We find that sEVs contribute to paracrine senescence.Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence   •Small extracellular vesicles (sEVs) mediate paracrine senescence•Inhibition of sEV biogenesis prevents paracrine senescence•in vivoMultivesicular bodies and CD63 are increased during senescence •MS analysis identifies IFITM3 as partially responsible for sEV-paracrine senescence loxP reporter systems confirm sEV-mediated paracrine senescence, while preventing sEV release averts senescence. Mass spectrometry and functional analysis show that the IFN protein, IFITM3, is partially responsible for this phenotype.Borghesan et al. show that the soluble fraction and small extracellular vesicles (sEVs) mediate paracrine senescence. RNA sequencing and  In fact, an enrichment in the number of senescent cells has been observed in vivo during both biological and pathological processes such as development, cancer, fibrosis, and wound healing . The activation of senescence is a response to different cellular stresses to prevent the propagation of damaged cells and has been shown to occur  healing . The SAS healing . The SAS healing .in vitro and in vivo have attributed the diverse functions of the SASP to individual protein components such as interleukin-6 (IL-6) or IL-8 to reinforce autocrine senescence  (30–120 nm) of endocytic origin, whereas microvesicles are formed by the shedding of the plasma membrane. Exosomes and microvesicles are secreted by all cell types and found in most bodily fluids. Both contain nucleic acids, proteins, and lipids that generally reflect the status of the parental cell and can influence the behavior of recipient cells locally and systemically . The incHere, we show that both the soluble and sEV fractions transmit paracrine senescence . The analysis of individual cells internalizing sEVs using a reporter system shows a positive correlation between the uptake of sEVs and paracrine senescence. We can also observe an increase in multivesicular body (MVB) formation in a mouse model of oncogene-induced senescence (OIS) and high CD63 staining in human lung fibrotic lesions enriched in senescent cells. sEV protein characterization by mass spectrometry (MS) followed by a functional small interfering RNA (siRNA) screen identify the interferon (IFN)-induced transmembrane protein 3 (IFITM3) within sEVs as partially responsible for transmitting senescence to normal cells.G12V in a 4-hydroxytamoxifen (4OHT)-inducible form (ER:EV or ER:H-RASG12V). These cells undergo senescence upon treatment with 200 nM 4OHT , and the accumulation of p53 by immunofluorescence (IF)  B and 1C.nce (IF) D. As obsls by IF B and S1Cls by IF D, sugges+/CD81+ (G12V (ex vivo mouse hepatic stellate cells (mHSCs) derived from an adult mouse harboring a doxycycline (Dox)-inducible construct to express shp53  in our sEV preparations F and S1G+/CD81+ J. Previo+/CD81+ . Thus, w1+  bre1+ ss shp53  , we performed RNA sequencing (RNA-seq) of HFFF2s treated with sEVs derived from iRAS and from HFFF2s treated with Etop, mimicking OIS and DDIS, respectively A. Gene Oignature B, with tignature C. Furtheignature D and S2Aignature . Treatmeignature D and S2Bignature C. Comparignature  (SN-PS) ignature E. ELISA ignature F and S2Dignature . SimilarCDKN2A, CDKN1A, components of the SASP, and integrin β3 subunit (ITGB3), which regulates senescence (TP53 (shp53) and CDKN2A (shp16) that prevent the establishment of senescence  and GW4869 (1 and 10 μM). Torin-2 (25 and 50 nM), an mTOR inhibitor that suppresses the SASP , was useCIP A. FurtheAX by IF D and S3EAX by IF D. As perF (INK4A E and 3F.INK4A and p21CIP) were detected  allele, which develops premalignant intra-epithelial neoplasias (PanIN) (wild-type (WT) or KrasG12D mice to electron microscopy staining and imaging and found that KrasG12D-derived PanIN showed an increase in MVB formation in comparison with WT epithelial ducts  , and pre (PanIN) B  . We subjal ducts E and 4F.al ducts B , and HFFF2s expressing GFP. First, we co-cultured both GFP+ and iRAS;CD63-ch+ cells in a 1:1 ratio with 200 nM 4OHT for 2 days, followed by replenishing with fresh media for 4 days , which release mCherryr 4 days A; after croscopy A and thecroscopy B. Furthecroscopy  show intF2 cells B. Overal+ HFFF2 incubated with CD63-sEV.A 3D modeling video shows that some sEV positive for CD63 can be observed in both the bottom and upper section of the GFPloxP reporter system described by + MCF7) and MCF7 expressing a loxP flanked DsRed construct (reporter MCF7), which when recombined allows the expression of a downstream GFP gene (+ reporter MCF7 cells (that have taken up Cre-sEV), we observed a positive correlation between GFP+ and Sudan Black+ cells  <0.01 in both OIS and DDIS. The Venn diagram analysis shows that 265 proteins are deregulated in sEVs in both OIS and DDIS  p < 0.05, and (3) >4 peptide fold change between control and senescent sEVs. After applying this cutoff, we selected the top 50 most abundant proteins upregulated in both DDIS and OIS (TP53 (sip53) and CDKN2A (sip16) as a positive control (green bars)  >2 log and OIS  and conden bars)  and detels by IF G and S6B p-γH2AX C. The IF p-γH2AX , and witIFITM and the related IFN-induced protein with tetratricopeptide repeats (IFIT) transcripts upon sEV incubation, but not when treated with N-SMase (Our previous data show that IFITM3 (1) is important for CM-mediated paracrine senescence E Acosta, and (3) N-SMase A.Figure + sEVs onto beads and detecting CD81 by FACS, we confirmed that iRAS cells released more IFITM3+/CD81+ sEV particles than the iC cells  and 10 elderly (∼80 years old) donors and determined the expression levels of IFITM3 protein by immunoblotting. The expression levels of IFITM3 were increased in 6 of 10 elderly human donors, while there was very little change in the expression levels of the sEVs derived from young donors I. FurtheIntercellular communication is an important mechanism by which cells interact with one another. It can be mediated in the form of soluble factors or extracellular vesicles . HoweverloxP reporter system to determine EV internalization shows a positive correlation between sEV uptake and paracrine senescence activation.Non-cell autonomous (paracrine) senescence via the SASP has been previously described as an important mechanism during senescence , althougINK4A. In fact, other investigators have found that IFN signaling can be mediated via EVs in neural stem cells .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ana O’Loghlen  were obtained from the Culture Collections . IMR-90 , MCF7  and HEK293T  were bought from ATCC. Breast fibroblasts (BF) were isolated from a female breast mastectomy and have been described elsewhere . All celPtf1aCre;lsl-KrasG12D mice were injected subcutaneously with two doses, two days apart, of 400 μg tamoxifen per 20 g mice (Tamoxifen (Sigma) stock 20 mg/ml in corn oil) to obtain acinar cell-specific activation of KrasG12D. Mice were euthanized 5 months following treatment. All mice experiments were done with approval from the Hebrew University Animal Care and Use Committee.6 week-old Young donors were all male with an age range between 29-36 years old, while old donors were a mix between male and female and age range between 70-92. None of the donors presented underlying diseases. The study was approved by the London - City & East Committee (10/H0704/73) and all donors gave informed written consent to participate.Human tissues were received from the Papworth Hospital Research Tissue Bank (REC 08/H0304/56+5).All cells were maintained in sEV-depleted FBS. FBS was depleted of sEV by overnight (ON) ultracentrifugation at 100,000 g at 4°C . The supernatant was removed and stored in 50 mL falcons at −20°C until required. CM was collected after 72h incubation with cells, unless specified otherwise.To isolate the different EV fractions, the protocol of differential ultracentrifugation  was modisEV isolated by serial ultracentrifugation were re-suspended in 1.5 mL of suspension buffer  . Next, sEV were mixed 1:1 with 60% stock solution of iodixanol/Optiprep . Then, 1.4ml 40% iodixanol, 1.3ml 20% iodixanol and 1.2ml 10% iodixanol were successively layered on top of the sEV suspension and tubes were centrifuged at 100,000 g ON, stopping without break. After centrifugation, ten fractions of 700μl were collected from the top of the tube. Fractions were washed with 15ml PBS at 100,000 g for 1h 20 min. The fractions were re-suspended in 50 μL of lysis buffer.CM was centrifuged at 350 g for 15min at 4°C to pellet cells. The supernatant was centrifuged at 2,000 g for 20min at 4°C and filtered with a 0.22μm filter. Size exclusion chromatography (SEC) using qEV columns  was used to the isolate sEV. Twelve eluted fractions were collected in sequential fractions of 1ml according to the manufacturer’s instructions. The particle and protein concentration of each fraction was then measured by NTA and MicroBCA. The fractions enriched in particles and lacking protein contaminants were pooled, centrifuged at 100,000 g for 1h 20 min and used for functional assays.6 cells. After 24h, iC and iRAS cells were treated with 200nM 4OHT for 48h in 10% media, washed, incubated with 0.5% media and allowed to produce new CM for 72h. The sEV obtained from these cells were used to treat 10-12 wells of a 96-well plate.HFFF2 fibroblasts were plated in a 100mm dish at 1x10Young HFFF2 were treated with the CM from several experiments previously centrifuged at low speed to discard dead cells and supplemented with FBS to reach 10% in the final volume. HFFF2 treated with isolated sEV were also supplemented with media containing 10% FBS, while in the experiments performed with MCF7, the cells were incubated with isolated exosomes resuspended in 0.5% FBS media.24h after plating HFFF2 in 96-well plates, senescence was induced by adding 200nM 4OHT. 48h after the cells were washed and supplemented with 0.5% FBS and the indicated inhibitors for 2-3 days, after which plates were washed again to remove the inhibitors from the media and supplemented with fresh media (0.5% FBS) for further 72h. For the experiments with the siRNA, reverse transfection with 50nM siRNA was performed and the media replenished (10% FBS) with 4OHT after 2 days and left for an additional 48h. After, the cells were washed to remove all siRNA from the media and incubated with 0.5% FBS fresh media for an additional 72h.8-109 particles. At least three repeated-measurements of 60 s were taken per each individual sample and the mean value was used to determine particle number. Static mode (without flow) was used for each analysis. The movement of each particle in the field of view was measured to generate the average displacement of each particle per unit time which was calculated using the NTA 3.0 software.Prior to the NTA analysis, the NanoSight LM10 equipped with a 405nm laser  was calibrated using Silica Microspheres beads (Polyscience). Samples to be measured were then diluted in PBS in order to obtain a particle number between 10For exosome characterization by flow cytometry, aldehyde/sulfate latex beads (Thermo Fisher) were coated with anti-CD63 or anti-IFITM3 antibody and incubated with the different CM overnight at 4°C in a rotation wheel. After extensive washing, anti-CD81-PE conjugated antibody was added for further 40min at RT, washed with PBS and acquired using NovoCyte Flow Cytometer  with a 488nm laser. Gates were set using the NovoExpress Software  to analyze single bead fluorescence. Isotype-matching coated beads were used as a negative control in all experiments.