Current commercially available serological tests for COVID-19 patients are detecting antibodies against SARS-CoV-2 nucleoprotein and spike glycoprotein. The antinucleoprotein and antispike antibodies can be accurately detected in patients during the mid or late stage of infection, and therefore, these assays have not been widely used for early diagnosis of COVID-19. In this study, we characterized the secretory property of a SARS-CoV-2 orf8 protein and proposed that orf8 secretion during infection facilitates early mounting of the B cell response. We demonstrated the presence of anti-orf8 antibodies in both symptomatic and asymptomatic patients during the early stage of infection, while the anti-N antibody is not detected. Our serological test detecting anti-orf8 antibodies may facilitate the development of early and accurate diagnosis for COVID-19. An accurate diagnostic test for early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the key weapon to control the coronavirus disease 2019 (COVID-19) pandemic. We previously reported that the SARS-CoV-2 genome contains a unique orf8 accessory gene absent from other human-pathogenic coronaviruses. Here, we characterized the SARS-CoV-2 orf8 as a novel immunogenic secreted protein and utilized it for the accurate diagnosis of COVID-19. Extracellular orf8 protein was detected in cell culture supernatant and in sera of COVID-19 patients. In addition, orf8 was found highly immunogenic in COVID-19 patients, who showed early seropositivity for anti-orf8 IgM, IgG, and IgA. We hypothesize that orf8 secretion during SARS-CoV-2 infection facilitates early mounting of B cell response. The serological test detecting anti-orf8 IgG antibody can be used for the early and accurate diagnosis of COVID-19. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already affected more than 17 million patients with more than half a million deaths within 7 months  and receptor-binding domain (RBD) of spike protein can be detected in some patient sera as early as 1 week after the onset of disease 24. There Sarbecovirus, genus Betacoronavirus. Unlike other human highly pathogenic coronaviruses, the SARS-CoV-2 genome contains a unique open reading frame 8 (orf8) gene (SARS-CoV-2 is a positive-stranded enveloped virus belonging to the subgenus f8) gene . In thisThe orf8 protein is absent in all human-pathogenic coronaviruses except the recent SARS-CoV-2, but it is present in some bat coronaviruses. We first examined the amino acid sequences of human SARS-CoV-2 orf8 and other orf8 proteins. The orf8 amino acid sequences of known bat SARS-CoV-like coronaviruses, recently discovered bat SARS-CoV-2-related coronaviruses, SARS-CoV and SARS-CoV-2 were segregated into three clades in the phylogenetic tree . In cont10.1128/mBio.02431-20.1FIG S1KT444582; bat SL-CoV WIV1, KF367457; bat SL-CoV Rs3367, KC881006; bat SL-CoV Rs672, FJ588686; bat SL-CoV RsSHC014, KC881005; bat SL-CoV Rp3, DQ071615; bat SARS-CoV HKU3-1, DQ022305; bat SARSr-CoV RaTG13, MN996532; bat SL-CoV ZXC21, MG772934; bat SL-CoV ZC45, MG772933; bat SL-CoV Rf1, DQ412042; bat SL-CoV YNLF-31C, KP886808; bat SL-CoV YNLF34C, KP886809), Paguma SARS-CoV , SARS-CoV , and SARS-CoV-2 . Multiple alignment analysis was performed, and the alignment of orf8 protein sequences was presented as a BoxShade diagram. There are three clades representing (i) bat SARS-like coronaviruses , (ii) human SARS-CoV-2 and its closest bat coronaviruses  and (iii) human SARS-CoV and its related paguma and bat coronaviruses . Black and gray boxes of amino acid letters represent identity and similarity, respectively. Download FIG S1, EPS file, 2.6 MB.Multiple alignment of orf8 amino acid sequences. Amino acid sequences of orf8 proteins are derived from bat SARS-like coronaviruses  Three-dimensional structure at 0° and 270° and top view of the mature orf8  is shown. β-Strands (green), predicted disulfide bonds (blue), amino acid loop (orange), and the external domain (light blue) are shown. (D) Overall topology of the predicted structure. Download FIG S2, EPS file, 2.5 MB.Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To characterize the novel SARS-CoV-2 orf8 protein, a basic overexpression model in mammalian cell lines was adopted to preliminarily test its function. In two human cell lines, lung epithelial A549 cells and embryonic kidney 293FT cells, ectopic expression of orf8 did not cause cell death or morphological changes . On the 10.1128/mBio.02431-20.3FIG S3FIG S3, EPS file, 2.5 MB.No morphological changes were observed in orf8-expressing A549 cells. A549 cells were transfected with empty vector or Flag-orf8 expression vector as described in the legend to Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We further characterized the secretion property of the orf8 protein by introducing mutations to the signal sequence . Three m10.1128/mBio.02431-20.5MOVIE S1Movie S1, AVI file, 8.0 MB.3D localization orf8 protein in lung A549 epithelial cells. Video illustration of Flag-orf8 staining in A549 cells. This 3D movie was exported from the same confocal field as illustrated in Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To validate the secretion of orf8 protein during infection and its clinical relevance, sera of COVID-19 patients post-symptom onset were analyzed for the presence of the orf8 protein. First, 14 of the most abundant serum proteins, including serum albumin and immunoglobulins, were removed. The presence of SARS-CoV-2 proteins in the depleted sera was then determined by liquid chromatography-mass spectrometry. Impressively, four unique peptides covering 90% of the orf8 amino acid sequence could be detected despite the small size (13 kDa) of orf8 . We alsoCurrently, there is insufficient knowledge as to when and what kind of immune responses were elicited by orf8. Since orf8 is a secreted viral protein that could be detected in sera of hospitalized COVID-19 patients, we questioned whether orf8 is immunogenic. Therefore, we tested for the presence of anti-orf8 antibodies in infected patients using our in-house orf8 enzyme-linked immunosorbent assay (ELISA). Serum samples were collected from 29 adult COVID-19 patients ≥28 days post-symptom onset  see . Moreove10.1128/mBio.02431-20.4TABLE S1Table S1, DOCX file, 0.02 MB.Cohort characteristics of patients. Characteristics of patients involved in this study were summarized in the table. Download Copyright © 2020 Wang et al.2020Wang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the There are several commercially available serological assays that can detect anti-N or anti-RBD antibodies in almost all PCR-positive serum samples from COVID-19 patients at ≥28 days post-symptom onset . HoweverDue to the limitation that anti-N/RBD antibodies are generally detected only at the mid or late stage of acute infection, for example, more than 10 days after disease onset –4, curre–Current studies have reported that COVID-19 patients develop neutralizing antibodies and high titer of SARS-CoV-2-specific antibody response , 12, 13.in vitro orf8 secretion assay suggested that orf8 could be readily secreted upon expression  in a SARS-CoV-2 genome in Singapore (45/191 screened samples) and Taiwan (one case) , 17. ThiThis study did not investigate cellular immunity to COVID-19, i.e., immune response involving T cells rather than antibodies. Some previous studies have shown the necessity of sufficient magnitude or durability of T cell response for antibody generation –21. A st–Although the anti-orf8 antibodies may aid in the diagnosis of COVID-19 disease, there is also great uncertainty about whether adaptive immune responses to orf8 are protective or pathogenic and whether this might influence susceptibility to COVID-19 disease. For example, the possibility that anti-orf8 antibodies contribute to host pathogenesis by forming immune complex with orf8 protein should be evaluated in future studies. Additionally, to date, the duration of SARS-CoV-2 RNA shedding has not been well characterized. One recent study of 191 COVID-19 patients reported that the median duration of viral shedding was 20 days in survivors  . In thisThe function of extracellular or secreted orf8 protein has not yet been reported. Some of the secreted viral proteins function as cytokine mimics , cytokine inhibitors , complement inhibitors  and inflammatory cell inhibitor (myxoma virus SERP-I) . It willIn summary, we provide experimental evidence that orf8 functions as a secreted protein that is highly immunogenic. To the best of our knowledge, this study is the first to report the secretory property of SARS-CoV-2 orf8 and the earlier induction and higher sensitivity of anti-orf8 antibodies compared to that of anti-N antibodies. Our findings suggest the ELISA described in this study can be directly applied for the early and accurate diagnosis of COVID-19.COVID-19 patients with respiratory samples positive for SARS-CoV-2 by reverse transcription-quantitative PCR (RT-qPCR) after admission to the Queen Mary Hospital in Hong Kong during January and July 2021 were included in this study. An in-house RT-qPCR targeting the SARS-CoV-2 RNA-dependent RNA polymerase-helicase gene region was performed as we previously described , 11. In Coding sequences of C-terminus Flag-tagged or His-tagged orf8 were subcloned into mammalian expression vector pCAGEN (Addgene plasmid no. 11160). Gene fragments for signal peptide mutation and L84S amino acid substitution were generated by PCR and subcloned into the same pCAGEN backbone vector.2. Expi293F cells were purchased from Thermo Fisher Scientific and used for the protein production. Suspension Expi293F cells were cultured in serum-free and protein-free Expi293 expression medium (Thermo