Panthera species are largely unknown to date. In this study, the gut bacterial, fungal, and viral metagenomic composition was assessed from three Panthera species  of Indian origin, which were consuming the same diet and belonged to the same geographical location. A non-redundant bacterial gene catalog of the Panthera gut consisting of 1,507,035 putative genes was constructed from 27 Panthera individuals, which revealed a higher abundance of purine metabolism genes correlating with their purine-rich dietary intake. Analysis with Carbohydrate Active enZyme (CAZy) and MEROPS databases identified enrichment of glycoside hydrolases (GHs), glycoside-transferases, and collagenases in the gut, which are important for nutrient acquisition from animal biomass. The bacterial, fungal, and viral community analysis provided the first comprehensive insights into the Panthera-specific microbial community. The Panthera gene catalog and the largest comparative study of the gut bacterial composition of 68 individuals of Carnivora species from different geographical locations and diet underscore the role of diet and geography in shaping the Panthera gut microbiome, which is significant for the health and conservation management of these highly endangered species.Majority of metagenomic studies in the last decade have focused on revealing the gut microbiomes of humans, rodents, and ruminants; however, the gut microbiome and genic information  of large felids such as  Fusobacterium, Clostridium, Sutterella, Fusibacter, Collinsella, Escherichia, Peptostreptococcus, and Phascolarctobacterium have been found abundant in the felids , leopard (Panthera pardus), and tiger (Panthera tigris), from Central India was carried out in this study. The individuals from these three species were on the same diet and location, which makes it a unique study to assess the influence of genetic factors in shaping the gut microbiome, while keeping diet and location constant. The structure of the gut bacterial community was determined using 16S rRNA amplicon and shotgun metagenomic sequencing. The fungal community was assessed using ITS1 region amplicon sequencing, and sequencing of virus-like particles (VLPs) revealed the viral community structure in the gut. A comparative analysis of gut microbial composition and functional profiles of Panthera species with other species of order Carnivora provided novel insights into the gut microbiome of these species.To elucidate the microbial composition and functional repertoire, metagenomic analysis of 21 individuals of three n = 3), Indian leopard (n = 9), and Bengal tiger (n = 9) were collected fresh in nature from the Van Vihar National Park, Bhopal, India, and brought to the laboratory at 4°C immediately after collection. Bacterial metagenomic DNA was isolated from all the fecal samples using QIAamp Stool Mini Kit  according to the manufacturer’s instructions.Fecal samples of healthy lion . The extracted fungal DNA was used for amplification of fungal ITS1 region.The fungal DNA was extracted by using Mo Bio Power Soil DNA isolation kit  with minor modifications. Approximately 200 mg of each fecal sample was added to the PowerBead tubes with 200 μl zymolyase 20 T solution (0.01% in 0.1 M sorbitol) . The tubes with samples were vortexed for a few seconds and incubated at 30°C for 30 min. After addition of 60 μl of Solution C1, the tubes were allowed to bead-beat at 4,600 r/min for 280 s. The tubes were then centrifuged at 10,000 × Fungal and bacterial DNA concentration was estimated by Qubit HS dsDNA assay kit , and quality was estimated by agarose gel electrophoresis. All the DNA samples were stored at −80°C until sequencing.Equal concentration of bacterial and fungal DNA (∼1 ng) was used for PCR amplification of bacterial 16S rRNA V3 hypervariable region and fungal ITS1 region, respectively. The amplification of bacterial 16S rRNA V3 region was performed using Illumina Nextera XT adapter-ligated eubacterial V3 region-specific primers, 341F and 534R, with three different base modifications . NucleotPrimer sequences for amplification of bacterial 16S rRNA V3 region are as below (the base inclusions are marked in bold): The underlined regions in all the primer sequences are the Illumina Nextera XT adapter overhangs, whereas the non-underlined regions are the primer sequences known to target eubacterial 16S rRNA V3 region or fungal ITS1 region, respectively.1.341F_ADA_2BTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTCCTACGGGAGGCAGCAG 3′5′ 534R_ADA_2BGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTATTACCGCGGCTGCTGGC 3′5′ 2.341F_ADA_3BTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATCCTACGGGAGGCAGCAG 3′5′ 534R_ADA_3BGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACTATTACCGCGGCTGCTGGC 3′5′ 3.341F_ADA_4BTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCATCCTACGGGAGGCAGCAG 3′5′ 534R_ADA_4BGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTATATTACCGCGGCTGCTGGC 3′.5′ The optimized PCR conditions were: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 69°C for 30 s, extension at 72°C for 30 s, and a final extension cycle at 72°C for 5 min. Paq5000 DNA polymerase  was used, and 5% dimethyl sulfoxide (DMSO) was added to the master mix to enhance the concentration of amplified product from the metagenomic template.The amplification of fungal ITS1 region was performed using Illumina Nextera XT adapter-ligated ITS1 region-specific primers, ITS1-ADA-F and ITS1-ADA-R .Primer sequences for amplification of fungal ITS1 region:ITS1-ADA-FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCT TGGTCATTTAGAGGAAGTAA 3′5′ ITS1-ADA-RGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGG CTGCGTTCTTCATCGATGC 3′.5′ 2 was added to the PCR master mix to enhance the amplification of ITS1 region.The optimized PCR conditions were: initial denaturation at 95°C for 15 min (polymerase was added after this stage), followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 66.5°C for 2 min, extension at 72°C for 2 min, and a final extension cycle at 72°C for 10 min. Paq5000 DNA polymerase  was used, and a final concentration of 2.5 mM MgClg for 10 min at 4°C. The supernatant was passed once through 0.45-μm pore diameter syringe filter and twice through 0.22-μm pore diameter syringe filter. Then, 20 μl lysozyme (10 mg/ml) was added to the filtrate and incubated at 37°C for 30 min, followed by 10 min incubation with 0.2 volume of chloroform. The samples were then centrifuged at 2,500 × g for 5 min at room temperature. The aqueous phase was collected and incubated with 3 U of DNase-I and 20 μl of 10 × DNase buffer for 1 h at 37°C. Further, the inactivation of DNase-I was done by incubating the samples at 65°C for 15 min. The samples were then incubated with 10 μl of 10% sodium dodecyl sulfate (SDS) and 1 μl of 20 mg/ml proteinase K for 20 min at 56°C, and further incubation for 10 min at 65°C with 35 μl of 5 M NaCl and 28 μl of cetyl trimethylammonium bromide solution (10% CTAB in 0.7 M NaCl). Equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added and centrifuged at 8,000 × g for 5 min at room temperature. The aqueous phase was collected and centrifuged at 8,000 × g for 5 min at room temperature with an equal volume of chloroform.The purification of VLPs and DNA extraction was carried out by minor modification in the protocol suggested by g for 1 min. The remaining steps for elution were performed using the DNeasy Blood and Tissue Kit  according to the manufacturer’s protocol, and finally the DNA was eluted in 20 μl of elution buffer. The extracted DNA was amplified using Illustra GenomiPhi V2 DNA amplification kit, according to the manufacturer’s instructions. The DNA was purified and eluted in 20 μl of elution buffer using DNeasy Blood and tissue kit’s elution protocol. Quality control assays were performed using 16S rDNA PCR to confirm the absent to negligible bacterial DNA contamination using the above protocol.The aqueous phase mixed with 500 μl ethanol was passed through DNeasy Mini spin column  at ≥6,000 × After evaluating the amplified products on 2% w/v agarose gel, the products were purified using Ampure XP kit . The libraries were prepared using Illumina 16S metagenomic library preparation guide and evaluated on 2100 Bioanalyzer using Bioanalyzer DNA 1000 kit  to estimate the library size and Qubit 2.0 flourometer using Qubit dsDNA HS kit  to estimate the library concentration. After this, 150 bp paired-end sequencing of both the libraries was performed on the Illumina NextSeq 500 platform  using NextSeq 500/550 v2 sequencing reagent kit  at the Next-Generation Sequencing (NGS) Facility, Indian Institute of Science Education and Research (IISER) Bhopal, India.The extracted metagenomic and virome DNA were used to prepare the sequencing libraries using Illumina Nextera XT sample preparation kit  by following the manufacturer’s protocol. Size of all the libraries was assessed on the Agilent 2100 Bioanalyzer using Agilent high sensitivity DNA kit  and were quantified on a Qubit 2.0 fluorometer using Qubit dsDNA HS kit  and by qPCR using KAPA SYBR FAST qPCR Master mix and Illumina standards and primer premix  following the Illumina-suggested protocol. Both the shotgun metagenomic and virome libraries were loaded on Illumina NextSeq 500 platform using NextSeq 500/550 v2 sequencing reagent kit , and 150 bp paired-end sequencing was performed at the NGS Facility, IISER Bhopal, India.Raw sequencing reads were processed to obtain high-quality reads for further analysis. First, the ambiguous bases in the 5′ or 3′ ends of the raw reads were trimmed using NGSQC toolkit (v2.3.3) , and thede novo clustering was performed at 97% identity using UCLUST and they were assigned using lowest common ancestor approach against the Greengenes database for 16S and ntf ITS-1 database for ITS1 amplicons. The operational taxonomic units (OTUs) with less than 100 sequences were filtered out from the analysis.The high-quality reads were then clustered at 97% identity against Greengenes database (v13_8) as reference using QIIME (v1.9.1) for 16S amplicons . The ntfde novo clustering of remaining reads was performed using similar criteria mentioned above.The bacterial community structure of nine Bengal tigers, nine Indian leopards, and three lions used in this study were compared with other datasets obtained from NCBI SRA: 13 North Chinese leopards and eight Amur leopards (SRR7229827-47), two Malayan tigers (SRR6147361 and SRR6147362), one caracal (SRR6841704), three cheetahs , five African wild dogs , four wolves , and five foxes (ERR1804913-17). The raw reads were processed to obtain high-quality reads for all samples as mentioned above. Data produced using Ion torrent were first converted to Illumina format using NGSQC toolkit and then processed further. For single-end reads, the step involving merging using FLASH was skipped. Closed reference followed by The alpha diversity of bacteria in each sample was assessed using Shannon indices and observed OTUs, and rarefactions were performed from 100 sequences to a maximum of 560,000 sequences using QIIME. The beta diversity was estimated using weighted UniFrac distances in QIIME. The alpha diversity of fungi in each sample was assessed using Shannon indices and observed OTUs, and rarefactions were performed from 100 sequences to a maximum of 10,000 sequences using QIIME. The principal component analysis (PCA) of taxonomic profiles of all samples was performed