Arabidopsis, as well as in abi1td, mkkk17, and mkkk18 mutants. We identified 10 novel miRNAs in WT after ABA treatment, while in abi1td, mkkk17, and mkkk18 mutants, three, seven, and nine known miRNAs, respectively, were differentially expressed after ABA treatment. One novel miRNA (miRn-8) was differentially expressed in the mkkk17 mutant. Potential target genes of the miRNA panel were identified using psRNATarget. Sequencing results were validated by quantitative RT-PCR of several known and novel miRNAs in all genotypes. Of the predicted targets of novel miRNAs, seven target genes of six novel miRNAs were further validated by 5′ RLM-RACE. Gene ontology analyses showed the potential target genes of ABA-responsive known and novel miRNAs to be involved in diverse cellular processes in plants, including development and stomatal movement. These outcomes suggest that a number of the identified miRNAs have crucial roles in plant responses to environmental stress, as well as in plant development, and might have common regulatory roles in the core ABA signaling pathway.miRNAs are involved in various biological processes, including adaptive responses to abiotic stress. To understand the role of miRNAs in the response to ABA, ABA-responsive miRNAs were identified by small RNA sequencing in wild-type  PYR/PYL/RCAR genes); the negative regulators of the pathway, the protein phosphatase 2C (PP2C) group A family; and the positive regulators SNF1-related protein kinases type 2 (SnRK2 genes). Other kinases such as MAP kinases function downstream of the ABA core pathway: MAPKKK17 and MAPKKK18 are regulated by the ABA core signaling module, and these activate C group MAPKs via MKK3; these downstream kinase modules probably contribute to the generation of a robust, long-term signal that maintains ABA-dependent responses under chronic stress conditions [MAPKKK17 and MAPKKK18 is induced by ABA, and these genes seem to have redundant functions in ABA signaling [Over the last few decades, there has been intensive research into the physiological effects of abscisic acid (ABA)  on plantnditions . Transcrignaling . Thus, pignaling . HoweverDCL-1 (dicer-like enzyme 1) in association with a dsRNA-binding protein, HYL-1. These duplexes are methylated by a dsRNA methylase, HEN1 (HUA ENHANCER 1), and loaded into AGO1 (Argonaute1) [HASTY [RISC), where they guide the cleavage of matching target mRNAs by AGO1, which possesses RNA-binding domains such as PAZ and PIWI [miRNAs are ~21-nucleotide (nt)-long, single-stranded small RNAs that play important monitoring roles in eukaryotes. miRNA genes code for long pri-miRNA (primary miRNA) transcripts with imperfect stem-loop secondary structures, which are transcribed by RNA polymerase II in plants ,9. Theseonaute1) ,11,12, a) [HASTY ,14, tranand PIWI . The effand PIWI . Stresseand PIWI ,18,19. IDimocarpus longan Lour.), bread wheat, Artemisia annua, Populus euphratica, Moringa oleifera, and Solanum lycopersicum [Arabidopsis somatic tissues by regulation of the LEC2-controlled pathway of auxin biosynthesis [MYB33 and MYB65 [MYB factors, MYB33 and MYB101 [Arabidopsis.Deep sequencing technology has led to the identification of numerous miRNAs in plant genomes from species such as Italian sweet pepper , tea, mupersicum ,28,29,30persicum ,32,33. mynthesis . A loss-nd MYB65 , while Ad MYB101 . After td MYB101 . HoweverIn the current study, we aimed to identify novel ABA-responsive miRNAs and their putative target mRNAs and to investigate whether and how the ABA response in Arabidopsis involves miRNA regulation of the ABA signaling pathway.ABI1, MKKK17, and MKKK18 knockout lines and compared them to the wild-type Col-0 control plants. The MAPKKK17/MAPKKK18 cascade is activated by the ABA core module. Moreover, MAPKKK18 activity is regulated by the ABA core element ABI1 PP2C. The MAPKKK18 and a close paralogue kinase, MAPKKK17, seem to exert partially redundant functions in the ABA signaling network [A. thaliana WT Col-0, abi1td, mkkk17, and mkkk18 seedlings were treated with 100 µM ABA solution for 4 h alongside untreated controls. Total RNA was then prepared as described in the Materials and Methods section and sent to Macrogen Inc.  for small RNA library construction and sequencing on an Illumina HiSeq 2500 system. Each of the 24 individual libraries produced an average of more than 10 million reads. Details of the miRNA statistics are given in Molecular and genetic studies have revealed that in response to environmental stress, complex signaling pathways are induced in plants, leading to a host of diverse responses . The str network ,4. ThereArabidopsis.Overall, the miRNAs ranged from 16 to 30 nt in size across all libraries of all genotypes . DiffereArabidopsis genome downloaded from the TAIR database. After these screening steps, we identified 225, 315, 212, and 148 known miRNAs and 51, 73, 50, and 18 novel miRNAs in the WT Col-0, abi1td, mkkk17, and mkkk18 libraries, respectively  were used to evaluate the expression levels of each miRNA . Table 2ndidates , and theIn addition, we identified a few mature miRNAs in WT Col-0 as novel and named them ath-miRn-1, ath-miRn-2, ath-miRn-3, and so on. A few of the conserved miRNA families identified in our sequencing data were present in all libraries, as shown in p-value < 0.05), 23 of the 85 known miRNAs were upregulated, whereas 45 were downregulated in WT. Among them, we identified ten novel miRNAs: six novel miRNAs were upregulated, and three were downregulated after ABA treatment  upregulated after ABA treatment. In mkkk17, five of seven DE known miRNAs were upregulated, two were downregulated, and one novel miRNA (miRn-8) was highly downregulated in this mutant in response to ABA. In the mkkk18 mutant, seven of nine DE known miRNAs were upregulated, one was downregulated, and one miRNA’s expression was considered unchanged after ABA treatment.After normalization of the reads as ‘reads per million’ and analysis of differentially expressed (DE) miRNAs  were expressed commonly only in mkkk17 and mkkk18 after ABA. In other cases, miRNAs were identified in mutants only; for instance, DE miR390a-3p and miR169k were highly downregulated only in mkkk17 and mkkk18 mutants, respectively, as shown in Among the statistically significantly regulated miRNAs, we identified representatives with genotype-specific expression patterns after ABA treatment. Some miRNAs were identified in both WT and one or more, but not all, mutant lines. Of the known and novel miRNAs that were differentially expressed in all genotypes, 86 miRNAs were WT-specific after ABA treatment A. One miTo further understand the post-transcriptional changes occurring after ABA treatment in WT and mutants, further analyses of the differentially expressed miRNAs were carried out. For example, the number of reads in ABA-treated libraries showed that the abundance of miRNA846 and miR156e in WT was lower than in the three mutants A,B. Thes® miRNA assays . All 15 miRNAs were tested against RNA samples from mock- and ABA-induced WT Col-0, abi1td, mkkk17, and mkkk18 mutants. Of the six known miRNAs, after ABA treatment, three were upregulated and three downregulated across all genotypes, while three of the novel miRNAs were downregulated and the remaining six upregulated. In almost all cases, the expression levels were consistent with the sequencing data: although the log2 fold changes for the various genotypes were occasionally different to those suggested by the NGS data, the trends were generally consistent, although not in all cases  were selected at random and subjected to qRT-PCR using TaqManll cases . Thus, qll cases . This shArabidopsis. Two other cleavage sites were located outside the complementary region. These findings verified cleavage sites in target genes AT1G73390.3/AT1G73390.4 for ath-miRn-1, AT5G40550.1 for ath-miRn-2, AT3G14070 for ath-miRn-4, AT1G56650, AT5G58490 for ath-miRn-6, AT3G15570 for ath-miRn-8, and AT2G29140 for ath-miRn-9. Among these target genes, a cleavage site for ath-miRn-9 was detected in the predicted miRNA complementary region, while for ath-miRn-8, cleavage sites were detected in the predicted miRNA complementary region and also upstream of this region   see . The resabi1td and mkkk17, and the target genes in these categories were not present at all in the mkkk18 mutant after ABA treatment. While the upregulated miRNA target genes in WT were more enriched in the biological process of ‘transcription, DNA-templated’ than in the mkkk18 mutants, no enrichment was observed for the upregulated miRNA target genes in the abi1td and mkkk17 mutants after ABA treatment. Interestingly, the top 10 enriched roles of the target genes of up- and downregulated miRNAs, i.e., the molecular processes ‘ion binding’, ‘protein binding’, ‘ATP binding’, ‘nucleotide binding’, ‘metal ion binding’, ‘DNA binding, ‘transferase activity’, ‘nucleic acid binding transcription factor activity’, and ‘kinase activity’ were much more enriched in WT Col-0 than in the abi1td, mkkk17, and mkkk18 mutants after ABA treatment  pathways. Previously, ABA has been reported to regulate lysine catabolism in bidopsis , and ourthaliana  and glutthaliana . Furtherthaliana . In the recovery . The preonfirmed . KEGG an8 mutant . OverallArabidopsis and other plants, miRNAs are involved in a wide range of physiological processes, such as phytohormone signaling, biotic and abiotic stress responses, plant development, and nutrient signaling pathways. Specific examples include the brassinosteroid response and the response to blue light in Arabidopsis, and pollen development and the heat stress response in Solanum lycopersicum. Other types of non-coding RNA include sesame, as well as in the cold response in grapevine, the drought response in mulberry, and the drought-responsive multi-tiered regulatory network in drought-tolerant rice [Arabidopsis. Here, we identified a number of miRNAs that are likely involved in this cascade and thus will be the subject of further detailed research.In ant rice ,54,55,56Dimocarpus longan Lour [RISC), which results in the degradation of the target mRNA or inhibition of its translation.An average of more than 8–10 million clean reads for each sample of sRNA was obtained by high-throughput sequencing, and known and novel miRNAs from the control and ABA-treated libraries were identified by bioinformatics. Small sRNA reads of 24 nt were the most predominant sequence length, followed by 21 nt sRNAs, which is consistent with the results of high-throughput sequencing in other plants, such as gan Lour  and tea gan Lour . Our resgan Lour ,59,60. mabi1td, mkkk17, and mkkk18 genotypes (both control and ABA-induced samples). Importantly, the highest number of miRNAs was identified in the abi1td mutant  regulates various biosynthetic pathways and is involved in the responses to light [MYB75 could be regulated by ABA via the novel miRNA (ath-miRn-6) in Arabidopsis. Another novel miRNA target gene highlighted in our study is PUM3 (AT2G29140), whose cognate protein was previously reported to be a mRNA-binding protein [Arabidopsis.In our study, DE novel miRNAs identified in WT were validated by qPCR, and cleavage sites of novel miRNAs in their target genes were also confirmed. However, 15 randomly selected (6 known and 9 novel) miRNAs were confirmed by stem-loop RT-PCR . Neverthet mRNAs . Indeed,eostasis . The trato light  and to fto light , for ins75 AT1G560 regulatB75 AT1G550 regulamkkk18 than in WT  expression and tolerance to drought stress [mkkk18 after ABA treatment  and is involved in anther development and pollination. AT2G02850 was experimentally confirmed to be cleaved by miR408 in bidopsis , and itsbidopsis . Our datbidopsis , and AP2PL genes ; our seqreatment A. ath-mipatterns , which dAGO1 [mkkk18 mutant after ABA treatment. The miRNA390-dependent transacting small interfering RNA pathway is regulated by auxin [mkkk17, but was unchanged in WT and other mutant lines, which suggests that the target gene of this miRNA might be overrepresented in mkkk17. Taken together, the data for these DE miRNAs indicate possible crosstalk between the auxin and ABA signaling pathways by regulation of the respective miRNA target genes.miR168 targets the mRNA of AGO1 , and NaCAGO1 , as doesAGO1 . It is aby auxin , and theby auxin ; miRNA39by auxin . In respArabidopsis can be proposed , as well as abi1td, mapkkk17td, and mapkkk18td mutants were used in this study. The abi1td [mkkk17 (SALK_080309C), and mkkk18 mutant lines were created by T-DNA insertion as described previously [® Reagent  according to the manufacturer’s protocol . ABA application was confirmed by the amplification of ABA-related marker mRNAs. Induction or downregulation of marker mRNAs was tested by real-time PCR analysis. All measurements were carried out in three biological replicates. An Agilent 2100 Bioanalyzer System  was used to confirm the RNA integrity (>8) of the miRNA samples.Seeds of WT e abi1td , mkkk17 eviously . After teviously  for two https://bedtools.readthedocs.io/en/latest/content/tools/merge.html, accessed on 30 October 2018). Additionally, we kept only miRNAs identified in at least two distinct samples and with a pre-miRNA sequence not shorter than 40 nt. miRNA* sequences were filtered out using Bedtools to find unique miRNA loci. The novel precursor sequences were used to construct secondary structures using RNA structure version 6.2 software (https://rna.urmc.rochester.edu/RNAstructure.html) with the default settings. Mature miRNAs were annotated further using the miRbase database  to identify both conserved and novel miRNAs. The raw sequencing data (20 GB) were deposited at NCBI GEO with the accession number GSE172377.Macrogen Ltd., Seoul, South Korea, carried out miRNA sequencing using the TruSeq Small RNA Library Prep Guide (Part #15004197 Rev. G protocol) on the Illumina HiSeq2500 platform to generate 50 bp paired-end reads. Adapters were clipped, and reads with no adapters and reads less than 16 nt long were discarded. The reads were then filtered for quality using the FASTX-toolkit, requiring that at least 95% of nucleotides had a Phred quality scores of 20 or more. Read cataloging was performed using a mirPRo stand-alone pipeline . Identifp-adjusted value, was defined as significantly down- or upregulated. To prepare the Venn diagram, an online webtool (http://bioinformatics.psb.ugent.be/webtools/Venn/) (accessed on 5 November 2018) was used to identify differentially expressed miRNAs that were shared between mock- and ABA-treated samples. Similarly, the effect of mutants was investigated by comparing untreated control samples of mutants  with WT. The interaction effect between genotypes was studied by principal component analysis to find the variance among samples.The raw counts were analyzed using the DESeq2 package  to find ® MicroRNA Reverse Transcriptase using TaqMan® MicroRNA Reverse Transcription Kit , according to the manufacturer’s protocol, along with 3 µL stem-loop reverse transcription primer  in a 15 µL reaction mixture was used. The TaqMan® miRNA assays were performed using TaqMan® Universal PCR Master Mix II with uracil N-glycosylase  according to the manufacturer’s protocol. The reaction was performed at 50 °C for 2 min , at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. Ct values for all miRNA primary transcripts and other transcript cDNAs were normalized to the SnoR66 and Actin8 TaqMan Assay cDNA fragment Ct value; both had the same trend of expression of miRNAs, so the qPCR results show results using SnoR66 as endogenous control. Differences in the relative transcript levels were calculated using the 2−ΔΔCt method. For each sample, the PCR amplification was repeated three times, and the average values of 2−ΔΔCt were used to determine the differences in gene expression by Student’s t-test. Three biological replicates were performed with similar results, and one replicate is shown. TaqMan® miRNA assay details are available in Total RNA from three independent biological replicates was extracted using Tri-reagent according to the manufacturer’s protocol . RNA samples were treated with DNaseI provided in the RNA Miniprep kit to remove genomic DNA. For qPCR analysis, 10 ng total RNA was used to synthesize cDNA from small RNAs. A total of 1.5 µL 10× reverse transcription buffer, 0.15 µL 100 mM deoxynucleotide triphosphates, 0.19 µL RNase inhibitor (20 units/µL), 1 µL TaqMan® . To examine miRNA-directed cleavage of predicted targets in vivo, we isolated total RNA using Tri-reagent, as described above, from seedlings of A. thaliana. We employed the 5′ RLM-RACE kit to amplify the 5′ UTR of target gene mRNAs. These target genes were selected based on the prediction value (from 0 to 3.5) of novel miRNA target genes. Combinations of three different primers were used in the 5′ RLM-RACE experiments as listed in ®  according to the manufacturer’s instructions. The amplicons were then cloned into the TA cloning vector, pGEMT-easy , and sequenced.Target mRNA cleavage sites for several novel miRNAs were experimentally mapped by RNA ligase-mediated rapid amplification of cDNA ends (5′ RLM-RACE). A modified procedure for 5′ RLM-RACE was followed using the FirstChoice RLM-RACE Kit−ΔΔCt method. For each sample, the PCR amplification was repeated three times, and the average values of 2−ΔΔCt were used to determine the differences in gene expression by Student’s t-test. Three biological replicates were performed for all target genes. All primers used in the qRT-PCR analyses are available in To quantify the identified target genes of novel miRNAs, qRT-PCR was performed using a published protocol  with min2(fold change)| > 1 or downregulated |log2 (fold change)| < −1) known and novel miRNAs were predicted using the online server psRNATarget (http://plantgrn.noble.org/psRNATarget/) [http://expath.itps.ncku.edu.tw/rnaseq/enrichment/Arabidopsis/enrichment_analysis.php, accessed on 25 August 2020) [p-value of <0.05, respectively, were considered.The putative target genes of upregulated (|logTarget/)  with defst 2020) , and theArabidopsis and its abi1td, mkkk17, and mkkk18 mutant lines, which are differentially expressed in response to ABA treatment. Both known and novel miRNAs were characterized and the expression profiles of a subset were validated by qRT-PCR; we also verified the potential target genes of selected novel miRNAs by 5′ RLM-RACE. A hypothetical model of the ABA-responsive miRNA regulatory network and their target genes in Arabidopsis was developed from the data and is presented schematically (Arabidopsis plants. Few of the novel Arabidopsis miRNAs detected in our study have previously been connected with the ABA response, especially in the WT Col-0 genotype. Therefore, it will be interesting to investigate the role of the ABA-responsive miRNAs that we report here, together with the proteins encoded by mRNAs targeted by these miRNAs, in plants responding to environmental challenges.Using RNA-Seq, we identified miRNAs in the WT Col-0 of atically . Overall


Matrix metalloproteinases (MMPs) cleave extracellular matrix proteins, growth factors, cytokines, and receptors to influence organ development, architecture, function, and the systemic and cell-specific responses to diseases and pharmacological drugs. Conversely, many diseases , viral infections (COVID-19), and cancer), cholesterol-lowering drugs (such as statins), and tetracycline-class antibiotics (such as doxycycline) alter MMP activity through transcriptional, translational, and post-translational mechanisms. In this review, we summarize evidence that the aforementioned diseases and drugs exert significant epigenetic pressure on genes encoding MMPs, tissue inhibitors of MMPs, and factors that transcriptionally regulate the expression of MMPs. Our understanding of human pathologies associated with alterations in the proteolytic activity of MMPs must consider that these pathologies and their medicinal treatments may impose epigenetic pressure on the expression of MMP genes. Whether the epigenetic mechanisms affecting the activity of MMPs can be therapeutically targeted warrants further research. MMPs arammation ,2.The transcriptional regulation of MMP activity involves transcription factors, DNA polymorphisms, and promoter/enhancer elements, and epigenetic mechanisms, which are the focus of this review. The concept of epigenesis finds its origin in the scientific debate regarding processes that drive development in cells, proposing that chemical reactions involving soluble cellular components can influence cell development . With ouChemical modifications of DNA and histones regulate gene expression by impacting the extent of DNA compaction and therefore the accessibility of transcriptional machinery to genes ,5. DNA cHere, we review the current evidence of epigenetic regulation of MMP expression in human diseases including atherosclerosis, arthritis, bacterial infection (tuberculosis), viral infection COVID-19), and cancer. We also examine evidence of the epigenetic regulation of MMP expression in response to commonly prescribed medicinal drugs with known, but poorly understood, MMP modulatory actions such as statins, which exert epigenetic pressure on many genes and inhibit MMP secretion ,17,18, a, and canVaried gene expression patterns of MMP family members across different cell types may be caused by the cell-type-specific expression of transcription factors and polymorphisms in the promoters of MMP genes, in addition to DNA methylation and histone modifications .MMP-1, −2, −9, −11, −14, −15, −16, −17, −21, −23, −24, −25, −26, and −28) and all four tissue inhibitors of metalloproteinases  contain CpG islands that are prone to DNA methylation by DNA methyltransferases [MMP9 promoter (hypermethylation of a promoter represses gene transcription) and concomitantly induced MMP9 gene expression in lymphoma cell lines [MMP1 expression is silenced by the methylation of a single CpG site that can be rescued by 5-aza-2′-deoxycytidine [Dmt-1 and Dmt3b and treatment with demethylating agents induced MMP3 gene expression without affecting MMP1 or MMP2 [MMP3 expression, but instead concentration-dependently increased MMP10 transcription [The promoter regions of at least fourteen MMP genes  [MMP9 expression [MMP2 and MMP9 expression [GRK6. This gene encodes G protein-coupled receptor kinase 6 (GRK6), an enzyme involved in repressing angiogenesis, tumor growth, and metastasis [MMP2 and MMP7 [GRK6 promoter is hypermethylated and GRK6 expression is repressed, which promotes cell migration and invasion [MMPs promote cancer progression through distinct mechanisms, including (i) activation of growth factors , (ii) insiveness . In breaription) , resultipression . Furtherpression . In gliopression . MMP gentastasis . GRK6 deand MMP7 . In lunginvasion . Thus, aMMP9 gene in a fibrosarcoma cell line (HT1080) and B-cell lymphosarcoma cell line (TBL3) infected with T. annulata [MMP9 mRNA levels in TBL3 cells were associated with greater H3K4me2 and H3K4me3 marks in the regulatory region of the MMP9 promoter. The elevated histone marks were determined to be the result of SMYD3, as it was the only methyltransferase significantly induced in TBL3 cells. SMYD3 induces transcriptional activation by binding to the 5′-CCCTCC-3′ or 5′-GGAGGG-3′ DNA sequences in the promoter region of genes including MMP9. Furthermore, HT1080 cells transfected with siRNAs against SMYD3 resulted in decreased cell migration and proliferation accompanied by decreased levels of MMP9 mRNA and protein. Similar results were achieved by knocking down only MMP-9. Therefore, SMYD3 acts as an epigenetic regulator of MMP9, and consequently affects cancer invasion.Cock-Rada et al. (2012) showed that H3K4 methyltransferase SMYD3 epigenetically regulates the transcription of the annulata . IncreasMMP9 has also been reported to be the target of miR-211 [MMP9 (pM) treatments was associated with anti-proliferative and apoptotic signaling in 4910 cancer stem cells and U87 glioma cells. Following treatment, an increase in the cleaved 35 kDa fragment of Caspase-9 was observed, which was indicative of Caspase-9 activation. This then triggered pro-apoptotic pathways following the effector caspase cleavage and activation. The overexpression of miR-211 also inhibits glioma cell invasion and migration via suppressing MMP9 expression. Other known targets of miRNAs in cancers include MMP2, MMP9, MMP13, MMP14, and MMP16 and their substrates such as type I, II, and IV collagens [ miR-211 . In mult miR-211 . An inveollagens ,23.MMP2 and MMP9 via microRNA-29b upregulation is a mechanism by which oxidized low-density lipoprotein (oxLDL) contributes to atherosclerosis [MMP2 and MMP9 following oxLDL treatment revealed significant demethylation in the two genes [MMP2 and MMP9 mRNA levels, indicating that MMP2 and MMP9 were repressed by DNA methylation. These results show that upregulated MMP2 and MMP9 expression resulted from decreased DNA methylation due to DNMT3b downregulation. Further, Chen et al. (2011) also determined that microRNA-29b was upregulated in a dose-and time-dependent manner in oxLDL treated SMCs [MMP2 and MMP9, and thus their expression, by regulating the expression of DNMT3b.Aberrant activity of MMPs contributes to the development and progression of atherosclerosis by influencing the local inflammatory response at the site of atherosclerosis plaque formation and by catalyzing plaque rupture, an event that ultimately culminates in myocardial infarction and heart failure . Recent clerosis . The oxLclerosis . Analysiwo genes . DNMT3b wo genes . DNMT3b ted SMCs . These rMMP9 gene and the aberrant upregulation of MMP9 expression is observed in Kawasaki disease, a pediatric vascular disease characterized by inflammation of the coronary arteries [MMP9 shows a strong negative correlation with mRNA expression levels, indicating that MMP9 is repressed by DNA methylation [Thus, DNA methylation leading to aberrant expression of MMPs can significantly influence the progression and development of cardiovascular diseases. Similarly, the hypomethylation of the arteries . The methylation . Furtherhylation   in osteoarthritis versus non-osteoarthritis chondrocytes [MMP13, 19% versus 53% for MMP9, and 43% versus 70% for MMP3 [MMPs modulate bone growth, development, and repair processes through their actions on extracellular matrix components, and dysregulated MMP expression and activity are thus implicated in bone diseases such as osteoarthritis (OA) and rheumatoid arthritis  Figure . A genomdrocytes ,50. The MMP13 expression in osteoarthritis chondrocytes [MMP13 expression via histone de-acetylation. The study also showed that the inhibition of HDACs by Trichostatin A (TSA) lowered the HDAC-4 and MMP-13 protein expression in OA chondrocytes, thereby confirming the role of HDAC-4 in regulating MMP-13. When overexpressed, MMP-13 can exacerbate OA [MMP13 gene in the OA chondrocytes [MMP13 mRNA, which contains a complementary sequence to the miR-27b seed sequence and downregulates MMP13 expression at the post-transcriptional level [β stimulation compared to chondrocytes that were not transfected with a miR-27b mimic, and inhibition of miR-27b increased MMP-13 protein expression in IL-1β–stimulated chondrocytes when compared to control OA chondrocytes. These findings indicate that miR-27b acts as a regulator of MMP-13 protein expression in human OA chondrocytes [MicroRNA-222 and microRNA-27b regulate drocytes . Song etdrocytes . Elevaterbate OA . Akhtar drocytes . MicroRNal level . MiR-27bdrocytes .MMP1 and MMP14 [MMP1, MMP3, MMP9, and MMP13 [MMP9 expression was not upregulated by interleukin-6 (IL-6) induction, while MMP1, MMP3, and MMP13 were upregulated. This occurs as a result of the IL-6-inducible transcription factor STAT3 being recruited to the promoters of MMP1, MMP3, and MMP13, but not to the MMP9 promoter [In rheumatoid arthritis (RA), abnormal histone modifications and DNA methylation both lead to aberrant upregulation of MMP gene transcription  Figure . Genome-nd MMP14 . In RASFnd MMP13 . Interespromoter . The difMMP9 expression in RA may also be epigenetically regulated by miRNAs. Wang et al. (2019) reported that the fibroblast-like synoviocytes from patients with RA exhibited elevated miR-145-5p expression and increased MMP9 mRNA and protein levels [MMP9 by binding the MMP9 promoter region [MMP9 in patients with RA [Furthermore, recent evidence suggests that n levels . The over region . These d with RA .Mycobacterium tuberculosis (Mtb) in pulmonary tuberculosis [MMP1 promoter region was reported in CoMTb-stimulated NHBEs compared to the uninfected controls. This accompanied a more-than-10 fold increase in RNA polymerase II binding to the MMP1 promoter [MMP1 gene expression.Epigenetic regulation of MMP-1 and MMP-3  modulates the immune response against rculosis ,58,59,60rculosis . No signrculosis . The inhrculosis . Howeverrculosis . Thereforculosis . HAT inhrculosis . These rpromoter . These rMMP1 gene expression [MMP2 gene expression, but rather the downstream effects of trichostatin A-induced RECK upregulation, which decreased CL-1 cancer cell invasion [MMP1 gene expression in rabbit primary synovial fibroblasts (RSF) [MMP1 and MMP13 genes, via downstream phosphorylation pathways [MMP1 expression, as reported in tuberculosis and the cytokine-induced expression of MMP-1/-13 proteins in human articular chondrocytes. The relationship between HDAC/HATs activity and MMP expression and activity is influenced by determinants of inflammatory status such as the levels of pro-inflammatory cytokines  and transcription factors .The effects of HDACs and HATs on MMP expression can be more complex and indirect, due to interrelated signaling cascades involving transcription factors and non-histone proteins that can also be substrates of the above-mentioned enzymes ,62,63. Spression ,64. To fpression , inducedpression . The HDApression . Trichosinvasion . The resinvasion . Barchowpathways ,68. Thespathways ,68. Intepathways , which cMMP9 gene expression in human HT1080 fibrosarcoma cells [MMP9, subsequently reducing histone H3 acetylation [MMP9 expression could reduce its pro-angiongenic, inflammatory, and metastatic roles  reported the inhibitory effect of the anti-proliferative cytokine interferon-β (IFN-β), on ma cells . IFN-β dtylation . IFN-β-itylation . Therefoic roles , and thuMMPs can contribute to infection-induced endotoxic shock through their altered expression in the disease state, in which they modulate the secretion of inflammatory cytokines and degrade the proteins of the endothelial basement membrane, such as type IV collagen . Such cl2O2 (a reactive oxygen species)-induced apoptosis in human hepatocytes through the p53-dependent pathway by epigenetically inhibiting its modulator p14 [Multiple viruses including the hepatitis C virus (HCV), the middle east respiratory syndrome coronavirus (MERS-CoV), and H5N1 avian influenza (HPAI) impact the epigenetic regulatory mechanisms in the genome of the host cell ,77,78. Hator p14 . The corator p14 . CoronavACE2 gene, leading to ACE2 overexpression, which may further increase the susceptibility of host cells to SARS-CoV-2 infection [MMP1, MMP3, and MMP13 [Likewise, the highly infective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease-19 (COVID-19), also impacts the host’s epigenome environment . The entnfection . In a renfection . In sevend MMP13  and regund MMP13 . Circuland MMP13 . These dnd MMP13 ,84,85.Cryptosporidium species (causing cryptosporidiosis) such as C. parvum, Vorinostat showed anti-parasitic in vitro and in vivo potential by targeting the HDAC of C. parvum [Elucidating the epigenetic mechanisms involved in chronic diseases and infections can be critical not only to understand the pathogenesis mechanisms of these diseases but also to design novel therapeutic interventions. For instance, in tuberculosis, the inhibition of HDACs using suberanilohydroxamic acid (SAHA), also known as the drug Vorinostat, resulted in an immune response by macrophages and subsequently T cells against Mtb . This im. parvum . Further. parvum . However. parvum . Therefo. parvum .T. denticola [T. denticola challenge of periodontal ligament (PDL) cells, the induced expression of the MMP2 gene was reversed with HDAC inhibition treatment using apicidin and trichostatin A, in addition to inhibitors of histone phosphorylases and DNA methyltransferases [The MMP-2-mediated degradation of the extracellular matrix (ECM) in periodontitis can be epigenetically induced by the spirochete enticola . During sferases . Thus, tp21 promoter [MMP gene expression is altered at the transcriptional and post-transcriptional levels by statins ,17, a fapromoter . The incpromoter . DNA hyppromoter . Karlic promoter . Furtherpromoter . Thus, spromoter ,92,94 meCCR7 promoter region and increasing histone H3 and H4 acetylation to the same region [The epigenetic effects of rosuvastatin, atorvastatin, and lovastatin are involved in atherosclerosis regression, where they induce the mRNA expression of the chemokine receptor CCR7 in mice RAW264.7 macrophages . Rosuvase region . Conseque region .Atorvastatin, Fluvastatin, and simvastatin show impacts on MMP gene expression ,98,99. TTreatment of human vascular endothelial cells with fluvastatin and lovastatin inhibited the expression of MMP-1, but not the expression of the tissue inhibitor of MMP-1 . FluvastClinicalTrials.gov Identifier: NCT03508232). Whether MMP inhibition or the epigenetic mechanisms leading to decreased MMP activity underlie the benefits of doxycycline in these cardiac infarct patients remains unclear. However, emerging evidence points at possible epigenetic mechanisms of doxycycline that merit consideration from a therapeutic point of view. For instance, in models of abdominal aortic aneurysm, a vascular disease associated with the overexpression of MMP-2 [MMP2 mRNA from 49 h to 28 h [MMP2 gene expression in cultured human skin keratinocytes [MMP2 and MMP9 mRNAs are targets of miR-486 [Tetracyclines exert antibiotic effects by binding to bacterial 30S ribosomal subunits to inhibit bacterial protein synthesis . In addiof MMP-2 , doxycycof MMP-2 , which w to 28 h . A conveinocytes . Furtherinocytes . Regulatinocytes  with rec miR-486 ,110,111.Various epigenetic mechanisms: DNA methylation, histone acetylation/methylation, and alterations in small non-coding RNAs can influence the expression of MMPs, tissue inhibitors of MMPs, and the transcription factors that regulate the expression of MMPs . Highly


To predict the main active ingredients, potential targets, and key pathways of Jiawei Chaiqin Wendan decoction treatment in vestibular migraine and explore possible mechanisms by network pharmacology and molecular docking technology.  The active ingredients and related targets of Jiawei Chaiqin Wendan decoction were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The corresponding genes of the target were queried by UniProt database, and the “drug-compound-target-disease” network was constructed by Cytoscape 3.7.2 software. GO functional enrichment analysis and KEGG pathway enrichment analysis were carried out by R software and Bioconductor, and column chart and bubble chart were drawn by Prism software and OmicShare database for visualization. Finally, the mechanism and potential targets of Jiawei Chaiqin Wendan decoction in the treatment of vestibular migraine were predicted. P < 0.05). KEGG pathway enrichment analysis screened 156 signal pathways (P < 0.05), involving PI3K-Akt signal pathway, AGE-RAGE signal pathway in diabetes complications, MAPK signal pathway, HIF-1 signal pathway, IL-17 signal pathway, etc. Molecular docking results showed that quercetin, luteolin, kaempferol, tanshinone IIa, wogonin, naringenin, nobiletin, dihydrotanshinlactone, beta-sitosterol, and salviolone have good affinity with core target proteins IL6, PTGS2, MAPK1, MAPK3, and CGRP1.  The “drug-compound-target-disease” network contains 154 active ingredients and 85 intersection targets. The key targets include AKT1, IL6, MAPK3, VEGFA, EGFR, CASP3, EGF, MAPK1, PTGS2, and ESR1. A total of 1939 items were obtained by GO functional enrichment analysis ( The active ingredients in Jiawei Chaiqin Wendan decoction may regulate the levels of inflammatory factors and neurotransmitters by acting on multiple targets such as IL6, MAPK3, MAPK1, and PTGS2, so as to play a therapeutic role in vestibular migraine. Vestibular migraine (VM) is a chronic and disabling disease that is common and has a genetic tendency; the main symptoms are recurrent dizziness/vertigo, which may be accompanied by nausea and vomiting with or without headache . StudiesThe diagnosis of VM is mainly based on the characteristics of the patient's clinical onset and lacks specific signs and objective diagnostic basis. In the past few decades, uncertainty about the diagnosis of VM has limited the progress of treatment . There iChaiqin Wendan decoction is made of Wendan decoction from Yao Sengyuan in the Southern and Northern dynasties. The modified Chaiqin Wendan decoction (JCWD) is an effective empirical prescription for the treatment of vertigo in clinic. This prescription is composed of Radix Bupleuri (Chaihu), Radix Scutellariae (Huangqin), Pinellia tuber (Banxia), Dried tangerine Peel (Chenpi), Fructus Aurantii Immaturus (Zhishi), Poria cocos (Fuling), Radix Paeoniae Alba (Baishao), Angelica sinensis (Danggui), Salvia miltiorrhiza (Danshen), Gastrodia elata (Tianma), Uncariae Ramulus Cumuncis (Gouteng), and Shi Cassia (Shijueming). At present, clinical observation indicates that Chaiqin Wendan decoction has a significant effect on the treatment of patients with dizziness and headache caused by Triple Burner Congestion –11. Therhttp://tcmspw.com/tcmsp.php) [http://www.uniprot.org/), and gene annotation was performed.The TCMSP (msp.php)  databasehttps://www.genecards.org/) and OMIM database (https://omim.org/) by retrieving “vestibular migraine” look up vestibular migraine related genes and remove duplicates. Through Venny2.1 software, the target of active ingredients of traditional Chinese medicine obtained above was intersected with disease-related genes.Using GeneCards , and the species was selected as “Homo sapiens,” with a medium confidence of 0.400 to predict the associated PPI network. Download protein-protein interaction TSV file, import Cytoscape3.7.2 software, and use CytoHubba plug-in to screen core target proteins.Multiple proteins were introduced into String11.0 (http://www.bioconductor.org) to GO functional annotations and KEGG pathway enrichment analysis. P < 0.05 was selected for functional annotation clustering. In order to study the biological effects of VM targets of drug therapy by regulating specific pathways, the top 10 items were screened according to the enrichment degree and a bar chart was drawn. Similarly, P < 0.05 was selected as the significant pathway for cluster analysis, and the top 20 items were screened according to the enrichment degree to draw a bubble plot.There is the core target gene ID through the R software and Bioconductor (https://www.ncbi.nlm.nih.gov/pubmed). The 3D structures of ligands were generated by Chem3D software, and their energy was minimized and exported to mol2 format. All compounds were saved as ligand parameter files in pdbqt format by AutodockTools-1.5.6 software. The 3D structure of the core target protein was downloaded from PDB database (https://www.rcsb.org/) and saved in PDB format. The solvent and water molecules in the target protein receptor molecules were removed by pymol software. Then, the protein receptor molecules were hydrogenated and calculated by AutodockTools software and saved in pdbqt format. After the ligand and protein receptor molecular preprocessing is completed, the appropriate box center and box lattice parameters are set by AutodockTools-1.5.6 software, the active pocket sites that small molecular ligands may bind to are included, and the parameter text file is set, and the other parameters remain default. Molecular docking was carried out by Autodock Vina, and 3D diagrams were drawn by PyMol software for the docking results with the strongest binding activity of each target protein to show the hydrogen bond and hydrophobic effect formed by it.The 2D structures of key compounds were downloaded in PubMed  and OMIM database (https://omim.org/) by retrieving “vestibular migraine” search related genes, a total of 826 were extracted after duplication. Through Venny2.1 software, the intersection of traditional Chinese medicine target and disease genes was obtained, and 85 genes related to JCWD treatment of VM were obtained, as shown in In GeneCards  and 915 edges, as shown in Eighty-five JCWD gene targets for VM treatment were introduced into String11.0 to construct a Protein-protein interaction network (PPI), and isolated nodes were removed, as shown in P < 0.05）, among which 1731 items of biological processes (BP) were closely related to the reactions of metal ions, blood circulation, reactive oxygen species, and oxidative stress, lipopolysaccharide, steroids, and bacterial origin molecules. The 141 items of molecular function (MF) are mainly related to protein phosphatase binding, nuclear receptor activity, and steroid receptor activity, transcription factor activity, cytokine activity, and receptor binding, NO synthase regulator activity, etc. 67 results of cell composition (CC) are mainly related to membrane microdomain, receptor complex, components, and inherent components of synaptic membrane and vesicle cavity, as shown in GO function enrichment analysis and KEGG pathway enrichment analysis were carried out by R software and Bioconductor database installation packages such as clusterProfiler and enrichplot. 1939 GO enrichment results were obtained  pathway, MAPK in the activator protein-1 pathway, and cytokine signal inhibitors in the signal transduction and transcriptional activator 3 pathway, regulating inflammatory mediators and playing an anti-inflammatory role. Luteolin can inhibit all proinflammatory cytokines, such as interleukin-1β, IL-2, IL-6, IL-8, IL-12, IL-17, TNF-α, interferon-β(IFN-β), and granulocyte-macrophage colony stimulating factor and upregulate anti-inflammatory cytokine IL-10 [κB pathway, inhibit the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1β and IL-6, and reduce the inflammatory response of spinal dorsal root neurons induced by lipopolysaccharide [κB-α and the activation of NF-κB induced by LPS by inhibiting the activation of NIK-IKK and MAPKs  signal pathways, so as to exert its anti-inflammatory activity [From the point of view of the active components of the drug, quercetin can exert its anti-inflammatory effect by inhibiting the production of IL-6, TNF-and VEGF . In addipathways . Kaempfepathways . Luteoline IL-10 . Wogoninccharide ,25. Tansactivity .α, IL-2, IL-6, and other proinflammatory factors [Protein-protein interaction (PPI) network was used to screen out 10 core genes by Matthews correlation coefficient (MCC) algorithm. They are VEGFA , MAPK3 (mitogen-activated protein kinase 3), MAPK1 (mitogen-activated protein kinase 1), EGF , EGFR , IL-6 (interleukin-6), PTGS2 (prostaglandin endoperoxide synthase 2), STAT3 , MMP9 , and MMP2 . Vascular endothelial growth factor (VEGF) has heparin active growth factor, which can play a role in vascular endothelial cells to cause the division and proliferation of vascular endothelial cells, thus forming new blood vessels and improving vascular permeability. The main causes of migraine include vasodilation and protein exudation. In the onset stage of migraine, patients' serum VEGF level is abnormally elevated, blood vessels dilate, and their permeability increases, eventually leading to extravasation of plasma protein . Epiderm factors .Through the analysis of KEGG pathway, it is found that MAPK signal pathway, HIF-1 signal pathway, and IL-17 signal pathway are the main treatment pathways, which play a synergistic anti-inflammatory effect. MAPK signaling cascades include three different regulatory pathways: extracellular signal-regulated kinase (ERK1/2), c-jun N-terminal kinase (JNK) and p38 protein (P38), which are responsible for mitosis, proliferation, differentiation, and survival of cells during development , as wellIn this study, the active components of JCWD and the potential targets and key pathways acting on vestibular migraine were predicted by the method of network pharmacology, which once again confirmed the process of multicomponent, multitarget, and multipathway treatment of diseases with traditional Chinese medicine compound prescription. The active components such as quercetin, kaempferol, luteolin, wogonin, and tanshinone II as in Jiawei Chaiqin Wendan decoction may regulate the levels of inflammatory factors and neurotransmitters (such as CGRP) by acting on IL6, MAPK3, MAPK1, PTGS2, and other targets, thus playing a role in the treatment of vestibular migraine. Due to the limitations of the database and the software itself, the above results need to be further verified by experiments.


KAT6A, is an autosomal dominant inherited genetic disorder characterized by various degrees of developmental delay, dysmorphic facial appearance, cardiac anomalies, and gastrointestinal problems.Arboleda-Tham syndrome (ARTHS), caused by a pathogenic variant of KAT6A gene  at 2 months of age. After a diagnosis of ARTHS, an expressive language delay was observed during serial assessments of developmental milestones.A baby presented multiple facial deformities including a high arched and cleft palate, with philtral ridge and vermilion indentation, a prominent nasal bridge, a thin upper lip, low-set ears, an epicanthal fold, and cardiac malformations. Whole exome sequencing (WES) revealed a heterozygous nonsense mutation in exon 8 of the KAT6A variant first identified in Korea. This case broadens the scope of clinical features of ARTHS and emphasizes that WES is necessary for early diagnosis in patients with dysmorphic facial appearances, developmental delay, and other congenital abnormalities.In this study, we describe a case with a novel The online version contains supplementary material available at 10.1186/s12920-021-01148-x. KAT6A (MIM 601408) with autosomal dominant inheritance ), length was 52.3 cm (1.68 SD), and head circumference was 32.5 cm (− 1.17 SD). At birth, her mother and father were both 37 years old. Prenatal examinations, including non-invasive screening for aneuploidies, did not reveal abnormalities. During a one-month hospitalization for respiratory distress syndrome and persistent pulmonary hypertension, she presented with craniofacial deformities, including a high arched and cleft palate with philtral ridge and vermilion indentation, and a subtle dysmorphic facial appearance with a prominent nasal bridge, a thin-tented upper lip, low-set ears, and epicanthal fold . The analysis showed a novel de novo heterozygous nonsense variant, Chr8:41834577G>A, KAT6A:NM_001305878.1:c.1312C>T:p.(Arg438*), in exon 8 of the KAT6A gene  karyotyping. WES was conducted when the patient was two months of age to search for pathogenic variants of genes causative of dysmorphic facial appearance, cleft palate, and cardiac anomalies idelines .Fig. 2Lord edition) did not indicate any developmental delays. However, a comprehensive developmental evaluation at the age of eight months with the Bayley Scales of Infant and Toddler Development, 3rd edition (Bayley-III) found a mild language delay. Currently, the patient received a variety of intensive rehabilitation therapies, twice a week.After diagnosis of ARTHS, developmental screening tests at the age of four months (Korean developmental screening test for infants and children; The Bayley Infant Neurodevelopmental Screener, 3KAT6A gene have been identified in solid tumors and patients with syndromic disease with developmental disorders of variable severity [KAT6A variants has gradually increased [Lysine acetyltransferase 6A, KAT6A, a member of the MYST family of proteins, forms a histone acetyltransferase complex and plays an essential role in regulating transcriptional activity and gene expression . KAT6A iseverity , 18, 20.ncreased , 21, 22.KAT6A were first reported in 2015, nearly 100 cases of ARTHS had delineated variable presentation, with a continuously expanding extent of symptoms [KAT6A, including 34 frameshift mutations, 41 nonsense mutations, 9 missense mutations, 4 splicing mutations, and 1 deletion mutation (Table KAT6A were mild [Since patients with syndromic developmental delay due to pathogenic variants of symptoms , 13. We ere mild . The varere mild .Table 1TKAT6A variants includes neurological, craniofacial, gastrointestinal, cardiac, and ocular features. Neurological manifestations, such as developmental delay, hypotonia, microcephaly, and seizure, are main characteristic features of patients with ARTHS [KAT6A variants have congenital heart disease, with ASD noted most frequently, followed by PDA (Table The spectrum of phenotypes with th ARTHS , 7. All th ARTHS , 7. Mostth ARTHS , 7, 13. th ARTHS , 8. Feedth ARTHS , 7, 22. KAT6A variants [KAT6A variants [The salient features of this case included dysmorphic facial appearance, cleft palate, and congenital heart disease, which are major features that strongly suggest multiple congenital abnormalities –26. As Wvariants , 13. WESvariants –31. BecaGenetic diagnosis at a younger age is important in syndromic cases because special treatments or surveillance programs are available for major symptoms. Most patients diagnosed with ARTHS have speech delays and intellectual disabilities, and early assessment of developmental delays can improve prognoses through proper treatment according to patient age –6. In thKAT6A has not been clearly identified, the number of patients found to have de novo pathogenic variants of KAT6A continues to increase. Therefore, clinicians must be familiar with the various phenotypic features of ARTHS for the diagnosis by exome sequencing. In our case, because a genetic diagnosis was confirmed at an early age, the unexpressed features that may develop later in life can be monitored. Moreover, further study of the variant is important since there had been no functional studies to confirm that this variant was truly deleterious. This report broadens the scope of clinical features of ARTHS that might be an unrevealed genetic cause of neurodevelopmental disorder and facial dysmorphism. WES is useful to identify any associated genetic disorders and provide personalized care for patients with facial dysmorphism and other congenital anomalies possibly associated with developmental delay.Even though the role of Additional file 1. Candidate variants identified by whole exome sequencing


Metagenomic sequencing and analysis showed large microbiome difference between wheat rhizosphere and bulk soil but small difference between HZn and LZn. Most of the rhizosphere-enriched microbes in HZn and LZn were in common, including many of the previously reported soil Zn-mobilizing microbes. Notably, 30 of the 32 rhizosphere-enriched species exhibiting different abundances between HZn and LZn possess the functional genes involved in soil Zn mobilization, especially the synthesis and exudation of organic acids and siderophores. Most of the abundant potential Zn-mobilizing species were positively correlated with grain Zn concentration and formed a module with strong interspecies relations in the co-occurrence network of abundant rhizosphere-enriched microbes. The potential Zn-mobilizing species, especially Massilia and Pseudomonas, may contribute to the cultivars’ variation in grain Zn concentration, and they deserve further investigation in future studies on Zn biofortification.An effective solution to global human zinc (Zn) deficiency is Zn biofortification of staple food crops, which has been hindered by the low available Zn in calcareous soils worldwide. Many culturable soil microbes have been reported to increase Zn availability in the laboratory, while the status of these microbes in fields and whether there are unculturable Zn-mobilizing microbes remain unexplored. Here, we use the culture-independent metagenomic sequencing to investigate the rhizosphere microbiome of three high-Zn (HZn) and three low-Zn (LZn) wheat cultivars in a field experiment with calcareous soils. The average grain Zn concentration of HZn was higher than the Zn biofortification target 40 mg kg An effeutrition . Achieviutrition . TherefoBacillus, Arthrobacter, and arbuscular mycorrhizal fungi (AMF) has been reported to increase soil available Zn and crop grain Zn concentration, especially in soils with low available Zn  16.3 g kg–1, total nitrogen (N) (Kjeldahl) 1.01 g kg–1, nitrate-N (1 M of KCl) 15.3 mg kg–1, ammonium-N (1 M of KCl) 0.1 mg kg–1, total phosphorus (P) (HClO4 + H2SO4) 0.78 g kg–1, available P (0.5 M of NaHCO3) 11.7 mg kg–1, total potassium (K) (NaOH) 14.2 g kg–1, available K (1 M of NH4Ac) 122 mg kg–1, total Zn (HF + HNO3 + HClO4) 74 mg kg–1, and available Zn (0.005 M of DTPA) 0.46 mg kg–1.The field experiment was located at the Agriculture Research Station of Northwest A&F University in Yangling, Shaanxi, China . This site is in the temperate continental monsoon climate zone and located on the southern edge of the Loess Plateau, with elevation of 520 m, annual atmosphere temperature of 12.9°C, annual precipitation of 579 mm, and annual potential evaporation of 1,400 mm. The soil type of the experimental filed is Eum-Orthic Anthrosol according to the soil taxonomy system of the United States Department of Agriculture (USDA), and the soil texture is silt clay loam. Basic soil chemical properties are as follows: pH (water:soil = 2.5:1) 8.23, cation exchange capacity  16.3 cmol kg–1 as urea (46% N), 100 kg P2O5 ha–1 as calcium superphosphate (16% P2O5), and 74 kg K2O ha–1 as potassium sulfate (51% K2O). Each plot consisted of five 2-m-long rows with the seed spacing of 2.5 cm and the row spacing of 20 cm. All cultivars were sown on October 3, 2018, and harvested on June 18, 2019. All the fertilizers were applied before sowing, no irrigation was applied during wheat growth period, and only pesticides were used when necessary.Based on our previous studies on screening hundreds of wheat cultivars by yield and grain Zn concentration , six culThe top 0- to 20-cm soil of the experimental field was sampled before sowing for the determination of basic soil physicochemical properties. At anthesis, the plant, rhizosphere, and bulk soil samples used for chemical analyses and metagenomic sequencing were collected. For each cultivar, 10 wheat plants were randomly selected and dug out from each plot, cut-off at the stem-root jointing part, and separated into ears, leaves, stems, and roots with adhered rhizosphere soils by sterile stainless tools and shaking. For bulk soil sampling, 10 points were randomly selected from the unplanted areas over 50 cm away from wheat plots in each block and sampled at 0- to 10-cm soil layer and then mixed into one sample using sterile stainless tools. At maturity, the plant samples used for chemical analyses and estimation of yield and biomass were collected. For each cultivar, the wheat plants of 30 ears without roots and soil were sampled by the same procedures used in plant sampling at anthesis, and all the remaining plants were harvested to estimate grain yield.One third of the samples of root with rhizosphere soil and bulk soil were transported to the laboratory in a dry ice box and stored in an ultra-low-temperature refrigerator at −80°C before metagenomic sequencing, and the other soil and plant samples were transported to the laboratory under air temperature for further processing. Firstly, the rhizosphere soils were separated from roots using a nylon brush; then, the separated rhizosphere soil and bulk soil samples were air-dried and ground to pass through a 1-mm sieve for chemical analysis; finally, the plant samples of separated root, stem, leaf, ear, glume, and grain were successively washed by tap water and distilled water, oven-dried at 65°C and weighed, and ground to powder using a ball miller  for chemical analysis.2–free water extraction (water:soil = 2.5:1) and a pH meter, and soil available Zn was extracted by 0.005 M of DTPA-TEA-CaCl2 solution and determined by an atomic absorption spectrometer (AAS). Grain yield and biomasses were expressed in oven-dried weight, and plant Zn concentration was determined by microwave digestion with HNO3 and H2O2 and an inductively coupled plasma mass spectrometry (ICP-MS), and then shoot Zn uptake was calculated.The chemical analyses of soil and plant samples were conducted according to the methods described by g and 4°C for 20 min to get the rhizosphere soil pellet, which was firstly frozen in liquid nitrogen and then stored at −80°C for DNA extraction. All the above operations were done using sterile tools under sterile conditions. Soil DNA was extracted using the DNeasy PowerSoil DNA Isolation Kit  according to the provided protocol with minor modifications. In short, 0.4 g of soil was weighed, added into the PowerBead Tube and mixed on a vortex, and incubated at 65°C in a water bath for 10 min before being subjected to the same procedures as described in the protocol. The extracted DNA was quantified by Invitrogen Qubit 4.0 and Qubit dsDNA HS Assay Kit  and quality checked by NanoDrop 2000  and agarose gel electrophoresis . The DNA concentration was diluted or concentrated to ∼ 25 ng μl–1 for the preparation of sequencing library.Rhizosphere soil samples used for metagenomic sequencing were separated from roots according to the methods described by The prepared DNA sample was fragmented into 350-bp fragments in 60-μl volume in a microTUBE by Covaris S220 ultrasonicator . Then, the fragmented DNA was transformed into sequencing library using the Truseq DNA PCR-Free Library Prep Kit  according to the provided protocol. Finally, the prepared library was sequenced by the Illumina Hiseq X ten  in the mode of paired-end 150 bp.https://github.com/kingforest93/Ta6_rhizo_meta_scripts.The raw reads were firstly checked for sequencing quality; and the ambiguous bases, low-quality bases, and adapter contamination were removed using fastp v0.20.0 . Then thTo identify the potential soil Zn-mobilizing species, we summarized the functional characteristics of the reported soil Zn-mobilizing microbes in published literature , selectet-test. The within-sample microbial alpha diversity (the Shannon–Wiener index) of species and OGs were analyzed using vegan v2.5-6 package  and non-metric multidimensional scaling (NMDS) using vegan and ggplot2 v3.3.2 package  method  method . Spearma) method . The co-) method . All figP < 0.05, –1, the six cultivars were classified into HZn and LZn groups, and the grain Zn concentration and shoot Zn uptake of HZn wheat were significantly higher than those of LZn wheat  of wheat rhizosphere was 4.3% lower than that of bulk soil  of rhizosphere-enriched species and eggNOG OGs showed no significant difference between HZn and LZn . PossiblP < 0.05, Comparisons of the relative abundances of rhizosphere-enriched eggNOG OGs between HZn and LZn wheat showed that 368 OGs were significantly enriched in HZn with the abundance ratio of HZn to LZn >1.5 and 86 OGs were enriched in LZn with the abundance ratio of HZn to LZn <0.5  among species, most of the relatively abundant potential Zn-mobilizing species closely related with each other and formed a module in the network, while the published Zn-mobilizing species scattered in the network  in wheat rhizosphere were analyzed by Spearman’s correlation of their relative abundances and illustrated in a co-occurrence network . Conside network . Besides network . TherefoHost plant genetic factors are thought to have much weaker influences on shaping rhizosphere microbiome than compartment niches do . We provPseudomonas has been reported to produce citric acids  and National Genomics Data Center and BIG Data Center (NGDC BIG) under BioProject accession PRJCA003344 (CRR196125 to CRR196152).The generated reads in the present study have been deposited in NCBI SRA under BioProject accession SW: conceptualization, data curation, formal analysis, investigation, methodology, visualization, and writing (draft and revision). ZG and LW: data curation, investigation, methodology, resources, visualization, and writing (revision). YZ: formal analysis, methodology, resources, and visualization. FJ: formal analysis, methodology, software, and visualization. XW: data curation, investigation, methodology, and resources. LY: formal analysis, methodology, resources, and visualization. BL, HL, HW, and AW: formal analysis, resources, software, and visualization. YR and CL: methodology, investigation, and resources. WF: conceptualization, funding acquisition, methodology, project administration, supervision, visualization, and writing (revision). ZW: conceptualization, funding acquisition, project administration, resources, supervision, and writing (revision). All authors contributed to the article and approved the submitted version.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.


Chemokines are a well-defined group of proteins with chemotactic properties. We focus on key chemokines that not only attract leukocytes to tumor sites but also shape their biological properties. We propose using stabilized forms of two of them: CXL9 and CXCL10, to enhance anti-tumor immunity and possibly transform cold tumors into hot ones. Additionally, we discuss the possibility of targeting or deleting a key subset of Tregs that are CCR8+ Tregs and are highly dominant at the tumor site of several cold tumors. This may convert these cold tumors into hot tumors, and thus extend the success of immunotherapy beyond its current limits.For the last decade, the most successful approach for treating cancer has been the use of monoclonal antibodies to immune checkpoint inhibitors (ICI), also known as immune checkpoint blockers (ICB). Unfortunately, many cancers do not respond to these treatments. “Hot” tumors are those that show signs of inflammation, meaning they have been invaded by effector T cells rushing to fight the cancerous cells. Evidence suggests that the limited success of ICI-based immunotherapies is related to attempts to treat patients with “cold tumors” that either do not contain effector T cells or in which these cells are markedly suppressed by regulatory T cells (Tregs). Chemokines are a well-defined group of proteins that were so named due to their chemotactic properties. The current review focuses on key chemokines that not only attract leukocytes but also shape their biological properties. CXCR3 is a chemokine receptor with 3 ligands. We suggest using Ig-based fusion proteins of two of them: CXL9 and CXCL10, to enhance anti-tumor immunity and perhaps transform cold tumors into hot tumors. Potential differences between CXCL9 and CXCL10 regarding ICI are discussed. We also discuss the possibility of targeting the function or deleting a key subset of Tregs that are CCR8+ by monoclonal antibodies to CCR8. These cells are preferentially abundant in several tumors and are likely to be the key drivers in suppressing anti-cancer immune reactivity.Over the last decade, monoclonal antibodies to immune checkpoint inhibitors (ICI), also known as immune checkpoint blockers (ICB), have been the most successful approach for cancer therapy. Starting with mAb to cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibitors in metastatic melanoma and continuing with blockers of the interactions between program cell death 1 (PD-1) and its ligand program cell death ligand 1 (PDL-1) or program cell death ligand 2 (PDL-2), that have been approved for about 20 different indications. Yet for many cancers, ICI shows limited success. Several lines of evidence imply that the limited success in cancer immunotherapy is associated with attempts to treat patients with “cold tumors” that either lack effector T cells, or in which these cells are markedly suppressed by regulatory T cells (T All of them suppress anti-tumor immune reactivity, and some of them directly support tumor growth [Chemokines are small proteins that have mostly been associated with directing leukocyte migration, and in affecting the dynamics of cancer, inflammation, and immune regulation ,2,3. As r growth ,12,13,14r growth . Yet, thr growth ,17.+ and CD8+ T cells [The breakthrough of using monoclonal antibodies to immune checkpoint inhibitors (ICI)  opened new therapeutic opportunities ,24,25,26 T cells ,32,34,37 T cells ,39,40,41 T cells ,46,47,48+ T cells or include massive accumulation of Tregs that suppress their activities [regs, it is likely that blocking their activity or depleting these cells from the TME would turn cold tumors into hot.It is believed that one of the major reasons for which the success of ICI is limited is that therapy is applied on diseases that either lack infiltration of effector CD8tivities ,34,35,36tivities ,50,51. Ttivities ,50,51,52regs [The current review focuses on two key chemokine pathways, with relevance to cancer immunotherapy. The first refers to the CXCR3-CXCL9/CXCL10 pathway ,54,55. Tregs . Both apCXCR4 is a chemokine receptor with a single ligand, CXCL12 , that also binds CXCR7 . The CXC+ monocytic cells has been mostly studied for two types of diseases: inflammatory autoimmune diseases, and cancer. Targeting this interaction restrained inflammatory autoimmunity by limiting the accumulation of inflammatory macrophages at autoimmune sites [CCR2 is a chemokine receptor to numerous ligands including CCL2, CCL7, CCL8, and CCL13. Of them, CCL2 is its major ligand and exclusively binds CCR2 . The rolne sites ,71,72,73ne sites ,81,82,83ne sites ,92,93,94ne sites . As targCCR5 is a chemokine receptor with three ligands: CCL3, CCL4, and CCL5. Of them, only CCL4 exclusively binds CCR5, whereas CCL3 also binds CCR1, and CCL5 also binds CCR1 and CCR3 . CCR5 anCXCR1 is a chemokine receptor with two ligands, CXCL6 and CXCL8. CXCL8 also binds to CXCR2 . The rol+ and CD8+ T cells, and to some extent by other cells, among them, macrophages [+ subset, CXCR3 is most abundant on effector T cells, but notably, it is also expressed by FOXp3+ regulatory T cells (Tregs) In humans, three isoforms were identified: CXCR3A that is reciprocal to the mouse CXCR3 and binds CXCL9, CXCL10, and CXCL11, CXCR3-B that binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4, and CXCR3-alt that only binds CXCL11 [CXCR3 is a chemokine receptor that is primarily expressed on CD4rophages , NK cellrophages ,168,169,rophages . Within s CXCL11 . The CXCs CXCL11 . Togethes CXCL11 ,174.high CD4+ T regulatory-1 cells (Tr1) [high CD4+ effector Th1 cells, and therefore targeted neutralization of CXCL10 may restrain T cell-mediated autoimmunity [+ effector T cells that include potentiation of IFNghigh effector cells via CXCL9 and CXCL10, and potentiation of FOXp3-negative IL10high in T-regulatory 1 (Tr1) cells via CXCL11 [Biased signaling  means that a single receptor may transmit different signal transduction pathways, usually in response to different ligands. It was first associated with G-protein coupled receptors long ago by Luttrell et al. . Very rels (Tr1) ,191. Thiimmunity ,193. Morimmunity . Indepena CXCL11 . This moa CXCL11 . While Ca CXCL11 , adminisa CXCL11 .Several studies, including our , have su+ effector-cytotoxic T cells [+ effector CD4+ and mostly effector -cytotoxic CD8+ T cells, and CXCR3+ NK cells to the tumor site [+ T cells thus enabling tumors to escape CXCL10 induced recruitment to the tumor site [+ TILs were abundant and high levels of CXCL9 and CXCL10 were secreted by stromal cells [high effector T cells away from the tumor site.For immunotherapy, it is believed that the key target cells for CXCL10/CXCL9 based therapies are effector T cells, mostly CD8 T cells ,198,199.mor site ,201,202.mor site . Along wmor site , or diremor site , or targmor site , may limal cells ,207. Thial cells ,207. The+ effector CD4+ T cells, effector CD8+ T cells, and NK cells that are then recruited to at the tumor sites to limit cancer. At this point, we do not exclude the possibility that following peripheral administration some of the injected CXCL10-Ig or CXCL9-Ig would enter the tumor site, and therefore may have an additional contribution to CXCR3+ cell recruitment to the TEM [Our collaborative study with Israel Vlodavsky and his team was among the first studies showing that systemic administration of CXCL10-Ig restrains cancer diseases . Aside f the TEM ,209.As both CXCL10 and CXL9 exclusively bind CXCR3 and are thought to attract, and possibly potentiate effector T cells, it is an open-end question whether except for redundancy they do differ in some biological functions, particularly functions that are related to effector T cell potentiation and combined immunotherapies? Chow et al. recently reported that CXCR3KO mice respond poorly to anti-PD-1 therapy. Their study suggests that the CXCL9-CXCR3 interaction is critical for successful anti-PD-1 immunotherapy . FinallyAmong the parameters that may assist in selecting target disease for CXCL10 or CXCL9 based therapies are results of pre-clinical trials in experimental models of various cancer diseases, and selection based on human data from cancer patients regarding the association of CXCL10/CXCL9 expression at the tumor site, or sera levels of these chemokines, and cancer prognosis. Diseases in which high expression of CXCL9 or CXCL10 correlated with a good prognosis, and low expression with a poor prognosis are the best potential candidates for CXCL10 or CXCL9 based therapy. Moreover, if the sera level of CXCL9/CXCL10 may predict success in CXCL9/CXCL10 based therapy it could be used for future precision medicine.ret transgenic mice for melanoma [Regarding animal preclinical studies, as CXCL9 and CXCL10 mostly affect anti-cancer immunity the immunocompetent models are favorable. Of these models, there are currently available transgenic models in which cancer is developed “spontaneously” such as TRAMP mice for prostate cancer , or ret melanoma . Cancer melanoma ,216,217,high a better response to chemoradiotherapy could be recorded, suggesting a synergistic beneficial effect of both [+ T cell recruitment and good prognosis [As for the association of CXCL9 and CXCL10 with cancer prognosis : more th of both . Another of both . An addi of both . Lastly, of both . These drognosis . ER-Negarognosis . Anotherrognosis .Are there diseases in which high CXCL10/CXCL9 could not be associated with a good prognosis and low CXCL10/CXCL9 with a poor prognosis? Few publications challenge the concept of high CXCL10 or CXCL9 and a good prognosis. This included pancreatic cancer, and myeloma, and Breast Cancer Registry ,227,228.+ and CD8+ T cells and promote their activities along with viral infections [+ dendritic cells within the TME [Chemokines and their receptors hold different biological functions, among them directing the immune system to generate effective responses against bacteria and viruses. Among these chemokines, the CXCR3 ligands CXCL9 and CXCL10 are of major interest as they direct targeted migration of effector CD4fections ,233,234. the TME . Importa the TME ,224,225.+ and CD8+ T cells [From a deterministic viewpoint of cancer evolution, cancer cells developed means of using chemokines and their receptors to support tumor growth, metastatic spread, and escape from immune eradication. Key examples are the CCR2-CCL2 pathway, CXCR4-CXCL12 pathway, CCR2-CCR3 ligands pathway. Many cancer cells express these receptors and produce their ligands that support tumor growth, metastatic spread, and recruitment of bone marrow-derived cells that are recruited at the TME to support tumor growth and suppress anti-tumor immunity. It is somehow puzzling that cancer cells also produce CXCL9 and CXCL10 and that this correlates with a better prognosis, in part due to the induction of anti-tumor CD4 T cells ,222,235. T cells . These c T cells .+ regulatory T cells; [regs), and those that are FOXp3-negative but produce high levels of IL-10, also known as T regulatory -1 cells (Tr1) [regs and their ability to limit anticancer effector T cells: 1. Blocking the migration and accumulation of Tregs at the tumor site. 2. Inhibiting their suppressive activities within the tumor site and 3. Depletion of Tregs within the tumor site. Of these approaches, depleting Tregs is likely to be the most dramatic and possibly effective way. Yet systemic depletion of Tregs may result in major impairment of immune regulation. For example, a loss-of-function mutation in the gene encoding FOXp3 leads to a very severe autoimmune syndrome in humans named immune deficiency poly-endocrinopathy enteropathy X-linked (IPEX) syndrome [Maintenance of immunological self-tolerance by suppressing self-reacting T cells, as well as restraining the activities of effector T cells in response to infectious stimuli, thus, limiting chronic inflammatory conditions, is largely regulated by CD4T cells; ,239. Thels (Tr1) ,239,240.ls (Tr1) ,242,243.ls (Tr1) . There asyndrome .regs to the tumor site, and also in their potentiation within this site. Tregs express several chemokine receptors among them: CCR8, CCR4, CXCR3, CCR2, CCR6, and CCR5 [regs to tumor sites and their potentiation there. Moreover, their selective accumulation within the tumor site may suggest that selective depletion of CCR4+ or CCR8+ Tregs may enhance anti-cancer immunity while having a very limited effect on Tregs in the periphery. This subject is further discussed below.Chemokines and chemokine receptors are thought to be involved in the selective migration of Tand CCR5 . Among tregs at the tumor site [regs, CCR4 is present in other leukocytes, among them CD4+ Th2 cells, NK cells, and macrophages [regs, and demonstrated the pivotal role of CCR4 in recruiting and inducing NK cells and Tregs to limit tumor development and metastatic spread [+, among them breast cancer. In human cancers, major target diseases are several solid tumors, B-cell lymphomas, T-cells lymphomas, and leukemia in which not only CCR4 is highly expressed within the tumor microenvironment by Tregs, NK cells, and tumor cells, but mostly in those that poor prognosis has been associated with high expression of CCR4 on these cells [regs recruitment , a small chemical class II antagonist that blocks the interaction between CCL22 and CCR4 (FLX-475 produced by FLx-Bio) in phase 1/2 clinical trials as monotherapy or in combination with anti-PD-1 (Merck), a humanized mAb (KW-0761) capable of inducing ADCC to CCR4+ cells in phase 1a monotherapy for solid tumors, in combination with anti-PD-1 (Merck) for B cell lymphoma (phase 1/2), in combination with anti-PD-L1 or anti-CTLA-4 (Astra-Zeneca) in Phase 1b for solid tumors, and combination with anti-PD-1 (BMS) for solid tumors .regs [+ Tregs as master drivers of the immune regulation [+ Tregs that is very low in the periphery increases along with the development of experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system (CNS). This study also observed that within the CNS CCR8+ Tregs are potentiated by CCL1, possibly in an autocrine manner, which makes them “driver” regulatory cells that restrain the progression of the disease [+ T cells, or include massive accumulation of Tregs that suppress their activities [CCR8 is a chemokine receptor mostly, but not exclusively, expressed by FOXp3+ Tregs ,267,268.regs , whereasregs ,271,272.regs , whereasregs ,271,272.gulation . In this disease . Indepen disease . Along w disease ,275,276.tivities ,279,280.+ T cells. The tumor cells have undergone many mutations that create neoantigens recognized by these T cells. For this reason, hot tumors typically respond well to immunotherapy treatment using checkpoint inhibitors [regs. Which are known to dampen the immune response and inhibit T cells trying to move into the tumor [regs via anti CCR4 or CCR8 mAbs.Tumors described as “hot” are those that show signs of inflammation, particularly massive infiltration and enrichment with effector T cells, the vast majority of the CD8hibitors ,289,290.hibitors ,295,296.hibitors . Many cahe tumor . One wayregs in the context of inflammatory autoimmunity [regs in human inflammatory autoimmunity, as it doses in cancer diseases [The concept of using Fc-based stabilized chemokines for therapy of either cancer or inflammatory autoimmunity is relatively new, and to our best knowledge has not been explored in human clinical trials yet. We first applied this technology thirteen years ago when generating stabilized CXCL12-Ig for therapy of autoimmunity within the CNS . The basimmunity . It is ydiseases . Stabiliregs. The current review focuses on two complementary approaches to possibly overcome this obstacle. The first includes the administration of two chemokines that potentiate the activity of effector CD4+ and CD8+ T cells and the other that selectively deplete CCR8+ regulatory T cells that are likely to be the dominant regulatory T cells within the TME of several human tumors, among them breast cancer. Future clinical trials will show if any of these approaches, or both could be used for cancer immunotherapy either as monotherapy or in combination with clasica ICI.The most successful approach for cancer therapy over the last decade has been the use of monoclonal antibodies (mAb) to immune checkpoint inhibitors (ICI). Yet for many cancers, ICI shows limited success. Several lines of evidence imply that this limited success is mostly associated with attempts to treat patients with “cold tumors” that either lack effector T cells, or in which these cells are markedly suppressed by T


Docking and MD simulations further gave insights into the binding modes of these ODCs with the XO protein. The results indicated that key residues Glu802, Arg880, Asn768, Thr1010, Phe914, and Phe1009 could interact with ODCs by hydrogen bonds, π-π stackings, or hydrophobic interactions, which might be significant for the activity of these XOIs. Four potential hits were virtually screened out using the constructed pharmacophore model in combination with molecular dockings and ADME predictions. The four hits were also found to be relatively stable in the binding pocket by MD simulations. The results in this study might provide effective information for the design and development of novel XOIs.Xanthine oxidase (XO) is an important target for the effective treatment of hyperuricemia-associated diseases. A series of novel 2-substituted 6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (ODCs) as XO inhibitors (XOIs) with remarkable activities have been reported recently. To better understand the key pharmacological characteristics of these XOIs and explore more hit compounds, in the present study, the three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking, pharmacophore modeling, and molecular dynamics (MD) studies were performed on 46 ODCs. The constructed 3D-QSAR models exhibited reliable predictability with satisfactory validation parameters, including q Gout is a clinical syndrome accompanied by some chronic and recurrent symptoms, such as pain, inflammation, and swelling ,2,3,4. SIn the past few decades, XO inhibitors (XOIs) have been widely used in the clinical treatment of HUA. Allopurinol, as a XOI pioneer, was discovered for HUA treatment in the 1960s . HoweverAs shown in To explore the structure–activity relationships (SARs) of these novel ODC XOIs and find more ideal candidates, in this study, 46 ODCs were selected for the integrated modeling studies. Three-dimensional quantitative SAR (3D-QSAR) models, including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), and pharmacophore models, were constructed using these ODCs. Meanwhile, molecular dockings were served to obtain the possible binding conformations of these XOIs and to explore their action mechanism. To explore novel XOI scaffolds, virtual screening was then carried out by pharmacophore model; molecular dockings; and predictions of absorption, distribution, metabolism, and excretion (ADME). Molecular dynamics (MD) simulations were subsequently performed to verify the rationality of the docking method, and to further analyze the effects and stability of hit compounds in the XO binding pocket. This study might provide important information and more alternatives for the design and development of novel XOIs.50s) were given in 2) of 0.897, an optimum number of components (ONC) of 7, a standard error of estimate (SEE) of 0.050, a non-cross-validated correlation coefficient (R2) of 0.983, an F-statistic values (F) of 229.50, and a predictive correlation coefficient (rpred2) of 0.948 in the partial least squares (PLS) analysis. The external validation parameters of the CoMFA model, including a root-mean-squared error (RMSE) of 0.074, a Δrm2 of 0.052, and a A dataset of 46 ODCs was selected for 3D-QSAR modelling, and their structures and biological activities represented by half-maximal inhibitory concentrations , and HBA fields were used to construct different CoMSIA models, and the q50s (−logIC50), predicted pIC50s, and their residuals were listed in 41 and 42, whose predicted activities based on the CoMSIA model were slightly lower than their actual activity. All residual values  were in the reasonable range. The statistic points of other compounds exhibited great linear correlation, indicating that the 3D-QSAR models possessed high quality for the activity prediction of ODCs.The obtained CoMFA and CoMSIA models were then applied to predict the bioactivities of the training and test compounds. The actual pIC44 as a reference molecule were shown in 1 position of compound 44 in both CoMFA and CoMSIA models indicated that bulky substituents at this position might be beneficial to the activity. This was supported by the activity orders as follows: 29 (iso-pentyl) > 28 (iso-butyl) > 27 (iso-propyl) and 4 (iso-pentyl) > 3 (iso-butyl) > 2 (iso-propyl). Another small green contour neared the meta-position of the phenyl ring (R1) in the CoMFA and CoMSIA models, demonstrating that big substituents for structural modification at this place might be favorable for the increment of activity. In the steric contours of the CoMFA model, a large green contour covering the R3 position of the pyrimidine ring highlighted the importance of large groups in this region. The fact that an imino substituent at the R3 position was better for the activity than an oxygen atom could explain this result, as illustrated in the activity orders: 43 (R3 = NH) > 32 (R3 = O) and 44 (R3 = NH) > 31 (R3 = O).The CoMFA and CoMSIA contour maps with the most potent compound 1 position, which were congruent with the following activity order: 5  > 8 (propinyl) and 4 (iso-pentyl) > 7 (iso-pentenyl), attributed to the existence of electropositive substituents. A medium red contour in the CoMFA model and a small red contour in the CoMSIA model appeared over the pata- or meta-position of the phenyl ring (R1), indicating that the occupation of electronegative groups at this region was advantageous for increasing the inhibitory activity. This result was in accordance with the experimental data: 20 (m-fluorobenzyl) > 12 (benzyl) and 36 (p-bromobenzyl) > 37 (p-tert-butylbenzyl). Two small blue contours in the CoMFA model and one small blue contour in the CoMSIA model were located near the pyrimidine ring, which were in accordance with the actual activity orders: 45 (R3 = NH) > 36 (R3 = O) and 46 (R3 = NH) > 38 (R3 = O), as the electropositivity of the oxygen is weaker than that of the nitrogen.The electrostatic maps of the CoMFA and CoMSIA models were shown in 1 position revealed that the substituents of hydrophobic groups in this position tended to increase the activity, such as the following activity orders: 13 (p-methylbenzyl) > 12 (benzyl), 38 (p-methylbenzyl) > 33 (benzyl), and 1 (methyl) > 26 (H). There was a white contour covering the R2 position of the pyrimidine ring, suggesting that the hydrophobic groups in this position might not be beneficial for the activity improvement. For instance, compounds 29, 31, 36, and 38 without substituent at the R2 position exhibited higher activity than the corresponding compounds 39, 40, 41, and 42 with a methyl group, respectively.Regarding the hydrophobic contours of the CoMSIA model a, the yeThe HBD and HBA contour maps of the CoMSIA model are shown in 1 position; (2) negative charged groups at the para- or meta-position of the phenyl ring at the R1 position; (3) hydrophilic groups at the R2 position; (4) bulky and/or positive charged groups at the R3 position; (5) a tetrazole ring or a cyano group at the phenyl ring as HBAs; (6) a carboxyl group of the pyrimidine ring as HBDs or HBAs; and (7) the nitrogen atom of the pyrimidine ring as HBDs.According to the analyses of the 3D-QSAR models, the appropriate substituents for improving the inhibitory activity of these ODC XOIs at specific positions might be concluded as follows: (1) bulky, positive charged, and/or hydrophobic groups at the R44, and 26 was highlighted in To validate the reliability of the docking method and explore the key interactions, the co-crystallized febuxostat was extracted and then redocked into the XO protein (PDB ID: 1N5X). As shown in 44 and the least active compound 26 are shown in 44 (10.69) was much higher than that of compound 26 (7.85), which was in agreement with their experimental activities. In general, we found that the docking score order of these XOIs was accordant with their inhibition potencies. Moreover, it was noted that compounds febuxostat, 44, and 26 have similar docking orientations and binding interactions in the pocket. Similar to febuxostat, compound 44 ) was also used for validating key residues interacted with the ligand febuxostat in XO protein [An online web service Protein Contacts Atlas ), and a portion of data was summarized in VS12 (ZINC71763654), VS16 (ZINC09234838), VS19 (ZINC59369278), and VS26 (ZINC16688904) were obtained through the following criteria: 150 < molecular weight (MW) < 500 g/mol, 20 < topological polar surface area (TPSA) < 130 Å2, high gastrointestinal (GI) absorption, inexistent blood–brain barrier (BBB) permeability, synthetic accessibility (SA) score < 4.5, Lipinski violations = 0, and lower inhibitory characteristics with cytochrome P450 (CYP450) [The best pharmacophore model was converted into a UNITY query for the virtual screening from the ZINC purchasable database. The screened compounds (QFIT > 35) from the pharmacophore were then docked into the XO protein, and 37 compounds (docking score > 10) were obtained. We found that several screened compounds contained a similar indole ring that corresponded to the benzene ring of febuxostat analogues, which might provide new ideas for designing novel XOIs. The ADME properties of screened hits, compound (CYP450) ,29,30,31VS12, VS16, VS19, and VS26 were summarized in VS12, VS16, and VS26 could form similar hydrogen bonds with residues Arg880, Thr1010, and Glu802, which were similar with the docking results of febuxostat and compound 44. In the previous docking results, residues Arg880, Thr1010, and Glu802 have also been proved to be essential for XOI binding. As for compound VS19, it could be observed to bind stably by hydrogen bonds with Arg880, Thr1010, Asn768, and Leu648. Compared with febuxostat and compound 44, the nitrogen-containing heterocyclic rings of compounds VS12, VS16, VS19, and VS26 were also observed to form π-π stacking interactions with Phe914 and Phe1009. These docking results demonstrated that the 4 screened compounds might have the potential to become novel XOIs, which might provide new ideas for designing novel XOI chemotypes.The chemical structures and docking scores of compounds 44, VS12, VS16, VS19, and VS26 were subjected to MD simulations based on the repaired XO protein. To further analyze the stability of these protein–ligand complexes, the time-dependent behavior of complexes XO-VS12, XO-VS16, XO-VS19, and XO-VS26 in the 50 ns simulation trajectories were analyzed using XO-febuxostat and XO-44 as the comparisons, and their results were shown in The best docking conformations of compounds febuxostat, 44, VS12, VS16, VS19, and VS26) with low RMSD values could become stable during MD simulations. Compared to febuxostat, the RMSD results manifested that compounds VS12, VS16 and VS26 might be more favorable for binding to XO.The RMSD values that could reflect the differences between the initial and current conformations were suited to evaluate the convergence and stability of the systems . The comThe root-mean-square fluctuation (RMSF) values c were us44, XO-VS12, XO-VS16, XO-VS19, and XO-VS26 fluctuated within 0–4, 0–6, 0–7, 0–4, 0–5, and 0–4, respectively, during the MD simulations. Furthermore, the hydrogen-bond numbers in complexes XO-febuxostat, XO-44, XO-VS12, XO-VS16, XO-VS19, and XO-VS26 maintained in 2, 3, 3, 2, 1, and 1, respectively. The above results suggested that compound VS12 might bind tightly with XO by more hydrogen bonds than the other virtual-screened compounds.The hydrogen-bond number were carried out to assess the stability of the complexes from another perspective . Figure 44, VS12, VS16, VS19, and VS26 in the XO protein were −97.04, −95.30, −88.31, −159.71, −95.91, and −150.84 kJ/mol, respectively. These results indicated that the binding strengths of compounds VS16 and VS26 might be stronger than those of compounds febuxostat and 44 [VS16 and VS26 with XO were more favorable due to their higher van der Waals interactions . In addition, VS16 exhibited relatively low polar solvation energy (ΔGPB: 109.67 kJ/mol), and VS26 showed relatively good electrostatic interaction (ΔEele: −40.34 kJ/mol), respectively. As for all 6 systems, the ΔEvdw played an important role in the ΔGbinding, the contribution of ΔEele was nearly counteracted by the ΔGPB, and the values of SASA energy (ΔGSA) in each complex were semblable. These results indicated that the nonpolar energy might be the main driving force for the binding of these XOIs. To understand the stability of complexes, the binding energies obtained during the MD simulations versus time were shown in VS12, VS16, VS19, and VS26 in the XO binding pocket were verified. The above analyses proved that the screened compounds might have powerful potential to be XOI hits.The binding free energy was calculated by the Molecular Mechanics-Poisson Bolzmann Surface Area (MM-PBSA) method, and the corresponding values are listed in t and 44 . It was All calculations and simulations were performed using SYBYL-X 2.1 software  running on Windows 7 workstations, unless otherwise specified. A dataset of 46 ODCs  was sele3 carbon probe (default probe atom) [44 with the highest potency was selected as a template, and the other molecules were superimposed onto the common skeleton (colored as magenta) by automatic alignment and manual adjustment. The alignment results of all ODCs were shown in The CoMFA and CoMSIA models were used to help us to better understand the relationship between the characteristics of molecular structures and their activities. SYBYL-X 2.1 software was used for the CoMFA and CoMSIA models. Different physicochemical properties used for the CoMFA and CoMSIA models are calculated by the same lattice boxes with the same spbe atom) . Thereinbe atom) . In CoMSbe atom) . Besidesbe atom) . Forty-sbe atom) . In this2, ONC, R2, F, and SEE, were calculated to assess the predictive ability of models [02, r0′2, k, k’, rm2, r’m2, rpred2, Δrm2, 02  and r0′2  are the correlation coefficients of regression lines with a zero intercept, and k  and k’  are the slopes of regression lines, respectively. rm2, rpred2, and RMSE are calculated according to the following Equations (1)–(3), respectively.PLS regression analysis method was performed for establishing the 3D-QSAR models. The first step detecting the reproducibility and robustness of the CoMFA and CoMSIA models was the internal validation. In the PLS analysis, a series of parameters, including qf models . To furtderation . r02 (pr(test), ypred(test), and 50 value of each test set compound, the predicted pIC50 value of each test set compound, and the mean pIC50 value of the training set compounds, respectively [2 > 0.5, R2 > 0.8, m2 < 0.2, pred2 > 0.6 [The yectively . An apprd2 > 0.6 .Bos taurus) and human (Homo sapiens) XO with approximately 90% sequence identity was shown in http://www.mrc-lmb.cam.ac.uk/pca/ (accessed on September 2020)) was used to explore the non-covalent contacts between ligand and protein [44, and the least active compound 26 were used for further analyses. The docking visualization was completed by PyMOL software .Molecular docking served as a helpful tool to obtain the reasonable binding conformations of bioactive molecules and to identify core residues in the active site of target protein. The crystal structure of bovine XO protein PDB ID: 1N5X), a very close homologue of human XO enzyme, was used for molecular docking by the surflex-docking package of SYBYL-X 2.1 with default parameters [N5X, a ve protein . After t protein . The RMS protein . Finallyhttp://dud.docking.org/r2/ (accessed on September 2019)) and 35 active ODCs ) [http://www.swissadme.ch (accessed on December 2019)) [A multi-stage virtual screening was carried out against the purchasable ZINC15 database (r 2019))  by the cr 2019)) . The comr 2019)) . In the r 2019)) . Among tr 2019)) ,29,30,31complex, Gfree-protein, and Gfree-ligand represent the free energy of protein–ligand complex, protein, and ligand, respectively [To further verify the stability of the docked hits compounds in the XO protein, 50 ns MD simulations were performed on different complexes using GROMACS 2016.5 software . The overall XO protein was a homodimer, in which one of subunit was selected for simulations. Residue deficiencies in 166–191 and 532–536 (loop region) were repaired by the protein loop search module of SYBYL-X 2.1 software, and the residues 1326–1332 were reasonably removed to solve the problem of the residues 1317–1325 deficiencies in the subunit terminal. The repaired models with good fit and high homology were further evaluated using PROCHECK and ProSa servers. Compared with the evaluation results of the original and optimal repaired proteins , the resVS12, VS16, VS19, and VS26) with ideal compatibility for the pharmacophore model, relatively high docking scores, and good ADME characteristics were retrieved by the multi-round virtual screening. The MD simulations also indicated hits VS12, VS16, VS19, and VS26 could bind well with the XO protein during the dynamic environment. We expect that these results will be helpful for the design and development of novel XOIs, and the screened compounds could provide more alternatives and ideas for XOIs.In this work, an integrated computational study, including 3D-QSAR models, molecular dockings, pharmacophore models, and MD simulations, was performed on 46 novel ODC XOIs. 3D-QSAR models with good statistical parameters were constructed to provide significant insights into the SARs of these ODCs. Molecular docking results indicated that residues Glu802, Arg880, Thr1010, and Asn768 could form hydrogen bonds with these XOIs, and residues Phe914 and Phe1009 could also form π-π interactions with them. These interactions might be essential for their affinity with the XO protein. The best pharmacophore model with eight features was inconsistent with the 3D-QSAR and docking results. Four hit compounds (