+ MCF7, were washed twice with PBS, analyzed and sorted into two populations (GFP+ and DsRed+) using FACS Aria Ill Cell Sorter (BD Biosciences). Cell debris was discriminated by the cell forward scatter (FSC) and side scatter (SSC) properties. FACS data sorter was generated using BD FACDIVA SoftwareTM v.8.0.1 (BD Biosciences).Reporter MCF7 incubated with sEV isolated from DMSO and Palbo treated CreMice were perfused with PBS followed by using fixative solution . Small tissue sections were dissected and placed under rotation in fixative solution for 2h at RT, followed by ON rotation at 4°C. After 2 days, the tissue was transferred to 0.1M cacodylate buffer, pH 7.4. After fixation, samples were rinsed several times with 0.1 M cacodylate buffer and post-fixed in 1% (v/v) osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 1.5h at 4°C. Samples were then en-bloc stained with 1% (w/v) aqueous uranyl acetate for 1h at RT, thoroughly washed and dehydrated through a graded ethanol series before infiltration with epoxy resin (TAAB). Finally, tissue samples were embedded on flat molds and polymerized at 70°C for 24h. Ultrathin sections (70-90nm) were cut using a Leica UC7 ultramicrotome mounted on 150 mesh copper grids and contrasted using Uranyless (TAAB) and 3% Reynolds Lead citrate (TAAB). Sections were examined at 120kV on a JEOL JEM-1400Plus TEM fitted with a Ruby digital camera (2k x 2k).2.5 μL of sEV resupended in PBS were placed on Formvar-coated grids and allowed to settle for 3-5min, without being allowed to dry. sEV were then fixed with 2% glutaraldehyde for 5min and washed three times with distilled de-ionised water. After washing, the grids were stained for 20min with 3% uranyl acetate: 2% methyl cellulose (1:9). Imaging of sEV was carried out using a JEOL JEM-1400Plus, operated at 120kV, fitted with a Ruby camera (2k x 2k).Cells were washed with PBS and fixed with 0.05% (w/v) glutaraldehyde (in PBS) for 15mins at RT. Cells were washed a second time with PBS and incubated with 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside  solution for 1h at 37°C. Cells were imaged after 12-24h using a light microscope (Nikon) at 20X magnification and single representative images of each well were taken. IF β-Galactosidase was performed according to the manufacturer’s instructions using the following commercial kit . Briefly, 33 μM of the β-gal substrate C12FDG  (F2756 Sigma-Aldrich) was added to the cells for 8h at 37°C, After, the cells were washed with PBS and fixed with 4% PFA.2. Cells were then washed in PBS and incubated 1h with secondary antibody, DAPI and Cell Mask Deep Red (Invitrogen). For confocal images, cells were mounted onto slides using Vectashield Mounting Medium (Vector Laboratories). Signals were visualized using a Zeiss LSM 880  provided with Airyscan for super-resolution acquisition.Cells grown in 96-well plates were washed with PBS and fixed in 4% paraformaldehyde for 15min at RT. Cells were then washed in PBS twice before been permeabilized and blocked for 40min with 0.2% Triton X-100 together with 1%BSA and 0.2% gelatin fish (Sigma). For IF staining, cells were incubated ON with the primary antibody and in the case of BrdU cell were treated with 0.5U/μl DNaseI and 3mM MgClCells were washed with PBS and lysed directly into the culture dish using TRIzol Reagent (Thermo Fisher). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher). qPCR reactions were performed using SYBR Green PCR Master Mix  on a 7500 Fast System RealTime PCR cycler (Applied Biosystems). Primer sequences are listed in The SN sample was diluted 1:2 prior to the analysis and sEV - isolated by ultracentrifugation - were suspended in 100μl of lysis buffer to determinate the concentration of TGF-β1, IL-6 and IL-8. Regarding TGF-β1, it was first activated in the samples. All samples were incubated with 1N HCl for 10min at RT. Then, the acidified samples were neutralized with 1.2 N NaOH/0.5 M HEPES. TGF-β1 was detected using the quantitative sandwich human TGF-β1 immunoassay  and human IL-6 and IL-8 immunoassays . Samples were measured at 540nm using Synergy HT Multi-Mode Microplate Reader.Stable retroviral and lentiviral expression was performed as in previous studies .TM P-40 0.5% (v/v); Triton X-100 0,1% (v/v)], and quantified using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific) or cell lysates were prepared with Lysis Buffer 6 (R&D Systems), supplemented with protease inhibitor cocktail (Thermo Fisher Scientific), and protein content was determined using the Advanced Protein Assay Reagent (Sigma-Aldrich). Lysates were diluted with 4X Laemmli Sample Buffer  and equal quantities of total protein were separated in SDS-PAGE gels, transferred to a PVDF 0.45 μm pore size membrane  and probed with different antibodies. Protein bands were detected using a SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and the ChemiDoc XRS+ System (Bio-Rad). Sequential detection of different proteins was performed following HRP inactivation with 30% H2O2 (Sigma-Aldrich) for 30 minutes at 37°C up to five times, or without inactivation where appropriate.sEV and cultured cells were lysed using the following lysis buffer [ leupeptin, pepstatin A and antipain; NonidetSA-β-Gal staining was performed in whole tissue, using the Senescence β-Galactosidase Staining kit , following the manufacturer instructions. Briefly, whole tissue was fixed at RT for 45min with a 2% formaldehyde and 0.2% glutaraldehyde, washed and incubated ON at 37°C with the staining solution containing X-gal in N-N-dimethylformamide (pH 6.0). Tissues were subsequently dehydrated and embedded in paraffin and sectioned. For the immunohistochemistry, 5μm paraffin sections were deparaffinized and re-hydrated, and slides were incubated with anti-CD63 . The immunohistological reaction was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB), and nuclei counterstained with hematoxylin. Positive signal for SA-β-Gal and CD63 was quantified with ImageJ.For RNA-seq, total RNA was extracted using TRIzol Reagent. First strand cDNA synthesis was performed with SuperScript III First-Strand Synthesis System. After purification using SPRI beads, the double stranded cDNA was ligated to in-house designed adapters (based on TruSeq Indexed adapters (Illumina)) using NEBNext Ultra II (NEB) followed by 15 cycles of amplification and library purification. Sequencing was performed on an Illumina NextSeq500, High Output run with 75bp paired-end at the Genomics Centre (QMUL).LC-MS/MS was done by coupling a nanoLC-Ultra 1D+ system (Eksigent) to an Impact mass spectrometer (Bruker) via a Captivespray source (Bruker) supplemented with a nanoBooster operated at 0.2 bar/min with isopropanol as dopant. Peptides were loaded into a trap column  for 10min at a flow rate of 2.5 μl/min in 0.1% FA. Then peptides were transferred to an analytical column  and separated using a 100 min effective curved gradient  at a flow rate of 250 nL/min. The gradient used was: 0-2min 2% B, 2-102 min 33% B, 102-112 min 98% B, 112-120 min 2% B. The peptides were electrosprayed (1.35 kV) into the mass spectrometer with a heated capillary temperature of 180°C. The mass spectrometer was operated in a data-dependent mode (130-1600 m/z), with an automatic switch between MS and MS/MS scans using a top 20 method . An active exclusion of 30 s was used. The precursor intensities were re-evaluated in the MS scan (n) regarding their values in the previous MS scan (n-1). Any m/z intensity exceeding 5 times the measured value in the preceding MS scan was reconsidered for MS/MS. Peptides were isolated using a 2 Th window and fragmented using collision induced dissociation (CID) with a collision energy of 23-56 eV as function of the m/z value.10ml of blood was collected in EDTA vacutainer tubes . Plasma was obtained by centrifuging the tubes for 5min at RT at 1500 rpm. sEV were isolated from 2ml of plasma and centrifuged at 4,000 g for 10min at 4°C. Platelet-free plasma was filtered (0.22μm) and ultracentrifugated at 100,000 g for 1h 20min. After, the pellet was washed two times with PBS at 100,000 g for 1h 20min. None of the donors presented any underling disease.Ct values were generated using the 7500 software version 2.0.6 (Applied Biosystems). Relative gene expression was calculated using the ΔΔCt method and normalized to a housekeeping gene, RPS14. The relative mRNA expression level changes were expressed as a fold change relative to the control or the senescent sample.Immunofluorescence images were acquired using IN Cell 2200 automated microscope (GE) and the IN Cell 2200 Developer software version 1.8 (GE) as previously . Regardi2 ratio > 2 or < −2 were considered as regulated.Raw files were processed with MaxQuant (v 1.5.3.30) using the standard settings against a human protein database  supplemented with contaminants. Label-free quantification was done with match between runs . Carbamidomethylation of cysteines was set as a fixed modification whereas oxidation of methionines and protein N-term acetylation as variable modifications. Minimal peptide length was set to 7 amino acids and a maximum of two tryptic missed-cleavages were allowed. Results were filtered at 0.01 FDR (peptide and protein level). Further statistical analysis was performed using Perseus (v1.5.5.2). A minimum of three LFQ valid values per group was required for quantification. Missing values were imputed from the observed normal distribution of intensities. Then, a two-sample Student’s T-Test with a permutation-based FDR was performed. Only proteins with a q-value < 0.10 and logGenomic mapping was performed by QMUL Genome Centre using Fastq files aligned to HG 19 using STAR aligner implemented in BaseSpace . BAM file outputs from STAR were annotated using Partek Genomic Suite (v6.6) and the RefSeq data base (RefSeq 21). Differential analysis was performed with Partek Genomic Suite (v6.6) running ANOVA.∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001Statistical analysis was performed using Student’s t test except were specified. P values represent the following: https://string-db.org/) for the proteomic dataset. For the RNA seq datasets a combination of DAVID Functional Annotation Bioinformatics Microarray Analysis (https://david.ncifcrf.gov/) and PANTHER - Gene List Analysis (http://www.pantherdb.org/) was used. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA software (Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard (https://www.broadinstitute.org/gsea)).Gene Ontology Analysis was performed using STRING: functional protein association networks (GSE131503.The accession number for the sequencing data reported in this paper is GEO: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD: 010379.


