Crystal structures of Csm6’ in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the ‘jaws’ of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA  CARF-family proteins include the ribonucleases Csx1/Csm6  RNase domains for cellular defence. cA4 binding to dimeric Card1 results in a rotation of the two monomers with respect to one another, coupled with activation of the nuclease domains  second messenger synthesis by the large Cas10 subunit of the effector complex . cOA, cosx1/Csm6 , PD-D/Exsx1/Csm6  and Can2sx1/Csm6 ,13 and tsx1/Csm6 . cA4 or  domains . In the  domains .Csx1/Csm6 enzymes cleave nucleic acid non-selectively, therefore collateral damage to host transcripts during the immune response slows cell growth , posing 4 activator are found in both the Csm6 and Csx1 groups  bound to a fluorinated analogue of the cA6 activator revealed the activated form of this family  and solved X-ray crystal structures in the presence and absence of cA6. This revealed a dramatic conformational change upon cA6 binding, with a pronounced rotation of the dimeric CARF domains (through 58°), transmitted via the 6H domain, to prise open the HEPN domains, like the opening of a jaw (see Video Abstract). Based on these structures and supporting biochemical and EPR (electron paramagnetic resonance) data, we propose a model for activation of Csm6 proteins by cA6, whereby a large rotation of the CARF domains results in activation of the HEPN domains.Here we carried out a biochemical characterization of S. thermophilus Csm6’, codon optimized for expression in Escherichia coli  and (C223A:C292A:N209C:D88C), were generated using the QuikChange Site-Directed Mutagenesis kit as per manufacturer's instructions (Agilent Technologies).A synthetic gene (g-block) encoding hia coli , was purhia coli  between csm6’ wild-type and mutant constructs were transformed into C43 (DE3) E. coli cells. StCsm6’ protein was expressed according to the standard protocol described previously . Briefly, 2–4 l of cell culture (in LB broth) containing the pEV5HisTEV-csm6’ plasmid was induced with 0.4 mM isopropyl-β-D-1-thiogalactoside (IPTG) at an OD600 of ∼0.6 and grown overnight at 16°C. Cells were harvested  and resuspended in lysis buffer containing 50 mM Tris–HCl pH 7.5, 0.5 M NaCl, 10 mM imidazole and 10% glycerol. Cells were lysed by sonicating six times, with cycles of 1 min on ice and 1 min rest intervals and the cell debris removed by centrifugation. StCsm6’ was purified with a 5 ml HisTrapFF column ; following loading of the supernatant following cell lysis, the column was washed with 20 column volumes (CV) of buffer containing 50 mM Tris–HCl pH 7.5, 0.5 M NaCl, 30 mM imidazole and 10% glycerol, and the protein was eluted with a linear gradient of buffer containing 50 mM Tris–HCl pH 7.5, 0.5 M NaCl, 0.5 M imidazole and 10% glycerol across 15 CV. Protein containing fractions were concentrated and the 8-His affinity tag was removed by incubating protein with Tobacco Etch Virus (TEV) protease (10:1 StCsm6’:TEV protease) overnight at room temperature.The pEV5HisTEV-Cleaved StCsm6’ was further purified by repeating the immobilized metal affinity chromatography step and collecting the unbound fraction. Size exclusion chromatography  was used to complete purification, with pure StCsm6’ protein eluted isocratically in buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl. The protein was concentrated using a centrifugal concentrator, aliquoted and frozen at –70°C.csm6’ gene was transformed into E. coli B834 (DE3) cells. Cells were grown in M9 minimal medium supplemented with Selenomethionine Nutrient Mix  and 50 mg l−1 (l)-selenomethionine (Acros Organics). The protein was purified by the same method described for native StCsm6’. StCsm6’ variants were expressed and purified as described for the WT protein.For seleno-methionine labelled expression, the plasmid containing the 3, cA4 or cA6)  in buffer containing 20 mM HEPES pH 7.0, 150 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM EDTA and 3 units SUPERase·In RNase inhibitor at 45°C. Control reactions incubating RNA in buffer without protein, and RNA with protein in the absence of cOA activator were also carried out. Reactions were stopped by adding phenol-chloroform and vortexing to remove protein, and 5 μl of reaction product was extracted into 5 μl 100% formamide xylene-cyanol loading dye. All experiments were carried out in triplicate and RNA cleavage was visualized by phosphor imaging following denaturing polyacrylamide gel electrophoresis (PAGE).To determine RNA cleavage by StCsm6’ and variants, 50 nM radiolabelled RNA oligonucleotide A1 (5′AGGGUAUUAUUUGUUUGUUUCUUCUAAACUAUAAGCUAGUUCUGGAGA) was incubated with protein (1 μM dimer) and cOA activator  was preincubated with buffer alone (control) or with StCsm6’, S105W or H336A variants (1 μM dimer) for 60 min at 45°C prior to adding 50 nM radiolabelled RNA and StCsm6’ (1 μM dimer) and incubating for a further 30 min at 45°C. Control reactions, with cOA not preincubated or treated with StCsm6’ (c1), and no cOA but RNA and StCsm6’ added after preincubation of buffer (c2), were carried out. Reactions were stopped by adding phenol-chloroform as detailed above and RNA cleavage was visualized by phosphor imaging after denaturing PAGE. RNA cleavage was quantified using the Bio-Formats plugin  and 125 nM RNaseAlert FAM™ reporter substrate (IDT) in a 30 μl volume in buffer containing 20 mM MES pH 6.0, 30 mM NaCl and 20% glycerol for 30 min at 45°C. Fluorescence intensity (excitation wavelength at 490 nm and emission at 520 nm) corresponding to RNA cleavage was measured during this period using a FLUOstar Omega microplate reader (BMG LABTECH) with gain set to 2100. Experiments were carried out in triplicate.StCsm6’ was incubated with a doubling series of cA6 (hereafter StCsm6’-cA6) in a 1:1.5 molar ratio of StCsm6’:cA6 and incubated at room temperature for 30 min prior to crystallization. Crystallization trials were performed using sitting drop vapour diffusion, with JCSG and PACT 96-well commercial screens (Jena Bioscience), set up with an Art Robbins Gryphon robot. Non-labelled StCsm6’-cA6 crystallized at a concentration of 12.5 mg/ml and the selenomethionine-labelled StCsm6’-cA6 (hereafter SeMet-StCsm6’-cA6) at 10 mg/ml. Following optimization using hanging drops in a 24 well plate, crystals were obtained from 1.85 M sodium malonate and 5% ethanol, over a reservoir volume of 600 μl, for StCsm6’-cA6, and 1.6 M sodium malonate, over a reservoir volume of 1 ml, for SeMet-StCsm6’-cA6. 3 μl drops in a 2:1 or 1:1 protein:mother liquor ratio were added to a silanized cover slip and sealed with high-vacuum grease  and incubated at room temperature. Crystals were harvested and cryoprotected with the addition of 20% PEG 1000 and 10% glycerol to the mother liquor, mounted on loops and vitrified in liquid nitrogen.StCsm6’ was mixed with cANo diffraction quality crystals were immediately forthcoming for apo StCsm6’; a crystal was observed after 6–12 months incubation in the initial 96 well crystallization screens, with the drop noticeably dehydrated. Apo-StCsm6’was crystallized from 0.95 M sodium citrate, 0.18 M sodium bromide and 0.1 M HEPES, pH 8. Crystals were harvested and cryoprotected with the addition of 20% PEG 1000 to the mother liquor, mounted on loops and vitrified in liquid nitrogen.6 were collected at Diamond Light Source (DLS) on beamline I04, at a wavelength of 0.9795 Å, to 2.61 Å resolution. Diffraction data were automatically processed through the Xia2 pipeline . MOLREP (6 using molecular replacement with the model generated by ARP/wARP (from the SeMet-StCsm6’-cA6 data) as the search model. Iterative cycles of REFMAC5 , restraints generated in JLigand . Successful phasing required the N- and C-terminal domains of StCsm6’ to be separated into two distinct search models. The model was refined as described above.X-ray data for apo-StCsm6’ were collected at DLS on beamline I03 and processed to 3.54 Å resolution using FAST_DP , which i6, the Molprobity score is 1.35; 99th centile. Ramachandran statistics are 97% favoured; 2% allowed; 1% outliers. For apo-StCsm6’, the Molprobity Clashscore is 3.14; 84th centile. Ramachandran statistics are 76% favoured; 18% allowed; 6% outliers. Data processing and refinement statistics are shown in 6) and 8PCW (apo-StCsm6’).Throughout refinement, the quality of both models was assessed and validated using PDB-Redo  and MolpS-[methyl] methanesulfonothioate (MTSL) spin label (Santa Cruz Biotechnology) overnight at 4°C, yielding the spin-labelled side chain R1. Excess MTSL was removed using a PD10 column.Purified protein  of StCsm6’ variants C223A:C292A:N209C:R85C (hereafter R85C/N209C) and C223A:C292A:N209C:D88C (hereafter D88C/N209C) in 20 mM Tris, 150 mM NaCl, pH 7.5, were incubated overnight at 4°C with DTT at a final concentration of 7.5 mM to freshly reduce cysteine residues. DTT was removed using a desalting column . Eluted protein solutions were concentrated to a volume of 1.5–2 mL and incubated with a 20-fold molar excess (10-fold with respect to cysteine residues) of m/z. Expected masses were obtained for both mutants before and after labelling (see Successful spin labelling was confirmed via electrospray ionization (ESI) mass spectrometry using the in-house mass spectrometry facility. ESI mass spectrometry was performed on samples before (control) and after spin labelling. Samples were diluted to 1 μM in 1% formic acid (FA). 10 μl per sample was injected onto the liquid chromatography (LC) system (Waters Xevo G2 TOF MS with Acquity HPLC) using a MassPrep cartridge column (Waters), applying a 5 minute gradient from 95% water, 5% acetonitrile to 5% water, 95% acetonitrile (eluents supplemented with 1% FA). Data were collected in positive mode from 500–2500 m/z and charged ion series deconvolution to 0.1 Da resolution was performed using the MaxEnt I algorithm utilizing a peak width at half height of 0.4 ling see . Labelliling see .6. 