Thisbehavior was observed for aryl acetylenes in their regioselectivecyclotrimerization to 1,2,4-(aryl)-benzenes. Two kinetically distinctregimes are observed dependent upon the substrate-to-catalyst ratio([RC≡CH]0/[1]0), referredto as the low ([RC≡CH]0/[1]0 < 40) and high ([RC≡CH]0/[1]0 > 40) regimes. Both showedsigmoidalkinetic response, with positive Hill indices of 1.85 and 3.62, respectively,and nonlinear Lineweaver–Burk replots with an upward curvature,which supports positive substrate cooperativity. Moreover, two alkynemolecules participate in the low regime, whereasup to four are involved in the high regime. The second-orderrate dependence on 1 indicates that binuclear complexesare the catalytically competent species in both regimes, with thatin the high one being 6 times faster than that involvedin the low one. Moreover, Eyring plot analyses revealedtwo different catalytic cycles, with a rate-determining step moreendergonic in the low regime than in the high one, but with a more ordered transition state in the high regime than in the low one.Enhancing catalytic activity through synergic effectsis a currentchallenge in homogeneous catalysis. In addition to the well-establishedmetal–metal and metal–ligand cooperation, we showcasehere an example of self- This self-activation by the substrate, alsotermed homotropic cooperativity, is characterized by kinetic patternsstrongly deviated from the typical hyperbolic Michaelis–Mentencurve and can be identified from kinetic studies.6Another option involvesthe substrates behaving as the main cooperativeactors.1, 3)(dipp)}],8 [Fe{N(SiMe3)(R)}2]−,9 and [Fe{N(SiMe3)2}2],10 as the sole previously reported examples benzenescatalyzed by a two-coordinate iron complex 1, 1. The poexamples 1.11 include isolated low-valent iron(0) complexessuch as tri-/tetra-coordinatedcomplexes with diphosphines,12N-heterocyclic carbenes,13 diolefins,14 and pyrimidinediimine ligands.15 This last example exhibits an unusual 1,3,5-regioselectivity;typically the major product is the unsymmetrical 1,2,4-arene. In otherinstances, hydride iron complexes formed in situ suchas [FeI(pincer)(H)],16 and [FeII(H)]17 have been proposedas the active species. Additionally, different combinations of ironsalts and reductants18 or activators,19 as well as a three-component system 20 also result in catalytically competent low-valent iron species.Related iron catalysts for this reaction2C6H3)2]  in C6D6 at 60 °C . The major product from the reaction was found to be the unsymmetrical1,2,4-tri(phenyl)benzene (2) along with the symmetrical1,3,5-tri(phenyl)benzene (3) and a small amount of unidentifiedoligomers with a ratio 2:3:others of 76:11:13.A noticeable decrease in the reaction time wasobserved with alarger substrate loading and identical initial concentration of theprecatalyst 22 The best fit for run 2 was obtained when plotting 1/[PhC≡CH] versus time, suggestive of a second-order reaction for thealkyne . Noticeably, an apparent fractional order (n) of 1.5 wasobtained for run 1. A straight line required plotting 1/([PhC≡CH])0.5versus time .A particularfeature of these two catalytic runs is their kineticprofiles, which were initially analyzed by using the general equationfor an nth-order reaction for the substrate.apparent reactionorder inPhC≡CH can be attributed to either the initial concentrationof phenylacetylene or to the [PhC≡CH]0/[1]0 ratio. In order to discriminate between these, additionalexperiments at similar [PhC≡CH]0 but with [PhC≡CH]0/[1]0 ratios of 36 and 100 were carriedout , which clearly indicates that the [PhC≡CH]0/[1]0 ratio plays a more importantrole than [PhC≡CH]0 .This change in the 23 for runs 1–3 , which were obtainedby shifting thetime scale of runs 3 and 1 to the point in which run 2 (with the highestsubstrate concentration) reaches [PhC≡CH] = 0.479 and 0.266M, respectively. As shown in Additional information was gathered from the time-adjustedprofilesV0) with the initial concentration of phenylacetylene at the same catalystconcentration ([1]0) (Table S1). Saturation kinetics with an increase of the initialconcentration of phenylacetylene was observed, but, to our surprise,the curve displayed significant deviation from the hyperbolic Michaelis–Mentenbehavior, showing a sigmoidal shape. This feature implies that theoverall catalytic process is cooperative in nature , and h (unitless), alsoknown as the Hill index, reflects the level of cooperativity. Valuesof h > 1 indicate positive cooperativity, whereas h < 1 indicates negative cooperativity. Noncooperativeprocesses correspond to h = 1, and The data were then analyzed using The major difference between the Michaelis–Mentenbehavior2 and tho24 for all of the data producedthe following parameters: Vmax = 0.040± 0.0016 M min–1, K0.5 = 1.17 ± 0.031 M, h = 3.62 ± 0.20, althoughit is not sufficient for [PhC≡CH] below 0.6 M (h) for Vmax = 0.040gives an average value of h = 2.3 (low (inred) and high (in green) (The best fitow 0.6 M 3a. Indeen green) 3b.high and low regimesindependently, the plots in Vmax = 0.040± 0.0016 M min–1 for the high regime was directly obtained from the data. However, Vmax for the low regime showed a largeerror (0.0078 ± 0.0039 M min–1) because ofthe lack of data at a high concentration of phenylacetylenein this regime. A more precise value of Vmax for this regime of 0.0063 ± 0.00019 M min–1 was independently estimated by alternative methods .If we fit the data to the Hillequation for the h) in both regimes indicatespositive cooperativity such that coordination of one molecule of phenylacetyleneto the catalyst facilitates the binding of the next, thus enhancingthe catalytic activity. Moreover, analysis of the data using the Lineweaver–Burkreplot showed the characteristic nonlinear upward curve for both regimes,again indicating positive cooperativity . The Hill index value of 1.85 in the low regime implies that cooperation is restricted to two molecules ofphenylacetylene, which increases to four molecules at high [PhC≡CH]0/[1]0 ratio .The positive Hill index (cat) capable of interactingreversibly with up to two molecules of substrate (S)(K1 and K2h > 1) means that K2 > K1. If the system were “infinitely” cooperative (K2 ≫> K1) such that only species cat and cat-S2 are present in the reaction,a value of h = 2 is expected. Thus, the observedvalue of 1.85 in the low regime indicates a highlevel of cooperativity.For a catalyticsystem (rate (S)3, the Hicat-S4 are involved, wherea value of h =4 would reflect “infinitely” cooperativebehavior involving four molecules of substrate and only the species cat and cat-S4 in thereaction media. Accordingly, the value of 3.62 estimated for the high regime is again indicative of a high level of cooperativity.The same applies if four equilibriato apparent partial reaction orders in the substrate from zeroto h can be observed depending on the relative valuesof K0.5 and [S]At this point, it is interesting to comment that, for kineticsfollowing low regime ([PhC≡CH]0/[1]0 < 40) and [1]0 ≈ 0.013 M, thedata is represented closer to Scenario A, where the reaction is operatingclose to the maximum partial reaction order. Consequently, the observedpartial reaction order in the alkyne of 1.50 , two different apparent partial reaction orders in the alkyne were observed:2 and 2.9 .The former, which contains a [PhC≡CH]0 ≈1.33 M, is in the region between both Scenario A and B where (K0.5)h is comparableto [S]h. This leads toa discrepancy of the partial reaction order of PhC≡CH to themaximum value of h (2 vs 3.82).For experimentsin the 0 to 0.47 M (at the same[PhC≡CH]0/[1]0 ratio of100), the experimental conditions approach Scenario A  and consequently,the apparent partial reaction order in the alkyneshould increase . Therefore,the value of 2.9  values,when fitting the data to the generalpower law V = k[S]n, are not directly related to the molecularityof any individual elementary step in the mechanism.It should be emphasizedthat noninteger (1.5) or excessively 1. For these studies, the standard ‘normalized time scale method‘25 cannot be used since it requires varying [1]0 at a fixed [PhC≡CH]0. This methodologyis associated with a change in the [PhC≡CH]0/[1]0 ratio, which in our case is associated witha change in the kinetics. Consequently, experiments were performedvarying [1]0 at fixed [PhC≡CH]0/[1]0 ratios of 20 and 100  and analyzed by the “adjusted normalized timescale method.”25 To thisend, the x-axis of the standard [substrate] vs t[cat]n plots was shiftedthe standard time adjustment (τ) multiplied by 0.0133  to the power ofthe order in the catalyst. A good overlap of the substrate profileswas observed for a partial reaction order of 2 for the catalyst  vs ln([1]0) forthe catalytic runsin the low regime (Table S2), which all displayed the same apparent order in[PhC≡CH] (n = 1.5), also indicated a partialreaction order in the catalyst of 2. A straight line with a slopeof 2.08 ± 0.00011 was obtained . Moreover, the y-intercept gives a value for kcat (low) = 35.5, which leads to Vmax = kcat ([1]0)2 = 0.0063 ± 0.00019 M min–1 for a [1]0 = 0.0133 M. Thisvalue produces a more precise sigmoidal curve for the low regime (Vmax = 0.04 Mmin–1 (kcat (high) = 226.1. These results allow usto conclude that the catalytically active species in the high regime reacts around 6 times faster than those in the low regime. Moreover, the homotropic cooperativity, involving up tofour molecules of alkyne, accounts for the considerable 54% decreaseof the reaction time (from 13 to 6 h) on increasing [PhC≡CH]0 from 0.26 to 1.33 M  of PMe3 as a catalyst poison. The reaction is slower in the presence ofPMe3 with a kobs (PMe3)/kobs (none) ratio (6.073 × 10–3/1.91 × 10–2) of 0.32 2, a value for kobs (PMe3)/kobs (none) of 0.36 (0.62) is expected.26Further support for the binuclear nature ofthe active specieswas established by an additional experiment using a substoichiometricamount . At alltemperatures studied, an apparent