Background: Unclassified primary antibody deficiency (unPAD) is a relatively novel inborn error of immunity (IEI) condition that can vary with time to more defined entities. Since long-term follow-up (FU) studies are scarce, we aimed to provide insight into the evolutionary clinical and immunological scenario of unPAD children to adulthood and identification of biomarkers of primary immune deficiency (PID) persistence. Methods: A total of 23 pediatric unPAD patients underwent clinical and immunological FU for a mean time of 14 years . Results: UnPAD diagnosis may change over time. At the last FU, 10/23 (44%) children matched the diagnosis of transient hypogammaglobulinemia of infancy and 13/23 (56%) suffered from a persistent PID. In detail, an unPAD condition was confirmed in 7/23 (30%) patients, whereas 3/23 (13%), 2/23 (9%), and 1/23 (4%) were reclassified as common variable immunodeficiency, selective IgA deficiency, and isolated IgM deficiency, respectively. Low IgA, low specific antibody response to pneumococcus, and lower respiratory tract infections at diagnosis were independently associated with IEI persistence. Conclusions: Long-term monitoring of unPAD patients is required to define their outcome and possible evolution towards a definitive IEI diagnosis. Primary antibody deficiencies (PADs) consist of a varied group of conditions with different genetic etiologies characterized by an impairment of B cell development, differentiation or class switch recombination leading to hypogammaglobulinemia, and/or defective antibody production . They rehttps://esid.org/Working-Parties/Registry-Working-Party/Diagnosis-criteria; accessed on 15 April 2023). A local ethical committee approved the study, and written informed consent was obtained from all participants or legal guardians.Twenty-three pediatric patients (12–36 months) with an initial diagnosis of unPAD, attending the Pediatric Immunopathology and Allergology Unit/ Regional Referral Center for PIDs at Policlinico Tor Vergata in Rome, the Department of Pediatrics of University of Pisa, and the Pediatric Immunology Center of Federico II University in Naples were enrolled in the study. All patients matched ESID diagnostic criteria for unPAD . In patients with allergy, skin prick tests (SPT) and serum IgE (sIgE) were performed. Serum IgE was tested by ImmunoCAP FEIA, whereas airborne and/or food allergen extracts were used for SPT, according to clinical phenotype. In a limited number of patients, genetic analysis (next-generation sequencing—NGS) for the main PAD-associated genes was also performed. The NGS panel included the following genes: ICOS, TNFRSF13B, TNFRSF13C, TNFRSF12, CD19, CD81, CR2, CD20, CD27, IL21, IL21R, LRBA, CTLA4, PRKCD, PLCG2, NFKB2, NFKB1, PIK3CD, PIK3R1, PTEN, VAV1, RAC2, BLK, IKZF1, IRF2BP2, BTK, CD40L, SYK, LYK, FYK, MYD88, IRAK4, TNFSF13B, TNFSF17, TNFRSF17, RELB, REL, IKBE, IKBA, IKBB, IKK-alpha, IKK-beta, MAP3K14, RELA, STK4, AKT, LAT, IL12RB1, IL12B, IFNGR1, IFNGR2, ISG15, EVER1, EVER2, AICDA, CD40, UNG, CD79A, CD79B, PAX5, TCF3, BLNK. Sanger sequencing was used to confirm genetic variants detected by NGS as previously reported [According to general practice, immunological work-up of these patients included serum Ig levels by nephelometry, serum IgG subclass values by radial immunodiffusion, extended T and B cell immunophenotype by fluorescence-activated cell sorting (FACS), and specific IgG antibody response to tetanus and pneumococcal vaccines by ELISA .Clinical and immunological data of patients were analyzed using Fisher’s exact test and the chi-squared test. A Twenty-three children with an initial diagnosis of unPAD,  and 9 females (39%) were included in the study. Positive family history for PIDs was reported in 6/23 (26%) of patients, of whom two had parental consanguinity. A positive family history for early deaths was present in one patient (4%).p = 0.028). Seven out of nine unPAD patients presented IgG subclass deficiency, with no correlation with age at diagnosis. A poor specific antibody response to tetanus and pneumococcus was detected in 4/18 (22%) and 7/19 (37%) patients, respectively. Standard immunophenotypic analysis was normal in all 23 patients. Extended B cell immunophenotyping was performed in 18/23 patients, and low expression of switched memory B cells and IgM memory B cells was observed in 10/18 (55%) and 1/18 (5%), respectively. Percentages of activated CD21 low B cells and transitional B cells were within age-matched reference values in all patients.At diagnosis, 22/23 (96%) unPAD patients (10/10 < 24 months and 12/13 > 24 months) were symptomatic. As detailed in All 23 patients underwent clinical and immunological follow-up every 6 months up to 4 years of age and then yearly for a mean time of 14 years . As reported in p value 0.04), mostly with URTI and LRTI . Moreover, isolated or combined IgG, IgA, and IgM defects were detected in 46%, 61%, and 54% of persistent PID patients, respectively, whereas associated IgG subclass deficiency was observed in 7/13 (53%) of them. A poor specific antibody response to tetanus and pneumococcus was found in 1/9 (11%) and 2/10 (20%) of persistent PID patients, respectively. When the extended B cell immunophenotype was performed in the persistent PID cohort, low switched memory B cells and low IgM memory B cells were detected in 7/13 (54%) and 2/13 (15%) of patients, respectively. The percentage of activated CD21 low B cells and transitional B cells were within age-matched reference values in all patients (data not shown).At the last follow-up, two patients reached age-appropriate Ig values at the age of 16 years and 17 years, respectively, joining the category of THI for a total of 10/23 (44%) patients A,B. Amonp value 0.03). Isolated or combined IgA deficiency and low anti-PCP antibody response were also found to be associated with a final diagnosis of persistent PID . The variables that showed a significant (p < 0.05) association with PID persistence in univariate analysis were evaluated in a logistic regression model for multivariate analysis. Low IgA, low specific antibody response to pneumococcus, and LRTI at diagnosis were observed to be independently associated with a persistent PID diagnosis.To identify potential predictive and/or prognostic markers of clinical outcome, a retrospective analysis of clinical and immunological findings at diagnosis of 13 patients with a final diagnosis of persistent PID vs 10 patients with a final diagnosis of THI was performed. As reported in Next-generation sequencing analysis was performed in 8/13 (61%) of patients with persistent PID. Mutations in TNFRSF13B were identified in 4/8 patients (50%), belonging to two families. As reported in The onset and clinical spectrum of unPAD patients is widely