using PCA–sklearn in Python. The genus with >1% abundance in either sample was considered in this analysis. The top two principal components were plotted.de novo assembly using metaSPAdes  . The reataSPAdes  for eachtaSPAdes . The reaA total of 11,345 viral genomes were downloaded from NCBI along with their taxonomic information. The taxonomic assignment of contigs at family level was performed using dc_megablast against the downloaded viral genomes as described previously .To examine the correlations among bacterial, fungal, and viral taxa, pair-wise Spearman correlation was calculated between (1) bacterial and fungal genera, (2) fungal genera and viral families, and (3) bacterial genera and viral families in R. The taxa with at least 1% abundance in mean values were used to find the correlations.Raw metagenomic reads of nine Bengal tigers, nine Indian leopards, and three lions generated in this study were processed to obtain high-quality reads for further analysis. Sequencing reads from six Amur tigers were also obtained from NCBI SRA database . First, the low-quality bases in the 5′ or 3′ ends were removed from the raw reads, and then ambiguous bases at both ends were trimmed using NGSQC toolkit (v2.3.3) . The reaDe novo assembly of all the samples was performed using high-quality reads with metaSPAdes (v3.13.0) (v3.13.0) . Genes wv3.13.0) . The reav3.13.0)  using a v3.13.0) .To assess the abundance of genes, reads were aligned to the gene catalog using Bowtie2 (v2.2.3) . For eacThe putative amino acid sequences which translated from the gene catalog were aligned against the Kyoto Encyclopedia of Genes and Genomes (KEGG), Carbohydrate Active enZyme (CAZy) , and MERPanthera species was carried out to study the bacterial, fungal, and viral community structure. A total of 21 individuals from three Panthera species, lion, leopard, and tiger, were included in this study  generated from amplicon sequencing of V3 hypervariable region of 16S rRNA gene  and leopard (24%) samples, and the genus Collinsella was the most abundant in tiger (14%) (Escherichia (24%) in their gut microbiome; however, in this study, its abundance was less than 1% in all the Panthera individuals , and phylum Firmicutes was most abundant in tiger (40%) and leopard (32%) . These per (14%) . A recenividuals . Other beviously , b. A laTrichosporon (mean abundance: 18%) was identified as the most abundant (>80%) genus in a few samples and less than 10% in most samples of the Panthera species. The genus Fusarium (10%) was found as the second most abundant genus in the Panthera gut. Other genera, including Cladosporium, Rhodosporidiobolus, Chaetomium, Phoma, Aspergillus, Cutaneotrichosporon, and Edenia, were also found abundant (>2%) in the gut. Species of genus Trichosporon and Aspergillus have been found abundant in felids.Analysis of ITS1 OTUs revealed the predominance of Ascomycota (50%) and Basidiomycota (32%) in the gut . Two othCaudovirales, Poxviridae, Mimiviridae, and Microviridae family of viruses. Only 13% of virome contigs could be assigned to a taxonomic level, indicating the enormous sequence variation present in these viruses. The viral genomic reads assignment and quantification revealed a higher abundance of Caudovirales viruses in the Panthera gut (32% in lion and 41% in leopard) (Microviridae (14%) and Poxviridae (6%) also showed high abundance in lion individuals. In tiger, 82% of the reads could not be assigned to any known viruses. Previous studies suggest that viruses belonging to Caudovirales and Microviridae are commonly found in the mammalian gut microbiome  and their higher abundance were identified in the Panthera feces. In Microviridae family, “Lynx canadesis associated microvirus CLP 9413,” previously identified in feline feces  . Microvicrobiome . Phage seviously . In thisra feces . A largene feces , was amoCollinsella and Fusarium (ρ = −0.5), Ruminococcus with Phoma and Culvularia (ρ = −0.5), and Sutrella with Aspergillus and Chaetomium (ρ = −0.6)  and Microviridae with Curvularia and Edenia (ρ = 0.6) were also observed.Pair-wise Spearman correlations were calculated among the bacterial genera, fungal genera, and viral families. The analysis revealed a significant negative correlation of  = −0.6) . PositivPanthera individuals lipids, glyco(amino)glycans, and nucleoside diphosphate sugars. Alpha-amylase was among the other abundant carbohydrate-active enzymes, which plays vital roles in the utilization of dietary starch and proteins in mammals , glycoside hydrolase (GH), and carbohydrate-binding modules (CBMs). Among the GTs, the subfamilies GT2 and GT4 were the two topmost abundant subfamilies identified in Panthera . The lys mammals . A highePanthera gut. The analysis identified a total of 24,526 unique MEROPS genes. These genes were highly enriched (>8% abundance) for Subfamily M23B unassigned peptidases, which are zinc metalloendopeptidases. Among other abundant enzymes were prolinases, carboxydipeptidases, and carboxypeptidases .Several studies suggest that geography and diet are the critical confounding factors that can alter the gut microbiota of mammals . The effPanthera gut including the Bengal and Amur tigers, where the Amur tigers were separated from the other individuals on PC1 . The observed differences in the gut metagenome of the same Panthera species belonging to different geographical locations reaffirm the key impact of geographical locations in shaping the gut microbiome composition  from 48 felids and 14 canids  species. Therefore, in this work, the bacterial, fungal, and viral species present in the gut are studied to have a complete understanding of the gut microbiota of hypercarnivores, specifically the Panthera species of Indian origin.Recent studies have revealed that the host-associated gut microbiota significantly influences the host health and, therefore, the understanding of gut microbiota of the endangered species is important for their better management and conservation . HoweverFusobacterium and Collinsella species observed in the Panthera individuals in this study was also reported previously in Amur tigers  produced by Fusarium and thus reduces the pathogenic potential of Fusarium species  genes of which 64% could be annotated using the KEGG database. The number of genes in the gene catalog is substantially lower compared to the human gut gene catalog, which consisted of 9.9 million genes   . The lowPanthera species gut. An important observation was enrichment in the purine metabolic pathways essential for uric acid degradation in the intestine. Its higher abundance corroborates with previous reports on the association of the pathway with purine-rich diet in these hypercarnivores  Bhopal. The protocol was approved by the Institute Ethics Committee.Panthera individuals. RS and SM performed the experimental procedures and sequencing. PM performed the computational analysis and created figures. PM and VS interpreted the results. All the authors have read and approved the final manuscript.AG provided the fecal samples and necessary information of the 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.


K5, L311RK5, R211PK14, I150VK18), the tetramer interface , or higher-order interactions needed for mature filament formation . The biochemical changes included altered hydrophobic and electrostatic interactions, and altered surface charge, hydrophobicity or contour. Together, these findings advance the genotype-structurotype-phenotype correlation for keratin-based human diseases.Keratin intermediate filaments constitute the primary cytoskeletal component of epithelial cells. Numerous human disease phenotypes related to keratin mutation remain mechanistically elusive. Our recent crystal structures of the helix 1B heterotetramer from keratin 1/10 enabled further investigation of the effect of pathologic 1B domain mutations on keratin structure. We used our highest resolution keratin 1B structure as a template for homology-modeling the 1B heterotetramers of keratin 5/14 (associated with blistering skin disorders), keratin 8/18 (associated with liver disease), and keratin 74/28 (associated with hair disorder). Each structure was examined for the molecular alterations caused by incorporating pathogenic 1B keratin mutations. Structural modeling indicated keratin 1B mutations can harm the heterodimer interface (R265P Intermediate filaments (IFs) work with actin microfilaments and microtubules to provide essential cytoskeleton functions within eukaryotic cells. IFs are aptly named because their diameter ~10 nm) is in between that of actin (~4–5 nm) and microtubules ~25 nm) . Q1. Q144]. fibrosis . Q169K8 nterface a. It mak Q153K18 c. Q169EKmutation e. This aK18 is located at the N-terminus of the K8/18 1B heterodimer  is a type II IF protein whose obligate IF partner has yet to be unambiguously identified. Prior work showed that K74 can pair with K14 and K18 . HoweverK74 was identified during sequencing of a consanguineous Pakistani family which had homozygous autosomal recessive pure hair and nail ectodermal dysplasia (PHNED) [K74 would interfere with keratin hetero-dimerization. However, our recent crystal structures of K1/K10 1B tetramers (wild-type and S233LK1) demonstrated that the sequence position of F274K74 forms part of the anchoring knob involved in the anchoring knob–hydrophobic pocket tetramer assembly mechanism  and mature filament assembly [F274S (PHNED) . Patientechanism a,b. Thisassembly .K74 mutation dramatically alters the knob-pocket interaction site, eliminating hydrophobic interactions and aromatic–aromatic interactions  known to cause human disease [With 54 genes and differential expression by time and tissue type, keratins play a significant, but complex role in human physiology. Their medical relevance is exemplified by the high numbers of IF-related diseases associated with keratin mutation . Their r disease . Therefo disease ,15.In the spirit of this paradigm, the work here examines nine pathologic keratin mutations that occur within the 1B subdomain of the keratin coiled-coil rod domain. Most keratin mutations associated with clinical disease are located in the 1A and 2B subdomains, with only a small number to date occurring in 1B. The molecular explanation for this difference is not clear, but evidence suggests the 1B domain is critical for establishing the tetramer building block of IFs, whereas the 1A and 2B domains may play a greater role in the higher-order packing interactions needed to form mature IFs ,16,48,49K5, L311RK5, R211PK14, I150VK18), some harm the tetramer interface , and some harm higher-order interactions needed for mature IF formation . Some mutations, like L311RK5, alter multiple interfaces. Three of the 1B mutations have a prominent change in molecular surface properties: Q169EK8 generates an acidic surface patch, L311RK5 eliminates a hydrophobic patch, and H128LK18 creates a new hydrophobic patch. Solvent accessible hydrophobic surface has been shown to cause aberrant aggregation for S233LK1 [K18 altered K8/K18 IFs in vitro by electron microscopy [K8, H128LK18, I150VK18) allocated a “benign” prediction by Polyphen-2 analysis   and cros


