Oligodendrocyte progenitor cells (OPCs), which differentiate into myelinating oligodendrocytes during CNS development, are the main proliferative cells in the adult brain. OPCs are conventionally considered a homogeneous population, particularly with respect to their electrophysiological properties, but this has been debated. We show, by using single-cell electrophysiological recordings, that OPCs start out as a homogeneous population but become functionally heterogeneous, varying both within and between brain regions and with age. These electrophysiological changes in OPCs correlate with the differentiation potential of OPCs; thus, they may underlie the differentiational differences in OPCs between regions and, likewise, differentiation failure with age.  •Oligodendrocyte progenitor cells (OPCs) acquire ion channels with age•OPCs become functionally heterogeneous both between brain regions and with age•NMDARs disappear in non-myelinating regions but remain in actively myelinating areas•Heterogeneity in OPC ion channel density indicates different functional states Spitzer et al. show that oligodendrocyte progenitor cells (OPCs) acquire ion channels and sensitivity to neuronal activity that differ between region and age. The onset and decline of ion channels follow developmental milestones. This heterogeneity indicates different functional states of OPCs. Glial cells, astrocytes, oligodendrocytes, and microglia are conventionally thought of as homogeneous cell types; however, recent findings show that glial cells, like neurons, have a different transcriptome and proteome depending on the brain region and with age and that the glial cell transcriptome, but not the neuronal one, is mostly altered by aging . How thede novo myelination, maintenance, and regeneration.The fourth type of glial cell, the oligodendrocyte progenitor cell (OPC), which expresses the proteoglycan NG2, is widely distributed throughout the adult brain and is the main proliferative cell present in the adult CNS . Its funin vitro and in vivo, shows that neuronal activity and glutamate signaling are regulatory signals for myelination and remyelination in young adults  knockin mice, where the expression of the reporter gene is regulated according to the endogenous NG2 gene, allowing unbiased sampling of this population. We report that OPCs start out as a homogeneous population but become functionally heterogeneous, varying both within and between brain regions and age. These electrophysiological changes correlate with the differentiation potential of OPCs.+ cells are Olig2- and NG2-positive throughout life, indicating that EYFP expression tightly follows expression of the NG2 protein ; in these mice, all parenchymal EYFP protein B. We vol protein ; then at protein ; then we protein ; and the protein A.Figure V), a stereotypic transient inward current that is evoked upon depolarization beyond −50 mV. This depolarization-evoked transient inward current was absent in OPCs at E13. In fact, at this time, no voltage-gated currents were detected in OPCs. We first detected a depolarization-evoked transient inward current, known to be TTX-sensitive NaV (V) currents, in a subset of OPCs at E18. Notably, KV currents were detected in 85% of cells, whereas NaV were detected in only 38% of OPCs at E18 (p = 0.04), indicating that KV channel expression starts before NaV expression. Although both the proportion of OPCs with functional NaV channels and their density sharply increased after birth, the proportion of OPCs with NaV channel expression lagged behind KV channels  and was detected in over 90% of OPCs recorded from the second postnatal week  following the end of the peak period of developmental myelination , but not a low membrane resistance, as is more reminiscent of OPCs that have started to differentiate (−7), starting low, peaking at the time when the myelination rate is highest in the forebrain, and then declining after the first month to a level similar to that of prenatal cells -evoked currents were undetectable  in a majority of OPCs at E18 , significantly more so compared with OPCs from P12 or older mice. However, at P12, OPCs had stronger signatures of proliferating  and committed OPCs (Myrf and Enpp6) and upregulated signal transduction but a reduced migratory molecular signature , differentiation, and metabolism at the time when myelination starts to decline  of OPCs isolated by PDGFRα magnetic activated cell sorting  at different ages: E16, P12, P80, and P310. We observed that embryonic OPCs had molecular signatures of migrating cells  and NMDAR subunits  significantly increased from embryonic to postnatal OPCs, and subsequently all remained constant to old age, with the exception of Grin3a and Scn3b. However, AMPAR and KAR subunits, apart from Grik3, remained constant from E16 to old age  have a 1.7-fold higher density of NaV than OPCs in G0/G1phase . There was no change detected in either NMDAR or AMPA/KAR densities between G2/M phase and G0/G1 phase of the cell cycle  compared with cycling OPCs (G2/M phase). To test for heterogeneity, we analyzed the variance in the dataset between non-cycling OPCs and cycling OPCs. The variance in the density of NaV channels and NMDARs was identical between both states , whereas the variance in AMPA/KAR densities differed greatly (p = 5 × 10−8), implying that AMPA/KAR densities in OPCs might differ between G1 and G0 phase of the cell cycle.To test whether ion channel expression changes with the cell cycle, we crossed NG2-EYFP mice with either Fucci2a mice, in which mCherry is expressed during GG1 phase A and 3G,G1 phase A and 3F.+ OPCs reached a constant level by the end of the first month, whereas in the CTX, the proportion of proliferating OPCs gradually decreased with age , AMPA/KAR (p = 0.79), and NMDAR (p = 0.59) densities and frequency of synaptic inputs (p = 0.12). Hence, at this time, OPCs have a similar sensitivity to neuronal activity. However, after the first postnatal week, OPCs diverged and varied significantly between gray and white matter, particularly in regard to AMPA/KAR (p = 4 × 10−4) and NMDAR (p = 0.02) densities (V channels (p = 0.29) and AMPA/KAR (p = 0.93) densities were unchanged between the two cortical layers (V channels (p = 0.47), AMPA/KAR (p = 0.39), and NMDAR (p = 0.70) densities or proportions  in OPCs located in either the anterior or posterior CC  neurons, mimicking youth (−6). Administration of growth and differentiation factor (GDF) 11 has been shown to recapitulate some of the rejuvenative effects of young blood in mice, although it has not yet been tested for myelination  from P150, when NaV and NMDAR density has declined. No differences were detected in NaV channel, AMPA/KAR, or NMDAR densities  compared with saline administration. These data indicate that when OPC properties have changed, as occurs with maturation, ion channel expression and myelination potential do not easily revert to that of neonatal OPCs.Age-related changes in the systemic milieu have been shown to lead to a decrease in ion channel function in hippocampal neurons, similar to the changes we detect in OPCs, and are linked to declining cognitive function. Similarly, the differentiation and myelination potential of OPCs also decreases with age. Parabiosis of young blood can reverse both of these age-related changes . Therefong youth A. The nelination . We thusV, AMPA/KAR, and NMDAR currents  and ML . Moreover, there was much greater variability (p < 1 × 10−15) in the NMDA-evoked currents in the SVZ compared with parenchymal OPCs, presumably indicating continuous cycles of early-born and old OPCs in the SVZ (Next we addressed whether the changes in ion channel expression we identified differ between white matter (CC) and a gray matter region that has some myelination (CTX) or a gray matter region that is never myelinated (molecular layer of the cerebellum [ML]) and the subventricular zone (SVZ), an area that provides a continuous supply of myelinogenic OPCs throughout life A. At P7,currents B–6E. Theents −3; E. In conents −3; D, and a  the SVZ E.Figure V, reminiscent of early embryonic OPCs (−3), whereas in the CTX, the AMPA/KAR density peaked at P20–P35  and NMDAR densities, which both peaked at a time when OPCs start to differentiate into myelinating oligodendrocytes and begin to express myelin genes  OPCs in S/G2/M phase have a higher density of NaV than OPCs in G0/G1 phase  in cells recorded in G0/G1 phase suggests that AMPA/KAR may differ between G1 and G0.For example, when OPCs first appear, at E13, they have no detectable voltage-gated ion channels or glutamate receptors and may therefore be considered as being in a naive state. The first ion channels to appear are KG1 phase H, (2) thG1 phase  at the tG1 phase E, (3) EdG1 phase , and (4)G1 phase . NMDARs n starts , and at n starts  and so mV channel, AMPA/KAR, and NMDAR densities drive the age-related changes in OPC behavior nor whether maintaining high densities of NMDARs and NaV, and low AMPA/KAR densities in OPCs would counteract the age-dependent decline in myelination. What is clear is that these changes in NaV channel, AMPA/KAR, and NMDAR densities will have a profound effect on how OPCs sense neuronal activity and on the effect neuronal inputs will have on OPCs. With increasing understanding of the regulation of gene expression and generation of novel biological engineering technology, it may become possible to manipulate NaV channel, AMPA/KAR, and NMDAR densities in OPCs postnatally. However, as postnatal NaV channels, AMPA/KAR and NMDAR gene expression is relatively stable; this indicates more that the detected changes in the density of these channels are post-transcriptionally and/or post-translationally regulated. How these changes are regulated is currently unknown, but presumably they are environmentally regulated .Further information and requests for resources and reagents should be directed to, and will be fulfilled by, the Lead Contact, Ragnhildur T. Káradóttir  Act 1986 and subsequent amendments. Use of animals in this project was approved by the Animal Welfare and Ethical Review Body for the University of Cambridge and carried out under the terms of UK Home Office Licenses P9B1FBC4B and 70/7715. All mice were maintained under a 14 h light:10 h dark cycle with food and water supplied -in mice  expressi-in mice , or Ki67-in mice  to the N2–5% CO2) aCSF solution and kept at RT after a 1h recovery period. aCSF contained (in mM): 126 NaCl, 24 NaHCO3, 1 NaH2PO4, 2.5 KCl, 2.5 CaCl2, 2 MgCl2, 10 D-glucose, pH 7.4. 