50% (v/v) deuterated glycerol (CortecNet) was used for cryoprotection. The samples with a final volume of 65 μl were transferred to 3 mm quartz EPR tubes which were immediately frozen in liquid nitrogen.Exchange of the spin labelled protein into deuterated buffer was performed by means of repeated steps of dilution followed by centrifugal concentration until a theoretical deuteration of at least 99.5% was reached. Samples for cryogenic CW and pulse EPR measurements were prepared at a final protein concentration of 50 μM (100 μM spin label). Both StCsm6’ mutants were prepared with and without 25 μM cAPDS experiments were performed at 50 K on a Bruker ELEXSYS E580 spectrometer with an overcoupled 3 mm cylindrical resonator (ER 5106QT-2w), operating at Q-band frequency (34 GHz), using a second frequency option (E580-400U). Pulses were amplified by a pulse travelling wave tube (TWT) amplifier (Applied Systems Engineering) with nominal output of 150 W. Temperature was controlled using a cryogen-free variable temperature cryostat (Cryogenic Ltd) operating in the 3.5 to 300 K temperature range.A) – τ1 – π(νA) – (τ1 + t) – π(νB) – (τ2 – t) – π(νA) – τ2 – echo) as described previously  experiments were performed with the 4-pulse DEER  pulse seeviously , with a PDS experiments were analyzed using DeerAnalysis2015 . PDS dat2 of 8000 ns were cut at 7300 for processing to remove artifacts at the end of the trace. Traces recorded with a τ2 of 6500 ns resulted in a best fit where the background fit had a positive and thus unphysical slope after the first validation. Therefore, data were cut iteratively by 10% of the initial trace length and Tikhonov regularization and first validation rounds were repeated until the best fit had a decaying background function. This resulted in a 10% cut for the D88R1/N209R1 + cA6 data and a 30% cut for the R85R1/N209R1 + cA6 data. Validation trials from the second validation round were pruned with a prune level of 1.15, where trials exceeding the root mean square deviation of the best fit by at least 15% were discarded. In all cases the regularization parameter α was chosen according to the L-curve criterion  version 2.0 within DeerAnalysis2022 (DEERNet  Spinach CW EPR measurements were performed using a Bruker EMX 10/12 spectrometer equipped with an ELEXSYS Super Hi-Q resonator at an operating frequency of ∼9.9 GHz (X-band) with 100 kHz modulation.Room-temperature CW EPR measurements were performed to assess labelling efficiency. Samples were recorded using a 100 G field sweep centred at 3455 G, a time constant of 20.48 ms, a conversion time of 20.12 ms, and 1707 points resolution. An attenuation of 20.0 dB (2 mW power) and a modulation amplitude of 0.7 G were used. Protonated StCsm6’ samples were measured in 50 μl capillaries at 50 μM protein (100 μM spin) concentration and double integrals were compared to MTSL as a standard. Labelling efficiency was ≥97% for D88R1/N209R1 and ≥87% for R85R1/N209R1, and samples showed negligible free spin label contribution.Cryogenic CW EPR spectra were obtained at 120 K. The temperature was controlled with an ER4141 VTM Nitrogen VT unit (Bruker) operated with liquid nitrogen and a quartz Dewar insert. Samples were recorded using a 200 G field sweep centred at 3256 G, a time constant of 20.48 ms, a conversion time of 20.00 ms, and 2000 points resolution. An attenuation of 40.0 dB (20 mW power) and a modulation amplitude of 1 G were used.6 bound constructs were simulated as dipolarly broadened spectra obtained by convoluting the apo spectrum with the dipolar broadening functions corresponding to a Gaussian distribution centred at 1.1 and 0.9 nm with widths of 0.2 and 0.35 nm for StCsm6’ R85R1/N209R1 and D88R1/N209R1, respectively.To estimate the dipolar broadening by short inter-spin distances in CW EPR, the spectra of cA6. R1 moieties were introduced at residues 85 or 88 and 209 of both chains of the StCsm6’ dimer using mtsslWizard ; QuikChange Site-Directed Mutagenesis kit ; M9 minimal medium supplemented with Selenomethionine Nutrient Mix ; (L)-selenomethionine ; Tobacco Etch Virus (TEV) protease (recombinantly over-expressed in-house); cA3, cA4, cA6 ; SUPERase·In RNase inhibitor ; 125 nM RNaseAlert FAM™ reporter substrate ; radiolabelled RNA oligonucleotide A1 (5′AGGGUAUUAUUUGUUUGUUUCUUCUAAACUAUAAGCUAGUUCUGGAGA) ; JCSG and PACT 96-well commercial screens ; Gryphon robot ; S-[methyl] methanesulfonothioate (MTSL) spin label ; Xevo G2 TOF MS with Acquity HPLC ; MassPrep cartridge column ; deuterated glycerol .S. thermophilus Csm6’, codon optimized for expression in Escherichia coli ; pEHisV5TEV vector (E. coli C43 (DE3) cells ; E. coli B834 (DE3) cells .Synthetic gene (g-block) encoding V vector ; E. coliFor kinetic analyses, all assays were repeated in triplicate. Means and standard deviations were calculated and shown along with original data points.Bio-Formats plugin of ImageJ , Fiji paS. thermophilus encodes two Csm6 proteins, denoted Csm6 (StCsm6) and Csm6’ (StCsm6’)  Figure . The kin) Figure , while S) Figure . As StCs6 Figure . Extendi6 Figure .6 activator  (PDB: 6TUG) (A DALI  search sB: 6TUG) , with a B: 6TUG) , this is6/cFA6 complexes are largely superimposable (RMSD of 2.1 Å over 162 Cα atoms for the monomer)  Figure . The corThe 6H domain, first predicted bioinformatically , links tStaphylococcus epidermidis   , with RMde 5YJC) . HoweverFobs-Fcalc map revealed electron density consistent with a molecule of cA6 bound to the StCsm6’ dimer, which was fitted into the model , with occupancies estimated as 0.50 (cA6) and 0.50 (both A3 > P)  . All intr Figure .6 and StCsm6’ were made exclusively to residues in the CARF domains, and were symmetrical with respect to each monomer in the dimer. cA6 formed hydrogen bonds with main chain atoms in D10, T11, R15, D19, A107, Q110, S133, H136, A137 and N139, and with side chain atoms in T11, S39, D73, K112, N139 and R167, as well as a stacking interaction between H77 and one of its adenosine moieties  sides of the cA6 ring to produce two A3 > P molecules. The observed position of cleavage in cA6 is consistent with the notion that the angle formed between the 2′-OH, P and O should be close to 180°. The mean of this angle at the two positions in cA6 where cleavage occurs is 172°, compared to 100° and 159° at the other equivalent positions in the cA6 ring  StCsm6’, we could evaluate the extent to which StCsm6’ and variants degraded cA6 at stage 1, and thus protected RNA from enzymatic degradation in the second stage of the assay , WT StCsm6’, S105W StCsm6’ (CARF variant) or H336A StCsm6’ (HEPN variant). The control reaction demonstrated that cA6 remaining in the reaction mixture after stage 1 could activate RNA degradation by StCsm6’ in stage 2. In contrast, when cA6 was pre-incubated with WT StCsm6’, RNA was protected from degradation at stage 2, across the different concentrations of cA6 tested. When cA6 was preincubated with the H336A variant at stage 1, RNA degradation was greatly reduced at stage 2, suggesting that this variant can still degrade cA6 similarly to the WT protein. In contrast, the S105W variant degraded only very small amounts of cA6 at stage 1. Thus, the CARF domain seems to be the major site for cA6 degradation in StCsm6’ under the conditions tested, which had low (nM) cA6 and an excess of enzyme. These data fit well with previous findings for StCsm6 where cA6 degradation was measured directly   Figure . This wa) Figure .6, with RMSD values of 1.4 Å or lower over the entirety of each domain , which are positioned at a similar angle in the two structures, but those in complex with cA6 are pulled inwards towards the dimer interface  . As seenproteins .6 bound states of StCsm6’ observed in the crystal structures, pulse dipolar electron paramagnetic resonance spectroscopy (PDS) was employed . As StCsm6’ is a homo-dimer, introduction of a single cysteine would yield a protein complex containing two spin labels. However, modelling revealed that in the dimer only minimal distance changes between any two identical amino acid residues in the two monomers are expected upon cA6 binding. Thus, double cysteine mutants were designed for which significant distance changes could be predicted . Despite the spin label distances in both states exceeding the models and hinting at a more expanded state in solution, the increase of short distance distributions was very clear in the PDS data. Results on an additional double cysteine mutant of StCsm6’, D88R1/N209R1, confirm this finding  . For R85 finding . These d6 binding to StCsm6’ is required for activation, we mutated residue D80 to a bulkier tyrosine residue. In the apo StCsm6’ structure, D80 sits in a solvent exposed position in the CARF domain   Figure . We hypoy Figure . This is6 activated family of Csm6 enzymes, which are broadly distributed in bacteria , sharing 34% identity at the amino acid level (6, although Csm6 is activated by lower levels of cA6 than Csm6’ (6 bound in the CARF domain ((S. thermophilus may relate to defence against anti-CRISPRs, as suggested previously (6 concentrations, or simply redundancy.id level . Each foan Csm6’ . Both de domain ( and thiseviously , differi6-bound forms of StCsm6’, which underlies the activation of nuclease activity upon binding cA6. These structural insights show the 6H domains, which connect the CARF and HEPN domains in StCsm6’, play a key role in facilitating a large rotational movement to open the ‘jaws’ of the HEPN domains and reorientate the active site.In summary, we have demonstrated a dramatic structural transition between the apo and cAgkad739_Supplemental_FilesClick here for additional data file.


