Komagataella phaffii T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from Aspergillus oryzae RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase xAor was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in  Xylanase are related to diverse groups of enzymes, hydrolyzing the main hemicellulose of plant cell walls—xylanes . XylanesAspergillus oryzae was chosen for further cloning and heterologous expression. Aspergillus oryzae is one of the most common multicellular fungi in biotechnology for having highly active enzymes with wide substrate spectrum; among them proteases, cellulases, amilases, pektinases, and β-galactosidases are the most notable [xAor belongs to GH10 family. The characteristic of GH10 family is the presence of TIM barrel (α/β)8, being a typical trait of class A enzymes. Active site contains conservative glycone subsites with high substrate affinity and less conservative sites with lower affinity. Glycone subsites from −2 to −1 and from −3 to −1 bind strongly to xylobiose and xylotriose, respectively. All the substrates, exceeding this size, are hydrolyzed from non-reducing end after three xylose monomers, with the presence of two consecutive non-substituted xylose monomers being essential for GH10 family [In this work, gene of endo-1,4-xylanase F1 of  notable . XylanasHemicellulases, such as xylanases, are the most important enzymes group with broad industrial application, including bioconversion lignocellulose in biofuel, SCP (single-cell protein), bio-whitening, fabric production, juices clarification, sewage purification, and ink-washing . ApplicaAspergillus fungi have an ability to decompose xylans [Various genus e xylans . Chemicae xylans . Xylanase xylans . Notwithstanding, it is of great importance that in most cases production of xylanases in host fungi is impeded by the low generation velocity and aeration complications in viscous media . ConcernxAor gene under control of AOX1 gene promoter and terminator with subsequent cultivation in bioreactor.In our laboratory, producer strain was constructed carrying four copies of xylanase Komagataella phaffii T07 strain deposed in National bioresource center in All-Russian collection of industrial microorganisms (VKPM) based in Kurchatov Institute Research Center (Accession Number #Y-4936) ) was obtained (#Y-4936) . Optimizobtained .xAor activity was calculated based on xylose calibration, wherein molar extinction coefficient was shown to be ε ≈ 0.00045 ± 0.00005 µMol −1 × cm−1. It was shown, that at pH = 7.5, enzyme activity was E = 258,240 IU/mL for culture liquid, containing a total of 3.1 g of xylanase. Xylanase 6 IU/(mg protein). For lyophilized cultural liquid after tangential centrifugation specific activity was 57.69 × 106 IU/(mg protein) with total amount of 1.8 g. Lyophilized xylanase after chromatographic purification showed activity of 129.53 × 106 IU/(mg protein) and has a total amount of 0.32 g of protein. Specific activity for culture liquid was shown to be 37.23 × 10pH-optimum of xylanase was 7.5, while working range was shown to be from pH = 3.0 to pH = 9.0, with still showing activity at pH = 2.5 and pH = 10.0 . The enzNominal mass of recombinant xAor was 35,552 Da, and matched calculated mass—35.41 kDa. Purity of studied sample was verified with electrophoretic analysis a,b. IsoePS (protein score) was shown to be 1492, which is statistically significant result. MS sequence coverage was 63% . To detep ≥ 0.95).Thus, mass-spectrometry of the studied sample showed the identity of the primary target sequence compared to the predicted one (4)2SO4. The induction stage began with 40 mL of methanol and lowering the temperature down to 27 °C. After the adaption of the culture to methanol and reduction of oxygen, periodical methanol replenishment was started, wherein replenishment was carried out every 20 min in the first three hours, and by the end of the cultivation process volume of added methanol increased twice. It is important to highlight that wherein abrupt increase of oxygen higher than 25% was shown, methanol was added. Periodically, samples were collected to monitor specific enzyme yield and biomass  is a methylotrophic yeast widely used in biotechnological industry due to its high secretory capacity [xAor was obtained from A. oryzae fungi, thus, expression in fungal organism is more preferable for correct processing of the target protein. Taking into consideration the abovementioned, strain K. phaffii T07 (Accession Number #Y-4936), previously discovered in our laboratory, was shown to be the most suitable candidate as a base for producer strain of xAor xylanase. capacity  and abilcapacity . Moreovecapacity . XylanasA. oryzae in Pichia pastoris was described in Yin et al.’s work [xAor from A. oryzae, consisted of 981 bp. after the deletion of signal peptides. xAor gene was inserted into four tandem copies since it was shown that multicopy plasmids with recombinant DNA tandem clusters are effective for increasing production of target protein in case of K. phaffii transformation [For the first time heterologous expression of 10th family xylanase Aoxyn10 from .’s work . Gene Aoormation . Extraceormation , while aAspergillus oryzae HML366 XynH1 are identical to A. oryzae RIB40 protein XP_001826985.1 of glycosyl hydrolases family 10, with the optimal specific activity being 476.9 U/mg at pH 6.0, while remaining active within pH 4.0–10.0 and at a temperature below 70 °C [Aspergillus oryzae F1 endo-1,4-xylanase, it was shown that pH optimum is 5.0, and temperature optimum is 60 °C [A. oryzae LC1 in expression system E. coli BL21 (DE3) had specific enzyme activity 1037.3 EU/mg, being 9.3 times higher, than in host organism. Recombinant xylanase XynF1 [It was shown that fungi xylanases have higher enzyme activity, comparing with bacterial ones, with most of them active at 50 °C and pH range from 4 to 6 . Describow 70 °C . For Aspis 60 °C . Extraceis 60 °C . BhardwaxAor was obtained. The ability of xAor producer strains to be grown in bioreactors and high specific activity of xAor xylanase even in culture liquid suggest that the strain obtained is suitable for industrial scale production.In conclusion, in this work, soluble, secreted xylanase Aspergillus oryzae (strain ATCC 42149/RIB 40) was chosen (Sequence ID: O94163). Codon composition was calculated according to the table of codons occurrence frequency, optimal for expression in K. phaffii. Signal peptides, detected with using algorithm SignalP-5.0 [After GenBank database analysis, sequence of xynF1 gene from nalP-5.0 . ColonieMalI, BamHI, and BglII at 37 °C for 30 min. Plasmid pPZL-ost-xyl-Aor, containing one xAor gene copy, was hydrolyzed with BglII restriction enzyme at 37 °C for 30 min, and then with alkaline phosphatase 16 °C for 120 min. Obtained DNA fragments with sticky ends were purified using KAPA Pure Beads , according to the manufacturer’s instructions, and were used in ligation reaction.For the construction of the plasmid with two tandem genetic construction copies, expression construction target fragment was amplified from the primary plasmid. Amplification was performed according to the next—95 °C 3 min; 5 cycles: 95 °C 10 s, 58 °C 15 s, 72 °C 80 s; 20 cycles: 95 °C 10 s, 63 °C 15 s 72 °C 80 s. Amplicon, purified with magnetic beads, was treated with restriction enzymes E. coli strain XL-blue cells were electoporated with obtained solution, after suspending in 1 mL of LB media and incubating for 45 min at 37 °C. 1/100 of cells were spread on agarized LB media with the addition of 20 µg/mL