Bombyx mori (BmGlv2) was synthesized using a prokaryotic expression system. Circular dichroism spectroscopy showed that the recombinant BmGlv2 has random coil structure, which is relatively stable at the temperatures ranging from 15 to 82.5 °C. Antimicrobial activity analysis revealed that BmGlv2 significantly inhibited the growth of gram-negative bacteria, Escherichia coli JM109 and Pseudomonas putida, by disrupting cell integrity. Western blotting and immunofluorescence analyses suggested that BmGlv2 absorbed on the cell surface after incubation, which might be the first step in the antibacterial process. Our results also proved that the cell wall component lipopolysaccharides (LPS) induce a conformational change in BmGlv2 from a random coil to α-helix. Subsequently, α-helical BmGlv2 would recruit more BmGlv2 and form higher aggregation state. Collectively, these findings expand our understanding of antibacterial mechanism of BmGlv2.Gloverin is one of the glycine-rich antimicrobial peptide exclusively found in Lepidoptera insects. It is generally activated through the innate immune system in insects. In this study, recombinant Gloverin2 from  Bombyx mori) is an important economic insect and a classic model for studying Lepidoptera insects as well. Like other members of Lepidoptera, silkworms have an innate immune system and lack the acquired immune system [Hyposphora cecropia (HcGlv) [E. coli D21f2. The inhibitory activity is not affected by heating at 100 °C for 10 min [Helicoverpa armigera showed activity against several E. coli that possess smooth lipopolysaccharides (LPS) [Plutella xylostella showed high activity against E. coli K12 [Trichoplusia ni Glv is active against E. coli D21f2, E. coli D22 strains and viruses. Only a few Glvs are known to exhibit inhibitory activity against gram-positive bacteria. For example, SeGlv in Spodoptera exigua can inhibit the growth of gram-positive bacterium (Flavobacterium sp.) [Manduca sexta is active against gram-positive Bacillus cereus and fungi (Saccharomyces cerevisiae and Cryptococcus neoformans) but inactive against E. coli strains with smooth LPS [Silkworm   and inhir 10 min . In hydrr 10 min ,10. Prevr 10 min ,12,13. Ges (LPS) . Glv frocoli K12  and Tricium sp.)  and MsGlooth LPS .E. coli JM109 [B. bassiana [B. mori cecropin A to against B. bassiana and the combined antimicrobial effect was stronger than the two alone [B. bassiana. Although numerous studies on the antimicrobial activity of Glvs have been reported and the pathways that induce Glvs are relatively well understood [E. coli and provide clues for better understanding of the mechanism of action of Glvs.Four Glv members have been identified in the silkworm, BmGloverin1, BmGloverin2 (BmGlv2), BmGloverin3 and BmGloverin4, which are famous marker genes in the immune response of the silkworm . It was li JM109 ; BmGlv2 bassiana . Since dbassiana . Similarwo alone , suggestderstood ,24,25,26The BmGlv2 cDNA encodes a 173 amino acid protein with a signal peptide composed of 18 amino acid residues, indicating that BmGlv2 might be secreted into the extracellular milieu. The molecular weight of full-length BmGlv2 is 16.8 kDa and the isoelectric point is 6.5. Protein blast analysis showed that BmGlv2 shares 74–86% identity with other BmGlvs in silkworm and a 52–62% identity with Glv from other Lepidoptera insects A. MultipE. coli BL21 (DE3) under the induction of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h and purified the protein by metal chelated affinity chromatography and gel filtration chromatography. The purity was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration analysis showed that the eluted peak of recombinant BmGlv2 is corresponding to ~42.3 kDa . As shown in E. coli JM109, P. putida, P. aeruginosa and E. coli DH5a were incubated with different concentrations of recombinant BmGlv2. The growth curve of bacteria was monitored by ultraviolet spectrophotometry. As shown in E. coli JM109 and P. putida at the concentration of 10 µM concentration. Significant inhibitory activity against E. coli JM109 was observed even at low concentrations (2.5 µM) of BmGlv2 (P. putida at low concentrations (2.5 and 5 µM), suggesting BmGlv2 has a higher binding affinity to E. coli JM109 than to P. putida. Moreover, BmGlv2 was inactive against P. aeruginosa  were adequately distributed and maintained cellular integrity  determines whether the LPS is rough or smooth. A long O-polysaccharide chain would render the LPS smooth, whereas the absence or reduction of O-antigen chain would make LPS rough [E. coli JM109, E. coli DH5α, P. putida and P. aeruginosa probably contains different O-polysaccharide chains and this might underlie the different sensitivities to BmGlv2.Similar to other Glv homologs, the positively charged BmGlv2 consists of a signal peptide sequence, N-terminal non-conserved region and C-terminal conserved domain. The C-terminal conserved domain represents the matured Glv (active form), which is normally activated by other innate immune factors in vivo . To obtaPS rough . PreviouPS rough . The LPSE. coli JM109 was further confirmed by Sytox green staining. Sytox green is a nucleic acid stain for distinguishing dead cells from live cells. It was found that almost half of E. coli cells were stained in the BmGlv2-treated group compared to that of the PBS-treated group, indicating these E. coli cells were killed by BmGlv2  and NCBI (available online: https://www.ncbi.nlm.nih.gov/). The sequences of Glvs were aligned using the clustax and GenDoc. The TMHMM Server v.2.0 (available online: http://www.cbs.dtu.dk/services/TMHMM/) and SignalP 4.1 Server  predicted the transmembrane helices and signal peptide of BmGlv protein. Primer premier 5.0 was used to design primers for BmGlv2. The ExPASy website (available online: https://web.expasy.org/compute_pi/) was used to predict the protein molecular weight and isoelectric point of BmGlv2. The Lepidopteran Glvs nucleotide sequences and amino acid sequences were downloaded from the silkworm genome database SilkDB (available online: Dazao) was provided by the Gene Resource Library of Domesticated Silkworm . The primers containing Xho I and Nco I sites were designed based on the conserved coding region of the BmGlv2 (CDS: BGIBMGA005658-TA) sequence of silkworm. The gene of BmGlv2 with complete ORF was cloned by the fat body cDNA of silkworm at day 3 of the 5th instar using the primers. Then the PCR products were ligated into the pMD-19T Vector . The verified correct recombinant plasmid BmGlv2-pMD-19T-simple and pET-21d vector was digested with Nco I and Xho I, then the BmGlv2-pET21d recombinant expression plasmid was constructed by ligation. The recombinant plasmid was confirmed by restriction and DNA sequencing.The diapause silkworm strain (BmGlv2-pET21d was transformed into E. coli Bl21 (DE3) cell and the positive bacterial colonies were inoculated into LB medium containing ampicillin (100 μg/mL) at 37 °C to OD600 = 0.6 to 0.8 and then protein expression was induced by 0.1 mM IPTG at 37 °C for 5 h. The bacterial cells were harvested by centrifugation at 6000× g for 20 min at 4 °C and then re-suspended in lysis buffer .The g for 25 min, the supernatant was collected and applied to Ni2+-NTA column  equilibrated with binding buffer . The target protein was eluted with 350 mM imidazole in 20 mM Tris-HCl, 200 mM NaCl, pH 8.0 and then was loaded onto a HiLoad 16/60 Superdex 200 column  pre-equilibrated with 10 mM phosphate, pH 6.0. The purified protein was estimated by SDS-PAGE then stored at −80 °C.After 6 min of sonication and centrifugation at 12,000× CD spectra were recorded on a MOS-500 circular dichroism spectrometer  with a 0.1 cm quartz cell at 25 °C in 190–250 nm using standard procedures. BmGlv2 concentration was 0.05 mg/mL in 10 mM phosphate, pH 8.0. The effects of temperature on the secondary structure of BmGlv2 were performed from 15 to 82.5 °C at a step of 2.5 °C. The concentration was 0.05 mg/mL. The percentages of secondary structure elements were estimated with the method of variable selection using a data base of 22 proteins with known secondary structure ,34.E. coli JM109, P. putida, P. aeruginosa and E. coli DH5α. After cultured in LB medium to OD600 of 0.4, the bacteria (160 μL) were incubated with 40 μL recombinant protein BmGlv2  in a 96-well plate at 37 °C with 80 rpm. The ultraviolet spectrophotometer  was used to measure the OD600 value every hour.Antimicrobial activity of recombinant BmGlv2 was estimated against four gram-negative bacterial E. coli JM109, fluorescent staining was performed. The recombinant BmGlv2 protein (10 µM) was incubated with E. coli JM109 (OD600 = 0.4) at 25 °C for 3 h with the shaking of 80 rpm. Set the BmGlv2 only and the E. coli only as a control group. The samples were washed 3 times and re-suspended with 200 µL PBS, pH 6.0, fluorescent staining was added according to the Sytox Green [To test the lethal effect of BmGlv2 to ox Green  , bacteria were re-suspended with 200 µL binding buffer . The recombinant BmGlv2  40 μL and 160 μL were mixed in a 1.5 mL centrifuge tube and incubated at room temperature for 3 h. After centrifuging at 3000× g for 3 min, the bacterial pellets were washed 5 times to remove the uncombined recombinant protein. Then 15% SDS-PAGE gel electrophoresis and Western blotting  analyzed the combining effect. For immunofluorescence staining assay, the E. coli JM109 cell treated by BmGlv2 was incubated with the normal goat serum  at 4 °C overnight. After washing 3 times with 10 mM phosphate, pH 6.0, the microorganism samples were incubated with BmGlv2 rabbit antibody (diluted 1:500 (v/v) with 10 mM phosphate, pH 6.0) and then incubated with fluorescein-conjugated goat anti-rabbit antibody Cy3 (diluted 1:500 (v/v) with 10 mM phosphate, pH 6.0). The recombinant BmGlv2 peptides on the bacterial cell surfaces were visualized using laser-scanning confocal microscopy .Gram-negative bacterial E. coli JM109. The 50, 10, 5 μL E. coli JM109 LPS  extracted by LPS extraction kit  was mixed with 150 μL recombinant protein BmGlv2  respectively. After incubation at 25 °C for 3 h, the secondary structure of BmGlv2 was performed by circular dichroism spectral. Spectra were recorded over a wavelength from 190 to 260 nm. Each spectrum was obtained by subtracting the signal from protein-free solution. Simultaneously, the incubated mixtures of BmGlv2 and LPS were loaded onto a HiLoad 10/300 Superdex 75 column  pre-equilibrated with 10 mM phosphate, pH 6.0 and the eluted sample was estimated by Western blotting.The circular dichroism spectral and gel filtration chromatography was used to explore the interaction between BmGlv2 and LPS of