Femoral head necrosis is responsible for severe pain and its incidence is increasing. Abnormal adipogenic differentiation and fat cell hypertrophy of bone marrow mesenchymal stem cells increase intramedullary cavity pressure, leading to osteonecrosis. By analyzing gene expression before and after adipogenic differentiation, we found that Microfibril-Associated Protein 5 (MFAP5) is significantly down-regulated in adipogenesis whilst the mechanism of MFAP5 in regulating the differentiation of bone marrow mesenchymal stem cells is unknown. The purpose of this study was to clarify the role of MAFP5 in adipogenesis and therefore provide a theoretical basis for future therapeutic options of osteonecrosis. By knockdown or overexpression of MFAP5 in C3H10 and 3T3-L1 cells, we found that MFAP5 was significantly down-regulated as a key regulator of adipogenic differentiation, and identified the underlying downstream molecular mechanism. MFAP5 directly bound to and inhibited the expression of Staphylococcal Nuclease And Tudor Domain Containing 1, an essential coactivator of PPARγ, exerting an important regulatory role in adipogenesis. Long-term use of glucocorticoids induces necrosis of trabecular bone and bone marrow, eventually leading to hip dysfunction2. The pathogenesis of SONFH involves increased adipogenesis and fat cell hypertrophy in marrow, increasing intraosseous pressure and inducing avascular necrosis3. However, there are no effective methods to prevent and treat SONFH, which thus necessitates hip replacement, the main treatment of end-stage SONFH at the present time. However, hip replacement is costly and causes marked trauma. Reversing the disorders of lipid metabolism, such as inhibiting adipogenesis of bone marrow mesenchymal stem cells (BMSCs), might improve SONFH treatment.Steroid-induced osteonecrosis of the femoral head (SONFH) is a common orthopedic disease with a high disability rate4. The differentiation and maturation of adipocytes is complex and involves multiple signaling pathways, such as Erk, Wnt, BMP, and hedgehog pathways7. These affect the transcriptional activity of key factors, such as peroxisome proliferator-activated receptor-g (PPARγ), and CCAAT/enhancer binding protein alpha (CEBPα), which promote the initiation and maturation of adipogenesis9. These mechanisms maintain normal adipogenic differentiation by complex mutual regulation. Among them, the PPARγ signaling pathway is important in adipogenic differentiation and maturation10. A hormonal adipogenic stimulus triggers expression of CCAAT/enhancer-binding proteins, inducing the expression of PPARγ and initiating a series of downstream adjustment processes11. Transcriptional activation of PPARγ and other nuclear hormone receptors regulates the participation of co-activators or co-repressors that link nuclear receptors with the basal transcription machinery. Previous studies indicated the special role of PPARγ in SONFH. Pravastatin was found to prevent SONFH by suppressing the expression of PPARγ and activating Wnt pathway at both the mRNA and protein levels13, and Huogu I formula has been specifically shown to inhibit fat formation-related gene expression and the occurrence of SONFH by inhibiting the expression of the PPARγ gene14. SND1 , also known as Tudor staphylococcal nuclease (Tudor-SN) or p100, is a co-activator of PPARγ, which, by directly binding PPARγ, promotes the transcription of downstream factors and adipogenic differentiation15.Fat cells in bone marrow are mainly derived from BMSCs17. Mutation of MFAP5 is associated with the pathology of thoracic aortic aneurysm and dissection in human18. Moreover, MFAP5 regulates the progression of ovarian cancer, breast cancer, and tongue cancer21. Previously, our research group found that MFAP5 promoted osteogenic differentiation of MSCs by activating the Wnt/β-catenin and AMPK signaling pathways22. However, the function of MFAP5 in regulating the differentiation of BMSCs remains unclear. By analyzing the data of adipogenic precursor cells and mature adipocytes, we found that the expression of MFAP5 was significantly down-regulated, implicating MFAP5 in adipogenesis. We verified the sequencing results and confirmed the function of MFAP5 in adipogenesis by silencing and overexpressing. In addition, we validated the mechanism of its regulation of BMSC differentiation. MFAP5 directly bound to and inhibited the expression of SND1, a novel coactivator of peroxisome PPARγ, suppressing the expression of downstream molecules of PPARγ including CD36 and Adipsin24. In summary, MFAP5 is involved in the regulation of adipogenic differentiation, and has potential as a therapeutic target for diseases caused by adipogenic over-differentiation, such as SONFH.MFAP5, also known as Microfibril-Associated Glycoprotein 2 (MAGP2), is a 25 kDa glycoprotein, present in the stroma and extracellular matrix of all tissues. MFAP5 secreted by mesenchymal stromal cells plays a key role in the hematopoietic and immune systemshttps://www.ncbi.nlm.nih.gov/). The GEO2R web tool was used to identify differentially expressed genes with screening criteria of an adjusted P < 0.05 and an absolute logFC value of > 1.5 after eliminating invalid and duplicate values. Next, the intersection of the differentially expressed genes was found and the top 30  differentially expressed genes were visualized.The gene expression profiles of undifferentiated and differentiated adipocytes, including GSE20696, GSE40565, and GSE119593, were acquired from the GEO database , L-Ascorbic acid , β-Glycerophosphate , Isobutylmethylxanthine  and Indomethacin  were purchased from Sigma Aldrich, USA. The antibody to MFAP5(DF13146) was from Affinity, USA. Antibodies to SND1(A5874), IgG (AC005), PPARγ(A19676), CD36(A19016), Adipsin (A8117), and β-actin(AC026) were from ABclonal, CHN. Goat anti-rabbit(7074) and -mouse(4410) antibodies from CST, USA were used as secondary antibodies.The 293 T (for lentivirus packaging), 3T3-L1, and C3H10 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai, and placed in high-glucose DMEM containing 10% FBS; the medium was exchanged every 3 days. 3T3-L1 and C3H10T1/2 cells were induced to differentiate when they reached 100% confluence in differentiation medium, . The differentiation medium was renewed every 2 days.Three plasmids  were purchased from GeneChem, China. Three shRNA sequences targeting mouse MFAP5 were designed for knockdown of MFAP5 expression  and 3 to SND1 .The full-length coding sequence of mouse MFAP5 was amplified and inserted into a lentiviral vector CMV-MCS-EGFP-SV40-Puro to overexpress MFAP5 in C3H10 and 3T3-L1 cells. To produce lentiviruses, a lentiviral vector (pLKO.1-puro or pCDHCMV-MCS-EF1-Puro), psPAX2, pMD2.G, and Lipofectamine2000 (Invitrogen) were mixed and added to 293 T cells in high-glucose DMEM without FBS. The cell density was about 80%. At 12 h after transfection, the medium was changed to high-glucose DMEM with 10% FBS. Two days later, the supernatants were collected and filtered through a 0.45 μm membrane (Millipore). Cells were infected with lentivirus using 6 μg/mL polybrene. Next, puromycin was used to screen for stably transfected cells at a concentration of 3 μg/mL for 3 days and 1 μg/mL for 1 week.Oil red O staining was performed to assay lipid accumulation. Differentiated cells were rinsed with PBS three times and fixed in 4% formaldehyde for 25 min at 21 °C. Saturated Oil red O stain (0.5% in isopropanol) was diluted 60% with ddH2O and added to wells for 1 h at 21 °C. The cells were rinsed in 75% ethanol to remove residual Oil red O stain, and stained cells were observed and photographed.−ΔΔCT method was used to analyze the data. β-actin was set as the internal reference to normalize gene expression between samples. The primers used for qRT-PCR were from PrimerBank (https://pga.mgh.harvard.edu/primerbank/) and are listed in Supplemental File RNAiso Plus  was used to extract total RNA according to the manufacturer's instructions. RNA samples (500 ng) were reverse transcribed into cDNA with Primescript™ RT Master Mix . Quantitative real-time PCR reactions were implemented in a total volume of 10 μL, comprising 5 μL of 2 × SYBR Premix Ex Taq, 1 μL of diluted cDNA, and 0.2 μM primers. The amplification program was as follows: 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 34 s at 55 °C, then melting curve analysis for 15 s at 95 °C, 1 min at 55 °C, 15 s at 95 °C and 15 s at 60 °C. Each sample was established three holes and detected in three times independently. The 2https://imagej.nih.gov/ij/).Culture medium was removed, and adherent cells were washed twice with cold PBS. Next, 60 μL of RIPA  buffer with 1% PMSF  were added to 60 mm dishes and the cells were scraped into EP tubes. Lysates were vortexed for 10 s every 5 min for 30 min at 0 °C and centrifuged at 12,000 rpm at 4 °C for 10 min to harvest supernatant. A BCA kit  was used to determine the total protein concentration. Protein samples were subjected to SDS-gel electrophoresis and transferred to PVDF membranes (Millipore), which were blocked using 5% non-fat milk for 2 h at room temperature. According to the molecular weight of different proteins, the blots were cut from the membranes into a single strip, and put into a 15 ml centrifuge tube containing primary antibody for incubation. Following incubation with the primary antibodies at 4 °C overnight, the PVDF membranes were rinsed with TBST three times for 10 min each and probed with a goat anti-rabbit or -mouse secondary antibody  at room temperature for 2 h, followed by detection using an ECL Kit . Images were analyzed using the Fluor Chem E system (Proteinsimple) and results were quantified using ImageJ software  buffer with 1% PMSF  were added to a 100 mm dish. Next, 1 mL syringes were used to pump cells for 10 min. Next, 40 μL of Protein L Magnetic Beads  were incubated with the corresponding antibody for 30 min at room temperature and washed four times in PBST. Protein samples and treated magnetic beads were incubated for 1 h at room temperature on a rotator. The magnetic beads were collected and rinsed four times in PBST, and eluted with 20ul loading buffer at 100 °C for 10 min.t-test was used to compare two groups, and one-way ANOVA for more than two groups. P < 0.05 indicated a significant difference.Statistical analysis was performed using GraphPad Prism 8.0 software for Windows. Numerical data are means ± SD from at least three replicates. The independent two-tailed Student’s 27. Oil red O staining and analyzing the mRNA levels of CEBPα, FABP4, and SREB1 at 0, 3, 7, and 10 d of adipogenic induction showed that C3H10 and 3T3-L1 had good adipogenic differentiation potential (Fig. As shown in Fig. 1A, 57 differentially expressed genes were at the intersection of GSE20696, GSE40565, and GSE119593; the top 30 genes were shown in Fig. An siRNA was used to knockdown expression of MFAP5 in C3H10 and 3T3-L1 cells. To enhance knockdown, three shRNA sequences were designed and used in combination with a lentivirus transfection system to establish stable expression cell lines. Western blotting Fig. A-C and qWe evaluated the role of MFAP5 in adipogenesis by Oil red O staining Fig. F and by 15. As shown in Fig. After ensuring the effect of MFAP5 on adipogenic differentiation, we further explored the underlying exact mechanism. Adipogenic differentiation involves complex pathway regulation. PPARγ requires auxiliary factors to regulate its downstream molecular transcription and trigger lipid accumulation, resulting in fat differentiation and maturation. Duan et al. reported that SND1 was an essential coactivator of PPARγTo confirm that MFAP5 suppressed adipogenesis by inhibiting the expression of SND1, we knocked down SND1 in MFAP5-silenced cells and assessed their adipogenic differentiation capacity (knockdown efficiency is shown in Supplemental File To validate the above results, we established MFAP5 overexpression C3H10 and 3T3-L1 cell lines and evaluated their gene expression profiles (Fig. 28. Here, we found that the expression of MFAP5 was significantly down-regulated during adipocyte differentiation and maturation. We verified the sequencing results and demonstrated the function of MFAP5 in adipogenesis by silencing or overexpression in C3H10 and 3T3-L1 