The effects of sintering temperature and composition on the phase formation, microstructure, and microwave dielectric properties were investigated. The results show that the microwave sintering process requires a lower sintering temperature and shorter sintering time of (1–x)CLT-xNMT ceramics than conventional heating methods. All of the (1–x)CLT-xNMT ceramics possess a single perovskite structure. With the increase of x, the dielectric constant (ε) shows a downward trend; the quality factor (Qf) drops first and then rises significantly; the resonance frequency temperature coefficient (fτ) keeps decreasing. With excellent microwave dielectric properties , the 0.65CLT-0.35NMT ceramic can be applied to the field of mobile communications.The (1– Qf value, near-zero temperature coefficient of resonance frequency, and low sintering temperature are strong candidates for 5G technology  ceramics were prepared by microwave sintering and an in-depth study was conducted of the effects of sintering process and component ratio on its phase composition, microstructure, and microwave dielectric properties.As an efficient sintering method for materials, microwave sintering can effectively reduce the sintering temperature, increase the sintering rate, and promote the grain refinement of ceramics, thus improving the microwave dielectric properties ,13. Up tx)Ca0.61La0.26TiO3-xNd(Mg0.5Ti0.5)O3  ceramics were prepared by the solid-state reaction method. The ingredients were proportioned according to the stoichiometric ratio. High-purity CaCO3 , La2O3 , Nd2O3 , MgO , and TiO2  powders were mixed by ball mill for 8 h and then dried for 24 h, ground, sieved (200 mesh), and calcined . Then, the calcined CLT and NMT powders were mixed by ball mill for 8 h, dried for 24 h, and sieved (200 mesh). After added 10 wt % of polyvinyl alcohol solution  as a binder, the mixed powders were pressed into columns with a diameter of 13 mm and a thickness of 2~6 mm and then these specimens were heated at 600 °C for 1 h to remove the PVA. Finally, these specimens were sintered in air in a microwave sintering furnace  .The (1–x)CLT-xNMT samples was conducted by X-ray diffraction . After the samples were polished and cleaned with ultrasonic cleaner, etched at 50 °C lower than sintering temperature for 30 min, their microstructures were observed by a scanning electron microscope .The density was measured by the Archimedes method. After crushed and ground, the phase analysis of (1–x)CLT-xNMT ceramic cylindric specimen was put in a metal cavity of vector network analyzer , in which high-frequency electromagnetic field can keep oscillating without radiation loss. The dielectric constant (ε) and quality factor (Q) were measured at 25 °C. The temperature coefficient of resonant frequency (fτ) was calculated by using the Equation (1):f1 and f2 represent the resonant frequency at T1 (25 °C) and T2 (85 °C), respectively.To measure the dielectric properties, polished (1–ρ) of (1−x)CLT-xNMT ceramics is shown in T), the ρ presents the tendency of increasing first. However, with the further increase of T, the ρ tends to decrease. It may be attributed to oversintering.The influence of sintering temperature on the density (ρ and relative density (rρ) of the (1−x)CLT-xNMT ceramic with x is shown in ρ increases with the increase of x, up to 5.457 (x = 0.60), mainly because the density of NMT ceramic (6.16 g/cm3) is higher than that of CLT ceramic (4.51 g/cm3). The rρ is all higher 95.5% with slightly floating and reaches 96.9% when x = 0.50.The relationship between the x)CLT-xNMT ceramics are illustrated in x = 0.35~0.60, indicating a perovskite structure without second phase. It should be pointed out that superlattice diffraction peaks were observed when x = 0.40 and 0.45. The enlarged part of 32.1~33.3°, as shown in the upper right corner of x)CLT-xNMT ceramics shift toward low angle with the increase of x. It suggests the increasing lattice constant of the identified perovskite structure.The XRD patterns of the  and unit cell volume (uV) of (1−x)CLT-xNMT ceramics are shown in x, which is in accordance with the XRD analysis. This trend depends on two factors: the decreasing vacancy concentration in A-site, the increasing Mg2+ content (r(Mg2+) > r(Ti4+), r(Mg2+) = 0.072 nm, r(Ti4+) = 0.061 nm when CN = 6) in B-site  ceramics were prepared by microwave sintering. The effects of sintering process and component distribution compare on its phase composition, microstructure, and microwave dielectric properties were investigated. Microwave sintering can effectively reduce the sintering temperature and the sintering time. The (1–x)CLT-xNMT ceramics have formed a perovskite structure. As x increases, the ε shows a downward trend, the Qf first drops to 8482 GHz and then rises to 32,637 GHz, and the fτ keeps decreasing. When x = 0.35, the comprehensive microwave dielectric performance is: ε = 51.3, Qf = 13,852 GHz, fτ = −1.9 × 10−6/°C . The (1–x)CLT-xNMT ceramics can be applied to the field of mobile communications.The (1–


Replacement of vitamin D (VD) among patients with chronic hepatitis C (CHC) before viral eradication has demonstrated a protective effect on serum markers associated with hepatic fibrogenesis. We therefore hypothesized that VD may facilitate further fibrosis amelioration following curative treatment with direct-acting antivirals (DAA).β1) and tissue inhibitors of matrix metalloproteinases 1 (TIMP-1)], and fibrolytic markers [matrix metalloproteinase 9 (MMP-9) and amino terminal type III procollagen peptide (P3NP)], were assessed at baseline and at 6 weeks. Serum 25(OH)D was analyzed by a chemiluminescence immunoassay. Serum hepatic fibrogenesis markers were measured using a quantitative sandwich enzyme-linked immunosorbent assay.This study was a randomized, double-blind, placebo-controlled trial conducted between February 2018 and August 2018. Patients with CHC and VD deficiency were randomized in a 1:1 ratio to either receive ergicalciferol or placebo over 6 weeks. Biochemical analysis indicators, including 25-hydroxyvitamin D (25(OH)D), fibrogenic markers ), p = 0.63), TIMP-1 (−5.5 ng/mL (95% CI [−26.4 –15.3]), p = 0.60), MMP-9 (122.9 ng/mL (95% CI [−69.0 –314.8]), p = 0.21), and P3NP (−0.1 ng/mL (95% CI [−2.4 –2.2]), p = 0.92) between the VD and placebo groups.Seventy-five patients with CHC and VD deficiency were randomly assigned to VD (Short-term VD supplementation after DAA treatment in patients with CHC does not improve serum fibrogenesis markers and may not expedite the residual liver fibrosis healing process. Future studies are warranted to evaluate the long-term effect of VD supplementation on hepatic fibrosis regression. This process consequently leads to a change in the fibrotic cascade and results in an excessive accumulation of extracellular matrix (ECM) within the liver parenchyma  infection remains a major health-care problem worldwide, leading to high morbidity and mortality from cirrhosis and hepatocellular carcinoma (HCC) . NotablyVitamin D (VD) deficiency/insufficiency is a global problem affecting approximately 1 billion people; its prevalence varies by age and ethnicity in different countries . A numbeGrowing experimental and clinical evidence has revealed an anti-fibrogenic effect of VD . Our pre® D level <30 ng/mL), and evidence of more than F2 liver fibrosis from either Fibroscan≥7.1 kPa , ultraso≥7.1 kPa , or magn≥7.1 kPa ). PatienData were collected as previously described in Vitamin D2  and the placebo were prepared in identical capsules of the same weight by the Department of Pharmacy at King Chulalongkorn Memorial Hospital. All investigators and patients were blinded to the type of medication used until the end of the study. Each capsule of VD2 contained 20,000 international units of ergocalciferol. The protocol for VD supplementation was based on the 25(OH)D level at baseline , which hβ1, TIMP-1, MMP-9, and amino terminal type III procollagen peptide (P3NP) levels at the end of the study. Pill counts and patient interviews were used to assess adherence to the prescribed medications. All participants were asked to refrain from outside-of-trial VD or multivitamin supplements and to maintain their normal activity. At the end of the study, patients who remained in a VD-insufficient state received VD supplementation as a standard of care.Baseline clinical characteristics, HCV genotypes, liver enzymes, and fibrosis-4 (FIB-4) and aspartate aminotransferase to platelet ratio index (APRI) scores were assessed. Blood samples were collected at two time points (baseline and at the end of the 6-week follow-up) and were kept at −70 °C until analysis. All blood samples were simultaneously analyzed for TGF-β1, TIMP- 1, MMP-9, and P3NP, relative to the effect in the placebo group at the 6-week follow-up.The primary outcome was the effect of VD supplementation on serum markers associated with hepatic fibrogenesis, including TGF- ® chemiluminescence analyzer following the manufacturer’s instructions. The final level is reported in ng/mL. According to the Endocrine Society Practice Guidelines (Serum 25(OH)D levels, a functional indicator of VD status, were measured using the Liaison 25(OH) vitamin D total assay ; the measurement was performed on the LIAISONidelines , VD insuβ1 were measured with a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique following the manufacturer’s instructions . Before the assay, the latent TGF-β1 contained in the patients’ serum was activated to the immunoreactive form using acid activation and neutralization. The results were calculated by reference to the standard curve.The serum levels of human activated TGF-® ELISA). The results were calculated by reference to the standard curve.The serum levels of human TIMP-1 were measured using the quantitative sandwich ELISA technique according to the manufacturer’s instructions (Quantikine® ELISA). The results were calculated by reference to the standard curve.The serum levels of human MMP-9 protein were measured by a quantitative sandwich ELISA according to the manufacturer’s instructions . The results were calculated by reference to the standard curve.