Feeble cellular responses induced by T cell-based vaccines are a major challenge for the development of an effective vaccine against Hepatitis C virus (HCV) infection. To address this challenge, the potential of N-terminal fragment of gp96 heat shock protein (rNT (gp96) as an adjuvant was evaluated and compared to that of the CpG (as a recognized Th1-type adjuvant) in the formulation of HCV core/NS3 antigens in three immunization strategies of protein/protein, DNA/DNA, and DNA/protein. Immunized mice were evaluated for elicited immune responses in week 3 (W3) and 11 post-immunizations. Our results demonstrated that the protein (subunit) vaccine formulated with rNT (gp96) in protein/protein strategy (core/NS3 + gp96) was significantly more efficient than CpG oligodeoxynucleotides (CpG ODN) formulation and all other immunization strategies in the induction of Th1-type cytokines. This group of mice (core/NS3 + gp96) also elicited a high level of anti-Core-NS3 total immunoglobulin G (IgG) with dominant IgG2a isotype at W3. Thus, the co-administration of recombinant NT (gp96) protein with rHCV proteins might be a promising approach in the formulation of HCV subunit vaccine candidates for induction of high levels of Th1 cytokines and humoral responses. Infection with Hepatitis C virus (HCV) turns to chronic hepatitis in the majority of the infected individuals that might result in liver cirrhosis or malignancies in time. Although recently invented direct-acting antiviral drugs (DAAs) can cure a high percentage of chronic hepatitis C (CHC) patients , only a HCV genome encodes for several structural  and non-structural proteins p7 to NS5) [ to NS5 . In geneIn the present study, we investigated and compared the adjuvant effects of NT (gp96) and CpG ODNs in the formulation of both recombinant proteins and DNA vaccines encoding HCV core and NS3 Ags (core/NS3) in various protein/protein, DNA/DNA, and DNA/protein immunization regimens in mice. We also studied the long-term efficacy of the vaccine candidates in all immunized mice groups.NheI and EcoRI restriction sites  to construct pET-28b-NT (gp96). Subsequently, to prepare pEGFP-Core-NS3-NT (gp96), the NT (gp96) sequence was amplified from pET-28b-NT (gp96) using KZ1/2 primer pairs and inserted into the EcoRI and ApaI restriction sites of pEGFP-N3 (Invitrogen)  A. The saion site B. The pcion site . The pre4 COS-7 cells per well were seeded on a four-well plate  and transfected when the cells reached a confluency of 75%. Five μg of pEGFP-Core-NS3, pEGFP-Core-NS3-NT (gp96), pEGFP-N3 (positive control), and pcDNA3.1 (negative control) plasmids were transfected using linear polyethyleneimine (MW = 25 kDa)  as described previously [E. coli M15 for pQE30-NT (gp96)) were grown in 2 xyt broth  to an optical density of 0.7–0.8 at 600 nm. The protein expression was induced with 1 mM IPTG at 16 °C for rNS3 and 37 °C for core and NT (gp96) proteins. The cell pellets were collected and stored at −20 °C until used for protein purification. The cells were lysed by lysozyme digestion followed by sonication. The protein samples were analyzed by SDS-PAGE in 10% (w/v) polyacrylamide gel-based on standard protocols . The protein bands were transferred onto a polyvinylidene difluoride (PVDF) membrane using a Bio-Rad wet blotting system, and the membrane was incubated with blocking solution (TBS 10× (pH 7.4), 0.1% tween 20, 2.5% BSA) overnight. After washing steps, the membrane was treated with mouse anti-His monoclonal antibody  and horseradish peroxidase-conjugated goat anti-mouse IgG antibody  as the first and second antibodies, respectively. Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit , and light emission was visualized by exposing the membrane to a Hyper Film ECL . The 6xHis-tag encoding recombinant core, NS3 and NT (gp96) proteins were purified by affinity chromatography on a Ni-NTA superflow column. The purification was performed under native conditions according to the manufacturer’s instructions . Column washing buffers ; pH 8) were used to wash the column sequentially. After washing steps, bound proteins were eluted with elution buffer . The eluted fractions were pooled, concentrated by ultrafiltration (Amicon), and dialyzed against PBS. The protein concentrations were determined by a BCA assay kit . The endotoxin level of the purified proteins was quantified by QCL-1000 Chromogenic Limulus amoebocyte lysate test according to the manufacturer protocol , which showed less than 25 endotoxin units per 50 µg of the purified protein . The purified rCore and rNS3 proteins were used as an immunogen in combination with purified rNT (gp96) protein and CpG ODN as an adjuvant in mice immunization procedures. The same proteins were also used as coating Ag for ELISA and stimulation of the splenocytes in cytokine.The expression of the Core-NS3 and Core-NS3-NT (gp96) proteins was tested in the COS-7 cell line. 5 × 10eviously . At 24 hn = 15 per group) obtained from the breeding stock maintained at Pasteur Institute of Iran. All experiments were performed according to the guidelines approved by the Ethical Committee of School of Public Health, Tehran University of Medical Sciences (Code of Ethics: 95-02-27-31344). Group1 (G1) was immunized twice with 10 μg rCore, and 10 μg rNS3 formulated with 10 μg rNT (gp96) adjuvant for both prime and boost immunizations ). Group2 (G2) was immunized three times with 80 μg of pcDNA-Core-NS3-NT (gp96) plasmid in PBS for both prime and boost immunizations /DNA-NT (gp96)). Group3 (G3) was vaccinated with 100 μg of pcDNA-Core-NS3-NT (gp96) plasmid in PBS for prime but twice with 10 μg rCore and 10 μg rNS3 formulated with 10 μg rNT (gp96) for boost immunizations /protein + rNT (gp96)). Group 4 (G4) received three times PBS as control. Group 5 (G5) was vaccinated three times with 80 μg of pcDNA-Core-NS3 plasmid in PBS for both prime and boost immunizations . Group 6A (G6A) was vaccinated with 10 μg rCore and 10 μg rNS3 in combination with 40 μg CpG ODN adjuvant (5/-TCC ATG ACG TTC CTG ACG TT-3/) for both prime and boost immunizations . Group 6B (G6B) was vaccinated with 10 μg rCore and 10 μg rNS3 with 40 μg non-CpG (5/-TCC AGG ACT TCT CTC AGG TT-3/) for both prime and boost immunizations . Group7 (G7) was immunized three times with 80 μg of empty pcDNA3.1(+) plasmid in PBS for both prime and boost immunizations . Group8 (G8) was immunized twice with 10 μg rNT (gp96) for both prime and boost immunizations / rNT (gp96)). Group9 (G9) was immunized twice with 10 μg rCore and 10 μg rNS3 for both and boost immunizations . All groups were vaccinated intramuscularly in the tibialis anterior muscle at a three-week interval, as summarized in Three different prime-boost regimens were carried out in ten groups of 6-week-old female BALB/c mice  was used to lyse the erythrocytes. After washing in DMEM, up to 2 × 106 splenocytes were seeded per well in 96-well plates in complete DMEM  and stimulated with rCore and rNS3 at a concentration of 5 μg/mL at 37 °C in 5% CO2. Cells with no treatment  and cells incubated with concanavalin A  were used as negative and positive controls, respectively. The cell culture supernatants were harvested after 72 h for IL-4 assay and 96 h for IFN-γ assay. The levels of cytokines were detected using a sandwich ELISA according to the manufacturer’s protocol . All assays were performed in duplicates. The lower detection limit of both IFN-γ and IL-4 was 2 pg/mL, and the concentration was calculated according to the standard curve.Three mice in each group were euthanized randomly at W3 and W11. The ACK (ammonium-chloride-potassium) lysis buffer  was carried out to evaluate statistical analysis. The significance of experimental differences was analyzed using one-way ANOVA (Tukey’s multiple comparison test) and Student’s As shown in E. coli was analyzed by SDS-PAGE  proteins in SDS-PAGE D–F and wSDS-PAGE G. SDS-PASDS-PAGE D,E. SDS-SDS-PAGE F. SDS-PASDS-PAGE G.p < 0.0001). Our results also revealed a significantly higher level of IFN-γ in G3 compared to G2, G5, G6A, and all control groups at W3 (p < 0.0001), but the same pattern was not observed for this group at W11. Moreover, in contrast to G3 and G6A, other vaccinated groups G1, G2, and G5 indicated an increase in IFN-γ level up to 1.2, 2.93, and 5.32-fold, respectively, at W11  was measured using the supernatant of Ag–stimulated splenocytes of all mice groups at W3 and W11. As shown in , at W11 A. We furp < 0.01, p < 0.001, p < 0.01, Although antibodies raised against HCV core and NS3 proteins, have no neutralization value, but to gain insights about the potential of rNT (gp96) as an adjuvant for induction of humoral responses, the level of specific Abs against rCore-rNS3 proteins in the sera of the corresponding mice groups were evaluated. As shown in The present study was designed to evaluate the capacity of recombinant protein NT (gp96) as an adjuvant to enhance immune responses in the formulation of HCV rCore and rNS3 proteins as an immunogen. To our knowledge, this is the first report on the utilization of recombinant gp96 protein as an adjuvant to develop a candidate HCV protein vaccine. In addition, the efficacy of three immunization regimens, including DNA/DNA, DNA/protein, and protein/protein for HCV rCore and rNS3 proteins, was compared in the BALB/c mice model. Our results indicated that immunization with a protein vaccine formulated with rNT (gp96) protein as an adjuvant in a homologues protein/protein + rNT (gp96) strategy (G1) could efficiently induce short and long-term Th1-type cytokines compared to other immunization regimens . Moreover, results of the present study indicated that NT (gp96) as a recombinant protein adjuvant along with HCV proteins (core/NS3) could induce more potent immune responses than the previously reported vaccine formulations comprising NT (gp96) fragment fused to HCV polytope DNA (PT) (PT-NT (gp96)) .Considering the importance of Th1 cytokine production for protection from HCV infection and prevention of chronicity, we evaluated our vaccine formulations for induction of these cytokines by cytokine assay. Of note, immunized mice in G1 not only elicited significantly higher levels of IFN-γ than other vaccinated groups at W3 and W11 but also showed an increase of IFN-γ (1/2 fold) production and a concomitant decrease in IL-4 (1/8 fold) at W11 compared to W3 A,B. ThesAs shown in It should be noted that the main “aim” of the present study was studying the potential of gp96 in combination with HCV core/NS3 Ags for induction of Th1-type cytokines as a “proof of concept” with no claim for protection from HCV infection by this immunogenic formulation. Nevertheless, for the next step, “pre-clinical studies,” a proper animal model and challenging studies might be considered.Overall, in this study, we provided the first report on the application of rNT (gp96) as an adjuvant for the induction of more potent immune responses against rCore and rNS3 Ags in a protein (subunit) vaccine candidate. Our preliminary findings suggested rNT (gp96) as a promising adjuvant for vaccination purposes to HCV and demonstrated that immunization with homologous protein/protein-rNT (gp96) strategy was capable of inducing the highest level of Th1-type cytokine. Despite recognizing no neutralization value for anti- Core/NS3 Abs, but our results indicated the potency of rNT (gp96) as an adjuvant for induction of high-level humoral responses, which might be considered in the formulation of HCV E2 protein . Therefore, collectively, the co-administration of recombinant NT (gp96) protein with rHCV proteins might be a promising approach in the formulation of HCV subunit vaccine candidates for the induction of high levels of Th1 cytokines and humoral responses.