zeocin and incubated at 37 °C all night.Ligation was carried out using highly active T7 DNA ligase  at 16 °C for 30 min. Ligation reaction was purified with magnetic beads, as described above. Next, competent BglII and BamHI. Then, the presence of the second copy and accuracy of orientation were confirmed by electrophoresis in 1% agarose gel. For the construction of the plasmid with four copies of expression construction, abovementioned assay was performed twice. To confirm the correct orientation of the second expression construction, obtained colonies were incubated in 4 mL of LB media with 20 µg/mL concentration of zeocin all night at 37 °C and 250 rpm. Next, plasmid DNA was extracted using QIAprep Spin Miniprep Kit , according to manufacturer’s guidelines. Aliquots of extracted plasmid DNA were treated with restriction enzymes K. phaffii yeasts, plasmid replication origin, and zeocin resistance gen under control of EM7 promoter for E. coli expression and TEF1 promoter for K. phaffii expression, was obtained. To sum up, plasmid carrying xAor gene under control of AOX1 promoter and terminator for inductive expression in x-OA-xyl-AsOr-pPZL-4 was electoporated into yeast cells, after transformants were cultivated for 2 h 30 °C in 1 mL 1 M sorbitol. Next, 1/5 of obtained cell suspension was spread on 1.5% agarized BD Difco media  with the addition of 200 µg/mL zeocin.Plasmid K. phaffii T07 strain without the inserted xylanase gene was used as a negative control. Plate was placed in thermo-shaker at 360 rpm, wherein every 24 h 200 µL 10% methanol was added to every well. On the third day of cultivation, aliquots were taken and centrifuged at 4000× g for 5 min to precipitate cells.The presence of xylanase expression was verified by inoculation of the obtained colonies in 24 deep well plate, wherein every colony was inoculated in separate well, containing 2 mL 1% yeast extract, 2% peptone with 0.3% glucose and 1% methanol (YPGM). Incubation was carried out at 30 °C, while no pH level observation was performed. 2, pH 9.0), and 5 volumes of 100 mg 4-nitrophenyl-β-D-xylanopyranoside solution was added to 10 mL 100 mM PBS. Cultivation was carried out at 37 °C. The course of the reaction was detected by Epoch (Agilent Technologies (formerly BioTek Instruments), Santa Clara, CA, USA) well spectrometer. Evaluation was performed every 10 min at 405 nm. Supernatant of K. phaffii T07 strain without the inserted xylanase gene cell culture was used as a negative control. Enzyme activity was detected by the release of the dye of synthetic substrate 4-nitrophenyl-β-D-xylanopyranoside. To detect enzyme activity in the well 1 volume of culture liquid, then 4 volumes of buffer  and cultivated at 250 rpm in shaker at 30 °C for 48 h.Cultivation of xAor xylanase was performed using ProLab  bioreactor. Respective 4)2SO4, 1.88 g/L CaSO4∙2H2O, 0.94 g/L NaCl, 3.75 g/L MgSO4∙7H2O, and 3.75 g/L KH2PO4. Bioreactor was previously sterilized for 45 min at 121 °C.Primary culture was aseptically inoculated in a bioreactor, with the salt media consisting of 32.5 g/l glycerin, 9.38 g/L  with 10 kDa pore filters. All the reaction were carried out at 5 °C. Concentrate was frozen at −70 °C and freeze-dried . Obtained half-product was dissolved in 10 mM sodium phosphate buffer (pH 7.5), purified from chromogenic additives by gel-filtration using Sephadex G-25 . Elution was carried out with 10 mM sodium phosphate buffer (pH 7.5). Protein purification was performed using ion-exchange chromatography on the column with anionite DEAE-Sepharose 6HF . Column was washed with 10 mM sodium phosphate buffer (pH 7.5), target protein was eluted with linear gradient 0.0–0.5 M NaCl in starting buffer. Purity grade in fractions with the highest enzyme activity were analyzed by electrophoresis in poly acryl-amide gel. Fractions, containing minimal amount of additional proteins were combined, desalted, and concentrated, using centrifugal filters with 10 kDa pore size and freeze-dried.Xylanase activity was determined by GOST 31488-2012 “Enzyme preparations. Methods of xylanase enzyme activity determination” using 1% oat xylane substrate . To do tTo evaluate the operating pH range, the following buffers were used: for pH 2.5–3.5—50 mM Gly-HCl buffer; for pH 4.0–5.5—50 mM sodium acetate buffer; pH 6.0–7.0—50 mM potassium phosphate buffer; for pH 7.5–11.0—50 mM Tris-Gly buffer. Samples with xylanase activity were dissolved in 50 mM buffer.The assay was performed as follows: 250 µL substrate in the respective buffer was heated for 5 min at 50 °C. Next, to the preheated substrate solution equal volume of enzyme solution, while in respective negative control—equal volume of buffer solution. Enzyme reaction was carried out for 10 min a t 50 °C. The reaction was interrupted by the addition of 750 µL 1% dinitrosalicylic acid. All samples were incubated for 10 min at a boiling water bath until coloring development. Optical density was measured at 546 nm.Temperature operating range of xAor xylanase was performed as described above with following modifications: enzyme reaction was carried out at pH = 7.5 in all cases, but incubation temperature varied. To perform an electrophoretic analysis, lyophilized sample was dissolved in 1 mL 62.5 mM Tris-HCl pH 6.8 with 2% phenylmethylsulfonyl fluoride and 1% sodium dodecyl sulfate (DSN), and treated with ultrasound on ice for 3 min 2 s impulses with 2 s pauses, using ultrasound probe with 91 Watt capacity . Next, sample was mixed with application buffer, consisted of 62.5 mM Tris-HCl pH 6.8, 25% glycerin, 2% DSN, 0.01% bromophenol blue, and 5% β-mercapthoethanol in 1:2 volume, and incubated at 100 °C for 5 min. ® Tetra Cell . Gel was colored with SYPRO™ Ruby , and visualized by gel-documenting system VersaDoc MP4000 .Sample was concentrated in 4% PAAG-DSN at 15 mA and separated in acrylamide/bis-acrylamide gel with 0.1% DSN, 0.38 mM Tris-HCl with pH 8.8, 0.05% TEIUD, 0.05% ammonium persulfate at 25 mA in electrophoretic chamber Mini-PROTEAN4HCO3 to the concentration of 5 mg/mL. Next, sample was with 20 mM TCEP, 40 mM chloroacetamide, 100 mM NH4HCO3, and 0.1% sodium deoxycholate buffer for 5 min at +4 °C at 1:20 volume, 5 min at 90 °C, and 20 min at RT in the dark. Tripsin was added at 1:100 volume and incubated for 16 h at 37 °C. Reaction was disrupted by the addition of 1% trifluoromethyl ketone. Samples were emulsified with the ethylacetate equal volume thrice for 5 min with organic phase removal. For the third extraction ethilacetate with 1% TFA was used. Sample, equal to 20 µg of protein, was purified, using StageTips, according to Rappsilber et al. [Lyophilized sample of culture liquid was dissolved in 100 mM NHr et al. . Sample Identification of MS2 was carried out using UltraFlex III MALDI tandem time-of-flight , MS/MS digest—LTQ-Orbitrap-XL-ETD . For the detection from optimized theoretical protein sequence Mascot software with Swissprot and ICIG_enzymes  databases were used.Xylanases have applications in food and beverages industry, animal nutrition, wood pulp bioleaching, and papermaking. Taking into consideration the wide working range of pH and temperatures enzyme activity, and the ability to be produced in a bioreactor, the authors suggest that xAor xylanase may have suitable industrial application. Authors suggest that with high activity in the culture liquid xylanase, xAor may be used for industrial applications without being purified or concentrated. In concussion, protocol for soluble, secreted xylanase suitable for industrial scale production was obtained.