β1 for the calculation, since it resulted in the largest sample size. The mean difference in the change in TGF-β1 level from baseline to week 6 in the VD supplementation arm versus the placebo arm as a control was 81 pg/mL, with a pooled standard deviation of 110. Thus, enrolling 30 patients per group would give 80% power to detect a difference in TGF-β1 change from baseline between treatment groups of this size or more, at a 2-sided significance level of 5%. We inflated the sample size to 37 per randomized arm to account for possible dropouts.Sample size calculations were based on data from our previous study on patients with CHC . Of the t-test to evaluate mean differences and 95% confidence interval (95% CI) in the change from baseline to week 6 between the VD and the placebo groups. As a sensitivity analysis, to ensure that outliers did not exert an undue influence on the obtained between-arm estimates, we summarized the data as the median and 25th and 75th percentile levels, and used quantile regression to compare the median serum level changes between randomized arms. Statistical analyses were performed with Stata 16.1 .Baseline characteristics, including clinical and laboratory data, are presented as percentage or mean ±  standard deviation (SD). In the initial analyses, the baseline characteristics of the patients were compared using t-tests or chi-square tests. Categorical variables were analyzed using the chi-square test or Fisher’s exact test, as appropriate. For the primary study outcome of fibrogenesis marker changes, we used the independent This study was reviewed and approved by the Ethics Committee and Institutional Review Board (IRB) at Chulalongkorn University, Bangkok, Thailand, and was performed in accordance with the Declaration of Helsinki (1989) of the World Medical Association (IRB number: 707/60). Moreover, this trial was registered with the Thai Clinical Trials Registry (TCTR) based on the World Health Organization criteria on 22 November 2017 (TCTR20171206003). Prior to participation, all patients enrolled in this study provided written informed consent for participation, as well as for publication.n = 37) or the placebo group (n = 38). Mean age was 60.4 ±  7.8 years, patients were mostly female , and a majority of the participants had cirrhosis . As shown in Between February 2018 and August 2018, 80 patients with CHC who achieved SVR after DAA treatment were screened for eligibility. Five patients were excluded from the study . A totalp < 0.001).Mean baseline serum 25(OH)D level was 17.2 ±  4.8 ng/mL and 16.6 ±  4.1 ng/mL in the VD and placebo groups, respectively. At the 6-week follow-up, the mean 25(OH)D level in the VD group increased to a normal level (41.8 ±  9.1 ng/mL), whereas a slight increase was observed in the placebo group (18.1 ±  4.6 ng/mL) . The meaOf note, three patients (8.1%) in the VD group still had a serum 25(OH)D level <30 ng/mL at 6 weeks after VD replacement; the 25(OH)D levels in two patients changed from an initial deficient state to an insufficient state after VD supplementation , while the remaining patient had a slightly increased VD level (from 21.8 ng/mL at baseline to 23.5 ng/mL) after VD replacement. All participants in the placebo group still had VD insufficiency/deficiency. All unused pills were returned at the last follow-up, and no patient reported missing any medications.β1 levels slightly increased from 16.3 ±  6.6 ng/mL at baseline to 16.8 ±  7.6 ng/mL at 6 weeks in the VD group, and increased from 14.3 ±  8.3 to 15.4 ±  6.8 ng/mL in the placebo group. However, there was no significant difference in the mean change from baseline between the VD and placebo groups (−0.6 ng/mL (95% CI [−2.8 –1.7]), p = 0.63).Mean and median differences in serum marker changes from baseline to 6 weeks are shown in p = 0.60).Similarly, mean serum TIMP-1 levels in the VD group slightly increased from 236.9 ±  50.0 ng/mL at baseline to 244.0 ±  66.0 ng/mL at 6 weeks. In the placebo group, mean serum TIMP-1 levels slightly increased from 231.9 ±  50.3 ng/mL at baseline to 244.5 ±  63.6 ng/mL at 6 weeks. There was no significant difference in the mean TIMP-1 change from baseline to 6 weeks between the VD and placebo groups (−5.5 ng/mL (95% CI [−26.4 –15.3]), p = 0.21).Regarding fibrolytic markers, mean MMP-9 levels showed an increasing trend in the VD group (576.2 ±  296.2 ng/mL at baseline and 684.3 ±  423.5 ng/mL at 6 weeks); median levels showed a consistent pattern. In contrast, mean MMP-9 levels in the placebo group were approximately the same at baseline and week 6, but the median levels increased. There was no significant difference in the mean change from baseline at week 6 between the VD and placebo groups (122.9 ng/mL (95% CI [−69.0–314.8]), p = 0.92).Mean P3NP levels slightly decreased from the baseline in both groups. There was no significant difference in the mean P3NP change score from baseline to week 6 between the VD and placebo groups (−0.1 ng/mL (95% CI [−2.4–2.2]), We conducted quantile regression, using the median as another measure of the central tendency, to assess whether extreme outliers in the dataset had a strong influence on the observed associations. In both VD and placebo groups, the median, 25th and 75th percentile values of each marker were very similar at baseline and week 6 visits , Table 4A subgroup analysis was performed according to the severity of VD deficiency/insufficiency, which showed no significant changes in serum hepatic fibrogenesis markers in both groups. Additional subgroup analyses based on sex, cirrhosis, liver fibrosis severity, and a 25(OH)D level <10 ng/ml at baseline also revealed similar results  and S2.In this randomized, double-blind, placebo-controlled study, 6-week VD supplementation in patients with CHC after HCV eradication by DAA did not help attenuate liver fibrosis as assessed by fibrogenesis markers. Additionally, the effect of VD supplementation did not vary according to baseline serum 25(OH)D levels and liver fibrosis severity.β1 results in fibrillary component accumulation, which leads to progressive liver fibrosis  significantly decreased in the VD supplementation group, whereas the levels of anti-fibrotic enzymes  were significantly higher in the VD supplementation group than in the placebo group  for a longer period could potentially help ameliorate liver fibrosis is warranted.10.7717/peerj.10709/supp-1Supplemental Information 1(A) Baseline VD levels. (B) Sex. (C) Presence of cirrhosis. (D) Liver fibrosis severity by FIB4 score. (E) Liver fibrosis severity by APRI score.Click here for additional data file.10.7717/peerj.10709/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10709/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10709/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.10709/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.10709/supp-6Supplemental Information 6Click here for additional data file.


In this study, improvement of the antimicrobial activity of BSI-9, against the European Committee on Antimicrobial Susceptibility Testing (EUCAST) strains of S. aureus, E. coli, A. baumannii, and P. aeruginosa was examined. This led to synthesis of eighteen peptide analogues of BSI-9, produced in four individual stages, with a different focus in each stage; cyclization point, hydrophobicity, cationic side-chain length, and combinations of the last two. Specifically the modified compound 11, exhibited improved activity against Staphylococcus aureus and Pseudomonas aeruginosa with MIC of 4 µg/mL and 8 µg/mL, respectively, compared to the original BSI-9, which had an MIC of 16–32 µg/mL. The emergence of multi-drug resistant bacteria is becoming a major health concern. New strategies to combat especially Gram-negative pathogens are urgently needed. Antimicrobial peptides (AMPs) found in all multicellular organisms act as a first line of defense in immunity. In recent years, AMPs have attracted increasing attention as potential antibiotics. Naturally occurring antimicrobial cyclic lipopeptides include colistin and daptomycin, both of which contain a flexible linker. We previously reported a cyclic AMP BSI-9 cyclo(Lys-Nal-Lys-Lys-Bip-O Klebsiella pneumonia, Acinetobacter baumannii, and Eschericia coli, where pan-resistance is now observed -1H-1,2,3-triazolopyridinium-3-oxid hexafluorophosphate, N-pyridin-1-ylmethylene]-N-methylmethanaminium hexa-fluorophosphate N-oxide) were purchased from GL Biochem Shanghai. DMF , DCM , and MeCN  were obtained from VWR. Chemistry: Disposable 5-mL polypropylene reactors fitted with a PTFE filter were acquired from Thermo Scientific TentaGel18, HPLC column (5 µm 10 × 250 mm) equipped with a WatersTM Cartridge Holder PKG (10 × 10 mm). Peptides were purified by preparative Reverse Phase Analytical High Performance Liquid Chromatography (RP–HPLC) on a preparative WatersTM XBridgeTM BEH130 CMicroflexTM [Bruker Corporation] FlexControl software [Bruker Daltonik GmbH] was used to obtain the spectra and the data were processed using flexAnalysis [Bruker Daltonik GmbH]. All reagents and solvents were used without further purification. ®. 96-well plates cell culture cluster, round-bottom, polypropylene plates polystyrene, flat-bottomed 96-well ELISA plate and V-shaped 96-well polypropylene plate were purchased from Corning® Inc., Costar®, Corning, NY, USA. Microseal® film was obtained from Bio-Rad Laboratories, Inc., Hercules, CA, USA. VersaMaxTM Tunable Microplate Reader  and the data were evaluated on Softmax® Pro (Molecular Devices LLC).Microbiology and Haemolysis: Non-cation adjusted Mueller–Hinton broth (MHB) media  resin, with a loading of 0.22 mmol/g. The resin was weighed out into a disposable 5-mL polypropylene reactor, fitted with a PTFE filter and swelled in DMF for a minimum of two hours or overnight, and washed with DMF (3×), DCM (3×), and DMF (5×). The base-labile Fmoc group was removed by treatment with 4 mL 20% piperidine/DMF for four minutes, and reiterated three times with a DMF wash in between each treatment. After the third deprotection, the resin was washed with DMF (3×), DCM (3×), and DMF (5×). For BSI-9 and analogues 1–4, the amino acids were coupled successively and a single amino acid coupling used 5 equivalents of amino acid, COMU, and Oxyma each, and 2 equivalents DIEA. Fmoc-protected amino acids were dissolved in DMF at a concentration of 0.6 M. COMU and Oxyma were weighed out per coupling and dissolved in fresh DMF at a 0.6 M concentration, just prior to coupling. The amino acid solution, COMU/Oxyma solution, and DIEA were combined and immediately transferred to the syringe, covered in tin foil and put on a shaker for 1.5–2 h. For analogues 5–18, HATU, HOAt, and DIEA were used as coupling reagents, as described above. The coupling solution was discharged and the resin was washed with DMF (3×), DCM (3×), and DMF (5×). The Fmoc group was removed with 4 mL 20% piperidine/DMF for 3 × 4 min, as described here earlier, and washed with DMF (3×), DCM (3×), and DMF (5×). The next amino acid was then coupled. Solid-phase peptide synthesis: Peptide analogues were synthesized in 4 stages, S1, S2, S3, and S4. Peptides were synthesized on a TentaGelThe Fmoc group of the last amino acid was removed . Next, the Dmb group was removed from the Asp by treatment with 3 mL 1.5% TFA/ DCM solution, 6 times for 5 min. The resin was washed with DMF, followed by DCM and finally ethanol (EtOH). The resin was freeze-dried and transferred to a reactor fitted with a PTFE filter and swelled in DMF for 2 h.4 was dissolved in minimum DMF, 6 equivalents of DIEA were added, and the reaction was allowed to proceed for 8 h or overnight. The resin was washed with DMF (5×). Afterwards, 3 equivalents of DMTMM·BF4 and 3 equivalents of DIEA were added and the reaction was left for 8 h or overnight. The resin was washed with DMF (3×), DCM (3×), and EtOH (5×). Cleavage of a small amount of resin was done to confirm sequence completion and cyclization. The resin was placed in the freeze-dryer until dry.Three equivalent DMTMM·BF2O:TIS (95:2.5:2.5) solution for 2 h. The cleavage solution was collected and concentrated with a gentle stream of N2, down to about 300 µL. The peptide was precipitated and washed with 4 mL cold (−20 °C) diethyl ether, 3 times. The residual ether was left overnight to evaporate. Next, the crude product was dissolved in 90% water