Aspergillus fumigatus oryzin, allergen Asp f 13, or alkaline proteinase ALP1, containing the sequence motif His–Asp–Ser of the subtilisin family, structure, and function at atomic detail. Given the resolution of the data (1.06 Å), we use fragment molecular replacement with ideal polyalanine α-helices to determine the first crystal structure of oryzin. We probe the catalytic serine through formation of an irreversible bond to a small molecule compound, specifically labeling it, describing the amino acid residues performing the catalytic function. Defined by a self-processed pro-peptide, the active site architecture shapes up pocket-like subsites that bind to and unveil the S1′–S4′ substrate binding preferences. We use molecular modeling to dock a model of the pro-peptide in the S1–S4 region and to dock collagen along the active site cleft. Opposite to the face harboring the catalytic serine, the enzyme binds to a calcium ion in a binding site created by backbone flipping. We use thermal unfolding to show that this metal ion provides structural stability. With no known host inhibitor identified thus far, this structure may hasten the progress of developing new therapeutic agents for diseases caused by pathogenic fungi.We report the molecular basis of  Aspergillus spp. are microorganisms commonly found in the environment that produce airborne fungal spores. Several hundred spores can be inhaled and eliminated per day without harm to the host; however, in individuals predisposed to infection through lung disease and in patients undergoing cancer chemotherapy or immunosuppressive therapy following organ transplants, aspergilli can cause invasive pulmonary aspergillosis, which can be a life-threatening condition2. Several studies have shown that patients with severe COVID-19 had a high risk of developing invasive fungal infections, including aspergillosis3. Among aspergilli, the World Health Organization has designated Aspergillus fumigatus as a critical priority pathogen4. Azoles are the first choice of drug for the treatment of aspergillosis; however, reports of antifungal resistance are increasing in the literature6 such that up to 20% of Aspergillus isolates exhibit de novo resistance to commonly used antifungal agents7. Aspergillosis is an increasingly difficult condition to diagnose and treat9. Recent data suggests that the pathogen's genetic pool for key metabolic and catabolic enzymes in human infection is more versatile than originally thought10. More research into available molecular targets will help us understand the complexities of this organism and its relation to host virulence, drug resistance, and target characterization with the goal of developing new therapeutics.Aspergillus spp. degradome includes 111 peptidase genes (about 1% of the genome) coding for 14 different types of peptidases11. The most studied of such peptidases is the alkaline serine peptidase oryzin . By a conserved sequence motif, and the Asp–His–Ser triad arrangement, oryzin belongs to the SB clan, S8A subfamily of subtilisins12. Initially, oryzin clinical isolates were found to hydrolyze elastin, a key protein component of the connective tissue, and fibrinogen, a blood-circulating glycoprotein, hence mechanistically this enzyme was characterized as elastinolytic or fibrinogenolytic14. Different Aspergillus strains including A. fumigatus, A. flavus, A. nidulans, and A. terreus showed a strong digestion of collagen mediated by abundantly secreted oryzin when grown in a collagen medium16. Another study showed oryzin efficiently cleaved complement components C3, C4, C5, and C1q, as well as immunoglobulin G, and that this was the only serine protease in culture supernatant performing this activity, such that it could be prevented by abrogating the oryzin gene17. Additionally, oryzin isolated from fungal spores was shown to detach cultured Vero cells suggesting oryzin's ability to remove attachment without impairing the cells18. In patients with asthma, Aspergillus may cause a spectrum of conditions from allergic bronchopulmonary aspergillosis to severe asthma with fungal sensitization. In murine models of fungal asthma, oryzin was found as an essential mediator for the recruitment of inflammatory cells and remodeling of the airways19, disrupting airway smooth muscle cell-extracellular matrix interactions, and eroding epithelial cell junctions22. Using a deletion mutant of the oryzin gene, Namvar et al.19 found that oryzin was indispensable for the initiation and progression of fungal asthma. In summary, oryzin activity on host proteins during infection serves as a promising route for the development of diagnostic tools and inhibitors to target this peptidase. However, the molecular basis for substrate recognition is largely unknown and is difficult to rationalize based on primary sequence alone. To provide a deeper insight into the function and structure of the Aspergillus secreted serine peptidase oryzin, we determined its first three-dimensional structure by X-ray crystallography. We obtained complexes with its pro-peptide segment, defined the substrate binding pockets, a tetrahedral irreversible adduct with the catalytic serine, captured the interactions between the key residues and calcium in catalysis, defined the metal ion binding site that provides structural stability.Fungal colonization of the lungs requires a vast supply of hydrolytic enzymes. 23. Typically, serine peptidases are synthesized as large multi-segmented polypeptides (zymogens) that are activated auto-catalytically and/or exogenously. Previous work with clinical isolates of oryzin-producing spores identified the Asp–His–Ser catalytic signature typical for serine peptidases of the subtilisin type26. Given the importance of serine proteases in key biological reactions carried out both inside and outside the cell, they are present among all taxonomic kingdoms, from archaea to animals  and the catalytic domain (residues 122–403), originating from a single polypeptide precursor , a causative agent of mycoses . Further, bacterial enzymes such as a proteinase K-like from Serratia spp.  and the aqualysin from Thermus aquaticus  are highly similar. In addition, the structure aligns to the human pro-protein convertase/subtilisin kexin type 9  at a rmsd 1.4 Å over 251 Cα atoms.Oryzin has a globular fold that conforms to the α/β three-layered sandwich type resembling eukaryotic and prokaryotic subtilisin-like peptidase enzymes Fig. a. A 3D sBacillus amyloliquefaciens . Interestingly, it also is similar to a natural subtilisin inhibitor from the fungus Pleurotus ostreatus . Contained in the C-terminal tail of oryzin, there is a common motif involving a stretch of negatively charged residues around position 113  Fig. b. These  PDB ID: cnq;39. I2 out of total ~ 10,080Å2) is occupied by pro-domain atoms. Although both the N– and C–termini are unstructured segments, the C-terminal residues Trp118-Leu119-Tyr120-Asp121 from the pro-peptide remain bound in the catalytic cleft. They reveal the S1 to S4 substrate binding pockets in oryzin  shows alanine, histidine, alanine, and tyrosine. In the model serine proteinase from B. amyloliquefaciens , the P4–P1 residues are tyrosine, arginine, alanine, and leucine. The comparison to structurally characterized counterparts indicate that substrate binding preferences are for subsites: (S4), bulky aromatic/aliphatic; (S3), large side chains, aromatic or otherwise; (S2), small aliphatic side chain; and (S1), large side chains, aliphatic or not. Interestingly, subsites S4 and S2 show a preference to bury aromatic/aliphatic side chains while also promoting strong hydrogen bonding interactions; in contrast, subsites S3 and S1 seem open to accommodate side chains with lower selectivity. In summary, these binding interactions illustrate a distinct pattern of substrate-binding preferences among different subtilisins. Next, we use molecular modeling to explore the C-terminal subsites and the binding of an oryzin protein substrate.Trp118 is buried deep in a hydrophobic pocket lined by several small amino acid side chains where strong hydrogen bonds are established between the main chain atoms of this residue and Ser225 Fig. b. The buTo delineate oryzin’s binding pockets beyond the S1 site, we performed molecular modeling using a peptide fragment with adjacent P1′–P4′ residues extending the pro-domain from C-terminal residue Asp121.Our model shows a transition from pocket-like on the non-prime subsites to a shallow plain on the prime subsites Fig. a. Coveri41. We use phenylmethanesulfonyl fluoride (PMSF), a chemical compound that sulfonylates serine to form a stable adduct, to introduce an oryzin-PMS crystal structure . In summary, the geometry around base His193, hydrogen bonded to Asp162 farther from the catalytic Ser349 Oγ, and the latter hydroxyl group pointing away His193 in the direction of Asn284 and the new C-terminal carboxyl, would suggest that the enzyme's structure represents an end-product state. We then use a peptide substrate to analyze the enzyme's activity after its activation through chemical unfolding.42. Though urea is a commonly used chemical denaturant, solutions of urea are not stable and are subject to decomposition. To circumvent this, we use guanidine hydrochloride (GuHCl). To determine the ideal denaturant concentrations for purification, we perform chemical unfolding of oryzin with differential scanning fluorimetry (DSF) monitoring changes in fluorescence as a function of GuHCl concentration. We determined that oryzin has two major unfolding events at 2.2 M and 3.8 M GuHCl 13 for the determination of oryzin enzymatic activity of peaks I and II and a non-chemically-denatured sample  to measure the unfolding of oryzin in the presence of calcium, by monitoring changes in fluorescence as a function of temperature. We found that oryzin unfolds at a lower Tm (the Tms are 58.38 (0.07) °C and 60.09 (0.02) °C in the absence and presence of calcium, respectively) compared to oryzin in the presence of calcium , these changes in Tm in the presence of calcium compares to what has been previously observed in subtilisin with similar magnitudes44. Similarly, calcium increases the transition temperature at which the protein aggregates resulting in an increase in the unfolding ΔTm of the protein. Oryzin begins to aggregate at 57.40 (0.12) °C and 59.39 (0.06) °C in the absence and presence of calcium, respectively, following the trend observed in the thermal unfolding experiment. As the effect of calcium is to structurally stabilize the molecule, the questions as to where the calcium binding site locates and what its binding means to the protein fold, are to be solved. To this end, we analyzed a third crystal form, oryzin-calcium, obtained with calcium as an additive in the crystallization cocktail.Earlier work demonstrated that ethylenediaminetetraacetic acid (EDTA), a compound typically used for sequestering metal ions, could render oryzin inactiveThe calcium ion is placed at the center of an almost regular octahedron formed by oxygen ligands Fig. b. Three P. cyclopium subtilisin, an alanine occupies the equivalent place of Glu269, the proline is further from Asp323, and its carbonyl O atom is replaced by a water molecule. The calcium ions are in the same position in both structures. Moreover, in human pro-protein convertase, the calcium site relates to the oryzin calcium site (they are further away to 2.5 Å). In the unbound form . Combining crystallography and in vitro assays, our work confirms that oryzin binds calcium, that calcium stabilizes the enzyme, that the metal binding site is rather flexible and solvent exposed further from of the catalytic machinery. Thermal unfolding analysis combined with crystallographic data shows that the binding of calcium results in local structural changes, without detriment for the overall fold. Together with earlier observations our work suggests that enzyme activity changes are due to extra cleavage events that would result in changes in the protein integrity or because of products of hydrolysis directly modulating the catalytic mechanism by physical hindrance. Further work will be needed to address the complexities of this serine hydrolase substrate recognition properties and progression to its mature state. Diseases caused by Aspergillus spp. remain a challenging problem45 and oryzin is well suited for target-based drug development46. In the human genome, serine proteases are a predominant class of enzymes47 making the design of Aspergillus-specific inhibitors a difficult endeavor. Structure-based screening in exploratory research of new inhibitors is well established50 and may assist in the design effort. Combined with the present structural work it will help progress research into new agents to block progression of aspergillosis.We report on the first crystal structure of A. fumigatus sequence  