Isatis indigotica accumulates several active substances, including C-glycosylflavonoids, which have important pharmacological activities and health benefits. However, enzymes catalyzing the methylation step of C-glycosylflavonoids in I. indigotica remain unknown. In this study, three O-methyltransferases (OMTs) were identified from I. indigotica that have the capacity for O-methylation of the C-glycosylflavonoid isoorientin. The Type II OMTs IiOMT1 and IiOMT2 efficiently catalyze isoorientin to form isoscoparin, and decorate one of the aromatic vicinal hydroxyl groups on flavones and methylate the C6, C8, and 3′-hydroxyl positions to form oroxylin A, wogonin, and chrysoeriol, respectively. However, the Type I OMT IiOMT3 exhibited broader substrate promiscuity and methylated the C7 and 3′-hydroxyl positions of flavonoids. Further site-directed mutagenesis studies demonstrated that five amino acids of IiOMT1/IiOMT2  were critical residues for their catalytic activity. Additionally, only transient overexpression of Type II OMTs IiOMT1 and IiOMT2 in Nicotiana benthamiana significantly increased isoscoparin accumulation, indicating that the Type II OMTs IiOMT1 and IiOMT2 could catalyze the methylation step of C-glycosylflavonoid, isoorientin at the 3′-hydroxyl position. This study provides insights into the biosynthesis of methylated C-glycosylflavonoids, and IiOMTs could be promising catalysts in the synthesis of bioactive compounds. It has lubility . Methoxylubility .O-methylation of flavonoids in plants is strictly controlled by O-methyltransferases (OMTs), and the reported plant flavonoid O-methyltransferases were classified into two types [Medicago sativa [Glycine max [Perilla frutescens [Oryza sativa [Arabidopsis thaliana [Catharanthus roseus [Triticum aestivum [Citrus depressa [Citrus reticulata [Ocimum basilicum, methylating hydroxyl groups at specific flavonoid positions [O-methylation of flavonoids without any metal cations. Cation-dependent OMTs (Type II OMTs) are smaller enzymes that range in size from 23 to 29 kDa and require divalent cations for their activity. Most Type II OMTs methylate caffeoyl coenzyme A (CCoAOMTs) during lignin biosynthesis [Mesembryanthemum crystallinum is the first Mg2+-dependent O-methyltransferase that can catalyze flavonols, aside from its traditional substrate caffeoyl-CoA [At4g26220 from A. thaliana methylates 4′-OH or 3′-OH of flavanones and dihydroflavonols [Plagiochasma appendiculatum methylates 6-OH of baicalein and scutellarein to form oroxylin A and hispidulin, respectively [Scutellaria baicalensis methylate norwogonin, baicalein, and luteolin to form wogonin, oroxylin A, and chrysoeriol, respectively [Vitis vinifera [Cyclamen, CkmOMT2 catalyzes 3′ or 3′,5′ O-methylation at the B ring of glycosylated anthocyanidins involved in flower coloration [Prunus persica [Solanum lycopersicum has a strong affinity for glycosylated anthocyanidins [C-glycosylflavonoids in plants.The wo types . Type I o sativa  (ChOMT acine max  (GmIOMT1utescens  (PfOMT3)a sativa  (ROMT-9)thaliana  (AtOMT1)s roseus  (CrOMT2)aestivum  (TaOMT2)depressa   from A. thaliana as a query, while three unigenes from two types of OMT were obtained as putative methyltransferases. The full-length cDNAs of the IiOMTs were amplified with the cDNA of I. indigotica leaves as a template, and were designated as IiOMT1, IiOMT2, and IiOMT3, respectively. The open reading frames (ORF) of the three IiOMT genes were 744, 699, and 1095 bp and were predicted to encode proteins of 257, 232, and 364 amino acids, respectively. The predicted amino acid sequences of the candidate IiOMT cDNAs corresponded to a 28.93-kDa protein, a 26.24-kDa protein, and a 39.74-kDa protein; all three proteins contained a conserved domain identified as methyltransferase (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).A BLASTP search of the IiOMTs in different organs of I. indigotica. Three IiOMT genes were constitutively expressed in leaves, stems, and roots of I. indigotica, with the strongest expression in the leaves . The expression level of IiOMT1 was relatively higher in the youngest leaves and L4 leaves. Expression levels of IiOMT2 and IiOMT3 exhibited an overall increasing tendency in young leaves; levels were highest in L4 leaves and then decreased in senescence leaves (IiOMTs in early-stage leaves (L1 to L3), slightly decreased expression in L4 leaves, and high accumulation in early senescence with lower expression levels of IiOMTs  from Populus tremuloides, and MsCCoAOMT (AAC28973.1) from M. sativa, respectively. Interestingly, IiOMT1 also shared high homology with SOMT-9 from G. max (soybean). SOMT-9 methylates substrates with ortho-hydroxyl groups at a 3′-OH position including phenylpropanoids, as well as coumarins and flavonoids [A. thaliana (AAB96879.1), which methylates flavonoids, coumarin, and aromatic esters. Furthermore, the OMTs in this subgroup, including AnthOMT from S. lycopersicum [V. vinifera, methylate anthocyanins and flavonoid glycosides at the 3′-OH and 5′-OH positions [A. thaliana, which can methylate flavonoids at the 3′-OH position [I. indigotica.For further insight into potential functions of IiOMTs, we aligned the three protein sequences with similar and previously characterized plant OMTs that have flavonoid or phenylpropanoid activity and generated a phylogenetic tree. Although IiOMT1 and IiOMT2 shared a high amino acid sequence identity of 54.0% and belonged to Type II OMTs, IiOMT1 and IiOMT2 were placed in the caffeoyl CoA-OMT (CCoAOMT) subgroup and the CCoAOMT-like subgroup, respectively . Additioavonoids . IiOMT2 persicum , AOMT anositions , 27. IiOposition . Thus, tin vitro, their coding sequences were expressed in E. coli Rosetta (DE3). These three IiOMT proteins with a hexahistidine (His) tag and a SUMO tag at the N-terminus were highly expressed, and then purified using Ni-affinity chromatography. The molecular weights of the IiOMT fusion proteins were consistent with their expected values  of isoorientin showed a signal at 3.95 ppm; meanwhile, it showed a signal at 56.7 ppm at the 13C NMR spectrum . The results suggested that these three IiOMTs could be involved in flavonoid biosynthesis in I. indigotica, while IiOMT1 and IiOMT2 likely contribute to isoscoparin biosynthesis.IiOMT activity was tested scoparin . To clarspectrum . Therefo product . Luteoli2+  . Under t2+  and C. T2+ . This inKm value of IiOMT3 for luteolin (41.49 μM) was the highest; however, the Kcat/Km value of IiOMT3 for luteolin (366.62 μM−1 s−1) was the lowest. The apparent Km and Kcat/Km values of IiOMT1 and IiOMT2 for luteolin were 113.5 μM and 3645.33 μM−1 s−1, and 77.03 μM and 1225.23 μM−1 s−1, respectively. Additionally, both IiOMT1 and IiOMT2 showed comparable affinity to isoorientin, whose values toward isoorientin were 75.56 and 62.08 μM for Km, and 4035.65 and 1289.91 μM−1 s−1 for Kcat/Km, respectively. In contrast, the catalytic efficiency of IiOMT3 for isoorientin  was relatively weaker. Overall, IiOMT1 was the more efficient enzyme for the methylation of luteolin and isoorientin compared with IiOMT2.The kinetic properties of IiOMTs were determined within the linear range of the enzymatic reaction. Each purified recombinant protein was subjected to a reaction at 45°C and an optimal pH in Tris–HCl buffer with various concentrations of luteolin and isoorientin. As shown in To explore the catalytic regioselectivity of IiOMTs, a total of 40 compounds with varied structures were used for methylation reactions containing only one hydroxyl group on a specific site, an ortho-hydroxyl group or a hydroxymethyl group on the A-ring or B-ring . Liquid O-methylation of flavone glycosides , and the conversion rates of IiOMT2 exceeded 98.9% for flavone glycosides. With baicalein as acceptor, IiOMT1 and IiOMT2 could convert baicalein (compound 7) into 6-methylflavone (oroxylin A) with SAM as a methyl donor, whereas IiOMT3 could produce 7-methylflavone (negletein)  and caffeic acid (38), which possess aromatic vininal dihydroxy groups but could not accept syringic acid (37) and ferulic acid (39) as substrates.To identify additional functions of the IiOMTs, we also tested other flavonoid substrates. Both IiOMT1 and IiOMT2 could also catalyze the gletein)  and D. Wgletein)  and S5B.Altogether, the results indicated that IiOMTs could efficiently methylate the 3′-OH position of vicinal hydroxyl group flavonoids at the B-ring, but IiOMT3 showed weaker or loss of catalytic capabilities toward substrates with introduced sugar molecules. Additionally, IiOMT1 and IiOMT2 methylated the C6- and 8-hydroxyl positions to form oroxylin A and wogonin, respectively, while IiOMT3 exhibited broader substrate promiscuity and methylated the C7-hydroxyl positions of flavonoids.Medicago CCoAOMT (PDB ID: 1sui) and IiOMT2 based on Sorghum CCoAOMT (PDB ID: 5kva) were created after optimal templates were identified. In both structures, the key residues of divalent metal ions were almost identical, but residues that interacted with the acceptors differed in each structure .The different catalytic activity of IiOMT1 and IiOMT2 led us to investigate their protein structure and key residues determining catalytic patterns. Homology models for IiOMT1 based on CoAOMT PD ID: 1suitructure . As showMg2+ ion . We analO-methylation. Additionally, the K176A mutation that affected enzyme activity was specific to IiOMT1 while the D175A that affected enzyme activity was specific to IiOMT2.Site-directed mutagenesis and enzyme assays demonstrated that five amino acid mutations of IiOMT1/IiOMT2  decreased and almost abolished the methylation activity to two optimal substrates  and D. FN. benthamiana leaves. As shown in To examine the localization of IiOMTs, the recombinant IiOMT fused with GFP in the C-terminal was transiently expressed in IiOMTs, we overexpressed IiOMTs under the control of the 35S promoter in N. benthamiana leaves. Leaves with an infiltrated suspension of A. tumefaciens with 35S empty vector were used as a control. The fluorescent signals of three IiOMT fusion GFP proteins were found to be distributed throughout the cytoplasm, suggesting that three IiOMTs were expressed in N. benthamiana  and wogonin (8-methylflavone), while the expression of IiOMT3 yielded negletein (7-methylflavone) in high abundance in N. benthamiana leaves , but decreased in more mature stages (L5) and then returned to the basal level. This observation may suggest that chryseoriol was an intermediate product during the biosynthesis of flavonoids and preferred to transform into downstream compounds, and thus the amount of chryseoriol returned to low-level accumulation in senescence leaves. In contrast, the amount of isoscoparin showed an increasing tendency with high expression levels of IiOMTs in early-stage leaves L1 to L3; accumulation remained high in early senescence, with lower expression levels of IiOMTs. This result indicated that isoscoparin was a main storage form of methylated C-glycosylflavonoids in planta. This notion was also supported by the observation that the amount of isoscoparin was still higher in early senescence than in mature leaves. Our finding also supported the idea that non-active glycosylated forms of compounds are stored to avoid autotoxicity in plants, which may release toxic aglycones in response to the attack [The process of biosynthesis and accumulation of e attack , 40.G. max, O. sativa, and C. reticulata; however, C-glycosyltransferases in plants typically use non-methylated flavonoid aglycones as acceptors, such as daidzein, apigenin, luteolin, or 2-hydroxynaringenin [O-methylation reactions after C-glycosylation during the biosynthesis of isoscoparin in I. indigotica. The Km values for IiOMT1 and IiOMT2 indicated that they prefer the substrate isoorientin, not luteolin. IiOMT1 and IiOMT2 catalyzing the O-methylation of isoorientin was consistent with the accumulation of large amounts of isoscoparin, but not chrysoeriol or isoorientin, in I. indigotica leaves . However, the enzyme activity of IiOMT1 was detectable