and 10% ACN and freeze-dried, ready for analysis and purification. Peptides were identified using Matrix Assisted Laser Desorption Ionization time of flight mass spectroscopy (MALDI–ToF MS). To this end, 1 µL peptide solution and 1 µL α-cyano4-hydroxycinnamic acid matrix  were spotted onto a target plate, followed by detection using a MicroflexTM. FlexControl software was used to obtain the spectra and the data were processed using flexAnalysis [Bruker Daltonik GmbH]. RP–HPLC was used to assess peptide purity. The system consisted of a WatersTM In-Line Degasser AF, a WatersTM 600 pump, a WatersTM 2996 Photodiode Array Detector, and a WatersTM Symmetry C18, 4.6 × 250 mm, 5 mm column. The data were processed using Empower3 software. Purity was ≥95% for all tested peptide. Peptide purification was done by dissolving the peptides in minimal H2O/can, and 300–500 µL was injected into the HPLC system to achieve separation. Cleavage was performed on half or all of the resin with a TFA:HE. coli ATCC 29522, S. aureus ATCC 29213, A. baumannii ATCC 19606, and P. aeruginosa ATCC 27853) were grown overnight on LB agar plates at 37 °C. Overnight cultures were prepared by inoculation of two to three colonies in 10 mL non-cation adjusted Mueller–Hinton broth (MHB) media . Bacterial inoculum for MIC testing was prepared as balanced cultures, as previously described [8 colony forming units (CFU/mL), and then diluted 1:200 in MHB, to give a final bacterial suspension of 5·105 CFU/mL, as described by Wiegand et al. [Bacterial strains  and negative (0%) controls, as described previously [A total of 150 µL of 5 µM melittin was added to the positive control wells 1-6H of a V-shaped 96-well polypropylene plate and left overnight. The melittin solution was removed and the positive control wells were washed 3 times with 150 µL PBS. Afterwards, a 2.5 µM melittin solution was added to the positive control wells. For the assay, 0-negative blood in EDTA was used. Blood was washed by mixing 3 mL of PBS with 1 mL of whole blood, and centrifuged for 8 min at 3000 rpm. The supernatant was discarded and 4 mL of PBS added, mixed gently, and centrifuged. This wash cycle was repeated but the last centrifuge velocity was raised to 4000 rpm. Next, 40 µL red blood cells (RBC) was suspended in 8 mL of PBS to achieve an RBC suspension of 0.5% volume/volume (v/v). Peptide solutions were prepared at 600 µM, two times the highest concentration. A total of 150 µL peptide solution was transferred to row A. Next, 75 µL PBS was transferred to wells B1 to G12, as well as H7-12 (negative control), and the peptides were serially diluted two-fold. Then, 75 µL RBC suspension was added to all wells and the plate was covered with a Microsealeviously .S. aureus ATCC 29213 or P. aeruginosa ATCC 27853. We used balanced cultures, as described in Oddo et al. [5, the cultures were back diluted into fresh MHB, heated to 37 °C from an exponentially growing culture. In the late exponential phase, cultures were simply kept in the exponential phase, until OD600 = 0.1, before experimentation.Time-kill experiments were performed on balanced exponentially growing cultures of o et al. . In brie11 showed activity against P. aeruginosa and S. aureus, but showed no activity against E. coli and A. baumannii in MIC assays. The most active analogue 11, had a bactericidal activity against P. aeruginosa and bacteriostatic activity against S. aureus, as determined in exponential and late exponential phase time-kill experiments. At 5× MIC, 11 was just as effective as colistin in killing P. aeruginosa. For S. aureus, 11 was significantly better than chloramphenicol at 5× MIC. The results from this study might be helpful in the design of novel cyclic antimicrobial peptides. We are currently pursuing the synthesis of fatty acid analogues of 11 to mimic known cyclic lipopeptide antibiotics such as colistin and daptomycin.The present study described the successful synthesis of 18 analogues of a cyclic peptide with a flexible linker. The analogues were synthesized in four stages, focusing on cyclization point, hydrophobicity, cationic side-chain length, and combinations of the last two. We found that compound


N-glycolyl chondroitin (Gc-CN) is a metabolite of N-glycolylneuraminic acid (Neu5Gc), a sialic acid that is commonly found in mammals, but not humans. Humans can incorporate exogenous Neu5Gc into their tissues from eating red meat. Neu5Gc cannot be biosynthesized by humans due to an evolutionary mutation and has been implicated in causing inflammation causing human diseases, such as cancer. The study Neu5Gc is important in evolutionary biology and the development of potential cancer biomarkers. Unfortunately, there are several limitations to detecting Neu5Gc. The elimination of Neu5Gc involves a degradative pathway leading to the incorporation of N-glycolyl groups into glycosaminoglycans (GAGs), such as Gc-CN. Gc-CN has been found in humans and in animals including mice, lamb and chimpanzees. Here, we present the biosynthesis of Gc-CN in bacteria by feeding chemically synthesized N-glycolylglucosamine to Escherichia coli. A metabolically engineered strain of E. coli K4, fed with glucose supplemented with GlcNGc, converted it to N-glycolylgalactosamine  that could then be utilized as a substrate in the chondroitin biosynthetic pathway. The final product, Gc-CN was converted to disaccharides using chondroitin lyase ABC and analyzed by liquid chromatography–tandem mass spectrometry with multiple reaction monitoring detection. This analysis showed the incorporation of GalNGc into the backbone of the chondroitin oligosaccharide. N-glycolyl chondroitin (Gc-CN) is a stable metabolite of Neu5Gc.E. coli K4.Metabolic engineering of E. coli has promiscuous enzymes involved in CN biosynthesis.E. coli K4 with GlcNGc affords Gc-CN.Feeding N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). An enzyme called cytidine monophosphate N-acetylneuraminic acid hydroxylase (Cmah), encoded by the CMAH gene, is responsible for converting CMP-Neu5Ac to CMP-Neu5Gc through the addition of an oxygen atom  were fed with dietary Neu5Gc and challenged with anti-Neu5Gc antibodies, they developed inflammation, and prolonged exposure to these conditions led to significantly higher cases of cancer   chondroitin sulfate and unsulfated chondroitin (CN). These GAGs are relatively stable and easy to isolate from serum and analyze making these better candidates for testing the role of dietary Neu5Gc in human diseases. The catabolism of Neu5Gc containing glycans involves six metabolic steps leading to the production of uridine diphosphate N-glycolylglucosamine (GlcNGc-UDP) and ultimately uridine diphosphate N-glycolylgalactosamine  -β-d glucuronic acid (GlcA) (1→3)-β-d-N-acetylgalactosamine , just like chondroitin. However, this CPS also contains an unwanted linked β-fructofuranose branching from the C3 position of GlcA  has been traditionally extracted from animals. Recent efforts to produce CS in microorganism have been successful. CS has been biosynthesized from ction He . In the E. coli K4 serotype O5:K4:H4  was engineered for the synthesis of chondroitin sulfate. The fructosyltransferase encoded by kfoE was deleted using λ red recombinase 2HPO4, 100 mL of 10× MOPS buffer, , 1 mL of 1 m MgSO4, 1 mL of 0.5 g/L thiamine HCl, 0.1 mL of 1 m CaCl2, 20 g/L glucose, with 12.5 mM GlcNGc. Micronutrient stock consisted of 0.2 g/L (NH4)6Mo7O24, 1.2 g/L H3BO3, 0.1 g/L CuSO4, 0.8 g/L MnCl2, and 0.1 g/L ZnSO4. E. coli K4 serotype O5:K4 (L):H4 was from American Type Culture Collection . All reagents for medium preparation were from Sigma Chemical Co. .Shake flask fermentations utilized rich defined medium developed from modified protocols  0.05 and transferred to a 250 ml Erlenmeyer flask and incubated at 37 °C with shaking at 220 rpm. The cultures were left to grow under the same conditions for an additional 48 h. The cell growth and chondroitin production was similar to that previously reported in our laboratory  and DNAse  was added. The sample was incubated at 37 °C for 1 h after which protease K  was then added, and the sample was incubated at 56 °C for 2 h. After a second precipitation from 80% cold ethanol, the dry pellet was collected, re-dissolved in water (~ 1 mL), and filtered using a 10 KDa spin column  to remove small peptides and salt. The chondroitin obtained was sufficiently free of other impurities or other polysaccharides to undertake disaccharide analysis.CPS was purified from the cell pellet by re-suspending in water and autoclaving in the liquid cycle for 15 min. The autoclaved solution was centrifuged, and the supernatant was collected. Autoclaved supernatant from the cell pellet and cell culture supernatant were precipitated with 80 vol% cold ethanol and stored in an explosion-proof refrigerator overnight at 20 °C. Both intracellular and extracellular chondroitin were recovered. The pellet was collected and re-suspended in buffer  was added to the sample. Recombinant chondroitin lyase ABC  was added to each sample and mixed. The samples were incubated at 37 °C for 3 days. Under these reaction conditions, chondroitin lyase ABC could depolymerize their GAG substrates (in amounts of over 100 µg) into GAG disaccharides. The samples were washed twice with 100 µL distilled water in 3K MWCO filter unit. The filtrates passing through the filter unit contained disaccharide products, and these were dried using a vacuum centrifuge and stored at − 20 °C for AMAC-labeling.Digestion buffer  and incubating at room temperature for 10 min, followed by the addition of 10 µL of 1 m aqueous NaBH4OAc aqueous solution, and the mobile phase B (MPB) was pure methanol. The concentration of MPB increased from 5 to 45% for 10 min, then rose to 100% MPB in the next 0.2 min, and a 4 min flow of 100% MPB was applied to elute all compounds. A triple quadrupole mass spectrometer equipped with an ESI source  was used as a detector. The online MS analysis was at the multiple reaction monitoring (MRM) mode. The conditions and collision energies for all of the disaccharides MRM transitions are listed in our previous publication  with a flow rate of 300 µL/min. Mobile phase A (MPA) was 50 mM NH2SO4 in methanol. NMR experiments were performed on a Bruker Advance III 600 MHz spectrometer  with Topspin 3.2 software (Bruker). N-glycolyl glucosamine (GlcNGc) was synthesized following a previously published strategy  was performed using Merck Kieselgel 60F254 pre-coated aluminum backed plates. Plates were visualized using 5% Hly Sinaÿ .O-acetyl-β-D-glucopyranose 1  was dissolved in CH2Cl2 (20 mL), then triethylamine  was added to the solution. The reaction mixture was cooled to 0 °C, and acetoxyacetyl chloride  was added. The resultant mixture was stirred for 3 h at room temperature. The reaction was monitored by TLC. When the reaction was completed, the mixture was diluted with EtOAc, and the organic phase was washed successively with water, 1 m NaOH, 0.1 m HCl and, brine. The organic phase was dried over MgSO4, filtered, and concentrated to yield a white crystalline solid. The material was recrystallized using a mixture of ethyl acetate and hexanes to yield the desired compound 2 as a white solid . 