Hordeum sativum L.). Plants of a “Hana-type” landrace (B1) were taller, flowered earlier and produced heavier, larger and more vigorous seeds that resisted ageing longer compared to a semi-dwarf breeding line (B2). Accordingly, a NAC domain-containing transcription factor (TF) associated with rapid response to environmental stimuli, and the TF ABI5, a key regulator of seed dormancy and vigour, were more abundant in B1 seeds. Drought significantly reduced seed yield in both genotypes, and elevated temperature reduced seed size. Genotype B2 showed partial thermodormancy that was alleviated by drought and elevated temperature. Metabolite profiling revealed clear differences between the embryos of B1 and B2. Drought, but not elevated temperature, affected the metabolism of amino acids, organic acids, osmolytes and nitrogen assimilation, in the seeds of both genotypes. Our study may support future breeding efforts to produce new lodging and drought resistant malting barleys without trade-offs that can occur in semi-dwarf varieties such as lower stress resistance and higher dormancy.High seed quality is key to agricultural production, which is increasingly affected by climate change. We studied the effects of drought and elevated temperature during seed production on key seed quality traits of two genotypes of malting barley ( Hordeum vulgare L.) is among the four most important crops globally (http://faostat.fao.org) and its caryopses, henceforth termed seeds, are used for staple food, for animal feed and in the brewing industry. Malting barley varieties must meet specific quality criteria, especially low lodging potential and high tolerance against diseases, high and stable seed yield, a seed size > 2.5 mm and for malting, the seed protein content should not exceed 12% (semi-dwarf (sdw) gene, such as the Sdw1/Denso gene  leads to its proteolysis, thereby alleviating the repression of seed germination by ABI5 , in the ‘Hana’ progeny, and a semi-dwarf breeding line HOR 4710 (B2) used in Northern Germany. These two genotypes were part of the “EcoSeed” mapping panel, which revealed that plant height, dormancy, number of seeds per spike and days to anthesis are heritable phenotypic traits . We hypot stress . ABI5 prt stress . Further by ABI5 . Nitric  by ABI5  and in A by ABI5 . Members by ABI5 . For exa by ABI5 . ANAC089 factors . ChangesHordeum vulgare L. convar. distichon (L.) Alef. var. nutans (Rode) Alef.), the Austrian landrace HOR 2110 (https://doi.org/10.25642/IPK/GBIS/18216 termed “B1”) and the breeding line HOR 4710 (https://doi.org/10.25642/IPK/GBIS/94164 termed “B2”) originally received from the VEG Saatzucht Boldebuck/Güstrow, were taken after 40 years of long-term storage at 0°C at the Federal ex situ Genebank for agricultural and horticultural plants of Germany (IPK Gatersleben). Seeds were multiplied in 2012 and the progeny was used for experiments in 2014. Of each genotype, 336 seedlings were grown in a greenhouse in pots  at a 23/15°C day/night cycle until anthesis. After half of the spikes had flowered, plants were kept for another seven days under the same conditions, and then randomly selected and subjected either to “control” conditions  or “elevated temperature” , or “drought” . After harvest, seeds were cleaned, dried at 20°C and 20% relative humidity (RH) for eight weeks (for after-ripening), and then stored at -18°C. The agronomic traits days to anthesis, days to maturity, number of spikes on the tillers and of seeds harvested, plant height, spike length, thousand seed weight (TSW) and seed grades  were evaluated.Caryopses (in which seed and fruit coats are fused), hereafter termed “seeds” for simplicity, of two barley genotypes  was scored daily and considered completed when the radicle had protruded by more than 2 mm. Root and shoot lengths of seedlings were recorded for untreated (non-stratified) seed lots. After 14 days, seeds were categorised for vigour testing into normal seedlings (NS), abnormal seedlings , and non-germinating seeds according to 10, T50), and the area under the germination progress curve  after 100 h were calculated using the curve-fitting module of the Germinator software  and multiplied by a known conversion factor, 5.7, for wheat grains , to estig for 30 min, 2 mL of the supernatant was used for MDA determination. Equivalents of MDA were calculated from the difference in absorbance at 532 and 600 nm using an extinction coefficient of 155 mM-1 cm-1.Malondialdehyde was determined as described in ® EDTA-free protease inhibitors (Sigma) followed by centrifugation for 10 min at 15,800 g at 4°C. Final protein concentration was determined by the Bio-Rad Protein Assay (Bio-Rad) based on the Bradford method  containing 1 mM PMSF and 1 x cOmpleted method . In totad method , and antd method , anti-Ac-1 ribitol as an internal standard and extracted for 10 min at 4°C by shaking  with an Eppendorf Thermomixer (Eppendorf AG). Insoluble material was removed by centrifugation at 20,000 g for 5 min. 25 µL were collected and dried for 150 min in a SpeedVac  and stored at -80°C. Seed samples were removed from the freezer and allowed to equilibrate with ambient temperature for 15 min before opening and drying in a SpeedVac for 1 hour before adding 10 µL of 20 g L-1 methoxyamine in pyridine to the samples, the blanks or the standard solutions of amino acids and sugars. After 90 min of continuous shaking in an Eppendorf Thermomixer at 28°C, 90 µL of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) were added and the reaction continued for 30 min at 37°C. After cooling, 50 µL were transferred to an autosampler vial. Two hours after derivatization, 1 µL of sample was injected in splitless mode into an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass spectrometer and separated on a Rxi-5SilMS column . The liner (Restek # 20994) was changed before each series of analysis and 10 cm of column were cut. Oven temperature ramp was 70°C for 7 min, then 10°C min-1 to 325°C for 4 min for a total run time of 36.5 min. Helium flow was 1.5231 mL min-1, injector temperature 250°C, transfer line temperature 290°C, ion source temperature 250°C and quadrupole temperature was 150°C. Samples and blanks were analysed in a randomized manner. Amino acid standards were injected at the beginning and end of each sample batch for monitoring of the derivatization stability. An alkane mix  was injected in the middle of the queue for external calibration of retention indices. Five scans per second were acquired. Raw Agilent data files were converted to NetCDF format and analysed with AMDIS (http://chemdata.nist.gov/mass-spc/amdis/), and metabolites identified according to their retention indices and mass spectra. Peak areas were then analysed using the QuanLynx software  after conversion of the NetCDF file to MassLynx (Waters) format. Two analytical replicates of each biological replicate (n=3) were analysed. The data is available as part of the Metabolomics Workbench repository . The ground, frozen samples were re-suspended in 1 mL of ice-cold (-20°C) water:acetonitrile:isopropanol (2:3:3 v/v/v) containing 4 mg LT002272;  under htpost-hoc test, as appropriate. For the metabolite profiling data, raw values were transformed via log1p transformation ) and imputation of missing values was performed using the R package ‘missForest’  by the Tukey or Dunn’s sForest’ . MetabolControls. The barley genotype B1 developed significantly taller plants , and DIMINUTO (HvDIM), both involved in brassinosteroid biosynthesis, and HvBRI1, which encodes a brassinosteroid receptor  gene expression associated with phosphate homeostasis and phosphorus mobilization from phytic acid (myo-inositol monophosphatase (CaIMP) gene was associated with increased seed size in chickpea . The DOI for this project (PR001453) is: http://dx.doi.org/10.21228/M8X13R. Further inquiries can be directed to the corresponding author.The original contributions presented in the study are included in the article/supplementary files. The GC-MS based metabolite profiling dataset was submitted to the Metabolomics Workbench repository (MN, LR, GCl, GCu, and MB conducted the main experiments together with CS, IS-V, and HR. MN, IK, EA, LR, GCl, GCu, CB, CS, TR, IS-V, HR, and OL analysed the data. MN and IK wrote the paper with inputs from all other authors. All authors contributed to the article and approved the submitted version.


Herein, black mesoporous rutile/anatase TiO2 microspheres with a homojunction and surface defects have been successfully synthesized by an evaporation-induced self-assembly, solvothermal and high-temperature surface hydrogenation method. The H500-BMR/ATM  materials not only possess a mesoporous structure and relatively high specific surface area of 39.2 m2 g−1, but also have a narrow bandgap (∼2.87 eV), which could extend the photoresponse to the visible light region. They exhibit high photocatalytic hydrogen production (6.4 mmol h−1 g−1), which is much higher (approximately 1.8 times) than that of pristine mesoporous rutile/anatase TiO2 microspheres (3.58 mmol h−1 g−1). This enhanced photocatalytic hydrogen production property is attributed to the synergistic effect of the homojunction and surface defects in improving efficient electron–hole separation and high utilization of solar light. This work proposes a new approach to improve the performance of photocatalytic hydrogen production and probably offers a new insight into fabricating other high-performance photocatalysts.The photocatalytic hydrogen evolution of TiO 2 microspheres with surface defects are fabricated and exhibit excellent solar-driven photocatalytic performance due to synergistic effect of the homojunction and surface defects favoring efficient e–h separation.Mesoporous rutile/anatase TiO Fortunately, converting solar energy into chemical energy can compensate for this problem, and it has high-energy-density, facilitating the virtues of energy storage and transportation. Among new energy sources, hydrogen energy has the advantages of being highly efficient and clean, produces no pollution, is easy to produce, is convenient for transportation and is renewable, which could make it the ideal energy carrier.6,10,13,14 Therefore, hydrogen energy has an increasingly important role in energy sources to replace fossil fuels. In recent years, semiconductor photocatalytic water splitting for hydrogen evolution technology has become a promising strategy because of its environmental friendliness, low-cost and cleanliness by using solar energy.The energy crisis and environmental pollution are two classic problems in today's world, which must be resolved in the near future.2) photocatalyst has been intensively investigated during the past several decades due to its cheapness, nontoxicity, and higher refractive index and stable physicochemical properties.15–20 Compared with single-phase TiO2, rutile/anatase mixed TiO2 (such as P25) enabled the transfer of electrons excited via solar light from rutile to anatase TiO2 because the energy of the anatase phase is slightly below the conduction band (CB), which can greatly suppress charge recombination and improve the photocatalytic performance.21–25 Previous studies have verified this result.52 However, although the rutile/anatase mixed phase of TiO2 can efficiently enhance its photocatalytic performance, TiO2 alone is limited to absorbing ultraviolet light, which covers less than 5% of the solar energy spectrum. Fortunately, Mao and co-workers reported a black TiO2 by a high-temperature surface hydrogenation method, which possessed a narrowed bandgap and extended photoresponse features.26 Since then, more and more researchers have been paying attention to the study of black TiO2, which has opened up a new era for TiO2 photocatalysis.6,10,27–32 Nevertheless, the most reported black TiO2 materials were generally nanoparticles, which were non-porous materials with a low surface area. Therefore, mesoporous black TiO2 is an ideal material for the efficient use of solar energy.The titanium dioxide .Here, we report a facile evaporation-induced self-assembly, solvothermal and high-temperature surface hydrogenation method to synthesize black mesoporous rutile/anatase TiO22.1106PPO70PEO106, Mw = 12 600 g mol−1) was purchased from Sigma-Aldrich company. All chemicals were analytical reagents and used directly without any further processing.Titanium tetrabutoxide (TBOT), acetic acid (HOAc) and tetrahydrofuran (THF) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Hydrochloric acid (HCl) and ethanol were obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. Pluronic F127  were slowly added to the mixed reaction solution with drastic stirring for 30 min at normal temperature. Thereafter, 3.4 mL of TBOT was slowly mixed under magnetic stirring for 2 h to obtain a golden liquid. Subsequently, the obtained golden solution was transferred to a blast drying oven to evaporate THF solvent at 40 °C for 500 min. Afterwards, the golden solution was transferred to a 50 mL autoclave and reacted at 80 °C for 1400 min. After that, the obtained samples were washed 3 times with ethanol and distilled, respectively, and then dried at 80 °C overnight. Then the as-synthesized samples were calcined at 500 °C for 3 h under an air atmosphere. Finally, the white powder samples were calcined at 400, 500 and 600 °C for 3 h under a hydrogen atmosphere to obtain black mesoporous rutile/anatase TiO2 microspheres .The mesoporous rutile/anatase TiO2.32 microspheres were measured by X-ray diffraction over the diffraction angle range (2θ) 20–60° with a Bruker-Norius D8 advanced diffractometer, using a CuKα (λ = 1.5406 Å) radiation source operated at 40 kV and 40 mA. The images of transmission electron microscopy (TEM) were obtained with a JOEL JEM 2100F instrument operated at 200 kV. The pictures from scanning electron microscopy (SEM) were obtained with a Hitachi S-4800 instrument working at 15 kV. Diffuse reflectance spectroscopy (DRS) was measured by a UV/vis spectrophotometer (Shimadzu UV-2550) in the range of 200–800 nm. Nitrogen adsorption–desorption isotherms at 77 K were collected on an AUTOSORB-1 (Quantachrome Instruments) nitrogen adsorption apparatus. All samples were degassed under vacuum at 150 °C for at least 5 h prior to the measurement. The Brunauer–Emmett–Teller (BET) equation was used to calculate the specific surface area. Pore size distributions were obtained using the Barrett–Joyner–Halenda (BJH) method from the adsorption branch of the isotherms. Surface photovoltage spectroscopy (SPS) tests were carried out with a home-built apparatus equipped with a lock-in amplifier (SR830) synchronized with a light chopper (SR540). The photoluminescence (PL) spectra were studied by a PE LS 55 spectrofluoro-photometer with an excitation wavelength of 319 nm. A scanning Kelvin probe (SKP) test  was executed to evaluate the work function at ambient atmosphere.The mesoporous rutile/anatase TiO2.4−2 from a 300 W Xe lamp and was measured by an on-line gas chromatograph. The measurement of the apparent quantum efficiency (AQE) for hydrogen evolution was performed using the same closed circulating system under bandpass filter (365 and 420 nm) irradiation from a 300 W Xe lamp .The photocatalytic hydrogen production tests were carried out in an on-line photochemical reaction system at room temperature . Classically, the photocatalysts (100 mg) were loaded with Pt (2 wt%) evenly dispersed in 80 mL of deionized water and 20 mL of methanol. The suspension of photocatalysts was sealed in the closed-gas circulation reaction cell. Prior to reaction, the reactor and the entire photocatalytic gas circulation system were degassed by a vacuum pump for about 40 min. Hydrogen evolution was studied under AM 1.5G irradiation with a power density of 100 mW cm2.52 for 60 min was used as the electrolyte. The photoanodes were prepared via a traditional spray coating method, using a glass rod to roll a paste containing 50 mg of the MR/ATM materials and 3 mL of ethanol on a transparent FTO glass-substrate with an effective diaphragm area of 1 cm2 , followed by calcining 2 h at 350 °C under a N2 atmosphere, then calcining at 500 °C hydrogen temperature to obtain H500-BMR/ATM material. An AM 1.5 power system was used as the light source.Photoelectrochemical performances were measured using a Princeton Versa STAT 3 in a standard three electrode configuration with H500-BMR/AMT and MR/AMT materials used as photoanodes, Ag/AgCl reference electrode, Pt foil as the counter electrode. And 1 M KOH solution thoroughly purged with N3θ = 27.3°, 36.1°, 39.1°, 41.3°, 44.0°, 54.3° and 56.7°, which can be indexed as (110), (111), (200), (111), (210), (211) and (220) indicating the rutile phase,13,33,34 and 2θ = 25.3° (101), 37.8° (004) and 48.1° (200) indicating the anatase phase,6,9,10 indicating that the as-prepared samples are rutile and anatase mixed phase. In addition, 2, Raman spectroscopy was also conducted. The peaks at 143, 437 and 610 cm−1 can be assigned to the classical vibrational of B1g, Eg and A1g, which are characteristic of the rutile phase.33,35,36 In addition, the peak at 238 cm−1 is caused by the multi-proton scattering process. It is worth pointing out that the Raman peaks at 513 cm−1 can be attributed to the typical anatase bands of A1g (B1g), further confirming that the mesoporous TiO2 microspheres are comprised of the rutile/anatase phase. Another interesting finding is that the peak at 513 cm−1 is gradually weakened as the hydrogenation temperature increases, indicating that the content of the anatase phase gradually decreases with increasing hydrogenation temperature. The result is in good agreement with the XRD analysis. The UV/vis diffuse reflectance in 2. Meanwhile, the visible-light absorption is gradually enhanced with an increase in the hydrogen calcination temperature, owing to the production of more surface defects.37 The surface disorder layer after hydrogenation of rutile/anatase TiO2 can form mid-gap states, which is beneficial to visible-light absorption. As shown in 38 And the bandgap of H500-BMR/ATM is only 2.93 eV, which is beneficial for visible light photocatalysis.As shown in 2 adsorption–desorption analysis on the calcined samples was performed. 6,13 Until the hydrogen temperature is increased to 500 °C, the adsorption–desorption curves show almost no significant change, indicating that the mesoporous structure can still be effectively and stably retained. When the hydrogen calcination temperature continues to increase to 600 °C, the adsorption–desorption isotherm changes, indicating that the structure of the sample calcined at this temperature has been changed, which is probably caused by partial pore collapse. The pore size distribution curve shows that the HX-BMR/ATM material has a narrow pore distribution, indicating that the MR/ATM material possesses relatively uniform pores, and the pore diameter is approximately 15 nm. The surface areas  specific area, N13,33,39–42 The results indicate that the as-prepared samples are a mixed phase of rutile and anatase, which coincides with the XRD and Raman analysis.The morphology of the as-synthetized mesoporous microspheres possessed a dehiscent configuration according to the scanning electron microscopy (SEM), as shown in 2 material. And other peaks are not found with calcination by high-temperature hydrogen, indicating that the sample structure was stable. 3/2 and Ti 2p1/2 orbitals for Ti4+, respectively.29,31 And the other two peaks located above at 457.2 eV and 462.9 eV are derived from Ti 2p3/2 and Ti 2p1/2 peaks for Ti3+. Moreover, it can be clearly seen that the Ti3+ peaks of the H500-BMR/ATM samples are significantly enhanced compared with the MR/ATM samples, indicating the effect of high-temperature hydrogenation.37 The O 1s spectra show three separated peaks . In order to further confirm the stability of the photocatalytic hydrogenation performance, cycle experiments were carried out within 24 h. After testing 8 cycles, the amount of photocatalytic hydrogen production did not decrease significantly, as shown in −1 at 365 nm, which is better than the performances at 420 nm (11.8 μmol h−1) and 520 nm (3.4 μmol h−1)  for different single wavelengths is 76.2, 8.4 and 0.8 at 365, 420 and 520 nm, respectively. The result exhibits that the mesoporous rutile/anatase TiO2 microspheres after hydrogenation manifestation have excellent visible light photocatalytic activity.In mol h−1) , which m45 As shown in 46 As shown in 47 The result indicates that the photogenerated charge carrier lifetime of H500-BMR/ATM (13.86 μs) is much longer than that of MR/ATM (11.59 μs), which proves that the hydrogenation process helps to enhance the separation of photogenerated charge carriers. The scanning Kelvin probe (SKP) is a measurement technique of vibration-capacitance-based non-contact lossless gas phase metal surface potential, which is used to measure the material's work function or surface potential.48 The work functions of H500-BMR/ATM and MR/ATM are ∼4.96 and ∼4.69 eV, respectively. This indicates that the Fermi level of H500-BMR/ATM is slightly lower than that of MR/ATM, and slightly detrimental to photocatalytic performance. However, the existence of rich surface defects on H500-BMR/ATM provides more active sites, which accelerates the transfer of photoelectrons to the surface, thereby greatly improving the separation efficiency of electrons and holes, thereby enhancing the photocatalytic hydrogenation performances.The separation of photogenerated electron–hole pairs is analyzed by surface photovoltage spectroscopy (SPS).−1) than Air-MR/ATM (14.3 μA cm−1) under AM 1.5 illumination, which exhibits a more conducive charge separation. In addition, both samples exhibited low current density under dark conditions, illustrating the higher separation efficiency of photogenerated electron–hole pairs under irradiation conditions. Furthermore, the transient chronoamperometry on the samples of H500-BMR/ATM and MR/ATM is shown in 3+ and surface oxygen vacancy defects, which are beneficial to improving the electron–hole pair transport and separation, furthermore enhancing the photocatalytic activity.The photoelectrochemical properties of H500-BMR/ATM and MR/ATM materials are tested by a Princeton VersaSTAT. The linear sweep voltammograms (LSV) in 2 has a slightly lower energy gap than anatase phase TiO2 (3.02 eV vs. 3.20 eV). When rutile and anatase phases are combined, a staggered band gap is formed and the homojunction effect can lead to an effective charge separation across the phase junctions . The results suggested that the existence of the homojunction and surface defects caused a narrowed bandgap and improved the separation efficiency of photogenerated electron–hole pairs, further enhancing the photocatalytic hydrogen evolution performance.In summary, we have demonstrated a facile method to prepare a black mesoporous rutile/anatase TiOThere are no conflicts to declare.RA-009-C9RA00633H-s001


Dipteryx alata Vog.) almond oil supplementation on vascular function, platelet aggregation, and thrombus formation in aorta arteries of Wistar rats. Methods: Male Wistar rats were allocated into three groups. The control group (n = 6), a Baru group receiving Baru almond oil at 7.2 mL/kg/day , and (iii) a Baru group receiving Baru almond oil at 14.4 mL/kg/day . Baru oil was administered for ten days. Platelet aggregation, thrombus formation, vascular function, and reactive oxygen species production were evaluated at the end of treatment. Results: Baru oil supplementation reduced platelet aggregation (p < 0.05) and the production of the superoxide anion radical in platelets (p < 0.05). Additionally, Baru oil supplementation exerted an antithrombotic effect (p < 0.05) and improved the vascular function of aorta arteries (p < 0.05). Conclusion: The findings showed that Baru oil reduced platelet aggregation, reactive oxygen species production, and improved vascular function, suggesting it to be a functional oil with great potential to act as a novel product for preventing and treating cardiovascular disease.Background: This study assessed the effects of Baru ( Dipteryx alata Vog., Fabaceae family) is an emerging nut from the central-western area of the Brazilian Savanna [Baru almond  with high severity , and thiDipteryx alata Vog.) on vascular function, platelet aggregation, superoxide anion production, and thrombus formation in aorta arteries of Wistar rats.Considering the beneficial cardiometabolic effects and tocopherol concentration in Baru almond oil, we have evaluated the effects of Baru almond oil  were used in this study. The rats were maintained in the Animal Production Unit of the Institute for the Research on Drugs and Medications (IPeFarM) of the Federal University of Paraiba (UFPB) in collective polypropylene cages  with controlled temperature (22 ± 1 °C), humidity (50–55%) and light-dark cycle (12 h), receiving water and diet ad libitum. The procedures followed the National Council for Control of Animal Experimentation (CONCEA) and International Principles for Biomedical Research. The experimental protocols were approved by an Institutional Animal Care Committee (CEUA-UFPB protocol number # 128/2016).Male Wistar rats (n = 6), receiving phosphate-buffered saline (PBS) as placebo; (ii) a Baru group receiving Baru almond oil at 7.2 mL/kg/day ; and (iii) a Baru group receiving Baru almond oil at 14.4 mL/kg/day . The placebo or Baru almond oil was administered daily with oral gavage for ten days. Baru oil (Lot B43) was purchased from Ybá Óleos Puros Company .Rats were grouped into: (i) a control group -tocopherol, y = 584,174x − 2 × 10−6, r2: 0.9993; and δ-tocopherol, y = 652,752x − 2 × 10−6, r2: 0.9975 [Serum tocopherol was determined by a high-performance liquid chromatography system equipped with an LC-20AT quaternary solvent pumping module, SIL-20A autoinjector, DGU-20A degassing system, RF-20A detector, and CBM-20A controller. A CLC-ODS (M) Shim-pack column and an aG-ODS-4 Shim-pack pre-column  for tocopherol determination were used . The ide: 0.9975 .3 solution [Anesthetized rats were coupled with an X1 ultrasound probe and a data acquisition system . The carotid artery flow was recorded using the LabChart software version 8.0 . After 10 min of baseline recording, a thrombus was induced by inserting a piece of Whatman No. 1 paper embedded with 5 μL of FeClsolution  into thesolution ,18,19. Tsolution . Subsequsolution .2•− in platelet-rich plasma (PRP), the blood was deposited in siliconized plastic tubes and centrifuged at 120 g for 10 min to obtain the PRP. Platelet-poor plasma (PPP) was obtained after second centrifugation at 2300× g for 15 min [Blood was collected from the inferior vena cava using sterile heparinized syringes. To determine platelet aggregation by light transmission and evaluate the production of Or 15 min ,22.7 platelets/mL) were stimulated with the adenosine 5′-diphosphate  or phorbol 12-myristate 13-acetate . For ADP stimulation, the recording time was 10 min, and for PMA, the recording time was 15 min . The degree of aggregation was expressed as a percentage of the maximum light transmission obtained with PRP [PRP samples  was added to 200 µL of the platelet suspension, protected from light, and placed at 37 °C for 30 min. After washout, the fluorescence was evaluated with a flow cytometer FACS CANTO II equipped with a 15-mW argon laser, λ = 488 nm . A total of 10,000 events were acquired in the FITC channel (564–606 nm) ,22, foll2 and 5% CO2 . All the rings were subjected to a basal tension of approximately 1.0 g during a stabilization period of 60 min, exchanging the nutrient solution every 15 min [−9 − 10−5 M), ACh  and SNP  were obtained. The last two were constructed shortly after contraction induced by Phe (1 μM) reached the tonic phase.The thoracic aorta artery was isolated and rings (1–2 mm) mounted in an organ bath (10 mL), suspended vertically, and attached to a force transducer . The tissues were maintained in Krebs solution at 37 °C and aerated with 95% Oy 15 min . After sy 15 min . Next, imax and pD2 values for the concentration–response curves were obtained through nonlinear regressions. Statistical analysis was carried out with Prism 6 software . A p-value of < 0.05 was considered significant.The results were described as mean ± standard deviation for parametric data or median (maximum-minimum) for non-parametric data. A Kolmogorov–Smirnov test was used to assess data normality. Parametric variables were analyzed with one-way ANOVA and a Tukey post hoc test. Non-parametric variables were compared with a Kruskal–Wallis test with a Dunn’s post hoc test. The EA typical chromatogram of the separation of tocopherols in Baru oil showed three well-defined peaks identified by external standards as δ-, (β + γ)- and α-tocopherol . The resp = 0.19) were similar among control and rats receiving Baru oil  and dry thrombus weight  were significantly reduced in rats receiving Baru oil at 14.4 mL/kg when compared to the control group  maximum effect (Emax) of phenylephrine in aorta rings with and without endothelium  decreased thrombus establishment, platelet aggregation, and superoxide anion production in rats. Additionally, Baru almond oil treatment for ten days effectively improved vascular function in aorta arteries, suggesting a great potential to be helpful in the treatment and prevention of cardiovascular disorders.The present study demonstrated that the administration of Baru almond oil  might be a safe and promising strategy to reduce platelet aggregation, thrombotic process, and improve endothelial function (In summary, our findings revealed that Baru almond oil (function .


Nigella sativa) and black pepper (Piper nigrum), respectively. The current study aimed to explore the potential chemo-modulatory effects, mechanisms of action, molecular targets, and binding interactions after TQ and PIP treatments and their combination with sorafenib (SOR) against human triple-negative breast cancer (MDA-MB-231) and liver cancer (HepG2) cells.Traditional herbal medicine has been used for centuries to cure many pathological disorders, including cancer. Thymoquinone (TQ) and piperine (PIP) are major bioactive constituents of the black seed (DNMT3B), histone deacetylase (HDAC3) and miRNA-29c expression levels. Finally, a molecular docking study was performed to propose potential mechanisms of action and binding affinity of TQ, PIP, and SOR with DNMT3B and HDAC3.We determined drug cytotoxicity by MTT assay, cell cycle, and death mechanism by flow cytometry. Besides, the potential effect of TQ, PIP, and SOR treatment on genome methylation and acetylation by determination of DNA methyltransferase (DNMT3B and HDAC3 expression and upregulation of the tumor suppressor, miRNA-29c. Finally, the molecular docking study has identified strong interactions between SOR, PIP, and TQ with DNMT3B and HDAC3, inhibiting their normal oncogenic activities and leading to growth arrest and cell death.Collectively, our data show that combinations of TQ and/or PIP with SOR have significantly enhanced the SOR anti-proliferative and cytotoxic effects depending on the dose and cell line by enhancing G2/M phase arrest, inducing apoptosis, downregulation of This study reported TQ and PIP as enhancers of the antiproliferative and cytotoxic effects of SOR and addressed the mechanisms, and identified molecular targets involved in their action.The online version contains supplementary material available at 10.1186/s12906-023-03872-6. Globalld Africa . On the tality 8.% after lChemotherapy is one of the major treatment patterns for cancer, used alone or in combination with other treatment modalities. Chemo-drugs kill cancer cells by interfering with the cell cycle regulating genes, therefore, inhibiting cell growth and proliferation, leading to cell death mainly by apoptosis –6. Cancehttp://ctdbase.org/). Also, it is used as a therapy for about 890 cancer clinical trials .Sorafenib (SOR), doxorubicin, and cisplatin are the most used chemotherapeutic agents suitable for patients with liver and breast cancer. Sorafenib is a protein kinase inhibitor that is active against various protein kinases. In 2008, the US FDA approved SOR (NEXAVAR®) for treating patients with liver and advanced kidney cancers [Natural products derived from plants have always been used to treat various diseases and most of the present anticancer drugs use active ingredients extracted from plant sources –13. ThesNigella sativa) [Nigella sativa L. is shown in Table Thymoquinone (TQ) is a major bioactive constituent of the black seed ( sativa) , it has  sativa) . Interes sativa) –21. The Piper nigrum), white and long pepper (Piper longum), it has several actions, including anti-inflammatory and anticancer properties [Piper nigrum [Piper longum [Piperine (PIP) is the major bioactive alkaloid found in black , and SOR was purchased from Cipla Ltd, India. All cell culture materials were obtained from Gibco .2.Human hepatocellular carcinoma HepG2 and breast cancer MDA-MB-231 cells were supplied from American Type Culture Collection (ATCC). Cells were cultured in a complete DMEM medium and incubated at 37 °C in an atmosphere containing 5% CO3 per well in a 96-well plate with 100 µl of complete fresh medium for 24 h before treatment with different concentrations of PIP (12.5–200 µM), TQ (25–400 µM) and SOR (6.25–100 µM) for 48 hs. Cell viability was measured by MTT as previously described [50 was calculated by nonlinear regression analysis of the dose–response curve in each cell line.HepG-2 and MDA-MB-321 Cells were cultured at 15 × 10escribed , and the50 values in the combination treatments of TQ and/or PIP with SOR, HepG2, and MDA-MB-231were treated with (IC10-IC50) doses of TQ and PIP, together with SOR (1.0 – 40.0), then incubated for 48 hs before performing the MTT assay as mentioned above.For the determination of IC50 values for all cellular and molecular analyses for each compound. Both cell lines were treated as follows: culture media or 0.1% dimethyl sulfoxide (DMSO), controls; single treatment with either TQ or PIP or SOR; double treatment with TQ + PIP or TQ + SOR or PIP + SOR and finally triple treatment with TQ + PIP + SOR. Treatment was performed 48 hs before the respective analysis, and experiments were repeated at least three times.Cells were treated with half of the predetermined calculated ICg for 5 min, resuspended using PBS, and 1.2 ml ethanol was added, and the tube was stored at -20 °C for 1 h then, cells were pelleted by centrifugation, the cell pellet was washed with PBS. 100 μl RNase A reagent was added to resuspend the cells and incubated at 37 °C water bath for 30 min then 400 μl propidium iodide (PI) staining solution was added, mixed, and incubated at 4 °C for 30 min. Finally, cells were analyzed using proper machine settings.Cell cycle distribution analysis was carried out by cell cycle assay kit, Elabscience Biotechnology Co., Ltd . Following trypsinization, cells were centrifuged at 300 × The influence of TQ, PIP, and SOR on apoptosis in HepG2 and MDA-MB-321 cells were quantified by flow cytometry. In brief, cells were collected, washed with PBS, resuspended in 500 μl of annexin V binding buffer, and added 5 μl of annexin V-FITC/PI solution. Cells were resuspended and darkly incubated at 22º C for 20 min before FACS analysis.DNMT3B), histone deacetylase (HDAC3) genes, and miRNA-29c were assessed by qRT-PCR. Total RNA was first isolated using the miRNeasy Mini Kit  and reverse‑transcribed to cDNA using the QuantiTect Reverse Transcription kit . Second, qRT-PCR was performed using the qPCR Master Mix kit . The qRT-PCR cycles consisted of 10 min at 95 °C, 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 15 s. The primers for DNMT3B, HDAC3, miRNA-29c, β-actin, and U6 genes are listed in Table DNMT3B and HDAC3 were calculated by the comparative 2−ΔΔCt method [β-actin as a housekeeping gene, while miRNA-29c was calculated using the U6 gene as an endogenous control.The mRNA levels of DNA methyltransferase (t method  using thhttps://www.rcsb.org/) and prepared by BIOVIA Discovery Studio  [To perform molecular docking of TQ, PIP, and SOR against DNMT3B (target site PDB ID: 6KDL), HDAC3 (target site PDB ID: 4A69), and vascular endothelial growth factor receptor-2 (VEGFR-2) (target site PDB ID:3V2A), we first downloaded from RCSB PDB database ( France) –30. The https://pubchem.ncbi.nlm.nih.gov/). The binding free energy, binding affinity (pKi), and the ligand efficiency of TQ, PIP, and SOR against prepared DNMT3B (6KDL-A) and HDAC3 (4A69-A) were determined using InstaDock software [In addition, the 3D structures of TQ, PIP, and SOR were obtained from the PubChem database . Data were represented as mean ± SEM of three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison tests were used to compare group differences. p < 0.05 was deemed to show statistical significance.Statistical analysis was performed using GraphPad prism 8.4.2  of TQ and PIP alone and in combination resulted in a significant decrease in the IC50 value for SOR, with the maximum reduction in IC50 (85.78%) detected in the combined treatment with the predetermined IC50 for TQ and PIP. Similarly, the same combined treatment made the SOR IC50 in MDA-MB-231 cells drop by 75.13%.The ICMicroscopic examination of cells after exposure to TQ, PIP, and SOR for 48 hs showed various degrees of cytotoxicity depending on the treatment and cell line Figs. A-B. HepGThe percentages of viable and dead cells were determined after 48 hs of treatment by double annexin V-FITC/PI staining Figs. A and 4A.The cell death mechanism (apoptosis vs. necrosis) was investigated using flow cytometry by analyzing the cells in (early + late) apoptosis vs. necrotic cells. The highest percentage of apoptotic cells were detected after treatment of HepG2 with PIP (31.77% ± 0.29) and MDA-MB-231 cells with TQ + PIP + SOR (55.0% ± 0.46) Figs.D and 4D.On the other hand, the highest percentage of necrosis was observed after treatment of HepG2 with TQ + PIP + SOR (90.97% ± 0.03) and MDA-MB-231 with PIP + SOR (56.33% ± 0.24) Figs.C and 4C.p < 0.0001) significant arrest compared to control cells (27.01% ± 0.5)  ,  Fig. B. Finall1), Fig. B.Fig. 5EDNMT3B/HDAC3 genes and the tumor suppressor miRNA-29c was examined by RT- PCR.The effect of TQ, PIP, and SOR treatment on the expression of DNMT3B expression analysis represented in Fig. DNMT3B expression almost entirely in MDA-MB-231 cells  in HepG2 cells and PIP single treatment (11-fold) in MDA-MB-231,  and MDA-MB-231 (58 folds), respectively, compared to control cells Figs. C and 7F.Ki: 6.45), (pKi: 5.79), and (pKi: 5.43) toward DNMT3B, HDAC3, and VEGFR-2, respectively. Also, PIP exhibited pKi of 5.21, 4.77, and 4.69 toward DNMT3B, HDAC3, and VEGFR-2, respectively, while TQ exhibited the lowest affinities . Co-crystallized ligands redocked in DNMT3B, HDAC3, and VEGFR-2 are shown in Figs. Molecular docking of TQ, PIP, and SOR against DNMT3B (6KDL-A), HDAC3 4A69-A), and VEGFR-2 (3V2A-A) are represented in Table A69-A, anTQ, PIP, and SOR interacted with the amino acid residues in the binding site of DNMT3B by 6, 3, and 11 bonds, respectively Table . HydrophEight hydrophobic interactions were recognized between TQ and PHE144 (2), PHE199 (2), PHE200 (2), and LEU266 (2) in HDAC3’s binding site , and epigenetic genome modifications. The cell cycle is vital in maintaining cell growth and tissue homeostasis, abnormalities in cell cycle progression results in serious diseases, including cancer . We showImpaired apoptosis is reported as one of the mechanisms leading to SOR resistance in cancer cells and the antiapoptotic protein, B-cell lymphoma 2, is suggested to modulate this phenomenon . Many reInterestingly, a combination of TQ or PIP with SOR increased the population of necrotic cells by 6.5 and 8 folds, respectively, compared to SOR alone treatment. Such data might be important in future combination therapy studies aiming to bypass apoptosis as a major cell death mechanism in SOR-resistant cancer cells. Bypassing cancer drug resistance by induction of necroptosis is under clinical investigation as a promising therapeutic mechanism against apoptosis-resistant cancer cells .HDAC3 and anti-DNMT3B effect of SOR, causing a significant drop in HDAC3 and DNMT3B expression levels. HDACs and DNMT3B act as transcriptional repressors for tumor suppressor genes, high mRNA levels of HDAC3 and DNMT3B are associated with poor prognosis in patients with different types of cancer [DNMT3B) inhibitors are currently under investigation for achieving a full cancer therapeutic potential [The current study showed that both TQ and PIP potentiated the anti-f cancer , 59. Comotential , 61. Sevotential , 63. SORotential –66. FurtIn the same way, TQ, PIP, and SOR can bind effectively to VEGFR-2 and inhibit angiogenesis. More research is needed to figure out how TQ, PIP, SOR, and their combinations affect cancer angiogenesis.The miR-29C has been identified as a crucial miRNA in several cancers; it regulates several oncogenic processes, including epigenetics. The miR-29 family was reported to function as tumor suppressor microRNA through sponging DNMT3A and DNMT3B. However, the biological activity of miR-29C in cancer development and progression is still disputed, most studies reported miR-29C as a tumor suppressor, and others suggested it acts as an oncogene . miR-29bThe molecular docking study was based on assessing the binding energy and binding affinity of ligand-receptor interactions as reflected by the docking score in kcal/mol. A lower docking score indicates a higher binding affinity. The molecular docking results showed a strong affinity of SOR to DNMT3B, and HDAC3, followed by PIP and then TQ.DNMT3B are thought to be involved in de novo DNA methylation in embryonic stem cells and early embryos. It was recently found that DNMT1, DNMT3a, and DNMT3B are overexpressed in several human tumors, compared to levels in corresponding normal tissues [DNMT transcription [DNMT3B with RNA interference or a selective DNMT3B inhibitor effectively suppressed the DNMT3B activity in vitro and in vivo. Our docking study showed strong binding between SOR, PIP, and TQ to DNMT3B, which inhibited DNMT3B activity. This finding agrees with another study that showed that DNMT3B inhibitor showed a synergistic effect with SOR in the SOR-resistant Hep3B cells [DNA methylation occurs almost exclusively in CpG islands in mammals catalyzed by DNA methyltransferases (DNMTs). DNMT1 has been shown to maintain methylation in somatic cells, and DNMT3a and  tissues –71. Preccription . Inhibit3B cells .Genome acetylation is one of the most important epigenetic modifications in promoters of genes that regulate the cell cycle, differentiation, cell growth, and survival in cancer . The aceThe present study showed that TQ and PIP enhance SOR's anti-tumor and cytotoxic effects against hepatocellular carcinoma and triple-negative breast cancer cells. In our study, we studied cell cycle arrest, cell toxicity, apoptosis, and necrosis induction, as well as molecular epigenetic mechanisms that reduce DNMT3B and HDAC3 expression and increase miR-29C expression. Additionally, the results of the molecular docking study validated and supported our in vitro findings.Additional file 1. Sorafenib anticancer potentials.Additional file 2. Cancer clinical trials of sorafenib.Additional file 3. The anticancer potential of Thymoquinone.Additional file 4. Thymoquinone anticancer clinical trials.Additional file 5. Piperine anticancer potentials.Additional file 6. Piperine anticancer clinical trials.


Stylosanthes guianensis (stylo) is largely unknown. This study aimed to determine the responses of Nramp genes to metal stresses and investigate its metal transport activity in stylo. Five SgNramp genes were identified from stylo. Expression analysis showed that SgNramp genes exhibited tissue preferential expressions and diverse responses to metal stresses, especially for manganese (Mn), suggesting the involvement of SgNramps in the response of stylo to metal stresses. Of the five SgNramps, SgNramp1 displayed the highest expression in stylo roots. A close correlation between SgNramp1 expression and root Mn concentration was observed among nine stylo cultivars under Mn limited condition. The higher expression of SgNramp1 was correlated with a high Mn uptake in stylo. Subsequent subcellular localization analysis showed that SgNramp1 was localized to the plasma membrane. Furthermore, heterologous expression of SgNramp1 complemented the phenotype of the Mn uptake-defective yeast (Saccharomyces cerevisiae) mutant Δsmf1. Mn concentration in the yeast cells expressing SgNramp1 was higher than that of the empty vector control, suggesting the transport activity of SgNramp1 for Mn in yeast. Taken together, this study reveals that SgNramp1 is a plasma membrane–localized transporter responsible for Mn uptake in stylo.Transporters belonging to the natural resistance-associated macrophage protein (Nramp) family play important roles in metal uptake and homeostasis. Although Nramp members have been functionally characterized in plants, the role of Nramp in the important tropical forage legume  Trace metals, such as manganese (Mn), iron (Fe), zinc (Zn), and copper (Cu), are essential elements for plant growth and productivity. These metal ions involve in multiple physiological and biochemical processes in plants, such as stabilizing the structure of many biomacromolecules and serve as cofactors for key proteins and enzymes . For exavia a variety of metal transporters, such as members of the natural resistance-associated macrophage protein (Nramp), Zn-regulated transporter/Fe-regulated transporter-like protein (ZRT/IRT), metal tolerance protein (MTP), and heavy metal ATPase (HMA) (Oryza sativa) (Medicago truncatula (Glycine max) (Hordeum vulgare) (Nicotiana tabacum)  . Among tse (HMA) . Nramp pse (HMA) . To datese (HMA) , rice (O sativa) , Medicaguncatula , soybeanine max) , barley vulgare)  and tobatabacum) .in vivo (Saccharomyces cerevisiae) mutant Δsmf1, which is defective in Mn uptake  for Cd (Fagopyrum esculentum) for Mn and Cd  for Cd , FeNrampn and Cd , and NtNn and Cd .Stylosanthes guianensis (stylo). Stylo is an acid soil adapted, pioneer forage legume, which is widely used for multipurpose including land reclamation and restoration, soil quality improvement, and fodder for farm animals . The conserved motifs of Nramp proteins were analyzed by MEME (https://meme-suite.org/meme/tools/meme) and Pfam (http://pfam.xfam.org/search/sequence) programs with default parameters.Five in stylo . The fullo roots . These f3)2, 3 mM KNO3, 0.25 mM KH2PO4, 0.5 mM MgSO4, 25 μM MgCl2, 80 μM Fe-Na-EDTA, 5 μM MnSO4, 0.5 μM ZnSO4, 1.5 μM CuSO4, 0.09 μM (NH4)6Mo7O24, and 23 μM Na2B4O7. The pH of the solution was adjusted to 5.8 every 2 days. Stylo seedlings were grown in a greenhouse at 25–32°C under normal sunlight conditions with a photoperiod of about 13h light. For expression analysis of SgNramps, root, stem, and leaf tissues were harvested at 21 days of growth, whereas flower and seed were harvested at 120 days and 140 days, respectively.In this study, the stylo cultivar “Reyan NO.5” was used for gene expression analysis. Stylo seeds were sown on moistened filter papers at 25°C in the dark for 2–3 days. The germinated seedlings were then transplanted into Hoagland solution as previously described . The macSgNramps to deficiencies of Mn, Fe, Zn, and Cu, the 14-day-old stylo seedlings precultured in half-strength Hoagland solution were transferred to nutrient solution without Mn, Fe, Zn, or Cu application for 7 days. Leaf and root were harvested and used to gene expression analysis. To analyze the effects of excess Mn, Fe, Zn, and Cu treatments on the transcripts of SgNramps, the 14-day-old stylo seedlings precultured in half-strength Hoagland solution were transplanted into fresh Hoagland solution supplied with 400 μM MnSO4, 800 μM Fe-Na-EDTA, 20 μM ZnSO4, or 10 μM CuSO4 for excess Mn, Fe, Zn, or Cu treatments, respectively. After 7 days of treatments, leaf and root were harvested and used for gene expression analysis. The stylo seedlings grown in full-strength Hoagland solution for 21 days were set as the control treatment.To investigate the responses of S. guianensis, including “Reyan NO.2”, “Reyan NO.5”, “Reyan NO.10”, “Reyan NO.13”, “Reyan NO.18,” “Reyan NO.20”, “Reyan NO.21”, “Reyan NO.22”, and “Reyan NO.24”, were used to evaluate the genotypic variations in SgNramp1 expression. Fourteen-day-old stylo seedlings precultured in half-strength Hoagland solution as described above were transplanted into fresh Hoagland solution supplied with 0.1 or 5 μM MnSO4 regarding as low Mn and normal Mn treatments, respectively. After 7 days of Mn treatments, roots were harvested for SgNramp1 expression and Mn concentration analyses.In addition, nine stylo cultivars belonging to via the HiScript III cDNA synthesis kit . The synthesized cDNA was further used to perform quantitative real-time polymerase chain reaction (qRT-PCR) analysis using SYBR Green Master mix reagent . qRT-PCR reaction was detected by a QuantStudio™ 6 Flex Real-Time equipment . Gene specific primers for qRT-PCR analysis are detailed in SgNramp was calculated relative to the housekeeping gene SgEF-1a , and then it was used to synthesize first strand cDNA  SgEF-1a . Three bAfter samples thoroughly dried in an oven for 7 days, the dried samples were digested with concentrated nitric acid at 140°C. Mn concentration in the digested solution was determined by atomic absorption spectroscopy. The standard reference material (GBW07603) was used to validate Mn determination as previously described .SgNramp1 was amplified by PCR using SgNramp1-GFP primers (pBWA(V)HS vector (Agrobacterium tumefaciens strain Gv3101 and were then transiently expressed in leaves of 5-week-old tobacco (Nicotiana benthamiana) seedlings according to Li et al. (2021). The PM marker (OsMCA1) (The open reading frame (ORF) of S vector . The con(OsMCA1)  fused wismf1 , the Fe uptake-defective mutant Δfet3fet4  and the Cd-sensitive mutant Δycf1  were obtained from the Euroscarf. The ORF of SgNramp1 was amplified using SgNramp1-pYES2 primers . The construct and empty vector were then introduced into the above yeast mutants using the LiOAc/PEG method. The yeast transformants were incubated in a liquid synthetic complete (SC–U/Glu) medium consisting of yeast nitrogen base, glucose, and amino acids without uracil at 30°C for about 6 h. After optical density (OD600) of the yeast cells reached to 0.6, the yeast cells were diluted to an OD600 of 0.2 and three 10-fold serial dilutions were prepared. Then, 10 μl of each dilution was spotted onto the induction SC–U plates containing 2% (w) galactose  and 1% raffinose according to smf1, the plate was added with 0, 20, and 25 mM Mn-chelator ethylene glycol tetraacetic acid (EGTA). For Fe transport analysis in Δfet3fet4, the plate was added with 0, 20, and 40 µM FeSO4. For assay on the tolerance to Cd in Δycf1, the plate was added with 0, 40, and 60 µM CdCl2. The yeast cells were incubated at 30°C for 2 d prior to photographing.In this study, the Mn uptake-defective mutant Δsmf1 was precultured in SC–U liquid medium containing 2% (w) Gal and 1% raffinose to the log phase. The precultured yeast cells were diluted to an OD600 of 0.05 and were then grown in SC-U liquid medium added with or without 20 mM EGTA. The OD600 value of yeast cells was measured from 0-48h. To determine Mn uptake in yeast, the yeast cells with an initial OD600 of 0.2 were incubated in SC–U liquid medium supplemented with 20 and 40 μМ MnSO4 for 24 h. To analyze kinetics of Mn uptake, the yeast cells with an initial OD600 of 0.2 were incubated in SC–U liquid medium supplemented with 0–10 μМ MnSO4 for 6 h. After treatments, the yeast cells were harvested and washed with 5 mM CaCl2 solution, followed by washing with deionized water twice. The collected cells were used for Mn determination. The Km and Vmax values of Mn uptake in yeast cells expressing SgNramp1 were estimated by Lineweaver-Burke plots of Mn concentrations at various Mn treatments after subtracting the Mn concentration in yeast cells transformed with pYES2 empty vector.To assess yeast growth in liquid medium, the transformed yeast strain Δt-test were analyzed by using SPSS13.0 .One-way ANOVA and Student’s Nramp genes were cloned in stylo, which were named as SgNramp1 to SgNramp5   Figure 2SgNramps in tissues of root, stem, leaf, flower, and seed of stylo were measured via qRT-PCR. Results showed that SgNramps exhibited different expressions in various tissues of stylo  was higher than those in Fe, Zn, and Cu stresses. For example, a total of 6, 3, and 1 SgNramp genes were regulated by deficiencies of Mn, Fe, and Cu in leaf and/or root, respectively, whereas the transcripts of 6, 4, and 3 SgNramp genes were regulated by excess Mn, Fe, and Cu treatments in leaf and/or root, respectively  was found between SgNramp1 expression and root Mn concentration in nine stylo cultivars under 0.1 μM MnSO4 condition (low Mn treatment) . Then, subcellular localization analysis was performed in tobacco epidermis cells through transiently expressed 35S:SgNramp1-GFP and empty vector. Results showed that the fluorescence of GFP alone in epidermis cells was found in the cytoplasm, nucleus, and PM, whereas GPF fluorescence of SgNramp1 was found to be co-localized with that of the PM marker in the epidermis cells  and carried the unique amino acid residues of Nramp proteins (Phaseolus vulgaris), and CsNramps from tea (Camellia sinensis)  . Furtherinensis) . ConsistM. truncatula, and OsNramp3 from rice were grouped into the same cluster in Group I  (AhNramp1 from peanut (Arachis hypogaea)  , AhNrampypogaea) , FeNrampuckwheat , and NtN tobacco . The rooand seed Figure 3SgNramp genes exhibited diverse responses to Mn, Fe, Zn, and Cu stresses as well as heavy metal toxicity in stylo  can be found in the article/ZC designed the research. XZ, RH, LW and GW performed the experiments and analyzed the data. YM and GL prepared the plant material for this work. XZ, RH and ZC wrote the manuscript. IR, GL and ZC discussed and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the National Natural Science Foundation of China , the National Natural Science Foundation of Hainan (321RC645), and the China Agriculture Research System of MOF and MARA (CARS-34).The authors appreciate Yufen Xing for technical help.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


These results demonstrate that erastin can exert cytotoxicity on cancer cells via ferroptosis, but has little effect on immune activity by itself. However, when combined with an OVV, erastin promoted antitumoral immunity and efficacy by increasing the number of activated dendritic cells and promoting the activities of tumor specific CD8+ T cells in the tumor. Ferroptosis is a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides. In this study, we explore the combination of a ferroptosis activator with an oncolytic vaccinia virus in tumor models. Erastin induced cell death in hepatoma, colon, and ovarian cancer cells, but not in melanoma cancer cells. Erastin, not the oncolytic vaccinia virus (OVV), induced the expression of key marker genes for ferroptosis in cancer cells. In hepatocellular carcinoma and colon cancer models, either erastin or OVV inhibited tumor growth, but a combination of the two yielded the best therapeutic effects, as indicated by inhibited tumor growth or regression and longer host survival. Immunological analyses indicate that erastin alone had little or no effect on systemic immunity or local immunity in the tumor. However, when combined with OV, erastin enhanced the number of activated dendritic cells and the activity of tumor-infiltrating T lymphocytes as indicated by an increase in IFN-γ A cancer immunotherapy breakthrough in the early 2010s, marked by objective clinical responses and longer survival in patients with advanced melanoma and other solid cancers, won the Nobel Prize in Medicine in 2018 ,4. Oncolytic virus (OV)-mediated cancer therapeutics are considered a novel type of immunotherapy ,6,7. OVsWe developed highly tumor-selective and potent oncolytic vaccinia viruses (VVs) for cancer virotherapy . An oncoOver the last two decades, an interesting concept regarding the immunological consequence of cell death was established: ICD of cancer cells . The ICD-. Cells treated with erastin are deprived of cysteine and are unable to synthesize the antioxidant glutathione. Ferroptosis is a regulated cell death nexus linking metabolism, redox biology, and disease, and may be useful as a target for therapeutics [Ferroptosis is a type of cell death induced by the small molecules erastin or RSL3, as discovered by the Stockwell group . It is capeutics .+ T cells regulate tumor ferroptosis during cancer immunotherapy [+ T cell effector function and impairs its antitumor ability [Ferroptosis’ functions and mechanisms of action in antitumor immunity remain to be elucidated. These functions and mechanisms may come from two groups of target cells: immune cells and cancer cells, respectively. On immune cells, a recent study by Wang et al. provided solid evidence that CD8otherapy . However ability . In addi ability . On tumo ability . These s ability ,36. The + T cells. These targeted immunological effects may explain the improved therapeutic efficacy.What a ferroptosis activator might do to a tumor and intact immune system, and if it can work synergistically or additively with an OV for improved immunotherapy, remain largely open questions. In this study, we hypothesize that an oncolytic VV, when combined with the ferroptosis activator erastin, may have additive, if not synergistic, effects for cancer therapy. A superagonist IL-15-armed oncolytic VV was used in this study simply because it is one of the most potent OVs in inducing antitumor immunity and therapeutic results ; we aime2.B16, Hepa1-6, MC38-luc, and ID8-luc tumor cells were all used in our previous studies. B16 (CRL-6322) and Hepa1-6 (CRL-1830) were originally obtained from American Type Culture Collection . MC38 is a murine colon adenocarcinoma cell line from a C57BL/6 mouse . We modiErastin was obtained from ApexBio . The chemical was stored at −20 °C and dissolved in DMSO as stock solution. The stock solution was diluted with 1× phosphate buffered saline (PBS) to appropriate concentration before use.These OVs were described and used in previous studies ,40. The 4 cells), MC38-luc (1.0 × 104 cells), ID8 (5.0 × 103 cells), and B16 (5.0 × 103 cells) were plated per well in 96-well plates. The next day, cells were cocultured with different concentrations of erastin . Cell viability was measured after treatments for 24 h using MTS assay .Hepa1-6 (1.0 × 10C57BL/6J (B6) female mice were purchased from the Jackson Laboratory  and housed in the University of Pittsburgh Animal Facility under pathogen-free conditions. The Institutional Animal Care and Use Committee at the University of Pittsburgh approved all animal studies and procedures.6 Hepa1-6 tumor cells or 5.0 × 105 MC38-Luc tumor cells in 60 µL volume subcutaneously (s.c.) on the right flank to establish tumor implants and were randomly divided into required groups. When tumor nodules reached ~5 × 5 mm, vvDD-IL15-Rα were intratumorally (i.t.) injected with 2.0 × 107 pfu/50 µL for only one injection and 20 mg/kg erastin was intraperitoneally (i.p.) injected daily for seven total injections. At some timepoints, mice were sacrificed to harvest s.c. tumor nodules as well as spleens for analyses. As for the rechallenge assay, naïve mice and B6 mice cured by dual therapy were s.c. injected with 2.0 × 106 Hepa1-6 tumor cells; the tumor formation rate was recorded after 12 days. Tumor growth was monitored via digital caliper every other day and tumor volume was calculated as: V (mm3) = 0.5 × (Length × Width2).B6 mice were injected with 2.0 × 103 in size, became ulcerated, and/or interfered with murine activity.Animal health status and survival was monitored closely. Mice were sacrificed when tumors reached 2000 mm5 MC38-Luc tumor cells in 60 µL volume s.c. on the right flank to establish tumor implants and randomly divided into required groups. Data are quantified . When tumor nodules reached ~5 × 5 mm, vvDD-IL15-Rα were i.t. injected with 2.0 × 107 pfu/50 µL for only one injection and 20 mg/kg erastin was i.p. injected daily for seven total injections. Tumor tissues were recovered eight days after the first treatment for further analysis.B6 mice were injected with 5.0 × 10We collected spleens and tumor tissues, weighed, and incubated them in RPMI 1640 medium that contained 2% FBS, 1 mg/mL collagenase IV  #C5138), 0.1 mg/mL hyaluronidase (Sigma #H6254), and 200 U/mL DNase I (Sigma #D5025) at 37 °C for 1–2 h to make single cell preparations. Following this, these single cells were blocked with α-CD16/32 Ab  , then stained with antibodies against mouse CD45  #103132; dilution 1:300), CD3 , CD4  #553729; dilution 1:300), CD8 , PD-1 , CD206 , CD86 ; CD11b , CD11c , CTLA-4 , F4/80 , Foxp3 , Gr-1 , IFN-γ , and NK1.1 . The intracellular FOXP3 and IFN-γ staining kits were obtained from BioLegend. A BD Accuri C6 cytometer was used for flow cytometry, and the machine associated software was used to analyze and process the data.™ cDNA SuperMix  and a Dyad® Peltier Thermal Cycler . qPCR was then performed using TaqMan analysis with PerfeCTa® qPCR SuperMix  and a StepOnePlus System . All primers for PCR were ordered from a commercial source . Relative gene expression was calculated by comparing to a housekeeping gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT1) as fold increase (2−ΔCT), where ΔCT = CT (Target gene) − CT (HPRT1).RT-PCR was performed according to the previously described procedure . Briefly6 cells/mL) were re-stimulated with 200-Gy irradiated MC38-luc or ID8 cells (2.0 × 105 cells/mL) in 200 μL RPMI 1640 medium supplemented with 10% FBS. After incubation at 37 °C, 5% CO2 for 24 h, microplates were thoroughly washed before incubating with biotinylated α-mouse IFN-γ Ab overnight . These plates were then processed using the protocol provided by the vendor . Data were collected and analyzed using an ImmunoSpot™ analyzer .Spleens were collected from MC38-luc tumor-bearing mice at 8 days post virus administration. These isolated splenocytes . One-way and two-way ANOVA were used to analyze the collected data and presented as means ± SD whenever suitable. Kaplan-Meier survival analysis and the log-rank test were used to analyze anima survival data. A We first explored the cytotoxicity of erastin to some murine cancer cells  represenp = ns). The combination of erastin and OV treatment maintained the induction of these three marker genes , ferritin light polypeptide 1 (FTL1), ferritin heavy chain 1 (FTH1), and NAD(P)H quinone dehydrogenase 1 (Nqo1) ,43,44. Wer genes . Intereser genes . Togethep ≤ 0.05 compared to vvDD-IL-15-Rα; p ≤ 0.0001 when compared to other groups). The capacity of tumor growth inhibition translated as longer mouse survival in the groups , yet the dual therapy obtained the best therapeutic efficacy assessed as inhibition of tumor growth   A. These n group) B.5 splenocytes (or ~50 spots/1.0 × 106 splenocytes) were observed. In the dual treatment group, this number went up to 120/1.0 × 106 splenocytes . Altogether, these data show that the dual treatment induced a significant tumor-specific antitumor memory immune response.In order to examine if the antitumor effects occur through immunological mechanisms, we set up another experiment using MC38-luc tumors. Eight days after therapy, tumors were harvested and weighed C. The tuUsing RT-qPCR assays  and late+ cells, CD8+ cells, expression of IFN-γ and TNF-α, and increased PD-1 and CTLA-4. When treatment only with OV was compared to the dual therapy, the only major difference was that IFN-γ and PD-1 in CD8+ T cells were further increased in dual therapy (p < 0.05).When the RT-qPCR data panel was analyzed, erastin had little effect on immune cells, but vvDD-IL15-Rα had a large of impact on immune cells, as expected. The OV treatment increased CD4+CD8+ T cells, PD-1+CD8+ T cells, CD86+CD11c+ cells, macrophages, MDSC, and the ratio of CD8+ T cells/FoxP3+CD4+ T cells. We did not observe any changes induced by erastin alone, yet all positive changes involved vvDD-IL15-Rα. When the two were combined, we observed further enhancement in IFN-γ+CD8+ T cells , colon cancer (MC38), and ovarian cancer (ID8). Not surprisingly, we found that one of the cell lines tested, melanoma B16, was resistant to the induction of ferroptosis by erastin. Can VV induce ferroptosis? Previous studies indicate that oncolytic VV induced cell death through dual modes ofapoptosis and necroptosis ,47, the In tumor models of syngeneic mice, we showed that either erastin or the OV exerted therapeutic efficacy on HCC and colon cancer. However, much improved efficacies were obtained when the two anti-tumor agents were combined on colon cancer and HCC. The Hepa1-6 tumor-bearing mice cured by vvDD-IL15-Rα and erastin were resistant to the rechallenge of Hepa1-6 cancer cells, suggesting that immune memory existed in these cured mice.+ and CD8+ T cells. However, the OV impacted the immune profile of the tumor tissues, mostly for pro-antitumor immunity. Interestingly, the addition of erastin to OV, i.e., the combination, made two types of significant changes. One significant change was an increase in macrophages and activated dendritic cells. The second was that even though no changes in the numbers of CD4+ and CD8+ T cells were observed, these CD8+ T cells were more active, as indicated by increasing IFN-γ and PD-1 expression. In addition, the ratio of CD8+ T cells over Treg increased, which is a good indicator of stronger antitumor immunity. In summary, both innate and adaptive immunity against cancer were promoted by the combinatorial treatment of erastin and OV.We then conducted immunological analyses. First, tumor-specific T cells in splenocytes were identified using ELISpot assays. We detected few if any tumor-reactive T cells in the spleens of mice treated with erastin. However, we detected a significant number of tumor-reactive T cells in the group treated with vvDD-IL15-Rα, and even more in the group that received the combination treatment. When flow cytometry and RT-qPCR analyses were conducted for immune cells isolated from the tumor tissues, we found that erastin by itself did not change the number of activated immune cells, including macrophages, dendritic cells, myeloid-derived suppressor cells (MDSCs), and CD4+ and CD8+ T cells and production of Th1 cytokines IFN-γ and TNF-α in the TME are increased after therapy by the OV or dual therapy with OV and erastin, quantities of MDSCs are also increased, as well as PD-1 and CTLA-4, the checkpoint molecules that can inhibit antitumor immunity. Our data show that PD-1+CD8+ T cells were increased by the OV treatment, and further promoted by the dual therapy (+ and CD8+ T cells, and other immune cells such as NK and macrophages [However, as quantities of CD4 therapy . We did rophages ,52,53,54+ T cell functions [+ T cells induced ferroptosis in cancer cells in vivo [+ T cells, activated through immunotherapy, downregulated the expression of SLC7A11 protein, which is required for the induction of ferroptosis. Further in vivo experiments revealed that T cells induce ferroptosis in cancer cells in ovarian tumor-bearing mice [There are some studies on ferroptosis in cancer cells and signaling pathways, and what types of drugs can induce ferroptosis . So far,unctions ,34. Inte in vivo . The CD8ing mice .+ T cells without increasing the number of these T cells in the TME. Our new findings suggest that this novel combination strategy is a promising therapeutic regimen that is warranted for clinical studies in human cancer patients.Our current study, to our knowledge, is the first one to directly apply a ferroptosis enhancer in vivo in combination with an OV and observe subsequent induction of antitumor immunity and improved therapeutic efficacy. We made two new key discoveries. The first is that induction of ferroptosis in cancer cells by itself changed little, if any, systemic or local immunity in the TME. This seemed to be contradictory to the previous observations made by others ,34. Howe+CD8+ and PD-1+CD8+ T cells. This combination regimen may become a novel strategy for cancer therapy. This combination also results in increased expression of PD-1 and CTLA-4 in the TME, providing the molecular basis for rational multiple combinations with ICB therapy in the future.Both erastin and OV show limited effects in eliciting antitumor immunity and therapeutic efficacy at the indicated doses in susceptible cancer models when used on their own. When combined, they work together to enhance immunotherapeutic efficacy by increasing tumor-specific IFN-γ


Toxoplasma rhoptry protein 38 (PruΔrop38) through its pathogenicity and immunoprotective efficacy in mice. Mice inoculated intraperitoneally with 1 × 103, 2 × 103, or 4 × 103 PruΔrop38 showed no visible signs, whereas mice inoculated with 1 × 103 parental Pru strain showed obvious wasting and bow-back, suggesting a significantly lower pathogenicity of PruΔrop38 in mice. Vaccination with 1 × 102 PruΔrop38 triggered a mixed Th1/Th2 response (Th1 response predominant), with higher IgG, IgG2a, and IgG1 levels in serum from week 3 to week 12, and a significant increase in IFN-γ, IL-12, and IL-10 in suspensions of splenocytes at 30 or 60 days post-immunization. All vaccinated mice survived when infected intraperitoneally with tachyzoites  or when infected orally with cysts (Pru or ME49). The brain parasite burden during Pru tachyzoite, Pru cyst and ME49 cyst challenges were significantly reduced in vaccinated mice. The duration of immunization showed that vaccination with PruΔrop38 could protect mice from challenge with different varied genotypes of Toxoplasma strains against different routes of infection. Collectively, these findings indicate that PruΔrop38 is an attenuated strain that provides long-term protective efficacy against acute or chronic toxoplasmosis in mice.Toxoplasmosis is a serious zoonotic disease that threatens human and animal health. Here, we evaluated the vaccine potential of the deletion of  Toxoplasma gondii is an obligate intracellular protozoan with a wide host range that can infect almost all warm-blooded animals and is capable of causing toxoplasmosis in both humans and animals [Toxoplasma infection is acquired by ingestion of water and food, contaminated with T. gondii oocysts or cysts. Ingestion of these life stages rapidly converts them into tachyzoites in the host for mass reproduction, leading to acute infection [ animals ,2. Toxop animals . Infecte animals . Toxoplanfection .Pyrimethamine and sulfadiazine are commonly available treatments for the control of toxoplasmosis, while failure rates and severe side effects of the drugs remain significant, targeting only the active stage of infection . VaccineT. gondii secretory proteins from three obligate secretory organs  have been shown to manipulate the host immune response [rop38 exhibited attenuated virulence in mice [rop38/29/19 [rop38 may have the potential to be a live-attenuated vaccine candidate for toxoplasmosis, we assessed the pathogenicity, immunogenicity and protective efficacy of PruΔrop38 in mice.The progress in the development of live-attenuated vaccines has always been facilitated by CRISPR/Cas9-mediated homologous recombination , and sevresponse . We foun in mice . Another38/29/19  comparedFemale CD-1 mice (6 to 7 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.  2021-0006). Immunogenicity and protection protocols were in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, Ministry of Science and Technology of China. The procedures were reviewed and approved by the Laboratory Animal Welfare and Animal Experimental Ethical Committee of China Agricultural University (Certificate No.: CAU-AW31901202-2-1).rop38, T. gondii type I strain RH, type II strain Pru, type III strain VEG, and ToxoDB #9 strain TgCatBj1 [2.Tachyzoites of PruΔTgCatBj1  were proT. gondii type II Pru and ME49 strain were maintained in CD-1 mice by oral passaging of cysts in mice.Cysts of rop38 in vivo, different doses of freshly egressed Pru (1 × 103 and 2 × 103) or PruΔrop38  tachyzoites were injected intraperitoneally (i.p.) into mice (five mice per group), followed by daily monitoring of body weight gain status, clinical symptoms [3 Pru caused obvious clinical signs [3, 2 × 103, 4 × 103, and 8 × 103 PruΔrop38 to determine the degree of reduced pathogenicity. Initial screening at different doses can also provide an understanding of the safety range for vaccine strain.To determine the virulence of PruΔsymptoms , and sural signs , so micerop38 strains, five mice per group were infected by i.p. injections of 1 × 102, 5 × 102, and 1 × 103 PruΔrop38 tachyzoites, respectively. Three mice from each group were selected for DNA extraction from spleen, kidney, liver, lung, heart, brain tissue, mesenteric lymph nodes and peritoneal fluid collection weekly after inoculation, in accordance with the instructions of Genomic DNA Extraction Kit . To screen for T. gondii, amplification was performed using the 529 [To determine the dynamic distribution of immunized PruΔ the 529 , ITS-1 [ the 529  and B1 [ the 529  genes, r2, 5 × 102, and 1 × 103 PruΔrop38 tachyzoites, and a group of 5 mice infected by i.p. injection of 1 × 103 Pru were used as control. Thirty days post-infection, the brains of mice infected with Pru and PruΔrop38 strains were harvested and homogenized in 2 mL volumes of sterile PBS using homogenizers. Cyst counts were performed on 200 μL of brain homogenates by DBA-FITC  staining [Toxoplasma and the 28S rRNA gene of the brain, respectively. Reaction conditions were 7 min incubation at 94 °C, followed by 45 cycles of 1 min at 94 °C, 1 min at 60 °C, 1 min at 72 °C and a final 10 min incubation at 72 °C using quantitative real-time PCR with the SYBR Green Master Mix  [Groups of 5 mice were infected by i.p. injection of 1 × 10staining . Cyst im, China)  , and mice were infected type I strain RH, type II strain Pru, type III strain VEG, and ToxoDB #9 strain TgCatBj1 tachyzoites by i.p. inoculation. To mimic the natural infection route, mice were also orally infected with Pru and ME49 tissue cysts. Four weeks after challenge, the number of brain cysts in mice challenged with Pru tachyzoites, Pru cysts and ME49 cysts was determined under microscopic examination.To evaluate the protective efficacy against toxoplasmosis, 30 and 60 days after vaccination, mice were challenged with different genotypes of Toxoplasma-specific IgG and its two subtypes IgG1 and IgG2a. Briefly, 96-well plates were coated with 100 μL of T. gondii soluble antigens from RH strain [Three mice from the vaccination and control groups were selected for sera extraction every week after inoculation, and the serum samples were stored at −20 °C until assayed by ELISA to measure the levels of H strain   and cultured in RPMI 1640  supplemented with 8% FBS. Finally, the suspensions were stimulated with TSA , Concanavalin A  as a positive control  or RPMI 1640 medium alone as a negative control at 37 °C and 5% CO2 environment. Culture supernatants were collected at different time points, where levels of interleukin (IL)-10, interferon gamma (IFN-γ), and IL-12 were detected at 72, 96, and 96 h, respectively [Mice spleens from the vaccination and control groups were collected to compare cytokine production of splenocytes. Briefly, spleens were aseptically removed from three mice 30 and 60 days post-immunization and pressed through a 70 μm cell strainer to remove tissue debris. Splenocytes were extracted and purified using Lymphocyte Separation Medium . Purified splenocytes were plated in 96-well plates . Statistical analyses were performed using unpaired Student’s 3 showed obvious wasting and bow-back, with a survival rate of 80%, and mice inoculated at a dose of 2 × 103 showed obvious signs with the death of all mice , and brain tissues with 1 × 102 inoculation , 25 (p < 0.001) and 31 (p < 0.001), respectively . These results suggest that PruΔrop38 significantly reduces the brain parasite burden compared to the parental Pru strain.To determine whether deletion of TgROP38 affects chronic infection in vivo at 30 days post-infection, we enumerated brain cysts in mice inoculated with 1 × 10 control D. The avectively E. The paectively F, being rop38 as an effective vaccine. To determine the humoral immune responses derived from PruΔrop38 immunization, we measured the antibody levels of specific anti-T. gondii IgG and IgG isotypes in the mouse serum. The safe immunization dose chosen was 1 × 102 tachyzoites by i.p. inoculation from the immunoprotective efficacy comparison of mice from four immunization doses in mice  at both time points, indicating that immunizing mice with PruΔrop38 induces a mixed Th1/Th2 response and that Th1-type cell-mediated immune responses trigger leading protection against challenge.Splenocytes harvested from vaccinated or control mice at 30 or 60 days post-immunization were stimulated with TSA, ConA or RPMI 1640 medium. The suspensions (stimulated with TSA) were measured by ELISA to assess the immunological memory and cellular immune responses induced by PruΔof IFN-γ D, IL-12 of IFN-γ E and IL-of IFN-γ F were ma2 RH, 1 × 105 VEG, 5 × 103 Pru, or 5 × 103 TgCatBj1 tachyzoites, exhibited significant weight loss and bow-back, and all mice died within the second week after challenge, with only 40% of mice surviving the 1 × 105 VEG infection (3 Pru (3 TgCatBj1 (p < 0.001) and 94.53% (p < 0.001) of their initial weight, respectively, and only one mice died with 1 × 103 TgCatBj1. In contrast, vaccinated mice challenged with the four genotypes of tachyzoites showed normal weight gain (p > 0.05) and the same mental status as blank control mice. Taken together, PruΔrop38 may provide efficient protective efficacy against varied genotypes of RH, Pru, VEG, and TgCatBj1 tachyzoites challenge in mice.To evaluate the protective efficacy against toxoplasmosis in mice, varied genotypes of tachyzoites  were used to infect mice intraperitoneally at corresponding doses. Control mice, challenged with lethal doses of 1 × 10nfection A,B. Besin . Mice were also infected with 10 Pru or 5 ME49 cysts (pathogenic dose) to assess the protective efficacy against chronic infection, and all control mice survived the observation period while their body weight dropped to a minimum of 95.17% (p < 0.001) (p < 0.001) (p > 0.05). Taken together, PruΔrop38 may also provide efficient protective efficacy in mice that survive during acute or chronic infection with Pru and ME49 cysts.We also evaluated the protective efficacy of PruΔf 20 Pru A,B or MEf 20 Pru C,D, with< 0.001) B and 94.< 0.001) D of thei3 Pru tachyzoites was 2062, 940, and 111, respectively (p < 0.001), 20 (p < 0.001), and 17 (p < 0.001), respectively. Additionally, the number of cysts in vaccinated mice challenged with 20 Pru cysts, 20 ME49 cysts, and 5 × 103 Pru tachyzoites  was 32 (p < 0.001), 24 (p < 0.001), and 25 (p < 0.001), respectively. Vaccination with PruΔrop38 has the ability to significantly reduce the brain parasite burden of the challenging type II strains and provide protection against T. gondii chronic infection.Another aspect of evaluation of the protective efficacy against chronic infection is the brain parasite burden, so we counted the number of brain cysts in mice challenged with type II strains. The average number of cysts in control mice challenged with 10 Pru cysts, 5 ME49 cysts, and 1 × 10ectively A–C. Howerop38, we finally assessed whether mice were able to resist T. gondii challenge 60 days after vaccination with PruΔrop38. When challenged with lethal doses of tachyzoites  or cysts (20 Pru or 20 ME49), control mice exhibited serious clinical signs , and all mice died at the second week  or cysts (10 Pru or 5 ME49), all control mice survived (p < 0.001), 93.16% (p < 0.001), 94.33% (p < 0.001), and 92.61% (p < 0.001) of the initial weight, respectively  and 35 (p < 0.001), and 24 (p < 0.001), respectively. In addition, the number of cysts in vaccinated mice challenged with 20 Pru cysts, 20 ME49 cysts, and 5 × 103 Pru tachyzoites  was 31 (p < 0.001), 36 (p < 0.001), and 26 (p < 0.001), respectively. Taken together, vaccination with PruΔrop38 remains able to significantly reduce the brain parasite burden of the challenging type II strains, providing long-term efficacy against acute or chronic toxoplasmosis in mice.The average number of cysts in control mice infected with 1 × 10ectively . HoweverT. gondii tissue cysts in food-producing animals and oocyst shedding in cats [T. gondii vaccines with varying degrees of protective immunity, the field of live-attenuated vaccine development has been carried out for safer vaccines for advantages in terms of protective efficacy [rop38 in mice [rop38 as an applicable vaccine candidate and assess its pathogenicity, immunogenicity and protective efficacy.Toxoplasmosis is a serious zoonotic disease that threatens human and animal health, and vaccination strategies are considered ideal to prevent infection. The objectives are to prevent congenital toxoplasmosis in both women and livestock, prevent/reduce  in cats . While vefficacy . Due to efficacy  and the  in mice , the objrop38 in vivo, besides, we also focused on clinical symptoms and body weight gain of mice after inoculation with different doses of Pru (1 × 103 and 2 × 103) or PruΔrop38  tachyzoites. No obvious signs (wasting and bow-back) or deaths were seen even when inoculated with 4 × 103 PruΔrop38, demonstrating the attenuated pathogenicity of the candidate to mice. T. gondii-specific genes were only detected in the brain from week 4 onwards, and deletion of TgROP38 significantly reduced the brain parasite burden compared to the parental Pru strain. These data indicate that PruΔrop38 conforms to a number of characteristics that make it an ideal live-attenuated vaccine. Pathological changes in organs and tissues were also observed, with some degree of enlargement in the spleen and MLN, and no obvious histological lesions observed in the remaining organ and tissue. The final vaccination dose (1 × 102 per mouse) was considered to significantly reduce pathogenicity and provide high biosafety in mice.Survival status was the primary factor determining the pathogenicity of PruΔrop38 induces strong humoral and cellular immune responses. The level of specific anti-T. gondii IgG and IgG two isotypes (IgG1 and IgG2a) revealed a significantly high level from week 3 to week 12 after vaccination, since the IgG1/IgG2a ratio was less than 1, PruΔrop38 induces a combined Th1/Th2 immune response, in which the Th1 response predominates. In contrast, IgG activated by B-cell response can regulate immune cells and complement to inhibit the infective capacity of T. gondii [As with live-attenuated vaccine candidates , immuniz. gondii .T. gondii by activating cytotoxic lymphocytes and natural killer cells, leading to protection against acute T. gondii infection [rop38 rapidly activated humoral and cellular immune responses to specifically recognize the Toxoplasma antigen.At 30 or 60 days post-vaccination, the secretion of IFN-γ, IL-12 and IL-10 in the immunized mice splenocytes significantly increased under the TSA stimulation, indicating a Th1-type cell-mediated immune response was induced predominantly. IL-12 and IFN-γ trigger multiple intracellular responses to clear nfection ,28. Howenfection . Immunogrop38 provides long-term efficacy against acute and chronic toxoplasmosis in mice. For Toxoplasma it is now apparent that, besides the three typical genotypes  described worldwide, there exist many atypical T. gondii genotypes, with a high degree of genetic diversity [rop38, with TgcatBJ1 being a ToxoDB#9 genotype strain isolated from stray cats [rop38 could provide effective protection to mice against challenge by tachyzoites of varied genotypes. However, intraperitoneal injection does not represent the natural route of infection, so Pru and ME49 cysts were orally challenged to objectively assess the protective efficacy, and PruΔrop38 could provide efficient efficacy in mice that survived during acute or chronic infection with Pru and ME49 cysts. Additionally, vaccination with PruΔrop38 was able to significantly reduce the brain parasite burden of the challenging type II strains in chronic infections. Taken together, PruΔrop38 is a potential live-attenuated vaccine candidate. However, the current study is limited to the mouse model, so more studies are needed to evaluate the effectiveness and safety of the vaccine candidate for food-producing animals or cats.PruΔiversity ,31. As aray cats . Severalray cats , TgC7 [3ray cats , and C77ray cats , as wellray cats . PruΔroprop38 was significantly less pathogenic in mice compared to the parental Pru strain. Vaccination with 1 × 102 PruΔrop38 triggered high levels of serum IgG, IgG2a, and IgG1 from week 3 to week 12, and a significant increase in IFN-γ, IL-12, and IL-10 in splenocyte suspensions at 30 or 60 days post-immunization. PruΔrop38 provides long-term efficacy against acute and chronic toxoplasmosis in mice and significantly reduces the brain parasite burden of the challenging T. gondii type II strains.In summary, virulence, dynamic distribution, and brain parasite burden assays indicated that PruΔ


Given the available clinical evidence through the literature search when the Japanese Clinical Practice Guidelines for Management of Sepsis and Septic Shock 2020 was being created, we suggested administering vitamin C to such patients. Recently, several randomized control trials have been published, some of which suggested the harmful effect of vitamin C in terms of mortality or persistent organ dysfunction. Therefore, we performed updated systematic reviews and meta-analyses. Accordingly, we revised our recommendation as “We suggest against administering vitamin C to septic patients (GRADE 2D: certainty of evidence = “very low”).”The online version contains supplementary material available at 10.1186/s40560-022-00641-4. To the editor,Given the available clinical evidence through the literature search when the Japanese Clinical Practice Guidelines for Management of Sepsis and Septic Shock 2020 (J-SSCG2020) was being created, J-SSCG2020 , 2 suggeRecently, Lamontagne et al. conducted a large multi-center RCT, including 872 septic patients, who required vasopressors, to evaluate the effect of high-dose vitamin C . This RCn = 3534). Thereby, the desirable anticipated effect was thought to be “trivial”. The estimated values of the effects on mortality were as follows: long-term mortality, namely more than 60 days, yielded a risk difference (RD) of 42 more per 1000 (95% CI: 8 more–83 more) , 28 or 30 days mortality yielded an RD of 34 fewer per 1000 (95% CI: 70 fewer–12 more) , in-hospital mortality yielded an RD of 20 fewer per 1000 (95% CI: 80 fewer–53 more) . Of these three mortalities, long-term mortality was chosen as the effect on mortality since we predetermined that the highest certainty of evidence was adopted. Subsequently, the estimated values of the other undesirable anticipated effects were as follows: the length of hospital stay yielded an MD of 0.24 days longer (95% CI: 0.97 days shorter–1.45 days longer) , and acute kidney injury yielded an RD of 6 more per 1000 (95% CI: 20 fewer–38 more) . Thereby, the undesirable anticipated effects were “moderate”. Thus, we presumed that administering vitamin C was inferior to the placebo or control. Judgment of values, acceptability, and feasibility were not changed, namely, “probably no important uncertainty or variability”, “probably yes”, and “probably yes”, respectively.In our updated meta-analysis, the estimated value of the desirable anticipated effect was as follows: the length of ICU stay yielded a mean difference (MD) of 0.25 days shorter : 0.72 days shorter–0.22 days longer) .”Additional file 1. PRISMA flow diagram, risk of bias summary, Forrest plot, funnel plot, and evidence to decision table.