encoding a truncated protein lacking the 20 amino acids from the secretion signal and containing a C-terminal His6-tag cloned into a pD444 vector with ampicillin resistance and IPTG inducible T5 promoter was purchased as a synthetic gene . After transformation of protease deficient E. coli strain BL21(DE3) , transformants were grown in lysogeny broth  containing ampicillin (100 µg/ml) at 37 °C in a shaking incubator at 225 rpm. The culture was grown until an optical density of 0.6–0.8 was reached and then transferred to a shaking incubator at 16 °C for 1 h before induction with 1 mM IPTG . After 3–4 h post induction, the culture was spun down at 10,000×g for 60 min at 4 °C, resuspended in 1/50 culture volume of buffer , and, after addition of lysozyme and Triton X-100, incubated in a rotator shaker at 4 °C for 10 min. Cells were lysed by sonication in a Fisherbrand Model 705 Sonic Dismembrator (ThermoFisher Scientific) three or four times for 30 s each on ice, and the lysate was cleared by centrifugation at 12,000×g for 60 min at 4 °C. The supernatant was mixed with 1 ml of Ni–NTA resin , incubated in a rotator shaker at 4 °C for 30 min, and the resin was applied on a plastic column. After washing with 10 bed volumes of 20 mM Tris–HCl, 500 mM NaCl, 20 mM imidazole, pH 8.5, elution of the His6-tagged protein was performed with an imidazole gradient, 1 bed volume of 10–500 mM imidazole, stepwise by gravity. The purity of the eluted fusion protein was analyzed by SDS-PAGE . We attempted to capture the intact uncleaved zymogen by using acidic pH buffers, but we consistently obtained the separate polypeptides (judged by SDS-PAGE) suggesting that the cleavage products must have appeared very early during protein expression. Fractions containing pure protein as determined by SDS-PAGE analysis were loaded onto a Superdex 75 column for size-exclusion chromatography using a Akta Pure  system. The protein was concentrated to 30 mg/ml using Amicon Ultra 10 kDa MWCO spin filters (Millipore Sigma), in buffer 0.1 M MES, 0.05 M NaCl, pH 5.6, and aliquots stored at − 80 °C. For chemically denatured protein purification, oryzin lysate was incubated with 2.2 M GuHCl  for 1 h at room temperature to allow for chemical unfolding of the protein. Protein was further purified following the protocol from above.A plasmid for expression of the 51 at the Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC National Accelerator Laboratory . The best diffracting sample was up to a Bragg spacing of 1 Å and data was collected taking advantage of the macroscopic properties of the crystal. To mitigate radiation damage, we employed a helical data collection strategy on a rod-shaped crystal using fine slicing by translating the microfocused beam along the longest edge of the crystal. The crystal is in space group P212121 with cell dimensions a: 60.60 Å, b: 70.76 Å, c: 78.55 Å; α, β, γ: 90° with one polypeptide chain in the asymmetric unit. Solving the phase problem using high-quality diffraction data to find one single polypeptide chain consisting of 382 aminoacids at ~ 1 Å resolution proved to be practicable using a method to place idealized peptide fragments via molecular replacement iterated with automated cycles of density modification. Phasing of the 1.06 Å data was with fragment location with ideal polyalanine α-helices as search fragments. Calculations were performed with ARCIMBOLDO_LITE52, a method which has previously been shown to successfully phase a 385-residues target protein at a lower resolution. This method uses Phaser53 for searching combined with density modification and autotracing with SHELXE54. ARCIMBOLDO_LITE runs in two macrocycles: first, Phaser sequentially places all requested copies of the search model(s) and partial structures are ranked and optimized with SHELXE. Similar solutions are clustered; then, top-ranked substructure solutions  are selected and subjected to density modification and autotracing with SHELXE. A promising substructure comprising 8 α-helices was expanded to trace a polyalanine-only model encompassing 367 residues (out of 382 from sequence). This solution gave a final CC of 49.89 and was subsequently chosen for refinement. This partial model was completed by successive rounds of manual adjustment and model building with COOT55 and refinement with REFMAC556. The electron density map was of excellent quality and allowed for unambiguously tracing the pro-domain, residues Arg27–Asp121 (21–26 missing), while the catalytic domain could be traced fully from Ala122–Ala403. Refinement progressed to convergence and reached an excellent agreement to the experimental data  PEG 1000, 12.5% (w/v) PEG 3350, 12.5% (v/v) MPD; L-glutamate, DL-alanine, glycine, DL-lysine, and DL-serine (each 0.02 M), in 0.1 M MOPS/HEPES, pH 7.5, with CaCl2 as an additive. The crystal is in space group P212121 with cell dimensions a: 59.25 Å, b: 66.07 Å, c: 86.79 Å; α, β, γ: 90° with one polypeptide chain in the asymmetric unit. Data at 1.65 Å Bragg spacing was collected at SSRL beamline BL12-2. The condition for the 1.06 Å crystal was repeated to obtain oryzin-PMS containing PMSF as an additive. The crystal is in space group P21 with cell dimensions a: 51.09 Å, b: 75.21 Å, c: 87.92 Å; α, γ: 90°, β, 101.30° with two polypeptide chains in the asymmetric unit. Data at 1.55 Å Bragg spacing was collected at SSRL beamline BL12-2. Further crystals were grown using CaCl2 as an additive to collect anomalous data at 7 keV (1.7715 Å). The 1.06 Å crystal structure was used as the search model for molecular replacement of these derivatives with Phaser. See Tables 58, scaled with SCALA59, and analyzed with different software routines within the CCP4 suite60. Graphics were rendered with Pymol30.Crystals were obtained using sitting-drop vapor diffusion for screening and sitting- or hanging-drop vapor diffusion for optimization at 6 °C or 16 °C. Crystallization experiments were setup using a Douglas Oryx8 Nanodrop dispensing robot . Diffraction-quality crystals were obtained from a solution containing 20% (w/v) polyethyleneglycol 550 monomethylether, 10% (w/v) polyethyleneglycol 20,000, sodium nitrate, sodium dihydrogen phosphate, ammonium sulfate (each 0.03 M), in 0.1 M MES/Imidazole buffer at pH 6.5. Crystals were harvested and cryocooled by plunging in liquid nitrogen with or without addition of cryoprotectant . Crystals were screened for their diffracting power at 100 K at beamlines BL14-1 and BL12-2Haloferax mediterranei (strain ATCC 33500/DSM 1411/JCM 8866/NBRC 14739/NCIMB 2177/R-4) ; P29143, Halolysin, Natrialba asiatica (strain ATCC 700177/DSM 12278/JCM 9576/FERM P-10747/NBRC 102637/172P1); P04072, Thermitase, Thermoactinomyces vulgaris; P00782, Subtilisin BPN', Bacillus amyloliquefaciens (Bacillus velezensis); P00780, Subtilisin Carlsberg, Bacillus licheniformis; Q3S3L6, Putative 36kDa protease, Lysinibacillus sphaericus (Bacillus sphaericus); U5J9E4, Peptidase_S8 domain-containing protein, Bacillus phage vB_BanS-Tsamsa; J9E4K3, Peptidase_S8 domain-containing protein, Wuchereria bancrofti; A0QQ47, Subtilase family protein, Mycolicibacterium smegmatis (strain ATCC 700084/mc(2)155) (Mycobacterium smegmatis); P59996, Proprotein convertase subtilisin/kexin type 9, Rattus norvegicus (Rat); Q8NBP7, Proprotein convertase subtilisin/kexin type 9, Homo sapiens (Human); A0A2R2JFW8, Protease, Penicillium cyclopium; P06873, Proteinase K, Parengyodontium album ; Q01471, Serine protease, Purpureocillium lilacinum (Paecilomyceslilacinus); B8N106, Alkaline protease 1, Aspergillus flavus (strain ATCC 200026/FGSC A1120/IAM 13836/NRRL 3357/JCM 12722/SRRC 167); P12547, Alkaline protease 1, Aspergillus oryzae (strain ATCC 42149/RIB 40) (Yellow koji mold); P28296, Alkaline protease 1, Neosartoryafumigata (strain ATCC MYA-4609/Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus); D5LGB3, Alkaline protease 1, Aspergillus versicolor; A1CIA7, Alkaline protease 1, Aspergillus clavatus (strain ATCC 1007/CBS 513.65/DSM 816/NCTC 3887/NRRL 1/QM 1276/107); F2E033, Predicted protein, Hordeum vulgare subsp. vulgare (Domesticated barley); P87184, Alkaline protease 2, Neosartorya fumigata (strain ATCC MYA-4609/Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus); Q8GB52, Extracellular subtilisin-like serine proteinase, Vibrio sp. PA-44; Q3HUQ2, Proteinase K, Serratia sp. GF96; P80146,Extracellular serine proteinase, Thermus sp. (strain Rt41A); P08594, Aqualysin-1, Thermus aquaticus;P16397, Bacillopeptidase F, Bacillus subtilis (strain 168). Alignment was done with Seaview38. Structure comparisons were done with DALI server31. All molecular modeling was performed using Coot and the model coordinates refined against reference parameters with REFMAC5 (version 5.8.0135) until deviation from ideal bond length, bond angle, planar restraints, chiral volume, reached convergence and Ramachandran outliers were minimized . Extension of the pro-domain beyond residue 121 was made exploiting the location of water molecules that hydrogen bond the protease to guide the tracing of the hydroxyl group of residues Thr124 and Thr125 and the main chain carbonyl of Gln126. The docking model was based on a synthetic triple-helix peptide containing a region from human collagen type III61. Molecular protein docking was made with Frodock version 2.062. Docking was validated by using the pro-domain for a docking prediction and comparison to the experimentally determined structure. As a test, the pro-domain's coordinates were randomly oriented and then submitted to docking. The comparison with the experimentally determined binary complex showed an excellent agreement.UniProtKB entries used for alignment: I3R794, Halolysin, 2 was used in serial dilutions between 50 mM and 1.2 µM. Additionally, MgCl2 was tested at concentrations between 6.25 and 50 mM, but the Tm for this metal ion was indistinguishable from protein control without metal. Tabulated data are the average value and standard deviation .Thermal unfolding measurements were performed using a Prometheus Panta instrument . The thermal unfolding was on a linear thermal ramp from 25 to 95 °C to the sample while reading out fluorescence and turbidity to analyze thermal stability and aggregation. To assess protein aggregation and precipitation backreflection was used for measuring light intensity loss due to scattering. Data recorded by the backreflection optics is termed turbidity. The unfolding profile plots the 350nm/330 nm fluorescence ratio as a function of temperature. The ratio is a measure for a spectral shift in the fluorescence emission profile of tryptophan residues. Non-aggregated oryzin exhibits a turbidity signal of under 100 mAU and then sharply goes up on melting. The overall degree of aggregation is calculated according to the Beer–Lambert law and expressed in attenuation units (AU), plotted as milli-AU. Turbidity data was measured in parallel to the thermal unfolding experiment on the same capillaries. The cumulant analysis was used for dynamic light scattering (DLS) data analysis and to assess sample homogeneity. It models the autocorrelation function (ACF) using an average diffusion coefficient to obtain a single averaged hydrodynamic radius rH. Heating rate was with a temperature slope of 0.5 °C/min. Experiments performed by triplicate. Oryzin stock concentration was at 0.4 mg/ml in 0.05 M Hepes buffer, pH 7.5 with 0.1M NaCl. CaClChemical unfolding measurements were performed using Prometheus Panta . Guanidine hydrochloride (GuHCl) was used as the chemical denaturant with 30 µL dilutions from 6 to 0.25 M prepared in Tris–HCl buffer. A 0.5 mg/mL stock of oryzin in 0.2 M Tris–HCl buffer, pH 8.0, was added to each sample and incubated for 1 h at room temperature. The unfolding profile plots the 350 nm/330 nm fluorescence ratio as a function of denaturant concentration. The ratio is a measure for a spectral shift in the fluorescence emission profile of tryptophan residues. The c50, denaturant concentration in which 50% of the protein is unfolded, is calculated.The substrate used to determine enzymatic activity of oryzin was Suc-Ala-Ala-Pro-Phe-pNA . Suc-AAPF-pNA 10 mM stock solutions were prepared in 0.2 M Tris–HCl buffer, pH 8.0, aliquoted, and stored at − 20 °C. Protein stocks were all diluted to the lowest concentration stock (0.2 mg/mL) using Tris–HCl, pH 8.0. Samples were prepared in a cuvette as 1 mL total volume as follows: 100 μL protein of interest, 50 μL of Suc-AAPF-pNA substrate, 850 μL Tris–HCl, pH 8.0 buffer. A blank was prepared using 50 μL of Suc-AAPF-pNA substrate and 950 μL Tris–HCl, pH 8.0 buffer. Samples were measured at 410 nm using a Nanodrop 2000c spectrophotometer  at 0, 0.02, 0.08, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 21, 23, 24 h, making sure to blank before each measurement. Substrate-enzyme activity could also be visualized by a color change from clear to yellow.Supplementary Information.


