Genomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants.® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant.SARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology , Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results.These data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management. As of March 7, 2022, over 447 million COVID-19 cases have been reported worldwide, resulting in over 6 million deaths (https://coronavirus.jhu.edu/map.html). By late 2020, it became apparent that the SARS-CoV-2 virus was acquiring mutations in the spike protein gene (S-gene) that impacted key biological and epidemiological properties of the virus, including viral transmissibility, infectivity, and immune escape (https://covid.cdc.gov/covid-data-tracker).Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in the Chinese province of Wuhan in November 2019  and was e escape . Subseque escape , followehttps://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html). The WHO also classifies several additional strains as variants of interest (VOI), based on characteristics such as genetic changes that are predicted or known to affect virus characteristics  (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants); the CDC has adopted the designation of variants being monitored (VBM) for such strains.At various times in 2020 and 2021, these SARS-CoV-2 variants have achieved the designation of variants of concern (VOC) as determined by the WHO and the United States Center for Disease Control and Prevention (CDC). A VOC is defined as a variant for which there is evidence of an increase in transmissibility, more severe disease , significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures (Whole genome sequencing (WGS), which has been employed by public health and other high-complexity laboratories, informs public health decisions by facilitating the tracking of SARS-CoV-2 transmission, outbreak detection, and contact tracking, and has been used to help trace the origin of the pandemic . With th® SARS-CoV-2 Alpha/Beta/Gamma and Alpha/Beta/Gamma+, including Omicron), P681R (PlexPrime® SARS-CoV-2 P681R Delta), and L452Q (PlexPrime® SARS-CoV-2 L452Q Lambda). We further show these assays permit the rapid and accurate identification of SARS-CoV-2 viruses designated as VOC or VBM. Implementation of such testing could facilitate widespread, ongoing monitoring of SARS-CoV-2 evolution and early detection of variants requiring an urgent public health response and appropriate patient management.In the present study, we describe the analytical performance characteristics of multiplexed assays for detection of the S-gene mutations E484K, N501Y, and S982A  at Quest Diagnostics and with variants previously assigned by WGS . In accovia the RdRp gene and mutations associated with these variants . Briefly, 200 µL of sample was extracted and eluted in 50 µL with the DNA and Viral RNA Small Volume extraction kit using the Pathogen Universal 200 protocol (Roche Diagnostics). For the PlexPrime® SARS-CoV-2 Alpha/Beta/Gamma, PlexPrime® SARS-CoV-2 P681R Delta, and PlexPrime® SARS-CoV-2 L452Q Lambda, a 10-µL aliquot of nucleic acid extract was added to a 15-µL reaction mix  and tested in a 96-well plate. For the PlexPrime® SARS-CoV-2 Alpha/Beta/Gamma+ with N501Y-Omicron assay, a second set of 0.5 µL of 20x SARS-CoV-2 variant oligo mix for N501Y-Omicron was added, instead of 0.5 µL of nuclease-free water. The PCR cycle was performed on an ABI 7500 FAST Dx Instrument . Cycling consisted of 48°C for 10 min and 95°C for 2 min, followed by 10 cycles of 95°C for 5 s and 61°C for 30 s (−0.5°C per cycle) and 40 cycles of 95°C for 5 s and 52°C for 50 s. For the PlexPrime® SARS-CoV-2 Alpha/Beta/Gamma assay and PlexPrime® SARS-CoV-2 Alpha/Beta/Gamma+ assay, data acquisition was set for E484K in channel A (FAM), RdRp in channel B (JOE), S982A in channel C (Texas Red), and N501Y in channel D (Cy5). For the PlexPrime® SARS-CoV-2 P681R Delta assay, data acquisition was set for RdRp in channel A (JOE) and P681R in channel B (Cy5). For the PlexPrime® SARS-CoV-2 L452Q Lambda assay, data acquisition was set for RdRp in channel A (JOE) and L452Q in channel B (Texas Red). Analysis of the results was performed using the instrument software and an Excel spreadsheet to calculate ΔCqs (quantification cycles) between the RdRp channel and each mutation channel. The cutoff ranges were evaluated through analytical sensitivity studies using extracted clinical specimens that had undergone WGS and were set to specifically determine the mutations present, as follows:The PlexPrime variant assays  allow simultaneous detection of the SARS-CoV-2 pathogen variants Table 1.ΔCq E484K = E484K Ct – RdRp Ct; cutoff range: -2.0 to 5.01 = N501Y Ct – RdRp Ct; cutoff range: -2.0 to 4.0ΔCq N501Y2 = N501Y Ct – RdRp Ct; cutoff range: -2.5 to 4.0ΔCq N501YΔCq S982A = S982A Ct – RdRp Ct; cutoff range: -2.0 to 5.0ΔCq P681R = P681R Ct – RdRp Ct; cutoff range: -2.0 to 5.5ΔCq L452Q = L452Q Ct – RdRp Ct; cutoff range: -2.0 to 5.01 N501Y in the Alpha/Beta/Gamma assay.2 N501Y in the Alpha/Beta/Gamma+ assay.If the RdRp cycle threshold (Ct) value was > 19 for the E484K, N501Y, and S982A mutations in the Alpha/Beta/Gamma assay or >26 in the Alpha/Beta/Gamma+ assay, the result was inconclusive and the PlexPrime assay was repeated from sample extraction. Because we observed that the E484K probe could cross-react with the uncommon E484Q mutation, a positive result for this probe was considered as positive for E484K/Q. If the RdRp cycle threshold (Ct) value was > 26 for the P681R Delta or the L452Q Lambda mutations, the result was considered inconclusive and the PlexPrime assay was repeated from the extraction step.https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/in-vitro-diagnostics-euas-molecular-diagnostic-tests-sars-cov-2). Primer and probes for the Quest SARS-CoV-2 assay are based on the CDC primer and probe sets 2019-nCoV_N1 and 2019_nCoV_N3 (https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf). Samples were quantitated using a standard curve generated by serially diluting the SARS-CoV-2 viral transcript containing the N gene in stabilizing buffer consisting of poly-A RNA in PBS. Sample dilutions were made in their respective negative sample matrix, including viral transport media  and phosphate-buffered saline (PBS). For each targeted mutation, 20 replicates were tested at 6 different concentrations. The limit of detection was defined as the lowest viral load (copies/mL) that resulted in 95% detection of the mutation in all replicates assayed.Positive samples were extracted and tested with the Quest Diagnostic SARS-CoV-2 qualitative RT-PCR assay. An IFU for the assay can be found at the FDA website  for the triplicate Ct values , a panel comprised of individual inactivated respiratory related pathogens  manufactured specifically for use as positive controls in nucleic acid tests  in two different collection media, UTM and PBS, were prepared to assess the sensitivity for the detection of spike gene mutations N501Y and S982A using the Alpha/Beta/Gamma+ assay. SARS-CoV-2 gamma (P.1) was selected for sensitivity studies of mutations N501Y and E484K in the Alpha/Beta/Gamma+ assay. Sensitivity for mutations was assessed using SARS-CoV-2 Delta (B.1.617.2) for the P681R mutation and SARS-CoV-2 lambda (C.37) for L454Q. Sensitivity for N501Y in the Alpha/Beta/Gamma+ assay was assessed using SARS-CoV-2 Omicron (BA.1). The limits of detection for the respective targets and media types ranged from 500 copies/mL for the N501Y and S982A mutation in lineage B.1.1.7 in UTM or PBS, to a maximum of 10,000 copies/mL for the E484K mutation in lineage P.1 diluted in UTM Table 2.The maximum percentage of CVs of the replicate Ct values for the RdRp gene and for 5 spike gene variants assessed for intra-assay reproducibility was <4% for all mutations and media types and viral load levels. Likewise, the maximum percentage of CVs assessed for inter-assay reproducibility in 3 runs was <7% for all mutations and media types and viral load levels , 16 Delta clinical specimens, 3 Alpha (lineage B.1.1.7), 4 Gamma (lineage P.1), and 4 Wuhan WT typed by WGS, as well as 5 high , 4 medium , and 5 low (2500 copies/mL) viral load diluted Omicron specimens Table 3.For the WGS data, the prevalence of the Delta lineage increased from 34.5% to 100% over the 3-month period in which these samples were collected Figure 1https://www.covid19treatmentguidelines.nih.gov/management/clinical-management/nonhospitalized-adults–therapeutic-management/). In this study, we have described and characterized the performance of a genotypic assay that can facilitate the rapid and accurate identification of VOC and VBM designated SARS-CoV-2 viruses.The surveillance of SARS CoV-2 variants during the pandemic has been primarily driven by WGS. While WGS remains the gold standard for genotyping, its availability is limited to laboratories with next generation sequencing and bioinformatics capabilities. Rapid variant typing by RT-PCR provides a high throughput method to enhance surveillance efforts as well as provide information that can guide infection control and treatment decisions in a timely manner .From June to August 2021, the Delta variant emerged in the US displacing the Alpha variant as the dominant circulating strain. The sampling of specimens with the Delta variant typing assay correlated with WGS Figure 1https://www.ama-assn.org/system/files/coronavirus-long-descriptors.pdf). At present, the clinical use case for a rapid genotyping assay is to guide antiviral therapy . With expanding therapies available for SARS-CoV-2, the utility of diagnostics to guide therapy will likely expand.There are limitations to utilizing an RT-PCR typing assay. Given that such assays focus on a select group of mutations, the results are preliminary and may require additional genotyping for confirmation or to provide a richer epidemiological picture. However, if the primary circulating strain is dominant in a population , the need for confirmation by sequencing may be minimal. Rapid SARS-CoV-2 viral evolution and the imperative to address diagnostic needs are challenging. As new variants emerge, there is a need to update the assay design for typing . DespiteIn summary, this study demonstrates the performance and utility of RT-PCR genotyping assays. Such assays could be more widely available to laboratories performing SARS-CoV-2 molecular testing if a commercially available solution were available with regulatory approval.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GB performed all experiments and participated in data analysis, study design, and manuscript review and editing. RK participated in data analysis, data review, manuscript preparation, and editing. EM participated in the study conception and design, data review, manuscript preparation, and editing. All authors contributed to the article and approved the submitted version.This study received funding from Quest Diagnostics. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. All authors declare no other competing interests.The authors are employees of Quest Diagnostics, a commercial reference laboratory that performs commercial SARS-CoV-2 testing and own shares of Quest Diagnostics stock.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.


