Plasmodium falciparum resistance to dihydroartemisinin–piperaquine (DHA–PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele.The rise in pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility.Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly.The transgenic line, B5-90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-ICThe online version contains supplementary material available at 10.1186/s12936-022-04148-z. Plasmodium falciparum infections in almost all endemic countries sample − [Ctpfpm2 − Ctpf β-tubulin]3D7. Parasites with copy number greater than 1.6 copies for pfpm2 and pfpm2 [To determine copy numbers of escribed . The ampnd pfpm2 , 16 werepfpm2/3 breakpoint PCR assay was performed as previously described [pfpm1. Primers BF_for and BR_rev amplified a 484 bp product surrounding the breakpoint at the 3′ end of pfpm3. Primers BF_for and AR_rev amplified the junction between the breakpoint, producing a 497 bp product in parasite isolates with pfpm2/3 amplifications. A pfpm2/3 single copy isolate is not expected to have the PCR product with these primers. One copy isolate was only noted when the control primer sets amplified a product, and the duplication PCR was negative. Two or more copies were annotated as > 1 copy of pfpm2/3 only when both the control and duplication primer sets generated a product. PCR reactions contained 12.5 µL GoTaq® Green Master Mix (Promega), 1 µL of each primer (10 µM stocks), 3 µL of DNA up to 25 µL final volume with water. PCR conditions were as follow: 95 °C for 2 min, followed by 30 cycles of 95 °C for 45 s, 50 °C for 30 s, 72 °C for 1 min, followed by a 5-min extension at 72 °C.The escribed . Three p50 and IC90) performed as previously published [50s and IC90s were estimated by nonlinear regression analysis using GraphPad Prism version 6.0 program. Samples having poor growth rate, as perceived by obtaining an OD ratio of < 1.7 between the no-drug test wells and the maximum tested drug concentration, were excluded from data analysis.Drug susceptibility testing used HRP-2 ELISA to measure 50% or 90% inhibitory concentration  and the dilution series were increased from 8 to 24 points, according to previously published reports , 24. Cul0–3 h was performed on 0–3 h post-invasion rings obtained from selected culture-adapted clinical isolates following published methods [d-sorbitol and 75% Percoll to obtain 0 to 3-h post-invasion rings which were adjusted to 0.5–1% starting parasitaemia with a 2% haematocrit in culture media (0.5% Albumax RPMI 1640 with 2.5% AB serum) and cultured in a 48-well microplate with 700 nM DHA and 0.1% DMSO in separate wells for growth control. The culture plate was then incubated for 6 h at 37 °C in modular incubator chambers and gassed with 5% CO2, 5% O2 and 90% N2 gas. Cells were then washed once, resuspended in drug-free medium, and cultured further for 66 h. Susceptibility to DHA was assessed microscopically on thin films by estimating the percentage of viable parasites, relative to control . For the controls, the RSA0–3 h was also performed on P. falciparum reference clones W2 (ART-sensitive control), IPC-4884 and IPC-5202  as ART-resistant control lines. A survival rate > 1% was deemed resistant for RSA.In vitro RSA methods  with sli0–3 h was performed on culture-adapted clinical isolates with 0–3-h ring-stage parasite cultures following a previously published method [d-sorbitol and 75% Percoll to obtain 0 to 3-h post-invasion. Synchronized ring parasites at 0.5–1% starting parasitaemia and 2% haematocrit were incubated with 200 nM PPQ or 0.5% lactic acid in water at 37 °C for 48 h in a 48-well microplate. The cultures were then washed once, resuspended in drug-free medium, and cultured further for 24 h. Susceptibility to PPQ was assessed microscopically on thin films by estimating the percentage of viable parasites in the similar manner as RSA. A survival > 10% was deemed resistant to PPQ.PSAd method . Brieflyd-sorbitol and parasitaemia levels were calculated, and cultures adjusted to 0.1% parasitaemia, 2% haematocrit in a final volume of 1 mL per well of a 12-well plate. Samples were then taken at t = 0, 24, 72, 120, and 168 h, fixed in 0.8% glutaraldehyde in PBS and stored at 4 °C for flow cytometry analysis. Culture media was replaced at 48, 71, and 120 h. Giemsa-stained thin films were also prepared as required for microscopic analysis.For longer-term replication assays, cultures were synchronized as described and resulting in ring stage cultures. The ring stage parasites were synchronized using 5% (w/v) Parasite samples were fixed in 0.8% glutaraldehyde in PBS and stored at 4 °C. Cells were prepared for analysis by staining with 2× SYBR Green I nucleic acid gel stain  for 30 min at 37 °C. Labelling was stopped with an equal volume of PBS and samples acquired using a CytoFlex  with CyEXpert software. Total RBC numbers were calculated using forward- and side-scatter while fluorescence was detected using the 530/630 blue detection laser. Fluorescence intensity was used to distinguish uninfected from infected RBCs, low fluorescence indicating uninfected cells and gating fixed accordingly. Data were analysed using FlowJo.Statistical analysis was performed using GraphPad Prism version 6.0 . The difference of the data between groups was assessed by nonparametric Mann–Whitney U test, as appropriate. Statistical significance was defined as a P value of < 0.05.pfexo gene from P. falciparum 3D7 (PF3D7_1362500) consists of 2274 bp with 2 exons and 1 intron. Exon 1 comprises nucleotides 1-949, while the exon 2 starts from nucleotides 1082–2274  site was also attempted to rule out a role for silent mutations in drug susceptibility. Eleven transfections were performed to introduce pfexoE415G mutation and 3 transfections for pfexoE415E mutation but only three transfections provided revived parasites. Two of the three transfections gave rise to the modified pfexo gene, and one transfection did not provide the modified parasites. Successful modification of the pfexo gene in the transfected population following the introduction of the targeting vector was observed around 21 days after transfection and confirmed by diagnostic PCR  and genomic sequences of transgenic lines  [pfexo gene. Regarding the two clinical isolates, both carried the PfK13-C580Y and PfEXO-E415G mutations. The ASAP-21 isolate harboured the novel PfCRT-F145I mutation while ASAP-168 did not. Genetic studies have identified copy number variations in the pfpm2 and pfpm3 genes that associate with clinical and in vitro PPQ resistance [pfpm2/3 amplification, the putative breakpoints lie near the 3′ end of both pfpm1 and pfpm3 so each amplification produces an intact extra copy of pfpm2 together with a new chimeric pfpm3 with its 3′ end replaced by the 3′ end of pfpm1  while B5 and B5-rexo-E415G-B8 lines showed no pfpm2/3 amplification as detected by the breakpoint assay   were inc50 values of B5-rexo-E415G-B8 and ASAP-168 parasites compared to B5 parasites was observed, while PPQ-IC50 value of ASAP-21 parasites was significantly higher than that of B5 parasites. The same trend was observed for PPQ-IC90 values in that there was no significant difference in the IC90 values for B5-rexo-E415G-B8 and ASAP-168 parasites compared to B5 parasites. The PPQ-IC90 of ASAP-21 exceeded the highest PPQ concentration used in this study, so the IC90 could not be reported.The transgenic parasites expressing E415G mutant of PfEXO and the clinical isolates were tested for their PPQ susceptibility Table . No signpfexo transgenic parasites and clinical isolates with PfEXO-E415G mutation have cross-resistance, parasite drug sensitivity was also assessed against a panel of antimalarial drugs  and proguanil (PG), where higher IC50 values were detected in comparison with the B5 parental line. The ASAP-21 parasite had significantly lower IC50 values for quinine (QN), chloroquine (CQ), atovaquone (ATQ) and doxycycline (DOX) but higher IC50 value for pyronaridine (PND) compared to the B5 parental line, while the ASAP-168 parasite had  increased drug sensitivity against dihydroartemisinin (DHA), mefloquine (MQ), QN, and ATQ. The reduced IC50 values of QN and ATQ were observed in both ASAP-21 and ASAP-168 parasites compared to the B5 parental line.To evaluate if the modified gs Table . No sign0–3 h and PSA0–3 h) and PPQ-bimodal dose response curve were performed , similar to that of the parental B5 parasites. This suggested that the presence of PfEXO-E415G mutation alone could not confer the PPQ resistance phenotype. The ASAP-21 and ASAP-168 parasites, containing the PfEXO-E415G mutation in combination with either PfCRT mutation or multiple copies of pfpm2, exhibited a % PSA survival rate higher than 10, indicative of PPQ-resistance.To gain a better understanding of the ART and PPQ resistance phenotypes, the survival assay   grew much more slowly than W2 (an Indochina clone exhibiting CQ resistance) and 3D7 (African origin) lines. Parasite B5-rexo-E415G-B8, harbouring pfexo-E415G mutation, seemed to grow slightly faster than its parental line B5. In the presence of PfK13-C580Y, PfEXO-E415G, novel PfCRT-F145I mutations and multiple copy number of pfpm2, ASAP-21 parasite had a severely retarded growth rate. ASAP-168, having the similar genetic background to ASAP-21 but without the PfCRT-F145I mutation, grew marginally better than ASAP-21 parasites. This observation implies an important contribution of the strain background toward parasite fitness, especially when PfK13-C580Y, PfCRT-F145I mutations and multiple copy number of pfpm2 were present.To assess whether the introduction of E415G mutation into PfEXO affects the parasite fitness, growth assays of B5-rpfk13 for ART resistance, have been widely employed as identification and prediction tools for the emergence of drug-resistant P. falciparum [P. falciparum malaria has been reported [pfexo cannot alone reduce PPQ susceptibility neither improve parasite survival rates that are characteristic of PPQ resistant isolates circulating in the Greater Mekong Subregion [Validated molecular markers, such as lciparum . While areported , 20, 29,ubregion .pfexo gene from P. falciparum (PF3D7_1362500) encodes a protein with a mass of 86.7 kDa with a 3′–5′ exonuclease domain at the C-terminus. In this study, the P. falciparum B5 line used for genome editing had one mutation (K614N) in exonuclease, located in the 3′–5′ exonuclease domain. Since no in vivo relevance of this mutation has yet been proved in this study, it is suggested that the prevalence of this mutation should be determined. Nucleases may be partially or fully redundant, depending on the pathway, and such redundancy might complement functional losses. Zhang et al. [P. falciparum genome, including exoribonuclease functional domain-containing proteins Dis3 and Rrp6, the latter of which was found in PfEXO of this study.Exonucleases are essential to genome stability, catalysing the removal of a single nucleotide monophosphate (dNMP) from the end of one strand of DNA and acting as a proof-reader during DNA replication . Exonuclg et al.  used bioPlasmodium genome editing can be achieved, and the approach has been employed to introduce point mutations into several P. falciparum genes [P. falciparum 3D7 or Dd2 lines, the Cambodia adapted-B5 line [P. falciparum in Cambodia. It has been previously shown that different phenotypes were observed when using different parasite backgrounds. Targeted gene disruption of either pfpm2 or pfpm3 in the 3D7 genetic background caused a only slight decrease in PPQ susceptibility [pfpm2 and pfpm3 overexpression in 3D7 did not alter the sensitivity of P. falciparum to PPQ [P. falciparum Dd2 parasites with copy number variation in pfpm2 were generated [pfpm2 amplification contributed to PPQ resistance, with a bimodal dose-response observed. In addition to a qPCR assay to detect pfpm2 copy number variation, the breakpoint assay, as described by Imwong et al. [pfpm2/3 copy number above the cut-off of 1.52, 88% were confirmed to have pfpm2/3 amplification by the breakpoint assay, while for those < 1.14 (the cut-off for a single copy number) and for an intermediate value between 1.14 and 1.52, the proportion of pfpm2/3 amplified parasites using the breakpoint SNP were 4% and 38%, respectively. In this study, the ASAP-168 parasite was found to have pfpm2 copy number variation of 1.11, lower than the cut-off for a single copy number, but the breakpoint assay confirmed the gene duplication of pfpm2. As for pfpm3 copy number variation, the detection by qPCR was not 100% concordant with and pfpm2/3 breakpoint assay for B5 and B5-rexo-E415G-B8 lines as qPCR assay showed the pfpm3 multiple copy for Cambodia isolates, which was different from the previous report by Ansbro et al. [pfpm3 and further investigation could be done. Therefore, it is important to also apply the breakpoint assay to isolates with copy number values < 1.52 for pfpm2, to capture all isolates with pfpm2/3 amplification.CRISPR/Cas9 approaches have transformed the speed and scale with which um genes . In this-B5 line  was empltibility  and pfpmm to PPQ . Howeverenerated , pfpm2 ag et al. , was caro et al. . It is n90 values, PPQ survival rates, and bimodal dose-response curves were used for assessing PPQ resistance in vitro [exo-E415G-B8) did not show a PPQ resistant phenotype. On the contrary, parasites with the combination of PfEXO-E415G mutation with either novel PfCRT mutations or pfpm2 multiple copies demonstrated reduced IC90 susceptibility, high PPQ survival rates, and a second peak of bimodal curve  show reduced PPQ susceptibility and involved in PPQ resistance. Nonetheless, when the novel PfCRT mutation was added on top of the pfpm2 multiple copies, the level of PPQ resistance increased tremendously as judged by the IC50 and IC90 values as well as AUC between 0.01 and 100 µM of PPQ in the bimodal dose-response curve. Bopp et al. [pfpm2 multiple copies grew much slower than those without mutations. It is likely that introducing PfEXO-E415G mutations may account for better fitness compared to parasites with the same genetic background. Even though the scope of this work could not address the function of PfEXO, it is evidently showed that the E415G mutation in PfEXO does not alter PPQ susceptivity, but rather affects parasite fitness.ICin vitro , 17, 48.rve Fig. . If the p et al. , 20. Sim90 or improve %PPQ survival, suggesting that this mutation is not associated with PPQ resistance. Additionally, this specific mutation resulted in parasites that grew better than those with the same background, highlighting the importance of genetic mutations toward parasite fitness and resistance.In summary, this study suggests that the E415G mutation in PfEXO could still be an important marker if used in conjunction with other markers. The insertion of the PfEXO-E415G mutation did not lead to an increased PPQ-ICAdditional file 1: Table S1. Oligonucleotideprimer sequences used in this study. Figure S1. DNA sequence alignment of exo gene from P. falciparum 3D7,B5 and B5-rexo-E415G-B8 parasites. Figure S2. Gene and amino acid composition of PfEXO. Figure S3. Schematic of P. falciparum plasmepsin 2/3 (pfpm2/3)gene duplication.