N6-methyladenosine (m6A) modification regulates mRNA stability and translation. Here, we show that transcriptomic m6A modification can be dynamic and the m6A reader protein YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) promotes mRNA decay during cell cycle. Depletion of YTHDF2 in HeLa cells leads to the delay of mitotic entry due to overaccumulation of negative regulators of cell cycle such as Wee1-like protein kinase (WEE1). We demonstrate that WEE1 transcripts contain m6A modification, which promotes their decay through YTHDF2. Moreover, we found that YTHDF2 protein stability is dependent on cyclin-dependent kinase 1 (CDK1) activity. Thus, CDK1, YTHDF2, and WEE1 form a feedforward regulatory loop to promote mitotic entry. We further identified Cullin 1 (CUL1), Cullin 4A (CUL4A), damaged DNA-binding protein 1 (DDB1), and S-phase kinase-associated protein 2 (SKP2) as components of E3 ubiquitin ligase complexes that mediate YTHDF2 proteolysis. Our study provides insights into how cell cycle mediators modulate transcriptomic m6A modification, which in turn regulates the cell cycle.The  It acce complex  or the e complex . In zebr complex . Loss of complex . Likewis complex , and Yth complex , suggest complex ,22. This complex .6A pathway has been implicated in regulating genes related to cell cycle. It was reported that transcripts involved in cell cycle regulation contain m6A modification in mouse embryonic stem cells (mESC) [6A demethylase, facilitates glioblastoma proliferation by demethylating the transcripts of forkhead box protein M1 (FOXM1), an important cell cycle regulator [6A pathway potentially plays a role in cell cycle regulation; however, mechanistic studies are still limited, and the impact of m6A readers on cell cycle remains unexplored. Here, we investigated changes in transcriptomic m6A during cell cycle and demonstrated a role of YTHDF2 in regulating phase-specific transcript degradation. Furthermore, we identified the proteolysis pathway of YTHDF2, which establishes a mechanistic interconnection between the m6A pathway and cell cycle.The ms (mESC) . Down-res (mESC) . AlkB Hoegulator . METTL14egulator . These s1 and S phases [6A modification may also change during cell cycle because m6A on mRNAs are deposited co-transcriptionally [6A changes during cell cycle, we synchronized HeLa cells to the stage of G1/S transition by double thymidine block and collected cells for m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq) from 0, 4, and 8 hours post release, which correspond to G1/S, S, and G2/M phases, respectively  of transcripts at all phases  to S (4 hours) phase and remained steady thereafter , an m6A demethylase by western blot. We found that METTL3 showed lower protein level at early G1/S (0 h) phase and M (10 h) phase, while METTL14 only decreased at M phase  data (see subsequently) revealed that the transcript levels of METTL3, METTL14, and FTO all remained relatively steady  displayed consistent slower proliferation  using unsynchronized cells followed by sequencing. Based on the YTHDF2 RIP sequencing (RIP-seq) data and the published YTHDF2 photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) data , we idenntargets . These r6A enrichment. We found that the 188 phase-specific YTHDF2 targets displayed significantly higher accumulation at 8 and 10 hours post release in the absence of YTHDF2, compared with the 515 nontargets , which is controlled by phosphorylation activities of Wee1-like protein kinase (WEE1) and membrane associated tyrosine/threonine 1 (MYT1), and dephosphorylation by cell division cycle 25 (CDC25) [YTHDF2 knockout cells, p-CDK1-Y15—but not p-CDK1-T14—is accordingly excessive in the knockout cells  . We foun (CDC25) ,42. Consut cells . RNA-seq M phase , consist M phase . Moreoveic entry . These r6A MeRIP-seq and PAR-CLIP data, we found that WEE1 transcripts indeed contain m6A modification mainly at the 3′ UTR region  and Really Interesting New Gene (RING) E3 ligases are among the major classes of ubiquitin ligases . The NEDTo rule out that RO-3306 treatment possibly activates NAE activity leading to downstream YTHDF2 degradation, we assessed the level of neddylated form of UBC12 (UBC12-NEDD8) catalyzed by NAE. No increase in UBC12-NEDD8 was observed after RO-3306 treatment . ConsistCUL1, CUL4A, CUL4B, and CUL7. Both CUL1 and CUL4A knockdown, but not CUL4B and CUL7, led to the overaccumulation of YTHDF2  is a known adaptor of CUL4A  and mighincrease , suggestincrease . These r6A participates in a variety of biological processes, such as cell differentiation, development, memory, and immunity. Although it has been implicated in cell cycle regulation, systematic and mechanistic studies are still lacking. In this study, we investigated whether transcriptomic m6A is impacted by cell cycle regulation and found that m6A-modified transcripts, especially the ones with phase-specific expression patterns, display dynamic changes during cell cycle. It has been reported that mRNA synthesis and decay show dynamic changes during yeast cell cycle [6A modification in promoting RNA degradation during cell cycle.Transcriptomic mll cycle ; however2 to M phase transition by increasing the ratio of the inactive CDK1 form—CDK1-Y15. Phosphorylation and dephosphorylation of regulators, such as CDK1 and WEE1, are important mechanisms that regulate mitosis [6A modification of cell cycle regulator transcripts represents another pathway to fine-tune cell cycle progression. Future epitranscriptomic studies of important cell cycle regulators in different cell types and biological processes could provide more insights.Further investigation found that YTHDF2 depletion leads to delayed mitotic entry. By meticulously comparing a number of key regulators gating mitotic entry, we found that WEE1, a suppressor of CDK1 by phosphorylation, is regulated by YTHDF2 by promoting WEE1 transcript decay. Therefore, overaccumulation of WEE1 protein upon YTHDF2 depletion leads to the delay of G mitosis ,60,61. H6A. However, whether CDK1 maintains YTHDF2 stability by directly phosphorylating YTHDF2 or through an indirect mechanism remains to be investigated in the future. Considering that CDK1 represses WEE1 by phosphorylation at serine 123 [By testing a number of small molecule inhibitors, we found that the YTHDF2 protein stability relies on CDK1 activity. YTHDF2 undergoes dramatic polyubiquitination and subsequent degradation upon CDK1 inhibition. This result indicates that CDK1 maintains YTHDF2 stability during cell cycle, which represses WEE1 at the RNA level via mrine 123 , CDK1, YAdopting a similar chemical biology approach, we interrogated the degradation pathway of YTHDF2. We found that YTHDF2 proteolysis occurs on proteasomes mediated by the CRL-NEDD8 pathway, which is known for regulating many cellular processes, including cell cycle progression . Screeni6A that serves as an additional level of regulation in cell cycle control, in addition to the well-known regulations through protein phosphorylation and degradation. Moreover, we identified a feedforward regulatory loop that consists of CDK1, YTHDF2, and WEE1 regulating mitotic entry. The recent study revealed that YTHDF2 is essential for AML cells and depletion of YTHDF2 leads to the apoptosis of AML cells [In summary, we revealed a novel regulatory mechanism involving mML cells , renderiML cells . TherefoML cells —or for eML cells ,22.2. NB4 cells were grown in RPMI medium 1640 (Gibco) with 10% FBS, 1% HEPES (Gibco), and 1% penicillin/streptomycin. MonoMac-6 cells were grown in RPMI 1640 supplemented with 10% FBS, 1% HEPES, 2 mM L-Glutamine (Gibco), 1% nonessential amino acids solution (Gibco), 1 mM sodium pyruvate (Gibco), 9 μg/ml insulin (MilliporeSigma), and 1% penicillin/streptomycin.Human HeLa and HEK 293T cell lines were purchased from ATCC and maintained in DMEM (Gibco) with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco) in a humidified 37°C incubator with 5% COPS100001) were ordered from OriGene.Human full-length YTHDF2 cDNA was cloned and inserted into the PiggyBac vector with a FLAG tag at the N-terminus. High-purity plasmids used for mammalian cell transfection and stable cell line preparation were prepared using the HiSpeed Plasmid Midi Kit (Qiagen). The WEE1 construct (Category Number RC209760) and empty vector control (Category Number Cells were resuspended 16 hours prior to both siRNA and plasmid transfections. siRNAs were transfected to cells at approximately 50% confluency using the transfection reagent Lipofectamine RNAiMAX (Invitrogen). Cells reaching approximately 70% confluency were transfected with plasmids using Lipofectamine 2000 (Invitrogen). Both siRNA and plasmid transfections were conducted following the manufacturer’s protocols. Transfected cells were harvested 48 hours post siRNA or plasmid transfections for the following experiments. For stable FLAG-YTHDF2 HeLa cell line generation, DMEM medium supplemented with 1 μg/mL puromycin was used for selection 48 hours post plasmid transfection. Cells were maintained in medium with 1 μg/mL puromycin for at least a week before experiments.YTHDF2 gene in line KO-4 having 39-bp deletion, resulting in the deletion of 13 amino acid residues.YTHDF2 knockout was performed using the Alt-R CRISPR-Cas9 System from Integrated DNA Technologies (IDT) in HeLa cells. Guide RNA sequences were designed using CHOPCHOP  and synt2 incubator, followed by measuring of the absorbance at 450 nm using the Synergy HTX Multi-Mode Microplate Reader (BioTek). Absorbance was measured for 3 additional days to calculate the relative cell proliferation. To measure the proliferation of cells with siRNA knockdown, cells were transfected with siRNAs using Lipofectamine RNAiMax (Invitrogen) at least 6 hours before trypsinization for cell counting and seeding into 96-well plates. At least 5 wells were measured to calculate the average absorbance for each cell line at each time point.Cells were suspended in medium and counted using the Countess Automated Cell Counter (Invitrogen). About 3,000 cells were seeded to each well of the 96-well plates. After the cells adhered to the plate, 10 μL of the Cell Counting Kit-8 (MilliporeSigma) solution was added to each well and incubated for 2 hours in the 37°C CO2 incubator for 14 hours and then washed with 10 mL PBS twice. Growth medium supplemented with 24 μM deoxycytidine was added to the cells and incubated for 9 hours. The amount of 200 μL of 100 mM thymidine stock solution was added to the cell culture and incubated for 14 hours. To release the block, cells were washed with 10 mL PBS twice, and 10 mL medium was added for cell cycle progression. Cells were collected at different time points for up to 10 hours for experiments.HeLa cells were synchronized to early S phase using the double thymidine block based on a previous protocol . BrieflySingle-cell suspensions were acquired by treating cells with trypsin solution at different time points post double thymidine block and mixed with equal volume of freshly prepared 4% paraformaldehyde (PFA) solution in PBS to reach a final concentration of 2% PFA. Cells were fixed at room temperature for 15 minutes, followed by washing twice with PBS. Cells were stained in 5 μg/mL Hoechst 33342 (MilliporeSigma) in PBS for 15 minutes at 37°C before flow cytometry. Ultraviolet light with 355-nm wavelength and 450/50-nm bandpass filters were used to quantify the DNA content of the