This phase 1 trial evaluated the safety, preliminary efficacy, and pharmacokinetics of surufatinib, a small molecular tyrosine kinase inhibitor, combined with toripalimab, a programmed cell death protein-1 antibody, in patients with advanced solid tumors.This is an open-label, dose-escalation and expansion study in patients with solid tumors who had failed standard therapies or had no effective treatment. In the dose-escalation stage, patients were treated with surufatinib, at dose levels of 200, 250, or 300 mg once daily (QD) in combination with toripalimab 240 mg, every 3 weeks (Q3W), to estimate maximum tolerated dose. Additional patients were enrolled in the dose expansion stage to further assess the efficacy, safety, and pharmacokinetics profile. Recommended phase 2 dose (RP2D) was determined based on the safety, tolerability, and preliminary efficacy from dose-escalation and expansion stages.From Feb 14, 2019 to Dec 20, 2020, 33 patients were screened, of which 30 patients were enrolled. One patient in the 300 mg cohort experienced dose limited toxicity, a grade 3 hyperthyroidism. The most frequent treatment-related adverse events of grade ≥ 3 were hypertension (20.0%), transaminases increased (13.3%), and blood bilirubin increased (13.3%). No treatment-related death or treatment discontinuation was identified. The RP2D was determined to be surufatinib 250 mg QD plus toripalimab 240 mg Q3W. Objective response rate was 24.1%  in this study.Surufatinib plus toripalimab was well tolerated, with no unexpected safety signals, and showed preliminary anti-tumor activity in patients with advanced solid tumors.Clinicaltrials.gov Identifier: NCT03879057; registration date: March 18, 2019.The online version contains supplementary material available at 10.1007/s00432-021-03898-8. During the past several years, immune checkpoint inhibitors (ICIs), such as antibodies against programed cell death protein-1 (PD-1) and programed cell death ligand-1 (PD-L1), have shown impressive clinical benefits in the treatment of solid tumors  inhibitors and PD-1/PD-L1 inhibitors could lead to an increase in anti-tumor activity, by provoking T-cell function and modulating suppressive immune cells as well as the stroma, in the tumor microenvironment  of 70%, 53%, and 36.0% in patients with renal clear cell carcinoma, advanced endometrial cancer and unresectable hepatocellular carcinoma, etc. , and colony-stimulating factor 1 receptor (CSF-1R). Surufatinib has demonstrated clinical activity in several solid tumors, especially in neuroendocrine tumor (NET) , or had not responded to currently available therapies. Enrolled patients had physical performance score of 0–1 [Eastern Cooperative Oncology Group performance status (ECOG PS) score]. Patients with hepatocellular carcinoma had Child–Pugh A or B7 hepatic function. Patients also had measurable target lesions [per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1], adequate bone marrow function and liver function, an expected survival time of more than 12 weeks. Patients who experienced unrecovered anti-tumor treatment-related toxicity or had other malignant diagnosis, central nervous system (CNS), or brain metastases were excluded. Detailed inclusion and exclusion criteria are available in Supplementary Table S1.This clinical trial was approved by Ethics Committee of Peking University Cancer Hospital and was conducted strictly in accordance with the Declaration of Helsinki and Good Clinical Practice. All patients provided written informed consent form (ICF) before enrollment. The study was registered at ClinicalTrials.gov with registration number NCT03879057.All patients enrolled received the study treatment in 3-week cycles. In the dose-escalation stage, a modified “3 + 3” design was applied. Patients were assigned to one of the three surufatinib dose cohorts and received surufatinib 200, 250, or 300 mg orally once daily  in combination with a fixed dose of toripalimab (240 mg intravenously) every 3 weeks (Q3W) to evaluate the maximum tolerated dose (MTD). Dose-limited toxicity (DLT) assessment was performed within 28 days of the first dose. In the dose expansion stage, 3–6 patients were additionally enrolled into the 3 dose cohorts to further assess the efficacy, safety, and pharmacokinetic (PK) profile. Recommended phase 2 dose (RP2D) was determined based on the safety, tolerability, and preliminary efficacy from dose-escalation and expansion stage.All patients received treatment until the first occurrence of disease progression, death, intolerable toxicity, or completion of the study. The dose of surufatinib could be adjusted according to the dose adjustment principle; however, dose reduction was not allowed for toripalimab.Tumor assessments (per RECIST v1.1) were performed by investigators once every 6 weeks (± 7 days) from administration of the first dose, and once every 12 weeks (± 7 days) after 48 weeks of the treatment until disease progression, death, intolerable toxicity, or fulfillment of other criteria for terminating the study treatment, whichever occurred first. Patients were followed every 12 weeks (± 7 days) after discontinuation of the treatment to record their survival status until death, lost to follow up, or withdrawal of informed consent, whichever occurred first. All complete responses (CRs)/partial responses (PRs) were confirmed by a consecutive tumor assessment at least 4 weeks after the initial assessment of CR/PR.Blood samples were collected for PK evaluation at multiple time points on Cycle 1 Day 1, Cycle 2 Day 1, and Cycle 3 Day 1 for surufatinib and toripalimab, respectively.For patients who signed an optional biomarker ICF, additional tumor samples were collected to evaluate the correlation between the efficacy of surufatinib and/or toripalimab and PD-L1 expression. PD-L1 expression was detected by immunohistochemistry staining with SP263 antibody using a Ventana  autostainer  and PK profile of surufatinib, combined with toripalimab, in patients with advanced solid tumors.The exploratory objective was to evaluate potential biomarkers that predict the efficacy of surufatinib and/or toripalimab.DLT events were based on the DLT evaluable population, including patients who received at least 1 cycle of treatment and completed all safety assessments within the DLT assessment window or discontinued early due to DLT in the dose-escalation stage. All safety analyses, progression-free survival (PFS), and overall survival (OS) statistics were based on the safety population, including patients who received at least 1 dose of any study drug. All efficacy endpoints, excluding PFS and OS, were analyzed based on the efficacy evaluable population, including all patients who received at least 1 dose of any study drug and had tumor assessment at baseline and post drug administration prior to initiation of new anti-cancer therapies. The PK analysis was based on the PK population, which included all patients who received at least one dose of the study drug, had at least one PK sample collected and analyzed, and had no protocol deviation that might affect the PK data.Two-sided 95% confidence interval (CI) of ORR and DCR were computed using the Exact (Clopper–Pearson) method. For time-to-event variables , median event time as well as the corresponding 95% CI were estimated using Kaplan–Meier method. All statistical analyses were performed using SAS 9.4 software. Adverse events (AEs) were graded by NCI CTCAE (Common Terminology Criteria for Adverse Events) v4.03, and coded using the Medical Dictionary for Regulatory Activities (MedDRA) v23.1. Treatment-related AEs (TRAEs) referred to AEs related to surufatinib or toripalimab, which were judged by the investigators. PK parameters were calculated by non-compartmental analysis using Phoenix WinNonlin v8.0.From Feb 24, 2019 to Dec 20, 2020 (cut-off date), 33 patients were screened, and 30 were enrolled, including 6 in 200 mg cohort, 12 in 250 mg cohort, and 12 in 300 mg cohort, respectively. By the data cutoff, 6 patients (20.0%) were still on study treatment, including 5/12 patients (41.7%) in the 250 mg cohort, and 1/12 patients (8.3%) in the 300 mg cohort. Twenty-four of the 30 patients (80.0%) had discontinued study treatment at cut-off date; most of these study treatment discontinuations were due to disease progression (19 patients).Demographic and baseline characteristics are shown in Table In the dose-escalation stage, 16 patients were enrolled. Fifteen of them were included in the DLT evaluable population (1 patient excluded due to other reason). Among them, 1 patient in the 300 mg cohort reported a DLT event, a grade 3 hyperthyroidism which was deemed related to toripalimab. This DLT event started on Day 16 from the first dose, and was recovered on Day 30 with the treatment of the supportive medication and dose interruption of both study treatments.As of the data cut-off date, 30 patients were included in the safety population. All patients had at least 1 TRAE of any grade. Of the 30 patients, 15 (50%) experienced 1 or more TRAE of grade ≥ 3. The most common (≥ 5% incidence) TRAE of grade ≥ 3 included hypertension [6 patients 20.0%) ], transaminase increased [4 patients (13.3%) (1 patient in the 200 mg cohort and 3 patients in the 300 mg cohort)], blood bilirubin increased [4 patients (13.3%) (1 patient in the 250 mg cohort and 3 patients in the 300 mg cohort)], vomiting [2 patients (6.7%) in the 300 mg cohort], asthenia [2 patients (7.1%) in the 300 mg cohort], and anaemia [2 patients (6.7%) (1 patient in the 250 mg cohort and 1 patients in the 300 mg cohort)] (Table 0.0% (1 pSix (20.0%) patients experienced treatment-related serious adverse events (SAEs), 4 of which were observed in the 300 mg cohort [4/12 patients (33.3%)]; the remaining 2 patients [2/12 patients (16.7%)] were in the 250 mg cohort. Treatment-related SAEs included blood bilirubin increased, blood glucose increased, decreased appetite, appendicitis perforated, hyperthyroidism, and gastrointestinal haemorrhage. No fatal treatment-related SAE was reported.TRAE caused dose interruption or dose reduction in 16 patients (53.3%), with a frequency of 58.3% (7/12 patients) in 300 mg cohort, which is similar with incidence from the other 2 cohorts .There was no TRAE that led to treatment discontinuation in this study.The incidence of immune-related AEs was 83.3% in the safety population and was comparable among all dose cohorts .0-τ) reached steady state on Cycle 2 Day 1 after 200 to 300 mg QD with accumulation ratio of 1.8, 2.0, and 2.1 compared to those on Cycle 1 Day 1, respectively. And the AUC0-τ at steady state increased with the dose ascending from 200 to 300 mg.The PK parameters of surufatinib were available for 30 patients for Cycle 1 Day 1, 27 patients for Cycle 2 Day 1, and 22 patients for Cycle 3 Day 1. The exposure of surufatinib in terms of area