Obesity has become a global pandemic. WDTC1 is a WD40-containing protein that functions as an anti-obesity factor. WDTC1 inhibits adipogenesis by working as an adaptor of the CUL4-DDB1 E3 ligase complex. It remains unclear about how WDTC1 is regulated. Here, we show that the TRiC/CCT functions as a chaperone to facilitate the protein folding of WDTC1 and proper function in adipogenesis. Through tandem purification, we identified the molecular chaperone TRiC/CCT as WDTC1-interacting proteins. WDTC1 bound the TRiC/CCT through its ADP domain, and the TRiC/CCT recognized WDTC1 through the CCT5 subunit. Disruption of the TRiC/CCT by knocking down CCT1 or CCT5 led to misfolding and lysosomal degradation of WDTC1. Furthermore, the knockdown of CCT1 or CCT5 eliminated the inhibitory effect of WDTC1 on adipogenesis. Our studies uncovered a critical role of the TRiC/CCT in the folding of WDTC1 and expanded our knowledge on the regulation of adipogenesis. Adipose, the Drosophila homolog of WDTC1, caused obesity in Drosophila  is an anti-obesity factor conserved from worms to mammals . Mutatioosophila . Wdtc1 hfat mass . In humafat mass . WDTC1 ifat mass . We havefat mass . MED20 dfat mass . HoweverThe tailless complex protein-1 (TCP-1) ring complex (TRiC), also known as complex containing TCP-1 (CCT), is the primary eukaryotic cytosolic chaperonin that is essential for cytosolic protein folding and assembly . The TRiHere, in an effort to purify WDTC1-interacting proteins, we identified the TRiC/CCT as a major binding partner of WDTC1. The TRiC/CCT binds WDTC1 primarily through the CCT5 subunit and facilitates the folding of WDTC1. We demonstrate that the TRiC/CCT is required for the inhibitory effect of WDTC1 on adipogenesis. Our findings uncover a role of the TRiC/CCT in regulating adipogenesis.We obtained Strep-Tactin Superflow (2-1206-002) and desthiobiotin (2-1000-001) from IBA life sciences; dexamethasone, isobutylmethylxanthine (IBMX), biotin, pioglitazone, bovine insulin, urea, SDS, DTT, DMSO, and Triton X-100 from Sigma-Aldrich; Dulbecco’s modified Eagle’s medium (DMEM) with low (1 g/L) or high (4.5 g/L) glucose, fetal and neonatal bovine serum, blasticidin, and puromycin from Thermo Fisher Scientific; donkey anti-rabbit IgG or anti-mouse IgG conjugated to horseradish peroxidase from Jackson Immuno Research; protease inhibitor cocktail from Roche Applied Science; and all other chemicals from local suppliers unless otherwise specified.Full-length cDNAs of human CCT1-8 subunits of the CCT were generous gifts from Dr. Jiahuai Han at Xiamen University, China. The coding regions of these genes were cloned into pcDNA3.3 (Thermo Fisher Scientific) with an N-terminal Flag tag. Mouse WDTC1 were cloned from a cDNA library prepared from testis mRNA of a C57BL6 mouse. A pIRESpuro-GLUE vector   was used2. To generate stable cell lines, HEK293T cells were set up at 5×104 cells per 10-cm dish on day 0. On day 1, cells were transfected with either 0.5 μg pIRESpuro-GLUE (pGlue) or WDTC1/pGlue using polyetherimide  at 2.4:1 (wt/wt) of PEI to plasmids. Starting from day 2, cells were selected with Medium A containing 1 μg/ml puromycin until colonies were formed, which were then screened by Western blot using the anti-HA antibody . A positive clone of either construct was subcloned and used for the current studies.HEK293T cells were cultured as previously described . In brie2. Cells were maintained at no more than 50% confluence.3T3-L1 preadipocytes were obtained from the American Type Culture Collection and cultured in Medium B  NCS, 100 U/ml penicillin, and 100 mg/ml streptomycin) at 37°C in an atmosphere of 8.8% COTo differentiate into mature adipocytes, 3T3-L1 cells were subjected to a standard cocktail hormone as previously described . In brie4 cells/35-mm dish. Cells were counted every day for 4 days.For the cell growth curve, control and CCT1/5 knockdown 3T3-L1 cells were set up at 1 × 10For Oil Red O staining, fully differentiated 3T3-L1 cells were washed with PBS and fixed in 2.5% glutaraldehyde at room temperature for 1 h. Cells were then treated with 60% isopropanol for 5 min and stained with freshly prepared Oil Red O (1.8 mg/ml) for 5 min. For quantification of triglycerides in 3T3-L1 adipocytes, cells were harvested in PBS containing 0.5% SDS, gradually heated up to 95°C, and kept at 95°C for another 5 min. The cooled samples were subjected to triglyceride measurement using a commercial kit from Wako Chemicals. The content of triglycerides was normalized to protein content, which was quantified by using a Pierce BCA kit (Thermo Fisher Scientific).5 cells per 60-mm dish. On day 2, cells were transfected with 1 μg lentiviral vector and two packaging plasmids, 0.75 μg psPAX2  and 0.25 μg pMD2.G , using polyetherimide . On day 3, cells were refed with Medium A. On days 4 and 5, lentiviral particles were collected from the media after centrifugation at 1,000 g for 5 min, concentrated at 70,000 g for 2 h or directly aliquoted, and stored at −80°C until further use.To produce lentivirus in HEK293T cells, on day 0, cells were set up at 2.5×10For lentiviral infections, 3T3-L1 preadipocytes were set up, cultured to 50%–70% confluence, and infected with lentivirus in the medium containing 8–10 μg/ml polybrene. Cells, 24 h after infection, were selected against 5 μg/ml puromycin and/or 5 μg/ml blasticidin for at least 48 h and then used for the described experiments.2, and 2.5 mM desthiobiotin). The eluted fractions were combined and incubated with 0.1 ml calmodulin beads at 4°C for 3 h, washed with 5 ml Buffer C , and eluted in 0.1 ml Buffer B. The extracts were subjected to silver staining and protein ID analysis by AB5600+ mass spectrometry.To purify WDTC1-interacting proteins, nuclear extracts were prepared from 10 of 10-cm dishes of stable cell lines of pGLUE or WDTC1/pGLUE as previously described . The nucTo express recombinant WDTC1, the coding region of WDTC1 was cloned into a pET28a vector and transformed into BL21(DE3)pLys bacterial cells. On day 0, a colony was inoculated in the LB medium and cultured at 37°C overnight. On day 1, the bacteria were scaled up at 1:100, cultured to an OD600 around 0.8, and induced with 1 mM IPTG for 3 h. Cells were then harvested and lysed in PBS containing 1 mg/ml lysozyme supplemented with protease inhibitors. As WDTC1 was expressed in the inclusion bodies, the pellet from the lysis was collected and thoroughly washed with PBS containing 1% Triton X-100 five times. The final product shows about 95% purity on SDS-PAGE.in vitro, purified WDTC1 proteins were denatured in 6 M urea in 10 mM HEPES pH 8.0 at 25°C overnight. On the next day, cytosolic fractions were prepared from cells infected with control shRNA, shCC1, or shCCT5. In brief, cells were harvested by spinning at 1,000 g for 5 min and resuspended in hypotonic buffer  to swell for 10 min on ice. Cells were homogenized in a Dounce homogenizer with 10 strokes using a loose-fitting pestle and spun at 1,000 g for 15 min. The resultant was supplemented with 0.11 volume of 10× cytoplasmic extract buffer  and spun at 100,000 g at 4°C for 1 h. The supernatant is the cytosolic fraction. The denatured WDTC1 proteins (100 ng) were diluted into cytosolic fractions and incubated at 25°C for 1 h. The samples were then centrifuged at 20,000 g for 10 min, and the supernatant and pellet fractions were subjected to SDS-PAGE and Western blot.To refold WDTC1 Western blot was carried out as previously described . The folp < 0.05; **, p < 0.01; and ***, p < 0.001. No data were excluded from any of the experiments.All the statistical analyses were performed using Student’s two-tailed paired t-test. The value represents mean ± SEM. Statistical details of all experiments can be found in the figure legends, including the exact number of cell samples. Asterisks (*) indicate levels of statistical significance. *, To search for potential interacting proteins of WDTC1, we generated a stable HEK293T cell line expressing Glue (SBP-HA-CBP)-tagged WDTC1 and performed tandem purification . When suThe chaperonin TRiC/CCT has been shown to facilitate the folding of WD40-domain containing proteins . As WDTCtetratricopeptide domains (TPR)  . To map ns (TPR)  and tranWe then determined which subunit of the TRiC/CCT interacts with WDTC1. We co-transfected WDTC1 with each of the Flag-tagged subunit of the TRiC/CCT into HEK293T cells and performed a Co-IP experiment. As shown in We then went on to check the effect of the TRiC/CCT on WDTC1. We generated a stable cell line in 3T3-L1 cells that overexpress WDTC1 and knocked down CCT1 or CCT5 to check their effect on the protein and RNA levels of WDTC1. Consistent with the work of To further confirm the results, we overexpressed WDTC1 in control and CCT1 or CCT5 knockdown 3T3-L1 cells, treated with cycloheximide, and examined the half-life of WDTC1. As shown in To examine how WDTC1 was degraded in cells lack of the TRiC/CCT, we treated CCT1 or CCT5 knockdown 3T3-L1 cells with MG132 or chloroquine and found that chloroquine, but not MG132, largely restored the level of WDTC1, suggesting a lysosomal degradation of WDTC1 in TRiC/CCT-depleted cells .in vitro refolding assay. We expressed WDTC1 in E. coli and purified it from the inclusion bodies (To directly test whether the TRiC/CCT was required for the folding of WDTC1, we carried out an n bodies . The purWe then went on to test the effect of the TRiC/CCT on the inhibitory effect of WDTC1 on adipogenesis. We overexpressed WDTC1 in 3T3-L1 cells, knocked down either CCT1 or CCT5, and then, subjected the cells to differentiation. After 8 days of differentiation, while the overexpression of WDTC1 suppressed differentiation, knockdown of either CCT1 or CCT5 largely restored the differentiation, as illustrated by lighter Oil Red O staining, intracellular triglyceride content, and the expression of the adipocyte markers . These rAdipogenesis is closely associated with obesity and related metabolic disorders . Over thIn addition, we demonstrated a clear role of the TRiC/CCT in regulating adipogenesis. The TRiC/CCT is a common molecular chaperone found in all eukaryotes, and it is involved in many cellular activities by helping the folding of its substrates . WD40-coMoreover, there are also some limitations of this study. In the adipogenesis study, we only choose 3T3-L1 preadipocytes, which, although widely applied in adipogenesis , cannot


