NCF1) missense variant, which is one of the most significant associated risk variants for lupus, and found that the substitution of arginine (R) for histidine (H) at position 90 in the NCF1 protein (NCF1 p.R90H) led to excessive activation of pDCs. A mechanism study demonstrated that p.R90H reduced the affinity of NCF1 for phospholipids, thereby impairing endosomal localization of NCF1. As NCF1 is a subunit of the NADPH oxidase 2 (NOX2) complex, this impairment led to an acidified endosomal pH and facilitated downstream TLR signaling. Consistently, the homozygous knockin mice manifested aggravated lupus progression in a pDC-dependent lupus model. More important, pharmaceutical intervention revealed that hydroxychloroquine (HCQ) could antagonize the detrimental function of NCF1 p.R90H in the lupus model and systemic lupus erythematosus samples, supporting the idea that NCF1 p.R90H could be identified as a genetic biomarker for HCQ application. Therefore, our study provides insights into the genetic control of pDC function and a paradigm for applying genetic variants to improve targeted therapy for autoimmune diseases.Plasmacytoid dendritic cells (pDCs) are a professional type I IFN producer that play critical roles in the pathogenesis of autoimmune diseases. However, both genetic regulation of the function of pDCs and their relationships with autoimmunity are largely undetermined. Here, we investigated the causality of the neutrophil cytosolic factor 1 ( Genetic evidence and clinical studies have revealed that the type I interferon (IFN-I) pathway is a primary pathogenic factor in a variety of autoimmune diseases –3. As thPDCs are crucial mediators of innate immunity , 5. WhenNCF1), also known as phoxp47, that was strongly associated with SLE ; OR = 2.61 and MAF = 0.0575 in White people of European descent; OR = 2.02 and MAF = 0.1570 in African Americans) and other autoimmune diseases, including primary Sjögren’s syndrome (SS)  and rheumatoid arthritis (RA) (OR = 1.65 in Koreans)  and other IFN-I–related autoimmune diseases deduced from Immunochip data. We identified what we believe to be a novel missense SNP, rs201802880, in neutrophil cytosolic factor 1 (Koreans) –18. NCF1Koreans) . Hence, In this study, we explored the function of NCF1 p.R90H in lupus by using knockin (KI) mice. The R-to-H change in NCF1 led to decreased phospholipid-binding affinity, less endosomal localization, and acidified endosomal pH, followed by increased cleavage of TLR7 and TLR9, resulting in excessive activation of pDCs and aggravated lupus progression. More important, our study of the therapeutic effect of hydroxychloroquine (HCQ) on KI mice identified a genetic biomarker for improving the treatment of SLE.Ncf1-KI mice to obtain pDCs with different alleles (https://doi.org/10.1172/JCI153619DS1). The base changed from G to A, corresponding to the amino acids from R to H. Splenic pDCs were isolated from WT (G/G), homozygous (A/A), and heterozygous (G/A) KI mice. Then, pDCs were stimulated with the TLR7/-8 ligand R848 and the TLR9 ligand CpG, respectively. As shown in As NCF1 p.R90H is strongly associated with an elevated IFN-I signature in SLE, it drew our attention to investigating the function of this SNP in pDCs. It was impossible to edit human primary pDCs directly, so we turned to constructing the Tnf, Il6, and Il12b, chemokines such as C-C motif chemokine ligand 3 (Ccl3), Ccl4, C-X-C motif chemokine ligand 9 (Cxcl), Cxcl10, and Cxcl11, and the costimulatory molecule Cd86 ((Trpm) 2 and catalase (Cat), and genes responsible for ROS generation, such as NAD(P)H quinone dehydrogenase (Nqo1) and heme oxygenase (Hmox1), were found to be downregulated in A/A pDCs  with R848 for 12 hours and then subjected them to RNA-Seq. Upregulated genes in A/A pDCs were mainly enriched in the TLR pathway, the PI3K/AKT pathway, and the JAK/STAT pathway . Gene seA/A pDCs . Taken tN-acetyl-l-cysteine  were added to the culture medium and pDC cytokine production was measured. Interruption of the energy supply fueled by FAO, glycolysis, or OXPHOS suppressed the production of IFN-I by pDCs, while ROS clearance significantly boosted pDC IFN-I generation P2], according to the structure resolution , 29. It In summary, NCF1 p.R90H resulted in impaired localization of NCF1 at the endosome, followed by lowered pH and more cleavage of TLRs, which resulted in overactivation of the TLR signaling pathway.Mx1, Irf7, Oas1, and Isg15, were increased in the renal tissues of KI lupus mice compared with WT lupus mice  challenge. Strategically, both WT and KI mice were subjected to IMQ to establish the lupus model, which has been proven to be dependent on the production of IFN-I and IFN-III in pDCs , 31. Thepus mice . Thus, wTo further clarify the immunological changes as a result of NCF1 p.R90H, we evaluated the proportion and activation of pDCs and lymphocytes in the spleen. In IMQ-induced lupus model, the percentage of pDCs decreased, which was similar to the finding that the number of circulating pDCs decreased as a result of migration to the skin in patients with SLE . We detehiCD62Llo activated CD4+ T cells increased at the expense of CD44loCD62Lhi naive CD4+ T cells, which decreased  B cell population and transitional 2 (T2)/follicular (Fo) B cells, together with an increase in transitional 1 (T1) B cells and an emergence of age-associated B cells (ABCs) –34. We fecreased . Intraceecreased . In contecreased . Taken tTo confirm whether pDCs were involved in the regulation of aggravated lupus symptoms in KI mice, we adopted a method similar to that used in a previous study by depleting pDCs in vivo using anti-PDCA1 antibodies . Mice weNCF1 expression in IFN-I–producing pDCs. Single-cell transcriptome profiling (scRNA-Seq) has enabled high-resolution mapping of cellular heterogeneity, development, and activation states in diverse systems , the importance of NCF1 has been revealed for various autoimmune diseases, including arthritis, experimental autoimmune encephalomyelitis (EAE), SLE, psoriasis, etc.  mice, for instance, will help us to reach more solid conclusions  classification criteria for SLE. The SLE Disease Activity Index 2000 (SLEDAI-2k) and the renal SLEDAI (RSLEDAI) score  were calculated on the basis of chart reviews. Patient information is provided in Ncf1-KI mice were generated using the CRISPR/CAS9 technique. The sgRNA sequence was 5′-ACGAGCCGCTGAGAGTCGCC-3′, and the donor sequence was 5′-CATGGGTCTCTGGCTCCCCCACCCAGCACCCAGGTGGTTTGATGGGCAACGAGCAGCTGAGTCCCACCAGGGCACTCTCACTGAATACTTCAACGGCCTCATGGGACTGCCCGTGAAGAT-3′. Cas9 mRNA, sgRNA, and donor DNA were coinjected into zygotes. Thereafter, the zygotes were transferred into the oviduct of pseudopregnant ICR  females at 0.5 dpc. Approximately 19–21 days after transplantation, all offspring of the ICR females (F0 mice) were identified by sequencing of tail DNA. Positive F0 mice were crossed with C57BL/6J mice to generate heterozygous KI mice. In addition, female mice at 10–12 weeks of age were used to carry out all experiments. All mice were bred and housed under specific pathogen–free conditions.The lupus model was induced according to the previous reports with minor modifications . BrieflyintSiglecH+PDCA1+ cells  and cultured in RPMI-1640  containing l-glutamine supplemented with 10% FBS ; 1% penicillin-streptomycin ; and 0.1% 2-mercaptoethanol . For Western blotting, FLT3L-induced pDCs were used. Murine BM cells were cultured with 200 ng/mL FLT3L (Peprotech) for 9 days, and pDCs were sorted as CD11cintSiglecH+. Human primary pDCs were first enriched from PMBCs using the Human Diamond Plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi Biotec) and then sorted by FACS. Cells were treated with 10 μg/mL R848 or 0.5 μM ODN2216 (InvivoGen), with or without the indicated inhibitors. The inhibitor concentrations were as follows: 2-DG, 1 μM; metformin, 1 μM; etomoxir, 200 μM; NAC, 1 mM; HCQ, 0.5 μM or 1 μM ; BafA, 100 nM; and 2-GBI, 100 μM.Mouse primary pDCs from spleens were isolated by FACS as CD11cMouse kidneys were fixed in formalin and embedded in paraffin, and tissue sections were stained with H&E. Pathology was assessed using Case Viewer software (3DHISTECH Ltd.) and scored in a blinded manner on a scale of 0–3 for increasing severity of glomerulonephritis as follows: (a) minimal damage to the glomerular basement membrane (GBM) and little to no cellular infiltrates; (b) intermediate degree of cellular infiltration with moderate damage to the GBM; (c) extensive cellular infiltration and severe destruction of the GBM, which may include the presence of casts indicating proteinuria.Murine IFN-α and IFN-β levels were detected using the Mouse IFN-α ELISA kit and the Mouse IFN-β ELISA kit (both from R&D Systems), respectively. Murine TNF-α and IL-6 were quantified using the ELISA MAX Deluxe Set Mouse IL-6 and ELISA MAX Deluxe Set Mouse TNF-α kits (BioLegend). Human IFN-α was determined with the Human IFN-α Multi-Subtype ELISA Kit (R&D Systems). Proteinuria was examined using the LBIS Mouse Urinary Albumin Assay Kit (Shibayagi). RF-IgM, RF-IgG, and anti-dsDNA antibody levels in serum were measured by ELISA . All detection was performed according to the manufacturer’s instructions.3, GE Healthcare) through gradient centrifugation and analyzed by FACS. The ROS levels were detected with the OxyBURST Green H2DCFDA kit (Thermo Fisher Scientific) according to the protocol. Mitochondrial ROS levels were detected with MitoSOX Red (Thermo Fisher Scientific) according to the manufacturer’s instructions. The antibodies and reagents used are listed in For mice, single splenic cells were prepared by digesting the tissue fragments with collagenase IV (MilliporeSigma) and DNase I (MilliporeSigma) and subjected to FACS analysis by Fortessa (BD Biosciences). Human PBMCs were isolated with Ficoll-Paque PLUS  by analysis of DEseq2 and a log2 fold change of 1 or greater or –1 or less were selected for further analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to determine the significant pathway of the selected genes. The RNA profiling data generated in this study are available in the NCBI’s Gene Expression Omnibus (GEO) database (GEO GSE176349).Total RNA was extracted by TRIzol Reagent . Next, RNA libraries were prepared using the VAHTS mRNA-Seq, version 2, Library Prep Kit for Illumina (Vazyme) and sequenced in a HiSeqx 10 system (Illumina) on a 150 bp paired-end run. Genes with a statistically significant Human primary PBMCs were obtained from a healthy 29-year-old woman. After sorting by FACS, approximately 12,000 pDCs were counted, and their viability was confirmed to be greater than 90%. Single-cell suspensions were loaded onto a 10X Genomics Chromium Single-Cell 3′ v2 chip to prepare the libraries according to the manufacturer’s protocol. The library quality was verified with an Agilent 2100 Bioanalyzer and Qubit fluorometric quantitation. Samples were sequenced on an Illumina NovaSeq 6000, with a sequencing depth of at least 50,000 reads per cell.The raw fastq data were processed with CellRanger (version 2.1.1) using GRCh38 annotation (version 1.2.0) . Output Actb and RPL13a mRNA by calculating 2–ΔCt, where Ct was the cycle threshold.cDNAs were prepared using reverse transcription  and amplified by real-time quantitative PCR with the primers shown in NCF1 genotyping was performed as described in our previous reports . Primer sequences are listed in  reports . Briefly6) were lysed using RIPA protein extraction buffer (Thermo Fisher Scientific). Cell lysates (10 μg/lane) were separated by 10% SDS-PAGE and