The objectives of this study were to characterize two endoglucanases TrepCel3 and TrepCel4, and determine the effect of the combination of them  on in vitro rumen fermentation characteristics. In this study, three nature lignocellulosic substrates  were evaluated for their in vitro digestibility, gas, NHEscherichia coli (E. coli) BL21 (DE3), and enzymatic characteristics were further characterized. The combination of TrepCel3 and TrepCel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability.Two endoglucanases’ genes were successfully expressed in 3-N and VFA production, and MCP synthesis during in vitro rumen fermentation.The maximum enzymatic activity of TrepCel3 was determined at pH 5.0 and 40 °C, while TrepCel4 was at pH 6.0 and 50 °C. They were stable over the temperature range of 30 to 60 °C, and active within the pH range of 4.0 to 9.0. The TrepCel3 and TrepCel4 had the highest activity in lichenan 436.9 ± 8.30 and 377.6 ± 6.80 U/mg, respectively. The combination of TrepCel3 and TrepCel4 exhibited the highest efficiency at the ratio of 60:40. Compared to maximum hydrolysis of TrepCel3 or TrepCel4 separately, this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4% for RS, 236.7% for wheat straw WS, 222.4% for LC and 131.1% for sugar beet pulp (SBP). Supplemental this combination enhanced the dry matter digestion (DMD), gas, NHThe TrepCel3 and TrepCel4 exhibited the synergistic relationship (60:40) and significantly increased the saccharification yield of lignocellulosic substrates. The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates. This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants. If possible, in the future, experiments in vivo should be carried out to fully evaluate its effect.The online version contains supplementary material available at 10.1186/s40104-022-00814-z. Lignocellulosic biomass is the most abundant, inexpensive, available and renewable raw material in nature for industrial biorefining and livestock farming , 2. Lignhttp://www.cazy.org), including GH5 to 10, 12, 26, 44, 45, 48, 51, 60, 61, 74, 124 and 131 [Cellulases are grouped into 17 glycoside hydrolase (GH) families based on the sequence and structure similarity of CAZy database  regarded as the best cellulase cocktail, which can produce beyond three times fermentable sugar than that of Palkonol MBW. Zhao et al. [Phialophora sp. G5 showed synergistical effect on the hydrolysis of filter paper. The saccharification yield could be improved by enzymatic cocktails contain lytic polysaccharide monooxygenases (LPMOs) that leave cellulose using molecular oxygen and an electron donor [The bio-degradation of lignocellulosic biomass is a complex process consists of these steps: pretreatment of biomass for enzymatic hydrolysis of the polysaccharides, and fermentation of fermentable sugars to bioethanol or VFA. Hence, cellulases with high hydrolytic ability are essential to decrease the loading amounts of enzymes, which could decrease economic cost . Enzymato et al.  presenteon donor , 39. Foron donor , 41. Bowon donor  showed tThe buffalo diet consists of a variety of recalcitrant lignocellulosic materials including aquatic plants, crops, grasses, herbs, microalgae, leaves and bark of trees . TherefoEscherichia coli (E. coli) DH5α  was used for the propagation of plasmid and E. coli BL21 (DE3)  was used for the expression of recombinant product. Plasmid pET-28a (+)  was used for In-Fusion cloning, and expression of recombinant protein. Carboxymethylcellulose (CMC), locust bean gum (LBG), lichenan, barley β-glucan, filter paper (Whatman Grade 40), p-nitrophenyl-β-D-glucopyranoside (pNPG) were purchased from Megazyme , General Electric Company  and Sigma-Aldrich . The RS, WS, LC and SBP were purchased from the local market .Three healthy, mature and rumen-cannulated buffaloes  were used in this study. The buffaloes were fed chopped green roughage ad libitum for one month, and had free access to drink fresh water. All animal procedures were performed in accordance with Institutional Animal Care and Use Committee of Nanjing Agricultural University . A total of 6 stitched nylon bags were filled with wheat straw  were placed into the buffalo rumen . All bags were retrieved at 24 h, followed by washing gently with phosphate buffered saline (PBS) buffer to remove the rumen contents attached to the bag and loosely attached microbes. The collected sample residues were frozen immediately in liquid nitrogen, and stored at − 80 °C until DNA isolation.Genomic DNA was extracted using the protocol as Cheng et al.  describehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The analysis of signal peptides was carried out by using the SignalP-6.0 online tool [https://www.ebi.ac.uk/Tools/msa/clustalo/). The phylogenetic tree was further constructed by MEGA-X according to the neighbour-joining method [http://www.ebi.ac.uk/interpro/search/sequence/). The homology modelling and secondary structure of these two candidate enzymes were done predicted by using the Phyre2 online tool [The sequences of TrepCel3 and TrepCel4 were submitted to NCBI GenBank . The TrepCel3 and TrepCel4 sequences were both synthesized by Sangon Biotechnology . The analyses of conserved domain were performed using the NCBI conserved domain database (CDD) /pET28a (+)/TrepCel3, and BL21 (DE3)/pET28a (+)/TrepCel4 were cultured in 6 mL Luria–Bertani (LB) medium containing 50 μg/mL kanamycin at 37 °C with shaking at 200 r/min overnight. The growing cells were then inoculated into in a 1-L flask containing 600 mL LB medium supplemented with 50 μg/mL kanamycin and incubated at 37 °C with shaking at 200 r/min. When the optical density at 600 nm of the culture reached 0.5 to 0.6, a final concentration of 0.5 mmol/L isopropyl β-D-thiogalactopyranoside (IPTG) was added, and then incubated for 24 h further in 16 °C, 150 r/min shaking conditions. Later, the cells were collected by centrifugation at 4 °C and 12,000 r/min for 30 min. The pellets were further resuspended in 40 mL of lysis buffer , disrupted by sonication , followed by centrifugation at 12,000 r/min for 15 min at 4 °C to obtain the supernatant. The collected supernatant was purified by Ni–NTA Fast Start Kit using standard protocols . Briefly, the cell lysate supernatant was applied into a Fast Start Column with 6xHis-tagged resin. The bound 6xHis-tagged protein was eluted by Native Elution Buffer . The quality and concentration of the purified fractions were measured by 12% SDS-PAGE gel, and Bradford method [Recombinant d method , respectCellulase activity was measured by the DNS method with CMC as a substrate . The rel2HPO4 (pH 3 to 7), 100 mmol/L Tris–HCl (pH 8 to 9) and 100 mmol/L glycine–NaOH (pH 10 to 12). The pH stability was evaluated by pre-incubating the enzyme at 4 °C for 16 h at different pH buffers, and then the residual activity was measured. The enzyme solution in 100 mmol/L citric acid-Na2HPO4 buffer with 2% (w/v) CMC was incubated in the different temperature (30 to 70 °C) for 10 min to determine the optimum temperature. The thermal stability was used by observing the residual enzyme activity after incubation of the enzymes in 100 mmol/L citric acid-Na2HPO4 buffer at 50 and 60 °C for 60 min without substrate. The residual enzyme activity was determined at different time intervals . All assays were measured by the DNS method at 540 nm, and the maximum enzyme activity was established as 100%.To determine the pH optima of enzymatic activity, purified TrepCel3 and TrepCel4 were incubated with 2% (w/v) CMC in various pH buffers, respectively. The buffers used were 100 mmol/L citric acid-Na+, Cu2+, Mg2+, Zn2+, Ca2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, NH4+ and Ba2+, 10 mmol/L PMSF, EDTA and β-mercaptoethanol, 1% (w/v) Tween 20, Tween 80, Triton X-100 and SDS, 2 mmol/L urea and guanidine hydrochloride on the enzymatic activity was measured in individual reactions by pre-incubating with enzyme for 60 min at RT using the DNS method [The effect of 10 mmol/L KS method –56. The 2, 0.99 g/L; MgCl2·6H2O, 6.09 g/L; KCl, 0.74 g/L; MgSO4·7H2O, 3.94 g/L [The NaCl resistance test of TrepCel3 and TrepCel4 was determined by different concentrations of NaCl (1 to 5 mmol/L); without adding NaCl was set as the control group . Besides3.94 g/L .pNPG and β-pNPG. The enzymatic activity was determined by measuring the release of reducing sugar or p-nitrophenol (pNP) from various substrates [2HPO4 buffer containing 2% (w/v) cellulose substrate, or 2 mmol/L pNPG at individual optimal pH and temperature for 5 min. Then 300 μL of DNS solution was added to end the reaction and boiled for 5 min, or the reaction mixture was stopped with 400 μL of 2 mmol/L Na2CO3. The released reducing sugar or pNP was measured as absorbance at 540 or 405 nm by using a TECAN Spark Multi-functional Microplate Reader , respectively. One unit of cellulase activity is defined as the amount of TrepCel3 or TrepCel4 producing 1 μmol of pNP per min per milliliter. For the kinetic parameters test of TrepCel3 and TrepCel4, Km and Vmax, were measured with the substrate  between 1 and 20.0 mg/L in citric acid-Na2HPO4 buffer by the Lineweaver–Burk method for 5 min. Each treatment was incubated in triplicate.The substrate specificity of purified cellulase was determined in the reaction mixtures with CMC, β-glucan, filter paper, LBG, lichenan, α-bstrates , 59. TheThe synergistic effect of TrepCel3 and TrepCel4 in the degradation of lignocellulosic substrates was further studied. Therefore, the effect of different ratios of TrepCel3 and TrepCel4  on the hydrolysis of CMC was firstly studied . Hence, 2HPO4 buffer, respectively. Each treatment was incubated in triplicate, and was in an incubator shaker at 60 r/min at 45 °C for 168 h [The RS, WS, LC and SBP were evaluated for the hydrolysis capabilities in lignocellulose biorefinery. The RS, WS and LC were grinded to the particle size of 2 to 3 mm, and SBP was milled and sieved through a 50-mesh sieve. These lignocellulosic substrates were treated by 2% NaOH treatment at 121 °C for 15 min, and then washed with distilled water to fully remove reducing sugars. The purified 50 