Neisseria species such as Neisseria meningitidis and Neisseria gonorrhoeae. The genes encoding the Maf polymorphic toxin system are found in specific genomic islands called maf genomic islands (MGIs). In the MGIs, the MafB and MafI encode toxin and immunity proteins, respectively. Although the C-terminal region of MafB (MafB-CT) is specific for toxic activity, the underlying enzymatic activity that renders MafB-CT toxic is unknown in many MafB proteins due to lack of homology with domain of known function. Here we present the crystal structure of the MafB2-CTMGI-2B16B6/MafI2MGI-2B16B6 complex from N. meningitidis B16B6. MafB2-CTMGI-2B16B6 displays an RNase A fold similar to mouse RNase 1, although the sequence identity is only ~ 14.0%. MafB2-CTMGI-2B16B6 forms a 1:1 complex with MafI2MGI-2B16B6 with a Kd value of ~ 40 nM. The complementary charge interaction of MafI2MGI-2B16B6 with the substrate binding surface of MafB2-CTMGI-2B16B6 suggests that MafI2MGI-2B16B6 inhibits MafB2-CTMGI-2B16B6 by blocking access of RNA to the catalytic site. An in vitro enzymatic assay showed that MafB2-CTMGI-2B16B6 has ribonuclease activity. Mutagenesis and cell toxicity assays demonstrated that His335, His402 and His409 are important for the toxic activity of MafB2-CTMGI-2B16B6, suggesting that these residues are critical for its ribonuclease activity. These data provide structural and biochemical evidence that the origin of the toxic activity of MafB2MGI-2B16B6 is the enzymatic activity degrading ribonucleotides.The Maf polymorphic toxin system is involved in conflict between strains found in pathogenic  For example, lantibiotics, polypeptides containing lanthionine, are used by Gram-positive bacteria such as Lactococcus lactis to kill or inhibit other bacterial cells3. Recent reports have shown that a family of multidomain toxins called polymorphic toxins (PT) are involved in interstrain competition in the same species6. Polymorphic toxins are characterized by modular domain arrangement with an N-terminal module for transportation and a C-terminal module for toxicity. This family of PTs includes contact-dependent growth inhibition (CDI) systems, colicins and S-type pyocins, rearrangement hot spot (RHS) repeat-containing proteins and effectors of type VI secretion system such as VgrG and Hcp13. The MafB toxin in Neisseria species also belongs to PTs family.Microorganisms have evolved strategies to obtain resources such as space and nutrients for their own survival. Simple strategies include killing other organisms or inhibiting their growth in the environment. The use of proteinaceous toxins is a typical example of restricting the growth of other organismsNeisseria spp. such as Neisseria gonorrhoeae (the gonoccocus) and Neisseria meningitidis (the meningococcus)14,. There are multiple types of MGIs, which are classified as MGI-1, MGI-2, MGI-3, MGI-4 and MGI-5, depending on the genes flanking the MGIs15. For example, MGI-1 is flanked by anmK (the gene for anhydro-N-acetylmuramic acid kinase) and an ORF that encodes a hypothetical periplasmic protein similar to the N-terminus of FfpC15. N. gonorrhoeae harbors the 5 types of MGIs, but N. meningitidis has only MGI-1, -2, and -315.The genes encoding MafB proteins are found in the specific loci called maf genomic islands (MGIs) on the chromosomes of pathogenic 16. A recent report showed that MafA located in the outer membrane (OM) is the transporter of MafB for the exposure of MafB on the extracellular surface of the OM17. It is assumed that the C-terminal domain (MafB-CT) of MafB on the OM is cleaved and transported to the target cells by an unknown mechanism17. The mafB gene is always immediately followed by a mafI gene that encodes a small polypeptide of approximately 100 amino acids18. Since MafB contains a specific domain (MafB-CT) in the C-terminal region that has toxic activity, a gene encoding an immunity protein, MafI, is necessary to protect MafB-producing cells from the toxic activity. Therefore MafB and MafI exist as MafBI modules in MGIs. As a feature of the polymorphic toxin system, several alternative MafBI modules encoding solely the C-terminal part of the toxin (MafB-CT) and its cognate MafI are found in MGIs downstream of the mafBI module encoding the full length toxin15. It is thought that the downstream alternative MafBI modules could serve used as alternative MafB toxins after genetic recombination. Alternative toxin production by recombination events has been reported19.MafA, which is known to function as an adhesion by binding to a specific glycolipid , is found upstream of MafB15. A sequence motif between the N-terminal DUF1020 domain and the C-terminal domain is conserved in different MafB toxins, and is used to divide MafB toxins into 3 classes15. Class 1, 2, and 3 MafB contain a VSFDF motif with a bacterial intein-like (BIL) domain, a VKYDT motif and a DWVKN motif, respectively. The sequence of the N-terminal DUF1020 domain in the three MafB toxin classes is conserved well with more than ~ 85% sequence identity, but the C-terminal toxin domains displays sequence variation with less than 35% identity, suggesting that the same class of MafB toxins can have different toxic activities15. The C-terminal region has conserved domains such as bacterial toxin 50 , novel toxin 17 , MafB19-like deaminase (pfam14437) and bacterial EndoU nuclease 15; thus, this domain causes cell toxicity through enzyme activity. Specifically, when RNase activity is used in the toxic enzyme activity of PTs, various RNase enzymatic activities such as EndoU nuclease activity cleaving after uridylates of RNA15, RNase activity cleaving RNA non-specifically20, RNase activity specific to ribosomal RNA21 and tRNase activity specific to tRNA22 have been shown to be the basis of the toxic activity of the toxin domain.MafB toxin consists of a signal peptide, an N-terminal DUF1020 domain with unknown function and a C-terminal toxin domainNeisseria spp. revealed the role of a novel polymorphic toxin system, MafB, in intrastrain competition and identified a number of MafB prototype proteins exhibiting a conserved N-terminal region but a highly variable C-terminal region15. However, it is known that the MafB-CT is the toxic domain that should have cell damaging activity. Although the toxic activity of the MafB-CT can be inferred in some cases by querying domain databases, there are still many MafB toxins for which the in silico analysis is not able to reveal the cause of toxicity. When the information obtained through sequence similarity investigation is limited, structural studies can provide additional insights into the enzymatic basis for toxicity. This is accomplished by searching for structural homologs with known enzymatic activity, because the overall protein structure rather than the sequences is conserved for similar biochemical functions23. For example, structural studies of the C-terminal domain (CdiA-CTYkris) of CdiA from Yersinia kristensenii, which is involved in contact-dependent growth inhibition and is also a polymorphic toxin, showed that CdiA has a domain similar to RNase A and uses ribonuclease for toxic activity even though the sequence similarity is very low20. MafB2MGI-2B16B6 is a polymorphic toxin present in MGI-2 in N. meningitidis strain B16B6 and it has been shown that MafB2-CTMGI-2B16B6 has toxic activity14. Although the amino acid sequences suggest the presence of a conserved toxin domain predicted to adopt the BECR (Barnase-EndoU-ColicinE5/D-RelE) fold, further supporting experimental data are limited. Accordingly, in order to gain insight into the enzymatic activity governing the toxicity of the C-terminal domain of MafB2MGI-2B16B6, we have determined the X-ray crystal structure of a MafB2-CTMGI-2B16B6/MafI2MGI-2B16B6 complex at 1.5 Å resolution and delineated the residues important for the toxic activity of MafB2-CTMGI-2B16B6 by using mutagenesis and cell toxicity assays. The structural and biochemical studies indicate that MafB2-CTMGI-2B16B6 is similar to the RNase A superfamily.An in silico analysis and subsequent experimental analysis using MGI-2B16B6, which is the second MafB protein encoded in maf genomic island 2 (MGI-2) from N. meningitidis B16B6, the X-ray crystal structure of the complex between the toxin domain of MafB2MGI-2B16B6, hereafter referred to as NmMafB2-CT, and the corresponding immunity protein, MafI2MGI-2B16B6, hereafter referred to as NmMafI2, was determined at 1.5 Å resolution with 18.0% Rwork and 20.8% Rfree using a single anomalous dispersion method  from N. meningitidis NEM8013 and the corresponding immunity proteins are identical except two amino acids, suggesting that strains B16B6 and NEM8013 are not conflicted by the toxin domain of MafB2  as the highest similar structure with a Z score of 4.4; this protein is the CDI toxin of Yersinia kristensenii and known as a novel member of the RNase A superfamily20. The structures of NmMafB2-CT2 and CdiA-CTYkris were overlapped with an rmsd of 2.3 Å over 66 Ca atoms 27; it was structurally similar with an rmsd of 2.6 Å over 63 Ca atoms. The amino acid sequences of the CdiA-CTYkris and MsqR have approximately 12% and 6% similarity, respectively, with NmMafB2-CT2. It has been shown that CdiA-CTYkris belongs to the RNase A superfamily with structural similarity to angiogenin and Mus musculus pancreatic RNase 1 and subsequent in vitro RNase assays20. The structures of NmMafB2-CT2 and RNase 1 also showed structural similarity, with a Z score of 3.0 Å and an rmsd of 3.9 Å over 76 Ca atoms 29, with a Z score of 3.5 and an rmsd of 2.9 Å over 70 Ca atoms, even though there was only approximately 6% sequence similarity. However, there are no β-sheet components in the structure of colicin D corresponding to β1, β4 and β5, which comprise central core β-sheets in the NmMafB2-CT2 structure  and two α-helices (α1 and α2) flanking on the one side of the central β-sheet overlapped quite well with each other, but there were no structural elements of the cytoplasmic domain of CzrB equivalent to the region (α3 and β4) of NmMafI2 Ykris, the latter of which was shown to be a member of the RNase A superfamily structurally and functionally, the structural superposition and structure-guided sequence alignment of NmMafB2-CT2 were performed with CdiA-CTYkris and mouse RNase 1. The nuclease sites of RNase A for RNA degradation consist of His12, Lys41 and His11932. His12 and His119 function as a base and acid for transphosphorylation reaction during RNA cleavage, respectively, and Lys41 is involved in stabilizing the intermediate of cyclic 2’-3’-cyclic nucleotides at the 5′ end32. His175 and Thr276/Tyr278 of CdiA-CTYkris correspond to His12 and His119 and Arg186/Ans191/Lys193 in the vicinity of Val192 correspond to Lys41 of RNase A , which emits a fluorescence when RNA oligonucleotides tethering a fluorophore and a quencher is cleaved, showed that NmMafB2-CT2, which was refolded after isolation from the NmMafB2-CT2/NmMafI2 complex, degraded the RNA oligonucleotide . RNase A as a positive control was also included for the hydrolysis assay. RNase A hydrolyzed cCMP, but NmMafB2-CT2 did not have hydrolysis activity . These results suggest that in contrast to the known RNase