In mice infected with SARS-CoV or MERS-CoV,both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailableNHC-prodrug (β-D-N4-hydroxycytidine-5′-isopropyl ester), improvedpulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoVyields in vitro and in vivo were associated with increased transition mutation frequencyin viral but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. Thepotency of NHC/EIDD-2801 against multiple coronaviruses and oral bioavailability highlightits potential utility as an effective antiviral against SARS-CoV-2 and other futurezoonotic coronaviruses.Coronaviruses (CoVs) traffic frequently between species resulting in novel diseaseoutbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agentof COVID-19. Herein, we show that the ribonucleoside analogβ-D-N Orthocoronavirinae  familycirculates in many avian and mammalian species. Phylogenetically, CoVs are divided into 4genera: alpha (group 1), beta (group 2), gamma (group 3) and delta (group 4). Three newhuman CoV have emerged in the past 20 years with severe acute respiratory syndrome CoV(SARS-CoV) in 2002, Middle East respiratory syndrome CoV (MERS-CoV) in 2012, and nowSARS-CoV-2 in 2019  is orally bioavailable ribonucleoside analogwith broad-spectrum antiviral activity against various unrelated RNA viruses includinginfluenza, Ebola, CoV, and Venezuelan equine encephalitis virus (VEEV)(50) of 0.15 μM and no observed cytoxicity insimilarly treated uninfected cultures across the dose range  . Using a recombinantMERS-CoV expressing nanoluciferase (MERS-nLUC)( >10 μM) . Thethef >10 μM . Wethenf >10 μM with an  qRT-PCR . Like th50 = 0.024μM), which correlated with reduced genomic (ORF1) and subgenomic (ORFN) RNA inpaired samples (50 = 0.14 μM)and in copies of genomic and subgenomic RNA in SARS-CoV- infected HAE  cell cultures. HAE model thearchitecture and cellular complexity of the conducting airway and are readily infected bymultiple human and zoonotic CoV, including SARS- and MERS-CoV . In MERS samples . We obsected HAE . Thus, N50  buthuman-infecting CoV are found in only the alpha and beta subgroups thus far . There i50 (21). Con50 . As our To explore the breadth of antiviral efficacy against zoonotic CoV, we performed antiviralassays in HAE with three zoonotic Bat-CoV: SHC014, HKU3, and HKU5. Closely related to thebeta 2b SARS-CoV, Bat-CoV SHC014 is capable of replicating in human cells withoutadaptation. TherefoIt has recently been shown that NHC treatment increases the mutation rate in viralgenomic RNA of RSV . We thenWe then examined the mutational spectra induced by NHC, which can be incorporated intoviral RNA as a substitution for either cytosine (C) or uracil (U). RNA-mutagenicantivirals may incorporate in both nascent negative and positive sense RNA during genomereplication . Adenine4-hydroxycytidine-5′-isopropyl ester), designed forimproved in vivo pharmacokinetics and oral bioavailability in humans and non-humanprimates  prevented body weight loss following SARS-CoV infection on 2 dpi and thereafter  as compared tovehicle treated animals . Treatme, After obtaining promising in vivo efficacy data with SARS-CoV, we investigated whetherEIDD-2801 would be effective against MERS-CoV. As the murine ortholog of the MERS-CoVreceptor, dipeptidyl peptidase 4 (DPP4), does not support viral binding and entry, all invivo studies were performed in genetically modified mice encoding a murine DPP4 receptorencoding two human residues at positions 288 and 330 (hDPP4 288/330 mice) preventedbody weight loss from 2 through 6 dpi  mRNA, a highlyup-regulated innate immune-related gene after MERS-CoV infection  and host interferon stimulated gene 15  against multiple emerging CoV in vitro and antiviral efficacy ofits prodrug, EIDD-2801, in mouse models of CoV pathogenesis. Coupling cell lines andprimary HAE cell cultures, we evaluated the antiviral activity of NHC against the threemost recently emerged human CoV: SARS-CoV, MERS-CoV, and SARS-CoV-2. For both SARS-CoV andMERS-CoV, the data presented for HAE studies are representative of those from 2-3 separatehuman donors. For SARS-CoV-2, the HAE were from a single human donor. We evaluated drugcytotoxicity in both Calu-3 2B4 and HAE cell cultures. Calu-3 and the SARS-CoV andMERS-CoV HAE studies were performed in biological triplicate. HAE studies with SARS-CoV-2and the SARS- and MERS-like bat CoV were performed with two wells per condition. Drugeffects were measured relative to vehicle controls in vitro and comparisons in vivo wereperformed to vehicle controls. We also aimed to determine the antiviral efficacy ofEIDD-2801 in mouse models of CoV pathogenesis. These studies were intended to provide thepreclinical data to justify nonhuman primate studies and human clinical trials. Mice wereage- and sex-matched and randomly assigned into groups before infection and treatment.Pathology was scored blinded by a board-certified veterinary pathologist. Primary data forall studies are provided in data file S1.Efficacy studies were performed in animal biosafety level 3 facilities at UNC ChapelHill. All work was conducted under protocols approved by the Institutional Animal Care andUse Committee at UNC Chapel Hill (IACUC protocol #16-284) according to guidelines set bythe Association for the Assessment and Accreditation of Laboratory Animal Care and theU.S. Department of Agriculture.4-hydroxycytidine  and its prodrug EIDD-2801  was supplied by EmoryUniversity Institute for Drug Discovery (EIDD). NHC was supplied as a 10 mM stock in DMSOand EIDD-2801 as a solid and solubilized in vehicle containing 10% PEG400, 2.5% CremophorRH40 in water  prior to use. RDV was solubilized in 100% DMSO andprovided by Gilead Sciences, Inc as previously described , 20% fetal bovine serum(Hyclone) and 1x antibiotic/antimycotic (Gibco). At Vanderbilt University Medical Center(VUMC), Calu-3 2B4 were propagated in DMEM supplemented with 20% FBS (Gibco), 100 U/mlpenicillin and streptomycin (Gibco), and 0.25 μM amphotericin B (Corning). At VUMC,VeroE6 cells were cultured in DMEM supplemented with 10% FBS (Gibco), 100 U/ml penicillinand streptomycin (Gibco), and 0.25 μM amphotericin B (Corning). At UNC, VeroE6cells were cultured in DMEM supplemented with 10% Fetal Clone II (Hyclone) and 1xantibiotic/antimycotic (Gibco). Murine delayed brain tumor (DBT) cells were maintained inDMEM supplemented with 10% FBS (Gibco), 100 U/ml penicillin and streptomycin (Gibco), and0.25 μM amphotericin B (Corning). Primary human airway epithelial (HAE) cellcultures were obtained from the Tissue Procurement and Cell Culture Core Laboratory in theMarsico Lung Institute/Cystic Fibrosis Research Center at UNC and are described morethoroughly below  of 0.08. Cells were infected and concurrently treated with NHC intriplicate per drug dilution for 1hr, after which viral inoculum was aspirated, cultureswere rinsed once and fresh medium containing drug or vehicle was added. At 48 hours postinfection, nanoluciferase expression as a surrogate for virus replication was quantitatedon a Spectramax plate reader (Molecular Devices) according to the manufacturer’sinstructions . For the 100% inhibition control, diluted MERS-nLUC wasexposed to short-wave UV light  for 6 min to inhibit the ability of the virus toreplicate. For the 0% inhibition control, cells were infected in the presence of vehicleonly. DMSO was kept constant in all conditions at 0.05%. Values from triplicate wells percondition were averaged and compared to controls to generate a percent inhibition valuefor each drug dilution. The IC50 value was defined as the concentration atwhich there was a 50% decrease in luciferase expression. Data were analyzed using GraphPadPrism 8.0. The IC50 values were calculated by non-linear regression analysisusing the dose-response (variable slope) equation (four parameter logistic equation): Y =Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)). To measure cell viability todetermine if there was any NHC-induced cytotoxicity, Calu-3 2B4 cells were plated andtreated with NHC only as described above. Cells were exposed to the same ten-pointdilution series created for the in vitro efficacy studies. As above, 0.05% DMSO-treatedcells served as the 0% cytotoxicity control. Wells without cells served as the 100%cytotoxic positive control. After 48 hours, cell viability was measured on a Spectramax(Molecular Devices) via Cell-Titer Glo Assay (Promega) according to themanufacturer’s protocol. Similar data were obtained in three independentexperiments.SARS-CoV-2 in Calu-3:Calu-3 2B4 cells wereadsorbed with MOI 0.1 PFU/cell of SARS-CoV-2 (2019-nCoV/USA-WA1/2020 strain) at37°C. Plates were manually rocked every 10 min to redistribute the inoculum. After30 min, virus inoculum was removed, cells were washed with Phosphate buffered saline (PBS)once to remove unbound virus, medium containing NHC or vehicle control (DMSO) was addedback onto the cells, and cells were incubated for 72 hours at 37°C.SARS-CoV-2 in Vero E6: Vero E6 cells were plated at 20,000 cells/well ina 96-well plate. 24hr later, medium containing a dose response of NHC was added concurrentwith SARS-CoV-2 (2019-nCoV/USA-WA1/2020 strain) at an MOI of 0.05. 48 hours postinfection, cell viability was measured by CellTiter Glo assay.SARS-CoV, MERS-CoV, and SARS-CoV-2 in HAE: Human tracheobronchialepithelial cells provided by Dr. Scott Randell were obtained from airway specimensresected from patients undergoing surgery under University of North Carolina InstitutionalReview Board-approved protocols (#03-1396) by the Cystic Fibrosis Center Tissue CultureCore. Primary cells were expanded to generate passage 1 cells and passage 2 cells wereplated at a density of 250,000 cells per well on Transwell-COL (12mm diameter) supports(Corning). Human airway epithelium cultures (HAE) were generated by provision of anair-liquid interface for 6 to 8 weeks to form well-differentiated, polarized cultures thatresembled in vivo pseudostratified mucociliary epithelium (table S1) and TaqMan Universal PCR Mix (LifeTechnologies). Results were then normalized as described above.Total RNA was isolated using the Zymo Direct-zol RNA MiniPrep Kit (Zymo Research Corp.)according to the manufacturer’s directions. Cells were treated with 1μMstaurosporine (Sigma-Aldrich) as a positive control. First-strand cDNA was generated usingSuperscript III reverse transcriptase (Life Technologies). For quantification of cellularmarkers of toxicity/apoptosis, real-time PCR was performed using commercially validatedTaqMan-based primer-probe sets (Mouse lungs were stored in RNAlater (ThermoFisher) at -80°C until processed viahomogenization in TRIzol (Invitrogen). Total RNA was isolated using Direct-zol RNAMiniPrep kit (Zymo Research). Previously published TaqMan primers were synthesized byIntegrated DNA Technologies (IDT) to quantify MERS genomic RNA : 5′-TGCTCCAACAGTTACAC-3′, Reverse: 5′-AAGCCCAGGCCCTACTATTAGC)(Cell supernatants were harvested in TRIzol LS reagent (Invitrogen), and RNA was purifiedfollowing phase separation by chloroform as recommended by the manufacturer. The RNA inthe aqueous phase was collected and further purified using PureLink RNA Mini Kits(Invitrogen) according to the manufacturer’s protocol. Viral RNA was