with flavones containing vicinal hydroxyl groups and hydroxymethyl groups. Sequence alignments of different plant Type II OMTs, including IiOMT1 and IiOMT2, demonstrated that the amino acid residues identified as putatively important for SAM binding and substrate binding were not the same . To test whether the amino acids located in conserved domains of IiOMT1 and IiOMT2 contribute to their distinct catalytic patterns, molecular docking and site-directed mutagenesis were performed. Although the five amino acids of IiOMT1/IiOMT2  indicate that those highly conserved in Type II OMTs play an important role in O-methylation, our molecular docking indicated that the Mg2+ coordination geometry differed for IiOMT1 and IiOMT2. These interactions, which are shared between Mg2+ and IiOMT1, can help stabilize the substrate for the reaction [2+ ion. However, D199 within 3.4 Å may have a weak interaction with the Mg2+ ion, meaning the replacement of conserved D199 with Ala did not abolish activity toward the substrates  do not methylate at a detectable level, and the D175 mutant retained its methylation activities. In IiOMT2, the side-chain oxygens of D149, D175, and N176 were involved in the chelation of the metal ion, which could mediate the deprotonation of the isoorientin 3′-hydroxyl group and contribute to facile transmethylation [O-methylation of isoorientin.Although IiOMT1 shared high sequence identity with IiOMT2 (>50%), they were grouped into different subgroups in the Type II OMTs. Their substrate specificity and regioselectivity were distinctly different. IiOMT2 showed strict substrate stereoselectivity, only accepted glycosylflavonoids or phenylpropanoids with aromatic vicinal hydroxyl groups as substrates reaction . The sidbstrates  and C. Chylation . We alsoC. roseus [Solanum habrochaites [O. sativa [M. sativa [G. max [O-methylation of flavonols and flavones, they have different substrate catalytic properties. For example, ROMT-9 exhibits relatively broad substrate regiospecificity for flavonoids, with a preference for the 3′-hydroxy group, but it did not perform catalytic activity for the 7-hydroxy group of flavones compared with IiOMT3. The identification of IiOMT3 from I. indigotica based on the similarity to AtOMT1, which shared 93.0% amino acid sequence identity, suggested the structural conservation of flavonoid methyltransferases [Flavonoid methylation is typically attributed to Type I OMTs. These enzymes often mediate the methylation of flavonols (such as CrOMT2 from . roseus  and ShMOochaites ), flavon. sativa ), or iso. sativa  and GmIO [G. max ). Thoughsferases . AlthougO-methyltransferases that could modify methylated C-glycosylflavonoids in I. indigotica. We determined that IiOMT1 could play a critical role in isoorientin modification and found that two types of O-methyltransferases catalyzed O-methylation at the 3′-OH position of luteolin in I. indigotica. Identifying these three IiOMTs provided novel insights into the biosynthesis of methylated C-glycosylflavonoids, which could be efficient biocatalysts capable of synthesizing methylated C-glycosylflavonoids and subsequently be used in medicinal treatments.In summary, we characterized two types of Isatis indigotica plants growing in the suburbs of Beijing were harvested in the autumn of 2017. Total RNA was extracted from different organs using Trizol reagent , and transcribed to cDNA with a PrimeScript™ RT Reagent Kit with gDNA Eraser. Full-length cDNAs of IiOMTs were amplified with specific primers using the cDNA of leaves as a template, and then cloned into pE-SUMO expression vectors  using a PrimeScript™ RT Reagent Kit with gDNA Eraser. Gene expression levels were detected with TB Green Premix Ex Tap™ II (Tli RNaseH Plus) on a Roche LightCycler 480. The primers of I. indigotica were freeze-dried and ground into powder. Fifty milligrams of each powdered sample was extracted with 1.5 mL methanol under sonication for 45 minutes and centrifuged . The supernatants were filtered with 0.22-μm filters using a Waters C18 column  with 0.1% formic acid–acetonitrile (A) and 0.1% acid–water (B) as mobile phases. The elution method was as follows: 5% A at the initial time; 25% A at 1 minute; 50% A at 4 minutes; 95% A at 4.5 minutes; 95% A at 6 minutes; 5% A at 7 minutes; 5% A at 9 minutes. Multiple monitoring methods were used to assess quantification, while the selected m/z transitions were 447.1 → 357.1 at 1.32 minutes for isoorientin, 461.1 → 341.1 at 1.52 minutes for isoscoparin, 285.0 → 133.0 at 2.33 minutes for luteolin, and 299.1 → 284.0 at 3.02 minutes for chrysoeriol.For ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) analysis, roots, stems, and leaves of http://www.ncbi.nlm.nih.gov/), and the amino acid sequences were analyzed with Vector NTI Advance 11.5.3. A phylogenetic tree was constructed with plant OMT protein sequences using MEGA version 7.0 (Supplementary Data Table S2). The neighbor-joining method was used with 1000-replicate bootstrap support.The nucleotide sequences of IiOMTs were compared by blastn at NCBI (Escherichia coli Rosetta strain (DE3) for fusion protein expression. E. coli Rosetta 1 (DE3) cells harboring IiOMT-pE-SUMO were cultured at 37°C until the OD600 reached 0.6, and then induced with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) at 16°C for 16 hours. The induced cells were harvested by centrifugation and then washed with sterile cold distilled water, centrifuged, resuspended with the buffer , and sonicated on ice. Recombinant protein purification was carried out with Ni-NTA resin and concentrated using an Amicon-Ultra-0.5 Ultracel-10 k membrance. The purity of the His-tag-fused IiOMTs was examined with 10% SDS–PAGE glue, and the protein concentration was determined by a Bradford Protein Assay Kit with bovine serum as the standard .The full-length cDNAs of IiOMTs were amplified with specific primers and then inserted into the pE-SUMO expression vector at BamHI/SalI sites . After s2, CaCl2, ZnCl2, MnCl2, or EDTA was added individually at the final concentration of 10 mM to estimate the metals’ inhibition of enzyme activity. For kinetic studies, recombinant IiOMTs were incubated in a final volume of 100 μL with 480 μM S-adenosyl-L-methionine (SAM). Luteolin and isoorientin concentrations ranging between 5 and 2500 μM were used for determination of the Michaelis constant (Km). Data were adapted to the Michaelis–Menten equation, and a non-linear regression program was used to calculate the Vmax and Km values.To test the optimal pH of IiOMT enzyme activity, assays were performed in 50 mM citric acid–sodium citrate buffer (pH 4.0–6.0), 50 mM sodium phosphate buffer (pH 6.0–8.0), 50 mM Tris–HCl buffer (pH 7.0–9.0), and 50 mM sodium carbonate buffer (pH 9.0–10.8) with isoorientin as the substrate. To determine the optimal temperature for IiOMT activity, +the reactions were incubated at 25–55°C. The optimal quantity of pure protein was assessed in the 2–3000 μg range. MgClThe enzymatic reactions (100 μL) included 480 μM SAM and 200 μM substrate. Pure proteins  were incubated at 45°C for 45 minutes. Double-volume methanol was then added to the assays to quench the reactions. The mixtures were clarified by centrifugation , and then the supernatant was filtered through a 0.22-μm nylon column and analyzed by liquid chromatography–mass spectrometry (UPLC/Q–TOF–MS). Separation was performed with a Waters ACQUITY UPLC HSS T3 . Mobile phases consisting of 0.1% (v/v) formic acid in acetonitrile (solvent A) and 0.1% (v/v) formic acid in water were used at a flow rate of 0.5 mL/minute and a UV wavelength from 200 to 400 nm. The elution gradient system was 10–21% A (0–3 minutes), 35–45% A (5–7 minutes), 55–65% A (9–12 minutes), 95% A (12.5–14 minutes), and 10% A (14–16 minutes).1H-nuclear magnetic resonance (NMR), and 13C-NMR. NMR spectra were recorded with a Bruker Ascend spectrometer and compared with the reported NMR spectrum for isoscoparin [1H-NMR  δ 7.50 , 7.47 , 6.93 , 6.64 , 6.51 , 4.90 , 3.95 , 3.87 , 3.73 . 13C-NMR  δ 166.1 (C-2), 104.3 (C-3), 184.1 (C-4), 162.1 (C-5), 109.3 (C-6), 165.1 (C-7), 95.3 (C-8), 158.8 (C-9), 105.2 (C-10), 123.6 (C-1′), 121.8 (C-2′), 149.5 (C-3′), 152.2 (C-4′), 116.8 (C-5′), 110.6 (C-6′), 75.3 (C-1′′), 72.6 (C-2′′), 80.1 (C-3′′), 71.8 (C-4′′), 82.6 (C-5′′), 62.9 (C-6′′), 56.7 (3′-OCH3).The reaction system was amplified in a total volume of 30 mL IiOMT2 crude protein with 450 μM isoorientin and 480 μM SAM. The reaction mixture was extracted with two volumes of methanol, and dissolved with 2 mL methanol after the organic phase was pressurized and dried. Mobile phases consisting of acetonitrile and water  were used to separate the reaction product in a Shimadzu UFPLC system at a flow rate of 10 mL/minute and a UV wavelength of 350 nm using a C18 20 × 250 mm column. The separated product was determined using UPLC/Q–TOF–MS, scoparin : 1H-NMR https://swissmodel.expasy.org/) based on the Medicago CCoAOMT (PDB ID: 1sui) and Sorghum CCoAOMT (PDB ID: 5kva) crystal structure, respectively. Molecular docking between IiOMT and isoorientin was investigated using AutoDockTools 1.5.6.To build the 3D structures of IiOMT1 and IiOMT2 binding with isoorientin (1), we modeled their homology using SWISS-MODLE (E. coli. The sequences of the IiOMT-pE-SUMO mutants were confirmed via sequencing and transformed into Rosetta (DE3) E. coli for heterologous expression.The site-directed mutagenesis of IiOMTs at the residues was performed via PCR with IiOMT1-pE-SUMO or IiOMT2-pE-SUMO as a template. Corresponding primers are listed in Supplementary Data Table S1. PCR products were digested with a DMT enzyme and transformed into DMT IiOMT and the empty vector pCAMBIA1300-cGFP were transferred into Agrobacterium tumefaciens strain GV3101. Transformants were cultured in LB medium containing 50 μg mL−1 kanamycin and 50 μg mL−1 rifampicin at 28°C, collected by centrifugation, and then resuspended in 10 mM MES buffer  to a final OD600 of 1.0. After incubation for 2 hours at room temperature, the mixed A. tumefaciens was injected into 6-week-old N. benthamiana leaves. After 2 days, the green fluorescent protein (GFP) fluorescence of N. benthamiana leaves was captured using a Zeiss LSM 510 META confocal microscope.The coding regions without IiOMT stop codons were cloned into the plant expression vector pCAMBIA1300-cGFP. Recombinant vectors carrying For substrate feeding studies, 100 μM of luteolin or isoorientin in 0.1% DMSO in water was infiltrated into previously agroinfiltrated leaves. Leaves were harvested 1 day later and freeze-dried for metabolite analysis.P-values were calculated using Tukey’s test. Non-linear regression analysis was carried out to calculate kinetic parameters using the Michaelis–Menten model.Statistical analyses were performed using GraphPad Prism 8.0 software. Data are presented as mean ± standard error of the mean unless stated otherwise. This work was supported by the Scientific and Technological Innovation Project of the China Academy of Chinese Medical Sciences , the National Natural Science Foundation of China , the Fundamental Research Funds for the Central Public Welfare Research Institutes from the China Academy of Chinese Medical Sciences (ZZ11-115), and a key project at central government level: The Ability to Establish Sustainable use of Valuable Chinese Medicine Resources (2060302).Y.T.: conceptualization, methodology, and original draft preparation. J.Y.: methodology and NMR analysis. Y.J., S.S., X.W., and R.W.: methodology, software and visualization. L.K.: chemical analysis. T.C.: bioinformatics analysis. D.L., J.G., and G.C.: data analysis and reviewing. L.H.: funding acquisition, project administration, and resources. J.T.: investigation, validation, writing, reviewing, supervision, and editing.All data supporting this research result can be obtained in this paper and in its supplementary materials published online.The authors declare no conflicts of interest.Horticulture Research online.supp_data_uhac140Click here for additional data file.


