The as-synthesized Cr3+ cation–doped perovskite nanocrystals displayed ∼45.45% decrease in the (100) phase intensity with an enhanced Bragg angle (2θ) of ∼15.01° compared to ∼14.92° of pristine perovskites while retaining their cubic  crystalline phase of pristine perovskites. During synthesis, an eco-friendly solvent, ethanol, was utilized as an antisolvent to grow nanometer-sized rod-like crystals. However, Cr3+ cation-doped perovskite nanocrystals display a reduced crystallinity of ∼67% compared to pristine counterpart with ∼75% crystallinity with an improved contact angle of ∼72° against water in thin films. Besides, as-grown perovskite nanocrystals produced crystallite size of ∼48 nm and a full-width-at-half-maximum (FWHM) of ∼0.19° with an enhanced lattice-strain of ∼4.52 × 10−4 with a dislocation-density of ∼4.24 × 1014 lines per m2 compared to pristine perovskite nanocrystals, as extracted from the Williamson–Hall plots. The as-obtained stable perovskite materials might be promising light-harvesting candidates for optoelectronic applications in the future.In this work, we developed a facile and environmentally friendly synthesis strategy for large-scale preparation of Cr-doped hybrid organometallic halide perovskite nanocrystals. In the experiment, methylammonium lead bromide, CH We developed a facile and eco-friendly synthesis strategy for large-scale preparation of undoped and Cr-doped hybrid organometallic halide perovskite nanocrystals. Due to diverse probability in shape and size, the nanomaterials might control their crystal phases during crystal growth based on reaction conditions such as solvents, reaction time, and temperature.18,19 Compared to metal oxides, PVNCs have been reported in limited studies to progress via controlled growth based on their phases, facets, sizes, and shapes, but no optimized technique has been available to date.20,21 In spite of enormous approaches, an optimized synthesis procedure is still a big challenge for researchers in the precise control of PVNCs with phase calibration and crystal geometry.22–24 Basically, the crystal-growth reactions of PVNCs showed a short time to make its reaction-mechanism really tough to understand with a controlled facet, design and shape till now.To fulfil the growing energy demand, a bulk architecture of semiconducting quantum dots (SQDs) with organic materials demonstrates various structural geometries such as zero-, one- and two-dimensional nanomaterials for light-emitting diodes (LEDs), photodetectors (PDs), photovoltaics (PVs), and sensor applications.25,26 Furthermore, the defect and strain of perovskite materials have been significantly relieved by various encapsulation techniques to suppress ion-migration or high density of defects due to solution-processed fabrication at low temperatures.27–29 In addition, semiconducting perovskite materials have been presenting many tunable properties using defect-engineering via chemical-doping, which introduces novel functional sites to control their physical and optoelectronic properties.28–30 During crystal growth, perovskite materials have a tunable lattice-strain due to chemical doping, solvent washing, thermal treatment and anti-solvent treatment for crystallization, which lead to improve fluorescence emission properties, charge mobility and electrical conductance.31,32 Furthermore, the ionic migration and accumulation of reactive species might create more defects in the crystal structure as well as adjacent functional layers, resulting in performance degradation of optoelectronic devices.33,34 Interestingly, the tunable energy band-gap of PVNCs makes them light-harvesting materials for efficient optical devices due to broad absorption in visible region based on different compositions of PVNCs.35,36 In continuation, the partial replacement of organic or inorganic components might affect the crystal distortion to reduce growth rate of crystals during crystallization.37 Herein, the antisolvent crystallization technique, a facile, and cost-effective approach, was adopted to control nucleation of crystals resulting in diverse crystal shapes and morphological phases. The partial replacement of Pb2+ cations (ionic radius of 119 pm) was observed by small-sized Cr3+ cations (ionic radius of 62 pm) with a high spin (t32ge0g) electronic configuration due to partially filled orbitals in an octahedral environment. Recently, highly fluorescent dual metal (Cr3+ and Mn2+) cation doped cesium-based perovskite nanomaterial, (Cr3+:CsPbCl3:Mn2+) as a quantum dot (QD) has been reported as light-harvesting material with ultraviolet absorption, longer Stokes shift, and excellent stability for solar cell applications.38 On Cr3+ cation (∼7.5 mol percent) doping, an excellent photoluminescence quantum yield (PL-QY) of ∼97% was achieved due to the synergistic effect of high energy transfer and passivation effect of Cr3+ cations, resulting in a superior performance of ∼22.35%.On a great concern to stability, the chemical reactivity of PVNCs with air and moisture is a major hurdle for their long-term and commercial applications due to their decomposition features upon heat and light irradiation during harsh environmental conditions.3+ cations as dopants were applied to grow metal-doped hybrid organic-inorganic metal halide perovskite nanocrystals (HOME PVNCs) with tuned optical, physical, structural, and crystal lattice properties. In the present paper, a cost-effective eco-friendly synthesis strategy has been used to grow HOME PVNCs as light-harvesting materials with or without metal ion doping which improve their thermal stability and tune strain properties for diverse applications under ambient conditions. It was revealed that smooth insertion of small-sized Cr3+ cations partially replaces Pb2+ cations of perovskite nanocrystals to boost their optical properties estimated in terms of Tauc plots and Urbach plots with high thermal-stability along with detainment of cubic geometry. Specifically, perovskite nanocrystals might suppress surface grain boundaries and recombination losses via crystal defects after metal cation doping. Hence, Cr3+ cation-doping might produce novel doped-organometallic halide perovskite rod-like nanocrystals with cubic-phase retention with slightly modified crystal lattice parameters. Owing to such excellent properties, the as-obtained doped nanocrystals might be largely applicable in various optoelectronic fields in the future.Herein, Crii) acetate (Pb(OCH3COO)2, 99.99%), hydrogen bromide , dimethylformamide , ethanol (75%), anhydrous toluene (99.8%), and n-hexane (99.5%) were purchased from Sigma-Aldrich Company, Republic of Korea.For all the experiments, the chemicals and reagents were obtained from commercial sources and used without further purification. Lead(λ = 1.5406 Å) for structure and bonding phases of perovskite materials. For perovskite films, UV-visible spectral analysis was measured (200−800 nm wavelength) on pristine ITO glass substrates using an Agilent Technologies instrument (Carey 5000 UV-vis-NIR spectrophotometer) equipped with a light source of deuterium and Tungsten lamps. In addition, field-effect scanning electron microscopic (FESEM) analysis was performed with perovskite crystals using a Hitachi instrument (SU 8230) in terms of high- and low-resolution images. Energy-dispersive X-ray spectroscopic (EDX) analysis was performed to find out doping weight-percentage (wt%) of metal components and their homogeneity in nanocrystals. While a Raman spectrometer (at λex 532 nm) was used to investigate various bands using a Nicolet Almeca XRA instrument of Thermo Fisher Scientific Company Ltd. The thermal properties of perovskite nanocrystals were investigated using a thermogravimetric analysis instrument  in 25–600 °C temperature range. A differential scanning calorimetry  instrument was applied to find various phase changes of perovskite materials before and after metal doping. X-ray photoelectron spectroscopic  instrument display various binding interactions with an Al Kα X-ray wavelength of ∼1486.6 eV (monochromatic) under ultrahigh vacuum for pristine and doped PVNCs. The crystalline phases and intermolecular distance were evaluated using a transmission electron microscope  in image and FFT patterns analysis for PVNCs.X-ray diffraction (XRD) analysis was performed using an X-ray diffractometer  having a wavelength of Cu Kα radiation (3COO)2·3H2O) was utilized in place of its bromide counterpart. However, a strong complexing agent, trihydrate lead salt, presents a complicated chemistry responsible for perovskite conversion in presence of water.39,40 In summary, the stoichiometric PVNCs formed as follows:For cost-effective lead precursors, lead acetate trihydrate 2 and MABr in 1 : 1 ratio, and appropriate amounts of Cr3+ cations (formulated as CH3NH3Crx)(Pbx)(1−Br3) were mixed with stirring to form a brown slurry. Different amounts  of hydrated chromium salt (Cr(iii)Cl3·6H2O) were added for experimental comparisons, and Pb2+ cations were partially replaced with Cr3+ cations. Subsequently, a HBr solution  was dropwise added to above-mentioned solution with stirring at room temperature. Then, an antisolvent, ethanol (∼10 ml), was added and stirred for 1 h under ambient conditions. Thus obtained doped perovskite solution was heated at ∼80 °C for 12 h to develop Cr-doped PVNCs as orange crystals. The as-obtained Cr3+-doped PVNCs were further washed several times with ethanol and dried at ∼80 °C for 6 h to evaporate excess solvent to get ultrapure Cr-doped PVNCs (The hybrid perovskite precursor solution was prepared by mixing Pb (OAc)ed PVNCs .N,N-dimethylformamide (DMF) solvent for pristine and doped sample nanocrystals. The precursor perovskite solutions were applied for thin film deposition by a drop-casting method for doped MAPbBr3 in DMF solutions in ambient atmosphere. The as-obtained thin films were treated at ∼100 °C for 10 min to get orange perovskite thin films in ambient air.The indium tin oxide (ITO) glass substrates were treated for sonication with detergent, acetone, water and isopropyl alcohol for up to ∼10 min each. The as-obtained perovskite materials were taken in appropriate amounts to make up precursor solutions in an anhydrous 3, X = Br) nanocrystals display excellent thermal and air stability due to their high thermal capacity with no phase transitions up to ∼200 °C.62 The degradation of perovskite is based on hydration of perovskite crystals to form mono-hydrate in a reversible reaction under humid conditions.41 Thus, PbBr2 might precipitate as a solid to rule out any possibility of re-formation of perovskite nanocrystals. In addition, water is a strong polar solvent, and forms quite stable complexes with hybrid perovskite components.42 In TGA plots, organic and inorganic components have been decomposed and interpreted for perovskite precursors of hybrid CH3NH3PbBr3 nanocrystals as follows:In hybrid perovskite-based research, the anti-solvent crystallization technique was applied widely due to their promising, rapid and excellent synthetic style to achieve large-scale production for wide-area fabrication devices. Methylammonium (MA)-based perovskite  due to decomposition of water molecules. In addition, the decomposition threshold of pristine perovskite nanocrystals at ∼262 °C (∼3% weight loss) is well suitable for perovskite nanocrystals for stable device applications. Moreover, the profile of pristine perovskite nanocrystals involves two major degradation steps including first degradation at ∼291 °C with a remaining weight of ∼88.58% from its original weight. In particular, pristine perovskite explores first degradation step of ∼11.42% weight loss corresponding to organic component methylammonium bromide  of as-grown pristine nanocrystals.42–44 In continuation, the major second degradation starts at ∼447 °C, which is attributed to degradation of PbBr2 (M2PbBr = 367.05 g mol−1) molecules, indicating that amine group is strongly bonded into hybrid perovskite crystal matrix. However, the weight-fraction (α) of pristine perovskite was also estimated to be about ∼16.8% at ∼300 °C temperature followed by major thermal degradation of ∼61.21% at ∼576 °C, which is attributed to melting of the PbBr2 precursor of perovskite nanocrystals.45 The precursor, lead acetate, itself might be removed at soft-bake low temperatures despite high risk of side-products in perovskite layer due to the high polarity. Thus, lead acetate might be converted into lead hydroxide or hydrate, PbO·H2O, to reduce lead quantity for perovskite conversion and retard the conversion-speed of precursors, as observed in EDX analysis.44,45 From Cr3+-doped perovskite nanocrystals, two small degradation steps have been observed at ∼260 °C with ∼4.96% mass-loss and ∼120 °C with ∼4.82% mass-loss, which are related to strong Cr bonding in perovskites and also found as small minima in percent derivative  with a high mass destruction of ∼11.42% which indicate their high thermal stability under ambient conditions.46 In addition, major thermal degradation of ∼74.82% weight loss might be related to melting of the PbBr2 precursor with a weight residue ∼15.41% of Cr3+-doped perovskite nanocrystals. From TGA plots, Cr-doped perovskite nanocrystals provide a low weight fraction (α) of ∼5.7% at ∼300 °C temperature due to excellent doping, revealing high thermal stability compared to their pristine counterparts. Hence, the decomposition of perovskite nanocrystals is very low up to ∼282 °C (∼4% weight loss) and ∼363 °C (∼8% weight loss), which display further better thermal stability after Cr3+ doping for optoelectronic applications. The as-doped PVNCs provide huge incentives as potential candidates for long lifetime, high thermal stability, and optical and electronic applications.47From the TGA spectrum  of pristrivative  curves dvia heating and cooling scans of doped and pristine perovskite materials. The glass transition (Tg) temperature of perovskite nanocrystals was reduced from ∼89 °C to ∼47 °C after Cr ion insertion. Furthermore, the melting transition (Tm) temperature of doped perovskites might reduce after metal doping compared to their pristine perovskite materials resulting in conversion of phases. For pristine perovskites, MAPbBr3 nanocrystals, heating scan shows a small crystalline peak at ∼300 °C with a strong melting transition peak at ∼321 °C. Upon cooling, a glass transition peak was observed at ∼89 °C along with three crystalline peaks  the is reaction model function. The shape and structural stability of perovskite crystals might govern through composition and stoichiometry of precursors. The k value was evaluated using the Arrhenius equation:E, A, R, and T are the activation energy (kJ mol−1), pre-exponential factor (min−1), standard gas constant (∼8.314 J K mol−1), and absolute temperature in Kelvin, respectively. Thus, the final equation is as follows:Under standard temperature conditions, the thermal decomposition kinetics of perovskite nanocrystals in a facile and simple approach has been established to investigate gradual mass-loss with temperature. Thus, activation energy values have been evaluated from TGA plots in model-free methods. The kinetic studies follow the rate equation as:For the dynamic TGA process, the heating rate is expressed as follows:Hence, the resulting equation can be found as follows:σT) against 1/T plots  images , (110), (200), (210) (211), (220) (221), (222) and (400) space groups, respectively. The XRD peak values are well matched with the spectrum of high-crystalline pristine cubic (ICSD card no. 00-069-1350) perovskite phase.55 The XRD peaks at ∼14.92° and ∼30.12° of the (100) and (200) space groups  were applied for doping perovskites, and XRD plots  along with a slight increment in the Bragg angle values. After efficient metal doping, the nucleation growth of perovskite crystals might control crystal assembly , peak broadening at FWHM value, Bragg angle, and X-ray wavelength of Cu kα radiation, respectively. Additionally, crystallinity  has been done by relatively small-sized Cr3+ cations (ionic radius 62 pm) resulting in substantial crystal-size contraction from ∼193 nm to ∼48 nm of rod-like nanocrystals after doping at low temperatures.57Furthermore, Cr3+-doped perovskite nanocrystals. Hence, the crystal imperfection and distortion in terms of crystal lattice strain (ε) were calculated using the following relation:The hybrid perovskite nanocrystals were analyzed by an integral method using W–H plots  to deterd) of XRD peaks induces peak broadening of high-crystalline materials.The line broadening of XRD peaks might be affected by size and lattice-strain of perovskite nanocrystals. The lowering of intermolecular distance  of crystalline materials can be obtained from the Williamson and Smallman relation:Furthermore, Crδ value of crystalline materials depends on crystallite size of perovskite nanocrystals. Thus pristine perovskite nanocrystals exhibit a dislocation density of ∼2.675 × 1013 lines per m−2. The size of pristine nanocrystal (∼193 nm) significantly reduced up to ∼48 nm after doping with Cr3+ cations. Hence, the δ value has been drastically improved up to ∼4.24 × 1014 lines per m−2 for Cr-doped perovskite nanocrystals. Thus, various intensity percentages have been depicted by pi-charts  and (200) with ∼31.06% and ∼19.87% have been reduced to ∼17.37% and ∼18.71% after doping. In addition, the (220) crystal phase has explored an increment of ∼23.83% in Cr3+-doped nanocrystals from ∼18.12% of pristine perovskite nanocrystals.59The i-charts  for prisvia doping-engineering. Henceforth, efficient incorporation of metal cations or replacement of any metal cations has provided very unique, useful and interesting features into doped nanomaterials.60 Owing to small ionic radius, various metal ions provide a great probability to insert into hybrid perovskite lattice in a relatively simple and facile approach, which might significantly improves their stability. However, crystal defect chemistry of perovskite nanomaterials has been drawing tremendous attention due to their robust, facile, cost-effective ways of handling doping techniques. Of course, partial replacement of Pb2+ cations might reduce its toxicity, resulting in production of eco-friendly perovskite nanocrystalline materials. The oxidation state of dopant chromium cation displays a stable electronic configuration in Cr3+ high-spin  state  octahedral field, which provides a low-cost, green and eco-friendly synthesis method at low temperatures.61In a crystal lattice, size of foreign materials is a decisive factor for its insertion 3 crystal lattices, the atomic positions might change in cubic crystal structure due to crystal phase conversion. From XRD spectra and EDX analysis, it can be revealed that transition metal  cations inserted into perovskite crystal lattice partially replace Pb2+ cations. The theoretical estimation of Goldschmidt's tolerance factor (t) and octahedral factor (μ) generally applied for a benchmark of stability, phase and geometry of perovskite-type nanomaterials. In addition, t determines the degree of crystal deformation after metal doping to induce structural stability and might be calculated as follows:− anion belong to RA, RB, and RX, respectively. Thus, effective ionic radii (reffective) of metal-doped perovskites were obtained after lead substitution by Cr3+ cations via chemical doping. These ionic radii are taken in a six-coordinate halide system, which translated directly as the bond-length of octahedron of 3D perovskite nanomaterials. Thus obtained tolerance factor (t) of nanomaterials should be 0.81 to 1.10 range to get ideal cubic crystal structure of perovskite-type materials.62 Thus, metal cation substitution of perovskite materials might enhance their structural stability of doped perovskite nanocrystal effective doping. Meanwhile, the B–X bond length plays a crucial role in band gap energy, which decreases slightly after Cr3+ cation doping. Hence, effective radii of Cr3+ cation-doped perovskite nanocrystals formulated as CH3NH3Cr0.12Pb0.88Br3 which might be precisely calculated as follows:RA(effective) and RB(effective), respectively. Then, x is doping percentage of Cr3+ cations, and RB′ and RB′′ belong to ionic radius of RCr3+ cations and Pb2+ cations, respectively. Hence, effective t of doped perovskite nanocrystals might be calculated as follows:In doped MAPbBrt value follows stability in 0.8 < t < 1 range related to cubic crystal phase after metal-doping with no change in crystal phase of perovskite materials. Hence, trivalent Cr3+ cations might insert into perovskite crystals due to their small size with high-polarity Cr–Br bonds compared to Pb–Br bonds, resulting in high stability of doped perovskite nanocrystals. Hence, the general formula of Cr-based perovskite nanocrystals is postulated as CH3NH3CrxPbx1−Br3 to evaluate the t value  energy can be calculated by using following equation:α, hν, and A are absorption coefficient, energy, and constant, respectively. Eg can be evaluated from following relation:The ITO-based thin films of pristine and doped CHNH3PbBr3  revealedNH3PbBr3  of UV sp3NH3PbBr3 by a color change of thin film from yellow to orange color of perovskites after annealing at 100 °C for 10 min. From UV plots  using the following equation:α0 is a constant. EU slightly depends on the localized states of low crystalline or amorphous materials and temperature. The logarithm of above-mentioned relation can be modified as follows:In thin films, the Urbach energy designates disorder of phonon states, which is governed by structural disorder and stoichiometric defects at surface. The Urbach energy has been observed as absorption of photons in a large number of amorphous materials, which is found below absorption band edge of compounds. The Urbach energy  is consiergy EU,  and estiU value  energy with some traces of lead metallic (Pb0) peaks. In addition, the peak intensity and binding energy of respective lead metal peaks were significantly lowered due to enhanced perovskite conversion in Cr-doped perovskite nanocrystals with no traces of lead oxide components.69 However, a peak binding energy of Pb+2 cations was lowered at ∼137.01 eV and ∼141.88 eV for Pb 4f7/2 and Pb 4f5/2, respectively, to produce enhanced ΔE of ∼4.88 eV after insertion of Cr+2 cations into perovskite nanocrystals. Thus, the binding energy has been reduced by ∼0.60 eV and ∼0.41 eV for Pb 4f7/2 and Pb 4f5/2 peaks, respectively after Cr3+ doping. It was demonstrated that PbBr2 formation after HBr insertion and its conversion into hybrid perovskite, CH3NH3PbBr3, occurred in a molecular exchange reaction between CH3NH3Br and lead acetate precursor with addition of ethanol as an antisolvent. In pristine perovskites, two Br 3d peaks  bond of nanocrystals. Along with this, the emergence of a low intensity peak at ∼401.67 eV corresponding to C–NH3 bond reveals a weak hydrogen-bonding possibility in MA+ (CH3NH3+) cations of perovskite nanocrystals. The variation in metal concentration histogram profiles  at ∼1593 cm−1 in crystal MAPbBr3 are quite weak and slightly red-shifted for Cr-doped perovskite nanocrystals  due to small size of cations used for insertion. Thus obtained Cr3+-doped PVNCs have negligible effect on the crystalline cubic phase (ICSD No. 00-069-1350) of pristine perovskite with an enhanced t ∼0.95 with μ of ∼0.58 for doped perovskite nanomaterials. The Cr3+ doped nanocrystal-based thin films display high hydrophobicity against water with high contact angle of ∼72° compared to ∼63° for their pristine counterpart. Owing to high thermal and humid stability, such PVNCs might be promising light-harvesting materials for optoelectronic applications in future.In summary, we have developed stable CrThe authors declare no competing financial interests.NA-004-D2NA00053A-s001


We hypothesize that such improvement would have its origin in the higher quality of S1 seeds resulting from plants whose seeds were primed. In this work, we characterised biochemically the whole-wheat flour of unprimed S1 offspring whose S0 seeds were hydro- and nutriprimed with Fe and/or Zn and compared it to the offspring of untreated S0 seeds (control). We identified and quantified 16 free amino acids and five soluble sugars per offspring using high-performance liquid chromatography and the Association of Official Analytical Chemists (AOAC) methods. The most abundant amino acids were glutamic acid and glutamine, proline, and glycine, presenting their highest contents in the offspring of seeds nutriprimed with 8 ppm Zn (0.351 mmol∙g−1), 8 ppm Fe + 8 ppm Zn (0.199 mmol∙g−1), and (0.135 mmol∙g−1), respectively. The highest contents of glucose (1.91 mg∙g−1 sample), ash , and crude protein (209.70 g∙kg−1 DM) were presented by the offspring resulting from 4 ppm Fe + 4 ppm Zn, 8 ppm Zn, and 8 ppm Fe + 8 ppm Zn, respectively. The highest total starch content (630.10 g∙kg−1 DM) was detected in the offspring of seeds soaked in 8 ppm Fe. The nutritional value of the flour of the S1 offspring resulting from nutripriming was significantly higher than the control. Overall, the novelty of our research is that seed priming can improve the quality of the wheat grain and flour, at least till the first offspring, without the need to repeat the presowing treatment. Beyond the study of subsequent generations, the unravelling of transgenerational mechanisms underlying the biochemical improvement of the offspring is approached.Seed hydropriming or nutripriming has been used for wheat biofortification. Previously, the untreated S1 offspring of bread wheat S0 seeds hydro- and nutriprimed with FeSO Triticum aestivum L.) is essential worldwide due to its nutritional and economic value, supplying one-third of the world’s population with more than half of their daily calorie intake [Bread wheat  and zinc (Zn) has been used to improve the germination, seedling emergence, vegetative growth, development, stress response, and nutritional quality of the grain ,7,8,9,10Seed priming with micronutrients (nutripriming) has been used to overcome micronutrient deficiency in crops ,17,18,19Our previous cytogenetic works revealed a certain degree of cytotoxicity in bread wheat seeds nutriprimed with 4 and/or 8 ppm of Fe and/or Zn  but alsoMost of the S1 offspring showed higher concentrations of each amino acid than the control .p < 0.05) among the S1 offspring  was found in the S1 offspring of S0 seeds primed with 4 ppm Zn  among the S1 offspring  lower concentration of sucrose, fructose, raffinose, and maltose than the control  among the S1 offspring , and total starch showed significant differences  the meristematic root cells showed a higher degree of cytotoxicity, and (ii) the mature S0 plants presented lower yield-related parameters; however, (iii) their whole-wheat-flour samples had a higher amount of total soluble protein content ,24. In aThe biochemical characterisation focused on the identification and/or quantification of free amino acids, soluble sugars, ash, crude protein (CP), and starch.We identified 16 free amino acids in the whole-wheat-flour samples of the S1 offspring. Eight of them  are essential amino acids  and are required in adequate amounts to the human diet ,33. The Beyond the improvement of the quality of whole-wheat flour, the amino acids also contribute to the proper development of the plant, given their crucial roles in various biological processes ,13. AminThe profile of the free amino acids present in wheat flour differs among varieties, but the high content of glutamic acid and/or glutamine is common ,39,40. A−1 dry matter), thus placing “Jordão” in the classification between MPC and HPC [In our previous study, the S0 seeds primed with 4 ppm Fe + 4 ppm Zn, 8 ppm Fe + 8 ppm Zn, and 8 ppm Zn, showed a higher content of total soluble protein than the hydroprimed ones used as a control in this study . Similar and HPC . The rem and HPC  and its  and HPC ,31.The content of total soluble sugars decreased in all S1 offspring except in the control. This decrease significantly differed between the control and the following S1 offspring: hydropriming, Zn priming, and 8 ppm Fe + 8 ppm Zn. Among the five sugars identified, only glucose increased relative to the control. This increase was significant in the S1 offspring of 4 ppm Fe + 4 ppm Zn, 8 ppm Zn, and 8 ppm Fe + 8 ppm Zn. Such an increase in glucose might be related to the stress responses of the plant during its life cycle, as previously reported for other wheat cultivars . Further2 net assimilation, maximizes the photochemical efficiency of photosystem II, and α-amylase activity under abiotic stress [The average sucrose, fructose, raffinose, and maltose concentrations in the S1 offspring was lower than in the control. Seed priming improves the seedling dry weight, leaf area, and leaf COc stress . TherefoThe lowest amounts of total starch, accompanied by the high content of glucose detected in the S1 offspring of S0 seeds hydroprimed and nutriprimed with Fe + Zn, reflected the enhanced activity of the enzymes responsible for starch breakdown, such as α- amylase, β-amylase, or α-glucosidase, also explaining their high CP content.In addition to the reduced amount of total sugars, we verified low antioxidant capacity (data not shown) and increased ash and CP concentrations. Our results are in concordance with previous studies, which reported that the increase in minerals (including Fe and Zn) in wheat flour could be accompanied by a significant decrease in the antioxidant capacity and carbohydrate content and by the augmentation of protein, fibre, ash, and fat concentrations ,63. FurtAll chemicals and reagents were of analytical grade unless specified otherwise. The amino acid standards: L-alanine (batch no. 66H0292), L-arginine (batch no. 16H1490), L-aspartic acid (batch no. 57H11401), glycine (batch no. 36H2503), L-histidine (batch no. 27H04061), L-isoleucine (batch no. 45H0145), L-leucine (batch no. 55H0786), L-norvaline (batch no. 105H1127), L-phenylalanine (batch no. 47H1485), L-serine (batch no. 57H0508), L-threonine (batch no. 87H0098), L-tyrosine (batch no. 57H11041), L-tryptophan (batch no. 27H12431), and L-valine (batch no. 35H0531), were purchased from Sigma-Aldrich . The amino acid standard L-glutamic acid (batch no. K14508091) was acquired from Merck , and the L-lysine (batch no. 13 ID 0627/0) was from Extrasynthese .The 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate  was acquired from Santa Cruz Biotechnology, Inc. . Acetonitrile  was purchased from Honeywell . Sodium hydroxide  and hydrochloric acid  were obtained from Panreac .Calcium disodium ethylene diamine tetraacetic acid (EDTA batch no. 11466302) was acquired from Scharlau .2B4O7, batch no. 129H0052), triethylamine , and phosphoric acid (batch no. SZBF0350V), were purchased from Sigma-Aldrich .Sodium acetate anhydrous (batch no. SZBF2210V), sodium tetraborate  variety “Jordão” that constitute the offspring of S0 seeds that were kindly provided by José Coutinho .4.7H2O, batch no. 20K044109, VWR International, Leuven, Belgium), or zinc sulfate heptahydrate , as described by [The S0 seeds were previously hydroprimed (soaking in distilled water), and nutriprimed with aqueous solutions of 4 ppm or 8 ppm of iron (II) sulfate heptahydrate . Per S1 offspring, we used three whole-wheat-flour samples. Therefore, the 24 whole-wheat-flour samples (8 treatments × 3 replicates) that were biochemically analysed corresponded to S1 seeds of 12 different plants per offspring.−1 HCl and tightly sealed with a cap. Hydrolysis was carried out at 110 °C for 24 h, after which samples were left to cool at room temperature (RT) and adjusted to pH 2.0 with NaOH. To each sample, 1 mL of 5 mmol∙L−1 of the internal standard L-norvaline solution was added and completed with distilled water till reaching a final volume of 5 mL in a volumetric flask. An aliquot of 1 mL was then filtered through a 0.22 µm filter and stored at 4 °C until initiating the analysis. For tryptophan and tyrosine, 0.025 g of each sample was added to 5 mL of 5 mol∙L−1 NaOH, and the hydrolysis occurred at 120 °C for 12 h. The samples were left to cool at RT, the pH was adjusted to 2.0 with HCl, and 100 µL of tramadol hydrochloride at 500 µg∙mL−1 was added. These solutions were completed with distilled water until a final volume of 50 mL within a volumetric flask. An aliquot of 500 µL was filtered through a 0.22 µm filter into an HPLC vial and stored at 4 °C until initiating the analysis. Processed samples and calibration curve standards were prepared according to the pre-column derivatization procedure using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) [−1 of sodium borate and 5 mmol∙L−1 of calcium disodium EDTA, pH 8.8) and 10 µL of AQC (3 mg.ml−1 in acetonitrile) were added and immediately mixed for 30 s. Vials were then tightly capped and stored at 50 °C for 10 min, followed by placement in the autosampler system maintained at 10 °C. For tyrosine and tryptophan, no derivatization procedure was necessary, and processed samples were directly injected into the HPLC system.To determine the free amino acid concentrations, except for tryptophan and tyrosine, 0.025 g of each flour sample was added to 5 mL of 6 mol∙Lte (AQC) , with sl−1 of sodium acetate, 17 mmol∙L−1 of triethylamine, 1 mmol∙L−1 of EDTA in water, pH 4.95 (phase A), acetonitrile/distilled water  (phase B) and water (phase C). The program started at 100% phase A, increasing to 33% phase B and 7% phase C, for 40 min; followed by an increase to 40% phase B and reduction of phase C to 0%, for 8 min; and finally increasing to 100% phase B in 0.5 min, and maintained for 5.5 min. The column was re-equilibrated for 10 min between injections. Fluorescence detection occurred at excitation 250 nm and emission 395 nm. For tyrosine and tryptophan, a gradient program consisting of 50 mmol∙L−1 NaH2PO4 (phase A) and acetonitrile (phase B) was used. The program started at 5% phase B, increasing to 60% phase B for 8 min, and maintained for 1 min. The program then returned to the initial conditions for 0.5 min, which were kept for another 3.5 min for column re-equilibration. The injection volume was 5 µL, with the column oven set to 40 °C. Fluorescence detection was performed according to a timetable: excitation at 274 nm, emission at 304 nm; changing to excitation at 280 nm, and emission at 340 nm for 3 min; changing to excitation at 202 nm, and emission at 296 nm for the 5 min mark of the chromatographic run.The amino acid separation was carried out on an ACE 5 C 18 column . For all amino acids, except for tyrosine and tryptophan, a ternary gradient program was employed, with the mobile phases being 140 mmol∙LThe chromatographic analyses were performed on a Thermo Scientific Dionex UltiMate 3000 Series system , composed of a RS quaternary pump, a WPS-3000RS autosampler (maintained at 4 °C), a TCC-3000RS column compartment (maintained at 35 °C), and a FLD-3400RS fluorescence detector . Results were interpreted on the Chromeleon software version 7.2 . The amino acid identification was performed accordingly by comparison with authentic standards and their quantification calibration curves prepared and analysed anew for every day of analysis.−1 of barium hydroxide was added to all mobile phases. After being filtered through a 0.22 µm membrane, the mobile phases were kept under a helium atmosphere (0.3–0.5 bar) during the entire time of analysis. The column oven was kept at 28 °C, and the injection volume was 5 µL. The sugars were detected by pulsed amperometric detection (PAD) using a 6041RS amperometric cell with gold working electrode through a quadrupole waveform: +200 mV (500 ms), −2000 mV (10 ms), +600 mV (10 ms) and −100 mV (10 ms). The chromatographic analyses were carried out on a Thermo Scientific Dionex UltiMate 3000 Series system , composed of an LPG-3400RS quaternary pump, a WPS-3000TRS autosampler (maintained at 4 °C), and an ECD-3000RS electrochemical detector with an incorporated column compartment. Results were interpreted using the Chromeleon software version 7.2 .An amount of 0.100 g of each wheat flour sample was added to 6 mL of distilled water, and the extraction was performed at 80 °C for 30 min. Then, each solution was transferred to a volumetric flask, and the volume was completed to 50 mL with distilled water. An aliquot of 1 mL was filtered through a 0.22µm filter and stored at 4 °C until initiation of the analysis. The sugar separation was performed with 30 mM NaOH in a Dionex CarboPac PA200 (3 × 250 mm i.d.) analytical column . To prevent the formation of carbonates, 2 mmol∙LThe 24 lyophilised-wheat-flour samples were analysed using the official procedures described by the Association of Official Analytical Chemists  for ash p-value significance was set for probabilities lower than 5% (p < 0.05).All results are presented as mean ± standard error (S.E.) values per S1 offspring (n = 3) indicated in The results achieved from the biochemical characterisation performed on the unprimed S1 offspring corroborated our experiment’s hypothesis in the sense that a high nutritional value was achieved in the whole-wheat-flour samples of those resulting from nutripriming with Fe and/or Zn, and hydropriming, relative to the control (unprimed S1 offspring of untreated S0 seeds). Furthermore, the results were consistent with our previous determination of total soluble protein content in the nutriprimed S0 seeds.Based on our previous and present research, we recommend the use of reduced concentrations of these two essential micronutrients, Fe and Zn, to prevent cyto- and phytotoxicity and, as demonstrated here, to improve the nutritional value of the grain and whole-wheat flour in the plants resulting from seed priming and, at least, in their first offspring.In conclusion, our work evidenced the effects of the transmission of seed priming from generation S0 to the S1 offspring without repeating the treatments on the S1 seeds. Future research regarding unravelling the mechanisms underlying the transgenerational transmission of the improved nutritional value of the grain and whole-wheat flour due to seed nutripriming is envisaged.


Verbena officinalis, has been reported to exhibit sleep-promoting and antioxidant activities. This study demonstrates the effects of verbenalin on amyloid-beta (Aβ) peptide generation in Swedish mutant amyloid precursor protein (APP)-overexpressing Neuro2a cells (SweAPP/N2a) and in Alzheimer’s disease (AD) animal models. We further performed molecular biological analyses of these in vitro and in vivo models of AD. The effects of verbenalin were assessed based on the expression of factors related to Aβ peptide production using Western blotting, enzyme-linked immunosorbent assay, and immunohistochemistry (IHC). The intracellular expression and release of APP protein were both decreased by verbenalin treatment in SweAPP/N2a cells. Thus, the production of Aβ peptides was decreased. Compared to those in AD transgenic (Tg) mice, IHC revealed that verbenalin-treated animals showed decreased Aβ and tau expression levels in the hippocampus. In addition, verbenalin restored the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus of AD animal models. These findings suggest that verbenalin may decrease Aβ formation both in vitro and in vivo. Verbenalin may also help improve the pathological hallmarks of AD.Verbenalin, among the major constituents of  Alzheimer’s disease (AD) is the most common chronic neurodegenerative disease and the leading cause of dementia worldwide. It is characterized by a reduction in thinking ability and independence in daily activities ,2. HippoVerbena officinalis, is classified as an iridoid glucoside and has a wide range of biological activities, including sleep-inducing, antioxidant, and hepatoprotective properties [Verbena officinalis have been reported to decrease Aβ-induced neurotoxicity such as neurite shorting and DNA condensation in primary cortical neurons [Verbenalin, a natural product found in operties ,13,14. I neurons . In a re neurons . Further neurons .This study investigated the effects of verbenalin on Aβ peptide production in vitro and in vivo. The effect of verbenalin the expression and secretion of APP proteins and Aβ peptide generation was investigated. In addition, verbenalin has previously restored the expression of brain-derived neurotrophic factor (BDNF) in the hippocampal region of AD animal models. Recent studies have shown that serum BDNF can be used as a biomarker for AD diagnosis in clinical practice since it reflects Aβ aggregation in the brain ,18. HoweThe pathological hallmark of AD is Aβ deposition in the brain; endoproteolytic processing of APP results in either neurotoxic Aβ or secreted ectodomain APPα. First, we examined APP protein expression levels in SweAPP/N2a cells. After 24 h of treating SweAPP/N2a cells with verbenalin, immunoblotting data revealed that APP expression was downregulated in the cell lysate and supernatant . FurtherNext, we investigated how verbenalin decreases Aβ peptide generation in an AD animal model. In particular, the hippocampus is among the first brain regions to be affected by AD, and hippocampal dysfunction is thought to underpin the key features of cognitive impairment. However, measuring hippocampal volume alone is not always sufficient to detect early AD . The corAs shown in Another pathological hallmark of AD is tau expression. Tau is a phosphoprotein abundant in mature neuronal axons and regulates microtubule stability. Hyperphosphorylated tau protein in NFTs can disrupt the microtubule network, impairing neuronal function and viability . IHC staNext, we attempted to determine how verbenalin reduced Aβ and tau expression in the hippocampus of B6Cg-Tg mice. BDNF is a neurotrophin, a protein that promotes the survival, function, and development of neurons ,23. AlthIn C57BL/6J mice, the PoDG and DG showed a moderate (++) staining response; therefore, the overall immunostaining intensity was 4. B6Cg-Tg mice showed a weak (+) response in DG and PoDG, resulting in an overall immunostaining intensity of 2. Donepezil-treated B6Cg-Tg mice demonstrated a more robust moderate (++) immunostaining response in the PoDG and DG, with an overall immunostaining intensity of 4. Compared to that in B6Cg-Tg mice, verbenalin administered animals displayed weak (+) immunostaining responses in CA1 and CA3 and moderate (++) immune responses in PoDG and DG. The overall immunostaining intensity was 6 .Aβ peptides are produced via APP proteolysis by β- and γ-secretases, and newly generated Aβ is either released into the extracellular environment or remains associated with the plasma membrane . AccumulIn the present study, verbenalin was chosen among the components of various herbal extracts for its ability to inhibit Aβ production (data not shown). Verbenalin, an iridoid glucoside, was shown to reduce both the intracellular and extracellular expression of APP, which is a precursor to Aβ . Further2 at 37 °C. Verbenalin was purchased from Sigma–Aldrich . SweAPP/N2a cells were plated in wells of plates of variable size and treated with a range of verbenalin concentrations.Swedish mutant APP overexpressing Neuro2a (SweAPP/N2a) cells were provided by Prof. Jae Yoon Leem (Woosuk University). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) and Opti-MEM supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin amphotericin B, and 1% L-glutamine in 5% COg for 15 min at 4 °C to remove debris. Proteins were then separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Subsequently, the membranes were incubated with 5% skim milk for 1 h and then with an APP antibody , followed by HRP-conjugated secondary antibodies . Specific proteins were visualized using enhanced chemiluminescent HRP substrate (Millipore) and analyzed using a luminescent image analyzer, LAS3000-mini . The relative intensity of each band was measured using the Multi-Gauge V 3.0 (Fujifilm). β-Actin was used as the loading control.SweAPP/N2a cells were washed with PBS and incubated with lysis buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 30 mM sodium pyrophosphate, 5 mM glycerol-2-phosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, protease inhibitor, and phosphatase inhibitors  for 30 min on ice. The cellular lysates were centrifuged at 20,000× g for 30 min. The pellet was washed with acetone to remove acid and centrifuged again at 20,000× g but for 15 min. After removing the supernatant, the pellet was resuspended in 2X loading buffer containing 63 mM Tris (pH 6.8), 10% glycerol, 2% SDS, 0.0013% bromophenol, and 5% β-mercaptoethanol. The dissolved proteins were denatured at 100 °C for 5 min.After collecting the culture media, the supernatants were centrifuged at 2000 rpm for 5 min to remove residual cells. The supernatant was then transferred to a new microcentrifuge tube and mixed with 20% trichloroacetic acid to precipitate proteins. After incubating the samples for 30 min on ice, the mixture was next centrifuged at 20,000× SweAPP/N2a cells were placed on culture plates. After overnight incubation, the cells were serum-starved and treated with verbenalin for 24 h. The Aβ 40 and Aβ 42 levels in the cell supernatant were measured using a commercial ELISA kit , according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a multimode reader, SpectraMAX i3x .All animal care procedures were approved by the Institutional Animal Care and Use Committee (IACUC) guidelines of Woosuk University (IACUC WS2020-06). Animals were maintained in a light-controlled environment . Food and water were provided ad libitum. In the present experimental study, male B6;C3-Tg85Dbo/Mmjax  and C57BL/6J mice were randomly allocated into five groups ; Group 4: B6Cg-Tg AD mice + verbenalin (200 mg/kg); Group 5: B6Cg-Tg AD mice + donepezil (5 mg/kg)). Verbenalin and donepezil were administered once daily for 35 d via oral gavage using a stainless-steel needle. The mice were then euthanized on day 36.2O2 + 1.5% normal goat serum. After washing a second time with 0.1 M PBS, the sections were incubated with Aβ , tau  and BDNF  at 4 °C. After overnight incubation, they were washed twice again with 0.1 M PBS for 10 min and then the incubated with secondary antibodies for 1 h. IHC staining was performed using the VECTASTAIN® Elite ® ABC-HRP Kit  for 30 min. After washing, visualization was performed with an 3-3′diaminobenzidine  for 10 min. The sections were then imaged under a microscope. Slides were evaluated for relative staining intensity and distribution within the hippocampus  and the results were recorded as follows: - negative, + weak positive, ++ moderate positive, and +++ strong positive.The sectioned brain tissues were attached to glass slides and washed with 0.1 M PBS for 15 min. The sections were then incubated for 30 min with 0.1 M PBS + 1% BSA + 0.2% Triton X-100 + 0.3% Ht-test or one-way analysis of variance (ANOVA). Asterisks indicate statistically significant differences between groups: * p < 0.05, ** p < 0.01, and *** p < 0.001.Statistical analysis was performed using SigmaPlot software 14.5 . All experimental data are expressed as the mean ± SEM. Significant differences were assessed using Student’s Among the primary focuses of treating AD should be the accumulation of Aβ and tau phosphorylation in the neurodegenerative processes associated with AD. Our findings demonstrate that verbenalin treatment decreased the generation of Aβ peptides in both in vitro and in vivo AD models. Additionally, verbenalin restored the neurotrophic factor BDNF, which was reduced in the hippocampal brain region of AD animal models. Verbenalin may thus be utilized as a potential supplement or at least as an adjuvant medication to prevent and manage disorders such as AD.


Extracellular vesicles (EVs) and their cargo have been studied intensively as potential sources of biomarkers in HIV infection; however, their DNA content, particularly the mitochondrial portion (mtDNA), remains largely unexplored. It is well known that human immunodeficiency virus (HIV) infection and prolonged antiretroviral therapy (ART) lead to mitochondrial dysfunction and reduced mtDNA copy in cells and tissues. Moreover, mtDNA is a well-known damage-associated molecular pattern molecule that could potentially contribute to increased immune activation, oxidative stress, and inflammatory response. We investigated the mtDNA content of large and small plasma EVs in persons living with HIV (PLWH) and its implications for viral replication, ART use, and immune status. Venous blood was collected from 196 PLWH, ART-treated or ART-naïve , and from 53 HIV-negative persons, all recruited at five HIV testing or treatment centers in Burkina Faso. Large and small plasma EVs were purified and counted, and mtDNA level was measured by RT-qPCR. Regardless of HIV status, mtDNA was more abundant in large than small EVs. It was more abundant in EVs of viremic than aviremic and control participants and tended to be more abundant in participants treated with Tenofovir compared with Zidovudine. When ART treatment was longer than six months and viremia was undetectable, no variation in EV mtDNA content versus CD4 and CD8 count or CD4/CD8 ratio was observed. However, mtDNA in large and small EVs decreased with years of HIV infection and ART. Our results highlight the impact of viral replication and ART on large and small EVs’ mtDNA content. The mechanisms underlying the differential incorporation of mtDNA into EVs and their effects on the surrounding cells warrant further investigation. Analysis of extracellular vesicles (EVs) is emerging as a diagnostic tool for tailoring therapeutic strategies for chronic diseases. Based on their biogenesis, these membranous secretions are classified as exosomes, microvesicles, or apoptotic bodies ,2. They Mitochondria are specialized organelles that play a major role in energy production for cell processes . They haAccumulating evidence suggests that mitochondria in persons living with HIV (PLWH) exhibit a dysfunctional state marked by altered mitochondrial membrane potential, reactive oxygen species accumulation, decreased adenosine triphosphate generation, loss of mass, and depletion of mtDNA . DecreasMitochondrial DNA can be liberated by active, stressed, apoptotic, or necrotic cells and circulate as cell-free DNA . MitochoTo investigate the possible relationship between EV mtDNA content and the health of PLWH, we purified the large and small EVs fractions in plasma of HIV-infected and uninfected participants, as we have published previously ,8,38, ann = 249) of 309 ± 155 nm for large EVs and 130 ± 39 nm for small ones (p < 0.0001). As observed by previous authors, the large EVs were more susceptible to lysis by detergents than small EVs  had no significant differential effect on mtDNA in large or small EVs C. SignifLevels of mtDNA in persons living with HIV reportedly vary with CD4 and immune activation markers ,25,26. IThe amount of mtDNA in EVs appears to differ among the categories of participants in this study . It was The group of MSM contained mostly Tenofovir-treated younger males with fewer years of infection and ART treatment . RefocusBased on our analyses, mtDNA levels in large and small EVs were inversely correlated with age, years since HIV infection, and years on ART . MoreoveHaving observed that EV-borne mtDNA is more abundant in viremic participants and given that mtDNA is considered pro-inflammatory, we used receiver operator characteristic curve analysis to test the performance of mtDNA as an indicator of patients with ongoing viral-replication or immune activation, which is characterized by a CD4/CD8 ratio <1 and a CD8 T cell count >500 cells/µL, as described previously . The conUsing the CD4/CD8 ratio and CD8 T cell count thresholds, the test result was significant only in the MSM group, with an area under the curve of 0.72–0.83. No significant results were observed for any other participant/patient subgroup . Based oIt has been reported previously that at least 90% of circulating non-cell-associated mtDNA is extracellular vesicle borne ,39. In tThe release of microvesicles carrying mitochondrial DNA, proteins, and other contents and even of whole mitochondria by several cell types after activation has been reported ,40,41. TAlthough mtDNA was more abundant in large EVs of viremic participants, its abundance had no significant correlation with viremia. Little is known about EV-borne mtDNA levels during HIV infection and even less about its distribution among plasma EV subtypes. Plasma mtDNA has been found significantly more abundant during acute HIV infection and in late presenters taking antiretroviral therapy for the first time than in healthy individuals or in long-term non-progressors, and a positive correlation with plasma viral load has been observed . More diAmong viremic participants, mtDNA was more abundant in large and small plasma EVs of ART-naive patients. Evidence is mounting that stress can trigger mtDNA release from the mitochondria to the cytosol or extracellular space, but how this occurs remains unclear . The preDespite viral replication, ART seems to decrease EV-borne mtDNA. These results suggest that ART somehow decreases cell mtDNA content or incorporation of mtDNA into EVs during HIV infection. It was found that after treatment with NRTIs, mitochondrial dysfunction emerges due to mtDNA depletion ,53. NRTIHowever, not all NRTIs contribute to mtDNA depletion. In vitro studies reported a hierarchy of mtDNA depletion for various NRTIs. Maximum inhibition was observed after treatment with Zalcitabine followed by Didanosine and Stavudine. Lamivudine, zidovudine, and abacavir were found to be weak inhibitors of gamma polymerase . In our We found an inverse correlation between EV-borne mtDNA and duration of HIV+ status or ART use and a weak inverse correlation with age among HIV+ participants. The duration of ART would be a confounding factor of the HIV status duration. A decline in plasma EV mtDNA with age has been noted previously . Long-teWe observed no correlation between EV-borne mtDNA and CD4 or CD8 T cell count or CD4/CD8 ratio among ART-treated non-viremic participants, unlike some studies in which plasma mtDNA appeared to be associated directly with CD4 T cell counts ,26. In aThere are some limitations to our study. Given the cross-sectional design, one cannot determine causality or the direction of the relationship . In addition, the size of some groups is small and would contribute to under or overestimating the significance of some analyses. Despite these methodological limitations, our results provide information that should be taken into account in further analysis of EVs in the context of HIV infection.In conclusion, our results showed the differential enrichment of EV subpopulations with mtDNA and the impact of viral replication or ART on this measurement. The mechanisms underlying the differential incorporation of mtDNA into EVs and their effects on surrounding cells or tissues warrant further investigation. The discovery of new biomarkers for monitoring the health of persons living with HIV remains an important challenge, and interest in the possible contribution of EV analysis  is growing. We believe that a longitudinal study of EV-borne mtDNA analysis taking into consideration the limitations of this study will lead to a better understanding of the mechanism, the implications of its concentration, and possibly its use as a biomarker for improved management of HIV infection.n = 196) were recruited by referral from 5 follow-up centers in Bobo-Dioulasso and Ouagadougou (Burkina Faso). These included the Yerelon clinics (Centre Muraz) of both towns, specialized in female sex worker follow-up; the Association African Solidarité (AAS) center for the follow-up of men who have sex with men (MSM); and dedicated follow-up day hospital centers—Centre Hospitalier Universitaire (CHU), Souro Sanou (Bobo-Dioulasso), and CHU Yalgado Ouédraogo (Ouagadougou). The HIV-negative participants (n = 53) were recruited in the Yerelon clinics, which also offer HIV testing.HIV-infected patients , which targets two highly conserved regions of the HIV-1 genome and has a detection limit of 20 copies/mL.The HIV-1 viral load was measured using the COBAS+ T and CD8+ T lymphocytes were obtained using a BD FACSCount™ System flow cytometer .Absolute counts of CD4g at room temperature. The plasma was centrifuged for 10 min at 3000× g to obtain platelet-free-plasma and stored at −80 °C until analysis. Thawed platelet-free plasma (250 µL) was treated with proteinase K  for 10 min at 37 °C, which reduces substantially the amount of non-EV protein  and RNA that otherwise remains present. The plasma was then centrifuged at 17,000× g at room temperature to pellet large EVs. The supernatant was mixed with 63 µL of ExoQuick  in an Eppendorf tube and maintained at 4 °C overnight. The large EV pellet was resuspended in 250 µL of microfiltered (0.22 µm pore size membrane) 1× phosphate-buffered saline (PBS)  and centrifuged for 30 min at 17,000× g. This supernatant was discarded, and the washed large EV pellet was resuspended in 250 µL of PBS and kept at 4 °C. Small EVs were pelleted as ExoQuick precipitate by centrifuging for 30 min at 1500× g, resuspended in PBS, centrifuged for 5 min at 1500× g (both supernatants were discarded) and then in 250 µL of PBS by vortex mixing, and kept at 4 °C. All relevant experimental data have been submitted to the EV-TRACK knowledgebase (EV-TRACK ID: EV220124) [EVs were purified as described previously ,7,8. BriEV size was estimated as hydrodynamic radius using a Zetasizer Nano S , which measures light scattering due to Brownian motion at a fixed position with an automatic attenuator and controlled temperature. The size distribution is obtained from the diffusion constants using the Einstein–Stokes equation . Two mea® microspheres, 15 µm, Polysciences, Inc., Warrington, PA, USA) was mixed in (5 µL) by vortex. EVs were counted using a flow cytometry method described previously [Purified EVs were stained with the lipophilic fluorescent carbocyanine dye DiD (DiIC18(5) solid or 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine 4-chlorobenzene sulfonate salt, Invitrogen™, Carlsbad, CA, USA) and the vesicular or cell-permeable dye CFSE . DiD and CFSE were diluted, respectively, 1/100 and 1/500 in filtered (0.22 µm) PBS 1X + EDTA to 100 µM. Diluted DiD solution was then mixed with EV suspension . After 5 min at 37 °C, 50 µL of CFSE were added . After 15 min (37 °C), the staining was fixed by adding 0.02% Pluronic F-127  solution, followed by 100 µL of 4% paraformaldehyde , holding for 20 min, and then adding 200 µL of filtered PBS 1X . Count bead suspension   and Triton 100-X . For this, isolated EVs were stained with DID and Cell Trace Violet Cell Proliferation kit  as performed for CFSE. After the fixation of staining with 0.02% Pluronic F-127, EVs were supplemented with SDS solution for final concentration in 200 µL of 0.025%, 0.075%, and 0.150%. For Triton 100-X, the final concentrations were 0.025% and 0.075%. These conditions were established based on previous studies  and prelm/v Tris base, 1% m/v sodium dodecyl sulfate, and 100 mM EDTA) plus proteinase K  was held at 55 °C for 10 min. UltraPure™ phenol/chloroform/isoamyl alcohol  was added (500 μL), followed by vigorous shaking then centrifuging at 12,000× g for 3 min at 4 °C. The aqueous phase (350 μL) was shaken with an equal volume of chloroform and centrifuged at 12,000× g for 3 min at 4 °C. The aqueous supernatant (200 μL thereof) was mixed thoroughly with 20 µL of 5.0 M NaCl, then 400 μL of ice-cold absolute ethanol and 1 μL of GlycoBlue™ co-precipitant , and held at −20 °C for 1 h. After centrifugation for 20 min at 12,000× g at 4 °C, the supernatant was removed and the pellet was washed with 700 μL of cold 70% ethanol, followed by centrifugation at 12,000× g for 8 min. Pellets were dried at room temperature and resuspended in 15 μL of TE 1X Buffer . The DNA was held at 4 °C overnight and quantified in 1 µL aliquots using a BioDrop-μLITE kit .A mixture of EV suspension (100 µL) plus lysis buffer  and specific primers. The targeted genes included human mitochondrial NADH dehydrogenase subunit 5. The primer sequences (Integrated DNA Technologies) were forward ACGCCTGAGCCCTATCTATTA-3′ (Tm = 54.9) and reverse 5′-GTTGACCTGTTAGGGTGAGAAG-3′ (Tm = 55). The amplification temperature profile was as follows: 15 min at 95 °C (enzyme activation) then 40 cycles of 15 s at 94 °C (denaturation), 30 s at 55 °C , 30 s at 72 °C (elongation). Reaction specificity was confirmed using the melt curve procedure  at the end of the amplification according to the manufacturer’s instructions.Quantitative real-time PCR was performed using a CFX384 Touch Real-Time PCR Detection System  with a SYBRA standard curve for mtDNA copy quantification was generated using total DNA obtained from human platelets and diluted in series. All samples were analyzed in triplicate, and a no-template control was included in every analysis. Quantity of mtDNA was expressed in picograms per nanogram of total DNA.Briefly, EV pellets, large or small, purified as described above, were mixed with 2.5% glutaraldehyde  plus 0.1 M sodium cacodylate  buffer pH 7.4, pipetted (10 µL) onto a nickel grid with carbon-coated formvar film, and held for 10 min. Excess liquid was removed by blotting. The grids were stained for 2 min with 2% uranyl acetate solution. Images were acquired at 80 kV using a FEI Tecnai Spirit G2  transmission electron microscope equipped with a bottom-mounted CCD camera.t-test for continuous variables. A p value less than 0.05 was considered statistically significant.All analyses were performed with GraphPad Prism 9.4.1 . Participant demographic and clinical characteristics were presented as a proportion or median with an interquartile range (IQR) and tabulated . Initial


Twenty-two university standard male rugby union players (20 ± 2 years) completed the study. Using a double-blind, randomized, and counterbalanced within-subject experimental design. Participants completed six experimental trials (three caffeine and three placebo) where force time characteristic of the Isometric Mid-Thigh Pull (IMTP), Countermovement Jump (CMJ) and Drop Jumps (DJ) were assessed followed by assessments of Chest Press (CP), Shoulder Press (SP), Squats (SQ), and Deadlifts (DL) Repetitions Until Failure (RTF at 70% 1 RM). ANOVA indicated that caffeine improved both the CMJ and DJ (p < 0.044) and increased RTF in all RTF assessments (p < 0.002). When individual caffeine trials were compared to corresponding placebo trials, effect sizes ranged from trivial-large favoring caffeine irrespective of a main effect of treatment being identified in the ANOVA. These results demonstrate for the first time that the performance enhancing effects of caffeine may not be repeatable between days, where our data uniquely indicates that this is in part attributable to between sessions variation in caffeine’s ergogenic potential.This study aimed to determine the effect of 3 mg.kg The ergogenic potential of caffeine to evoke improved physical and cognitive performance has been firmly established ,2,3. NumDespite well-established and overwhelmingly reported beneficial effects, there remains a wealth of evidence indicating limited or no acute caffeine effects on measures of muscular strength and power ,5,8,12 aWhere caffeine has been shown to elicit beneficial effects on muscular strength and power performance, effects are generally small in magnitude ,5,6,7 anAlmost exclusively, previous work examining the acute effects of caffeine has compared performance in a single treatment trial to that of a controlled trial  ,30,31, w−1 acute caffeine ingestion on muscular strength, power, and strength endurance, (ii) to determine if 3 mg.kg−1 caffeine on muscle functions is repeatable across multiple trials.Given that the effect of caffeine may be influenced by assessment mode, contraction modality and maybe muscle group-specific ,33,34, tn = 8) , illness (n = 3), or for reasons unstated (n = 7) leaving a sample of 22 (Mean ± SD Age (years) 20 ± 2; Height (cm) 181 ± 7; Body Mass (kg) 91 ± 23). Participants completed a caffeine consumption questionnaire [Following ethics approval from the host institute P94037] and written informed consent, 40 apparently healthy participants from the Coventry University Men’s Rugby Union team participated in the study. Participants completed a health screen questionnaire at each visit and were excluded if suffering from a musculoskeletal injury that prevented safe completion of the exercise trials, consumed psychoactive medication, or had any other underlying contradictions to exercise. From the original sample, 18 dropped out due to injury  or a placebo . In each case, the required dose was determined for each individual and transferred to a transparent gelatin capsule . 3 mg.kg−1 is regularly cited as a minimal dose needed to evoke a physical performance enhancement [Participants were asked to abstain from caffeine and intense physical exercise for 12 and 48 h, respectively, in accordance with previous work . When prancement  and prevancement ,6,7.Participants completed a 1 RM for Chest Press (CP), Shoulder Press (SP), Squats (SQ) and Deadlifts (DL), assessments that have been used previously to evaluate acute effects of caffeine on muscle function . ParticiInitially, participants removed shoes and heavy clothing, and measures of height (cm) and body mass (kg) were taken using a stadiometer  and electronic weighing scales  respectively. 1 RM was then reassessed and then participants familiarised to the CMJ, DJ, IMTP, and RTF testing procedures.2/2 g) [−2). Using the attempt that elicited the greatest JH, reactive strength index modified (RSI mod) (JH /Time to TO), Peak Force (PF)  prior to TO), Peak Power (PP), Concentric Impulse (Ns) and Eccentric Impulse (Ns) were determined as be the procedures outlined by Chavda et al. [CMJ’s were performed on two PASCO force plates  sampling at 1000 Hz. Participants were instructed to have their arms akimbo (fixed on the hips) and to remain this way during the movement. Participants started on the force plates in a standing upright position and executed the movement following at least 1 s of quiet standing . Partici2/2 g) ,45,46. Wa et al.  with PF DJs were performed on two bilateral PASCO force plates sampling at 1000 Hz. Participants started standing upright on a box 45 cm off the ground compensating for the height of the force plate (5 cm) resulting in a 40 cm drop height. Participants with arms akimbo remained in this position through the entire jump. Participants were instructed to jump as high and as fast as possible and executed the movement by stepping off the box with their dominant leg leading and upon landing, instantaneously performed a maximal vertical jump. A successful DJ was determined by the arms being akimbo, stepping off the box leading with the dominant leg, and upon landing on the two force plates simultaneously. Jumps that deviated from this were repeated. Participants completed 3 successful jumps, separated by a minimum of 30 s rest. Backwards integration (BI) of raw unfiltered vertical force time data, as per the method outline by Wade et al. , was useIMTPs were performed on floor integrated triaxle force plates  sampling at 1000 Hz. IMTP was assessed using a custom-built steel rack fixed to the ground. Participants were asked to stand with a knee angle of 135–145° and a hip angle of 140–150° ,50 with Following the warm-up outlined in the 1 RM protocol, participants completed an RTF assessment of CP, SP, SQ, and DL. Participants completed two sets of RTFs at 70% 1 RM as per previous work ,26,55. PPrior to completing the exercises outlined above, participants ingested either a caffeine or a placebo treatment 45-min prior to experimental trials  as maxim2) was reported with as a measures of effect size [p < 0.05.Data analysis was performed using Statistical Package for the Social Sciences (IBM SPSS Statistics Version 25) and Excel (Microsoft Windows Version 16.41 2020). In order to identify a potential training effect from continuous bouts of resistance exercise pre-and-post-experimental 1-RM performance was evaluated using paired t-test. Test-re-test reliability of each variable was measured between all placebo trials  and caffeine trials . For each comparison, Interclass correlation (ICC) was performed, and 95% CI determined, with reliability being classed as: Poor (<0.50), Moderate (0.50–0.74), Good (0.75–0.89), and Excellent (0.90<) . Coefficect size  and was ect size . Effect ect size . Hedge’sect size . Data wep = 0.437 (ES = 0.22), SP: p = 0.149 (ES = 0.39), SQ: p = 0.051 (ES = 0.61), DL: p = 0.162 (ES= 0.44).Upon completion of the study 1 RM performance did not statistically differ to that prior to completion of the experimental trials in any of the assessed lifts  or Trial*Time (p > 0.228 ηp2 < 0.068) interactions. For RTE Mental and FAS there were no Treatment*Trial interactions (p > 0.068 ηp2 < 0.239). However, there was a Treatment*Trial (p = 0.019 ηp2 = 0.171) interaction for RTE Physical with pairwise comparison indicating higher RTE Physical in the caffeine trials (p < 0.013 d > 0.17). Furthermore, RTE Physical, RTE Mental, and FAS there was an interaction between Treatment*Time (p < 0.050 ηp2 > 0.170). Pairwise comparison for RTE Mental revealed an increase pre-and post-ingestion in the caffeine trial (p < 0.033 d > 0.26) but RTE Physical and FAS was only higher with caffeine post-ingestion (p < 0.006 d > 0.24). For RTE mental and FAS there was no main effect of Trial (p > 0.064 ηp2 < 0.123).For RTE Physical, RTE Mental, and FAS there were no significant Treatment*Trial*Time (Test re-test reliability of CMJ measures ranged from good to excellent across both treatments .p < 0.873 ηp2 < 0.096) or no main effect of Trials (p < 0.923 ηp2 < 0.100). For eccentric impulse (Ns) there was no effect of treatment (p = 0.771 ηp2 = 0.004). However, JH (m), RSI (mod), PF (N.kg), PP (W.kg), and concentric impulse (Ns) were all higher following caffeine treatment (p < 0.044 ηp2 > 0.179).In all cases there was no significant interactions between Treatment*Trial (Test re-test reliability of DJ measures ranged from poor to moderate across both treatments .p < 0.924 ηp2 < 0.027) and no main effects of Trial (p < 0.876 ηp2 < 0.047). For, RSI and PF there was no effect of Treatment (p < 0.633 ηp2 < 0.094) however, JH and PP were higher in the caffeine (p < 0.042 ηp2 > 0.183). In all cases there was no Treatment*Trial interactions  interaction, main effects of Trials (p < 0.732 ηp2 < 0.052) or Treatment (p < 0.705 ηp2 < 0.125) for any of the measured variable.There was no Treatment*Trial , Treatment*Trial (p < 0.846 ηp2 < 0.065), Trial*Set (p < 0.956 ηp2 < 0.006), and Treatment*Set  interactions. However, for SQ RTF there was a Treatment*set (p = 0.048 ηp2 = 0.174) interaction with pairwise comparison indicating caffeine trials had higher RTF (p = 0.001 d < 0.28) as well higher performance in Set1 compared to Set2 in both cases (p = 0.001 d < 0.25). In all cases there was no main effect of Trial (p < 0.975 ηp2 < 0.119) apart from SP RTF (p = 0.047 ηp2 = 0.136) with Trial 1 having a greater RTF. In all other cases RTF were greater in Set1 compared to Set2 (p = 0.001 ηp2 > 0.710) and there was a main effect of Treatment (p < 0.002 ηp2 > 0.365) where performance in the caffeine trial was improved.For RTF CP, SP, DL, SQ there were no Treatment*Trial*Set (Test–retest reliability of Set1 and Set2 RPE was poor or moderate .p < 0.896 ηp2 = 0.055), Treatment*Set (p > 0.107 ηp2 < 0.129), or Trial*Set (p < 0.768 ηp2 < 0.102) and Treatment*Trial interactions (p < 0.514 ηp2 < 0.075) apart from SQ which had a significant Treatment*Trial interaction (p = 0.019 ηp2 = 0.173). Pairwise comparison indicated trial 2 Set1 RPE was significantly higher in the Placebo treatment than the Caffeine treatment (p = 0.032 d = 0.098). All exercises had no main effect of Treatment (p < 0.402 ηp2 < 0.115). CP, SP, and DL had no significant main effect between Trials (p < 0.720 ηp2 < 0.183). In all cases, RPE was higher following completion of the second set (p = 0.000 ηp2 < 0.842).In all cases there was no Treatment*Trial*Set . Recent meta-analyses have identified the benefits of caffeine on measures of muscular strength and power, inclusive of CMJ, DJ, and RTF [−1 caffeine had no effect on IMTP performance of non-specifically resistance trained female but contradicts work by Harty et al. [The present study identified some overall treatment effects during measures of muscular function , and in some cases, there was no main effect of treatment but caffeine effects prevalent when single caffeine trials were compared to placebo . Although, it must be noted these effects were predominantly small , however, there were trivial to large effects when specific caffeine trials were compared to placebo . Conversely, in other cases, trivial to large effects were prevalent for specific comparisons in the presence of a main effect of treatment . Given that acute caffeine ingestion is typically associated with small significant increase in performance , it is ePoor between session reliability is unlikely the only cause of the varied response to acute caffeine ingestion as such effects were still prevalent in cases where between session reliability was good/excellent. For example, CMJ JH, PF, PP, and concentric impulse were highly reliable and demonstrated a main effect of treatment, However, when individual caffeine trials were compared to placebo, effect sizes varied between trivial to small. Given that experimental trials were conducted in identical conditions, it is speculated that the variation in response may be a related to participants mood, motivation, diet and prior activity, factors which may influence caffeine erogenicity.As previously outlined, caffeine has been shown to effect mood (i.e., alertness, vigor, arousal, and decreased levels of fatigue ,26,86 anFurther contributing factors to the between session variation in caffeine’s effect may be attributable to fatigue state and pre-exercise diet. Although evidence is limited with respect to effects on physical performance, caffeine has been shown to be more effective for cognitive function following cognitive fatigue or sleep deprivation ,88. WhilAlthough not without opposition , there iThe present study offers valuable insight into the repeatability of acute caffeine consumption on muscular strength and power performance but does not come without limitations. The ergogenic potential of caffeine has been shown to be influenced by variation in CYP1A2 and ADORA2A genes ,93, factIt is clear from the present work, that if only a single caffeine trial was compared to single placebo trial, as is common in this field of work , a numbe−1 effects on measures of muscular strength and power. Collectively these data demonstrate a contractile mode specific effect of caffeine on ingestion on specific measures of muscular strength and power. However, results from this study indicate for the first time that an individual is unlikely to see a performance enhancing benefit on every occasion where caffeine is consumed. Our data infer that benefits of caffeine ingestion may only be prevalent in athlete specific optimized mood and cognitive states. As such, athletes should seek to identify the condition where caffeine ingestion is likely to evoke the greatest performance enhancing benefits, and where this is not the case, consider the benefits of supplementation against well reported side effects. Furthermore, future work evaluating the ergogenic effects of caffeine on sports performance should assess response over multiple trials in order to more precisely evaluate its effects and prevent misleading results.The present study examined the repeatability of acute caffeine consumption of 3 mg.kg