Aedes albopictus and Culex pipiens. The pathogenicity and virulence against the two species of mosquitoes were evaluated by using 30 isolates of Beauveria bassiana, an entomopathogenic fungus isolated from Korea that has high thermotolerance and UV-B tolerance. Regarding pathogenicity, 23 isolates were pathogenic to Ae. albopictus and 12 isolates were pathogenic to Cx. pipiens; Ae. albopictus adults were more susceptible to B. bassiana than Cx. pipiens adults. Among the isolates, 6 isolates that were simultaneously pathogenic to the two species of mosquitoes were used to evaluate virulence and conidia productivity. B. bassiana CN6T1W2 and JN5R1W1 had higher virulence than the other isolates, and they were more virulent in Ae. albopictus than inCx. pipiens. The conidia productivity of B. bassiana JN5R1W1 on millet grain medium was higher than that of B. bassiana CN6T1W2. Based on these results, B. bassiana JN5R1W1 was selected as the most efficient isolate for the simultaneous control of the two mosquito species. B. bassiana JN5R1W1 can be used effectively in the development of fungal insecticides to simultaneously control Ae. albopictus and Cx. pipiens adults with similar distribution areas.This study was conducted to determine the optimal entomopathogenic fungus for the simultaneous control of the adults of two mosquito species,  Aedes albopictus (Skuse) (Diptera: Culicidae), transmits many viral diseases, such as yellow fever, dengue and chikungunya. It is also believed to have the potential for Zika transmission among humans due to the host capacity of the Zika virus  (Diptera: Culicidae), is a temperate Eurasian nocturnal mosquito that has established populations throughout temperate North America  and Lysinibacillus sphaericus  were transferred to sterile PCR tubes and immediately placed in a thermal cycler adjusted to 45 °C. Aliquots (20 μL) were removed after 4 h and plated (without spreading) on Sabouraud dextrose agar  with 0.5 μg/mL of benomyl (95% active ingredient) (SDA-B)  from each isolate was plated (without spreading) on SDA-B. Conidia were immediately exposed to irradiances of 0.2 J/cm2 in a UV irradiation chamber  at 25 °C  was sprayed into each cylinder using an SD tower sprayer  capped with netting material. To evaluate the pathogenicity of the fungus, 10 mosquito adults aged 1–2 days were placed in the cylinder, and 1 mL of a conidial suspension . Millet grains for the production of conidia were prepared according to a previous report . The conidia on the millet grains were collected daily and counted using a haemocytometer.Conidia were produced in Petri dishes on Italian millet grains . The lethal time for the treatments was analysed with probit analysis. Data were expressed as the mean ± standard error (SE), and statistical significance was set at the conventional α < 0.05 level.B. bassiana isolates used in this study showed low thermotolerance but high UV-B tolerance  showed 100% mortality against both species of mosquito adults. Regarding lethality, all isolates were found to be slightly more pathogenic to Ae. albopictus than to Cx. pipiens adults. The LT50 values of B. bassiana CN6T1W2 and JN5R1W1 in Cx. pipiens were 4.3 and 4.4 days, respectively, which were lower than the values for the other 4 isolates, which were all more than 5.2 days. B. bassiana CN6T1W2 and JN5R1W1 also showed the lowest LT50 values for Ae. albopictus at 4.8 and 4.5 days, respectively. The LT90 values of B. bassiana CN6T1W2 and JN5R1W1 were also significantly lower than those of the other isolates. Thus, B. bassiana CN6T1W2 and JN5R1W1 were found to be the most virulent for both species of mosquitoes at the same time.The virulence of 6 isolates of nt Table . MoreoveB. bassiana that were simultaneously pathogenic to the two species of mosquitoes was evaluated on millet grain medium. All the isolates showed conidia productivity of 1.6–2.3 × 109 conidia/g at 7 days after inoculation; there was no significant difference in productivity  and 4.4 days (Cx. pipiens). Although there are few report of entomopathogenic fungal virulence on Ae. albopictus and Cx. pipiens adults, similar report has shown that M. anisopliae showed 3.1–4.1 days against Ae. aegypti adult   and Cx. pipiens (B) adults showing sporulation of B. bassiana.


Drosophila melanogaster with different wavelengths of blue light. Specifically, the lethal effect on eggs increased at shorter light wavelengths . In contrast, wavelengths from 405 to 466 nm had similar lethal effects on larvae. A wavelength of 466 nm had the strongest lethal effect on pupae; however, mortality declined as pupae grew. A wavelength of 417 nm was the most harmful to adults at low photon flux density, while 466 nm was the most harmful to adults at high photon flux density. These findings suggest that, as the morphology of D. melanogaster changes with growth, the most harmful wavelength also changes. In addition, our results indicated that reactive oxygen species influence the lethal effect of blue light. Our findings show that blue light irradiation could be used as an effective pest control method by adjusting the wavelength to target specific developmental stages.Short-wavelength visible light (blue light: 400–500 nm) has lethal effects on various insects, such as fruit flies, mosquitoes, and flour beetles. However, the most toxic wavelengths of blue light might differ across developmental stages. Here, we investigate how the toxicity of blue light changes with the developmental stages of an insect by irradiating  The responses of insects to light have been studied and applied to various pest control methods. In particular, phototaxis has been well studied in insects –5. By idDrosophila melanogaster, as a model insect because of its short life cycle. Furthermore, because it is possible to rear large numbers of D. melanogaster with ease, the experiments of this study could be conducted efficiently. In addition, D. melanogaster is suitable for future research to clarify the mechanism that generates differences in the effective wavelength among developmental stages or species, because many mutants are produced and cell culture is straightforward.However, while many methods use light to control pests, these methods target the behavior of insects. While the lethal effects of ultra-violet light on insects have been reported over the last five decades –19, our D. melanogaster individuals were purchased from Sumika Technoservice Co.  for use in our experiments. The flies were the same strain as the flies used in our previous study (FBrf0227020) [Wild type FBrf02270 20]. Th. ThD. me®, ISL-150 × 150 Series, CCS Inc., Kyoto, Japan) and power supply units (ISC-201-2) were used for light radiation. Insects were irradiated with LED light in a multi-room incubator . The emission spectra  and DD conditions. We also compared the relationships between the lethal effects and the number of photons among the six wavelengths. Ten replications (Petri dishes) were performed for each light dose and wavelength. The actual measurement value of the number of photons is shown in Five pairs of mated adult flies were released onto 10 ml culture medium (the same as the rearing culture medium) in a glass Petri dish . The females were allowed to lay 10 eggs on the medium within 6 h of being placed in the incubator . Each Petri dish with 10 eggs was sealed with Parafilm and was placed in the incubator equipped with the LED lighting unit. The eggs were irradiated with different wavelengths of LED light for 48 h at 25 ± 1 °C. After irradiation, the number of newly hatched larvae was counted under a stereomicroscope. Considering the possibility of delayed hatching, the Petri dish was kept under dark conditions for three additional days after the initial count. Then, the number of hatching larvae was counted again. Lethal effects at 4.0 × 1018 photons·m-2·s-1 were compared among the six wavelengths  and DD conditions. We also compared the relationships between lethal effects and the number of photons among the six wavelengths. Ten replications (Petri dishes) were performed for each light dose and wavelength. The actual measurement value of the number of photons is shown in Ten final-instar larvae  were co18 photons·m-2·s-1 were compared among the six wavelengths  and DD conditions. Ten replications (Petri dishes) were performed for each light dose and wavelength. The actual measurement value of the number of photons is shown in The pupae passed through 15 growth stages . Photogr18 photons·m-2·s-1 were compared among the six wavelengths  and DD conditions. Ten replications (Petri dishes) were performed for each light dose and wavelength. The actual measurement value of the number of photons is shown in Five pairs of adults were collected from the plastic rearing boxes within 12 h of emergence and were released onto 10 ml culture medium (the same as the rearing culture medium) in a glass Petri dish . The Petri dish was placed in the incubator equipped with the LED lighting unit. The adults were irradiated with different wavelengths of light for 12 days at 25 ± 1 °C. Every 3 days, we counted the number of dead adults and replaced the Petri dish containing the culture medium with a fresh one. Lethal effects of irradiation at 1.0, 5.0, and 10.0 × 102O2 was measured by conversion to luminescence of luciferin using ROS-Glo™ H2O2 Assay . The amount of luminescence was measured with luminometer  and represented