reaction orderin phenylacetylene of 2 was observed for a ratio of [PhC≡CH]0/[1]0 = 100, but of 1.5 with a ratioof [PhC≡CH]0/[1]0 = 20 . Consequently, temperature does notaffect the apparent reaction order in PhC≡CH,which is mainly controlled by the [PhC≡CH]0/[1]0 ratio.The effect of temperature was analyzed by performingthe catalysisat 40, 80, and 100 °C for both regimes. At room temperature (≈25°C), the catalysis was found to be very slow achieving only a32% conversion after 16.5 h (kobs/T) vs 1/T overthe temperature range of 40–100 °C give the activationparameters ΔH≠ = 8.31 kcalmol–1, ΔS≠ = −41.50 cal mol–1 K–1 and ΔH≠ = 13.98 kcal mol–1, ΔS≠ = −27.40cal mol–1 K–1 for the high and low regimes, respectively, confirmingthe presence of two different catalytic cycles  intermolecularcompetition27 was monitored to determinethe isotope effect. This method was chosen mainly because cyclotrimerizationof PhC≡CH and PhC≡CD occurs under exactly the same conditions,avoiding experimental errors, and thus the ratio of the products (PH/PD) can be measuredby NMR spectroscopy with good precision. Small values of PH/PD of 1.07 and 1.09, forthe high and low regimes, respectively,were observed. Therefore, neither the rate-determining step nor thepreequilibria detected by the sigmoidal behavior  were followed by 1H NMR spectroscopy.In both cases, the slow transformation of PhC≡CH into the correspondingtrimers takes place, whereas complex 1 was the only observedorganometallic species, a clear indication that K1 in our case is small.To help identify the species formed prior tothe rate-determiningstep, stoichiometric reactions with two and four equivalents of PhC≡CH.30 These reactions serve as a basis for the reactivity observed asthere can be no cleavage of the acetylene C–H bond during thereaction. Inserting the terphenyl into the acetylene can open up thecoordination sphere, allowing more acetylene to bind. Thus, startingfrom complex 1, coordination of the first molecule ofPhC≡CH would give the tricoordinated species A.31 At this point, molecular models revealedthat there is not enough space in A to accommodate asecond molecule of phenylacetylene. Consequently, insertion of theacetylene into Fe–C bond is proposed to provide access to thesecond molecule of PhC≡CH. This insertion reaction is associatedwith a small change in the C–H bond (change of hybridizationof the carbon from sp to sp2) and therefore a small KIEis expected as observed. Through this second equilibrium, species B would be formed. Since B is less crowded than A, equilibrium 2 is expected to be shifted to the right (K2 ≫ K1).From the literature, reversible acetylene insertionhas been shownto be possible using various metals , allowing then the easyentry of the fourth acetylene to yield D. The valuesof the Hill indexes of 1.85 and 3.62 agree with this proposal, withmainly complex 1 and intermediate B in the low regime, but 1 and D in the high regime. In this scenario, a low value for K1 is required to account for complex 1 asthe only observable species even with a ratio [PhC≡CH]0/[1]0 = 150, the highest experimentallyfeasible.At a high concentration of phenylacetylene, a third molecule ofPhC≡CH could reproduce the reaction to give intermediate low and high catalytic cycles,which areboth binuclear according to the second partial reaction order in complex 1. Among the wide range of possibilities to form binuclearcomplexes from B and D, a reasonable proposalinvolves further interaction of both with complex 1,which could give the dimer-low and dimer-high depicted in high regime.The next point concerns the catalytically active speciesin the 32 a picturealso found in mononuclear iron complexes and iron(0) nanoparticles.However, a novel binuclear alternative as proposed herein has beenrecently reported for iron cyclotrimerization,8a as well as in related metal complexes.33In general terms, mononuclear catalystshave been mainly reportedfor alkyne trimerizations,Table S3). In addition, 3C6H4C≡CH and 4-BrC6H4C≡CH) increases the reaction rate, whereas the oppositetrend was observed with the electronically richer alkynes 4-MeC6H4C≡CH and 4-tBuC6H4C≡CH. In the latter case (in pink),the catalysis slows down after 45% conversion, most likely due toan unknown impurity that deactivates the catalyst.The scope of the reaction was extended to other terminalaryl acetylenes.In all cases, the catalysis is regioselective to the correspondingunsymmetrical 1,2,4-cyclotrimers (85–97%) (V0) vs Hammett constants(σp)34 reveals alinear correlation  is noted to be slightly faster than with the more stericallyhindered isomer, 2-MeC6H4C≡CH  immediately reactswith 1 at roomtemperature to afford a gummy precipitate, insoluble in common organicsolvents, which is presumably polymeric. Internal alkynes such asPhC≡CPh did not undergo cyclotrimerization at 60 °C, probablydue to steric effects.In addition, the highly electron-deficientmethyl propiolate .As described above for phenylacetylene,the alkynes shown in 0/[1]0 ratioin such a way that two alkyne molecules cooperate at ratios <40,whereas up to four molecules cooperate at ratios >40. Values fortheHill index of 1.85 and 3.62 strongly support a noteworthy high levelof positive substrate cooperation in both scenarios. Moreover, activespecies in the high regime were found to react ca. 6 times faster than those involved in the low regime. Accordingly, two sets of parameters for the high and low regimes, respectively, confirming the presenceof two different catalytic cycles, were obtained from Eyring analyses.The rate-determining step in the low regime was foundto be more endergonic than in the high one, witha more ordered transition state in the high regionthan in the low one. We believe that our findingsreveal unique features of the kinetic behavior of alkynes, providingkey insights into detecting and analyzing these atypical phenomenain reactions catalyzed by organometallic complexes.In summary, this work demonstrates an unprecedented caseof homotropiccooperativity of alkynes in regioselective [2+2+2]-cycloadditionsto 1,2,4-(aryl)-benzenes. The kinetic behavior is strongly dependenton the [RC≡CH]


The designed material presented favorable features of peroxidase-like ability and photodynamic property, which endowed it with fascinating antimicrobial capacity. Compared with free MoS2 nanosheets, the MoS2/Ag nanosheets (denoted as MoS2/Ag NSs) exhibited better antibacterial performance against Staphylococcus aureus by the generated reactive oxygen species (ROS) from both peroxidase-like catalysis and photodynamic, and the antibacterial efficiency of MoS2/Ag NSs could be further improved by increasing the amount of Ag. Results from cell culture tests proved that MoS2/Ag3 nanosheets had a negligible impact on cell growth. This work provided new insight into a promising method for eliminating bacteria without using antibiotics, and could serve as a candidate strategy for efficient disinfection to treat other bacterial infections.Pathogenic microbial infections have been threatening public health all over the world, which makes it highly desirable to develop an antibiotics-free material for bacterial infection. In this paper, molybdenum disulfide (MoS Bacterial infection, a major case resulting in pathological disorder, remains challenging to treat in terms of its highly horrible morbidity and mortality . With thDue to the minimal likelihood of developing drug resistance and indeed the reliance on photo-responsive materials to carry out the operation , the pho2 has been utilized in the departments of catalysis   , hastenie (H2O2) . Therefo2/Ag nanosheets (denoted as MoS2/Ag NSs) with rapidly adjustable antibacterial properties was constructed by a simple chemical exfoliation and xenon lamp reduction method  layered bulk crystals, n-Butyllithium (2.5 M in cyclohexane) were purchased from Sigma Aldrich (United States). Polyvinylpyrrolidone (PVP) with the average MW 58000 was purchased from Aladdin Reagent Company (China). AgNO3 was obtained from Macklin. Ag nanoparticles were successfully grown on MoS2 nanosheets by 300 W xenon lamp (PLS-SXE100) irradiation. 3--2,5-diphenyl tetrazolium bromide (MTT) was provided from Sigma Biochemical Technology Co., Ltd. . LIVE/DEAD™ Baclight™ Bacterial Viability Kit (L7012) was purchased from Invitrogen (China). Cell counting Kit-8 (CCK-8) was purchased from Beyotime (China). Staphylococcus aureus and L929 cells were supplied by the Henan Eye Institute.Molybdenum disulfide . UV-Vis-NIR spectra were detected with a spectrophotometer from Agilent Technologies . Fluorescence pictures of microbial were observed with a Nikon 80i fluorescence microscope.Scanning electron Microscope (SEM) images were captured Field Launch Scanning Electron Microscope . Transmission electron microscope (TEM) images taken on by field emission transmission electron microscope . The diameter of particles and zeta potential of MoS2 nanosheets  were immersed in a solution of n-butyllithium  and kept in glove boxes for 48 h to obtain lithium intercalation compounds. The precipitate at the bottom was washed three times with hexane after taking of the upper layer of n-butyllithium, followed by adding 50 mL of water and sonicated for 30 min to produce a homogeneous suspension. The large-size nanosheets were removed by centrifuging at 5,000 rpm for 10 min. The supernatant was further centrifugation products at 5,000–12000 rpm were collected and washed three times with DI water to obtain MoS2 nanosheets.The similar approach outlined in the prior research was used to prepare the MoSnosheets . The spe2 suspension in triplicate, and the AgNO3 (1 mg/mL) solution with various volumes  was added to the mixture. After stirring evenly, the mixture was lit under a xenon lamp at 300 W for 30 min. The crude products with different silver content were denoted as MoS2/Ag1, MoS2/Ag2, and MoS2/Ag3 and were washed two times with DI water to get the MoS2/Ag nanosheets