Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has α-mannosidase activity, however, we conclusively show that it cleaves only β-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant Bs164, and multiangle light scattering we found that it is a trimeric retaining β-mannosidase, that is susceptible to several known mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164, the first known structure of a GH164, at 1.91 Å resolution. Bs164 is composed of three domains: a (β/α)8 barrel, a trimerization domain, and a β-sandwich domain, representing a previously unobserved structural-fold for β-mannosidases. Structures of Bs164 at 1.80–2.55 Å resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family ( N-glycans (Leishmania (http://www.cazy.org)6–Mannose is an essential component of protein human -glycans , storage-glycans , and manishmania , and hasishmania . Consideishmania  8 barrel with catalytic residues on β-strands 4 and 7 placing this family in clan GH-A. NMR analysis revealed a retaining mechanism and site-directed mutagenesis confirmed the assignment of the nucleophile and acid/base catalytic residues. Structures of inhibitor complexes show the conserved catalytic machinery involved in substrate recognition and catalysis and provide insight into the likely conformational itinerary for mannoside hydrolysis. Taken together, this work provides a thorough biochemical basis for the β-mannoside hydrolysis catalyzed by GH164 enzymes.Here we present the detailed biochemical and structural analysis of B. saylersiae, four genes originate from the Alistipes genus, commonly found in the human gut . A single peak was observed with a calculated molecular mass of 227 kDa, signifying that Bs164 forms trimers in solution.The Bs164 we assayed the purified enzyme with a variety of synthetic aryl glycosides and mannooligosaccharides . Bs164 shows no activity toward d-xylose, d- or l-arabinose, d-galactose, d-glucose, or l-fucose containing substrates. Furthermore, no activity was observed toward α-d-linked mannosides, contradicting the initial report of enzyme activity by Helbert et al. , which we attribute to pNP being a slightly better leaving group. This enzyme is also able to hydrolyze β-linked mannooligosaccharides and shows comparable specificity constants for the hydrolysis of mannobiose, mannotriose, and mannotetraose  over the course of time . Each trimer-donut has an outer diameter of ∼100 Å and an internal diameter of between 30 and 35 Å. The individual Bs164 chains contain three clearly defined domains: a modified (β/α)8 barrel (residues 22–389), a domain containing a 7-membered mixed β-sheet sandwiched between α-helices (390–609), and a β-sheet domain (610–681) (B). This domain architecture is quite similar to that seen for family GH42 enzymes  is indeed a GH42 β-galactosidase from Bacillus circulans sp. alkalophilus (PDB code 3TTY) also with trimeric quaternary structure (We solved the structure of solution A. Each t610–681) B. This d enzymes , but is tructure .8-fold of domain A is found in the catalytic domain of many glycoside hydrolases and is a distinctive feature of clans GH-A, -D, -K, and -R. Domain A also contains a helix-containing subdomain, atypical of (β/α)8-folds but also observed in the GH42 family (B). We speculate that the presence of this domain helps, in part, to stabilize the trimeric structure. Domain A shows highest similarity, by means of a Dali search (Rahnella sp. (PDB ID code 5E9A) with RMSD of 3.2 Å and complete query coverage (5E9A) and acid/base residue (Glu-157 in PDB 5E9A) are conserved in Bs164 and the putative catalytic nucleophile and acid/base are Glu-297 and Glu-160, respectively. Comparison of amino acid conservation within the GH164 family also shows that this active site is completely conserved across the family (The (β/α)2 family , which i2 family B. We spei search , to the coverage . Overlaye family . The posBs164 is a shallow pocket that has a much more open + binding subsites than is seen in the exo-mannosidases BtMan2A . The chloride present is observed in all 6 chains in the crystal structure and is observed in all of the inhibitor complexes described below. This chloride is present at a site adjacent to the active site and one of the coordinating arginines, Arg-73, and forms a direct interaction with the catalytic nucleophile. Additionally, Arg-75 projects into the active site and the two η-nitrogens form hydrogen bonds with the 2- and 3-hydroxyls of the sugar residue bound at the −1 subsite, as will be described below.In addition to the active site, domain A also contains a chloride-binding site. The chloride ion coordinates Gln-45, Thr-110, and two arginine residues (Arg-73 and Arg-75) in a penta-coordinate, distorted square pyramidal geometry see D. The chBs164 interacts over a surface area of 1380 Å2 per protomer that includes seven residues involved in direct hydrogen bonds and two salt bridges between chains. The corresponding interface in domain A consists of the loops connecting the last two pairs of β-sheets and α-helices. This trimerization also likely has a role in catalysis as the interaction of residues Ser-342–Ala-345 and Ala-347 in domain A with the domain B of the neighboring protomer appears to help position the active site residues Arg-341 and Glu-346 that interact directly with the substrate, as shown in the inhibitor-bound structures described in the next section.Domain B bears a striking resemblance to the trimerization domain of GH42 enzymes. In GH42 structures , 18, 20 Unlike the first two domains, function of the C-terminal β-sandwich domain is much more difficult to infer from the structure. This domain is also present in GH42 enzymes , 18, 20,Bs164 in complex with 2,4-dinitrophenyl 2-deoxy-2-fluoro-mannoside (DNP-2FM), noeuromycin, and mannoimidazole through soaks of Bs164 crystals. Mannoimidazole and noeuromycin act as competitive inhibitors of Bs164 with inhibition constants (iK) of 470 ± 60 μm and 340 ± 40 nm, respectively. The inhibition constant for noeuromycin is well in line with previous reports of β-mannosidase inhibition (iK = 1.4 μm)  . The flu 1.4 μm) . All thr subsite .4RE2) (2VMF) (1UZ4)  , GH2 (PD) (2VMF) , and GH5) (1UZ4) . The O4   indicating that the conformational itinerary is centered, as for most β-mannosidases, around a B2,5 transition state. The structure of the noeuromycin complex, in which a 1S5 conformation is observed (A), lends further support to this itinerary. Observations from other β-mannosidases  with the 2F positioned axially, instead of equatorially as would be expected in a OS2 conformation. The comparatively low resolution of the 2FM structure (2.55 Å) makes it difficult to equivocally confirm the conformation of the glycosyl-enzyme intermediate that appears to refine as 4C1/E5. We endeavor to further explore the energetic consequences of this 4C1/E5 conformation and whether this structure is representative of the true glycosyl-enzyme intermediate or an energetic relaxation of this intermediate to a position that is not along the conformational itinerary.The inhibitor-bound structures of tinerary , the outsee Ref. . The strsee Ref. , 28, 30,ive site B indicatobserved A, lends osidases  suggest Bs164, the prototypical GH164, adds to our growing understanding of the mechanisms underpinning enteric carbohydrate degradation. Our interrogation of this enzyme has shown that it exists as a donut-shaped homotrimer, a new domain architecture and quaternary structure for β-mannosidases, and employs two conserved glutamic acid residues in a retaining mechanism. The structural analysis of Bs164 in the presence of inhibitors revealed a host of active-site interactions and suggested a boat-like transition state. Further investigation of how the genes co-localized within GH164 containing operons, promises to reveal how these loci function in concert to enable degradation of mannose containing polymers within the human microbiome.The capacity of commensal bacterial to digest dietary carbohydrates relies on a broad range of enzymes, tailored to their specific target. Examination of d-mannopyranoside and methylumbelliferyl β-d-mannopyranoside were purchased from Sigma-Aldrich. Mannobiose, mannotriose, and mannotetraose were purchased from Megazyme . Mannoimidazole and noeuromycin were kind gifts from Professor Spencer Williams and Professor Robert V. Stick, respectively. 2,4-Dinitrophenyl 2-deoxy-2-fluoro-β-d-mannopyranoside was a kind gift from Professor Stephen G. Withers (University of British Columbia).Paranitrophenyl β-Bs164 (GenBankTM accession EIY59668.1) was predicted to contain a signal peptide with a cleavage site between amino acids 21 and 22 by signalP 5.0  was synthesized by and cloned into a pET-28 vector by GenScript . Plasmid was transformed into chemically competent BL21(DE3) gold cells (Agilent) and plated on LB agar containing 50 μg/ml of kanamycin. A single colony was used to inoculate 5 ml of LB media containing 50 μg/ml of kanamycin. After overnight growth at 37 °C with shaking at 180 rpm this starter culture was used to inoculate NZYTech autoinduction media (NZYTech) containing 50 μg/ml of kanamycin. Expression cultures were grown at 37 °C with shaking at 250 rpm for 6 h, the temperature was then decreased to 20 °C and cultures were incubated for an additional 22 h. Expression cultures were harvested by centrifugation  and cell pellets were stored at −80 °C until purification.The gene encoding nalP 5.0 . A codonm HEPES, 30 mm imidazole, 200 mm NaCl, pH 7.4) with additional protease inhibitor (4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 mm) lysozyme, and DNase. Resuspended cells were then lysed by passage through a cell-disruptor homogenizer at 25 kpsi. Lysed cells were centrifuged  and the supernatant was decanted from the cell debris. Clarified supernatant was loaded directly onto a 5-ml His tag Excel column . Bound protein was washed with 7.5 column volumes of buffer A then eluted with a linear gradient of 0–100% buffer B  over 20 column volumes. Eluted protein was concentrated with a 30-kDa cut-off Amicon centrifugal filter unit and further purified by gel filtration  in buffer C . The purity of eluted protein was analyzed by SDS-PAGE and the peak fractions were pooled and concentrated with a 30-kDa cutoff Amicon centrifugal filter unit. Concentrated protein was washed with buffer D  then diluted to 30 mg/ml with buffer D and flash frozen with liquid nitrogen until use. Protein concentrations were determined spectrophotometrically using a calculated A280 extinction coefficient of 128,480 m−1 cm−1. For biochemical studies, the N-terminal His6 tag was cleaved from Bs164 through overnight incubation with a 1:100 ratio (by mass) of 3C-protease. Cleaved Bs164 was then purified by gel filtration and concentrated as detailed above. The average yield of purified protein was ∼30 mg/liter of culture.To purify the protein cell pellets were resuspended in 120 ml of Buffer A  and the cell pellet was resuspended in 40 ml of M9 media containing 50 μg/ml of kanamycin. The resuspended cells (1 ml) were then used to inoculate a 1-liter culture of M9 media containing 50 μg/ml of kanamycin. This culture was incubated at 37 °C with shaking at 250 rpm until mid-log phase (A600 = 0.5). At this point lysine (100 mg/liter), phenylalanine (100 mg/liter), threonine (100 mg/liter), isoleucine (50 mg/liter), leucine (50 mg/liter), valine (50 mg/liter), and selenomethionine (50 mg/liter) were added to the culture, which was incubated for another 15 min at 37 °C. The incubation temperature was then lowered to 20 °C and isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 0.5 mm. After incubation for 18 h the cultures were harvested as for the unlabeled protein. The selenomethionine protein was purified as for the unlabeled protein except for the addition