with RLU (Relative Luminescence Unit). The RLU of irradiation at 1.0, 5.0, and 10.0 × 1018 photons·m-2·s-1 was compared among the six wavelengths  and DD conditions. Thirty replications were performed for each light dose and wavelength. The actual measurement value of the number of photons is shown in One pupa of P5(i)-25 stage  was collThe temperature of the inner space of the Petri dishes was measured using a button type temperature logger . The measurements were completed during irradiation with the strongest light intensity of each wavelength in each experiment. The temperature data of each developmental stage are summarized in Mortality was analyzed using the Steel-Dwass test. The RLU was analyzed using Tukey’s test. The calculations were performed using R version 3.0.3.D. melanogaster eggs. Ten replications were performed for each light dose and wavelength. In our previous study, while irradiation with 467 nm at 3.0 × 1018 photons·m-2·s-1 showed no lethal effects on egg stage, lethal effects were detected at > 4.0 × 1018 photons·m-2·s-1 [18 photons·m-2·s-1  on ·m-2·s-1 . Therefo·m-2·s-1 .18 photons·m-2·s-1. In contrast, mortality at 494 nm remained low (up to 26%), even when the number of photons was raised to 9.6 × 1018 photons·m-2·s-1.A wavelength of 405 nm (the shortest wavelength) had the most lethal effect, with 95% mortality. In contrast, 494 nm (the longest wavelength) showed the lowest lethal effect, with 18% mortality. The mortality of irradiated eggs tended to increase as the wavelength shortened, except for at 439 nm. Mortality at 439 nm was slightly lower than that at 417 and 454 nm. Next, we irradiated the eggs at the same wavelengths but with different intensities . For all wavelengths, mortality increased as the number of photons increased . Mortali18 photons·m-2·s-1 killed >90% of larvae before adult emergence, whereas, mortality at 5.0 × 1018 photons·m-2·s-1 was <60% [18 photons·m-2·s-1 because the mortality is too high. Therefore, we initially irradiated D. melanogaster larvae with different wavelengths of visible light  at 5.0 × 1018 photons·m-2·s-1 , with mortality significantly differing to that of DD conditions (12%). In contrast, mortality at 494 nm was 20%, but was not significantly different to DD. Next, we irradiated larvae at several light intensities . MortaliD. melanogaster for 7 days in our previous study [18 photons·m-2·s-1 (Ten replications were performed for each light dose and wavelength. According to Bainbridge & Bownes (1981) , pupal gus study , to inve·m-2·s-1 .Wavelengths of 417 to 466 nm had strong lethal effects . Mortali18 photons·m-2·s-1, to investigate how the lethal effect changes with the number of photons  and P5 (97%). Compared to DD, there was no significant difference in the mortality of P2–4 at 417, 439, and 454 nm  at 454 nm was not significantly different to DD (5%) (When the number of photons was reduced to 8.0 × 10d 454 nm . Compared 454 nm . The hig DD (5%) . Mortali DD (5%) .18 photons·m-2·s-1 shortened adult longevity about one third [D. melanogaster adults initially at 1× 1018 photons·m-2·s-1 because it was difficult to control the number of photons below 1× 1018 photons·m-2·s-1.Ten replications were performed for each light dose and wavelength. In the experiment using adults, we irradiated different wavelengths of blue light for 12 days continuously, and we calculated the accumulated mortality every 3 days. In our previous study, irradiation with 467 nm at 1× 10ne third . In the 18 photons·m-2·s-1, none of the wavelengths showed any lethal effect on males for up to day 6 of irradiation. However, on day 9, mortality was 54% at 417 nm. On day 12, lethal effects were recorded at 439 nm  and 417 nm . No lethal effect was detected for any of the other wavelengths throughout the 12-day irradiation period . At 5.0 × 1018 photons·m-2·s-1, 439 nm showed the highest RLU (4.06 × 107). At 10.0 × 1018 photons·m-2·s-1, 439 nm showed the highest RLU (5.58 × 107).H2O2 between wavelengths, multiple comparisons were carried out at 10.0 × 1018 photons·m-2·s-1 , while 456 nm was not lethal. This difference might be due to the use of different experimental conditions. In our previous study, pupae were irradiated with 3.0 × 1018 photons·m-2·s-1 for 7 days. In contrast, pupae were irradiated with 10.0 × 1018 photons·m-2·s-1 for 24 h in the current study , 5.2 days (5.0 × 1018 photons·m-2·s-1), and 2.95 days (10 × 1018 photons·m-2·s-1). However, adult sex was not distinguished when calculating this average longevity. Thus, we recalculated the average longevity of each sex. Under control conditions, male longevity was 55.9 days and female longevity was 59.3 days. At 1.0 × 1018 photons·m-2·s-1, male longevity was 18.5 days and female longevity was 20.3 days. At 5.0 × 1018 photons·m-2·s-1, male longevity was 4.9 days and female longevity was 5.5 days. At 10 × 1018 photons·m-2·s-1, male longevity was 2.9 days and female longevity was 3.0 days. These results suggest that female longevity is longer than that of males. Because adult females have naturally longer longevity than adult males, the lethal effect of light irradiation might not be visible when using 1.0 × 1018 photons·m-2·s-1 for 12 days (D. melanogaster [18 photons·m-2·s-1 laid much fewer eggs than non-irradiated (control condition) females. This result suggests that blue light irradiation could suppress the next generation.In the irradiation test for each wavelength of blue light on adults, the effective wavelength differed with the number of photons and sex. At 1.0 × 10 photons . Average 12 days . In our As observed with pupae, damage might differ between protracted irradiation with weak light versus short irradiation with strong light. In addition, the lethal effects of blue light on adults were similar to those recorded on pupae Figs  and 6. F2O2, a kind of ROS) in the pupae of D. melanogaster (P5) rose after irradiating them with blue light. The level of H2O2 rose as the number of photons increased at all tested wavelengths . Therefore, ROS generation in the insect body and the lethal effect of blue light on insects might be related. A preliminary experiment of our laboratory showed that proliferation of cultured D. melanogaster cells was inhibited by irradiation with blue light. Thus, blue light irradiation might directly damage insect cells and cause the lethal effects. Thus, some sort of endogenous factor in cell could relate the lethal effect. In addition, a recent report showed that blue light exposure decreases melanin contents and impairs immune function of Bactrocera dorsalis [Our results clearly showed that effective wavelengths differ with respect to the growth stage of the insect. In addition, the results of our previous study demonstrated that the effective wavelengths that had a lethal effect were species-specific . Therefoelengths . Further of H2O2 . This sp·m-2·s-1 . When wadorsalis . These fGalerucella grisescens, Clogmia albipunctatus, Liriomyza sativae, and so on. For example, the eggs of Galerucella grisescens, the strawberry leaf beetle, are killed by irradiation at 438 nm; however, the pupae are not killed by this wavelength [Culex pipiens molestus (unpublished). Whether the effective wavelength changes might depend on the species of insect. Thus, to use blue light irradiation for pest control, we must first identify the effective wavelengths of each developmental stage for each targeted species.This study showed that the effective wavelength of the lethal effects of blue light varies with the growth stage of insects. This finding is important toward providing baseline information for establishing pest control methods using light irradiation. Effective pest control could be realized by clarifying the most lethal wavelength of the targeted growth stage. For example, the eggs and pupae could be targeted because these stages do not move. Adult insects that live in limited spaces could also be targeted, such as sanitary insect pests or stored grain insect pests. Our group is investigating the relationship between the lethal effects of blue light and developmental stages for over 10 insect species, such as velength . This prvelength  only comIn conclusion, the findings of this study could contribute toward advancing pest control techniques, because they clarify how the lethal effects of light are caused. Furthermore, this study provides important information on insect photobiology.S1 TableData are the mean ± standard error of each five measurements before and after the experiment.(DOCX)Click here for additional data file.S2 TableData are the mean ± standard error of each five measurements before and after the experiment.(DOCX)Click here for additional data file.S3 TableData are the mean ± standard error of each five measurements before and after the experiment.(DOCX)Click here for additional data file.S4 TableData are the mean ± standard error of each five measurements before and after the experiment.(DOCX)Click here for additional data file.S5 TableData are the mean ± standard error of each five measurements before and after the experiment.(DOCX)Click here for additional data file.S6 Tablea Data are the mean of each five measurements before and after the experiment. b Data are the mean ± standard error of the 24 h period during irradiation.(DOCX)Click here for additional data file.S7 Tablea Data are the mean of each five measurements before and after the experiment. b Data are the mean ± standard error of the 24 h period during irradiation.(DOCX)Click here for additional data file.S8 Tablea Data are the mean of each five measurements before and after the experiment. b Data are the mean ± standard error of the 24 h period during irradiation.(DOCX)Click here for additional data file.S9 Tablea Data are the mean of each five measurements before and after the experiment. b Data are the mean ± standard error of the 24 h period during irradiation.(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigPuparia of P5–P14 were removed to clearly display pupal growth.(TIF)Click here for additional data file.