Astragalus grows mainly in drought areas. Cycloastragenol (CAG) is a tetracyclic triterpenoid allelochemical extracted from traditional Chinese medicine Astragalus root. Phospholipase C (PLC) and Gα-submit of the heterotrimeric G-protein (GPA1) are involved in many biotic or abiotic stresses. Nitric oxide (NO) is a crucial gas signal molecule in plants.Arabidopsis thaliana , the results showed that low concentrations of CAG induced stomatal closure, and high concentrations inhibited stomatal closure. 30 µmol·L−1 CAG significantly increased the relative expression levels of PLC1 and GPA1 and the activities of PLC and GTP hydrolysis. The stomatal aperture of plc1, gpa1, and plc1/gpa1 was higher than that of WT under CAG treatment. CAG increased the fluorescence intensity of NO in guard cells. Exogenous application of c-PTIO to WT significantly induced stomatal aperture under CAG treatment. CAG significantly increased the relative expression levels of NIA1 and NOA1. Mutants of noa1, nia1, and nia2 showed that NO production was mainly from NOA1 and NIA1 by CAG treatment. The fluorescence intensity of NO in guard cells of plc1, gpa1, and plc1/gpa1 was lower than WT, indicating that PLC1 and GPA1 were involved in the NO production in guard cells. There was no significant difference in the gene expression of PLC1 in WT, nia1, and noa1 under CAG treatment. The gene expression levels of NIA1 and NOA1 in plc1, gpa1, and plc1/gpa1 were significantly lower than WT, indicating that PLC1 and GPA1 were positively regulating NO production by regulating the expression of NIA1 and NOA1 under CAG treatment.In this study, using the seedlings of GPA1 and PLC1 acted upstream of NO.These results suggested that the NO accumulation was essential to induce stomatal closure under CAG treatment, and The online version contains supplementary material available at 10.1186/s12870-023-04555-7. Astragalus strictus root tissues on the following wild plants of Tibet: Medicago lupulina, Elymus nutans, Oxytropis microphylla, Festuca ovina, Stipa purpurea, Stipa capillacea, Kobresia littledalei, and Eragrostis nigra [Arabidopsis thaliana  [Allelopathy mainly refers to plants releasing allelopathic substances into the environment through leaching, volatilization, root secretion, leaf decomposition, and residual strain. These allelopathic substances inhibit or promote the growth of plants, secreting those allelopathic substances or their neighboring plants . The allis nigra . It has haliana) , 8.3 [3 is phosphorylated to generate IP6, which mediates the release of Ca2+ [6 of PLC regulates the change of intracellular Ca2+ level, thereby reducing the intracellular turgor pressure and finally causing stomatal closure [2+ oscillation by ABA and reduce the expression level of PI-PLC, which also weakens the sensitivity of tobacco leaves to ABA [A large number of phospholipases catalyze the hydrolysis of phospholipids in nature. According to the position of the phospholipid cleavage bond in the substrate, phospholipases are divided into phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD) . PLC, as3 . DAG is  of Ca2+ . Then, i of Ca2+  found th closure . The PLCs to ABA . Althouggpa1 mutant guard cells, indicating that Gα-submit of the heterotrimeric G-protein (GPA1) is involved in the regulation of plant anion channels. In the stomatal response triggered by ExCaM, Gα can activate the Ca2+ channel by promoting the formation of hydrogen peroxide (H2O2) and Nitric oxide (NO) in A. thaliana guard cells to induce stomatal closure [The heterotrimeric G protein consists of three subunits of α, β, and γ. It plays roles in plant seed germination, seedling growth and development, and plant reproduction-related processes . It also closure . Zhang e closure  showed tNO is a vital gas signal molecule in plants, which regulates various physiological processes of plants, including seed germination , photomoA. thaliana and the roles that PLC1, GPA1, and NO play in this process. In addition, the results reveal the allelochemical effect of the triterpenoid CAG on A. thaliana and provide a theoretical basis for the future application of triterpenoid allelochemicals in stomata. The study will lay a foundation for agricultural production in the future.Until now, the allelopathic effects of CAG in plants have not been reported. In this work, results analyzed the effects of CAG treatment on stomatal movement in A. thaliana seedlings treated with different concentrations of CAG for 5 days is shown in Fig. –1 CAG treatment. But after 30–60 μmol·L–1 CAG, the leaves gradually turned yellow. These results reflected that low concentrations of CAG could promote A. thaliana seedling growth while high concentrations of CAG can inhibit A. thaliana seedling growth.The growth status of A. thaliana leaves. The stomates treated with the different concentrations of CAG at different time are shown in Fig. −1 CAG was significantly lower than that of the control group (0 µmol·L−1 CAG). Stomatal apertures decreased significantly after 30 µmol·L−1 CAG treatment for 1.5 h. Therefore, 30 µmol·L−1 CAG treatment for 1.5 h was selected for further experiments.And then to determine the effect of CAG on stomatal movement in PLC1 and GPA1 and the enzymes activity levels of PLC and GTP hydrolysis were increased markedly at 30 µmol·L−1 CAG treatment, indicating that PLC1 and GPA1 involved in CAG-induced stomatal movement -4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium , 100 μM NG-nitro-L-Arg methylester  and 100 μM tungstate  treatments  significantly promoted stomatal closure in all the lines under CAG treatment Fig. a. Under  WT Fig. b. But th WT Fig. c, d. TheMoringa oleifera promoted the growth of Lepidium sativum at the lowest tested concentration, while the highest tested concentration inhibited the growth of Lepidium sativum [Parthenium hysterophorus L. showed an allelopathic effect by stimulating the growth of other plant species at low concentrations and suppressing the growth at high concentrations [Astragalus plants have been reported to inhibit seed germination, radical elongation, and seedling growth of many other plants [A. thaliana  genetic background. The T-DNA insertion mutants of plc1 (SALK_025769c), gpa1 (SALK_001846), noa1 (CS6511), nia1 (CS6936), nia2 (CS2355) were obtained by the Arabidopsis Biological Resource Center (https://abrc.osu.edu). Cycloastragenol was purchased from Shanghai Shifeng Biological Co., Ltd., with a purity of ≥ 98%.All of the A. thaliana seeds were cultured according to the study with minor modifications [The ications . Seeds wPLC1 complementation lines and overexpressed lines, full-length cDNA of PLC1 was amplified using by high-fidelity DNA polymerase  and subcloned into p-DONR vector. Confirmed construct was transformed into Escherichia coli and PLC1 was cloned into the destination vector pBIB-35S-PLC1-GFP vector via LR reaction. After verification of the construct by using traditional Sanger sequencing, the construct was transformed to plc1 mutant and WT via an Agrobacterium tumefaciens-mediated floral-dip method.Transgenic plants were generated according to the study with minor modifications . For the−1 PBS (pH 7.4), and centrifuged at 4000 rpm for 15 min at 4 ℃. The supernatant was used for assay. PLC activity measurement was performed using the PLC assay kit  according to the description of the manual.PLC activity were measured according to the study with minor modifications , 43. 0.2A. thaliana seedlings by 1.8 mL 0.01 mol·L−1 PBS (pH 7.4). The homogenate was centrifuged at 4000 r·min−1 for 15 min at 4 °C. The GTP hydrolysis activity was detected by a GTP hydrolysis assay kit . The manipulation followed the protocol of the GTP hydrolysis assay kit.GTP hydrolysis activity was measured as described previously . BrieflyTotal RNA extraction, reverse transcription PCR and qRT-PCR analysis were measured as described previously with minor modifications . Total RNO production was detected by specific fluorescent probes 4,5-diaminofluorescein diacetate  . EpidermArabidopsis seedlings were incubated in MES-KCl buffer  under light for 2 h to induce stomatal opening. Subsequently, incubated in MES-KCl buffer alone or MES-KCl buffer containing 30 µM Cycloastragenol, 200 µM c-PTIO, 100 µM L-NAME or 100 µM Na2WO4 under the same light conditions for 30 min-2 h. Subsequently, the abaxial epidermis of Arabidopsis thaliana was placed onto a slide and photographs were taken using the Leica DM4000B. The aperture width of each stomatal pore was determined from the image. To avoid the potential effects of the circadian rhythm on stomatal aperture, stomatal assays were always started at the same time of day. In each experiment, 30 pairs of guard cells of each plant line were measured, and three replications were maintained for each stomatal assay experiment. Stomatal apertures were digitized using a Dn-3 Image Analysis System . The difference at the level of P < 0.05 was regarded as significant.Leaves were selected from the same position and the similar size at the rosette leaves for stomatal aperture measurements. Stomatal apertures were measured as described previously with minor modifications , 46. In P-value < 0.05 was considered statistically significant. Origin 9.0 drawing software was used for drawing.Three technical replicates were performed for each experiment. The data showed as the mean ± standard error (SE). The statistically significant differences were analyzed with SPSS version 17.0, and error bars were determined based on Duncan’s multiple range test. Additional file 1: Fig. S1. Identification of plc1/gpa1 double mutant. Molecular analysis of WT, plc1, gpa1, and plc1/gpa1, primers LP, RP and LB (LBb1.3) were used to target the flanking sequences of the T-DNA. Fig. S2. PCR amplification of CDS of PLC1. Fig. S3. Construction of 35S-AtPLC1-GFP recombinant vector. Fig. S4. Screening of PLC1 transgenic trains. Fig. S5. PCR results of generation seedings. Fig. S6. RT-qPCR results of transgenic. Table S1. List of gene primers for qRT-PCR. Table S2. List of primers used for PCR identification.