of 5 mm DTT to buffers A, B, and C and the addition of 0.5 mm Tris(2-carboxyethyl)phosphine to buffer D.Selenomethionine-labeled protein was expressed using the same bacterial strain as for the WT protein. This strain was used to inoculate 40 ml of LB media containing 50 μg/ml of kanamycin, which was grown overnight at 37 °C. This culture was then centrifuged  protocol. For each mutant generated, PCR was first performed for 12 cycles with one of the sense or antisense primers ; these two reactions were subsequently pooled, and an additional 18 cycles of PCR were performed. PCR products were digested with DpnI (New England Biolabs) and transformed into chemically competent BL21(DE3) gold cells. Purified plasmids were sequenced to confirm the correct mutation prior to expression. Both the nucleophile and acid base variants were purified as for the WT protein.Mutagenesis was performed using a modified QuikChangeBs164 activity assays, unless otherwise stated, were performed at 25 °C in a buffer containing 80 mm MES (pH 5.5), and 160 mm NaCl, and were initiated by the addition of the appropriate amount of enzyme. Purified Bs164 was initially tested for activity on the following aryl glycosides: pNP α-l-arabinofuranoside, pNP α-l-arabinopyranoside, pNP α-l-fucopyranoside, pNP β-d-galactopyranoside, pNP α-d-glucopyranoside, pNP β-d-glucopyranoside, pNP α-d-mannopyranoside, pNP β-d-mannopyranoside, and pNP β-d-xylopyranoside. Purified enzyme was added  to a solution of 1 mm substrate. These assays were incubated at 25 °C and stopped after 6 h with a 1:1 ratio of 1 m Na2CO3 (pH 11.2) and absorbance at 400 nm was measured with a CLARIOstar Plus plate reader (BMG Labtech).All m MU β-Man, 160 mm NaCl, and either MES (pH 5–6.5) or HEPES (pH 6.5–8.5). Reactions were limited to this pH range as enzyme denaturation was observed above pH 8.5 and below pH 5. Fluorescence was monitored continuously  and depletion curves were fit to the equation: A = fA(1-e−kt), where k = [E] · kcat/KM, to give the specificity constant at each pH.The optimal pH for activity was determined with MU β-Man using substrate depletion kinetics. Purified enzyme was added to solutions containing 50 μpNP β-Man and MU β-Man using substrate concentrations covering the range of 1/5 to 5 times the eventual KM determined. Assays were performed in 96-well-plates and each plate included either pNP or MU standards in an identical buffer system for concentration calibration. MU β-Man hydrolysis was monitored continuously (λex = 365 nm and λem = 450 nm), whereas pNP β-man hydrolysis was measured after stopping the assay with a 1:1 ratio of 1 m Na2CO3 (pH 11.2).Kinetic parameters were determined for m mannobiose were incubated at 25 °C and aliquots were removed over a period of 30 min. These aliquots were added to an equal volume of freshly prepared bicinchonic acid reagent containing: 400 mm sodium carbonate (pH 11.2), 2.5 mm bicinchonic acid, 1.25 mm CuSO4, and 2.5 mml-serine. This mixture was heated to 80 °C for 10 min, and then cooled. The absorbance at 563 nm was measured with a CLARIOstar Plus plate reader (BMG Labtech) and compared with a standard curve of mannose to calculate the concentration of reducing sugars. Data were fit to linear equation to give the specificity constant.The specificity constant for the hydrolysis of mannobiose, mannotriose, and mannotetraose was determined using an assay for reducing sugars employing bicinchonic acid. Reactions containing between 0.1 and 4 miK) values Michaelis-Menten parameters were determined for Bs164 hydrolysis of pNP β-Man in reactions containing either mannoimidazole or noeuromycin. Assays were started with the addition of enzyme to the assay mix. Parameters were determined for at least 3 different inhibitor concentrations. Inhibition constants were calculated according to a competitive inhibition model for both inhibitors. All kinetic parameters were fit with the software program Origin 2019.To determine inhibition constant (m MES (pH 5.5), 200 mm NaCl as buffer. Shimadzu LC Solutions software was used to control the HPLC and Astra V software for the HELEOS-II and rEX detectors. Data were analyzed using the Astra V software. Molecular weights were estimated using the Zimm fit method . Work was conducted at room temperature (20 ± 2 °C). Sample injection volume was 100 μl at a protein concentration of 5 mg/ml. The samples were separated on a Superdex S200 10/300  using 80 mt method  with 1 dm MES, pH 5.5, and 160 mm NaCl), pNP β-mannoside and Bs164 were lyophilized. Buffer and enzyme were resuspended in D2O, whereas pNP β-mannoside was resuspended in 10% d6-DMSO in D2O. The final reaction mixture contained 4.5 mmpNP β-Man, 9 mm MES (pH 5.5), 145 mm NaCl, and 0.9% (v/v) d6-DMSO. NMR spectra were collected before the addition of enzyme and every 5 min after the addition of Bs164 to a final concentration of 120 μg/ml. NMR data were collected on a Bruker 700 MHz Avance Neo spectrometer equipped with a 5-mm triple resonance cryoprobe. 1-D proton spectra were recorded with a 30-degree excitation pulse, an interscan delay of 10 s, and a total acquisition time of 3 min. Sample temperature was maintained at 298 K, and referencing was relative to disuccinimidyl suberate.Prior to NMR experiments buffer , 12% (w/v) PEG 3,350, a condition that was used for further optimization. Crystals formed in 0.1 m ammonium tartrate dibasic at a pH range from pH 5.5 to 7.0. The optimized crystals were grown in maxi 48-well-plates using the sitting-drop vapor-diffusion method at 20 °C with 100 mm ammonium tartrate dibasic (pH 7.0), 13% (w/v) PEG 3,350 with a protein:well solution ratio of 500:500 nl. Crystals were soaked in well solution containing 25% (v/v) ethylene glycol before flash cooling in liquid nitrogen. Inhibitor complexes were obtained by soaking crystals in well solution containing 10 mm of the inhibitor for 3–4 h before flash cooling in liquid nitrogen. For the structures with DNP-2FM solid substrate was added to the drop containing crystals, which were then soaked overnight. The overnight soaking with crystals of DNP-2FM gave inhibitor complexes, but did appear to reduce diffraction quality. Refinement statistics are given in HisBs164 selenomethionine derivative, mannoimidazole, noeuromycin, and DNP-2FM diffraction data were collected at Diamond beamlines, and data were processed with the CCP4i2 suite (Bs164 was solved by MAD phasing of a selenomethionine derivative using 6 datasets: two at the peak wavelength (0.9794Å) using 0.1° oscillation and χ angle of 25° or 0°, for 360°; two at a remote wavelength (0.9777 Å) using 0.1° oscillation and χ angle of 25° or 0°, for 360°; and two at the inflection (0.9797 Å) using 0.1° oscillation and χ angle of 25° or 0°, for 360°. Crank2 (Bs164 were solved by molecular replacement using the unliganded coordinates as the search model in Phaser (i2 suite . The unl. Crank2  was used. Crank2 . Complexn Phaser . StructuAll 17 GH164 sequences currently available in the CAZy database  accesse and amin6T5O, 6T75, 6T6G, and 6T7G.The atomic coordinates and structure factors have been deposited in the Protein Data Bank using accession codes Z. A. data curation; Z. A. formal analysis; Z. A. and G. J. D. validation; Z. A. investigation; Z. A. visualization; Z. A. methodology; Z. A. writing-original draft; G. J. D. conceptualization; G. J. D. resources; G. J. D. supervision; G. J. D. funding acquisition; G. J. D. project administration; G. J. D. writing-review and editing.


Here, we asked how such mutations affect body stiffness by comparing force-indentation curves in dpy-10(e128) and wild type worms. On average, dpy-10(e128) worms have a steeper force-indentation relationship, leading to a higher body stiffness . Average stiffness values were 2.5 ± 0.9 N/m, SD, (n = 24) for dpy-10(e128) and 0.9 ± 0.3 N/m, SD, (n = 25) for wild type (N2) hermaphrodites. Qualitatively, we observed that some of the dpy-10(e128) worms had a disrupted cuticle and their bodies spontaneously eviscerated after being immobilized to an agarose pad. Finally, we confirm the finding that dpy-10(e128) worms are shorter  and fatter  than wild type worms with an average body length of 712 ± 68 µm  compared to wild type N2 worms 900 ± 68 µm  and an average body width at the pharynx of 42 ± 5.6 µm  compared to wild type N2 worms 36 ± 4 µm  . DPY-10 is a collagen protein in the nematode’s cuticle. Mutations in the dpy-10(e128) are twice as stiff as wild type: to reach the same indentation, a much higher force was necessary to be applied to dpy-10(e128) than to wild type worms . Prior measurements of body stiffness with the same method revealed that dpy-5(e61) worms are softer than wild type worms . Thus, body shape does not predict body stiffness. Why is one Dpy animal softer than wild-type and another one stiffer? We propose that dpy-10 is stiffer than wild type because it has an increased internal glycerol concentration and increased pressure, that is absent from dpy-5(e61) (Wheeler and Thomas 2006).From these results, we conclude that the bodies of Preparation of worms: Age-synchronized C. elegans nematodes were grown on standard OP50 growth plates at 20–23 °C. Late L4 or young adult hermaphrodites were immobilized on 8% agarose pads using WormGlu (GluStitch). During the experiments the worms were continuously superfused with extracellular saline.Force-Indentation measurements: The measurements were performed with the FALCON system described in Eastwood et al, 2015. In brief, we used piezoresistive cantilevers to deform the worm’s cuticle and to read out the respected indentation and force. Measurements were performed in displacement clamp. The cantilever had a spring constant kc of 1.3 N/m and a resonant frequency of 12.2 kHz. A glass microsphere (10 ± 1-μm diameter) was adhered to the cantilever tip. Cantilevers were mounted to a piezoelectric actuator. Because the device was placed directly above the worm’s surface before each experiment, sample indentation depth (z) was calculated as z = xt – xc,where xt is the total actuator displacement as measured by the built-in sensor on the actuator and xc is the cantilever deflection as measured by the piezoresistor, as described . Force was calculated as F= xt*kc. For each animal, we applied 15 displacement steps (0.5 – 12.5µm) in three replicates. The cantilever was positioned 154 ± 37 μm behind the pharynx. Data acquisition occurred with HEKA software and a sampling frequency of 10 kHz. Analysis was performed in Matlab.dpy-10(e128); N2. Strains available at the CGC.Extracellular saline: in mM: NaCl (145), KCl (5), MgCl2 (5), CaCl2 (1), Hepes (10), adjusted to pH 7.2 with NaOH. 8% agarose (wt/vol) dissolved in extracellular saline, pH adjusted to 8.01 with NaOH.CB128:


Mycobacterium tuberculosis (Mtb) infects macrophages and eventually leads to their death. Zhang et al. find that type I IFN signaling contributes to the death of Mtb-infected macrophages through an apparently novel mechanism. Mycobacterium tuberculosis (Mtb), the major cause of tuberculosis (TB). Once phagocytized, Mtb resists killing by macrophages, replicates inside them, and leads to their death, releasing Mtb that can infect other cells. We found that the death of Mtb-infected mouse macrophages in vitro does not appear to proceed by a currently known pathway. Through genome-wide CRISPR-Cas9 screening, we identified a critical role for autocrine or paracrine signaling by macrophage-derived type I IFNs in the death of Mtb-infected macrophages in vitro, and blockade of type I IFN signaling augmented the effect of rifampin, a first-line TB drug, in Mtb-infected mice. Further definition of the pathway of type I IFN–mediated macrophage death may allow for host-directed therapy of TB that is more selective than systemic blockade of type I IFN signaling.Macrophages help defend the host against  Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), appears to have coevolved with humans over at least 70,000 yr and is estimated to latently infect about a quarter of the global population. Mtb causes clinically active TB in ∼10 million people each year and kills an average of three people a minute ” locus . In C57BL/6 (B6) congenic mice carrying the “susceptible” allele of the Sst1 locus, heightened tissue damage has been related to increased production of IL-1 receptor antagonist (Macrophages produce type I IFNs in vitro when infected with Mtb and in vivo in Mtb-infected hosts . In mous)” locus  that impfections Preprinttagonist . HoweverMtb establishes chronic infection chiefly by parasitizing macrophages. After macrophages phagocytize the bacterium, Mtb inhibits maturation of the phagosome into acidified phagolysosomes , promotiMycobacterium marinum–infected zebrafish in vivo and M. marinum–infected macrophage cell lines in vitro without conforming to the current model of necroptosis , leads to death of the macrophages , but theTNFα is induced by mycobacterial infection in vitro and in vivo and plays a dual role, either protecting or harming the host . TNFα pl–like pseudokinase , RIPK3, and mixed-lineage kinase domainse MLKL; . Howeverse MLKL; . Consistse MLKL; , and GSKse MLKL; , a RIPK3se MLKL; .Recently, ferroptosis was reported to play an important role in Mtb-induced macrophage cell death . HoweverInhibitors of poly(ADP-ribose) polymerase 1, such as BMN 673, ABT-888, and rucaparib, are often used to inhibit parthanatos, a type of regulated cell death related to hyperactivation of poly(ADP-ribose) polymerase 1, a component of DNA damage response machinery . These tAutophagy can also contribute to cell demise in certain settings . HoweverCasp1−/− mice) were found not to be involved in Mtb-induced macrophage cell death , we identified 11 potential candidate genes whose deletion was protective . Among ttogether , suggest.7 cells  and confgher MOI .Ifnar1−/− mice was greatly delayed but not completely abolished compared with WT BMDMs, without a detectable difference in Mtb uptake or growth at either a low or high MOI , a component of the 6-mo regimen used for drug-sensitive TB. Prophylactic and ongoing weekly administration of anti-IFNAR1 mAb was complemented by a daily dose of 3 mg/kg Rif beginning on day 28 after infection. Although Rif alone had little effect on Mtb burden by day 37 , the combination of Rif with anti-IFNAR1 afforded a greater reduction in CFU in lungs and livers than Rif alone, suggesting the potential value of combination therapy .6 CFU  beginning on day 14 after infection, when the lung Mtb burden had reached ∼106 CFU . About t6 CFU . In thes6 CFU  and lowe6 CFU . There w6 CFU , but blo7 CFU. Mice in the control groups succumbed within 30 d, while anti-IFNAR1 significantly prolonged survival  was filtered through a 0.22-µm sterile unit with a polyethersulfone membrane, aliquoted, and frozen at −30°C. 2 d before Mtb infection, RAW264.7 cells were preadapted to DMEM supplemented with 2% FBS, 2 mM L-glutamine, 10 mM Hepes, pH 7.5, and 1 mM sodium pyruvate and maintained in this medium for the duration of infection unless otherwise stated.RAW264.7 , HEK293T , HT-1080 , and L929  were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 10 mM Hepes, pH 7.5, and 1 mM sodium pyruvate at 37°C in a 5% COg. The cells were centrifuged at 193 g for 8 min at room temperature , and the pellet was resuspended and cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 10 mM Hepes, pH 7.5, 1 mM sodium pyruvate, and 20% LCM at 37°C in a 5% CO2 incubator for 6–7 d. On the day of harvest, adherent cells were washed once with ice-cold PBS and incubated in cold PBS + 1 mM EDTA at 4°C for 10 min. Cells were dislodged by gentle pipetting, collected by centrifugation as above, resuspended in cold PBS, washed again the same way, and then cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 10 mM Hepes, pH 7.5, 1 mM sodium pyruvate, and 10% LCM.To prepare BMDMs, mouse femurs were flushed with DMEM and the cell-containing supernatant was collected after allowing debris to settle at 1 Casp1−/− (#032662), Gsdmd−/− (#032410), Stat2−/− (#023309), Casp1−/−Casp11−/− (#016621), and Cas9 knockin (#026179) mice  were purchased from The Jackson Laboratory. Casp11−/− mice were kindly provided by J. Blander. Cas9 knockin mice were mated with WT C57BL/6 mice, and the resulting offspring carrying one Cas9 allele (Cas9-het) were used for electroporation of gRNA amplicons. Female 129S2 mice (#476) were purchased from Charles River Laboratories, rested for 2 wk, and then infected at the age of 8 wk. All mice were housed in a specific pathogen-free facility. When infected with Mtb, mice were housed in a BSL3 vivarium. All mouse experiments were approved by and performed in accordance with requirements of the Weill Cornell Medicine Institutional Animal Care and Use Committee.WT C57BL/6 (#000664), WT C57BL/6N (#005304), LentiGuide plasmid (#52963) was purchased from Addgene. Plasmids with gRNA insertions were verified by Sanger sequencing. Mouse IFN-γ (#11276905001), TNFα (#T0157), CHX (#C4859), Rif (#R3501), Nec-1 (#N9037), Erastin (#E7781), RSL3 (#SML2234), and puromycin (#P8833) were purchased from Sigma. Mouse IFN-αA (#12100-1), mouse IFN-β (#12405-1), and Mouse IFN-β ELISA Kit, High Sensitivity (#42410-1) were from PBL Assay Science. BMN 673 (#A4153), ABT-888 (#A3002), rucaparib (#A4156), and z-VAD-FMK (#A1902) were from Apexbio. Fer-1 (#17729) was from Cayman Chemical. Smac mimetic birinapant (#501015095) was from Fisher Scientific.For neutralization on cells, mouse IgG1 κ isotype control (#16-4714-82) was from Affymetrix, and anti-mouse IFN α/β receptor I  was from Life Technologies. For use in mice, mouse monoclonal anti-human IFN-γ Rα chain  and mouse monoclonal anti-mouse IFNAR1  were purchased from Leinco Technologies. Antibodies for β-actin (#sc-47778) and RIPK3 (#sc-374639) were from Santa Cruz Biotechnology. Antibodies for cleaved caspase-3 , caspase-3 (#9662S), cleaved caspase-8 , caspase-8 (#4927S), phospho-RIP1 , MLKL (#37705S), and phospho-MLKL  were from Cell Signaling Technology. Phospho-RIPK3  and ATG16L1 (#ab187671) antibodies were from Abcam. RIPK1 antibody (#610459) was from BD Biosciences. Anti-FLIP  was from Adipogen Corporation.2 incubator. For infection of macrophages, Mtb cultures reaching an OD580 of 0.3–0.5 were centrifuged at 3,082 g, and the pellet was resuspended in PBS + 0.02% tyloxapol. The suspension was enriched for single cells by centrifugation at 123 g for 10 min with the brake set to 0. Mtb cell number was calculated by assuming 0.1 OD580 = 5 × 107 bacteria/ml. The required number of bacteria was centrifuged for 10 min at 3,082 g, resuspended in mammalian cell culture medium for infection of cells, and added to macrophages to initiate infection. 4 h after the addition of Mtb, macrophage monolayers were washed twice with PBS prewarmed to 37°C, and then fresh medium was replaced. No antibiotics were used at any time in the preparation, infection, or postinfection incubation of macrophages. For measuring extracellular bacterial burden, cell culture supernatant was removed for serial dilution and plating. For measuring intracellular bacterial burden, adherent cells in 24-well plates were washed twice with PBS and then lysed with 100 µl of 0.5% Triton X-100 in PBS at room temperature for 5 min. 400 µl PBS was then supplemented, and cellular content was mixed well by pipetting up and down. The lysates were used for serial dilution and plating on 7H11+glycerol+OADC agar plates.Mtb H37Rv was used in this study. Mtb was grown in Middlebrook 7H9 medium containing 0.5% glycerol, 10% oleic acid–dextrose–catalase  supplement, and 0.02% tyloxapol at 37°C in a 5% COCell viability was measured in two ways, as indicated in the figure legends. The viability of BMDMs was assayed by measuring ATP levels using a Promega CellTiter-Glo kit according to the manufacturer’s instructions. The viability of RAW264.7 cells was assayed using a Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay kit in the Total Cell Number Assay mode with slight modification. The remaining adherent cells were washed once with PBS, then lysed in the kit’s 1 × lysis buffer at 37°C for 45–60 min. An aliquot of the lysate was used for the lactate dehydrogenase enzymatic assay as instructed. Percentage viability was normalized to that of uninfected cells.g at 4°C for 5 min, and the supernatant was passed through a 0.22-µm cellulose acetate filter before being removed from the BSL3 facility. Neat samples were used for the IFN-β ELISA following the manufacturers’ instructions.At indicated times after Mtb infection of macrophages, cell culture supernatant was centrifuged at 21,130 7) stably expressing Cas9 protein were infected with the lentivirus library at an MOI of 0.3 with 8 µg/ml of polybrene. Cells were harvested 72 h later, reseeded at a density of 105 cells/ml, and treated with 5 µg/ml puromycin to remove noninfected cells. About 8 d later, the resulting mutant cells were harvested, and 3 × 108 cells were infected with Mtb at an MOI of 10 while another 108 cells served as control. 4 h after Mtb infection, cells were washed twice with prewarmed PBS to remove extracellular Mtb, and fresh medium was replaced. 5 d after infection, when ∼5% of the cells remained, the medium was replaced with fresh medium (10% FBS) containing 10 µg/ml Rif and 12.5 µM moxifloxacin to kill Mtb and allow surviving host cells to recover. After another 7 d, when cells were ∼90% confluent, cells were harvested and genomic DNA was prepared as described  followed the distributor’s guide. The lentivirus library was prepared as described . RAW264.escribed . The scrhttps://portals.broadinstitute.org/gpp/public/analysis-tools/crispr-gene-scoring) using the Negative Binomial Distribution (STARS) method with the threshold set to a standard 10%. Because we are only interested in the biological meaning of positive selection, we used the positive selection result to calculate the average score and average −log10 of three replicates. Candidate genes were determined based on the criterion that they were identified in at least two of the three independent experiments. The volcano plot of average −log10(p) versus average score was generated, and top hits were highlighted in red.The gRNA was amplified as in 5 RAW264.7 cells were transfected with 0.5 µg gRNA-expressing pSpCas9(BB)-2A-eGFP plasmid, and 48–72 h after transfection, eGFP-positive cells were sorted by flow cytometry on a BD Biosciences FACS Aria II or Influx sorter and cultured till confluency. Genomic DNA was prepared from a subset of cells, and gRNA efficiency was validated by SURVEYOR assay. Cells were then sorted singly into individual wells of 96-well plates. The genotype of each clone was verified by sequencing of the PCR fragments. The gRNA sequence for Ifnar2 is 5′-ACATAACAATACGAAATTCC-3′.The generation of CRISPR-Cas9 knockout cell lines was adapted from a published approach . 