with different silver contents.40 mg PVP was added to 20 mL of synthesized MoS2/Ag3 NSs was determined . Each well plate was injected with 7 to 8 103 L929 cells for 24 h in a thermostatic incubator set to 37°C with 5% CO2, and then the wells were filled with 100 μL of the MoS2/Ag3 NSs solution for another 24 h. Subsequently, a fresh medium (100 μL) containing 10% CCK-8 agentia was added. This medium was then cultured for 4 h in a thermostatic incubator. Enzyme labeling  was used to detect the optical density and cell viability at 450 nm.Using the Cell Counting Kit-8, the cytotoxicity of MoSStaphylococcus aureus (S. aureus) was used as the bacterial model. Frozen strains were first resuspended and transferred to columbia blood agar plates to be incubated overnight in a 37°C incubator, and then took a single colony by inoculation loops and transferred it to 4 mL LB medium and incubated for 12 h at 220 rpm on a shaker. Bacterial suspension was diluted to 107 CFU/mL for inhibition experiments. Microdilution, plate counting, fluorescence staining, and scanning electron microscopy were used to examine the antibacterial activity and mechanism of MoS2/Ag nanosheets against S. aureus.S. aureus was mixed with different concentrations  of sample groups MoS2, MoS2/Ag1, MoS2/Ag2, MoS2/Ag3 in 96-well plates, respectively. All groups were irradiated with a 660 nm laser for 10 min and treated with hydrogen peroxide (H2O2) at a final concentration of 100 μM. Furthermore, S. aureus treated with H2O2 under laser irradiation was taken as the control. To further determine the effect of H2O2 or laser on the antimicrobial properties of the materials, four groups of each kind of material: I) bacteria control, II) bacteria + H2O2, III) bacteria + material, IV) bacteria + material + H2O2 were treated without or with NIR laser (660 nm) irradiation for 10 min. All experiments were carried out according to the following procedure, after culturing at 37°C for 3 h, 100 µL of diluted bacterial suspension was taken out and evenly spread on the blood agar plates and placed at 37°C for 12 h. The antibacterial ability of the material was determined by the colonies that grew on the blood agar plates. Each experiment was conducted at least three times.The plate colony counting assay was employed to investigate the antibacterial performance. Firstly, 600) of various holes, the bacterial survival rate was examined by comparing the optical density data.The premise of the test is that tetrazole is converted to blue crystalline methionine by the enzyme succinate dehydrogenase of live cells . The perS. aureus treated with the material and hatched in a constant temperature incubator at 37°C for 4 h. The survival rate of S. aureus was obtained by detecting the value of optical-density at 600 nm for 0 and 4 h with a micropore meter.Briefly, MTT solution  was mixed with 2/Ag3 nanosheets (30 μg/mL) were mixed with a suspension of S. aureus treated by laser or H2O2 (100 μM), respectively. After being exposed to a 660 nm laser for 10 min, the cells were placed in an incubator at a constant temperature for 3 h, and the combination was then incubated at ambient temperature for 15 min without illumination. The stained S. aureus suspension (10 μL) was put on a slide, covered with the coverslip, and the excess dye was removed. Finally, bacterial staining images were observed by using a fluorescence microscope.SYTO™-9 is a green fluorescent nucleic acid dye that can penetrate cell membranes. PI is a nuclear stain that can penetrate the damaged cells, making the nucleus red. PBS or MoSS. aureus under different conditions were collected by centrifugation . The S. aureus was fixed with 2.5% glutaraldehyde for 12 h and washed twice with PBS at room temperature. Next, the bacteria were treated with 10%, 25%, 50%, 75%, and 100% ethanol successively and gradually dehydrated for 15 min. For SEM observation, the final bacterial solution was dropped onto a silica substrate and sprayed with gold.2/Ag nanosheets was schematically stated in 2 nanosheets were fabricated and obtained according the previous procedure  images clearly showed that the MoS2/Ag3 NSs exhibited the uniform nanosheet morphology with a size around 200–400 nm . The TEM images disclosed that the Ag NPs were successfully dispersed on the surface of MoS2 nanosheets (2 (100) and Ag (220), respectively, indicating Ag NPs were successfully loaded onto the surface of nanosheets. Promisingly, the selected area electron diffraction (SAED) pattern of MoS2/Ag3 NSs was characterized by the presence of bright diffraction spots with regular hexagon . The XPS survey spectra revealed the presence of six elements  in MoS2/Ag3 NSs (2 nanosheets were found to lack the Ag element (2 nanosheets (2/Ag3 NSs exhibited two distinct peaks at 372.98 eV and 366.98 eV corresponding to Ag 3d3/2 and Ag 3d5/2 of Ag (0) , respectively (1/2 and S 2p3/2 of S (II)   (Qiao etf Ag (0) , which iectively . The chaectively  originatf S (II) , respectf S (II)  were use/Ag3 NSs .2/Ag NSs with the highest silver content (MoS2/Ag3) by CCK-8 experiment in L929 cells. The viability of L929 cells was still higher than 85% after the treatment with MoS2/Ag3 at the concentration of 50 μg/mL, showing that excellent biocompatibility and the great potential for in vivo and in vitro antibacterial applications.As shown in S. aureus was further appraised by plate counting method. After co-incubation with the designed materials, it was found that the group of H2O2 or laser irradiation had a negligible antimicrobial effect against S. aureus  or in the presence of H2O2, we kept up with the plate-counting antibacterial experiment with MoS2/Ag nanosheets at the concentration of 30 μg/mL. It has been reported that H2O2 (100 μM) together with near-infrared irradiation exhibited hardly inhibitory effect  was utilized to carry out the live-dead bacteria staining experiment for the sake of investigating the antibacterial mechanism. All S. aureus could be stained by green fluorescence (SYTO™-9) and only damaged S. aureus presented red fluorescence (PI). Fluorescence images showed , or combination of H2O2 and NIR irradiation (10 min). Since the red fluorescence in the H2O2 condition only was practically identical to that of a single laser irradiation in 2/Ag3 NSs could kill off large numbers of bacteria in the presence of H2O2, implying that the synergistic effect made S. aureus unable to survive.MoSs showed  that mem2/Ag3 NSs, surface morphologies of antibacterial with different treatment were detected by SEM. It was observed that the bacteria of the PBS group maintained a smooth and unbroken membrane, meanwhile the S. aureus treated by H2O2 or NIR revealed no evident difference with the PBS group, suggesting the intact structure of S. aureus (S. aureus were observed in the MoS2/Ag3, MoS2/Ag3 + H2O2, and MoS2/Ag3 + NIR groups (2/Ag3 + H2O2 + NIR group (2/Ag3 under the NIR laser irradiation in the presence of H2O2.To further appraise the antimicrobic capacity of MoS. aureus . HoweverR groups . PromisiIR group , which ain vitro antibacterial assays described above, we hypothesized a multi-level synergistic antibacterial mechanism . The sterilization mechanism of Ag NPs was mainly to puncture the cell membrane of their tiny size (2 were stimulated to change into the conduction band and produce electron-hole pairs (2 could facilitate electron transport and prevent the compounding of electron-hole pairs, generating a significant amount of photoelectrons and holes (1O2 when reacted with oxygen (2O2 could be catalyzed to produce ROS for the sake of the peroxidase-like ability of MoS2 (Inspired by the results of the chanism . The stiny size . When irle pairs . Additiond holes . Virtualh oxygen . Importa of MoS2 . As a re2/Ag was designed by a simple method for synergistic photodynamic and peroxidase-like catalytic antibacterial treatment. The developed MoS2/Ag nanosheets may effectively inactivate bacteria by producing poisonous ROS supported to assault the membranes. Importantly, MoS2/Ag3 nanosheets exhibited an antimicrobial efficiency of 99.88% against S. aureus within 10 min under 660 nm illumination in the presence of H2O2. Owing to the modification of PVP, the biocompatibility of MoS2/Ag3 NSs was significantly improved. This particular type of photo-responsive material demonstrated prominent effectivity in antimicrobial treatment with minimal cytotoxicity, which could serve as a promising candidate of antibiotic-free treatment of bacterial infections.In conclusion, a facile artificial nanosheet of MoS