1mM Kynurenic acid was added to block glutamate receptors, which might be activated during the dissection procedure. For embryonic acute brain slices, the brains were embedded in agarose and sliced coronally in 225 μm thick sections as above.Acute brain slices (225 μm thick) were prepared either from the cerebellum  or the forebrain  from transgenic mice in ice-cold (∼1°C) oxygenated , 0.005 strychnine (to block glycine receptors), 200μM BaCl2 (only in some instances to block potassium conductance), pH set to 7.4 with NaOH, continuously bubbled with 100% O2. OPCs were whole-cell clamped with electrodes containing a recording solution that comprised (in mM) of either 130 K-gluconate or 130 Cs-gluconate, 4 NaCl, 0.5 CaCl2, 10 HEPES, 10 BAPTA, 4 MgATP, 0.5 Na2GTP, 2 K-Lucifer yellow, pH set to 7.3 with KOH (or with CsOH). Final osmolarity was ∼290mOsm. For activation of glutamate receptors, KA 30 μM  or NMDA 60 μM (Tocris) was bath applied.Slices were superfused at 22 ± 1°C with HEPES-buffered external solution containing (in mM): 144 NaCl, 2.5 KCl, 10 HEPES, 1 NaHFor whole-cell patch-clamp experiments parenchymal EYFP positive OPCs were selected in the brain area of interest (EYFP positive cells located close to or on blood vessels were excluded). In embryonic slices, OPCs were selected in the developing CTX at a sufficient distance from nearby blood vessels. When unspecified CTX OPCs were mainly recorded in cortical layers 5-2. When unspecified, OPCs in the CC were in and around the anterior CC. Patched cells were confirmed as OPCs by their post-recording dye-fill morphology, which overlaid EYFP, and by post hoc antibody labeling against the proteoglycan NG2 to identify oligodendrocyte progenitors (106/107 tested labeled for NG2 (Millipore) or GFP/EYFP (Abcam)). For identification of OPCs location: CTX Layer 1 was identified as the area between the edge of the slice and layer 2 marked with Cux1 antibody labeling; CTX layer 5/6 was marked with CTIP2 immunoreactivity; dorsal SVZ was identified with doublecortin labeling and the adjacent ventricle; anterior CC was identified at bregma 0 and posterior CC was identified overlaying the hippocampus A–S4D. ReSynaptic events were detected based on threshold (amplitude > 2xSD) and included when decay time τ (10%–90%) was longer than its rise time. For event detection and analysis of frequency, amplitude and decay time τ pClamp 10.3 (Molecular Devices) and the Strathclyde Electrophysiology Software package WinEDR V3.3.7 WinWCP V4.6.2 were used. Voltage-gated ion channels, series resistance, and membrane capacitance was analyzed using custom-written MATLAB scripts (MathWorks).EYFP-NG2 knock-in mice were implanted intraperitoneally with osmotic minipumps  at P150 for continuous i.p. delivery at a flow rate of 0.11 μl/h containing GDF11  or saline. After 4-weeks delivery mice were used for electrophysiological recordings, only cells responding to an agonist were used for comparisons, the proportion of cells responding did not differ between conditions. The use of minipumps ensured reduced stress responses of the animals and consistent dosing compared to i.p. injections which require daily handling.Mouse OPCs were isolated from EYFP-NG2 knock-in mice at different ages, as stated in manuscript, using magnetic cell sorting . First myelin debris were removed from single-cell suspension by myelin removal beads , and subsequently, OPCs were isolated using microbeads conjugated to monoclonal PDGFRα . Myelinating co-cultures were made as previously described . BrieflyAll the mice used were wild-type C57BL/6 mice (Charles River Laboratories) at the ages detailed in the manuscript. OPCs were isolated as described above for co-cultures, except from the E15.5 and P12 time points, where the myelin removal step was omitted. For the E15.5 and P12 time-points, each sample (n = 3) was a pool of 2-3 brains, all other time-points were isolated from one brain per sample . After OPC enrichment RNA was extracted using RNeasy Micro Kit (QIAGEN) according to manufacturer’s instructions. The library was prepared with SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Clontech) according to manufacturer’s instructions. Data was read on a HiSeq4000 a total of > 350M 150 bp strand-specific paired-end reads were generated.Trim Galore!https://www.bioinformatics.babraham.ac.uk/projects/trim_galore). Reads were aligned to the mouse reference genome GRCm38/mm10 using Tophat  allowing for one mismatch and selecting uniquely mapping reads only. Normalization was performed and differential expression of genes was statistically evaluated by using the R Bioconductor DESeq2 package (https://bioconductor.org/packages/release/bioc/html/DESeq2.html).RNA-seq reads were pre-processed and quality-trimmed using https://david.ncifcrf.gov). GO terms were represented according to their semantic similarity using REVIGO (http://revigo.irb.hr).Gene ontology (GO) term enrichment analysis for differentially expressed genes was performed using DAVID (De Novo Software)  according to manufacturer’s instructions. Cells were fixed in Fixation Buffer (BioLegend) for 15 min, permeabilized with Intracellular Staining Permeabilization Wash Buffer (BioLegend) for 10 min. After washing, cells were stained with DAPI (1μg/ml) for 10 min. Cells were washed again and acquired on a BD LSRFortessa flow cytometer (BD Bioscience) equipped with four lasers, a blue (488-nm), a red (640-nm), a violet (405-nm) and a yellow/green (561-nm) laser. The EYFP positive cells were detected by filtration through a 530/30 nm band pass (BP) filter and DAPI through a 450/50 nm BP filter. Analysis was performed with FCS Express 6 Flow (oftware) .225μm thick slices were cut as described above, and fixed in 4% paraformaldehyde. Antibody labeling was performed as described previously . BrieflyTo measure proliferation with immunohistochemistry, 3-6 slices from an average of 2 animals were quantified for each time point. Images were taken on a confocal microscope. 6 z stacks (3 in the cortex and 3 in the CC) were taken randomly in each slice. Z stacks were taken in order to include all EYFP+ cells in a field of view. Analysis was performed manually on the maximum projection of each stack, with the number of EYFP+ Ki67+ cells divided by the number of EYFP+ cells. Experimenters were not blind to age during quantification. All data collected was included in the statistical analysis, which was performed with a one-way ANOVA for each brain region.All statistical analysis was performed in GraphPad Prism or SPSS software or manually calculated. Data are shown as mean ± s.e.m. For electrophysiology results, numbers of cells are indicated on bars. For flow cytometry analysis , number GSE121083 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE121083).All data have been deposited in NCBI’s Gene Expression Omnibus  and are Flowrepository.org (https://flowrepository.org/id/RvFrXKqGrE6oSqbz3pUcrGHhBsNIcmlH5nBR0azMBaDS8vTjRXww0B6IbjNgEFkr)All flow cytometry data has been deposited in V current, KV current and K+ conductance analysis scripts are available upon request.Cell capacitance, Na


P. aeruginosa is a soil dwelling bacterium and a plant pathogen, and it also causes life-threatening infections in humans. Thus, P. aeruginosa thrives in diverse environments and over a broad range of temperatures. Some P. aeruginosa strains rely on the CRISPR-Cas adaptive immune system as a phage defense mechanism. Our discovery that low temperatures increase CRISPR adaptation suggests that the rarely occurring but crucial naive adaptation events may take place predominantly under conditions of slow growth, e.g., during the bacterium’s soil dwelling existence and during slow growth in biofilms. Pseudomonas aeruginosa, quorum sensing (QS) induces the CRISPR-Cas defense system at high cell density when the risk of bacteriophage infection is high. Here, we show that another cue, temperature, modulates P. aeruginosa CRISPR-Cas. Increased CRISPR adaptation occurs at environmental  temperatures compared to that at body  temperature. This increase is a consequence of the accumulation of CRISPR-Cas complexes, coupled with reduced P. aeruginosa growth rate at the lower temperature, the latter of which provides additional time prior to cell division for CRISPR-Cas to patrol the cell and successfully eliminate and/or acquire immunity to foreign DNA. Analyses of a QS mutant and synthetic QS compounds show that the QS and temperature cues act synergistically. The diversity and level of phage encountered by P. aeruginosa in the environment exceed that in the human body, presumably warranting increased reliance on CRISPR-Cas at environmental temperatures.Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) systems are adaptive defense systems that protect bacteria and archaea from invading genetic elements. In  Bacteria have evolved defense systems to fend off bacteriophage viruses that prey upon them. One of these systems, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) system, provides acquired and heritable sequence-specific immunity against previously encountered viruses and plasmids , 2. CRISNaive CRISPR adaptation to a foreign genetic element that a bacterium has not previously encountered is rare in type I CRISPR-Cas systems . HoweverNaive adaptation requires cleavage of the newly infecting DNA, the incorporation of a short fragment as a new spacer in the CRISPR array, transcription and processing of the new crRNA, and formation of a crRNA-Cas complex to scan the genomic and foreign DNA to pinpoint the foreign DNA and target it for cleavage. These steps take time. During that time, the phage is executing its parasitic program, either lysogenizing the host, in which case adaptation would lead to host suicide, or generating numerous copies of the phage genome as the phage prepares to lyse the host cell. With respect to the phage lysis program, it could be difficult for the CRISPR-Cas machinery to keep pace (degrading phage genomes as new phage genomes are produced), possibly a contributing feature underpinning why naive adaptation is so infrequent. Indeed, in line with this argument, defective phage particles capable of infecting but not killing host bacteria have been shown to increase adaptation frequency . In thiscas expression, CRISPR-Cas activity, and CRISPR adaptation in P. aeruginosa UCBPP-PA14 (here denoted PA14), enabling CRISPR-Cas function to increase in step with increasing bacterial cell density  for successful defense against a foreign invading element prior to cell division. If, simultaneously, the slow-growth conditions were unfavorable for phage propagation, it would give the CRISPR-Cas system the chance to accomplish the adaptation program prior to the cell becoming overwhelmed by replicating phage, making this mechanism particularly effective.P. aeruginosa is an environmental bacterium and an opportunistic human pathogen that causes nosocomial infections and chronic lung infections in patients with cystic fibrosis (CF) (sis (CF) . PA14, tsis (CF) . PA14 hasis (CF) , 23.P. aeruginosa with respect to its ability to successfully reside both in the soil/plants and in mammalian hosts. These niches vary dramatically in many respects, notably for our present work, with regard to temperature and phage exposure  the CRISPR-targeted plasmid is present at higher copy number at low temperatures than at high temperatures, fostering higher rates of priming, and hence, an increase in potential adaptation events; (ii) CRISPR-Cas complexes are more abundant at low temperatures than at high temperatures, which again would increase the chances for adaptation; and (iii) the reduced bacterial growth rate at low temperatures compared to high temperatures could provide CRISPR-Cas complexes additional time to perform all of the steps necessary to achieve immunity and hence enable higher numbers of adaptation events to occur prior to each cell division. Any combination of these three mechanisms is also possible and could contribute to the connection between low temperature and high CRISPR adaptation frequency.rpoB gene as the control. First, we examined the possibility that temperature affects the relative copy number of the incoming plasmid. To do this, we measured, at different temperatures, the relative copy number of pHERD30T, the empty high-copy-number vector backbone for the pCR2SP1 seed plasmid . PA14 cevir was not affected by fusion of the 3×FLAG tag to Csy4  across the virulent phage DMS3mvir . DMS3mvir is targeted by PA14 CRISPR2 spacer 1. A functional CRISPR-Cas apparatus protects PA14 cells against this phage  of 1. We treated the culture with gentamicin to arrest protein synthesis, divided the culture into two aliquots, and incubated one aliquot at 23°C and the other at 37°C for two more hours. Csy4 levels remained constant at both temperatures  strain carrying a CRISPR spacer targeting JBD44a were grown to an OD600 of 1 at 37°C (A) or 23°C (B) and infected with phage JBD44a at a multiplicity of infection (MOI) of 30. PFU were quantified over time. SD represents 3 replicates. Cells grown at 23°C adsorb phage at a higher rate than those grown at 37°C. Download FIG S3, TIF file, 0.01 MB.Temperature affects phage adsorption independently of CRISPR-Cas. WT PA14, a ΔCRISPR ΔCopyright © 2018 Høyland-Kroghsbo et al.2018Høyland-Kroghsbo et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To measure the influence of growth rate on the ability of CRISPR-Cas to eradicate a foreign genetic element, we assayed CRISPR-Cas effectiveness in eliminating the CRISPR-targeted plasmid called pCR2SP1  when PA1600 of 1. In spite of CRISPR targeting of pCR2SP1, after rapid growth, 22% of the plasmids were retained compared to time zero. In contrast, following slow growth, only 0.03% of the plasmids were retained. Thus, CRISPR-Cas interference was >600-fold more effective during slow