heterogeneous, and knowledge of their natural history is still scarcely investigated. Some children might be identified at an early age and initially diagnosed as unPAD to enter the THI definition when normalization of their immunoglobulin levels occurs within 4 years of age. Some others may develop over time a defined antibody defect, reaching the definitive diagnosis of a classified IEI. However, at unPAD diagnosis, no markers are currently identified to allow the distinction between patients who will achieve a condition of transient hypogammaglobulinemia from those who will persist in the same condition or will develop other humoral defects, which would be crucial to timely initiate appropriate monitoring and treatment.The aim of our study was to analyze the clinical, immunological, and genetic characterization of a cohort of children who received an early diagnosis of unPAD and their long-term clinical and immunological monitoring and outcome. To our knowledge, this is the first prospective study describing the evolution of unPAD children towards adulthood, with the clinical and immunological evaluation of their long-term follow-up.Except for one asymptomatic child, all symptomatic children suffered from recurrent infections, mostly of the respiratory tract, and to a lesser extent from allergy and autoimmunity . Indeed, as shown for other PAD patients ,19,20,21In a previous paper, we reported that in children with an initial suspicion of THI, nowadays pertaining into the category of unPAD, a milder clinical and immunological profile and a benign course over time was observed in comparison to those children who did not normalize their IgG levels . In the Moreover, the asymptomatic unPAD patient who was diagnosed at 15 months, whose brother was suffering from CVID, over time developed recurrent respiratory infections, receiving the final diagnosis of a selective IgM deficiency. It is still unclear why some patients may be asymptomatic. Functional immunological factors as well as epigenetic or environmental factors might be compensative for a certain time.When we investigated at diagnosis potential markers of dissection between PID persistence vs THI, we observed that in addition to LRTI, low IgA and low specific antibody response were independently associated with a persistent condition. Conclusively, we demonstrated that a subgroup of children with unPAD shares common B cell abnormalities with patients suffering from a range of antibody defects, mostly CVID, according to previous reports ,26,27. WOur study, despite the limited cohort, is the longest FU study analyzing a pediatric cohort of unPAD patients. A continuous long-term clinical and immunological monitoring of unPAD children is necessary to define their outcome and possible evolution towards a definitive IEI defect. We focused on distinct clinical-immunological markers, suitable for identifying patients at higher risk of PID persistence. Furthermore, as the genetic and functional characterization of patients with persistent hypogammaglobulinemia might provide us with valuable information on the pathogenic role of distinct molecules, potentially indicating specific treatment, we emphasized the need to structure national and international networks for the diagnostic approach and monitoring of the heterogeneous condition of unPAD patients.


Hordeum vulgare L.) and wheat (Triticum aestivum L.), changes of transcript levels and endogenous IAA content. Hence, the effect of light-involved auxin signaling on root growth and development is definitely a hot issue to explore in the horticultural studies now and in the future.The root is an important organ for obtaining nutrients and absorbing water and carbohydrates, and it depends on various endogenous and external environmental stimulations such as light, temperature, water, plant hormones, and metabolic constituents. Auxin, as an essential plant hormone, can mediate rooting under different light treatments. Therefore, this review focuses on summarizing the functions and mechanisms of light-regulated auxin signaling in root development. Some light-response components such as phytochromes (PHYs), cryptochromes (CRYs), phototropins (PHOTs), phytochrome-interacting factors (PIFs) and constitutive photo-morphorgenic 1 (COP1) regulate root development. Moreover, light mediates the primary root, lateral root, adventitious root, root hair, rhizoid, and seminal and crown root development via the auxin signaling transduction pathway. Additionally, the effect of light through the auxin signal on root negative phototropism, gravitropism, root greening and the root branching of plants is also illustrated. The review also summarizes diverse light target genes in response to auxin signaling during rooting. We conclude that the mechanism of light-mediated root development via auxin signaling is complex, and it mainly concerns in the differences in plant species, such as barley ( Ranunculus asiaticus L., and the effect was stronger under red:far red (R:FR) 3:1 light . Mo. MoA. thormation . PHYs alormation . In Nicon shoots  26]. Wh. WhA. thelopment ,27,28. Telopment . PhyB muaponicus . Hence, A. thaliana, hypocotyl phototropism is able to be regulated by CRYs. The PhyA cry1 cry2 mutant had a defect in phototropism , GmCRY1 was necessary for nodulation in a dark environment and inhibited the effect of BL on nodulation  receptors, which regulate different phases during plant growth and development. They are flavoproteins, which have significant homology to photolyases. CRYs functioned by transducing BL energy into a signal that can be recognized by the cellular signaling machinery . In A. totropism  31]. CR. CRA. th with WT  33]. Th. ThA. ththaliana  30]. Un. UnA. ththaliana  34,35].,35.A. thh the WT , indicath the WT . In soybdulation  38]..A. thaliArabidopsis, root phototropism was regulated by PHOT1, which was active at a wide range of BL intensities  transcript levels . Re. ReArabiototaxis  [4]. HenArabidopsis . Un. Un−2 s−ongation  48]. Bo. Bo−2 s−thaliana  50]. In. In−2 s−ormation  51]. In. In−2 s−ongation . PIF4 anongation .cop1-4 and cop1-6 mutants was decreased in dark condition whereas the root length of these mutants was enhanced in light condition compared with the WT , wh, wh3− upArabidopsis 1, PIN2, AUXR1, YUC1 and YUC4  resulted in shorter primary roots under light and larger primary root elongation in darkness, which suggested that the phenotype of the med18 root phenotype could be induced by light  in and YUC4  58]. Wh. WhA. thresponse  [58]. Ovresponse  [58]. Thby light  59]. Th. ThA. thncreased  [59]. Heeir1-4 PIN2: PIN2: VEN mutants enhanced the number of root hairs in light-grown roots (LGRs) compared with dark-grown roots (DGRs), suggesting that root hair emergence