transferred onto an Immobilon-P polyvinylidene difluoride membrane (MilliporeSigma). Then, the membrane was blocked with SuperBlock TBS blocking buffer (Thermo Fisher Scientific) and incubated with the antibodies listed in pDCs . The structures of NCF1 90R and 90H were analyzed using UCSF Chimera.The PX domain of NCF1 was obtained from the RCSB Protein Data Bank with the deposition ID 1KQ6  was constructed and transduced into lineage–c-Kithi BM progenitors.For catalase overexpression, full-length cDNA of murine catalase was subcloned into the site between EcoRI and NotI enzymes of the pMYs-IRES-Puro plasmid (Cell Biolabs) and transduced into the platinum-E cell line to produce a retrovirus. LineageCells were fixed with 4% paraformaldehyde (PFA) for 15 minutes and treated for 10 minutes with PBS containing Triton X-100 (0.25%). The samples were then incubated with mouse IgG–blocking reagent (BioLegend), followed by the addition of goat anti-NCF1 antibody  and rabbit anti-EEA1 antibody  for 1 hour and then washing. AF488 donkey anti–goat IgG antibodies (1:100) were then stained for 30 minutes and washed. Finally, AF647 goat anti–rabbit IgG antibodies (1:100) were incubated for 30 minutes at room temperature. To detect the colocalization of NCF1 and LAMP1, AF647 rat-mouse antibody  and a goat anti-NCF1 antibody  were incubated for 1 hour and washed. Then, AF488 donkey anti–goat IgG antibody (1:100) was stained for 30 minutes at room temperature. After that, cells were incubated with DAPI  for 5 minutes. Cells were then detached onto glass dishes and viewed and photographed on a confocal fluorescence microscope . All immunofluorescence was assessed in a blinded manner by 2 observers throughout the analysis process. The colocalization of NCF1 and EEA1 and of NCF1 and LMAP1 was calculated with ImageJ software (NIH).PDCs were washed and incubated with prewarmed (37°C) lysosensor probe–containing medium  for 1 hour. Then, the cells were washed and subjected to confocal analysis.P value of less than 0.05 was considered significant. Data are presented as the mean ± SEM. Statistical analysis was performed with a 2-tailed, unpaired t test. Comparisons among multiple groups were performed using 1-way ANOVA with Tukey’s multiple-comparison test. The single-cell data were analyzed with the Wilcoxon test. Differences between survival curves were estimated by log-rank test.For immunofluorescence assays, approximately 30–50 cells per treatment group from at least 3 independent experiments were analyzed to calculate the fluorescence in each cell. All data were analyzed using GraphPad Prism . A All human experiments were conducted in accordance with Declaration of Helsinki principles. Written informed consent was obtained from participants prior to inclusion in the study. Human studies were approved by the ethics committee of Renji Hospital of the Shanghai Jiao Tong University School of Medicine. All animal experiments were approved by the animal care committee of Renji Hospital.NS, HZ, and Z Ye designed the project. YM, JM, and CY performed the experiments, and HZ, YM, YH, and CY analyzed the data. Jun Li, CL, and GL established the mouse model. HD, Jia Li, and Z Yin collected the human samples. HZ, YM, CY, JM, LW, and NS prepared the manuscript.


We also demonstrated reduced production of high-affinity antibody, but increased production of low-affinity antibody in the NSD1B KO mice. Further analysis revealed that loss of NSD1 promoted the development of B1 cells by increasing the expression of Rap1b and Arid3a. In conclusion, our data suggest that NSD1 plays an important role in regulation the development of B1 and B2 cells, and the process of germinal center formation and high-affinity antibody production.B cells, which consist of two well-defined populations: B1 and B2 cells, which can produce antibodies that are essential for host protection against infections, through virus neutralization, opsonization and antibody-dependent cellular cytotoxicity. Epigenetic modifications, such as DNA methylation and histone modification could regulate immune cell differentiation and functions. In this study, we found a significant reduction of GC response in the B cell specific knockout of H3K36 methyltransferase NSD1 (Mb1-Cre B cells could produce antibodies which are essential for host protection against infections, through opsonization of pathogens for efficient phagocytosis by macrophages, virus neutralization and antibody-dependent cellular cytotoxicity , 2. The NSD1 (nuclear receptor SET (su(var)3–9, enhancer-of-zeste, trithorax) domain containing protein-1), which is isolated and characterized in 2001, belongs to the NSD protein lysine methyltransferase (KMT) family (via interactions with H3K36 methylation and RNA polymerase II  family . This fa) family . NSD1 coerase II . Loss oferase II , 19 and erase II , head anerase II , neuroblerase II . NSD1 coerase II , which sB KO) mice to reveal the function of NSD1 in the development of B cells and GC formation. We found out that NSD1 did not affect the development of B2 cells in bone marrow, but was required for the generation of follicular B cells in the spleen. The loss of NSD1 in B cells reduced the formation of GC and production of high-affinity antibody, but increased the production of low-affinity antibody. Further analysis revealed that the loss of NSD1 promoted the development of B1 cells in the peritoneal cavity and spleen by increasing the expression of Rap1b and Arid3a.In previous study, we identified H3K36me2 methyltransferase Nsd2 was required for GC B cell adhesion to follicular dendritic cell expressed adhesion molecules . HoweverTo generate NSD1 conditional knockout mice, a loxp site and aFNFL (Frt-Neo-Frt-Loxp) cassette were engineered to flank exon 6-8 of the NSD1 gene to generate the “floxed” NSD1 allele  of Nanjing Medical University. All the mice were maintained on C57BL/6J background and the control mice were littermate control. 7 – 9 weeks old male and female mice were used for experiments, except using 1 week old mice to explore the early development of B1 cells in the spleen.Bone marrow, spleen, mesenteric lymph node (mLN) and Peyer’s Patch (PP) cells were isolated as described previously . In brie8 sheep red blood cells (SRBCs). For serum immunoglobulin assay, mice were immunized i.p with 100 mg of NP-KLH or NP-Ficoll which were precipitated with aluminum hydroxide gel adjuvant .For induction of germinal centers, mice were immunized i.p. with 2×10An ELISA assay was performed as described previously . In brie+ B cells were sorted from the spleen of NSD1B WT and NSD1B KO mice with a BD Aria II. Sorting efficiency was tested, and only cells with high purity (>98%) were used for RNA extraction and library construction. Sequencing were done by BGI Co., Ltd. on an Illumina Hiseq platform and 150bp paired-end module. Then we mapped the sequence reads to mm10 reference genome using Bowtie2 software and Fragments Per Kilobase per Million were calculated with Cufflinks. A threshold of fold change > 1.2 and p < 0.05 was used to determine the differential expression of genes.B220+ B cells or B220+ Gl7+ Fas+ GC B cells from the spleen of NSD1B WT and NSD1B KO mice were sorted into PBS containing 0.5% FBS before RNA extraction with a BD Aria II. cDNA was synthesized with HiScript III 1st Strand cDNA Synthesis Kit  followed by PCR with primers 5’- CTCAGCAGCAGATCCCAAT -3’ and 5’- CTTCCTGAGGCGTTTCTTCT -3’ for NSD1 (181-bp PCR product), 5’-AAATGCGACTTGGAAGATGA -3’ and 5’-TTCCCAGGCACTGGAGTT -3’ for Rap1b (173-bp PCR product), 5’-TGGACCTTTGAGGAGCAGTT-3’ and 5’-GATGGAGGTAGGCAGGTTGA-3’ for Arid3a (255-bp PCR product), and 5’-TCTGCTACGTGGTGAAGAGGAG-3’ and 5’-CCAGTCTGAGATGTAGCGTAGG-3’ for AICDA (119-bp PCR product). The HPRT gene was used as a control to generate a 312-bp PCR product with primers 5’-GGGGGCTATAAGTTCTTTGC-3’ and 5’-TCCAACACTTCGAGAGGTCC-3’.B220B WT and NSD1B KO mice were fixed with ice-cold acetone for 10 mins at room temperature. Then the sections were incubated with 2% BSA for 2 hr at room temperature. After that, sections were incubated with Gl7-FITC (BioLegend) and IgD-PE (BioLegend) for 2 hr at room temperature then incubated with DAPI for 5 mins at room temperature. Images were captured using OLYMPUS BX53 microscope.For immunofluorescence staining, cryosections of the spleens from SRBCs immunized NSD1B WT and NSD1B KO mice were sorted and pooled together, respectively, for genomic DNA extraction using a DNA Microprep kit (QIAGEN Cat: #56304). After nested PCR amplification, the VH186.2 H chains and JH4 were sequenced and analyzed with Vector NTI software to determine the frequency of somatic mutations. VH186.2 sequences and JH4 intronic sequences were analyzed with ImMunoGeneTics (IMGT) V-QUEST (http://www.imgt.org/) and only sequences of good quality were considered for mutation analysis.The mutation analysis was performed as described , 27. In P-values are denoted in figures by *, P < 0.05; **, P < 0.01 and ***, P < 0.001.Student’s t tests were used for statistical analysis. The data in figures are displayed as the mean ± SEM unless otherwise indicated. fl/fl) were mated with Mb1-Cre transgenic mice to specifically remove NSD1 in B cells. Q-PCR analysis confirmed the significantly decreased expression of NSD1 in NSD1 B cell conditional knockout  mice  mice , andthe CD43- pre-B (Fr. D IgD-IgM-), immature B (Fr. E IgD+IgM-), and mature B cells (Fr. F IgD+IgM+)  and T3 (B220+CD93+ IgMlow CD23+) cells, and a slight decrease of T1 (B220+CD93+IgMhiCD23-) cells in the spleen from NSD1B KO mice (+CD23+CD21low) in the spleen of NSD1B KO mice, but not marginal zone B cells (B220+CD23-CD21high)  cells or marginal zone B (MZB) cells were inspected . And we B WT and NSD1B KO mice were immunized with SRBCs and the germinal center B  cells in the spleen from these mice were analyzed 7 days after immunization. The frequency of GC B cells decreased significantly in the NSD1B KO mice compared with the WT control (+ CD86-), but increased light zone  of GC B cells in the spleen from NSD1B KO mice in further analysis  and VH186 (≥10) in the GC B cells sorted from the spleen of NSD1B KO mice. The frequency of GC B cells with higher-affinity mutations (W33L/K59R/99G)   also decB KO mice 21 days after immunization, which was consistent with SRBCs immunization  from NSD1B KO mice than the NSD1B WT mice (+CD19-CD5+) and B1b (B220+CD19-CD5-) cells  and B2 (CD19+B220+) cells in the peritoneal cavity from NSD1B WT and NSD1B KO mice by flow cytometry, and found increased B1 but decreased B2 cells in NSD1B KO mice, but there was no difference of the distribution of B1a and B1b cells  between the NSD1B WT and NSD1B KO mice. Rap1b  and anti-CD40 (5μg/ml), we found increased levels of activated marker CD86 and MHC II staining in B cells from NSD1B KO mice than NSD1B WT mice on 8h, 16h and 24h after activation  can be found below: NCBI under accession ID: GSE208327.The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University. Written informed consent was obtained from the owners for the participation of their animals in this study.SZ, MC, XW and ZC conceptualized the project and designed the experiments. MC, SZ, HZ, HMZ, TX and ZC performed the experiments. YW, XW and ZC analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (Grant No. 31970828 to XW and 81701542 to ZC), the National Key R&D Program of China (Grant No. 2018YFC1003900 to XW) and the Jiangsu Outstanding Young Investigator Program (Grant No. BK20200030 to XW).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.