growth than during rapid growth . During slow exponential growth, between an OD600 of 0.025 and 0.2, the doubling time is 61.4 min (R2 = 0.9791), and between OD600 of 0.2 and 0.7, the doubling time is 185.3 min (R2 = 0.9546). n = 3. Download FIG S4, TIF file, 0.01 MB.Growth curves for PA14 grown at 150 rpm (slow) and 250 rpm (rapid). Colonies of PA14 carrying the pHERD30T plasmid were inoculated in LB broth and grown at 37°C with rapid (250 rpm) or slow (150 rpm) shaking. Growth was measured as the ODCopyright © 2018 Høyland-Kroghsbo et al.2018Høyland-Kroghsbo et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02184-18.5FIG S5600 of 1. Here, we show data for cultures grown at 250 rpm for an extended time, a time equal to that required for the cultures grown at 150 rpm to reach OD600 of 1. The data show that increased growth time at 250 rpm, which enables the culture to enter stationary phase, causes increased loss of both the untargeted pHERD30T plasmid and the CRISPR-targeted pCR2SP1 plasmid. Download FIG S5, TIF file, 0.05 MB.Plasmid loss after extended growth. The data in this figure extend the analyses shown in Copyright © 2018 Høyland-Kroghsbo et al.2018Høyland-Kroghsbo et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 12-homoserine lactone (3OC12-HSL), which activates the receptor LasR. LasR activates expression of many genes, including those encoding the second QS system, rhlI and rhlR (–4-homoserine lactone (C4-HSL) that, when bound to RhlR, activates a second wave of QS genes  exhibited reduced CRISPR-Cas expression, interference, and adaptation compared to WT PA14. Complementation of the ΔlasI ΔrhlI mutant via exogenous supplementation with the 3OC12-HSL and C4-HSL AIs restored CRISPR-Cas function to WT levels  or 150 rpm (denoted slow growth). One hundred percent denotes no plasmid loss. SD represents 3 replicates. . Download FIG S6, TIF file, 0.2 MB.The effect of QS on temperature-dependent Csy4 regulation and growth rate regulation of CRISPR-Cas activity. (A) Western blot of PA14 Csy4-3×FLAG grown at 37, 30, 23, and 15°C. Top, abundance of GroEL, which was used as the endogenous control. Bottom, abundance of Csy4-3×FLAG. Quantitation of the relative abundance of Csy4-3×FLAG normalized to GroEL is shown below the blot. (B) Retention of the control parent plasmid pHERD30T (left) and the CRISPR-targeted plasmid pCR2SP1 (right) in WT PA14 and the ΔCopyright © 2018 Høyland-Kroghsbo et al.2018Høyland-Kroghsbo et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the lasR mRNA, which in turn affects QS-controlled virulence in a mouse model of infection  Relative lasR mRNA levels normalized to 5S RNA were measured by qRT-PCR in PA14 and in the isogenic ΔCRISPR Δcas mutant strain under different QS states . (B) As in panel A, showing lasB mRNA, encoding the virulence factor elastase, the transcription of which is activated by LasR. Error bars represent SD from n = 3 replicates. . Unlike the results obtained by Li et al.  of lasI and rhlAB-R, the latter of which controls the expression of rhlR. Thermoregulation of lasI and rhlR increases their expression at 37°C compared to 30°C, and consequently, causes increased production of QS-regulated virulence factors  using HindIII and XbaI restriction sites  containing gentamicin (30 μg/ml) and irgasan (100 μg/ml), followed by recovery of mutants on M9 medium containing 5% (wt/vol) sucrose. Candidate mutants were confirmed by PCR and sequencing. A PA14 strain harboring a CRISPR spacer matching phage JBD44a was generated by cloning 1,581 bp of JBD44a gene gp33 using native HindIII sites into the adaptation-promoting pCR2SP1 seed plasmid  or the solvent dimethyl sulfoxide (DMSO) was used. Growth of bacterial cultures was measured by OD600, where 1 unit equals 109 CFU/ml.PA14 and mutants were grown at the indicated temperatures in LB broth or on LB solidified with 15 g agar/liter. LB was supplemented with 50 μg/ml gentamicin where appropriate. For AI supplementation assays, 3OClasI ΔrhlI mutant were transformed with pCR2SP1 seed, as described previously  or 2 μM 3OC12-HSL plus 10 μM C4-HSL (designated AI) was added. The plates were incubated at 37, 30, 23, and 15°C until the colonies were 1 mm in diameter. Single colonies were tested for population-wide integration of new CRISPR spacers by PCR using DreamTaq Green PCR master mix (Thermo Fisher); primers used were designed to anneal upstream of the CRISPR2 array and inside the second spacer, which enabled the detection of expansion of this array. The PCR products were subjected to agarose gel electrophoresis, and band intensities were analyzed using the ImageQuant TL software .WT PA14 and the Δeviously , and plarpoB.PA14 harboring pHERD30T, the empty vector backbone for the pCR2SP1 seed plasmid, was grown at 37, 30, 23, and 15°C on LB agar supplemented with gentamicin (50 μg/ml). Total DNA was extracted from individual colonies that were 1 mm in size using a DNeasy blood and tissue kit (Qiagen). qPCR was performed using PerfeCTa SYBR Green FastMix, Low ROX (Quantabio) with primers specific for pHERD30T and chromosomal csy4-3×flag and ΔlasI ΔrhlI csy4-3×flag mutant strains were streaked onto LB medium and grown at 37, 30, 23, or 15°C until individual colonies reached 1 mm in diameter. Single colonies were harvested and lysed with BugBuster protein extraction reagent (Millipore), following the manufacturer’s instructions. Fifty micrograms of protein was separated by SDS-PAGE on a 4 to 20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad) and blotted onto a polyvinylidene difluoride (PVDF) membrane . The membrane was incubated for 1 h with monoclonal Anti-FLAG M2-peroxidase (HRP) antibody , monoclonal anti-RpoB antibody , both at a 1:3,000 dilution, or polyclonal anti-GroEL antibody  at a 1:15,000 dilution in Tris-buffered saline with Tween 20 (TBST) and 5% skim milk. Anti-rabbit antibody  was used as the secondary antibody for detection of the anti-RpoB and anti-GroEL antibodies. The membrane was washed in TBST and was developed using SuperSignal West Femto maximum sensitivity substrate .The PA14


As measured by atomic emission spectroscopy, MNCb also demonstrated better delivery efficiency to tumors (3.79% ID) than MNCl (2.94% ID) and MNRd (1.21% ID). Nevertheless, MNCl overperformed its counterparts in tumor imaging, providing contrast enhancement in 96% of studied malignancies, whereas MNCb and MNRd were detected by MRI in 73% and 63% of tumors, respectively. Maximum MR contrasting efficiency for MNCb and MNCl was around 6-24 hours after systemic administration, whereas for MNRd maximum contrast enhancement was found within first 30 minutes upon treatment. Presumably, MNRd poor MRI performance was due to low r2-relaxivity and rapid clearance by lungs (17.3% ID) immediately after injection. MNCb and MNCl were mainly captured by the liver and spleen without significant accumulation in the lungs, kidneys, and heart. High biocompatibility and profound accumulation in tumor tissues make MNCb and MNCl the promising platforms for MRI-based tumor diagnostics and drug delivery.Magnetic resonance imaging (MRI) is a powerful technique for tumor diagnostics. Iron oxide nanoparticles (IONPs) are safe and biocompatible tools that can be used for further enhancing MR tumor contrasting. Although numerous IONPs have been proposed as MRI contrast agents, low delivery rates to tumor site limit its application. IONPs accumulation in malignancies depends on both IONPs characteristics and tumor properties. In the current paper, three differently shaped Pluronic F-127-modified IONPs  were compared side by side in three murine tumor models . Orthotopic B16 tumors demonstrated more efficient IONPs uptake than heterotopic implants. Magnetic nanocubes (MNCb) had the highest r2-relaxivity in vitro (300 mM In recent decade, as nanotechnology and materials science have progressed, nanomaterials have been mass produced and widely applied in various fields of medicine. Nanoparticles (NPs) are known to extravasate through fenestrations in abnormal neovasculature and accumulate in malignant tissues due to poor lymphatic drainage typical for tumors . This phIron oxide nanoparticles (IONPs) remain among the best choice for bioimaging studies being nontoxic and biodegradable –11. HoweCore size (dC) of the iron oxide nanoparticles plays a very important role in determining its saturation magnetization and dictates relationship between T1 and T2 relaxation times when used as MRI contrast agents , 11. ThuNanostructures with a high length to width aspect ratio (gold nanorods and iron oxide “nanoworms”) have shown longer blood circulation times over the spherical counterparts , 21. Ther2) is 3- to 5-fold higher compared with individual nanocrystals [r1) is also enhanced in multicore nanoparticles [r1 and r2 properties, there are only few works focusing on its MRI contrasting properties for tumor diagnostics [Nanoparticles magnetic performance is also shape dependent. Thus, colloidal nanocrystal clusters have been suggested as perspective nanoplatform for bioimaging . Magneticrystals , 30 and crystals , 28. Morarticles , 31. Desgnostics . Since mgnostics .Uncoated IONPs are often colloidally unstable and get rapidly eliminated by the macrophages. Different types of natural and synthetic polymers have been used to improve the pharmacokinetic performance of the IONPs . PluroniApart from nanoparticles' properties, biological factors also determine IONPs pharmacokinetics and biodistribution. Variations in the tumor-targeted delivery of the IONPs through the EPR effect have been attributed to differences in animal models, mice strain, and biological diversity of the tumors , 37. Altβ-FeOOH nanorods were obtained by hydrolysis of FeCl3 water solution in the presence of brunched high-weight polyethyleneimine . First, 2 mL of PEI was dissolved in 100 mL of deionized water and then 2.54 g of FeCl3 was added to the obtained solution. The mixture was heated at 80°C under magnetic stirring for 2 h. After cooling the mixture to room temperature, the pH was adjusted to 7.0 by adding 2 M NaOH water solution. The precipitate was separated by centrifugation and washed several times with deionized water. 15 mL of β-FeOOH nanorods with iron concentration 3 mg/mL was mixed with 50 µL of hydrazine hydrate. The mixture was placed in hermetic ampoule, which was then undergone by 3 microwave irradiation cycles . The final product was separated from the solution by permanent magnet and washed with deionized water several times. 1 mL of MNRd was mixed with 4 mL of Pluronic F-127 water solution (4 mg/mL). The obtained solution was stirred by vortex for 1 h at room temperature. The final product was centrifuged using 100 kDa centrifugal tubes and washed 2 times with deionized water to purify MNRd from unbound Pluronic F-127.MNCb and MNCl were synthesized as previously described , 39. MNRTEM images of IONPs were taken on JEOL JEM-1400 (120 kV) microscope. All samples were prepared by dropping a water dispersion of synthesized samples onto a carbon-coated copper grid (300 mesh) and subsequent evaporating of the solvent. The average diameter of the samples and size distribution were evaluated using ImageJ software. At least 1000 IONPs were analyzed for each sample.μL test tubes at 18°C on a ClinScan 7T MRI system. Image acquisition was performed in Spin Echo mode with the following parameters: MRI system TR = 10,000 ms, TE = 8, 16, 24, and 240 ms, ﬂip angle = 180°, resolution 640 × 448 pixels, and ﬁeld of view (FOV) = 120 × 82.5 mm2. Signal intensities from regions of interest were manually measured by ImageJ software, and the T2-relaxation time was calculated by ﬁtting the signal from images with different TE. T2-relaxivity values were calculated using a linear ﬁtting of 1/T2 relaxation times to iron concentration. The slope of the ﬁtting curve represents the R2-value for IONPs covered with Pluronic F-127 used for MR imaging.The T2-relaxation rate of water protons in the presence of IONPs covered with Pluronic F-127 was measured in 500 Hydrodynamic size and zeta-potential of IONPs were determined by Malvern Zetasizer Nano ZS. The concentration of IONPs water solution was 0.5 mg/mL for each sample.2.All cell lines were purchased from the American Type Culture Collection . 