A family enzymes and CdiA-CTYkris, NmMafB2-CT2 does not hydrolyze the the nucleoside 2',-3'-cyclic phosphodiester bond, rather NmMafB2-CT2 has only transphosphorylation activity for degrading RNA. It is unlikely that the loss of hydrolysis activity was due to incorrect folding during the refolding NmMafB2-CT2 isolated from the denatured solution because the binding affinity of the refolded NmMafB2-CT2 to NmMafI2 was comparable with that of CdiA-CTYkris to CdiIYkris and the refolded NmMafB2-CT2 had ribonuclease activity , the toxin domain was obtained by refolding after being purified in complex with its cognate immunity protein33. These results suggest that it is possible to determine the potential functionally functional important residues  by measuring the growth curve after initiating expression of a polypeptide with toxin activity. Accordingly, to determine the functional important residues of NmMafB2-CT2 that were identified by the fold similarity analysis, alanine mutants were generated and expressed under the control of L-arabinose. And then the toxic effect of each mutant on cell viability was determined by counting the number of clones that grew from the cell cultures on LB plates. The cell viability of cells expressing wild-type NmMafB2-CT2 was markedly reduced compared to that of the same culture in which no L-arabinose was added for expression, suggesting that NmMafB2-CT2 exhibits toxic activity, probably owing to RNA-degrading enzymatic activity . During the gene synthesis of NmMafB2-CT, a single point mutation (Gly to Arg) at residue 376 was introduced inadvertently. The G376R mutant was used for the structural determination. The G376R mutant gene of NmMafB2-CT was subcloned into a modified pET28a (pSKB3) vector with a His6-tag on the N-terminus and a TEV cleavage site using NdeI and EcoRI restriction enzyme sites. The gene of NmMafB2-CT with the original amino acid sequences was then obtained with site directed mutagenesis using two primers  of MafB2MGI-2B16B6, hereafter referred to as NmMafB2-CT2, was designed because the SDS-PAGE analysis of crystal samples revealed that NmMafB2-CT was degraded during crystallization  of MafB2rs Table . The NmMrs Table .Pfu DNA polymerase using a protocol based on the QuickChange II site-directed mutagenesis kit (Agilent Technologies). The forward and reverse primers for each mutant are listed in Table Site-directed mutagenesis was performed with E. coli BL21 Star (DE3) cells (Invitrogen). The NmMafB2-CT/NmMafI2 complex was expressed by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM once the OD600nm of cells grown at 37 °C in LB medium was about ~ 1.0 and incubating the cultures for 15 h at 20 °C. The cell cultures were harvested by centrifugation at 5000 g at 4 °C. The cell pellet was resuspended in buffer A  and 30 mM imidazole) and lysed by sonication. The lysate was centrifuged at 15,000 g for 30 min and the supernatant was loaded onto a 5 ml HisTrap crude column (Cytiva) equilibrated with buffer B . The complex was eluted using a linear gradient of 30 to 500 mM imidazole. TEV protease at a target protein to protease ratio of 50:1 (w/w) was added to remove the N-terminal 6xHis-tag during dialysis at 4 °C overnight with buffer C . The NmMafB2-CT/NmMafI2 complex without the His-tag was then purified by collecting the flow through fractions on a 5 ml HisTrap crude column (Cytiva) equilibrated with buffer B. Finally, the complex was purified with a Superdex 200 (Cytiva) column equilibrated in 10 mM Tris–HCl pH 8.0, 100 mM NaCl and 2 mM β-ME. The absorbance at 280 nm was measured, and the protein concentration was calculated using the extinction coefficient of 24,410 M-1 cm-1 obtained from ProtParm available at the ExPASy web site (http://web.expasy.org/protparam). The selenomethionine (Se-Met) labeled NmMafB2-CT/NmMafI2 complex was expressed using the PASM autoinduction medium34 and purified with the same protocol as the native protein.The two plasmids, the G376R mutant of NmMafB2-CT with a TEV-cleavable N-terminal 6xHis-tag and NmMafI2 with no affinity tag, were coexpressed in 35. The crystallographic data statistics are summarized in Table Initial crystallization conditions were screened in 96-well MRC Crystallization Plates (Molecular Dimension) using commercial crystallization kits such as Crystal Screen 1 and 2, Index and Salt from Hampton Research  and Wizard Classic 1 and 2 from Regaku Reagents . The G376R mutant of the NmMafB2-CT/NmMafI2 complex (0.8 µl) at a concentration of 10 mg/ml was mixed with anequal volume of reagent with a Mosquito robot (SPT Labtech), and then the drops were equilibrated against 70 µl of reservoir solution at 22 °C. The initial condition was optimized using the hanging-drop vapor-diffusion method with VDX plates (Hampton Research). The final plate-shaped crystals for X-ray diffraction were obtained in the wells containing 0.25—0.3 M magnesium formate and 25–30% polyethylene glycol (PEG) 3350 .The Autosol wizard in the -1 cm-1; NmMafI2, 18,450 M-1 cm-1.The two plasmids with different antibiotic resistances, one encoding NmMafB2-CT2 with no tag and the other encoding NmMafI2 with a C-terminal 6xHis-tag, were coexpressed. The complex was purified using a His-tag column with the same protocol as that for the NmMafB2-CT/NmMafI2 complex. NmMafB2-CT2 was isolated by collecting the flow through fractions using a 5 ml HisTrap crude column (Cytiva) in denaturing buffer  and then refolded by dialysis against 20 mM Tris pH 8.0, 100 mM NaCl and 2 mM β-ME overnight. After further dialysis aginst with 20 mM HEPES pH 7.5, 50 mM NaCl, 2 mM β-ME, NmMafB2-CT2 was purified further by linear NaCl gradient elution from a 5 ml Hitrap-SP column (Cytiva), that was equilibrated with 20 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM EDTA, and 2 mM β-ME. Finally, NmMafB2-CT2 was dialyzed against 10 mM Tris–HCl pH 8.0, 100 mM NaCl and 2 mM β-ME. NmMafI2 with a C-terminal 6xHis-tag was expressed and purified with the same protocol as that for the NmMafB2-CT/NmMafI2 complex omitting the steps of removing the N-terminal 6xHis-tag. The protein concentration was determined using the following extinction coefficient at 280 nm calculated by ProtParam program: NmMafB2-CT2, 4470 MThe affinity between NmMafB2-CT2 and NmMafI2 was measured at 20 °C using a Nano ITC calorimeter . All samples were prepared in the same buffer . Titration was performed by injecting 1 μl of NmMafI2 (~ 1.3 mM) into the cell containing NmMafB2-CT2 (~ 0.2 mM) 50 times at 250 s intervals. The NanoAnalyze program (TA Instruments) was used to calculate the interaction parameters after fitting the data with the independent binding sites model. The titration curve used for the program analysis was baseline-corrected with the buffer used in the titration.2O were also included. All measurement were repeated three times.In vitro hydrolysis activity was assessed spectroscopically in 20 mM Tris pH 7.5 and 150 mM NaCl buffer using 3 mM cCMP. The cCMP solution was incubated with 20 µM NmMafB2-CT2 in a final volume of 80 µl at room temperature. And then the absorbance at 296 nm was measured for 10 min. As a positive control, bovine pancreatic RNase A at a concentration of 20 µM was included in the hydrolysis activity assay. To examine the dependency of metal atoms, metal atoms at a concentration of 0.2 mM was included in the reaction buffer. RNase activity was measured by a fluorophotometer using an RNaseAlert Lab Test Kit (Invitrogen). The reaction mixtures in the final 50 µl volume containing 10 µg of protein were prepared in black 96-flat well plates. The fluorescence intensity was measured at 37 °C for 30 min using a Tecan Spark microplate reader (Tecan) with emission (Em) at 530 nm and excitation (Ex) at 485 nm with a 20 nm bandwidth for both wavelengths. As negative controls, BSA and RNase-free H39 using NdeI and HindIII restriction enzyme sites in such a way that no His-tag was expressed. The mutants were transformed into BL21 Star (DE3) cells, and each clone was cultured in 3 ml of LB medium containing 50 µg/ml chloramphenicol and 0.2% L-glucose at 37 °C overnight. The cultures were then diluted into 3 ml of fresh LB medium containing 50 µg/ml chloramphenicol and 0.2% L-arabinose at an OD600 of 0.1 and cultured further for 6 h at 37 °C. Finally, the cultures were diluted and then plated on LB plates containing 50 µg/ml chloramphenicol to check cell viability.All mutants were generated with the protocol described above using pET28a-MafB2-CT2 as a template and each forward and reverse primers Table . Each muSupplementary Information.


Halomonas HA1 and Marinobacter HA2. These strains required high concentrations of NaCl to ensure bacterial growth, especially Halomonas HA1 strain. Notably, both isolates can degrade phenol at optimal pH values, between 8 and 9, with the ability to grow in pH levels up to 11, like what was seen in the Halomonas HA1 strain. Moreover, both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta-cleavage pathway, while Marinobacter HA2 uses the 2,3 ortho-cleavage pathway as indicated by universal primers for 1,2 and 2,3 CTD genes. Interestingly, Marinobacter HA2 isolate eliminated the added phenol within an incubation period of 72 h, while the Halomonas HA1 isolate invested 96 h in degrading 84% of the same amount of phenol. Phylogenetic analysis of these 1,2 CTD (catechol dioxygenase) sequences clearly showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae. In comparison, 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Furthermore, semi-quantitative RT-PCR analysis proved the constitutive expression pattern of both dioxygenase genes. These findings provide new isolates of Halomonas sp. and Marinobacter sp. that can degrade phenol at high salt and pH conditions via two independent mechanisms.Haloalkophilic bacteria have a potential advantage as a bioremediation organism of high oil-polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotype script, biochemical characters, and sequence analysis of bacterial-16S rRNA were used to identify the bacterial isolates; The online version contains supplementary material available at 10.1007/s00284-022-02981-8. Phenol is an important industrial chemical that is utilized as an intermediate substance for chemical products such as xylenols and oil refining . PhenoliAerobic phenol degradation includes two highly conserved enzyme systems known as intradiol-cleaving and extradiol-cleaving enzymes; both of them use NADH as an electron donor and molecular oxygen to cleave aromatic rings. Another shared requirement for these two enzymatic systems is the using of non-heme iron to establish the functional enzymatic structure required for binding substrate .; The inIn this study, we aimed to isolate and identify phenol degradation haloalkaliphilic bacteria from the Hamra-lake depression in Wadi El-Natrun, located in the Sahara desert, 90 km north-west of Cairo, Egypt. Notable, this alkaline and hypersaline lake aggregate has a pH value between 8.5 and 11 and was considered as hypersaline and alkaline aquatic ecosystem rich in sulfate, chloride, carbonates, and sodium .3. The media was supplemented with 3 M NaCl and 2.5 mM phenol as a sole carbon and energy