At 30 °C, Pst DC3000 triggered significantly weak and relatively slow Ca2+ influx in plant cells, as compared to that at 18 °C. Increased temperature contributed to an enhanced disease susceptibility in plants; the enhanced disease susceptibility is the result of the compromised stomatal closure induced by pathogens at high temperature. A Ca2+ receptor, AtSR1, contributes to the decreased plant immunity at high temperatures and the calmodulin-binding domain (CaMBD) is required for its function. Furthermore, both salicylic acid biosynthesis (ICS) and salicylic acid receptor (NPR1) are involved in this process. In addition to stomatal control, AtSR1 is involved in high temperature-compromised apoplastic immune response through the salicylic acid signaling pathway. The qRT-PCR data revealed that AtSR1 contributed to increased temperatures-mediated susceptible immune response by regulating SA-related genes in atsr1, such as PR1, ICS1, NPR1, as well as EDS1. Our results indicate that Ca2+ signaling has broad effects on the molecular interplay between changing temperatures as well as plant defense during plant–pathogen interactions.Changing temperatures are known to affect plant–microbe interactions; however, the molecular mechanism involved in plant disease resistance is not well understood. Here, we report the effects of a moderate change in temperature on plant immune response through Ca Extreme temperatures cause adverse impacts on plant growth and development, which can lead to significant crop losses all over the world . In receNonexpresser of Pathogenesis-Related Genes 1 (NPR1)) and activating the biosynthesis of SA cyt (nM) = [X + (X∗55) − 1]/(1 − X)/0.02, where X = (L/Lmax)1/3.The calcium spikes in leaves were measured with AEQ-based calcium assay ,78,79. TPst DC3000 was cultured in King’s B medium , containing 50 ug/mL rifamycin and 25 ug/mL kanamycin, overnight at 28 °C. The cells were harvested (until an OD600 = 0.5 was reached) by centrifugation , washed twice by autoclaved water, and diluted into the desired density as described below.2. The 4- or 5-week-old WT and mutated Arabidopsis plants were dipped in bacterial suspension with gentle shaking for 5 min and kept under high humidity in the dark overnight. At 1 h after inoculation (as day 0) and 3 days after inoculation , the leaf samples were harvested for disease resistance test. The leaf samples were weighed and ground in 1 mL autoclaved water; serial dilutions were dropped on King’s B medium containing antibiotic as described above. Then, 48 h after being grown at 28 °C, the bacterial colony forming units (c.f.u.) were calculated.For dip inoculation: the cells were diluted to OD600 of 0.1, containing 0.05% Silwet L-77 in 10 mM MgClPst DC3000 at OD600 of 0.001 in 10 mM MgCl2, using 1 mL needleless syringe for time course induction (for testing the gene expression of defense-related genes) and disease resistance test. At 1 h after inoculation (as day 0) and 3 days after inoculation , the leaf samples were harvested for disease resistance test. The leaf sample was weighed and ground in 1 mL autoclaved water; serial dilutions were dropped on King’s B medium containing antibiotic as descripted above. Then, 48 h after growth at 28 °C, the bacterial colony forming units (c.f.u.) were calculated. Data were shown as an average of six biological replicates; the results are presented as mean ± S.D.For infiltrated inoculation: the leaf inoculation was performed as previously described . BrieflyArabidopsis plants described previously [2, 10 mM Tris, pH 5.8) to expose to white light (150–200 μE·m−2·s−1) for at least 3 h, at 18 or 30 °C, respectively. An equal volume (10 mL) of pathogen  was added into the peeled leaf samples with stomatal opening solution, at 18 or 30 °C, respectively. The stomatal movement was imaged and counted using light microscopy (200×). The stomatal aperture was measured using ImageJ software. Data are calculated as the average of 10 stomatal aperture for the leaf sample from 4 individual seedlings.Stomatal aperture was measured in epidermal peels excised from the abaxial side of leaves of 4- or 5-week-old WT or mutated eviously . In ordeArabidopsis seedlings were used to measure gene expressions. At one day post inoculation, 100 mg leaf tissues were harvested from control and infected leaf samples at different temperatures of different genotypes and immediately frozen in liquid nitrogen. The frozen tissues were ground to powder in 1.5 mL Eppendorf tubes. Total RNA was prepared using TRIzol Reagent (Invitrogen) based on the manufacturer’s protocol, followed by DNase-I (Roche) treatment. In total, 2 µg total RNA were used to synthesize cDNA with an oligo (dT) primer and random hexadeoxynucleotides primer . The cDNA was diluted 10 times and 1 μL/reaction (10 μL) was used as a template. Real-time PCR was performed on a MyiQTM single-color real-time PCR detection system using SYBR Green Supermix (Bio-Rad). Target gene expression levels were normalized to that of AtUBQ5 (AT3G62250). A minimum of two technical replicates and four biological replicates were used for each sample.Four-week-old WT and mutated p < 0.05), while the same letter indicates no statistically significant difference.Results were analyzed using Microsoft Excel. Error bars in all of the figures represent standard error . Number 2+ transients triggered by pathogen were compromised at high temperature. High temperature contributed to enhanced plant susceptibility in both stomatal defense and apoplastic immune response. AtSR1/CAMTA3, as a Ca2+/CaM receptor, is involved in increased temperature-mediated stomatal defense and apoplastic immunity. In addition, the contribution of AtSR1 to the temperature-modulated plant immune response requires functional CaMBD in AtSR1. This and other studies indicate that Ca2+ signaling acts as a general defense response to pathogen infection in the context of temperature.Our results suggest that Ca


Multiple inherent molecular interactions of natural polyphenols enable the                    assembly of quaternary ordered superstructures.                                         Soft structures in nature, such as supercoiled DNA and proteins, can organize                    into complex hierarchical architectures through multiple noncovalent molecular                    interactions. Identifying new classes of natural building blocks capable of                    facilitating long-range hierarchical structuring has remained an elusive goal.                    We report the bottom-up synthesis of a hierarchical metal-phenolic mesocrystal                    where self-assembly proceeds on different length scales in a spatiotemporally                    controlled manner. Phenolic-based coordination complexes organize into                    supramolecular threads that assemble into tertiary nanoscale filaments, lastly                    packing into quaternary mesocrystals. The hierarchically ordered structures are                    preserved after thermal conversion into a metal-carbon hybrid framework and can                    impart outstanding performance to sodium ion batteries, which affords a                    capability of 72.5 milliampere hours per gram at an ultrahigh rate of 200                    amperes per gram and a 90% capacity retention over 15,000 cycles at a current                    density of 5.0 amperes per gram. This hierarchical structuring of natural                    polyphenols is expected to find widespread applications.                             Hierarchical self-assembly with long-range spatial arrangement is ubiquitous in                nature and is of widespread interest for engineering materials with enhanced                properties  into hierarchical metal-phenolic mesocrystals with                ordered quaternary structure ,                a heterotetracyclic molecule found in fruits, vegetables, and tree bark] with                bismuth ions (Bitructure .                                       3)3] in N-methyl-2-pyrrolidone (NMP) at room temperature. The complexation of                    EA and Bi3+ ions started as amorphous complexes, which leads to the                    formation of spherical disordered aggregates due to the rate-limiting step of                    addition of the last component in solution. The nanoscale aggregates sintered                    and ordered themselves upon equilibration toward the most stable conformation                     with aging. Scanning electron microscopy (SEM) showed                    a morphology transition from aggregated spherical particles to mesocrystals                    during a 7-day aging process (I) versus log(q) plot showed a slope of −3.51, elucidated a                    fractal surface  spectra n3+ 53+] and four                        [Bi(NMP)43+] were adopted as the optimized                    coordination numbers for the complexes, as the results showed a similar                    stability . Tetracoordinate  and pentacoordinate  structures were selected as model complexes and                    applied directly to a reported unit cell of EA·H2O for                    crystal structure simulation (d2 = 4.26 Å) are favored over the dipole-dipole                    interaction of bound NMP in the two perpendicular axes (average d3 = 3.25 Å)  calculations on                    Bi-EA–NMP complexes under the B3LYP/6-311 theoretical model. The optimal                    coordination number of Bi(NMP)n3+ . Because 3.25 Å) .                3+ ion content was observed by inductively coupled plasma                    optical emission spectrometer (ICP-OES), which suggested that the                        Bi3+ ions were not readily be replaced by Na+ ions                    (table S1). Specifically, urea and Tween 80 solvents used for breaking hydrogen                    and hydrophobic bonds, respectively, did not lead to disassembly of the                    mesocrystals . Different cations and solvents were also                    examined for the preparation of metal-phenolic mesocrystals, including                        Fe3+, Al3+, Cu2+, and Co2+ ions                    and ethanol (EtOH), ethyl acetate (EtOAc), toluene, and N,N-dimethyl formamide (DMF);                    however, no mesocrystal particles were observed for any of these experiments                    (fig. S10). Collectively, these results confirm the unique assembly mechanism                    and preparation condition of Bi-EA mesocrystals and the importance of using NMP                    as the solvent.The presence of π-π interactions in the hierarchal assemblies is                    also supported by the bathochromic shifts in the ultraviolet-visible (UV-Vis)                    and fluorescence emission spectra of the Bi-EA mesocrystals when compared to                    those of EA molecule and nonstructured Bi-EA complexes (fig. S7) (                                        2                        g−1 due to the graphitization of the supramolecular                    structures (fig. S14). The isotherm curves of the Bi-EA mesocrystals and HBiC                    were both type I  and type IV                    , indicating the existences of both                    micropores and mesopores in the Bi-EA mesocrystals and HBiC. The micropores,                    mesopores, and macropores contained in HBiC could increase the contact areas                    between the HBiC electrode and electrolyte, which further shorten the diffusion                    distance of Na+ ions and facilitate their transportation (table S3).                    