under the concentration–time curve for a dosing interval  was similar, which may indicate no obvious impact of surufatinib administration on the toripalimab exposure in patients.The number of patients for the toripalimab PK analysis was 30, 25 and 22 for Cycle 1 Day 1, Cycle 2 Day 1, and Cycle 3 Day 1, respectively. The exposure of toripalimab in terms of AUCThe major PK parameters of surufatinib and toripalimab are listed in Supplementary Table S2.Twenty-nine of 30 enrolled patients were evaluable for tumor response. By the data cutoff, there were 1 patient with a confirmed complete response (CR), 6 patients with confirmed partial responses (PRs), and 16 patients with stable disease (SD) as their best overall response (BOR) among the evaluable patients , most of whom were in the 250 mg cohort (9 patients). There were 1 patient with a confirmed CR and 4 patients with confirmed PRs with response trends that were continuing at the cut-off date , and 3 patients with SD .At the data cutoff, tumor progression or death was observed in 22 patients , and overall median PFS (mPFS) was 4.0 months (95% CI 2.6‒5.5). The mPFS was 2.0 months (95% CI 1.2‒not reached) for the 200 mg cohort, 5.5 months (95% CI 2.7‒not reached) for the 250 mg cohort, and 4.1 months (95% CI 1.2‒9.5) for the 300 mg cohort . The overall 6 month PFS rate was 30.3% (95% CI 14.5‒47.9%). Median DoR, TTR, and OS were not reached at the cut-off date, and the overall 6-month OS rate was 76.7% (95% CI 57.2‒88.1%).Tumor tissues from 27 patients at baseline were available for PD-L1 expression analysis. Among the 14 PD-L1-positive patients, 1/14 (7.1%) patient achieved a confirmed CR and 4/14 patients (28.6%) achieved confirmed PRs, while 6/14 (42.9%) reported SD. In the 13 patients with negative PD-L1 status, 10/13 (76.9%) achieved their BOR as SD or better, including 2/13 patients (15.4%) with confirmed PRs, and 8/13 patients (61.5%) with SD.The results of this phase 1, open-label study showed that the combination treatment of surufatinib and toripalimab was reasonably well tolerated. There was no new safety signal noted in this study.Most TRAEs in this study were mild (grade 1 or 2), and were manageable with supportive care medications and dose adjustments. No treatment-related discontinuation or death was identified. The incidence of TRAEs of grade ≥ 3 in surufatinib and toripalimab combination was 50%, with hypertension being the most common TRAE. The grade ≥ 3 toxicity profile observed in this study was consistent with previously reported safety data for surufatinib or toripalimab monotherapies  were in 250 mg cohort, which was accompanied by the highest ORR (45.5%). In spite of the efficacy differences among the different dosage groups, which might due to multiple factors such as different tumor types and baseline characteristics, the efficacy results indicated that 250 mg QD surufatinib was the most effective dose in this combination therapy. Based on the observed promising preliminary efficacy results, as well as the favorable safety profile for 250 mg cohort, the RP2D was determined to be 250 mg QD surufatinib plus 240 mg Q3W toripalimab.Previously, in 2 phase 3 trials of surufatinib in advanced well-differentiated NET, ORRs were 19.2% (95% CI 12.2‒28.1) and 10.3% (95% CI 5.6‒17.0) in the pancreatic and extrapancreatic NET, respectively, and DCRs were 80.8% (95% CI 71.9‒87.8) and 86.5% (95% CI 79.3‒91.9), respectively  , and platelet-derived endothelial growth factor (PDGF)  patients  has been initiated to further evaluate the efficacy and safety profile of this combination in advanced solid tumors, which mainly include NEN, biliary tract cancer, gastric cancer, thyroid cancer, small cell lung cancer, soft-tissue sarcoma, endometrial carcinoma, and esophageal squamous cell carcinoma.In summary, surufatinib plus toripalimab was well tolerated with no unexpected safety signals observed and showed preliminary anti-tumor activity in patients with advanced solid tumor, particularly in NEN patients. This combination warrants further evaluations in clinical trials.Supplementary file1 Supplementary Figure S1 Progression-free survival (Kaplan-Meier plot). Abbreviations: CI, confidence interval; PFS, progression-free survival; SUR, surufatinib; TOR, toripalimab; Qd, once daily; Q3W, every 3 weeks. (TIF 4994 KB)Supplementary file2 (DOCX 30 KB)Below is the link to the electronic supplementary material.


Influenza virus neuraminidase (NA)-targeting antibodies are an independent correlate of protection against influenza. Antibodies against the NA act by blocking enzymatic activity, preventing virus release and transmission. As we advance the development of improved influenza virus vaccines that incorporate standard amounts of NA antigen, it is important to identify the antigenic targets of human monoclonal antibodies (mAbs). Here, we describe escape mutants generated by serial passage of A/Netherlands/602/2009 (H1N1)pdm09 in the presence of human anti-N1 mAbs. We observed escape mutations on the head domain of the N1 protein around the enzymatic site  and also detected escape mutations located on the sides and bottom of the NA . This work increases our understanding of how human antibody responses target the N1 protein.IMPORTANCE As improved influenza virus vaccines are being developed, the influenza virus neuraminidase (NA) is becoming an important new target for immune responses. By identifying novel epitopes of anti-NA antibodies, we can improve vaccine design. Additionally, characterizing escape mutations in these epitopes aids in identifying NA antigenic drift in circulating viruses. Orthomyxoviridae and contain a segmented, negative-sense RNA genome. Two of the genomic segments encode the glycoproteins present on the viral surface, the hemagglutinin (HA) and the neuraminidase (NA)  , 4. The ase (NA) –5. The Nse (NA) ––9. To fue (NA) –––12.Seasonal influenza virus vaccines are the first line of defense against infection . Typical17–21–Residues critical for NA-inhibiting antibodies were first characterized using murine antibodies –29. The –30–in vitro experiments. Each mAb’s neuraminidase inhibition (NAI) activity, measured using an enzyme-linked lectin assay (ELLA), and neutralization activity, measured by a plaque reduction neutralization assay (PRNA), were first determined against the wild-type A/Netherlands/602/2009 (H1N1)pdm09 strain. All mAbs, aside from 1000-3C05 and 294-16-009-A-1D05, had NAI activity  of each mAb. For NAI-active mAbs, the NAI IC50 was used, while the PRNA IC50 was used for NAI-inactive mAbs. At each subsequent passage, we doubled the amount of mAb present in the medium. EMVs were detected after 4 to 10 passages (2× IC50 to 128× IC50) (EMVs were produced by passaging the wild-type A/Netherlands/602/2009 (H1N1)pdm09 virus with each antibody in Madin-Darby canine kidney MDCK) cells. We began with multiplicities of infection (MOIs) of 0.01 and 0.25 times the 50% inhibitory concentration (IC8× IC50) . mAbs EM8× IC50) . We did 8× IC50) . S364N, 8× IC50) . N270D a8× IC50)  and C. NK cells. 8× IC50) , and R438× IC50) , 30. The8× IC50) . Mutatio8× IC50) .We next used the mAbs to evaluate the impact of each NA mutation on antibody binding, NAI, and neutralization activities. Using immunofluorescence assays, we identified that the N270D mutation impacted most of the antibodies in our panel, including those that produced other EMVs . The N8850) and 300-16-005-G-2A04 (30-fold change in the NAI IC50) (50)  and completely escaped from 294-16-009-A-1C02. Both the Q313R EMV and rNA completely escaped 294-16-009-A-1C02 without causing resistance to other mAbs . However, the N369T rNA did not exhibit resistance to any mAbs in the panel. The mutation R430Q did not have a strong impact on any mAb NAI activity  . The N2750) (50) . The Q31her mAbs . S364N aher mAbs . The S36activity  and B. Wactivity . This viactivity . No EMVsactivity .50 values in a plaque reduction neutralization assay, where each plaque present is counted regardless of its size. The N88D EMV completely escaped mAbs 1000-3C05 and 294-16-009-A-1D05  of each EMV to assess fitness changes in vivo. Since both Q313R EMV and S364N EMV had similar changes in binding, NAI, and neutralization activities to Q313K and S364N/N369T EMVs, only one was chosen for mLD50 experiments. Wild-type A/Netherlands/602/2009 (H1N1)pdm09 virus had an mLD50 of 1 PFU , and can utilize Fc effector functions  were obtained from the American Type Culture Collection (ATCC) and propagated using complete Dulbecco’s modified Eagle medium (cDMEM) piperazin-1-yl]ethane-1-sulfonic acid (HEPES) [Gibco]).A/Netherlands/602/2009 (H1N1)pdm09 was grown in our laboratory by injection of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (Charles River Laboratories) and incubation at 37°C for 2 days. All mAbs were identified and isolated previously and provided by Patrick Wilson . They we5 cells/mL in a 12-well, sterile cell culture plate and incubated overnight at 37°C with 5% CO2. The following day, virus was diluted to an MOI of 0.01 (1 × 103 PFU) in 1× minimal essential medium (MEM)  supplemented with 1 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin. Antibodies were then added to the virus at a concentration that was 0.25 times the neuraminidase inhibition (NAI) 50% inhibitory concentration (IC50). For the mAbs without NAI activity (1000-3C05 and 294-16-009-A-1D05), the plaque reduction neutralization assay (PRNA) IC50 was used instead. The virus-antibody mixture was incubated with shaking for 1 h at room temperature (RT). MDCK cells were washed with 1× phosphate-buffered saline , and the mixture was then added to the cells. They were then incubated at 37°C with 5% CO2 for 2 days. The supernatant was collected and stored at −80°C until further use. For subsequent passages, MDCK cells were plated as described above, infected with a 1:10 dilution of the previous passage in 1× MEM with TPCK-treated trypsin (1 μg/mL), and incubated for 40 min at 37°C with 5% CO2. In the meantime, mAb was diluted in 1× MEM with TPCK-treated trypsin to a concentration that was doubled from the previous passage . After 40 min, diluted mAb was added to the virus-infected cells and left for 2 days at 37°C with 5% CO2. The cell culture supernatant was screened for escape mutant viruses by plaque assays with 128× IC50 of mAb present in the agarose overlay. Individual plaques (3 to 6 per EMV) were chosen and propagated in SPF eggs for 2 days at 37°C as described above.Escape mutant viruses were generated using the H1N1 virus A/Netherlands/602/2009 (H1N1)pdm09 as the maternal strain. MDCK cells were plated at 6 × 10RNA was isolated from egg allantoic fluid using the E.Z.N.A. viral RNA extraction kit (Omega Bio-Tek) according to the manufacturer’s instructions and then underwent next-generation sequencing. Sequences were assembled using a pipeline