Background and Aims: The macrophage “don’t eat me” pathway CD47/SIRPα is a target for promising new immunotherapy. We hypothesized that a soluble variant of SIRPα is present in the blood and may function as a biomarker. Methods: Monocyte derived macrophages (MDMs) from human buffy-coats were stimulated into macrophage subtypes by LPS and IFN-γ (M1), IL-4 and IL-13 (M2a), IL-10 (M2c) and investigated using flow cytometry. Soluble SIRPα (sSIRPα) was measured in cell cultures and serum by Western blotting and an optimized ELISA. Serum samples were obtained from 120 healthy individuals and from 8 individuals challenged by an LPS injection. Results: All macrophage phenotypes expressed SIRPα by flowcytometry, and sSIRPα was present in all culture supernatants including unstimulated cells. M1 macrophages expressed the lowest level of SIRPαand released the highest level of sSIRPα (p < 0.05). In vivo, the serum level of sSIRPα increased significantly (p < 0.0001) after an LPS challenge in humans. The median concentration in healthy individuals was 28.7 µg/L , and 20.5 µg/L in an IFCC certified serum reference material. The protein was stable in serum for prolonged storage and repeated freeze/thawing. Conclusions: We demonstrate that sSIRPα is produced constitutively and the concentration increases upon macrophage activation both in vitro and in vivo. It is present in human serum where it may function as a biomarker for the activity of tumor-associated macrophages (TAMs), and for monitoring the effect of immunotherapy. T-cell based immunotherapy has revolutionized the treatment of certain cancers; however, in most cancers significant response rates are still not reached. Research is therefore focused now on ways to involve the innate immune system in cancer therapy . Tumor-aAn important signal system is the CD47/signal regulatory protein alpha (SIRPα) pathway, which is the target for several inhibitory drugs that are currently being investigated in clinical trials including soluble variants of SIRPα to outcompete macrophage binding to CD47 on the tumor-cells . CD47 ishttps://www.uniprot.org/uniprot/P78324, accessed on 20 June 2022). In humans, the SIRP gene family consists of SIRPα, SIRPβ1, SIRPβ2, SIRPγ, and SIRPδ and is clustered on chromosome 20p133 [SIRPα is an approximately 90 kDa glycoprotein with four possible N-linked glycosylation sites  from five healthy donors were obtained from the blood bank at the Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark (project no. 0094). According to Danish law, the use of anonymized buffy coats does not require specific ethical approval.Monocytes were isolated from the buffy coats as described previously . In shorFor monocyte-derived macrophage (MDM) differentiation, the isolated monocytes were cultured in non-treated T-75 flasks in complete maturation media ), 10 ng/mL macrophage colony-stimulating factor (M-CSF), and 1 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF)  for 5 days. Media were changed every 2–3 days.After MDM differentiation, the MDMs were stimulated with either 100 ng/mL LPS and 20 ng/mL IFN-γ (M1 stimulation), 10 ng/mL IL-4 and 10 ng/mL IL-13 (M2a stimulation), 10 ng/mL IL-10 (M2c stimulation)  or left untreated (M0) for 24 h to promote MDM polarization.3. Each subtype was split and stained with live/dead fixable dye near IF (ThermoFisher Scientific) and anti-SIRPα FITC , to assess SIRPα expression. Alternatively, in order to validate the MDM polarization, with: live/dead dye and anti-CD45 AF700 ; anti-CD11b BV510 ; anti-CD80 V450 ; anti-CD163 PE ; anti-SIRPα FITC ; anti-TLR2 PE-Vio770 ; and anti-CD206 APC . Cells were incubated with antibodies at 4 °C for 30 min, after which they were washed in stain buffer and fixed in PBS with 0.9% formaldehyde.After harvesting the MDM subtypes from the wells, unspecific antibody binding was blocked using human IgG  at a final concentration of 100 µg/mL. The cells were stained with antibodies in a stain buffer consisting of PBS with 0.5% BSA and 0.09% NaN® Comp Bead Kit anti-REA (Miltenyi Biotech), and ArC™ Amine Reactive Compensation Bead Kit for Live/dead (ThermoFisher). Data processing was performed using FlowJo 10.7 for Windows . The gating strategy for both panels is presented in The cells were analyzed on a Navios Flow Cytometer . Spectral overlap compensation was performed using single-stained antibody capture beads, BD™ CompBeads Plus (BD Biosciences), MACSv/v) Tween 20, 0.2% (w/v) bovine serum albumin Sigma A-4503), pH 7.2).One hundred microliters of a polyclonal anti-human SIRPα antibody  was coated onto microtiter wells (Nunc Maxisorp) and incubated at 4 °C overnight. The wells were washed three times in PBS, and subsequently blocked with 100 µL PBS-albumin  was diluted to 0.4 mg/L in PBS-albumin, added, and incubated for 1 h.TM FC).After washing, 100 μL of avidin-lysozyme mixture  + 6 μL avidin-POD ) was added and incubated for 1 h. The wells were washed and 100 μL TMB One  was added. After incubating for 20 min, 50 μL of 1 M phosphoric acid was added, and the plates were read at 450/620 nm in a microtiter plate reader . Recombinant human SIRPβ1  and recombinant human SIRPγ  were used to test for specificity.A standard curve spanning a range from 0.125-8 µg/L was prepared from a recombinant human SIRPα . A human serum quality control sample ) was included in all runs, diluted 1:100. One hundred and twenty human serum samples were obtained from the blood bank at the Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark (project no. 304) and diluted 1:100. The concentration of sSIRPα was also determined in a certified reference material (ERMSerum (diluted 1:50) and MDM culture supernatants were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis , according to the supplier’s instructions. Proteins were blotted onto PVDF membranes, blocked for 1 h, and incubated overnight at 4 °C with sheep anti-human SIRPα antibody . The membrane was subsequently incubated for 1 h at room temperature (RT) with rabbit anti-sheep horseradish peroxidase, and developed using enhanced chemiluminescence. BioRad, Hercules, CA, USA, Precision Plus Protein™ WesternC™ Blotting Standards, catalogue no. #1610376, were used.We measured sSIRPα in plasma samples from a previously conducted human randomized crossover trial  investigating eight young, lean, healthy male volunteers for six consecutive hours following LPS injection . A singlThe concentrations of the soluble endocytic receptors sCD163 (haptoglobin-hemoglobin receptor) and sCD206 (mannose receptor) were measured using in-house ELISA as described ,18.Multiple comparisons were performed by ANOVA followed by Tukey’s multiple comparison test. Non-parametric tests were used to investigate the effects of freeze–thaw cycles, storage temperature, storage time, and time to centrifugation. For sample stability before centrifugation and freeze–thaw stability, the Friedman test was used. The stability of pipetted samples, at different storage temperatures for up to one month, was tested with Wilcoxon matched-pairs signed rank test. A parametric 95% reference interval was established by calculating the mean ±1.96 σ from log-transformed data. Statistical analysis was performed using GraphPad Prism 8 and Analyse-it for Microsoft Excel.The expression and shedding of SIRPα was investigated in human MDMs polarized into inflammatory M1  or anti-inflammatory M2a , and M2c (IL10) macrophage subtypes. As expected, TLR-2 and CD80 were upregulated in M1 cells, CD206 upregulated in M2a cells, and CD163 upregulated in M2c cells .p = 0.0087) between subtypes, with the lowest expression on M1 MDMs  which was comparable to sCD206 , but lower than for sCD163 . The level of sCD163 reached maximum already after 3 h, whereas sSIRPα and sCD206 showed a weaker and more prolonged response.To investigate if LPS mediated SIRPα shedding could be demonstrated in vivo, we measured the concentration of sSIRPα in consecutive blood samples from eight healthy individuals injected with a bolus of LPS in a randomized crossover design a. We com2 = 0.99, covering a concentration range of 0.015–3.7 µg/L. Mean recovery of recombinant SIRPα added to patient samples was 92% . Neither rhSIRPβ nor rhSIRPγ  added to serum samples reacted in the assay (<0% recovery at tested concentrations). We determined the concentration of sSIRPα in a certified reference material from IFCC (ERM®-DA470k/IFCC) to 20.5 µg/L +/− 1.4 µg/L .We explored five commercially available antibodies against sSIRPα and established and optimized an ELISA using a polyclonal coating-antibody and a monoclonal biotinylated secondary antibody. Within run imprecision was 3.3% CV . Intermediary (between run) imprecision was 5.9% CV . The LOD was below 0.015 μg/L. The assay was linear in serum dilutions ranging from 1:10 to 1:2430, Rp = 0.95). In pools of serum, sSIRPα was stable for more than 24 h at RT and 4 °C before centrifugation of blood-samples . Men had marginally higher levels than women .Soluble SIRPα was measured in serum from 120 healthy blood donors  and was clearly detectable in all samples. The samples had levels between 17.4 and 54.6 µg/L (median 28.7) and showed a log-Gaussian distribution. A parametric 95% reference interval was established from log-data to 19.8 µg/L (90% CI 18.8–20.8) to 41.1 µg/L (90% CI 39.2–43.1). There was no positive correlation between sSIRPα and age ; it is stable, and it can be measured using the described validated assay. It is produced constitutively by MDMs, and the concentration of sSIRPα increases upon macrophage activation both in vitro and in vivo. sSIRPα may therefore function as a biomarker for the activity of TAMs and for monitoring immunotherapy, which is now being investigated in several clinical studies.