The peaks of Raman spectrum centered at 1350 and 1590 cm−1                    were assigned to the D and G bands, respectively, and the corresponding ID/IG value of 0.87 implied a partially graphitized                    N-doped carbon matrix with high conductivity  under inert atmosphere                    at 800°C for 2 hours . Energy-uctivity  uctivity . The N 1                c axis                        [d(003) = 3.95 Å] for                        Na+ insertion. Last, such a N-doped carbon matrix should be                    conducive to enriching Na+ trapping, thus enhancing the sodium                    storage capabilities.The chemistry and morphology of the HBiC composite suggest a promising candidate                    as anode for SIBs, which is given by the expected efficient cost, nontoxicity,                    and sustainability of the naturally abundant starting materials. Moreover, the                    nanostructured carbon matrix could lead to more favorable contact with the                    electrode-electrolyte interface and provide more accessible sodium storage                    sites. The Bi nanoparticles are promising components of the composite, due to                    the large lattice fringes of Bi along the                                         −1 in the voltage window of 0.01 to 1.5 V (versus                        Na/Na+) were first carried out to investigate the electrochemical                    reactions of Na3Bi ↔ NaBi ↔ Bi (fig. S16), which was                    consistent with the results of charge-discharge plateaus (fig. S17) , the average rate capacity reached                    317, 315, and 297 mA·hour g−1, respectively (−1 (83% capacity                    retention versus 317 mA·hour g−1 at 1.0 A                        g−1), even with a sharp increase in the testing current                    to 50 A g−1. At a 100 A g−1                    discharge-charge rate (6.5 s to full charge or discharge), more than 210                    mA·hour g−1 was achieved, while the ultrahigh                    charge-discharge rate of 200 A g−1  led to a capacity of 72.5 mA·hour g−1.                    While lacking hierarchically ordered structure , the carbon                    sheet–based Bi materials (Bi-C) showed low-rate capacities at various                    current densities and poor cycling stability . Although the                    carbon nanotube–based Bi materials (Bi-CNTs) presented some ordering,                    derived from the assembled CNTs , its rate capacities at                    various current densities and cycling stability were also observed to be                    inferior , suggesting that the multiscaled porosity of HBiC                    and uniformly distributed ultrafine Bi nanoparticles were additionally                    important. Full recovery of the capacity (318 mA·hour                        g−1) was obtained with the subsequently reducing test                    current to 1.0 A g−1, which suggested an excellent tolerance                    for rapid Na+ ions insertion-extraction reactions derived from the                    hierarchical structure of HBiC. However, when the Bi content was reduced to                    46.7%, the rate capabilities of HBiC-1 were inferior with the increased current                    densities, lastly reaching zero at a current of 50 A g−1                    (fig. S20). The capacity of HBiC-4 with a higher Bi content (78.9%) also decayed                    to zero, as the current density exceeded 100 A g−1, although                    its capacity was higher to 550.2 mA·hour g−1 than                    that of HBiC (540.1 mA·hour g−1) at the low current                    density of 0.1 A g−1. The rate performance of the HBiC,                    especially at current densities exceeding 100 A g−1, is the                    highest ever reported for Bi-based anode materials in SIBs (Cyclic voltammetry (CV) tests with HBiC at a scan rate of 0.1 mV                        s                + ions (fig. S21). The average diffusion                    coefficient of Na+ ions was calculated as 1.36 ×                        10−9 cm−1 s−1                    (figs. S22 and S23), thus delivering into this high charge-discharge rate                    capability. According to DFT calculations, the Na+ adsorption was                    modeled on the (001) crystal plane of Bi, which was the most readily exposed to                    the real environment, for the lowest surface energy (0.18 J                        m−2) (table S5 and fig. S24), where we selected two                    stable sites (S1 site and S2 site) (fig. S25). The Na+ preferred to                    migrate from an S1 site to an S2 site for a low adsorption energy of                    −0.67 eV and further to another S1 site . Moreover, the charge-discharge                    voltage profiles had overlapped over 100 cycles, indicating the stability and                    reversibility of the HBiC (fig. S28).The redox process in CV curve of HBiC showed fast reaction kinetics and depended                    on the diffusion of Na                −1 over 15,000 cycles at a                    high current density of 5.0 A g−1 (90.6% of capacity                    retention) (−1 at a current density of 1.0 A g−1                    after 1000 cycles (fig. S29). Furthermore, a high capacity of 302                    mA·hour g−1 over 1700 cycles at 2.0 A                        g−1 was also tested. This outstanding stability could be                    ascribed to the fact that the hierarchically ordered structure of the HBiC could                    potentially relieve the stresses resulting from swelling during the alloying                    process to facilitate rapid Na+ ions diffusion, thus delivering a                    high sodium ion storage performance (fig. S30). The HBiC presented a high                    reversible capacity of 306.9 mA·hour g−1 after 1035                    cycles at 5.0 A g−1 with a mass loading of 2.72 mg                        cm−2 (fig. S31). At mass loadings of 3.89 and 6.08 mg                        cm−2, the corresponding capacities were still maintained                    at 307.6 and 301.7 mA·hour g−1 after 450 cycles at                    1.0 A g−1, respectively (fig. S32). Even at a high mass                    loading of 9.35 mg cm−2, a capacity of 284 mA·hour                        g−1 was observed after 450 cycles, corresponding to a                    reversible areal specific capacity of 2.66 mA·hour                        cm−2. This is fundamentally comparable to commercial                    lithium ion battery anodes as electrodes in commercial cells, which commonly                    show an areal specific capacity of 2.5 to 3.5 mA·hour                        cm−2 and a mass loading of 9.0 to 9.5 mg                        cm−2  . The HBi                3Bi evolved in the voltage window of 0.50 to 0.01                    V. These results demonstrated that Bi was first alloyed via sodiation to NaBi                    and then to Na3Bi, which was consistent with our CV results. During                    the charge process, the XRD pattern also verified the gradual reduction of                        Na3Bi to NaBi and lastly to Bi. These results indicated the                    completely reversible transformations among Bi, NaBi, and Na3Bi                        .The volume variation of the incorporated Bi nanoparticles in HBiC during the                    initial sodiation-desodiation process was monitored by in situ TEM . This va                + cell was assembled by coupling the HBiC anode with a                    commercial Na3V2(PO4)3 (NVP)                    cathode, denoted as HBiC//NVP, and measured in the 1.6- to 3.2-V voltage window                    (fig. S34). The HBiC//NVP existed a reversible capacity of 256 mA·hour                        g−1 after 100 cycles test at 1.0 A g−1                    without any capacity decay. Moreover, the reversible capacities were maintained                    at 260, 258, 255, 253, 253, 246, 240, and 229 mA·hour                        g−1 at 0.1, 0.2, 0.5, 0.8, 1.0, 2.0, 3.0, and 5.0 A                        g−1, respectively. With the increasing in current                    density, the reversible capacities and voltage plateaus were found to be                    slightly reduced due to small polarization in the full cells arisen from the                    fast kinetics (fig. S35). Moreover, HBiC//NVP could deliver an energy density of                    76.6 watt-hour (Wh) kg−1 at 29.5 W kg−1.                    Even at 1445.6 W kg−1, the energy density was still 66.2 Wh                        kg−1. Comparison on electrochemical parameters of                    specific energy/power densities between the assembled HBiC//NVP full cell and                    other prototype instances are shown in a Ragone plot (fig. S36). These results                    suggested that HBiC//NVP could be a potential electrode material having high                    power, high energy densities, and high output voltage.A full Na                3+ enable the self-assembly of                    hierarchical mesocrystals with quaternary structure in a spatiotemporally                    controlled manner. The relative strong π-π interaction among                    EA-Bi layers facilitates the formation of supramolecular threads that further                    assemble into nanoscale filaments driven by dipole-dipole force and, eventually,                    superstructure into hierarchical mesocrystals. The hierarchical structure of the                    mesocrystals was preserved after thermal transformation into a Bi-carbon hybrid,                    presenting outstanding sodium energy storage performance. This work probes the                    fundamentals of hierarchical self-assembly by exploring a new class of natural                    building blocks apart from nucleobases and peptides and provides new simple                    manufacturing pathways of complex micro-macroscale hybrid materials and devices                    with enhanced performance.Our work shows that the multiple inherent molecular interactions of the simple,                    natural building blocks EA and Bi                                                        14H6O8; 97%,) was purchased from Acros                    (Belgium). Bi(NO3)3 pentahydrate                         and NMP (99%)                    were purchased from Chron Chemicals Co. Ltd. (China). Tannic acid  was purchased from                    Sigma-Aldrich. Commercial CNTs were purchased from Chengdu Organic Chemicals Co.                    