Herein, we report a three-dimensional hierarchical assembly structure composed of an ultrathin Ru shell and a Ru-Ni alloy core as a catalyst functioning under universal pH conditions. Compared with the typical Ir/C-Pt/C system, superior catalytic performances and excellent durability of the overall water splitting under universal pH have been demonstrated. The introduction of Ni downshifts the d-band center of the Ru-Ni electrocatalysts, modulating the surface electronic environment. Density functional theory results reveal that the mutually restrictive d-band interaction lowers the binding of  and  for easier O-O and H-H formation. The structure-induced eg-dz  •A facile method for the synthesis of 3D hierarchical assembly Ru-Ni NA catalyst•Superior catalytic reactivity and stability of water splitting in universal pH•The introduction of Ni can modify the d-band and surface electronic environment•Minimization of the surface Coulomb repulsion leads to barrier-free catalysis process Catalysis; Energy Materials; Materials Science Water splitting consists of the hydrogen evolution reaction (HER) and oxygen evolution reaction (OER), both of which require efficient catalysts to reduce the overpotential for practical applications (2 and oxygen (O2) under universal pH conditions is still a significant challenge.Hydrogen  consisting of ultrathin nanosheets as subunits and explore their high performances for overall water splitting under universal pH conditions. The distinctive hierarchical NA structures are highly beneficial for enhancing electrochemical energy conversion. We found that the introduction of Ni into Ru largely downshifts the d-band center of the Ru-Ni NAs and effectively modulates the surface environment for HER. After air treatment at 350°C, the newly generated abundant RuO3), nickel(II) acetylacetonate (Ni(acac)2), phloroglucinol, tetramethylammonium bromide, polyvinylpyrrolidone (PVP), and benzyl alcohol into a glass vial. After capping the vial, the mixture was ultrasonicated for approximately 1 h. The resulting homogeneous mixture was then heated from room temperature to 160°C and maintained at 160°C for 5 h using an oil bath. Ru-Ni NAs with different Ru/Ni ratios  have been readily achieved by tuning the Ru/Ni precursor amount ratios  acetylacetonate (Ru(acac)t ratios A–S1C.3Ni3 NAs were further carried out to determine the 3D assembly structure  plane of Ru and the (100) plane of the Ru-Ni alloy, respectively  plane of Ru. Lattice fringes of the (100) Ru-Ni alloy with interplanar distances of 0.231 and 0.232 nm were also observed in the Ru3Ni2 NAs and Ru3Ni1 NAs, respectively . −2, the overpotentials of Ru3Ni3 NAs, Ru3Ni2 NAs, Ru3Ni1 NAs, Ru NAs, and commercial Pt/C were 39, 42, 44, 62, and 90 mV, respectively, versus the reversible hydrogen electrode (RHE), and the Ru3Ni3 NAs showed the smallest value. The Tafel slope is an intrinsic property of the catalyst that is determined by the rate-limiting step of the HER  and Ru NAs (76.0 mV dec−1)  B. The Ru.1 M KOH F, which 3Ni3 NAs, Ru3Ni2 NAs, Ru3Ni1 NAs, and Ru NAs were 39 and 96 mV, 39 and 115 mV, 46 and 112 mV, and 55 and 122 mV at a current density of 10 mA cm−2 in 0.5 M H2SO4 and 0.05 M H2SO4, respectively. The Tafel slopes of the Ru3Ni3 NAs, Ru3Ni2 NAs, Ru3Ni1 NAs, and Ru NAs were 53.9 and 67.1 mV dec−1, 53.5 and 64.0 mV dec−1, 54.2 and 66.8 mV dec−1, and 81.6 and 79.6 mV dec−1 in 0.5 M H2SO4 and 0.05 M H2SO4, respectively. The Ru-Ni NAs showed a much better HER performance than the Ru NAs, indicating the vital role of Ni in improving the HER performance. After the working electrode was cycled for 6,000 cycles, the Ru3Ni3 NAs exhibited the best durability under acidic conditions with potential increases of only 62 and 39 mV in 0.5 M H2SO4 and 0.05 M H2SO4, respectively. In addition, after the 12-h chronopotentiometry test at 5 mA cm−2 in 0.5 M H2SO4 and 0.05 M H2SO4, the Ru3Ni3 NAs showed only potential increases of 36 and 49 mV, respectively (The HER properties of the Ru-Ni NAs under acidic conditions were further investigated. 2SO4 and 1 M KOH). The TEM images show that the hierarchical structures were largely preserved  and alkaline (1 and 0.1 M KOH) electrolytes. The commercial Ir/C catalyst was chosen as the reference because Ir is considered to be the benchmark catalyst for OER  and commercial Ir/C (407 mV)  B. The Taectively D. All thtry test , which d 1 M KOH .3Ni3 NAs after air treatment at 250°C for 1 h and cathodic catalyst Ru3Ni3 NAs after air treatment at 350°C for 2 h was used to study the potential application of Ru-Ni NAs in overall water splitting under universal pH conditions. The Linear Sweep Voltammetry (LSV) plots of Ru3Ni3 NAs and Ir/C-Pt/C under different pH conditions are presented in 3Ni3 NAs are much lower than those of Ir/C-Pt/C. The Ru3Ni3 NAs show an overpotential of 280 mV in 0.5 M H2SO4, which is considerably lower than that of Ir/C-Pt/C (370 mV). The Tafel slope of the Ru3Ni3 NAs is only 96.9 mV dec−1, whereas that of Ir/C-Pt/C is as high as 150.1 mV dec−1 , Ru4+ (orange line), and Ru0 (dark yellow line) (4+ fractions in the Ru3Ni3 NAs (57.00%), Ru3Ni2 NAs (44.46%), and Ru3Ni1 NAs (44.57%) were much higher than those in the Ru NAs (29.89%)  and Ni3+ (orange line)  A (Li et ow line) . It was (29.89%) , which cge line) . Ni3+ isformance . This rex+ (purple line), Ru4+ (orange line), and Ru0 (dark yellow line)  . It was  Ru NAs.  and indiC in air C and 5D.C in air . Pt is rC in air C and 5D,C in air .F) ]→[O*+2(H++e−)] (∼0.4 eV). An additional similarly energetic increase (1.50 eV) was found for the formation of *OOH, indicating that the O* on the Ru-Ni still stays active to oxidize OH under lower overpotential. The splitting of H for the [HOO*+3(H++e−)]→[O2+4(H++e−)] transformation is very active. Compared with the pathway at U = 1.23 V, we confirm the overall overpotential  is almost the same within the range of 0.200–0.300 V. Further calculations of the O2 dissociation confirmed that the combined O-O on the Ru-contained surface will be easily dissociated and enter into the surrounding solution conditions  is shown in absorption process in 2+ and O-pσ orbitals will facilitate the ion transfer. The distorted structure prompts Ru2+ (d6) to change from a low-spin state (t2g6eg0) to an intermediate-spin state (t2g5eg1), where the eg1 can point to the intermediate with high bonding possibility. We also find that the absorption energy of further absorption on vertical oxygen molecule will be lowered nearly 1 eV, which can be attributed to the Jahn-Teller effect from the extra oxygen molecule to the c-axis of the distorted octahedral unit, which decreases the whole energy. Electrons on t2g can be further excited to eg and then form a high-spin state (t2g4eg2) with energy decrease. Overall, the Ru-Ni catalytic system is found to be efficient in HER performance from acidic to the basic condition. Thus, the Ru-Ni (NAs) system exhibits a high catalytic reactivity for water splitting based on the DFT calculations. We have also made a detail comparison for the preliminary absorption behavior on the cubic Ru-Ni (111) and hexagonal close packed (hcp) Ru-Ni (001) surface to elucidate the experimental treatment and related analysis. The discussions and analysis cover the following sections: energetics, electronic structures, orbital energetic behaviors, and adsorption analysis  calculations to elucidate how the downshift effect of the Ru-Ni NAs is related to the high performance of water splitting for both the OER and HER. The Ru-Ni NA system was modeled by a hexagonal lattice (hex-Ru-Ni) based on the Ru local symmetry. It shows a good metallic behavior with uniform isotropic conductivity across the Fermi level (EF) A. The d-F) B due to e the EF B, regard pathway C shows tnditions  and S7.  surface D. At the surface E. We alsanalysis .Figure 62-decorated Ru-Ni NAs treated at 350°C in air provided additional active sites for the OER. The combined structural and electronic engineering leads to superior electrocatalytic performance for overall water splitting under universal pH conditions, and the performance is much better than that of the commercial Pt/C and Ir/C, demonstrating an unprecedented class of nanocatalysts with exceptional activity and excellent stability for electrochemical water splitting.In summary, for the first time, we have demonstrated a facile method for the synthesis of 3D Ru-Ni NAs, which leads to favorable 3D Ru-Ni superstructures with fully exposed active sites. The valence band spectra and DFT calculations revealed a change in the d-band center in the Ru-Ni NAs after the introduction of Ni, resulting in the transformation to a favorable surface environment for the OER and HER. The RuOOur work has demonstrated a novel bifunctional catalyst for water splitting in the universal environment from experimental and theoretical perspectives. Based on the combination of XPS and DFT as an effective approach, electronic environment modulation has been interpreted as the key factor that facilitates both HER and OER. However, an in-depth understanding of the oxidation states of the catalyst is still an open challenge because of the complex charge transfer induced by the overlap between the metal orbitals as well as the correspondingly accurate characterization. The site-to-site sampling and analysis of surface oxidation sites is of great significance for precise understanding of the catalyst reactivity. Therefore, we will keep working on further development and perfection on related theoretical exploration and advancement.All methods can be found in the accompanying


Conus textile by gene cloning, which has 4/6 inter-cysteine loop spacing and selectively inhibits α3β4 nicotinic acetylcholine receptor (nAChR) subtype. However, TxID is susceptible to modification due to it containing a methionine (Met) residue that easily forms methionine sulfoxide (MetO) in oxidative environment. In this study, we investigated how Met-11 and its derivatives affect the activity of TxID using a combination of electrophysiological recordings and molecular modelling. The results showed most TxID analogues had substantially decreased activities on α3β4 nAChR with more than 10-fold potency loss and 5 of them demonstrated no inhibition on α3β4 nAChR. However, one mutant, [M11I]TxID, displayed potent inhibition at α3β4 nAChR with an IC50 of 69 nM, which only exhibited 3.8-fold less compared with TxID. Molecular dynamics simulations were performed to expound the decrease in the affinity for α3β4 nAChR. The results indicate replacement of Met with a hydrophobic moderate-sized Ile in TxID is an alternative strategy to reduce the impact of Met oxidation, which may help to redesign conotoxins containing methionine residue.