cells for flow cytometry.Small molecule inhibitors were dissolved in DMSO as stock solutions. Inhibitors were added to cell cultures with final concentrations and incubation times as indicated in each figure legend. For proteasome or E3 ligase inhibition assays, cells were incubated with 10 μM MG-132 (Selleckchem) , 100 μM g for 5 minutes. Cell pellets were resuspended with 3 volumes of immunoprecipitation lysis buffer  and incubated on ice for 20 minutes. Cell lysates were then centrifuged at 16,000g for 15 minutes at 4°C. We saved 50 μl of cell lysate as input and incubated the rest with pre-equilibrated 50 μl anti-FLAG M2 magnetic beads (MilliporeSigma) by rotating at 4°C for 2 hours. After incubation, beads were subjected to washing 6 times with lysis buffer and boiled in NuPAGE LDS Sample Buffer (Invitrogen) with 5% 2-mercaptoethanol (2-ME). After magnetic separation, the supernatant was transferred as immunoprecipitation sample for western blotting, together with 1% input.A stable line of HeLa cells expressing FLAG-YTHDF2 was collected from 15 cm plates and pelleted by centrifuge at 400g for 15 minutes at 4°C, the supernatant was transferred and boiled in NuPAGE LDS Sample Buffer (Invitrogen) with 5% 2-ME (MilliporeSigma). Samples were loaded to a NuPAGE 4% to 12% Bis-Tris Protein Gel (Invitrogen) for electrophoresis at a constant voltage of 160 V until the front dye reached the bottom of the gel and were transferred to 0.2 or 0.45 μM nitrocellulose membranes (BioRad) using the Trans-Blot SD semi-dry transfer cell (BioRad), followed by blocking with 5% nonfat milk (BioRad) in TBST. Antibody concentrations used for western blots were adjusted based on the manufacturer’s instructions.For samples collected for western blot, cells were lysed using RIPA buffer (Invitrogen) containing 1:100 protease inhibitor cocktail (Invitrogen) and phosphatase inhibitor cocktail (Invitrogen) on ice for 20 minutes. After centrifuging at 16,000Total RNA was purified from cells with the TRIzol reagent (Thermo Scientific). Polyadenylated RNA was purified from total RNA using the Dynabeads mRNA DIRECT kit (Thermo Scientific) according to the manufacturer’s protocol. mRNA concentrations were measured by Qubit fluorometer (Invitrogen) with the Qubit RNA HS Assay Kit (Invitrogen).The amount of 300 ng of isolated polyadenylated RNA was further processed using RiboMinus Transcriptome Isolation Kit (Invitrogen). The eluted RNA was then digested by nuclease P1 (MilliporeSigma) followed by dephosphorylation by alkaline phosphatase (Invitrogen). The samples were then used for LC-MS/MS. For detailed procedures for sample process and mass spectrometry, refer to the previous report .Total RNA was extracted from HeLa cells at different time points post release from double thymidine block and was subjected to polyadenylated RNA isolation using the Dynabeads mRNA DIRECT kit (Thermo Scientific). The amount of 100 ng of mRNA samples was used for RNA-seq library construction using the TruSeq stranded mRNA sample preparation kit (Illumina) by following the manufacturer’s protocol. High-throughput libraries for 2 biological replicates were sequenced using Illumina HiSeq 4000 with single-end 50-bp read length at The University of Chicago.g, and washed once with cold PBS. The cell pellets were lysed with 3 volumes of lysis buffer  on ice for 20 minutes. The lysates were centrifuged at 16,000g for 15 minutes; 50 μL cell lysate was saved as input and mixed with 1 mL TRIzol for RNA extraction; and 20 μg of YTHDF2 antibody was added to the rest of the lysate and incubated by rotating for 2 hours at 4°C. Afterward, 40 μL of Pierce Protein A/G Magnetic Beads was washed twice with lysis buffer and added to the lysate and was incubated by rotating for 1 hour at 4°C. After separation using a magnetic rack, beads were washed with ice-code lysis 6 times. Beads were then mixed with 1 mL TRIzol and recovered for RNA, which was saved as immunoprecipitation. RNA extracted from input was further subjected to mRNA purification using the Dynabeads mRNA DIRECT kit (Thermo Scientific). Input mRNA of 100 ng and immunoprecipitation RNA samples with 2 biological replicates were used for RNA-seq library preparation.Unsynchronized HeLa cells with 90% confluency in 15 cm plates were collected by cell lifters, pelleted by centrifuge for 5 minutes at 400HPRT1 primers were designed and synthesized from IDT  according to the manufacturer’'s protocol. The resultant DNA-free RNA was reverse-transcribed using the PrimeScript RT Reagent Kit . The cDNA products were then used for real-time qPCR on the LightCycler 96 System (Roche) using FastStart Essential DNA Green Master (Roche). Gene-specific and housekeeping gene from IDT . qPCR an6A MeRIP-seq library preparation was adapted from the previous published protocol [6A immunoprecipitation using 15 μg anti-m6A antibody (Synaptic Systems) in immunoprecipitation buffer  for 2 hours at 4°C. The amount of 50 μl pre-washed protein A/G beads was added and incubate by rotating for 2 hours, followed by washing 3 times using immunoprecipitation buffer. The m6A-containing RNA fragments from beads were eluted twice using 100 μl elution buffer  for 1 hour while rotating at 4°C. Eluted RNA was then precipitated in 0.3 M sodium acetate (pH 5.2) and 75% ethanol with GlycoBlue (Invitrogen) at −80°C overnight. RNA was recovered from the pellet by centrifuge at 16,000g for 20 minutes at 4°C. Both input and immunoprecipitation RNA samples were subjected to RNA-seq library construction.The mprotocol . Briefly6A-seq libraries was performed using FastQC version 0.11.5. After trimming adapter using Cutadapt version 1.1.5 [The quality control of sequencing reads of RNA-seq, RIP-seq, and mon 1.1.5 , we thenon 1.1.5 . For RNAon 1.1.5 . For RIPon 1.1.5 .6A-seq data analysis, the m6A-enriched regions in each m6A-immunoprecipitation sample were identified by MACS2 version 2.1.0 with the option of “—nomodel” [6A distribution on transcripts was performed using the MetaPlotR pipeline [6A peak visualization [6A peak differential analysis with a cutoff of P < 1 × 10−3. Genes with differential m6A enrichment at different phases of the cell cycle were subjected to GO analysis using DAVID [For mnomodel” , using cnomodel” . Metagenpipeline . IGVtoollization . The R png DAVID . GO termng DAVID .S1 Fig6A modifications across 3 phases of the cell cycle. Color key represents the −log of enriched GO terms. (C) Correlation heatmap representing pairwise comparison of m6A enrichment for each replicate at 3 phases. (D) Western blot of METTL3, METTL14, and FTO at different time points post synchronization. (E) Expression levels of METTL3, METTL14, and FTO at different time points post synchronization from the RNA-seq data. Underlying data for this figure can be found in (A) Workflow of cell synchronization by double thymidine block and time points for cell collection after release. (B) GO terms of common genes with m(TIF)Click here for additional data file.S2 Fig6A peak enrichment. (B) Cumulative distribution of gene expression changes with differential m6A enrichment at different phases. Left panel shows cumulative distribution by comparing the expression level of transcripts with up- or down-regulated m6A from G1/S to S phase. Right panel shows that from S to G2/M phase. The x-axes indicate the log2 fold change of gene expression level in the next phase compared with the previous phase. P values were calculated using the Mann-Whitney test. (C) GO terms for increased m6A peaks at S phase compared with G1/S. (D) GO terms for decreased m6A peaks at S phase compared with G1/S. (E) GO terms for increased m6A peaks at G2/M phase compared with S. TUBB4B is an example that is related to “microtubule-based process” with higher m6A at G2/M phase. (F) GO terms for decreased m6A peaks at G2/M phase compared with S. SMAD3 is an example that is related to regulation of transcription with reduced m6A from S phase to G2/M phase. Underlying data for this figure can be found in (A) Correlation analysis of gene expression levels and m(TIF)Click here for additional data file.S3 FigYTHDF2 knockout. (B) Rescue of YTHDF2 knockout cell lines by FLAG-YTHDF2 transfection. Two knockout cell lines KO-1 and KO-2 were randomly selected for transfection and proliferation assay. (C) Cell proliferation assays for HeLa cells with YTHDF2 siRNA knockdown compared with the siRNA control. Underlying data for this figure can be found in (A) Design of crRNAs for CRISPR-Cas9 for (TIF)Click here for additional data file.S4 FigP values were calculated using the Mann-Whitney test. Underlying data for this figure can be found in (A) Intersection for a confident YTHDF2 targets in HeLa cells between YTHDF2 RIP-seq and PAR-CLIP data. PAR-CLIP results are from Wang and colleagues . The 4,6(TIF)Click here for additional data file.S5 FigWEE1 revealed by RNA-seq in wild-type and knockout cells at different time points post release from G1/S phase. (C) Western blot of WEE1 at different time points post synchronization in wild-type and YTHDF2 knockout HeLa cells. The right panel shows the normalized values of WEE1 quantified by ImageJ. (D) Effect of WEE1 overexpression in HeLa cells. Left panel shows cell proliferation of HeLa cells transfected with Myc-WEE1 compared with the empty vector control. The right panel shows flow cytometry analysis results of each phase during cell cycle. The percentages of each phase were quantified using FlowJo. (E) siRNA knockdown of YTHDF2 and METTL3 in HeLa cells. The left panel shows RT-qPCR results with two-sided Student t test . The right panel shows western blot results of each protein. (F) Flow cytometry results of each phase in the cell cycle upon YTHDF2 or METTL3 knockdown. Underlying data for this figure can be found in (A) Quantification of WEE1 and p-CDK1-Y15 by ImageJ from (TIF)Click here for additional data file.S6 FigYTHDF2 at each time point, which were normalized to the value at 0 hours. (B) RO-3306 induces YTHDF2 degradation in HEK 393T cells within 3 hours. The concentrations of RO-3306 are indicated. (C) Purvalanol A, a different CDK1 inhibitor, induces YTHDF2 degradation. Concentrations and incubation times are as indicated. (D) Detection of YTHDF2 levels after the treatment of relevant inhibitors, including mTOR and ERK inhibitors. (E) Western blot of YTHDF2 and apoptosis markers PARP and Caspase-3 (cleaved form) after CDK1 inhibitor treatment in NB4 and MonoMac-6 cells. Underlying data for this figure can be found in (A) The level of YTHDF2 at different time points post synchronization. The black line indicates protein level changes of YTHDF2 quantified by ImageJ. The red dots indicate transcript levels of (TIF)Click here for additional data file.S7 FigCUL4B, CUL7, SKP1, and CDT2. Underlying data for this figure can be found in (A) Western blot of YTHDF2 after cells incubated with different concentrations of MLN4924 for 24 hours. p27 is a positive control. (B) Detection of the conjugated form of UBC12-NEDD8 after sequential treatment of RO-3306 and MLN4924. Cells were pretreated with RO-3306 for 3 hours before MLN4924 incubation for 3 hours, with concentrations indicated in the figure. (C) Detection of the conjugated form of UBC12-NEDD8 after treating cells with 10 μM RO-3306 for indicated hours. (D–F) Western blot of YTHDF2 after siRNA knockdown of (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


