In vitro GCV sensitivity was assessed by MTT assay for monitoring time- and dosage-dependent cytotoxicity. The capability for neural differentiation in stable glioblastoma clones during GCV treatment was assessed by performing immunocytochemistry for nestin, GFAP, and βIII-tubulin. Under GFAP promoter control, the U251 stable clone exhibited GCV sensitivity, while the neuronal N2A clones were nonreactive. During GCV treatment, cells underwent apoptosis on day 3 and dying cells were identified after day 5. Nestin was increasingly expressed in surviving cells, indicating that the population of neural stem-like cells was enriched. Lower levels of GFAP expression were detected in surviving cells. Furthermore, βIII-tubulin-positive neuron-like cells were identified after GCV treatment. This study established pGFAP-HSVtk-P2A-EGFP plasmids that successfully ablated GFAP-positive glioblastoma cells, but left neuronal N2A cells intact. These data suggest that the neural differentiation of glioblastoma cells can be promoted by treatment with the HSVtk/GCV system.Glioblastoma is a malignant brain tumor with poor prognosis that rapidly acquires resistance to available clinical treatments. The herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system produces the selective elimination of HSVtk-positive cells and is a candidate for preclinical testing against glioblastoma via its ability to regulate proliferation and differentiation. Therefore, in this study, we aimed to establish a plasmid encoding the HSVtk/GCV system driven by a glial fibrillary acidic protein (GFAP) promoter and verify its possibility of neural differentiation of glioblastoma cell line under the GCV challenge. Four stable clones—N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk—were established from neuronal N2A and glioblastoma U251 cell lines.  Glioblastoma is a devastating, uniformly lethal primary brain tumor. The standard treatment for glioblastoma is surgery followed by radiotherapy and chemotherapy, but tumor recurrence is observed in most patients . In factPrevious studies revealed that malignant tumors are initiated by a population of tumor cells sharing similar biological properties with normal adult stem cells , 4. ThesGanciclovir (GCV) is an antiviral medication used to treat infections from the herpes family. GCV is first phosphorylated to GCV monophosphate by a viral kinase encoded by the cytomegalovirus (CMV) gene. Afterwards, GCV is metabolized to the triphosphate form by guanylate kinase and phosphoglycerate kinase. GCV-5′-triphosphate inhibits the replication of viral DNA. This inhibition includes a selective inhibition of the viral DNA polymerase and leads to disruption of DNA synthesis and inhibition of cell proliferation –16. SincAs mentioned, HSVtk-positive cells can be selectively ablated by the HSVtk/GCV system. However, controlling HSVtk expression in mammalian cells is also essential to avoid any influence on other nontarget cell populations. For this purpose, the delivery of the HSVtk/GCV system using embryonic stem cells is a possible approach. Embryonic stem cells have the potential for use in cell replacement therapies, but the transplantation of differentiated cells harbors the risk of teratoma formation. This problem was overcome by establishing a negative selection system that permitted selective removal of undifferentiated stem cells . The HSVGlial fibrillary acidic protein (GFAP), a type III intermediate filament protein, is expressed mainly in astroglia , 24, andEcoRI at the 5′-end and SalI at the 3′-end. The forward oligo was GAATTCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT-3′5′- and the reverse oligo was GTCGACAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCC-3′5′-. Next, 100 μM of forward and reverse primers were dissolved in annealing buffer  and annealed in a thermo-cycler . The P2A oligo was then subcloned into pEGFP-N3  at restriction enzyme sites: EcoRI at the 5′-end and SalI at the 3′-end. After pEGFP-N3-P2A was constructed, HSVtk was inserted upstream of P2A. The clone with a full-length 1131-bp HSVtk was purchased from Addgene . The primers used to clamp HSVtk were 5′-CTGCTCGCCGGGATCCATGGCTTCGTACCCCTGC-3′ and 5′-CCATGGTGGCGTCGACAGGTCCAGGGTTCTCCTC-3′. An In-Fusion® HD cloning kit  was used to fuse HSVtk and linearized pEGFP-N3-P2A with Xhol at the 5′-end and EcoRI at the 3′-end. The sequences of P2A and HSVtk were checked by DNA sequencing provided by Genomics Inc.  and confirmed in frame. The plasmid pCMV-HSVtk-P2A-EGFP was constructed and confirmed by DNA sequencing.P2A, a 66-bp self-cleaving peptide, was synthesized by oligos annealing with restriction enzyme cutting sites 5′-GGACTCAGATCTCGAGCCCACCTCCCTCTCTGTG-3′ and a reverse primer 5′-GATCCCGGGCCCGCGGAGGTCCAGGGTTCTCCTC-3′. An In-Fusion HD® cloning kit was used to fuse the GFAP promoter and linearized pCMV-HSVtk-P2A-EGFP together. The sequence of the GFAP promoter was confirmed by DNA sequencing provided by Genomics, Inc.After pCMV-HSVtk-P2A-EGFP was constructed, the CMV promoter was replaced by the GFAP promoter to construct pGFAP-HSVtk-P2A-EGFP. Plasmid pAAV-GFAP-hChR2(H134R)-mCherry with a full-length 2207-bp GFAP promoter was purchased from Addgene . The GFAP promoter region was clamped by a forward primer 2. Medium was refreshed every 2~3 days.The NIH/3T3 cell line , a mouse fibroblast cell line, was cultured in Dulbecco modified Eagle medium  supplemented with 10% fetal bovine serum  and 1% antibiotic-antimycotic solution . The Neuro-2A (N2A) cell line , derived from a spontaneous neuroblastoma tumor of a strain A albino mouse, and the U251 cell line , derived from a malignant glioblastoma tumor, were cultured separately in Minimum Essential Medium α  supplemented with 10% heat-inactivated FBS , 1% of nonessential amino acid , and 1% Anti-anti . All cell lines were seeded onto cell culture dishes  and maintained in a 37°C humidified incubator with 5% CO5) were seeded on 12-well cell culture plates 1 day before plasmid transfection. In this experiment, Lipofectamine® LTX with PLUS™ reagents  were used. Both NIH/3T3 and U251 cell lines were transfected with 2 μg of pCMV-HSVtk-P2A-EGFP or pGFAP-HSVtk-P2A-EGFP plasmid. Since the transfection efficiency of the two plasmids into N2A cells was low using Lipofectamine, N2A cells were transfected using the Neon® electroporation transfection system  and cell density was increased to 5 × 106 cells/ml. Before transfection, N2A cells were trypsinized to individual cells dissolved in buffer R, and 2 μg of pCMV-HSVtk-P2A-EGFP or pGFAP-HSVtk-P2A-EGFP plasmid DNA were added. Electroporation parameters for N2A cells were 1200 V, 50 ms once. Cells were reseeded into 12-well cell culture plates containing 1 ml electroporation transfection medium per well.NIH/3T3 and U251 cells . Cells that emitted strong green fluorescence were identified as successfully transfected with promoter expression. The promoter expression level was calculated as followed:the number of green fluorescent cells / total cell number under phase image).Promoter expression level =  was added to the medium to select successfully transfected U251 cells. G418 sulfate selection lasted for 4 weeks and the cell population was also enriched by fluorescence-activated cell sorting . Thereby, the U251-pCMV-HSVtk and U251-pGFAP-HSVtk stable clones were established. For N2A stable clone selection, 500 μg/ml G418 sulfate were applied to transfected N2A cells. After 4 weeks of G418 selection, surviving cells with green fluorescence of the N2A-pCMV-HSVtk cells were enriched by FACS and surviving cells of N2A-pGFAP-HSVtk cells were selected. Thus, the N2A-pCMV-HSVtk and N2A-pGFAP-HSVtk stable clones were established for use in further studies. After stable clones were established, G418 was still present for the maintenance of all stable clone cells, but not in the experimental cells.For protein extraction, cells were trypsinized and homogenized with radioimmunoprecipitation assay buffer , 1 × phosphatase inhibitor , and 1 × protease inhibitor . Cell lysates were centrifuged at 14,000 rpm for 30 minutes at 4°C, and supernatant was collected. Protein samples with 1 × protein sample buffer were boiled at 97°C for 10 minutes and then placed onto ice immediately. The prepared protein samples were stored at –20°C for further analysis. Protein samples (40 μg/lane) were resolved in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), separated at 120 V for 2 hours, and transferred to polyvinylidene fluoride (PVDF) membranes  at 100 V for 90 minutes. Subsequently, the PVDF membrane with transferred proteins was blocked with a blocking buffer  for 1 hour at room temperature. After blocking, the PVDF membrane was incubated with primary antibodies see  diluted 4) were seeded in 12-well cell culture plates. Cells were incubated for 12 hours for cell adhesion and treated with 0, 1, 5, or 10 μg/ml GCV for 5 days. After treatment, cells were placed in serum-free media containing 5 μg/ml MTT  and incubated at 37°C for 1 hour. The purple crystals formed by the cells were dissolved in dimethyl sulfoxide . The level of cell viability was determined by measuring the absorbance at 550 nm with an ELISA reader .Stable clone cells (1.5 × 104) were seeded onto poly-L-lysine-coated 18-mm round coverslips in 12-well culture plates. After 12 hours of cell-adhesion, cells were treated with 5 μg/ml of GCV for 1, 3, 5, and 7 days. Treated cells were washed by 1 × DPBS and fixed using 4% paraformaldehyde . Specimens were incubated in a nuclear dye of PI /Hoechst 33342  mixture diluted in 1 × PBS for 5 minutes, washed three times in 1 × PBS, and mounted with Fluoro-Gel .U251-pCMV-HSVtk or U251-pGFAP-HSVtk cells  was added to induce apoptosis in the positive control groups. All images were acquired using a DMR fluorescence microscope (Leica) and a LSM880 confocal microscope .The TUNEL assay was performed with a cell death detection kit  to detect DNA fragmentation generated during apoptosis. For cell apoptosis analysis, 1.5 × 104) were seeded onto poly-L-lysine-coated 18-mm round coverslips in 12-well culture plates. After 12 hours of cell-adhesion, cells were treated with 5 μg/ml of GCV for 1, 3, 5, and 7 