We investigated the diagnostic values and artifact severities of 3D-T1W sequences in the diagnosis of cerebrospinal fluid (CSF) leakage.We retrospectively reviewed 3-tesla contrast-material enhanced MR cisternography exams of 22 patients with suspected CSF leakage in 4 years. The presence of the artifacts on 3D-T1W data was evaluated using a 4-point scale . Agreements between CSF leakage results of the 3D-T1W sequences and consensus decisions were evaluated via kappa values. Artifact scores were analyzed by Fisher’s exact test.The most compatible techniques with the consensus diagnoses were fat-saturated 3D-T1W-SPACE and 3D-T1W-VIBE sequences. The most artifact containing the 3D-T1W sequence was 3D-MPRAGE.3D-SPACE and 3D-VIBE are more successful in evaluating CSF leakages compared to 3D-MPRAGE. 3D-SPACE has lower artifact scores compared to 3D-VIBE and 3D-MPRAGE sequences. Contrast-material enhanced MR cisternography (CE-MRC) provides physiologic and morphologic details of the skull base without radiation, and it has quite a high accuracy for cerebrospinal fluid (CSF) leakages . RoutineGenerally, CE-MRC examinations are made in the prone position. Therefore, more motion artifacts are observed in CE-MRC images compared to routine MR exams obtained in the supine position. All isotropic 3D-T1W sequences still lack clinical validation for CE-MRC in patients with CSF leakages. In this retrospective study, we compared CSF leakage detection capabilities and artifact scores of three commercially available 3D T1W MRI sequences at 3-tesla (3T) in patients with suspected CSF leakage. According to our knowledge, there is no comparative study about the artifacts and diagnostic roles of 3D T1W MRI sequences at 3T in patients with suspected CSF leakage.According to respective institutional guidelines, an institutional review board approval was not required for this type of retrospective study. All the procedures being performed were part of the routine care. Informed consent was obtained from all subjects before the exams. In pediatric cases, informed consent was obtained from both parents and the patient. Patients who have (or suspected) otorhinorrhea and underwent 3T CE-MRC in 4 years period were included in this retrospective study. After a retrospective analysis of these patients and the CE-MRC images, 22 patients with CE-MRC obtained with 3D-T1W sequences were included in the study.Patients who have CSF leakage or otorhinorrhea suspicion based on neurosurgeon, otolaryngologist, or neurologist evalualtion, and patients with CE-MRC exam and 3T 3D-T1W  images were included in the study.Patients without detailed clinical and follow-up information were exluded from the study.All CE-MRC exams were acquired on a 3T MRI unit  with a 20-channel head coil. After obtaining noncontrast material-enhanced 3D-T1W and heavily 3D-T2W (noncontrast MR cisternography) images, lumbar puncture was performed by using a 22-gauge needle at the L3–L4 level under sterile conditions. Subsequently, 2 mL of saline mixed with 0.5 mL of gadobutrol was injected intrathecally for all patients. During transfer to the 3T MR unit, the patient is kept in bed in a prone position. Three hours after the gadobutrol injection, CE-MRC images were obtained in a prone position . All patients were clinically observed in the hospital for 24 h after the procedure. Details of CE-MRC acquisitions are given in MRC images were evaluated on a PACS workstation by two neuroradiologists (6 and 15 years of neuroradiology experience) that were blinded to clinical data using a Leonardo workstation . Curved, freehand, or oblique multiplanar reconstructions and maximum intensity projection images were generated from 3D datasets. An extension of spontaneously hyperintense CSF on 3D heavily T2W images (for NCE-MRC) and/or hyperintense contrast-material-enhanced CSF on postcontrast 3D T1W images (for CE-MRC) from the cerebral cistern into the paranasal sinuses, nasal cavity, or middle ear cavity was regarded as the positive finding for CSF leakage for each sequence, as stated in the literature . Pre- anThe final or definite diagnosis of the presence of CSF leak was established according to the physical exam, clinical, beta-2-transferrin test, thin-section CT, CT cisternography, and CE-MRC findings. If present, surgical and endoscopic results were used to confirm the diagnosis. Any disagreement related to the final diagnosis was resolved by consensus of the authors. All cases were followed during regression of the symptoms after CE-MRC exams and surgical and endoscopic therapies to verify the CSF leakage presence/localization and complications.Moreover, the presence of the artifacts on 3D-T1W data was evaluated using a 4-point scale . Follow-up clinical exam and imaging data were used to evaluate changes after the intrathecal gadolinium administration.Data were analyzed using SPSS v.21.0 software . Age and follow-up time related to the cases were given as mean, standard deviation, median, and minimum-maximum values. Agreement between techniques was evaluated via kappa values. Artifact scores of sequences were given as number and frequency. Artifact scores of the sequences were compared via Fisher’s exact test. Some 3D-T1W images or sequences of some patients were not available related to the PACS or hospital information system updates. These sequences were not included in the statistical analysis. p < 0.05 was accepted as statistically significant.CSF leakage was not detected in 5 cases. The arachnoid cyst was detected in 2 cases. In 11 cases, there were also CTC examinations obtained in the same week with CE-MRC. Indication of CE-MRC exam was rhinorrhea in 19 cases and otorrhea in 3 cases. CSF leakage was detected in 17 cases based on consensus sessions . Eight of these had trauma history, and one of them had postoperative leakage. The most common leakage locations were cribriform plate (in 6 cases) and ethmoid sinuses (in 5 cases) . The ageAll cases were followed for at least 3 years. No major complication was detected in the patients. Minor complications  were observed in three patients. None of the patients with negative CE-MRC reports had a new finding or recurrence of rhinorrhea during this follow-up period.In patients with CSF leakage, determination of the leakage site in the preoperative period is crucial for increased surgical success rate and reducing the possible complications ,7. Altho3D-T1W data were relatively newly introduced at 3T. 3D-T1W sequences still lack validation for clinical use in patients with suspected CSF leakage. We observed that the multiplanar reconstruction capabilities of 3D-T1W sequences were very useful for the demonstration of CSF leaks in complex-shaped tiny regions.Three-tesla isotropic 3D-T1W-SPACE has a shorter acquisition time and a larger coverage area (including the whole cranium) than multiplanar 2D-T1W spin-echo-based acquisitions . IsotropIt was reported that 3D-T1W-SPACE and 3D-VIBE have higher contrast-to-noise ratio values and visual conspicuity compared with 3D-MPRAGE . Subtly This study has several limitations. Our first and most important limitation is the low number of study cases. The other limitation is the absence of surgical or beta-2-transferrin test results in all cases of CSF leakage. These limitations are also present in many studies about cisternography or CSF leakages . Future In conclusion, 3D-T1W-SPACE and 3D-VIBE are more successful in evaluating CSF leakages compared to 3D-MPRAGE. 3D-T1W-SPACE is less susceptible to movement or susceptibility artifacts compared to 3D-VIBE and 3D-MPRAGE.


Picrophilus torridus is a euryarchaeon that can thrive under conditions of extremely low pH (0.7), and thus far no reports have been published regarding DNA methylation in this extremophile. This study reports the first experimentation examining DNA methylation in P. torridus. We find the genome to carry methylated adenine (m6A) but not methylated cytosine (m5C) residues. The m6A modification is absent at GATC sites, indicating the absence of an active Dam methylase even though the dam gene has been annotated in the genome sequence. Two other methylases have also been annotated in the P. torridus genome sequence. One of these is a part of a Type I restriction-modification system. Considering that all Type I modification methylases characterized to date target adenine residues, the modification methylase of this Type I system has been examined. The genes encoding the S subunit (that is responsible for DNA recognition) and M subunit (that is responsible for DNA methylation) have been cloned and the recombinant protein purified from E.coli, and regions involved in M-S interactions have been identified. The M.PtoI enzyme harbors all the motifs that typify Type I modification methylases, and displays robust adenine methylation in in vitro assays under a variety of conditions. Interestingly, magnesium is essential for enzyme activity. The enzyme displays substrate inhibition at higher concentrations of AdoMet. Mutational analyses reveal that Motif I plays a role in AdoMet binding, and Motif IV is critical for methylation activity. The data presented here lays the foundation for further research in the area of DNA methylation and restriction-modification research in this most unusual microorganism.DNA methylation events mediated by orphan methyltransferases modulate various cellular processes like replication, repair and transcription. Bacteria and archaea also harbor DNA methyltransferases that are part of restriction-modification systems, which serve to protect the host genome from being cleaved by the cognate restriction enzyme. While DNA methylation has been exhaustively investigated in bacteria it remains poorly understood in archaea.  The primary DNA methylation mark in eukaryotes is m5C, while in bacteria the m6A methylation mark is the most widely prevalent. The marking of DNA by methylation has two distinctive roles: one, it modulates DNA interactions with proteins, thereby having an impact on DNA replication, DNA mismatch repair and gene expression, and second, it protects the bacterial and archaeal genome from self-destruction by their own restriction systems  as the methyl-group donor and are either orphan methyltransferases or a component of restriction-modification systems. Orphan methyltransferases (which do not have cognate restriction endonucleases) are associated with methylation events that regulate DNA-related transactions like replication and transcription. The Dam methylase which targets adenine methylation in the sequence 5′-GATC-3′ is the best known of these . Methylavia their S (specificity) subunit, modify the DNA at the recognition sequence via their M (modification) subunits, and cleave DNA through their R (restriction) subunits hundreds of base pairs away. While the restriction endonuclease exists in active form only as part of the multisubunit 1S2M2R enzyme, the modification methylase can also exist in active form independent of the R subunits, as a complex with the S subunit which it depends on for DNA sequence recognition (M2S1).The Types I-III R-M systems differ in their subunit structure and organization, recognition sequence and cleavage site, and cofactor requirements. In the widely found Type II R-M systems, the modification methylase and the restriction endonuclease functions reside in independent enzymes that recognize the same palindromic sequence, with the endonuclease cleaving DNA within the (non-methylated) recognition sequence or just beyond. Type I and Type III R-M systems are complex multisubunit enzymes, with the modification methylase and restriction endonuclease functions residing in the same enzyme albeit different subunits . Type IIThermococcus onnurineus where over 2,000 sites of m6A were detected in addition to several sites of m4C  sequencing has led to the analysis of the DNA methylomes of several bacteria and archaea. In a landmark effort, s of m4C , and theectively . The DNATCT/C-3′ , and theTCT/C-3′ .Thermoplasma acidophilum  was cultured as described earlier  and PtS-R (5′-TACTCGAGATCACCACTTATTTTCAC-3′), with the proof-reading enzyme Phusion DNA polymerase . The amplicon was cloned into pUC19, followed by subcloning into the BglII-XhoI sites of the pETDuet-1 vector , thus obtaining the pET-Duet/PtS clone. The ~1.7 kb gene encoding the M subunit was similarly amplified using the primers PtM-F (5′-TAGAATTCGATGGCAGATTTAAACTGG-3′) and PtM-R (5′-TAGCGGCCGCTTC ATAGTAACCAAG-3′), the amplicon cloned into pUC19, and then subcloned into the EcoRI-NotI sites of pET-Duet and pET-Duet/PtS, thus obtaining plasmids pET-Duet/PtM and pET-Duet/PtSM, respectively.The ~1.3 kb gene encoding the S subunit was amplified off 2.3.