The O PA-2 and A22 IRQ vaccines, containing 10 μg and 5 μg of antigen, respectively, provided protection against homologous viruses in pigs. This is the first report demonstrating that the antigens obtained from the pilot-scale production of O PA-2 and A22 IRQ are viable candidate vaccines. These results will pave the way for industrial-scale FMD vaccine production in South Korea.Foot-and-mouth disease (FMD) is a highly infectious disease affecting cloven-hoofed animals and causes significant economic losses to the livestock industry. The Type O PanAsia-2 (O PA-2) vaccine strain is protective against a wide range of serotype O FMD virus (FMDV) strains in East Asia, and A22 Iraq/24/64 (A22 IRQ) is the most widely used vaccine strain in FMD vaccine antigen banks. The aim of this study was to produce antigens from O PA-2 and A22 IRQ viruses using a 100 L bioreactor and evaluate the protective efficacy of varying antigen concentrations in pigs. More than 2 μg/mL of the antigen was recovered from the O PA-2 and A22 IRQ virus-infected supernatants. Further, inactivation of O PA-2 and A22 IRQ by binary ethyleneimine revealed that the viral titers decreased below 10 Aphthovirus genus within the Picornaviridae family. This virus encompasses seven recognized serotypes: O, A, Asia 1, C, South African Territories (SAT) 1, SAT 2, and SAT 3  , by the Animal and Plant Quarantine Agency (APQA) and the Korea Research Institute of Bioscience & Biotechnology for use in suspension culture with serum-free media—were adapted for growth in Cellventog, 20 min, 4 °C). These supernatants were used to quantify FMDV particles (146S) and viral titers.The O PA-2 and A22 IRQ strains were obtained from O PAK/44/2008 (GenBank accession no. GU384682) and A22 Iraq/24/64 (GenBank accession no. AY593764.1), respectively. The two FMD vaccine strains were recombinant viruses in which P1 of the recombinant O1 Manisa backbone was replaced with P1 of O/PAK/44/2008 or A22/Iraq/24/64 and termed O PA-2 and A22 IRQ, respectively [5 cells/mL. The bioreactor was equipped with probes to monitor and regulate the temperature and dissolved oxygen levels, maintaining them at 37 °C and 50% air saturation, respectively. The pH was controlled within the range of 7.2–7.4 by adding CO2 and a 1 M NaOH solution. The agitation speed was set at 100 rpm. Samples were collected every 24 h to assess the cell density and viability. When the cell density reached approximately 3 × 106 cells/mL, 30 L of fresh Cellvento BHK-200 cell culture medium was added to the bioreactor. Subsequently, O PA-2 or A22 IRQ was introduced to the bioreactor along with fresh medium at MOIs of 0.001 and 0.05, respectively. The supernatants infected with O PA-2 and A22 IRQ were collected at 16 and 24 hpi, respectively. These supernatants were then transferred to a 250 L inactivation tank  and inactivated using 3 mM binary ethyleneimine . The inactivation process took place for 24 h at 26 °C, employing two individual tanks. Hourly samples were collected from the inactivation tank during the 0 to 6 h period to evaluate the inactivation kinetics of each strain. The remaining BEI was neutralized by adding 10% of a 1 M sodium thiosulfate solution , resulting in a final concentration of 2%. The inactivated viruses were treated with 50% Polyethylene Glycol (PEG) 6000 (Sigma-Aldrich) solution into a final 7.5% concentration of PEG. The solution was added with NaCl (0.5 M) and then incubated for 16 h at 4 °C. Subsequently, they were harvested using a continuous centrifuge  operating at 16,400 rpm and a flow rate of 42 L/h. The resulting pellet was resuspended in 50 mM Tris containing 300 mM KCl (pH 7.6) and stored at −70 °C until further use.Cellvento BHK-200 cell culture medium was prepared using a mixer . A total of 70 L of the medium was then transferred to a 100 L bioreactor (Sartorius) with an initial cell density of 3 × 1050) per milliliter [Viral titers were determined using adherent BHK-21 cells via endpoint titration according to the Spearman-Kärber method, which was expressed as the tissue culture infectious dose affecting 50% of the culture (TCIDlliliter .v/v) by vigorous inversion for 5 min. After mixing, the mixture was centrifuged at 3000× g for 15 min at 4 °C, and the aqueous phase located above the organic solvent was collected. This process was repeated twice, and the resulting samples were centrifuged at 16,000× g for 10 min to obtain the supernatants. The samples were then treated with benzonase (Sigma-Aldrich) at a final concentration of 0.025 units/μL and incubated at 37 °C with shaking for 1 h. After the incubation, the samples were centrifuged at 16,000× g for 10 min at 4 °C to obtain a clear sample. To further refine the samples, they were filtered through 0.22 μm Millex-GV filters (Merck KGaA) and loaded onto a size-exclusion high-performance liquid chromatography (SE-HPLC) column for quantification of 146S intact virus particles. The SE-HPLC analysis utilized a TSKgel G4000PWXL (300 × 7.8 mm) column  combined with a TSKgel PWXL GuardCol (40 × 6.0 mm) guard column (TOSOH Bioscience) using an Agilent 1260 Infinity II system . The mobile phase consisted of 30 mM Tris-HCl and 400 mM NaCl at pH 8.0, and the flow rate was set at 0.5 mL/min. The area under the target peak was integrated using the OpenLAB chromatography data system (CDS) ChemStation Edition Rev. C01.10, and the amount of 146S antigen (g/mL) was calculated based on a previous study [The supernatant was mixed with chloroform  in a 1:1 ratio (BEI was prepared by dissolving BEA (bromoethylamine hydrobromide) in 10 mL of a solution of 0.2 N sodium hydroxide  to achieve a concentration of 0.1 M. The solution was placed in a shaking incubator at 37 °C and 100 rpm for 1 h. The pH of the solution was adjusted to a range of 8.5–9. A fresh solution was prepared prior to each use. Samples were collected every hour from 0 to 6 h from the inactivation tank to assess the virus inactivation rate for each strain. The graph for each strain was extrapolated as a linear line to analyze the kinetics of FMDV inactivation. The remaining BEI was neutralized with 10% of the volume using 1 M sodium thiosulfate to a final concentration of 2%.g for a duration of 4 h. The band located between the sucrose layers of 30% and 35% was collected and further subjected to ultracentrifugation at 100,000× g for 4 h. The resulting pellet was resuspended again and subjected to dialysis at 4 °C against a solution of 50 mM Tris and 300 mM KCl (pH 7.6) to eliminate any residual sucrose. A small amount of the purified FMDV suspension was applied to formvar-coated grids and negatively stained with 1% uranyl acetate. Finally, the FMDV particles were observed using a transmission electron microscope .The inactivated antigen, concentrated using Polyethylene Glycol (PEG), was carefully placed on sucrose gradients (15–45%) and subjected to ultracentrifugation at 100,000× n = 5 for all groups) were immunized with a monovalent O PA-2 vaccine (1 mL) containing 20, 15, and 10 µg of antigen, respectively. The control group consisted of two unvaccinated pigs. Each group of pigs was intramuscularly administered vaccine containing various payloads of antigens followed by homologous virus challenge using O PA-2 (1 × 105 TCID50/0.1 mL) in the heel bulb at 28 days post vaccination (dpv). Blood samples were collected at 0, 7, 14, 21, and 28 dpv. During the experiment, one pig each from groups 2 and 3 died of pleuropneumonia and Glasser’s disease, respectively. For the A22 IRQ vaccine, the same experiment was performed with the same number of pigs as described above. However, the virus challenge was conducted with A22 IRQ. If pigs showed clinical signs of FMD, they were immediately isolated to prevent further virus challenge to the pigs not showing clinical signs. The clinical score was calculated by the following parameters: the presence of vesicles on the foot, which was contingent upon the number of affected feet (up to a maximum of 4 points); and observable mouth lesions on the tongue (1 point), gums and/or lips (1 point), or snout (1 point) (maximum score = 3). Protection was defined as all regions of the pigs, except for the injection site, showed no clinical signs throughout the experimental period, within 7 days following viral challenge. Clinical scores were calculated by summing the scores corresponding to FMD lesions or signs (maximum score = 7) [The antigens O PA-2 and A22 IRQ, as described in ore = 7) .50. Following an incubation for 1 h at a temperature of 37 °C, 50 μL of LFPK cells  (0.5 × 106 cells/mL) was added to each well. The plates were sealed and incubated at 37 °C with 5% CO2 for 2–3 days. The VN titer was determined using the Spearman-Kärber method and reported as a log10 value.The virus neutralization (VN) test was conducted in accordance with the procedures outlined in the WOAH terrestrial manual . Prior tWe produced FMD vaccine antigens in a 100 L bioreactor in triplicate. Virus titration, quantification of 146S particles, and inactivation kinetics were performed three times, and the mean and standard deviation of all values were reported. VN titers and inactivation kinetics were analyzed using GraphPad Prism version 9  for visual representations.7 TCID50/mL and 1.71 × 107 TCID50/mL, respectively  VN titers, starting from 21 dpv . Similar to the O PA-2 vaccine, pigs in groups 1–3  were immunized with monovalent A22 IRQ vaccine (1 mL) containing 15, 10, and 5 µg of antigen, respectively. Pigs in all groups were challenged at 28 dpv with A22 IRQ. VN titers were measured for up to 7 days after the virus challenge. In the 15 µg antigen group, all pigs had more than a 1.65 log10 VN titer at 28 dpv. On the other hand, in the 10 µg and 5 µg antigen groups, there was one pig each with a VN titer less than 1.65 log10 on day 28  at the time of virus challenge but had no clinical signs, indicating that they were still protected from the virus. Our results corroborate with those of previous reports showing that some animals can be protected with lower levels of VN titers [Similarly, the antigen dose range of A22 IRQ was adjusted from 5 to 15 µg based on a previous report that more type O antigen is required than type A to achieve an equivalent potency . In thisN titers .In conclusion, this is the first report demonstrating a pilot-scale production of recombinant O PA-2 and A22 IRQ antigens that can protect pigs against virus challenge at dosages of 10 µg and 5 µg, respectively. These results will pave the way for industrial-scale FMD vaccine production in South Korea.


BCL2L13, CEP135, E2F4, IP6K2, KDM6A, SMS, and XPO4), four candidates for docetaxel , and nine candidates for cabazitaxel . We generated single-gene C4 knockout clones/populations for all genes and could validate effect on treatment response for five genes . Altered enzalutamide response upon IP6K2 and XPO4 knockout was associated with deregulation of AR, mTORC1, and E2F signaling, and deregulated p53 signaling (IP6K2 only) in C4 mCRPC cells. Our study highlights the necessity of performing individual validation of candidate hits from genome-wide CRISPR screens. Further studies are needed to assess the generalizability and translational potential of these findings.Enzalutamide, docetaxel, and cabazitaxel treatment resistance is a major problem in metastatic castration resistant prostate cancer (mCRPC), but the underlying genetic determinants are poorly understood. To identify genes that modulate treatment response to these drugs, we performed three genome-wide CRISPR/Cas9 knockout screens in the mCRPC cell line C4. The screens identified seven candidates for enzalutamide ( While organ-confined PC is curable, advanced metastatic PC generally has a poor prognosis3. Androgen deprivation therapy constitutes the backbone for first-line treatment of advanced metastatic PC. However, resistance invariably develops, and patients enter a second phase of disease known as metastatic castration-resistant PC (mCRPC)4. Despite castrate levels of serum androgens, the androgen receptor (AR) remains a key driver of mCRPC5. The importance of AR signaling has prompted development of second-generation androgen signaling pathway inhibitors, including enzalutamide, that directly target AR6. Other approved therapeutics for mCRPC include the microtubule-stabilizing chemotherapeutics docetaxel and cabazitaxel, which exert their effects through G2M cell cycle arrest and induction of apoptosis7.Prostate