Nursing home residents continue to experience significant COVID-19 morbidity and mortality  were included to assess VE among nursing home residents during March 29–June 15, 2022, with a maximum of 60-days of follow-up through July 25, 2022. The population included nursing home residents from 196 nursing homes in 19 statesInternational Classification of Diseases, Tenth Revision, Clinical Modification diagnosis code U07.1 or SARS-CoV-2 rapid antigen or reverse transcription–polymerase chain reaction test result), 2) immunosuppressive condition, 3) “do not resuscitate” orders, 4) acute hospitalization during the preceding 90 days, 5) time since last COVID-19 vaccination, 6) length of stay in the nursing home, 7) history of any influenza vaccination during the previous influenza season, 8) age, and 9) number of Charlson index comorbidities . Among these residents, 9,503 (99.7%) served as controls for ≥1 day of follow-up and 3,245 (34.1%) residents received a second booster dose during the study period and were eligible to be included in the treatment group. In the matched analysis, 1,902 residents were matched 1:1 with controls; 1,343 residents were excluded because they could not be matched to a control. Residents in the matched group had a mean age of 78 years, a median length of stay of 880 days, a median 196 days since the last COVID-19 vaccination, four Charlson comorbidities, and 35.5% were male. Observed characteristics between matched groups were <0.1 standard mean differences . CompareCompared with a first booster dose only, 60-day VE of a second mRNA COVID-19 vaccine booster dose was 25.8% (95% CI = 1.2–44.3) against infection, 60.1% (95% CI = −18.8−91.5) against hospitalization, 89.6% (95% CI = 45.0–100.0) against death, and 73.9% (95% CI = 36.1–92.2) against the severe composite outcome of COVID-19–associated hospitalization or death .In this analysis, comparing the relative effectiveness of a second booster dose of COVID-19 mRNA vaccines with a single booster dose among eligible nursing home residents in 19 states, VE of a second booster dose against the severe composite outcomes of SARS-CoV-2–associated hospitalization or death was 73.9% and 89.6% for death alone. VE against SARS-CoV-2 infection during a period crossing both Omicron subvariants BA.2 and BA.2.12.1 (March–June 2022) and BA.4 and BA.5 (July 2022) predominance was 25.8%. The findings in this report are subject to at least five limitations. First, the point estimates for the findings in the current study are similar to those estimated in previous studies; however, too few hospitalization events were observed to definitely attribute a reduction to vaccination. A recent study from Israel provided similar VE estimates during B.1.617.2 (Delta) variant circulation for a second booster dose  in a long-term care setting  and 90% against death alone compared with receipt of a single booster dose.Efforts should be made to ensure that nursing home residents remain up to date with recommended booster doses of COVID-19 vaccines.


Gypothamnium pinifolium Phil. (Asteraceae) is a small shrub that grows in the Paposo Valley of the II Antofagasta Region of Chile. This initial study is of the high-resolution phenolic fingerprinting, antioxidant activity, the relaxation effects in rat aorta, the inhibitory enzyme potential, plus the antiproliferative activity of the ethyl acetate and n-hexane extract from G. pinifolium and its two major isolated secondary metabolites . The study involves using ultra-high-performance liquid chromatography todiode array detection coupled with Q-Orbitrap mass spectrometry analysis (UHPLC-PDA-Orbi-trap-MS), in which various compounds were identified, including specific coumarins. The n-hexane extract showed total phenolic and flavonoid contents of 517.4 ± 12.5 mg GAE/100 g extract and 72.3 ± 3.7 mg QE/100 g extract, respectively. In addition, the antioxidant activity of the n-hexane extract was assessed using in-vitro assays such as bleaching of DPPH and ABTS , FRAP , and ORAC . Furthermore, the inhibition against cholinesterases (acetylcholinesterase (AChE) 4.58 ± 0.04 µg/mL, butyrylcholinesterase (BChE) IC50: 23.44 ± 0.03 µg/mL) and tyrosinase (IC50: 9.25 ± 0.15 µg/mL) enzymes of the n-hexane extract, and main compounds  were measured. The antiproliferative potential of the extracts and the two principal compounds against several solid human cancer cells was investigated. All of them showed good activity against cancer cells. Label-free live-cell imaging studies on HeLa cells exposed to the isolated coumarin and the diterpene enabled the observation of cell death and several apoptotic hallmarks. Our results indicate that G. pinifolium Phil. is a valuable source of secondary metabolites with potential activity against noncommunicable diseases. In recent years, the use of extracts rich in phenolic compounds from native shrubs or plants has been increasing due to their potential to ameliorate or support the treatment of noncommunicable or chronic diseases (NCDs). The main types include cardiovascular disease, cancer, chronic respiratory disease, and diabetes (WHO) . These dMitochondrial alterations, inflammation, plus oxidative stress, are molecular mechanisms that perform a central role in the initiation and progression of various NCDs . CardiovNatural products represent a family of diverse molecules with a large range of bioactivities reported . It has Weinmannia trichosperma cav. it was possible to perform the study of its main component, isoastilbin − ion at m/z: 151.03960 was identified as vanillin (C8H7O3−), peak 9 with a parent ion at m/z: 343.15549 as the vanillyl derivative: 3,4-divanillyltetrahydrofuran (C20H23O5−), peak 3 as camphoric acid (C10H15O4−) and peak 4 as jacareubin (C18H13O6−) following, the isomer peaks 5 and 6 and 15 with pseudomolecular ions at m/z: 313.14478, 313.14490 and 313.14389, were identified as aphillodenticulide isomers . Fi. Fi20H17oserone CH31O5−, pent-labda-8,13-E-diene-15-ol * (C19H22O6−) [Peak 12 was identified by spiking experiments as n-hexane and ethyl acetate extracts of G. pinifolium. For the n-hexane and ethyl acetate extracts, the total phenolic content was 517.4 ± 12.5 and 538.4 ± 4.7 mg of gallic acid/100 g dry extract, respectively. Although the TPC results were similar between both extracts, for the total flavonoid content, the ethyl acetate extract obtained a higher result (465.8 ± 27.5 mg of quercetin/100 g dry extract), six times greater compared to the n-hexane extract (72.3 ± 3.7 mg of quercetin/100 g dry extract). For the antioxidant activity, the ethyl acetate extract also showed better results for DPPH, ABTS, FRAP, and ORAC assays compared with the n-hexane extract.Results corresponding to total phenolic and flavonoid contents and antioxidant activity measured using the different in-vitro assays are summarized in G. pinifolium would produce a potential hypotensive effect. The relaxation in the aorta (intact) with 100 μg/mL for n-hexane and ethyl acetate extract was 63 ± 7% and 64 ± 7%, respectively. Furthermore, with the highest concentration used of 1000 μg/mL, the relaxation was 140 ± 4% and 121 ± 5% for the n-hexane and ethyl acetate extract (50) was not significantly different in the presence of G. pinifolium n-hexane extract (177 ± 1 μg/mL) or GP EtOAc extract in intact aortic rings (135 ± 1 μg/mL).The vascular relaxation observed suggests that  extract B. The haG. pinifolium extracts produce a good relaxation effect at 100 µg/mL in the pre-contracted intact aorta. Although the ethyl acetate extract showed higher antioxidant activity  than the n-hexane extract, the relaxation effect was similar in both extracts. Therefore, the biological activity should be due to other causes  and not by an increased antioxidant activity leading to increased bioavailability of the endothelial nitric oxide, which is an important vasodilator molecule.1 and 2 showed a slight relaxation effect in aortic rings  and 2 (diterpenoid derivative) was not significantly different.Compounds ic rings . Only atG. pinifolium extract in the intact rat aorta may occur by a synergist effect among the compound 1 and 2 or that the extract may have another coumarin more potent is responsible for this activity because the percentage of relaxation effect of G. pinifolium extract (1 or 2 (Coleus forskohlii Briq. (Lamiaceae) in different animal models have also been made. Through a vasodilator effect, it can reduce normal or high blood pressure, in addition to exerting a positive ionotropic action on the cardiac muscle, and can also produce a significant inhibition of platelet aggregation [Marrubium vulgare L. (Lamiaceae) contains marrubiin and marrubenol, compounds that are labdane diterpenes, in addition to an interesting mixture of phenolic compounds [ent-labdane 2, ent-labda-8,13-E-diene-15-ol, showed no vascular relaxation in the intact aorta  . G. piniG. pinifolium. The main results of the present study are summarized in 50 values (µg/mL). In the AChE inhibition assay, G. pinifolium showed an IC50 of 4.58 ± 0.04 µg/mL and 6.43 ± 0.03 µg/mL for n-hexane and ethyl acetate extracts, respectively, and for BChE inhibition assays the IC50 results were 23.44 ± 0.03 µg/mL and 33.25 ± 0.02 µg/mL for n-hexane and ethyl acetate extracts, respectively. On the other hand, in a similar way to G. pinifolium, the fruits (pulp and seeds) of Greigia sphacelata (Ruiz and Pav.) Regel (Bromeliaceae) showed potent inhibition of AChE for the pulp and seeds, the IC50 for AChE was 4.49 ± 0.08 µg/mL and 4.38 µg/mL, respectively [Himantormia lugubris from Antarctica, a good inhibition for AChE with an IC50 of 12.38 µg/mL [G. pinifolium extracts used in this research are more potent. Also, for Artemisia copa Phil. (Asteraceae), ethanolic extract showed a potent anticholinesterase activity, with IC50 of 3.92 ± 0.08 µg/mL for AChE and 44.13 ± 0.10 µg/mL for BChE [G. pinifolium extract the IC50 values showed a less potent activity, the same occurs if we compare the results for G. sphacelata for which the IC50 for pulp was 73.86 ± 0.09 µg/mL and for seeds 78.57 ± 0.06 µg/mL [H. lugubris the enzyme inhibition against BChE was 31.54 ± 0.02 µg/mL, a value close to G. pinifolium ethyl acetate extract and less potent that n-hexane extract [Weinmannia trichosperma Cav., the in vitro inhibitory effects against 5-hLOX, AChE, and BChE were investigated for the aqueous extract, and interesting activity as a 5-hLOX inhibitor was found. Additionally, the results for inhibitory activities for AChE and BChE were 3.13 ± 0.03 µg/mL and 2.94 ± 0.08 µg/mL, respectively [Previously, for other species from Chile, our group found good enzymatic inhibitions against AChE, BChE, and tyrosinase; in some cases, these results are similar to those obtained for ectively . Recentl38 µg/mL  was founfor BChE , for thi06 µg/mL , and for extract . Likewisectively .H. lugubris showed moderate activity with an IC50 of 22.32 ± 0.21 µg/mL, and in comparison with the potent inhibition produced by G. pinifolium n-hexane and ethyl acetate extracts with IC50 values of 9.25 ± 0.15 µg/mL and 12.32 ± 0.21 µg/mL, respectively, showed a better capacity for inhibiting this enzyme [Ovidia pillopillo (Gay) Meissner (Thymelaeaceae) ethanolic extract, the IC50 value for tyrosinase inhibition was 9.92 ± 0.05 µg/mL, a potent and close result to that produced by G. pinifolium n-hexane extract.Furthermore, for tyrosinase inhibition, s enzyme . RecentlG. pinifolium is an interesting source of compounds, phenolics, and specially coumarins, with the potential to inhibit enzymes related to NCDs, so this report highlights the importance of these metabolites for the prevention of neurodegenerative diseases.These results confirm that For a better understanding of the results, molecular docking studies were performed, as discussed below.n-hexane and ethyl acetate extracts from the G. pinifolium (Asteraceae). The latter allows us to get insights into the intermolecular interactions and energy descriptors between the derivatives and the corresponding enzyme catalytic sites, considering the inhibition assays obtained and shown in Some of the major identified metabolites and proposed biosynthetic epimeric derivatives were selected to test them as potential acetylcholinesterase, butyrylcholinesterase, and tyrosinase inhibitors by performing docking assays, according to the ultra-high performance liquid chromatography-photodiode array detection hyphenated with Orbitrap mass spectrometry analysis (UHPLC-PDA-Orbitrap-MS) of the nor-1,2-secolycoserone, which displayed binding energy of −13.396 kcal/mol. This result agrees with the experimental inhibition assays obtained and shown in n-hexane and ethyl acetate extracts exhibited low half-maximal inhibitory concentration (IC50) values. Compared to galantamine, extracts’IC50 value is slightly higher, which could be explained by the more complex profile of extracts, with several active metabolites that can compete among them for the acetylcholinesterase catalytic site. This fact is supported by the similar IC50 range values of extracts compared to galantamine . The third hydrogen bond interaction occurs between the oxygen atom of the carbonyl group at the side chain of cyclolycoserone and the hydrogen atom (–H) of the –OH functional group of tyrosine (Docking results showed that cyclolycoserone interacts through three hydrogen bonds with the residues of Gly117 and Tyr130 (two of them through the hydroxyl group (–OH) at the 2tyrosine A. Althoutyrosine B.nor-1,2-secolycoserone shows different (and more) interactions within the acetylcholinesterase catalytic site, such as four hydrogen bond interactions with Tyr121, Tyr130, Gly119, and Ser200. Moreover, this compound shows two π-π interactions with Phe288 and Phe290, but also two T-shaped interactions with the residues of Trp233 and His440, wherein the former interaction with the phenyl ring of 2-nor-1,2-secolycoserone produces the T tip of the interaction, while with His440 it is exactly the opposite, being the T tip the imidazole ring of this amino acid , which could be attributed to the presence of three hydrogen bond interactions with Gly117, Gly116, and His438 through the oxygen atoms of the 3,4-dihydro-2H-pyran moiety and the carbonyl function of its side chain respectively  at the 2H-chromene skeleton respectively. Besides, 8-epi-gypothaminol performs a T-shaped interaction between His263 and its phenyl core , as well as some hydrophobic interactions that allow for fitting properly in the tyrosinase catalytic site. In the same way, ent-labda-8,13-E-diene-15-ol , HBL-100 and T-47D (breast), HeLa (cervix) and WiDr (colon). The GI50 values showed growth inhibition in all cell lines in the range of 7.6–20 µg/mL. Accordingly, we analyzed the two main products contained in the extract. Thus, isolated compounds 1–2 were tested against the same cell panel. Results are shown in 2 (GI50 4.5–10 µM) resulted in more active than 1 and with similar potency to the standard anticancer drug cisplatin. This result is consistent with a previous study, where compound 1 was reported to induce cell growth inhibition in the cancer line MCF-7 after 24 h of drug exposure (IC50 = 15 µM) [As part of our interest in the bioprospection of microorganisms ,38,39 an= 15 µM) .1. In the case of terpene 2, the SwissTargetPrediction database  provided a hint on predicted macromolecular targets. The analysis revealed low probability against all targets, which implies that compound 2 holds a novel structure as a bioactive substance. Thus, no initial hint is available, and the mechanism of action should be further studied from the beginning.According to Zhu, J.J. & Jiang , some co1 and 2 on HeLa cells were studied in detail using label-free continuous live-cell imaging  can induce death in HeLa cells earlier than coumarin 1 (yellow line). Thus, both compounds might have different modes of action, and most probably, they will have different cellular targets. The LC50 values for compounds 1–2 against HeLa cells are >100 and 69 ± 27 µM, respectively. We speculate that this might explain the observed differences in apoptosis kinetics.The effects of compounds  imaging . Live-ceallmarks . The LCDallmarks , i.e., tG. pinifolium Phil. Through UHPLC-PDA-Orbitrap-MS, it was possible to detect 26 metabolites. Besides, this is the first research on the inhibitory activity (against cholinesterase and tyrosinase) and antiproliferative screening of this shrub and its main components in the specified group of cancer cell lines. Enzymatic activity results showed a potent inhibition. Furthermore, the extracts plus pure compounds showed antiproliferative activity and relaxant effect in the rat aorta. This study highlights the potential of G. pinifolium extracts to become a product with nutritional and health-promoting properties with potentiality in chronic noncommunicable diseases. It is still necessary to elucidate which are the minor bioactive compounds that are responsible for the activities studied and to develop bioassay-guided fractionation with G. pinifolium extracts for isolation and identification to correlate the minor compounds to these interesting bioactivities.For the first time, the antioxidant activity, the hypotensive effect, enzymatic inhibitory, and the antiproliferative potential have been studied, complemented with the full UHPLC phenolic fingerprinting of


The author would like to make the following corrections to his published paper . The corUrolitus syn. nov. is placed as a junior synonym of Sepedophilus, and Palporus stat. nov. is raised to a distinct genus from a subgenus of Tachyporus sensu. nov., and †Mesotachyporus syn. nov. is treated as synonym of the latter.” with “Urolitus syn. nov. is placed as a junior synonym of Sepedophilus. Additionally, Palporus stat. nov. is raised to a distinct genus from a subgenus of Tachyporus sensu. nov., and †Mesotachyporus syn. nov. is synonymized with the latter.”.On page 1, in the Abstract, replace the sentence “On page 1, in the Introduction, replace the sentence “Among one extinct and 32 extant subfamilies : 231 syn. nov.” with “=Megarthropsini Cameron, 1919 [101]: 231 syn. nov. , usually widest between basal 1/4 to middle  (B) 58B: 50-3)” with “Pronotum  of typical Tachyporinae sensu nov. form except in Nepaliodes , usually widest between basal 1/4 to middle ”.On page 90, in “Section 3.2. Systematic Part”, 3.2.13. Tribe Tachinusini Fleming, 1821 stat. rev., sensu nov., replace the text “Pronotum  of typical Tachyporinae sensu nov. from except in Bobitobus Tottenham, 1939 stat. rev., the author erroneously used “comb. rev.” instead of “comb. nov.” for the Bobitobus species on both pages. All instances of “comb. rev.” should be replaced with “comb. nov.” throughout the text, except Bobitobus pulchellus  based on the previous taxonomic treatments [On pages 109 and 110, in “Section 3.2. Systematic Part”, 3.2.16. Genus eatments .On page 113, in “Section 3.3. Identification Keys”, 3.3.4. Key to Extant Genera of Vatesini, replace the text “5. Body minute (length: ca. 1.0 mm) ” with “5. Body minute (length: ca. 1.2 mm) ”.On page 116, in “Section 3.3. Identification Keys”, 3.3.6. Key to Extant Genera of Mycetoporinae, replace the text “7. Labial palpomere 3 longer than labial palpomeres 2 and 3 combined  … 8” and its subsequent text “- Labial palpomere 3 shorter than labial palpomeres 2 and 3 combined  … 10” with “7. Labial palpomere 3 longer than labial palpomeres 1 and 2 combined  … 8” and “- Labial palpomere 3 shorter than labial palpomeres 1 and 2 combined  … 10”, respectively.Nitidotachinus and Leucotachinus (both Tachinusini).” with “Meanwhile, they are completely absent in the rest of Tachyporinae, except for Nitidotachinus (Tachinusini), Leucotachinus (Tachinusini), Tachinoporus (Vatesini), and Vatesus berghoffae (Vatesini).”.On page 139, in “Appendix B. List of Characters”, Character 109, replace the sentence “Meanwhile, they are completely absent in the rest of Tachyporinae, except The author apologizes for any inconvenience caused and states that the scientific conclusions are unaffected. The rest of the manuscript does not need to be changed. The original article has been updated.