4T1, mouse breast cancer cells, and CT26, mouse colon carcinoma cells, were cultured in RPMI-1640 medium (Gibco). B16-F10, mouse melanoma cells, were cultured in DMEM medium with 4.5 g/L glucose (Gibco) and SC-1, feral mouse embryo cells-in DMEM with 1 g/L glucose (Gibco). All culture media were supplemented with 10% FBS and 2 mM L-glutamine (Gibco), and for CT26 cell line, 10 mM HEPES (Helicon) and 1 mM sodium pyruvate (Gibco) were added. Cells were maintained at 37°C in a humidified incubator supplied with 5% COµg/mL. PBS and DMSO (25%) served as negative and positive controls, respectively. After 48 h incubation with IONPs, cells were washed with PBS, and 20 μL of MTS reagent  was added to each well with 100 μL of culture medium. After 4 h incubation at 37°C in darkness, 100 μL of culture medium with MTS from each well was carefully replaced in new plates to avoid the presence of nanoparticles in the analyzed solution. The absorbance of the obtained solution was measured at 490 nm using Thermo Scientific Multiskan GO spectrometer. Experiments were performed in triplicates.SC-1 cells were plated at concentration of 25,000 cells per well in 96-well plates. After 48 h, IONPs were added to the cells at final concentration 1–200 µg/mL iron) were added to cells for 6 or 24 h. Cells incubated in culture medium or in medium with PBS were used as controls. To detect ROS in cells after incubation with IONPs, unfixed cells were washed with HBSS (Gibco), supplemented with 2 mM L-glutamine and 10 mM HEPES (Helicon) , and stained with 2 µM H2DCFDA solution (Life technologies) in HBSS for 30 min at 37°C in darkness. Then, cells were carefully washed with HBSS 3 times for 5 min. The obtained samples were analyzed at fluorescent microscope EVOS (Life technologies), objective PlanFluor 20x/0.45. Experiments were performed in duplicates.Cells were plated in the wells of Stripwell 96-well plates (Corning) at concentration of 25,000 cells per well and cultured at 37°C in a humidified incubator. After 48 h IONPs (200 µg/mL iron) were added to cells for 6 and 24 h. Cells incubated in free culture medium or in medium with PBS were used as controls. Cells were washed with HBSS supplemented with L-glutamine and HEPES and intravitaly stained with DNA Nuclear Green DCS1 dye (Abcam) for 40 min at room temperature in the darkness, and washed twice with HBSS. The photos were captured by fluorescent microscope EVOS (objective PlanFluor 20x/0.45) and analyzed in ImageJ software. Experiments were performed in duplicates.Cells were plated in the wells of Stripwell 96-well plates. After 48 h, IONPs , 5 mg/kg IONPs were intravenously (i.v.) injected.All animal experiments were approved by N.I. Pirogov Russian National Research Medical University bioethical committee . Six- to eight-week-old female BALB/c and C57BL/6 mice were obtained from Andreevka Animal Center . At the time of use, animals were between 7 and 11 weeks old and weighed 20–22 g. 4T1, CT26, and B16 tumors were established by subcutaneous injection into the hind flanks of 1 × 10n=5 for each group). The following regimens were used: (a) fat-suppressed T2-weighted turbo spin-echo (TSE) ) and (b) T2∗-weighted gradient echo (GRE) ). Images were processed in RadiAnt DICOM Viewer.For in vivo studies, images were obtained using a 20-cm volumetric coil as a transmitter and a 4-segment surface coil as a receiver of the RF signal. Tumor-bearing mice were anaesthetized with 2% isoflurane and scanned before and 1 h, 6 h, and 24 h after i.v. injection of 5 mg/kg MNCb, MNRd, and MNCl (n=5 for each group) by cardiac perfusion with 30 mL PBS under anesthesia. Liver, spleen, kidneys, lungs, and heart were collected, weighted, and digested in aqua regia during 24 h. Quantification of the iron concentration was carried out by atomic emission spectroscopy  using the calibration curve for the standard samples in 0.1-1 mg/mL concentration range. Untreated animals (n=5) were used as control for measuring endogenous iron levels. Mean iron levels in control organs were subtracted from corresponding iron levels in MNCb-, MNRd-, and MNCl-treated groups to get IONPs-associated iron concentration (µg/g tissue). IONPs delivery efficiency calculations were based on iron concentration in the tissues, organ mass, and injected dose. For tumor-bearing animals, iron concentration in organs and tumors was measured in untreated and IONPs-treated (5 mg/kg) groups 24 h upon i.v. injection as described above.Tumor-free mice were injected with 5 mg/kg IONPs and sacrificed 1 h or 24 h after injection  and standard error of mean (SEM) values were made in GraphPad Prism 5. Data were analyzed using the analysis of variance (ANOVA) test, The synthesis and detailed physicochemical characterization of Pluronic F-127-modified MNCb and MNCl have been previously described , 39. DatAll three IONPs had a comparable crystallite size and surface charge. As expected, MNCl core diameter was larger than those of MNCb and MNRd and the differences between hD were even more profound due to IONPs geometry. Magnetic measurements showed that MNCl had the highest value of saturation magnetization . The XRD spectra and characteristics of the obtained samples are presented in The XRD measurements of MNCb, MNRd, and MNCl showed that all samples can be attributed to magnetite phase Feµg/mL concentration . Consistent with MTS results, no difference in ROS production  tumor-free animals Figures . One houTumors are known to affect organs vascularization, morphology, and function through cancer cells dissemination and/or stimulating myeloid-derived suppressor cells maturation and recruitment. For instance, spleen weights were significantly higher for 4T1-bearing animals when comparing with controls and animals with CT26 tumor implants . Therefore, IONPs biodistribution may differ between tumor-free and tumor-bearing animals. To check it, iron concentration was measured in mice with implanted 4T1, CT26, and B16 tumors 24 h after IONPs injection . Qualitap=0.12) with maximum IONPs delivery rate in B16 tumors (4.1% ID) and minimum in 4T1 tumors (0.8%ID) . MNCb, MNRd, and MNCl delivery efficiency for all tumor types was 3.79% ID, 2.94% ID, and 1.21% ID, respectively .It is known that nanoparticles accumulate in solid tumors in EPR-dependent manner. To address this point, iron levels were measured in 4T1, CT26, and B16 tumors 24 h after IONPs injection . Indeed,T2 regimen, and then tumors were imaged with T2∗ settings . Each animal was scanned using settings .IONPs performance in MRI was assessed by percentage of tumors with enhanced contrast  and typeConsistent with biodistribution data, the best tumor enhancing contrast was revealed in B16 model. IONPs were detected in 88% of the tumors, primarily by diffuse signal decrease in tumor tissues . 4T1 tumAmong studied IONPs, MNCl demonstrated the best MRI-contrast properties . DiffuseMRI was used to track the dynamics of IONPs accumulation in malignant tissues and to identify the optimal time point for tumor visualization. The most profound tumor contrasting corresponded to 6-24 hours after MNCb and MNCl injection, whereas MNRd accumulation in malignancies reached its maximum within first 30 minutes upon i.v. administration Figures , but 4T1Although extensive work yielded many valuable insights on the contributions of particle size and surface chemistries to in vivo tumor imaging, information on the biological influence exerted by particle shape is relatively lacking . VisualiDespite numerous studies on IONPs in vivo imaging, only few of them compare particles under the same conditions , 24. MosT2-relaxivity estimation is probably due to different degrees of freedom that a tissue can impose on the MRI signal. For instance, distribution profiles throughout the tissues  may affect intensity and type of signaling . Nanorods had the lowest r2-relaxivity, the less efficient accumulation in tumors that resulted in poor MRI performance in tumors.Among the three IONPs, MNCb exhibited the highest relaxivity rates in vitro and the best delivery efficiency to tumor site. However, MNCl overperformed MNCb in contrasting malignant tissues. Discrepancy between in vitro and in vivo Despite some variability between mice models, the overall results clearly indicate that the liver and spleen captured the majority of IONPs consistent with multiple previous reports , 24, 45.IONPs accumulation via EPR effect varies greatly among tumors depending on the location, size, and structure/histology. Thus, rapid-growing tumors have more leaky vessels contributing to NPs enhanced extravasation. The ratio between tumor parenchyma and stroma determines tumor tissue consistency and tension, therefore affecting lymphatic drainage . Using sTumor accumulation dynamics varies significantly for different nanoparticles. Thus, the highest uptake for gold nanospheres and nanocages was observed at 24 hours after i.v. administration, while nanodisks reached accumulation peak in tumor tissues at 6 hours after injection . StudyinDelivery efficiency to tumor site is a key parameter for IONPs-based cancer diagnostics and therapy. According to recent comprehensive review on nanoparticles delivery, only around 0.7% ID accumulates in tumor after systemic injection . In the µg/mL in agreement with numerous reports [µg/mL in 20 g mice. This dose was well tolerated consistent with previous studies where i.v. injection of 5, 10, 20 and even 100 mg/kg iron was found safe in rodents [µg/g for MNCb, MNRd, and MNCl, respectively. These concentrations are significantly lower than the level required for cirrhosis and/or hepatocellular carcinoma development (4000 µg/g). Collectively, the data suggest that studied IONPs are biocompatible and safe for biomedical purposes.It is commonly accepted that IONPs are highly biocompatible, which makes them attractive for medical applications , 51, 52. reports , 45, 53. rodents . In earl rodents . Biodistr2-relaxivity and delivery to malignant tissues resulted in efficient MR contrasting of multiple tumors in mice. In contrast, MNRd low in vitro r2-relaxivity coupled high accumulation in lungs and suboptimal uptake by tumors limited its contrasting efficiency. Overall, the results suggest that synthetized MNCb and MNCl are promising for cancer diagnostics and drug delivery.All studied IONPs were found safe and biocompatible for in vivo applications. MNCb and MNCl high