α-Conotoxin TxID was discovered from  Conus genus, which are disulfide-constrained peptides targeting a range of ion channels and receptors TxID increased remarkably and its retention time shifted from 15.48 min to 11.42 min  and charged amino acid  and these analogues (1 μM) were tested at α3β4 nAChR A. The [M50 for [M11I]TxID on α3β4 nAChR subtype was 69.09 nM, which retained comparable activity compared to native peptide. Surprisingly, the activities of [M11L]TxID and [M11V]TxID reduced dramatically with an IC50 of 256.7 nM and 980.3 nM respectively  act by blocking different isoforms of nAChRs, which locate in the neuromuscular junction or central nervous system broadly ,6. Methiectively . EI demoectively ,16. Addiectively ,26. The ectively . Pu14a cectively . Recentlectively . TherefoIncreasing evidences indicate that Met residue in proteins is the same as cysteine which serve as antioxidants and impact the structure of proteins . Here, wConus aulicus, selectively inhibits α3β4 nAChR with an IC50 of 750 nM [Conus regius venom, inhibiting α3β2, α3β4, and α7 nAChRs [To date, several α-CTxs and its analogues have been identified to antagonize α3β4 nAChR ,32,33,34f 750 nM . Alaninef 750 nM . α-CTx R7 nAChRs . Another7 nAChRs . CombiniOwing to its potent activity, the TxID has a potential in the development of a novel drug. Unfortunately, the conversion of Met to MetO is readily to happen during TxID synthesis and oxidation steps as well as under oxidative environment in vivo, which causes the loss of activity in the inhibition of α3β4 nAChR. Furthermore, conotoxins containing Met require low-temperature storage in the entire supply chain which increases the inconvenience and cost . These dIn summary, we firstly investigated how the conversion of Met to MetO affected the efficacy of TxID at the α3β4 nAChR. The substitution of Met of TxID by an Ile maintained the comparable activity at the α3β4 nAChR. Homology models and dynamic simulation suggested the molecular mechanism of interaction between different analogues of TxID and α3β4 nAChR. These findings provide further information for understanding the function of Met residue in α-CTx TxID.2O; Solvent A was 0.1% TFA in H2O. The purity of the TxID and its analogues were determined by monitoring absorbance at 214 nm during HPLC (≥95% purity). ESI-MS mass spectrometry was utilized to confirm the identity of the products. The linear peptides of TxID and its mutants were synthesized as previously described . Two dis2O, which was incubated with 5 mL of 10% hydrogen peroxide (H2O2) in 20 min at room temperature. Then reaction products were separated using semi-preparative RP-HPLC with a linear gradient as above conditions. The purity of oxidative products was identified by Waters ACQUITY UPLC™ System with BEH300 C18 column  . The molecular mass was measured on a Waters TQD mass spectrometer equipped with an electrospray ionization source .Met is easily modified to methionine sulfoxide under oxidative conditions. α-CTx TxID (0.2 mg) firstly dissolved in 100 μL of 60% acetonitrile in ddHXenopus oocyte with a Drummond microdispenser , and incubated at 17 °C. Oocytes were injected within 1 day of harvesting, and recordings were made 2–4 days post-injection.Plasmid DNAs encoding rat nAChR subunits were kindly provided by Stefan H. Heinemann . The plasmids were linearized with appropriate restriction enzymes. Then capped RNA (cRNA) for the various subunits were made using transcription kit of the mMessage mMachine SP6  in vitro. The cRNA was purified using MEGAclear™ kit (Ambion). The concentration of each cRNA was determined using Smart Spec™ plus Spectrophotometer  at 260 nm. cRNA of the various subunits was combined to give ~50 ng/μL of each subunit cRNA. 50.6 nL of this mixture was injected into each 2·6H2O, 1.8 mM CaCl2·2H2O, 5 mM HEPES, pH 7.1–7.5) containing 1 μM atropine and 0.1 mg/mL bovine serum albumin (BSA). For α9α10 subtype, the ND96 contained no atropine. Two electrode voltage clamp recordings from oocytes were carried out at room temperature using an Axoclamp 900A amplifier  at a holding potential of −70 mV. The continuous gravity perfused with standard ND96 solution and stimulated with 1-s pulses of ACh once every minute. For screening of receptor for toxin concentration 10 μM and lower, once a stable baseline was achieved, we added 5 μL of different concentration toxin to the chamber and waited for 5 min, then applied perfusion system, during which 1-s pulses of 100 μM ACh were applied every minute until a constant level of block was achieved. The electrophysiology data were recorded and analyzed using Clampfit 10.2 software . The results were acquired with at least three oocytes. Oocytes were voltage-clamped and exposed to ACh and CTxs as described previously . Briefly50)nH}, where nH is the Hill coefficient, by nonlinear regression analysis using GraphPad Prism 5.0 .The data and statistical analysis in this study comply with previous strategy . An averAplysia californica acetylcholine-binding protein) with the potent mutant TxIA(A10L) (PDB ID: 2BR8) [Homology models of the extracellular ligand binding domain of Rat α3β4 nAChR were generated using Modeler9v14 as previous method ,41. The D: 2BR8)  and crysD: 2BR8)  were adohttp://www.pymol.org/).Molecular dynamics (MD) simulations were performed for the α3β4 nAChRs with TxID and its two analogues. The structure of methionine sulfoxide was built with GaussView V.5 and Amber tools 14. These models were implemented using the GROMACS5.1  with the


A major hurdle for exploring RNA interference (RNAi) in a therapeutic setting is still the issue of in vivo delivery of small RNA molecules (siRNAs). The chemical modification of polyethylenimines (PEIs) offers a particularly attractive avenue towards the development of more efficient non-viral delivery systems. Here, we explore tyrosine-modified polyethylenimines with low or very low molecular weight  for siRNA delivery. In comparison to their respective parent PEI, they reveal considerably increased knockdown efficacies and very low cytotoxicity upon tyrosine modification, as determined in different reporter and wildtype cell lines. The delivery of siRNAs targeting the anti-apoptotic oncogene survivin or the serine/threonine-protein kinase PLK1  oncogene reveals strong inhibitory effects in vitro. In a therapeutic in vivo setting, profound anti-tumor effects in a prostate carcinoma xenograft mouse model are observed upon systemic application of complexes for survivin or PLK1 knockdown, in the absence of in vivo toxicity. We thus demonstrate the tyrosine-modification of (very) low molecular weight PEIs for generating efficient nanocarriers for siRNA delivery in vitro and in vivo, present data on their physicochemical and biological properties, and show their efficacy as siRNA therapeutic in vivo, in the absence of adverse effects. The induction of RNA interference (RNAi) by small interfering RNAs (siRNAs)  has grea® (Patisiran), was approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) in 2018. ONPATTRO® is a lipid-based siRNA nanoparticle designed to downregulate transthyretin in the liver for treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis (hATTR) [While it is thus necessary and sufficient to deliver siRNAs to the cell for inducing RNAi-mediated gene knockdown, the physicochemical properties of siRNAs, i.e., relatively high molecular weight, high polyanionic charge density, hydrophilic properties, and their instability/susceptibility to nuclease degradation, largely prevent their use in an unmodified form for therapeutic approaches . Beyond  (hATTR) ,15. Desp (hATTR) .Polymeric reagents are another well studied class of materials for the delivery of nucleic acids . A hN-acetyl-leucine was used for the delivery of miR34a, significantly improving bone regeneration in an orthodontic in vivo model [The chemical modification of PEI with amino acids offers another very promising approach for nucleic acid delivery in vivo. In previous studies, the grafting of arginine, lysine, or leucine onto PEI showed increased β-galactosidase activity in tumor-bearing mice upon i.v. injection of modified PEI/pDNA complexes . In anotvo model . The incvo model ,49.It becomes apparent from these studies that for the successful modification of cationic polymers with amino acids a balanced hydrophobicity and hydrophilicity is of great importance. This may then also allow for exploring lower molecular weight PEIs which are insufficiently active in the unmodified form, especially for the delivery of small nucleic acid molecules like siRNAs, but show significantly improved bioactivities upon introducing modifications. In a recent study, we selected a low molecular 10 kDa branched PEI for tyrosine modification. This new PEI derivative (P10Y) showed excellent in vitro siRNA transfection efficacies and increased complex stability upon serum contact. Moreover, P10Y/siRNA complexes decreased tumor growth in an aggressively growing melanoma xenograft mouse model ,51. Base2. HCT116, PC3, G55T2 cells were grown in IMDM medium, H441 and MV3 cells were grown in RPMI 1640 medium, and Saos-2 cells were grown in McCoy’s 5a medium. All media were supplemented with 10% FCS and 2 mM alanyl-glutamine.Cell culture plastics and consumables were from Sarstedt . Cell culture media were from Sigma-Aldrich  and fetal calf serum was purchased from Biochrom . The cell lines HCT116 , H441 (lung adenocarcinoma), PC3 (prostate carcinoma), Saos-2 (osteosarcoma), and MV3 (melanoma) were obtained from ATCC/LGC Promochem , and G55T2 (glioblastoma) was a kind gift from Katrin Lamszus . All cell lines were cultivated in a humid atmosphere at 37 °C and 5% COReporter cell lines were stably transduced by lentiviral transduction. The EGFP/luciferase plasmid pCCLc-MNDU3-Luciferase-PGK-EGFP-WPRE was a gift from Fernando Fierro  ) and theN-Boc-tyrosine-OH, N-hydroxysuccinimide, EDC·HCl were from Carbolution Chemicals . Dry N,N-Dimethylformamide (DMF) was from VWR , Trifluoroacetic acid (TFA), ethanol, and methanol were from Carl Roth .Branched polyethylenimines were obtained from the following suppliers: 2 kDa PEI , 5 kDa PEI , and 10 kDa PEI . N-Boc (tert-butyloxycarbonyl) protected tyrosine  and N-hydroxysuccinimide  were dissolved in 3 mL dry DMF in a glass vial, followed by addition of EDC·HCl , and stirred for 4 h under a nitrogen atmosphere at RT. In a second vial, PEI  was dissolved in 3 mL dry DMF, and the pre-activated tyrosine mixture was added and further stirred under nitrogen for 3 d. The reaction mixture was purified by dialysis  against methanol for 10 h with intermediate solvent replacement to remove by-products. Next, the methanol was removed in vacuo and the viscous polymer was dissolved in 3 mL TFA, prior to stirring overnight for Boc-deprotection. Excess TFA was removed by co-evaporation with ethanol. Finally, the crude polymer was dissolved in 0.1 M HCl and excessively purified by dialysis against 0.05 M HCl for 24 h, then against water for 48 h. Lyophilization yielded the tyrosine-modified PEIs as yellowish fluffy powders. The degree of functionalization (~25–30% based on ethylenimine monomer) was confirmed by 1H-NMR  as described previously [For the tyrosine modification, eviously ,51.The polyplexes were prepared based on polymer/siRNA mass ratios. Typically, the P5Y- and P10Y-based complexes were prepared at a mass ratio of 2.5 unless indicated otherwise. For P2Y, a mass ratio of 10 was used. For standard transfection experiments in a 24 well plate, 30 pmol (0.4 µg) siRNA per well was diluted in 12.5 µL HN buffer -1-piperazineethanesulfonic acid), pH 7.4). In a separate tube, the calculated amount of the polymer was diluted in 12.5 µL HN buffer and the siRNA solution was added to the polymer solution, thoroughly mixed by pipetting up and down and incubated for 30 min at RT. For serum stability studies, the prepared polyplexes were additionally incubated with different volumes of FCS.w/v) glucose, 10 mM HEPES pH 7.4. The complexation was performed as described above.For in vivo experiments, the polyplexes were prepared as above, with slight modifications. Per dose for injection, 10 µg siRNA was diluted in 75 µL HN buffer and 25 µg (mass ratio 2.5) of the polymer (P5Y) was diluted in 75 µL 5%  and phase analysis light scattering (PALS) with a Brookhaven ZetaPALS system . Polyplexes containing 5 µg siRNA in 250 µL were prepared as described above and diluted to 1.7 mL with millipore water. The data were analyzed using the manufacturer’s software, with applying the viscosity and refractive index of pure water at 25 °C. For the size determination, polyplexes were measured in five runs with a run duration of 1 min per experiment. Zeta potentials were analyzed in ten runs, with each run containing ten cycles using the Smoluchowski model. Additionally, polyplex sizes were analyzed by nanoparticle tracking (NTA) using a NanoSight LM10  equipped with a 640 nm sCMOS camera, software NTA 3.0, and a siRNA concentration of 1 µg/mL.v/v) for 1 h. The complexes were then subjected to the polyanion heparin at different concentrations (units), as indicated in Complex stabilities were determined by a heparin displacement assay. The complexes based on the different tyrosine-modified PEIs were prepared at their optimal mass ratios as described above, prior to further incubation in FCS . The medium was aspirated and the cells were lysed with 300 µL Luciferase Cell Culture Lysis Reagent  for 30 min at RT. In a test tube, 10 µL lysate was mixed with 25 µL substrate and the luminescence was immediately measured in a luminometer .