cells32. As mesenchymal stem cells, C3H10 cells are more primitive than 3T3-L1 cells and have multidirectional differentiation ability. Both cell lines are used in adipogenic differentiation research. Our findings showed that MFAP5 inhibits adipogenesis by suppressing an essential coactivator of PPARγ.Normal adipogenesis or adipocyte differentiation, which is regulated by a cascade of sequentially acting chromatin-modifying coregulators and transcription factors, plays an important role in balancing cell ratios in bone marrow. In older adults, the differentiation of adipocytes is enhanced, causing increased intraosseous pressure, avascular necrosis, and inhibition of homologous cell differentiation34. Maija et al.35 reported that MFAP5 was highly expressed in adipose tissue, and the expression of MFAP5 decreased during adipocyte differentiation in SGBS cells. However, they focused on adipose tissue inflammation and did not conduct an in-depth study of changes in gene expression or the role of MFAP5 in regulating adipocyte differentiation. During tumor development, MFAP5 may participate in the notch1, notch2, and Akt signaling pathways, which are related to adipocyte differentiation38. Therefore, MFAP5 may be involved in the regulation of adipogenesis.MFAP5 is a component of extracellular elastic microfibrils, implicated in cardiovascular progression, breast cancer, carcinogenesis, and alveolar elastogenesis39. After binding to ligands, activated PPAR combines with 9-cis-retinoic acid retinoid X receptors to form a heterodimer, then binds to the peroxisome proliferator response element of a target gene, activating its transcription40. PPARs play an important regulatory role in physiological processes such as fat synthesis, lipid metabolism, insulin sensitivity, and particularly the synthesis of enzymes involved in fatty acid β-oxidation41. According to their structure and function, PPARs are divided into three subtypes: PPARα, PPARβ/δ, and PPARγ42. Based on their promoter structure and mRNA splicing mode, PPARγ genes can be divided into PPARγ1, PPARγ2, PPARγ3, and PPARγ4. Among them, PPARγ1, PPARγ3, and PPARγ4 encode the same protein43. Compared with other types of PPARs, PPARγ is the most adipocyte-specific; its expression is high in adipose tissue and adipose cell lines, but low in other tissues and cell lines44. Activated PPARγ regulates the expression of adipocyte-related genes and promotes the differentiation and increases the number of adipocytes. Co-activators—including SND1, SRC-1, PRIP, p300 and TATA—are necessary for transcriptional activation of target genes47. Adipocyte differentiation is inhibited in the absence of these co-activators48. Duan et al.15 knocked out the expression of SND1 in 3T3-L1 cells and cultivated them in adipogenic induction medium for 8 days. Adipogenic differentiation almost completely stopped compared to control cells.The ligand-activated transcription factor PPAR, a nuclear receptor of the steroid, thyroid, and retinoic acid receptor superfamily, is the master regulator of adipogenesisMFAP5 directly binds to SND1, as determined by co-immunoprecipitation, suggesting that the two proteins interact. In MFAP5-knockdown cells, the expression of SND1 was significantly inhibited. Notably, the downstream genes of PPARγ were markedly inhibited during adipogenesis in MFAP5-knockdown C3H10 and 3T3-L1 cells. Next, we knocked down the expression of SND1 in MFAP5-silenced cells, followed by adipogenic induction. As expected, the promotion of adipocyte differentiation by MFAP5-silenced cells was reversed. Therefore, SND1 is implicated in the negative regulation by MFAP5 of adipogenic differentiation.MFAP5 directly binds to and inhibits the expression of SND1. Our findings expand the upstream molecules of the PPARγ signaling pathway and suggest molecular targets for related research. MFAP5 knockdown may facilitate the development of novel therapeutic strategies for diseases caused by excessive adipogenic differentiation by inhibiting adipogenic differentiation of BMSCs in the femoral head of patients on long-term glucocorticoids.Supplementary Information.


In flowering plants, pollination, pollen tube growth, and fertilization are regarded as the first hierarchical processes of producing offspring. However, their independent contributions to fruit set and development remain unclear. In this study, we examined the effect of three different types of pollen, intact pollen (IP), soft X-ray-treated pollen (XP) and dead pollen (DP), on pollen tube growth, fruit development and gene expression in “Micro-Tom” tomato. Normal germination and pollen tube growth were observed in flowers pollinated with IP; pollen tubes started to penetrate the ovary at 9 h after pollination, and full penetration was achieved after 24 h (IP24h), resulting in ~94% fruit set. At earlier time points , pollen tubes were still in the style, and no fruit set was observed. Flowers pollinated with XP followed by style removal after 24 h (XP24h) also demonstrated regular pollen tubes and produced parthenocarpic fruits with ~78% fruit set. As expected, DP could not germinate and failed to activate fruit formation. Histological analysis of the ovary at 2 days after anthesis (DAA) revealed that IP and XP comparably increased cell layers and cell size; however, mature fruits derived from XP were significantly smaller than those derived from IP. Furthermore, there was a high correlation between seed number and fruit size in fruit derived from IP, illustrating the crucial role of fertilization in the latter stages of fruit development. RNA-Seq analysis was carried out in ovaries derived from IP6h, IP24h, XP24h and DP24h in comparison with emasculated and unpollinated ovaries (E) at 2 DAA. The results revealed that 65 genes were differentially expressed (DE) in IP6h ovaries; these genes were closely associated with cell cycle dormancy release pathways. Conversely, 5062 and 4383 DE genes were obtained in IP24h and XP24h ovaries, respectively; top enriched terms were mostly associated with cell division and expansion in addition to the ‘plant hormone signal transduction’ pathway. These findings indicate that full penetration of pollen tubes can initiate fruit set and development independently of fertilization, most likely by activating the expression of genes regulating cell division and expansion. To prepare DP, the anther cones were dried at 100°C for 2 h. IP were prepared by drying the anther cones at 35°C for 6 h. Finally, to make XP, fresh anther cones were first subjected to a soft X-ray irradiation  of 1000 Gy for 72.15 minutes followed by drying at 35°C for 6 h. The protocol for soft X-ray irradiation was derived from our work (unpublished data) at the University of Tsukuba.2.3Flowers were emasculated one day before anthesis (-1 DAA) to avoid self-pollination. The emasculated flowers were then pollinated on the next day (0 DAA) using the pollen types described in section 2.2. Styles were removed from IP-pollinated flowers 3 h, 6 h, 9 h, 12 h, and 24 h after pollination . For XP- and DP-pollinated flowers, styles were removed 24 h after pollination . In all cases , ovaries that remained after style removal were left to stand on the plants (10 plants per each treatment) for further histological and RNA-Seq analyses as well as assessment of fruit set ratios. The ovaries were collected at 2 DAA  and from 2–10 DAA . Ovaries from emasculated but unpollinated flowers (denoted as E) with style removal 24 h after anthesis were also collected at the same timepoints to represent control treatments.2.43PO4 buffer. The stained pistils were then mounted in 100% glycerol on a slide and observed under a UV microscope . At least three pistils were observed for each of the seven treatments.Pistils were collected 3 h, 6 h, 9 h, 12 h, and 24 h after pollination with IP pollen, and 24 h after pollination with XP and DP. The pistils were then fixed in 3:1 ethanol (100%): acetic acid (100%) solution for 12 h, washed in 70% ethanol and finally softened in 5 N NaOH for 12 h. Softened pistils were washed five times in distilled water and stained overnight in the dark with 0.01% aniline blue solution in K2.5Fruit set ratios were evaluated using five different plants and a total of 21-30 flowers for each style removal  treatment. The ratios were expressed as the percentage of ovaries which remained on the flowers and showed an increase in size at 10 DAA.2.6Histological analyses were carried out using Technovit 7100  according to the protocol of 2.7® Plant Mini Kit  according to the manufacturer’s instructions. Treatment with DNase I  was carried out to remove genomic DNA contamination. mRNA was then purified from the total RNA using a poly(A) mRNA magnetic isolation module kit (New England BioLabs). The pure mRNA was used to construct paired-end libraries for Illumina using the Ultra™ II directional RNA library prep kit (New England BioLabs), and sequencing was performed on an Illumina NovaSeq 6000 platform .Samples for RNA-Seq analysis included emasculated but unpollinated ovaries (E) as a control , and ovaries from IP6h, IP24h, DP24h, and XP24h style removal treatments. These samples were selected based on pollen tube growth observations; IP6h represented partial pollen tube growth (no penetration into the ovaries and hence no fertilization), IP24h represented full pollen penetration into the ovaries with fertilization, XP24h represented full pollen tube penetration without fertilization, while DP24h represented pollination only (no pollen tube growth and no fertilization). All samples were collected 48 h after pollination (2 DAA), and each sample contained 5 ovaries with three replications. Total RNA was extracted from the ovary samples using the RNeasy2.8RNA-Seq data were analysed on the Galaxy main server2.9P < 0.05. All statistical analyses were performed using Statistical Tool for Agricultural Research (STAR), version 2.0.1  test at on 2.0.1 .33.1In vivo pollen tube growth assays, following pollination of tomato flowers with IP, XP and DP, were crucial in this study to determine the independent effects of pollination, pollen tubes, and fertilization on fruit setting. For IP-pollinated flowers, pollen tubes elongated to approximately one-third and three-quarters of the styles after 3 h and 6 h  that developed from XP24h and IP24h ovaries. As shown in 2 = 0.85) between these two phenotypes  were also determined , while there were 11, 65, and 178 uniquely up-regulated genes in IP6h, XP24h, and IP24h samples, respectively  and cyclin B2_7 (Solyc03go32190). Likewise, five members of the expansin gene family and one gene encoding an expansin precursor (Soly02g088100) showed up to 3.6-fold higher expression in XP24h and IP24h ovaries  of ARF7 and ARF5, which are key transcription factors that regulate fruit set and early fruit development. In addition, XP24h and IP24h ovaries displayed increased expression of two genes that positively regulate gibberellin synthesis (GA20ox1 and GA20ox2), and repression of GA2ox4, a negative controller of gibberellin catabolism , particularly in XP24h and IP24h ovaries (GA20ox1 (log2FC = 3.09), NCED1 (log2FC = -2.75), GA2ox2 (log2FC = -2.26), AGL6 (pat-k) (log2FC = -2.17), and ARF7 (log2FC = -2.02).In this study, pollination by X-ray-irradiated pollen resulted in a considerably high fruit set ratio (78.3%) of parthenocarpic fruits . We therIAA9 which is an early auxin-responsive gene remained unchanged  have been unsuccessful to date, as irradiation with X-ray or gamma-ray on dried pollen severely affected pollen germination, reduced fruit set, and resulted in very tiny parthenocarpic fruits  of fruit development. In addition, our results suggest that the contribution of cell expansion to fruit development is greater than that of cell division from 4 DAA onwards, which is a much earlier timepoint than previous models  can be found below: DNA Data Bank of Japan (DDBJ) database (DRR461225-DRR461239).All authors contributed to the article and approved the submitted version. Conceptualization: LT, HE, KS. Experimental design: LT, HE, KS. Data curation: LT, MK. Data analysis and visualization: LT, KS, OM. Writing-original draft preparation: LT, KS. Writing-review and editing: LT, HE, KS, OM, MK. Supervision: HE, KS. Resources: HE.