required auxin with direct root light . Au. AuPhyscOryza sativa L.). During the development of seminal roots, the very low-fluence responses (VLFR) and low-fluence responses (LFR) were controlled by PhyA and PhyB (Oryza sativa root architecture associated 1 (OsRAA1) gene in the Taichung Native 1 (TCN1) seed root was induced by light in rice. The constant white light induced the seminal roots of TCN1 to become shorter and wavier . Mo. MoOryza−2 s−1) of BL illumination, the absence of BL receptor CRY1 and PHOT2 genes showed normal root negative phototropism in A. thaliana , the root cap needs light to generate a positive geotropic response. Under continuous white light (~36 µmol m−2 s−1) irradiation for ≥1–2 h, the content of IAA was increased in maize root tips, especially in the transition zone  and ARF19 , HY1 which was produced in the pericycle cells of branches or local xylem could mediate lateral root branching /INDOLE ACETIC ACID 7 (IAA7) and SOLITARY ROOT (SLR)/IAA14 (HY1 induced the transcript level of ranching  73]. Fu. FuHY5 aR)/IAA14  74]..HY5 and PIN3. Some auxin-signaling-related target genes, such as PIN1, PIN2, PIN3, LAX3, ARF19, HAT2, SUAR23, SAUR28 and IAA29, have also been demonstrated in root development. Light not only affects the growth and development of primary roots, root hair, lateral roots, and adventitious roots through auxin signals, but also affects phototropism, gravitropism, root greening, and root branching. Despite continuous advancements, the molecular mechanism underlying the crosstalk between light and auxin signaling during rooting still needs to be illustrated and completed. Therefore, further research is required to investigate these mechanisms. Finally, the question of whether there any other photoreceptors that can be used to regulate root development via the auxin-signaling pathway should be considered. In future, related work should be carried out to improve our knowledge regarding the crosstalk between light and auxin signaling during rooting in plants.Light, as one of the most important environmental factors, can affect root growth and development via the auxin-signaling pathway. Over the past several decades, there has been an increasing number of studies conducted on the light-mediated root development via auxin signal. In this review, we summarized the role of light-regulated auxin signaling in root growth and development. First, some photoreceptors, such as PHYs, PHOTs and CRYs are involved in root development, for example, the primary root, root hair, adventitious root and lateral root development. Additionally, other TFs including PIFs, HY5, COP1and MYB73/77 are associated with light response during root development. Auxin, as an essential plant hormone, can interact with these photoreceptors to regulate root growth and development. For example, the BL receptor PHOT1/PHOT2 might be involved in the induction of adventitious rooting under BL conditions by increasing the transcript level of the auxin-signaling gene


Tricholoma matsutake strain 2001, which was isolated from a mushroom fruiting body in South Korea. The genome has 80 contigs, a size of 162.6 Mb, and an N50 value of 5,103,859 bp and will provide insight into the symbiotic association between T. matsutake and Pinus densiflora.We report the high-quality genome sequence of  Tricholoma matsutake, a symbiotic pine mushroom, has declined due to climate change and newly occurring pine tree diseases  stock center and was one of the strains showing the best mycorrhization rate  Link v8.0 with default parameters, and the SMRTbell adapters were trimmed on the PacBio Sequel instrument.Genomic DNA was extracted using the DNeasy plant minikit  following the manufacturer’s protocol and evaluated by 1% agarose gel electrophoresis and the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit . The genomic DNA was sheared using a g-TUBE , and small fragments were removed using the BluePippin size selection system . The size-selected library, with an average size of 20 kbp, was prepared with the SMRTbell DNA library construction protocol and sequenced using the Pacific Biosciences (PacBio) Sequel platform . This long-read sequencing produced 2,017,716 subreads , with a read Short-read sequencing on the Illumina NovaSeq 6000 platform was used to correct errors in the long-read assembly (DNA Link). The DNA sample quality was evaluated using 1% agarose gel electrophoresis and the Qubit dsDNA HS assay kit (Thermo Fisher Scientific). The DNA library was prepared using the Illumina TruSeq Nano DNA library preparation protocol, in which high-molecular-weight genomic DNA was randomly sheared and processed by blunt-ending, phosphorylation, and adapter ligation at both ends. Ligated DNA was PCR amplified, and the quality of the libraries was verified using a 2100 Bioanalyzer . After quantitative PCR (qPCR) using SYBR Green PCR Master Mix , indexed libraries were combined in equimolar amounts and subjected to whole-genome sequencing (WGS) with the 2 × 150-bp sequencing protocol. The sample was sequenced, with a total size of 14,191,932,810 bp and 93,986,310 reads.The genome was assembled using NextDenovo v2.4.0 with the long-read sequences. NextPolish v1.3.1 was used for error correction with default parameters . The genT. matsutake strain 2001 has 80 contigs, a size of 162.6 Mb, a GC content of 45.5%, and an N50 value of 5,103,859 bp (T. matsutake will provide insight into the symbiotic association between T. matsutake and Pinus densiflora and allow future studies using genomic approaches.The genome of 3,859 bp . Of the GCA_026213095.1, WGS accession number JALPZM000000000, BioProject accession number PRJNA831907, and BioSample accession number SAMN27770844.The genome data generated in this study were submitted to NCBI with GenBank assembly accession number


