Fusarium oxysporum, severely reduces ornamental quality and yields. WRKY transcription factors are extensively involved in regulating disease resistance pathways in a variety of plants; however, it is unclear how members of this family regulate the defense against Fusarium wilt in chrysanthemums. In this study, we characterized the WRKY family gene CmWRKY8-1 from the chrysanthemum cultivar ‘Jinba’, which is localized to the nucleus and has no transcriptional activity. We obtained CmWRKY8-1 transgenic chrysanthemum lines overexpressing the CmWRKY8-1-VP64 fusion protein that showed less resistance to F. oxysporum. Compared to Wild Type (WT) lines, CmWRKY8-1 transgenic lines had lower endogenous salicylic acid (SA) content and expressed levels of SA-related genes. RNA-Seq analysis of the WT and CmWRKY8-1-VP64 transgenic lines revealed some differentially expressed genes (DEGs) involved in the SA signaling pathway, such as PAL, AIM1, NPR1, and EDS1. Based on Gene Ontology (GO) enrichment analysis, the SA-associated pathways were enriched. Our results showed that CmWRKY8-1-VP64 transgenic lines reduced the resistance to F. oxysporum by regulating the expression of genes related to the SA signaling pathway. This study demonstrated the role of CmWRKY8-1 in response to F. oxysporum, which provides a basis for revealing the molecular regulatory mechanism of the WRKY response to F. oxysporum infestation in chrysanthemum.Chrysanthemum Fusarium wilt, caused by the pathogenic fungus  Fusarium wilt is a severe soil-borne disease that causes yellowing and wilting of plant leaves by damaging their vascular bundles. Fusarium oxysporum is the culprit of wilt disease, which usually invades the roots and multiplies the vascular bundles. F. oxysporum can block the vascular bundle and water cannot be transported, eventually leading to plant death [nt death ,2,3. Thent death . Intracent death . Among tArabidopsis, almost all class III WRKY transcription factors respond to biotic stress, suggesting that class III WRKY transcription factors may have evolved under biotic stress [Arabidopsis, AtWRKY8 and AtWRKY33 have enhanced resistance to Botrytis cinerea, whereas AtWRKY57 increases the sensitivity to B. cinerea [CsWRKY25 can improve resistance to Penicillium digitatum by regulating ROS production and PR gene expression in citrus [CaWRKY40 interacts with CaMPK9 to enhance the stability of CaWRKY40, and CaWRKY40 positively regulated the transcription of CaWRKY33 to improve resistance to F. oxysporum in chickpea [CmWRKY53 may reduce aphid resistance by regulating the production of secondary metabolites [CmWRKY15-1 has been shown to enhance resistance to Puccinia horiana Henn via the SA signaling pathway in chrysanthemum [MdWRKY15 can bind to the W-box on the downstream MdICS1 promoter, thus enhancing the accumulation of endogenous SA and improving apple resistance to Botryosphaeria dothidea [WRKY transcription factors are a family of transcription factors that are unique to plants and are widely distributed. WRKY transcription factors are widely involved in the regulation of plant growth, development, senescence, organ synthesis, and various hormone-mediated signaling pathways . More imc stress . WRKY prc stress . In rece cinerea ,14,15. Wn citrus . CaWRKY4chickpea . Similarabolites . In addiabolites . More reanthemum , and MdWdothidea .Genetic engineering techniques have become essential tools for studying plant transcription factors. The fusion of dCas9 with a transcriptional activation domain enables the upregulation of target gene expression and is referred to as CRISPR activation. The VP64 fusion of the TAD tetramer of VP16 with the dCas9 protein is called dCas9-VP64. This complex is often capable of recruiting regulatory transcription factors. In mammals, dCas9-VP64 is often used to activate target genes. dCas9-VP64 consists of a C-terminal fusion of the VP64 activation domain of the dCas9 protein. This protein binds to the gene promoter regions mediated by gRNA and binds to PoIII through the VP64 activation region to activate gene transcription ,23,24. TF. oxysporum infection. In the previous study of 15 WRKY transcription factors in chrysanthemum, Song found that CmWRKY8 (KC615362) responded to the infestation of F. oxysporum, and it was inferred that CmWRKY8 might be related to the resistance of chrysanthemum regarding F. oxysporum [CmWRKY8-1 and studied its function in response to F. oxysporum using transgenic technology. Finally, we observed that CmWRKY8-1 responds to F. oxysporum infestation by regulating the SA signaling pathway in chrysanthemum.Chrysanthemums are among the top ten traditional flowers in China and one of the world’s four most famous cut flowers. Chrysanthemums have a profound history and culture of cultivation and are widely used in floral ornamental and medicinal cultivation . Althougxysporum . To testCmWRKY8 (KC615362) was found to respond positively to F. oxysporum inoculation in chrysanthemums ‘Jinba’, suggesting that CmWRKY8 may be involved in resistance to F. oxysporum in chrysanthemums [CmWRKY8-1. CmWRKY8-1 contains 27 negatively charged residues and 33 positively charged residues. The molecular weight is 27,704.47 Da, and the theoretical pI is 8.80. The instability index is computed as 48.14, which classifies the protein as unstable. The grand average hydropathicity is −1.079, which makes it a hydrophilic protein. CmWRKY8-1 contains a WRKY domain of approximately 60 amino acids that contains a WRKYGQK motif and a zinc finger motif (C-X7-C-X23-H-X1-C), belonging to class III WRKY transcription factors  content in chrysanthemums increases, triggering systemic defense [F. oxysporum, we sprayed exogenous SA. The experimental group was inoculated 24 h after spraying with 200 μm SA, and the control group was inoculated 24 h after spraying with sterile water. By observation, we found that chrysanthemums sprayed with SA showed greater resistance , four transcripts of EDS1 , one transcript of NPR1 (evm.TU.scaffold_11524.9), and two transcripts of AIM1  were lower in CmWRKY8-1 transgenic lines. To verify the authenticity of the transcriptome data, we validated the DEGs using qRT-PCR against the SA pathway described above. The results showed that the trends of transcript expression changes obtained by qRT-PCR were consistent with the transcriptomic data  in plants ,38. The ncreased . Our exp sprayed B. In soy pathway . In a st pathway . Silencipression . In wood in vivo . It has ith NPR1 . In the culation A. In theWT lines B. TherefWT lines C. RNA-seic lines . A GO anic lines B. In sumF. oxysporum will accumulate in the soil, resulting in the frequent occurrence of chrysanthemum Fusarium wilt. We expect to apply transgenic chrysanthemum to field cultivation, screen the resistant germplasm of chrysanthemum by transgenic means, and cultivate resistant and high-quality varieties. We also hope to link the environment, molecule and F. oxysporum to study the disease resistance mechanism of chrysanthemum, improving the quality of chrysanthemum from many angles.Under the conditions of high temperature, high humidity and continuous cultivation, large amounts of v/v) mixture of soil and vermiculite. Chrysanthemums were grown in a greenhouse with a photoperiod of 16 h/8 h (light/dark), a temperature of 25 °C, and a humidity of 70%.The chrysanthemum variety ‘Jinba’ used in this experiment was provided by the Chrysanthemum Germplasm Resource Preserving Center, Nanjing Agricultural University . Chrysanthemum cuttings were planted in a 1:2  and stored at −80 °C. Before inoculation, the preserved fungal cakes were inoculated on PDA plates and cultured at 28 °C for six days. Then, 10 fungal cakes with a diameter of 0.7 cm were cut and inoculated into 500 mL PDB medium at 28 °C and 180 rpm, and the culture was shaken for 5 days. Spore concentration was determined using a blood counting plate. For inoculation, chrysanthemum roots were cut lightly and soaked in a spore suspension at a concentration of 107 CFU ml−1 for 30 min. When each plant finally colonized, the seedlings were inoculated with 1 × 107 spores per gram of substrate. The chrysanthemums were cultured in an environment of 16 h/8 h (light/dark), 28 °C and 80% humidity in a photo-culture chamber.https://www.arabidopsis.org/, accessed on 10 October 2022) and the NCBI web site . CmWRKY8-1 was used to perform multiple sequence alignments of homologous sequences using DNAMAN6, and MEGA X was used to construct phylogenetic trees using the adjacency method. The online tool ExPASy  was used to predict the physicochemical properties of CmWRKY8-1.Total RNA was extracted from snap-frozen roots of the cultivar ‘Jinba’ using an RNA extraction kit (HuaYueyang), and reverse transcription was performed using a rapid reverse transcription kit (TaKaRa) to obtain cDNA as a template for gene cloning. Primers  were desCmWRKY8-1 was constructed using the pORE-R4 (35S::GFP) vector. pORE-R4-CmWRKY8-1 and 35S::D53-RFP were transferred to the Agrobacterium tumefaciens strain GV3101. They were then transiently cotransformed into Nicotiana benthamiana leaves [The ORF of a leaves , and 35SCmWRKY8-1 [CmWRKY8-1 vector was constructed. pCL1 (positive control), pGBKT7 (negative control), and pGBKT7-CmWRKY8-1 were transformed into the yeast cells Y2HGold. pCL1 was coated in SD/Leu-media, and the pGBKT7 and pGBKT7-CmWRKY8-1 vectors were coated in SD/Trp-media and incubated for 3–5 days at 28 °C in an inverted position. Finally, colonies were picked and spotted according to different concentration gradients plates onto SD/Ade-His-media with and without X-α-gal, and incubated overnight at 28 °C in an inverted position. Colonies were observed and photographed.A yeast assay was performed to examine the transcriptional activity of mWRKY8-1 . The pGBCmWRKY8-1 was conducted by the chrysanthemum protoplast system. High-concentration plasmids 35S::GAL4DB-CmWRKY8-1, 35S::GAL4DB-AtARF5, 35S::GAL4DB, and 5×GAL4-LUC plasmids were prepared. Chrysanthemum protoplasts were extracted from the leaves of young one-month-old chrysanthemum histoculture seedlings by referring to the transformation method of Arabidopsis protoplasts [CmWRKY8-1 and 5×GAL4-LUC were cotransformed into chrysanthemum protoplasts to determine whether CmWRKY8-1 had transcriptional activation or repression activity. The positive control group was 35S::GAL4DB-AtARF5+5×GAL4-LUC. 35S::GAL4DB empty vector+5×GAL4-LUC in the negative control group and 35S::GAL4DB-CmWRKY8-1+5×GAL4-LUC in the experimental group. LUC luminescence were measured using a GLOMAX®-20/20 instrument.The detection of the transcriptional activation activity of toplasts . 35S::GA−ΔΔCt method [Quantitative primers  were dest method .CmWRKY8-1 under different stress treatments. For the inoculation treatment, the experimental group was treated with F. oxysporum, whereas the control group was treated with sterile water. Root samples were collected at 0 h, 3 h, 6 h, 12 h, 24 h, 72 h, and 120 h after treatment with F. oxysporum. For the SA treatment, the experimental group was sprayed with 200 μm SA, while the control group was sprayed with sterile water.40 days old seedlings were used to determine the expression patterns of CmWRKY8-1 was fused to the VP64 (4 × VP16) protein to produce the transcriptional activator R4-FVuv-CmWRKY8-1. The vector was introduced into Agrobacterium tumefaciens strain EHA105 and ‘Jinba’ was transformed by Agrobacterium-mediated transformation [CmWRKY8-1 specific primers (CmWRKY8-1 quantitative primers.ormation . Transge primers  for DNA-Twenty seedlings of 40 days old wild-type and transgenic chrysanthemums were used for inoculation treatment. The disease severity index (DSI) was calculated on days 8 and 12 after inoculation, and the phenotypes were photographed and recorded. The DSI was calculated according to the following formula: DSI = ∑ (number of diseased plants per level × number of corresponding levels) ×100 /  .Root samples were taken from transgenic lines and WT lines on the 8th day after inoculation. The enzyme activity in chrysanthemums was measured using POD, CAT, PAL, and PPO kits . In the meantime, the endogenous SA content was determined by a Plant Hormone Salicylic Acid ELISA Kit .CmWKRY8-1 transgenic lines were planted in a greenhouse at Nanjing Agricultural University for 40 days. After inoculation at 0 h, 3 h, and 72 h, roots of WT lines and CmWKRY8-1 transgenic lines were sampled with three biological replicates. Total RNA was extracted using a TRIzol reagent kit  and assessed using an Agilent 2100 Bioanalyzer . Transcriptome sequencing was performed using an Illumina Novaseq6000 made by the Gene Denovo Biotechnology Co. . The reads were filtered and aligned using Bowtie2 (version 2.2.8) [Seedlings of the WT lines and n 2.2.8) . Paired-n 2.2.8) . Differen 2.2.8) . The tran 2.2.8)  and Genen 2.2.8) . The quan 2.2.8) .t-tests to determine significant differences.Statistical analyses were performed using SPSS version 25.0. All data were analyzed using analysis of variance (ANOVA) and CmWRKY8-1, and found that it was responsive to F. oxysporum infestation. By overexpressing CmWRKY8-1-VP64, we found that the resistance of chrysanthemums to F. oxysporum was reduced. Moreover, the expression of genes related to the SA signaling pathway and endogenous SA content was decreased in CmWRKY8-1 transgenic lines. RNA-seq analysis showed that the expression of DEGs of the SA signaling pathway was decreased in CmWRKY8-1 transgenic lines. Therefore, we demonstrated that CmWRKY8-1 responds to F. oxysporum infection by regulating the SA signaling pathway.In conclusion, we identified a WRKY gene in chrysanthemums,


The aim of this study was to compare changes in the metabolite levels of ex-smokers and nonsmokers using a metabolomics approach, accounting for the weight gain in ex-smokers. Volunteer ex-smokers and nonsmokers were recruited from two cohorts Shijingshan (174) and Xishan (78), respectively, at a 1 : 1 ratio for age and sex. Nontargeted metabolomics was performed on the volunteers' blood samples using liquid chromatography-mass spectrometry, and multivariate statistical analysis was performed using principal component analysis and orthogonal partial least squares discriminant analysis. Enrichment analysis was used to identify Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with differential metabolites and weighted gene co-expression network analysis and maximal correlation coefficient (MCC) algorithms were used to identify key metabolites. The results revealed no significant differences between the distribution of blood metabolite levels in the ex-smokers and nonsmokers. The biosynthesis of valine, leucine, and isoleucine was determined to be associated with differential metabolites, and five key metabolites were identified. Further analysis revealed differences in weight gain and regained metabolite levels in ex-smokers, and 10 differential metabolites were identified that may be associated with weight gain in ex-smokers. These findings suggest that quitting smoking restores metabolites to almost normal levels and results in weight gain. The identified key metabolites and metabolic pathways may also provide a basis for clinical studies. Tobacco use is a major risk factor for disability and premature death, and it imposes a global burden of disease . In 2019Smoking-related diseases are a significant burden on global public health, and smoking accounts for an average of five million lives per year . In 2019Due to the excessive disease burden, a large number of smokers are deciding to cease smoking. However, it has been found that compared to nonsmokers, previously heavy smokers have a significantly increased risk of disease five years after quitting . Other sRecently, the relationship between the metabolome and disease has attracted attention. The metabolome is the product of gene expression in an organism and is present in cells, tissues, organs, or organisms. The metabolome constitutes a vast network of metabolic reactions, and the differences between the metabolites of patients and normal subjects can be analyzed to identify disease biomarkers and develop diagnostic methods. Hence, in this study, we aimed to compare the metabolites of ex-smokers with those of nonsmokers and to identify the possible metabolic pathways contributing to any differences and exploring the potential metabolome-related mechanism of weight gain after quitting.The volunteer participants were first divided into two cohorts (Shijingshan and Xishan), and then into two groups, ex-smokers and nonsmokers matched 1 : 1 according to age and sex within each cohort. The Shijingshan cohort contained 87 ex-smokers and 87 nonsmokers, and the baseline survey was conducted in 2010 and the resurvey was conducted in 2015. The Xishan Gun Factory cohort contained 39 ex-smokers and 39 nonsmokers and was first surveyed at in 2007 and resurveyed in 2012. The participants were defined as the nonsmokers who were never smokers at the two surveys with the 5-year interval and were defined as the ex-smokers who were former smokers at the two surveys. The participants' age range was 35–75 years old, and they did not have cardiovascular diseases, stroke, lung, or liver disease. In addition, the weight gain was defined as the 5-year difference of weight ≥2 kg measured at the resurvey subtracted by the weight measured at the first survey. Blood samples were collected separately, and plasma samples were obtained by centrifugation and stored at −70°C.μm, Waters, UK). The column temperature was set at 35°C and the flow rate was 0.4 ml/min. The mobile phases used were phase A: water (0.1% formic acid) and phase B: acetonitrile (0.1% formic acid). It was translated with https://www.DeepL.com/Translator (free version).The plasma samples were pretreated and the supernatant was obtained by a protein precipitation method. The supernatant was analyzed using liquid chromatography (HPLC). A SCIEX, UK liquid phase system was used for data acquisition, and the column used was an ACQUITY UPLC T3  and orthogonal partial least squares discriminant analysis (OPLS-DA) on all the metabolic characteristics of the test groups. PCA was mainly used to visualize differences in distribution between the ex-smoker and nonsmoker groups, and OPLS-DA was mainly used to select differential metabolic characteristic ions that distinguished the two groups.P < 0.05. Metabolite identification and annotation were based on HPLC and used reliable in-house and online databases.The metabolic phenotypes of the ex-smoker and nonsmoker groups were assessed using KEGG pathway enrichment analysis to observe the differences between the metabolite levels of the two groups. Pathway enrichment analysis was performed using all metabolic signatures with a threshold of β = 22 was selected as a soft threshold for network development. Co-expression modules were identified and their characteristics were analyzed. The adjacency matrix was transformed into a topological overlap matrix network to build gene dendrograms and module colors. Hierarchical clustering of modules was performed by calculating the differences in module Eigen objects and merging similar modules. The metabolite-metabolite module-phenotype  axes were constructed to resolve the metabolite changes and biological process differences found in the ex-smoker groups. The correlation between modules and phenotypes was calculated using the Pearson method, and the modules with the highest correlation were selected to identify the top five key metabolites using the MCC plug-in of Cytoscape software and to analyze the metabolite-related pathways.After excluding samples that lacked clinical phenotypes from the Shijingshan cohort, the metabolite profiles of 166 samples were included in a weighted gene co-expression network analysis (WGCNA), and The multivariate statistical methods PCA and OPLS-DA were used to analyze the nonsmoker and ex-smoker groups of the Shijingshan cohort. The results are shown in We then analyzed the relationship between smoking cessation and weight gain in the Xishan cohort. As shown in Figures Based on the 174 Shijingshan cohort samples used for metabolite detection analysis, 93 differential metabolites were identified and used for KEGG pathway analysis. As seen in With phenotypes  used as feature profiles and metabolic profiles used as network construction objects, the metabolic profiles were analyzed for weighted metabolite co-expression regulatory networks using the WGCNA algorithm. All metabolites were divided into five color modules , and theMetabolomics is used to comprehensively analyze metabolites to provide a systematic view of the biological processes of diseases. It supports disease diagnosis, pathogenesis, and interventions by detecting potential biomarkers and metabolic pathways . In thisThe multivariate statistical analysis showed that the difference in metabolite levels between the ex-smoker group and the nonsmoker group was not significant, reflecting that metabolic levels may return to normal levels after five-year smoking cessation.This study also examined the effect of smoking cessation on body weight by analyzing body weight changes in ex-smokers of Xishan cohort from 2007 to 2012. It was found that there was a more significant difference in the data distribution between the weight-gain group and the non-weight-gain group after quitting. The relationship between smoking cessation and weight gain was reported in the last century ; those wBased on this information, we used WGCNA to identify metabolites associated with both smoking status and BMI changes in phenotypes in the Shijingshan cohort. The five most relevant metabolites were Nb-palmitoyl tryptamine, palmitic acid, ganolucidic acid C, hovenidulcigenin A, and argenteane. Palmitic acid is a saturated fatty acid, and an elevated blood concentration leads to an inflammatory response . The remThe metabolic pathways affected by lifestyle behaviors can be understood by detecting changes in blood metabolites of ex-smokers and nonsmokers. Based on this conclusion, we performed a KEGG pathway enrichment analysis of differential metabolites in the Shijingshan cohort, and the results pointed to a unique pathway: the biosynthesis of valine, leucine, and isoleucine. Leucine increases protein synthesis, stimulates insulin release, and is an important metabolic signal in skeletal muscle . IsoleucCurrently, there is an increasing interest in using metabolomic analysis to study smoking-related diseases; however, there are few studies on the effect of smoking cessation on metabolites. Here, we analyzed the metabolites of ex-smokers and nonsmokers from a metabolomics perspective using HPLC techniques and multivariate statistical analysis and concluded that metabolite levels generally return to normal after smoking cessation. The KEGG pathway associated with the differential metabolites was identified using enrichment analysis. Five key metabolites were identified using the WGCNA and MCC algorithms. It was also concluded that there were differences in weight gain in the ex-smoker population, which will be needed to elucidate the mechanism underlying weight gain after smoking cessation in the future.


In total, 27 males  were recruited and attended one familiarization session and two experimental sessions (EXP 1 and EXP 2). The force–velocity profile during maximal BPT and BPTbounce (randomized order) under different loads (30–60 kg) was established (EXP 1), and the effect of varying external barbell descent velocity cues “slow, medium, and as fast as possible”  on the power output for each technique (BPT and BPTbounce) was examined (EXP 2). Comparing two BPT techniques (EXP 1), BPTbounce demonstrated 7.9–14.1% greater average power , 6.5–12.1% greater average velocity , and 11.9–31.3% shorter time to peak power  across the loads 30–60 kg than BPT. The cueing condition “fast” (EXP 2) resulted in greater power outcomes for both BPT and BPTbounce than “slow.” No statistically significant differences in any of the power outcomes were observed between “medium” and “slow” cuing conditions for BPT (p = 0.097–1.000), whereas BPTbounce demonstrated increased average power and velocity under the “medium” cuing condition, compared to “slow” . No statistically significant differences were observed in barbell throw height comparing BPT and BPTbounce under each cuing condition (p = 0.225–1.000). Overall, results indicate that both bouncing the barbell and emphasizing barbell descent velocity be considered to improve upper body power in athlete and non-athlete resistance-training programs.The aims of this study were to compare power output during a bench press throw (BPT) executed with (BPT Barbell bench press is one of the most frequently used resistance exercises for developing upper body strength and mechanical power , particuThe traditional bench press technique adopted during power training is characterized by a large acceleration at the beginning of the barbell lift (ascent phase) . Howeverbounce) or the ballistic BPT during the bench press action  . Performs action .bounce during bench press throw (BPT) has not been explored.Traditionally, it is recommended that the barbell should only lightly touch the chest and not rebound  off it . Theoretbounce than BPT, and that higher velocity during the barbell descent phase would increase the power outcomes.Therefore, the present study aimed to characterize the acute effects of performing BPT with and without the bounce technique on mechanical power and barbell kinematics, and second, to examine whether externally cueing lowering velocity had an impact on power output in resistance-trained males. Based on the findings from previous studies , we hypoα = 0.05 and β = 0.80, the sample size of 24 subjects appeared to be necessary to detect significant differences in mean power between BPT bounce and BPT. In total, 27resistance-trained men  were recruited. To be included, participants had to be free of injury, pain-free during maximal lifting, performing bench press as part of their weekly training routine, and with a 1-RM bench press of at least their own body weight. Participants were informed verbally and in writing regarding the implications and potential side effects of participating in the experiment, and were asked to refrain from any strenuous activity 48 h prior to testing. The study was conducted in accordance with the Declaration of Helsinki and confirmed by the Norwegian Centre for Research Data (ref. 288211).With reference to Loken et al.  and withbounce techniques were performed across a range of loads  to ensure correct BP lifting and bouncing techniques were used. Each participant was given two to three attempts at each load for both BPT techniques. In EXP1, subjects performed maximal effort BPT, using both techniques in a randomized order, with loads ranging from 30 to 60 kg. All participants achieved peak power for loads in the 30–60 kg range. In EXP2, participants performed BPT and BPTbounce under three externally cued lowering velocity conditions: “slow,” “medium,” and “as fast as possible” , using the loads established in EXP1 corresponding to individual peak power output for each BPT technique. In addition, participants’ 1-RM for bench press (BP) was measured using the traditional BP technique.The study used a within-subjects cross-sectional design. Participants visited the test location three times . The bench press throw (BPT) was conducted in a Smith machine . In the familiarization session, BPT and BPTParticipants attended the lab three times over a period of 2 weeks; each visit was separated by 4–5 days. In the first session, participants were familiarized with BPT performed with and without the bounce technique. Session two examined the participants’ force–velocity profile across a range of loads (EXP 1) and session 3 (EXP 2) investigated the effect of different barbell lowering cues on BPT, with and without barbell bounce. Before entering the lab, participants completed a 5-min general warm-up (jogging or cycling). The warm-up continued in the lab with dynamic stretches for the pectoralis, anterior deltoid, and triceps brachii muscles, followed by 10 B P repetitions at 20kg, four repetitions at 50% of self-reported 1-RM, and two repetitions at 75% of self-reported 1-RM. Participants used their preferred grip- and feet-width, which were measured initially and then controlled before each subsequent lift in all sessions .bounce involved completing two to three trials (loading range: 20–70 kg) to lift the barbell using each technique. In BPT trials, participants were instructed to lower the barbell, lightly touch the chest (sternum position), and immediately press upward aiming for the maximal voluntary velocity of the barbell to the point of projection . Similar instructions were used for BPTbounce trials, with the additional instruction to “bounce” the barbell off the sternum. For both techniques, participants were given the following instructions: “the aim is to lift the bar as fast as possible and lower the barbell fast, but with control.” For BPT, trials were rejected if the barbell bounced or if the lowering phase terminated at a visible distance (≥2 cm) above the chest. For BPTbounce, trials were rejected if the bar did not clearly make contact and then bounce off the chest. For both techniques, trials were rejected if the hips lifted off the bench, or if any hesitation occurred in the transition between the lowering and lifting phases.Familiarization with the BPT and BPTbounce was determined across the range of loads used. Typically, maximum power in BPT is produced with a load corresponding to approximately 50% of 1-RM (bounce) under each loading condition, beginning with the lowest load. Immediate feedback on power output was used to motivate participants toward maximal effort. The average lower velocity was 0.99–1.04 m s−1 for BPTbounce and 0.59–0.64 m s−1 for BPT. Rest between loads ranged from 1 to 3 min with 3 min rest between techniques. Three acceptable trials were performed at each load; however, only the trial with the highest average power  was used in further analyses.In EXP1, power output for BPT and BPT of 1-RM . Therefo of 1-RM . Particibounce. “Fast” corresponded to the same velocity achieved in the familiarization session and EXP1. A lift was not accepted if a participant increased the lowering velocity of the bar during the last part of the descent phase, that is, did not maintain a steady lowering velocity. In EXP 2, the load used corresponded to participants’ highest average power output for each BPT technique obtained in EXP1. The participants executed BPT and BPTbounce in a randomized order under each of the three lowering instructions.In EXP2, the effect on the power output of cueing three lowering velocities: “slow,” “medium,” and “fast” was examined for both BPT and BPTAfter completing lifts using both techniques under all three lowering cues, a bench press 1-RM test was performed in the Smith machine at 90% of self-reported 1-RM, with 2.5–5.0 kg added stepwise, until the participant and test leader agreed that 1-RM was achieved. 1-RM was obtained within two to five attempts. A 5-min rest separated each trial.r = 0.993, CV = 2.54) and displacement  when compared with Qualisys Motion Capture Systems .To calculate power output, a linear encoder  was attached to the barbell in both experimental sessions  to identify barbell peak velocity (pV), average velocity (aV), time to peak velocity (tpV), peak power (pP), average power (aP), time to peak power (tpP), and vertical displacement and velocity during the barbell lifting phase. The linear encoder had a resolution of 0.019 mm and a sampling rate of 200 Hz. Data were analyzed with the commercial software . Unpublished data from the Norwegian School of Sport Sciences show that the encoder is reliable and valid for average velocity  for both BPT and BPTversus BPTbounce) using a paired t-test using SPSS statistical software . To determine the effects of the three lowering cues, a two-way split-plot repeated analyses of variance (ANOVA) [within-subject factor: lowering cue ] x [between-subject factor: condition (BPT and BPTbounce)] was used. When differences were detected with ANOVA, paired t-tests with Bonferroni post hoc correction were applied. The magnitude of the effect was determined using Cohen’s d and interpreted according to the following scale: 0.0–0.2 , 0.2–0.5 , 0.5–0.8 (moderate), and >0.8 (large) . Barbell lowering velocity and power output at each load were compared  . All datbounce compared to BPT , and loads tested  represented intensities of 29.1% (±3.9), 38.8% (±5.2), 48.5% (±6.5), and 58.3% (±7.9), respectively, of the participants’ bench press 1-RM. There were no differences in barbell lowering velocity across the loads for BPTbounce (p = 0.666–0.901) or BPT (p = 0.280–0.622); however, the BPT lowering velocity was lower for all loads than BPTbounce (p < 0.001–0.007).The participants’1-RM in bench press was 105 ± 16 kg corresponding to a relative strength (1-RM load/body weight) of 1.32. The load corresponding to the greatest average power out was 5.7% greater using the BPTbounce demonstrated 7.8–14.1% greater average power , 6.5–12.1% greater average velocity , and 11.9–31.3% shorter time to peak power  across 30–60 kg than BPT . For the variables’ peak velocity and time to peak velocity, no differences were observed between the two techniques at 30 and 60 kg (p = 0.057–0.875); however, BPTbounce elicited 2.9 and 2.8% greater peak velocity and 4.7 and 7.9% shorter time to peak power at 40 kg  and 50 kg , respectively, than BPT , average velocity , and time to peak power , whereas for peak power, peak velocity, time to peak velocity, barbell lowering distance, and lowering velocity, no interaction  or main effect of condition  was observed, but there was a main effect for lowering cue (F = 197.623–8.465 p ≤ 0.001–0.003). For barbell throw height, no interaction  or main effect  was observed. All post hoc tests are presented in There was a significant interaction between condition and lowering cue for the following outcomes: average power  BPTbounce displayed greater average and peak power, and barbell velocity than BPT for the loads 40, 50, and 60 kg; 2) lowering the barbell “fast” demonstrated resulted in higher average and peak power, average and peak barbell velocity, independent of BPT technique, than “slow”; and 3) independent of lowering cue, BPTbounce displayed greater average power than BPT.The aim of this study was to compare the effects of BPTbounce at 40 and 50 kg, greater power and velocity were displayed at 30 and 50 kg. In the BPTbounce technique, the barbell is brought down against the chest before being re-accelerated into the lifting phase. This action may improve the transition of energy from the descending to the ascending phase, resulting in greater acceleration and barbell velocity than the traditional BPT technique  and 46%  of 1-RM. This may explain why 30 kg did not demonstrate any advantage for the bounce technique for the outcomes of peak power, peak velocity, and time to peak velocity. Alternatively, lighter loads may result in less compression  of the thoracic cage and therefore reduce the potential advantage  of the bounce technique. Of interest, Cronin et al.  velocity, to access performance gains attributed to SSC-related mechanisms, as proposed here.In general, and supporting our hypotheses, cueing barbell descent velocity at different speeds using external verbal instruction had an impact on power outcomes, with the largest effect observed for comparison between “fast” and “slow,” which is not unexpected. Furthermore, for both BPT techniques, performance was superior  using the cue to lower “fast,” which supports the practice of using external verbal encouragement to enhance power outcomes during RT with the bench press. No difference was observed between the “medium” and “slow” velocity cueing conditions for BPT, a finding which could be of value in applied settings, as it suggests that lowering the bar more slowly may not result in power reduction, which could benefit less experienced practitioners, who may be technically less adept at throwing the bar, aiming to use this technique to enhance strength adaptations. Unlike for BPT, using BPTbounce improves the power profile during BTP (bounce). However, as none of the participants conducted bench press using the bounce technique regularly, it is possible that participants’ focus was directed towards the descending phase (barbell lowering) and not necessarily on the transition between movement phases. Therefore, lack of familiarization with technical aspects of actions examined  and individual differences in responsiveness to external auditory cues (lowering instructions) could have influenced the results. Still, previous studies have demonstrated highly acceptable reliability for power and velocity outcomes in BPT, at similar loads and participants’ training status as the present study  in addition to eliciting greater stretch–reflex activation, increasing storage of energy in the tendons, promoting neurosensory pre-activation, and enhancing cross-bridge kinetics . It shouring BTP . It is int study . Despiteversus 4 s lowering phase. In the present study, lowering time under the “slow” instruction was 1.25 s and under the “medium” instruction was 0.8 s, which are closer to, and less than the “fast” condition examined by Pryor et al.  on throwing velocity, power output, and strength among handball players, and found no difference between the groups after 8 weeks. The authors speculated that the relative 1-RM strength level of the bounce group was too low to exploit potential gains from utilizing the bounce technique.No difference in barbell throw height was found under any cueing condition, or comparing between the two BPT techniques. For both techniques, loads that elicited the greatest average power in the trial phase were used, which could explain why no significant differences in barbell throw height were found, as the load for BPTnt phase , but maynt phase , which int phase . For exant phase  demonstrnt phase . The pront phase  demonstrnt phase . Furthernt phase  examinedbounce. Furthermore, although all participants were resistance-trained, they did not use the bounce technique in their regular training; therefore, familiarization with both a novel technique and external velocity cueing may have required more than the single session allocated. In addition, as the present study only included resistance-trained males, findings cannot be generalized to other populations. Small sample size, large variation between individual participants in training exposure and technical capacity, and conservative post hoc corrections may increase the risk of a type II error, when comparing the effect of varying lowering velocities on force profile outcomes. Of note, none of the participants experienced injuries as a result of this study, but some reported minor chest soreness from the bouncing technique.Even though the present study presents novel findings, some limitations need to be addressed. Loads corresponding to the greatest average power output (EXP1) were used to examine the impact of varying lowering velocity cues (EXP2). It is plausible that using other loads in EXP2, results might have been different, although we deliberately used mean power and not peak power to prescribe loads in EXP2. Several investigators have argued that peak power is a more reliable measure than mean power ; howeverbounce elicited greater average power, average velocity, and time to peak power than BPT, and may therefore be superior, if high power output throughout the BP action is the desired outcome of prescribing BP training. Our findings suggest that if the bounce technique is preferred to throwing the barbell, technical proficiency should be sufficient to perform the descent phase action at a higher velocity, as power outputs were significantly greater at medium than controlled (1.5 s) descent velocity of the barbell for this technique. Overall, lowering the barbell at higher velocity increased power outputs across all variables, and seems to be of more importance than whether BPT with or without the bounce technique is adopted. In conclusion, while athletes involved in throwing-related sports may benefit from bouncing the barbell, irrespective of technique, emphasizing velocity during barbell descent is recommended to maximize power output.At loads of 30–60 kg, BPT


Cork particle size also had a significant effect on the mechanical properties of the composites. Both the flexural and impact results showed first a rise and then a fall with a decrease in the cork size. TGA results indicated cork particles and nanocellulose could have a synergistic enhancing effect on the thermal stability of the rosin-based epoxy resin. This work can add value to rosin and cork waste and widen the industrial applications of the epoxy resin.In order to develop a bio-based epoxy resin with high mechanical and thermal performance, cork particles and nanocellulose were introduced into the rosin-based epoxy resin to improve the toughness, stiffness and thermal stability. The flexural properties of the epoxy composites indicated that the strength and modulus were reduced when the content of cork particles was relatively high (>3%) due to the low stiffness and modulus of cork itself. However, the flexural performance was significantly improved after the addition of 1% nanocellulose. In contrast to the flexural properties, the impact toughness results showed the synergistic toughening effects of nanocellulose and cork particles on the rosin-based epoxy resin. The highest impact toughness of 13.35 KJ/m In order to reduce crude oil consumption and the corresponding environmental impact, the use of sustainable resources to develop biopolymers has recently attracted much attention. Epoxy resin is the most widely used thermosetting resin at present, and has been widely used in the fields of coatings, encapsulation, composites and electronic applications ,3,4,5,6.Quercus surber or Quercus variabilis, is a natural and closed-cell biological material with a set of excellent properties, such as light texture, elasticity, admirable insulation and flame retardancy [Cork, which is a part of the bark obtained by cutting broad-leaved tardancy ,18. Sometardancy . Hence, tardancy . Many sttardancy , polyprotardancy , epoxy rtardancy , poly and nanocrystal cellulose (NCC) were prepared using Chinese white poplar flour as the raw material. All chemicals were of analytical grade and used without any further purification. The qualitative characteristics of all components are presented in w/w) to be purified. The system was heated to 60 °C and continuously stirred for 15 min. Finally, the sample was filtered and washed with distilled water to neutralize it, and dried for 24 h to obtain white powdered acrylopimaric acid.An amount of 250 g rosin and 1.25 g hydroquinone were added to a four-necked round-bottom flask equipped with a thermometer, an electric stirrer, a condensing reflux tube and a dropping funnel at constant pressure. Nitrogen was slowly added into the flask and the reaction system was heated. When heated to 220 °C, 45 mL acrylic acid was added into the flask dropwise through the feeding funnel over half an hour, and the crude acrylopimaric acid could be obtained by resting the whole system for 4 h, then cooling to 170 °C. The collected acrylopimaric acid was introduced into the sodium hydroxide ethanol solution at a concentration of 10%  mold. Finally, the samples were placed in an oven at 160 °C for 2 h for the epoxy curing, then the temperature was increased to 180 °C for 1 h. When the epoxy resin was fully cured, the composites were de-molded and used for characterization.The as-prepared CNF and NCC were solvent-exchanged to anhydrous ethanol by successive centrifugation, and the sediments were fully dispersed by sonication to form CNF and NCC ethanol suspensions. The rosin-based epoxy resin was heated to 50 °C to reduce its viscosity, mixed with the CNF or NCC suspension, and stirred to form a uniform mixture. Then the same content of curing agent, methyl hexahydrophthalic anhydride, as the pure resin polymer was added to the mixture, followed by stirring for 30 min under ultrasonic conditions. The content of CNF or NCC was fixed at 0.2%, 0.5%, 1.0% and 1.5% of rosin-based epoxy resin. The mixture was stirred under vacuum for 15 min at 70 °C to remove the ethanol solvent, then poured into the PTFE mold and placed in an oven for the curing process. The temperature was set at 160 °C for 2 h, then increased to 180 °C for 1 h. When the epoxy resin was fully cured, the composites were de-molded and used for characterization.The ethanol solvent-exchanged CNF or CNC suspension was mixed with the rosin-based epoxy resin and curing agent and sonicated for 30 min to form a uniform mixture. The content of CNF or CNC was fixed at 1% of the pure epoxy resin polymer. Then, cork particles with different contents were added to the mixture and continuously stirred under vacuum at 70 °C for 15 min to evaporate the solvent. The cork/nanocellulose/epoxy resin ternary composite was obtained after it was poured into a PTFE mold and placed in the oven for curing. The temperature was set at 160 °C for 2 h, then increased to 180 °C for 1 h. When the epoxy resin was fully cured, the composites were de-molded and used for characterization.−1 and a resolution of 8 cm−1.The FTIR spectra of the synthesized rosin-based epoxy resin before and after curing were recorded on a FTIR instrument  equipped with a Germanium Attenuated Total Reflectance (ATR) in the wavenumber range of 400–4000 cmThe as-prepared CNF/NCC water suspension was freeze dried until the moisture content of the mixture became stable. The freeze-dried nanofibers were observed using an FE-SEM . The samples were sputter-coated with gold for 30–60 s to avoid charging. The acceleration voltage was 3 kV and the coating current was 10 mA.The TEM observation was conducted on a Hitachi-H7650  at an acceleration voltage of 100 kV. A droplet of CNF/NCC suspension was deposited on a copper grid coated with a thin carbon film, followed by staining with phosphotungstic acid solution.The pure cork particles with different sizes were observed using an FE-SEM . The samples were sputter-coated with gold for 30–60 s to avoid charging. The acceleration voltage was 3 kV and the coating current was 10 mA.Pure resin polymer, cork/epoxy, nanocellulose/epoxy and cork/nanocellulose/epoxy composites were subjected to mechanical tests . The flexural tests were carried out using a universal materials testing machine  according to ASTM D 638 with the crosshead speed of 1 mm/min and support span of 40 mm. Six defect-free specimens were selected for each test. The impact toughness was measured according to ASTM D 256 with an Izod impact test machine  at room temperature. At least six specimens were tested for each measurement.The fracture surfaces of the cured composites were observed using an FE-SEM . The samples were frozen in liquid nitrogen then quickly broken. Before observation, the surfaces of the sample was sprayed with gold for 30~60 s to reduce the discharge.−1) from 30 to 600 °C at a scanning rate of 10 ℃ min−1. Pure cork, rosin-based epoxy resin, pure nanocellulose, cork/epoxy, nanocellulose/epoxy and cork/nanocellulose/epoxy composites were subjected to the TGA test.TGA was performed using an STA 449 F3 Jupiter  under nitrogen 2− group. The peak at 1240 cm−1 is due to the stretching vibration of C–O in the group –COOH, and the peak at 1146 cm−1 is due to the skeleton vibration of C–O–C. In addition, the peaks at 910 cm−1 and 848 cm−1 are also the characteristic peaks of the epoxy group. The absorption peaks in the infrared spectrum met the molecular structure and functional chemical groups of the epoxy resin, so it could be proved that the synthesized sample was the rosin-based epoxy resin. From the spectrum of the rosin-based epoxy resin after curing, it could be seen that the absorption peaks were almost the same as the epoxy before curing, except for the absorbance of peaks at 910 cm−1, 848 cm−1 and 3498 cm−1. The absorbance of peaks at 910 cm−1 and 848 cm−1 are much lower than those on the spectrum of the epoxy resin before curing, and these two peaks nearly disappeared. However, the absorbance of peak at 3498 cm−1 is higher than that of the epoxy before curing. These changes indicated the curing reaction of the rosin-based epoxy resin.The FE-SEM and TEM images of poplar NCC and CNF are shown in The microstructure of the pure cork particles with different sizes is revealed in The flexural strengths and moduli of the cork/nanocellulose/epoxy resin composites are presented in The effects of cork particle size on the flexural performance of the composites are presented in Similarly, the effects of nanocellulose content on the flexural strength and modulus of composites are indicated in 2. When adding 1% cork particles, the impact toughness of the composite was increased to 7.65 KJ/m2, an increase of 42.72% compared to the resin matrix, indicating that the addition of cork particles significantly improved the toughness of rosin-based epoxy resin. When the content of cork particles was 2%, 3%, 4% and 5%, the impact toughness of the composites was 9.55 KJ/m2, 10.23 KJ/m2, 10.56 KJ/m2 and 9.47 KJ/m2, respectively. In contrast to the flexural properties, the impact toughness of the composites showed an obvious upward trend with an increase in cork particles when the content was less than 4%. The toughening effect of the cork particles can be attributed to their closely arranged cell structure. Cork exhibits an alveolar structure without empty spaces between contiguous cells; therefore, these cells are closed units and the interior is full of air [l of air . When th2, an increase of 168.84% compared to the neat rosin-based epoxy resin. Within a certain range, cork particles of smaller size have larger specific surface area and contact area with the bonded epoxy matrix compared to cork particles of larger size. Therefore, smaller cork particles have better wettability to the epoxy resin, leading to a more effective transfer of the stress load from the matrix to the cork particles. However, with a further decrease in cork particle size (200–300 mesh), the impact toughness of the composite had a decline in the trend. This reduction can be ascribed to the damage to a large amount of cork cells. It was reported that cork cells had good impact behavior only when a group of intact cells were present [The effect of cork particle size on the impact toughness of the composites with 3% cork is shown in  present . During 2, an increase of 149.07% compared to the neat epoxy resin. It could also be observed that the impact toughness of the composites with CNF was significantly higher than that of the composites with NCC. This is because CNF has a higher aspect ratio and larger specific surface area than the NCC particles. The strong network structure formed by the long CNF can act as a crack stopper and suppress the crack propagation.The thermal stability of pure cork, pure rosin-based epoxy resin, pure nanocellulose, cork/epoxy, CNF/epoxy and CNF/cork/epoxy composites measured by TGA in the temperature range of 25–600 °C is presented in 2, an increase of 149.07% compared to the neat epoxy resin. Cork particle size also had an important effect on the mechanical properties of composite. The flexural and impact performance of the composites both exhibited a rise followed by a fall with a decrease in cork size. In addition, CNF exhibited a better reinforcing effect on the epoxy resin than NCC due to the higher aspect ratio. TGA results indicated that both the addition of cork particles and nanocellulose could improve the thermal stability of the rosin-based epoxy resin.In this work, the synergistic toughening and strengthening effects of cork particles and nanocellulose on the rosin-based epoxy resin were investigated in detail. The flexural results indicated that the MOR and MOE of the epoxy composites were obviously reduced when the cork content was more than 3% because of the low stiffness of cork itself. However, the flexural performance was improved significantly after the addition of 1% nanocellulose, suggesting a network structure-strengthening effect of nanocellulose. The impact toughness results showed the synergistic toughening effects of nanocellulose and cork particles on the rosin-based epoxy resin. The highest impact toughness of the composite with 1% CNF and 3% cork particles was 13.35 KJ/m


EGFR  is overexpressed in a variety of human cancers  and therefore is regarded as an ideal target for cancer therapy or imaging purposes. In the current study, we produced a scFv-based near-infrared probe  and evaluated its ability in recognizing and imaging of EGFR-overexpressing tumors in a mouse model. Like the molecular probe consisting of its parental antibody  and IRD800CW, cet.Hum.scFv-IRDye-800CW was able to recognize EGFR-overexpressing tumors in mice. cet.Hum.scFv-IRDye-800CW was found to be superior to the cetuximab-based probe in imaging of mouse tumors. The tumor-to-background ratio and blood clearance rate were higher when cet.Hum.scFv-IRDye-800CW was used as an imaging probe. Surgical resection of the primary solid tumors in patients plays an important role in cancer therapy. Positive margins remaining after an incomplete resection significantly contribute to the disease recurrence , 2. TradMany studies have highlighted the clinical benefits of fluorescence-guided surgery of patients with solid tumors, where molecular probes made from antibody molecules and a fluorescent dye are used , 7–16. FCurrently, there are four major EGFR-targeting monoclonal antibodies, namely, panitumumab, nimotuzumab, cetuximab, and necitumumab. Panitumumab, nimotuzumab, and cetuximab have been conjugated to various tracers, and the resultant conjugates are currently under clinical trials . For insTumor-detecting agents are of great interest for surgical oncologists. OTL-38 is an FDA-approved agent that is used for detection of ovarian cancer during fluorescence-guided surgery . AntibodIn our previous work, we produced and evaluated the binding capacity of a germline-humanized recombinant anti-EGFR scFv . In the Cetuximab  was prepared from Red Cross Pharmacy of Gorgan . IRDye800CW-NHS was purchased from LI-COR Biosciences, Lincoln (USA). Diaminobenzidine (DAB) tablet set (cat. no. T0440) and TMB  (cat. no. D4293) were purchased from Sigma-Aldrich. Recombinant human EGFR protein (cat. no. ab155726) and anti-alpha tubulin antibody (cat. no. ab15246) were purchased from Abcam. HRP-Protein L (cat. no. M00098) and Ni-NTA Agarose (cat. no. 30210) were purchased from Qiagen (Germany) and GenScript Biotech (USA), respectively.A humanized single-chain variable fragment (scFv) was the product of our previous study. We named the scFv as cet.Hum.scFv. The scFv-encoding sequence (consisting of heavy-chain variable domain- (VH-) encoding sequence, linker-encoding sequence, and light chain variable domain- (VL-) encoding sequence) has been inserted in the cloning region of pET22b(+) bacterial expression vector between NcoI and XhoI restriction sites. Expression of the sequence results in the production of a 27 kDa scFv in VH-linker-VL format .Escherichia coli (E. coli) BL21 (DE3) cells using the heat shock method. E. coli cells were cultured in a conical flask containing 50 mL Luria-Bertani (LB) medium supplemented with 100 μg/mL ampicillin and allowed to grow for 3 h at 37°C. When OD600 reached to 0.5, IPTG  was added to the flask  to induce recombinant cet.Hum.scFv expression. After an overnight growth at 18°C, the medium was centrifuged at 4000 g for 15min and the resultant bacterial pellet was suspended in lysis buffer  for sonication. Sonication was carried on ice for 30 cycles with 30 s intervals at amplitude 80. cet.Hum.scFv was purified using Ni-NTA agarose according to the company's (Qiagen) protocol and stored at −20°C for subsequent use. The Bradford assay was used to quantify cet.Hum.scFv concentration in the solution.For recombinant protein expression, recombinant pET22b(+)-cet.Hum.scFv vector was transformed to 2 in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (pen/strep), and 25 μg/mL gentamicin. A-431 and U-87 MG were used as high and low EGFR-expressing cell lines, respectively.A-431 and U-87 MG (U-87) cell lines were obtained from the Cell Bank of Pasteur Institute . A-431 is a human skin cancer cell line overexpressing EGFR . U-87 isThe cells were lysed by sonication (5 s) while being suspended in modified lysis buffer  , 41. Celμg/mL), cetuximab (30 μg/mL), or anti-alpha tubulin (20 μg/mL) for 2 hours at room temperature (RT). After another round of washing with TBS, the membranes (those reacted with cet.Hum.scFv or cetuximab in the previous stage) were reacted with HRP-Protein L solution  for 1 hour at RT. Finally, the membranes were reacted with freshly prepared DAB solution to visualize the spots.A-431 and U-87 cell lysates were run on an SDS-PAGE, and separated proteins were transferred to PVDF membranes. To prevent unspecific reactions, the membranes were immersed in blocking buffer  and 0.05% Tween-20). After an overnight incubation at 4°C, the membranes were washed with TBS buffer and reacted with cet.Hum.scFv  according to the manufacturer's guidelines , 43. BriA280 and A774)  × MW Protein × Dilution Factor. The degree of labeling was measured using the following formula: DOL = [A774/εIRDye800CW] ÷ [(A280 − (0.03 × A774))/εProtein]. The correction factor for the absorbance of IRDye800CW at 280 nm  is 0.03. εProtein is the molar extinction coefficients for the protein. MW protein is the molecular weight of the protein. The dilution factor is the dilution of the labeled conjugate prior to measurement with a spectrophotometer. The molar extinction coefficient of IRDye800CW is 240,000 M−1 cm−1, and the molar extinction coefficients for the proteins (ɛProtein) are 53,860 M−1 cm−1 (for scFv) and 217,440 M−1 cm−1 (for cetuximab). ELISA data was analyzed using GraphPad Prism 8 . The tumor-to-background ratio and fluorescent signal intensity were calculated using ImageJ (https://imagej.net/).The concentration of cet.Hum.scFv-IRDye800CW and cetuximab-IRDye800CW was calculated using the following formula: protein concentration (mg/mL) = [(A280 − (0.03 × A774))/μg/mL and 51.3 μg/mL, and the DOL ratio was 1.983 and 2.128, respectively.cet.Hum.scFv expression was carried out as described in our previous work and purified using NI-NTA resin . As expeKd values of cet.Hum.scFv-IRDye800CW and cetuximab-IRDye800CW were calculated to be 21 nM (±0.5) and 24.3 nM (±0.9), respectively. Unlabeled cet.Hum.scFv and cetuximab had Kd values of 5.3 ± 1.3 and 4.9 ± 1.8 nM, respectively. No significant difference was found in Kd values between the cet.Hum.scFv and cetuximab (p > 0.05). No significant OD values were obtained when the antibodies reacted with U-87 MG cells  (p ≤ 0.05)  . 1 h aft Figures . The tum Figures .In the current study, we compared the abilities of a humanized cet.Hum.scFv (approximately 27 kDa) and its full-length parental antibody  to see which one is better for tumor imaging when conjugated to IRDy800CW near-infrared fluorescent dye. The ability of IRDye800CW-conjugated cetuximab in detecting EGFR-overexpressing tumors has already been reported in both preclinical and clinical studies, and it has been found to be a suitable tumor-detecting agent , 21. In Cetuximab-IRDye800CW was also unable to detect U-87 MG tumors at all. These results confirm that cet.Hum.scFv-IRDye800CW has a higher tumor penetration ability than RDye800CW-conjugated cetuximab. Considering the specificity of cet.Hum.scFv-IRDye800CW toward EGFR-overexpressing cells and also the less tissue penetration ability of infrared wavelengths , it will be useful only for imaging of superficial human cancers that overexpress EGFR. Squamous cell carcinoma of head and neck (SCCHN) is a group of cancer that usually overexpresses EGFR molecules. These cancers are also superficial enough to be imaged using infrared dye-conjugated antibodies like cet.Hum.scFv-IRDye800CW.Kd values of both cet.Hum.scFv and cetuximab, unexpectedly, increased after IRDye800CW conjugation. Although it is usual, Kd rising following dye conjugation has already been reported. Bernhard and colleagues have reported that Kd values of (scFv)2, scFv-Fc, and an IgG increased after IRDye800CW conjugation but did not point out how this shifting in Kd value occurs.Previous studies have shown that fluorophore conjugation can lead to affinity reduction –55. Fluohttps://www.licor.com/bio/reagents/irdye-800cw-nhs-ester), IRDye800CW binds to proteins via their side chain primary and secondary amino groups. The dye  in antibody molecules are responsible for antigen recognition. There are a number of amino acids in CDR loops of both cet.Hum.scFv and cetuximab which have primary or secondary amino group(s) at their side chains. Based on the information provided by LI-COR  as well as some infrared dyes like 5-aminolevulinic acid (5-ALA). IRDy800CW has near-infrared spectrum absorption and emission wavelengths (774-794 nm). So, autofluorescence observed with 5-ALA and visible light fluorescent dyes does not occur tangibly when working with IRDy800 CW . Altoget


There has always been a debate on the optimal timing of endoscopy in patients with acute variceal bleeding (AVB).This study aimed to examine the relation between the timing of endoscopy and the short-term outcomes of patients with AVB.Patients with AVB who underwent endoscopy within 24 h after admission at our tertiary care center from 2014 to 2022 were evaluated retrospectively. The primary outcomes were the 6-week mortality and re-bleeding. The secondary outcomes included the total number of blood units transfused, the length of hospital stay, and the need for salvage therapy. We used Cox proportional hazards model to analyze the predictors of 6-week mortality in all patients as well as in those who were at high risk of further bleeding or death.P value = 0.132) and 6-week re-bleeding rate . In multivariate analysis, time to endoscopy was independent of 6-week mortality , but the time between the beginning of bleeding and endoscopy (within 12 h) was significantly associated with low 6-week mortality . Time to endoscopy was still not associated with 6-week mortality in patients at high risk for further bleeding or death .A total of 312 patients were enrolled. Among them, 170 patients (54.49%) underwent urgent endoscopy (< 6 h), and 142 patients (45.51%) underwent early endoscopy (6–24 h). There were no significant differences between the urgent-endoscopy group and the early-endoscopy group, regarding the 6-week mortality (16.47% vs. 10.56%; Endoscopy performed within 6 h of admission, rather than within 6 to 24 h, did not improve six-week clinical outcomes in patients in stable condition with AVB and even those who were at high risk of further bleeding and death. Acute variceal bleeding (AVB) is a common and life-threatening complication of cirrhosis. Although there have been significant improvements in diagnostic and therapeutic modalities for the management of AVB in the recent several decades  2], the, the2], Current European consensus and American guidelines recommend emergency endoscopy to be performed within 12 h of admission –10, whilThis study aimed to explore the clinical outcomes in patients with AVB according to different timing to endoscopy. In addition, we aimed to investigate the clinical predictors of short-term mortalities, especially in patients at high risk for further bleeding or death.Medical records of esophagogastric varices bleeding patients undergoing endoscopy from our tertiary hospital were retrospectively reviewed between July 2014 and May 2022. The diagnosis of cirrhosis was established according to the previous history or on the combination of laboratory and imaging tests. The inclusion criteria were as follows: (1) Emergency patients admitted via the emergency room (ER) with upper gastrointestinal bleeding (UGIB); (2) Esophagogastric varices confirmed as the source of bleeding; (3) Endoscopy was performed within 24 h; (4) aged > 18 years. The exclusion criteria were as follows: (1) Severe dysfunction of a major organ ; (2) UGIB from other than variceal bleeding ; (3) Prior acute variceal bleeding (AVB) in the past three months; (4) Loss to follow-up within 6 weeks after AVB. This study protocol was approved by the Institutional Ethics Committee of Shanghai East Hospital and was performed in accordance with the Declaration of Helsinki.When a patient with suspected AVB arrived at the ER, adequate fluid resuscitation, a vasoactive drug with somatostatin, and a prophylactic antibiotic were administered immediately. Packed red blood cell (PRBC) was given in the case of persisting bleeding or hemoglobin < 7 g/dl.The endoscopy procedures were performed by five senior endoscopists with more than 3 years experience in the field of endoscopy. The timing of endoscopy depended on hemodynamic status, the discretion of the endoscopists on duty, and the patient’s will.Endoscopic therapy was performed under the circumstances as follows: (a) Active bleeding of the varices; (b) Presence of adherent blood clot or the “white nipple” sign; (c) Presence of blood in the upper gastrointestinal tract and the varices as the only potential bleeding source; (d) In the case of the presence of varices without blood in the upper gastrointestinal tract, the endoscopist decided whether to perform the therapeutic endoscopy. The varices were treated by endoscopic band ligation (EBL), injection of a tissue adhesive , sclerotherapy, or with the combination of hemoclips according to the discretion of the endoscopist.Two independent investigators screened the medical records, including demographic, endoscopic reports, laboratory results, and clinical data. Model for End-stage Liver Disease (MELD) score , the GlaThe timing of endoscopy was defined as the interval between the hospital arrival and the initial endoscopic procedure, while the bleeding time was regarded as the duration between the beginning of the presence of bleeding and the initial endoscopic procedure. Endoscopy performed within 6 h of admission was defined as urgent endoscopy, and endoscopy performed within 6–24 h of admission was considered as early endoscopy.The primary outcomes of this study were the 6-week mortality and re-bleeding rates. The secondary outcomes were the total number of blood units transfused, the length of hospital stay, and the need for salvage therapy .P value less than 0.05 was considered significant. Hazard ratios (HRs) and 95% confidence intervals (CIs) were also calculated. All statistical analyses were performed by SPSS version 20.0 .Means with standard deviations and frequencies with percentages were used for descriptive statistics. Statistical differences between the groups were investigated by the chi-square test and Fisher’s exact test for categorical variables and the Student’s t-test for continuous variables. Cumulative 6-week survival rates were estimated by Kaplan–Meier method and compared by log-rank test. Predictors of 6-week mortality were assessed by Cox’s proportional hazards model. A Overall, 352 AVB patients meeting the inclusion criteria from our tertiary center were admitted. Among them, according to the exclusion criteria, 40 patients were excluded. Finally, the remaining 312 patients were enrolled. Among them, 170 patients (54.49%) underwent urgent endoscopy, and 142 patients (45.51%) underwent early endoscopy.As shown in Table P value = 0.051), gender , Glasgow-Blatchford score , CTP score , MELD scores , etc., did not differ significantly between the two groups. The median time to endoscopy was significantly different between the two groups .The baseline patient characteristics, including age (n = 43). Concerning the 6-week mortality, there were no significant differences between the urgent-endoscopy group and the early-endoscopy group . There were no significant differences in the 6-week re-bleeding rate between the urgent-endoscopy group and the early-endoscopy group . The overall 5-day re-bleeding rate was 4.49% (n = 14). The number of transfusions per patient  was significantly higher in the early-endoscopy group than in the urgent-endoscopy group. The length of hospital stay  and the rate of need for salvage therapy  were not significantly different between the urgent-endoscopy group and the early-endoscopy group. . However, the bleeding time shorter than 12 h (vs. more than 12 h) was independently associated with low short-term mortality . The other independent risk factors of 6-week mortality were as follows: the presence of HCC , the presence of encephalopathy , high Glasgow-Blatchford score  and high MELD score .By multivariate analysis, clinical predictors associated with 6-week mortality were explored, as shown in Table n = 138), multivariate analysis revealed HCC  and encephalopathy  as significant predictors of 6-week mortality. However, time to endoscopy was not associated with 6-week mortality .  is not significantly superior to early endoscopy (6–24 h) regarding the 6-week mortality or the 6-week re-bleeding rate. This phenomenon could be explained by the fact that basic resuscitation and medical treatments have a major influence on the patient’s outcome in the early stages of treatment , 10, 19.To explore the potential benefits of urgent endoscopy, we selected high-risk patients to perform the subgroup analysis. We applied the Glasgow–Blatchford score as a measure of risk, which has been shown to correlate with mortality . An obseTo the best of our knowledge, five retrospective studies have investigated the optimal timing of endoscopic treatment in AVB patients. Two studies published in Taiwan in 2009  and 2012Most of the previous studies defined “urgent endoscopy” as within 12 h of admission. This study is one of the very few studies to compare the short-term outcomes in patients with AVB between urgent endoscopy (< 6 h) and early endoscopy (6–24 h) with a big sample size. Actually, it is difficult in many countries to perform urgent endoscopy within 6 h of admission. In the randomized controlled trial aiming to explore the timing of endoscopy for acute upper gastrointestinal bleeding, patients assigned to undergo endoscopy within 6 h were regarded as urgent-endoscopy group. However, in this urgent-endoscopy group, the time from presentation to gastroenterologic consultation was 7.4 ± 6.2 h; the time from gastroenterologic consultation to endoscopy was 2.5 ± 1.7 h; time from presentation to endoscopy was 9.9 ± 6.1 h . In the It is essential to select appropriate outcomes in studies of endoscopy. Since the long-term prognosis of patients with AVB is mainly associated with their basal liver function, it is inappropriate to demonstrate the precise effect of a single-point endoscopy by a long-term prognosis. Therefore, the endpoints were set to be the composite outcomes at 6 weeks in this study .This is the first study to report the bleeding time (the duration between the start of bleeding and endoscopy), which might be more precise and valuable than the timing to endoscopy . Furthermore, we found that the bleeding time within 12 h was independently related to low 6-week mortality, while the timing to endoscopy was not associated with 6-week mortality. The inconsistency might be explained that basic resuscitation and medical treatments immediately after bleeding, other than timing to endoscopy, played an important role in short-term outcomes of patients with AVB. Endoscopy is a relatively invasive treatment, compared with other medical treatments. If unnecessary urgent endoscopies are frequently performed, medical staff fatigue and medical costs may increase dramatically . FurtherDespite these originalities, this study has some limitations. First, some bias may exist due to the retrospective nature of this study. Conducting patient randomization could address this limitation, but it would be problematic in these acutely ill patients because of their unstable vital signs and obvious ethical concerns. Second, the MELD score was recorded soon after the admission, but it might have changed after resuscitation and pharmacological treatment. Re-calculate the MELD score before the endoscopy might be more appropriate in predicting the risks. Third, those who had persistent hypotensive shock despite initial resuscitation were not included. Therefore, our trial findings should not be generalizable to AVB patients with persistent hypotensive shock. Forth, the Fibroscan value is not available in the present study, which might be useful in the multivariate analysis.In conclusion, we found that the time to endoscopy does not appear to be associated with short-term mortality in patients with AVB and those who were at high risk for further bleeding or death. However, the bleeding time more than 12 h is an independent risk factor of high short-term mortality in patients with AVB. Our results should be taken into consideration when making clinical decisions. A multi-center prospective study with a bigger sample size from different countries is in demand to explore the optimal timing for endoscopy.


Interestingly, the UBC-858 and SSR CNMPF-13 markers were unique to Grand Nain and Ardhapuri cultivars, respectively, which could be used for clonal fidelity analysis. Furthermore, the analysis of banana fruit volatilome using headspace solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GCMS) revealed a total of fifty-four volatile compounds in nine banana cultivars with 56% of the total volatile compounds belonging to the ester group as the significant contributor of aroma. The study assumes significance with informative biomarkers and signature volatiles which could be helpful in breeding and for the authentic identification of commercial banana cultivars.Banana is an important fruit crop in the tropics and subtropics; however, limited information on biomarkers and signature volatiles is available for selecting commercial cultivars. Clonal fidelity is a major contributor to banana yield and aroma; however, there are no useful biomarkers available to validate clonal fidelity. In this study, we performed the molecular profiling of 20 banana cultivars consisting of diploid (AA or AB) and triploid (AAA or AAB or ABB) genomic groups. We screened 200 molecular markers, of which 34 markers  yielded unequivocally scorable biomarker profiles. About 75, 69, and 24 allelic loci per marker were detected for RAPD, ISSR, and SSR markers, respectively. The statistical analysis of molecular variance (AMOVA) exhibited a high genetic difference of 77% with a significant FST value of 0.23 ( Musa spp.) is one of the world’s major fruit crops belonging to the Musaceae family. It is native to the tropical region of Southeast Asia1. India ranks first in banana production, with 31.5 million tons accounting for 26.43% of global output2. Banana is rich in carbohydrates, vitamin B, and a good source of sodium, potassium, calcium, and magnesium4. It is a perennial with a faster relative growth rate than other fruit crops and produces fruits throughout the year. Banana cultivars are propagated asexually via micropropagation or suckers5.Banana  and Musa balbisiana Colla (B genome); both belong to the section Eumusa. The banana cultivars possess good genetic diversity with genome groups of AA, AB, AAA, AAB, ABB, AABB, AAAB, or ABBB type resulting from inter or intra-specific hybridization of M. acuminata and M. balbisiana6. Morphological data have suggested that Musa spp. is diverse and showed some well-defined unifying characteristics, indicating the genomic constitution. Complex genome structure and phylogenetic relationship among the hybrid cultivars and wild types need further exploration. Due to its narrow genetic base, banana cultivation is prone to pests and diseases7. Further, this is exacerbated by devastating extremities of abiotic stresses induced by climate change and global warming8. Identifying genotypes with high potential is critical for an effective, economical, and environmentally friendly approach to boosting banana productivity. The wild germplasm (BB-rich genomes) possesses hardiness and could act as potential donors in developing high-performing cultivars through molecular breeding10. In this regard, the utility of molecular tools in selecting and developing biotic and abiotic stress-resistant genotypes is of paramount importance in banana breeding programs12.The cultivated edible 11. Such molecular markers can be valuable for assessing genetic diversity to complement traditional breeding methods. Moreover, the genetic content (molecular identity information) is highly stable and not influenced by environmental factors; hence differences among genotypes could be discerned at the DNA level. Molecular markers are being widely used in plants to detect the genetic basis of variation, genetic relationships within germplasm, identification of duplicate accessions, and genetic fidelity testing. Several marker-assisted traits have been used in banana breeding13. Restriction fragment length polymorphism (RFLP)14; random amplified polymorphic DNA (RAPD)9 amplified fragment length polymorphism (AFLP)17; and microsatellites or simple sequence repeats (SSRs) and inter-simple sequence repeats (ISSRs)18 have been employed in the characterization and evaluation of genetic diversity in Musa species. Amongst these markers, SSRs show a higher degree of polymorphism and codominant inheritance mode and are reproducible19.Researchers and farmers essentially require authentic high-yielding cultivars to maximize the output. However, traditional methods based on the phenotypic selection are complex and irreproducible to identify authentic cultivars. Hence, it is necessary to search for and develop novel molecular biomarkers which are stably inherited and unperturbed by environmental deviations20. Despite the limited literature on banana fruit volatiles and their contribution to aroma22, the role of banana volatiles has been indicated in the resistance mechanism to some important fungal diseases like Sigatoka23 and insect pests. The study of volatile components from banana fruits is also very important to assess the effect of changing climatic conditions on fruit quality*1. In light of these, we sought to understand the characteristic volatilome of each banana cultivar. Profiling of volatile compounds representing characteristic aroma and flavor of banana germplasm by using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography and mass spectrometry (GC–MS)25 provides an additional tool for characterization of banana germplasm which could be utilized for decoding the flavor of diverse banana cultivars and breeding for enhanced sensory quality26.Fruit volatile  is the vital contributor to banana appeal and aroma characteristics which influence fruit delicacy, quality, and consumer preference. Banana fruits are rich in flavor and aroma with the occurrence of more than 250 volatile compounds of several chemical classes, including esters, ketones, terpenes, and aldehydesIn the present study, we have used various molecular markers to evaluate the genetic diversity among commercially grown banana cultivars belonging to different genomic groups. This study revealed the identification of cultivars-specific biomarkers which may help to establish genetic relationships. Additionally, the HS-SPME and GC–MS were used to develop the volatile profile of the genotypes. Such markers will enable plant breeders to select banana cultivars with better aroma, flavor, and desirable characteristics, besides helping researchers and farmers verify commercially grown varieties’ genetic fidelity.27 followed by RNase treatment. The qualitative and quantitative analysis of genomic DNA was done using a spectrophotometer and by resolving on 0.8% agarose gel. The Genomic DNA was diluted to 50 ng/µL and subsequently used for RAPD, ISSR, and SSR fingerprint analysis.The leaf samples of twenty different germplasm of banana comprising diploid and triploid genomes were collected from the Banana Research Station, Nanded affiliated to Vasantrao Naik Marathwada Krishi Vidyapeeth (VNMKV), Parbhani Table . The stu2(10×), 0.5 µL dNTP mix (10 mM), 0.33 µL Taq DNA polymerase (3 U/µL), 2 µL Primer (10 µm/µL) and 1 µL template DNA (50 ng/µL). ISSR PCR reaction of 25.0 µL comprised 2.5 µL 10× PCR assay buffer with MgCl2, 0.5 µL dNTP mix (10 mM), 0.4 µL Taq DNA polymerase (3 U/µL), 1 µL Primer (10 µm/µL) and 1 µL template DNA (50 ng/µL). The SSR genotyping were performed in 25.0 µL reaction with 2.5 µL 10× PCR assay buffer, 1.5 µL MgCl2(25 mM), 0.5 µL dNTP mix (10 mM), 0.5 µL Taq DNA polymerase (3 U/µL), 1.5 µL each forward and reverse primer (10 µm/µL) and 1.5 µL template DNA (50 ng/µL). The RAPD PCR was carried out in Eppendorf thermal cycler for 5 min at 94 °C, followed by 40 cycles of 45 s at 94 °C, 1 min at 36 °C, 1 min at 72 °C with a final extension time of 10 min at 72 °C. The thermal cycler conditions for ISSR genotyping were initial denaturation at 94 °C for 5 min followed by 40 cycles of 45 s at 94 °C, 1 min at 52 °C, 1 min at 72 °C with a final extension time of 10 min at 72 °C. A thermal cycler for SSR PCR was carried out at 94 °C for 4 min, 35 cycles of 94 °C for 40 s, 54 °C for 30 s, and 72 °C for 40 s followed by a final extension of 10 min at 72 °C. The RAPD and ISSR PCR amplified products were resolved on 1.5% agarose gel at 100 V. Whereas, SSR PCR products were resolved on 2.5% agarose gel at 100 V, followed by ethidium bromide staining. The images were captured by a gel documentation system .A total of 200 primers were used for pre-screening and of which 11 RAPD, 11 ISSR, and 12 SSR primers were used for genotyping twenty banana cultivars /. Genetic relationship among the germplasm was estimated with the dendrogram constructed through an unweighted pair group of arithmetic means using (UPGMA)29 and NTSYS pc 2.02 software. Further, the population genetic structure was analyzed based on SSR amplicons score as allele size (bp) using the Bayesian clustering method and STRUCTURE v.v 2.3.3 software30. The admixture model and correlated allele frequencies were applied for the estimation of ancestry fractions of each cluster. The value of K (range 1–10) was estimated using five independent runs and a burn-in period of 20,000 followed by 200,000 MCMC (Markov Chain Monte Carlo) repetitions. The web-based software STRUCTURE HARVESTER version 0.6.92 was used to determine the optimum K value using the log probability of data, LnP(D) based on the rate of change in LnP(D) between successive K31.All visible and unambiguously scorable fragments amplified by the RAPD, ISSR, and SSR primers were scored as scorable fragments. Amplification profiles of the germplasm were compared, and bands of DNA fragments were scored as present (1) or absent (0). The DNA fingerprint data of the primers were utilized to estimate genetic similarity based on a number of shared amplification products32. The 430 GC analyzed the volatile compounds, and 210 MS (Varian) gas chromatograph-mass spectrometer (GC–MS) equipped VF-5 MS column with helium as the carrier gas at a flow rate of 1.0 mL/min was used for detection of m/z ratio of fragmented ions and derived spectrum. The injection port was lined with a 0.75 mm, i.e., splitless glass inserter, and maintained at 200 °C. The oven temperature was programmed to rise from 50 to150 °C at 2 °C/min and the total GC run time was 55 min. MS transfer line was maintained at 290 °C, ionization energy was 70 eV, and the mass range was 50–550 m/z. The volatile compounds were identified by comparing the results obtained with the reference mass spectra from the NIST library  and retention index values obtained using a standard mixture of alkanes (C8–C20).The unripe banana fruits of available banana cultivars were collected from the Banana Research Station, Nanded, and kept for ripening at 20 °C.The pulp of fully ripe banana fruits was used for volatile analysis. A blended pulp juice from the three banana fruits of each banana cultivar was prepared separately, and 16 g of juice in a 30 ml glass vial tightly sealed with a Teflon septum with a plastic cap was used for volatile analysis. Optimized SPME parameters, i.e., sample equilibration for 15 min, adsorption for 60 min at 25 °C, and desorption time of 10 min, were used for GCMS sampling33. Here we employed three different markers—RAPD, ISSR, and SSR to search the cultivar-specific biomarkers. The molecular marker provided genetic information and assisted in finding out biomarkers specific to Grand Nain and Ardhapuri cultivars. Additionally, we performed the volatile profiling of banana cultivars to characterize the compounds that contribute to the characteristic aroma of the banana fruits.Various molecular markers have been used extensively to screen the diversity and develop biomarkers for clonal identity in crops, including bananaRAPD profiling of twenty banana cultivars produced polymorphic DNA fingerprint patterns and generated 725 polymorphic amplicons. Most RAPD primers produced 100% polymorphism . The ISSR-827 was yielding 0.7 kb unique fragment in banana cultivar Grand Nain to confirm the presence M. acuminata calmodulin-like protein mRNA, partial CDS (765 bp) (AF414128.1) in M. acuminata Grand Nain (AAA) banana cultivar. Thus ISSR-827 yielding ⁓750 bp fragment was identified as a genic biomarker specific to the Grand Nain banana cultivar. The ISSR marker UBC-858 displaying 0.4 kb unique fragment in Ankur and 0.2 kb in Grand Nain is identified as a genic microsatellite biomarker as it was showing similarity with the M. acuminata subsp. malaccensis transcription factor bHLH112 (LOC103998874), transcript variant X2, mRNA, M. acuminata subsp. malaccensis abscisic acid 8'-hydroxylase 3-like (LOC103999378), transcript variant X1, mRNA, M. acuminata subsp. malaccensis indole-3-pyruvate monooxygenase YUCCA2-like (LOC103984381), mRNA etc., with E value 0.023 and confirmed its genic nature. Further, the ISSR-825 profile yielded a specific fragment of 1.0 kb , mRNA (XM_009421366.2) (1370 bp), M. acuminata subsp. malaccensis uncharacterized LOC103999495 (LOC103999495), mRNA (1004 bp)( XM_018818029.1). The banana cultivar Ardhapuri produced a specific fragment of 0.5 kb in the DNA fingerprinting analysis using SSR primer CNMPF-8, and a 0.9 kb fragment was observed in the fingerprint of SSR CNMPF-9 ; and M. acuminata subsp. malaccensis uncharacterized LOC103989204 (LOC103989204), transcript variant X3, mRNA 925 bp linear mRNA (XM_009407999.2). The identified SSR and ISSR biomarkers were genic markers and represented transcripts of the Musa genome  and Ankur (ABB) were clustered separately Fig. A. Group The clustering analysis helped to obtain a dendrogram based on the SSR fingerprinting data of twenty banana cultivars Fig. C. The SSConsequently, we combined the RAPD, ISSR, and SSR fingerprinting data of twenty banana cultivars to construct a dendrogram Fig. D. The coThe two-and three-dimensional PCA based on genetic variance was performed, which helped to categorize banana germplasm into two distinct groups similar to the groups or clusters obtained during dendrogram analysis of the combined molecular marker system Fig. A,B. PCA p > 0.001) was obtained, suggesting the presence of overall high genetic differentiation among and within populations. Further, it was observed that 23% of genetic variation separated the four sub-populations, and 77% of the total variation existed within the sub-populations , contributing to the flavor of these cultivars. Although several compounds such as hydrocarbons, carboxylic acid, chlorine-containing ethers, and diverse functional groups were detected in the volatiome analysis, previous studies suggested that these compounds do not contribute to banana aroma26.Banana fruits are characterized by specific aroma profiles (volatilome), and considerable effort is needed to investigate aroma profiles during the ripening and post-ripening stages or storage of banana cultivars38. Further, it can reduce the number of years consummated from pollination to pre-assessment of first fruit setting and yield, assessing several pollination cycles prior to screening for other biotic and abiotic stresses. Another advantage of MAB is the reduced cost of genetic screening and the wide range of sensing technology that can easily complement phenotyping12.Genetic diversity and the development of genotype-specific markers are crucial for successful breeding and crop improvement. Banana breeding is a time-consuming and laborious process considering the requirements of time for crop cultivation, field size, and human resources. Therefore, the inclusion of marker-assisted breeding (MAB) can speed up the process of germplasm improvement and make it appealing to develop better performing commercial varieties17. However, misnomers are frequently grown as commercially cultivars, mislabelled with different names in local languages. To avoid such mislabeling and inappropriate branding, the characterization of misnomers (or synonyms) using molecular markers is essentially required. Thus a quick, simple, reproducible, and cost-effective method for cultivar identification and validation is required, which can be further developed as a certification method for cultured tissue plantlets of bananas. Moreover, it could also help in germplasm preservation and documentation of novel genetic resources39. Therefore, molecular markers hold promise and might help to encourage banana cultivation and smooth trade of authentic genetic material among banana-growing regions like the Marathwada region of Maharashtra, India. The present study offers unique information on molecular markers and volatile profiles for commercially grown cultivars from the Marathwada region, a significant banana cultivation belt within India. This particular region has a growing concern with several misnomers. Hence, a desired molecular marker, a short unique genomic DNA sequence or fragment that carries a conserved inter-specific motif, would be beneficial for appropriate varietal identification. Moreover, such DNA markers possess minimum intra-specific separation to enable the classification of several cultivars and species40; this approach is exploited in different species and cultivars of horticultural crops, including grapes17, pineapple41, and figs42. Our results have demonstrated that ISSR primer UBC-858 was found to be unique to Grand Nain, whereas the SSR maker CNMPF-13 was able to confirm the specific loci from the Ardhapuri cultivar, which could be further developed as biomarkers for DNA barcoding the plantlets produced through tissue culture for use in commercial cultivation or breeding programs. Since these DNA markers possessed strong discriminatory power, it could be helpful in the validation of uniformity among the plant population. The study showed that all the phenotypic characteristics in cultivated Musa sp. are geographical location and climate-specific. An earlier analysis of 68 banana accessions performed using RFLP of ITS fragments (digested with RsaI), produced steady, reliable, and distinctive patterns of polymorphic DNA in Musa sp. cultivars44. Likewise, ITS1–5.8S-ITS2 sequences45 have also been successfully exploited in molecular diversity analysis of Musaceae.Chronically several limitations are faced by banana and other horticultural crop breeders as well as farmers from the tropics or subtropics, for example, with the widespread occurrence of misnomers25. The distinctive chemical nature of aroma and flavor are formed by combination of several volatile small molecules. In the case of banana, its agreeable and classic aromatic smell has been studied relatively recently, and there have been over 150 volatiles found to bequeath a unique aroma32.The aesthetic value of fruits is primarily defined by the constituent volatiles (volatilome), which contribute to the unique aroma and flavor. Moreover, the aroma or flavor of fruits generates significant appeal driving the urge for consumer acceptance, consumption and usage26. In our study, we observed that over 56% contribution to banana flavor is contributed by esters and in cultivar, Grand Nain esters contributed to 78% of the volatiles. Similarly, cultivars namely Ardhapuri, Karpurvalli, Ankur, and Borkee Baista represented significant portion of volatiles belonging to ester compounds. Suggesting dominance of the ester molecules in contribution towards characteristics aroma in these cultivars. Additionally, alcohols and carboxylic compounds were also significant constituents of banana aroma. The SPME method used to extract volatiles is simple, quick, robust, and solvent-free24. The method was efficient for the recovery of volatiles and could be optimized quickly for any fruit volatilome analysis46. Since some of the volatile compounds are reported to impart resistance against plant pathogens47, additional research is warranted on extensive spatial and temporal volatilome analysis from banana crops to establish the underlying mechanism of volatile biosynthesis and genetic control of resistance against certain pathogens47. In the future, novel technologies such as CRISPR-Cas48 and nanotechnological applications52 could help to enhance banana crop improvement.In the present study, we report over 33% of the volatiles from fruits of nine banana cultivars. This volatiles belonged to several classes of small molecules like esters, ketones, terpenes, and aldehydes. Previously, isoamyl and isobutyl esters and ketones have been reported to be dominant in bananaSince banana is a polyploid and vegetatively propagated horticultural plant, genetic diversity and the development of genotype-specific markers are crucial for successful breeding and crop cultivation. The present study revealed a high polymorphism across the 20 banana genotypes evaluated with 11 RAPD, 11 ISSR, and 12 SSR primers. Phylogenetic and PCA clustered 20 banana genotypes into two significant clusters at 62% similarity level and 34% cumulative variation. AMOVA distributed 23% variation among the populations and 77% within the sub-populations with a significant FST value of 0.23, representing a high level of genetic differentiation. Interestingly, ISSR marker UBC-858 and SSR primer CNMPF-13 yielded a unique fingerprint for Grand Naine and Ardhapuri cultivars, respectively, and could be utilized as cultivar-specific biomarkers for clonal fidelity testing in the tissue culture industry. The volatile profiling revealed the presence of several volatiles in banana cultivars and highlighted the role of ester compounds in the characteristic aroma. Thus, the food processing industry could exploit the contribution of ester-like compounds in banana flavor.Supplementary Information.