designed at the Icahn School of Medicine at Mount Sinai as described previously . To iden5 cells/mL in a sterile, 12-well plate and incubated overnight at 37°C with 5% CO2. Next, virus samples were serially diluted in 1× MEM from 10−1 to 10−6. MDCK cells were washed with 1× PBS and then infected with 200 μL of each virus dilution. Virus was incubated for 40 min at 37°C with 5% CO2, with rocking every 10 min. Afterward, virus was aspirated and immediately replaced with 1 mL of an agarose overlay containing 2× MEM, 0.1% diethylaminoethyl (DEAE)-dextran, 1 μg/mL TPCK-treated trypsin, and 0.64% Oxoid agarose. Plates were incubated for 2 days at 37°C with 5% CO2. Cells were then fixed using a 3.7% solution of paraformaldehyde (PFA) and incubated at 4°C overnight. For plaque visualization, the overlay was removed, and cells were stained with a solution containing 20% methanol and 0.5% crystal violet.Plaque assays were performed using a standard protocol. MDCK cells were seeded 24 h previously at 8 × 10o-phenylenediamine dihydrochloride (OPD) substrate solution (Sigma-Aldrich) was added to each well for the reaction at RT for 10 min. The reaction was stopped by the addition of 50 μL of 3 M hydrochloric acid (HCl). The optical density at 490 nm was measured using a Synergy 4 plate reader (BioTek). The area under the curve (AUC) was calculated in GraphPad Prism 9. Percent binding was determined by comparing the wild-type rNA AUC to mutant rNA AUC values.Recombinant NA proteins of A/California/7/2009 (H1N1) with mutations were produced using the well-established baculovirus expression system, which has been described in detail previously . Immulon5 cells/mL in a sterile, 96-well plate and incubated overnight at 37°C with 5% CO2. The following day, cells were checked for >99% confluence and washed with 1× PBS. Virus was diluted to an MOI of 5 in 1× MEM and added to each well (100 μL/well). Plates were incubated overnight at 37°C with 5% CO2. The following day, cells were fixed using 200 μL of 3.7% PFA and incubated overnight at 4°C. Next, the PFA was removed, and cells were blocked with 1× PBS containing 3% nonfat milk (American Bio) for 1 h at room temperature. The blocking solution was then removed and replaced with 1% nonfat milk. Primary mAbs were diluted to 300 μg in 1× PBS and added to the 1% milk at a 1:10 dilution, for a final concentration of 30 μg per well. Primary antibodies were incubated with shaking for 1 h at room temperature. Plates were then washed 3 times with 1× PBS. The secondary antibody Alexa Fluor 488 goat anti-human IgG(H+L) (Invitrogen) was diluted to 1:500 in 1% milk and added to the plates, and the mixture was incubated for 1 h at room temperature in the dark with shaking. The plates were then washed 3 times with 1× PBS. To prevent cells from drying out, 50 μL of 1× PBS was added to each well. Plates were visualized using the Celigo S adherent cell cytometer (Nexcelom Bioscience) with the 2-channel target 1+2 (merge) setting. The exposure time, gain, and focus  for the 96-well plate were automatically determined by the machine. Fluorescence was calculated using the default analysis settings, and percent fluorescence was determined based on the wild-type signal. We performed 2 independent assays; however, representative images from only 1 assay are shown here. The mAb CR9114 was included as a positive control to illustrate that all viruses had similar infectivities. This mAb is a pan-influenza A virus (IAV) HA stalk mAb  in a sterile 96-well plate. Once diluted, an additional 1:1 ratio of the sample diluent was added to the plate. This mixture was incubated for 1 h at room temperature with shaking. After 1 h, the fetuin-coated plates were washed 3 times with PBST using the AquaMax 3000 automated plate washer. Diluted virus was immediately added to the plates, and the plates were then incubated at 37°C with 5% CO2 for 18 h (overnight). The following day, plates were washed 6 times with PBST. Peanut agglutinin  was diluted to 5 μg/mL in conjugate diluent buffer  and added to the washed plates. This mixture was incubated for 2 h in the dark at room temperature. The PNA was then removed, and plates were washed 3 times with PBST. SigmaFast OPD (Sigma) was diluted in water. OPD was added at 100 μL per well, and the mixture was incubated for 3 min at room temperature. The reaction was stopped by adding 50 μL of 3 M hydrochloric acid, and the absorbance (at 490 nm) was then immediately determined using a Synergy H1 hybrid multimode microplate reader (BioTek). Prism 7.0 was used to determine the effective concentration of each virus that would yield detectable NA activity. Each enzyme-linked lectin assay (ELLA) was done in triplicate.Flat-bottom Immulon 4HBX microtiter plates (Thermo Scientific) were coated with 25 μg/mL of fetuin (Sigma) diluted in 1× PBS, at 100 μL per well and incubated overnight at 4°C. The next day, viruses were serially diluted (3-fold) in sample diluent buffer . The following day, we performed the ELLA procedure as described above. The IC50 and 95% confidence intervals were determined using a nonlinear regression [log(inhibitor) versus response-variable slope (4 parameters)] in Prism 7.0. Each NAI assay was performed in duplicate. Significance between IC50 values for each EMV and the irrelevant IgG control virus was calculated using 2-way analysis of variance (ANOVA). The fold change in the mAb NAI IC50 was calculated by dividing the EMV IC50 by the IC50 of irrelevant IgG control virus B. Fold changes in NAI IC50s for rNAs were calculated by dividing the rNA IC50 by the wild-type rNA IC50.To determine the NAI activity of each mAb, flat-bottom Immulon 4HBX microtiter plates (Thermo Scientific) were coated with 25 μg/mL fetuin (Sigma) diluted in 1× PBS and incubated overnight at 4°C. The following day, antibodies were diluted in sample diluent buffer and then serially diluted 1:3 in a sterile 96-well plate. Virus or rNA was diluted to 2 times the effective concentration determined by an ELLA and added to mAbs at a 1:1 ratio. This mixture was incubated for 1 h at room temperature with shaking. The fetuin-coated plates were washed 3 times with PBST as described above. Virus-mAb dilutions were transferred to the fetuin-coated plates and incubated at 37°C, with 5% CO5 cells/mL in 12-well plates. The following day, mAbs were diluted to 100 μg/mL in 300 μL 1× MEM and serially diluted 1:5 in 1× MEM to a final concentration of 0.032 μg/mL. Each virus was then diluted in 1× MEM to 1 × 103 PFU, and 50 μL was added to each antibody dilution. This virus-mAb mixture was incubated for 1 h at room temperature with shaking. Afterward, MDCK cells were washed with 1× PBS and then immediately infected with 200 μL per well of the virus-mAb mixture. The plates were incubated for 40 min at 37°C with 5% CO2, rocking every 10 min. In the meantime, the overlay was prepared. Antibodies were diluted to 100 μg/mL in 625 μL of 2× MEM and then serially diluted as described above. Next, a solution containing 1× DEAE-dextran and 1 μg/mL of TPCK-treated trypsin in sterile water for injection (Gibco) was added at 180 μL to each antibody dilution. When the infection finished, 360 μL of 2% Oxoid agarose was added to the overlay mixture in small batches to prevent solidification before being transferred to cells. The inoculum was removed and immediately replaced with the overlay so that the mAb dilution in the overlay was the same as the concentration in the inoculum. The plates were then incubated at 37°C, with 5% CO2, for 2 days. Cells were fixed and stained as described above. When determining plaque numbers per well, all plaques present were counted regardless of their size. The IC50 and 95% confidence intervals were determined using a nonlinear regression [log(inhibitor) versus response-variable slope (4 parameters)] in Prism 7.0. Each PRNA was done in duplicate. Significance between neutralizing IC50 values for each EMV and the irrelevant IgG control virus was calculated using 2-way ANOVA. The fold change in the mAb PRNA IC50 was calculated by dividing the EMV IC50 by the IC50 of irrelevant IgG control virus B.MDCK cells were plated at 8 × 1050) for each EMV was determined using female BALB/c mice  in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai. Each virus was diluted from 105 to 101 in 1× PBS, and 3 mice per dilution were infected (50 μL per mouse). Weight loss and survival were monitored daily for 14 days postinfection. Mice that lost more than 25% of their initial body weight were euthanized.The 50% mouse lethal dose (mLD


Immunotoxins do not only bind to cancer-specific receptors to mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. The plant glycoside SO1861 was previously reported to enhance the endolysosomal escape of antibody-toxin conjugates in non-hematopoietic cells, thus increasing their cytotoxicity manifold. Here we tested this technology for the first time in a lymphoma in vivo model. First, the therapeutic CD20 antibody obinutuzumab was chemically conjugated to the ribosome-inactivating protein dianthin. The cytotoxicity of obinutuzumab-dianthin (ObiDi) was evaluated on human B-lymphocyte Burkitt’s lymphoma Raji cells and compared to human T-cell leukemia off-target Jurkat cells. When tested in combination with SO1861, the cytotoxicity for target cells was 131-fold greater than for off-target cells. In vivo imaging in a xenograft model of B-cell lymphoma in mice revealed that ObiDi/SO1861 efficiently prevents tumor growth (51.4% response rate) compared to the monotherapy with ObiDi (25.9%) and non-conjugated obinutuzumab (20.7%). The reduction of tumor volume and overall survival was also improved. Taken together, our results substantially contribute to the development of a combination therapy with SO1861 as a platform technology to enhance the efficacy of therapeutic antibody-toxin conjugates in lymphoma and leukemia. ObiDi was separated from unconjugated obinutuzumab and a part of unconjugated dianthin by cation exchange chromatography . Bound proteins were eluted with a gradient of 1.5% to 10% of 2 M NaCl, 20 mM HEPES buffer with a constant flow rate of 0.5 mL/min at 18 °C. Fractions with protein content were analyzed by SDS-PAGE -bis(4-methoxy6-nitro)benzene sulfonic acid hydrate  assay. Cells were seeded in a 96-well plate  in 100 µL of RPMI-1640 medium without phenol red supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin. After 24 h of cell proliferation, either 25 µL/well medium or 25 µL/well medium containing SO1861 at a final concentration of 1 µg/mL was added. Furthermore, 25 µL/well medium containing immunotoxin (ObiDi), obinutuzumab or dianthin was added at final concentrations from 0.1 fM to 100 nM. Raji and Jurkat cells were incubated in the presence of the compounds for further 72 h and finally cell proliferation was determined applying the XTT assay. A