LMS) have been shown to synthesize PS without control, but why the uncontrolled synthesis would lead to LMS is unknown. Here we investigated the effect of PSS1LMS on osteoclasts (OCs) to elucidate the causative mechanism of LMS. PSS1LMS did not affect the expression of OC-related genes but inhibited the formation, multinucleation, and activity of OCs. Especially, OCs expressing PSS1LMS showed abnormal patterns and dynamics of actin podosome clusters, which have roles in OC migration and fusion. PSS1LMS did not affect the level of PS but changed the acyl chain compositions of PS and phosphatidylethanolamine, and decreased the level of phosphatidylinositol. The introduction of a catalytically inactive mutation into PSSLMS canceled the changes in phospholipids and the phenotypes observed in OCs expressing PSS1LMS. A gain-of-function mutant of PSS2 (PSS2 R97K) also impaired OC formation and caused changes in phospholipid composition similar to the changes caused by PSS1LMS. Our results suggest that uncontrolled PS synthesis by PSS1LMS causes changes in the quantity or fatty acid composition of certain phospholipid classes, impairing OC formation and function, which might be a cause of osteosclerosis in patients with LMS.Phosphatidylserine (PS) is an acidic phospholipid that is involved in various cellular events. Heterologous dominant mutations have been identified in the gene encoding PS synthase 1 (PSS1) in patients with a congenital disease called Lenz-Majewski syndrome (LMS). Patients with LMS show various symptoms, including craniofacial/distal-limb bone dysplasia and progressive hyperostosis. The LMS-causing gain-of-function mutants of PSS1 (PSS1 Phosphatidylserine (PS) is a major phospholipid class constituting cell membranes. PS has a negative charge in its polar head group, through which PS interacts with proteins in various biological processes , 2. PS iPTDSS1, were found in patients with a congenital disease called Lenz-Majewski syndrome (LMS) (PTDSS1 in five unrelated patients (LMS) showed elevated PS synthetic activity, accompanied by elevated PE formation  . LMS is me (LMS) . The patpatients . That repatients , 13, 14 ormation , which iormation . Intereshibition . Howeverhibition . TherefoThe most striking symptoms of LMS are bone dysplasia and sclerosis. In clinical reports, many patients showed craniofacial osteosclerosis, which could result in nasal airway obstruction or hydrocephalus by blocking cerebrospinal fluid at the sclerosing craniovertebral junction , 16. NasOsteoclasts (OCs) are the main cells involved in destroying bone. Impairment of OC function results in osteopetrosis or osteosclerosis , 18. The14C(U)] (MC-265) was purchased from Moravek, Inc. Brain PS was purchased from Sigma-Aldrich. Recombinant murine macrophage colony-stimulating factor (MCSF) (315-02) was purchased from PeproTech. Glycerol, [14C(U)] (NEC441X) was purchased from PerkinElmer. 12:0/13:0 PC, PE, PS, and phosphatidylinositol (PI) were from Avanti Polar Lipids.L-Serine, serine (0.5 μCi/ml) for 3 h. For PS treatment, OCs were treated with 20 μM PS 2 h before and during [14C]serine labeling. Phospholipids were extracted from cells by the method of Bligh and Dyer  staining kit  according to the manufacturer’s protocol. TRAP-positive cells were imaged with an EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific) and counted manually using ImageJ.6 cells/well at day 0. On day 1, the cells were treated with retrovirus to express PSS1. The medium was changed every 2 days until day 7. On day 5, 0.85 μl of concentrated HCl was added to every 1 ml of medium to give the pH around 7 to promote activity  at 0.2 × 10activity . On day Total RNA was extracted from cells with ISOGEN II (Nippon Gene) and reverse transcribed with High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s protocol. Quantitative RT-PCR analysis was performed with a LightCycler 480 II (Roche) or LightCycler 96 (Roche), using SYBR Premix Ex Taq II (Takara Bio) or KAPA SYBR FAST One-Step qRT-PCR Kit . The primers used in RT-PCR are listed below.Nfatc1 fwd: tccaaagtcattttcgtgga, rvs: ctttgcttccatctcccaga.mouse Acp5 fwd: cgtctctgcacagattgcat, rvs: aagcgcaaacggtagtaagg.mouse Ctsk fwd: cgaaaagagcctagcgaaca, rvs: tgggtagcagcagaaacttg.mouse Dcstamp fwd: cgaagctccttgagaaacga, rvs: ggactggaaaccagaaatgaa.mouse Atp6v0d2 fwd: aagcctttgtttgacgctgt, rvs: gccagcacattcatctgtacc.mouse Gapdh fwd: aggtcggtgtgaacggatttg, rvs: tgtagaccatgtagttgaggtca.mouse Actb fwd: atgaagatcaagatcattgctcctc, rvs: tgtccaccttccagcagatgtmouse Total RNA was extracted from cells with ISOGEN II (Nippon Gene) at days 3 and 6. Cells from four independent wells were pooled to generate one sample of total RNA. The extracted RNA was purified with a High Pure RNA Isolation kit (Roche), and a microarray analysis was conducted by Clarion S Assay, mouse (Thermo Fisher Scientific). The resulting data were analyzed with Transcriptome Analysis Console software (Thermo Fisher Scientific). The microarray data can be accessed at the Gene Expression Omnibus (GEO) repository (GSE207351).BMCs were seeded on glass coverslips, which were pre-coated with collagen type I  according to the manufacturer’s protocol. On day 1, the cells were treated with retrovirus to express PSS1-GFP. Generally, cells became mature OCs on day 4 and were fixed with 4% paraformaldehyde  in PBS for 15 min at room temperature. After a three-time wash with PBS, cells were permeabilized with 0.1% Triton X-100  in PBS for 5 min at room temperature. The cells were washed again and then blocked with 3% bovine albumin serum (BSA) (Sigma-Aldrich) in PBS. The primary antibodies were diluted in 3% BSA-PBS and incubated with the cells overnight at 4°C. On the next day, the cells were incubated with secondary antibodies, fluorescent phalloidin (Invitrogen), or DAPI (Sigma-Aldrich) for 1 h at room temperature and mounted with PermaFluor . For 2xPH (evt2)-His staining, the cells were permeabilized with 50 μg/ml digitonin in PBS for 5 min at room temperature. 2xPH (evt2)-His was incubated together with anti-His primary antibody. Expression and purification of recombinant protein 2xPH (evt2)-His was described previously .Confocal microscopy was performed using a TCS SP8 (Leica) with a 63 × 1.2 Plan-Apochromat water immersion lens.LMS-GFP or fluorescent Lifeact. They became mature OCs on day 4, and live imaging was conducted. The cells were imaged in xyt mode at 37°C with 5% CO2 using a TCS SP8.BMCs were seeded in IWAKI 35 mm glass base dishes , which were precoated with collagen type I. The cells were treated with retrovirus to express PSS1OCs were collected with lysis buffer . After incubation at 4°C for 20 min, the lysate was centrifuged at 12,000 rpm at 4°C to remove the insoluble fraction. The supernatant was used as the protein extract. Protein concentrations were determined by the BCA assay (Pierce). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA in TTBS buffer  and incubated with primary antibodies overnight at 4°C. On the next day, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody . The proteins were detected by enhanced chemiluminescence  using LAS4000 .Phospholipids were extracted from cells by the method of Bligh and Dyer . When OCThese procedures were conducted as described previously . The OCsOCs were prepared in the same way they were prepared for live imaging. They were photobleached with 100% laser power for 2 s. Then, images were collected every 0.582 s for 1–2 min.WT-HA or FLAG-human PSS1LMS-HA and selected with 10 μg/ml blasticidin S. HeLa cells were seeded in 6-well plates. To analyze de novo synthesis of phospholipids, the cells were labeled with 1.0 μCi [14C]glycerol or 10 μCi [32P]orthophosphate per well for 2 or 4 h, respectively, on the next day. Phospholipids were extracted by the method of Bligh and Dyer. The extract was applied to thin layer chromatography and eluted with chloroform: methyl acetate: 1-propanol : methanol: 0.25% KCl = 25: 25 : 25: 10: 9. The radioactivity was detected with imaging plates and Typhoon FLA 9000  and normalized by total protein content.HeLa cells were infected with retrovirus solutions to express FLAG-human PSS114C]glycerol per well for 12 h on the day after seeding. Then the medium was washed out, and the radioactive label was chased for up to 0, 2, 8, or 24 h. Phospholipids analysis was done as described above.To analyze the degradation of phospholipids, the cells were labeled with 0.3 μCi  and PLCγ2 at Y1217 [pPLCγ2 (Y1217)], the indicators of their activity, by western blotting. As a result, they were unchanged or increased in PSS1LMS-expressing cells  to total Src was elevated. When we detected pSrc (Y416) by immunofluorescence, PSS1LMS-expressing cells showed an increased signal , 34:1, and 38:4 PS were higher than those in the non-transduced and PSS1WT-expressing cells, while the levels of 36:1, 40:5, and 40:4 PS were lower. Similar differences were observed for PE. Unexpectedly, PI, whose synthesis pathway is different from that of PS, was decreased without any change in its composition of molecular species. The amounts of PI monophosphate (PIP) and PI bisphosphate (PIP2) were also measured using supercritical fluid chromatography – MS/MS -His staining pattern, and the signal of 2xPH (evt2)-His was separated from that of PSS1-GFP, which localized to ER and nuclear membrane.Sohn 3T cells . When OC3T cells , 35, the3T cells . NeitherLMS is the cause of the LMS phenotypes. To address this issue in the OC system, we mutated the amino acid residues crucial for PSS1’s catalytic reaction. Eight mutations of PSS1 were previously shown to inactivate the enzyme  has been reported to be resistant to feedback inhibition by PS  or PSS2RK. PSS2WT-expressing OCs exhibited multinucleated osteoclast formation, as did non-transduced OCs  were prone to bone fractures and their bone biopsy specimens lacked osteoclasts  by PSS1LMS. However, other increased molecular species, such as 32:1, 32:0, 36:4, and 38:4 PS, were not especially abundant in PC. A specific portion of PC molecular species might be used in PS synthesis by PSS1. PE is synthesized by the both CDP-ethanolamine pathway and the PS decarboxylation pathway. Ethanolamine phosphotransferase 1 (EPT1), which is a major enzyme in the CDP-ethanolamine pathway, contributes to the synthesis of PE species such as 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4 PE , the phosphorylated derivatives of PI, are explicitly recognized by a number of actin-associated proteins . The datasets generated and