The complexity of the metazoan proteome is significantly increased by the expression of small proteins (<100 aa) derived from smORFs within lncRNAs, uORFs, 3′ UTRs and, reading frames overlapping the CDS. These smORF encoded proteins (SEPs) have diverse roles, ranging from the regulation of cellular physiological to essential developmental functions. We report the characterization of a new member of this protein family, SEP53BP1, derived from a small internal ORF that overlaps the CDS encoding 53BP1. Its expression is coupled to the utilization of an alternative, cell-type specific promoter coupled to translational reinitiation events mediated by a uORF in the alternative 5′ TL of the mRNA. This uORF-mediated reinitiation at an internal ORF is also observed in zebrafish. Interactome studies indicate that the human SEP53BP1 associates with components of the protein turnover pathway including the proteasome, and the TRiC/CCT chaperonin complex, suggesting that it may play a role in cellular proteostasis.  •TP53BP1 mRNA transcripts from an alternative promoter carry a uORF within the 5′ TL•The uORF promotes delayed reinitiation events at an internal overlapping ORF (ioORF)•The ioORF expresses a 50 aa Small ORF Encoded Protein (SEP53BP1)•The SEP53BP1 interactome includes the proteasome and the TRiC/CCT chaperonin Protein; Molecular biology; Cell biology The differential recruitment of mRNA populations onto polysomes permits a rapid response to changes in the cellular environment. As such, it is a key process in the maintenance of homeostasis, and perturbations in its control are associated with numerous disorders. Translation can be subdivided into four main steps: initiation, elongation, termination and subunit recycling. Most regulation is exerted at initiation, and this has been confirmed in translational profiling studies covering the entire mammalian transcriptome.,,,The 5′ TL contains a number of features that can regulate the translational readout during both PIC recruitment and subsequent scanning.,,,,Alterations in the 5′ TL arise because of the use of alternative promoters (AP), transcriptional start site (TSS) heterogeneity and alternative splicing,,,,in-cis to modulate downstream initiation events, or have distinct biological function(s).,,cis-acting peptides that are responsive to environmental signals and have been coined “peptoswitches”.We have discussed uORFs as translational regulatory elements. However, transcriptome analysis has identified thousands of yet non-annotated small open reading frames (smORFs) with the potential to encode biologically active peptides or SEPs (smORF-encoded proteins/peptides) smaller than 100 aa.,,,,,Both leaky scanning and reinitiation permit access to internal AUGs. When in-frame with the principle ORF these give rise to N-terminally truncated proteins.PRNP also expresses a novel 64–81 aa polypeptide (depending on species) from a smORF, referred to as Alt-PrP.,,,Concerning the smaller SEPs, their implications for the human proteome are beginning to be appreciated.TP53BP1 gene, uses two promoters . Luciferase based reporter assays revealed that the V3 5′ TL was more repressive than V1/2 with regards to downstream initiation events because of the uORF.In our earlier work, we reported on a differential RNA-seq analysis comparing the tumoural MCF7 and non-tumoural MCF10 cell lines.isoforms A. These  is poor .55 Basedb-53BP1. Examination of the human sequence reveals that the next start codon downstream AUGb-53BP1 opens a smORF, +1 relative to the 53BP1 ORF, that would encode a polypeptide of 50 aa that we named SEP53BP1  (UGA/UGC) thereby fusing the uAUG to the 53BP1 ORF  A. Transit codon) B. This pt codon) C. To mon3BP1 ORF C. This ethe uAUG C. This wthe uAUG A.b-53BP1. Consistent with a reinitiation model, this increased initiation events at the AUGb-53BP1 relative to AUG-SEP53BP1 by between 4 and 5-fold (b-53BP1 means that initiation events occur mainly on AUG-SEP53BP1. This would direct the expression of the smORF-encoded peptide of 50 aa (SEP53BP1).,The results demonstrate high levels of initiation at the AUG-SEP53BP1 in transcripts carrying the V3 5′ TL and this is mediated by its small uORF. However, we wanted to ascertain to what extent reinitiation played a role in 53BP1 expression in the V3 context. For this, we introduced a 30 nt spacer element between the uORF and the AUGd 5-fold D. This od 5-fold C, leads TP53BP1 genes . Using the T-COFFEE software,BC129236.1 and NM_001080170: longer than the human) whose stop codon is 15 nt upstream of the first AUGa-53BP1 (similar to human) (Z) would express an “SEP-like” polypeptide (a-53BP1 and AUG-ioORF is 400 nt in zebrafish compared to 97 nt in the human V3 mRNA. Within this 400 nt region there is a second AUG in the 53BP1 ORF (AUGb-53BP1) that could express an N terminally truncated (Δ121 aa) 53BP1 protein . These downstream initiation events can give rise to N-terminal truncated forms of the 53BP1 protein and the expression of smORFZ. However, unlike the human V3 transcript the configuration of the single zebrafish 5′ TL assures robust expression from all initiation sites , we noted an extensive overlapping reading frame in the 5′ end of the o human) C. As in o human) C.62 uORFypeptide B and 2C. protein C. To exa protein C. To mon protein C. We als53BP1/LP C. In thee smORFZ C, lane 2rt sites C, lane 3 is poor C. Thus amutation C, lane 4a-53BP1: C, lane 5a-53BP1: C. We conUG-ioORF C, lane 7on sites C, lane 2on sites C.Z are observed in other fish species, for example carp (41 codons) and electric eel (58 codons). In this context, it is worth noting that despite the problems associated with 5′ TL annotation,Internal ORFs analogous to smORFATGa…), and inserted this into a pcDNA3 vector. The CMV promoter driven expression would generate an mRNA transcript with a ∼70 nt 5′ TL derived from vector sequences   C. The trb-53BP1, consistent with the repressional nature of the uORF, and marginally reduced initiation events further downstream, at AUG-SEP53BP1 and AUG-402  (construct 3) and GCGuORF/GCG53BP1(b) double mutant (construct 4: hereafter referred to as Long 5′ TL-3HA) gave very similar expression profiles with the notable exception of the AUG-402 whose product levels were reduced in the latter. This would suggest that its utilization is also coupled to delayed reinitiation. Such a mechanism would allow the 40S to bypass the AUG-SEP53BP1 whose context is relatively good , we fused a 3HA tag to the latter’s C-terminal (V3Δ3′-3HA: scanning A. The GCely good A. Furthein-vitro from the same plasmid constructs (ACOXL gene (407 nt). Its 68% G/C is the same as that found in the Long 5′ TL, the Kozak context remains unchanged and it contains no AUG codons. However, this construct produced SEP53BP1 levels equivalent to the Short 5′ TL in transient transfection assays, despite the fact that the AUG-SEP53BP1 appeared to be less leaky , which is predicted to remove the major RNA structural elements as confirmed by the change in the ΔG, and region 1–350 (Δ2) E. These TP53BP1 P2 promoter activity will permit the expression of a novel SEP. However, at this point in the study it was necessary to detect the endogenous protein, and determine the cellular compartment(s) in which it accumulates as a route toward function. We had already observed that V3 transcript levels are regulated in a cell-specific manner (http://sysbio.sysu.edu.cn/rpfdb/index.html). The image in https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39561).,,7) with a subsequent enrichment by immunoprecipitation before immunoblotting. This probably reflects a low intracellular concentration combined with the technical difficulties associated with the detection of small proteins by immunoblotting.TP53BP1 gene using a previously published siRNA whose target site overlaps the SEP smORF , in a manner that assured that the cell was imaged in its entirety. Pictures were analyzed using the Imaris software. The first few seconds of each video shows the original pictures collected, with the endogenous SEP53BP1 staining red and the nuclei staining blue. This was followed by a 3D reconstruction of the nucleus using the surface function within Imaris. In this representation, the cytoplasmic SEP53BP1 stains magenta and nuclear SEP53BP1 stains light blue.Z stacking was performed using a Zeiss LSM 800 confocal scanning microscope equipped with a Plan Apochromat 63 x/ 1 40 Oil DIC M 27 objective. For the Raji cells, 30 slices were taken (6.09 μm), in a manner that assured that the cell was imaged in its entirety. Pictures were analyzed using the Imaris software. The first few seconds of each video shows the original pictures collected, with the endogenous SEP53BP1 staining red and the nuclei staining blue. This was followed by a 3D reconstruction of the nucleus using the surface function within Imaris. In this representation, the cytoplasmic SEP53BP1 stains magenta and nuclear SEP53BP1 stains light blue.,The punctate staining in the cell suggested that the SEP53BP1 was interacting with intracellular structures and/or assembling into aggregates. To explore the former, we performed a sedimentation analysis using cytoplasmic extracts prepared from HEK293 T cells transiently expressing SEP53BP1. It revealed that despite its small size (50 aa); undetectable amounts remained in the upper fraction (fraction 10). A significant amount smeared through fractions 9 to 4, with fractions 4–6 corresponding to the 20S/26S zone based on the sedimentation of the α4 subunit of the proteasome  and untagged versions of SEP53BP1 either alone or together. We observed that on co-expression we could IP the untagged version of SEP53BP1 with the HA Ab, suggesting multimer formation B. This o,To gain insights into function we employed a yeast-2-hybrid (Y2H) screen to identify partners. SEP53BP1 was used as a prey, and screened against a peptide library generated from a human B cell Lymphoma_RP1. Around 51 million interactions were tested and 5 genes  gave interactions with good confidence levels A. The seTo further probe the interactome, we performed CoIP-MS starting from HEK293T extracts transiently expressing SEP53BP1-3HA. In total, 945 proteins (min. 2 peptides per protein) were identified and quantified using intensities across all samples. After subtraction of the empty vector control and further data mining, we obtained 74 hits . String ,,www.openprot.org).,,,,,,,,,,,,,,,It is increasingly evident that the complexity of the metazoan proteome is considerably increased by the expression of SEPs (<100 aa), which until recently escaped detection using conventional biochemical procedures. They are also referred to as small protein,TP53BP1 gene, the main CDS of which expresses a protein that plays a central role in non-homologous DNA repair.TP53BP1 becomes a new member of the mammalian dual coding or polycistronic genes.,,,TP53BP1 specific region(s) upstream of the SEP53BP1 smORF. This extends beyond the conventional Kozak consensus.trans-acting factors in the cytoplasm.6A modifications within in the 5′ TL control ribosome scanning and subsequent start codon selection.In this manuscript, we have identified a new member of the SEP family expressed from a smORF within the ,,,D. melanogaster,,,,TP53BP1 gene expression in metazoans.The number of annotated mammalian