® Acoustic Focusing Cytometer . Finally, 20,000 events in the vital gate were analyzed using the Attune® software (V2.1.0).The EGFP expression was determined by flow cytometry after 72 h. The cells were trypsinized, centrifuged for 3 min at 3000 rpm, and resuspended in 0.5 mL PBS, 1% BSA. The cells were measured in an AttuneFor uptake experiments, complexes were prepared as described above , but usi® 647-labeled siRNA (2 µg siRNA/well). After 36 h, the siRNA uptake and intracellular distribution was analyzed by confocal microscopy .To visualize the complex uptake by confocal microscopy, PC3 wildtype cells were seeded at a density of 200,000 cells onto glass coverslips in a 6 well plate. The next day, the cells were transfected with complexes of the different tyrosine-modified PEIs containing Alexa Fluor For proliferation assays, cells were seeded at a density of 2000 cells (PC3 and Saos-2) or 1000 cells (MV3) per well of a 96 well plate in 100 µL medium on the day before transfection. For endpoint cell viability assays, PC3 or G55T2 cells were seeded at a density of 7000 cells per 96 well in 100 µL medium. The number of metabolically active cells after transfection was determined by using a colorimetric assay . Briefly, after aspirating the medium, 50 µL of a 1:10 dilution of WST-8 in serum-free medium was added to the cells and incubated at 37 °C for 1 h. The absorbance was measured at 450/620 nm in a plate reader.Acute cell damage caused by the polyplexes was determined by measuring the extent of lactate dehydrogenase (LDH) release, using the Cytotoxicity Detection Kit  according to the manufacturer’s manual. Briefly, conditioned medium from transfection experiments (negative control siRNA) was collected after 24 h. For the determination of the maximum LDH release, cells were lysed with Triton X-100 at a final concentration of 2%, and conditioned medium from untreated cells served as negative control. In a 96 well plate, 50 µL sample medium was mixed with 50 µL reagent mix and incubated for 30 min in the dark. The reaction was stopped with 50 µL 1 M acetic acid and the absorption at 490/620 nm was measured using a plate reader. For blank value correction, fresh fully supplemented medium was mixed with the reagent and subtracted from all values.Total RNA was isolated using a combined TRI reagent and silica column protocol. Per 24 well, cells were lysed in 500 µL TRI reagent  and incubated for 5 min at RT. The lysate was transferred into a 1.5 mL tube and 200 µL chloroform was added, mixed well by shaking and incubated for 5 min at RT. The samples were centrifuged at 12,000 g for 15 min and the upper aqueous phase (~300 µL) was pipetted into a new 1.5 mL tube containing 300 µL 100% ethanol. After mixing, the sample was loaded onto the column , prior to centrifugation at 8000 g for 1 min. The flow-through was discarded. The column was washed twice with 450 µL 3 M sodium acetate pH 5.2, and once with 70% ethanol by centrifugation at 8000 g for 1 min; the collection tube was emptied after each step. For the drying step, the collection tube was replaced by a new one, and the columns were centrifuged at 12,000 g for 2 min. For RNA elution, 15 µL DEPC-water, pre-warmed to 65 °C, was added onto the column and incubated for 2 min, before finally eluting the RNA by centrifugation at 8000 g for 2 min. RNA concentrations and purities were measured in a NanoDrop 2000c .2O, and 0.5 µL RevertAid™ H Minus M-MuLV Reverse Transcriptase (200 U/µL) were added. The cDNA synthesis mixture was incubated at 25 °C for 10 min, 42 °C for 60 min, and heat denatured at 70 °C for 10 min.The total RNA was reverse transcribed with the RevertAid™ H Minus First strand cDNA synthesis Kit . In brief, 1 µL random hexamer primer was added to 1 µg RNA diluted in 10 µL DEPC-treated water, followed by an incubation step for 5 min at 65 °C and cooling to 4 °C. Then, 4 µL 5× reaction buffer, 2 µL 10 mM dNTP mix, 2.5 µL DEPC-treated ddH® Green FastMix ROX  and 1 µL 5 µM forward and reverse primer mix  was used, applying the ΔΔCt method . The cDN6 cells in 50 µL were mixed with 50 µL complexes containing different siRNA amounts and incubated for 1 h at 37 °C. For absorption measurements, 25 µL of each sample were transferred into a 96 well plate in triplicates. Erythrocytes incubated with buffer served as negative control and erythrocytes treated with Triton X-100 to a final concentration of 1% served as positive control (= 100%). The absorbance of the samples was measured at 550 nm using a plate reader.To determine the hemolytic activity of the complexes, whole blood from healthy mice was diluted in Ringer’s solution and erythrocytes were purified by several centrifugation and washing steps at 5000 rpm for 5 min, until the supernatant became clear. After the final washing step, the cells were taken up into physiological NaCl solution. Then, 1 × 106 cells/mL) were mixed with 50 µL aqueous solution containing 7.5 µg P5Y/3 µg siCtrl, or 750 kDa branched PEI (1 and 3 µg) as positive control. After incubation for 2 h at 37 °C, the samples were mounted onto glass slides and examined microscopically.To evaluate erythrocyte aggregation, 50 µL of the purified cells (~1 × 106 PC3 cells in 150 µL PBS subcutaneously (s.c.) into both flanks of mice. When tumors reached a size of ~100 mm3, the mice were randomized into different treatment groups (6–9 tumors/group). The polyplexes were prepared as described above and amounts corresponding to 10 µg siRNA were i.p. injected every 2–3 days over 14 days.Athymic nude mice  were kept at 23 °C in a humidified atmosphere, 12 h light/dark cycle, with standard rodent chow and water ad libitum. Animal studies were performed according to the national regulations and approved by the local authorities . Tumor xenografts were established by injecting 3 × 106 HCT116-Luc cells were s.c. injected for the induction of tumor xenografts described above. Mice were treated five times every 2–3 days with P5Y/siRNA complexes (siLuc2 = siCtrl and siLuc3 = Luciferase specific siRNA). Tumors were excised and lysed with Luciferase Cell Culture Lysis Reagent (app. 100 mg tumor tissue/1 mL buffer), prior to homogenization using an ULTRA-TURRAX®. Homogenates were repeatedly centrifuged at 13,000 rpm for 5 min and the supernatants were transferred into fresh tubes until the lysate became clear. Luminescence was determined as described above. Relative light units (RLU) were normalized for total protein concentration of the lysates using the BCA assay  according to the manufacturer’s protocol.For in vivo luciferase knockdown experiments, 3 × 10For the determination of blood serum markers, healthy nude mice were i.p. injected with P5Y/siLuc3 complexes containing 10 µg siRNA every 2–3 days, with a total of four times over 8 days. Three hours after the last injection, the blood was collected. Untreated mice served as negative control. The serum was diluted 1:20 with water and serum levels of various analytes were determined using an AU480 .For the analysis of the immunostimulatory cytokines TNF-α and INF-γ, P5Y/siLuc3 complexes (10 µg siRNA) were i.v. injected twice within 24 h into immunocompetent C57BL/6 mice. Four hours after the last injection, the blood was collected. Mice treated with lipopolysaccharides (LPS) 50 µg in 150 µL (single injection) served as positive control and untreated mice served as negative control. The serum levels of TNF-α and INF-γ were measured using ELISA kits  following the manufacturer’s instructions.t-test or One-way ANOVA, and significance levels are * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and # = not significant, with at least n = 3.Statistical analyses were performed by Student’s 1H-NMR analysis as described  revealed a ~30% decrease in siRNA fluorescence when switching from P5Y to P10Y, while siRNA delivery in P2Y complexes was poor . This waThe very profound siRNA transfection efficacies of P5Y were also confirmed in other cell lines like Saos-2 osteosarcoma cells D. A 1.5-For physicochemical characterization, complexes with different polymer/siRNA mass ratios were subjected to Zetasizer and Nanosight measurements. The hydrodynamic diameters determined by dynamic light scattering (DLS) were around 200–400 nm, with a slight trend towards smaller complexes with lower P5Y/siRNA mass ratios A.The diameter for the optimal mass ratio of 2.5 was additionally characterized by nanoparticle tracking (NTA) of which the main peak was at 200 nm and a second small peak at 400 nm B. The meHigh biological activity may well be associated with increased cytotoxicity or other adverse effects, which were therefore assessed next. Lactate dehydrogenase (LDH) release assays, however, revealed no increase of LDH levels over background (untransfected cells). This was true for both, P5Y/siRNA and P10Y/siRNA complexes and was found in two reporter cell lines . Upon termination of the experiment at day 6 after treatment start, tumors were harvested and luciferase activities were analyzed from tumor lysates. Some variations were found between individual samples, especially in the negative control group. Still, the comparison of luciferase activities between the specific (P5Y/siLuc3) and the non-specific negative control (P5Y/siCtrl) group revealed a ~25% reduction of luciferase expression already after three injections , as indicated by identical tumor growth curves , blood glucose (Gluc), urea, albumin, or LDH A. Other PEIs and other cationic polymers are promising with regard to nucleic acid delivery, but they are often associated with toxicity. This is particularly true for higher molecular weight PEIs, while their lower molecular weight counterparts suffer from poor efficacy ,22,23. TThe fact that PEIs offer relatively easy access to chemical modification is a particularly attractive approach for further improvement of physicochemical and biological properties of the polymer and its complexes. Tyrosine may be considered as among the best candidate amino acids for chemical modification, due to its ability to balance between hydrophilicity (primary amines necessary for nucleic acid complexation) and hydrophobicity . ManSerum stability is an important factor for nucleic acid delivery systems, and we demonstrate high serum compatibility of our complexes based on tyrosine-modified PEIs (see Results and ). This mIndeed, we could demonstrate in this study that tyrosine-modification allows for using very low, otherwise inactive PEIs. High complexation efficacies even at very low mass ratios are particularly relevant in the case of small oligonucleotides like siRNAs, considering the lack of binding cooperativity and their high rigidity . Our res