1 h NMR , δ (ppm) 6.18 , 5.68 , 5.17–5.10 , 4.52 , 4.39 , 4.30 , 4.23 , 4.10 , 3.78 , 2.14 , 2.09 , 2.07 , 2.02 , 2.01 . 13C NMR , δ (ppm) 171.3, 170.6, 169.7, 169.6, 169.2, 167.5, 92.4, 73.0, 72.1, 67.6, 62.6, 61.6, 52.7, 20.8, 20.7, 20.5. ESI (m/z): [M−H]− calcd. for C18H24NO12 446.1298, found m/z 446.1296.The hydrochloride salt of 2-amino-2-deoxy-1,3,4,6-tetra-O-acetyl-2-acetyloxyacetamido-2-deoxy-β-d-glucopyranose 2  in MeOH (50 mL) at 0°C, 0.5 m NaOMe (5 mL) was added dropwise. The solution was stirred 2 h at room temperature. Amberlite IR 120 h+ resin was added to neutralize the reaction and was filtered. The filtrate was concentrated under reduced pressure to afford the desired product as a white solid . 1 h NMR  δ (ppm) 5.12 , 4.04 , 3.86 , 3.79 , 3.75 , 3.72 , 3.42 , 3.39 . ESI (m/z): [M−H]− calcd. for C18H24NO12 236.0770, found m/z 236.0772.To a solution of 1,3,4,6-tetra-We exhaustively digested chondroitin samples with chondroitin lyase ABC to afford GAG disaccharides and remove any other minor impurities, such as residual lipopolysaccharides. These digests were then purified and AMAC labeled for LC–MS/MS-MRM to verify the presence of Gc-CN. A low capillary temperature and spray voltage was maintained to minimize in-source fragmentation of AMAC-labeled Gc-CN oligosaccharides in MRM , by using chondroitin ABC lyase. The resulting disaccharide was then analyzed by an ultrasensitive and reliable method for disaccharide analysis LC–MS/MS MRM with a limit of detection (LOD) < 1 ng/mL and limit of quantification (LOQ) < 4 ng/mL  was able to transport GlcNGc along with glucose and GlcNAc into the cell. GlcNGc, taken up into the cell through the phosphotransferase system (PTS), was phosphorylated by N-acetyl-d-glucosamine kinase (NagK) (Uehara and Park GlmM), responsible for the conversion of glucosamine-6‐phosphate to glucosamine‐1‐phosphate  (Mengin-Lecreulx and van Heijenoort KfoA)   (Mengin-Lecreulx and van Heijenoort N-acetylglucosamine (UDP-GlcNAc) (Mengin-Lecreulx and van Heijenoort glmU gene to inactivate the acetyltransferase while maintaining uridyltransferase activity. Such a mutation should stop the cells from making GalNAc, so that all of the cell’s resources can be diverted to GalNGc synthesis. Modulating this gene could also be useful in generating Gc-CN standards. Such modulation has been reported (Pompeo et al. These preliminary studies showed about 1% conversion to Gc-CN. The exact concentration of synthesized Gc-CN could not be determined due to the absence of standards. Comparatively, a Gc-CN could be chemically synthesized in a 10% yield (Bergfeld et al. NGc Fig.  with theNGc Fig. . E. coliE. coli. While the levels of Gc-CN residues are relatively low, they are similar to the Gc-CN content expected on human consumption of red meat. Enhancement of incorporation of GalNGc into chondroitin might be achieved through optimized feeding of GlcNGc, by knocking out enzymes in the biosynthetic pathway or through the protein engineering of bifunctional biosynthetic enzymes. Future studies will examine all three of these approaches.In conclusion, we demonstrate the first production of Gc-CN in an engineered strain of


Bacteroides fragilis protects animals from intestinal diseases, such as ulcerative colitis, and its capsular polysaccharide plays a key role in reducing inflammation. B. fragilis strain ZY-312 was isolated from the feces of a healthy breast-fed infant, and the zwitterionic capsular polysaccharide zwitterionic polysaccharide, TP2, was extracted. In rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced enteritis, TP2 at an optimal dose of 2.5 mg/kg could significantly alleviate enteritis and reduced the degree of intestinal adhesions, the intestinal ulcer area, and the incidence of ulcers in rats. To understand the underlying mechanism, TP2 was labeled with Fluorescein isothiocyanate and orally administered at a dose of 2.5 mg/kg in rats. TP2 was mainly distributed in the cecum and colorectum, but it was not detected in the blood and other organs except that a compound with a molecular weight greater than that of TP2-FITC was found in liver tissue. During the absorption, distribution, metabolism, and excretion, TP2 was indigestible. These results were further confirmed by investigation in the simulated gastric, intestinal fluid, and colonic fluid with fecal microbiota in vitro, where TP2 remained unaltered at different time points. Furthermore, flora composition was analyzed in simulated colonic fluid with TP2 added and it was found that TP2 increased the abundance of Faecalibacterium, Enterococcus romboutsia, and Ruminococcaceae, whereas the abundance of the phylum Proteobacteria represented by Sutterella, Desulfovibrio, and Enterobacteriaceae was decreased. However, the amount of short-chain fatty acids in the simulated colonic fluid was not changed by intestinal flora post-TP2 addition. In conclusion, these findings confirmed that TP2, a capsular polysaccharide of B. fragilis, protects against ulcerative colitis in an undegraded form.The prominent human symbiont  Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon and rectum. In the past decades, UC has become a global disease. The highest incidence of UC was reported in North America and Europe, ranging from 8.8 to 23.14/100,000 and 0.97 to 57.9/100,000 individuals, respectively . TraditiThe pathogenesis of UC was related to the overreaction of the immune system to intestinal flora, which, in turn, triggers a series of inflammatory events that may damage and destroy the intestinal wall . The gutBacteroides fragilis NCTC 9343. It improved inflammatory diseases, such as enteritis, multiple sclerosis, and asthma, in animal models by inducing the secretion of Foxp3+Tregs by anti-inflammatory IL-10  that might produce ZPS (B. cellulosilyticus DSM 14838) or their lysates induce abundant IL-10 in vitro and protect against colitis in a trinitrobenzenesulfonic acid (TNBS)-induced enteritis model (The best-studied ZPS was polysaccharide A (PSA), extracted from ry IL-10 . Many baduce ZPS . These bis model . The uniB. fragilis strain (named ZY-312) from a healthy infant’s feces . The total sugar content of TP2 was 98%, the protein content was 1%, and the nucleic acid content was 0.5%.Fluorescein isothiocyanate (FITC) with >90% purity was purchased from Beijing Bailingwei Technology Co., Ltd. Salazosulfapyridine, pepsin, and trypsin were purchased from Sigma . 2,4-dinitrobenzenesulfonic acid (DNBS) was purchased from Tokyo Chemical Industry Co., Ltd .Wistar rats  were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. for DNBS-induced colitis studies, which were approved by PharmaLegacy Laboratories IACUC .For the pharmacokinetic study of TP2, Wistar rats  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd . This study was approved by the Animal Ethics and Welfare Committee of Guangzhou Boji Medical Biotechnological Co., Ltd. , in accordance with the relevant ethical principles and guidelines set by the Animal Welfare Act and the NIH Guidelines for the Care and Use of Laboratory Animals.After 48 h of fasting, the rats were randomly divided into six groups (n = 8 each group). The DNBS group received 0.5 ml DNBS (50 mg/ml) in 30% ethanol administered intrarectally after Zoletil (25 mg/kg) anesthesia to induce enteritis at day 0, and then received normal saline from days 1–7; the NS group received 30% ethanol intrarectally at day 0, and then received normal saline from days 1–7; the SSZ group received 300 mg/kg sulfasalazine for 7 days consecutively after enteritis was induced. Sulfasalazine is the first-line drug for the treatment of UC  and servThe body weight (BW) and fecal consistency of the rats were recorded daily. The fecal consistency was scored based on the following visual grading scale: 1) formed, stool maintains its shape, brown, score = 1; 2) semi-formed or soft, does not pour, yellow, score = 2; 3) liquid, pours more easily, yellow, score = 3. On the day after the final dose was administered, the rats were anesthetized and euthanized, and the colorectum was collected. The intestinal adhesion was scored based on the following visual grading scale: 1) no adhesion, score = 0; 2) slight adhesion, score = 1; 3) severe adhesion, score = 2. The ulcer area (ulcer area = length × width), ulcer score, and incidence of ulcer coefficient were recorded.g for 10 min at 4°C. The retentate was collected and stored in the dark at −20°C. The labeled TP2 was termed TP2-FITC.TP2 (200 mg) and FITC (20 mg) were solubilized in PBS , and the mixture was reacted at room temperature for 24 h in the dark. Then free FITC was removed with 0.02 mol/L PBS (pH 8.3), using a 10 kDa ultrafiltration tube  by centrifugation at 4,000 × 2, 2.5 μm, Shodex, Tokyo, Japan). The excitation and emission wavelengths of FLD were 494 and 520 nm, respectively.The analysis was performed on an Agilent 1260 system coupled with FLD, on which a 100-μL volume of sample was eluted with 20 mmol/L PBS (pH 7.0) at a flow rate of 1.0 ml/min with the column temperature maintained at 30°C  were sacrificed. The excitation and emission wavelengths of the imaging system were 495 and 515 nm, respectively.Wistar rats were randomly divided into TP2-FITC groups (n = 15), FITC groups (n = 15), and blank control groups (n = 3). A dose of 2.5 mg/kg of TP2-FITC was orally administered to the TP2-FITC group. The FITC group was administered FITC solution, and its fluorescence intensity was the same as TP2-FITC, as evaluated by plotting FITC concentration vs. fluorescence intensity. PBS served as the blank control. The optical images of TP2-FITC were acquired using the imaging system . g for 10 min at 4°C, and half of the volume was mixed with 11% NaOH solution, followed by centrifugation at 10,000 × g, 4°C for 4 min. The tissues were ground in PBS buffer. The supernatant was collected by centrifugation at 10,000 × g at 4°C for 10 min. Finally, the samples were analyzed using a GPC-FLD.Wistar rats  were randomly divided into TP2-FITC groups (n = 12) and blank control groups (n = 2). A dose of 2.5 mg/kg of TP2-FITC was orally administered to the TP2-FITC group. PBS solution served as a blank control. The rats (n = 2 each group) were sacrificed, and blood, urine, feces, and major tissues including liver, spleen, kidney, stomach, small intestine, cecum, and colon, were collected at 1, 2, 4, 6, 12, and 24 h after oral administration. Plasma was mixed with a 20% trichloroacetic acid (TCA) solution at a volume ratio of 5:2 to remove the impurities. The supernatant was collected after centrifugation at 10,000 × 2PO4 (6.8 g/l), and the pH was adjusted to 6.8 with 0.1 mol/l NaOH. The simulated colonic fluid was prepared as reported previously  and pepsin (10 g/l), while the simulated intestinal fluid consisted of trypsin (10 g/l) and KHeviously . The fecuent use . During g for 10 min at 4°C. The reaction was stopped with the addition of 11% NaOH, and the subsequent supernatant collected by centrifugation was analyzed by GPC-FLD. The samples were stored at –80°C for subsequent assays.TP2-FITC solution was added to simulated gastric fluid , simulated intestinal fluid , and simulated colonic fluid , and the final concentration of TP2-FITC solution was 0.25 mg/ml. A volume of 1 ml fluid was withdrawn at different time points