days. Cells were fixed with 100% methanol and 4% paraformaldehyde and blocked with 1% of FBS in phosphate buffer saline with 0.1% Triton X-100 (PBS-T) for 1 hour. Thereafter, cells were incubated with primary antibodies  were calculated as differentiation rates of U251-pGFAP-HSVtk and U251-pCMV-HSVtk.After treatment with 5 μg/ml of GCV for 7 days, U251-pCMV-HSVtk and U251-pGFAP-HSVtk cells were supplemented with fresh growth medium for 3 days. The remaining cell populations were regarded as recovery subclones. Subsequently, PI/Hoechst 33342 staining was performed to investigate this cell population. In addition, immunocytochemistry of βIII-tubulin, GFAP, and nestin were conducted to study the expression patterns of neural markers in the recovery cell subclones.t-test was performed, with a p value < 0.05 regarded as statistically significant. All data were expressed as mean ± standard error of the mean (SEM).GraphPad Prism® 4.0 software  was used to analyze the data. When comparing two conditions, a AseI-XhoI restriction sites using an In-Fusion cloning kit.HSVtk and P2A were subcloned into pEGFP-N3, and named as pCMV-HSVtk-P2A-EGFP, to serve as a positive control vector in which the HSVtk and the reporter gene EGFP were separately expressed . P2A, a To examine further the promoter specificity of pCMV-HSVtk-P2A-EGFP and pGFAP-HSVtk-P2A-EGFP in non-glial and glial cell lines, transient transfection into three cell lines—NIH-3T3 (mouse fibroblast cell line), N2A (mouse neuroblastoma cell line), and U251 (human glioblastoma cell line)—was conducted, and green fluorescent cells were analyzed on day 2 . A high To produce stable clones that constitutively expressed HSVtk, 500 μg/ml and 400 μg/ml of G418 were applied to N2A and U251, respectively. During 14 days of selection, cells with a strong EGFP signal formed colonies, which were isolated and amplified for further FACS enrichment. Cells that expressed strong green fluorescence were sorted and further enriched by cell sorting and regarded as candidates for stable clones. There was no significant difference in the morphology or cell doubling time between parent cells and the transfected cells used in this selection. We established four stable clones—N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk—for use in subsequent experiments, as described below.To verify the protein expression of these stable clones, the levels of HSVtk protein expression were determined by western blot . Stable To investigate the sensitivity of the stable clones to GCV and determine the optimal working concentration for all cell lines, the cell viabilities in different GCV concentrations were tested. All stable clones were treated with 0, 1, 5, or 10 μg/ml GCV for 5 days, and then an MTT assay was performed to evaluate cell viabilities. Live-cell phase images and immunocytochemistry were used to examine cell morphology and EGFP expression. Phase images collected using an inverted microscope revealed that N2A parent cells  and tran50 at 5 μg/ml GCV treatment. Therefore, the optimal dosage of GCV chosen for ablating stable clones in subsequent experiments was 5 μg/ml.U251 parent cells sustained their proliferation in the presence of treatment with different concentrations of GCV . The numIn summary, the experimental stable clone, U251-pGFAP-HSVtk, and the positive-control stable clone, U251-pCMV-HSVtk, displayed a dose-dependent response to GCV, while only N2A-pCMV-HSVtk was sensitive to GCV. N2A-pGFAP-HSVtk cells remained intact because the GFAP promoter would not be expressed in N2A neuronal cells. These data demonstrate that the GFAP promoter can specifically drive the HSVtk/GCV system and selectively ablate glioblastoma cell lines, while keeping neuronal cell lines intact.A previous study demonstrated that the HSVtk/GCV system triggered cells to undergo apoptosis rather than necrosis . TherefoTo identify dead cells and evaluate cell viability, U251-pCMV-HSVtk and U251-pGFAP-HSVtk cells treated with GCV for 7 days were subjected to PI/Hoechst double nuclear staining . Very weEnlargement and process extension of surviving  cells, which are morphological features of cell differentiation, were observed in U251-pGFAP-HSVtk and U251-pCMV-HSVtk cells during GCV treatment. Therefore, immunocytochemical staining for nestin, GFAP, and βIII-tubulin was performed to characterize the identity of the survival cells in the U251-pGFAP-HSVtk  and U251For identification of neuronal differentiation, the expression of βIII-tubulin, a neuronal marker, was investigated. Weak βIII-tubulin expression was detected by day 3 of GCV treatment , and surU251-pCMV-HSVtk cells, which served as a positive-control, stable clone for U251-pGFAP-HSVtk cells, were also immunostained for nestin, GFAP, and βIII-tubulin . SimilarThe survival rates and the expression ratio of the nestin-, GFAP-, and βIII-tubulin-positive cells in the U251-pGFAP-HSVtk  and U251In this study, the GFAP promoter, an astrocyte-specific promoter, was used to restrict HSVtk gene expression in the glioblastoma cell line, U251. The specificity of the GFAP promoter in pGFAP-HSVtk-P2A-EGFP was confirmed by comparison with the control plasmid, pCMV-HSVtk-P2A-EGFP. An extremely low number of EGFP-positive cells was detected when NIH-3T3 and N2A cells were transfected with pGFAP-HSVtk-P2A-EGFP. A previous study demonstrated that a different GFAP promoter could be expressed in various kinds of cell lines, and the expression level of the GFAP promoter was 1.3% and 3.5% in N2A and NIH-3T3 cell lines, respectively . In addiIn this research, the GFAP promoter was applied to control the expression of the HSVtk gene in glioblastoma cell lines, while the cytotoxicity of GCV was limited in neurons and fibroblasts. Our result could correspond to the previous study which showed that the HSV-TK gene driven by human GFAP promoter could selectively kill C6 and U251 glioma cell lines, with no effect on the ovarian cells . BesidesOur results also demonstrated that the HSVtk/GCV system promoted neural differentiation of U251 cells and enriched the neural stem-like cell population after 7 days of GCV treatment. We demonstrated that cells with a high GFAP expression level were killed, while βIII-tubulin-positive neuron-like cells were identified after day 5 of GCV treatment. Previous studies have also revealed that some drugs can induce neural differentiation in other glioblastoma cell lines. The novel small molecule, CG500354, was reported to induce growth arrest of GBM-derived cells and neural differentiation via the cAMP/CREB signaling pathway. In contrast to our results, CG500354 treatment decreased the number of nestin-positive neural progenitor cells and increased the number of GFAP- and Tuj1-(βIII-tubulin)-positive neural cell types . FurtherTo test the HSVtk/GCV system on another genomic background, another human primary glioblastoma cell line, U87, was examined in our laboratory (unpublished data). The HSVtk/GCV system driven by the GFAP promoter could ablate GFAP-positive glioblastoma cell lines and promote neural differentiation within 5 days of GCV treatment. A similar trend found in U87 cells indicated that this system represents a promising preclinical test for glioblastoma.In this study, we showed that cell number of GFAP-positive cells would decrease during the treatment of GCV, and increase after 3 days of GCV withdraw. We demonstrated that GFAP promoter could specifically drive the HSVtk/GCV system and selectively ablate GFAP-positive cells. One reason for the increase of GFAP-positive cells after 3 days recovery may be due to the differentiation of the nestin-positive neural stem cell. Previous studies reported that cancer stem cells could be identified in U251 glioma cell line , 44 and We observed that the nestin-positive neural stem cell increased after the GCV treatment and βIII-tubulin-positive neuron-like cells gradually increased after 5 days of GCV treatment and decreased after 3 days recovery. We suggested that the increase of βIII-tubulin-positive neuron-like cells during GCV treatment may be further differentiated from nestin-positive neural stem cell which increased from day 3 of GCV treatment. Because previous study suggested that cellular origin may affect lineage differentiation propensity of human induced pluripotent stem cells , nestin-The HSVtk/GCV system can cause the death not only of HSVtk-positive cells but also of the surrounding HSVtk-negative tumor cells, which is called bystander effect and is another important feature of this system . BystandWhile the precise mechanisms underlying the neural differentiation are not known, some possible mechanisms are suggested by other studies. Autophagy is involved in differentiation, survival, and cell death, and the PI3K/AKT/mTOR pathway was reported to promote growth and proliferation over differentiation of adult stem cells, and neural stem cells, specifically . InactivIn conclusion, this study established pGFAP-HSVtk-P2A-EGFP plasmids that could successfully ablate GFAP-positive glioblastoma cell lines, while neuronal N2A cells remained intact. The data also suggested that the neural differentiation of glioblastoma cells could be promoted after challenge with the HSVtk/GCV system.in vitro. We demonstrated that this system could selectively ablate GFAP-positive glioblastoma cell lines, while it left the neuronal N2A cells intact and promoted neural differentiation after challenge with the HSVtk/GCV system  CMV promoter of pCMV-HSVtk-P2A-EGFP was substituted by 2207 bps GFAP promoter from another plasmid, pAAV-GFAP-hChR2(H134R)-mCherry, at restriction enzyme cutting sites of AseI and XhoI. HSVtk and EGFP in this clone were controlled by GFAP promoter, which is a specific promoter of glia cell. Kanamycin/neomycin resistance gene was used for both kanamycin selection of E.coli colony and G418 sulfate selection of stable clone in the further experiments.