analyzed during this study are available from the corresponding author on reasonable request.This article contains The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Poly(glycolic acid-co-butylene succinate)(PGBS) and poly(PGBT) copolymers with high nGA were synthesizedfor comparison. DSC analysis revealed that PGBAT21 (nGA = 21%) and PGBAT92 were semicrystalline, melting between102.8 and 163.3 °C, while PGBAT44, PGBAT86–89, PGBT80,and PGBS90 were amorphous, with Tg valuesfrom −19.0 to 23.7 °C. These high nGA copolymers showed similar rates of hydrolysis to PGA, whereasthose containing <50% GA showed almost no mass loss over the testingperiod. Their mechanical properties were highly dependent upon theircrystallinity and improved significantly after annealing. Of the high nGA copolymers, annealed PGBS90  possessed excellentmechanical properties with a modulus of 588 MPa, tensile strengthof 30.0 MPa, and elongation at break of 171%, a significant improvementon PGA’s elongation at break of 3%. This work demonstratesthe potential of enhancing PGA’s flexibility by introducingminor amounts of low-cost diols and diacids into its synthesis.Poly(glycolic acid) (PGA) is a biodegradable polymerwith highgas barrier properties, mechanical strength, and heat deflection temperature.However, PGA’s brittleness severely limits its applicationin packaging, creating a need to develop PGA-based copolymers withimproved elasticity that maintain its barrier properties and hydrolyticdegradability. In this work, a series of PGBAT (poly(glycolic acid- However, the recent development of large-scale, continuousPGA production processes has lowered its cost and enabled its applicationin packaging.5Widespread plastic pollutionhas encouraged the development ofbiodegradable plastics for packaging. Currently, most biodegradableplastics are limited by their poor barrier properties, low heat resistance,and slow degradation rates in natural environments. Poly(glycolicacid) (PGA) is both an established and emerging biodegradable polymerwith excellent barrier properties, high tensile strength, high heatdeflection temperature, and fast degradation rate that outperformsmost commercial biodegradable polymers.8 Alternatively, copolymerizationcan reduce PGA’s brittleness and melting temperature. Traditionally,industrial PGA production has relied upon ROP. However, copolymerizationof PGA via ROP is limited by the lack of commercially available cyclicmonomers  and their relatively high cost. Additionally, these comonomershave low reactivity ratios compared to glycolide, leading to increasedreaction times and temperatures.11 Comparatively, a wide varietyof low-cost diols and diacids are available, which could be copolymerizedwith glycolic acid via polycondensation, allowing for the tailoringof the copolymer’s structure.Despite its excellent tensilestrength and barrier properties,PGA’s application in packaging is hindered by its brittleness.A further problem of PGA is its susceptibility to degradation duringsynthesis and processing, which is partly due to its high meltingpoint and poor thermal stability. To improve its flexibility, PGAcan be blended with PBAT.Mn (2120 g mol–1) PGET ) copolymer containing 36% glycolicacid with a Tm of 166°C via the melt polycondensation of bis(2-hydroxyethyl) terephthalateand glycolic acid oligomers using Sb2O3.12 Zhou et al. prepared PLET with Mw values of 22,600–56,000 g mol–1 through a one-pot ROP polycondensation method from lactide, ethyleneglycol, and terephthalic acid using Sn(Oct)2 and a titaniumcatalyst.13 These PLETs contained 10–50%lactic acid and displayed decreases in the Tg, Tm, crystallinity, and tensilestrength as the lactic acid content was increased. Nakayama et al.synthesized alternating PGEGT polyfrom the polycondensation of ethylene diglycolate and terephthaloyldichloride at 0 °C in a tetrachloroethane and pyridine mixture.This copolymer’s molecular weight was low , but it showed increased Tg (48 °C) and decreased Tm (209 °C) compared to PGA.14Various researchers haveexplored methods of polymerizing hydroxyacids with diols and diacids, such as ethylene glycol and terephthalicacid. Olewnik et al. produced a low 15 Minor amounts of pentaerythritoland trimethylolethane were incorporated to introduce branching andachieve firmer materials. High-molecular-weight  copolymers of PLAand PBAT have also been produced by reacting a PBAT prepolymer anda PLA prepolymer together in a vacuum reactor.16Hot melt adhesives containing approximately 85 wt % of glycolicacid were synthesized from glycolic acid, adipic acid, and ethyleneglycol.17 Thesecopolymers possessed high molecular weights  and excellentmechanical properties . These were prepared either via the polycondensationof two oligomers or the direct esterification and polycondensationof glycolic acid, diacids, and diols using tin and titanium catalysts.Recently, a process for improving the marine biodegradabilityofPBS, PBAT, PET, and PBT via copolymerization with 10–30 mol% of glycolic acid was disclosed.co-butylenesuccinate) (PGBS), poly (PGBT), poly (PGET), poly (PGBAT), and poly(glycolicacid-co-butylene furanoate) (PGBF) containing 0–60%glycolic acid have been synthesized and examined in detail.23 They all showed good mechanical properties and were semicrystallineat low glycolic acid percentages but became amorphous as the glycolicacid content increased above 30 mol %. Poly(lactic acid-co-butylene furanoate) (PLBF), poly(lactic acid-co-butylene succinate) (PLBS), poly(caprolactone-co-butylene furanoate) (PCBF), and poly(caprolactone-co-butylene succinate) (PCBS) have also been reported and displayedsimilar trends as the copolymer ratio was adjusted.27Following this, poly 2-ethylhexanoate, and titanium butoxide were purchased fromVWR, Alfa Aesar. Zirconium(IV) acetylacetonate was purchased fromStrem Chemicals U.K., Ltd. Irgafos 126, Irgafos 168, and Joncryl ADR4468 were from BASF.Glycolic acid, dimethylterephthalate, adipic acid, succinic acid, bismuth(III) subsalicylate,antimony(III) oxide, zinc acetylacetonate, zinc acetate, methanesulfonicacid, phosphoric acid, DMSO-2.22.2.12 , and a magnetic stir bar were added to a 250 mL single-neckround-bottom flask attached to a vacuum distillation setup. The mixturewas purged with N2 three times and heated at 160 °Cwith stirring for 6–8 h until the reaction mixture solidified. Mn was calculated as 494.8 g mol–1 via end-group analysis from the 1H NMR spectra .Glycolic acid , Sn(Oct)2.2.24 , and a magnetic stir barwere added to a 250 mL single-neck round-bottom flask attached toa vacuum distillation setup. The mixture was purged with nitrogenthree times and stirred at 160 °C for 1 h. Adipic acid  was then added, and the mixture was stirred at 180 °Cfor 4 h. Mn was calculated as 647.6 gmol–1 via end-group analysis from the 1H NMR spectra (CDCl3).Dimethyl terephthalate , 1,4-butanediol , Ti(OBu)2.2.32O3 , and Irgafos 126  to a single-neck round-bottomflask attached to a vacuum distillation setup. The mixture was heatedto 190 °C and stirred under nitrogen for 30 min. The pressurewas then gradually reduced to 30 mbar over 30 min. After a further30 min, the pressure was further reduced to ≤0.1 mbar and leftfor 4–5 h. The reaction was considered finished once the magneticstirrer bar could no longer stir the mixture. The molten polymer wasremoved and analyzed without further purification.Using PGBAT80 as an example, we added OGA , OBAT, Sb2.2.44 , and a magnetic stirrerbar were added to a 250 mL single-neck round-bottom flask attachedto a vacuum distillation setup. The mixture was purged with nitrogenthree times and stirred at 170 °C for 30 min (until the mixturesolidified). Then, adipic acid , 1,4-butanediol, glycolic acid , and Sn(Oct)2  were added, and the mixturewas heated at 180–210 °C for 4 hours, with the temperaturebeing increased at 10 °C per hour. After this, Sb2O3 (0.1 wt %) and Irgafos 126 (0.2 wt %) were added, andthe pressure was gradually reduced to 30 mbar over 30 min. After afurther 30 min, the pressure was further reduced to ≤0.1 mbarand left for 4–5 h. The reaction was considered finished oncethe magnetic stirrer bar could no longer stir the mixture. The moltenpolymer was removed and analyzed without further purification.Using PGBAT90 as an example, dimethyl terephthalate, 1,4-butanediol, , Ti(OBu)Copolymers synthesized for the annealing experiments in section 4.2.3were synthesized using twice this scale (∼60 g) and a mechanicalstirrer; roughly 30–35 g of polymer was obtained. Polymer yieldwas calculated on the basis of the mass of polymer collected and themass of sublimed glycolide.2.32.3.1nGA ≤ 50 mol % was performedusing CHCl3 as the eluent. SEC was carried out using anAgilent Infinity II 1260 MDS instrument equipped with differentialrefractive index (DRI), viscometer (VS), dual-angle light scatter(LS), and multiple wavelength UV detectors. The system was equippedwith 2× PLgel Mixed C columns (300 mm × 7.5 mm) and a PLgel5 μm guard column. The eluent was CHCl3 run at 1mL/min at 30 °C. Poly(methyl methacrylate) standards  were used to create a third-order calibration between 1,020,000and 1,840 g mol–1. Analyte samples were filteredthrough a 0.22 μm nylon membrane before injection.SEC of copolymerscontaining 2.3.2nGA ≥ 50 mol % was performedusing DMF as the eluent. SEC was carried out using an Agilent InfinityII 1260 MDS instrument equipped with differential refractive index(DRI), viscometry (VS), dual-angle light scatter (LS), and multiplewavelength UV detectors. The system was equipped with 2× PLgelMixed D columns (300 mm × 7.5 mm) and a PLgel 5 μm guardcolumn. The eluent was DMF containing 5 mmol of NH4BF4 run at 1 mL/min at 60 °C. Poly(methyl methacrylate)standards  were used to create a third-order calibrationbetween 1,020,000 and 1,840 g mol–1. Analyte sampleswere filtered through a 0.22 μm nylon membrane before injection.SEC of copolymerscontaining 2.3.3nGA value was determined via 1H NMR spectroscopy. NMR spectra were obtained with a BrukerAvance III HD 400 MHz Spectrometer. Copolymers containing nGA ≤ 50 mol % were analyzed using CDCl3 as a solvent, and those with nGA ≥ 50 mol % were analyzed using d6-DMSO. nGA values were calculated usingIGA = Integrationof glycolic acid units, IA = intergradationof adipic acid units, and IT = integrationof terephthalic acid units .The 2 flow at a heating rate of 10 °C min–1.DSC experiments were performed using a Mettler-ToledoDSC 1 with 5–10 mg of sample in a 40 μL aluminum DSCpan. Samples were scanned from −40 to 230 °C under