Melatonin, a multi-regulatory molecule, stimulates root generation and regulates many aspects of plant growth and developmental processes. To gain insight into the effects of melatonin on adventitious root (AR) formation, we use cucumber seedings subjected to one of three treatments: EW (hypocotyl exposed and irrigated with water), SW (hypocotyl shaded and irrigated with water) and SM (hypocotyl shaded and irrigated with 100 µM melatonin). Under shaded conditions, melatonin induced significant AR formation in the hypocotyl. To explore the mechanism of this melatonin-induced AR formation, we used transcriptome analysis to identify 1296 significant differentially expressed genes (DEGs). Comparing SM with SW, a total of 774 genes were upregulated and 522 genes were downregulated. The DEGs were classified among different metabolic pathways, especially those connected with the synthesis of secondary metabolites, with hormone signal transduction and with plant-pathogen interactions. Analyses indicate exogenous melatonin increased contents of endogenous auxin, jasmonic acid, salicylic acid, cytokinin and abscisic acid levels during AR formation. This study indicates melatonin promotes AR formation in cucumber seedings by regulating the expressions of genes related to hormone synthesis, signaling and cell wall formation, as well as by increasing the contents of auxin, cytokinin, jasmonic acid, salicylic acid and abscisic acid. This research elucidates the molecular mechanisms of melatonin’s role in promoting AR formation in the hypocotyl of cucumber seedings under shaded conditions. Cucumber plants have poor ability to take up water and minerals because of their weak root regeneration and shallow roots system, which seriously reduces yield. The hypocotyl of cucumber seedlings has the ability to generate AR , therefoCKX) increases AR formation in transgenic tobacco (Adventitious roots (ARs) can emerge from a hypocotyl, a stem, a leaf or some other plant organ, with emergence regulated by environmental signals and/or by hormones . Many ho tobacco . It is r tobacco  and Arab tobacco . Moreove tobacco . Moreove tobacco .Melatonin is a natural molecule that exists widely in plants , which iPrevious studies have shown that melatonin is an important modulator of gene expression related to hormone production, as well as of the metabolism of auxin, CK, JA, SA and ethylene and other endogenous hormones . For exaTo better understand how melatonin induces AR formation in the hypocotyls of cucumber seedlings under shaded conditions, we first analyze the effects of melatonin on AR formation and hormone content in cucumber hypocotyls. Then, combined with transcriptomics analysis, we explore the mechanism of AR formation induced by exogenous melatonin under shaded hypocotyl conditions. This research aimed to explore the effects of melatonin on AR development in cucumber seedlings, and to provide a basis for revealing the mechanism of melatonin in regulating AR formation in cucumber seedlings.Cucumis sativus L.) cultivar ‘Xinyan 4’ were soaked in 55°C water for 12 h to trigger germination, then placed in a growth chamber under conditions of photoperiod , light intensity (300 µmolm-2 s-1), temperature (28/18°C) and relative humidity (75%). When the second true leaf was fully expanded, seedlings were divided randomly into three groups. Each group was subjected to one of three treatments: EW , SW  and SM  and then cultivated for three days. The hypocotyls were then isolated and analyzed for hormone content and subjected to transcriptome analysis. Each treatment was repeated three times, with 180 seedlings per treatment.Seeds of cucumber  according to the method described previously . BrieflyThe RNA was extracted from the hypocotyl tissue (three replicates per treatment). The RNA-Seq was commissioned to Metware Biotechnology Co. Ltd. . Sequencing libraries were constructed using Illumina’s NEBNext UltraTM RNA Library Prep Kit . Firstly, magnetic beads with oligo (dT) were used to enrich mRNA, and the mRNA were broken into fragment by adding fragmentation buffer. Then added M-MuLV Reverse Transcriptase, random hexamer primer, RNase H and DNA Polymerase I to synthesize double-stranded cDNA. Library was constructed after purification and amplification. Sequencing was carried out on the Illumina HiSeq platform.http://cucurbitgenomics.org/organism/20). The gene expression levels were calculated as fragments per kb of transcript per million reads (FPKM) values. We used the DESeq2 package to conduct differentially expressed genes (DEGs) analyses. Genes with |log2FC|≥1 and padj ≤ 0.05 were considered to be DEGs. KEGG pathway analysis and GO analysis were carried out for all DEGs.Clean data were produced by removing reads of low-quality, containing poly-N and adapter sequences from raw reads. HISAT2 tools was used to map the clean data to the cucumber reference genome sequence (−(ΔΔCt) method. Specific primers of 16 genes randomly selected from the DEGs were listed in The qRT-PCR was carried out on a Step One Plus Real-Time Fluorescent Quantitative PCR system  using a SYBR Premix EX Taq kit . Quantification was evaluated using the 2After three days of treatment, AR primordia on hypocotyls could be seen in the SW and SM treatments, with SM showing more AR primordia than SW Figure 1Three cDNA libraries were obtained by sequencing RNA extracted from the EW, SW and SM hypocotyls. Each library consisted of more than 7.1 Gb of clean data. The Q20 percentage was more than 95%, and the error rate was less than 0.04%, indicating high sequencing quality Table 1.2 = 0.8283 . Hierarchical cluster analysis was carried out to observe the overall expression pattern of DEGs, with red and green bands representing high and low genes expression levels, respectively Figure 3We carried out a KEGG enrichment analysis of the DEGs to identify the main pathways enriched in the melatonin-induced AR development process. Between EW and SW, the top 20 enriched KEGG pathways were mainly related to biosynthesis of secondary metabolites, metabolic pathways, photosynthesis, MAPK signaling pathways and phenylpropanoid biosynthesis Figure 3TDC, YUCCA and TAA), decomposition (DAO), transport (PIN7) and signaling . The contents of IAA and indole-3-acetyl-L-aspartic acid (IAA-Asp) in SW increased significantly, while indole-3-acetyl-L-valine methyl ester  and 3-Indoleacetonitrile (IAN) were significantly decreased in SW relative to in EW. Whereas compared with SW, the contents of IAA-Val-Me and IAN increased in SM 3.4-time and 3.1-times, respectively. Moreover, the contents of indole-3-carboxylic acid (ICA) and of indole-3-carboxaldehyde (ICAld) in SM also increased significantly (TDC) (CsaV3_1G036910), PIN7(CsaV3_3G000190), small auxin-up RNA (SAUR71) (CsaV3_1G009830), SAUR36 (CsaV3_6G001030), SAUR21 (CsaV3_7G000720) and 2-oxoglutarate-dependent dioxygenase (DAO) (CsaV3_7G032850) were significantly upregulated in SW relative to in EW. Also, the expressions of these genes (except SAUR21) in SM were upregulated further relative to those in SW (GH3.17) (CsaV3_4G002130), SAUR32(CsaV3_4G031250), Flavin-containing monooxygenase (YUCCA) (CsaV3_3G017920 and CsaV3_6G012670) and tryptophan aminotransferase (TAA) (CsaV3_1G001250 and CsaV3_1G001250) were significantly downregulated in SW. Among these, GH3.17 was upregulated, while SAUR32 was downregulated in SM relative to those in SW. YUCCA and TAA which did not change significantly in SM (DAO (CsaV3_4G000540 and CsaV3_7G032860) but inhibited the expression of auxin/indole-3-acetic acid (AUX/IAA26) (CsaV3_3G048460) , conjugation (UGT76C1), decomposition (CKX) and signal transduction (AHP) were also regulated by melatonin under shaded conditions. Compared with that in EW, the contents of 2-Methylthio-cis-zeatin riboside (2MeScZR) and cis-Zeatin riboside (cZR) were significantly increased in SW, while that of N6-isopentenyladenine (IP) was significantly decreased. The level of 2-Methylthio-cis-zeatin (2MeScZ) in SM was increased 5.97-times relative to that in SW (CKX) (CsaV3_2G001450 and CsaV3_4G036030) in SW were downregulated relative to those in EW, while in the SM treatment these genes were significantly upregulated. In SW, however, compared with in EW, the expressions of histidine kinase (AHP6) (CsaV3_4G001620) and UDP-glycosyltransferase (UGT76C1) (CsaV3_1G035800) were upregulated, the expressions of these genes in SM were further upregulated relative to those in SW (tRNA-IPT (CsaV3_3G013770) and CKX (CsaV3_5G006200) were significantly inhibited under shaded conditions, while AHP1 (CsaV3_1G039330) was induced by melatonin , conjugation (UGT74F2) and signaling (PR). Compare with EW, the SW treatment significantly upregulated the expressions of chorismate mutase (CM) (CsaV3_5G038370), prephenate aminotransferase (PPA-ATs) (CsaV3_4G000360) and UDP-glycosyltransferase (UGT74F2) (CsaV3_6G015940), while the expressions of these genes in SM were upregulated further relative to in SW (PR (CsaV3_7G007550 and CsaV3_7G007610) and of transcription factor TGA , while it downregulated the expression of phenylalanine ammonia-lyase (PAL) (CsaV3_6G036550) relative to in SW  and SA under shaded conditions, with contents of SA increasing by 10.9-times relative to that in SW (LOX) and signaling (JAZ and MYC2) was significantly affected. Transcriptome analysis showed that compared with in EW, the expressions of eight lipoxygenase (LOX) genes  were upregulated in SW and the expressions of these genes were even further upregulated in SM (JAZ) (CsaV3_1G041270) and did not significantly change those in SM (LOX (CsaV3_2G023200 and CsaV3_4G023890) and one transcription factor MYC2 (CsaV3_6G014370) were significantly upregulated in SM, relative to in SW , N-[(-)-Jasmonoyl]-(L)-valine , jasmonoyl-L-isoleucine (JA-ILE) and cis(+)-12-Oxophytodienoic acid (OPDA). It also significantly promoted accumulations of JA, N-[(-)-Jasmonoyl]-(l)-phenalanine (JA-Phe) and 3-oxo-2-(2-(Z)-Pentenyl) cyclopentane-1-butyric acid (OPC-4) (ERF1) (CsaV3_3G022370 and CsaV3_7G027380) in SW were upregulated. The expressions of these genes were even further upregulated in SM (ACO (CsaV3_3G012240 and CsaV3_3G023650) in SW were significantly downregulated, and these genes did not change significantly in SM (ACO (CsaV3_1G005350 and CsaV3_3G012290) and ACS (CsaV3_6G000890) were significantly upregulated in SM, while S-adenosylmethionine synthase (SAMs) (CsaV3_6G039940) was significantly downregulated (The content of 1-Aminocyclopropanecarboxylic acid (ACC) in SW decreased significantly relative to in EW, but it did not change significantly in SM (ABA2), metabolism  and signal transduction (CDPK) were also regulated by melatonin. We found that the expressions of β-glucosidase (β-Glu)  and ABA 8′-hydroxylase (CsaV3_7G008610) were