In the present study, to obtain better knowledge of the biological functions of the MTPs family, 20 potential EgMTPs genes were identified in Eucalyptus grandis and classified into seven groups belonging to three cation diffusion facilitator groups  and seven groups. EgMTP-encoded amino acids ranged from 315 to 884, and most of them contained 4–6 recognized transmembrane domains and were clearly prognosticated to localize into the cell vacuole. Almost all EgMTP genes experienced gene duplication events, in which some might be uniformly distributed in the genome. The numbers of cation efflux and the zinc transporter dimerization domain were highest in EgMTP proteins. The promoter regions of EgMTP genes have different cis-regulatory elements, indicating that the transcription rate of EgMTP genes can be a controlled response to different stimuli in multiple pathways. Our findings provide accurate perception on the role of the predicted miRNAs and the presence of SSR marker in the Eucalyptus genome and clarify their functions in metal tolerance regulation and marker-assisted selection, respectively. Gene expression profiling based on previous RNA-seq data indicates a probable function for EgMTP genes during development and responses to biotic stress. Additionally, the upregulation of EgMTP6, EgMTP5, and EgMTP11.1 to excess Cd2+ and Cu2+ exposure might be responsible for metal translocation from roots to leaves.Metal tolerance proteins (MTPs) as MeThe online version contains supplementary material available at 10.1186/s12870-023-04240-9. They greactions .+, Cd+2, Cr+2, Pb+2, Al+2, and Hg+2, have no known functions in plants and can also cause plant cell toxicity at very low concentrations [Moreover, a deficient concentration of non-essential metal ions, including Agtrations . In additrations , 6. Theytrations .Diverse remediation approaches with mechanical or physio-chemical strategies are utilized to reclaim heavy metal-contaminated soil with inefficient mechanical, physical, and chemical strategies, expensive equipment, and poisonous chemicals which can deteriorate the soil ecosystem and introduce of secondary pollutions . Therefo2+/H+ integral membrane ion transport proteins, which act as divalent cation transporters in the transportation of some ions such as cadmium, iron, zinc, manganese, cobalt, and nickel, from the cytosol into organelles [2+ and Zn2+, and the EcZitB protein from E. coli, which mainly transports Zn2+ and Cd2+. Two groups contain Fe/Zn-CDF transporters  and the third encloses only the Mn-CDF transporter (MTP8-like sequences) which shares many conserved residues, suggesting that they are derived from a common ancestor [Plants have numerous physiological and molecular mechanisms as a part of the response-specific metal toxicity to precisely regulate cellular concentrations of metal ions, i.e. metal efflux and uptake, chelation, storage, trafficking, and detoxification/sequestration . In planganelles . The CDFganelles . The firancestor . These tancestor .Oryza sativa [Triticum aestivum [Populus trichocarpa [Medicago truncatula, Arachis hypogaea [Nicotiana tomentosiformis [Vitis vinifera [Arabidopsis thaliana (AtMTP1 and AtMTP3) were considered to be Co2+ and/or Zn ions transporters by their excess transferring of ions into the vacuole [CDF transporters in plant cells are commonly considered metal tolerance proteins (MTPs), which can be clustered in seven distinct phylogenetic classes, according to the exclusivities in transported special metals: Zn-CDFs in groups 1–4, 5, and 12 function as MTP1-MTP4, MTP5, and MTP12, groups 6 and 7 function as Fe/Zn-CDFs containing MTP6 and MTP7, and Mn-CDFs are placed in group 8 (MTP8) and 9 (MTP9-MTP11) . MTP proa sativa , Triticuaestivum , Populuschocarpa , Medicaghypogaea , Nicotiasiformis , and Vitvinifera . Previou vacuole .+2, Co+2, Zn+2, and Fe+2, resulting in the homeostasis of these metals in plant cells [Vitis viniera, the VvMTP1- VvMTP12 of the cell vacuole, participate in the transportation of metal ions producing environmental stress responses, particularly hyperosmotic stress [ShMTP gene from Stylosanthes hamata resulted in Mn tolerance in Arabidopsis. Additionally, the members of the heavy metals tolerance genes family were identified in P. trichocarpa, and their influence on reaction to heavy metals was screened [Regarding rice OsMTP1, it has been reported that MTP1 is presumably involved in the translocation of non- and essential divalent metals like Cdnt cells . Moreovent cells  as AtMTPnt cells , 26. In c stress . Howeverc stress  reportedscreened .Eucalyptus grandis (2n = 4x = 40), one of the top 500 species of Eucalyptus widely grown on saline and alkaline lands across the world, has received great attention to produce high-valued medicinal oil by aromatic leaves and timber [Eucalyptus grandis seedlings have a high capacity to accumulate heavy metal pollution in their root, as an adaptation system with hardly detrimental conditions, which would enable them to survive and bioaccumulate in response to heavy metals [Eucalyptus grandis provided an opportunity to analyze the EgMTP gene family at the genome-wide level.d timber . Previouy metals . Its comEucalyptus, according to our knowledge, there is a research gap in identifying the EgMTP genes family in Eucalyptus grandis and discovering their structure and evolutionary relationships of these sequences. To facilitate the perception of the potential roles of Eucalyptus MTPs, the expression profiling of known genes was evaluated in response to two important divalent metals (Cd2+ and Cu2+). This research aims to analyze the evolution of the MTP gene family in Eucalyptus grandis and understand further functional characterization of the MTP gene family in both plant compartments (roots and leaves) and response to heavy metal stress in a plant cell, which will provide a novel research condition to understand the molecular mechanism of metal transport and homeostasis and eventually will help to identify the tolerant- heavy metal cultivars or species in future works.Irrespective of several researches on evaluating the variation of metal tolerance and accumulation in the well-known species of MTP gene family in the genome of E. grandis was performed based on the MTP gene sequences in A. thaliana [O. sativa [E. grandis genome. Finally, twenty MTP proteins of Eucalyptus, based on high similarities with the Arabidopsis MTP families, were designated for subsequent analysis. The candidate genes were nominated with a particular name, i.e. EgMTP1.4, EgMTP1.6, EgMTP1.5, EgMTP1.2, EgMTP1.1, EgMTP1.3, EgMTP11.2, EgMTP12, EgMTP2, EgMTP5, EgMTP1.7, EgMTP9.2, EgMTP7, EgMTP9.1, EgMTP9.3, EgMTP8.1, EgMTP4, EgMTP6, EgMTP8.2, and EgMTP11.1. The features of the EgMTPs protein sequences were investigated in details (Table EgMTP9.3) to 884 (EgMTP12), and the molecular weight of EgMTP proteins was from 36.07 kDa (EgMTP9.3) to 97.32 kDa (EgMTP12). Most of the EgMTPs showed a low isoelectric point from 5.03 (EgMTP11.1) to 8.62 (EgMTP9.3), 17 EgMTP members with low isoelectric point (pI < 7) and 3 EgMTP members with a relatively high isoelectric point (pI > 7). The results of predicted TMD numbers with cytosolic N and C termini exhibited a variable range, in which most of the EgMTP protein sequences had 4–6 TMDs , which contained seven groups, i.e. groups 1–4, 5, 6, 7, 8, 9, and 12 based upon their phylogenetic relationship and previous results reported by Montanini et al. [MTPs were placed in the Zn-MTP sub-families, containing 11 EgMTPs , and then Mn-MTP sub-families, including EgMTP8.1/8.2, EgMTP9.1/9.2/9.3, and EgMTP11.1/11. Finally, two members containing EgMTP6 and EgMTP7 were clustered in the Zn/Fe-MTP sub-family  and 4  