anN2 flow at a heatingrate of 10 °C min–1.TGA experiments were performed using a Mettler-Toledo TGA1 instrumentwith ∼10 mg of sample in an alumina crucible. Samples werescanned from 25 to 500 °C under an N2.3.4–1 with a 10 kN load cell,and the room temperature was 21 °C. The gauge length was setto 40.4 mm. Five tensile tests were performed for each sample, andthe mean and standard deviation were reported.0.5mm polymer films were prepared by hot-pressing at 100–190 °C.Samples were held at the same temperature without pressure for 2 minto soften the polymer and then pressed at 40 kN for 3 min. PGBAT21–44were hot-pressed at 150 °C; PGBAT86–89, PGBT80, and PGBS90–93were hot-pressed at 100 °C; and PGBAT92 was hot-pressed at 190°C. The films were cut according to DIN 53504S2. Tensile testingwas performed by using a Shimadzu Autograph AGS-X tester. The extensionrate was set to 10 mm min2.3.50.5 cm ×1 cm pieces were then cut and immersed in 1 mL of pH 7 phosphate-bufferedsaline (PBS) solution at 60 °C. After degradation, each samplewas washed with distilled water and dried overnight at 30 °C.Each sample was tested in triplicate.33.12O3.29 Using a similar method  and a butylene furanoate oligomer (OBF) at 190–210°C using 0.15 wt % of Sbr method 1a, PGBAT2O3 isan effective polycondensation catalyst, it is a known carcinogen.Therefore, a catalyst screen was performed to identify whether analternative could be used. However, the results  found that only Sb2O3 yielded a sufficiently high Mw . Catalyst screening was alsocarried out to examine the effect of glycolic acid esterificationon the polycondensation of PGBAT, and Sn(Oct)2 was foundto yield the highest Mw value . However, while thesecatalysts yielded high molecular weights, the resulting PGBATs weredark brown. Antioxidants were screened for their ability to reducethis discoloration, and Irgafos 126 was found to be the most effective. It reduced dispersityand Mw values but not Mn, suggesting it prevents radical-induced chain branchingreactions, which may lead to the high Mw values observed without an antioxidant.While SbTable S4) found that whenPGBATs with high nGA values were synthesizedvia the polycondensation of oligomers, polymer yields were low, dueto the formation and sublimation of glycolide throughout the reaction.Additionally, these copolymers displayed poor thermomechanical properties(Table S5). The synthesis of PGBAT viathe direct esterification and polycondensation of monomers provedto be an easier and more effective method, resulting in higher yieldsand less glycolide formation, meaning that the PGBAT’s nGA was close to its feed value (Table S6). It was also found that for high nGA PGBAT, a lower excess of 1,4-butanediol toadipic acid and dimethyl terephthalate (B:A + T) was required to yieldhigh Mw values (Table S7). For a feed value of nGA =90%, reducing the excess of 1,4-butanediol from 1.25:1 to 1.05:1 increasedthe PGBAT’s B:A+T ratio from 1:0.83 to 1:1 and increased the Mn from 16,100 to 21,700 g mol–1 (Table S7).Initial work  and monomodaldistributions . Polymer yielddecreased as the nGA increased since moreglycolide formed as a side product. From Figure S4, it can be seen that the copolymers darkened in appearanceas the nGA increased, suggesting the discolorationis due to the thermal degradation of glycolic acid segments in thepolymer.Following these studies, the effectsof increasing the glycolic acid content on the thermomechanical propertiesand hydrolysis rate of PGBAT were investigated. nGA feed valuesof 90% were also synthesized to observe how changing the diacid influencedthe copolymers’ properties. Compared to PGBAT86–92,PGBT required a longer polycondensation time to obtain a highly viscousmelt and had a lower yield and a lower nGA of 80%. Therefore, further reaction optimization is likely requiredfor this copolymer. Conversely, PGBS’s yield was higher, andit showed less discoloration.PGBS and PGBT with Tg ranged from −19 to 20 °C and increasedas more glycolic acid was incorporated. All PGBAT copolymers displayedmelting peaks (61.3–166.9 °C) in their first heating cycle,but only PGBAT21 and PGBAT92 showed melting peaks in their secondheating cycle. Thus, PGBAT21 and PGBAT92 were semicrystalline, whereasPGBAT44–PGBAT89 were virtually amorphous and had very slowcrystallization rates, which is likely due to their lack of structuralregularity. This agrees with similar trends observed with increasing nGA in PGBS and PGBF.29 Unlike PGA, PGBAT92 showed acold crystallization peak during its second heating cycle but no crystallizationduring cooling, indicating its slow crystallization rate. Since PGBAT86–92displayed lower melting temperatures (132.8–166.9 °C)and crystallinity (ΔHm = 0.6–32.5J g–1) than PGA , introducing these comonomer units can be used tolower PGA’s Tm and crystallinity,DSC analysis was carried out usingsamples from the reaction flask,which were stored at room temperature for 5 days. The results 1 revealeTg (15.7 °C), whereas PGBT80’s Tg was higher (23.7 °C). This shows thatremovingthe aromatic terephthalic acid units lowers these copolymers’ Tg, while increasing the aromatic content raisesit.Compared with PGBAT92, PGBS91 displayed a lower Tg was just below room temperature, and it showeda weak melting peak in its first DSC heating cycle but not in itssecond, which suggests the copolymer crystallizes slowly. Poly(butylenecarbonate-co-furandicarboxylate) copolymers withsimilar thermal properties have been shown to crystallize during roomtemperature storage, as do poly(3-hydroxybutyrate) and PGBS.31 Therefore, the observed changesin the mechanical properties of PGBAT86 may be caused by a similarphenomenon.Subsequently, PGBAT films were prepared by hot-pressing;thesewere then cut into dumbbell-shaped specimens for tensile testing.After hot-pressing, the PGBAT86–89 films appeared soft andsticky but became firmer after storage at room temperature. Therefore,PGBAT86 was stored at 21 °C for 6 weeks, and the changes in itsmechanical properties were examined 2. PGBAT8nGA furtherincreased from 86 to 89 to 92%, the tensile strength increased from2.12 to 3.79 to 6.14 MPa, and the elongation at break fell from 36.8to 28.1 to 18.6%. This is the result of the Tg increasing from 11.2 to 20.0 °C and the polymer chainsbecoming stiffer and more rigid as more glycolic acid is added. PGA’stensile strength of 124 MPa was significantly higher, but its elongationat break (3%) was much lower. Therefore, minor amounts of butyleneadipate and butylene terephthalate units can be copolymerized withPGA to improve its flexibility at the expense of reductions in strength.Tensile testing was then performed on all copolymers3, and thTg (23.7 °C) was higher than the room temperature (21 °C),so it was in its glassy state during testing while all of the othercopolymers were in their rubbery state.Due to the absence of aromatic units, PGBS91 was asofter, tackiermaterial with a tensile strength of 0.57 ± 0.09 MPa and an elongationat break of 496 ± 33%. PGBT80 displayed significantly improvedelongation at break (285 ± 140%) and higher tensile strength(8.56 ± 1.40 MPa) compared to PGBAT86–92. This is likelybecause, unlike the other copolymers, PGBT80s nGA was variedfrom 0–60%; polymers had very slow degradation rates at 0–50%of glycolic acid but showed rapid increases at 60% of glycolic acid.29In order to comparethe hydrolytic stability of these copolymerswith that of PGA, hydrolytic degradation tests were performed by measuringthe mass loss of samples in PBS buffer at 60 °C. Tm or Tcc in DSC first heating cycle)and was heated well above its Tg (23.7°C).PGBS91 displayed faster hydrolysis than PGBAT86–92,andthis was attributed to its lower molecular weight. The mass loss ofPGBT80 could not be determined since it melted during testing; thiswas because it was completely amorphous  revealed these copolymers’ nGA values were close to thefeed value (90%), and SEC analysis showed that they are monomodal. Additionally, as was observedbefore, PGBS displayed less discoloration and required a shorter reactiontime than PGBAT .Under these conditions, the Hm values, resulting in a crystallinity increasefrom 1.8 to 15.5% for PGBS90 and from 0.7 to 8.0% for PGBAT88. Afterannealing, these copolymers’ appearances changed , turning from transparent to opaque,which further indicates a transition from an amorphous state to asemicrystalline one. These copolymers had melting temperatures of127.6–141.0 °C, well below that of PGA (225.1 °C),so they can be processed at significantly lower temperatures. TGAshowed they have Td 5% values of271–280 °C, so they have a wide processing window (Tm – Td).In the DSC 5, thesenGA with high strength and increased flexibility over PGA.The mechanical properties of PGBS90 and PGBAT88 changedsignificantlyafter annealing 6. Amorph1H NMR spectrum , it was calculated that PGBS90 contained approximately75 wt % of PGA. Compared to compatibilized PGA blends containing similarPGA quantities, PGBS90 displayed superior elongation at break. 70/30PGA/PBAT blends were reported with an elongation at break of 45% anda tensile strength of 46 MPa.33 70/30 PGA/PCLblends showed an elongation at break of 40.3% and a tensile strengthof 49.6 MPa.34From the 37 Therefore, it was investigated whether annealinginfluenced the rates of hydrolysis. There were no significant differencesin the hydrolysis rate between the annealed and amorphous samplesof PGBS90 and PGBAT88, It is known that annealingPGA can produce more ordered crystallinestructures, which prevent the intrusion of water molecules, improvinghydrolytic stability.4nGA valueincreased, elongation at break fell, and copolymers became more amorphous.At very high nGA values (86–92%),PGBAT displayed improved elongation at break over PGA (18.6–36.8%vs 3%) but reduced tensile strength (2.12–6.12 MPa vs 124 MPa).PGBAT, PGBS, and PGBT copolymerscontaining glycolic acid as theirmajor component were successfully synthesized by melt polycondensation.The synthesis of PGBAT from the direct esterification and polycondensationof monomers produced higher yields and molecular weights than thosefrom the polycondensation of oligomers. Additionally, the ratio ofdiol to diacid was found to significantly affect the molecular weight.In PGBAT21–92, the mechanical properties were highly dependenton the comonomer ratio. As the Mw 49,400 g mol–1) and PGBS90  were largely dependentupon their crystallinity. In their amorphous states, these copolymerswere weak, elastomeric materials ; however, after annealingat 100 °C, they became semicrystalline and displayed increasedstrength (11.8–30.0 MPa) and decreased elongation (85.9–171%).Hydrolysis tests performed at 60 °C in PBS buffer showed thatthese copolymers degrade at similar rates to PGA during the earlystages of degradation but then proceed to degrade much more rapidlythan PGA. Overall, this work shows that high nGA PGBS and PGBAT copolymers can be synthesized and annealedto yield materials with high strength and improved flexibility overPGA.The mechanical properties of PGBAT88 (