2 × 105Atg16l1-a: 5′-ACCGAACTGCACAAGAAGCG-3′; and Atg16l1-b: 5′-GGGTCTGGTTGGCTACCTCG-3′. If the intensity and purity of the PCR products were good, then the rest of the amplified DNA was purified using a Qiagen PCR purification kit and eluted with 200 µl double distilled H2O. 20 µl of 3 M NaAc (pH 5.2) followed by 500 µl ethanol was added to precipitate DNA at −80°C for at least 1 h. After centrifugation at 18,506 g at 4°C for 20 min, the DNA pellet was washed twice with 70% ethanol and allowed to air dry. DNA was then solubilized using 12 µl sterile double distilled H2O, and the concentration was checked using Nanodrop. The Mouse Macrophage Nucleofector Kit (Lonza) was used to electroporate 3 µg DNA into 2 × 107 freshly isolated bone marrow cells from Cas9-het mice on an Amaxa Nucleofector II machine following the manufacturer’s instructions. Electroporated cells were differentiated in 20% LCM-containing DMEM as above and harvested on day 7 for use.LentiGuide plasmids with the correct gRNA insertion were used as templates to amplify hU6 promoter-gRNA fragments in a 50 µl × 8 PCR reaction system using NEB Phusion polymerase. The following primers were used: forward: 5′-ACCCAGAGAGGGCCTATTTC-3′; reverse: 5′-CTGTCCCTGTAATAAACCCG-3′. The gRNA sequences used are negative control: 5′-GAACTCGTTAGGCCGTGAAG-3′ ; Atg16l12 incubator. Log phase cultures were centrifuged, and the pellet was resuspended in PBS + 0.05% Tween-80. A suspension enriched in single cells was prepared as above. The required volume was centrifuged for 10 min at 3,082 g, and the pellet was resuspended in PBS. This Mtb inoculum was used to infect mice using a Glas-Col Inhalation Exposure System.Mtb H37Rv was grown in Middlebrook 7H9 medium containing 0.5% glycerol, 10% albumin–dextrose–saline supplement, and 0.05% Tween-80 at 37°C in a 5% COThe schedule of administration of antibodies, Rif, or Veh is illustrated in the figures. Antibodies were administrated by intraperitoneal injection. For the first dose (2.5 mg), antibodies were diluted to 6.25 mg/ml in PBS, and 400 µl was injected. For later weekly doses, 0.5 mg of antibodies in 200 µl was injected. The volume of PBS injected as a control matched the volume used to administer antibodies.Rif was solubilized in Veh (0.5% carboxymethylcellulose and 0.25% Tween-80) by sonication. 200 µl Rif or Veh was delivered to mice by oral gavage once a day for 5 consecutive days/week.2, and lung homogenates in PBS were plated on 7H10 agar supplemented with 0.5% glycerol and 10% OADC. CFU were counted 3 wk later to determine the initial bacterial load. On indicated days, mice were euthanized with CO2, and lungs, livers, and spleens were harvested. Each organ (except the upper lobe of the left lung) was homogenized in PBS, serially diluted, and plated on 7H10 agar for determination of CFU at 3 wk. The upper lobes of the left lungs were fixed in 10% formalin.On day 1 after infection, two to five mice were euthanized with COt test) using Prism (GraphPad Software). Mouse survival assays were analyzed by log-rank (Mantel-Cox) test. P values were noted as follows: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.Results of cell viability assays, ELISAs, and bacterial growth assays in macrophages are represented as means ± SD. Mtb burden in mouse organs and changes in mouse body weight are represented as means ± SEM. Data were analyzed by two-tailed Mann-Whitney test of ranks (unpaired, nonparametric Table S1lists oligonucleotide sequences for the CRISPR-Cas9 screen.Click here for additional data file.


Sophora tonkinensis with various pharmacological activities. This study aims to evaluate its in vitro and in vivo inhibitory potential against the nine major CYP enzymes. Of the nine tested CYPs, it exerted the strongest inhibitory effect on CYP2C9-mediated tolbutamide 4-hydroxylation with the lowest IC50 (Ki) value of 0.966 ± 0.149 μM (0.503 ± 0.0383 μM), in a competitive manner. Additionally, it strongly inhibited other CYP2C9-catalyzed diclofenac 4′-hydroxylation and losartan oxidation activities. Upon 30 min pre-incubation of human liver microsomes with SPN in the presence of NADPH, no obvious shift in IC50 was observed, suggesting that SPN is not a time-dependent inactivator of the nine CYPs. However, oral co-administration of SPN had no significant effect on the pharmacokinetics of diclofenac and 4′-hydroxydiclofenac in rats. Overall, SPN is a potent inhibitor of CYP2C9 in vitro but not in vivo. The very low permeability of SPN in Caco-2 cells , which suggests poor absorption in vivo, and its high degree of plasma protein binding (>99.9%) may lead to the lack of in vitro–in vivo correlation. These findings will be helpful for the safe and effective clinical use of SPN.(‒)-Sophoranone (SPN) is a bioactive component of  Sophora tonkinensis, is used in traditional Chinese medicine for the treatment of acute pharyngolaryngeal infections and sore throat  is the drug concentration in the receiver chamber.The apparent permeability coefficient (Pn = 6) and (2) SPN and diclofenac co-administration (n = 6). SPN was suspended in dimethylsulfoxide:PEG400:distilled water  and administered by oral gavage at a dose of 75 mg/kg in a volume of 5 mL/kg. Fifteen minutes after oral administration of SPN, 2 mg/kg diclofenac was dissolved in normal saline and administered by oral gavage. Approximately 0.25 mL of blood from each rat was collected into an Eppendorf tube before diclofenac dosing (0 min), and at 3, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, and 480 min post-dosing. The blood samples were immediately centrifuged at 13,000× g for 5 min at 4 °C. The plasma samples were divided into two Eppendorf tubes by 50 μL and stored at −80 °C until LC-MS/MS analysis. After the experiments, the rats were euthanized with CO2.In this study, we investigated whether SPN, an in vitro potent inhibitor of CYP2C9, affects the pharmacokinetics of diclofenac in rats. Male Sprague–Dawley rats  were purchased from Orient Bio , and the protocol for pharmacokinetic interaction studies in rats was approved by the Institutional Animal Care and Use Committee (IACUC-CUK) at The Catholic University of Korea . The procedures used for housing and handling were previously reported . Before The plasma or liver microsomal protein bindings were performed using a rapid equilibrium dialysis device and cellulose membranes with a molecular weight cutoff of 8000  . The ratThe human liver microsomal incubation mixtures  without NADPH generating system were used to determine the unbound fraction of SPN. Other procedures were similar to those of plasma protein binding assay.18, 50 mm × 2.0 mm i.d.; 3 μm particle size; Phenomenex, Torrance, CA, USA) maintained at 40 °C. The mobile phase consisted of distilled water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B), with a flow rate of 0.5 mL/min. The gradient elution program used was as follows: (1) Mobile phase A was set to 95% at 0 min, (2) a linear gradient was run to 5% in 2.6 min, and (3) a linear gradient was run to 95% in 3.0 min and re-equilibrated for 2 min. The total run time was 5 min. The optimized compound-dependent parameters of the metabolites of the nine P450-selective substrates and the internal standard are listed in Metabolites of nine P450-selective substrates were analyzed using a Shimadzu Nexera X2 UPLC system coupled to an LCMS-8050 triple quadruple mass spectrometer  equipped with an electrospray ionization interface as previously described with a slight modification ,18. Sepam/z 312 > 231 and m/z 437 > 207.1 for quantification of 4′-hydroxydiclofenac and losartan carboxylic acid, respectively. HPLC conditions were the same as those in the cocktail assay.The auto-optimized mass transitions were 18 column . The isocratic mobile phase consisted of 0.1% formic acid in distilled water (A) and 0.1% formic acid in acetonitrile (B) , with a flow rate of 0.3 mL/min. The transitions were m/z 296.0 > 214.0 for diclofenac, m/z 312 > 231 for 4′-hydroxydiclofenac, and m/z 277 > 111 for the internal standard. The data acquisition was computed using LabSolutions LCMS Ver.5.6 (Shimadzu Corporation). The calibration curves for diclofenac and 4′-hydroxydiclofenac were linear (r ≥ 0.996) over the concentration range of 20–5000 ng/mL.The plasma concentrations of diclofenac and 4′-hydroxydiclofenac were determined by a previously reported LC-MS/MS method  with somr ≥ 0.995) over the concentration range of 1–250 ng/mL.The LC-MS/MS condition for the determination of SPN in plasma was the same with a previously reported method . The cal50 values were calculated via nonlinear least-squares regression analysis from logarithmic plots of inhibitor concentration versus percentage of activity remaining after inhibition, using SigmaPlot . The Ki values were determined from the equations for a single substrate single inhibitor model and the software available in the SigmaPlot Enzyme Kinetics module. Competitive, non-competitive, uncompetitive, or mixed inhibition models were evaluated and ranked according to the best fit based on Akaike Information Criterion (AIC) values. For visual inspection, the data were presented as Dixon plots.The IC∞) or the last measured time (AUCt). The logarithmic trapezoidal rule was used during the declining plasma level phase and the linear trapezoidal rule was used for the rising plasma-level phase. The peak plasma concentration (Cmax) and time to reach Cmax (Tmax) were read directly from the experimental data. Statistically significant differences were recognized at p < 0.05.Pharmacokinetic parameters were calculated by a non-compartmental analysis using WinNonlin Professional software  that used the total area under the plasma concentration–time curve from time zero to infinity  in human liver microsomes are shown in d values ,19,20,210.149 μM . SPN sho0.149 μM ; the res0.149 μM .50 values of 0.879 ± 0.0888 μM and 0.455 ± 0.0486 μM, respectively,  and found that SPN also markedly inhibited their activities, with ICctively, .50 value for CYP2C9, to characterize the type of reversible inhibition of CYP2C9 by SPN, enzyme kinetic experiments were performed in the presence of various concentrations of SPN and tolbutamide, or diclofenac. Otherwise, identical samples containing a known potent CYP2C9 inhibitor (sulfaphenazole), were included in the analysis. Representative Dixon plots of CYP2C9 inhibition by SPN and sulfaphenazole in human liver microsomes are shown in Ki values of 0.503 ± 0.0383 μM and 0.587 ± 0.0470 μM  with pre-incubation indicates time-dependent inactivation [50 was observed for inhibition of the nine CYPs  × 10−6 cm/s and (0.172 ± 0.0488) × 10–6 cm/s at 10 μM and 50 μM, respectively, . These results indicated that SPN is poorly absorbed in vivo. The Papp values from B-to-A were (0.101 ± 0.00444) × 10−6 cm/s at 10 μM (n = 3) and (0.152 ± 0.0353) × 10−6 cm/s at 50 μM (n = 3). SPN was not a substrate for efflux transporters, that is, P-gp and BCRP, as the efflux ratio (B-to-A/A-to-B) is less than 2. The Papp of propranolol, a reference high permeable compound, from A-to-B and B-to-A were (26.8 ± 3.31) × 10−6 cm/s and (21.5 ± 2.19) × 10−6 cm/s, respectively, , similar to the reported values [A bi-directional permeability assay using Caco-2 monolayer cells was performed to predict the intestinal absorption of SPN. SPN showed very low permeability in both directions (from A-to-B and B-to-A). The calculated Pd values ,25.S. tonkinensis herbs indicate that a recommended daily dose for an adult human with the body weight of 60 kg were to be 6–10 g [S. tonkinensis samples were found to be approximately 2.53 mg/g (0.0253%). Reflecting this content, the dosage in rats, 0.620–1.03 g/kg of the dried herb, might be consistent with 15.7–26.1 mg/kg in terms of SPN. Thus, in this study, the SPN dose of 75 mg/kg was used in rats, which is approximately 2.87- to 4.87-fold greater than the recommended human dose.We conducted pharmacokinetic studies to investigate the effects of SPN on the pharmacokinetics of diclofenac in rats. Findings in the literature on the dried e 6–10 g , which ce 6–10 g . He et ae 6–10 g  reportedp value of 0.215) in the presence or absence of SPN (max of SPN was found to be 33.7 ± 14.8 ng/mL (0.0732 ± 0.0321 μM) at approximately 60–75 min post-dose , the plasma concentrations of SPN are too low to inhibit CYP2C9-mediated metabolism of diclofenac in vivo. Overall, the co-administration of SPN did not alter the pharmacokinetics of diclofenac and 4′-hydroxydiclofenac.The mean plasma concentration-time profiles of diclofenac and 4-hydroxydiclofenac after oral