In the original article, there was an error. The psychometric scale used to measure the depression scores was the “Beck Depression Inventory” and not the “Beck Depression Inventory II.”Materials and Methods, subsection Sociodemographic and Psychological Scales:A correction has been made to the “Subjects filled a questionnaire to characterize gender, age, educational level, handedness, and ethnic origin. Weight and height were also measured to prevent the inclusion of participants with an unhealthy body mass index. Subjects were assessed with the 10-items Perceived Stress Scale (PSS-10)  = 3.30, p = 0.003, effect size d = 1.27]. No statistically significant differences were found for BAI  and BDI  between groups.”“The stress group revealed higher levels of perceived stress (mean ± standard deviation 15.07 ± 5.23) than the control group (8.64 ± 5.27) as assessed by PSS-10 [Abbreviations section:Lastly, a correction has been made to the “ACTH, adrenocorticotropic hormone; BAI, Beck Anxiety Inventory; BDI, Beck Depression Inventory; dlPFC, dorsolateral prefrontal cortex; fMRI, functional magnetic resonance imaging; GLM, general linear model; MNI, Montreal Neurological Institute; PSS-10, 10-items Perceived Stress Scale; vmPFC, ventromedial prefrontal cortex.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Here, we synthesized ZnONPs and TiO2NPs simply by mixing ZnO or TiO2 solution with a lemon fruit extract at room temperature and showed their antibacterial activities against Dickeya dadantii, which causes sweet potato stem and root rot disease occurring in major sweet potato planting areas in China. Ultraviolet–visible spectrometry and energy dispersive spectroscopy determined their physiochemical characteristics. Transmission electron microscopy, scanning electron microscopy, and X-ray diffraction spectroscopy revealed the nanoscale size and polymorphic crystalline structures of the ZnONPs and TiO2NPs. Fourier-transform infrared spectroscopy revealed their surface stabilization groups from the lemon fruit extract. In contrast to ZnO and TiO2, which had no antibacterial activity against D. dadantii, ZnONPs and TiO2NPs showed inhibitions on D. dadantii growth, swimming motility, biofilm formation, and maceration of sweet potato tuber slices. ZnONPs and TiO2NPs showed similar extents of antibacterial activities, which increased with the increase of nanoparticle concentrations, and inhibited about 60% of D. dadantii activities at the concentration of 50 µg∙mL−1. The green synthetic ZnONPs and TiO2NPs can be used to control the sweet potato soft rot disease by control of pathogen contamination of seed tubers.Edible plant fruits are safe raw materials free of toxicants and rich in biomolecules for reducing metal ions and stabilizing nanoparticles. Zinc oxide nanoparticles (ZnONPs) and titanium dioxide nanoparticles (TiO Nanoparticles (NPs) with at least one dimension in the range of 1–100 nm have high surface-to-volume ratios and display exceptional physical, chemical, and biological properties compared to their bulk counterparts. Nanotechnology dealing with the synthesis, development, and applications of NPs has brought revolutions in catalysis, cosmetic, medicine, food, agriculture, and environment technologies ,3,4.2) NPs (TiO2NPs) [Nanoparticles were conventionally synthesized by various physical and chemical methods, such as ultrasonication, microwave irradiation, laser vaporization, wet impregnation, and sol–gel methods ,6,7, whiTiO2NPs) .2NPs are the most manufactured nanomaterials used in consumer products, such as sunscreens, toothpastes, cosmetics, and paints [2NPs show remarkable antibacterial activities against Gram-positive and Gram-negative pathogens to humans [2NPs have rarely been used against phytopathogenic bacteria. Only a few studies have shown antibacterial activities of ZnONPs and TiO2NPs against phytopathogenic Xanthomonas [ZnONPs and TiOd paints ,20. Moreo humans ,21,22,23thomonas ,25,26.Dickeya and Pectobacterium within the family Pectobacteriaceae [Dickeya species caused rice foot rot [Dickeya and Pectobacterium. Meanwhile, large-scale use of effective antibiotics is no longer allowed in fields due to the risks of selection of antibiotic-resistant bacterial pathogens of humans or animals [Gram-negative bacteria belonging to the genera eriaceae  are broaeriaceae . They caeriaceae ,30. In tfoot rot ,32, maizfoot rot , potato foot rot ,35,36, sfoot rot ,38, banafoot rot ,40, threD. dadantii recently broke out in major sweet potato planting areas in China [2NPs with lemon fruit extract and determine their antibacterial activities against D. dadantii and potentials to control the disease.Bacterial stem and root rot disease of sweet potato caused by in China ,38 and rDickeya dadantii strain CZ1501, which causes stem and root rot disease of sweet potato, was isolated from a diseased stem of a sweet potato plant (Ipomoea batatas) grown in Hangzhou, Zhejiang Province, China. Dickeya dadantii was cultured in the nutrient medium  with or without agar (15 g per liter).Citrus limon) fruits bought from the supermarket were washed with tap water and Millipore water, cut into pieces, and dried at 60 °C for 10 h. Dry lemon pieces were ground into powder and mixed with Millipore water , and then stirred continuously at 100 rpm at 60–70 °C for 4 h. After cooling to room temperature, the suspension was filtered through muslin cloth and then Whatman No. 1 filter paper; the extract  was used for the synthesis of NPs or stored at −80 °C.Fresh lemon   were used for synthesis of ZnONPs and TiO2NPs, respectively. ZnO solution (0.5 M) and TiO2 solution (0.5 M) were prepared by dissolving ZnO (4.07 g) and TiO2 (4.00 g) separately in ethylene glycol (10 mL) (Sinopharm) and adding Millipore water to 100 mL. ZnONPs and TiO2NPs were separately synthesized using a protocol modified from a previous study [g for 10 min and the pellets were washed with Millipore water and then freeze-dried with an Alpha 1-2 LDplus . The freeze-dried NPs were stored at −80 °C or prepared as stock solutions (50 mg∙mL−1) for further analyses.ZnO and TiOus study . The met2NP powder were analyzed by Fourier transform infrared (FTIR) spectroscopy to detect groups responsible for synthesis and stabilizing ZnONPs and TiO2NPs as previously described [https://chem.libretexts.org/) were used to interpret the FTIR spectra. The elements of ZnONPs and TiO2NPs were detected by energy dispersive X-ray spectroscopy [2NPs were observed and analyzed by transmission electron microscopy (TEM), scanning electron microscopy, and X-ray diffraction spectroscopy [Dry lemon powder, ZnONP powder, and TiOescribed . Bruker troscopy . The siztroscopy .Dickeya dadantii CZ1501 grown to mid-exponential phase was adjusted with the nutrient broth to about 5 × 108 CFU∙mL−1 before use.D. dadantii were first detected by the diffusion assay with agar plates [Dickeya dadantii suspension was inoculated into the nutrient agar to 107 cells∙mL−1. Wells (7 mm in diameter) were made in the agar plates with sterilized steel punchers. Fifty microliters of lemon fruit extract (10 mg∙mL−1), ZnO (0.5 M), TiO2 (0.5 M), ZnONP (50 µg∙mL−1), and TiO2NP (50 µg∙mL−1) were loaded into the wells and incubated at 30 °C for 24 h. Antibacterial activities were determined by the diameters of the clearing zones formed around the wells.Antibacterial activities against r plates . DickeyaD. dadantii growth by ZnONPs and TiO2NPs at different concentrations were further determined in nutrient broth [2 was added into nutrient broth to a final concentration of 50 µg∙mL−1. ZnONPs or TiO2NPs was added into nutrient broth to final concentrations of 12, 25, and 50 µg∙mL−1. Dickeya dadantii suspension (100 μL) was inoculated into the nutrient broth (5 mL) (1 × 107 CFU∙mL−1) without (control) or with lemon fruit extract, ZnO, TiO2, ZnONPs, or TiO2NPs and grown at 200 rpm and 30 °C for 24 h. Optical density at 600 nm of the cultures was measured using a SpectraMax spectrophotometer  [Antibacterial activities against nt broth . Lemon fCA, USA) .Dickeya dadantii suspension in nutrient broth (1 × 107 CFU∙mL−1) without or with lemon fruit extract, ZnO, TiO2, ZnONPs, or TiO2NPs prepared as described above was transferred into wells (200 μL of suspension in each well) of 96-well microplates  and incubated at 30 °C for 24 h. Biofilm formed by D. dadantii was stained by crystal violet and quantified by absorbance at 590 nm as previously described [escribed .D. dadantii was determined with semisolid nutrient agar (0.3% (w/v)) [2 was added into the semisolid nutrient agar to the final concentration of 50 µg∙mL−1. ZnONPs or TiO2NPs was added into the semisolid nutrient agar to final concentrations of 12, 25, and 50 µg∙mL−1. Dickeya dadantii suspension (5 µL) was spotted onto the center of the semisolid nutrient agar plates and incubated at 30 °C for 48 h. The diameters of the halo-like colonies of D. dadantii were measured [The swimming motility of % (w/v)) . Lemon fmeasured .D. dadantii was determined with sweet potato tuber slices [v/v) ethanol, washed with sterile distilled water, and cut into slices (10 mm in thickness). The tuber slices were also surface-sterilized with 70% (v/v) ethanol, washed with sterile distilled water, and immersed in distilled water (control), lemon fruit extract (50 µg∙mL−1), ZnO solution (50 µg∙mL−1), TiO2 solution (50 µg∙mL−1), ZnONP solutions , or TiO2NP solutions  for 1 h, and then air-dried in Petri dishes for 1 h. Afterwards, D. dadantii suspension (5 µL) was spotted in punctures at the center of the tuber slices and incubated at 30 °C for 24 h. Diameters of the maceration zones around the punctures were measured [In vivo antibacterial activity against r slices . Sweet pmeasured .2NPs on D. dadantii cell structure was determined by TEM. Dickeya dadantii suspension (100 μL) was inoculated into the nutrient broth (5 mL) (1 × 107 CFU∙mL−1) without (control) or with ZnONPs (50 µg∙mL−1) or TiO2NPs (50 µg∙mL−1) and grown at 200 rpm and 30 °C for 4 h. Dickeya dadantii cells were harvested and prepared for TEM as previously described [The effect of ZnONPs and TiOescribed .p < 0.05.All data in each experiment were subjected to one-way analysis of variance and means were compared by the Duncan’s multiple range test using the SPSS software version 16 . The significance was set at 2NPs were synthesized by constant mixing of ZnO or TiO2 solution with the lemon fruit extract at room temperature. ZnONPs displayed a characteristic surface plasmon resonance peak at 388 nm in the range of 350–420 nm , O (17.51 wt%), and C (3.81 wt%) in the ZnONP powder d and Ti P powder e.2NPs , 34.46° (0 2 2), 36.20° (1 0 1), 47.58° (1 0 2), 58.64° (1 1 0), 62.90° (1 0 3), and 67.99° (1 1 2) , 37.80° (0 0 4), 48.09° (2 0 0), 54.35° (1 0 5), and 62.94° (2 0 4) (2NPs (JCPDS Card no. 21-1272) while the four characteristic reflection peaks at 2θ values of 27.47° (1 1 0), 36.11° (1 0 1), 41.27° (1 1 1), and 56.65° (2 2 0) (2NPs (JCPDS Card no. 21-1276). The average particle sizes of the ZnONPs (60.8 nm) and TiO2NPs (41.5 nm) were calculated using the Debye–Scherrer formula: D = Kλ/(βCosθ), where D is the average particle size, K is the Scherrer constant (0.9), λ is the X-ray wavelength (0.15406 nm), β is the full width at half maximum of the X-ray diffraction peak, and θ is the Bragg angle.Electron microscopy revealed polymorphic ZnONPs a,c and T2NPs b,d in di2NPs a,c whiler shapes b,d; ZnONcroscopy c,d, as p (1 1 2) e. This X (2 0 4) f indicat (2 2 0) f indicat−1), ZnO (0.5 M), and TiO2 (0.5 M), the raw materials for synthesis of ZnONPs and TiO2NPs, did not generate clearing zones around them in the nutrient agar containing D. dadantii cells and thus did not inhibit the growth of D. dadantii. In contrast, the products ZnONPs (50 µg∙mL−1) and TiO2NPs (50 µg∙mL−1) generated clearing zones with diameters of 30.0 ± 0.7 mm and 28.5 ± 0.5 mm, respectively, around the wells (including the well diameter 7 mm) and thus inhibited the growth of D. dadantii or killed D. dadantii. In nutrient broth, ZnONPs and TiO2NPs significantly inhibited D. dadantii growth and the extents of inhibition increased with the increase of the concentrations  of ZnONPs and TiO2NPs, whereas lemon fruit extract, ZnO, or TiO2 (50 µg∙mL−1) did not inhibit D. dadantii growth  did not inhibit D. dadantii growth and swimming in the semisolid medium, whereas ZnONPs and TiO2NPs significantly inhibited D. dadantii growth and swimming and the extents of inhibition increased with the increase of the concentrations of ZnONPs and TiO2NPs  did not inhibit the biofilm formation, whereas ZnONPs and TiO2NPs significantly inhibited the biofilm formation and the extents of inhibition increased with the increase of the concentrations of ZnONPs and TiO2NPs  did not inhibit the tissue maceration by D. dadantii, whereas ZnONPs and TiO2NPs significantly inhibited the tissue maceration and the extents of inhibition increased with the increase of the concentrations of ZnONPs and TiO2NPs . -Dickeya dadantii cells grown without NPs had intact cell envelopes and dense cytoplasm filled in the cells  while Zn2+ may be chelated by citrate through two carboxyl groups and one hydroxyl group and form a pentabasic ring and a hexahydric ring [2+ may occur in the mixture and stabilize Zn2+ [2 solutions and lower the energy needed to form the ZnO and TiO2 crystals [2NPs.We made ZnONPs and TiObic acid , and amibic acid . The O–Hbic acid . Citric bic acid ,45,46. Ebic acid ,47. Unliolutions . Dissoluric ring . Perhapsize Zn2+ . Citric crystals . Togethe2 with no antibacterial activity against D. dadantii, ZnONPs and TiO2NPs showed distinct antibacterial activities against D. dadantii growth, swimming motility, biofilm formation, and maceration of sweet potato tuber slices. ZnONPs and TiO2NPs showed similar extents of antibacterial activities against D. dadantii and an increase of antibacterial activities with the increase of NP concentrations, and