Patients with locoregionally uncontrolled breast tumors are frequently referred for breast palliative radiotherapy (PRT) to mitigate symptoms. We analyzed the outcomes following breast PRT to optimize PRT according to risk groups.We reviewed 133 patients who underwent breast PRT. A median total dose of 45 Gy was prescribed with an equivalent dose in 2 Gy fractions  of 53 Gy. The Cox proportional hazards model was used to analyze the prognostic factors of local control (LC).p = 0.055) for group 2, but no improvement for group 3 (p = 0.300).Most (90.2%) had polymetastatic disease (> 5 lesions), and 48.9% had bone metastasis. With a median follow-up of 17.2 months, the 2-year LC and overall survival (OS) rates were 49.4%, and 48.3%, respectively. Multivariable analyses demonstrated progressive or mixed responses outside the breast and > 2 lines of previous therapy as adverse features for clinical outcomes. Group 1 (0 risk factors) showed favorable 2-year LC and OS of 63.9%, and 72.8%, respectively, whereas group 3 (2 risk factors) showed the worst outcomes of 0%, and 6.8%, respectively. Breast PRT with EQD2 ≥ 63 Gy showed a significant benefit in LC for group 1 and marginal benefit (Breast PRT showed favorable LC outcomes in patients with stable disease outside the breast and treated with ≤ 2 lines of systemic treatment. Our findings warrant future clinical trials investigating the role of higher than palliative dose and early intervention of PRT in stage IV patients.The online version contains supplementary material available at 10.1186/s13014-023-02357-7. Recent advances in systemic treatments, which have improved overall outcomes in stage IV breast cancer with uncontrolled primary tumors have motivated physicians to integrate locoregional treatment in these patients . HistoriIn patients with unresectable breast tumors, polymetastatic status, or recurrent stage IV breast cancer, breast palliative RT (PRT) is administered with the aim of alleviating breast pain, bleeding, and discharge symptoms . In addin = 28), (2) PRT was not completed (n = 5), or (3) follow-up data were unavailable (n = 3). Among 5 patients who did not complete PRT, 3 patients refused to receive any further treatments and 2 patients showed rapidly progressive systemic disease resulting in early administration of systemic treatments. A total of 133 patients were identified in the final analysis. The requirement for informed consent was waived owing to the retrospective nature of the study.After receiving approval from the institutional review board (SMC 2023–01-030), we retrospectively reviewed 169 patients who underwent PRT for locoregionally uncontrolled breast cancer between January 2010 and June 2021 at the Samsung Medical Center. Patients were excluded from the final analysis if: (1) re-irradiation was performed for local recurrence  at diagnosis and PRT. Based on international consensus guidelines for advanced breast cancer statements, oligometastatic disease was defined as ≤ 5 lesions involved at the time of PRT . In addin = 51, 38.3%), and (2) treatment of radiologic progression without symptoms . When summarized by treatment year, the number of PRT cases has recently increased  or whole breast/chest wall  were treated for PRT planning. The gross lesion was defined as gross tumor volume, and the median gross tumor volume was 613.5 cm3 . Overall, a total dose of 45.0 Gy  was prescribed; an equivalent dose in 2 Gy fractions  was 53.2 Gy . Details of dose scheme are summarized in Additional file n = 56, 42.1%) followed by 54 Gy in 18 fractions , 48 Gy in 10 fractions , and 60 Gy in 15 fractions . Most patients were treated with three-dimensional conformal RT , followed by intensity-modulated RT  and proton beam therapy . Regarding systemic treatment, 46 patients (34.6%) received PRT between cytotoxic chemotherapy cycles or concurrent cytotoxic chemotherapy, and 27 (20.3%) and 10 (7.5%) patients received concurrent endocrine or anti-HER2 targeted therapy, respectively. Fifty patients (37.6%) did not receive systemic treatment within one month before and after PRT.With an interval of 12.3 months (interquartile range [IQR] 5.5–27.5) from the diagnosis of metastatic disease, patients were referred for PRT due to the following reasons: (1) relief of symptoms  .Response after PRT was assessed based on RECIST. Local control (LC) was calculated from the date of PRT to the date of local failure. Overall survival (OS) was calculated from the date of PRT to death from any cause or last follow-up. LC, and OS rates were estimated using the Kaplan–Meier method. The Cox proportional hazards model was used for multivariable analysis of factors affecting LC, and OS; only factors with statistical significance in the univariable analysis were included. The optimal cut-off value for gross tumor volume and EQD2 was evaluated using the R package “MaxStat," which iteratively tests all possible cut-off points to find the one achieving the maximum rank statistic . A two-sn = 65, 48.9%). The median two lines  of systemic therapy were administered before PRT. Most of the patients  had polymetastatic disease at the time of breast PRT. Specifically, 65 patients (48.9%) had single-organ metastatic disease, followed by 38 (28.6%) and 30 (22.6%) patients with 2 or 3–4 organ involvements, respectively. Regarding disease status at PRT, more than half  of the patients had progressive stage IV disease, whereas 30 patients (22.6%) and 27 patients (20.3%) received PRT at de novo stage IV and recurrent stage IV disease, respectively. In addition, 104 patients (78.2%) had stable disease outside the breast, whereas 17 (12.8%) and 12 (9.0%) patients had progressive disease and mixed responses, respectively at sites other than the breast.The patient and tumor characteristics are summarized in Table The overall response rate for breast tumors following PRT was 74.4%; complete, partial response, stable disease, and progressive disease was observed in 9.0%, 65.4%, 18.8%, and 6.8%, respectively. Among 51 patients (38.3%) with symptomatic disease, 5 (9.8%) and 34 (66.7%) patients experienced disappeared and decreased pain/bleeding, respectively; 12 (23.5%) showed no response after PRT. In addition, among 82 (63.7%) patients with asymptomatic radiologic progression, 7 (8.5%) and 53 (64.6%) showed complete remission and partial response, respectively, whereas 22 (26.8%) showed stable or progressive disease. There were no grade 3 or more toxicities related to PRT. During a median follow-up of 17.2 months , the 2-year LC and OS rates were 49.4%, and 48.3%, respectively , those with de novo stage IV disease , and those with stable disease outside the breast  showed favorable LC and OS outcomes . Additionally, either hormone receptor positive tumor treated with endocrine therapy or HER2 positive tumor treated with anti-HER2 therapy showed superior OS outcomes than others (Table Patients treated with ≤ 2 lines of systemic therapy (P = 0.001), progressive disease or mixed responses at other sites , and breast PRT < 63 Gy  were associated with inferior LC , group 2 , and group 3 . A significant difference in clinical outcomes was observed between the risk groups  was further evaluated in subgroup analyses based on risk factors. In these analyses, ≥ 63 Gy showed a statistically significant benefit in LC . In addition, Bourgier et al. reviewed 308 patients with stage IV disease, showing that PRT could provide long-term LC in 85% of patients, with better OS outcomes than surgical resection [Various modalities for local treatment can be employed to control breast tumors and alleviate local symptoms in patients with stage IV breast cancer. Breast PRT or surgical resection, with or without postoperative RT, can be adopted as for local therapeutic modalities. Based on the theory of primary cancer as a persistent source of circulating tumor cells, it is thought that PRT to the primary tumor can eradicate the circulating tumor cells, potentially leading to the control of metastatic disease outside the breast in selective patients , 24. Sevesection . Furtheresection . In theivs. 68.9%, P = 0.048). Based on the results of our study, potential candidates for PRT with curative intent could be expanded to include patients treated with 1–2 lines of systemic treatment. Adopting PRT in not only de novo stage IV patients but also in the currently suggested low-risk group could result in a favorable median survival of 43.8 months, with a 3-year estimated OS of 59.1%.As demonstrated above, previous studies have presented outcomes following PRT, mainly focused on patients with de novo stage IV breast cancer. Nonetheless, there is a paucity of data concerning the role of breast PRT in cases of recurrent or progressive stage IV disease. In one study, Mauro et al. evaluated the role of PRT at the primary site in 125 patients with stage IV disease [Meanwhile, surgical resection of the breast tumor, with or without postoperative RT, exhibited favorable LC in stage IV breast cancer –19, 25. P = 0.06) in locally incurable inflammatory breast cancer[P = 0.028) [Regarding the prescription dose, limited data is available to suggest optimal PRT schedules , 28–30. = 0.028) . ConsistThe interpretation of the current study has several limitations owing to its retrospective nature. First, the heterogeneity and retrospective nature of the current cohort hindered a comprehensive analysis to assess the beneficial impact of PRT. Although uniform EQD2 was calculated for analysis, a large variety of dose scheme , 36 Gy in 6 fractions (62 Gy in EQD2), or 35 Gy in 5 fractions (67 Gy in EQD2) can be recommended for PRT in low- or intermediate-risk groups for long-standing LC. Given the dismal outcomes observed in the high-risk group (group 3), it is postulated that a short course of RT, delivering around 30 Gy, may be necessary to achieve symptoms relief. Further confirmatory, prospective, randomized controlled trial is crucial to validate these results.Additional file 1: Supplementary Data.