(A) HSVtk (1131 bps) gene was subcloned into pEGFP-N3 at (TIF)Click here for additional data file.S2 Figst, 2nd, and 4th week were demonstrated by western blot and β–actin served as an internal control in all groups. The western blot images were quantified by ImageJ software (n = 3). (A) Before transfection, N2A parent cells showed no HSVtk expression level. After transfection and selection, the HSVtk expression levels of N2A-pCMV-HSVtk were stable within 4 weeks. Thus, N2A-pCMV-HSVtk was a stable clone and could be used for further experiments. (B) The U251 parent cells showed no HSVtk expression before transfection. U251-pCMV-HSVtk showed stable expression levels of HSVtk within 4 weeks and could be used as a control stable clone for glioblastoma. (C) HSVtk expression could not be detected in N2A-pCMV-HSVtk, since GFAP promoter was not activated in N2A cell line. (D) U251-pGFAP-HSVtk expressed HSVtk stably and continuously within 4 weeks.HSVtk protein expression levels of 4 stable clones, N2A-pCMV-HSVtk, U251-pCMV-HSVtk, N2A- pGFAP-HSVtk, and U251-pGFAP-HSVtk, at the 1(TIF)Click here for additional data file.S3 Fig2O2 for 1 hour before staining and the majority of the cell nuclei were TUNEL/Hoechst double positive. (I-L) U251-pGFAP-HSVtk cells without GCV treatment were regarded as negative control group and cell nuclei were TUNEL negative. Scale bar = 50 μm.(A-D) Fluorescence confocal images of apoptosis obtained from TUNEL assay. U251-pGFAP-HSVtk cells were treated with 5 μg/ml of GCV for 3 days. Both TUNEL- and Hoechst- positive cells were identified (C) and dying cells had rounded morphology in DIC image (D). (E-H) Positive control group was treated by 10 μM H(TIF)Click here for additional data file.S4 FigThe survivals of U251-pGFAP-HSVtk (A-F) and U251-pCMV-HSVtk (G-L) cells were determined by PI/Hoechst co-staining for cell nuclei after 5 μg/ml GCV treatment for 7 days. PI/Hoechst co-localized and condensed cell nuclei were identified at the 5th day and the 7th day of GCV treatment in both U251-pGFAP-HSVtk  and U251-pCMV-HSVtk  groups. No PI signal was detected after 3 days of recovery (F and L). Scale bar = 50 μm.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Pneumocystis. However, the dynamics and diversity of the T cell immune repertoire in human immunodeficiency virus (HIV)-negative Pneumocystis pneumonia (PCP) remains unclear. In this study, single-cell RNA and single-cell T cell receptor (TCR) sequencing were applied to cells sorted from lung tissues of mice infected with Pneumocystis. Our findings demonstrated the clonal cells were mainly composed of CD4+ T cells in response to Pneumocystis, which were marked by highly expressed genes associated with T cell activation. Mice infected with Pneumocystis showed reduced TCR diversity in CD4+ T cells and increased diversity in CD8+ T cells compared with uninfected controls. Furthermore, Th17 cells were mostly clonal CD4+ T cells, which exhibited the phenotype of tissue-resident memory-like Th17 cells. In addition, Pneumocystis-infected mice showed biased usage of TCRβ VDJ genes. Taken together, we characterized the transcriptome and TCR immune repertoires profiles of expanded T cell clones, which demonstrate a skewed TCR repertoire after Pneumocystis infection.T cell responses play critical roles in host adaptive immunity against  Pneumocystis pneumonia (PCP) is a life-threatening complication in human immunodeficiency virus (HIV)-negative immunocompromised patients with more abrupt clinical manifestation, worse prognosis, and higher mortality than HIV patients  has facilitated in dissecting the heterogeneity of immune cells. Furthermore, single-cell T cell receptor (TCR) sequencing (scTCR-seq) can effectively identify clonal cells and characterize their relationships. In infectious diseases, the combination of scTCR-seq and scRNA-seq allows for simultaneous analysis of paired TCR sequences and the transcriptome to track T cell clones . WaickmaPneumocystis-specific CD4+ T cells in HIV patients using spectratyping; however, changes in TCR repertoires and clonal expansion in multiple T cell subsets in PCP remains unclear.Pneumocystis infection using scRNA-seq coupled with scTCR-seq. We comprehensively analyzed the composition and characteristics of clonal T cells, diversity of TCR repertoire, distribution of CDR3 length, and usage patterns of TCRβ VDJ gene segments and combination at different time points post-infection. Our study characterized the dynamics and diversity of TCR profiling across different stages of Pneumocystis infection, which contribute to elucidating host adaptive immunity and discovering novel therapeutic targets.In this study, we quantitatively tracked the expanded T cell clones and revealed their phenotype during P. murina  as we previously reported  were injected through the trachea of each mouse. Control mice were transtracheally injected with 100 μL PBS. To determine the Pneumocystis burden, real-time PCR was conducted to determine the copy number of Pneumocystis rRNA from right lung lobes by TaqMan assays. The primers were 5′-AGGTGAAAAGTCGAAAGGGAAAC-3′ and 5′-AAAACCTCTTTTCTTTCACTCAGTAACA-3′. The sequence of probe was 5′-FAM-CCCAGAATAATGAATAAAG-MGBNFQ-3′. Pneumocystis burden would increase until the third week, and then decrease in the fourth week after generation of the PCP model. Based on the previously reported Pneumocystis burden and our results  were housed under specific pathogen-free conditions at the Beijing Institute of Respiratory Medicine . C.B-17 SCID mice were inoculated with reported . For the results , we desig for 6 min and incubated with Percp-Cy5.5 anti-mouse CD45 antibody (BD) and Ghost DyeTM Red 780 (Tonbo) for 15 min on ice. BD FACS ARIA II cell sorter was applied to sort CD45+ cells from lung cell suspensions pooled from three mice.Single cell suspensions were isolated from mouse lungs by collagenase digestion following recently published protocols . Briefly® II Automated Cell Counter with AO/PI reagent). After assessing the quality of libraries by Agilent 4200, sequencing libraries were loaded onto an Illumina Novaseq platform. Cell Ranger (v.3.0.2) was applied to demultiplex align reads (mouse GRCm38/mm10 as reference genome) and produce gene-barcode matrices. The Cell Ranger aggr pipeline was used to combine data from all five samples.Single-cell 5′ RNA-Seq libraries were generated using Single Cell 5′ Library and Gel Bead Kit and Chromium Controller (10 × Genomics) following automated cell counting and a sample quality test  and t-distributed stochastic neighbor embedding (t-SNE) projections. Clusters were annotated based on the Seurat-based cluster-specific marker gene list and the expression of canonical marker genes, including T cells , naïve CD4+ T cells , effector CD4+ T cells (Cd4 and Cd44), naïve CD8+ T cells , effector CD8+ T cells (Cd8a and Cd44), γδT cells (Tcrg-C1), double-positive T cells , NKT cells (Nkg7 and Klrc1), and cycling cells (Mki67 and Stmn1).Quality control and cell clustering were performed using Seurat (version 3.1.2) . If the We performed Scrublet to exclude cell doublets . We calcFull-length TCR VDJ segments were generated using the Chromium Single-Cell V(D)J Enrichment kit following the manufacturer’s protocol. The Cell Ranger vdj pipeline was applied to assemble the TCR sequences and identify the CDR3 sequence and TCR genes. Then, the cells were filtered according to the following steps: (1) Cells annotated as T cell clusters in scRNA-seq were kept and (2) Cells that possessed productive TCR α and β chains were incorporated into the analysis. If more than one α or β chain were detected in a cell, then we retained the chain with the highest UMIs . We defivia chord diagrams using the circlize package (version 0.4.8). To explore the common and unique V-J and V-D-J gene combinations, we obtained a list of specific V-J and V-D-J gene combinations for all samples, then utilized the Venn Diagram package (version 1.6.20) in R software (version 3.6.1) to display the relationship of those combinations in each sample.V-J gene combinations were visualized P value was < 0.05 based on the Wilcoxon rank sum test. Gene pathway analyses were implemented using the DAVID website1.The FindMarkers function was used to identify the differentially expressed genes among cell clusters. Genes were considered differentially expressed when the logFC > 0.25, expressed in >25% of cells in the cluster and the adjusted + T cells’ residency and migration were obtained from previous studies , naive CD8+ T cells (C2), effector CD4+ T cells (C3), effector CD8+ T cells (C4), double-positive T cells (C5), NKT cells (C6), cycling T cells (C7), and γδT cells (C8)  . All fivlls (C8) . The expnfection . Effectodamental .+ T cells, 36.6% were CD8+ T cells, and 18.6% were cycling T cells , and the length distributions of TCRβ CDR3 were nearly identical in both infected and uninfected tissues  is the most variable region of the TCR . Therefo tissues .+ T cells were the most expanded clonal cells after Pneumocystis infection, we performed sub-clustering analysis of CD4+ T cells from all five samples and identified six subclusters , TRBV13-2 (9.8%), TRBV3 (11.9%), TRBV1 (11.1%), and TRBV3 (10.7%), respectively  were shared with uninfected controls, and 52 (21.6%) were unique  were shared with uninfected controls and 135 (36.6%) were unique J gene usage for PCP, several key matters require validation. First, we only obtained data from mice infected with Pneumocystis infection at the single-cell resolution and to track the clonal cells uniting TCR clonotype and transcriptome phenotype, thereby providing novel insights to improve our understanding of adaptive immune response in PCP.In conclusion, investigations on T cell immunity have always been an important subject in PCP. Due to the lack of studies on the TCR repertoire of PCP, we performed scTCR-seq and scRNA-seq to comprehensively reveal the reconstructed TCR repertoire during GSE157627.The sequence data in this study were deposited in the Gene Expression Omnibus (GEO). The accession number of scTCR-seq data is GSE156843 and that of scRNA-seq is The animal study was reviewed and approved by Capital Medical University Animal Care and Use Committee.H-QY conducted the experiments and drafted the manuscript. H-QY and Y-SW performed the data analysis. KZ and Z-HT designed the experiments, supervised the study, and revised the manuscript. 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.