solution (50 µL/well) of XTT at 1 mg/mL and of phenazine-methosulfate  at 8 µg/mL was pipetted to the cells and the 96-well plate was incubated at 37 °C for 2 h. Absorbance was measured directly from the media at 450 nm by the SpectraMax 340PC Absorbance Microplate Reader . Untreated cells served as control and were set to 100% viability. IC6) in 100 µL PBS. Paralysis of the hind legs or weight loss of >20% in the course of the experiments were used as end points. All animal experiments have been performed according to the institutional guidelines with permission from the competent authorities. The mice were housed in individually ventilated cages under a constant day and night cycle (12 h each) and had free access to water and animal feed. All animals were monitored daily for well-being during the entire experiment.Eight-week-old female SCID CB17 mice  were inoculated intravenously with luciferase-expressing Raji lymphoma cells  start of treatment one day after tumor cell inoculation as frequently described in the literature and (b) start of treatment two weeks later. Mice received either (i) 15 µg SO1861 in 100 µL PBS subcutaneously into the neck in combination with 1.8 µg ObiDi in 100 µL PBS intraperitoneally 1 h later, (ii) 1.8 µg ObiDi as in experiment (i), (iii) 1.8 µg obinutuzumab in 100 µL PBS intraperitoneally or (iv) 100 µL PBS as mock-treated control. The therapy lasted four weeks with alternating treatment intervals of 3 and 4 days.Tumor development was quantified by in vivo imaging using an IVIS Lumina imaging system . Mice were anesthetized with isoflurane. Luciferin  was injected intraperitoneally with 375 mg/kg body weight and mice imaged 5 min later. Total flux values that correlate directly with tumor mass were measured weekly. Images were analyzed with Living Image 2.0 software . n = 3). Blood was collected in S-Monovette  1.2-mL K3 EDTA sterile tubes and was analyzed by Labor 28 GmbH, Berlin, Germany, for blood parameters analysis, including cholinesterase, ferritin, glutamate dehydrogenase (GLDH), asparagine aminotransferase (ASAT), alanine aminotransferase (ALAT) and creatinine.Four days after two treatments with 15 µg SO1861 in 100 µL PBS s.c. and 10 µg ObiDi in 100 µL PBS i.p. approximately 1.2 mL of blood was collected by cardiac puncture in isoflurane-anesthetized pretreated mice and compared with non-treated animals (Saponaria officinalis L. by methanol extraction and further purified by high-performance liquid chromatography, as described elsewhere [SO1861 was gained from the dried roots of the common soapwort lsewhere ,43,80.


We then asked whether the TCRβ locus chromatin structure is altered by hypergravity exposure. ChIP studies performed on four Vβ segments of the murine double-negative SCIET27 thymic cell line, which corresponds to the last maturation stage before V(D)J recombination, revealed increases in H3K27me3 after 2× g exposure. Finally, we evaluated the implication for the EZH2 methyltransferase in the regulation of the H3K27me3 level at these Vβ segments by treating SCIET27 cells with the GSK126-specific inhibitor. These experiments showed that the downregulation of H3K27me3 contributes to the regulation of the Vβ germline transcript expression that precedes V(D)J recombination. These data show that modifications of H3K27me3 at the TCRβ locus likely contribute to an explanation of why the TCR repertoire is affected by gravity changes and imply, for the first time, EZH2 in the regulation of the TCRβ locus chromatin structure.Gravity changes are major stressors encountered during spaceflight that affect the immune system. We previously evidenced that hypergravity exposure during gestation affects the TCRβ repertoire of newborn pups. To identify the mechanisms underlying this observation, we studied post-translational histone modifications. We first showed that among the four studied post-translational histone H3 modifications, only lysine 27 trimethylation (H3K27me3) is downregulated in the thymus of mice exposed to 2×  During T lymphopoiesis, cells go through highly regulated differentiation steps that lead to the production of functional CD4+ and CD8+ T-cells. Four main differentiation stages can be identified using specific cell surface markers: the early double negative (DN) (CD4-CD8-), double positive (DP) (CD4+CD8+), single positive CD4 (SP4) (CD4+CD8-CD3+TCR+) and single positive CD8 (SP8) (CD4-CD8+CD3+TCR+) stages. The diversity of T-cell receptors (TCRs), composed of one heavy chain (TCRβ) and one light chain (TCRβ), is generated via the V(D)J recombination mechanism occurring at specific differentiation stages between the DN2 and DN3 stages for TCRβ at the DP stage for TCRβ. This V(D)J recombination process associates a variable (V) and a joining (J) gene segment to create an α chain gene and a variable (V), a diversity (D) and a joining (J) gene segment to create a β chain gene. V(D)J recombination relies on recombination signal sequences (RSS) located at the 3′ end of V, on both sides of D and at the 5′ end of the J segments, as well as on specific proteins  proteins). Before V(D)J recombination, unrearranged V gene segments and J-C regions of the TCR loci are transcribed in a stage-specific manner ,2,3,4. GIn addition to germline transcription, epigenetic and, more precisely, posttranslational histone modifications contribute to T lymphopoiesis ,6 and V(g) [During space missions, astronauts are exposed to different types of stresses, i.e., microgravity during flight, hypergravity at launch and landing, radiation, confinement, isolation, sleep deprivation and disrupted circadian rhythm, which can negatively affect their health ,16. Indeg)  or underg) , suggestg) . Howeverg)  and loweg) . These tg) for 3 weeks to allow a comparison with the only previous study to have analyzed the effects of gravity change (2× g during 3 weeks) on the TCRβ chain repertoire and V(D)J recombination [g gravity. Moreover, we demonstrated that EZH2 could contribute to V(D)J recombination regulation. Indeed, we showed that EZH2 inhibition in the SCIET27 cell line modified the expression of Vβ germline transcripts and that these changes correlated with a decrease in the H3K27 trimethylation on 5′ and 3′ ends of affected Vβ segments.In this study, we wondered whether gravitational stress could impact V(D)J recombination by altering the H3K27me3 histone posttranslational modification and EZH2 methyltransferase activity. To this end, we exposed adult male mice to hypergravity  toward an increase in Vβ12-1 germline transcripts in the thymus of the 2G mice (Knowing that chromatin compaction contributes to the regulation of V(D)J recombination, we then asked whether chromatin compaction is changed under hypergravity exposure. To address this question, we quantified, by semi-quantitative PCR, some germline transcripts that are associated with chromatin opening. Three germline transcripts were detected among the eight that were tested. We evidenced a trend Knowing that the methyltransferase EZH2 is essential for T lymphopoiesis and that it is one of the two enzymes responsible for H3K27 methylation, we then asked whether its thymic expression is altered under hypergravity. As shown in However, because few data exist concerning the contribution of EZH2 to H3K27methylation in the thymus, we wished to ensure that EZH2 is involved in this process. To this end, we treated thymocytes overnight with GSK126, which specifically inhibits EZH2 activity, and then assessed the level of H3K27me3 by Western blotting. As shown in Altogether, these results show that only some post-translational histone modifications are globally affected by hypergravity in the thymus. Furthermore, the decrease in H3K27me3 suggests a global alteration in the thymocytes’ chromatin structure in response to hypergravity, which could be explained by an alteration in the EZH2 level.g during 18 h and performed ChIP experiments to evaluate the H3K27me3 level on the promoter and RSS regions of four Vβ segments   A. This cnd Vβ14) B, suggesdetected . Altogetp = 0.0538 and 0.063, respectively) (p = 0.0577) toward an increase in Vβ13-1 germline transcripts  B. We thenscripts C. Note tAltogether, these results suggest that in some cases EZH2, via regulation of the H3K27me3 level on the Vβ segments, might contribute directly to germline transcript expression but that, in other cases (Vβ14), other actors are required. Indeed, we could not detect Vβ14 germline transcripts but could detect a decrease in H3K27me3 on its promoter and RSS regions. This observation could be due to the fact that a histone tail can carry different types of modifications acting together to generate histone code.Only a few studies have investigated the effects of spaceflight on lymphocyte development ,33,34,35g induced an increase in the murine thymus mass but decreased its cellularity. This increase in the thymus mass was also observed in male pups developed under hypergravity (2G) conditions [We found that 21 days spent at 2× nditions ,36, incrnditions  or remainditions ,39. Suchnditions , who didnditions . Horie anditions  also notnditions , nor in nditions ,43,44. Anditions  but the nditions ,46,47. TA likely explanation for a global lymphocyte decrease is a modification of thymic microenvironment, which is essential for T-cell development. Different cell types contribute to this microenvironment, including thymic epithelial cells (TECs) whose number and functions have been shown to be reduced in hypergravity mice . AdditioAt the molecular level, epigenetics contributes to V(D)J recombination regulation ,12,13,14Different enzymatic activities are implicated in the regulation of H3K27 methylation. PRC2, via EZH2 or its homolog EZH1, adds methyl groups, while KDM6a and KDM6b demethylate this lysine . In thisFinally, we showed that in the SCIET27 cell line, hypergravity induced a global increase in EZH2 protein level, which was not correlated with an increase in H3K27me3. This absence of correlation could be due to a modification of the balance between the methylase (EZH2/EZH1) and demethylase (KDM6a/b) activities that coexist within cells. To our knowledge, no data currently exist concerning KDM6a/b’s function in the thymus. Thus, it would be very interesting to test this hypothesis in the future. Another hypothesis is a modification of EZH2’s function. Indeed, EZH2 can methylate H3K27, but it can also act on nonhistone proteins, such as talin  or promyNote that this study is limited by the fact that we could not quantify the glucocorticoids produced within the thymus; the lack of quantification of hormones, such as ACTH, CRF and catecholamines; the difficulty in detecting apoptosis in the thymus; a limited study of cell cycle; and the fact that the balance between methylase (EZH2/EZH1) and demethylase (KDM6a/b) activities was not studied.In conclusion, our results