Im-3m) to 2D hexagonal (p6mm) symmetries. More interestingly, the resultant mesoporous nanofibers can be assembled into 3D hierarchical cryogels on a large scale. The 1D nanoscale of the mesoporous nanofibers, in combination with small diameter (~65 nm), high aspect ratio (~154), large surface area (~452 m2 g−1), and 3D open mesopores (~6 nm), endows them with excellent performances for sodium ion storage and water purification. Our methodology opens up an exciting way to develop next-generation ordered mesoporous materials for various applications.One-dimensional (1D) nanomaterials have sparked widespread research interest owing to their fascinating physicochemical properties, however, the direct self-assembly of 1D porous nanomaterials and control over their porosity still presents a grand challenge. Herein, we report a monomicelle oriented self-assembly approach to fabricate 1D mesoporous nanostructures with uniform diameter, high aspect ratio and ordered mesostructure. This strategy features the introduction of hexamethylenetetramine as a curing agent, which can subtly control the monomicelle self-assembly kinetics, thus enabling formation of high-quality 1D ordered mesostructures. Meanwhile, the micellar structure can be precisely manipulated by changing the reactant stoichiometric ratio, resulting in tailorable mesophases from 3D cubic ( One-dimensional materials have interesting physiochemical properties, but control over porosity is still a significant challenge. Here, the authors report a self-assembly method for the fabrication of 1D materials with an ordered mesostructure. Over the past years, there is increasing interest in the topological design of nanomaterials with delicate controls on their morphology, structure, and composition14. As an illustrative example, one-dimensional (1D) nanomaterials offer a number of fascinating advantages because of their high-aspect-ratio structural feature, combined with high surface area, flexibility, quantum confinement effects, and rich surface functionalities, which enables the formation of a 3D interconnected open texture with low material-consuming but high stretchability22. These intriguing physical and chemical characteristics endow them huge potential in many high-tech applications, including adsorption, catalysis, sensor, biomedicine, energy conversion and storage, etc30. Additionally, introducing porous structure into 1D nanomaterials can not only provide abundant active sites and interfaces for reaction but also alleviate mechanical stress and accommodate volumetric changes, which are highly desirable for many mass diffusion-limited applications32.Low-dimensional nanomaterials have attracted widespread attention owning to their fascinating mechanical, optical, and electrical merits resulted from the reduction of dimensions33, self-templating35, hydrothermal carbonization37, self-assembly39, electrospinning42, and biomass conversion43. However, most of samples prepared using these approaches limited to small pore size or irregular porosity. As a result, the capacity of the guest materials that can be loaded in the pores and the efficiency of mass/ions transport are quite restricted by their pore size and structure. In this regard, bringing ordered mesoporous structures into 1D nanofibers may achieve optimized, enhanced, or even new-yet-unexpected properties. Several recent studies have indicated that the 1D ordered mesostructures can be achieved by assembling the block copolymers in confined spaces, such as anodic aluminum oxide (AAO) films, or via a nano-casting route to duplicate the porous structure from original 1D templates45. Such syntheses showed the ability in morphology control but were also known for their obvious shortcomings, including time-consuming, low yield, and environmental risk. In addition, the obtained materials usually suffered from uncontrollable mesophase. The development of a simple but powerful self-assembly approach templated by amphiphilic block copolymers to simultaneously achieve ordered mesostructure and 1D morphology is highly interesting and desirable. The presence of tunable mesophases within nanofibers, such as 3D open mesoporosity, can further offer broad accessibility for mass transport and significantly improve the loading capacity for guest species48. Despite significant advances in synthetic methodology, the direct fabrication of ordered mesoporous nanofiber with tailorable mesophases and porosities still remains challenging.Up to now, tremendous research efforts have been dedicated to developing methodology for synthesizing 1D porous nanomaterials, such as nanocasting106-PPO70-PEO106) as the structure-directing agent, hexamethylenetetramine (HMT) as the curing agent, and phenolic resol (Mw = 500−2000) as the carbon source under hydrothermal condition, and followed by carbonization in N2 atmosphere  were fabricated via a kinetically driven monomicelle oriented self-assembly strategy by using F127 triblock copolymer  method , pyrrolic N (399.7 eV), graphitic N (400.8 eV), and oxidized N  image of the as-made OMPFs shows remarkable uniform 1D morphology with an average length of ~10 μm  as a catalyst, uniform carbonaceous nanofibers can be formed but without ordered mesoporous structure , the ordered mesostructure is disappeared and irregular solid particle is appeared    Fig. , which i3m) Fig. . By care3m) Fig. . Interes3m) Fig. . When thmm) Fig. . The TEMmm) Fig. . The chamm) Fig. , suggestmeso-building block  (Step 1c). The corresponding morphological evolution is confirmed by the time-dependent experiments . After the freeze-drying and carbonization processes, uniform OMCFs were generated with a 2D hexagonal symmetry (p6mm) (Step 2c). However, a higher F127/Resol mass ratio could cause a change in micellar morphology and consequently induce mesoporous nanofibers to be bend and increase in dimension  hierarchical porous cryogel on a large-scale through a unidirectional ice-templating strategy56 Fig. . The opt56 Fig. . The SEM56 Fig. , which i56 Fig. . The top56 Fig. . The thi56 Fig. . Magnifi56 Fig. , endowin60% Fig. , suggestles Fig. . When beles Fig. , indicatles Fig. .Fig. 5St−1, respectively, at the current density of 0.1 A g−1 2F3 (NVPF) as a cathode material, respectively. The galvanostatic charge/discharge profiles of the FIBs shows two working plateaus at ~3.5 (discharge status) and 3.8 V (charge status), respectively, and delivers a specific capacity of 105 mAh g−1 at 0.1 A g−1 based on the NVPF cathode material   in the original seawater source show 4 orders of concentration decrement after desalination  Fig. . In cont−2) Fig. . The wat−2) Fig. . It is cion Fig. , which aThe exceptional performance of OMCFs for practical application can be attributed to their fascinating nanostructure and chemical properties, including nanoscale dimension, 1D morphology, high aspect ratio, 3D ordered open mesostructure, amorphous framework, and rich O, N-dopants. Firstly, the nanoscale dimension and 3D ordered open mesostructure of OMCFs offer much shorter migration distances for mass transfer in comparison with bulk materials, allowing organic molecules and metal ions to easily penetrate into the whole nanomaterials. Secondly, the 1D morphology and high aspect ratio of OMCFs can construct hierarchical porous networks integrating nanoscale and the microscale morphologies effects, which not only provides a 3D continuous pathway for mass transfer and electron transport but also offers multi-level pore structures and large stacking space to buffer the mechanical stress/strain and volume change during reaction processes. Thirdly, the big pore size, thin pore walls, and amorphous frameworks can offer large confined spaces, accessible surfaces/interfaces, and abundant active sites for guest molecule storage and reaction. Fourthly, the homogeneous rich N, O-dopants in OMCFs can further enhance the electronic conductivity and tailor interfacial wettability, thus additionally advance their performance.Im-3m) to 2D hexagonal (p6mm) symmetries. The resultant OMCFs show small diameter (~65 nm), high aspect ratio (~154), large surface area (~452 m2 g−1), 3D large open mesopores (~6 nm), and favorable nitrogen dopants (~4.1 wt%). Impressively, the OMCFs can be assembled into hierarchical porous cryogel on a large scale. As a result, the OMCFs and their derived 3D cryogels deliver excellent rate capability (163 mAh g−1 at 10.0 A g−1) for SIBs and great water purification performance (2.4 kg m−2 h−1) for seawater desalination, respectively. We believe that this work presents flexible and versatile tools to prepare interesting 1D ordered mesoporous nanomaterials for various advanced applications and, more impressively, gains insights into the monomicelle self-assembly chemistry and science.In summary, we have demonstrated a simple but efficient monomicelle oriented self-assembly approach to fabricate the OMCFs with well controlled 1D morphology and mesoporous structures. In this synthesis, we have shown the possibility of modulating the monomicelle self-assembly kinetics through a HMT hydrolysis induced process, which provides an opportunity to simultaneously realize 1D morphology and ordered mesostructure controls. By regulating the reaction parameters such as the reactant stoichiometric ratio, it is convenient to systemically tailor the ordered mesophase of the prepared OMCFs from 3D cubic . In a typical process, 1.0 mL of resol precursor solution, 0.24 g of Pluronic F127, and 0.08 g of HMT were added into 80 mL of deionized water under stirring at 300 rpm to obtain the F127/Resol monomicelle system. The stirring bar was 2 cm in length. Then, the above mixture was placed into a 100 mL Teflon-lined stainless-steel autoclave for hydrothermal treatment at 100 °C for 24 h. After the autoclave was cooled, the as-made polymeric composites were collected by centrifugation and washing with water and ethanol. Finally, the freeze-dried polymeric composites were further calcined at 800 °C for 2 h under NThe hierarchical OMCFs cryogel was prepared through an ice-templating method. Typically, 1.0 g of ordered mesoporous polymeric nanofibers were dispersed in 300 mL of deionized water by stirring at 10000 rpm for 30 min, yielding uniform nanofiber dispersion. Next, the obtained dispersion was transferred into a designed Teflon mold, which was subsequently frozen in a liquid nitrogen tank and then dried for 48 h to generate the hierarchical polymeric nanofibers (OMPFs) cryogel. Finally, the OMPFs cryogel was carbonized at 800 °C for 2 h to produce the OMCFs cryogel.Supplementary InformationPeer Review File