quantifiedusing one-step quantitative reverse transcription PCR (qRT-PCR) on a StepOnePlus Real-TimePCR system (Applied Biosystems) by TaqMan Fast Virus 1-Step Master Mix chemistry (AppliedBiosystems). SARS-CoV-2 N gene RNA was amplified using forward(5′-GACCCCAAAATCAGCGAAAT) and reverse (5′-TCTGGTTACTGCCAGTTGAATCTG) primersand probe (5′- FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1) designed by the United StatesCenters for Disease Control and Prevention . Copy numbers were interpolated from a standard curve produced with dilutions ofN gene RNA. Briefly, SARS-CoV-2-N positive control plasmid DNA  wasamplified using forward (5′-TAATACGACTCACTATAGGGATGTCTGATAATGGACCCCA) and reverse(5′- TTAGGCCTGAGTTGAGTCAG) primers, resulting in a 1280 nucleotide fragmentcontaining a T7 promoter. The PCR product was purified by column (Promega) and in vitrotranscribed using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) according to themanufacturer’s protocol. Transcribed RNA was purified using RNeasy mini kit(Qiagen) according to the manufacturer’s protocol, and serial 10-fold dilutionswere quantified as described above.10 combinations) fromwhich Illumina indexed libraries are made. We used a multiplexed Primer ID library prepapproach and MiSeq sequencing to investigate the presence of mutations in the viralgenomes and murine mRNA. We designed cDNA primers targeting multiple regions on the viralgenome and murine mRNA, each with a block of random nucleotides (11 bp long) as the PrimerID . Viral RNAwas extracted using QIAamp viral RNA kit. A pre-amplification titration of templates wasperformed to estimate the amount of template to use. We used SuperScript III to make cDNAwith multiplexed cDNA primers based on the regions to be sequenced. We used 41R_PID11 forthe pilot sequencing and titration determination. For the MERS-CoV sequencing, wemultiplexed nsp10_PID11, nsp12_PID11 and nsp14_PID11 for the cDNA reaction; for the murinemRNA sequencing, we used mixed primers of nsp10_PID11, ifit3_PID11, isg15_PID11. Afterbead purification, we amplified the cDNA with a mixture of forward primers (based on thedescribed schemes) and a universal reverse primer, followed by another round of PCR toincorporate Illumina sequencing adaptors and barcodes in the amplicons. Aftergel-purification and quantification, we pooled 24 libraries for MiSeq 300 base paired-endsequencing. The TCS pipeline version 1.38 (https://github.com/SwanstromLab/PID) was used to process the Primer IDsequencing data and construct template consensus sequences (TCSs) to represent eachindividual input templates, and the sequences of each region in the pool wasde-multiplexed. The RUBY package viral_seq version 1.0.6  was used to calculate the mutation rateat each position. NCBI SRA Accession numbers for sequence data are as follows: PRJNA613261 and incubated at37°C for 1hr after which wells were overlaid with 1X DMEM, 5% Fetal Clone 2 serum,1X antibiotic/antimycotic, 0.8% agarose. Two days after, plaques were enumerated togenerate a plaque/ml value. Lung hemorrhage is a gross pathological phenotype readilyobserved by the naked eye driven by the degree of virus replication where the colorationof the lung changes from pink to dark red . First, we performed prophylactic dose escalation studies for both SARS- andMERS-CoV to determine the most efficacious dose of EIDD-2801 per virus. For SARS-CoV, incohorts of equivalent numbers of male and female 20-29 week old SPF C57BL/6J (Stock 000664Jackson Labs) mice (n = 10/dose group), we administered vehicle  or 50, 150 or 500 mg/kg EIDD-2801 by oral gavage 2 hours prior tointranasal infection with 1E+04 PFU mouse-adapted SARS-CoV strain MA15 in 50μl.Mice were anaesthetized with a mixture of ketamine/xylazine prior to intranasal infection.Vehicle or drug was administered every 12hr for the remainder of the study. Body weightand pulmonary function by whole body plethysmography were measured daily. On 5 dpi,animals were sacrificed by isoflurane overdose, lungs were scored for lung hemorrhage, andthe inferior right lobe was frozen at −80°C for viral titration via plaqueassay. Briefly, 500,000 Vero E6 cells/well were seeded in 6-well plates. The followingday, medium was removed and serial dilutions of clarified lung homogenate were added perplate , -2 hours (n= 10), +12 hours (n = 10), +24 hours (n = 10), +48 hours (n = 10). The numbers of mice pergroup for the MERS-CoV therapeutic studies were as follows: Vehicle (n = 9), -2 hours (n =9), +12 hours (n = 9), +24 hours (n = 7), +48 hours (n = 10). As described above, each daymouse body weight and pulmonary function were quantitated. On 5 dpi for SARS-CoV and 6 dpifor MERS-CoV, animals were humanely sacrificed and tissues were harvested and analyzed asdescribed above. In addition, for the MERS-CoV study, lung tissue was harvested and storedin RNAlater (Thermo Fisher) at -80°C and then thawed, homogenized in Trizol reagent(Invitrogen) and total RNA was isolated using a Direct-zol RNA MiniPrep kit (ZymoResearch). This total RNA was then used for Primer ID and qRT-PCR.Pulmonary function was monitored once daily via whole-body plethysmography . Mice intended for this analysis were randomly chosenprior to the initiation of the study. Briefly, after a 30-min acclimation time in theplethysmograph, data for 11 parameters was recorded every 2 s for 5 min.Two different and complementary quantitative histologic tools were used to determine ifantiviral treatments diminished the histopathologic features associated with lung injury.Both analyses and scoring were performed by a Board Certified Veterinary Pathologist whowas blinded to the treatment groups.American Thoracic Society lung injury scoring tool. In order to helpquantitate histological features of ALI observed in mouse models and increase theirtranslation to the human condition, we used the ATS scoring tool (Diffuse Alveolar Damage (DAD) tool. The second histological tool toquantitate lung injury was reported by Schmidt et al.. Protein similarity scores werecalculated using Blosom62 matrix. The accession numbers used were: PDCoV (KR265858), AIBV(NC_001451), HCoV-229E (JX503060), PEDV (NC_003436), MHV (AY700211), HCoV-HKU1 (DQ415904),HCoV-NL63 (JX504050), HCOV-OC43 (AY903460), HKU5-1 (NC_009020), MERS-CoV (JX869059),HKU9-4 (EF065516), 2019-nCoV (MN996528), HKU3-1 (DQ022305), SHC014 (KC881005), WIV1(KF367457), SARS-CoV (AY278741). Amino acid conservation scores of coronavirus RdRp weregenerated using ConSurf Server (https://consurf.tau.ac.il/) using theprotein alignment described above and visualized on the SARS-CoV RdRp structure (PDB:6NUR) in PyMol (version 1.8.6.0) protein sequence alignments and phylogenetic trees weregenerated using Geneious Tree Builder in Geneious Prime (version 2020.0.5) and visualizedusing Evolview , two-way ANOVA was performed with the suggested multiplecomparison test as advised by Prism. For comparative data with for a single timepoint Kruskal-Wallis or one-way ANOVA was performed with the suggestedmultiple comparison test. For each test, a p-value <0.05 was considered significant.Specific tests are noted in each figure legend.


Dear Editor,3. However, cytokine release syndrome (CRS), the most prominent toxicity of CAR-T cell therapy, could be serious and even life-threatening5. Tocilizumab, an IL-6 receptor antagonist, has been widely used to treat CRS-related toxicities7, although corticosteroids could effectively abrogate CRS as well5, several earlier cases showed steroids may inhibit CAR T-cell persistence and their antimalignancy efficacy8, making corticosteroid therapy in CRS be often reserved for failure of tocilizumab or for neurologic toxicity since tocilizumab could not cross the blood-brain barrier. In our center, we use corticosteroids instead of tocilizumab as the first-line agent to manage CRS, here, we assessed the influence of steroids on the treatment effect and kinetics of CAR-T cells by comparing the difference between two groups of B-ALL patients who did (42 cases) or did not (26 cases) accept steroids.Chimeric antigen receptor (CAR) T-cell therapy has been demonstrated as a promising immunotherapeutic approach for treating the patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL)9 patients treated by CAR-T therapy were from our clinical trials of ChiCTR-OIC-17013623 (CD19 for B-ALL), ChiCTR-ONC-17013648  and ChiCTR-OIC-17013523 (CD22 for B-ALL), these trials were approved by Beijing Boren Hospital institutional review board and informed consents were obtained. The lentiviral vectors encoding second generation CARs composed of CD3ζ and 4-1BB were used to produce CAR-T cells, a single dose or 2 fractionated doses  of T-cells were infused. The construction of CD19- and CD22-specific CARs, the CAR-T cell manufacture and details of clinical protocols have been described in our published papers11 and on the ChiCTR website. Treatment effects were evaluated on day 30 after T-cell infusion and then monthly in follow-up patients. Minimal residual disease (MRD) was detected by multiparameter flow cytometry (FCM) and real-time quantitative PCR for fusion genes. Extramedullary diseases (EMD) were examined using PET-CT, CT or MRI. The dynamic monitoring of CAR-T cells was performed through flow cytometric quantitation of FITC + CD3 + T cells12. B-cell aplasia (BCA) was assayed by FCM and defined as less than 3% CD19- or CD22-positive lymphocytes6. SPSS 23 software was used to analyze data and tests were two-sided, comparison of means was performed using T test or Mann Whitney U-test when continuous variables were abnormal distribution, categorical variables were compared by the chi-square test. P-value < 0.05 was considered to be statistically significant.Relapsed/refractory B-ALL2/d or more higher up to methylprednisolone 10 mg/kg/d. Once CRS was improved, steroids were rapidly reduced and stopped. This study using corticosteroids to treat CRS was approved by Beijing Boren Hospital institutional review board.Dexamethasone or methylprednisolone or both  were administrated for CRS when (1) continuous high fever not being released by antipyretics; (2) moderate-severe organ disfunction. Dexamethasone was applied in most cases especially for patients with neurologic symptoms; methylprednisolone was preferred for patients with pulmonary or liver dysfunction, and patients accepting high dose steroids. Steroids were usually started with low dose (dexamethasone 2–5 mg/dose) and would be increased if symptoms were not resolved, for severe CRS, steroids could be escalated up to dexamethasone 20 mg/m13. Five (7.4%) patients presented grade II (4 cases) and III (1 case) neurologic toxicity. Graft-versus-host disease (GVHD) induced by CAR-T therapy occurred in 6 (19.4%) of 31 post-hematopoietic cell transplantation (post-HCT) patients  were excluded. The median age was 15  years, with 28 (41.2%) adults and 40 (58.8%) children younger than 18 years, 22 (32.4%) patients presented with EMD, bone marrow blasts in patients without EMD varied between 5%–96.5%. Thirty-one (45.6%) cases had an HCT. Fifty-four (79.4%) patients received CD19- and 14 (20.6%) received CD22-specific CAR-T therapy. CRS occurred in 94.1% (64/68) of patients, 10 (14.7%) cases experienced grade I, 44 (64.7%) experienced grade II and 10 (14.7%) experienced grade III CRS2/d dexamethasone or equivalent), the average days of high-dose steroid administration was 4  and 91.3% (21/23) of patients took high-dose steroids no more than one week  patients were administrated steroids, including all of 10 with grade III CRS, 68.2% (30/44) with grade II CRS and 2 patients with no CRS but GVHD (1 case) or neurotoxicity (1 case), the duration of steroid use was 1-16 days (78.6% ≤ 7 days). Whereas 26 (38.2%) patients did not accept any steroids only supportive cares. Fourteen cases received CD22 CAR-T cells happened to be equally distributed in steroid and non-steroid group (each has 7 patients). In steroid group, 23 (54.8%) patients were given high-dose steroids  and MRD negative CR was 79.2% (19/24). In the steroid group of 42 patients, 40 obtained CR and 2 were in PR, the rate of CR was 95.2% (40/42) and MRD negative CR was 80.0% (32/40). Obviously, there was no difference between steroid and non-steroid group in CR rate (p = 0.344) or in MRD–CR rate (p = 0.249) , the rest of 24 patients achieved complete remission (CR), the CR rate, composed of both CR and CR with incomplete count recovery (CRi)p = 0.0302 on D11; p = 0.0053 on D15; p = 0.0045 on D20 and p = 0.0028 on D30), except for day 7 when CAR-T cells began to expand (p = 0.9815). These data demonstrated that steroids did not suppress the proliferation of CAR-T cells in PB, on the contrary, the T-cell expansion in steroid group was much greater, the reasonable explanation is that higher expansion of CAR-T cells led to the higher grade of CRS which hence needed to be controlled by steroids.Then, we analyzed the expansion of CAR-T cells in peripheral blood (PB) by assessing the quantity of CAR-T cells using FCM Fig.  on day 715, we investigated the existence of CAR-T cells in BM and CSF by detecting them once or twice during day 14-35 after cell infusion. In steroid group, the percentages of patients with detectable CAR-T cells were 85.2% (23/27) in BM and 68.6% (24/35) in CSF; while in non-steroid group, the percentages of patients with CAR-T cells were 78.6% (11/14) in BM and 57.9% (11/19) in CSF, there were no significant differences between two groups   and cerebrospinal fluid (CSF)12, we hence monitored BCA monthly in evaluable patients. Since most of this cohort of patients underwent transplantation or received other therapies after CART, BCA was only observed in some patients and the number of patients gradually decreased over time. At 2- and 3-month, 100%  and 84.6%  of patients presented BCA in steroid group, compared to 75%  and 50%  of patients in non-steroid group, the percentages of patients with BCA in steroid group were higher than those in non-steroid group but there were no significant differences between 2 groups  hs) Fig. . In late7). Very recently, two studies showed that early intervention with corticosteroids for CRS16 or neurotoxicity17 does not impact on the antitumor potency of CD19 CAR -T cells, which support our results, the potential mechanisms need to be further explored.In conclusion, our data revealed that corticosteroids even high-dose steroids did not influence treatment outcomes of CAR-T cells and their proliferation and duration. The possibility of our results differing from previous observation could be: we used high-dose steroids for short term  whereas they used longer time (Davila ML et al mentioned 3 cases accepted 7-21 days of high-dose steroidssupplimentary table1supplimentary table2supplimentary table3supplimentary table4supplimentary table5ethics approval


NCT-547 was an effective inhibitor of tumor growth and angiogenesis, and no toxic outcomes were found in initial hepatic and renal analysis. Our findings suggest that NCT-547 may have applications in addressing trastuzumab resistance in HER2-positive breast cancer.Trastuzumab resistance in HER2-positive breast cancer is associated with a poorer prognosis. HSP90 is thought to play a major role in such resistance, but N-terminal inhibitors of this target have had little success. We sought to investigate the utility of NCT-547, a novel, rationally-designed C-terminal HSP90 inhibitor in the context of overcoming trastuzumab resistance. NCT-547 treatment significantly induced apoptosis without triggering the heat shock response (HSR), accompanied by caspase-3/− 7 activation in both trastuzumab-sensitive and -resistant cells. NCT-547 effectively promoted the degradation of full-length HER2 and truncated p95HER2, while also attenuating hetero-dimerization of HER2 family members. The impairment of cancer stem-like traits was observed with reductions in ALDH1 activity, the CD24The online version contains supplementary material available at 10.1186/s12943-020-01283-6. HSP90 is an important protein chaperone that responds to stress conditions by maintaining the integrity of protein synthesis and folding for cellular homeostasis . HER2 isTrastuzumab resistance has been correlated to both EGFR/HER2 and HER2/HER3 heterodimers generating aberrant compensatory signaling, rendering anti-HER2 therapy ineffective [HSP90 inhibitors developed in recent years have primarily targeted the N-terminal domain of HSP90. However, no candidates have been approved to date, due to issues including poor solubility and organ impairment caused by off-target toxicity . HSF-1 ip < 0.01, Fig. We previously synthesized the C-ring truncated deguelin derivative L80 as a C-terminal HSP90 inhibitor and demonstrated that it elicits anti-metastatic activity in TNBC via suppression of STAT3 signaling . NCT-547The expression levels and phosphorylation of HER2, HER3 and EGFR were significantly reduced, together with Akt downregulation in both BT474 and JIMT-1 cells following NCT-547 challenge  have been implicated in drug resistance and metastatic relapse. A positive correlation has been reported between overexpression of HSP90 and ALDH-positive BCSCs . ImpaireCompelling evidence suggests that HSR induced by N-terminal targeting HSP90 inhibitors is a major obstacle that impedes anti-tumor activity . HER2 prp < 0.001, Fig. To confirm the interplay between NCT-547 and C-terminal binding site of HSP90, we conducted a specific HSP90α C-terminal inhibitor screening assay. Compared with geldanamycin, NCT-547 significantly restrained C-terminal HSP90 activity (***Stable HER2- and p95HER2-overexpressing cell lines were generated from MDA-MB-231 TNBC cells lacking HER2 expression to examine whether NCT-547 could effectively degrade HER2 and p95HER2. While HER2-overexpressing cells showed both expression of ECD- and ICD-HER2, the p95HER2-overexpressing cells expressed ICD-HER2 specifically . Cell vip < 0.001, Fig. p < 0.001, Fig. p < 0.001, Fig. To confirm the physiological relevance of our in vitro observations, we examined the impact of NCT-547 on tumor growth in trastuzumab-resistant xenografts. The growth of JIMT-1 tumors was significantly inhibited by treatment with NCT-547, and tumor burden in the NCT-547-treated group was less than the control counterparts (***There was no significant decline in body weight observed (Additional file Our observations of the novel rationally-designed C-terminal HSP90 inhibitor NCT-547 suggests that it may have potential to address limitations in the treatment of trastuzumab-resistant HER2-positive breast cancer Fig. j. FurtheAdditional file 1. Materials and Methods.Additional file 2. Experimental Procedure for the Synthesis of NCT-547.Additional file 3. Supplementary Figures.


FBW7, which block degradation of the pro-survival Bcl-2 family protein Mcl-1. We tested if Mcl-1 inhibition can be used to develop a precision combination therapy for overcoming regorafenib resistance.Intrinsic and acquired resistance to targeted therapies is a significant clinical problem in cancer. We previously showed that resistance to regorafenib, a multi-kinase inhibitor for treating colorectal cancer (CRC) patients, can be caused by mutations in the tumor suppressor FBW7-mutant and -wild-type (WT) CRC cells and tumors, and in those with acquired regorafenib resistance due to enriched FBW7 mutations. Furthermore, translational potential was explored by establishing and analyzing FBW7-mutant and -WT patient-derived organoid (PDO) and xenograft (PDX) tumor models.Methods: Small-molecule Mcl-1 inhibitors were tested on CRC cells with knock-in (KI) of a non-degradable Mcl-1. Effects of Mcl-1 inhibitors on regorafenib sensitivity were determined in in vitro and in vivo, including those with FBW7 mutations. Importantly, Mcl-1 inhibition also sensitized FBW7-mutant PDO and PDX models to regorafenib. In contrast, Mcl-1 inhibition had no effect in FBW7-WT CRCs.Results: We found that highly potent and specific Mcl-1 inhibitors such as S63845 overcame regorafenib resistance by restoring apoptosis in multiple regorafenib-resistant CRC models. Mcl-1 inhibition re-sensitized CRC tumors with intrinsic and acquired regorafenib resistance FBW7 mutations might be a potential biomarker predicting for response to the regorafenib/Mcl-1 inhibitor combination.Conclusions: Our results demonstrate that Mcl-1 inhibitors can overcome intrinsic and acquired regorafenib resistance in CRCs by restoring apoptotic response.  Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US RAS and RAF mutations F-box and WD repeat domain-containing 7 (FBW7) are intrinsically resistant to regorafenib. FBW7 is a tumor suppressor and E3 ubiquitin ligase frequently mutated in CRCs FBW7 hot-spot mutations at R505, R465 and R479 FBW7 mutations in intrinsic and acquired resistance to regorafenib suggests that Mcl-1 is an attractive target for developing a precision combination therapy for overcoming regorafenib resistance in FBW7-mutant CRCs.Regorafenib inhibits the RAS/RAF/MEK/ERK axis, which is aberrantly activated in most CRCs due to prevalent Targeting pro-survival Bcl-2 family proteins is a promising therapeutic strategy and has led to the approval of the Bcl-2-selective inhibitor Venetoclax FBW7 mutations in promoting Mcl-1-dependent resistance to regorafenib, as well as how this resistance can be reversed by Mcl-1 inhibition. Our study provides a compelling rationale for developing a precision combination therapy on CRCs that are refractory to regorafenib treatment using Mcl-1 inhibitors.In this study, we evaluated the effects of Mcl-1 inhibitors on regorafenib sensitivity by using isogenic cell line, xenograft, patient-derived organoid (PDO) and patient-derived xenograft (PDX) models. Our results revealed a striking efficacy of Mcl-1 inhibitors in sensitizing regorafenib-resistant CRCs to regorafenib. We demonstrate the critical role of Mcl-1-KI) were generated by homologous recombination as described previously FBW7-KO HCT116 cells was obtained from Horizon Discovery 2 in McCoy's 5A modified media (Invitrogen) supplemented with 10% defined FBS (HyClone), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). For drug treatment, cells were plated in 12-well plates at 20-30% density 24 hours before treatment. Anticancer agents including regorafenib, sorafenib, S63845, AZD5991, and AMG176 were dissolved in dimethylsulfoxide (DMSO) and diluted to appropriate concentrations with cell culture medium.Human CRC cell lines, including