SEP and alt-ORF proteins has been steadily increasing thanks mainly to more robust proteomic and translatomic (i.e. ribo-profiling) analysis pipelines. However, one limitation of these approaches is that they provide limited if no information on the nature of the translational mechanism regulating the proteins expression. Rarely do these studies examine the nature of the regulatory elements within the 5′ TL, elements that are crucial particularly in the context of a dual coding gene in which startsite selection can be controlled. Compounded with this, is the complexity that arises from 5′ start heterogeneity and promoter switches that alter the nature of the 5′ TL.The human SEP53BP1 protein is predicted to have a highly ordered structure . As pred,As an avenue to function, we performed interactome studies using both a Y2H approach and coIP-MS. The Y2H study revealed that SEP53BP1 associates with components of the cellular protein turnover apparatus, including the α4 subunit of the 20S proteasome barrel . The SIDThe coIP-MS analysis also revealed interactions with a number of heat shock proteins and cellular chaperones  will also confirm that SEP53BP1 is not merely a neutral translational by-product that arises from the regulation of intracellular 53BP1 levels via the dual promoter configuration.The studies on the translational regulation of SEP53BP1 expression have been performed by transient over-expression using reporter constructs. Despite the fact that these novel reporters permitted monitoring of initiation events at multiple, out-of-frame sites, it remains to be determined if all observations apply to the endogenous mRNA whose intracellular levels are much lower. In addition, it is unclear how 53BP1 sequences upstream of the AUG-SEP53BP1 can actually improve the efficiency and non-leakiness of the start codon. The regulation of the alternative Joseph.Curran@unige.ch).Further information and requests for resources and reagents should be directed to Joseph A. Curran (Plasmids generated in the study will be available upon request.2 chamber. Cells were cultured in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 1% penicillin/streptomycin (P/S) and 10% fœtal bovine serum (FBS) for HEK293T, HEK293, MDA-MB-231, and HeLa cells. MCF7 were cultured DMEM F-12 medium, 1% P/S and 10% FBS supplemented with 10 μg/mL human recombinant insulin and 0.5 nM estradiol. MCF10A were cultured DMEM F-12 medium containing 1% P/S, 5% horse serum heat inactivated, 10μg/mL Epidermal Growth Factor (EGF), 1 μM Dexamethasone, and 5μg/mL human recombinant insulin. THP-1 and Raji cells were cultured in RPM1 1640 1X (+L-Glutamine) supplemented with 0.05mM 2-mercaptoethanol and 10% fœtal bovine serum. MRC-5 cells were grown in Eagle’s Minimum Essential Medium  supplemented with 10% FBS, 1% P/S, 1X amino acids and 1mM sodium pyruvate.HEK293T, HeLa, MCF7, MCF10A, THP-1 and Raji cells were grown at 37°C in a humidified 5% COTransfections of HEK293T cells were performed using Lipofectamine 3000 when the cells were 70–80% confluent. Eight hours post-transfection, the medium was replaced with normal growth medium, and lysates were usually prepared at 24h post-transfection.Clones were prepared in a pcDNA3 backbone. All mutations and deletions were introduced by PCR. The oligos employed are listed.2, 20 mM HEPES and 2 mM DTT gradients were prepared manually in SW41 rotor tubes. Cells were treated for 5 min with 50 μg/mL cycloheximide and then collected in cold PBS containing 100 μg/mL cycloheximide. Cells were pelleted and lysed in polysome lysis buffer  NP-40, 100 μM cycloheximide, 1% aprotinin, 1 mM AEBSF and 100U/mL of RNasin) supplemented with protease cocktail inhibitor EDTA-free, on ice for 20 min. Lysates were cleared by centrifugation  and the supernatants loaded onto the gradients. These were centrifuged for three and a half hours at 35,000 rpm and 4°C. They were collected through an UV-lamp and an Absorbance detector. One mL fractions were recovered using a Foxi Junior Fraction Collector (Isco). RNA was isolated from each fraction by adding an equal volume of TriZol. Samples were mixed and incubated on ice for 15 minutes before addition of 0.3 volumes of chloroform. After centrifugation, the upper phase was collected, and the RNA precipitated with 0.7 volumes of isopropanol. The RNA pellet was resuspended in water.For polysome profiling, 20–60% sucrose (Sigma) in 100 mM KCl, 5 mM MgClThe RT-PCR was performed using 250 ngs of total or polysomal RNA that were reverse transcribed using 50 U of Superscript II in a total volume of 25 μL at 42 °C for 1 h. Relative mRNA levels were evaluated by semi-quantitative PCR using the Pfu polymerase as detailed previously.Gels were prepared as follows: Separating gel: 16% Acrylamide (Acrylamide/bis-acrylamide 19:1), Gel buffer , 8.75%(v/v) Glycerol, 0.04% ammonium persulphate (APS), and 0.05% TEMED.Stacking gel: 4.2% Acrylamide (Acrylamide/bis-acrylamide 19:1), Gel buffer , 0.2% APS, and 0.2% TEMED.Migration was performed in the following running buffer: 100mM Tris base, 100mM Tricine, 0.1% SDS at pH8.3.2, 1mM DTT, 2mM ATP, protease inhibitor) or CSH buffer  Triton X-100). Whole cell extracts were prepared by resuspending the cell pellet in either X2 sample buffer  glycerol, 10% (v/v) β-mercaptoethanol, 5% SDS, 0.025% (w/v) bromophenol blue) for SDS-PAGE, or X2 sample buffer Novex  glycerol, 4% SDS, 0.00075% Coomassie Blue G, 10% β-mercaptoethanol) for the Tricine gels.Cytoplasmic extracts were prepared in either hypotonic lysis buffer  using two peptides that spanned most of the smORF A. AntiboExtracts were prepared following the protocol outlined in Terenin and coworkers.2, and 20 mM HEPES pH 7.4 prepared in an SW60 tube. Gradients were centrifuged for 16hat 30,000 rpm in a SW60 rotor at room temperature. After centrifugation, 10 X 400 μL fractions were collected from the bottom of the tube. Proteins were recovered by methanol/chloroform precipitation, resuspended directly in 40μL of X2 sample buffer and analysed by immunoblotting.Glycerol gradients were prepared as previously described.in vitro transcribed using the mMessage mMachine™ T7 Transcription Kit and purified by LiCl precipitation according to the manufacturer’s instructions. Transcripts were capped using the vaccinia capping system according to the suppliers’ protocol and polyadenylated using the polyA tailing kit. The mRNA quality was controlled on a 2% agarose gel. RNA (100 ng) was translated either in a 50% extract wheat germ extract (WGE) or a HEK293T hypotonic cell extract. For WGE, RNAs were translated for 90minat 25°C in a final volume of 20 μL. For HEK293T hypotonic cell extracts, RNAs (100 ng) were translated for 2 hrsat 30°C in a final volume of 10μL. The following translation energy mix buffer was added . The reactions were stopped by the addition of Laemmli SB buffer  paraformaldehyde (PFA), permeabilised with 0.3% (v/v) Triton/PBS and then incubated in blocking buffer (2% w/v BSA/PBS) for 60 minutes at RT. Blocking buffer was replaced by the SEP53BP1 primary antibody (1:100 dilution in blocking buffer) overnight at 4°C. Cells were washed X3 with PBS and incubated in the dark for 60 minutes at RT with Alexa Fluor 594 goat anti-rabbit IgG (H+L) (1:500 dilution in blocking buffer). Coverslips were washed X3 with PBS. Nuclei were stained with DAPI (1:1000 in PBS) at RT for 10 minutes in the dark. Coverslips were mounted using Prolong Diamond Antifade Mountant and sealed with nail polish.Confocal images were collected on a Zeiss LSM800 confocal scanning microscope equipped with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. Pictures were analysed using the ImageJ software. Z-seriesvideos in THP1 and RAJI cells are shown as maximum z-projections, and gamma, brightness, and contrast were adjusted  and were generated using the Imaris software.7:50–60% confluence) were transfected with either a scrambled siRNA or a siRNA targeting the TP53BP1 gene using the RNAiMAX reagent. The sequences of the siRNA molecules are indicated in the MCF7 cells (∼3x10https://www.hybrigenics-services.com/) against the Human B cell Lymphoma library.Y2H was performed by Hybrigenics Services  containing 2mM ATP. 500 μg of protein were pre-incubated with α4 antibody overnight at 4°C. The next day, the lysates were incubated with 10 μL of Dynabeads Protein G for 1hat 4°C. Beads were washed X3 in hypotonic lysis buffer, resuspended in X2 sample buffer and analysed by SDS-PAGE.HEK293T cells were co-transfected with pcDNA3 clones expressing SEP53BP1 and UBQLN4. They were lysed in hypotonic lysis buffer. 500 μg of protein were pre-incubated with UBQLN4 antibody overnight at 4°C. The next day, the lysates were incubated with 10 μL of Dynabeads Protein G 1hat 4°C. Beads were washed X3 in hypotonic lysis buffer, resuspended in X2 sample buffer and analysed by SDS-PAGE.4OAc. Biological duplicates were prepared at one-week intervals. The immobilisation of SEP53BP1-3HA to the beads was confirmed by immunoblotting using 1/50th of the final wash bead suspension. The beads were stored at 0°C before sending to the Functional Genomics Centre Zurich (FGCZ) for LC-MS analysis.HEK293T cells seeded on 3 × 10 cm petri dishes were transfected with either a pcDNA expressing SEP53BP1-3HA or the empty vector. At 24 h post-transfection, cells were lysed in 0.5 mL/dish of CSH buffer. The pooled extracts (1.5 mLs containing ∼15 mg of protein) were incubated ON with 30 μL of anti-HA magnetic beads. Beads were washed x3 CSH and x2 with 50 mM NHThe LC-MS data was processed using the FragPipe proteomics pipeline. The protein quantification results were extracted from the combined_protein.tsv file . In ordehttps://www.encodeproject.org) selecting only human datasets that had undergone validation and initial filtering. Mean and standard deviations were calculated directly on EXCEL. The numbers of samples are indicated in the figure legend.Transcriptome data was extracted from the ENCODE site (