cancer (PC) is the second leading cause of cancer-related death among men in the Western world8. Known mechanisms of enzalutamide resistance include AR re-activation by mutations, genomic amplification, or alternative splice variants (e.g. AR-V7), intra-tumoral androgen synthesis, and AR-bypass via the glucocorticoid receptor6. Known taxane resistance mechanisms include activation of drug efflux pumps such as ABCB19 and upregulation of the CCL2 chemokine11. A better understanding of the genes that contribute to treatment response in mCRPC is needed to guide more personalized treatment selection, which in the future could help improve response rates and patient survival, while also reducing side effects.Unfortunately, primary and acquired resistance commonly occurse.g. treatment resistance. Genome-wide libraries that consist of thousands of unique single guide RNAs (sgRNAs) can be employed to target and knockout each protein-coding gene in human cell lines. By comparing the individual sgRNA abundance between treatment vs. vehicle at a given time point, significantly enriched or depleted sgRNAs can be identified, unveiling the corresponding gene(s) that modulate cellular response towards treatment12.The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has emerged as a powerful tool for genome editing and high-throughput screening of the genetic factors underlying Here, we performed three genome-wide CRISPR/Cas9 knockout screens in the mCRPC cell line C4 to identify novel genes involved in enzalutamide, docetaxel, and cabazitaxel resistance, respectively.13. The three AR positive cell lines exhibited sigmoidal dose–response curves for enzalutamide, with C4 being less sensitive (IC50: 45 µM) than LNCaP (IC50: 25 µM) and LAPC-4 (IC50: 19 µM)  or without AR expression (PC3 and DU145), respectivelyBased on this, the C4 cell line was selected for the genetic screening as it (i) showed intermediate resistance to enzalutamide and thus should be suited for identification of genes that confer increased drug resistance upon knockout (positive hits) as well as genes that confer decreased drug resistance upon knockout (negative hits), (ii) is easy to culture and expand, and (iii) is amenable to lentiviral transduction. C4 also responded to docetaxel and cabazitaxel in a dose-dependent manner Fig. C, and he14 at an MOI of ~ 0.3, and subjected to puromycin selection for 7 days. Aliquots of the library-transduced C4_Cas9 cell population were treated with IC50 and IC90, respectively, of enzalutamide, docetaxel, or cabazitaxel for 7 or 21 days , indicating homogenous sgRNA read count distribution within each sample : CEP135 and IP6K2 , BCL2L13, E2F4, KDM6A, SMS, and XPO4 , LMO7 and NCOA2 , DRG1 and ZNF268 , PRKAB1 and TCEA2 , and ARHGAP11B, DRG1, FKBP5, FRYL, RP2, SMPD2, and ZNF585B  . Generation of single-gene knockout clones was not successful for the remaining 7 candidates .Next, we generated single-gene knockout cell lines for each candidate by transfection of the C4_Cas9 cell line with the best-performing sgRNA (highest read count difference between vehicle and treatment in the screen) for each gene. Using FACS Fig. A, we sor16. Mean indel frequency for the 19 knockout populations was 69.1% (range: 40.0–86.5%)  Fig. B, while 5%) Fig. D. Consis5%) Fig. C and E. 5%) Fig. .Next, we performed 10-point dose–response assays to validate the phenotypic effect of knockout for each of the 19 candidate genes identified in the CRISPR/Cas9 screens. Cell viability after drug treatment for each single-gene knockout population/clone was determined using the alamarBlue assay and normalized to vehicle-treated controls.IP6K2 knockout increased resistance in C4 cells, while XPO4 knockout decreased resistance  showed statistically significant shifts in IC50, concordant with the screen results  showed statistically significant shifts in IC50 , docetaxel (NCOA2), and cabazitaxel screen   and decreased resistance upon XPO4 knockout (negative hit), resulting in a validation rate for enzalutamide screen hits of 2/7 (29%). None of the two positive or two negative hits for docetaxel  and decreased resistance upon DRG1 and RP2 knockout (negative hits), resulting in a validation rate for the cabazitaxel screen hits of 3/9 (33%).In summary, out of two positive and five negative hits for enzalutamide Fig. B, we valIP6K2, XPO4, DRG1, PRKAB1, and RP2) displayed similar viability and proliferation under vehicle-treated conditions in alamarBlue  or matched vehicle for 6 days and performed RNA sequencing  treatment in wildtype and IP6K2 or XPO4 knockouts, respectively. For all three cell lines, the Androgen Response Signature was depleted by enzalutamide treatment ent Fig. A,B, suppmTORC1 Signaling Signature, the G2M Checkpoint Signature, and the E2F Targets Signature  identified several pathways related to mTORC1 and p53 signaling  as well as pathways involving apoptosis and AR signaling  , although the E2F Targets Signature was more depleted in XPO4 knockout cells than in wildtype cells upon enzalutamide treatment. Moreover, the Mitotic Spindle Signature was depleted in XPO4 knockout cells upon enzalutamide treatment, but not in wildtype cells  and E2F signaling (e.g. E2F Targets) in addition to pathways related to AR signaling  , suggesting that these pathways mediate the changes in enzalutamide resistance observed in C4 cells upon IP6K2 and XPO4 knockout, respectively.In summary, transcriptome analyses of IP6K2 and XPO4) and three genes with cabazitaxel resistance . Neither of these five genes have been associated with enzalutamide or cabazitaxel resistance before. We also sought to validate altered cellular drug resistance upon knockout of five other candidate genes identified from the enzalutamide screen , four candidate genes from the docetaxel screen , and six other candidate genes from the cabazitaxel screen . However, none of these 15 candidate genes were validated, resulting in an overall validation rate of 5/20 (25%). This highlights the necessity of performing individual validation of candidate hits from genome-wide CRISPR/Cas9 knockout screens. While further studies are warranted and should assess the generalizability and translational potential of our findings, we propose that the extended gene hit lists from our three genome-wide screens , which responds to ATP depletion upon cellular stress by increasing ATP production or inhibiting ATP consumption32. PRKAB1 has not previously been associated with taxane resistance, and it is currently unclear whether PRKAB1 has an oncogenic or tumor suppressor effect in PC, as previous studies have shown contrasting results. For example, one study reported that PRKAB1 is overexpressed in metastatic vs. primary PC, indicating an oncogenic function, whereas another study showed that deletion of the PRKAB1 gene in mouse models conferred earlier prostate adenocarcinoma development, indicating a tumor suppressor function34. Our results are predominantly in line with a tumor suppressor function in C4 mCRPC cells, as we showed that PRKAB1 knockout increased cabazitaxel resistance.Furthermore, our results showed that RP2 knockout was associated with decreased cabazitaxel resistance in C4 cells  and three novel genes implicated in cabazitaxel resistance  in C4 mCRPC cells.In summary, this study identified two novel genes implicated in enzalutamide resistance . Accordingly, future studies should include validation in additional mCRPC models, such as other PC cell lines. For further in-depth analyses, cell line studies may include both 2D and 3D culture experiments as well as xenograft studies in mice. Future studies may also investigate the association between the five validated candidate genes and drug response in mCRPC patient-derived organoids.While such further studies are warranted, they are considered to be beyond the scope of the current study, a main strength of which is that it is the first report of genome-wide CRISPR/Cas9 knockout screens for genes involved in enzalutamide, docetaxel, and cabazitaxel resistance in PC cells. As such, this report also provides the scientific community with extended gene lists of positive and negative hits from the three screens (Supplementary Data 2. LAPC-4 cells were propagated in RPMI 1640 + / + and stimulated with 10 nM R1881 (Sigma-Aldrich). All cell lines were authenticated by short tandem repeat (STR) profiling (IdentiCell) and repeatedly tested negative for mycoplasma .C4, LNCaP, PC3, and DU145 cells were propagated in RPMI-1640 medium with L-glutamine (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 100 U/mL penicillin and 100 μg/mL streptomycin (RPMI 1640 + / +) (Life Technologies) at 37 °C in humidified atmospheric air with 5% CO37, with minor modifications. In brief, C4 cells were transduced with LentiCas9-Blast  at a multiplicity of infection (MOI) ~ 1.0 and selected by blastidicine (Sigma-Aldrich) for 5 days. The resulting C4_Cas9 population, with stable Cas9 expression, was transduced with the Brunello library14  at an MOI ~ 0.3 and selected by puromycin (Sigma-Aldrich) for 7 days. Library-transduced cells were expanded and split into several arms that were exposed to, respectively, IC50 and IC90 of enzalutamide , docetaxel , or cabazitaxel  treatment for 7 or 21 days depending on toxicity  v. 0.5.9.238 for sgRNA read mapping, normalization, and ranking  in the IC50 and IC90 screen for each drug  was subjected to Reactome pathway analysis2O to 20 µL. The reaction was incubated in a thermocycler under the following conditions: 10 cycles of (5 min at 37 °C then 10 min at 23 °C), hold for 30 min at 37 °C, hold for 15 min at 75 °C, and store at 4 °C. The product was chemically transformed into XL2-Blue ultra-competent bacteria (AH diagnostics) and seeded onto LB agar plates containing 100 µg/mL of ampicillin. Bacterial clones were checked for correct sgRNA insert by Sanger sequencing using a primer located in the U6 promoter region of the PX458 plasmid  and single cell clones (scc) for all 19 candidate genes identified in the screens  cells were then transfected with the corresponding cloned PX458 plasmid using FuGENE HD Transfection Reagent (Promega). After 72 h, GFP-positive cells were isolated by FACS and either seeded as single cells directly into 96-well plates containing 200 µL conditioned RPMI 1640 + / + medium to establish single cell clones, or sorted as populations into 50 µL FBS in Eppendorf tubes and later transferred to flasks for cultivation. Following FACS, all cells were tested negative for mycoplasma.16.Genomic DNA was harvested from C4_Cas9 and all knockout cell lines using the NucleoSpin Tissue Kit (Macherey–Nagel) according to the manufacturer’s instructions. Subsequently, candidate gene genomic knockout was validated by Sanger sequencing of the DNA , and total RNA was purified using the RNeasy mini kit (Qiagen). cDNA was synthesized using the SuperScript II reverse transcriptase kit (Life Technologies). RT–qPCR was performed using SYBR Green PCR Master Mix (Life Technologies)  and incubated for 24 h. A 10-point drug dilution series was added to the cells and incubated for 3 days , 6 days  or 3 days (docetaxel and cabazitaxel: C4_Cas9 and knockout cell lines), respectively. Cell viability was determined by incubation with 10% alamarBlue Cell Viability Reagent (Thermo Fisher Scientific) for 4 h, followed by fluorescence readout at 530 nm/620 nm using the Synergy HT reader (BioTek). Results were normalized to matched vehicle controls and presented as percent viability using GraphPad Prism v. 8.3.1. All experiments were performed in technical triplicates and repeated at least twice.Viability of untreated cells was determined for C4_Cas9 and the knockout populations C4_pop_IP6K2, C4_pop_XPO4, C4_pop_DRG1, C4_pop_PRKAB1, and C4_pop_RP2. In brief, cells were seeded at a density of 6000–8000 cells/well in 96-well plates and incubated for 72–96 h. Cell viability was determined using the alamarBlue Cell Viability Reagent, as described above. All experiments were performed in technical triplicates and repeated at least twice.C4_Cas9 and the knockout populations C4_pop_PRKAB1 and C4_pop_RP2 were seeded at a density of 7000–8000 cells/well in ePlate16 wells (ACEA Bioscience). Cell proliferation was measured using the xCELLigence Real-Time Cell Analyzer  with cell indices recorded every 15 min for 144 h. All experiments were performed in technical triplicates and repeated twice.C4_Cas9 and the knockout clones C4_scc_IP6K2_3 and C4_scc_XPO4_2 were seeded in TC-25 flasks and incubated for 24 h in duplicates. Cells were exposed to enzalutamide (IC50) treatment  was performed in the JavaGSEA Desktop Application (v. 4.0) using the Hallmark MSigDB collection. Log2-transformed counts per million (CPM) values were used as input. Gene sets with FDR < 0.05 after 1000 permutations were considered significant. GraphPad Prism (v. 8.3.1) was used to present the normalized enrichment score (NES) of deregulated gene sets.edgeR (v. 3.34.1)22 R (v. 4.1.0) package was used for differential gene expression analysis of CPM values. Genes that were differentially expressed in enzalutamide- as compared to vehicle-treated C4_scc_IP6K2_3 and C4_scc_XPO4_2 cells, but not in enzalutamide- as compared to vehicle-treated C4_Cas9 cells  pathways/ontologies were extracted from five databases: KEGG 2021 Human, Elsevier Pathway Collection, MSigDB Hallmark 2020, GO Biological Process 2021, and GO Molecular Function 2021.The Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