The T cell in vitro culture system provides a very efficient model to study compounds’ therapeutic activity and mechanism of action. Tinospora cordifolia (Willd.) Hook.f. & Thomson (Family. Menispermaceae) is one of the widely used drugs in Ayurveda (ancient Indian system of medicine) for various ailments such as inflammatory conditions, autoimmune disorders, and cancer as well as for promoting general health. In vitro and in vivo studies on immune cells comprising dendritic cells, macrophages, and B cells suggest its immune-modulating abilities. However, to date, the effect of T. cordifolia on individual purified and polarized T cell subsets has not been studied. Studying drug effects on T cell subsets is needed to understand their immunomodulatory mechanism and to develop treatments for diseases linked with T cell abnormalities. In this study, we examined the immunomodulatory activity of T. cordifolia on primary CD4+ T cells, i.e., Th1, Th17, and iTreg cells. An aqueous extract of T. cordifolia was non-cytotoxic at concentrations below 1500 µg/ml and moderately inhibited the proliferation of naive CD4+ T cells stimulated with anti-CD3ε and anti-CD28 for 96 h. T. cordifolia treatment of naive CD4+ T cells differentiated under Th17-polarizing conditions exhibited reduced frequency of IL-17 producing cells with inhibition of differentiation and proliferation. For the first time, in-depth genome-wide expression profiling of T. cordifolia treated naive CD4+ T cells, polarized to Th17 cells, suggests the broad-spectrum activity of T. cordifolia. It shows inhibition of the cytokine-receptor signaling pathway, majorly via the JAK-STAT signaling pathway, subsequently causing inhibition of Th17 cell differentiation, proliferation, and effector function. Additionally, the molecular docking studies of the 69 metabolites of T. cordifolia further substantiate the inhibitory activity of T. cordifolia via the cytokine-receptor signaling pathway. Furthermore, in vitro polarized Th1 and iTreg cells treated with T. cordifolia extract also showed reduced IFN-γ production and FoxP3 expression, respectively. This study provides insight into the plausible mechanism/s of anti-inflammatory activity of T. cordifolia involving T cells, mainly effective in Th17-associated autoimmune and inflammatory diseases.Naive CD4 Tinospora cordifolia (Willd.) Hook.f. & Thomson (Family Menispermaceae) (TC) plant, also known as Guduchi, is widely used in ayurvedic medicine (ancient Indian system of medicine) and other traditional medicine for treating various ailments through the restoration of homeostasis  myeloid cells: monocytes, macrophages, and dendritic cells, or 2) lymphoid cells: T cells and B cells . TC acti in vivo . G1-4A,  in vivo . Immunom in vivo . Bone ma in vivo . -α in vivo . RR1 is ytokines . TC reveytokines . Recent ytokines .-12 and IFN-γ concomitantly signal through the transcription factors STAT4, STAT1, and T-bet, to differentiate naive T cells towards the Th1 lineage  cells based on the expression of signature cytokines and lineage-specific master transcription factors. Th1 cells are characterized based on their interferon-γ (IFN-γ) production. IL lineage . T-bet i lineage . These c lineage . They ar lineage . Th2 cel lineage . The pro lineage . Treg is lineage .via the JAK-STAT signaling pathway, followed by the differentiation of naive CD4+ T cells to Th17 cells and induction of several Th17-associated genes such as IL-17, Rorc, and IL-23R. In addition, cytokines such as IL-21 act as autocrine factors and promote the differentiation of Th17 cells through a positive feedback mechanism. Similarly, for terminal differentiation and maintenance, Th17 cells require IL-23  was reconstituted by mixing 100 mg of the lyophilized plant material with 1 ml Milli-Q, mixed well, and kept at room temperature for 1 h followed by overnight incubation at 4°C. The next day, the aqueous extract was filtered through a 0.45 µm filter and used for assays at different concentrations.T.cordifolia extract consisted of 0.1% formic acid in water (A) and acetonitrile (B), and the gradient system was 0.1–1 min, 100%–100% B; 1–12 min, 100%–30% B; 12–14 min, 30%–30% B; 14–16 min, 30%–100% B; 16–20 min, 100%–100% B. The sample injection volume was 5 μl. The diode array detector was set to monitor at 254 and 280 nm, and the online UV spectra were recorded in the scanning range of 190–400 nm. The mass spectrometer was operated in positive electrospray ionization mode, and mass spectra were recorded by scanning the mass range of m/z 50–1500 in MS positive mode. Nitrogen was used as drying, nebulizing, and collision gas. The drying gas flow rate was 12 L/min. The heated capillary temperature was set to 350°C, and the nebulizer pressure was at 45 psi. The source parameters capillary voltage (VCap), fragmentor, skimmer, and octapole voltages were set to 3500 V, 175 V, 65 V, and 750 V, respectively. The accurate mass data of the molecular ions were processed through the Mass Hunter version B 04.00 .The chemical fingerprinting of the commercially procured TC was performed using the LC-MS technique at SAIF, CSIR-CDRI, Lucknow, India . BrieflyC57BL/6 mice were obtained from the animal facility of CSIR- Institute of Genomics and Integrative Biology (IGIB), New Delhi, India. Mice aged between 7 and 10 weeks were used for all experiments. All mice were bred and maintained under specific pathogen-free conditions at the animal facility of IGIB, as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). All animal experiments were performed in compliance with protocols approved by the Institutional Animal Ethics Committee of CSIR-IGIB, Delhi, India.Monoclonal antibodies specific to the following mouse antigens were purchased from Thermo Fisher Scientific, eBioscience, United States: CD4 Alexa488 (GK1.5), Foxp3 eFluor450 (FJK-16s), IL-17A APC (eBio17B7), CD8 PE (Ly-2), CD25 PE (PC61), and IFN gamma PE (XMG1.2).−CD25− cells were isolated using the MACS column technique. Briefly, single-cell suspension isolated from the spleen and lymph nodes of mice was resuspended in the MACS buffer, anti-CD8 PE and anti-CD25 PE antibodies were added and incubated for 15 min at 4°C. Cells were washed and resuspended in MACS buffer, and anti-PE microbeads, mixed and incubated for 20 min at 4°C. Cells were subjected to MACS MS columns in a MACS separator, and the flow-through consisting of CD8−CD25− cells were collected.CD8+ T cells were isolated from the spleens and lymph nodes of mice by enrichment with the Dynal Mouse CD4 Negative Isolation Kit . The isolated CD4+ T cells were incubated with an anti-CD25 PE antibody to isolate CD4+CD25− T cells. CD4+CD25− T cells were isolated using the MACS column, as mentioned above. The purity of CD4+CD25− T cells was >90%.CD45 CD4+CD25− T cells were cultured for 4 days with plate-bound anti-CD3ε , anti-CD28 , anti-IFN-γ , anti-IL-4 , rhTGFβ1 , rmIL-6  and rmIL-1β . For iTreg cell induction, 5 × 104 CD4+CD25− T cells were cultured for 4 days in the presence of plate-bound anti-CD3ε (5 µg/ml), anti-CD28 (1 µg/ml), rhIL-2  and rhTGFβ1 (2 ng/ml). On day 2, rhIL-2 (200 U/mL) was added again. For Th1 cell culture differentiation, 1–1.5 × 105 CD4+CD25− T cells were cultured for 5 days with plate-bound anti-CD3ε , anti-CD28 , rmIL-12 , anti-IL-4 . The cultured cells were treated with 500, 1000, 1500, and 2000 µg/ml of AETC or Milli-Q as a vehicle control on day 0.TexMACS™ RPMI 1640 GlutaMAX  medium was used for CD4, iTreg, and Th1 cell cultures, and IMDM  for Th17 cell cultures. The medium was supplemented with 10% heat-inactivated FCS , 500U penicillin-streptomycin , and 50 µM β-mercaptoethanol . For Th17 cell induction, 1–1.5 × 10+CD25− T cells were isolated as mentioned above and labeled using 5 µM CellTrace Violet Cell Proliferation Kit . Briefly, the isolated CD4+CD25− T cells were washed and resuspended in warm PBS. Naive T cells were labeled using 5 µM CellTrace Violet and incubated in the dark for 15 min in a 37°C water bath. The labeled cells were centrifuged, washed with a warm culture medium, and resuspended as 1 × 106 cells/mL. The cells were either stimulated with anti-CD3ε and anti-CD28 and cultured as such or differentiated to Th17 cells following the Th17 differentiation protocol mentioned above. The cultured cells were treated with 500, 1000, 1500, and 2000 µg/ml of AETC or Milli-Q as a vehicle control on day 0. On day 4, cells were harvested and stained with ethidium monoazide (EMA), followed by surface staining with anti-CD4. Cell proliferation was determined by quantifying the dilution of CellTrace Violet on CD4+ live cells by flow cytometry using LSRII  or FACSAria .CD4For analysis of surface markers, cells were stained with the specific surface marker for each phenotype in PBS containing 0.25% BSA and 0.02% azide. Dead cells were excluded by EMA . For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate  and ionomycin  for 2 h, followed by Brefeldin A  for 2 h and stained using the Foxp3/Transcription Factor Fixation/Permeabilization Kit  according to the manufacturer’s instructions. Cells were acquired on FACSAria  or LSR II , and data were analyzed with FlowJo software .+CD25− T cell were isolated from splenocytes as mentioned above and were cultured with 500, 1000, 1500, and 2000 µg/ml of AETC or Milli-Q as a vehicle control on day 0 for 24 and 96 h. Cells were washed with PBS containing 0.25% BSA and 0.02% azide and stained with a live dead stain, either propidium iodide (PI) or EMA , to analyze cell viability using the flow cytometry technique. Cells were acquired on FACSAria  or LSR II , and data were analyzed with FlowJo software .CD4Th17 cell culture was performed as mentioned above, and cultured cells were treated with 500, 1000, 1500, and 2000 µg/ml of AETC or Milli-Q as a vehicle control on day 0. On day 4, cells were washed with PBS and fixed in 70% ethanol at 4°C overnight. Further, the cells were centrifuged at 380 × g for 5 min at room temperature. The supernatant (ethanol) was removed, and the cells were resuspended in PBS and centrifuged again. The supernatant was removed, the cell pellet was resuspended in PBS, and 10 µl of RNase A  was added. The samples were incubated for 4 h at 37°C. Finally, 10 µl of PI (1 mg/ml) was added, and the cells were incubated for an additional 2–3 min in the dark at 37°C. The cell cycle distribution was determined by flow cytometry for DNA content using LSR II . The data were finally presented as percentages of the cells in the G0/G1, S, and G2/M phases using Flowjo software.