Δ537-576-R (5′-TAGCGGCCGCTTTATTGTCTATATATAAAG-3′). The ~1.6 kb amplicon thus obtained was cloned into pJET vector followed by subcloning into the EcoRI-NotI sites of pETDuet/PtS, yielding the plasmid pETDuet/PtSMΔ537-576. The NPPW and xxGxxG mutants were created by overlap PCR. To mutate the NPPW motif, the N-terminal part of the gene was amplified using primers PtM-F and PtM-N360A-W363A-R (5′-CTGGTTCGCTGGAGGCGCCG CAAC-3′) while the C-terminal part of the gene was amplified using primers PtM-N360A-W363A-F (5′-GTTGCGGCGCCTCCAGCGAACCAG-3′) and PtM-R. The full length amplicon obtained by amplification with PtM-F and PtM-R primers was cloned into pJET vector followed by subcloning into the EcoRI-NotI sites of pETDuet/PtS, yielding the plasmid pETDuet/PtSMN360A-W363A. To mutate the xxGxxG motif, the N-terminal part of the gene was amplified using primers PtM-F and PtM-G284S-R (5′-CCTGCAGTTGAACATGCCGG-3′) while the C-terminal part of the gene was amplified using primers PtM-G284S-F (5′-CCGGCATGTTCAACTGCAGG-3′) and PtM-R. The full length amplicon obtained using primers PtM-F and PtM-R was cloned into pJET vector followed by subcloning into the EcoRI-NotI sites of pETDuet/PtS, yielding the plasmid pETDuet/PtSMG284S.All mutageneses were carried out through PCR using Phusion DNA polymerase. For creating PtM mutants the plasmid pUC/PtM was used as template. The C-terminal deletion mutant was created by PCR using the PtM-F primer in combination with PtM2.4.211-222(A-G) was created using Overlap PCR. The N-terminal fragment of the mutant amplicon was produced using primers PtS-F and PtS211-222(A-G)-R (5′-GGCCCCGGCCC CAGCTCCACCTGCCCCTGCTCCTGCCAACTGTATTTCCTT-3′) and the C-terminal fragment of the mutant amplicon was produced using primers PtS211-222(A-G)-F (5′-GCAGGAGCAGGGGCAGGTGGAGCTGGGGCCGGGGCCTTCTGAAGGCATC-3′) and PtS-R. The full-length mutated gene was amplified using PtS-F and PtS-R and ligated into the BglII-XhoI sites of pET-Duet vector to create plasmid pET-Duet/PtS211-222(A-G). This was followed by cloning the PtM gene into the EcoRI-NotI sites of pETDuet/PtS211-222(A-G) to create plasmid pETDuet/PtS211-222(A-G)M.M.PtoI/S2.5.E.coli BL21 Codon Plus cells  that had been transformed with their pET-Duet clones. Expression of the wild type or mutant proteins was induced in cells growing exponentially (OD600 = 0.6–0.8) at 37°C using 1 mM IPTG. Cells were allowed to grow for a further 3 h at 37°C before harvesting by centrifugation and resuspension in lysis buffer  containing protease inhibitors and lysozyme. This was followed by incubation of the cell suspension on ice for 20 min and lysis by sonication. After clarification of the lysates by high speed centrifugation they were subjected to cobalt-affinity chromatography using TALON metal affinity resin (Clonetech). The lysates were loaded on the column resin, the column extensively washed (with 100 mM Tris.Cl (pH 8), 1 M NaCl, 10% glycerol), and the bound proteins eluted using 100 mM Tris.Cl (pH 8), 250 mM imidazole, 10% glycerol. The M subunit of M.PtoI was similarly purified from bacteria transformed with pET-Duet/PtM.The M.PtoI enzymes were expressed in 2.6.P. torridus cultures as described above. For this, 1 μg genomic DNA was digested with EcoRI and then denatured with 0.3 N sodium hydroxide for 30 min. This was followed by neutralization with 1 M ammonium acetate before spotting on Hybond N+ nylon membrane  using the Bio-Dot apparatus . The membrane was baked at 80°C for 2 h before probing with anti-m6A antibody  or anti-m5C antibody . This was done by blocking the baked dot blots with 10% skim milk (Difco Laboratories) for an hour at room temperature, washing the blots twice with 1X PBS-T, and then incubating them overnight at 4°C with the anti-m6A or anti-m5C antibodies (1:1000 dil in 1X PBS). Following three washes with 1X PBS-T the blots were incubated with HRP-labelled secondary antibody  for an hour and developed using a chemiluminescence method.Dot blot assay was used to analyze genomic DNA that was isolated from 2.7.−dcm−E.coli dam strain . Reactions (10 μl) were typically carried out in a Tris-acetate based buffer  containing 100 μM S-adenosyl methionine , 12 mM 2-mercaptoethanol, 20 mM sodium chloride, 1 μg DNA and 1.4 μM M.PtoI, at 55°C for 1 h. Reactions were stopped with 100 mM EDTA. Reactions were analyzed by dot blot assay as described above. The blots were quantified using ImageJ analysis, and the ratios of m6A with reference to E.coli genomic DNA spotted on the same blot was determined. Each experiment was done thrice. The graphs depict average values of three experiments with error bars representing standard deviation.DNA methylation assays were performed using a dot blot-based assay with lambda DNA (as the substrate) that had been isolated from phage particles hosted by an 2.8.CD spectroscopy was carried out as earlier . Briefly2.9.protein homology/analogy recognition engine version 2.0) online server  regardless of the pH at which it is growing, although displaying lower levels of methylation at pH 0.7  we exploited the property of the DpnI restriction enzyme that cleaves methylated but not unmethylated GATC sites. Genomic DNA isolated from Picrophilus cells grown in medium of pH 1.0 was thus subjected to DpnI digestion, and it was observed that while E.coli genomic DNA was cleaved by the enzyme the P. torridus genomic DNA was not dam gene, the euryarchaeon has genes encoding a Type II R-M system and a Type I R-M system. All Type I modification methylases characterized to date are m6A methylases, and thus we investigated the modification methylase of the P. torridus Type I R-M system. The three genes of the P. torridus Type I R-M system encoding for R, M and S subunits lie in a single operon on the lower strand of the circular genome (An examination of the r genome . The inv3.2.The M.PtoI genes (designated herewith as PtM and PtS for M and S subunit genes respectively) were cloned by amplification using primers designed against the published PTO0078 and PTO0077 sequences (encoding M and S subunits respectively), as described in Materials and Methods. The pUC clones obtained were sequenced for verification. The sequences of the M and S subunits of M.PtoI were compared with those of typical Type I modification methylases EcoKI and EcoR124I as well as the identified archaeal Hvo MTase (HVO_2270–71), using Clustal Omega and BLASTp analyses .5 Whilem6A and m4C methylases are typified by the presence of nine conserved motifs, identified more than 25 years ago by analyzing the sequences of over 40 DNA MTases . By exam3.3.While the structures of the individual subunits of Type I R-M systems have been solved in case of several enzymes, the holoenzyme (M2S1) structures of only two Type I modification methylases are available to date. The EcoKI modification methylase structures (pdb IDs: 2Y7C and 2Y7H) were proposed based on single particle electron microscopy constructions at 18 Å resolution . The morThe most reliable model obtained for the M subunit used the EcoR124I enzyme as template , over ~97% coverage (amino acids 6–106 and 121–437). The S subunit of Type I systems harbors two TRDs (target recognition domains) that are oriented inverted with respect to each other. Each TRD carries a globular domain and an alpha-helical dimerization domain  through a four-helix bundle that comprises the C-terminal helical domains of the two M subunits  was expressed in E.coli using the pET-Duet expression vector which allows the simultaneous expression of two proteins from two separate T7lac promoters, with one of the two proteins being expressed in fusion with the His tag at the N-terminus. The M and S subunits were thus simultaneously expressed in BL21 Codon Plus E. coli cells from the pETDuet/PtSM clone (as described in Methods) such that M was His-tagged. The cell lysates were subjected to cobalt affinity-based chromatography. SDS-PAGE analysis revealed the eluate to carry both subunits in stoichiometric amounts, leading us to conclude that the M.PtoI holoenzyme (M2S1) assembles in the host bacterium, allowing the S subunit to co-purify with the His-tagged M  (described in “Methods”). When the recombinant M.PtoI/S211-222(A-G) protein was purified from E.coli using cobalt affinity chromatography it was found that there was a substantial increase in the ratio of M:S in the eluate fractions  were replaced by a 12 amino acid stretch of alternate glycine and alanine residues to create M.PtoI/Sractions .E.coli cells from respective pET/Duet clones carrying either M or S genes. As all M subunits were tagged with His at their N-terminus, they were immobilized on Co-affinity resin. Whole cell lysates harboring recombinant S subunit (wild type or mutant) were added to the reaction, and after washing off the unbound fractions, the bound fractions (M subunits plus any interacting S subunits) were eluted using imidazole and analyzed. As seen in Δ537-576 subunit (lane 6). The mutant S211-222(A-G) subunit interacts with M subunit to a significantly lesser extent than wild type S (compare lane 7 with lane 5). These data confirm the role of the C-terminus of M subunit and residues 211–222 of S subunit in maintaining M-S interactions.M-S interactions in wild type and mutant M.PtoI proteins were also checked using purified proteins. For this, M and S subunits were separately expressed in 3.4.−dcm− E.colidam strain. The reactions were analyzed in dot blot assays using anti-m6A antibody, as described in Methods. Activity assays were initiated at 55°C for 1 h in Tris-acetate buffers whose pH ranged over 3 to 7, in the presence of sodium chloride ranging over 0–100 mM. As seen in The ability of the purified M.PtoI to methylate DNA at adenine residues was analyzed using unmethylated lambda DNA isolated from phage that had been grown in a 3.5.E.coli cells from pETDuet/PtSMG284S and pETDuet/PtSMN360A-W363A, respectively. The mutant enzymes M.PtoI/MG284S and M.PtoI/MN360A-W363A were purified  and the asparagine side chain of the NPPW motif possibly polarizes the amino group, promoting the direct transfer of the methyl group from AdoMet to the adenine N6 position  does not appear to be harboring endogenous AdoMet when purified from E.coli since no catalysis is detected at 0 μM AdoMet  is methylated by a Type I adenine modification methylase  exhibit DNA methylation. We find evidence for m6A but not m5C methylation in P. torridus, and the absence of GATC methylation indicates that the organism lacks an active Dam. Our results suggest that the Type I R-M system annotated in the P. torridus genome sequence is active and mediates m6A methylation. We were unable to analyze the in vivo role of M.PtoI as Picrophilus species are not amenable to genetic manipulations. Attempts to carry out complementation analyses in E.coli dam− strain  did not succeed as the M and S subunits did not get expressed (from the tetracycline-driven promoter system in pASKIBA43PLUS) in this strain, which is deficient in argU, ileY, and leuW tRNA genes (Taken together, the data presented here establish that even organisms growing in such extreme conditions (as NA genes . Future The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SS and MG designed the project. PG and AS performed the research and prepared the figures. PG, AS, MG, and SS analyzed the data. SS wrote the paper. All authors contributed to the article and approved the submitted version.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.