administration of diclofenac (2 mg/kg) in the absence or presence of oral co-administration of SPN (75 mg/kg) in rats are illustrated in e of SPN . In the ost-dose C. Given n = 3, each). Similarly, when 10 and 50 μM SPN were added to the rat plasma, the free fractions were 0.0380 ± 0.0102% and 0.0531 ± 0.0149%, respectively, . After adding 10 and 50 μM SPN to rat and human plasma, free fractions remained relatively unchanged, suggesting that SPN has no binding saturation in plasma.SPN was extensively bound to plasma proteins, regardless of species. The free fractions (%) of SPN at 10 and 50 μM in human plasma were 0.0457 ± 0.00612% and 0.0927 ± 0.0400%, respectively, , at a microsomal protein concentration of 0.1 mg/mL.SPN also exhibited marked non-specific bindings to human liver microsomes, although to a lesser extent than those in human plasma. The unbound fractions of SPN at 10 and 50 μM were calculated to be 0.621 ± 0.0405% and 0.724 ± 0.170%, respectively  were 53.9 ± 3.53% and 53.3 ± 4.00%, respectively  in human liver microsomes. Of the nine tested CYP isoforms, SPN exerted the strongest inhibitory effect on CYP2C9 activity, with the lowest IC0.149 μM . In addiectively . Althougectively . No appaectively . SPN alsectively . The inhKi value of 2.17 ± 0.29 μM. They only focused on the in vitro inhibitory potentials of several flavonoids against CYP3A4 activity. To the best of our knowledge, to date, bufalin has not been used as the in vitro probe substrate for the CYP3A4 activity, and the reference material of 5′-hydroxybufalin is not commercially available. Because of the presence of several binding regions within the CYP3A4 active site, multiple probe substrates are often used for in vitro CYP3A4-mediated drug–drug interaction studies, including midazolam, nifedipine, and testosterone [50 values by SPN were reported to be 5.62–38.4 μM [50 value of 31.5 ± 4.79 μM, which showed a higher percentage inhibition compared to midazolam (data not shown). Altogether, the in vitro CYP3A4 inhibition by SPN seemed to be substrate-specific.The reversible inhibition of SPN-mediated CYP3A4 activity was less consistent with the published literature. Li et al.  reportedosterone . In that–38.4 μM . AdditioS-warfarin, phenytoin, tolbutamide, losartan, and several anti-inflammatory drugs [Generally, alterations in the activities of hepatic CYPs through in vitro inhibition or induction represent the major mechanisms underlying pharmacokinetic drug–drug interactions ,12,13. Iry drugs ,35. Cons1 value , which representing the predicted AUC ratio in the presence or absence of inhibitor. Where, Imax,u  is maximal free plasma concentration of the inhibitor and Ki,u is the unbound in vitro inhibition constant. However, little information is yet to be reported on the Cmax values of SPN after oral administration of SPN. As stated in the Introduction, from our previous study, the Cmax of SPN was reported to be 13.1 ng/mL in rats after oral dosing of 12.9 mg/kg SPN in rats [max of SPN was found to be 33.7 ± 14.8 ng/mL (0.0732 ± 0.0321 μM) at 60–75 min , respectively. Considering these values, the R1 value of SPN for the inhibition of CYP2C9 in vitro was calculated as 1.0124  or 1.0106  which are both below the EMA and US FDA cut-off criteria of R1, 1.02 [4′-hydroxydiclofenac/AUCdiclofenac, which indicated a causal factor for the evaluation of the capacities of CYP2C9 activity in vivo, did not show significant differences (0.799 ± 0.167 versus 0.904 ± 0.0534) in both groups  across the Caco-2 cell barrier has been shown [app values < 1–2 × 10−6 cm/s; moderate permeability (20–80% absorbed) to Papp values < 2–10 × 10−6 cm/s; and high permeability (80–100% absorbed) to Papp values > 10 × 10−6 cm/s [app value of (26.8 ± 3.31) × 10−6 cm/s in our assay. SPN exhibited a very low permeability with mean Papp values of 0.115 × 10−6 cm/s (0.429% of propranolol Papp) and 0.172 × 10−6 cm/s (0.642% of propranolol Papp) at 10 and 50 μM, respectively, indicating that it is poorly absorbed in vivo. SPN was not a substrate for efflux transporters, that is, P-gp and BCRP, as the efflux ratio (B-to-A/A-to-B) is less than 2.To explain the lack of in vitro–in vivo correlation, we assessed two factors that could limit the accuracy of in vitro models in predicting metabolic drug interactions in vivo, which were SPN’s degree of plasma protein binding and its permeability in Caco-2 cells. We found that SPN was extensively bound in both human and rat plasma proteins (>99.9%) with a mean unbound fraction value of 0.0574% in the range of 10 and 50 μM. Thus, taking the plasma protein binding of SPN into account, the unbound maximum concentrations of SPN in plasma might be 0.0420 ± 0.0184 nM, which is much lower than the unbound reported ,39,40. Treported . Montelu–0.15 μM . Howeveren shown ,25. A reOverall, SPN is a potent inhibitor of CYP2C9 in vitro but not in vivo. This apparent discrepancy is due to the extensive plasma protein binding and very low permeability of SPN, which resulted in poor oral absorption. These approaches could help in making more reliable in vitro–in vivo extrapolations about the risk of in vivo inhibition potential. In conclusion, these findings have provided useful information on the safe and effective use of SPN in clinical practice.


This study explores the feasibility of I2-IK-enhanced micro-CT to evaluate the surgical margin for tongue squamous cell carcinoma (TSCC) specimens and to further seek optimal staining parameters.A positive surgical margin (PSM) following oral cancer resection results in local recurrence and poor prognosis. Mono-block tumor specimens, especially from the tumor base, are difficult to evaluate. This inaccurate sampling ultimately leads to a false pathological diagnosis. Lugol’s iodine (Ivia I2-IK-enhanced micro-CT. The optimal I2-IK concentration and staining time were determined before clinical application using tissue shrinkage, micro-CT image quality, and effect on pathological diagnosis as assessment criteria. Next, 6 TSCC specimens were used to verify the process feasibility of surgical margin imaging with the optimal parameters. Finally, the possible reason by which I2-IK could enhance micro-CT imaging was validated in vitro.Rabbit tongue tissues and human TSCC samples were imaged 2-IK staining influenced specimen shrinkage, micro-CT image quality, and pathological image quality in a concentration- and time-dependent manner. After comprehensively considering these indicators, 3% I2-IK staining for 48 and 12 h were found to be optimal for rabbit tongue tissues and TSCC samples, respectively. This method could provide a detailed 3-D structure of TSCC samples compared with H&E sections. Moreover, tumor and normal tissues could be differentiated by their glycogen content, which has high affinity with I2-IK.I2-IK-enhanced micro-CT could, thus, indicate the tumor margin and assist pathological sampling in patients with TSCC postoperation.I Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide, especially in developing countries . SurgicaThe principle of en-bloc resection has been standard in surgical oncology. Surgical margins on mono-block specimens after tumor resection need to be evaluated for solid organ cancers. In fact, the positive surgical margin (PSM) rate for oral cavity cancer has been reported to be 12.75%—the highest overall PSM rate among all-site tumors . TherefoSerial whole-mount sectioning is an effective way to examine mono-block specimens; it is also helpful to decide reasonable treatment options based on the NCCN guidelines. However, this is impractical for patients with TSCC and for clinicians because of its high cost and time consumption . Near-in2-IK)-enhanced micro-CT is an emerging method to solve this dilemma as it can show the fine anatomic structure of various tissue samples in various animals  sections  were purchased from the Comparative Medical Center of Yang Zhou University. After the rabbits were euthanized, a total of 27 tissues from the front of the tongue were cut off; 15 of these were used for I2-IK-enhanced micro-CT imaging. The remaining 6 samples from 2 patients were used to further verify the feasibility of I2-IK enhanced micro-CT to evaluate the status of the surgical margin in TSCC.For human TSCC sample acquisition, this study was first approved by the medical ethics committee of the Institute Affiliated Stomatology Hospital, the Nanjing University Medical School. Then, 21 samples were acquired from 3 patients with TSCC. The samples were acquired from the junction between the tumor and normal tissues. All 3 patients provided a signed informed consent form. Fifteen samples from the same patient were used to determine the optimal parameters for I2-IK  was purchased from Shandong lvying Chemical Technology Co. Ltd., China. Various concentrations including 1%, 3%, 5%, and 7%  of I2-IK were prepared by diluting the I2-IK mother liquor with 4% formalin solution. All I2-IK solutions were preserved at room temperature away from light. After fixation with formalin for 12 h, both rabbit tongue tissues and human TSCC samples were stained with different concentrations of I2-IK for 72 and 48 h, respectively, in 10-ml brown bottles. During the staining period, these samples were scanned by micro-CT every 12 h.IA small animal micro-CT  was used for all CT imaging. The micro-CT scan parameters were as follows: power: 8 W, voltage: 60 V, electric current: 133.3 μA, detector mode: binging 2*2, slice thickness: 50 μm, and repetition time: 75 ms. All CT image data were processed using the SeProcessPro Version.1 software . Contrast-to-noise ratio (CNR) , which r2-IK-enhanced micro-CT imaging. Then, a series of 4-µm sections were prepared according to the CT image data. For H&E staining, all sections were stained using an automatic pathological section staining machine. For pan-CK IHC staining, paraffin sections were deparaffinized and dehydrated using a series of graded ethanol solutions. For antigen retrieval, the sections were heated in a microwave oven with a 10-mM citrate buffer solution (pH = 6) for 10 min. Endogenous peroxidase activity was quenched by incubating the sections in 0.3% H2O2 for 5 min. After blocking with 3% bovine serum albumin for 1 h at room temperature, the sections were incubated with anti-pan-CK  primary antibodies overnight at 4°C and gently washed three times with PBS. Sample sections were then incubated with goat-antimouse secondary antibody  for 2 h at room temperature. The signal was developed using the HRP substrate 3, 3’-diaminobenzidine (DAB). Nuclear counterstaining was performed using hematoxylin.All rabbit tongue tissues and human TSCC samples were embedded in paraffin using the standard method after IThe quality of H&E sections was scored by two experienced pathologists in a blinded manner according to the Chinese standards for the quality control of pathological sections. The scoring criteria are shown in p = 0.829) . Before H&E staining, the maximum area of the tissues was calculated. Then, the maximum tissue sections were cut and used for the subsequent staining process. After H&E staining, we calculated the area of the pathological section.5 SCC9high glucose and SCC9low glucose cells in 5 ml were seeded on 6-well plates and incubated overnight under a 5% CO2 atmosphere at 37°C. Next, the treated cells were washed twice with PBS and fixed in a formalin solution for 30 min. Then, the treated cells were incubated with various concentrations of I2-IK  for 1 h. Finally, the treated cells were observed by optical microscopy. The staining intensity was calculated using Image J .The human TSCC line SCC9 was purchased from Procell Life Science & Technology Co., Ltd. . Cells were cultured in DMEM/high-glucose medium and DMEM/low-glucose medium, respectively, supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin, and 