® GeneChip™ Command Console software. Raw data was automatically extracted using the Affymetrix data extraction protocol in the AffymetrixGeneChip® Command Console® Software (AGCC). A comparative study between the vehicle control and the AETC treated samples was done using the Transcriptome Analysis Console (TAC) software. TAC was used to import. CEL files, perform normalization, remove batch effects, and analyze differentially expressed genes. The genes with absolute log2 fold change ≥2 or ≤2 and p-value < .05 were considered differentially expressed. The raw data files have been submitted to Gene Expression Omnibus (GEO) with the accession number GSE201104.Total RNA from Th17 cultured cells, treated with 500, 1000, and 1500 µg/ml of AETC or Milli-Q as a vehicle control on day 0, was isolated as per the manufacturer’s protocol for the RNAeasy Mini Kit . Isolated RNA integrity was checked on a 1% agarose gel followed by Nanodrop quantification . The Affymetrix Gene Chip MTA 1.0 array was used for genome-wide expression analysis according to the manufacturer’s instructions. In brief, total RNA, 250 ng for each sample, was quantified and hybridized to microarray chips following the sequential steps described in the protocol. After hybridization, microarray chips were washed and stained using the Affymetrix Gene Chip Fluidics Station 450. The stained chips were finally scanned using an Affymetrix Gene Chip Scanner 3000 . The signal values were further evaluated using the Affymetrixp-value was < .05 in the Enrichr tool. To identify specific gene sets modulated by AETC, Gene set enrichment analysis (GSEA) was performed using the GSEA (gsea-msigdb.org) tool. A pre-ranked analysis was done using a list of genes ordered from most positive to most negative gene expression. The hallmark genes dataset was selected for the query, and datasets with >500 and <15 genes were excluded. A minimum cut-off of p-value < .05, and corrected for FDR <25% was set for the output.Functional analysis was performed using the Enrichr (maayanlab.cloud/Enrichr) tool. For gene ontology and pathway analysis, we examined gene enrichment in Biological Processes, KEGG, BioPlanet, and WikiPathway. The gene set enrichment was considered significant when the Total RNA from Th17 cultured cells, treated with 1500 µg/ml of AETC or Milli-Q as a vehicle control on day 0, was isolated according to the manufacturer’s protocol for the RNAeasy Mini Kit . Complementary DNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s instructions. Quantitative PCRs (qPCRs) were performed using SYBR Green . Primers used to quantify RORC, HIF1-α, IL-23R, CD3, IL-17A, IL-17R, IL-7R, SLC2A3, GLUT1, IFN-γ, PHD2, HK2 and VEGF were as shown in RORC, HIF1-α, IL-23R, CD3, IL-17A, IL-17R, IL-7R, SLC2A3, GLUT1, IFN-γ, PHD2, HK, and VEGF were determined by evaluating the threshold cycle (Ct) of the target gene after normalization against the Ct value of ACTB.Primer sequences for CD3, IL-17R, IL-7R, SLC2A3, VEGF, and PHD2 were obtained through OriGene. The levels of https://www.rcsb.org). The active regions of all proteins were identified using the COACH meta-server, and these results were compared and validated with the results obtained from the CASTp web server . Data are presented as mean ± SEM or mean ± SD with d-glucopyranosyl-(1→2)-β-d-glucopyranoside] (Rt 7.86) m/z 601 [M + H2O]+, jatrorrhizine (Rt 10.2.) m/z 338 [M]+, cordifolide A (Rt 8.48) m/z 621 [M + Na]+ (Compounds reticuline (Rt 10.62) m/z 330 [M + H]+, tinocordiside (Rt 8.14) m/z 419 [M + Na]+, 20β-Hydroxyecdysone (Rt 10.83) m/z 481 [M + H]+, cordifolioside A (Rt 5.82) m/z 503 [M + Na]+, tinosporinone (Rt 5.24) m/z 342 [M]+, 1,2-dihydroxyaporphine (R)-form, O1-Me, N-de-Me, N-formyl, 2-O-[β-M + Na]+  were ide+ T cells were treated with increasing doses of AETC  in the presence of stimulation (anti-CD3 and anti-CD28 mAb) to assess the cytotoxic effect of AETC on non-polarized CD4+ T cells. We found no cytotoxic activity of AETC in CD4+ T cell cultures at doses ranging between 500 and 2000 μg/ml, compared with the vehicle control at 24 h of incubation , was performed. AETC treatment at 1500, 1000, and 500 µg/ml showed an upregulation of 1406, 771, and 229 genes, respectively (via JAK-STAT, neutrophil-mediated immunity and neutrophil degranulation, interleukin-mediated signaling pathway, cellular response to interleukin-21, regulation of toll-like receptor 3 and 4 signaling pathway were enriched in 1500 µg/ml of AETC downregulated genes (STAT5B, STAT3, AHR, TGFBR2, RARA, IL-21R, IL-6ST, JAK3, MAPK3, VEGFA, and SLC2A3, important for Th17 cell differentiation and homeostasis, were downregulated (IL-17A, IL-17F, IL-1A, IL-2, IL-22, IL-3, and IL-9, important cytokines secreted by activated T cells and Th17 cells, were observed to be upregulated (IL-2, IL-7, ITGB2, IL-24, CCR7, PARP8, TGFBR2, HDAC4, CXCR4, STAT4, TNFRSF14, IL-12RB2, FOSL2, BCL6, CD27 FAS, CD68, IL-7R, PIK3CG, CDKN1B, IL-1R2, were significantly underexpressed in the AETC treated Th17 cells (MAP3K1, NFATC3, CD3G, ARFGAP3, PIK3CG, CTLA4, PLCG1, CARD11, PDK1, genes of the T cell receptor signaling pathway, were also downregulated (IL-17R, IL-7R, SLC2A3, and CD3 and enhanced expression of IL-17 and IFN-γ. Additionally, we studied genes RORC, HIF1-α, PHD2, GLUT1, HK2, IL-23R, and VEGF relevant for Th17 polarization and development. Genes RORC, HIF1-α, PHD2, GLUT1, HK2, IL-23R, and VEGF exhibited reduced expression at 1500 μg/ml of AETC treatment relative to the vehicle control (To understand the molecular mechanism of TC inhibition, transcriptomic analysis of naive CD4ectively . While 8ectively  comparedectively . We obseectively . In accoed genes . At 1000ed genes . Th17 ceegulated . Interesegulated . Genes i17 cells . MAP3K1,egulated . The upregulated . Gene seegulated . Signifi control .+ T cells, we further studied the effect of AETC on naive CD4+ T cells differentiated into Th1 cells. AETC significantly reduced IFN-γ production in naive CD4+ T cells induced under Th1 polarizing conditions at concentrations of 500 μg/ml and above compared to the vehicle control (After investigating the effect of AETC on Th17 polarized naive CD4 control .+ T cells differentiated under iTreg polarizing conditions and treated with AETC exhibited a significant downregulation of FoxP3 expression at concentrations of 500 μg/ml and above compared to the vehicle control (Naive CD4 control .The secondary metabolites of TC showed a good binding affinity with the proteins and receptors involved in T cell proliferation . The comThe binding affinity score of Apigenin-6-C-glucosyl and Pinoresinol for both TGFβ and TGFβR were numerically above −6 kcal/mol , considevia hydrogen bonds as shown in via three hydrogen bonds, two Pi-Alkyl bonds with Ile 28, Leu 113, and one Alkyl bond with Thr 102 residue of IL-23R (The analysis showed Apigenin-6-C-glucosyl interacted by several hydrogen bonds with TGFβR ; Table 2f IL-23R .A detailed study was performed to understand further the interactions of compounds in TC extract with the receptor IL-17R. The possible cavities/binding domains in the IL-17R proteins were analyzed using CASTp and COACH meta servers, and five binding regions were identified. The amino acid residues in the binding cavities are shown in + T cells and the cytokine-receptor signaling pathway, resulting in reduced effector function of polarized Th17 cells. In addition, AETC was found to be non-cytotoxic until 1500 μg/ml. In previous reports, TC extract had shown increased lymphocyte proliferation in mouse spleen in vivo and mouse splenocytes in vitro (+ T cell proliferation in Th17 cell culture at concentrations above 1000 μg/ml. The reduced proliferation of the CD4+ T cells could be a probable cause leading to a relative increase of the CD4− cells in the Th17 cell culture. The population present in the CD4− cell, whether a single or a mixed cell phenotype and the rationale for the increased proliferation of these unknown phenotypes needs further investigation.This study identified a novel mechanism of action of TC aqueous extract, which constrains the proliferation of naive CD4in vitro . Howeverin vitro . Our resetc., is well established (−CD25− splenocyte Th17 cell culture. In some experiments, we also observed inhibition of IL-17 even at doses of 500 μg/ml of AETC in naive cell culture, but this was inconsistent. Since CD8−CD25− cells mimic a complex cell culture system consisting of various immune cells; it was interesting to study the activity of AETC in such a system. Our results indicated that the inhibitory activity of AETC is also maintained in the presence of other immune cells. The immune cells in CD8−CD25− cell Th17 culture synergizing the modulatory activity of AETC remains to be studied. Analysis of separate generations of daughter cells in the AETC treated Th17 culture indicated a significant reduction in the IL-17 producing cells from the second generation at doses above 1500 μg/ml. This may indicate the development of Th17 cells being affected at an early phase by treatment with AETC. Since cell proliferation inhibition is associated with cell cycle arrest, our study did not observe a significant change in the different phases of the cell cycle until 1500 µg/ml. An increased frequency of cells was observed in the S phase and a reduced number in the G2 phase of the cell cycle at 2000 µg/ml of AETC. This indicated increased DNA replication but reduced cell division in the cell, which could be a possible reason for a significantly reduced cell number at 2000 µg/ml of AETC. In line with our study, it is reported that a methanol extract of TC effectively ameliorated autoimmune arthritis in the rat model, wherein TC downregulated the pro-inflammatory cytokines, mainly IL-17 and IL-1β (The importance of IL-17 producing Th17 cells in the pathogenesis of many inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, psoriasis, ablished . This stnd IL-1β .+ Th17 cells treated with AETC to understand the probable mechanisms of action. Our transcriptomic analysis of AETC treated Th17 cells shows the downregulation of genes and pathways key to the differentiation of Th17 cells and the pathogenesis of Th17-associated inflammatory and autoimmune diseases (RORC, HIF1-α, PHD2, IL-23R, and VEGF with downstream inhibition of genes HK2, GLUT1, and GLUT3 of the glycolytic pathway on TC treatment demonstrates the anti-inflammatory activity of TC.We performed genome-wide expression profiling of naive CD4diseases . The dowA decreased IL-17 mRNA expression is generally correlated to lower IL-17 protein expression. Although our current study observed no change to a slightly higher expression of IL-17 mRNA, there was inhibition of IL-17 at the protein level. Since the current study involves a multi-component herbal drug, the rationale behind this observation requires further extensive investigation. However, the phenotypic effect on Th17 cells, reduced proliferation, and inflammation, supported by reduced expression of a cascade of genes involved in the cytokine-receptor signaling pathway, is in concordance with the reported effect of TC on autoimmune mouse model studies.Leishmania donovani and treated with TC extract resulted in a substantial decrease in parasite load coupled with a reduced percentage of Treg cells (+IFN-γ+ producing cells (+ T cell culture was observed in our study in the same direction. The in vitro model utilized in the present study consisted of a uniform naive CD4+ T cell culture; this could be a possible reason for observing a significant inhibitory effect on IFN-γ producing cells in our study.Our results show a significant reduction in the expression of FoxP3 in iTreg culture, which concord with the study wherein mice infected with eg cells . IFN-γ peg cells . Althougng cells . A consiin silico molecular docking study, wherein the binding affinity of the TC components with Th17-associated proteins and receptor molecules suggests TC as a potential therapeutic candidate exhibiting anti-proliferative activity on polarized Th17 cells, resulting in a controlled immune response under inflammatory conditions. Some compounds, namely Apigenin-6-C-glucosyl, Pinoresinol, Acetylasimilobine, Cordifolioside-B, Secoisolariciresinol-9, Formylasimilobine, Cordifolide-A showed an efficient binding affinity for the protein molecules as well as their receptors involved in the Th17-associated immune pathways (The results observed with TC whole extract were further supported by an pathways . Based oetc., intensifies the use of T cells as a model system to unravel the molecular and therapeutic aspects of the associated diseases. In addition to its health-promoting and homeostasis-restoring activities, TC is clinically used for treating various complex disorders, autoimmune conditions, and cancers (in vitro culture model system in the current study. We have used a whole extract of TC for our study due to its wide availability and clinical use. However, it would be interesting to study the active compounds of AETC individually or in combination for their activity on immune cells through novel approaches like network pharmacology (T cells are a critical immune system component, eliciting a specific immune response against a foreign particle, and they are essential in maintaining immune homeostasis. The importance of T cells in autoimmune disorders, cancer, and complex disorders such as asthma, diabetes, cardiovascular diseases,  cancers . Therefomacology .in vitro model systems and clinical trials.In the present study, primary mouse T cells were polarized into individual T cell subsets under defined polarizing conditions. The observation of reduced frequency of IL-17 and IFN-γ secreting cells in Th17 and Th1 cell subsets, respectively, and reduced expression of FoxP3 in iTregs indicate that under stimulating conditions, TC could elicit its activity on different T cell subsets. The