These cells can be distinguished from other B cells by their higher expression of the adenosine receptor 2a. Here we address whether A2A receptor activation can affect CD11c+T-bet+ B cells. We show that administration of the A2A receptor agonist CGS-21680 depletes established CD11c+T-bet+ B cells in ehrlichial-infected mice, in a B cell-intrinsic manner. Agonist treatment similarly depletes CD11c+T-bet+ B cells and CD138+ B cells and reduces anti-nuclear antibodies in lupus-prone mice. Agonist treatment is also associated with reduced kidney pathology and lymphadenopathy. Moreover, A2A receptor stimulation depletes pathogenic lymphocytes and ameliorates disease even after disease onset, highlighting the therapeutic potential of this treatment. This study suggests that targeting the adenosine signaling pathway may provide a method for the treatment of lupus and other autoimmune diseases mediated by T-bet+ B cells.CD11c +T-bet+ B cells have been linked with different autoimmune diseases, but targeting these cells has been challenging. Here the authors use an adenosine 2A receptor agonist to deplete these B cells and to inhibit or reverse autoimmune symptoms and pathology in mice.CD11c CD11c+T-bet+ B cells develop in infections characterized by type I immunity, such as malaria, HIV, influenza, and hepatitis C4. CD11c+T-bet+ B cells generated in these infections are driven by Toll-like receptor (TLR) signals, and by CD40L, IFN-γ, and IL-21 produced by T-bet+CD4+ T cells8. CD11c+T-bet+ B cells can function as memory cells that are capable of self-renewal and that differentiate following secondary challenge9. Moreover, elimination of CD11c+ B cells following Ehrlichia muris infection ablated the switched-antibody recall-response10. CD11c+T-bet+ B cells are involved in the immune response to many other diseases. During malarial infection, CD11c+T-bet+ B cells generate pathogen-specific antibodies and are associated with reduced parasite-density13. CD11c+T-bet+ B cells generated during influenza infection account for 20–30% of the antigen-specific B cells3. In HIV infection, the CD11c+T-bet+ B cell population contains more HIV-specific B cells than classical memory B cells2. In addition to their function during infection, CD11c+T-bet+ B cells are also involved in pathogenic immune responses in diseases such as rheumatoid arthritis (RA), Sjøgren’s Syndrome, and systemic lupus erythematosus (SLE)17.CD11c+ B cells are key mediators of disease etiology in SLE, a relapsing autoimmune disease that affects approximately 5 million people worldwide. SLE commonly manifests as facial rash, fatigue, joint pain, and disease flares, followed in some cases by end-stage renal disease and organ failure18. CD11c+T-bet+ B cells contribute to SLE pathogenesis, as the cells correlate with autoantibody titers and disease severity23. CD11c+T-bet+ B cells are responsive to TLR7 stimulation, and exhibit reduced expression of the negative proinflammatory regulators NFKBIA, TNFAIP3, TRAF5, and TRAF4, which likely contributes to their generation during SLE16. It is also known that CD11c+T-bet+ B cells in humans and mice can give rise to antibody-secreting cells (ASCs), and it has been proposed that SLE-derived CD11c+T-bet+ B cells are poised to differentiate into plasmablasts (PBs)16. Given their propensity to generate ASCs, CD11c+T-bet+ B cells likely contribute to SLE disease pathogenesis via their differentiation into ASCs and the production of autoreactive antibodies27. CD11c+T-bet+ B cells may also contribute to SLE pathogenesis via the activation of CD4+ T cells, as these B cells can also function as potent antigen presenting cells28. CD4+ T follicular helper cells (TFH), in turn, can drive the formation of autoreactive germinal center (GC) B cells and ASCs, which can give rise to autoantibodies and immune complexes, ultimately leading to systemic inflammation and glomerulonephritis31. Previous work has clearly established that T-bet+ B cells can contribute to disease pathogenesis in SLE, as elimination of T-bet expression in B cells substantially reduced disease burden in lupus-prone mice26. Another study similarly demonstrated that depletion of CD11c+ B cells reduced anti-Smith antibodies in mice immunized with a TLR7 agonist25. These studies highlight the importance of CD11c+T-bet+ B cells in the pathogenesis of SLE, and suggest that targeted depletion of these cells will reduce disease severity in autoimmune diseases wherein these cells have been implicated; however, no such pharmacological treatments currently exist.T-bet+T-bet+ B cells elicited by E. muris infection, Winslow et al. identified a number of genes that were highly expressed, relative to CD11c-negative B cells. Among these was the gene encoding the G-protein-coupled-receptor A2A receptor, Adora2a, which was detected at levels 10-fold higher in CD11c+T-bet+ B cells, compared to CD11c-negative B cells32. The A2A receptor is one of several receptors for extracellular adenosine and is expressed on most leukocytes, as well as neurons and endothelial cells35. Extracellular adenosine can be produced via the catabolism of ATP and NAD+ by ectoenzymes such as CD73, CD39, and CD38, all of which are highly expressed on CD11c+T-bet+ B cells36. Adenosine signaling via the A2A receptor can be immunosuppressive, by increasing regulatory T cell (Treg) generation, inhibiting effector T cells (Teff) and TFH proliferation, and blocking the formation of GC B cells40. Given its immunomodulatory ability, the A2A receptor has been widely studied for possible anti-tumor effects, as some tumors are known to generate large quantities of extracellular adenosine43. Adenosine production by these tumors is thought to impair anti-tumor responses by inducing anergy among Teff cells, and by increasing the generation of Treg cells and their production of the immunosuppressive cytokines TGFβ and IL-1044. Consistently, A2A receptor-deficient mice showed improved tumor rejection compared to A2A receptor-competent controls, although these mice were prone to develop autoimmunity35. A2A receptor deficiency also exacerbated severe experimental autoimmune encephalitis in mice, the result of increased macrophage and lymphocyte activation and increased IFN-γ secretion45.As part of studies of CD11c2A receptor has been implicated in other autoimmune diseases such as RA48. Lymphocytes from RA patients have been shown to express high levels of A2A receptor, and in vitro stimulation of this receptor inhibited the production of TNF, IL-1β, and IL-6 by these lymphocytes48. A2A receptor agonism has been shown to impair the production of autoantibodies and reduce disease severity40. In lupus-prone mice, treatment with the A2A receptor agonist CGS-21680 significantly improved kidney function, although the mechanism whereby this occurred was not known49. Other studies have demonstrated that the A2A receptor is correlated with disease severity in SLE patients, and that stimulation of this receptor in vitro reduces the production of proinflammatory cytokines by lymphocytes50. These studies highlight the importance of A2A receptor signaling during autoimmunity and suggest that pharmacological targeting of this receptor may aid in the treatment of diverse diseases.The A+ B cells. We demonstrate that administration of the A2A receptor agonist CGS-21680, can deplete CD11c+T-bet+ B cells in both E. muris-infected and lupus-prone mice. Moreover, A2A receptor agonist treatment of MRL/lpr mice significantly reduces disease severity, compared to vehicle-treated controls when administered either early or late relative to the onset of disease. Our work thus describes a targeted pharmacological approach for the elimination of pathogenic CD11c+T-bet+ B cells and highlights the therapeutic potential of A2A receptor agonists in the treatment of SLE, and possibly other autoimmune diseases.In the present study, we address the effect of adenosine signaling on T-bet2A receptor is expressed at least ten-fold higher in E. muris-elicited CD11c+T-bethi B cells, relative to CD11c-negative B cells32. While A2A receptor signaling is known to be immunomodulatory, its effects on the maintenance of CD11c+T-bethi memory B cells had not been addressed. Therefore, we treated E. muris-infected mice with CGS-21680 once daily from days 30–37 post-infection, after the CD11c+T-bethi B cell population is fully developed10. CD11c+ B cells and T-bethi B cells are referred to interchangeably as it has been shown that these cells represent the same population at this time point in E. muris infection5. CGS-21680 treatment eliminated nearly all the CD11c+T-bethi B cells in E. muris-infected mice within 7 days post-administration  present on day 30 post-ehrlichial infection, possibly memory TFH1 cells, were not depleted following CGS-21680 treatment  mice, which provide a well-developed polygenic model for SLE52. CD11c+T-bet+ B cells were substantially reduced in the spleens of 20-week-old MRL/lpr mice that were treated with CGS-21680 twice weekly starting at 8 weeks of age  in the spleens and lymph nodes of the same MRL/lpr mice  . This modest effect was likely due to the inherent variability among MRL/lpr mice, the contribution of other A2A receptor tolerant cell types to disease, or to A2A receptor desensitization following repeated agonist treatment . However, these findings may have been confounded by heterogeneity within the sample groups, incomplete B cell depletion, and by the high levels of corticosteroids administered to both groups of patients77. Current B cell-targeted treatments lack specificity, and act by eliminating most B cells in patients 78. Pan-depletion of B cells also eliminates suppressive B cells, and it has been proposed that loss of these B cells contributes to the spike in symptoms observed among some MS patients receiving anti-B cell therapy79. Because of the adverse effects of current B cell-targeted therapies, there is a need for novel treatments with limited and tolerable side-effects. Therapies that target the adenosine signaling pathway may therefore provide a promising avenue for treatment of SLE and other autoimmune diseases. Indeed, the A2A receptor agonist Regadenoson  is already used for myocardial imaging in patients unable to undergo exercise-induced stress testing80. Because of its wide use, side effects from Regadenoson treatment have been thoroughly documented and are well-tolerated in humans, at least under the conditions of current usage. Side effects include, headache, chest pain, dizziness, and disruption of the blood-brain barrier, although most of these symptoms resolve within 15 min following treatment81. Therapeutic treatment for autoimmunity will likely require repeated agonist administrations, and although the dose and frequency of administration required to limit disease in humans is not yet known, modest side effects may be well-tolerated in otherwise healthy patients. Additional research is required to determine the efficacy of A2A receptor agonists in humans, but our research suggests that this class of drugs represents a promising approach for the treatment of autoimmune diseases caused in part by CD11c+T-bet+ B cells.Strategies to target B cells have been effective at ameliorating disease in RA and MS, although the efficacy of such methods in the treatment of SLE and yet other autoimmune diseases remains uncertaincre (B6.Cg-Tg(Cd4-cre)1Cwi/BfluJ), Mb1cre (B6.C(Cg)-Cd79atm1(cre)Reth/EhobJ), MRL/lpr (MRL/MpJ-Faslpr/J), SLE123 , and Rosa26eYFP (B6.Cg-Gt(ROSA)26Sortm3(CAG-EYFP)Hze/J) mice were obtained from The Jackson Laboratory . T-bet-creERT2 mice were generated by Dr. Lin Gan at the University of Rochester, Rochester, NY. Adora2aflox  mice were provided by Dr. Joel Linden, La Jolla Institute for Immunology, La Jolla, CA. All mice, except for the MRL/MpJ-Faslpr/J, were on the C57BL/6J background. All mice were housed and bred in the SUNY Upstate Medical University Animal Care Facility , in accordance with institutional guidelines for animal welfare. All mice used for experiments were at least 6 weeks old, and both male and female mice were used unless otherwise stated. All studies involving animals were approved by the SUNY Upstate Medical University Institutional Animal Care and Use Committee NYSDOH Unit A073 IACUC number 311.C57BL/6J, CD44E. muris bacterial copies, as determined by qPCR, and as previously described (probe sequence: 56-FAM/AGGGATTTC/ZEN/CCTATACTCGGT/3IABkFQ)82. CGS-21680 hydrochloride and Istradefylline were purchased from Cayman Chemical . Regadenoson  was purchased internally from SUNY Upstate Medical University. E. muris-infected and lupus-prone mice were injected intraperitoneally with 50 μg of CGS-21680 , 132 μg of Istradefylline (6.6 mg/kg), or 50 μg of Regadenoson (2.5 mg/kg) in 200 μl PBS containing 5% DMSO, without anesthesia.Mice were infected intraperitoneally with 5–10 × 10Spleens and lymph nodes were disaggregated using a 70 μm cell strainer . Erythrocytes were removed by incubation with ACK lysis Buffer . Cells were treated with anti-CD16/32 (2.4G2) and mouse cells were incubated with the following antibodies: PerCpCy5.5-conjugated anti-CD19 , Alexa Fluor 700-conjugated anti-CD19 , APC-eFluor 780-conjugated anti-CD11c , Brilliant Violet 785-conjugated anti-B220 , V500-conjugated anti-B220 , Alexa Fluor 647-conjugated anti-T-bet , PerCpCy5.5-conjugated anti-T-bet , FITC-conjugated anti-PD-1 , PerCpCy5.5-conjugated anti-CD4 , Alexa Fluor 700-conjugated anti-CD3 , Brilliant Violet 421-conjugated anti-CXCR5 , PE-conjugated anti-CD138 , Brilliant Violet 421-conjugated anti-IgM , PE-conjugated anti-FoxP3 . LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific) or Ghost Dye Violet 510 (TONBO biosciences) were used to stain for viability.The cells were stained at 4 °C for 30 min, washed, and analyzed. For intracellular staining, surface-stained cells were fixed/permeabilized for 40 min at 4 °C, using the Transcription Factor Buffer set Fixation/permeabilization buffer (BD Pharmingen), washed, stained at 4 °C for 30 min, washed, and analyzed. Unstained cells were used to establish the flow cytometer voltage settings, and single-color positive controls were used to adjust compensation. The data were acquired on a BD Fortessa flow cytometer using Diva software (BD Bioscience) and were analyzed with FlowJo software version 10.7 (BD Bioscience.). All flow cytometry analyses were performed on freshly harvested cells.Livers were perfused with PBS and disaggregated using a 70 μm cell strainer . Cells were gradient separated using 40% Percoll (Sigma Aldrich) and erythrocytes were removed by incubation with ACK lysis Buffer .Urine albumin concentration was determined using Fisherbrand 10-SG Urine Reagent Strips according to the manufacturer’s instructions.83. Gomerulonephritis was assessed using a scale from one to six based on mesangial cellularity and expansion, the presence of patent capillary loops, glomeruli size, karyorrhexis, crescent formation, and sclerosis. Interstitial nephritis was graded on a scale from one to four based on the prevalence of lymphocytes and plasma cell infiltrates in the perivascular area and/or in the interstitial space.Kidneys were harvested from mice and fixed in 4% PFA for at least 48 h at room temperature. Fixed kidneys were paraffin embedded, cut into 5 μm sections, stained with hematoxylin and eosin (H&E), and blindly scored by a single pathologist at Histowiz  or by a certified pathologist at the University of Pittsburgh Medical Center, using a previously described metric84. Flat-Bottom Immuno plates (Thermo Scientific) were coated with 100 μl of poly-L-lysine (50 μg/ml) (Sigma Aldrich), followed by 100 μl of yeast RNA (15 μl/ml) (Fisher Scientific). The plates were blocked with fetal bovine serum and incubated with serum (1:100 dilution in wash buffer), overnight at 4 °C. RNA-specific antibodies were detected with alkaline phosphatase-conjugated goat anti-mouse antibodies (1:1000) and background removed using no serum controls. .Anti-RNA ELISAs were performed as described in Blanco et al.Anti-Smith and Anti-dsDNA ELISAs were performed using the Bio-Rad Anti-Sm (cat # 96SM) and Anti-dsDNA (cat # 96DS) EIA Kits (Bio-Rad), according to the manufacturer’s instructions, except that anti-Smith and anti-dsDNA antibodies were detected using alkaline phosphatase-conjugated goat anti-mouse secondary antibodies (1:1000) .Statistical analyses were performed using Prism 9 (GraphPad). The statistical tests that were performed are indicated in the figure legends. The column in each of the plots indicates the arithmetic mean of the dataset, and upper and lower bounds indicate the standard deviation of the dataset. Data were analyzed for normality using the Shapiro–Wilk test and statistical tests were chosen based on the normality of the data. An effect size of 2.02 was estimated based on previous data and assuming a power of 80% and a significance level of 0.05.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


TM, i.e., tezacaftor, elexacaftor and ivacaftor rescued trafficking and function of both of these mutants. These in-vitro findings suggest that patients harbouring H609R or I1023_V1024del, alone or with I148T, may benefit clinically from treatment with TRIKAFTATM.The rare Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutations, c.1826A > G (H609R) and c.3067_3072delATAGTG (I1023_V1024del), are associated with severe lung disease. Despite the existence of four CFTR targeted therapies, none have been approved for individuals with these mutations because the associated molecular defects were not known. In this study we examined the consequences of these mutations on protein processing and channel function in HEK293 cells. We found that, similar to F508del, H609R and I1023_V1024del-CFTR exhibited reduced protein processing and altered channel function. Because the I1023_V1024del mutation can be linked with the mutation, I148T, we also examined the protein conferred by transfection of a plasmid bearing both mutations. Interestingly, together with I148T, there was no further reduction in channel function exhibited by I1023-V1024del. Both H609R and I1023_V1024del failed to exhibit significant correction of their functional expression with lumacaftor and ivacaftor. In contrast, the triple modulator combination found in TRIKAFTA CFTR) gene. The CFTR protein is an ion channel that mediates chloride and bicarbonate transport in the epithelial cells of multiple organs including the lungs, pancreas and intestine -CFTR constructs. After18h, the cells were treated with DMSO (0.1%), 3 µM VX-809, and 3 µM VX-661 + 3 µM VX-445  for 24 h at 37 °C. Then, the cells were loaded with a blue membrane potential dye dissolved in chloride free buffer ,20.HEK293 were lysed in modified radioimmunoprecipitation assay (RIPA) buffer  with protease inhibitor cocktail  for 10 min in ice . Solublep-values < 0.05 were considered significant. Data with multiple comparison were assessed with Tukey’s multiple-comparison test with α = 0.05. Data are represented as mean ± S.D. GraphPad Prism 8.0 software  was used for all statistical tests. One-way ANOVA was conducted and In order to study the molecular consequences of the H609R and I1023_V1024del mutations, we used HEK293 cells to overexpress those mutations for biochemical and functional studies. We also determined whether existing therapies were effective in improving their function, thus providing insights into personalized medicine for CF patients. CFTR channel function was measured using the membrane depolarization assay (FLIPR) and we measured the CFTR chloride channel response the addition of forskolin and correctors/potentiators, as well as inhibitors. Recently, we showed that VX-661 + VX-445 treatment significantly rescued F508del-CFTR channel activity and protein processing to close to fifty-percent of Wt-CFTR, in HEK293 and primary nasal epithelial cells . TherefoWestern blot studies demonstrated that H609R, I1023_V1024del and I148T/I1023_V1024del mutations induced a folding defect by diminishing the relative amount of complex CFTR produced  in comparison to this ratio measured for Wt-CFTR A,B. RelaAs shown in Since the I148T mutation can be linked with I1023_V1024del [3199del6] in CF patients , we thenTM).In this study we showed that the defective protein processing and channel activity caused by the H609R and I1023_V1024del mutations could be partially rescued by the FDA-approved drug combination of tezacaftor (VX-661), elexacaftor (VX-445) and ivacaftor  [H609R is an R domain localized missense mutation highly prevalent in the CF population of Ecuador (19%) ,31,32. Muginosa) . Furtheruginosa) . As expeuginosa) . Both miuginosa)  and has Interestingly, unlike F508del-CFTR, there was no significant potentiated activity conferred by VX-770 and forskolin in the case of the H609R and I1023_V1024del mutant proteins, despite partial rescue by VX-809 of their processing defects. These findings suggest that there are functional defects conferred by H609R and I1023_V1024del. These functional defects were partially repaired following treatment with the triple combination, suggesting that the novel modulator: VX-445 exerts a distinct effect that enables superior functional rescue for the H609R and I1023_V1024del mutants. Overall, the magnitude of the functional rescue with VX-661, VX-445 and VX-770 for both of these mutations, H609R and I1023_V1024del is approximately 50% of WT-CFTR and is comparable to the activity of rescued F508del-CFTR recorded via membrane depolarization assays in HEK293 cells . Such fi


Escherichia coli invasive isolates obtained from a tertiary-care hospital in Budapest, Hungary.This study was carried out to determine the prevalence and the genetic background of extended-spectrum β-lactamase-producing E. coli isolates were collected from Central Hospital of Southern Pest. The antimicrobial susceptibility testing was performed according to the EUCAST guidelines. The possible clonal relationships were investigated by core genome (cg)MLST (SeqSphere +) using whole-genome sequencing (WGS) data of isolates obtained from Illumina 251-bp paired-end sequencing. From WGS data acquired antimicrobial resistance genes, virulence genes and replicon types were retrieved using ResFinder3.1, PlasmidFinder2.1, pMLST-2.0, VirulenceFinder2.0 and Virulence Factors Database online tools.Between October–November 2018, all invasive ESBL-producing E. coli isolates proved to be resistant to third-generation cephalosporins and ESBL-producers in the study period. Full genome sequence analysis showed that five E. coli isolates belonged to the ST131 clone: two to C1-M27 subclade with blaCTX-M-27 and three to C2/H30Rx subclade with blaCTX-M-15. One isolate belonged to ST1193 with blaCTX-M-27. According to cgMLST, all C2/H30Rx isolates formed a cluster , while the blaCTX-M-27-producing C1-M27 isolates differed at least 35 alleles from each other. Both C2/H30Rx and C1-M27 ST131 isolates harbored similar antimicrobial resistance gene sets. However, only C2/H30Rx isolates had the qnrB and aac(3)-IIa. The isolates carried similar extraintestinal virulence gene set but differed in some genes encoding siderophores, protectins and toxins. Moreover, only one C2/H30Rx isolate carried salmochelin siderophore system and showed virotype B. All isolates showed resistance against ceftriaxone, cefotaxime, and ciprofloxacin, and the C2/H30Rx isolates were also resistant to gentamicin, tobramycin, and ceftazidime.Overall, six E. coli, five belonged to the ST131 clone. This study indicates, that the C2/H30Rx and C1-M27 subclades of the ST131 appear to be the dominant clones collected in a Hungarian hospital.Out of six ESBL-producing  Escherichia coli increased the most, in terms of the number of infections and the number of deaths in Europe [E. coli (ExPEC) harbor various virulence genes that allow them to cause severe extraintestinal infections, including bloodstream infections, urinary tract infections, pneumonia, and neonatal meningitis [Recently, among the most worrying multidrug resistant bacteria the burden of disease caused by third-generation cephalosporin-resistant n Europe . Its gloningitis .E. coli ST131 high-risk clones connected with extended-spectrum β-lactamase (ESBL) production and fluoroquinolone resistance causing multidrug-resistant infections belong to the most prevalent clade C associated with fimH30. Within C/H30, two subclades were emerging in the 2000s: C1/H30R subclade with fluoroquinolone resistance (FQ-R) and C2/H30Rx subclade with blaCTX-M-15, and FQ-R [blaCTX-M-27 of C1/H30R became dominant in Asia and showed global distribution like the C2/H30Rx did before [blaCTX-M-15 gene is associated with the C2/H30Rx subclade and the blaCTX-M-27 encoding F1:A2:B20 plasmid is associated with the C1-M27 subclade [E. coli are ST10, ST38, ST69, ST155, ST315, ST405, ST410, ST648, and ST1193 [and FQ-R . In the d before . The expsubclade . The othE. coli has risen in Hungary from 5.1% in 2006 to 22.6% in 2018 [E. coli from Hungary, and its genomic epidemiology and no prospective study was made before.Previous studies have shown that the proportion of third-generation cephalosporin-resistance among invasive  in 2018 , 12. HowE. coli collected from a Hungarian hospital; and to perform genomic typing of these isolates in order to characterize their genetic background.This study was designed to determine the incidence of third-generation cephalosporin-resistant invasive E. coli were collected in the Central Hospital of Southern Pest. An isolate was considered invasive if it has been isolated from blood or cerebrospinal fluid [Central Hospital of Southern Pest National Institute of Hematology and Infectious Diseases is a tertiary-care hospital in Budapest, Hungary, with a capacity of approximately 1400 beds. Between October–November 2018, all invasive consecutive non-duplicate clinical isolates of ESBL-producing The culturing from clinical samples, the preliminary antibiotic susceptibility tests and the identification of isolates were performed in the clinical microbiological laboratory of the hospital. All isolates were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry .The ability of ESBL-production were determined by Double Disc Synergy Test (DDST) for all isolates. The DDST confirmation test was performed by the “ESβL Detection Disc Set”  according to the manufacturer’s instruction.The antibiotic susceptibility test of putative ESBL-producing isolates was performed to ceftriaxone, ceftazidime, cefotaxime, gentamicin, amikacin, tobramycin, fosfomycin, ceftazidime/avibactam, tigecycline, ertapenem by MIC Test Strips ; and to imipenem, meropenem, ciprofloxacin, colistin by broth microdilution and interpreted using EUCAST guidelines  and the cultures were incubated at temperatures of 35–37 °C for 16–20 h.E. coli were calculated per 1000 hospital admissions or 1000 patient–days, respectively.The incidence rate or incidence density of invasive infection caused by E. coli was defined as isolates attributed to the same clone with ≤ 10 differing alleles (CT threshold) within a group. The ST131 isolates were compared to three international ST131 E. coli genomes (C1-M27: EC81009 [http://genomicepidemiology.org/). In order to determine sequence similarity (coverage and identity), the isolates were aligned to the pEC-81009 , to the uk_P46212 plasmid  and pU1 (F1:A1:B16) reference plasmid sequences [The DNA extraction from the bacterial cultures was performed with the DNeasy UltraClean Microbial Kit  according to the manufacturer's instruction. Genomic DNA analysis of the isolates was performed by whole-genome sequencing (WGS) using Illumina MiSeq 251-bp paired-end sequencing. Raw data were processed by Ridom SeqSphere + and assembly was performed using Velvet 1.1.04 . The qua EC81009  and H105 EC81009 , C2/H30R EC81009 ) obtaine EC81009  online t, virotype B , virotype C , and virotype D .Virulence factor genes were retrieved using VirulenceFinder2.0  and ViruThe raw reads are available on the Sequence Read Archive (SRA) database under the BioProject number PRJNA683640.E. coli were isolated from haemoculture in the study period. Six from 25 isolates (24%) proved to be resistant to third-generation cephalosporins and were obtained from patients with bloodstream infection. The incidence density and the incidence rate of third-generation cephalosporin-resistant E. coli were 0.11 per 1,000 patient-days and 0.79 per 1,000 hospital admission, respectively. The phenotypic detection tests showed that all of them were ESBL-producer. Out of the six isolates collected from the Central Hospital of Southern Pest, two isolates originated from the Haematology and Stem Cell Transplantation ward, two from the Infectious Diseases ward, one from the Intensive Care Unit and the last one from the 1st Internal Medicine ward. The median age of the patients was 70 years (range 38–90), and the sex ratio was 1:1.Twenty-five According to the both EUCAST guideline versions the isolates were resistant to ceftriaxone, cefotaxime, and ciprofloxacin but remained susceptible to colistin, fosfomycin, ceftazidime-avibactam, and carbapenems Table . The Ec1The whole-genome sequencing and genome assembly results met all quality requirements for all isolates. The assembled draft genome size of the isolates was 5 325 328 bp for Ec1, 5 309 727 bp for Ec2, 5 298 859 bp for Ec3, 5 041 503 bp, for Ec4, 5 070 035 bp for Ec5 and 5 115 835 bp for Ec6.E. coli isolates could be assigned to two different sequence types (ST) by MLST. Of the six isolates, five (Ec1-Ec5) belonged to the globally dominant ST131 clone (O25:H4 serotype), which comprised two distinguished groups of strains with different characteristics. The first group contains two isolates with blaCTX-M-15 and one isolate with blaCTX-M-15 and blaTEM-1B while the two other isolates harbored blaCTX-M-27 ESBL-genes. The remaining isolate belonged to the ST1193 with blaCTX-M-27. Analysis of genetic relatedness between the strains was performed by cgMLST. The five ST131 isolates were compared to one C2/H30Rx (JJ2434) and two C1-M27  international ST131 ESBL-producing E. coli isolates, to determine the subclade-specific relatedness of the isolates within the ST131 , while the C1-M27 isolates producing blaCTX-M-27 differed at 35 alleles from each other. The ST1193 isolates showed ≥ 2209 allele differences from any others.According to cgMLST Fig. , all theqnrB (FQR) and aac(3)-IIa (aminoglycoside-R) which were present only in C2/H30Rx isolates. The ST1193 isolate harboured L416F mutation in parE, and also had no qnrB and aac(3)-IIa genes. Three plasmid families were detected: IncF, IncQ, and Col-like. The IncF replicons were present in every isolate, but only the C1-M27 ST131 isolates  carried the Group1 replicons . The coverage of the Group1 (F1:A1:B20) reference plasmid by the reads of Ec4 and Ec5 was 96%, and 72%, respectively  and several toxin genes.Table E. coli was 0.11 per 1,000 patient-days and the incidence rate was 0.79 per 1,000 hospital admission. There are no previous data on the incidence of third-generation cephalosporin resistant E. coli invasive infections in Hungary.In our study, the incidence density of invasive infection caused by third-generation cephalosporin-resistant E. coli was 0.03/1,000 patient-days in Europe. There was significant difference in incidence density between northern region and southern region of Europe: 0.002/1,000 patient-days and 0.06/1,000 patient-days, respectively. In the eastern region of Europe (including Hungary) the incidence density was similar to the average  [De Kraker et al. investigated changes in the trends of blood stream infections for major bacterial pathogens by analysing the European Antimicrobial Resistance Surveillance System database between 2002–2008. In 2008 the average incidence density of invasive infections caused by third-generation cephalosporin resistant nt-days) . These fnt-days)  and invent-days)  in similE. coli isolates (24%) proved to be third-generation cephalosporin-resistant and ESBL-producer. In 2010, Pál T. et al. found 26 ESBL-producers among 117 E. coli isolates (22.2%) from bloodstream infections collected in three university hospitals in Hungary between March and November 2010, where nine of them belonged to ST131 clone carrying exclusively blaCTX-M-15 [E. coli was similar in our study, however, the rate of ST131 was higher (83.3 vs. 34.6%). Furthermore, in our study two of the five isolates harbouring blaCTX-M-27 gene belonged to the C1-M27 subclade. The first ESBL-producing invasive E. coli isolates belonged to the C1-M27 subclade were detected in 2015 in Hungary . In 2018, according to an observational study of Jánvári et al., 44.6% (75/168) of invasive ESBL-producing E. coli isolates investigated at the National Public Health Center belonged to the ST131 clone, where the ratio of C2/H30Rx and C1-M27 was 1 to 0.8 [In our study, six from the 25 CTX-M-15 . The rat1 to 0.8 .E. coli clone in invasive infections. The three ST131 C2/H30Rx isolates formed a relatively close cluster , and this could suggest an undetected outbreak affected two hospital wards . However, based on the results of plasmid replicon analysis it was unlikely.In this study, ST131, including the C1-M27 (2 isolates) and the C2/H30Rx subclades (3 isolates) proved to be the dominant multidrug-resistant All isolates were resistant to ceftriaxone and cefotaxime. The isolates with CTX-M-15 ESBL were resistant to ceftazidime, while the CTX-M-27 expressing isolates remained susceptible, probably because these enzymes hydrolyze ceftazidime poorly. Moreover, the C2/H30Rx isolates showed resistance to tobramycin and gentamicin probably because they have AAC(3)-IIa aminoglycoside-modifying enzyme .E. coli in Hungary. This clone has been widely spread in Asia [E. coli isolates in the Tertiary Care University Hospital in Korea [E. coli ST1193 was 0.6% (3/495) among all E. coli infections [E. coli from urine samples belonged to ST1193 [We found one isolate that belonged to the emerging ST1193 global high-risk clone . To the  in Asia , 32. Thein Korea . In Eurofections . In a Soo ST1193 .E. coli clones, however, the exact role of these plasmids is not clear [It has been known that the narrow-host-range IncF type plasmids have been evolved along with fluoroquinolone-resistant ST131 H30 clades. The F2:A1:B- plasmid is associated with the C2/H30Rx subclade, whereas the F1:A2:B20 plasmid is strongly associated with the C1/H30R subclade . The preot clear . In our ot clear . The covE. coli isolates have a wide range of virulence-coding genes, including adhesins, invasines, iron-uptake factors, protectins, toxins that enable them to bind to the host cells, invade the host tissues and avoid host defense systems [sat, iha, iucD, ompT, usp [sat, iha, vat, iutA, usp which were associated with ST1193 isolate [iucABCD), yersiniabactin (irp2), and the Ec2 isolate also carried the salmochelin (iroBCDEN) operon. The Ec2 showed virotype B, while the other isolates of both subclades of ST131 showed virotype C. The global disseminated virotype C has been described as the most prevalent E. coli ST131 virotype [It is not completely understood, which key factors determine the successful dissemination of the ST131 C2/H30Rx and C1/H30R (C1-M27) clones worldwide, besides the antibiotic genes, but the virulence armament probably has an important part in it. The ST131 extraintestinal  systems , 37. Sev systems . We idenmpT, usp –40. The  isolate , 41. Thevirotype , 42. Thevirotype , 43. Blavirotype .It has been described that ST131 clones can transmit among human and animal hosts . Based oE. coli isolates in a Hungarian hospital. It highlights the dominance of the ST131 clones and the C1-M27 and C2/H30Rx subclone in invasive infections. Although, further national studies are needed with a larger collection of isolates belonging to the two subclades to determine the possible factors  that can influence their dissemination.This study has several limitations, like small sample size and a two months time limit. However, it gives an insight into the incidence and genomic characteristics of ESBL-producing


GIPR variants, R190Q (rs139215588) and E288G (rs143430880) linked to lower body mass index (BMI) in carriers. At the molecular and cellular level, both variants displayed reduced G protein coupling, impaired arrestin recruitment and internalization, despite maintained high GIP affinity. The physiological phenotyping revealed an overall impaired bone strength, increased systolic blood pressure, altered lipid profile, altered fat distribution combined with increased body impedance in human carriers, thereby substantiating the role of GIP in these physiological processes.Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) are involved in multiple physiological systems related to glucose metabolism, bone homeostasis and fat deposition. Recent research has surprisingly indicated that both agonists and antagonists of GIPR may be useful in the treatment of obesity and type 2 diabetes, as both result in weight loss when combined with GLP-1 receptor activation. To understand the receptor signaling related with weight loss, we examined the pharmacological properties of two rare missense  GIP, altrations .The GIPR is not only expressed in pancreatic islet cells and adipocytes but has a wide expression profile including, but possibly not limited to, the heart, spleen, lung, central nervous system, and thyroid cells . AdditioGIP is associated with the pathophysiology of obesity and type 2 diabetes mellitus (T2D) and have therefore been the focus of therapeutic interest for many years. It is currently debated whether to use GIPR agonists or -antagonists in combination with GLP-1 agonists to treat obesity and T2D, as both combinations show promising results . ClearlyGIPR are associated with obesity , two rare variants in GIPR were identified  have revealed that common variants in the  obesity  and impa obesity . With th obesity , the GIPentified . These mentified , the free height .GIPR variants. First, we investigated experimentally the GIP receptor binding and activation properties of the two variants, and secondly, we linked our findings to human physiology by assessing summary data of previously published studies and online portals.Here we combine molecular pharmacological phenotyping with the physiological consequences of carrying these two rare NM_000164) was synthesized and purchased from GenScript  along with the GIPR mutations: R190Q, E288G and the double mutant R190Q-E288G. For the real-time internalization assay, the N-terminally SNAP-tagged GIPR was synthesized and purchased from Cisbio  and R190Q and E288G were introduced into the wild-type GIPR by site-directed mutagenesis according to quick-change protocol, using primers:The human GIPR that was inserted into pcDNA 3.1 plasmid (GenBank accession number: GCGGCCATTCTCAGCCAGGACCGTCTGC (forward for R190Q), GCAGACGGTCCTGGCTGAGAATGGCCGC (reverse for R190Q), CGCAGTGCTGGGGCCGCAACGA AGTCAAGGC (forward for E288G), GCCTTGACTTCG TTGCGGCCCCAGCACTGCG (reverse for E288G).Human GIP(1-42) was purchased from Caslo ApS . HEK293 and COS-7 cells were both purchased from ATTC . Cell medium for HEK293 was purchased from Thermo Fisher Scientific  and the cell medium for COS-7 cells were prepared in-house. Other chemicals were purchased from standard commercial sources.2 and 37°C in Dulbecco’s Modified Eagle Medium (DMEM) 1885 supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 180 units/mL penicillin and 45 g/mL streptomycin. HEK293 cells were cultured at 10% CO2 and 37°C in DMEM GlutaMAXTM-I supplemented with 10% FBS, 180 units/mL penicillin and 45 g/mL streptomycin. Both cell lines were transfected using the calcium phosphate precipitation method -based assay . In brie125I-GIP(1-42). The following day, the cells were assayed by competition binding for 3-h at 4°C using ∼15–40 pM of 125I-GIP(1-42) and increasing concentrations of GIP(1-42) in binding buffer . After incubation, the cells were washed in ice-cold binding buffer and lysed with 200 mmol/L NaOH with 1% SDS for 30 min. The samples were analyzed by the Wallac Wizard 1470 Gamma Counter.Transiently transfected COS-7 cells expressing either wild-type GIPR, R190Q, E288G or R190Q;E288G were seeded in a clear 96-well plate 1 day after transfection. The number of cells added per well was adjusted aiming for 5–10% specific binding of To measure β-arrestin 2 recruitment, HEK293 cells were transiently transfected with either wild-type GIPR, R190Q, E288G or R190Q;E288G and the donor Rluc8-Arrestin-3-Sp1, the acceptor mem-linker-citrine-SH3 and GPCR kinase 2 (GRK2) to facilitate β-arrestin 2 recruitment. Two days after transfection, the cells were washed with PBS and re-suspended in PBS with 5 mmol/L glucose. Subsequently, 85 μL of the cell suspension solution was transferred to its respective wells on a white 96-well isoplate followed by the addition of PBS with 5 μmol/L coelenterazine-h. After a 10 min incubation of the cells with coelenterazine-h, increasing concentration of endogenous GIP(1-42) were added and luminescence was measured by the Berthold Technologies Mithras Multilabel Reader (Rluc8 at 485 ± 40 nm and YFP at 530 ± 25 nm).2, 1 mM MgCl2, 20 mM HEPES and 0.1% BSA . 50 μM pre-heated fluorescein-O’-acetic acid (acceptor) was added to all wells, except wells where only donor signal was measured. The 384-plates were incubated at 37°C for 5–10 min prior to ligand addition. Then, the cells were stimulated with increasing doses of GIP(1-42), that was pre-heated at 37°C, and donor signal and internalization rate were measured every 4 min for 90 min at 37°C in PerkinElmerTM Envision 2014 multi-label Reader.HEK293 parental cells transiently expressing the SNAP-tag GIPR or the variant, SNAP-tag-R190Q or—E288G were seeded in white 384-well plate after transfection, at a density of 20.000 cells per well. The following day, the medium was removed and fresh medium was added to all wells. The next day, the assay was carried out by labeling all SNAP-tagged cells with 100 nmol/L Taglite SNAP-Lumi4-Tb (donor) in OptiMEM for 60 min at 37°C. Subsequently, the cells were washed 4 × with HBBS supplemented with 1 mM CaClGIPR R190Q and E288G on relevant phenotypes. Data on bone mineral density (BMD) and bone fracture risk have been contributed by p-values P.NI and P.I were used, respectively, as recommended by the authors. The data was downloaded from http://www.gefos.org/?q=content/data-release-2018. The BMD and fracture risk summary data derive from analyses performed in UK Biobank . Summary statistical data on body composition, obesity risk, physical activity, and cardiovascular events were derived from GeneATLAS   have been contributed by http://diagram-consortium.org/downloads.html. Results included two models either not including BMI as a covariate or adjusted for BMI (BMI adj.). The lipid levels association results were derived from summary data of an exome-wide meta-analysis  contributed by http://csg.sph.umich.edu/willer/public/lipids2017EastAsian/. Blood pressure and hypertension were investigated based on summary data derived from a meta-analysis of rare variants associated with blood pressure measures in European individuals  performed by https://app.box.com/s/1ev9iakptips70k8t4cm8j347if0ef2u. Data on myocardial infarction include summary statistics  contributed by the CARDIoGRAMplusC4D Consortium ] are derived from a meta-analysis of three GWAS, namely UK Biobank (interim release), CARDIoGRAMplusC4D 1000 Genomes-based, and the Myocardial Infarction Genetics and CARDIoGRAM Exome  . These snsortium . Data onAM Exome . Data on2.67  domain in position 67 of the GIPR, hence denoted R190gen bond .s is the main signaling pathway for the GIPR, we assessed the impact of these two mutations either separately or in combination. This was done by measuring intracellular cAMP accumulation in transiently transfected HEK293 cells in response to increasing concentrations of GIP. Both variants displayed reduced signaling capacity compared to wild-type GIPR with a markedly decreased (>250-fold) potency of GIP with EC50-values of 10 nM for R190Q and 3.6 nM for E288Q, compared to the wild-type GIPR with an EC50-value of 4.2 pM (max) of 75% of that of wild-type GIPR at 1 μM, whereas E288G reached 90%. The double mutant, R190Q-E288G resulted in a complete loss of activation through Gαs (As Gαf 4.2 pM . R190Q rough Gαs .125I-GIP(1-42) as radio-ligand for the wildtype plus all three GIPR variants. Both single mutations displayed reduced binding capacity (Bmax) with 30% of the wild-type GIPR for R190Q, and only 13% for E288G, while the double mutant exhibited minimal binding (< 1%) (D) of GIP were, however, not affected substantially as GIP bound with an affinity (KD) of 5.0 nM and 3.9 nM for R190Q and E288G, respectively, while it bound the wild-type GIPR with an affinity of 2.7 nM  . The binf 2.7 nM .max of 9.0% for R190Q, 8.6% for E288G, and 12% for the double mutant compared to wild-type GIPR. There was, however, no major difference with respect to the potencies of the receptors’ ability to recruit β-arrestin 2; R190Q had an EC50 of 0.76 nM while E288G had an EC50 value of 0.23 nM compared to wild-type GIPR with an EC50 of 0.88 nM. The double mutant, however, displayed an EC50-value of 11 nM  and E288G (rs143430880) diminished receptor activation, we were interested in linking these functional consequences with phenotypes in humans. At first, we searched for the largest genetic studies to gather available results of the two p-value = 0.002) and R190Q showed similar effect size (–0.057 SD), but this was not statistically significant  and fracture risk in carriers of R190Q and E288G using summary data from a study in UK Biobank with a total sample size of 426,824 individuals . Interesnificant . None offracture .GIPR variants were associated with higher systolic blood pressure , although the diastolic blood pressure was not significantly different between carriers and non-carriers , yet with no changes in low-density lipoprotein (LDL), triglycerides or total cholesterol   . Only R1 57,232) .GIPR variants affect risk of T2D in summary data from a study of coding variants in T2D    . In a mo = 0.04) .GIPR variants, R190Q and E288G, impact adiposity, we evaluated adiposity-related traits using UK Biobank results from the GeneATLAS portal  , lower hip circumference , lower waist circumference , lower fat percentage , lower fat mass  and fat-free body mass  (GIPR variant carriers significantly decreased risk of obesity (data not shown).To further assess how the two 452,264) . We foun452,264) , however452,264) . Interes.057 SD) . Furtherp-value < 10–4 for both GIPR variants were related to adiposity , result in impaired GIPR function at the molecular level which in turn seems to impact human physiology and pathophysiology regarding adiposity, bone health and the cardiovascular system  than on activation, when substituted with an alanine  of the receptor recognizes the C-terminal of the endogenous peptide hormone that in turn allows the N-terminal part of the ligand to position itself into the transmembrane domain (TMD) . While s alanine . This is alanine . We, in  alanine . AltogetGIPR variants showed a significantly increased overall bone fracture risk, possibly due to low statistical power. The common GIPR variant, E354Q (rs1800437), showed similar effects of lowered BMD along with increased risk of non-vertebral fractures  can be found in the article/HK, KS, MR, and NG: study design, manuscript writing—original draft. HK, MR, AS-U, and CK: functional studies. KS and NG: human genetic studies. AH: structural modeling. HK, KS, LG, AH, NG, and MR: manuscript writing—reviewing and editing. All authors revised the manuscript and approved the final version. All authors contributed to the article and approved the submitted version.MR was co-founder of Antag Therapeutics and Bainan Biotech. AS-U was co-founder and CEO of Antag Therapeutics. LG was co-founder of Antag Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Genomic epidemiology of SARS-CoV-2 is imperative to explore the transmission, evolution, and also pathogenicity of viruses. The emergence of SARS-CoV-2 variants of concern posed a severe threat to the global public health efforts. To assess the potential consequence of these emerging variants on public health, continuous molecular epidemiology is of vital importance. The current study has been designed to investigate the major SARS-CoV-2 variants and emerging mutations in virus structural and non-structural proteins (NSP) during the fourth wave in September 2021 from the Punjab province of Pakistan. Twenty SARS-CoV-2 positive samples have been collected from major cities were subjected to next-generation sequencing. Among the 20 whole genomes (GenBank Accession SRR16294858-SRR16294877), 2 samples failed to be completely sequenced. These genome sequences harbored 207 non-synonymous mutations, among which 19 were unique to GISAID. The genome sequences were detected: Delta 21I, 21J variants (B.1.617.2). Mutation’s spike_F157del, spike_P681R, spike_T478K, spike_T19R, spike_L452R, spike_D614G, spike_G142D, spike_E156G, and spike_R158del have been detected in all samples where K1086Q, E554K, and C1250W were unique in spike protein. These genomic sequences also harbored 129 non-synonymous mutations in NSP. The most common were NSP3_P1469S (N = 17), NSP3_A488S (N = 17), NSP3_P1228L (N = 17), NSP4_V167L (N = 17), NSP4_T492I (N = 17), NSP6_T77A (N = 17), NSP14_A394V (N = 17), NSP12_G671S (N = 18), and NSP13_P77L (N = 18). The mutation, F313Y in NSP12, detected in the current study, was found in a single isolate from Belgium. Numerous other unique mutations have been detected in the virus papain-like protease (NSP3), main protease (NSP5), and RNA-dependent RNA polymerase (NSP12). The most common non-synonymous mutations in the spike protein were subjected to stability analysis, exhibiting a stabilizing effect on structures. The presence of Delta variants may affect therapeutic efforts and vaccine efficacy. Continuous genomic epidemiology of SARS-CoV-2 in Pakistan may be useful for better management of SARS-CoV-2 infections. The deadly SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) posed a major effect on public health, disrupting the global healthcare system. SARS-CoV-2 has the ability to exhibit a high mutation frequency due to the presence of single-stranded RNA, posing major issues to public health. Molecular epidemiology is required to study the virus evolutionary stages in specific geographic locations and also to establish how the virus may affect the vaccine efficacy and drug response. The virus genome encodes 4 structural and 16 non-structural proteins (NSP) ,3,4,5. AAmong the several highly contagious VOCs, a large number of mutations have been detected. The B.1.1.7 lineage has been thought to be 40–80% more contagious ,11. HoweAmong the different SARS-CoV-2 variants, B.1.617 lineage was discovered in India ,19,20. TTo ensure a better public health policy, it is critical to continuously monitor and identify the rapidly evolving variations in circulating isolates of SARS-CoV-2 in populations. The current study was designed to investigate the major circulating VOC and also the emerging mutations in structural proteins and NSP among the SARS-COV-2 patients from major cities in the Punjab province of Pakistan. Twenty SARS-CoV-2 positive samples have been collected and whole-genome sequencing was performed through Ion Torrent next-generation technology. All the samples harbored numerous mutations in NSP and structural proteins, including S protein. The ethical clearance for the current study was obtained from the Ethical Review Committee for Medical and Biomedical Research at The University of Lahore (IMBB/UOL/21/1379).The nasopharyngeal swab method was used to collect samples from COVID-19 infected individuals, as recommended by the World Health Organization (WHO) . All theThe proper specimen collection procedure under laboratory testing for coronavirus disease (COVID-19) interim guidance by WHO was followed for SARS-CoV-2 samples collection and processing. The nasopharyngeal swabs were collected in 2 mL of vial transport medium  and placed at 4 °C for further use. All these samples were confirmed with TaqPath™ COVID-19 CE-IVD RT-PCR Kit .RNA extraction was performed from nasopharyngeal swabs using a standard volume of 200 ul by MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit  and . The extracted RNA was stored at −80 °C for downstream application. The real-time quantification and copy number determination of nucleic acid was performed using TaqPath™ 1-Step RT-qPCR Master Mix  and the relevant copy number was determined according to the CT and control reactions in quantification. Only samples with relevant CT of 18–28 values were chosen. Using a minimum input amount of 10 ng/uL of the RNA sample, RNA to cDNA reversed transcription was performed with the SuperScript™ VILO™ cDNA Synthesis Kit .The library was prepared manually using Ion AmpliSeq™ Library Kit . Amplification was performed with a 2-pool Ion AmpliSeq™ SARS-CoV-2 Research Panel . The library was partially digested and ligated with Ion Xpress™ Barcode Adapters 1–96 Kit . Library purification and quantification steps were carried out with GenDx-AMPure® XP beads  and TaqMan™ Quantitation Kit . The libraries were diluted to a final concentration of 40 pM, loaded on Thermo Fisher Scientific Ion Chef™ Instrument for PCR, and loaded on Ion S5 530 and 510 . Sequencing was accomplished on Ion GeneStudio™ S5 System at LabGenetix, Lahore, Pakistan. The fastQ base sequence file quality was assessed using the FastQC (v0.11.8). Trimmomatic tool (v0.39) was used to remove the low-quality reads (Q < 30) and index adapter sequences were utilized to improve sample multiplexing. Sequenced reads were aligned with the reference . The duplicated PCR reads were removed with Picard Tools (v2.21.6). Mapping problems due to the presence of small Indels were removed with a Genome Analysis Toolkit, “RealignerTargetCreator” and “InDelRealigner” (GATK v. 3.3.0) to analyze the mapped read. To improve the accuracy of variant calling, GATK tool “HaplotypeCaller” was applied for realignment of sequenced reads via local de-novo assembly of haplotypes in the regions showing variation. https://www.gisaid.org/epiflu-applications/covsurver-mutations-app/ (accessed on 28 September 2021)) on 15–20 September 2021, collected during the fourth wave of infection. The CoVsurver research tool has been developed with GISAID , aiding researchers to identify and interpret the amino acids (aa) changes in coronavirus genomes. The server rapidly aligns the query genome sequences in fasta format with reference SARS-CoV-2 and screen coronavirus genomes for aa changes to identify any special epidemiological relevance. All mutations in structural proteins of SARS-CoV-2 were separated and arranged in the form of excel sheets. The statistical analysis was performed using EpiData Analysis [All the 20 whole genome sequences in fasta format were aligned with reference (NC_045512) using CoVsurver application  along with a good resolution of results. To compute the SARS-CoV-2 wild type and mutant protein stability and flexibility, the PDB file of proteins were retrieved from the Protein Data Bank [Some of the most common mutations were analyzed for their thermodynamic effect on S protein through DynaMut server . The serata Bank  and uploIn DynaMut, changes upon point mutation on free energy of protein folding, combining the impacts of mutation stability of protein and also the dynamic properties were computed by ENCoM, Bio3D, and DUET computational servers, generating a more robust predictor of energies.Genomic sequences in the current study were subjected to MAFT server  and NextTwenty nasopharyngeal swab samples were collected during the fourth wave of the pandemic in September 2021 from SARS-CoV-2 patients . Among thttps://www.ncbi.nlm.nih.gov/bioproject/PRJNA770504 (accessed on 28 September 2021)). The average length of sequenced genomes was 29,586 nucleotides (9691 amino acids). The sequence information, including length and clade, is shown in Among the 20 samples (GenBank Accession No. SRR16294858-SRR16294877), samples 7 and 10  . All the genomic samples were of GK clade. Mutations E554K (S1 domain of S), K1086Q, and C1250W (S2 domain of S)  were uniAll the samples were detected as Delta variants. Mutation details in S and other structures are provided in Among the 207 mutations, 26 were detected in S protein . The mosSimilarly, nucleocapsid (N) also harbored nine different kinds of mutations . Among tDelta variant harbors L452R, P681R, and T478K in S protein. These mutations, along with others, present in all genomic samples, exhibited a stabilizing effect on the S protein structure , and mayThe spike_G142D mutant exhibited a little destabilizing affect , E156G , L452R , P681R , and T19R . The sequences harbored 129 mutations in NSP in which 13 were unique to GISAID  Supplem. The higThe mutation details in NSP are shown in The highest frequency of mutations was detected in virus NSP3  in whichNSP5 (main protease), harbored three non-synonymous mutations in a very low frequency. Four mutations were detected in NSP6 in which T77A was detected with the highest frequency (N = 17). Virus NSP12, which is also called RdRp (RNA-dependent RNA polymerase), plays an essential role in replication. Mutations NSP12_G671S (N = 18) and NSP12_P323L (N = 16) were the most common, present in the interface and RdRp domain . A single mutation P77L in NSP13 was detected in all genomic isolates (N = 18). Forty-one mutations were detected in NSP14, including five non-synonymous mutations in which NSP14_A394V (N = 17) NSP14_M72I (N = 6) were the most common. The recent emergence of different SARS-CoV-2 variants posed severe health issues. The α is one of the identified variants that first emerged in the UK and became one of the predominant lineages worldwide. Pakistan reported its first case with α variant in December 2020 , triggerAmong the different variants, the Delta variant of SARS-CoV-2 is more deadly and highly transmissible with severe disease signs and symptoms. This variant has been detected in 77 countries as of 24 June 2021 ,32. The In the current study, all the genomic samples were detected as Delta variants when analyzed on GISAID CoV-Surver on 15 to 20 September 2021, collected during the fourth wave of infection. Mutations T478K, T19R, L452R, F157del, E156G, P681R, D614G, R158del, and G142D were the most common, present in all genomics isolates. The T478K mutation present in RBD of S protein is involved in interaction with human ACE2 ,34. The A previous study during the third wave from Pakistan reported that the Delta variant is prevalent in 45% of cases followed by β (46%) . Very liAmong the structural proteins, the S protein harbored two mutations in the RBD region (L452R and T478K), four in the NTD , and one at the furin-cleavage site (P681R) and S2 region (D950N), detected in 12 virus genome sequences . This clMutations Arg158 and Phe-157/del were also detected in all genomic isolates , presentThe Delta S protein P681R mutation has an important role in the variant replacement of the α-to-Delta. The Delta S P681R mutation present at the furin-cleavage site exhibited stabilizing effects , separatConsidering the circulation of the Delta virus with specific mutations and a high transmissible rate, the Pakistani national health authority needs to take timely measures against it. Further, molecular epidemiological studies with insight into genomic analysis of the virus are needed to screen the most prevalent variants and specific mutations that may affect the diagnosis and vaccine potency, to adopt potential measures in the future.Emerging mutations in NSP may affect the virus transmission and pathogenicity. Recently, a synonymous mutation (F106F) in NSP3 along with other signature mutations exhibited a virus fitness effect . In CoV-The NSP6_L37F mutation, which was very common in the earlier infections, were associated with asymptomatic cases. NSP6 reduces autophagic ability, important for viral infections and promotes cell death . MutatioPreviously, mutation P323L in RdRp was the most common in Pakistani isolates . This muVaccine efficacy may be reduced in previous VOCs; however, BNT162b2 retained its potency against β VOC . OmicronIn conclusion, Delta variants with some unique mutations, are circulating during the fourth wave of the pandemic, in major cities in the Punjab province of Pakistan. The structural proteins and NSP harbored numerous mutations, present in different functional domains. Mutations spike_K1086Q, spike_E554K, and spike_C1250W were unique to GISAID. The most common mutations in the S protein exhibited a stabilizing effect, facilitating the binding affinity for S protein RBD with human ACE2. Geographic specific vaccines and drugs may be designed for better management of COVID-19 in the future. The geo-climate distribution of the mutations may decipher higher uniqueness and disease severity in underdeveloped countries, including Pakistan. The effect of these mutations on virus pathogenicity may be experimentally verified to access the effect on virus severity and vaccine efficacy. Continuous molecular epidemiology is required to screen geographic-specific mutations for better understanding of the virus pathogenicity, diagnosis, and treatment of COVID-19.