and neutralized with 0.2 mol/ml NaOH. After the reaction was terminated, the reaction was mixed with 20% TCA at a volume ratio of 5:2, and the supernatant was harvested by centrifugation at 10,000 × g at 4°C for 5 min to collect the supernatant that was analyzed by gas chromatography using an fluorescence detector.TP2 solution  was added to 15 ml of simulated colonic fluid and fermented for 0, 3, 6, 12, and 24 h in an anaerobic tube at 37°C. An equal volume of normal saline was added to simulated colonic fluid as control. Subsequently, 0.6 ml of the sample was mixed with 0.1 ml of 50% sulfuric acid and 0.5 ml ether, followed by centrifugation at 12,000 /min for 5 min. The temperature of fluorescence detector inlet was 250°C. The flow rate of hydrogen and air was 40.0 ml/min and 300 ml/min, respectively, and the loading volume was 8 μL.Chromatographic conditions were as follows: agilent DB-WAX capillary column ; nitrogen was used as the carrier gas, and the flow rate was 13.7 ml/min with a split ratio of 1:10. The initial temperature was 70°C for 1 min, followed by an increase from 10°C/min to 160°C for 9 min and then increased to 210°C at 30°CTP2 solution was added to 15 ml of the above-mentioned simulated colonic fluid to maintain the final concentration of TP2 solution at 0.25 mg/ml and 2.5 mg/ml, while an equivalent volume of physiological saline was added to simulated colonic fluid as control. The samples from each group were fermented in an anaerobic tube at 37°C for 24 h.16S rRNA gene was amplified from DNA samples using 515F and 806R primers. All libraries were sequenced using the Illumina HiSeq 2,500 platform .The 16S sequencing and analysis of samples were performed using Novogene . The total genomic DNA of samples was extracted using the CTAB/SDS method as described in Molecular Microbial Ecology Manual . The V4 p < 0.05 indicated statistical significance.Data are expressed as mean ± SD and analyzed by GraphPad Prism 5. The chromatograms were processed with Origin Pro 8. Each group was analyzed by analysis of variance (ANOVA), and p < 0.01), indicating that DNBS successfully induces diarrhea. Compared to the DNBS group, sulfasalazine failed to reduce the score of fecal consistency, while 2.5 mg/kg TP2 reduces the score, albeit not significantly  , intestiThe SCFAs, including acetic acid , propion16S rDNA sequencing. The Shannon index was used to estimate the diversity of microorganisms in the samples. The results , Pearl River S&T Nova Program of Guangzhou (No. 201906010034), Pearl River S&T Nova Program of Guangzhou (No. 201806010010), and R&D Plan for Key Areas in Guangdong Province (No. 2019B020204003).LZ, GK, YL, DL, HH, XY, CW, and YW were employed by Guangzhou ZhiYi Biotechnology Co. Ltd. The property of B. fragilis ZY-312 and polysaccharide TP2 belongs to Guangzhou ZhiYi Biotechnology Co. Ltd. Any use of ZY-312 and TP2 without permission of Guangzhou ZhiYi Biotechnology Co. Ltd. will be illegal.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TabZIP15 gene encoding a 396 amino acid (aa) polypeptide in the fungus Trichoderma asperellum ACCC30536 was cloned and characterised. The protein includes a basic region motif (NR-x2-QR-x2-R) and has a pillar-like structure. The 25 basic region/leucine zipper transcription factors (TFs) identified in the T. asperellum genome were divided into YAP (14 TFs), ATF2 (5), GCN4 (2), Zip1 (2), BRLZ (1) and u1 (1) subfamilies based on conserved domains. T. asperellum was cultured in minimal media (MM) control, C-Hungry and N-Hungry medium , and differential expression analysis showed that 14 TabZIP genes (including TabZIP15) were significantly altered under both conditions; TabZIP23 responded strongly to N-Hungry media and TabZIP24 responded strongly to C-Hungry media. However, only YAP genes TabZIP15, TabZIP12 and TabZIP2 were significantly upregulated under both conditions, and expression levels of TabZIP15 were highest. T. asperellum was also cultured in the presence of five fungal pathogenic toxins, and RT-qPCR results showed that TabZIP15 was significantly upregulated in four of the five toxin stress conditions .The  Trichoderma are important biological control agents, and their biological control mechanisms have been investigated in detail3. Species in the Trichoderma genus can readily adapt to their environment, propagates and grow, hence their use worldwide. Genome sequencing programs have targeted Trichoderma4 and the genomes of 16 Trichoderma species have been published (https://mycocosm.jgi.doe.gov/mycocosm/home/), laying the foundation for molecular biological analysis.Members of the fungal genus 5. These proteins possess two distinctive structural features located on a contiguous alpha helix; (1) a basic region of ~ 16 amino acid (aa) residues comprising a nuclear localisation signal followed by an invariant N-x7-R/K motif that is responsible for interacting with DNA and nuclear import, and (2) a heptad repeat of leucines or other bulky hydrophobic amino acids positioned exactly nine amino acids from the C-terminus that creates an amphipathic helix which regulates dimerisation5. The DNA binding and dimerisation mechanism in fungi has been studied in Saccharomyces cerevisiae, and the results revealed that Met4 and Met28 form a heteromeric complex with a centromere binding factor to activate transcription7.Basic region/leucine zipper (bZIP) transcription factors (TFs) are one of the largest and most diverse TF families, and proteins with bZIP domains are present in all eukaryotes8. Recent studies showed that members of the bZIP TF family play diverse regulatory roles in filamentous fungi. Eight bZIP TFs in Ustilaginoidea virens are involved in stress tolerance and pathogenicity9. In Aspergillus spp., bZIP TFs including AtfA, NapA, AflR, RsmA and Apyap1 have been demonstrated to be the key factors responding to oxidative, osmotic, environmental and drug stresses13. In other fungi, such as Botrytis cinerea, Neurospora crassa, Fusarium graminearum and Magnaporthe oryzae, bZIP TFs are known to be involved in responding to oxidative stress and pathogenicity15. These findings suggest that bZIP TFs play an important role in stress responses in several fungi, and a similar regulatory function can be inferred in other fungal species. However, most of the research on fungal bZIP proteins has focused on pathogens, which bZIP proteins in biological control agents such as Trichoderma spp. have received little attention. Phytopathogen toxins are metabolites in pathogens that can harm plants and other fungi. To better understand the detoxification mechanism of Trichoderma that is triggered in response to phytopathogen toxins, the roles of bZIP TFs should be investigated under different stress conditions.The mechanism of bZIP TFs has been analysed in various fungus speciesTabZIP15 TF gene of Trichoderma asperellum ACCC30536 was cloned, and characterised alongside another 24 similar bZIP TFs in the Joint Genome Institute (JGI) database. The basic biochemical characteristics of these bZIP TFs were investigated, and expression levels were analysed based on RT-PCR data obtained for T. asperellum under three conditions at 72 h. Furthermore, transcription levels of TabZIP15 were measured by RT-qPCR following exposure to toxins produced by five pathogenic fungi . The results provide theoretical support for the analysis and development of TFs from T. asperellum.Herein, the T. asperellum ACCC30536 was obtained from the Agricultural Culture Collection of China. The five phytopathogens were R. solani, F. oxysporum, S. sclerotiorum, A. alternata and C. chrysosperma, and they were stored at the Laboratory of Forestry Protection, Northeast Forestry University, Harbin, China. T. asperellum was cultured on potato dextrose agar (PDA) slant culture medium at 28 °C for 7 days and stored at 4 °C. A. alternata, S. sclerotiorum, R. solani, C. chrysosperma and F. oxysporum were inoculated in 200 mL of 1/4, 1, 1/2, 1 and 1/2 strength PD medium and cultured in shake flasks for 10 days at 28 °C with shaking at 200 rpm. The medium was filtered using 0.2 μm filters  to remove spores, and the filtered fermentation liquid from each pathogen was combined, mixed, and stored in tubes at − 20 °C.TabZIP15  were designed using Primer Premier 6.0 software 16. Extraction of T. asperellum DNA was performed as described previously17, and thermal cycling for PCR was performed at 94 °C for 5 min, followed by 35 cycles at 94 °C for 25 s, 56 °C for 30 s, and 72 °C for 30 s, and followed by 72 °C for 7 min. The PCR product was purified and ligated into the pGEM-T vector  and sequenced . Conserved domain prediction and identification of other homologous proteins was performed using the Blastp tool18 from the National Center for Biotechnology Information (NCBI). Multiple sequence alignment was conducted using the Clustal X program 19, and three-dimensional structure prediction was carried out by SWISS-MODEL ( https://swissmodel.expasy.org/)20.Primers for cloning T. asperellum bZIP protein (TabZIP protein) sequences were obtained from the T. asperellum CBS 433.97 v1.0 JGI database (https://genome.jgi.doe.gov/)21, and conserved domains were predicted using the NCBI tool Blastp. Proteins without conserved bZIP domains were omitted. The molecular weight (MW) and isoelectric point (pI) of each bZIP protein was calculated using ExPASy (https://www.expasy.org/)22, and gene structural information for exons and introns was obtained from the JGI database (https://genome.jgi.doe.gov/)21 and analysed by the Gene Structure Display Server 2.0 website (https://gsds.cbi.pku.edu.cn/index.php)23. A phylogenetic tree was constructed via the maximum likelihood method with 1,000 bootstrap replicates using the MEGA 7.0 program24. Motifs were searched using the MEME suite (http://meme-suite.org/tools/meme)25 with the site distribution set as ‘zoops’, and the number of motifs set as ‘3’. Conserved domains and motifs were analysed by the MEME suite and TBtools26.Full-length 2PO4, 5 g/L (NH4)2SO4, 600 mg/L CaCl2·2H2O, 600 mg/L MgSO4·7H2O, 5 mg/L FeSO4, 2 mg/L CoCl2, 1.6 mg/L MnSO4, 1.4 mg/L ZnSO4 and 0.1% (w/v) glucose. MM + A comprised MM plus 5% (v/v) fermentation liquid from A. alternata. MM + S comprised MM plus 5% (v/v) fermentation liquid from S. sclerotiorum. MM + R comprised MM plus 5% (v/v) fermentation liquid from R. solani. MM + C comprised MM plus 5% (v/v) fermentation liquid from C. chrysosperma. MM + F comprised MM plus 5% (v/v) fermentation liquid from F. oxysporum, C-Hungry was MM without a carbon source, and N-hungry was MM without a nitrogen source. Differential expression of 25 bZIP family genes was analysed using T. asperellum ACCC30536 transcriptome data obtained previously27, and the heatmap was drawn by TBtools26. Expression of TabZIP15 genes was analysed by RT-qPCR under five pathogenic toxin stress conditions. Spores from T. asperellum were inoculated into 200 mL 1/4 strength PD broth medium at a final concentration 1 × 104 spores/mL and cultured at 28 °C with continuous shaking at 200 rpm for 48 h. The resulting mycelia were filtered, washed, transferred to MM for 2 h, then transferred into medium containing different pathogenic toxins for 72 h, three replicates were set. Mycelia were