show for the first time that the level of a specific histone modification, H3K27me3, is decreased in the murine thymocytes following hypergravity exposure and that these alterations will probably induce epigenome modifications that contribute to a decrease in the absolute T-cell number. In the future, it will be interesting to study more extensively the epigenomes of various thymocyte subpopulations to improve our understanding of alterations in T lymphopoiesis in response to gravity changes, especially under simulated or real microgravity conditions, and pay more attention on the TCR loci structure to understand how its modification will impact the TCR repertoire’s creation.Experimental procedures were carried out in conformity with the French National Legislation and the Council Directive of the European Communities on the Protection of Animals Used for Experimental and Other Scientific Purposes (2010/63/UE). Experiments were approved by the French Ministry of Research (authorization 04827), and authors complied with the ARRIVE guidelines. The mice used in this study were 7-week-old male C57BL6/J mice purchased from Charles River . For acclimation to animal house conditions, mice were housed for a week in standard cages  in a quiet room under constant conditions  with free access to standard food and water. Animals were anesthetized using 5% isoflurane and then put to death by cervical dislocation.g (Standard cages (36 cm × 20 cm × 14 cm) containing four mice were placed in a large radius centrifuge (radius of 1.80 m)  with a rg . Mice weAnti-CD4 PE (RM4-5), anti-CD8a PECy7 (53-6.7), anti-TCRβ FITC (H57-597), anti-CD3 APC (17A2) and the respective isotype controls were purchased from Biolegend . Anti-GAPDH (G9545) and anti-EZH2 (07-689) used for Western blot, and anti-EED (AA19), anti-H3K27me3 (07-449), anti-H3K4me2 (07-030) and anti-H3K9ac (07-352) antibodies were purchased from Merck Millipore . Anti-Suz12 (D39F6) and anti-H3K9-panmethyl (4069) antibodies were purchased from Cell Signaling . The anti-γH2A.X (39118) and the anti-H3 C-terminal (39164) antibodies were obtained from Active Motif . Anti-PARP1 antibody (GTX100573) came from Gentex . The EZH2 inhibitor GSK126 was purchased from Interchim  .The SCIET27 cell line was kindly provided by I. Screpanti  and I. Aifantis  .5 fresh cells were stained for 30 min in PBS−2% FCS at 4 °C and then washed in PBS−2% FCS. Data acquisition was performed using a Gallios Beckman Coulter flow cytometer, and data analysis was performed using FlowJo software .Thymuses were dissociated in PBS−2% FCS, and cells were counted using a Scepter Cell Counter . To identify DN, DP, SP4 and SP8 subpopulations, cells were stained with anti-CD4 PE, anti-CD8a PE-Cy7, anti-TCRβ FITC and anti-CD3 APC . This stSerum corticosterone concentration was measured in duplicate using a commercial ELISA kit , according to manufacturer’s instructions, and serum samples were prepared from blood collected between 8 and 10 a.m.The SCIET27 cell line was cultured in IMDM  supplemented with 0.1 mM penicillin, 0.1 mM streptomycin  and 10% FCS  according to Aifantis et al. 2001 .6 cells/mL in IMDM medium supplemented with 10% FCS, 0.1 mM penicillin and 0.1 mM streptomycin in the absence or presence of 2 µM GSK126 inhibitor and incubated overnight at 37 °C and 5% CO2.For EZH2 inhibition, SCIET27 cells were plated at 1.5 × 106 cells/mL in IMDM medium supplemented with 10% FCS, 0.1 mM penicillin and 0.1 mM streptomycin in the Petaka®G3 culture device . Then, the Petaka®G3 devices were placed overnight in a centrifuge (radius of 10.5 cm) with a rotational speed of 132 rpm producing a gravity vector of 2× g, which was placed in an incubator set at 37 °C . In both cases, RNA was reverse transcribed using random primers, RNaseOut and MML-V reverse transcriptase , following the manufacturer’s instructions.RNA was extracted from the thymus of hypergravity and control mice using the RNeasy kit  and from 10 × 10The qPCRs were performed in triplicate using Takyon No ROX SYBR MasterMix blue dTTP  and a Mastercycler Realplex2 Real-Time PCR System . The cycling program was: 5 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 45 s at the annealing temperature indicated in Semiquantitative PCR amplifications were performed because some Vβ segments have highly homologous DNA sequences that prevent the design of specific primers that generate 150–200 bp amplicons, a prerequisite to perform real-time PCR. To ensure the specificity of our amplifications, we had to design primers in leader and V-encoding exons, thereby generating amplification products >200 bp. These polymerase chain reactions (PCRs) were done using 0.625 U of Taq Polymerase (Thermo Fisher) and 0.7 µM of each primer. The PCR program was 5 min at 95 °C, followed by cycles of 45 s at 95 °C, 45 s at the annealing temperature indicated in TCRβ mRNAs were amplified by 5′-RACE PCR using the SMARTer™ RACE cDNA amplification kit . Briefly, for each experiment or treatment, total RNA from the thymus or thymocytes of 8 or 12 mice (depending on the experiment) was mixed in equimolar quantities, and 200 ng of this mix were reverse transcribed according to the manufacturer’s instructions. Then, TCRβ mRNAs were preamplified via two successive PCRs using Advantage 2 Taq DNA polymerase . The first PCR was performed using the GSP1 primer  annealin2, 0.1 mM EGTA, 5% glycerol, 0.5% NP40) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail following the manufacturer’s instructions (Thermo Fisher) at a ratio of 10 mg of tissue for 100 µL of total buffer. This lysis was performed on ice for 30 min. Then, tubes were centrifuged at 13,500× g for 15 min at 4 °C, and supernatants were collected. Proteins were extracted from dissociated thymocytes using the All prep DNA/RNA/Protein Mini Kit (Qiagen) following the manufacturer’s instructions.Proteins were extracted from thymus lysed in total buffer , 0.02% NaN3 (w/v), 5 mM sodium butyrate, 1× PBS) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher) at a ratio of 15 mg of tissue for 500 µL of TEB buffer for 10 min on ice with regular mixing. Histones were extracted from 5 × 107 dissociated thymocytes by mixing them with 750 µL of TEB buffer and incubating the obtained solution on ice for 10 min. Then, in both cases, samples were centrifuged at 800× g for 10 min at 4 °C. Pellets were washed with TEB buffer  and incubated overnight with 0.2 M HCl at 4 °C . A final centrifugation at 6000× g for 10 min at 4 °C was performed, and the supernatants containing histones were recovered.Histones were extracted from thymuses homogenized in TEB buffer  membranes . Membranes were incubated with primary antibodies overnight at 4 °C and then with corresponding HRP-coupled secondary antibodies for 1 h at room temperature. Blots were stripped and probed again as necessary. An anti-GAPDH antibody or an antibody directed against the C-terminal tail of histone H3 was used as a loading control for total proteins or histones, respectively. Pierce ECL Western blotting substrate (Thermo Fisher) was used for immunodetection, and signals were visualized by chemiluminescence using a Fusion FX7 camera (Vilbert-Lourmat). Signal intensity was quantified using ImageJ software (NIH) and normalized over the corresponding loading control.g, 5 min, 4 °C), the cells were washed in 1× PBS. Then, cell prelysis was performed as previously described [6 cell/mL in lysis buffer  supplemented with Protease Inhibitor Cocktail . Cell lysates were diluted five times in sonication buffer  supplemented with protease inhibitors; 300 µL of cell lysate were sonicated for 3 cycles of 30 s using the ultralow program of the Bioruptor Pico sonication device  and then centrifuged for 10 min at 4 °C at maximum speed. Supernatant containing chromatin was collected. For immunoprecipitation, 50 µL of Dynabeads protein A  were washed 3 times with IP Buffer  and blocked with IP Buffer containing 5% BSA and 0.2 µg/µL of salmon sperm  for 2 or 12 h at 4 °C. Then, 250 µL of chromatin were diluted with 250 µL of IP buffer supplemented with 2.5% BSA and protease inhibitors. Chromatin was precleared by adding 20 µL of blocked Dynabeads and incubating for 1 h at 4 °C. Collected chromatin was then incubated overnight at 4 °C with 1.5 µg of antibodies  or isotype control ). Before adding antibodies, 2.5 µL of chromatin (input control) was collected and stored at 4 °C. Finally, immunoprecipitation was performed. A total of 30 µL of blocked Dynabeads were added to chromatin and incubated for 20 min at room temperature. Dynabead complexes were washed twice with low salt wash buffer (0.1% SDS (v/v), 1% Triton X-100 (v/v), 2 mM EDTA, 20 mM Tris-HCl pH 8.1), twice with high salt wash buffer (0.1% SDS (v/v), 1% Triton X-100 (v/v), 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), twice with LiCl wash buffer , 1% sodium deoxycholate (v/v), 1 mM EDTA, 10 mM Tris pH 8.1) and twice with TE buffer . Crosslinking was reversed by incubating Dynabeads complexes in elution buffer ) overnight at 65 °C, followed by incubation at 37 °C for 1 h with proteinase K 0.5 mg/mL. DNA fragments were purified using NTB Buffer and the NucleoSpin Gel and PCR Clean-up kit  following the manufacturer’s instructions. Quantitative PCRs (qPCR) were performed in triplicate using Takyon No ROX SYBR MasterMix blue dTTP  and a Mastercycler Realplex2 Real-Time PCR System . The cycling program was 5 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 45 s at the annealing temperature indicated in ChIP was performed as described in Ghislin et al., 2012  with somescribed . Cells wt-tests were performed. When the variance and/or the distribution was not confirmed, Mann–Whitney nonparametric tests were performed. For more than two group comparisons, the homogeneity of variance and normality of distribution were checked using the Levene and Shapiro–Wilk tests, respectively. If these two sets of test criteria were met, ANOVA tests followed by post hoc Tukey–Kramer tests were performed for two by two comparisons. In the other cases, Kruskal–Wallis tests followed by post hoc Dunn tests were performed. Here, p values <0.05 indicate significance. All in vivo results are shown as the mean ± standard error of the mean (SEM). All in vitro results are shown as the mean ± standard deviation (SD).Statistical analyses were performed using StatView software . For two-group comparisons, the homogeneity of variance was assessed with Fisher’s test and the normality of distribution was assessed with the Kolmogorov–Smirnov test. When both criteria were validated,