Fisher Scientific) and incubated at 37°C with 8% CO2 and shaking at 125 rpm.Human embryonic kidney 293FT cells were purchased from Invitrogen (USA), and human lung epithelial A549 cells were obtained from ATCC (USA) (ATCC CCL-185). Both cell lines were cultured in Dulbecco’s modified Eagle medium  with 10% fetal bovine serum (DMEM/10% FBS) and incubated at 37°C with 5% COFor 293FT, Flag-tagged orf8 expression plasmid was transfected using GeneJuice transfection reagent  according to the manufacturer’s instructions. Cells or culture supernatant was harvested according to experiment conditions. For A549, Flag-tagged orf8 expression plasmid was transfected using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. At 5 h posttransfection, cells were washed with phosphate-buffered saline (PBS) two times and replenished with fresh DMEM/10% FBS. Cells or culture supernatant was harvested at the indicated time points.Escherichia coli. Therefore, the Expi293 expression system (Thermo Fisher Scientific) was used for mammalian recombinant orf8 protein production. Expi293F cells were transfected with pCAGEN-orf8-His using Expifectamine 293 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Five or 6 days posttransfection, serum-free medium carrying secreted orf8 protein was harvested and purified by nickel-nitrilotriacetic acid (Ni-NTA) agarose  or anti-His affinity resin (Genscript). Recombinant His-orf8 protein was eluted using 200 mM imidazole in 500 mM NaCl or eluted with low pH buffer.Recombinant orf8 is highly insoluble and cannot be expressed in Transfected cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer  with EDTA-free protease inhibitor cocktail (Roche). Clarified protein lysates were mixed with 5× protein sample buffer  and denatured at 95°C. Proteins were then separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene fluoride membranes, and detected by WesternBright ECL reagent . Primary anti-Flag and anti-β-actin antibodies from Sigma-Aldrich (USA), and secondary anti-mouse and anti-rabbit antibodies from GE Healthcare (USA) conjugated with horseradish peroxidase (HRP) have been used.g for 20 min to remove cell debris. The clarified supernatants were then applied to equilibrated anti-Flag M2 magnetic beads for immunoprecipitation as described above.Human lung epithelial A549 cells and human embryonic kidney 293FT cells were transfected with Flag-tagged orf8 mammalian expression plasmids. At 48 h posttransfection, transfected cells were harvested in immunoprecipitation (IP) lysis buffer  containing EDTA-free protease inhibitor cocktail (Roche) and clarified by centrifugation. Cell lysates were then immunoprecipitated using anti-Flag M2 magnetic beads (Millipore), followed by elution with 1× protein sample buffer with boiling. For immunoprecipitation from culture supernatant, the conditioned culture media were harvested and centrifuged at 1,000 × 18 spin columns (Thermo Fisher Scientific), followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. MS data were further searched for matches to all amino acid sequences derived from SARS-CoV-2 .Serum samples of RT-qPCR-confirmed COVID-19 patients were handled in biosafety level 3 laboratory. Before heat inactivation, 14 abundant proteins, including albumin, IgG, IgA, IgM, IgD, IgE, fibrinogen, and transferrin, were removed by the High-Select Top14 abundant protein depletion spin column (Thermo Fisher Scientific). Depleted sera were mixed with final 1× protein sample buffer and denatured at 98°C for 10 min. Inactivated sera were loaded into 12% SDS-PAGE. The protein was stained with Coomassie blue and excised from the gel. The gel slice was then dehydrated with acetonitrile followed by overnight in-gel trypsin digestion. The peptides were then column cleaned using Pierce CA549 cells seeded on coverslips were transfected with pCAGEN-Flag-orf8. Cells were then fixed with 1:1 (vol/vol) acetone-methanol at −20°C, PBS washed, and stained with the indicated antibodies. Antibodies used for immunofluorescence staining were as follow: anti-Flag (Sigma-Aldrich), anti-calnexin , anti-α-tubulin (Abcam), anti-mouse labeled with Alexa Fluor 488 (Abcam), anti-rabbit labeled with Alexa Fluor 594 (Abcam). The coverslips were then mounted on glass microscope slides with Vectashield Vibrance mounting medium . Image acquisition was performed with a Zeiss LSM-880 confocal microscope with 63× oil objective (Zeiss). Z-stack acquisition was performed using ZEN software (Zeiss).Total RNA was isolated from cultured cells using RNAiso Plus  following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized by reverse transcribing 1 μg purified RNA using PrimeScript RT reagent kit with gDNA Eraser (TaKaRa). RT-PCR was performed using DreamTaq Green DNA polymerase (Thermo Fisher Scientific).KT444582; bat SL-CoV WIV1, KF367457; bat SL-CoV Rs3367, KC881006; bat SL-CoV Rs672, FJ588686; bat SL-CoV RsSHC014, KC881005; bat SL-CoV Rp3, DQ071615; bat SARS-CoV HKU3-1, DQ022305; bat SARSr-CoV RaTG13, MN996532; bat SL-CoV ZXC21, MG772934; bat SL-CoV ZC45, MG772933; bat SL-CoV Rf1, DQ412042; bat SL-CoV YNLF-31C, KP886808; bat SL-CoV YNLF34C, KP886809), Paguma SARS-CoV , SARS-CoV , and SARS-CoV-2 . Multiple alignment analysis was performed using on-line CLUSTALW software , and the alignment of orf8 protein sequences was drawn using BoxShade program . Phylogenetic analysis of orf8 amino acid sequences was performed using MEGA X . The topology was drawn based on the predicted structure.SARS-CoV-2 orf8 protein structure prediction was done using comparative modeling with RosettaCM  on the RSerum samples were run on the Abbott Architect instrument using the Abbott SARS-CoV-2 IgG assay following the manufacturer’s instructions. The assay is a chemiluminescent microparticle immunoassay for qualitative detection of IgG in human serum or plasma against the SARS-CoV-2 nucleoprotein. The Architect platform requires a minimum of 100 μl of serum or plasma. Qualitative results and index values reported by the instrument were used in analyses.E. coli and recombinant orf8 protein purified from Expi293 cells were coated with 100 ng and 250 ng of protein per well in 50 mM coating buffer (pH 9.6 Na2CO3/NaHCO3) on ELISA plates  and incubated overnight at 4°C. Plates were blocked with 0.5% (wt/vol) bovine serum albumin (BSA) and 0.5% gelatin in 0.05% Tween 20 (Sigma-Aldrich) in PBS at room temperature for 1 h and washed one time in 0.05% Tween 20 (Sigma) in PBS. Diluted sera were added and incubated for 1 h at 37°C. Plates were washed three times and incubated with HRP-conjugated goat anti-human IgM , goat anti-human IgG , or goat anti-human IgA  for 1 h at 37°C. The color was developed using trimethyl borane (TMB) solution (Thermo Fisher Scientific), and absorbance was measured at 450 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific). We used archived anonymous serum samples as the negative control. The cutoff for seropositivity was set as the mean value of 100 control serum samples plus two times the standard deviation.Antigen-specific antibodies were examined by enzyme-linked immunosorbent assay (ELISA). Briefly, recombinant N protein purified from t test was used for analysis of two groups, and one-way analysis of variance (ANOVA) was used for analysis of multiple groups. Nonparametric data were analyzed by a Mann-Whitney test. P values of less than 0.05 were considered statistically significant. Data in this study were indicated as means with standard deviations.All analysis was performed using GraphPad PRISM software. A paired or unpaired


The 3D spheroids exhibited altered expression of EMT markers associated with metastasis such as E-cadherin, vimentin and Snail, compared to 2D monolayer cells. TKI treatment did not result in significant changes of EMT markers, however, specific inhibition of FGFR signaling by BGJ398 showed more favorable molecular-level changes than treatment with the multi-RTK inhibitor Dovitinib. This study provides evidence for the first time that FGFR1 plays an essential role in the proliferation of PCa CSCs at a molecular and cellular level, and suggests that TKI targeting of FGFR signaling may be a promising strategy for AR-independent CRPC.Metastatic castrate-resistant prostate cancer (CRPC) remains uncurable and novel therapies are needed to better treat patients. Aberrant Fibroblast Growth Factor Receptor (FGFR) signaling has been implicated in advanced prostate cancer (PCa), and FGFR1 is suggested to be a promising therapeutic target along with current androgen deprivation therapy. We established a novel  Prostate cancer (PCa) is the fifth leading cause of cancer-related deaths among men worldwide . PCa is in vivo with high efficiency. Cancer stem cells have been characterized with respect to biomarker expression, such as cell surface markers, functional markers such as self-renewal genes, and intracellular enzyme activity, which may be responsible for drug resistance . 25 μg or 30 μg of total protein was separated by 10% or 12.5% SDS-PAGE followed by transfer to Immobilon-P membrane. Immunoblotting reagents were from the following sources: antibodies against p-FGFR (Tyr653/654), FGFR1 (D8E4), FGFR4 (D3B12), p-VEGFR (19A10), VEGFR2 (55B11), p-AKT (D9E), AKT (9272), p-MAPK (D13.14.4E), p44/42 MAPK (Erk1/2), OCT4 (2750), CD133 (D2V8Q), Androgen Receptor (D6F11), and β-actin (4967) antibodies were from Cell Signaling Technology; FGFR2 (C-8), FGFR3 (B-9), and STAT5 (C-17) were from Santa Cruz Biotechnology; ALDH7A1 (CAT:ABO11656) was from Abgent; HRP anti-mouse, HRP anti-rabbit, and Enhanced Chemiluminescence (ECL) reagents were from GE Healthcare. Other reagents included: Dovitinib, BGJ398 and PD166866 were from Selleckchem.