collected at 0, 4, 8, 12, 24, 48, and 72 h. At each time point, the biomass was calculated (averaged from three replicates), and 25 mL mycelium cultures were collected and stored at − 80 °C. Total RNA was extracted from mycelia28 using TRIzol reagent , digested with DNaseI , and reverse-transcribed into cDNA using a PrimeScript RT Kit  according to the manufacturer’s instructions.Minimal media (MM) contained 15 g/L NaHTabZIP15 in T. asperellum were measured by RT-qPCR, and calculated according to the 2−ΔΔCt method29 using cDNA as template, with actin, α-tubulin and β-tubulin as reference genes. Three RT-qPCR replicates were performed per cDNA sample. Primers for RT-qPCR 30.Expression levels of t tests using SPSS19.031. Statistical significance of differences between groups was determined by Turkey’s multiple range tests (p < 0.05).All experimental data were subjected to analysis of variance (ANOVA) and independent sample TabZIP genes encode proteins ranging from 177 to 753 aa in length, with an average length of 408 aa. The molecular weight of TabZIP proteins ranges from 19.63 to 80.93 kDa, with an average of 44.7 kDa. The pI ranges from 4.66 to 10.05, and the average pI is 6.65. TabZIP TF genes in T. asperellum are distributed on scaffold 1 to scaffold 41, and some chromosomes have more than one TabZIP TF gene  in MM, C-Hungry and N-Hungry media, respectively. Genes were clustered based on their expression, and TabZIP15, TabZIP13 and TabZIP23 were highly expressed in all three conditions, while TabZIP18, TabZIP24 and TabZIP 25 were highly expressed in MM and N-Hungry media, and TabZIP6 and TabZIP20 were highly expressed in MM and C-Hungry media.Expression of the 25 red Fig. . Interes2 value > 1) change in expression level relative to MM was considered significant. The results showed that six TabZIP genes were significantly upregulated and six were significantly downregulated in C-Hungry media  were significantly upregulated in both N-Hungry and C-Hungry media, while TabZIP6, TabZIP11, TabZIP20 and TabZIP25 were the only genes significantly downregulated in these two conditions. During the stress response process, upregulated genes may act as positive response factors. Thus, we focused on TabZIP15, TabZIP12 and TabZIP2, and TabZIP15 displayed the highest expression, hence this gene was subjected to further analysis.In the differential expression analysis, a two-fold  includes six exons and five introns. The TabZIP15 cDNA  encodes a 396 aa polypeptide with a calculated molecular weight of 45.05 kDa, and a pI of 5.54. TabZIP15 is a member of the bZIP-YAP family of TFs  occurred at 24 h. Expression of TabZIP15 was upregulated at 4 h in MM + A, at 4 h in MM + C, at 8 h in MM + F, at 4 h in MM + R, and at 12 h in MM + S, with peak expression levels at 48 h (26.55-fold), 48 h (26.98-fold), 48 h (24.74-fold), 12 h (26.98-fold) and 24 h (24.42-fold), respectively. Analysis of differential expression (relative to controls) showed that TabZIP15 was highly expressed when T. asperellum was exposed to toxins from A. alternata, C. chrysosperma, F. oxysporum and R. solani, but expression in MM + S media did not differ significantly from that in MM media.Expression of Trichoderma genus have been widely used as biological control agents, and the biocontrol mechanism has been investigated3. bZIP proteins regulate various biological processes13 and are present in all eukaryotes5. Thus, bZIP TFs may regulate biological control processes in T. asperellum. Herein, 25 TabZIP TFs were identified in T. asperellum, and multiple sequence alignment revealed relatively low sequence similarity (data not shown). Seventeen sequences sharing homology with TabZIP15 were identified using Blastp , which simulated Trichoderma nutrient competition and interaction with the pathogens27, TabZIP19 and TabZIP21 have similar motif arrangements to TabZIP15, hence they may be expected to share similar expression modes, but the RNA-Seq data showed that they did not have similar expression modes. Similarly, TabZIP9 and TabZIP10 are in the same clade as TabZIP15, and they share similar phylogenetic relationships, hence they may be expected to share similar expression modes, but again, RNA-Seq data showed that they did not have similar expression modes  have a conserved bZIP-ATF2 domain, and they may perform similar functions, but this should be further investigated  or N-Hungry medium , and a homolog of this protein is reportedly involved in fungal development and differentiation38. Meanwhile one u1 and one Zip1 were significantly downregulated in both N-Hungry and C-Hungry media. A homolog of Zip1 was reportedly induced under stress conditions39, but u1 proteins have not been investigated, and further studies are needed. Thirteen genes were highly expressed in MM media (Log2RPKM > 6), and they may be involved in normal physiological regulation  were significantly upregulated in both C-Hungry and N-Hungry media , which suggests that they may be important for resistance against pathogens. TabZIP12 and TabZIP2 were both significantly upregulated. TabZIP12 expression levels were upregulated 22.31- and 21.85-fold in C-Hungry and N-Hungry media respectively, while TabZIP2 expression levels were upregulated 22.14- and 21.58-fold in C-Hungry and N-Hungry media respectively. However, the RPKM value for TabZIP12 in MM and C-Hungry media was 45 and 224, respectively; compared with 22 and 97 for TabZIP2. The RPKM value for TabZIP12 in MM and N-Hungry media was 45 and 163, respectively, compared with 22 and 66 for TabZIP2. Thus, the RPKM values for both genes were not high, and can be easily influenced by experimental error40, but fold-changes in expression were relatively large, hence further investigation is clearly needed.Our transcriptome data showed that 16 of the 25 TabZIP15) was chosen for further analysis, and its expression was measured in the presence of five pathogenic toxins by RT-qPCR. The results confirmed our hypothesis . Our results reveal some of the properties of bZIP TFs in T. asperellum, identify potential biological control-related bZIP TFs, and will guide further research in this area.Herein, we identified 25 Supplementary Table S1.


With adopting BTPV-4F as the rear cell acceptor material, the resulting tandem devices reached a high power conversion efficiency of over 16.4% with good photostability. The results indicate that BTPV-4F is an efficient infrared-absorbing narrow bandgap acceptor and has great potential to be applied into tandem organic solar cells.Tandem organic solar cells are based on the device structure monolithically connecting two solar cells to broaden overall absorption spectrum and utilize the photon energy more efficiently. Herein, we demonstrate a simple strategy of inserting a double bond between the central core and end groups of the small molecule acceptor Y6 to extend its conjugation length and absorption range. As a result, a new narrow bandgap acceptor BTPV-4F was synthesized with an optical bandgap of 1.21 eV. The single-junction devices based on BTPV-4F as acceptor achieved a power conversion efficiency of over 13.4% with a high short-circuit current density of 28.9 mA cm Development of tandem organic solar cells has been limited by the choice of near-infrared absorbing materials for the rear cell. Here, the authors report a simple strategy to extend the conjugation length of acceptor Y6 and broaden its absorption range to near-infrared region. A tandem organic solar cell with efficiency of 16.4% was achieved. Due to its advantages of light weight, flexibility, and low-cost solution processability, OSCs could realize commercialization in the near future8. “Tandem” architecture has been widely adopted as a simple and reliable methodology to achieve higher performing OSCs with better utilization of near-infrared (NIR) solar energy13. For a single-junction OSC with a specific absorption region and bandgap, the trade-off between thermalization loss of photon energy and spectrum unitization is limited on a certain level16. While in the series-connected tandem OSCs, the absorption spectrum wavelength region can be effectively extended by employing a wide bandgap sub-cell to harvest high energy photons and another narrow bandgap sub-cell for utilizing low energy photons. At the same time, the open-circuit voltage (Voc) of the tandem OSCs is the summation of those of the two sub-cells19. And the thickness of each sub-layers can be easily tuned separately to match a balanced absorbance in each wavelength region and provide maximized photon-to-energy efficiency21.Organic solar cells (OSCs) have drawn much attention in the past decade for its potential application as a reliable clean energy source71BM was then adopted as the rear cell active layer and P3HT:ICBA as the front cell active layer to fabricate tandem OSC, leading to an NREL certified power conversion efficiency (PCE) of 10.6%, which was the first time of the PCE of the OSCs exceeding 10%22. Later on, in order to broaden the absorption, Hou et al. synthesized IEICO-4F acceptor with fluorine substituted end groups23. Combined with J52-2F:IT-M as front cell, the tandem OSC reached PCE of 14.9%24. By introducing Cl atoms into small molecule acceptor based on BDT units, Forrest et al. synthesized BT-CIC acceptor with absorption range over 900 nm and the PCE of tandem OSCs reached 15%26. Recently, Chen et al. adopted PTB7-Th:O6T-4F:PC71BM ternary system as a rear cell material with absorption spectra over 1000 nm and a high PCE of 17.3% was reported for the tandem OSCs27.In 2013, Yang et al. designed the narrow bandgap polymer donor PDTP-DFBT with NIR absorption through molecular modification. PDTP-DFBT:PC28. One main limitation for the development of tandem OSCs comes from the lack of efficient low bandgap materials for rear cells, which mostly determines the effectiveness of utilizing the NIR region of the solar spectrum32. Based on previously reported theoretical simulations, the most suitable active layer materials for rear cells should possess an NIR absorption edge around 1050–1150  nm33. However, absorption onset of most reported narrow bandgap acceptors could not surpass 1000 nm without losing substantial external quantum efficiencies (EQEs) response in the NIR region36. Therefore, to meet the requirements raised by tandem OSCs, more effort needs to be devoted to developing ultra-narrow bandgap materials that can simultaneously offer a suitable absorption spectrum with high short-circuit current densities (Jsc) and low energy loss.At the current stage, the PCE of tandem OSCs still holds no advantage over single-junction OSCs40. The A-D-A molecules are composed of a fused ring central donor (D) unit and two strong electron-accepting (A) end groups with an ethylene double bond π-bridges between them. Our group extended the absorption edge of the A-D-A structured small molecule acceptors to 850–900 nm by inserting another ethylene double bond π-bridges between the central fused ring D unit and the A end groups42, indicating that using two enthylene double bonds π-bridge is an effective way to further redshift the absorption of the A-D-A acceptors. By inserting an electron-accepting (A′) core in the central fused ring D unit, Zou et al. recently synthesized an A-DA′D-A small molecule acceptor Y6 with absorption edge extending to ca. 950 nm41. And the OSCs with Y6 as acceptor and wide bandgap polymer donor demonstrated high PCE of over 16%45. Based on the above consideration, in this work, we designed and synthesized a new low bandgap acceptor BTPV-4F , and the expected most suitable bandgap for front cell materials should be around 1.6 eV in matching with the bandgap of ca. 1.2 eV for the rear cell, based on the simulation. Recently, Hsu et al. reported a carbazole based acceptor with two fluorine on the IC end groups called DTC(4Ph)-4FIC40. The PM6: DTC(4Ph)-4FIC device shows high Voc of 0.95 V and over 800 nm EQE response. In order to simultaneously get ideal bandgap and higher Voc, we accordingly designed and synthesized a carbazole based small molecule acceptor m-DTC-2F with single fluoro-substitution IC end groups and meta-alkyl-phenyl side chains  and onset reduction potential (Ered), HOMO energy level (EHOMO) and the LUMO energy level (ELUMO) were determined according to the equation ELUMO/HOMO = −e (Ered/ox + 4.36) (eV) where the unit of Ered/ox is V vs. Ag/AgCl.  