current study, supported by other reported immunomodulatory activity of TC on B cells, DCs, and macrophages, suggest that TC tends to restore immune homeostasis by eliciting its activity on various immune cells . Howeverin vitro cell culture systems which mimics an extreme inflammatory condition wherein TC exhibited its significant inhibitory activity. Another interesting observation from our study is the downregulation of CXCR4, a receptor for HIV entry into T cells, as the second most downregulated gene in the transcriptomic profile.In addition to TCR and co-stimulatory signals, T cell activation requires cytokine receptor signals . Cytokin+ T cells and secretion of IL-17 in the Th17 polarized cell culture system. This activity was observed via the cytokine-receptor signaling pathway involving the JAK-STAT signaling cascade, suggesting the potential immune therapeutic application of TC in Th17-associated inflammatory diseases. The docking results provide insight into the active ingredients involved in the immunomodulatory activity of TC on Th17 cells; however, the validation of the activity of individual components remains to be further elucidated. In addition, AETC showed inhibition of FoxP3 expression and secretion of IFN-γ in iTreg and Th1 cell subsets, respectively.In light of the above results, it can be concluded that AETC has potent immunomodulatory properties. AETC inhibited the proliferation of CD4in vitro, in vivo, and clinical trial studies together suggests its immune homeostasis maintaining capacity depending upon the disease condition type. Further, a comprehensive study on other immune subsets is needed to understand and validate the biological mechanism of AETC action. This work integrates the ancient knowledge of Ayurveda with the modern tools of science, resulting in deciphering the multi-dimensional activity of the herb TC on T cells.The broad spectrum activity of TC


Lkb1 (Liver Kinase B1) has known roles in the differentiation of airway epithelial cells during embryonic development. However, the effects of Lkb1 in adult lung epithelial progenitor cell regeneration and its mechanisms of action have not been determined. In this study, we investigated the mechanism by which Lkb1 regulates lung epithelial progenitor cell regeneration. Organoid culture showed that loss of Lkb1 significantly reduced the proliferation of club cells and alveolar type 2 (AT2) cells in vitro. In the absence of Lkb1, there is a slower recovery rate of the damaged airway epithelium in naphthalene-induced airway epithelial injury and impaired expression of surfactant protein C during bleomycin-induced alveolar epithelial damage. Moreover, the expression of autophagy-related genes was reduced in club cells and increased in AT2 cells, but the expression of Claudin-18 was obviously reduced in AT2 cells after Lkb1 knockdown. On the whole, our findings indicated that Lkb1 may promote the proliferation of lung epithelial progenitor cells via a niche-dependent pathway and is required for the repair of the damaged lung epithelium.Lung homeostasis and regeneration depend on lung epithelial progenitor cells.  The lung epithelium protects the lungs from environmental insults. The maintenance of lung tissue homeostasis and regeneration depend on healthy lung epithelial progenitor cells . Two majLKB1), a tumor suppressor encoded by serine/threonine kinase 11 (Stk11), was first associated with Peutz-Jeghers syndrome and was involved in the control of embryonic development, tissue homeostasis, stem cell function, energy metabolism, and apoptosis [Lkb1 causes embryonic lethality and, in adult mice, contributes to weight loss and disrupted metabolism [Lkb1 deletion [Lkb1 in periosteal mesenchymal progenitor cells induces osteoblastogenesis by activating the mammalian target of rapamycin complex 1 (mTORC1) [Lkb1 in intestinal epithelial cells leads to impaired immune barriers and increased bacterial susceptibility [Lkb1 knockout in the renal epithelium activates chemokines and recruits inflammatory cells, leading to polycystic kidney disease [Lkb1 deletion promotes tumor cell proliferation by regulating mitogen-activated protein kinase (MAPK) signaling [Lkb1 is required for the normal ciliated cell differentiation in both embryonic and adult lungs [Lkb1 deficiency in the embryonic lung epithelium results in weight loss and tamoxifen-induced death within 5 weeks. Lkb1 regulates airway goblet cell metaplasia by mediating interactions between airway progenitors and macrophages [Liver kinase B1  . The lostibility . Lkb1 kn disease . Lkb1 deignaling  and indult lungs . Our prerophages .Lkb1 may promote club cell proliferation via an autophagy-dependent pathway, while promoting AT2 cell proliferation in an autophagy-independent manner. Taken together, our results proposed a niche-dependent regulatory mechanism by which Lkb1 may affect airway and alveolar progenitor cell proliferation during homeostasis and lung injury.In conclusion, our results suggested that Lkb1 in the embryonic lung disrupts the lung epithelial structure and decreases the epithelial cell abundance in the adult lung [Lkb1 regulates the proliferation of lung epithelial progenitor cells in adult mice. Based on comprehensive analyses of published single-cell transcriptome sequencing data  [LKB1 expression in various cell populations  in normal human lung tissues   ,24,25,26 tissues A–C. We fd cells) D–F. Simial cells G–L. ThesLkb1 in airway club cells or alveolar AT2 cells to investigate the function of Lkb1 in the regeneration of lung epithelial progenitor cells in adult mice. We created CreER; Lkb1f/fScgb1a1 mice by crossing f/fLkb1 mice with CreERScgb1a1 mice, in which Lkb1 was conditionally deleted in airway club cells  vs. 10.873 ± 3.261 (Control), p = 0.001) and colony size (146.200 ± 13.580 (Lkb1 KO) vs. 175.591 ± 11.076 (Control), p = 0.003) of club cells sorted from CreER; Lkb1f/fScgb1a1 mice were obviously decreased  vs. 1.050 ± 0.020 (Control), p = 0.001) and colony size (101.806 ± 36.761 (Lkb1 KO) vs. 122.730 ± 39.257 (Control), p = 0.001) were reduced in the deletion of Lkb1  vs. 29.302 ± 7.109 (Control), p = 0.007) that in the absence of Lkb1 , but the GFP+ cells could not proliferate normally to form clones after Lkb1 deletion , Col1α, and α-SMA, between mice with and without Lkb1  was injected intratracheally to induce alveolar epithelium injury in stration D. The bostration E,F. Therout Lkb1 G. Immunof/f mice H. These Lkb1 may regulate the proliferation of lung epithelial progenitor cells via autophagy, we isolated mouse club cells and AT2 cells using FACS, and RT-PCR was conducted to analyze the expression of Atg-related genes. We found that the levels of autophagy markers, including Atg5 (0.003 ± 0.001 (Lkb1 KO) vs. 0.007 ± 0.004 (Control), p = 0.013), Atg6 (0.005 ± 0.001 (Lkb1 KO) vs. 0.017 ± 0.002 (Control), p = 0.001), Atg7 (0.004 ± 0.001 (Lkb1 KO) vs. 0.007 ± 0.002 (Control), p = 0.042), and Atg12 (0.005 ± 0.002 (Lkb1 KO) vs. 0.020 ± 0.006 (Control), p = 0.001) were lower in club cells isolated from CreER; Lkb1f/fScgb1a1 mice than in those isolated from control mice  vs. 0.005 ± 0.002 (Control), p = 0.017), Atg6 (0.017 ± 0.001 (Lkb1 KO) vs. 0.008 ± 0.004 (Control), p = 0.003), Atg7 (0.008 ± 0.002 (Lkb1 KO) vs. 0.004 ± 0.002 (Control), p = 0.027), and Atg12 (0.020 ± 0.004 (Lkb1 KO) vs. 0.006 ± 0.002 (Control), p = 0.002), were higher in AT2 cells isolated from CreER; Lkb1f/fSftpc mice  vs. 0.512 ± 0.026 (Control), p = 0.004) and organoid cultures (0.060 ± 0.044 (Lkb1 KO) vs. 0.201 ± 0.097 (Control), p = 0.023) after Lkb1 deficiency , were provided by Dr. Hongbin Ji . CreERScgb1a1 and CreERSftpc mice were obtained from the Jackson Laboratory . To induce Lkb1 knockdown in the airway epithelial progenitor cells, CreERScgb1a1 mice were crossed with f/fLkb1 mice to create CreERScgb1a1; f/fLkb1 mice. Similarly, CreERSftpc; f/fLkb1 mice were created by crossing CreERSftpc and f/fLkb1 mice. The heterozygous mice were mated for 3–5 generations to obtain homozygous mice for experimental study. All experimental mice at 8–12 weeks of age were randomly assigned to groups. Mice were anesthetized using 1% sodium pentobarbital (50 mg/kg). All procedures involving animals were reviewed and approved by the Tianjin Haihe Hospital Animal Care and Use Committee (2021HHKT-018).The experimental mice were retained in a specific pathogen-free (SPF) facility at Tianjin Haihe Hospital (SYXK (Jin) 2021–0002). The mice were exposed to a 12 h light/dark cycle and had free access to food and water. f/fLkb1 and CreERScgb1a1; f/fLkb1 mice every other day for three times. After tamoxifen injection was completed, the mice rested for seven days and then were intraperitoneally injected with naphthalene  dissolved in corn oil (250 mg/kg)  to induce mouse airway epithelium injury. After naphthalene treatment, mouse lung tissues were collected on days 0, 2, and 20 for histological analyses.200 mg/kg tamoxifen  was intraperitoneally injected into f/fLkb1 and CreER; Lkb1f/fSftpc mice received tamoxifen  every day for five consecutive days to induce Lkb1 knockout in mouse AT2 cells. Mice were anesthetized using 1% sodium pentobarbital and received an intratracheal injection of bleomycin (BLM)  at a dose of 2U/kg. Control animals only received phosphate-buffered saline (PBS) . Lung tissues were separated for histological analysis on day 14 after BLM administration.-CD34-CD45-EpCAM+CD24+Sca-1+ club cells and CD31-CD34-CD45-EpCAM+CD24-Sca-1- AT2 cells were sorted for organoid culture or RNA analyses.Mouse lung single-cell suspension was obtained as previously described . Briefly3 cells/well) or AT2 cells (2 × 104 cells/well) were mixed with MLg fibroblasts in Matrigel /basic medium (1:1) that contained DMEM/F12 , 10% FBS, 1% insulin-transferrin-selenium (ITS) (Sigma-Aldrich), 100 IU/mL penicillin, 100 µg/mL streptomycin and SB431542 (Sigma-Aldrich). The cell mixtures were then added to Transwell filter inserts  in 24-well plates containing 410 µL medium. Organoid cultures were maintained in an incubator with 5% CO2 at 37 °C, and the medium was renewed every other day. Organoid cultures were observed through an IX73 inverted fluorescence microscope . Clones with diameters greater than 50 μm were counted, and colony-forming efficiency (CFE) was measured by counting the number of colonies in each well as a proportion of the implanted cells 10 days after seeding. Organoid cultures were embedded in Tissue-Ted optimal cutting temperature (O.C.T.) compound  for immunofluorescence or lysed for RNA analyses.Sorted club cells  and incubated with anti-CYP2F2 , anti-surfactant protein C (SPC) antibody , anti-Podoplanin Monoclonal antibody , anti-Ki67 , and anti-ABCA3 . Samples were then incubated with the secondary antibody . Then, lung sections were mounted with Fluoromount G containing 4-6′-diami-dino-2-phenylindole (DAPI) ). The sections were observed under a IX73 inverted fluorescence microscope.Total RNA was extracted from lung tissues or sorted lung epithelial progenitor cells using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. 0.2 μg of total RNA was used for reverse transcription. Quantitative real-time PCR was performed using SYBR Green Supermix  and a Light Cycler 96 Real-Time PCR system . The PCR conditions were: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. Gene expression was measured relative to the level of the endogenous reference gene, mouse β-actin. The primer sequences used for qPCR are shown in The single-cell RNA sequencing data of human lung samples were downloaded from GEO database and analyzed using Seurat package on R platform. The cells expressed fewer than 3 genes, and the genes expressed in less than 200 cells were considered as low-quality cells and genes and removed from data matrices. The expression of genes was normalized using LogNormalize method: gene expression values for each cell were divided by the total number of transcripts of that cell and multiplied by 1000, and the results were then natural-log transformed using log1p. The cell types were annotated using canonical marker genes after dimension reduction and clustering based on KNN and SNN algorithm .t-tests were used to evaluate differences between the experimental and control groups. P < 0.05 was considered statistically significant .All data are displayed as mean ± standard deviation (SD). Student’s Lkb1 may promote the proliferation of lung epithelial progenitor cells via a niche-dependent pathway, and is required for the repair of the damaged lung epithelium.In conclusion, our findings indicated that