significantly upregulated by shading, and the expressions of these genes (except of ABA 8′-hydroxylase) were further upregulated by melatonin under shaded conditions, while ABA 8′-hydroxylase was inhibited by melatonin (β-Glu (CsaV3_1G042490) was downregulated by shading, while it was upregulated by melatonin under shaded. Melatonin also significantly induced the expressions of short-chain dehydrogenase (ABA2) (CsaV3_5G002010) and calcium dependent protein kinase (CDPK) (CsaV3_3G038220 and CsaV3_6G038920) under shaded conditions (The content of ABA was significantly decreased by shading but significantly increased by melatonin under shading (expansin (CsaV3_7G001490 and CsaV3_7G001530), nine peroxidase (POD)  and cellulose synthase (CesA) (CsaV3_1G033150) were upregulated. The SM treatment further upregulated the expressions these genes (except CesA) (expansin (CsaV3_7G001510 and CsaV3_7G001520), of ten POD  and cellulose synthase-like (CSL) (CsaV3_7G011490), and downregulated the expressions of xyloglucan glycosyltransferase (CsaV3_5G037390) and CesA (CsaV3_2G009180 and CsaV3_6G007440) significantly  (CsaV3_1G036910) and PIN7 (CsaV3_3G000190), whereas it inhibited the expressions of genes related to auxin synthesis, such as YUCCA (CsaV3_3G017920 and CsaV3_6G012670) and TAA(CsaV3_1G001250)  treatment further activated the expression of TDC (CsaV3_1G036910) and PIN7 (CsaV3_3G000190) (DAO (CsaV3_4G000540 and CsaV3_7G032860), which convert IAA to bound state. It is clear that the increased level of IAA induced by hypocotyl shading is required for the formation of AR  and SAUR36 (CsaV3_6G001030) were upregulated, GH3.17 (CsaV3_4G002130) was downregulated by hypocotyl shading, and the expressions of these genes were upregulated by melatonin under hypocotyl shading conditions (AUX/IAA26 (CsaV3_3G048460) under hypocotyl shading conditions  were downregulated by hypocotyls shading (UGT76C1 (CsaV3_1G035800) and glycosylate free-state IP to the bound state. However, melatonin downregulated the expression of tRNA-IPT (CsaV3_3G013770) and upregulated the expressions of CKX (CsaV3_2G001450 and CsaV3_4G036030) under hypocotyl shading conditions , while SM treatment activated AHP6 expression further  and PPA-ATs (CsaV3_4G000360) (UGT74F2 (CsaV3_6G015940) was also upregulated by melatonin, which glycosylated SA to SAG, resulting in a significant increase in SAG in the SM plants. These results indicate that the significant increase in AR induced by exogenous melatonin may be related to increased levels of SA. This is consistent with the effect of SA in cucumber hypocotyl AR induction (PR1 (CsaV3_7G007550 and CsaV3_7G007610) and of transcription factors TGA   was significantly downregulated by hypocotyl shading, and the expression of MYC2 (CsaV3_6G014370) was significantly upregulated by melatonin under hypocotyl shading conditions  were downregulated by hypocotyl shading (ACO (CsaV3_1G005350 and CsaV3_3G012290) and ACS (CsaV3_6G000890), while it downregulated the expression of SAMs (CsaV3_6G039940) under hypocotyl shading conditions (ERFs) are common mediators of root developmental. It has been found that ERFs are upregulated during AR development in cuttings (ERF1 (CsaV3_3G022370 and CsaV3_7G027380) under hypocotyl shading conditions  were significantly upregulated by hypocotyl shading and melatonin further upregulated these genes under hypocotyl shading conditions, while ABA 8′-hydroxylase was inhibited by melatonin (CDPK (CsaV3_3G038220 and CsaV3_6G038920) under hypocotyl shading conditions  are the main load-bearing components of cell walls. AtCSLD3 is very important for the synthesis of primary cell wall in the root hair region and plays vital roles in root hair growth. In affected . Peroxidaffected . From ounditions . The expell wall . In tranncreased . In our Hypocotyl shading was necessary to induce AR formation, while exogenous melatonin application significantly promoted AR formation in cucumber seedlings under hypocotyl shading conditions. To gain a deeper understanding of the underlying regulatory mechanisms, RNA-seq and hormone analyses were carried out. Our results show that exogenous melatonin induced the expressions of auxin, SA, JA, ethylene and ABA synthesis and signaling related genes. It also inhibited the expressions of CK signaling genes, as well as significantly increasing the auxin, CK, JA, SA and ABA contents Figure 7https://www.ncbi.nlm.nih.gov/bioproject/PRJNA717318. The original contributions presented in the study are publicly available. This data can be found here: YW, ZZ and LZ carried out experimental work. LL, XH, YZ and LH performed the data analyses. YW wrote the manuscript. HZ and ML designed the experiments and assisted with writing the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by Shanxi Province Key R&D Plan (202102140601013), the National Key R&D Program of China (2019YFD1001900), Shanxi Province Applied basic Research Program (201801D221304), Science and Technology Innovation Fund of Shanxi Agricultural University .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.


We report the coding-complete sequences of rhinovirus types C48, A46, A39, and C56, determined from nasopharyngeal swabs from three individuals with influenza-like symptoms in the United States. One sample showed a coinfection of rhinovirus types A46 and C48. Enterovirus (family Picornaviridae). This genus includes 15 species, among them Rhinovirus A, Rhinovirus B, and Rhinovirus C. Enterovirus genomes encode a large polyprotein, which is autocatalytically cleaved into three smaller proteins (P1 to P3) that are further cleaved into 11 proteins: 4 structural proteins (VP1 to VP4) and 7 nonstructural proteins (2A to 2C and 3A to 3D). In humans, rhinoviruses cause the common cold and trigger approximately 50% of asthma flare-ups and exacerbations of chronic obstructive pulmonary disease . To determine the viral etiology of the clinical presentation, RNA was extracted from 200 μl of the resuspended sample using the high pure viral RNA kit . The RNA was used to prepare libraries using the TruSeq stranded total RNA LT kit with the Ribo-Zero human/mouse/rat kit . The 2 × 150-bp libraries were sequenced on a NovaSeq 6000 instrument at Psomagen Inc. (USA). All bioinformatic tools were run with default parameters unless otherwise specified. The demultiplexed reads were trimmed using Trimmomatic v0.39  to 7,102 nt; coverage depth, 54× to 540×; GC content, 39% to 42.8%) from the three samples had four near-complete genomes (based on the complete coding region for viruses in the genus Enterovirus) of rhinoviruses . Isolates AZ6_4 and AZ6_15 were identified in the same sample (S6) as a coinfection. Based on blastn analysis, we identified the four rhinovirus sequences as part of the species Rhinovirus A and Rhinovirus C. Datasets of representative coding-complete or partial sequences of rhinovirus A and C genotypes available at GenBank were assembled into two datasets, together with those from this study and two enterovirus D sequences (GenBank accession numbers D00820 and AY426531) as an outgroup. These two datasets were aligned using MAFFT v7  of 840 nucleotides (VP1 cleaved protein coding region) that shares 92.5% identity with the sequence we report here (AZ6_4).In our comparative analysis of the four genotypes, the 11 cleaved protein coding regions share >89% nucleotide identity , determiMW679007, MW679008, MW679009, and MW679010. The raw reads have been deposited under BioProject accession number PRJNA701833 and SRA accession numbers SRR13720058, SRR13720059, and SRR13720060.The raw reads and rhinovirus contigs generated in this study have been deposited in the NCBI databases under GenBank accession numbers


Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs  using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications.IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate. Since February 2013, a novel H7N9 avian influenza virus (AIV) has caused 1,568 confirmed human infections and 616 deaths, with an ~40% case fatality rate  virus was detected, while the high-pathogenicity avian influenza (HPAI) H7N9 virus emerged in late 2016, causing up to 100% mortality in infected chickens . Given tNeuraminidase (NA) inhibitors, such as zanamivir (Relenza) and oseltamivir (Tamiflu), are the major antivirals that have been recommended for the treatment of severe infection with AIV in humans. However, the rapid emergence of NA inhibitor-resistant H7N9 viruses highlights the need for new anti-influenza drugs or therapeutics , includiWe previously identified four H7N9 human IgG antibodies, namely, L4A-14, L3A-44, K9B-122, and L4B-18, from humans naturally infected with H7N9 AIV  with HA and NA from H7N9 AIV and the six internal genes from PR8 (A/Puerto Rico/8/34 [H1N1]) virus. All four mAbs showed HI activity against H7N9 AIV, with stronger HI activity being observed with L4A-14, followed by L3A-44 and K9B-122 and L4B-18 showing relatively lower HI titers . The MN To select the mAb escape mutants, an H7N9 Anhui/13 AIV stock was 10-fold serially diluted and then incubated with 1 mg/mL of each mAb, and the antibody-virus mixture was then inoculated into 10-day-old embryonated eggs. The HA-positive allantoic fluid from the embryonated eggs inoculated with the lowest concentration of virus was selected for viral HA gene sequence analysis.The results showed that the propagation of virus in the presence of the monoclonal antibodies gave rise to the following substitutions: N to D at residue 149 (N149D) for L4A-14, G to E at residue 133 (G133E) for K9B-122, A to T at residue 125 (A125T) for L3A-44, and L to Q at residue 217 (L217Q) for L4B-18 . These substitutions are positioned within the respective footprints of the antibodies that selected them .To confirm the functional role of these substitutions in the escape mutants, each of the identified mutations was engineered into the wild-type HA of Anhui/13 reverse genetic (RG) virus that contained the six internal genes of the PR8 virus. The HI assay showed that the mutant virus with HA substitutions G133E and A125T completely lost HI activity with mAb K9B-122 and L3A-44, respectively . The L21To investigate the cross-reactivity of the H7N9 HA mutants with the individual mAbs, all of the selected escape mutants were tested by an HI assay with the panel of mAbs. Additionally, we included mutant A151T because it introduced an N-linked glycosylation site at amino acid residue 149, a regular strategy for influenza viruses to escape neutralizing antibodies by attaching glycans, which can sterically block the binding of antibodies to multiple antigenic sites , 15, 16.To investigate the cross-reactivity of the mAbs against recent H7N9 AIV isolates, we performed HI assays against both LPAI (A/Hong Kong/125/2017 [HK125]) and HPAI (A/Guangdong/17SF003/2016 [GDSF003]) viruses from epidemic wave 5. Additionally, we included reassortant Anhui/13 with A125T, A151T, and L217Q substitutions in HA, as these three mutations have been found in recent H7N9 isolates from poultry in China as well as H7N9 virus that is serially passaged in the presence of homologous ferret serum . mAb L4AIt is inevitable that escape mutants will emerge following the therapeutic use of mAbs. Therefore, it is critical to assess the risk of these mutants to public health before their application as therapeutics for human H7N9 AIV infection. Ideally, the escape mutant should not be able to evade infection- or vaccine-induced immunity. To this end, we checked the HI and MN titers of the mAb escape mutants with both chicken and ferret postinfection sera. Additionally, we included chicken antiserum raised against inactivated H7N9 virus to mimic chicken postvaccination serum. In agreement with the results of a previous study, L217Q resulted in a significant drop in the HI titer against both ferret postinfection serum (16-fold) and chicken postvaccination serum (8-fold)  (17). HoTo conclude, the A125T, G133E, and L217Q substitutions in HA resulted in significant decreases in cross-reactivity to both chicken and ferret postinfection sera; however, only the L217Q mutant showed reduced cross-reactivity against chicken postvaccination serum compared to wild-type Anhui/13. All of the postinfection and postvaccination sera tested here demonstrated comparable neutralizing profiles among wild-type Anhui/13, mAb L4A-14 escape mutant N149D, and mutant A151T, having completely lost HI activity against mAb L4A-14.To evaluate the effects of mAb escape mutations in the HA on virus replication fitness, we generated a series of reverse-genetics-based mutant viruses carrying the escape mutations A125T, G133E, N149D, and L217Q by site-directed mutagenesis. We did not include the A151T mutant for further analysis because it did not emerge under mAb pressure. The mutant with A151T substitution in HA also showed a significant drop in viral thermal stability as well as reduced receptor binding to human-like receptors. Therefore, it is unlikely to pose an increase zoonotic risk  cells and MDCK-SIAT1 (SIAT) cells, which have been modified to express a higher density of α2,6-sialyltransferase . SimilarReceptor binding affinity is one of the key determinants of the interspecies transmission of influenza viruses. To examine the impacts of mAb escape mutations on virus receptor binding properties, we utilized biolayer interferometry to characterize the receptor binding of these viruses to the avian-like and human-like receptor analogues 3′-sialylacetyllactosamine (3SLN) and 6′-sialylacetyllactosamine (6SLN). In agreement with previous reports, H7N9 Anhui/13 showed comparable binding to both the 3SLN and 6SLN receptor analogues, and the L217Q mutant showed a considerable increase in binding to avian 3SLN (~432-fold), with only a slight decrease (~2-fold) in binding to 6SLN analogues  15, 19), 1915, 1The pH of fusion has been shown to play an important role in host adaptation and transmission . ConsistIn addition to receptor binding and the pH of fusion, HA thermal stability also plays a vital role in AIV evolution , 14. WilIn our previous study, we isolated four potent monoclonal antibodies from humans naturally infected with H7N9 AIV. To gain in-depth knowledge of the therapeutic potential of these antibodies, we mapped the antigenic epitopes of these antibodies by escape mutant selection methods. In addition to the neutralizing capabilities, we also evaluated the potential risk to humans of mAb-induced escape mutants by assessing their replication fitness, receptor binding, pH of fusion, thermal stability, and potential to evade host adaptive immunity. Overall, the L4A-14 mAb showed robust neutralizing capabilities and broad cross-reactivity to H7N9 AIVs. Moreover, its escape mutant N149D demonstrated reduced thermal stability while retaining strong antigenic cross-reactivity with both postinfection and postvaccination sera, making the L4A-14 mAb a suitable therapeutic candidate for further investigation.in vitro to predict the evolution of influenza viruses in vivo, as seen for human mAbs to seasonal H1N1  in H2, H3, and H4 avian influenza viruses –26, whic–We previously showed that the amino acid at position 217 is a key mediator of H7N9 AIV antigenicity. Both HI and MN assay results support this finding, demonstrating that the L217Q substitution in HA resulted in a significant reduction in antigenic cross-reactivity with both ferret and chicken postinfection sera as well as chicken postvaccination serum. In contrast, Wang et al. concluded that L217Q had a minimal impact on the antigenicity of H7N9 AIV ; howeverin ovo. Additionally, we assessed the potential zoonotic risk and the viral fitness consequences of the identified mutations. Our data revealed that the L4A-14 mAb could be a potential therapeutic candidate for human H7N9 infections, with escape mutants posing a lower risk to human health and, therefore, lower pandemic potential.To conclude, we evaluated the potential of utilizing human-derived mAbs raised against H7N9 AIV as an infection therapy. However, immune pressure, including mAb therapy, will inevitably induce escape mutations. As such, we identified and mapped the H7N9 HA by immune escape The embryonated chicken egg work was carried out in accordance with the guidance and regulations of the United Kingdom Home Office under project license number P68D44CF4. All of the influenza virus-related work was carried out under biosafety level 2 conditions.The reassortant H7N9 viruses containing HA and neuraminidase (NA) from low-pathogenicity avian influenza (LPAI) H7N9 (A/Anhui/1/2013) virus (referred to as wild-type Anhui/13) and the internal gene segments from A/Puerto Rico/8/34 (H1N1) (PR8) were generated as previously described . The rea2 atmosphere.Madin-Darby canine kidney (MDCK) cells, MDCK-SIAT1 (SIAT) cells, human embryonic kidney HEK293T cells, and Vero cells (ATCC) were maintained with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Gibco) and 100 U/mL penicillin-streptomycin (Gibco) at 37°C under a 5% COThe monoclonal antibodies were produced in ExpiCHO-S cells and purified by protein affinity chromatography. Basically, the ExpiCHO-S cells were cotransfected with antibody light and heavy chain plasmids using an ExpiFectamine CHO transfection kit according to the manufacturer’s protocol. The supernatants were filtered and purified using HiTrap protein G HP antibody purification columns .Postinfection ferret antiserum raised against LPAI H7N9 (A/Anhui/1/2013) virus was kindly provided by John McCauley . The postinfection chicken antiserum was raised previously in our laboratory by infection of specific-pathogen-free (SPF)-derived Rhode Island Red chickens  with reassortant LPAI H7N9 (A/Anhui/1/2013) virus . The serMDCK or MDCK-SIAT1 cells were infected with H7N9 AIV at a multiplicity of infection (MOI) of 0.001 and incubated at 37°C for 1 h. The cells were then washed once with phosphate-buffered saline (PBS)  and replenished with DMEM containing 2 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin. The supernatants were taken at 15, 24, 48, and 72 h postinfection and stored at −80°C before titration by a plaque assay in MDCK cells. The plaque assay was carried out as previously described . MDCK ceTen-day-old embryonated chicken eggs were infected with 100 PFU of the indicated reassortant H7N9 AIV. The eggs were incubated at 35°C for 48 h before allantoic fluid was harvested for hemagglutination (HA) assays and plaque assays.The HA titration assays were performed using 1% chicken red blood cells (RBCs) as described in the WHO animal influenza training manual . BrieflyTwenty-five microliters of human mAbs was 2-fold serially diluted in 96-well V-bottom plates (Greiner) with PBS prior to being mixed with an equal volume of H7N9 AIVs (8 HA units). The concentrations of mAbs L4A-14, K9B-122, L3A-44, and L4B-18 were 1.0 mg/mL, 1.5 mg/mL, 0.8 mg/mL, and 1.0 mg/mL, respectively. The antibody-virus mixtures were incubated for 1 h at room temperature and then mixed with 50 μL of 1% chicken RBCs. The plates were incubated for a further 30 min at room temperature before the hemagglutination inhibition (HI) titer was recorded as the reciprocal of the dilution that completely inhibited hemagglutination. For the data in The reassortant H7N9 AIVs were diluted with embryonated chicken egg allantoic fluid to 32 HA units/50 μL. The viruses were then left at 4°C as a control or heated at 50°C, 50.7°C, 51.9°C, 53.8°C, 56.1°C, 58.0°C, 59.2°C, and 60°C using a PCR thermal cycler (Bio-Rad) for 30 min before HA assays.The pH of fusion for H7N9 AIV was determined by syncytium formation assays as previously described . BrieflyMonoclonal antibodies  or polyclonal antisera were 2-fold serially diluted and then mixed with an equal volume of 100 PFU of reassortant H7N9 AIV in serum-free medium. MDCK cells were washed once with PBS before infection with the virus-serum or virus-antibody mixture for 1 h at 37°C, and the cells were then washed once with PBS and replenished with DMEM containing 2 μg/mL TPCK-treated trypsin. The cells were further kept at 37°C for 72 h before crystal violet staining. Virus microneutralization (MN) titers were expressed as the reciprocal of the highest dilution of antisera/antibodies that blocked virus infectivity in MDCK cells. The MN titers are normalized to 1 mg/mL in Three microliters of the sucrose-gradient-purified viruses  was mixThe influenza virus receptor binding affinity was measured on an Octet Red instrument (FortéBio). The equilibrium responses for virus binding were plotted as a function of the amount of sugar immobilized on the biosensor calculated from the response during the sugar loading step . BrieflyP values of <0.05 were considered significant.Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). One-way analysis of variance (ANOVA) was used to test differences between different groups for