Francisella novicida. The purification yield of the recombinant proteins was up to 3.6-fold greater than that of FnCas12a. In cell-free DNA cleavage assays, both mgCas12a proteins showed the higher cleavage efficiencies when Mn2+ was provided with KCl (< 100 mM) than tested other divalent ions. They were able to tolerate ranges of pH points and temperature, and showed the highest cleavage efficiencies at pH 8.0 and 50 °C. In addition, mgCas12a proteins showed 51% less crRNA-independent and 56% less crRNA-dependent non-specific nuclease activity upon prolonged incubation than did FnCas12a. Considering their greater yield in protein preparation and reduced non-specific nuclease activity, our findings may expedite the use of Cas12a especially when genome editing needs to be practiced with the form of ribonucleoproteins.CRISPR/Cas9 and Cas12a belonging to the Class II CRISPR system are characterized by a single-component effector protein. Despite unique features of Cas12a like DNA cleavage with 5′ staggered ends and a single crRNA, Cas12a has not been adopted in biotechnological applications to the similar extent as Cas9. To better understand the CRISPR/Cas12 systems, we selected two candidates, designated mgCas12a-1 and mgCas12a-2, from an analysis of the human microbiome metagenome (mg) and provided biochemical characterization. These new Cas12a proteins shared about 37% identity in amino acid sequences and shared the same direct repeat sequences in the crRNA with FnCas12a from  Cas9 and Cas12a belong to the class 2 CRISPR system and consist of a single effector Cas endonuclease that can be guided by a single RNA to cleave specific target DNA sequences5. Despite their similarities, they differ in that type II Cas9 from Streptococcus pyogenes Cas9 (SpyCas9) recognizes the protospacer adjacent motif (PAM) 5′-NGG-3′ and requires both a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA), which are processed by the host RNase III6. Successful enzymatic cleavage by SpyCas9 generates blunt-ended DNA double-strand breaks (DSBs) via its HNH and RuvC domains8. By contrast, type V Cas12a from Acidaminococcus sp. (AsCas12a) recognizes the PAM 5′-TTTV-3′, utilizes only a crRNA, and produces 5′ staggered-ended DSBs via the RuvC domain10. Studies have focused on improving the fidelity while reducing the off-target effects of Cas12a13 and Cas916, and on developing their applications as base editors18, prime editors19, transcriptional regulators23, and nucleic acid detectors27.Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins comprise a bacterial and archaebacterial adaptive immune system against mobile genetic elements from phages or conjugating bacteriaAcidaminococcus sp. (AsCas12a), Francisella novicida (FnCas12a), and Lachnospiraceae bacterium (LbCas12a) are actively in use. Here, we report two new members of Cas12a protein family identified from a survey of the human metagenome, mgCas12a-1 and mgCas12a-2  and their enzymatic characteristics including crRNA and divalent metal ion compatibility, salt, pH and temperature-dependent target DNA cleavage activities.Of the many members of the Cas12a protein family, those identified from Cas12a possesses a unique feature called, “collateral DNase activity”. Activated by Cas12a ribonucleoprotein (RNP) cleaving a target DNA, this non-specific DNase activity degrades surrounding DNA fragments. Researchers recognized this unique characteristic of Cas12a and developed nucleic acid detection methods. In this study, we aimed to further understand various non-specific DNase activities including collateral DNase activity, by comparing such activities among five different Cas12a orthologs. Through cell-free in vitro DNA cleavage assays, we found that both mgCas12a-1 and 2 possess more than 50% reduced non-specific DNase activities than FnCas12a did. In addition, we have also detected remaining non-specific DNase activities even in deactivated version of the mgCas12a proteins.28. We identified all proteins encoded by genes located within 15 kb of the CRISPR arrays and saved them in a local database for a BLASTp search. We then queried the local database using 16 known Cas12a proteins as baits  human metagenome database. Using this search pipeline   and 53% (mgCas12a-2) cleavage efficiency. Several other metal ions, such as CaCl2 and FeCl2, resulted in only marginal activity  of potassium or sodium. In the KCl-based assays, we observed the highest cleavage efficiency for mgCas12a-1 (80% cleavage of the substrate DNA) in the presence of 100 mM KCl and for mgCas12a-2 (78%) with 50 mM KCl Fig. d.After metal ions and salt concentrations, we determined the effect of pH on mgCas12a activity. The calculated pI values for mgCas12a-1 and mgCas12a-2 were 6.91 and 6.15, respectively. We thus tested cleavage efficiency for each mgCas12a RNP complex at pH values 5.0, 6.0, 7.0, and 8.0 and obtained the highest activity at pH 8.0 Fig. b.Figure We also tested nine different temperatures  for in vitro cleavage assays. We incubated pre-assembled mgCas12a RNP complexes and target DNAs at each temperature. Over 60% of the substrate DNA was cleaved by mgCas12a-1 at temperatures above 20 °C, suggesting a broad spectrum of temperature tolerance  without any help of MBP in the bacterial (Escherichia coli) production system. Based on SDS-PAGE analysis of fractions purified on immobilized affinity chromatography columns and final Cas12a protein yield measurements, we estimated that mgCas12a-1 shows the highest yield, followed by AsCas12a, mgCas12a-2, LbCas12a, and FnCas12a  at their N termini increases levels of protein production35.Our DNA in vitro cleavage assay exhibited that the assembled mgCas12a RNPs in reaction buffer does not need any other components when creating double strand breaks in the target DNA Figs. , 4, 5, 6The PCR-amplified DNA treated with mgCas12a effector protein only was not cleaved by the T7E1  were downloaded on November 3, 2017; CRISPR arrays and spacers were identified using MetaCRT with default parameters; and contigs with a CRISPR region were selected. Putative proteins were predicted using Prodigal with metagenome parameters; those within 15 kb upstream and downstream of the CRISPR region were selected, and their predicted protein sequences were saved and uploaded into a local BLASTp database. Amino acid sequences of 16 Cas12a were downloaded from the UniProt database on the same day. Conserved motifs across Cas12a proteins were identified based on protein alignments of structurally identified Cas12a sequences using MAFFT 37 with default parameters and saved for later use as query. A local BLASTp was run with the conserved Cas12a motifs defined above as query, and putative Cas12a proteins with a size between 800 and 1500 amino acids were retained. An online BLASTp was run against non-redundant protein sequences with all candidates for preliminary annotation. Sequences not starting with methionine were excluded. All new putative Cas12a proteins were aligned using MAFFT version 7 online using default parameters, from which a phylogenetic tree was drawn using the neighbor-joining method with pairwise deletion and 100 × bootstrap in MEGA7. Based on the results of the preliminary annotation, candidates annotated as putative Cas12a proteins and whose encoding genes were adjacent to CRISPR arrays were selected. Gene fragments due to low-quality alignments were discarded after structure-based comparison between sequences using ESPript 3.038. A phylogenetic tree of all new Cas12a proteins and 16 previously characterized Cas12a proteins was constructed using FastTree 2.1, 1000 × bootstrap, with default parameters and visualized using iTOL v6 after an alignment with MAFFT online.Metagenome nucleotide sequence data from the open access NCBI FTP BLAST databasemgCas12a-1, mgCas12a-2 , AsCas12a (Addgene #102565), FnCas12a (Addgene #102565), and LbCas12a (Addgene #102566) were cloned in-frame between the sequence encoding a polyhistidine tag and a bipartite nuclear localization signal (BPNLS) in a modified pET28a vector. The catalytic residues of each mgCas12a protein were determined by multiple amino acid sequence alignment of AsCas12a, FnCas12a, and LbCas12a. Catalytically inactive mutants were generated by PCR mutagenesis using Gibson Assembly Master Mix .The coding sequences of Cas12a were individually cloned into the pET28a vector. An 1-mL aliquot of an overnight-grown culture of Rosetta2 (DE3) pLysS cells containing individual pET28a-Cas12a constructs  was added to each culture; and cultures were incubated for an additional 20 h at 18 °C to allow protein production. Each culture was harvested by centrifugation , and the resulting cell paste was resuspended in 50 mL lysis buffer , followed by lysis via sonication . Each supernatant was then separated from the cell lysate by centrifugation  and filtered through 0.5-µm and 0.45-μm syringe filters  to remove any remaining cell debris and impurities. The filtered supernatant was then applied to an equilibrated fast protein liquid chromatography (FPLC) system  and flowed through a nickel column . Nickel column–bound proteins were then eluted with elution buffer  in a fill-in step gradient manner from 14 to 100%. Fractions were separated by SDS-PAGE to identify those with Cas12a protein; positive fractions were combined for concentration using a protein concentrator . The concentrated proteins were injected into a desalting column  equilibrated with desalting buffer  to ensure the long-term stability of the eluted proteins. The purity of each eluate was analyzed by SDS-PAGE  were initially amplified from genomic DNA extracted from HEK293T cells and individually cloned into a pUC-based cloning vector  to provide a template for PCR amplification. The target amplicons were purified  before in-tube DNA cleavage assays. All crRNAs used in this study were synthesized by IDT . Sequences of target DNAs, crRNAs and oligonucleotides are listed in Supplementary Tables 39 to determine the cleavage efficiency of each Cas12a RNP complex. Prism GraphPad was used for statistical analysis and data plotting.Target DNA #1, #2 and #3 (5 cells) using 4D-Nucleofector  with the CM-130 program.The purified mgCas12a-1 (126 pmol) and synthetic full-length crRNA1 or crRNA3  were mixed and incubated at room temperature for 20 min for RNP assembly. After adding 1 µL Alt-R® Cas12a (Cpf1) Electroporation Enhancer , the RNP complexes were individually transfected into the HEK293T cells  following the manufacturer’s instructions. Using the genomic DNA as a template, PCR amplification was performed with HsCCR5 gene-specific primers to amplify the substrate DNA for T7E1 assay. Purified PCR products (300 ng) in NEBuffer 2 (NEB) were denatured (95 °C for 10 min), and re-annealed by gradually cooling from 95 to 85 °C at − 2 °C/s and 85 to 25 °C at − 0.1 °C/s and then held at 4 °C using a thermocycler, and then the PCR products were treated with 10 U of T7 endonuclease 1  in a 20 µL final reaction at 37 °C for 15 min. The products were analyzed on a 2.0% agarose gel. The DNA band intensity was determined using ImageJ software.Supplementary Figures.Supplementary Tables.


