Acer rubrum L.) is a type of colorful ornamental tree with great economic value. Because this tree is difficult to root under natural conditions and the seedling survival rate is low, vegetative propagation methods are often used. Because the formation of adventitious roots (ARs) is essential for the asexual propagation of A. rubrum, it is necessary to investigate the molecular regulatory mechanisms of AR formation in A. rubrum. To address this knowledge gap, we sequenced the transcriptome and small RNAs (sRNAs) of the A. rubrum variety ‘Autumn Fantasy’ using high-throughput sequencing and explored changes in gene and microRNA (miRNA) expression in response to exogenous auxin treatment. We identified 82,468 differentially expressed genes (DEGs) between the treated and untreated ARs, as well as 48 known and 95 novel miRNAs. We also identified 172 target genes of the known miRNAs using degradome sequencing. Two key regulatory pathways , Ar-miR160a and the target gene auxin response factor 10 (ArARF10) were selected based on KEGG pathway and cluster analyses. We further investigated the expression patterns and regulatory roles of ArARF10 through subcellular localization, transcriptional activation, plant transformation, qRT-PCR analysis, and GUS staining. Experiments overexpressing ArARF10 and Ar-miR160a, indicated that ArARF10 promoted AR formation, while Ar-miR160a inhibited AR formation. Transcription factors (TFs) and miRNAs related to auxin regulation that promote AR formation in A. rubrum were identified. Differential expression patterns indicated the Ar-miR160a-ArARF10 interaction might play a significant role in the regulation of AR formation in A. rubrum. Our study provided new insights into mechanisms underlying the regulation of AR formation in A. rubrum.Red maple ( Acer rubrum, a large deciduous tree in family Aceraceae Juss1, is often used to beautify urban gardens due to its strong adaptability and rapid growth2. However, this tree struggle to root under natural conditions, and the survival rate of naturally rooted seedlings is low3. Therefore, some red maple varieties, such as the A. rubrum hybrid ‘Autumn Fantasy’, are primarily vegetatively propagated using softwood cuttings4.5. AR formation is a developmental process wherein new roots are generated spontaneously (or in response to certain stimuli) from stems, leaves, or the non-pericycle tissues of older roots6. In particular, AR formation provides the basis for clonal multiplication, a technique that is utilized for the breeding and propagation of many crop and forestry plants7. Auxin, which is formed in roots and stems, and which is involved in stem cell maintenance and differentiation, effectively induces AR formation9. Auxin dosage, gradient, and response are all important for plant root growth10. Synthetic auxins, such as indole-3-butyric acid (IBA), have been used to induce AR for almost 80 years12. However, the molecular mechanisms regulating hormone-induced AR development in forest tree species remain incompletely understood.AR formation is a critical aspect of cultivation for most forest species that are vegetatively propagated from elite genotypesArabidopsis thaliana and Populus13, only a few genes that regulate AR formation have been identified in horticultural plants14, and most of these encode TFs14. In total, 35 genes encoding TFs were shown to exhibit significant changes in expression level during AR growth and development in Populus15. However, the functions of most of these genes remain unknown.Although molecular studies have identified several genes associated with AR in model plant species, such as A. thaliana16. In particular, ARFs are the TFs that regulate the expression of auxin response genes10. Studies of A. thaliana and Populus ARF mutants have shown that ARFs play important roles in plant growth and development. For example, in A. thaliana, single mutations in ARF7 or ARF19 reduced the numbers of both lateral and adventitious roots, and the numbers of lateral and adventitious roots were even more drastically reduced in ARF7/ARF19 double mutants17. Compared with wildtype (WT) poplars, transgenic poplars overexpressing ARF17.1 (Pro35s::PeARF17.1) had numerous stems, no obvious trunk, and an increased number of ARs18. ARFs were strongly inhibited by members of the equally large Aux/Indole-3-Acetic Acid (Aux/IAA) protein family  and its core inhibitor TOPLESS19. Key to the regulation of auxin signaling is the ubiquitin-26S proteasome system20-dependent disassembly of Aux/IAA proteins by a four-member family of SCF E3 ligases assembled with transport inhibitory response 1 (TIR1) F-box proteins or its relatives AuxinBinding F-Box 1 (AFB1), AFB2, and AFB320. Gretchen Hagen 3 (GH3) acyl acid amido synthase has also been reported to help regulate levels of plant hormones, including auxin21.By contrast, several genes and proteins associated with AR have been characterized in 16. miR160, in conjunction with auxin, was shown to regulate the gene expression of ARF10, ARF16, and ARF17; plants overexpressing miR160c produced shorter roots22. miR167-IAA-Ala Resistant3 (IAR3) also influences lateral root development24. In cherry radish, the upregulation of miR160-ARF16, in conjunction with lengthier photoperiods, was shown to drive rapid root formation25. Using high-throughput sequencing, it was shown that the target genes of miR160 and miR390 were related to auxin signaling and involved in the formation of apple rootstock26. Finally, miR396 regulated the transition of root stem cells into transit-amplifying cells by interacting with growth-regulating factor (GRF) in A. thaliana27. miRNAs may also influence AR growth by targeting ARFs10. For example, in A. thaliana, miR160a, miR160b, and miR160c, which have the same mature sequence, target ARF10, ARF16, and ARF17, respectively22. By targeting ARFs, the miR160 family affects the auxin signaling pathway, and thus plays an important regulatory role in root growth and development16.AR development is also regulated by miRNAs, including miR160, miR167, and miR396A. thaliana and poplar, the AR-associated functions of many miRNAs remain unknown in most horticultural tree species. To address this knowledge gap, we aimed to systematically identify candidate miRNAs that may be involved in AR development in A. rubrum. In addition, to investigate the AR-associated regulatory miRNA network, we aimed to characterize the expression profiles of miRNAs and their targets during A. rubrum root development. To this end, we constructed RNA-seq, sRNA-seq, and degradome libraries from the ARs of A. rubrum ‘Autumn Fantasy’. We then used bioinformatics approaches to identify AR-associated regulatory pathways, to identify TFs and miRNAs associated with those regulatory pathways, and to characterize the expression profiles of the identified miRNAs and their targets. Finally, to further explore the regulation of ArARF10 by Ar-miR160, we overexpressed Ar-miR160a in A. thaliana and assessed produced root numbers and lengths. Our study showed that the Ar-miR160-ArARF regulatory network plays crucial roles in the growth and development of ARs. These results are of great scientific significance, as they clarify the molecular mechanism of AR formation in A. rubrum, as well as provide a basis for the improvement of A. rubrum rooting ability through molecular breeding to increase its utility in landscape applications.Despite these previous studies in model plans such as After 30 days of growth, all IBA-treated groups (100–500 mg/L IBA) had developed ARs, while only a small amount of AR development was observed in the control group  were remained. The percent GC content was greater than 38.16%, the AT and GC ratios were within a reasonable range, and there was no separation. We identified 82,468 DEGs  between the control and IBA300. Of these, 69,777 were upregulated and 12,691 were downregulated. Cross all DEG sequences, 31,385 CDS were detected, and 1417 Unigene-encoding transcription factors were predicted. These sequencing data were submitted to the SRA database with the accession numbers SRR13808891, SRR13808890, SRR13808889, and SRR13808888.In total, approximately 51.22–52.42 million high-quality clean reads from the control and IBA300 groups were obtained using sRNA-seq. Most of the sRNAs were 21–24 nt long, with 24 nt sequences being the most common across all samples Fig. . The reaAnalysis of the sRNA-seq data showed that identified 48 known Table  and 95 nA. rubrum, we first identified the mRNA transcripts targeted by miRNAs in the total RNA samples using high-throughput sequencing. We obtained 34,373,253 short reads from the control RNA samples and 28,061,482 short reads from the IBA300 RNA samples, representing the 5′ ends of uncapped, poly-adenylated RNAs. After initial processing, equal numbers of 20 and 21 nt sequence reads remained. In total, 47.66% of the unique reads were successfully mapped to the A. rubrum transcriptome. We identified 172 mRNAs targeted by the known miRNAs and 243 miRNA-mRNA pairs using CleaveLand (Table To generate a miRNA-cleaved target library (degradome) from nd Table . These sAr-miR172a-2 targeted AP2 (CL321.Contig7_All), at position 2063 nt; Ar-miR160a-5P targeted ARF (CL3897.Contig1_All), at position 1793 nt; Ar-miR156f targeted SPL (CL2023.Contig7_All), at position 1497 nt; Ar-miR171 targeted SCL (CL2444.Contig4_All), at position 1501 nt. All peaks were category 0. Three of the validated differentially expressed miRNAs  had previously been shown to target transcription factors.The T-Plot of five conserved miRNAs and one predicted novel miRNA is shown in Fig. Across all DEGs, GO analysis indicated that 14,451 were significantly enriched in 30 GO terms between the control and IBA300 groups . KEGG mapping indicated that 59 terms and 12 metabolic pathways were significantly enriched in these DEGs, with the largest numbers of DEGs associated with the MAPK signaling pathway and the plant hormone signal transduction pathways Fig. . The logAux/IAA5 (Unigene3732_All), SAUR11 (Unigene73966_All), Aux/IAA4 (Unigene5767_All), GH3 (CL4559.Contig2_All), and ARF18-1 (Unigene5190_All). The expression levels of 24 genes  were increased in IBA300 as compared to the control  were significantly differentially expressed between the control group and the IBA300 group, including We also performed GO and KEGG analyses of the miRNA target genes. We found that the GO term “DNA binding pathway (GO: 0003677)” was significantly enriched in the target genes (p = 0.003258). KEGG pathway analysis also showed that 10 of the targets of the 220 differentially expressed miRNAs that played a significant regulatory role in the plant hormone signaling pathway belonged to three gene families  Fig. .A. rubrum growth, especially during rooting.Nine miRNAs were selected for qRT-PCR validation. All nine miRNAs were significantly downregulated in the IBA300-treated samples as compared to the control Fig. , which wA. rubrum roots, we quantified the expression levels of six miRNAs and six target genes that were verified to interact by degradome sequencing, and where the target gene was also differentially expressed in the hormone transduction pathway. After IBA treatment, these six miRNAs showed inverse expression patterns to the corresponding six target genes  to 3510 bp (ArARF19-3). These genes encoded polypeptides of 612–1170 amino acids, with predicted molecular masses of 67.17–131.19 kDa and theoretical pIs of 5.30–8.39  phylogenetic tree, which included 18 ana Fig. A. The ARIII Fig. C. UnsurpIII Fig. B.ArARF10 (CL3897.Contig1_All) and ArARF18-1 (Unigene5190_All); Ar-miR171d-1 targeted ArSCL6-1 (Unigene23148_All) and ArSCL6-2 (CL2444.Contig4_All); and Ar-miR156f targeted ArSPL-2 (CL2023.Contig1_All)  values for the precursors of our three focal miRNAs  were − 87.10 kcal/mol, − 72.90 kcal/mol, and − 34.70 kcal/mol, respectively. The precursor sequences had typical stem-loop structures Fig. . High-thArARF10 in N. benthamiana protoplasts showed that, while the green fluorescent protein (GFP) control was dispersed throughout the cell   Fig. G. Howeve WT Fig. H. Overex28. Our results suggested that 300 mg/L IBA most effectively induced rooting in A. rubrum cuttings. It was found that the efficiency of AR formation was a Gaussian distribution that correlated with the concentration of IBA, indicating that, as in many other woody plants, auxin might play an important role in AR formation in A. rubrum.The plant hormone auxin is commonly used to promote vegetative propagation in woody plants that are difficult to rootA. rubrum in response to IBA treatment, we first identified 9507 genes that were differentially expressed in IBA-treated A. rubrum ARs compared to untreated A. rubrum ARs. KEGG pathway analysis suggested that these genes were associated with a total of 134 metabolic pathways. In particular, genes in the tryptophan metabolism, anthocyanin biosynthesis, and nitrogen metabolism pathways were generally downregulated in response to IBA treatment.To further investigate the molecular mechanisms regulating AR formation in 29. Here, genes in the pathway responsible for synthesizing the auxin precursor tryptophan were generally downregulated after exogenous IBA treatment. Thus, we speculated that exogenous auxin might inhibit the synthesis of internal auxin in A. rubrum. However, the changes in expression profiles of genes in the plant hormone signal transduction pathway suggested that appropriate exogenous auxin might prolong AR development, improving rooting.Because auxin has such a profound effect on plant growth and development, it is likely that certain checks must be in place to prevent excessive responses. Consistent with this, it has been shown that the PIN auxin efflux carrier genes transport excess auxin out of the cell via a feedback regulation loop31. A previous transcriptomic study in carnation indicated that sucrolytic enzymatic activity was regulated at the transcript level during AR induction32. Consistent with this, we found that 384 of the DEGs were enriched in the “starch and sucrose metabolism” (ko00500), while several genes associated with the sucrose metabolism, including α-glucosidase 3 (Unigene42466_All) and glucoamylase 1 (Unigene10485_All), were significantly upregulated at the transcriptional level. This implied that the energy provided by the sucrose metabolism is crucial for the development of ARs in A. rubrum.AR development is characterized by high energy requirementsA. thaliana proteome20, are a large and complex system of degraded proteins that play a prominent role in hormone regulation. Among the UPS, the SCF group, consisting of the Skp1, CUL1, and SCF E3 subunits34, was the most numerous and well-characterized36. Auxin mediates the binding of Aux/IAA to TIR1/AFBs, and then brings the complex to the SCF for ubiquitination and subsequent degradation37. Several studies have shown that auxin promotes the SCFTIR1/AFB degradation of Aux/IAA proteins39. Here, most of the ubiquitination-associated DEGs were implicated in the ubiquitination and degradation of Aux/IAA by the SKP1-CUL1-SCF complex, mediated by auxin. In particular, genes that mediate the ubiquitination of Aux/IAA proteins, including SKP1 (Unigene7049_All), CUL1 (Unigene20934_All), and SCF E3 F-box (Unigene17129_All), were significantly upregulated in response to exogenous auxin treatment. This result suggested that auxin-mediation ubiquitination and degradation of Aux/IAA might facilitate AR formation in A. rubrum.The multisubunit ubiquitin ligases of the ubiquitin–26S proteasome system (UPS), which comprise 6% of the Aux/IAA family are short-lived nuclear proteins that play a critical role in suppressing the expression of ARF-activated genes40. Aux/IAA proteins have been known to bind to ARFs and prevent the activation of auxin-responsive genes in the absence of auxin41. At high levels of auxin, Aux/IAA proteins are ubiquitinated by interaction with the TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) (Unigene20637_All) receptor and subsequently degraded by the 26S proteasome43. The SCFTIR1/AFB-Aux/IAA-ARF signaling system thus allows for a diversity of auxin responses. Interestingly, our transcriptome sequencing results indicated that this signaling regulatory system in A. rubrum ARs was significantly affected by treatment with exogenous auxin. Altering the transcription of genes in the auxin signaling pathway might be a genetic strategy used by A. rubrum in response to auxin alterations. Analysis of these genes might help to identify those that play a key role in AR development.Members of the ArARF genes in the AR transcriptome of A. rubrum, most of which contained an amino-terminal DNA binding domain, a middle region that functioned as the activation or repression domain, and a carboxy-terminal dimerization domain. Four of these genes were significantly upregulated in response to IBA treatment: ArARF5 (CL11296.Contig1_All), ArARF17 (CL5608.Contig3_All), ArARF18-1 (Unigene5190_All), and ArARF19-1 (CL1642.Contig1_All). The nuclear targeting of the ArARF10 protein was consistent with transcriptional regulation and transcriptional activation functions. Structural domain function analysis implied that ArARF10 promoted AR development in A. rubrum by activating downstream auxin-responsive genes. These results indicated that ArARFs might play different roles during AR development in A. rubrum cuttings.We identified 14 putative A. rubrum, Longan, Citrus sp., and A. thaliana suggested that ARF proteins fell into three major clades, consistent with previous evolutionary analyses47. Consistent with this, previous studies have shown that ARF proteins include a highly conserved region of about 100 amino acid residues in the N-terminal region, corresponding to the DNA-binding domains48. Motif analysis identified 10 unique motifs across the 18 ArARF proteins49. Notably, ArARF8, ArARF10, and ArARF6-2 exhibited a high degree of homology with DiARF8, DiARF1, CsARF6, CsARF8, and CsARF10. In addition to the high degree of domain conservation and homology in ARF proteins among species, numbers of ARF copies are similar between A. rubrum and other species in the Sapindales, including Longan, and Citrus sp.50. This indicated that species in this order might undergo similar whole-genome duplications, and it was speculated that ARF diversification might represent an adaptation to environmental changes51.Phylogenetic analysis of the ARF proteins from 52, miRNA interactions with TFs and other targets might also influence the regulatory networks that control the root transcriptome and might play a key role in the eventual translation of mRNAs53. Using sRNA sequencing, we identified six differentially expressed miRNAs associated with plant hormone signal transduction. miR171, SCL6-II, SCL6-III, and SCL6-IV are expressed ubiquitously in plants55. Transgenic plants overexpressing MIR171c (35Spro–MIR171c) and scl6-II scl6-III scl6-IV triple mutant plants exhibited pleiotropic phenotypes56, including alterations in shoot branching, plant height, chlorophyll accumulation, root development, flower architecture, and leaf shape and pattern. We found that Ar-miR171 cleaved ArSCL in A. rubrum. Ar-miR171 was significantly downregulated after exogenous IBA treatment, suggesting that the expression of miR171/SCL might be related to auxin activity in A.rubrum. miR171/SCL may play an important role in AR development. In Arabidopsis, SPL was targeted by miR156, while SPL3, SPL9 and SPL10 were involved in suppressing lateral root growth57. In addition, both miR156 and SPLs were sensitive to auxin signaling58. In our work, we found that Ar-miR156 was sensitive to exogenous IBA and targeted ArSPL, suggesting that miR156/SPL might be involved in AR development in A. rubrum.Although ARF TFs have been shown to play a critical role in the regulation of root development and auxin correspondenceArARF10 and ArARF18-1. Ar-miR160 and its target gene ArARF10 showed opposite expression patterns during AR formation in the presence of exogenous auxin. Most miRNAs in the miR160 family regulate plant development through the targeted cleavage of ARFs. Transgenic A. thaliana and Medicago truncatula overexpressing miR160 had shorter roots than their WT counterparts59, and a study in Longan showed that Dlo-miR160 negatively regulated DiARF10, DiARF16, and DiARF17 to affect hormone signaling and somatic embryogenesis60. In developing ARs of poplars, peu-miR160a was downregulated, while its target (peARF17.1) was upregulated61; AR formation in transgenic poplar overexpressing peu-miR160a was inhibited, whereas AR formation in polar overexpressing PeARF17.1 or PeARF17.2 was promoted18. Finally, A. thaliana overexpressing miR160 exhibited reduced AtARF10 and AtARF16 expression, shortened roots, and abnormal root cap development, as well as uncontrolled cell division and root apical meristem (RAM) differentiation22. Considering the highly conserved nature of the miR160 family62, we speculated that Ar-miR160 plays an important role in AR development through the targeted cleavage of ARF genes.Ar-miR160a-5p and Ar-miR160b-1 targeted and cleaved miR160 in AR development, the specific effects of miR160 expression tend to vary among species. For example, miR160 overexpression in poplar suppressed AR development and elongation, but overexpression of miR160a in A. thaliana promoted AR development16. Here, A. rubrum seedlings overexpressing Ar-miR160a generally had fewer ARs than the WT. Similar results were observed in poplar seedlings overexpressing Peu-miR160a18.Despite this evidence supporting the important role of A. thaliana overexpressing ArARF10 had more ARs than the WT; GH3.6 expression was also significantly upregulated in the transgenic plants. GH3 genes are highly responsive to exogenous auxin63; auxin stimulates AR development by inducing the expression of the GH3.3, GH3.5, and GH3.6 genes via the positive regulators ARF6 and ARF8. Consistent with this, the three-null mutant gh3.3gh3.5gh3.6 had fewer ARs than its wild-type counterpart64. These results suggested that, in A. thaliana, high levels of auxin led to the overexpression of ArARF10, stimulating AR formation. Interestingly, although ArARF10 was functionally similar to AtARF8 and AtARF6, ArARF10 overexpression did not upregulate GH3.3 and GH3.5, implying that auxin regulation pathways might differ between A. rubrum and A. thaliana. Our examination of Ar-miR160-ArARF10 function in AR development in A. rubrum revealed differences with its role in AR development in A. thaliana. AtARF17 suppresses AR development in A. thaliana16, whereas ArARF10 played a positive role in AR development in the model. Interestingly, this difference was also present between Populus and A. thaliana. PeARF17.1 plays a similar role to ArARF1018.In this study, we identified two pathways involved in the auxin response and analyzed the ArARF genes in those pathways. We characterized the TF ArARF10, and separately verified the role of the Ar-miR160a-ArARF10 interaction in AR development. We found that, during AR development, Ar-miR160 acted as a negative regulator to cleave the positive regulator ArARF10; this process was also integrated into the plant hormone signaling pathway and the ubiquitin mediated proteolysis pathway. Based on our results, we propose a model clarifying the