Drosophila. This study provides a valuable reference for the full use of Ziziphus jujube, as well as a new direction in food development.Ultrasonic‐assisted extraction is a rapid and effective extraction method that uses ultrasound energy and solvents to extract target compounds from various plant matrices. In this study, the ultrasonic‐assisted extraction conditions of sour jujube were optimized. A five‐level central composite design (CCD) with four variables was used to evaluate ultrasonic treatment variables influencing the total saponin content (TSC), total flavonoid content (TFC), and total phenolic content (TPC) extracted from sour jujube. The solvent concentration, extraction time, ultrasonic power, and solid‐to‐liquid (S/L) ratio were optimized using aqueous ethanol and methanol solutions as extraction solvents. A central composite design (CCD) was used for an in‐depth study, and then the optimal value that could produce the maximum TPC, TFC, TSC, and four in vitro antioxidant activities , phosphomolybdic acid reduction method, and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging activity) was determined. Hydrogen peroxide‐induced oxidative stress experiment confirmed that the Jujube extract could have an antioxidant role in vivo. The relationship between the contents of three compounds and the antioxidant activity in vitro and in vivo was further studied. The results showed that optimizing methanol and ethanol extraction process parameters could improve target components’ extraction efficiency. Under the optimum conditions, the TFC and TPC yields of sour jujube by ethanol are better than methanol, while the yield of TSC by methanol is better than ethanol. In vivo data showed that Jujube extract protects against the adverse effects of oxidative stress and improves the life span of female and male  1.Sour jujube could be explored as a new dietary supplement to be a potential antioxidant. 2.sour jujube extracts exter excellent antioxidant in vivo and in vitro tests. 3.Determines the preparation process of the antioxidant effective parts of sour jujube and the antioxidant activity of different parts of different origins.  It is widely distributed in Shanxi, Gansu, Liaoning, and other provinces of China. Besides being used as food, the fruit has also been used as medicine for more than two thousand years. Semen ziziphi spinosae, a seed of sour jujube, is an important traditional Chinese medicine  is a tree species of Free radicals are a major factor associated with a variety of chronic degenerative diseases, tumor, cardiovascular disease, and age‐related functional decline, such as Parkinson’s and Alzheimer's diseases . An analytical high‐performance liquid chromatograph (Waters 2695‐2998), ultrasonic extraction equipment , an analytical balance , an electrothermal constant temperature blast drying oven , and a multifunctional microplate reader  were utilized.2.2Drosophila specimens, a wild‐type Canton S Drosophila melanogaster, were provided by the Core Facility of Drosophila Resource and Technology, CEMCS, CAS.Sour jujube fruits were obtained from a farm in Yangquan city, Shanxi Province, China, in September 2020. Other sour jujube fruits producing areas were Hotan (Xinjiang), Lvliang (Shanxi), Dunhuang (Gansu), Linyi (Shandong), and Chaoyang (Liaoning). The 2.3Sour jujube samples were prepared as follows: 0.5 g of jujube was added to 20, 40, 60, 80, and 100% (v/v) ethanol. The following solid‐to‐liquid (S/L) ratios were used: 1:10, 1:30, 1:50, 1:70, and 1:90 g/ml; the temperatures were: 20, 35 50, 65, and 80°C; extraction time was: 10, 30, 50, 70, and 90 min; ultrasonic power was: 100, 250, 400, 550, and 700 W. A group without sample addition was defined as a control group and was also prepared at the same time.2.42.4.1Variance analysis was conducted to evaluate five extraction variables: (1) extraction time, (2) extraction solvent concentration, (3) solid‐to‐liquid (S/L) ratio, (4) ultrasound power, and (5) temperature. There were four central points in this model. The main effects of the variables on the TSC were determined by calculating the difference between the lower and higher levels of the measured mean. The t‐test was used to examine the significance of the effect of each variable on the TSC.2.4.21, ultrasonic power X2, solvent concentration X3, and solid‐to‐liquid ratio (S/L ratio) X4. Each variable was examined at five different levels, including factor point , axial points , and the central point (0)  ratio had a significant effect on the extraction rate of antioxidant components; yet, this needed further optimization. Thus, a four‐factor five‐level CCD was run 30 times to determine the optimal values of important extraction factors and their effects on TSC, TFC, TPC, and antioxidant activity . The tested variables were: extraction time X2.4.3The Total saponin content (TSC) of the extracts was determined by the jujuboside A reagent method  is the total content of saponin compounds (mg/g), C is the concentration of jujuboside A obtained established from the calibration curve (mg), V is the volume of ethanol or methanol used (ml), m is the weight of sour jujube (g), and n is the dilution ratio.2.4.4The Total flavonoid content (TFC) of the extracts was determined by the rutin reagent method  is the total content of flavonoid compounds (mg/g), and C, V, and m are the same as defined for TSC determination.2.4.5The Total phenolic content (TPC) of the extracts was determined by the Folin–Ciocalteu method  is the total content of phenolic compounds (mg/g), and C, V, and m are as defined for TSC determination.2.4.6− was determined based on the salicylic acid method as previously described , H2O2 solution (6 mmol/L), and salicylic acid ethanol solution (6 mmol/L) were added to the sample or 1 ml of the positive control vitamin C (0.8 mg/ml). After mixing well, the samples were placed in a water bath at 37°C for 1 h, and the absorbance was measured at 510 nm. The blank contained 1 ml of distilled water, the control contained no distilled water, and the other steps were the same as described above. The –OH scavenging capacity of the extracts was calculated according to Formula OHSforcin, . A totalg) for 10 min, 2.5 ml of supernatant was collected, 2.5 ml of anhydrous ethanol and 0.5 ml of FeCl3  were added, and the absorbance was measured at 700 nm. Distilled water was used as the blank control. The inhibition rate of FRAP activity was calculated according to Formula The total reduction capacity was determined by the potassium ferric cyanide reduction method, also known as the ferric‐reducing antioxidant power (FRAP)   was used to describe the effect of ultrasonic power, extraction time, S/L ratio, and solvent concentration on the TPC, TFC, TSC, and four antioxidant activity assays. The optimal levels of these four extraction variables were also further investigated by this method.3.23.2.12) was used to provide additional information about model fitness. The R2 values for TSC, TFC, TPC, %DPPHsc, FRAP, PA, and %OH − sc with methanol and ethanol extraction were 90.0%, 90.1%, 92.3%, 95.2%, 95.7%, 98.1%, 92.1%, and 90.4%, 91.2%, 92.3%, 95.8%, 97.8%, 98.6%, and 91.5%, respectively, which suggests that the model fits the results well. The p‐value for the lack of fit was not significant (p > .05), which confirms the validity of the obtained model.The results were analyzed by fitting a second‐order polynomial model, and the regression coefficients for linear, interaction, and quadratic terms were determined by employing the least‐squares method. Analysis of variance (ANOVA) was used to determine an adequate and significant matrix. The coefficient of determination  sour jujube extraction methods are shown in Tables 1 = 84.6, 20.4, 10.0, and 90.0 min; X2 = 166.3, 555.1, 57.7, and 54.6 W; X3 = 94.3%, 24.0%, 57.7%, and 54.6%; and X4 = 1:41.2, 1:11.3, 1:10.0, and 1:10.0 g/ml, respectively. The quadratic regression coefficients of X2, X4, X1X4, X2X3, X2X4, X12, X22, X32, and X42 in TSC; X3, X22, X32, and X42 in TFC; X1, X3, X3X4, X12, X22, X32, and X42 in TPC; X2, X3, X4, X2X3, X3X4, X22, X32, and X42 in %DPPHsc; X1, X2, X3, X4, X1X3, X1X4, X2X3, X2X4, X3X4, X12, X22, and X32 in FRAP; X3, X4, X1X2, X1X4, X2X3, X32, and X42 in PA; and X1, X4, X1X4, X12, X22, and X32 in %OH−sc were significant. The predictive model results are shown in Formula The optimal extraction variables of these different bioactive groups with ethanol in the highest % DPPHsc, FRAP, PA, and %OH − sc were X1 = 10.0 min, X2 = 100.0 W, X3 = 20.0%, and X4 = 1:55.9 g/ml; the highest extraction method for TFC was: X1 = 90.0 min, X2 = 582.3 W, X3 = 30.9%, and X4 = 1:60.2 g/ml; the highest extraction method for TPC was: X1 = 90.0 min, X2 = 587.5 W, X3 = 55.6%, and X4 = 1:47.2 g/ml.Methanol extraction resulted in 80.6–145.1 