S156P with an additional E protein S156P mutation were successfully rescued and characterized. Growth kinetics studies showed that the two chimeric viruses replicated to similar titers as the parental 180P virus in cells. Animal studies also revealed that the virulence and neuroinvasiveness of the 180P/JEV-prM-E chimeric virus was decreased in mice inoculated intracerebrally (i.c.) and intranasally (i.n.), respectively, compared to the wild-type JEV strain. However, the chimeric 180P/JEV-prM-E virus was still more virulent than the parent 180P vaccine in mice. Additionally, the introduction of a single ES156P mutation in the chimeric virus 180P/JEV-prM-ES156P further attenuated the virus, which provided complete protection against challenge with a virulent JEV strain in the mouse model. These results indicated that the FX2010-180P could be used as a promising backbone for flavivirus vaccine development.Since its outbreak in 2010, Tembusu virus (TMUV) has spread widely throughout China and Southeast Asia, causing significant economic losses to the poultry industry. In 2018, an attenuated vaccine called FX2010-180P (180P) was licensed for use in China. The 180P vaccine has demonstrated its immunogenicity and safety in mice and ducks. The potential use of 180P as a backbone for flavivirus vaccine development was explored by replacing the pre-membrane (prM) and envelope (E) genes of the 180P vaccine strain with those of Japanese encephalitis virus (JEV). Two chimeric viruses, 180P/JEV-prM-E and 180P/JEV-prM-E The encoded polyprotein is cleaved into three structural proteins  and seven non-structural proteins by viral and host proteases , dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV) . The Flaroteases . The strormation , while tormation . Previouavivirus , and theavivirus . The E pFlavivirus genus in the Flaviviridae family, was first isolated from mosquitoes in Malaysia in 1955  and JEV attenuated vaccine SA14-14-2 was prepared as described previously (Baby Hamster Syrian Kidney cells (BHK-21) and Vero-E6 cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) , supplemented with 10% fetal bovine serum (FBS) , 100 U/mL of penicillin, and 100 μg/mL of streptomycin  at 37°C in a 5% COeviously . The prM2.2.S156P viruses, the plasmids p180PT7–1-455, pJEV-prM-E, and pJEV-prM-ES156P were constructed, respectively.To generate the templates for PCR-based virus rescue, four plasmids p180PT7-1-976, p180PE957–2459, p180P2433–3831, and p180P3656-10991 containing the overlapping fragments of the FX2010-180P were generated and were used as templates for full-length genome amplification . To resc2.3.in vitro using mMESSAGEmMACHINE® T7 Kit  and purified by lithium chloride precipitation. The RNAs were then transfected into BHK-21 cells at 70%–80% confluency, cultured in T25 cm2 flasks, with an amount of 5 μg using Lipofectamine LTX& Plus Reagent . The cell culture medium was changed to DMEM containing 2% FBS at 6 h post-transfection. The virus released into the supernatant was collected when 70%–80% transfected cells start showing apparent cytopathic effects (CPEs). To rescue the 180P/JEV-prM-E or single-site mutant chimeric 180P/JEV-prM-ES156P viruses, plasmids p180PT7–1-976 and p180PE957-2459 were replaced by the p180PT7-1-455, pJEV-prM-E, and pJEV-prM-ES156P plasmids during rescues, respectively. The rescued viruses were amplified on BHK-21 cells for further study  of 0.001. The infected cells were incubated with DMEM containing 2% FBS at 37°C, 5% CO2. The supernatant was collected every 12 h post-infection and titrated on BHK-21 cells.To determine the viral replication of rescued viruses 2.5.S156P mutant virus and its parental virus (180P/JEV-prM-E) were used to infect BHK-21 cells at 0.0001 MOI. The cells were incubated for 2 h at 37°C and 5% CO2. An equal volume of agarose and 2× DMEM were added to each well, fully mixed, and allowed to solidify at 4°C. The plates were then inverted and incubated at 37°C and 5% CO2 for 4 days. After formaldehyde fixation and crystal violet staining, typical plaque formation was observed at the appropriate amount of virus infection. Additionally, the diameter of 10 randomly selected plaques was measured for statistical analysis.The 180P/JEV-prM-E2.6.S156P chimeric viruses in mouse, groups of 3 weeks-old female BALB/c mice were inoculated intracerebrally (i.c.), intranasally (i.n.), or intraperitoneally (i.p.) with 104.0 TCID50 of each virus at volume of 30 μL(or at volume of 50 μL for 180P/JEV-prM-ES156P), 30 μL (or at volume of 50 μL for 180P/JEV-prM-ES156P), and 100 μL, respectively. And the control group was inoculated with PBS. In each group, three mice were euthanized on 4, 6, and 8 dpi by CO2 inhalation, respectively, and the tissue samples of brain, liver, spleen, lung, and kidney were collected for viral titration. Body weight data, survival data and clinical scores  of the remaining five mice in each group were recorded daily until 14 dpi. And the neurovirulence and neuroinvasiveness of each virus were evaluated based on all the data.To test the virulence and neuroinvasiveness of parental 180P, wildtype JEV, JEV attenuated vaccine SA14-14-2, and the 180P/JEV-prM-E and 180P/JEV-prM-ES156P in mice, two groups of eight 3-week-old female BALB/c mice were intranasally immunized with 104.0 TCID50 of 180P/JEV-prM-ES156P mutant virus at volume of 50 μL and JEV attenuated vaccine SA14-14-2 at volume of 30 μL. Another control groups of consisting of eight mice were used as a negative control group . On day 21 post-inoculation, mouse blood was collected and serum was separated. To detect the JEV and TMUV specific antibodies in serum, IFA was conducted on the BHK-21 cells transfected with JEV E, TMUV C, prM and E protein expression plasmids. All animals were then challenged intranasally with 104.0 TCID50 of wildtype JEV. Three mice were euthanized on 6 dpi by CO2 inhalation and the tissue samples of brain, spleen and lung were collected for viral titration. Body weight data of the remaining five mice were recorded daily until 14 dpi.To test the efficacy of the 180P/JEV-prM-EAll animal studies were approved by the institutional Animal Care and Use Committee of Shanghai Veterinary Research Institute (SHVRI) and conducted in the Biological Safety Level 2 (BSL2) facility with permit number was SHVRI-SZ-20200506-02.2.7.To determine the virus load, the mouse tissue samples were weighed and homogenized in sterile PBS to yield homogenates. Tissue homogenates were clarified by centrifugation at 12,000 × g, 4°C for 10 min, and 10-fold serially diluted supernatants were inoculated onto 70%–80% confluent BHK-21 cells in 96-well plates at 37°C for 2 h. After adsorption, cells were washed and cultured with DMEM containing 2% FBS at 37°C. The virus titer was calculated by the method of Reed-Muench.2.8.t-test, posed ANOVA. p < 0.05 was considered to be significant.Statistical analyses were performed using GraphPad Prism version 7.0 for Windows . Significant differences were calculated using Student’s 3.3.1.To investigate the potential of using 180P as a backbone for developing flavivirus vaccines, the 180P/JEV-prM-E chimeric virus containing prM and E genes of JEV were rescued in backbone of the 180P . Growth 3.2.To further evaluate the pathogenesis of 180P/JEV-prM-E in mice, neuroinvasive ability of the viruses were evaluated in mice inoculated intraperitoneally and intranasally. The results showed that intraperitoneal inoculation did not result in any weight loss or mortality in mice , whereasNeurovirulence was evaluated through intracerebral inoculation, which resulted in weight loss in the 180P/JEV-prM-E group with 60% mortality on 14 dpi . In contVirus distribution analysis in infected mice revealed that the viruses were mainly detected in the brains. In the i.p. group, only JEV was detected in brain of infected mice on 6 and 8 dpi, while no virus was detected in the spleen or lung tissues. Neither 180P/JEV-prM-E nor 180P were detected in the i.p. infected mice . In the 3.3.S156P with the glycosylation site deletion was successfully rescued  or intranasally (i.n.) with either 180P/JEV-prM-Eor death ,B. In coice died . In contice died . All theS156P nor JEV vaccine strain SA14-14-2 were detected in any tissues of intraperitoneally (i.p.) or intranasally (i.n.) infected mice  recombinant vaccines against JEV  and DENVJapanese encephalitis virus is still a threat to the public health, according to data from the Chinese Bureau of Disease Control and Prevention, between 2009 and 2021, China had reported 18,597 cases of JEV infection, resulting in 757 deaths and a mortality rate of 4.1%. The flavivirus E protein is a main structural protein that plays an important role in binding and entry, and prM/E proteins are the main neutralizing antigens of JEV . StudiesS156P with the potential glycosylation site deletion were rescued successfully. In the mouse model, the neurovirulence and neuroinvasiveness of the chimeric virus 180P/JEV-prM-E was significantly decreased compared with the wildtype JEV. However, the 180P/JEV-prM-E is still more virulent than the parental 180P. Notably, when the glycosylation site was deleted in the 180P/JEV-prM-ES156P E protein, the neurovirulence was further reduced and neuroinvasiveness was completely abolished. These finding suggest that the absence of the glycosylation site further weakens the 180P/JEV-prM-ES156P virus. And 180P/JEV-prM-ES156P could provide complete protection against challenge with wildtype JEV in mouse model. However, the limitation of this study is that although point mutation virus 180P/JEV-prM-ES156P could protect immunized mice from death after challenge, it cannot prevent wildtype JEV replication in the brain. Overall, in this study, we demonstrated that 180P can be used as a backbone to develop JEV attenuated vaccine candidate, which provided a new vector tool for the development and preparation of flavivirus vaccines.In this study, we investigated whether 180P could be used as a backbone for developing flavivirus vaccines, the chimeric virus 180P/JEV-prM-E and 180P/JEV-prM-EThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Shanghai Veterinary Research Institute (SHVRI) with permit number was SHVRI-SZ-20200506-02.BX and XL conceived of the study and participated in its design and coordination. BX participated in laboratory work with the help of DY, QT, CY, and ZZ. BX drafted the manuscript and ZL and QL modified it. ZL directed the project. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (32102654 and 32072862), the Shanghai Agriculture Applied Technology Development Program, China (2021-02-08-00-12-F00746), the Shanghai Sailing Program (no. 21YF1456500), and the Agricultural Science and Technology Innovation Program (CAAS-ZDRW202203).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. 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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is causing a worldwide pandemic since the spring of 2020. In Osaka, the second biggest prefecture in Japan, we identified a novel SARS-CoV-2 variant from a coronavirus disease 2019 (COVID-19) patient that was detected by polymerase chain reaction (PCR) using E primers, but not by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) using the N1 and N2 primer-probe sets recommended by CDC. We analyzed the S and N gene regions by reverse-transcription and nested PCR using the S and N specific primers, and DNA sequencing, and found that this BA.5 variant contained point mutations in the probe sequences of both the N1 and N2 primer-probe regions. This finding led us to affirm the importance of monitoring the genome sequence of the SARS-CoV-2 variants continuously. Coronavirus disease 2019 (COVID-19) was first noted in November 2019 in Wuhan, China, and WHO declared a global pandemic on March 11, 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for causing the serious life-threatening COVID-19, and its genome sequence had been analyzed and determined . AccordiIn mid-October 2022 in Osaka, the third biggest prefecture in Japan, we examined a patient, a female in her 40s, who suffered from fever and a sore throat. We obtained the sputum sample from the patient and analyzed it by Xpert Xpress SARS-CoV-2  that uses primer-probe sets in the E gene (the primer sequence was not disclosed by the company) and N gene (N2). RT-qPCR for E primers showed Ct-value of 29.1, but, unexpectedly, RT-qPCR for N2 did not show positive result. Then we examined the sample by SARS-CoV-2 direct detection RT-qPCR kit  that uses the N1 and N2 primer-probe sets recommended by CDC, but could not get a positive result. Next day, we re-examined the same sample by using TRCReady SARS-CoV-2  and got a positive result. However, we observed some abnormal changes in the amplification curve, namely showing a slower rising pattern than usual. The patient’s symptoms of fatigue, low-grade fever, sore throat, and cough lasted three days and were relieved without sequelae. Although we could diagnose the patient as COVID-19 positive, since we identified such abnormal findings in the sputum sample, we decided to further analyze the sample more in detail.First, we analyzed the S gene region of the same sputum sample from the patient Figure . We prepThen we hypothesized the presence of unknown mutations in the N gene and designed two primer sets for reverse transcription and nested PCR that cover the N1 and N2 regions, respectively , but not C29200T (in N2), by SARS-CoV-2 direct detection RT-qPCR kit . We confirmed that it could show a positive result, but showed an abnormal amplification curve presumably because the N1 probe set could not work while the N2 probe could still work . BF.5 variants carrying C29272T are rare in Japan, and any BF.5 variants carrying both C29200T and C29272T have been identified neither in Japan nor in the world . We think that this novel “stealth” Omicron variant, carrying triple mutations in the N1 and N2 regions, could have been generated sporadically from BF.5 in Osaka, Japan.We analyzed a SARS-CoV-2 sample found in Osaka, Japan, which could not be detected by using RT-qPCR kits using the N1 and N2 primer-probe sets, and identified mutations, G22775A, A22786C, G22813T, T22917G, G22992A, C22995A, A23013C, T23018G, A23055G, A23063T, and T23075C in the S gene and mutations A28271T, C28311T, A28330G, del(28362-28370), A28402G, C29200T, C29272T, and A29510C in the N gene. Variants carrying all of these mutations except C29200T and C29272T are mostly designated as BF.5 (covSPECTRUM: https://cov-spectrum.org/). These incidences may be underestimates since the genome analysis should have been performed by using PCR-positive samples. Thus, our present study points out a risk that some COVID-19 patients may be overlooked if they are examined only by an RT-qPCR system using the N1 and N2 primer-probe sets.Also, our present study demonstrated that SARS-CoV-2 variants carrying C28311T, A28330G, and C29200T may not have been detected by RT-qPCR using the N1 and N2 primer-probe sets. SARS-CoV-2 variants carrying C28311T, A28330G, and C29200T have been identified with incidences of 0.23% (80% of them were designated as BF.5) and 0.06% of the total sequences in Japan and in the world, respectively (as of March 1, 2023, covSPECTRUM: https://cov-At present, we do not know how broadly the “stealth Omicron” identified in this study is spreading in Japan. Based on the current finding, we propose that it is important to keep monitoring the variant at molecular levels, namely PCR and genome sequencing, and the diagnostic kit should be continuously updated as new variants emerge. Unlike RATs, which require more time-consuming efforts to be adapted to new mutations, RT-qPCR tests will easily be adapted to them by designing new primer-probe sets. Also, we recommend the PCR test should be redundant, namely examining other domains at the same time to avoid leaving patients undiagnosed.


Through AT1R, angiotensin II increases proinflammatory cytokines contributing to fibrosis, angiogenesis, proliferation, and migration of immune cells to the skin. In contrast, AT2R suppresses the effects mentioned above. Many studies show that angiotensin receptor blockers (ARBs) and angiotensin-converting enzymes (ACEi) reduce the proinflammatory cytokines and fibrogenic factors including transforming growth factor β (TGF-β), Connective tissue growth factor (CTGF), and IL-6. This review article provides a detailed research study on the implications of ARBs in wound healing, hypertrophic scar, and keloids. We further discuss the therapeutic potentials of ARBs in autoimmune and autoinflammatory skin diseases and cancer, given their anti-fibrotic and anti-inflammatory effects.Renin-angiotensin system (RAS) components such as angiotensin II, angiotensin receptors (AT These inflammatory mediators stimulate fibroblast cells to produce fibrin and extracellular matrix. The proliferation phase happens and lasts for three weeks. During the early stages of proliferation, the number of infiltrated inflammatory cells dwindle while collagen, granulation tissue, angiogenesis, and epithelialization accrete. Over the remodeling phase, collagen production and degradation are balanced and modified into mature collagen . Prolong healing . 1R is rapidly increased. The same trend but at a slower rate also happen for AT2R. The process then continues with a distinct decline in the expression of AT1R but a dramatic up-regulation of AT2R. To a lesser extent, AT1R has increased during the remodeling phase. Hence, AT2R dominancy is apparent in the later phase  for 14 days speeded up the epidermal repair in a streptozotocin-induced diabetic wound in mice  , (TGF-β)S , 25, andS . It has S . Therefo1R and angiotensin II concentrations are higher in keloid and hypertrophic scar tissue than in normal skin in humans  has been shown to reduce TNF-α-induced IL-6 expression in human skin fibroblasts  while being poorly expressed in basal cell carcinoma (BCC)  . A randoma (BCC) . 1R  mice, it was reduced in AT1R-deficient mice, and TCV-116, a selective AT1R blocker, decreased melanoma growth and angiogenesis in WT mice  suppressor effect following ectopic expression of AGTR1 (encoding AT1R) in melanoma cell lines lacking endogenous expression of AT1R (47). Moreover, the inhibitory effect of losartan on the growth of murine melanoma confirms the oncogenic properties of AT1R . Hematogalsartan . AT1R ha WT mice . Besidesigration . Althouge effect -9, a recSome studies have shown the opposite effects of ARBs and ACEi drugs in the occurrence of skin cancer, which mARBs in autoinflammatory/autoimmune skin diseases1R has proinflammatory effects mediated through several chemokines and cytokines and induces specific inflammatory signaling pathways (reactive oxygen species (ROS), nuclear factor-κB, IL-6, and TGF-β) (Ang II via ATd TGF-β) , 49-51. d TGF-β) . ACE I/D (insertion (I) and deletion (D)) gene is reported in psoriatic patients which might contribute to the high levels of serum ACE  [A(1-7)], Nor-Leu-3 angiotensin (1-7), the other RAS-modifying reagent, in the MRL-lpr mouse model of SLE. Their findings demonstrated that the RAS-modifying therapies significantly reduced the onset and severity of skin rashes  mice, and AT1R antagonist decreased VEGF and the wound healing process (69). On the other hand, activation of AT2R alleviates angiogenesis, and the release of proinflammatory cytokines  contributes to skin fibrosis  is an agonist of non-peptide AT2R owned by vicore pharma (Sweden) Which is in phase II clinical trials with approved anti-inflammatory effects.Furthermore, there is a growing body of evidence in favor of the therapeutic effects of ARBs in the context of autoimmunity and autoinflammation. ARBs are commonly used to inhibit AT2R agonists for dermatological diseases. The topical formulation of ARBs would be a better therapeutic option for skin disorders. Still, the epidermal layers prevent drugs from penetrating deeper into the skin (1Rs are different (Further studies recommended using ATthe skin . The solifferent . Therefoifferent . K H, GH B, H K, S N, E KH, A D, and A KH participated in all aspects of the main file draft. N S participated in the revision of the article.The authors declare that there are no conflicts of interest.