mechanisms underlying the promotion of AR growth by exogenous auxin , the miRNA Ar-miR160a, and its target gene ArARF10, were involved in the auxin response. Experiments overexpressing ArARF10 or Ar-miR160a showed that ArARF10 increased AR development, while Ar-miR160a inhibited AR development. Our results may help to clarify the key regulatory pathways and genes associated with AR formation in woody plants, and provide a basis for the eventual improvement of A. rubrum rooting success.In this study, AR development in This study including sample collection was conducted following China’s Biodiversity Conservation Strategy and Action Plan (2011–2030) (Index number: 000014672/2010-00714) and the Seed Law of the People’s Republic of China (2015 Revised Version), which together permit use of biological resources by Chinese researchers for scientific purposes.A. rubrum hybrid ‘Autumn Fantasy’ trees. The branches were divided into 8–10 cm cuttings, each containing two nodes. Cuttings were disinfected with 0.5% potassium permanganate solution, and then soaked in 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, or 500 mg/L IBA for 1 h to induce AR formation (n = 50 branches per group). The depth of each cutting was 3 cm. Control cuttings were soaked in clean water for one hour (n = 50). Cuttings were then grown in a greenhouse under a 16 h light (25 °C)/8 h dark (18 °C) photoperiod. After 30 days of growth, roots were washed and the numbers of ARs were recorded. The ARs from branches soaked in 300 mg/L IBA and in clear water were stored at − 80 °C for RNA sequencing (RNA-seq), small RNA sequencing (sRNA-seq), and degradome sequencing.We collected full and semi-woody annual branches (60 cm in length) from Total RNA was isolated from the CK and IBA300 groups using Trizol Reagent  following the manufacturer’s instructions. After verification of RNA sample quality using a Nanodrop 2000 , mRNA was reverse transcribed using PrimeScriptTM . Finally, the double-stranded cDNA was purified using a DNA purification kit  to generate high quality cDNA. Libraries were prepared from 300 to 500 bp size-selected fractions following adapter ligation and agarose gel separation. The libraries were sequenced using a paired-end read protocol with 100 bp of data collected per run on the Illumina HiSeq 2000.The sRNA libraries were constructed using a NEBNext Multiplex Small RNA Library Prep Set for Illumina and sequenced on a BGISEQ-500 system. mRNA was captured using magnetic beads. Mixed biotinylated random primers and mRNA were used for reverse transcription. After library preparation was completed, the constructed library was sequenced using an Illumina HiSeq2500, with a single-ended read length of 1 × 50 bp.67. The transcripts were clustered and redundant transcripts were eliminated using Tgicl to obtain Unigenes68. The assembled transcripts were quality-checked by comparison with conserved genes using the single-copy direct homology database BUSCO69. Potential duplicate molecules were removed from the aligned BAM format records. Fragments per kilobase of exon per million fragments mapped (FPKM) values were used determined relative gene expression levels in Cufflinks70. Novel genes were identified based the transcripts newly discovered using Cufflinks. Genes encoding peptides with less than 50 amino acid residues were filtered. DEGs were detected using DEGseq; gens were considered differentially expressed when thefold change in relative expression was ≥ 2 and the Q-value was ≤ 0.00171.The raw transcriptome sequencing data were filtered by removing adapters and low quality sequences. The clean data were assembled using Trinity version 2.0.672. Fold-change in miRNA expression levels were calculated using Transcripts Per Kilobase Millions (TPMs)73. DEGseq was used to identify differentially expressed miRNAs between IBA300 and CK71. The P-values calculated for each gene were adjusted to Q-values to correct for multiple testing using two alternative strategies74. miRNAs were considered differentially when fold change was ≥ 2 and Q-value was ≤ 0.001.The raw sRNA sequencing data were filtered by removing adapters and low-quality data to generate clean reads. Unique sRNAs were aligned to the miRNA precursors of corresponding species in miRBase to obtain a miRNA count. We used miRA, which identifies miRNAs based on the ability of the miRNA precursors to form hairpin secondary structures, with default plant parametersto predict novel miRNAs76. The mRNA sequences of target genes paired with the sRNA sequences of A. rubrum were predicted by Targetfinder, with a prediction score cutoff of four. The target genes corresponding to the predicted miRNAs and the mRNAs in the degradome were combined and analyzed to identify shared mRNAs targeted by the miRNAs. Degradome peak classifications and scores, as well as the prediction results, were plotted77. The MFE values for these precursors were predicted using the classic algorithm of Zuker and Stiegler78.Standard sequences were compared to the degradome sequences using reads per million (RPM) to remove redundancy. The Needle program in the EMBOSS package was applied to derive all sequences that matched the sequences in the previous sRNA library, and then the columns were scored according to the plant miRNA/target pairing criteria81. We investigated the GO enrichment of DEGs using the Gene Ontology Consortium (http://geneontology.org/); p-values were FDR corrected using DEGseq71, and terms with Q-value ≤ 0.05 were considered significantly enriched in the DEGs. We identified KEGG pathways significantly associated with the DEGs based on relative expression levels. Hierarchical clustering of expression data was performed using TBtools82.DEGs were annotated by searching the sequences against the NR, Swiss-Prot, GO, KEGG databases using BLASTA. thaliana (https://www.arabidopsis.org/) against the transcript of A. rubrum using Tblastn, with the following settings: matrix, BLOSUM62; expect value, < 1e − 005; gap-existence, 11; gap-existension extension, 1; and filter, low-complexity. Previously published ARFs from A. thaliana, C. sinensis, and D. longan were identified using a BLASTP search with the score value set to ≥ 100 and e-value set to ≤ e−1083. Next, the Pfam database (http://pfam.xfam.org/) was used to determine whether each candidate ARF sequence belonged to the ARF gene family84. To exclude overlapping genes, all candidate ARF genes were aligned with ClustalW and checked manually. The SMART (http://smart.embl-heidelberg.de/) and InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) web servers were used to examine the conserved domains of the identified A. rubrum genes. Obtained ArARF genes were named based on their A. thaliana homologs. Protein characteristics, including molecular weight (MW), isoelectric point (pI), and length, were predicted using the online ProtParam tool.We searched 23 protein sequences from A. thaliana ARFs, 16 D. longan ARFs, and 18 citrus ARFs . Neighbor-joining (NJ) phylogenetic analysis of the 74 ARF protein sequences was performed using MEGA785, with 1000 bootstrap (BS) replicates. The best-fit model of nucleotide substitution  was JTT + G + I. All positions with less than 80% site coverage were eliminated; that is, fewer than 20% alignment gaps, missing data, and ambiguous bases were allowed at any position86. To investigate the structural differences among ArARF genes, conserved motifs in the predicted ARF proteins were investigated using online MEME analysis (http://meme.nbcr.net/meme/), with an optimum motif width of 6–50. The maximum number of motifs was set to 1087. Results were visualized using TBtools88.To explore inter- and intra-specific phylogenetic relationships among ARF proteins, we constructed a dataset of ARF proteins including the ArARFs identified in this study, as well as 22 89, Actin6 was used as the internal control90. PCR volumes (20 μl) contained 1 μl of 20× diluted cDNA, 5× SYBR buffer, and 0.25 μM forward and reverse primers; the gene-specific primers used for qRT-PCR are given in Table −ΔΔCt method while the relative fold-change in ArARF10 expression was calculated using the 2−ΔCt method91.qRT-PCRs were performed using a CFX Connect TM Real-Time System. Each total RNA sample (2 μg) was digested with RNase-free DNase I (Invitrogen) to remove genomic DNA contamination and subsequently reverse transcribed into cDNA using an Oligo (dT)18 primer. For the qRT-PCR of the miRNAs, U6 snRNA was used as the internal control. For the qRT-PCR analyses of the miRNA target genesArARF10 were cloned from the cDNA of ARs from A. rubrum. The subcellular location of ArARF10 was determined by transfecting GFP-tagged ArARF10 into 3–4-week-old Nicotiana benthamiana protoplasts. The full-length cDNA of ArARF10 was fused in-frame with GFP cDNA and ligated between the CaMV 35S promoter and the nopaline synthase terminator. The fluorescence signals in the tobacco protoplasts were examined under a confocal laser scanning microscope (Axio Lab A1)92. The transcriptional activation of ArARF10 was investigated by transforming the pGBKT7 construct (in which ArAFR10 was fused with the GAL4 DNA-binding domain) into yeast strain AH109. Yeast strain AH109 contains the His-3 reporter genes. The transformants were streaked on the SD/Trp- and SD/Trp-/His-media. After incubation at 28 °C for 3 days, the growth of the transformants was evaluated. The transformed yeast cells were grown on synthetic defined (SD) media (with or without His); yeast with active His-3 reporter genes were able to grow on synthetic defective plates.The full-length cDNA sequences of the target gene Ar-miR160a and ArARF10 affected the growth and development of A. thaliana. The Ar-miR160a-5p precursor was cloned from A. rubrum genomic DNA. The precursor sequence of Ar-miR160a-5p and the ORFs of ArARF10 were cloned into the entry vector pTOPO-T. After verification by sequencing, the fragments of the Ar-miR160a-5p precursor and ArARF10 inserted into the entry vector were transferred to the destination vector pCAMBIA1301 via an LR reaction. The constructed vectors (Pro35S::Ar-miR160a and Pro35S::ArARF10) were driven by the CaMV 35S promoter. The pCAMBIA1301 vector contained a GUS reporter gene initiated by the 35S promoter.We next tested whether the overexpression of Pro35S::Ar-miR160a and Pro35S::ArARF10) were introduced into WT A. thaliana (Columbia-0) using electroporation and Agrobacterium-mediated transformation. Seeds of the plants exhibiting transgenic phenotypes were sterilized and sown on 1/2 Murashige and Skoog (1/2 MS) medium, as described previously93. Plates were incubated at 4 °C for 48 h for stratification and then exposed to light for several hours to induce germination. Plates were next wrapped in three layers of aluminum foil and placed in the dark until the seedlings had an average hypocotyl length of 6 mm (48 h). The A. thaliana primordium was excised using a sterile blade, preserving about 6 mm of the hypocotyl, and then the seedlings were exposed to a natural photoperiod (16 h light/8 h dark) to induce AR formation. AR lengths and numbers were measured using ImageJ94 8 days after transfer to lighted conditions. For each biological replicate, at least ten seedlings were analyzed, and each experiment was repeated at least three times. One-way ANOVAs, combined with Tukey's multiple comparison tests, were performed using GraphPad Prism 8 to analyze differences in means and variances among genotypes.The constructs . After overnight staining at 37 °C in the dark, seedlings were decolorized in 70% ethanol, examined, and photographed.After eight days of cultivation, transgenic and WT Supplementary Information.