EgMTPs, respectively. Chromosomes 02, 10, and 11 had two genes each, each of the four chromosomes  possessed only one gene, whereas no EgMTP was recognized in the chromosomes 08 and 09. Noticeably, all EgMTP genes on the same chromosome did not separate at a high distance. Six paralogous pairs of EgMTP were located on chromosome 05, which contained EgMTP1.1 to EgMTP1.6 in the tandem arrangement with above 80% similarity percentage. As indicated in Table MTP gene family members between chromosomal pairs of E. grandis was caused by the segmental duplication event, like what occurred in EgMTP8.1/EgMTP8.2 from the chromosomes 02/11 and EgMTP1.5/EgMTP1.7 from the chromosomes 04/05. Additionally, tandem duplication was detected among almost more EgMTP genes, which included EgMTP1.5/ EgMTP1.3, EgMTP1.5/ EgMTP1.1, EgMTP1.5/ EgMTP1.4, EgMTP1.5/ EgMTP1.2, EgMTP1.5/ EgMTP1.6, EgMTP1.3/ EgMTP1.1, EgMTP1.3/ EgMTP1.4, EgMTP1.3/ EgMTP1.2, EgMTP1.3/ EgMTP1.6, EgMTP1.1/ EgMTP1.4, EgMTP1.1/ EgMTP1.2, EgMTP1.1/ EgMTP1.6, EgMTP1.4/ EgMTP1.2, EgMTP1.4/ EgMTP1.6, and EgMTP1.2/ EgMTP1.6. The ratio of Ka and Ks of all gene duplication pairs was less than 1 (Table ) in 20 nucleotide sequences covered T/U to C. The mean intra- and inter-group differences among the three sub-families showed the maximum difference between Mn-MTPs and Zn-MTPs and the minimum difference in Mn-MTPs.The distribution of the mes Fig. . The maxEgMTP family, the organizations of exon–intron of EgMTP genes were investigated . They were annotated to Pfam database to have cation efflux function as six transmembrane domains (TMDs) and C-terminal ions transport domain (CTD) using the MEME tool , while the other 13 members of EgMTP proteins contained motif 8 encoded ZT_dimer, PF16916 (the zinc transporter dimerization domain). All the EgMTPs were members of the Mn-MTP sub-family, composed of eight motifs with zinc transporter dimerization function Also, the MTP1.1 to MTP1.6 members of the Zn-MTP sub-family included a ZT dimer which contained a Zn transporter dimerization domain. The greatest number of the motifs (eight motifs) were found in MTP1.1 to MTP1.6, which also contained three CDF and Zn transporter dimerization domains, while MTP5 had only one motif.It was predicted thatool Fig. . Figure EgMTP genes, a search for cis-elements related to different stress responsiveness in the 1.5 kb of 5'-flanking region of most EgMTP genes was also observed by the PlantCare program (Supplementary Table Scis-regulatory elements (CREs) in the upstream regions of whole EgMTP genes, which were divided into four main categories based on the regulation of gene expression in response to different factors, i.e. including several cis-elements in core promoter regions and light-, developmental-, hormone-, and environmental stress-responsive cis-elements , except for EgMTP1.2 and EgMTP6, were more abundant than the other phytohormone-responsive elements. Most of the cis-acting elements in the Mn-MTP sub-family were phytohormone-responsive, while the elements in the Zn/Fe-MTP and Zn-MTP sub-families were light-regulated. A few cis-active motifs, including CAT-box, HD-Zip 1, and GCN4, participated in the regulation of the developmental process in a number of EgMTP genes. Different stress-responsive CREs, i.e. circadian, ARE, MBS, TC-rich repeat, GC-motif, LTR, and O2 site were also identified in most EgMTP gene promoters. (Putative low-temperature responsive element (LTRE), anaerobic response elements (ARE), and O2 site (involved in the O2 regulation) were found more abundant than other stress-responsive CREs. Circadian , MBS (MYB binding site involved in drought-inducibility and flavonoid biosynthesis), TC-rich repeats (anaerobic response elements), and GC-motif (oxidative responsive element) were also other stress-responsive CREs in EgMTP genes. Totally, four hundred and twenty-five stress-responsive CREs were found in all EgMTP genes, indicating that the expression rate of EgMTP genes at mRNA level may be possibility affected by various stresses.To better understand the post-transcriptional mechanism of EgMTP coding sequence-based miRNAs showed a total of 36 miRNAs and 14 EgMTPs were predicted to be the targets for cleavage inhibition  in the cation transmembrane transporter and zinc ion transmembrane transporter (17/85%). The total scores of MTP protein functions in various biological workflows were 8/40% in cellular cadmium, 8/40% in zinc, and 8/40% in iron homeostasis. Diverse genetic indexes such as ENC, RSCU, CBI, SChi2, etc. , EgMTP2 , EgMTP1.6 , EgMTP8.1 (CGU-R), EgMTP1.4, EgMTP1.2, EgMTP1.1 (CCC-P), EgMTP9.2, and EgMTP9.3 (GAC-D). Finally, the codon usage pattern of EgMTP genes was exhibited by the heatmap analysis 4-(AAAT)3 and (ATC)4-(CAT)4 sequences, respectively. Among them, EgMTP1.7 and EgMTP5 contained seven SSRs; (TTG)4- (AATT)4- (ATTT)3- (AATT)3- (TTTA)3-(TGATG)3-(GGTCAC)3 and (CT)8-(TG)8-(AAG)4-(TCA)4-(AAAG)3- (TTAT)3-(GAGCA)3, respectively. Three SSRs were identified in EgMTP12 and EgMTP4 and four and five SSRs were predicted in EgMTP9.3 and EgMTP8.1, respectively. The highest SSR type belonged to the tri-nucleotide SSRs with 22SSRs, followed by di-nucleotide SSR with 13 SSRs, tetra-nucleotide SSR with 20 SSRs, and penta-nucleotide SSR with 3 SSRs.The analysis of SSR markers showed that the 57 SSRs were predicted in 18 es Table . Almost MTP genes of F1 hybrid E. grandis × E. urophylla  and E. grandis TAGoo14 clonal genotype in different tissue samples was drawn  of inoculated E. grandis with C. austroafricana showed a higher EgMTP gene expression in control plants. It was concluded that the EgMTP family members were not essentially affected by C. austroafricana, whereas, the positive expression correlation of EgMTPs was observed in the stems of E. nitens inoculated with P. cinnamomi. When two biotic conditions were mentioned, the highest gene expression was observed in EgMTP11.2 and the lowest gene expression was seen in EgMTP1.4 and EgMTP1.3. Also, in comparison with the resistant genotype, the stems of susceptible E. grandis in the samples seem to be uninfected and infested with L. invasa, which reveals a higher gene expression. This demonstrated the negative correlation of the EgMTP genes expression with gall wasp caused by L. invasa.In this study, RNA-Seq data from the three types of biotic stresses were examined for different lar Fig. C. HoweveEgMTP genes regulation in response to excessive heavy metal treatment stress, the relative expression of the three genes EgMTP5, EgMTP6, and EgMTP11.1 at different concentrations of CuSO4 and Cd (NO3) in root and leaf tissues was investigated using qRT-PCR analysis  and steadily incremented at 100 mM , followed by boosting excessive concentration level at 300 mM  in the treated leaves. With regard