Rabies, a highly fatal zoonotic disease, is a significant global public health threat. Currently, the pathogenic mechanism of rabies has not been fully elucidated, and no effective treatment for rabies is available. Increasing evidence shows that the tripartite-motif protein (TRIM) family of proteins participates in the host’s regulation of viral replication. Studies have demonstrated the upregulated expression of tripartite-motif protein 21 (TRIM21) in the brain tissue of mice infected with the rabies virus. Related studies have shown that TRIM21 knockdown inhibits RABV replication, while overexpression of TRIM21 exerted the opposite effect. Knockdown of interferon-alpha and interferon-beta modulates the inhibition of RABV replication caused by TRIM21 knockdown and promotes the replication of the virus. Furthermore, our previous study revealed that TRIM21 regulates the secretion of type I interferon during RABV infection by targeting interferon regulatory factor 7 (IRF7). IRF7 knockdown reduced the inhibition of RABV replication caused by the knockdown of TRIM21 and promoted viral replication. TRIM21 regulates RABV replication via the IRF7-IFN axis. Our study identified TRIM21 as a novel host factor required by RABV for replication. Thus, TRIM21 is a potential target for rabies treatment or management. Rabies is a zoonotic infectious disease caused by the rabies virus (RABV), with a dysfunctional central nervous system (CNS) being the main manifestation. The mortality rate of rabies is almost 100% without effective treatment. There are approximately 59,000 people worldwide who die from rabies annually ,3,4. At Innate immunity is the host’s first line of defence against viral infection. Type I interferon (IFNα/IFNβ), the main effector of innate immunity, plays an important antiviral role ,6,7,8. SDifferent types of post-translational modifications (PTMs) are critically involved in the control of the immune system. Additionally, PTMs regulate protein activity, stability, folding, localization, and interaction with other molecules. PTMs enhance several antiviral immune responses, such as phosphorylation, ubiquitination, dephosphorylation, and deubiquitination ,22,23,24To identify additional host factors that can regulate RABV infection, transcriptomic analysis of mouse brains infected with RABV was performed. We found that several TRIM proteins, especially TRIM21, were significantly differentially expressed in mouse brain tissue after RABV infection. This study focused on the role of TRIM21 in RABV infection. Our results showed that TRIM21 promotes RABV infection by ubiquitinating and degrading IRF7, inhibiting the type I IFN response.p < 0.01)  A. To con < 0.01) B and pro < 0.01) C. The reTRIM21 plays an important regulatory role in viral infection ,35,36,37Type I interferon plays a crucial role in the clearance of RABV during the infection ,38. NextIRF7 is known to play a crucial role in interferon induction during RABV infection . Next, wTRIM21 has been reported to be involved in the ubiquitination of IRF7 . To furtWithout proper treatment, the mortality rate from rabies can reach 100%. Therefore, it is important to understand the mechanism of RABV infection, particularly the host factors that participate in RABV infection. In this study, we found that RABV infection significantly upregulated the expression of TRIM21. Interestingly, TRIM21 was found to facilitate RABV infection. Studies have shown that numerous TRIM family proteins display antiviral activity despite many viral infections enhancing their expression. TRIM22 expression is upregulated under influenza A virus (IAV) and hepatitis C virus infections, and this protein inhibits the replication of these viruses ,43. TRIMHost antiviral activity involves either inhibition of the viral replication by restricting binding, entry, synthesis, assemble, and budding of the virus or clearing the virus using the innate immune response. TRIM21 regulates the innate immune response under viral infection through several mechanisms ,50,51,52Viruses and their products can be recognized by several PRRs. The recognition induces the expression of IFN-I genes, activating the cellular antiviral response. Different pathways downstream of PRRs, including TLRs, RLRs, and DNA sensors, can transduce signals that converge on several key molecules, such as IRF family members. In most cases, IRF3 and IRF7 are required as base IRFs. IRF3 initiates the initial transcription of IFN-I and IRF7, and it is important for signal amplification of IFN-I transcription ,55,56. AStudies have shown that FADD can form a complex with TRIM21 to jointly regulate the ubiquitination of IRF7 . After IIn conclusion, this study identified TRIM21 as a novel host factor that regulates RABV infection . This stNA cells  were cultured in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (FBS) (Gibco). HEK 293T cells were cultured in DMEM medium with 10% FBS. RABV fixed HEP-Flury strain  was propagated in NA cells.TRIM25 gene from NA cells was cloned into pCAGGS-HA using the primers: TRIM21-F: 5′-AGCTCATCGATGGTACCCGGGCCATGTCACCCTCTACAACCTCAAAAAG-3′ and TRIM25-R: 5′-ATTAAGATCTGCTAGCTCGAGTCACATCTTTAGTGGACAGAGCTTTAG-3′ for overexpression of TRIM21. The IRF7 gene from NA cells was cloned into pCAGGS-flag using the primers: IRF7-F: 5′-GCGAATTCATGGCTGAAGTGAGGGGGGTC-3′ and IRF7-R: 5′-GCCTCGAGTCAAGGCCACTGACCCAGGTC-3′ for overexpression of IRF7. The sequences of siRNA targeting interested proteins and the non-targeting control (NC) used in this study are shown in HA-tagged pCAGGS (pCAGGS-HA), flag-tagged pCAGGS (pCAGGS-flag), and Myc-tagged ubiquitin (pcDNA3.1-Myc-Ub) plasmids were purchased from Wuhan Miaoling Biotechnology . The RABV titrations were conducted using direct fluorescent antibody assays (dFA) as previously described . BrieflyTreated NA cells were fixed with 80% acetone for 30 min at −20 °C. Cells were washed three times with PBS and stained with FITC-labeled anti-RABV-N antibodies for 60 min at 37 °C followed by the staining of DAPI. Antigen-positive foci were observed and photographed under a fluorescence microscope. ImageJ was used to calculate the number of fluorescent spots in three different fields of view and to perform a difference analysis.A monolayer of NA cells or HEK 293T cells were transfected with recombinant plasmid or siRNA using Lipofectamine 3000 transfection reagent  according to the manufacturer’s instructions. An empty vector (pCAGGS-HA) was used as the control for plasmid transfection, while non-targeting control siRNA (NC) was used as the control for siRNA transfection. For more plasmids or siRNAs transfection, plasmid or siRNA was transfected at 12 h post the previous plasmid or siRNA transfection.Co-immunoprecipitation (Co-IP) was performed using the Pierce™ Crosslink Magnetic IP/Co-IP Kit  according to the manufacturer’s protocol. Briefly, following transfection with the indicated plasmids for 36 h, the whole cell lysates were prepared and used for immunoprecipitation with the indicated antibody-conjugated magnetic beads at 4 °C for 10 h. Normal IgG was used as a control. The IP products and whole cell lysates were analyzed by Western blotting (WB) with the indicated antibodies.Western blotting was performed as previously described . BrieflyTreated cells were harvested and total RNA was extracted using TRizol reagent  according to the manufacturer’s protocol. Reverse transcription (RT) was performed using the Transcriptor First Strand cDNA Synthesis Kit . Each reaction was performed in triplicate using SYBR Green Master Mix (Vazyme Biotech). Quantitative real-time PCR (RT-qPCR) was performed in a CFX connect Real-Time System . The levels of TRIM21 mRNA, IFNα mRNA, IFNβ mRNA, and RABV genomic RNA (gRNA) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences of TRIM21 were used: forward, 5′-GCTCCCTCATTTACACCTTCTCG-3′, reverse, 5′-GGCTCCTGACCATCACATCTTTAG-3′. The primer sequences of IFNα, IFNβ, gRNA, and GAPDH were described previously .t test. p < 0.05 was considered statistically significant.Data were analyzed using GraphPad Prism 6 software . Statistical significance was determined using a Student’s


Vdr(R270L/H301Q) rats was significantly lower than in wild-type (WT) rats. Meanwhile, Vdr(H301Q) rats had calcium concentrations falling between those of Vdr(R270L/H301Q) and WT rats. GM rats exhibited markedly elevated plasma parathyroid hormone and 1,25D3 levels compared to those of WT rats. An analysis of bone mineral density in the cortical bone of the femur in both GM rats revealed significantly lower values than in WT rats. Conversely, the bone mineral density in the trabecular bone was notably higher, indicating abnormal bone formation. This abnormal bone formation was more pronounced in Vdr(R270L/H301Q) rats than in Vdr(H301Q) rats, highlighting the critical role of the VDR-dependent function of 1,25D3 in bone formation. In contrast, neither Vdr(H301Q) nor Vdr(R270L/H301Q) rats exhibited symptoms of alopecia or cyst formation in the skin, which were observed in the Vdr-KO rats. These findings strongly suggest that unliganded VDR is crucial for maintaining the hair cycle and normal skin. Our GM rats hold significant promise for comprehensive analyses of vitamin D and VDR functions in future research.Vitamin D has been known to exert a wide range of physiological effects, including calcemic, osteogenic, anticancer, and immune responses. We previously generated genetically modified (GM) rats and performed a comparative analysis of their physiological properties to elucidate the roles of vitamin D and vitamin D receptor (VDR). In this study, our primary goal was to investigate the manifestations of type II rickets in rats with the VDR(H301Q) mutation, analogous to the human VDR(H305Q). Additionally, we created a double-mutant rat with the VDR(R270L/H301Q) mutation, resulting in almost no affinity for 1,25-dihydroxy-vitamin D3  or 25-hydroxy-vitamin D3 (25D3). Notably, the plasma calcium concentration in  Vdr(R270L) rats highlights the action of VDR-dependent 1,25D3, and (2) a comparison of Vdr(R270L) and Cyp27b1-KO rats emphasizes the action of VDR-independent 1,25D3. (3) A comparison of Vdr(R270L/H301Q) and Vdr-KO rats reveals an unliganded VDR action, and (4) a comparison of Vdr(R270L) and Vdr(R270L/H301Q) rats demonstrates a VDR-dependent effect of 25D3. Recently, we unveiled that a VDR-expressing adenoviral vector (AdV) is a promising tool for elucidating the function of a VDR [Vdr(R270L/H301Q) rats showed no alopecia, whereas Vdr-KO rats exhibited alopecia [Vdr(H301Q), and Vdr(R270L/H301Q) rats in terms of their plasma Ca, PTH, and 1,25D3 levels and bone structures. The unliganded VDR function is discussed based on the remarkable differences in hair cycling and skin senescence between Vdr(R270L/H301Q) and Vdr-KO rats. Our GM rat series, including Vdr(H301Q) and Vdr(R270L/H301Q) rats, may have strong implications for elucidating the molecular mechanisms of vitamin D and VDR action  to form a heterodimer with RXR, subsequently binding to the vitamin D response element (VDRE) and regulating the expression of various genes. A mutation causing a loss of function in the VDR gene results in the inactivity of VDR-dependent vitamin D actions, leading to symptoms like bow legs/knock knees due to osteodystrophy and alopecia due to hair follicle dysplasia, known as vitamin D-dependent type II rickets (VDDRII) . Bone dyof a VDR . In partof a VDR ,11. Usinalopecia . These rR action .25D3 and 