Total knee arthroplasty is a reliable procedure able to reduce pain and disability in patients suffering from osteoarthritis. However, a considerable percentage of patients still experiences unsatisfactory results. Medial pivot total knee arthroplasty has been introduced in the clinical practice to overcome problems related with classic design implants and better mimic native knee kinematics. The aim of this study was to analyze survivorship and clinical and radiographic outcomes of medial pivot implants.A systematic research was conducted in eight different databases. Thirty-four studies met the inclusion criteria and were included in the analysis. Data on objective and patients-reported outcomes, radiographic alignment, and survivorship were collected and analyzed. Revision rate was expressed as revision per 100 components years.A total of 3377 procedures were included. Mean follow-up was 85.7 months . The revision per 100 components years was 0.19, which corresponds to a revision rate of 1.9% after 10 years. Mean post-operative range of motion was 117.3 ± 0.4°. Mean clinical and functional Knee Society Score were, respectively, 85.9 ± 1.1 and 84.7 ± 3.5 at final follow-up. Post-operative femorotibial alignment was 177.1 ± 0.5°. Alfa and beta angles were 95.7 ± 0.1° and 89.2 ± 0.1°, respectively. Gamma and delta angles were 2.3 ± 0.6° and 86.7 ± 0.4°.Medial pivoting implants provided excellent survivorship and low revision rate, as well as good-to-excellent results in term of objective and patient-reported clinical outcomes, and reliable correction of radiographic parameters. More high-quality studies with long-term follow-up are needed to clarify the role of medial pivoting implants.The online version contains supplementary material available at 10.1007/s00402-021-04210-6. Total knee arthroplasty (TKA) represents a safe and reliable procedure to reduce pain and functional limitation caused by end-stage osteoarthritis (OA) . The incDespite the continuous studies to develop new prosthetic designs with advanced kinematic concepts, unsatisfactory results are still reported in 20% of patients undergone TKA . The impNative kinematic of the medial compartment of the knee is a “ball-and-socket” mechanism, with medial femoral condyle constrained in a pivot motion, while the lateral femur is free to translate posteriorly through a complete arc of flexion .The medial pivot design was introduced in 1994 miming the physiological knee kinematic, to ensure greater efficacy of extensor mechanism in full range of motion (ROM) , 9. ThisSeveral studies showed promising mid-term results of medial pivot TKA , but theThe purpose of this systematic review is to analyze survivorship and clinical and radiographic outcomes of medial pivot design TKA.Cochrane Handbook of Systematic Reviews of Interventions [A systematic review of the literature has been performed, following ventions  and Prefventions  for studA systematic search from January 1st, 1990, until October 1st, 2020, was performed in the following databases: the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE/PubMed, Embase, Scopus, the Science Citation Index Expanded from Web of Science, ScienceDirect, CINAHL, and LILACS. The research was conducted using the following keywords alone and in all the various combinations: “Medial pivot knee”, “TKA design”, “medial congruent knee”, and “medial stabilized knee”.Clinical studies reporting objective and patient-reported outcome of medial pivot design TKA were considered eligible for inclusion. There was no quality restriction for study inclusion. Case report, technical notes, editorial commentaries, ex vivo, biomechanical, pre-clinical, and clinical studies without adequate quantitative or qualitative data were excluded. Studies that did not report clear clinical-functional data or survivorship of primary medial pivot TKA were excluded from this research.Two reviewers independently screened each title and abstract collected from the primary electronic search. In case of relevant title and abstract, the full-text version was obtained.All references of each study were screened to find any additional relevant paper potentially missed with the first review process. The two reviewers independently followed the same checklist to screen all studies and evaluate the eligibility criteria. Disagreements were resolved through discussion with third reviewer.The primary endpoints of this analysis were the survivorship and clinical outcome of medial pivot design TKA and revision rate. Secondary endpoints were radiological alignment and complications.The level of evidence of included studies were evaluated through the adjusted Oxford Centre For Evidence-Based Medicine 2011 Levels of Evidence . The quaThe risk of bias was assessed with “Risk Of Bias” (Rob 2.0) for Randomized Trial (RCT) and “Risk of bias in non-randomized studies” (ROBINS-I)  to evaluRelevant conflict of interest having potential impact on study design and results were considered and reported.A stepwise analysis on study design, aim of study, level of evidence, journal, number of procedures included in the study, prosthetic implant used, indication to TKA, mean age, body mass index (BMI), follow-up, and patellar resurfacing. Disparities in data extraction were discussed and resolved by consensus meeting between the authors. When studies reported mixed cohorts of patients, data extraction was selectively focused on procedures involving medial pivot implants.α), femoral beta angle (β), sagittal femoral gamma angle (γ), and sagittal tibial delta angle (δ) were collected. The femoral and tibial radiolucent lines (RLL) were also noted, and divided into major and progressive and minor and non-progressive, according to the knee society total knee arthroplasty roentgenographic evaluation and scoring system [Radiological data reported as pre- and post-operative femoral tibial angle (FTA), implant alignment with alpha angle  were finally included in this systematic review.2. The mean follow-up was 85.8 months . Patellar replacement was described in 23 studies [The mean age at surgery was 69.9 ± 4.4 years and the mean BMI was 29.9 ± 1.0 kg/m studies , 42–46, The revisions per 100 CY were 0.19, which correspond to a revision rate of 1.9% after 10 years Table . The cauThe pre-operative mean ROM was 103.4 ± 1.5 (CI 95% 103.3–103.5°). Mean pre-operative KSS and KSS for function were 38.7 ± 1.7 (CI 95% 38.6–38.9) points and 45.8 ± 4.4 (CI 95% 45.7–45.9) points, respectively, and mean pre-operative OKS was 38.6 ± 8.6 (CI 95% 38.2–39.0) points. Considering post-operative results, mean ROM was 117.3 ± 0.4° (CI 95% 117.29–117.31), clinical and functional KSS were 85.9 ± 1.1 (CI 95% 85.88–85.92), and 84.7 ± 3.6 (CI 95% 84.6–84.8). WOMAC and KOOS values were 39.1 ± 7.6 (CI 95% 38.8–39.4) and 84.9 ± 2.2 (CI 95% 84.6–85.2), OKS was 28.2 ± 5.7 (CI 95% 27.9–28.5), and FJS mean value was 68.5 ± 1.0 (CI 95% 68.4–68.6).Mean ROM of RCTs improved from 112.2 ± 7.2° (CI 95%111.5–112.9) preoperatively to 115.9 ± 1.6° (CI 95% 115.7–116.1) at final follow-up, while clinical and functional KSS varied from 34.0 ± 5.1 (CI 95% 33.3–34.7) and 44.7 ± 0.2 (CI 95% 44.6–44.8) before surgery to 87.7 ± 1.0 (CI 95% 87.6–87.8) and 78.1 ± 2.6 (CI 95% 77.8–78.4). OKS improved from 25.9 ± 0.9 (CI 95% 25.7–26.1) to 35.9 ± 1.5 (CI 95% 35.7–36.1).Detailed functional outcomes are reported within Table Eight studies , 43, 48 Minor (< 2 mm) and non-progressive femoral radiolucent lines were found in 146 (7.5%) knees on the femoral side and in 175 (9.0%) cases on the tibial side. Major or progressive femoral RLL were reported in 11 (0.5%) cases. Two studies , 35 repoPost-operative outliers were 116 (24.3%) cases . Regarding RCTs, the FTA varied from 188.1 ± 5.7° (CI 95% 187.4–188.8) after the procedure to 175.9 ± 2.3° (CI 95% 175.2–176.6) after 9.1 years.Post-operative alfa and beta angles were 96.6 ± 2.2° (CI 95% 96.3–96.9) and 88.6 ± 0.1° (CI 95% 88.5–88.7), while gamma and delta values were 2.8 ± 0.4° (CI 95% 2.7–2.9) and 86.5 ± 0.5° (CI 95% 86.4–86.6). In RCT, studies were not reported any case of RLL or AL. Details of radiological measurements are reported within Table The main complications were 30 (1.0%) cases of stiffness, 25 (0.9%) cases of deep vein thrombosis, 17 (0.6%) PJIs, 17 (0.6%) superficial wound infections, 16 (0.6%) cases of persistent pain, 16 (0.6%) retarded wound healings, and 11 (0.4%) periprosthetic fractures.Other reported complications were 7 (0.2%) cases of pulmonary thromboembolism, 6 (0.2%) AL, 6 (0.2%) peroneal neurapraxias, 4 (0.1%) cases of persistent knee swelling, 3 (0.1%) cases of patellar fractures, 3 (0.1%) cases of knee instability, 1 regional pain syndrome, and 1 patellar tendon rupture. In RCT studies, 14 (3%) cases of stiffness and 4 PJI were reported. Of these, three patients needed reoperation.The aim of this systematic review was to summarize the literature evidence on survivorship and clinical–radiological outcomes of the medial pivoting design TKA. To the best of our knowledge, this is the first systematic review of the literature with a detailed ROM report, patient-reported and objective outcome measures, radiological outcomes, and complications of patients who underwent medial pivot TKA.Although knee replacement is one of the most performed surgical procedures worldwide, some concerns are related to the relatively high percentage of unsatisfactory outcomes , 50. NatThe most important finding of this research is the excellent overall survivorship of medial pivoting design TKA. In fact, the revisions per 100 CY were 0.19, corresponding to a revision rate of 1.9% after 10 years Fig. . Only 51Furthermore, medial pivot TKA showed good-to-excellent results in term of objective and patient-reported outcomes measures Table . The RCTA potential conflict of interest was disclosed in ten studies , 45, 48 Hossain et al.  found beThis study has several limitations. First, there is a low level of evidence among included studies, since 22 papers were classified as level of evidence IV. Moreover, only one research was considered at low risk of bias precluding strong conclusions on the results of the included studies. No quality restriction was applied to obtain the largest population of medial pivot TKA. There are possible selection biases deriving from different diagnosis and high heterogeneity in TKA indications that include population .Heterogeneous RCTs studies have been included where the medial pivoting design was compared to the conventional posterior stabilized and cruciate retaining TKA or compared to other models of the medial pivot TKA. All reported outcome measures were highly heterogeneous resulting in a difficult systematic analysis. To reduce bias, the largest number of procedures available in the literature were included and variables gathered from RCTs were analyzed separately.Conflict of interests were disclosed in several included studies, and this aspect can overestimate the medial pivot TKA outcomes. Only six studies , 42, 45 Medial pivoting design TKAs provided high survivorship of implants, with a revision rate of 1.9% after 10 years. Moreover, good-to-excellent results were obtained in term of objective and patient-reported outcomes measures. Radiological evaluation of studies showed excellent post-operative correction of axial deformities. Three RCTs demonstrated better functional outcomes of medial pivoting designs when compared to the conventional TKA. Only one RCT showed worse results in the medial pivoting group, whereas the remaining RCTs demonstrated non-significant differences between groups. However, several limitations and biases affect this review and further high-quality studies are needed to clarify the role of medial pivoting implants in TKA.Supplementary file1 (DOCX 20 kb)Below is the link to the electronic supplementary material.