HCT116, DLD1, Lim1215, Lim2405, RKO, SW837, SW48, LoVo, SW1463, HCT-8 and LS411N, and the mouse CRC cell line CT26 were obtained from ATCC. HCT116 cells with knock-in of the Mcl-1 phosphorylation site mutant S121A/E125A/S159A/T163A -5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay using the MTS Assay Kit (Promega) as described Cells seeded in 96-well plates at a density of 1×10Western blotting was performed using antibodies listed in Binding of Mcl-1 inhibitors to endogenous Mcl-1 was analyzed by CETSA based on a published protocol Cell lysis, preparation of cell lysates, and IP using 1-2 µg of IP antibodies  was perfAdherent and floating cells were harvested after treatment. Apoptosis was measured by counting condensed and fragmented nuclei after nuclear staining with Hoechst 33258 (Invitrogen) as previously described Mcl-1-KI cell lines and detect FBW7 hotspot mutations in regorafenib-resistant CRC cells and patient-derived samples, genomic DNA was isolated by using ZR-96 Quick-gDNA Kit (ZYMO Research) according to the manufacturer's instructions. One μL out of 50 μL genomic DNA preparation was amplified by PCR using previously described cycle conditions Mcl-1 KI: 5'-GGGTCTTCCCCAGTTTTCTC-3'/5'-AATGAACCCCCTTACCTTGG-3'; FBW7 R465: 5'-CCCAACTTCCCATTCCCTTA-3'/5'-ATTAGTATGCCCCTGCAACG-3'; and FBW7 R479/R505: 5'-GGTGGAGTATGGTCATCACAAA-3'/5'-CAAAACGCTATGGCTTTCCT-3'.To verify Patient-derived CRC organoids were established using surgically resected CRC tissues from the Pitt Biospecimen Core (PBC) at University of Pittsburgh as described Before treatment, organoids were digested into small clumps and seeded into 24-well or 96-well plates at appropriate density and cultured for 2 days. After treatment, organoid cell viability was analyzed by using the CellTiter-Glo® 3D Cell Viability Assay Kit (Promega) according to the manufacture's protocol. Active caspase 3 in organoids was analyzed by immunostaining as described ad libitum. Cell line xenografts were established by subcutaneously injecting 4×106 HCT116, Mcl-1-KI, or HCT116-R cells into both flanks of 5-6-week-old female Nu/Nu mice (Charles River). Syngeneic tumors were established by injecting 5×105 CT26 cells in to into both flanks of 5-6-week-old BABL/cJ mice (Jackson Laboratory). PDX tumors were established and propagated in 5-6-week-old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratory) as described FBW7-mutant and microsatellite stable (MSS) tumor (T4N0M1) in the sigmoid colon of a 77-year-old male. PDX2 was established from a FBW7-WT and MSS tumor (T2N0) in the right colon of a 69-year-old male. Xenograft tumors reached ~60 mm3 in size before treatment.All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Mice were housed in a sterile environment with micro isolator cages and allowed access to water and chow 2. Ethical endpoint was defined as a time point when a tumor reached 1.5 cm or more in any dimension.Tumor-bearing mice were randomized into different groups and treated with regorafenib  with or without a combination with S63845 . S63845 was dissolved in 25 mM HCl, 20% hydroxypropyl-β-cyclodextrin (Sigma). Regorafenib was dissolved in Cremephor EL/95% ethanol (50:50) as a 4× stock solution and diluted to the final concentration with sterile water before use. Six mice were included in each group. Tumor growth was monitored by calipers, and tumor volumes were calculated according to the formula 1/2×length×widthTumor tissues were dissected and fixed in 10% formalin and embedded in paraffin. Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling  and active caspase 3  immunostaining was performed on 5 μm paraffin-embedded tumor sections as described P values were calculated by the Student t test between two groups or one-way ANOVA in three or more groups and considered significant if P< 0.05. The mean ± s.d. was displayed in the figures.Statistical analyses were performed using GraphPad Prism Ⅵ software. Mcl-1-KI cells relative to the parental HCT116 cells. Treating Mcl-1-KI cells with the Mcl-1 inhibitor S63845, AZD5991, or AMG176 completely restored regorafenib sensitivity in Mcl-1-KI cells, indicated by regorafenib inhibitory concentration 50 (IC50)  of an Mcl-1 phosphorylation site mutant (S121A/E125A/S159A/T163A) in regorafenib-sensitive HCT116 CRC cells A blocks s Figure B-C. Mcl-s Figure D, and acs Figure A 9, 23. Mcl-1-KI cells. However, this binding was disrupted by treatment with S63845  on the parental HCT116 cells. This analysis showed that treatment with S63845 or AMG176 markedly protected endogenous Mcl-1 from heat-induced denaturation, indicating strong binding of these inhibitors to Mcl-1  HCT116 cells generated by homologous recombination FBW7-mutant cells, FBW7-KO cells were much less sensitive to regorafenib and deficient in regorafenib-induced apoptosis and caspase activation  alone significantly suppressed the growth of the parental HCT116 tumors and induced Mcl-1 degradation; but had little effect on Mcl-1-KI and HCT116-R tumors  using surgical specimens from 12 CRC patients. Upon targeted genomic sequencing for FBW7 mutational hotspots including R505, R465 and R479, we identified a PDO with a heterozygous dominant R505C mutation  (Figure FBW7-mutant PDO (Figure FBW7-WT PDO with or without regorafenib (FBW7 mutated CRCs.To explore the translational potential of Mcl-1 inhibitors in CRC, we tested if the regorafenib/S63845 combination is efficacious against patient-derived CRC samples containing orafenib A-B. Thesin vivo models, PDX models better recapitulate heterogeneity, histology, and molecular alterations of patient tumors FBW7 (FBW7-mutant PDX (Figure FBW7-mutant PDX (Figure FBW7-mutant PDX (Figure Compared with other ors FBW7 C. RegoraX Figure A-B. RegoX Figure C. S63845X Figure D. CollecMetastatic CRC is one of the most deadly cancers characterized by poor prognosis and low five-year survival rate of just 11% Our results from isogenic cell line, xenograft, and patient-derived models demonstrate that inhibiting Mcl-1 is an effective approach for re-sensitizing CRCs with intrinsic and acquired resistance to regorafenib. Regorafenib is a multi-kinase inhibitor approved for treating CRC and other gastrointestinal malignancies FBW7-mutant CRCs suggest that FBW7 status is critical in determining the response to the regorafenib/Mcl-1 inhibitor combination. FBW7 is frequently mutated in human cancers including 10-15% of CRCs FBW7 mutations have a broad functional role in determining therapeutic responses of cancer cells FBW7 encodes an F-box protein that functions as a substrate receptor for SCF-type of ubiquitin ligase complexes FBW7 mutations to explore their use as a biomarker of therapeutic response.The observed differences between WT and FBW7 hotspot mutations can predict for poorer response to regorafenib. FBW7 mutations may appear during the course of regorafenib treatment and indicate an acquired resistance mechanism, or they may be present in regorafenib-naïve patients and signal intrinsic resistance to regorafenib. Anticancer therapies including targeted therapies often generate highly heterogeneous patient responses and are associated with substantial toxicities. Hepatotoxicity and other side effects have been observed in regorafenib-treated patients KRAS and BRAF mutations have been routinely used to exclude anti-EGFR therapy in CRC patients FBW7 mutations in liquid biopsies The current study highlights multiple areas of translation. First, our results suggest that the presence of -FBW7-driven regorafenib resistance and help to develop a precision therapy for improving CRC treatment. The potential use of this strategy needs to be further assessed in clinical studies to determine the safety and efficacy of this combination, as well as the utility of FBW7 mutations and Mcl‐1 level to predict therapeutic response.In conclusion, we demonstrate that Mcl-1 inhibitors can be used to overcome mutantSupplementary figures and table.Click here for additional data file.


Forsythia suspensa, displays antioxidant activity. This study examined the neuroprotective effects of IFY and its underlying mechanisms. In the L-glutamate (L-Glu)-induced apoptosis of HT22 cells, IFY increased cell viability, inhibited mitochondrial apoptosis, and reduced the intracellular levels of reactive oxygen species (ROS), caspase-3, -8 and -9 after 3 h of pretreatment and 12–24 h of co-incubation. In the APPswe/PSEN1dE9 transgenic (APP/PS1) model, IFY reduced the anxiety of mice, improved their memory and cognitive ability, reduced the deposition of beta amyloid (Aβ) plaques in the brain, restrained the phosphorylation of the tau protein to form neurofibrillary tangles, inhibited the level of 4-hydroxynonenal in the brain, and improved phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway-related mitochondrial apoptosis. In Aβ1-42-induced U251 cells, IFY relieved the mitochondrial swelling, crest ruptures and increased their electron density after 3 h of pretreatment and 18–24 h of co-incubation. The improved cell viability and mitochondrial function after IFY incubation was blocked by the synthetic PI3K inhibitor LY294002. Taken together, these results suggest that IFY exerts a protective effect against AD by enhancing the expression levels of anti-apoptosis proteins and reducing the expression levels of pro-apoptosis proteins of B-cell lymphoma-2 (BCL-2) family members though activating the PI3K/AKT pathway.Improving mitochondrial dysfunction and inhibiting apoptosis has always been regarded as a treatment strategy for Alzheimer’s disease (AD). Isoforsythiaside (IFY), a phenylethanoid glycoside isolated from the dried fruit of  As one of the chronic neurodegenerative disease, Alzheimer’s disease (AD) attacks more and more elderly people , and is Aβ has been found in the brain mitochondria of AD patients and AD mice  and inteForsythia suspensa (Thunb.) Vahl (Lianqiao in Chinese) )), factor-related apoptosis ), beta-site APP cleaving enzyme 1 (BACE1 (a key enzyme required for AD-related Aβ synthesis [p < 0.01])), amyloid precursor protein ), presenilin-1 (PS-1 (leading to increasing toxicity of APP via producing a large amount of neurotoxic Aβ deposits [p < 0.05])), caspase-3 (a well-known apoptotic executor )), as seen in Label-free quantitative proteomics is considered a reliable tool and is used as a fairly precise measure of relative protein abundance in complex protein mixtures . Compare signals  )), AKT (phospho T450 (dilution 1:2000 [56 kDa])), AKT (dilution 1:2000 (56 kDa)), BCL-2 (dilution 1:1000 (26 kDa)), AIF (dilution 1:2000 (57 kDa)), PI3K (dilution 1:1000 (85 kDa)), glyceraldehyde-3-phosphate dehydrogenase (GAPDH (dilution 1:2000 [36 kDa])), followed with an incubation on horseradish peroxidase-conjugated secondary antibody at a dilution of 1:5000 including goat anti-rabbit IgG (H+L) and goat anti-mouse IgG (H+L) at 25 °C for 4 h. An electrochemiluminescence kit was used to visualize the protein bands under the imaging system . The quantitation of the results was accomplished by Image J software .U251 cells . The statistical significance was determined using SPSS 16.0 software and using a one-way analysis of variance, then post hoc multiple comparisons were performed (Dunn test).