1,25D3 were purchased from Cayman Chemical Company . The other chemicals were commercially available and of the highest quality.Vdr(H301Q) and Vdr(R270L/H301Q) rats were generated using the CRISPR-Cas9 genome editing system as previously described [Jcl:Wistar rats were purchased from Nihon Clea Co., Ltd. . The escribed . Embryo escribed . All aniVdr(H301Q) (15 weeks), Vdr(R270L/H301Q) (15 weeks), and Vdr-KO (14 weeks) male rats, treated with 4% PFA , and fixed at 4 °C for 15 h. The resultant samples were then filled with O.T.C compound , frozen in liquid nitrogen, and then cut to a thickness of 20–25 μm using a cryostat microtome . The resulting cryosections were mounted on glass slides. The 4% PFA solution was dropped onto the sample glass, incubated at room temperature for 10 min, and then washed with water for 10 min. Hematoxylin was dropped onto the sample glass, incubated at room temperature for 15 min, and then washed with water for 10 min. Eosin–alcohol was dropped onto the sample glass, incubated for 2 min at room temperature, and washed with water for 2 min, 70% EtOH for 2 min, 80% EtOH for 2 min, 90% EtOH for 2 min, and 100% ethanol. An additional 2 min wash with EtOH was followed by two washes with xylene. The obtained HE-stained samples were observed using a phase contrast microscope  (Skin samples were prepared from WT (15 weeks), , Japan) .Vdr(H301Q), and Vdr(R270L/H301Q) male rats, and white adipose tissue was removed. The resultant skin samples were incubated for 18 h at 4 °C in 14 mL of CnT-07 epithelial growth medium  containing 5 mg/mL collagenase and washed with PBS to remove excess collagenase. Next, the resulting skin samples were incubated in 5 mL of Tryp Express  for 15 min at 37 °C, and then 25 mL of CnT-07 medium was added, and the samples were vigorously pipetted to disassociate cell–cell adhesions. The resulting cell solution was centrifuged at 2000 rpm for 5 min to obtain a pellet containing keratinocyte cells. Next, the pellet was suspended in 4 mL of CnT-07 medium containing penicillin–streptomycin and amphotericin B at final concentrations of 100 units/mL and 0.5 μg/mL, respectively, and the collected cells were cultured in a 6 cm collagen-coated dish. After 2 days of culture, the medium was changed from CnT-07 medium to Keratinocyte Growth Medium 2 .Back skin was excised from 15-week-old WT, Vdr(H301Q), or Vdr(R270L/H301Q) male rats were cultured in 6 cm collagen-coated dishes . After washing the cells with PBS, a CnT-07 medium containing 10 nM of 1,25D3 was added to observe the ligand-dependent nuclear translocation of VDR. At 120 min after the 1,25D3 addition, the cells were fixed with 4% PFA  for 15 min at 4 °C. PBS containing 0.01% Triton-X was used for cell permeabilization, and 0.1% BSA/PBS was used for blocking to prevent non-specific detection. For the immunofluorescence detection of VDR, the Anti-VDR antibody (D2K6W) Rabbit mAb  and Alexa Flour 488 goat anti-rabbit IgG  were used as the first and secondary antibodies, respectively [Primary culture cells prepared from the dorsal skin of the WT, The back skin was excised and homogenized using a Minilis personal homogenizer . The resulting tissue lysate containing 10 mg of protein was applied to each lane of the gel and subjected to SDS-PAGE on a 4–20% linear gradient polyacrylamide/SDS gel. After electrophoresis, the proteins in the gel were electrotransferred onto a PVDF membrane. The PVDF membrane was then incubated in Tris-buffered saline with 0.05% Tween 20 (TBS-T) containing 5% skim milk, followed by anti-VDR antibody   or anti-β-Actin Rabbit Ab  [3 was chosen to distinguish between calcified and non-calcified tissues. Then, 3D images of the femur were constructed from the scanned images using VGSTUDIO 3.2 software .A computed tomography (CT) analysis of the left femur was performed to examine the morphological characteristics and bone mineral density (BMD) of the rat bones . The femThe plasma Ca concentration was measured using a Calcium E-Test Wako . The plasma PTH concentration was measured using a Rat Intact PTH ELISA Kit . The plasma 1,25D3 concentration was measured using a 1,25-D3 ELISA Kit  . t-test. The criterion for significance was p < 0.05.The statistical significance of differences in Figures and Tables was analyzed using Student’s Vdr(H301Q), Vdr(R270L/H301Q), and Vdr-KO phenotypes in rats fed a CE-2 diet containing 1.15% Ca at 15 weeks after birth was documented in our previous studies [Vdr-KO rats, hair loss progressed over time, and the elasticity and softness of the skin markedly decreased, resulting in the wavy appearance of their skin (Vdr(H301Q) and Vdr(R270L/H301Q) rats did not show alopecia [Vdr(H301Q) and Vdr(R270L/H301Q) rats revealed normal hair follicles and skin maintenance without dermal cyst formation  or VDR(R270L/H301Q) in the back skin of the corresponding rats was examined using a Western blot analysis. Prominent VDR expression was observed in both GM rats . The levVdr(H301Q), or Vdr(R270L/H301Q) rats. The remarkable nuclear translocation of VDR was observed in WT and Vdr(H301Q) upon adding 10 nM of 1,25D3 (Vdr (R270L/H301Q) under similar conditions. These results are consistent with our previous study [Primary keratinocytes were prepared from WT, f 1,25D3 . In contVdr(H301Q), and Vdr(R270L/H301Q) rats at 10, 12, and 15 weeks of age. The Ca levels in both GM rats were significantly reduced compared with the WT rats (Vdr(H301Q) and Vdr(R270L/H301Q) rats were significantly higher than in WT rats  and Vdr(R270L/H301Q) rats (Vdr(H301Q) and Vdr(R270L/H301Q) rats were not significantly different from those of the WT rats. However, the bone length of the GM rats was significantly shorter than that of the WT rats. In contrast, the maximum bone width of the GM rats was significantly shorter than that of the WT rats. The bones of the Vdr(R270L/H301Q) rats were morphologically more abnormal compared to those of the Vdr(H301Q) rats. It is noted that these morphologic features were also observed in Vdr(R270L), Vdr-KO, and Cyp27b1-KO rats, whereby the rupture of the epiphyseal cartilage growth plate and the disorganization of cartilaginous growth plates were observed [A μCT analysis revealed abnormal morphological changes in the cortical and trabecular bones of the femurs of the 1Q) rats . As showobserved . The disVdr(H301Q) and Vdr(R270L/H301Q) rats showed decreased cortical BMD among all sections in the femur, and the latter showed more reduced BMD than the former. In the WT rat femurs, the cortical BMD was higher at the diaphysis than at the epiphysis, resulting in a gentle, mountain-shaped BMD distribution, as shown in Vdr(H301Q) and Vdr(R270L/H301Q) rats was substantially different from that in the WT rats, which showed a flat curve (Vdr(H301Q) and Vdr(R270L/H301Q) rats showed increased trabecular BMD in all sections in the femur, with the latter showing a higher BMD than the former. Vdr(H301Q), and Vdr(R270L/H301Q), whereas the BMD of the trabecular bone increased in the same order.The femur trabecular bone was highly distributed in the epiphysis in the WT rats, which showed a U-shaped curve when the trabecular BMD was plotted from proximal to distal along the major axis of the femur B. In conat curve B. Both VVdr(R270L/H301Q) rat serves as an optimal model animal for analyzing the unliganded VDR function. As described in the Introduction, (3) comparing Vdr(R270L/H301Q) with Vdr-KO rats, we can observe unliganded effects of VDR. The Vdr-KO rats developed alopecia, whereas the Vdr(R270L/H301Q) rats did not. These results strongly support the idea that unliganded VDR contributes to the maintenance of the hair cycle. Although the molecular mechanism of hair loss in Vdr-KO rats remains unknown, it is possible that the complex formation of VDR with both RXR and Hairless (Hr) may be involved in the activation of Wnt signaling by interacting with β-catenin and Lef1 [Vdr(R270L/H301Q) rats did not exhibit hair loss or dermal cyst formation. These results suggest that unliganded VDR in keratinocytes plays a pivotal role in regulating hair follicle regression, thereby inducing the transition from the catagen to the telogen phase of the hair follicle by promoting apoptosis of the epithelial strand. VDR is expressed in the nuclei of normal epithelial strand-forming cells during the catagen phase, implying that it promotes cell death in epithelial strands through transcriptional regulation. This speculation, combined with our results, leads to the hypothesis that unliganded VDR promotes the death of epithelial strands through transcriptional regulation in the nuclei. We intend to confirm this in future studies. Recently, a novel model of hair follicle development was reported [We successfully generated rats expressing VDR(R270L/H301Q), which exhibited almost no binding affinity for 1,25D3 and 25D3. Two amino acid substitutions in the ligand-binding pocket of VDR nearly completely abolished its ligand binding ability ,11. A Weand Lef1 ,13,14,15and Lef1 . This suand Lef1  demonstrand Lef1  reportedreported ; howeverVdr(H301Q) and Vdr(R270L/H301Q) rats revealed the cumulative VDR-dependent effects of 1,25D3 and 25D3. The plasma calcium level in the Vdr(R270L/H301Q) rats was lower than that in the Vdr(H301Q) rats. In contrast, the plasma PTH and 1,25D3 levels in the Vdr(R270L/H301Q) rats were higher than those in the Vdr(H301Q) rats (Vdr(R270L/H301Q) rats was more aberrant than in the Vdr(H301Q) rats. These findings indicate that type II rickets symptoms in the Vdr(R270L/H301Q) rats were more severe, likely due to the lower plasma Ca levels. As shown in 1Q) rats . Bone foVdr(R270L) [Vdr(R270L/H301Q) rats revealed the VDR-dependent effects of 25D3. However, no significant differences were observed in the plasma Ca levels and bone mineral density of the cortical and trabecular bones between the Vdr(R270L) [Vdr(R270L/H301Q) rats in this study. This suggests that the VDR-dependent effects of 25D3 are non-essential for maintaining plasma Ca levels and bone formation. Therefore, the observed difference between the Vdr(H301Q) and Vdr(R270L/H301Q) rats appears to be attributed to variations in plasma Ca levels due to the VDR-dependent effects of 1,25D3. Vdr-KO rats. These results strongly support the idea that unliganded VDR is essential for the regulation of hair follicle cycling and skin homeostasis. A proteome analysis of back skin proteins of the WT and Vdr-KO rats at 28 weeks of age revealed a significant decrease in various proteins related to oxidative phosphorylation, including NADH dehydrogenase, cytochrome c oxidase, and ATP synthetase in Vdr-KO rats. These findings suggest that enhanced skin aging in Vdr-KO rats is closely associated with mitochondrial dysfunction. Multi-omix analyses are expected to unveil the roles of vitamin D and VDR in various organs and tissues. Comparing the r(R270L)  and Vdr(r(R270L) ,9 and VdVdr(H301Q), and Vdr(R270L/H301Q) rats. However, if the effect of 1,25D3MARRS (ERp57) is important rather than VDR, it is possible that there is not much difference among the three groups. Thus, our GM rats are likely to become increasingly important for comprehensive analyses of vitamin D and VDR functions.It is known that 1,25D3 induces rapid Ca influx into cells via VDR ,20 and/oThe unliganded VDR in keratinocytes is essential for the regulation of hair cycling and skin homeostasis. On the other hand, VDR-dependent 1,25D3 action is vital for maintaining plasma calcium levels and normal bone formation.