Scientific Reports 10.1038/s41598-019-55389-z, published online 19 December 2019Correction to: The original version of this Article contained errors.In the Abstract,“Since improvement of obstetric care at the hospital level needs quantitative evidence, using routinely collected health data we contrasted the performance of the 11 maternity centres  of an Italian region, Friuli Venezia Giulia (FVG), during 2005–15, after removing the effect of several factors associated with different delivery modes (DM): spontaneous vaginal delivery (SVD), instrumental vaginal delivery (IVD), overall CS (OCS) and urgent/emergency CS (UCS).”now reads:“Since improvement of obstetric care at the hospital level needs quantitative evidence, using routinely collected health data we contrasted the performance of the 11 maternity centres  of an Italian region, Friuli Venezia Giulia (FVG), during 2005–15, after removing the effect of several factors associated with different delivery modes (DM): spontaneous vaginal delivery (SVD), instrumental vaginal delivery (IVD), overall CS (OCS) and urgent/emergency CS (UCS).”“Although the overall CS rate in FVG during 2005–15 was 24.2%, well below the corresponding average Italian national figure (38.1%), the variability of DM rates across FVG maternity centres could be targeted by policy interventions aimed at reducing the recourse to unnecessary CS.”now reads:“Although the overall CS rate in FVG during 2005–15 was 24.2%, well below the corresponding average Italian national figure (38.1%), the variability of DM rates across FVG maternity centres could be targeted by policy interventions aimed at further reducing the recourse to unnecessary CS.”Furthermore, in Figure 1 the number of vaginal deliveries was omitted. As a result the Figure legend,“Flowchart displaying the criteria applied to the initial database to obtain the final number of hospital births available for the analysis. SVD  =  spontaneous vaginal deliveries; IVD  =  instrumental vaginal deliveries; CS  =  cesarean sections.”now reads:“Flowchart displaying the criteria applied to the initial database to obtain the final number of hospital births available for the analysis. VD = Vaginal Deliveries; SVD  =  spontaneous vaginal deliveries; IVD  =  instrumental vaginal deliveries; CS  =  cesarean sections; PCS: Planned CS; UCS = Urgent/Emergency CS”In Figure 7 the number of Placenta previa/ abruptio placenta/ante-partum Hemorrage was incorrectly reported as 752. As a result, in the Methods section under subheading ‘Data Cleaning’,“87 births by SVD and 15 births by IVD associated with placenta previa/abruptio placenta/ante-partum hemorrage have maintained their original DM, but recoded as not having any placenta previa/abruptio placenta/ante-partum hemorrhage.”now reads:“Placenta previa/abruptio placenta/ante-partum haemorrhage delivering by SVD (N = 136) and by IVD (N = 22) have been excluded from the analysis, for a total 158 missing data.”Under the subheading ‘Conceptual Framework’,“4. Obstetric conditions, shown in Fig. 7: oligohydramnios; polyhydramnios; eclampsia/pre-eclampsia; placenta previa/abruptio placenta/ante-partum hemorrhage; non- reassuring fetal status; fetal anomalies; cord prolapse; PROM; Rh immunization, obstructed labour (except shoulder girdle dystocia); labour analgesia; labour mode; presentation; any medical assisted fertilization.”now reads:“4. Obstetric conditions, shown in Fig. 7: oligohydramnios; polyhydramnios; eclampsia/pre-eclampsia; placenta previa/abruptio placenta/ante-partum hemorrhage; non- reassuring fetal status; Congenital malformations at birth; cord prolapse; PROM; Rh immunization, obstructed labour (except shoulder girdle dystocia); labour analgesia; labour mode; presentation; any medical assisted fertilization.”Under the subheading ‘the database’,“We used the following ICD-9 codes to retrieve the obstetric conditions associated with each childbirth:Polyhydramnios: 657.0;Oligohydramnios: 658.0;Antepartum hemorrhage, abruptio placentae and placenta previa: 641.(0-1-2-3-8-9);Obstructed labour (except shoulder girdle dystocia): 660.(0-1-2-3-5-6-7-8-9);Non-reassuring fetal status: 656.3;Fetal anomalies: 655.9;Cord prolapse: 663.0;Premature rupture of membranes (PROM): 658.1;Eclampsia/pre-eclampsia: 624.(4-5-6-7);Rh iso-immunization: 656.1.”now reads:“We used the following ICD-9 codes to retrieve the obstetric conditions associated with each childbirth:Polyhydramnios: 657.0;Oligohydramnios: 658.0;Antepartum hemorrhage, abruptio placentae and placenta previa: 641.(0-1-2-3-8-9);Obstructed labour (except shoulder girdle dystocia): 660.(0-1-2-3-5-6-7-8-9);Non-reassuring fetal status: 656.3;Cord prolapse: 663.0;Premature rupture of membranes (PROM): 658.1;Eclampsia/pre-eclampsia: 642.(4-5-6-7);Rh iso-immunization: 656.1.”Finally, the Supplementary File that originally accompanied this Article contained mathematical inaccuracies in the rounding of some estimates in Supplementary Tables S1 and S2.These errors have now been corrected in the PDF and HTML versions of the Article and in the accompanying Supplementary Information file.


Taken together, our data support recent clinical observations indicating a benefit of chemoimmunotherapy compared to monotherapy in breast cancer and suggest potential underlying mechanisms.Despite the success of immune checkpoint blockade in cancer, the number of patients that benefit from this revolutionary treatment option remains low. Therefore, efforts are being undertaken to sensitize tumors for immune checkpoint blockade, which includes combining immune checkpoint blocking agents such as anti-PD-1 antibodies with standard of care treatments. Here we report that a combination of chemotherapy (doxorubicin) and immune checkpoint blockade (anti-PD-1 antibodies) induces superior tumor control compared to chemotherapy and immune checkpoint blockade alone in the murine autochthonous polyoma middle T oncogene-driven (PyMT) mammary tumor model. Using whole transcriptome analysis, we identified a set of genes that were upregulated specifically upon chemoimmunotherapy. This gene signature and, more specifically, a condensed four-gene signature predicted favorable survival of human mammary carcinoma patients in the METABRIC cohort. Moreover, PyMT tumors treated with chemoimmunotherapy contained higher levels of cytotoxic lymphocytes, particularly natural killer cells (NK cells). Gene set enrichment analysis and bead-based ELISA measurements revealed increased IL-27 production and signaling in PyMT tumors upon chemoimmunotherapy. Moreover, IL-27 signaling improved NK cell cytotoxicity against PyMT cells  The idea to engage the immune system in the fight against cancer was already proposed in the early twentieth century but was then mainly disregarded . Over a 2 × π/6. For all animal experiments the guidelines of the Hessian animal care and use committee were followed .Female mice expressing the polyoma virus middle T oncoprotein (PyMT) under the Mouse Mammary Tumor Virus (MMTV) promoter in a C57BL/6 background were used. In the PyMT model, mice spontaneously develop tumors in each mammary gland starting from 8 weeks after birth. Mice were divided into four groups according to treatment  + anti-PD-1, and DOX + IgG1). For animals receiving immune checkpoint blockade only, treatment was initiated (day 0) once the first tumor reached a size of 0.6 cm in diameter. Antibodies were administrated i.p. at a concentration of 20 mg/kg (on day 0) and 10 mg/kg . All mice received either anti-mouse PD-1 antibody  or anti-mouse IgG1  diluted in sterile 0.9% NaCl. In the model with the combination of chemotherapy and immune checkpoint blockade treatment started once the first mammary tumor reached a size of 1 cm in diameter. Doxorubicin  diluted in sterile 0.9% NaCl was administrated i.p. (5 mg/kg) once a week for 5 weeks. One day after doxorubicin administration, mice were treated with 10 mg/kg of either anti-mouse PD-1 antibody  or anti-mouse IgG1 (BioXCell). Mice were monitored three times a week for up to 5 weeks after initial treatment. Tumor size was determined by tumor palpating. The tumor volume was calculated using the formula: V = length × widthTumor single cell suspensions were generated using the Tumor Dissociation Kit and the gentleMACS™ Dissociator  using standard protocols. The following anti-mouse antibodies were used for staining of single cell suspensions: anti-CD3-PE-CF594, anti-CD4-BV711, anti-CD8-BV650, anti-CD11c-BV711, anti-CD19-APC-Cy7, anti-CD45-AlexaFluor700, anti-CD49f-PE-CF594, anti-CD146-AlexaFluor488, anti-CD326-BV711, anti-Ly6C-PerCP-Cy5.5, anti-NK1.1-BV510 , anti-CD31-PE-Cy7, anti-CD117-APC-eFluor780 , anti-CD90.2-PE, anti-MHC-II-APC (both from Miltenyi Biotec), anti-CD11b-BV605, anti-CD324-AlexaFluor647, anti-F4/80-PE-Cy7, anti-GITR-FITC, anti-Ly6G-APC-Cy7, anti-SiglecH-FITC, and anti-γδTCR-APC . NK/PyMT cell co-culture samples were stained with the following antibodies. anti-CD25-PE-Cy7, anti-CD69-BV605, anti-CD107a-PE and anti-NK1.1-APC . Samples were acquired with a LSR II/Fortessa™ flow cytometer (BD Biosciences) and analyzed using FlowJo software V10 (BD Biosciences). All antibodies and secondary reagents were titrated to determine optimal concentrations. CompBeads (BD Bioscience) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one (FMO) controls were used. The instrument calibration was controlled daily using Cytometer Setup and Tracking (CS&T) beads (BD Bioscience).Total RNA was isolated from snap frozen PyMT tumors using the peqGOLD Total RNA Kit . RNA samples were analyzed on a 2100 Bioanalyzer using Agilent RNA 6000 Nano chip . Library preparation was performed using the SMARTer® Stranded Total RNA Sample Prep Kit–HI . Quantity and quality of the cDNA libraries were determined by Qubit™ dsDNA HS Assay Kit  and Agilent High Sensitivity DNA chip (Agilent Technologies). Libraries were sequenced on a NextSeq 500 sequencer  using V2 chemistry . Sequencing data were analyzed using the SeqBox software . In brieThe METABRIC data set  was usedTM 7-Color Fluorescent Immunohistochemistry (IHC) Kits . The following anti-mouse antibodies were used: anti-αSMA , anti-DIO2 , anti-GSN , anti-MMP3 , anti-Pan-Cytokeratin , anti-PD-L1 , and anti-PDK4  in an automated staining using the BOND RX Automated IHC Research Stainer . Stained tumor sections were scanned using Vectra® 3 automated quantitative pathology imaging system and analyzed using inForm® software V2.3 (both Akoya). Marker expression in the cytoplasm was quantified with the inForm® software using a positivity or 4-bin (0–3+) scoring algorithm  between individual treatment groups as an input, enriched biological processes were identified using Gene Set Enrichment Analysis (GSEA) version 4.0.0 .Tumor interstitial fluids were obtained by manual cryopulverization and subsequent incubation with 1:2 tumor weight/volume of 2 × PBS for 3 h at 4°C under rotation. The LEGENDplex™ mouse inflammation panel (Biolegend) was used to determine cytokines levels in the tumor supernatants. To quantify protein levels in NK/PyMT cell co-culture supernatants, ELISA kit for PRF1  as well as the mouse IFN-γ Flex Set  were utilized according to the manufacturer's instructions. Bead-based array samples were acquired by flow cytometry and analyzed using FlowJo V10.NK cells were isolated from spleens of either wildtype (WT) or IL-27 receptor α (IL-27Rα) KO mice using the EasySep™ Mouse NK Cell Isolation Kit . NK cells used as effector cells were co-cultured for 4 h at 37°C with PyMT target cells at different effector cell-target cell ratios, as indicated. Both NK cells and PyMT cells were labeled with different fluorescent dyes  and dead PyMT cells were identified using 7-AAD staining (Miltenyi Biotec). Living (7-AAD-negative) PyMT cells were subsequently determined via flow cytometry.Cd25, sense: 5′-CAAGAACGGCACCATCCTAAA-3′, anti-sense: 5′-TCCTAAGCAACGCATATAGACCA-3′; Cd69, sense: 5′-AAGCGATATTCTGGTG AACTGG-3′, anti-sense: 5′-ATTTGCCCATTTCCATGTCTGA-3′; Prf1, sense: 5′-CTG CCACTCGGTCAGAATG-3′, anti-sense: 5′-CGGAGGGTAGTCACATCCAT-3′. Rps27a served as internal control. Data were analyzed using QuantStudio™ (Thermo Fisher Scientific).RNA was isolated as described above followed by cDNA transcription using the Sensiscript® cDNA synthesis kit . The following murine primers were used: t-test as indicated. For the latter two data were pre-analyzed to determine normal distribution and equal variance with D'Agostino–Pearson omnibus normality test. Differences in patient survival were analyzed using Log-rank (Mantel–Cox) test. Statistical analysis was performed with GraphPad Prism V8. Differences were considered significant at p < 0.05. Asterisks indicate significant differences between experimental groups .Data are presented as means ± SEM. Statistical comparisons between two groups were performed using either two-way ANOVA, Mann-Whitney test or unpaired two-tailed Student's We and others previously observed that anti-PD1 therapy was poorly effective in the PyMT mouse model of mammary carcinoma , 15. Senper se did not alter the abundance of PD-1-expressing T cells within the total immune cell population  using Phenoptics™ multiplex IHC staining . Interespulation . Within pulation . Furtherpulation . Moreovep < 0.1; log2 fold change in expression >1). Only 4 genes were found to be significantly altered between the IgG1 control and the anti-PD-1 monotherapy group, and 19 genes were altered when comparing the DOX/IgG1 with the DOX/anti-PD-1 group, while 93 genes were differently regulated between the DOX/IgG1 and the IgG1 group (DIO2), gelsolin (GSN), matrix metalloproteinase 3 (MMP3) and pyruvate dehydrogenase kinase 4 (PDK4)  . Accordi4 (PDK4) . DIO2 pr4 (PDK4) . DIO2 wa4 (PDK4) , and in 4 (PDK4) . PDK4 re4 (PDK4) , and PDK4 (PDK4) , while h4 (PDK4) . GSN and4 (PDK4) , whose t4 (PDK4) , 31. In 4 (PDK4)  and in b4 (PDK4) . MMP3 de4 (PDK4) , 35, and4 (PDK4) .To validate the impact of these four selected target genes upon chemoimmunotherapy at protein level, PyMT tumor sections of all four treatment groups were stained using Phenoptics™ multiplex IHC staining. Therefore, tumor sections were stained for the four specific prognostic markers, as well as for Pan-Cytokeratin (Pan CK) as an epithelial/tumor marker, alpha-smooth muscle actin (α-SMA) as a stromal marker and were counterstained with DAPI . Tumor tp ≤ 0.05, FDR q ≤ 0.25). DOX monotherapy (+ IgG1) induced the most prominent changes (58 gene sets induced) when compared to IgG1 treatment alone, with a number of pathways being induced by DOX treatment that suggest changes in intracellular signaling events  to identify gene sets that were differentially regulated between individual treatment groups within the Molecular Signatures Database  in the METABRIC cohort. There was also no difference in the expression of the four genes irrespective of whether patients did or did not receive chemotherapy, while patients receiving hormone or radiotherapy actually expressed lower levels of these genes. Thus, the four gene signature predicts survival independent of prior standard of care treatment. It will be interesting to see how its expression is affected in patients receiving immune checkpoint blockade in the future. Importantly, to the best of our knowledge, an impact of these proteins in anti-tumor immunity or lymphocyte function has not been reported, indicating that the success of sensitizing for immune checkpoint blockade may be determined, at least partially, independently of a direct impact on cytotoxic lymphocytes.GSE149479.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI under accession number The animal study was reviewed and approved by Hessian animal care and use committee were followed .ES-F, CO, and AW conceptualized and designed research. ES-F, AF, and AW developed methodology. ES-F, AF, CO, MB, and AH performed experiments and acquired data. ES-F, TS, and AW analyzed and interpreted results. KT and BB provided technical and material support. BB and AW supervised research and all authors participated in writing the manuscript. All authors contributed to the article and approved the submitted version.KT was employed by ONO Pharmaceutical Co., LTD. 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.


Scientific Reports 10.1038/s41598-020-58029-z, published online 24 January 2020Correction to: This Article contains an error in Equation 5.should read:In addition, this Article contains an error in the Methods.“The electron-beam lithography process (VISTEC variable-shape VB6B) was optimized using 85 nm of negative tone resist from TOK (OEBR-CAN038) on top of 30 nm of silicon antireflective coating (SiARC) ISX412 and 130 nm of spin on carbon (SOC) HM8102.”should read:“The electron-beam lithography process (VISTEC variable-shape SB3054) was optimized using 85 nm of negative tone resist from TOK (OEBR-CAN038) on top of 30 nm of silicon antireflective coating (SiARC) ISX412 and 130 nm of spin on carbon (SOC) HM8102.”


We engineer two distantly related arenaviruses, Pichinde virus and lymphocytic choriomeningitis virus, for therapeutic cancer vaccination. In mice, life-replicating vector formats of these two viruses delivering a self-antigen in a heterologous prime-boost regimen induce tumor-specific CTL responses up to 50% of the circulating CD8 T cell pool. This CTL attack eliminates established solid tumors in a significant proportion of animals, accompanied by protection against tumor rechallenge. The magnitude of CTL responses is alarmin driven and requires combining two genealogically distantly related arenaviruses. Vector-neutralizing antibodies do not inhibit booster immunizations by the same vector or by closely related vectors. Rather, CTL immunodominance hierarchies favor vector backbone-targeted responses at the expense of self-reactive CTLs. These findings establish an arenavirus-based immunotherapy regimen that allows reshuffling of immunodominance hierarchies and breaking self-directed tolerance for efficient tumor control.Therapeutic vaccination regimens inducing clinically effective tumor-specific CD8  Engineered arenaviruses induce potent tumor self-specific CD8 T cell (CTL) responseCombinations of distantly but not closely related arenavirus vectors eliminate tumorsVector backbone-targeted CTL responses compete against tumor self-reactive CTLsOptimized vector combinations reshuffle immunodominance to break self-tolerance Therapeutic tumor vaccination should break self-tolerance. Assessing combinations of engineered arenavirus vectors, Bonilla et al. find that distantly related vector combinations reshuffle T cell immunodominance hierarchies to break self-tolerance and eliminate established solid tumors, whereas closely related vectors interfere because of immunodominance of anti-vector CD8 T cells rather than antibodies. Tumor-infiltrating CTLs in several tumor types are associated with clinical outcome,7Cytotoxic CD8,+ T cell frequencies by modalities such as adjuvanted peptides,,,,,,Therapeutic vaccination for CTL induction holds great promise for cancer therapy,,Anti-vector immunity can inhibit viral delivery systems, impeding their re-administration to augment immune responses. A single immunization with vaccinia virus or modified vaccinia virus Ankara (MVA) elicits neutralizing antibody (nAb) responses in the majority of individuals,,,For several decades, members of the Arenaviridae family have found widespread use in basic immunological research because of their capacity to induce CTL responses of exceptional magnitude, functionality, and longevity.,,We and others have developed replication-attenuated, tri-segmented, arenavirus-based vector formats  segment encodes for the viral polymerase L and the matrix protein Z, whereas the short (S) segment carries the envelope glycoprotein (GP) and nucleoprotein (NP) genes, separated by intergenic regions, respectively A. We andin vivo replication, whereas r3LCMV underwent inter-segmental recombination, reverting to a non-transgenic bisegmented WT-like virus.NP and SGP segments of r3PICV-GFP ,,Intracranial inoculation of mice is the standard model to assess the neurovirulence of LCMV-based vectors.+ T cell pool, with only limited contraction over a 1-month-period  and NP118 in BALB/c mice (H-2Ld restricted). Interestingly, the responses of C57BL/6 and BALB/c mice, respectively, were substantially lower in animals receiving heterologous artPICV-artLCMV immunization compared with homologous artLCMV prime-boost  and WT control mice. E7-specific responses to artPICV-E7E6 and artLCMV-E7E6 prime were indistinguishable in the two strains of mice B. More i20–27 . Control animals were either given vaccinia virus expressing an irrelevant transgene (Vacc-lacZ) as preimmunization or were left unimmunized. As expected, VACC-NP396 preimmunization, but not VACC-lacZ, induced a CTL response to NP396 C, wherea20–27 D. When s20–27 E. Converive mice F. Thus, ive mice G.3; TC-1, day 8). artPICV-artLCMV heterologous therapy delivering the respective tumor antigen, P1A or E7E6, was compared with homologous artPICV-artPICV or artLCMV-artLCMV prime-boost, all administered at an interval of 7 days (P815 model) or 10 days (TC-1 model). Homologous artPICV and homologous artLCMV immunotherapy afforded clear tumor volume control compared with untreated control animals  or had reached an average critical volume  and granzyme B-expressing (cytotoxic) P1A-specific, tumor-infiltrating CTLs in mice with heterologous artARENA immunotherapy .Further information and requests for resources and reagents should be directed to the Lead Contact, Daniel D. Pinschewer ,Il1rl1−/− mice on C57BL/6J background have been described,AGRAG mice  were immediately euthanized irrespective of tumor size and diameter. Study sample sizes in animal experiments were chosen based on experience in our labs with respect to group sizes readily revealing biologically significant differences in the experimental models used. The groups were neither randomized nor were experiments conducted in a blinded fashion.The PICV virulence study in guinea pigs was conducted at Meditox (Czech Republic) and was approved by the Institutional Animal Care and Use Committee (IACUC) and the Committee for Animal Protection of the Ministry of Health of the Czech Republic. Dunkin Hartley guinea pigs were from Charles River, France, and weighed 370 – 520 g at the start of the study.BHK-21 cells, HEK293T cells were purchased from ECACC , P815 mastocytoma cells (TIB-64), NIH 3T3 and BSC40 cells from ATCC. FreeStyle 293-F suspension culture cells were purchased from Invitrogen/ThermoFisher. LCMV-GP-expressing BHK-21 cells (BHK-21-GP) and 293T-GP cells have previously been described.,95 served as primary antibody. mAb 17.1.C6-9The titration of LCMV, PICV, MOPV and derived vectors by immunofocus assay has been described,Recombinant vaccinia viruses expressing the NP396 miniepitope or lacZ, respectively  have been described.The neutralizing capacity of immune serum was determined by immunofocus reduction assays.Intracranial LCMV infection was administered through the skull and mice developing signs of terminal disease were euthanized in accordance with the Swiss law.The wellbeing of guinea pigs undergoing PICV or artPICV infection was monitored twice daily during the entire study and clinically scored. Moribund animals were sacrificed. Humane endpoints were hypothermia  and/or body weight loss ≧20%.6 per mouse) were implanted subcutaneously in the right flank. Tumor growth was assessed three times per week. The longest and the shortest diameter were determined using a caliper. Tumor volumes (mm3) were calculated as ½ (length∗widthˆ2). When tumor volumes exceeded 1500 mm3 or when the longest median tumor diameter exceeded 20 mm, mice were euthanized in accordance with the Swiss law.P815 cells  were generated by RT-PCR cloning  and by gene synthesis (transgenic S segments of vectors). cDNAs of the full-length cancer-testis antigen P1A (comprising the immunodominant LPYLGWLVF epitope), a non-oncogenic fusion protein consisting of the complete HPV16 E7 and E6 sequences (comprising the immunodominant epitope RAHYNIVTF),The reverse genetic engineering of LCMV-WT, r3LCMV and artLCMV vectors using a polymerase I- / polymerase II-based plasmid system has been described.Infections and immunizations of mice with arenaviruses and arenavirus-based vectors were performed at a dose of 10e5 PFU i.v. unless specified otherwise. Vaccinia virus vectors were given at an intravenous dose of 2x10e6 PFU.Blood-brain-barrier leakage was assessed by detecting IgG deposits in mouse brain parenchyma.+ area in Cognition Network Language (Definiens Developer D software). For representative images, white balance was adjusted and contrast was linearly enhanced using the tools “levels,” “curves,” “brightness” and “contrast” in Photoshop CS6 (Adobe).Cryosections of 10μm were fixed with 4% PFA for 15 min, endogenous peroxidases were inactivated and tissue sections were incubated with HRP-labeled-anti-mouse-IgG . Bound peroxidase polymers were visualized using polymerized 3.3′-diaminobenzidine . The stained sections were scanned using a Panoramic Digital Slide Scanner 250 Flash II at 200x magnification. Quantifications were performed on scanned slides applying a custom-programmed script to detect the DABViral RNA was extracted from cell culture supernatant and from serum of infected mice using the QIAamp Viral RNA Mini Kit . RNA from spleens of mice was extracted using Tri Reagent (Sigma Aldrich). Reverse-transcription PCR was performed with the One Step RT-PCR kit (QIAGEN) and gene-specific primers. Amplified products were gel-purified for Sanger sequencing (Microsynth).A mammarenavirus genealogy tree was built based on S segment sequences of the following viruses and GenBank accession numbers: Allpahuayo virus (AY081210.1), Amapari virus (AF485256.1), Junin virus (AY358023), Bear Canyon virus (AY924391), Sabia virus (U41071), Pichinde virus (K02734), Chapare virus (EU260463), Cupixi virus (AF512832), Flexal virus (AF512831), Gairo virus (KJ855308), Guanarito virus (AY129247), Ippy virus (DQ328877), Lassa virus (AF181854.1), Latino virus (AF512830), Loei River virus (KC669698), Lujo virus (FJ952384), Luna virus (AB586644), Lunk virus (AB693150), Machupo virus (AY129248), Mariental virus (KM272987), Merino Walk virus (GU078660), Mobala virus (AY342390), Mopeia virus (AY772170), Okahandja virus (KM272988), Oliveros virus (U34248), Parana virus (AF485261), Pirital virus (AF485262), Ryukyu virus (KM020191), Solwezi virus (AB972428), Souris virus (KP050227), Tacaribe virus (M20304), Tamiami virus (AF485263), Wenzhou virus (KJ909794), Whitewater Arroyo virus (AF228063). To build the phylogenetic tree, we used the software package BEAST2Antibodies against CD8 (53-6.7 or Ly-3.2), CD45R/B220 (RA3-6B2), Klrg1 (2F1), CD127 (A7R34), CX3CR1 (SA011F11), CD27 (LG3A10), CD43 (1B11), GrzB (GB12), Ki67 (solA15), IFN-γ (XMG1.2), TNF (MP6-XT22), IL-2 (JES6-5H4) CD11b (M1/70) CD11c (HL3) CD19(1D3) Nkp46 (29A1.4) and CD4 (RM4-5) were from Biolegend, BD Biosciences/PharMingen and eBioscience/ThermoFisher. To assess intracellular levels of the transcription factor Tcf1, primary antibody binding  was detected using donkey anti-rabbit IgG PE (Poly4064-eBisocience). Eomes (Dan11mag) and T-bet (4B10) were detected using the eBioscience™ FOXP3 transcription factor staining kit (Invitrogen).+B220– lymphocytes. The H-2Db tetramer loaded with the NP396 epitope and conjugated to PE was obtained through the NIH Tetramer Core Facility, the H-2Ld tetramer loaded with the P1A epitope and conjugated to PE as well as the H-2Kb tetramer loaded with the B8R epitope and conjugated to PE were purchased from the University of Lausanne Tetramer core facility. For tumor-infiltrating CTL analyses, cells expressing CD11b, CD11c, CD19 or NKp46 were excluded. For detection of E7- (RAHYNIVTF epitope) and NP118- (RPQASGVYM epitope) specific CTLs the corresponding dextramers (Immundex) were used analogously (for simplicity referred to as “tetramer” in the text). Splenic single-cell suspensions were prepared by mechanical disruption and were counted using the respective single-use chambers in a Immunospot S6 device (C.T.L.). Total numbers of peptide-MHC tetramer-binding CTLs were back calculated. Cytokine profiles after restimulation with overlapping peptide sets spanning the E7 and E6 proteins of HPV16, respectively (JPT) were determined in intracellular cytokine assays as previously described.Dead cells were excluded with Zombie UV Fixable Viability Kit . P1A epitope- (LPYLGWLVF), B8R epitope- (TSYKFESV) and NP396 epitope- (FQPQNGQFI) specific CTLs were identified by peptide-MHC class I tetramers after gating on CD8For TIL analysis, tumors were dissected and digested with accutase (Sigma Aldrich), Collagenase IV (Worthington), Hyaluronidase (Sigma Aldrich) and DNaseI (Sigma Aldrich) for 60 min at 37°C, followed by red blood cell lysis. Single cell suspensions were filtered using a cell strainer (70 μM).For statistical analysis, GraphPad Prism software  was used unless stated otherwise. Differences between two groups were generally assessed using unpaired two-tailed Student’s t tests. To compare one group against multiple other groups we used one-way Analysis Of Variance (ANOVA), followed by Dunnett’s post hoc test G–7J. Two3, termination criterion for 3 for Tumor volume curves were compared as described.P values of p < 0.05 were considered significant (∗), p < 0.01 (∗∗) as highly significant.


C. elegans ortholog TMEM-39 from an unbiased RNAi screen and show that deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. We propose that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved.Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. Here, we report that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. We identify the  C. elegans tmem-39 that regulates ER stress response and collagen secretion. Human TMEM39A directly interacts with SEC23A, a core component of the COPII vesicle coating complex responsible for vesicular cargo secretion to the Golgi apparatus. The function of TMEM-39 proteins in collagen secretion appears highly conserved and independent to the ER stress response and the autophagy pathway. Our results provide insights into functions and mechanisms of TMEM39 proteins in collagen secretion and suggest it as a plausible target for tissue fibrotic diseases.As the most abundant protein in animals, collagen plays diverse roles and its dysregulation impacts aging and many fibrotic disorders. It is important to understand how premature collagen proteins in the ER are processed and secreted, as many other aspects of collagen regulation have been elucidated in mechanistic detail. In this paper, we have characterized a novel conserved family of TMEM39 proteins, including human TMEM39A and  Collagen is the major molecular component of connective tissues, and the most abundant protein in animals . CollageSpecific mechanisms underlying the secretion of procollagen still remain poorly understood. In general, specialized intracellular vesicles defined by the coat protein complex II (COPII) transport most secreted proteins, including procollagen, from the ER to the Golgi apparatus ,10. Sec2Caenorhabditis elegans produces over 180 collagen members that constitute the cuticle and basement membranes, encodes conserved homologs of COPII/TRAPP proteins, yet lacks apparent TANGO1 homologs  V, ire-1(zc14) II; zcIs4 V, dmaIs10  X, dmaIs40 , nIs617 , kuIs55 [lon-3::GFP], kaIs12 [col-19::GFP], qy24 [emb-9p::emb-9::mNG] III and tmem-39(dma258) I. Transgenic strains dmaEx169 , dmaEx153 , and dmaEx152  were generated as extrachromosomal arrays as described , caIs618 [eff-1p::eff-1::GFP], dnSi4 [gna-1p::GFP + Cbr-unc-119(+)], juEx1111 [spon-1::vGFP], lrp-1(ku156)eqIs1 [lrp-1p::lrp-1::GFP] I; rrf-3(pk1426) II, muIs49 [egl-20::GFP+unc-22(+)], nIs590 [fat-7p::fat-7::GFP], nuIs26 [cat-1::GFP], osIs60 , osIs66 [myo-3p::eGFP::wrk-1], sqIs11 [lgg-1p::mCherry::GFP::lgg-1+rol-6(+)], osIs77 , pwIs503 [vha-6p::mans::GFP+Cbr-unc-119(+)], qyIs44 [emb-9p::emb-9::mCherry], rhIs23 [GFP::him-4], veIs13 [col-19::GFP + rol-6(+)] V; let-7(mn112) unc-3(e151) X; mgEx725 [lin-4::let-7 + ttx-3::RFP], vkEx1243 [nhx-2p::ubiquitin-V::mCherry+myo-2p::GFP], vkEx1256 [nhx-2p::cpl-1::YFP], vkEx1260 [nhx-2p::cpl-1::YFP], vkEx1879 [nhx-2p::cpl-1(W32A Y35A)::YFP] and xnIs96 [hmr-1p::hmr-1::GFP].Translational fluorescent reporters used by C. elegans strains were maintained in standard nematode growth medium (NGM) plates with seeded E. coli at 20°C [ at 20°C . Worm st at 20°C . For RNADigital automated epifluorescence microscopes  and SPE confocal microscope (Leica) were used to obtain fluorescence images. Animals at the same stage were randomly picked from the plate, and transferred to a 4% agar pad with 10 mM sodium azide and 1 mM levamisole in M9 solution  on a slide for imaging. Identical setting and conditions were used to compare experimental groups with controls. ImageJ was used for the quantification of worm body length at L4 stages.HEK293T cells were transfected with the indicated plasmids, following the instruction of TurboFect Transfection Reagent . After transfection for 48 hr, cells were lysed on ice for 30 min in cell lysis buffer  with protease inhibitor cocktail (SIGMA 11836153001). After centrifugation at 13,000 rpm for 15 mins at 4°C, supernatants were collected and precleaned by control magnetic beads  for 30 mins at 4°C, and followed by immunoprecipitation with GFP-Trap agarose beads  for 2 hr at 4°C. After washing with 1XPBS for 4 times and cell lysis buffer for 1 time at 4 degree, the bound proteins were eluted with 1xSDS Laemmli Sample Buffer with 10% β-mercaptoethanol and analyzed by immunoblotting.Animals at the same stage from the control and experiment groups were picked (N>30) into 20 μL Laemmli Sample Buffer with 10% β-mercaptoethanol and lysed directly for Western blot analysis. Protein samples were run with 15% Precast Protein Gel , except that COL-101::GFP samples were run with 7.5% Precast Protein Gel , and then transferred to the nitrocellulose membrane . The membranes were blotted by antibodies against GFP , mCherry , Tubulin  and H3 .For subcellular fractionation, three plates (6cm dish) of adult-stage animal pellets were washed with M9 buffer three times for worm samples, were resuspended in 500 μL of RIPA lysis buffer  with 10 mM phenylmethylsulfonylfluoride (PMSF) and protease inhibitor cocktail . Then, pellet samples were disrupted by TissueRuptor (motor unit “8” for 1 min) and incubated for 45 min in a 4°C cold room. The lysate was centrifuged at 12,000 rpm for 20 min, the supernatant was collected as the supernatant fraction, and the pellet was resuspended in 500 μL of RIPA lysis buffer with 10 mM PMSF and protease inhibitor cocktail as the precipitation fraction. Then, 20 μL Samples added with 4× Laemmli sample buffer were subject to Western blot analysis, as described above.act-1 for C. elegans samples. Results were presented as fold changes to respective references. Statistical significance was determined with t-test, using GraphPad Prism 7. Primer sequences are listed in Worm total RNA was extracted by following the protocol of Quick-RNA MiniPrep kit . cDNA was reverse transcribed by the reverse transcriptase mix kit . Using SYBR Green Supermix , the real-time qPCR was performed on the Roche LightCycler96  system. Ct values of specific genes were normalized to the housekeeping gene levels: tmem-39(dma258) animals at L4 stages were recognized by a white crescent in the presumptive vulval region. Two days later, animals at adult stage were transferred to the 6 well tissue culture plate (Fablab FL7105), which is supplied with 2 mL distilled Water and 40 animals in each well . The number of animals shown intestine exposure was counted as sensitive to osmotic stress treatment. Each group was with at three biological replicates (n = 40).Wild-type and tmem-39 (dma258) mutants were maintained in the normal NGM plates for at least 2 generations at 20°C. Two L4 stage worms were picked into indicated RNAi plate for synthetic lethality test as described [Wild-type and escribed . Seven dFly strains included: UAS-Cg25C:RFP.2.1/CyO; Lsp2-Gal4/TM6B, and UAS-CG13016_dsRNA (Vienna Drosophila Resource Center ID# 42509/GD). Lsp2-Gal4 is specifically expressed in the fat body cells. Flies expressing Collagen:RFP in fat body were crossed to either wild type or UAS-CG13016_dsRNA flies. Wandering-stage third instar larvae were picked out. Fat body was dissected and fixed in 4% PFA, stained with DAPI, and mounted for imaging by confocal microscopy.The cDNA coding sequences of the first and second cytoplasmic loop domain of human TMEM39A were cloned into the pGBKT7 vector and screened against a normalized universal human cDNA library , following instruction of the Matchmaker Gold Yeast Two-Hybrid System . Verification of positive colonies was achieved by co-transforming wild-type or YR-mutant TMEM39A loop domain (in pGBKT7 Vector) with genes of interest (in pGADT7 Vector) following the instruction of YeastMaker Yeast Transformation System 2  as well as plasmids from re-cloned cDNA.TMEM39A full-length cDNA in the FUGW plasmid backbone, and the ER localization marker mCherry-ER-3 (Addgene: 55041) for 2 days. After 1xPBS washing for once, cells were treated by 4% formaldehyde solution for 10 mins. With 1xPBS washing for three times, cells were treated with 0.2% Triton X-100 in 1xPBS solution for 15 mins. Following 1xPBS washing for three times, the cover slide with cell samples was sealed on the microscope slide with Fluoroshield Mounting Medium with DAPI  for imaging by confocal microscopy.Hela cells were seeded in 24-well plates with cover glass, each with three replicates . Cells were transiently transfected with GFP-tagged human S1 Fig(A-B) Multiple sequence alignment of TMEM39A from major representative animal species (by COBALT program), with conserved domains indicated in bars (A). First cytoplasmic loop domain in green frame with dotted line, WS and YR residues indicated in the second cytoplasmic loop domains in blue frame with dotted line (B).(DOCX)Click here for additional data file.S2 Figwrk-1, (B) gna-1, (C) hmr-1, (D) mans, (E) eff-1, (F-G) cpl-1, (H) mig-23, (I) fat-7, (J) ced-1, (K) egl-20, (L) ubiquitin-V, (M) Y73E7A.8, (N) spon-1, (O) cat-1, (P) SP12, (Q) lgg-1, (R) F23H12.5, (S) lrp-1, (T) T19D2.1 and (U-W) emb-9 in wild-type animals by control and tmem-39 RNAi, wild-type and tmem-39(dma258) mutant (V) at 20°C (n = 3–4 for each reporters). Scale bars: 20 μm.(A-V) Exemplar fluorescence images showing translational reporters for (A) (DOCX)Click here for additional data file.S3 Figtmem-39 RNAi (A-B). Exemplar Western blot analysis of COL-19::GFP proteins from different fractions of wild type and mutant animals (C). wt, wild-type. mut, mutants. IB, immunoblotting. Arrows indicate procollagen monomers; triangles indicate mature monomers and cross-linked COL-19::GFP.(A-C) Exemplar Western blot analysis of COL-19::GFP proteins from total lysates of wild type animals with control and (DOCX)Click here for additional data file.S4 Figcol-101 and (B) lon-3. In wild-type animals at 20°C (n = 3–4 for each reporters). The area in the inset indicates longer exposure for enhanced fluorescence intensity. Arrows indicate decreased COL-101::GFP abundance but largely intact cuticle furrows in tmem-39(dma258) mutants. Scale bars: 20 μm. (C) Exemplar images of COL-101::GFP in wild-type and tmem-39(dma258) animals for Western blot analysis with 15% SDS-PAGE. (D) Osmotic stress sensitivity of wild-type and tmem-39(dma258) animals after treatment with distilled water after indicated time points. (E) Synthetic lethality test for wild-type and tmem-39(dma258) with genes involved in the ER associated degradation (ERAD) pathway. The score was assigned into 0 to 6 . Each group was with at three biological replicates.(A-B) Exemplar fluorescence images showing translational reporters for (A) (DOCX)Click here for additional data file.S5 Fig(A-B) Two independent repeats of co-immunoprecipitation and Western blot analysis of interaction between mCherry-labeled TMEM39A cytoplasmic loop domain and GFP-labeled Sec23A Ct fragment in HEK293T cells. Cells were transfected with expression vectors, lysed for immunoprecipitation by GFP-TRAP, and blotted with antibodies against GFP and mCherry.(DOCX)Click here for additional data file.S6 Figcol-19::gfp translational reporter for (A) control, (B) sar-1, (C) sec-24.1, (D) sec-24.2, (E) sec-31, (F) trpp-6, (G) trpp-8, (H) npp-20, (I) sec-12, (J) rab-1 and (K) tmem-131 RNAi in wild-type animals at 20°C. Scale bars: 20 μm.(A-J) Exemplar fluorescence images of (DOCX)Click here for additional data file.S7 Fighsp-4p::GFP transcriptional reporters for (A) control, (B) sec-24.1, (C) sar-1, (D) npp-20, (E) tmem-131, (F) sec-24.2, (G) sec-31, (H) pdi-2, (I) trpp-8 and (J) uso-1 RNAi in wild-type animals at 20°C. Scale bars: 20 μm.(A-J) Exemplar fluorescence images of (DOCX)Click here for additional data file.S8 Figwww.treefam.org). Domain architectures of DCTN6 family proteins (right). Arrows indicate conserved bacterial transferase hexapeptide domains. (B) Exemplar fluorescence images of hsp-4p::GFP transcriptional reporter for control and dnc-6 RNAi in wild-type animals at 20°C. Scale bars: 20 μm. (C-D) Exemplar confocal fluorescence images with indicated phenotypic penetrance (C) and Western blot analysis (D) of control, dnc-6 and tmem-39 RNAi in wild-type COL-19::GFP animals. Scale bars: 20 μm. IB, immunoblotting. The arrow indicates premature monomers; triangles indicate mature monomers and cross-linked COL-19::GFP.(A) Cladogram of phylogenetic tree for the DCTN6 protein family from major representative eukaryotic species (adapted from (DOCX)Click here for additional data file.S9 Figcol-19 translational reporters for (C) control, (D) dlst-1, (E) uggt-1, (F) cdc-48.1, (G) ire-1 and (H) xbp-1 RNAi in wild-type animals at 20°C. Scale bars: 20 μm. (I-G) Independent repeats of Western blot analysis of COL-19::GFP with control, manf-1 and sdf-2 for RNAi in wild-type animals. Arrows indicate procollagen monomers; triangles indicate mature monomers and cross-linked COL-19::GFP.(A-B) Independent repeats of Western blot analysis of COL-19::GFP in control RNAi and ER proteostasis gene in wild-type animals. Arrows indicate procollagen monomers; triangles indicate mature monomers and cross-linked COL-19::GFP. (C-H) Exemplar fluorescence images of (DOCX)Click here for additional data file.S10 Figwww.treefam.org). Domain architectures of SAC1 family proteins (right). (C-F) Independent repeats of Western blot analysis of COL-19::GFP in sac-1 and sac-2 RNAi (B-C), let-363 RNAi (D), and atg-5 RNAi (E-F) treated animals. Arrows indicate procollagen monomers; triangles indicate mature monomers and cross-linked COL-19::GFP.(A) Cladogram of phylogenetic tree for the SAC1 protein family from major representative Eukaryotic species (adapted from (DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Vascular smooth muscle cells (VSMC) are stromal cells of the blood vessels and their differentiation is thought to be essential during atherosclerosis. Cathelicidin-related antimicrobial peptides (CRAMP) are suggested to play a role in the development of atherosclerosis. Even so, the relationship of CRAMP and VSMC remains unclear. The present study was to determine whether CRAMP regulates VSMC phenotypic transformation and underlying mechanisms. We demonstrated that CRAMP could reverse platelet-derived growth factor-BB (PDGF-BB)-induced VSMC phenotypic transformation, evidencing by increasing α-smooth muscle actin (α-SMA), smooth muscle 22α (SM22α) and decreasing of proliferation and migration. Further studies showed that CRAMP inhibited nuclear factor κB (NF-κB)-induced autocrine of interleukin-6 (IL-6), which further activated of janus kinase 2 (JAK2)/signal transducer and activator 3 (STAT3). Meanwhile, our data showed that CRAMP can significantly inhibit PDGF-BB enhanced intracellular reactive oxygen species (ROS) level which further affected the NF-κB signaling pathway, indicating that CRAMP can regulate the phenotypic transformation of VSMC by regulating oxidative stress. These results indicated that CRAMP regulated the differentiation of VSMC by inhibiting ROS-mediated IL-6 autocrine, suggesting that targeting CRAMP is a potential avenue for regulating the differentiation of VSMC and treatment of atherosclerosis. Cardiovascular disease, the world’s leading cause of death, claiming an estimated 17.9 million lives each year, is a group of heart and vascular diseases such as atherosclerosis . VSMC phWith the development of research, more and more factors are considered to affect the phenotypic transformation of vascular smooth muscle. The stimuli triggering the phenotypic modulation include injury, mechanical force, oxidative stress, intracellular and extracellular microenvironment and molecules, and cell-cell and cell-matrix interactions ,10,11,12Antimicrobial peptides (AMPs) are short cationic molecules that are components of innate immunity and can defend against invading pathogens . AMPs haIn this study, we discovered that CRAMP could modulate phenotype in VSMC. Furthermore, we found that CRAMP alleviated platelet-derived growth factor-BB (PDGF-BB)-induced proliferation, migration and phenotypic transformation of VSMC through modulating ROS/NF-κB, which was dependent on IL-6 autocrine. These findings contribute to our understanding of the mechanism of Cathelicidin peptides on regulating atherosclerosis.The antimicrobial peptide CRAMP (SP-CRPL-5) was purchased from Innovagen, Sweden. PDGF-BB (CYT-740) was obtained from ProSpecbio , dissolved in sterile deionized water, and stored at −20 °C. Anti-α-smooth muscle actin (α-SMA), anti-smooth muscle 22α (SM22α) and anti-interleukin-6 (IL-6)  (ab9324) antibody were purchased from Abcam technology . Anti-signal transducer and activator (STAT3), anti-phospho-STAT3 (Tyr705), anti-extracellular signal-regulated kinase 1/2 (ERK1/2) and anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-inhibitor of nuclear factor kappa-B kinase (IKKα/β) (Ser176/180) (#2697) were purchased from Cell Signaling Technology Inc. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AP0063) and Goat anti-Rabbit IgG (H&L)-horseradish peroxidase (HRP) (BS12478) antibodies were obtained from Bioworld, USA. Anti-IL-6 (A0286), anti-phospho-janus kinase 2 (JAK2) (Y1007/1008) (AP0531), anti-β-actin (AC026), anti-IκBα (A19714), anti-phospho-inhibitor of nuclear factor kappa-B (IκBα) (AP0707), anti-NF-kB p65 (A2547) antibodies were purchased from ABclonal. Anti-JAK2 (17670-1-AP), anti-Histone-H3 (17168-1-AP), anti-IKK (15649-1-AP), anti-NADPH oxidase (NOX)1 (17772-1-AP), anti-NOX2 (19013-1-AP), anti-NOX4 (14347-1-AP) were purchased from Proteintech, Manchester, UK.2. The primary VSMC were identified using smooth muscle α-actin antibody. For all experiments, VSMC (2–5 passages) were used following by quiescence for 12 h.VSMC were harvested from normal rat aortas using the explant technique. The VSMC were cultured routinely in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum (FBS), supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C with a humidified atmosphere of 5% COCells were seeded into 96-well plates and allowed to adhere for 24 h. After being treated with different doses of CRAMP for 48 h, cells were subjected to viability detection by using the 3--2,5-diphenyl tetrazolium bromide (MTT) assay kit  according to the manufacturer’s specifications. In brief, cells in each well were incubated with 10 μL MTT working solution at 37 °C for 4 h. The absorbance of each well at 490 nm was measured using an Epoch Microplate Reader .g, 4 °C. Samples incubated with a sodium dodecyl sulfate (SDS) sample loading buffer were heated on the boiling water bath for 5 min, then subjected to 12% SDS-polyacrylamidegel (PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes. After being blocked in 5% fat-free milk at room temperature (RT) for 1 h, membranes were incubated overnight at 4 °C with primary antibodies, followed by HRP-conjugated secondary antibodies for 1 h at RT. Finally, membrane-bound antibodies were detected using a chemiluminescence reagent. The total protein content of loading was monitored by reprobing the same blots with loading control.Tissues and cells were lysed by using lysis buffer and centrifuged at 13,000× C57BL/6 mice were purchased from JOINN Laboratories (Suzhou). CRAMP knockout C57BL/6 mice were preserved in our laboratory. All animal experimental procedures were approved by the Jiangnan University Experimental Animal Management and Animal Welfare Ethics Committee (IACUC Issue Number: JN. No20191015c0401218). Mice were raised under conventional controlled conditions  and had free access to a standard diet and tap water. All mice were allowed to acclimate to these conditions for at least 2 days before inclusion in experiments.TM EdU Apollo®488 In Vitro Imaging Kit  instructions. Briefly, cells were cultured in 96-well plates, after treatment, EdU  was added and incubated for 12 h. Following, VSMC cells were fixed with 4% paraformaldehyde. After 30 min, 1× Apollo® staining reaction liquid was added (100 μL/well), and cells were incubated for 30 min at room temperature; After 10 min of permeabilization with 0.5% Triton X-100, the cells were stained with 1× Hoechst 33,342 (100 μL/well) for 30 min. The ratio of EdU-positive cells (EdU-stained cells/Hoechst-stained cells × 100%) was determined using a fluorescence microscope .Thus, 5-Ethynyl-2′-Deoxyuridine (EdU) was used to detect the proliferation of VSMC cells according to the Cell-LightIL-6 ELISA detection kit (SBJ-M0657) were purchased from SenBeiJia Biological Technology Co., Ltd. . Labeled antibodies and the biotin were co-incubated with the test samples. The optical density (OD) values of the samples were detected using a BioTek microplate reader. Standard curves were plotted according to standard OD values, and test sample concentrations were calculated from the standard curve.ROS was detected by using the commercial assay kit. Briefly, cell extracts were incubated with ROS specific dye, 2, 7-dichlorofuorescin diacetate (DCFH-DA), at 37 °C for 30 min, and then were centrifuged, washed and suspended in PBS. ROS were detected by using Epoch Microplate Reader  at 525 nm and Fluorescence microscope. Dihydroethidine hydrochloride  was topically applied to the freshly cut frozen aortic sections (10 μm) for 30 min at 37 °C to reveal the presence of ROS as red fluorescence (585 nm) by Fluorescence microscopy.p < 0.05; ** p < 0.01; *** p < 0.001.All statistical analysis was carried out by using GraphPad Prism software. Error bars for in vitro and in vivo analysis represent the standard deviation among intra-class data collected from more than 3 independent experiments. Data were analyzed by using analysis of variance (ANOVA). Statistical significance was determined using unpaired Student’s two-tailed t-test for two data sets and using a one-way ANOVA and followed by least significance difference multiple comparison tests. Statistical significance was defined as * VSMC phenotypic change to dedifferentiated state was a key step in arterial neointimal hyperplasia during the formation of restenosis . To inveWe then detected the effects of CRAMP on VSMC proliferation and migration. As showed in Activation of ERK1/2 and STAT3 plays an effective role in VSMC phenotypic switching ,33,34,35As IL-6 was a classical activator of JAK2-STAT3 signaling , we hypoNF-κB is an important transcription factor regulating IL-6 expression ,38 and pIntracellular ROS accumulation is critical for NF-κB activation . Our res−/−) mice, and the proportion of intimal hyperplasia in the CRAMP−/− mice was higher than that in the WT mice  and CRAMP knockout / rapidly accelerated fibrosarcoma (Raf)/ mitogen-activated protein kinase kinase (MEK)/ extracellular regulated protein kinases (ERK) kinase cascade, which leads to Elk-1 phosphorylation and the inhibition of SMC, specific marker genes, via the displacement of myocardin from smooth muscle cell-specific prompters . Our preThe NOX catalytic subunits-NOX1, NOX2, and NOX4-share a conserved structure and associate with p22phox on the cell membranes and are expressed in the vascular wall cells in rodents and humans . NOX1 isHerein, we have demonstrated that CRAMP modulates phenotypic transformation in VSMC. One potential mechanism by which this occurs is via CRAMP/autocrine IL-6/JAK2/STAT3 cascade; However, we also provided evidence that CRAMP regulates ROS and ROS-mediated NF-κB activation. These data suggest critical mechanism by which CRAMP regulates oxidation stress and cell differentiation. Given the reported relevance of CRAMP in atherosclerosis, the present findings which demonstrate a strong impact of CRAMP on the activation of ROS/NF-κB/IL-6 studies and provide new insights into the mechanisms by which CRAMP can regulate oxidation signaling in cardiovascular diseases like atherosclerosis.


Cell Death and DiseaseCorrection to: 10.1038/s41419-020-03210-5 published online 21 November 2020The original version of this article unfortunately contained a mistake. In the original publication, Supplementary Tables Supplementary Table 1Supplementary Table 2


We sequenced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from deep throat saliva samples of three imported cases in Hong Kong by Nanopore sequencing. Epidemiological and clinical features of these coronavirus disease 2019 (COVID-19) cases were presented for genomic epidemiology studies. We sequenced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from deep throat saliva samples of three imported cases in Hong Kong by Nanopore sequencing. Epidemiological and clinical features of these coronavirus disease 2019 (COVID-19) cases were presented for genomic epidemiology studies. Coronaviridae and genus Betacoronavirus) is causing an ongoing coronavirus disease 2019 (COVID-19) pandemic. There were 1,109 COVID-19 cases in Hong Kong as of 12 June 2020 , with most cases reported in March to April 2020. We report here the genome sequences of SARS-CoV-2 strains detected from three imported cases in Hong Kong.Severe acute respiratory syndrome coronavirus 2  and case 2  are a couple who had symptom onset on 10 March 2020 after returning from the United States to Hong Kong and tested positive on 14 March. Case 1 was relatively asymptomatic apart from fever, but case 2 showed symptoms and signs of pneumonia during hospitalization. They were enrolled in a clinical trial of interferon beta-1b, lopinavir-ritonavir, and ribavirin (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w). Briefly, cDNA was synthesized using a SuperScript III first-strand synthesis system  with random hexamers. Two multiplex PCRs of 49 amplicons (V1 panel) were performed using AmpliTaq Gold DNA polymerase , combined, purified using AMPure XP beads , and quantified using a Qubit double-stranded DNA (dsDNA) high-sensitivity assay . Each genome was sequenced separately. For each sample, a separate Nanopore sequencing library was prepared using a ligation sequencing kit , sequenced using a new MinION R9.4.1 flow cell (FLO-MIN106D) with MinKNOW live base calling version 19.12.2 (fast model), and analyzed following the ARTIC nCoV-2019 bioinformatics protocol. Briefly, filtered reads  were mapped to the Wuhan-Hu-1 reference strain (GenBank accession number MN908947.3) using Minimap2 version 2.17 (parameter -x map-ont) and subjected to primer soft-clipping using align_trim (version 1.1.0) (https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html) or BAMClipper version 1.1.1 (parameter -u 40 -d 40) (https://github.com/nextstrain/ncov) on 12 June 2020 (default GTR substitution model and nucleotide substitution rate of 0.0008 per site per year). Default parameters were used for all software unless otherwise specified.Deep throat saliva samples treated with Sputasol  were subjected to nucleic acid extraction using eMAG . The full genome was amplified following the ARTIC nCoV-2019 V1 protocol (0 -d 40)  , 0.534-Gbp reads for HKSH0004 , and 7.740-Gbp reads for HKSH0007 . All three SARS-CoV-2 genomes were sequenced at a mean depth of >3,500×, reference coverage of 92% to 96%, length of 27,384 to 28,723 nucleotides, and GC content of 38%. Phylogenetic analysis showed that they descended from European samples  and were distinct from most other Hong Kong sequences . AlthougMT510744, MT510745, and MT517419, BioProject number PRJNA634965, BioSample numbers SAMN15013847, SAMN15013848, and SAMN15028143, and SRA numbers SRR11844878, SRR11844877, and SRR11852109.These sequences were deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database  and in NCBI GenBank under the accession numbers


Serine-threonine kinase receptor-associated protein (STRAP) functions as a regulator of both TGF-β and p53 signaling that participates in the regulation of cell proliferation and cell death in response to various stresses. Here, we demonstrate that STRAP acetylation plays an important role in p53-mediated cell cycle arrest and apoptosis. STRAP is acetylated at lysines 147, 148, and 156 by the acetyltransferases CREB-binding protein (CBP) and that the acetylation is reversed by the deacetylase sirtuin7 (SIRT7). Hypo- or hyperacetylation mutations of STRAP at lysines 147, 148, and 156 (3KR or 3KQ) influence its activation and stabilization of p53. Moreover, following 5-fluorouracil (5-FU) treatment, STRAP is mobilized from the cytoplasm to the nucleus and promotes STRAP acetylation. Our finding on the regulation of STRAP links p53 with SIRT7 influencing p53 activity and stability. Serine/threonine kinase receptor associated protein (STRAP), known as the transforming growth factor-β1 (TGF-β) signaling negative regulator protein, is a 38kD protein that contains a WD40 domain ,3. It haSirtuin7 (SIRT7) is a member of the sirtuins, which has Nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase activity . More anThe tumor suppressor p53 plays a critical role in cellular processes, including cell growth, cell differentiation, and cell death, in response to a wide range of cellular stresses . In thisIn our previous report, we purified the SIRT7 complex by immunoprecipitation and mass spectrometry. STRAP was found in the complex . To veriThe STRAP–SIRT7 interaction indicated that STRAP could be a novel substrate of SIRT7. Therefore, we first examined whether STRAP could be acetylated. We used HDAC inhibitor Trichostatin A (TSA) and Sirtuins inhibitor nicotinamide (NAM) to detect the acetylation status of STRAP. STRAP acetylation was more significant in cells treated with NAM than in cells treated with TSA A, indicaBased on the previous results, we then explored whether SIRT7 deacetylates STRAP. We first co-transfected Flag-STRAP and different amounts of HA-SIRT7 into HEK293T cells. Western blotting showed that STRAP acetylation levels decreased with increasing amounts of SIRT7 transfection A. We theInterestingly, the acetylation level of STRAP decreased with SIRT7 and NAD+ co-incubation C, suggesTo explore the role of STRAP acetylation in the regulation of p53-mediated signaling pathway, HCT116 (wild-type), HCT116 (p53 -/-), and H1299 cells were transfected with STRAP-WT (WT) and STRAP-3KR (3KR), respectively. In HCT116 cells, expression of STRAP-WT caused the obvious upregulation of p53 as well as its targets, including p21 and Bax. Conversely, STRAP-3KR decreased this effect A. HoweveWe further investigated the regulation mechanism of STRAP acetylation on p53. Half-life assay was performed by using empty vector (Vector), STRAP-WT (WT), STRAP-3KR (3KR), and STRAP-3KQ (3KQ) constructs. As compared with the control, the expression of STRAP-WT, STRAP-3KR, or STRAP-3kQ all partly increased the p53 half-life in HCT116 cells, while STRAP-3KR decreased the p53 half-life as compared with STRAP-WT. The result showed that deacetylated STRAP can reduce the stability of p53 relative to wild-type STRAP A,B. We tA recent report showed that 5-fluorouracil (5-FU) induces radio-sensitivity via SIRT7 degradation, which promotes cell death during cancer cell radiotherapy . To analSTRAP is known as a negative regulator in the TGF-β signaling pathway, participating in the regulation of cell growth, cell differentiation, and apoptosis in response to various stresses . Here, whttp://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) [STRAP acetylation has not been reported previously. A recent study revealed that STRAP stability and activity is mainly regulated by phosphorylation at threonine and serine residues . Our stuorm.cgi) . We obtaorm.cgi) . The reaSTRAP is considered a prognostic factor  due to tDuring our investigation of the functions of STRAP acetylation, we also tried to examine its influence on the TGF-β pathway. However, we found that the STRAP acetylation neither affects its interaction with Smad7 nor causes any change in the TGF-β signaling pathway. It is interesting to see the differential regulation on STRAP acetylation within p53 and TGF-β signaling. It is possible that STRAP acetylation targets the p53 pathway and other post-translational modifications, such as phosphorylation, ubiquitination regulates TGF-β signaling pathway. Further investigation into the differential regulation of STRAP post-translational modifications on different targets and its regulating mechanisms would be worthwhile.SIRT7 regulation of p53 activity has been reported by several groups with differing controversial results. In vivo and in vitro loss of SIRT7 showed hyper-acetylation of p53, increases the rate of apoptosis, thereby promoting cell survival ,24,25. T2OS cells were cultured in DMEM (Invitrogen), supplemented with 10% (v/v) FBS, 100 U/mL penicillin/streptomycin, and incubated at 37 °C in humidified air with 5% CO2. The HCT116 cell line was obtained from Weiguo Zhu. The U2OS cell line was provided by Junjie Hu.HEK293T, HCT116 (wild type), HCT116 (p53 -/-), H1299, and UThe following primary antibodies were used: The mouse antiSIRT7 (sc-365344), rabbit anti-SIRT7 (sc135055), anti-p53 (FL) (sc-6243), anti-p53 (DO-1) (sc-126), anti-p21 (sc-271532), anti-actin (sc-8432) and anti-tubulin (sc-73242) were purchased from Santa Cruz Biotechnology . The anti-STRAP (18277-1-AP) was purchased from Proteintech , the anti-GAPDH (#5174S) was purchased from Cell Signaling Technology , the anti-FLAG (F3165) was purchased from Sigma–Aldrich , the anti-HA (#26183) was purchased from Pierce , the anti-pan-acetyllysine (#9441L) was purchased from Cell Signaling Technology .pcDNA3.1-Flag/HA-STRAP/SIRT7 was obtained by subcloning STRAP/SIRT7 sequence into pcDNA3.1-Flag/HA vector. The mutant constructs were generated by PCR amplification of STRAP/SIRT7 with specific primers. STRAP full-length (FL), STRAP  were subcloned into the pGEX4T-3 vector.STRAP siRNA 1#: gggugcaacacugaauaag; siRNA 2#: uuacgcauauaugacuuga .Protein/protein interaction was evaluated by Co-IP according to standard methods. Cell pellets were lysed in BC100 buffer (20 mM Tris-HCl (pH 7.9), 0.2% NP-40, 100 mM NaCl, and 20% glycerol) containing 1 mM Dithiothreitol (DTT), 1 mM Phenylmethanesulfonyl fluoride (PMSF), and protease inhibitor cocktail , centrifuged, and used for immune-precipitation. Western blotting was then performed to detect protein expression, as indicated in immune-precipitates.For the pull-down assay, 1 μg of GST, GST-STRAP, or GST-p53 protein was incubated with Flag-tagged SIRT7/STRAP protein in 100 μL interaction buffer at 4 °C overnight in BC100, then incubated with glutathione–sepharose beads (50% slurry) for an additional 2 h. Bound GST fusion proteins were then resolved by SDS-PAGE and analyzed by Western blotting using antibodies as indicated.For the acetylation assay, cells were transfected with STRAP alone or co-transfected with STRAP and CBP for 24 h, treated with 1 μM TSA and 5 mM NAM  for an additional 6 h before harvest. Cell extracts were incubated with anti-Flag M2 beads, and bound proteins were analyzed by Western blotting. For the deacetylation assay, cells were transfected with STRAP alone or co-transfected with STRAP and different amount of SIRT7 for 24 h, whole-cell lysates were prepared in BC100 supplemented with protease inhibitors. Cell extracts were incubated with anti-Flag M2 beads, and immune-precipitated proteins were analyzed with different antibodies by Western blotting.The in vitro reactions were performed as described . RecombiThe in vitro reactions were performed as described . Purifien = 3, mean ± S.D.) was statistically analyzed.A luciferase reporter assay was performed, as described . HCT116 A ubiquitination assay was performed, as described . HCT116 For all localization experiments, HCT116 cells were cultured on glass coverslips treated with or without 5-FU at 40–60% confluence. The cells were fixed in 4% paraformaldehyde for 10 min at Room temperature (RT), and then washed with PBS plus 0.2% Triton X-100 for 10 min and blocked for 1 h at 4 °C. Primary antibodies used were anti-STRAP (1:200) or anti-SIRT7 (1:200), and secondary antibodies used were Alexa Fluor-488 anti-mouse (1:1000) or Alexa Fluor-594 anti-rabbit (1:1000). Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) . Fluorescent images were taken and analyzed using a confocal laser microscope (Leica).In summary, we have shown that STRAP can be acetylated by CBP and deacetylated by SIRT7. Moreover, following 5-FU treatment, STRAP is mobilized from the cytoplasm to the nucleus and promotes STRAP acetylation. Our finding on the regulation of STRAP links p53 with SIRT7 influences p53 activity and stability.