Korean medicine (KM) interventions are attractive for children with idiopathic short stature (ISS). We investigated the real-world clinical practice patterns of KM doctors in ISS treatment.  The survey targeted KM doctors who have treated patients with ISS in KM clinical settings for >3 years. We included questions on the diagnosis and treatment patterns, effects, cost, and opinions of doctors on KM treatment for ISS. A frequency analysis was performed. n = 57, 98.3%), “height, weight, and growth rate of child” , and “amount of meals, digestive function”  were frequently used as indicators for diagnosis and treatment. The most frequently used KM interventions were “herbal medicine” , “acupuncture” , “moxibustion” , “dry cupping” , and “physiotherapy” . Herbal medicines were generally prescribed to tonify the spleen or kidney, and the most frequently used individual herbs were Acanthopanacis cortex, Astragali Radix, and Cervi parvum cornu. The most common acupuncture points were ST36, GB34, SP6, EX-LE5, and LI4.  There were 58 respondents, and “heights of parents” ( This study showed the use of KM for ISS in real-world clinical settings. In the future, well-designed clinical studies to verify the effect of KM treatment on ISS based on real-world clinical practice patterns should be actively performed. Idiopathic short stature (ISS) is defined as when the individual's height is two standard deviations or more below the corresponding average height for a given age, sex, and population group, in the absence of any systemic, endocrine, nutritional, or chromosomal abnormality . ApproxiIn South Korea, Korean medicine (KM) treatments, including acupuncture, moxibustion, and herbal medicine, also known as East Asian traditional medicine (EATM), one of the most well-known types of complementary and integrative medicine, have been widely used as an alternative treatment method to support child growth. According to a retrospective study in 2016, growth retardation was the chief complaint among children and adolescents who visited the department of Korean Pediatrics in a KM Hospital . SeveralThe aim of this study was to investigate the clinical practice patterns of KMDs in ISS treatment and their therapeutic effects and costs in real-world clinical settings.https://www.mediresearch.co.kr), which is a professional survey research company. The company sent a questionnaire survey via mobile phone to KMDs affiliated with KM clinics or hospitals, based on the cooperation of the Association of Pediatrics of KM. The company recruited participants with consideration of the ratio between specialists in pediatrics and general KM practitioners. The participants were enrolled voluntarily and they were informed that responses were required for all our questions in the questionnaire. The survey was conducted anonymously between November 24 and December 23, 2020.This study used a cross-sectional survey design to assess the clinical practice patterns of KMD for ISS treatment in South Korea. The survey was conducted by Mediresearch ; purpose of herbal prescription; and frequently used individual herbs and acupoint.Effectiveness and cost: mean height growth (cm) before and after KM treatment, according to sex and age, and cost of each intervention.Opinions on KM treatment for ISS: Korean-Western medicine collaborations for the treatment of growth disorders; essential KM interventions that should be included in health promotion program for children's growth; limitations of KM treatment for children's growth; and the appropriate herbal medicine for insurance.The questionnaire consisted of five categories. Variable descriptions are as follows:n) and percentage (%). Microsoft Excel 2016  was used for all analyses.Frequency analysis was conducted for each question, and the ranking was confirmed by indicating the number of responses .n = 27, 46.6%), followed by “≥10 and <30” . The most frequent average age of ISS patients was prepubertal , followed by preschool   men and 27 (46.6%) women, participated in this study. Most respondents were aged between 30 and 49 years (86.2%). In addition, most of their clinical experience was evenly distributed between 5 and 30 years (94.8%). The average monthly number of ISS patients was “<10”  .n = 57, 98.3%), ‘height, weight, and growth rate of child' , and “amount of meal, digestive function” , followed by “pattern identification” , “growth plate test (X-ray)” , “pulse diagnosis” , “abdominal diagnosis” , and “tongue diagnosis” . The top comorbidity that was assessed for when diagnosing and treating ISS was “digestive diseases” , followed by “respiratory diseases” , and “allergic diseases”   .n = 58, 100%) as an intervention in ISS treatment, followed by “acupuncture” , “moxibustion” , “dry cupping” , and “physiotherapy” . “Electroacupuncture,” “pharmacopuncture,” and “chuna” were each selected by 20 respondents (34.5%). The herbal medicine administration period typically ranged from 1 to 6 month: “≥1 and <2 months” ; “≥2 and <3 months” ; and “≥3 and <6 months” . The frequency of other interventions was generally “once a week” in a clinical setting  for “tonifying the spleen,” 19.0% (n = 11) for “tonifying the spleen and the kidney,” 12.1% (n = 7) for “tonifying the kidney,” and 10.3% (n = 6) for “tonifying the liver and the kidney.” The frequently used herbs for ISS treatment were “Acanthopanacis cortex” , “Astragali Radix” , “Cervi parvum cornu” , and “Rehmanniae Radix preparata” . The most frequently used acupuncture points for ISS treatment were “ST36 (Zusanli)” , “GB34 (Yanglingquan)” , “SP6 (Sanyinjiao)” , “EX-LE5 (Xiyan)” , and “LI4 (Hegu)”   .According to the response of KMDs, the mean (standard deviation) of growth height after KM treatment was 6.03 (0.98) cm before puberty and 9.03 (1.56) cm during puberty in boys; and 5.83 (1.05) cm before puberty and 8.36 (1.71) cm during puberty in girls .n = 45, 77.6%) ranged from 156 to 268 dollars per 15 days. The daily cost of herbal medicine was between 10.4 and 17.9 dollars.The distribution of the cost of each intervention is described in n = 45, 77.6%) as an essential KM intervention that should be included in a health promotion program for pediatric growth disorders. The top herbal medicines requiring cover by health insurance for ISS treatment were Yukmijihwang-tang , Sogunjung-tang , Yukgunja-tang , Sipjeondaebo-tang , and Sagunja-tang  (Twenty-eight KMDs (48.3%) responded that ‘KM treatment is more cost-effective than GH.' By contrast, 22 KMDs (37.9%) responded that ‘It is best to combine herbal medicine, acupuncture, and GH.' Herbal medicine (decoction) was selected the most  .This survey investigated the clinical practice patterns of KM treatment for ISS, including a detailed evaluation of diagnosis, treatment, effectiveness, and costs. Furthermore, we sought the opinions of KMDs regarding their use of these treatments. We have also previously reported a network meta-analysis  and an oPattern identification based on KM theory could be a significant method for treating ISS because it is a primary growth disorder with an unknown cause. ISS is typically regarded as a problem with digestive disorders or lack of nourishment in KM . This isTaken together, these data show that herbal medicine is regarded as an essential ISS treatment intervention. Furthermore, acupuncture and moxibustion are also necessary for ISS treatment. The administration period for herbal medicines was between 2 and 6 months, and the frequency of other interventions was once a week. Yukmijihwang-tang (六味地黃湯) , the mosAcanthopanacis cortex (五加皮), followed by Astragali Radix (黃芪) and Cervi parvum cornu (鹿茸). The effects of a complex herbal mixture containing Acanthopanacis cortex, Astragali Radix, and Dipsaci Radix (續斷) on height growth have been studied previously and the study showed that height gain was significantly higher in the herbal mixture group than in the placebo group after 24 weeks [Cervi parvum cornu is the most popular supplement for growth promotion and continues to grow in popularity [The most frequently used individual herbs for ISS treatment in clinical settings were 24 weeks . Additiopularity . In addipularity .All the acupoints frequently used for the treatment of ISS, except GV20 (Baihui) and CV12 (Zhongwan), were located below the knee to the toes . This coSeveral studies have conducted the economic evaluation of GH treatment on ISS , 25. FurAccordingly, we collected costs and height growth data in this survey, although it was not sufficient to make any conclusions. Nonetheless, we estimated that three months (the most responsive treatment period) of treatment with herbal medicine, acupuncture, and moxibustion would cost in the range of USD 1,554–1,824. The frequency and cost of acupuncture and moxibustion were assumed to be as once a week and USD 9 per session, respectively. The difference in height growth before and after KM treatment was 1.93 cm (before puberty) and 2.53 cm (puberty) in boys; and 1.69 cm (before puberty) and 2.22 cm (puberty) in girls . TherefoCurrently, KM interventions for ISS are not covered by health insurance in South Korea. This is reported as the primary limitation of KM treatment for growth disorders. Several health promotion programs have reported using KM interventions for child growth in public health centers . Health There are several limitations in this study. First, the number of participants was small and not representative of the entire KMD population. This survey aimed to recruit KMDs who focused on pediatric care with >3 years of clinical experience with ISS rather than more respondents to investigate clinical patterns in detail. The total number of KMDs is approximately 22,000, and there are only about 130 pediatric specialists focusing on KM in South Korea . In addiThis study analyzed the current KM clinical practice for the treatment of ISS, focusing on diagnosis indices and treatment patterns. The effects and costs of height growth and opinions of KMDs were also investigated. In the future, studies evaluating the efficacy and cost-effectiveness of KM treatment for ISS should be performed based on real-world clinical practice.


Margaritaria nobilis is a shrubby species widely distributed in Brazil from the Amazon to the Atlantic Rainforest. Its bark and fruit are used in the Peruvian Amazon for disinfecting abscesses and as a tonic in pregnancy, respectively, and its leaves are used to treat cancer symptoms. From analyses via UHPLC-MS/MS, we sought to determine the chemical profile of the ethanolic extract of M. nobilis leaves by means of putative analyses supported by computational tools and spectral libraries. Thus, it was possible to annotate 44 compounds, of which 12 are phenolic acid derivatives, 16 are O-glycosylated flavonoids and 16 hydrolysable tannins. Among the flavonoids, although they are known, except for kaempferol, which has already been isolated from this species, the other flavonoids  are being reported for the first time in the genus. Among the hydrolysable tannins, six ellagitannins present the HHDP group , one presents the DHHDP group (5), and four contain oxidatively modified congeners . Through the annotation of these compounds, we hope to contribute to the improved chemosystematics knowledge of the genus. Furthermore, supported by a metric review of the literature, we observed that many of the compounds reported here are congeners of authentically bioactive compounds. Thus, we believe that this work may help in understanding future pharmacological activities. Margaritaria nobilis, for a time, was classified as belonging to the genus Phyllanthus¸ which chemical-pharmacological knowledge is widely disseminated −; and methyl gallate [M−H]− at m/z 183, fragmenting into m/z 168 due to loss of methyl radical [M−H−CH3•]−•, followed by m/z 124 due to loss of CO2.The main phenolic compounds identified in is class . For exaO-coumaroylgalactaric acid [M−H]− at m/z 355, due to loss of coumaric acid and coumaroyl, produced the fragments at m/z 191 [M−H−C9H8O3]− and 209 [M−H−C9H6O2]−, respectively. Similarly, the compound [M−H]− at m/z 385, identified as O-feruloylgalactaric acid, by the loss of ferulic acid and feruloyl, produced the fragments at m/z 191 [M−H−C10H10O4]− and 209 [M−H−C10H8O3]−, respectively.The compound 18) was identified, which presented itself as a [M−H]− ion at m/z 301, and in its MS/MS spectrum it was observed loss of characteristics of 18 Da (H2O), 28 Da (CO) and 44 Da (CO2). This justify the fragments at m/z 283 [M−H−H2O]−, 229 [M−H−CO−CO2]−, 201 [M−H−2CO−CO2]− and 185 [M−H−CO−2CO2]− −•, 313 [M−H−2CH3•]− and 298 [M−H−3CH3•]−• − ion at m/z 433, identified as ellagic acid O-xyloside (13), producing the fragment m/z 300 by homolytic cleavage of the O-xyloside bond [M−H −C5H9O4•]−•, and the fragment m/z 301 by the neutral loss of the glycosidic moiety [M−H−C5H8O4]−.Methylated ellagic acid derivatives could be identified due to loss of methyl radical (−15 Da). For example, compound [M−H]1 and 4, which were previously isolated from M. nobilis − ion at m/z 285, produced the fragments at m/z 255, 227 and 151, as reported in the literature [O-glycosylated derivatives. The compound [M−H]− at m/z 447, identified as kaempferol 3-O-glucoside (Isomer 30 and 33), showed fragments at m/z 285 [M−H−C6H10O5]− and 284 [M−H−C6H11O5•]−•, in addition to the characteristic fragments of its aglycone. Similarly, compounds 24, 25, 34 and 35 presented product ions [M−H−308 Da]−, [M−H−294 Da]−, [M−H−278 Da]− and [M−H−278 Da]−, indicating the loss of the O-rhamnosyl-glucoside, O-xylosyl-glucoside, O-rhamnosyl-xyloside and O-xyloside moiety, respectively (Kaempferol (terature . These fectively .40), observed as a [M−H]− ion at m/z 301, produced the fragments at m/z 273, 257, 229, 179 and 151, as reported in the literature − and 300 [M−H−C6H11O5•]−•, in addition to the characteristic fragments of its aglycone. Similarly, compounds 10, 14, 15, 28 and 29 presented product ions [M−H−324 Da]−, [M−H−294 Da]−, [M−H−308 Da]−, [M−H−278 Da]− and [M−H−132 Da]−, indicating the loss of the O-glucosyl-glucoside, O-xylosyl-glucoside, O-rhamnosyl-glucoside, O-rhamnosyl-xyloside and O-xyloside moiety, respectively. In addition to these, a compound [M−H]− at m/z 477 was identified as an isomer of methyl quercetin 3-O-glucoside (41), differing only by the presence of a methoxyl in the B ring of quercetin, producing the fragments at m/z 315 and 314 referring to cleavages in the O-glycosidic bond  are being reported for the first time in the genus.It is noteworthy that glycosylation at the 3-spectrum . The preterature . However nobilis , the oth8, 23 and 32, the number of galloyl groups can be calculated by adding n × C7H4O4 (152 Da) to the glycosidic moiety which, in this study, basically consisted of a glucose C6H12O6 (180 Da) and a dideoxyglucose C6H12O4 (148 Da). In the negative mode MS/MS spectra, gallotannins derivatives produced characteristic fragment ions, such as [M−H−152 Da]− and [M−H−170 Da]−, denoting neutral losses of galloyl and gallic acid groups, respectively − , one containing DHHDP group (5), two isomers containing Che group (7 and 17) and four containing modified congeners oxidatively . Here, the ion [M−H]− at m/z 925 taken as an example, fragmented into m/z 755 [M−gallic acid]−, 615 [M−H−C13H10O9]−, 605 [M−H−HHDP−H2O]−, 309 [C13H10O9−H]− and 301 [Ellagic acid−H]−, which allowed its identification as an isomer of phyllanthusiin C (12), already isolated from the species Phyllanthus myrtifolius and P. urinaria (Phyllanthaceae) − and 275 [Urolithin−H]−, confirming the presence of a modified sugar as shown in For example, the [M−H]− ion at m/z 763 indicates an ellagitannin of the Galloyl-Cinnamoyl-HHDP-glucose type (43), which is confirmed by the neutral losses of 170 Da , 148 Da (cinnamic acid), 130 Da (cinnamoyl) and 302 Da (Ellagic acid), in addition to neutral losses of H2O (18 Da) as shown in Analysis of the MS/MS spectrum  of the [− at m/z 857 showed the fragments at m/z 169 [Gallic acid−H]−, 275 [Urolithin−H]− and 301 [Ellagic acid−H]−, indicating the presence of galloyl and HHDP groups in the structure of the deprotonated molecule, as well as the loss of 242 Da suggests a galloyl-methylacetate group. From the fragmentation proposal shown in 22)  followed by 54 Da (C3H2O) from the deprotonated molecule forming, respectively, the ions m/z 879 and 825, which loses the residue of this group forming the ions m/z 615 and 209. Thus, from the fragmentation proposal presented in 39) . The remaining hydrolysable tannins are being reported for the first time in the genus.Research on the annotated hydrolysable tannins, carried out in a database of natural products, such as KNApSAcK and Dictionary of Natural Products, confirmed the presence of these compounds in the Phyllanthaceae family, especially in the  nobilis , and wasMargaritaria nobilis in traditional medicine, only one study was performed on antimycobacterial evaluation [Despite reports of the use of aluation , as wellaluation .M. nobilis leaves.In view of this, as an alternative to the use of the barks, which compromises the integrity and perpetuation of the species, we preferred to evaluate the leaves in view of their high availability and rapid natural replacement, with the perspective that it may have interesting compounds as much as those already observed at the bark. Based on the results obtained, a search was carried out in the scientific literature on the pharmacological activities already attributed to compounds (or their class) that were putatively identified in the ethanolic extract of 15) produces antinociceptive effects involving central modulation of the vIPAG downstream circuit partially by an opioidergic mechanism [O-glucoside (21 and 26) showed comparable antinociceptive activity to the reference compound indomethacin [As result, studies with extracts of plant species rich on glycosylated flavonoids show pharmacological activities such as analgesic and anti-inflammatory . For exaechanism . A mixtumethacin .42) and its glycosylated derivatives are widely distributed in nature and have several biological activities. A review of kaempferol discussed the anti-inflammatory effects and mechanisms of action of this substance, confirming its potential to improve inflammation under both in vitro and in vivo conditions [Kaempferol  and its nditions , gastropnditions , anti-arnditions , anti-canditions  and neurnditions .18) is a polyphenol widely investigated for its pharmacological properties, mainly against toxicity and liver diseases, which can be justified by its antioxidant capacity, in addition to reducing the lipid profile and lipid metabolism, altering pro-inflammatory mediators and decrease factor activity (kB). In addition to being detected in its free form, ellagic acid can be released by the hydrolysis of ellagitannins under physiological conditions [Ellagic acid , geraniin (5) and chebulagic acid (7 and 17).The hydrolysable tannins are subdivided into gallotannins and ellagitannins. In our analyses, three gallotannins and several ellagitannins were identified. We mention here those that were detected with the highest degree of ionization, which are the isomers of: corilagin . Ethyl alcohol (99%) was purchased from Êxodo Científico . Acetonitrile Grade LC-MS and formic acid were purchased from Merck . Ultrapure water was obtained by a Direct-Q 5 system .Margaritaria nobilis were collected in the forest region of the municipality of Bragança/PA, Brazil, under the coordinates (1°02′08″ S and 46°49′41″ W). The botanical identification was carried out at the Embrapa Amazônia Oriental institution, by the botanist Nascimento, E.A.P., with an exsiccata deposited in the IAN herbarium, in the same institution, under registration number 191496. After the botanical certification, the material was washed with 0.1% sodium hypochlorite solution (NaCℓO) to eliminate micro-organisms , then with distilled water to remove residues and sprinkled with absolute ethanol for asepsis. Then, the material was dried in a circulation oven  at 45 °C until constant weight.Approximately 1 (one) kilogram of green and homogeneous leaves of mature specimens of The dried leaves were ground in a ball mill  until obtaining a semi-fine powder granulometry (60–100 µm). The crushed material was subjected to a 48-h extraction divided into two 24-h batches, using ethanol (99%) as solvent in the proportion of 4 L of solvent for each 1.0 kg of dry and crushed material. Subsequently, the volumes were pooled and concentrated in a rotary evaporator . The concentrate was oven dried at 40 °C to constant weight.2O:MeOH 2:8 (v/v) system to retain interferences, especially fat and chlorophyll present in the leaves. For this, a C18 analytical cartridge  was used with 50 mg of stationary phase and a volume of 1 mL, previously conditioned with 1 mL of MeOH and 1 mL of ultrapure water. After SPE treatment, a 3-mg aliquot was solubilized in 1 mL of a 2:8 H2O:MeOH system, followed by filtration with a 0.22 µm hydrophilic syringe filter  for analysis.The extract (10 mg) was subjected to a pre-treatment by solid phase extraction (SPE) in a HThe matrix was analyzed in an ultra-performance liquid chromatography system coupled to an ESI-QToF Xevo G2-S mass spectrometer  with an electrospray ionization (ESI) source operating in negative ionization mode. The mass scan had a range of 100 to 1200 Da and leucyin-enkephalin was used as the Lockspray reference mass.2) at 800 L/h and desolvation temperature at 450 °C, cone gas flow (N2) at 50 L/h, source temperature at 120 °C. The capillary and sampling cone voltages were set to 2.0 kV and 80 V, respectively.UHPLC analysis was performed on a BEH C18 column  Waters. The column and autoinjector temperatures were maintained at 40 and 25 °C, respectively. The chromatography run was performed with ultrapure water (solvent A) and acetonitrile (solvent B), both acidified with 0.1% formic acid. The gradient method was defined as follows: 0 min—10% B; 2 min—20% B; 30 min—50% B. The flow rate was 300 µL/min, and the injection volume was 2.00 µL. The total ion chromatogram was acquired using Masslynx V4.1 software . The mass spectrometry parameters were set to the following: desolvation gas flow (Nm/z) and an extraction tolerance of 2 Da. The differentiation of molecular ions, adducts and fragment ions were performed by chromatographic deconvolution with 3 Da isotope tolerance and 6 Da isotope extraction tolerance. The MS/MS isolation window width was 1 Da, and the scaled normalized collision energy (NCE) was set to units of 10, 20, 30, 40 and 50 eV.Data-dependent acquisition  was performed on the five most abundant ions detected in full-scan MS (top 5 experiments per scan). The ion peaks were detected at charge states +1 and +2 with the inclusion of the 10 most intense ion peaks with a charge state tolerance of 0.2 Da  was used for the deconvolution of the chromatogram. The S/N intensity window was used as the S/N estimator with a signal-to-noise ratio set to 15, a minimum feature height of 5.0 × 103, a coefficient area limit of 50, a peak duration ranging from 0.01 to 1.0 min and an RT wavelet range of 0.01 to 0.1 min, an m/z interval for MS2 scan pairing of 0.02 Da and an R/T interval for MS2 scan pairing of 0.2 min were also used. Isotopes were detected using the isotope peak grouper with an m/z tolerance of 0.02 Da, an RT tolerance of 0.2 min (absolute) and the maximum load set to 2 and the representative isotope used was the most intense. Finally, using the peak list lines filter option, features without an associated MS2 spectrum were removed, also using the parameter consecutive minimum peaks as 1 and minimum peaks in an isotope pattern as 1 as well. Finally, a manual validation step was performed to exclude false features, such as fragments from the ionization source [2 spectra, resulting in a final list containing 151 features.UHPLC-MS/MS data were converted from standard .raw format  to .mzML format using MSConvert 3.0.2 software . The resn source  and feathttps://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp) (accessed on 1 April 2022). The precursor ion mass and MS/MS fragment ion tolerances were both set at 0.02 Da. A molecular network was then created in which the edges were filtered to have a cosine score above 0.65 and more than 4 corresponding peaks. The edges between two nodes were kept in the network only if each of the nodes appeared in each of the other 10 most similar top nodes. The molecular family size was set to a maximum of 100, and the lowest scoring borders were removed from the molecular families until the molecular family size was below this threshold. The spectra on the network were searched against the GNPS spectral libraries [https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=72419d61c18f424d9544b41bc32c87e9 (accessed on 7 April 2022). From the .mgf and .csv files obtained from processing the raw data with MZmine 2.53, a Molecular Network was created using the Feature-Based Molecular Networking workflow  on the Gibraries . The libibraries . MoleculMargaritaria (M. nobilis species and, adjunct to the molecular network created, served as a guide for the putative identification of the compounds present in the matrix under study. MS/MS spectra that did not have any correspondence on the GNPS platform were annotated using Sirius 4 software, in addition to being compared with spectral data present in the scientific literature.An extensive search in the scientific literature was carried out in order to build an internal database for the genus aritaria , which rMargaritaria, as well as supported by high-performance computational tools, we were able to establish a chemical profile for the ethanolic extract of M. nobilis leaves. In our results, 44 compounds were annotated; among these, we highlight compounds ellagic acid, galloyl-HHDP-glucose, quercetin 3-O-glucoside and galloyl-Che-HHDP-glucose that, in the first instance, may support the understanding of expected pharmacological activities for the species. We also highlight that by UHPLC-MS, we were able to analyze trace compounds that in conventional methods would not be verified. We emphasize that monitoring the availability of these compounds is also important, since the magnitude of the bioactive profile of this species can change dramatically due to seasonality.From a workflow based on previous chemical reports from species of the genus Finally, we understand that, through this work, we contributed to the knowledge of the chemical profile of the leaves of this species, providing valuable information for the understanding and certification of pharmacological activities that will be studied in the future.


COVID-19 vaccination for general population started on April 12, 2021, in Osaka, Japan. We investigated public attitudes toward vaccination and associated factors of vaccine hesitancy during the third state of emergency.st dose]”, “Received [2nd dose]”, or “Want to receive vaccines”). Multivariable Poisson regression analysis was conducted to examine the associations between hesitancy and population characteristics.An internet-based, self-reported, cross-sectional survey was conducted in June 2021, using the smartphone health app for residents of Osaka aged ≥18 years. Respondents were asked about their attitudes toward COVID-19 vaccine. Responses “Don’t want to receive vaccines” or “Don’t know” were defined as vaccine hesitancy (vs. “Received [1st dose)”, “Received (2nd dose)”, “Want to receive vaccines”, “Don’t want to receive vaccines”, “Don’t know”, and “Don’t want to answer” were 14.6%, 3.8%, 70.6%, 4.3%, 6.1%, and 0.5% among men; and 11.3%, 6.0%, 64.9%, 6.2%, 11.0%, and 0.6% among women. Factors associated with vaccine hesitancy included being a woman , age 18–39  and 40–64 years , living alone , non-full-time employment and unemployment (aPRs ranged 1.12 to 1.49 vs. full-time employment), cardiovascular diseases/hypertension , and pregnancy (women of reproductive age only) .23,214 individuals  were included in the analysis. Proportions that answered “Received (1Most respondents expressed favorable attitudes toward COVID-19 vaccination while hesitancy was disproportionately high in certain populations. Efforts are needed to ensure accessible vaccine information resources and healthcare services. Coronavirus disease 2019 (COVID-19) has evolved into a global public health threat. Besides non-pharmaceutical measures , vaccines are expected to play a significant role in ending the COVID-19 pandemic by establishing herd immunity [Osaka, the third most populated prefecture (8.8 million)  in Japandelay in acceptance or refusal of vaccination despite availability of vaccination services” [As getting vaccinated is a personal choice, the decision-making process is affected by a variety of intrapersonal, interpersonal, and social contexts. People’s health beliefs including perceived barriers and perceived benefits are considered as key determinants of COVID-19 vaccine hesitancy, and these are directly associated with modifiable or nonmodifiable factors such as gender, education, age, geographical locations, occupation, marital status, and race/ethnicity . Vaccineervices”  is a majIn light of the above, objectives of the present study are to 1) assess public attitudes toward COVID-19 vaccine and 2) examine prevalence and associated factors of vaccine hesitancy using large-scale data.Asmile”. The app was launched in 2019 as part of the Osaka Health and Fitness Support Project and is available for all residents of Osaka aged 18 years or older [Asmile are reported elsewhere [An internet-based, self-reported, cross-sectional survey was conducted during June 1–20, 2021, using the smartphone health app “or older . Upon dolsewhere .Asmile to all users to investigate the changes in health behaviors and attitudes during the third state of emergency in Osaka. Of approximately 246,000 users as of June 2021, 23,460 individuals responded to the survey. After excluding 246 individuals whose basic demographic information  unverified, a total of 23,214 respondents  were included in the analyses. The study was approved by the Institutional Review Board of the Osaka International Cancer Institute . The data were deidentified before use.The study population of the present study was individuals aged 18 years or older residing in Osaka prefecture. The survey questionnaire was developed by the Osaka prefectural government in collaboration with the Osaka International Cancer Institute. Web-based questionnaires were distributed within the smartphone app st dose)”, “Received (2nd dose)”, “Want to receive vaccines”, “Don’t want to receive vaccines”, “Don’t know”, and “Don’t want to answer”. Vaccine hesitancy was defined as either of the responses “Don’t want to receive vaccines” or “Don’t know”. Those who answered “Don’t know” likely intended to wait for some time to see how the vaccine rollout went or simply had not considered taking the vaccine at the time of survey. Thus, they were assumed to delay decision-making and intake of the vaccine, meeting the aforementioned definition of vaccine hesitancy [Respondents were asked “Free-of-charge COVID-19 vaccine rollout has started. Have you already received the COVID-19 vaccine? If not, do you want to receive it?” Response categories included “Received (1esitancy .The assessed population characteristics included sex (man/woman), age (18-39/40-64/65+ years old), number of household members (1/2/3/4+ including self), employment status (full-time/part-time/contractor/self-employed/unemployed/other), presence of morbidities or any health conditions that are listed as potential risk factors for severe illness from COVID-19 infection  .Descriptive analyses were conducted to assess public attitudes toward COVID-19 vaccination and the prevalence of vaccine hesitancy. Multivariable Poisson regression analysis was conducted to examine the associations between vaccine hesitancy and population characteristics by excluding individuals who answered “Don’t want to answer” to the vaccine attitude question (N = 128). All abovementioned variables except pregnancy were included in the main model. A separate model was fitted for women of reproductive age (18–49 years) only to control for pregnancy and all other covariates. Variance inflation factors were computed for all independent variables in the models to examine correlations, and all values were confirmed to be below 5.0. All analyses were performed using R version 4.1.0.Of the 23,214 respondents, the proportions of man and woman were 36.5%  vs. 63.5% , respectively, and individuals aged 18–39 years accounted for 10.3% . The respondents were biased toward women and the middle-aged compared to the sampling source    or the est dose)”, “Received (2nd dose)”, “Want to receive vaccines”, “Don’t want to receive vaccines”, “Don’t know”, and “Don’t want to answer” were 14.6%, 3.8%, 70.6%, 4.3%, 6.1%, and 0.5% among men overall; and 11.3%, 6.0%, 64.9%, 6.2%, 11.0%, and 0.6% among women overall with substantial variations across age groups  . By age,Multivariable analysis revealed that women were more likely to report vaccine hesitancy than men . Individuals aged 18–39 years and 40–64 years had 6.99 times (95%CI = 5.99–8.15) and 4.27 times (95%CI = 3.73–4.90) higher likelihoods of reporting hesitancy, respectively, than older individuals aged 65+ years. Compared to respondents living in a household of 4+ members, those living alone were more likely to report vaccine hesitancy . With full-time workers as the referent, all other types of employees or the unemployed had increased likelihood of vaccine hesitancy. The highest aPR was observed among contractors  followed by unclassified (“other”) individuals  and the self-employed . Respondents who reported having cardiovascular diseases or hypertension had lower likelihood of vaccine hesitancy  compared to those who did not have the disease. Among women, pregnant individuals had 1.35 (95%CI = 1.03–1.76) times higher likelihood of vaccine hesitancy.During June 1–20, 2021, in Osaka, over 80% of respondents in both sexes reported having received at least one does of COVID-19 vaccine or intending to receive one. Vaccine hesitancy was reported by 14.7% of overall respondents with higher likelihood among women, and younger, non-full-time working or unemployed, or pregnant individuals. The present study provides a profile of public attitudes toward COVID-19 vaccination to inform the ongoing rollout strategies.st, 2021 has been approved by the Japanese health ministry [Aligned with findings from previous assessments , 14–17, The associated factors of COVID-19 vaccine hesitancy found in the present study were largely consistent to those from previous studies –16. OkubThe present study is subject to several limitations. First, the internet-based sampling led to biased demographic distribution of the respondents. Although such bias was addressed by stratified analyses and multivariable adjustment, the results may not be representative of the entire Asmile users or the general population of Osaka. Specifically, the data collection was conducted on a voluntary basis that may have resulted in biased estimation of vaccine hesitancy. Given that the respondents were assumed to be more health-conscious and had higher internet literacy than those who did not participate in the survey, they possibly had keener perceptions on susceptibility, severity, barriers, and benefits regarding the COVID-19 vaccine. Since these perceptions contribute to determine the attitude toward the vaccine , vaccineDuring June 1–20, 2021, the majority (≥80%) of respondents in both sexes reported having received the COVID-19 vaccine or intending to receive one. Likelihood of reporting vaccine hesitancy was higher among women and younger, non-full-time working or unemployed, or pregnant individuals. Coordinated efforts are needed through effective communications and community-based interventions to ensure accessible vaccine information resources and healthcare services.


Abdominal aortic aneurysm (AAA) is a lethal peripheral vascular disease. Inflammatory immune cell infiltration is a central part of the pathogenesis of AAA. It’s critical to investigate the molecular mechanisms underlying immune infiltration in early-stage AAA and look for a viable AAA marker.CIBERSORT were used to assess immune infiltration in early-stage experimental AAA. The scRNA-seq datasets were then utilized to analyze AAA-related gene modules of monocytes/macrophages infiltrated into the early-stage AAA by Weighted Correlation Network analysis (WGCNA). After that, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis for the module genes was performed by ClusterProfiler. The STRING database was used to create the protein-protein interaction (PPI) network. The Differentially Expressed Genes (DEGs) of the monocytes/macrophages were explored by Limma-Voom and the key gene set were identified. Then We further examined the expression of key genes in the human AAA dataset and built a logistic diagnostic model for distinguishing AAA patients and healthy people. Finally, real-time quantitative polymerase chain reaction (RT-qPCR) and Enzyme Linked Immunosorbent Assay (ELISA) were performed to validate the gene expression and serum protein level between the AAA and healthy donor samples in our cohort.In this study, we download several mRNA expression datasets and scRNA-seq datasets of the early-stage AAA models from the NCBI-GEO database. mMCP-counter and Thbs1, Clec4e, and Il1b were identified as key genes among the hub genes in the modules, and the high expression of Clec4e, Il1b, and Thbs1 was confirmed in the other datasets. Then, in human AAA transcriptome datasets, the high expression of CLEC4E, IL1B was confirmed and a logistic regression model based on the two gene expressions was built, with an AUC of 0.9 in the train set and 0.79 in the validated set. Additionally, in our cohort, we confirmed the increased serum protein levels of IL-1β and CLEC4E in AAA patients as well as the increased expression of these two genes in AAA aorta samples.Monocytes/macrophages were identified as the major immune cells infiltrating the early-stage experimental AAA. After pseudocell construction of monocytes/macrophages from scRNA-seq datasets and WGCNA analysis, four gene modules from two datasets were identified positively related to AAA, mainly enriched in Myeloid Leukocyte Migration, Collagen-Containing Extracellular matrix, and PI3K-Akt signaling pathway by functional enrichment analysis. This study identified monocytes/macrophages as the main immune cells infiltrated into the early-stage AAA and constructed a logistic regression model based on monocytes/macrophages related gene set. This study could aid in the early diagnostic of AAA. Abdominal aortic aneurysm (AAA) was defined as having diameters 1.5 times greater than normal (or which measure > 3 cm), which is a life-threatening aortic disease characterized by permanent, localized dilations of the abdominal aorta. AAA is an important cause of morbidity and mortality in developed countries . Early d+ T cells, NK cells, and others, played a significant role in the development of AAA For datasets GSE152583 and GSE164678, R package “Seurat” v4.0 was used for quality control, normalization, CCA integration, and TSNE dimensional reduction. Cells were filtered out by nFeature_RNA < 200, nFeature_RNA > 4,000, nCount_RNA > 25,000, and percent. mt > 10. After dimensional reduction, marker genes for different cell types were used for cluster identification, and the marker genes used were consistent with the original publications . For GSEFor datasets GSE51227, GSE109639, GSE17091, GSE57691, and GSE47472, R package “Limma” was used for data normalization. Samples from AOD patients in GSE57691 were excluded as those samples were not relevant to the purpose of our study. Boxplots were generated to confirm the normalization effect by R package “ggplot2” .CIBERSORT, with a reference gene set came from ImmuCC , a method developed recently to quantify immune cell populations for the mouse, was employed to evaluate infiltration of immune cells for the mouse aorta samples by using R package “mMCPcounter” . The othm ImmuCC , 13. TheR package “WGCNA” was used for the Weighted Gene coexpression Network Analysis of monocytes/macrophages populations from datasets GSE152583 and GSE164678. Firstly, monocytes/macrophages populations gene expression matrices were subset, and then we constructed pseudo cells by combining cells in the same sample and clusters. Ten cells were combined as one pseudo cell. After that, high variable genes were selected for further analysis.2 with a confidence score of 0.4, and the disconnected nodes in the network were hidden.The R package “clusterProfiler” (version 4.0.2) was adopted for the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology (GO) functional annotation to explore the biological functions of genes in the modules that were associated with the disease. By setting up cor.geneModuleMembership > 0.8 and cor.geneTraitSignificance > 0.4 for dataset GSE152583, cor.geneModuleMembership > 0.8 and cor.geneTraitSignificance > 0.3 for dataset GSE164678, hub gene in the modules were selected. Then the PPI network of hub genes was constructed by the STRING database2 | FC| ≥ 1 and P < 0.05 were set as cut-offs for GSE152583, while FC ≥ 1.3, FC ≤ 0.7 and P < 0.05 were set as cut-offs for GSE164678, and the results were visualized by volcano map using R package “ggscatter.”According to previous comparative analysis, Limma-voom was an ideal method for DEG analysis of scRNA-seq data . Thus, tMultivariate logistic regression analysis was performed using the glm function in R package “stats.” AAA samples and control samples were used as categorical responsive values, and gene expression values were used as continuous predictive variables. Visualization of logistic regression analysis by dynamic nomogram was constructed through R package “DynNom.” Hosmer-Lemeshow goodness-of-fit test was used for calibration examination. Receiver operating characteristic (ROC) curve analysis was generated to evaluate the model to distinguish AAA and normal aorta samples by R package “pROC.”For the protein-specific enzyme-linked immunosorbent assay (ELISA), a total of 38 patients diagnosed as AAA and 18 age and gender matched healthy controls were enrolled in the study from the First Hospital of China Medical University. The diagnosis of all patients was confirmed by computed tomography angiography (CTA), The exclusion criteria included subjects with chronic aortic dissection, congenital heart disease, severe vascular stenosis, autoimmune diseases, infectious diseases, malignant tumors, hematological system diseases, previous aortic surgery or received non-steroidal anti-inflammatory drugs or steroids. Approximately 5 mL fasting blood sample was collected from each participant using standardized sterile tubes. All samples were centrifuged immediately at 3,000 r/min for 10 min at 4°C, and the serum was separated, and stored at −80°C until analysis.For the mRNA expression detection, 10 patients diagnosed as AAA and 10 age and gender matched healthy controls were enrolled in the study. Fresh infrarenal AAA wall tissue samples were collected from patients undergoing open elective aneurysmectomy, and control infrarenal aortas were obtained from organ donors. All the aortic tissues were put into liquid nitrogen in 30 min after collection and stored at −80°C.Written and informed consent to participate in this study was obtained from all subjects. The baseline characteristic data of the subject involved are presented in RNAiso Plus reagent  was used to extract total RNAs, and the concentration and purity of total RNA were detected by a nanometer photometer (IMPLEN). After that, reverse transcription was performed using PrimeScript RT reagent Kit with gDNA Eraser . Real-time quantitative polymerase chain reaction (RT-qPCR) was conducted using TB Green® Premix Ex Taq  on an ABI Q3 7500 Real-Time PCR System (ABI). ACTB was used as internal controls, and the relative expression level of the target gene was calculated as 2−ΔCt, where −ΔCt = . Statistical analysis was conducted with GraphPad Primer 8.0 . Primer sequences used in the study were listed in Aortic specimens were ground in liquid nitrogen, and IL-1β and CLEC4E concentrations in the serum were measured using a commercial ELISA kit according to the manufacturer’s instructions .U-test was used for pairwise comparison of data that did not followed a normal distribution, while the Student’s t-test was used to evaluate normally distributed data. Correlation analyses were performed by the Spearman test and visualized by R package “corrplot.” P < 0.05 was considered statistically significant.All statistical analyses were completed in the R language (Version 4.0.2). The continuous variables were presented as means ± standard deviations. The Shapiro-Wilk normality test was used to test whether the continuous variables conformed to a normal distribution. The Mann–Whitney 2 induced AAA dataset (GSE164678), we got 15 cell clusters and classified them as monocytes/macrophages (4 clusters), fibroblasts (4 clusters), smooth muscle cells (2 clusters), B cells (1 cluster), endothelial cells (1 cluster), dendritic cells (1 cluster), NK-T cells (1 cluster), and neutrophils (1 cluster) , then we identified these clusters by marker genes and classified them into 10 different cell types, which were monocytes/macrophages (4 clusters), smooth muscle cells (4 clusters), fibroblasts (2 clusters), NK-T cells (2 clusters), endothelial cells (1 cluster), dendritic cells (1 cluster), B cells (1 cluster), erythrocytes (1 cluster) and neural cells (1 cluster). Similarly, from the CaClcluster) . DespiteCIBERSORT was used to estimate the immune cell composition in the sample, among the various immune cell types, monocytes and macrophages got the most enrichment ratios in the early stage AAA samples  created by three different methods. Microenvironment Cell Population counter (mMCP-counter) immune infiltration analysis was used to analyze the differences of the immune cell infiltration levels between different early-stage AAA and control samples. As shown in  samples . All the2 induced AAA dataset (GSE164678), while for the peri-adventitial elastase incubation induced AAA dataset (GSE152583), 84 pseudocells with 1,960 high variable genes were generated from 938 monocytes/macrophages cells. Sample clustering dendrogram for the pseudocells generated from the two datasets was shown in R2 = 0.9) to comply with the scale-free network, and β = 6 was used for the CaCl2 induced AAA dataset , GO analysis revealed that genes within the module are associated with the following BPs including “response to interferon-beta,” “defense response to virus,” and “response to Virus”; the following CCs including “extracellular Matrix,” “collagen-containing extracellular matrix” and “extracellular matrix component”; the following MFs including “protein kinase regulator activity,” “heparin binding” and “kinase regulator activity.” While, KEGG enrichment analysis revealed genes in the module were associated with “PI3K-Akt signaling pathway,” “Epstein-Barr virus infection” and “Viral protein interaction with cytokine and cytokine receptor” . In addiCxcl16, Ifi30, AF251705, Ctss, Cd72, Fcgr4 were identified as hub genes for the yellow module. As for the yellow module in the elastase induced AAA dataset, genes in this module were involved in “myeloid leukocyte migration,” “leukocyte migration” and “leukocyte chemotaxis” for BPs; “lysosome,” “extracellular matrix” and “collagen-containing extracellular matrix” for CCs; “chemokine receptor binding,” “chemokine activity” for MFs. What’s more, KEGG enrichment analysis revealed genes in the module were associated with “NF-Kappa B Signaling Pathway,” “Chemokine Signaling Pathway” and “TNF signaling pathway” . Cxcl16,Thbs1, Csf2rb, Il1b, Cd44, Clec4d, Adam8, and Clec4e. PPI network of hub genes in this module is shown in In terms of genes within the blue module, “positive regulation of cytokine production,” “leukocyte migration,” “extracellular structure organization” were the most enriched pathways for BPs; “extracellular matrix structural constituent,” “receptor ligand activity,” and “cell adhesion molecule binding” were the most enriched for CCs; while “extracellular matrix,” “collagen-containing extracellular matrix” and “receptor complex” were the most enriched pathways for MFs. Meanwhile, “PI3K-Akt signaling pathway,” “Focal adhesion” and “Cytokine-cytokine receptor interaction” were the most enriched pathways for KEGG analysis . Hub gen2 induced AAA dataset (GSE164678), only the yellow module was positively associated with AAA development. GO enrichment analysis revealed that genes in this module were associated with “cell Chemotaxis,” “myeloid leukocyte migration,” and “leukocyte migration” for BPs; “lysosome,” “lytic vacuole,” and “cell-cell junction” for CCs, “cell adhesion molecule binding,” “integrin binding” and “cytokine receptor binding” for MFs. KEGG enrichment analysis revealed “Rap1 signaling pathway,” “Osteoclast differentiation” and “Leukocyte transendothelial migration” pathways were related to genes in this module   . Then wee chosen . After trophages . The elerophages . Furthernduction .P-values of IL1B and CLEC4E were < 0.05 in the logistic regression model. P-values of the Hosmer-Lemeshow test were > 0.05. The area under the curve (AUC) value was used to assess the discrimination of the models. As a result, the AUC of the combined diagnostic method was 0.9  and non-AAA aortas from healthy donors (n = 10). Patients and the control group were age and sex-matched . IL1B was increased in AAA patients  compared with controls . To our surprise, CLEC4E protein was detectable in the serum of all the participants with a high level of expression. Also, the expression of CLEC4E protein was higher in AAA serum than that in control serum .To further validate the high expression of the two genes in human AAA samples, we collected aortic tissues from AAA patients , is a pattern recognition receptor that belongs to the C-type lectin receptor family. Antigen-presenting cells such as macrophages, neutrophils, DCs, and B cells express MINCLE, which can bind a variety of PAMPs generated from the fungal microbiome, including -mannose, lipidic species, and certain endogenous self-ligands such Sin3A-associated protein 130 (SAP130)  , 27. In clerosis . Hence, IL1B, which encoded interleukin-1, is a well-known inflammatory gene involved in various diseases. Due to ROS-mediated inflammasome activation, Il-1 was observed to be increased in the early stages of experimental AAA  than that by elastase induced AAA (day 7), we suggest that the difference in IL1B expression pattern stems from the different timing of sampling. In AAA patients, elevated plasma and aortic wall Il-1β levels were reported in the previous studies (ntal AAA , 30. Somntal AAA . Consist studies , 33. It  studies .IL1B and CLEC4E in the early-stage AAA patient cohorts.There were several limits to our study. First of all, although we used datasets from different AAA models, there was still a certain difference between the pathology of the early-stage experimental AAA and that of human AAA. Secondly, Due to the lack of clinical information in the dataset, risk factors such as gender and smoking status were not considered. Lastly, because it didn’t meet the surgical indications, it was difficult for us to obtain blood and tissue samples from early-stage AAA patients. Thus, we couldn’t evaluate the diagnostic performance of Clec4e, Il1b, and Thbs1 as key monocytes/macrophages related genes. After that, a logistic regression diagnostic model was established based on CLEC4E and IL1B, which can distinguish AAA patients from the control group well.This study downloaded scRNA-seq data and transcriptome data of experimental AAA and human AAA samples from the GEO database. Through multiple bioinformatics analysis methods based on the data, we identified macrophages as the main immune cells infiltrated in the early stage AAA. Moreover, we identified https://www.ncbi.nlm.nih.gov/geo/, GSE152583, GSE164678, GSE166676, GSE51227, GSE109639, GSE17091, GSE57691, and GSE47472.The datasets presented in this study can be found in online repositories. The names of the repository and accession numbers can be found below: The studies involving human participants were reviewed and approved by Ethics Committee at the Chinese Medical University, The First Affiliated Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study.SC and SX designed the study. YL collected the data and materials. SC and YL performed the data analysis. SC wrote the manuscript. YJ and BJ contributed to essential reagents and tools. BJ and SX revised the manuscript. All authors contributed to the article and approved the submitted version.


This study proposes a 3D conductive multichannel carbon framework (MCF) with homogeneously distributed vertical graphene nanowalls (VGWs@MCF) as a multifunctional host to efficiently regulate Li deposition and accelerate Li+ transport. A novel electrode for both Li|VGWs@MCF anode and LFP|VGWs@MCF (NCM811|VGWs@MCF) cathode is designed and fabricated using a dual vertically aligned architecture. This unique hierarchical structure provides ultrafast, continuous, and smooth electron transport channels; furthermore, it furnishes outstanding mechanical strength to support massive Li deposition at ultrahigh rates. As a result, the Li|VGWs@MCF anode exhibits outstanding cycling stability at ultrahigh currents and capacities . Moreover, full cells made of such 3D anodes and freestanding LFP|VGWs@MCF (NCM811|VGWs@MCF) cathodes with conspicuous mass loading (45 mg cm–2 for LFP and 35 mg cm–2 for NCM811) demonstrate excellent areal capacities (6.98 mAh cm–2 for LFP and 5.6 mAh cm–2 for NCM811). This strategy proposes a promising direction for the development of high‐energy‐density practical Li batteries that combine safety, performance, and sustainability.Lithium (Li) dendrite formation and poor Li + ion transport. The dual vertically aligned architecture electrodes using VGWs@MCF offers significant boost in both ultrahigh rates and capacities (40 mA cm–2 and 40 mAh cm–2) for LMBs.Homogeneously distributed vertical graphene nanowalls with abundant exposed edges, as a highly conductive coating layer, are successfully fabricated on 3D multichannel carbon framework (VGWs@MCF), which can effectively regulate Li deposition and accelerate Li Lithium metal anodes (LMAs) are considered to be a promising anode material for next‐generation rechargeable batteries because of their ultrahigh theoretical specific capacity (3860 mAh g–1), extremely low electrochemical potential (−3.040 V vs standard hydrogen electrode), and low gravimetric density (0.534 g cm–3). Since the 1960s, tremendous attention has been given to LMAs. However, their practical utilization is hindered by several intrinsic defects, including dendritic formation during deposition, instability of the Li metal interface, and infinite volume fluctuation during repeated cycles, causing a fracture in the solid electrolyte interphase (SEI) film, leading to the formation of dendrites and “dead” Li. This uncontrollable and dendrite‐forming growth of Li metal can lead to battery short circuits, low cycling coulombic efficiencies, poor cycle lifespans, and safety hazards, which have significantly impeded the application of LMAs.The ever‐growing demand for high‐energy‐density and safe energy storage systems requires the development of battery techniques that provide high specific capacity, stable cycling performance, and a long lifespan.2, 3 which prevents fresh Li metal from further reacting with the electrolyte. Second, prefabricated artificial SEI layer possesses good chemical stability and mechanical properties, benefitting the adaption of the large volume change of the Li metal during the plating and stripping process. Third, employing solid inorganic or polymer electrolytes can partly relieve several safety issues, such as leakage, poor chemical stability, and flammability for liquid electrolytes. Finally, constructing three‐dimensional (3D) conductive scaffolds as the host matrix can not only regulate Li plating and accommodate volume changes but also decrease the local current density due to the enlarged surface area. These strategies have contributed to improving the performance of LMAs; however, the problems of dendrite growth and poor cycling stability remain major concerns in the case of high current density and high areal capacity operation of LMAs.For decades, several strategies have been proposed to solve the notorious issues of LMAs, including the following four approaches. First, inducing additives in the electrolyte is instrumental in stabilizing the SEI films,18 whi Meanwhile, 3D frameworks can effectively support Li and accommodate the stress and strain caused by the large volumetric expansion that occurs during repeated Li plating/stripping cycles, suppressing the formation and growth of Li dendrite. Among the various 3D architecture materials, some typical micro‐/nanostructured frameworks, including metal‐based, carbon‐based, and metal/carbon hybrid frameworks, have been demonstrated to be effective in eliminating the problems mentioned above. Metals, such as stainless steel, Cu, Ni, and Ti, have been widely used to develop 3D‐nanostructured Li hosts. Researchers have developed various nanostructures on the Cu‐metal‐based Li hosts such as porous Cu, copper nanowires (Cu NWs), 3D pie‐like structures, vertically aligned Cu, and Cu foams. Some 3D porous Cu‐Sn, Cu‐Zn, and Cu‐Al alloys have also been used in LMAs. However, the corrosion of metal‐based 3D hosts is difficult to avoid, and their performance under high current density and high capacity is not optimistic. Furthermore, the 3D conductive carbon architecture has been considered an ideal Li host owing to its light weight, flexibility, high mechanical and chemical stability, high electronic conductivity, low cost, and natural abundance. Various porous carbon host matrices, such as carbon nanotubes (CNT), carbon nanofibers (CNFs), hollow carbon nanospheres, graphite carbon foams, multilayer graphene, graphene‐CNT hybrids, reduced graphene oxide, biomass porous carbon, and metal‐organic frameworks, have been employed to regulate the electron and ion distributions, realize a uniform Li plating and stripping morphology and accommodate volumetric Li expansion without any mechanical destruction. Additionally, other metal/carbon hybrid frameworks, such as CNTs, graphene, and metal‐organic frameworks (MOFs) deposited on various 3D metal frameworks allow the bottom‐to‐top deposition of Li and stabilize the SEI. These novel architectures have been developed to improve the performance of LMAs; however, less attention has been paid to Li metal in the case of high current density and high Li storage capacity.The design and fabrication of 3D conductive structures as a host matrix for LMAs play a critical role in reducing the local current density and promoting uniform Li plating/stripping owing to an increase in the electroactive surface area compared with interface engineering design, including electrolyte additives, artificial SEI, protective layers, and separator modification.31 Mea Meanwhile, a higher Li‐ion flux at the host surface, resulting in high Li‐ion diffusion resistance in the bulk electrolyte among the 3D hosts, weakens the effect of the 3D hosts, leading to an overlying deposit of Li and Li dendrites. Hence, various surface modifications are proved to be highly effective in reducing Li nuclear energy and guiding Li deposition on the modified 3D matrix, such as those with active coating materials  and suitable seeding sites . Although significant efforts have been made to introduce lithiophilic materials into various 3D hosts, induce uniform Li nuclei, and alleviate dendrite formation, several issues remain to be addressed. First, in terms of the synthesis process, most 3D conductive hosts are complex and costly, and the inhomogeneous, complicated, and uneconomical preparation technology for the design of lithiophilic‐coating layers limit their large‐scale applications. Second, conventional 3D hosts with relatively large pore sizes (>10 µm) cannot efficiently dissipate large current densities due to their limited surface areas, deteriorating the high‐rate performance of LMAs. Third, the performance of LMAs with high current density and high areal capacity has long been neglected; analysis of this performance would be truly beneficial to the commercialization process. Therefore, it is essential to choose a simple, facile, and economical preparation technology for designing a 3D conductive host with functional surface modification for high current density/areal capacity LMAs.Some strategies, such as electrochemical deposition, molten Li infusion, and mechanical pressurization, have received increasing attention for achieving high‐energy and power density LMBs to host Li metal into 3D structures. However, there is a large nucleation barrier for the Li deposition on different 3D host matrices due to its “lithiophobic” nature.50 Mea3O4‐nanoparticle‐anchored vertical graphene nanowalls with abundant oxygen and nitrogen doping were grown on multichannel carbon framework (VGWs@MCF) from carbonized wood as a highly mechanical Li host for dendrite‐free LMAs. Vertically aligned LiFePO4 (LFP) and LiNi0.8Co0.1Mn0.1O2 (NCM811) were fabricated using a facile vacuum filtration method to facilitate Li+ transport and reduce Li+ diffusion resistance to minimize the Li+ transport tortuosity in both the anode and cathode, enhancing the high areal capacities and rate performance. Both fast‐charging Li|VGWs@MCF symmetric cells and LFP|VGWs@MCF (NCM811|VGWs@MCF) full cells were fabricated based on the dual vertically aligned electrode structure. Particularly, a 3D conductive MCF with homogeneously distributed vertical graphene nanowalls (VGWs@MCF) host offers the following advantages: (i) 3D VGWs with well‐ordered structure and edge‐enriched surface growth on the carbonized wood matrix via a chemical vapor deposition (CVD) technique significantly regulate the local current distribution and maximize Li storage due to their enlarged specific surface area and porous structure. (ii) The uniform distribution of ZnO and Co3O4 nanoparticles and N, O, dual‐doped graphene nanowalls endow the VGWs@MCF host with excellent lithiophilicity, guaranteeing uniform Li deposition behavior with a small overpotential. (iii) The VGWs@MCF host possesses high electric conductivity, mechanical strength, and abundant microchannels, which are beneficial for suppressing Li dendrite growth and reducing the risk of short‐circuiting under high current densities and capacities. Additionally, the vertically aligned LFP (NCM811) electrode enabled by simply infiltrating commercial cathode materials into the channels obtained ultrahigh mass loading of electroactive materials and low‐tortuosity pathways for Li+ ion transport, demonstrating a high capacity and outstanding rate performance. Consequently, stable cycling performance of the assembled symmetric cells over 5200 h was achieved at a current density of 1.0 mA cm–2 with an areal capacity of 1.0 mAh cm–2. Remarkably, excellent cycling stability was maintained over 800 h under ultrahigh current densities and areal capacities of 40 mA cm–2 and 40 mAh cm–2, respectively, with a low overpotential of ≈100 mV. Moreover, the LFP| Li|VGWs@MCF full cell delivered a high capacity of 6.98 mAh cm–2 at 0.1C, which is comparable to those of other reported high‐performance LFP cathodes. The NCM811|Li|VGWs@MCF full cell also exhibited an improved cycling life of over 400 cycles at a high rate of 1C with a discharge capacity of 2.61 mAh cm–2. Hence, the insights gained from this work will open new opportunities for developing LMAs with high current densities and capacities.This study proposes a dual vertically aligned electrode configuration with high conductivity and mechanical stability to develop a dendrite‐free LMA and high‐capacity cathode. In this design, 3D hierarchical ZnO, Co22.1Figure3) and carbon dioxide (CO2) mixture  and CH4. When the concentration of CH4 was significantly high, the graphene nanowalls became granular, whereas the thickness of the graphene nanowalls increased when the concentration of hydrogen was low , D peak (≈1356 cm–1), and 2D peak (≈2727 cm–1) were observed in the spectrum of VGWs@MCF, which is a signature of graphene; and it can be attributed to the introduction of graphene nanowalls. The 2D peak was absent in original carbonized wood. The presence of high‐intensity D′ (≈2445 cm–1) and D′′ (≈2939 cm–1) peaks indicates the formation of many sharp edges in the VGWs. The intensity ratio of I2D/IG was 0.61, revealing the property of the graphene nanowalls with few layers. The intensity ratio (≈3.26) of ID/IG for the MCF was higher than that of the VGWs@MCF (≈1.24), which is primarily related to the defects caused by NH3 etching within the MCF. Raman mapping  analysis was performed to investigate the chemical state properties of VGWs@MCF. The full spectrum of VGWs@MCF proves the existence of C, N, and O elements  and bare Cu (55 mV)  of 259.4 Ω than Li|MCF and bare Li. The Rct values dropped to 150.4, 189.4, and 164.5 Ω after the first cycle for bare Li, Li|MCF, and Li|VGWs@MCF anodes, respectively. Moreover, the resistances of Li|VGWs@MCF anodes decreased to 68.5 Ω after 20 cycles. However, the resistance of Li foil significantly increased due to the crack of SEI and the formation of dead Li. Both the SEI layer resistance (4.5 Ω) and charge transfer resistance (11.7 Ω) for the Li|VGWs@MCF anodes after 100 cycles of Li plating and stripping were significantly lower than those of the Li foil anode (2208.0 Ω for SEI layer resistance and 660.7 Ω for charge transfer resistance)  and deposition capacity (20 mAh cm–2), the high CE (over 98%) of VGWs@MCF demonstrated excellent plating and stripping stability  distributions, which is beneficial for Li deposition. The circles in Figure + ions on the upper surface were larger than those within the microchannels of the MCF electrode, resulting in the preferential aggregation of Li on the surface of the MCF skeleton  is schematically summarized to clarify the advantages of vertical graphene nanowalls grown on this 3D multichannel carbon framework during the Li plating and stripping process. n Figure  and 2g–kn Figure  and 4l, w Figure . The disw Figure . This re2.40.8Co0.1Mn0.1O2 (NCM811) as cathode were assembled to demonstrate the potential use of the Li|VGWs@MCF anode in practical applications. The 3D multichannel frameworks for Li metal inspired us to develop ultrathick 3D vertically aligned electrodes of LFP or NCM811 cathodes with a large thickness of 800 µm and ultrahigh active material loading of 45 mg cm–2 by infiltrating the commercial cathode materials into the channels of the 3D VGWs@MCF. Furthermore, a vertically aligned LFP or NCM811 cathode was introduced into 3D MCFs with abundant VGWs to facilitate Li+ transport and reduce Li+ diffusion resistance, improving the rate performance and long‐term cycling lifespan. Therefore, we employed a 3D VGWs@MCF structure for both the anode and cathode in the full battery design of dual vertically aligned electrodes , increased with increasing LFP mass loading when the mass loading was up to 45 mg cm–2, which is consistent with the SEM results , which also proved that the LFP particles were successfully pumped into the graphene channels. As shown in Figure –2, respectively. As the rate increased, the full cell of Li|VGWs@MCF/LFP|VGWs@MCF‐45 exhibited high discharge capacities of 6.61, 6.38, 6.06, 5.79, 5.53, 5.32, 4.8, and 4.456 mAh cm–2 at 0.1, 0.2, 0.5, 1, 2, 4, 6, and 8 C, respectively. Even at a high rate of 10 C, a high specific capacity of 4.21 mAh cm–2 was maintained for the dual vertically‐aligned cell, while the Li/LFP cell only delivered a capacity of 0.54 mAh g–1 at 5 C. The cell‐level gravimetric and volumetric energy densities of the LFP/VGWs@MCF electrodes were calculated based on the detailed components of cells  and electrode–electrolyte interface resistance  for the LFP|VGWs@MCF electrode compared with the conventional LFP electrode (Rct of 483 Ω and Re of 9 Ω)  was observed for the LFP|VGWs@MCF‐45 electrode   electrode, and 5.6 mAh cm–2 for the NCM811 (35 mg cm–2) electrode. Even at a high current of 6 C, areal capacities of 4.8 mAh cm–2 for LFP and 1.3 mAh cm–2 for NMC811 were maintained. These results contribute to the improved Li+ transport kinetics by the dual vertically aligned electrode structure in Li|VGWs@MCF and LFP|VGWs@MCF (NCM811|VGWs@MCF). This study provides a new strategy for designing high current density and areal capacity electrodes, achieving a series of dendrite‐free metal anodes. Furthermore, it paves the way for energy storage devices with high energy and power densities.A dual vertically aligned electrode using hierarchical 3D MCF hosts with VGWs was designed and fabricated by depositing Li metal and filtrating cathode materials into an optimized 3D conductive network structure. On the anode side, 3D VGWs@MCF possessed high conductivity and sufficient space to promote uniform interfacial Li4Methods and any associated references are available in the online version of the paper.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supporting Information Video 1Click here for additional data file.


Chrysanthemum × grandiflorum (Ramat.) Kitam. ‘Lilac Wonder’ and ‘Richmond’ leaf explants were cultured on the modified MS medium supplemented with 0.6 mg·L−1 6-benzylaminopurine (BAP) and 2 mg·L−1 indole-3-acetic acid (IAA) and treated with AgNPs . AgNPs strongly suppressed the capability of leaf explants to form adventitious shoots and the efficiency of shoot regeneration. The content of primary and secondary metabolites  and the activity of enzymatic antioxidants (superoxide dismutase and guaiacol peroxide) in leaf explants varied depending on the AgNPs treatment and age of culture. Phenotype variations of ex vitro cultivated chrysanthemums, covering the color and pigment content in the inflorescence, were detected in one 50 mg·L−1 AgNPs-derived and five 100 mg·L−1 AgNPs-derived ‘Lilac Wonder’ plants and were manifested as the color change from pink to burgundy-gold. However, no changes in inflorescence color/shape were found among AgNPs-treated ‘Richmond’ chrysanthemums. On the other hand, the stem height, number of leaves, and chlorophyll content in leaves varied depending on the AgNPs treatment and the cultivar analyzed. A significant effect of AgNPs on the genetic variation occurrence was found. A nearly two-fold higher share of polymorphic products, in both cultivars studied, was generated by RAPD markers than by SCoTs. To conclude, protocols using leaf explant treatment with AgNPs can be used as a novel breeding technique in chrysanthemum. However, the individual cultivars may differ in biochemical response, the efficiency of in vitro regeneration, genetic variation, and frequency of induced mutations in flowering plants.Novel and unique properties of nanomaterials, which are not apparent in larger-size forms of the same material, encourage the undertaking of studies exploring the multifaced effects of nanomaterials on plants. The results of such studies are not only scientifically relevant but, additionally, can be implemented to plant production and/or breeding. This study aimed to verify the applicability of silver nanoparticles (AgNPs) as a mutagen in chrysanthemum breeding.  The natus Voss. . On the Fukuhara . In Alliof seeds .Vicia faba L. root meristem cells. The genotoxicity increased with the increasing concentration of AgNPs, from 12.5 to 100 mg·L−1. In another experiment, the 10, 20, 40, and 50 mg·L−1 AgNPs-treated root tip cells of Triticum aestivum L. exhibited various types of chromosomal aberrations [Despite many perspectives and benefits arising from the enormous progress in nanotechnology development, nanomaterials at higher concentrations may also have adverse effects that have not been sufficiently explored during the implementation of the innovative agro-nanotechnologies in crop improvement. A good understanding of nanomaterials’ influence on plants at the morphological, histological, physiological, biochemical, and molecular levels is of paramount importance for assessing nanomaterials toxicity, evaluating environmental risks, food safety, and human health. The genotoxic effects, which are often difficult to predict, in particular, need deeper research ,5,8. Aftrrations .+) released from AgNPs can interact chemically or physicochemically with nucleic acids and induce DNA damage [The main mechanism underlying the AgNPs phytotoxicity is the overproduction of reactive oxygen species (ROS) leading to oxidative stress in plant cells, lipid peroxidation, damage to cell membranes, proteins, and DNA ,8,15. MoA damage . HoweverA damage . The mosA damage ,19,20. HA damage  or mutatA damage .Chrysanthemum × grandiflorum (Ramat.) Kitam. is one of the most economically important and favored floricultural crops worldwide, ranking second in the cut flower trade, following the rose. Chrysanthemums are mainly valued for their multi-colored inflorescences characterized by an exceptionally long flowering period [g period . Their ig period ,22.3) almost exclusively cause single base substitutions [The most frequently used chrysanthemum breeding methods are generative crossing and mutagenesis . Howeveritutions ,25. In citutions ,23,26.−1 AgNPs [Mutation induction with X- or Gamma-rays is, unfortunately, associated with the use of specialized devices located in medical centers or scientific institutions. The application of nanoparticles as a new chemical mutagen added into/onto the culture medium could make chrysanthemum breeding relatively easy and be routinely performed in in vitro plant laboratories, without the need for sophisticated equipment used in the new breeding technologies (NBTs). Despite the first report on the genotype and phenotype variation in chrysanthemum as a result of adventitious shoots regeneration on internodes inoculated on media supplemented with 5, 10, and 20 mg·L−1 AgNPs , the sur−1 AgNPs ,26,27. A−1 AgNPs .Chrysanthemum × grandiflorum (Ramat.) Kitam. ‘Lilac Wonder’ and ‘Richmond’ cultivars, induced as a result of in vitro application of silver nanoparticles colloid at the concentration of 50 and 100 mg·L−1. The selected cultivars were used previously in breeding programs due to their genetic and phenotypic uniformity and non-chimeric structure [This study aimed to analyze the biochemical activity of leaf explants cultured in vitro, evaluate the effectiveness of in vitro adventitious shoots regeneration, and identify genetic and phenotypic variation among ex-vitro-cultivated plants in tructure ,12,26. T−1). Chrysanthemum ‘Lilac Wonder’ explants produced callus more intensively than ‘Richmond’ explants. In both cultivars tested, adventitious shoot regeneration occurred indirectly. The formation of the first adventitious shoots was observed in the fourth week of culture on ‘Lilac Wonder’ explants in all experimental combinations and on ‘Richmond’ control explants. Conversely, in ‘Richmond’, 50 mg·L−1 and 100 mg·L−1 AgNPs-treated leaves started to regenerate first shoots in the fifth and sixth week of culture, respectively , compared to non-treated explants forming, on average, almost 5 shoots. The same tendency resulting from silver nanoparticles application was observed in chrysanthemum ‘Richmond’ whose control leaf explants regenerated 1.35 shoots, whereas explants treated with 50 and 100 mg·L−1 AgNPs formed only 0.28 and 0.02 shoots, respectively  negatively affected the capability of explants to produce chlorophyll b, total chlorophylls, and carotenoids. As for the interaction between the AgNPs concentration and the week of culture, in ‘Lilac Wonder’, the highest content of chlorophyll a, chlorophyll b, total chlorophylls, and carotenoids was reported in the first week of culture for 0–100 mg·L−1 AgNPs treatments and in the second culture week for 50 mg·L−1 and 100 mg·L−1 AgNPs treatments, while the interaction between the control object and the third week of culture was characterized with the lowest content of these metabolites. Similarly, in ‘Richmond’, the highest values for chlorophyll a, chlorophyll b, total chlorophylls, and carotenoids contents were reported in the first week of culture, in the whole range of the tested AgNPs concentrations, and the second week of culture but for 0 mg·L−1 and 50 mg·L−1 AgNPs treatments. Explants treated with 100 mg·L−1 AgNPs and analyzed in the third week of culture had the lowest content of chlorophyll a, chlorophyll b, total chlorophylls, and carotenoids. No significant differences in the main effects and interactions between the tested experimental factors were observed for the values of chlorophyll a/b and chlorophylls/carotenoids ratios in ‘Richmond’, and chlorophylls/carotenoids ratio in ‘Lilac Wonder’. The highest chlorophyll a/b ratio was found in the third week of culture in 100 mg·L−1 AgNPs-treated ‘Lilac Wonder’ leaf explants   and the lowest for the interaction of 100 mg·L−1 AgNPs × first week of culture (27.65 U)  in ‘Lilac Wonder’ leaf explants were observed depending on the silver nanoparticles treatment and irrespectively of the culture duration A. Howevectively) D. On thectively) B, whereactively) E. As for27.25 U, C and in 21.96 U, F. On the culture A,D. As f culture B,E. In ‘27.65 U) C. Considectively F.−1, and 100 mg·L−1 AgNPs-derived plants in ‘Richmond’ and also for all standard and 100 mg·L−1 AgNPs-treated ‘Lilac Wonder’ plants. As for the control and 50 mg·L−1 AgNPs-derived ‘Lilac Wonder’ chrysanthemums, 59% and 56.5% of them, respectively, survived the acclimatization process. Losses in the number of acclimatized plants were caused by fungal contamination and the weaker ability of plants to adapt to ex vitro conditions during acclimatization in the greenhouse. Flowering was not significantly influenced by AgNPs treatment and occurred in 74.6–96.0% ‘Lilac Wonder’ chrysanthemums and 68–100% ‘Richmond’ chrysanthemums . The mutation was phenotypically manifested by the change of inflorescence color—from pink to light pink. This plant was also of chimeric structure because part of one ligulate floret was covered with a narrow sector (stripe) of burgundy-gold color. Five mutants with changed color of the whole inflorescence were observed among 100 mg·L−1 AgNPs-treated chrysanthemums; the frequency of mutant/mutation occurrence amounted to 10%. Three plants were characterized by changed inflorescence color to burgundy-gold . The mutation identified in Individual No. 4 and Individual No. 5 was related to inflorescence color change to light burgundy-gold. No mutations concerning inflorescence shape were found among the flowering ‘Lilac Wonder’ chrysanthemums. On the other hand, all flowering ‘Richmond’ chrysanthemums formed typical purple-pink inflorescences, regardless of experimental treatment. However, the shape of one plant from the control treatment  was changed from full and flat to full and irregular  with the highest number of leaves . Nonetheless, chrysanthemums from these two experimental objects contained less chlorophyll in leaves (22.02–22.39 CCI) than the standard plants (22.97 CCI). No significant differences were found between the standard plants and 100 mg·L−1 AgNPs-derived plants in terms of the stem length, the number of leaves, and chlorophyll content. On the other hand, ‘Richmond’ plants regenerated on explants treated with silver nanoparticles at the concentration of 50 mg·L−1 produced the shortest stems (49.74 cm) during ex vitro cultivation as compared to the other treatments (61.91–70.00 cm). The highest number of leaves was reported on the stems of standard chrysanthemum (42.11) and 100 mg·L−1 AgNPs-derived plants (50). No differences were found in chlorophyll content depending on nanoparticles treatment. The size of inflorescence diameter was not affected by AgNPs application, both in ‘Lilac Wonder’ (7.02–7.59 cm) and in ‘Richmond’ (7.7–9.08 cm)  .loci were polymorphic). As for the cultivar ‘Richmond’, the S1 primer produced 12 amplicons per sample. On the other hand, primer R5 generated only two amplicons in ‘Lilac Wonder’.A total of 9563 scorable bands were detected by five RAPD and five SCoT primers in 153 plants . Among tAn over two-fold higher heterozygosity index (H) was reported in the ‘Richmond’ plants compared to ‘Lilac Wonder’, according to both RAPD and SCoT markers . A simil−1) after 50 mg·L−1 AgNPs treatment, 100 mg·L−1 AgNPs treatment (only in ‘Lilac Wonder’), and in the standard plants of ‘Lilac Wonder’. The results of the grouping UPGMA analysis of the studied populations varied depending on the marker system used (Polymorphic plants could be found in the untreated control plants (0 mg·Ltem used .−1) was the most distant from the remaining populations placed in a single cluster divided into two sub-clusters (standard and AgNPs-treated). On the other hand, according to the SCoT analysis, the population of chrysanthemums treated with 100 mg·L−1 AgNPs was significantly different from the other three populations grouped in a single cluster  from the control and 50 mg·L−1 AgNPs objects significantly different from the standard.As for the cultivar ‘Richmond’, four groups were distinguished by the RAPDs, with five plants (representing three genotypes) obtained as a result of 50 mg·LPT = −0.029; The AMOVA analysis confirmed a significant influence of the experimental treatments on the occurrence of interspecific genetic variation in most experimental objects , except for the SCoT analysis in chrysanthemum ‘Lilac Wonder’ . The regeneration of adventitious shoots is related to the acquisition of pluripotency by the callus cells and their further dedifferentiation and redifferentiation [Micropropagation through adventitious shoots regeneration from non-meristematic explants combined with mutagen treatment is a commonly exploited technique in chrysanthemum breeding, allowing for a restitution, from a single mutated explant cell, of genetically homogenous plants with a changed phenotype. The application of mutagens usually limits the regeneration capacity of the treated explants ,26. For ntiation ,26. Thisntiation . Apparenntiation  and Milentiation .−1, 10 mg·L−1, or 20 mg·L−1 AgNPs. In calli, the concentration of chlorophylls and polyphenols was stable, regardless of AgNPs concentration, but the production of carotenoids was enhanced with 20 mg·L−1 AgNPs-treatment. In contrast, in shoots, the content of chlorophylls was lower with 10 mg·L−1 and 20 mg·L−1 treatments, but no differences in carotenoids and phenolic compounds were found [Similarly to our study, variable results concerning the response of plant cells at the biochemical level after AgNPs treatment were also reported earlier in chrysanthemum ‘Lilac Wonder’ calli and adventitious shoots regenerated on 10-week-old internodes cultivated on media supplemented with 5 mg·Lre found . Usuallyre found ,6,30. More found  and usuare found . To summ−1) application during different stages of a cryopreservation protocol in Lamprocapnos spectabilis ‘Valentine’ revealed that the activities of SOD, APX, and CAT were significantly higher when AuNPs were added at higher concentration into the preculture (SOD) or recovery media , but no differences were found in GPOX activity [Solanum lycopersicum L. ‘Poranek’, Raphanus sativus L. var. sativus ‘Ramona’, and Brassica oleracea var. sabellica ‘Nero di Toscana’, the three-week-old seedlings developed from seeds treated with 50 and 100 mg·L−1 AgNPs did not differ in terms of SOD activity. The 100 mg·L−1 AgNPs treatment resulted in an almost two-fold higher GPOX activity in tomato but decreased the activity of this enzyme in radish. As for kale, a significant increase in GPOX activity was found with both 50 and 100 mg·L−1 AgNPs concentrations. These results are in line with our findings and clearly show that nanoparticles interact differentially with individual plant species and that elevation/reduction in activity or inactivation of particular enzymes can occur over time [Studies on gold nanoparticles  was tested, and a different method of NPs application was used; AgNPs were added into a regeneration medium at lower concentrations of 5 mg·L−1, 10 mg·L−1, and 20 mg·L−1 [−1 AgNPs, while three mutants with light pink inflorescence, one mutant with burgundy-gold, and one mutant with dark pink inflorescences were identified among 20 mg·L−1 AgNPs-derived plants. Moreover, one of the light pink mutants presented a changed inflorescence type, and the second light pink mutant was of chimeric structure with a red stripe on one ligulate floret. The frequency of mutant/mutations occurrence amounted to 1%/1% for 10 mg·L−1 and 6.3%/8.9% for 20 mg·L−1, respectively, and it was lower than in the present study with the higher AgNPs concentrations tested (2.3%/2.3% at 50 mg·L−1 and 10%/10% at 100 mg·L−1). This comparison shows that in the same cultivar tested, different results in mutation induction can be obtained depending on the explant type used, AgNPs concentration, and the method of their application. One should also keep in mind that mutation induction is a random phenomenon and can also occur spontaneously during the in vitro and/or ex vitro chrysanthemum growth, as in the present study, among flowering control shoots in ‘Richmond’.In the present experiment, ‘Lilac Wonder’ cultivar treated with 50 mg·L0 mg·L−1 . ConsequIn chrysanthemum breeding with the use of γ-radiation, white-flowering chrysanthemum ‘Albugo’ produced eight mutants with a yellow inflorescence, while dark pink ‘Satinbleu’ produced six mutants of pale pink inflorescences . The app−1 AgNPs-derived flowering ‘Lilac Wonder’ chrysanthemums had the longest stems compared to standard plants. However, 50 mg·L−1 AgNPs-derived ‘Richmond’ chrysanthemums developed the shortest stems. No AgNPs influence on the inflorescence diameter, both in ‘Lilac Wonder’ and in ‘Richmond’, was found. Miler and Kulus [2) produced longer shoots (by 39%) and inflorescences of greater diameter (by 21.5%) compared to the control. An interesting effect of enhanced growth, stimulated by a low dose of irradiation, was observed in ‘Profesor Jerzy’ and ‘Karolina’ chrysanthemum cultivars. Plants originating from ovaries irradiated with 5 Gy high-energy photons had the longest stems; 10 Gy-derived plants were medium sized, while 15 Gy-derived plants were the shortest [The spectrophotometric analysis of pigments in ligulate florets of mutants obtained in the present study revealed that variations of inflorescence color were a result of both quantitative and qualitative differences in the content of anthocyanins and carotenoids. These findings are in agreement with those reported for AgNPs-derived mutants in ‘Lilac Wonder’  and for nd Kulus  reportedshortest . Such deIn the present study, a nearly two-fold higher share of polymorphic products, in both cultivars studied, was generated by RAPD markers than by SCoTs. Our findings are in agreement with those reported by Lema-Rumińska et al.  and Kulu−1). Our findings confirm the genotoxic character of nanoparticles, also described by other authors [Lamprocapnos spectabilis (L.) Fukuhara plants at 100 mg·L−1 concentration [The results of the AMOVA analysis showed a significant influence of AgNPs on the genetic variation occurrence during in vitro adventitious organogenesis from leaf explants in chrysanthemum. A similar phenomenon was reported by Tymoszuk and Kulus  in the a authors , particuntration .Interestingly, a significant effect of the genotype factor on the efficiency of mutation induction was observed. According to both marker systems analyzed, chrysanthemum ‘Richmond’ generated significantly more new genotypes than ‘Lilac Wonder’. This cultivar was also characterized by higher values of nearly all polymorphic indices described in −1 sucrose and 8 g·L−1 Plant Propagation LAB-AGARTM . The medium was supplemented with plant growth regulators (PGRs): 0.6 mg∙L−1 6-benzylaminopurine (BAP) and 2 mg∙L−1 indole-3-acetic acid (IAA)  to stimulate the regeneration of adventitious shoots. The medium pH was adjusted to 5.8. Next, 40 mL of the medium was poured into 350 mL glass jars sealed with plastic and autoclaved at 105 kPa and 121 °C for 20 min.For in vitro culture, the modified MS medium  was used−1 . The AgNPs solutions were sterilized with the use of syringe filters . Next, the nanocolloids were poured onto the culture medium—2 mL of AgNPs colloid at each tested concentration per culture jar. Explants inoculated on the regeneration medium without AgNPs (0 mg·L−1) were used as the control. Silver nanoparticles were manufactured by Nanoparticles Innovation NPIN s.c. . The AgNPs were produced by the seeded-mediated growth method [TM, Hillsboro, OR, USA) at accelerating voltage 30 kV were 20 ± 3 nm. The DLS size distribution graphs and STEM images of the used silver nanoparticles are presented in Tymoszuk and Miler [Leaves, dissected from plantlets cloned by the single-node method on the modified MS medium without PGRs or AgNPs, were used as explants. Four leaves were placed in a vertical position per each culture jar filled with the medium for adventitious shoots regeneration. Immediately after inoculation in the medium, the explants were treated with silver nanoparticles at the concentration of 50 and 100 mg·Lh method ,49 and cnd Miler  and Tymond Miler .−2·s−1.In vitro cultures were incubated in the growth room at a constant temperature of 23 ± 1 °C, under a 16 h photoperiod, using Philips TLD 36W/54 fluorescent lamps emitting cool daylight . The photosynthetic photon flux density was set at 35 μmol mObservations of the dynamics of adventitious shoots regeneration on all inoculated leaf explants depending on the experimental treatment were performed weekly for nine successive weeks. In the 10th culture week, the shoot induction rate of explants and the mean number of shoots per one inoculated explant were estimated.−1 IAA for two weeks. Additionally, 25 shoots multiplied in vitro via the single-node method on the modified MS medium without PGRs and AgNPs were rooted for further ex vitro cultivation to form a genotype/phenotype standard of ‘Lilac Wonder’ and ‘Richmond’ plants.Regenerated adventitious shoots  were cut off from leaf explants and transferred onto the modified MS rooting medium supplemented with 2.0 mg·L−1 AgNPs. The whole-leaf explants were used as fresh tissue samples. Chlorophylls and carotenoids were extracted using 100 mg samples and 100% acetone  according to Lichtenthaler’s [v/v) , as described by Harborne [In the first, second, and third weeks of the in vitro culture, biochemical analyses were performed to assess the oxidative stress response of leaf explants after the application of 0, 50, and 100 mg·LHarborne . The samHarborne  with galTo determine the enzymatic activity, samples (100 mg) were prepared as described by Homaee and Ehsanpour . The obtmax): for anthocyanins (cyanidin-3-glucoside)—at 530 nm; for chlorophyll a and b—at 645 and 662 nm; for carotenoids—at 470 nm; for phenolics—at 765 nm; for proteins—at 595 nm; for SOD—at 560 nm; and for GPOX—at 470, respectively. The contents of the plant pigments and the phenolic compounds were calculated per 1 g of sample fresh weight (FW). The enzymatic activity U (μmol·min−1) was calculated per 1 mg of protein.The spectrophotometric analyses were performed using the SmartSpec PlusTM spectrophotometer  at specific wavelengths (λv/v). Plants were regularly sprayed with water and covered with perforated transparent foil and geo-cover. Next, the plants were transferred to plastic pots  filled with peat substrate . In the early growing period, plants were cultivated vegetatively from July to mid-August in the natural photoperiod to achieve the shoot height of about 20 cm and develop the appropriate number of leaves. Starting from 15 August, further cultivation was conducted in 10 h short-day conditions to induce the generative development and bring plants to full flowering .Rooted plantlets were acclimatized in a glasshouse, in June, in natural light conditions, for two weeks. They were planted in plastic trays filled with a mixture of peat substrate  and perlite  , and AgNPs-treated plants. The color of the inner and outer sides of ligulate florets of fully developed inflorescences was established using the Royal Horticultural Society Colour Chart catalog (RHSCC) [At the full flowering stage, the mutants  and mutations  were identified. The frequency of mutant and mutation occurrence was determined against the total number of flowering plants. The mutants were distinguished by defining the color and shape of the inflorescences of the standard , control (0 mg·L (RHSCC) . Moreove (RHSCC)  and Harb (RHSCC)  protocolThe genetic fidelity of AgNPs-treated plants was assessed using randomly amplified polymorphic DNA (RAPD)  and star® NP80 . The isolated DNA was stored at 4 °C.Total genomic DNA was extracted from fresh leaf tissues. The Genomic Mini AX Plant SPIN Kit  reagents and materials were used to isolate DNA. The concentration of DNA was measured and standardized with the NanoPhotometerTM Express DNA Ladder  100–5000 bp DNA marker was used as a size reference.The DNA samples were used as a template for the PCR analysis with a total of 10 primers (5 RAPD and 5 SCoT). PCR was performed in the BioRad C1000 Touch thermal cycler  in the 25 µL reaction solution. The composition of the reaction solution, PCR profiles, and electrophoretic separation of amplified DNA fragments were described in detail in Lema-Rumińska et al. . The PCRloci were counted. Values of heterozygosity index (H), polymorphic information content (PIC), effective multiplex ratio (E), marker index (MI), discriminating power (D), and resolving power (R) were investigated for every primer and marker system used, independently for each cultivar [The banding patterns were recorded as 0–1 binary matrices, where ‘1’ indicates the presence and ‘0’ the absence of a given fragment followed by statistical analysis. For every primer tested, the numbers of monomorphic, polymorphic, and specific/unique cultivar .p ≤ 0.05. For data expressed as a percentage, the Freeman–Tukey double-arcsine transformation was used. Tables with results provide numerical data, with the alphabet indicating the homogeneous groups. All statistical analyses were performed with the use of the Statistica 13.3 software .The experiment was set up in a completely randomized design. Each treatment in the experiment with in vitro cultures consisted of 15 jars . One explant was considered as one repetition. All biochemical analyses performed at the in vitro and ex vitro stages were repeated six times. The obtained data were presented as mean ± standard deviation (SD) and subjected to a one-way or two-way analysis of variance (ANOVA) and post hoc Tukey’s test at the significance level of −1; 25 from 50 mg·L−1; and 25 from 100 mg·L−1 AgNPs treatment, respectively) and 43 ‘Richmond’ plants , as well as 25 standard plants of each cultivar were included in the genetic stability analysis. The coefficient of genetic distance based on the Nei and Li algorithm [−1; control) and treated with AgNPs (50 and 100 mg·L−1). The dendrograms were created based on agglomerative hierarchical clustering (AHC) with the unweighted pair group average method (UPGMA) using Statistica 13.3. Population groups were distinguished based on the analysis of molecular variance (AMOVA) and principal cluster analysis (PCoA) estimates using GeneAlEx 6.5 software [A total of 75 ‘Lilac Wonder’ plants (25 from 0 mg·Llgorithm  was calcsoftware  with thesoftware .−1 and 100 mg·L−1 limited the capacity of leaf explants to form adventitious shoots, affected the biosynthesis of primary and secondary metabolites, and modulated the activity of antioxidant enzymatic defense system in the tested chrysanthemum cultivars, similarly to the action of mutagens used in plant breeding but in a cultivar-specific way. The impact of AgNPs on the genetic variation occurrence during in vitro adventitious shoot organogenesis in the two tested cultivars and the later identification of mutations in the plants’ phenotype during ex vitro cultivation in ‘Lilac Wonder’ allow concluding that the proposed innovative and, at the same time, relatively easy-to-perform method of AgNPs use for mutation induction can find practical application in chrysanthemum breeding. Nevertheless, the response of individual cultivars to AgNPs treatment varies, and the changes induced at the genetic level are not always manifested in the phenotypic traits of chrysanthemum inflorescence or plant architecture during the final stage of cultivation. Future studies could focus on the use of NPs of a different type, size, and concentration to evaluate their mutagenic potential in different chrysanthemum cultivars or plant species and implement other methods for the assessment of cyto- and genotoxic effects of nanoparticles on plant cells.The results of the conducted experiment provide a better understanding of the multifaced influence of AgNPs on plants at the biochemical, genetic, and phenotypic levels. Silver nanoparticles applied at the concentrations of 50 mg·L