mg/g in TSC from 10.1 to 26.4 mg/g for TFC, from 11.0 to 25.3 mg/g for TPC, from 10.12% to 85.61% for %DPPHsc, from 0.49 to 2.99 mmol/L for FRAP, from 0.22 mmol to 1.34 mmol for PA, and from 0.71% to 38.29% for %OH−sc. The highest TSC values were obtained using the following experimental conditions: X1 = 90.0, 10.2, 82.9, and 90.0 min; X2 = 104.7, 687.1, 700.0, and 100.0 W; X3 = 94.4%, 20.1%, 23.1%, and 42.8%; and X4 = 1:28.9, 1:18.0, 1:70.0, and 1:10.0 g/ml, respectively. The quadratic regression coefficients of X2, X4, X1X3, X2X3, X22, X32, and X42 in TSC; X1, X2, X3, X1X3, X3X4, X22, X32 and X42 in TFC; X1, X3, X1X2, X22, X32, and X42 in TPC; X2, X3, X4, X32, and X42 in % DPPHsc; X1, X4, X1X2, X1X3, X2X4, X3X4, X12, X32, and X42 in FRAP; X3, X4, X32, and X42 in PA; and X1, X4, X1X4, X12, and X32 in %OH − sc were significant. The predictive model results are shown in Formula The optimal extraction variables of these different bioactive groups with ethanol in the highest %DPPHsc, FRAP, PA, and %OH − sc were XBy optimizing the extraction conditions, under various factors, the TFC and TPC extracted by ethanol were higher than those extracted by methanol, while the TSC extracted by methanol was higher.By optimizing the extraction conditions, under the influence of various factors, the DPPH radical‐scavenging capacity, total antioxidant capacity, and total reducing capacity of the methanol extract were higher than those of the ethanol extract, while the hydroxyl‐scavenging capacity of the ethanol extract was more optimal.3.2.2The optimized extraction process for obtaining maximum values of TSC, TFC, TPC, and antioxidant activities  was obtained based on the analysis of the results of the response surface methodology study. The optimal extraction process results are shown in Table 3.3In this experiment, the extraction conditions, namely, extraction time, ultrasonic power, solvent concentration, and S/L ratio, were optimized to obtain bioactive substances in sour jujube by ultrasound. As shown in Table 3.4The ultrasonic extraction of Yangquan (Shanxi) jujube flesh, shells, and seeds was carried out with ethanol as extraction solvent. The content of TPC, TFC, and TSC, as well as the biological activity, was compared. As shown in Table 3.52O2 oxidative stress models were created to provide the condition of functions declining and damaging. As shown in Figure Drosophila compared with that of the control group.To study the antiaging effect of sour jujube extract, the H3.618 column  with a mobile phase of acetonitrile–aqueous 0.1% formic acid solution. The following gradient elution program was used: 0 ~ 26 min, 10 ~ 20% acetonitrile; 26 ~ 30 min, 20 ~ 23% acetonitrile; 30 ~ 43 min, 23 ~ 26% acetonitrile; 43 ~ 45 min, 26 ~ 37% acetonitrile; 45 ~ 47 min, 37% acetonitrile; 47 ~ 54 min, 37 ~ 39% acetonitrile; and 54 ~ 63 min, 39 ~ 100% acetonitrile. The flow rate was 1.0 ml/min, the column temperature was 25°C, the injection volume was 10 μl, and the ultraviolet (UV) detection wavelengths were 227 nm and 335 nm. A representative HPLC‐UV chromatogram is shown in Figure After being treated with a macroporous resin column, the concentrated sample solution was analyzed by high‐performance liquid chromatography (HPLC). Separation was achieved on a Waters Symmetry C4This study optimized the ultrasonic extraction process parameters to extract bioactive substances from sour jujube. Two extraction solvents, methanol and ethanol, were used to optimize the extraction parameters screened by CCD. The results showed that optimizing methanol and ethanol extraction process parameters could improve target components’ extraction efficiency. Under the optimum conditions, the TFC and TPC yields of sour jujube obtained by using ethanol were better than those obtained by using methanol, while the yield of TSC obtained by using methanol was better than that obtained by ethanol.Ultrasonic‐assisted extraction is a rapid and effective extraction method that has a higher extraction rate and detection efficiency than traditional extraction methods, and provides a good solution to the selectivity of target compounds , which results in a shortened life span  concentration, ultrasonic power, and S/L ratio, which are ultrasonic extraction variables, were optimized to achieve maximum bioactive substance contents  and optimal antioxidant activities  of sour jujube. Compared to methanol, ethanol was generally more effective in extracting solvent bioactive compounds. In addition, the antioxidant activities of sour jujube in vitro were related to the TSC, TFC, and TPC. Sour jujube extract can be considered as a source of natural antioxidants, which can improve the oxidative damage caused by free radicals, and is safer than synthetic antioxidants. Therefore, greater attention should be paid to the more widespread use of sour jujube.This research was funded by the Science Foundation for Youth of Heilongjiang Province (LH2019H106), Heilongjiang Province Postdoctoral Research Start‐up Grant (LBH‐Q16214), Heilongjiang Provincial Science Foundation Project (H2018056), Heilongjiang University of Traditional Chinese Medicine “Excellent Innovative Talent Support Program” Project (2018RCD03), National Natural Science Foundation of China (No. 81973439), and this work was also supported by The National Natural Science Foundation of China (No. 82003974) for the development of traditional Chinese medicine.The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.All animal studies were approved by the Animal Experimental Ethical Committee of Heilongjiang University of Chinese Medicine.Appendix S1Click here for additional data file.


The SARS-CoV-2 spike (S) protein engages ACE2 for cell entry, and the S protein/ACE2 interface is an important target for neutralizing antibodies. In the course of the COVID-19 pandemic, SARS-CoV-2 variants have emerged that harbor mutations in the S protein, which confer neutralization resistance and allow viral spread in immunologically nonnaive populations. The most prominent example is the highly mutated Omicron variant, which infects convalescent or vaccinated individuals with unprecedented efficiency . Here, wThe B.1.640.2 variant was detected in November 2021 in a vaccinated adult patient who had returned from Cameroon and exhibited mild respiratory symptoms . B.1.640pp) can enter cell lines commonly used for SARS-CoV-2 research. The S proteins of variant B.1, which circulated early in the pandemic, and the Omicron variant (sublineage BA.1) served as controls. All particles entered Huh-7 (human liver) and Vero cells (African green monkey kidney) robustly and at comparable rates, and entry efficiency was not increased by the expression of TMPRSS2, an S protein-activating host cell protease  was reduced, while the entry of Omicronpp was augmented, compared to that of B.1pp  and Calu-3 (human lung) cells was equal to or more efficient than that measured for B.1pp, while the entry of Omicronpp was reduced (Fig. For analysis of the B.1.640.2 S protein, we employed rhabdoviral pseudotypes, which faithfully mimic SARS-CoV-2 entry and antibody-mediated neutralization . We firsase Fig. . Further1pp Fig. . Finallyced Fig. , as prevpp by soluble ACE2 (Fig. We next analyzed the binding of the B.1.640.2 S protein to ACE2, employing S protein-transfected cells and soluble ACE2. The S proteins of B.1 and the Omicron variant bound to ACE2 efficiently, as expected, while binding to B.1.640.2 S protein was markedly reduced Fig. . The redCE2 Fig. , which ipp and B.1.640.2pp by most antibodies (Fig. pp (Fig. pp with significantly reduced efficiency compared to B.1pp, and neutralization of Omicronpp was further reduced (Fig. pp was again markedly diminished compared to that of B.1pp.Finally, we examined antibody-mediated neutralization. The analysis of monoclonal antibodies that are in development or have already been used for COVID-19 therapy revealed robust inhibition of B.1ies Fig. . Bamlani pp Fig. , in keep pp Fig. . Furtherced Fig. . Similarced Fig. . Neutralced Fig. . HoweverThe S protein of variant B.1.640.2 harbors more mutations than its counterparts in the preceding variants of concern, Alpha, Beta, Gamma, and Delta. These mutations had a moderate impact on cell entry but markedly decreased ACE2 binding, potentially due to Y449N [Supplementary material