Therefore, NPL1010 and NPL3008 are potentially valuable inhibitors of BMP signaling that act through selective inhibition of Chordin cleavage.BMP signaling is critical for many biological processes. Therefore, small molecules that modulate BMP signaling are useful for elucidating the function of BMP signaling and treating BMP signaling-related diseases. Here, we performed a phenotypic screening in zebrafish to examine the in vivo effects of N-substituted-2-amino-benzoic acid analogs NPL1010 and NPL3008 and found that they affect BMP signaling-dependent dorsal–ventral (D–V) patterning and bone formation in zebrafish embryos. Furthermore, NPL1010 and NPL3008 suppressed BMP signaling upstream of BMP receptors. BMP1 cleaves Chordin, an antagonist of BMP, and negatively regulates BMP signaling. Docking simulations demonstrated that NPL1010 and NPL3008 bind BMP1. We found that NPL1010 and NPL3008 partially rescued the disruptions in the D–V phenotype caused by  BMP signaling is related to numerous biological phenomena. During development, the activity gradient of BMP signals is essential for dorsal–ventral (D–V) patterning and is tightly regulated by a multistep functional mechanism controlling the levels of ligands and antagonists in vertebrates . BMP sigRecently, the regulation of BMP signaling in the hippocampus was reported to be involved in the depression phenotype and aging-dependent decline in cognitive function ,5,6. TheBMPs are secreted glycoproteins that belong to the transforming growth factor β (TGF-β) family. BMPs interact with two types of receptor serine/threonine protein kinases known as type I  and type II  receptors ,8,9. BMPBMP signaling is negatively or positively modulated in several steps in its signal cascade. Among them, BMP ligand antagonists Chordin (Chrd), Noggin, Gremlin, etc., directly interact with BMP ligands and interfere with their binding to BMP receptors to negatively regulate BMP signaling . MoreoveSeveral small molecules have been developed to artificially modulate BMP signaling and activity. Dorsomorphin, an inhibitor of BMP type I receptors, blocks the phosphorylation of Smads and inhibits BMP signaling ,14. UK38In previous research, we screened Disheveled1(Dvl1)-PDZ domain inhibitors by performing virtual screening and identified N-substituted-2-amino-benzoic acid analogs . We furtWe performed phenotype screening using zebrafish embryos to explore N-substituted-2-amino-benzoic acid analogs that had been identified from in silico screening for their capacity to bind to the PDZ domain of Dvl1. Among them, ten candidate compounds  were appn = 36) with an elongated A-V axis phenotype; 100 μM NPL1010, 82% (n = 34) with an elongated A-V axis phenotype; 2.5 μM NPL3008, 10% (n = 10) with an elongated A-V axis phenotype; and 5 μM NPL3008, 70% (n = 10) with an elongated A-V axis phenotype). At 24 hpf, NPL1010 or NPL3008 treated embryos exhibited reductions in the ventral tail fin and tail length at 24 hpf. These phenotypes are characteristic features of dorsalization  were transfected into the cells in each well using polyethyleneimine (PEI). In the CA-Alk6 and Smad overexpression experiment, 20 ng of pCDNA3-CA-Alk6, 15 ng of pcDNA3-FLAG-Smad1, and/or 15 ng of pcDNA3-FLAG-Smad5 were also transfected with PEI. For measuring Wnt signaling activity, HEK293 cells were seeded into 96-well plates at a density of 3 × 104 cells/well in DMEM ) and cultured for 24 h. Twenty nanograms of pCS2+ mouse Wnt1 or pCS2+, 70 ng of TOP Flash or FOP Flash reporter plasmid, and 4 ng of pGL4.74 [hRluc/TK] were transfected into the cells in each well using polyethyleneimine (PEI). After plasmid transfection, the cells were incubated at 37 °C with 5% CO2 for 3 h. NPL1010 and NPL3008 were added at 5, 12.5, 25, and 50 μM, and dorsomorphin was added at 1, 5, 12.5 μM. After 21–24 h of incubation at 37 °C with 5% CO2, the culture medium was removed, the cells were washed with 100 µL/well of 1× PBS, and 25 µL of 1× Passive Lysis Buffer  diluted to 1/5) was added to each well. The cells were lysed by shaking at room temperature for 20 min, and the lysates were transferred to 1.5 mL tubes. After centrifugation at 15,000× g for 2 min at 4 °C, 10 µL of the supernatant were transferred to a white 96-well plate . Fluc- and hRluc-dependent luminescence was measured by sequentially adding 40 µL/well of luciferin buffer  and 50 µL/well of Renlite buffer  and detected using the GloMax®-Multi Detection System (Promega). Each sample was measured in duplicate. The Fluc activity was normalized to the hRluc activity.For measuring BMP signaling activity, C2C12 cells were seeded into 96-well plates at a density of 5 × 106 cells/dish, simultaneously reverse-transfected with 20 µg of pCS2+ Chrd-Myc [2 for 24 h. After incubation, the medium was removed, cells were washed with 1× PBS, and the medium was replaced with Opti-MEM (Thermo Fisher Scientific). After 48 h of incubation under the same conditions, the culture supernatant was collected and stored at −80 °C.HEK293T cells were seeded on collagen -coated 10 cm dishes at a density of 3.0 × 10Chrd-Myc , and incFor this experiment, 18.4 µL of OptiMEM (Thermo Fisher Scientific) was added to a 1.5 mL tube, and 18 µL of conditioned medium containing Chrd-Myc was added to each tube. A stock solution of each compound was diluted with DMSO to 5, 10, 25, and 50 mM. The solution of each compound was further diluted 1:40 with Opti MEM, and 1.6 µL of the diluted solution was added to each sample. The 10 mg/mL rhBMP1 stock solution was diluted 1:10 with OptiMEM, and 2 µL of diluted rhBMP1 were added to each sample and mixed. After incubation at 37 °C for 1 h, an equal volume (40 µL) of 2× sample buffer  was added to the samples, and they were then heated at 98 °C for 5 min.2O2). Equal volumes of each solution were mixed immediately before use. Membranes were imaged using an EZ-capture MG instrument . Quantitative densitometry was used to measure the levels of protein fragments with a CS Analyzer (ATTO).The prepared samples were separated on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gels and transferred to PVDF membranes . The membranes were blocked with 0.3% skim milk  in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and incubated with an anti-c-Myc mouse monoclonal antibody  overnight at 4 °C. Then, the membranes were washed three times with TBST and incubated with HRP-conjugated goat anti-mouse IgG (H + L)  at RT for 1 h. The membranes were washed three times with TBST and incubated with the ECL solution  was mixed with 4–8 ng of recombinant human BMP1 and leupeptin  in assay buffer . NPL1010, NPL3008, or UK383,367 was added to the mixture . The total volume of the reaction mixtures was adjusted to 20 µL. The mixtures were incubated at 37 °C for 1 h, an equal volume (20 µL) of 2× sample buffer was added to the mixtures, and mixed samples were heated at 98 °C for 5 min. Heated samples were separated on 10% SDS–PAGE gels and stained with Oriole Fluorescent Gel Stain  according to the manufacturer’s instructions. Stained gels were imaged using ImageQuant LAS4000 , and the relative amount of proteins was analyzed using CS Analyzer (ATTO). BMP1-dependent cleavage of COL3A1 was calculated from the amount of each COL3A1 fragment by subtracting BMP1-independent degradation of COL3A1 based on the molecular weight of each fragment.Zebrafish were raised and maintained under standard conditions with the approval of the Chiba University Institutional Animal Care and Use Committee . Zebrafish embryos were obtained from the natural spawning of wild-type adults.2, and 0.33 mM MgSO4) and exposed to several concentrations of compounds from 3 to 11 hpf or 3 to 24 hpf; the phenotype was observed after incubation. Images were captured using a stereomicroscope  and Axio Cam MRc . For examination of the NPL1010 and NPL3008 effects on fin morphogenesis and bone mineralization, the chorion was removed at 1 dpf, and embryos were treated with NPL1010 or NPL3008 from 1 dpf to 3 or 5 dpf.One to five zebrafish embryos were placed in 24-well plates with 1 mL of E3 medium  twice at RT for 5 min and stained with coloration solution  and 2.3 µL of NBT  per 1 mL of NTMT) for approximately 2 h to overnight at RT or 4 °C in the dark. After color development was complete, the embryos were washed with 1× PBSTw three times for 5 min at RT and stored in 4% PFA/1× PBS at 4 °C. Images were captured using a stereomicroscope (Leica MZ16) and Axio Cam MRc (Zeiss). The measurement of the angle of the chrd expression domain was performed using Fiji software [The embryos were collected in a 1.5 mL tube and fixed with 4% paraformaldehyde (PFA)/1× phosphate-buffered saline (PBS) at 4 °C overnight. The embryos were dechorionated and dehydrated by sequential treatment with 25% methanol/1× PBS supplemented with 0.1% Tween 20 (1× PBSTw), 50% methanol/1× PBSTw, and 75% methanol/1× PBSTw. The embryos were incubated with each solution for 5 min at room temperature (RT). Then, the embryos were treated with 100% methanol at −30 °C for 30 min. The embryos were hydrated by sequential treatment with 75% methanol/1× PBSTw, 50% methanol/1× PBSTw, and 25% methanol/1× PBSTw for 5 min at RT each. The embryos were washed with 1× PBSTw for 5 min at RT twice and prehybridized with hybridization buffer , 0.1% Tween 20, and 50 µg/mL heparin) at 65 °C for 1 h. Then, the prehybridization HYB was replaced with HYB containing a probe, and the embryos were hybridized at 65 °C overnight. The embryos were washed with 100% HYB, 50% HYB/2× SSC supplemented with 0.1% Tween 20 (2× SSCTw), and 2× SSCTw at 65 °C for 15 min each, followed by two washes with 0.2× SSCTw at 65 °C for 30 min. Then, the embryos were treated with 1× MABDT (1× maric acid buffer (MAB), 1% DMSO, and 0.1% Tween 20) at RT for 15 min and a blocking solution , 1× MAB, 10% FBS, 0.1% Tween 20, 1% DMSO) for 1 h at room temperature. Then, the embryos were incubated