The persistence of pHNSHP24 improved after 36 days evolution due to the plasmid-borne mutation A51G in the 5′UTR of gene traJ. This mutation largely increased the infectious transmission of the evolved plasmid, presumably by impairing the inhibitory effect of FinP on the expression of traJ. We showed that increased conjugation rate of the evolved plasmid could compensate for the plasmid loss. Furthermore, we determined that the evolved high transmissibility had little effect on the mcr-1-deficient ancestral plasmid, implying that high conjugation transfer is vital for maintaining the mcr-1-bearing plasmid. Altogether, our findings emphasized that, besides compensatory evolution that reduces fitness costs, the evolution of infectious transmission can improve the persistence of antibiotic-resistant plasmids, indicating that inhibition of the conjugation process could be useful to combat the spread of antibiotic-resistant plasmids.Conjugative plasmids play a vital role in bacterial evolution and promote the spread of antibiotic resistance. They usually cause fitness costs that diminish the growth rates of the host bacteria. Compensatory mutations are known as an effective evolutionary solution to reduce the fitness cost and improve plasmid persistence. However, whether the plasmid transmission by conjugation is sufficient to improve plasmid persistence is debated since it is an inherently costly process. Here, we experimentally evolved an unstable and costly mcr-1) plasmid under laboratory conditions and found that increased conjugation rate was crucial for the persistence of this plasmid. Interestingly, the evolved conjugation was caused by a single-base mutation, which could rescue the unstable plasmid from extinction in bacterial populations. Our findings imply that inhibition of the conjugation process could be necessary for combating the persistence of antibiotic-resistance plasmids.Conjugative plasmids play a key role in the spread of antibiotic resistance, and they are well-adapted to the host bacteria. However, the evolutionary adaptation of plasmid-bacteria associations is not well understood. In this study, we experimentally evolved an unstable colistin resistance ( Bacterial conjugation is an important mechanism of HGT . It tranccharide -11. Sogenicity . Some regenicity -16. ThPseudomonas fluorescens that target the two-component system GacA/GacS or the hypothetical protein PFLU4242 reduce the fitness cost of pQBR plasmids and increase their permissiveness  in the replication initiation protein encoded by an IncP-1β plasmid reduced the plasmid cost and improved long-term plasmid survival  (E. coli C600(pHNSHP24) was used to investigate plasmid-host adaptation in the presence of colistin. Here, we chose the transconjugant C600(pHNSHP24) to perform a laboratory evolution experiment instead of the native strain because E. coli SHP24 contains four other plasmids besides the pHNSHP24 ( in 2013 . pHNSHP2 plasmid . The per14 days) , E. colipHNSHP24 .E. coli C600 carrying pHNSHP24 was cultured consecutively for 36 days. We monitored plasmid persistence following 7, 14, and 36 days of serial transfer in Luria-Bertani (LB) broth containing colistin. As shown in To investigate whether plasmid-host adaptation could enhance the persistence of pHNSHP24, we performed a plasmid evolution experiment in liquid medium containing colistin . E. coliE) from the E1-1 clone to the ancestral host E. coli C600A (HA), and the resulting strain was named E. coli C600A(pHNSHP24-36D)(HAPE). The plasmid-free evolved strain E1-1 was obtained from the plasmid persistence experiment. Then, the ancestral plasmid pHNSHP24 (PA) was transferred into the plasmid-free strain E1-1 designated as E. coli C600E (HE), and the resulting strain was named E. coli C600E(pHNSHP24)(HEPA). Then, we determined the plasmids (PE and PA) persistence in HA or HE. As shown in E. coli C600A and E. coli C600E shows dramatic improvement in persistence compared to the ancestral plasmid pHNSHP24. In contrast, we did not detect improved persistence of pHNSHP24 or pHNSHP24-36D in E. coli C600E compared with E. coli C600A, indicating the evolution occurred in the plasmid, not the chromosome. We also compared the plasmid persistence profiles of HAPA, HEPE, HAPE, and HEPA, using the HT plasmid population dynamics model, as described in previous studies  showed a significant difference with PA-bearing strains  . However, the plasmid persistence profiles of host HAPA or HAPE were indistinguishable from that of the host HEPA or HEPE, respectively (ΔBIC=19.17 or 19.47), further implying that the improved persistence was attributable to plasmid evolution.Then, we purified the lineage E1 evolved for 36 days by streaking on LB plates containing colistin and randomly picked three clones  to determine the plasmid persistence. As shown in  studies , 31. We cDmt (cytosine-specific DNA methyltransferase gene) and the upstream sequence (A51G) of traJ in pHSNHP24-36D. Besides, we also sequenced the plasmids pHNSHP24-7D and pHNSHP24-14D purified from the clones that evolved for 7 and 14 days in the E1 lineage and found that the two plasmids only contained the C1999T mutation in cDmt . However, there were no significant differences in the conjugation transfer rate between pHNSHP24 and pHNSHP24-14D (P = 0.4171). In addition, we confirmed that the cDmt gene has little effect on the conjugation of pHNSHP24 and pHNSHP24-36D plasmids  or mutated traJ (A51G) under the control of its native promoter to test their ability to conjugate. As expected, the complementation of mutated traJ significantly outperformed the transfer rate of traJ (wild type)-complemented pHNSHP24traJ  of traJ or mutated traJ with a promoterless lacZ, yielding PtraJ-lacZ and PtraJ(A51G)-lacZ, respectively. As shown in tra genes in both BW25113(pHNSHP24) and BW25113(pHNSHP24-36D) by qPCR. Expectedly, the transcription level of tra genes, including traY, traA, traC, traG, traD, and traI, in BW25113(pHNSHP24-36D) strongly increased compared with that of BW25113(pHNSHP24)  . These rtraJ is negatively regulated by the antisense RNA FinP, which interacts with the leader region of the traJ transcript and prevents the translation of traJ  is located at SLIIc . Fi. FitraJ at SLIIc , we specat SLIIc . We hypotraJ, we changed the loop size of SLIIc by introducing mutations in the 5′UTR of traJ (+51 or +56) -lacZ and PtraJ(C56U)-lacZ, respectively. As shown in PtraJ(C56U)-lacZ and PtraJ(A51G)-lacZ increased significantly compared with PtraJ-lacZ(wild-type). Likewise, the mutation A51C is predicted to create the same loop size as in pHNSHP24 and PtraJ(A51C)-lacZ shows similar activity to PtraJ-lacZ  probably impaired the inhibitory effect of FinP on traJ. Notably, the PtraJ(C56U)-lacZ fusion shows lower activity than PtraJ(A51G)-lacZ implying that the stability of SLIIc also affects the inhibitory effect of FinP, since the SLIIc harboring A51G, which contains GC base pair, is probably more stable than the SLIIc harboring C56U, which contains AU base pair.To test the effect of the loop structure of SLIIc on the expression of  or +56)  and fuseraJ-lacZ . These rE. coli BW25113. As shown in E. coli BW25113, while the growth rate of BW25113(pHNSHP24-36D) was lower than BW25113(pHNSHP24). Then, we also evaluated the fitness of BW25113(pHNSHP24-36D) relative to BW25113(pHNSHP24) in a competition assay. To facilitate the screening of BW25113 harboring pHNSHP24-36D in competition cultures, we introduced a kanamycin resistance gene at the cDmt locus since the deletion of cDmt in pHNSHP24-36D did not affect fitness (cDmt::kan) instead of BW25113(pHNSHP24-36D) for competition assays, since both strains show comparable fitness (cDmt::kan) showed a significant reduction in fitness relative to BW25113(pHNSHP24)  , indicat−6 to 1.99 × 10−5, are negligibly low. The model-based conjugation frequency (γ) of BPE (evolved plasmid pHNSHP24-36D) is more than eight times of BPA ; however, its cost is twofold of BPA. To better evaluate the role of the model-based conjugation frequency (γ) and cost (σ) on plasmid persistence, we introduced a concept of the persistence threshold γ/θ described by Ponciano et al. , fitness cost (σ) as well as conjugation rate (γ) , 31, 39.cDmt in pHNSHP24-36D did not affect the plasmid transmission ability and fitness cost (cDmt::kan) instead of BW25113(pHNSHP24-36D) for plasmid invasion in competitive co-cultures. As shown in cDmt::kan invaded the cell population faster than pHNSHP24, as expected from the results of the persistence assay.We also performed a plasmid invasion experiment to determine the invasion abilities of ancestral and evolved plasmids. The invasion abilities of pHNSHP24-36D and pHNSHP24 were compared individually or in competitive co-cultures. For each plasmid individually, the evolved plasmid pHNSHP24-36D invaded most cells after 24 h , indicatess cost ; Fig. S2mcr-1 on plasmid persistence since mcr-1 usually exerts a fitness cost to host bacteria. We constructed in-frame deletion of mcr-1 on pHNSHP24 and pHNSHP24-36D and monitored the persistence of pHNSHP24 mcr-1::kan and pHNSHP24-36Dmcr-1::kan. Interestingly, the deletion of mcr-1 improved the persistence of pHNSHP24 considerably, even better than the A51G mutation of pHNSHP24-36D, while the deletion of mcr-1 slightly improved the persistence of pHNSHP24-36D  at 37°C for 12 h. Bacterial growth was measured OD600 every hour by Multiskan spectrum microplate spectrophotometer .The bacterial strains used in this study are listed in E. coli C600 harboring pHNSHP24 plasmid (GenBank accession number: CP065023) was cultured serially for 36 days in the presence of colistin (2 μg/mL). The E. coli C600(pHNSHP24) strain was grown in LB broth containing colistin overnight at 37℃, then 3μL culture was transferred into 3 mL LB broth containing colistin and shaken at 37℃ each day. The plasmid persistence was measured following 7, 14, and 36 days of serial transfers. Briefly, the evolved cultures or strains were cultured serially in the absence of colistin for 14 days. The cultures which were passaged for 4, 7, 10, and 14 days were spread on non-selective LB agar plates with proper dilution, then 50 colonies were randomly selected to confirm the presence of plasmid by PCR using primers FrepB-F/FrepB-R, mcr-1-F/mcr-1-R, and pO111-F/pO111-R (The /pO111-R .The plasmid persistence profile describes the plasmid persistence over time (from days 0 to 10). The plasmid persistence profile was analyzed and compared using plasmid population dynamics models as described previously , 31. The is segregational loss frequency and σ is plasmid cost. This formula states that plasmid loss due to segregation and cost must be balanced by a high transfer frequency for the plasmids to persist in the populations. It should be noted that γ described here is a model-based conjugation transfer frequency, and it is not the same as the experimental estimated transfer rate γ estimated by the conjugation experiment based on the Simonsen method. All these analyses were conducted using the R package “StabilityToolkit” (https://github.com/jmponciano/StabilityToolkit/blob/master/RunningStabToolsPack.zip) created by Ponciano et al.  and clones that evolved for 7 and 14 days in E1 lineage were extracted and subjected to perform whole-genome sequencing  and Nanopore sequencing . Nanopore reads and Illumina reads were combined to produce a traJ with their native promoter were amplified from pHNSHP24 and pHNSHP24-36D using primers pro-traJ-F/pro-traJ-R, respectively, and cloned into pHSG575 to generate pHSG575-traJ and pHSG575-traJ(A51G). The upstream region of traJ with its native promoter (−217 to +242) was amplified from pHNSHP24 or pHNSHP24-36D using primers lacZ-traJ-F/lacZ-traJ-R and cloned into the HindIII site of pHGR01 plasmid which contains a promoterless lacZ gene to produce PtraJ-lacZ and PtraJ(A51G)-lacZ. The traJ (A51C) mutation in PtraJ-lacZ was constructed by lacZ-traJ-F/mu-24traJ(A51C)-R primers and mu-24traJ(A51C)-F/lacZ-traJ-R primers. The traJ(C56U) mutation in PtraJ-lacZ was constructed by lacZ-traJ-F/mu-24traJ(C56U)-R primers and