Scientific Reports 10.1038/s41598-020-58507-4, published online 31 January 2020Correction to: This Article contains an error in Figure 4 where the labels PAX8 and PTEN on the x-axis were reversed. The correct Figure 4 appears below as Figure


S. cerevisiae, Dot1 modulates the meiotic checkpoint response triggered by synapsis and/or recombination defects by promoting Hop1-dependent Mek1 activation and Hop1 distribution along unsynapsed meiotic chromosomes, at least in part, by regulating Pch2 localization. However, how this protein regulates meiosis in metazoans is unknown. Here, we describe the effects of H3K79me depletion via analysis of dot-1.1 or zfp-1 mutants during meiosis in Caenorhabditis elegans. We observed decreased fertility and increased embryonic lethality in dot-1.1 mutants suggesting meiotic dysfunction. We show that DOT-1.1 plays a role in the regulation of pairing, synapsis and recombination in the worm. Furthermore, we demonstrate that DOT-1.1 is an important regulator of mechanisms surveilling chromosome synapsis during meiosis. In sum, our results reveal that regulation of H3K79me plays an important role in coordinating events during meiosis in C. elegans.Epigenetic modifiers are emerging as important regulators of the genome. However, how they regulate specific processes during meiosis is not well understood. Methylation of H3K79 by the histone methyltransferase Dot1 has been shown to be involved in the maintenance of genomic stability in various organisms. In  C. elegans DOT-1.1, a conserved histone methyltransferase that catalyzes histone H3 lysine 79 (H3K79) methylation, regulates chromosome pairing, synapsis and recombination, thereby promoting normal meiotic progression and chromosome segregation. Moreover, we show that DOT-1.1 promotes a meiotic checkpoint function set in place to survey chromosome synapsis through a mechanism that appears to be distinct from the one described in budding yeast. Altogether, our study reveals a link between the regulation of H3K79me and the normal progression of meiosis.Sexual reproduction relies on the production of sperm and eggs by the specialized cell division program called meiosis. Errors during meiosis often lead to miscarriages, infertility, and birth defects. Therefore, understanding the mechanisms regulating meiosis has major implications for human health. Chromatin modifiers have emerged as important regulators of a wide-range of cellular processes. However, despite their importance, their roles during meiosis remain poorly understood. Here, we show that  Meiosis is an essential cell division program for all sexually reproducing organisms. It halves the genome’s content by following one round of DNA replication with two successive rounds of cell division, meiosis I and II, to generate haploid gametes (i.e. sperm and oocytes). A series of well-orchestrated events ensure accurate homologous chromosome segregation at meiosis I while preserving sister chromatid associations until meiosis II . Namely,Alterations in the chromatin landscape are mediated in part by post-translational modifications of histones which form octamers wrapped by DNA (one H3/H4 heterotetramer and two H2A/H2B dimers) to form the building blocks of chromatin, the nucleosomes . Post-trdisruptor of telomeric silencing in yeast , rad-54(ok615)/ht2[bli-4(e937)let-?(q782)qls48] ; LGII, pch-2(tm1458); LGIII, zfp-1(gk960739); LGIV, ced-3(n1286); LGV, syp-1(me17)/nt1[unc-?n754]let-?gls50). The zfp-1(gk960739) mutant has been outcrossed at least eight times from the VC40040 strain generated by the Million Mutation Project [ribed in . The N2 nt1unc-?n4let-?glsAge-matched  individual hermaphrodites were placed on regular NGM plates and assessed for embryonic lethality, sterility and the percentage of males among their progeny. Worms were moved every 24 hours to new NGM plates for four consecutive days. The total number of fertilized eggs laid, hatched, the number of progeny that reached adulthood, and the frequencies of male progeny, were scored.Whole mount preparations of dissected gonads and immunostainings were performed as in  with somQuantitative analysis of pHIM-8 foci consisted of scoring the number of foci observed per nucleus for nuclei in all seven zones composing the germline. Between 4 and 6 gonads were scored for each genotype .To evaluate chromosome morphology in oocytes at diakinesis, whole worms were Carnoy’s fixed and then stained with DAPI as in . Images Imaging was performed using an IX-70 microscope (Olympus) with a cooled CCD camera  controlled by the DeltaVision system (Applied Precision). Images were collected using a 100x objective with or without auxiliary magnification (1.5x) and Z-stacks were set at 0.2 μm thickness intervals. Image deconvolution was done using the SoftWoRX 3.3.6 program (Applied Precision) and processed with Fiji ImageJ ,80.dot-1.1 ced-3, ced-3 and zfp-1 are as follows: zone 1 (n = 84), zone 2 (n = 80), zone 3 (n = 85), zone 4 (n = 93), zone 5 (n = 95), zone 6 (n = 76), and zone 7 (n = 93). Statistical comparisons were performed using the Fisher’s exact test.Probe for the 5S rDNA locus (center of chromosome V) was generated and labeled as in . QuantifQuantitative analysis of RAD-51 foci for nuclei in all seven zones composing the germline was performed as in . The avezfp-1, syp-1 and pch-2 mutants as well as in the corresponding double and triple mutants was performed as in [Acridine orange (AO) staining of apoptotic germ cells in wild type (N2), ed as in . BrieflyAdult worm lysates were resolved via SDS-PAGE on a 4 to 12% gel (Invitrogen NuPAGE Bis-Tris gel) and transferred to a 4.5-μm nitrocellulose membrane (Bio-Rad) at 100 mA for 1 h. The membrane was blocked using 3% (wt/vol) BSA in PBS with 0.01% Tween for 1 h, followed by overnight incubation at 4°C with an anti-ZFP-1 C-terminus-specific antibody , dilutedS1 FigHigh-resolution images of early pachytene nuclei (zone 4) from wild-type germlines co-stained with H3K79me1 , H3K79me2  or H3K79me3 , AcK (magenta) and DAPI (blue). In wild-type gonads, AcK is enriched on autosomes compared to the X chromosomes, while H3K79me1,-2,-3 signals are detected on all chromosomes. Yellow arrowheads indicate X-chromosome based on nearly absent AcK signal. Scale bar, 5 μm.(TIF)Click here for additional data file.S2 Figzfp-1(gk960739) removes the first 109 amino acids from the long isoform.The top band represents the long isoform (above 100 kDa) and the lower band corresponds to the short isoform (predicted at 65 kDa). The deletion in (TIF)Click here for additional data file.S3 Fig(A) Whole mounted gonads of wild-type and zfp-1 worms co-immunostained with SUN-1(pS8) (green) and DAPI (blue). zfp-1 mutant worms showed an extension in SUN-1-positive signal compared to wild type suggesting defects in meiotic progression. Asterisks indicate the premeiotic tip and white arrows show the direction in which nuclei move proximally in the germline (meiotic progression). Hatched lines demarcate the first (left) and the last (right) rows with nuclei showing SUN-1 (pS8) signal. At least 30 gonads from two independent biological repeats were analyzed for each genotype. Scale bars, 5 μm. (B) Top left, high-resolution images of early pachytene nuclei (zone 4) co-stained with HIM-8 (magenta) and DAPI (blue). Yellow arrow indicates nucleus with unpaired HIM-8 signal. Scale bar 5 μm. Bottom left, histogram representing the percentage of nuclei with paired HIM-8 signals scored at different zones along the germline in wild-type and zfp-1 worms. X chromosomes were scored as paired when the two HIM-8 signals were ≤ 0.75 μm apart from each other. * P<0.05, *** P<0.001, Fisher’s exact test. Top right, high-resolution images of early pachytene nuclei (zone 4) stained with DAPI and hybridized with a FISH probe against the 5S rDNA locus located near the center of chromosome V (green). Yellow arrow indicates nucleus with unpaired FISH signals. Scale bar 5 μm. Right bottom, histogram representing the percentage of nuclei with paired FISH signal (5S rDNA) scored at different zones along the germline in wild type and zfp-1. Chromosomes were scored as paired when two signals were ≤ 0.75 μm apart from each other. *P<0.025, ***P<0.0005, Fisher’s exact test. (C) Left, histogram indicating the percentage of nuclei that exhibit complete synapsis as a function of meiotic progression in wild-type and zfp-1 gonads. Nuclei showing complete overlapping signal of the lateral element component HTP-3 and the central region component SYP-1 along all chromosomes were considered as nuclei with complete synapsis. 4 gonads from two biological repeats were scored for wild type (n = 154) and zfp-1 (n = 173); n = number of nuclei scored. ***P<0.001, Fisher’s exact test. TZ, transition zone; EP, early pachytene; MP, mid-pachytene; LP, late pachytene. Right, high-resolution images of mid-pachytene nuclei co-immunostained with SYP-1 (green), HTP-3 (red), HIM-8 (yellow) and DAPI (blue). Unlike wild-type worms, 5.78% of nuclei (10/173) in zfp-1 mutants exhibited DAPI-stained regions lacking SYP-1 signal at the pachytene stage (arrow). 10% (1/10) of these nuclei exhibited discontinuities in HTP-3 signal and in 70% (7/10) the unsynapsed chromosome corresponded to the X chromosome as determined by presence of HIM-8 signal (arrowhead). Scale bar, 5 μm.(TIF)Click here for additional data file.S4 Fig(A) High-resolution images representative of mid-pachytene nuclei (zone 5) immunostained for RAD-51 (magenta) and co-stained with DAPI (blue). Scale bar, 5 μm. (B) Histogram shows the mean number of RAD-51 foci/nucleus (y-axis) scored along each zone in the germlines (x-axis) of the indicated genotypes. Between 4 and 6 gonads were scored per genotype. A significant decrease in levels of RAD-51 foci were observed for zones 3 to 5 in zfp-1 germlines compared to wild type and for zones 3 to 7 in zfp-1; syp-1 germlines compared to syp-1. Error bars represent SEM for technical repeats from two biological replicates. ***P<0.0003 by the two-tailed Mann-Whitney test, 95% C.I., after Bonferroni correction. TZ, transition zone; EP, early pachytene; MP, mid-pachytene; LP, late pachytene. (C) Table shows the percentage of -1 oocytes at diakinesis displaying each one of the indicated defects in chromosome morphology. n = number of -1 oocytes scored. Decond. = decondensation. Aggreg. = aggregates.(TIF)Click here for additional data file.S1 Tablerad-54 backgrounds, germ cell apoptosis counts and pHIM-8 foci counts.Raw data for plate phenotyping, RAD-51 foci counts in wild-type and (XLSX)Click here for additional data file.S2 Tablerad-54 backgrounds, and germ cell apoptosis.P-values for comparisons among all genotypes used in this study using the two-tailed Mann-Whitney test, 95% C.I. P-values for RAD-51 foci counts in wild-type and (XLSX)Click here for additional data file.S3 TableList of strains used in this study.(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


DNFB induced features of allergic reactions in rat basophilic leukemia (RBL2H3) cells. HHT suppressed effect of AD on the expression of Th1/Th2 cytokines. HHT inhibited passive cutaneous anaphylaxis and passive systemic anaphylaxis. MiR-183-5p, increased by antigen stimulation, was downregulated by HHT in RBL2H3 cells. MiR-183-5p inhibitor suppressed anaphylaxis and AD. B cell translocation gene 1 (BTG1) was shown to be a direct target of miR-183-5p. BTG1 prevented antigen from inducing molecular features of in vitro allergic reactions. AD increased the expression of NF-κB, and NF-κB showed binding to the promoter sequences of miR-183-5p. NF-κB and miR-183 formed positive feedback to mediate in vitro allergic reactions. Thus, HHT can be an anti-allergy drug. We present evidence that NF-κB-miR-183-5p-BTG1 axis can serve as target for development of anti-allergy drug.Homoharringtonine (HHT) is a drug for treatment of chronic myeloid leukemia. However, the role of HHT in allergic inflammations remains unknown. Mouse model of atopic dermatitis (AD) induced by 2, 4,-dinitroflurobenzene (DNFB) and anaphylaxis employing 2,4-dinitropheny-human serum albumin (DNP-HSA) were used to examine the role of HHT in allergic inflammations. HHT inhibited  Homoharringtonine (HHT) exerts apoptotic effects against acute lymphoblastic leukemia (ALL) cells and chronic myeloid leukemia (CML) cells by decreasing expression of B cell lymphoma-6 (Bcl-6) . It decrHHT in combination with hsp90 inhibitor IPI504 synergistically inhibits leukemia cells by exerting apoptotic effects . AbivertMYC gene  Acute Myeloid Leukemia (AML), by decreasing the expression of c-myc (HHT binds to NF-κB repressing factor (NKRF) . Second MYC gene . HHT binMYC gene . ApoptotMYC gene . HHT decof c-myc .Anti-cancer drug candidate, AZD7762, suppresses IgE-mediated degranulation of mast cells and passive cutaneous anaphylaxis by inhibiting functions of Lyn and Fyn . Imatiniin vitro allergic reactions. MiR-183-5p mediated atopic dermatitis (AD) and anaphylaxis. NF-κB was responsible for the increased expression of miR-183-5p during allergic reactions. BTG1 served as a target of miR-183-5p and inhibited in vitro allergic reactions. The mechanisms of anti-allergic effects by HHT merit further investigation. We provide evidence NF-κB-miR-183-5p-BTG1 axis can be a target for the development of anti-allergy drugs.In this study, we examined roles of HHT in allergic inflammations. HHT exerted negative effects on atopic dermatitis and anaphylaxis. MiR-183-5p was one of those miRNAs that was regulated by HHT during DNFB was purchased from Sigma-Aldrich . Anti-mouse and anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibodies were purchased from Thermo Pierce . All other antibodies used in this study were purchased from Santa Cruz Biotechnology . The jetPRIME transfection reagent was purchased from Polyplus . Oligonucleotides and primers used in this study were commercially synthesized by Bioneer Company .Female BALB/C mice that were five week old were purchased from Nara Biotech . Animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (KW180823-1).2 at 37°C. Human keratinocyte HaCaT cells were purchased  and expanded in Dulbecco's modified eagle medium  containing 8% fetal bovine serum  at 37°C with 5% CO2.Isolations of mast cells and macrophages were performed according to the standard procedures with slight modifications . CultureThe microRNA (miRNA) expression analysis was performed by using miRNA Array III kit  following the manufacturer's instructions.TargetScan program identified targets of miR-183-5p.