Sodium bituminosulfonate (Ichthyol® light) was tested in double-dilution concentration steps ranging from 0.03 g/L to 256 g/L. For time-kill analysis, two reference and two clinical S. aureus strains were tested with different concentrations of sodium bituminosulfonate , 2× MIC, 4× MIC, 16× MIC and 256× MIC). For MSSA isolates, MBC50, MBC90 and the MBC range were 0.5 g/L, 1.0 g/L and 0.125–1.0 g/L; (MBC/MIC ratio)50, (MBC/MIC ratio)90 and the range of the MBC/MIC ratio were 4, 4 and 1–8, respectively. Among MRSA isolates, MBC50, MBC90 and the MBC range amounted to 0.5 g/L, 1.0 g/L and 0.06–1.0 g/L; (MBC/MIC ratio)50, (MBC/MIC ratio)90 and the range of the MBC/MIC ratio were 2, 4 and 1–8, respectively. Time-kill kinetics revealed a bactericidal effect after 30 min for sodium bituminosulfonate concentrations of 16× MIC and 256× MIC. The bactericidal activity against MSSA and MRSA was demonstrated for sodium bituminosulfonate. The killing was very rapid with the initial population reduced by 99.9% after only short incubation with concentrations of 16× MIC and higher.Antibiotic resistance is increasing worldwide making it necessary to search for alternative antimicrobials. Sodium bituminosulfonate is a long-known substance, whose antimicrobial inhibitory activity has recently been re-evaluated. However, to the best of our knowledge, the bactericidal mode of action of this substance has not been systematically characterized. The aim of this study was to investigate the in vitro bactericidal activity of sodium bituminosulfonate by determining the minimal bactericidal concentrations (MBC), as well as the rapidity of bactericidal effect by time-kill curves. Clinical isolates of methicillin-susceptible (MSSA,  Staphylococcus aureus (MRSA) have been made in certain countries, in the WHO priority list of antibiotic-resistant bacteria, MRSA is still ranked as a high priority pathogen [®, light” [S. aureus.The burden of bacterial resistance against antibiotics is steadily increasing ,2. The dpathogen . In 2019pathogen . Despitepathogen ,7. In thpathogen . Such repathogen . Sodium , light” , has bee, light” ,13,14. T, light” . To the mecA gene was detected in 19 strains, whereas one strain possessed the mecC gene. All MSSA isolates tested mecA/mecC-negative. For MSSA as well as for MRSA clinical isolates, the MIC50 and MIC90 values were 0.125 g/L and 0.25 g/L, respectively. The MIC range was 0.06–0.5 g/L for MSSA and 0.06–0.25 g/L for MRSA. The MBC50 and MBC90 values were equal for MSSA and MRSA and amounted to 0.5 g/L and 1.0 g/L, respectively 90 and the range of the MBC/MIC ratio are shown in Of 20 clinical MRSA strains, the ectively . MBC ranS. aureus ATCC 29213 by general QC testing were within the ranges recommended by EUCAST and CLSI [The MICs of vancomycin determined for and CLSI ,16.6 CFU/mL and 1.3 × 106 CFU/mL for the reference strains MSSA ATCC 29213 and MRSA ATCC 43300, respectively, 1 × 106 for clinical MSSA and 1.5 × 106 for clinical MRSA strains. At concentrations of 16 × MIC and 256 × MIC, a bactericidal efficacy of 99.9% inoculum reduction by sodium bituminosulfonate was already achieved after 30 min of incubation. Regrowth was observed at 1 × MIC, 2 × MIC and 4 × MIC within 4 to 24 h, whereas killing without regrowth was documented at 16 × MIC and 256 × MIC  were used, as well as two reference strains (MSSA ATCC 29213 and MRSA ATCC 43300). The presence of mec genes in MRSA strains was confirmed using a loop-mediated isothermal amplification assay  according to the user’s manual [For the determination of minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs), 40 clinical isolates of ® light) was provided by the Ichthyol-Gesellschaft Cordes, Hermanni & Co. .Sodium bituminosulfonate  with an incubation time of 18 ± 2 h at 35 °C in ambient air. Sodium bituminosulfonate was tested in double-dilution concentration steps ranging from 0.03 g/L to 256 g/L. As no quality control (QC) ranges exist for the susceptibility testing of sodium bituminosulfonate, MIC determination was additionally performed for S. aureus ATCC 29213 with vancomycin to control for the overall performance of the testing procedures.For MIC determination, a broth microdilution method was used according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) and the International Organization for Standardization (ISO) ,34,35. TFor the determination of MBCs, 10 µL samples from the clear wells were sub-cultured on tryptic soy agar plates  and colonies were counted after overnight incubation at 35 °C. The minimal concentration needed to kill at least 99.9% of the initial inoculum was considered as the MBC, according to CLSI . All MICS. aureus, a time-kill kinetic methodology was used. The initial liquid culture prepared in 10 mL tryptic soy broth (TSB) from overnight growth on Columbia blood agar was incubated for 3 h at 35 °C and 160 rpm. After adjusting to the 0.5 McFarland turbidity standard and dilution, 5 mL of a suspension containing approximately 106 CFU/mL was transferred into glass flasks with different concentrations of sodium bituminosulfonate . The suspensions were incubated at 35 °C and 160 rpm for 0.5 h, 1 h, 4 h, 8 h and 24 h. After incubation, 200-µL samples were collected and serially diluted, followed by plating of 10 µL in triplicate on TSA plates. After overnight incubation at 35 °C, the colonies were counted, and average values were calculated. A growth control without antimicrobial substance, as well as a sterile control, were used in each experiment. The time-kill curves were performed in triplicate.To determine the rapidity of the bactericidal effect of sodium bituminosulfonate against


The catalytic performance for hydrogen evolution reaction (HER) was evaluated on the NiMo/Ti and Au(NiMo)/Ti catalysts in an alkaline medium. It was found that among the investigated NiMo/Ti and Au(NiMo)/Ti catalysts, the Au(NiMo)/Ti-3 catalyst with the Au loading of 5.2 µg cm−2 gives the lowest overpotential of 252 mV for the HER to reach a current density of 10 mA·cm−2. The current densities for HER increase ca. 1.1–2.7 and ca. 1.1–2.2 times on the NiMo/Ti and Au(NiMo)/Ti catalysts, respectively, at −0.424 V, with an increase in temperature from 25 °C to 75 °C.In this study, NiMo catalysts that have different metal loadings in the range of ca. 28–106 µg cm Among various candidates to fulfill energy demands, hydrogen (H2) can be a potential substitute fuel for effective energy production and storage. H2 is a clean, economical renewable energy source and an excellent energy storage medium with excellent energy conversion efficiency, higher gravimetric energy density than gasoline (120 vs. 44 MJ kg−1), eco-friendliness, and zero carbon dioxide emission [2, an important chemical feedstock widely used in petroleum refining and ammonia synthesis, is industrially produced via coal gasification and steam reforming reaction under harsh conditions, resulting in the emission of greenhouse gases and micro-pollutants [2 production that gained intense research interest in the last decades, as electricity-driven water splitting generates green H2.Although fossil fuels, such as coal, oil, and natural gas, are the main energy sources and are widely used to meet energy needs, the increasing emissions of pollutants, carbon dioxide CO, and othemission ,4. H2, allutants ,6. Among2 production by electrocatalytic water splitting in alkaline media is limited by the sluggish hydrogen evolution reaction (HER) kinetics and enormous electricity consumption. The HER mechanism of electrocatalytic water splitting includes three main steps, i.e., Volmer, Heyrovsky, and Tafel reactions, as shown below, in alkaline media [It is well-known that Hne media :Volmer r3O+ in the acid electrolyte, which involves additional energy to break the H–O–H bond [The sluggish HER kinetics in alkaline media is mainly due to the fact that in the Volmer reaction, the proton source comes from the water molecule instead of HO–H bond . To dateO–H bond ,9. In thO–H bond ,14,15,16O–H bond ,18,19, iO–H bond ,21, tungO–H bond ,23, and O–H bond ,25, phosO–H bond ,27,28, nO–H bond , and sulO–H bond , were inO–H bond . A numbeO–H bond , electroO–H bond ,33,34, h−2 at a small overpotential of −63 mV in 1 M KOH electrolyte [−2, respectively, in 1 M potassium hydroxide (KOH) solution [According to Engel–Brewer valence bond theory, whenever metals of the left half of the transition series (such as Ni and Co) are alloyed with metals of the right half of the transition series metals , a synergistic effect can be anticipated in terms of hydrogen evolution activity . The synctrolyte . Heterossolution .This study presents a simple and low-cost procedure to fabricate efficient catalysts for HER. Three-dimensional (3D) binary Ni-Mo catalysts with different total metal loadings supported on a titanium (Ti) surface (denoted as NiMo/Ti) were prepared via the electrodeposition method, whereas for the decoration of the prepared NiMo/Ti catalysts with a small amount of Au crystallites, the galvanic displacement method was used.4 (99.995%) were purchased from Sigma-Aldrich  Supply. H2SO4 (96%), HCl (35–38%), nickel sulfate hexahydrate , sodium molybdate dihydrate , and NaOH (98.8%) were purchased from Chempur Company . Ultra-pure water with a resistivity of 18.2 