Until recently, many phytoremediation studies were focused solely on a plants ability to reclaim heavy metal (HM) polluted soil through a range of different processes, such as phytoextraction and phytostabilization. However, the interaction between plants and their own rhizosphere microbiome represents a new research frontier for phytoremediation. Our hypothesis is that rhizomicrobiome might play a key role in plant wellness and in the response to external stimuli; therefore, this study aimed to shed light the rhizomicrobiome dynamics after an organic amendment  and/or HM pollution , and its relation with plant reclamation ability. To reach this goal we set up a greenhouse experiment cultivating in pot an elite black poplar clone (N12) selected in the past for its excellent ability to reclaim heavy metals. N12 saplings were grown on a soil amended with compost and/or spiked with high Zn doses. At the end of the experiment, we observed that the compost amendment strongly increased the foliar size but did not affect significantly the Zn accumulation in plant. Furthermore, the rhizomicrobiome communities (bacteria and fungi), investigated through NGS, highlighted how α diversity increased in all treatments compared to the untreated N12 saplings. Soil compost amendment, as well as Zn pollution, strongly modified the bacterial rhizomicrobiome structure. Conversely, the variation of the fungal rhizomicrobiome was only marginally affected by soil Zn addition, and only partially impaired by compost. Nevertheless, substantial alterations of the fungal community were due to both compost and Zn. Together, our experimental results revealed that organic amendment increased the bacterial resistance to external stimuli whilst, in the case of fungi, the amendment made the fungi microbiome more susceptible. Finally, the greater microbiome biodiversity does not imply, in this case, a better plant wellness or phytoremediation ability, although the microbiome plays a role in the external stimuli response supporting plant life. Heavy metal (HM) contamination of the environment has increased to levels that are harmful for living organisms, mainly because of anthropogenic activities. HMs are non-degradable pollutants, and, thus, they persist indefinitely in diverse environmental matrices. Among HMs, zinc (Zn) can be included; it has an atomic number of 30 and atomic weight of 65.38, it is the 24rd most abundant element on earth, and it is an essential trace element for all living beings, including plants. Zinc is a constituent of many proteins, it is also an enzyme cofactor and it is fundamental for optimum plant growth and development . HoweverPopulus has several features that are suitable for effective soil reclamation, such as rapid growth, a deep and spreading root system, marked adaptability to different pedoclimatic conditions and remarkable capability to vegetative reproduction, which makes its propagation quite easy. In addition, poplars are highly tolerant of different contaminants, and a large clonal variability in metal-resistance or accumulation traits  soil amendment. Compost may be easily obtained from artificially controlled microbial degradation of organic wastes . ComposSeveral studies have clarified the structure and variation of root-associated microbiomes in different plants species employed in bio- phytoremediation . At presPopulus nigra L. that originated from an Italian natural population, was selected for this experimentation because during a field trial on a multi-metal polluted site (–1 soil dry weight (DW). The pot trial included the following four experimental groups: CNT (3 plants on unpolluted soil); Zn450 (3 plants on Zn polluted soil); CMP (3 plants on unpolluted and CMP amended soil); CMP + Zn450 (3 plants on Zn polluted and CMP amended soil).The experiment was conducted using N12 poplar cuttings (12) sprouting separately in single pots and grown in a greenhouse to minimize the impact of other environmental stress factors. Pots were filled using an agricultural soil , T0 soilted site  it was sAt the end of experimentation, leaf area and length, average and maximum width were recorded on five completely expanded leaves of each poplar plant, using a portable leaf area meter . The total number of measured leaves is 60.At the end of the experimentation (about 3 months after planting), whole intact saplings were harvested from each pot. Roots, carefully washed with distilled water to eliminate soil sediments, stems and leaves were collected and separately dried at 75°C to constant weight, for measurements of their DW and for estimation of Zn content. At the start of the experimentation, soils, CMP amended or not, were analyzed to evaluate the main physical and chemical features. For pH determination, 10 g of soil were placed in 25 mL of deionized water and shaken for 2 h. Soil organic carbon and Organic Matter Content (OMC) were determined using the 3 65%; HF, 50% = 2:1v/v) in a microwave oven  using the following digestion program: 1 min at 250 W, 1 min at 0 W, 5 min at 250 W, 4 min at 400 W, 4 min at 600 W, 5 min at 250 W. The method of Dried soils were pulverized in a planetary ball mill , while plant organs were pulverized in a mortar  using liquid nitrogen, or reduced to ash (stems) by baking at 550°C for 2 h. For each plant organ, three biological replicates were analyzed. All the matrices (soils and plant organs) were digested with an acid mixture . Tubes were vortexed at maximum speed for 15 s and then shaken for 1 h at room temperature to release the majority of the microorganisms adhering to the roots. Roots were recovered and transferred to a new sterile 50-mL tube with 25 mL of physiological saline (0.9% NaCl), sonicated at low frequency for 5 min to further disrupt tiny soil aggregates and recover the attached microbes. The roots were then removed from this solution. The solutions containing fine sediment and microorganisms were pooled and centrifuged for 20 min at 5000 rpm to pellet the microorganisms. Pelleted microorganisms were washed twice with physiological saline, and finally resuspended in 20 mL of the same solution. Aliquots of 1 ml were flash-frozen in liquid nitrogen and stored at −80°C until processing. Aliquots of rhizosphere solutions (100 μL) were mixed in the lysis buffer of the DNA EXTRACT-N-AMP kit , following the supplier instructions, for a rapid DNA extraction.Microbial DNAs were extracted in triplicate from rhizosphere solution of each experimental group and PCR amplified. The V3-V4 region of the bacterial 16S rRNA genes was amplified with 341F (5′-CCTACGGGRSGCAGCAG-3′) and 909R (5′-TTTCAGYCTTGCGRCCGTAC-3′) specific primers, the following PCR thermal profile was used: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 1 min and elongation at 72°C for 1 min. with additional final elongation step at 72°C for 5 min. The fungi ITS2 region was amplified using the ITS3 (5′-GCATCGATGAAGAACGCAGC) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers and the following PCR thermal profile was employed: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and elongation at 72°C for 1 min, with additional final elongation step at 72°C for 5 min. The amplicon libraries were processed following the manufacturer’s instructions  and sequenced by BMR Genomics .1 with plugins-demux2 dada2  OTUs database, in the case of 16S rDNA4, which has been trimmed to include the V3-V4 region of 16S rRNA gene, bound by the 341F/909R primer pair, was used. While, the classifier, for fungi ITS2 DNA sequences, was pre-trained on UNITE database version 7–99%, and applied to paired-end sequence reads to generate taxonomy tables. Taxonomic and compositional analyses were conducted by using plugins feature-classifier5 x2 dada2  and featx2 dada2 . α - andx2 dada2  diversitssifier5 , taxa6 aposition .P-values were then obtained using 999 permutations. Linear Discriminant Analysis (LDA) Effect Size (LEfSe) was used to detect the bacterial taxonomic biomarkers across the different treatments  and the lowest in leaves  when compared to CNT . Quality filtering, denoising and the removal of chimeric sequences reduced this number to 108,387. The ASV was 436 and the rarefaction curves are reported in d to CNT .p < 0.05). Correlation analyses revealed that Observed OTUs were negatively correlated with total sapling biomass  and total sapling Zn accumulation . In particular, the number of Observed OTUs was negatively correlated with root  and stem biomass , and stem Zn accumulation .The α diversity was greater in the case of CMP, CMP + Zn450 and Zn450 compared to CNT (p ≤ 0.1).The β diversity was calculated in order to highlight the differences among the experimental groups using the Dice metrics. The results  showed tThe taxa bar plot was carried out at three different hierarchic levels: phylum, class and family. At phylum level , the resThe Firmicutes phylum was predominant in CNT and its relative abundance was reduced in the other three experimental groups, whilst, Proteobacteria phylum increased its relative abundance as well as Actinobacteria and Acidobacteria phyla. All the other phyla showed a relative low frequency and were strongly influenced by Zn addition in the case of the Zn450  or CMP + Zn450 . At the class level, Gammaproteobacteria  dominateAt the family level , 62 OTUsIn the case of CMP group, the most represented families were Xanthomonadales, Pseudomonadales and Betaproteobacteriales, and they were not affected by the Zn addition (CMP + Zn450 vs. CMP). However, CMP amendment favored the presence of Aeromonadales, Desulfuromonadales, Tistrellales, whilst Zn addition and CMP amendment (CMP + Zn450) modified the rhizosphere microbiome favoring the presence of Corynebacteriales, Gaiellales, Salinisphaerales, and Pseudonocardiales families.A qualitative and quantitative analysis of the rhizosphere microbiome was carried out in order to evaluate the differences, in terms of the classes or families, that were attributable to CMP amendment and/or Zn addition. In particular, the Venn analysis  revealedp < 0.05, log10 LDA score > 3.5) among the different experimental groups. In particular, members belonging to Bacillales family  may be considered a marker of CNT microbiome, whilst Actinobacteria  and Acidobacteria (Subgroup_6) were for Zn450. The CMP and CMP + Zn450 rhizosphere microbiome were characterized by the presence of Gemmatimonadales and Verrucomicrobiales, or Pseudomonadales and Cellvibrionales (Gammaproteobacteria) classes, respectively.In order to investigate whether some bacteria phylum, class or family was a potential marker of the diverse rhizosphere microbiome, a LEfSe analysis was performed . SeventyITS sequencing generated 1,184,489 gene sequences and, after quality filtering, denoising and removal of chimeric sequences, 1,015,382 genes sequences remained. The ASV was 718 and the rarefaction curves are reported in p < 0.05), with the exception of the comparison between CMP and Zn450. In addition, each treatment significantly affected the Shannon indices (p < 0.05) when compared with CNT, with the exception of CMP. Although for this index, the comparison between CMP and Zn450 was not statistically significant . On the contrary, the Pearson coefficient highlighted that Shannon diversity and Zn addition were strongly negatively correlated . A similar result in terms of correlation was obtained estimating the correlation between Zn addition and Chao 1 , or Simpson index .Furthermore, the comparison of Observed OTUs and Simpson indices revealed statistically significative differences among all treatments (nificant . Correlap ≤ 0.1).The b diversity was calculated in order to highlight the differences among the experimental groups using Dice metrics. The results  highlighThe taxa bar plot was reported at three hierarchic levels: phylum, class and family. At phylum level  AscomycoAt the class level , the difThis trend was confirmed when the third hierarchic level was considered . The mosThe fungi community was investigated from qualitative and quantitative points of view, aiming to identify those fungal families that were specifically associated to the treatments of the different experimental groups. At the family level, the Venn analysis  revealedIn particular, the CMP group was enriched by fungal families belonging to the Sordariomycetes and Rhizophydiales classes; whilst Agaricomycetes, Eurotiales, Atheliales, and Leotiomycetes were present when the CMP + Zn450 was considered. Interestingly, five families, Cantharellales, Mortierellales, Agaricales, Pezizales and Pleosporales, were present in all experimental theses.A LEfSe analysis , as in tp < 0.05, log10 LDA score > 3.5) were enriched among the different experimental groups. Dothideales and Pleosporales families  can be considered as markers of the CNT; Rhizophydiales (phylum Chytridiomycota. class Rhizophydiomycetes), Mortierellales , Cantharellales and Agaricales (both belonging to the phylum of Basidiomycota class Agaricomycetes) were markers of CMP, while Eurotiales  was maker of CMP + Zn450. No one specific marker was identified for Zn450 group.Only 47 root ASVs . The CMP supplied can significantly modify physical and chemical properties of the soil of CMP group, and in fact it slightly increased organic matter content and CEC respect to CNT. However, the amendment had no effect on Zn bioavailability and uptake by the poplar saplings. On the contrary, it favored Zn soil stabilization. The hazard of inorganic contaminants, such as metals and metalloids, arises from their absorption and accumulation in the cells. In fact, although Zn is an essential micronutrient for all living organisms, an excess of it can be toxic .Zn mobility in soil may be altered by the presence of some oxidized forms of Fe, Ca, Al, Mn, and P, and also by organic matter. Soil organic amendments can reduce solubility and mobility of metals, immobilizing them and/or decreasing their leaching . VariousHowever, the supply organic amendments to the soil can modify CEC, which, in turn, improves soil trace and oligo element  retention. Some studies have revealed that CMP, applied alone or in combination with others , increased the CEC of the soil due to the input of stabilized organic matter, which is rich in functional groups (such as carboxylic and phenolic acid groups) released into the soil exchange sites . ParadelIn our study, we found that N12 saplings were able to accumulate Zn in all of the organs analyzed : the highest concentration of Zn was observed in the roots followed by the stems and leaves. The binding of contaminants at root cell walls, or their accumulation and storage into the vacuole, are recognized avoidance mechanisms for trace metals in plants . MoreoveOur results are consistent with previous findings where the application of CMP did not increase Zn concentrations in the plant organs or it decreased it slightly . Soares P. nigra N12 clone is suitable for Zn phytoextraction , ectomycorrhizal fungi (EMF), and dark septate endophytes (DSE). The AM fungi comprise about 150 species of zygomycetous fungi, and EM fungi include about 6.000 species that are primarily Basidiomycetes, along with a few Ascomycetes and Zygomycetes .–1) depending on the species, strains, or even the type of growth media . For all of the other experimental groups, Ascomycota and Basidiomycota phyla were abundantly represented (about 95%). Only the less represented classes of CNT, such as Lecanoromycetes, Agaricomycetes and Leotiomycetes, belonging to the phylum of Ascomycota, increased in the case of Zn450. Fungi, especially those belonging to Ascomycetes and Basidiomycetes, are able to degrade very complex organic compounds including cellulose and lignin, but many of them also live as root symbionts (mycorrhizas) and obtain simple sugars from their plant partners . The ranth media . HøilandThe fungal community composition of CNT and CMP differed in relation to four classes: Pezizomycetes, Dothideomycetes, Agaricomycetes, and Mortierellomycetes. The CMP reduced the relative frequencies of the first two classes (both belonging to Ascomycota phylum), whilst it increased in the case of Agaricomycetes (Basidiomycota phylum) and Mortierellomycetes (Mortierellomycota phylum). Furthermore, these four classes were also the ones that were more sensitive to Zn addition in the case of CMP + Zn450. In fact, when CMP + Zn450 was compared to CMP, the relative frequency of Agaricomycetes and Mortierellomycetes, which are characteristic of the CMP, was reduced by the Zn addition. These differences in the relative abundance of specific OTUs, due to the different ecologic driven force (CMP and/or Zn), were also investigate trough LEfSe analyses in order to identify specific markers in each experimental group. The CNT fungal rhizosphere microbiome was characterized by the presence of Dothideales and Pleosporales families , whilst CMP was characterized by the presence of Rhizophydiales , Mortierellales , Cantharellales and Agaricales (both belonging to the phylum of Basidiomycota class Agaricomycetes). In particular, Eurotiales family  was identified as maker for CMP + Zn450. This family was recognized as a taxon able to tolerate organic pollution; in fact, Phytoremediation is one of the most widely studied green technologies for soil and water reclamation. In the last 20 years, many studies have focused on the identification of the best plant species to accumulate, extract, degrade or volatilize, inorganic or organic contaminants in specific pedoclimatic conditions. However, although these studies have improved knowledge and yielded interesting results, a thorough understanding of the interactions between plants and soil/rhizosphere microorganisms, and of roles that microbes play in phytoremediation processes, is still far away. In addition, soil amendment with CMP represents both a source of organic matter and of microorganisms, both of which are useful to improve the physico-chemical soil properties  and also increase the biodiversity. The resistance and resilience of soil microbiomes in turn can improve plant tolerance to contaminants and promote their growth. In this study, CMP amendment was able to improve biomass of N12 poplar saplings, in particular that of leaves, and, mostly, it counterbalanced the negative effects of Zn soil addition on their biomass. A greater biomass due to CMP amendment allowed a lower Zn accumulation in the different plant organs preserving plant health and, at the same time, improving the total Zn content in the N12 poplar saplings. Our experimental results demonstrated that CMP amendment induced different effects on bacterial and fungal rhizosphere microbiome; in fact, in the case of bacteria, CMP amendment deeply affected the rhizosphere microbiome, which in turn was more resilient to the presence of Zn. In contrast, in the case of fungal rhizosphere microbiome, CMP amendment strongly modified the community structure. Moreover, the Zn addition mainly affected the fungal rhizosphere microbiome of saplings grown on CMP amended soil. Our study confirmed that the rhizosphere microbiome is characterized by a complex network of relationships through which plants obtain several advantages. In our opinion, further studies on phytoremediation should be focused on these relationships with the goal of understand how these networks may improve phytoremediation effectiveness and opening the route to phytoremediation phase 2.0.https://www.ebi.ac.uk/metagenomics/, PRJEB39028 and https://www.ebi.ac.uk/metagenomics/, PRJEB39025.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: FG, AC, and SC: conceptualization. FG and AC: formal analysis, investigation, methodology, and validation. AC, SC, and MT: funding acquisition. AC: supervision. All authors: writing – original draft, review, and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In “Using an Electronic App to Promote Home-Based Self-Care in Older Patients With Heart Failure: Qualitative Study on Patient and Informal Caregiver Challenges” :e15885) the authors noted two errors.In the originally published article, Sahr Wali was listed as the Corresponding Author. The Corresponding Author address was listed as:Institute of Health Policy, Management and EvaluationDalla Lana School of Public HealthUniversity of Toronto155 College StToronto, ON, M5T 3M6CanadaPhone: 1 416 978 4326Email: sahr.wali@mail.utoronto.caIn the corrected version, the Corresponding Author has been changed to Catherine Demers, with the Corresponding Author address:Department of Health Research Methods, Evidence and ImpactMcMaster University237 Barton St EHamilton, ON, L8L 2X2CanadaPhone: 1 905 525 9140 ext 73324Email: demers@hhsc.caAn Acknowledgements section has also been added to the original paper with the following text:This work was completed at McMaster University.The correction will appear in the online version of the paper on the JMIR Publications website on November 11, 2020, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories.


Pigeonpea is an important economic crop in the world and is mainly distributed in tropical and subtropical regions. In order to further expand the scope of planting, one of the problems that must be solved is the impact of soil acidity on plants in these areas. Based on our previous work, we constructed a time series RNA sequencing (RNA-seq) analysis under aluminum (Al) stress in pigeonpea. Through a comparison analysis, 11,425 genes were found to be differentially expressed among all the time points. After clustering these genes by their expression patterns, 12 clusters were generated. Many important functional pathways were identified by gene ontology (GO) analysis, such as biological regulation, localization, response to stimulus, metabolic process, detoxification, and so on. Further analysis showed that metabolic pathways played an important role in the response of Al stress. Thirteen out of the 23 selected genes related to flavonoids and phenols were downregulated in response to Al stress. In addition, we verified these key genes of flavonoid- and phenol-related metabolism pathways by qRT-PCR. Collectively, our findings not only revealed the regulation mechanism of pigeonpea under Al stress but also provided methodological support for further exploration of plant stress regulation mechanisms. Cajanus cajan L.) is a kind of economic crop that grows in the semiarid tropics. In India, it is the second most important food legume. Pigeonpea is a hardy plant with strong growth and good stress tolerance. It can be used as food, feed, fuel wood, green manure, and hedges. In addition, it also plays an important role in insect reproduction, soil and water conservation, and so on [Pigeonpea  levels and aluminum (Al) toxicity are two major adverse factors in acidic soil, which, together, restrict crop growth ,5,6,7. ACcSTOP1, which is a C2H2-type transcription factor, regulates Al tolerance genes in pigeonpea [2 [Many Al-related works in legumes have focused on gene regulation and transcriptional analysis. For Al stress, igeonpea . Anotheronpea [2 .Arabidopsis, as a model plant, has also provided a research basis for Al stress [Arabidopsis, rice, tomato, and pigeonpea has been found to cause aluminum-dependent root growth inhibition [STOP1 in Arabidopsis and CcSTOP1 in pigeonpea have been proved to play an important role in regulating Al resistance [Arabidopsis, the regulation of resistance to bacterial and aluminum toxicity has been found to respond to Al and bacterial regulation at the same time [Aluminum in soil not only affects plants in special areas, such as pigeonpea, but is also found in the growing environment of many crops, such as rice and tomato ,12,13,14l stress ,16,17. Asistance ,16. The ame time .Cajanus cajan L.) and its wild relative Cajanus platycarpus (Benth.) Maesen under infection of Helicoverpa armigera (Hübner), many important genes have been identified [Arabidopsis [The availability of high-throughput sequencing technologies  and the entified . The canentified . There abidopsis ,25. TherIn the current study, we analyzed the whole transcriptome changes under aluminum response by time series RNA sequencing (RNA-seq) in pigeonpea. Three replicates ensured the credibility of the data. The correlation analysis of differentially expressed genes (DEGs) in time points for different responses showed that the differences between two time points were also obvious under Al stress. Further clustering analysis divided all the DEGs into 12 clusters. To mine the Al-response-related core genes, we performed functional analyses in independent clusters. The regulation of metabolic processes showed enrichment among all the related functions and had differences between clusters. Global pathway analysis coupled with differential expression characterization of the DEGs revealed some new factors that have profound effects on Al stress. In addition, we verified these key genes related to metabolism by qRT-PCR. Collectively, our results provide vital metabolic characteristics for the study of Al stress in pigeonpea and important research methods regarding time series data.In a sterile environment, pigeonpea seeds were first sterilized with 75% alcohol for 30 s, then sterilized with sodium hypochlorite for 6 min, and finally, the pigeonpea seeds were washed 5 times in sterile distilled water for 30 s each time. Pigeonpea seeds were then planted in sterile medium containing MS base salts, sucrose, and agar in a greenhouse. The temperature in the greenhouse was a constant 24 °C, the light conditions were light/dark cycles for 16 h/8 h, and the illumination was 5000 lux. When the pigeonpea seedlings were 3 weeks old, we selected pigeonpea seedlings with consistent growth status for Al treatment. According to our previous research, we performed Al treatment at a concentration of 50 μM . Sampleswww.bioinformatics.babraham.ac.uk/projects/fastqc/). The adapter and low-quality reads  were removed by Cutadapt [Three biological repeats were performed for four time points. Libraries were sequenced on a HiSeq 2000 system (Illumina) using the 150 bp Paired-End protocol. The outputs for 12 samples were about 84 G paired-end reads (2 × 150 bp). The reads were initially tested by fastQC  for the 12 samples was performed using the R package Psych. Consistency analysis between two samples was performed using the R packages Hmisc and Corrplot.The cluster analysis was conducted using Mev . The K-mThe DEGs were annotated with a gene ontology (GO) number by script, and then the comparison was performed using WeGO . The DEGArabidopsis genes’ protein library, we found the sequencing similar genes. The evalue was less than 0.01. The max_target_seqs was set to 1. Then, the sequencing similar genes were used to map to the Arabidopsis annotation in Mapman. The pigeonpea genes were selected by enrichment results.We used Mapman  to do thhttp://www.sangon.com/newPrimerDesign) and primer 5.0 to synthesize the primers, and the primer information for qRT-PCR is listed in Roots of pigeonpea were ground into powder in liquid nitrogen, total RNA was extracted from the samples using the CTAB method , while dC.cajan_V1.0 reference using STAR [Under Al stress, the root growth of legumes is inhibited ,11. It cing STAR . Then, tIn addition to the comparison between the treated samples and the control, we also made a comparative analysis among three Al processing samples, among which included 6062 DEGs at 3 and 0 h (group 1), 8073 DEGs at 6 and 0 h (group 2), 9231 DEGs at 12 and 0 h (group 3), 2368 DEGs at 6 and 3 h (group 4), 4782 DEGs at 12 and 3 h (group 5), and 1578 DEGs at 12 and 6 h (group 6) . ThroughTo further mine the core genes in the Al response, we separated those total DEGs into 12 clusters by their dynamic regulation patterns using Mev  Figure A. The dyAccording to the DEG cluster analysis, we analyzed the differential expression of genes in each cluster at different time points in a Heatmap pattern B. Similap-value simultaneously  and Fe(II)-dependent oxygenase superfamily, the chalcone and stilbene synthase family, the HXXXD-type acyl-transferase family, the laccase gene family, the NAD(P)-binding Rossmann-fold superfamily, the oxidoreductase family acting on NADH or NADPH, the senescence-related gene family, the flavonoid 3-o-glucosyltransferase family, and the UDP-glycosyltransferase superfamily. In order to further verify the relationship between these genes and metabolism, we selected two genes from each gene family separately based on transcriptome data: a positive regulatory gene with the highest expression level at 12 h and a negative regulatory gene with the highest expression levels at 0 h. According to this principle, we screened 12 genes, and then we used qRT-PCR to detect the relative expression of these genes under Al stress. The results are shown in Among these nine gene families, all gene families except the laccase family were related to flavonoids. Among them, the 2OG and Fe(II)-dependent oxygenase superfamily, the chalcone and stilbene synthase family, the HXXXD-type acyl-transferase family, the flavonoid 3-O-glucosyltransferase family, and the UDP-glycosyltransferase superfamily had been reported and they were related to flavonoids. In plants, the 2OG and Fe(II)-dependent oxygenase superfamily plays an important role in many biosynthetic pathways, including biosynthetic pathways that lead to collagen, β-lactam antibiotics, and many modified amino acids and peptides . Some irIn addition, we found that all core genes related to phenolics under aluminum stress belonged to the laccase family. Regarding the effect of laccase on phenols, it is generally believed that laccase participates in the anabolic metabolism of phenolic polymers, leading to the morphogenesis of plant fibers. Detailed analysis of phenolic compounds in plant tissues clearly showed that laccase mediated the polymerization of low-molecular-weight natural phenols in apoplastic bodies. In addition, laccase purified from plant tissues can use monolignol as a substrate and subsequently produce a coupling reaction to form lignin, but whether laccase in plants can convert monolignol to lignin has not yet been clearly reported ,41,42,43In summary, the core genes related to flavonoids and phenols that we have screened provide some clues for analyzing the mechanism of flavonoids and phenols in aluminum stress in pigeonpea. All the analysis methods in this study present a useful reference for exploring the relationship between plant secondary metabolites and stress resistance.For the analysis of stress response, the time series sequencing method has been used in many works to study changes at the transcriptional level ,25,44,45Under biotic and abiotic stresses, plants have developed complicated signaling pathways to adapt. Currently, most research on this subject focuses on gene families in the signaling pathways ,46,47,48Through the functional comparative analysis of important grouping genes, we found that there were significant differences in important metabolism-related pathways . This reA certain number of the important genes related to metabolism found in our study were downregulated , which wIn summary, we found that genes that had dynamic changes among the response time points under Al stress in pigeonpea. Further functional analysis showed that many pathways contribute to the stress responses. The metabolism-related pathways were the most significant differential pathways between DEGs. The core genes in metabolism-related pathways, containing flavonoids and phenols, were verified to demonstrate a vital role under Al stress. This finding sheds new light on Al stress in pigeonpea and other plants.


Nausea and vomiting are the most common complications following chemotherapy and usually lead to decreased quality of life. Acupuncture therapy is an effective method for chemotherapy-induced nausea and vomiting (CINV), the effects and safety have been observed by many clinicians and demonstrated in a systematic review, which was included in the Cochrane Library in 2014. After several years, new studies have occurred and an updated systematic evaluation is needed. This protocol describes a method for performing a systematic review and meta-analysis to further evaluate the beneficial effects and safety of acupuncture for CINV.A searching strategy will be carried out mainly in eight databases in English and Chinese, Cochrane Central Register of Controlled Trials, PubMed, Embase, China National Knowledge Infrastructure, the Chinese Scientific Journal Database, the Wanfang database, China Doctoral Dissertations Full-text Database, and China Master's Theses Full-text Database. Only randomized controlled trials related to acupuncture for CINV will be included to enhance the effectiveness. The effective percentage will be used as primary outcome. Changes in the symptoms of nausea and vomiting, like severity, duration, and frequency as well as quality of life will be assessed as secondary outcome. Side effects and adverse events will be used as safety evaluations. To ensure the quality of the systematic evaluation, study selection, data extraction, and quality assessment will be independently performed by 2 authors, and the third author will deal with any disagreement. The Review Manager V.5.3.3 s will be used to perform the data synthesis and subgroup analysis.There are additional studies, further explanations and more subgroup analyses compared with the previous systematic analysis to determine the effects and safety of acupuncture for CINV.The result of this systematic review may offer clinicians stronger evidence to assist patient in relieving CINV.There is no need to acquire ethical approval for individuals come from literatures instead of recruiting directly. The findings of this review will be reported in peer-reviewed publications and/or presented at relevant conferencesCRD42016045223. However, nearly most of patients suffer from chemotherapy-induced nausea and vomiting (CINV),–7 which can lead to a series of harmful consequences, such as electrolyte disturbances, dehydration, and lack of appetite. Furthermore, more severe complications, such as bleeding due to injury to the digestive tract mucosa, infection, and even death, may occur. Therefore, CINV not only influences the quality of life (QoL) but also physiological-psychological functions, and even result in large costs and bad compliance.With high incidence of cancer worldwide, the burden of the disease is increasing. to date, commonly prescribed medicines for nausea and vomiting are: 5-hydroxytryptamine-3 serotonin receptor antagonists, tachykinin NK1 receptor antagonist, steroids, histamine receptor-blocking agents, dopamine receptor-blocking agents. However, these medicines for nausea and vomiting do not have sufficient curative effects; moreover, increasing the dosage to achieve better efficacy may result in other side effects, such as headache, dizziness, and constipation. Conversely, acupuncture treatment for nausea and vomiting has advantages that include efficacy–15 low cost, convenience, and safety.Although the mechanisms underlying CINV are not clear enough,,17 In 2009, the Cochrane Library system evaluated 67 types of diseases, of which 7 kinds including CINV, were confirmed to have been effectively treated with acupuncture. Acupuncture may have antiemetic effects mediated via regulation of visceral nerves, transmitters, and affection of gastrointestinal rhythm.–21Historically, acupuncture has played an important role in protecting the health of Chinese people for more than 2000 years and is increasingly being recognized and is gaining popularity in some Western countries.Accordingly, this report presents a protocol for a systematic review of the prevention of CINV with acupuncture to ensure its curative effects and safety.2 and further evaluate effects and safety of acupuncture on CINV. Moreover, we aim to explore whether acupuncture can improve the QoL and compliance of chemotherapy patients.The objective of this study is to update the systematic review3The methods of this systematic review will be guided by the preferred reporting items for systematic reviews and meta-analyses statement.3.13.1.1Randomized controlled trials related to CINV will be included for meta-analysis in this systematic review.3.1.2Participants affected by CINV will be included in the meta-analysis regardless of their race and sex. The age range will be set to 18 to 80 years for better compliance and responses. Participants with nausea and vomiting secondary to motion sickness or nervous system disease will be excluded. Additionally, patients with mental disorders will be excluded. The specific criteria are as follows:3.1.2.1(1)Diagnosed with cancer with an indication for chemotherapy and an expected survival time of more than 3 months.(2)Age between 18 and 80.(3)Either gender.(4)Good physical condition, with a Kamofsky score >60.(5)Absence of digestive system diseases.3.1.2.2(1)Sever cardiac insufficiency, hepatic or kidney failure, or serious uncontrolled infection.(2)Central nervous system pathology involving tumors, digestive tract obstructions with hydrothorax or ascites or any other disease that may cause nausea and vomiting.(3)Metal disorders or any other diseases influence compliance.3.1.3Cancer is a unique disease, which requires that patients in both treatment and control group undergo basic treatment. In the treatment group, the intervention will be acupuncture or acupuncture with antiemetic drugs, acupuncture can be of any form as mentioned in the search strategy. Under normal conditions, patients in control group receive common drugs that prevent CINV nausea and vomiting instead of being left completely untreated. In the control group, no active treatment or sham acupuncture for the purpose of blinding are encouraged.3.1.4, the frequency and dosage of remedial antiemetic medications, the QoL rating scale score, and any other clinical assessments.The primary outcome will be the effective percentage in the treatment. The secondary outcomes will include the appetite grading may refer to simplified nutritional appetite questionnaireAll reported side effects and adverse events will be included as safety outcomes. The common side effects of acupuncture, such as local pain, hematomas, and fainting, as well as complications, such as electrolyte disturbances, deprivation of body fluid, lack of appetite, bleeding resulting from digestive tract mucosal injuries, infection, and death, will also be recorded and evaluated respectively.3.23.2.1http://apps.who.int/trialsearch/) and Current Controlled Trials .We will search for relevant studies in the following databases: Cochrane Central Register of Controlled Trials, PubMed, Embase, China National Knowledge Infrastructure, the Chinese Scientific Journal Database, the Wanfang database, China Doctoral Dissertations Full-text Database, and China Master's Theses Full-text Database. Additionally, we will search for as much information as possible from the following sources: the reference lists of the included studies, unpublished conference proceedings, and ongoing trials, which will be identified using the World Health Organization International Clinical Trials Registry Platform (3.2.2The following terms will be used for the search:A.Search strategy to identify “nausea and vomiting”:(1)Nausea(2)Vomiting(3)Emesis(4)Sick(5)Naupathia(6)Lack of appetite(7)Or/1–6B.Search strategy to identify “chemotherapy”:(8)Chemotherapy(9)Chemotherapeutics(10)10 Or/8–9C.Search strategy to identify acupuncture interventions:(11)Acupuncture(12)Electroacupuncture(13)Body acupuncture(14)Auricular acupuncture(15)Ear acupuncture(16)Scalp acupuncture(17)Intradermal needle(18)Fire needle(19)Elongated needle(20)Warm needle(21)Dry needle(22)Or/11-213.33.3.1All the authors in our team will be trained regarding the purpose and process of the review. The selection work will require at least 3 independent authors, 2 of whom will perform the study selection, and the third author will adjudicate any disagreement throughout the process. Specifically, 2 authors will first select studies by reading titles, abstracts, and if necessary, the full texts. Then, to ensure the authenticity of the collected studies, the authors may search the related published protocols or contact either the first author or the corresponding author.3.3.2Data extraction will also require 3 independent authors. Two will conduct the extraction and cross-checking work from the included studies, and the third author will adjudicate any disagreements during the process. All extracted data will be put into an extraction form which is developed according to the recommendations of the Cochrane Handbook. The extraction form will consist of general information items: the author, work location, publication date, journal, participants, characteristics of the interventions, outcomes, randomization, allocation concealment, incomplete data, blinding, selective reporting, adverse events, follow-up, sources of funds, and conflicts of interest.3.3.3The 2 independent authors mentioned above will first evaluate the risk of bias separately and then cross-check their findings. The third author will help resolve any potential disagreements. We will grade risk of bias as “high risk,” “low risk,” and “unclear risk” of bias using the “Risk of bias” tool from the Cochrane Handbook (V.5.1.0). We will assess the following types of bias:(1)selection bias, including random sequence generation and allocation concealment;(2)performance bias, including blinding of the investigators, participants, and operators;(3)detection bias, including the blinding of the outcome assessment, and in particular, the blinding of the data assessor;(4)attrition bias, which refers to incomplete data/differential dropout;(5)reporting bias, including selective reporting, whether or not all data are reported; and(6)other bias, including conflicts of interest, such as sponsorships from companies, and deficiencies in the follow-up involving some of the participants not being contacted again.3.3.4The results of the studies will be integrated and presented as risk ratios with 95% confidence intervals (95% CI) for dichotomous data and as mean differences or standardized mean difference with 95% CIs for continuous data.3.3.5Because studies are not regulated by a particular fixed format, the data presented in each article may not be consistent. In such cases, the authors may contact either the first or corresponding author to ask for the required data by via telephone or email.3.3.6χ2 test for heterogeneity will be used to calculate statistical heterogeneity. Significant heterogeneity will be identified by P > .1, or I2 < 50%. As described in the Cochrane Handbook, there are 4 categories of heterogeneity as determined by the P-value: Little or no heterogeneity, P < 40%; Moderate heterogeneity, 30% < P < 60%; Substantial heterogeneity, 50% < P < 90%; Considerable heterogeneity, 75% < P < 100%. At the same time, we will use fixed-effect model if I2 < 50% or P > .10, otherwise, we may consider random effect model and descriptive analysis.The Mantel–Haenszel 3.3.7If 10 or more trials are selected for the meta-analysis, we will analyze the potential publication bias by generating funnel plots. Quantitative analysis will be carried out by using Egger test.3.3.8Meta-analysis will be performed with Review Manager (V.5.3) statistical software provided by Cochrane Collaboration. A fixed effect model will be carried out if there is little significant heterogeneity among the trials, or a random effect model will be established.3.3.9A single meta-analysis is difficult to analyze if included studies contain obvious differences, analysis of the data in a single meta-analysis will be difficult. In such cases, subgroup analyses will be performed, and the studies will be distinguished according to the following:(1)type of acupuncture therapy,(2)treatment timing ,(3)grade of nausea and vomiting,(4)types of chemotherapy drugs, and(5)types of symptoms including acute vomiting, delayed vomiting, and anticipatory vomiting.3.3.10To evaluate the robustness and stability of the pooled outcomes, sensitivity analysis is needed by sequentially eliding each trial. When a low-quality study is identified, we may perform the meta-analysis with and without that study and subsequently compare the 2 results to determine whether the low-quality study should be included. The final result will depend on the sample size, missing data, risk of bias and quality of methods of each study.3.3.11 will be used to evaluate the quality of evidence as 4 levels: very low, low, moderate, and high.Grading of recommendations assessment, development and evaluation4 the studies included in the update did not include those studies since 2006. However, due to the rapid pace of acupuncture development, several new studies testing and verifying the curative effect of acupuncture on CINV were performed during this period. Therefore, updating the systematic review is necessary.Acupuncture have exact effects on CINV, a systematic review of acupuncture-point stimulation for CINV was published over 10 years ago, and although that review was revised in 2014 by the same team, by the following. ① Only 11 studies were included in the older systematic review, we will search for and include additional studies to obtain more data for the new analysis. ② The previous review only reported a significant reduction in the proportion of patients who experienced acute vomiting and a marginally significant change in the proportion of patients with reduced acute nausea severity, whereas the new systematic review will attempt to further explain these findings and report more aspects. ③ For delayed vomiting or nausea, no outcomes were significantly improved by noninvasive stimulation techniques. Accordingly, we will include additional studies to enable a reasonable subgroup analysis to demonstrate a more definite conclusion. ④ Study selection, data extraction, and quality assessment will be independently performed by 2 reviewers, and any disagreements will be adjudicated by a third reviewer. This process will improve the quality of the analysis and the review. ⑤ Notable dissimilarities may have been caused by the “dose,” 1 point was stimulated until Deqi was elicited. Therefore, in our review, “dose” will be considered one of the main influential factors in the evaluation of the curative effects of the treatments. ⑥ For the evaluation of safety, our review will search for and collect additional data from recent studies. Furthermore, we will focus on how to prevent the occurrence of CINV, the treatment of cancer patients with acupuncture before chemotherapy rather than the use of acupuncture as a method to relieve existing pain.The updated review will differ from the former oneDespite these efforts, limitations in this systematic review will still exist. For example, we will only search English and Chinese databases. Additionally, as there are different types of acupuncture interventions, as mentioned above, and different types of nausea and vomiting, such as acute, delayed, and postoperative nausea and vomiting, subgroup analyses will be needed. In the present, we cannot quantify the stimulation of acupuncture.T-T M, T Z, X-M W and L-P W conceived of and designed this study. T-T M and T Z developed the search strategy and wrote the first draft of the protocol. C-F D and B-R Z will independently perform the specific work, such as screening the potential studies, performing the data extraction, assessing the risk of bias, entering the data into RevMan, and completing the data synthesis. X-M W will make the final decision when any disagreement arises. T Z will ensure that no errors occur during the review. G-L Z, L-P W, and X-M W revised the protocol. All authors have read and approved the final manuscript.


Zinc-regulated transporters, Iron-regulated transporter-like Protein (ZIP) genes to regulate their expression. Furthermore, the expression of histone deacetylases (HDACs) varies greatly in response to individual and combined metals, and HDACs expression showed a negative correlation with ZIP transporters. Our study highlights the implication of DNA methylation and histone acetylation in regulating the metal stress tolerance dynamics through Zn transporters and warns against the excessive use of Zn fertilizers in metal contaminated soils.The persistent nature of lead (Pb) and cadmium (Cd) in the environment severely affects plant growth and yield. Conversely, plants acquire zinc (Zn) from the soil for their vital physiological and biochemical functions. However, the interplay and coordination between essential and toxic metals for their uptake and translocation and the putative underlying epigenetic mechanisms have not yet been investigated in maize. Here, we report that the presence of Zn facilitates the accumulation and transport of Pb and Cd in the aerial parts of the maize plants. Moreover, the Zn, Pb, and Cd interplay specifically interferes with the uptake and translocation of other divalent metals, such as calcium and magnesium. Zn, Pb, and Cd, individually and in combinations, differentially regulate the expression of DNA methyltransferases, thus alter the DNA methylation levels at the promoter of  Plants are exposed to diverse fluctuating environmental challenges during their development that can affect their fitness, survival and yield . Among tZIP family members may also uptake Fe, Mn, and Cd [ZIP transporters [ZIP transporters are localized in plasma membrane and endoplasmic reticulum, and ZIPs showed differential expression under Zn and Fe treatment [ZIPs could be functional transporters of Zn and Fe. Therefore, the exploitation of maize ZIPs would provide an excellent tool to maintain the desired amount of essential Zn and Fe in grains, which would be helpful to address the sustainable solution of malnutrition as well as to ensure the desired crop yield.Essential metals are distributed to different cells and organelles of plants depending on their needed concentration, through a variety of metal transporters . Among t, and Cd ,16,17. Msporters ,19. MaizIn response to fluctuating environmental and developmental cues, the gene expression is regulated by chromatin architecture, thus controls various cellular and physiological processes . In thisHDACs). Histone deacetylation has been reported to play an important role in plant growth and development, flowering, seed development, and to deal with biotic stress and abiotic stress including salt, cold, and drought stresses [HDACs in cotton [HDACs play a role in plant development [ZIP transporters in this particular interplay has not been investigated yet. We hypothesized that divalent metals  interfere with each other’s uptake and translocation due to the un-specific nature of divalent Zn transporters. Further, we also hypothesized that heavy metals alter the epigenetic landscape of maize plants, which in return regulates the expression of Zn transporters.DNA methylation cross-talks with histone acetylation for gene expression regulation . Histonestresses ,31,32. Pn cotton ,34, indielopment ,36,37,38ZIP transporters, which are under the control of DNA methylation and histone acetylation.By applying several metals to the maize plants, we found that Zn, Pb, and Cd combinations not only interfere with each other’s accumulation and mobility to aerial parts, but also interfere with the uptake and translocation of other divalent metals . Moreover, our results indicate that the interplay among Zn, Pb, and Cd is regulated by As expected, we found that the exposure of plants to Pb, Cd, or Zn leads to an accumulation of the respective metals into the roots, shoots, and leaves , showingInterestingly, at the same concentration, plants preferentially accumulate Pb rather than Cd and Zn compared with control. Among Pb, Cd, and Zn treatments, only Zn concentration was significantly higher in leaves and shoots compared with control. This result indicates that Zn is more mobile to aerial parts of the maize plant than Pb and Cd . ContrarWe further analyzed if the combination of metals alters their accumulation or their transport throughout the plant. Therefore, we checked the metal accumulation in the different parts of the seedling after combined treatments, e.g., Pb + Cd, Pb + Zn, Cd + Zn, and Pb + Cd + Zn. To our surprise, the combinations of Pb with Zn or with both Zn and Cd, enhanced the total Pb accumulation in plants compared with the single Pb treatment . MoreoveWe then investigated whether the total accumulation and mobility of Zn are altered with the addition of Cd and Pb . We founWe next investigated whether Pb, Cd, and Zn dynamics can influence the accumulation of these other divalent metals as well as their transport to aerial parts . Total CWe further analyzed if the combination of Pb, Cd, and Zn alters the accumulation of Ca and Mg as well as their transport in aerial parts of the plants. We found that the levels of Ca and Mg in shoots and leaves were different between the Pb/Cd/Zn individual treatments and their combinations . For example, in the presence of Pb + Zn, Ca and Mg accumulation in leaves was further increased compared with Zn treatment alone. These results show that Pb, Cd, and Zn dynamics influence the Ca and Mg mobility to aerial parts of the plants. However, we found that K levels did not change in response to Pb, Cd, and Zn treatments , suggestMetal stress has been reported to alter the activity of Peroxidase (POD), Superoxide dismutase (SOD), and Catalase (CAT) antioxidant enzymes ,40. We tZIP transporters may play a role in the transport of Pb/Cd to aerial parts of plants. We therefore investigated the expression of ZIP transporters in response to Zn, Pb, and Cd applied alone and in combinations. The results show that ZIP transporters present a differential expression in response to different metals, applied alone and in combinations (Iron-Regulated Transporter 1 (IRT1) expression was increased, whereas the expression of other ZIP transporters was decreased compared with control. In response to Pb treatment, the expression of IRT1, ZIP1, and ZIP6 was increased compared with control, whereas the expression of ZIP2, ZIP3, and ZIP4 was decreased compared with control. In response to Cd, the expression of IRT1, ZIP2, and ZIP8 was increased compared with control, whereas the expression of other ZIP transporters was decreased compared with control. The expression of some genes was either increased or decreased in response to Pb, Cd, and Zn, but their levels were significantly different in many cases, e.g., IRT1 expression was highest in response to Pb; ZIP2 and ZIP8 expressions were highest in response to Cd; and ZIP1 and other ZIP gene expression was lower in response to Cd than that of Zn.The increased concentration of Pb/Cd in shoots and leaves with the addition of Zn led us to hypothesize that inations . In respZIPs is altered when Pb, Cd, and Zn were combined. To our surprise, we found that the dynamics of ZIPs expression were changed in response to Pb + Cd, Pb + Zn, Cd + Zn, and Pb + Cd + Zn. In the case of Zn + Pb, the expression of IRT1 and ZIP1 was increased compared with control, but still lower than that of Pb alone. Furthermore, the expression of ZIP6 did not change compared with control, while the expression of ZIP2, ZIP3, ZIP4, ZIP5, and ZIP7 was similar to that of Zn alone. These results indicate that Zn encountered the increased expression of IRT1, ZIP1, and ZIP6 compared with Pb alone. In the case of Cd + Zn, similar to Cd, the expression of IRT1, ZIP2, and ZIP8 was increased compared with control, whereas the expression of other ZIPs was decreased. However, increased expression of ZIP2 and ZIP8 was lower than that of Cd alone, suggesting that Zn antagonizes the increased expression of ZIP2 and ZIP8 from Cd. In addition, the expression of other ZIP genes was at the similar level than that of Cd and/or Zn alone. In the case of Pb + Cd, the expression of ZIP2 and ZIP8 was increased compared with control, but their expression was lower than that of Cd treatment, suggesting that Pb antagonized the expression of ZIP2 and ZIP8 compared with Cd. However, the expression of other ZIP transporters was similar to that of Cd in Pb + Cd treatment. Furthermore, we combined all three metals and found that, similar to Cd and Zn + Cd, the expression of IRT1, ZIP2, and ZIP8 was increased compared with control but still was lower than that of Cd, whereas the expression of other ZIPs was decreased to levels similar to Pb, Cd, and/or Zn levels. Together, these results show that, in addition to Zn, the expression of ZIP transporters is also regulated by Pb and Cd. Furthermore, metal combinations differently regulate the expression of IRT1, ZIP1, ZIP2, ZIP6, and ZIP8 compared with individual metals.We next investigated whether the expression of ZIP expression). The R2 value for the MLR equations between Pb, Cd, and Zn uptake models and ZIPs expression were 0.82, 0.80, and 0.95, respectively. This indicates a good fit of the model for all cases (ZIP5 and ZIP6 (23%); for Cd with ZIP4 (36%); and for Zn with ZIP6 (26%)  method was applied to find the linear relationship between explanatory variables  and response variables (ll cases B. The hiP6 (26%) C. This fHDACs could also play a role in plant adaptation under these metal stresses. Therefore, we checked the expression of all HDACs in maize in response to Pb, Cd, or Zn applied alone and in combination ; for Cd with HDA102 (27%); and for Zn with HDA106 (47%)  C. This fZIP transporters through the regulation of DNA methyltransferases in maize. Therefore, we checked the expression of maize DNA methyltransferases [Methyltransferase 1 (MET1), MET2a, and MET2b was decreased compared with control, whereas the expression of MET3a and MET3c was increased ; for Cd with Met4 (29%); and for Zn with Met1 (29%)  C. This fZIP transporters. We employed Chop-PCR to detect the non-methylated DNA levels at the promoter of some selected candidates, i.e., IRT1, ZIP1, ZIP2, ZIP6, and ZIP8  is a measure of the strength of the association between two variables. We therefore calculated the Pearson correlation between the expression of sferases . The expZIP transporters to regulate their expression.In response to fluctuating environment, chromatin landscape is achieved through post-transcriptional histone modifications to regulate the gene expression. Here, we studied the dynamic metal interactions among Zn, Pb, and Cd in maize and found that the presence of Zn facilitates the accumulation and transport of Pb and Cd in the aerial parts of the maize plants. Furthermore, we showed that Pb, Cd, and Zn alone and in combinations alter the expression of DNA methyltransferases, which consequently change the DNA methylation levels at the promoter of some Met2b and Met1 ; for Cd with Met4 and Met3a ; and for Zn with Met1 and Met3c . This indicates the specific regulation of each DNA methyltransferase in response to Pb, Cd, and Zn. It also indicates that one DNA methyltransferase could be regulated by more than one metal. In response to metal stress, changes in DNA methylation have been reported in wheat, Vicia faba, rape seedlings, white clover, industrial hemp hyper-accumulators plants, and Arabidopsis [Vicia faba [IRT1, ZIP1, ZIP2, and ZIP6 confirm the DNA hypermethylation at these loci in response to Cd. Furthermore, DNA methylation levels were also slightly increased in response to Zn or Pb at specific ZIP transporters. These results indicate the specific DNA methylation changes in response to Pb, Cd, and Zn. Notably, we also observed further increase in DNA methylation levels at ZIP transporters in response to the combination of metals, e.g., DNA methylation levels at IRT1, ZIP1, ZIP2, and ZIP6 in response to Pb + Cd. This indicates that the combination of metals could produce different DNA methylation levels compared with individual metals. However, further studies are required to investigate the genome wide DNA methylation status as well as targets of DNA methyltransferases in response to metals applied individually or in combination.Arabidopsis MET1 ) and plant specific Chromomethylases (CMTs) are reported to maintain the CG and CHG methylation, respectively ,42. Morebidopsis ,44,45,46bidopsis ,26, whilHDACs also responds differently to the individual metals as well as the combination of metals. Furthermore, MLR analysis also suggests the specific regulation of HDACs in response to Pb, Cd, and Zn uptake. These observations suggest that Pb, Cd, and Zn could cause different histone acetylation profiles. Interestingly, our Pearson correlation analysis showed a negative correlation between the expression of HDACs/DNA methyltransferases and the expression of IRT1, ZIP1, ZIP3, ZIP5, ZIP6, and ZIP7. This suggests that altered DNA methylation and histone acetylation levels, alone or together, could regulate the ZIPs gene expression. However, the expression of ZIP2 and ZIP8 showed a positive correlation with the expression of HDACs and DNA methyltransferases, suggesting that the regulation of these ZIP transporters could be independent of HDACs and DNA methyltransferases. However, further studies are required to validate the crosstalk between histone acetylation and DNA methylation to regulate the ZIPs gene expression.Similar to DNA methylation, the expression of all ZIP transporters to transport Pb and Cd. Notably, we also observed that Pb and Cd also regulate the expression of ZIP transporters, e.g., IRT1, ZIP1, and ZIP6 expression increased in response to Pb, while the expression of ZIP2, ZIP3, and ZIP4 decreased compared with control. Furthermore, MLR analysis also suggests that Pb, Cd, and Zn specifically regulate the expression of certain ZIP transporters. Interestingly, the ZIP family has been reported to uptake Fe, Mn, and Cd [ZIP metal transporters. Moreover, Arabidopsis IRT1, a ZIP transporter, has also been reported to transport multiple metals, including Fe, Zn, Mn, and Cd [ZIP transporters may also transport Pb/Cd. Therefore; we propose that heavy metals alter the DNA methylation and histone acetylation levels through the activity of DNA methyltransferases and histone deacetylases , Yellow Stripe-Likes (YSLs), etc. [ZIP metal transporters that specifically uptake Zn and block Pb and Cd is desired and need further investigations. Zn is being applied as a fertilizer. In the light of our results, if the agriculture land is already polluted with divalent metals, the application of Zn could make toxic metals more mobile to aerial parts of plants. Furthermore, the presence of Pb/Cd in the soil can block the Zn accumulation and transport to aerial parts of the plants. However, how plant imports preferentially one metal over the other metals, and how the metal combinations change metal accumulation and mobility dynamic need further investigations.Our results also show an interesting plant preference of metal accumulation and mobility to different parts of the plants. To our surprise, the addition of Zn facilitates the accumulation of Pb and Cd to the aerial parts of the plants. Furthermore, Pb, Cd, and Zn dynamics seem specific to divalent metals, as K levels did not change after treatments. There are two possible explanations for this phenomenon. Firstly, Pb and Cd could be potentially co-transported with Zn. Secondly, Pb and Cd could allosterically regulate the ability of some , and Cd ,16,17,47, and Cd ,49. Thesetylases . The ress), etc. ,51,52. IZea Mays L. cv. NK-8441 Syngenta) were placed on a filter paper humidified with 0.5 mM solution of CaSO4 and placed in incubator at 28 °C for 48 h to germinate. After germination, two-day-old young seedlings of similar size were transferred to hydroponics medium containing 0.2 mM KH2PO4, 1 mM K2SO4, 2 mM Ca(NO3)2, 2 mM CaCl2, 0.5 mM MgSO, 0.2 mM FeSO4 . 7H2O, 5 µM H3BO3, 2 µM MnSO4, 0.5 µM ZnSO4, 0.3 µM CuSO4, and 0.01 µM (NH4)2Mo7O24. Hydroponics medium was changed twice a week. The heavy metal treatment was applied at three leaf stage by adding Cd (100 µM), Pb (100 µM), and Zn (100 µM) alone or in combination using completely randomized design (CRD). Cd, Pb, and Zn were added as CdCl2, Pb(NO3)2, and ZnSO4, respectively, and their concentrations were selected based on previously published reports [−2 s−1 PAR)/10 h dark, and a relative humidity of 65% ± 5%.Maize seeds  was used to make a final volume of 10 mL. The digested plant material from different parts of the plants was filtered  and analyzed for Pb, Cd, and Zn concentrations through the atomic absorption spectrophotometer , as suggested by A.O.C.A  protocol [Two weeks after treatment with Pb, Cd, or Zn, freshly harvested maize plants were washed with double distilled water (distilled water passed through Millipore filters) to remove any soil particles and air-borne pollutants. The plants were divided into roots, shoots, and the whole 4th leaf for atomic absorption analysis. After taking their fresh weight, the samples were placed in an oven at 60 °C for 72 h until a constant dry weight was attained using weight balance . Plant materials were ground (<0.2 mm) and about 150 mg of dried plant material were used for the analysis of Pb, Cd, and Zn. The material was burned into ashes at 550 °C for 5 h in the furnace and digested with 2 mL of 4 M HNOprotocol . The insg for 5 min, the supernatant was collected in eppendorf tubes and used for the analysis of antioxidant enzymes. Catalase, peroxidase and superoxide dismutase activities were determined as described previously [2O2 at 240 nm through spectrophotometer , while the activity of POD was measured by using guaiacol as substrate. The reaction mixture contained 0.1 M phosphate buffer of pH 7, 1% guaiacol, 0.4 M H2O2, and the enzyme extract. Change in absorbance per unit time was measured at 470 nm. SOD activity was measured by photoreduction of nitroblue tetrazolium (NBT). Reaction mixture comprised 50 mM phosphate bufer of pH 7.8, 0.1 mM EDTA, 20 mM L-methionine, 750 µM NBT, 20 µM riboflavin, and the enzyme extract. The amount of protein was also determined according to the protocol of Lowry [Green leaf samples were harvested from plants and quickly immersed in liquid nitrogen. The frozen samples (0.5 g) were homogenized in freshly prepared potassium phosphate buffer (100 mM potassium phosphate pH 7 and 0.1 mM EDTA). After centrifugation at 4 °C and 13,000× eviously . The CATof Lowry .5 (ZmUBQ5) was used as an internal reference gene to normalize the data. Primer sequences for qRT-PCR are listed in After 48 h of treatment with Pb, Cd, and Zn applied alone and in combinations, total RNA from roots was extracted by using Trizol  according to the manufacturer’s instructions. DNase I treatment was performed with DNAse , and reverse transcription was performed with Superscript III  using the gene-specific primers as described previously . RT-qPCRChop-PCR was performed as previously described . Brieflyp ≤ 0.05. In the next step, correlation between studied parameters was found by using Pearson Correlation. Multiple linear regressions (MLR) analysis was constructed between dependent variables  and predictors . The MLR equation was used as described below [Each treatment was applied in three biological replicates and there were three technical replicates in each biological replicate regarding the biochemical analysis. Data of means from each biological replicates were subjected to theGeneral Linear Model (GLM) to know the effect of treatments on each studied parameter. Furthermore, after doing analysis of variance (ANOVA), post hoc multiple comparison (least significant difference test) was performed to compare and rank the treatments means value by using alphabets. Statistical analyses were performed at significance level of ed below (1)Y=b0 0 is the value of multiple regression constant, b1-xth is the value of regression constant of studied parameters, and x1–xth represents the studied variables of ZIP expression, DNA methyltransferases, and HDACs expression.Y is the predicted value of the dependent variable , b2 value represents the coefficient of multiple linear regression and it denotes worthwhile competence between predicted and quantified values. A higher R2, more than 0.75, is considered a good fit model of MLR for metal uptake and studied parameters. Cumulative percentage contributions (CPC) represents the contribution of studied parameters against heavy metal uptake and calculated by using the following equation;The RB is the cumulative percentage contribution of studied parameter, bi is the value of MLR coefficients of each studied parameter, and Ʃbi is the value of sum of MLR coefficients of all studied parameters.Mobility Index was calculated by using the following formula;All the statistical analyses were performed using IBM SPSS statistical software.ZIP transporters, which are most likely implicated in the transport of Pb, Cd, and Zn. This study demonstrates that epigenetic regulators may play an important role against the tolerance of heavy metals in maize. However, further studies, such as on genome wide DNA methylation changes and histone acetylation changes in response to metal stress alone and in combination, will advance our understandings regarding their role in the tolerance to metal stress.This study show that Zn, Pb, and Cd interfere with each other’s accumulation and transport to aerial parts of the maize plants. Further, the interplay among Zn, Pb, and Cd specifically alters the uptake and translocation of divalent calcium and magnesium but not the monovalent potassium. Finally, we showed that DNA methyltransferases and histone deacetylases together regulate the expression of


The CONUT score was the most important variable for OS, with OS rates of 70.0% and 29.0% in the low and high CONUT groups, respectively. Additionally, the median survival time was longer in the low CONUT group than in the high CONUT group . The CONUT score was the most important prognostic variable, rather than albumin–bilirubin grade and BCLC stage, in patients with HCC treated with LEN. Accordingly, immuno-nutritional status may be an important factor in the management of patients with HCC treated with LEN.We aimed to investigate the impact of the controlling nutritional status (CONUT) score, an immuno-nutritional biomarker, on the prognosis of patients with hepatocellular carcinoma (HCC) treated with lenvatinib (LEN). This retrospective study enrolled 164 patients with HCC and treated with LEN  stage B/C 93/71). Factors associated with overall survival (OS) were evaluated using multivariate and decision tree analyses. OS was calculated using the Kaplan–Meier method and analyzed using the log–rank test. Independent factors for OS were albumin–bilirubin grade 1, BCLC stage B, and CONUT score <5 (hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.58–5.31,  Hepatocellular carcinoma (HCC) is a common malignancy, and one of the leading causes of cancer deaths worldwide . The proLenvatinib (LEN) is an oral MKI  that is Recently, immuno-nutritional status has been reported to be associated with outcomes in patients with HCC ,13,14. T.The CONUT score has been reported to predict the risk of postoperative complications of grades III–V , as wellThis retrospective study was carried out in five institutions: Kurume University Hospital , Yokokura Hospital , Iwamoto Internal Medical Clinic , Omuta City Hospital , and Yanagawa Hospital . The protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the ethics committees of the Kurume University School of Medicine , Yokokura Hospital, Omuta City Hospital, Yanagawa Hospital, and Iwamoto Internal Medical Clinic. An opt-out approach was employed to obtain informed consent from the patients, and personal information was protected during data collection.The inclusion criteria for this study were as follows: (1) diagnosis of intermediate- or advanced-stage HCC, based on the Barcelona Clinic Liver Cancer (BCLC) staging system ; (2) agen = 13). A total of 164 patients were enrolled in the study : aspartate aminotransferase, alanine aminotransferase, albumin, total bilirubin, total cholesterol, and white blood cells.The CONUT scores, used to assess immuno-nutritional status, were calculated from the following three parameters, as previously described ,26: (1) HCC was diagnosed using a combination of tests for serum tumor markers, such as alpha-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP), and imaging modalities, such as ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI). HCC was classified using the BCLC staging system.After obtaining written informed consent from each patient, treatment with LEN was initiated. The administration dose of LEN is determined based on body weight, according to the manufacturers’ instruction. LEN was orally administered at a dose of 12 mg/day in patients with bodyweight ≥60 kg, or 8 mg/day in patients with bodyweight <60 kg, and it was discontinued when any unacceptable or serious adverse events (AE) were observed.Therapeutic response was evaluated, using CT or MRI, 4–6 weeks after the initiation of treatment with LEN, and thereafter at intervals of 2–3 months until death or study cessation. The therapeutic response was evaluated using the Modified Response Evaluation Criteria in Solid Tumors (mRECIST) . We alsoAE were assessed based on the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0 . In accoThe primary endpoint of this study was the OS of the patients.p-value of <0.05 was considered statistically significant.All data are expressed as the frequency or median (range). All statistical analyses were carried out using statistical analysis software . OS was calculated using the Kaplan–Meier method and analyzed using the log–rank test. Univariate and multivariate analyses were conducted using the Cox proportional hazards model, to identify factors associated with OS. We also performed decision tree analysis to identify factors associated with OS, as previously described . Correla2. The etiology of HCC was hepatitis C virus in 48% of patients (78/164), and 39% of patients (64/164) had an ALBI grade of 1. The median total cholesterol level and lymphocyte count were 171 mg/dL and 4600/μL, respectively. The frequencies of CONUT scores of 0–1 , 2–4 , 5–8 , and ≥9  were 29.3% (48/164), 49.3% (81/164), 20.8% (34/164), and 0.6% (1/164), respectively  were female . The medectively . The medectively .One month after treatment, a complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) were observed in 3% (5/164), 30% (49/164), 41% (68/164), and 26% (42/164) of patients, respectively . The oveUsing univariate analysis, ALBI grade 1, CONUT score <5, BCLC stage B, and AFP ≤200 ng/mL were selected as variables. Using multivariate analysis, ALBI grade 1, CONUT score <5, BCLC stage B, and AFP ≤200 ng/mL were identified as independent factors for OS .Kaplan–Meier survival analysis for all patients treated with LEN is shown in In this study, the survival rate in all subjects was 61% at the time of study cessation. To determine the profiles associated with survival rates, decision tree analysis was performed, which revealed that the CONUT score was the splitting variable for survival rate. Although the survival rate was 29% in patients with CONUT scores ≥5, the survival rate was 70% in patients with CONUT scores <5 . In patiKaplan–Meier survival analysis according to CONUT score is shown in There was a significant negative correlation between CONUT scores and total lymphocyte count a. SignifThe duration of treatment with LEN according to CONUT score is shown in p = 0.001). We examined the impact of the parameters of the CONUT on discontinuation of LEN due to AE. There was no significant difference in total lymphocyte count and total cholesterol level between the discontinuation and no discontinuation due to AE groups (Cessation of treatment with LEN due to severe AE occurred in 45% (74/164) of all patients. Cessation of treatment was significantly more frequent in the high CONUT group than in the low CONUT group (39% in the low CONUT group vs. 69% in the high CONUT group; E groups . HoweverE groups .Through this study, we demonstrated that a CONUT score <5, ALBI grade 1, BCLC stage B, and AFP ≤200 ng/mL were independently associated with OS in patients with HCC treated with LEN. Moreover, through decision tree analysis, we revealed that the CONUT score was the initial splitting variable for survival rate. These results indicate that, alongside liver function and tumor factors, immuno-nutritional status may be an important factor in the management of patients with HCC treatment with LEN.Treatment with LEN is a recommended therapy for unresectable HCC, according to the clinical guidelines for HCC in the United States, Europe, and Japan ,5,6,32. In this study, a CONUT score <5, ALBI grade 1, BCLC stage B, and AFP ≤200 ng/mL were identified as independent factors for OS, using multivariate analysis. Previous studies have reported that ALBI grade, AFP level, and BCLC stage are independent factors for OS in patients with HCC treated with LEN ,9, indicIn this study, we investigated the prognostic importance of each factor for OS, using decision tree analysis. We found that the CONUT score was the initial splitting variable for survival rate in patients with HCC treated with LEN, followed by BCLC stage and ALBI grade. Thus, the CONUT score may be the most important prognostic factor in patients treated with LEN. Daitoku et al. report that the CONUT score is a prognostic factor in patients with metastatic colorectal cancer treated with chemotherapy . In factThe CONUT score is based on total lymphocyte count, total cholesterol level, and serum albumin level. All three of these factors showed significant correlations with total CONUT score in this study. All three are also well-known nutritional parameters. Protein energy malnutrition has been reported to downregulate protein/hormone and antibody synthesis ,37, leadThe cessation of treatment due to severe AE was significantly more frequent in the low CONUT group than in the high CONUT group in our study. This suggests that a lower CONUT score may be associated with better OS through the longer duration of treatment with LEN. We examined the impact of the parameters of the CONUT on discontinuation of LEN due to AE. We found that serum albumin level, but not total lymphocyte count or total cholesterol level, was significantly higher in the no discontinuation due to AE group than in the discontinuation due to AE group. Since lenvatinib is known to mainly bind to human serum albumin , a higheThis study could inform the experimental design of future prospective trials in this clinical context. Our results may indicate the importance of evaluation for nutritional state of patients, using CONUT as a base line and throughout the study. In addition, cholesterol is a precursor for bile acids and various other bioactive molecules, such as oxysterols , which cThis study has several limitations. First, the study was retrospective. Second, we did not evaluate patient histories of previous and subsequent treatment for HCC. Third, we did not evaluate sarcopenia, which has a significant relationship with both nutrition and prognosis. To prove a prognostic impact of the CONUT score in patients with HCC treated with LEN, it would be necessary to perform a prospective study, including analysis of various host and tumor factors.In this study, we showed that a CONUT score <5, ALBI grade 1, BCLC stage B, and AFP ≤200 ng/mL were independently associated with better prognosis in patients with HCC treatment with LEN. Moreover, we revealed that the CONUT score was the most important variable for OS. Accordingly, immuno-nutritional status should be an important factor in the management of patients with HCC treatment with LEN. Future work will focus on nutritional therapy for the improvement of CONUT scores in patients with HCC treated with LEN.


BCR-ABL1, resulting from t, is the oncogenic driver of chronic myeloid leukemia and the therapeutic target of the disease. Molecular studies have been the gold standard modality for patient assessment since the advent of tyrosine kinase inhibitor therapy. In spite of that, there are cytogenetic abnormalities that can render the disease unresponsive to conventional therapy, thus making cytogenetics an important component of patient management guidelines.We present a case of a Tajik, Afghan patient with chronic myeloid leukemia with del(6)(q23.3q27), t, monosomy 11, monosomy 12, and marker chromosome who, despite having typical clinical and hematological disease with initial response to therapy, progressed to blast crisis very early and thus required special interventions.Cytogenetic monitoring is an important pillar in the management of patients with chronic myeloid leukemia that cannot be ignored. It should therefore be a part of patient management not only during diagnosis but also during management. We present an unusual cytogenetic abnormality in a patient with chronic myeloid leukemia that resulted in early disease progression. ABL) from chromosome 9q34 with the breakpoint cluster region (BCR) gene on chromosome 22q11.2 [BCR-ABL1, the oncogenic driver of chronic myeloid leukemia (CML), results from a balanced translocation, t9;22)q34;q11.2), involving a fusion of the Abelson gene  abnormalities, including the major route cytogenetic abnormalities that can affect management as well as prognosis . We presOur patient was a 42-year-old Tajik, Afghan man with no known medical illness who presented to our institution with lethargy, anorexia, pallor, and progressive abdominal distension that had developed over a duration of 4 months. On examination, the patient was pale, not in distress, and had no lymphadenopathy. His abdominal examination revealed gross splenomegaly crossing the midline with the liver just two finger breadths in the subcostal region. His complete blood count revealed moderate anemia, thrombocytosis, and hyperleukocytosis , showing predominance of granulocytes with a bimodal peak of mature neutrophils (68%) and myelocytes (33%). The patient’s blast count was 4% with normal basophil count. The clinical impression at that time was CML in CP based on the initial clinical and hematological evaluation. The patient was counseled to proceed with cytogenetic analysis for confirmation and prognostication of disease, but, due to financial constraints, he opted to start on TKI therapy. Accordingly, the patient was started on conventional imatinib therapy 400 mg/day. Initially, he responded well with resolution of hyperleukocytosis and improvement in hemoglobin and platelet count toward normal, but he did not achieve complete hematological remission. A repeated complete blood count analysis revealed moderate anemia, mild thrombocytopenia, and the presence of >90% blast cells in a total white cell count of 62,000/μl. After detailed counseling, the patient agreed to proceed with cytogenetic analysis, in which the analyzed metaphases of all 20 cells revealed 45,XY, presence of Ph chromosome 9q34;22q11.2), and additional complex chromosomal abnormalities, including deletion of chromosomes 6q23.3 to q27, monosomy 11, monosomy 12, insertion 12p13.3, and a marker chromosome, as shown in Fig. 1.2, and CML, once an indolent but indefinitely progressive disease of the hematopoietic system, has now become a success story in the field of hemato-oncology . Almost The field of molecular genetics has enabled physicians not only to identify the disease burden but also to tailor management in accordance with measurable residual disease , 13. AllTargeting deep molecular response has enabled identification of individuals who can successfully achieve complete molecular remission with successful discontinuation of therapy . Novel, Mutational studies have now become a commonplace practice in the management of patients with CML . The preDespite all the mentioned progression in molecular studies, cytogenetics plays a very important role and thus has always been incorporated in CML management guidelines . ChromosOur patient, despite having the characteristic clinical and hematological profile of CML, progressed to blast crisis early during therapy for harboring complex chromosomal abnormalities that were in addition to the pathognomonic Ph. Thus, the role played by cytogenetic analysis in the management of patients with CML along with the modern molecular modalities is absolutely not replaceable. Therefore, when feasible, cytogenetic studies must be included in the analysis of patients for whom molecular tests are being done.We present a case of a patient with CML who had an unusual “complex” cytogenetic abnormality in addition to Ph that resulted in early blast crisis. To our knowledge, this is the first case report of a patient with CML harboring del(6)(q23.3q27), t, monosomy 11, monosomy 12, and marker chromosome in addition to Ph.


Although schizophrenia patients have been reported to manifest deficits in cognitive flexibility and lower processing speed , there still remain a few matters that require further investigation. We have therefore formulated three research aims: 1) to examine the factor structure of CTT in schizophrenia patients and healthy controls, 2) to compare different CTT performance measures in the two groups, 3) to investigate the relationship between these measures and selected psychopathological symptoms in the patient group.Sixty-seven patients with paranoid schizophrenia and 67 healthy controls, matched for gender, age, number of years of education, and overall cognitive functioning underwent assessment of cognitive flexibility and processing speed with the CTT.Factor analysis of CTT variables based on the principal component method revealed a four-factor solution in both groups. Compared with healthy controls, the patients performed poorer on CTT 1 time, CTT 2 time, 2-1 difference, prompts in CTT 2, and had higher regression factor scores for Factor 1 . Furthermore, significant links were found between some CTT measures, and negative and disorganization symptoms.Schizophrenia patients exhibit problems with speed of perceptual tracking and executive processes dependent on processing speed. Our results may be useful for the development of neuropsychological diagnostic methods for schizophrenia patients. It seems that, compared to other CTT indices, CTT 1 time, CTT 2 time, and 2-1 difference are more appropriate measures of cognitive performance in schizophrenia patients. Cognitive flexibility is defined as the ability to flexibly transition between at least two processes or tasks . ProcesThese processes can be measured with two corresponding neuropsychological tests—the Color Trails Test  and theAlthough the CTT and TMT are reported to measure various neuropsychological domains, data concerning their factorial validity when applied in clinical samples is still scarce. Internal structures of CTT investigated in the normative vs. traumatic brain injury patient samples proved somewhat divergent, although both yielded four-factor solutions, reflecting the speed of perceptual tracking and sequencing . To our To date, the CTT was used less often than the TMT to assess response time-dependent mental processes in schizophrenia patients , 11. A PAssessment of cognitive flexibility based on CTT or TMT performance times as a single outcome measure is bound to be limited. In turn, a more qualitative approach can offer much more useful clinical information , as suggAlthough deficient cognitive flexibility and processing speed have been reported in schizophrenia, their association with psychopathology  remainsConsidering all these findings and inconsistencies, we have formulated the main objective of this study, i.e., to examine the factor structure of CTT in schizophrenia patients and healthy controls. Our secondary aims were to compare different CTT performance measures in the two groups and explore their links with selected psychopathological symptoms in the patient group.Sixty-seven inpatients of three psychiatric wards were diagnosed with paranoid schizophrenia according to ICD-10 diagnostic criteria  by threSixty-seven persons without mental or neurological disorders, matched for gender, age, and number of years of education were recruited through information spread by students of the local universities. Inclusion and exclusion criteria for healthy controls were the same as those for patients .Polish Version of CTT by Łojek and Stańczak , 13 was The Positive and Negative Syndrome Scale  was uset-test. Cohen’s d was used to determine the magnitude of effect size measures , CTT 2 time (p < 0.001), and 2-1 difference (p < 0.001). The effect sizes (d) of the analysed processes were found to be 0.98–1.26, thus suggesting large effect sizes.As shown in p < 0.01) only in CTT 2. The effect size (d) of the analysed errors was found to be 0.58, indicating medium effect sizes.Compared to healthy controls, schizophrenia patients required significantly more prompts (p < 0.001) were significantly higher in the patients than the controls. The effect size (d) of the analysed scoring was found to be 0.97, indicating large effect sizes.Regression factor scores for Factor 1  and 2-1 difference , predicting about 7 and 6% of variance, respectively. Disorganization symptoms were significant predictors of CTT 2 time , 2-1 difference , and regression factor scores for Factor 1 , predicting 13, 14, and 4% of variance, respectively. Excited symptoms were a significant predictor of near-misses , predicting 8% of variance. We identified no other significant predictors of any CTT indices.Negative symptoms were significant predictors of CTT 2 time , suggesting that the error and prompt variables might tap factors dissociable from those based on the time variables. In addition, these qualitative variables may offer an alternative take on the speed vs. accuracy trade-off of CTT performance. Respondents may attempt to increase their speed at the expense of committing errors, or reduce the number of errors at the expense of elevated time scores. Our results are thus similar to those reported in the traumatic brain injury and normative samples .p-value correction, the differences were no longer statistically significant, which is consistent with other reports  and the Pomeranian Medical University in Szczecin (FSN-337-06/2016 and FSN-246-05/2017). The project was also financed by the Polish Minister of Science and Higher Education’s program named “Regional Initiative of Excellence” in 2019–2022 (002/RID/2018/19).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.