PP2C genes were identified in peanut, which were unevenly distributed across the 20 chromosomes, with segmental duplication in 78 gene pairs. AhPP2Cs could be divided into 10 clades (A-J) by phylogenetic analysis. AhPP2Cs had experienced segmental duplications and strong purifying selection pressure. 22 miRNAs from 14 different families were identified, targeting 57 AhPP2C genes. Gene structures and motifs analysis exhibited PP2Cs in subclades AI and AII had high structural and functional similarities. Phosphorylation sites of AhPP2C45/59/134/150/35/121 were predicted in motifs 2 and 4, which located within the catalytic site at the C-terminus. We discovered multiple MYB binding factors and ABA response elements in the promoter regions of the six genes (AhPP2C45/59/134/150/35/121) by cis-elements analysis. GO and KEGG enrichment analysis confirmed AhPP2C-A genes in protein binding, signal transduction, protein modification process response to abiotic stimulus through environmental information processing. Based on RNA-Seq data of 22 peanut tissues, clade A AhPP2Cs showed a varying degree of tissue specificity, of which, AhPP2C35 and AhPP2C121 specifically expressed in seeds, while AhPP2C45/59/134/150 expressed in leaves and roots. qRT-PCR indicated that AhPP2C45 and AhPP2C134 displayed significantly up-regulated expression in response to salt stress. These results indicated that AhPP2C45 and AhPP2C134 could be candidate PP2Cs conferring salt tolerance. These results provide further insights into the peanut PP2C gene family and indicate PP2Cs potentially involved in the response to salt stress, which can now be further investigated in peanut breeding efforts to obtain cultivars with improved salt tolerance.Plant protein phosphatase 2C (PP2C) play important roles in response to salt stress by influencing metabolic processes, hormone levels, growth factors, etc. Members of the PP2C family have been identified in many plant species. However, they are rarely reported in peanut. In this study, 178  PKs prie family .PP2Cs are highly conserved in evolution and regulate stress signaling pathways in a targeted manner . Most PPArabidopsis (76)  are divided into clades A-J  , cotton sis (76)  and ricesis (76) . In Arabades A-J . In cladades A-J . ABI1 anades A-J . Four meades A-J . POLTERGades A-J . The funt stress .PP2C gene family members have been reported to participate in plant salt stress regulation directly. (AtPP2CG1 (At2G33700), a PP2C gene from Arabidopsis, is fully expressed under salt stress and positively regulates salt tolerance in an ABA-dependent manner  and OsPP2C51 (Os05g49730) are significantly expressed under 150mM NaCl stress (OsPP2C08 (Os01g46760) is reported to be distinctly expressed in response to saline stress at the seedling stage compared to other PP2Cs (Many irectly. . AtPP2CGt manner . At3G633ore size . Among Ol stress . OsPP2C0er PP2Cs . In riceer PP2Cs .Arachis hypogaea L.) is an important oilseed and food crop worldwide. Cultivated peanut evolved from a cross between Arachis duranensis (A) and Arachis ipaensis (B) which subsequently underwent chromosome doubling to form the modern heterotetraploid  with a total genome size of approximately 2.7 Gb (Arabidopsis homeodomain-leucine zipper transcription factor (AtHDG11) to enhance salt tolerance, the identification of crucial intrinsic proteins that regulate salt stress in peanuts, the overexpression of the sodium/proton antiporter gene (AtNHX1) to improve salt tolerance, the vacuolar H+-pyrophosphatase gene (AVP1) . Moreover, their expression patterns under salt stress were analyzed from publicly available RNA-Seq data and qRT-PCR. AhPP2C45 and AhPP2C134 displayed significantly up-regulated expression under salt stress. They were considered as strong candidates for PP2Cs mediating salt stress signaling in peanut. These results provide theoretical support for biological functions of the peanut PP2C gene family under salt stress. Also, identification of AhPP2C45/134 furnish new research threads for mechanism study of salt response differences between peanut germplasms, which play important roles in selection of salt tolerance germplasms and breeding.Peanut (y 2.7 Gb . The maiy 2.7 Gb . There he (AVP1) . Compare22.1A. hypogaea cv. Tifrunner and their annotated gene models were downloaded from PeanutBase1 (-20) were used to construct a peanut-specific hidden Markov model using hmmbuild from HMMER v3. The resultant peanut-specific HMM was used to search the protein sequences once again, and proteins that conformed to the criteria were submitted to NCBI-CDSProtein and gene sequences of nutBase1 . Protein2.2Phylogenetic trees were constructed with MEGA11 software  using th2.3AhPP2Cs were mapped with Map Gene 2 Chrom web v2AhPP2C gene duplications were identified based on a sequence similarity of their CDS ≥75% and a sequence overlap of ≥75% of the longest sequence of the pair. Gene duplications were depicted using a circos diagram , isoelectric point, were calculated using Expasy ProtPrarm tools2.5AhPP2Cs were predicted and visualized using Gene Structure Display Server 2.0AhPP2Cs in subclades AI and AII, cis-acting elements were searched for in the 1500 bp region upstream of their start codons using PlantCAREThe gene structures of clade A 2.6AhPP2Cs was used to predict miRNA target sites  and reverse transcribed into cDNA using Advantage RT-for-PCR Kit  following the manufacturer’s instructions. The specific primers were designed using Beacon Designer 7.9. SYBR Green real-time PCR was performed according to the guidelines of the PCR machine. The relative expression levels were calculated by the 2t method . Statist2.9AhPP2Cs was determined from RNA-Seq data obtained for 22 different tissues at different developmental stages during peanut growth . After screening for the presence of the typical PP2C domain (PF00481), 178 genes were retained. A phylogenetic tree was created after multiple sequence alignment of the 178 AhPP2Cs 76 Arabidopsis PP2C genes. The 178 AhPP2Cs were divided into the 10 clades, A-J, as reported in other plants before, while 6 AhPP2Cs did not belong to any reported PP2Cs clades  was zero, indicating strong purifying selection.As shown in  species . A gene 3.3AhPP2C genes were regulated by miRNAs during translation, we identified 22 miRNAs targeting 57 genes , plant hormone signal transduction (04075), environmental information processing (A09130), MAPK signaling pathway – plant (04016) , while AhPP2C121 and 59 did not. AhPP2C45 contained the maximum number of cis-elements (21), while AhPP2C35 contained the minimum number of cis-elements (4). The differences in number and kind of elements may lead to functional difference of AhPP2Cs.In higher plants, PP2C is involved in many stress resistance responses and is induced by a variety of abiotic stresses.  and AII Table 2.3.9AhPP2Cs were extracted and shown in AhPP2Cs diverse with differing levels of tissue specificity. AhPP2C17, 59, 150, 134, 45, 15 and 101 expressed at relatively high levels in several of the 22 different tissues. In contrast, the overall expression levels of AhPP2C54, 35 and 121 were lower, and mainly detected in seeds tissues. The remaining genes (7) were moderately expressed in all tissues. Considering AhPP2Cs of subclades AI and AII, AhPP2C121 and 35 were only expressed in seeds, while the other four genes were also expressed in leaves and roots. The above results indicated that clade A AhPP2Cs exhibited different expression patterns.Based on the transcriptome data of 22 peanut tissues , the exp3.10AhPP2Cs in seedlings under salt stress used publicly available RNA-Seq data from leaves and roots. The heatmap of relative expression is shown in AhPP2C45, 134, 59 and 150 were up-regulated compared with the control, but this was only significant in AhPP2C45 and 134. In roots, AhPP2C45 and AhPP2C134 were also up-regulated, while AhPP2C59 and AhPP2C150 were down-regulated relative to the control. The expression of AhPP2C121 was not detected in roots or leaves with or without salt stress.An initial assay of the expression of subclade AI and AII AhPP2Cs  in leaves and roots were analyzed by qRT-PCR  was identified as a potential candidate gene conferring improved salt tolerance through the integration of genome-wide polymorphism information with QTL mapping and expression profiling data (OsPP2C51 (Os05g49730) in Arabidopsis revealed that it could positively regulate ABA-induced rice seed germination under conditions of salt stress . ABI2 (AT5G57050.1) as a SOS2-interacting protein can affect salt stress response in Arabidopsis  was up-regulated  that have more weight in their phylogenetic placing. In addition, the peanut AhPP2Cs that closely clustered with PP2Cs conferring salt tolerance also shared similar motifs and gene structures with their Arabidopsis and rice counterparts  and Os05g51510 (OsPP2C53) and two bidopsis . Mutatioulation. . OsPP2C5 control . Microaregulated . Salt toterparts Figure 6cis-elements, two of them, ABRE/ABRE4/ABRE3a and MYB binding factor are related to salt tolerance. ABRE/ABRE4/ABRE3a has been discovered in previous studies of PP2Cs in stress  contained both cis-elements and having the maximum number of both cis-elements. The greater number of salt tolerance-related cis-elements AhPP2Cs have, the greater expression levels under salt stress. The number of salt tolerance-related cis-elements may therefore be correlated with either high or low gene expression in response to salt stress.Additionally, we found nine n stress , howeverThe potential phosphorylation sites and protein structures of the AhPP2Cs were also examined. The conserved catalytic core domain of PP2Cs contains a central β-sandwich with a pair of α-helices on either side of each β-sheet . Within cis-acting elements analysis of AhPP2C-A, AhPP2C45 and AhPP2C134 had similar structures to the reported PP2C genes associated with salt tolerance in rice and Arabidopsis and contained more cis-elements that related to salt tolerance. According to the salt stress effects on the expression, AhPP2C45 and AhPP2C134 had significant differences compared with the control. These results indicated that AhPP2C45 and AhPP2C134 could be candidate PP2Cs conferring salt tolerance. The study of AhPP2Cs in response to salt stress may be used in conjunction with phenotypic data to screen for more salt tolerant genotypes and salt tolerant varieties, providing a crucial foundation for the next step breeding.Based on phylogenetic, gene structure, motif and 5AhPP2C genes which distributed across the 20 chromosomes, with segmental duplication in 78 gene pairs. Phylogenetic tree, gene and protein structures, cis-elements and expression analysis indicated that AhPP2C45 and AhPP2C134 could be candidate PP2Cs conferring salt tolerance. These results provide important information for the research of salt-stress mechanism, selection of salt-resistant germplasm and breeding of salt-tolerance cultivar in peanut.In this study, we identified 178 The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The public repository of NCBI under the biological project accession number, PRJNA560660.FL conceived the study, designed the experiments, supervised and complemented the writing. YW provided suggestions and supervision. ZW and LL produced the figures and wrote the original article. All authors discussed the results and commented on the article. All authors contributed to the article and approved the submitted version.


Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide. E2Fs are a group of transcription factors involved in the carcinogenesis and progression of various cancers. However, the exact roles of each member of E2F family in the development and progression of HNSCC are still unknown.RNASeq and clinical follow-up information were extracted from The Cancer Genome Atlas (TCGA). The expressions of E2Fs and their roles in HNSCC progression were explored using the R software and the cBioPortal database.Our results showed that the mRNA levels of E2Fs were significantly higher in HNSCC tumors than in normal tissues. E2F1, E2F3, E2F4, E2F6, and E2F7 were identified as reliable diagnostic markers. E2Fs (except for E2F3) expressions were closely related to the clinical features (excluding metastasis) of HNSCC. High E2F6 mRNA expression was an independent risk factor for the OS of female HNSCC patients. In addition, high E2F4 expression could lead to poor prognosis in HNSCC in both males and females, high expressions of E2F5, E2F6, and E2F7 were associated with poor OS of female HNSCC patients, while high E2F2 and E2F8 expressions were positively correlated with the OS of male HNSCC patients. Interestingly, E2Fs expressions had stronger associations with immune cell infiltrations in male HNSCC patients than in female HNSCC patients.The expressions of E2Fs were found to be correlated with the progression of HNSCC. E2F1, E2F3, E2F4, E2F6, and E2F7 could be good diagnostic molecules for HNSCC. In addition, E2F6 was an independent risk factor for the prognosis of female HNSCC patients. It is rms, etc. . Dependims, etc. , 8. Findms, etc.  because ms, etc. . Althougms, etc. . Recent ms, etc. . HoweverE2Fs are a group of transcription factors encoded by a family of genes with highly homologous and conserved DNA binding and retinoblastoma (RB) protein-binding domains –15. ThisThe developments of microarray and sequencing technologies for analyzing multiple databases have revolutionized biological and biomedical studies and made RNA and DNA research more efficient . To the https://tcga.xenahubs.net) as previously described [The Cancer Genome Atlas (TCGA) is publicly available and open-ended, based on which we retrieved RNASeqV2 data from the TCGA-HNSC (head and neck squamous cell carcinoma) project . The oral squamous cell carcinoma cell lines SCC15 and TSCCA were purchased from Shanghai Zhongqiao Xinzhou Technology Co., Ltd. . HOK, SCC15, and TSCCA cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin at 37 °C with 5% CO2. Total RNA was isolated using an RNA Isolation Total RNA Extraction Reagent  according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using an RT Master Mix for qPCR II (gDNA digester plus) kit . RT-PCR was conducted on the CFX Connect Real-Time System  using an SYBR Green qPCR Master Mix (No ROX) . The experiments were conducted in more than three independent replicates. The primers for PCR analysis are listed in Table https://www.cbioportal.org/) [cBioPortal was used as previously described  . The HNSAs described above, the top 200 expression-correlated genes of E2F6 in HNSCC were obtained by utilizing the “co-expression” module of the cBioportal database, with the same parameters described above. Moreover, the correlations between E2F6 expressions with the top 5 expression-correlated genes were further explored via Spearman correlation analysis. Subsequently, the top 200 expression-correlated genes were input to operate gene enrichment analysis. Specifically, the Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis and Gene Ontology (GO) enrichment analysis of correlated genes of E2F6 was performed using the R statistical software.P-value was 0.05 . ROC, COX regression, Kaplan–Meier, and immune infiltration analyses were performed using the “pROC”, “survival”, “survminer”, and “GSVA” R packages. Enrichment analyses were performed using the “org.Hs.eg.db” and “clusterProfiler” R packages. The visualizations were conducted by the “ggplot2” R package.The R statistical software  was used for statistical analyses. The significance threshold of the The clinical characteristics and gene expression data of 502 HNSCC primary tumors and 44 adjacent normal samples were downloaded from the TCGA database. The patients’ clinicopathological details, including age, gender, TNM stages, clinical stage, histologic grade, smoking, and HPV status are summarized in Table P < 0.05) . Moreover, in the paired comparison, the expressions of E2F1, E2F3, E2F4, E2F6, and E2F7 in HNSCC patients were significantly higher than those of adjacent normal samples (P < 0.001) . Figure P > 0.05)  Fig. A-H and tP > 0.05)  Fig. C. These 05) Fig. H.P < 0.05)     of female HNSCC patients . Briefly, the correlation between immune cell infiltrations and E2Fs seemed to be stronger in males than in females. Most E2Fs expressions could be negatively associated with the infiltration level of neutrophils in both female (excluding E2F2 and E2F8) and male HNSCC (excluding E2F4) patients (P < 0.01). In males, E2F2 and E2F8 expression might be positively associated with infiltration levels of activated B cells, activated CD8 + T cells, effector memory CD8 + T cells, immature B cells, natural killer (NK) T cells, etc. However, a negative trend was found in most correlations between E2F4 expression and infiltration levels of all immune cells , FA Complementation Group L (FANCL), NADH:Ubiquinone Oxidoreductase Complex Assembly Factor 7 (NDUFAF7), WNK Lysine Deficient Protein Kinase 2 (WNK2), and Perilipin 3 (PLIN3)  Fig. C-G. The To further investigate the relevant molecular mechanism of E2F6 associated with the survival of HNSCC patients, the top 200 co-expressed genes associated with E2F6 in HNSCC were used to perform KEGG and GO pathway enrichment analyses.KEGG and GO analyses on the genes co-expressed with E2F6 in HNSCC revealed 3 pathways, 2 valid molecular functions (MF), 5 cellular components (CC), and 2 biological processes BP; Fig. H. SpecifOur present study showed that the mRNA expressions of E2Fs were up-regulated in the TCGA-HNSCC database, which was consistent with the activation of the E2F signaling pathway in patients with OSCC recurrence after radiotherapy in a previous study . The dys+ cells, and natural killer (NK) cells generally correspond to improved survival in HNSCC [+ cells, and NK cells in males and this could be correlated with the suppressive function of E2F4 but improved effects of E2F2 and E2F8 on the OS of male HNSCC patients. Interestingly, the infiltration level of neutrophils was negatively associated with most E2Fs expressions in both females and males. This result indicated that neutrophil infiltration might be related to the regulation of the tumor cell cycle.The tumor microenvironment (TME) in HNSCC consists of tumor cells, endothelial cells, CAFs, and immune cells. Generally, HNSCC has a high degree of immune infiltration, although the extents and compositions of immune cell infiltration could be different in different anatomic sites and etiology. Our study showed the degree of E2Fs-correlated immune infiltration in male HNSCC patients was higher than in female HNSCC patients. Importantly, it was reported that elevated levels of B cells, CD8in HNSCC . Our resGenetic alteration, co-expressed genes, and enrichment analyses were performed to explore the molecular mechanisms and determine the molecular evidence that could support the effects of E2F6 on the prognosis of HNSCC patients. Our results showed that genes related to mismatch repair, acetyltransferase complex, and somatic mutation could be regulated by E2F6 expression. As reported, mismatch repair deficiency could promote hypermutability and lead to the phenomenon called microsatellite instability. Therefore, it could affect cell proliferation and increases the probability of tumor development . MeanwhiThere were some limitations in our study that should be addressed. First, although our COX regression and survival analyses showed the potential role of E2F6 in female HNSCC patients, the effects of all genes of the E2F family in HNSCC should be more extensively studied. Second, gender-disaggregated survival analyses indicated that E2Fs might play different roles in patients of different genders. Thus, more detailed research disaggregated by gender needs to be carried out in the future. Third, infiltration analysis results suggested that E2Fs expressions had stronger associations with immune cell infiltrations in male HNSCC patients. However, the effects of E2Fs expressions on the immune microenvironment in HNSCC patients with different genders and the specific reasons remained unclear. Lastly, the effects of E2F6 on the progression of HNSCC could be partially interpreted in the genetic alterations and GO/KEGG enrichment analysis and the specific underlying regulatory mechanisms and molecular pathways involved need to be explored and verified.In conclusion, our results suggested that the mRNA expressions of E2Fs were significantly correlated with the carcinogenesis and progression of HNSCC. More specifically, E2F1, E2F3, E2F4, E2F6, and E2F7 might be reliable diagnostic molecules, and E2Fs (excluding E2F3) were closely related to the clinical features (except for metastasis) of HNSCC. We found that E2F6 was an independent risk factor for the survival of female HNSCC patients. In addition, E2F expression has a stronger predictive effect on the prognosis of women than men in HNSCC.Altogether, this study provided new insights into the early diagnosis and prognostic prediction of HNSCC, which could be of great significance in improving the detection rate, treatment, and prognosis estimation of HNSCC patients.