Methods: We retrospectively retrieved NBS data and analyzed total homocysteine, methylmalonic acid and methyl citrate on stored NBS dried blood spots (DBS) of 70 infants diagnosed with symptomatic B12 deficiency and compared them to 646 matched and 434 unmatched DBS controls to evaluate the Austrian and Heidelberg B12 NBS algorithms. Results: The sensitivity of NBS in detecting infants later diagnosed with symptomatic B12 deficiency at median age 10.9 weeks was ≤10%. Total homocysteine was higher in DBS for the unmatched controls who were born in hospitals providing N2O compared to in hospitals not providing N2O, with median total homocysteine 4.0 µmol/L compared to 3.5 µmol/L . Conclusion: NBS algorithms were unable to identify most infants diagnosed with symptomatic B12 deficiency after the neonatal period. Being born in hospitals providing N2O may impact total homocysteine at NBS.Background: The sensitivity of newborn screening (NBS) in detecting infants that later develop symptomatic vitamin B12 deficiency is unknown. We evaluated the predictive value using NBS algorithms in detecting infants that later were clinically diagnosed with symptomatic B12 deficiency. Furthermore, we investigated whether being born in a hospital using nitrous oxide (N Twenty-eight (40%) had either S-B12 < 148 pmol/L or S-holoTC < 35 pmol/L, 34/67 (51%) had S-B12 < 200 pmol/L and 62/70 (89%) had either S-B12 < 200 pmol/L or S-tHcy > 10 µmol/L. Sixty of 66 (91%) had tHcy ≥ 8 µmol/L The mothers (n = 60) had median S-B12 254 pmol/L [187–342]. In a subgroup of 30 infants with S-B12 < 160 pmol/L (n = 20) or holoTC < 35 pmol/L (n = 10), median S-B12 was 144 [129–188] pmol/L, S-holoTC 31 [25–39] pmol/L, S-tHcy 12 [11–16] µmol/L, S-MMA 1.34 [0.43–2.42] µmol/L and the median of 25 maternal S-B12 was 229 [183–287] pmol/L.2O was available as birth analgesia at 25 (74%) of these hospitals. In total, 239/434 (55%) of unmatched controls were born in hospitals providing N2O. tHcy was higher for the unmatched controls who were born in hospitals providing N2O compared to in hospitals not providing N2O, with tHcy = 4.0 µmol/L compared to 3.5 µmol/L , while mean MMA was 0.26 µmol/L compared to 0.21 µmol/L, respectively (p = 0.131). The clinical cases and the matched controls were all born at two hospitals which provided N2O [We applied the same percentiles for tHcy as the Austrian published NBS algorithm . The tHcided N2O . DescripFirst-tier pathways identified clinical cases in 19% using the Heidelberg algorithm  and 5.7%p < 0.001, p < 0.001). Of the second-tier tests, tHcy had the strongest correlation  with serum or plasma tHcy at diagnosis of symptomatic B12 deficiency. NBS tHcy had the best diagnostic accuracy among the second-tier tests with AUC = 0.665, followed by MMA with AUC = 0.639. Methionine and methionine/phenylalanine did not correlate with diagnostic markers  at NBS. However, at diagnosis of symptomatic infant B12 deficiency, maternal dose of N2O was associated with both tHcy and MMA. We therefore suggested that N2O is a risk factor for later presenting symptomatic infant B12 deficiency [When authors of published NBS programs have reported high sensitivities and specificities for infant B12 deficiency ,6, a bioficiency ,16.Presenting symptoms in three of the five NBS positive infants were spells of apneas, absences, or seizures, and two of five showed abnormal eye contact. These are potentially life-threatening symptoms that could have been prevented with NBS for B12 deficiency. Half of the cases with B12 < 160 or holoTC < 35 pmol/L had head lag at pull-to-sit and a third had tremor, which were significant findings compared to clinical controls. The yield of NBS was doubled in this subgroup with a stricter B12 definition, although the sensitivity remained ≤10%. We speculate in a difference in sensitivity of having symptoms from low B12 between different genotypes of the B12 dependent enzymes, and then there is a risk that the more sensitive and vulnerable infants will be missed in NBS.2O was available at the hospital of birth or not; however, data on the individual mothers receiving N2O or not was not retrieved for this cohort. This information would probably have strengthened the association between mothers N2O intake and tHcy in DBS as we have shown in a recent study [Our study was original in the design of combining clinical cases with symptomatic B12 deficiency with their respective NBS results and re-analysis of DBS. In the unmatched 450 DBS controls, we only had access to whether Nnt study . Maternant study , and thint study , but it To summarize, NBS showed a low sensitivity for symptomatic B12 deficiency in our cohort of infants presenting beyond the neonatal period. However, NBS may still play an important role in detecting and treating breastfed newborns with B12 deficiency but the shortcomings of NBS in detecting all infants prone to develop B12 deficiency should be acknowledged and sustain awareness of B12 deficiency as a cause of subtle and overt neurological symptoms in infancy.