X-ray photoelectron spectroscopy (XPS) and Raman results showed that the C=O proportion and defect degree (ID/IG) rose to 75% and 1.53, respectively. The obtained CNT-O-H possessed a superior performance towards peroxydisulfate (PDS) activation, and the degradation efficiency of tetracycline (TC) in the CNT-O-H/PDS system was increased to 75.2% from 56.2% of the raw CNTs/PDS system within 40 min. Moreover, the activity of CNT-O-H after use could be easily recovered with re-calcination. In addition, the CNT-O-H/PDS system exhibited high adaptabilities towards wide solution pH (2–10), common coexisting substances and diverse organic pollutants. Singlet oxygen (1O2) was confirmed to be the dominant reactive oxygen species (ROS) generated in the CNT-O-H/PDS system. It was inferred that surface C=O groups and defects of CNTs were the key site to activate PDS for TC degradation. Carbon nanotubes (CNTs) were considered a promising activator for persulfates due to their high electrical conductivity, large specific surface area and low toxicity. The functional groups and surface defects of CNTs could significantly affect their activation performance. In this study, CNTs with high C=O ratio and defect density (CNT-O-H) were prepared through a facile treatment of raw CNTs with HNO Tetracycline (TC), one of the most common antibiotics, is widely used in the treatment and prevention of various diseases and infections due to its broad-spectrum antibacterial property ,2,3. How2-hybridized atom configuration have been reported to effectively activate persulfates for organic pollutant degradation [Recently, advanced oxidation processes (AOPs) ,10,11,12radation ,30.2SO4, H3PO4, KMnO4 and H2O2 in sequence, the CNTs could effectively activate PMS to degrade acetaminophen, and the pyridinic N and surface carbonyl groups of CNTs were dominate active sites [2 plasma technique to graft oxygen-containing functional groups (like C–O and O–C=O) onto vertically-aligned CNTs, and the O–C=O fraction could increase to around 20% from 3.7% of pristine CNTs. Additionally, the defect degree of CNTs could be adjusted by ethanol-flame treatment [It was reported that surface groups and defects of CNTs would remarkably affect the catalytic performance towards persulfate activation ,32. Seveve sites . Zhao etve sites  used thereatment , atom doreatment  and ultrreatment . However3 oxidation and calcination at 800 °C. This method could effectively promote the C=O proportion and defect degree of CNTs, facilitating PDS activation without transition metal components and complex equipment. A series of characterization techniques including X-ray diffractometer (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy were utilized to analyze the changes of structure, morphology and chemical component of CNTs after modification. The performances of CNTs before and after treatment were comparatively evaluated by activating PDS to degrade TC. Further, the effects of activator dosage, PDS dosage, initial solution pH and temperature on TC degradation were investigated in detail. In addition, the adaptability of modified CNTs and their regeneration were explored for potential practical application. Additionally, quenching experiments and electron paramagnetic resonance (EPR) tests were conducted to determine the dominant ROS contributing to TC degradation, and an activation mechanism of modified CNTs towards PDS was proposed on the basis of XPS and EPR tests.Herein, a facile strategy was developed to modify raw CNTs through a combination process of HNO3, 99.5 wt%), sodium nitrate , ethanol , sulfuric acid , nitric acid , sodium sulfate , hydrochloric acid , sodium hydroxide  and p-benzoquinone  were purchased form Sinopharm Chemical Reagent Co., Ltd. . Humic acid , tetracycline , potassium peroxydisulfate , furfuryl alcohol , 4-hydroxy-TEMPO , 5,5-dimethyl-pyridine N-oxide  and 4-amino-2,2,6,6-tetramethylpiperidine  were provided by Aladdin Reagent Co., Ltd. . All chemicals were of analytical grade and used without any purification in the experiments.Raw CNTs (97 wt%) were purchased from Shenzhen Nanotech Port Co., Ltd. . Melamine, Rhodamine B , methyl orange , methyl blue , chlortetracycline hydrochloride , sodium chloride , sodium bicarbonate  were added to a round-bottomed glass flask and stirred at 80 °C for 48 h. After oxidation, the slurry was taken out, cooled, centrifuged and washed with water and ethanol several times until the pH was neutral. Then, the wet solid was dried at 60 °C in a vacuum oven overnight, and the collected sample was denoted as CNT-O. Further, CNT-O was heated in a tube furnace under an argon atmosphere from room temperature to 800 °C with a heating rate of 5 °C/min and kept at 800 °C for 2 h. After cooling to room temperature, the final product was obtained and labeled as CNT-O-H.−1  was carried out to determine chemical functional groups. The electron paramagnetic resonance (EPR) signals were detected on a Bruker X-band A200 spectrometer . The electrochemical measurements were tested via a CHI660 electrochemical workstation with a standard three-electrode system .Phase structures were characterized by a Philips/X’Pert PRO X-ray powder diffractometer (XRD) equipped with Cu Kα radiation (λ = 0.15406 nm) , and the morphologies of samples were explored with a Tecnai G2 20 transmission electron microscopy (TEM) with a working voltage of 200 kV . The composition and chemical state of samples were examined with a Kratos/Axis Ultra DLD-600 W X-ray photoelectron spectrometer . The specific surface area and pore size distribution were determined with a Micromeritics ASAP2020 surface area analyzer . The defect and disorder degree of materials were analyzed with a LabRAM HR800 Raman spectroscopy . A Bruker VERTEX 70 Fourier transform infrared spectroscopy (FT-IR) equipped with a KBr beam splitter in the regular scanning region of 4000–400 cm2SO4 and measured with a pH meter. Common matrix species were introduced into the initial TC solution to determine the adaptability of CNT-O-H. After magnetically stirring for 30 min to reach adsorption equilibrium, the degradation experiment was initiated by adding PDS under the corresponding conditions. At set intervals, about 3 mL of the suspension was collected and filtered through a 0.22 μm pore-size syringe in order to remove the insoluble residue. The TC concentration in solution was measured with a UV-Vis spectrometer  at the wavelength from 280 to 700 nm. According to Lambert-Beer law, the degradation efficiency of TC was calculated by the difference between C0 (concentration of TC after adsorption) and Ct (concentration of TC at a selected time), according to the following Equation (1):The degradation reaction was carried out in a glass beaker containing 100 mL of 50 mg/L TC solution and a different amount of activator. The initial pH of TC solution could be adjusted using 0.1 M NaOH or HThe morphologies of raw CNTs, CNT-O and CNT-O-H were examined with the TEM technique. As shown in −1, originating from the D-(defect) and G-(graphitic) bands, respectively [D/IG) were estimated as 1.19, 1.21 and 1.53 for raw CNTs, CNT-O and CNT-O-H, respectively. The increase of ID/IG ratio might be attributed to the generated defects from the decomposition of some groups at 800 °C. Clearly, the surface defects were significantly promoted in the carbon network of CNTs after heat treatment, similar to other reported results [The defect degrees of CNTs before and after treatment were determined with a Raman microscope. As illustrated in ectively . The rat results . It was  results . −1 and C=O at 639 cm−1 remarkedly increased in CNT-O, which was attributed to the introduction of oxygen-containing groups after acid oxidation [−1 was attributed to the stretching vibration of the O–H groups, resulting from the absorbed moisture [The functional groups of raw CNTs, CNT-O and CNT-O-H were checked with an FT-IR spectrometer and shown in xidation . Howeverxidation . The bromoisture ,46. Appa2/g) and pore volume (1.27 cm3/g), which would be conductive to the interaction between CNT-O-H and persulfate. The pore size distribution curves of CNTs before and after modification were calculated with the Barrett-Joyner-Halenda (BJH). As shown in The nitrogen adsorption/desorption isotherms for raw CNTs, CNT-O and CNT-O-H were shown in Further, XPS was employed to examine the surface chemical components of raw and modified CNTs. The high-resolution C 1s spectra of all CNTs  could beActivities of raw CNTs, CNT-O and CNT-O-H were evaluated in PDS activation for TC degradation. First, the adsorption experiments showed that the removal efficiencies of TC by raw CNTs, CNT-O and CNT-O-H were 32.2%, 33.1% and 39.8%, respectively , demonst−1, which was only a quarter of k of raw CNTs (0.024 min−1). After heat treatment, the k was 0.049 min−1 much higher than those of raw CNTs and CNT-O. In addition, the chemical oxygen demand (COD) removal rates in the CNT-O-H/PDS system were estimated to be about 44.2%, 47.9% and 54.6% at 1.5 h, 3 h and 4.5 h, respectively  was calculated to be 16.09 kJ/mol, indicating that high temperature was conducive to TC degradation in the CNT-O-H/PDS system.The ability of the CNT-O-H/PDS system for TC degradation was also examined under different temperature b. The deinset in b, and thFrom −, 10 mg/L The adaptability of CNT-O-H/PDS system was explored using common matrix species and different organic pollutants. First, 100 mg/L Cln extent a, accompn extent . A relatIn addition to TC, the degradation performances of the CNT-O-H/PDS system towards MO, MB, RhB and CTC were investigated. As illustrated in D/IG value dropped significantly from 1.53 of fresh CNT-O-H  after every run. As depicted in  CNT-O-H a to 1.11 CNT-O-H , indicat CNT-O-H c to 48%  CNT-O-H . The resTo regain the activation ability of CNT-O-H, the collected CNT-O-H was re-calcined at 800 °C under Ar atmosphere. Surprisingly, the TC degradation efficiency of regenerated CNT-O-H reverted to over 70%, even recycling for four runs. Based on above results, CNT-O-H possessed high regeneration ability and reusability after a facile re-calcination process to recover its activation performance.1O2 was the main ROS responding for TC degradation. Aiming to identify the probable ROS generated in the CNT-O-H/PDS system, quenching experiments were conducted under a set of different chemical quenchers a. It cou1O2 generated in the CNT-O-H/PDS system. As presented in 1O2 were observed in the CNT-O-H/PDS system, and the signal intensities were much higher than those of individual PDS. This verified that 1O2 generated in the CNT-O-H/PDS system, which was consistent with the results of quenching experiments. While no obvious 1O2 was the dominant ROS accompanied with a small amount of Further, EPR analyses were performed to confirm Although persulfates could self-decomposition to form ROS , this fu−, 10 mg/L 1O2, resulting from the interaction between PDS and active sites (C=O groups and surface defects) of CNT-O-H. This study provided a simple strategy to prepare CNTs with high C=O ratio and defect degree for persulfate activation, which possessed great potential in the practical treatment of organic-polluted wastewater.To sum up, the CNTs with high C=O ratio and defect degree (CNT-O-H) were successfully obtained via treating raw CNTs with nitric acid oxidation and calcination at 800 °C, which exhibited an enhanced performance towards PDS activation; 0.2 g/L CNT-O-H and 0.4 g/L PDS could degrade 75.2% of 50 mg/L TC at room temperature within 40 min. More importantly, The CNT-O-H/PDS system had strong anti-interference ability to common matrix species (100 mg/L Cl


Our previous study found that Cystathionine-γ-lyase (CTH) and H2S are correlated with the occurrence and development of Clinical Mastitis (CM) in Holstein cows. However, the functions and regulatory mechanisms of CTH/H2S are still unknown. In this study, the inflammatory mammary cell model based on the MAC-T cell line was established by Lipopolysaccharide (LPS)-induced manner to further explore the function and regulatory mechanism of CTH/H2S in cows with CM. In the inflammatory MAC-T cell, the CTH expression and H2S production were both repressed in an LPS-dose dependent manner, which demonstrated that CTH/H2S is related to the progression of inflammation. The inhibition of CTH/H2S using a selective CTH inhibitor, β-cyano-l-Alanine (BCA), promoted LPS-induced inflammation response and the expression of inflammatory cytokines. However, this was reversed by the H2S donor NaHS, demonstrating that H2S can protect cells from inflammatory damage. Intriguingly, interleukin-8 (IL-8) showed an inverse expression pattern correlated with the H2S-mediated cell protection effect during the inflammation process, and the inhibition test using a selective IL-8 receptor antagonist, SB225002, showed that IL-8 signaling plays a critical role in mediating endogenous H2S synthesis, and CTH/H2S exerts its anti-inflammation via IL-8-mediated signaling. This study provided support for the prevention and treatment of CM and the development of a novel anti-inflammatory strategy.Hydrogen sulfide (H Clinical mastitis (CM) is one of the greatest diseases impacting high milk yield and results in huge economic losses for the global dairy industry ,2. CM, a2S), as an endogenous gaseous signaling molecule, plays an important role in animal physiological regulation and exerts anti-bacterial activity [2S can lead to a variety of diseases related to the nervous system, cardiovascular system, digestive system and urinary system [2S is involved in many physiological and pathological processes, including apoptosis, inflammatory and adaptive immunity such as monocyte and polymorphonuclear cell apoptosis, leukocyte adhesion and infiltration, T cell activation, proliferation, and inflammatory cytokine production [2S exerts its anti-inflammatory effects mainly via increasing the levels of anti-inflammatory and cytoprotective molecules and inhibiting the secretion of inflammatory cytokines [2S may serve as a dual role modulator in the inflammatory process. Some reports showed that H2S can inhibit aortic atherosclerotic plaque and neutrophil aggregation in lung tissue, so as to reduce pulmonary edema and increase pulmonary capillary permeability [2S stimulates leukocyte activation and trafficking to promote a pulmonary inflammatory response in the model of septic shock, caused by cecal ligation and perforation [2S may serve as a dual role modulator in the inflammation process.Hydrogen sulfide , 3-mercaptopyruvate sulfotransferase (3-MST), and cysteine aminotransferase (CAT) [2S system plays a part in both health and diseases, and CTH-derived H2S is implicated in the regulation of multiple signal physiological effects, including immune response, cell differentiation, cancer, aging and the injury process [2S synthesis [2S-mediated inflammation regulation in mammary glands with CM is still unclear.Cystathionine-γ-lyase (CSE/CTH) is a major endogenous Hunctions . Togethese (CAT) ,14. Thesse (CAT) . The CTH process ,16. In aynthesis . However2S are correlated with the occurrence and development of CM in Holstein cows [2S in Holstein cows is still unknown. This study aimed to find the expression pattern of CTH and the production property of H2S in the Lipopolysaccharide (LPS)-induced inflammation cell model, and uncover the molecular regulatory mechanism of CTH/H2S in the inflammation process, attempting to provide support for the prevention and treatment of CM and the development of an anti-inflammatory strategy.Our previous study identified a series of genes related to sulfide metabolism, including CTH and CBS in mammary glands with CM, and uncovered that CTH and Hein cows . However2S in mastitis in cattle, the bovine mammary alveolar cell inflammatory model was established using LPS in MAC-T cells. Expressions of multiple inflammatory factors, including Interleukin-1β (IL-1β), Interleukin (IL-8), Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), and Toll Like Receptor 4 (TLR4) were evaluated in both mRNA and protein levels after treatment, with scalar concentrations of LPS using a real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot (WB) assay, respectively. Cell viability was evaluated by a Cell Counting Kit-8(CCK-8) assay. Normal MAC-T cells were set as controls. As shown in IL-1β and IL-8 increased gradually, in an LPS dose-dependent manner compared to the controls. In addition, the transcripts of IL-6, TLR4 and TNF-α increased with the increase of the LPS concentration compared to the control group , qRT-PCR and WB assays, respectively, in LPS-induced MAC-T cells. Normal MAC-T cells were set as controls. As shown in 2S synthesis, the result suggested that CTH and CBS are expressed in MAC-T cells, and H2S can be produced in the bovine mammary alveolar cell.H process ,20. To u2S concentration showed that a MAC-T cell can produce H2S, with an average concentration of 822 μM in supernatant , a CTH-specific reversible inhibitor [2S donor to trigger H2S release in cells [2S function. CTH and CBS expression was evaluated by qRT-PCR and WB assays. Endogenous H2S production in supernatant was determined by a Micro H2S Content Assay Kit, and cell viability was evaluated by CCK-8 assay. Normal MAC-T cells without treatment were set as controls. As shown in CTH expression both at the mRNA level and protein levels in MAC-T  establishing a link between inflammation and mineralization in vascular calcification [2S stimulates autophagy by blocking Mammalian target of rapamycin (mTOR) signaling in sepsis-induced acute lung injury [2S promotes activation of Nuclear factor-E2 related factor2 (Nrf2) and the consequent expression of antioxidant proteins to alleviates liver damage [2S can suppress the P2X7 receptor (P2X7R) to attenuate NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome-mediated neuroinflammation in rats [2S can inhibit inflammation through multiple pathways, but the molecular targets of its action and its mixed effects are not fully understood. In addition, H2S has a regulatory effect on the release and function of various inflammatory factors. Therefore, regardless of whether H2S has pro-inflammatory or anti-inflammatory effects, the development of H2S systemic administration using inhibitors of H2S synthesis or through H2S donors can provide new avenues for the treatment of inflammatory diseases. This study revealed a novel downstream target of hydrogen sulfide in the IL-8 signaling pathway. All these findings help to understand how hydrogen sulfide works, and provides a basis for hydrogen sulfide as a drug development and application.Intriguingly, the inflammatory cytokine, IL-8, showed an inverse expression pattern correlated with the H process , raisingignaling . Neverthfication . H2S stig injury . H2S pror damage . Endogen in rats . These f2 at 37 °C in a DMEM culture medium  containing 10% fetal bovine serum . The cells were grown to 80% confluence, and cells were passaged or treated with different compounds such as LPS , β-cyano-L-Alanine , NaHS , or/and SB225002 . The cells were seeded at 1 × 104 cells/well in 96-well plates or 1 × 105 cells/cm3 in different dishes. Before treatment, the culture medium was changed to DMEM without FBS and phenol (Gibco) for 12 h. The MAC-T cells were treated with or without LPS, BCA, NaHS, or/and SB225002 for 24 h. The appropriate concentration gradient was selected based on literature reports [MAC-T cells were purchased from the ATCC Corporation , and maintained in dishes or plates in a humidified atmosphere of 5% CO reports ,37,38 an2S concentration of cells culture supernatants were tested following the manufacturer’s instructions of the Micro H2S Content Assay Kit . All H2S detection was evaluated under a wavelength of 665 nm using a microplate reader . All experiments were conducted in triplicate and repeated at least three times.The endogenous HThe viability of MAC-T cells was measured after treatment with or without LPS (0–200 μg/mL), BCA (0.5–4.0 mM), NaHS(30 μM) and SB225002 (0–10 µM) using a CCK-8,  according to the manufacturer’s instructions. Briefly, the cells were seeded in 96-well plates and incubated for 24 h. The cells were treated with different concentrations of LPS, BCA, NaHS and SB225002 for 24 h. 10 µL of CCK-8 was added to the cells and allowed to incubate for 2 h before measuring the OD at 450 nm with a ReadMax 1900 microplate reader . All concentration gradient selections are based on the literature search and adjusted according to reagent conditions. The cell viability experiments were performed at least in triplicate.MAC-T cells were plated in a 35 mm dish with cover slips to a monolayer. IF assay was performed as previous described . In briepro Taq HS Premix  following the manufacturer’s protocols. The amount of transcripts in each sample were normalized using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. Primers used for qRT-PCR assay  according to the manufacturer’s protocols. RNA quantify was monitored by a NanoDrop-8000 , and RNA integrity was assessed by 1% denaturing gel electrophoresis . The cDNA was synthesized using the Evo M-MLV RT Kit . The qRT-PCR was performed with 2×SYBR Green CR assay  were desw/v) of skimmed milk in a Tris-HCl buffer  at room temperature. Co-incubation of blocked PVDF overnight at 4 °C with the following primary antibodies: rabbit anti-IL-1β , mouse monoclonal anti-IL-8 , rabbit anti-CBS , mouse monoclonal anti-CTH , and anti-β-actin . After three washes, the membranes were incubated with the corresponding secondary antibody conjugated to horseradish peroxidase  for 1 h at room temperature. Blot bands were visualized using the Gel Imaging System  and then digitized using Image-Pro Plus 6.0 . β-actin was used as a control. All immunoblot assays were performed at least in triplicate.After treatment, MAC-T cells were harvested for protein extraction using a Radio-immunoprecipitation assay buffer  with 1 mM phenylmethylsulfonyl fluoride  and a protease inhibitor . The protein concentration was determined following a Bicinchoninic Acid (BCA) protein assay kit . A WB assay was performed as previous described . Brieflyp < 0.05 were considered as significant.All statistical procedures were performed using Statistical Package for the Social Sciences 22.0 for Windows . Graphs were constructed using Prism 5.0 . One-way ANOVA  and post hoc Tukey’ test were used for finding significant differences between groups. 2S and significantly increase the expression of IL-8, IL-β, TLR4 and other inflammatory factors. The relative expression levels of CTH mRNA and protein and the content of H2S were inhibited in a LPS dose-dependent manner, and were negatively correlated with the relative expression levels of IL-1β and IL-8. Cells were treated with a CTH inhibitor BCA, and the results indicated that CTH inhibited the development of inflammation by regulating the LPS-induced reduction of H2S synthesis and the expression of IL-8 in MAC-T cells. Inhibition assays using the selective IL-8 receptor antagonist SB225002 suggest that IL-8 signaling plays a critical role in mediating endogenous H2S synthesis, and that CTH/H2S exerts its resistance through IL-8-mediated signaling. These results can provide new ideas and methods for the prevention and treatment of clinical mastitis and the development of anti-inflammatory drugs.In MAC-T cells, LPS can significantly reduce the content of H


The host interactome of influenza viral proteins is ever-expanding. In this work, we report the identification of host heterogeneous nuclear ribonucleoprotein C (hnRNP-C) as an interacting partner of influenza A virus nucleoprotein (NP). We confirmed that this interaction exists across different influenza A subtypes and strains. Using biochemical methods, we determined that hnRNP-C interacts with NP via its C-terminal auxiliary domain. Further, we determined that the hnRNP-C is a negative regulator of influenza viral growth. Its interaction with NP is implicated in the promotion of host cell apoptosis during viral infection. It is the first time that the interaction between influenza nucleoprotein and host heterogeneous nuclear ribonucleoprotein C is characterized in detail. Overall, these findings not only characterize the interaction between NP and its host interacting partner hnRNP-C but also clarify the functional significance of this interaction. This work may lead to a new therapeutic target for the development of anti-influenza drugs. The influenza virus, an infamous respiratory virus pathogenic to humans, belongs to the orthomyxoviridae family, and its genome contains eight negative, single-stranded RNA gene segments wrapped along the ribonucleoproteins, encoding at least thirteen proteins and polypeptides including the nucleoprotein (NP), which performs multiple structural and functional roles, such as the formation of viral ribonucleoprotein (RNP) with PA, PB1 and PB2, and the encapsidation of viral RNA. NP is an important protein for the stabilization of positive-sense complementary RNA during the replication process, via the formation of a complementary ribonucleoprotein (cRNP) [NP has also been found to interact with multiple host factors including a-actinin 4, TRIM22, clusterin, heat shock protein 40, and nuclear factor 90 ,4,5,6,7.3(C2) heterotetramer.hnRNP-C is a member of a family of more than 20 heterogeneous nuclear ribonucleoproteins (hnRNPs). It resides in the host cell nucleus and is involved in RNA processing such as mRNA splicing, mRNA retention and packaging, maintenance of mRNA stability, and mRNA export. It has also been reported to be involved in translation initiation by binding to internal ribosomal entry sites on mRNA ,14. hnRNhnRNP-C is implicated in numerous viral infections, including poliovirus, hepatitis delta virus, Ebola virus, dengue virus, the human papillomavirus type 16, MERS-CoV, and SARS-CoV-2 ,18,19,20In order to identify the interacting partners of NP, we immobilized purified H5N1 NP protein onto N-hydroxysuccinimide (NHS) beads and allowed H292 lysates to flow through the beads. The proteins captured by NP were resolved via 2D electrophoresis. hnRNP-C was found, along with nucleophosmin (NPM) and beta-actin, which were previously identified as the interacting partners of NP ,26.To confirm the NP–hnRNP-C interaction in a cellular context upon infection, HEK293T cells overexpressing myc-tagged hnRNP-C1 were infected by the A/WSN/1933(H1N1) virus. Viral NP was co-immunoprecipitated by myc-tagged hnRNP-C1 A, lane 4Since both NP and hnRNP-C were found to bind RNA ,27, we pWe then set forth to define the NP-binding region on hnRNP-C by constructing various hnRNP-C truncation fragments to evaluate the extent of their in vitro pull-down by NP. Full-length hnRNP-C and its fragments were passed through NP, which was immobilized on NHS-Sepharose beads. For C-terminal truncations, only fragment 1–251 was weakly captured by NP but not the fragments 1–130 and 1–217. For N-terminal truncations, fragments 161–293, 216–293, and 251–293 were all captured by NP to various extents . It could be shown that when hnRNP-C was transiently knocked down, viral growth was enhanced at as early a time point as 6 h post-infection  showed sTo investigate the functional significance of the NP–hnRNP-C interaction, a fragment corresponding to residues 216–293 of hnRNP-C (216–293 fragment) was constructed to perturb the NP–hnRNP-C interaction by competing with the full-length hnRNP-C protein for binding to NP. In the HEK293T cells expressing the 216–293 fragment and infected with the A/WSN/1933(H1N1) virus, full-length myc-tagged hnRNP-C precipitated around 40% less NP than the amount in cells expressing GFP control A, confirWe identified and characterized the interaction between influenza nucleoprotein (NP) and host heterogeneous nuclear ribonucleoprotein C (hnRNP-C) in this work. We found that the NP–hnRNP-C interaction did not involve RNA and that the C-terminal region on the auxiliary domain of hnRNP-C was responsible for NP binding. A C-terminal fragment from hnRNP-C was able to perturb the NP–hnRNP-C complex formation and suppress the influenza RNP activity.NP has been shown to interact with hnRNP-A2/B1 via its glycine-rich domain but not the RNA-binding domain . LikewisNP is the most abundant protein component of the ribonucleoprotein (RNP), which is responsible for the transcription and replication of viral proteins. Hence, RNP activity would directly correlate with viral fitness. Using a minigenome luciferase reporter assay, we were able to investigate RNP activity as luciferase signal readout. In this assay, PA, PB1, PB2, and NP subunits were co-transfected into HEK293T cells along with the luciferase reporter, driven by an influenza promoter. An EGFP plasmid driven by a CMV promoter was also transfected as an internal control. A minigenome RNP was thereby reconstituted in cells for the transcription of the luciferase gene. Normalized luciferase signal served as a reflection of RNP activity. Utilizing this system, we demonstrated suppressed RNP activity upon hnRNP-C overexpression and, in contrast, enhanced activity during hnRNP-C depletion.hnRNP-C was reported to be involved in various viral infection conditions. During poliovirus infection, it interacts with the 3′-end of the negative RNA strand and promotes positive-strand synthesis . hnRNP-CIn our work, we showed that hnRNP-C itself is a negative regulator of RNP activity and viral growth. However, the NP–hnRNP-C interaction is indispensable for proper RNP activity. These two results may seem contradictory at first glance. Nevertheless, based on our findings, we postulate that the influenza NP exploited hnRNP-C to modulate host apoptosis. Consistent with this postulation, it has been reported that the apoptosis-induced enlargement of nuclear pores would facilitate progeny RNP nuclear export ; therefoIn summary, we characterized, for the first time, the NP–hnRNP-C interaction, and these results provide a better understanding of NP host interactome and give additional insights as to how the influenza A virus acts upon the host cell apoptosis machinery.pRHisMBP-NP  and the 2. All culture media and supplements were obtained from Gibco unless otherwise stated.A549, Beas2B, H292, MDCK, and HEK293T cells were obtained from ATCC. A549 and H292 were maintained in RPMI-1640 medium. MDCK was maintained in a minimal essential medium (MEM). HEK293T was maintained in a Dulbecco’s modified Eagle’s medium (DMEM). Beas2B was maintained in a DMEM with Ham’s F-12 supplement. All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 ug/mL streptomycin. The cells were cultured at 37 °C under 5% COAnti-myc , anti-actin (Genscript), and anti-GFP (JL-8) (Takara Bio USA) were obtained commercially. Horseradish peroxidase (HRP)-conjugated anti-mouse, and anti-rabbit secondary antibodies were purchased from Bio-rad, Hercules, CA, USA. Anti-hnRNP-C was purchased from Santa Cruz Biotechnology, Dallas, TX, USA. NP anti-serum was raised in-house and described previously .The nucleoprotein of A/HK/156/97 (H5N1) was expressed as a maltose binding protein (MBP) fusion protein as described previously . After RFor infection experiments, the culture medium was aspirated, and the cells were washed twice with PBS. The A/WSN/1933 (H1N1) virus was diluted in a serum-free medium and was used for infection at the multiplicity of infection (MOI) as stated. The serum-free medium was used as a mock infection control. After 1 h incubation at room temperature, the virus was replaced with a fresh serum-free medium, and the cells were incubated at 37 °C for virus propagation. Supernatants were collected at 24 h post-infection for titer determination or further experiment.Co-immunoprecipitation was performed in HEK293T cells by transfecting relevant plasmids, as stated in the text. The cells were lysed through sonication in an IP buffer . After clearance of cell debris via centrifugation, immunoprecipitation was performed by the addition of appropriate antibodies at room temperature for 4 h or at 4 °C overnight, followed by incubation with Protein A beads for 1 h. After extensively washing with the IP buffer, the beads were boiled in SDS-loading dye and subjected to SDS–PAGE and Western blotting. In the case of infection as a source of NP, myc-tagged hnRNP-C1 was transfected to HEK293T. Then, 12–16 h later, the cells were washed with PBS and changed to a serum-free DMEM, after which they were infected with the A/WSN/1933 (H1N1) virus at an MOI of 0.01 for 12–16 h. To test for the effect of the hnRNP-C fragment of residues 216–293, the plasmid encoding GFP–216–293 fragment or the control pEGFP-C1 was pre-transfected to HEK293T 24 h prior to infection. myc-tagged hnRNP-C was expressed in HEK293T separately. At 24 h post-infection, the cells were harvested, sonicated, and cleared. Lysates with myc-hnRNP-C were mixed with the lysates with GFP or GFP–216–293 to allow for immunoprecipitation at 4 °C overnight.hnRNP-C and its truncations were expressed in the pET28a vector or in the pETZ2_1a vector (for fragment 251-293 only) in BL21(DE3)pLysS cells. A list of primers used for cloning these truncations is included in −∆∆Ct method.The culture medium was removed, and the cells were collected in 500 μL TRIzol Reagent (Invitrogen). The total RNA extracted was used in first-strand DNA (cDNA) synthesis using M-MLV  for real-time PCR, according to the manufacturer’s protocol. Real-time PCR was performed in Applied Biosystems 7300 (Perkin Elmer) using the following primers: hnRNP-C, forward 5′-GCA GGT GTG AAA CGA TCT GC-3′ and reverse 5′-TTA CTT TCC AGA CTT GGA AGA TCC-3′; CASP8, forward 5′-AAG CAA ACC TCG GGG ATA CT-3′ and reverse 5′-GGG GCT TGA TCT CAA AAT GA-3′; BIM, forward 5′-GAG ATA TGG ATC GCC CAA GA-3′ and reverse 5′-CAA TGC ATT CTC CAC ACC AG-3′; BCL2L13, forward 5′-CTG AGC CAG CCA GTG ACA TA-3′ and reverse 5′-TCC AGG GTA TTC CTC CTC CT-3′; GAPDH, forward 5′-GAG TCA ACG GAT TTG GTC GT-3′ and reverse 5′-GAC AAG CTT CCC GTT CTC AG-3′. PCR reactions constituted of a 3 min hot start at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 sec and amplification at 60 °C for 30 s, and extension at 72 °C for 15 s. All RT-qPCR results represent the mean from at least three independent experiments. The relative quantification of mRNA levels was performed with the comparative Ct method using GAPDH as the reference gene and the 2For the endogenous hnRNP-C knockdown, Beas-2B or HEK293T cells were transfected with human hnRNP-C1/C2 siRNA (Santa Cruz Biotechnology) using Lipofectamine RNAiMAX . Non-targeting siRNA  was used as a transfection control. For the transfection of one well on a 24-well plate, 0.06 nmol of siRNA was transfected.t-test in GraphPad Prism.The system has been described previously , which wA549 cells were pre-seeded on 12 mm diameter glass slides in a 24-well plate. hnRNP-C was depleted by specific siRNA, as described above. The cells were harvested by removal of the medium and washed three times with cold PBS. Diamidino-2-phenylindole (DAPI) at 1 µg/mL was added to each well for 5 min in the dark. Afterward, the cells were washed three times with cold PBS, and propidium iodide at 1 µg/mL was added for staining in the dark for 20 min. The cells were then washed three times with cold PBS and mounted onto microscopy slides using a DAKO mounting medium. The cells were visualized with a Leica TCS SP8 Confocal Microscope System. For comparison purposes, acquisition parameters remained identical for all slides.The samples were resolved on SDS–PAGE gels and electrotransferred to a PVDF membrane. After blocking for 1 h with 5% non-fat dry milk in a TBS-T buffer , the blots were probed overnight at 4 °C with primary antibodies as stated. The blots were then incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h followed by detection with ECL chemiluminescence reagent  and exposure on X-ray films or detection with a ChemiDocMP+ imager (Bio-rad).


In conclusion, this study indicated that USP44 enhanced the growth of T-ALL through interacting with WDR5 and repressing its ubiquitination. This study highlights the potential use of USP44 as a therapeutic target of T-ALL.T-cell acute lymphoblastic leukemia (T-ALL) is a common hematologic malignancy. Based on the data from GSE66638 and GSE141140, T-ALL patients depicted a higher USP44 level. However, its role in T-ALL is still unclear. In the present study, we investigated the role of USP44 in T-ALL growth. USP44 overexpression elevated the proliferation of CCRF-CEM cells, while USP44 knockdown suppressed the proliferation of Jurkat and MOLT-4 cells. In addition, USP44 accelerated the cell cycle progression, with boosted cyclinD and PCNA levels. However, USP44 knockdown induced apoptosis in Jurkat and MOLT-4 cells, with an upheaval among cleaved caspase-3 and PARP levels. Mechanistically, USP44 co-localized and interacted with WDR5, leading to the repression of its ubiquitination and degradation. Interestingly, WDR5 overexpression abolished the apoptosis induced by USP44 knockdown. Consistently, the T-cell acute lymphoblastic leukemia (T-ALL), a common malignancy in the hematologic system, is arisen from genetic lesion accumulation during T-cell development, leading to the differentiation arrest and abnormal proliferation of immature progenitors. During T-ALL, leukocytosis initiates and infiltrates within the lymph nodes and other organs et al. demonstrated a high USP44 level in T-ALL Ubiquitination, a form of post-translation modification, leads to protein degradation. The ubiquitination/deubiquitination system is critical in various physiological events by participating in intracellular signaling pathways http://www.hitpredict.org/), WDR5 may bind with USP44. However, whether WDR5 mediates the functions of USP44 is unknown.The WD repeat protein family members regulate various cellular events and are considered as potential targets for disease treatment In this study, the role of USP44 in T-ALL was explored. It was revealed that USP44 accelerated the growth of T-ALL cells by interacting with WDR5 and suppressing its ubiquitination and degradation. This study depicted a potential role of USP44 as a candidate target for treating T-ALL.GEO DataSets (GSE66638 and GSE141140) were utilized to analyze the expression of USP44 within T-ALL samples and normal cells.2 at 37 °C.Human T-ALL cell lines Jurkat, MOLT-4 and CCRF-CEM were procured from iCell Bioscience Co., Ltd  and cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum . Human HEK-293T cells were procured from ZhongQiaoXinZhou Biotechnology Co., Ltd  and grown in DMEM medium  with 10% FBS. All the cells were cultured under a humidified atmosphere with 5% COLV-shUSP44-1#: CCGGCTTCAAAGTGAAGATCAACTTTCAAGAGAAGTTGATCTTCACTTTGAAGCTTTTT;LV-shUSP44-2#: CCGGGCACTGTGTGGACTGCAACATTCAAGAGATGTTGCAGTCCACACAGTGCCTTTTT;LV-shNC: CCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTT.USP44 CDS was inserted into a pLVX-IRES-puro plasmid  and infected into CCRE-CEM cells through a lentiviral infection system. Blank pLVX-IRES-puro plasmid served as the negative control. USP44 shRNAs were inserted into a pLVX-shRNA1 plasmid  and infected into Jurkat and MOLT-4 cells through a lentiviral infection system. No-targeting sequence inserted into a pLVX-shRNA1 plasmid served as the negative control. The sequence information was as follows:The cells were selected using puromycin . HA-Ubiquitin, Flag-WDR5, and His-USP44 overexpression plasmids  were transfected into HEK-293T cells, and WDR5 overexpression was transfected into Jurkat cells using Effectene Transfection Reagent  according to the manufacturer's protocol. Finally, the cells were collected for subsequent experiments after 48h.-ΔΔCT method and normalized to GAPDH.Total mRNA was extracted from the cells using TRIpure Reagent . After cDNA synthesis using BeyoRT II M-MLV  based on the instruction, quantitative real-time PCR was carried out using 2×Taq PCR MasterMix  and SYBR Green  on an Exicycler 96 PCR system . CDNA served as the template. The following primers were used: forward primer for USP44, 5'-CAA CTT ATG ATA TGC CAC CTA-3'; reverse primer for USP44, 5'-GTA CCC AGA ACC CTC CT-3'; forward primer for WDR5, 5'-CAC CTG TGA AGC CAA ACT-3'; reverse primer for WDR5, 5'-GAG GCA GAA ACA AGA AGG-3'; forward primer for GAPDH, 5'-GAC CTG ACC TGC CGT CTA G-3'; reverse primer for GAPDH, 5'-AGG AGT GGG TGT CGC TGT-3'. The thermal circulation is as follows: 94 °C for 5 min; 94 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s, 40 cycles; then 72 °C for 2.5 min and 40 °C for 1.5 min. It was melting form 60 °C to 94 °C, 1 °C/1 s. Gene expression was determined based on the 23/well). According to the instruction, the cell viability was assessed using CCK-8 reagent  at indicated time points. Absorbance at 450 nm was measured with a microplate reader .After infection or transfection for 48 h, the cells were seeded into 96-well plates . The supernatants were harvested through centrifugation at 10000 xg for 5 min at 4 °C. After measuring the protein concentration using a BCA protein assay kit , 20 μg of whole cell lysates were subjected to SDS-PAGE, followed by electroblotting onto the polyvinylidene fluoride membranes . After blockade using 5% skim milk and washing using TBST buffer, the membranes were incubated with primary antibodies overnight at 4 °C. It was followed by washing using TBST buffer and incubating with corresponding secondary antibodies  at 37°C for 1 h. Then the targeted proteins were washed using TBST buffer and visualized by enhanced chemiluminescence . The following primary antibodies were utilized: anti-USP44 , anti-Cyclin D1 , anti-PCNA , anti-cleaved caspase-3 , anti-cleaved PARP , anti-WDR5 , and anti-GAPDH . The densitometric quantification of blots was undergone using the Gel-Pro-Analyzer software.Based on the instruction, the cell lysates were incubated with bead-USP44 or bead-WDR5 for immunoprecipitation after measurement of protein concentration. The immunoprecipitates were washed with PBS, eluted with 50 μl elution buffer and subjected to western blot as described above. The antibodies used for the western blot were USP44 (1: 300) and WDR5 (1: 300).HA-Ubiquitin, Flag-WDR5, and His-USP44 were co-transfected into HEK-293T cells. After 48 h, the cells were treated using 5 μM MG132  for 6 h to repress the proteasomal degradation. After that, the cells were harvested, and the ubiquitination status of WDR5 was determined through immunoprecipitation, as description above. Briefly, the whole cell lysates were immunoprecipitated with bead-flag and subjected to western blot. The antibodies utilized in western blot were flag , his  and HA .For cell cycle determination, the cells were incubated with 10 μM BrdU  for 40 min after indicated treatment and then harvested, fixed, permeabilized and labeled using a FITC-BrdU determination kit  and a 7-ADD cell cycle determination kit  based on the protocol. After that, the cells were analyzed through a flow cytometer . For determinating cell apoptosis, the cells were harvested, washed using PBS and stained with a cell apoptosis determination kit  following the protocol. Thereafter, the cell apoptosis was determined with a flow cytometer.The femurs and tibias of mice were harvested to obtain the bone marrow. The spleen tissues of mice were also harvested. Cells in the bone marrow and spleens were incubated with CD45 primary antibody  at 4 °C for 30 min. Later, the cells were analyzed with a flow cytometry.Cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% TritonX-100 and blocked using 1% BSA. Thereafter, the cells were incubated with antibodies against WDR5  or USP44  at 4 °C overnight, followed by incubating with Cy3-conjugated  or FITC-conjugated secondary antibodies  at room temperature for 60 min. DAPI  was incorporated to stain the nuclei. After washing with PBS, cell images were visualized using a fluorescence microscope . The co-localization of USP44 and WDR5 was analyzed with Image Pro Plus software.In order to determine the half-time of WDR5 in Jurkat cells with USP44 knockdown, the cells were treated with 10 μg/ml CHX  to block protein translation. After treatment with CHX for 0, 2, 4, and 8 h, the cells were collected, and western blot was conducted to determine the WDR5 protein level.5). At the end of the study, mice were sacrificed. The bone marrow and spleens were obtained to determine CD45+ cells. The images and weight of spleens were recorded. The lung and liver tissues were harvested for hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining using CD3. All the experiments were conducted following the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of Shengjing Hospital of China Medical University.Six-week-old NOD-SCID mice were procured from Beijing Huafukang Biotechnology Co., Ltd  and fed within a standard condition. USP44 knockdown cells (LV-shUSP44-1#) were injected into mice through the caudal vein  at 4°C overnight. It was followed by incubation with peroxidase-conjugated secondary antibodies  for 60 min at 37 °C. The slices were visualized using DAB  and counterstained with hematoxylin . The images were captured under a microscope.The lung and liver tissues were harvested, fixed in 4 % paraformaldehyde, embedded in paraffin, and cut into 5-μm slices. After deparaffinage, rehydration and antigen retrieval, the slices were treated with 3% Ht test or one-way analysis of variance followed by Tukey's post hoc test. A P-value < 0.05 was considered statistically significant.All the results were represented as mean and SD. Comparison between the groups was analyzed using Student's Based on the data from GSE66638 and GSE141140, the USP44 level in T-ALL patients was higher than in normal cells , the percentage of CD45+ cells in the bone marrow decreased when USP44 was knockdown (Figure + cells in the spleens was reduced (Figure in vivo.Jurkat cells with/without USP44 knockdown were injected into NOD-SCID mice to verify the function of USP44 on the progression of T-ALLSeveral USPs are involved in leukemia. For instance, USP15 deletion injures leukemic progenitor function and derepresses antioxidant responses through the Keap1-Nrf2 pathway In our study, USP44 contributed to the growth of T-ALL cells. As the cell cycle is a critical regulator of cell growth, we also investigated the role of USP44 in the cell cycle modulation and depicted that USP44 accelerated cell cycle progression. Noticeably, overexpression of USP44 increased the percentage of cells in S phase, in which phase DNA synthesis is processed, indicating that USP44 might promote the synthesis of DNA. In addition, we showed that knockdown of USP44 increased the percentage of cells in G2/M phase. USP44 has a role in mitosis regulation USP44 also regulated apoptosis of cancer cells in a cell-dependent way. Interestingly, USP44 induced the apoptosis of colon cancer cells WDR5, an oncogene often found in cancers, is associated with the proliferation, cell cycle, and apoptosis of cancer cells Besides, USP44 and WDR5 are also associated with the metastasis of cancer, such as the migration and invasion capability of cancer cells and vasculogenic mimicry in vivo and their infiltration in the livers and lungs. Significant research demonstrated that USP44 interacted with WDR5 and repressed its ubiquitination and degradation, contributing to the role of USP44 in T-ALL. Our results depicted that USP44 could become a potential therapeutic target for treating T-ALL.Collectively, in the present study, USP44 contributed to the growth of leukemia cells and suppressed their apoptosis. It also regulated the progression of T-ALL


Increased myeloid subsets, including CD11c+ DCs and F4/80+ macrophages with higher MHC II and CD80 expression in bone marrow, spleen, and liver, were detected when mice reached remission after anti-CD19 CAR T treatment. Compared to mice without anti-CD19 CAR T administration, intrinsic T cells were triggered to produce more IFN-γ and TNF-α. However, some lymphoma mice relapsed by day 42 after therapy, which coincided with CAR T-cell recession, decreased myeloid cell activation and increased Treg cells. Elevated intrinsic T cells with high PD-1 and TIGIT exhaust signatures and attenuated cytotoxicity in TME were associated with the late-stage relapse of CAR T-cell treatment. In summary, the cellular compositions of TME as allies of CAR T cells may contribute to the anti-tumor efficacy at the initial stage, whereas anti-CD19 CAR T-cell disappearance and host response immunosuppression may work together to cause lymphoma relapse after an initial, near-complete elimination phase.Anti-CD19 chimeric antigen receptor (CAR) T cells represent a highly promising strategy for B-cell malignancies. Despite the inspiring initial achievement, remission in a notable fraction of subjects is short-lived, and relapse remains a major challenge. Tumor microenvironment (TME) was proved to be aroused by CAR T cells; however, little is known about the dynamic characteristics of cellular components in TME especially during the different phases of disease after anti-CD19 CAR T-cell treatment. We took advantage of an immunocompetent model receiving syngeneic A20 lymphoma cells to dissect the changes in TME with or without CAR T-cell injection. We found that anti-CD19 CAR T-cell treatment attenuated the symptoms of lymphoma and significantly prolonged mice survival through eradicating systemic CD19 Chimeric antigen receptor (CAR) T cellular therapy targeting the B-cell surface protein CD19 has produced remarkable outcomes in the treatment of B-cell malignancies . ApproveRecently, evidence showed that two-thirds of tumor lysis events were directly performed by CAR T cells, which suggested CAR T-cell cytotoxicity was a major player in driving tumor regression but also suggested the existence of additional mechanisms elicited by CAR T cells in the tumor microenvironment (TME) . AlthougThe paradoxical role of the TME during stages of tumor progression is dependent on its cellular components, which are highly heterogeneous and versatile . The gen+ myeloid subsets and cytokine producing endogenous T cells accumulated in initial-remissive mice following anti-CD19 CAR T-cell administration. When the mice were resistant to CAR T cells, declining host immune responses characterized by reduced activity of macrophages and DCs as well as increased immunosuppressive Treg cells were investigated. Our results identify the potential mechanisms of lymphoma relapse as not only the disappearance of anti-CD19 CAR T cells but also intrinsic T-cell dysfunction in TME. Therefore, redressing the immunosuppressive TME represents an attractive strategy to prevent relapses after therapy.To address this question, we took advantage of the immunocompetent mice bearing A20 lymphoma cells to dissect the changes of immune cells in TME, which probably will be remodified by anti-CD19 CAR T cells. We illustrated here that effective CD11bFemale BALB/c mice, between 6–8 weeks of age and 22–25 g in weight, were obtained from the Beijing Vital River Laboratory Animal Technology. Mice were housed at a constant temperature of 25 ± 2 °C and a relative humidity of 55% in the animal house. These are specific pathogen free conditions at Xuzhou Medical University. Animal experiments were approved by the Medical Ethics Committee of Xuzhou Medical University, Xuzhou, Jiangsu, China. All mice were included in the experiment, and best efforts were made to minimize animal suffering. All the experimental procedures used in this study were carried out according to the experimental animal protocol approved by the ethics committee.+ cell line was preserved in our laboratory, which was cultured in RPMI-1640 medium containing β-mercaptoethanol , and the CD19 expression was detected by flow. BALB/c mice received a sublethal dose of irradiation (3.5 Gy) that was used as a conditioning regimen, and a B-cell lymphoma model was established by intravenously injecting A20-GFP+ cells (0.5 × 106/mouse) 4 h later. The mice were monitored daily for survival and every 3 days for body weight changes. The clinical scores of the model were assessed based on three parameters, including weight loss, activity, and paralysis. Each parameter contains a severity scale, and the maximum score is 15, which is shown in . The CAR construct is composed of the anti-murine CD19 single-chain fragment variable domain, the transmembrane and intracellular domains of CD28, and the CD3z intracellular domain. The retroviral vector also encodes green fluorescent protein (GFP) to evaluate transduction efficiency and identify CAR T cells. DH5α competent cells were used in the plasmid amplification, and the extraction was done following the instructions of the plasmid extraction kit (Omega). The concentration and purity of the anti-CD19 CAR plasmid were tested, and the plasmid was stored at −20 °C. Plasmids (15 μg) and 45 μl of X-tremeGENE 9 DNA transfection reagent (Roche) were transfected into Plat-E cells to package retroviral anti-CD19 CAR. The fluorescence intensity of GFP was investigated with a fluorescence microscope . Retrovirus particles were filtered and stored at −80°C.+ T cells of BALB/c mice were isolated and purified using EasySep negative selection reagents according to the manufacturer’s instructions (StemCell Technologies). T-cell purity was >95%. Purified T cells (2 × 106/ml) were activated in a 24-well plate pre-coated with anti-CD3 mAb  and anti-CD28 mAb  in the presence of hIL-2 . Spin infections were performed 36 h after T-cell activation using retroviral particles (3 ml/well) supplemented with HitransG P virus infection enhancer . T cells were cultured for 2 additional days in the presence of hIL-2. Infected T cells were collected and the percentage of CD3+GFP+ CAR T cells was detected by flow cytometry. Three days after the lymphoma model was established, mice were randomly divided into two groups that were either given a single dose of anti-CD19 CAR T cells (1 × 106/mouse) through the tail vein or not. The CAR-T-cell treatment day was set as day 0.Splenic CD3g for 4 min at 4 °C to pellet nonhematopoietic cells. The supernatant was transferred to new tubes and spun at 460 g for 10 min. The cell pellet was resuspended in RPMI-1640, and lymphocytes were separated on a 25% Optiprep gradient (StemCell).Bone marrow (BM), spleen, and liver were harvested on days 9, 15, and 42. BM cells were isolated by flushing the femurs and tibias of tumor-bearing mice. The spleen was ground, and single-cell suspensions were prepared by filtering the cells through 200 um cell strainers. Livers were excised, minced, and passed through a cell strainer. Suspensions were centrifuged at 30Single-cell suspensions from BM, spleen, and liver were stained with the following mAbs purchased from Biolegend: CD3 PE , CD4 PE/Cyanine7 , CD4 BV421 , CD8 APC/Cyanine7 , CD11b PB , F4/80 BV605 , MHCII AF700 , CD19 PE-cf594 , FOXP3 AF647 , CD11c PE-cf594 , IFN-γ PE/Cyanine7 , TNF-α APC , CD25 PE , PD-1 PE/Cyanine7 , TIGIT PE , and Tim-3 BV605 . CD3 EV450  and CD80 PE  were purchased from Elabscience and BD Bioscience, respectively. Granzyme B eFlourTM450  was purchased from Thermo Fisher Scientific. For intracellular cytokine staining, cells were stimulated with PMA  and Ionomycine  in the presence of Brefeldin A  at 37 °C for 4 h. Flow cytometry was performed using a BD LSRFortessa Fortessa Flow Cytometer (RRID : SCR_019601). Data were analyzed using FlowJo software .6/ml) from BALB/c mice were stained with Cell Trace Far Red Cell Proliferation Kit reagent  and incubated in a 37°C water bath for 20 min. Then cells were washed in RPMI 1640 complete medium and incubated in a 37°C water bath in the dark for 5 min. After centrifugation at 400g for 5 min, dye labeled splenocytes were re-suspended to the working concentration (5 × 105/ml), and co-cultured with A20 (5 × 105/ml) and CAR-T cells (1 × 106/ml) at the ratio of 1:1:2. Experimental groups were set as CAR-T + A20 + SP, A20 + SP, and SP alone. After 48 h of incubation, the cells were collected for flow cytometry detection. The dye stained spenocytes (APC+) were distinguished from in vitro-induced CAR-T cells (APC−GFP+CD3+) and activated T cells (APC−GFP−CD3+).Splenocytes . Data conformed to a normal distribution and were presented as the mean ± standard deviation. The Student’s t test was used for comparison between two groups if the variance was homogeneous, and the non-parametric test was used for unequal variance. A one-way or two-way ANOVA was performed on multiple groups. Survival rates were compared using the log-rank test. A With the aim to delineate the effect of anti-CD19 CAR T cell therapy, we used a syngeneic mouse model of A20 lymphoma as others and our previously described , 19. As + cells and GFP+ CD19+ lymphoma cells were detected. Representative flow data of BM in + cells were reduced in mice with CAR T cells. Importantly, GFP+CD19+ malignant cells were dramatically eliminated by anti-CD19 CAR T cells, which was consistent with the remissive responses of CAR T-treated mice. Compared with A20 mice, the statistically analyzed data demonstrated that systemic CD19-expressing cells, and especially GFP+ CD19+ lymphoma cells, were significantly cleared after anti-CD19 CAR T-cell treatment  was significantly better in lymphoma hosts receiving anti-CD19 CAR T cells, and the benefits of CAR T-cell administration were enlarged in progress free survival (PFS)  were dyed with Celltrace Far Red fluorescein to distinguish them from anti-CD19 CAR T (H-2d) or A20 cells in the co-cultured system. The gating strategy for FACS data is shown in + T cells were found both in splenocytes plus A20 combined with or without CAR T cells than that in splenocytes alone group, which were consistent with the reduced CD4+ T cells in vivo. However, obvious increased CD25 expression and IFN-γ secretion of splenic CD4+ T cells were detected under the CAR T-cell induced A20 lysis condition . Furthermore, we confirmed the persistence of CAR T cells in the BM, spleen, and liver on days 9, 15, and 42 post-injection. However, CAR T cells became largely undetectable after treatment for 42 days  ultimately relapsed at the late time-points. By day 42 after therapy, the GFPhigh expressed CD4+ T cells in relapsed BM, whereas the total PD-1 fluorescence intensity was slightly decreased both in BM and spleen. By contrast, significantly increased PD-1 levels in CD8+ T cells were shown in relapsed BM. Consistent with the expression pattern of PD-1, TIGIT fluorescence intensity was obviously elevated in CD8+ T cells, and an increased trend was found in CD4+ T cells. However, there was no statistically significant difference in TIM3 between mice that achieved remission and relapse , and regulatory T cells. TAMs are essential cellular components of TME and take part in diverse physiological and pathological processes , 24. TAM+ T cells, and injected CAR T cells are associated with the lack of a durable response that results in relapse.Abundant reports showed that the memory-like or exhausted characteristics of CAR T cells were related to therapeutic success or failure, respectively , 31. ShoIn conclusion, we report the involvement of TME after anti-CD19 CAR T-cell treatment in lymphoma mice. The increased immunosuppressive functions of cellular components in TME are presented in the late phase of CAR T administration, which might promote disease relapse.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Medical ethics committee of the Xuzhou Medical University, Xuzhou, Jiangsu, China.KX and KZ conceived of experiments, analyzed data, and revised the paper. KZ and CR conceived and executed experiments, analyzed data, and wrote the paper. KZ and CR contributed equally to this study. LZ, DT, and XC assisted in performing experiments. YW provided technical advice. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Rosa S-locus controlling self-incompatibility (SI); (ii) test the genetic linkage of the S-locus with other loci controlling important ornamental traits, such as the continuous-flowering (CF) characteristic; (iii) identify the S-alleles (CS) of old Chinese CF cultivars  and examine the changes in the frequency of cultivars with Sc through the history of breeding; (iv) identify wild species carrying the Sc-alleles to infer wild origins of CF cultivars. We identified a new S-RNase (C2S) of Rosa chinensis in a contig from a genome database that has not been integrated into one of the seven chromosomes yet. Genetic mapping indicated that C2S is allelic to the previously-identified S-RNase (C1S) in chromosome 3. Pollination experiments with half-compatible pairs of roses confirmed that they are the pistil-determinant of SI. The segregation analysis of an 1F-population indicated genetic linkage between the S-locus and the floral repressor gene KSN. The non-functional allele ksn is responsible for the CF characteristic. A total of five S-alleles (C1–5S) were identified from old CF cultivars. The frequency of cultivars with CS dramatically increased after the introgression of ksn from Chinese to European cultivars and remains high (80%) in modern cultivars, suggesting that S-genotyping is helpful for effective breeding. Wild individuals carrying CS were found in Rosa multiflora (C1S), Rosa chinensis var. spontanea (C3S), and Rosa gigantea , supporting the hypothesis of hybrid origins of CF cultivars and providing a new evidence for the involvement of Rosa multiflora.This study aims to: (i) identify the  The rose is one of the globally most popular ornamental plants, with a long history of breeding and cultivation. It is important not only economically, but also culturally. More than 30 000 rose cultivars have been developed  mainly bS-locus controlling self-incompatibility (SI) in roses is essential for improving breeding at the diploid level, it has not been fully elucidated. The gametophytic SI is controlled by a single S-locus with multiple alleles, and when one of the two S-alleles of the pistil matches those of pollen, the pollen is recognized as self and is rejected [S gene of Rosaceae encodes an extracellular ribonuclease called S-RNase, which acts as a cytotoxin in self-pollen tubes. The pollen S gene is involved in detoxifying non-self-S-RNases and encodes an F-box gene [Petunia [F-box genes are involved in pollen specificity, and each targets a subset of non-self-S-RNases for detoxification. The SI of Rosaceae may adopt this “non-self-recognition by a multiple factors” system [F-box gene system in Prunus [R. chinensis “Old Blush”, two different regions have been proposed as the S-locus [et al. [S-locus because its S-RNase  has a stronger similarity to the Prunus S-RNase than the S-RNase36 identified by Hibrand-Saint Oyant et al. [et al. [S-locus-like region in the genome of Rosa rugosa, whose the S-RNase is orthologous to the C1 S-RNaseS of Old Blush. Du et al. [S-RNase controlling SI in Fragaria, which is also more similar to the C1 S-RNaseS rather than S-RNase36. These results indicate that the C1 S-RNaseS is the true S gene controlling SI in Old Blush. However, validation of the Rosa S-locus by pollination experiments has been conducted with only a few individuals [et al. [C1 S-RNaseS of Old Blush from the genome database of Raymond et al. [et al. [S-RNase36 identified by Hibrand-Saint Oyant et al. [C1 S-RNaseS, which has two introns. As the two genome databases of Old Blush represent different haplotypes of chromosome 3 [S-RNase in the genome database of Hibrand-Saint Oyant et al. [S-RNases in Old Blush and to confirm the Rosa S-locus with strong evidence from a number of pollination experiments.Although identification of the rejected . The pisbox gene . The “co[Petunia , where m” system , except n Prunus . In the  S-locus , 15, bot [et al.  identifiu et al.  identifimosome 3 , there sS-locus with other loci controlling important ornamental traits. In the chromosome 3 where the Rosa S-locus is assumed to be located, there are other important loci controlling valuable traits for ornamental plants, such as CF [S-locus. The genetic linkage of the S-locus and other loci may have important consequences for rose breeding. For example, the CF characteristic is controlled by a single recessive locus on the chromosome 3, and the underlying gene is the non-functional allele (ksn) of RoKSN [ksn will be targets in the cross breeding of CF roses. But, if the ksn alleles of parental roses are strongly linked with the same S-allele, breeding of CF roses with the ksn-homozygote will be hampered by SI, where the pistil rejects the pollen with the same S-allele linked to ksn.The second objective of this study is to (ii) test the genetic linkages of the ch as CF , double ch as CF , thornlech as CF , and resch as CF . These pof RoKSN , 10, a fof RoKSN . Roses hS-allele (CS) of old Chinese CF cultivars and to clarify the changes in the frequency of rose cultivars with CS during the history of rose breeding. The ksn allele conferring the CF characteristic in modern rose cultivars originated from old Chinese cultivars around 200 years ago [ksn from China to Europe, the frequency of rose cultivars carrying the Sc should increase but may progressively decrease during the 200 years’ history of rose breeding due to the hybridizations with European cultivars. However, if Sc is genetically linked with ksn, the strong artificial selection on ksn [ksn-associated increase in the frequency of Sc during the history of rose breeding.The third objective of this study is to (iii) determine the ears ago , 24. Theears ago . After tCS as markers. The introduction of Chinese CF cultivars is one of the most revolutionary events throughout the history of rose breeding [S-locus [S-allele specific marker is useful to pinpoint the candidate wild ancestors of old cultivars.The fourth objective of this study is to (iv) identify candidate genotypes for wild ancestors of old Chinese CF cultivars by using breeding , 25, andbreeding . Howeverbreeding –32. The breeding , 34, and[S-locus , we expeS-RNase genes in the genome databases of Old Blush [R. multiflora [R. rugosa [R. multiflora, and R. rugosa  high expression levels in the pistil  si S-RNase , and  in the S-locus of R. rugosa [S-RNase gene agrees well with the assumption that this gene encodes the true S-RNase controlling SI in roses .The 3D c3D_SC1”  is the s  of the genome database of Hibrand-Saint Oyant et al. [S-RNase-like genes from the genomedatabases and pistil transcriptomes of R. multiflora and R. rugosa  expressed one 3D gene and one 0A gene, while the other three plants  expressed no 0A genes but two 3D genes , “The Fairy” (TF), has two 0A genes, one of which is identical to the C2Rc0A_S  of Old Blush and the other called 21S . The RW has an 1wS / xS genotype of the 3D S-RNase . The 0A and 3D S-RNase genes are mapped to homologous positions of chromosome 3 of the female (TF3) and male (RW3) genetic linkage maps, respectively (S gene from map cM of the integrated map (IT3) indicates that the S gene is located on 41.5 Mbp of the chromosome 3 (RC3) in the genome database of Hibrand-Saint Oyant et al. [et al. [C1 S-RNaseS is located at 5.5 Mbp of the Chr3 close to the estimated position  is in an orthologous position to the C1 S-RNaseS position in Chr3 (5.5 Mbp) and is located in a gap of synteny between the two genome databases (The two haploid genome databases of oriented . The estatabases .F-box genes flanking the S-RNase. This prediction was confirmed by the genome database search and transcriptomes of the stamens of Old Blush (F-box genes and one S-RNase (C1S). Homologous F-box genes were identified in 41.3–41.9 Mbp region in RC3, and three other F-box genes were also identified in the contig RC0 containing the C2 S-RNaseS  and R. rugosa  genome databases, and they were expressed in the stamens but not in the pistils  show close sequence identities with the end regions of the contig RC0. Block-a is 11 220 bp with a 98.8% identity between the RC3 and RC0. Block-b is 11 382 bp with a 97.5% identity. The C2 S-RNaseS in the contig RC0 appears to be integrated into the poly-N region of RC3. Furthermore, SLF7 is present in the contig RC0, and its orthologous genes were identified in the S-locus regions of R. rugosa and R. multiflora .pulation  and founS-RNase genes as the pistil-determinant of SI, pollination experiments on the pairs of diploid roses that share the same S-alleles were performed  were selected and pollinated by pollen collected from the plants with C1S / yS (where yS is another anonymous genotype). After seed maturation, the S-genotypes of the seeds were determined, and the genotypic frequency of pollen fertilizing the ovule were calculated. A total 111 seeds were analyzed, and results show that no C1S pollen fertilized the ovules . These pollination tests with half-compatible pairs were performed for R. multiflora plants sharing C1 S-RNaseS-like genes, i.e. Rm3D genes, 7S, 9S, 11S, or 13 S-RNaseS  with the pollen of Old Blush (C1S / C2S) supported the prediction that the C2S pollen of Old Blush was rejected by the pistil of TF by analyzing 60 seeds  was tested. The KSN gene (controlling CF) has a 13.5Mbp distance from the S-locus, and the AP2-like gene (controlling DF) has a 9.0 Mbp distance from the S-locus  introduced into Europe in the 18th century and examine their fate during the past 200 years’ history of rose breeding, we first analysed the S-genotypes of old Chinese CF cultivars  as Old Blush. The other CF cultivars, such as Slater’s Crimson China, R. chinensis “Mutabilis”, and R. chinensis, have either C1S or C2S, indicating that they have unidentified S-alleles. These unidentified CS-alleles were amplified with RT-PCR using mRNA prepared from their pistils with degenerate primers for S-RNase in roses and sequenced  and full (C5S) S-RNase cDNA sequences were obtained and analysed via BLASTX searches against a local protein database of the Rosa S-RNase and S-RNase-like proteins, shown in SC3–5 sequences are closest to the 0A S-RNase proteins , and Sanguinea = C2S / C3S , copiaksn  [nullksn  [nullksn allele (copiaksn / nullksn), whereas Slater’s Crimson China and R. chinensis are homozygous for one non-functional allele (copiaksn / copiaksn), and Sanguinea is homozygous for the other non-functional allele (nullksn / nullksn). Other old Chinese cultivars with once-flowering (OF) behaviour all have the wild KSN allele (WKSN). The ap2 allele  of AP2-like [The genotyping of the enotypes . Three K allele) , and ksn allele)  were disl allele . Old BluAP2-like , 11 exisksn and ap2 and their associated CS  in roses bred from 1850–1900, slightly decreasing to 83% in roses bred from 1900–1940, but not changing much until recently (ksn in the period II (86%) is not significantly different from that in the period V (100%) . The frequency of rose cultivars with Sc in the period II (95%) is not significantly different from that in the period V (82%) .The frequency of roses carrying 980–2020 . On the recently . Due to C1S and C2S are major CS alleles, and the frequency of roses with the C3S increased during the 19th century (C4S and C5S were found with low frequency (<10%) in modern cultivars.The  century . In contap2 showed the same trend of frequency changes with breeding periods as ksn . In the RNA-seq analysis of pistil-expressed genes, it was already found that three R. multiflora plants  have C1 S-RNaseS (C1= Rm3D_S in C1S (= C1Rc3D_S). The cDNA sequence of C1Rm3D_S is also 100% identical to C1Rc3D_SBy screening a total of 95 plants from 25 wild  species  with SC-entified . Positivbrunonii . The seqC2S-specific primers (R. gigantea (SRR6175515) has 100% identical sequences to C2 S-RNaseS of Old Blush  the low or undetectable expressions of 3C gene in the pistil  in this study. Vieira et al. [S-RNase based on the results of no fruit set of a few individuals pollinated by other individuals with the same S-genotypes. This cannot exclude the possibility that inbreeding depression results in no fruit set. We provide strong evidence that the 3D S-RNase is the pistil determinant of the SI in roses by genotyping more than 400 seeds produced by a number of half-compatible pairs of roses (The  S-locus . This ree pistil  and (ii)e pistil . Vieira   in the contig RC0 of the Old Blush genome database  cannot be created if ksn remains to link with a same S-allele. The 20% of recombination frequency  in modern cultivars, although it tended to decrease from 95% of cultivars from1850–1900  in modern cultivars (C4S (R. chinensis) and C5S (Mutabilis) are still useful breeding materials to introduce these rare S-alleles into CF cultivars.As we expected, 850–1900 . Due to ultivars , originaAs the SI can break down with polyploidization , breedinCS-alleles of old Chinese cultivars by screening wild roses with the CS-specific primers (R. chinensis var. spontanea and R. gigantea in the section Chinenses (Indicae) and the introgression from R. multiflora in the section Synstylae , Prunus dulcis (AAL35960.2), Prunus avium , and Prunus persica (BAF42768.1) as queries, the Old Blush genome databases [S-RNase. All genomic regions with significant hits , including the regions without any annotations, were listed and manually annotated to infer the coding regions. The other genome databases of Rosa multiflora [R. rugosa [S-RNase genes of Old Blush as queries to identify their orthologous genes. For the phylogenetic reconstruction of S-RNase-like genes in roses, deduced amino acid sequences were aligned using MUSCLE and then converted back to DNA sequences. The maximum-likelihood phylogenetic tree was constructed from the nucleotide protein-coding sequence alignment by FastTree [By using lcis AAL3960.2, Prstica AAA9841.1, P. rugosa , 39 wereFastTree , 38 usinS-RNase in the pistil and to isolate new S-RNase alleles, RNA sequencing (RNA-seq) was used. Flower buds one or a few days before anthesis were collected from three individuals of Old Blush, eight individuals of R. multiflora, and one individual of R. rugosa. Pistils and stamens were collected from the buds and immediately frozen with liquid nitrogen. Total RNAs were extracted using a commercial kit according to the protocol described in Dubois et al. [R. multiflora and R. rugosa plants without any specific reference genome databases, the RNA-seq reads were assembled first, followed by the construction of a local database of mRNA sequences, and the database was blasted using the Old Blush genes as queries to identify orthologous genes. The assembly of RNA-seq data was conducted by using the velvet and tadpole algorithm, with default parameters in Geneious Prime 2020.To confirm the expression of candidate s et al. , and RNAS-RNase in the contig that is not assigned to seven chromosomes was performed by using an 1F diploid mapping population (FW) [S-RNase were added to the previous map to estimate the genomic position of the S-gene (Genetic mapping of a newly-identified ion (FW) , 47, 48.e S-gene .S-RNase in the Old Blush, R. multiflora, and R. rugosa genomes, and the F-box genes were identified by a Blast search of the ORFs.ORFs longer than 1kbp were extracted from the 1Mbp genomic regions surrounding the S-allele  were used for pollination experiments to validate the candidate S-RNase gene. Old Blush (C1S / C2S), The Fairy (C2S / 21S), R. chinensis “Single white-eye” (C1S / 12S), and 11 wild individuals of R. multiflora were selected based on their S-genotypes. Before anthesis, flower buds were bagged to prevent open pollination. Petals and anthers were removed at the balloon stage, and outcross pollen grains were put on the exposed stigma. Pollinations were carried out from April to May in 2018 and 2020, and matured fruits were collected from September to October of the same years. The fruits were opened in the laboratory, the achenes (seeds) were collected, and the S-genotypes of the seeds were determined by PCR  and DF (AP2-like). The genotyping of the S-locus, KSN, and AP2-like was performed by PCR (Two ions (FW  and 94 /ions (FW ) were usd by PCR , and recS-alleles, mutated-alleles ksn, wild-allele WKSN, or the mutated-allele ap2 by using EmeraldAmp PCR Master Mix (TaKaRa) with thermal cycling: (1) 2 min of 95°C; (2) 30 s of 95°C; (3) 30 s of Tm°C; (4) 20–50 s of 72°C; and (5) go back to step (2) 29 times. Information on primer sequence and annealing temperature (Tm) for PCR is available from S-alleles originally linked with ksn, we used three old Chinese CF cultivars, Slater’s Crimson China, Mutabilis, and Rosa chinensis, extracted RNAs from their pistils, and performed RT-PCR with primer sets designed on conserved sites of S-RNase. Partial or full sequences of three new S-alleles were determined and named as C3S, C4S, and C5,S and specific primers were designed for each (A total of 153 rose cultivars were selected from a wide range of breeding ages and classifications, and young leaves were collected from 20 rose gardens and nurseries in Japan from 2014–2020 . DNA wasfor each .S-alleles as the old Chinese CF cultivars, the wild ancestral species were inferred. A total of 25 Rosa species were surveyed (R. chinensis, named R. chinensis var. spontanea, is naturally distributed. Field collections of wild plants in southwestern China were performed during the flowering seasons of 2018 and 2019. Forty wild plants of seven species were sampled, and S-genotyping was performed by PCR. The nucleotide sequences of the PCR products were determined to confirm the results.By screening wild rose species carrying the same surveyed , with a surveyed , where aThis study was supported by the JSPS KAKENHI No. 24688004 and 17H04616. We would like to express our appreciation for Cheng Tingting and Jin Tang in Sichuan University, and Ryosuke Sakamoto in Osaka Institute of Technology, for their helpful support during the fieldwork in China. We are also appreciative of the support from The Rose Culture Institute, Japan, and Drs. Masaki Ochiai and Kunio Yamada at Gifu University, for providing rose materials.K.K. designed the project and wrote the manuscript. K.K., Y.U., W.L., and G.W. conducted fieldwork in China. K.K., S.M., M.L., and T.D. performed pollination experiments. K.K., T.H., and S.O. analyzed candidate genes, L.H.O., F.F., M.L., and T.D. provided mapping populations. All authors are involved in final manuscript editing.Sequence data are available in Supplementary data Tables. NGS read data of RNA-seq are available from DDBJ, BioProject PRJDB12320, with accession numbers from DRR321244 to DRR321267.The authors declare no conflicts of interest associated with this manuscript.Horticulture Research  online.Web_Material_uhac155Click here for additional data file.