H10, H11 and H12 (H10–H12) subtypes of the avian influenza virus (AIV) are associated with waterfowl. Although these subtypes of AIV are infrequently detected in nature, they can undergo reassortment with other AIV subtypes. Few H10–H12 subtypes of AIV have been isolated from wild birds in China. In this study, 12 AIV isolates of H10–H12 subtypes were identified via routine surveillance of wild birds in Shanghai, China from 2016 to 2019, including two H10, three H11 and seven H12 isolates. Sequence and phylogenetic analyses revealed that the genomic segments of the 12 isolates are highly diverse. These 12 isolates are closely related to those in the Eurasian lineage and share a high degree of sequence identity with those from wild birds and domestic ducks in countries in the East Asian–Australasian Flyway, including Japan, Korea, Bangladesh, Vietnam and China. However, parts of the genomic segments of two H12N2 isolates (NH112319-H12N2 and NH101807-H12N2) belong to the North American lineage, suggesting intercontinental reassortment among H12 AIVs in Eurasia and North American. To better understand the ecological and phylodynamic features of H10–H12 subtypes in wild birds, a large-scale surveillance of AIVs in wild birds is warranted. Avian influenza viruses (AIVs) isolated from birds can be divided into H1–H16 and N1–N9 subtypes based on the antigenic characteristics of the two surface proteins, hemagglutinin (HA) and neuraminidase (NA) , especiaH10–H12 subtypes of AIV are also waterfowl-associated, but are infrequently detected in nature; however, they can reassort with other subtypes of AIV . It has Waterfowl play an important role in AIV transmission because AIVs in the feces of infected birds are excreted into water and can be transmitted by migratory birds to other birds and mammals ,19. ShanWith the permission and supervision of the Shanghai Wildlife Protection and Management Office, a total of 6944 throat and anal swab samples were collected from wild birds in the Nanhui Dongtan wetland  and the Jiuduansha Natural Reservation Zone  in Shanghai, China from 2016 to 2019. All wild birds were released after sample collection. Each swab was placed in 2 mL of viral transport medium (VTM) composed of Hank’s balanced salt solution (pH 7.4), amphotericin B (15 µg/mL), penicillin G (100 units/mL), streptomycin (50 µg/mL) and bovine serum albumin (1%) and stored at −80 °C until used.All experiments were conducted under biosafety level (BSL)-2 conditions. The swab-containing tubes were swirled, and the supernatants were collected after centrifugation. AIV RNAs were extracted using the MagMAX™ Pathogen RNA/DNA Kit  with the Magmax-96 Express instrument (Applied Biosystems). After extraction, the samples were screened for the presence of AIVs by real-time reverse transcription PCR (qRT-PCR) with primers and probes  specific to the matrix gene using a 7500 real-time PCR instrument (Applied Biosystems). The positive samples were transcribed into cDNA using the Uni12 primer (5ʹ-AGC AAA AGC AGG-3ʹ) and PrimeScript™ II 1st Strand cDNA synthesis kit . AIV subtypes were determined using primers specific to HA and NA genes ,22, and https://github.com/ddarriba/jmodeltest2) based on the Akaike information criterion (AIC). Phylogenetic trees were constructed using the maximum-likelihood (ML) method with the software package PhyML version 3.0 (http://www.atgc-montpellier.fr/phyml/) with 100 bootstrap replicates.The sequences obtained were spliced and analyzed using the DNAMAN 6.0 software. The sequences of related AIV strains were downloaded from NCBI and GISAID databases. The models of nucleotide evolution for each gene of these viruses were developed using the software package jModelTest version 2.1.10 (http://www.ncbi.nlm.nih.gov) and the Global Initiative on Sharing Avian Influenza Data (www.gisaid.org). After excluding duplicated data, a total of 3000 H10–H12 isolates were found up to September 10, 2020. The total number of H10–H12 isolates each year was increased since 2000, but the percentage of H10–H12 isolates out of the total number of AIVs detected per year is relatively constant. The number of H10 isolates was greater than that of H11 and H12 isolates  samples tested positive for AIVs by qRT-PCR. Among the positive samples, 12 H10–H12 AIV isolates were identified, including two H10, three H11 and seven H12 isolates. Of these 12 isolates, one H11 isolate was from Gruiformes and designated A/Eurasian Coot/Shanghai/PD112440/2016 . The remaining 11 isolates were from Anseriformes and were designated A/Common Teal/Shanghai/JDS120613/2018 (JDS120613-H10N4), A/Mallard/Shanghai/JDS120662/2018 (JDS120662-H10N4), A/Common Teal/Shanghai/PD112452/2016 (PD112452-H11Nx), A/Eurasian Wigeon/Shanghai/NH101834/2017 (NH101834-H11N2), A/Common Teal/Shanghai/NH101807/2017 (NH101807-H12N2), A/Mallard/Shanghai/JDS110851/2017 (JDS110851-H12N5), A/Common Teal/Shanghai/NH102615/2018 (NH102615-H12N2), A/Common Teal/Shanghai/NH110165/2018 (NH110165-H12N2), A/Common Teal/Shanghai/NH112319/2018 (NH112319-H12N2), A/Mallard/Shanghai/NH011204/2018 (NH011204-H12N5) and A/Common Teal/Shanghai/JDS110203/2019 (JDS110203-H12N8). The full genomes of these 12 isolates were sequenced and the sequences were deposited in the GenBank. The accession numbers are shown in The amino acid sequence motifs of the HA cleavage site were found to be ELTQGR↓GLF for JDS120613-H10N4, ELMQGR↓GLF for JDS120662-H10N4, PAIASR↓GLF for the three H11 isolates and PQAQDR↓GLF, PQAQGR↓GLF and PQVQNR↓GLF for the seven H12 isolates , This obAnas crecca) and Mallard (Anas platyrhynchos), respectively, in the Jiuduansha Natural Reservation Zone in 2018. Except for the polymerase acidic (PA) gene of JDS120613-H10N4, the entire genomic sequences of these two isolates were determined. Results of sequence identity analysis showed that these two isolates share 92.9% to 99.3% nucleotide sequence identity among the seven genomic segments. A BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed that the HA genes of these two H10N4 isolates are closely related to that of A/duck/Mongolia/709/2015(H10N7) and that their NA genes are highly related to the NA genes of isolates A/duck/Mongolia/258/2011(H8N4) and A/garganey/Bangladesh/38,920/2019(H7N4). The matrix (M) gene of JDS120662-H10N4 is highly related to that of the H6 N2 AIV found in Hubei, China and the other internal genes of the two H10N4 isolates share >98.36% identity with H10, H7, H11, H8, H12 and H3 isolates found in Hokkaido, Mongolia, Egypt, Bangladesh, Georgia and Tottori (Two H10N4 isolates (JDS120613-H10N4 and JDS120662-H10N4) were found in Common Teal  and Common Teal (Anas crecca), respectively. The other isolate, NH101834-H11N2, was found in Eurasian Wigeon (Anas penelope) in the Nanhui Dongtan wetland in Pudong in 2017. Except for the NA gene of PD112452-H11Nx, the whole genomic sequences of these three isolates were determined. The two 2016 isolates (PD112440-H11N9 and PD112452-H11Nx) are almost identical with the nucleotide sequence identity of the six genes ranging from 99.1% to 100%. In contrast, the PA genes share only 93.7% nucleotide sequence identity. Except for the NA gene, the 2017 isolate, NH101834-H11N2, share a relatively low nucleotide identity (91.0~97.6%) with the two 2016 isolates in the other seven genomic segments. These three H11 isolates exhibit a very high sequence identity with the duck and wild bird AIVs circulating in Vietnam, Japan, Korea, South Africa and China . The 2017 isolate, NH101834-H11N2, is grouped in another sublineage and is closely related to H11N3 and H11N9 AIVs circulating in domestic and Mandarin Ducks in Bangladesh and South Korea a. The NAAnas crecca). The other two isolates were from Mallard (Anas platyrhynchos) in the Jiuduansha Natural Reservation Zone and the Nanhui Dongtan wetland in Pudong. The entire genome of each of these seven H12 isolates was sequenced and analyzed. Results showed that there is a low degree of identity in the nucleotide sequences of the eight gene segments . This finding indicates that these H12 isolates are highly diverse. A BLAST (https://www.ncbi.nlm.nih.gov/blast/) search showed that the HA, PB2 and PB1 genes of NH112319-H12N2 and the PB1 gene of NH101807-H12N2 share a high degree of sequence identity with those of isolates A/Mallard/Alaska/AH0029066S.1.A/2016 (H12N5) and A/Mallard/California/3070/2012 (H11N2) in the North American sublineage. The other gene segments of these H12 isolates share >97% identity with those of H12 AIVs isolated from wild birds and ducks in the East Asian–Australasian Flyway regions   .Results of phylogenetic analysis showed that the seven HA genes of these H12 isolates belong to two sublineages. The isolate NH112319-H12N2 is in the North American lineage, while the other six isolates are in the Eurasian lineage a. Two H1Results of phylogenetic analysis showed that the NA genes of these H12 isolates are clustered in the Eurasian lineage. Isolates NH101807-H12N2 and NH112319-H12N2 are clustered with the AIVs found in Japan and Korea in a small sublineage. The other two H12N2 isolates and the H5 N2 isolates found in Korea form a sister sublineage b. The N5The PB2 and PB1 genes of isolate NH112319-H12N2 and the PB1 gene of isolate NH101807-H12N2 are clustered in the North American lineage. In contrast, all the other internal genes belong to the Eurasian lineage and form a separate clade with other H12 AIV isolates form migratory waterfowl, domestic aquatic birds and chickens in Japan, Korea, Mongolia, Bangladesh, Vietnam and China . These rBased on the results of phylogenetic analyses of the H10–H12 isolates, the reassortment relationships of these viruses were evaluated by tracing the phylogenetic positions of their genomic segments in the phylogenetic trees. In this analysis, if the lines of the same color linking the same subtypes are parallel each other, the isolates are considered as having the same phylogenetic position, whereas if the line cross each other, the isolates are considered as having different genomic compositions. As shown in The increased awareness that wild birds are the natural hosts of AIVs  has promAmong the twelve H10–H12 isolates  investigated in this study, 11 isolates were from Anseriformes, suggesting that they are major reservoirs of these rare AIV subtypes . The totIt is well known that mutations in HA and NA genes—or reassortment of genomic fragments of influenza virus—can cause the virus to escape host immunity, leading to a large-scale outbreak . Some keResults of phylogenetic and reassortment analysis of the H10–H12 isolates suggest that reassortment is a frequent occurrence among these isolates. For example, the two H10N4 isolates were found in birds at the same location in Jiuduansha Natural Reservation Zone in 2018, but parts of their internal gene segments belong to different sublineages . This fiResults of genetic analyses of the 12 H10–H12 isolates revealed that there is a high degree of diversity of the AIVs circulating in wild birds. This situation may pose a threat to public health due to the frequent reassortment among wild birds and poultry. Because Shanghai is an important ecological site for AIVs in China and around the world, monitoring and studying the rare AIV subtypes in this region is of great importance. In the future, AIV surveillance in the migratory flyways should be strengthened to provide a timely and effective strategy to control AIV infections throughout the world.