to the root tissue, the highest frequencies of EgMTP5, EgMTP6, and EgMTP11.1 transcripts were observed under C3 circumstances , while at lower levels were achieved C1  and C2 , respectively  treatment as Cu2+ stress, as the transcript amounts in EgMTP5, EgMTP6, and EgMTP11.1 gradually increased, showing the peak values of ~ 2.91, 4.02, and 5.95 until C3, respectively.To get an initial insight into sis Fig. . On the EgMTP5, EgMTP6, and EgMTP11.1 genes, and vice versa.According to the results, the greater the excessive heavy metal stress, the higher the transcript abundance of MTP gene family has previously been reported in diverse plant species, including Medicago truncatula [N. tabacum [V. vinifera [P. trichocarpa [Arachis hypogaea [Solanum lycopersicum [MTP family in E. grandis and concurrent assessment of the effect of different concentrations of heavy metals on the transcript amounts of the genes known to be expressed in response to metal stress.Plant metal tolerance proteins are membrane divalent transporters that play a substantial role in the specific transport of different types of heavy metal ions as part of the response-specific metal toxicity. In addition to their pivotal function in maintaining mineral plants, changes in their transcript amounts can be considered environmental biomarkers in examining the heavy metal pollution level. The identification of uncatula , N. taba tabacum , V. vinivinifera , P. tricchocarpa , Arachishypogaea , and Solpersicum . HoweverEgMTP genes were determined, selected, and nominated according to both their respective orthologous relationship and high similarities with the Arabidopsis MTP families. The diversity of physicochemical characteristics of the EgMTP genes, including length of CDS, molecular weight, pI, and subcellular localization, is consistent with previous reports demonstrating that the MTP gene family members play a role in several pathways and biochemical networks leading to resistance against metal stress [EgMTPs were localized in the vacuole membrane, so it could be concluded that EgMTP might act as cation transporters [Herein, 20 putative l stress , 33, 36.sporters .EgMTPs have a close evolutionary relationship with other plant species based on amino acid similarities and may have the same tasks [EgMTP gene family members have orthologous relationships with AtMTPs, except AtMTP3 , in which about eleven, seven, and two MTPs belong to three major substrate-specific sub-families, i.e. Zn-MTPs, Mn-MTPs, and Zn/Fe-MTPs, respectively , which go along with the report of prior research reporting similar results for the MTP families in various plant species [EgMT12 with 12 TMDs, including CitMTP12 [EgMTPs were predicted to be localized in the vacuoles as primary sites for various processes like detoxification, stopping, and storage of metals. This result was validated by previous studies, demonstrating the VvMTP and TaMTP members were localized in the vacuoles [EgMTP proteins, overall, the numbers of cation efflux domain were highest in the 19 EgMTPs proteins, while 13 members of the EgMTP proteins contained specific motifs encoding the zinc transporter dimerization domain  were predicted as miR473a-5p targets and were shown to regulate the genes in drought-responsive genes in Populus plants [EgMTP genes, including EgMTP2 and EgMTP1.7, were suppressed by miR156, playing dominant roles in regulating plant development and detoxification of heavy metals, such as cadmium, aluminum, manganese, and arserinc in Brassica napus, Oryza sativa, Glycine soja, Populus vulgaris, and Brassica juncea [EgMTP2 was also predicted as the target of miR395p-3p, regulating sulfate starvation and balancing the ion concentration in plants [Eucalyptus genome and clarify their functions in regulating metal tolerance in Eucalyptus plants. Gene ontology is an essential analysis to anticipate the recognized subcellular localization, molecular function, and biological process in organisms [EgMTP protein molecules in cation transmembrane transporter, especially zinc ion, which is suggestive of a substantial role in the transporting of heavy metals  to 1 (maximum codon bias) [The codon usage pattern (CUP) analysis was originally proposed to predict the evolutionary pattern of genome and is used to evaluate the accuracy or efficiency of translation. Our results showed the ENC indices with the values 57 to 59.7, suggesting that the synonymous codons were possibly utilized to code the amino acids of the EgMTPs belonging to the Mn-MTPs sub-family , while ts codons . Most ofon bias) .EgMTPs were predicated to be tri-nucleotide SSRs with 22SSRs, followed by di-nucleotide SSR with 13 SSRs, tetra-nucleotide SSR with 20 SSRs, and penta-nucleotide SSR with 3 SSRs . For both leaves and roots, compared to the control, the transcription rate of the three genes slightly exhibited some positive alterations under excess Cu2+ and Cd2+ concentrations. In agreement with previous studies [EgMTPs were more affected by metal ion exposure in the roots than in the leaves, suggesting its important role in the translocation of divalent metals. Previous results have shown that SlMTP genes exhibit distinctive responses in either plant leaves or roots in response to different concentrations of heavy metals. The upregulation of EgMTP5, EgMTP6, and EgMTP11.1 might contribute to the Cu+2 and Cd+2 transposition via suppression of vacuolar and vesicular ions exposed to excessive metal. SlMTP1, SlMTP3, SlMTP4, SlMTP8, SlMTP10, and SlMTP11 display maximum expression responses when exposed to different concentrations of heavy metals [2+, Ni2+, Co2+, Cd2+, Fe2+, and Mn2+ [EgMTP genes were determined in Eucalyptus grandis, which were phylogenetically clustered in seven groups  as a property of three main substrate-specific clusters . It seems all EgMTP genes experience expansion and gene loss by gene duplication events, among which EgMTP1.5/ EgMTP1.7 and EgMTP8.1/ EgMTP8.2 might be segmentally duplicated genes. Moreover, we ascertained different CRE elements associated with light, phytohormone, and abiotic stresses in the 5’-flanking regions of EgMTP genes, indicating that they might take part in activated signaling pathway under different environmental stimuli. Also, a total of 36 miRNAs corresponding to 14 EgMTPs were identified, which play dominant roles in regulating plant development and heavy metal detoxification. Our results revealed that most of EgMTPs participated in processes related to heavy metal transportation and ion homeostasis. The transcription activity of EgMTP5, EgMTP6, and EgMTP11 genes seems to be positively upregulated under excessive Cu2+ and Cd2+ concentrations. Our results showed the essential roles of EgMTP proteins in the absorption and translocations of heavy metals in