-ΔΔCT method (ΔCT = CTmiR − CTreference).Total miRNA was isolated with the miRNeasy Micro Kit . The extracted miRNA was reverse transcribed using a miScript II RT Kit . The expression level of miR-183-5p was quantified with SYBR Green Master Mix  Relative expression of miRNA was calculated using the 2For miR-183-5p knockdown, cells were transfected with 10 nM oligonucleotide (inhibitor) with Lipofectamine 2000 (Invitrogen) The sequences used were 5′-AGUGAAUUCUACCAGUGCCAUA-3′ (miR-183-5p inhibitor) and 5′-TAACACGTCTATACGCCCA-3′ (control inhibitor).The pGL3 3′-UTR-BTG1 construct was made by cloning a 947-bp mouse BTG1 gene segment encompassing 3′-UTR into the XbaI site of pGL3 luciferase plasmid. The mutant pGL3 3′-UTR-BTG1 construct was made with the Quick Change site-directed mutagenesis kit (Stratagene). pFlag-BTG1construct was made by PCR amplification and cloning into Flag-tagged pcDNA3.1 vector.MiR-183-5p promoter sequences, specific primers of miR-183-5p promoter-1 sequences [5′- GGCCCAGAATCTACTGATAGTG -3′ (sense) and 5′- TAAGTCTCTCTGGAGCTGGTG -3′ (antisense)], miR-183-5p promoter-2 sequences [5′- CACCAGCTCCAGAGAGACTT -3′ (sense) and 5′- AGAGGCCCAGAAGGTAAGAC -3′ (antisense)], miR-183-5p promoter-3 sequences [5′- GTCTTACCTTCTGGGCCTCT -3′ (sense) and 5′- GACTGATTTCTTGGGTTTGCAG -3′ (antisense)], and miR-183-5p promoter-4 sequences [5′- AGCCCCGTCTTTCTCCTT -3′ (sense) and 5′- CAGACCCTACAGAGAGGTCA -3′ (antisense)] were used for detection of binding of NF-κB to the promoter sequences of miR-183-5p.Skin samples were fixed with 10% neutral buffered formalin, and embedded in paraffin. Sections (5 μm thickness) were stained with hematoxylin and eosin (H&E) or toluidine blue for leukocyte infiltration or mast cell infiltration and degranulation, respectively.Immunohistochemical staining of tissues was performed using an established avidin-biotin detection method . The following primary antibodies were used: anti-HDAC3 ; anti-MCP1 ; anti-CD163 ; anti-TSLP ; anti-BTG1 , and anti-inducible nitric oxide synthase (iNOS) . After washing, biotinylated secondary antibody was applied at 1:100 or 1:200 dilutions for 1 h. Color was developed with diaminobenzidine . Sections were counterstained with Mayer's hematoxylin. The sections incubated without primary antibody served as controls. To visualize tissue mast cells, the sections were stained with 0.1% toluidine blue (Sigma) in 0.1 N HCl for 15 min.Cells were fixed with 4% paraformaldehyde (v/v) for 10 min and then permeabilized with 0.4% Triton X-100 for 10 min. Cells were incubated with primary antibody specific to CD163 , iNOS , or NF-κB  for 2 h. Anti-rabbit Alexa Fluor 488 (for detection of iNOS) or anti-goat Alexa Fluor 546 (for detection of CD163 and NF-κB) secondary antibody (Molecular Probes) was added to cells and incubated for 1 h. Fluorescence images were acquired using a confocal laser scanning microscope and software (Fluoview version 2.0) with a X 60 objective .Symptoms of atopic dermatitis (AD) were induced by using DNFB, as previously described . Twenty-The BALB/C mice were given an intradermal injection of 2, 4-dinitrophenol (DNP)-specific IgE (0.5 μg/kg). After 24 h, the mice were intravenously injected with DNP-human serum albumin (HSA) (250 μg/kg) and 2% (v/v) Evans blue solution. After 30 min of DNP-HSA challenge, the mice were euthanized, and the 2% (v/v) Evans blue dye was extracted from each dissected ear in 700 μl of acetone/water (7:3) overnight. The absorbance of Evans blue was measured at 620 nm. To determine the effect of miR-183-5p on the passive cutaneous anaphylaxis (PCA), BALB/C mice were given an intradermal injection of IgE and intravenous injection of the indicated inhibitor (each at 100 nM). The next day, BALB/C mice were given an intravenous injection of PBS or DNP-HSA along with Evans blue solution for determining the extent of vascular permeability accompanied by PCA.To determine effect of miR-183-5p on passive systemic anaphylaxis, BALB/C mice were intravenously injected with control inhibitor (100 nM) or miR-183-5p inhibitor (100 nM). The next day, BALB/C mice were given an intravenous injection of IgE (0.5 μg/kg). The next day, BALB/C mice were intravenously injected with DNP-HSA (250 μg/kg). Changes in rectal temperatures were monitored by using a digital thermometer.Data were analyzed and graphed using the GraphPad Prism statistics program . Results are presented as means ± SE. One–way ANOVA was carried out when multiple comparisons were evaluated. Values were considered to be significant at p value less than 0.05.in vitro allergic reactions has not been reported.To identify the chemical compounds that inhibit allergic inflammations, we screened chemical library, consisting of 1019 chemicals derived from natural products. We identified several compounds that prevented antigen (DNP-HSA) from increasing β-hexosaminidase activity in rat basophilic leukemia cells (RBL2H3). Among these compounds, homoharringtonine (HHT) showed the most potent effect on β-hexosaminidase activity (data not shown). HHT, in a concentration-dependent manner, prevented antigen from increasing levels Histone deacetylase 3 (HDAC3), Suppressor of cytokine signaling 1 (SOCS1) and Transglutaminase II (TGaseII) in RBL2H3 cells (HHT exerted a negative effect on passive cutaneous anaphylaxis (PCA) employing BALB/C mouse , TH2 (IL-31), and TH17/TH22 (IL-23p19/IL-8/S100A12) mRNA expression are molecular features of human AD , TLR2, and TLR4 . SOCS1 regulates AD by forming a negative feedback loop with miR-122a-5p  . HO-1 suin vitro allergic reactions. The roles of BTG1 in allergic inflammations have not been reported.TargetScan analysis predicted BTG-1 as a target of miR-183-5p. MiR-183-5p inhibitors acts as a negative regulator of BTG1 and suppresses endothelial cell tube formation . BTG1 acIn this study, we found that miR-19a-3p was also regulated by HHT in antigen-stimulated RBL2H3 cells. MiR-19a-3p targets BTG1 and regulates apoptosis of castration-resistant prostate cancer cells . MiR-10aAutophagy is necessary for degranulation of mast cells . Mast ceAnaphylaxis involves cellular interactions among mast cells, macrophages, and endothelial cells . In thisHigh expression of histamine H1repceptor (H(1)R) is found in patients with allergic rhinitis . HHT is www.bindingdb.org) predicts that Egl nine homolog1 (PHD2), porphobilinogen synthase, Glycosylasparaginase (GA), Histone lysine demethylase or Methyl-accepting chemotaxis protein(McpS) can bind to HHT. It will be necessary to examine roles of these proteins in allergic inflammations.For better understanding the mechanism of anti-allergic effect of HHT, identification of proteins that bind to HHT will be necessary. For this, biotin labeling of HHT will make it possible to identify HHT-binding proteins. Incubation of cell lysates with biotin-labeled of HHT, followed by electrospray-ionization quadrupole time-off light mass spectrometry (ESI-Q-TOF MS) will make it possible to identify HHT-binding proteins . Bindingvia non-specific mechanisms and cause side effects (Chemotherapy can cause skin toxicities such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) . It is p effects . In orde effects . HHT-aci effects . ApplicaIn this study, we showed potential of HHT as an anti-allergic drug. We also presented evidence that NF-κB-miR-183-5p-BTG1 axis mediates AD and anaphylaxis. For better understanding mechanism of anti-atopic effect of HHT, it would be necessary to identify molecules that regulate the expression levels of genes that are involved in NF-κB-miR-183-5p-BTG1 axis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of Kangwon National University.DJ and HSJ conceived and designed this study. MK, HC, and YKw performed experiments and analyzed the data. DJ and HSJ drafted and revised the manuscript. YKi provided suggestion for the experiments. All authors contributed to the article and approved the submitted version.This work was supported by National Research Foundation Grants , a grant from the BK21 plus Program.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.


C. elegans Wnt ligands, with known roles in neuron migration, axon guidance and nerve ring placement ). CWN-2 is highly expressed in the SMDD neurons , and therefore we were interested in determining whether CWN-2 regulates SMDD axonal development.CWN-2 is one of five cwn-2(ok895) deletion allele to analyze SMDD axonal development. We found that loss of cwn-2 caused a defective SMDD axonal phenotype, where axons do not extend along the dorsal sublateral cord, suggesting that the axons did not exit the nerve ring due to defects in axon outgrowth or guidance . Expressing cwn-2 using this promoter partially rescues the defective axonal phenotype , one Ror receptor (CAM-1) and one Ryk receptor (LIN-18) . We analyzed the effect of loss-of-function mutations for each receptor and found that loss of cam-1, but not the other receptors, caused defective SMDD axonal development . The SIA, SIB, RMED/V and SMDD motor neuron cell bodies are all positioned in the nerve ring and extend posteriorly-directed axons . Therefore, our data show that CWN-2 and CAM-1 have a common role in controlling axon extension in posteriorly-directed lateral or sublateral axons.Next, we investigated which Wnt receptor controls SMDD axonal development. There are six Wnt receptors encoded in the elopment . CAM-1 wC. elegans strains were grown using standard growth conditions on NGM agar at 20 °C and fed with Escherichia coli OP50. Neuroanatomical reporter strains used – rhIs4 [Pglr-1::GFP] and rpEx1739[Pctbp-1a::GFP]. Homozygous mom-5 mutant animals were selected based on the Unc phenotype caused by the linked unc-13(e1091) mutation. Day two adult hermaphrodites were anesthetized with 0.2% levamisole hydrochloride on 5% agarose pads for anatomical scoring. Schematics of axonal defects are shown instead of images as bright GFP expression in multiple cell bodies around the pharynx obscure imaging of axon defects. Refer to  for images of the SMDD axons posterior to the pharynx. ‘Defective SMDD axonal phenotype (%)’ indicates the percentage of SMDD axons that do not extend along the dorsal sublateral cord. Two SMDD axons (L and R) were scored per animal. For cwn-2(ok895) rescue experiments, transgenic animals were compared to the cwn-2(ok895) mutant. Three biological replicates were performed, and statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons tests.Pctbp-1a::cwn-2 rescue construct was generated by cloning the cwn-2 1083 bp cDNA sequence, amplified from C. elegans cDNA, into a Pctbp-1a::GFP vector to replace the GFP sequence . Rescue constructs were injected into rhIs4; cwn-2(ok895) mutant backgrounds at 2 ng/μl with Punc-122::GFP (20 ng/μl) as injection marker.Cloning was performed using Takara In-Fusion® restriction-free cloning. The StrainsrhIs4 [Pglr-1::GFP] IIIHRN169 rhIs4 III; cwn-2(ok895) IVHRN498 rhIs4 III; cwn-2(ok895) IV; rpEx2027 [Pctbp-1a::cwn-2] line #1RJP4553 rhIs4 III; cwn-2(ok895) IV; rpEx2028 [Pctbp-1a::cwn-2] line #2RJP4554 rhIs4 III; cwn-2(ok895) IV; rpEx2029 [Pctbp-1a::cwn-2] line #3RJP4555 mig-1(e1787) I; rhIs4 IIIRJP4485 lin-17(n671) I; rhIs4 IIIRJP4486 rhIs4 III; cfz-2(ok1201) VRJP4534 cam-1(ks52) II; rhIs4 IIIRJP4688 rhIs4 III; lin-18(e620) XRJP4715 rpEx1739 [Pctbp-1a::GFP]RJP4076 cwn-2(ok895) IV; rpEx1739 [Pctbp-1a::GFP]RJP4598 mom-5(zu193) unc-13(e1091)/hT2 III; rpEx1739 [Pctbp-1a::GFP]RJP4624


Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo-Cy5 (S-Cy5), have been covalently attached to a well-characterized CXCR4-targeted self-assembling protein nanoparticle (known as T22-GFP-H6). The biodistribution of labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles has been then compared to that of the non-labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22-GFP-H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non-target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non-target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss-interpretations of the actual nanoparticle biodistribution. Appropriate biodistribution is one of the most sought properties in nanomedicine since selective drug accumulation in target tissues is expected not only to enhance its effectiveness but also to reduce undesired side effects ,2. BeingOne of the most used approaches is based on the chemical conjugation of small molecular weight fluorescent probes such as ATTO, Alexa Fluor or Cyanine molecules, in order to monitor their fluorescent signal accumulation in target and non-target tissues upon intravenous administration ,13,14,15Most of the studies described above analyze the biodistribution of small molecular weight molecules and peptides; however, whether fluorescent dye labeling changes the biodistribution of larger actively targeted nanoscale particles remains still poorly explored, despite its critical value in the design of targeted drug delivery systems.To address this issue, we have chemically conjugated two different widely known small molecular weight fluorescent probes such as ATTO488 (ATTO) and Sulfo-Cyanine5 (S-Cy5) to the well-characterized T22-GFP-H6 targeted protein nanoparticle. We have then tracked the biodistribution of the original unlabeled T22-GFP-H6 nanoparticle (measuring its intrinsic green fluorescence) and the resulting two labeled variants (T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5) upon intravenous administration in animal models bearing human, CXCR4+ diffuse large B-cell lymphoma (DLBCL) or colorectal cancer (CRC).T22-GFP-H6 is a modular fusion protein composed by an N-terminal cationic peptide T22, a potent ligand of the cell surface cytokine receptor CXCR4 ,27 overeTherefore, labeling such a well-characterized targeted nanoparticle, that display intrinsic GFP fluorescence, with some of the fluorescent probes most widely used in nanomedicine, should allow us to precisely determine the impact of the conjugation process over the original nanoparticle’ biodistribution. Thus, this work could help in making decisions on the suitability of such labeling strategies to study the biodistribution of targeted nanoscale drug delivery systems.Escherichia coli Origami B  at 20 °C overnight upon addition of 0.1 mM Isopropyl-β-d-thiogalactopyranoside (IPTG). Protein nanoparticles were then purified from the soluble fraction of sonicated bacterial cells by IMAC affinity chromatography with HisTrap Chelating HP 5 mL column  in an ÄKTA pure system . Purified proteins were finally dialyzed against sodium carbonate with salt buffer . Protein purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot immunodetection with anti-His monoclonal antibody . Protein integrity was verified by MALDI-TOF mass spectrometry and final concentration determined by Bradford colorimetric assay in a UV/light spectrophotometer  at 595 nm.The protein T22-GFP-H6 was produced as described . In shor3, 333 mM NaCl, 0.1 mM NiCl2, pH = 8). The eluent was monitored by an in-line UV–Vis detector, a Dawn Heleos MALS detector and Optilab rEX RI detector . All data were then analyzed with an Astra 6.0.2.9 software (Wyatt technology Corporation), and the average molar masses were calculated in duplicate using both UV (ε: 1.099 mL/(mg.cm)) and RI (dn/dc: 0.185 mL/g) signals as protein concentration sources.Size exclusion chromatography coupled to a Multi Angle Light Scattering (SEC-MALS) was used to calculate the average molar mass of self-assembled T22-GFP-H6 nanoparticles. For that, protein samples were injected in a Superdex 200 increase 10/300 GL column  and run in a degassed Nickel enriched sodium carbonate with salt buffer (166 mM NaHCON-hydroxysuccinimide ester (NHS-ester) conjugated ATTO488  and Sulfo-Cyanine 5  fluorescent dyes were covalently bound to T22-GFP-H6 protein nanoparticles through exposed external lysines, by ester-amine reaction. For that, an excess of fluorescent probes was incubated in presence of T22-GFP-H6 protein (2:1 dye to protein monomer molar ratio) and sodium carbonate with salt buffer in a one-pot reaction for 1 h and subsequently dialyzed against sodium carbonate with salt buffer in order to remove not reacted fluorescent dye. Conjugation efficiency was then checked by MALDI-TOF mass spectrometry.Volume size distribution and Z-potential of T22-GFP-H6 protein nanoparticles and T22-GFP-H6-ATTO nanoconjugates was determined in a Zetasizer Nano ZS  by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) respectively at 633 nm. All samples were measured in triplicate. T22-GFP-H6-S-Cy5 nanoconjugates cannot be analyzed by DLS or ELS, since Sulfo-Cyanine 5 fluorescence (excited at 633 nm) prevents reliable light scattering measurements. Ultrastructural morphometries of T22-GFP-H6, T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 (size and shape) were determined at nearly native state with field emission scanning electron microscopy (FESEM). Drops of 3 µL of diluted samples at 0.3 mg/mL were directly deposited on silicon wafers  for 30 s, excess of liquid blotted, air dried and immediately observed without coating with a FESEM Zeiss Merlin  operating at 1 kV and equipped with a high resolution in-lens secondary electron detector. In a qualitative approach, representative images of a general fields and nanostructure details were captured at two high magnifications . In a quantitative approach, nanoparticles and nanoconjugate average size were analyzed by Image J software  from FESEM images.Excitation and emission spectra of T22-GFP-H6, T22-GFP-H6-ATTO, T22-GFP-H6-S-Cy5 and free ATTO and sulfo-Cy5 dyes were recorded in a Cary Eclipse spectrofluorimeter . A quartz cell with 10 mm path length and a thermostated holder was used. λem was set at 510 nm (GFP), 523 nm (ATTO) or 662 nm (S-Cy5) for excitation spectra, and λex was set at 450 nm (GFP), 498 nm (ATTO) or 645 nm (S-Cy5) for emission spectra. Dye samples were buffered in carbonate buffer with salt pH 8.Fo and F correspond to fluorescence intensity in absence or presence of [Q] quencher (Sulfo-Cy5).The effect of the free dyes was also studied on the fluorescence properties of T22-GFP-H6. The fluorescence emission spectra of T22-GFP-H6 (0.2 mg/mL) were recorded in the presence of ATTO (0–7 µM) or Sulfo-Cy5 (0–7 µM) (λex = 488 nm). The quenching of T22-GFP-H6 fluorescence induced by Sulfo-Cy5 was evaluated with the Stern–Volmer theory . The linin vitro model to carry out the evaluation of receptor specificity of CXCR4-targeted nanoparticles. It was then also used for the generation of DLBCL mouse models. Patient-derived M5 CRC tumor tissue that displays a highly compact solid tumor with a moderate CXCR4 overexpression and a high content of stromal cells that interact with CXCR4+ tumor cells was also used for the generation of CRC mouse models since it maintains the tumor microenvironment observed in humans. A human derived Toledo Diffuse Large B-Cell lymphoma (DLBCL) cell line displaying very high CXCR4 overexpression and a scarce stroma was selected as 2 at 37 °C for 24 h. Cells were then incubated in presence of 10 nM T22-GFP-H6, T22-GFP-H6-ATTO or T22-GFP-H6-S-Cy5 for 1 h. For competition assays, a potent CXCR4 receptor antagonist AMD3100  was incubated 1 h before sample addition. Incubation plates were then treated with a “harsh” trypsin digestion (1 mg/mL for 15 min) in order to remove externally attached protein, and the internal cell fluorescence was finally analyzed in a FACS Canto flow cytometer  using a 15 mW air-cooled argon ion laser at 488 nm and a D detector (530/30 nm bandpass filter) for GFP and ATTO fluorescence and a laser at 635 nm and a B detector (660/20 bandpass filter) for Sulfo-Cy5 fluorescence. All samples were analyzed in duplicate and free ATTO488 and Sulfo-Cyanine 5 molecules  were also incubated as controls.Toledo lymphoma cells were incubated in 96-well plates in RPMI medium  containing 10% of feta bovine serum (Gibco) in humidified atmosphere and 5% CO2 at 37 °C for 1 h in absence  or presence (for unspecific protein binding) of the CXCR4 antagonist AMD3100. Cells were then cooled down to 4 °C for 1 h before adding T22-GFP-H6, T22-GFP-H6-ATTO or T22-GFP-H6-S-Cy5 nanoparticles at different concentrations (from 5 nM to 3000 nM) and incubated for an additional hour at 4 °C. Membrane attached protein was finally analyzed in a FACS Canto flow cytometer (Becton Dickinson) using a 15 mW air-cooled argon ion laser at 488 nm and a D detector (530/30 nm bandpass filter) for T22-GFP-H6 and T22-GFP-H6-ATTO and 635 nm laser and B detector (660/20 bandpass filter) for T22-GFP-H6-s-Cy5. All experiments were performed in duplicate and Propidium iodide was added to all samples in order to select living cell population. KD and NS parameters were calculated by binding saturation, one site-total nonlinear regression equation using Graphpad prism 8 software:y corresponds to fluorescence signal, Bmax is the fluorescence signal at saturation, X correspond to sample concentration, KD is equilibrium dissociation constant, NS is the slope of non-specific binding and Background correspond to system background fluorescence signal. Goodness of fitting for each of the samples is as follows: T22-GFP-H6 (R2: 0.9638), T22-GFP-H6-ATTO (R2: 0.9728) and T22-GFP-H6-S-Cy5 (R2: 0.9889).CXCR4 receptor binding affinity in terms of equilibrium dissociation constant (KD) and slope of nonspecific binding (NS) was calculated for CXCR4-targeted T22-GFP-H6, T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles in CXCR4+ Toledo Lymphoma cell line by Saturation binding experiments. For that, Toledo cells were incubated in 96-well plates in RPMI medium (Gibco) containing 10% of fetal bovine serum (Gibco) in humidified atmosphere and 5% CO6 cells/100 µL saline solution per flank. Twenty-four days later, when the SC tumors of mice reached approximately 600 mm3, 300 µg of T22-GFP-H6 (n = 3) nanoparticles and T22-GFP-H6-ATTO (n = 3) and T22-GFP-H6-S-Cy5 (n = 3) nanoconjugates were injected intravenously (i.v.) to different mice (200 µL). Finally, a mouse was injected i.v. with 200 µL of sodium carbonate with salt buffer as a negative control (n = 3). Mice were euthanized after 5 h, and tumors and liver, kidney, lung, pancreas and spleen were excised to measure their fluorescence in IVIS® Spectrum .NOD SCID (NOD.CB17-Prkdcscid/NCrCrl) female mice (4 weeks old) were obtained from Charles River Laboratories. Mice were housed in microisolator units and kept under specific pathogen-free (SPF) conditions with sterile water and food ad libitum. After 1 week of quarantine, NOD SCID mice were subcutaneously injected with Toledo cells in 2 flanks at 10 × 103, mice received one single 300 μg i.v. bolus of T22-GFP-H6 (n = 3), T22-GFP-H6-ATTO (n = 3) or T22-GFP-H6-S-Cy5 (n = 2) in sodium carbonate with salt buffer. Control animals received the same buffer (n = 2), 0.25 μg of free ATTO488 (n = 2) or 0.0347 μg of free Sulfo-Cy5 (n = 2). At 5 h, mice were euthanized and subcutaneous tumors and organs  were collected. Biodistribution of T22-GFP-H6 nanoparticles, and ATTO488- and Sulfo-Cy5-labeled nanoconjugates in tumor and non-tumor organs was determined by measuring the emitted fluorescence in ex vivo tissue sections (3 mm thick), using the IVIS® Spectrum (Perkin Elmer) platform.Five-week-old female Swiss Nu/Nu mice, weighing 18–20 g  were used and maintained in specific pathogen-free conditions. To generate the subcutaneous (SC) mouse model, we implanted subcutaneously 10 mg of the patient-derived M5 colorectal (CRC) tumor tissue from donor animals in the mouse subcutis. When tumors reached approximately 500 mm® Spectrum imaging platform. Those showing a fluorescence emission over the tissue background auto-fluorescence were selected.All in vivo experiments and procedures in both colorectal cancer and DLBCL mouse models were approved and conducted in accordance with guidelines by the institutional animal Ethics Committee of Hospital de Sant Pau . Furthermore, based on the results obtained in previous biodistribution assays, we used the sample size and power calculator  with the two-sided test for two independent means to determine sample size. The results established that the minimum number of animals per group to be used in both models was 2. Time points for the biodistribution studies were selected based on previous assays. In them, T22-GFP-H6 showed the peak of protein uptake in tumor at 5 h post-injection, and fluorescence was then progressively