In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO3+) or nodulated by Frankia (NOD+) were exposed to a gradient of salt concentrations  and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD+) to 314 (600 mM NaCl/KNO3+), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species.Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N Climate change is unequivocally driving major environmental struggles associated with greenhouse gas (GHG) emissions and their impact on global warming ,2 and seHigh salinity is strongly associated with ionic and osmotic stresses , which d2-fixing Frankia bacteria, which promotes biomass accumulation and soil fertility [Actinorhizal plants are a group of perennial dicotyledonous angiosperms covering three orders , eight families, and 25 genera . This grertility ,18. TherCasuarina glauca Sieb. ex Spreng. (family Casuarinaceae), a fast-growing tropical tree originating in the southeastern coastal regions of Australia, is considered the model species [C. glauca well suited for basic research. In addition, the biology of its root-nodule symbiosis with Frankia has been studied extensively [C. glauca has an outstanding capacity to endure extreme environments, particularly saline conditions, as it tolerates seawater levels of salt and is commonly planted in areas with a shallow, saline water table [Frankia. Such studies revealed that C. glauca withstands up to 400 mM NaCl with remarkably low tissue dehydration, associated with significant osmotic adjustments that minimize the impact of salt on cell water content and the photosynthetic machinery [C. glauca to endure high NaCl concentrations might be coupled or not to the presence of symbiotic Frankia [Within the actinorhizal group,  species . Its smaensively . Moreoveer table ,21,22,23achinery , osmolytachinery ,26, and achinery . Moreove Frankia ,28,29,30The availability of high-throughput techniques, with a high level of precision and at increasingly affordable prices is currently one of the most straightforward strategies to understand molecular changes of plants in response to salinity at the system level. Among them, RNA-Seq is the main choice for studying phenotypic variations, through the detection of differentially expressed genes (DEGs) between specific environmental conditions . AdditioC. glauca (branchlets), one of the first stress targets, to unravel the molecular mechanisms underlying stress tolerance, in complement to previous studies in this species, thus contributing to halophyte research. For that, C. glauca plants either supplied with chemical nitrogen (KNO3+) or nodulated by Frankia (NOD+) were exposed to a gradient of salt concentrations  and RNA-Seq was performed.In the present work, we have analyzed the transcriptome of the photosynthetic organs of + plants and between 24.5 and 27.4 million clean reads (from 33.5 to 38.7 million raw reads) in KNO3+ plants  in NOD+ plants . Minimum+ plants . More th+ plants  with alm+ plants .3+ plants (thereafter referred as 200- KNO3+); (ii) control and 200 mM NaCl NOD+ plants (thereafter referred as 200- NOD+); (iii) 400 and 600 mM NaCl KNO3+ plants (thereafter referred as 400- and 600- KNO3+); and (iv) 400 and 600 mM NaCl NOD+ plants (thereafter referred as 400- and 600- NOD+). PC1 accounted for 86% of the total variance, with a clear distinction between the control and 200 mM NaCl samples from the 400 and 600 mM NaCl samples. PC2 (9% variance) distinguished KNO3+ from NOD+ plants  showed a clear separation of samples in four different quadrants: (i) control (0 mM NaCl) and 200 mM NaCl KNO+ plants .3+ plants expressed a total of 19,913 genes in the control and an average of 19,645 genes in samples exposed to salinity  DEGs were downregulated and ca. 13%  upregulated  increased with increasing salinity A. All DEsalinity . From thegulated .+ plants expressed a higher number of genes compared to KNO3+ plants, with a total of 20,278 genes at 0 mM NaCl and an average of 19,780 genes in samples exposed to salinity , 0.5% (104), and 1.3% (254), respectively in 200, 400, and 600 mMNOD+. At 200 mM NaCl, the number of up-and down-regulated DEGs was similar. At 400 and 600 mM NaCl, the percentage of downregulated vs. upregulated DEGs was 60% (62) vs. 40% (42), and 85% (217) vs. 15% (37), respectively.NODsalinity . From th3+ plants, the number of treatment-specific downregulated DEGs was 1 (0.3%), 69 (18%), and 175 (45%) at 200, 400, and 600 mM NaCl, respectively. Additionally, two (0.5%) DEGs were commonly found in 200- and 400 KNO3+, six (2%) in all set of conditions, and 133 (35%) in 400- and 600 KNO3+. Together, the two highest salt concentrations accounted for ca. 98% (175 + 133 + 69) of all downregulated DEGs. Among the upregulated DEGs, 11 (18%) were activated in both 400- and 600- KNO3+, 17 (27%) were specific to 400 mM NaCl and 34 (55%) were specific to 600 mM NaCl , 62 (400 mM), and 218 (600 mM). Among these, 27 (11%) were specifically found in 400 NOD+, and 183 (75%) in 600 NOD+ (+. Only 1 (0.4%) DEG was commonly found in 200- and 400 NOD+, 2 (0.8%) in all sets of conditions, and 32 (13%) in 400- and 600 NOD+. Together, the number of specific DEGs in 400- and 600- NOD+ accounted for 99% (27 + 32 + 183) of the downregulated pool. Among the upregulated DEGs, only 1 (1.5%) was found in 200 NOD+, 30 (44%) in 400 NOD+, and 25 (37%) in 600 NOD+  of the treatment-specific DEGs.The number of DEGs specific to only one salt concentration , increased with the salinity in both plant groups B. In KNO mM NaCl B. In NOD600 NOD+ C. Specif600 NOD+ D. The tr3+ and NOD+ plants, no common DEGs were observed in samples exposed to 200 mM NaCl  vs. 72 (23%), and 224 (47%) vs. 119 (25%), at 200, 400, and 600 mM NaCl, respectively  DEGs were uniquely mapped to UniProtKB/Swiss-Prot database proteins, two of which corresponded to yet uncharacterized proteins , related to stress signal transduction pathways, and GT-3B (log2FC: −3.81), a salt responsive transcription factor. In 400 KNO3+ the top responsive DEGs were WAXY (log2FC: 3.46) and XCP1 (log2FC: −6.88), respectively. The first is associated with starch biosynthesis, while the second is related to programmed cell death and proteolysis. In 600- KNO3+, XCP1 (log2FC: −6.88) was also the most downregulated DEG, and PLT6, which is related to glucose import, the most upregulated one (log2FC: 4.55). The top 10 most down- or upregulated DEGs for KNO3+ plants are summarized in In KNOproteins . Consideproteins . In 200 3+ plants exposed to 600 mM NaCl included an association with response to hypoxia (GO:0001666), DNA (GO:0003677), carbohydrate metabolism (GO:0030246), metal ion- (GO:0046872) and mannose binding (GO:0005537), leaf senescense (GO:0010150), cell death (GO:0008219), cell wall organization biogenesis (GO:0071554), and iron homeostasis (GO:0055072) and -transport (GO:0006810). The top upregulated DEGs were related to the biosynthesis of (−)-secologanin, the precursor of monoterpene indole alkaloids (GO:1900994), abscisic acid-activated signaling (GO:00097389), alkaloid metabolism (GO:0009820), plant-type hypersensitive response (GO:0009626), regulation of stomatal opening (GO:1902456), glucose import (GO:0046323), UDP-glycosyltransferase (GO:0008194), antiporter activities (GO:0015297), chitin catabolism (GO:0006032), and chaperone cofactor-dependent protein refolding (GO:0051085)  .+ plants, 43% (136 out of 313) DEGs were uniquely mapped to known proteins, four of which uncharacterized  which is involved in PAMP-triggered immunity (PTI) signaling, followed by GDH2 (log2FC: 3.32) that catalyzes the production of GABA, and CYP78A5 (log2FC: 2.89) required for the promotion of leaf and floral growth and the prolongation of the plastochron. The only downregulated gene was GLPR2.8 (log2FC: −3.25), involved in light signal transduction and calcium homeostasis via the regulation of calcium influx into cells. In 400 NOD+, the most responsive DEGs were GDH2 (log2FC: 5.09) encoding a glutamate dehydrogenase, and RL5 (log2FC: −7.79) related to DNA binding. In 600 NOD+, the most up- or downregulated DEGs were PEPR1 (log2FC: 4.59) encoding a Leucine-rich repeat receptor-like kinase involved in PTI signaling, and RL5 (log2FC: −7.79), respectively. The top 10 most responsive DEGs in the NOD+ group are summarized in In NODcterized . Consideectively . Among t+, one including the 0 mM and the 200 mM NaCl groups, and the other including the 400 mM and 600 mM NaCl groups. However, unlike the situation in KNO3+ plants, a clear separation of control and 200- NOD+ was observed in the first cluster. Similar to KNO3+ plants, the top downregulated DEGs in NOD+ plants exposed to 600 mM NaCl were associated with response to hypoxia, DNA- and mannose binding, cell death and cell wall biogenesis and organization , alkaloid metabolism (GO:0009820) and regulation of stomatal opening (GO:1902456)  . Additio1902456) .RL5 and FLA12, both related to plant-type secondary cell wall biogenesis. Among the top upregulated DEGs, only the sulphur deficiency induced gene SDI1 was common to both groups. At 600 mM NaCl, half of the top downregulated DEGs were common to both KNO3+ and NOD+, namely RL5, Lectin, LAC22, LECRK91, and LRK10L-1.4. These genes were respectively involved in the following biological processes: DNA binding, iron ion homeostasis and transport, lignin catabolic process, defense response to bacterium and oomycetes, positive regulation of cell death, positive regulation of hydrogen peroxide metabolic process, cellular response to hypoxia and phosphorylation. Regarding the top upregulated DEGs, only two were common to KNO3+ and NOD+ plants, namely DTX56, associated with the cellular response to carbon dioxide and regulation of stomatal opening, and HSP70, involved in cellular response to unfolded protein and chaperone cofactor-dependent protein refolding. Overall, differential expression values, estimated by log2FC, were always higher in downregulated than in upregulated DEGs in both plant series.Only two out of the top 10 most downregulated DEGs in samples exposed to 400 mM NaCl were found in both plant groups, namely 3+ plants, Clust tool showed 10 different clusters , cell wall organization (GO:0071555), external encapsulating structure organization (GO:0045229), defense against pathogenic bacteria (GO:0050829), polysaccharide biosynthetic process (GO:0000271), leaf abscission (GO:0060866), cell periphery (GO:0071944), and anchored component of plasma membrane (GO:0046658). In NOD+ plants enriched DEGs corresponded to glutamate dehydrogenase (NAD+) (GO:0004352), terpenoid catabolic process (GO:0016115), and cell periphery (GO:0005886) , ATP binding (GO:0005524), purine ribonucleoside triphosphate binding (GO:0035639), adenyl ribonucleotide binding (GO:0032559) and drug binding (GO:0008144), protein phosphorylation (GO:0006468) and defense response, incompatible interaction (GO:0009814). In KNO3+ plants, DEGs were also enriched in carbohydrate derivative binding (GO:0097367) and abscission (GO:0009838). In NOD+ plants, there was an enrichment in all-trans-beta-apo-10’-carotenal cleavage oxygenase transcription (GO:0102251), 9-cis-10’-apo-beta-carotenal cleavage oxygenase activity (GO:0102396), phospholipase transcription (GO:0004620), purine ribonucleotide binding (GO:0032555), as well as processes involving the interaction between organisms (GO:0051704), response to hypoxia (GO:0001666), defense response to bacteria (GO:0042742), positive regulation of defense responses (GO:0031349), lipid catabolic process (GO:0016042), leaf abscission (GO:0060866), and cell death (GO:0008219)  performed in gProfiler for both plants, found a few sets of enriched GO terms including DEGs in samples exposed to 400- and 600 mM NaCl with FDR < 0.05. Due to their low number, no enriched GO terms were found for DEGs in samples exposed to 200 mM NaCl. In 400 KNO0005886) . In 600-0008219) .3+ plants were enriched for the transcription of genes involved in the following pathways: glycosyltransferase family 8, GIY-YIG catalytic domain/nuclease superfamily, UDP-glucoronosyl and UDP-glucosyl transferase, KOW motif, and the unkown function domain DUF2828. Furthermore, a set of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was also enriched for this condition, namely cyanoamino acid metabolism, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism. NOD+ plants were enriched in transcripts of AAA-type ATPase, chalcone/stillbene synthase, flavinoxireductase/NADH oxidase, glutamate/phenylalanine/leucine/valine dehydrogenase, and phosphatase 2C family. All enriched pathways are listed in Through ShinyGO, enriched DEGs were observed only at 600 mM NaCl in both plant groups. In both InterPro and Pfam databases, KNO3+ plants, three DEGs associated with enriched biological processes were mapped in the PPI network, namely SAG101, CRT3 and TAO1 . A significant positive correlation between % GC and the frequency of optimal codons (Fop) was found (r = 0.57), whereas a significant negative correlation was obtained between GC3 and the effective number of codons (Nc) (r = −0.62). On the contrary, the upregulated genes in KNO3+ plants showed different features. In this group, GC was 50.02% with 51.03% GC3. Moreover, statistical analyses between GC and Fop along with GC3 and Nc did note demonstrate statistical significance. This indicated distinct genetic volatility among the upregulated genes present in KNO3+ plants. Moreover, COG analysis revealed “cellular processing and signaling” as a major functional category followed by “information storage and processing” and “metabolism” in NOD+ plants  and nodulated (NOD+) C. glauca plants. RNA-Seq analysis yielded an average of ca. 25 and ca. 26 million clean reads for KNO3+ and NOD+ plants, respectively, corresponding to 86,202 unigenes with a N50 size of 2792 bp and 41% GC content. These values are within the range of those reported in similar studies in Casuarina, which vary according to the species, environmental condition, and plant organ. For example, in Casuarina equisetifolia seedlings exposed to cold, ca. 21 million reads corresponding to ca. 118,000 unigenes and a N50 value of 2827 bp have been reported [C. equisetifolia roots from plants exposed to salt, these values were ca. 75–95 million clean reads comprising ca. 53,000–72,000 unigenes [C. glauca exposed to salt. These results, together with the high mapping coverage of our data  indicates that a high-quality transcriptome assembly has been generated for downstream analyses.The impact of salt stress in me scale . Altogetreported . Developreported . On the unigenes . Finallyunigenes  reported3+ plants, and 20,278 in the control and an average of 19,780 genes in NaCl-treated NOD+ plants), the percentage of significant DEGs was remarkably low, ranging from 6 (200 NOD+) to 314 (600 KNO3+), mostly downregulated  clearly separate two groups, (i) control and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants . With a egulated ; Table 1ectively . Neverthven days  and >400ven days . Howevere system .Frankia is residual [A minor proportion of DEGs was common to both plant groups, in response to 400 (10%) and 600 mM NaCl (28%) , while nresidual  may explresidual .C. glauca branchlets’ proteome [C. glauca has a strong stress-responsive system with an extensive set of constitutive defense mechanisms [3+) and abscisic acid (ABA)-activated signaling ; Myb transcription factors and jasmonic acid signaling (400 NOD+), regulation of sulphur utilization, transporter activity, and protein refolding , and response to cellular CO2/regulation of stomatal opening . It is widely known that the mechanisms used by plants to cope with salt stress are highly dependent on a coordinated set of events that regulates growth, metabolism, and homeostasis [C. equisetifolia [Spartina alterniflora [Puccinellia nuttalliana [Considering the two key stress concentrations, i.e., 400 and 600 mM NaCl, the stress-responsive genes were in general related to regulatory processes in both plant groups. The results are rather different from those reported for the proteome , and metproteome ,26,27, wproteome ,25,26,27chanisms  complemechanisms ,40. Despeostasis ,44,45. Ietifolia , Spartinrniflora , and Puctalliana , transcrArabidopsis and Medicago in response to various abiotic stresses [C. glauca [C. glauca responses to high salinity through the regulation of signal transduction pathways and associated mechanisms leading to stress tolerance [The top up-regulated genes common to both plant groups were the sulphur deficiency induced gene, SDI1, the gene encoding protein detoxification 56, DTX56, and the gene encoding the 70 KDa heat shock protein, HSP70 . The accstresses  and thatstresses . Given t. glauca ,30, an u. glauca ,54,55, aolerance . Finallyolerance , droughtolerance , and heaolerance .+ plants, glutamate dehydrogenase (GDH) 2 is upregulated under all three salinity levels tested , ion homeostasis, and photosynthetic functioning [+), a polygalacturonase (400 KNO3+), an arabinogalactan (400 NOD+ and 400 KNO3+), a cellulose synthase (600 KNO3+), a laccase, (600 NOD+ and 600 KNO3+), and a regulator of cell expansion, cobra-like protein (600 NOD+).One of the mechanisms used by ctioning . In thisctioning , or cystctioning , is not ctioning ,71. Acco+ and KNO3+ plants (400 and 600 mM NaCl). These TFs form a small family in Arabidopsis [Ricinus communis) by Han et al. [As mentioned above, the expression of several genes encoding TFs putatively involved in signal transduction was differentially regulated under salt stress. The striking effect here, however, was the consistent downregulation of RADIALIS-LIKE SANT/MYB TFs (RADIALIS-LIKE 3 and RADIALIS-LIKE5) in both NODbidopsis , and onebidopsis . The funn et al. , who sug3+) and 11 (NOD+) gene clusters . Despite the differences between the two plant groups, likely related to the nitrogen source [C. glauca is able to balance the transcriptional activity at 200 mM NaCl to levels similar to that of the control  medium under the conditions previously described by Zhong et al. [Frankia casuarinae Thr (NOD+) or supplemented with mineral nitrogen (KNO3+) and maintained in a walk-in growth chamber  under environmental controlled conditions of temperature (26/22 °C), photoperiod (12 h), relative humidity (70%), external CO2 levels (380 μL L−1), and irradiance (ca. 500 μmol m−2s−1). Salt stress was gradually imposed through the addition of 50 mM NaCl per week to the nutrient solution until concentrations of 200, 400 and 600 mM were obtained, respectively. Control plants were supplemented only with mineral nutrients without the addition of NaCl. In all cases, the nutrient solution was renewed twice per week. To ensure the same age of all plants at the time of sample collection, i.e., one week after the exposure to each NaCl concentration, stress implementation was imposed sequentially from the highest to the lowest salt condition, i.e., the 600 mM NaCl group was treated first, followed by the 400 mM group (when the 600 mM group surpassed 200 mM NaCl), and the 200 mM group (when the 400 mM group surpassed 200 mM NaCl). Branchlets from four plants per treatment were harvested, frozen immediately in liquid nitrogen, and stored at –80 ºC until RNA extraction.g et al.  and Tromg et al. . Six-mon+ and KNO3+ plants exposed to the three stress levels, as well as from control plants was extracted using the GeneJet Plant RNA Purification Kit  and digested with dsDNAse  to remove genomic DNA, as per manufacturer’s instructions. The quality of the RNA samples was verified by electrophoresis in 1% agarose–Tris acetate EDTA buffer containing GelRed Nucleic Acid Gel Stain , by evaluating the integrity of the 28S and 18S ribosomal RNA bands and absence of smears, as well as with a bioanalyzer , by determining the RNA integrity number (RIN > 8.5). Libraries were prepared with the TruSeq RNA Sample Prep Kit v2  and sequenced on an Illumina HiSeq 2000 platform  at Macrogen . Raw data is available in NCBI Sequence Read Archive (SRA) BioProject SymbSaltStress under accession PRJNA706159 .Total RNA from branchlets of NODThe sequenced raw reads were assessed for integrity, quality and contamination through the application of the following tools: FastQC version 0.11.8 to analyze reads quality  and FastTo identify differentially expressed genes (DEGs), a set of R packages were applied, namely edgeR version 3.26.8 , DESeq v3+ and NOD+ plants, respectively. The results were adjusted with the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR) [To study the effect of salinity, these three tools were used to identify DEGs at each salinity level  compared to the control (0 mM NaCl), for KNOte (FDR) . A filtete (FDR) .The Clust version 1.8.10 command-line tool was applied to visualize the expression patterns of the detected DEGs and to find co-expressed genes . This isThe Basic Local Alignment Search Tool (BLAST) version 2.9.0 command line application from the NCBI C++ Toolkit  was usedAn over-representation analysis (ORA) was implemented by g:GOSt functional profiling tool from gProfiler website, which was applied using g:SCS tailored algorithm that uses a minimum hypergeometric test (Fisher’s exact test). ShinyGO version 0.61 webtool, which is also based on hypergeometric distribution followed by FDR correction was used to retrieve Protein-Protein Interactions (PPIs) from the STRING database . In bothhttp://www.ncbi.nlm.nih.gov/COG, accessed on 1 September 2022). A detailed study on biological networks maintained by DEGs was performed.The downstream analysis included the codon and amino acid usage analysis of DEGs. Compositional constrain (GC and GC3), effective number of codons (ENc), and frequency of optimal codons (Fop) were determined using CodonW software. Biosynthetic energy costs of DEGs were obtained through DAMBE software. The Cluster of Orthologue analysis (COG) was performed using an in-house python-based program where information regarding different COG families was taken from the COG database (


Piezo1-regulated bone-related diseases and to explain the potential mechanisms using bioinformatic analysis. The differentially expressed genes (DEGs) in tendon, femur, and humerus bone tissue; cortical bone; and bone-marrow-derived macrophages were identified with the criteria of |log2FC| > 1 and adjusted p-value < 0.05 analysis based on a dataset from GSE169261, GSE139121, GSE135282, and GSE133069, respectively, and visualized in a volcano plot. Venn diagram analyses were performed to identify the overlapping DEGs expressed in the above-mentioned tissues. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, protein–protein interaction (PPI) analysis, and module analysis were also conducted. Furthermore, qRT-PCR was performed to validate the above results using primary chondrocytes. As a result, a total of 222 overlapping DEGs and 12 mostly overlapping DEGs were identified. Key Piezo1-related genes, such as Lcn2, Dkk3, Obscn, and Tnnt1, were identified, and pathways, such as Wnt/β-catenin and PI3k-Akt, were also identified. The present informatic study provides insight, for the first time, into the potential therapeutic targets of Piezo1-regulated bone-related diseasesPIEZO1 is a mechano-sensitive ion channel that can sense various forms of mechanical stimuli and convert them into biological signals, affecting bone-related diseases. The present study aimed to identify key genes and signaling pathways in  Mechano-sensitive ion channels are a type of ion channel closely related to biomechanical signal transmission. Once discovered, they attracted widespread attention from scholars all over the world . Under aPiezo1 in different bone tissues.The load borne by knee cartilage in daily activities is about three-times the body weight. Chondrocytes perceive and respond to mechanical load stimulation to maintain a steady state in the cartilage . The excIl11 and Chadl were identified as robust OA risk gens and attractive therapeutic targets via RNA sequencing in subchondral bone and cartilage [En1 was determined as the key gene of bone density and fracture using whole-genome sequencing and verified by an En1 conditionally knocked-out mouse [Piezo1 in tendon, femur, and humerus bone tissue, cortical bone, and bone-marrow-derived macrophages and identified the key genes and signal pathways regulated by Piezo1. Furthermore, functional enrichment analysis and protein–protein interaction (PPI) analysis was performed. The most overlapping DEGs were further verified by qRT-PCR in primary chondrocytes of mice. This study may reveal the potential mechanism of Piezo1 in bone-related diseases, suggesting its upstream and downstream therapeutic targets and providing evidence for the further development of effective treatment for bone-related diseases.With the rapid development of high-throughput technologies, such as microarray, second-generation sequencing, and single-cell sequencing, bioinformatics analysis has been widely applied to identify key genes and signal pathways in various diseases to provide accurate diagnosis and personalized treatment. For instance, the OA miRNA interactome and its related pathways were elucidated by performing RNA sequencing and differential expression analysis of miRNA and mRNAs in lesioned OA cartilage in humans by comparing it with preserved cartilage from the same patient . Il11 anartilage . En1 wasut mouse . The Cirut mouse . Howeverut mouse ,21. TherPiezo1-mutant tendon samples were retrieved from a mouse dataset obtained using the Illumina NextSeq 500 platform (GSE169261). Two wild-type femur and humerus bone tissue samples and two Piezo1-knock-out bone tissue samples were retrieved from a mouse dataset obtained with the Ion Torrent S5 platform (GSE139121). Four cortical bone samples of wild-type mice and four cortical bone samples of Piezo1-knock-out mice were retrieved from a mouse dataset obtained with the Illumina HiSeq 2500 platform (GSE135282). Five wild-type bone-marrow-derived macrophages samples and six Piezo1-knock-out bone-marrow-derived macrophages samples were retrieved from a mouse dataset obtained with the Illumina HiSeq 2500 platform (GSE133069).The gene expression profiling in tendon, femur and humerus bone tissue, cortical bone, and bone-marrow-derived macrophages was obtained from GEO datasets GSE169261, GSE139121 , GSE1352https://www.ebi.ac.uk/, data set GSE169261 was accessed on 20 September 2021; data set GSE139121 was accessed on 6 April 2020; data set GSE135282 was accessed on 7 December 2019; data set GSE133069 was accessed on 21 August 2019) and assessed to detect sequencing failures using FastQC (V 0.11.9), and lower-quality reads were filtered or trimmed using trimmomatic (V 0.39). HISAT2 (V 2.2.1) was applied to map sequencing reads to a population of mice genomes based on the Hierarchical Graph FM index (HGFM) [The raw fastq data were obtained from the EBI database (x (HGFM) . SequencPiezo1 mutant tissues, respectively. The criteria for a DEG were |log2FC| > 1 and adjusted p-value < 0.05. The results were visualized in volcano plots.The original gene expression profiles were analyzed to identify the upregulated or downregulated DEGs in Piezo1 mutant tissues.A Venn diagram was used to identify the most overlapping DEGs in different tissues including tendon, femur and humerus, cortical bone, and bone-marrow-derived macrophages. Overlapping DEGs were identified when they appeared in at least two of the tissues. All the DEGs were identified by comparing the gene expression profiles between wild-type tissues and p-value < 0.05.GO enrichment analysis and KEGG pathway analysis were performed using the R package clusterProfiler (V 4.0.5) with overlapping DEGs. GO enrichment analysis and KEGG pathway analysis were performed with the thresholds of a https://string-db.org/, accessed on 30 March 2022) was used to construct the PPI network [The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database  plugin on the Cytoscape (V 3.9.0) platform. The parameters set to identify enriched functional modules were as follows: Degree Cutoff = 2, Node Score Cutoff = 0.2, K-Core = 2 and Maximum. Depth = 100. Modules with the MCODE score ≥4 were identified as significant modules and were further evaluated for GO enrichment analysis and KEGG pathway analysis with the threshold of a Piezo1. The total RNA was extracted from the cells using TRIzol reagent (Invitrogen). cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (TaKaRa). qRT-PCR was conducted to amplify cDNA using an SYBR Premix Ex Tag Kit (TaKaRa) and a 7500 Real-time detection system . The primers used in this work were designed by Sangon Biotech Co., Ltd. , and the sequences of these primers are listed in Gapdh and presented in a heatmap after normalization (log10 transformation).To verify the expression of key genes from the forementioned analysis, mice primary chondrocytes were isolated and cultured . Yoda 1 For the GSE169261 dataset, a total of 766 genes was identified by DESeq2 (V1.32.0), of which 311 were upregulated and 455 were downregulated. For the GSE139121 dataset, 241 genes were identified by DESeq2 (V1.32.0), of which 136 were upregulated and 105 were downregulated. For the GSE135282 dataset, 311 genes were identified by DESeq2(V1.32.0), of which 60 were upregulated and 251 were downregulated. For the GSE133069 dataset, 1653 genes were identified by DESeq2(V1.32.0), of which 767 were upregulated and 886 were downregulated. The distribution of the gene expression for each dataset is visualized in the corresponding volcano plot A–D.Lcn2, Creb3l1, Dkk3, Clec5a, Tspan32, Mt1, and Hpgd were differently expressed in bone-marrow-derived macrophages, femur and humerus, and tendon. Fxyd2 and Tnnt1 were differently expressed in bone-marrow-derived macrophages, cortical bone, and tendon. Cox8b, Art1, and Obscn were differently expressed in femur and humerus, cortical bone, and tendon.As shown in the Venn diagrams, 222 overlapping DEGs were identified , of whicThe GO enrichment analysis results are presented in KEGG pathway analysis results are shown in Ttn, Acta 1, Tcap, Myoz2, My12, Obscn, My13, Srl, Smpx, and Trdn.A total of 578 interactions were obtained with interaction scores > 0.4 using the STRING database. The PPI network was then constructed and presented with the Cytoscape (V.3.9.0) platform . In addiA total of seven modules, including three significant modules , were obtained through MCODE analysis . Among tPiezo1 in chondrocytes, Quantitative Real-time PCR assay (qRT-PCR) was performed to evaluate the relative expression of the putative Piezo1-related key genes that were identified in the aforementioned results in mice primary chondrocytes. Yoda 1 is a specific agonist of PIEZO1, which affects the sensitivity and inactivation kinetics of mechanically induced responses [Piezo1 was remarkably overexpressed in Yoda-1-treated chondrocytes. Significantly upregulated Creb 311 and Mt 1 was observed in Piezo1-overexpressed chondrocytes. In addition, Dkk, Icn 2, Clec 5a, Hpgd, and Tnnt were downregulated in Piezo1-overexpressed chondrocytes  is a key regulator of synovial injury and bone erosion during autoimmune joint inflammation. The activation of CLEC5A can increase the recruitment of inflammatory macrophages to the joint, promote bone erosion, and enhance the expression of pro-inflammatory cytokines and chemokines in myeloid cells. Moreover, clinical symptoms of autoimmune joint inflammation can be reduced by blocking CLEC5A receptors, suggesting that CLEC5A receptors may be a promising therapeutic target for immune-mediated bone disorders ,34.The Wnt/β-catenin signaling pathway plays an important role in inflammatory regulation and cell fate determination, which were studied comprehensively ,36,37. ACREB3L1 is a transcription factor that activates genes involved in the assembly of the collagen-containing extracellular matrix . CREB3L1Tnnt1 and Piezo1 may be closely related, playing a key role in maintaining bone balance, which deserves deep research.Tnnt1 is a protein associated with troponin that is critical for the generation of the mechanical forces required for muscle contraction, and loss of Tnnt1 leads to skeletal muscle atrophy . Thus, wPiezo1-upregulated chondrocytes in this project, Mt1 was also remarkably upregulated, which shows a consistent function in osteoporosis and osteosclerosis. In 2008, HPGD was first discovered to be able to lead to autosomal recessive primary hypertrophic osteoarthropathy, which reduces its metabolite levels by causing elevated levels of prostaglandin E2 (PGE2) [Mt1 acts as a melatonin receptor and binds to melatonin to prevent oxidative stress, inflammation, and mitochondrial dysfunction to play an anti-aging role. The expression of melatonin and its receptors decreases with age, which can lead to osteoporosis . From th2 (PGE2) ,52. HowePiezo1.According to KEGG pathway analysis based on overlapping DEGs, apart from cardiac muscle and cardiovascular diseases, general biological function, and malaria, pathways enriched in mineral absorption were shown to play key roles in several bone-related diseases, such as bone loss  and osteTnnt1 is a hub gene and located in Module 3, whose function has been discussed before. In Module 2, the PI3k-Akt signaling pathway is closely involved in the inflammatory response to induce MMPs, Adamts, IL-1β, and TNF-α in osteoarthritis [Piezo1 is still not clear yet, indicating a promising direction when performing osteoarthritis research.From the results of PPI network and module analysis, a total of 578 interactions, 47 hub genes, and 3 significant modules were obtained. Taking most overlapping DEGs together, rthritis ,56. HowePizeo1 and our identified genes and pathways in different bone tissues in a diseased state, such as osteoarthritis, osteoporosis, or osteosclerosis, should also be considered and confirmed.The present study also has some limitations. Although we intended to include as many datasets as possible, only four expression profiles met the criteria, which is far from enough to extend to full and more reliable profiles in order to illustrate the function of Piezo1 and its related genes and signaling pathways. In addition, only primary chondrocytes were used to carry out qRT-PCR validation experiments. The relative expression of our identified genes in other components needs to be further validated. Moreover, the expression of Piezo1 mutant in different bone tissues. A total of 12 of the most overlapping genes were identified. Among them, key genes, such as Lcn2, Dkk3, Obscn, and Tnnt1, along with their biological function, especially the relationship with Piezo1, represent a gap in the research so far. Their downstream mechanisms and specific effects on the different joint components also need to be explored. Signaling pathways, such as the Wnt/β–catenin and PI3k–Akt signaling pathways, have been well investigated, even under clinical trials. However, it is still unclear whether Piezo1 is involved or even plays a critical function in these pathways. Our study provides insight, for the first time, in the identification of potential therapeutic targets of Piezo1 mutant bone tissue by comprehensively analyzing them based on bioinformatics.In conclusion, this project provides a comprehensive bioinformatics analysis of key genes and signaling pathways with the


Based on the expression data of the 215 candidate genes located in these QTL intervals, we performed a weighted gene co-expression network analysis (WGCNA). A combined use of KEGG pathway enrichment and eigengene-based connectivity (KME) values identified the EC induction-associated module and four hub genes . Gene-based association analyses validated that the variations in Zm00001d028477 and Zm00001d034388, which were involved in tryptophan biosynthesis and metabolism, respectively, significantly affected EC induction ability among different inbred lines. Our study brings novel insights into the genetic and molecular mechanisms of EC induction and helps to promote marker-assisted selection of high-REC varieties in maize. The ability of immature embryos to induce embryogenic callus (EC) is crucial for genetic transformation in maize, which is highly genotype-dependent. To dissect the genetic basis of maize EC induction, we conducted QTL mapping for four EC induction-related traits, the rate of embryogenic callus induction (REC), rate of shoot formation (RSF), length of shoot (LS), and diameter of callus (DC) under three environments by using an IBM Syn10 DH population derived from a cross of B73 and Mo17. These EC induction traits showed high broad-sense heritability (>80%), and significantly negative correlations were observed between REC and each of the other traits across multiple environments. A total of 41 QTLs for EC induction were identified, among which 13, 12, 10, and 6 QTLs were responsible for DC, RSF, LS, and REC, respectively. Among them, three major QTLs accounted for >10% of the phenotypic variation, including  Zea mays L.) is an important food and forage crop worldwide. Breeding superior varieties is the guarantee of high and stable yield in maize. Traditional genetic modification methods have a long cycle and are limited by natural variations [Maize , BABY BOOM (BBM), and WUSCHEL2 (WUS2). The expression of LEC2 was significantly upregulated during somatic embryogenesis in Arabidopsis explants induced in vitro, and a close link was observed between auxin and the LEC2 activity [BBM and WUS2 genes produced higher transformation frequencies in many previously reported non-transformable maize inbred lines [WUS2, WOX2, was confirmed as the candidate gene regulating the capacity of EC regeneration [ZmMYB138 and ZmSAUR15, which modulated EC formation by participating in the hormone signal transduction pathway [ZmSAUR15 encodes a small auxin-upregulated RNA, which negatively mediates the capability of maize EC formation by affecting IAA biosynthesis and cell division in immature embryo-derived callus [ZmMYB138 promotes the formation of EC through GA signal transduction in maize immature embryos [In addition to QTLs, several genes were reported to influence EC formation in maize, including activity . The oveed lines ,17. In aneration . Our pre pathway . ZmSAUR1 embryos .Recently, a weighted gene co-expression network analysis (WGCNA) was developed to identify the hub genes in gene co-expression modules according to the gene expression patterns derived from transcriptome data . WGCNA hTo evaluate the phenotypic performances of EC induction capability among the IBM Syn10 DH population, we investigated the REC, DC, RSF, and LS for this population and its parents, B73 and Mo17, across three environments. The EC had a loose and fragile structure and formed globular nodules on the callus surface a,c, wherAmong the IBM Syn10 DH population, the REC ranged from 0 to 52.17%, 0 to 58.61%, and 0 to 56.48% under the three environments of Xishuangbanna (XSBN), Ya’an (YA), and Chongzhou (CZ), respectively, with the coefficients of variation (CVs) being 2.00, 2.90, and 2.67. However, most of these recombinant lines cannot form EC (with the REC = 0). The skewness and kurtosis of REC were > 1 under each environment , suggestp < 0.01). The broad-sense heritability (H2) of the four EC induction-related traits varied between 83.17 and 88.27%  to 11.54% (qLS1-1 in XSBN)  values. Finally, a total of 41 QTLs were identified for the four EC induction-related traits under the three environments and by using the BLUP values. These QTLs were distributed on 10 chromosomes, and the phenotypic variation explained (PVE) by a single QTL ranged from 4.01 (in XSBN) . Three QqDC1-2 and qDC9-3 were separately co-detected across CZ and the BLUP model, whereas qDC7-1 was repeatedly identified under YA and by using the BLUP values  was co-identified across XSBN and BLUP, whereas the others were environment-specific QTLs  had a PVE > 10%. Moreover, three QTLs were separately repeatedly detected under multiple environments or across environment(s) and BLUP (qLS1-1 and qLS3-2) were individually repeatedly detected in XSBN and using BLUP, which explained 74–11.54% and 7.34–9.20% of phenotypic variation across different environments, respectively (qLS10-1) was simultaneously identified across XSBN, YA, and BLUP, with the PVE ranging from 4.01 to 6.98%. Moreover, nine QTLs showed negative additive effects, suggesting that the increased LS was mainly contributed by the alleles from Mo17  was detected in XSBN, which explained 11.45% of the phenotypic variation  was validated by evaluating co-segregation between genetic variations in the QTL interval and REC phenotype using six high-REC lines (Mo17 and five other lines with REC >10%) and six low-REC lines (REC approximately 0). A total of 10 fragments covering 1035–3300 nucleotides in qREC6-1 were amplified by PCR among these 12 lines. In fragment 1 (F1), only one SNP (G/A) was found; all six high-REC lines contained the G-allele, and the six low-REC ones contained the A-allele | > 1, and Q < 0.05. We then conducted a WGCNA using the expression data of the 122 genes. Finally, these 122 genes were grouped into six co-expression modules, namely green (14 genes), turquoise (32 genes), brown (24 genes), yellow (15 genes), blue (26 genes), and red (11 genes)  . Remarka1 genes) a,b. Sinc), brown  genes, y1 genes) . General1 genes) d.p < 0.05) associated with the REC, DC, RSF, and LS in Zm00001d028477, and 20 SNPs were significantly (p < 0.05) associated with the four traits in Zm00001d034388 (p < 0.05) associated with LS , and non-Stiff Stalk (NSS) germplasms previously genotyped using a maize 56K SNP Array . Cluster analysis based on the genotypes showed that these 75 lines were classified into six different subgroups with 1–19 lines in each subgroup . The DC,1d034388 . Meanwhi with LS . Among t with LS . For Zm0 with LS . Two SNP with LS . p < 0.05) lower REC (5.50%) than that (22.60%) of the group with the T-allele (p < 0.05) higher DC (0.35 cm) relative to that (0.21 cm) of the group with the G-allele (p < 0.05) higher RSF (87.72%) than that (74.90%) in Hap3 (p < 0.001) larger LS value (1.04 cm) than that (0.59 cm) of Hap2 (p < 0.05) higher than that (0.47 cm) of Hap2 lines (p < 0.01) lower than that (85.99%) of those containing the G-allele , one for RSF (qRSF9-1), and three for LS . Moreover, high recombination rates and high-density markers in segregation populations facilitate fine mapping of the genetic loci controlling target traits [qREC1-1, qREC4-1, qREC5-1, qREC6-1, qREC7-1, and qREC7-2). Compared to the larger intervals reported in previous studies on QTL mapping of EC induction [Abundant phenotypic variations and high heritability estimates are both important to perform QTL mapping of complex traits . In the t traits . The IBMt traits . In the nduction ,13, the In contrast to LS, RSF, and DC, REC did not conform to a normal distribution among the IBM Syn 10 DH population , which wTo date, most research on maize EC has focused on REC, callus weight, callus color, callus texture, callus proliferation ability, callus browning tendency, and callus regeneration ability ,30,31,32The results of QTL mapping are susceptible to environmental influences , and QTLZmSAUR15, WUS2, BBM, LEC, SERK, and ZmMYB138 [ZmSAUR15) involved in auxin signal transduction improved and inhibited the induction of maize EC [Up to now, only a few genes have been proven to affect EC induction in maize, such as ZmMYB138 ,17,19,39ZmMYB138 , and theZmMYB138 . A numbeZmMYB138 ,43,44. Omaize EC ,46,47. TThe IBM Syn10 DH population used for QTL mapping was derived from a cross between the lines B73 and Mo17, as described in our previous study . In brieThree immature ears were harvested from the middle row per line at 12 d after self-pollination for each replicate. For each line, 108 immature embryos with 1.2–1.5 mm in length were collected from the middle sections of the three immature ears and evenly incubated among three Petri dishes with a modified N6 inducting medium [The RSF, which was calculated as  × 100%, was investigated after 7 d of incubation. The LS (mm) and DC (mm) were measured after 10 d of incubation. The REC, which was equal to  × 100%, was scored after 30 d of incubation. The criteria for identification of EC were in accordance with the previous description . H2) of each trait was calculated following the previous description [The average value of each trait among three replicates was taken as the phenotype value for each line. The SPSS software  was utilized to analyze the phenotypic data, including the range, mean value, CV, skewness, kurtosis, and correlation of each trait across three environments. Variance analysis incorporating genotype, environment, and genotype–environment interaction was conducted with the general linear model (GLM) in the SPSS 26.0 software. The broad-sense heritability (cription . To reduA bin map with high-density markers was constructed for the IBM Syn10 DH population in our previous study . This bihttps://www.snapgene.com/). The genomic DNA was extracted from the young leaves of B73, Mo17, 5 high-REC IBM Syn10 DH lines , and 5 low-REC IBM Syn10 DH lines  using the CTAB method . In the https://ngdc.cncb.ac.cn/gsa, accessed on 1 July 2022). Briefly, transcriptomes of the embryos were analyzed at 0 d, 5 d, 10 d, and 15 d after callus induction culture in the four maize lines. The clean reads were obtained by filtering the raw sequencing reads using the fastp software  [2(fold change) between each induction stage  and 0 d, and the threshold of DEGs was set to 1. The TPM of the DEGs were then subjected to a WGCNA using the WGCNA package in R software [https://www.omicshare.com/tools/Home/Soft/pathwaygseasenior, accessed on 8 may 2021, GENE DENOVO Biotechnology Co., Ltd., Guangzhou, China). The co-expression network of the hub genes was drawn using the Cytoscape software  [Expression data of the gene models located in the detected QTL intervals were obtained from our RNA-Seq data of four maize lines  during EC induction  . Gene exle, USA) .For each hub gene, the sequences of the gene body and its 2000 bp upstream were amplified by PCR in 80 randomly selected lines from our previously reported association panel [


The zinc finger proteins (ZNFs) are essential transcription factors, and the genes encoding them constitute about 3% of the entire human genome. They are involved in the development of several tissues, and any alterations in their structure may promote chronic conditions like diabetes and tumorigenesis. Lately, their role in the development, progression, and metastasis of Oral Squamous Cell Carcinoma (OSCC), Epithelial Dysplasia, Oral Lichen Planus, and Periodontitis has been found. The present review aims to describe their role in various oral conditions. Electronic databases like Medline (PubMed) and Scopus were searched for original studies related to the role of ZNFs in various oral conditions. It yielded 48 studies included in the review. It was found that the ZNFs influenced chronic conditions like Oral Cancer and Periodontitis. They act both as tumor suppressors and oncogenes and have an anti-inflammatory effect. The knowledge from the present review may be utilized in designing drugs that prevent unusual expression of specific ZNFs. Besides, they may be applied as prognostic markers due to their high expression specificity in some tumors. Xenopus laevis, they are one of the largest groups of proteins . , 5. 4, 5 α-helix . Some ofThe ZNFs are involved in the development of several tissues, and any alterations in their structure may result in chronic conditions like neurodegenerative disorders, diabetes, and tumorigenesis . Lately,κB) and Activator protein-1 (AP-1) signaling [About 90% of the oral cancers in the world are OSCC managed by either surgery, radiation, or coadjutant therapy . They haignaling . The ZNFignaling –35.κB in periodontitis. Besides, it blocks osteoclastic differentiation. The keystone periodontopathogen, Porphyromonas gingivalis , promotes tumorigenesis by disrupting epithelial cell pathways. Additionally, it stimulates ZEB1 expression in immortalized gingival epithelial cells along with Snail Family Transcriptional Repressors (SNAI) 1and 2. They promote a mechanistic convergence between the tumorigenic potential and periodontal diseases owing to their ability to control inflammatory responses [P. gingivalis hinders apoptosis and increases the cell cycle progression. It disrupts local inflammatory responses and increases the epithelial-mesenchymal transition (EMT). As a result, the epithelial cells transform and develop a motile phenotype, producing self-renewing tumor-initiating cells. In malignant tumors, transcription factors like ZEB1 and ZEB2, SNAI1 and SNAI2, and Twist-related protein (TWIST) 1 and 2 may lead to migratory and invasive cancer cells [The ZNFs may be important therapeutic targets in periodontitis , 36–39. esponses , 38. Theer cells . In thisThe research publications on the influence of ZNFs in various oral conditions were identified with the help of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. “Zinc finger protein” And “Oral” AND “Disease” OR “Oral squamous cell carcinoma” OR “Oral cancer” OR “Periodontitis” were the keywords applied in the databases like Medline (PubMed) and Scopus. The initial search leads to 197 articles . After sIn total, 54 original research articles were read, and six articles were omitted. Finally, 48 original studies were included , 12–57. Among the 48 studies, twenty-two studies reported results from the tissue samples obtained from human subjects , 56, 57,The various ZNFs reported in the included studies were Gli, ZEB1, ZEB2, ZNF582, zinc finger associated with squamous cell carcinoma (ZASC1), repressor element-1 silencing transcription factor (REST), A20, KLF, gut-enriched Krüppel-like factor (GKLF), ZNF185, Snail, Slug or SNAI2, Myeloid zinc finger 1 (MZF1), Krüppel-like zinc finger transcription factor 9 (ZF9), zinc finger AN1-type containing-4 (ZFAND4), ZNF750, Zinc finger protein X-linked (ZFX), Keratin 17 (KRT17), interferon regulatory factor (IRF), PR domain zinc finger protein1 (PRDM1), nucleus accumbens-associated protein 1 (NAC1), zinc finger and BTB domain-containing 7A protein (ZBTB7A), monocytic leukemia zinc finger protein (MOZ-) related factor (MORF), ZNF718, Homeobox A4 (HOXA4), and ZNF57 –57.The ZEB1 and ZEB2 , 49, 54 The role of ZNFs was primarily observed in the progression and inhibition of OSCC, followed by other oral conditions like lichen planus, epithelial dysplasia, and periodontitis , 35–54. The ZNFs of the C2H2 domain play an essential role in cancer progression by controlling the transcription of downstream genes concerned with proliferation, apoptosis, migration, and invasion [Evidence shows that ZNFs are involved in oral cancer progression , 35. EMT(1) ZEB-1. ZEB-1, a transcription factor, is significant for the invasion and metastasis of various human cancers, including oral cancer [l cancer , 63. It l cancer , 64, 65.l cancer , 66. In l cancer , 67.P gingivalis, increases the ZEB1 expression as a dual-species community with Fusobacterium nucleatum (F. nucleatum) or Streptococcus gordonii (S. gordonii) [P. gingivalis [P. gingivalis increased ZEB1 levels in gingival tissues samples from OSCC [P. gingivalis in OSCC [The overexpression of miR-429 inhibited OSCC cell lines growth and vice versa due to inhibition of ZEB1 expression; i.e., the two were negatively correlated . Furtherordonii) . Howeverngivalis . Even inrom OSCC . Therefo in OSCC .In OSCC, the ZEB1 expression, metastasis, and poor prognosis were inversely related to the downregulation of miR-101, which inhibits cancer cell proliferation, apoptosis resistance, migration, and invasion in vitro. As miR-101 directly affects the ZEB1, it could be a potential therapeutic target for OSCC .The ZEB-1 and the E-cadherin expression were inversely related . They weIt was also evaluated in oral lichen planus, but its role was unclear in the condition .(2) ZEB2. The ZEB2, or Smad Interacting Protein 1 (SIP1), is a transcription factor and a protein related to the transforming growth factor-β (TGF-β) signaling cascade [ cascade . It inte cascade . It is a cascade . Further cascade .In OSCC, a direct interaction between miR-200b, ZEB2, and Kindlin-2 mRNA was observed . The expBesides, a study showed that in OSCC, the miR-345 induced cell cycle arrest in the G1 phase [(3) Gli1 and Gli2. The Gli-ZNF, a glioma-associated oncogene, is a protein encoded by the Gli-1 gene in humans [n humans . The Glin humans . They arβ, and Wnt signaling) in controlling the parathyroid hormone expression (PTH) suggested that the canonical Hedgehog and TGF-β signaling increased the PTH expression and mandibular invasion in a Gli2-dependent manner [Evaluation of the role of regulators of Gli2  ZNF 750. The ZNF750 is a lineage-specific tumor suppressor gene related to squamous cell carcinoma (SCC) [ma (SCC) . It is aβ, NF-κB, p53, Notch, Hedgehog, Peroxisome Proliferator activated receptor (PPAR), and hypoxia signaling [β [An RNA sequence profiling for the genes and pathways involved in tumor suppression by overexpression of ZNF750 in OSCC showed augmentation of cell cycle-associated genes, which stimulated cell cycle arrest in the G0/G1 phase, a critical factor in the antitumor effect on OSCC cells . Anotherignaling . It was ignaling . It suppaling [β . As ZNF7aling [β . It indualing [β . Finallyaling [β .(5) ZNF 582. The ZNF 582 is located at chromosome 19q13.43 and contains one Krüppel-associated box (KRAB) AB domain and nine zinc finger motifs [r motifs , 73. Togr motifs . The PAXr motifs . In primr motifs . The pror motifs . Furtherr motifs .(6) Snail1 and Snai2 (Slug). Snail, encoded by the SNAI1 gene, and Slug, encoded by the SNAI2 gene, are ZNFs belonging to the Snail family. They are expressed in the skeletal stem or stromal cells during the pre- and postnatal states [l states . During l states .In OSCCs, the cells from primary carcinoma showed the phenotype of squamous epithelial cells, including E-cadherin and laminin-332 (laminin-5), while those from the recurrent tumors exhibited characteristics of dedifferentiated tumors with EMT . The SnaFurther, a direct interaction between DNA binding protein inhibitor-ID2 and Snail1 was found . The ID2F. nucleatum [The Slug expression also modifies the adherens and desmosomal junctions. Long-term P gingivalis infection may increase Slug levels and other EMT-associated transcription factors, like Snail and ZEB-1, due to its ability to promote EMT phenotype and cell migration, which was slightly enhanced in cases of coinfection with ucleatum . Besidesucleatum .(7) A20. A20 was initially recognized as a tumor necrosis factor- (TNF-) α induced primary response gene in human umbilical vein endothelial cells, which encodes a novel ZNF [ovel ZNF . Severalovel ZNF .The A20 RNA was expressed in 76% of undifferentiated nasopharyngeal carcinoma and 80% of poorly differentiated SCC . However(8) KLF. The KLF family comprises diverse homologous genes that function as DNA-binding transcriptional regulators, which control cell proliferation, differentiation, and migration and maintain pluripotency [ipotency . They haipotency . An actiipotency . The KLFipotency , 30.The KLF4 is a zinc finger transcriptional factor highly expressed in differentiated, postmitotic cells in both gut and skin epithelium and the lung, testis, thymus, cornea, cardiac myocytes, and lymphocytes . As it hHowever, it may act as an oncogene in some specific cancers. For instance, its expression was significantly decreased in oral cancer . Its supBesides KL4, the KLF7 promotes migration and EMT in human OSCC. Its overexpression changed their migratory behavior and triggered EMT and lymph node metastasis through the expression of Snail .(9) ZFAND4. The zinc finger, ZFAND4 also known as AN1 ubiquitin-like homolog (ANUBL1), is one of the most upregulated genes identified in recurrent OSCC [ent OSCC , 78. Itsent OSCC , 79.Increased cytoplasmic expression of ZFAND4 was observed in about 21% of the subjects with OSCC . Its ove(10) Other ZNFs. Some other ZNFs that were evaluated in very few studies ZASC1 [es ZASC1 , REST [4es ZASC1 , GKLF [4es ZASC1 , ZNF185 es ZASC1 , MZF1 [4es ZASC1 , ZFX [47es ZASC1 , IRF [39es ZASC1 , and NACes ZASC1 .The ZASC1 is a ZNF transcription factor localized on chromosome 3q26, which carries oncogenes frequently altered in neoplasms . An examAnother ZNF evaluated was REST, the Neuron-restrictive silencer factor (NRSF). It is a major transcriptional repressor for neuron-specific genes in nonneuronal and neuronal progenitor cells. Higher expression of REST was found in the human OSCC-KB cell line, while its knockdown reduced the cell viability due to apoptosis and DNA fragmentation, suggestive of its contradictory roles in tumor suppression and cancer progression . It evenSome studies found the downregulation of a ZNF185 in head and neck cancers . The MZFAnother protein, the NAC1, which controls several cellular functions, was considered a strong predictor of OSCC. It was also a potential marker for distinguishing oral epithelial dysplasia from OSCC .P. gingivalis stimulate destructive host responses, including leukocyte recruitment and proinflammatory cytokine  release [κβ pathway enhances proinflammatory gene expression and destructive mediators release [κβ pathway activation via inflammatory cytokine, toll-like, and NOD-2 receptors [β, TNF-α, and LPS [Chronic periodontitis (CP) is an immunoinflammatory disease triggered by the dental plaque biofilm that destroys tooth-supporting tissues leading to tooth loss , 80. The release , 81, 82. release , 83. Fur release . The ZNFeceptors , 84. Bes and LPS , 85–87.  and LPS , 88, 89.κβ, protein kinase Cα, Akt, and GSK-3β pathways activation  gene, ZF9, and muskelin genes [β, and influenced cellular adhesion, respectively. They altered neutrophil functions in aggressive periodontitis [Another ZNF, the ZF9, activates TGF-in genes . Even thdontitis .An animal study investigating the role of diabetes in periodontal disease and alveolar bone loss evaluated Gli homologs encoding Gli1, Gli2, and Gli3 zinc finger transcription factors . The ostα mRNA expression was initially upregulated, followed by the alveolar bone loss at the third and seventh days in streptozocin-induced diabetic mice. The Gli1 and collagen type VI-α1 were downregulated in the gingiva of the ligated site with reduced alkaline phosphatase activity and enhanced levels of tartrate-resistant acid phosphatase positive multinucleated cells [Further, the TNF-ed cells . Anothered cells .P. gingivalis enhanced its expression via the pathways related to β-catenin and Forkhead Box O1 (FOXO1). However, S. gordonii antagonized ZEB2 expression by suppressing FOXO1 even in the presence of P. gingivalis. It was considered a homeostatic commensal, capable of modifying the activity of P gingivalis by altered host signaling.Polymicrobial communities influence the ZEB2, a transcription factor involved in EMT and inflammatory responses in periodontitis . It was Another ZNF, the MORF, was related to impaired function and prolonged stress of the endoplasmic reticulum and defective osteogenic differentiation of periodontal ligament stem cells .Lately, it has been suggested that immune system modulation through gene regulation mechanisms plays a significant role in the progression and susceptibility of periodontitis. The epigenetic mechanisms like epigenetic DNA methylation, histone modification, and miRNA coordinate the gene regulation. Besides, DNA methylation in the blood helps detect treatment response predictors in several diseases, including periodontitis. In periodontitis, the DNA methylation patterns of ZNF718, HOXA4, and ZNF57 genes were observed in peripheral blood leukocytes. The ZNF718 and HOXA4 were hypermethylated, while ZNF57 was hypomethylated. The ZNF718 is associated with metabolic syndrome and diabetes, the HOXA4 acts as a transcription factor during embryogenesis and inhibits abnormal remodeling of vascular smooth muscle cells during inflammation, and ZNF57 is related to antigen presentation. Due to their immune regulatory and antigen processing functions, leukocyte DNA methylation could be applied to evaluate systemic immune-related epigenetic patterns in periodontitis [The zinc finger structures could be applied for engineering proteins that target specific genes.Combining zinc fingers with other effector domains can enable genome manipulation .Fusion of zinc finger peptides to repression or activation domains could switch the genes off or on. It could manage conditions like genetic diseases, cancer, or viral infection.They could boost the expression of beneficial genes to generate advantageous characteristics in living organisms.The ZNFs are essential for tissue hemostasis and disease as they influence the recruitment of chromatin modifiers, cofactors, or structural proteins . The ZNFTechnology has been developed to understand and manipulate ZNFs utilizing their specific ability to bind RNA. It has led to the development of synthetic ZNFs, which can activate, repress, or create defined changes to user-specified genes in human cells, plants, and other organisms . They alAnother metal ion can replace the Zn in zinc fingers as an inhibition method. For instance, gold, platinum, cobalt, and selenium complexes may be developed as zinc finger inhibitors for therapy . HoweverThe present review provides an insight into the realm of ZNFs, particularly their influence on conditions like oral cancer and periodontitis. As the ZNFs affect cancer development, progression and metastasis, and inflammatory pathways, they can influence the disease severity. They function as tumor suppressors or oncogenes and have anti-inflammatory effects. Drug therapy to target specific ZNFs may be utilized to avoid their expression. Owing to the high specificity in function and expression of some ZNFs in tumors, this class of proteins could be applied as prognostic markers.


CAMP) gene product LL-37 as prominent AMPs expressed by oral cells and tissues. HNP, HBD, and LL-37 expression can be induced by micronutrients , nutrients, macronutrients , proinflammatory agonists, thyroid hormones, and exposure to ultraviolet (UV) irradiation, red light, or near infrared radiation (NIR). Localized AMP expression can help reduce infection, inflammation, and pain and help oral tissues heal. The use of a specific inducer depends upon the overall objective. Inducing the expression of AMPs through beneficial foods would be suitable for long-term health protection. Additionally, the specialized metabolites or concentrated extracts that are utilized as dosage forms would maintain the oral and intestinal microbiome composition and control oral and intestinal infections. Inducing AMP expression using irradiation methodologies would be applicable to a specific oral treatment area in addition to controlling local infections while regulating inflammatory and healing processes.Antibiotics are often used to treat oral infections. Unfortunately, excessive antibiotic use can adversely alter oral microbiomes and promote the development of antibiotic-resistant microorganisms, which can be difficult to treat. An alternate approach could be to induce the local transcription and expression of endogenous oral antimicrobial peptides (AMPs). To assess the feasibility and benefits of this approach, we conducted literature searches to identify (i) the AMPs expressed in the oral cavity; (ii) the methods used to induce endogenous AMP expression; and (iii) the roles that expressed AMPs may have in regulating oral inflammation, immunity, healing, and pain. Search results identified human neutrophil peptides (HNP), human beta defensins (HBD), and cathelicidin AMP ( Candida species to colonize, promote the development of antibiotic-resistant microorganisms, and result in complications associated with hypersensitivity reactions and allergic disorders [Antibiotics are used throughout all disciplines of dentistry for the treatment of both soft and hard tissue infections . These tisorders ,2,3.Antimicrobial peptides (AMPs) have been recently proposed as alternatives to antibiotics for treating oral infections . HoweverHost-directed therapy (HDT) is a concept proposed to boost innate immune mechanisms for controlling antibiotic-resistant microorganisms ,11. InclCAMP) gene product LL-37 [In this study, we hypothesized that this approach could be used to induce AMP transcription and expression in the oral cavity. We started by conducting an initial literature search to identify AMPs expressed in the oral cavity. Human neutrophil peptide (HNP)-1, -2, and -3 defensins , the humct LL-37  were fouct LL-37 . Next, wTo date, between 3000  and 5000AMPs are expressed in cells and tissues of the oral cavity . We searIn oral tissues, HNP-1-3, HBD1-2, and S100A7 are produced by the tongue ,24,25, HDEFB1 and DEFB4B transcription and HBD1 and HBD2 expression were detected by RT-PCR in pulp tissue and detected by immunohistochemical staining in the cytoplasm of odontoblasts [AMPs are also transcribed and expressed in deeper sites and dental pulp, as these tissues have the capacity to produce AMPs ,32,33,34toblasts .Of the more than 2290 proteins detected in saliva ,36, apprAMPs in cells, tissues, and salivary tissues are induced by a variety of nutritional, hormonal, or physical stimuli , Table 1Selenium ,43,44, zExperimentally, trace elements induce AMP transcription and expression . ChickenCalcium is an abundant mineral found in many food groups, including vegetables and dairy products. Calcium can be consumed as a supplement to improve skeletal, cardiovascular, and neuromuscular health and is commonly involved in cellular signaling as an enzyme cofactor or signal transducer .DEFB4B and DEFB103A transcription and HBD2 and HBD3 expression [Experimentally, calcium is known to have direct effects on innate and adaptive immune functions . For exapression . One modpression  featured1–B12, C (ascorbic acid), D, and E .Vitamins are micronutrients essential for normal growth and development. They are not synthesized but are required in small quantities in the diet. They form a diverse family containing vitamins A (retinoic acid), BVitamins are involved in AMP transcription and peptide expression . Vitamin1 (thiamin), B2 (riboflavin), B5 (pantothenic acid), B6 (pyridoxine), B7 (biotin), B9 (folic acid), or B12  in AMP transcription and peptide expression. However, vitamin B3 (niacin) promoted the expression of porcine β defensin (pBD) 2, protegrin 1–5, and PR39 in the gastrointestinal tract of weaned pigs, which contributed to their elimination of enterotoxigenic Escherichia coli K88 infections [Little information is available on the roles of vitamins Bfections ,55.DEFB1 transcription in normal keratinocytes [Porphyromonas gingivalis lipopolysaccharide (LPS) [Vitamin C (ascorbic acid) induced inocytes , and vitde (LPS) .2) and cholecalciferol (vitamin D3) [Vitamin D has pleotropic activities, and it is important in the cellular uptake and metabolism of calcium, phosphate, and other minerals necessary for mineral metabolism and bone development ,109. Vit3 comes from the dietary intake of fish, eggs, and vegetables high in vitamin D or dairy products and fruit juices fortified with vitamin D [3 by ultraviolet B (UVB) as a result of normal exposure to sunlight [3 can also come from the photochemical conversion of lumisterol and tachysterol [3 then becomes vitamin D3 and is hydroxylated in the liver to form 25(OH)D and hydroxylated in the kidneys to form 1,25(OH)2D3  [2D3  is the physiologically active form [Some vitamin Ditamin D . Howeversunlight ,109,111.hysterol ,109,111.CAMP gene expression [2D3-dependent gene expression [2D3 induces antimicrobial peptide gene expression in isolated human keratinocytes [Important to this review is the ability of vitamin D to regulate antimicrobial innate immune responses . This repression ,66, and pression . The propression : 1,25 [2D3  then combines with VDR and VDRE [CAMP and DEFB4B transcription, resulting in LL-37 and HBD2 expression [Signaling  occurs tcitriol) ,112,113.and VDRE . This copression .6 (>500 mg/d) can result in photosensitivity and neurotoxicity; supplements of vitamin E (800–1200 mg/d) can result in diarrhea, weakness, and blurred vision; and excess supplements of vitamin A can result in a loss of bone mineral density with an increase in fracture risk [The use of micronutrients  to induce endogenous AMP transcription and expression is unlikely to have side effects if such micronutrients are obtained as consumable nutrients in foods. However, if consumed in excess or misused as supplements, micronutrients can induce a variety of off-target side effects. An estimated 10,176 of 32,000 (31.8%) emergency department visits per year (2004–2013) were estimated to result from adverse events related to the consumption of micronutrients . Within ure risk .Many nutrients and macronutrients can induce AMP transcription and expression ,17. ThesDEFB1 transcription in normal keratinocytes [CAMP transcription and LL-37 expression [CAMP transcription and LL-37 expression in the colonic epithelial cell line T84, THP-1 monocytes, and macrophages [Glucose, lactose, and complex polysaccharides such as β-glucans all induce the expression of AMPs . Glucoseinocytes . Lactosepression . Lactoserophages . LL-37 tβ-glucans have potent immune regulatory functions : they reDEFB1 transcription and HBD1 expression. The administration of l-isoleucine induced a significant increase in HBD3 and HBD4, which was associated with decreased bacillary loads and tissue damage in animals infected with the Mycobacterium tuberculosis antibiotic-sensitive strain H37Rv and the M. tuberculosis strains of MDR clinical isolates, suggesting that the induction of HBDs might aid in controlling this infection [Amino acids regulate gene expression via the mTORC1, AMPK, and MAPK signaling pathways ,118. Aminfection . Inductinfection .Pyroglutamyl peptides form a family of bioactive molecules ,79 contaFirmicutes) [PyroGlu peptides are thought to enhance the production of AMPs in the gastrointestinal tract to help maintain normal intestinal microbiota . They remicutes) .DEFB1 transcription and HBD1 expression [DEFB1 transcription and HBD1 expression. Induced DEFB1 transcription was related to c-myc over-expression, which suggested that DEFB1 transcription may be regulated via c-myc signaling [Bovine serum albumin (BSA) has also been found to regulate pression . BSA is pression . It is cpression . When adignaling .Free fatty acids (FFAs) form a large family of carboxylic acids of saturated or unsaturated aliphatic hydrocarbon chains. FFAs are formed as breakdown products from the hydrolysis of fats and oils and readily occur in the oral cavity, in the gastrointestinal tract, and on the skin. FFAs are grouped by size into those containing five or fewer carbons (short-chain fatty acids or SCFAs), six to eleven carbons (medium chain fatty acids or MCFAs), or twelve or more carbons (long-chain fatty acids or LCFAs).CAMP transcription and LL-37 expression [CAMP transcription in VA10, HT-29, A498, and U937 cell lines [DEFB1 transcription was increased in the lung epithelial cell line VA10, but decreased in the monocytic cell line U937 [CAMP gene expression in HT-29 and U937 cell lines [FFAs have very potent innate immune properties, including direct antimicrobial activities against viruses, bacteria, and fungi  and the pression . In labopression ,85,86,87pression ; and LCFpression  but modepression . Sodium pression  and indupression . Phenylbine U937 . Valeratll lines . Hexanoall lines  and laurCAMP transcription through the ERK1/2 and c-Jun N-terminal kinase (JNK) signaling pathways [DEFB4B transcription  [DEFB1, DEFB4B, and CAMP transcription and HBD1, HBD2, and LL-37 expression in a variety of cell types [Lipoproteins, lipoteichoic acid, triacyl bacterial lipopeptides, and fungal zymosans are TLR2 and TLR6 ligands ; double  (rHagB)  are TLR4ll types ,96. UnmeDEFB1, DEFB4B, and CAMP transcription and HBD1, HBD2, and LL-37 expression in a variety of cell types [Proinflammatory cytokines including IL-1β and TNF-α bind and signal via IL1R , TNFR-1,ll types ,96. Agonll types  and NF-κll types  signalinThe use of proinflammatory agonists such as LPS to induce endogenous AMP transcription and expression is unlikely to be a popular or feasible approach. Systematic LPS administration can systemically induce inflammation, proinflammatory cytokine production, fever, and septic shock . LPS preCAMP transcription [Thyroid hormones regulate metabolism and immune functions , and an cription . LL-37 ecription .CAMP gene [CAMP and DEFB4B transcription and LL-37 and HBD2 expression [There are a number of possible mechanisms for this phenomenon, one of which is receptor mediated. T3 and T4 bind to cellular thyroid hormone receptors (TRs), which are ligand-activated transcription factors (TFs). TRs then bind to thyroid hormone response elements (TREs) together with retinoid X receptors (RXR) in the promotor regions of their target genes to regulate gene expression. A good example is the presence of TR/TRE binding sites in the promoter region of the AMP gene . Anotherpression .While thyroid hormones are associated with changes in the oral cavity, altering thyroid hormone concentrations to induce endogenous AMP transcription and expression would not likely be a valid approach. Any advantages would be offset by numerous and more severe consequences that could be associated with metabolic complications .AMP transcription and expression is induced in cells and tissues after exposure to radiation within select ranges in the electromagnetic spectrum ,140,141.CAMP and DEFB1 transcription [The use of UVC irradiation is relatively new compared to that of other areas in the electromagnetic spectrum. UVC irradiation (100–280 nm) alone is directly antimicrobial and rapidly kills microorganisms but is not toxic to oral cells ,144. Irrcription .UVC is thought to signal through the p38 MAPK, ERK1/2, and JNK pathways . AbbreviDEFB4B, DEFB103A, RNASE7, S100A7, and CAMP transcription and HBD2, HBD3, RNase7, S100A7, and hCAP18/LL-37 expression [2) induced DEFB1 and DEFB4B transcription, whereas transformed human keratinocyte A431 cells did not [CAMP transcription [UVB irradiation (280-315 nm) alone is directly antimicrobial but also induces cells and tissues to express AMPs . In cultpression ,104. For did not . In tiss did not ; upregul did not , a poten did not ; and indcription .In clinical studies, UBV irradiation induces AMP expression in normal human skin and regulates AMP expression in the skin of individuals with atopic eczema, psoriasis, dermatitis, and vitiligo . UVB incUVB is thought to signal through the p38 MAPK, ERK1/2, and JNK pathways . AbbreviDEFB1 transcription, whereas 10 and 20 min of UVA irradiation reduced DEFB1 transcription [UVA (340–400 nm) is a longer wavelength that also regulates AMP transcription and expression in keratinocytes. In human keratinocytes, Cruz Diaz et al. found that 5 min of UVA irradiation induced cription .DEFB1, DEFB4B, and DEFB103A transcription levels were higher in localized scleroderma compared to normal skin. After treatment with UVA, clinical scores and areas of treatment improved. DEFB1 transcription decreased in localized scleroderma compared to normal skin. DEFB103A transcription decreased in localized scleroderma but increased in normal skin [Clinically, UVA can regulate AMP transcription, which is correlated with clinical improvement of localized scleroderma . Prior tmal skin .2+ to PRKCA through RAS to ERK1/2. ERK1/2 then signals through RPS6KA5 to STAT1. Finally, UVA can activate JNK directly. Activated (phosphorylated) JNK then signals to AP-1 and STAT1.UVA signals through the p38 MAPK, ERK1/2, and JNK pathways . AbbreviCAMP and DEFB1 transcription and LL-37 expression in immortalized gingival fibroblasts infected with P. gingivalis [The red-light region occurs within the visible spectrum at 625–740 nm. Lasers emitting light at 625 nm can induce ngivalis .DEFB4B transcription in oral fibroblast cells than in oral keratinocytes [NIR occurs just outside the visible spectrum at 800 to 2500 nm. NIR at 810 nm induces higher inocytes . Transcrinocytes .Using irradiation to induce AMP transcription and expression would be an attractive approach. Irradiation can be strictly controlled in the clinic and used locally in the oral cavity. This measure can avoid systemic or long-term over exposure. The dental community has been using UV irradiation in the oral cavity for over 50 years to whiten teeth and polymerize compounds used in restorations and fillings . Since t3 [3 and LPS [CAMP gene [Increases in AMP transcription and expression are induced in cells after exposure to multiple AMP inducers . Co-expo3 , and vit and LPS  were allAMP gene . The colAMP gene .DEFB4B and DEFB103A transcription. Depending on the combination of proinflammatory cytokines and HBD examined, DEFB4B and DEFB103A transcription can vary from a 4- to 150-fold increase.Synergy also occurs among proinflammatory cytokines . VariousInduced HNPs, HBDs, and LL-37 have additional roles beyond their antimicrobial activities. Their local presence can regulate the intensity of inflammation; regulate innate and adaptive immune functions, and accelerate angiogenesis, vasculogenesis, and wound healing activities. We searched the PubMed literature database using innate immunity, chemotaxis, cell migration, proliferation, cytokine response, gene expression, microbial antigens, pathway signaling, adaptive immunity, antibody response, Th1, Th2, wound healing, angiogenesis, cytotoxicity, apoptosis, and pain together with antimicrobial peptides, defensins, cathelicidins, HBD, HNP, and LL-37 as search terms. These terms were linked in various combinations using Boolean operators to identify AMPs reported to also have roles in inflammation, immunity, healing, and pain. Appropriate articles were downloaded and read by the authors. The information from this search was used to construct Although there are wide ranges in the concentrations of AMPs used in many studies , Table 2AMPs regulate inflammatory processes in part by (i) altering proinflammatory agonist binding to cells and (ii) altering intracellular cytokine signaling pathways. Regulation occurs in both directions. In some circumstances, AMPs increase the expression of proinflammatory cytokines in stimulated cells ,156,157,P. gingivalis recombinant fimbrillin A (rFimA) [P. gingivalis rHagB [AMPs can directly bind to microbial agonists ,164,165, (rFimA) ; HNP-1,2is rHagB ,163; andis rHagB . The binis rHagB . HNP-1 ais rHagB . Histatiis rHagB ,173.2-Lipid A [2-lipid A-treated macrophages [+ monocytes, dendritic cells, B-lymphocytes, and T-lymphocytes all produced different profiles of intracellular cytokines [+ monocytes [AMPs alter the intracellular signaling pathways. As an example, HBD3 rapidly enters cells and once in the cytoplasm, prevents the expression of genes related to the activation of proinflammatory responses to LPS/KDO-Lipid A . The inhrophages . LL-37-tytokines . Scott eytokines . Additioonocytes . LL-37 aonocytes .Once expressed, AMPs regulate important innate and adaptive immune-related activities ,177,178.In adaptive immunity, AMPs induce adaptive immune responses, enhance antibody responses, and increase cell survival . LL-37 , cells involved in allergic inflammation (mast cells and eosinophils), cells involved in angiogenesis , and some cancer cell lines .A new and exciting area of AMP functions is the potential of AMPs to be antinociceptive. While inducers of endogenous AMP expression are not yet known to be directly antinociceptive, it is tempting to speculate that they may have stimulatory activities on the MAPK  and NF-κB signaling pathways that produce AMPs and cytokines involved in pain reduction . BlockagRecent work in a variety of fields has demonstrated that small peptides with antimicrobial activities are also involved in nociceptive and antinociceptive responses. These peptides come from insects ,186,187,The use of a specific inducer would depend upon the overall objective ,16,17,18Inducing the expression of AMPs through micronutrients, nutrients, and macronutrients  would beInducing the expression of AMPs by irradiation would be primarily applicable to clearing focal infections. Treatments would likely be highly focused and controlled in a clinical setting. Expressed AMPs would have direct antimicrobial activities and regulate local defenses to attenuate inflammation, ameliorate pain, and help oral tissues heal. Applications include controlling infections in the oral cavity and include root canals. The wavelength and dose of UV irradiation can be easily controlled to facilitate healing and tissue regeneration, and treatments would not be toxic at therapeutic levels.Dental procedures often have accompanying pain. Treating dental infections, as well as reducing the associated pain, would have popular benefits. However, the ability of AMPs to be antinociceptive is not yet fully known and remains under investigation.


Brucella abortus strain 2308, isolated from an abortion storm on a dairy farm in India. Bacteria were isolated in pure culture from the placentas of aborted calves, and next-generation sequencing (NGS) revealed that the genome sequence length of the isolated strain is 3,285,606 bp, with a GC content of 57.25%, an N50 value of 296,426 bp, and an L50 value of 4, containing 3,119 coding DNA sequences (CDSs), 49 tRNAs, 1 transfer-messenger RNA (tmRNA), and 3 rRNA genes.The present report communicates the first complete genome sequence of  Brucella-positive animals are culled and sacrificed  in 2019 with the goal of eradicating brucellosis by 2030 using a Brucella abortus strain 19 vaccine. It is a live vaccine with long-lasting immunity and persistent antibodies , 48 contigs, 48 scaffolds, 30,901 open reading frames (ORFs), and 3,330 genes, with an average length of 853,828 nt, including 51 tRNAs, 1 transfer-messenger RNA (tmRNA), and 3 rRNA genes, as identified by annotation using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 4.11  under the accession number SRR11526759.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


Triterpenoids, important secondary plant metabolites made up of six isoprene units, are found widely in higher plants and are studied for their structural variety and wide range of bioactivities, including antiviral, antioxidant, anticancer, and anti-inflammatory properties. Numerous studies have demonstrated that different triterpenoids have the potential to behave as potential antiviral agents. The antiviral activities of triterpenoids and their derivatives are summarized in this review, with examples of oleanane, ursane, lupane, dammarane, lanostane, and cycloartane triterpenoids. We concentrated on the tetracyclic and pentacyclic triterpenoids in particular. Furthermore, the particular viral types and possible methods, such as anti-human immunodeficiency virus (HIV), anti-influenza virus, and anti-hepatitis virus, are presented in this article. This review gives an overview and a discussion of triterpenoids as potential antiviral agents. Viral infections are currently the main infectious disease worldwide , accountViruses usually spread from one region to another, and in the past 10 years, the large-scale outbreaks of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), Ebola, and other viruses have caused considerable threats to human health. Moreover, the residual effects of viral infections cause considerable deleterious effects, pain, and inconvenience to patients. For example, the “cytokine storm” triggered by influenza virus infection can cause lung inflammation and acute respiratory distress syndrome, which is an important cause of mortality ,5. The nPresently, vaccines and screening antiviral drugs are the main drugs used for the prevention and treatment of human viral infections . However5H8) units. Studies reported that triterpenoids have a wide range of pharmacological effects and important biological activities, especially anti-inflammatory, hypoglycemic, antitumor, and antiviral activities . Th. ThSchisely (48) , isolate) > 32.8 . The tri2.8 ..Schisand8), betulinic acid , betulin (17), and lupeol (18) are representative pentacyclic triterpenoids that are widely found in nature with a broad antiviral spectrum (18), extracted from Hemidesmus indicus R. Br., has an inhibitory effect on HIV-1 RT-related ribonuclease, with an IC50 of 11.6 μM -7-aminoheptyl]-carbamoyl] methane), with an EC50 value of 0.0026 μM . Fu. FuGanodprotease  [47]. Gand 48 μM  [48].29), ginsenoside Rb2 , ginsenoside Rg3 , ginsenoside Rg1 , and ginsenoside Rh1 , showed strong inhibitory activity against HIV (29) eliminated HIV-1-infecting macrophages through the AKT pathway, whereas G-Rh1 (33) mainly eliminated HIV-1 infection by inhibiting the pyruvate dehydrogenase kinase 1 (PDK1)/Akt pathway . GL. GL40].  protein . Furthernfection .3) (9)  with OA (8) or UA (10) greatly improved the anti-influenza-virus activity of the compound, and compounds Y3 and Y5, obtained after the partial acetylation of galactose, showed the best anti-influenza-virus activity, with EC50 values of 14.2 and 15.1 μM, respectively . OA. OA1) stactivity , is a 3Cectively . Compoun6.25 μM)  Table 31 stronglViral hepatitis can be caused by a variety of hepatitis viruses, which are usually divided into A, B, C, D, and E types. The A and E forms of the virus cause acute infections and can be transmitted through the fecal–oral route. The B, C, and D viruses mainly cause chronic infections, which are transmitted through blood and body fluids. Recently, traditional Chinese medicine (TCM) practices and formulations have been widely explored for the treatment of viral hepatitis and, in particular, the pentacyclic triterpenes have been a focus.1) achieved a concentration-dependent inhibition of the replication of HAV in PLC/PRF/5 cells (29) and G-Rg1 (32) have been shown to significantly inhibit HAV in vitro (The hepatitis A virus (HAV), which is the main cause of hepatitis A, is a food-borne disease, and vaccination is the most effective preventative treatment. Early research has shown that GL (/5 cells  78]. Gi. Gi1) acin vitro  79]..1) achieChronic hepatitis B is a long-term progressive disease caused by HBV infection, which leads to hepatitis, cirrhosis, and even liver cancer. Presently, the main drugs for the treatment of chronic hepatitis B are IFN in combination with nucleoside drugs such as lamivudine, adefovir dipivoxil, and clevudine. The low treatment success rate and severe side effects of IFN and the increased resistance to nucleoside drugs has limited the clinical application of these drugs . Triterp1) effectively controlled HBV replication in chronic HBV carriers . Cl. Cl1) ef000-fold . GL-3, a-IV, 49)  is an efed cells . G-Rg3 , liver cirrhosis, and liver cancer, and approximately 185 million people are infected with the HCV. During the HCV replication process, NS3/4A serine protease mediates the cleavage of polyprotein precursors, which promotes the maturation and function of nonstructural proteins. Clinically, >95% of CHC patients are treated with antiviral drugs that act against NS3/4A protease, which can induce a sustained virological response. However, some patients with liver cirrhosis still develop hepatocellular carcinoma, and there is serious drug resistance to these agents, which have multiple side effects. Triterpenoids have been proven to have obvious advantages in the treatment of HCV .8), was isolated from Dipsacus asperoides and exhibited a good anti-HCV activity . UA. UA12) (articles  92]. Mo. Mo12) (dulators  93]..12) (10)Herpesviruses are double-stranded DNA viruses with icosahedral symmetry, belonging to the Herpesviridae family. Most herpes viruses can cause human infections, including herpes simplex virus (HSV)-1 and HSV-2, the African lymphocyte Epstein–Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and human cytomegalovirus (HCMV) . The her5) and cicloxolone sodium  are derivatives of triterpenoid GL (1), found in glycyrrhiza plants, that inhibit HSV-1 and HSV-2 (1) effectively inhibits the adsorption of HSV to rat brain capillary endothelial cells and polymorphonuclear leukocytes, thereby significantly reducing the inflammatory response caused by the HSV infection of host cells . GL. GL5) anhe virus . Moreovec effect  against phocytes . Further effects . 20(S)-Gof 35 μM  102]..5) and c8) and UA (10) have anti-guinea pig cytomegalovirus (GPCMV) effects . I. I5) hasprotease  111]..5) has aNoV is the main pathogen causing nonbacterial acute gastroenteritis in humans and acute diarrhea in infants and young children . NoV con49) has been shown to upregulate the expression of IFN-γ mRNA, which inhibits CV . F. F29) exarticles .1) inhibits the cellular penetration of PRRSV and significantly reduces its proliferation and the expression of its encoded protein . G. G1) inh32) inhibited PRRSV infection in MARC-145 cells and porcine alveolar macrophages [32) showed extensive inhibitory activity against PRRSV-2, including the highly pathogenic (HP)-PRRSV XH-gD and JXA1, NADC-30, and the classic strain VR2332. G-Rg1 (32) reduced the expression of the pro-inflammatory cytokines IL-1β, IL-8, IL-6, and TNF-α and reduced the activation of NF-κB signaling triggered by PRRSV infection (10), compound 15  inhi2) inhibits HRSV adsorption and internalization and the stimulation of IFN secretion [1), isolated from licorice, exhibited no obvious inhibitory effect on HRSV.HRSV is the main pathogen that causes bronchiolitis and pneumonia in infants and young children. However, there is currently no vaccine to prevent HRSV infection . 18β-GA ecretion . HoweverThe rapid mutation of viruses enables them to easily develop resistance to drugs, which limits the use of currently available drugs. Therefore, the development of broad-spectrum antiviral drugs is important, especially for newly emerging viruses, such as SARS-CoV-2. Traditional herbal medicines and plant-based natural compounds are rich resources of new antiviral drugs. Many traditional herbal medicines possess antiviral activities against a plethora of viral strains, exerting their antiviral activities on the virus life cycle, including viral entry, replication, assembly, and release as well as the virus–host-specific interactions, allowing them to theoretically lead to the discovery of broad-spectrum antivirals.Triterpenoids are the focus of considerable attention because of their novel chemical structure and unique mechanism of action. In recent years, an increasing number of studies have reported on the effects of triterpenoids on viruses. This present article systematically summarizes the recent advances in the study of the types, structural characteristics, and potential antiviral mechanisms of triterpenoids. The investigation of the antiviral mechanisms of these triterpenoids leads to the conclusion that they are mainly exerted through the inactivation of viral particles, the prevention of viral replication and entry into host cells, and the regulation of immunity. The present review aims to provide evidence to support the effectiveness of the strategy of discovering new antiviral drugs from triterpenoids.However, there are still some obstacles to developing broad-spectrum antiviral drugs from triterpenoid compounds. The targets and mechanisms underpinning some natural products remain unclear, potential side effects have not been fully investigated, and the activities of triterpenoids are usually moderate. Advanced technology for chemical modification, investigations into underlying biological mechanisms, and progress in drug design will facilitate future developments in the field. In addition, the structure of triterpenoids determines that they have high polarity and poor bioavailability, which limits their biofilm permeability and absorption. Therefore, it is necessary to improve the dissolution and absorption ability of triterpenoids by chemical or pharmaceutical methods. For example, the esterification or amidation structure modification of triterpenoids can increase their lipid solubility, membrane permeability, and oral absorption rate. In addition, new pharmaceutical dosage forms and new technologies can also improve the oral bioavailability of triterpenoids, such as oral self-emulsifying systems, liposomes, microspheres, microcapsules, nanomicelles, etc. Furthermore, appropriate administration routes can also improve the bioavailability of triterpenoids, for example, transdermal drug delivery systems, colonic administration, nasal mucosa administration, pulmonary inhalation administration, etc.