with anti-digoxigenin-AP Fab fragments  diluted 1/5000 in blocking solution at RT for 3 h. The embryos were washed with 1× PBSTw eight times at RT for 15 min and held at 4 °C overnight. The embryos were washed with NTMT  at room temperature overnight. After staining, embryos were treated with 1.5% H2O2 and 1% KOH at room temperature for 20 min. Then, the embryos were cleared by sequential treatment with 0.375% KOH, 25% glycerol; 0.375% KOH, 50% glycerol; and 0.125% KOH, 75% glycerol at room temperature for 1–3 days and stored in 100% glycerol at 4 °C. Images were captured using a stereomicroscope (Leica MZ16) and Axio Cam MRc (Zeiss).The fertilized eggs were dechorionated at 1 dpf and treated with DMSO, NPL1010 (2.5 or 5 μM in E3 medium), NPL3008 (2.5 or 5 μM in E3 medium), or dorsomorphin (5 μM in E3 medium) until 5 dpf. Staining was performed as described . Briefly, 5 dpf embryos were collected in a 1.5 mL tube and fixed with 4% PFA for 2 h. Then, the embryos were dehydrated with 50% ethanol at room temperature for 10 min and stained with staining solution  for the docking study was modeled using the SWISS-MODEL server with the human BMP1 structure as the template (PDB: 6BSL). Then, hydrogen atoms were added and subjected to further docking experiments with AutoDock Vina v1.1.2 according to the instructions . The griAlphaFold simulation was performed using AlphaFold Colab notebook on Google Colaboratory ,43. The Statistical analyses were performed using Prism 8 (GraphPad Software). Information about error bars, sample size, and statistical test methods is described in the corresponding figures and figure legends.


BackgroundIncreased posterior tibial slope (PTS) is an important risk factor for non-traumatic graft failure and revision surgery after anterior cruciate ligament reconstruction. If a tibial posterior slope is an important factor for graft failure after anterior cruciate ligament reconstruction, does it affect clinical outcomes? This study aimed to evaluate the association between PTS and clinical outcomes after anterior cruciate ligament reconstruction.Material and methodsPatients undergoing arthroscopic anterior cruciate ligament reconstruction with hamstring tendons in the clinic were evaluated retrospectively. Inclusion criteria were: patients with at least an 18-month follow-up period who were evaluated with the Tegner Lysholm scoring system, aged between 18 and 40 years, with only an anterior cruciate ligament rupture. PTSs were measured from the lateral radiographs of the knees. The patients were divided into two groups with a PTS of 10° or less.ResultsThe mean Tegner Lysholm score was 86.8 ± 8.9. The mean PTS was 9.7° ± 1.5°. In total, 14 and 15 patients had a PTS of above 10° and below 10°, respectively. The mean age and follow-up time of patients were 28.5 ± 5.3 years and 24.6 ± 7.2 months in the group with a PTS of above 10° and 30.2 ± 5.3 years and 24.2 ± 5.18 months in the group with a PTS of below 10°, respectively. Tegner Lysholm scores were 88.2 ± 8.8 and 85.6 ± 9.1 in the group with values above 10° and below 10°, respectively. Statistically, there was no significant difference between the clinical outcomes of both groups.ConclusionPTS does not affect the clinical outcomes of patients who underwent arthroscopic anterior cruciate ligament reconstruction in the early period. Arthroscopic anterior cruciate ligament (ACL) reconstruction is one of the most common orthopedic surgical procedures. There are defined reasons for revision surgery after ACL reconstruction. Age, sex, body mass index, joint laxity, inability to open the tunnels in the appropriate position, graft thickness, inadequacy of the materials used in graft fixation, and tibial posterior slope are some of these ,2.Recent studies have reported that increased posterior tibial slope (PTS) is an important risk factor for non-traumatic graft failure and revision surgery after ACL reconstruction ,4. IncreThe purpose of this study was to evaluate the association between PTS and clinical outcomes after ACL reconstruction. The PTS of the patients was determined according to the preoperative lateral knee radiographs, and the clinical outcomes of both groups were compared by dividing them into two groups: those below and above 10°.This study was conducted at Bahcesehir University, VM Medicalpark Pendik Hospital. This study was approved by the ethics committee of the local institute (Decision No. 2021-12/06). All patients provided informed consent. In this study, 62 patients who underwent arthroscopic ACL reconstruction with hamstring tendons in the clinic were evaluated retrospectively between 2016-2018. The inclusion criteria were as follows: patients with at least an 18-month follow-up period who were evaluated with the Tegner Lysholm scoring system at the last control, aged between 18 and 40 years, with only ACL rupture, and who underwent lateral knee X-ray before surgery. Patients with a follow-up period of less than 18 months who were not evaluated with the Tegner Lysholm scoring system, aged under 18 years and over 40 years, with chondral and meniscal tears with ACL tears, and who did not undergo a lateral knee X-ray in the preoperative period were excluded from this study. Twenty-nine patients who met the inclusion criteria were included in this study. PTSs were measured from the lateral radiographs of the knees obtained preoperatively. The patients were divided into two groups with a PTS of 10°or less.Surgical techniqueAll ACL reconstructions were performed using hamstring tendons as autografts. After diagnostic arthroscopy, the semitendinosus and gracilis tendons were harvested. Tendons were prepared in a four-strand style. The femoral tunnel was prepared using the anteromedial portal approach. The tibial tunnel was prepared using the arthroscopic control technique. The prepared autograft was passed through tunnels; femoral side fixation was performed with an endobutton; and tibial side fixation was performed with a bioabsorbable screw and staple.Post-operative follow-up and physical therapyThe patients used an angle knee brace for the first two weeks of the post-operative period. Up to 90° of flexion of the knee was allowed. After the first two-week period, the use of angle-adjustable knee braces was terminated, and a physical therapy program was started to strengthen the quadriceps and increase the knee joint’s range of motion. The same physical therapy program was applied to all patients. Early and late complications among the patients were determined. Treatments for complications were arranged. Patients were evaluated with the Tegner Lysholm score of the final controls.Measuring method of PTSA line extending proximal to the tibial plateau was drawn at the posterior tibial cortex. The second line was drawn parallel to the medial tibial plateau. The angle between the two lines was measured. The measured angle was accepted as the posterior tibial slope  Figure .Statistical analysisThe suitability of the data for normal distribution was tested, and because they were not normally distributed, the non-parametric Mann-Whitney U test was used. A p-value <0.05 at the 95% confidence interval was considered statistically significant.A total of 62 patients underwent ACL reconstruction with the hamstring tendon technique. There were 12 male (80%) and 3 female (20%) patients in group 1, and 11 male (78.6%) and 3 female (21.4%) patients in group 2. The mean follow-up durations were 24.6 ± 7.2 and 24.2 ± 5.1 months for groups 1 and 2, respectively. The mean ages of the patients were 28.5 ± 5.3 and 30.2 ± 5.3 years in groups 1 and 2, respectively. The mean BMIs were 27.2 ± 3.6 and 26.3 ± 3.8 kg/m2 in groups 1 and 2, respectively. There was no significant difference in age, BMI, or follow-up duration between the two groups (p>0.05).The mean age of the patients in the study was 29.4 ± 5.3 years. The mean follow-up period was 24.4 ± 6.1 months. The mean Tegner Lysholm score was 86.8 ± 8.9. The mean PTS was 9.7° ± 1.5°. The lowest PTS was 7.8°, and the highest PTS was 12.5°. In total, 14 and 15 patients had a PTS of above 10° and below 10°, respectively. The mean tibial slope of the 1st group was 8.7° ± 0.9° (7.8°-9.1°), and that of the 2nd group was 11.1° ± 0.8° (10.2°-12.5°).Tegner Lysholm scores were 88.2 ± 8.8 and 85.6 ± 9.1 in the group with a PTS above 10° and in the group with a PTS below 10°, respectively. Statistically, there was no significant difference between the clinical outcomes of both groups (p>0.48)  and anterior cruciate ligament (ACL) injuries has garnered attention in recent studies, as PTS is believed to be a potential contributing factor to ACL injury risk. PTS is measured as the posterior inclinations of the medial and lateral tibial plateau, with typical values being 7° for the medial plateau and 5° for the lateral plateau . It's hySeveral studies have explored the connection between PTS and ACL injuries. A meta-analysis reported an association between both medial and lateral PTS and ACL injuries . HoweverVarious studies in the literature have reported different angle values as risk factors for ACL injury and the need for revision surgery after ACL reconstruction. For instance, some studies suggest that a PTS above 12° is associated with a higher risk of ACL injury and reconstruction failure ,17,18. AThe reasons for inadequacies after ACL reconstruction are multifactorial, including surgical technical errors, re-injury, and biological deficiencies . High laThis study is limited by the absence of a conservatively treated control group and the small number of participants.In conclusion, the posterior tibial slope has no effect on the clinical outcomes of patients who underwent arthroscopic anterior cruciate ligament reconstruction in the early treatment period. In the future, we anticipate that studies with longer follow-up periods and larger sample numbers will contribute to the existing body of literature.