Newcastle disease (ND) is a highly contagious viral disease of poultry causing significant economic losses worldwide. Vaccination is considered the most reliable approach to curb the economic menace that is ND, but the thermolabile nature of Newcastle disease virus (NDV) vaccination poses a significant threat to its protective efficacy. This study aimed to profile the thermostability of NDV isolates from duck (As/Km/19/44) and parrot (As/WB/19/91) and evaluate their immunogenic potential in chicks. Fusion protein cleavage site (FPCS) and phylogenetic analysis demonstrated the lentogenic nature of both the isolates/strains and classified them as class II genotype II NDV. The characterized NDV isolates were adapted in specific-pathogen-free (SPF) chicks by serially passaging. Biological pathogenicity assessment of chicken-adapted As/Km/19/44 (PSD44C) and As/WB/19/91 (PSP91C) revealed both the isolates to be avirulent with a mean death time (MDT) of more than 90 h and an intracerebral pathogenicity index (ICPI) ranging from 0.2 to 0.4. Both of the NDV isolates displayed varied thermostability profiles. PSD44C was the most thermostable strain as compared to PSP91C and the commercially available LaSota vaccine strain. The immunogenicity of PSD44C and LaSota was significantly higher than PSP91C. Based on these results, it is concluded that NDV isolate PSD44C is more thermostable and immunogenic when administered intraocularly without any adverse effects. Therefore, PSD44C is suitable for further research and vaccine development. Avian orthoavulavirus 1 (AOAV-1) and formerly designated as Avian avulavirus 1 (AAvV-1) or Avian paramyxovirus 1 (APMV-1), is a highly contagious devastating viral disease of poultry worldwide. The causative agent is classified under the genus Orthoavulavirus, within the subfamily Avulavirinae, of the family Paramyxoviridae [Newcastle disease (ND), caused by oviridae . A wide oviridae .Vaccination remains the most valuable implement for effective prevention and control of ND throughout the world. Most currently available vaccines, particularly the live vaccines, are thermolabile, demanding cold chain maintenance from manufacture to dispensation. These vaccines must be stored at a temperature range of 2–8 °C to remain effective , which iAnas platyrhynchos) and parrot (Eclectusroratus) were adapted to chickens through serial passage. The chicken-adapted As/Km/19/44 (PSD44C) and As/WB/19/91 (PSP91C) was evaluated for thermostability and immunogenicity.Addressing the need to develop a thermostable vaccine, several attempts based on reverse vaccinology, the use of chemical stabilizers and the addition of stabilizing adjuvants followed by freeze drying have been made ,7,8,9. Nv/v) chicken red blood cells (RBC). The identity of the HA-positive allantoic fluid was confirmed by a hemagglutination inhibition (HI) test [fusion (F) gene of NDV [The virus isolates described in this study were isolated under DBT Twinning project tilted “An integrated omics approach to characterize circulating Newcastle disease virus and intervention strategies to control Newcastle disease in North East India”, Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Khanapara, Assam, India from cloacal swab and intestinal content of duck (As/Km/19/44) and parrot (As/WB/19/91), respectively, in 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECE)  through the allantoic cavity route . BrieflyHI) test  using NDe of NDV . The hypAn HI serological test was performed on HA-positive allantoic fluids following standard beta-procedure . BrieflyFusion-Forward (5′-TTGATGGCAGGCCTCTTGC-3′) and Fusion-Reverse (5′-GGAGGATGTTGGCAGCATT-3′) were used to amplify a 363 bp genome fragment containing a part of F gene of NDV [−1) and visualized in an image documentation system.The presence of NDV in the allantoic fluid was further reconfirmed by RT-PCR. In brief, RNA was extracted from HI-positive allantoic fluids using a Qiagen RNeasy Extraction Kit  following the protocol provided by the manufacturer. The extracted RNA was subjected to two steps RT-PCR using Revert Aid (M-Mulv) (Thermo Fisher). The primer e of NDV . The RT-F gene of NDV. The F gene sequences of several NDV isolates were retrieved from the GenBank®  and multiple sequence alignment was performed using ClutalW . The primers were Fcds-Forward (5′-ATGGGCTCCAGACCTTCTACC-3′) and Fcds-Reverse (5′-TGYGTTCAYRYTYTTGTRGTGGCTCTCATC-3′).The primers were designed to amplify a 1618 bp full-length F gene of NDV was amplified by RT-PCR as previously described. The optimized thermal profile was as follows: reverse transcription at 50 °C for 30 min, initial denaturation at 94 °C for 5 min, 35 cycles with denaturation at 94 °C for 30 s, annealing at 56 °C for 1 min, extension at 72 °C for 1 min 30 s and final extension at 72 °C for 5 min. The amplified products were visualized by electrophoresis on 1% agarose gel stained with ethidium bromide (0.5 µg mL−1).The total RNA was extracted and full-length F gene sequences were aligned with the BioEdit program using the Clustal W algorithm and analyzed using BLAST. The amino acid motif at the F protein cleavage site area of the NDV isolates was also determined.Sequencing was performed by outsourcing at 1st BASE DNA sequencing division, Malayasia. The full-length F gene sequence was constructed by the neighbor-joining method using the sequences under study and from NDV strains with various genotypes published in GenBank. The significance of the deduced phylogenetic tree was verified by bootstrap analysis of 1000 replicates. Nucleotide sequences of commercial vaccine strains LaSota (GenBank acc. AF077761) and B1 (GenBank acc. AF309418) were included in this phylogenetic analysis.The phylogenetic and molecular evolutionary analyses were conducted using MEGA X . The nucF gene of NDV [The NDV isolates from duck (As/Km/19/44) and parrot (As/WB/19/91) were serially passaged in SPF chicks up to six passage levels. Briefly, each of the isolates were inoculated into three 5-day-old SPF chicks via the intraocular and intranasal route. Three days post infection, the chicks were sacrificed and trachea samples were collected. The identity of the virus was confirmed by RT-PCR amplification of e of NDV . The vir5(Log 2 5/50 µL) by intracerebral route. The chicks were observed every 24 h for 8 days and scored as follows: (0) if normal, (1) if sick and (2) if dead. The ICPI was obtained by calculating the mean score per bird per observation over the 8-day period.The pathogenicity of the chicken-adapted PSD44C and PSP91C was determined on the basis of mean death time (MDT) in eggs and intracerebral pathogenicity test (ICPI) in day-old chicks . MDT was50) [Seven paired sealed vials containing 1 mLaliquots each of the chicken-adapted PSD44C, PSP91C and LaSota were prepared for two different sets of temperature (40 °C and 56 °C). One pair of each set was left on ice while the other six for each temperature treatment were kept at 40 °C and 56 °C . At time50)  using th50) . BrieflyCalculation of proportionate distance (PD) between the two dilutions above and below the 50% end point:The immunogenicity of chicken-adapted PSD44C and PSP91C was evaluated and compared with commercially available LaSota vaccine strain . A safety test was performed as per standard protocol before immunogenicity studies . Briefly1, T2 and T3 (n = 20). Water and feed were given ad libitum. The chicks of the T1, T2 and T3 groups were immunized against chicken-adapted PSD44C and PSP91C and commercially available NDV LaSota vaccine at 7 days of age via the intraocular route with a standard dose of 106 EID50 per chick [A total of sixty 1-day-old SPF chicks were randomly divided into 3 groups: Ter chick . HI titeThe thermal stability of PSD44C and PSD91C at different temperature–time combinations was analyzed statistically by regression analysis and compared with commercial vaccine strain LaSota. In order to determine the half life, nonlinear regression (curve fit) followed by exponential one phase decay analysis was completed. Two-way ANOVA with interaction was also performed along with a critical difference (CD) test for pair-wise comparison of mean serum antibody titer in chicks, keeping time and treatment as factors. Statistical analysis was performed using Graphpad prism 9.3.0.The Institutional Animal Ethics Committee, Assam Agricultural University, Khanapara, Guwahati-781022, Assam reviewed and approved this study .F gene of NDV was successfully amplified in the HI-positive allantoic fluids by RT-PCR  and parrot (As/WB/19/91) gave a positive reaction to the HA test and the hemagglutinating activity was inhibited by NDV-specific hyperimmune serum in the HI test. A 363 bp fragment covering a part of y RT-PCR .F gene of both the isolates was amplified with the designed primers (F gene sequences showed that both the isolates shared the cleavage site motif 112GRQGRL117 with an amino acid residue leucine (L) at position 117, indicating its lentogenicity  and parrot (As/WB/19/91) were serially passaged in SPF chicks in order to facilitate adaptation in chicken. After the six passages, chicken-adapted NDV from duck (PSD44C) and parrot (PSP91C) were recovered from the inoculated chicks and propagated in ECE for further use.The MDT of PSD44C and PSP91C was 116.50 ± 4.646 and 95. 