inhibited about 60% of D. dadantii growth, swimming motility, biofilm formation, and maceration of tuber slices at the concentration of 50 µg∙mL−1.In contrast to ZnO and TiO2 have known photo-oxidizing and photocatalytic activities and generate reactive oxygen species (ROS). ZnONPs and TiO2NPs adhering to the cell surface can generate extracellular ROS and induce intracellular generation of ROS, leading to oxidative stress on cells, distortion and damage of cell membranes, leakage of intracellular contents, and eventually cell death [2 and their NPs on D. dadantii were processed in the dark; photo-oxidization and photocatalysis may not participate in the antibacterial mechanisms of ZnONPs and TiO2NPs against D. dadantii. ZnONPs and TiO2NPs may induce osmotic stress on bacterial cells in the dark and induce membrane depolarization and loss of membrane integrity, resulting in cellular leakage [2+ may be dissolved from ZnONPs and expose its heavy metal toxicity to cells.The distinct antibacterial activities of metal and metal oxide NPs were achieved by their smaller sizes and larger surface-area-to-mass ratios and generation of oxidative stress on bacterial cells ,50. The ll death ,50. In t leakage , as reve2NPs produced in this study show polymorphic structures and surface defects with numerous edges and pits. Such abrasive surface texture not only has more reactive surface sites but also tends to disrupt cell membranes and abrade biofilms. D. dadantii regulates plant surface colonization via regulation of flagella-mediated motility and biofilm formation. D. dadantii movement and attachment to plant surfaces and formation of biofilms on plant surfaces, in intercellular spaces, and in xylem vessels are essential for survival and completing disease cycles [2NPs adhering to bacterial cell surfaces may inhibit bacterial movement and attachment to plant surfaces, inhibit bacterial growth, and damage bacterial cells and biofilms.The ZnONPs and TiOe cycles ,53,54,55D. dadantii by mixing AgNO3 solution with cell-free bacterial culture supernatants for 48 h [We previously produced AgNPs against for 48 h . That en2NPs instead of AgNPs is to avoid using toxic Ag+. However, the green synthetic ZnONPs and TiO2NPs have properties distinct from ZnO and TiO2, such as the antibacterial activity against D. dadantii, and raise the concern about NP toxicity to plants and humans. NPs can be taken up and accumulated in plants and enter the food chains of animals and human, and thus pose a risk to human health [The reason we used lemon fruit extract to produce ZnONPs and TiOn health ,56,57.2NPs may have positive and negative impacts on plants, depending on not only NP properties , concentration, and exposure time but also plant properties (susceptible or tolerant to NPs) and development stages. Generally, ZnONPs and TiO2NPs in excess are harmful to plants, while in traces can be beneficial for plants [2NPs or equivalent amounts of Zn2+, Cu2+, or Ce4+ at three concentrations . Only the application with the highest concentration of Zn or Cu, which is unlikely to occur in the environment, caused adverse outcomes, that is, reduction in tuber biomass, metal accumulation, and dietary intake regardless of the chemical forms of the metals added to the substrate. The concentrations of metals were higher in the peels than in the inner tuber flesh of sweet potato. Under such conditions, metal oxide NPs pose no greater risk to sweet potato yield or food safety than do the ionic metals [2 at 25, 75, or 125 mg∙L−1 under continuous stirring for 30 min and found that the Cu concentration in internal tissues of tubers treated with CuONPs was similar to that in control tubers, suggesting no risk of Cu contamination in peeled tubers. The peel of sweet potato tubers restricts the inward radial transfer of metal NPs. Thorough cleaning of tuber surfaces and peeling of tubers can effectively reduce the consumer exposure to metal NPs or ionic metals [ZnONPs and TiOr plants ,57,58. Br plants  grew swec metals . Bonillac metals  immersedc metals .2, which are generally recognized as safe substances, and extracts from edible lemon fruits, which are free of toxicants and rich in biomolecules, as safe raw materials to reduce health and environmental risks of the sources for the production of NPs. We developed a simple, rapid, cost-effective, and ecofriendly method to produce green ZnONPs and TiO2NPs simply by mixing ZnO or TiO2 solution with the lemon fruit extract. Actions of carboxylic acids, amino acids, and polyols in the lemon fruit extract may lead to the formation of the polymorphic ZnONPs and TiO2NPs. In contrast to ZnO and TiO2, the ZnONP and TiO2NP products effectively inhibited D. dadantii growth, swimming motility, biofilm formation, and maceration of sweet potato tubers and likely combat the bacterial pathogen via multiple mechanisms. The multiple bacteriocidal and bacteriostatic mechanisms would make it difficult for the pathogen to develop resistance to the NPs. The latently infected seed tuber is one of the major sources of the soft rot disease of sweet potato. The green ZnONPs and TiO2NPs appear to be promising materials to treat the seed tubers to avoid and reduce the pathogen contamination and to produce healthy crops. A study is needed to clarify if ZnONPs and TiO2NPs may contaminate the sweet potato tuber flesh and can be used to preserve and increase the shelf life of sweet potato tubers without exposing consumers to excess metals. More studies are needed before bringing the ZnONPs and TiO2NPs to the field to control the soft rot disease and promote sweet potato growth.We used ZnO and TiO


GAS is among the most common human pathogens and causes a wide variety of diseases, likely more than any other microorganism. The diverse clinical manifestations of GAS may be attributable to its large repertoire of virulence factors that are selectively and synergistically involved in streptococcal pathogenesis. To date, GAS vaccines have not been successful due to multiple serotypes and postinfection sequelae associated with autoimmunity. In this study, five conserved virulence factors that are involved in GAS pathogenesis were used as a combined vaccine. Intranasal immunization with this vaccine induced humoral and cellular immune responses across GAS serotypes and protected against mucosal, systemic, and skin infections. The significance of this work is to demonstrate that the efficacy of GAS vaccines can be achieved by including multiple nonredundant critical virulence factors and inducing local and systemic immunity. The strategy also provides valuable insights for vaccine development against other pathogens. Group A streptococcus (GAS) species are responsible for a broad spectrum of human diseases, ranging from superficial to invasive infections, and are associated with autoimmune disorders. There is no commercial vaccine against GAS. The clinical manifestations of GAS infection may be attributable to the large repertoire of virulence factors used selectively in different types of GAS disease. Here, we selected five molecules, highly conserved among GAS serotypes, and involved in different pathogenic mechanisms, as a multicomponent vaccine, 5CP. Intranasal (i.n.) immunization with 5CP protected mice against both mucosal and systemic GAS infection across serotypes; the protection lasted at least 6 months. Immunization of mice with 5CP constrained skin lesion development and accelerated lesion recovery. Flow cytometry and enzyme-linked immunosorbent assay analyses revealed that 5CP induced Th17 and antibody responses locally and systemically; however, the Th17 response induced by 5CP resolved more quickly than that to GAS when challenge bacteria were cleared, suggesting that 5CP is less likely to cause autoimmune responses. These findings support that immunization through the i.n. route targeting multiple nonredundant virulence factors can induce immunity against different types of GAS disease and represents an alternative strategy for GAS vaccine development, with favorable efficacy, coverage, duration, and safety. Group A streptococcus (GAS) species are leading human bacterial pathogens with diverse clinical manifestations, ranging from mild skin infections, such as impetigo, to serious conditions, such as necrotizing fasciitis, streptococcal toxic shock syndrome, and scarlet fever. Repeated GAS infections are linked to autoimmune sequelae, including acute glomerulonephritis and rheumatic heart disease , 2; howeThe upper respiratory tract (URT) mucosa is a common site of GAS colonization. Parenteral immunization does not protect against mucosal infection , because5–The diverse clinical manifestations of GAS are attributed to their large arsenal of virulence factors , which c11–The goal of the present study was to evaluate the efficacy of immunity induced by a combined multiple-component vaccine, 5CP, in protection against different types of GAS infection and the associated immune responses. We demonstrate that i.n. immunization with 5CP protects against mucosal and systemic infection, independent of GAS M serotypes. In addition, 5CP-induced immunity constrained skin lesion development, promoted lesion recovery, and provided protection in a subcutaneous invasive disease model. These findings suggested that i.n. immunization with multiple virulence factors may be an optimal strategy for GAS vaccine development.Five conserved GAS virulence factors were formulated as a multicomponent vaccine, designated 5CP, including SrtA , the adh10.1128/mBio.02600-19.1FIG S1FIG S1, PDF file, 0.09 MB.Purified recombinant proteins were analyzed by SDS-PAGE. Lane 1, molecular weight marker; lane 2, SLO; lane 3, CEP-5; lane 4, SpyAD; lane 5, SCPA; lane 6, SrtA. Download Copyright © 2019 Bi et al.2019Bi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Enzyme-linked immunosorbent assay (ELISA) analyses were also performed to determine whether these antibodies could recognize GAS. Serum IgG from mice immunized with 5CP displayed levels of bacterial cell binding similar to those from GAS infection-experienced mice . The IgAT helper type 17 (Th17) cells can function as B cell helpers. They induce the formation of germinal centers and invoke strong proliferative responses in B cells, leading to pronounced antibody production , 21. In + IL-17A+ cells were increased in nasal-associated lymphoid tissue (NALT) from 5CP-immunized mice, 5 days after the last immunization, with lower levels than those observed for GAS infection-experienced mice (+) T cells were detected in three or four of the five human PBMC samples , indicating that the bacteria were disseminated through the body (P < 0.0001) at the end of the experiment, comparable to survival rates in infection-experienced mice (P < 0.01) (Systemic protection was further tested the body . Mouse sced mice . Immuniz < 0.01) . These r7 CFU of GAS M1 (strain 90-226), and skin lesion size was measured daily for 14 days. After 3 or 4 days, lesion sizes reached maxima of 37.46 ± 4.53 and 34.28 ± 4.89 mm2 in 5CP-immunized and GAS infection-experienced mice, respectively, relative to 52.78 ± 4.08 mm2 in CpG-treated control mice. Further, while lesion size continually increased to a peak value (57.49 ± 6.39 mm2) in control mice on day 6, lesion size decreased to 27.90 ± 5.67 and 26.66 ± 8.40 mm2 in 5CP-immunized and infection-experienced mice, respectively  are the primary source of these antibodies and primarily reside in the bone marrow (BM) . Similar in NALT . ELISAs  in NALT  and g, w in NALT  and i.4 CFU, while there were 1.0 × 106 CFU in adjuvant immunized mice . (b to d) Two weeks after the last immunization, antibodies in serum and mouthwash samples were measured by ELISA. Levels of serum IgG (b) and IgA in mouth wash directed to 5CP (c). Levels of IgA in mouth wash directed to GAS (d). Data are presented as means ± SD (n = 5). Statistical significance was determined by two-tailed Student’s t test and indicated as follows: **, P < 0.01; ns, not significant. Download FIG S2, PDF file, 0.2 MB.Subcutaneous immunization with 5CP did not provide protection against group A streptococcus (GAS) mucosal infection. Mice were intranasally (i.n.) or subcutaneously (s.c.) immunized with 5CP or CpG as described in Materials and Methods. IR, immunization route. (a) Two weeks after the last immunization, mice were i.n. challenged with GAS M1. CFU in NALT were determined 24 h after challenge. Data are presented as means ± SEM  Ethics Committee. The protocols were approved by the Committee on the Ethics of Animal Experiments of IMCAS (permit APIMCA2019030). Mice were bred under specific-pathogen-free conditions in a laboratory animal facility at IMCAS. All animal experiments were conducted under isoflurane anesthesia, and all efforts were made to minimize suffering. The collection and use of samples from patients were approved by the Ethics Committee of Beijing Children’s Hospital and Beijing Red Cross Blood Center.2. Overnight cultures  were washed with and resuspended in PBS and then used for infection or challenge. CFU were verified by plating on blood agar plates.GAS serotype M1 (strain 90-226) was obtained from the University of Minnesota. Serotypes M12 and M49 were clinical isolates obtained from Beijing Children’s Hospital. All strains were maintained on sheep blood agar and grown in THB-Neo broth at 37°C in 5% COEscherichia coli BL21(DE3) for expression. Recombinant proteins were purified as previously described (DNA sequences for the selected antigens were amplified from GAS serotype M1 (strain 90-226). Recombinant SrtA and SCPA were cloned into the pET28a vector as previously described , 6. Recoescribed . Lipopol8 to 1 × 108/mouse) of live GAS M1 (strain 90-226) was used for infection. Control mice were administered PBS or 10 μg of CpG-OND in PBS. Mice were immunized or infected three times at 1-week intervals. For the mucosal challenge model, mice were i.n. challenged with GAS serotype M1 (strain 90-226) or serotype M12 at a sublethal dose of 2.0 × 108/mouse, 2 weeks after the last immunization. CFU in NALT were determined 24 h postchallenge, as previously described (8/mouse) of GAS serotype M1 (strain 90-226) or M49, and weight loss and survival were monitored twice per day for 14 days. Mice with weight loss of 25% of the starting body weight were euthanized and recorded as dead. Mortality was an anticipated outcome and approved by the animal ethics committee. Surviving mice were humanely euthanized at the end of the experiment. For the skin abscess model, i.n. immunized mice were anesthetized and s.c. challenged on the shaved lower back with 3 × 107 CFU/mouse of GAS M1 . The size of dermonecrotic lesions was measured daily. For the invasive subcutaneous infection mouse model, immunized mice were s.c. challenged with 1 × 108 CFU/mouse of GAS M1 , and survival was monitored for 14 days.Female BALB/c mice (aged 4 to 6 weeks) were purchased from Vital River Laboratory Animal Technology, whose colonies were all introduced from Charles River Laboratories. Mice were anesthetized with an isoflurane/oxygen mixture and i.n. inoculated with 50 μg of 5CP (10 μg of each recombinant protein) with 10 μg of CpG-oligodeoxynucleotides  as adjuvant, in a 10-μl volume/mouse (5 μl per nostril). For subcutaneous (s.c.) immunization, 50 μg of 5CP, with 10 μg CpG-OND 1826 as adjuvant, in 50-μl volume/mouse was s.c. injected into the upper back. A low dose , and human PBMCs were obtained from Beijing Red Cross Blood Center .7/ml of heat-killed (HK) GAS M1 serotype (strain 90-226) or 5 μg/ml of single recombinant antigen in PBS and incubated overnight at 4°C. Aliquots (100 μl) of each serum sample were added to each well of plates and incubated at 37°C for 2 h. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or goat anti-mouse IgA were used to detect mouse antibodies. HRP-conjugated goat anti-human IgG was used for detection of human serum IgG. IL-17A levels in the supernatants of NALT homogenate were measured using ELISA kits  as previously described  for 5 days. Differentiated HL-60 cells were assessed by flow cytometry to confirm that ≥55% and ≤20% of the total cells were CD35+ and CD71+ , respectively. OPKA reactions were performed in round-bottomed 96-well plates with heat-inactivated (56°C for 30 min) serum from immunized mice, 1 × 103 CFU of GAS in opsonization buffer, 4 × 105 differentiated HL-60 cells, and 5% baby rabbit complement . Control reactions were conducted using heat-inactivated serum from naive mice without differentiated HL-60 cells. Reaction mixtures were incubated for 1.5 h at 37°C with 200 rpm shaking. CFU were determined by plating serial 10-fold dilutions on blood agar plates. The bacterial growth rate was calculated as mean CFU of test serum well/mean CFU of control well.HL-60 opsonophagocytic killing assay (OPKA) was conducted as previously described . Briefly5 BM or splenic cells, 2 × 105 NALT cells, or 2 × 105 human PBMCs, per well) were seeded in 96-well plates. Cells were cocultured with HK GAS (multiplicity of infection of 10) or each antigen from 5CP individually (20 μg/ml) for 24 h. Antigen-specific LLPCs in the BM, NALT, and spleen were quantified by ELISPOT assays using specific antibody-secreting cells as described previously  with Tukey’s posttest was used to analyze the statistical significance of differences among more than two groups. Survival curve was analyzed using the log rank test. When two independent variables were compared, data were analyzed by two-way ANOVA with multiple-comparison test as stated. A P value of <0.05 was considered significant.Statistical analyses were performed using GraphPad Prism software (version 7.0). The CFU were log


Cereals like wheat, rice, corn, barley, rye, oat, and millet are staple foods in many regions around the world and contribute to more than half of human energy requirements. Scientific publications contain evidence showing that apart from energy, the regular consumption of whole grains is useful for the prevention of many chronic diseases associated with oxidative stress. Biological activities have mostly been attributed to the presence of glucans and polyphenols. In recent years however, food proteins have been investigated as sources of peptides that can exert biological functions, promote health and prevent oxidative stress. This review focuses on the role of hydrolyzed proteins and peptides with antioxidant properties in various models and their mechanisms which include hydrogen or electron transfer, metal chelating, and regulation of enzymes involved in the oxidation-reduction process. The majority of bioactive compounds are located within whole grains, bran or germ rich milling fractions. Many studies therefore have been performed to investigate the beneficial effect of whole grains or milling fractions for the general well-being, and for the reduction of oxidative stress and associated disease conditions such as cardiovascular diseases, diabetes, obesity, and cancer [Cereals are major sources of energy for populations around the world. Taxonomic classification places them in the Gramineae or Poaceae family which is divided into seven major subfamilies of grasses, d cancer .The beneficial effects of cereals have mostly been attributed to the presence of fibers (e.g. glucans), a group of polysaccharides that are resistant to digestive enzymes in the gastro-intestinal tract, meanwhile their health promoting effects go beyond fibers. In women for example, the health effects of whole grains on heart disease remained even after controlling the intake of fibers 22.1Oxidative stress occurs as a result of an imbalance between the amount of oxidants produced and the antioxidant defense mechanism. There are endogenous and exogenous oxidants. Two groups of oxidants commonly referred to as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the main contributors. However, chlorine, bromine, and sulfide reactive species are contributors as well to the alteration of the redox balance. Most oxidants are unstable and short-lived chemical species generated under various conditions in foods and physiological conditions.2.1.1Processes that generate oxidants in food include the length of storage and temperature, heat treatment, the amount of available oxygen, the presence of transition metals, or oxidative enzymes. Thermal oxidation of lipids, for example, form unstable primary oxidation products (i.e. hydroperoxides) through initial proton removal and oxygen consumption In biological systems, the primary cellular sources of ROS and RNS are the mitochondria where oxidative phosphorylation occurs. The mitochondria electron transport chain is composed of four multi-protein complexes I-IV and comprises a series of electron carriers such as ubiquinone, cytochromes, flavoproteins, and iron-sulfur proteins. The domains are arranged spatially according to their redox potentials, which vary from −0.320 to +0.380 V 2.1.22·−), hydroxyl (HO·), nitric oxide (NO·), nitrogen dioxide (NO2·), and peroxyl (ROO·), as well as non-radical species like singlet oxygen (1O2), hydrogen peroxide (H2O2), ozone (O3), hypochlorous acid (HOCl), nitrous acid (HNO2), peroxynitrite (ONOO−), dinitrogen trioxide (N2O3), and hydroperoxide (ROOH) [1O2, O2·−, or HO· through successive reactions generate aldehyde compounds that are responsible for off-flavor characteristics of many food products [· and ROO· can decrease the nutritional value of proteins by reacting with nucleophilic groups, such as the side-chain amine group of lysine or the sulfhydryl group of cysteine in free amino acids, peptides, and proteins [Common ROS and RNS include free radicals like superoxide anion (Oe (ROOH) , 29. Oxiproducts , 23. Pheproteins , 21, 30.ROS released by mitochondria are believed to play an important role in conditions associated with oxidative stress such as the aging process, neurodegeneration (e.g. Parkinson's disease), and atherosclerosis , 31. Thi2.1.32·ˉ) radicals There is no known beneficial role of oxidants in foods, but they exist in biological systems. At low or moderate levels, ROS and RNS play important role in the maturation process of cellular structures and maintenance of the host defense system 2.2Proteins represent about 6–15% of cereals grains. In general, there are two main groups of proteins: prolamins and globulins 2.2.1The initial step is the extraction of proteins which can be achieved by solubilisation of cereal flours in solution of salts such as KCl and NaCl, or at alkaline conditions (pH 9–10) , 44. The2.2.2One of the well known and commonly investigated properties of antioxidant molecules is their ability to scavenge radicals. It is, therefore, not surprising that data on the quenching of radicals by hydrolyzed cereal proteins exist in the literature.2.2.2.12·− (0.6 mg/mL) radicals 2·−, and HO· radicals with EC50 values of 1.3, 0.4 and 0.1 mg/mL, respectively 50 values for wheat proteins fermented with Bacillus Subtilis B1 were 3.2 mg/mL, 6.0 mg/mL and 7.5 mg/mL, respectively 2·− and HO· radical scavenging peptides from wheat germ while Proleather FG-F was better for releasing peptides that quenched DPPH radicals. For rice dreg proteins, protamex hydrolysates had better DPPH activity (EC50 8.7 mg/mL) than those from alcalase, neutrase, flavourzyme, and trypsin treatments (EC50 9.9–14.0 mg/mL) 50 0.05 mg/mL) and hydroxyl radical (EC50 2.0 mg/mL) scavenging activities, as well as a weak superoxide anion radical scavenging activity 50 6.8 mg/mL) compared to values of hydrolysates prepared with a combination of two proteases comprising papain, flavourzyme, neutrase, protamex, or trypsin 50 of 1.57 mg/mL for DPPH radical scavenging activity of a trypsin digest. Treatment of rice proteins with neutrase, alcalase, and flavourzyme produced hydrolysates with ABTS· scavenging activities (89–151 μg ascorbic acid (AA) equivalent/mL) relative to treatments with microbial cell extracts (220–280 μg AA eq/mL) Wheat germ proteins hydrolysed with Proleather FG-F, a protease from Bacillus subtilis, scavenged 81% DPPH (1.6 mg/mL) and 75% O· (EC50 0.8–7.5 mg/mL), O2·− (EC50 12.5–12.8 mg/mL), and DPPH (EC50 1.0–1.26 mg/mL) radicals [· radicals with values of 65.6–191.4 μM TE/g 50 values from 0.5 to 3.8 mg/mL or 48–58%, at 0.5 mg/mL, and quenched O2·− by up to 40% at 0.5–1.0 mg/mL [2·− and HO· scavenging activities of barley gluten alcalase hydrolysates were stronger than those of flavourzyme