mu-24traJ(C56U)-F/lacZ-traJ-R. Deletion mutants of pHNSHP24 and pHNSHP24-36D were constructed using the λ Red recombination system (traJ gene was done on pHNSHP24 using primers knock-traJ-F/knock-traJ-R and check-traJ-F/check-traJ-R. Deletion of the cDmt gene was done on pHNSHP24-36D and pHNSHP24 using primers knock-cDmt-F/knock-cDmt-R and check-cDmt-F/check-cDmt-R.Plasmids used in this study are listed in n system . DeletiolacZ gene of E. coli BW25113 to construct the kanamycin-resistant E. coli BW25113::kan using λ Red recombination system. The kanamycin-resistant gene fragment with homology extension was cloned from the pKD4 plasmid using primers insert-kan-F/insert-kan-R. The mutant was verified by PCR using primers check-kan-F/check-kan-R.The kanamycin-resistant gene was inserted in the truncated E. coli BW25113::kan was used as recipient (R). Thus, donors (D), recipients (R), and transconjugants (T) could be estimated by selective LB plates containing colistin (2 μg/mL) and/or kanamycin (30 μg/mL). The donor and recipient were individually cultured in 5 mL LB broth overnight at 37℃. The overnight cultures were adjusted to the same density (OD600=1), and 3 µL of each culture was transferred into tubes containing 3 mL LB broth. At the start point of mating, 100 µL mixture was sampled to quantify the total initial cell densities (N0). The mating cultures were incubated at 37℃ with shaking for 12 h. During the exponential growth phase, the optical density was measured at tb and ta to calculate the growth rate of the mating cultures, using the following formula , recipients (R), and transconjugants (T) have the same growth rate. However, if they do not have the same growth dynamics, it could lead to biased estimates  were plated to colistin-containing and kanamycin-containing plates. The mating cultures with serial dilutions  were plated to plates containing both colistin and kanamycin. The number of donors (D) was calculated by the following formula: donors (D)=colistin-containing plate counts minus two antibiotics-containing plate counts. The number of recipients (R) was calculated by the following formula: recipients (R)=kanamycin-containing plate counts minus two antibiotics-containing plate counts. The number of transconjugants (T) was counted from two antibiotics-containing plates. The total cell densities (N)=donors (D)+recipients (R)+transconjugants (T). The transfer rate (At the endpoint time (12 h) of mating, the mating cultures with proper dilution were spread on selective LB plates to quantify the total cell densities (N), donors (D), recipients (R), and transconjugants (T). The mating cultures with serial dilutions (1:10 formula :10 S1dt–log10 S1d0)/(log10 S2dt–log10 S2d0) was used to calculate relative fitness (RF), where RF is the relative fitness of S1 strain (compared to S2 strain), S1dt and S1d0 represent the densities of cells (CFU/mL) of S1 and the end and beginning of the competition, and S2dt and S2d0 represent the densities of cells of S2 and the end and beginning of the competition. All competition experiments were carried out with three biological replicates.Competition experiments were used to measure the relative fitness of strains harboring pHNSHP24 or its derivatives. The overnight cultures of the two strains were diluted 1:1,000 in LB broth and mixed at a 1:1 ratio. The mixture was incubated at 37°C with shaking (180 rpm) for 24 h and then the mixed population was again 1:1,000 diluted into fresh LB broth. This procedure was repeated until the competition experiment lasted for 4 days. The samples of each competitive mixture with proper dilution were spread on LB agar every 24 h. The properly diluted cultures were plated to antibiotic-free, kanamycin-containing, or colistin-containing LB agar plates. The formula RF= logcDmt::kan) overnight cultures, respectively. The 2 mL mixed cultures grew in a 50-mL tube at 37℃ with shaking (45 rpm), and diluted 1:100 into fresh media every 24 h for 96 h. Viable counts were performed at 24, 48, 72, and 96 h. For each time point, dilutions of the cultures were plated to non-selective, colistin-containing and kanamycin-containing media. Cells containing pHNSHP24 or pHNSHP24-36D grew on non-selective and colistin-containing plates but were killed by kanamycin-containing media. Cells containing pHNSHP24-36DΔcDmt::kan grew on all plates. Plasmid-free cells were killed by both antibiotics. Therefore, the number of cells containing pHNSHP24, pHNSHP24-36D, and pHNSHP24-36DΔcDmt::kan were counted from colistin-containing plates, colistin-containing plates, and kanamycin-containing plates, respectively. The number of plasmid-free cells BW25113 was calculated by the following formula: BW25113 (plasmid-free) cells=antibiotic-free plate counts minus colistin-containing/kanamycin-containing plate counts. The experimental procedure for a mixture containing BW25113, BW25113(pHNSHP24), and BW25113(pHNSHP24-36DΔcDmt::kan) is consistent with the steps described above. The cell numbers of three strains were calculated by the following formula: BW25113 (plasmid-free) cells=antibiotic-free plate counts minus colistin-containing plate counts, BW25113(pHNSHP24) cells=colistin-containing plate counts minus kanamycin-containing plate counts, BW25113(pHNSHP24)-36DΔcDmt::kan cells=kanamycin-containing plate counts.Plasmid invasion assays were performed according to the method described in the previous study . All plaA260/A280 between 1.8 and 2.2 were used for cDNA synthesis. About 1 µg total RNA was used for reverse transcription using Goldenstar RT6 cDNA Synthesis Mix , according to the production protocol. qPCR was performed using the TB Green Premix Ex Taq II reagent  and Bio-Rad CFX96 Real-Time PCR Detection System . The reaction mixture contained 10 µL of TB Green Premix Ex Taq II, 1 µL of cDNA (diluted 1:10), 0.5 µL of each primer (10 µM), and 8 µL of double distilled water. The amplification was performed as the following conditions: 95℃ for 5 min, 40 cycles of 95℃, 30 s; 60℃, 30s; 72℃, 20s, followed by the melting reaction from 65℃ to 95℃. The relative gene expression levels were calibrated by the 2−ΔΔCt method and gapA gene was used as reference gene. The tenfold gradient-diluted cDNA mixture from all samples was amplified for construction of standard curve. The standard curve was used for calculating the amplification efficiency (E) and correlation coefficient values (R2) of the primers (R2) range from 0.98 to 1. The relative expression level of genes was obtained by the 2−ΔΔCt method , according to the product manual. Then, the integrity of total RNA was confirmed by agarose gel electrophoresis and quantified by NanoDrop 2000c. The RNA samples with 23S/16S rRNA ratio≈2.0 and the  primers . The ampt method . All explacZ fusion vectors were cultured in LB broth containing kanamycin overnight at 37℃. Overnight cultures diluted 1:10 into fresh media with shaking (180 rpm) at 37℃ for 4 h. Cells were collected and resuspended in lysis buffer  and lysed by sonication. The lysates were centrifuged at 12,000× g for 10 min at 4℃ to obtain supernatants, adding 4 μL of the supernatant to Bradford Protein Assay Kit  to determine protein concentration. About 50 μL of the supernatant was taken to mix with 950 μL of the lysis buffer. Whereafter, recording began immediately after the addition of 0.2 mL of o-nitrophenyl-β-D-galactopyranoside (4 mg/L), with all reactions performed at room temperature. Reactions were terminated when the color of mixtures turned yellow by the addition of 500 μL of 1 M Na2CO3. The optical density at 420 nm (OD420) was measured by Multiskan spectrum microplate spectrophotometer . The units of activity were calculated as previously described (Cells carrying escribed .


The hydration reaction of γ-C2S slurry activated with chemical activators  was enhanced by 1% nano-SiO2. The hydrate reaction rate was determined by isothermal calorimetry, and the hydrated samples were characterized by XRD, TGA/DTG, SEM-EDS, and 29Si MAS/NMR. The results revealed a substantial enhancement in the hydration activity of γ-C2S due to the presence of the alkaline activator. Furthermore, nano-SiO2 did not alter the composition of γ-C2S hydration products, instead providing nucleation sites for the growth of hydration products. Incorporating nano-SiO2 promoted the formation of C-(R)-S-H gel with a low calcium-to-silica ratio and increased its polymerization levels, resulting in more favorable structures. Among all the activators used in this study, potassium salts had a better activation effect than sodium salts. After 28 days of curing, the degree of hydration reaction in the KC+Si group was 48% and about 37% for the NHC+Si group. Whereas, the KH+Si and NH+Si groups only reached approximately 20% after the same hydration duration.This paper investigated the combined effect of chemical activators and nano-SiO Nevertheless, they are only retained at the terminus of the C-S-H gel structure, leading to a lower retention capacity than potassium ions [1 and Q2, which influences the degree of silicate polymerization.The MCL serves as an effective indicator of the degree of silicate polymerization within the C-(R)-S-H phase. As shown in tructure . As a retructure . In contium ions ,35. Cons2 induces the decalcification reaction due to its enhanced reactivity. This reaction produces a lower C/S ratio within the C-(R)-S-H structure. The degree of silicate polymerization in the C-S-H phase is closely related to the C/S ratio [2 can significantly enhance the degree of silicate polymerization of C-(R)-S-H gels, causing the structures to be favorable and thereby contributing to the improved mechanical properties [Combined with EDS data, incorporating nano-SiO/S ratio . C-(R)-Soperties ,37. In aoperties .2S. The results prove that the reactivity of γ-C2S activated with chemical activators can be significantly enhanced by nano-SiO2. The main conclusions of this study are summarized as follows:2CO3 activator, followed by the K2CO3 and KOH activators, and the lowest cumulative heat release is for the NaOH activator. The cumulative exothermic capacity of the chemically activated samples can be significantly increased with the inclusion of nano-SiO2.The highest total heat release during the first 3 days of hydration is observed with the Na2S slurry activated with chemical activators are C-(R)-S-H gels, calcium carbonate, and portlandite. Nano-SiO2 primarily serves as a seed to provide nucleation sites for the growth of γ-C2S hydration products. It does not alter the hydration product type but reduces the Ca/Si ratio of the hydrated product (C-(R)-S-H gel). The morphology of C-(R)-S-H gels is influenced by the Ca/Si and the type of chemical activator.The primary products of γ-C29Si NMR spectroscopy shows that the C-(R)-S-H phase mainly comprises Q1 (end chain) and Q2 (bridging silicate tetrahedra) with a structure similar to C-S-H. Variations in the calcium–silica ratio impact the degree of C-(R)-S-H polymerization. The decalcification reaction induced by nano-SiO2 can effectively increase the degree of C-(R)-S-H polymerization and form favorable structures.2CO3 exhibits the most effective activation effect on γ-C2S, while NaOH displays the worst. It is confirmed by measurement methods  that KC+Si-activated γ-C2S has the highest total hydration after 28 days of sample reaction. Under the combination of K2CO3 and nano-SiO2 (KC+Si group), the 28-day hydration degree of γ-C2S slurry can reach up to 48%. The NHC+Si group achieves approximately 37% hydration, while the KH+Si and NH+Si groups are only about 20% after the same hydration duration.KThis study investigates the combined effect of chemical activators and nano-silica on the hydration behavior of γ-C2S can be transformed into reactive minerals by the combined effect of chemical activation and crystal species activation. This will provide a low-pollution and low-risk way to enhance the activity of environmentally friendly cementitious materials  and a theoretical basis for the preparation of belite-based low-carbon gelling materials.In this sense, it is concluded that the non-hydraulic γ-C