MΩ·cm−1 was used for preparing the solutions. All chemicals were of analytical grade and used directly without further purification.Titanium foil (99.7% purity) and HAuCl2+ and Mo6+ ion on the surface of the Ti electrode, followed by a spontaneous Au displacement from the Au (III)-containing solution. Before the deposition of the NiMo catalysts, the Ti plates were pretreated in diluted H2SO4 (1:1 vol) at 70 °C for 3 s. NiMo catalysts were electroplated on the Ti surface (1 × 1 cm) from a bath containing 0.03 M Na2MoO4 along with 0.1, 0.2, and 1.0 M NiSO4 in an acidic condition (1.5 M H2SO4 and 1 M HCl). The chronopotentiometry was used for the NiMo coatings deposition on the Ti surface. The plating of coatings was carried out at the current of 0.1 mA and 1 mA for 3 min at each current. The Au crystallites were deposited on the prepared NiMo/Ti electrodes by their immersion into 1 mM HAuCl4 + 0.1 M HCl solution for 10 s. After plating, the samples were taken out, thoroughly rinsed with deionized water, and air-dried at room temperature.The catalysts were prepared by a facile, two-step process that involves electrodeposition of NiThe morphology and composition of the prepared catalysts were investigated by scanning electron microscopy (SEM) TM 4000 Plus .XRD patterns of pure Ti sheet, Ni-Mo/Ti, and Au-Ni-Mo/Ti catalysts were measured using an X-ray diffractometer D2 PHASER . The measurements were conducted in the 2θ range of 10–90°.Ni 231.604 nm, λMo 202.031 nm, and λAu 267.595 nm.The metal loadings were determined by inductively coupled plasma optical emission spectrometry (ICP–OES) analysis. The ICP–OES spectra were recorded using an Optima 7000DV spectrometer  at wavelengths of λA conventional three-electrode electrochemical cell was used for electrochemical measurements. The fabricated NiMo/Ti and Au(NiMo)/Ti catalysts were employed as working electrodes, a Pt sheet was used as a counter electrode, and a calomel electrode was used as a reference. All potentials in this work were converted to the reversible hydrogen electrode (RHE) scale using the following Equation (5):2. Linear sweep voltammograms were recorded in a 1 M NaOH solution and always deaerated by argon (Ar) for 20 min prior to measurements. HER polarization curves were recorded from the open circuit potential (OCP) to −0.42 V (vs. RHE) at a polarization rate of 10 mV·s−1. Polarization curves were recorded at several temperatures from 25 to 75 °C, and temperatures were set with a water jacket connected to a LAUDA Alpha RA 8 thermostat. Stability was studied by recording chronoamperometry (CA) curves for HER at a potential of −0.22 V (vs. RHE) for half an hour. All electrochemical measurements were performed with a Metrohm Autolab potentiostat  using the Electrochemical Software (Nova 2.1.4).Current densities were calculated using the electrodes’ geometric area of 2 cm4 + 0.1 M HCl solution for 10 s at room temperature.In this study, we investigated the electrocatalytic activity of prepared 3D binary NiMo/Ti and ternary Au(NiMo)/Ti catalysts for HER. These catalysts were deposited on the Ti surface (1 × 1 cm) using an electrochemical bath. The optimal conditions for different 3D binary catalyst depositions were determined and are given in 4 + 0.1 M HCl solution for 10 s, the porous leaf-like alloy structure was immediately covered with a tiny globular surface  in the prepared catalysts are quite different and vary from 23.9 up to 106.2 µgmetalcm−2. It should be noted that Ni and Mo amounts increase in the NiMo coatings by increasing the Ni2+ concentration in the plating solution, whereas the Mo amount was kept the same. Calculated Mo:Ni and Au:NiMo mass weight ratios are given in It can be seen that the formed 3D binary NiMo/Ti catalysts contained ca. 82.8–93.7 wt.% of Ni, whereas those 3D ternaries Au(NiMo)/Ti catalysts possessed 76.6–87.9 wt.% of Ni. The total metal loadings  in –1 in a potential range from open-circuit potential (OCP) up to −0.42 V (vs. RHE) for HER, at several temperatures from 25 up to 75 °C  using Au-decorated ternary Au(NiMo)/Ti-3, Au(NiMo)/Ti-2, and Au(NiMo)/Ti-1 catalysts, and relatively lower −34.81, −26.5, and −21.75 mA·cm−2 current densities were recorded at the same potential via using 3D binary NiMo/Ti-3, NiMo/Ti-2, and NiMo/Ti-1 catalysts at 25 °C, respectively (−2 (η10) were found in a gradual increasing order for both Au(NiMo)/Ti and NiMo/Ti catalysts as follows:For instance, the current densities of −49.84, −40.73, and −36.58 mA·cmectively . OverpotAu(NiMo)/Ti-3 (−252 mV) < Au(NiMo)/Ti-2 (−298 mV) < Au(NiMo)/Ti-1 (−308 mV)NiMo/Ti-3 (−288 mV) < NiMo/Ti-2 (−344 mV) < NiMo/Ti-1 (−349 mV).2+ concentration in the plating solution /Ti catalysts, Tafel slope values of 143.8, 98.7, and 131.2 mV·dec−1 were determined at Au(NiMo)/Ti-1, Au(NiMo)/Ti-2, and Au(NiMo)/Ti-3 catalysts, respectively (0) reflects the electrode’s intrinsic activity for HER. The exchange current density (j0) was calculated for HER at all six catalysts by extrapolating the Tafel plots, η vs. log j. Thus, the j0 value of 0.144, 0.011, 0.076, 0.075, 0.006, and 0.005 mA·cm−2 were calculated for Au(NiMo)/Ti-3, Au(NiMo)/Ti-2, Au(NiMo)/Ti-1, NiMo/Ti-3, NiMo/Ti-2, and NiMo/Ti-1 catalysts, respectively /Ti-3 coating was ca. 2–28 times higher than that determined for the rest of the studied catalysts.HER polarization curves were then further used for constructing the Tafel plots and calculating the Tafel slope. Tafel slope values of 99.6, 100.5, and 130.4 mV·decectively . The detectively . It is w−2 at 50 s) (−2) and ca. 5 times lower with Au(NiMo)/Ti-1 catalysts (−2.02 mA·cm−2). In the case of the binary NiMo/Ti-3 catalyst, a comparatively lower current density was recorded (−6.13 mA·cm−2 at 50 s) along with a 3–5 times lower value for NiMo/Ti-2 (−2.06 mA·cm−2) and NiMo/Ti-1 (−1.23 mA·cm−2) catalysts.Another crucial criterion for an advanced electrode material is its electrochemical stability. Chronoamperometric measurements with all six catalysts were carried out in 1 M NaOH at −0.22 V for 2 h. Initially, in the first 50–200 s, a decrease in current density was observed for all investigated catalysts. However, after approximately 500 s, the current densities settled down and remained apparently parallel throughout the experiments. CA results confirm the result of LSV analysis in terms of the ternary Au(NiMo)/Ti-3 catalyst, giving the highest current density during HER (−10.36 mA·cmat 50 s) . A more A comparison of HER parameters generated using herein-tested NiMo/Ti and Au(NiMo)/Ti catalysts in an alkaline medium with some electrodes reported in the literature is given in −2 were prepared using a simple and low-cost metal electrodeposition method. The decoration of the prepared NiMo/Ti catalysts with a small amount of Au-crystallites in the range of ca. 1–5 µg cm−2 was carried out using the galvanic displacement method.In summary, NiMo and Au(NiMo) catalysts supported on a titanium surface were studied as electrocatalysts for HER in an alkaline medium. NiMo/Ti catalysts with different total metal loadings in the range of ca. 28–106 µg cm−2 exhibits the highest current density, as well as exchange current density during HER in a 1 M NaOH solution. Moreover, the Au(NiMo)/Ti-3 catalyst also displays excellent HER performance with an overpotential of 252 mV at a current density of 10 mA·cm−2.It was determined that, among the investigated catalysts, the Au(NiMo)/Ti-3 catalyst with the Au loading of 5.2 µg cm


However, this did not entail relevant changes in fatty acid content or in the abundance of volatile organic compounds from oxidised lipids. Increasing the RLE dose only improved its antioxidant efficacy for some oxidation indexes. Thus, the oxidative deterioration of these types of fish emulsion can be naturally controlled with rosemary extracts rich in diterpenes.The use of natural plant extracts with standardised antioxidant properties is a growing strategy to stabilise food products. The use of a rosemary lipophilic extract (RLE), obtained from the by-product of high-yield selected plants and rich in polyphenols , is here proposed. Four RLE doses  were tested in a salmon pâté formulated with sunflower oil and linseed, which was pasteurised (70 °C for 30 min) and subjected to storage at 4 °C and 600 lux for 42 days. Rosemary diterpenes resisted pasteurisation without degrading and showed antioxidant activities during the shelf-life of pasteurised pâté. RLE addition led to increased peroxide value (from 3.9 to 5.4 meq O Fish emulsions, such as salmon pâté, represent a valuable dietary source of polyunsaturated fatty acids (PUFA), in particular, of some essential long-chain n-3 PUFA, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) . The intNowadays, “green label” formulations for fish products often include “generally