In conclusion, IGF2BP1 was a potent pyroptosis inducer in septic AKI through targeting the MIF component of NLRP3 inflammasomes. Inhibiting IGF2BP1 could be an alternate pyroptosis-based treatment for septic AKI.Septic acute kidney injury (AKI) is characterized by inflammation. Pyroptosis often occurs during AKI and is associated with the development of septic AKI. This study found that induction of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to a higher level can induce pyroptosis in renal tubular cells. Meanwhile, macrophage migration inhibitory factor (MIF), a subunit of NLRP3 inflammasomes, was essential for IGF2BP1-induced pyroptosis. A putative m6A recognition site was identified at the 3′-UTR region of E2F transcription factor 1 (E2F1) mRNA via bioinformatics analyses and validated using mutation and luciferase experiments. Further actinomycin D (Act D) chase experiments showed that IGF2BP1 stabilized E2F1 mRNA dependent on m6A. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) indicated that E2F1 acted as a transcription factor to promote MIF expression. Thus, IGF2BP1 upregulated MIF through directly upregulating E2F1 expression via m6A modification. Experiments on mice with cecum ligation puncture (CLP) surgery verified the relationships between IGF2BP1, E2F1, and MIF and demonstrated the significance of IGF2BP1 in MIF-associated pyroptosis  Acute kidney injury (AKI) caused by sepsis has a high death rate, which is associated with systemic inflammation RNA methylation on the sixth N atom of adenylate is defined as N6-methyladenosine (m6A), which is the most prevalent form of RNA internal modification in vivo and in vitro was generated by performing cecum ligation puncture (CLP) surgery on mice or treating HK2 cells with LPS. In CLP mice, declined renal function was associated with the elevation in IGF2BP1 expression and pyroptosis. Further study demonstrated that E2F1 modified by IGF2BP1 transcriptionally promoted MIF-related pyroptosis though triggering the assembly of NLRP3 inflammasomes. Inhibiting IGF2BP1 might be an attractive and alternative pyroptosis-based treatment in the future for treating septic AKI.Here, we hypothesized that the elevation of IGF2BP1 in septic AKI caused renal damage and enhanced NLRP3 inflammasome activity via macrophage migration inhibitory factor (MIF) to exacerbate pyroptosis. The septic AKI model In this study, lipopolysaccharide  and MIF antagonist  were bought from Sigma-Aldrich . Cycloheximide  and 3-Deazaadenosine  were purchased from MedChemExpress . VX765 (#inh-vx765i) was purchased from Invivogen . The sh-IGF2BP1, sh-METTL3, sh-MIF, sh-E2F1, and sh-NC were obtained from Genepharma company . Lentiviruses expressing sh-IGF2BP1 and nonsense control (NC) were purchased from Tsingke . The E2F1 overexpression vector was constructed using p-ENTER, while the overexpression plasmids for MIF or METTL3 were constructed in pcDNA3.1.Patients with AKI admitted between December 2021 and July 2022 at The First Affiliated Hospital of Nanjing Medical University were included. The control participants were recruited from a healthy population that received a health checkup at The First Affiliated Hospital of Nanjing Medical University between February and July 2022. The critical elements of the clinic are listed in Table 7 TU/mouse) facilitated lentivirus-mediated gene transfer in the kidneys. The CLP operation was conducted one week after the injection. In vivo tests were conducted in accordance with Chinese regulations on the use and care of laboratory animals.The Committee on the Ethics of Animal Experiments at Nanjing Medical University approved the animal experiments (IACUC-2111039). Mice feeding and model building were performed in the animal biosafety level-3 laboratory (ABSL-3 Lab) of Nanjing Medical University. The male C57BL/6 J mice  were purchased from Vital River Laboratory Animal Technology  and randomly divided into groups. For the septic AKI model, mice were subjected to the CLP surgery 2. LPS was dissolved in PBS and kept at -20 °C. HK2 cells were treated with serum-free DMEM/F12 for 8 h in advance and then replaced with new culture medium containing LPS of specified concentration or PBS solution of the same volume. To block protein synthesis, HK2 cells were incubated with 100 μM CHX Human Kidney-2 (HK2) cells were purchased from the Cell Bank of the Chinese Academy of Sciences  and cultured in DMEM/F12  supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin  at 37 °C with 5% COThe blood of mice was centrifuged at 1500 r/min at 4 °C, and the separated serum was stored at -80 °C. Serum creatinine (Scr) and BUN were detected to reflect renal function via commercialized kit reagents. Kidney tissues embedded with paraffin were cut into slices, followed by being immersed in 4% paraformaldehyde for hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining. The frozen kidney tissues were stored at -80 °C for protein or mRNA detection.Before the immunostaining, antigen retrieval for paraffin-embedded tissues in a citrate antigen retrieval solution was conducted using microwave heating. For IHC, kidney tissues were incubated with antibodies against IGF2BP1 , MIF , or E2F1 . Immunofluorescence staining of kidney sections was performed with the antibody against IGF2BP1 , while those of HK2 cells were performed with primary antibodies against MIF , IGF2BP1 , and NLRP3 . Cell nuclei were stained with DAPI. Immuno-stained samples were visualized using a Leica Thunder microscope system.Using the DynaBeads mRNA Purification Kit , poly (A) RNA was purified from total RNA for dot blot. Poly(A) RNAs were diluted to concentrations of 250 ng/μl and 100 ng/μl. Each dilution was spotted twice (2 ul) on positively charged nylon membranes . The membranes with poly(A) RNAs were crosslinked using a Stratalinker 2400 Crosslinker. Then, the membranes were incubated for 5 min in wash buffer prior to 1-h blocking. The membranes were incubated in blocking buffer with diluted anti-m6A antibodies overnight at 4 °C. The levels of total m6A were detected using enhanced chemiluminescence after typical immunoblotting techniques including treatment with secondary antibodies.WB was conventionally performed. The primary antibodies used were as follows: anti-IGF2BP1 , anti-MIF , anti-E2F1 , anti-NLRP3 , anti-Caspase1 , anti-Cleaved-Caspase1 , anti-ASC , and anti-GAPDH .For MeRIP-qPCR experiments, total RNA of HK2 cells with different treatments was extracted. After fragmentation, RNA was incubated with anti-m6A or IgG antibodies using the GenSeq® m6A MeRIP kit  for immunoprecipitation. Enrichment of m6A-containing mRNA was measured using a qPCR assay. For Co-IP experiments, collected HK2 cells were homogenized in lysis buffer from the IP/CoIP kit  on ice. The cell lysates were co-incubated with selected antibodies at 4 °C overnight and then added with protein agarose A/G beads to reduce non-specific bindings. The precipitate was washed three times before WB. The antibodies employed were the same as those used in WB.The thawing of samples was conducted at room temperature. Human IL-1β (#ab214025), IL-18 (#ab215539), Caspase 1 (#ab219633), IL-6 , and TNF-α  levels in the culture supernatant were measured with commercialized sandwich enzyme-linked kits from Abcam . Mouse MIF , Scr , IL-1β  levels in mice were determined. Each sample was measured in quadruplicate or sextuplicate.The extracellular LDH was examined to detect pyroptotic cell death using the LDH Cytotoxicity Assay Kit . Briefly, the culture supernatant was centrifuged at 400 × g for 5 min and then loaded into a new 96‐well plate, mixed with 60 μL LDH detection working fluid. After incubation at room temperature for 30 min in the dark, the absorbance at 490 nm was detected using a microplate reader .For qPCR, total RNA was isolated using the EZNA ® Total RNA Kit I . cDNA was synthesized with the PrimeScript RT Master Mix kit . The relative level of mRNA expression was quantified with an SYBR Kit  and normalized to that of GAPDH expression. Primers used for qPCR were listed in Cell viability was determined using the cell viability assay . Briefly, HK2 cells were seeded into the 96-well plates and added with CCK-8 (10 ul/per well) for further incubation for 2 h at 37 °C. Optical density (OD) was detected at 450 nm using a microplate reader .The MIF promoter was cloned into the pGL3-basic plasmids  for promoter study and named pGL3-MIF. The wild-type (Wt) and mutant (Mut) PCR products containing the E2F1 binding motif in the MIF promoter were synthesized and cloned into the pGL3-basic plasmids , which were named E2F1-Wt and E2F1-Mut respectively. To explore the mechanism by which IGF2BP1 stabilized E2F1 mRNA via the m6A site in the 3'-UTR region, adenosine (A) in the m6A motif was replaced by cytosine (C) and then cloned into pmir-GLO reporter plasmids using Tsingke . Transfections were carried out using Lipofectamine-3000 transfection reagent . Each luciferase-containing plasmid (400 ng) and internal control plasmid  were co-transfected. The dual-luciferase reporter gene assay kit  was applied to detect luciferase activities. The activities of firefly luciferase were normalized to those of Renilla luciferase.For EMSA, the Wt and Mut short oligonucleotide probes were synthesized by GenePharma . The probes were biotin end-labeled using the Biotin 3' End DNA Labeling Kit . The labeled and unlabeled probes were annealed to double-stranded probe DNA. EMSA assays were carried out using the LightShift Chemiluminescent EMSA kit . For the super shift assay, anti-E2F1  antibody or IgG  were added to the nuclear extracts. All mixtures were separated on 6% native polyacrylamide and then transferred onto positively charged nylon membranes . After crosslinking the DNA by UV, the biotin-labeled DNA was identified using chemiluminescence. Probe sequences used for EMSA were listed in ChIP was conducted using a Magna ChIP® A/G Chromatin Immunoprecipitation Kit . Protein-DNA complexes were incubated with anti-E2F1  or IgG  antibodies coupled with protein beads at 4 ℃ overnight. Next, DNA was purified and precipitated for subsequent PCR analysis after elution and reverse cross-linking. The PCR was performed using Rapid Taq Master Mix . On a 2% agarose gel, the products were separated by electrophoresis. 10% of the initial sheared chromatin DNA served as the input control after reverse cross-linking and purification. In addition, ChIP DNA was detected by quantitative PCR using an SYBR Kit  using the StepOnePlus™ Real-Time PCR System . Primers used for ChIP were listed in http://blast.ncbi.nlm.nih.gov/Blast.cgi) http://www.softberry.com/berry.phtml?topic=index&group=programs&subgroup=promoter) https://services.healthtech.dtu.dk/service.php?Promoter-2.0) https://jaspar.genereg.net/) http://genome.ucsc.edu/) http://cistrome.org/db) https://rmvar.renlab.org/) Database searches to find homologies between sequences were performed using the BLAST algorithm  were considered statistically significant while N.S. meant non-significance.GraphPad Prism software (version 7.00) was used for statistical analyses and data visualization, with each point representing independent data. in vitro.Worse renal function in the CLP mice was confirmed as the serum creatinine (Scr) level was much higher in the CLP mice than in the Sham ones A. Total To explore the relationship between IGF2BP1 and pyroptosis in LPS-stimulated HK2 cells, the IGF2BP1 gene was knocked down by shRNA, and verified via WB  for further analysis . JASPAR E2F1 increases MIF transcription and is necessary for IGF2BP1 to promote MIF Figure ; hence, Given that IGF2BP1 is widely considered as a reader for m6A modifications, we investigated whether m6A mediated IGF2BP1 involvement in elevating E2F1 mRNA stability in septic AKI. After knocking down METTL3, a major m6A writer, we found that control cells exhibited greater E2F1 mRNA stability than sh-METTL3-expressing HK cells Figure D, indicaIn order to further confirm that IGF2BP1 affected MIF in an m6A-dependent manner, we introduced DAA, an S-adenosylhomocysteine hydrolase inhibitor and an m6A methylation inhibitor We mined the DART-seq data GSE54365) 5 6A). WeDespite the fact that E2F1 mRNA can be m6A methylated, it was unclear whether IGF2BP1 stabilized E2F1 mRNA via the putative m6A region. The E2F1 3′-UTR region containing the m6A site was a cloned downstream of the firefly luciferase coding region of the pmir-GLO plasmids  can be stabilized by IGF2BP1 In this study, the m6A reader, IGF2BP1, was a pyroptosis inducer through indirectly targeting MIF in AKI. The elevation of MIF via E2F1 improved our knowledge that E2F1 and MIF were indispensable for IGF2BP1 to induce pyroptosis. Inhibiting IGF2BP1 could be an alternative strategy in the future to supplement pyroptosis therapy based on small chemical molecules to improve the treatment of AKI.Supplementary figures and table.Click here for additional data file.


Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naïve individuals and also in the event of a pandemic outbreak.Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naïve individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naïve cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage FcγR2a and FcγR3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-γ-producing CD4 + T-cell responses. Our study provides key insights into the immune responses induced by WPV vaccination in immunologically-naïve animals.Influenza viral infections cause significant morbidity and mortality. Although current split virus (SV) vaccines are the most effective way to combat seasonal influenza, their efficacy can be modest. We investigated immune responses directed towards inactivated whole influenza virus particle vaccine (WPV) formulations in a non-human primate model as an important step towards clinical testing in humans. Our analysis of 46 immune parameters found that compared to SV vaccination, WPV-induced antibody responses were significantly increased in scope and magnitude, together with higher frequencies of influenza-specific B-cell and CD4 A/H1N1 and A/H3N2 influenza A viruses (IAV), together with influenza B viruses (IBV), are responsible for the majority of seasonal influenza outbreaks in the human population . In counInfluenza vaccination that induces neutralizing antibodies against viral hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins is the most effective way to control the impact of seasonal influenza. However, despite this, up to 5 million cases of severe infections and 243,000 to 640,000 deaths still occurred worldwide annually in pre-COVID-19 years . The mosFirst introduced in the 1940’s, vaccination with inactivated whole influenza virus particle vaccines (WPV), although effective at conferring protection, were often associated with local and systemic adverse effects typified by excessive inflammatory responses and fever –16. ConcThe All-Japan Influenza Vaccine Study Group was established to develop safe and immunogenic WPVs for use in humans, with the involvement of all four of Japan’s seasonal influenza vaccine manufacturers. WPV formulations prepared by each manufacturer has been shown to induce stronger neutralizing antibodies and innate immune responses in mice compared to SV formulations consisting of the same vaccine strains. To predict the potential efficacy of these WPV formulations in humans before transitioning to clinical trials requires evaluations in animal models that bear close genetic and physiological similarities to humans versus other species such as rodents. In this regard, non-human primates are ideal as their immune responses elicited by infection or vaccination can closely reflect those in humans. However, the ability to perform in-depth analysis of the immune mechanisms that underpin the generation of protective immunity in non-human primates, particularly against influenza, is limited by the availability of reagents and assays suited to this task. We have recently established detection methods for analysing and understanding immune responses in influenza virus-infected cynomolgus macaques . In thisWe first compared the immunogenicity of monovalent WPV and SV A/Singapore/GP1908/2015 (H1N1) formulations containing equivalent amounts of HA by vaccinating naïve cynomolgus macaques via the sub-cutaneous route , the mosTo determine antibody responses following immunization with WPV and SV, we firstly performed hemagglutination-inhibition (HI) assays. Significantly higher A/H1N1-specific HI antibody titres were induced by WPV after both first and second doses  against the vaccine-matched virus strain P<0.001;  in compaTo simultaneously define multidimensional antibody features elicited after WPV and SV immunization, a 28-parameter multiplex bead array was performed , 27. TriHigher IgA responses to A/H1N1 HA (P<0.001) were also induced after each dose of WPV compared to SV. When FcγRs were examined across different antigen specificities, one WPV dose was sufficient to induce significantly higher levels of FcγR2a and FcγR3a-binding antibodies to A/H1N1 HA (both P<0.001) and HA Stem  compared to an equivalent dose of SV, and remained higher after the second dose. The increased binding to FcγR2a and FcγR3a soluble dimers for HA and HA Stem-specific antibodies suggests that, in addition to their potential ability to neutralize virus infectivity, they can facilitate antibody-mediated activity such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). As these were vaccine monovalent formulations, vaccination with either vaccine type did not induce responses against A/H3N2 HA and NA.s = 0.816) and HA Stem (rs = 0.721) in WPV-vaccinated macaques after two vaccine doses (day 56). Interestingly, when we examined correlations with FcγR usage, HI titres were associated with FcγR2a-binding responses of HA Stem-specific antibodies (rs = 0.970) as well as FcγR3a-binding responses to HA-specific antibodies (rs = 0.850), again suggesting their potential to facilitate multiple mechanisms of antibody-mediated protection.Analysis of 17 humoral immune parameters revealed key antibody features that were significantly elevated for WPV in comparison to SV, after both single or double dose immunization, including FcγR3a HA, FcγR2a HA, IgG HA, IgA HA, IgG HA stem, FcγR3a HA stem, FcγR2a HA stem and H1N1 HI antibody titres . Having To further analyze the magnitude of antibody responses in each macaque, we collated a heatmap based on the fold-change in HI antibody titres and A/H1N1 antigen-specific antibody responses after the first and second doses (on day 28 and day 56 respectively), relative to their baseline levels day 0; . When weHaving shown strong antibody responses elicited by monovalent WPV, we subsequently compared immune responses induced by quadrivalent WPV and SV containing A/H1N1 (A/Singapore/GP1908/2015), A/H3N2 (A/Singapore/INFIMH-16-0019/2016), B/Yamagata (B/Phuket/3073/2013) and B/Victoria (B/Maryland/15/2016) strains. Macaques were first inoculated with either vaccine on day 0 (n = 8 per vaccine type). On day 28, half of the animals from each group (n = 4) were immunized with the same vaccine used for the first dose, while the remaining animals received a mixed vaccine regimen .Increased IFN-α levels were detected after the first WPV dose compared to SV (P = 0.001 at 6 and 24 hours)  which waSignificant increases in antibody responses were observed more so after a single dose of WPV than SV (on day 28 compared to day 0) across different antigen specificities . This trIn animals that received the mixed vaccine regimens, there was a consistent trend in animals vaccinated with WPV followed by SV (WPV⇢SV) to exhibit higher responses (on day 49), particularly against A/H1N1 HA and HA Stem and the HA of B/Yamagata, in comparison to the opposite regimen (SV⇢WPV) . CompariIncreased antibody responses induced by WPV also translated into elevated HI antibody titres against each of the viral strains used in the formulation. Significantly higher titres were detected against A/H1N1 (P = <0.001) and A/H3N2 (P = 0.004) after one WPV dose compared to SV . In factA ranked comparison of antibody responses according to HI antibody titres against each strain revealed once again that double-dosed WPV vaccinated animals with the highest titres also had the strongest IgG levels as well as dimeric FcγR2a and FcγR3a binding antibodies, especially to the HA of A/H1N1 in comparison to SV-vaccinated animals . Antibod- CD19+ B cells in cynomolgus macaques [+ B cells specific for A/H1N1 HA  (We have recently established the use of recombinant antigen HA (rHA) probes to define antigen-specific class-switched IgDmacaques . Using t07/2009)  could be07/2009) .As much of the human population has pre-existing influenza-specific immunity, we next determined the immunogenicity of A/H1N1 WPV and SV immunization regimens in macaques previously infected with influenza virus to understand how pre-existing immunity affects vaccine responses. Macaques were first infected with the seasonal strain, A/Yokohama/91/2007 (H1N1), and subsequently vaccinated 56 days later with monovalent A/Singapore/GP/1908/2015 (H1N1) WPV or SV . As cont10-fold increases in IgG levels to A/H1N1 HA, NP and HA Stem with similar trends observed for FcγR2a and FcγR3a usage (Comparable antibody responses induced by the first A/Yokohama/91/2007 (H1N1) infection (on day 56) were detected in all macaques prior to vaccination and typified by ~2log3a usage . Vaccina3a usage . No diff+ B cells in WPV-vaccinated animals . Immune responses induced by both SV and WPV vaccination were sufficient for viral clearance as detectable viral titres were only observed in nasal swabs from PBS but not SV- and WPV-vaccinated animals following A/Narita/1/2009 infection . In contnfection .+ and CD8+ T cells (+CD4+ T cells (P = 0.0203). There was also a strong positive correlation between IFN-γ+CD4+ T cells with rHA+ B cell frequencies  detected amongst all groups  by performing intracellular cytokine staining of PBMCs following an overnight stimulation with live A/Singapore/GP1908/2015 , 29, 30. T cells  was dete T cells . In comp T cells , with sil groups , in lineulations , suggestWhile SV formulations have been instrumental in reducing global disease burden caused by seasonal influenza viruses, their effectiveness, especially in immunologically naïve individuals, remains suboptimal. Amongst the many different formulations of influenza vaccines that have been evaluated, studies have indicated that the use of WPV can be of a great benefit –23. ThisWhile methods to analyse influenza-specific immune responses in mice and humans are well established, similar techniques have been less developed for non-human primates. Here, we have applied an array of techniques and use of cross-reactive reagents to comprehensively probe influenza-specific responses in vaccinated cynomolgus macaques. In immunologically-naïve settings, stronger humoral responses were observed in WPV-immunized animals. The multiplex array used here to simultaneously measure 28 antibody features revealed enhanced antibody responses induced by monovalent and quadrivalent WPVs, directed against a broad range of representative viral antigens expressed by A/H1N1, influenza B and to a lesser extent A/H3N2. As the multiplex array uses plasma at a specific dilution, an important consideration is that the volume used is sufficient for capturing differences in responses across all antigens examined. In the case of NA, this has resulted in a high baseline level but nonetheless still provided a sufficient window to detect the differences. The most prominent augmentation in antibody responses were linked to IgG antibodies directed against the HA of A/H1N1 and influenza B, and to some degree, the HA Stem of A/H1N1. Not surprisingly, in the case of HA-specific IgG, HA inhibition of each strain strongly correlated with these antibody responses, indicating their potential to inhibit viral entry. Given that antibodies directed to the conserved HA Stem region are noted for their ability to cross-react with different HA subtypes within the same antigenic group , our datIn addition to binding via the antigen-binding fragment (Fab) region of the antibodies, the crystallizable fragment (Fc) region of antibodies plays a key role in recruitment of complement and activation of cellular effector mechanisms, most notably via ADCP by FcγR2a  and ADCC+ T cell responses and frequencies of HA-specific B cells, a higher proportion of which expressed IgG, suggesting a more mature response and antibodies which could have better binding affinities and/or avidities. An important point to note is that differences in vaccine-induced antibody responses between groups at later time points were observed by ELISA and not in the multiplex array. This could be because the antibody levels were relatively high and beyond the detection limits of the array. Given the disrupted nature of the antigens in SV compared to intact antigens in WPV, there could be additional differences in the epitopes bound by these antibodies, which we are unable to detect as both the ELISA and array measures responses against whole antigens. A further note to consider is that the antigens used in the ELISA and array are not identical to those in the vaccines and using better matched antigens could reveal more specific differences.In the context of infection induced immunity, higher titers of HI antibodies were induced by WPV vaccination and further boosted following a secondary influenza virus infection. This was associated with increased IgG HA and HA Stem-specific antibodies, virus-specific CD4+ T cell populations, it is reasonable to speculate that follicular helper T cells also contribute to the enhanced responses observed by providing the necessary signals required, and/or acting synergistically with BCR cross-linking, to drive germinal center and plasmablast responses [The enhanced immunogenicity of WPV formulations is likely attributed to the presence of viral RNA, providing stimulation of innate immune responses via TLR7 recognition and signaling , 42. Altesponses . Notablyesponses .Overall, our results support other studies which show that WPV formulations are immunogenic , 22, 24 Macaca fascicularis) weighing 2 to 3.5 kg and aged 2 to 3 years old, were purpose-bred in Cambodia and imported and maintained at Shin Nippon Biomedical Laboratories, Ltd.All animal experiments were approved by the Institutional Animal Care and Use Committee (Approved ethics number IACUC435-001 and IACUC435-002) and was performed in accordance with the animal welfare by laws of SNBL, which is accredited by American Association for Accreditation of Laboratory Animal Care (AAALAC) International. Healthy female and male cynomolgus monkeys (The influenza virus strains A/Singapore/GP1908/2015(IVR-180) (H1N1), A/Singapore/INFIMH-16-0019/2016(IVR-186) (H3N2), B/Phuket/3073/2013 (Yamagata linage) and B/Maryland/15/2016 (NYMC BX-69A) (Victoria lineage), were provided by the National Institute of Infectious Diseases (NIID) in Japan. A/Yokohama/91/2007 (H1N1) was provided by the Yokohama City Institute of Public Health. Viruses were propagated in 10-day old embryonated chicken eggs or in MDCK cells. Collected allantoic fluids or culture supernatants were stored at -80°C until use.WPVs and SVs used in this study were manufactured and provided by KM Biologics Co. Ltd. (Japan). Monovalent formulations were produced from A/Singapore/GP1908/2015(IVR-180) (H1N1). Quadrivalent formulations were produced with strains used in 2017–2018 seasonal vaccines in Japan; A/Singapore/GP1908/2015(IVR-180) (H1N1), A/Singapore/INFIMH-16-0019/2016 (IVR-186) (H3N2), B/Phuket/3073/2013 (Yamagata linage), and B/Maryland/15/2016 (NYMC BX-69A) (Victoria lineage). WPVs were prepared from highly purified virions and inactivated with formalin and/or β-propiolactone according to standard methods used by the manufacturer. SVs were prepared by disrupting purified virions with ether in accordance with manufacturing licenses and protocols for producing seasonal vaccines. HA protein concentrations of WPV and SV were quantified using the single-radial-immunodiffusion method.All vaccine doses were administered at amounts equivalent to 15μg protein of the HA. For prime and boost experiments comparing monovalent formulations , macaque5 TCID50/ml (in a volume of 1ml) of pre-pandemic strain of influenza A/Yokohama/91/2007 (H1N1) on day 0 followed by vaccination with monovalent WPV or SV or PBS on day 56. All macaques were then challenged with 2×105 TCID50/ml (in a volume of 2ml) of the 2009 pandemic stain A/Narita/1/2009 (H1N1) on day 77. Blood samples were collected on day 0, 56 and 77 prior to each intervention as well as on day 85. All blood samples were collected in heparin tubes  from the femoral vein and plasma and peripheral blood mononuclear cells (PBMCs) separated and stored at -80°C until use. Rectal temperatures were measured in non-anesthetized animals using a C402 or C405 digital thermometer .For experiments examining how previous influenza virus infection affects vaccine-induced responses , macaquePlasma were treated with receptor-destroying enzyme  at a plasma:RDE ratio of 1:3 and incubated for 16–18 hours at 37°C and then at 56°C for 1 hour. RDE-treated plasma were serially diluted two-fold with PBS in 96-well microplates. The diluted plasma were mixed with 8 hemagglutinin units of virus antigen and incubated at room temperature for 30 min. Chicken red blood cells (0.5%) were added to the antigen-plasma dilution mixtures and incubated at room temperature for a further 30 min. HI titres were expressed as reciprocals of the highest plasma dilutions that showed complete hemagglutination.6 PBMC were infected with live influenza A virus at a multiplicity of infection (MOI) of 6 or with media alone in serum-free media at 37°C for 1 hour. FBS was added  and cells incubated for a further 3 hours. Golgi Plug  was then introduced at a final concentration of 1 μg/ml to inhibit protein export and cells incubated for a further 16 hours. Cells were harvested and stained with surface antibodies (Stimulation of macaque PBMCs with live influenza virus was performed as previously described . Cryopretibodies  in 50 μltibodies  in 50μl +CD19+CD20+ cells within live events (+ within class-switched (IgD-) B cells.Recombinant HA (rHA) probes specific for the A/California/07/2009(H1N1)pdm09 HA were generated and used for staining HA-specific B cells as described , 48, 49.e events . InfluenCytokine levels in plasma samples from vaccinated macaques were analyzed using a BD Human Cytometric Bead Array kit according to the manufacturer’s instructions and as previously described .Antigens were covalently coupled to magnetic carboxylated beads using a two-step carbodiimide reaction as previously described , 27. BriAntigens (2μg/ml PBS) were coated onto Nunc MaxiSorp flat bottom 96-well plates (Thermo Fisher Scientific) for a minimum of 24 hours. Removal of antigen was followed by blocking of wells using PBS (containing w/v 10% BSA) for 1 hour. Plasma was serially diluted across each plate in PBS (containing v/v 0.05% Tween and w/v 5% BSA) and incubated for 2 hours at room temperature. For the detection of IgG, wells were incubated with alkaline phosphate-conjugated mouse anti-human IgG  at a 1:1000 dilution for 1 hour and developed with pNPP substrate (Sigma) for 2 hours. Absorbance of wells were determined at 405nm on a Multiskan plate reader (Labsystems). All wells were washed extensively between steps using PBS (containing 0.05% Tween). Endpoint titers were determined by interpolation from a sigmodial curve fit  and expressed as the reciprocal of the highest dilution of plasma required to produce an absorbance value of 0.4.Prism 9  was used to perform statistical analyses. Where appropriate, data normality was tested using a Shapiro-Wilk test to inform the statistical analysis. For non-parametric analysis between 2 groups, an unpaired two-tailed Mann-Whitney test was used; for analysis between 3 or more groups, the Kruskal-Wallis test or two-way ANOVA with Tukey’s correction for multiple comparisons was used. A P value less than 0.05 was considered statistically significant.S1 FigMacaques (n = 9 per group) were vaccinated with monovalent A/Singapore/GP1908/2015 (H1N1) WPV or SV followed by a second dose 28 days later. Animals were monitored for (A) amount of food consumed per day as well as (B) body weight and (C) temperature throughout the course of the experiment.(TIFF)Click here for additional data file.S2 FigMultiplex antibody responses (mean±SD) from (A) (TIFF)Click here for additional data file.S3 Fig(A) Plasma from animals vaccinated with monovalent A/Singapore/GP1908/2015 (H1N1) WPV or SV (day 28) followed by a second dose 28 days later (day 56) in (TIFF)Click here for additional data file.S4 Fig(A) Multiplex antibody responses (mean±SD) from (TIFF)Click here for additional data file.S5 Fig+ CD20+class switched (IgD-) rHA+cells and could be further defined by IgM or IgG expression. (B) CD8+ and CD4+ expressing PBMCs were gated for CD3 expression to define IFN-γ and/or TNF-α cytokine producing CD3+CD8+ and CD3+CD4+ T cells.(A) Detection of A/H1N1 virus-specific B cells in PBMCs were analysed by gating CD19(TIFF)Click here for additional data file.S1 DataRaw data for all the figures and supplementary figures.(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Multi-parametric MRI (mpMRI) of the prostate is one emerging tool for early detection of clinically-significant cancer. Standard acquisition protocols provide a slice thickness of 3 mm in T2-weighted TSE imaging. However, thin-slice imaging might be superior for the assessment of the prostate parenchyma. The main disadvantage of thin-slice imaging is related to prolongation of acquisition time. In this study, similar acquisition times could be enabled by deep learning image reconstruction of thin-slice imaging as compared to standard 3 mm T2 imaging. The results demonstrate superior image quality and diagnostic confidence in thin-slice imaging.DLR) of the prostate as compared to conventional T2w TSE imaging (T2S). Materials and Methods: Thirty patients were included in this prospective study at one university center after obtaining written informed consent. T2S (3 mm slice thickness) was acquired first in three orthogonal planes followed by thin-slice T2DLR (2 mm slice thickness) in axial plane. Acquisition time of axial conventional T2S was 4:12 min compared to 4:37 min for T2DLR. Imaging datasets were evaluated by two radiologists using a Likert-scale ranging from 1–4, with 4 being the best regarding the following parameters: sharpness, lesion detectability, artifacts, overall image quality, and diagnostic confidence. Furthermore, preference of T2S versus T2DLR was evaluated. Results: The mean patient age was 68 ± 8 years. Sharpness of images and lesion detectability were rated better in T2DLR with a median of 4 versus a median of 3 in T2S (p < 0.001 for both readers). Image noise was evaluated to be significantly worse in T2DLR as compared to T2S . Overall image quality was also evaluated to be superior in T2DLR versus T2S with a median of 4 versus 3 (p < 0.001 for both readers). Both readers chose T2DLR in 29 cases as their preference. Conclusions: Thin-slice T2DLR of the prostate provides a significant improvement of image quality without significant prolongation of acquisition time.Objectives: Thin-slice prostate MRI might be beneficial for prostate cancer diagnostics. However, prolongation of acquisition time is a major drawback of thin-slice imaging. Therefore, the purpose of this study was to investigate the impact of a thin-slice deep learning accelerated T2-weighted (w) TSE imaging sequence (T2 Prostate cancer is one of the most common cancer diseases in men . HoweverTherefore, the aim of this study was to investigate the impact of a DLR thin-slice T2-weighted turbo spin echo (TSE) sequence of the prostate on image quality, artifacts, and diagnostic confidence.This monocentric prospective study was approved by the institutional review board. Informed consent was obtained from all participants. Thirty patients who were referred for mpMRI due to suspicion or relapse of prostate carcinoma between September 2021 and April 2022 were included in this study. Exclusion criteria were MRI unconditional implants.fit, Siemens Healthcare; Erlangen, Germany). The acquisition protocol consisted of standard T2-weighted TSE imaging (slice thickness: 3 mm) in 3 planes (T2S) followed by thin-slice (slice thickness: 2 mm) DLR axial T2-weighted TSE imaging (T2DLR). Subsequently, the remaining sequences of the mpMRI protocol were acquired  imaging). Acquisition time for axial T2S was 4:12 min as compared to 4:37 min for T2DLR. Detailed acquisition parameters are displayed in All examinations were performed using a 3T clinical MRI scanner (MAGNETOM Vida and VidaS and 30 datasets of T2DLR). As thin-slice imaging was only performed in the axial plane, coronal and sagittal T2S was not available for study readings. All datasets were evaluated regarding the following criteria by both radiologists using a Likert-scale ranging from 1–4, with 4 being the best image impression: extent of noise , sharpness , artifacts , lesion detectability , overall image quality , and diagnostic confidence . Furthermore, suspicion of extraprostatic growth was assessed. PI-RADS and T2 scoring were performed, including lesion size measurement of the most suspicious lesion in cases of PI-RADS ≥ 3. For reading, PI-RADS Version 2.1 was applied. After a time gap of 4 weeks, a second reading was performed regarding the preference of T2S versus T2DLR. For this reading session, both image datasets (without identifying information) were shown simultaneously next to each other.All imaging datasets were evaluated in a blinded random order reading independently by two different radiologists with three and six years of experience in prostate imaging. A dedicated workstation  was used for study readings. Datasets consisted of axial T2-weighted imaging, diffusion-weighted imaging (DWI) and DCE imaging forming 60 datasets . The Wilcoxon signed rank test for paired data was used for comparison of image quality parameters. Inter-reader agreement was calculated using Cohen’s kappa. Thirty patients were enrolled in this study and successfully examined. In one patient, post prostatectomy was revealed after imaging status. The mean patient age was 68 ± 8 years . The median prostate specific antigen (PSA) level was 7.5 ng/mL with an interquartile range (IQR) of 2.7–12 ng/mL. Twenty patients underwent biopsy, which revealed no malignancy in seven cases. Four patients underwent prostatectomy after the MRI examination. Further details are shown in S to 0.80 for T2DLR. The results from the more experienced reader 1 are displayed in the following section for better readability using median and IQR in parentheses. All results are available within Inter-reader agreement ranged from 0.69 for T2DLR as compared to T2S, with a median of 3.5 (3–4) versus 4 (4–4) (p < 0.001), respectively. However, the sharpness of the images and the extent of artifacts were evaluated to be better in T2DLR, with a median of 4 (4–4), versus T2S with a median of 3 (3–3) (both p < 0.001)  .DLR, with a median of 4 (4–4) versus a median of 3 (3–4) in T2S (p < 0.001). The assessment of overall image quality resulted in superior ratings for T2DLR, with a median of 4 (4–4), as compared to a median of 3 (3–3) for T2S (p < 0.001). Diagnostic confidence levels were also rated higher for T2DLR, with a median of 4 (4–4) in comparison to a median of 3 (3–4) for T2S (p < 0.001). For image examples, see Lesion detectability was also rated to be superior in T2DLR as their preference in 29 cases.Both readers chose T2S and T2DLR) and in seven cases a T2 score of 5 (both T2S and T2DLR). In seven cases reader 2 diagnosed a T2 score of 4 (both T2S and T2DLR) and in eight cases a T2 score of 5 (both T2S and T2DLR). Reader 1 chose a PI-RADS 4 score in ten cases (both T2S and T2DLR), whereas reader 2 chose a PI-RADS 4 score for nine lesions (both T2S and T2DLR). No significant difference was found between the sequences or between the readers. Further details are shown in In eight cases reader 1 diagnosed a T2 score of 4 . Additionally, this investigation was limited by the reconstruction algorithm, as well as scanners from one vendor. Furthermore, additional sequences should be investigated in future studies with thin-slice imaging, e.g., DWI or DCE imaging.This study demonstrates the feasibility of thin-slice T2-weighted TSE imaging of the prostate, including DLR, within a similar acquisition time. The smaller slice thickness and improvement in image quality might lift prostate MRI to the next level.