μL (0.5 mg/mL) TrepCel3, TrepCel4 and the combination of them were incubated with the substrates  soaked in 20 mL citric acid-Naor 168 h . The pro3-N and microbial protein (MCP) were described by Weatherburn [3-N, MCP and lactate concentration was measured by using a TECAN Spark Multi-functional Microplate Reader  based on the spectrophotometer method. The residue was filtered using nylon bags for the determination of DMD.The effect of the combination of TrepCel3 and TrepCel4 on rumen microbial fermentation of RS, WS and LC was investigated by an in vitro experiment. Rumen fluid was collected from three ruminally-fistulated Hu sheep  fed a diet consisting of 700 g/kg alfalfa and 300 g/kg concentrates before morning feeding. Rumen samples were then squeezed through four layers of sterile cheesecloth. The in vitro study was performed in 120-mL bottles containing 600 mg agricultural straws, and 60 mL buffered rumen fluid that was composed of 45 mL buffer solution and 15 mL filtered rumen fluid . Each trtherburn , Carro etherburn , and Makt-test was conducted to determine the results of in vitro rumen fermentation experiment. IBM SPSS Statistics 20  was used for statistical analysis. Significance was defined as P < 0.05.Statistical analysis was done by the one-way ANOVA with LSD multiple range test was performed to evaluate the significance of the hydrolysis of RS, WS, LC and SBP. Independent samples Based on the metagenome data for identifying new endoglucanases with valuable industrial and livestock farming applications, in this study, two novel endoglucanases were mined from the metagenome data of buffalo rumen using computational screening, and named TrepCel3 and TrepCel4, respectively. After the in-silico screening, TrepCel3 and TrepCel4 were cloned, expressed, purified and characterized.Treponema sp. based on the non-redundant (NR) protein database (GenBank: MBR4789481.1), while showed up to 52.6% identity with an endoglucanase from Treponema bryantii based on UniProt database (Accession: A0A1H9EK09). The results of phylogenetic tree showed that TrepCel3 was classified into the GH5 family  with 100% confidence and 92% coverage  length of TrepCel3 consisted of 1131 base pairs (bp), and the N-terminal 19 amino acids were predicted to be a putative signal peptide. TrepCel3 encoded a polypeptide of 357 amino acids with a theoretical molecular weight (Mw) of 39.8 kDa and isoelectric point (pI) 5.04, and only contained a module domain  based on NR protein database, while showed up to 81.0% identity with an endoglucanase from Treponema bryantii based on UniProt database (Accession: A0A1H9JN96). As shown in Fig. Butyrivibrio proteoclasticus (PDB: 4NF7). The predicted secondary structure of TrepCel4, contained 35% α-helix and 12% β-strand  vector to construct recombinant plasmids , which was consistent with the endoglucanase Cel-1 from buffalo rumen [2+ increased enzymatic activity with approximately 36%, which suggested that Co2+ was important for TrepCel3. Similarly, in previous studies, Co2+ could also strongly enhance enzymatic activities sun as endoglucanase XacCel8 [The effects of a range of metal ions and chemical reagents on enzymatic activities of TrepCel3 and TrepCel4 were measured as shown in Fig. lo rumen , and thelo rumen . For Tre XacCel8  and EG5C XacCel8 . Differe XacCel8 , while T XacCel8 , 76. The XacCel8 , and 18. XacCel8 . Further XacCel8 . TherefoGenerally, endoglucases with excellent specificities are of worthwhile interest for industrial applications where extremophilic conditions like high temperature and salt concentrations. In addition, for industrial applications, seawater is abundant and amply available in nature, and usually used as an efficient solvent for hydrothermal pretreatment of plant biomass . EnzymatAlkalibacterium sp. SL3 was even inhibited by 0.25 mmol/L NaCl [If possible, cellulolytic enzymes applied in textile industries or biofuel synthesis that should be tolerant to various salts. A novel alkali thermostable endoglucanase PersiCel4 from sheep rumen metagenome exhibited high tolerance to the high concentration of NaCl (5 mmol/L) . Howeverl/L NaCl .pNPG and β-pNPG. Based on the result assays, TrepCel3 and TrepCel4 had higher ability to hydrolyze CMC, but no ability to hydrolyze pNPG, which indicated that TrepCel3 and TrepCel4 were both endoglucanases not β-glucosidases.The substrate activities were measured in the presence of various substrates. The results of the specific activity assays were shown in Fig. Vmax) values of TrepCel3 for barely β-glucan, lichenan and CMC were 527.3 ± 22.69, 883.8 ± 25.59 and 228.3 ± 15.80 U/mg, respectively. The determined Km (Michaelis–Menten constant) values of TrepCel3 for barely β-glucan, lichenan and CMC were 4.9 ± 0.73, 7.1 ± 0.58 and 3.6 ± 0.43 mg/mL. The Vmax values of TrepCel4 for barely β-glucan, lichenan and CMC were 441.4 ± 9.32, 772.3 ± 48.65 and 219.7 ± 13.11 U/mg, respectively. The Km values of TrepCel4 for barely β-glucan, lichenan and CMC were 3.9 ± 0.14, 5.0 ± 0.47 and 2.3 ± 0.38 mg/mL, respectively. Compared with some reported studies, TrepCel3 and TrepCel4 had higher Vmax values (CMC as substrate) than enzymes such as AcCel12B [Km values could reflect the affinity of an enzyme towards its various substrate. Based on the Km values, TrepCel3 and TrepCel4 both had the highest affinity against CMC instead of β-glucan and lichenan. Besides, Km values of TrepCel3 and TrepCel4 (CMC as the substrate) were lower than that of AcCel12B [The kinetic parameters examined for barely β-glucan, lichenan and CMC were shown in Table AcCel12B . The Km AcCel12B , which iWhen TrepCel3 and TrepCel4 were incubated together with CMC, a strong synergy effect was observed in line with the production of reducing sugar compared with the released reducing sugar by TrepCel3 and TrepCel4, separately. The combination of TrepCel3 and TrepCel4 at the ratio of 60:40 showed the highest efficiency Fig. . As we mP < 0.05). Production of reducing sugar of the hydrolysis of WS and LC at 24 and 168 h was similar with RS . The enzyme treatments showed greater gas production volume of RS, WS and LC by 21.6%, 23.4%, and 15.5%, respectively (P < 0.05), while did not affect fermentation fluid pH (P > 0.05). The concentrations of TVFAs, acetate, propionate and other VFAs  were significantly affected by the addition of enzyme (P < 0.05), resulting in a significant increase in the acetate:propionate ratio . When enzyme incubated with RS, butyrate concentration was significantly increased (P < 0.05), but when incubated with WS or LC, the butyrate concentration was not significantly affected (P > 0.05). Compared to the control, acetate and propionate molar proportions were significantly greater in enzyme-added lignocellulosic residues (P < 0.05), while butyrate molar proportion was significantly lower (P < 0.05). With RS, the addition of enzymatic combination did not affect the molar proportion of other VFAs (P > 0.05), other VFAs were significantly decreased by enzyme in WS and LC (P < 0.05). The supplementation of enzyme significantly increased the NH3-N concentration of RS and WS group (P < 0.05), while did not affect the NH3-N concentration of LC group (P > 0.05). Application of enzyme significantly increased the MCP concentration of RS, WS and LC group, and the lactate concentration of RS group (P < 0.05).As shown in Table Trichoderma longibrachiatum and a cellulase from Aspergillus niger) could significantly improve the in vitro fermentation parameters of diet  including NDF and ADF apparent digestibility [3-N concentration is range from 7.7 to 17.3 mmol/L, which is regarded as N source for MCP synthesis [3-N is relatively low because of the low crude protein content of straw fiber. Supplemental the enzyme also increased the production of NH3-N for RS and WS. Vallejo et al. [3-N concentration compared with the control group. The quantity of dietary nitrogen breakdown and nitrogen absorption by ruminal bacteria may be responsible for the increased generation of NH3-N [Trichoderma longibrachiatum and a cellulase from Aspergillus niger) could significantly improve VFA production [Desulfovibrio. However, in this study, we did not investigate the change of fibrolytic bacterial community. Rumen butyrate is an energy source or metabolized to β-hydroxybutyrate by rumen epithelial cells, and it can also be regarded as a primer for short fatty acid synthesis [The EFEs can increase the nutrient digestibility of agricultural residues because it can disrupt the basic structure of straw fiber , improvetibility . Gas protibility  presentetibility . In thistibility . Currenttibility . The neuynthesis . Howeverof NH3-N . The carof NH3-N , 61, whiof NH3-N . Brieflyoduction . Supplemoduction . In the oduction . Li et aoduction  found thynthesis . Butyratynthesis . As we aynthesis  also fou2+ and Co2+ ions (10 mmol/L), degradation efficiencies of TrepCel3 and TrepCel4 increased, respectively. TrepCel3 and TrepCel4 were active at pH 5.0 and 6.0, respectively. Enzymatic activities of TrepCel3 and TrepCle4 could be improved by 3 and 5 mol/L NaCl, respectively. Compared to TrepCel3 and TrepCel4 separately, the combination of them was capable of producing high concentrations of reducing sugar from agricultural feedstocks. The enzymatic combination of TrepCel3 and TrepCel4 gave the highest efficiency at the ratio of 60:40. This enzymatic combination improved in vitro rumen fermentation of natural lignocellulosic feedstocks by increasing the DMD, VFA, NH3-N and lactate production, and microbial protein synthesis. This enzymatic combination has the potential to be a feed additive to improve agricultural straw utilization in biotechnological and livestock farming applications.In this study, two novel endoglucanases from buffalo rumen metagenome were successfully cloned, expressed, and characterized. When incubated with MnAdditional file 1: Fig. S1a. The predicted secondary structure of TrepCel3. Fig. S1b. The predicted secondary structure of TrepCel4.Additional file 2: Table S1. The constituent components in rice straw, wheat straw, leymus chinensis and sugar beet pulp.