source. The water samples were added to HGM media in a ratio of 1:10/v and incubated at 30 °C for 2 weeks. The successive growth media was carried out using 50 µl of diluted culture, which spread on agar plates with the same media. A single colony was inculcated individually on new agar plates with the same growth media for biochemical characterization.Water samples were collected from Hamralake in Wadi El Natrun, Egypt , in which the pH is 10.0 and water salinity is 300 g/L. Halophile growth medium (HGM) was prepared as previously described , and thehttp://blast.ncbi.nlm.nih.gov/Blast.cgi). Based on high-scoring BLAST hits, the phylogenetic tree was performed using the (MEGA 7.0.26) software [Genomic DNA was extracted from the pure culture using GeneJET Genomic DNA Purification Kit (Thermo Scientific). PCR amplification of the 16S rRNA gene was carried out using Bact 27f (5′-AGAGTTTGATC(A/C)-TGGCTCAG-3′) and Bact 1492r (5′-TACGG(C/T)-ACCTTGTTACGACTT-3′) . Accordisoftware .3 or Na2CO3) were applied separately to determine the best conditions for bacterial growth in the phenolic condition. The primary culture was prepared by growing bacteria on mineral salt media described above supplemented with 0.3% yeast extract. Then the cells were harvested by spin down at 3000 rpm and 5 °C for 15 min and washed twice with 50 mM phosphate buffer (pH 7), and resuspended in liquid HGM media supplemented with 2.5 mM phenol with an initial optical density of 0.05 (OD600). Triplicate samples were centrifuged at regular intervals over an incubation period with optimal environmental conditions, and the OD600 was used to measure phenol concentration in parallel. Further, phenol concentrations in the samples were measured using a modified amino antipyrine method [g for 10 min. A volume of 300 µL of the supernatant was added to 6 µL 4-amino-antipyrine (2% w/v) and 6 µL potassium ferricyanide (8% w/v). After an incubation period of 10 min, the solution was mixed with 2 mL of chloroform. The absorbance level of the organic phenol was estimated at 505 nm. Phenol concentration was calculated according to the standard curve with standard phenol concentrations.HGM medium amended with 2.5 mM phenol, as a sole carbon source, was used to monitor phenol degradation by bacterial isolates. Different temperatures, NaCl concentration, and pH  and DOG R (5′-TTATGGACGCGCTTGCAGCTC-3′). These primers were deduced depending alignment of high similarities 1,2 CTD genes. The amplification program was conducted in 35 cycles, including 94 °C for 30 s, annealing at 60 °C for1 min, and extension at 72 °C for 1 min. The complete 2,3-cat gene sequence of Marinobacter HA2 isolate was isolated using C5 and C31 primers designed according to conserved sequence alignment of high similarity 2,3 cat genes. The primers sequence C5 (′5-ATGAAAAAAGGTGTAATGCGTCC-3′) and C31 (′5-GTTCAGYRYVCGRTCGTGG TAG-3′)  were used. The amplification program was conducted in 35 cycles, including 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min. The PCR product was electrophoresed using 1% agarose gel with 0.01% ethidium bromide and visualized with UV illumination. The DNA sequence was carried out using the amplified products and was sequenced using a 3100 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s protocols. The conserved domain analysis was performed using the program of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).The presence of intradiol 1,2 CTD was detected in isolated strains using degenerative primer for the highly conserved region (~ 400 bp), cat1 (5′-ACCATCGARGGYCCSCTSTAY-3′) and cat3 (5′-GTTRATCTGGGTGGTSAG-3′) , as previously described . The det–3, 10–4, and 10–5 of cDNA were prepared in sterilized distilled water [Halomonas HA1) and primers C5 and C31 .Total RNA was purified from 5 ml of bacterial culture grown in a mineral salt media containing phenol (2.5 mM), glucose (5 mM), or the medium contains both of them. RNA was extracted using the GeneJET RNA Purification Kit (Thermo Scientific) and eluted in 50 µl of RNase-free water , 21. Acced water , 23. Fort-test was used to determine the significance of phenol degradation by bacterial isolates. P ≤ 0.05 was considered statistically significant (*), while P ≤ 0.01 was considered highly significant (**).The Student’s two-tails H. salifodinae BC7 while HA2 16S rRNA sequence analysis revealed almost 98.2% similarity with M. alkaliphilus. Growth parameters of HA1 isolate showed obligatory salt requirement of 1%, while the bacterial growth can be sustained up to 20% NaCl with optimal growth at 8% NaCl. In addition, the pH tolerance of the strain extended up to pH 11 with optimal growth at pH 9. The optimal growth temperature was 35 °C with the ability to grow up in 50 °C. In contrast, the Marinobacter HA2 isolate showed a lower tolerance for adverse growth conditions. The best growth layout was obtained at 30 °C, pH 7, and 4% NaCl .The morphological investigation of phenol-degrading isolates revealed the presence of two different isolates; HA1 and HA2 that shared Gram-negative rods with differences in colonies color and shape. The biochemical characters demonstrated a distinguished pattern of urease production, which was negative for HA1 isolate. The full-length 16S rRNA (1500 bp) of both strains were sequenced and deposited under GenBank accession numbers KT223026 (HA1) and KU323642 (HA2). The phylogenetic analysis of HA1 demonstrates a similarity of 98% with the 16S rRNA gene sequence of Halomonas HA1 was grown at a temperature of 35 °C, pH 9, and 10% NaCl, while the optimal growth conditions for Marinobacter HA2 included a temperature of 30 °C, pH 7.5, and 4% NaCl. A sterile media supplemented with phenol and bacterial culture without phenol was severed as control. Both types of controls show a steady record of phenol content and absence of growth respectively. Marinobacter HA2 strain completely degraded the added phenol in 72 h. In comparison, the Halomonas HA1 isolate was able to degrade only 70% of phenol by the same period. The correlated bacterial cell growth  was 0.22 and 0.24 for Marinobacter HA2 and Halomonas HA1, respectively . The protein identity was 79% and 76% with 1,2-CTD of H. heilongjiangensis and H. pacifica. Marinobacter 2,3 CTD sequence with 299 amino acids was obtained using the degenerative primers set and submitted to the GenBank under accession number AMY26490.1 .Universal primers for the 1,2 CTD gene were used to isolate a gene sequence encoding for 303 amino acid residues that was submitted to the GenBank under accession number AMY26491.1. The amino acid sequence of isolated 1,2 CTD demonstrates the main characters of the conserved 1,2 CTD domains. The conserved domain of substrate binding sites of Leu 73, Ile 105, and Gly 107 showed high corresponding to all intradiol dioxygenases. Other similarities to halophilic 1,2 CTD include Val 200 replacement of the Tyr 200 residue and the presence of Gly 77 instead of Ala as previously reported . The preHalomonas sp. and 2,3-CTD Marinobacter sp. were used for RT-PCR. As shown in Fig. RNA was isolated from a bacterial culture grown on a minimal saline media supplemented with either phenol or glucose as a sole carbon source, or media supplemented with both of them. Primers for the conservative domain of 1,2-CTD Halomonas and Marinobacter contain several members of haloalkaliphilic species, and many of them are known for their ability to degrade aromatic compounds at high pH [Halomonas HA1 isolate. The high tolerance for salt concentration is a common attribute in this family which may refer to a particular proton translocation system in which NaCl used for glucose adsorption [Halomonase sp. can be adapted at a high salt concentration [P. putida has both cleavage pathways as reported by Basak et al., [Halomonas HA1 exploits the 1,2 phenol meta-cleavage pathway while Marinobacter HA2 uses the 2,3 ortho-cleavage pathway as indicated by universal primers for 1,2 and 2,3 CTD genes. Marinobacter HA2 isolate can eliminate the added phenol within an incubation period of 72 h. In comparison, Halomonas HA1 isolate required 96 h to degrade 84% of the same amount of phenol. The presence of the phenol meta-cleavage pathway of Marinbacter HA2 could interpret the variation of the phenol degradation rate of both strains. The functionality of the 2,3 catechol dioxygenase pathway in phenol degradation may clarify the gene outspread among microbial isolates in environments of phenol-contaminated sediments [Halomonas HA1 isolate showed more growth potential under stress conditions. Halomonas genus was represented in the isolated phenol decomposer consortium [H. organivorans was proved to catabolize different concentrations of low molecular weight aromatic compounds at 10% NaCl concentration [H. salina showed phenol degradation efficiency of 66% at 10% NaCl. The catechol 1,2 CTD gene sequences alignment in Fig. Halomonas sp..The 1,2-CTD of Halomonas HA1 showed segregation in a distinct cluster including the p. putida gene. The bacterial phylogenetic estimation based on the 1,2 CTD sequence was previously reported [Marinobacter 2,3 CTD. The identified protein shares the main conservative domains with other extradiol dioxygenases. The isolated sequence shows high similarity with taxonomically related M. excellens and other members of Microbacteriaceae, as shown in Fig. Halomonas HA1 1,2 CTD and Marinobacter HA2 2,3 CTD showed a constitutive gene expression pattern. Many phenol-degrading operons in proteobacteria expression are activated by inducers substances of targeted pathway [P. pseudoalcaligenes is mainly utilized with phenol and salicylate inducers [P. stutzeri [Both genera of  high pH , 26. Grosorption . A specintration . Under antration . The metntration . The metntration , 32. How et al., . In the ediments . Accordinsortium . The biontration . The phyreported , 38. In reported . Comparereported , 38. A d pathway , 41. Phe pathway , 43. Theinducers . In someinducers . The coninducers . Evidencstutzeri .Halomonas HA1 and Marinobacter HA2, were isolated from Hamralake in Wadi El Natrun, Egypt. These isolates can degrade phenol at high salt and pH conditions. The two strains proved to follow different strategies for phenol degradation. A New 1,2dioxygenase enzyme has been isolated from Halomonas isolate, and its sequence analysis showed an interesting evolutionary intermediate linkage. However, Marinobacter isolate revealed the 2,3 catechol dioxygenase activity. Semi-quantitative RT-PCR demonstrated that the expression of both dioxygenases in different isolates was constitutive and not induced by phenol.Phenol is a common industrial pollutant, and its accumulation in the soil causes a severe threat to underground water. There is an urgent need to isolate phenol microbial decomposers that are adapted for high salt and alkaline conditions of industrial wastes. The information about gene structure and expression is necessary to understand degrader gene evolution and regulation. We sought to isolate and identify new isolates of phenol degradation bacteria in the present work. Here two bacterial species, Supplementary file1 (DOCX 1395 KB)Below is the link to the electronic supplementary material.