Histones posttranslational modification represent an epigenetic mechanism that regulate gene expression and other cellular processes. Quantitative mass spectrometry used for the absolute quantification of such modifications provides further insight into cellular responses to extracellular insults such as infections or toxins. Methamphetamine (Meth), a drug of abuse, is affecting the overall function of the immune system. In this report, we developed, validated and applied a targeted, MS-based quantification assay to measure changes in histone H3 lysine 14 acetylation (H3K14Ac) during exposure of human primary macrophages to HIV-1 infection and/or Meth.9KSTGGKAPR17 peptides and the corresponding stable isotope labeled (SIL) heavy peptide standards of the same sequences. The histone samples were propionylated (Poy) pre- and post- trypsin digestion so that the sequences of the monitored peptides were: K[Poy]STGGK[1Ac]APR, K[Poy]STGGK[1Ac]APR-heavy, K[Poy]STGGK[Poy]APR and K[Poy]STGGK[Poy]APR-heavy. The absolute amounts of the acetylated and unmodified peptides were determined by comparing to the abundances of their SIL standards, that were added to the samples in the known concentrations, and, then used for calculation of H3K14Ac stoichiometry in CIC, CIM and MIM hMDM.The quantification assay was developed and validated to determine H3K14Ac stoichiometry in histones that were isolated from the nuclei of control (CIC) and exposed to Meth before (CIM) or/and after (MIM) HIV-infection human monocyte-derived macrophages (hMDM) of six donors. It was based on LC–MS/MS measurement using multiple reaction monitoring (MRM) acquisition of the unmodified and acetylated form of lysine K14 of histone H3 9KSTGGKAPR17 peptides, respectively. The LLOQ was 0.5 fmol/µL and the linear range of the assay was from 0.5 to 2500 fmol/µL. The absolute abundances of the quantified peptides varied between the donors and conditions, and so did the H3K14Ac stoichiometry. This was rather attributed to the samples nature itself, as the variability of their triplicate measurements was low.The assay was characterized by LLOD of 0.106 fmol/µL and 0.204 fmol/µL for unmodified and acetylated H3 The developed LC–MS/MS assay enabled absolute quantification of H3K14Ac in exposed to Meth HIV-infected hMDM. It can be further applied determination of this PTM stoichiometry in other studies on human primary macrophages.The online version contains supplementary material available at 10.1186/s12014-023-09438-5. Post-translational modifications (PTMs) of histones are one part of highly complex epigenetic regulatory mechanisms  regulatiChanges in hPTMs are orchestrated by histone modifying enzymes and reflect cells’ response to a variety of stimuli. These stimuli can be related to differentiation, response to environmental changes including insults by all types of infections , toxins,Mass spectrometry proved to be an efficient tool for analysis of PTMs of proteins . MethodsA previous study from our group resulted in a qualitative map of hPTMs of the resting hMDM  Fig. 1)1). In th.The hMDMs used in this study were obtained by differentiating the monocytes from six healthy, HIV-1-, HIV-2- and hepatitis- seronegative human donors. The leukapheresis from normal donors was done in core facilities, similarly as it was in Macur et al. and GrabMonocytes were obtained from leukapheresis and purified by counter-current centrifugal elutriation as previously described . DiffereDigested histone CIC, CIM, and MIM samples were derived from the six human donors included in this study and designated as D160, D164, D222, D406, D410, D423. A separate set of histones from cultured hMDMs from another 10 different donors was pooled together to create biological sample matrix for the development and validation of the LC–MS/MS method.2SO4 by gentle pipetting. The samples were incubated with constant rotation for 2–4 h at 4 ℃. Then, they were centrifuged at 3400 rcf at 4 ℃ for 5 min. The supernatants were transferred to new tubes. The centrifugation and transfer steps were repeated twice to remove any insoluble material. Next, the histones were precipitated by adding chilled 100% trichloroacetic acid (TCA)  with ½ volume of TCA to the collected supernatant to obtain a final TCA concentration of 33%; the samples were mixed by pipetting. The mixtures were incubated on ice for at least 1 h. Then, the mixtures were centrifuged at 3400 rcf for 5 min; and the supernatants were carefully (without scraping the sides or the pellet) removed by aspiration. By using a glass Pasteur pipette, the tubes were rinsed with ice-cold acetone + 0.1% HCl , centrifuge at 3400 rcf for 2 min; and supernatants were carefully aspirated. The rinse and centrifuge step were repeated using 100% ice-cold acetone. The obtained histone pellets were dried with a vacuum centrifuge. Then, they were dissolved with 100 µL ddH2O (double distilled water), centrifuged at 3400 rcf for 2 min, and the supernatants were transferred to new tubes. The samples were dried in a vacuum centrifuge. The histone pellets were stored in – 80 ℃ until further use.The cell pellets were washed in nuclear isolation buffer (NIB) without NP-40 Alternative  in the 1:10 cell pellet to buffer ratio (v/v). Supernatant was removed by centrifugation at 700 rcf for 5 min. The cell pellets were placed on ice and lysed by adding NIB with 0.2% NP-40 Alternative in the 1:10 cell pellet to buffer ratio (v/v). The cell lysates were left on ice for 5–10 min and then centrifuged at 1000 rcf for 5–10 min at 4 ℃. The nuclei pellets were then washed by gently resuspending them in 500 µL NIB complete without NP-40 Alternative at 1:5 (v/v) ratio. Next, the samples were centrifuged at 1000 rcf for 5 min at 4 ℃ then supernatants were removed. The wash step was repeated two times to completely remove NP-40 Alternative. The cell nuclei were resuspended in 1:5 (v/v) chilled 0.2 M H4HCO3 (Sigma-Aldrich), pH 8.0. Then, a propionylation reagent was freshly prepared by mixing propionic anhydride (Sigma-Aldrich) with acetonitrile  in the ratio 1:3 (v/v). Caution should be taken when working with propionic anhydride as it is a combustible and corrosive substance. The propionylation reagent was added to samples in 1:4 (v/v) ratio, after which NH4OH (Sigma-Aldrich) was quickly added to the samples with a ratio of 1:5 (v/v) to re-establish pH 8.0 to the solution. The samples were mixed immediately by vortexing, the pH was checked, and they were incubated at room temperature for 15 min. Next, the samples were dried, resuspended in 40 µL of 50 mM NH4HCO3, and the propionylation step was repeated to ensure complete derivatization, as it was done by Sidoli et al. K14[Poy]APR and K9[Poy]K14[1Ac]APR peptides, we chose three of the best performing peptides for the final LC–MS/MS method, as indicated in CPTC Assay Development Guidelines ver.1.0 K14[Ac]-heavy and K9[Poy]K14[Poy]-heavy) were used by the software for calculation of the absolute quantities (fmol/µL) of the endogenous K9[Poy]K14[Ac] and K9[Poy]K14[Poy] peptides in the donors’ CIC, CIM, and MIM samples  coupled with a QTRAP 6500+ mass spectrometer , and controlled by Analyst software (SCIEX). The samples (4 μL) were injected in three technical replicates via an autosampler kept at 8 ℃ into the Omega  reversed-phase high performance liquid chromatography (RP-HPLC) column with SecurityGuard ULTRA Cartridge . 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (ACN) (both LC–MS grade from Honeywell Inc.) at a flow rate of 0.2 mL/min were used for separation of the samples’ components using a linear gradient of 5% to 50% solvent B in 25 min, then ramped to 95% in 3 min, after which the column was re-equilibrated with 5% solvent B for 10 min. Electrospray ionization of the samples was achieved in TurboV Ion Source (SCIEX) operated at a positive 5,5 kV spray voltage, with curtain gas at 35 psi, ion source gas 1 and 2 at 45 psi, temperature 450 ℃, CAD collision gas (CAD) flow medium and then analyzed in MRM mode. We used an absolute quantification (AQUA) approach  to deter ver.1.0 . The det ver.1.0  and is anative_mod is absolute abundance of native modified peptide, Absnative_unmod is absolute abundance of native unmodified peptide. The calculated H3K14Ac stoichiometries for three technical replicates of each of analyzed conditions  for each donor were averaged to obtain average H3K14Ac stoichiometries in CIC, CIM, and MIM samples for donors. Additionally, the CIC, CIM, and MIM H3K14Ac stoichiometries for all six donors were also averaged, to obtain average stoichiometries for each of the analyzed conditions in all donors’ samples together. The SDs and % coefficient of variation (CVs) were calculated as well  and unmodified (K9[Poy]K14[Poy]) peptides determined in Skyline  to create those reversed calibration curves. The max LLOQ bias (relative error) and LLOQ CV were set to 15% and qualitative ion ratio threshold set to 30%. The LOD was calculated from the blanks injected before the calibration curve as the average plus 3 times the standard deviation (SD) of the signal of the blank (3 × SD of the blank). The LLOQ was determined as “the lowest concentration of peptide at which the imprecision of the assay (expressed as the CV) is < 20% “ [2 [Features available in Skyline software were used to prepare the reversed calibration curves for H3 K9[Poy]K14[Poy]-heavy and K9[Poy]K14[Ac]-heavy peptides and calculate figures of merit: lower limit of detection (LLOD) and lower limit of quantification (LLOQ) to determine sensitivity of the assay. The reversed calibration curves were created by plotting nominal analyte concentration (fmol/µL) versus heavy:light peak area ratio for each of the heavy peptides studied  were used by the software for calculation of the absolute quantities (fmol/µL) of the K9[Poy]K14[Ac]-heavy and K9[Poy]K14[Poy]-heavy peptides in the series of the SIL standards dilutions used for reversed calibration curve preparation.Global and untargeted identification of hMDM hPTMs have been published previously . The sum13C, 15N) are incorporated in one or more amino acids that result in the mass shift of the SIL peptide(s), therefore, distinguishing them from unlabeled native peptides in mass spectrometry analysis. In our case the C-terminal arginine (R) of the SIL standards of the analyzed endogenous peptides was 13C-15N labelled and doubly charged, resulting in mass shift of 5 m/z units (10 Da)  in both, acetylated and non-acetylated peptide, while the K14 was acetylated (Ac) or propionylated (Poy) in modified and non-modified peptides, respectively. Therefore, the peptides quantified in this study and their SIL standards were: K9[Poy]K14[Ac], and K9[Poy]K14[Ac]-heavy for acetylated K14 and K9[Poy]K14[Poy], and K9[Poy]K14[Poy]-heavy for unmodified K14 . The LC–MS/MS method for their quantification was developed using standard solutions of the custom-synthetized native light and reference heavy peptides.2 values of 0.9998 and 0.9997  equal to 5.48% for peptide K9[Poy]K14[Poy]APR-heavy and 4.22% for peptide K9[Poy]K14[1Ac]APR-heavy . Based on the absolute abundances of the native modified K9[Poy]K14[Ac] and unmodified K9[Poy]K14[Poy] peptides, we calculated stoichiometry of the K14Ac in the donors’ samples as a percentage of total amount—a sum of K14 modified  and unmodified  fractions—of the investigated H3 peptide . AlthougIn this work, we focused on change in acetylation (Ac) of lysine 14 (K14) in Histone H3.At first we focused on development and validation AQUA LC–MS/MS method and tested how it can be used to investigate changes in hMDM. The obtained absolute quantities of modified and acetylated H3 peptide 3–17 enabled us to calculate stoichiometry of this PTM in HIV-infected and/or Meth treated at different experimental points hMDM. Ac influences transcription regulation leading to many intracellular effects. Lysine residues are positively charged on the side chains while acetylation cause neutralization of the charge. This leads to the change in negatively charged DNA and positively charged histones. Thus, we may expect that modification of H3 K14Ac is participating in cell cycle regulation, cell proliferation, and apoptosis cellular differentiation, DNA replication and repair, nuclear import, and neuronal repression .2 0.9998 and 0.9997, respectively)  monitored for histone H3 K9[Poy]K14[Poy], K9[Poy]K14[Poy]-heavy, K9[Poy]K14[Ac] and K9[Poy]K14[Ac]-heavy peptides investigated in the present study, with manually optimized declustering potential (DP), collision energy (CE) and cell exit potential (CXP). Table S2. LC-MS/MS method validation – peptide results. Table S3. LC-MS/MS method validation – transitions results. Table S4. The LC-MS/MS results for all investigated donors' CIC, CIM and MIM samples with absolute abundances of the endogenous K9[Poy]K14[Ac] and K9[Poy]K14[Poy]. Table S5. MRM transition ratios for standard dilutions and donors’ samples. Table S6. Stoichiometry of H3K14[Ac] in donors’ CIC, CIM and MIM samples.Additional file 2: Figure S1. Reversed calibration curve for K[Poy]STGGK[Ac]APR-heavy peptide. Figure S2. Reversed calibration curve for K[Poy]STGGK[Poy]APR-heavy peptide. Figure S3. MRM chromatograms for K[Poy]STGGK[Ac]APR-heavy peptide from matrix blank spiked with 50fmol/µL K[Poy]STGGK[Ac] peptide. Figure S4. MRM chromatograms for K[Poy]STGGK[Poy]APR-heavy peptide from matrix blank spiked with 50fmol/µL K[Poy]STGGK[Poy] peptide.


Pina-D1a/Pinb-D1b, accounting for 32.0% of the total tested varieties, while the CIMMYT wheat lines were mainly carrying Pina-D1b/Pinb-D1a with 45.8% of the total collection. The distribution frequencies of subunit 1/2* and 5 + 10 were 47.0% and 42.5%, respectively, in CIMMYT varieties, however they were only 31.4% and 13.7% respectively of the Chinese wheat tested varieties. In addition, the proportion of phytoene synthase (PSY) allele, PPO allele and LOX active allele were roughly the same between Chinese and CIMMYT varieties. Based on the present study, we found that Pina gene had a greater impact on grain hardness value than Pinb gene; The influence of PPO-A1 gene on polyphenol oxidase activity was more significant than PPO-D1 gene. The high protein content of varieties mostly containing hardness genes and 1/2*/5 + 10 subunit combinations. Based on the present study, we found that the quality gene distribution of Chinese and CIMMYT varieties was quite different, for instance, the high-quality HMW-GS subunits of Chinese varieties were lower than CIMMYT lines. It will be much useful for Chinese wheat breeders to develop good quality wheat variety by crossing with 3 good strong gluten CIMMYT wheat lines by molecular marker-assisted selection.In order to understand the difference of quality for Chinese and CIMMYT wheat varieties (lines), we selected 153 wheat germplasm from both China and CIMMYT to explore the contribution relationship of different allelic variation combinations to wheat quality through genotyping and phenotyping, including grain hardness, polyphenol oxidase (PPO) activity, lipoxygenase (LOX) activity, yellow pigment (YP) content and protein content. In terms of flour milling quality, Chinese wheat varieties were mainly carrying  Wheat is one of the most important food crops in the world. China is the largest wheat producer and consumer worldwide, accounting for about 1/6 of the global wheat yield production. Although China has high production and inventories of wheat, there is still a large gap of good quality wheat resulting in a huge import . With thHa, which is controlled by Pina and Pinb (Pins) genes, respectively. The loss of Pina protein or the mutation of Pinb protein gene can cause the change of wheat from soft to hard , polyphenol oxidase (PPO) activity, and grain lipoxygenase (LOX) activity mainly affect the quality of wheat flour. In China, the color of flour is a crucial trait of wheat quality evaluation. Phytoene synthase (Psy) is a key enzyme of yellow pigment in flour, which plays a significant role in yellow pigment in flour. Previous studies showed that one of the major QTL on wheat chromosome 7A showed significant association with yellow pigment content, which can explain 60% of YP phenotypic variation . PPO actAt present, the low-quality wheat inventory is high, while the high-quality wheat gap is large. It has become an important issue to explore the quality differences of core germplasm at home and abroad, accelerate the cultivation of high-quality wheat, and improve the structural supply-demand relationship in China. At the present study, we collected 153 wheat germplasm from Chinese and CIMMYT and genotyped with molecular makers of known genes and phenotyped them with the related traits. The objectives of this study are 1) to analyze the differences of wheat quality between Chinese and CIMMYT wheat germplasm; 2) to identify good quality wheat germplasm for wheat breeder to develop new wheat varieties; 3) to figure out the best quality gene combinations for achieving good quality wheat varieties.Ppo-A1b and Ppo-D1a, respectively, those were correlated with the lower PPO activity. The frequencies of Psy-A1b and Zds-A1a were 32.7% and 31.4%, respectively, and they were associated with the lower YP content. The proportions of hardness genes, Pina-D1b and Pinb-D1b, were 48.4% and 32.7%, respectively, while LOX-B1b was accounted for 78.4% that was relevant to the lower LOX activity. The frequencies of 1/2* and 5 + 10 were 78.4% and 56.2%, respectively, association with the HMW-GS subunits  values have been used to define the hard wheat (>62) and soft wheat (<46) . Through studies . The Chi studies . Grain h studies . This reGlu-D1 site has the greatest effect, while Glu-B1 and Glu-A1 have less influence. The different subunits at different sites showed different effects on the processing quality of wheat flour. 1, 2*, 5 + 10 are high-quality subunits for improving the quality of gluten . A standard polymerase chain reaction (PCR) was performed by −1 primer, adding ddH2O to 10 μL. The PCR amplification process was as follows: predenaturation at 94°C for 5 min; denaturation at 94°C for 1 min; annealing at 50°C–66°C for 1 min; extension at 72°C for 2 min. And it was 30–35 cycles in total. Then the PCR amplification was prolonged for 10 min at 72°C and stored at 4°C. The PCR products were detected by 1.2% agarose gel electrophoresis. KASP markers were assayed by RT-PCR . PCR system could be summarized as follows: DNA 1 μL, KASP Master Mix  2.5 μL, primer 0.25 μL and ddH2O 1.25 μL, 95°C pre-denaturation for 15 min, 94°C denaturation for 15 s, annealing at 65°C for 60 s, then each cycle decreased by 0.8°C, a total of 10 cycles, finally reduced to 57°C, 94°C denaturation for 20 s, 57°C annealing for 60 s, a total of 40 cycles. The primers used were synthesized by Beijing Tsingke Biotech Co.Genomic DNA of 153 wheat varieties was isolated from their leaf using the modified cetyltrimethyl ammonium bromide (CTAB) method . 153 wheUse wheat hardness tester JYDX100 × 40 to determine the hardness value HI of wheat samples according to the national standard GB/T21304 .Use a near-infrared instrument (NIR) 2600XT-R to determine the protein content of wheat in terms of AACC39-25.The LOX activity was determined using the method reported by The PPO activity was determined using the method reported by AACC 14–50 has been slightly modified (Approved Methods of AACC. Method 14–50.); Pour 0.5 g of flour into a 10 mL centrifuge tube; add 2 mL of water-saturated n-butanol extract (volume ratio 5:1); stir well and shake for 30 min; centrifuge with Eppendorf at 4,000 r/min for 10 min; 200 μL supernatant is taken from pipette and poured into a 96-well microtiter plate; use a water-saturated nbutanol solution as a control; use a microplate reader to measure the absorbance of the supernatant at 436.5 nm·A, Yellow pigment content = 30.1*A .https://www.sas.com/zh_cn/home.html) and Microsoft Excel, incorporating descriptive statistics, phenotypic correlation analysis and analysis of variance (ANOVA).The phenotype data and genotype test results were processed and analyzed using data processing software SAS9.2 //2*PANDORA/5/WAXWING/3 /BL1496/MILAN//PI610750/4/FRNCLN/6/KACHU/BECARD//WBLL1*2/BRAMBLING and KACHU*2/3/ND643//2*PRL/2*PASTOR/4/KACHU/DANPHE as well as three higher weak gluten Chinese wheat varieties, such as EEN 1, Luo 6010 and Emai 9721. These five wheat germplasm will be used for wheat breeders to develop wheat varieties of better quality through marker-assisted selection.