Both CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are activated by the chemokine CXCL12 yet evoke distinct cellular responses. CXCR4 is a canonical G protein–coupled receptor (GPCR), whereas ACKR3 is intrinsically biased for arrestin. The molecular basis for this difference is not understood. Here, we describe cryo-EM structures of ACKR3 in complex with CXCL12, a more potent CXCL12 variant, and a small-molecule agonist. The bound chemokines adopt an unexpected pose relative to those established for CXCR4 and observed in other receptor-chemokine complexes. Along with functional studies, these structures provide insight into the ligand-binding promiscuity of ACKR3, why it fails to couple to G proteins, and its bias toward β-arrestin. The results lay the groundwork for understanding the physiological interplay of ACKR3 with other GPCRs. Structures of an atypical chemokine receptor show a novel ligand pose and shed light on its plasticity and arrestin bias. S1). A nanobody that restricts the flexibility of the elbow arm between the variable and constant domains of the light chain . Two Fabs that bound to opposite sides of the complex were chosen to aid the structure determination process. CID25 binds to an extracellular epitope that includes elements from both ACKR3 and CXCL12, and CID24 binds to the cytoplasmic pocket of the active receptor . Fab binWT–CID24 complex , whereas its N terminus penetrates deep into the receptor orthosteric pocket . A cholesterol molecule in the analogous site is also present in structures of CXCR2 and other class A GPCRs  > 1 μM], but readily bind CXCR4   entries 7SK5 and 7SK6] indicated that CXCL12of CID25 , furtherth CXCR4 . Strong 5.38, ACKR3-D1794.60, and ACKR3-Y200ECL2 and its backbone carbonyl close enough to form a hydrogen bond with the hydroxyl of Y2686.51 , placing its side chain near the side chains of ACKR3-E213Y2686.51 . P2 of CWT, CXCL12LRHQ recruits arrestin with unchanged potency and efficacy  at the N terminus followed by three residue substitutions (RHQ) relative to CXCL12ractions . The majWT. However, we observed density for the agonist in the cryo-EM maps, resulting in the first six residues of CXCL12WT being disordered in both the NB-CID25-ACKR3·CXCL12WT·CCX662 and CID25-ACKR3·CXCL12WT·CCX662-CID24 complexes. To eliminate any ambiguity, we also determined the 3.8-Å structure of ACKR3·CCX662-CID24. In each of the three structures, CCX662 binds in the orthosteric pocket, with its core occupying a similar volume as the first few residues of CXCL12LRHQ . When compared to the chemokine-bound structures, several ACKR3 side chains are rearranged in the CCX662 complex. For example, ACKR3-Q3017.39 is reoriented toward the entrance of the pocket and forms no hydrogen bonds with CCX662, and the H1213.29 side chain adopts two distinct conformers. Still, CCX662 recruits arrestin with almost equal efficacy as CXCL12  for 174 Cα atoms; 2 of accessible surface area (2AR (WT·CCX662 complex in the absence of CID24 (table S1), which may be the first structure of a receptor in an active-state conformation without cytoplasmic cleft binding partners or stabilizing crystal contacts. The open active-like conformation of the TM helices is maintained, but all the ICLs and H8 exhibit disorder  on the intracellular side of TM6 at a position where all other chemokine receptors have shorter hydrophobic side chains (6.40N) and rhodopsin (M2576.40Y) increases constitutive receptor activity and, in the case of CXCR1, affinity for bound chemokine . Similarly, the Y3157.53A mutation reduced chemokine affinity and impaired the ability of ACKR3 to recruit arrestin . Mun mutant  (36). ThBecause CCX662 can efficiently recruit arrestin but does not activate G proteins (fig. S10D), arrestin bias probably has little to do with the unique pose observed for CXCL12 in the ACKR3 complexes . On the P2G) convert the chemokine into an antagonist  (7.39 (corresponding to CXCR4−E2887.39), and to a lesser extent ACRK3-Y2686.51, results in increased constitutive activity (fig. S10A). Thus, it seems that distortion of the binding pocket by steric bulk may be sufficient for ACKR3 activation, as has been suggested for the viral chemokine receptor US28 (7.39 and H1213.29) as well as a segment of TM5 at the base of the orthosteric pocket, may facilitate binding of diverse ligands and allow for distortion-driven activation. The absence of a disulfide bond between the receptor N terminus and ECL3 also likely contributes to the deformability of the orthosteric pocket by diverse ligands.Beyond determining the basic architecture of ACKR3 and how it interacts with CXCL12, a goal of this work was to gain insight into the atypical promiscuous activation of ACKR3 by diverse ligands. To date, a nanobody and a small-molecule antagonist are the only known ACKR3 ligands that do not promote arrestin recruitment  293 (fig. S10D) and other cells . After 48 hours, single white colonies were picked and grown overnight in 5 ml of LB medium with kanamycin (50 μg/ml), gentamicin (7 μg/ml), and tetracycline (10 μg/ml). Sf9 cells were cultured in ESF 921 medium (Expression Systems) at 27°C with shaking at 140 rpm. Bacmid DNA was isolated and mixed with transfection medium and X-tremeGene HP DNA for 30 min at room temperature before addition to 2.5 ml of 2.0 × 106/ml Sf9 cells in a 24–deep well plate. The transfection mixture was then incubated at 27°C for 96 hours while shaking at 300 rpm. After 96 hours, the plates were centrifuged (20 min at 2000g) and the P0 virus containing supernatant was collected. Four hundred microliters of P0 virus was added to 40 ml of 2.0 × 106/ml Sf9 cells and incubated at 27°C with shaking at 130 rpm for 48 hours, after which the cells were again spun down (20 min at 2000g), and the P1 virus–containing supernatant was collected and stored at 4°C. Expression of ACKR3 or coexpression of ACKR3 and chemokine was achieved by adding P1 virus to Sf9 cells at a density of 2 × 106 to 3 × 106/ml with a multiplicity of infection (MOI) of 6 for each virus. After 48 hours of infection, cell pellets were collected and stored at −80°C.Production of baculoviruses  was performed using the Bac-to-Bac Baculovirus Expression System (Invitrogen) as described previously , 10 mM MgCln-dodecyl-β-d-maltoside (DDM)/CHS]. CCX662 (ChemoCentryx)  and 20 mM imidazole were then added to the soluble supernatant, and the sample was incubated overnight at 4°C. The next morning, the receptor-bound resin was pelleted by centrifugation for 5 min at 350g and washed with 25 ml of wash buffer 1  and centrifuged again. The resin was then transferred to Poly-Prep Chromatography columns (Bio-Rad) and washed with an additional 20 ml of WB1, followed by 20 ml of WB2 , and finally eluted with elution buffer (WB2 with 250 mM imidazole). The elutions were concentrated, and imidazole was removed with a desalting column and then buffer exchanged to low-salt buffer . Protein concentrations were determined by A280 using extinction coefficients of 77,000 and 85,000 M−1 cm−1 for ACKR3 and ACKR3·CXCL12 complexes, respectively. Receptors were aliquoted, snap-frozen, and stored at −80°C until use.To purify ACKR3 or ACKR3·CXCL12 complexes, thawed membranes were incubated with iodoacetamide (2 mg/ml) and protease inhibitors for 30 min at 4°C, before diluting 2× with solubilization buffer  and sonicated at room temperature, and soluble materials were removed by centrifugation at 12,000g for 15 min, after which the pellet was again dissolved in lysis buffer and inclusion bodies were collected by centrifugation at 6000g for 15 min. The inclusion bodies were resuspended by dounce homogenization in equilibration buffer  and sonicated once more to extract chemokine. Insoluble material was removed by centrifugation at 6000g for 15 min, and the supernatant was passed over a Ni–nitrilotriacetic acid (NTA) column equilibrated in equilibration buffer. The column was washed with equilibration buffer followed by wash buffer , and the chemokine was eluted with 50 mM acetate (pH 4.0) and 6 M guanidine-HCl. The chemokine elutions were pooled, dithiothreitol was added to 4 mM final concentration, and then the sample was incubated for 10 min at room temperature before adding the solution dropwise into refolding buffer . The refolding solution was incubated with minimal stirring for 4 hours at room temperature and then dialyzed against 20 mM tris (pH 8.0) and 50 mM NaCl with two buffer changes. After dialysis, precipitate was removed by centrifugation at 8000g for 30 min, CaCl2 was added to a final concentration of 2 mM, and the sample was incubated with enterokinase at 37°C for 2 to 4 days to cleave the N-terminal purification tag. To remove uncleaved chemokine and cleaved tag, the sample was applied to a Ni-NTA column equilibrated with 50 mM tris (pH 8.0). The column was washed with 50 mM tris (pH 8.0), and cleaved chemokine was eluted with wash buffer. As a final purification step, the sample was purified on a reversed-phase C18 column equilibrated with 75% buffer A [0.1% trifluoroacetic acid (TFA)] and 25% buffer B  and eluted by a linear gradient of buffer B (33 to 45%). The resulting protein was lyophilized for 48 hours and stored at −80°C until use. The purity of the E. coli expressed chemokine was assessed by SDS–polyacrylamide gel electrophoresis followed by Coomassie staining. As shown in fig. S14, both CXCL12WT and CXCL12LRHQ used for biochemical assays are of high purity. The higher molecular weight band in CXCL12LRHQ corresponds to uncleaved material that was not fully separated by high-performance liquid chromatography. Because this material was only used for the titration in LRHQ was added to both −/+CID25 samples and the conclusion that the Fab has no effect on ACKR3 activation remains valid.CXCL12MSP × 2 + εACKR3/εACKR3Δ332 + εCXCL12(8730 M−1 cm−1) = εND,ACKR3/εND,ACKR3Δ332.Fab selections were performed with CXCL12 in complex with full-length ACKR3 and a C-terminally truncated version. ACKR3·CXCL12 and ACKR3Δ332-CXCL12 complexes were expressed and purified as described above except that DDM/CHS was substituted for LMNG/CHS in the wash and elution buffers. The final samples  were concentrated to 32 and 18 μM for the ACKR3 and ACKR3Δ332 complexes, respectively. The concentration was determined from absorbance at 280 nm using the calculated extinction coefficients of the ACKR3·CXCL12-containing NDs  and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)  were combined in chloroform in a 3:2 POPC:POPG molar ratio, dried to a thin lipid film, and solubilized with ND buffer  containing 200 mM cholate. MSP1E3D1 was expressed and purified using established methods as previously described (The ACKR3Δ332·CXCL12 complex was reconstituted into biotinylated MSP1E3D1 NDs as previously described (A450 higher than 0.2 (three times higher than average background level in this assay) were considered as positive hits, and Fab fragments corresponding to those wells were sequenced. Among 144 clones tested, 21 clones were positive and 5 unique CDR sequences of Fabs were subcloned into a pRH2.2 expression vector . The sequences of the Fabs used in our structure determinations are shown in fig. S15, with their CDRs highlighted in red.The ACKR3·CXCL12 complex was reconstituted into egg PC:POPG (8:2 molar ratio) MSP1D1 biotinylated NDs (E. coli BL21 Gold cells in 2xYT medium supplemented with ampicillin (100 μg/ml) at 37°C until the OD600 reached 0.8 to 0.9. Expression was then induced with 1 mM IPTG for 4 hours at 37°C. Cells were harvested by centrifugation, and pellets were frozen at −80°C until further processing. For Fab purification, cells were resuspended in lysis buffer  using a high-pressure microfluidizer (Avestin) and heated to 60°C to precipitate bacterial proteins and any residual proteolytic degradation fragments of Fabs. The resulting lysate was loaded