BSI decline associated with OS (p = 0.0194). PSA-RR(≥ 50%) was 43%(6/14) in CTC−/ARV7−, 19%(5/26) in CTC+ ARV7−, and 12%(1/8) in CTC+/ARV7+ ( p > 0.05). AR-V7 in CTCs at baseline not associated with OS. AR-V7 was not associated with CBZ resistance in CTCs. Reductions in BSI and PSA in early stages of CBZ treatment may predict OS.Androgen receptor splice variant-7 (AR-V7) expression in circulating tumor cells (CTCs) in metastatic castration-resistant prostate cancer (mCRPC) is associated with abiraterone and enzalutamide resistance. We determine whether cabazitaxel (CBZ) is equally effective in AR-V7-positive and -negative CRPC and whether AR-V7-positive patients retain CBZ sensitivity. This is the first prospective, open-label, Asian validation study of CBZ in Japanese patients with mCRPC after docetaxel . Primary endpoint was prostate-specific antigen response rate (PSA-RR); secondary endpoints included overall survival (OS), bone scan index (BSI) PSA-RR (≥ 50% decline from baseline) for CTC−/ARV7−, CTC+ /ARV7−, and CTC +/ARV7+ groups. PSA-RR ≥ − 30% was 38% (18/48) and ≥ − 50% was 26% (12/48). BSI-change rate ≥ − 30% was 19% (9/41) and ≥ − 50% was 17% (8/41). Median OS was 13.7(12.2–18.9) months. PSA decline in early CBZ treatment associated with OS ( Approximately 20–40% of the patients with CRPC have primary resistance to new anti-androgen agents such as enzalutamide and abiraterone3. Moreover, virtually all patients who initially respond to these agents develop secondary resistance over time. One plausible explanation for this resistance could be the presence of AR splice variants5. Although truncated AR in these splice variants is incapable of binding ligand, it is constitutively active as a transcription factor. One of the variants by which the AR can become ligand independent is the androgen receptor splice variant-7 (AR-V7). AR-V7 lacks a ligand-binding domain and remains functionally active even in the absence of androgen, which can lead to a poor prognosis7. AR-V7 expression in circulating tumor cells (CTCs) from patients with CRPC has recently been linked to resistance to abiraterone and enzalutamide5.Androgen receptor (AR) signaling is an important survival pathway for castration-resistant prostate cancer (CRPC) cells. In most CRPC cells, the AR maintains its function as a nuclear receptor in a ligand-independent manner, allowing it to evade androgen deprivation therapy8. In addition, Onstenk et al. reported that cabazitaxel (CBZ) response may be independent of the AR-V7 status of CTCs from patients with mCRPC, and that CBZ could be a viable treatment option for patients with AR-V7-positive CTCs9. However, there are limited data on the efficacy of taxane-based chemotherapy for AR-V7-positive CRPC.Theoretically, taxane-based chemotherapy should be effective against CRPC with constitutively active AR because its anti-tumor effect is exerted through a different mechanism, as a tubulin-stabilizing agent. Antonarakis et al. reported that the detection of AR-V7 in CTCs from men with metastatic CRPC (mCRPC) was not associated with primary resistance to taxane chemotherapy.This prospective, real-world study aims to investigate if CBZ is equally effective in AR-V7-positive and AR-V7-negative CRPC, and if AR-V7-positive patients retain CBZ sensitivity. This first-of-its-kind Asian validation study is an open-label, single-arm, single-center  cohort study to assess the efficacy of CBZ in Japanese patients with mCRPC with or without AR-V7-positive CTCs who have demonstrated disease progression on docetaxel treatmentA total of 55 patients with mCRPC were enrolled and received CBZ, with the majority of patients (n = 48) receiving more than four cycles of CBZ. Table Initial treatments for mCRPC included: enzalutamide, abiraterone, radium-223 chloride, flutamide, ethinylestradiol, estramustine phosphate, and docetaxel. Prior to CBZ treatment, all patients had received docetaxel; the median (range) was 6 (1–39). Prior to enrollment, 17 patients (35%) had received abiraterone, 30 patients (62%) had received enzalutamide, and 10 patients (21%) had received both enzalutamide and abiraterone (Table p = 0.19) and no significant difference in time to CRPC and the OS .The median observation period for all 48 patients treated with CBZ was 13.7 (7.3–34.6) months. The median OS for all patients was 13.7 months  , and eight of these 34 samples (16%) had detectable AR-V7-positive cells (Table p = 0.662)  and ≥ 50% in 12 patients (25%)   of the patients did not show a PSA-lowering response to chemotherapy. Overall survival was significantly worse in patients who did not have a PSA-lowering response in the early stages of CBZ treatment .Findings from the CTC analyses showed that median OS was 13.9 months  in the AR-V7-positive group, 13.4 months  in the CTC-positive/AR-V7-negative group, and 16.7 months  in the CTC-negative/AR-V7-negative group .  achieved a ≥ 30% reduction, and eight patients (17%) achieved a ≥ 50% reduction after 12 weeks of CBZ treatment  of G3 or higher developed and the dose of CBZ was reduced in six cases, pegfilgrastim prevented severe FN and treatment-related death in the majority of patients. Although CBZ administration was not discontinued, blood transfusions were required in four patients with G3 anemia and one patient with G3 thrombocytopenia . Four paOnstenk et al., i.e., all patients had previously received docetaxel chemotherapy, and the effect of CBZ treatment was evaluated based on CTC status10. The median OS rate in this real-world study, which included patients with poor performance status or severe comorbidities 14.This study was conducted in Japanese patients with mCRPC in a setting similar to the CABARESC study by es Table , was 13.8. In this study, we found that the efficacy of CBZ was independent of AR-V7 status in both patients who were CTC-positive and -negative , or inclusion into clinical trials (43%)16. Zhang et al. reported that AR-V7-positive patients with mCRPC benefit from taxane chemotherapy regimens rather than additional AR targeted therapies17. However, Belderbos et al. found that AR-V7 status in CTCs had no prognostic value in patients with mCRPC, who progressed after docetaxel and/or enzalutamide or abiraterone and were started on CBZ as the next line of treatment18. However, the authors noted that further prospective trials are needed18, and this current study adds to that evidence base.This study is the first to prospectively investigate the correlation between the presence or absence of CTCs and AR-V7 status to determine the effect of CBZ treatment in Japanese patients with mCRPC. Recent studies have reported that the presence of AR-V7 in CTC is associated with resistance to the novel AR-targeted therapies, such as abiraterone or enzalutamide, but not to taxane-based chemotherapyive Fig. . These five Fig. . This su19, we collected and analyzed CTCs using AdnaTest kit. According to recent findings, the number of CTCs must be considered when describing the relationship between AR-V7 status and prognosis in CTCs20. Even though AdnaTest measured CTC to be negative, CellSearch detected 69.5% of CTCs20, implying that AdnaTest was less sentitive than CellSearch. However, in this study, we used AdnaTest as a “Point Of Care Test,” focusing on the simplicity and feasibility of the CTC analysis. In this study, we demonstrated that AR-V7 in CTCs could be detected in any laboratory without the use of expensive equipment, and the results can be applied in the context of precision medicine. It was also considered that AdnaTest would be useful in clinical practice as a POCT tool that could easily evaluate AR-V7 status even if the CTC detection sensitivity was slightly inferior. Thus, we used the AdnaTest to perform the following three-group analysis: CTC-negative/AR-V7-negative (29%), CTC-positive/AR-V7-negative (54%), and CTC-positive/AR-V7-positive (17%)  Table . Antonarion Fig. . One reaWe propose that the effect of CBZ on bone metastases is also independent of CTCs and AR-V7 status Fig. a. Furthe. reported that reduction of BSI (ΔBSI) after abiraterone administration correlated with survival time in patients with mCRPC23. A retrospective study by Anand et al. also showed that ΔBSI after enzalutamide administration was correlated with survival in patients with mCRPC and bone metastases24. However, a subsequent retrospective study by Miyoshi et al. reported no such correlation between ΔBSI and OS after 16 weeks of CBZ administration25. Although the findings of Miyoshi et al. differ from our findings, there are differences between the studies: prospective versus retrospective study design; slightly larger patient population (48 versus 32 patients); early versus late treatment line with CBZ in mCRPC; and a higher rate of visceral metastases. While there were not many cases of visceral metastases in this study, over 90% of patients had bone metastases; it is, therefore, theoretically convincing that the effect of CBZ on bone metastases contributed to the prolongation of survival.In a retrospective study, Reza et alThere were no new safety concerns identified in this cohort of Japanese patients with mCRPC receiving prophylactic pegfilgrastim, suggesting that tolerability to CBZ in Japanese patients is comparable to that of mCRPC patients in Western countries.21. Additionally, other androgen receptor variants, such as AR-V1, AR-V3, and AR-V9, could have been analyzed for completeness26.This study has a number of limitations as well. First, because CBZ was most frequently administered in patients with relatively advanced stages of mCRPC, it was challenging to evaluate all patients until the end of the study period (seven patients withdrew prematurely). Second, the false positive rate due to benign bone degeneration and medication-related osteonecrosis of the jaw might have affected the analyses in patients with a low percentage of bone metastases (BSI < 1%). Third, and most important, we used AdnaTest exclusively for CTC collection and analysis procedures. If we had used a highly sensitive device capable of counting CTCs, such as CellSearch, the number of CTC-negative groups would have significantly decreased. It may have also contributed to a higher survival rate in the CTC-negative group. Lastly, the full-length andogen receptor (AR-FL) could have been analyzed alongside AR-V7 as an additional associated marker in mCRPCThe findings of this Japanese cohort study confirm previously reported outcomes for CBZ in patients with mCRPC from Western countries who were previously treated with docetaxel. The detection of AR-V7 in CTCs was not associated with CBZ resistance. Findings also suggest that the BSI-reducing response using bone scintigraphy and the PSA-reducing response during the initial stages of CBZ administration could be important predictors of CBZ efficacy on OS rate. CTC-based AR-V7 detection has the potential to be used as a treatment selection biomarker in patients with mCRPC. AdnaTest would be useful in clinical practice as a POCT tool for evaluating AR-V7 status.2 of CBZ every 3–4 weeks until disease progression or the occurrence of unacceptable toxicity. To reduce the incidence of severe febrile neutropenia, Granulocyte Colony Stimulating Factor  was administered prophylactically to all patients beginning with the first cycle of CBZ.Patients with mCRPC who had progressed on docetaxel were recruited from Juntendo University Hospitals (between February 2017 and August 2020) for phase 2, a non-randomized, open-label, single-arm, estimation-based study. The baseline AR-V7 status of CTCs was determined, and all patients received CBZ irrespective of AR-V7 status. Patients received 20 mg/mThis study was approved by the Institutional Review Board of Juntendo University Hospital (admission number: 14–052), and all experiments were carried out in accordance with approved guidelines. All participants provided written informed consent.27. The extracted mRNA was subjected to reverse transcription using the Sensiscript Reverse Transcriptase Kit (QIAGEN). Reverse transcription polymerase chain reaction (RT-PCR) was used to detect PSA, AR-V7, and AR in CTCs. PSA and AR were amplified using the AdnaTest PrimerMix ProstateDetect . These experiments confirmed that all samples were positive for PSA. Thus, we concluded that PSA positivity is a common denominator in this study and defined successful CTC detection as positive PSA expression. The primer set and PCR conditions for AR-V7 RT-PCR are as follows: AR-V7 primer set designed to yield 125-bp AR-V7-specific band—5′-CCATCTTGTCGTCTTCGGAAATGTTA-3′ and 5′-TTTGAATGAGGCAAGTCAGCCTTTCT-3′ . Amplified PCR products were electrophoresed and visualized by the DNA 1 K Experion™ automated electrophoresis system . To evaluate gene expression, the fluorescence intensity scale was set to “scale to local” (default setting), and any visible bands with detectable peaks under these conditions were considered positive.The AdnaTest  was used to detect CTCs in accordance with the manufacturer’s protocol: 5 mL of blood was collected in EDTA-3 K tubes, RNA was extracted using antibody-conjugated magnetic beads (AdnaTest ProstateCancerSelect), and mRNA was then extracted using AdnaTest ProstateCancerDetectThe primary end point was PSA response rate (PSA-RR), and the primary objective was to compare PSA-RR between CTC-negative, AR-V7-positive, and AR-V7-negative (CTC-positive) cohorts. PSA-RR was defined as a ≥ 30% and ≥ 50% decline in PSA level from baseline to 12 weeks of CBZ treatment, or earlier if treatment was discontinued. Secondary end points included overall survival in patients with or without PSA decline 3 months following CBZ treatments, with or without CTCs and AR-V7 status at baseline, and the relationship with or without decline of Bone Scan Index (BSI) by bone scintigraphy. The safety of CBZ in Japanese patients with mCRPC was assessed using CTCAE 4.0 protocol.The Kruskal–Wallis test was used for statistical analysis of patient background. Survival was analyzed using Cox regression models and visualized in Kaplan–Meier plots, and OS curves were generated using the Kaplan–Meier method. The PSA-RR (≥ 50% and ≥ 30% decline in PSA from baseline PSA following chemotherapy) was compared for three groups: CTC-negative/AR-V7-negative; CTC-positive/AR-V7-negative; and CTC-positive/AR-positive; and analyzed using the chi-square or Fisher’s exact tests. Statistical differences between the three groups were compared using the log-rank test. Supplementary Information 1.Supplementary Information 2.


In aging societies, dementia risk increases with advancing age, increasing the incidence of dementia-related degenerative diseases and other complications, especially fall risk. Dementia also escalates the care burden, impacting patients, their families, social welfare institutions, and the social structure and medical system.In elderly dementia, traditional card recognition rehabilitation (TCRR) does not effectively increase one’s autonomy. Therefore, from the usability perspective, we used the Tetris game as a reference to develop an interactive somatosensory game rehabilitation (ISGR) with nostalgic style for elders with mild cognitive impairment (MCI). Through intuitive gesture-controlled interactive games, we evaluated subjective feelings concerning somatosensory game integration into rehabilitation to explore whether the ISGR could improve the willingness to use and motivation for rehabilitation among elders with MCI.A total of 15 elders with MCI  underwent 2 experiments for 15 minutes. During experiment 1, TCRR was performed, followed by completing the questionnaire of the System Usability Scale (SUS). After 3-5 minutes, the second experiment (the ISGR) was conducted, followed by completing another SUS. We used SUS to explore differences in impacts of TCRR and ISGR on willingness to use among elders with MCI. In addition, we further investigated whether the factor of gender or prior rehabilitation experience would affect the rehabilitation willingness or not.t28=–4.62, P<.001). Furthermore, the ISGR individual item scores of “Willingness to Use” , “Easy to Use” , “System Integration” , and “Easy to Learn”  were better than TCRR. The somatosensory game was easier to learn and master for females than for males . Besides, the ISGR was easier to use  and learn  for those without prior rehabilitation experience. The result indicates that for elders with no rehabilitation experience ISGR was easier to use and simpler to learn than TCRR.The novel ISGR made the elderly feel interested and improved their willingness for continuous rehabilitation. According to the overall SUS score, the ISGR had better overall usability performance (73.7) than the TCRR (58.0) (Regardless of prior rehabilitation experience, the ISGR developed in this study was easy to learn and effective in continuously improving willingness to use. Furthermore, the adoption of a nostalgic game design style served the function of cognitive training and escalated interest in rehabilitation. The ISGR also improved user stickiness by introducing different game scenarios and difficulties, increasing long-term interest and motivation for rehabilitation. For future research on the adoption of interactive somatosensory games in rehabilitation, additional rehabilitation movements can be developed to benefit the elderly with MCI. Owing to social changes and medical advancement, the global demographic structure is becoming an aging society. The World Health Organization (WHO)  has repoAmong various treatment methods for dementia, traditional rehabilitation such as card recognition training utilizes commercially available playing cards for cognitive training. However, its long-term use can neither enhance the autonomy of the elderly nor increase user stickiness. On the contrary, digital games have now been introduced as an emerging cognitive training mode and are referred to as “serious games” . Unlike This study found that existing card-based rehabilitation not only failed to promote the autonomous use among the elderly, but also triggered hand movements only 3-5 times per minute. Therefore, this study developed a nostalgic game (Tetris) combined with gesture detection technology, allowing elders use their finger movements to control the interactive game. Concurrently, the nostalgic game stimulated the elder’s memory (returned to their childhood days) as well as increased their frequency of finger movement (8-10 times per minute), thereby effectively improving user motivation as well as achieving a cognitive training effect. However, for the elderly, the operation of interactive somatosensory game rehabilitation (ISGR) was more complicated than that of the traditional card recognition rehabilitation (TCRR). Therefore, whether the operation of ISGR would compromise the willingness to use for elders’ rehabilitation remains an unsolved question. Would the factors such as prior rehabilitation experience and gender be affected by their rehabilitation motivation? To answer these questions, the System Usability Scale (SUS)  was adopThe SUS was developed by Brooke  to exploThis study aimed to develop an interactive somatosensory game to promote the rehabilitation motivation of elders with MCI, to ensure they were satisfied with the ISGR and could continuously use the ISGR. Sections of the paper included discussions of relevant literature on dementia, the design process of the interactive somatosensory game, usability experiments, and analysis and discussion on the SUS.In this section, a literature review and analysis of the importance of dementia rehabilitation, the application of gesture recognition to dementia treatment, and the efficacy of nostalgia therapy on cognitive impairment were conducted. Furthermore, this section will discuss the development status of the application of gesture-based interactive somatosensory games in the rehabilitation.The cognitive function includes emotion, personality, perception, memory, abstract judgment, and the ability to express things, and MCI refers to the transitional stage during which a normal person develops dementia (increasing with advancing age) . During Despite being a disease, dementia is thought of by many family members as a common aging phenomenon and the importance of its treatment is therefore often neglected. Once the severity of dementia and cognitive impairment elevates, the difficulty of care increases correspondingly. Furthermore, as the patient ages, the risk of fall also rises, with a study reporting that the risk of fall of elderly people with dementia was substantially higher than that of those without . While aThe incidence of dementia has been on the rise in recent years, making it a common disease among the elderly. Furthermore, its prevalence increases with advancing age . HoweverRecent studies have verified the correlation between the fingers and the brain. Fried et al  reportedFascinating Dermatoglyphics by Chen [The book  by Chen  mentione by Chen  is shownThumb: Corresponding to the prefrontal lobe, it affects motivational functions .Index finger: Corresponding to the postfrontal lobe, it affects conceptual functions .Middle finger: Corresponding to the parietal lobe, it affects somatosensory functions .Ring finger: Corresponding to the temporal lobe, it affects auditory functions .Little finger: Corresponding to the occipital lobe, it affects visual functions .Stimulating the Thumbs to Make the Brain Younger by Hasegawa [The book Hasegawa  mentioneHasegawa . FurtherWith the advancement of science and technology, the cost of production is becoming increasingly lower, making technology products more accessible and their implementation in medical care an important development goal. At present, multiple gesture recognition sensors are commercially available, of which controller-based visual recognition technologies, such as leap motion (LM) and Kinect (Microsoft), are the most common tools for hand rehabilitation. The therapeutic value of these technological tools has been well verified in previous studies ,33. SmerTreatment methods of dementia can be divided into drug treatment and nondrug treatment; the latter includes nostalgia therapy, music therapy, serious training, art therapy, among others . The goaNostalgia therapy uses a set of planned nursing activities to remind the elderly of people, events, and things that have special meanings to them and guide them to tell their experiences, thereby helping them realize that their life is meaningful and valuable and regain old memories . It can Autobiographical memory is a very important memory for humans, the loss of which can lead to a disconnection between the past and the present and more severely, the loss of personal identity . ResearcThis study aims to explore the integration of interactive somatosensory games into cognitive training and to investigate the feasibility of applying the interactive somatosensory game developed in this study to the rehabilitation of the elderly.The interactive somatosensory game developed in this study incorporated simple operations and easy-to-learn gameplay, and integrated the gesture recognition technology of LM, which distinguished it from traditional Tetris games. Gesture recognition was used to control the number and the movement of the blocks. This not only made it easier for the elderly to operate, but also facilitated the movement and rehabilitation of their hands through hand exercise, thereby improving the cognitive ability of the elderly and preventing degenerative diseases. The following sections provide an in-depth discussion on the 3 major aspects of the game design, that is, gesture recognition technology, game interface design, and game system design.With the development of science and technology and the rise of human-computer interaction, motion recognition technology has received substantial attention. As a key technology in motion recognition, gesture recognition captures the dynamic gestures of human hands through visual sensor technology. In recent years, as the development cost has decreased and technology has rapidly developed, many dynamic gesture capturing tools have emerged in the market. This study adopted the LM gesture capture tool, which realizes gesture recognition through dual cameras and 3 infrared LEDs . LM can The game interface design developed in this study was presented in a nostalgic style, providing players with a unique experience through cultural scenes of Taiwan in the 1960s, such as traditional Taiwanese grocery stores, classic movie posters. The background of this game was based on nostalgic designs created by Chang , who reiOnce the interactive somatosensory game was initiated, the player was first shown the main screen A before The clinical trial of the experiment was described in this section, including research patients and the experimental design . The SUS questionnaire with a 5-point scale was adopted to collect quantitative information.This study interviewed a total of 15 patients at a Clinic in Tainan, Taiwan, consisting of 7 males (5 with rehabilitation experience and 2 without) and 8 females (2 with rehabilitation experience and 6 without). The average age of the patients was 78.4 years. The inclusion criteria were elderly people over 60 years old who were diagnosed with MCI or were prone to dementia. Participants were also required to be able to cooperate with researchers, follow game instructions, and complete a subjective assessment after rehabilitation. The experimental location of this study is shown in The experiment adopted a single-group design, that is, all patients would participate in both TCRR and ISGR. The questionnaire, composed of 10 questions (items), was designed based on the modified SUS . Each quThe TCRR used commercial poker cards for rehabilitation. During the experiment, researchers would cover the cards and place them on the table. The elders could choose any 2 cards and flip them over each time. If the 2 cards were paired (having the same pattern), the elders would gain the opportunity to continue revealing other cards, or otherwise the 2 cards would be flipped back over. The rehabilitation would end when all cards were revealed . In thisPrior to the formal start of the experiment session, the researcher would first explain the operation of the game verbally, followed by 1 or 2 practices to ensure that the elderly understood the operations correctly. This process would take approximately 3 minutes. During the experiment, the game scenario was randomly selected, and the elderly would use gestures to perform the ISGR. Upon completion, the researcher would ask the elderly about his/her subjective feelings of the ISGR through the 5-point scale. The elderly would verbally answer the questions and the researcher would assist in filling out the records.This study was approved by the National Cheng Kung University Human Research Ethics Committee .t test was adopted to analyze whether there were any differences in the willingness to use between TCRR and ISGR. This was realized by conducting 2 SUS surveys, with the first enquiring the elderly about their intention to use (willingness to use) the TCRR, and the second enquiring the same group about their intention to use (willingness to use) the ISGR. t28=–4.62, P<.001). The result shows that the ISGR has better overall usability performance (average +) than the TCRR (poor) [The purpose of this study was to investigate whether the ISGR could improve the willingness and motivation for rehabilitation among the elderly with MCI. The study recruited 15 patients, consisting of 7 males and 8 females with an average age of 78.4 years (please see the “Methods” section). Subsequently, the independent (unpaired) sample R (poor) ,48.average benchmark (items 2 and 8) [average level  and 5 reaching the good level . Only items 4 and 6 need to improve further. This result indicates that the ISGR presents an average or good experience for elders with MCI when interpreting single items from the SUS [To further investigate, 2 and 8) ,48, thust value of TCRR and ISGR was –0.64  and –0.15 , respectively, both of which indicated insignificant differences. This result showed that for elders, their willingness to use the ISGR was not significantly affected by prior rehabilitation experience. Hence, regardless of prior rehabilitation experience, the elderly demonstrated consistent subjective feelings of use (willingness to use) toward both TCRR and ISGR, that is, their intention to use and motivation for ISGR would not be compromised because of complicated operations. This finding was further supported by the elders’ answers to item 3, “I thought the TCRR/ISGR was easy to use.” The t value of TCRR and ISGR for this question was –0.41  and 1.08 , respectively, again showing insignificant differences. Further analysis suggested that despite insignificant differences, the average score of ISGR (4.20) was considerably higher than that of TCRR (3.33). This result revealed the fact that the elderly believed the ISGR was easier to operate/use (please refer to the following sections for more details).One objective of the study was to determine the degree of acceptance of the novel ISGR among the elderly who previously went through TCRR, and to further understand whether prior rehabilitation experience would affect the willingness to use the novel rehabilitation method. Therefore, the 2 rehabilitation methods were analyzed separately for those with and without rehabilitation experience, the results of which are shown in t value of TCRR was –0.54 , whereas that of ISGR was 2.71 , which showed significant differences. The results suggested that the novel ISGR developed in this study was easier to learn for females. Based on the findings, it was concluded that while the willingness to use  and ease of use  of the ISGR were not affected by the gender factor, this was not the case for the ease to learning (item 7). The results indicated that females were more willing to learn new things than males were; this should be investigated in future research.In this section, the 2 rehabilitation methods were analyzed separately for patients of different gender  to see if the gender factor exhibited any effects on the willingness to use the TCRR or ISGR. In the previous sections, effects of “prior rehabilitation experience” and “gender” were analyzed. The results showed that prior rehabilitation experience did not affect the elderly’s willingness to use (item 1) and ease of use (item 3). However, the ease of learning (item 7) was affected by the gender factor. In this section, elders with and without rehabilitation experience were divided into separate groups to analyze their respective subjective feelings (willingness to use) of the 2 rehabilitation methods , item 3 , item 5, “I found the various functions in this TCRR/ISGR were well integrated” , item 7 , and item 10, “I needed to learn a lot of things before I could get going with this TCRR/ISGR” . The results suggested that the novel ISGR proposed in this study showed better “willingness to use,” “ease of use,” “integration,” “ease of learning,” and “system complexity” than TCRR. The elders indicated that they were more willing to use the novel method continuously, which was of great significance for the long-term rehabilitation of patients with dementia.From t12=–4.26, P<.001 and t14=–7.00, P<.001, respectively) and item 5 on “integration” . This is consistent with the previous finding that “Regardless of prior rehabilitation experience, the elderly consistently showed improved willingness to use the novel ISGR over the TCRR and believed that the integration of ISGR was better.”Further analysis was performed by dividing participants into those with and without prior rehabilitation experience. t14=–2.50, P=.20) and item 7 on “ease of learning” . This finding indicated that in terms of “ease of use” and “ease of learning,” the ISGR was easier for the elderly, especially for those without prior rehabilitation experience to master and operate. Thus, if an individual was exposed to the 2 rehabilitation methods for the first time, he or she would find the ISGR much easier to learn and use, which would in turn significantly affect the willingness of the elderly to continue rehabilitation. The results also echoed the conclusions of previous sections.Comparison between the 2 rehabilitation methods suggested that for the elderly without prior rehabilitation experience . This result suggested that regardless of prior rehabilitation experience, the elderly believed that it took them a decent amount of time to learn and become familiar with the TCRR before they were willing to continue rehabilitation. By contrast, the elderly could rapidly master the novel ISGR (because they could use intuitive gesture recognition during rehabilitation) and were more willing to use it in the long term.For item 10 on “system complexity” (I need to learn a lot...), the 2 rehabilitation methods showed significant differences  in the future.


Subcutaneous immunotherapy (SCIT) is an effective therapy for allergic rhinitis (AR), but some AR patients still do not benefit from it. Nasal nitric oxide (nNO) and inducible nitric oxide synthase (iNOS/NOS2) act important roles in AR. This study aims to explore the abilities of serum NOS2 and nNO in predicting the clinical efficacy of SCIT in AR patients.  We recruited 40 healthy controls (HCs) and 120 AR patients in this study. Serum NOS2 and nNO levels were compared between the two groups. In the AR group, patients underwent and finished 1-year of SCIT, and divided into the effective and ineffective groups, and the relationships between serum NOS2 and nNO levels and efficacy of SCIT were evaluated.  The serum NOS2 and nNO levels were higher in AR patients than HCs. In the effective group, the serum NOS2 and nNO levels were increased than the ineffective group. ROC curves presented that a combination of serum NOS2 and nNO exhibited promising predictive ability in predicting the clinical efficacy of SCIT.  Serum NOS2 and nNO levels were enhanced in AR patients and might affect the efficacy of SCIT. The combined use of serum NOS2 and nNO levels could be a reliable and useful method for predicting the clinical efficacy of SCIT. Allergic rhinitis (AR) is one of the most common chronic inflammatory diseases of the upper airways, which is characterized by Th2 type inflammatory disease mediated by immunoglobulin E (IgE) . Recent Inducible nitric oxide synthase (iNOS/NOS2) catalyzes the production of nitric oxide (NO) and plays an essential role in metabolic and inflammatory processes , 13. StuDer f) and/or Dermatophagoides pteronyssinus (Der p) (at least ++) and/or sIgE level for Der f or Der p >0.35 IU/ml; and (3) moderate-severe AR. Exclusion criteria for the patients include (1) age<18 years; (2) with other inflammatory diseases or autoimmune diseases; (3) with active asthma; (4) had a history of immunotherapy; (5) treated with antibiotics, corticosteroids, or antiallergic drugs within 4 weeks before the study; and (6) pregnant condition. Demographic and clinical information of the subjects were collected, and serum samples were collected before SCIT. In the AR group, 94 of 120 AR patients received standard SCIT for 3 years. All participants were asked to complete several questionnaires about their symptoms at baseline and during the whole schedule of SCIT.We recruited 120 AR patients and 40 age- and sex-matched healthy controls (HCs) from December 2019 to January 2020. All patients met the diagnostic criteria of AR referring to the allergic rhinitis and its impact on asthma (ARIA) guidelines . The criFive ml of fresh venous blood was collected from all subjects and stored at room temperature for 1 hour. All blood samples were centrifuged at 4°C (3000 rpm for 10 min), and supernatants were collected and stored in aliquots at -80°C for subsequent experiments.Serum samples were thawed and centrifuged before use. Serum NOS2 levels were measured by the ELISA kit commercial  according to the manufacturer's instructions. Briefly, the target antigen was immobilized by passive uptake on a 96-well polystyrene microtiter plate. A blocking buffer was added to saturate all unbound sites and then incubated with an antigen-specific unlabeled primary antibody. An antihuman enzyme-coupled secondary antibody was subsequently added to bind to the primary antibody. Generally, the secondary antibody is conjugated to horseradish peroxidase (HRP) and detected with an enhanced chemiluminescence substrate (ECL) .Participant held a filter with one nostril blocked, and another nostril was unblocked during the entire measurement. Every subject was requested to inhale air through the filter and then emit a whistle for at least 10 seconds without stopping. Before the examination, smoking and strenuous exercise were unallowed for at least one hour, and intranasal corticosteroids or oral antiallergy medication should be suspended for more than 3 days. The nNO levels were measured by the Nano Coulomb Breath Analyzer (Sunvou-CA2122).Der f and Der p at a 1 : 1 ratio. According to the manufacturer's instructions, SCIT consists of two phases: initial treatment phase and maintenance treatment phase. To achieve long-term efficacy, a treatment course of 3 years is recommended. The initial treatment phase started with the minimum dose of low concentration NHD NO.1 and gradually increased to the maximum dose of high concentration NHD NO.3. The injection interval was generally 7-14 days. For NO.1 and NO.2, the doses were increased from 0.1, 0.2, 0.4, to 0.8 ml. For NO.3, the dose was increased from 0.1, 0.2, 0.4, 0.6, 0.8, to 1.0 ml. In the maintenance treatment phase, patients were injected with 1.0 ml of NO.3 with an injection interval of 4 to 6 weeks. SCIT was conducted in the outpatient department under the guidance of allergy experts, and all patients were observed for >30 minutes before leaving. All adverse reactions were recorded throughout the whole treatment.SCIT was conducted as previously described . All AR After 1-year follow-up, the symptoms and medication consumption were recorded throughout the course of treatment. The early clinical efficacy of SCIT was assessed based on the improvement of clinical symptoms and the reduction of medication consumption after 1 year of treatment. The symptoms of all AR patients were scored using the widely accepted total nasal symptom score (TNSS) and visual analogue score (VAS). The sum of medication consumption in the previous week was defined as the medication score (MS), which was recorded according to the recommendations of the World Allergy Organization: 1, 2, and 3 points for oral or intranasal antihistamines, intranasal glucocorticoids, and oral glucocorticoids, respectively . The sumt-test was used for normally distributed variables, and the Mann–Whitney U test was applied for nonnormally distributed variables. Categorical data were expressed as frequencies and percentages, and the difference was compared using the Chi-square test. Receiver operating characteristic (ROC) curves were performed to evaluate the potential value of serum NOS2 combined with nNO in predicting the clinical efficacy of SCIT. Bilateral P values below 0.05 were considered statistically significant. All statistical analyses were performed using SPSS 19.0 statistical software .Numerical variables were expressed as mean ± standard deviation (SD), Student's A total of 160 individuals were included in this study, including120 cases in the AR group, and 40 cases in the HC group. As shown in P < 0.001, P > 0.05, Figures P < 0.05), but no statistically different in the AR with AC and without AC subgroups in Figures P > 0.05). To further explore the relationship between serum NOS2 and nNO levels and clinical variables, the correlation analysis was performed on the associations among age, BMI, disease duration, baseline VAS, and baseline TNSS. The results showed that serum NOS2 and nNO were inversely correlated with age and BMI, respectively, and serum NOS2 and nNO were positively correlated with each other . Accordingly, NOS2 was derived from airway epithelial cells which were gradually deteriorated with age [NOS2 was reported to be expressed in the nasal epithelial cell and associated with the production of NO, which contributed to aggravating eosinophilic inflammation . Previouwith age . Meanwhiwith age . Given tSCIT was demonstrated to be an effective treatment for AR, but a certain proportion of patients still responds poorly to this therapy , 43. TheThe present study has several limitations. Firstly, the follow-up period was only 1 year, and the therapeutic evaluation might be biased and not representative. Secondly, the sample size was relatively small, and the participants were recruited from a single medical center, which might increase the risk of selection bias. Therefore, further investigation with a larger population and longer follow-up time was necessary to confirm the results of this study.In conclusion, this study had certain strengths in terms of novel design. Our results proved that serum NOS2 and nNO were not only involved in the pathological process of AR but also affected the efficacy of SCIT. Combined use of serum NOS2 and nNO levels seems to be a reliable and useful method for predicting the clinical efficacy of SCIT in AR patients.


The superior germplasm identified for curcuminoids content were as follows; curcumin (CL 157 – 2.17% and CL 272 – 2.13%), demethoxycurcumin (CL 253 – 1.45% and CL 157 – 1.31%), bisdemethoxycurcumin (CL 216 – 1.24% and CL 57 – 1.11%) and total curcuminoid content (CL 157 – 4.55% and CL 272 – 4.37%). Clustering based on dendrogram, grouped 200 accessions into seven clusters. Among seven clusters, the maximum number of accessions were grouped into cluster II while cluster VII showed maximum mean value for majority of the traits. Correlation analysis revealed a significant relationship between the traits where the total curcuminoid content is significantly and positively correlated with the primary rhizome core diameter and length of the secondary rhizome. The selection of these particular traits may result in the identification of germplasm with high total curcuminoid content. Taken together, it is the first report on the large screening of turmeric accessions for variation in the rhizome yield traits and curcuminoids content. The genetic diversity revealed in this study could be useful for further crop improvement programs in turmeric to develop new varieties with high rhizome yield coupled with high curcuminoids content.Turmeric is an important commercial crop widely grown in Asia due to its pharmacological and nutritional value. India is the centre of turmeric diversity and many turmeric accessions have good rhizome yield, varying curcuminoids content and are well-adapted to various agro-climatic zones. In the present study, we unravel the diversity among 200 Indian turmeric accessions based on rhizome yield traits and curcuminoids content. Clustering and correlation studies were also performed to group the turmeric accessions and to observe the relationship between the traits. Results revealed the presence of large variability among turmeric accessions including the major traits such as yield (24.77 g p Curcuma longa L.) belongs to the genus Curcuma (Zingiberaceae) and, is a commercially important crop because of its pharmacological, nutritional, religious, and cultural significance. The crop is indigenous to India and has a very long history of cultivation in Asia. The genus Curcuma contains about 80 species all over Asia. The other important species related to turmeric are C. amada, C. angustifolia, C. aromatica, C. caesia and C. zedoaria (n = 3x = 63), which can be vegetatively propagated using its underground rhizomes (Turmeric (zedoaria . Turmerirhizomes . Curcumirhizomes .C. longa collections), it is due to both rhizome yield and curcumin content, the rhizome yield in second group (Southeast Asian C. longa collections) while it is curcumin content in third group (C. aromatica and other Curcuma species).Genetic improvement work on turmeric is typically limited to germplasm selection as hybridization is unsuccessful in many cases. To assess the genetic diversity, genetic relationship among the germplasm is the backbone of any crop improvement program. This can support breeding activities by both farmers and plant breeders. Many studies used the rhizome yield traits to assess the genetic diversity and there are only few studies which characterized the germplasm based on the curcuminoids content. For instance, research findings of Curcumin content in rhizomes may vary depending on the germplasm. Studies focusing on estimating individual curcuminoid content by screening large number of germplasm might help in selecting the germplasm with high individual curcuminoids content. This is required because when the price is fixed based on curcumin content, farmers are willing to grow varieties with high curcumin content. Depending on demand in markets, traders are even willing to buy turmeric rhizomes with high curcumin content . EarlierIndia is the centre of turmeric diversity in particular Southern India . Indian turmeric accessions exhibit good rhizome yield, varying curcuminoids content, and has potential to adapt different agro-climatic zones. As the farmers and industries are not aware of the characteristics of existing germplasm, particularly with regard to total or individual curcuminoid content, they are facing difficulty in recognizing the superior germplasm. With this backdrop, the objective of the present study was to explore the diversity among 200 turmeric accessions for rhizome yield traits and curcuminoids content. In addition, clustering and correlation studies were performed to understand the relationship between the traits.. longa accessions along with two check varieties viz., CO2  and CIM PITAMBER . The turmeric accessions were originated from different parts of India . The experiment was laid out in Augmented Block Design (ABD) with 20 blocks. Each turmeric accession was planted in two rows in a 3 m × 15 cm Figure 1viz., fresh rhizome yield (g p-1), dry recovery (%), number of mother rhizomes per plant, number of primary rhizomes per plant, number of secondary rhizomes per plant, weight of mother rhizomes (g p-1), weight of primary rhizomes (g p-1), weight of secondary rhizomes (g p-1), length of mother rhizome (cm), girth of mother rhizome (cm), length of primary rhizome (cm), girth of primary rhizome (cm), primary rhizome diameter (cm), primary rhizome core diameter (cm) and length of secondary rhizome (cm) according to the DUS guidelines given by Protection of Plant Varieties and Farmers Rights Authority, Government of India. For dry recovery, one kilogram of fresh rhizomes from each turmeric accession and check varieties were sliced to a thickness of 2 mm. It was further spread on the floor for drying under a glass house until the moisture content was sufficiently reduced to less than 10% (approximately seven days until the sliced rhizomes are brittle enough). Finally, the dry recovery percentage was calculated using the following formula,Rhizomes were harvested when the plant leaves were completely withered. After harvesting, rhizomes were evaluated for 15 traits, 1 is the fresh weight of the rhizomes and W2 is the dry weight of the rhizomes.Where W-1), demethoxycurcumin (1 mg mL-1) and bisdemethoxycurcumin (0.5 mg mL-1) were prepared in acetone and were loaded on a TLC plate. Chromatography was performed on a pre-activated (110°C) silica gel TLC plate 60 F254 (20 x 10 cm). Standards and samples were applied to the plate as 8 mm wide bands with an automatic TLC applicator, Linomat 5 . The TLC plates were developed using a CAMAG twin trough glass tank (20 X 10 cm), which was pre-saturated with mobile phase chloroform-methanol (95:5) for one hour, and each plate was developed to a height of about 8 cm. TLC was run at 25 ± 5°C and 50% relative humidity. After development, the TLC plates were removed, allowed to dry for a few minutes, and spots were visualized under a UV chamber  at 366 nm. Curcuminoids were quantified with a CAMAG TLC Scanner 4 equipped with Vision CATS software and a computer. The slit width is kept at 6 x 0.45 mm. A wavelength of 436 nm is used with absorption/reflection detection mode.Powdered samples of 200 accessions and two check varieties (0.2 g each) were extracted separately in 10 mL of acetone for 12 hours, filtered, and evaporated until near dryness. The individual sample extracts were re-dissolved with 2.0 mL of acetone for quantification. Stock solutions of curcumin .UHPLC was performed with Schimadzu N series UHPLC system equipped with binary solvent manager, auto sampler (SIL – 40C XS), a quaternary pump (LC 40 DXS), column manager and a PDA detector. The sampling speed maintained at 5 µL secAll the data of traits were statistically analyzed. Variability and diversity were computed as per the methods suggested by -1 with an average of 357.74 g p-1. The maximum yielding accession was CL 180, whereas the minimum yielder was CL 5 , weight of mother rhizomes per plant (46.77%), weight of primary rhizomes per plant (38.83%), number of primary rhizomes per plant (38.29%), number of secondary rhizomes per plant (37.51%), number of mother rhizomes per plant (36.69%) and yield per plant (33.43%) , dry recovery (21.58%), number of mother rhizomes per plant (4.65), number of primary rhizomes per plant (16.48), number of secondary rhizomes per plant (19.42), weight of mother rhizomes (133.17 g p-1), weight of primary rhizomes (257.65 g p-1), weight of secondary rhizomes (126.03 g p-1), length of mother rhizome (5.59 cm), girth of mother rhizome (9.28 cm) and length of primary rhizome (8.50 cm). Cluster I showed maximum mean value for the girth of primary rhizome (6.96 cm), primary rhizome diameter (2.30 cm), primary rhizome core diameter (1.41 cm), length of secondary rhizome (4.19 cm), bisdemethoxycurcumin content (0.88%) and total curcuminoid content (3.52%). Among the clusters, the maximum mean value for demethoxycurcumin content was 1.04%, and the value was the same for both cluster I and cluster III, whereas the maximum mean value for curcumin content (1.61%) was obtained in cluster III.All the 200 Cclusters Figure 5The first six principal components accounted for about 78.88% of the total variance Table 4.The per cent contribution of each character to the total divergence was obtained by ranking the individual trait. The trait, weight of secondary rhizomes per plant contributed the maximum to the divergence, i.e., 10.25%, followed by weight of mother rhizomes per plant (9.54%) and weight of primary rhizomes per plant (7.92%) Figure 7Pearson correlation analysis was performed between 19 characters of turmeric germplasm, and correlation coefficients were given in -1), but the dry recovery percentage of CL 180 was significantly low when compared with the accession having maximum dry recovery (29.18%). But, the accession with maximum dry recovery percentage (CL 9) is a low yielder (119.23 g p-1). CL 161 is one of the top three high-yielding accessions and also possessed values that are significantly superior over the means of dry recovery percentage, number of mother rhizomes per plant, and the weight of primary rhizomes per plant. In addition, this CL 161 was found to have the maximum weight of mother rhizomes per plant (229.28 g p-1). Similar research on 19 turmeric genotypes at high altitude region of Andhra Pradesh was executed by The evaluation of genetic variation can provide information about the uniqueness and distinctness of germplasm accessions, which is very important in the effective conservation and utilization of particular germplasm accession . DescripLavandula angustifolia. Similar outcomes were also attained by The germplasm exhibited wide variation for curcuminoids content viz., curcumin (0.41% to 2.17%), demethoxycurcumin (0.38% to 1.45%), bisdemethoxycurcumin (0.37% to 1.24%) and total curcuminoid content (1.26% to 4.55%). In an experiment, . longa accessions, the data on both rhizome yield traits and curcuminoids content were analyzed using cluster analysis and PCA. The distribution pattern of accessions among the clusters reflected the significant variability in the germplasm. Clustering based on both rhizome yield traits and curcuminoids content has divided the germplasm into seven clusters. Among the seven clusters, cluster VII registered maximum mean value for fresh rhizome yield (516.85 g p-1) whereas cluster I recorded maximum mean value for total curcuminoid content (3.52%). Interestingly, cluster VII which recorded maximum mean for fresh rhizome yield was also having third highest mean value for total curcuminoid content (3.32%). Similar trend was observed in cluster I which recorded maximum total curcuminoid content was also found to have third highest mean for fresh rhizome yield (413.05 g p-1). So, it is suggested to go with clustering or grouping of the germplasm based on both rhizome yield traits and curcuminoids content together as this kind of clustering results in selection of accessions with high yield along with high curcuminoid content. This kind of clustering will be helpful to find out the superior accessions in a condition where the price is based on curcuminoid content and also on rhizome weight basis. In PCA, PC1, and PC2 together constitute around 45% of the variation. Yield is the major contributor to PC 1 and is confirmed by CL 180 (High yielding accession), which was placed at the extreme right corner of the biplot, and CL 5 (Low yielding accession), which was placed at the extreme left corner of the biplot. Thus, the contrasting accessions were placed in the opposite position to each other. The outcome of PCA was consistent with the result of cluster analysis. Similar results were obtained by Grewia tenax by using dendrogram and PCA. Cluster analysis and PCA disclosed the complex relationship between the accessions with equal effectiveness (To explore the diversity among 200 Ctiveness .i.e., less than 25 and only seven parameters were considered for divergence studies. As the number of traits under study increases, automatically the per cent contribution of each trait to the total divergence decreases. In the present study, 15 rhizome yield traits and four qualitative traits were considered for evaluating the per cent contribution to the total divergence. Thus, the per cent contribution of traits to the total divergence was comparatively low. The selection of parents mainly depends on the contribution of traits towards divergence. The traits showing the maximum contribution towards divergence can be used further in breeding programs to improve the traits in the existing germplasm. Correlation studies were done to identify the relationship between individual traits, especially between total curcuminoid content and rhizome yield traits. Results revealed that total curcuminoid content had a significant positive correlation with primary rhizome core diameter and length of the secondary rhizome. The curcuminoid content is majorly present in the primary rhizome core diameter. So, automatically when the primary rhizome core diameter is more, the percentage of curcuminoid content will be more. A research report on turmeric by The weight of secondary rhizomes per plant, weight of mother rhizomes per plant and weight of primary rhizomes per plant contributed maximum to the total divergence. The traits showing the maximum contribution towards divergence can be used further in breeding programs to improve the traits in the existing germplasm. Research findings of The original contributions presented in the study are included in the article/MD, SN, VK, RM, and SB contributed to the study conception and design. Material preparation and data collection were done by MD, SN, VK, and SB. Data was analyzed by MD, SS and KA. The first draft of the manuscript was written by MD, KA, BM and SN. All the authors commented on previous versions of the manuscript. All authors contributed to the article and approved the submitted version.


The submicroscopic level focuses on particles. The representative level includes pictorial and symbolic representations to visualize substance in its nature. Students often have problems separating these levels and conceptually transfer each of the three levels to the other. Therefore, teachers need to use chemical terminology correctly when teaching the substance-particle concept. Augmented reality (AR) connects the real and virtual worlds. The observer physically moves in a real environment that integrates virtual elements. This can be effective for learning when chemical processes that are invisible are made visible. The simultaneous presentation should avoid split attention and offers new possibilities to interactively deal with multiple external representations ((M)ER). The question arises whether AR has a positive effect on the use of technical language. With an AR app on the tablet and on the hololens, chemical processes of a real experiment are represented by AR visualizations. In this study, the chemistry terminology of chemistry teachers (N = 30) was captured using a pre-post survey. Each test includes five tasks elaborated by thinking aloud. Therefore, the AR app was piloted. The thinking-aloud protocols to acquire the use of the chemical terminology are evaluated in MAXQDA.Chemistry as a whole is divided into three levels. The  According to This study focuses on the learning effectiveness of an AR learning environment (on tablet or hololens) to promote the use of chemical terminology, i.e., dealing with (M)ER. Therefore, a learning environment was designed that is aimed at teachers to expand their professional knowledge. The target is to be able to use innovative digital technologies in the subject lessons with students in a perspective and didactically reflected way. The following questions will be investigated in the research project:(1) Can the AR learning environment be used to promote reflective use of technical language at the substance and particle level from a teaching perspective among chemistry teachers?It is hypothesized that the use of AR learning environment promotes the integration of the representation level when observing a real experiment and improves in this context the substance-particle concept understanding.(2) Can the interactive use of augmented representational forms in the learning environment, with regard to the use of tablet or hololens, describe different elaboration profiles?It is hypothesized that the use of the AR learning environment on a tablet has a positive effect on the use of chemical terminology. The simultaneous linking of AR representations with the content of the real experimental environment is expected to initiate cognitive processing. In comparison, the use of the simulation should disrupt cognitive processing and improve chemical terminology to a much lesser extent. By avoiding split attention, AR is expected to support the construction of mental models and thus largely shape elaboration behavior.or hololens.Besides this, (M)ER can be controlled in a self-regulated way. Different elaboration profiles are expected when interacting with augmented (M)ER on the tablet N = 30). Experimental group 1 consists of 10 subjects working with AR learning environment on the tablet. Furthermore, experimental group 2 works with the same AR learning environment on a hololens. The control group consists of 10 other subjects working with a content equivalent simulation-based learning environment on the tablet.The subjects are teachers from German secondary schools who teach chemistry ER. The tasks refer to the donor-acceptor principle. They always focus on the construction, interpretation, and translation of (M)ER. In order to analyze the effect of AR on technical language and the related construction of mental models about redox reactions, the method of thinking aloud is used. For this purpose, subjects’ utterances are recorded during the processing of the tasks and the AR learning environment.In order to investigate technical language via the elaboration behavior of virtual representations in the AR environment among chemistry teachers , all subjects in the study participate in a pre-post survey: Before starting the learning environments, subjects are informed about what they need to pay attention to when completing the tests and thinking aloud. This is followed by the completion of the pre-test. Teachers are then briefly instructed on how to use the digital device (AR or simulation-based technology) on the tablet/hololens. Afterward, the experimental group 1 resp. 2 works on the AR learning environment on the tablet resp. hololens and the control group on the simulation-based learning environment. The simulation-based learning environment is designed to be similar in content to the AR environment but has—compared to the AR app—on the tablet a detrimental split attention effect from a cognitive psychology perspective . During The thinking-aloud protocols of processing the test tasks will be analyzed with qualitative content analysis according to If AR is to be applied in the classroom, the teaching and learning offer must be accepted by the teachers . AcceptaN = 18). Natural scientists, (prospective) chemistry teachers, science educators, and software developers were interviewed, all of whom use digital media regularly. Half of the subjects consisted of teachers.In March 2021, the review of the AR learning environment (acceptance/usability) took place  had not been completed. Help buttons were missing and particle modeling was undeveloped.All subjects processed the AR learning environment using a tablet. During the interaction with the AR learning environment, the participants had to explain the processes on the particle level with different representations. Subsequently, the questionnaire on the acceptance and usability of the AR learning environment was completed by the subjects.During the piloting of the AR learning environment, scales according to A reliability analysis of the AR learning environment provided predominantly good to excellent internal consistency values:I would use the AR learning environment in my own chemistry classes.”) with a total of 7 items has a Cronbach’s alpha of 0.73. The eight Usability scales  with 4–17 items per scale also show a Cronbach’s alpha between 0.668 and 0.904. Furthermore, all scales on the characteristics of the learning environment have mean values above the mean scale level  to be used in the main study.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of the Technical University of Munich. The patients/participants provided their written informed consent to participate in this study.MR was a Ph. D. student under the supervision of CN at TUM. In the context of the doctoral project, the presented study was carried out. Both authors contributed to the article and approved the submitted version.