Backgroud: Nowadays, biofilms that are generated as a result of antibiotic abuse cause serious threats to global public health. Such films are the primary factor that contributes to the failure of antimicrobial treatment. This is due to the fact that the films prevent antibiotic infiltration, escape from innate immune attacks by phagocytes and consequently generate bacterial resistance. Therefore, exploiting novel antibacterial agents or strategies is extremely urgent.Methods: Herein, we report a rational construction of a novel biofilm microenvironment (BME)-responsive antibacterial platform that is based on tungsten (W)-polyoxometalate clusters (POMs) to achieve efficient bactericidal effects.Results: On one hand, the acidity and reducibility of a BME could lead to the self-assembly of POMs to produce large aggregates, which favor biofilm accumulation and enhance photothermal conversion under near-infrared (NIR) light irradiation. On the other hand, reduced POM aggregates with BME-induced photothermal-enhanced efficiency also exhibit surprisingly high peroxidase-like activity in the catalysis of bacterial endogenous hydrogen peroxide (H2O2) to produce abundant reactive oxygen species (ROS). This enhances biofilm elimination and favors antibacterial effects. Most importantly, reduced POMs exhibit the optimal peroxidase-like activity in an acidic BME.Conclusion: Therefore, in addition to providing a prospective antibacterial agent, intelligent acid/reductive dual-responsive POMs will establish a new representative paradigm for the areas of healthcare with minimal side effects. Bacterial infection diseases remain a major public health concerns to worldwide because of the high morbidity and mortality. Recently, numerous strategies that take advantage of a unique bacterial-infected microenvironment as an important regulated target to facilitate the destruction of biofilm structure and to degrade the EPS have attracted widespread attention 2O2), which is an endogenously active molecule, plays a critical role in physiological and pathophysiological processes such as wound disinfection. However, the concentration of H2O2 that is traditionally used is in the range of 166 mM to 1 M (volume ratio: 0.5-3 %) 2O2, and increase antimicrobial resistance under single antibacterial methods. In this regard, the development of new biocompatible and intelligent peroxidase-like nanostructures with BME stimuli-responsive character and the performance of the combination of multiple antibacterial substances are highly desirable for enhancing antibacterial efficiency and for achieving better antibacterial outcomes with potential therapeutic effect and decreased inevitable side-effects.Among several antibacterial agents, hydrogen peroxide (H10O36 NCs) for the regulation of BMEs to achieve efficient bactericidal effects and wound healing. Owing to the unique self-adaptive electronic structure and chemical versatility of POMs, such as polyanionic feature, rigid framework, monodispersed size, versatile properties, and biofunctionality, this representative W-based POM can self-assemble into larger aggregates in bacteria under the activation by the BME via protonation-triggered hydrogen bonding formation. This promotes effective adhesion to bacteria that have negatively charged cell surfaces within biofilms. Meanwhile, a reductive BME can “regenerate” the strong NIR absorption of POM through the chemical conversion of GSH-promoted W (VI) to W (V), which significantly increases their photothermal conversion performance. Most importantly, the obtained reduced POM aggregates in the BME have self-adaptive enhanced photothermal conversion, they catalyze endogenous hydrogen peroxide (H2O2) to generate abundant hydroxide radicals (•OH) via intrinsic peroxidase-like catalytic activity. This increases the obliteration of biofilm and cause bacteria to be more vulnerable to damage; they also can strengthen the antibacterial effect via the combination of NIR laser-induced hyperthermia with a low endogenous concentration of H2O2. Therefore, we believe that this work will advance the development of these acid/reductive-responsive POMs in the field of enzymatic catalysis and areas of healthcare.Herein, we report a representative acid/reductive-responsive inorganic paradigm as a result of tungsten (W)-based POM clusters (GdW2WO4·2H2O), gadolinium chloride hexahydrate (GdCl6·6H2O), glutathione (GSH), and L-ascorbic acid (AA) were obtained from Alfa Aesar Reagent Company . Tetramethyl benzidine (TMB), o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 3, 3'-diaminobenzidine (DAB), horseradish peroxidase (HRP), propidium iodide (PI) and calcein (AM) were all obtained from Aladdin Company . P-Phthalic acid (TA) was obtained from the Chengdu area of the industrial development zone Xindu Mulan. Crystal violet staining solution was obtained from Scientific Phygene. All of the other chemical agents were used as received without any modification.All of the reagents and solvents were used as received without any modification. Sodium tungstate dehydrate (Na9[Gd(W5O18)2]·×H2O nanoclusters (GdW10O36 NCs) were fabricated via a facile recrystallization method that has previously been described 2WO4·2H2O was dispersed in 20 mL of ultrapure water under continuous stirring at room temperature to form a homogeneous solution. A solution of acetic acid was then added dropwise until the pH was in the range of 7.40-7.50. Subsequently, 1.25 mmol/mL GdCl6·6H2O  aqueous solution was added dropwise to this system under continuous stirring and heated to 85 °C until a transparent solution formed. Finally, crude crystals were isolated from solution when the mixtures were cooled to room temperature. To obtain the reduced GdW10O36 nanoclusters (reduced GdW10O36 NCs), another solution of glutathione with a different concentration or a solution of glycol was injected dropwise into the system, which was irradiated with a hand-held ultraviolet lamp. After irradiation for 12 h, water and ethanol were added three times to precipitate the reduced clusters, which were then dried in a freeze dryer to obtain reduced cluster powders.Na10O36 NCs (33 μg/mL) dispersed in tris buffer solution containing Na2HPO4·12H2O (0.2 M) and citric acid (0.1 M) to convert H2O2 (33 mM) into •OH radicals in the absence or presence of 808 nm NIR laser irradiation for 30 min. UV-Vis spectrophotometer  was used to monitor the absorbance change of the characteristic peak at 652 nm or 450 nm. In addition, TA was used as a probe to react with the generated •OH to form TAOH, which is highly fluorescence. Fluorescence spectroscopy  was then used to record fluorescence spectra of samples after different treatments. Also the color change of peroxidase-like reduced GdW10O36 NCs was also photographed after 30 min of co-incubation at room temperature.TMB (1 mM) and OPD (1.85 mM) were chosen to evaluate the capacity of reduced GdWE.coli (ATCC 25922) and S.aureus (ATCC 29213) bacteria were planted in a Luria-Bertani (LB) agar plate and then moved to 10 mL of LB with shaking for 8 h at 37 °C. When the logarithmic phase (A600 = 0.4) was achieved, Bacteria were collected via centrifugation at a speed of 8000 rpm for 1 min. The bacteria that collected at the bottom were washed and resuspended in phosphate buffered solution , which was attenuated to ~ 106 colony-forming units (CFU∙mL-1) for subsequent experiments. To evaluate the dose-dependent antibacterial ability of reduced GdW10O36 NCs against E.coli and S.aureus bacteria, a series of reduced GdW10O36 NCs at different concentrations was mixed with 500 μL of the as-prepared bacteria solution for 30 min. Finally, bacterial strains with a concentration of 103 CFU∙mL-1 were cultured in solid medium for 24 h using the spread plate method to count the number of the bacterial colonies.10O36 NCs with respect to E.coli and S.aureus bacteria, the following treatments were used on eight separate groups: (I) Control, (II) Reduced GdW10O36, (III) H2O2, (IV) Reduced GdW10O36 + H2O2, (V) Control + NIR, (VI) Reduced GdW10O36 + NIR, (VII) H2O2 + NIR, and (VIII) Reduced GdW10O36 + H2O2 + NIR. The final concentration of bacteria was 1.0 × 106 CFU mL-1, that of reduced GdW10O36 NCs was 200 μg mL-1, and that of H2O2 was 200 μM. After 10 min of co-incubation, groups V, VI, VII, and VIII were irradiated with an 808 nm NIR laser at a power density of 1.0 W cm-2 for 15 min. Then, an infrared thermal imager  was used to monitor the temperature. When co-incubation is conducted for 30 minutes, 100 μL of bacterial suspension in all of the groups was transferred to an agar culture plate for continuous cultivation for 12 h at 37 °C. All of the experiments were repeated in parallel three times.To investigate the antibacterial property of peroxidase-like reduced GdWE.coli and S.aureus bacteria were used in different experimental groups and were incubated with fluorescing propidium iodide (30 μM) at 25 °C (avoiding light) for 15 min. The stained bacterial suspension (5 μL) was dropped onto a microslide that was then covered a coverslip, and an inverted fluorescence microscope (Carl-Zeiss) was used to visualize the sample. For further visual observations of the specific morphology of the bacteria after different treatments, E.coli and S.aureus bacteria were obtained via centrifugation at 8,000 rpm for 1 min and fixed with 4.0 % glutaraldehyde solution overnight at 4 °C. After three repeated rinsings with PBS, a graded series of ethanol solutions  was used to dehydrate bacterial cells for 10 min. Finally, the obtained specimens were set on a silicon glide, sprayed with gold, and imaged under an ultrahigh-resolution field emission SEM .Propidium iodide (PI)  was chosen to evaluate the dead fluorescent staining of bacteria. In the assay, E.coli and S.aureus bacteria were washed three times with PBS (0.01 M) to discard the floating samples, and then 0.9 % saline solution was used to suspend the above-mentioned bacteria to an OD600 value up to 0.1. The diluted bacterial suspension (100 μL) was then co-incubated with 900 μL of LB or TSB medium for E. coli and S. aureus bacteria in a 96 well-plates. The samples were then continuously cultured and for 48 h at 37 °C. After that, fresh medium was substituted every 12 h. To evaluate the combined antibacterial effect of reduced GdW10O36 on bacterial biofilm, separate experiment groups were used: (I) PBS, (II) Reduced GdW10O36, (III) H2O2, (IV) Reduced GdW10O36 + H2O2, (V) PBS + NIR, (VI) Reduced GdW10O36 + NIR, (VII) H2O2 + NIR, and (VIII) Reduced GdW10O36 + H2O2 + NIR. After different disposes, bacterial biofilms were set aside in a shaker at 37 °C for 12 h. Then, 0.9 % physiological saline was used to rinse the biofilm three times to remove the samples and dead bacteria. Next, 500 μL of anhydrous alcohol was immediately used to fix the biofilm in each group, which was further cultured at 4 °C for 15 min. Thereafter, 300 μL of 1 % crystal violet