1% (v/v) streptomycin for 1 week. Then, 5×10Six tumor tissues, muscle tissues, and mucosal tissues from patients with TSCC were acquired to determine the glycogen content using the liver/muscle glycogen assay kit .2-IK. One-way ANOVA was used to compare the volume shrinkage and quality of pathological sections with different concentrations of I2-IK. Student’s t-test was used to compare the glycogen content between tumor tissues, muscle, and mucosa. P < 0.05 was considered statistically significant.Statistical analysis was performed using the SPSS statistical software . Values are presented as mean ± standard derivation (SD). Multivariate repeated-measures ANOVA was used to compare the CNR and volume shrinkage of tissue blocks with different concentrations of I2-IK concentration . The mean volume of the fresh tissue blocks was 0.3867 cm3 ± 0.0284 cm3. Before I2-IK staining, all 15 blocks of tissues were fixed with formalin solution for 12 h, and the mean volume shrinkage was 22.3% ± 2.6% , whereas 1% and 3% I2-IK staining for 72 h had little influence compared with that in the control group (P > 0.05) . The H&E imaging quality and the area alteration of tissue sections (4 μm) were analyzed to evaluate the influence of I2-IK for 48 h was the optimal staining parameter for micro-CT imaging of rabbit tongue tissues.After comprehensive consideration of the above-mentioned evaluation indicators, we could take a preliminary view that 3% I2-IK concentrations and staining time on the volume change of TSCC samples, 12 samples were acquired and fixed in formalin solution for 12 h. The mean volumes of fresh samples were 0.8966 ± 0.0628 cm3, and the tissue after formalin fixation shrinkage was 16.42% ± 2.93%  for 48 h. During the staining period, the samples were scanned by micro-CT every 12 h, and the volumes were determined by 3-D reconstruction. In detail, the mean volume changes in the 1% group were -13.7% ± 2.0%, -16.9% ± 1.8%, -18.4% ± 1.8%, and -20.1% ± 1.8%, respectively; the mean volume changes in the 3% group were -13.5% ± 4.8%, -17.4% ± 4.8%, -19.4% ± 4.4%, and -20.8% ± 3.9%, respectively; the mean volume changes in the 5% group were -19.3% ± 2.0%, -24.5% ± 1.6%, -27.1% ± 1.9%, and -28.6% ± 1.7%, respectively; and the mean volume changes in the 7% group were -20.5% ± 3.5%, -26.3% ± 2.2%, -28.6% ± 2.4%, and -30.5% ± 2.4%, respectively  , phosphotungstic acid (PTA), and osmium tetroxide are the commonly used contrast agents at present. Among these, I2-IK is the most widely used for its advantages of low cost, convenient use, nontoxicity, and so on  technology, the combination of deep machine learning and a biomedical image processing algorithm like texture matching and nonrigid registration algorithms may provide a perfect solution  treatment, tissue penetration of doxorubicin into the peritoneal tissue improved by 3.8-fold , the Jiangsu Province Science & Technology Department (BE2018618), and the Nanjing Medical Science and Technique Development Foundation (QRX17174).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Platelet activation plays a key role in cardiovascular diseases. The generation of mitochondrial reactive oxygen species (ROS) has been described as a critical step required for platelet activation. For this reason, it is necessary to find new molecules with antiplatelet activity and identify their mechanisms of action. Mitoquinone (MitoQ) is a mitochondria-targeted antioxidant that reduces mitochondrial overproduction of ROS. In this work, the antiplatelet effect of MitoQ through platelet adhesion and spreading, secretion, and aggregation was evaluated. Thus MitoQ, in a non-toxic effect, decreased platelet adhesion and spreading on collagen surface, and expression of P-selectin and CD63, and inhibited platelet aggregation induced by collagen, convulxin, thrombin receptor activator peptide-6 (TRAP-6), and phorbol 12-myristate 13-acetate (PMA). As an antiplatelet mechanism, we showed that MitoQ produced mitochondrial depolarization and decreased ATP secretion. Additionally, in platelets stimulated with antimycin A and collagen MitoQ significantly decreased ROS production. Our findings showed, for the first time, an antiplatelet effect of MitoQ that is probably associated with its mitochondrial antioxidant effect. The association between cardiovascular diseases (CVD) development and mitochondrial damage is well known . This mi+); that is the lipophilic cation that stores several hundred-fold in the mitochondria [Mitoquinone decyl triphenylphosphonium) is the pioneer molecule specifically designed to decrease mitochondrial oxidative stress that has been evaluated in clinical trials ,12. Mitochondria ,16. One chondria ; as overchondria .MitoQ is attached to the matrix-facing surface of the inner mitochondrial membrane (mainly in the hydrophobic core of the membrane) that is determined by the membrane potential, and is constantly reprocessed to ubiquinol by complex II of the respiratory chain . The actp < 0.001; p < 0.001; p < 0.001). Similarly, it was observed that MitoQ 10 μM (8.5% ± 2.2%) induced a significant increase in PS exposure on the platelet membrane when compared to the basal control (p < 0.05), which suggests that MitoQ 10 μM could induce apoptosis on platelets. Meanwhile, in MitoQ at 1 and 5 μM there was no significant increase in PS on platelets . Under these conditions, MitoQ 1 and 5 μM significantly decreased platelet deposition on collagen surfaces , respectively. MitoQ did not affect TRAP-6-induced P-selectin expression. Similarly, MitoQ 2.5 and 5 μM attenuated the effect of collagen-induced CD63 (35.9% ± 3.5%) expression to 21.6% ± 5.1% and 5.7% ± 2.6%, respectively (p < 0.05). Furthermore, MitoQ 5 μM significantly decreased the expression of CD63 stimulated by convulxin (52.1% ± 11.5%) and PMA (92.2% ± 2.4%) with values of 34.3% ± 3.4% and 72.4% ± 17.3%, respectively (p <0.05). Meanwhile, MitoQ did not affect TRAP-6 stimulated CD63 expression (p < 0.001). Additionally, the platelet antiaggregant effect of MitoQ was confirmed on increasing collagen concentrations (p < 0.001), but with lower potency regarding collagen. In the activation with TRAP-6 5 μM (88% ± 4.74%) the antiaggregant effect was only observed by MitoQ at 5 μM . Finally, the aggregation by PMA 100 nM (85.7% ± 2.9%) was only significantly decreased by MitoQ 5 μM . Similarly, MitoQ inhibited collagen and ADP-induced platelet aggregation in PRP samples . Likewise, collagen-induced platelet ATP secretion was inhibited by MitoQ 2.5 and 5 μM . In MEF cells, treatment with antimycin A produced an increase in superoxide, measured by the Mitosox probe (fold change 1.45 ± 0.31), which was blocked by the addition of MitoQ 0.1 µM   . Besides < 0.05) In this article, the antiplatelet activity of MitoQ is reported for the first time. To evaluate compounds in platelets, it was necessary to study whether they had the cytotoxic capacity and to determine concentrations that adequately demonstrate their effect. The most common methods are centered on the quantification of the cytoplasmic activity of enzymes released by impaired cells and platelet viability by labeled with calcein-AM ,24. In tThe activation of platelets comprises of different steps, such as adhesion, spreading, secretion, and aggregation . Here itMitoQ induces important protection against oxidative stress-mediated mitochondrial dysfunction and apoptosis . The eliSince antimycin A inhibited collagen-induced platelet aggregation and secretion , the antv/v) and centrifuged at room temperature (RT) for 10 min × 200 g in order to collect platelet-rich plasma (PRP). Then PRP was centrifuged for 8 min × 900 g. Platelets pellet was suspended in calcium-free Tyrode’s buffer: ACD (proportion 5:1 v/v) and this was centrifuged again for 8 min × 900 g and platelets were resuspended in calcium-free Tyrode’s buffer and adjusted to the assays by a hematological counter  and used within 3 h for MitoQ  assays.Platelets were obtained by phlebotomy from healthy donors (two weeks without drugs) that had previously accepted informed consent, as previously reported . Briefly6 platelets/mL) were incubated with MitoQ  for 10 min at 37 °C. Ethanol 0.5% was used as a vehicle for MitoQ. After that, viability was determined by Accuri C6 flow cytometer  using calcein-AM [g for 8 min and evaluated with lactate dehydrogenase (LDH) cytotoxicity kit  [Washed platelets  . A solut6 platelets/mL) were preincubated for 5 at 37 °C min with CaCl2 (2 mM), and MitoQ  or positive control. The Annexin V positive control used was a combination of agonists TRAP-6 5 µM + Collagen 1 ug/mL. Then, 50 μL of the sample was diluted with 150 μL of annexin V binding buffer and incubated for 5 min. Finally, 30 µL of platelets were labeled with 2 µL of FITC Annexin V antibody and incubated in the dark for 30 min. The samples were acquired and analyzed in the Accuri C6 flow cytometer .The externalization of PS in platelets was determined by the Accuri C6 flow cytometer . Washed 6 platelets/mL) were previously incubated with CaCl2 (2 mM) and MitoQ  for 5 min at 37 °C. Following this, platelet aggregation was stimulated by collagen 1 µg/mL, convulxin 5 ng/mL, TRAP-6 5 µM or phorbol 12-myristate 13-acetate (PMA) 100 nM and was measured for 5 min at 37 °C. ATP secretion was detected using the chronolume reagent.Platelet aggregation and ATP secretion were evaluated by light transmission and lumi-aggregometry, respectively  . Washed The effect of MitoQ  on P-selectin and CD63 platelet expression were measured by the Accuri C6 flow cytometer in washed platelets incubated with anti-CD62-PE or anti-CD63-PE for 30 min at RT. Platelets were identified with anti-CD61-FITC .Washed platelets were preincubated with rotenone 20 μM plus adenosine 5′-diphosphate (ADP) 0.2 μM and were evaluated on collagen surface platelet deposition and platelet diameter using fluorescence microscopy . For this, a collagen film was prepared by heat fixation (37 °C) on a slide . Then, 10 µL of platelet suspension previously incubated with the anti-CD61 FITC antibody was added to the collagen film. For platelet deposition, an objective 63×, in vivo camera system with the filter of the COLIBRI system at 470 nanometers (blue light), and 20% intensity was used. Nine images were randomly selected for each condition and fluorescence images were analyzed using ImageJ software . The platelet diameter was measured using the ZEISS ZEN imaging software. Using an increase of 200%, the horizontal diameter of 20 (random) platelets arranged in the field was measured in microns (µm).6 platelets/mL) using dihydroethidium (DHE) 10 µM [ROS production was determined in washed platelets (50 × 10E) 10 µM . The labE) 10 µM .6 platelets/mL) and a control of mitochondrial depolarization carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) 1 μM was used [Mitochondrial membrane potential was determined using the potentiometric probe tetramethylrhodamine, methyl ester, perchlorate (TMRM) 100 nM, and analyzed by the Accuri C6 flow cytometer. MitoQ was evaluated at 1, 2.5 and 5 µM on washed platelets .p values < 0.05 were considered significant.The obtained results were presented as the mean ± standard deviation (SD) and analyzed using Prism 8.0 software . Differences between three or more conditions were analyzed using ANOVA, and subsequently analyzed by Tukey’s post-hoc test. In this study, an antiplatelet effect of MitoQ via inhibition of platelet adhesion and spreading, secretion, and aggregation has been described for the first time and this may be associated with its mitochondrial antioxidant effect. However, further research studies on animal models and in human clinical trials are needed to confirm the antiplatelet mechanism of MitoQ, which will evaluate potential applications  and therapeutic potentials.