Ltd., Chinese Academy of Sciences. Aluminum chloride hexahydrate                        , cupric chloride                        , cobalt chloride , iron(III)                    chloride , commercial bismuth citrate , NaCl (99%), urea (99%), and                    Tween 80 (99%) were purchased from Adams-beta (China). EtOH, EtOAc, toluene, and                    DMF solvents were purchased from General Reagents. All these materials were used                    as received. Milli-Q water with a resistivity of 18.2 megohm·cm was                    obtained from Merck Elix Advantage water purification system. All solutions were                    freshly prepared for immediate use in each experiment.EA (C                                        3)3·5H2O (0.52                    mmol) were dissolved in NMP (25 ml) under ambient condition without stirring.                    After aging for 7 days, a light yellow precipitate was obtained and collected by                    centrifugation (5000 rpm for 5 min). The precipitate was further washed with NMP                    (1 × 50 ml) and EtOH (2 × 50 ml). The Bi-EA mesocrystals were                    collected by centrifugation (5000 rpm for 5 min) and dried at 40°C under                    vacuum. The corresponding HBiC was obtained by annealing EA-Bi mesocrystals at                    800°C for 2 hours under argon atmosphere with a heating rate of                    5°C min−1.EA (0.26 mmol) and Bi(NO                3)3·5H2O was replaced with                        FeCl3, AlCl3·6H2O,                    CuCl2, and CoCl2, respectively. For different solvent, NMP                    was replaced with DMF, EtOH, EtOAc, and toluene, respectively.The preparation of materials with different cations and solvents were conducted                    in similar methods as that of Bi-EA mesocrystals. For different cations,                        Bi(NO                3)3·5H2O (0.26 and 1.04 mmol)                    were dissolved in NMP (25 ml) under ambient condition without stirring,                    respectively. After aging for 7 days, the corresponding precipitates                    (EA-Bi–1 and EA-Bi–4) were further washed and collected as                    abovementioned treatment. Last, the HBiC-1 and HBiC-4 were obtained after                    carbonized as following the above thermal treatment process, respectively.The preparation of HBiC-1 and HBiC-4 were described as follows: EA (0.26 mmol)                    and Bi was added. After mixing,                    the 1.5 ml of Bi3+ in NMP solution was further added with well                    mixing. Then, the precipitates were collected, and after annealing at                    800°C for 2 hours in argon atmosphere with a heating rate of 2°C                        min−1, the Bi-CNT materials were prepared.Fifty micrograms of CNTs were dispersed into 30 ml of distilled water, and the                    1.0 ml of TA solution (40 mg ml                                        −1. SEM images                        were observed by a Hitachi S-4800 field-emission scanning electron                        microscope (Japan). TEM characterizations were performed using JEOL 2100F.                        XPS  of the as-prepared                        composites were recorded using Mg Kα x-rays (hv = 1253.6 eV) and a pass energy of 31.5 eV. AFM observation                        was carried out by Bruker MultiMode .                            N2 adsorption-desorption isotherms and pore size                        distributions were characterized using a Kubo-X1000 analyzer (Beijing                        Builder Electronic Technology Co. Ltd.). The specific surface area was                        analyzed using the Brunauer-Emmett-Teller method. The pore size distribution                        was derived from the desorption branches of isotherms using the                        Barrett-Joyne-Halenda model. Positive ion mode MS was obtained using the                        Bruker solanX70 FT-MS. Sample                         was injected into ESI ion source. Water/acetonitrile (v/v = 1:1) was used as                        effluent solvent. The thermogravimetric analysis was performed by Shimadzu                        DTG-60H  with a heating rate of 10°C                            min−1 in air. The contents of metal ions were tested                        by ICP-OES. UV-Vis spectra were acquired on a PerkinElmer LAMBDA 1050                        spectrophotometer. Fluorescence spectra were acquired on a Hitachi                        Fluorescence Spectrophotometer F-7000.The crystal structure and phase analysis were identified by XRD . SAXS measurement was performed with Bruker NANOSTAR U SAXS . FT-Raman spectra were taken on a Raman                        spectrophotometer  with a 532-nm laser                        excitation in the range of 200 to 2000 cm                                                    −2 per slice. The NVP                        cathode was prepared by blending of NVP powder (Guangdong Card New Energy                        Technology Co. Ltd.), acetylene black, and polyvinylidene fluoride  in a mass ratio of 9.0:0.5:0.5 in NMP and then coating on an Al                        foil. The corresponding area loading was about 6.0 mg                            cm−2. For the Na+ ion half cells, CR2016                        coin-type cells were assembled in an argon-filled glove box. Sodium metal                        was used as the reference electrode and counter electrode, while Celgard                        2325 (polypropylene/polyethylene/polypropylene) severed as the separator.                            NaPF6 in 1,2-dimethixyethane (1.0 M) was used as the                        electrolyte. A battery test system  was used to test the                        electrochemical performances with a voltage window of 0.01 to 1.5 V (versus                            Na/Na+) for HBiC anode and 2.0 to 3.8 V for NVP cathode. The                        HBiC anode and NVP cathode were paired up in a mass ratio of 1:6 in CR2032                        coin-type cells, of which electrochemical performances were performed                        between 1.6 and 3.2 V. CV was carried out on a PARSTAT multichannel                        electrochemical workstation .The working electrodes were prepared by a coated Cu foil with a slurry,                        containing the HBiC composite [80 weight % (wt %)], acetylene black , sodium carboxymethylcellulose binder                        , and deionized water. After                        drying at 80°C for 12 hours, the coated Cu foil was punched into                        disks with a diameter of 12 mm, and the areal loading of active materials                        was approximately 0.9 to 1.2 mg cm                                                    −1. Data                        were collected in the 2θ range of 20° to 50° with a                        step width of 2.5° min−1.The in situ XRD of the HBiC was carried out by a Rigaku Ultima IV power XRD                        with Cu Kα radiation at 40 kV and 40 mA. A homemade battery with an                        x-ray transparent Be window was connected with a LANNE CT2001 battery                        instrument with a current density of 100 mA g                                                    2O on the Na metal surface served as a solid                        electrolyte. Once the contact between HBiC and Na2O was                        established, a constant bias of −2.0/2.0 V was applied to the HBiC                        and Na metal to drive sodiation and desodiation reactions.The dynamic sodiation-desodiation processes were conducted by in situ TEM                        using a nanobattery system (Nanofactory TEM holder operated at 200 kV). HBiC                        was attached to a Cu tip, and a piece of Na metal was mounted on a W tip.                        The native Na                                                    q range was 0.0711 to 2.28                            nm−1, corresponding to the sphere size in the range                        of 1.38 to 44.2 nm; the number of smearing points around each q was 25; the minimum uncertainty estimate was 1%;                        the target number of bins was 100; the variance of Gaussian beam profile was                        0.0115 nm−1; the convergence criterion was 1; the number                        of repetitions was 10; and the fitted data that showed the section diameters                        of the filament were from 1.38 to 44.2 nm with the number of bins of 50.The SAXS data were analyzed by McSAS package 1.3.1, with a minimal assumption                        Monte Carlo method. A sphere model was used to fit the collected data. The                                                                                G , lanl2dz at 298.25 K.                        Dispersion corrections were computed with Grimme’s D3(BJ) method in                        optimization (Ei), which was obtained by Eq. 1The quantum chemical calculations in this work were performed using Gaussian                        09 program package (                    Ea +                                b). Simulated                        XRD patterns were calculated with powder diffraction reflex module.Crystal unit modeling of tetracoordinate and pentacoordinate complexes was                        built with Material Studio v8.0. A reported unit cell data (                                                                                <sec sec-type="supplementary-material">            <title>Supplementary Material</title>            <supplementary-material position="float" content-type="local-data">                <label>20210908-1</label>                <media xlink:href="sciadv.abh3482.v1.pdf">                    <caption>                        <p>Click here for additional data file.</p>                    </caption>                </media>            </supplementary-material>        </sec>


Sunitinib, a recently-approved, multi-targeted Tyrosine Kinases Inhibitor (TKi), prolongs survival in patients with metastatic renal cell carcinoma and gastrointestinal stromal tumors, however a dose related cardiotoxicity was well described. Polydatin  is a monocrystalline compound isolated from Polygonum cuspidatum with consolidated anti-oxidant and anti-inflammatory properties, however no studies investigated on its putative cardioprotective and chemosensitizing properties during incubation with sunitinib. We investigated on the effects of polydatin on the oxidative stress, NLRP3 inflammasome and Myd88 expression, highlighting on the production of cytokines and chemokines  during treatment with sunitinib. Exposure of cardiomyocytes and cardiomyoblasts (AC-16 and H9C2 cell lines) and human renal adenocarcinoma cells (769‐P and A498) to polydatin combined to plasma-relevant concentrations of sunitinib reduces significantly iROS, MDA and LTB4 compared to only sunitinib-treated cells (P<0.001). In renal cancer cells and cardiomyocytes polydatin reduces expression of pro-inflammatory cytokines and chemokines involved in myocardial damages and chemoresistance and down-regulates the signaling pathway of NLRP3 inflammasome, MyD88 and NF-κB. Data of the present study, although  Tyrosine kinase-targeted therapies (TKi) have revolutionized the treatment of many cancers in the last years , 2. ThesPoligonum Cuspidatum plant, native to Asia but currently very widespread also in America and Europe (Polydatin (trans-resveratrol-3-O-glucoside) is a natural compound belonging to the stilbenes class of the polyphenols . It is ed Europe . Polydatd Europe , 17. Sevd Europe –20.Polydatin is a well-established nutraceutical with anti-inflammatory properties , 22. As ®, LGC Standards) and cultured in Gibco® Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS)  and Penicillin-Streptomycin  in a humidified incubator at 37 °C with 5% CO2. Cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Human renal adenocarcinoma cells 769‐P and A498 were obtained from American Type Culture Collection . A498 cells were cultured in DMEM, while 769‐P cells were cultured in RPMI. All media was supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 0.1 mg/ml streptomycin.Human cardiomyocytes (AC-16 cells) and cardiomyoblasts (H9C2 cells) were purchased from American Type Culture Collection  or treated for 48 h with polydatin  at 50,100,200 and 400 µM or Sunitinib  at 5,10,25 and 50 µM alone or combined to polydatin. After treatments, adherent cells were washed three times with PBS at pH 7.4 and incubated with 100 μL of an MTS solution (0.5 mg/mL in cell culture medium) for 4 h at 37°C, as described in literature . Absorba® ATP Assay System  according to the manufacturer’s instructions. Briefly, cardiomyocytes and renal cancer cells were untreated (control) or treated for 48 h with polydatin (100 and 200 µM) or Sunitinib (10 µM) alone or combined to polydatin. After treatments, 100 μl of lysis/assay solution provided by the manufacturer was added to confluent cell cultures in 96-well plates. After the plates were shaken for 1 min and incubated for 10 min at 23°C, luminescence was measured in a microplate luminometer . Data were expressed as relative units (r.u) according to literature  or treated for 48 h with polydatin (100 and 200 µM) or Sunitinib (10 µM) alone or combined to polydatin and cultured with CaspaseGlo 3/7 reagent  for half an hour at 37°C, according to literature . CaspaseCardiomyocytes and renal cancer cells were untreated (control) or treated for 12 h with polydatin (100 and 200 µM) or Sunitinib (10 µM) alone or combined to polydatin. After treatments, cells were lysed through triton X-100  and leukotriene B4  expression in cell lysates was determined through ELISA  following the supplier’s instructions ; data weIntracellular reactive oxygen species (iROS) and lipid peroxidation are key mediators of several cardiac dysfunctions induced by TKi and doxorubicin . CardiomCardiomyocytes and renal cancer cells were untreated (control) or treated with polydatin (100 and 200 µM) or Sunitinib (10 µM) or both in combination for 12 h. After treatments, cells were harvested and lysed in lyses buffer . Lysates were then centrifuged, the supernatants were collected and analyzed for quantification of NLRP3 inflammasome, Myd88 (myddosome) and NF-Kb. Quantification of MyD88 myddosome was performed through human and rat MyD88 ELISA Kit (ab171341), Abcam, Milan, Italy; NLRP3 inflammasome was quantified through Human and rat NLRP3 ELISA Kit (OKEH03368), Aviva Systems Biology, San Diego, CA, USA) –37. BrieThe expression of IL-1β, IL-6, IL-8, CXCL-2, TGF-β and IL-18 in cardiomyocytes and human renal cancer cells was performed through ELISA method, as described elsewhere . BrieflyPolydatin is the natural bio derivate of resveratrol therefore, as control cardiomyocytes and renal cancer cells were unexposed (control) or exposed to resveratrol  (100 and 200 µM) or Sunitinib (10 µM) alone or combined to resveratrol for 12 h. After incubation period, expression of NLRP3 inflammasome, IL-1β and IL-18 were determined through ELISA method described before.All cell-based assays were performed in triplicates and results are presented as mean ± Standard Deviation (SD). Statistical significance was analyzed by Student’s t test using Sigmaplot software . p-value < 0.05 indicates a significant difference between two data values.50 value was 11,5 ± 2,2 µM, in agree with other in vitro studies . However, cytotoxicity results are slightly different from the literature  Figure 3Intracellular reactive oxygen species (iROS) are involved in chemoresistance to TKi and in cardiotoxicity mediated by several anticancer drugs like daunorubicin, cisplatin, doxorubicin, 5-fluorouracil and alkylating agents , 35. A rin vitro studies recently published  which is crucial for post-hypoxia cell survival. Thus, sunitinib may induce cardiomyocyte hypertrophy and death , 66. ThePolygonum cuspidatum  are natural bioactives with anti-inflammatory, anticancer and chemo preventive properties. Several clinical trials demonstrated the safe and useful of CAM in cancer patients –82. Polyspidatum , with inspidatum , 85. A rspidatum , 87. In spidatum . Other sspidatum , 90.As summarized in in vitro’’ investigation, points out for the first time that polydatin reduces iROS production and lipid peroxidation as well as the expression of pro-inflammatory chemokines through MyD88 and NLRP3 mediated pathways; based on the results obtained, further in vivo studies in renal cancer-bearing mice treated with sunitinib are suggested.We demonstrated that sunitinib increased IL-1β, IL-6,IL-8 and IL-18 expression in cardiomyocytes and renal adenocarcinoma cells and that polydatin is able to significantly reduce their expression. In conclusion, previous studies have investigated the mechanisms of cardiotoxicity mediated by sunitinib indicating that it might promotes endothelial dysfunction, myocytes death and apoptosis through the disruption of mitochondrial homeostasis . HoweverThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Conceptualization, VQ, MB, and NM. Methodology, VQ, SB, MI, and AP. Software, EC and RT. Validation, GB and NM. Formal analysis, SB, MI, and MB. Investigation, writing—original draft preparation, VQ. writing—review and editing, NM, GB and MB. Visualization: RT, NM, MB, and GB. Supervision, NM and GB. Funding acquisition, GB and NM. All authors contributed to the article and approved the submitted version.This work was funded by an “Ricerca Corrente” grant from the Italian Ministry of Health. Cardiotossicitá dei trattamenti antineoplastici: identificazione precoce e strategie di cardioprotezione” Project code: M1/5-C.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatin–pDNA coacervate in its center. Thermal stabilization enhances the core’s colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications. Nucleic acid-based therapies are currently moving with vast strides towards increasingly broader clinical application. DNA and various forms of RNA , as well as antisense oligonucleotides ,2, have Given the physicochemical properties of mRNA and its inherent instability that renders its intact, intracellular translocation, and successful translation quite challenging, the life-saving impact of such mRNA-based vaccines was mostly possible via their incorporation into efficient nanocarriers ,8. In thMessenger RNA, while being structurally less stable than pDNA, if it can successfully evade endo-lysosomal digestion and reach the cytosol in an intact form, it can be rapidly translated in the cytosol without the need for nuclear translocation . As oppoExamples of the importance of such targeted co-delivery can be found in the context of anti-cancer vaccines, where studies have demonstrated that untargeted delivery of NA-based adjuvants, such as TLR4 and CD40 encoding mRNA, caused an enhancement in tumor resistance to tumor-specific cytotoxic T cells and neoangiogenesis, respectively ,27; whilPlasmid DNA, on the other hand, while being more challenging to deliver and translate due to the additional requirement of crossing the nuclear membrane , has a lCo-delivering mRNA and pDNA can benefit from the rapid onset, transient expression of mRNA, and the adjustable expression of pDNA, creating distinct expression patterns for one or more transgenes. Such an approach could be of value in achieving sequential expression of the same therapeutic protein at two different time points analogous to conventional multi-dosing or as a tool for co-expression of synergistic therapeutic proteins and/or peptides with varying half-lives or expression target cells. Within a vaccination context, this could mean a prolonged antigen expression or co-expression of body-own immunostimulatory protein(s) as an adjuvant to enhance vaccine efficacy.For simultaneous co-delivery of mRNA and pDNA using just one and the same nanocarrier, we first prepared gelatin–pDNA coacervates. It is important to set this work apart from that of Morán et al., who reported a system based on gelatin type B instead of gelatin type A, and relied on electrostatic interaction between the negatively charged gelatin B and protamine to encapsulate the nucleic acid species. This system could be formulated either in the absence or presence of the NA cargo . The NA ® MedellaPro® < 100, porcine gelatin, 228 g Bloom, pharmaceutical-grade was purchased from GELITA® Deutschland GmbH, Eberbach, Germany. Protamine sulfate was purchased from Sigma-Aldrich, Darmstadt, Germany. JetMessenger (JetM) and JetPrime (JetP) were purchased from Polyplus-transfection®, Illkirch, France. Branched polyethyleneimine (PEI), Mw~25,000, was purchased from Sigma-Aldrich Darmstadt, Germany. Lipofectin was purchased from Invitrogen, Thermo Fisher Scientific, Darmstadt, Germany. Purified water was obtained from a Milli-Q water purification system , and is referred to as MQ water. DLin-MC3-DMA was purchased from MedChemExpress , Cholesterol was purchased from Sigma-Aldrich Darmstadt, Germany, DSPE-PEG 2000 and DPPC were a kind gift from Lipoid GmbH, Ludwigshafen, Germany.Gelatin GELITA®, Heidelberg, Germany. Disodium dihydrate ethylenediamine tetra-acetic acid (EDTA-Na2) was purchased from Roth GmbH+Co. KG, Karlsruhe, Germany. DNA loading dye (6×) was purchased from Thermo Fisher Scientific, Waltham, MA, USA. DNA Ladder 250–10,000 bp was purchased from PEQLAB Biotech GmbH, Erlangen, Germany. Ethidium bromide 10 mg·mL−1 was purchased from Sigma-Aldrich, Darmstadt, Germany. Live/dead fixable stain (568/583) was purchased from PromoCell GmbH, Heidelberg, Germany. Quant-iT™ PicoGreen™ dsDNA Assay Kit and RiboGreen™ RNA Assay-Kit, DNase I, DNase I buffer, 50 mM EDTA, RNase A, and Ribolock were purchased from Thermo Fisher Scientific, Darmstadt, Germany. Bovine collagen type I solution, Purecol was purchased from CellSystems, Troisdorf, Germany.Agarose research grade was purchased from Serva® mCherry mRNA was purchased from Tri-Link BioTechnologies LLC, San Diego, CA, USA.Plasmid DNA encoding AmCyan fluorescent protein (pAmCyan1-C1) was purchased from Clontech Laboratories Inc., Mountain View, CA, USA. The plasmid was propagated in Subcloning Efficiency™ DH5α E. coli competent cells , then isolated and purified using Qiagen EndoFree Plasmid Mega Kit  as per manufacturer’s protocol. CleanCapMurine dendritic cell line DC2.4 was purchased from Millipore Corporation, California, USA. Cells were cultured in RMPI-1640 supplemented with 10% FCS, 1× non-essential amino acids , 1× HEPES buffer solution (1M), and 0.0054× β-mercaptoethanol 100×, purchased from Merck, Darmstadt, Germany. RPMI-1640, FCS, NEAA, Trypsin-EDTA (0.25%), and HEPES were all purchased from Gibco, Thermo Fisher Scientific, Darmstadt, Germany.−1) with pAmCyan (100 µg·mL−1) in a ratio of 1:1 v/v under three different mixing temperatures . For further optimization of the mixing conditions, the appropriate mass ratio between gelatin and pDNA was investigated. A solution of pAmCyan in MQ water (100 µg·mL−1) was added to gelatin type A solutions in MQ water with different concentrations  at 37 °C (Gelatin–pDNA coacervates (CoAc) were prepared using the complex coacervation technique , where aat 37 °C . The twoat 37 °C . Assessm30 was selected given its small size, low PDI, and negative zeta potential for further experiments. Thermal stabilization was performed in four subsequent heating–cooling cycles per sample. Each cycle started with CoAc incubation at 55 ± 0.5 °C for 30 min, followed by rapid cooling to 0 ± 0.5 °C for 5 min. The resulting gelated particle is referred to as thermally stabilized coacervate (TS-CoAc), while particles that were not subjected to thermal cycling (unstabilized) are referred to simply as coacervate (CoAc).CoAc−1) was used to coat the preformed CoAc30 or TS-CoAc30, to the final protamine sulfate to gelatin mass ratio of 1:5 w/w (taking into consideration the two-fold dilution of gelatin’s mass contribution during the coacervation step). Protamine sulfate solution was mixed with the preformed particle cores under laminar flow conditions in a microfluidic setting, using a meander chip with two inlets, one for each component at a total flow rate of (2 mL·min−1). The formed core-shell system was left to stand for at least 24 h at 4 °C. The core-shell particle was then surface-loaded with 1 µg mCherry per 170 µg of particles, and mixed by simple pipetting to a final pDNA: mRNA mass ratio of 5:1 per particle. The particles were further allowed to stand for 15 more minutes before use.Protamine sulfate solution (0.3 mg·mL−1) of non-coated particles and (925 µg·mL−1) of coated particles. Particle size is given as intensity-based z-average. Moreover, the colloidal stability of P- CoAc and P- TS-CoAcs was studied for three weeks at 4 °C storage temperature, where dynamic light scattering was used to assess changes in particle size or PDI over time.Samples were characterized for particle size, PDI, and zeta potential using Zetasizer Nano-ZS , utilizing 4 mW He−Ne laser at a wavelength of 633 nm and a backscattering angle of 173° at 25 °C. Samples were measured in concentrations of (1550 µg·mLv/v) at 37 °C for 4 h. For each sample, particle count (particle·mL−1) was recorded at zero time and after 4 h of incubation in RPMI-1640. Data was collected from three videos, 30 s each, where the camera level was manually set to 14 during all captures. NanoSight 3.3 software was used to process the videos at a detection threshold of 5.NTA  was used to assess the effect of thermal stabilization on the colloidal stability of the gelatin–pDNA core in a cell culture medium; this is where both non-stabilized CoAc and thermally stabilized TS-CoAc were incubated in RPMI-1640, in a ratio of  was prepared by dissolving gelatin in MQ water at 55 °C. From the gelatin stock, CoAc was prepared as previously described. A portion of the resultant CoAc was then thermally cycled to prepare TS-CoAc, as previously described. CD spectra of all samples were recorded at 37 °C on a Jasco 810 spectropolarimeter  in a 0.1-cm path-length quartz cell. Samples were scanned in and blanked to MQ water. Each sample was scanned 15 rounds per measurement, and eventually, the spectra for each were obtained after subtracting the contribution of the MQ water blank.Gelatin solution (3 mg·mL−1) or coated particles (925 µg·mL−1) were used, where 10 µL samples were mounted on a copper grid , and allowed to dry overnight before visualization. The TEM measurement was performed at an accelerating voltage of 200 kV.Unstained CoAc and TS-CoAc particles, with or without protamine sulfate coating, were visualized using TEM . Either non-coated particles  in Dnase I working buffer at 37 °C for 30 and 60 min. Particle samples were compared to samples of equivalent masses of naked mRNA and pDNA subjected to the same treatments. Following the pre-stated incubation periods, 20 µL samples or controls were drawn from the reaction mixture and the reaction was quenched using 3 µL 50 mM EDTA and 1 µL Ribolock , then the nucleic acids were released from the particles via digestion with Trypsin  for 30 min at 37 °C, followed by addition of high molecular weight Heparin . Samples were loaded onto 1.3% w/v agarose gel  containing ethidium bromide (0.3 µg·mL−1) , in TBE buffer (1×) and run for 90 min at 90 mV. The gel was visualized under UV-light .Investigating the effect of thermal stabilization on pDNA protection was performed by Dnase I challenge, comparing P-CoAc without and with thermal stabilization. In the same assay, the protection of the surface-bound mRNA against RnaseA was also monitored. mRNA-loaded P-TS-CoAc or P-CoAc (NA load of 1.5 µg mRNA and 7.5 µg pDNA) were incubated with 0.008 U·mL−1) for 150 min at 37 °C, then in a high molecular weight Heparin solution . Samples were loaded onto a 0.7% w/v agarose gel and run for 60 min at 60 mV. Data were normalized to intact supercoiled pDNA as a control.As further confirmation of the difference in pDNA shielding effects between thermally stabilized and unstabilized cores in a more physiologically relevant medium, sample volumes from uncoated CoAc and TS-CoAc equivalent to 3 µg pDNA were incubated with 10% FCS in HBSS for 3 h at 37 °C. The activity of serum nucleases was then quenched using EDTA . pDNA was then released from the sample using subsequent treatment with Trypsin , HEPES (1%), NEAA (1%), β-mercaptoethanol (0.0054%). Cells were grown for 48 h reaching confluency of approximately 80% before being treated with either samples or controls. Samples were either P-CoAc or P-TS-CoAc, both using a concentration of 170 µg particles per well, equivalent to NA concentrations of 1 µg mCherry and 5 µg pAmCyan per well. As negative controls, untreated cells and cells treated with either naked mRNA or pDNA were used. Cells treated with commercial transfection reagents were used as positive controls. For single transfection, JetMessenger was used for mRNA, whereas JetPrime was used for pDNA. As an additional positive control, a solid lipid nanoparticle (SLN) inspired by the current clinical standard of mRNA/siRNA delivery was prepared. Briefly, an aqueous solution of mRNA and pDNA in a mass ratio of 1:5 (pH = 4) was mixed with an ethanolic solution of the following lipids, DLin-MC3-DMA, DPPC, Cholesterol, and DSPE-PEG2000, in a molar ratio of 50:10:38.5:1.5 and at a final N/P ratio of 0.5 [W branched PEI is a golden standard among the polymers in terms of transfection efficacy, despite its cytotoxicity. Thus, PEI-NPs were prepared and included as a polymeric reference sample, using PEI: pDNA: mRNA mass ratio equivalent to the protamine sulfate: pDNA: mRNA mass ratio of 30:5:1 originally present in TS-CoAc. As an internal control, the same protamine sulfate concentration that was used for CoAc or TS-CoAc coating was used to formulate a protamine sulfate coacervate with pDNA, to which mRNA was added immediately before cell treatment, in the same pDNA: mRNA mass ratio used for either TS-CoAc or CoAc. Samples were incubated with cells for 6 h under shaking at 250 RPM, then removed and replaced with fresh medium and further incubated for 48 h. Cell harvesting was performed following washing twice with HBSS, where cells were detached using Trypsin-EDTA (100 µL), followed by the addition of 2% FCS in HBSS (900 µL). Samples were centrifuged at 4 °C and 300× g for 5 min, the pellet was rewashed in 1 mL HBSS then re-suspended and fixed in paraformaldehyde (4% w/v). Transfection efficiency was analyzed using flow cytometry , using the PE-Texas red channel for mCherry and AmCyan channel for pAmCyan. Flowjo version 10.6.1 was used for data processing.Both transfection efficiency and cytotoxicity of the system were assessed in murine dendritic cell line DC2.4. Transfection efficiency of pAmCyan and mCherry was assessed in DC2.4 murine dendritic cell line, passages 6 to 8. Briefly, cells were seeded at a density of 50,000 cells per well in 24 well plates, in RPMI-1640 containing FCS . Cells were incubated with the samples for 6 h under shaking at 250 RPM, following which the samples were removed, and the cells detached as previously described. As a positive control, cells killed by heating at 70 °C post detachment were used. Following the last washing step and before fixation with paraformaldehyde (4% w/v), cells were stained with live dead fixable stain 568/583  according to the manufacturer’s protocol. The used kit stains only dead cells using cell membrane-impermeable amine-reactive peptides that can be detected on the PE-emission filter. The percentage of dead cells in the different samples was then measured by flow cytometry using the PE-A channel. Cell viability was calculated according to Equation (1).Cytotoxicity of P-TS-CoAc was assessed in DC2.4, passages 10 to 12. Cells were seeded as previously described and then treated with P-TS-CoAc in a concentration of (340 µg·mLv/v) paraformaldehyde  in HBSS for 5 min at r.t. After rinsing with HBSS, cells were mounted and stored at 4 °C until CLSM analysis . Image acquisition was conducted on a Leica TCS SP8 confocal imaging microscope with a 25× water immersion objective (Fluotar VISIR 25×/0.95) at 1024 × 1024 resolution. For AmCyan, fluorescence was detected between 495–550 nm , for mCherry, fluorescence was detected between 683–784 nm , both using a HyD detector. Images were then processed with the Leica Application Suite (LAS) X software.DC2.4 cells were seeded in 8 well glass bottom µ-slide , precoated with 1 mg/mL bovine collagen type I solution, Purecol , at a density of 25,000 cells/well. The transfection procedure was performed as previously described. Immediately before visualization, the cells were washed twice using HBSS and fixed for 5 min with 4% .Data were analyzed using Graph Pad Prism 8 for Windows  and generally presented as the mean of individual values , with standard deviation indicated by the error bars. (N) refers to the number of experiments, (n) refers to the number of samples per experiment. A one-way ANOVA was performed for all test samples, followed by Tukey’s post hoc test for assessment of inter-group individual differences. Data were considered statistically significant at a level of significance of Either non-stabilized coacervates (CoAc) or thermally stabilized coacervates (TS-CoAc) were prepared according to the scheme illustrated in , with thw/w). It was observed that a gelatin to pDNA mass ratio of 30:1 (CoAc30) or 70:1 (CoAc70) led to the formation of coacervates possessing significantly smaller diameters  and smaller polydispersity indices (PDI)  compared to higher or lower ratios, which possessed particle diameters and PDIs of at least 257 nm and 0.32 in case of CoAc100 (50) showed the largest particle diameter of 1772 and a PDI of 0.52 (30 and CoAc70 showed slightly negative (−5.5 mV) and positive (1.8 mV) zeta potentials, respectively, while CoAc50 showed a zeta potential of almost zero (We combined pAmCyan (pDNA) and gelatin via electrostatic interaction in Milli-Q purified water to form a coacervate-based core. We varied the gelatin to a pDNA (pAmCyan) ratio first  and five CoAc100 c,d. The  of 0.52 c,d. CoAcost zero b.30 and CoAc70, despite not having the highest packing density, or storage modulus is more kinetically stable than the high-density coacervates formed at the point of absolute charge neutralization (CoAc50), which are more liable to aggregation over time due to their surface neutrality [30 and CoAc 70 showed acceptable particle sizes and PDIs as opposed to CoAc50. It is worth mentioning that upon changing the plasmid size between 2.6 to 7.2 Kbp, such CoAc could still be assembled at an acceptable size and PDI at a mass ratio of 30:1 gelatin to pDNA  used for gelatin type A (Bloom number 228) and pDNA (4.7 kbp), the gelatin to pDNA mass ratios of 30:1, 50:1, and 70:1 provided the three coacervates with the smallest zeta potentials. Above 70:1 and below 30:1, a surplus of the positively charged gelatin or negatively charged pDNA existed in the coacervates. The repulsive forces between molecules of the similarly charged predominant polyion reduced the packing density of coacervate and its storage modulus, observed as an increase in particle size and PDI, in accordance with what was previously described by Arfin et al. . A slighutrality . This ex to pDNA .100 showed a smaller diameter of 190 nm and PDI of 0.26 when assembled at 37 °C compared to coacervates assembled at 55 °C or 23 °C showing diameters of 541 nm and 246 nm, and PDI of 0.611 and 0.35, respectively  using NTA  as opposed to the non-stabilized system (P-CoAC) with regards to the core loaded pDNA A trend in enhanced protection of the pDNA against serum nucleases following thermal stabilization was observed. In 10% serum incubation for 3 h, TS-CoAc maintained 31.9% of intact pDNA, compared to 17.4% of CoAc particles .Circular dichroism data g showed Gelatin type A is the acidic denaturation product of collagen. Being a denatured protein, gelatin possesses low antigenicity , thus reThermal stabilization relies on the conversion of gelatin–pDNA coacervate in the case of CoAc to an anisotropic nanogel state (TS-CoAc), a phenomenon previously reported by Rawat et al. , but remA microfluidic system was adopted for coating the cores under laminar flow conditions. This technique deposited a homogenous coat across the whole particle population without any visible compromise to core integrity, as observed in the TEM images .We could not detect a distinct core-shell structure in the case of P-CoAc c. MeanwhFollowing mRNA surface-loading on P-TS-CoAc, particle size and PDI showed no discernible difference from unloaded P-TS-CoAc for up to one week. This further supports the assumption that the system remains intact and is taken up as a unit by the cells, while no mRNA-protamine sulfate coacervate sub-populations are formed .® technology, where it shows promise as an NA vaccine delivery tool [Protamine is another peptide commonly used in the field of vaccination, that has been widely employed by CureVac AG in their RNactiveery tool ,64,65,66ery tool . Protamiery tool ,68. All 30 and TS-CoAc30 showed no considerable difference in EE % of pDNA (12) than pDNA (1.076 × 1012) molecules per 170 µg of particles. Moreover, on the particle level, based on the average particle count obtained using NTA (data not shown) each P-TS-CoAc particle packed approximately 5318 pDNA molecules and 9312 mRNA molecules. Calibration curves used in the establishment of these assays are provided in (Entrapment efficiency (EE%) assessed using PicoGreen assay  showed t of pDNA . RiboGrevided in .−1), the same (170 µg·mL−1) or half of (85 µg·mL−1) the particle concentration used in transfection efficiency studies showed cell viabilities of 91.9%, 97.1%, and 97.7%, respectively, following 6 h incubation  cells treated with either the two-fold  and pDNA (pAmCyan). The transfection efficiency and level of protein expression of both pAmCyan and mCherry significantly surpassed all other test groups except for the JetM single transfection of mCherry transfection. Yet in the case of double transfection, despite the insignificant difference in transfection efficiency between JetM and both P-TS-CoAc and P-CoAc, JetM failed to cause any discernible transfection with pAmCyan a,b. FiguProtamine sulphate-NA coacervate serving as an internal control showed inconsistent transfection of both pDNA and mRNA , which wProtamine combines an ability to promote cytosolic delivery of mRNA, as well as nuclear translocation of pDNA via four specialized nuclear localization signal-like sequences in its structure ,70, settMoreover, the confocal microscopy images of the highest performing treatments reveal that cells treated with P-TS-CoAc displayed visibly more consistent transfection patterns in the case of mRNA b and pDNThis study shows an approach to produce nanocarriers based on gelatin, a pharmaceutically established biopolymer, using a mild and straightforward preparation technique to load polynucleic acid cargos suitable for gene delivery. The shown improvement of colloidal stability by thermal stabilization could be essential for the further development of a product with sufficient storage stability. As expected for a system made from a biocompatible, biodegradable protein material, no cytotoxicity was observed in the concentration range successful for transfection.A core-shell system was prepared by taking advantage of an intrinsic property of the two core components, gelatin and pDNA, to form an irreversible gel when heated together. The stability of this gel-core allowed for the deposition of a protamine sulfate shell. We loaded mRNA on the particle’s shell while maintaining pDNA in the core. Transfection of both nucleic acids was observed with comparable transfection efficiencies from both pDNA and mRNA when used in a mass ratio of 5:1, as opposed to clinical, experimental, and commercial transfection reagents, where such co-transfection was not feasible.We here present a proto-type NA carrier with unique co-transfectional capabilities. A vast pool of applications can be based on or expanded off the concept, both in the areas of vaccine delivery, as well as protein replacement therapies. In this study, we employed commercial fluorescent reporter molecules of rapid onset of expression and long-expression product half-life. Using selected combinations of nucleotides, we think the interesting possibility of gene expression at varying time scales could be achievable and should be further studied. In future studies, the potential of this system to optimize the expression kinetics and location of NA cargos could be explored. Besides clinical applications, this system could also serve as a research tool to study differences between expression kinetics of more than one NA cargo in parallel.


WB was also examined for RBC, leukocyte, and platelet viability and indices. LA caused dose-responsive hemolysis, and Ca2+-dependent PS exposure, elevated erythrocyte sedimentation rate (ESR), cytosolic Ca2+ overload, cell shrinkage and granularity, oxidative stress, accumulation of lipid peroxides, and stimulation of casein kinase 1α (CK1α). In WB, LA disrupted leukocyte distribution with elevated neutrophil-lymphocyte ratio (NLR) due to selective toxicity to lymphocytes. In conclusion, this report provides the first evidence of the pro-eryptotic potential of LA and associated mechanisms, which informs dietary interventions aimed at CVD prevention and management.Cardiovascular diseases (CVD) are a leading cause of mortality worldwide, and dietary habits represent a major risk factor for dyslipidemia; a hallmark of CVD. Saturated fatty acids contribute to CVD by aggravating dyslipidemia, and, in particular, lauric acid (LA) raises circulating cholesterol levels. The role of red blood cells (RBCs) in CVD is increasingly being appreciated, and eryptosis has recently been identified as a novel mechanism in CVD. However, the effect of LA on RBC physiology has not been thoroughly investigated. RBCs were isolated from heparin-anticoagulated whole blood (WB) and exposed to 50–250 μM of LA for 24 h at 37 °C. Hemoglobin was photometrically examined as an indicator of hemolysis, whereas eryptosis was assessed by Annexin V-FITC for phosphatidylserine (PS) exposure, Fluo4/AM for Ca Cardiovascular diseases (CVD) are a leading cause of mortality and disability worldwide . In factDietary intakes are a major risk factor for these underlying conditions ,2. GuideWhile recommendations to limit intakes of saturated fats are widespread, these recommendations fail to consider the varying health effects of different saturated FAs in the diet ,15. LaurOils rich in LA, however, may still be harmful in terms of CVD through other mechanisms . The comCell membranes are a complex amalgam of proteins, lipids, and carbohydrates . While pHydrophobic and amphipathic compounds have long been known to exhibit a dual interaction with the cell membrane. Earlier studies on red blood cells  revealed that anesthetics cause hemolysis in isotonic solutions but prevent hypotonic lysis . This isIn light of the growing appreciation of the role of RBCs, hemolysis, and eryptosis in CVD, this study was initiated to investigate the cytotoxic potential of dietary medium-chain fatty acid, LA, in human RBCs, and to identify the underlying molecular mechanisms.2-EDTA-anticoagulated whole blood (WB) was used [This study was approved by the Ethics Committee/IRB of the College of Medicine, King Saud University (Project No. E-20-4544) and conducted according to the Declaration of Helsinki. Heparinized blood samples were collected from 12 healthy volunteers and RBCs were isolated by centrifugation at 3000 rpm for 20 min and repeated washing in phosphate-buffered saline (PBS) . For comwas used .2DCFDA), BODIPY 581/591 C11, calcium ionophore ionomycin (IMC), pan-caspase inhibitor zVAD(OMe)-FMK (zVAD), p38 MAPK inhibitor SB203580, casein kinase 1α (CK1α) inhibitor D4476, reduced glutathione (GSH), cyclooxygenase (COX) inhibitor aspirin (Asp), glucose transporter 1 (Glut1) inhibitor WZB117, and erythropoietin (EPO) were obtained from Solarbio Life Science , whereas AmplexTM Red Reagent  was obtained from Thermo Fisher Scientific . LA and PA were made as 100 mM stock solutions in dimethylsulfoxide (DMSO) and stored at −80 °C. Ringer solutions were prepared essentially as described elsewhere [2 when present.LA (CAS: 143-07-7), palmitic acid , Annexin-V-FITC, Fluo4/AM, 2’,7’-dichlorodihydrofluorescein diacetate  [Control and experimental cells were pelleted by centrifugation at 13,300× CA, USA) . Cells s2 for 15 min at room temperature away from light and then examined at an excitation light of 488 nm and emission light of 512 nm.Cells were stained with 1% Annexin V-FITC and examined with CytoFLEX™ flow cytometer  as previously reported . A totalControl and experimental cells in WB were allowed to vertically sediment in Westergren tubes at room temperature for 60 min away from light, and the distance traveled in millimeters was then recorded .Cytosolic calcium was detected with 2.5 μM of Fluo4/AM as per standard protocols .Forward scatter (FSC) and side scatter (SSC) of light were measured by flow cytometry as surrogates for cell size and surface granularity, respectively .2DCFDA as previously reported [2 and 5 μM of H2DCFDA for 30 min at 37 °C away from light. DCF was excited by the blue laser at 488 nm and the green fluorescence was detected at 512 nm.Total reactive oxygen species (ROS) were labeled with 5 μM of Hreported . Control2O2 was determined by AmplexTM Red as previously reported [The generation rate of Hreported , while Sreported .Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were measured using colorimetric kits (Solarbio Life Science) as previously described .Detection of 2-nitro-5-mercaptobenzoic acid from Ellman’s reagent ; DTNB) was used as a surrogate for GSH and oxidized glutathione (GSSG) content as documented elsewhere .Homogeneous 50 μL aliquots of control and LA-treated cells were washed in Ringer solution, resuspended in 150 μL of 5 μM of BODIPY 581/591 C11 staining solution, and incubated at 37 °C in the dark for 30 min. Cells were then washed twice, excited by 488-nm blue laser, and fluorescence of emitted light was captured at 530 nm ,48.Oxidized protein content was determined following derivatization by 2,4-dinitrophenylhydrazine (DNPH) as per .Control and 100 μM LA-treated WB were examined for reticulocyte, RBC, white blood cell (WBC), and platelet viability and indices on a Sysmex XN Series hematology analyzer , as previously described .n = 9–18). Flow cytometry data as arbitrary units (a.u.) or percentages were analyzed by FlowJo™ Software v10.7.2 , and GraphPad Prism v9.2.0  was used for statistical analysis. Two groups were analyzed by unpaired, two-tailed Student t-test, whereas multiple means were analyzed by one-way ANOVA and either Dunnett’s or Tukey’s post-hoc test, or by two-way ANOVA followed by Šidák correction. In all cases, a cutoff p-value of <0.05 was set for statistical significance. Asterisks * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001) indicate significantly different values from control conditions whereas # (p < 0.05), ## (p < 0.01), and ### (p < 0.001) indicate statistical significance compared to LA-treated cells.Data are represented as means ± standard error of the mean (SEM) of at least nine determinations from three independent experiments  or with 50, 100, and 250 μM of LA for 24 h at 37 °C, and hemolysis was then measured.As shown in 2+ in LA-mediated hemolysis by incubating control and experimental cells in standard and Ca2+-free Ringer solutions. 2+ (63.49 ± 8.44% vs. 62.12 ± 11.27%). Moreover, we investigated the possible interaction of LA with IMC by exposing the cells to 50 μM of LA with and without 0.25 μM of IMC for 24 h at 37 °C . Likewise, the percentage of eryptotic cells significantly decreased from 27.37 ± 7.41% in the presence of Ca2+ to 9.44 ± 3.13% upon elimination of Ca2+ , 38.06 ± 3.39% (100 μM), and 56.90 ± 7.94% (250 μM).Eryptosis is triggered by disturbances in Ca2+ was secondary to the influx of extracellular Ca2+. 2+ exhibited significantly reduced Fluo4 fluorescence (29.26 ± 6.38) in comparison to those exposed to 100 μM of LA in the presence of Ca2+ (51.51 ± 6.73 a.u.). This was also reciprocated when the percentage of LA-treated cells with increased Ca2+ was examined with (48.34 ± 7.26%) and without (26.43 ± 7.07%) extracellular Ca2+  as seen in Another morphological hallmark of eryptosis is membrane blebbing and acanthocytosis. We thus investigated changes in SSC patterns following exposure to 100 μM of LA. 2+ elevation, and the formation of acanthocytes secondary to Ca2+ influx.Altogether, these observations suggest that LA-induced eryptosis is accompanied by significant loss of cellular volume independently of CaA pivotal mechanism leading up to eryptosis is oxidative stress. Cells were therefore assessed for ROS levels following exposure to 100 μM of LA. In 2+ significantly rescinded LA-induced ROS accumulation of 24.71 ± 6.94 folds to 5.58 ± 1.08 folds as depicted in Moreover, removal of Ca2O2 and SOA radicals were significantly increased in LA-treated cells from 10.44 ± 0.61 to 16.88 ± 0.30 nmol/g Hb and from 40.19 ± 1.26 to 56.18 ± 0.82 nmol/g Hb, respectively. Accordingly, the activities of antioxidant enzymes responsible for quenching these two radicals were also found to be significantly enhanced in LA-treated cells. As DCF is a general ROS indicator, we were prompted to identify specific free radicals responsible for the imbalanced redox status instigated by LA. As seen in In light of the oxidative stress observed upon LA exposure, we sought to determine the glutathione status and the role played by this non-enzymatic antioxidant in the presence of LA. As shown in Next, we examined the possible role of ROS in LA-induced eryptosis and hemolysis by supplementing the cells with 20 μM of GSH. As results in Furthermore, given the crosstalk between oxidative stress and inflammation, we evaluated whether Asp would rescue the cells from the hemolytic effect of LA. A major consequence to ROS accumulation is peroxidative damage to lipid molecules. In 2+ significantly decreased LA-induced BODIPY fluorescence (11.81 + 0.61 folds to 5.32 + 2.35 folds), as shown in Oxidative injury to proteins manifests as carbonylation of amino acid residues, and PCC content was thus determined and also found to be significantly increased upon treatment with 100 μM of LA (338.9 ± 8.12 to 422.8 ± 2.72 pmol/g Hb) as seen in These results point to a central role for oxidative stress in LA-induced cell death.In order to screen for signaling pathways involved in LA-induced cell death, hemolysis was measured following incubation of RBCs with 100 μM of LA in presence or absence of 100 μM of zVAD, 100 μM of SB203580, 20 μM of D4476, or 2 mU/mL of EPO, for 24 h at 37 °C.Inhibition of caspase did not significantly ameliorate LA-induced hemolysis (72.99 ± 3.05% to 69.34 ± 4.11%; Taken together, these observations seem to indicate that LA-induced cell death may be mediated through CK1α stimulation.3 cells/μL to 0.81 ± 0.07 × 103 cells/μL; Based on the cytotoxicity of LA to RBCs, we sought to determine the range of other peripheral blood cells susceptible to LA. To this end, WB was treated with the vehicle control or with 100 μM of LA and examined for indices of cellular subsets. In light of the reduced percentage of lymphocytes and the emerging role of NLR as a stress marker, it was then of interest to probe possible disturbances in NLR of LA-treated WB. Indeed, treatment with LA caused a significant elevation in NLR from 2.10 ± 0.13 to 2.93 ± 0.27, as seen in Collectively, these observations reveal that LA is selectively toxic to lymphocytes which disturbs peripheral WBC proportions reflective of significant systemic stress.2+-dependent premature RBC death associated with elevated ESR, dysregulated ionic homeostasis, cell shrinkage and granularity, oxidative injury and accumulation of peroxides, and stimulation of CK1α. These outcomes demonstrate the importance of LA and its pathophysiological mechanisms, particularly in terms of its potential impact on the risk of CVD.CVD remains a leading cause of death worldwide, for which hypertriglyceridemia is a recognized risk factor . In thisDespite the scarcity of data regarding blood levels of LA, in one small study, values of up to 37 μM were detected in apparently healthy participants . HoweverExcessive hemolysis  may leadAn independent risk factor for cardiovascular complications, anemia increases the risk of death by more than two times among patients with kidney disease and CVD compared to those without anemia  and has Our results also introduce a novel role of LA in human RBCs, which is the stimulation of eryptosis . Under pDue to severely disrupted structural integrity, eryptotic cells exhibit a significantly increased propensity to adhere to endothelial wall, promoting blood stasis, and adversely affecting blood rheology ,67. This2+ influx (2+ channel activity in PS externalization (2+ as a cofactor for activity [2+ and subsequent disturbance of these Ca2+-dependent enzymes culminates in PS externalization and overt eryptosis.Calcium regulates essential cellular processes, most notably death and survival. We found that the eryptotic potential of LA is mediated through Ca+ influx , possibllization E–G. Scraactivity . Increas2+ levels, among which K+ channels are the most notable [2+ activity increases, the opening of Ca2+-responsive K+ channels forces the escape of KCl and water, which explains the loss of cellular volume observed in our study (2+ (2+ (2+ influx (Many channels and transporters are also sensitive to fluctuations in cytosolic Ca notable . When Caur study . It is itudy (2+ C. This itudy (2+ F,G, whic (2+ (2+ F,H. Acan+ influx .Oxidative stress and lipid peroxidation contribute to atherosclerosis by oxidation of LDL-C particles that accumulate in the subendothelium and trigger an inflammatory immune response and plaque formation ,35,36. NAnimal studies ,84, systOur results also seem to point to a potential role for CK1α in LA-induced RBC death C, which 2+ influx through disrupted ion channel activity. These observations provide a working platform for a more thorough understanding of the pathophysiology of individual FAs and, thus, the optimization of dietary interventions for CVD prevention and management.In conclusion, this study presents novel mechanistic insights into the nature of interactions between LA and erythrocytes with important implications for CVD risk and development. Importantly, the eryptotic properties of LA and associated underlying mechanisms are orchestrated by Ca


Candida albicans differentiates from the white to opaque phenotype, a prerequisite for mating and a process accompanying colonization of the lower gastrointestinal tract and skin. In analyses of white cell populations induced to synchronously differentiate from the white to opaque phenotype, opaque commitment occurs at approximately the same time as evagination and chitin ring formation in the process of daughter cell formation, several hours after the master switch gene WOR1 is upregulated. Mutational analyses of transcription factor binding regions P1, P2, P3, P4, and P6 of the WOR1 promoter reveal that individual deletion of any of the five transcription factor binding regions does not eliminate morphological differentiation to the opaque cell phenotype under opaque-inducing conditions, but individual deletion of P2, P3, or P4, blocks opaque commitment and maintenance of the opaque phenotype after transition to noninducing conditions. These results suggest that commitment occurs at the level of the WOR1 promoter and that morphological differentiation can be dissociated from phenotypic commitment.During induced differentiation, the process often involves a commitment event, after which induced cells, when returned to noninducing conditions, continue to differentiate. The commitment event is rarely identified.  When a cell is induced to differentiate, there is a transition time to establish the new phenotype. Most differentiations involve changes in regulatory molecules during this period, which activate or suppress structural and physiological genes involved in generating the new phenotype. Since most studies of cellular differentiation involve comparisons between an initial phenotype and terminal phenotype, the transitional events that take place in establishing the new phenotype in many cases remain undescribed in space and time or, in the case of transient events, remain unidentified. In addition, attention is rarely paid to the commitment event, defined as the time at which a cell, induced to undergo a phenotypic transition, will continue to transition to the new phenotype when returned to noninducing conditions.Candida albicans, an opportunistic fungal pathogen  binding regions along the WOR1 promoter. Finally, we demonstrate that cells lacking any one of three TF binding regions can express the opaque cell phenotype under inducing conditions, but cannot commit to the opaque phenotype, demonstrating that expression of the opaque phenotype can be dissociated from commitment to the opaque phenotype.pathogen , undergopathogen , 3, hostpathogen –9, biofiathogen –, and genathogen ––14. Liketo white , 16, in MTL-homozygous cells of strain WO-1 (MTLα/α) or P37005 (MTLa/a) were grown to stationary phase at 25°C in air (0.04% CO2) on nutrient agar containing glucose as the carbon source (Gluc-agar). At stationary phase under these conditions, over 95% of cells accumulated in the unbudded white phase (N-acetylglucosamine (GlcNAc) rather than glucose as the carbon source (GlcNAc-agar) and incubated at 25°C in 12% CO2 rather than in air  phenotypic homogeneity of the initial stationary phase population of white cells; (ii) cellular changes that can be used to assess synchrony ; (iii) an adequate degree of synchrony; (iv) an adequate degree of mass conversion to the opaque phenotype; and (v) temporal parallelism of alternative daughter cell formation between test (opaque) and control (white) populations. All of these requisites were achieved in the protocol outlined in 17–2; T50 of evagination and filament ring formation. Examples of dishes in the commitment assay are presented in For control cultures initially plated under noninducing conditions  in panel A and in RT-PCR gels in panel B. Because of such temporal changes and conditional variation in TDH3 expression, and presumably other genes commonly used for data normalization, we have presented WOR1 expression data using semiquantitative RT-PCR in TC value, threshold cycle in PCR. Download FIG S1, PDF file, 0.3 MB.Since the expression of housekeeping genes, commonly used as a reference to normalize qRT-PCR analyses, can change over time as unbudded white cells exit stationary phase under noninducing or opaque-induced conditions, they could not be used to normalize Copyright © 2021 Conway et al.2021Conway et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the WOR1 promoter  were generated in both the α/α strain WO-1 and the a/a strain P37005 (2 (GlcNAc/high CO2-inducing)  have been shown to bind at four regions along the ansition –22, the nsition –, and thensition ––33, we cregion 5 . All of n P37005  3, 34)..WOR1 proain WO-1 . Howeverain WO-1 . In contain WO-1 . Therefo2). These strong opaque-inducing conditions proved to be of limited use to assess switching differences between binding site mutants, since 100% of white stationary-phase cells of strains WO-1, ΔP1, ΔP2, ΔP3, ΔP4, and ΔP6 switched to opaque after 5 days of incubation. Milder opaque-inducing conditions were required for a finer analysis of the role of the binding sites in induced switching to opaque. Milder conditions were achieved by applying only one of the two inducing agents  and reducing the CO2 levels from 12% to 5%.In preliminary experiments, we evaluated white-to-opaque switching under the same opaque-inducing conditions employed for commitment experiments in WO-1 , wild type WO-1 and deletion mutants ΔP1, ΔP2, ΔP6, and ΔP1–2 underwent mass switching from white to opaque , the ΔP2, ΔP3, ΔP4, ΔP1–2, and ΔP1–3 derivatives of WO-1 did not switch, whereas WO-1 WT, ΔP1, and ΔP6 exhibited high levels of switching . In cont2-inducing conditions, WT and all WO-1 promoter deletion derivatives except ΔP1–3 underwent mass switching to opaque . WT, ΔP1, and ΔP6 cells underwent commitment with the same time kinetics as evagination, but the remaining three deletion derivatives  did not undergo opaque commitment, even though all three evaginated with the same time kinetics as WT cells  (2 (opaque-inducing conditions) for 72 h. These cells were then replated on Gluc-agar at 25°C in air (noninducing conditions), incubated for 24 h, and the colonies assessed for maintenance of the opaque phenotype   . To verimitment) . The inimitment) . When whmitment) . To testmitment) , 38. Themitment) . When opmitment) , those fmitment) , indicatWOR1 expression, we measured the levels of WOR1 expression in WO-1 WT, ΔP1, ΔP2, ΔP3, ΔP4, and ΔP6 by semiquantitative RT-PCR in the initial white cell preparations, after 72 h under opaque-inducing conditions, and then after 24 additional hours after replating under noninducing conditions  of the WOR1 transcript has been shown to play a role by reducing translation efficiency . To distinguish opaque colonies and sectors, the vital stain phloxine B, which differentially stains opaque cells red  were considered a single evagination event. In summary, % evagination =  × 100.2. After 5 days, the proportions of white and opaque colonies were measured (6 cells/plate) and incubated under noninducing conditions or on GlcNAc-agar at 25°C and 12% CO2 (“opaque-inducing conditions”). At specific time intervals, cells were resuspended in one ml of sterile distilled H2O, microscopically imaged for cell phenotype, and replated on Gluc-agar containing phloxine B and incubated at 25°C in air. Cultures were incubated for 5 days and scored for the proportion of opaque colonies.White cells were grown on Gluc-agar at 25°C in air (“noninducing conditions”) for a minimum of 5 days to reach stationary phase. To assess the frequency of white-opaque switching, white cells were replated at approximately 200 cells/plate on Gluc-agar or GlcNAc-agar containing phloxine B and incubated at 25°C in air or in 5% COmeasured . To asse2O, and then stained with calcofluor . Stained cells were imaged with a Leica TCS SP8 confocal microscope.Cells were fixed with 2% paraformaldehyde in 1× phosphate-buffered saline (PBS), washed three times with sterile distilled H6 heat-killed WO-1 white cells to remove antibodies to surface antigens common to white and opaque cells , incubated for 1 h at 4°C, and then stored at –80°C. Immediately prior to RNA extraction, cell samples were homogenized by mechanical lysis with acid-washed glass beads in a bead beater. DNA contamination was removed from samples by DNase treatment using the TURBO DNA-free kit (Ambion). The RNA samples were cleaned using the RNeasy minikit (Qiagen). The RNA content of each sample was quantified using a NanoDrop One spectrophotometer . All RT-PCRs performed for semiquantitative analyses were carried out using a Superscript III OneStep RT-PCR kit  in accordance with the manufacturer’s instructions. The primers used for analysis of 10.1128/mBio.02320-21.2TABLE S2Table S2, PDF file, 0.04 MB.Primers used in this study Copyright © 2021 Conway et al.2021Conway et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To avoid saturation in our RT-PCRs and in pixel density analyses, we performed a reaction dilution series using the RNA from the time point sample that yielded the highest band intensity in the initial calibration reactions. The RNA concentrations assessed in our dilution series were 0.0625 ng, 0.09375 ng, 0.125 ng, 0.1875 ng, 0.375 ng, 0.5 ng, 0.75 ng, 1.0 ng, 1.5 ng, and 2.0 ng. Based on the results, we chose to carry out all RT-PCRs with 0.5 ng of RNA, since the resulting signal was within the linear range.WOR1 locus were generated in strains WO-1 (α/α) and P37005 (a/a) (SAT1)  using the primer pairs listed in C. albicans genome targeted for deletion.Deletion derivatives of the 05 (a/a) . The vec) (SAT1) , was useSAT1 gene. Incorporation of the deletion construct at the appropriate locus was verified by PCR amplification, as was the subsequent excision of the recyclable cassette from the genome. The recyclable flipper construct encodes a site-specific recombinase, caFLP, under the control of a maltose-inducible promoter, allowing the selection cassette to be excised from the genome by growth of verified transformants in YP-maltose liquid medium.The generation of homozygous deletion derivative mutants was carried out in a stepwise manner with the first successful transformation producing a heterozygous deletion derivative and the second successful transformation producing the desired homozygous deletion derivative. Transformations were performed as described by De Backer et al. . Transfo