Bacillus sp. 8A6 can completely degrade the feather in 20 h, which is an efficient keratin degrader reported so far. Comprehensive transcriptome analysis continuously tracks the metabolism of Bacillus sp. 8A6 throughout its growth in feather medium. It reveals for the first time how the strain can acquire nutrients and energy in an oligotrophic feather medium for proliferation in the early stage. Then, the degradation of the outer lipid layer of feather can expose the internal keratin structure for disulfide bonds reduction by sulfite from the newly identified sulfite metabolic pathway, disulfide reductases and iron uptake. The resulting weakened keratin has been further proposedly de-assembled by the S9 protease and hydrolyzed by synergistic effects of the endo, exo and oligo-proteases from S1, S8, M3, M14, M20, M24, M42, M84 and T3 families. Finally, bioaccessible peptides and amino acids are generated and transported for strain growth. The keratinase has been applied for soybean hydrolysis, which generates 2234 peptides and 559.93 mg/L17 amino acids. Therefore, the keratinases, inducing from the poultry waste, have great potential to be further applied for producing bioaccessible peptides and amino acids for feed industry.Keratin is a recalcitrant protein and can be decomposed in nature. However, the mechanism of keratin degradation is still not well understood. In this study, The online version contains supplementary material available at 10.1186/s13068-023-02426-9. More than 10 million tons of feather wastes are annually accumulated from the poultry farm , 3. MeanN-acetylglucosamine and serine or threonine in the non-coiled head structure of the keratin filament dimer [Feathers  . Currentnt dimer , 10.B. licheniformis BBE11-1 and its mutants, which showed that cysteine catabolism can mediate the catalytic procedure of keratinases via the secreting the sulfite [Streptomyces sp. SCUT-3 grew in the feather and indicated that the disulfide bond reduction is the first key step for feather degradation. Then the proteases hydrolyzed the feather to peptides and amino acids for the bacteria nutrients. Iron addition and aeration have been discussed to improve the feather degradation efficiency. These studies have mainly elucidated the thiolysis mechanism of keratin degradation. However, it is still unclear how the keratin degraders acquire nutrients and energy in an oligotrophic feather medium for rapid adaptation and proliferation in the early stage. Meanwhile, the full elucidation of the mechanism of sufficient keratin degradation is still needed for efficiently refining the keratin waste.Unfortunately, feathers cannot be completely degraded by the sole synergistic effect of different types of keratinases. It has been reported that the blending of the purified recombinant keratinases cannot well degrade the feather , 12. The sulfite . The sup sulfite . Li et a sulfite  were theBacillus sp. 8A6, was recently identified to completely degrade feathers in our previous work [Bacillus sp. 8A6 grew in the feather medium. In addition, soybeans are the major sources for food and feed proteins [Bacillus sp. 8A6 were further applied for soybean protein hydrolysis. Relatively high amounts of bioaccessible peptides and amino acids after the degradation revealed the potential for applying the keratinase in feed industry.The efficient keratin degrader, ous work . This stproteins , 24. Howproteins , 26. Alkproteins . TherefoBacillus sp. 8A6 was obtained from the Bacillus Genetic Stock Centre at Ohio State University . The LB medium  was used for cell growth. The feather medium contained 0.75 g/L NaCl, 1.75 g/L K2HPO4, 10 mM MOPS, 0.25 g/L MgSO4·7H2O, 0.055 g/L CaCl2, 0.010 g/L FeSO4·7H2O, 0.005 g/L ZnSO4·7H2O and different concentration of feathers ).Bacillus sp. 8A6 was inoculated into the sterile feather medium and growth under the optimal conditions for 0, 8, 20 h. Cells were collected for RNA extraction. The cells from three time points were mixed and RNA was extracted from the mixture. After grinding cells into powder with liquid nitrogen, RNA was extracted by the instruction of the RNAprep pure Bacteria Kit . Then, the RNA was reverse transcribed to cDNA using reverse transcriptase  according to the manufacturer's instructions. The reaction system was as follows: 4 μl 5X PrimeScript RT Master Mix , 10 μl total RNA, and 6 μl RNase Free ddH2O. The reaction conditions were as follows: 37 °C for 15 min, 85 °C for 5 s and then 4 °C.After the single-factor and response surface method (RSM) optimization, the optimal conditions were inoculation amount of 1.83 mL, feather concentration of 3.905% (w/v) and pH 9.21  . The reaction system was as follows: 10 μl TB Green® Premix Ex Taq™ II, 1 μl each of forward and reverse primers, 1 μl cDNA, and 7 μl ddHBacillus sp. 8A6 genome sequences (Genbank: accession number QFZE00000000) via software Bowtie2 (v2.2.6) [ p-value less than 0.05. And topGO was used to plot DAG. The Kyoto Encyclopedia of Genes and Genomes (KEGG) web server was used to extract enzyme codes and the KEGG pathways. The software used in this analysis was EdgeR. The results were measured according to the difference significance criteria  for screening. Cluster of Orthologous Groups of proteins (COG) database was used to predict the function of proteins or protein assemblies [The RNA was sent to Genewiz company  for Illumina sequencing. The raw data were filtered by Cutadapt (v 1.9.1) and mapped to the (v2.2.6) . Then HT(v2.2.6)  was used(v2.2.6) , 32, and(v2.2.6) , which wsemblies . COG clasemblies .The protease activity was analyzed by using Azo-casein as substrate as described with few modifications . 20 μl 1The keratinase activity was detected as described . 0.4 g kThe optimum pH of protease was determined according to the method of protease activity analysis described above with some modifications that crude enzyme solution was diluted by buffer at a pH range from 6 to 10 . And the substrate Azo-casein (50 mM) was also prepared with buffers at pH 6–10.Similarly, in the determination of the optimum temperature of enzymes, diluted crude enzyme solution and azo-casein were prepared by above optimum pH buffer. The reaction system was incubated at the range of 30–65 °C.The crude enzyme solution was diluted 2.5 times with buffers of pH 6–11  and incubated in the 4 °C for 1 h or 4 h. And then diluted it 2 times again for the analysis of the residual activity of protease by Azo-casein as substrate. Similarly, the crude enzyme solution diluted 2.5 times with 50 mM pH 9 Tris–HCl buffer was incubated at the temperature range of 30–65 °C for 1 h or 4 h and the residual activity of protease was measured as above.The surface morphology at different times in the degradation process of feathers was investigated by SEM, including 0 h, 8 h and 20 h. The feather culture was harvested at 0 h, 8 h and 20 h, and the feather was washed three times with phosphate buffer saline . The remaining steps of sample preparation follow the literature .Bacillus sp. 8A6 keratinase or NY100 with the same enzyme activity, then the system was suspended with 10 mL sodium carbonate buffer (pH 9) and incubated at 60 °C for 1 h with shaking at 250 rpm. Besides, the system without Bacillus sp. 8A6 keratinase or NY100 was used as a negative control. After centrifugation at 12,000 rpm for 10 min, the supernatant was analyzed for amino acids and short chain peptides by Nano LC–MS/MS in Standard Testing Group Co. Ltd. .The biodegradability of SPI was investigated. The SPI degradation system contained 1 g SPI and 2 mL p < 0.05 was regarded as statistically significant.The statistical analyses were executed by GraphPad Prism 8.3.0. In this experiment, the mean ± standard deviation (SD) of more than three sets of data were compared, and checked the significance between each of the two groups by one-way ANOVA. *Bacillus sp. 8A6 in the medium with feather as the sole carbon and nitrogen source  [B. licheniformis, B. subtilis, B. cereus, B. thuringiensis, B. aerius, Bacillus sp. CL18, etc. (24–84 h degradation with extra nitrogen source in the medium) [Pseudochrobactrum sp. IY-BUK1 (3 days), and even microbial consortia KMCG6 (3 days) [Bacillus sp. 8A6 achieved 136.5 U/mL and it was considerably higher than that of other strains [Bacillus sp. 8A6 grown in the feather