Perovskite semiconductors hold a unique promise in developing multijunction solar cells with high-efficiency and low-cost. Besides design constraints to reduce optical and electrical losses, integrating several very different perovskite absorber layers in a multijunction cell imposes a great processing challenge. Here, we report a versatile two-step solution process for high-quality 1.73 eV wide-, 1.57 eV mid-, and 1.23 eV narrow-bandgap perovskite films. Based on the development of robust and low-resistivity interconnecting layers, we achieve power conversion efficiencies of above 19% for monolithic all-perovskite tandem solar cells with limited loss of potential energy and fill factor. In a combination of 1.73 eV, 1.57 eV, and 1.23 eV perovskite sub-cells, we further demonstrate a power conversion efficiency of 16.8% for monolithic all-perovskite triple-junction solar cells. Integrating several different perovskite absorber layers in a multi-junction solar cell imposes a great processing challenge. Here, the authors demonstrate a versatile two-step solution process for fabricating monolithic all-perovskite triple-junction solar cells. The record power conversion efficiency (PCE) of single-junction perovskite solar cells (PSCs) has now increased to 25.2%, approaching the state-of-the-art inorganic PV cells of 29.1% and the Shockley-Queisser (S-Q) efficiency limit of ~33%7. Further increase of efficiency of PSCs alongside high-throughput and low-cost manufacturing processes provides enormous potential for commercializing perovskite PV technologies8.Over the last decade, hybrid perovskites have been under the spotlight of the photovoltaic (PV) research community for their excellent optoelectronic characteristics, cost-effectiveness as well as solution processability9. By strategically stacking two or more light-absorbing layers with complementary bandgaps, monolithic multijunction solar cells can effectively mitigate these losses and raise the theoretical efficiency limit to 68%11. In practice, III–V crystalline semiconductors have demonstrated high PCEs of 39.2% and 37.9% in a six- and triple-junction solar cell, respectively, however their intricate and costly deposition processes prohibit large-scale applications13. Alternative technologies such as inexpensive organic semiconductors have also been exploited for multijunction solar cells9. Nevertheless, given the lack of comparably high-performing organic absorber layers over a wide range of bandgaps, suboptimal PCEs of 17.4% for tandem14, 11.6% for a triple cell15, and 7.6% for a quadruple-junction cell16 have been reported in such multijunction approach. Perovskite semiconductor, by virtue of its cost-effectiveness and widely tunable bandgaps17, holds a unique promise for the development of all-perovskite multijunction solar cells. The bandgap of Pb-based perovskite can be continuously tuned from 1.5 eV to 2.3 eV by substituting I with Br19, and bandgaps as narrow as 1.2 eV are obtained when mixing Pb- with Sn-based compounds21. Device and optical modeling23 have suggested that a monolithic tandem with 1.8 eV wide-bandgap and 1.2 eV narrow-bandgap perovskite materials can reach a feasible PCE of 33.4%. Moreover, the monolithic all-perovskite triple-junction solar cell comprising 2.0 eV, 1.5 eV, and 1.2 eV absorbers leads to an even higher PCE of 36.6%22. To date, tremendous research effort has been made for all-perovskite tandem devices29, with PCEs up to 24.8% achieved by Tan and co-workers30. In comparison, all-perovskite triple-junction solar cells remain largely unexplored, with only a proof-of-concept 6.7% triple cell demonstrated by Snaith and co-workers31.Fundamentally, the PCE of single-junction solar cells is limited by the thermalization loss of photons with energy higher than the bandgap and the transmission loss of photons with energy lower than the bandgap8. Between series-connected sub-cells, the interconnecting layers (ICLs) should serve as a physical barrier to protect the underlying layer from solvents used for the subsequent layers26. Also, reflective losses and parasitic absorption of the ICLs need to be minimized such that more low-energy photons can reach the next narrower-bandgap absorbing layers10. Besides, the ICLs should possess a large sheet resistance while retaining sufficient mobility such that selected holes and electrons from the adjacent perovskite layers can recombine efficiently27. For all-perovskite multijunction cells, examples of solution-processed ICLs are p-doped cross-linked poly(triarylamine) (PTAA)/n-doped phenyl-C61-butyric acid methyl ester (PCBM)32 and poly:polystyrene sulfonate (PEDOT:PSS)/indium tin oxide (ITO) nanoparticles31. In comparison, using sputtered ITO and indium zinc oxide (IZO) as the recombination layers has enabled higher efficiency in tandem devices28, albeit the increased lateral shunt pathways and optical losses in the near-infrared region27. Moreover, a thin metal oxide layer such as SnO2 and Al-doped ZnO (AZO) prepared by atomic layer deposition (ALD) was necessary to prevent sputter damage28. It has been previously demonstrated that tuning the growth conditions can yield compact and conductive ALD layers, which alone prevent solvent damage and allow for fast charge transport after depositing a thin TCO layer34. Recently, Tan and co-workers30 utilized TCO-free ICLs based on C60/ALD-SnO2/Au/PEDOT:PSS for all-perovskite tandem solar cells. Nevertheless, conventional ALD technique requires vacuum and is limited by a low deposition rate. Atmospheric pressure spatial-ALD (SALD) can be done at a much higher deposition rate while preserving conformal and pinhole-free depositions, which is closer to the industrial manufacturing requirements38. Furthermore, to maximize the performance of multijunction solar cells, stringent bandgap and thickness optimizations are needed to balance light absorption and match current density among sub-cells12. However, integrating several very different perovskite layers in such a complex multijunction cell could impose significant processing challenges, as they typically require very specific film formation strategies to achieve high efficiencies in single-junction solar cells39. To this end, using a simple and yet effective fabrication method suitable for various perovskite compositions and bandgaps would greatly benefit the development of all-perovskite multijunction solar cells.It is not trivial to fabricate monolithic all-perovskite multijunction solar cells, bearing in mind the optical and electrical losses inherent to the complex cell design and the processing and compatibility issues encountered in depositing widely different materials on top of each other2/PEDOT:PSS, we achieve PCEs of above 19% for monolithic all-perovskite tandem solar cells consisting of 1.73 eV and 1.23 eV absorber layers. Following the same strategy, we demonstrate efficient and reproducible all-perovskite triple-junction solar cells combining 1.73 eV, 1.57 eV, and 1.23 eV absorber layers. The best-performing triple-junction device shows a very promising PCE of 16.8%, with a short-circuit current density (Jsc) of 7.4 mA cm–2, an open-circuit voltage (Voc) of 2.78 V, and a fill factor (FF) of 0.81.Here we present a versatile two-step solution process for high-quality perovskite thin films. With only minor changes in the processing conditions, we fabricate efficient single-junction PSCs with bandgaps of 1.73 eV, 1.57 eV, and 1.23 eV. Through optimization of the ICLs based on fullerene/spatial ALD (SALD) grown SnOz(FA0.66MA0.34)z1–Pbx1−SnxIy(1–z)3–Bry(1–z) . By changing the molar ratio of precursor solutions, perovskites based on wide bandgap Cs0.1(FA0.66MA0.34)0.9PbI2Br, medium (mid) bandgap FA0.66MA0.34PbI2.85Br0.15, and narrow bandgap FA0.66MA0.34Pb0.5Sn0.5I3 are obtained. In a two-step deposition route, inorganic salts  dissolved in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) are first spin-coated to obtain an intermediate precursor film, on which organic salts  dissolved in isopropanol are spin-coated and followed by thermal annealing to accelerate the transition to perovskite crystals  spectra indicate bandgaps of 1.73 eV for Cs0.1(FA0.66MA0.34)0.9PbI2Br, 1.57 eV for FA0.66MA0.34PbI2.85Br0.15, and 1.23 eV for FA0.66MA0.34Pb0.5Sn0.5I3 perovskite absorbers, respectively  diffraction peak towards higher angle is consistent with a decrease in the cubic lattice constant from 6.324 Å to 6.303 Å for narrow bandgap FA0.66MA0.34Pb0.5Sn0.5I3 (Sn incorporation), and to 6.194 Å for wide bandgap Cs0.1(FA0.66MA0.34)0.9PbI2Br  images reveal a compact and pinhole free surface morphology for all perovskite films, with an average grain size span from ~250 nm for both wide and mid bandgap absorbers, to ~500 nm for narrow bandgap perovskite /Ag design was used for narrow bandgap PSCs. All perovskite layers are 400–450 nm thick. Figure J–V) curves and external quantum efficiency (EQE) spectra of representative PSCs with various perovskite bandgaps. The corresponding PV parameters are summarized in Table Jsc of 17.3 mA cm–2, a Voc of 1.13 V, and an FF of 0.74. In comparison, the device based on 1.57 eV mid bandgap exhibits an increased Jsc of 21.1 mA cm–2, a decreased Voc of 1.08 V, and an FF of 0.80, resulting in a PCE of 18.3%. The changes in Jsc and Voc are attributed to the decreased perovskite bandgap, in line with the redshifted EQE onset from 720 nm to 810 nm. As expected, further decreasing the bandgap to 1.23 eV leads to a higher Jsc of 28.0 mA cm–2, a lower Voc of 0.78 V, and an FF of 0.73, yielding a PCE of 15.9% for the narrow bandgap PSC. Meanwhile, all the devices show low hysteresis between reverse, forward, and stabilized J–V scans /PEDOT:PSS layers were used as the ICLs. Similar to previous reports33, we found that the SALD-SnO2 deposited on a fullerene layer can significantly reduce H2O and DMF permeation, which prevents damage to the wide bandgap perovskite film caused by the solution processing of PEDOT:PSS as well as narrow bandgap perovskite layers /SALD-SnOlls Fig. . The cor60 layer (20 nm), which shows comparable PCE in single-junction PSCs , which is caused by the change in optical interference after replacing the PCBM by C60  at the ALD-SnO2/PEDOT:PSS interface, similar to a previous study30. As shown in Fig. 60/SALD-SnO2/Au/PEDOT:PSS ICLs exhibits an improved PCE of 19.7% under reverse scan, thanks to a higher FF of 0.82, together with a Jsc of 12.7 mA cm–2, and a Voc of 1.91 V. Notably, the extra Au layer reduces transmission in the near-infrared, which further reduces the Jsc in the 1.23 eV back sub-cell  by a thermally evaporated Ccan Fig. . In thisC60 Fig. . On the ell Fig. .J–V measurement, the tandem shows a PCE of 19.2% (6.76 mm2 aperture