Eucalyptus species, which could be useful in selecting a variant with high resistance to heavy metals and improving modified cultivars which act better in stressful environments in future.Based on the results obtained above, most of  studies , we founy metals . Previouy metals , 18 by iand Cd NO. For botand Mn2+ . In concEgMTP genes in Eucalyptus grandis, the protein sequences of Arabidopsis thaliana  from the Arabidopsis Information Resource (http://.arabidopsis.org) [Oryza sativa  from Rice Genome Annotation Database (http://rice.plantbiology.msu.edu/) [−10 was investigated via Phytozome database, and these sequences were used as queries to compare with the Eucalyptus genome [https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan) for Hidden Markov Model (HMM) profiling of PF01545, the cation efflux domain, on the Pfam website. Finally, the HMMER-EMBL-EBI database was used to confirm the predicted domain [To identify sis.org)  and Oryzsu.edu/)  were obtEucalyptus, Populus, Arabidopsis, and Oryza were aligned by ClustalX 2.0.8 with default parameters [MTP gene family from Eucalyptus was nominated as EgMTP based on sequence similarities, and Matrix Global Alignment Tool (MatGAT) software was used to confirm the evolutionary relationship with AtMTPs and OsMTP in Arabidopsis and Oryza, respectively [The MTP protein sequences of rameters . To disprameters . The ideectively .http://web.expasy.org/protparam) [The physiochemical features of MTP family protein sequences were analyzed to obtain molecular weights (kDa), isoelectric points (pI), etc. by ProtParam tool (otparam) . Furtherotparam) . TMD, mootparam) . Also, totparam) . The funEucalyptus genetic database was utilized to identify the location of the EgMTP genes on chromosomes, which were used to create a location image, as linkage groups on different chromosomes using TBtools genetic mapping software. Furthermore, the plant genome duplication database (PGDD) was analyzed to identify segmental duplication. Tandem duplication was considered with the following parameters: maximum distance 20 genes with > 75% coverage and > 75% similarity in aligned sequences [The equences , 89. Theequences . The proequences  along wiequences .EgMTP genes was carried out to investigate the organization of intron/exon and the number of exons and introns in these genes using both sequences of gDNA and CDS by the gene structure display server (GSDS) [cis-acting regulatory elements (CREs) of the EgMTP gene promoter were identified using PlantCARE database (http://www.bioinformatics.psb. ugent.be/webtools/plantcare/html/) [EgMTP gene family using the BatchPrimer3v1.0 server [The structural analysis of the predicted r (GSDS) . All cise/html/) . Also, t0 server . miRNAs 0 server .https://string-db.org) [All identified EgMTP protein sequences were used to analyze GO annotation according to all the three GO sub-vocabularies, i.e. cellular component, biological process, and molecular function, using STRING software (-db.org) . Codon u-db.org) . Finally-db.org) .EgMTP genes of six organs/tissues types  from E. grandis TAG0014 clonal genotype were identified. Also, the expression profiles of MTPs tension wood xylem, upright control xylem (cambium), immature xylem, mature xylem, phloem, young leaves, mature leaves and shoot tips tissues from F1 Hybrid, E. grandis × E. urophylla , were identified using RNA-seq data obtained from EucGenIE (http://eucgenie.bi.up.ac.za) [Eucalyptus species were obtained from the EucGenIE database under the following conditions. The compatible third-generation E. nitens seedling stems were inoculated with Phytophthora cinnamomi, and mock inoculation was performed with sterile cV8 agar plugs over five-week post-inoculation from 1.5 cm above and below the inoculation sites [E. grandis, GC540, and TAG5 genotypes were grown in a field cage insectarium, respectively. After four months, the E. grandis clonal replicates were split into uninfected and infested Leptocybe invasa groups. This required the gall wasp and RNA of three biological replicates of six plants which were individually extracted from leaf midrib tissue harboring insect ovipositor sites after seven days [E. grandis ZG14 and the incompatible (resistant) clone E. grandis TAG5 were inoculated with Chrysoporthe austroafricana  strain CMW2113 causing Eucalyptus stem canker [MTP gene expression was also analyzed as a unit of normalized transcripts per million (TPM) (fragments per kilo-base). The changes in the gene expression of EgMTP genes were analyzed using Pearson correlation and a complete linkage algorithm.The expression profiles of the p.ac.za) –103. Theon sites . Also, tven days . In anotm canker . Subsequ2 values of different vegetative organs/tissues and were clustered into a heat map by MeV 4.0 software [Developmental and stress-associated expression data were used to draw hierarchical clustering according to logsoftware .E. grandis were obtained from the Seed and Plant Improvement Institute of Iran (SPII). First, the seeds were subjected to the disinfection process, submerged in 70% alcohol for 15 s, and then presoaked in benomyl 1 × 1000 solution for 20 min. Then, to eliminate any interaction related to soil and to control all conditions required for accessing any nutrients, the germination of seeds was carried out in 1–2 mm silica that was thoroughly washed and sterilized in an oven and transferred into individual pots containing PittMoss soil to continue their growth with optimum circumstances for 2 additional months. Different heavy metal concentrations of 50, 100, and 300 mM of CuSO4 and 10. 50 and 200 mM of Cd (NO3)2 were performed on the two-month-old seedlings treated in normal Hoagland's nutrient solution. The experiment was performed in a completely randomized design with three replications. The sampling of leaves and roots of plantlets was performed after 18 days of metal exposure. Then, they were harvested and directly preserved in liquid nitrogen and stocked at -80 °C until further analyses.In general, the seeds of http://frodo.wi.mit.edu/primer3/) was used to analyze the specific primers of the three candidate genes: EgMTP5, EgMTP6, EgMTP1, and Elongation factor S-II was used as a reference gene  reagent was utilized for the total RNA isolation from all treated seedlings (roots and leaves), and subsequently, first-strand cDNA synthesis was performed using a Kiagene Kit . Primer3 software (specimen .Table 5Ap ≤ 0.05). Also, the values were expressed as mean ± standard deviation (SD).Experimental results were the means of three replicates of heavy metal treatments. The data were analyzed by analysis of variance (ANOVA) to assess the significant differences between treatments using Duncan's multiple range tests (Additional file 1: Supplementary Table 1.