reduced by proteolysis leading to complete degradation of the nanoparticle . To sele® Spectrum (Perkin Elmer) correlate with the amount of administered protein accumulated in each tissue. Emitted FLI signals were first digitalized, displayed as a pseudocolor overlay and normalized by the organ size to be expressed as radiant efficiency ((p/sec/cm2/sr)/μW/cm2). FLI values were calculated subtracting the auto-fluorescence from the negative control and finally expressed as tissue/tumor ratio to compare the biodistribution between different nanoparticles. Data were also expressed as a percentage of distribution among the analyzed organs. The excitation–emission filters used to measure the FLI of each nanoparticle and nanoconjugate were selected in a preliminary experiment where different sets of bandpass (BP) filters were tested and the spectral regions where samples showed the maximum fluorescence signal selected: T22-GFP-H6 was excited by 465/30 BP filter (450–480 nm) and recorded with 520/20 BP filter (510–530 nm). T22-GFP-H6-ATTO was excited by 500/30 BP filter (485–515 nm) and recorded by 540/20 BP filter (530–550 nm). Finally, T22-GFP-H6-S-Cy5 was excited by 640/30 BP filter (625–655 nm) and monitored by 700/20 BP filter (690–710 nm).The fluorescent signals (FLI) measured using the IVISin vitro experiments were determined by Student t-tests using SigmaPlot 10.0 . For the in vivo experiments t-test and Mann–Whitney U-tests were used to analyze differences in biodistribution between nanoparticle and nanoconjugate groups for each tested organ. Differences between groups were considered significant at p < 0.05. All in vivo statistical analyses were performed using SPSS version 22.0 package , and values were expressed as mean ± standard error of the mean  and T22-GFP-H6-ATTO (GFP and ATTO fluorescence) and by red fluorescence emission for T22-GFP-H6-S-Cy5 (S-Cy5 fluorescence). The size of T22-GFP-H6-S-Cy5 could only be determined by image analysis of high-resolution electron microscopy since Cy5 fluorescence also interfered with DLS and ELS measurements.T22-GFP-H6 is a fully fluorescent 30.6 kDa modular protein that spontaneously self-assembles into 12 nm toroid-like structure containing around 10 copies of the polypeptide  Figure . AdditioWe then tested whether fluorescent labeling could significantly disturb the CXCR4-mediated endosomal cell internalization of nanoparticles in CXCR4+ human Toledo lymphoma cells. In this context, all labeled and non-labeled nanoparticles showed very efficient cell internalization upon in vitro incubation B althougOnce proved that nanoparticle labeling did not alter their receptor specificity for internalization, we studied the proteolytic and structural stability of nanoparticles in serum. In this sense, no protein degradation was detected upon incubation of labeled and unlabeled nanoparticles in human serum for 24 h. No significant alterations in nanoparticles hydrodynamic size were either detected, suggesting strong structural stability and no interactions with serum proteins .We then finally moved forward to in vivo biodistribution studies in a Toledo cell line-derived subcutaneous CXCR4+ DLBCL mouse model to determine if the labeling of nanoparticles could significantly modify their original biodistribution pattern. The intravenous administration of control T22-GFP-H6 nanoparticles, which shows enough intrinsic fluorescent for ex vivo tracking, resulted in their stable accumulation in target tumor cells at 5 h post-administration (74% ± 5% TFU). In contrast, they showed very low uptake in main non-target organs such as spleen, liver, kidney or lung (range 0.2–3% TFU), with only moderate signal in pancreas (20% ± 6% TFU)  as previThe same comparative biodistribution study was performed in an alternative M5 tumor cell line-derived subcutaneous CXCR4+ colorectal cancer mouse model in order to determine if the previously observed biodistribution changes were replicated in a completely different tumor model. Similarly, as found in the DLBCL model, the administration of the original unlabeled T22-GFP-H6 nanoparticles resulted in a high accumulation in tumor (57% ± 7% TFU) with only a moderate accumulation in pancreas at 5 h post-administration (20% ± 3% TFU). However, the administration of T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 also resulted in a drastic reduction of tumor uptake to the range of 20% ± 8%–22.9% ± 0.8% TFU, and in a significant accumulation of labeled nanoparticles in liver (range: 32.5% ± 0.9%–54% ± 12% TFU) and kidney (range: 11% ± 0.5%–30% ± 1% TFU) .To discard possible unspecific signal accumulation produced by free fluorescent dye molecules coming from nanoconjugate leakage in blood, we intravenously administered an equivalent fluorescent amount of free ATTO488 or Sulfo-Cy5 molecules that resulted in no accumulation in tumor or any other organ C.Developing tracking strategies for nanoparticles is a key issue in nanomedicine and especially in targeted drug delivery systems. However, whether the processes for labeling drugs or their vehicles can significantly alter their tissue accumulation pattern needs to be considered since disrupted distributions can lead to misinterpretations of the material original behavior. In this sense, we have shown here how some of the most widely used labeling molecules such as small molecular weight fluorescent probes can significantly change the biodistribution of a very well characterized active targeted protein nanoparticle namely T22-GFP-H6 ,34. We cOur results are in tune with other reported biodistribution patterns where different types of nanoparticles, including carbon nanotubes , self-asObserved changes in nanoparticle biodistribution patterns could not be related with their supramolecular structure properties since no changes over nanoparticle size, shape or surface charge was observed upon conjugation B,C. NanoIn this regard, the conjugation chemistry used for T22-GFP-H6 labeling does not seem to be the problem, since the covalent binding of small molecular weight drugs such as FdU, using the same lysine/amine chemistry, resulted in a perfectly functional T22-GFP-H6-FdU nanoconjugate with highly selective tumor accumulation .On the other hand, the contribution of fluorescent dye leakage from labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoconjugates and their subsequent unspecific tissue accumulation in the observed biodistribution pattern are completely discarded since the lysine-amine covalent binding of fluorochromes to the protein, which is widely used in nanomedicine including marketed products such as ADC Kadcyla , resultsThe physicochemical properties of conjugated fluorescent molecules could also be an additional factor driving the fate of nanoparticles. In fact, alternative distribution patterns have been already reported for untargeted peptides when attached to several fluorescent probes with different physicochemical properties . In thisIn this sense, although the reported hydrophobicity  and the membrane interaction factor  are significantly different for both ATTO and Sulfo-Cy5 molecules  Supplem, none ofOverall, as the exact mechanisms involved in the observed modification of biodistribution are still unclear, alternative tracking strategies such as nuclear imaging or labeling molecule encapsulation need to be considered, while new fluorescent dye molecules aimed at having low or null impact on nanoparticle biodistribution and targeting are being developed. In this sense, the incorporation of radioisotopes in the protein building block is still considered the best alternative to study their biodistribution since it maintains nanoparticles molecular structure and physicochemical properties while allowing its radioactive emission based tracking as already seen in ADCs ,63,64. MTracking-oriented labeling of nanoparticles can significantly alter their in vivo pharmacokinetic profiles, which can induce misleading interpretation of target specificity, as demonstrated here by the use of small molecular weight fluorescent molecules. The chemical conjugation of widely used fluorescent probes to a very well characterized actively targeted nanoparticle with highly specific biodistribution profile, reduces its tumor targeting capacity while dramatically increasing its liver and kidney accumulation. Although the exact mechanisms involved in this multifactorial process are still unclear, this is a highly relevant issue that needs to be considered in the design of nanoparticles as targeted drug delivery systems. In this sense, different labeling strategies need to be evaluated in order to ensure that particle pharmacokinetic properties are not modified during the process inducing dramatic misinterpretation of their tissue biodistribution.


In this article we show that genes under control of a normally Isopropyl β-gp2> is able to precisely regulate protein production rates on a cellular level in an l-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to l-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting.Here, we show that BL21-AI<gp2> outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.In all cases tested, BL21-AI< Escherichia coli (E. coli) is a promising strategy to further optimize soluble recombinant protein production (RPP) levels. This approach is of particular interest for membrane proteins (MP) or other difficult-to-express proteins (e.g. proteins rich in disulfide bonds) that tend to overwhelm host cell capacities ) (Coli Genetic Stock Center #12504) and BL21-AI  [malB+]K-12(λS) araB::T7RNAP-tetA)) (ThermoFisher Scientific) which was used as reference system.Experiments in this study were performed with gp2> was generated by linear double-stranded DNA(dsDNA) cartridges, containing the expression unit from pROCOLI(gp2) fused to a chloramphenicol acetyltransferase resistance gene (CatR), which were integrated into the bacterial chromo- some at the attTn7 site of E. coli BL21-AI according to Stargardt et al.  and mean specific GFP yield [rfu/mg] represents duplicate samples. Figure S13. Product formation kinetics and flow cytometry analysis of single‑cell expression of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing PstA-GFP fusion protein during fed-batch like cultivation. Figure S14. Process characteristic showing product formation kinetics and flow cytometry analysis of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing PstA-GFP fusion protein during fed-batch like cultivation. Figure S15. Product formation kinetics and flow cytometry analysis of single‑cell expression of E. coli strains BL21-AI  and BL21-AI<gp2>   expressing YliF-GFP fusion protein during fed-batch like cultivation. Figure S16. Process characteristic showing product formation kinetics and flow cytometry analysis of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing YliF-GFP fusion protein during fed-batch like cultivation. Figure S17. Product formation kinetics and flow cytometry analysis of single‑cell expression of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing YdiK-GFP fusion protein during fed-batch like cultivation. Figure S18. Process characteristic showing product formation kinetics and flow cytometry analysis of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing YdiK-GFP fusion protein during fed-batch like cultivation. Figure S19. Product formation kinetics and flow cytometry analysis of single‑cell expression of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing YhhJ-GFP fusion protein during fed-batch like cultivation. Figure S20. Process characteristic showing product formation kinetics and flow cytometry analysis of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing YhhJ-GFP fusion protein during fed-batch like cultivation. Figure S21. Product formation kinetics and flow cytometry analysis of single‑cell expression of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing YfbF-GFP fusion protein during fed-batch like cultivation. Figure S22. Process characteristic showing product formation kinetics and flow cytometry analysis of E. coli strains BL21-AI  and BL21-AI<gp2>  expressing YfbF-GFP fusion protein during fed-batch like cultivation. Figure S23. Western Blot showing overexpression of YhdY-GFP during fed-batch cultivations of E. coli strain BL21-AIand BL21-AI<gp2> . Figure S24. Western Blot showing overexpression of PstA-GFP during fed-batch cultivations of E. coli strain BL21-AI and BL21-AI<gp2> . Figure S25. Western Blot showing overexpression of YliF-GFP during fed-batch cultivations of E. coli strain BL21-AI and BL21-AI<gp2> . Figure S26. Western Blot showing overexpression of YdiK-GFP during fed-batch cultivations of E. coli strain BL21-AI and BL21-AI<gp2> . Figure S27. Western Blot showing overexpression of YfbF-GFP during fed-batch cultivations of E. coli strain BL21-AI and BL21-AI<gp2> . Figure S28. Western Blot showing overexpression of YhhJ-GFP during fed-batch cultivations of E. coli strain BL21-AI and BL21-AI<gp2>.