WEE1, a negative regulator of G2/M transition, was highly expressed at 12 °C compared to 18 °C. This finding suggested that cells at 12 °C are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phospho-histone H3 (pH 3) immunostaining. Inhibition of WEE1 during diapause at 12 °C resulted in cell cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18 °C-diapaused embryos. These findings suggest that diapause at low temperatures leads to WEE1 upregulation, which arrests the cell cycle at the G2/M phase, promoting the perseverance of embryonic cytoarchitecture and future SRD. In contrast, WEE1 is not upregulated during diapause at higher temperature, leading to continuous proliferation and maladaptive morphology associated with poor survivability. Combining HREM-based analysis with RNAseq and molecular manipulations, we present a novel mechanism that regulates the ability of diapaused avian embryos to maintain their cytoarchitecture via cell cycle arrest, which enables their SRD.Avian blastoderm can enter into diapause when kept at low temperatures and successfully resume development (SRD) when re-incubated in body temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular level. To determine how temperature affects embryonic morphology during diapause, high-resolution episcopic microscopy (HREM) analysis was utilized. While blastoderms diapausing at 12 °C for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18 °C, much thicker blastoderms with higher cell number were observed. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially expressed cell cycle regulatory genes. Among them,  The avian embryo at oviposition, termed blastoderm, corresponds to the blastocyst stage in mammalian embryos . HoweverAt oviposition, the blastoderm consists of 60,000–130,000 cells distributed in an outer ring termed the area opaca (AO), which forms extra embryonic structures, and an inner disk known as the area pellucida (AP), which is composed of an epithelium layer, termed epiblast, that gives rise to the embryo proper ,6,7,8. FThe cell cycle is regulated by multiple regulators at different phases and checkpoints of the cell cycle. At the G1 phase, the tumor suppressor retinoblastoma protein (pRb) interacts with the E2F transcription factor to inhibit cell cycle progression . PhosphoWEE1 was significantly increased at 12 °C diapaused embryos in comparison to the 18 °C ones. Further investigation of the role of WEE1 demonstrated that during diapause at lower temperatures, cell cycle is predominantly arrested at the G2/M transition via WEE1, which is associated with better maintenance of cell viability, blastoderm cytoarchitecture and the ability to SRD. These findings are the first to uncover molecular processes that are involved in diapause phenomenon in avian.Based on our recent findings, which showed that blastoderms that diapause at 12 °C or 18 °C have a different morphology and ability to undergo cell division , in thisFreshly laid Ross (308) broiler eggs from young flock age (28–45 weeks) were collected from a commercial farm and stored at 18 °C or 12 °C up to 28 d in a cooled incubator . The temperature and relative humidity of the cooled incubator was continuously monitored throughout the experiment. Following diapause, blastoderms were isolated as previously described . BrieflyEmbryos were fixed overnight in 4% PFA, washed with PBS and dehydrated in a series of methanol concentrations . Following dehydration, the embryos were impregnated with 50% methanol and 50% JB4 dye mix solution, overnight, on a rotating shaker at 4 °C. JB4 dye mix contains solution A , 12.5 mg/mL catalyst , 2.75 mg/mL Eosin B  and 0.5 mg/mL Acridine orange , dissolved overnight at 4 °C and then filtered. The 50% methanol and 50% JB4 dye mix solution was replaced with 100% JB4 dye mix solution, and impregnation continued for 24 h at 4 °C. For curing the resin at mounting, 30 µL/mL of solution B  was added to the JB4 dye mix, followed by curing the resin overnight at 4 °C in a plastic embedding mold . Polymerized sample blocks were further hardened by overnight baking at 70 °C.Cooled hardened sample blocks were mounted in the HREM machine , and block surface images were serially taken every 2.76 µm section throughout the entire sample in 2700 × 1800 image pixel resolution. Depending on the experimental design, selected plastic sections obtained during HREM sectioning were preserved by placing them on DDW drops on histological slides and letting them dry at room temperature. Images of the plastic section were captured using a microscope . Image of reference 1000 µm graticule  was taken for each block sectioned to allow for accurate size measurements. All the images acquired from HREM imaging were processed, 3D reconstructed and analyzed as previously described ,28 usingEmbryos from four distinct storage conditions  were processed for RNAseq gene expression profiling. Each group consisted of 4 biological repeats, thus 16 blastoderms were isolated from eggs as previously described . Isolate−∆∆ct method. Briefly, samples from diapaused embryos were the test samples, and the 0 d-unstored fresh embryos were used as the calibrator samples. The Ct value of the target genes  of each sample group were normalized to that of the reference GAPDH gene. Then, the GAPDH-normalized Ct value of test sample group was again normalized with calibrator samples. Finally, the expression ratio was calculated using 2−∆∆ct method. Thus, the result obtained is the fold of change in expression of the target gene in the tested sample relative to the calibrator sample. The comparison of fold change gene expression level was analyzed by one-way ANOVA statistical tool. The list of primers that were used to analyze the expression of genes is given in Three isolated embryos were pooled for RNA extraction. Subsequently, cDNA was prepared using Promega kit . The primers were designed according to the sequence information from the NCBI database using Primer3 Input (version V. 0.4.0) software . qRT-PCRFresh, fresh +6 h incubation for positive control, 7 d/18 °C, 7 d/12 °C, 28 d/18 °C and 28 d/12 °C stored embryos were treated with 0.2 mg/mL BrdU  in PBS, by injecting 100 µL in volume to at least 4 embryos through a shell window. Following injection, the eggshell window was sealed using Leukoplast  and placed back to its respective condition for 6 h. The embryos were isolated, fixed in 4% PFA overnight at 4 °C and dehydrated in methanol . Dehydrated samples were stored at −20 °C in 100% methanol. Whole mount embryo samples underwent DNA denaturation steps by incubating them in 50% formadide , in 4XSSC in DDW, for 2 h at 65 °C under slow agitation. Subsequently, samples were rinsed in 2XSSC for 15 min and incubated in 2N HCL at 37 °C for 30 min, followed by neutralization in 0.1 M sodium borate (pH 8.5) for 10 min. The samples were then washed 6 times, 15 min each, in TBS  and blocked for 1 h with 3% Normal Bovine serum in TBS containing 3% triton-X-100 . Following blocking, the embryos were incubated with rat anti-BrdU IgG2a  overnight at 4 °C, washed 3 times in TBS for 15 min each, at room temperature, rinsed once in TBST for 15 min and incubated for 2 h with F(ab)2 donkey anti-rat IgG-Cy3 antibody . The embryos were washed in TBS stained with 4′,6-diamidino-2-phenylindole (DAPI)  for 30 min at RT. Stained embryos were mounted between two coverslips using fluorescence mounting medium  and scanned using a confocal microscope . The DAPI-stained nuclei and BrdU-positive cells were counted manually to determine the percentage of BrdU incorporating cells. The embryos were isolated and fixed in 4% PFA overnight, washed with PBS, permeabilized with 0.2% triton in PBS (PBST), blocked in 10% NGS in PBST at 4 °C for 3 h, and immunohistochemistry steps were carried out by incubating the samples with primary anti-pH 3 antibody  overnight at 4 °C as previously described ,40,41. TFirstly, 15% pluronic gel  was prepared in PBS at 4 °C and mixed with MK-1775 to a final concentration of 500 nm  ,46,47. T2 and 10 mM CaCl2) for 10 min, fixed in 4% PFA and washed in DDW. The TUNEL staining was performed according to manufacturer’s instructions of TUNEL detection kit; then, the samples were stained with DAPI, washed and mounted with coverslip and mounting media . The intensity of TUNEL-positive cells and pH 3-positive cells was quantified as previously described [Whole mount embryos were immunostained with anti-pH 3, followed by dehydration as described above. The embryos were cleared in xylene and processed for paraffin 7 µm sections as described before [escribed ,49,50. t-test, one-way ANOVA and two-way ANOVA. Data were presented as mean ± SEM. Statistical tests were performed using JMP (John’s Macintosh Project) software  at significance level (p < 0.05).Data were analyzed using the statistical tests, The cytoarchitectural structure of prolonged diapaused embryos was investigated by three-dimensional 3D) HREM imaging D HREM im,51,52. Tp = 0.0021, *** p = 0.0007, respectively). To investigate whether the thickening of blastoderm tissues after prolonged diapause at 18 °C was due to cell hypertrophy or to cell proliferation, we further sectioned the embryos and stained their nuclei with DAPI (p < 0.0001), suggesting that cell proliferation majorly accounts for the thicker tissue in embryos diapaused at 18 °C.In addition, using multi planar view (MPV) and segmentation tool enabled the visualization of the XY and YZ planes of the blastoderm G–I and vith DAPI K to alloith DAPI L, our reTo identify at which time point the cellular changes are initiated, we compared freshly laid embryos A,D,G witp = 0.1152), poly-ingressing clusters in embryos diapaused for 7 d at 18 °C are significantly larger (p < 0.0001). Combined with the transparency test  freshly laid embryos, which serve as a reference control, (ii) embryos diapaused for 7 d at 18 °C (7 d/18 °C), (iii) embryos diapaused for 28 d at 18 °C (28 d/18 °C) and (iv) embryos diapaused for 28 d at 12 °C (28 d/12 °C). The differences in molecular pathways between 28 d/12 °C and 28 d/18 °C groups were analyzed using enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways A. This regulated B. Notabl d/12 °C , furtherCyclin E1 (CCNE1), Cyclin A1/2 (CCNA1/2) and Cyclin B2 (CCNB2) were found be downregulated in the 28 d/18 °C group compared to the other groups , while WEE1 expression was upregulated in the 28 d/12 °C group (* p = 0.0485 compared to 28 d/18 °C group). These results, which confirmed the RNAseq data, indicated a deceleration in cell cycling following 28 d of diapause at 18 °C. However, the morphological changes observed in the 18 °C group prompted us to further determine whether there was a shift in cell cycling from proliferation to deceleration state in the 18 °C group and whether the maintained cytoarchitecture at 12 °C was associated with safeguarding of cell cycle progression through higher WEE1 expression. As a first step to experimentally examine the cell cycle state of embryonic cells, BrdU incorporation assay was performed in order to quantify the number of cells in the S phase of the cell cycle. Different experimental groups were treated with BrdU: 1). As a positive control, fresh embryos were incubated at 37.8 °C for 6 h, treated with BrdU and further incubated for 6 h before fixation; 2). As a starting reference point that refers to the embryonic state before diapause, fresh embryos were treated with BrdU for 6 h at room temperature before fixation; 3). For the 7 d time point, embryos were diapaused at 18 °C or 12 °C for 7 d, then treated with BrdU for 6 h and fixed; and 4). For the 28 d time point, embryos were diapaused at 18 °C or 12 °C for 28 d, treated with BrdU for 6 h and fixed (p = 0.001), while at 12 °C, BrdU incorporation remained stable throughout the 28 d of diapause and was comparable to fresh embryos  and in embryos diapaused at 18 °C . However, the quantification of discrete M sub-phases revealed that fresh embryos consisted of cells in all mitotic phases, while embryos diapaused at 12 °C were in the prophase stage (p < 0.0001), which corresponds to the G2/M transition (p ≤ 0.002) and in all mitotic sub-phases during 28 d, which distributed similarly along the different stages , in MK-1775-treated embryos, the cells were found in more advanced stages, from the pro-metaphase M3/M4 stages and onward (p = 0.0111). These results strongly support a regulatory role of WEE1 in preventing cell cycle progression in embryos diapaused at low temperature.The above results revealed that during diapause at 12 °C, the majority of M phase cells are arrested in prophase, which correlates with the high expression of activity ,47,54,55p < 0.0001). Moreover, in control embryos, the poly-ingressing cell clusters were smaller and discreetly arranged (p < 0.0001). These results suggest that during