 0.07 at the wavelength of 532 nm. It showed that the band at 785 cm−1 was almost independent of temperature, as reported in the literature. Therefore, we preferred to assign the 785 cm−1 band to A1g octahedral breathing mode. In addition, the 304 cm−1 band representing the tetragonal structure disappeared above 60 and 0 °C for x = 0.04 and 0.07 samples, respectively, indicating that the tetragonal-to-cubic transition temperature  decreased with increasing Sm content.Two additional 785 and 840 cmn BaTiO3 ,28,29, a−xZrx)O3 ,31,32,33= Y, Yb)  ceramics= Y, Yb)  claimed = Y, Yb)  assigned−1 band increased gradually with Sm content. We attributed this band to the Raman charge effect, which arose from an internal deformation of the BO6 octahedron induced by the occupation of trivalent ions  at equivalent Ba sites in the BaTiO3 lattice [−1 band was first reported in La-doped BaTiO3 by Kchikech and Maglione [−1 in Nd-doped Ba0.77Ca0.23TiO3 ceramics, and the intensity of this mode increased linearly with Nd content. −1 band did not change as a function of temperature, and persisted up to 150 °C. It indicated that the tetragonal–cubic phase transition did not affect the Raman charge effect, as reported in the literature [ lattice . The 840Maglione , and itsMaglione  also obsterature . −1. BS7T ceramic with a cubic structure  as a Kramer ion is EPR-active, and showed the sextet hyperfine structure of 55Mn (I = 5/2) [0.99Mn0.01)O3 ceramics with mixed tetragonal and hexagonal phases, the coexistence of Mn3+ and Mn4+ was determined by EPR technique [3+ ions in all samples. Here, the sextet signal was noticed in all samples, indicating that the Sm3+ donor induced the conversion of high valence state Mn to divalent Mn. Thus, Mn ions existed as two valence states, Mn2+ and Mn4+, in BSTM with a tetragonal structure (x ≤ 0.06). This result was in good agreement with previous experimental results. Wang et al. [2+ and Mn4+ coexisted in MnCO3-modified (Ba0.85Ca0.15)(Zr0.1Ti0.9)O3 ceramics with tetragonal phase using XPS.As seen from the EPR spectra at 30 °C, a clear sextet signal with ing Mn2+ ,44. Mn2+I = 5/2) . In Ba and dielectric loss (tan δ) as functions of temperature for BSTM measured at 1 kHz are shown is Tc), which was related to a ferroelectric –paraelectric (cubic) phase transition. With increasing Sm content, the maximum permittivity (ε′m) increased from 5480 to 15,220 and the room temperature permittivity (ε′RT) increased from 2080 to 13,810. The Curie temperature Tc decreased to room temperature, and the sample with x = 0.07 satisfied the EIA Y5V specification /ε’RT within +22 to −82% in the temperature range of −30 to 85 °C) with tan δ < 0.03. Cai et al. [3+ ions on Ba sites in BaTiO3 led to the fall of Tc. Park et al. [3 ceramics using the liquid-mix method and solid-state reaction, and found that Tc moved to lower temperatures with the increase of Sm content. The δ was less than 0.05 over a range from 1 to 106 Hz for x = 0.07. In addition, the Curie temperature determined by dielectric measurements was in good agreement with the temperature range of the phase transition presented in variable temperature Raman , and all of the Mn ions existed as Mn2+ in a cubic phase (x = 0.07). The dielectric properties were investigated as functions of frequency (1–1 × 106 Hz) and temperature (−75 to 200 °C). The Curie peak shifted towards a lower temperature, and the x = 0.07 sample satisfied EIA Y5V specification with ε′RT = 13,810 and tan δ < 0.03. BSTM ceramics as normal ferroelectrics exhibited a near frequency-independent stability over a frequency range of 1–1 × 106 Hz, implying that ionic displacement polarization plays a dominate role in BSTM ceramics.Sm and Mn co-doped BaTiO