The lipidome of SARS-CoV-2-infected Caco-2 cells was markedly different from that of mock-infected samples, with most of the changes involving downregulation of ceramides. In COVID-19 patients' plasma samples, a total of 54 lipids belonging to 12 lipid classes that were significantly perturbed compared to non-infected control subjects' plasma samples were identified. Among these 12 lipid classes, ether-linked phosphatidylcholines, ether-linked phosphatidylethanolamines, phosphatidylcholines, and ceramides were the four most perturbed. Pathway analysis revealed that the glycerophospholipid, sphingolipid, and ether lipid metabolisms pathway were the most significantly perturbed host pathways. Phosphatidic acid phosphatases (PAP) were involved in all three pathways and PAP-1 deficiency significantly suppressed SARS-CoV-2 replication. siRNA knockdown of LPIN2 and LPIN3 resulted in significant reduction of SARS-CoV-2 load. In summary, these findings characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.Viruses exploit the host lipid metabolism machinery to achieve efficient replication. We herein characterize the lipids profile reprogramming  Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses that can infect a wide range of human and animal species in vitro and in vivo. Pathway analysis and downstream validation studies identified phosphatidic acid phosphatase (PAP) as essential host factors for efficient SARS-CoV-2 replication and may be a potential treatment target for COVID-19.Virus-targeting antivirals such as monoclonal antibodies and enzyme inhibitors may become ineffective when resistant virus strains emerge The use of clinical specimens was approved by the Institutional Review Board of the University of Hong Kong / Hospital Authority Hong Kong West Cluster. Plasma samples were collected from COVID-19 patients whose respiratory tract specimens tested positive for SARS-CoV-2 and blood donors who tested negative for SARS-CoV-2 by quantitative reverse transcription-polymerase chain reaction (RT-qPCR).2. From day 1 to 3, cells were cultured in StemPro34 SFM (Invitrogen) with 50µg/ml ascorbic acid (AA) (Sigma), 2mM Gluta-MAX (Invitrogen), 10ng/ml BMP4, and 10ng/ml human recombinant activin-A (Invitrogen). From day 4 to 7, 5µM Wnt inhibitor IWR-1, a Wnt inhibitor (Tocris) was added. From day 8 to 14, cells were cultured under normoxia in RPMI 1640 medium (Invitrogen) supplemented with 2mM Gluta-MAX, 1×B-27 supplement (Invitrogen) and 50µg/mL AA. The cells were then dissociated with Accutase and seeded as monolayer in desired culture vessels for 3 days before infections. Vero-E6 (ATCC® CRL-1586™), and human colonic  cells were maintained in Dulbecco's modified eagle medium  culture medium supplemented with 10% heat-inactivated fetal bovine serum , 100U/mL penicillin, and 100μg/ml streptomycin. A549-hACE2-TMPRSS2 cells (Invivogen) were cultured according to the protocol provided by the manufacturer. All cell lines were confirmed to be free of mycoplasma contamination by PlasmoTest (InvivoGen).SARS-CoV-2 wild-type  strain was isolated from respiratory tract specimens of laboratory-confirmed COVID-19 patients in Hong Kong Lipid extraction was performed on the subjects' plasma samples for liquid chromatography-mass spectrometry (LC-MS) analysis as we previously described with slight modifications 2 at 37°C. Archived clinical isolates of SARS-CoV-1 and SARS-CoV-2 isolated from patients were available at the Department of Microbiology, The University of Hong Kong. The viruses were cultured in Caco-2 cells in serum-free DMEM supplemented with 100 U/mL penicillin and 100µg/mL streptomycin. Cell-based lipid extraction and inactivation of virus infectivity were according to a previously described protocol Caco-2 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100U/mL penicillin, and 100µg/mL streptomycin and incubated in 5% COoC and 4oC, respectively. The injection volume was 7µL for negative and 5µL for positive. The applied chromatography gradients and mobile phase composition were described in Table S1. The mass spectrometer was operated in MSE mode and the data were acquired in both positive and negative modes. The capillary voltage was maintained at 2.5kV (positive mode) and 2.0kV (negative mode). Mass spectral data were acquired over the m/z range of 100 to 1500. MS and MS/MS acquisition were operated in the same parameters. Collision energy was applied at the range from 30 to 45eV for obtaining fragmentation pattern that provide identification and structural elucidation of the significant lipids. A total of 14 lipid internal standards were applied for sample preparation and LC-MS analysis for monitoring the lipids coverage and extraction efficiency including of Arachidonic acid-d8, Platelet-activating factor C-16-d4 (PAF C-16-d4), PE (17:0/17:0), PG (17:0/17:0), PC(17:0/0:0), C17 Sphingosine, C17 Ceramide, SM (d18:1/17:0), PC (12:0/13:0), Cholesterol (d7), TG (17:0/17:1/17:0) d5, DG (12:0/12:0/0:0), DG (18:1/2:0/0:0), PE (17:1/0:0) and commercial standards used for lipids identification. They all were purchased from Cayman Chemical  and Avanti Polar Lipids, Inc . Moreover, to check the stability of the LC-MS system during the analytical batch, QC samples were injected at the beginning of the run and after every 6 or 8 samples for monitoring the system variation. QC samples were pooled and prepared by mixing equal aliquots of all the biological samples Untargeted lipidomics analysis was performed as previously described http://www.metaboanalyst.ca) and SIMCA-P V12.0  were used for univariate and multivariate analyses, respectively. Prior to statistical analysis, the data matrix needs to perform QC or DNA-based normalization for better comparison http://mona.fiehnlab.ucdavis.edu/), METLIN database (http://metlin.scripps.edu/), and LIPID MAPS (http://www.lipidmaps.org/). For confirmation of lipid identity using authentic chemical standards, the MS/MS fragmentation patterns of the chemical standards were compared with those of the candidate lipids measured under the same LC-MS condition. MetaboAnalyst and KEGG mapper were used to perform significant lipids pathway analyses All lipidomics data was processed to a usable data matrix by the MS-DIAL software for further analysis 5 cells per well were transfected with a 500µL mixture containing 60nM siRNA, 2µL Lipofectamine RNAiMAX (Thermo Fisher), Opti-MEM (Invitrogen), and antibiotic-free cell culture medium. At 24h after third transfection, the medium was replaced, and the cells were infected with SARS-CoV-2 (MOI=0.10). At 24h or 48h post-infection (hpi), the cells were collected into RLT lysis buffer , followed by total nucleic acid extraction. The viral RNA load was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The values obtained using scrambled siRNA-transfected cells were set as negative controls. For gene overexpression assay, LIPIN3 plasmids  were transfected using Lipofectamine 3000 reagent (Thermo Fisher) into Caco-2 cell in 24-wells plate and collected the cell lysate samples in 48hpi (MOI=0.10), followed by nucleic acid extraction. The viral RNA load was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The values obtained using mock-transfected cells were set as negative controls.Caco-2 cells and A549-hACE2-TMPRSS2 cells in 24-well plates containing about 10Normality check was performed for the lipid features of each study group using Shapiro-Wilk test. False discovery rate (FDR) multiple test correction was performed for univariate analysis Table S2). Principal component analysis (PCA) score plots were used to illustrate the cell lipidome upon viral or mock infection at different time points. The lipidome of SARS-CoV-2-infected samples was markedly different from that of SARS-CoV-1-infected or mock-infected samples at 8hpi  and more similar at 24hpi . Corroboratively, the heatmap demonstrated more differentially perturbed lipids between SARS-CoV-2-infected samples and SARS-CoV-1-infected or mock-infected samples at 8hpi  than at 24hpi . At 8hpi, most of the changes in SARS-CoV-2 infection were ceramides with downregulation, whereas upregulation of multiple lipids, such as cardiolipins (CL) and phosphatidylethanolamines (PE), were observed in SARS-CoV-1 infection . Interestingly, at 8hpi, we showed that around 90% (17/20) of the lipids that were significantly different between SARS-CoV-2 and SARS-CoV-1 were also downregulated ceramides that belong to the sphingolipid metabolism pathway (Table S5). Thus, the suppressed sphingolipid metabolism was a hallmark to distinguish SARS-CoV-1 or SARS-CoV-2 at the early stage of infection. At 24hpi, most of the ether lipids were downregulated in SARS-CoV-2 and SARS-CoV-1 infections. Pathway analysis revealed that glycerophospholipid metabolism , glycerolipid metabolism , sphingolipid metabolism  and ether lipid metabolism  were the four most significantly perturbed pathways after SARS-CoV-2 infection .Lipidomics analysis was performed in human colorectal Caco-2 cells infected with either SARS-CoV-2 or SARS-CoV-1 . There were 20 male and 17 female COVID-19 patients and 24 male and 20 female non-infected control subjects. The mean age was 55.4 and 57.5 years for the COVID-19 patients and non-infected control subjects, respectively. Our analysis detected a total of 622 known lipid features in positive mode and 311 known lipid features in negative mode. The coefficient of variation (CV) of all spike standards in all subjects and the quality controls (QC) samples were lower than 25% and 10%, respectively (Table S6) Table S7).To characterize the lipid profile changes after SARS-CoV-2 infection in human and to validate the pathway perturbations identified in our cell culture model, we conducted LC-MS-based untargeted lipidomics analysis on the plasma samples of 37 COVID-19 patients and 44 non-infected controls  Figure ). Around two-thirds  of the perturbed lipids, including mainly Ether PC, Ether PE, and PCs, were downregulated, and the other one-third  of the perturbed lipids, including five Cers, five PEs, and four SMs, were significantly upregulated .These lipids belonged to 12 lipid classes, including carnitine (Car), ceramides (Cer), ether-linked lysophosphatidylethanolamine (EtherLPE), ether-linked phosphatidylcholine (EtherPC), ether-linked phosphatidylethanolamine (EtherPE), n-acyl-lysophosphatidyl-ethanolamine (LNAPE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamines (PE), phosphatidylinositols (PI), and sphingomyelin (SM) . All 54 significantly perturbed lipids (Table S7) were used to perform ROC curve analysis and the top 10 lipids ranked by area under ROC curve (AUROC) were selected to generate the ROC curve plot . The 10 lipids consisted of 4 Ether PCs, 2 Ether PEs, 2 PCs, 1 Cer, and 1 LNAPE . The ROC curve plot based on the top 10 lipids exhibited excellent discrimination between the plasma samples of COVID-19 patients and healthy controls. Corroborative with our pathway analysis results, these lipids also belonged to the glycerophospholipid, sphingolipid, and ether lipid classes. Collectively, these findings showed that glycerophospholipid, sphingolipid, and ether lipid metabolisms were markedly perturbed in both cell culture model and COVID-19 patients' plasma samples.Next, we conducted pathway analysis which revealed sphingolipid metabolism , glycerophospholipid metabolism , and ether lipid metabolism  to be the three dominant perturbed pathways in COVID-19 patients' plasma samples . Among the three pathways, glycerophospholipid metabolism is the central pathway that links the other two pathways. In the glycerophospholipid pathway, glycerone-P is firstly metabolized to sn-Glycerol 3-phosphate, then further metabolized to lysoPA, and finally converted to PA, DG, and other glycerophospholipids. Importantly, PAP-1/2 is involved in all three pathways. We therefore further investigated the effects of PAP-1/2 on SARS-CoV-2 infection. To determine if PAP-1 and/or PAP-2 affect SARS-CoV-2 replication, siRNA knockdown and gene overexpression assays were applied to SARS-CoV-2-infected Caco-2 cells. No significant change in viral genome copies was observed when the PAP-2-encoding genes , as well as other phosphatidic acid phosphatases-coding genes (PLPP4 and PLPP5) were individually knockdown . Next, we investigated the PAP-1-encoding genes LPIN2 and LPIN3, as LPIN1 is poorly expressed in Caco-2 cells Figure ) despite similar knockdown efficiency . In addition, siRNAs directed against LPIN2 or LPIN3 were introduced into virus-permissive human lung epithelial cells (A549-hACE2-TMPRSS2 cells) with favorable knockdown efficiency to validate the efficacy of PAP-1 inhibition . Similarly, the viral genome copies in the lysates of siLPIN2-treated (P<0.01) or siLPIN3-treated (P<0.01) cells were remarkably decreased, and also higher dependence of SARS-CoV-2 on LPIN3 than LPIN2 . In a single cycle virus growth experiment, siLPIN3 but not siLPIN2 significantly decreased intracellular viral genome copies (P<0.001) . LPIN3-overexpression assay demonstrated no significant effect on SARS-CoV-2 replication . Collectively, these results indicated that the PAP-1 enzyme is important for the efficient replication of SARS-CoV-2.A globally reprogrammed lipids pathway map based on the identified perturbed pathways was constructed (in vitro and in vivo. We identified glycerophospholipid metabolism, sphingolipid metabolism, and ether lipid metabolism to be the dominantly perturbed pathways. Glycerophospholipid metabolism is the central pathway that links the sphingolipid and ether lipid metabolism pathways. Recent studies demonstrated marked differences between the glycerophospholipid composition of purified influenza virions and that of uninfected host cells, which may facilitate viral processing during infection Many viruses including coronaviruses induce lipid metabolism remodeling in host cells to facilitate their own replication PAP-1 has important roles in the mediation of lipid metabolism that affects virus replication. As shown in figure 2+-dependent PAP-1/2 is involved in all three lipid metabolism pathways dominantly perturbed in SARS-CoV-2 infection. Mammalian cells contain two different isotypes of PAP. PAP-1 but not PAP-2 is MgIn summary, our findings identified PAP-1 as a key regulatory enzyme of the glycerophospholipid remodeling in SARS-CoV-2 infection. PAP-1 and/or lipin 3-targeting drug compounds may be a potential host-based treatment strategy for COVID-19.Supplementary figure and tables.Click here for additional data file.


BAT26 is a highly sensitive MSI marker that defines the mismatch repair (MMR) status with high sensitivity and specificity. However, isolated BAT26-only instability is rare and has not been previously reported. Of the 6476 cases tested using pentaplex MSI polymerase chain reaction, we identified two BAT26-only instability cases (0.03%) in this study. The case #1 patient was diagnosed with endometrial adenocarcinoma without MMR germline mutations. The endometrial tumor showed BAT26-only instability, partial loss of MLH1/PMS2 protein expression, and a high programmed cell death ligand 1 (PD-L1) combined positive score (CPS = 8). The tumor exhibited a somatic phosphatase and tensin homolog (PTEN) R303P missense mutation and loss of the PTEN protein. On a comprehensive cancer panel sequencing with ≥500 genes, the tumor showed an MSI score of 11.38% and high tumor mutation burden (TMB) (19.5 mt/mb). The case #2 patient was diagnosed with colorectal carcinoma with proficient MMR and PTEN protein loss without PTEN alteration, as well as a high PD-L1 CPS (CPS = 10). A pathogenic KRAS A146T mutation was detected with an MSI score of 3.36% and high TMB (13 mt/mb). In conclusion, BAT26-only instability is very rare and associated with PTEN protein loss, high TMB, and a high PD-L1 score. Our results suggest that patients with BAT26-only instability may show good responses to immunotherapy.Detecting microsatellite instability (MSI) in advanced cancers is crucial for clinical decision-making, as it helps in identifying patients with differential treatment responses and prognoses.  Microsatellite instability-high (MSI-H) is a well-recognized phenomenon resulting from defective mismatch repair (dMMR) . Low-levBAT26 (a poly(A) tract localized in the fifth intron of the mutS homolog (MSH)-2) [BAT26 is specific and sensitive in identifying MSI-H cases [BAT26 locus, and the BAT26-only MSI test showed 94% sensitivity and 98% specificity for the identification of tumors with MMR deficiency, suggesting BAT26 as the best single marker to identify tumors with MSI-H [BAT26-only instability was identified in two cases (0.03%), which is very rare, and there have been no previous reports of such cases in the literature.The mononucleotide marker (MSH)-2)  is an ex-H cases ,6. Cicekth MSI-H . In our BAT26-only instability cases associated with high tumor mutation burden (TMB), phosphatase and tensin homolog (PTEN) protein loss, and high PD-L1 expression via a comprehensive cancer panel test and immunohistochemistry.Herein, we report two rare BAT26-only instability with a partial loss of MLH1/PMS2 expression and a high PD-L1 combined positive score (CPS = 8). The tumor showed an “immune-excluded” microenvironment (immune cells aggregating at the tumor boundaries). NGS revealed the PTEN R303P alteration, while IHC demonstrated the PTEN protein loss (KRAS (G12A) and AT-rich interaction domain 1A (p.Q1519Pfs*13 and p.D1850Tfs*33) mutations . The endometrial tumor showed ein loss . Cooccurutations . The tuml tumors . The patBAT26-only instability. After the cancer diagnosis, a low anterior resection was performed. A histopathological examination of the surgically resected sample revealed moderately differentiated adenocarcinoma. Pathological staging identified the cancer as stage IIIA with invasion into the muscularis propria (pT2) and regional lymph node metastasis to 3–21 regional lymph nodes (pN1b). The tumor was pMMR with a high PD-L1 CPS score (CPS = 10) and an immune-excluded tumor microenvironment. The tumor showed a loss of the PTEN protein without a PTEN alteration  with teration .KRAS A146T mutations. The MSI score was 3.36%, and the tumor showed high TMB (13 mt/mb). The EPCAM R153T variant from ClinVar was annotated as nonpathogenic but showed an intact EPCAM protein expression. Two variants (Q419K and Q629R) were classified as benign or likely benign within MSH2. Two variants (P18L and G25D) of uncertain clinical significance (VUS) were observed in mutY DNA glycosylase. The mononucleotide MS markers at MYB and lysosomal associated membrane protein 1 loci were unstable  for the data analysis and review of the medical records.Of the 6476 cases between December, 2017 and February, 2020, we identified two cases with The tumor-rich areas were circled by pathologists on hematoxylin and eosin-stained 4-μm-thick slides of formalin-fixed, paraffin-embedded tissues for dissection. The MSI status was determined via multiplex PCR, which amplified five quasi-monomorphic mononucleotide repeat markers , as previously described ,29.IHC staining was performed on paraffin-embedded 3-µm-thick tissue sections for MSH2, mutL homolog 1 (MLH1), MSH6, PMS1 homolog 2 (PMS2), epithelial cell adhesion molecule (EPCAM), programmed cell death ligand 1 (PD-L1), and PTEN ,30. An Ihttp://genome.ucsc.edu/) (accessed on 1 March 2022). Mutation allele frequencies below the predefined thresholds were considered to be wildtypes.Library preparation was performed using the hybrid capture-based Illumina TruSight Oncology 500 , as previously described . Data ou


Increasingly, older adults are receiving hematopoietic cell transplant (HCT) and chimeric antigen receptor T-cell therapy (CAR-T), intensive therapies for treatment of hematologic cancers which typically require prolonged hospital admissions. Older adults are at high risk of increased healthcare utilization and complications of prolonged hospitalization . We identified patients age ≥70 years who received HCT or CAR-T in a primary outpatient transplant program at Vanderbilt University Medical Center between 1/1/19 and 12/31/20. Healthcare utilization, including all visits and admissions, was captured from the start of conditioning chemotherapy through early post-therapy. Thirty-eight patients met inclusion criteria; 26 (68%) received autologous HCT (autoHCT), 7 (18%) allogenic HCT , and 5 (13%) CAR-T. Twenty-four patients (63%) had high HCT-Comorbidity Index (HCT-CI). Eighteen (69%) autoHCT, 6 (86%) alloHCT, and no CAR-T patients had at least one unplanned admission. The median number of total hospital days (LOS) was 7.5 (2-14), 8 (4-62), and 9 (7-9) days, respectively. One-year mortality was 12% (3) in autoHCT, 43% (3) in alloHCT, and 0% in CAR-T. Low performance status and high HCT-CI did not correlate with LOS  or number of outpatient visits . In conclusion, most patients who received auto- or alloHCT in a planned primary outpatient setting experienced at least one unplanned admission. LOS duration varied widely with shorter LOS among autoHCT patients. Further research is needed to identify factors among older adults (≥70 years) at risk of increased healthcare utilization during HCT or CAR-T.


Correction: BMC Pregnancy Childbirth 20, 191 (2020)https://doi.org/10.1186/s12884-020-02878-zFollowing publication of the original article , the folThe first paragraph of the results should read:A total of 162 patients [66 (40.7%) nulliparous and 96 (59.3%) multiparous women] were recruited .Table 3 shows that there was a statistically significant weak positive correlation between cervical length and gestational age at delivery in nulli-parous women.


ATG12 and glutaminolysis through GLS1. In transfection assays, mimics of miR-23b-3p demonstrated reduced gene expression for both ATG12 and GLS1, decreased cell viability, and increased cell apoptosis of sorafenib-resistant HepG2 cells, whereas the antimiRs of miR-23b-3p demonstrated contrasting results. Conclusion: Our study highlights the cytoprotective role of autophagy and glutamine addiction modulated by miR-23b-3p (tumor suppressor), suggesting new approaches to curb sorafenib resistance in HCC.Background: Hepatocellular carcinoma (HCC) is the second most common malignancy with increasing cancer deaths worldwide. HCC is mainly diagnosed at its advanced stage, and treatment with FDA-approved sorafenib, the multikinase inhibitor drug, is advised. Acquired resistance against sorafenib develops through several pathways involving hypoxia, autophagy, high glycolysis, or glutaminolysis. Small non-coding RNAs, similar to microRNAs (miRNAs), are also known to affect sorafenib resistance in HCC. However, there is a lack of information regarding the significance of differentially expressed miRNA (if any) on autophagy and glutamine regulation in sorafenib-resistant HCC. Methods: The expression of autophagy and glutaminolysis genes was checked in both parental and sorafenib resistant HepG2 cell lines by real-time PCR. MTT and Annexin/PI assays were also performed in the presence of inhibitors such as chloroquine (autophagy inhibitor) and BPTES (glutaminolysis inhibitor). Next generation sequencing and in silico analysis were performed to select autophagy and glutamine addiction-specific microRNA. Selected miRNA were transfected into both HepG2 cells to examine its effect on autophagy and glutamine addiction in regulating sorafenib-resistant HCC. Results: Our in vitro study depicted a higher expression of genes encoding autophagy and glutaminolysis in sorafenib-resistant HepG2 cells. Moreover, inhibitors for autophagy (chloroquine) and glutaminolysis (BPTES) showed a diminished level of cell viability and augmentation in cell apoptosis of sorafenib-resistant HepG2 cells. NGS and real-time PCR demonstrated the downregulated expression of miR-23b-3p in sorafenib-resistant cells compared to parental cells. In silico analysis showed that miR-23b-3p specifically targeted autophagy through  Hepatocellular carcinoma (HCC) is the second most common malignancy increasing cancer-associated mortality worldwide ,2. An agThe death-promoting effect of Sorafenib-induced autophagy has been reported by recent studies . AlthougGLS1 (glutaminase) is associated with advanced disease stages , the number of green autophagy dye0stained vesicles significantly (p = 0.0321) increased with a difference of 224 ± 69.42 compared to the presence of glutamine (The green autophagy dye-stained vesicles (markers of autolysosomes and autophagic compartments) were also examined using a CYTO-ID Autophagy detection kit. The increased autophagic flux was directly proportional to the increased number of green autophagy dye-stained vesicles detected through green emitting fluorescence using flow cytometry. The number of green autophagy dye-stained vesicles was significantly  decreased compared to non-treated resistant cells (50) significantly (p = 0.0010) increased cell apoptosis (10.57 ± 1.216-fold higher) of resistant cells than in non-treated HepG2 cells (We performed an MTT assay using BPTES (glutaminase inhibitor), where the cell viability of BPTES-treated sorafenib-resistant cells was significantly  . The miR-23b-3p was also observed to target GLS1  with a target score of 90  . The higre of 90 . Further0  increased with a fold change of 4.59 ± 0.6566  increased with a fold change of 6.13 ± 0.4131  decreased the extracellular expression of miR-23 with a fold change of 0.3027 ± 0.05773 compared to resistant cells without sorafenib treatment (The extracellular expression of miR-23b-3p (miR-23) was evaluated in the presence of increasing concentrations of sorafenib (4 and 6 µM). The increasing concentration of sorafenib decreased the miR-23 extracellular expression. Higher concentrations of sorafenib (6 µM) significantly (reatment .p = 0.0001) decreased the cell viability with a mean difference of 77.43 ± 4.741 in HepG2 (R) cells compared to non-treated resistant HepG2 cells)  . Hence, p = 0.0127) increased miR-23b-3p (miR-23) expression compared to the negative control (NC). However, the transfection of antimiR (miR-23a) significantly (p = 0.0234) decreased miR-23b-3p expression compared to the negative control (p = 0.0282) stable up to 72 h  or inhibitor (antimiR) in HepG2 (P) cells where scrambled miRNA was used as the negative control (NC). The transfection of mimic (miR-23m) significantly  (30 nM) significantly (p = 0.0031) decreased the expression of ATG12 in resistant HepG2 cells compared to non-transfected resistant cells  decreased in sorafenib-resistant HepG2 cells transfected with miR-23b-3p mimic (miR-23m) (30 nM) compared to resistant cells without miR-23m transfection (The antimiR (miR-23a) increased nt cells A. Similasfection B.p = 0.0224) increased cell apoptosis percentage with a mean difference of 6.367 ± 1.761 compared to sorafenib-resistant cells without mimic transfection  cells. The transfection of miR-23a enhanced the proliferation rate, whereas the presence of miR-23m decreased the proliferation rate in HepG2 (R) cells compared to non-treated sorafenib-resistant cells A,B. The sfection C,D.ATG7, ATG12, Beclin1, and LC3II in HepG2 (R) cells compared to HepG2 (P) cells indicated the protective role of autophagy in sorafenib-resistant HepG2 cells. Moreover, decreased cell proliferation and increased cell apoptosis of sorafenib-resistant HepG2 cells in the presence of chloroquine (the autophagy inhibitor) also confirmed the protective role of autophagy in sorafenib-resistant HCC. Similarly, studies performed by Manov et al. [HCC is one of the predominant causes of cancer-associated deaths . The FDAv et al.  and Shimv et al.  and Zhaiv et al. . HoweverGLS1) in HepG2 (R) cells. In ovarian cancer progression, the higher expression of GLS1, the vital gene of glutaminolysis, has also been discussed [® Autophagy Detection Kit evaluated higher numbers of autophagy-stained vesicles, suggesting that detrimental glutamine might enhance autophagy pathways in sorafenib-resistant HCC, which is similar to a study performed by Zhu et al. [Altered tumor metabolism, including high glycolysis and glutaminolysis, is also crucial in cancer resistance. We demonstrated a higher glutaminase expression  and GLS1 (glutamine addiction). The association between miR-23b-3p and ATG12 has been discussed in gastric cancer cells [ATG12 and autophagy or GLS1 and glutamine addiction on sorafenib resistance in HCC has not been discussed. Our NGS data and extracellular expression demonstrated downregulated levels of miR-23b-3p in HepG2 (R) cells, suggesting the tumor-suppressing role of miR-23b-3p. Similarly, the downregulated expression of miR-23b-3p as a predictor has been discussed in HCC progression [ATG12 and glutamine addiction through GLS1 in sorafenib-resistant HCC.We also correlated the importance of differentially expressed microRNAs (miRNAs) in association with autophagy and glutamine addiction in sorafenib-resistant HCC. A recent study showed that the sistance . Our nexer cells ; metaboler cells . Howevergression . Furthergression . HoweverATG12 and glutamine addiction through GLS1 in sorafenib-resistant HepG2 cells. Transfection assays validated the tumor-suppressing effect of miR-23b-3p and modulated the expression of both ATG12 and GLS1 in sorafenib-resistant HepG2 cells, curtailing sorafenib resistance in HCC.Our study determined the cytoprotective role of both autophagy and glutamine addiction in HepG2 (R) cells. The NGS and in silico analysis confirmed miR-23b-3p as differentially expressed miRNA targeting cytoprotective autophagy through


Clinical information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in patients with inborn errors of immunity (IEI) during the current Coronavirus disease 2019 (COVID-19) pandemic is still limited. Proper DNA repair machinery is required for the development of the adaptive immune system, which provides specific and long-term protection against SARS-CoV-2. This review highlights the impact of SARS-CoV-2 infections on IEI patients with DNA repair disorders and summarizes susceptibility risk factors, pathogenic mechanisms, clinical manifestations and management strategies of COVID-19 in this special patient population. Coronaviruses (CoVs) belong to the subfamily Coronaviruinae, in the entity of the Nidovirales. Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is an enveloped, single positive-strand RNA (ssRNA) virus. SARS-CoV-2 can infect mammals and lead to lethal human diseaseExcept for SARS-CoV-2, influenza virus and respiratory syncytial virus (RSV) are the main pathogens associated with severe viral respiratory disease worldwideIn recent studies, innate immunity has shown its importance to combat SARS-CoV-2 infection. The antiviral innate immune response against SARS-CoV-2 can be initiated by endosomal RNA receptor recognition when Toll-like receptor 7/8 (TLR7 and TLR8) recognize SARS-CoV-2 endocytosed single-strand RNA (ssRNA). Double strand RNA (dsRNA) of the virus in the cytoplasm can be sensed by the cytosolic RNA sensor, RIG-1/MDA-5 (Retinoid-inducible gene/ melanoma differentiation-associated gene 5). Subsequently, the adaptor proteins, myeloid differentiation factor 88 (MYD88) and mitochondrial antiviral signaling protein (MAVS), are recruited, triggering and amplifying downstream signaling pathways through activation of several transcription factors, such as nuclear factor kappa B (NF-κB) and interferon response factor 3/7 (IRF3/7)+ T cells function by stimulating B cells, helping/activating CD8+ T cells, and recruiting more innate immune cells to the infectious sites+ T cells, is crucial for the development of protective immunity against severe viral infections+ T cells are responsible for the clearance of SARS-CoV-2-infected cells, and a virus-specific CD8+ T cell response is associated with milder COVID-19 symptomsThe central antigen-presenting cells in viral infections, plasmacytoid dendritic cells (pDCs), are responsible for activating antigen-specific adaptive immune cells in the respiratory tract, thus linking the innate and adaptive immune systems. Therefore, genetic defects in essential functional genes of pDCs have also been identified in patients with severe respiratory infections, including SARS-CoV-2B cells respond to virus infection during the second week after the symptom onset in COVID-19 patients, and produce antibodies against various structural proteinsDNA damage occurs frequently in human cells due to exposure to physiological and pathological events, including oxygen radicals, ionizing radiation or during DNA replication, genomic rearrangements, meiosis and adaptive immune development ,22 . SiThe MRN complex, MRE11 (meiotic recombination 11)-RAD50-NBS1 (Nijmegen breakage syndrome 1), is a crucial factor involved in both the HR and NHEJ pathways –32 . ThThe critical roles of ATM in cell cycle regulation and DDR have been described previouslyDNA repair defects in human have been described to predispose to growth retardation, genomic instabilityAs mentioned above, type I IFN immunity is critical for the innate immune response against SARS-CoV-2. The type I IFN pathway can be induced by viral pathogens recognized by members of the TLRs family. TLRs localized in endosomes can recognize dsRNA (TLR3), ssRNA (TLR7/8), and CpG DNA (TLR9), respectively –64 . ThDNA repair processes may also play a critical role in the adaptive immunity against SARS-CoV-2, where three main immunological diversification mechanisms require efficient and regulated DNA repair function, T- and B-cell receptor generation (V(D)J recombination), immunoglobulin class-switch recombination (CSR) and somatic hypermutation (SHM)Both CSR and SHM occur in the germinal center during late B cell development. CSR is initiated by activation-induced cytidine deaminase (AID) through cytosine (C) to uracil (U) deamination ,75 . ThTLR3,UNC93B1,TRIF,TRAF3, andTBK1) [+ T cells  [IFIH1, encoding MDA5) deficiency [IRF7,IRF9,STAT1, andSTAT2) [MCM4,CTPS1, andFCGR3A)GATA2,TMC6,TMC8,CIB1, andCXCR4)IFNAR1,IFNAR2,STAT2, andIRF9) and more recently, life-threatening SARS-CoV-2 infection  [Inborn errors of immunity (IEIs) are a group of diseases characterized by an increased susceptibility to infections and immune dysregulation due to genetic defects affecting the development or/and function of the immune system. There are over 450 IEI-related genes classified into ten categories based on the International Union of Immunological Societies (IUIS) classificationdTBK1) ,86 . WhdCD70) –91 , Fuiciency –94 . OtSTAT2) ,96 , vadIRF3) –102 . ORAG1/2,DCLRE1C,PRKDC,NHEJ1,LIG4,ATM,NBS1,BLM,DNMT3B,ZBTB24,CDCA7,HELLS,PMS2,RNF168,MCM4,OLE1/2,NSMCE3,ERCC6L2 andGINS1RAG1/2,DCLRE1C,PRKDC,NHEJ1, andLIG4 result in isolated non-syndromic combined immunodeficiency  while defects in other genes cause syndromic combined immunodeficiencies  [Supplementary Figure S1). Of note, DNA repair-deficient patients are at a high risk to develop severe complications after varicella-zoster virus (VZV) (~25%), rubella virus  and cytomegalovirus  infections. Other viral infections affecting a minority of patients include Epstein-Barr virus (EBV), Herpes simplex virus (HSV), rotavirus (RVs), adenovirus (HdAV), influenza virus (IV), metapneumovirus (HMPV), human respiratory syncytial virus (RSV), hepatitis B virus (HBV) and John Cunningham virus (JCV). The most common realm of viral infections in this group of IEI are Duplodnaviria (54%) and Riboviria (42%), and the majority are dsDNA (65%) and ssRNA viruses (26%) [Among the known IEI, 21 autosomal recessive DNA-repair disorders have been identified. DNA repair-associated genes include cells) ,106 . Ts (26%) –120 .RAG1 andRAG2 help to create site-specific DSBs, and the RAG complex seals hairpins during antigen receptor recombination. Patients withRAG1/RAG2 mutations thus have no or low level of V(D)J recombination activity, which leads to a reduction in B- and T-cells numbers and may present as severe CID (SCID) or Omenn syndrome (OS) [RAG1/RAG2 mutationsRAG mutations lead to a ‘leaky’ SCID with a higher rate of DNA lesions [Among the known DNA repair-related genes in IEIs,me (OS) ,121 . Dlesions ,124 .PRKDC gene, encoding the catalytic subunit of a nuclear DNA-PK, drives the broad antiviral response in innate immunity. Human adenovirus 5 and HSV have been shown to block the DNA-PK innate pathwayPRKDC showed both affected DNA lesion sensing and adaptive immune lymphocyte development and later presented with a T-B-SCID phenotypeTheATM results in A-T, which is a syndromic autosomal recessive disorder. Several studies have indicated that DNA damage due to ATM deficiency leads to an increased production of type I IFN [As mentioned above, ATM is a central element for the DNA damage response. Loss-of-functione I IFN ,128 . Ie I IFN ,130 , wNBNNibrin, also known as NBS1 or NBN, encodes a protein involved in the DNA-damage kinase activation in the DNA repair process and the MRN complex. NBS is a rare autosomal recessive DNA repair disorder characterized by predisposition to malignancy, ionizing radiation sensitivity and dysregulated cell cycle checkpoint and apoptosis due to mutations inDNMT3B deficiency (ICF1) who had otitis, bronchopneumonia, sepsis, Candida infection orPneumocystis jirovecii infectionDNMT3B (DNA Methyltransferase 3 Beta) can be recruited to DNA damage sites by proliferating cell nuclear antigen (PCNA)BLM lead to Bloom syndrome (BS) which is characterized by growth retardation, genome instability and predisposition to malignancy. The BS patients may also be associated with mild immunodeficiency, with low levels of immunoglobulinsBloom’s syndrome protein, which is involved in HR, may inhibit replication fork progressionTaken together, DNA repair defects are in general related to an enhanced susceptibility to viral infections and worse outcomes due to poor cellular immunity and subsequent reduced viral control.https://covid19.who.int/). The case fatality ratio (CFR) has decreased gradually from 10% to 2% in unvaccinated individuals since the very beginning of the pandemic. However, around 10%–20% of those with a confirmed COVID-19 diagnosis become seriously ill and require medical treatment and oxygen therapyAccording to the World Health Organization (WHO) SARS-CoV-2 has infected over 510 million people around the world (ATM (n=4),NBS (n=1) andDNMT3B (n=1) mutationsAlthough the impact of the pandemic is not yet clear in DNA repair-deficient patients, a previous study on a national IEIs cohort showed that patients with DNA repair disorders contributed about 15.8% (3/19) of all IEI cases diagnosed with COVID-19, and showed a mortality rate of 33.3% (1/3)syndromic CIDs, and 9% patients had non-syndromic CIDs (Supplementary Table S1). The median age at the time of SARS-CoV-2 infection was 9.3 years. To date, 44 patients had A-T (81%) [ATM,RAG1/RAG2 andDNMT3B patient groups, respectively (Supplementary Table S1). One possibility is that patients with specific DNA repair disorders, such as A-T patients, develop severe SARS-CoV-2 less frequently, potentially due to an enhanced activation of innate immunity, which is crucial in the early anti-viral response.Based on the current reported SARS-CoV-2-infected IEI patients, among the 54 patients with a DNA repair disorder , 91% of patients hadT (81%) –149 ; 3T (81%) ,151 ; 1T (81%) ,144 andT (81%) ,146 . Octively . Life-th) exists based on the current observation. Overall, patients with DNA repair defects, especially those at a young age, are at a much higher risk of severe complications, compared to children without pre-existing IEI. It is of particular concern given the note of rapidly increased hospitalization rates among non-vaccinated infants and children due to the circulation of new COVID-19 variant(s). It also highlights the potential benefit of this group of patients from advanced treatments such as passive immunotherapy  [DNA repair disorders are considered to show an intensified innate immune response and reduction of adaptive immunity against SARS-CoV-2 infection. Severe COVID-19 cases are rare in selected DNA repair disorder patients (such as A-T), which may be due to the over-activation of type I IFNs. On the other hand, a higher COVID-19 mortality rate in specific monogenic DNA repair defects  ,154 and


We prospectively investigated the immunogenicity of heterologous ChAdOx1 nCoV-19-BNT162b2 mRNA vaccination in an LTR cohort. (2) Methods: Forty-nine COVID-19 naïve LTRs received a two-dose regimen ChAdOx1 nCoV-19 vaccine. A subset of 32 patients received a booster dose of BNT162b2 mRNA vaccine 18 weeks after the second dose. (3) Results: Two-doses of ChAdOx1 nCoV-19 induced poor immunogenicity with 7.2% seropositivity at day 180 and low neutralizing capacities. The BNT162b2 mRNA vaccine induced significant increases in IgG titers with means of 197.8 binding antibody units per milliliter (BAU/mL) (95% CI 0–491.4) and neutralizing antibodies, with means of 76.6 AU/mL (95% CI 0–159.6). At day 238, 32.2% of LTRs seroconverted after the booster dose. Seroneutralization capacities against Delta and Omicron variants were found in only 13 and 9 LTRs, respectively. Mycophenolate mofetil and high-dose corticosteroids were associated with a weak serological response. (4) Conclusions: The immunogenicity of a two-dose ChAdOx1 nCoV-19 vaccine regimen was very poor in LTRs, but was significantly enhanced after the booster dose in one-third of LTRs. In immunocompromised individuals, the administration of a fourth dose may be considered to increase the immune response against SARS-CoV-2. Lung transplant recipients (LTRs) are considered at higher risk of developing severe SARS-CoV-2 pneumonia due to chronic immunosuppressive regimen and underlying comorbid conditions ,2,3.Impaired immunogenicity following a two-dose regimen anti-SARS-CoV-2 vaccine has been observed in solid organ transplant recipients (SOTRs), mainly focusing on mRNA-based vaccines ,5,6,7,8.Heterologous vaccination  has been shown to induce strong immune responses, inducing improved humoral and cellular immune responses in the general population and immunocompromised individuals ,9,10. BaHowever, data regarding the immunogenicity of a third vaccine dose and heterologous vaccination in LTRs are scarce. In the present study, we aimed at evaluating the immune response to the two-dose regimen of ChAdOx1 nCoV-19 vaccine in a prospective cohort of LTRs up to 6 months after the first administration. In addition, we investigated the 28-day impact of the heterologous third dose (BNT162b2 mRNA vaccine) on the immune response in a subset of LTR participants.®, Pfizer/BioNtech, Mainz, Germany) was administered 18 weeks after the second dose, on 7 October 2021 (at day 210). Fifty-four LTRs were vaccinated against COVID-19 with a two-dose regimen ChAdOx1 nCoV-19 vaccine  on 7 March 2021 (day 0). The second dose was administered 12 weeks later, on 6 June 2021 (day 84). The third heterologous dose of BNT162b2 mRNA COVID-19 vaccine (ComirnatyFive LTRs with previous SARS-CoV-2 infection (documented by a positive PCR and/or positive antibodies against the nucleocapsid (NCP)) were excluded. Forty-nine LTRs without previous SARS-CoV-2 infection were considered COVID-19-naïve before the study and enrolled in the present ongoing prospective and non-interventional clinical trial, approved by an ethical commission  . Among tAll vaccinated and enrolled LTRs provided written informed consent prior to data and specimen collection. Demographic and clinical data were retrieved from their electronic medical record at baseline.Serum samples were collected before the administration of the first vaccine dose  (n = 49) and 28 (n = 47), 84 (n = 49), 112 (n = 44), 180 (n = 42), 210 (n = 32) and 238 (n = 28) days after the first dose.A total of 291 sera were aliquoted and stored at −80 °C until analysis. Sera at days 84 and 210 were collected prior to the administration of the second ChadOx1 nCov-19 and booster BNT162b2 dose, respectively.® Anti-SARS-CoV-2 NCP qualitative ECLIA, Roche Diagnostics, Machelen, Belgium) were measured to document a previous infectious episode. According to the manufacturer’s instructions, the positivity threshold cutoff was set to 1.Total antibodies against the SARS-CoV-2 Nucleocapsid (NCP) . All samples were analyzed using anti-SARS-CoV-2 IgG quantitative immunoassays  against the trimeric spike S1 protein. The results are expressed as binding antibody units per milliliter (BAU/mL), and results < 2 and >4000 BAU/mL (lower and upper limits of quantification) were rounded to 2 and 4000 BAU/mL for statistical analyses. The positivity cut-off was set to ≥17.8 BAU/mL as reported by the manufacturer. Anti-SARS-CoV-2 responses were assessed and performed on a VITROSThe neutralizing capacity of antibodies (NAbs) was estimated as previously described . BrieflyAll serum samples (n = 291) were assessed using a pseudovirus neutralization test (pVNT) for wild-type SARS-CoV-2 . Samples with positive NAbs for the wild-type virus (n = 45) were also assessed for the Delta and the Omicron variants. A sample was considered negative if the half maximal inhibitory concentration (IC50) value of that sample was less than 1:20 dilution .10 ) and descriptive statistics were used to analyze the demographic and clinical data. Statistical significance across longitudinal collection time-points was assessed on raw data by the Kruskal–Wallis test followed by Dunn’s multiple comparison tests. The Mann–Whitney test was performed to evaluate the impact of immunosuppressive regimen on anti-SARS-CoV-2 IgG titers. A chi-square test or a Fisher’s exact test was used to determine if there was a significant relationship between two variables. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism .Quantitative data regarding the serological response are expressed in logThe baseline characteristics of the study population are summarized in Regarding anti-SARS-CoV-2 IgG titers, no significant modification was observed regarding anti-SARS-CoV-2 IgG titers after two doses of ChadOx1 nCov-19 vaccine in LTRs  a. At dayFor sVNT NAbs titers, no significant difference was observed after the two-dose regimen of ChAdOx1 nCoV-19 vaccine in LTRs . Before vaccination (day 0), one LTR presented positive NAbs titers (14.4 AU/mL), while negative having anti-nucleocapsid titers. This was considered a false-seropositive patient, probably due to interference because NAbs titers did not progress throughout the follow-up. At day 112, five LTRs (11.4%) displayed detectable NAbs titers, with discordant results for two LTRs, which had negative anti-SARS-CoV-2 IgG titers . None ofThe results of the pseudovirus neutralization test showed that four patients (8.1%) already presented positive wild-type SARS-CoV-2 neutralizing titers with a mean dilution titer of 63.5 (95% CI 18–145) before the first vaccine dose. At day 84, 18.4% of LTRs had positive wild-type SARS-CoV-2 neutralizing titers with a mean dilution titer of 56.8 (95% CI 25.0–88.5). After two doses of the ChAdOx1 nCoV-19 vaccine (day 112), 27.3% of LTRs showed positive wild-type SARS-CoV-2 neutralizing titers with a mean dilution titer of 415.7 (95% CI 13.6–845.0). After the heterologous dose (day 238), 21.4% of LTRs had positive wild-type SARS-CoV-2 neutralizing titers with a mean dilution titer of 110 (95% CI 0–305.6) a. The saThis study highlights that the two-dose regimen of ChAdOx1 nCoV-19 vaccine induces poor immunogenicity in LTRs, as witnessed by the low neutralizing capacities against wild-type and specific SARS-CoV-2 variants. Indeed, 92.8% of the included LTRs failed to mount detectable antibody response following this vaccination regimen. A third heterologous dose with the BNT162b2 vaccine, administered in a subset of LTRs, elicited higher rates of both seroconversion and positive neutralizing antibodies against SARS-CoV-2, observed in 32.2% of triple-vaccinated LTRs. Our findings are consistent with previously published data demonstrating weak immune responses after vectored or mRNA vaccines ,7,16, coThe immunosuppressive regimen appears to be a key player in preventing the development of immune response. Among SOTRs, LTRs are distinguished by a heavier immunosuppressive regimen, potentially explaining the discrepant response to vaccination observed in our study population compared with other studies. For instance, Schmidt et al. reported 35.3% seropositivity in SOTRs after completion of the two-dose ChAdOx1 nCoV-19 vaccine versus 7.2% of LTRs in our study . Our datFacing the weak immune response after the completion of a primary vaccination course with vectored or mRNA vaccine in SOTRs, booster doses (third and even a fourth) have been proposed by national and international competent authorities ,24 to reOur study has several limitations. Firstly, the national vaccination strategy negatively impacted the recruitment of a two-dose ChAdOx1 nCoV-19 vaccinated control group, although information about immunogenicity after a two-dose ChAdOx1 nCoV-19 regimen in the general population can be found in several previous studies ,34,35,36We showed that a two-dose regimen of ChAdOx1 nCoV-19 vaccine provides poor immunogenic effects on humoral response in LTRs. Our data, specifically collected in LTRs, indicated that a third heterologous dose of BNT162b2 (booster) elicited higher titers of IgG and NAbs after homologous two-dose ChAdOx1 nCoV-19 vaccine. Nevertheless, the rate of three-dose non-responders remained substantial at day 238 with 67.8% of seronegative LTRs in our study population and was even more important regarding Omicron SARS-CoV-2 neutralizing capacities. These results support the strategy of administering a fourth SARS-CoV-2 vaccine dose in immunocompromised patients, a decision that was implemented in January 2022 by the Superior Health Council of Belgium.


Pten, develop age-related lymphoproliferative diseases and humoral autoimmunity not seen in wildtype animals. Platelet-specific Pten-deficient mice have aberrant T cell activation, excessive T follicular helper (Tfh) cell responses and accumulation of platelet aggregates in lymph nodes. Transferred Pten-deficient platelets are able to infiltrate into the peripheral lymphoid tissues and form more aggregates. Moreover, Pten-deficient platelets are hyperactive and overproduce multiple Tfh-promoting cytokines via activation of the PDK1/mTORC2-AKT-SNAP23 pathway. Pten-deficient platelets show enhanced interaction with CD4+ T cells and promote conversion of CD4+ T cells into Tfh cells. Our results implicate PTEN in platelet-mediated immune homeostasis, and provide evidence that hyperactive platelets function as an important mediator in autoimmune diseases using mouse models.Beyond a function in hemostasis and thrombosis, platelets can regulate innate and adaptive immune responses. Hyperactive platelets are frequently associated with multiple human autoimmune diseases, yet their pathogenic functions in these diseases have not been fully established. Emerging studies show an essential function of the phosphatase and tensin homolog (PTEN) in maintenance of immune homeostasis. Here, we show that mice with platelet-specific deletion of  FH cells and systemic autoimmune pathology.Platelets have been shown to alter immune cell function but it is not clear if this includes autoimmunity. Here the authors show that phosphatase and tensin homolog (PTEN) deficient platelets promote autoimmunity in mouse models through excessive activation of T Platelets promote activation and differentiation of CD4+ helper T cells5, and induce immunoglobulin class-switching and B-cell antibody responses7. Platelets and megakaryocytes have a function in processing and presenting both foreign and self-antigens via MHC class I molecules, and regulate the activation and function of CD8+ cytotoxic T-lymphocytes9. Platelets are involved in surveying and collaborating with macrophages via the platelet-adhesion receptor GPIb, rapidly helping eradicate blood-borne bacterial infection in the liver10. Platelets are able to regulate the maturation and cytokine secretion of dendritic cells (DCs), establishing the communication between innate and adaptive immune responses6. It has been believed that platelets are the most numerous circulating cell type harboring immune regulatory functions.Anucleate blood platelets are well-known to participate in hemostasis and thrombosis, and their aberrant activation is implicated in multiple human cardiovascular diseases. Platelets can also affect multiple types of innate and adaptive immune cells to regulate immune and inflammatory responses11. Activated platelets release the contents of their a-granule and secrete a variety of cytokines, chemokines, growth factors, and inflammatory mediators, including P-selectin, TGF-β, IL-6, IL-21, IFN-γ, and non-neuronal serotonin15. The inflammatory cytokines IL-6 and IL-21 are critical for T follicular helper (Tfh) cell differentiation and proliferation18, and IFN-γ promotes the generation and effector function of Th1 cells19. Moreover, platelets from the patients with systemic lupus erythematosus (SLE) displayed increased IFN pathway signatures and enhanced activation15. And also, platelets express and secrete a plethora of critical immune molecules such as CD40, CD40 ligand (CD40L), and MHC class I molecules, which allows them to directly influence innate and adaptive immune responses during the immune disease processes9. Of note, activated platelets are the major source of soluble CD40 ligand (sCD40L) in the circulation20. CD40 is expressed on multiple immune cells including CD8+ T cells, CD4+ T cells, B cells, and DCs23. The CD40L-CD40 interaction has been recognized to be of critical importance in adaptive immune response and inflammation in the infectious diseases like atherosclerosis24. Moreover, CD40-sCD40L ligation activates platelets and subsequently enhances platelet-leukocyte adhesion, which is important in the recruitment of leukocytes to sites inflammation23.Platelets can produce over 1100 proteins as identified by proteomics26. Most importantly, the factors involved in direct interactions include platelet P-selectin, CD40/CD40L and GPIb, as well as PSGL-1, CD40L/CD40 and Mac-1  on other immune cells28. The direct interaction between platelets and immune cells is also important for their activation and functions29. Platelets can bind to CD40L-positive T cells, triggering granule release and recruiting T cells through the secreted RANTES29. Platelets form aggregates with monocytes via surface P-selectin, consequently inducing monocyte tissue factor expression in patients with severe COVID-1927. Platelets form aggregates with CD4 T cells via P-selectin and CD166, regulating pathogenic CD4 T-cell functions during autoimmune neuroinflammation12. Platelets form aggregates with Treg cells via sCD40L and P-selectin under infection conditions30. The aggregations alter the transcription profiles of Treg cells, promoting them to enter the lung to resolve pulmonary inflammation30.In addition, platelets can physically interact with various immune cells by a variety of factors31, rheumatoid arthritis, and systemic lupus erythematosus32. Compared with healthy individuals, the activation of platelets is increased in patients with SLE. These patients have increased levels of thromboxane and βthromboglobulin, an indicator of sustained platelet activation. In SLE patients, the increased levels of platelet surface P-selectin34 and platelet-derived soluble CD40L are correlated with the SLE disease activity index (SLEDAI) score36. In addition, platelets from SLE patients are also more readily aggregated ex vivo than those from healthy donors, suggesting that in SLE, platelets are primed or hyperactive in vivo34. However, the cellular and molecular mechanisms of hyperactive platelets in regulating autoimmune diseases, such as SLE, have not been demonstrated.Hyperactive platelets are frequently observed in various human autoimmune diseases such as multiple sclerosis37. It is well established that PTEN regulates many cellular processes including survival, differentiation, energy metabolism, cellular architecture, and mobility37. Recently, it is increasingly clear that PTEN also has important functions in maintenance of immune homeostasis37, although the underlying mechanisms remain to be further explored. Our previous study showed that platelets require PTEN to restrain collagen-induced activation and aggregation by inhibiting lipid kinase PI(3)K-AKT signaling, and extend the bleeding time38. Therefore, PTEN-deficient platelets are hyperactive and platelet-specific Pten-deficient (Ptenfl/flPf4-Cre) mice are suitable to be used as an ideal model to explore the functions of hyperactive platelets in vivo.Phosphatase and tensin homolog (PTEN) acts as a tumor suppressor frequently mutated in diverse cancersPten-deficient mice develop lymphoproliferative and autoimmune diseases mainly by inducing excessive Tfh cell responses compared with wild-type controls. Mechanistically, Pten-deficient platelets oversecrete various Tfh-promoting cytokines through AKT-SNAP23 signaling compared to Pten-sufficient platelets. Pten-deficient platelets are able to infiltrate into lymphoid tissues and convert CD4+ T cells into Tfh cells via physical contact. Our study establishes a potentially causative function of hyperactive platelets in autoimmunity. The linking of platelets to Tfh cells could provide a therapeutic approach for treating autoimmune diseases.In this work, we demonstrate that platelet-specific Pten-deficient (Ptenfl/flPf4-Cre) mice. Ptenfl/flPf4-Cre mice were undistinguishable from the wild-type littermates by 5 weeks of age. Beginning at 2 months, Ptenfl/flPf4-Cre mice spontaneously developed lymphoid hyperplasia  controls  eventually developed severe lymphadenopathy  were positive for PF4-Cre (GFP+) in the pLNs and spleens from wild-type Rosa26mT/mGPf4-Cre mice as well as Rosa26mT/mGPtenfl/flPf4-Cre mice , mTORC2-mediated AKT Ser473 phosphorylation, and membrane-bound P-selectin than WT counterparts in response to thrombin . Activated platelets release sphingosine-1-phosphate (S1P) and further promote VE-cadherin expression on HEVs52. This axis-mediated platelet extravasation is critical for maintenance of HEV integrity52. Moreover, the released S1P, a bioactive metabolite of sphingosine, has a pivotal intrinsic function in platelet activation and can further activate platelets53. It is possible that excess extravasated platelets are activated and further infiltrate into the tissues. In recent, accumulating evidences have demonstrated that activated platelets infiltrate into tissues to regulate inflammatory responses30. We hypnotized that PTEN deficiency promoted platelets to infiltrate into the lymphoid tissues. We utilized the Rosa26mTmGPf4-Cre reporter mice in which platelets are indicated by PF4-Cre driven GFP expression. We observed that Rosa26mT/mGPtenfl/flPf4-Cre mice displayed an increase in the number and size of GFP-positive platelet aggregates in their lymph nodes, especially in the T-cell zones  or Pten-deficient (Rosa26mT/mGPtenfl/flPf4-Cre) platelets were transferred into WT mice via tail vein injection  around high endothelial venule (HEV)nes Fig. . Moreovells Fig. . InteresLNs Fig. . To furtion Fig. . More Ptner Fig. , indicat55. The importance of hyperactive platelets in the pathogenesis of autoimmune diseases has not been established. In this study, we deleted Pten in platelets to generate mice with hyperactive platelets. These mice developed lymphoid hyperplasia, excessive Tfh cell responses, and spontaneous inflammatory diseases. Our work demonstrates a crucial function of PTEN for platelet to maintain immune homeostasis, and implies hyperactive platelets as an important mediator in human autoimmune diseases. Mutations and single nucleotide polymorphisms in PTEN have been reported in SLE patients by whole-exome sequencing57. It is likely that these genetic alterations may affect PTEN expression and function in platelets, subsequently causing platelet-mediated disruption of immune homeostasis. It is interesting to elevate the association of inflammatory cytokines with hyperactive platelets or PTEN malfunction in SLE patients. It is also a fruitful field for further research to analyze the status of platelets in patients with autoimmune diseases. Furthermore, the demonstration of inflammatory cytokines or infiltrating platelets in these patients would develop a therapeutic approach to treating such autoimmune diseases as SLE.Emerging studies have shown that hyperactive platelets are frequently associated with various human autoimmune diseases52. The interaction activates platelets via CLEC-2 signal to maintain the HEV integrity52. CLEC-2 signal can accelerate platelet activation and aggregate formation58, providing the potential mechanisms underlying an increase in the number and size of platelet aggregates in T-cell and B-cell zones of lymph nodes in Ptenfl/flPf4-Cre mice. Of course, collagen, ADP, ATP, and other platelet agonists may contribute to hyper-activation and aggregation of Pten-deficient platelets in the lymphoid tissues. Importantly, WT mice generated more platelet aggregates and Tfh cell responses in the lymph nodes after transfer of Pten-deficient platelets compared to Pten-sufficient platelets. These results support this notion that PTEN expression limits platelets to infiltrate into lymphoid tissues and form aggregates.It is well established that platelets can extravasate and interact with FRCs around HEV under physiological conditionsPten-deficient platelets secreted more inflammatory cytokines, including IL-6, IL-17B/F, IL-21, and IFN-γ. Some of these cytokines have been proved to promote T-cell activation, Tfh, and GC B-cell differentiation 38 have been described. Rosa26mT/mG (Jax007676) mice were from Nanjing Biomedical Research Institute of Nanjing University. The Rosa26mT/mG reporter mice we used contain a loxP-flanked (‘floxed’) tandem dimer Tomato gene (mT) and STOP cassette followed by the gene encoding GFP (mG) at the ubiquitously membrane-targeted Rosa26 locus. In this lineage tracing by Cre-Loxp system, PF4-Cre-mediated excision of the floxed STOP cassette results in descendible and constitutive expression of GFP. According to different experimental purposes, these mice were crossed to generate Ptenfl/flPf4-Cre, Rosa26mT/mGPf4-Cre, Rosa26mT/mGPtenfl/flPf4-Cre, respectively. All mice were crossed on C57BL/6 background. The development of these lymphoproliferative and autoimmune diseases was monitored by frequent visual examination, flow cytometry, and histopathological analyses. Ptenfl/flPf4-Cre or Rosa26mT/mGPtenfl/flPf4-Cre mice were analyzed at 3–8 months of age, according to different design in each experiment, and their age- and sex- matched littermate Ptenfl/fl and Rosa26mT/mGPf4-Cre mice were used as control groups.All experiments were performed in strict accordance with protocols approved by Institutional Animal care and Use Committee (IACUC) of Shanghai Jiao Tong University School of Medicine. All mice were maintained in identical standard conditions  and in isolated ventilated cages in specific pathogen-free (SPF) facilities. Bone marrow cells were flushed from tibias and femurs with sterile FACS buffer (PBS supplemented with 2% FBS and 2 mM EDTA). Splenic and lymphoid cells were gently grinded against the cell strainer by the plunger from a syringe and single-cell suspensions were filtered through fine mesh. In regard to the detection of DCs, splenic tissues were mechanically and enzymatically digested with 1 mg/ml collagenase IV and 50U/ml DNase I for 30 min at 37 °C with rotation. Cells were counted and stained with antibodies in FACS buffer, followed by incubation for 30 min on ice. Staining reagents included fluorescein isothiocyanate (FITC) anti-B220: clone RA3-6B2, allophycocyanin (APC) anti-IgM: clone II/41 (RUO), phycoerythrin (PE) anti-CD24: clone M1/69 (RUO), FITC anti-CD41: clone MWReg30, Percp-Cy5.5 anti-BCL6: clone K112-91, PE-Cy7 anti-CXCR5: clone 2G8, APC anti-IL4: clone 11B11, APC-Cy7 anti-IL17a: clone TC11-18H10, BV421 anti-IFN-γ: clone XMG1.2, FITC anti P-selectin: clone RB40.34, PE-Cy7 anti-Gr-1: clone RB6-8C5, APC anti-Mac1: clone M1/70, PE anti-CD41: clone MWReg30, PE anti-CD138: clone 281-2 were all purchased from BD Biosciences; Pacific Blue anti-CD4: clone GK1.5, Percp-Cy5.5 anti-CD19: clone 6D5, APC anti-CXCR5: clone L138D7, APC anti-CD44: clone IM7, APC-Cy7 anti-CD62L: clone MEL-14, PE-Cy7 anti-CD43: clone 1B11, Pacific Blue anti-GL7: clone GL7, PerCP-Cy5.5 anti-F4/80: clone BM8, APC-Cy7 anti-CD8: clone 53-6.7, PerCP-Cy5.5 Streptavidin were all purchased from Biolegend; APC anti-Fas/CD95: clone 15A7, AF700 anti-Foxp3: clone FJK-16s, APC anti-Cd42d (GPV): clone 1C2, APC anti-CD45: clone 30-F11, APC anti-CD41: clone eBioMWReg30, PE anti-CD62L: clone MEL-14, Biotin AH anti-mouse CD11c: Clone N418, FITC anti-mouse MHC II (I-A/I-E): clone M5/114.15.2, APC Anti-mouse CD86: Clone GL1, PE anti-mouse CD80: clone 16-10A1, PE-Cy7 anti-mouse CD11b: Clone M1/70 were all from eBioscience. Nonspecific binding was blocked with anti-CD16/CD32 (Biolegend): clone 93. Samples were collected on a LSR Fortessa cytometer (BD Biosciences) and then analyzed with FlowJo software. Dead cells and non-singlet events were excluded by 7-amino-actinomycin D staining  and the forward scatter area (FSC-A) versus height (FSC-H) characteristics.+ T cells and submitted for RNA sequencing using the sequencing platform BGISEQ-500 (BGI). Three biological replicates of each genotype were sequenced. Sequence reads were mapped to mouse genome (mm9) by using bowtie 2 (v 2.3.4.2) with default settings, and reads mapped to multiple positions (MAPQ < 10) were discarded. Gene expression levels were measured by RPKM (reads per kilobase of exon per million reads) by RSEM (v1.2.30). Significantly differentially expressed genes were identified by edgeR (v3.20.9) with the following criteria: Benjamini-Hochberg corrected P value < 0.01 and fold-change > 2. Lists of genes differentially expressed by twofold or more were analyzed for functional enrichment using the Ingenuity Pathways Analysis . Differentially expressed genes between CD4+ T cells of pLNs from Ptenfl/fl and Ptenfl/flPf4-Cre mice were selected by Limma (v3.42.2). Gene Ontology (GO) Categories enriched analysis was performed by cluster Profiler (v3.14.3). GSEA was performed using the GSEA tool (v4.2.3) (Broad Institute). The gene signature upregulated in Tfh cells relative to their expression in non-Tfh cells were from published data (GEO accession code: GSE21379). The RNA-seq data has been deposited into the GEO series database (GSE143698).Total RNA was harvested from the lymphoid CD4Ptenfl/flPf4-Cre mice and age-matched Ptenfl/fl littermates (control) were fixed in 4% paraformaldehyde and embedding with paraffine. Sections (4–6 μm in thickness) were cut with a Leica RM2235 Microtome and then examined by Hematoxylin-and-eosin staining. For immunohistochemical analysis, tissue sections were incubated with biotinylated Peanut Agglutinin  and the signals were detected using VECTASTAIN ABC (Avidin-Biotin Complex) peroxidase-based kits from Vector Laboratories (Cat: PK-7100). Immunohistochemistry was performed according to standard procedures.Tissues from Rosa26mT/mGPf4-Cre) or Pten-deficient (Rosa26mT/mGPtenfl/flPf4-Cre) platelets were transferred into Ptenfl/fl mice through tail vein injection. After 2 days, Ptenfl/fl mice were sacrificed and lymph nodes instantly frozen in Tissue-Tek optimum and stored at −80 °C until further processing or sections were cut as mentioned above.Lymph nodes were dissected and instantly frozen in Tissue-Tek optimum cutting temperature compound . Frozen tissues were stored at −80 °C until further processing. Sections (10 μm in thickness) were cut with a Leica Cryostat, mounted on Superfrost Plus glass slides. Tissue cryosections were air-dried for 1 h before and after fixation in 4% Paraformaldehyde or cold acetone for 10 min, and then were rehydrated for 10 min in PBS. Slides were stained overnight at 4 °C in a humidified chamber with the following diluted primary antibodies: anti-B220 (from Biolegend): clone RA3-6B2, PNA (from Vector), anti-CD4 (from Biolegend): clone GK1.5, anti-CD31 (from BD Biosciences): clone MEC13.3, Biotinylated anti-LYVE (from R&D Systems). After washing, secondary antibodies, Alexa Fluor 647 conjugated Goat anti-Rat IgG (Life Technologies), PE-conjugated Goat anti-rat IgG  or APC Streptavidin (BD Biosciences) were applied for 1 h at room temperature. Slides were mounted with the Prolong Gold Antifade reagent . GFP-labeled WT (Immunoglobulin subclasses in plasma were detected by specific ELISA kits (SouthernBiotech 5300-4B) according to the manufacturer’s protocol. To detect anti-dsDNA autoantibodies in plasma, high-binding ELISA plates were coated with 2 μg/ml dsDNA  from calf thymus. Coated plates were blocked, and subsequently incubated with diluted samples overnight at 4 °C in TBS with 1% BSA. Bound anti-dsDNA antibodies were detected with AP-conjugated anti-mouse IgG  and streptavidin-HRP  followed by TMB substrate solution (eBioscience 00-4201-56). Absorbance was measured at 450 nm.8/ml) for further experiments. Platelet aggregation was measured using the light transmission aggregometry70. Platelet purity (>99%) was routinely detected by flow cytometry with platelet-specific CD41 antibody staining. Adenosine 5′-diphosphate (ADP), apyrase, and PGE1 were purchased from Sigma-Aldrich . α-Thrombin was from Enzyme Research Laboratories . Collagen was from CHRONO-PAR. Washed Ptenfl/fl, Ptenfl/flPf4-Cre platelets or Rosa26mT/mGPf4-Cre, Rosa26mT/mGPtenfl/flPf4-Cre platelets were transferred into Ptenfl/fl mice via tail vein injection.The whole blood was collected from abdominal aorta of indicated mice. Platelets were prepared using a centrifuge to separate the platelet-rich plasma from the whole blood, followed by a second centrifugation to concentrate platelets, and then suspended in the Tyrode’s buffer (3 × 10Ptenfl/fl and Ptenfl/flPf4-Cre platelets at a concentration of 3 × 107/ml were incubated with FITC-labeled P-selectin (BD Biosciences) and 0.1 U/ml of thrombin for 20 min at room temperature in a final volume of 100 μL Tyrode’s buffer, and P-selectin binding was measured by flow cytometry. The indicated inhibitors were added to Ptenfl/fl and Ptenfl/flPf4-Cre platelets and incubated for 5 min before stimulation with thrombin. AKT inhibitor SH6 and PDK1 Inhibitor II were purchased from Calbiochem. mTORC2 inhibitor PP242 was purchased from Sigma.Washed Ptenfl/fl and Ptenfl/flPf4-Cre platelets in response to thrombin, respectively. Prepared platelet purity was routinely detected by flow cytometry with platelet-specific CD41 antibody staining (>99%). The potential contamination of other immune cells in prepared platelets used for these cytokine assays was excluded with leukocyte-specific CD45 antibody staining (<0.05%). The manufacturer’s recommended protocol was used to perform this assay. Cytokine protein profiles were quantified by determining band intensities with National Institutes of Health ImageJ software.A RayBio Mouse Cytokine Antibody Array kit  was used to detect a panel of 46 secreted cytokines and chemokines that stored in resting platelets or released from activated Washed platelets prepared and lysed in cell lysis buffer supplemented with Protease and Phosphatase Inhibitor Cocktail (Roche Life Science). Primary antibodies included anti-AKT-Thr308 , anti-AKT-Ser473 (CST), anti-SNAP23-Ser95 (GenScript), anti-Sin1-Thr86 (CST), anti-SNAP23 (abnova), anti-PTEN (CST), anti-CD62P antibody (Abcam) clone: EPR22850-190 and anti-GAPDH (CST) were diluted in 1× TBS buffer (supplemented with 5% BSA or skim milk), followed by incubation at 4 °C overnight.9/ml. The prepared platelets had a leukocyte contamination rate < 0.05% as assessed by detecting CD45+ events with flow cytometry. CD4+ T cells were isolated using EasySep™ Mouse CD4 negative isolation kit from STEMCELL Technologies (Cat:19852) according to the manufacturer’s instructions. The isolated T cells were > 98% CD4 positive and were suspended in DMEM at the cell density of 5 × 106/ml.Washed platelets were prepared as described above. Afterward, the platelets were pelleted and resuspended in DMEM culture medium at the concentration of 1.25 × 10+ T cells were cultured in the absence or presence of Ptenfl/fl, Ptenfl/flPf4-Cre platelets or PTEN inhibitor bisperoxovanadium  pretreated platelets in DMEM containing 5% FBS, 2 µM L-glutamine, 1 µM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. CD4+ T-cell activation was induced by coated anti-CD3 mAb  and soluble 0.3 µg/ml anti-CD28 mAb (Invitrogen 16-0281-85). The cell density ratio of T cells to platelets were adjusted to 1:100, 1:200 or 1:400, respectively. T cells and platelets were co-cultured 4 days at 37 °C with 5% CO2. Co-cultured cells were collected and detected for T-cell phenotyping by flow cytometric analysis.CD4All company names and catalog numbers of antibodies or commercial reagents used in this study were provided in the Supplementary Table t test was used to compare endpoint means of different groups. Experiments were independently repeated at least three times. P values less than 0.05 were considered as statistically significant. Error bars on bar graphs represent the standard error of mean (SEM). Total 3–10 mice were used in each experimental group to define meaningful differences between groups. If differences between groups were closed to statistical significance (P = 0.1–0.05), more mice were used for a more rigorous test of statistical analysis.Data were analyzed with GraphPad Prism 7. Two-tailed unpaired Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic of coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded to develop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP. These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able to detect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of SARS-CoV-2 and biological cell response against it.The online version contains supplementary material available at 10.1186/s12985-022-01948-2. While human coronaviruses (CoV) are widespread in humankind as a pathogen for the common cold, severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 cause severe acute respiratory syndrome and have extremely high mortality rates –4. AmongSARS-CoV-2 is a positive-sense single-stranded RNA virus that has a large RNA genome (~ 30 kb) and shares high sequence homology to SARS-CoV ~ 80%) and MERS-CoV (~ 50%) . A serie0% and MEAlthough a few antibodies (Abs) against nsp12 of SARS-CoV-2 are commercially available, these Abs are all polyclonal and not highly specific. In addition, these polyclonal Abs cannot discriminate between nsp12 of SARS-CoV and SARS-CoV-2. To study the molecular mechanism underlying the RdRP reaction of SARS-CoV-2, monoclonal antibody (mAb) against nsp12 of SARS-CoV-2, which has not been developed so far, could be powerful tools. Furthermore, the mAb that could specifically detect SARS-CoV-2 nsp12, but not that of SARS-CoV, would be highly potential to examine difference between the RdRP reactions of SARS-CoV and SARS-CoV-2. In this study, we have established novel mouse mAbs against SARS-CoV-2 nsp12 (RdMabs), and evaluated whether they could be applied for western blotting, immunoprecipitation, and immunostaining analyses. In addition, we examined the specificity of these mAbs, especially focusing on whether they discriminate between nsp12 of SARS-CoV and SARS-CoV-2.P3X63Ag8U.1 (P3U1) and SV40-transformed human embryonic kidney cell line HEK-293T (293T) cells were obtained from the American Type Culture Collection . P3U1 cells were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium  that was supplemented with 10% heat-inactivated fetal bovine serum , 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B . 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, streptomycin (100 μg/ml), and penicillin (100 U/ml).Anti-β-actin (AC-15) mouse mAb  and anti-FLAG mouse mAb (M2) (Merck) were used for western blotting analysis. mAbs against nsp12 of SARS-CoV-2  were developed in this study (see below).34AFDIYNDKVAGFAKFLKTNC53, 74RHTFSNYQHEETIYNLLKDC83, and 248TRALTAESHVDTDLTKPYIC266, which were keyhole limpet hemocyanin (KLH)-conjugated  were immunized intraperitoneally (i.p.) with Imject Alum (Thermo Fisher Scientific Inc.) into BALB/c mice (100 μg of each peptide/one mouse). The procedure included three additional immunization procedures (100 μg of each peptide), followed by a final booster intraperitoneal injection (100 μg of each peptide) 2 days before its spleen cells were harvested. The harvested spleen cells were subsequently fused with P3U1 cells, using polyethylene glycol 1500 . Then, hybridomas were grown in an RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific Inc.). Cultured supernatants were finally screened using enzyme-linked immunosorbent assay (ELISA) for the detection of nsp12 peptides [Three female BALB/c mice (6-weeks-old) were purchased from CLEA Japan . The animals were housed under specific pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all animal experiments (Permit number: 2019NiA-001). We designed three peptides (#1–3) of nsp12 as immunogens to immunize mice around the NiRAN domain . The SARS-CoV and -CoV-2 nsp12 genes  cloned into the plasmid vectors were codon optimized for expression in human cells.To overexpress SARS-CoV and -CoV-2 nsp12 transiently, 293T cells were transfected with plasmid vectors carrying a FLAG-tagged nsp12 expression cassette, pCMV-FLAG-CoV-nsp12 and -CoV-2-nsp12 using Lipofectamine 2000 (Thermo Fisher Scientific). After 72 h of incubation, cells were lysed in RIPA buffer  for western blotting analysis.Western blotting assay was performed as previously described . Briefly7 cells were lysed in 1 ml of Lysis buffer A , and 150 mM NaCl). After sonication, lysates were cleared of insoluble material by centrifugation at 21,000×g at 4 °C for 15 min. FLAG-nsp12 proteins were co-immunoprecipitated using Pierce Protein A Plus Agarose (Thermo Fisher Scientific) and 20 µg of anti-nsp12 mAb (RdMab-2) from the lysate, and then precipitated immune complexes were eluted in 2 × SDS loading buffer ). The eluted proteins were detected by western blotting analysis. To detect the precipitated FLAG-nsp12 proteins, anti-FLAG mouse mAb  and Mouse TrueBlot ULTRA Anti-Mouse Ig HRP  were used for western blotting analysis.Immunoprecipitation assays were performed as previously described . BrieflyImmunofluorescence cell staining was performed as previously described . BrieflyStructural analyses using electron cryo-microscopy have shown that SARS-CoV-2 nsp12 protein has a closed, right-handed structure, which consists of palm, finger, and thumb domains  and discontinuous transcription of various sizes of subgenomic RNAs (sgRNAs) , 21. RegWhile several rabbit polyclonal antibodies against nsp12 of SARS-CoV-2 are now commercially available from Cell Signaling Technology, Proteintech, GeneTex, and so on, no mAb against nsp12 of SARS-CoV-2 have been reported. Notably, Yamada et al.  reportedIn this study, we designed three peptides #1–3) for producing mAbs, focusing on the structure around the NiRAN domain in the nsp12, because SARS-CoV-2 nsp12 has a characteristic β-hairpin motif in the NiRAN domain, which was not found by structure analysis of SARS-CoV nsp12 . Intrigu–3 for prTaken together, we have successfully developed three novel mouse mAbs against SARS-CoV-2 nsp12 , which are suitable for western blotting, immunoprecipitation, and immunostaining analyses. These novel anti-nsp12 mAbs were able to discriminate between nsp12 of SARS-CoV and SARS-CoV-2. Generally, elucidation of specific protein functions critically depends on the ability to recognize the target protein specifically, and we obtained mAbs that could specifically recognize SARS-CoV-2 nsp12. These novel anti-nsp12 mAbs could be utilized for investigation of the RdRP reaction of SARS-CoV-2 and responses against it in host cells. In this study, we could develop mAbs against SARS-CoV-2 nsp12 as an initial step. We hope that further studies using these anti-nsp12 mAbs will lead to elucidation of the RdRP reactions of SARS-CoV-2 and development of more effective anti-viral strategies.Additional file1. Figure S1: Screening of anti-nsp12 mouse monoclonal antibodies using the culture supernatant of hybridomas. Figure S2: Selection of anti-nsp12 mouse monoclonal antibodies to discriminate between nsp12 of SARS-CoV and SARS-CoV-2.


TP53 is conserved from nematode to human. In humans the TP53 gene is found mutated in a majority of cancers and therefore, the p53-induced cell death pathway is dysfunctional. As such, it is of the utmost importance to determine mechanisms and models to test for ways to induce p53-independent cancer cell death. The small, transparent, nematode C. elegans is a whole animal with a germline stem cell tumor model that presents such an opportunity. We used this model with a well-studied p53 mutant that increases germline tumor size to test for ways to induce p53-independent cell death. Herein, we report that two p53-independent death inducers, a nucleoside analogue and a PARP inhibitor, are capable of inducing C. elegans germline tumor cell death. This suggests new targeted drugs can be tested in this model for p53-independent cancer cell killing.The tumor suppressor gene TP53 gene is mutated in over 50% of human cancers, and the C. elegansp53-1(cep-1) gene encodes the ortholog CEP-1. CEP-1 is activated by ultraviolet type C (UVC)-induced DNA damage and activates genes that induce germline apoptosis. UVC treatment of gain-of-function glp-1(ar202gf)/Notch tumorous animals reduces germline stem cell numbers , while UVC treatment of double-mutant cep-1/p53(gk138);glp-1/Notch(ar202gf) increases DNA damage adducts and stem cell tumor volume. We compared UVC-induced mitotic stem cell death and animal lifespans for the two different C. elegans tumorous strains. C. elegans stem cell compartment death has never been observed, and we used engulfed small stem cells, notable by green fluorescent puncta, to count cell death events. We found UVC treatment of glp-1(ar202gf) animals increased stem cell death and increased lifespan. However, UVC treatment of double-mutant cep-1/p53(gk138);glp-1/Notch(ar202gf) animals decreased stem cell death, increased tumor volume, and decreased animal lifespan. There are pharmacological agents that induce p53-independent cell death of human cells in culture; and two notable protocols are the PARP-trapping agents of temozolomide plus talazoparib and the nucleoside analogue 8-amino-adenosine. It is important to determine ways to rapidly test for pharmacological agents able to induce p53-independent cell death. We tested feeding cep-1/p53(gk138);glp-1/Notch(ar202gf) nematodes with either 8-amino-adenosine or temozolomide plus talazoparib and found both were able to decrease tumor volume. This is the first comparison for p53-independent responses in cep-1/p53(gk138);glp-1/Notch(ar202gf) animals and showed UVC DNA damage increased tumor volume and decreased lifespan while PARP inhibition decreased tumor volume. The  The cep-1(gk138) I glp-1(ar202gf) III bcls39[P(lim-7)ced-1::GFP + lin-15(+)] V (JBC7) strain was constructed by the Bargonetti Laboratory: cep-1(gk138) I bcls39[P(lim-7)ced-1::GFP + lin-15(+)] V (JBC1) strain males were crossed with cep-1(gk138) I glp-1(ar202gf) III (JBC2) strain hermaphrodites, yielding an F1 population. F1 progeny (F2 generation) laid at 15 °C of worms were selected if F2 worms laid at 25 °C developed full-body tumors, resulting in sterility. The selection process was repeated with the offspring of the selected F2 worms at 15 °C, and epifluorescence microscopy was used to identify a brood in which 100% of worms screened tested positive for germline GFP expression. The strain bcIs39[P(lim-7)ced-1::GFP+lin-15(+)] (MD701) were also used  and glp-1(ar202); bcls39[P(lim-7)ced-1::GFP + lin-15(+)]. These are the animals used throughout these studies.CEP-1 induces germ cell apoptosis and promotes DNA repair in response to stress, including stress following UVC-induced DNA damage ,6,7,33. lifespan ,30,31,32repeats) ,26,42. Trepeats) . To compThese strains were created to establish a model in which lifespan and tumor development could be monitored while also monitoring cell death by using the CED-1::GFP fusion protein to mark dying cells. The CED1::GFP is a strong marker for cell death and can be scored for cell death that is both apoptotic and necrotic ,45. We aglp-1(ar202gf) genotype causes animals maintained at 25 °C to have a shorter lifespan  and glp-1(ar202gf); bcls39[P(lim-7)ced-1::GFP + lin-15(+)] were monitored for CED-1::GFP fusion-labeled protein dying cells. Imaging for the CED-1::GFP fusion protein is a sensitive method for visualizing the somatic sheath cell that encircles early apoptotic corpses in the transition zone of the germline during engulfment  and glp-1(ar202gf); bcls39[P(lim-7)ced-1::GFP + lin-15(+)] allow for the comparison of complex outcomes that influence tumor size in the presence or absence of CEP-1/p53 signaling. We reasoned that comparison of these two strains would enable the evaluation of chemotherapeutic protocols found in preclinical trials to activate p53-independent cell death pathways  worms to double-mutant cep-1(gk138);glp-1(ar202gf);bcls39[P(lim-7)ced-1::GFP + lin-15(+)] worms. Interestingly, we observed an increased lifespan in cep-1; glp-1 double-mutant animals at 25 °C but not at 15 °C. This follows a previous observation that metabolic stress in the presence of wild-type CEP-1/p53 can reduce nematode lifespan, and the loss of normal functioning CEP-1/p53 extends lifespan  worms (cep-1(gk138);glp-1(ar202gf);bcls39[P(lim-7)ced-1::GFP + lin-15(+)] worms had reduced cell death ] worms . In contll death . As we hll death . Interesll death .C. elegans, different CEP-1/p53 mutants that are transcriptionally inactive are characterized by a separation of function in maintaining genomic fidelity, with cep-1/p53(gk138) remaining more viable than cep-1/ p53(lg12501)  V) that is useful for detection of pharmacologically induced p53-independent cell death in mitotic germ cells. Furthermore, this strain is useful for assessing the ability of pharmacological agents to reduce C. elegans germline tumor size. We conclude that studying the C. elegans germline tumor model with and without functional p53 enables the influence of whole animal cell stress to be assessed. The reduction in lifespan seen in gain-of-function glp-1 (Notch) animals might be due both to the burden of a large germline size as well as the metabolic stress signaling that comes from activation of the CEP-1/p53 pathway. A recent report delves in to the complexity of p53 and aging, showing that autophagy suppresses the p53 pathway to reduce the pace of aging [C. elegans reduces axonal degeneration, which coordinates with the lack of a change in tumor volume in the presence of a functional CEP-1/p53 pathway [C. elegans.We conclude that we have constructed a of aging ,53. We p pathway ,55. It r