staining solution was added to each well for 30 min of biofilm staining. Then, 0.01 M PBS was used to wash the biofilm in each well again, and the biofilms were then dried at room temperature. Last, each well was blended with anhydrous ethanol (500 μL) and co-incubated for 30 min. For the biofilm in each group, a multi-function microplate reader  was used to monitor the OD590 value, and a digital camera was used to record the color.First, For visually observations of biofilms using CLSM, biofilms were disposed with different methods. They were then cultured with Calcein-AM for 20 min and rinsed with 0.9 % NaCl to replace the discarded bacteriologic medium. An A1R-si Meta Inverted CLSM was then used to collect photographs with a size of 212.1 μm×212.1 μm and a framework size of 805×805. All of the samples were imaged with the same experimental facilities and analyzed using Zen 2011 software (Carl Zeiss). Each sample was independently measured in parallel three times.2O2, (III) reduced GdW10O36 NCs, (IV) reduced GdW10O36 NCs + NIR, (V) H2O2 + reduced GdW10O36 NCs, and (VI) H2O2 + reduced GdW10O36 NCs + NIR with three mice in each group. Then, an E.coli (ATCC 6538) bacterial suspension (1×105 CFU mL-1) was infected four times on each mouse's back to produce subcutaneous abscesses with a wound area of ~78 mm2 after anesthesia. Twelve hours later, 5 μL of reduced GdW10O36 NCs (100 μg/mL) and H2O2 solutions (100 μM) were added dropwise onto the subcutaneous abscess at each infected site in each corresponding group. The mouse in groups IV and VI was then further irradiated under an 808 nm NIR laser at a power density of 1.0 W cm-2 for 15 min; meanwhile, a thermal infrared imager was used to monitor temperature changes. Toward the end of all of the treatments, the wound site of each mouse was photographed every other day, and the wound tissues were collected and fastened with 4% paraformaldehyde solution for histological analysis with Hematoxylin and eosin (H&E) staining, Masson's trichrome staining and Giemsa staining.First, female BALB/c mice  were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. and separated into six groups: (I) no treatment, (II) H10O36 NCs) according to a previously described method  and Keggin-type macroanionic units. The successful fabrication was confirmed by Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy  and ethylene glycol (EtOH), which are reducing agents, were used as patterns for incubating with GdW10O36 NCs at different concentrations. As seen in Figure 10O36 NCs was colorless and had no absorption in the near infrared (NIR) region. However, the color of the solution gradually changed to blue after reduction with GSH and EtOH, This color change was simultaneously accompanied by a corresponding strong characteristic absorption in the NIR region, and this gradually became stronger with an increase in the GSH, EtOH, or GdW10O36 NCs concentration. Consistent with this, the relative residual amount of AA and GSH gradually decreased with GdW10O36 NCs oxidation, as shown in 10O36 NCs. Beyond that, to obtain fundamental insight into the redox-activated behavior of these POM clusters, X-ray photoelectron spectroscopy (XPS) was recorded. The XPS results reveal the oxidation state of W in GdW10O36 NCs before and after the reduction process. As seen in Figure 10O36 NCs were reduced by GSH to form reduced GdW10O36 NCs, and this confirms that the reduced GdW10O36 NCs were successfully synthesized. This phenomenon is the results of multiple reversible steps of electron exchange between delocalized electron density and occupiable cation site of W (V), which are present because of the increased reduction of GdW10O36 NCs 10O36 NCs have admirable acidic/reductive dual-responsive capability that enables the efficient regulation of the acidic BME and promotes oxidation of endogenous GSH, which play a critical role in the bacterial antioxidant defense system.To demonstrate the feasibility of our assumption, we first fabricated W-based polyoxometalate clusters GdW0O36 NCs troscopy . GdW10O310O36 NCs had a navy blue color and strong absorption in the NIR region. Thus, the photothermal effects of samples were carefully investigated. The results indicate that the photothermal effect of reduced GdW10O36 NCs depended on laser power intensity and GSH concentration. With an increase in the NIR laser power density, the temperature of this photothermal material increased  and 3,3,5,5-tetramethylbenzidine (TMB) as peroxidase substrates for evaluating the peroxidase-like activity of reduced GdW10O36 NCs or the commonly used enzyme HRP. The results indicate that reduced GdW10O36 NCs and HRP can both efficiently catalyze •OH radical generation to form a strong fluorescence intensity with TA or a characteristic absorption peak at 652 nm with the TMB substrate  under the irradiation of an 808 nm laser to improve antibacterial activities for synergitic wound therapy. Thus, we used 3,3,5,5-tetramethylbenzidine (TMB), OPD and TA as substracts to record the production of hydroxyl radicals (•OH) that originate from the effective photothermal conversion of the reduced GdW10O36 NCs to promote the low concentration of H2O2 species decomposition. As expected, the fluorescence intensity is significantly enhanced in the presence of reduced GdW10O36 NCs compared the fluorescence intensity of the pure H2O2 species. This indicates that reduced GdW10O36 NCs powerful catalysts for the prodution of •OH from H2O2 because of their optimal peroxidase-like catalytic activity. More interestingly, when reduced GdW10O36 NCs were treated successively with H2O2 for 10 min and irradiation from an 808 nm laser for 30 min, an apparent increase in fluorescence intensity was monitored in this system. In contrast, only a relative weak fluorescence signal was obtained in the absence of either H2O2 or irradiation from an 808 nm laser  and reduced GdW10O36 concentration (200 μg/mL), the single H2O2 group and reduced GdW10O36 NCs group both exhibited negligible antibacterial effects towards E. coli. The survival percentages were 85.4% and 64.1%, respectively, which indicates that peroxidase catalysis alone cannot achieve ideal antibacterial effects. Correspondingly, similar experimental results were also confirmed in S. aureus bacteria , which act as a barrier in preventing antibiotic permeation into the bacterial cells; consequently, bacterial drug-resistance is produced and antibiotic treatment failure occurs. Therefore, anti-biofilm activity is an important characteristic of antibacterial agents for achieving desirable antibacterial effects. Herein, the crystal violet staining method was used to investigate biofilm formation of reduced GdWs Figure B, D. The10O36 NCs with antibacterial activity have for promoting wound healing in vivo, E. coli bacteria-infected wound-disinfected mice were first designed and then divided into six groups:(I) PBS, (II) H2O2, (III) Reduced GdW10, (IV) Reduced GdW10 + NIR, (V) Reduced GdW10 + H2O2, and (VI) Reduced GdW10 + NIR + H2O2. The concentration of reduced GdW10O36 NCs was fixed at 100 μg/mL, and that of H2O2 was fixed at 100 μM; these are much less than the concentrations that are commonly used (0.5-3 % and about 166 mM to 1 M) 10O36 NCs or H2O2 alone exhibited serious inflammatory reactions with ulceration, erythema, and edema on day 4. Also, the relative wound area was only reduced 81.54±3.09%, 84.48±3.69% and 75.94±3.63%, respectively. In contrast, the group that treated with reduced GdW10O36 NCs in the presence of irradiation with an 808 nm laser or with H2O2 alone, the relative wound area only decreased to 71.88±2.81% and 70.79±3.77%, respectively. This indicates that there was a weak inhibition effect of photothermal or peroxidase-like catalytic effect alone that was induced by reduced GdW10O36 NCs. Surprisingly, the reduced GdW10O36 NCs + NIR + H2O2-treated group was effective against wound infection and effectively accelerated wound healing and scab formation after 4 days of treatment. Also, the wound was sharply crusted, and the relative area was reduced to 9.18±2.70% after 10 days of treatment. Accordingly, histological analysis containing hematoxylin and eosin and masson and giemsa staining revealed that the level of inflammation in the abscesses that were treated with reduced GdW10O36 NCs in the presence of irradiation from an 808 nm laser or of H2O2 alone slightly decreased over time, which is contrary to the untreated group. More importantly, the skin of the mice was almost completely recovered on day 10 after treatment with reduced GdW10O36 NCs + NIR + H2O2  are relatively clear, complete, and regenerated. In contract, intact established collagen fibers and dermal layer were observed in the reduced GdW10O36 + H2O2 + NIR group, and this implies that the wounds have completely healed. Additionally, Giemsa staining was used to test the amounts of adherent bacteria after 10 days of treatments. The results indicate that the amount of bacteria in the reduced GdW10O36 + H2O2 + NIR group decreased significantly in comparison to the control and monotherapy groups. This confirms the positive effects of reduced GdW10O36 during wound treatment. In brief, this antibacterial platform efficiently inhibits bacterial proliferation, and also promotes differentiation of fibroblasts and regeneration of skin. Therefore, all of these results demonstrated that reduced GdW10O36 NCs have great potential for accelerating wound healing in the E. coli-infected abscesses because of their superior photothermal efficiency and surprisingly high peroxidase-like activity.To further investigate the potential that reduced GdW2O2 with low concentrations to generate·OH. More importantly, in the presence of bacterial endogenous H2O2, an enhanced antibacterial effect can be achieved via the strengthened photothermal effect and intrinsic peroxidase-mimic activity of reduced POMs. Therefore, the intelligent pH/GSH dual-responsive tungsten (W)-polyoxometalate cluster resolves the problem of severe biofilm infections and also achieves an unprecedented antibacterial efficacy. In brief, the particular acidic/reductive dual-responsiveness of POMs is anticipated to be a powerful tool for illustrating significant guidance on the future design of BME-specific antibacterial nanoplatforms with efficient antibacterial effects.In brief, we successfully developed an antimicrobial platform using the intelligent tungsten (W)-polyoxometalate cluster paradigm. This system features pH/GSH dual-response via acidic BME-triggered self-assembly and reductive BME-driven strengthened NIR absorption. Thus, it results in the rapid elimination of biofilm and exhibits remarkable antibacterial activity upon exposure to NIR laser radiation. Additionally, the intrinsic peroxidase-mimic activity of reduced POMs that are generate in a BME can catalyze the decomposition of endogenous HSupplementary figures.Click here for additional data file.