The silver conductive ink we prepared with 1030H has good dispersion performance with a particle size of 50–100 nm, as well as good storage stability and excellent adhesion. Additionally, the printing performance and conductivity of the silver conductive ink prepared with n,n-dimethylformamide (DMF): proprylene glycol monomethyl ether (PM) (1:1) as solvent are better than those of the silver conductive ink prepared by DMF and PM solvent. Cured at a low temperature of 160 °C, the resistivity of 1030H-Ag-82%-3 conductive ink is 6.87 × 10−6 Ω·m, and that of 1030H-Ag-92%-3 conductive ink is 0.564 × 10−6 Ω·m, so the low-temperature curing silver conductive ink has high conductivity. The low-temperature curing silver conductive ink we prepared meets the printing requirements and has potential for practical applications.In the field of flexible electronics manufacturing, inkjet printing technology is a research hotspot, and it is key to developing low-temperature curing conductive inks that meet printing requirements and have suitable functions. Herein, methylphenylamino silicon oil (N75) and epoxy-modified silicon oil (SE35) were successfully synthesized through functional silicon monomers, and they were used to prepare silicone resin 1030H with nano SiO Over the last decade, there has been growing interest in printed electronics for the creation of flexible electronic circuits and devices using printing technology ,3,4,5,6.Printed electronics have been applied to sensors ,13, tranConductive inks based on silver ,26,27, cThe quality and performance of silver conductive inks are generally dependent on the components used in their preparation. The application of silver conductive ink is limited by many factors, such as a complex ink preparation process, as well as potential conflicts between ink stability and solid content. Low solid content decreases the thickness of printed tracks, leading to high pattern resistance. Additionally, the sintering temperature of commercial silver conductive ink is high (higher than 200 °C) . As a reIn the field of flexible electronics manufacturing, inkjet printing technology has always been a research hotspot, and it is key to developing low-temperature curing conductive inks that meet printing requirements and have suitable functions. In this work, methylphenylamino silicon oil (N75) and epoxy-modified silicon oil (SE35) were successfully synthesized through functional silicon monomers, and 1030H silicone resin was prepared as the resin binder for low-temperature curing silver conductive ink. The addition of 1030H silicone resin suppressed the aggregation of nano silver particles, making the prepared low-temperature curing silver conductive ink have good dispersion performance with a particle size of 50–100 nm, as well as good storage stability and excellent adhesion. In addition, we discussed the effect of different solvents on the performance of silver conductive ink. Among them, the optimal solvent is n,n-dimethylformamide (DMF): proprylene glycol monomethyl ether (PM) (1:1). The silver conductive ink prepared with the solvent (DMF:PM (1:1)) not only meets the printing requirements but also can be cured at a relatively low temperature, with a curing temperature below 200 °C. The pattern printed with silver conductive ink has high conductivity. The low-temperature curing silver conductive ink we prepared has potential for the fabrication of conductors on flexible substrates as circuit microelectrodes.2), N,N-dimethylformamide (DMF), and proprylene glycol monomethyl ether (PM) were bought from Aladdin, China, and used as obtained. Nano silver powder was bought from BroadCON, China, and used as obtained.Phenyltrimethoxysilane (FEOS), diethoxydimethylsilane (DMDES), diphenyldimethoxysilane (DMDPS), (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)dimethoxymethylsilane (APDMS), tetraethyl orthosilicate (TEOS), hexamethyldisiloxane (MM), 3-glycidyloxypropyltrimethoxysilane (GOPTS), nano silica (SiO2O was removed under vacuum to obtain methylphenylamino silicone oil (N75). The hydrogen value of the synthesized N75 was 0.68 mol/100 g, and the synthetic process is shown in Under the protection of nitrogen gas, 7.4 g of DMDES, 12.6 g of DMDPS, 1.76 g of APTMS, 2.9 g of APDMS, 3.16 g of FEOS, 1.03 g of TEOS, and 4.54 g of MM were charged in a 250 mL flask equipped with a stirrer, reflux condenser, and constant pressure titration funnel. Then, 0.04 g potassium hydroxide was dissolved in 7 g of ethanol–water solution (mass ratio 1:1), stirred and added to the flask. Under the catalysis of potassium hydroxide, the mixture was stirred at 60 °C for 4 h. The system was then slowly heated to 130 °C to remove the generated alcohol and excess water, resulting in a transparent viscous oil. A certain amount of cyclohexane was added to the oil and then 732 cation exchange resin was used to remove the strong base catalyst from the reaction system. The resulting liquid was filtered and distilled to remove the cyclohexane at 140 °C. H2O was removed under vacuum to obtain epoxy-modified silicone oil (SE35). The epoxy value of the synthesized SE35 was 0.35 mol/100 g, and the synthetic process is shown in Under the protection of nitrogen gas, 5.55 g of DMDES, 9.45 g of DMDPS, 1.76 g of GOPTS, and 2.27 g of MM were charged in a 250 mL flask equipped with a stirrer, reflux condenser, and constant pressure titration funnel. Then, 0.02 g of potassium hydroxide was dissolved in 3.5 g of ethanol–water solution (mass ratio 1:1), stirred and added to the flask. Under the catalysis of potassium hydroxide, the mixture was stirred at 60 °C for 4 h. The system was then slowly heated to 130 °C to remove the generated alcohol and excess water, resulting in a transparent viscous oil. A certain amount of cyclohexane was added to the oil and then 732 cation exchange resin was used to remove the strong base catalyst from the reaction system. The resulting liquid was filtered and distilled to remove the cyclohexane at 140 °C. H2 was added and stirred for 1 h to obtain 1030H silicone resin. The 1030H silicone resin undergoes a curing reaction at 120 °C, and the reaction product of N75 and SE35 is shown in 5.00 g of methylphenylamino silicone oil (N75), 7.42 g epoxy-modified silicone oil (SE35) , and 14.71 g of DMF solvent were added to a beaker and stirred for 1 h. Then, 2 w% of nano SiOThe silver conductive ink was prepared by using 1030H silicone resin as the resin carrier and nano silver powder as the conductive filler, which was uniformly dispersed in the solvent ). The formulations for the silver conductive ink are shown in The prepared silver conductive ink was printed on a 50 μm thick polyethylene terephthalate (PET) film using an inkjet printing process. The Micro-Electronic Printer purchased from Shanghai CCI Instrument Co., Ltd., Shanghai, China, (including an ink box with 16 nozzles with diameters of 20 µm arranged in a row) was used to print silver conductive patterns. The number of nozzles used for inkjet printing can be controlled using Bits Assembler (software for controlling Micro-Electronic Printer). In addition, the inkjet process of each nozzle is controlled via each piece of piezoelectric ceramics. The pattern printed had a thickness of 10 μm, a length of 10 cm, and a width of 1 mm. The printed silver conductive ink was then cured to ensure that the conductive ink was stably attached to the film. The curing conditions were drying at 90 °C to volatilize solvent to achieve semi-curing, hot pressing at 145 °C for 5 min in a film laminator, and then curing at 160 °C for 2 h to obtain printed and cured conductive patterns. The preparation and printing process of silver conductive ink is shown in −1 at ambient temperature. The nuclear magnetic resonance (NMR) measurements were carried out on an Avance NEO 600 (Bruker) at 500.13 MHz (1H). The spectra were referenced to the solvent signal (CDCl3: δ (1H) = 7.26 ppm). Thermogravimetric analysis (TGA) data were recorded on a Mettler tga 2 instrument over the range of 25 to 800 °C, with a heating rate of 5 °C min−1 under a flow of N2 (20 mL min−1). SEM and EDX were acquired on a Schottky Field Emission Scanning Electron Microscope ZEISS MERLIN Compact operating in variable pressure mode with an accelerating voltage of 15 keV.Fourier transform infrared spectra (FT-IR) were obtained using a Niolet iN10 instrument with an ATR attachment, operating over 400–4000 cmMethylphenylamino silicone oil (N75) was synthesized by the hydrolysis and co-condensation reaction under the catalysis of Potassium hydroxide . Epoxy-m−1 was assigned to the characteristic stretching vibration of C-H, corresponding to the benzene ring. The peak at 2958 cm−1 was attributed to the saturated C-H stretching vibration, including -CH2- and -CH3. while the peaks at 1429 cm−1 and 1258 cm−1 were the characteristic absorption peaks of -Si-CH2- and -Si-CH3, respectively [−1 was the characteristic absorption peak of the organic silicon framework Si-O-Si [−1, which was attributed to the N-H characteristic absorption peak, and a peak at 2807 cm−1, which was attributed to the characteristic absorption peak of C-NH [−1, which was attributed to the characteristic absorption peak of the epoxy group in SE35. In summary, N75 and SE35 were successfully synthesized. In the spectrum of N75-SE35, its FT-IR spectrum contained characteristic absorption peaks of both N75 and SE35. It can be clearly seen that the intensity of the characteristic absorption peak of the epoxy group at 905 cm−1 was significantly reduced, indicating that N75 and SE35 have undergone the curing reaction to generate N75-SE35.To confirm the occurrence of the reaction to synthesize N75-SE35, FT-IR was carried out to compare the chemical structures of N75, SE35, N75-SE35, and the spectra are shown in  Si-O-Si . The FT- of C-NH . In the 1H-NMR spectra of N75 and SE35 are shown in 1H-NMR spectrum of N75, the signals at 0.09 ppm and 3.37 ppm were attributed to (CH3)3SiO1/2 and (CH3)2SiO, respectively. The signals at 1.19 ppm and 1.37 ppm were attributed to N-H from NH2(CH2)2NH(CH2)3Si. The signal at 7.55 ppm was typically attributed to C-H from the benzene ring. The signals at 0.48 ppm, 2.67 ppm, and 3.64 ppm were assigned to NH2(CH2)2NH, NH(CH2)3Si, and CH3SiO3/2, respectively. For the 1H-NMR spectrum of SE35, the signal at 0.09 ppm was attributed to the -CH3 of Si(CH3)3. The signals at 2.62 ppm and 2.80 ppm were attributed to C-H from the epoxy group. The signals at 0.52 ppm, 1.58 ppm, 3.41 ppm, and 3.71 ppm were assigned to CH2OCH2, CH2CH2O, CH3Si, and (CH3)2SiO, respectively [1H-NMR and FT-IR analysis, the chemical structure of N75 and SE35 were in good agreement with the anticipated structure.The ectively ,46,47. BConductive ink is a key material in electronic printing technology. After printing, the ink functions like a wire by providing a conductive connection. The quality of flexible electronic devices is influenced by the properties of the conductive ink, such as its dispersion uniformity, conductivity after printing, and adhesion to the substrate material. Silver conductive inks with different formulations exhibited various performance characteristics after printing .Inkjet printing requires a specific particle size for conductive ink particles, as particles that are too large will clog the nozzles. However, in silver conductive ink, the silver nanoparticles tend to aggregate, leading to particles that are too large. Thus, suppressing the aggregation of silver nanoparticles in silver conductive inks is a challenging issue. In this study, 1030H resin was designed and synthesized to suppress the aggregation of silver nanoparticles.