Triplicate 2-L samples of spiked plasma were SD in a lab drier set at an outlet temperature of 80°C or 71°C. The final dried samples were stored at 4°C or 20°C for 14 d. Liquid and SD samples were analyzed for ASFV infectivity in two mirror 24-well plaques containing VERO cells monolayers. Wells were inoculated with different dilutions of SDPP dissolved 1:9 in PBS. One plaque was immediately frozen at -80°C and the other was incubated at 37°C for 3 d. Each dilution was replicated 9 times. After incubation both plaques were analyzed for ASFV by qRT-PCR. Results indicated that the SD process inactivated between 3.2 to 4.2 Logs ASFV TCID50/mL and 2.53 to 2.75 Logs TCID50/mL when the outlet temperature were 80°C and 71°C respectively. All SD samples stored at 4°C or 20°C for 14 d were absent of infectious ASFV. The combination of SD and post drying storage at both temperatures for 14 d was able to inactive >5.18 ±0.08 Log10 of ASFV inoculated in liquid porcine plasma, demonstrating that the manufacturing process for SDPP can be considered safe regarding ASFV.African swine fever virus (ASFV) is a dsDNA virus that can cause high mortality in pigs of all ages. Spray-dried porcine plasma (SDPP) is a highly digestible ingredient used in feed because it benefits performance, gut function and immunity. The objectives were to test if the spray-drying (SD) conditions along with post-drying storage of product for 14 days can inactivate ASFV inoculated in liquid plasma. Fresh liquid porcine plasma was inoculated with ASFV (BA71V) to a final concentration of 10 Spray-dried porcine plasma (SDPP) is a highly digestible, high-protein ingredient that is widely used in feed because it benefits growth performance, feed efficiency, intestinal integrity, and immune parameters –3.https://www.eapa.biz/quality-safety) and been adopted by most SDPP manufacturers.The manufacturing process of SDPP begins with the collection of blood from healthy animals declared fit for slaughter for human consumption by the veterinary authorities. Blood is collected into containers with anticoagulant, chilled, and centrifuged to separate plasma. Liquid plasma is then concentrated and spray-dried at 80°C throughout its substance to produce SDPP , 4. SpraAfrican swine fever virus (ASFV) is an enveloped dsDNA virus belonging to the Asfarviridae family [e family  that cane family , 16. At e family –17.10 TCID50/mL [The prevalence of African Swine Fever (ASF) has increased concerns that feed ingredients could represent a risk factor for spreading ASF. Spray-drying has shown an ASFV inactivation capacity of 4.11 ± 0.20 LogCID50/mL  resultinCID50/mL . HoweverThe objective of the present study was to determine the level of ASFV inactivation in liquid plasma artificially contaminated with ASFV by the safety steps involved in commercial SDPP production including spray-drying in combination with storage for 14 days at different temperatures.6.9 TCID50/mL measured by immunoperoxidase monolayer assay (IPMA).ASFV strain Badajoz-71 adapted to Vero cells (ASFV-BA71-V)  was provThe plasma used for this experiment was obtained from pigs slaughtered in European porcine abattoir facilities from animals inspected and approved as fit for slaughter for human consumption by official veterinary authorities. Blood was collected in stainless steel containers, with an anticoagulant (sodium tripolyphosphate), refrigerated and transported to the APC Europe facilities . Plasma was separated in less than 24 hours from collection by commercial centrifugation. Eight liters of fresh plasma were UV-C irradiated at 10000 J/L for this study using the UV-C SurePure SP-1 device as previously described , 22 to i6.9 TCID50/mL) achieving a viral theoretical titer of 105.9 TCID50/mL.Three 2-L batches of UV-C irradiated liquid porcine plasma were used for the study. From each batch, 200 mL of the 2 L were collected and stored at –80°C for later analysis. The remaining 1800 mL of liquid plasma per batch were inoculated with 200 mL of ASFV-BA71-V stock solution (103 h-1 (at 20°C) and 0.2 lh-1, respectively. The airflow through the feed nozzle adjusted to 0.7 m3h-1 (at 20°C). Residence time was estimated to be 0.41 s.The spray-drying process was conducted using a laboratory spray-drier  simulating industrial process conditions. The laboratory spray-drier was set to an inlet air temperature of 200 ± 5°C and outlet air temperature set at 80 ± 1°C or at 71 ± 1°C as previously described . AirflowBefore processing and collecting dried samples for analysis, the lab spray-dryer processing parameters were stabilized by running it with non-inoculated UV-C irradiated liquid porcine plasma at the target temperature for 10 min. After this time, the sample collector for the spray-dryer was replaced with a clean one and the previously collected powder was discarded. This preliminary drying time assures that the system was stabilized at the desired processing parameters before the ASFV inoculated plasma was dried and samples for analysis were collected.Each dried inoculated sample was distributed in twenty-seven samples of 0.5 g in 0.5 cm glass tubes (inner diameter). Nine samples were obtained directly without receiving additional heat post drying. To simulate the longer retention time in commercial spray driers, an additional 9 samples were placed in a water bath at 90±3°C for 30 seconds  and another nine samples for 60 seconds . The temperature of the spray dried samples during the 30 s and 60 s post-drying heat treatment was monitored with temperature probes. In each simulation, three of these samples were analyzed directly, three samples were stored at 4°C for 14 days and another 3 samples at 20-22°C (room temperature) for 14 days .Spray-dried samples were reconstituted by adding 4.5 mL of PBS to 0.5 g of plasma sample (ratio SDPP 1:9 PBS). A ten-fold dilution series was done from native samples to -3 dilution. Each dilution was inoculated in 9 wells, obtaining 9 replicates of the same sample. This procedure was done by duplicate to obtain two identical mirror 24 wells plates. Of these two identical plates, one of them was immediately frozen at -80°C and the other one was incubated at 37°C for 3 days. After the 3-day incubation period, the plates were frozen and thawed and the frozen plates were thawed also.50/mL including standard error. Standard curves were established at each outlet temperature by regressing Log10 TCID50/mL SDPP on CT results  when stored either at 4°C or 20°C for 14 days were completely inactivated . Our datSpray-dried plasma (SDP) is a functional protein source that significantly improves daily gain, feed intake, production efficiency and reduces post weaning lag , 25–29. Porcine respiratory and reproductive syndrome virus (PRRSV), Pseudorabies virus (PRV), and Porcine epidemic diarrhea virus (PEDV), have been shown to be effectively inactivated by spray-drying [Escherichia coli or Salmonella enterica [Several viruses of concern to the swine industry, including the y-drying –12, as wenterica , 8.Swine vesicular disease virus and Foot and mouth disease virus (FMDV) in cooked meat products (“Council Directive 2002/99/EC of 16 December 2002). In the present study we tested the commercial outlet air temperature conditions of 80°C and 71°C. Under these conditions we observed higher ASFV inactivation at 80°C ) than 71°C (range 2.53±0.12–2.75±0.04 LRV), results that agrees with publications indicating that pathogen inactivation during spray-drying is related to the inlet and outlet air temperatures, especially the outlet temperature [The phenomena involved in spray-drying inactivation of these pathogens rely on the rapid desiccation , cytoplaperature . These rperature .10 TCID50/mL inactivation at 80°C or 2.65±0.08 Log10 TCID50/mL at 71°C partially agrees with results obtained by Fischer et al., 2021 [10 TCID50/mL loss of viability when SDPP was stored at 4°C. The differing results between our study and theirs may be that in our study we inoculated ASFV into the liquid plasma before drying, whereas they spiked ASFV on previously dried porcine plasma. Therefore, the initial viral titer after spray drying in our study was lower than the initial viral titer of the spiked SDPP used in the study by Fischer et al. (2021). In addition, we started the storage period just after spray-drying of the liquid plasma without time for the virus to recover from the heat and desiccation effects associated with the spray-drying process. In contrast, the current results are consistent with the inactivation of 2 Log10 TCID50 observed by Dee et al., 2018 [In previous studies spray-drying at 80°C for 60 s, was able to inactivate 4.11 Log TCID50/mL of ASFV . In the l., 2021 , who achl., 2018  over 30 10 TCID50/pig. Considering this minimum infective dose of ASFV in the feed, the proven inactivation capacity of the spray-drying process (> 4 log TCID50/mL) and the subsequent inactivation during post-drying storage (inactivation between 1.95 to 5.7 Log10 TCID50/mL) within two weeks at room temperature, the manufacturing process for SDPP has a high inactivation capacity against ASFV.Furthermore, in a previous study  it was d10 TCID50/mL of ASFV result in a safe feed ingredient for use in swine feed.The overall results demonstrated that spray-dry process at 80°C or 71°C and the subsequent storage at room temperature for 14 days inactivates >5.18 ±0.08 Log