digests, however, DPPH activity was lower · radical scavenging activities of 343–608 μM TE/g and inhibited the formation of O2·− by 20–36% and HO· by 10.2–14.1%, depending on the hydrolysis time and the concentration of pepsin · (408–712 μM TE/g) activities, but lower HO· (2.4–11.2%) and higher O2·− (24–58%) scavenging activities · activities were 269 and 434 μM TE/g, respectively while DPPH radicals quenching were 20–35% at 1 mg/mL Corn gluten proteins treated with various proteases possess antioxidant activities against HOradicals , 60. Hyd.0 mg/mL , 63. In ·, and O2·− activities relative to fractions with higher molecular weights [· activities after conjugation of wheat gluten hydrolysates  with glucosamine · in scavenging activity of pepsin and pancreatin hydrolysates upon deamidation with citric acid · activities of corn protein hydrolysates [Fractionation of hydrolysed proteins can concentrate activities within certain fractions. Wheat pepsin (<3 kDa) and papain (<5 kDa) gluten hydrolysates had higher DPPH, HO weights , 67, 68.olysates , 72. Oth2.2.2.2The number of studies performed on pure peptides is limited. This is likely because it is labor intensive to fraction and isolate individual peptides using available chromatography and membrane technologies. Meanwhile, many peptides have been identified in hydrolysates or fractions with antioxidant activities but only a few have been tested .Table 1S· radical scavenging activities. Amongst them, GLVYI and YHNAP were the most potent with values of 0.67 and 0.61 μM TE/μM peptide, respectively while DVNNNANQLEPR had the least activity, 0.14 μM TE/μM 2·− and HO· radicals were 371, 3977, 666 and 3629 mmol glutathione/mol peptide, respectively 50 0.15 ± 0.01 mg/mL) 50 0.144–0.161 mg/mL) and ABTS (EC50 0.1070–0.658 mg/mL) scavenging activities 50 value of 37.63 μM 50 values of 0.116 and 0.39 mg/mL in the DPPH and O2·− tests Seven peptides derived from oat proteins were evaluated for ROO2.2.3There are two main mechanisms by which antioxidant molecules can deactivate free radicals: hemolytic or hydrogen atom transfer (HAT) and single electron transfer (SET). Both will produce identical end-products despite the difference in mechanisms Tyrosine containing peptides can act mainly through a HAT mechanism while cysteine, tryptophan and histidine peptides act mainly via SET mechanisms. During HAT there is a hemolytic separation of the proton bound to the heteroatom as illustrated in ·) by H-donation. It has been found that the reactivity is determined by the bond dissociation energy (BDE) of the H-X group in the antioxidant compound [· scavenging activity than YHNAP. It should be noted that the second peptide in addition to tyrosine, contains histidine, which is another proton donating amino acid · activity of the oat peptides shows that the presence of proton donating amino acids and their actual location on the sequence are important. In the hydroxyl radical scavenging assay, the presence of cysteine and its location in tripeptides  derived from rye secalin was critical for their degree of activity. The mechanism occurred mainly through the homolytic cleavage of thiol (S—H) bonds as computed using density functional theory The proton can be directly transferred between the reacting molecules, or can be involved in a solvent assisted proton loss (SAPL) depending on pH and acid-base properties compound , 81. PepDPPH and ABTS assays are usually classified as SET reactions, meanwhile, these indicator radicals can be neutralized by reduction via electron transfers but also by quenching via the HAT mechanism r > 0.831) with their DPPH and ABTS radical scavenging activities Residues of amino acids that are important for HAT mechanisms are also important for SET assays. In the absence of peptide sequences, the amount of some aromatic amino acids in hydrolysates can be used to determine the importance SET-based assays. For example, the tyrosine and phenylalanine content of rice bran hydrolysate and ultra-filtered fractions had positive correlations  2·−, E = 940 mV) is, therefore, not strong enough to attract hydrogen from unsaturated fatty acids ·, E = 1060 mV) can. Typical oxidative reactions of lipids are displayed in Oxidation of lipids in food and biological systems can be initiated by radical species but also by the presence of transition metals such as ferrous and copper ions. Transition metals are pro-oxidants and they decrease the oxidative stability of lipids through the decomposition of hydroperoxides into free radicals 2+-induced LDL oxidation mainly through their chelating mechanisms 2+Hydrolyzed proteins and peptides like other antioxidant molecules can inhibit the oxidation of lipids in foods or biological systems through three established mechanisms. They can work through direct radical scavenging as described above and prevent proton abstraction from the lipid. Alternatively, they can donate protons to covert peroxyl radicals like those formed at C-9 and C-13 of linoleic acid  to form Peptides can also reduce lipid hydroperoxides to relatively nonreactive lipid hydroxides through non-radical reactions. Gluten incubated with linoleic acid hydroperoxides and monolinolein hydroperoxides showed that most sulfhydryl groups were converted to a disulfide and other oxidation products 2.32O2-induced oxidative stress by 63.7% 2O2-induced cell death with an increase viability of 37% Models other than in vitro chemistry and food-based models have been used to assess the antioxidant properties of cereal hydrolyzed proteins and peptides. In general, studies on cell and animal models are limited compared to in vitro tests. Rat adrenal gland pheochromocytoma (PC12) cells exposed to wheat germ protein alcalase hydrolysates maintained their integrity (i.e. no cytotoxicity) and, at 1 mg/ml, they prevented HIn rats treated with non-steroidal anti-inflammatory drugs, oxidative stress often increased and cause tissue damage in the small intestine. Daily intragastric administration of a low molecular weight wheat peptide fraction (140–1000 Da) for one month reduced edema and oxidative stress in the small intestine 2.3.12·− radicals into H2O2 which is then converted in the peroxisome to water by CAT. In the cytoplasm and extracellular environments, GSHPx converts H2O2 and organic peroxides into water or alcohol derivatives while Prx acts on all peroxides and peroxynitrite (ONOO−) The mechanisms of antioxidant molecules at the cellular level can be through radical scavenging, regulation of the activity of redox enzymes or regulation and antioxidant response elements (AREs). Most ARES are genes activated by the nuclear factor erythroid 2 related factor 2 (Nrf2) and play a critical role in redox homeostasis, phase II metabolism, and cytoprotection during oxidative stress Most studied cellular mechanisms of hydrolyzed cereal proteins and peptides include regulation of antioxidant enzymes, available thiol groups, intracellular reactive oxygen species, and reduction of lipid oxidation. The hepatoprotective effect of a corn peptide fraction in mice was associated with a decrease in activities of amino acid transferases  in serum while in the liver there was a decrease lipid oxidation and an increase in SOD activity and glutathione levels 3Cereal peptides and hydrolyzed proteins have been shown to reduce oxidative stress in chemical-based assays, animal models, cell cultures, and food systems. Overall, only a small number of pure peptides have been investigated at a cellular level. There is, therefore, a need for more studies of diseases or conditions associated with oxidative stress in different types of cells and in animal models. There is a lack of human studies, even though peptides from digested food proteins are generally recognised as safe. Finally, more chemistry-based studies are also needed to investigate modifications that can occur to peptides due to temperature or high pressure.All authors listed have significantly contributed to the development and the writing of this article.National Science and Engineering Research Council of Canada (Grant No: 371908).This work was supported by the The authors declare no conflict of interest.No additional information is available for this paper.


N-vinyl secondary amines of various naturally occurring materials using readily available solid acetylene reagents . Pyrrole, pyrazole, indoles, carbazoles, and diarylamines were successfully vinylated in good yields. Cross-linked and linear polymers were synthesized from N-vinyl carbazoles through free radical and cationic polymerization. Post-modification of olanzapine (an antipsychotic drug substance) was successfully performed.We developed a simple and efficient strategy to access  N-heterocyclic compounds, amines, amino-acids, and their biopolymers are pivotal to a vast majority of biochemical processes. Effective modification of secondary amines by the insertion of a vinyl group leads to demanded N-vinyl derivatives with a variety of new applications. Vinylated products are interesting both as biologically active cores thieno[2n) (m.p. 188–190 °C).An amine 1.0 mmol), crushed KOH , anhydrous KF , and granulated calcium carbide  were added to a reaction tube (7 mL) with 1 mL of DMSO. After stirring at room temperature for 5 min, water  was added, the tube was sealed, and the mixture was heated at 130 °C for 4 h with vigorous stirring. After cooling to 25 °C, the mixture was extracted with hexane (4 × 4 mL). Combined extracts were treated with 5% aqueous NaOH, then with brine, with water, and finally dried over Nat. 64 °C ), N,N-di52–54 °C ), N-(β-n70–82 °C ), 9,9′-d.0 mmol, 2m and 2n, methyl tert-butyl ether (MTBE) was used for extraction instead of hexane. N-vinyl derivatives were isolated by flash column chromatography with system hexane/MTBE (5/1) as an eluent with gradient elution. Compounds characterization is reported in the In case of compounds 2 should be implemented (see corresponding regulations).Caution: The experimental procedures described in the present study involve the evolution of gaseous acetylene upon the reaction of water with calcium carbide—the necessary safety requirements for experiments with gases, acetylene, and CaC1l and 5 mg (0.03 mmol) of AIBN were added to a Schlenk tube containing 1 mL of dry and degassed toluene. The tube was sealed, and the reaction was performed with stirring under an inert atmosphere at 70 °C for 48 h. Then, the reaction mixture was precipitated in methanol (20 mL). The crude product was dissolved in chloroform (1 mL) and again precipitated in methanol (20 mL). After the residue was washed with hexane (3 × 3 mL), the work-up procedure was repeated. The residue was dried under a reduced pressure for two days at 40 °C to obtain a yellow solid , Đ = 1.50).To start, 276 mg (1.4 mmol) of 1l was placed into a Schlenk tube under an inert atmosphere. Then, 1 mL of degassed dry toluene was injected into the tube. After three degassing cycles, a toluene solution of boron trifluoride diethyl etherate (2 mol %) was injected into the tube under stirring at −40 °C. After 24 h in a cryostat, the toluene solution was poured into methanol. The yellow solid was collected through a filter, washed with hexane, dissolved in chloroform, and precipitated in methanol again. The powder was dried under a reduced pressure for two days at 40 °C , Đ = 1.61).To start, 276 mg (1.4 mmol) of N-vinyl derivatives. Calcium carbide was utilized as a vinylation agent to avoid high pressure equipment when producing required amounts of acetylene and thereby simplify the synthetic procedure. The application of KF/KOH as a base was important for efficient transformation. The reaction turned out to have a good substrate scope, and various pyrrole, pyrazole, indoles, aryl amines, and carbazoles were successfully involved in the transformation. Regular and cross-linked polymers were obtained by radical and cationic polymerization of the studied carbazole- and bicarbazole-containing substrates. N-vinyl olanzapine was synthesized, and its molecular structure was established for the first time.In conclusion, a new strategy of vinylation was developed, and a variety of secondary amines were effectively converted into