−3) and tend to agglomerate in conductive inks. Silver conductive ink may experience varying degrees of delamination after long-term transportation and storage. Therefore, silver needs to have certain storage stability after uniform dispersion in conductive ink. To explore the storage stability of the prepared silver conductive ink, silver conductive inks with different formulations were stored at room temperature (25 °C) for 60 days, and the delaminating phenomenon was observed and recorded by taking photographs, as shown in The conductive filler in silver conductive ink is silver nanoparticles. Compared to the density of solvents and resins, silver nanoparticles have a high density  mixed solvent system is preferred for silver conductive ink.The conductivity of printed patterns is an important indicator of conductive ink. Silver conductive ink was printed on a PET film, dried at different temperatures to achieve semi-curing by evaporating the solvent, and then hot-pressed at a pressure of 1 MPa and a temperature of 145 °C for 5 min. Then, it was heated in an oven from 120 °C to 160 °C at a rate of 10 °C/15 min and maintained at 160 °C for 1 h to obtain the printed pattern. We studied the effect of drying temperatures on the conductive properties of printed patterns, and the results are shown in −6 Ω·m to 171 × 10−6 Ω·m, the resistivity of 1030H-Ag-82%-2 prepared with PM increased from 11.8 × 10−6 Ω·m to 410 × 10−6 Ω·m, and the resistivity of 1030H-Ag-82%-3 prepared with DMF:PM (1:1) mixed solvent increased from 5.27 × 10−6 Ω·m to 210 × 10−6 Ω·m. The high drying temperature accelerates the volatilization rate of the solvent, leaving air holes on the printed pattern, which increases the distance between the silver nanoparticles and reduces the conductivity of the silver conductive ink. However, the low drying temperature slows the volatilization rate of the solvent, which doubles the drying time. So, the optimal drying temperature was 90 °C. At a drying temperature of 90 °C, the conductivity of 1030H-Ag-82%-3 conductive ink (6.87 × 10−6 Ω·m) was better than that of 1030H-Ag-82%-1 conductive ink (9.55 × 10−6 Ω·m) and 1030H-Ag-82%-2 conductive ink (18.9 × 10−6 Ω·m).−6 Ω·m to 1.74 × 10−6 Ω·m, the resistivity of 1030H-Ag-92%-2 prepared with PM increased from 0.716 × 10−6 Ω·m to 5.15 × 10−6 Ω·m, and 1030H-Ag-92%-3 prepared with DMF:PM (1:1) increased from 0.253 × 10−6 Ω·m to 1.35 × 10−6 Ω·m. The conductivity of 1030H-Ag-92%-3 conductive ink (0.564 × 10−6 Ω·m) was better than that of 1030H-Ag-92%-1 conductive ink (0.724 × 10−6 Ω·m) and 1030H-Ag-92%-2 conductive ink (0.774 × 10−6 Ω·m) under the same conditions, at a drying temperature of 90 °C.−6 Ω·m, so the low-temperature curing silver conductive ink has high conductivity. Compared with silver commercial conductive inks, our silver conductive inks had a lower curing temperature and higher adhesion. This research provides new insights for preparing low-temperature curing silver conductive ink, and hence will encourage more research studies to achieve extended practical applications.In summary, the resin binder of silver conductive ink (1030H silicone resin) was prepared with methylphenylamino silicone oil and epoxy-modified silicone oil that were successfully synthesized through functional silicone monomers. Additionally, low-temperature curing silver conductive inks were prepared with silver nanoparticles as conductive fillers, 1030H silicone resin as the resin binder, and DMF, PM, or DMF:PM (1:1) as solvent. The low-temperature curing silver conductive inks with DMF:PM (1:1) as solvent exhibited desirable dispersibility with particle sizes of 50–100 nm, good storage stability, and excellent adhesion. The printing performance and conductivity of the silver conductive ink prepared with DMF:PM (1:1) as solvent were better than those of the silver conductive ink prepared by DMF and PM solvent. Cured at a low temperature of 160 °C, the resistivity of low-temperature curing silver conductive inks with DMF:PM (1:1) as solvent was 0.564 × 10


As the basic building blocks of modern electronics, field-effect transistors (FETs) use gate electrodes to control the conductivity of channel materials through the electrical polarization of dielectrics . The per2O3, was innovatively introduced as the buffer layer between the 2D semiconducting MoS2 and the ultra-thin atomic-layer-deposited high-κ HfO2 layer  value of 73 mV/dec and an on/off ratio of 5 × 107  . Specifiyer Fig. . Sb2O3 cng bonds . Moreoveays Fig.  fabricat107 Fig. , which aThe research work  demonstr


However, this approach is challenging to realise due to the difficulty of implementing a solution process to convert a robust and complex solid (lignocellulose) together with a barely soluble and stable gas (CO2). Herein, we present the complete oxidative valorisation of lignocellulose coupled to the reduction of low concentration CO2 through a three-stage fractionation–photocatalysis–electrolysis process. Lignocellulose from white birch wood was first pre-treated using an acidic solution to generate predominantly cellulosic- and lignin-based fractions. The solid cellulosic-based fraction was solubilised using cellulase (a cellulose depolymerising enzyme), followed by photocatalytic oxidation to formate with concomitant reduction of CO2 to syngas (a gas mixture of CO and H2) using a phosphonate-containing cobalt(ii) bis(terpyridine) catalyst immobilised onto TiO2 nanoparticles. Photocatalysis generated 27.9 ± 2.0 μmolCO g2TiO−1  and 147.7 ± 12.0 μmolformate g2TiO−1 after 24 h solar light irradiation under 20 vol% CO2 in N2. The soluble lignin-based fraction was oxidised in an electrolyser to the value-added chemicals vanillin (0.62 g kglignin−1) and syringaldehyde (1.65 g kglignin−1) at the anode, while diluted CO2 (20 vol%) was converted to CO (20.5 ± 0.2 μmolCO cm−2 in 4 h) at a Co(ii) porphyrin catalyst modified cathode  at an applied voltage of −3 V. We thus demonstrate the complete valorisation of solid and a gaseous waste stream in a liquid phase process by combining fractioning, photo- and electrocatalysis using molecular hybrid nanomaterials assembled from earth abundant elements.The simultaneous upcycling of all components in lignocellulosic biomass and the greenhouse gas CO 2 as an attractive opportunity to synthesise sustainable and valuable chemicals.This work demonstrates the simultaneous upcycling of all components in lignocellulosic biomass together with the greenhouse gas CO By harnessing the power of renewable energy sources, such as solar and wind, the conversion of non-edible biomass and CO2via photo- or electrochemical approaches presents an opportunity to produce sustainable fuels and chemicals.3The renewable generation of valuable chemicals and fuels is a critical step towards a sustainable chemical industry.4,5 Cellulose and hemicellulose consist mainly of polysaccharide made from glucose, xylose, mannose and arabinose. Lignin is made of different polymerised aromatic units, and its utilisation remains challenging due to the robustness of its polyaromatic structure, which requires harsh conditions to break down the polymer .6,7 Unlike cellulose, lignin-to-chemical conversion technologies remain scarce.4,5,7,8 An important linkage in lignin, often found between its aromatic polymeric backbone, is the β-O-4 bond between two phenyl rings, which serves as an ideal target to depolymerise lignin into smaller aromatic fragments.9–11 The selective depolymerisation of all lignocellulose components and their subsequent chemical transformation would enable large-scale access to aliphatic and aromatic renewable feedstock chemicals.Lignocellulosic biomass such as wood, is abundant and cheap and consists predominantly of three polymeric components: cellulose, hemicellulose and lignin.2 can be used as an abundant carbon source to produce energy-rich chemicals such as CO, formate, hydrocarbons or alcohols.12 However, photo- and electroreduction of CO2 are predominantly performed in the presence of pure CO2, where concentrated CO2 streams have to be generated involving additional energy input.13–15 To alleviate the energy demand of the process, it is desirable to perform catalytic reactions at lower CO2 concentrations .16–20 The challenge of using low concentration CO2 streams lies in maintaining high product selectivity and catalytic activity compared to reactions employing pure CO2 streams.17 Molecular CO2 reduction catalysts display an increased product selectivity compared to most heterogeneous electro- or photocatalysts.16,21,22Apart from ubiquitous biomass sources, the greenhouse gas CO2 together is their simultaneous conversion in photo- or electrocatalytic processes.23–25 This strategy opens the possibility to couple biomass oxidation with CO2 reduction in a single process driven by sunlight or renewable electricity. The coupling of productive half-reactions thereby allows the conversion of a solid and a gaseous waste stream into valuable products such as CO, syngas, formate and aromatic chemicals, which can be more attractive than conventional systems performing overall water splitting to generate H2 and O2 from water. Additionally, the oxidation of biomass-derived substrates is thermodynamically less demanding than water oxidation, thereby facilitating the catalytic conversions, as well as the generation of value-added products.26–28 This combined approach also allows for the isolation of products in different phases and compartments, which can help with product separation. Conventional approaches such as water splitting generate explosive H2 and O2 mixtures in the reactor headspace.An attractive approach to utilise biomass and CO2 streams has recently been reported using a TiO2 nanoparticle with an immobilised CO2 reducing cobalt(ii) bis(terpyridine) catalyst containing phosphonate anchors (CotpyP) (TiO2∣CotpyP). Photoexcitation of this hybrid TiO2∣CotpyP photocatalyst reduced aqueous CO2 to syngas, while cellulose-derived glucose was simultaneously oxidised to formate and arabinose. TiO2∣CotpyP could operate for 24 h and be recycled up to three times.29 However, only concentrated CO2 and pure cellulose have been used, and no strategy for lignin separation and utilisation were reported in this previous study.The valorisation of cellulose and CO2 reduction, which has been achieved by employing molecular CO2 reduction hybrid nanomaterials  with a molecular cobalt(ii) porphyrin (CoPL) catalyst immobilised on a multiwall carbon nanotube (MWCNT) cathode. Thus, we demonstrate the complete valorisation of lignocellulose and low concentration CO2, which has been enabled by a precious-metal free fractionation–photocatalysis–electrolysis process.Herein, we report the valorisation of all components in lignocellulose coupled to low concentration COaterials . First, 2SO4 hydrolysis:6 glucose (34.7 ± 1.6 wt%), xylose (20.3 ± 1.0 wt%), mannose (1.8 ± 0.1 wt%), arabinose (0.9 ± 0.1 wt%) and lignin (17.6 ± 1.4 wt%). Further details are provided in the Experimental section, †The composition of dried and extracted white birch wood used in this study was determined by quantifying its sugar and lignin composition following standard characterisation procedures using H7 The dioxane/HCl/HCOOH mixture solubilises lignin and the liquid fraction predominantly consisted of lignin (41.2 ± 2.1 wt%) along with xylose (6.9 ± 1.3 wt%), glucose (2.4 ± 0.6 wt%), mannose (1.3 ± 0.3 wt%) and arabinose (2.5 ± 0.7 wt%). The second most abundant component xylose (derived from hemicellulose) is partially converted to furfural under these fractioning conditions  and xylose (6.7 ± 1.6 wt%) with some lignin (5.4 ± 1.2 wt%).To effectively utilise the different components of lignocellulose, their individual components are first separated by acid hydrolysis. Lignocellulose was pre-treated in a dioxane/HCl/HCOOH mixture at 80 °C for 3 h (500 mg in 5.9 mL) to obtain a liquid (or liquor) and a solid fraction.via cellulase pre-treatment.29 The depolymerised glucose and cellobiose are suitable electron donors in semiconductor suspension systems,31 and their valorisation has been previously reported.32,33 Specifically, the solid fraction was enzyme pre-treated with cellulase (0.05 mgcellulase mgsolid−1) to generate soluble sugars, predominantly glucose (39.3 ± 4.7 mM) and cellobiose (8.7 ± 2.1 mM) after 24 h incubation at 37 °C in an aqueous sodium acetate buffer solution (50 mM) at pH 5.29 With respect to white birch, 10.2 ± 0.4 wt% and 4.2 ± 0.6 wt% were converted to glucose and cellobiose, respectively  was added to a photoreactor containing a TiO2 suspension  in 2 : 1 MeCN : cellulase-treated solid fraction aqueous solution (3 mL). The photoreactor was sealed with a rubber septum and purged with 100% CO2 or 20% CO2  at a flow rate of 15 mL min−1 for 15 min. The sealed and stirred photoreactor was irradiated with a solar light simulator  for 24 h. The UV in the full solar spectrum is necessary to photoexcite electrons from the valence to the conduction band of TiO2.29 The gaseous products (H2 and CO) in the headspace (4.74 mL) were quantified by gas chromatography (GC), and HCOO− formed in the solution from glucose photooxidation was quantified by ion chromatography.The lignocellulose-derived sugar solution was then utilised for COcatalyst .29 In a 2, 69.9 ± 4.0 μmolCO g2TiO−1, 109.8 ± 8.0 μmol2H g2TiO−1  and 153.7 ± 4.0 μmolformate g2TiO−1 were formed. Under 20 vol% CO2, the formation yields were 27.9 ± 2.0 μmolCO g2TiO−1, 141.7 ± 27.9 μmol2H g2TiO−1  and 147.7 ± 12.0 μmolformate g2TiO−1  to H2 and CO2 to CO by CotpyP as well as the oxidation of glucose/cellobiose by TiO2 to formate are two-electron processes with an expected 1 : 1 stoichiometric ratio for (H2 + CO) : formate, which is close to the observed ratios.29,34–36After 24 h of photocatalysis under 100 vol% CO gTiO2−1 . The obt13CO2 and cellulase enzyme pre-treated cellulose were performed to confirm that the CO originates from CO2 reduction . Analysis of the gas headspace after photocatalysis by transmission IR spectroscopy reveals that 13CO produced by TiO2∣CotpyP was only formed when 13CO2 was used as the carbon source , using an electrochemical flow setup that allowed continuous purging of the electrolyte solution with a given gas composition.38Low concentration COlyst see . CoPL waL were prepared, following a reported procedure  suspension containing 2.37 mg MWCNT mL−1 and 0.1 mM CoPL onto carbon paper (CP) , which is denoted as CP∣CoPL. Electrocatalysis with CP∣CoPL was performed in a two-compartment electrochemical cell with a three-electrode setup. Pt foil was used as the counter electrode (CE) and Ag/AgCl (sat. KCl) as reference electrode (RE), a Nafion membrane separating the cathode and anode chambers, with the catholyte (0.1 M NaHCO3 in H2O) under a constant gas flow (9 mL min−1) of CO2 and N2 regulated by mass flow controllers. The generated gaseous products during electrochemical experiments (H2 and CO) were measured via online GC  the catholyte chamber was continuously purged with pure N2 for 30 min, after which the CO2 concentration was gradually increased to 10, 20, 50 and 100 vol% every 45 min  likely caused by the chemical equilibrium between carbonic acid (H2CO3) and CO2. Upon increasing the CO2 concentration (in vol%), the current density gradually increased, from approximately −0.8 mA cm−2 (10 vol%), to −1.2 mA cm−2 (20 vol%), to −1.5 mA cm−2 (50 vol%) and to −1.8 mA cm−2 (100 vol%). The H2 formation rate remained constant at ∼0.06 μmol2H min−1 at all CO2 concentration steps, whereas the CO formation rate and CO selectivity increased from ∼0.12 μmolCO min−1 and ∼65% (10 vol%), to ∼0.24 μmolCO min−1 and ∼80% (20 vol%), to ∼0.35 μmolCO min−1 and ∼90% (50 vol%) and to ∼0.50 μmolCO min−1 and ∼93% (100 vol%)  may be explained by the high affinity of CoPL to CO2.28 The carbon source of CO was previously confirmed for CoPL through 13CO2 isotopic labelling experiments.28,37During chronoamperometry (CA) experiments, under an applied potential of −1.2 V y 45 min . Under p00 vol%) . The low2 gas flow concentrations: (1) CoPL exhibits a high product specificity with around ∼80% CO selectivity even under 20 vol% of substrate CO2. (2) The CO formation rate at 20 vol% CO2  = 8.3 min−1; single-pass CO2 conversion yield39 = 4.7%) corresponds to roughly half the activity with respect to 100 vol% CO2 , thus indicating that decreasing five-fold the CO2 concentration only reduces two-fold the CO formation rate (CO)3(triethanolamine)], which operates in a DMF/triethanolamine solvent mixture at variable CO2 concentrations  under comparable flow conditions,22 CP∣CoPL is three orders of magnitude more active . Without taking into account that CO2 is more soluble in organic solvents than in water (e.g. ∼180 mM in DMF vs. ∼33 mM in water at 25 °C), these differences in performance could be tentatively associated to the catalytic mechanism of CO2 reduction, which enables cobalt porphyrins to achieve higher TOF than rhenium bipyridine electrocatalysts.22,37When comparing CP∣CoP9–11The electrooxidation of the liquid fraction or liquor, containing predominantly lignin and obtained from pre-treating lignocellulose (250 mg), was studied in a two-compartment electrochemical cell with a three-electrode setup. For this purpose, the anodic conditions were initially optimised using the lignin model substrate 1--2-(2-methoxyphenoxy)propane-1,3-diol, which contains a β-O-4 linkage between two phenyl rings that mimics those ubiquitously found in lignin see .9–11−1, 0.1 mL cm−2) containing Nafion 117 (2 vol% of a 5 wt% solution) to achieve a high surface area MWCNT layer with ∼20 μm thickness  in the presence of 10 mM of lignin model substrate in 0.1 M Na2CO3 in 1 : 1 MeCN : H2O achieved high current densities (∼8 mA cm−2) at +1 V vs. Ag/AgCl (sat. KCl), while in absence of lignin, and under the same conditions, the current densities were lower (∼3 mA cm−2)  spectroscopy in CDCl3 with mesitylene as internal standard  with an applied voltage (Uapp) of −3 V for 4 h. The anolyte comprised of 0.1 M Na2CO3 in a 1 : 1 MeCN : H2O solvent mix containing lignin (obtained from pre-treating 250 mg lignocellulose), and the catholyte had 0.1 M NaHCO3 in H2O. The catholyte was constantly purged at 9 mL min−1 with 20 vol% CO2 . The gaseous products on the cathodic side were monitored by online GC.38Having established the optimised conditions  corresponding to a CO selectivity of 78 ± 2% and a FY2CO+H of 59 ± 6%  and methoxy regions (3.8–4.0 ppm) can be observed in the 1H NMR spectra  and the crude product was analysed by  Fig. S20, which sllin see  and 4c,  spectra .402, has been previously reported.41–43 For instance, these Pb/PbO2 anodes were able to generate different lignin-derived products, such as vanillin (5.83 g kglignin−1) and syringaldehyde (9.30 g kglignin−1), via electrooxidation/electrohydrogenation of bamboo-derived lignin when used with Cu cathodes in 1 M NaOH solution.41 Despite the difficulty to compare this previously reported system with our CP∣MWCNT∣∣CP∣CoPL system due to the different experimental conditions , the metal-free CP∣MWCNT anodes were able to generate yields of vanillin and syringaldehyde within the same order or one-order of magnitude lower than those reported for the metal/metal oxide based Pb/PbO2∣∣Cu system.In comparison to CP∣MWCNT, the use of heterogeneous anodes for lignin oxidation based on metals/metal oxides, such as toxic Pb/PbO2 to formate using photoelectrochemical systems.44,45 In comparison, our work presents a unique and successful three-stage approach that shows that complete valorisation of lignocellulose is possible through the combination of fractionation and utilisation of photocatalysis and electrocatalysis to oxidise the resulting solid fraction to formate and liquid fraction to aromatics and reduce diluted CO2 and water to CO and H2. Although this system is a proof-of-concept demonstration, different factors would need consideration for practical implementation. These include optimisation of catalyst performance, scalability,46,47 integration of a carbon capture step,48 potential limitations such as catalyst stability, the engineering design of reactors49,50 for lignocellulose fractionation,46 photocatalysis51 and electrolysis,52 and the separation of products for further use.53,54Previous work has shown that lignin valorisation can be coupled with hydrogen evolution or reduction of CO2. Fractioning lignocellulosic wood provides sugar-based solid and lignin-based soluble fractions that are photo- and electrocatalytically converted with immobilised molecular cobalt(ii) catalysts to produce CO, formate, vanillin and syringaldehyde. The presented results set a precedent in integrating renewable processes for complete waste feedstock valorisation under ambient pressure and temperature. Moreover, our molecular hybrid systems were able to convert low CO2 concentrations (10–20 vol% CO2), which paves the way towards utilising CO2 concentrations nearing those found in flue gas (4–10 vol% CO2). However, to utilise actual flue gas additional CO2 purification steps are necessary to eliminate impurities,55 such as SO2, SO3, NOx, O2 and particulate matter depending on the type of fuel used. Learning from the use of low CO2 concentration also brings us a step closer towards operating directly from atmospheric CO2 (420 ppm).20,56We report a combined fractionation-photocatalysis-electrolysis process for the complete valorisation of lignocellulose and low-concentration CO57 in the valorisation of waste streams, which can be expanded beyond CO2 reduction in future developments to perform chemistry in a more sustainable and circular manner. As biomass and CO2 emerge as the most scalable and readily available sustainable carbon sources to defossilise the chemical industry,58 this work aims to inspire new approaches in their practical valorisation.Hence, this work demonstrates a proof-of-concept strategy to valorise challenging multicomponent waste streams simultaneously through solar-driven and electrochemical redox processes. This work further highlights the potential of molecularly engineered hybrid materials2O (Sigma Aldrich), d(+)-glucose , d(+)-cellobiose (Acros), Cellulase from Trichoderma reesei ATCC 26921 (Sigma Aldrich), sodium acetate trihydrate , hydrochloric acid , 1--2-(2-methoxyphenoxy)propane-1,3-diol , Vanillin (Thermo Scientific), syringaldehyde (Thermo Scientific), vanillic acid (Sigma Aldrich), syringic acid (Thermo Scientific), fufural (Sigma Aldrich), 2-methoxyphenoxyacetic acid (Alfa Aesar), 3,4-dimethoxybenzaldehyde (Acros), 4-hydroxybenzoic acid (Thermo Scientific), white birch (Merck), dioxane , formic acid , sulfuric acid , multiwalled carbon nanotubes , carbon paper , Nafion solution (5 wt% Sigma Aldrich), NaHCO3 (Merck), EtOAc , THF , Na2CO3 (Merck), MgSO4  CDCl3 (Sigma Aldrich), 50 wt% H2SO4 (Fluka), Na2CO3 64 mM and NaHCO3 20 mM  were purchased from commercial sources and used as received. MilliQ® grade H2O was used for all the experiments. TiO2 powder P25  was obtained from Evonik; [Co2](BF4)2 (denoted as CotpyP)35 and CoPL 37 were synthesised according to reported procedures. Reaction gases (CO2 and N2) were purchased from BOC. Ag/AgCl reference electrode was stored in a saturated NaCl solution  and anion exchange membrane , Nafion 117 membrane (Sigma Aldrich), bipolar membrane , were stored in MilliQ® grade H2O.Acetonitrile , D1H NMR spectroscopy was recorded on a Bruker DPX 400 MHz spectrometer with the chemical shifts (δ) of the 1H NMR spectrum being referenced against the residual solvent signal . Scanning electron microscopy was performed on a TESCAN MIRA3 FEG-SEM instrument. Before measuring the samples, they were sputtered with 10 nm of platinum.2 and CO under static conditions were analysed by a Shimadzu Tracera GC-2010 Plus with a barrier discharge ionization detector. The GC-2010 Plus was equipped with a ShinCarbon micro ST column (0.53 mm diameter) kept at 40 °C using helium carrier gas. The response factors for the gases were determined by calibration with known amounts of H2 and CO. Typically, 50 μL of headspace gas from the photoreactor was injected using an air-tight syringe . Formate was analysed by ion chromatography (IC) on a Metrohm 882 compact IC plus chromatography system equipped with a Metrosep A Supp 5 – 150/4.0 column using an aqueous Na2CO3 (3.2 mM) and NaHCO3 (1 mM) solution as eluent. The response factor of formate was determined by calibration with known amounts of aqueous formate solutions. Sugar concentrations  were determined by high performance liquid chromatography (HPLC) on a Waters Breeze system equipped with a refractive