diapause at 12 °C, the increased levels of WEE1 arrest the cells at the G2/M, thereby inhibiting proliferation and thickening of the blastoderm, while following WEE1 inhibition with MK-1775, the cells progress with the cell cycle, which results in blastoderm thickening, similar to the phenomenon observed during diapause at 18 °C, when WEE1 expression levels are low.To test whether the activity of WEE1 is coupled with the thinner blastoderm’s phenotype observed in the 12 °C diapausing embryos A, such earranged B,H, whilarranged C,C’ researranged . Furtherp < 0.0001) and the number of cells entering the M phase . Notably, TUNEL-positive cells were also pH 3-positive at mitotic phases, M4 and M5 (WEE1 expression during diapause at 12 °C may serve as a survival mechanism for embryos diapaused at low temperatures by preventing accelerated cell proliferation and death, and thus, maintaining the cytoarchitectural integrity of the blastoderm. As a G2/M checkpoint regulator, WEE1 was found to allow the extension of time spent in repairing DNA damage prior to mitosis . Thus, t4 and M5 D. These The freshly laid blastoderm consists of epithelial cells in the AP region that adhere to each other to form a single-layer epithelial sheet . This isPreviously, 2D imaging strategies were mainly used to study morphology of blastoderms , which lWEE1 [Xenopus, it has been shown that the inhibition of Wee1 activity causes gastrulation defects by impairing key morphogenetic movements involved in gastrulation [To identify the mechanisms underlying the morphological changes in blastoderms that diapause at different temperatures, RNAseq was performed, and this revealed that cell-cycle-related genes are distinctly regulated at the two temperatures tested, 12 °C or 18 °C. The KEGG pathway enrichment analysis further suggested that the G2/M transition regulatory gene WEE1  is proviWEE1 . HoweverWEE1 . Thus, prulation .WEE1 [WEE1 at 12 °C, could be the response to DNA damage, which occurs at lower temperatures. This in turn leads to better embryonic survivability following diapause at 12 °C, as revealed by the lower number of Annexin-positive cells at lower temperature compared with higher temperature [WEE1 and the similarities between the highly proliferating embryonic cells and related diseases in human patients may render the chick embryo as a valuable model organism for studying potential therapeutic approaches.One of the main roles of the G2/M checkpoint is to assure that no DNA damage exists prior to cell division . ConsequWEE1 ,80, resuWEE1 . MoreoveWEE1 . Thus, operature . Thus, tWEE1, allowing embryos to withstand the colder temperature for a longer duration of diapause. WEE1 is an evolutionarily conserved tyrosine kinase that controls the cell cycle from yeast to humans [WEE1 expression is dependent on minute changes in environmental temperature; thus, warmer springs may lead to increased risk of chick mortality of wild birds [Laying of eggs is a common feature of oviparous animals, such as insects, birds, fish, reptiles and amphibians. However, the number of eggs laid varies among these organisms as part of their reproductive strategy ,84,85,86o humans ,104, andld birds . ConversWEE1 expression is not upregulated in embryos diapaused at 18 °C, suggesting that the enhanced and abnormal tissue volume at this temperature is due to progression of mitosis. Altogether, our findings provide, for the first time, a missing link between cytoarchitectural changes, cell proliferation and cell survival mechanisms in diapaused embryos.In summary, this study suggests that embryonic survival at 12 °C is associated with maintaining blastoderm cytoarchitecture and cell viability as a result of cell cycle regulation by an arrest in G2/M phase mediated by WEE1. In contrast, WEE1 at 12 °C, but not at 18 °C. The inhibition of WEE1 kinase activity in embryos in diapause at 12 °C resulted in increased tissue size and higher staining for cell death marker, similar to embryos diapaused at 18 °C. By combining HREM imaging technology with RNAseq and molecular manipulation, this revealed a new role of WEE1 in safeguarding mitotic entry to regulate cytoarchitecture and cell survival in chick embryos during diapause.In conclusion, avian embryos must adapt to low temperature exposure at the very initial stage of their development, by undergoing diapause. This allows embryos to suspend their development for a long duration and still survive after exiting from it. In this study, we used HREM image analysis to quantify the morphological changes in embryos diapaused at different temperatures, 12 °C and 18 °C. In particular, we found that the cytoarchitecture of the embryos diapaused at 12 °C was better preserved than that of embryos diapaused at 18 °C. This characteristic was coupled with an arrest in the cell cycle at the G2/M stage, by upregulation of


Glycine max L. accumulates a large amount of isoflavonoid compounds, which is beneficial for plant defense, plant-microbe symbiotic interactions, and human health. Several CYP450 subfamily genes are involved in the flavonoid biosynthetic pathway in plants. In the present study, we found 24 CYP82 subfamily genes were differentially expressed in various tissues of soybean, in Phytophthora sojae-infected soybean varieties and in soybean hairy roots treated with cell wall glucan elicitor. Six of them  were co-expressed with other known isoflavonoid pathway genes in soybean. Their enzymatic activity in yeast feeding assays showed that only GmCYP82D26 was able to convert naringenin to daidzein with both aryl migration and dehydration function. When GmCYP82D26 was over-expressed in soybean hairy roots, the contents of the two major isoflavonoid aglycones in soybean (daidzein and genistein) were reduced, but total flavonoids were not affected. When GmCYP82D26 was suppressed by RNAi in the hairy roots, daidzein content was decreased but genistein content was increased, with unchanged total flavonoid content. GmCYP82D26 was found to be localized in the endoplasmic reticulum at subcellular level when transiently expressed in tobacco leaf epidermis. GmCYP82D26 gene was preferentially expressed in roots, with low expression level in other tissues in soybean. Homology modeling and molecular docking showed that GmCYP82D26 could form hydrogen bond with both HEM and naringenin at C5–OH and C4 carbonyl. All these results indicated that GmCYP82D26 possesses new and dual enzymatic activity, which bridges the two branches (daidzein and genistein branch) of isoflavonoid pathway in soybean. The single-strand cDNAs were synthesized using Fast King gDNA Dispelling RT Super Mix . The sequence information of six soybean CYP82 genes  were obtained based on Phytozome 12.0. The full-length open reading frames of these six genes were amplified by using the phusion high-fidelity DNA polymerase  with primers listed in BamH I and Sma I for GmCYP82A2 and GmCYP82C20; BamH I and Kpn I for GmCYP82A3; Sma I and Kpn I for GmCYP82A4; BamH I and Sac I for GmCYP82A23; Sma I and Sac I for GmCYP82D26), and the digested fragments were then ligated to pYeDP60 digested with the same restriction endonucleases.Roots of 3-week-old seedlings were used for total RNAs isolation by using TRIzol-AS. cerevisiae WAT11. Yeast cells were grown at 28 °C for 2 days on plates with SGI + Trp medium containing 20 g/L glucose. PCR positive colonies were selected and initially grown in 1 ml SGI + Trp liquid medium with 20 g/L galactose medium at 28 °C for about 12 h. The cells were harvested by centrifugation and resuspended in the same medium to induce the expression of target proteins. Dihydromyricetin, eriodictyol, liquiritigenin, naringenin, apigenin, kaempferol, daidzein, genistein, 2′-hydroxygenistein and 3,9-dihydroxypterocarpan  with final concentration of 10 μM, were then supplemented individually to the yeast cell cultures. After 24 h cultivation, the yeast cells were centrifuged, extracted twice with ethyl acetate, frozen-dried, and dissolved in 80% MeOH for further analysis.The six resulting expression vectors, and the empty vector pYeDP60 as control, were transformed into yeast 2.5Six GmCYP82 proteins and coordinates of the HEM protoporphyrin group were modeled by using I-TASSER . Naringe2.6GmCYP82D26 was cloned into plasmid pCAMBIA1302 digested with Nco I and Bgl II. The resulting GmCYP82D26-GFP fusion construct was transformed into A. tumefaciens strain GV3101, which was co-transformed by infiltration in N. benthamiana, with A. tumefaciens strain GV3101 containing the ER marker fused to mCherry, at a ratio of 1:1 cell culture. GFP and mCherry fluorescence were assayed after 48–72 h, using a Leica TCS SP5 confocal microscope as described previously [The open reading frame of eviously . Fluores2.7GmCYP82D26 gene in soybean, its open reading frame for over-expression, and the 147 bp sequence in 3′ un-translated regions for RNAi were subcloned into plasmid pTOPO-ENTR/D . These two fragments were confirmed by sequencing and cloned respectively into plant overexpression vector pK7WG2D and RNAi vector pK7GWIWG2II 2D with Gateway LR Clonase II Enzyme Mix according to the manufacturer's protocols.For the functional characterization of Agrobacterium rhizogenes strain ARqua I strain, and used for infection of soybean cotyledon to generate hairy roots as described previously [These two resulting vectors were individually transformed into eviously  with som2.8−ΔΔCT method and normalized with SUB3 gene as a reference [Gene expression levels in various tissues, including roots, stems, leaves, flowers, pods 10 days after flowering and seeds 20 days after flowering, or hairy roots, were determined by RT-qPCRs. Total RNAs from these tissues were isolated and reverse transcribed into cDNA as above. All RT-qPCRs were carried out using a 2 × RealStar Green Fast Mixture  on ABI 7500 real-time Detection System . Gene-specific primers were shown in eference . Data we2.9Enzymatic reactions and hairy root extracts were filtered and analyzed on UPLC  with a Eclipse XDB-C18 column  maintained at 30 °C. Separation was performed with 0.1% (v/v) formic acid/water (A) and 100% methanol (B) at 1 mL/min flow rate. The linear gradient was 5%–80% B over 16 min, 80%–100% B from 16 to 18 min, 100% B for 2 min, and equilibrated at 5% B for 1 min. The flavonoid compounds were detected at 280 nm with a diode array detector. Flavonoids contents were measured by comparing area of individual peak with standard curves obtained with standard compounds (daidzein and genistein).m/z spectra were collected from 500 to 1000 as described previously [The enzymatic products were also detected on LC-MS/MS on an ion-trap TOF mass spectrometer attached to UPLC . Separation was performed on a Eclipse Plus C18 column  using the same solvent system as described above for UPLC. The linear gradient was 5%–60% B over 5 min, 60%–100% B from 5 to 10 min, 100% B for 2 min, and equilibrated at 5% B for 0.1 min. The flow rate was maintained at 0.4 mL/min, and the column temperature was maintained at 30 °C. Flavonoid compounds were detected under negative mode, and the eviously .2.102O and 15 μL 5% NaNO2, and stood for 5 min. Then 15 μL 10% AlCl3 was added, mixed well and stood for 10 min. Afterwards, 100 μL 1 M NaOH and 120 μL ddH2O were added, mixed well and stood for 15 min. Finally, 180 μL reaction was measured at wavelength of 510 nm using the microplate reader spectrophotometrically at 510 nm. Absorbance values were converted into total flavonoids using a standard curve with quercetin.Total 50 μL flavonoid extracts extracted with 80% methanol as above was sequentially mixed with 200 μL ddH33.1GmCYP82A2, GmCYP82A3, GmCYP82A4, GmCYP82A18, GmCYP82A19, GmCYP82A20, GmCYP82A22, GmCYP82A23, GmCYP82A24, GmCYP82A25 and GmCYP82A26), four of GmCYP82C subfamily , five of GmCYP82D subfamily , one of GmCYP82J subfamily (GmCYP82J3) and three of GmCYP82L subfamily . Multiple sequence alignment at amino acid level showed that all 24 members shared high sequence similarity in two characteristic regions, E-R-R triade and the heme-binding domain (FXXGXRXCXG)  . The E-RG. max, Arabidopsis thaliana, Scutellaria baicalensis, Gossypium hirsutum, N. tabacum, Ocimum basilicum, Eschscholzia californica, Papaver somniferum and Mentha × piperita) showed that these CYP82 proteins could be clustered into seven clades (B–H) and GmCYP93C5 was clustered into a separate clade A . Total twelve genes  were relatively highly expressed in flowers, seven genes  were relatively highly expressed in roots. In addition, GmCYP82A25 was highly expressed in 1 cm pods and in seeds of 14 and 21 days after flowering, GmCYP82C1 in seeds of 14 days after flowering, GmCYP82D25 and GmCYP93A41 in seeds of 14 days after flowering, GmCYP82J3 in pod shell of 10 days after flowering, whereas GmCYP82L5 was not expressed in all tested tissues (The