The ELUMO and EHOMO of m-DTC-2F were estimated to be −3.89 and −5.67 eV, and those values of BTPV-4F were −4.08 and −5.39 eV, respectively. Both cases in DFT calculation and cyclic voltammetry measurements, BTPV-4F shows relatively higher EHOMO, similar ELUMO and narrower bandgap than Y6. Such a trend indicates that the insertion of double bonds can effectively upshift the HOMO level and reduce the bandgap of acceptors. In considering the complementary absorption and well-matched energy levels of the donor and acceptor materials 47. The optimized weight ratios of the photovoltaic materials in the active layers are 1:1.5 for the binary PTB7-Th:BTPV-4F active layer and 1:1.5:0.15 for the ternary PTB7-Th:BTPV-4F:PC71BM active layer. Figure 71BM (1:1.5:0.15). The blend films exhibit strong absorption in the NIR region. The fabrication conditions of the OSCs were optimized and the results are listed in Supplementary Tables J–V) curves of the optimized devices under the illumination of AM1.5 G, 100 mW cm−2, and the detailed photovoltaic parameters are listed in Table Voc of 0.65 V, a high Jsc of 28.3 mA cm−2, and an FF of 65.9%. After introducing PC71BM as the third component, the best PCE of the ternary OSC based on PTB7-Th:BTPV-4F:PC71BM with a weight ratio of 1:1.5:0.15 reached 13.4% with simultaneously enhanced Voc of 0.67 V, Jsc of 28.9 mA cm−2, and FF of 69.3%, compared to those of the binary device. The enhanced Voc could be due to the reduced nonradiative recombination loss by introducing PC71BM, which can be widely observed in OSCs based on acceptors with the BTP central core47. The EQE spectra of the binary and ternary devices are shown in Fig. Jsc from the EQE spectra of the binary and ternary devices are 27.83 and 28.31 mA cm−2, respectively, which is in good agreement with the Jsc values measured from the J–V curves within 2% mismatch.To study the photovoltaic performance of the narrow bandgap acceptor BTPV-4F, we fabricated the inverted structure OSCs with BTPV-4F as acceptor and PTB7-Th as polymer donorA→B = 3.3 ± 0.1 ps and τB→GS = 58.4 ± 3.2 ps (ca. 60 ps), so we think the lifetime is 60 ps). In the PTB7-Th:BTPV-4F blend film, after excitation at 850 nm, a GSB signal at 737 nm was apparent, which coincides with the absorption peak of PTB7-Th. This indicates the hole transfer from the acceptor BTPV-4F to the donor PTB7-Th. Fitting of the kinetic curves gave a fast charge transfer lifetime of 2 ps, indicating a high yield in charge generation and efficient photocurrent generation in the PTB7-Th:BTPV-4F systems.To investigate the charge transfer dynamics of the active layer, femtosecond transient absorption spectroscopy (fs-TA) measurement was carried out on the pristine BTPV-4F film and the PTB7-Th:BTPV-4F blend film. Immediately following excitation of pristine BTPV-4F, we observed a broad ground state bleach (GSB) with a peak at 871 nm and two excited state absorption (ESA) signals at 610 nm and 990 nm  and BTPV-4F has the narrowest optical bandgap (1.21 eV) among all the reported works related to the narrow bandgap acceptors. Obviously, BTPV-4F exhibits great superiority in both optical bandgap and photovoltaic performance, which would be a promising acceptor for constructing the rear cell in high-performance tandem OSCs.Figure Jsc vs. light intensity (Plight) α. The α value should be 1 when there is no bimolecular recombination in the active layer48. For the BTPV-4F based OSCs, α values are calculated to be 0.975 and 0.998 for the binary and ternary devices respectively, indicating more efficient transportation of carriers and neglectable bimolecular recombination in the ternary device. The relationship between photocurrent density (Jph) and effective voltage (Veff) of the binary and ternary OSCs was further characterized to study the charge dissociation behavior of the devices48. As shown in Supplementary Fig. Jph/Jsat ratios are 0.982 for the binary device and 0.986 for the ternary device; under maximal power output conditions, the ratios are 0.839 and 0.858, respectively. The results indicate the ternary devices have the higher both exciton dissociation and charge collection efficiency.To further characterize the BTPV-4F based OSCs, charge carrier recombination behavior was studied by measuring the dependence of µh and µe) of PTB7-Th:BTPV-4F binary systems are estimated to be (9.4 ± 0.6) × 10−4 cm2 V−1 s−1 and (7.5 ± 0.7) × 10−4 cm2 V−1 s−1, and the best values are 1.1 × 10−3 cm2 V−1 s−1 and 8.3 × 10−4 cm2 V−1 s−1, respectively. As for the PTB7-Th:BTPV-4F:PC71BM ternary system, the corresponding hole and electron mobilities are (9.2 ± 0.4) × 10–4 cm2 V−1 s−1 and (9.1 ± 0.6) × 10–4 cm2 V−1 s−1, and the best values are 1.0 × 10–3 cm2 V−1 s−1 and 9.8 × 10–4 cm2 V−1 s−1, respectively. The increased electron mobilities in ternary blends could be accountable for the higher FF in the ternary devices.The charge carrier mobilities of the active layers were estimated using the space charge limited current (SCLC) method  peaks are observed in the in-plane direction and (010) peaks are found in the out-of-plane (OP) direction  was conducted to determine the molecular packing of the BTPV-4F based films. Two-dimensional (2D) patterns and line-cut profiles for neat films are shown in Supplementary Fig. The phase separation morphology of the blend films was also studied by atomic force microscopy (AFM). As shown in Fig. 71BM system was used as active layer materials in rear cells. To ensure the ideal match of Jsc with the rear cell, PM6:m-DTC-2F was used as a front cell active layer material. As shown in Supplementary Fig. m-DTC-2F films covers the short wavelength region from 300 nm to 770 nm, which is complementary to the absorption of the rear cell. Additionally, the PM6:m-DTC-2F based single-junction devices exhibit a high Voc of ~ 1 V and a high EQE response, giving a current of 17.1 mA cm−2 as shown in Supplementary Fig. Voc of 1.65 V, a Jsc of 14.5 mA cm−2, and FF of 0.685, as shown in Fig. Voc of the tandem solar cell is 1.65 V, which is only 0.01 V less than the sum of the Voc values of the two sub-cells, indicating a well-formed effective tunneling junction in the interconnection layer. In this work, m-DTC-2F has a Voc loss of ca. 0.61 eV, which is one of the lowest values in the reported materials with a similar optical bandgap of around 1.6 eV. However, in the current stage of tandem OSCs, higher percentage of Voc loss still originates from the wide bandgap materials in front cells. Future works could focus more on the development of high-performance wide bandgap materials to further boost the efficiency of tandem OSCs.Finally, the optimized rear sub-cell was used in fabricating organic monolithic inverted-structured tandem solar cells. According to the analysis of the single-junction device result, PTB7-Th:BTPV-4F:PCJsc of front and rear sub-cells are 14.27 and 14.32 mA cm−2, respectively, indicating the highly balanced current generation in each sub-cell. The high and well-balanced Jsc of the tandem OSCs is mainly attributed to the complementary absorption range of both sub-cells and the carefully tuned thickness of each active layer. The detailed thickness dependence of photovoltaic performance is listed in Supplementary Table To understand the current matching behavior between front and rear sub-cells in the tandem OSCs, EQE spectra of the sub-cells were measured, as shown in Fig. 49, as shown in Fig. n and extinction coefficient k of all the layers used in the tandem OSCs were measured by a spectroscopic ellipsometer. Based on the optical simulation, an theoretically optimal value of over 14.8 mA cm−2 was obtained when the optimal thicknesses of the active layers in the front and rear cells were 138 and 99 nm, respectively. The simulation result of the optimal thickness is consistent with our experimental results in the J–V measurements.The optical light-field simulations were carried out to determine the optimal thicknesses of the sub-cells in experiments50. To date, very few works studied the photostability of tandem OSCs. Considering the commonly used light‐emitting diode (LED) lamps fail to completely cover the infrared region of the absorption spectrum of the rear cell, a solar simulator with a metal halide lamp was used as the testing light source with illumination intensity equivalent to 1 sun. The illumination spectrum of the light source is included in Supplementary Fig. 71BM device shows excellent stability with 91% PCE remaining after 500 h aging. This result indicates that introducing double bond into non-fullerene acceptors is an advantageous way of constructing stable-structured low bandgap molecules with good photostability. However, the front cell based on PM6:m-DTC-2F shows obvious burn-in from the beginning and PCE just remains 40% after 500 h aging. Interestingly, thanks to the excellent stability of rear sub-cell, the stability of total tandem OSC is much improved even with the relatively unstable front sub-cell and the T80 lifetime is over 200 h. The relatively weak light distribution would surely show lower decay rate of sub-cells, which causes the tandem devices to exhibit better overall light stability over single-junction sub-cells. It is reasonable to conclude that the tandem structure is superior in improving photostability of OSCs, and a more stable front cell materials in the future would lead to significantly more enhanced tandem OSCs stability.Long‐term photostability is critical for the commercialization of this photovoltaic technologyEgopt of 1.21 eV, because of its extended molecular conjugation length. The OSC based on PTB7-Th:BTPV-4F:PC71BM  demonstrated a record Jsc of 28.9 mA cm−2 and overall PCE of 13.4%, with a broad EQE response wavelength range from 300 to 1050 nm. BTPV-4F shows a high potential for application in a rear sub-cell of tandem OSCs. Then we synthesized a medium bandgap acceptor m-DTC-2F with a bandgap of 1.61 eV, and select PM6: m-DTC-2F as the front sub-cell active layer for well-matching absorption of the front and rear sub-cells to fabricate the tandem OSCs. Due to the efficient utilization of solar spectrum in the range of 300−1050 nm and the reduced energy losses of two sub-cells, high PCE of 16.4% is achieved for the tandem OSC, which is one new case among all the best-performing tandem PSCs to date. we believe that the PCE of tandem OSCs can surpass 18% if FF can be promoted to over 75% by optimizing front cell material systems. So, in the next step we should design high-performance front cell material systems with high FF (ca. 75%) in thick film device and low energy loss (ca. 0.55 eV). Furthermore, the BTPV-4F based OSC shows excellent photostability with 91% PCE remaining after 500 h aging. All of the results indicate that inserting the double bonds π-bridge into the A-DA’D-A organic semiconductor acceptors is a simple and effective way to construct NIR organic acceptors with high performance and stability.In summary, we introduced one more double bond π-bridge into the A-DA’D-A structured non-fullerene acceptor Y6 and synthesized a new ultra-narrow bandgap acceptor BTPV-4F. BTPV-4F shows significantly redshifted absorption with an 71BM) were purchased from Solarmer Materials Inc. The polymer donor PTB7-Th was purchased from 1-Material Inc. BTP-CHO was purchased from Derthon Optoelectronic Materials Co., Ltd. Other chemicals and solvents were obtained from J&K, Energy Chemical, and Sigma Aldrich Chemical Co., respectively. All of the reagents and commercial compounds were used as received. The synthetic routes of acceptor BTPV-4F and m-DTC-2F are shown in Fig. The polymer donor PM6 and -Phenyl-C71-butyric acid methyl ester . 