Moreover, the large binding constant (3.44 × 1014 M−1) between Al3+ and the sensor was calculated by fluorescence titration experiment. In addition, the synergistic effect mechanism due to photoinduced electron transfer (PET) and C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N isomerization was deduced according to the fluorescence behavior. In addition, the fluorescence imaging in living cells was studied systemically, which exhibited high fluorescence sensing activity toward Al3+.In the study, a highly selective and sensitive fluorescence sensor derived from nitrogen heterocycle was synthesized and characterized. By the fluorescence experiments, it was found to show a higher response toward Al In the study, a highly selective and sensitive fluorescence sensor derived from nitrogen heterocycle was synthesized and characterized. Thus, the monitoring of metal ions is of great significance. Among these common metals, aluminium is a non-essential metal element for bio-organisms. According to the reports of the World Health Organization, the average daily human intake of aluminium is approximately 3–10 mg.8 At present, aluminium is a proven neurotoxin, and the abnormal content of aluminium can cause many health hazards such as osteomalacia, Alzheimer's disease, and breast cancer. It can also damage the brain and kidneys.9–11 In addition, beyond the permissible concentration criterion of aluminium, the environment is adversely affected because it can enter the living organisms through the biosphere to induce the death of plants, soil pollution, and even diseases in humans.12–17 Due to the inadequate understanding of aluminium, it remains an important research topic. Thus, there is a considerable need for designing highly selective fluorescence sensors, which can dynamically and in real-time detect and rapidly analyze the aluminium element in biological and environmental system. In the living organism's environment, aluminium is mainly in the form of ions. Its ionic form also widely exists in the natural environment. Moreover, aluminium ion mainly exists in the aqueous system under physiological pH value condition. Therefore, it is a significant task to design the fluorescence sensor for Al3+ in aqueous media. To date, some fluorescence sensors for aluminium ion derived from organic molecules have been designed and reported.18–21 Consequently, considerable efforts need to be devoted for developing fluorescence sensors for Al3+ in aqueous media.22,23Recently, the highly selective and sensitive fluorescence chemsensors for detecting metal ions have attracted significant attention.3+, Co2+, and Cu2+ can interfere with the aluminium binding.24,25 Thus, it is very important to construct sensitive fluorescence chemosensors for aluminium ion in aqueous media. In continuation of our research topic based on metal ion fluorescence sensors,26,27 in the present investigation, a novel nitrogen heterocycle is developed to increase the selectivity and sensitivity for detecting Al3+. According to the spectral analysis, the fluorescence sensor exhibited high sensing property for Al3+ over other metal ions in the aqueous media (pH = 7.2). Compared with some reported fluorescence sensors for Al3+, the sensor derived from the heterocyclic compound exhibited unusual double ion coordination structures. Also, in the probe there are two highly efficient fluorophores, which are helpful in enhancing the luminescence efficiency. Moreover, the sensing mechanism was deduced to be due to the synergistic effect of photoinduced electron transfer (PET) and CN isomerization, which arose from the coordination interaction between Al3+ and the sensor.When aiming at the rational development of highly selective fluorescence sensors for targeting aluminium ion, the choice of binding molecules is the dominant factor because in a complicated system many metal ions such as Fe3+, Cu2+, Zn2+, Cd2+, Hg2+, Mg2+, K+, Mn2+, Co2+, Cr3+, Fe3+) were investigated in C2H5OH/H2O (4 : 1) solution. As shown in 3+ lead to the significant blue fluorescence signal. Except for the fluorescence increase for Al3+, other metal ions could not cause obvious fluorescence changes in L. It proved that the interaction between Al3+ and L gave rise to the large enhancement of fluorescence. The fluorescence selectivity behavior primarily indicated that L could act as a fluorescence probe for Al3+ under C2H5OH/H2O environment and physiological pH. This aqueous condition was an obvious advantage of the sensor. Moreover, to illustrate the high selectivity of L toward Al3+, the fluorescence images were also taken  was shown in 3+, the intensity of the absorption peaks at 230 nm, 275 nm and 320 nm decreased significantly. Simultaneously, the absorption intensity at 250 nm, 300 nm and 375 nm increased. The characteristic changes in the peaks (red shift) clearly indicated the coordination interaction between L and Al3+. This is because the coordination between the sensor and Al3+ lead to the energy increase of the n–π* transition from L, which resulted in the change in the UV-Vis spectrum. In addition, the new absorption band at 375 nm corresponded to the pale yellow color of the L–Al3+ solution  due to the reactive hydrogen of the hydroxide radical disappeared. In addition, the chemical shift (δ = 7.87) due to the reactive hydrogen of the imino group saw a marked change with a decreasing trend. These changes in the NMR signals indicated that the two groups (–OH and –NH–) could be acting as the coordination sites for Al3+. Also, in the process of coordination, the reactive hydrogen atom from the –OH is released. Moreover, the coordination modes were demonstrated by the HRESI-MS titration 2 + L–H+ + (NO3−)3) and 724.4231 ((Al3+)2 + L–H+ + (NO3−)3 + 2Na+) were accompanied by the generation of a new metal complex molecule, which emerged from the 1 : 2 coordination mode between L and Al3+.To confirm the coordination ratio between L and Alith Al3+ , the cheitration . The MS 3+ demonstrated the sensor's ability for sensing Al3+ in biological systems. The HeLa cells were treated with L for 30 min and the fluorescence signal was recorded. The HeLa cells showed no fluorescence signals. Then, the aqueous solution of Al3+ was added to the HeLa cells treated with a solution of L. As shown in 3+. The experiment demonstrated that L could act as a highly selective fluorescence sensor for Al3+ in biological systems. Moreover, the cytotoxicity (HeLa cells and HepG2 cells) of L was tested by CCK8 methods. As shown in 3+ in a low concentration aluminium ion solution.The fluorescence microscopy images of HeLa cells with L and L–AlAll the chemicals containing solvents were of reagent grade and were used without further purification. The HeLa cells were provided by the Bioeagle Technology Limited Corporation, Wuhan, P. R. China.The synthetic routine is shown in 28The precursor (P) of L was synthesized and characterized by the reported method and was used without further characterization.3OH/H2O (v : v = 1 : 2) gave the target product L which was dried under vacuum. Yield: 75%, mp: 260–261 °C. 1H NMR , δ 8.10–8.12 , δ 7.93–7.99 , δ 7.87 , δ 7.75–7.78 , δ 7.67–7.69 , δ 7.60–7.63 , δ 7.56–7.58 , δ 7.30–7.33 , δ 6.75–6.79 , δ 6.44–6.46 , δ 6.27–6.31 , δ 3.71 , δ 2.31 . 13C NMR . The solid powder of L exhibited a relatively regular columnar configuration as seen in the SEM images (A methanol solution (5.0 mL) of quinoline-2-carbaldehyde  was added to another methanol solution (10 mL) containing P . Then, the solution was refluxed for 10 h and cooled to room temperature, which lead to the appearance of a white precipitate. The mixture was filtered and dried under vacuum. Recrystallization from CHM images .λ = 0.71073 Å) by using φ–ω scan mode. Semi-empirical absorption correction was applied to the intensity data using the SADABS program. The structure was solved by direct methods and refined by full matrix least-square on F2 using the SHELXTL-97 program. All non-hydrogen atoms were refined anisotropically. All H atoms were positioned geometrically and refined using a riding model. Details of the crystal parameters, data collection, and refinements for L are summarized in Moreover, the X-ray diffraction measurement for L (size: 0.40 mm × 0.10 mm × 0.10 mm) was performed on a Bruker SMART APEX II CCD diffractometer equipped with a graphite monochromatized Mo Kα radiation  solution. To evaluate the influence of pH on the fluorescence, the fluorescence intensity of L at various pH values in the presence of Al3+ was tested  of the solution was controlled by the HEPES buffer solution. Stock solutions (1.0 × 10−3 M) of metal ions  were prepared in double-distilled water. The stock solution of 1.0 × 10−3 M was prepared in C2H5OH/H2O (4 : 1) and the pH was controlled by the HEPES buffer solution (pH = 7.2). In the titration experiments, each time 2 mL aqueous solution containing 10 μL solution of L (1.0 × 10−3 M) was filled in a quartz optical cell of 1 cm optical path length. Then, 10 μL of Al3+ stock solution was added to the compound solution with a micro-pipette. Spectral data were recorded 0.5 min after the addition. In the selectivity experiment, the test samples were prepared by mixing appropriate amounts of the metal ion stock solution into 2 mL C2H5OH/H2O solution of L (10 μL). For fluorescence measurements, excitation wavelength was at 350 nm.All spectroscopic measurements were performed in Cs tested . Under s3+ was calculated by the linear Benesi–Hildebrand expression.29,30where, I is the change in fluorescence intensity in the presence of Al3+ at 451 nm, Ks is the stability constant, and [L] and [M] is the concentration of L and Al3+, respectively. I0 is the fluorescence intensity of L in the absence of Al3+. On the basis of the plot of 1/(I − I0) versus 1/[Al3+], the stability constant can be obtained.The binding constant for L–AlNMR spectra were recorded on a Varian VR400-MHz spectrometer with TMS as the internal standard. The melting points of the compounds were determined on a Beijing XT4-100X microscopic melting point apparatus. The UV-Vis spectra were recorded on a Perkin-Elmer Lambda-35 UV-Vis spectrophotometer. Fluorescence spectra were obtained on a Cary Eclipse spectrophotometer at room temperature. The fluorescence image of the HeLa cells was obtained using Olympus FV1300 laser confocal fluorescence microscope. The crystal data were collected by the Bruker Smart-Apex-II instrument. The SEM images were obtained by F250 thermal field emission scanning electron microscope.−1 penicillin, and 100 U mL−1 streptomycin and were maintained at 37 °C in a humidified atmosphere (5% CO2 and 95% air). The cells were seeded in a six orifice plate and allowed to adhere for 12 h before treatment. Then, the cells were incubated with fresh media containing 100 μL of the sensor solution (dissolved in DMSO/C2H5OH (1 : 4), 1 mM). After 0.5 h, the growth medium was removed, and the cells were washed with saline solution several times. The cover glass was then mounted on a microscope glass slide and was studied under a microscope. Then, 200 μL fresh water media containing Al(NO3)3  was added to the above cell solution containing L. After the cells were incubated for 0.5 h, they were washed with saline solution several times, and were then studied under a microscope. Imaging was performed using an Olympus FV1300 laser confocal fluorescence microscope (excitation wavelength: 355 ± 10 nm).The HeLa cells were cultured in a flask in Dulbecco's modified Eagle's medium supplemented with a heat-inactivated bovine serum (10%), 100 U mLThe cytotoxicity of L towards the HeLa and HepG2 cells was evaluated by the following procedure. The cultured cells were placed in a sterilized phosphate buffer solution. After that, the cells were treated with the L solution (DMSO solvent) at various concentrations . After incubation for 12 h, 100 μL of CCK8 culture medium solution was added to the above solution, and incubated for 0.5 h. The absorption was measured at 450 nm. The cytotoxicity of L was evaluated by measuring the cell viability, which was achieved with a hemacytometer under a microscope. The cytotoxicity of the solvent was deducted.3+ derived from a nitrogen heterocyclic compound. The fluorescence response mechanism due to the synergistic effect of photoinduced electron transfer (PET) and CN isomerization was concluded by the fluorescence and energy change processes accompanying the no-fluorescence to intense blue fluorescence states. By structure analysis, it was found to exhibit the unusual double ion coordination configuration. Moreover, the sensor showed high sensitivity for sensing Al3+ in C2H5OH/H2O media. In addition, the cell fluorescence imaging was also investigated with HeLa cells, which exhibited high penetration depth. The coordination sites on the sensor L for binding Al3+ were confirmed by NMR and MS titrations. Such investigations endowed the potential application of sensor for tracking and detecting of Al3+ in biological and environment fields.In summary, we have fabricated a highly selective fluorescence sensor for AlThere are no conflicts to declare.RA-009-C8RA10036E-s001RA-009-C8RA10036E-s002