expression profiles of soybean  tissues . These dArabidopsis and soybean [CYP82 subfamily genes and known isoflavonoid pathway genes based on soybean affymetrix genome array (GDS3242)   and tranGDS3242) . In a pr control . It was reatment , therefod by WGE .CYP82 subfamily genes and known isoflavonoid pathway genes under infection of P. sojae in soybean cultivars varying in P. sojae resistance, based on the data retrieved from soybean affymetrix genome array (GDS3242)  were up-regulated after P. sojae treatment among 8 soybean cultivars   were co-expressed with isoflavonoid pathway genes and they were induced by WGE and P. sojae treatment, thus these six genes were selected as candidates for further analysis.We also performed expression analysis of soybean GDS3242) . It was ultivars . In addi GmG4DT) . Combine3.3Arabidopsis NADPH cytochrome P450 reductase gene (ATR1) [Six candidate GmCYP82 proteins  were expressed in yeast strain WAT11, which contains an e (ATR1) . The yeaAs a result, one new product was detected at retention time of 13.1 min in the feeding assay expressing GmCYP82D26 protein with naringenin as substrate  B, whereHowever, no new peak was produced for the other five proteins  with all the tested substrates .In summary, GmCYP82D26 showed activity towards naringenin to produce daidzein with strict substrate selection, while other five soybean CYP82 subfamily members  showed no activity toward any of these tested ten substrates.3.4GmCYP82D26 in soybean hairy roots, as well as suppressed its expression by RNAi technology. The positive soybean hairy roots were selected by GFP signals under microscope owing to the presence of the GFP selection marker in both the vectors used for over-expression and RNAi  marker A. Theref3.7To investigate potential catalytic mechanism of GmCYP82D26, its three-dimensional structure model was obtained through homology modeling with naringenin as substrate together with other five GmCYP82 proteins . The docAmong the other five proteins, GmCYP82A2 and GmCYP82A4 could not form hydrogen bond with HEM, or with C4 carbonyl and 5-OH of naringenin. GmCYP82A3 could form hydrogen bond with HEM, but not with C4 carbonyl or 5-OH of naringenin . Althoug4P. sojae and M. phaseolina [Soybeans is rich in isoflavones, which are precursors of phytoalexins with antibacterial and antiviral effects, and antiherbivore activities that protect plants from pathogen infections ,49. One aseolina ,51. In aaseolina . Furtheraseolina . In partGmCYP82A2, GmCYP82A3, GmCYP82A4, GmCYP82A23, GmCYP82C20 and GmCYP82D26) showed tissue-specific and inducible expression patterns as other isoflavonoid pathway genes with known functions  and flavone 8-hydroxylase (F8H), respectively. SbCYP82D1.1 had broad substrate specificity for flavones such as chrysin and apigenin, which was responsible for synthesis of both baicalein and scutellarein in roots and aerials parts of S. baicalensis, respectively. SbCYP82D2 can accept only chrysin as substrate to produce norwogonin with high substrate specificity, although minor 6-hydroxylation activity could also be detected [Among these six recombinant soybean CYP82s tested for enzymatic activity, only GmCYP82D26 showed activity toward naringenin but not other nine representative flavonoid/isoflavonoid substrates , which iyl group . SbCYP82detected ,55. UnliIn soybean, except for GmCYP82D26, the other five soybean CYP82 proteins did not exhibit any activity towards the tested flavonoid/isoflavonoid substrate although they could also be docked with naringenin , and theMore importantly, it was found for the first time that GmCYP82D26 can catalyze naringenin to daidzein with both aryl migration and dehydration function , which iⅤ = O in the initial stage, followed by the migration of the side chain aryl (B-ring) from C-2 to C-3, and a hydroxyl is introduced at C-2 to produce C2-hydroxy isoflavanone by FeⅣ-OH, which can be further dehydrated with hydrogen at C-3 by HID or automatically, and finally form isoflavones [For the known CYP93 family proteins in soybean, GmCYP93A1 encodes pterocarpan 6α-hydroxylase (P6αH) , GmCYP93flavones ,61. It iflavones . Soybeanflavones . Gly78 aflavones . As showGmCYP82D26 was localized in the endoplasmic reticulum when transiently expressed in tobacco epidermal cells , which wGmCYP82D26 showed the highest expression levels in roots as verified by both transcriptome and qRT-PCR analyses (GmCYP82D26 was up-regulated upon P. sojae treatment (in vitro and in vivo assays confirmed that GmCYP82D26 was involved in daidzein biosynthesis. The contents of daidzein in the hairy roots silencing GmCYP82D26 were decreased compared with the control (GmCYP82D26 was increased but daidzein was decrease, indicating GmCYP82D26 bridge the two branch pathways and balance the content of daidzein and genistein in soybean. Therefore, GmCYP82D26 could be utilized for the conversion of naringenin to daidzein by using one-step synthetic biology approach.analyses . Meanwhireatment . Further control B. The prYaying Xia: Methodology, Investigation, and, Writing – original draft. Chunfeng He: Methodology, and, Investigation. Su Yan: Methodology, and, Investigation. Jinyue Liu: Investigation. Haijun Huang: Investigation. Xue Li: Investigation. Qian Su: Investigation. Wenbo Jiang: Methodology. Yongzhen Pang: Conceptualization, Resources, Writing – review & editing, and, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


An effective prophylactic vaccine would be beneficial for controlling and eradicating hepatitis C virus (HCV) infections. However, the high diversity across HCV genotypes is a major challenge for vaccine development. Selection of the appropriate immunogen is critical to elicit broad HCV neutralizing antibodies (NAbs). To increase the antigenic coverage of heterodimer glycoproteins, we designed and produced recombinant E1E2 antigens for genotypes 1a/1b/2a/3a/6a from an IgG Fc-tagged precursor protein in FreeStyle 293-F cells. The recombinant E1 and E2 antigens were localized and associated with the endoplasmic reticulum and co-purified from membrane extracts. By examining the interactions with HCV entry co-receptors and the blockade of HCV infection, we found that these purified Fc-E1E2 proteins displayed correct folding and function. Mouse immunization results showed that each recombinant E1E2 antigen could elicit a pangenotypic antibody response to itself and other genotypes. We also found that the pentavalent formula triggered a relatively higher and more uniform NAb titer and T cell response than monovalent antigens. Taken together, our findings may provide a useful strategy for the vaccine development of HCV and other viruses with highly heterogeneous surface glycoproteins. TherHCV belongs to the family of Flaviviridae. Unlike the other flavivirus genus members, such as Dengue virus and Zika virus, the envelope glycoprotein of HCV is composed of E1 and E2, which usually form heterodimers on the viral surface. Despite containing the high mutation regions HVR1 and HVR2, which are prone to mutating and evading the host immune response , E2 mediin vivo  and protein subunit vaccines. Moreover, vaccines tend to be multivalent. A quadrivalent genotype 1a/1b/2a/3a HCV VLPs vaccine  was reported to produce broader HCV-specific immune responses , 25, butantibody –31. The The aim of this study was to explore a suitable antigen vaccine for the induction of broad-spectrum neutralizing antibodies and an effective cellular immune response against HCV. Thus, we adopt either monovalent or pentavalent formulations of the full-length E1E2 heterodimer from the HCV strains H77 (GT1a), Con1 (GT1b), J6 (GT2a), S52 (GT3a) and HK6a (GT6a) to maximize the simulation of the natural conformation of envelope proteins to improve the breadth and potency of immunity, which represents the majority of HCV cases worldwide. HCV E1E2 proteins were functional and elicited HCV-specific polyclonal neutralizing antibodies in mice. Narrow superiority in the immune response between pentavalent and monovalent E1E2 suggested that conserved epitopes in the full-length E1E2 dimer may be the key elements for a promising HCV prophylactic vaccine.All human hepatocyte Huh7.5.1 cells were provided by Dr. Francis Chisari (Scripps Research Institute), HEK-293T cells were obtained from ATCC, HeLa cells were obtained from ATCC, and CHO cells stably expressing human CD81 (NCBI RefSeq. NM_004356.4), or SRB1 (NCBI RefSeq. NM_001367981.1) were maintained in DMEM supplemented with penicillin, streptomycin, and 10% fetal bovine serum . FreeStyle 293-F cells purchased from ATCC were maintained in SMM 293-TII expression medium .in vitro, purified with a MEGAclear Kit (Ambion), and then transfected into naïve Huh7.5.1 cells with TransMessenger Transfection Reagent (Qiagen). The supernatant that contained virus was collected and used to inoculate naïve Huh7.5.1 cells. This supernatant collection/naïve Huh7.5.1 cell inoculation process was repeated to produce a large volume HCVcc stock and was filtered  from the codon-optimized sequences of H77 , Con1 , J6 , S52  and HK6a . A human IgG1 Fc fragment was inserted and fused to the N-terminus of E2 to obtain an affinity-tagged form of E1E2, and a Flag-tag was added at the N-terminus of E1 to facilitate the detection of E1 and then inserted into the pLVX lentiviral vector. Packaged lentiviruses were generated in HEK-293T cells and transduced into FreeStyle 293-F cells to obtain HEK293F cells that stably expressed Fc-E1E2 under puromycin pressure.Recombinant Fc-E1E2 was purified from FreeStyle 293-F cell extracts using protein A Sepharose 4 Fast Flow  and then concentrated with a 50,000 molecular weight cutoff centrifugal filter unit . For cell extracts preparation, FreeStyle 293-F cells were collected by centrifugation at 1,000 × g for 5 min, washed twice with PBS, and ultrasonic in hypotonic solution , then centrifugation was performed at 1,000 × g for 10 min at 4°C, the collected supernatant was then centrifuged at 20,000 × g for 30 min at 4°C to obtain precipitation. The pellets were then lysed with lysis buffer . The lysate was further clarified by centrifugation at 20,000 × g for 25 min at 4°C and removed impurities through 0.45μM filter.All animal studies were reviewed and approved by the Laboratory Animal Management and Ethics Committee at the Institute of Pathogen Biology, Chinese Academy of Medical Sciences. Female BALB/C mice were purchased from HuaFuKang Biotechnology Co., Ltd.  (6 to 7 weeks old) and were cared for in accordance with institutional guidelines. Eighty female mice (20-22 g) were randomly divided into 8 groups (10 mice per group) and were injected intramuscularly at weeks 0, 2, 4 and 8 with 5 μg of the monovalent Fc-E1E2 protein of H77, Con1, J6, S52 and HK6a, respectively, the human IgG1 Fc protein  or the pentavalent Fc-E1E2 cocktails  mixed with 75 μg of alum  and 7.5 μg of monophosphoryl lipid A (MPLA Vaccigrade) . Adjuvant mixed with the PBS group was set up as a control. Serum was collected at weeks 0, 9.5, 42, and 46. Sera were heat inactivated by incubation at 56°C for 1 h and kept at -80°C until use.Monolayer cells were lysed on ice with lysis buffer  supplemented with 1% protease inhibitor cocktail (Sigma–Aldrich). Then, the lysate was cleared by centrifugation, boiled in loading buffer and loaded onto an 8 to 12% polyacrylamide gel. After electrophoresis, the separated proteins were transferred onto a nitrocellulose membrane , blocked with 10% milk for at least 1 h, and incubated with the diluted primary antibody overnight at 4°C. Secondary antibodies conjugated to IRDye 680 RD or 800CW were used for detection by the Odyssey Infrared Imaging System (LI-COR Biosciences).The final concentration of Fc-E1E2 and human IgG Fc was 10 μM in PBS. The CD spectra were acquired on a Jasco spectropolarimeter  using a 1-nm band width with a 1-nm step resolution from 195 to 260 nm at room temperature. After correction by subtraction of a blank, data were averaged over three accumulations.To analyze the capacity of Fc-E1E2 to bind to the HCV entry factor CD81, 96-well enzyme immunoassay (EIA)/radioimmunoassay (RIA) flat-bottom plates  were coated overnight with recombinant CD81 large extracellular loop , and the plates were blocked with TBST buffer  containing 5% bovine serum albumin (BSA) for 1 to 3 h at 37°C. Then, the cells were incubated with different doses of Fc-E1E2 or BSA for 2 h at 37°C. The cells were incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG H&L antibody  for colorimetric analysis at 450 nm in a 96-well plate reader.To analyze the binding of AR3A antibody  to the FThe surface plasmon resonance experiments were performed at room temperature using a BiaCore T200 with CM5 sensor chips . The surfaces of the sample and reference flow cells were activated with a 1:1 mixture of 0.1 M NHS (N-hydroxysuccinimide) and 0.1 M EDC propyl-N-ethylcarbodiimide) at a flow rate of 10 μL/min. The reference flow cell was left blank. All the surfaces were blocked with 1 M ethanolamine, pH 8.0. The running buffer was HBS-EP . For the binding affinity assays, CD81-LEL was diluted in 10 mM sodium acetate buffer, pH 5.0, and immobilized on the chip at approximately 150 response units. Fc-E1E2 from H77, Con1 J6, S52 and HK6a at gradient concentrations  flowed over the chip surface. After each cycle, the sensor surface was regenerated with 10 mM glycine-HCl pH 2.5. The data were fitted to a 1:1 interaction steady state binding model using BIA evaluation 1.0 software.We performed flow cytometry analysis using CHO cells stably expressing CD81 or SR-BI. Briefly, 10 μg of Fc-E1E2 or BSA was incubated with parental CHO cells or with CHO cells expressing HCV receptors for 2 h at room temperature. After washing with PBS, the cells were incubated with PE-conjugated goat anti-human IgG Fc secondary antibody  and then analyzed on a BD LSRII flow cytometer . The results were analyzed with FlowJo software.5 HeLa cells. After 24 h, the cells were fixed with paraformaldehyde and permeated with 0.25% Triton X-100 in PBS, then incubation with 5% BSA in PBS for 1.5 hours, the cells were incubated with primary antibodies overnight at 4°C, followed by immunostaining with Alexa Fluor 594 or Alexa Fluor 488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories), or directed by Alexa Fluor 647- conjugated primary antibodies (Abcam). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured on a Leica SPW5 confocal microscope and analyzed using Leica confocal microscopy.Immunostaining of HeLa cells was performed using antibodies against Ezrin, Erp57 (with HA tag), P4HB, E1, and E2. Briefly, Fc-E1E2 plasmids were transfected into 1 × 104 cells per well for 12 h. The cells were treated with serially diluted Fc-E1E2 protein or BSA protein for 1 h at 37°C, and then approximately 1 x 103 focus-forming units (FFU)/ml JFH1 HCVcc was added to Huh-7.5.1 cells. After 2 h, the protein-virus mixture was changed to complete DMEM. Forty-eight hours post-infection, the cells were fixed in the original wells with paraformaldehyde and permeated with Triton X-100, followed by immunostaining with the mouse anti-HCV core antibody  and IRDye secondary antibody . Images were obtained on an Odyssey Infrared Imaging System .Huh7.5.1 cells were seeded in 96-well plates at a density of 2 × 10To measure the E1E2-specific antibody responses in serum samples by ELISA, 96-well enzyme immunoassay/radioimmunoassay flat-bottomed plates were coated overnight with 100 ng/well Fc-E1E2  from H77, Con1, J6, S52, and HK6a cells and blocked in 5% BSA. Antisera from vaccinated mice were diluted in TBST containing 1% BSA and added to the plates for 2 h at 37°C followed by the addition of HRP-conjugated anti-mouse IgG secondary antibodies diluted 1:12000. Then washed 5 times in TBST buffer, TMB substrate was added to the plates to develop the color. After adding termination solution, absorbance was read at 450 nm. An absorbance with an optical density (OD) unit more than 0.1 above the prevaccination serum samples was used as the cutoff. The end point titers of the serum were defined as the reciprocal of the maximum serum dilution that had an absorbance above the cutoff.4 per well for 12 h, and the serum samples were heat inactivated at 56°C for 1 h. Serially diluted sera were then mixed with JFH1 HCVcc in complete DMEM to approximately 1.2 x 103 FFU/ml and incubated at 37°C for 1 h. The virus-serum mixture was added to Huh-7.5.1 cells for 4 h at 37°C and replaced with complete DMEM. After 48 h, the cells were fixed and core immunostained on an Odyssey Infrared Imaging System . Immunofluorescence assays were performed to quantify HCV infection by counting the number of foci.For the JFH1 HCVcc neutralization assay, we performed in-cell Western blotting to assess the neutralization of the antisera. Briefly, Huh7.5.1 cells were seeded in a 96-well plate format at 2 × 10For the HCVpp neutralization assay, HCVpp in the presence of 8 g/ml polybrene and 1 μl of 2 M HEPES (pH 7.55) was premixed with heat-inactivated diluted sera for 1 h at 37°C, and then it was added to Huh7.5 cells for 6 h and replaced with fresh culture medium. Forty-eight hours post-infection, cells were processed using the Bright-glo luciferase assay system . Neutralization activity was calculated by comparing the luciferase activity of immune sera to that of a prevaccination serum control at the same dilutions.Neutralization activity was calculated using the following formula: percent neutralization= (pre - post)/pre x 100, where pre - post represents the luciferase activity done or the number of foci after incubating with either the pre- or postvaccination sera.Serum neutralization experiments usually use two duplicate wells for each mouse. Each neutralization curve data set was normalized by the lowest dilution to define the real value for 100% neutralization. After the transformation to neutralization, the highest dilution of sera was set to 0% neutralization. Then, the neutralization curves were fitted with a nonlinear regression model to calculate the ID50 values.Spleens were collected from mice when the animals were euthanized, and isolated splenocytes were stimulated with Fc-E1E2, medium, or human IgG Fc and then analyzed by ELISPOT. Briefly, 96-well polyvinylidene difluoride (PVDF) plates (Millipore) were precoated with anti-mouse IFN-γ capture antibody  or anti-mouse IL-4 capture antibody  at 4°C overnight. Plates were blocked with 200 μL/well of Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin for 2 h at 37°C, and freshly isolated splenocytes were added to the plates. Fc-E1E2, human IgG Fc (control), medium (negative control), or concanavalin A (positive control) was diluted in complete RPMI 1640 medium as a stimulus at a final concentration of 10 μg/mL after incubation for 48 h. Subsequently, the plates were incubated with biotinylated anti-mouse IFN- detection antibody  or biotinylated anti-mouse IL-4 detection antibody  diluted in PBST for 2 h and then with alkaline phosphatase (AP)-conjugated streptavidin (Mabtech) diluted 1:1000 in PBS for 1 h. After washing, nitroblue tetrazolium (NBT)/BCIP (5-bromo-4-chloro-3-indolyl phosphate) substrate (Promega) was added to the plates and incubated for 15–30 minutes to develop the color. The cytokine-secreting cell spots were imaged and counted on a CTL Immunospot reader (Cellular Technology Ltd.)P>0.05, *P<0.05, **P<0.01, ***P<0.001. All P value analyses were calculated by GraphPad Prism V.5.0.Significant differences were calculated with one-way analysis of variance followed by Tukey’s multiple comparison test. Significant values are shown as follows: no significant difference Most subunit HCV vaccine studies are based on E2 alone. However, E1 plays an auxiliary role in stabilizing the conformation of E2. Moreover, E1 and E2 form a heterodimer that is an immunogen targeted by neutralizing antibodies (NAbs). To obtain a recombinant E1E2 heterodimer for the vaccine study, we designed and synthesized eukaryotic constructs expressing full-length E1E2 from genotypes H77 (GT1a), Con1 (GT1b), J6 (GT2a), S52 (GT3a) and HK6a (GT6a) with reference to previous studies . The selThese E1E2 heterodimers were purified using one-step protein A Sepharose affinity purification from membrane protein extracts and were analyzed by Coomassie brilliant blue staining . The purA number of cell surface coreceptors, such as the tetraspanin CD81, scavenger receptor class B type I (SR-BI), claudin-1 (CLDN1) and occludin (OCLN), have been reported to be involved in essential HCV cell entry by interacting with viral glycoproteins (E1 and E2) . TherefoNext, the association between purified E1E2 and the HCV coreceptor CD81-LEL was examined. All five E1E2 proteins reacted in a dose-dependent manner with CD81-LEL in enzyme-linked immunosorbent assay (ELISA) analysis ; thus, t6 at week 9.5, and there was a slight decrease at week 42, indicating that antibody responses were elicited in mice and could last for almost the entire lifespan of mice. Interestingly, the antibody titers of each monovalent E1E2 protein showed no significant difference toward either homogenotypic or heterogenotypic antigens, suggesting that antigen design of this recombinant full-length E1E2 dimer can display conserved intergenotypic epitopes and induce a diversity of antibodies. In addition, because bNAbs mainly target conserved conformational epitopes, we performed native Western blotting to determine the antigen cross-reactivities of these immunized sera. All the immunized sera cross-reacted with native E1E2 proteins from five subgenotypes  was represented as the reciprocal value of the dilution. As shown in 50 of pentavalent ranged from 349.3 to 722.7), especially for UKN2B2.8. H77-immunized sera showed sensitive neutralization reactivity against H77 and Con1 strains. UKN2A2.4 and UKN4.11.1 showed relatively sensitive to all immunized sera than other HCVpp strains. Notably, the immune sera groups did not show obvious isotype specificity against homotypic strains. These results suggested the possibility of cross neutralization activity in all E1E2-immunized sera, especially in pentavalent sera. Limited to the HCVpp panel breadth in our research, the cross-neutralization ability of immunized sera needs to be further assessed using more HCVpp subgenotypes. Idealized analyses between the diverse HCVpp and cell culture-derived HCV (HCVcc) panels should be done in parallel. However, limited by the experimental platform, only an HCVcc  neutralization assay with immunized sera at week 9.5  system to analyze the breath of immunized sera neutralization, which consisted of H77 (GT1a), Con1 (GT1b), UKN2A2.4 (GT2a), UKN2B2.8 (GT2b), S52 (GT3a), UKN4.11.1 (GT4c) and SA13 (GT5a). Due to COVID-19 restrictions, immune serum from 10 to 41 weeks could not be obtained in time, and only a small amount of 9.5-week serum was collected to detect the change in antibody titers. Therefore, pre- and week 46 serum samples were evaluated for their ability to neutralize HCVpp entry into Huh7.5.1 cells. Twofold serial dilutions of sera were examined, and the inhibitory dose to achieve the 50% inhibitory dilution  and the National Natural Science Foundation of China (81871667).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compared to the setup using a laser pointer, the setup using a He-Ne laser gave better results. In the setup using the He-Ne laser, there were correlations with a coefficient of determination R2 ≥ 0.7 at illumination angles of 30°, 45°, and 60°, whereas in the setup using a laser pointer, there were correlations with R2 ≥ 0.7 at illumination angles of 30° and 45°. Mean characteristic features had more correlations with R2 ≥ 0.7 in the case of the angle of illumination of 45° (7 out of 36 correlations) and 60° (11 out of 82 correlations), while R-normalized characteristic features had more correlations with R2 ≥ 0.7 in the case of the angle of illumination of 30° (9 out of 38 correlations) for the setup using the He-Ne laser. Correlation (GLCM) had more correlations with R2 ≥ 0.7 in the case of the setup using a laser pointer . Roughness parameters aS and qS had more correlations with R2 ≥ 0.7 for an illumination angle of 30° (1 out of 2 correlations each), and pS and zS had more correlations with R2 ≥ 0.7 for an illumination angle of 45° (4 out of 19 correlations each) in the case of the setup using a laser pointer. The novelty of this work is (1) being a correlation study between 3D surface roughness and speckle pattern using a He-Ne laser and spatial filter, and (2) being a comparison study between two experimental setups on the correlation between 3D surface roughness and speckle pattern.Correlation between 3D surface roughness and characteristic features extracted from laser speckle pattern was done using an inexpensive laser pointer and a digital single lens reflex (DSLR) camera in previous research work. There had been no comparison work done between the experimental setup which uses a laser pointer, which has a diode laser as the laser source, and the experimental setup, which uses a He-Ne laser as the laser source. As such, in the current work, a comparison study between two experimental setups was carried out. One experimental setup was using a He-Ne laser, spatial filter, and charged coupled device (CCD) camera, while another experimental setup was using a laser pointer and DSLR camera. The laser beam was illuminated at angles of 30°, 45°, and 60° from the horizontal. When a laser beam falls on the surface, the beam gets scattered, and the scattered beam undergoes interference and produces speckle patterns which are captured using a camera. Using a Matlab program, the gray level