onto a 5-ml HiTrap MabSelect Sure column  equilibrated with 20 mM sodium phosphate (pH 7.4) and 500 mM NaCl, followed by washing with 10 column volumes of equilibration buffer. Fab fragments were eluted with 0.1 M acetic acid and loaded onto a 1-ml Resource S column , washed with 50 mM sodium acetate (pH 5.0), and eluted with a linear gradient (0 to 100%) of buffer containing 50 mM sodium acetate (pH 5.0) and 2 M NaCl. Fab concentration was determined from the absorbance at 280 nm using extinction coefficients of 91,260 and 89,200 M−1 cm−1 for CID24 and CID25, respectively. Fractions containing pure Fabs were pooled, dialyzed into phosphate-buffered saline (PBS), flash-frozen in liquid nitrogen, and stored at −80°C.Expression of Fabs was carried out as described previously   values were determined from the peak in the first derivative of the traces taken from melting curves analysis using the RotorGene Q software.The effect of Fabs on the thermostability of ACKR3 in complex with CXCL12LRHQ was combined with Fab (CID24 or CID25) in SEC buffer  to final ACKR3 and Fab concentrations of 5.7 and 8.8 μM, respectively. After incubation for 16 hours, samples were loaded onto a Superdex 200 10/300 GL column equilibrated with SEC buffer. Elution of the complexes was followed from absorbance at 280 nm. The top four fractions of the peak corresponding to ACKR3-CXCL12LRHQ-Fab complex were concentrated to 100 μl.For characterization of ACKR3-Fab complexes via SEC, ACKR3·CXCL12WT, ACKR3·CXCL12LRHQ, or ACKR3·CXCL12WT·CCX662  was mixed with Fabs at a molar ratio of 1:1.5 and incubated with anti-FLAG M2 magnetic beads (Sigma-Aldrich) at 4°C for 2 hours. The magnetic beads were then collected with a magnetic rack separator and washed with 20 column volumes of wash buffer  three times. The complex was eluted with one column volume of elution buffer (wash buffer supplemented with 0.1 mg/ml 3× FLAG peptide) three times. For CXCL12-bound complexes, 60 μM CXCL12 was added to the wash and elution buffers. For samples containing the anti-Fab nanobody (2.Purified ACKR3·CXCL12The image processing flowcharts for each dataset are shown in fig. S2. Beam-induced motion was corrected and binned twofold with MotionCor2 in Appion. The motion-corrected micrographs were imported to cryoSPARC (http://swissmodel.expasy.org). These models, along with crystal structures of CXCL12 (PDB entry 6SHR) and the anti-Fab nanobody (PDB entry 6WW2), were rigid body–docked into the cryo-EM map using Chimera and manually adjusted in COOT. The resulting model was refined using phenix.real_space_refine implemented in Phenix  . Briefly, single cysteines were introduced into ACKR3 and CXCL12WT  pfastBac constructs (described above) by QuikChange. Baculoviruses were then prepared for the ACKR3 and CXCL12 cysteine mutants as described above. The proteins were coexpressed by adding both receptor and chemokine baculoviruses, each at an MOI of 6, to 2.5 ml of Sf9 cells at a density of 2.0 × 106/ml in a 24–deep well block. The cells were incubated at 27°C with shaking at 300 rpm for 48 hours, after which 90 μl of cells was transferred to Protein LoBind tubes (Fisher Scientific) and 10 μl of 5 μM CXCL12LRHQ was added to displace any noncovalently bound CXCL12WT. After incubating for 2 hours at room temperature, 10 μl of cells was transferred to a 96-well assay plate and incubated with 10 μl of anti-HA phycoerythrin-conjugated antibody  [diluted 1:100 in tris-buffered saline (TBS) + 4% (w/v) BSA] for 30 min at 4°C. Following incubation, 180 μl of TBS was added to each well and samples were analyzed using a Guava benchtop mini-flow cytometer (EMD Millipore). Data analysis was performed using FlowJo software version 10 . The geometric mean fluorescence intensity (GMFI) for each cross-link was compared to a simultaneous experiment using an established efficient cross-linking construct, ACKR3-R197CECL2·CXCL12-Y7C  glucose. A total of 100,000 cells were aliquoted into each well of a 96-well, white, clear-bottom plate  and buffer was added to a final volume of 90 μl per well. Cells were incubated for 40 min at 37°C, 10 μl of serially diluted CXCL12 was added, and the plate was incubated for 20 min at 37°C. β-Arrestin2 expression was quantified from GFP10 fluorescence at 510 nm (400-nm excitation) on a SpectraMax M5 fluorescent plate reader (Molecular Devices). The bottom of the plate was covered, 5 μM Deep Blue C (coelenterazine-400a) (VWR) was added, and BRET was measured on a Victor X Light multilabel plate reader (PerkinElmer) by dividing GFP10 emission at 515 nm with RlucII emission at 410 nm. Following the experiment, total luminescence was measured to check receptor-RlucII expression.To characterize the Y2571.39A, Y2686.51A, and Q3017.39A, cells were washed with PBS, resuspended in Tyrode’s buffer , and diluted to 1.25 × 106/ml. Cells (80 μl) were aliquoted into a white, clear bottom, tissue culture 96-well plate  and incubated for 45 min at 37°C. The GFP10–β-arrestin2 fluorescence levels were then measured with a SpectraMax 5M fluorescence plate reader . Next, Prolum purple  was added to a final concentration of 5 μM alone or along with CID25. After incubating for 5 min, the total luminescence was measured for all wells using a VictorX Light multilabel plate reader (PerkinElmer) or Tecan Spark (Tecan). Next, CXCL12WT or CXCL12LRHQ was added at the indicated concentrations and the plate was measured at 410 and 515 nm for experiments on VictorX. For experiments using the Tecan Spark, emissions were collected using default BRET2 settings . The BRET ratios (515 nm/410 nm) were calculated for three independent experiments and normalized to WT ACKR3 responses and merged. Titrations were fit to a sigmoidal dose-response model using SigmaPlot 11.0 (Systat Software Inc.). Significance was assessed using the extra sum-of-squares F test.For testing the effects of CID25 and mutations ACKR3-Y51Recruitment to tag-less receptors was observed by enhanced bystander BRET2 between RlucII-tagged β-arrestin2 and plasma membrane–bound rGFP-CAAX as previously described -Gβ1, 500 ng of Venus(1-155)-Gγ2 , and 500 ng of receptor or empty vector DNA, all in pcDNA3.1 expression vectors. After 48 hours, the cells were washed and replated as above and allowed to re-adhere for 45 min at 37°C. After the Venus fluorescence was measured using a SpectraMax plate reader (same setup as the β-arrestin 2 assay) and the white tape was applied, 10 μM coelenterazine h (VWR) was added to each well and the initial basal BRET1 values were measured using a Tecan Spark luminescence reader (Tecan) using default BRET1 settings  for 5 min. Next, chemokine or small molecules were added at final concentrations of 100 nM and 1 μM, respectively, and the BRET1 signal was tracked for an additional 10 min. Experiments were conducted three times in triplicate, and representative results are shown. Significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test.Activation of the heterotrimeric G proteins was observed with a BRET1 assay as previously described  3:2 molar ratio NDs as described above except that the step where Talon resin was added to remove empty NDs was omitted. NDs (200 fmol empty NDs or NDs containing ACKR3), Fabs , streptavidin-coated polyvinyltoluene scintillation proximity assay beads , and buffer  were added to a white 96-well low-binding surface OptiPlate (PerkinElmer) in a total volume of 89 μl. The plate was shaken for 30 min at room temperature to allow immobilization of the NDs onto the beads. 125I-CXCL12  was diluted in 120 mM NaCl, 60 mM Hepes (pH 7.5), and 0.2% (w/v) BSA. Radioligand (10 μl) was added to the plate at a final concentration of 61 pM, and association was measured on a TopCount NXT device (PerkinElmer) in 1-min reads at 20°C. After reaching equilibrium, dissociation of 125I-CXCL12 was initiated by addition of 1 μl of 10 mM  ACKR3 agonist VUF11207 (ACKR3 was reconstituted into biotinylated MSP1E3D1 POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-


The synthesis also varies the content of potassium nitrate deposited on superfine iron oxide with a diameter of 3 nm (Nanocat®) to reach atomic ratios of 100 Fe:x K (x = 0–5). The structure, composition, and properties of the synthesized materials are investigated by X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, Fourier-transform infrared, Raman spectroscopy, inductively coupled plasma-atomic emission spectroscopy, and X-ray photoelectron spectroscopy, as well as transmission electron microscopy, with energy-dispersive X-ray spectroscopy and selected area electron diffraction. The hematite phase of iron oxide retains its structure up to 700 °C without forming any new mixed phase. For compositions as high as 100 Fe:5 K, potassium nitrate remains stable up to 400 °C, but at 500 °C, it starts to decompose into nitrites and, at only 800 °C, it completely decomposes to potassium oxide (K2O) and a mixed phase, K2Fe22O34. The doping of potassium nitrate on the surface of α-Fe2O3 provides a new material with potential applications in Fisher–Tropsch catalysis, photocatalysis, and photoelectrochemical processes.Potassium is used extensively as a promoter with iron catalysts in Fisher–Tropsch synthesis, water–gas shift reactions, steam reforming, and alcohol synthesis. In this paper, the identification of potassium chemical states on the surface of iron catalysts is studied to improve our understanding of the catalytic system. Herein, potassium-doped iron oxide (α-Fe Potassium hydroxide (KOH) , or pota on iron , iron-ma on iron , iron-co on iron , and red on iron . This K2obutanol . Alternaynthesis .3) has been calcined with gold-titanium-silicate [2) into propanol and long-chain hydrocarbons, respectively. For applications in Fisher–Tropsch synthesis, KNO3 has been calcined in the range of 300–500 °C with iron-based catalysts [2, the in situ generation of the K2O promoter was attempted over copper catalysts from KNO3, calcined at 400 °C [3 precursor to dope catalysts with small concentrations of K2O has been assumed to proceed at specific calcination temperatures, although typical instrumental limitations may have prevented the verification of the chemical and electronic states of interest.Interestingly, potassium nitrate  can change with increasing temperature. For example, at each temperature increment, the decomposition products that are generated can vary significantly, affecting the catalytic behavior of the material. Therefore, in this work, we perform a systematic study to optimize the calcination temperature of KNO3 (as the only precursor) on superfine iron oxide with a diameter of 3 nm (Nanocat®) and monitor the decomposition products. The surface-loaded potassium–iron oxides are synthesized by IWIM, with atomic ratios of 100 Fe:x K . In other words, we study the chemical and electronic states generated from variable amounts of KNO3 precursor with a fixed iron content. The most highly potassium-doped sample, 100 Fe:5 K, was selected to characterize its calcination in air from 400 °C to 800 °C, to understand how the decomposition proceeds.An additional, almost unnoticed problem associated with calcination when it is used to create catalysts with low loadings of the potassium precursor is that the decomposition behavior of the precursor  or electronic (K2O) promoters [Overall, evaluating the influence of the promoter effects  in the romoters ,38,39) c® superfine iron oxide  of hematite phase (α-Fe2O3) was dried at 110 °C for 3 h in an oven . The raw material has a Brunauer–Emmett–Teller (BET) surface area of 244.24 m2 g–1, a particle size of 3 nm, and a bulk density of 0.05 g cm–3. For the potassium precursor, KNO3 was obtained commercially . Both Nanocat® and KNO3 were used to obtain a series of α-Fe2O3 materials loaded with potassium, following the IWIM. For the syntheses, 15.0 mL solutions of KNO3 were