25 ± 2.868 and its ICPI was 0.28 ± 0.039 and 0.453 ± 0.028, respectively. Both the isolates were confirmed as lentogenic strains, according to their MDT and ICPI values .The results of thermostability testing and half life values of PSD44C, PSP91C and LaSota at 40 °C and 56 °C are tabulated in 10EID50) of PSD44C, PSP91C and LaSota were ,  and , respectively. PSD44C retained its HA activity and infectivity for an extended period of time and resulted in a half life of 206.50 and 205.00 min, respectively, as compared to PSP91C  and LaSota .The initial HA titer (Log2/50 µL) and infectivity (Log10EID50) of PSD44C, PSP91C and LaSotawere ,  and , respectively. PSD44C retained its HA activity and infectivity for an extended period of time and resulted in a half life of 38.39 and 34.36 min, respectively, as compared to PSP91C (16.79 and 14.59 min) and LaSota (19.20 and 19.19 min).The initial HA titer (Log2/50 µL) and infectivity  as compared to group T2 immunized with PSDP91C at different dpi (2) antibody levels of all the chicks immunized with PSD44C, T1 (3.20 ± 0.14), PSP91C, T2 (2.30 ± 0.11) and LaSota vaccine strain, T3 (3.30 ± 0.11). In all the three immunized groups, the HI (log2) antibody titer values increased steadily with each day and reached the titer peak at 21 dpi  which were maintained up to the end of the study period of 28 days . In the present study, there were no postvaccinal reactions in the immunized groups. Taken together, our results reflected that PSD44C is a better immunogenic than PSP91C and displayed a similar antibody profile as the commercial LaSota vaccine strain in chickens.Upon inoculation with ten times the single dose of both the chicken-adapted PSD44C and PSP91C intraocularly, the chicks did not show any local and/or systemic adverse reactions or death throughout the observation period of 21 days, revealing the isolates to be safe for immunization studies. Before immunization, chicks of all study groups were sero-negative. Post immunization, the chicks of groups Trent dpi . At 7 dp112GRQGRL117 with an amino acid residue leucine (L) at position 117 which is a typical feature of lentogenic strain [The lentogenic NDV strains from class I and genotypes I and II of class II are commonly isolated from apparently healthy domestic poultry and wild birds ,23,24,25c strain . The F cc strain . The seqc strain ,31. AnalThe European Union stated in their Commission Decision 93/152/EEC that for routine ND vaccination programs the viruses used as live NDV vaccines were to be tested under specific conditions and have an ICPI of less than 0.4 or 0.5, depending on the dose of vaccine given. Similarly, the OIE Biological Standards Commission recommended in 2000 that principal vaccines should have an ICPI <0.7. OIE proclaimed that the first principle to consider when selecting a strain for a live NDV vaccine is to determine its pathogenicity. In the present study, the biological pathogenicity assessment of the origin of chicken-adapted NDV isolates of duck (PSD44C) and parrot (PSP91C) showed that both the isolates were lentogenic with MDT more than 90 h and ICPI ranging from 0.2 to 0.4. Earlier, pathogenicity assay-like MDT in eggs and the intravenous pathogenicity test (IVPI) have been used , but the25.73 ± 0.18) and commercial LaSota vaccine strain (Log25.86 ± 0.19) elicited a significantly (p < 0.0001) higher antibody response than the chicken-adapted PSP91C (Log23.45 ± 0.11) in chicken. The OIE defines a “protective” antibody titer as Log2 4 or more against 4HAU of antigen. Thus, PSD44C might be a better thermostable immunogen in comparison to PSP91C and LaSota vaccine strain.Vaccination is one of the effective measures to curb the economic menace that is ND. Despite immunization regimens against ND using live vaccines or both live and inactivated vaccines , NDV conIn the present study, the thermostability of the chicken-adapted NDV isolates from duck (PSD44C) and parrot (PSP91C) was evaluated at 40 °C and 56 °C, and compared with commercially available LaSota vaccine strain on the basis of cumulative infectious titer drop and HA activity. Our results showed that PSD44C was the most thermostable as compared to PSP91C and LaSota vaccine strain. Further, the immunogenicity of PSD44C and LaSota was significantly higher than PSP91C. Therefore, it is concluded that NDV isolate PSD44C is more thermostable and immunogenic when administered intraocularly without any adverse effects and is suitable for further research and vaccine development.


Notably, LRRC8A is upregulated by NSUN2-mediated m5C modification. m5C modified-LRRC8A mRNA is bound by the RNA binding protein YBX1 followed by the increased RNA stability. Moreover, loss of NSUN2 suppresses the proliferation and metastasis of CC cells, and NSUN2 expression is positively correlated with LRRC8A expression in CC. Altogether, our study demonstrates that the NSUN2-m5C-LRRC8A axis is crucial and would be a potential therapeutic target for CC.Cervical cancer (CC) is one of the most common gynecological malignancies with poor prognosis for advanced CC patients. LRRC8A is a volume-regulated anion channel protein involved in cellular homeostasis, but its role in CC remains largely unknown. In this study, we found that LRRC8A is elevated in CC and associated with poor prognosis. LRRC8A maintains cell survivals under the hypotonic condition, and promotes tumorigenesis through apoptosis suppression  Cervical cancer (CC) is one of the most common gynecological malignancies throughout the world and has emerged as a prominent public health focus. According to the statistics in 2020, there are over 600,000 new cases and 340,000 deaths globally each year Maintenance of a relatively constant cell volume is the critical cell function to keep cellular homeostasis. Cell proliferation and apoptosis are associated with normotonic alterations of cell volume 5C) RNA modification, as an abundant modification, plays an important role in RNA metabolic processes, such as RNA stability, RNA processing, RNA export, and mRNA translation 5C modification has been linked to various diseases including cancer 5C deposited in 3' UTR of heparin binding growth factor (HDGF) mRNA enhances its stability, and therefore exhibits the essential oncogenic role 5C-modified GRB2 mRNA, promotes its expression, and ultimately activates PI3K/AKT and ERK/MAPK oncogenic signaling pathways in esophageal squamous cell carcinoma 5C modification exists in 94% of mRNA in HeLa cells, indicating that the regulation of RNA fate by m5C modification may be an important mechanism leading to the occurrence and development of cervical cancer. However, whether or not the m5C modification affects tumorigenesis by regulating cell homeostasis is little known.Recently, the association between abnormal RNA modifications and tumorigenesis has attracted widespread attention 5C modification and upregulated in CC cells. Higher expression of LRRC8A predicts poor prognosis for CC patients. Depletion of LRRC8A induces apoptosis under the hypotonic condition and suppresses tumorigenesis of CC cells. The m5C metyltansferasse NSUN2 mediates m5C modification of LRRC8A mRNA and increases its stability. Our study demonstrates that the NSUN2-m5C-LRRC8A axis is crucial for tumorigenesis of CC.Our results revealed that LRRC8A mRNA is subjected to m2 in a humidified atmosphere. Cells where indicated were treated with the AKT phosphorylation agonist SC79 .All cervical squamous cell carcinoma and normal cervical tissue specimens were obtained from patients undergoing surgery in Daping Hospital of the Army Military Medical University from 2020-03-01 to 2020-05-30. All subjects were informed the consent, and all these specimens were pathologically verified and reviewed by the institutional board of the Third Affiliated Hospital of Chongqing Medical University (2022033). HEK293T and HeLa cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) high glucose medium , and SiHa cells were cultured in minimum Eagle's medium (MEM) . Medium was supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin-streptomycin , and cells were incubated at 37°C with 5% CO. For LRRC8A overexpression, full-length LRR8CA coding sequence was cloned into pCDNA3.1 vector (Youbio). Overexpression of NSUN2 was conducted by the full-length NSUN2 coding sequence cloned into pCDH vector (Addgene). NSUN2-WT and NSUN2-MUT  expression plasmids were constructed by using the pEnter vector (Addgene), and the empty vector was used as the negative control.For LRRC8A and NSUN2 knockdown, all shRNAs including shCtrl (scramble), sh1 and sh2 were cloned into pLKO.1 backbone with puromycin selection. The related sequences of shRNAs were shown in the supplementary tablePlasmid transfection was conducted by using jetPRIME transfection . After 24 hours or later, the levels of mRNA and protein were measured. Lentiviral vectors were transfected into HEK293T cells with packaging vectors psPAX2  and pMD2.G  by using jetPRIME transfection . Infectious lentivirus particles were harvested at 48 hours after transfection.Trizol reagent  was used to extract total RNA from HeLa cells and SiHa cells. For mRNA expression analysis, qRT-PCR was performed by using SYBR Green Master Mix (Vazyme) on QuantStudioDx instrument (Life Technologies). A typical cycling condition included 95°C for 5 min followed by 40 cycles at 95°C for 10 s, 60°C for 30 s. GAPDH was used as the internal standard control. Each sample runs in triplicate. Primers used in qRT-PCR were listed in the supplementary table.HeLa and SiHa cells were treated with lysis buffer containing protease inhibitors  for western blot and IP . Cells were lysed on ice for 30 min, and the lysate was obtained by centrifugation at 12,000 rpm for 15 min. The concentration of proteins was measured by using a bicinchonininc acid BCA protein assay kit . Then the proteins were resolved by 10% SDS-PAGE and transferred on PVDF membranes . The membrane was blocked with 5% non-fat milk in TBS/Tween-20 for 1 hour at room temperature. After blocking, the membranes were blotted with primary antibodies against LRRC8A , NSUN2 , Flag-tag , YBX1 , Caspase-3 , p-AKT , AKT , p-PI3K , PI3K , or GAPDH  overnight at 4°C. After being washed with TBS/Tween-20, the PVDF membranes were incubated with chemiluminescent secondary antibody .After dewaxing the paraffin sections of tissues, heat induced antigen retrieval was conducted. The slides were blocked with goat serum  at 37°C for 30 min, and then incubated with primary antibodies against LRRC8A , NSUN2 , Caspase-3 , Ki67  at 4°C overnight. On the next day, the slides were incubated with the biotinylated secondary antibody at room temperature for 45 min, and then subjected to the DAB kit . The sections were stained with hematoxylin, decolorized with hydrochloric acid and ethanol, dehydrated and then mounted. The comprehensive score of immunostaining was evaluated independently by two professional pathologists.For cell proliferation assay, 1500 HeLa or 2500 SiHa cells were planted in 96-well plates with 200 μl fresh medium. Cell viabilities were measured after adding Cell Counting Kit-8  for 2 hours. The stably transfected cells were plated into 96-well plates and incubated at 37°C for 6 hours until adherence and were referred to as 0 hour. Cell numbers were counted on 24, 48, 72, 96 hours. For the colony formation assay, transfected cells were seeded into 6-well plates. HeLa cells were plated with 1500 cells per well, and SiHa cells were plated with 2500 cells per well with 2 ml fresh medium and grown for 2 weeks. The colonies were fixed with 4% paraformaldehyde. After stained with crystal violet, the visible colonies were counted.HeLa or SiHa cells were incubated with Annexin V and PI  according to the instructions given by the manufacturer. The stained cells were subjected to flow cytometry. BD Accuri C6 flow cytometer  was used to analyze apoptosis and the percentage of Annexin V+ PI- and Annexin V+ PI+ cells was calculated as the apoptosis rate.4; SiHa, 8 × 104) and 250 μl serum-free medium were all added into the upper chamber of a transwell coated with or without Matrigel . The lower chamber was added with 650 μl cell culture medium containing 10% FBS. After incubation for 24 hours, the chambers were collected and stained by 0.5% crystal violet. The cells passing through the membrane were counted in three random fields.Migration and invasion assays were performed by using transwell chambers . Stably transfected cells  were injected subcutaneously into the thigh of BALB/c nude mice . The length and width of the subcutaneous tumor was measured every 3 or 4 days. After injection for 22 or 24 days, the mice were euthanized, and the tumor weight was measured. The tumors were isolated and fixed in 4% formalin for IHC analysis. Animal studies were approved by the Ethics Committee of the Third Affiliated Hospital of Chongqing Medical University (2022033).Stably transfected HeLa cells . After cross-linked by ultraviolet and blocked by PBST+5% milk for 1 hour, the membranes were incubated with the primary antibody against m5C  overnight at 4°C. On the next day, the membrane was washed 3 times with PBST, and incubated with the secondary anti-rabbit antibody for 1 hour. Then the membrane was washed again with PBST. Finally, we used the chemiluminescence system to visualize the membrane. RNA levels were normalized with methylene blue staining to ensure consistency.mRNA was purified by mRNA Isolation System IV kit . The mTotal RNA was isolated using TRIzol Reagent . cDNA libraries were constructed by using NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs). All samples were sequenced on illumina Nova platform. Using STAR version 2.7.8a, all the clean reads were aligned to homo sapiens GENCODE GRCh38.v35 and counted the reads by featureCounts version 2.0.3. R package DESeq2 was used for differential expressed genes identification.7 HeLa cells or 4 × 107 SiHa were lysed in RIP lysis buffer . After incubation for 30 minutes on the ice, the lysate was harvested by centrifugation at 12,000 rpm for 10 min. The specific antibody (3 μg) or control IgG (3 μg) was incubated with the supernatant lysate overnight at 4°C. Next day, Dynabeads Protein A beads  were added into the lysate and incubated at 4°C for 3 hours. After washed three times with RIP lysis buffer, the co-precipitated RNA was extracted with Trizol Reagent . RNA was isolated and RT-qPCR was performed as described previously.About 2 × 105C antibody (3 μg/ml), Dynabeads Protein A beads  were added and incubated at 4°C for 3 hours. After washes, RNA was isolated, and RT-qPCR was performed as described previously.Total RNA was extracted by Trizol reagent  and then treated with RNase-free DNase set. After immunoprecipitated with anti-mTo test RNA stability, transfected HeLa or SiHa cells were seeded into 12-well plates. 5 µg/ml of actinomycin D  was added into each well and incubated for 0, 2, 4, 6 hours. Total RNA was extracted at the indicated time, and mRNA level was analyzed by qRT-PCR.All continuous data were shown as the mean ± SD and the significance of differences was evaluated by two tailed t-tests between two groups. It is considered statistical significant when p < 0.05, and in all cases, the p values were remarked as follows: ***P < 0.001, **P < 0.01, *P < 0.05, but statistically insignificant when P > 0.05. The growth rates difference was determined by ANOVA with repeated measures analysis of variances. The correlation between LRRC8A and NSUN2 expression in cervical cancer was analyzed by Spearman rank correlation analysis. All statistical analyses were performed using Graphpad Prism 8.We firstly analyzed the mRNA levels of LRRC8 subunits (LRRC8A-E) in vivo. Compared with control group, xenograft tumors obtained from mice which burdened cells with LRRC8A knockdown were smaller and lighter  by introducing point mutations in releasing and catalytic sites (cysteine 271 and 321)  and an m21) Fig. A. Then, 21) Fig. B. NSUN2-21) Fig. C-E. The 21) Fig. A-D. Furt21) Fig. E. NSUN2-C8A Fig. I. RIP asC8A Fig. J, I. Tak5C-LRRC8A axis is crucial for cervical tumorigenesis.To further confirm the synergetic carcinogenic effect of LRRC8A with NSUN2 in CC, LRRC8A was re-expressed in NSUN2 knockdown cells Fig. A, and thin vitro and in vivo experiments, we confirmed that LRRC8A depletion significantly inhibited the proliferation, migration and invasion of CC cells and promoted apoptosis.LRRC8A was identified as an essential component of VRAC which plays a pivotal role in cell volume regulation in vitro and in vivo3- cotransporter NBCn1 (Slc4a7) is increased in ErbB2-induced breast cancer tissue compared to normal, and disrupting NBCn1 expression delays ErbB2-induced breast cancer development Our results demonstrated that LRRC8A promotes tumorigenesis in CC. Consistently, LRRRC8A was demonstrated to be related with growth and metastasis of hepatocellular carcinoma and gastric cancer and prognosis in colon cancer 5C sites. RNA m5C modification is a widespread post-transcriptional RNA modification and participates in various cellular and physiological processes. NSUN2 is the main enzyme catalyzing m5C formation and plays an important role in the regulation of RNA fate, such as RNA stability, RNA processing, RNA export, and mRNA translation 5C modification, we established RNA-seq in NSUN2 knockdown CC cells and found that LRRC8A was downregulated upon NSUN2 knockdown. Correlation analysis showed LRRC8A expression was positively correlated with NSUN2 expression in CC. m5C methyltransferase NSUN6 was also demonstrated to mediate m5C methylation on mRNA 5C level in mRNAs in HeLa cells 5C and NSUN2 depletion could decrease the level of m5C modification on LRRC8A mRNA. Also, RIP-seq in SiHa cells as well as RIP-PCR assays both confirmed that NSUN2 could specifically interact with LRRC8A mRNA. YBX1 is an m5C reader protein, and we found that YBX1 knockdown led to lower mRNA level of LRRC8A in CC cells. Since m5C modification was reported to be associated with regulating the stability of mRNA 5C deposition and upregulates its expression in CC.RNA modification is an important post-transcriptional regulation. By analyzing RNA-BisSeq data of HeLa cells 5C level of mRNA. Thus, we explored the role of NSUN2 in CC cells. Results demonstrated that NSUN2 played a tumor-promoting role in CC. Depletion of NSUN2 significantly inhibited cell growth and metastasis, whereas ectopic expression of LRRC8A in NSUN2-depleted cells could largely rescue the tumor-suppressive effect of NSUN2 knockdown. Considering that HPV infection is an important causative factor of CC, we test whether NSUN2-LRRC8A regulation was associated with HPV infection. Results showed that the mRNA levels of NSUN2 and LRRC8A had no statistical difference in HPV+ CC compared with HPV- CC tissues according to analyzed two GEO datasets (5C modification and HPV-infection needs more investigations.Notably, gene ontology analysis showed that differentially expressed genes in NSUN2-loss cells significantly enriched PI3K-Akt signaling pathway. PI3K/AKT pathway plays vital roles in various cellular processes and is involved in many diseases such as malignant tumors datasets C. Interein vitro and in vivo. The m5C modification enhances LRRC8A mRNA stability and upregulates its expression followed by PI3K/AKT signaling pathway activation in CC (Fig. 5C-LRRC8A axis in tumorigenesis of CC, and it might be a potential target for CC treatment.In conclusion, elevated expression of LRRC8A in cervical cancer predicts a poor prognosis and promotes tumorigenesis Supplementary figures and table.Click here for additional data file.


Ovis aries (Oa)OX40L or OaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. While OaOX40L administration mainly increased TNF-α and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-γ producing cells, we detected greater effects on adaptive immunity after OaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-γ production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+ T cells (CD8+ CD62L- CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+ CD62L- CD27+). Moreover, recall anti-OVA CD8+ T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+ T cell effector differentiation and long-term activity. In this work we characterized the in vivo adjuvant potential on the humoral and cellular immune response of OaOX40L and OaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant.The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressing  The ultimate purpose of vaccination is to generate potent and long-lasting protection against diseases. Traditional vaccination approaches were often considered to be successful in controlling pathogens based on their capacity to induce neutralizing antibodies. There is nonetheless now increasing evidence that activation of T cells is a requisite to achieve long-term success for any vaccine. Ideally, T cell activation with viral antigens should promote a robust expansion of naïve precursor T cell, their differentiation to effector cells, and the survival over time of these virus-specific effector cells as high frequency memory T cells so that optimal surveillance against subsequent infections is achieved. T cells, in addition to carrying out effector mechanisms such as cytotoxicity, modulate the immune response through helper mechanisms that aim to eradicate the pathogen. Optimal T cell activation is a complex process that involves antigen recognition on MHC molecules and secondary costimulatory signals provided by different molecules (ligands) that drive the activation and survival of the antigen-specific T cell . Thus, tReceptors and ligands of the tumor necrosis factor (TNF) family are key in controlling co-stimulation of many types of immune reactions regulated by T cell help , like me+ T cells (via the phosphatydil-inositol-3 kinase (PI3K) pathways .OX40L is expressed on the surface of antigen-presenting cells , such as T cells , B cells T cells , nature pathways . The OX4The canonical and the alternative NF-κB pathways are also activated by the interaction of another pair of TNF superfamily molecules , CD70/CDThe use of these TNF/TNFR as adjuvants in classical vaccination has been proposed due to their involvement in immune responses against viral infections . In a mu+ and CD8+ T cells and on a subset of CD335+ NK cells, while being present at low levels on circulating B cells (Ovis aries (Oa)] OX40L and CD70 soluble molecules, respectively, that allow the activation of these costimulatory signaling routes of TNFR superfamily in sheep  and HEK293 cells (ATCC CRL-1573) were grown in Dulbecco’ minimal essential medium , supplemented with 10% Foetal Bovine Serum (FBS) , 2 mM L-glutamine, 1%100x non-essential amino-acids (AANE), 1 mM sodium pyruvate and 100 U/mL Penicillin/100 μg/mL Streptomycin . Final stocks of the recombinant adenoviruses AdOX40L, AdCD70 and AdDsRed were purified and titrated after sequential rounds of growth on HEK293 cells using standard protocols as described before  and storThe procedure to obtain recombinant adenoviruses has been described in detail before .Rubia del Molar” breed sheep  were inoculated intramuscularly with 108 infectious units (IU) of recombinant adenovirus expressing OaOX40L or OaCD70, or the control adenoviral vector AdDsRed, respectively, at the time of immunization with 20 mg/sheep OVA and bled at days 0, 15, 30 and 90 post-inoculation . Recall responses to OVA were assessed by inoculating the same antigen amount three months later (3MD0), and blood samples obtained at days 7 and 30 after the second OVA inoculation (3MD7 and 3MD30). PBMCs and serum samples were obtained for analysis of the adaptive immunity induced to OVA and obtained as described in  coated for 1 h at room temperature with 100 μL/well of 20 μg/mL OVA  in 0.1 M carbonate buffer (pH 9.6). Serial dilutions of sera from inoculated sheep were added to the plates and incubated for 1 h at room temperature, after blocking with blocking buffer  Tween-20 and 4% (wt/vol) skimmed milk) for 1 h at room temperature and washing five times with 1% (vol/vol) Tween-20 in PBS. The presence of OVA-specific IgG or IgM was detected using a secondary donkey anti-sheep IgG conjugated to HRP  or rabbit anti-sheep IgM also conjugated to HRP . After washing 10 times with 1% (vol/vol) Tween-20 in PBS, signal was developed using TMB Liquid Substrate System  and the reaction was stopped with 3 M sulfuric acid before reading. OD was determined at 450 nm on a FLUOstar Omega  ELISA plate reader. All IgG/IgM measurements were made in triplicate, and assays were only considered valid when SDs were below 10% of the average. IgG/IgM binding to OVA was considered positive only when the OD obtained was at least twice the OD obtained with the preimmune serum from the same sheep.6 cells/mL, and 1:2 dilutions were performed in RPMI medium  inactivated FBS) down to a concentration of 1x105 cells/mL; 100 μL/well of each cell suspension (in triplicate) was incubated overnight at 37°C, 5% (vol/vol) CO2. After discarding the cells and washing with PBS, membranes were incubated with anti-sheep IgG conjugated to HRP  for 3 h at room temperature. After five washes in PBS, membranes were developed using 3,3′,5,5′- tetramethylbemzidine (TMB) substrate . Once spots were formed, membrane were washed with abundant distilled water and allowed to dry in the dark. Results were expressed as the number of antigen-specific cells (ASCs) per 106 PBMCs.MSIPS4510 plates  were activated using sterile 35% (vol/vol) ethanol for 1 min, and after thorough washing with sterile water, incubated overnight at 4°C with 20 μg/mL OVA  in 0.1 M carbonate buffer, pH 9.6. Plates were blocked with PBS containing 4% (wt/vol) dried skimmed milk for 2 h at 37°C. Fresh PBMCs were suspended at 5x105 cells per well and incubated with 20μg/mL OVA, PBMC medium as negative control or Concanavalin- A (Con-A) (1.25 μg/ml) as positive control for 48 h at 37°C, 5% CO2. After discarding the cells and washing with PBS, membranes were incubated with biotin-labelled anti-bovine IFN-γ antibody  diluted at 0.25 μg/ml in PBS +0.5% FCS for 2 h. After 5 washes in PBS, membranes were incubated for 1 h with streptavidin conjugated to alkaline phosphatase  diluted 1:10,000 in PBS+ 0.5% FBS. Membranes were washed thoroughly first in PBS and then in distilled water before ELISPOT assay reactions were developed using Sigma FAST BCIP/NBT . Once spots were formed, membranes were washed with abundant distilled water and allowed to air dry in the dark. All cultures were performed in triplicates and ELISPOT assays were considered valid only when average spot counts were below 25 for control cultures and standard deviations in positive wells below 15% of the average counts.Ovine IFN-γ ELISPOT assays were performed using MSIPS4510 plates . Membranes were activated using sterile 35% ethanol for 1 min, and after thorough washing with sterile water, incubated overnight at 4°C with 5 μg/ml anti-bovine IFN-γ antibody . Plates were blocked in RPMI  for 2 h at room temperature. Sheep PBMCs were then plated at a density of 2–3x10+ cells within the CD4+ or CD8+ T cell compartment was measured and data presented as IFN-γ production above the background of unstimulated cells (net IFN-γ). Gating strategy for IFN-γ+ events is detailed in To characterize PBMC populations during the course of the experiment, the following antibodies were used: anti-ovine CD4 (clone 44.38), CD8 (clone 38.65), and WC1 (clone 19.19) ; anti-human CD14 (clone TÜK4), CD16 (clone KD1)  and CD27 (clone LG3.A10) ; and anti-bovine B cell marker (clone BAQ44A)  and CD62L (clone CC32) . Cells were labeled as described in . To asse-ΔΔCT method  and cDNA generated by reverse transcription using oligo dT primers with SuperScript III reverse transcriptase . Transcription levels of TNF-α, IL-12, IL-10, IL-1β, IL-4, IL-6, and IL-2 were evaluated by real-time PCRs performed in a Light Cycler 480 System instrument  in a Light Cycler 480 SYBR Green I Master Reagents  using primers and RT-qPCR conditions detailed in T method . A meltiData handling and statistical analyses was performed using Prism 6.0 software . Statistical tests used to compare data are indicated in the figure legends.+, CD8+, WC1+, B cell marker+, CD14+ and CD14- CD16+ (NK cells) cells in PBMCs obtained from the three inoculated sheep groups over the course of the experiment. AdOX40L and AdCD70 administration does not trigger unspecific PBMC expansion in CD4+, CD8+, WC1+ or B cell marker+ populations (+ cells (monocytes) (+CD14- cells (putatively NK cells) in the AdCD70 group 30 days after antigen exposure at D30 and 3MD30  were inoculated and bled as described in M&M with human recombinant adenovirus (Ad) expressing either of these two molecules (AdOX40L or AdCD70) or none (AdDsRed) concomitantly with OVA antigen. OVA-specific IgGs production was measured in the sera of inoculated sheep at days 0 (D0), 15 (D15), 30 (D30) and 90 (3MD0) post first inoculation (pi) and at days 7 (3MD7) and 30 (3MD30) post OVA booster (pb). Anti-OVA IgGs were detected after the first inoculation at D15 and D30 in all groups with higher values in the costimulatory inoculated groups than in the control group with statistically significant differences between the OaCD70 costimulated group and control sheep at D30  for 106 PBMC=43,92; blue OaCD70: mean number of IFN-γ SFC/106 PBMC=73,57) than in the control group . Interestingly, the AdCD70 inoculated group showed the highest numbers of IFN-γ producing T-cells on days D15, 3MD7 with statistically significant differences as compared to the control group at day 3MD30  and at day 7 post-booster (3MD7)  Figure 5+ and CD8+ T cells over time. To this end, the expression of the activation marker CD62L (which is lost upon activation) and the activation/memory marker CD27  was analyzed in CD4+ and CD8+ T cells obtained from OVA immunized sheep at day 0, 15, 30, 3MD0 (pre-booster with OVA), 3MD7 (7 days post booster), and 3MD30 (30 days post booster) for each group. A significant reduction in CD8+ CD62L- CD27+ cells was detected in the OaCD70 group at day 15 when compared to day 0 (+ CD62L- CD27- cells (+ T cells (CD8+ CD62L- CD27-) which results in decreased number of early activated cells (CD8+ CD62L- CD27+). No significant differences were detected in activated CD4+ cells, nor in CD8+ CD62L+ cells  with the exception of IL-2, that slightly increased from D15 to day 3MD7 in all groups. This increase in IL-2 in the OaCD70 group could be linked to the increased anti-OVA T cell activity detected at this timepoint  was detected 30 days after the combined OaOX40L and OVA inoculation and 7 days after OVA booster    at the tne IL-1β , when cone IL-1β . OverallOaCD70 delivery with the immunizing antigen could improve antibody titers to the antigen and prolong the presence of antibodies in the periphery. Moreover, OaCD70 improved CD8+ T cell recall responses to the antigen and increased in PBMC the expression of the Th1-polarizing cytokine IL-12. OaCD70 concomitant delivery with the immunizing antigen thus appears to improve cellular and humoral immunity. To develop effective vaccines against pathogens a combination of immunoglobulins and long-lasting memory CD8+ T cells should be generated. Receptors and ligands of the TNF family play important roles in controlling lymphocyte activation and survival during viral immunity. Therefore, activation of these costimulatory pathways could prove attractive in veterinary medicine. The present work furthers our understanding of the OX40-OX40L and CD27-CD70 pairings in sheep. Activation of these costimulatory pathways using a recombinant adenovirus as delivery system did not produce acute toxicity in vivo and improved to some extent adaptive immunity to the model antigen OVA. Further work is nonetheless required to dissect the effects of these molecules in sheep. Ultimately the use of costimulatory signals provided by these TNF-TNFR could help improve vaccination protocols.Our data indicate that The original contributions presented in the study are included in the article/The animal study was reviewed and approved by with the recommendations in the guidelines of the Code for Methods and Welfare Considerations in Behavioural Research with Animals  and all efforts were made to minimize suffering. Experiments were approved by the Committee on the Ethics of Animal Experiments (CEEA) (Permit number: 10/142792.9/12) of the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and the Comisión de Ética Estatal de Bienestar Animal (Permit numbers: CBS2012/06 and PROEX 228/14). Written informed consent was obtained from the owners for the participation of their animals in this study.JR, NS, and VM designed the experiments. VM directed the work. CM, JR, VM, and SM collected the samples at the experimental farm. DR-M, MA, AL-L, JR, and VM performed the experiments. NS, JR, and VM wrote the manuscript. All authors contributed to the article, read and approved the submitted version.This work was funded by AGL2015-64290R, RTI2018-094616-B-100 and PID2021-124872OB-I00 from the Ministerio de Ciencia (Spain), grant S2018/BAA-4370-PLATESA2 from Comunidad de Madrid .The authors wish to thank Dr. Alejo for helpful discussions and all the members of the Experimental Farm “La Chimenea” (IMIDRA) for their invaluable help with the animals.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.


Dear Editor,The intestinal epithelium is a rapidly self-renewing tissue for absorbing nutrients and providing barrier functions, and its homeostasis is orchestrated by several signaling pathways  DDB1fl/fl;Villin-CreERT2 mice, and five-day tamoxifen (TAM) administration led to the complete ablation of DDB1 throughout the intestinal epithelium , the small intestine of KO mice showed hemorrhage with shortened length, while the length of large intestine was unaltered  mice were used to label intestinal stem cells (ISCs) and treated with TAM as above. At day 4, the number of Lgr5+ crypts in the KO small intestine was reduced dramatically  induced knockout in vitro   were isolated and subjected to RNA-sequencing. The upregulated genes induced by ing Fig. . Indeed,ing Fig. . ConsistNA Fig. SA, indicaNA Fig. SB and S6C2) Fig. SB and S7CIn summary, using the genetic mouse models and organoids, we demonstrate that DDB1 plays a critical role in the fast homeostatic renewal of the intestinal epithelium, which is achieved by reducing CDK inhibitor expression, preventing cell cycle arrest in the G1 phase and thus ensuring normal cell proliferation.Additional file 1: Supplementary Methods. Figure S1.DDB1 deficiency leads to mouse lethality with shortened small intestine. Figure S2.DDB1 deletion causes reduced crypts in the small intestine. Figure S3.DDB1 deficiency impairs cell proliferation and enhances cell death in the intestine. Figure S4. Ablation of DDB1 reduces Lgr5+ ISCs. Figure S5. Decrease of goblet cells and enteroendocrine cells in the small intestine upon DDB1 deletion. Figure S6. Inhibition of p21 by UC2288 partially rescues the phenotypes caused by DDB1 deletion in organoids. Figure S7.DDB1 deletion inhibits cell proliferation induced by ALK3 KO. Table S1. Differentially expressed genes of small intestinal crypts after DDB1 deletion at day 2.


Brassica napus anthers exposed to high temperature for 5, 15, and 30 min to decipher the rapid transcriptional reprogramming associated with the unfolded protein response. Functional classification of the upregulated transcripts highlighted rapid activation of the ER-UPR signaling pathway mediated by ER membrane-anchored transcription factor within 5 min of heat stress exposure. KEGG pathway enrichment analysis also identified “Protein processing in ER” as the most significantly enriched pathway, indicating that the unfolded protein response (UPR) is an immediate heat stress-responsive pathway during B. napus anther development. Five minutes of heat stress also led to robust induction of the cytosolic HSF-HSP heat response network. Our results present a perspective of the rapid and massive transcriptional reprogramming during heat stress in pollen development and highlight the need for investigating the nature and function of very early stress-responsive networks in plant cells. Research focusing on very early molecular responses of plant cells to external stresses has the potential to reveal new stress-responsive gene networks that can be explored further for developing climate change resilient crops.Climate change associated increases in the frequency and intensity of extreme temperature events negatively impact agricultural productivity and global food security. During the reproductive phase of a plant’s life cycle, such high temperatures hinder pollen development, preventing fertilization, and seed formation. At the molecular level, heat stress-induced accumulation of misfolded proteins activates a signaling pathway called unfolded protein response (UPR) in the endoplasmic reticulum (ER) and the cytoplasm to enhance the protein folding capacity of the cell. Here, we report transcriptional responses of  Extreme temperature events associated with climate change manifesting as heatwaves significantly threaten crop productivity and global food security . In partHeat stress disturbs cellular protein-folding machinery causing an accumulation of misfolded/unfolded proteins, which can be toxic to cells. The stress-induced disturbance in protein homeostasis leads to the activation of protein quality control pathways, especially in the cytoplasm  and endoplasmic reticulum  . A classbZIP60  and anthers containing uninucleate micropores (referred to as “A2” hereafter)  either consists of anthers containing microspore mother cells (A1) from 20 to 25 buds of size > 0.7 mm from ten plants or anthers containing uninucleate microspores (A2) from 10 to 15 buds of size 1–1.5 mm from ten plants. Differential expression analysis identified rapid, extensive transcriptional reprogramming within 5 min of heat stress with 5,901 and 10,692 genes identified as differentially expressed  in A1 and A2, respectively   were colectively . Functioectively , pointinBrassica napus  is an allotetraploid species resulting from the hybridization of B. rapa  and B. oleracea  genomes approximately 7,500 years ago  across the contrasts . Further experimental validation is required to establish their functional roles in B. napus.The homologs were further categorized based on their significant differential regulation  transcripts indicates that the bZIP28 arm of the UPR signaling was activated within 5 min in response to heat stress exposure. Compared to bZIP28, bZIP17 homologs showed slight upregulation at 30 min in A1 and A2 . Thus, the heat stress responsiveness of UPR signaling genes is dependent on the duration and intensity of heat stress and varies across different plant cell types.Upregulation of 1 and A2 . Similarexposure . In contt stress . In Arabesponder . In anotB. napus anthers, the two BiP2 homologs  were upregulated gradually at all-time points. In contrast, BiP1 homologs responded after 15 min of heat stress exposure . Interestingly, in microspore mother cells containing anthers A1, BiP3 homologs  were not expressed under control or heat-stressed conditions. BiP3 homologs were expressed in haploid microspore containing anthers A2. However, their expression was downregulated upon exposure to heat stress. This observation in B. napus heat-stressed anthers contradicts the data reported in heat-stressed Arabidopsis reproductive tissues. Heat stress treatment (37°C for 3 h) of Arabidopsis reproductive tissues (flowers from stage 1–9 and stage 10–12) resulted in the upregulation of BiP3  in locally heat-stressed Arabidopsis leaves , which retains it on the ER membrane . In resp of BiP3 . BiP3 als leaves . On the s leaves . The difBiPs under non-stressed conditions was higher in anthers containing microspores. In Arabidopsis, bip1bip2bip3 triple mutation results in pollen abortion due to defective mitosis1 during microsporogenesis  transcripts showed variable differential expression in A1 and A2 heat-stressed B. napus anthers. In the meiotic stage anthers (A1), there was no significant change in the level of transcripts encoding the S1P homologs. In microspore stage anthers (A2), two S1P homologs (BnaA03g07680D and BnaC03g09690D) transcripts were up-regulated after 30 min of heat stress. The other two S1P homologs  were either non-significant or downregulated. Contrary to S1P, in meiosis stage anthers, A1, S2P  transcripts were upregulated after 15 min of heat stress. In the A2 stage, S2P proteases were upregulated even after 5 min of heat stress exposure. The upregulation of bZIP28/17 and the Golgi resident protease S2P transcripts further supports the activation of this arm of UPR signaling as a rapid heat stress response of B. napus. The cytosolic-facing components of bZIP17/bZIP28 are released from the Golgi membrane by S2P, allowing them to be transported into the nucleus to upregulate the expression of stress-responsive genes , CALRETICULIN (CRT) 1/2/3, THERMOSENSITIVE MALE STERILE 1 (TMS1/ERDJ3A), ERDJ3B, HSP70-17, PROTEIN DISULFIDE ISOMERASE-LIKE (PDIL) 1-1/1-2/1-3, P58IPK, STROMAL CELL-DERIVED FACTOR 2-LIKE PROTEIN PRECURSOR (SDF2), SENSITIVE TO SALT1 (SES1), SHEPHARD (SHD/HSP90-7), TUNICAMYCIN INDUCED 1 (TIN1), and UTR1/3 , an essential gene for male gametophytic thermotolerance in Arabidopsis  and Calreticulin (CRT) present in the ER bind transiently to newly synthesized glycoproteins as they pass through the ER. PDILs catalyze disulfide bond formation during the folding process. Additionally, the UDP-glucose transporters, UTR1 and UTR3, transport nucleotide-sugars (UDP-Glu) into the ER lumen required for glycosylation   were strongly upregulated within 5 min of heat stress in both A1 and A2  . CNXI, Cexposure . CNX ande growth . Additio1 and A2 . In Arabelopment .NAC062 and NAC089 encode type-II membrane proteins localized in the plasma membrane and ER membrane, respectively, while NAC103 encodes a soluble protein found in the nucleus. The expression of NAC103 homologous gene BnaC03g50150D was downregulated in A1 and A2 B. napus anthers after 30 min of heat stress   . The traB. napus anthers, NAC062 transcripts  upregulated following 15 min of heat stress  and NAC089 (NAC089c) cytoplasmic NAC domains from the membrane, followed by their translocation to the nucleus to induce the expression of genes involved in protein folding and programmed cell death (PCD), respectively. In heat-stressed A1 and A2 t stress . The cans leaves .B. napus gene homologous to NAC089 (BnaA02g05540D) upregulated within 5 min of heat stress exposure was among the early responders of heat stress in both A1 A2 B. napus anthers (B. napus genes homologous to NAC089 target genes such as WRKY33 (BnaA03g17820D) and BCL-2-ASSOCIATED ATHANOGENE 6  were also significantly upregulated within 5 min of heat stress. They showed further gradual upregulation with heat stress exposure in A1 and A2 B. napus anthers , and mammalian origins  in A1 and A2 anthers. After 30 min of heat stress exposure, the expression levels were over 50 folds higher in heat-stressed vs. non-stressed anthers (Likely Rapid ResponderABX Inhibitor-1 (BAXI-1), a conserved ER-localized suppressor of ER stress-induced PCD, modulates the threshold for cell death activation in response to diverse stressors, including excessive heat , 2009. It stress . Overexp origins . Heat st anthers .GAAP2  and GAAP5  encoding genes in anthers at both anther developmental stages  resident in membranes of Golgi complex confers resistance to a broad range of PCD inducers. Golgi localization of these proteins is unusual as apoptotic regulators are mainly localized in the cytosol, the ER and the mitochondria . In our l stages . GAAP3 wtivation . Our dats leaves  further B. napus has the largest reported HSF gene family (64 HSFs)  . In A1 ae points . Five mi100-fold . HSFA2, s leaves . Furthert stress .B. napus anthers. Most HSPs are significantly upregulated within 5 min of heat stress , a critical reactive oxygen signaling pathway component. In heat-stressed B. napus anthers, A1 and A2, APX2  transcripts were significantly upregulated after 5 min of heat stress exposure  in B. napus A1 and A2 anthers were also drastically upregulated after 5 min of heat stress. More than 100-fold upregulation was observed after 30 min of heat stress exposure  and ROF2 (FKBP65) in A1 and A2 heat-stressed anthers  levels in A2 and A1 were very low (∼2 TPM) but increased drastically upon heat stress exposure , an important cytosolic co-chaperone that mediates the HSP70-HSP90 interaction  .The RNA-Seq datasets presented in this study are deposited at the NCBI Sequence Read Archive (BioProject ID: NL analyzed the sequencing data. All authors contributed to the writing of the manuscript and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


H16 avian influenza viruses mainly circulate in wild migratory gulls worldwide, and the infection risks in poultry and mammals remain largely unknown. In this study, we isolated a novel H16N3 virus from migratory gulls in eastern China in 2021. Genetic analysis indicated that the H16N3 virus originated from the H16 and H13 viruses that circulated in wild birds. This H16N3 virus has not adapted to replicate in chickens, ducks, or mice, although it can be transmitted between inoculated and contacted birds. The circulation of H16Nx viruses in the Northern Hemisphere indicates that we should strengthen active surveillance to monitor their prevalence and evolution in migratory gulls and their introduction into other migratory and domestic waterfowl.IMPORTANCE Migratory wild birds are natural reservoirs of H16 viruses and play a key role in the global prevalence of these viruses. Here, we found that H16 viruses predominantly circulate in migratory gulls and that the gull H16N3 virus cannot replicate efficiently in chickens, ducks, or mice without prior adaptation. These findings contribute to our understanding of the ecology, evolution, and biological properties of H16 viruses and will guide avian influenza surveillance in birds. Avian influenza viruses are classified into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes according to differences in antigenicity and genetic profiles. To date, several important subtypes, mainly H5N1, H5N6, H5N8, H7N7, H7N9, H9N2, H10N3, and H10N8, which originated in wild birds or poultry have caused substantial economic losses to the poultry industry and pose an increasing threat to human health 18. Identi9–Larus ridibundus (125 isolates), Larus glaucescens (114 isolates), Larus argentatus (36 isolates), Larus canus (12 isolates), and others (4 isolates). Sixteen of the 307 isolates were detected in other migratory birds, including mallards, ruddy turnstones, northern pintails, dunlins, and ducks . We . We 9). nd ducks . These sFrom October 2017 to February 2022, we conducted annual avian influenza virus surveillance in wetland of migratory wild bird habitats along the East-Australasian migratory flyway in eastern China. The samples were identified by PCR with specific M and HA  primers. The suspected H5- or H7-positive samples were transferred to an enhanced animal biosafety level 3 (ABSL-3) facility in the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences for further virus identification and isolation, while the remaining suspected positive samples were injected into 10-day-old embryonated chicken eggs to isolate the viruses in an ABSL-2 laboratory at Liaocheng University. One H16N3, four H3N8, one H6N1, and six H13N6 viruses were isolated from 312 fecal dropping samples from migratory gulls in the Yellow River delta wetland on 10 October 2021. We named the virus A/gull/W1359/Shandong/2021(H16N3), sequenced the open reading frame (ORF) of the virus by Sanger sequencing, and characterized its molecular markers (GISAID accession numbers EPI2181372 to EPI2181379). Amino acid sequences of SINER/GLF were observed at the cleavage site in HA, which is typical of viruses with low pathogenicity in poultry. Several amino acid substitutions that contribute to increased virulence or transmissibility of avian influenza viruses were detected in the H16N3 virus, including PB1 207K, PB1 436Y, PA 515T, M1 30D, and M1 215A (2) of 6 to 7 at 24 or 48 h postinfection (hpi) . The chickens and ducks were inoculated intranasally with the virus at 106 50% egg infective doses (EID50) in a volume of 100 μL and were euthanized on day 3 postinfection (p.i.), and tissues from lung, trachea, spleen, liver, kidney, pancreas, intestine, rectum, bursa of Fabricius, and brain were collected to determine viral titers. No virus was detected in any organs or tissues of the infected chickens (2) ranging from 1 to 4 at 10, 15, and 21 days p.i. (2) of 2 and 1 at 10 and 15 days p.i., respectively . The accession numbers of eight gene sequences are EPI2181372 to EPI2181379.The whole-genome sequences of the H16N3 virus reported in this study have been deposited into GISAID (


It is unknown whether other residues of SMO are involved in the esterification between D95 and cholesterol. In this study, we find that the SMO-CRD(27–192) can undergo cholesterylation. In addition to D95 and Y130, the residues critical for cholesterol modification include Y85, T88, T90, W109, W119, K133, E160 and F166. T88, W109, W119 and F166 also seem to be involved in protein folding. Notably, we find that Y85 and K133 form a cation-π interaction whose disruption abolishes cholesterylation and ciliary localization of SMO. This study highlights the mechanism and function of cholesterol modification of SMO.The Hedgehog (Hh) signaling pathway is critical for embryonic development and tissue renewal. The G protein-coupled receptor (GPCR)-like protein Smoothened (SMO) is the central signal transducer in the Hh pathway. Cholesterol binds and then covalently links to the D95 residue of cysteine-rich domain (CRD) of human SMO. The cholesterylation of CRD is critical for SMO activation. SMO cholesterylation is a Ca The Hedgehog (Hh) signaling pathway controls embryonic development and tissue homeostasis –3 . AbeCholesterol plays an essential role in the regulation of SMO. The human SMO protein is composed of a signal peptide (1–26 aa), an extracellular/luminal cysteine-rich domain (CRD) (27–192 aa) and a seven-transmembrane (7TM) domain. The R451 residue in TM6 forms a cation-π interaction with the W535 residue in TM7, thereby locking SMO in an inactive state. This cation-π interaction, however, is broken upon SMO activation. The W535L mutation constitutively activates SMO and causes sporadic basal cell carcinoma2+-boosted autoreaction that requires an intramolecular ester bond formed between the side chains of D95 and Y130, which serves as a high-energy intermediateWe previously showed that SMO is covalently modified by cholesterol through an ester linkage between the 3-beta hydroxyl group of cholesterol and the side chain of D95 in human SMO (D99 in mouse SMO) ,21 . WeTo understand the biochemical mechanism of SMO cholesterylation, we systematically mutated 66 residues in CRD that contain the functional groups in their side chains. We found that the cation-π interaction between the Y85 and K133 residues is essential for SMO cholesterylation and function.4 (C8027), Vc (A7631) and TBTA (678937) were from Sigma. Cholesterol probe (CP) and biotin alkyne were synthesized by Shanghai Bio Bond Pharmaceutical Co., Ltd The mouse mAb anti-SMO clone E5 (sc-166685) was from Santa Cruz . The mouse mAb anti-FLAG  was from Sigma . The rabbit pAb anti-ARL13B (17711-1-AP) was from proteintech . The Alexa Fluor 488 goat anti-mouse secondary antibody (A11001) and Alexa Fluor 555 donkey anti-rabbit secondary antibody (A31572) were from Thermo Scientific . The donkey anti-mouse HRP-conjugated secondary antibody (715-035-150) was from Jackson ImmunoResearch . CuSO2.HEK293T and NIH3T3 cells  were cultured in DMEM containing 10% FBS , 100 units/mL penicillin, and 100 μg/mL streptomycin sulfate at 37°C in 5% COThe open reading frame of human SMO cDNA was amplified from HEK293T cell, and pCMV-SMO-WT was constructed by inserting cDNA into pcDNA3. pCMV-SMO(1–259)-FLAG, pCMV-SMO1–233)-FLAG and pCMV-SMO(1–192)-FLAG were generated by PCR, followed by insertion of a 3×Flag sequence after the truncation of SMO. All mutants of SMO plasmids were constructed by the quick-change method [3-FLAG an4, 2.5 mM Vc, 1 mM TBTA in sequence and incubated at 27°C, 1000 rpm for 1.5 h. After the click chemistry reaction, 30 μL streptavidin agarose beads were added and incubated at 4°C for 6 h to pulldown the SMO-CP-biotin. The resulting pellet beads were washed with RIPA buffer and boiled at 95°C for 10 min then mixed with membrane solubilization buffer for SDS-PAGE [The experiments were performed as previously describedDS-PAGE ,27 .g for 5 min, then washed with PBS to remove the redundant medium, followed by homogenization in the RIPA buffer , which contained protease inhibitors g for 2 min. Samples were then mixed with an equal volume of the membrane solubilization buffer, and incubated at 37°C for 30 min. Protein samples were resolved by SDS-PAGE and transferred to the PVDF membrane. Membranes were blocked with 2% BSA in TBST, incubated with indicated primary and secondary antibodies. Immunoreactivity was detected by using Pierce ECL substrate (Thermo Scientific).Cells were harvested by centrifugation at 1000SMO-KO NIH3T3 cells were washed with PBS, fixed with 4% PFA at room temperature for 30 min, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 2% BSA. Then, cells were stained with the anti-SMO antibody (1:1000) and anti-ARL13B antibody (1:1000) in 2% BSA and then fluorescent secondary antibodies at room temperature for 1 h. Confocal images were obtained by Leica microscope SP8 . Fluorescence quantification showing the localization of SMO mutants on cilia were performed using Fiji softwareFor SMO mutants analysis, SMO-KO NIH3T3 cells were plated in 24-well plates and transfected with the plasmids expressing SMO, GliSBS-firefly luciferase and pEGFP-N1 in a ratio of 2 : 2 : 1 with LTX reagent (Thermo Scientific)The sequence analysis was performed by using the clustal software to align SMO-CRD sequencesP<0.05 were considered statistically significant.Data are presented as mean±SD. Statistical analysis was performed in GraphPad Prism 8 software using one-way ANOVA.The topology of human SMO is shown in–259), SMO(1–233) and SMO(1–192) were all efficiently conjugated by CP (–E). These results are consistent with our previous report showing that SMO cholesterylation is a Ca2+-boosted autoreactionWe confirmed that SMO could be modified by CP B. The trd by CP B–E. TheTo understand the molecular details of SMO cholesterylation, we individually mutated the residues in CRD of human SMO that contain functional groups in the side chains. A total of 66 residues, including 15 acidic ones , 18 basic ones , 9 aromatic ones  and 24 polar ones , were mutated to alanine.Supplementary Figure S1. SMO-CRD contains three tyrosine residues and three lysine residues . Consistently, their residence is far from the cholesterol-binding groove in the CRD  can be conjugated by cholesterol (–E). These results demonstrate that E160 is critical for cholesterol linkage but this effect is not related to R485.We examined the effect of acidic residues in CRD on SMO cholesterylation. The D95 residue is the cholesterol modification site and D95A mutation prevented CP linkage A,B. Besiesterol E, E160A esterol B–E. TheWe analyzed whether any threonine or serine residues in CRD are required for SMO cholesterylation.Based on how alanine substitution affects cholesterylation and protein glycosylation, we divided the 66 residues examined earlier into three categories. Class 1 included the residues whose mutation abolished cholesterylation but not glycosylation appearance of SMO A, in redSmo-deficient NIH3T3 cells. ShhN induced the ciliary localization of SMO(Y85F) and SMO(K133R), both of which could be highly cholesterylated, as that of WT SMO. The Y130A, Y85A, K133A, and E160A mutants, like the D95A mutation, were barely translocated to primary cilium  that harbor functional groups. We analyzed the effects of 66 residues on SMO cholesterylation. Besides D95 and Y130, eight additional residues  were identified to be critical for cholesterol modification. The F166, W109, W119 and T88 residues also affect glycosylation of SMO. Therefore, it is possible that their impairment of cholesterylation is a secondary consequence of protein misfolding. The distance of other residues to D95 is more than 10.8 Å B, suggesAlthough we do not know how mutations of E160 and T90 abolish SMO cholesterylation, our results suggest Y85 and K133 form a cation-π pair that is required for cholesterol conjugation to SMO and ciliary localization of SMO. The cation-π interaction is a major force determining molecule interaction and protein conformation. In the SMO protein, a short α-helical structure proceeding the C295 residue in SMO is stabilized by a cation-π interaction