co-occurrence matrix (GLCM) characteristic features, such as contrast (GLCM), correlation (GLCM), energy (GLCM), entropy (GLCM), homogeneity (GLCM), and maximum probability, and non-GLCM characteristic features, such as mean, standard deviation (STD), uniformity, entropy, normalized R, and white-to-black ratio (W/B), were extracted and correlated with 3D surface roughness parameters. The coefficient of determination (R Surface roughness refers to finely spaced irregularities formed during the machining process . SurfaceOn the surface of soft materials, the stylus tip could cause a scratch. White light interferometers ,5, the fStatistical properties ,9,10, waCurrent vision approaches extract characteristics to correlate with 2D roughness parameters ,20,24,25Two samples with five surfaces each were machined on a CNC 5-axis milling machine  with a four flute high-speed steel (HSS) end mills cutter with a diameter of 12 mm. One of the most essential advantages of high-speed steel is its ability to cut through materials at high speeds. Because of the alloy’s unique combination of hardness, wear resistance, and high-temperature characteristics, one may make use of this benefit. HSS tools are also less expensive than carbide tools, making them an excellent choice for high-mix, low-volume applications. Sample 1 and sample 2 are the names of the two reference samples. aS);Arithmetic mean height (qS);Root-mean-square height (pS);Maximum peak height (vS);Maximum valley depth (zS);Maximum height (10zS);Ten point height (skS);Skewness (kuS);Kurtosis (dqS);Root-mean-square gradient (drS).Developed interfacial area ratio  of 4, 5.6, 8, 11, and 16. The CCD camera was fixed relative to the machined surface. This laser speckle pattern was captured using a CCD camera  fitted with a TV zoom lens (1:1.2/12.5–75) and an extension tube of size 20 mm. The viewing direction of the CCD camera was placed normal to the machined surfaces. The images were stored in Tag Image File Format (TIFF). The CCD camera and the machined surface together could be rotated. Experiments were conducted for the combination of illumination angle of the laser beam of 30°, 45°, and 60°, and camera aperture sizes  and converted from an RGB image to a grayscale image. f-number of 16, and shutter speed of 1/100 s for experimental setup 2. In the case of experimental setup 2, using MATLAB 2021a software, the speckle pattern image was cropped to a size of 101 × 101 pixels (maximum possible size) and converted from an RGB image to a grayscale image. 2) from the correlation study between the extracted characteristic features and 3D surface roughness were evaluated.The grayscale image was not subjected to any filtering process to avoid the loss of data caused by filtering. Characteristic features based on the histogram, such as mean intensity, root-mean-square intensity, energy, entropy, and texture-based parameters, such as normalized roughness, and gray level co-occurrence matrix (GLCM)-based parameters, such as maximum probability, correlation, contrast, energy, homogeneity, and entropy, were extracted from the grayscale images. To differentiate the energy and entropy descriptors that are obtained from histogram-based and GLCM-based parameters, energy and entropy descriptors based on GLCM shall be addressed as energy (GLCM) and entropy (GLCM). From the binary image, the white-to-black pixels ratio was obtained as a characteristic feature. Coefficients of determination  featuresEquations (1)–(12) ,27 that m obtained from original image f of size M × N, given by Equation (1).f is the gray value of the pixel at coordinate .○Standard deviationMean of the gray value of the image σ of an image is given by Equation (2).jr is the jth gray level;L is the total possible gray level value;p(jr) is the probability of occurrences of jr;m is the mean of gray values of the image.○EnergyThe standard deviation jr is the jth gray level;L is the total possible gray level value;p(jr) is the probability of occurrences of j.r○EntropyThe energy descriptor, which is also known as uniformity, measures how pixel values are distributed, along with the gray level range, and can be calculated for the grayscale image using Equation (3).jr is the jth gray level;L is the total possible gray level value;p(jr) is the probability of occurrences of jr.○The normalised descriptor of roughnessTexture featuresThe entropy descriptor provides information about the complexity of the image, as given by Equation (4).R is as given in Equation (5).2σ is variance;L is the total possible gray level value.Gray level co-occurrence matrix (GLCM)The normalised descriptor of roughness ir and jr occur in image f in the position specified by the displacement vector . In this work, as in the MATLAB software, default values of the displacement vector and orientation of d = 1 and θ = 0° were used. The matrix is normalized as given in Equation (6).gN is the normalized gray level co-occurrence matrix;g is the element of the gray level co-occurrence matrix.Histogram-based texture descriptors do not provide any information about the spatial relationship among pixels. This information can be obtained using the gray level co-occurrence matrix (GLCM). The matrix holds the information of the number of times pixels with intensities gN.○Maximum probability (GLCM) is given by Equation (7).○Correlation (GLCM) is given by Equation (8).iµ is the mean of the row sums of gN;jµ is the mean of column sums of gN;iσ is the standard deviation of row sums of gN;jσ is the standard deviation of column sums of gN.The following texture-based features are computed using a normalized GLCM, ○Contrast (GLCM) is given by Equation (9).○Energy (GLCM) is given by Equation (10).○Homogeneity (GLCM) is given by Equation (11).○Entropy (GLCM) is given by Equation (12).○Total white pixels to total black pixels ratio (W/B).From the binary image, the following characteristic features were extracted:2 ≥ 0.7 for an illumination angle of 30° for experiment setup 1. There are 38 correlations with an R2 ≥ 0.7. GLCM characteristic features account for 11 of the 38 correlations, while non-GLCM characteristic features account for 27 of the 38. 2 ≥ 0.7. A characteristic feature, R normalised, had 9 out of 38 correlations with R2 ≥ 0.7. 2 ≥ 0.7. The 3D roughness parameter 10zS had 15 out of 38 correlations. 2 ≥ 0.7 occurs for each f-number setting. The camera setting with f-number 8 had 19 correlations with R2 ≥ 0.7. Maximum probability (GLCM) vs. drS had the highest R2 of 0.8742.2 ≥ 0.7 for an illumination angle of 45° for experiment setup 1. It was found that there were 36 correlations with R2 ≥ 0.7. Of these, 11 out of 36 correlations were GLCM characteristic features, and 25 out of 36 correlations were non-GLCM characteristic features. 2 ≥ 0.7. The mean characteristic feature had 7 out of 36 correlations with R2 ≥ 0.7. 2 ≥ 0.7. The 3D roughness parameter S10z had 15 out of 36 correlations. 2 ≥ 0.7 occurs for each f-number setting. The camera setting with f-number 5.6 had 15 correlations with R2 ≥ 0.7. The highest R2 was for maximum probability (GLCM) vs. Sdq, with R2 = 0.9297.2 ≥ 0.7 occurred for an illumination angle of 60° for experiment setup 1. It was found that there were a total of 82 correlations with R2 ≥ 0.7. Of these, 39 out of 82 correlations were with GLCM characteristic features, while 43 out of 82 correlations were with non-GLCM characteristic features. 2 ≥ 0.7. The mean characteristic feature had 11 out of 82 correlations with R2 ≥ 0.7. 2 ≥ 0.7. The 3D roughness parameter 10zS had 24 out of 82 correlations with R2 ≥ 0.7. 2 ≥ 0.7 occurs for each f-number setting. The camera setting with f-number 5.6 had 30 correlations with R2 ≥ 0.7. The highest R2 value was for maximum probability (GLCM) vs. drS, with R2 = 0.9384.2 ≥ 0.7 for an illumination angle of 30° for experiment setup 2. It was found that there were two correlations with R2 ≥ 0.7 at the camera setting with an f-number of 8 and shutter speed of 1/200 s. There was no correlation for non-GLCM characteristic features. The only characteristic features with correlation were correlation (GLCM) and roughness parameters aS and qS, each having a correlation of 1 out of 2. The highest R2 was for correlation (GLCM) vs. qS, with R2 = 0.7438.2 ≥ 0.7 for an illumination angle of 45° for experimental setup 2. It was found that there were 19 correlations with R2 ≥ 0.7. An f-number of 8 with a shutter speed of 1/50 s had two correlations with R2 ≥ 0.7. An f-number of 16 with a shutter speed 1/100 s had eight correlations with R2 ≥ 0.7. An f-number of 22 with a shutter speed of 1/100 s had two correlations with R2 ≥ 0.7. An f-number of 22 with a shutter speed 1/200 s had seven correlations with R2 ≥ 0.7. In total, 13 out of 19 correlations were GLCM characteristic features, and 6 out of 19 correlations were non-GLCM characteristic features. The correlation (GLCM) characteristic feature had 4 out 19 correlations, and roughness parameters pS and zS each had 4 out of 19 correlations. The highest R2 is for energy (GLCM) vs. 10zS, with R2 = 0.8955.2 ≥ 0.7 at illumination angles of 30°, 45°and 60°, whereas experimental setup 2 had correlations with R2 ≥ 0.7 only at illumination angles of 30° and 45°. In the case of experimental setup 1, there were more non-GLCM characteristic features compared with GLCM characteristic features. The mean characteristic feature had more correlations with R2 ≥ 0.7 for illumination angles of 45° (7 out of 36 correlations) and 60° (11 out of 82 correlations), and R-normalized had more correlations with R2 ≥ 0.7 in the case of illumination angles of 30° (9 out of 38 correlations). Roughness parameter 10zS had more correlation with R2 ≥ 0.7 for all of the illumination angles  in the experimental setup 1. For experimental setup 2, correlation (GLCM) characteristic features had more correlations with R2 ≥ 0.7 . Roughness parameters aS and qS had more correlations with R2 ≥ 0.7 for an illumination angle of 30 (1 out of 2 correlations each), and pS and zS had more correlations with R2 ≥ 0.7 for an illumination angle of 45 (4 out of 19 correlations each) for experimental setup 2. The reason for better correlation using experimental setup 1 compared to experimental setup 2 could be due to the expansion of the laser beam, which reduced the intensity of the laser beam that fell onto the surface. In this way, pixel saturation can be avoided. Another reason could be due to cleaning the laser beam of noise using a spatial filter. Experimental setup 1 had correlations with R2 ≥ 0.7, and in the case of experimental setup 2, there were no correlations in the case of an illumination angle of 60°. The illumination angle of 60° gave the highest number of correlations with R2 ≥ 0.7 in the case of experimental setup 1 (82 correlations), and the illumination angle of 45° gave the highest number of correlations with R2 ≥ 0.7 in the case of experimental setup 2 (19 correlations). Mean characteristic features had more correlation with R2 ≥ 0.7 in the case of an angle of illumination of 45° and 60°, and R normalized characteristic features had more correlation with R2 ≥ 0.7 in the case of an angle of illumination of 30° for experimental setup 1. Correlation (GLCM) had more correlation with R2 ≥ 0.7 in the case of experimental setup 2. Roughness parameters aS and qS had more correlation with R2 ≥ 0.7 for an illumination angle of 30°, and pS and zS had more correlation with R2 ≥ 0.7 for an illumination angle of 45° in the case of experimental setup 2. A spatial filter that cleans the laser beam and then expands the cleaned beam could be the reason for the better result in the case of experimental setup 1. Previous works focus more on correlation studies involving 2D roughness parameters, while current work is focused on the correlation involving 3D surface roughness and characteristic features extracted from the laser speckle pattern. The current work shows that although experimental setup 1 gives better results compared to experimental setup 2, experimental setup 2 uses inexpensive components and is simple compared to experimental setup 1, and there is room for improvement in future work whereby the experimental setup 2 should be carried out by replacing the DSLR camera with a webcam. The novelty of this work is that it is (1) a correlation study between 3D surface roughness and speckle pattern using a He-Ne laser and spatial filter; and (2) a comparison study between two experimental setups on the correlation between 3D surface roughness and speckle pattern.From the results, it can be seen that there are good correlations between characteristic features and 3D surface roughness in both the experimental setups. Experimental setup 1 gives a better correlation compared to experimental setup 2. In the case of experimental setup 1, all the illumination angles had correlations with R