prepared in deionized water at concentrations of 0.0213, 0.0426, 0.0852, 0.1277, and 0.2130 M. Each solution was added slowly to 5.0 g of α-Fe2O3 placed in a rotating 250.0 mL round bottom flask using a customized West-type condenser provided with a polytetrafluoroethylene stopcock for dropwise addition. Rotation of the flask at a rate of 20 rpm was provided with a rotavapor . The resulting materials were dried at 110 °C overnight in porcelain crucibles. These dried samples were then calcined in a muffle furnace  at 400 °C for 5 h in air. By varying the amount of KNO3 loaded, materials with atomic ratio 100 Fe:x K  were obtained. In addition, the 100 Fe:5 K material was further calcined in air for 5 h at 500 °C, 600 °C, 700 °C, and 800 °C.Nanocat3, Acros Organics, Morris Plane, NJ, USA, ACS reagent, 65%) was added. The vessels were then closed and placed into a microwave digestion system , where the digestion was carried out with a power of 1.3 kW using a temperature ramp reaching 200 °C in 25 min, which was held at 200 °C for an extra 10 min. The digested samples were then allowed to cool for at least 3 h before opening the vessels. After cooling, the digested sample was augmented to a final volume of 10.0 mL with 10% nitric acid, from which 1.0 mL aliquots were diluted 10 times with 10% nitric acid to prepare stock solutions for potassium analysis. A further 1000-times dilution of the stock solution in 10% HNO3 was used for the iron analysis.Samples of 0.2500 g were weighed accurately into a Teflon MARSXpress vessel and 10.0 mL of nitric acid (HNO–1) were used to create calibration curves with solutions of concentration at ½, 1, 5, 10, and 50 ppm in 10% HNO3. Five parts per million (ppm) standards were used as check standards, and 1 ppm yttrium in 10% HNO3 served as an internal standard . Samples were analyzed in triplicate. The operating conditions of the ICP-OES instrument were as follows: 1.20 kW radio-frequency power, 15.0 L min–1 plasma flow rate, 1.50 L min–1 auxiliary flow rate, 3 mL min–1 sample uptake, 35 s read delay, and 0.9 L min–1 nebulizer gas flow rate. The analysis of potassium and iron in the previous samples was performed by inductively coupled plasma optical emission spectroscopy . The analysis for iron considers an average for emission maxima at 234.35, 238.20, 239.56, and 259.94 nm, while for potassium, the maximum at 766.49 nm is used. Multi-elemental standards of iron and potassium  patterns of the samples were obtained with B8 Advance Bruker AXS diffractometer with Cu Kα incident radiation (λ = 0.15418 nm). XRD data were recorded from 10° to 90°, at a scan rate of 1° min–1 and a heating rate of 10 °C min–1 from 30 to 800 °C.A TA Instruments Q5000 was used for the thermogravimetric analysis (TGA) of samples heated under a nitrogen atmosphere, at a flow rate of 25 mL minThe X-ray photoelectron spectroscopy (XPS) surface analysis was recorded with a pass energy of 50 eV at a step size of 0.1 eV and X-ray beam size of 400 μm, using a Thermo Scientific spectrometer (K-Alpha) with an Al Kα anode  excitation source.4 laser was used as the excitation source (532 nm), with 2 mW power and a 50 μm aperture slit for Raman characterization.The Raman spectra (200 scans) were recorded using a DXR Raman microscope (Thermo Scientific). A diode-pumped Nd:YVOTransmission electron microscopy (TEM) analysis was carried out using a Talos F200X instrument (Thermo Scientific) operated at an acceleration voltage of 200 kV. Images were recorded with Ceta 16M camera and Velox software (FEI). Selected-area electron diffraction patterns (SAED) and energy-dispersive X-ray spectroscopy (EDS) with four silicon drift detectors were also investigated from the electron micrographs. Before analysis, the as-prepared particles were dried in an oven for 24 h at 120 °C. The dry particles were then suspended in ethanol and sonicated for 30 min prior to being dropped onto a TEM grid with a lacy support film, which was allowed to air-dry for about 6 h.2O3 and the materials doped with increasing potassium content under a calcination temperature of 400 °C. The diffraction peaks of bare α-Fe2O3 (100 Fe:0 K) centered at 24.2°, 33.1°, 35.6°, 54.1°, 62.5°, and 63.8° are labeled, respectively, as (012), (104), (110), (116), (214), and (300) on the reflections at the bottom of 2O3 at 2θ values of 24.2°, 33.1°, 35.6°, 40.8°, 49.5°, 54.1°, 62.5°, 63.8°, 69.8°, 72.1°, 75.6°, 77.9°, 80.9°, 83.2°, 85.2°, and 88.7°, corresponding to diffraction from the planes (012), (104), (110), (113), (024), (116), (112), (214), (300), (208), (1010), (217), (036), (128), (0210), (134), and (226).3 or KNO2, either because these species are well-dispersed over the surface and pores of α-Fe2O3 without forming detectable aggregates, or because very low concentrations of these species remain after IWIM. Similarly, the negative detection of K2O or any other mixed oxide phase(s)  and (2) red crosses for the hexagonal phase of K2Fe22O34, (JCPDS #031-1034), respectively. A close-up of the top panel of 2O and K2Fe22O34.phase(s) , even fo3) and its decomposition products, potassium nitrite (KNO2), and potassium superoxides,  were not observed in 2O can also absorb water and form potassium hydroxide [Potassium nitrate  of the K-doped α-Fe2O3 can be interpreted by applying Scherrer’s equation:k = 0.9 is the shape factor, λ = 0.15418 nm is the X-ray wavelength, β is the full width at half-maximum for the (104) reflection in radians, and θ is the Bragg angle. 2O3 (100 Fe:0 K) increases to 16.1 nm for the 100 Fe:½ K material. For the further addition of potassium, the crystallite size remains almost the same at a constant calcination temperature of 400 °C. However, increasing calcination temperature leads to significant growth, up to a maximum of 36.6 nm at 800 °C.The effects of KNOhkld (Equation (2), for the Miller indices, hkl) [n is a positive integer and λ is the incident wavelength. Solving the equations for 110d and 104d for the planes (110) and (104) produces the lattice parameters of a = b, and c, which are reported in a and c for undoped and K-doped α-Fe2O3 materials, calcined at different temperatures  , with Breratures .2O3 lattice. This is also evident from the larger cation size of K+ (138 pm) than Fe3+ (65 pm) [+ ions form K2O and a mixed phase of K-Fe oxides, K2Fe22O34.Therefore, the crystallographic structure of hematite is largely preserved under the potassium loadings and calcination temperatures explored, indicating that potassium is not incorporated into the bulk defects of the α-Fe (65 pm) . Instead2O3, with an appreciably strong thermal stability of up to 800 °C.The TGA and first derivatives of the thermogram curves of the 100 Fe:5 K material at various calcination temperatures are displayed in 3 to KNO2 (peak B at 624.5 °C) and KNO2 to K2O (peak A at 568.9 °C) [2 can also recombine with gaseous products (NO2 and NO) to reform KNO3 [Two weight loss regions are identified in 68.9 °C) ,44,45. Torm KNO3 .3 is known to be stable as a liquid (m.p. 334 °C) at least to 530 °C, the presence of a basic metal oxide, such as ZrO2, was shown to drastically reduce its decomposition temperature, e.g., to 340 °C [3(l) ⇌ KNO2(l) + ½ O2(g) [2O and NOx (NO + NO2), grows [While pure KNOo 340 °C , which i ½ O2(g) . Thus, a), grows , to then2 is still present. The complete decomposition of KNO3 and KNO2 at 800 °C is noticeable in x K materials in the range of 0 ≤ x ≤ 5 is reported in the The assignment of peaks A and B in The results from the ICP-OES analyses, reporting the elemental composition of the materials, are presented in x K materials for increasing potassium content, calcined at 400 °C. Among these eight peaks for the undoped material, there are two A1g modes centered at 219.3 cm–1 (peak 1) and 487.2 cm–1 (peak 5), four Eg modes at 244.6 cm–1 (peak 2), 281.3 cm–1 (peak 3), 400.0 cm–1 (peak 4), and 608.2 cm–1 (peak 6), one mode for the forbidden Eu mode, showing the presence of disorder in the hematite lattice at 661.8 cm–1 (peak 7), and one mode for the two-magnon scattering band centered at 1302.5 cm–1 (peak 8) [–1.The Raman spectra of the undoped material in (peak 8) ,50,51,522O3. 3 in the doped materials, as evidenced by the Raman mode at 1055 cm–1 (peak 9) from the symmetric stretching vibration of 2O or K2Fe22O34 could be observed by Raman spectroscopy due to the limitations of the technique, which are overcome by surface-sensitive XPS analysis.These shifts could be due to the phonon confinement effects from the nanoscale size of undoped hematite particles with a size of 3.0 nm . A changing chemical bonding effect by the potassium doping is excluded, as no shifts are registered for increasing potassium loadings. The previous observation agrees with the XRD data in  of NO3− . Peak 9 2O3 after calcination at various temperatures is provided by XPS analysis. Qualitative elemental analysis is possible due to our analyzing the surveyed and high-resolution XPS spectra, which yield the electronic state and chemical bonding of the elements. For example, the XPS survey spectra of 100 Fe:x K for x = 0, 1, and 5 are shown in 2O3 only shows oxygen and iron , the doped materials  (denoted as Fe–O and labeled as peak 1) in α-Fe2O3, lattice hydroxyl (peak 2), and surface hydroxyl (peak 3), respectively.The surface composition of undoped and K-doped α-Fes XPS spectra do not show any new features of oxygen related to a possible K–O bond. The high-resolution XPS spectra of Fe 2p (right panel in p3/2 (peak 4) and Fe 2p1/2 (peak 6), respectively. The doublet (peaks 4 and 6) arises due to the spin-orbit coupling in the Fe 2p states. There are also two additional satellite peaks at binding energies of 718.0 eV and 732.1 eV, assigned to Fe 2p3/2 (peak 5) and Fe 2p1/2 (peak 7), respectively. These features of Fe 2p are in good agreement with the Fe(III) states reported for α-Fe2O3 (hematite) [p states was discarded, as there are neither spin-orbit splitting nor satellite peaks for Fe(II) states in both doped and undoped materials. The binding energies of peaks 1–7 are not shifted in the K-doped materials, discarding the incorporation of potassium into the lattice of α-Fe2O3.With K-doping, the O 1ematite) ,54,55. Tp3/2 (peak 8) and K 2p1/2 (peak 9), respectively. The binding energy difference of 2.7 eV for peaks 8 and 9 is due to the spin-orbit coupling of potassium cations in potassium nitrates [p has been observed in these spectra, which indicates that the added potassium is not interacting chemically with α-Fe2O3. The presence of nitrate, remaining at this low calcination temperature, is also confirmed in the high-resolution XPS spectra for nitrogen  and N 1s  signals at various calcination temperatures. Peak 10 is observed at 400, 500, and 600 °C. In addition, a peak at 402.8 eV for the nitrite (peak 11) [p neither show a shift with calcination temperature nor demonstrate the presence of new peaks in the high-resolution XPS spectra   is clear spectra . Thus, toss in p  may be ooss in p . Therefoectively . The O 12O3 are shown in 2O3, the average diameter of the particles is about 2.56 nm.The size, morphology, and microstructure of the materials are characterized using TEM and high-resolution TEM (HR-TEM). Individual hexagonal particles of undoped and K-doped α-Fed012 of α-Fe2O3 within ±0.002 nm for the materials with or without potassium . In addition, the interplanar spacings of other major diffraction peaks remain unchanged for both undoped and doped materials  line. TEM images for 100 Fe:5 K material calcined at various temperatures are displayed in The compositional analysis of iron and potassium using ICP-OES matches the nominal content. However, the EDS analysis shows a significant deviation from the nominal values. As described earlier in XRD and Raman analysis, potassium ion is not incorporated into the iron oxide lattice due to the larger size of K content ,62 than  content . Figure 2O3.At the macroscopic level, the increase in particle size can be ascribed to the reduction in total surface energy caused by the calcination temperature. The calcined 100 Fe:5 K material retains the hematite phase up to 700 °C (see the XRD and Raman spectra above) without forming any new phase, as shown in the SAED patterns, for example at 600 °C in 2O and K2Fe22O34. The transformation of KNO3 into KNO2 starts at 500 °C, and these species completely decompose at 800 °C. The current study verifies the finding that the remaining potassium produces K2O at 700 and 800 °C. This work confirms the importance of studying the chemical and electronic states of alkali metals such as potassium dopants in the host materials before using them as catalysts. We recommend the 100 Fe:5 K material calcined at 800 °C for catalytic applications of K2O/α-Fe2O3. Future work with potassium nitrate-doped iron oxide in the catalysis and photocatalysis fields can potentially improve multiple processes, if explored carefully, by ensuring the state of potassium species.The hematite phase of the materials remains stable upon the addition of potassium nitrate for iron oxide, with atomic compositions as high as 100 Fe:5 K up to 700 °C. However, XRD confirms that at 800 °C, two new phases are formed, K