index detector and a Rezex 8% Ca2+ Monosaccharide 300 × 7.80 mm HPLC column using 2.5 mM H2SO4 as the eluent with a flow rate of 0.5 mL min−1 (at 75 °C). The response factors for the sugars were determined by calibration of aqueous sugar solutions with known amounts of sugar.Gaseous H6 For the compositional analysis of white birch, the sample was first dried at 105 °C overnight followed by cooling to room temperature under static vacuum in a desiccator containing CuSO4 as drying agent. The dried solid was extracted three times with an ethanol : water mixture (4 : 1 v/v and 20 mL g−1) followed by water (20 mL g−1) by sonication for 30 min. The sample was then dried at 105 °C overnight followed by cooling to room temperature under static vacuum in a desiccator containing CuSO4 as a drying agent.Klason lignin and sugar determination was performed following a reported procedure.2SO4 at room temperature. The suspension was stirred periodically (every 15 min with a glass rod) for 2 h and 145 mL of MilliQ® grade H2O was added, followed by refluxing the suspension at 120 °C for 4 h. The suspension was filtered on a tared frit. The solid was dried at 105 °C overnight and the mass of the filtered solid was determined gravimetrically to determine Klason Lignin content. The filtrate was diluted with H2O to 250 mL and concentrated to 50 mL at 80 °C. The solution was then analysed by HPLC to determine the sugar content .To determine the Klason lignin content and sugar concentration, to 250 mg of the solid (dried and extracted white birch or solid after white birch pre-treatment in dioxane/HCl/HCOOH) was added 3.75 mL of a 72 wt% HTo determine the Klason Lignin and sugar content of the liquor from white Birch pre-treatment in dioxane/HCl/HCOOH, the liquor obtained from pre-treating 500 mg white Birch was used following the same procedure as with dried and extracted white Birch.7 To 500 mg of dried and extracted white birch was added dioxane (4.5 mL), conc. HCl (0.42 mL), formic acid (0.36 mL) and water (0.64 mL). The suspension was then stirred for 5 h at 80 °C and filtered and the residue was washed with dioxane until the solution became clear (solid/cellulose fraction). The combined filtrate was dried at 60 °C under vacuum and extracted with THF followed by drying at 40 °C to obtain a dark oil (liquid/lignin fraction).Lignocellulose fractioning was performed following a reported procedure.2O (20 mL g−1) to remove residual HCOOH and dried at 105 °C overnight. To the washed and dried solid fraction (150 mg) in an aqueous sodium acetate solution  at pH 5 (adjusted by the addition of HCl) at 37 °C was added cellulase (15 mg in 0.75 mL) in a 50 mM aqueous sodium acetate solution at pH 5. The suspension was incubated at 37 °C for 24 h, followed by 15 min at 90 °C and filtration through a syringe filter (0.2 μm). The filtered solution was stored at −4 °C. The pre-treated cellulose fraction solution contained glucose (39.3 ± 4.7 mM) and cellobiose (8.7 ± 2.2 mM) as determined by HPLC. Control experiments with the recovered solid after treatment of white Birch with dioxane/HCl/HCOOH under the same condition in the absence of cellulase did not show the formation of glucose or cellobiose.The obtained solid after treatment of white birch with dioxane/HCl/HCOOH was washed three times with H−1) and 2 vol% of a 5 wt% Nafion solution was sonicated for 15 minutes. The suspension was then dropcasted on a defined area of carbon paper (0.1 mL cm−2) that was masked with Teflon tape followed by overnight drying at room temperature. The electrode was then taped to a metal rod using a copper tape and the metal rod and copper tape were wrapped with Parafilm.A suspension containing MWCNT in ethanol (1.67 mg mL−1) was sonicated for 10 min. Subsequently, this ink was diluted 25% by adding 0.4 mM CoPL in DMF to achieve 2.37 mg MWCNT mL−1 and 0.1 mM CoPL. This ink was further sonicated for 10 min and then dropcasted on a defined area (masked with Teflon tape) of carbon paper (0.1 mL cm−2), followed by drying overnight at room temperature. The electrode was then taped to a metal rod using a copper tape and the metal rod and copper tape were wrapped with Parafilm.A suspension containing MWCNT in DMF  was added 2 mL of MeCN and 1 mL of the solution obtained from cellulase pre-treatment in a glass photoreactor  equipped with a magnetic stir bar. 50 nmol of CotpyP  was added, and the photoreactor (3 mL solution with 4.74 mL headspace) was capped with a rubber septum and purged with CO2  controlled by mass flow controllers for 15 min at 15 mL min−1, followed by stirring for 15 min in the dark. The photoreactor (kept at 25 °C and stirred at 600 rpm) was then irradiated with a calibrated solar light simulator  equipped with an air mass 1.5 global (AM 1.5G) filter and a water filter to remove infrared radiation. The photocatalytic process was monitored by analysing the headspace after 6 and 24 h by gas chromatography to monitor H2 and CO formation. Formate in the solution ) was analysed at the end of the photocatalytic studies (after 24 h) by ion chromatography.In a typical experiment, to TiO2-to-CO conversion yield (%) of TiO2∣CotpyP was calculated by dividing the mol of CO produced after 24 h by the mol of CO2 in the photoreactor headspace and multiplying the product by 100. The mol of CO2 was obtained using the ideal gas law equation (pV = nRT), where p is 1 atm, V is the volume of the reactor headspace in L multiplied by the molar fraction of CO2, R is the ideal gas constant (0.082 atm L mol−1 K−1) and T is 298.15 K.Photocatalytic CO2 which was suspended in 2.95 mL of 2 : 1 v : v MeCN : pre-treated cellulose solution. The molecular catalyst CotpyP  and 0.025 mL H2O (to reach 3 mL) was added and the photoreactor was capped with a rubber septum. The photoreactor was then degassed for one min (vacuum at 10−2 mbar) after which 13CO2 (1 bar) was introduced. The photoreactor (kept at 25 °C and stirred at 600 rpm) was then irradiated . The headspace was then transferred to an air-tight evacuated IR cell  and the background (IR cell under vacuum) corrected IR spectrum was recorded to detect 12CO and 13CO.To a suspension of cellulose (300 mg) in a 50 mM aqueous sodium acetate solution (6 mL) at pH 5 (adjusted by the addition of HCl) at 37 °C was added cellulase (15 mg in 1.5 mL) in a 50 mM aqueous sodium acetate solution at pH 5. The suspension was incubated at 37 °C for 24 h, followed by 15 min at 90 °C and filtration through a syringe filter (0.2 μm). The filtered solution was stored at −4 °C. The pre-treated cellulose solution contained glucose (53 ± 2 mM) and cellobiose (26 ± 1 mM) as determined by HPLC. To a glass photoreactor vial  equipped with a magnetic stir bar was added 5 mg of TiO3 solution in H2O was prepared and used as the anolyte and catholyte separated by a Nafion membrane. CA was performed with CP∣CoPL as working electrode, Pt foil as counter electrode and Ag/AgCl as reference electrode. The electrochemical cell was capped with rubber septa and the catholyte was purged for 30 minutes with N2  or the desired gas composition (for CA at a given gas composition (20 or 100 vol%) for 4 h) at a flow rate of 20 mL min−1 controlled by mass flow controllers (Brooks) to remove oxygen. Afterwards the flow rate was reduced to 9 mL min−1 and a potential of −1.2 V vs. Ag/AgCl was applied. CA was run at −1.2 V vs. Ag/AgCl for 4 h at constant gas flow of 20 or 100 vol% or during screening different gas composition, the electrocatalytic activity was measured for 30 min under N2 followed by increasing the CO2 concentration to 10, 20, 50 and 100 vol% every 45 min. Electrochemical experiments were carried out at room temperature. The formed gaseous products (H2 and CO) were measured by online GC measurement (injection every 4.5 min) using an Shimadzu Tracera GC-2010 Plus gas chromatograph equipped a barrier discharge ionization detector.38 Calibration was performed by determining the response factor by flowing a calibration gas with known CO and H2 composition under the same condition (9 mL min−1).Electrochemical experiments were performed on an Ivium Compactstat electrochemical analyser controlled by the Iviumsoft software. In a typical three-electrode setup experiment, a 0.1 M NaHCO3+ potentiostat. In a typical experiment, 0.1 M Na2CO3 solution in MeCN : H2O (1 : 1 vol%) was used as the electrolyte. To the electrolyte was added 0.01 M of the lignin model substrate or the lignin fraction from pre-treating 250 mg white birch in dioxane/HCl/H2O. The solution was then used as anolyte (8 mL) in electrocatalysis with 0.1 M Na2CO3 solution in MeCN : H2O (1 : 1 vol%), and separated from the catholyte by a Selemion anion exchange membrane, and the electrochemical cell was capped with rubber septa. Cyclic voltammetry (CV) or CA were performed with CP∣MWCNT as working electrode, Pt foil as counter electrode and Ag/AgCl (sat. KCl) as reference electrode. CV scans were run from −0.2 to 1.0 V vs. Ag/AgCl followed by a backwards scan to −0.2 V vs. Ag/AgCl with a scan rate of 50 mV s−1. CA experiments were performed for 4 h at 1.0 V vs. Ag/AgCl. Electrochemical experiments were carried out at room temperature. To analyse the products after electrocatalysis, the anolyte was acidified to a pH of 3 with 0.1 M HCl, extracted with EtOAc (3 × 5 mL), dried over MgSO4, filtered and dried at 40 °C under vacuum to obtain a light brown solid. The solid was further analysed by 1H NMR spectroscopy in CDCl3 with mesitylene as internal standard. Control experiments were performed without an applied potential and stirring the anolyte containing the lignin model substrate or lignin fraction for 4 h followed work up and analysis by 1H NMR spectroscopy in CDCl3.Electrochemical experiments were performed in three-electrode configuration on a PalmSens MultiEMStat2CO3 solution in MeCN : H2O (1 : 1 vol%) was prepared and used for the electrolyte. To the electrolyte was added 0.01 M of the lignin model substrate or the lignin fraction from pre-treating 250 mg white birch in dioxane/HCl/H2O. The solution was then used as anolyte (4 mL). A 0.1 M NaHCO3 solution in H2O was used as catholyte. The anolyte and catholyte were separated by a bipolar membrane. Electrolysis was performed with CP∣MWCNT and CP∣CoPL as anode and cathode, respectively. The electrochemical H-type cell was capped with two rubber septa and the catholyte was purged for 30 minutes with 20 vol% CO2  at a flow rate of 20 mL min−1 controlled by mass flow controllers (Brooks) to remove oxygen. Afterwards the flow rate was reduced to 9 mL min−1 and a potential of Uapp = −3 V was applied and run for 4 h. Electrochemical experiments were carried out at room temperature. The formed gaseous products (H2 and CO) on the cathode side were measured by online GC measurement (injection every 4.25 min) using Shimadzu Tracera GC-2010 Plus gas chromatograph equipped a barrier discharge ionization detector.38 Calibration was performed by determining the response factor by flowing a calibration gas with known CO and H2 composition under the same condition (9 mL min−1). To analyse the products, after electrocatalysis the anolyte was acidified to a pH of 3 with 0.1 M HCl, extracted with EtOAc (3 × 5 mL), dried over MgSO4, filtered and dried at 40 °C under vacuum to obtain a light brown solid. The solid was further analysed by 1H NMR in CDCl3 with mesitylene as internal standard. Control experiments were performed under the same conditions but without the lignin model substrate or lignin fraction dissolved in the anolyte.Electrochemical experiments were performed in two-electrode configuration on an Ivium Compactstat electrochemical analyser controlled by the Iviumsoft software. In a typical experiment a 0.1 M Na2 conversion yield (%) of CP∣CoPL was calculated by dividing the rate of CO formation (mol min−1) by the flow rate of CO2 (mol min−1) and multiplying the product by 100. The flow rate of CO2 was obtained by transforming mL min−1 to mol min−1 using the ideal gas law equation (pV = nRT), where p is 1 atm, V is the flow rate of CO2 in L min−1 , R is the ideal gas constant (0.082 atm L mol−1 K−1) and T is 298.15 K.The electrochemical single-pass COx̄) and standard deviation (σx̄) expressed as x̄ ± σx̄ withwhere n is the number of measurements and xi the individually determined value.Experiments were performed in triplicates and the results are represented with the mean (There are no conflicts to declare.GC-025-D3GC03258B-s001