medium and the growing properties in LB medium  , B. lich medium) –47, Pseu h , B. l strains –51. SEM Bacillus sp. 8A6 before (0 h) and after being inoculated in the feather medium for 8 h and 20 h, respectively  of fatty acid β-oxidation, proton-transporting ATP synthase, inorganic phosphate transmembrane transporter and iron uptake. According to the FPKM statistics, the sulfur cluster binding functional genes were also enriched during this period, which might relate to the sulfite synthesis and exportation for breaking down the feather disulfide bonds. The resulting loose feather could be easily accessed and further hydrolyzed by the induced proteases/peptidases. When the cells grew in the feather medium for 20 h, the GO terms changed greatly  degradation were identified and all of them were up-regulated , complex IV , complex V  and F0 unit (a & b)) were also up-regulated when the strain grew in the feather medium for 8 h. The complex II & IV can push the protons across the cell membrane into the cytoplasm, resulting in the formation of electromotive force which can further provide the free energy for ATP synthesis by the up-regulated complex V. Therefore, when the Bacillus sp. 8A6 started to grow in the feather medium, the oxidative phosphorylation was stimulated to provide sufficient ATP for cell growth and metabolism , homocysteine S-methyltransferase (EC2.1.1.10), methionine synthase (EC2.1.1.13) and 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (EC2.1.1.14) were highly up-regulated for l-methionine synthesis. The l-methionine was further converted to methylmercaptan and sulfite by the up-regulated alkanesulfonate monooxygenase (EC1.14.14.5). Meanwhile, the sulfite reductase (NADPH) (EC1.8.1.2) which was down-regulated for sulfite accumulation. The sulfite was then exported to exocellular to lose the feather structure. When the cells were inoculated into the feather medium for 20 h, the sulfite metabolism was down-regulated and the sulfite was converted to sulfide  were identified and up-regulated. Meanwhile, the sulfite exporter gene tauE instead of sulfite efflux pump gene (ssu) was found when bacteria Streptomyces sp. SCUT-3 grew in feather medium [Bacillus sp. 8A6 grew in feather medium, the 4 pathways are purine metabolism, 2-oxocarboxylic acid metabolism, microbial metabolism in diverse environments and glycine, serine and threonine metabolism. The new up-regulated sulfite metabolic pathways for Bacillus sp. 8A6 in our study could further provide potential strategies for construction efficient engineering Bacillus strains for keratin decomposition.The main challenge for sufficient keratin decomposition is the abundant cross-linked disulfide bonds in/among the keratin filaments . It is n8 h Fig. A. l-Cystionase EC.4.1.13, r medium . HoweverBacillus sp. 8A6 grew in the feather medium for 8 h , cysteine proteases , serine proteases , unknown proteases (U57), glutamic proteases (2G5), aspartic proteases , threonine proteases (T3), and 3 unclassified proteases  for the aspartate/glutamate/glutamine transporter, gene (TcyM) for cystine transporter and genes  for dipeptide transporter were up-regulated. Meanwhile, the biotin transporter  and the iron transporter  were also up-regulated. Li et al. [Bacillus sp. 8A6 inoculated in the feather medium for 8 h. The enzymes such as 3-dehydroquinate dehydratase (EC4.2.1.10) and 3-phosphoshikimate 1-carboxyvinyltransferase (EC2.5.1.19) were up-regulated for shikimate and chorismate synthesis after the active pentose phosphate pathway. Then the key enzymes for all the aromatic amino acids  were equally up-regulated  and temperatures (30–65 °C)  Fig. A and B. ures 30–6 °C  Fig. C. In the°C) Fig. D, betterB. licheniformis PWD-1 have been used in animal feed and significantly improved the crude protein digestibility [Bacillus sp. 8A6 keratinases were the first attempt to be used for improving soybean protein digestibility. The results showed that efficient keratinase could be applied for soybean protein hydrolysis, resulting in the formation of functional peptides and amino acids. The released functional peptides and amino acids after the degradation by Bacillus sp. 8A6 keratinases and well-known soybean degradation commercial protease NY100 were analyzed. During the hydrolysis of soybean protein, Bacillus sp. 8A6 keratinases produced 2234 short peptides  with a relative concentration of 1.14 × 1011, higher than commercial NY100, which produced 1839 short peptides  at a relative concentration of 8.34 × 1010. On the contrary, negative control (CK) produced 108 short peptides  with a relative concentration of 3.49 × 109. Comparatively, the species and concentration of short peptides produced by Bacillus sp. 8A6 keratinases and commercial NY100 separately increased nearly 20-fold and 1–2 orders of magnitude compared to CK, proofing the superiority of Bacillus sp. 8A6 keratinases. Further analysis of amino acids released during hydrolysis, it was found that 17 functional amino acids, including 6 essential amino acids  were produced after 1 h of degradation at 60 °C  and methionine (66.05 mg/L) for NY100 were the two most abundant amino acids, with almost twice the amount of abundant amino acids released by CK. More importantly, the most abundant amino acids produced by Bacillus sp. 8A6 keratinases and NY100 each contained one essential amino acid, namely phenylalanine and leucine. Meanwhile, the amino acid production of Bacillus sp. 8A6 keratinases and NY100 both reached superior levels  at 1 h of hydrolysis, whereas the amino acid production of CK only reached 105.46 mg/L. In addition, Bacillus sp. 8A6 keratinases degrade soybeans to produce more amino acids than papain [Bacillus sp. 8A6 keratinase can not only improve the apparent digestibility of the soybean protein, but also have the potential to reduce or eliminate the antigenicity of soybean protein [Keratinases can be used as additives in animal feed to improve digestibility . The comtibility , 71. In n papain . This ef protein . TherefoIn this study, we proposed the feather degradation mechanism as follows according to the comprehensive transcriptome analysis Fig. : (1) fatAdditional file 1. Table S1. Primers used in this study. Table S2. Response-surface analysis of the conditions when Bacillus sp. 8A6 grew in feather medium. Table S3. Quality analysis of the raw RNA sequencing data when Bacillus sp. 8A6 grew in feather medium for 0, 8 and 20 h. Table S4. Quality analysis of the clean RNA sequencing data when Bacillus sp. 8A6 grew in feather medium for 0, 8 and 20 h. Table S5. Up-regulated keratinases, disulfide reductases for the group 0 h VS 8 h when Bacillus sp. 8A6 grew in feather medium. Table S6. Up-regulated keratinases, disulfide reductases for the group 8 h VS 20 h when Bacillus sp. 8A6 grew in feather medium. Figure S1. Four factors that affect protease activity when the Bacillus sp. 8A6 grew in feather medium. (A) Effects of different temperatures; (B) Effects of different pH; (C) Effects of different feather concentration; (D) Effects of inoculation amount . Response surface 3D plots reveals the interaction of factors affecting protease activity, (E) pH and inoculation amount; (F) inoculation amount and feather concentration; (G) feather concentration and pH. Figure S2. The OD600 of Bacillus sp. 8A6 when grown in feathermedium at different time points. Figure S3. Four factors that affect protein concentration. (A) Effects of different temperatures; (B) Effects of different pH; (C) Effects of different feather concentration; (D) Effects of inoculation amount . Figure S4. Pearson correlation coefficient correlation analysis (A) and principal component analysis (B) among the three RNA-seq samples when Bacillus sp. 8A6 grew in feather medium for 0, 8 and 20 h. Figure S5. Volcano plot of the differentially expressed genes for the groups 0 h VS 8 h (A) and 8 h VS 20 h (B) when Bacillus sp. 8A6 grew in feather medium. Up-regulated genes are shown as red dots and down-regulated genes are shown as blue dots. The significant difference means that the expression level of the genes are 2 times different and the qvalue  ≤0.05. Figure S6. Hierarchical clusters of differentially expressed genes according to the log10(FPKM+1) value when Bacillus sp. 8A6 grew in feather medium for 0, 8 and 20 h. The up-regulated genes were indicated as red color and the down-regulated genes were indicated as blue color based on the color key. Six main clusters were enriched and highlighted by different colors next to the phylogenetic tree. Figure S7. Gene Ontology (GO) analysis of the molecular function, cellular component and biological process for the groups 0 h VS 8 h (A) and 8 h VS 20 h (B) according to the -log10 when Bacillus sp. 8A6 grew in feather medium. The higher -log10 indicates the more significant for enrichment of the GO term. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the pathways for the groups 0 h VS 8 h (C) and 8 h VS 20 h (D) according to the -log10 when Bacillus sp. 8A6 grew in feather medium. 30 significantly different pathways are shown according to the Q-value. Figure S8. Cluster of Orthologous Groups of proteins (COG) analysis of the gene function for the groups 0 h VS 8 h (A) and 8 h VS 20 h (B) according to the gene numbers when Bacillus sp. 8A6 grew in feather medium. The up-regulated genes were indicated as red color and the down-regulated genes were indicated as blue color. Supplemental peptide list 1. Short peptides of Bacillus sp. 8A6 keratinases. Supplemental peptide list 2. Short peptides of NY100. Supplemental peptide list 3. Short peptides of CK.