H-carbazol-9-yl)ethyl]phosphonic acid monolayeras a hole-selective contact.A triple-cation perovskite is used as the absorber layer. Thermallyevaporated C60 and atomic layer deposited SnO2 layers serve to create an electron-selective contact. The cellsuse an ITO top electrode with an antireflective MgF2 coating.The optimized cell fabricated on a polymer-coated steel substratereaches a power conversion efficiency of 16.5%, which approaches the18.4% efficiency of a p–i–n reference superstrate-configurationcell that uses a similar stack design. Optical simulations suggestthat the remaining optical losses are due to the absorption of lightby the ITO top electrode, the C60 layer, the Ti bottomelectrode, and reflection from the MgF2 coating in almostequal amounts. The major loss is, however, in the fill factor as aresult of an increased sheet resistance of the top ITO electrode.An efficient substrate-configurationp–i–n metal-halideperovskite solar cell (PSC) is fabricated on a polymer-coated steelsubstrate. The optimized cell employs a Ti bottom electrode coatedwith a thin indium tin oxide (ITO) interlayer covered with a self-assembled[2-(9 These industrial buildings often employ coated steel as thebuilding skin. Hence, it is of interest to consider steel as a substratefor fabricating photovoltaic cells. Because of their low cost, lightweight, high efficiency, and compatibility with a variety of substrates,15 The highest efficiency reported for such substrate p–i–nPSCs is 12.8%, employing a Cu foil bottom electrode and a Ag nanowiretransparent top electrode.11 Interestingly,the p–i–n substrate-configuration is now commonly inuse for wide-bandgap PSCs as part of two-terminal monolithic tandemsolar cells with a crystalline Si bottom cell.28 Moreover, for p–i–nPSCs, several charge-selective contacts are known that provide lowresistive losses and excellent energetic alignment with the perovskiteactive layer.31 A recent study demonstrated self-assembled monolayers(SAMs) consisting of [2-(9H-carbazol-9-yl)ethyl]phosphonicacid (2PACz) on indium tin oxide (ITO) as the hole-selective contactin combination with a thermally evaporated C60 layer asthe electron-selective contact.31Most PSCs fabricated on metalfoils use a n–i–p cellarchitecture and reports for p–i–n stacks are scarce.20 and excellent chemical resistance. Ni-plated steel substratesare commonly used in batteries.32 The planarizationlayer serves as an insulating layer. An opaque titanium electrodecovered with a thin sputtered ITO layer to enable binding of the phosphonicacid anchoring groups of the 2PACz monolayer serves as a hole-collectingelectrode. For electron collection, we use a C60 layercovered with a SnO2 buffer layer that prevents damage tothe underlying stack during sputter deposition of the transparentITO top electrode.34 Optimized devices on steel substratesreach an efficiency of 16.5%. While the cell outperforms the presentrecord performance for p–i–n and n–i–pcells on opaque substrates, the efficiency is still lower than the18.4% efficiency obtained for the corresponding superstrate p–i–nsolar cell. Optical modeling is used to analyze parasitic opticallosses.Herein, we demonstrate an efficient substrate-configuration p–i–nPSC on steel coated with a polyamide-imide (PAI) planarization layer.We chose to use a Ni-plated steel substrate because it is relativelysmooth and presents moderate macroscopic roughness22.1–1. The solution was sonicated for 30 min beforedeposition. For the active layer, a triple-cation Cs0.05(MA0.17FA0.83)0.95Pb(I0.83Br0.17)3 perovskite was used as described bySaliba et al.36 PbI2 (576 mg) and PbBr2 (550.5mg)  were dissolved separatelyin a mixture of N,N-dimethylformamide and dimethyl sulfoxide . Then, 0.936 mLof the PbI2 solution was added to FAI (200 mg) (GreatcellSolar), and 0.702 mL of the PbBr2 solution was added toMABr (99.7 mg) (Greatcell Solar). Finally, 0.833 mL of the PbI2-FAI solution, 0.167 mL of the PbBr2-MABr, and50 μL of CsI  of a stock solution of389.7 mg mL–1 in DMSO  were mixed. As the electron-transport layer (ETL), a combinationof C60  and a spatial atomic layerdeposition (ALD) SnO2 layer or bathocuproine (BCP)  was used. The ITO sputter target (purity 99.95%) for top electrodeswas purchased from Angstrom Engineering. As the antireflective coating,MgF2  was used.All materials andreagents were purchased from commercial sources.Solutions were stirred at 60 °C overnight before the spin coating,unless stated otherwise. 2PACz  was dissolvedin absolute ethanol  at a concentration of 0.33mg mL2.2–7 mbar. Pre-patterned ITO (180 nm) glasssubstrates (Naranjo Substrates) were cleaned by sonication in acetone(15 min), scrubbing and sonication in sodium dodecyl sulfate solution in water (10 min), rinsing in deionized water, and sonicationin 2-propanol (15 min). Prior to device preparation, the glass substrateswere blow-dried with nitrogen and activated by UV-ozone treatment(30 min). Ni-plated battery steel  substrateswere cleaned in 2-propanol and blow-dried with N2. On thesteel substrate, a wire bar-coated PAI  planarizationlayer was used. The PAI film was cured in air at 265 °C for 15min and cut to 3 × 3 cm2 samples for further use.Prior to the bottom electrode deposition, the samples were sonicatedin 2-propanol for 15 min and blow-dried with N2. The solarcell fabrication on the planarized steel substrates was identicalto the fabrication of glass. For substrate-configuration PSCs, a 200nm patterned Ti bottom electrode was deposited (2 Å s–1) onto the glass/ITO and PAI-coated steel substrates via electron-beamdeposition. A 10 nm patterned ITO interlayer was deposited (∼0.3Å s–1) onto the Ti bottom electrode via magnetronsputtering under an Ar/O2 flow.All thermallyevaporated films were deposited under high-vacuum conditions at ∼5× 10–1 acceleration) for 30 s and heat-treatedat 100 °C for 10 min.The 2PACz solutionwas statically spin-coated onto the ITO interlayer at 3000 rpm 0.95Pb(I0.83Br0.17)3 perovskite film(∼520 nm thick) was processed using a ramped spin-coating deposition.The perovskite precursor solution was deposited statically onto thehole-transport layer (HTL) at 4000 rpm (800 rpm s–1) for 35 s. 10 s prior to the end of the spin-coating program, 300μL of anhydrous ethyl acetate  was deposited.The perovskite film was then annealed in a glovebox at 100 °Cfor 60 min and cooled to room temperature.The Cs60 layer was deposited (2 Å s–1) as the ETLon the perovskite films via thermal evaporation. Forsubstrate-configuration cells, a 45 nm thick SnO2 was grownon the C60 layer by spatial ALD as described elsewhere.35 Tetrakis(dimethylamino)tin(IV) was the Sn sourceand H2O was used as the coreactant. Both vessels were keptat room temperature while flowing 500 sccm of Ar through them. Processingwas done at 100 °C with a nominal growth of 0.125 nm/cycle, determinedon a silicon wafer. After SnO2 deposition, the sampleswere transferred into a N2-filled glovebox for ITO sputtering.The ITO top electrode (180 nm) was deposited (∼0.3 Å s–1) using radio frequency sputtering under Ar/O2 flow. The antireflective MgF2 coating (90 nm)was deposited (2 Å s–1) via thermal evaporation.The superstrate-configuration cell of the device was finalized bya BCP (8 nm) layer and a 100 nm Ag top electrode which were deposited(2 Å s–1 both) via thermal evaporation. Theactive area (0.09 cm2 or 0.16 cm2) of the cellswas determined by the overlap of the ITO or Ti bottom electrode andthe transparent ITO or Ag top electrode.A 20 nm C2.3J–V) characteristicswere measured using a Keithley 2400 source meter. During the J–V measurements, light from a tungsten-halogen lampwas filtered using a Schott GG385 UV filter and a Hoya LB120 daylightfilter to mimic the AM1.5G spectrum (100 mW cm–2). For ITO/MgF2 side and glass ITO side illuminated solarcells, a black shadow mask with an aperture area of 0.0676 or 0.1296cm2 was employed to define the illuminated cell area. Duringthe fast J–V sweep measurements, the sourcemeter swept the voltage either from +1.5 to −0.5 V (reversescan) or from −0.5 to +1.5 V (forward scan) at a scan rateof 0.25 V s–1. Light soaking preconditioning ofthe solar cells was performed by exposing the cell area to continuousillumination of simulated AM1.5G (100 mW cm–2) lightfor a given time, followed by a fast sweep measurement. For the stabilized J–V measurement (slow sweep measurements), the open-circuitvoltage (Voc) of the solar cell was firsttracked for 5 min under constant illumination. Then, a reverse sweepfrom Voc +0.04 V to −0.04 V wasperformed with a step size of 0.04 V in which the current densitywas measured at each voltage step after a stabilization time of 5s.All sampleswere stored and measured in a nitrogen-filled glovebox without anyfurther exposure to air or any preconditioning, unless stated otherwise.The current density–voltage , which was modulatedwith a mechanical chopper  before passingthrough a monochromator . The spectral responseof the device was recorded as a voltage from a pre-amplifier  using a lock-in amplifier  and was calibrated by a reference silicon cell. To accuratelydetermine the short-circuit current density , a green LED  was utilized as a light bias during the EQE measurementto provide the solar cell with approximately 1 sun equivalent illuminationintensity.External quantum efficiency (EQE) measurements were performedin a N2.4n) and extinction coefficient (k) used for the differentmaterials are shown in the Supporting Information.Optical simulationswere performed using the transfer-matrix method with Setfos 5.0 (FluximAG). The wavelength-dependent refractive index (3Rp), determined with surfaceprofilometry on a 2 × 2 mm2 surface area, of the PAI-coatedsteel substrates is 510 nm. Locally, the surface is smoother and Rp is 10 nm when measured with atomic force microscopyon a 5 × 5 μm2 area. Electron-beam evaporationwas used to deposit a Ti (200 nm) bottom electrode. The Ti electrodewas covered with a magnetron-sputtered ITO (10 nm) interlayer thatprovides a surface capable of binding the phosphonic acid groups of2PACz to create a hole-selective contact between the perovskite absorberand the Ti electrode. The 2PACz monolayer (∼1 nm) forms a conformalcharge-selective dipole layer on the ITO interlayer with negligibleresistive losses.31 A triple-cation perovskite(Cs0.05(MA0.17FA0.83)0.95Pb(I0.83Br0.17)3, where MA is methylammoniumand FA is formamidinium) (520 nm), was deposited from a precursorsolution in a 4:1 (v/v) mixture of DMF and DMSO using ethyl acetateas the antisolvent to induce crystallization, followed by thermalannealing.36 Thermally evaporated C60 (20 nm) served as the ETL. To enable top illumination ofthe substrate-configuration cells, a transparent ITO/MgF2 top contact was used. ITO (180 nm) was deposited by magnetron sputteringand MgF2 (90 nm) by thermal evaporation. To protect theperovskite and C60 layers during magnetron sputtering,a SnO2 (45 nm) layer was deposited on top of C60 via ALD. The yield of working cells suffered from a suboptimal surfacewetting of the perovskite precursor solution on the HTL. This alsoresulted in variations in J–V characteristicsand thus in the PCEs. An extra UV-ozone surface treatment of the ITOinterlayer before applying the 2PACz HTL improved the quality of theperovskite film. Cells that were not affected by suboptimal film formationperformed well. For comparison, a conventional superstrate cell onglass covered with ITO (170 nm) was made in a similar stack designbut employing a thermally evaporated opaque top contact consistingof BCP (8 nm) and Ag (100 nm)  and indicate that the perovskitelayers formed on ITO/2PACz do not strongly depend on the choice ofthe substrate underneath .To investigatethe effect of the substrate on the performance,we built cells on glass and steel substrates ((100 nm) 1, cell CVoc) of 1.11 V,a short-circuit current density (Jsc)of 19.8 mA cm–2, and a fill factor (FF) of 0.72, however, provides a significantly higherPCE of 18.4%, mainly because of an improved Jsc (20.8 mA cm–2) and higher FF (0.81). Devicestatistics are shown in Figure S2 (SupportingInformation).The substrate cell on glass (cell A) reaches 15.8% PCE with anopen-circuit voltage  and efficiency (PCEEQE) that are withina small margin from the values obtained from the J–V characteristics  in the superstrate-configuration cell compared to the ITO top electrode(∼45 Ω sq–1) in the substrate-configurationcells. The principal reason for this difference is that thermal annealingat 350–550 °C in air, as commonly used to increase theconductivity of sputtered ITO on glass,38 is not compatiblewith the perovskite absorber and the organic charge transport layers.A noticeable difference between the two configurationsis the higherFF for the superstrate cell (0.81) than for the substrate cells (0.72–0.75).The difference is, at least in part, caused by the lower sheet resistanceof the ITO bottom electrode (∼16 Ω sqVoc of the substrate cells is slightlylower than for the superstrate cell (1.11 vs 1.13 V). Voc as a function of photon flux for both cells in a semilogarithmicplot. The ideality factor n = 2.10 for the substratecell is higher than n = 1.69 for the superstratecell and suggests that charge recombination dynamics at open circuitare dominated by trap-assisted recombination. The steep decrease of Voc observed for the substrate cell at the lowestphoton flux (<1015 cm–2 s–1) is ascribed to leakage current, originating from shunts betweenthe top and bottom electrodes via pinholes in the active layer. Boththe suboptimal wetting of the precursor solution and rough Ti/ITObottom electrode compared to a smooth glass/ITO electrode contributethis difference.The Jsc between the substrateand superstrate cells we modeled the distributionof light of the device stack using the transfer-matrix formalism,using the thickness and the wavelength-dependent refractive indexand extinction coefficient of all layers as input parameters . Inthe modeling, we omitted the ∼1 nm 2PACz monolayer becauseit has a negligible effect. –2 s–1 nm–1] as a function of wavelength.To better understandthe difference in q and integrating over a fixed wavelength regime. The current lossesas a consequence of optical losses of the substrate-configurationcell add up to 4.4 mA cm–2, which is more than the3.4 mA cm–2 for the superstrate reference cell (–2) and parasitic absorptionby the ITO top electrode (1.0 mA cm–2), the C60 layer (1.3 mA cm–2), and the Ti bottomelectrode (1.2 mA cm–2). The main optical lossesof the superstrate-configuration cell are due to reflection (2.3 mAcm–2) and absorption by the ITO bottom electrode(0.8 mA cm–2). We note that the reflection lossof the superstrate cell can be reduced to 1.6 mA cm–2 when depositing a ∼100 nm antireflective MgF2 coatingon the glass substrate. The resulting total optical loss in that casewould be reduced to 2.8 mA cm–2.The contributions to the photocurrentand the losses can be expressedin current densities by taking as the product of reflected or absorbedphoton flux (Φ) with the elementary charge nce cell 2. The ma–2, compared to 21.7 mA cm–2 for the superstrate cell  monolayer serves as the ETL, while the HTLis composed of a thin thermally evaporated layer of TCTA (tris(4-carbazoyl-9-ylphenyl)amine)(10 nm) covered with MoO3 (15 nm). The TCTA/MoO3 HTL causes less parasitic absorption and provides marginally higher Jsc (20.2 vs 19.9 mA cm–2).Also, the Voc is somewhat higher (1.15V vs 1.11 V), possibly because the SnO2/PCBA layer providesa better energetic alignment with the perovskite than C60. The main difference is in the FF which is significantly lower forthe n–i–p cell (FF = 0.64 vs 0.75). When comparing theFFs for n–i–p and p–i–n substrate cellson smooth glass instead of rough steel, the difference is much less(0.70 vs 0.72). This suggests that the origin for the difference inFF on steel is not primarily related to different HTLs and ETLs butrather due to a different sensitivity to surface roughness. This seemsto be higher for the n–i–p cell than for the p–i–ncell. In accordance, the FF of the n–i–p cells is furtherreduced to 0.60 when increasing the roughness of the steel substrate,12 while there is no loss in FF for the p–i–ncell when going from smooth glass to rougher steel  is only slightlyhigher than that of a standard superstrate p–i–n referencecell (3.3 mA cm–2). The difference is mainly dueto increased parasitic absorption by the C60 ETL and theTi bottom electrode. Strategies to further increase the PCE of substrate-configurationcells can therefore focus on reducing the optical losses in the ETL,enhancing the reflection of the bottom electrode, and using a lessresistive transparent top contact.In summary, an efficient p–i–nPSC has been fabricatedon a steel substrate coated with a polymer planarization layer incombination with a transparent top electrode. The substrate-configurationcell was fabricated on a Ti bottom electrode covered with a thin ITOinterlayer to enable binding of 2PACz as a self-assembled hole-selectivemonolayer. Although the processing of these cells must be improvedto increase the yield of efficient devices, the best cell made ona polymer-coated Ni-plated battery steel substrate achieved a PCEof 16.5%. Compared to the corresponding superstrate p–i–ncell on glass with a PCE of 18.4%, the main loss is in the FF (0.75compared to 0.81) due to the high sheet resistance of the ITO topelectrode. Optical simulations reveal that the total optical lossof the substrate cell (4.4 mA cm


Nepovirus. In this work, BRV transmission in the system Ribes ex vitro–Ribes in vitro was applied for the first time. Triple infection of BRV identified in blackcurrant cv. Gojai was used for phylogenetic analysis and inoculation assay. Transmission of BRV was successful due to its stability in the inoculum for up to 8 days at 4 °C; all BRV isolates were infectious. Our suggested inoculation method through roots was applied in six Ribes spp. genotypes with 100.0% reliability, and the expression levels of defence-related gene PR1 to biotic stress was observed. The prevalence of the virus in microshoots after 2–14 days post-inoculation (dpi) was established by PCR. In resistant genotypes, the BRV was identified up to 8 dpi; meanwhile, infection remained constant in susceptible genotypes. We established that BRV transmission under controlled conditions depends on the inoculum quality, post-inoculation cultivation temperature, and host-plant susceptibility to pathogen. This in vitro inoculation method opens possibilities to reveal the resistance mechanisms or response pathways to BRV and can be used for the selection of resistant Ribes spp. in breeding programs.Blackcurrant reversion virus (BRV) is the most destructive currant-infecting and mite-transmitted pathogen from the genus  Cecidophyopsis spp. (Acari: Eriophyidae). The virus is the causative agent of blackcurrant reversion disease (BRD). The pathogen (BRV), vector (C. ribis), and disease (BRD) complex causes significant yield losses in blackcurrant plantations worldwide . Mi. Mi40]. 2SO3), 0.08% 2-mercaptoethanol, and 0.2% Tween 20. The inoculum was filtered through 0.22 µm pore-size sterile filters for microorganism contamination.The inoculum was prepared from 3.0 g of ground symptomatic blackcurrant cv. Gojai leaves and 10.0 mL of 0.1 M phosphate buffered saline (PBS) buffer (pH 7.5) enriched with 2.0% polyvinylpyrrolidone (PVP), 0.2% sodium sulphite . Virus RNA was evaluated by the PCR method using 3 primer pairs from different sites of the RNA2 3′ NTR regions in the BRV genome .20 primer was used in the reaction.Total RNA was isolated from 100.0 µL of inoculum or 0.1 g fresh tissue of microshoots. Plant samples were homogenized in liquid nitrogen. For extraction of total RNA, we used a GeneJET Plant RNA Purification Mini Kit  according to manufacturer’s protocol. Total RNA was used directly as a template for cDNA synthesis with a Maxima H Minus First Strand cDNA Synthesis Kit , and oligo d(T)Ribes spp. microshoots after mechanical inoculation in vitro. BRV-specific oligonucleotide primers and their characteristics are presented in 2O, 2.5 µL 10× Taq buffer + (NH4)2SO4 + MgCl2, 2.0 µL 25 mM MgCl2, 2.0 µL 2 mM dNTP mix, 0.1 µL of each 0.1 µM forward and reverse primers, 0.2 µL Taq DNA polymerase , and 2.0 µL cDNA (about 100 ng). The amplification reaction was performed in a Mastercycler X50a  under the following conditions: 95 °C for 3 min, 35 cycles at 95 °C for 30 s, 40 s at temperature suitable for primers (w/v) agarose gel using electrophoresis and visualized by ethidium bromide staining and UV illumination. The size of the PCR products was determined with O’GeneRuler 1kb DNA Ladder .The detection of BRV was carried out by polymerase chain reaction (PCR) in the inoculum and in the  primers , 72 °C fJET 1.2 blunt vector using a CloneJET™ PCR Cloning Kit . The strain E. coli JM107 was transformed using TransformAid Bacterial Transformation Kit . Ten samples of plasmids with BRV insertions were sequenced using a Big Dye Terminator v 3.1 Cycle Sequencing Kit and performed on a 3130 Genetic Analyzer .Amplified fragments for sequencing with primer pair BCP11/P2  were purBefore inoculation, virus-free rooted microshoots were placed in the dark for 48 h at 21 ± 2 °C. Under sterile conditions, roots of 4-week-old microshoots were shortened with a scalpel to a length of 4 mm and soaked for 2 min in sterile tubes with fresh inoculum containing BRV . InoculaPR1 gene in mRNA of inoculated blackcurrant genotypes in vitro were analysed using quantitative real-time RT-PCR (qPCR). Reaction was performed in a 11.0 µL reaction volume containing 5.95 μL H2O, 1.25 μL 10× Taq buffer + (NH4)2SO4 + MgCl2, 1.0 μL 2 mM dNTP mix, 1.0 μL 25 mM MgCl2, 1.0 µL cDNA, 0.1 μL Taq DNA polymerase , 1.0 μL 20× EvaGreen dye , and 0.1 μL of each forward and reverse primer [Changes in the putative e primer . The anaSequences of the three Lithuanian BRV isolates were uploaded into the NCBI database . A percent identity matrix among the 3′ NTR of RNA2 sequences of BRV was created by the Clustal 2.1 program. The phylogenetic tree was constructed comparing 3 Lithuanian BRV sequences with 21 sequences of homologous region of BRV with reference to the NCBI database. Phylogenetic analysis was performed using the maximum likelihood method implemented in the PhyML program; a bootstrap analysis with 100 replications was performed . Relativ