Using the clinical samples as a template, the limit of detection (LOD) was 4.91 × 10−5 ng per reaction ), which means that this method was one of the most sensitive detection assays for CaPVs. A total of 85 clinical samples from CaPV-infected animals  and 50 clinical samples from healthy animals were used to test and compare the diagnostic results using the Synergy Brands (SYBR) Green-based PCR method recommended by the World Organization of Animal Health (WOAH). Both diagnostic sensitivity (DSe)  and diagnostic specificity (DSp)  results of the real-time quantitative PCR (qPCR) and SYBR Green PCR were 100%, and the kappa value (κ) was 1.0 . In summary, the assay established based on TaqMan probes was advantageous in high specificity, sensitivity, and general applicability and could be a competitive candidate tool for the diagnosis of CaPV in clinically suspected animals.Capripox viruses (CaPVs), including sheep pox virus (SPV), goat pox virus (GPV), and lumpy skin disease virus (LSDV), are the cause of sheep pox (SPP), goat pox (GTP), and lumpy skin disease (LSD) in cattle. These diseases are of great economic significance to farmers, as they are endemic on farms and are a major constraint to international trade in livestock and their products. Capripoxvirus (CaPV) infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) method for identifying CaPV in goats, sheep, and cattle. Clinical samples were tested and verified. The developed assay was highly specific for target viruses, including GPVSPV and LSDV, which had no cross-reaction with other viruses causing similar clinical symptoms. An artificially synthesized positive control plasmid using the CaPV 32 gene inserted into the vector pMD19-T was used as a template, and the correlation coefficient of the linear regression curve ( Capripoxvirus (CaPV) in the subfamily Chordopoxvirinae, family Poxviridae /[length (bp) × 109 × 650 (g/mol)]. The calculated copy number was 5.8 × 1010 copies/µL. PAC with copy number concentration was used as a template for detection limit (LOD) studies.The 550 bp sequence of the CaPV P32 gene (Accession no. MG458377.1) was inserted into the commercial vector pMD19-T to construct the plasmid, which was digested by Sac I and used as a linearized plasmid for positive amplification control (PAC). The synthesis of the PAC plasmid was completed by Sangon Biotech Co., Ltd. . The purified PAC plasmid was quantitatively determined using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific) with a concentration of 34.45 ng/µL. The Avogadro number . Each dilution level was analyzed 6 times and run under the condition of repeatability 4 times; the dilution series needed to be prepared again before each run. Each dilution level was measured 6 times, with 24 data points for each dilution point. The average Ct value obtained at each dilution level was plotted with PAC plasmid concentration, and linear regression analysis was performed using GraphPad Prism [−1/slope − 1) was used to calculate the qPCR efficiency and was expressed as a percentage. For each target, the slope of the regression curve should be between −3.9 and −2.9, and the corresponding qPCR efficiency should range from 80% to 120%. Meanwhile, the correlation coefficient 2R of the linear curve was an index to measure the linearity of the qPCR reaction. The 2R of each target should be ≥0.98 [To calculate the amplification efficiency of the qPCR, seven series of dilution levels were prepared using the CaPV artificial positive amplification control (PAC) plasmid as a template, including 5.8 × 10ad Prism  , with inputs of the corresponding number of sample materials, the number of repetitions, and the number of positive results in qPCR detection. In this study, the LOD95% of two types of plasmid PACs (in copies of the genome) and the GPV clinical sample DNA template (in units of DNA concentration (ng/µL) were identified.The analytical sensitivity of the qPCR method, which was presented by LOD95% of CaPV using a qPCR-assay-based TaqMan probe, 8 series of dilution levels were prepared, including 5.8 × 104, 5.8 × 103, 5.8 × 102, 5.8 × 101, 5.8, 2.9, 0.58, 0.058 copies/µL, and the test was repeated at each dilution level not less than 8 times. The minimum number of copies per analysis was determined. In addition, the GPV clinical sample DNA was continuously diluted using healthy animal tissue DNA to prepare 9 series dilution levels as the template, including stock solution (10 ng/µL), 101 × dilution (1 ng/µL), 102 × dilution (10−1 ng/µL), 103 × dilution (10−2 ng/µL), 104 × dilution (10−3 ng/µL), 105 × dilution (10−4 ng/µL), 106 × dilution (10−5 ng/µL), 107 × dilution (10−6 ng/µL), and 108 × dilution (10−7 ng/µL). There were at least eight replicates per concentration. The maximum dilutive level for each clinical sample tested was determined.To determine the LODThe specificity and cross-reactivity of the CaPV test assay were evaluated using qPCR. Undiluted sample material DNA/RNA was obtained using 9 closely related viruses and bacteria, including clinical samples or culture materials of differential viruses and bacteria of goat pox virus, sheep pox virus, lumpy skin disease virus, foot-and-mouth disease virus, peste des petits ruminants disease virus, bovine viral diarrhea virus, Brucella, Mycobacterium tuberculosis, and Bacillus anthracis, were tested using a CaPV exclusion assay based on the P32 gene. DNA/RNA analysis was repeated no less than 3 times for each sample material.DSe) and specificity (DSp) of the qPCR assay were evaluated using clinical samples from naturally CaPV-infected animals  and healthy animals. The diagnostic performance was compared with the SYBR Green qPCR method recommended by the WOAH [The diagnostic sensitivity  with thirty-nine representative entry numbers were selected  to analy6, 5.8 × 105, 5.8 × 104, 5.8 × 103, 5.8 × 102, 5.8 × 101, and 5.8 copies/µL, and they were analyzed using TaqMan probe qPCR. A linear regression curve was plotted between the threshold cycle value  and PAC plasmid concentration (in copy number). According to the equation E = 100 (10−1/slope − 1), the analysis efficiency was determined as the slope of the regression line, showing a good linear relationship between the Ct value and PAC plasmid concentration  plasmid as a template, including 5.8 × 10ntration . The cor95% of PACs were expressed as the copy number per analysis, where each copy represents one genome copy of the virus particle. Eight series of dilution levels were prepared using the CaPV artificial positive amplification control (PAC) plasmid as a template, including 5.8 × 104, 5.8 × 103, 5.8 × 102, 5.8 × 101, 5.8, 2.9, 0.58, and 0.058 copies/µL (eight replicates for per concentration), to evaluate the LOD95% of CaPV using a real-time qPCR assay. The expansion of PACs was linear. The results of probabilistic regression analysis showed that the LOD95% of CaPV using the qPCR assay was 3.80 copies/response, with a 95% confidence interval of 1.88–38.19 copies/response , 101 × dilution (1 ng/µL), 102 × dilution (10−1 ng/µL), 103 × dilution (10−2 ng/µL), 104 × dilution (10−3 ng/µL), 105 × dilution (10−4 ng/µL), 106 × dilution (10−5 ng/µL), 107 × dilution (10−6 ng/µL), 108 × dilution (10−7 ng/µL), and 109 × dilution (10−8 ng/µL), as templates (eight replicates per concentration) to evaluate the LOD95% of CaPV using the qPCR assay—the amplification using the clinical sample DNA as the template was linear. The results of the probabilistic regression analysis show that the LOD95% of CaPV determined using the qPCR assay was 4.91 × 10−5 ng/µL . The 95% confidence interval was 1.60 × 10−5–2.13 × 10−3 ng  and samples from six non-targeted, undifferentiated viruses and bacteria  were used. The specificity of the real-time qPCR assay for CaPV was evaluated . ExclusiDSe) and diagnostic specificity (DSp) of the real-time qPCR method for the diagnosis of CaPV were calculated using probability regression analysis. Samples from 85 CaPV-infected animals  and 50 healthy animals  were used for clinical verification. These clinical samples were confirmed using the SYBR Green qPCR method recommended by the WOAH.The diagnostic sensitivity , 29 SPV-infected sheep  and 30 LSDV-infected cattle  were clinically validated, and all tests were positive . We haveDSe) and specificity (DSp) of the real-time qPCR method for CaPV clinical samples were 100%  and 100% , respectively. Kappa values were 1.0   were clinically verified, and all tests were negative . At the  95% CI) .In this study, a real-time qPCR assay was developed to detect GPV, SPV, and LSDV simultaneously and to exclude multiple other pathogens from samples containing multiple pathogens.At the beginning of development, we considered the coverage of CaPV isolates worldwide in terms of sequence retrieval and the design of primer probes. Since the P32 protein of CaPV has high sequence homology among GPV, SPV, and LSDV, it is a common structural protein with high specificity and strong immunogenicity in CaPV isolates . Therefo95% of two types, respectively. Probabilistic regression analysis shows that for the assay of qPCR, the LOD95% was 3.80 copies per response , 4.91 × 10−5 ng per response . The maximum dilution level of DNA detected in clinical samples was a 106 × dilution level. Others also reported that the LOD of the CaPV detection method reached 10 copies [In terms of sensitivity (LOD), this study used continuous diluents of DNA from clinical samples of CaPV and plasmid PACs as templates to investigate the LOD0 copies ,32,35. TdSe evaluation of 135 clinical samples showed 100%  dSe and 100%  dSe and DSp compared to the SYBR Green PCR recommended by the WOAH. The Kappa value of 1.0  indicated that the two methods are comparable.The CaPV exclusion test showed 100% specificity for all serotypes/thesolates of the target viruses tested  and did not cross-react with clinical samples or culture materials of other viruses and bacteria. The Capripox (CaP) is a major infectious disease affecting the healthy development of the ruminant breeding industry in China . CurrentE) of the method was 96.06%. The LOD95% of serial dilutions of DNA from plasmid PACs and CaPV clinical samples were 3.80 copies per reaction  and 4.91 × 10−5 ng per reaction . The highest dilution level for detecting DNA in clinical samples was the 106 × dilution level. Non-targeted differential viruses and bacteria were not detected using this method. Compared with the SYBR Green PCR recommended by the WOAH, the diagnostic sensitivity and specificity were both 100% . The κ value was 1.0 .The study describes a real-time quantitative PCR method based on a TaqMan probe for identifying CaPV in goats, sheep, and cattle and its validation in clinical samples. The amplification efficiency (In summary, the established qPCR method is highly sensitive, specific, and reproducible and can be used as an effective tool for detecting CaP disease in ruminants early.