area), with negligible hysteresis between reverse and forward scans  and 1.23 eV back sub-cell (0.78 V). This low potential energy loss, together with a high FF of 0.81, implies the fast recombination of charges from adjacent sub-cells in the ICLs27. As discussed, our tandem cell performance is limited by the low Jsc. Compared to a Jsc of 28.0 mA cm–2 obtained for 1.23 eV single-junction PSC, the summed EQE spectrum of both sub-cells is generally lower in the near-infrared range and shows a current density of 26.8 mA cm–2  and ITO substrate, reflectance, optical interference, and insufficient 1.23 eV absorber layer thickness for light absorption 0.9PbI2Br/C60/SALD-SnO2/Au/PEDOT:PSS/PTAA/FA0.66MA0.34PbI2.85Br0.15/C60/SALD-SnO2/Au/PEDOT:PSS/FA0.66MA0.34Pb0.5Sn0.5I3/C60/BCP/Ag. In such a device stack, the 1.73-eV perovskite absorber was further reduced to ~100 nm to approach a current matching condition , with a Jsc of 7.4 mA cm–2, a Voc of 2.78 V, and an FF of 0.81. The triple cell performance is higher than that of single-junction PSCs prepared in the same batch  of the triple device is close to the summed Voc value (2.86 V) of the corresponding single-junction PSCs, suggesting the effectiveness of such ICLs in our triple-junction design. Furthermore, a statistical summary of eight triple cells illustrates a narrow distribution of PCEs, which demonstrates the good reproducibility of our approach to fabricate triple-junction cells , the summed EQE of all three sub-cells in a triple device (24.7 mA cm–2) suggests a considerable loss in the near-infrared, which accounts for a loss in photocurrent of 2.3 mA cm–2  and good photo-stability would significantly advance high-efficiency all-perovskite triple-junction solar cells.Under the current design, the PCE of our triple-junction solar cell is limited by the Voc of all three sub-cells (Voc = 2.78 V) and low-resistivity loss (FF = 0.81). Further improving the performance of ~2 eV wide bandgap perovskite, will be essential for the future development of high-efficiency all-perovskite triple-junction solar cells. During the submission process, we became aware of a related work on multi-junction perovskite solar cells42.In summary, we have reported a universal two-step solution process to fabricate PSCs with bandgaps of 1.73 eV, 1.57 eV, and 1.23 eV. By optimizing solvent-robust and low-resistivity ICLs, PCEs of above 19% are achieved in monolithic all-perovskite tandem solar cells with 1.73 eV and 1.23 eV absorbers. Furthermore, our strategy enables us to fabricate efficient and reproducible monolithic all-perovskite triple-junction solar cells. A triple device comprising a 1.73 eV, 1.57 eV, and 1.23 eV sub-cells shows a promising PCE of 16.8%, with very low potential energy drop of only 80 mV in comparison to the summed 0.1(FA0.66MA0.34)0.9PbI2Br, 876 μL DMF (99.8%) and 86.4 μL DMSO (99.9%) were sequentially added to 34.8 mg CsI  and 553 mg PbI2  to form a ~1.25-M precursor solution, before stirring at 60 °C overnight. The small portion of DMSO was added to form a 1:1 molar ratio of PbI2:DMSO. The order of solvent addition does not change the device performance. In all, 34.9 mg FABr (Greatcell Solar) and 16.0 mg MABr (Greatcell Solar) were dissolved in 1 ml isopropanol . Thinner Cs0.1(FA0.66MA0.34)0.9PbI2Br films were prepared by reducing the precursor concentrations. For a 300-nm-thick layer, 1.1 M CsI-PbI2 and 0.297 M FABr-MABr solutions were used. For a 100-nm-thick layer, 0.4 M CsI-PbI2 and 0.095 M FABr-MABr solutions were prepared. For the 1.57 eV FA0.66MA0.34PbI2.85Br0.15 precursor solutions, 553 mg PbI2 was dissolved in 876 μL DMF and 86.4 μL DMSO. 54.0 mg FAI (Greatcell Solar), 14.3 mg MAI (Greatcell Solar), and 7.6 mg MABr were dissolved in 1 ml isopropanol (0.471 M). All the solutions above were kept at 60 °C overnight. For the 1.23 eV FA0.66MA0.34Pb0.5Sn0.5I3 precursor solutions, 276.5 mg PbI2 and 223.4 mg SnI2  mixture were dissolved in 876 μL DMF and 86.4 μL DMSO, and 10 mol% SnF2  was added with respect to SnI2. The solution was stirred at 60 °C for 1 h and filtered by a PTFE syringe filter (0.22 µm). In all, 53.5 mg FAI and 25.6 mg MAI mixture were dissolved in 1 mL of isopropanol and stirred at 60 °C for 1 h. We note that all materials except PEDOT:PSS and PTAA (stored in ambient) were stored in a dry N2 glovebox. While it was crucial to weigh Sn-containing compounds in a dry N2 glovebox, other compounds showed little influence on the device performance when weighed either in dry N2 or in an ambient atmosphere. All solvents for spin coating were stored in an N2 glovebox. All solutions were prepared and stirred in the same glovebox and were cooled to room temperature before use.All materials were purchased from commercial sources and used as received unless stated otherwise. For 1.73 eV Cs–1 of PTAA in toluene  was spin-coated onto the ITO substrate at 5700 rpm for 30 s and annealed at 100 °C for 10 min in the N2 glovebox. After cooling down, the Pb-containing precursor solution was spin-coated at 3000 rpm (with a 2000 rpm/s acceleration) for 30 s, followed by the dynamic spin-coating of FA/MA-based precursor solution at a speed of 3000 rpm for 30 s. The substrate was immediately annealed at 100 °C for 30 min. For the devices using PCBM as the electron transport layer (ETL), 20 mg mL–1 of PCBM  in 1 mL chlorobenzene  and chloroform  mixture (1:1 volume ratio) was spin-coated at 1000 rpm for 60 s, followed by thermal annealing at 100 °C for 30 min. Finally, 1 nm LiF (0.2 Å/s) and 100 nm Al (2 Å/s) were thermally evaporated to complete the device fabrication. For devices with C60  as the ETL, 20 nm C60 (0.5 Å/s), 8 nm BCP  (0.5 Å/s), and 100 nm Al (2 Å/s) were thermally evaporated in a sequence. A similar approach was used for 1.23 eV narrow bandgap PSCs. PEDOT:PSS  was filtered by a PVDF filter (0.45 μm), spin-coated on the cleaned ITO substrate at 3000 rpm for 60 s and annealed at 140 °C for 15 min in ambient condition. In the N2 glovebox, the PbI2/SnI2 precursor solution was spin-coated at 3000 rpm (with a 2000 rpm acceleration) for 30 s. After drying the wet precursor film at room temperature for 30 min, FAI/MAI precursor solution was dynamically spin-coated on top at 3000 rpm for 60 s, followed by thermal annealing at 100 °C for 30 min. Afterward, 1 mg mL–1 NH4SCN  dissolved in isopropanol was spin-coated on top at 5000 rpm for 30 s. Finally, 20 nm C60 (0.5 Å/s), 8 nm BCP (0.5 Å/s), and 100 nm Ag (2 Å/s) were sequentially evaporated on top. The cell area was determined by the overlap of the top and bottom ITO electrodes (0.09 or 0.16 cm2). For tandem and triple devices, the same procedure was used to fabricate different perovskite sub-cells. Between 1.73/1.57 eV sub-cells and 1.57/1.23 eV sub-cells, after evaporation of 20 nm C60, the samples were transferred in the air to a homemade spatial ALD reactor as described previously43. Tetrakis(dimethylamino)tin(IV) bubbler was used as tin source while H2O was used as co-reactant. Both vessels were kept at room temperature flowing 500 sscm of argon through them. The process was carried out at 100 °C having nominal growth per cycle GPC of 0.125 nm/cycle, determined on silicon wafer. The final thickness of the layer was 45 nm. The substrates were then transferred back to the thermal evaporator for the deposition of a 1-nm Au layer. For Au evaporation, a shadow mask with aperture slightly larger than the cell area was applied. The samples were then exposed in air to spin-coat PEDOT:PSS at a speed of 3000 rpm for 60 s, and annealed at 100 °C for 15 min.Pre-patterned ITO glass substrates  and a daylight filter (Hoya LB120) was used to simulate the solar spectrum, the light intensity was calibrated by Si photodiode to be ~100 mW cm–2. A black shadow mask with an aperture slightly smaller than the cell area was used (0.0676 or 0.1296 cm2). For the fast J–V measurements, a Keithley 2400 source meter was used to sweep the voltage from +1.5 V (–0.5 V) to –0.5 V ( + 1.5 V) at a scan rate of 0.25 V s–1 in reverse (forward) scan. For the stabilized J–V tests, the solar cell was first monitored at Voc for 5 min, followed by a reverse voltage sweep from (Voc + 0.02) V to –0.02 V at a step size of 0.02 V. During the voltage sweep, a Keithley measures the current density for 5 s at each voltage point. From the stabilized J–V curves, the voltage at the maximum power point was extracted and was applied to the cell during steady-state power output tracking. In the EQE measurements, a modulated  tungsten-halogen lamp  was used as the light source. The signal of solar cells was amplified by a current preamplifier  and measured by a lock-in amplifier . The spectral response was then transformed into EQE using a calibrated silicon reference cell. For single-junction PSCs, to mimic the one-sun condition for the J–V measurements, additional LED bias light  was used to generate a photocurrent close to Jsc in the cell during the EQE measurement. We note that for our single-junction PSCs, the difference between non-biased and light-biased EQE spectra is negligible. For tandem solar cells, a 530-nm bias light was used to measure the EQE response of 1.23 eV back sub-cell, whereas a 940-nm bias light was used for the EQE of 1.73 eV front sub-cell. In a triple-junction solar cell, the 530 nm bias light was used for the 1.23 eV back sub-cell, the 730-nm bias light was used for the 1.73 eV front sub-cell, and a combination of 530 + 940 nm bias light was used to measure the 1.57 eV middle sub-cell. We also studied the effect of voltage bias by applying the sum of the Voc of the optically biased sub-cells on our triple-junction solar cells during the EQE measurements and found that the difference between voltage biased and non-voltage biased EQE spectra is insignificant.The λ = 1.5405 Å). UV-vis-NIR absorption measurements were performed by PerkinElmer Lambda 900 UV-vis-NIR spectrophotometer. Steady-state photoluminescence spectra were measured by Edinburgh Instruments FLSP920 double-monochromator luminescence spectrometer, with a near-infrared photomultiplier (Hamamatsu).SEM images were recorded by a FEI Quanta 3D FEG microscope, using a 5-kV electron beam and a secondary electron detector. XRD patterns were obtained by a Bruker 2D phaser (Cu Kα radiation, 44.To ensure the reliability of any conclusions drawn from optical modeling, component materials of the semi-transparent PSCs were prepared individually on glass and characterized with a J.A. Woollam ellipsometer to acquire optical constants. Furthermore, measured R and T of each material were compared to those simulated with its optical constants for validation.Optical modeling was carried out with the GenPro4 programFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary