Background: C1q/tumor necrosis factor-related protein 3 (CTRP3) has been reported to be a crucial regulator in myocardial infarction. Nevertheless, the potential molecular mechanism of CTRP3 in ischemia/reperfusion (I/R) injury remains largely unclear.in vivo myocardial I/R injury model was constructed to investigate the effect of CTRP3.Methods: The cell model of myocardial I/R injury was established by oxygen-glucose deprivation/reoxygenation (OGD/R) of rat cardiomyocyte H9C2. Expression of CTRP3 and lysosomal-associated membrane protein 1 (LAMP1) was detected in H9C2 cells treated with oxygen-glucose deprivation/reoxygenation (OGD/R). H9C2 cells were transfected with overexpression plasmids of CTRP3 (pcDNA-CTRP3) and LAMP1 (pcDNA-LAMP1), or CTRP3 small interfering RNA (si-CTRP3) or/and pcDNA-LAMP1, and cell proliferation, apoptosis and oxidative stress were testified. Co-IP assay was performed to validate the relationship among CTRP3, LAMP1 and JIP2. The role of CTRP3 and LAMP1 in JIP2/JNK pathway was evaluated with Western blot assay. Furthermore, in vivo.Results: Overexpression of CTRP3 and LAMP1 both significantly promoted cell proliferation, inhibited apoptosis and the production of reactive oxygen species (ROS), malondialdehyde (MAD) and cardiac troponin (cTn-I), while silencing CTRP3 exerted the opposite effects, and LAMP1 overexpression reversed the effect of silencing CTRP3 on the aspects above. CTRP3 interacted with LAMP1, and both CTRP3 and LAMP1 bound with JIP2. SP600125 (JNK inhibitor) could restore the effects of CTRP3 or LAMP1 overexpression on the expression of JIP2 and phosphorylated-JNK (p-JNK), proliferation and apoptosis. Moreover, overexpression of CTRP3 improved cardiac I/R injury Conclusion: CTRP3 alleviates cardiac I/R injury by elevating LAMP1 and activating JIP2/JNK signaling pathway, which may serve as a potential therapeutic target for I/R injury. Myocardial ischemia caused by coronary artery occlusion leads to increased loss of cardiomyocytes and is a main cause of elevated morbidity and mortality in myocardial infarction . ReperfuC1q tumor necrosis factor (TNF)-related protein-3 (CTRP3) belongs CTRP family and is a newly found group of proteins homologous to adiponectin (APN) collateral system . It is mLysosomal associated membrane protein 1 (LAMP1) is a glycosylated lysosomal membrane protein that belongs to lysosomal associated membrane protein family and could protect the lysosomal membranes from intracellular proteolysis . Chen etHerein, this study aimed to clarify the effects of CTRP3 and LAMP1 on myocardial I/R injury and the underlying mechanisms, with a view to providing biomarkers for clinical prevention of myocardial I/R.2 at 37°C . To establish the model of I/R injury in vitro, cells were cultured in glucose-free DEME at 37°C in a hypoxic incubator  for 24 h. After oxygen-glucose deprivation, cells were grown in complete medium at normal incubator  for 6 h.The rat embryonic cardiomyocytes H9C2 were got from the cell bank of Shanghai Biology Institute, Chinese Academy of Science  and maintained in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum (FBS) in a humidified condition containing 5% COCTRP3 and LAMP1 overexpression plasmid (pcDNA-CTRP3 and pcDNA-LAMP1), small interfering RNA targeting CTRP3 (si-CTRP3) and LAMP1 (si-LAMP1), as well as their negative control (vector and scramble) were constructed by Genepharma Biotech . H9C2 cells were transfected with above plasmid vector with lipofectamine 2000 . After transfection for 48 h, cells were given OGD/R treatment, and then cells were collected for subsequent experiments.−ΔΔCT method. The primer sequences were described as follow: CTRP3, F, 5′-AGC GTC TCT GGG TTG TCT TG-3′; R, 5′-TGA GCT TCT GCC CCA ACA AA-3′; LAMP1, F, 5′-AGG CGG TGA GAT CTA GAC GA-3′; R, 5′-AAG AAT AGT GTT GGC GGG GG-3′.Total RNA from cells was extracted with TRIzol reagent  following the manufacturer’s instructions. A TaqMan Reverse Transcription Kit  was used to reversely transcribe RNA to complementary DNA. RT-qPCR was run on a 7000 ABI System by using SYBR Green PCR Kit . GAPDH was used as an internal control. The relative quantitative expression was determined by using the 23 cells/well on 96-well plates. After OGD/R treatment, cells were incubated with 20 μL of MTT solution (0.5 mg/mL) for 4 h at 37°C. Then, the supernatant was removed, and 150 μL of dimethyl sulfoxide were added to each well. The absorption was measured at 490 nm by using microplate reader .Cell viability was detected by using MTT assay. H9C2 cells were seeded at 5 × 104 cells/well. Transfected cells were incubated with 20 μmol/L EdU reagent for 2 h, washed twice with phosphate Buffer solution (PBS) and fixed with 4% phosphate buffered paraformaldehyde for 15 min. Subsequently, the cells were stained with 100 μL of fresh Apollo reaction cocktail, and nuclei were stained with 100 μL of Hoechst 33342. The percentage of EdU positive cells was counted by using fluorescence microscope (LEICA).EdU detection was performed by using an EdU immunofluorescence staining kit  according to the manufacturer’s protocol. Sterilized slides were put into 24 well plates, and H9C2 cells were seeded into each well at a density of 3 × 10Apoptosis of H9C2 cells was detected by using the flow cytometry and a Annexin phycoerythrin/7-aminoactinomycin D (V-PE/7-AAD) Apoptosis Detection Kit . After treatment, cells were collected and digested with trypsin without ethylenediamine tetraacetic acid. Then, the cells were washed three times with cold PBS and resuspended in 100 μL of binding buffer. The cell suspension was incubated with 5 μL of Annexin V-PE and 5 μL of 7-AAD staining solution at room temperature for 10 min in a dark room. The apoptotic ratio of H9C2 cells was assessed within 1 h by using flow cytometry .Total protein was extracted with RIPA lysis buffer. Protein concentration was determined by using the Bradford method. Equal amounts of protein were electrophoresed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk and then incubated with primary antibodies, including anti-CTRP3 , anti-LAMP1 , anti-JIP2 , anti-p-JNK , anti-JNK  and anti-GAPDH  at 4°C overnight. The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody . GAPDH was used as an internal control. The signals were detected by using an ECL kit . Subsequently, the protein bands were analyzed with Image J 1.43 software.The production of ROS was detected by using Dichlorofluorescein diacetate (DCFH-DA) assay. Briefly, cells were incubated with 10 μmol/L DCFH-DA at 37°C in the dark for 20 min. Then, the cells were washed twice with PBS, and the fluorescence intensity was detected by using a fluorescence spectrophotometer (BioTek).4 cells/well. Myocardial tissues were homogenized and the supernatant was obtained. The activity of MDA was measured by using MDA activity kit .H9C2 cells were seeded in 96-well plates at a density of 1 × 10H9C2 cell culture medium and myocardial tissue abrasive fluid were collected by centrifugation at 4°C for 15 min. The concentration of cTn-I in the supernatant medium was determined by using enzyme-linked immunosorbent assay kits  according to the manufacturer’s instructions. The absorbance of the samples was measured at 450 nm with a standard microplate reader .Cells were lysed by utilizing Co-IP lysis buffer containing a protease inhibitor . The lysates were collected and mixed with 2 μg of primary antibody or immunoglobulin G (IgG). Then, the mixture was incubated with protein A/G beads overnight at 4°C. The beads were washed three times and boiled in loading buffer for 5 min. All samples were analyzed by Western blotting using various antibodies.n = 8 mice of each group): Sham group, I/R+ lentiviral vectors (LVs) group and I/R+ CTRP3 overexpressed lentiviral vectors (LV-CTRP3) group. Three days before operation, mice myocardium was injected with LV-CTRP3 (2 mg/kg). Mice were anesthetized by inhalation of 2% isoflurane, and an oblique incision was made in their left chest wall skin to reveal the heart. The left anterior descending coronary artery was ligated with a polypropylene 6–0 suture. After 30 min occlusion, the sutures were loosened for 24 h to establish the myocardial I/R model. Sham operated mice underwent left chest wall incision surgery, but the left anterior descending coronary artery was not ligated. This study was approved by the ethics committee of the Affiliated Hospital of Yanan University. All animal experimental procedures were in accordance with the guide for the care and use of laboratory animals of the National Institutes of health.C57BL/6J mice (weight 17–25 g) were purchased from Animal Laboratory of the Fourth Military Medical University and all mice were kept in a temperature and humidity-controlled room . Mice were randomly divided into 3 groups . Left ventricular end systolic diameter (LVESD), left ventricular end diastolic diameter (LVEDd), left ventricular end systolic volume (LVSV), and left ventricular end diastolic volume (LVDV) were measured and averaged over 3 consecutive cardiac cycles. As indicators of cardiac function, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) values were converted using the Simpson method with the following formulas: LVEF = (LVDV-LVSV)/LVDV × 100%; LVFS = (LVDD-LVSD)/LVDD × 100%. Experiments were performed 3 times and averaged.Mice were anesthetized with inhalation of 2% isoflurane after 24 h of reperfusion, and the hearts were subsequently excised and frozen for 30 min. Heart tissues were cut into 1-mm-thick sections and incubated with 1% 5-triphenyltetrazolium chloride (TTC) at 37°C for 15 min, with white being the infarct zone (INF) and red and white areas being the high-risk zone (AAR). Infarct size was expressed by using the following formula: Infarct size = INF/AAR × 100%.TUNEL assay was used to detect apoptosis in heart tissues. Myocardial sections were prepared and then were incubated with 50 μL TUNEL mixture at 37°C for 1 h. After washing with PBS, sections were stained with DAPI. Myocardial apoptosis was observed by fluorescence microscopy. The apoptotic rate was calculated as the number of apoptotic cells (green)/the number of total cells (blue) × 100%.t-test was performed for comparison between two groups. ANOVA was used to compare with multiple groups. A P-value of <0.05 was considered statistically significant.Data were presented as the mean ± SEM. SPSS 23.0 software  was used to perform all analyses. Student’s Animal studies were performed in compliance with the ARRIVE guidelines. All animals received humane care according to the National Institutes of Health (USA) guidelines.P < 0.05). Similarly, the protein level of CTRP3 was also remarkably restrained in H9C2 cells in response to OGD/R treatment (P < 0.05).To explore the role of CTRP3 in myocardia I/R injury, we assessed the expression of CTRP3 in H9C2 cells after OGD/R treatment by using RT-qPCR and Western blotting. The results showed that the mRNA expression of CTRP3 was significantly decreased in H9C2 cells in response to OGD/R treatment (P < 0.05). MTT and EdU assay results showed that CTRP3 overexpression accelerated the growth of H9C2 cells after OGD/R treatment, while silence of CTRP3 inhibited cell proliferation (P < 0.05). In addition, flow cytometry verified that the apoptosis ability of H9C2 cells was notably suppressed after CTRP3 overexpression, while increased after CTRP3 silence (P < 0.05). Moreover, upregulation of CTRP3 reduced ROS, MDA and cTn-I content, and silencing CTRP3 notably facilitated ROS, MDA and cTn-I production in H92C cells after OGD/R treatment (P < 0.05).To investigate the biological effect of CTRP3 in OGD/R injury, H9C2 cells were transfected with pcDNA-CTRP3 or si-CTRP3. The results of Western blotting indicated that transfection of pcDNA-CTRP3 was efficient to elevate CTRP3 protein level, and the introduction of CTRP3 siRNA caused a markedly decrease of CTRP3 level in H92C cells (P < 0.05). Subsequently, the interaction between CTRP3 and LAMP1 was validated by using Co-IP assay (P < 0.05).To further detect how CTRP3 participated in OGD/R injury, proteins that could interact with CTRP3 were investigated. LAMP1 has been reported to be a putative receptor of CTRP3 , hence, IP assay . BesidesP < 0.05). LAMP1 overexpression significantly promoted cell proliferation after OGD/R injury by using MTT and EdU assays (P < 0.05). Moreover, cell apoptosis following OGD/R injury was conspicuously reduced by LAMP1 overexpression (P < 0.05). Likewise, upregulation of LAMP1 markedly cut down the production of ROS, MDA and cTn-I in H9C2 cells after OGD/R treatment (P < 0.05). These data indicated that LAMP1 overexpression improved OGD/G injury in H9C2 cells.Then, the effect of LAMP1 in H9C2 cells was investigated. The protein level of LAMP1 was notably increased after H9C2 cells transfected with pcDNA-LAMP1 compared with OGD/R treatment (P < 0.05). Flow cytometry analysis demonstrated that enforced expression of LAMP1 could restore the CTRP3 deficiency-mediated cell apoptosis (P < 0.05). Furthermore, absence of CTRP3 promoted ROS, MDA and cTn-I production in H92C cells subjected to OGD/R, which was markedly countervailed by LAMP1 overexpression (P < 0.05).To verify whether CTRP3 regulated the biological functions of H9C2 cells dependent on LAMP1 during OGD/R injury, H9C2 cells were transfected with CTRP3 siRNA alone or together with pcDNA-LAMP1. Silencing CTRP3 significantly reduced the protein expression of LAMP1, attenuated the proliferation ability of H9C2 cells, and LAMP1 overexpression reversed this alteration caused by CTRP3 knockdown (https://thebiogrid.org) and MGnify website (http://www.ebi.ac.uk/metagenomics) predicted that CTRP3 may serve as a binding partner of JIP2. Furthermore, Co-IP assay suggested that CTRP3 physically interacted with JIP2 (http://www.hitpredict.org/) found that LAMP1 could bind to protein numb homolog (NUMB), which in turn interacted with JIP2. Co-IP confirmed the association between LAMP1, NUBM and JIP2 (P < 0.05). Moreover, the effects of SP600125 on H9C2 cell proliferation and apoptosis were also investigated. JNK inhibitor (SP610025) reversed the promotive effects of CTRP3 or LAMP1 overexpression on the upregulation of JIP2 and p-JNK expression (P < 0.05). Overexpression of CTRP3 and LAMP1 effectively increased cell proliferation and decreased cell apoptosis, while SP610025 treatment could reverse these effects (P < 0.05). These results proved that CTRP3 alleviated OGD/R injury via LAMP1/JIP2/JNK pathway.The JNK pathway is known to associate with myocardial I/R injury and JNK-interacting protein-2 (JIP2) is closely related to JNK. BioGRID website (ith JIP2 . More inand JIP2  and 6C. P < 0.05), and protein expression of CTRP3, LAMP1, JIP2 and p-JNK (P < 0.05) were significantly lower in mice subjected to myocardial I/R surgery. Moreover, myocardial infarct volume (P < 0.05), apoptosis (P < 0.05), and the contents of ROS (P < 0.05), MDA (P < 0.05) and c-Tn1 (P < 0.05) were increased after I/R injury. However, mice injected to LV-CTRP3 could reverse the inhibitory effects of I/R injury on LVEF and LVFS levels and protein levels of CTRP3, LAMP1, JIP2 and p-JNK, and the promotive effects on myocardial infarct volume, apoptosis, and the contents of ROS, MDA and c-Tn1 (P < 0.05).A mouse model of myocardial I/R was established to explore the role of CTRP3 on myocardial injury. The levels of LVEF and LVFS (Myocardial infarction is a severe disease associated with high mortality, and reestablishing the blood supply to the ischemic myocardial tissue is the most feasible treatment. However, prone to myocardial I/R injury is prone to occur . Previouin vitro by blocking the phosphorylation of signal transducer and activator of transcription 3 by LAMP1 [Current evidence confirmed that LAMP1 was a routinely used as a lysosome marker and closely correlated with autophagy . Upregulby LAMP1 . Our stuNUMB plays an important role in cardiac progenitor cell differentiation and cardiac morphogenesis and serves as a potential candidate gene for cardiac regeneration and treatment of congenital heart disease , 33. MitJNK pathway was involved in cellular fate such as proliferation, migration, invasion and apoptosis , 37. HowIn conclusion, the present study highlighted the protective roles of CTRP3 in myocardial I/R injury, manifested by promoting cell proliferation and suppressing cell apoptosis and oxidative stress via upregulating LAMP1 and activating JIP2/JNK signaling pathway. Therefore, CTRP3 may be a promising therapeutic target for the treatment of myocardial I/R injury.


Z-score of <2.58 confirmed that viral quasispecies detected at T0 and T1 were closely related for both Pt1 and Pt2, while the Z-score for Pt4 was suggestive of possible reinfection. NGS data analyses indicate that the Z-score may be a useful parameter for distinguishing late relapse from reinfection.The risk of hepatitis C virus (HCV) recurrence after direct-acting antiviral (DAA) treatment is <0.5%. However, the distinction between HCV RNA late relapse and reinfection still represents a challenge in virological diagnostics. The aim of this study was to employ next-generation sequencing (NGS) to investigate HCV RNA recurrence in patients achieving a sustained virologic response (SVR) at least six months post-treatment. NGS was performed on plasma samples from six HCV-positive patients (Pt1–6) treated with DAA. NGS of HCV NS5B was analyzed before treatment (T0), after HCV RNA rebound (T1), and, for Pt3, after a second rebound (T2). Reinfection was confirmed for Pt5, and for the first rebound observed in Pt3. Conversely, viral relapse was observed when comparing T0 and T1 for Pt6 and T1 and T2 for Pt3. Z-scores were calculated and used to predict whether HCV-positive patient samples at different time points belonged to the same quasispecies population. A low  Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, with approximately 71 million chronically infected individuals worldwide ,3.Several phases have occurred in the history of antiviral therapy for the treatment of chronic HCV hepatitis. The first drug used in the treatment was interferon (IFN) with sustained virologic response (SVR) rates of less than 10%. In the late 1990s, ribavirin was combined with IFN, resulting in SVR rates of 35–40% in treated patients. An important turning point came with the introduction of pegylated interferon (peg-IFN) where SVR rates greater than 50% were achieved. The introduction of new direct-acting antivirals (DAAs) revolutionized HCV treatment, leading to the possibility of HCV elimination .HCV recurrence (both reinfection and late relapse) is defined as the reappearance of HCV RNA in treated patients after achieving SVR. Reinfection is suspected in cases of a recurrence of HCV infection after more than 12 or 24 weeks post-SVR . The risAn unresolved question is how long the follow-up time should be after the end of treatment (EOT) before a DAA-treated patient is considered cured of HCV. The international guidelines indicate that 12 weeks after stopping therapy is sufficient. Moreover, Sarrazin et al.  demonstrNGS can be a useful tool to detect the degree of similarity of viral strains between and within individuals. To our knowledge, few studies have employed NGS to distinguish between late relapse and HCV reinfection, especially for PWIDs and transmission networks between individuals with high-risk behaviors .The aims of the present study were to perform virological characterization of HCV-positive patient-derived samples by NGS to distinguish between late relapse and reinfection in DAA-treated patients with HCV RNA recurrence after achieving SVR24.The institutional ethics committee at the INMI L Spallanzani Hospital approved the protocol for the described study , which was conducted in accordance with Good Clinical Practice, as defined by the principles of the Declaration of Helsinki. All patients provided written informed consent to be included in the study.Plasma samples from six HCV-infected patients (Pt1–Pt6) treated with different DAA regimens at the INMI L Spallanzani Hospital and who presented with late viral rebound were considered for NGS analysis.The first patient Pt1,  was a 54The second patient Pt2,  was a 58Pt3  was a 49Pt4  was a 51Pt5  was a 43Pt6  was a 59HCV RNA levels were assessed using the HCV Abbott RealTime HCV Assay with a lower limit of detection (LLOD) of 12 IU/mL .HCV genotype was determined using the Real-time HCV genotype II assay .Viral RNA was extracted from plasma using the QIASYMPHONY DSP virus/Pathogen kit with an automated Nucleic acid QIASYMPHONY instrument . For HCV RAS analyses, the NS3 region was amplified using nested PCR using primers specific for GT1a or GT1b, as previously described ,20. SpecThe HCV NS5A region was amplified using nested PCR and pan-genotypic primers, as previously described .The HCV NS5B region was amplified using nested PCR and pan-genotypic primers, as previously described . To obtaResultant HCV amplicons  were sequenced directly using Prism BigDye in an ABI3100 DNA automated Sequencer .HCV NS3, NS5A, and NS5B sequences were submitted to Geno2pheno [HCV] v. 0.92  systems Each patient-derived Sanger sequence was aligned, using CLUSTAL W program, with reference sequences , available from GenBank. The best fit model was identified (considering The Bayesian information criterion (BIC) and the Akaike information criterion (AIC)) and a phylogenetic analysis performed using the maximum likelihood method with Kimura 2-parameter model+G, implemented in MEGAX software . To evalNGS was employed to analyze the HCV NS5B region from samples derived from six patients (Pt1–Pt6) collected before treatment (T0) and after HCV RNA rebound (T1). For Pt3, sequencing was also performed on a sample obtained after the second rebound (T2). The Ion-Torrent S5 NGS platform was used to analyze 13 samples collected from six patients. After purification of amplicons by AMPure XP beads  and quantitation with high-sensitivity dsDNA assay on Qubit 2.0 fluorometer , libraries were prepared using 100 ng of amplified DNA using the Ion Xpress Plus Fragment Library kit  following manufacturer’s instructions. Resultant libraries were quantified on the Bioanalyzer 2100  using a high-sensitivity kit and pooled to obtain a concentration of 100 pM.For each sample, reads of less than 500 nt in length and with low mean quality (Phred < 20) were discarded. Pollux v.1.0.2 ,26 was aMaximum likelihood phylogenetic analyses were performed using IQ-TREE : transitFor each patient, the genetic distance between and within each sample was calculated, and intra-sample nucleotide diversity (π) was estimated as the mean genetic distance within each sample ,31.The Mann–Whitney U test  was adopTo calculate the Kimura-2-parameter distance and to plot in three-dimensional scale the genetic distances of every representative sequence of the quasispecies present in each sample with reference sequences denoting GT1a (EF407457.1), GT1b (EU781827.1), and GT3a (X76918.1), the “scatterplot3d” package was employed using home-made R script. GT1a, ZGT1b, and ZGT3a scores were calculated for each patient with respect to reference sequences using the following equation:Z > 2.58, it possible to consider T1 and T2 as two different quasispecies populations with a significance level α = 0.01.With the assumption that distances between quasispecies and reference sequences follow a normal distribution, Z-scores were calculated for each patient to test the hypothesis that HCV sequences at different times belonged to the same quasispecies population. In particular, distance Z0 and T1 . For Z >Frequencies of intra-sample Single Nucleotide Variants (iSNV) and intra-sample Single Aminoacidic Variants (iSAV) were collected for each sample by using a home-made python script.NGS analyses of the HCV NS5B region were performed on 13 samples from six patients (Pt1–Pt6) collected at baseline  and at the first available plasma sample with HCV RNA recurrence. Pt3 experienced a second rebound in HCV RNA, and thus the corresponding sample (T2) was included in the analysis. High-throughput sequence data were deposited in the NCBI’s Sequence Read Archive (SRA) and are accessible under the Project Number [PRJNA720653].The mean number of raw reads obtained for each sample evaluated by NGS was 220,106 . A phylogenetic analysis was performed to compare all clustered sequencing reads representing each patient sample . PatientEach analyzed time-point for Pt1 (light and dark blue sequences) and Pt2 (light and dark red), respectively, displayed a similar sequence distribution in GT1a. This was also apparent for Pt4 (light and dark grey) and Pt6 (light and dark brown) in GT1b and GT3a, respectively. However, Pt6 appeared to have two distinct variant species in both Pt6_T0  and in Pt6_T1 .All observed Pt3 variants (green) clustered with GT1a. Interestingly, while Pt3 T0 sequences (light green) formed a cluster unrelated to sequences detected in T1 and T2 , T1 and T2 variants appeared to be phylogenetically related and associated with the same independent cluster. This was exemplified by three variants detected in Pt3_T1  that were also detected in Pt3_T2 .Based on Sanger sequencing results, Pt5 (violet) appeared to experience reinfection with HCV as sequences from T0 (light violet) and T1 (dark violet) clustered with GT1a and GT1b, respectively. NGS phylogenetic analysis supported the Sanger sequencing results since no GT1b variants were detected in the T0 sequence reads. This hypothesis was also supported by the patient’s declared risky lifestyle. Moreover, phylogenetic analysis based on Sanger sequences confirmed NGS results related to Pt3 and Pt5 .By using phylogenetic analysis, it was confirmed that Pt5 was reinfected with a different genotype while Pt3 was reinfected with a distantly related GT1a strain (between T0 and T1). In contrast, the same variant was detected in T1 and T2 samples for Pt3, and T0 and T1 samples for Pt6, supporting the hypothesis that these patients experienced a late viral relapse.p-value <0.001 for all samples , GT1b (EU781827.1), and GT3a (X76918.1). These distances were mapped and displayed using a three-dimensional plot to better visualize the distribution of quasispecies at different time points .GT1a, ZGT1b, and ZGT3a scores are reported in Z-scores were also calculated for each patient sample with respect to reference sequences representing GT1a (EF407457.1), GT1b (EU781827.1), and GT3a (X76918.1). Z-scores were calculated to test the hypothesis that HCV sequences at different time points belonged to the same quasispecies population, as indicated in the Materials and Methods section. ZGT1a and ZGT1b scores were −90.37 and 71.09, respectively  suggested viral quasispecies detected at T0 and T1 were closely related for both Pt1 and Pt2. Genetic distances of the quasispecies detected in Pt1 samples appeared interspersed in the three-dimensional plot, while genetic distances of quasispecies detected in Pt2 samples were only partially separated  position: a glutamine (Q) at NS5B aa 206 was detected in all (100%) quasispecies sequences at T0, which was replaced with either an arginine (R)  or a stop codon   at T1. IIn Pt2, NS5B sequence analysis revealed substitution enrichment at two aa positions when comparing quasispecies detected at T0 and T1: at aa 300, Q300  or R300  was detected in T0 sequences, while only R300 (CGA) was detected at T1. At aa 327, either alanine (A)  or tyrosine (Y)  was detected in T0 sequences, while only A327 was detected at T1 . HoweverIn Pt3, differences in NS5B sequences were detected at five aa positions between T0 and T1, while no substantial differences were detected between the T1 and T2 time-points . In partIn Pt4, emergence of a single NS5B substitution was detected at T1 when compared with NS5B sequences at T0: T181 (ACC) was detected at T0, while an asparagine (N) (AAC) was detected at T1 .For Pt5, NS5B sequence analysis revealed twenty aa differences between the T0 and T1 time-points. This was expected, since GT1a sequences were detected at T0 and only GT1b sequences were detected at T1 . In Pt6, K304 (AGC) was the predominant species (99.71%) at T0, while R304 (AAG) was a minor variant (0.29%). This was reversed at T1: R304 was enriched (89.24% of quasispecies sequences) compared with K304 (10.76%) .In this study, virological characterization of HCV-positive patient-derived samples by NGS was performed to discriminate between late relapse and reinfection in six DAA-treated patients experiencing late HCV RNA recurrence after achieving SVR24. Phylogenetic analysis of Sanger sequences supported the hypothesis that the observed viral rebounds in Pt3 (between T0 and T1) and Pt5 (between T0 and T1) were reinfection events . The behThe NGS and phylogenetic analyses confirmed that Pt5 and Pt3 (between T0 and T1) experienced reinfection events and that Pt3 (between T1 and T2) subsequently experienced a late relapse. Pt6 relapsed; however, HCV RNA, although low, never became undetectable during treatment. Additionally, variants detected in Pt3_T1 and in Pt6_T0 were also detected in Pt3_T2 and Pt6_T1 sequences, respectively.The recurrent HCV RNA observed post-SVR was harder to define in sample sequences from Pt1, Pt2, and Pt4. The frequency analysis of substitutions in each patient sample did not distinguish between late relapse and reinfection. Pt2 exhibited a 100% enrichment at T1 of an aa detected at 50% in T0 (R300 and A327). Pt1 and Pt4 each had only one emergent substitution detected, which was related to a nucleotide change (100% change by NGS) in the second position of the codon. These substitutions may be linked to the selection of variants associated with DAA RAS (Pt1) or due to infection with a new viral strain contracted post-SVR (Pt4).p-value <0.001) in patients assessed for potential late relapse and reinfection, differentiation by this approach was inadequate. Utilization of the Z-score, however, appeared to provide a method to discriminate between a reinfection (Pt5 and Pt3 (between the T0/T1 time-points)) and viral rebound ). In particular, the Z-score for the viral rebound group, except for Pt4, was <2.58, suggesting that this cut-off may be used to discriminate between reinfection and late relapse. Interestingly, Pt4 had a Z-score of 3.68, suggestive of a possible intra-familial reinfection.In an effort to find a genetic distance threshold able to discriminate between late relapse and reinfection events, genetic distances between and within each sample were calculated for each patient. Since the mean genetic distances between T0 and T1 (or T1 and T2 for Pt3) were statistically significant  and gastrointestinal mucosa (GIM) cells. In particular, stromal and neuroendocrine cells have exhibited HCV antigen positivity . The exiLate RNA recurrence in this case may comprise quasispecies less similar between T0 and T1, such as in Pt4. Our NGS data analysis, although preliminary and of limited sample size, indicates that employment of viral genetic distance to discriminate between a reinfection and late relapse  may be mNGS analysis plays an essential role in confirming a late relapse in treated HCV-positive patients who achieved SVR. However, analysis of a larger number of cases is required to determine with accuracy an appropriate parameter suitable for distinguishing a late relapse from reinfection. Furthermore, measuring HCV RNA in PBMCs from DAA-treated patients who were HCV RNA negative at EOT may provide insight into whether the persistence of HCV RNA in extrahepatic reservoirs elicits a late relapse. Interestingly, patients retreated with certain three-DAA regimens did not experience viral recurrence (data not shown), suggesting that these regimens may have greater efficacy on extrahepatic sites as well.


Canonical nonsense-mediated decay (NMD) is an important splicing-dependent process for mRNA surveillance in mammals. However, processed pseudogenes are not able to trigger NMD due to their lack of introns. It is largely unknown whether they have evolved other surveillance mechanisms.6A levels than their cognate protein-coding genes, associated with de novo m6A peaks and motifs in human cells. Furthermore, pseudogenes have rapidly accumulated m6A motifs during evolution. The m6A sites of pseudogenes are evolutionarily younger than neutral sites and their m6A levels are increasing, supporting the idea that m6A on the RNAs of pseudogenes is under positive selection. We then find that the m6A RNA modification of processed, rather than unprocessed, pseudogenes promotes cytosolic RNA degradation and attenuates interference with the RNAs of their cognate protein-coding genes. We experimentally validate the m6A RNA modification of two processed pseudogenes, DSTNP2 and NAP1L4P1, which promotes the RNA degradation of both pseudogenes and their cognate protein-coding genes DSTN and NAP1L4. In addition, the m6A of DSTNP2 regulation of DSTN is partially dependent on the miRNA miR-362-5p.Here, we find that the RNAs of pseudogenes, especially processed pseudogenes, have dramatically higher m6A RNA modification in cleaning up the unnecessary processed pseudogene transcripts to attenuate their interference with the regulatory network of protein-coding genes.Our discovery reveals a novel evolutionary role of mThe online version contains supplementary material available at 10.1186/s13059-021-02402-2. It degrades mRNAs with premature translation termination codons (PTCs), which are usually generated splicing , 2. NMD splicing . In yeassplicing . Howeversplicing . Althougsplicing , intronlsplicing , 6.PTENP1 [BRAFP1 [HMGA1 [PPM1K [nNOSP [As non-functional copies of closely related protein-coding genes, pseudogenes are one of the major class of substrates of NMD due to their accumulated nonsense mutations in the evolution , 8. AlthPTENP1 , BRAFP1  [BRAFP1 , and HMG1 [HMGA1 . Some ot1 [PPM1K , or workK [nNOSP .There are three classes of pseudogenes according to the unique biogenesis mechanisms: unitary pseudogenes, unprocessed pseudogenes, and processed pseudogenes . Unitary6A) RNA modification is reported in recent years as a novel pathway of degrading RNAs [6A is a reversible and prevalent internal RNA modification in mRNAs and long noncoding RNAs (lncRNAs). It is installed on “DRACH” motifs of RNAs [6A methyltransferases complex with METTL3 as the catalytic subunit [6A can be specifically regulated through a variety of RNA binding proteins and co-transcriptionally through transcription factors as well as H3K36me3 histone modification [6A modification on mRNAs, m6A readers such as YTH domain-containing proteins can specifically read the m6A and regulate various post-transcriptional processes of host mRNAs [6A have been reported in a variety of physiological and pathological processes [N6-methyladenosine  .If de novo formation of m6A sites on pseudogenes, we took advantage of INSIGHT  to estimate ρ, which represents the proportion of negatively selected m6A sites on pseudogenes. Our estimate of ρ of the m6A sites on pseudogenes was 0.2 ~ 0.3  and unprocessed pseudogenes  respectively , which represent the genes with higher essentiality and stronger functional constraint in the evolution, suggesting that important genes are more likely regulated by the modification of their processed pseudogene transcripts   and moderately methylated (0.6 > m6A level ≥ 0.3)  processed pseudogenes had dramatically lower gene expression than lowly methylated (m6A level < 0.3) processed pseudogenes  and lowly methylated processed pseudogenes  , the same cell type as GM12878, from 462 European participants of 1000 genome project [To further address whether m project , 38. We 6A of pseudogenes affects the crosstalk between pseudogenes and their cognate protein-coding genes, we compared the correlation coefficients of the gene expressions between pseudogenes and their cognate protein-coding genes in three categories of gene pairs with different m6A levels of pseudogenes. We found that the processed pseudogenes with higher m6A levels had significantly lower correlation coefficients   Fig. c, suggesnes Fig. d.6A of pseudogenes affects the expression of their cognate protein-coding genes, we selected two representative processed pseudogenes DSTNP2 and NAP1L4P1 for further validation in GM12878 cell line.To experimentally test whether the mDSTNP2 can regulate its protein-coding cognate DSTN. We found knockdown of DSTNP2 by siRNA significantly down-regulated mRNA as well as the protein expression of DSTN  [TUSC2P (tumor suppressor candidate-2 pseudogenes) promotes TUSC2 function by binding multiple microRNAs, and ectopic expression of TUSC2P and TUSC2 inhibits cell proliferation, survival, migration, invasion, and colony formation, and increases tumor cell death [via the piRNA pathway in the germline [Though these competitive endogenous RNAs transcribed from pseudogenes are deleterious at the early stage of evolution, it is also possible that some of them become functionally important. For example, the lncRNA  (mESCs) . The cel (mESCs) . The TUSll death . Pseudoggermline . The Pangermline . In prinvia m6A RNA methylation. How does this mechanism evolve? In this study, we found a significantly higher probability of obtaining novel m6A motifs on sense strand than antisense strand, strongly suggesting that obtaining m6A sites on pseudogenes tends to be adaptive and accumulating in the evolution. In this way, the elevated m6A on processed pseudogenes are the results of positively selected mutations that produce novel m6A sites. Alternatively, we cannot rule out that there is an unknown specific mechanism, such as RBP-mediated specific regulation of m6A [6A RNA modification. However, whether and how the cells mark processed pseudogenes is still unknown and requires further investigation.Here, we found a novel mechanism of RNA surveillance for processed pseudogenes n of m6A , 50, to 6A played in evolution especially human evolution are still elusive. Ma et al. reported that newly acquired m6A modifications in humans were under positive selection [et al. had contradictory viewpoint that these newly acquired m6A sites were neutral and most m6A sites in protein-coding regions were nonfunctional and nonadaptive [et al. reported that a significant fraction of 3′ UTR m6A sites were under negative selection and recently gained 3′ UTR m6A in humans were positively selected [6A on human transcribed pseudogenes are evolutionarily young and evolved under positive selection, indicating that m6A plays more important and widespread roles than expected in human evolution.The roles of melection . Howeveradaptive . Recentlselected . Our stu6A RNA modification, it plays important roles in diverse physiology process which need to remove existing RNAs. It removes the RNAs of current cell status to facilitate cell fate transition [6A mediated RNA decay. In addition, nonsense mutations and frame-shift mutations that occur on intronless protein-coding genes can also produce poisonous truncated proteins, and we found the RNAs of protein-coding genes with single exons and the limited number of exons also showed significantly higher m6A levels than genes with a larger number of exons, suggesting that m6A-mediated RNA degradation may also play roles for RNA surveillance of protein-coding genes with single or a few of exons  [6A sites that are under negative selection. The rejected substitution scores calculated by GERP++ [6A sites. The ages of individual m6A sites were determined using a phylostratigraphy approach [6A peaks on pseudogenes were used as evolutionally neutral control sites.The SRAMP  and WhislemenTs)  to estimy GERP++  were useapproach  with pai2. Both cell lines were obtained from GuangZhou Jennio Biotech Co., Ltd., authenticated and tested for the absence of mycoplasma contamination using Myco-Blue Mycoplasma Detector (Vazyme).HEK293T (ATCC® CRL-3216™) cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (Corning), supplemented with 10% FBS  at 37 °C with 5% CO2. GM12878 cells were cultured in Roswell Park Memorial Institute Medium 1640 (Corning), supplemented with 15% FBS  at 37 °C with 5% CO4 GM12878 cells were seeded in a TC-untreated plate and transduced with viral supernatants in the presence of polybrene (8 μg/μL). Twenty-four hours after transduction, cells were selected with puromycin (2 μg/mL).For lentiviral production, 293 T cells were seeded in 6 cm cell culture plates, and 24 hours later, cells were transfected with 6.4 μg of lentiviral backbone, 4.8 μg of psPAX2 (Addgene #12260), and 1.6 μg of pMD2.G (Addgene #12259) using LipoFiter reagent (HANBIO). Lentiviral supernatants were harvested at 48 h and 72 h after transfection and filtered through using a 0.45μm PVDF filter (Millipore) and concentrated using PEG. 5 × 106A motif mutant DNA sequence of DSTNP2 and NAP1L4P1 genes were synthesized by GENEWIZ company. Lentiviral expression plasmids were generated using ClonExpress II One Step Cloning Kit , by combining PCR-amplified cDNA and EcoR I/BamH I digested pCDH-CMV-MCS-EF1α-CopGFP-T2A-Puro (SBI) backbone.Wild type and mCells were seeded in TC-untreated plates and transfected with specific miRNA inhibitors and inhibitor NC or siNC and specific siRNA using LipoFiter reagent (HANBIO). RNA samples and protein samples were harvested at 72 h after transfection for qRT-PCR.Proteins were extracted from cells by incubating with RIPA buffer  on ice for 10 min and insoluble fraction was removed by centrifugation. Twenty micrograms of extracted protein was separated on 15% SDS-PAGE and transferred to PVDF membrane. Membranes were blocked in 5% BSA in Tris-Buffered Saline with 0.01% Tween 20 (TBS-T) at room temperature for 1 h and incubated overnight with primary antibodies diluted in 1% BSA/TBS-T at 4 °C, followed by incubating with HRP conjugated secondary antibody diluted in TBS-T for 1 h at room temperature, and visualized using Clarity™ Western ECL Substrate (Bio-Rad). The following antibodies were used for immunoblotting: DSTN , GAPDH .-∆∆CT method. The primers of pseudogenes and their cognate protein-coding genes, which had been appended in Additional file Total RNA was extracted using the NucleoZol RNA reagent (MACHEREY-NAGEL). And fraction RNA was separated using the Cytoplasmic and Nuclear RNA Purification Kit (NORGEN). One microgram of DNA-free RNA was then reverse-transcribed using HiScript III RT SuperMix for qPCR (+gDNA wiper) Vazyme, R232). qPCR was carried out using the ChamQ Universal SYBR qPCR Master Mix  and performed in an LC480 Real-Time PCR System (Roche). Fold-change was calculated using the 232. qPCR RNA-seq libraries of the input, cytoplasmic, and nuclear RNAs of GM12878 with and without METTL3 knockdown were prepared using the VAHTS® mRNA-seq V2 Library Prep Kit for Illumina from Vazyme and sequenced on Illumina HiSeq 2500 platform to generate 150 bp paired-end reads.18S was used as the reference gene and fold-change was calculated using the 2-∆∆CT method.The final concentration of 5 μg/mL Actinomycin D  was added to cells to assess RNA stability. After incubation for indicated time points, the cells were collected, and RNA samples were extracted for reverse transcription and qPCR. Additional file 1: Figure S1. The optimization of conventional HISAT2 mapping procedure. Figure S2. Comparisons of m6A between pseudogenes and their cognate protein-coding genes in GM12878 and H1 cell line. Figure S3. Comparisons of m6A between pseudogenes and their cognate protein-coding genes in H1 cell line. Figure S4. Comparisons of pseudogenes m6A among 5′UTRs, CDSs, 3′UTRs and GC contents in their cognate protein-coding genes in GM12878 cell line. Figure S5. Positive natural selection of the m6A on pseudogenes in H1 cell line. Figure S6. m6A facilitates the cytosolic degradation of cognate protein-coding genes of processed pseudogenes in GM12878 cell line. Figure S7. m6A facilitates the cytosolic degradation of processed pseudogenes in H1 cell line. Figure S8. m6A facilitates the cytosolic degradation of cognate protein-coding genes of processed pseudogenes in H1 cell line. Figure S9. The gene regulatory network and primer design principles of processed pseudogenes and cognate protein-coding genes. Figure S10. The uncropped bots of western blots in Fig. Figure S11. Experimental validation of m6A on processed pseudogene NAP1L4P1 affects the expression of NAP1L4 in GM12878 cell line. Figure S12. Correlation between m6A levels and exon numbers of coding genes.Additional file 2: Table S1. The list of pseudogenes and their cognate protein-coding genes. Table S2. The m6A levels of pseudogenes and their cognate protein-coding genes in GM12878 cell line. Table S3. The m6A levels of pseudogenes and their cognate protein-coding genes in H1 cell line. Table S4. The primers of processed pseudogenes  and their cognate protein-coding genes . Table S5. The list of collected GEO datasets. Table S6. The list of collected EBI datasets.Additional file 3. Review history.


Streptococcus pneumoniae 19A (ST19A) and 19F (ST19F) are among the prevalent serotypes causing pneumococcal disease worldwide even after introduction of a 13-valent pneumococcal conjugate vaccine (PCV13). Synthetic glycoconjugate vaccines have defined chemical structures rather than isolated polysaccharide mixtures utilized in marketed vaccines. Ideally, a minimal number of synthetic antigens would cover as many bacterial serotypes to lower cost of goods and minimize the response to carrier proteins. To demonstrate that a chimeric oligosaccharide antigen can induce a protective immune response against multiple serotypes, we synthesized a chimeric antigen (ST19AF) that is comprised of a repeating unit of ST19A and ST19F capsular polysaccharide each. Synthetic glycan epitopes representing only ST19A, and ST19F were prepared for comparison. Semisynthetic glycoconjugates containing chimeric antigen ST19AF induced high antibody titers able to recognize native CPS from ST19A and ST19F in rabbits. The antibodies were able to kill both strains of pneumococci. Chimeric antigens are an attractive means to induce an immune response against multiple bacterial serotypes. Chimeric antigens are an attractive means to induce an immune response against multiple bacterial serotypes. The chimeric semisynthetic glycoconjugate ST19AF induced antibodies with opsonic activity able to kill ST19A and ST19F bacteria in rabbits. Streptococcus pneumoniae (Sp), are a major cause of morbidity and mortality in toddlers and older adults worldwide.2,3 Despite the introduction of a 13-valent pneumococcal conjugate vaccine (PCV13) in 2010, ST19A remains a major pathogen worldwide4–8 that is associated with 14% of all IPD cases.9 Since the efficacy of PCV 13 against ST19A is being debated4,10,11 the development of new vaccine approaches covering ST19A and ST19F is desirable.Invasive pneumococcal diseases (IPD) caused by 12 The ST19 CPS is among the most labile polysaccharides used for vaccine production as the phosphate diester groups can be cleaved during purification and conjugation resulting in severely suppressed immunogenicity.13,14 Phosphate diesters in the polysaccharide of Salmonella typhimurium and Clostridium difficile PSII are crucial for inducing a strong immune response to the native polysaccharide.15,16 Synthetic oligosaccharide conjugates containing well-defined antigens, have proven very effective in preventing bacterial infections caused by encapsulated bacteria,17–27 and the conjugation process does not impair antigens having phosphate diester groups. The saccharide to protein ratio (glycan loading) is an important parameter for glycoconjugate vaccine development. The novel strategy described here, helps to reduce the amount of protein used and may simplify the formulation of multicomponent vaccines against bacterial pathogens.Currently glycoconjugate vaccines contain polysaccharides isolated from bacterial cell cultures, that are conjugated to proteins to create heterogeneous vaccine compositions.d-ManpNAc-(1 → 4)-α-d-Glcp disaccharide linked to C2 (ST19F 1) or C3 of L-Rha (ST19A 2) respectively  and ST19A (4) via a phosphodiester linkage to form the chimeric oligosaccharide antigen ST19AF (5) (Almost 100 19AF (5) . A termi19AF (5) .3, 4, 5 requires the stereoselective installation of two 1,2-cis glycosidic linkages and the stereoselective formation of an α-phosphodiester linkage to the trisaccharide  in 82% yield . Selective levulinoyl ester (Lev) cleavage (92%) using hydrazine acetate followed by inversion of the 2-hydroxyl group was achieved by conversion of the corresponding sulfonyl diimidazole and subsequent nucleophilic displacement with tetrabutylammonium azide to obtain 16 (82% over two steps). The inversion of stereochemistry was confirmed by examination of the 1H–1H and 13C–1H coupling constants45,46 to H-1 at δ 4.43  and 1JC–H = 163.2 Hz. Conversion of the azide to the corresponding acetamido group was obtained by treatment with freshly activated Zn–Cu couple to give 17 in 74% yield. Removal of the p-methoxyphenyl group using ceric ammonium nitrite (CAN) gave hemiacetal 18. The installation of a stereoselective H-phosphonate at the reducing end of phosphate diester 19 was achieved with phosphorous acid and an excess of sterically bulky 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinane41 in pyridine at 50 °C to obtain thermodynamically more stable α-H-phosphonate 6 . The selectivity may result from conversion of the more reactive β-glycosyl H-phosphonate to the thermodynamically more stable α-phosphonate by SN2 displacement with excess phosphorous acid.47 The protected phosphodiester-linked trisaccharide 19 was prepared by coupling H-phosphonate 6 with 5-azido pentanol using pivaloyl chloride in pyridine as a condensing agent followed by oxidation with iodine in water–pyridine. Hydrogenation of 19 provided deprotected trisaccharide sodium salt 3 after passing through Dowex 50W X4 Na+ resin as a single diastereomer.Antigen construction of ccharide . Trisacc2% yield . Regiose4 followed a similar synthetic approach , which was subjected to reductive benzylidene acetal cleavage to obtain 21. Disaccharide 21 containing a 4-hydroxyl group was coupled with glycosyl imidate 11 to give trisaccharide 22 . Cleavage of the Lev group followed by stereoselective inversion of the C2-hydroxyl group in 23 yielded the corresponding azide 24 . Reduction of the azide to the corresponding acetamido derivative using Zn–Cu couple afforded 25. Hydrolysis of pentenyl glycoside delivered hemiacetal 26, which was converted into the azidopentyl linked phosphonate derivative 27via the H-phosphonate method. Hydrogenation using Pd/C in EtOAc : MeOH : H2O (3 : 2 : 1) provided deprotected trisaccharide as triethylammonium salt that was converted into corresponding sodium salt using Dowex 50W X4 Na+ resin to obtain white solid 4 as a single diastereomer .The synthesis of ST19A trisaccharide approach . Orthogo25 and 6 in hand, the synthesis of the chimeric antigen 5 commenced . Acetate ester cleavage furnished acceptor 7 that was condensed with ST19F H-phosphonate 6 to afford hybrid ST19AF 32. Finally, hexasaccharide 32 was deprotected by hydrogenolysis to provide the disodium salt of antigen 5 following exchange with Dowex 50W X4 Na+ resin. The antigens 3, 4, and 5 were obtained as white solids following purification by reverse phase C18-column HPLC and lyophilization.With trisaccharides ommenced . Reducti3–5 were conjugated to CRM197 with an average of seven, five and five oligosaccharides attached per CRM197 molecule respectively as determined by MALDI analysis. Rabbits were immunized with conjugates containing the chimeric ST19AF, or the ST19A or ST19F antigens formulated with aluminum hydroxide. The conjugates induced strong anti-glycan antibody titers after delivery of three vaccine doses at two week intervals. The long term immune response was confirmed following a boost three and a half months after the last immunization (day 133). High antibody titers were faster attained showing that memory B cells were activated and able to generate antibodies against the vaccine antigen (day 144)  . Rabbitsday 144) . Increasvia MHC class II  and ST19F (CPS19F) to determine the cross-reactivity of antibodies produced against synthetic or native antigens. Chimeric ST19AF generated antibodies that recognize both CPS. Anti-CPS19A antibody titer was highest after three doses (day 35). ST19A antigen at the reducing-end of the chimeric structure may favor a better B-cell presentation due to the proximity to the carrier protein presented class II .50 ST19Fdetected . The absbacteria .in vitro, was determined by an opsonophagocytic killing assay (OPKA) with sera of rabbits immunized with three doses of ST19A, ST19F, ST19AF, Prevnar13®, or PBS and aluminium hydroxide  was performed in strict accordance with the NIH/OLAW Animal Welfare Assurance, identification number F16-00178 (A5755-01) and was authorized by LALLF MV  in accordance to TierSchG 7221.3-2-002/19. The immune response was analyzed by glycan microarrays and ELISA. The functional attribute of the immune response was monitored by OPKA using HL-60 cells. Bacteria serotypes ST19A and ST19F were isolates from Charité Infektiologie und Pneumologie department, Universitätsmedizin Berlin. Detailed materials and methods can be found in ESI Appendix.†None of the authors declare a conflict of interest.SC-011-D0SC02230F-s001


Notch-Delta signaling regulates many developmental processes, including tissue homeostasis and maintenance of stem cells. Upon interaction of juxtaposed cells via Notch and Delta proteins, intracellular domains of both transmembrane proteins are cleaved and translocate to the nucleus. Notch intracellular domain activates target gene expression; however, the role of the Delta intracellular domain remains elusive. Here, we show the biological function of Delta like 1 intracellular domain (D1ICD) by modulating its production. We find that the sustained production of D1ICD abrogates cell proliferation but enhances neurogenesis in the developing dorsal root ganglia (DRG), whereas inhibition of D1ICD production promotes cell proliferation and gliogenesis. D1ICD acts as an integral component of lateral inhibition mechanism by inhibiting Notch activity. In addition, D1ICD promotes neurogenesis in a Notch signaling-independent manner. We show that D1ICD binds to Erk1/2 in neural crest stem cells and inhibits the phosphorylation of Erk1/2. In summary, our results indicate that D1ICD regulates DRG development by modulating not only Notch signaling but also the MAP kinase pathway. Delta1 mRNA also shows a ‘salt and pepper’ pattern among the neighboring cells. The proliferating cells in the developing nervous system subsequently undergo neuronal differentiation, thus gradually increasing the number of neurons. Later, the dividing cells give rise to satellite glial cells similar to mouse DRG. Based on these studies, it has been proposed that Notch signaling regulates proliferation and differentiation during DRG development via a lateral inhibition mechanism  development . During echanism .Hes and Hey  by enhancing TGF-β signaling by binding to Smad2 and/or Smad3 proteins  ligands  and four different Notch receptors. Upon binding with the DSL ligands (except Dll3) that are expressed on the surface of neighboring cells, the Notch receptor is first cleaved by a disintegrin and metalloprotease complex (ADAMs) followed by γ-secretase . The rel and Hey . It has  and Hey . Few stuproteins . Jag1 inproteins . Moreoveproteins . Contrarproteins . TherefoIn the present study, we generated two different genetically modified mice: one that overexpresses D1ICD using the Cre-loxP system and another that fails to produce D1ICD by deletion of genome sequences that are essential for the cleavage of Dll1. We show that, during DRG development, D1ICD overproduction promotes neurogenesis and inhibits proliferation, while inhibition of D1ICD production showed the opposite phenotype. With regards to the underlying molecular mechanism, we find that D1ICD acts as a component of the lateral inhibition by cooperating with Numb to repress Notch signaling, and that D1ICD also represses the MAP kinase pathway by inhibiting Erk1/2 phosphorylation.Fig. S1A,B). NCC-specific expression of 3×HA_D1ICD_Flag was achieved by crossing the mice with a Wnt1-Cre mouse, which induces recombination in cranial, cardiac and trunk NCCs  . Furthermore, we observed that D1ICD overproduction reduced the cell numbers and the DRG size at E12.5 . However, no difference in the ratio of apoptotic cells was observed in the D1ICD overproducing DRG compared with the control DRG  , indicating a reduction in the cell proliferation rate at E12.5. These results suggest that induced D1ICD inhibits cell proliferation after E10.5, when second-wave neurogenesis starts.In order to analyze the role of D1ICD in DRG development, we first examined the expression pattern of Dll1 protein and Notch activity using specific antibodies. We focused on the trunk level DRG at E12.5, as it is well documented that the differentiation to neuron or glia from the common multipotent stem cells is regulated by Notch signaling at this stage . Dll1 exig. S1B) . The rectrol DRG C,D. To eig. S1B) . We obseig. S1B) E,F. Addiig. S1B) G,H and nFig. S1A) (Notch1 and lunatic fringe (Lfng), both of which are expressed in the migrating NCCs and enhances Notch1-Dll1 binding . We also evaluated the Notch signaling transduction ability of NC-Dll1 by co-culturing cells expressing NC-Dll1 with NIH3T3 cells expressing Notch1, Lfng and transfected TP1 luciferase Notch-reporter. We observed no difference in the Notch signaling transduction ability between the wild-type Dll1 and NC-Dll1 . In addition, to reproduce the Notch-Delta signal transduction found in native DRG development, we isolated neural crest stem cells (NCSCs) using FACS from E12.5 DRG obtained by crossing Wnt1-Cre mice with GFP-reporter mice. NCSCs can differentiate into neurons, glia and myofibroblasts upon withdrawal of treatment with growth factors . This result indicates that the function of NC-Dll1 as the Notch ligand is not very different from that of wild-type Dll1, even in NCSC. Based on these results, we generated a mouse line producing NC-Dll1 by deleting the 48 bp genome sequence through homologous recombination using the CRISPR-Cas9 system . We obtained two independent heterozygous F0 NC-Dll1 mice, which were indistinguishable from the wild-type mice. We then analyzed DRG development in the homozygous NC-Dll1 mice, which were viable and showed no obvious abnormalities except hyperactive behavior. Importantly, we observed contrasting phenotypes in the DRG at E12.5 between the NC-Dll1 mice and the D1ICD-expressing mouse. The DRG cell number and the cell proliferation rate, as measured by Ki67 immunostaining, were increased in the NC-Dll1 mice at E12.5  . To conf binding . In west factors . Thus, wat E12.5 I-L. TakeOverproduction of D1ICD in DRG resulted in deceased cell proliferation without affecting cell death, suggesting that D1ICD-expressing cells might have entered a premature differentiation pathway. To examine the effect of sustained D1ICD production in DRG neurogenesis, we performed immunostaining for several differentiation markers. During DRG development, the first wave neurogenesis occurs in migrating NCCs until E10.5 . It has Tuj1 mRNA expression per Bfabp mRNA expression decreased in the NC-Dll1 DRG, indicating that the repression of D1ICD production promoted cell differentiation toward glial cells . A Delta-expressing neuronal precursor cell activates Notch signaling in the neighboring cells, leading to the induction of the downstream target genes Hes and Hey. The cells with active Notch signaling (Notch-active cells) proliferate or differentiate to glia. Delta expression is suppressed in the Notch-active cell; therefore, Notch signaling is suppressed in the cells adjacent to the Notch-active cells. As both Notch and Delta are membrane-bound proteins, the Notch- and Delta-mediated signaling usually requires close cellular proximity between adjacent cells; however, each adjacent cell is capable of sending the signal to the next. Therefore, the lateral inhibition regulates the cell number and differentiation in the whole DRG. To determine whether D1ICD participates in the Notch signaling pathway, we analyzed Notch activity-induced embryos produced by crossing the Wnt1-Cre with the CAG-CAT-D1ICD mice. We found that the NICD signal was absent in most of the D1ICD-induced cells but was strongly observed in the GFP-negative neighboring cells . Next, we looked for endogenous Dll1 protein expression under the control of Notch signaling by immunostaining using an anti-Dll1 N-terminal region antibody. Endogenous Dll1 signal was detected only in the GFP-positive cells, indicating that endogenous Dll1 expression was repressed in the Notch-active neighboring cells via the lateral inhibition mechanism . However, Notch-active cells were more frequently found near D1ICD-overproducing cells than around the control cells expressing only GFP . We transfected the siRNAs and Hey1-reporter plasmid set  into the cells at 24 h and 48 h after cell seeding, respectively . These results indicate that the lateral inhibition mechanism also worked properly even in NC-Dll1, and the upregulation of Notch activity is because of a lack of Notch activity modulation by D1ICD rather than disruption of the lateral inhibition mechanism. Taken together, these results suggest that D1ICD functions as a component of the lateral inhibition mechanism by repressing Notch signaling in the same cell.To further investigate the possible role of D1ICD, we examined the Notch activity in NC-Dll1 DRG that lacks D1ICD production. We found that the ratio of Notch active cells was increased in NC-Dll1 DRG . Notch aTuj1 mRNA expression was increased in D1ICD-expressing NCSCs compared with control NCSCs, although the expression of glial marker Gfap and myofibroblast marker α-smooth muscle actin  was not different . These results suggest that D1ICD promotes neurogenesis in NCSCs. Next, we conducted the differentiation assay using exactly the same NCSC population as the material by withdrawing growth factors and treating with the γ-secretase inhibitor compound E, to elucidate the possible function of D1ICD independent of Notch signaling. We confirmed that Notch signaling was inhibited by compound E treatment, because Hes1 mRNA expression was decreased . D1ICD significantly increased Tuj1 expression even in the NCSCs treated with compound E . These results indicate that D1ICD promotes neuronal differentiation independently of endogenous Notch signaling.Next, we investigated a hypothesis that D1ICD could promote neuronal differentiation in a Notch signaling-independent manner because it is reported that D1ICD promotes neuronal differentiation in NSCs by activating TGF-β/activin signaling through binding to Smad proteins . To exammpound E A, althouFig. S5C,D). To determine the possible binding partners of D1ICD, we performed immunoprecipitation using anti-HA and anti-Flag antibodies in NCSCs overexpressing the D1ICD, followed by mass spectrometry (IP-MS/MS). As the amount of D1ICD protein in the cells was too low, we could not detect any protein, including D1ICD, by MS. Thus, we used HEK293T cells transfected with 3×Flag_D1ICD, and precipitated and detected the D1ICD-binding proteins by IP-MS/MS using the anti-Flag antibody (Tables S1 and S2). The immunoprecipitated proteins were functionally annotated using the Database of Annotation, Visualization, and Integrated Discovery (DAVID) (Table S3). We detected membrane-associated guanylate kinase, WW and PDZ domain-containing protein (MAGI) 1 and MAGI3 proteins that are known D1ICD-binding proteins . In addition, the protein levels of ERK1 and ERK2 did not change, but the level of phosphorylated ERK1 and ERK2 (pErk1/2) was decreased  . To confproteins , indicatproteins . Furtherproteins . Therefoecreased B,C. We aecreased D. These in vivo, we performed immunostaining for pErk1/2 at E12.5. We found that the ratio of pErk1/2-positive cells among GFP-positive cells was decreased in the E12.5 DRGs overproducing D1ICD from E10.5 using UBC-CreERT2 , Notch signaling promoted cell proliferation and glial differentiation by repressing proneural genes and their downstream target Dll1. On the other hand, in the neighboring Notch-inactive cells, the expression of proneural genes and Dll1 was upregulated, resulting in neuronal differentiation. This balance is tuned by Notch signaling via lateral inhibition mechanism among the DRG cells . D1ICD inhibits Notch signaling in coordination with Numb. As a result, the expression of proneural genes is elevated to promote neuronal differentiation, together with the upregulation of Dll1. Therefore, the increased Dll1 strongly activates Notch signaling in the neighboring cells. Furthermore, our study reveals that D1ICD promotes neuronal differentiation in NCSCs in a Notch signal-independent manner  or presence .Collectively, we propose a model in which D1ICD plays a crucial role in DRG development via two mechanisms: the modulation of the lateral inhibition mechanism by inhibiting Notch signaling; and repression of the MAP kinase pathway by inhibitingErk1/2 phosphorylation (in vitro transcription (Thermo Fisher Scientific) using pX330 plasmid (Addgene plasmid #50718). The gRNA sequences were CCCAGGGGAAGGGCCCGCCC and UCACUGAGGUCCACCACCAU. The Oligo DNA sequence for homologous recombination is GCTATGGCGGCCCCAACTGCCAGTTTCTGCTCCCTGAGCCACCACCAGGGCCCTTCCCCTGGGTGGCCGTGTGTGCCGGGGTGGTGCTTGTCCTCCTGCT. The NC-Dll1 allele was detected by PCR. The animals had access to a standard chow diet and water ad libitum, and were housed in a pathogen-free barrier facility with a 12 h light:12 h dark cycle.The wild-type mice used in this study were MCH strain (CLEA Japan). Wnt1Cre and UBC CreERT2 were provided by S. Iseki  and P. Chambon , respectively. Transgenic mice that constitutively express loxP-CAT-loxP-3xHA_D1ICD_Flag under the control of the CAG promoter and a mouse that harbors non-cleavable Dll1 (NC-Dll1) via a specific 48 bp deletion of essential genome sequence for cleavage were generated in our laboratory by nucleotide injection into fertilized eggs. Homologous recombination was stimulated by CRISPR. Cas9 mRNA and gRNA were generated by Pregnant mice were injected intraperitoneally with 0.5 ml tamoxifen  dissolved in corn oil (20 mg/ml) at E10.5. Embryos were sampled at E12.5.Each Dll1 in pcDNA3.1 and NC-Dll1 in pcDNA3.1 was transfected into NIH3T3 cells expressing Notch1 and Lfng using Lipofectamine LTX (Thermo Fisher Scientific). 24 h after transfection, whole cells extracts were collected by Sample Buffer Solution with 2-ME  for western blotting. Reporter assays were carried out by the co-transfection of reporter plasmids TP1-luciferase  and pRL-TK (Promega) with Dll1 or NC-Dll1 into Notch1 and Lfng-expressing NIH3T3 cells . Cell lyThe Hey1 promoter construct was provided by Hiroki Kokubo . Luciferase is expressed under the control of Hey1 promoter activity. The constructs and pRL-TK (Promega) were transfected with either or both D1ICD in pc DNA3.1 and NICD in pcDNA3.1 into HEK293T cells by Lipofectamine LTX (Thermo Fisher Scientific). Cell lysates were then used for the luciferase assay using Dual luciferase reporter assay system (Promega).Numb and Numbl expression was knocked down using Stealth RNAi siRNA for human Numb  and Numbl (HSS113716), respectively. Transfection of NIH3T3 cells with the siRNA was started by the addition of Lipofectamine RNAiMAX complex (Invitrogen) containing siRNA to the culture medium. As a negative control for the siRNA treatment, Medium GC Stealth RNAi Negative Control  was used.Embryos were fixed with 4% PFA/PBS overnight at 4°C, submerged in sucrose/PBS at 4°C. The trunk of the embryo was dissected, then embedded in OCT compound (Sakura Finetek) and frozen. Following antigen retrieval, frozen sections (14 μm) were incubated with primary antibodies against cleaved Notch1 , the Dll1C-terminus , Dll1 N-terminus  and pERK1/2 . Sections were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody  and enhanced using a Tyramide signal amplification system (Perkin-Elmer). GFP, p75, Sox10, Tuj1, BFABP and ki67 were detected using chick anti-GFP , rabbit anti-p75 , goat anti-Sox10 , mouse anti-Tuj1 , rabbit anti-BFABP  and mouse ki67  primary antibodies. Bound primary antibodies were visualized using secondary antibodies: Alexa Fluor 488 goat anti-chicken IgG, Alexa Fluor 488 donkey anti-goat IgG, Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 594 donkey anti-goat IgG, Alexa Fluor 594 donkey anti-mouse IgG, Alexa Fluor 594 donkey anti-rabbit IgG . Images were acquired using the Olympus Bx51 microscope, and captured using a CCD camera and cellSens standard software (Olympus). Image settings such as brightness and contrast were changed using Photoshop CS5 extended.Table S4. The details of the quantification are described in the supplementary Materials and Methods.Because DRG development shows differences along the anteroposterior axis, counting data for comparison were collected from the sections at the thoracic level, which was determined by the appearance of the heart . Each scTrunk DRGs at E12.5 were dissected out and digested in 0.1% collagenase (Sigma), 0.1% dispase (Roche) and 0.05% DNase I (Roche) in HBSS supplement with 10% FBS and 1% penicillin and streptomycin for 30 min at room temperature. Isolated NCSCs were cultured in DMEM/F12 medium supplemented with N2, B27, primocin, 10 ng/ml EGF, 20 ng/ml FGF, 50 ng/ml IFG and 50 ng/ml heparan sulfate on the dish coated with ornithine and fibronectin. NCSCs were plated at 1000 cells/well on an ornithine- and fibronectin-coated eight-well chamber slide, and differentiated for 3 days with DMEM/F12 supplemented with N2, B27 and primocin.NCSCs were passaged using accutase (Thermo Fisher Scientific) and filtering with strainers (40 μm). GFP-expressing NCSCs were isolated by FACS Aria (BD Biosciences). FACS data were analyzed with BD FACSDIVA software.NCSCs were transfected with 3×HA_D1ICD_Flag in pcDNA3.1 using ViaFect transfection reagent (Promega). After 24 h, NCSCs were treated with 1 μM of the protease inhibitor epoxomicine. Nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific).HEK293T cells were transfected with 3×Flag_D1ICD in pcDNA3.1. Transfected cells or NCSCs (±3×HA_D1ICD_Flag) were treated with IP lysis buffer (Thermo Fisher Scientific) with proteasome inhibitor cocktail  and immunoprecipitated with anti-Flag M2 resin or anti-HA resin (Sigma), respectively. The proteins were eluted with 3×Flag peptide (Sigma) or SDS-PAGE sample buffer .Immunoprecipitated proteins and NCSC lysate were separated by SDS PAGE. For immunoblotting, antibodies used were as follows: anti-HA, anti-Numb, anti-Erk1/2 and anti-pErk1/2 , anti-β-actin , anti-Numbl, anti-Lamin B1 and anti-GAPDH , and anti-Dll1 IC-termius anti-acetylated α tubulin, anti-Cdk2 and anti-Cdk4 . Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson ImmunoResearch. Each of the protein bands were visualized. For the detection of Numb, Erk1/2 and pErk1/2 protein, the same amount of each cell lysate protein was loaded. The Numb signals were calculated as fold change from D1ICD-expressing NCSCs compared with control NCSCs normalized with β-actin signal. The fold change of pErk1/2 was calculated from the phosphorylated Erk1/2 signals per Erk1/2 signal using same cell lysates.To identify D1ICD-interacting proteins, HEK293T cells were transfected either with 3×Flag-tagged mouse D1ICD or empty vector, and immunoprecipitation was performed using an anti-FLAG antibody. To omit nonspecific interactants, we (1) performed two independent experiments, (2) picked up proteins reproducibly identified from both FLAG-D1ICD IPs and (3) omitted proteins identified from one of the control IPs. Thus, the resultant list of the interactants represents specific binders. Preparation for mass spectrometric analyses was performed as previously described . ProteinLiquid chromatography mass spectrometric (LC-MS) analyses were performed essentially as previously described . A Nanofhttps://david.ncifcrf.gov/).The data were analyzed using Mascot in Proteome Discoverer 1.3 . Maximum missed cleavage sites were set to 2, and the precursor and fragment mass tolerances were 10 ppm and 20 mmu, respectively. Oxidation (Met), pyroglutamate conversion (Gln) and phosphorylation  were searched as variable modifications. Peptide identification was filtered at FDR<0.01. Gene Ontology (GO) analyses were performed using DAVID database (Table S5).Total RNA was extracted from the NCSCs and pooled DRGs isolated from the thoracic and lumbar levels using RNeasy MICRO Kit (Qiagen). RNA samples were subsequently used for cDNA synthesis using rimeScript RT Reagent Kits (Takara). For quantitative PCR reactions on cDNAs, PowerUp SYBR Green Master Mix  was used together with gene-specific primers (see


P2RX7 gene expression in the peripheral blood of tuberculosis patients compared to healthy donors. Lung leukocytes of mice infected with hypervirulent mycobacteria also showed increased expression of the P2X7 receptor. P2X7 blockade in mice with advanced tuberculosis recapitulated in many aspects the disease in P2X7-deficient mice. P2X7-directed therapy reduced body weight loss and the development of inflammatory and necrotic lung lesions, as well as delayed mycobacterial growth. Lower TNF-α production by lung cells and a substantial reduction in the lung GR-1+ myeloid cell population were observed after P2X7 inhibition. The effector CD4+ T cell population also decreased, but IFN-γ production by lung cells increased. The presence of a large population with characteristics of myeloid dendritic cells, as well as the increase in IL-6 production by lung cells, also indicate a qualitative improvement in the pulmonary immune response due to P2X7 inhibition. These findings support the use of drugs that target the P2X7 receptor as a therapeutic strategy to improve the outcome of pulmonary tuberculosis.The risk of developing severe forms of tuberculosis has increased by the acquired immunodeficiency syndrome (AIDS) epidemic, lack of effective drugs to eliminate latent infection and the emergence of drug-resistant mycobacterial strains. Excessive inflammatory response and tissue damage associated with severe tuberculosis contribute to poor outcome of the disease. Our previous studies using mice deficient in the ATP-gated ionotropic P2X7 receptor suggested this molecule as a promising target for host-directed therapy in severe pulmonary tuberculosis. In this study, we assessed the effects of P2X7 pharmacological blockade on disease severity. First, we observed an increase in  The P2X7 receptor detects ATP released during stress or cell death and activates the NLRP3 inflammasome, leading to mature IL-1β and IL-18 secretion and cell death by pyroptosis. Notably, prolonged stimuli of the P2X7 receptor induce necrotic cell death due to formation of large pores in the cell membrane. The P2X7 receptor has been previously suggested as a promising target candidate for host-directed therapies in severe pulmonary tuberculosis. In this study, we provide proof of concept for this approach in mice infected with hypervirulent mycobacteria. P2X7-directed therapy administered over a short period of time in mice with advanced pulmonary tuberculosis was effective in reducing disease severity. This therapeutic strategy can be particularly useful, combined with anti-microbial drugs, to interrupt the vicious cycle of uncontrolled inflammatory response and damage to lung tissue in severe forms of the disease.Mycobacterium tuberculosis infection reached 1.4 million worldwide, making TB one of the top ten causes of global death (M. tuberculosis infection increases the risk of developing severe forms of TB (Tuberculosis (TB) is an airborne infectious disease that remains as one of the major causes of health threat . In 2019al death . Multiplal death . TB contal death . Failurems of TB . Severe M. tuberculosis infection.This emerging scenario has encouraged the combined use of standard anti-microbial treatments for TB with host-directed therapies based on anti-inflammatory interventions. This therapeutic approach directly targets the inflammatory response triggered by the infection to prevent and repair tissue damage, promote pathogen elimination and reduce disease sequelae . Acceler2+ and efflux of K+ cause cytoplasmic ionic changes, which lead to NLRP3-inflammasome activation and culminates in mature IL-1β and IL-18 secretion and cell death through pyroptosis  and active TB (N = 9). Array quality control was applied using arrayQualitymetrics  mice were bred at the isogenic mouse facility, ICB, USP. After infection, mice were maintained in micro isolator cages with Mycobacterium bovis MP287/03 strain (The frozen bacilli were thawed and grow in Middlebrook 7H9 medium enriched with 10% (vol/vol) ADC  , 0.4% (mass/vol) sodium pyruvate  and 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), and maintained at 37°C for 7 days until mid-log phase (OD 0.6 – 0.9). Bacterial concentration was determined using a spectrophotometer at 600 nm. Mice were anesthetized intraperitoneally (i.p.) with ketamine  and xylazine  and infected intratracheally (i.t.) with ~100 bacilli of the 3 strain .in vivo pharmacological blockade of the P2X7 receptor, mice were injected i.p. every 2 days with brilliant blue G  (45 mg/Kg/mouse in 300 µL of PBS), starting on day 21 of infection.For The harvested lung lobes were washed with sterile PBS and weighed. The lung relative mass was calculated by dividing the mean of lung weight in experimental mice by the mean of lung weight in uninfected controls. The right lung upper lobe was maintained in 10% buffer formalin, photographed and subsequently embedded in paraffin. Histological sections of approximately 4-5 μm were stained using the hematoxylin-eosin (HE) method for tissue morphological analysis and the Ziehl Neelsen (ZN) method for mycobacterial visualization. The microscopic analyses were performed with a Leica microscope (Germany), and images were captured with a Nikon camera (Japan).The lung lobes were dissected and digested with collagenase type IV  in RPMI 1640 medium  at 37°C for 40 minutes under agitation (200 rpm) . The lun6 cells/well) were seeded in round-bottom 96-well plates and stained using fluorochrome-labeled monoclonal antibodies to CD45 (30-F11), CD11b (M1/70), CD11c (N418), GR1 (RB6-8C5), CD4 (RM4.5), CD44 (IM7), CD69 (H1.2F3), P2X7 (1F11), lineage . Live/dead dye (Thermo Fisher Scientific) was used to stain dead cells, as described in data sheet. Cells were fixed with 4% paraformaldehyde and analyzed with the LSRFortessa™ flow cytometer (BD Bioscences – USA) and the FlowJo 10.4.2 software (BD Biosciences). The gate strategy for analysis of CD11b+ myeloid cells and CD4+ T cells are shown in the Lung cells  OADC   and 0.4% (mass/vol) sodium pyruvate, at 37°C for 21 days. CFUs were counted visually.6 cells/well) harvested from the lungs were cultured in sterile round-bottom 96-well plates in complete RPMI 1640 medium enriched with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate and 0.05% gentamicin for 48 hours at 37°C and 5% CO2. The supernatants were collected, filtered and the concentrations of TNF-α, IL-6, IL-10 and IFN-γ cytokines were determined using the appropriated Mouse ELISA kit, as described in data sheet .Cells   population to the lungs  family members, the ironment . Accumulironment . By inhiP2RX7-/- macrophages infected with hypervirulent mycobacteria are more resistant to cell death induced by high levels of extracellular ATP and release fewer bacteria to the extracellular milieu than wild-type macrophages , IL-8, CC-ligand 3 (CCL3) and CXC-ligand 2 (CXCL2), as well as the production of pro-inflammatory cytokines, such as TNF-α  grants: 2015/20432-8 (MRDL), 2019/24700-8 (IS-C), 2017/11030-9 (PCS), 2019/27139-5 (JCSS) and 2020/09043-8 (CCBB); and by National Council for Scientific and Technological Development (CNPq) grants: 408909/2018-8 (MRDL), 303810/2018-1 (MRDL) and 140666/2018-4 (GA-S). All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia phage C600M2  and Escherichia phage CL1 , which were isolated from a wastewater treatment plant in Qatar. Both Escherichia phage C600M2 and Escherichia phage CL1 genomes contain 128 protein-coding genes and 26 tRNAs.We report the genome sequences of  Escherichia coli (C and K-12), we enriched and purified bacteriophages present in wastewater samples collected from Doha West Wastewater Treatment Plant (WWTP) in the State of Qatar, prior to the start of subsequent treatment processes. Escherichia phage C600M2 was isolated from stage 1  in selected growth media for 48 h at 37°C at 220 rpm. For phages isolated using Escherichia coli K-12 C600, peptone-yeast extract-1 mM CaCl2 (PYCa) was used as the growth and enrichment medium. PYCa top agar was used for pour plating onto PYCa  agar plates to form a uniform layer. For phages isolated using Escherichia coli C, LB supplemented with 0.2% glucose and 1 mM CaCl2 was used as the growth and enrichment medium. LB top agar with CaCl2 was used for pour plating onto tryptone-potassium-calcium chloride (TKC) plates to form a uniform layer. Phages were selected based on the morphology and diameter of the plaques, and plaques were picked for at least five rounds of purification  method with some modifications . The whohttps://doi.org/10.1184/R1/16965004) related to the SPAdes-assembled contigs were identified from the INPHARED bacteriophage database (https://github.com/RyanCook94/inphared), and only the read sequences that mapped to those identified genomes were extracted with the BWA tool  were annotated for functional proteins using DNA Master  and the Escherichia phage C600M2 and Escherichia phage CL1 shared 99.92% identity and were 95.99% and 96.27% identical, respectively, to Escherichia phage SSBS18_WS_10_728 (GenBank accession number MT322327.1), which was inferred using the ANIb subcommand in the pyani v0.2 Python module  under the SRA accession numbers SRX12131445 and SRX12131444, respectively, as part of BioProject number PRJNA762188.The complete genome sequences of


In populations with high rates of human immunodeficiency virus (HIV)-coinfection, the nature of the relationship between human papillomavirus (HPV)-16 and -18  antibodies and cervical cancer is still uncertain. We measured the association between seropositivity to HPV  proteins and cervical cancer among black South African women with and without HIV co-infection.We used questionnaire data and serum collected from consecutively recruited patients with a newly diagnosed cancer from the Johannesburg Cancer Study from 1346 cervical cancer cases and 2532 controls (diagnosed with other non-infection related cancers). Seropositivity to HPV proteins was measured using a multiplex serological assay based on recombinant glutathione S-transferase (GST) fusion proteins. We measured associations between their presence and cervical cancer using unconditional logistic regression models and evaluated the sensitivity and specificity of these HPV biomarkers.Among controls, HIV-negative women from rural areas compared to urban had significantly higher HPV seroprevalence, HPV16 E7 (8.6% vs 3.7%) and HPV18 E7 (7.9% vs 2.0%). HPV16 E6 and E7 antibodies were positively associated with cervical cancer in HIV-positive (Adjusted Odds Ratio (AOR) = 33; 95% CI 10–107) and HIV-negative women . In HIV-positive women, HPV E6/E7 antibodies had low sensitivity (43.0%) and high specificity (90.6%) for cervical cancer detection. In HIV-negative women, HPV E6/E7 antibodies sensitivity was 70.6% and specificity was 89.7%.Our data show that HPV  antibody positivity is associated with cervical cancer in both HIV-positive and HIV-negative women. Nonetheless, being HIV-positive plays an important role in the development of cervical cancer.The online version contains supplementary material available at 10.1186/s13027-022-00418-2. Globally, in 2018, about 70% of cervical cancers were attributable to high-risk human papillomavirus (HPV) types 16 and 18 [The HPV early (E6 and E7) oncoproteins and the late (L1) protein are encoded by the HPV genome. The HPV oncoproteins play an important role in the tumorigenesis of cervical cancer . HumoralPrevious studies have shown that HPV types 16 and 18 L1, E6 and E7 antibody proteins are associated with cervical cancer susceptibility –13 howevLess is known about the role of high-risk HPV oncoproteins among cervical cancer patients in black African women who are HIV-positive and HIV-negative. In South Africa, despite high HIV prevalence (24.1%) among black South African women , studiesThe study population were participants recruited into the Johannesburg Cancer Study (JCS) between 1995 and 2016. The aims of JCS included measuring the relative importance of known and emerging risk factors for cancer in a black African population in Johannesburg, South Africa. The details of the JCS have been described elsewhere . BrieflyThe JCS is amenable to a case–control design and analysis by selecting controls that are unrelated to the exposures of interest. The current study was restricted to women aged 25 to 54 years because a large proportion of the older women did not have serology samples available. Cases were 1346 women with newly diagnosed cervical cancer (C53). Controls were 2532 women with newly diagnosed infection unrelated cancers (breast (C50) (n = 1953), colon (C18-20) (n = 197), oesophagus (C15) (n = 48), endometrium (C54-55) (n = 75), lung (C33-34) (n = 76), pancreas (C25) (n = 21) and minor cancer types (n = 162) ) in Heidelberg, for antibody testing for HPV16 and 18  using a multiplex serological assay. This was based on a glutathione S-transferase (GST) capture immunoassay in combination with fluorescent beads on a Luminex platform , 20 and Data on demographic characteristics were summarized using frequencies and percentages for the categorical variables, medians and the interquartile range (IQR) for the continuous variable that was not normally distributed. A Pearson's Chi-squared test  and t-test (for age) were used for the comparison of demographics between cases and controls. To assess cross-reactivity between the proteins, a tetrachoric correlation coefficient rank test  with BonWe used an unconditional logistic regression model to assess the association by calculating adjusted Odds Ratios (AOR) and 95% confidence intervals between HPV16 and 18 (E6 and E7) and L1 proteins seropositivity and the risk of cervical cancer. We stratified by HIV status to assess if the seroprevalence of the HPV proteins differs in HIV-positive and HIV- negative patients. We adjusted for age, education level, number of sexual partners, marital status, period of interview and place of residence. To assess whether the clinical performance of HPV-16 and -18 antibodies as a diagnostic marker for cervical cancer we performed the same in HIV-positive and HIV-negative cancer patients. We calculated the sensitivity, specificity of the various HPV antibodies for detecting cervical cancer using the "diagti” command in STATA. In addition, the area under the ROC curve (AUC) of the receiver operator characteristics was computed to compare the performance of the best combination of HPV E6 and E7 antibodies to discriminate cervical cancer from controls . At least two-thirds of both cases (63.5%) and controls (72.4%) had a secondary school education  aged 25–54 years were included  and controls HPV16 E6 (3.0%) as compared to HIV-positive women cases HPV16 E6 (26.8%) and controls HPV16 E6 (2.8%)  and for combined HPV18 E6&E7 0.2%) . Among HIV-negative controls, the prevalence of HPV16 E7 antibody was higher among women that lived in rural areas (8.6%) as compared to women who lived in urban areas (3.7%)  . Among HIV-positive controls, the prevalence of HPV16 E6 &E7 antibodies decreased with an increase in number births up to 2 births   antibodies by demographic and sexual/reproductive factors (5) Table . A similan areas .7% , HPV16 E6 , HPV16 E7  and HPV16 L1 . For HPV18, antibody seroprevalence ORs for cervical cancer ranged from AOR = 38.61 (95% CI 15.13–98.53) for combined HPV18 E6&E7, AOR = 8.94 (95% CI 6.64–12.03) for HPV18 E7, AOR = 4.67 (95% CI 3.30–6.50) for HPV18 E6 and AOR = 1.31 (95% CI 1.10–1.56) for HPV18 L1  in HIV-negative, and 33.10 (95% CI 10.22–107.20) in HIV-positive women , and L1 protein and calculated their association with cervical cancer. While the seroprevalence of HPV16 L1 in the previous JCS study among HIV-seronegative women was higher  than in The higher seroprevalence of HPV16 E6, HPV16 E7 and HPV18 E7 in women from rural areas might indicate a greater number of women with early undetected cervical or related cancer lesions. In rural areas of South Africa, women have poorer access to health care services, are less likely to present themselves to cervical cancer screening services and have less access to cancer diagnostic facilities, which are mainly found in tertiary hospitals in urban areas .The high seroprevalence of HPV16 E6 35.9%) antibodies in cervical cancer cases found in our study are similar to the seroprevalence of HPV16 E6 (32%) in cervical cancer cases reported from the United Kingdom (35%) and India and Algeria (32%) .9% antib. We founAntibodies to the E6 and E7 oncoproteins are late markers of invasive cervical cancer . In our Individuals with suppressed immune systems are susceptible to chronic infection, including HPV . Thus, HUnexpected findings in our study were that HPV-16 and -18 (E6 and E7) oncoproteins seropositivity and cervical cancer risk were higher in HIV-negative cancer patients compared to HIV-positive cancer patients. Similar findings have been observed in previous studies of HPV antibodies among HIV-positive and HIV-negative patients –38. In oIn the new guidelines for screening cervical cancer, the World Health Organization (WHO) has included HPV antibodies and oncoproteins as a future potential screening test . We, theThere are strengths to our study. The seroprevalence of HPV antibodies was conducted on a large sample size, and we used a high throughput multiplex serology so all tests were done under the same laboratory conditions. The laboratory tests were conducted ‘blind’ without prior knowledge of the case/control status of the participants. Nonetheless, our study has some limitations. Serology data on exposure and cervical cancer diagnoses  were collected at the same time. We did not have tumor HPV Deoxyribonucleic Acid (DNA) data to determine the causative type(s). Since only 50–70% of women with detectable HPV DNA in the cervix seroconvert , our stuOur data contribute to the evidence on the importance of HPV-16 and -18 E6 and E7 oncoproteins antibodies in discriminating cervical cancer from controls in a black South African population. HPV L1 antibodies show to be exposure markers of past HPV infection. HPV E6 and E7 show to be important markers of invasive cervical cancer. Furthermore, antibodies to HPV-16 and-18 E6 and E7 seropositivity are strongly associated with cervical cancer in both HIV-positive and HIV-negative patients.Additional file 1: Table S1: Comparison of HPV16 and 18  antibodies in cervical cancer Cases and Controls among young and older women. Table S2: Seroprevalence of HPV16 and 18 L1. Table S3: Seroprevalence of antibodies against HPV 16 and 18  by HIV-Status. Table S4: Clinical performance of HPV16 and 18 antibodies as a diagnostic marker for cervical cancer. Fig. S1: Age-adjusted seroprevalence of HPV related antibody markers in cervical cancer cases and other infection unrelated cancer controls and p-value for heterogeneity among the infection unrelated cancer controls . The seropositivity of the age-adjusted HPV L1 and (E6 and E7) antibodies in each of the infection unrelated cancer patients (controls) was similar, (p-heterogeneity > 0.05). Fig. S2: Correlation between HPV16 and 18 proteins L1, E6 and E7 antibodies and HIV antibodies, R-values > 0.8 shows high correlation. (* Significance at 0.05). The colour indicates the intensity of correlation. Green indicate positive values, red indicate negative values, orange shows R-values that are greater than 0.05 and dark orange indicate R-values < 0.05 but not statistically significant.