recognised-as-safe” (GRAS) natural preservatives. Among these, phenolic compounds present in fruits, vegetables, beverages , plants, seaweed and some herbs show antioxidant and antimicrobial activities, considered as emerging natural additives for fish products . PhenoliRosmarinus officinalis L.) is considered a source of phenolic antioxidants, being particularly rich in some hydrophobic diterpenes such as carnosic acid and carnosol, which can act as free radical scavengers in lipid matrixes . Salmo salar) loins were purchased from Nordlaks Produkter AS  and stored at −20 °C for 48 h. Thawed loins (at 4 °C/24 h) without skin and bones were cut in cubes (2 cm3) using a knife and cooked in water  at 90 °C for 10 min under stirring in a cooking robot . Before homogenising, cooked salmon cubes were minced for 10 min, skimmed milk was dissolved in water at 50 °C and linseed was hydrated at 1:2 (w:v) in a Silent Crusher homogeniser at 9500 rpm for 1 min. All ingredients  were homogenised together for 10 min. Once paste was obtained, sterile glass jars (33-mL capacity) were manually filled and pasteurised in a water bath at 70 °C for 30 min. The jars were cooled in a water bath at 5 °C for 30 min and then kept for up to 42 days in a refrigerated display cabinet  at 4 °C, 30% RH and white continuous illumination (600 lux). The antimicrobial efficiency of these pasteurisation conditions was checked in a previous trial [Salmon pâté ingredients are shown in us trial . SamplesCrude protein content was determined by the Kjeldahl method  . Crude apH was determined with a MicropH 2001 pH meter  and a Cat. 52-22 combined electrode . CIELab colour was measured using a CR-200/08 Chroma Meter II  with a D65 illuminant, 2° observer angle and 50-mm aperture size. The results were expressed as CIELab values: lightness (L*), redness (a*) and yellowness (b*).v/v) and 0.003% (w/v) BHT and then mixed with 1 M KCl. The organic phase was separated and dried in a Hei-vap rotavapor  at 30 °C and 337–470 mm Hg pressure. Fat content was gravimetrically determined  and stored at −80 °C until analyses.Pâté fat was extracted by the Folch method with some modifications . First, 2/kg fat.PV indicated the amount of hydroperoxides of fat pâté (AOCS Cd 8-53) . In brieCD and TC were determined according to Pegg (2001) , with soTBARS were measured according to Botsoglou et al. (1994)  and calcFA were identified and quantified as described by Cardenia et al. (2015) . Twenty 2 for COP purification. Both sterol and COP fractions were derivatized with 1 mL of a silylating mixture , left standing at 40 °C for 20 min, dried under a nitrogen stream, re-dissolved in n-hexane  and injected into a Fast gas-chromatograph/mass-spectrometer (Fast GC/MS) as reported by Cardenia et al. (2012) [Sterols were extracted and purified according to Cardenia et al. (2015) . About 2. (2012) , with sl. (2012) . Nine in6.VOC were determined by headspace solid phase-micro-extraction (HS-SPME) according to Ortuño et al. (2021) , with soTwenty-two polyphenols were determined in RLE , includiSalmon pâté contained 12 g/100 g total protein, 19 g/100 g total lipids and 61 g/100 g moisture, contributing with 270.6 kcal/100 g . Data ret n-9 as main FA (18.3–22.2 g/100 g fat), followed by PUFA (17.3–20.2 g/100 g fat), with C18:2c n-6 (11.8–13.8 g/100 g fat) and C18:3α n-3 (2.1–2.7 g/100 g fat) as major FA, and by SFA (9.2–10.6 g/100 g fat), with C16:0 as predominant FA (3.6–4.6 g/100 g fat). Levels of EPA and DHA were around 1 and 1.4 g/100 g fat, respectively. RLE addition did not affect the FA levels on day 0 and 42, while chill storage only led to some changes in minor FA. TFA content was similar for the untreated and the RLE pâtés on day 0 and 42. Consequently, nutritional FA ratios (n-6/n-3 and P/S) were unaffected by RLE addition or chill storage.Twenty-two FA were quantified (g FA/100 g fat) . The mosThe most abundant sterol was cholesterol (33.7–41.7 mg/100 g pâté), followed by β-sitosterol (12.9–17.8 mg/100 g pâté), campesterol (2.3–3.9 mg/100 g pâté) and stigmasterol (1.6–2.2 mg/100 g pâté) . CampestA total of twenty VOC were identified in the headspace of salmon pâté including several hydrocarbons , alcohols -2-penten-1-ol, 1-octen-3-ol, eugenol, and benzyl alcohol), aldehydes -octanal and 2,5-bis(trimethyl)-benzaldehyde), ketones , organic acids (2-amino-4-methyl-benzoic acids) and terpenes (D-limonene and α-pinene). The relative abundance of most of these VOC was unaffected by the addition of RLE or chill storage, with some exceptions . Of thesw:w) of carnosic acid plus carnosol, a percentage widely exceeded by the RLE used in this experiment. Assessment of the stability of carnosic acid and carnosol provides an idea of how rosemary antioxidants behave in food matrices. Under oxidising conditions, carnosic acid is transformed into carnosol [Recovery of rosemary polyphenols depends on the raw materials, solvents and operating conditions applied to obtain the extracts ,40. Diffcarnosol ,43, whiccarnosol . In genecarnosol ,41. In tcarnosol  found thcarnosol ,23.The formulation used for salmon pâté in the present study enabled the reduction of fat content and caloric intake compared to commercial fish pâtés  ,45,46. SRosemary polyphenols, containing benzene rings able to act as free radical scavengers, fulfilled their antioxidant role delaying lipid oxidation in the pâté; however, in quantitative terms, the addition of up to 210 mg/kg of rosemary polyphenols does not seem enough to protect a fatty fish emulsion  against oxidation. Lipid oxidation reactions involve three stages: initiation, propagation and termination. Some techniques measure the loss of initial reactants , others the formation of primary oxidation products (such as hydroperoxides and conjugated dienes) and others the formation of secondary oxidation products  . ThermalTBARS is a widely used index with technological (flavour and rancidity) and nutritional (toxicity) implications that measures the levels of aldehydes and other secondary oxidised lipids in the pâté. Lipid oxidation is enhanced by the thermal treatment and favoured by fat unsaturation and the presence of oxygen. Salmon pâté presented an incipient lipid oxidation just after pasteurisation. The antioxidant role of RLE during pâté preparation was modest, since it only slightly inhibited the formation of MDA and was not dose-dependent. Salmon was cooked before adding RLE, which would explain why there were no marked differences regarding MDA levels among the different freshly pasteurised pâtés. TBARS formation continued during further retail display. On day 42, MDA level practically doubled in the pâté formulated without RLE, reaching TBARS values near 2 mg MDA/kg, the threshold value from which rancidity is sensorially detected in cooked meat . In contThe antioxidant effect of RLE was also confirmed for cholesterol. The COP profile found in this study is similar to that reported by Echarte et al. (2004)  for salmRLE addition did not initially affect pâté colour, as also reported in pork liver pâté with RLE . It is uThe pH of pâté was slightly lower when RLE was incorporated into its formulation. Similar results were found in pork liver pâté made with RE  and withA relevant part of the VOC identified in headspace corresponded to C5–C8 aliphatic aldehydes and alcohols. Hexanal has been identified as the most abundant VOC in sunflower and linseed oil blends, together with other VOC -2,4-heptadienal) that contribute to oil aroma . In addiFood applications for rosemary extracts require standardising their polyphenol content. The crop of high-yielding selected rosemary plants can help in improving and standardising these extracts. As reported in the present study, the rosemary lipophilic extract used is rich in polyphenol antioxidants, particularly diterpenes, and provides good results in salmon pâté, a product prone to oxidation. Rosemary diterpenes resist pasteurisation without degrading and can act as antioxidants during the shelf-life of pasteurised pâté. As a result, lipids are stabilised against oxidation and salmon pâté contains less secondary oxidation compounds, in all likelihood being healthier. However, this did not entail relevant changes in the fatty acid content or relative abundance of volatile organic compounds from lipids. Therefore, oxidative deterioration of these types of fish emulsions can be naturally controlled with rosemary extracts rich in diterpenes, thus representing a valid alternative for the formulation of clean label seafood products. It is not necessary to reach the maximum quantity of rosemary diterpenes (150 mg/kg) authorized for fish products to obtain a suitable antioxidant effect in this PUFA-enriched pâté. Most of the oxidation indices improved in the salmon pâté when a third of the aforementioned dose was used.