1H NMR  δ (p.p.m.): 9.73 , 7.78 , 6.53 , 4.61 , 3.00 , 2.12–1.96 , 1.86 , 1.22–1.61 , 0.81–1.15 , 0.49–0.71 . 13C NMR  δ (p.p.m.) 192.48, 147.52, 143.72, 142.62, 142.06, 136.89, 133.30, 132.48, 125.79, 125.40, 125.14, 112.21, 55.12, 40.15, 31.91, 29.88, 29.70, 29.65, 29.61, 29.55, 29.43, 29.33, 28.38, 27.62, 27.60, 23.16, 22.68, 14.11, 13.68, 10.13, 10.11.To a solution of the mixture of BTP-CHO  and tributylphosphonium bromide  in anhydrous tetrahydrofuran was added sodium hydride  under an argon gas atmosphere, and the resulting turbid solution was stirred at room temperature for 16 h. After completion of the reaction, the reaction was quenched using a 10% HCl solution under cooling and the reaction mixture was brought to acidic pH and stirred at room temperature for 4−5 h. The contents of the reaction flask were concentrated, and the organic contents were extracted into ethyl acetate. The organic layer was washed with water followed by brine, dried with anhydrous MgSO1H NMR : δ (p.p.m.) 8.65–8.47 , 7.87–7.58 , 4.67 , 3.02 , 2.12-1.96 , 1.86 , 0.52–1.51  13C NMR : δ (p.p.m.) 187.21, 157.40, 147.50, 146.85, 145.19, 145.03, 144.32, 137.69, 136.98, 136.62, 134.94, 132.80, 127.82, 123.42, 122.22, 115.06, 114.84, 114.36, 113.15, 112.51, 77.22, 69.46, 55.38, 40.17, 31.92, 30.18, 29.75, 29.69, 29.65, 29.62, 29.55, 29.47, 29.34, 28.67, 27.72, 27.70, 23.24, 22.76, 22.69, 14.11, 13.72, 10.28, 10.26. HRMS (TOF) m/z calcd. for [M]+ C86H90F4N8O2S5 1502.5726, found 1503.5757.BTPV-CHO , 2-malononitrile , pyridine (0.5 mL) and chloroform (20 mL) were dissolved in a round bottom flask under nitrogen. The mixture was stirred at room temperature overnight. Then, the mixture was purified with column chromatography using dichloromethane to give BTPV-4F . 51. Compound 1  and 2- malononitrile  were dissolved in CHCl3 (25 mL) under a nitrogen atmosphere. 0.6 mL pyridine was added and refluxed for 12 h. Then the mixture was purified using column chromatography on silica gel employing petroleum ether/CHCl2 (1:1) as an eluent, yielding a dark blue solid m-DTC-2F . 1H NMR  δ 8.91 , 8.71 , 8.38 , 8.04–7.80 , 7.71 , 7.57 , 7.42 , 7.21–6.90 , 4.62 , 2.52 , 2.32 , 2.12 , 1.55-1.48 , 1.34–1.18 , 0.83-0.77 . 13C NMR  δ 187.18, 187.00, 168.00, 165.45, 161.60, 161.51, 159.08, 158.12, 147.22, 144.12, 144.09, 143.37, 142.35, 142.25, 140.61, 140.13, 140.05, 139.96, 138.74, 134.61, 134.15, 133.14, 128.39, 128.12, 127.30, 125.82, 125.73, 124.99, 124.42, 121.80, 121.56, 121.46, 121.39, 118.75, 118.43, 114.51, 114.36, 114.32, 112.95, 112.69, 104.91, 102.34, 77.33, 77.22, 77.01, 76.70, 69.61, 62.99, 57.58, 35.99, 33.67, 31.75, 31.62, 31.44, 29.40, 29.34, 29.15, 28.93, 27.17, 22.59, 14.06, 14.04. HRMS (TOF) m/z calcd. for [M]+ C113H117F2N5O2S2 1677.8460.5726, found 1678.8622.The intermediate compound 1 was prepared according to the literaturen-butylammonium hexafluorophosphate ([nBu4N]+[PF6]–) (0.1 M) in CH3CN employing a computer-controlled CHI660C electrochemical workstation, glassy carbon working electrode coated with films, an Ag/AgCl reference electrode, and a platinum-wire auxiliary electrode. The potentials were referenced to a ferrocenium/ferrocene (FeCp2+/0) couple using ferrocene as an internal standard.The NMR spectra were measured using Bruker AVANCE400 MHz spectrometer. Electrochemical measurements were carried out under nitrogen in a solution of tetra-m-DTC-2F was fabricated with the device architecture of ITO/ZnO/PM6:m-DTC-2F/MoO3/Ag. The ZnO precursor solution is prepared by dissolving 0.14 g of zinc acetate dehydrate  and 0.5 g of ethanolamine  in 5 ml of 2-methoxyethanol . A thin layer of ZnO is deposited through spin-coating the ZnO precursor solution on precleaned ITO glass at 5000 rpm and baked subsequently at 200 °C for 2 h. Then the device is transferred into a glove box filled with nitrogen, in which the active layer of PM6:m-DTC-2F (1:1.5 w/w)  is spin-coated onto the ZnO layer at 1500 rpm. After that, the active layer is annealed at 110 °C for 10 min for the devices with thermal annealing treatment. The thickness of the active layer is ca. 100 nm. Finally, a layer of ca. 10 nm MoO3 and then a Ag layer of ca. 100 nm is evaporated subsequently under high vacuum.The inverted structure device of PM6:71BM was fabricated with device architecture of ITO/ZnO/active layer/MoO3/Ag. The ZnO precursor solution is prepared by dissolving 0.14 g of zinc acetate dehydrate  and 0.5 g of ethanolamine  in 5 ml of 2-methoxyethanol . A thin layer of ZnO is deposited through spin-coating the ZnO precursor solution on precleaned ITO glass at 5000 rpm and baked subsequently at 200 °C for 2 h. Then the device is transferred into a glove box filled with nitrogen, in which the active layer of PTB7-Th:BTPV-4F:PC71BM (1:1.5:0.15 w/w)  is spin-coated onto the ZnO layer at 3000 rpm. After that, the active layer is annealed at 110 °C for 5 min for the devices with thermal annealing treatment. The thickness of the active layer is ca. 100 nm. Finally, a layer of ca. 10 nm MoO3 and then a Ag layer of ca. 100 nm is evaporated subsequently under high vacuum.The inverted structure device of PTB7-Th:BTPV-4F or PTB7-Th:BTPV-4F:PCm-DTC-2F /MoO3/M-PEDOT/ZnO NPs/PTB7-Th:BTPV-4F:PC71BM /MoO3/Ag. The PM6:m-DTC-2F active layers were fabricated via the same process as the single cells with different thicknesses. Subsequently, 10 nm MoO3 was evaporated under high vacuum. M-PEDOT:PSS solution was prepared by mixing PEDOT:PSS with 1 vol% nonionic ethoxylated fluorosurfactant (Zonyl FSO). The M-PEDOT:PSS solution was then spin-casted  and annealed at 130 °C for 2 min. Then, ZnO nanoparticles in IPA (20 nm) was spin-coated and annealed at 120 °C for 5 min in the glove box. Then, the PTB7-Th:BTPV-4F:PC71BM active layers were fabricated via the same process as the single-junction devices with different thicknesses. Finally, a layer of 10 nm MoO3 and then a Ag layer of 100 nm is evaporated subsequently under high vacuum. The device area of the OSCs was 5.0 mm2, which was defined by optical microscope (Olympus BX51). In order to accurately measure the photocurrent, mask with an area of 4.80 mm2 was used to define the effective area of the OSCs. The devices with or without mask showed consistent photovoltaic performance values with relative errors within 0.3%.The tandem OSCs were fabricated with an architecture of ITO/ZnO/ PM6:J–V) characteristics of the OSCs were measured in a nitrogen glove box with a Keithley 2450 Source Measure unit. Class AAA Solar Simulator  with a 450 W xenon lamp and an air mass (AM) 1.5 filter was used as the light source. Output spectrum of the solar simulator is provided in Supplementary Fig. −2 by a SRC-2020 reference cell with a KG2 filter window. The mismatch factor is calculated as 1.0048. The input photon to converted current efficiency (IPCE) was measured by Solar Cell Spectral Response Measurement System QE-R3-011 . The light intensity at each wavelength was calibrated with a standard single-crystal Si photovoltaic cell. To measure the rear and front cell, light bias obtained by 550 nm short wave pass filters and 850 nm long wave pass filters were selected to excite (saturate) the front and rear cells, respectively.The current density–voltage  from Nanosensors using tapping mode.0) of the film with a hard X-ray energy of 10 keV. X-ray irradiation time was 30–60 s, dependent on the saturation level of the detector. Beam center was calibrated using AgB powder and the sample-to-detector distance was about 280 mm. The π-π coherence lengths (L) are estimated based on the Scherrer Equation (L = 2πK/FWHM), where K is the shape factor (here we use 0.9), and FWHM is the full width at half maximum of the (010) diffraction peaks.Grazing-incidence wide-angle X-ray scattering (GIWAXS) measurements were conducted at Advanced Light Source (ALS), Lawrence Berkeley National Laboratory, Berkeley, CA at the beamline 7.3.3. Data were acquired at the critical angle  supplied laser beams centered at 1030 nm with pulse duration of ~180 fs, pulse repetition rate of 33 kHz, and a maximum pulse energy of 0.3 mJ. The output of the amplifier was split into two streams of pulses. One was used to drive an optical parametric amplifier  to obtain the pump beam. The residual stream was directed into an ultrafast spectroscopic system  to generate the white light continuum probe beam. In the spectrometer, the pump chopped at 150 Hz frequency was spatially and temporally overlapped with the probe beam on the sample. Pump wavelength was set to 850 nm to selectively excite the BTPV-4F acceptor. Excitation energy of the pump pulse was set to 2 μJ/cm3/Ag for hole-mobility and ITO/ZnO/active layer/PDINO/Al for electron mobility. The hole and electron mobilities are calculated according to the SCLC method equation: J = 9µɛrɛ0V2/8d3, where J is the current density, µ is the hole or electron mobility, V is the internal voltage in the device, εr is the relative dielectric constant of active layer material, ε0 is the permittivity of empty space, and d is the thickness of the active layer.The charge carrier mobilities were measured with the device structure of ITO/PEDOT:PSS/active layer/MoO2 without moisture (water and oxygen < 0.5 ppm). Solar Simulator with a metal halide lamp was used as a light source with intensity equivalent to 1 sun, which was calibrated by matching the device performance to those measured under AM1.5 G with the ultraviolet portion removed by filtering and a Keithley 2400 source meter. The initial exposure time is defined as time 0 h. The temperature of the cells was kept under 40 °C by a fan during measurements.Devices were sealed in glass fronted chambers under NFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary