Chimeric antigen receptor (CAR) T cell therapy has shown promising responses in patients with refractory or relapsed aggressive B-cell malignancies that are resistant to conventional chemotherapy or stem cell transplantation. A potentially combinatorial therapeutic strategy may be the inhibition of anti-apoptotic Bcl-2 family proteins, overexpressed in most cancer cells. In this study we investigated the combination of 3rd-generation CD19.CAR-T cells and the BH3 mimetics venetoclax, a Bcl-2 inhibitor, or S63845, a Mcl-1 inhibitor, under three different treatment conditions: pre-sensitization of cancer cells with BH3 mimetics followed by CAR-T cell treatment, simultaneous combination therapy, and the administration of BH3 mimetics after CAR-T cell treatment. Our results showed that administration of CAR-T cells and BH3 mimetics had a significant effect on the quantity and quality of CD19.CAR-T cells. The administration of BH3 mimetics prior to CAR-T cell therapy exerted an enhanced cytotoxic efficacy by upregulating the CD19 expression and pro-apoptotic proteins in highly sensitive tumor cells, and thereby improving both CD19.CAR-T cell cytotoxicity and persistence. In simultaneous and post-treatment approaches, however, the quantity of CAR-T cells was adversely affected. Our findings indicate pre-sensitization of highly sensitive tumor cells with BH3 mimetics could enhance the cytotoxic efficacy of CAR-T cell treatment. Chimeric antigen receptor (CAR) modified T cells have revolutionized the field of immunotherapy for cancer . CD19.CASeveral strategies, from defining the best starting material, developing novel CAR constructs and improving the manufacturing process up to combining CAR-T cell therapy with other anti-tumor agents, are intensively studied to optimize the clinical efficacy of CAR-T cell therapy , 5. EspeA characteristic of CLL and NHL is the overexpression of anti-apoptotic Bcl-2 family proteins. Hence, the inhibition of the anti-apoptotic machinery of cancer cells as a promising therapeutic option has driven the development of compounds that antagonize the function of anti-apoptotic proteins termed BH3 mimetics, which have shown meanwhile remarkable efficacy in clinical studies . Thus, cIn our study, two BH3 mimetic compounds venetoclax, a Bcl-2 selective inhibitor, and S63845, a Mcl-1 selective inhibitor, were combined with CAR-T cells. The cytotoxic efficacy was evaluated in pre-treatment, simultaneous and post-treatment systems.U698M, Daudi, 380, Nalm6, K562, and MeC2 cell lines used in this study were purchased from German Collection of Microorganisms and Cell Cultures (DSMZ). The culture condition and cell maintaining for each cell line were according to official guidelines of the DSMZ. All cell lines were used in logarithmic growth phase and mycoplasma were excluded by polymerase chain reaction (PCR).Primary tumor cells were derived from ten newly diagnosed, untreated B-CLL patients who have signed informed consent at Heidelberg University Hospital. Buffy Coats from consenting healthy donors (HDs) were obtained from the Heidelberg Blood Bank. Peripheral blood mononuclear cells (PBMCs) were separated by a ficoll gradient, and then frozen in liquid nitrogen. Sample collection and analysis were approved by the Ethics Committee of the University of Heidelberg (S-254/2016).Dead cells were excluded by EasySep™ dead cell removal kit (STEMCELL Technologies) according to manufacture instruction. Untouched living cells were used for further experiments.Manufacture of CD19.CAR-T cells mainly consisted of retroviral vector (SFG CD19.CD28/4-1BB/ζ) production, T cell transduction and CAR-T cell expansion. The retroviral vector used in this study was produced at Baylor College of Medicine in Houston . The gen® Reagent (Thermo Fisher Scientific), followed by removal of genomic DNA. Complementary DNA (cDNA) was reverse transcribed using the PrimeScript™ RT Reagent Kit (TAKARA). q-PCR was carried out with SYBR Green master mix (TAKARA) on StepOnePlus Real-Time PCR system. The expression of Mcl-1 and Bcl-2 was determined by Mcl-1 primers and Bcl-2 primers, respectively. The gene expression was normalized to GAPDH. The list of used primers is provided in Total RNA was isolated with TrizolDead cells were removed by magnetic beads sorting. Living cells were used for further protein extraction. Total protein was isolated using radioimmunoprecipitation buffer (RIPA) (Thermo Fisher Scientific). Samples mixed with loading buffer (Sigma-Aldrich) were heated to 95°C for 5 min to denature the protein. Each sample was electrophoresed on NuPAGE 4–12% bis-tris gel (Invitrogen™) and transferred to nitrocellulose membrane (Carl Roth). Membranes were incubated at 4°C overnight with the following primary antibodies: anti-human Mcl-1 (abcm), anti-human Bcl-2 (abcm), anti-human Bcl-xl antibody (abcm), anti-human BAK antibody (abcm), and anti-human β-actin antibody (Santa Cruz). The blots were then probed with HRP-linked anti-rabbit IgG antibody  and anti-mouse IgG antibody . The reactions were developed using the ECL™ prime western blotting detection reagent . Membranes were imaged on FUSION imaging system , and band intensities were quantified with ImageJ program.To test the activity and sensitivity of Mcl-1 inhibitor S63845 and Bcl-2 inhibitor venetoclax (Medchem express), cells were seeded into 384-well plates in the presence of 10-point 1:3 serial dilutions of compounds starting from 10 μM. Luminescence was measured after 48-h co-culture using the CellTiter Glo reagent (Promega). Results were normalized to the values of cells that had been treated with 0.1% PBS (vehicle) in culture medium for 48 h.To assess the effect of inhibitors on both CD19.CAR-T cells and tumor cells, three different co-culture systems including pre-, simultaneous, and post-treatment system, were established. The number of residual tumor cells and T cells in cultures were quantified every five days using flow cytometry. Subsequently, CD19.CAR-T cells were re-challenged with the identical number of fresh tumor cells until no or only a few CAR-T cells were left in the co-culture systems.4 venetoclax pre-treated 380 cells and 3 × 103 S63845 pre-treated U698M cells were introduced into the treatment system and co-cultured with 1.5 × 104 CD19.CAR-T cells.The tumor cells were pre-treated by venetoclax or S63845 in a series of concentrations for 24 h. After dead cell removal and washing out the BH3 mimetics, 6 × 104 CD19.CAR-T cells were co-cultured with Daudi cells at an E:T ratio of 1:1 in the absence or presence of different concentrations of venetoclax or S63845. When CAR-T cells were re-challenged with fresh tumor cells, the same amount of BH3 mimetics was added as well.1.5 × 10After 24-h co-culture of CD19.CAR-T cells with Daudi cells, different concentrations of BH3 mimetics were introduced to the system. Subsequently, the same amount of BH3 mimetics was always added after 24-h re-challenge by fresh tumor cells.Marker expression was evaluated by multicolor flow cytometry. Cells were harvested and stained with Near-IR, followed by incubation with different combinations of antibodies, as shown in Following Near-IR staining, CAR-T cells from day 7 were stained with antibody cocktail for 30 min at room temperature (RT) in the dark, then fixed using fixation buffer (R&D system) before acquisition.After 24-h stimulation by Daudi cells at an E:T ratio of 1:1 in the absence or presence of different concentrations of BH3 mimetics, CD19.CAR-T cells were harvested and stained with activation marker in addition with other surface maker antibodies for 30 min at RT in the dark.Near-IR and Annexin V FITC were used to check the cell viability. After Near-IR and surface marker staining, cells were re-suspended in Annexin V binding buffer and incubated with Annexin V for 15 min at RT in the dark. Then cells were washed and re-suspended. Acquisition was performed immediately after adding 50 μl Counting Beads (Thermo Fisher Scientific). 5,000 counting beads were acquired for each sample.Cells from 24-h co-culture with Daudi cells (E:T = 1:1) in the absence or presence of BH3 mimetics were stained with Near-IR, followed by fixation and permeabilization. Afterwards, cells were washed and stained with antibody cocktails consisting of surface marker and anti-apoptotic Bcl-2 family antibodies for 30 min at RT in the dark.Following Near-IR staining, activated CD19.CAR-T cells, which were stimulated with Daudi cells in the presence of monensin (Invitrogen) and brefeldin A (Invitrogen), were fixed and permeabilized, then finally stained for 30 min with surface marker and cytokine antibodies at RT in the dark.Cells were harvested and stained with Near-IR to exclude dead cells, followed by staining specific tumor markers in addition with other surface maker antibodies for 30 min at RT in the dark. Acquisition was performed immediately after adding 50 μl Counting Beads (Thermo Fisher Scientific). 10,000 counting beads were acquired for each sample.Following Near-IR staining, CD19.CAR-T cells were stained with exhaustion markers in addition with other surface maker antibodies for 30 min at RT in the dark.3 BH3 mimetics pretreated living tumor cells or 5 × 103 target tumor cells in the absence or presence of different concentrations of BH3 mimetics, using effector to target (E:T) ratio of 30:1. After a 3-h co-culture, supernatants were harvested, and fluorescence of Calceim AM was measured by a PerkinElmer plate reader. The maximal release and spontaneous release were determined by culturing the target cells with 2% Triton X-100 (Sigma-Aldrich) in PBSS and PBSS alone, respectively. The percentage of lysis was calculated following the formula: [experimental release - spontaneous release]/[maximal release - spontaneous release] × 100.The potency of CD19.CAR-T cells was determined by a standard Calcein AM assay. Briefly, living tumor cells were labeled with Calceim AM reagent. Then CD19.CAR-T cells were plated in triplicate with either 5 × 1050 values were calculated using nonlinear regression algorithms in IBM SPSS v22. The difference among concentrations of inhibitors was calculated using Tukey’s multiple comparisons test in GraphPad Prism 8.0 and paired t-test in IBM SPSS v22. In all tests, a p value < 0.05 was considered to be statistically significant.Statistical analysis was performed using IBM SPSS 22 for Windows and GraphPad Prism 8.0. The ICThe cytotoxic activity of venetoclax and S63845 was tested in a panel of six leukemia and lymphoma cell lines using the CellTiter Glo assay. The cell density used in this assay could ensure the cell growth rate to reach the exponential phase after 48 h of culture (50 < 0.1 µM), and five cell lines  were low sensitive (IC50 > 1 µM) (50 < 0.1 µM) to S63845, two  were moderately sensitive (0.1 µM < IC50 < 1 µM) and three  were low sensitive (IC50 > 1 µM)  and S63845 .One out of six tested cell lines (380 cells) showed a high sensitivity to venetoclax (ICp < 0.05) (+ tumor cells but negligible activity against CD19- K562 cells (in vitro.The CD19 CAR molecule could be efficiently transduced on T cells (78.9% ± 7.32)  Daudi cells could stably stimulate CD19.CAR-T cells to proliferate . Both BH3 mimetics did not reduce the number of challenging procedures , showing a disability of expansion and multi-round killing of CD19.CAR-T cells. Those adverse effects by BH3 mimetics on CAR-T cells might be explained by direct induction of apoptosis, sensitization of CAR-T cells to BH3 mimetics via increasing levels of inhibitor targeting proteins, and inhibition of T cell proliferation by Bcl-2 up-regulation . The patients/participants provided their written informed consent to participate in this study.AS, MS, LW, and MY designed the research project. MY performed the experiments. SW and WG collected and stored the samples. MY and LW acquired and analyzed the data. MS, AS, MY, LW, BN, TS, LS, M-LS, AH-K, JH, CK, and VK discussed the data and the organization of the manuscript. LW, AS, and MY wrote the manuscript. All authors critically reviewed the manuscript, MS, AS, CM-T, LZ, and PD edited the manuscript. AS, MS, and LW supervised the work. All authors contributed to the article and approved the submitted version.MS received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite, and BMS, as well as a co-founder and shareholder of TolerogenixX Ltd. AS received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt, and is a co-founder of TolerogenixX LtD. AS and LW are part- or full-time employers of TolerogenixX Ltd. LS was employed by Takeda Pharma Vertrieb GmbH & Co. KG.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling editor declared a past collaboration with one of the authors TS.


Our recent study showed high rate of HBsAg seroconversion achieved in inactive HBsAg carriers (IHCs) treated with peginterferon (PEG-IFN). To better understand the immune-mediated component to the HBsAg seroconversion, we investigated the role of B cells in this study. A total of 44 IHCs were given 48 weeks of PEG-IFN. Fifteen cases achieve HBsAg seroconversion (R group), whereas 29 failed (NR group). The proportion of total B cells and plasma B cells were measured before and during treatment. We found that the proportion of total B cells and plasma B cells was no significant between R group and NR group at baseline, but significantly higher in R group than NR group during PEG-IFN treatment, even when the exact age-, sex-, and treatment period-match was made. In conclusion, we demonstrated the increase of total B cell and plasma B cells during PEG-IFN treatment favored HBsAg seroconversion for IHC, and B cells may play a role in HBV seroconversion. Inactive HBsAg carriers (IHCs) characterized by effective control of HBV replication and sustained HBeAg seroconversion. Patients in this phase have detectable circulating HBsAg, absence of HBeAg, and presence of anti-HBe, as well as undetectable or low levels of HBV DNA. Our recent study showed high rate of HBsAg seroconversion for IHCs treated with peginterferon (PEG-IFN) . Li et aThe immune response of B cells is undoubtedly an important part of the body's protection against viral and bacterial infections by neutralizing, conditioning, antibody dependent cytotoxic effects and so on –5. HowevWe enrolled IHC patients at Beijing You’an Hospital from Jan 1 to April 30, 2018. Peginterferon α-2a was administered 135μg/week for 48 weeks. The final date of follow up was July 16, 2019. At baseline and every 12 weeks thereafter during the treatment, detection of HBsAg levels, liver function, routine blood tests, and HBV DNA was performed, and peripheral blood mononuclear cells (PBMCs) were separated and stored at -80°C. We had access to information that could identify individual participants during and after data collection.Inclusion criteria for IHC was HBsAg positive for more than 6 months, serum HBsAg levels <1000 IU/mL, HBeAg-negative/anti-HBe-positive, anti-HBc positive, HBV DNA<20IU/mL, absence of previous antiviral therapy within 1 year.Exclusion criteria: human immunodeficiency virus or hepatitis C virus coinfections; history of autoimmune diseases; hepatic dysfunction; serious metabolic disease; cardiac or pulmonary or renal disease; evidence of liver carcinoma or other neoplastic diseases; contraindication for interferon.Responder group (R): achieving HBsAg seroconversion. Non-responder group (NR): failed to achieve HBsAg seroconversion. Healthy control group (HC): HBsAg negative, vaccinated against HBV, and anti-HBs level >200 IU/L.HBsAg and anti-HBs levels were detected using the HBsAg quantitative Elecsys . The lower limit of quantitative HBsAg determination was 0.05 IU/mL, and anti-HBs >10 IU/L was defined as anti-HBs positive. HBV DNA levels were determined using the fluorescence quantitative polymerase chain reaction (FQ-PCR) method with a lower detection limit of 20IU/mL (Roche).+), naïve B cells (CD19+CD27-), memory B cells (CD19+CD27+), and plasma B cells (CD19+CD27hiCD38hi). . All monoclonal antibodies were purchased from BD . B cells were divided into four subsets: total B cells (CD19CD38hi). .Statistical analyses were performed using SPSS Version 24. All continuous data were present as mean ± standard deviation (SD). Categorical data were presented as number and percentages. Fisher’s exact or chi-square tests and t-test were conducted for the categorical and continuous variables, respectively, to compare the difference between variables and HBsAg seroconversion. The significance level was set at P<0.05 for all statistical analyses.This study protocol was approved by the institutional ethics board of Beijing You’an Hospital, Capital Medical University (No. JYKLZ 2017 [24]).A total of 44 IHC patients were given 48 weeks of PEG-IFN. Fifteen cases achieve HBsAg seroconversion (R), whereas 29 failed (NR). There were no significant differences in sex, age, ALT, WBC and HBV DNA among R, NR and HC groups. The HBsAg level in R group was lower than that in NR group (p<0.001). Patients in R group experienced different periods of time to get HBsAg seroconversion. One case got it at 12 week after PEG-IFN therapy, and 7 cases, 2 cases, 5 cases obtained it at 24 week, 36 week and 48 week. The patients’ demographic and clinical data are listed in +) and plasma B cells (CD19+CD27hiCD38hi) were analyzed by flow cytometry. The proportion of total B cells was (8.9±0.7)% in 44 IHCs at baseline, significantly higher than 12 healthy persons (3.2±0.4)%, P<0.001, P = 0.005. The proportion of total B cells  and 29 unachieved (NR group). The proportion of total B cells was (7.9±0.7)% in R group, and ((8.6±1.0)% in NR group at baseline. There was no significant difference. Because patients in R group (n = 15) experienced different periods of time to get HBsAg seroconversion, the comparison time points were set as the last time point before HBsAg seroconversion was detected (Pre-seroconversion point (Pre-P)) and the first time point when seroconversion of HBsAg was detected (Post-HBsAg seroconversion point (Post-P)). Given the fact that age and gender significantly affect the distribution of NK cells, 15 patients in NR group were compared with the 15 patients in R group after matching the age, sex, and course of treatment.P = 0.001), but no difference at Post-P , P = 0.019; 4.3±1.1vs 1.4±0.2, P = 0.016), The results showed that the proportion of total B cells in R group was significantly higher than NR group at Pre-P  P = 0.040; 4.2±0.9 vs 1.4±0.2, P = 0.014), The results showed the proportion of total B cells in R group were significantly higher than NR group at Pre-P (12 weeks of PEG-IFN treatment) and Post-P (24 weeks of PEG-IFN treatment),  compared to the NR group. Considering the differences in clinical characterization and length of time to achieve HBsAg seroconversion, we further selected seven patients who achieved HBsAg seroconversion at 24 weeks, and seven matched NR cases with for comparison. The results also showed the proportion of total B and plasma cells were significantly higher in HBsAg seroconversion patients than NR group at Pre-P and at the first time point when seroconversion of HBsAg was detected (Post-P).Consistent with our study, Xu X et al comprehensively investigated the frequency, phenotype, and function of peripheral B-cell subsets from chronic hepatitis B patients in different phases. They found the frequency of HBsAg-specific B cells were significantly decreased in CHB patients compared with healthy controls. More importantly, they reported IFN-α might preferentially activate B cells, and the reversal of B-cell functional impairment was associated with HBsAg seroconversion . ReconstTaken together, we found that the increases in total B cell and plasma B cells during the course of PEG-IFN treatment. The peaks of both total B cell and plasma cell levels coincided with HBsAg seroconversion, strongly suggesting the important role of B cells in HBV virus clearance. Our study highlights the therapeutic role of neutralizing anti-HBs against chronic HBV infection.S1 Data(XLSX)Click here for additional data file.


Introduction: Multiple studies have suggested an association between cytomegalovirus (CMV) infection and essential hypertension (EH). MicroRNAs (miRNAs) play a critical role in the development of EH by regulating the expression of specific target genes. However, little is known about the role of miRNAs in CMV-induced EH. In the present study, we compared the miRNA expression profiles of samples from normal and murine cytomegalovirus (MCMV)-infected C57BL/6 mice using high-throughput sequencing analysis. Methods: We collected the thoracic aorta, heart tissues, and peripheral blood from 20 normal mice and 20 MCMV-infected mice. We identified differentially expressed miRNAs in the peripheral blood samples and predicted their target genes using bioinformatics tools. We then experimentally validated them using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the target genes with double luciferase reporter gene assay. Results: We found 118 differentially expressed miRNAs, among which 9 miRNAs were identified as potential MCMV infection-induced hypertension regulators. We then validated the expression of two candidate miRNAs, mmu-miR-1929-3p and mcmv-miR-m01-4-5p, using qRT-PCR. Furthermore, the dual-luciferase reporter gene assay revealed that the 3′-untranslated region (UTR) of endothelin A receptor (Ednra) messenger RNA (mRNA) contained a binding site for mmu-miR-1929-3p. Collectively, our data suggest that MCMV infection can raise the blood pressure and reduce mmu-miR-1929-3p expression in C57BL/6 mice. Moreover, we found that mmu-miR-1929-3p targets the 3′-UTR of the Ednra mRNA. Conclusion: This novel regulatory axis could aid the development of new approaches for the clinical prevention and control of EH. Hypertension is one of the most common chronic diseases, affecting more than 1 billion people worldwide . ApproxiHCMV is a ubiquitous β herpes virus that has the largest known genome among human herpesviruses (230 kb) encoding a variety of factors that modulate the host immune response ,13. HCMV. have shown that the HCMV-encoded miRNA hcmv-miR-UL112 was up-regulated in hypertensive patients, and the blood pressure of the mice significantly increased 9‒12 days after adenoviral transfection of hcmv-miR-UL112 [www.mirbase.org) [MiRNAs are highly conserved, single-stranded, non-coding small RNAs (19‒25 nucleotides in length) that negatively regulate the expression of their target genes at the post-transcriptional level, by directly binding to the 3′-untranslated region (UTR) or 5′-UTR of their target messenger RNAs (mRNAs) . PreviouiR-UL112 . It has ase.org)  and can Our previous studies found that HCMV infection is associated with EH in patients of Kazakh and Han ethnicity in Xinjiang, and is associated with EH progression and target organ damage in patients of Han ethnicity, further supporting the correlation between HCMV and hypertension ,25. We tad libitum, and were kept at the Shihezi University School of Medicine animal facility. After adaptive feeding for 1 week, the mice were divided into the following two groups: the MCMV infection group  and the control group . All animal experiments were conducted at the Medical College of Shihezi University and performed according to the protocols approved by the Animal Experimental Ethical Inspection of First Affiliated Hospital, Shihezi University School of Medicine .All experiments in the present study were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal model was established as previously reported. A total of 40 C57BL/6 mice (4 weeks of age) were provided by the Institutional Animal Research Committee of Shihezi University School of Medicine. All mice were housed under pathogen-free conditions in individually ventilated cages, placed in a temperature-controlled environment with a 12-h day/12-h night cycle. Mice received food and water g for 20 min) and then further purified with ultracentrifugation (20000 rpm for 1 h) through a 15% sucrose cushion in a virus standard buffer . The virus pellet was resuspended in 0.5–1.0 ml of VSB, aliquoted, and stored at −80°C. Viral titers were determined with standard plaque assay in NIH 3T3 cells. Both the MCMV Smith strain and NIH 3T3 cells were donated by the Department of Neurovirology, Wuhan Institute of Virology .MCMV was maintained in NIH 3T3 cells. NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum and 1% antibiotic solution (100 U/ml of penicillin and 100 µg/ml of streptomycin). The virus was collected from the supernatant of infected NIH 3T3 (infection rate: 100%) by centrifugation  through intraperitoneal injection (30 mg/kg), after which they were fixed on the operating table. An incision was made along the midline of the neck and the neck tissues were separated to expose the common carotid artery. The vagus nerves surrounding the carotid artery were separated carefully. A silicone tube  was implanted near the artery (diameter: ∼0.5 mm) and a cannula was ligated using 6-0 silk threads to monitor the blood pressure. Then, the carotid artery was put back into position. Finally, the mice were killed by decapitation under pentobarbital anesthesia.The total RNA was isolated from thoracic aorta samples using miRNeasy Mini Kit . The total RNA isolation from peripheral blood monocytes for sequencing and validation analysis was performed using the miRcute miRNA isolation kit with the enrichment for small RNAs, following the manufacturer’s instructions . RNA quality was measured using the Agilent Bioanalyzer 2100 . Paired-end libraries were synthesized by using the QIAseq miRNA Library Kit  following the QIAseq miRNA Library Kit Guide. The products were then purified and enriched by PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer  and validated using the Agilent 2100 bioanalyzer  to confirm the insert size and calculate the library concentration. A cluster was generated by cBot with the library diluted to 10 pM and then sequenced on the Illumina Hiseq Xten .The library construction and sequencing were performed by Sinotech Genomics Co., Ltd .P-value ≤0.05 were used as criteria for the identification of differentially expressed miRNA genes. We then performed gene ontology (GO) enrichment analysis to annotate gene functions, according to three categories: biological process (BP), cellular component (CC), and molecular function (MF). Next, we performed an enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) to systematically analyze, annotate, and visualize gene functions. Potential targets of the differentially expressed miRNAs were predicted using miRanda (http://www.microrna.org/microrna/home.do), miRDB (http://www.mirdb.org/), and TargetScan (http://www.targetscan.org/). Only the genes identified by all three software were selected as target genes.After removing the adoptor sequences and the contaminated reads, the clean reads were processed for letter anlysis. Sequence files (fastq) were mapped to the reference genome (GRCm38) using Bowtie. Small RNA was classified by Unitas software. The miRNA abundance was expressed as counts of exon model per million mapped reads. Differential expression analysis for mRNA was performed using DESeq software. A minimum of two-fold difference between groups and a Ct−ΔΔ method [The integrity and purity of total RNA samples were evaluated using PCR and agarose gel electrophoresis. Total RNA samples of suitable quality were reverse transcribed into cDNA using the miRcute Plus miRNA First-Strand cDNA kit . The qPCR conditions were the following (Supplementary Table S1): 95°C for 15 min; 5 cycles at 94°C for 20 s, 65°C for 30 s, and 72°C for 34 s; 45 cycles at 94°C for 20 s and 60°C for 34 s. All primers were synthesized by Shanghai Living Creature (China) (Supplementary Table S2). Gene expression levels were calculated using the 2t method .Ednra) gene was amplified by PCR and cloned downstream the pmirGLO Luciferase  multiple cloning site by digestion with the SpeI and HindIII restriction enzymes. The resulting construct was called pEdnra-wild-type (Wt). The miR-1929-3p binding site on the Ednra mRNA was mutated and cloned in the pmirGLO Luciferase plasmid to create the pEdnra-Mutant (Mut) vector. The Wt and Mut vectors were each transfected with and without miR-1929-3p into the HEK293 cells. Additionally, a dual-luciferase reporter gene assay was performed using the dual-luciferase reporter assay system .The full-length 3′-UTR of the endothelin A receptor . Ednra sense 5′-CCTGGCAGGAAACAATGTCAAAGTGGCCAAATGAGCTGATTGTGTTAAGTGAGATGTAGTTACACC-3′ antisense 5′-TCGAGGTGTAACTACATCTCACTTAACACAATCAGCTCATTTGGCCACTTTGACATTGTTTCCTGCCAGGAGCT-3′; si-Ednra sense 5′-CGCAGAACAUCAAAGAAGATT-3′, antisense 5′-UCUUCUUUGAUGUUCUGCGTT-3′; negative control siRNA sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′.t test was used for comparing the differences between the two groups. A P-value <0.05 indicated a statistically significant difference.The SPSS 21.0 statistical software  was used for statistical analysis. Data were presented throughout the study as mean ± standard deviation. The P<0.01; To confirm the MCMV infection, we measured with quantitative reverse transcription polymerase chain reaction (qRT-PCR) the MCMV IE1 gene expression in the thoracic aorta of mice infected with the virus . The results confirmed that the infection was successful A. Then, P<0.05; We compared miRNA expression profiles in the peripheral blood of mice infected with MCMV with uninfected mice using a high-throughput sequencing approach. We found 118 significantly differentially expressed miRNAs, of which 91 were up-regulated and 27 were down-regulated . We isolated monocytes by performing Ficoll density gradient centrifugation and found that in peripheral blood monocytes of mice infected with MCMV [in vitro, we still found that MCMV infection is able to down-regulate the expression of mmu-miR-1929-3p in infected cells .qRT-PCR was used to validate the expression levels of the nine differentially expressed miRNAs in independent samples from different tissues  could be a potential target of mmu-miR-1929-3p (P<0.05), while no significant difference was observed upon transfecting the mednra-miR1929-MUT vector or an NC mimic (P>0.05) . Thus, we experimentally validated that Ednra is a target of mmu-miR-1929-3p.Using a bioinformatics approach, we found that endothelin receptor type A  B. At thein vivo the biological mechanism of viruses of the CMV genus. MCMV has been extensively used in cardiovascular studies [In the present study, we observed that MCMV infection caused the differential expression of various miRNA, originating both from the host and the virus, which could play a role in the pathogenesis of hypertension. In particular, we found that the MCMV-encoded mcmv-miR-m01-4-5p miRNA increased, while the host-encoded mmu-miR-1929-3p miRNA decreased. However, as the target genes regulated by mcmv-miR-m01-4-5p did not rank among the top ten hypertension-related genes in the bioinformatic analysis, we focused on mmu-miR-1929-3p for the subsequent analysis. CMV infection is usually latent, although it undergoes periodic reactivation events, by bypassing the host immune response . Althoug studies . Much of studies . A previ studies . Similar studies . In thisin vitro studies, CMV-encoded miRNAs play an important regulatory role in viral replication, but the association between host-encoded miRNAs, HCMV-encoded miRNAs, and hypertension remain unclear. Here, we identified 118 differentially expressed miRNAs. Among them, the expression of two miRNA encoded by MCMV was up-regulated. These results suggest that both MCMV- and host-encoded miRNA may be involved in the insurgence of MCMV-mediated hypertension.It has been widely recognized that miRNAs play an important role in regulating cardiovascular functions ,34. Pati. demonstrated that miR-181a-5p binds to the 3′-UTR of the renin mRNA to reduce its gene expression. In hypertensive individuals, high expression of renin mRNA is accompanied by a decreased expression of miR-181a in the kidney [cis-degradation of cytotoxic t-antigen mRNA, which encodes for a major viral regulator, in a process thought to help to evade the host immune response [trans-activator gene. The removal of miR-ul112-3p target sites in the IE72 mRNA increased IE72 expression [in vivo [Ednra, and experimentally validated the finding with the dual-luciferase assay.MiRNAs are known to be effective regulators of gene expression, by binding to target mRNA, blocking their translation by degrading the mRNA or inhibiting the translation process . ACE inhe kidney . In ratse kidney . By inhie kidney . In thisresponse . The HCMpression . One of [in vivo . HoweverEDN1, EDN2, and EDN3) and two receptors (encoded by Ednra and Ednrb), belonging to the seven-fold transmembrane domain receptor family of G protein-conjugated receptors [Ednra gene spans over 40 kb and contains 8 exons and 7 introns [Ednra increases vasoconstriction tension and may be associated with an increase in endothelin-1 (ET-1) [Ednra mRNA and thus might be involved in MCMV-induced hypertension. Future studies are required to identify potential biomarkers of CMV-mediated hypertension.In mammals, the endothelin signaling system (ligand plus receptor) consists of three ligands (encoded by eceptors . The Edn introns . In hype1 (ET-1) . ET-1 isA previous study identified different virus- and host-encoded miRNAs whose target genes are related to the inflammatory response, vasoconstriction, and vascular remodeling  . The disin vivo and elucidated at the cellular and molecular level.The present study presents some limitations. In particular, the effect of mmu-miR-1929-3p in the occurrence and development of hypertension and target organ damage still need to be further validated Ednra may be the target gene of mmu-miR-1929-3p. Thus, mmu-miR-1929-3p can be promising drug targets and/or potential biomarkers for the prevention and management of hypertension.In conclusion, we identified mmu-miR-1929-3p as a novel miRNA down-regulated in mice infected with MCMV. We also experimentally validated that Click here for additional data file.


Hsp104 is a hexameric AAA+ yeast disaggregase capable of solubilizing disordered aggregates and amyloid. Hsp104 couples ATP hydrolysis to polypeptide translocation through its central channel. Substrate binding by Hsp104 is mediated primarily by two conserved tyrosine residues in nucleotide binding domain (NBD) 1 and NBD2. Recent structural studies have revealed that an additional tyrosine residue (Y650) located in NBD2 appears to contact substrate and may play an important role in Hsp104 function. Here, we functionally analyze the properties of this proposed Hsp104 –substrate interaction. We find that Y650 is not essential for Hsp104 to confer thermotolerance. Supporting these findings, in a potentiated Hsp104 variant background, the Y650A mutation does not abolish potentiation. However, modulation of this site does have subtle effects on the activity of this potentiated Hsp104 variant. We therefore suggest that while Y650 is not essential for Hsp104 function, its modulation may be useful for fine-tuning Hsp104 properties. Protein disaggregases engage and dissolve proteins that aggregate and form amyloid. Protein aggregation is implicated in a range of diseases, and so it is important to understand the structure and mechanism of disaggregases. Hsp104, a member of the Hsp100 family, is a hexameric AAA+ protein disaggregase from yeast that is highly conserved in bacteria and eukaryotes, though it is absent in metazoa. Hsp104 assembles into a ring structure with a central channel through which substrate is threaded. ATP hydProtein misfolding underpins numerous disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and diabetes, 11. BecC. elegans model of PD[To enhance Hsp104 activity, efforts have been made to tune Hsp104 to boost its activity and modulate its substrate-specificity–23. Thesdel of PD. A majordel of PD, 19, 20.H641A showed a loss of ~50% activity relative to ClpBWT[In order to apply rational protein design principles to further engineer Hsp104, it is important to comprehensively understand its structure and mechanism. Recently, a series of near-atomic level resolution cryo-electron microscopy (cryo-EM) structures of Hsp104 were reported. These sto ClpBWT. Howeverto ClpBWT. It is tHere, to investigate the functional role of this putative substrate binding region, we employed a series of yeast-based assays. Using a combination of thermotolerance, temperature sensitivity, and disease-substrate toxicity assays, we find that removal of the tyrosine 650 side chain from this pore loop does not ablate Hsp104 function as occurs when the canonical pore loop residues are mutated. However, the Y650A mutation does subtly modulate the function of a potentiated Hsp104 variant. Our results suggest that modulation of this site may prove useful for tuning Hsp104 activity.Fig 1)[Fig 1)[Substrate binding by Hsp104 is mediated primarily by Y257 and Y662, located in nucleotide binding domain 1 (NBD1) and NBD2, respectively. These 12 residues project into the central channel of Hsp104 to bind substrate and mediate its translocation via a ratchet-like mechanism , 28. An Y650A would display impaired thermotolerance. We constructed Hsp104Y650A, Hsp104Y662A, and Hsp104Y257A-Y662A in W303Δhsp104 yeast with Hsp104 expression under the control of the heat-shock element (HSE), which is present in the promoter region of Hsp104. This promoter allows low basal expression of Hsp104 that is significantly enhanced following stress. Following a brief pre-treatment at 37°C to induce the heat shock response and enhance Hsp104 expression, we exposed the cells to a severe heat shock at 50°C for 0 to 30 minutes. Following outgrowth, we noted that the strain expressing Hsp104Y650A displayed thermotolerance nearly identical to that of Hsp104WT. Strains expressing Hsp104Y662A and Hsp104Y257A-Y662A did not confer thermotolerance, and displayed growth similar to the vector strain, which does not express Hsp104 . We confirmed expression of each variant via immunoblotting, and observed that the expression pattern was consistent with Hsp104 expression being driven by the HSE promoter . Our findings demonstrate that residue Y650 plays little role in conferring thermotolerance, suggesting that if the Y650 residues contact substrate, this interaction is not essential for maintaining substrate remodeling activity.Upon exposure to environmental stress, Hsp104 expression is upregulated to broadly counter protein aggregation and promote cell survival, thereby conferring thermotolerance, 7. FollY257F-A503V-Y662F, in which the Y257F and Y662F pore loop mutations are introduced into the potentiated Hsp104A503V background, still rescues the toxicity of TDP-43, FUS, and α-syn[A503V. Hsp104Y257F-A503V-Y662F confers thermotolerance at levels similar to Hsp104WT, while Hsp104A503V is impaired in thermotolerance. Additionally, Hsp104Y257F-A503V-Y662F confers diminished temperature sensitivity relative to Hsp104A503V. Enhanced thermotolerance and diminished temperature sensitivity in yeast likely reflects decreased off-target effects and suggests that modulation of the Hsp104 pore loops is a viable way to tune disaggregase properties[Potentiated Hsp104 variants confer a therapeutic gain of function and robustly rescue the toxicity of TDP-43 and FUS (implicated in ALS) as well as α-synuclein (implicated in PD), 29. It and α-syn. Howeverroperties.A503V background: Hsp104A503V-Y650A, Hsp104A503V-Y662A, and Hsp104Y257A-A503V-Y662A. We chose to introduce alanine rather than phenylalanine mutations, anticipating that this strategy would more robustly demonstrate if these residues played a role in substrate binding. While a Y650F mutation would likely maintain a rescue regardless of the role of Y650 in substrate binding, ablating Y650 via mutation to alanine should impair the capacity of Hsp104A503V to confer a rescue if Y650 binds substrate. We employed the 416GAL vector to drive galactose-inducible expression of the genes, and we co-expressed these variants with TDP-43, FUS, and α-synuclein. Potentiated Hsp104A503V robustly rescued toxicity of all three substrates, while Hsp104A503V-Y662A and Hsp104Y257A-A503V-Y662A did not rescue the toxicity of any of these substrates. Hsp104A503V-Y650A still strongly rescued toxicity, indicating that the variant retained substrate binding, and supporting our results in the thermotolerance assays. However, we did note a modest decrease in rescue of TDP-43 toxicity when compared to Hsp104A503V. Here the rescue decreases from a score of 3 for Hsp104A503V to a score of 2 for Hsp104A503V-Y650A . However, Hsp104A503V-Y650A shows only a subtle decrease in rescue of FUS and α-synuclein toxicity as compared to Hsp104A503V, and the average score for this variant is equal to that of Hsp104A503V against FUS and α-synuclein . We confirmed consistent expression of each of the Hsp104 variants and disease substrates using immunoblotting . Our findings suggest that while the Y650 residue is not crucial for mediating substrate remodeling, modulation at this position could be a useful way to tune the properties of potentiated Hsp104 variants.We therefore designed experiments to both corroborate our findings that Y650 does not drive substrate remodeling, and also test if modulation of Y650 might be a way to tune Hsp104 substrate-specificity. We constructed a series of variants in the potentiated Hsp104A503V background subtly diminishes the toxicity rescue conferred by Hsp104A503V, we decided to perform a final functional test to assess the temperature sensitivity of these variants. While Hsp104A503V confers thermotolerance nearly similar to Hsp104WT  [A503V also confers a temperature sensitive phenotype at 37°C, displaying impaired growth when Hsp104A503V is expressed during a prolonged heat stress treatment. This temperature sensitive phenotype is diminished for Hsp104Y257F-A503V-Y662F [A503V, as do the verified substrate binding residues Y257 and Y662. However, we found that Hsp104A503V-Y650A displayed a similar temperature sensitive phenotype to Hsp104A503V, while Hsp104A503V-Y662A and Hsp104Y257A-A503V-Y662A displayed diminished temperature sensitivity . These results support the idea that Y650 does not play a crucial role in substrate remodeling in contrast to the essential roles played by residues Y257 and Y662.Because the introduction of Y650A into the potentiated Hsp104 , 30, Hsp3V-Y662F , 18. We Y662A and Hsp104V663G are defective in thermotolerance[A503V background to generate Hsp104A503V-E648A and Hsp104A503V-K649A. We tested these variants for suppression of TDP-43, FUS, and α-Syn toxicity and found that, like with Hsp104A503V-Y650A, ablating the side chains of E648 and K649 only subtly modulates the potentiation of Hsp104A503V . Our findings suggest that while modulation of this pore loop region might be a useful way to tune the properties of Hsp104A503V, this pore loop region does not play a major role in Hsp104 –substrate remodeling.It has been demonstrated that residues neighboring key substrate-binding residues Y257 and Y662 play an important role in Hsp104 substrate binding. For instolerance. To asseY650A is fully functional in thermotolerance, indicating that Hsp104 can still remodel substrate without the Y650 side chain. To investigate the role of this site further, we tested Hsp104A503V-Y650A in toxicity suppression assays. We find that the Y650A mutation does not abolish activity, supporting our findings in the thermotolerance assays, but that it does subtly decrease the rescue of TDP-43, FUS, and α-syn toxicity by potentiated Hsp104A503V. Our findings are in contrast to recent work with the bacterial homolog of Hsp104, ClpB. ClpBH631A, in which this third pore loop is ablated, shows impaired activity in a luciferase reactivation assay[Amyloid and aggregate dissolution by Hsp104 is driven by substrate binding and translocation through its central pore. Binding is mediated via residues Y257 and Y662 leading to translocation via a ratchet-like mechanism[ion assay. These dhsp104 [All yeast were WT W303aΔ, ade2-1). Yeast w, ade2-1). pRS416G600nm = 1.5, serially diluted, and spotted in duplicate onto synthetic dropout media containing glucose or galactose. Plates were analyzed after growth for 2–3 days at 30ºC. Each experiment was repeated with three independent transformations.Yeast were transformed according to standard protocols using polyethylene glycol and lithium acetate. Hsp104 hsp104 yeast were transformed with the indicated pHSE-Hsp104 plasmid. Yeast were grown to saturation overnight at 30°C in glucose dropout media. Cultures were normalized to A600nm = 0.3 and grown in glucose dropout media for an additional 4h. Cultures were then normalized to A600nm = 0.6 and incubated at 37°C for 30 min. Cultures were then heat shocked at 50ºC for 0-30min and then cooled for 2min on ice. Cultures were serially diluted and spotted on glucose dropout media, and the plates were incubated at 30ºC for 2–3 days. Experiments were repeated with three independent transformations.W303Δhsp104 yeast were transformed with the indicated pRS416GAL-Hsp104 plasmid. Yeast were diluted and grown in synthetic raffinose medium overnight. Cultures were grown in synthetic raffinose medium to A600nm = 2.0 and spotted onto SD-Ura or SGal-Ura media and incubated at 30ºC or 37°C. Plates were analyzed after 48-72h of growth. Experiments were repeated with three independent transformations.W303Δ600nm = 0.6, 3ml cells were harvested, treated in 0.1M NaOH for 5 min at room temperature, and cell pellets were then resuspended into 1x SDS sample buffer and boiled for 4min. Lysates were cleared by centrifugation at 14,000 rpm for 2min and then separated by SDS-PAGE , and transferred to a PVDF membrane. Membranes were blocked in Odyssey Blocking Buffer (LI-COR). Primary antibody incubations were performed at 4ºC overnight. Antibodies used: anti-GFP monoclonal (Roche Applied Science), anti-TDP-43 polyclonal (Proteintech), anti-FUS polyclonal (Bethyl Laboratories), anti-Hsp104 polyclonal (Enzo Life Sciences), and anti-PGK monoclonal (Invitrogen). Membranes were imaged using a LI-COR Odyssey FC Imaging system.Yeast were grown and induced in galactose containing medium for 5h from overnight cultures supplemented with raffinose. For plasmids under the control of the HSE promoter, cells were grown as for the thermotolerance assay and harvested after the incubation at 37ºC. Cultures were normalized to AS1 Supporting Information(DOCX)Click here for additional data file.


The sensitivities of the multiplex influenza A/B/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A/B/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A/B/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses.Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher/fluorescence oligonucleotides connected by a 5′ backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A/H1 and A/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 10 For theTo confirm the possibility of cross-reactivity with other infectious viruses, 41 respiratory virus samples, including 4 RSV A, 4 RSV B, 4 adenovirus, 4 PIV(1–4), 9 coronavirus , 4 HBoV, 4 HEV, 4 HRV, and 4 MPV samples, were tested using the multiplex influenza A/B/IC RT-LAMP assay, WHO RT-PCR primer set, and RealStar® Influenza RT-PCR Kit 2.0  33, 34], 3433, 3Since many respiratory viruses, including influenza viruses, can cause similar symptoms, it is difficult for clinicians to distinguish one virus from another . Given tOur results showed that the multiplex influenza A/B/IC RT-LAMP assay had 100% analytical specificity for the identification of influenza A/H1, A/H3, and B viruses, and there was no cross-reaction with other genetically or clinically related control viruses tested in this study. The multiplex influenza A/B/IC RT-LAMP assay for influenza A clinical samples (n = 93) had a sensitivity and specificity of 94.62% and 100%, respectively. However, the multiplex influenza A/B/IC RT-LAMP assay showed a 90% sensitivity for influenza A/H1 because out of the 11 specimens that were positive for influenza A/H1, only 10 were determined by the assay to be positive. Further testing with additional clinical specimens is needed to evaluate the clinical performance characteristics of the multiplex influenza A/B/IC RT-LAMP assay to address the issue of the small sample size used (n = 10). In addition, the internal control signal showed in a very low sensitivity for influenza A clinical samples but not influenza B samples and negative clinical samples. This result might be that LAMP amplification reagents were consumed for the amplification of influenza A or influenza A amplification products interrupt the amplification of the internal control. Interestingly, the multiplex influenza A/B/IC RT-LAMP assay had a lower detection sensitivity for diluted clinical samples than the RealStar® Influenza RT-PCR Kit 2.0 but higher sensitivity for original clinical samples. These results suggest that the multiplex influenza A/B/IC RT-LAMP assay had a higher sensitivity for various influenza genetic sequences while the RealStar® Influenza RT-PCR Kit 2.0 had a higher sensitivity for specific influenza genetic sequences.RT-LAMP analysis is one of the most promising diagnostic tools for use in the field since it does not require sophisticated and expensive equipment or skilled personnel . The mulIn this study, we developed a multiplex real-time RT-LAMP assay that can diagnose influenza A and influenza B with one step. The multiplex influenza A/B/IC RT-LAMP assay using the probe-quencher that compensates for the disadvantages of LAMP, such as false positive diagnoses, shows similar sensitivity and specificity to the WHO RT-PCR primer set. Since LAMP takes less time (within 60 min) than conventional RT-PCR, the multiplex influenza A/B RT-LAMP assay can be used as an efficient method in on-site molecular diagnostic kits.S1 FigInfluenza A/B virus clinical samples, non-infection human serum RNA and distilled water (DW) were tested by RT-LAMP assays with monoplex influenza A RT-LAMP primer set (A), monoplex influenza B RT-LAMP primer set (B) and monoplex IC (actin beta) RT-LAMP primer set (C). Lane M: DNA ladder marker, Lane 1: Influenza A/H1, Lane 2: Influenza A/H1N1, Lane 3: Influenza A/H3N2, Lane 4: Influenza B, Lane 5: Non-infection human serum RNA and Lane 6: DW (negative control).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


A stabilized loop turn of VP2, once in contact with SCARB2, can enhance interaction with the host SCARB2 receptor for viral entry. Our findings here could facilitate early detection of severe cases infected with EV-A71 in clinical medicine. In addition, it opens up the opportunity of functional studies via infectious cDNA cloning, site-directed mutagenesis, and animal models in the future.Infection with the re-emerging enterovirus 71 (EV-A71) is associated with a wide range of disease severity, including herpangina, encephalitis, and cardiopulmonary failure. At present, there is no FDA-approved therapeutics for EV-A71. Early diagnosis for the high-risk children is the key to successful patient care. We examined viral genome sequences at the 5′ untranslated region (UTR) and the capsid protein VP1 from 36 mild and 27 severe cases. We identified five EV-A71 mutations associated with severe diseases, including (1) the 5′ UTR mutations C580U, A707G, C709U; (2) a VP1 alanine-to-threonine mutation at position 280 (280T), and (3) a VP1 glutamic acid-to-(non-glutamic acid) at position 145 [145(non-E)]. SCARB2 is a known entry receptor for EV-A71. Based on a recent cryoEM structure of the EV-A71-SCARB2 binding complex, VP1-280T is near the binding interface between the VP1-VP2 complex and its entry receptor SCARB2. A  Reemergence of non-polio enterovirus is a new threat to the children . Enterovin vivo. As summarized in The broad spectrum of disease manifestations is related in part to the differences in host immunity , 2019. IIn contrast to both mouse and monkey models, VP1-145E does not appear to be associated with severe sequelae using human samples. Instead, amino acids 145Q had been observed in 2 out of 9 severe diseases . In addiEV-A71 clinical isolates were kindly provided by Section of Clinical Virology and Molecular Diagnosis, Department of Laboratory Medicine, Changhua Christian Hospital, Taiwan. Biosafety Committee approval number BSF 005 20080030 from Academia Sinica, Taiwan.Clinical presentations of mild cases include herpangina or HFMD only. In addition to herpangina and HFMD, symptoms of severe cases include myoclonic jerks, meningitis, encephalitis, acute flaccid paralysis, pulmonary edema, cardiopulmonary failure, and death.Human rhabdomyosarcoma (RD) cells (ATCC CCL-136) were cultured in DMEM (Dulbecco’s modified Eagle medium) with 10% fetal bovine serum  and 1% penicillin-streptomycin. Viral strains of EV-A71 were isolated from throat swabs of patients by the Section of Clinical Virology and Molecular Diagnosis, Department of Laboratory Medicine, Changhua Christian Hospital, Taiwan. Virus preparation was as described previously .Procedures for RT-PCR are as described elsewhere . The seq6/well) and cultured with Dulbecco’s Modified Eagle medium  with 10% fetal bovine serum  and 1% penicillin-streptomycin (Gibco) at 37°C overnight. Culture medium was removed and cells were washed once with phosphate buffer saline (PBS) before incubation with EV-A71 virus VP1-280A or VP1-280T (M.O.I = 10) at 4°C for 1 h. Unbound virus was removed by washing with PBS three times. Total RNAs of cells were extracted with a WelPrep cell/tissue RNA kit (Wel-GENE), and were used for reverse transcription by a High-Capacity cDNA reverse transcription kit (Applied Biosystems). The synthetic cDNA was subjected to real-time quantitative PCR (qPCR) analysis by an ABI 7500 system with a Power SYBR green PCR master kit (both from Applied Biosystems). Specific primers for VP1 were CTAGAGGGTACCACCAATCC (forward) and AACCTGGCCAGTAGGAGT (reverse). The primer sequences of GAPDH  were used as the internal control: ACCCAGAAGACTGTGGATGG (forward) and TCAGCTCAGGGATGACCTTG (reverse). The amounts of viral RNA were normalized to the levels of GAPDH.SH-SY5Y, HTB-10 and RD cells were seeded in 6 well plate , San Diego, CA United States. The PyMOL software is from PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.t-test. Mutation frequencies of viral genome were analyzed by the Chi-square test and the Fisher’s Exact test. ∗P < 0.05; ∗∗P < 0.001.Relative EV-A71 binding assay were analyzed by Student’s MT348284 – MT348346; 5′ UTR sequences: MT360921 – MT360983; full-length EV-A71 genome: MT360984 – MT360998.VP1 sequences: p = 0.006; Fisher Exact test p = 0.0166)  . The mutp = 0.004; Fisher Exact test p = 0.0043) (Capsid protein VP1 (891 nt) has been used as a standard reference for genotyping. As shown in  0.0043) . These 2n = 3653, as of April, 2020) deposited at different times from different countries worldwide. For most of these GenBank sequences, no information is available regarding the degree of disease severity of their source patients from the sequence-donating laboratories. Here, we consider these pooled and non-stratified GenBank sequences as a convenient baseline for comparison with our data. It is striking to note that VP1-145(non-E) occurs only at a baseline frequency of 13% (481/3653) in the GenBank database  . We founEV-A71 can bind to SCARB2 or PSGL-1 (P-selectin glycoprotein ligand-1) receptors for entry . The strWe compared the binding activities between VP1-280A and VP1-280T viruses with human rhabdomyosarcoma cell line RD, and human neuroblastoma cell lines HTB10 and SH-SY5Y . No diffcis-elements important for both IRES-mediated cap-independent translation and the replication origin for genome multiplication. While nucleotide 580 falls within the stem-loop VI and IRES , such as VP1-280T, could also contribute to the pathogenesis. The potential linkage relationship of these severity-associated mutations is shown in It is noteworthy that in our mild cases, 67% (24/36) also contained a 145(non-E) . It is qVP1 amino acid 145 has been considered as a key residue for binding to heparan sulfate and PSGL-1 receptor . While sFirst, we used the same RD cells to amplify EV-A71 from both severe (n = 27) and mild cases (n = 36). There is no reason that the same RD cells preferentially select for 145(non-E) variant, only when the virus is from severe cases, but not when the virus is from mild cases. Why should the RD cells discriminate between viruses originating from severe versus mild cases? Second, the phenomenon observed in the RD-A cell line  contained VP1-145E .The datasets presented in this study can be found in online repositories. The names of the repository and accession number(s) can be found below: C-SC, C-CL, and CS: experimental design. Y-YY, C-YL, and J-SL: virus isolates. C-SC and C-CL: conduct the experiments. C-SS, M-JH, C-SC, and CS: bioinformatics. CS and C-SC: writing. All authors: data analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brassica napus double haploid population were analyzed. Five germination-related indexes, including germination percentage (GP), root length (RL), shoot length (SL), fresh weight (FW), and root-to-shoot length ratio (R/S) under control and drought stress, were calculated, and the drought stress index (DSI), including DSI-GP, DSI-RL, DSI-SL, DSI-FW, and DSI-R/S, was determined using the quantitative trait loci (QTLs) analysis based on high-density genetic linkage map. The phenotypic analysis indicated that the R/S is an effective morphological trait in the determination of drought tolerance in the seedling stage. Thirty-nine identified QTLs were observed for these traits and then integrated into 36 consensus QTLs, in which 18 QTLs were found to affect the DSI of four traits . Based on the co-linearity between genetic and physical maps of B. napus, 256 candidate genes were detected, and 128 genes have single-nucleotidepolymorphisms/insertion–deletion (SNP/InDel) variations between two parents, some of which were associated with the drought stress tolerance . The present results laid insights into drought tolerance and its genetic bases in B. napus.Drought stress is one of the most environmental abiotic stresses affecting seed germination and crop growth. In the present study, the genetic characteristics of seed germination under drought stress in a  In the future, it is expected that drought will probably increase in certain areas of the world. Unfortunately, global climate change could lead to increased drought in many parts of the world and thus could have a major impact on crops , which belongs to Brassicaceae, is one of the important crops, with soybean and oil palm considered as the largest oil crops in the world  or 15% BEG6000 solution (drought stress treatment) was added (The seeds of two parents (KenC-8 and N53-2) and KNDH lines were disinfected firstly with 70% ethanol and prepared for seed germination test. Germination test was carried out in a seed germination box (L × W × H = 12 cm × 12 cm × 5 cm). Firstly, two layers of filter paper were placed on the bottom of the dish and then 15 ml of ddHas added . Fifty sas added .Quantitative trait locus analysis was performed as described by bzh” reference B. napus genome (SRP156346).2Candidate genes were detected according to the description of s genome . The detThe distribution of germination-related traits under drought in the two parents and 300 DH lines in KH populations is shown in As shown in Using the high-density SNP linkage map and phenotypic data of seedling traits under control and drought conditions, a total of 39 QTLs were detected in KN DH populations . Thirty-For QTL analysis under control conditions, two QTLs (qRLK-6-1 and qSLK-11-1) were detected for RL and SL under control conditions on A06 and C01, respectively. The two QTLs have a negative effect on RL and SL, accounting for 5.56 and 5.86% of phenotypic variation (PV), respectively. No QTLs were detected for R/S, FW, and GP under control conditions.For QTL analysis under drought conditions, a total of 16 QTLs were detected for three different traits  under drought conditions . CompareFor QTL mapping for DSI, 18 QTLs affecting DSI of four traits  were identified on different chromosomes, including A06, A07, A10, C01, and C02 , and no B. napus, 256 candidate gene homologs with 234 genes involved in drought tolerance in Arabidopsis thaliana were detected  associated with both RL and SL under drought stress, which encoded a protein involved in cellular responses to abscisic acid. BnaC01g32600D was located on chromosome C01 within the two DSI-QTLs (qDSI_RL-11-2 and qDSI_SL-11-3) associated with both RL and SL under drought stress and encoded for an ortholog of the A. thaliana gene AT3G20340 involved in oxidative stress. BnaA06g02020D was located within QTLs qR/S-6-2 and qRLK-6-2, associated with R/S under drought and RL under control conditions, respectively, and orthologous to the A. thaliana gene “LysM-containing receptor-like kinase 3 (LYK3),” which functions in the abscisic acid-activated signaling pathway. BnaC01g34530D in QTL qSLK-11-1 was associated with SL under control, and it was orthologous to the A. thaliana gene “HALOTOLERANCE DETERMINANT 3 (HAL3A).” BnaC03g32780D within QTL qR/S-13-1 on chromosome C03 associated with R/S under drought was orthologous to the A. thaliana gene “ABI BINDING PROTEIN4 (AFP4).” BnaC03g37030D in QTL qR/S-13-1 was orthologous to the A. thaliana gene “DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2 (DREB2).” BnaC03g44440D was located in QTL qR/S-13-1 associated with R/S under drought conditions and orthologous to the A. thaliana gene “RING/U-box superfamily protein (XERICO).” BnaC03g45915D within QTL qR/S-13-1 was orthologous to the A. thaliana gene “LOW-TEMPERATURE-INDUCED 65, (RD29B).” BnaC03g45990D in QTL qR/S-13-1 associated with R/S under drought stress was an ortholog of the A. thaliana gene “COLD REGULATED 413 PLASMA MEMBRANE1 (COR413-PM1).” BnaC03g46570D was detected in QTL qR/S-13-1, orthologous to the A. thaliana gene “ASCORBATE PEROXIDSE 3 (APX3).” BnaC03g49530D in QTL qR/S-13-1 associated with R/S under drought conditions and orthologous to the A. thaliana gene “ABA REPRESSORE1, (ABR1).” BnaC03g64160D was detected within QTL qR/S-13-1 and was an ortholog of the A. thaliana gene “A. THALIANA MYB 4 (ATM4).” BnaC09g24550D in QTLs qR/S-19-1 and qRL-19-1 was associated with R/S and RL under drought stress, respectively, and orthologous to the A. thaliana gene “A. THALIANA INDUCAR OF CBP EXPRESSION 1 (ATICE1).” Two genes, BnaC09g27300D and BnaC09g27320D, were also located within QTL qR/S-19-1 and RL-19-1, and orthologous to the A. thaliana genes “RUBISCO ASSEMPLY FACTOR 2 (RAF2)” and “ATYY1”, respectively. BnaC09g36060D was detected in QTL qR/S-19-2 associated with R/S and orthologous to the A. thaliana gene “METHYLTHIOALKYLMALATE SYNTHASE 2 (MAM2)” ” . These iB. napus and QTLs related to drought tolerance. It was revealed that the R/S was sensitive to drought stress, and it was consistent with the results of B. napus by B. napus is likely to resist the effect of drought through increasing water uptake and reducing its loss. The increase in RL was essential to enhance water uptake, and the decrease in SL resulted in water loss  and were involved in abscisic acid stimulus. Two QTLs, qDSI_RL-11-2 and qDSI_SL-11-3, were located in the same position on C01 and associated with the BnaC01g32600D gene, which was orthologous to the gene for oxidative stress. Moreover, qDSI_RL-11-4 and qDSI_SL-11-3 were also located near each other and associated with B. napus gene BnaC01g30750D that is orthologous to ICE1, which is involved in ABA signaling and cold stress. However, QTLs qRL-11-3 for RL and qDSI_RL-11-2 affecting DSI were detected in the same region on C01. These results suggested the importance of these regions on chromosome C01 for drought tolerance.Several QTLs related to important drought-tolerant traits were detected on C01; therefore, this chromosome deserves specific importance in drought resistance in A. thaliana genes were associated with drought tolerance. BnaC03g44440D was located in QTL qR/S-13-1 that is associated with R/S under drought conditions and orthologous to the A. thaliana gene XERICO. XERICO improved drought tolerance in A. thaliana. BnaC03g37030D was located in QTL qR/S-13-1 and orthologous to the A. thaliana gene DREB2. Previous studies have reported that DREB overexpression enhanced drought tolerance in many plants such as A. thaliana, tobacco, and apple (BnaA02g09290D within QTL qSL-2-4 was orthologous to the A. thaliana gene MAPKKK15, which was reported to be involved in the drought tolerance mechanism in potato (BnaC09g27300D and BnaC09g27320D were located within QTL qR/S-19-2 and qRL-19-1 and orthologous to the A. thaliana genes RAF2 and ATYY1, respectively. The AtYY1 gene was expressed under ABA, salt, and dehydration conditions, and overexpression of AtYY1 decreased ABA sensitivity in A. thaliana (RAF2 and SDIR1 complex had an essential role under stress by mediating the ABA signaling pathway. In addition, BnaC03g45915D within QTL qR/S-13-1 was orthologous to the A. thaliana gene RD29B, which was highly expressed under desiccation and played a vital role in drought tolerance (BnaC01g35030D, BnaC01g32600D, and BnaC01g30750D, which indicated the role of these genes in drought tolerance in B. napus. In the end, we assumed that the above genes that were orthologous to drought stress tolerance genes in A. thaliana may be strongly associated with drought tolerance in B. napus. Further investigation and validation of these genes are underway to confirm their function in the drought tolerance in B. napus.The problem of selecting effective genes from hundreds of genes within QTLs is the biggest challenge, especially with the presence of many unfunctional genes . For thind apple . In thisn potato . The twothaliana . Furtherolerance . Our resB. napus. Few studies used R/S as a phenotyping trait to QTLs in B. napus before. According to the QTL analysis, 39 QTLs associated with drought tolerance were identified and integrated to 36 consensus QTLs. Moreover, 256 drought tolerant-related candidate genes were identified, of which 128 genes have SNP/InDel variations between the two parents. In addition, our results indicated that specific regions on chromosome C01 were important for improved drought tolerance in B. napus due to the presence of many QTLs associated with drought tolerance. However, further studies are required to further confirm the candidate genes related to drought tolerance in B. napus.To illustrate the complexity of drought stress mechanisms and identification of genes underlying these mechanisms, we have used many genetic and genomic technologies. In this study, we used R/S as a phenotyping trait in QTL mapping and gene identification for drought tolerance at seedling stages in The original contributions presented in the study are included in the article/The authors declare that the experiments comply with the current laws of the country in which they were performed.MG and HC conducted most of the experiments and data analysis for the overall study, and wrote and modified the manuscript. HL and WZ conducted parts of data analysis and provided a guide to the use of the related software. GL and ML designed and conceived the overall study and revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.NAD Furthermore, NAD+ is used as substrate for lysine deacetylases, ADP-ribosylating enzymes, DNA ligase, and for NADP+ biosynthesis. Increasing evidence supports that NAD+ also acts as an important signaling metabolite and that NAD+ is used for 5′-modified mRNA forming a caplike structure in bacteria  in which nicotinic acid adenine dinucleotide (NaAD) is amidated to produce NAD+   . Conservfunction .glnB gene, some organisms can encode additional PII gene paralogues. In Proteobacteria, the second PII gene is named glnK and is encoded in an operon along with the amtB gene lutamine . These mlutamine .The nucleotides ATP and ADP bind competitively to the three nucleotide-binding sites located in the clefts formed between each PII subunit . The thrThe PII proteins also respond to the levels of glutamine. However, the mechanism of regulation by glutamine is not universal. In Proteobacteria, PII proteins are subject to a cycle of reversible uridylylation of a Tyr residue located at the apex of a solvent-exposed loop, namely T-loop. This response is mediated by the glutamine-sensitive bifunctional uridylyltransferase/removing enzyme GlnD . On the Gln enzymes are negatively feedback-inhibited by physiological levels of NAD+, and this regulatory mechanism is conserved in distantly related bacteria. We show that PII proteins act as a dissociable regulatory subunit of dimeric NadE2Gln in bacteria. Complex formation between PII and NadE2Gln relieves the NadE2Gln inhibition by NAD+, thereby acting as a switch to coordinate NAD+ production with nutrient availability in prokaryotes.Here we show that type 2 NadEA. brasilense. This ligand fishing GlnZ-affinity column was then challenged with A. brasilense protein extracts in the presence of MgATP. A blank nickel column, without His-GlnZ, was used as a negative control. After extensive washes with buffer containing MgATP, both columns were washed with buffer containing MgATP plus 1.5 mm 2-OG. Two consecutive fractions of 1.5 ml (fractions 1 and 2) eluted with 2-OG were collected and analyzed by label-free LC-MS/MS. The rationale of this approach is that, with the addition of 2-OG, GlnZ would assume a different conformation and, therefore, release proteins that were specifically retained in the column because of direct physical interaction with GlnZ.We used Ni-NTA column loaded with N-terminal His-tagged GlnZ in an attempt to identify novel PII-binding proteins in the diazotrophic α-Proteobacterium Gln, that has been studied previously by our group   .Gln complex has been previously suggested by bioinformatic analysis . When the pulldown assays were performed in the presence of MgATP and saturating 2-OG concentrations, no interaction between GlnZ and AbNadE2 could be detected (A).The formation of a PII-NadEanalysis . Hence, el beads A. When tdetected A.dK of 300 nm in the presence of ATP and a dK of 150 nm in the presence of ADP .The interaction between GlnZ and AbNadE2 was then quantitatively analyzed using Biolayer interferometry. The His-tagged GlnZ was mobilized on Ni-NTA tips and exposed to various AbNadE2 solutions in presence of different effector molecules. The binding curves obtained with increasing concentrations of AbNadE2 enabled the estimation of a l-glutamine, ATP, and NaAD. The formation of NAD+ by AbNadE2 was determined spectrophotometrically after its conversion to NADH by alcohol dehydrogenase, as described previously . This feature seems to be a general characteristic of dimeric NadE2Gln as we also noted this effect with orthologous enzymes from the β-Proteobacterium Herbaspirillum seropedicae (HsNadE2Gln) and from the Cyanobacteria Synechocystis sp. (ScNadEGln) . On the other hand, the presence of DTT had no effect when l-glutamine was substituted by ammonium as N donor for the reaction . These data suggest that NadE2Gln enzymes require a reducing environment for the full activity of the glutaminase domain.We reported previously and confirmed here that AbNadE2 is more active in a reducing environment in the presence of DTT using glutamine as N donor  could play a role in the regulation of AbNadE2. Hence, AbNadE2 activity was measured by monitoring PPi released in the presence of these metabolites. We found that NAD+ acted as a potent inhibitor of AbNadE2, diminishing the enzyme activity to ∼17%. The other tested metabolites showed no effect (A). The presence of NAD+ inhibited AbNadE2 in a dose-dependent hyperbolic curve with an estimated iK of 1 mm (B).Given the role of NadE in NADo effect A. The pr of 1 mm B.+ (+ could inhibit NadE activity by competing with the NaAD substrate (competitive inhibition). The type of inhibition can be distinguished by the kinetic constants, which were, therefore, determined. The presence of NAD+ did not affect the AbNadE2 NaAD mK, but instead, this metabolite altered AbNadE2 Vmax (from 0.09 μmol s−1 to 0.04 μmol s−1 in the presence of NAD+) . The l-glutamine mK was also unaffected in the presence of 1 mm NAD+ . These data indicate the presence of an allosteric NAD+ inhibitory binding site on AbNadE2.The structural similarities between NaAD and NAD+  led us tm NAD+ (B). However, GlnZ was able to partially relieve the inhibitory effect on AbNadE2 activity when NAD+ was present under physiological relevant levels (between 0.5 and 2.5 mm). The AbNadE2 NAD+ inhibition curve changed from hyperbolic to sigmoidal in the presence of GlnZ (B), increasing the IC50 from 1 mm in the absence of GlnZ to 2.5 mm in its presence. The AbNadE2 NAD+ IC50 in the presence of GlnZ is near the reported intracellular NAD+ concentration in Escherichia coli of 2.6 mm . The estimated Kact was 640 nm, which is in the same range as the dK for the GlnZ-AbNadE2 complex estimated using Biolayer interferometry (B).When NADnt curve A. The eserometry B.A), we thought whether 2-OG could impair the ability of GlnZ to relive AbNadE2 NAD+ inhibition. When AbNadE2 activity was measured in the presence of 2.5 mm NAD+ and GlnZ was first equilibrated in buffer containing 2-OG before combining with AbNadE2, 2-OG impaired the ability of GlnZ to relive AbNadE2 NAD+ inhibition in a 2-OG–dependent manner (B). The response to 2-OG was hyperbolic with a iK for 2-OG of 15 μm (B). The iK for 2-OG is in the same range as the iK for 2-OG–dependent inhibition of GlnZ-AbNadE2 complex formation, as determined by pulldown or Biolayer interferometry (C and D).Given the negative effect of 2-OG on the formation of the GlnZ-AbNadE2 complex A, we thot manner B. The reof 15 μm B. The Kierometry , C and DiK of 1 mm . Note that although the experiments depicted in Fig. 4B reflect the ability of 2-OG to inhibit GlnZ-AbNadE2 complex formation, the assay in Fig. 4F reflects the ability of 2-OG to dissociate a pre-formed GlnZ-AbNadE2 complex. Pulldown assays indicated that concentrations of 2-OG up to 2 mm were not enough to fully dissociate a pre-formed GlnZ-AbNadE2 complex . On the other hand, 2 mm 2-OG completely abolished GlnZ-AbNadE2 complex formation . These data imply that once the GlnZ-AbNadE2 complex is formed, 2-OG is much less effective to impair the interaction between these proteins and, hence, to relieve AbNadE2 NAD+ inhibition.When AbNadE2 activity was measured by mixing GlnZ and AbNadE2 before the addition of 2-OG, a different response to increasing 2-OG levels was observed. The effect of 2-OG was much less pronounced, with 2-OG showing a in vivo . Furthermore, GlnZ-UMP3 could not interact with AbNadE2, as indicated by pulldown assays (data not shown). Hence, the uridylylation of GlnZ abrogates its ability to interact with AbNadE2 and to relieve the NAD+ inhibition.Under nitrogen-limiting conditions, GlnZ is predominantly found in fully uridylylated form in vivo . Using ihibition A. FurtheAFig. S5). In agreement with the pulldown data, the GlnZ variant ΔQ42-S54 was able to relieve the AbNadE2 NAD+ inhibition despite the presence of 2-OG (A).Most of the known interactions between PII proteins and their target proteins occur via the T-loop of the PII protein. Indeed, this is the region that suffers the most prominent conformational change upon 2-OG binding . We test of 2-OG A.A. brasilense encodes a second PII paralogue, namely GlnB, which is 67% identical to GlnZ at the amino acid sequence . In agreement, GlnB was much less effective than GlnZ in relieving the AbNadE2 NAD+ inhibition (A). These data suggest that both A. brasilense PII paralogues can interact with AbNadE2, but a more stable complex is apparently formed with the GlnZ paralogue.sequence . To detehibition A. These + and PII proteins is conserved among NadE2Gln in bacteria, the NadE2Gln enzymes from the β-Proteobacterium H. seropedicae and the Cyanobacteria Synechocystis sp. were purified to homogeneity and tested for enzymatic activity in the presence of NAD+ and the GlnZ/GlnB orthologue from the respective organism. In both cases, NadE2Gln was inhibited by NAD+ in a dose-dependent manner within the physiological range (B and C). For both organisms, the presence of PII relieved NadE2Gln NAD+ inhibition, although the PII effect was less pronounced in the case of Synechocystis sp NadE2 (B). These data suggest that both the inhibition by NAD+ and regulation by PII are conserved mechanisms to regulate NadE2Gln in bacteria.To determine whether regulatory interplay between NADal range , B and Csp NadE2 B. These i.e. containing similar domain architecture, a glutaminase domain fused to NAD synthetase domain, PIRSF accession 006630). These sequences are distributed in 1772 eukaryotes, 17,578 bacteria, and 222 Archaea. Hence, glutamine-dependent NadE enzymes are widespread throughout nature.Inspection of the PIRSF database  identifiPII-nadE genetic islands in prokaryotes. A total of 1300 nonredundant PII-nadE islands were identified (Table S1). Most of the PII-nadE islands identified belonged to β- and γ-Proteobacteria, 834 and 436 counts, respectively (Table S1). PII-nadE islands were identified in distantly related prokaryotes such as the bacteria phylum Firmicutes, Nitrospirae, Actinobacteria, Deinococcus-Thermus, Ignavibacteriae; and the Archaea phylum Euryarchaeota, Crenarchaeota (Table S1). Interestingly, in A. brasilense and Synechocystis sp., the PII gene is not linked nadE; PII nevertheless regulated the NadEGln function (B and 5C). Hence, NadEGln regulation by PII can occur despite the absence of a genetic link of respective genes in these cases. The bioinformatic analysis reinforces that the formation of the PII-NadE complex may be a conserved feature in bacteria and may also be present in Archaea.The conservation of gene order can be used as a fingerprint of proteins that physically interact . We havefunction B and 5C.PII-nadE islands were separated into three major groups using CLANS , ADP-ribosylating enzymes, DNA ligase, and NAD+ kinase and may act as a signaling metabolite , H. seropedicae (β-Proteobacteria), and Synechocystis sp (Cyanobacteria) are negative feedback–inhibited by physiologically relevant concentrations of NAD+ . We speculate that bacterial NadE2Gln may be universally inhibited by NAD+. Kinetic analysis with AbNadE2 showed that NAD+ does not compete with the binding of substrate NaAD but reduces the enzymes Vmax . This clearly indicates that NadE2Gln is feedback-inhibited through an allosteric inhibitory binding site for NAD+.Here we show that the glutamine-dependent dimeric NadE2 from distantly related bacteria such as from A. brasilense . The presence of GlnZ changed the response of AbNadE2 to its feedback inhibitor NAD+. Instead of a hyperbolic inhibition curve with a iK of 1 mm, the GlnZ-AbNadE2 complex displayed a sigmoidal inhibition curve with an IC50 of 2.5 mm. Thereby, GlnZ relived the AbNadE2 NAD+ inhibitory effect in particular under physiologically relevant NAD+ concentrations (B). The sigmoidal dose-response curve indicates the cooperativity of the NAD+ allosteric binding sites in the GlnZ-AbNadE2 complex (Hill coefficient 2.2). The presence of high 2-OG levels or GlnZ uridylylation impaired the formation of the AbNadE2-GlnZ complex, thereby preventing GlnZ to revert AbNadE2 NAD+ inhibition (D and 5A).Initially, we used a PII protein affinity column to successfully identify AbNadE2 as a novel target of the PII protein GlnZ in the diazotrophic α-Proteobacterium trations B. The sihibition D and 5A.Gln to regulate NAD biosynthesis according to the metabolic state sensed by GlnZ (+ feedback inhibition and low availability of the l-glutamine substrate (A). Upon an ammonium shock, glutamine levels rise . This is of particular importance in the nitrogen-fixing bacterium A. brasilense, which possesses a mechanism to inactivate the abundant enzyme nitrogenase by ADP ribosylation as an immediate response to ammonium shock . Apparently, the PII affinity for 2-OG binding is reduced within the PII-NadE2 complex compared with free PII. This may result in a “memory effect” such that once the GlnZ-AbNadE2 complex is formed, it would become resistant to small and transient fluctuations in 2-OG concentrations. That the affinity of PII proteins toward the effector molecules is differentially modulated through complex formation with various targets has already been described at a structural level for cyanobacterial PII protein  or Synechocystis sp (Cyanobacteria) (B and C). Hence, the model depicted in PII-nadE pair is conserved in widely distant prokaryotes of bacterial and archaeal origin (Table S1) suggests that the PII-NadE interaction may be widespread in prokaryotes. This is in analogy to the conserved PII-amtB genetic pair, whose protein products were shown to physically interact in a broad range of prokaryotes, including bacteria and Archaea  , B and C Archaea , 35. Ourwith PII . This hyGln as a novel PII target protein supports the notion that PII may function as a central orchestration unit of metabolism to tune the flux trough key metabolic pathways according to the metabolic state determined by the availability of nutrients and energy supply.The unique ability of the PII protein to sense and integrate the levels of essential metabolites such as ATP, ADP, 2-OG, and glutamine was capitalized by nature in such a way that PII regulates a range of key metabolic enzymes, transporters, and transcriptional regulators by direct protein– interactions . In addi+ and its derivatives  must increase to keep pace with cell growth, providing enough cofactors for daughter cells. Indeed, single-cell fluorescence measurements indicate a peak of NADH accumulation during the progress to cell division in E. coli , and NAD+ (sensed by NadE2) through the formation of the PII-NadE2 complex, constitutes a novel metabolic hub that may act to balance the levels of core cellular metabolites.The NADignaling , 39. Impfication , sirtuinfication , and ADPfication , suggestA. brasilense protein extract, 400 ml of the A. brasilense 2812 strain was cultivated on NFbHP to an A600 nm of 2. Cells were collected by centrifugation, resuspended in 20 ml of buffer containing 50 mm Tris-HCl, pH 8, 100 mm KCl, 20 mm imidazole, 5 mm MgCl2, 1 mm ATP and disrupted by sonication. The soluble fraction  was divided into two aliquots, one was the control fraction and the other the GlnZ affinity ligand fishing.To prepare the E. coli BL21(DE3) carrying the pMAS3 plasmid that expresses the A. brasilense His-GlnZ protein was cultured in LB medium at 37 °C until A600 nm of 0.5. Then, 0.3 mm IPTG was added, and after 4 h, cells were harvested by centrifugation and resuspended in buffer containing 50 mm Tris-HCl, pH 8, 100 mm KCl, and 20 mm imidazole. Cells were disrupted by sonication and the soluble fraction obtained after centrifugation .For the preparation of the His-GlnZ extract, 300 ml of 2+ were used. The first one was equilibrated with buffer containing 50 mm Tris-HCl, pH 8, 100 mm KCl, and 20 mm imidazole and loaded with His-GlnZ extract, washed with 20 ml of buffer containing 50 mm Tris-HCl, pH 8, 100 mm KCl, and 50 mm of imidazole to remove unspecific bound proteins and equilibrated with 5 ml of buffer containing 50 mm Tris-HCl, pH 8, 100 mm KCl, 50 mm imidazole, 5 mm MgCl2, 1 mm ATP. This column is prepared to bind the putative targets of GlnZ. The second column was equilibrated with 5 ml of buffer containing 50 mm Tris-HCl, pH 8, 100 mm KCl, 5 mm MgCl2, 1 mm ATP, and 50 mm imidazole. This column is a control for the unspecific binding of proteins to the column resin, thus, providing a control fraction of proteins eluted in the ligand fishing assay but not bound to GlnZ. Both columns were loaded with A. brasilense protein extract and washed with 20 ml of buffer containing 50 mm Tris-HCl, pH 8, 100 mm KCl, 5 mm MgCl2, 1 mm ATP, and 50 mm imidazole.For interaction assay, two Hi-trap chelating columns (1 ml)  charged with Nim 2-OG and 50 mm Tris-HCl, pH 8, 100 mm KCl, 5 mm MgCl2, 1 mm ATP, 50 mm imidazole. Two fractions of 1.5 ml (namely Fractions 1 and 2) were collected from the control and His-GlnZ columns and analyzed by label-free quantitative LC-MS/MS as described previously (A. brasiliense database from June 2012 (8122 sequence entries). The log enrichment ratio of each protein in the His-GlnZ column/control column in each fraction was plotted using GraphPad Prism 7 .Proteins bound to both columns were eluted with 3 ml of buffer containing 1.5 meviously . ProteinSynochocystis sp. strain PCC 6803, encoding dimeric NadE2Gln, was retrieved from CyanoBase (Synochocystis genomic DNA as a template with the forward primer 5′-ATCACCATCACCATCACGATTACGATATCCCAACTAGTGAAAACCTGTATTTTCAGGGCGCTAGCCATATGTTTACCATTGCCCTTGCCCAGCTTAATC and the reverse primer 5′-CAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGCCGCGGATCTTAACTGCCTTGGGGATGGAAAG.The sequence of the slr1691 gene from yanoBase . AmplifiglnD sequence from A. brasilense was retrieved from NCBI database. The amplification by PCR was performed using A. brasilense genomic DNA as a template with the forward primer 5′-CTAGTGAAAACCTGTATTTTCAGGGCGCTAGCCATATGCTCTCCACCCGCGCCGCCTCCGCCGACGCGTCCGACGCCAAGGACGCCGGCACAGCCAACATCCCCAACAAG and the reverse primer 5′-CCAAGGGGTTATGCTAGTTATTGCTCAGCGGCCGCGGATCCCTCATGCGGACGGATCGGCGAGCGCGTGCAGCAGCCGCTCGCGGATCTGGGCCAGCTTGTTC. Both amplified fragments were Gibson assembled into the pTEV5 vector previously cut with NdeI and BamHI (New England Biolabs).The glnZΔTloop was obtained from pMSA4Δ42-54 (The gene A4Δ42-54  and subcTable S2)  were sequenced to confirm the integrity of the inserted genes (able S2) , 48–53.E. coli Lemo21 (DE3) cells (New England Biolabs) in LB medium. The TEV protease was overexpressed in E. coli Rosetta (DE) pLysS (Novagen). Typically, E. coli strains carrying the expression plasmids (Table S2)  , 48–53 wm Tris-HCl, pH 8.0, 100 mm KCl, 20 mm imidazole) and cells were disrupted by sonication , followed by centrifugation at 30,000 × g for 30 min to remove cell debris and insoluble material. The soluble fraction (approximately 40 ml) was loaded onto a 1-ml HisTrap HP Ni-NTA column . The column was washed with 10 ml  and proteins were eluted using a linear gradient of imidazole (100 to 500 mm) in the same buffer. Eluted fractions were analyzed by 15% SDS-PAGE, and the fractions containing the protein of interest were pooled and dialyzed in storage buffer . For purification of His-tagged NAD synthetase and GlnD proteins , the same procedure was applied with the exception that the buffers contained 100 mm NaCl instead of KCl.For purification of His-tagged PII proteins , His-HsGlnK, or His-ScPII), cell pellets were resuspended in sonication buffer . TEV protease was purified as described previously . The Hiseviously , 13. Theescribed . All them Tris-HCl, pH 8, 50 mm KCl, 10 mm MgCl2, and indicated substrate concentrations. NadEGln activity was determined by coupling the production of NAD+ to the NADH-forming oxidation of ethanol using alcohol dehydrogenase, and photometric detection NADH (A340 nm). For enzyme assays, in which NAD+, NADH, NADP+, and NADPH were tested as potential effectors of NadEGln, the enzyme activity was measured using a discontinuous assay over the linear phase of the reaction. Reactions were started by the addition of l-glutamine and stopped at different times on ice by adding 100 mm EDTA. The pyrophosphate product was detected using the EnzChek Pyrophosphate Assay Kit (Thermo Fisher). All the assays were performed in triplicate using a Spark microplate reader (Tecan). The initial velocity data were fitted in the equation indicated in each figure using GraphPad Prism software package.Reactions were performed at 30 °C in 50 mIn vitro protein complex formation was assayed using His-Magnetic beads (Promega). All reactions were conducted in interaction buffer containing 50 mm Tris-HCl, pH 8, 100 mm NaCl, 5 mm MgCl2, 0.05% Tween 20 (v/v), 10% glycerol (v/v), 20 mm imidazole in the presence or absence of effectors  as indicated in each experiment. Five microliters of beads were equilibrated in 200 μl of interaction buffer. The beads were recovered and resuspended in 400 μl of buffer followed by the addition of 20 μg of His-PII and 40 μg of untagged NadE protein, in this order. After 5 min at room temperature with gentle mixing, the beads were washed three times with 200 μl of interaction buffer. Bound proteins were eluted by boiling the beads in SDS-PAGE sample buffer. Proteins were analyzed by Coomassie Blue–stained 15% SDS-PAGE.m Tris-HCl, pH 7, 50 mm KCl, 10 mm MgCl2, 2 mm ATP or ADP, 2.5 mm NAD+, and 1 mg/ml BSA). The Ni-NTA biosensor was first dipped into a solution of 2 μg/ml of His6-tagged GlnZ for 150 s until a binding signal of ∼2 nm was obtained (Loading). The sensor was briefly washed in binding buffer and then transferred to the analyte solution containing AbNadE2 at different concentrations for 150 s to record the association curve. Finally, the sensor was dipped into the interaction buffer for 300 s to monitor the dissociation. In parallel, a reference sensor was subjected to the same procedure, except that no His-tagged protein was bound, to determine the background of unspecific analyte binding. In a series of binding assays, AbNadE2 concentrations of 30 to 2000 nm (dimer concentration) were used to determine the dK of the GlnZ-AbNadE2 protein complex.To quantify the kinetic parameters of the PII-NadE complex an Octet K2 Bio-layer Interferometry System (FortéBio) was used. The purified proteins His-GlnZ and untagged AbNadE2 were diluted in the interaction buffer (50 miK) for 2-oxoglutarate on GlnZ-NadE complex formation, interaction assays using 2 μg/ml of 6xHis-tagged GlnZ and 400 nm AbNadE2 solutions were performed in the presence of different concentrations of 2-oxoglutarate (0 to 480 μm). Therefore, the Ni-NTA biosensor was loaded with His-GlnZ solution and then transferred into the AbNadE2 solution to measure the association. In a series of experiments, increasing concentrations of 2-OG were added to the interaction buffer to determine the inhibitory effect on association. The experiments were carried out in duplicate and analyzed with the Octet Data Analysis software using Savitzky-Golay filtering. The fitting of the curve was done with a 2:1 heterogeneous ligand model. Curves were then plotted in GraphPad Prism7 software.To determine the inhibition constant  at 10 mm total concentration was performed. The reaction buffer consisted of 50 mm Tris-HCl, pH 8, 50 mm KCl, 10 mm MgCl2, 2 mm ATP, and 0.2 μm of the indicated enzyme .To determine the dependence of NadE activity on the redox environment, continuous enzymatic assays were performed in the presence of saturating concentrations of 2 mftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/assembly_summary.txt and ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/archaea/assembly_summary.txt.  Only one genome sequence per species (or unknown species isolate) was downloaded to prevent data redundancy, giving priority to sequences obtained from type-strains and to those that represented complete genomes.Metadata of genome sequences from Archaea and Bacteria available in the NCBI Assembly database were obtained in the following addresses: A. brasilense as query (ACF77123.1). Only hits presenting e-values equal to or lower than 1e-5 were considered. A sequence window containing the PII homolog sequence with its respective 10 kb upstream and 10 kb downstream sequences was defined as a “PII island.” Genome assemblies not presenting this sequence interval (contigs too short to contain 20 kb of adjacent sequences) were discarded. All protein sequences encoded in the “PII islands” were recovered, and they composed a database named as “PII neighbors DB.”Positions of PII protein homologs were identified in the genome sequences through tblastn searches, using the GlnB protein of A. brasilense (UniProt G8AIW8) and (UniProt G8ASI0) were searched for in the “PII neighbors DB” with blastp, using 1e-5 as e-value cutoff. All NadE-like protein sequences were recovered using fasgrep from FAST tools (nadE homolog gene and its closest neighboring PII homolog (midpoint of the PII island) was computed. Only nadE homologous sequences with the midpoint to PII homolog lower than 2000 bp were kept. Duplicated NCBI protein entries were removed. The remaining sequences were retrieved from NCBI and subjected to clustering analysis based on all against all BLAST+ similarities to detect orthologous groups using CLANS (https://github.com/zyxue/ncbitax2lin),Homologs of the glutamine-dependent NadE proteins of ST tools  and dereST tools . The disng CLANS . The taxluciano.huergo@gmail.com.Additional data are available upon request: Prof. Luciano F. Huergo, Setor Litoral, UFPR, A. R. S. S., E. C. M. G., E. M. S., L. F. H., and K. F. conceptualization; A. R. S. S., L. M. P. P., L. F. H., and K. F. data curation; A. R. S. S., E. C. M. G., F. H. S., G. A. d. S., L. F. H., and K. F. formal analysis; A. R. S. S., E. C. M. G., E. P., F. H. S., G. A. d. S., L. F. H., and K. F. investigation; A. R. S. S., G. A. d. S., L. F. H., and K. F. methodology; E. C. M. G., M. B. R. S., L. S. C., L. M. P. P., L. F. H., and K. F. supervision; F. O. P., M. B. R. S., L. S. C., E. M. S., L. F. H., and K. F. funding acquisition; F. O. P., E. M. S., L. F. H., and K. F. project administration; F. H. S. and G. A. d. S. software; F. H. S., G. A. d. S., L. F. H., and K. F. writing-review and editing; L. F. H. resources; L. F. H. writing-original draft.