The dysregulation of intracellular and extracellular environments as well as the aberrant expression of ion channels on the cell membrane are intricately linked to a diverse array of degenerative disorders, including intervertebral disc degeneration. This condition is a significant contributor to low back pain, which poses a substantial burden on both personal quality of life and societal economics. Changes in the number and function of ion channels can disrupt the water and ion balance both inside and outside cells, thereby impacting the physiological functions of tissues and organs. Therefore, maintaining ion homeostasis and stable expression of ion channels within the cellular microenvironment may prove beneficial in the treatment of disc degeneration. Aquaporin (AQP), calcium ion channels, and acid-sensitive ion channels (ASIC) play crucial roles in regulating water, calcium ions, and hydrogen ions levels. These channels have significant effects on physiological and pathological processes such as cellular aging, inflammatory response, stromal decomposition, endoplasmic reticulum stress, and accumulation of cell metabolites. Additionally, Piezo 1, transient receptor potential vanilloid type 4 (TRPV4), tension response enhancer binding protein (TonEBP), potassium ions, zinc ions, and tungsten all play a role in the process of intervertebral disc degeneration. This review endeavors to elucidate alterations in the microenvironment of the nucleus pulposus during intervertebral disc degeneration (IVDD), with a view to offer novel insights and approaches for exploring therapeutic interventions against disc degeneration. With the growing population and aging trend, there has been a rapid increase in the number of individuals experiencing disabilities as a result of lower back pain ,2. The in rhythm . Comparek in CEP . The CEPk in CEP . Furtherk in CEP . Due to k in CEP ,7,8. Thek in CEP ,11,12,13k in CEP ,15,16, wk in CEP ,18. Aquak in CEP ,33,34,35k in CEP ,37,38,39k in CEP ,44,45,462+ can be regarded as a versatile messenger that operates at various levels, including cellular, subcellular, and extracellular domains [2+ plays a crucial role in transmitting signals for various fundamental physiological processes such as cell cycle regulation, survival, programmed cell death, migration, and gene expression [2+ homeostasis plays a pivotal role in regulating critical cellular processes such as survival, growth, differentiation, metabolism, and apoptosis [human diseases are associated with dysregulation of Ca2+ homeostasis, including developmental disorders [2+ storage organelles, releasing Ca2+ through either the 1,4,5-triphosphate receptor (IP3R) or the ryanodine receptor (RyR) in response to various stimuli that generate intracellular Ca2+ signaling. This signaling pathway is responsible for regulating nearly every aspect of cellular function [Intracellular calcium signaling is widely recognized as one of the most extensively investigated second messenger pathways . Calcium domains . As a sepression . The maipoptosis . Severalisorders , hyperteisorders , cardiovisorders , diabeteisorders , Alzheimisorders  and cancisorders ,57. The function ,59.2+ is precisely regulated by a variety of processing enzymes, proteins, channels, and transporters that work together to fine-tune the time- and spatially-specific Ca2+ signal [2+ that has accumulated in the endoplasmic reticulum and other cell membranes [2+ across the endoplasmic reticulum membrane and induces Ca2+ release from both endoplasmic reticulum and mitochondria, thereby elevating cytoplasmic Ca2+ concentration [2+ release pathway located in the endoplasmic reticulum of cells, which regulates the flow of Ca2+ from intracellular reservoirs such as the endoplasmic reticulum into the cytoplasm. Activation of RyR occurs through an intracellular increased Ca2+-induced Ca2+ release (CICR) mechanism [2+ ATPase (SERCA) actively reuptakes released Ca2+ back into the sarcoplasmic/endoplasmic reticulum to maintain proper Ca2+ homeostasis [2+ from the ER into the cytoplasm, while SERCA is responsible for actively transporting Ca2+ back into the ER from the cytoplasm. Together, they ensure a coordinated balance of Ca2+ outflow from and inflow into the endoplasmic reticulum [The concentration of Ca+ signal . Inositoembranes . IP3R fantration . The RyRechanism . The sareostasis . The IP3eticulum ,65.2+ in CEP tissues increases concomitantly with the progression of intervertebral disc degeneration. The vertebrae in direct contact with CEP are likely to be the primary source of elevated Ca2+ levels within CEP. As the medium’s Ca2+ concentration rises, there is a corresponding concentration-dependent decrease in the expression of collagen I, collagen II, and aggrecan in CEP cells, along with an increase in the activity of catabolic enzyme ADAMTS-5. Additionally, in the case of an increase in Ca2+, there will be a decrease in the diffusion of glucose into CEP and NP tissues, as well as a reduction in the expression of proteoglycan in CEP tissues, leading to CEP calcification. However, calcium-induced disc degeneration can be reversed through co-culturing with cells using calcium-sensitive receptor (CaSR) antagonists or by suppressing the expression of CaSR [2+ concentration in disc degeneration.The calcium ion, being a crucial regulator of cellular function, also plays a pivotal role in the process of intervertebral disc degeneration. Studies have demonstrated that the concentration of Ca of CaSR . The stu of CaSR . Regardl2+ not only functions extracellularly, but also serves as a crucial intracellular second messenger. The studies have demonstrated that within the articular cartilage tissue and IVD, when the pH value falls within the physiological range, Ca2+ exhibits a remarkably strong affinity towards ASICs and can bind to the lateral side of the ASICs channel pore, consequently inducing channel closure. Upon pH reduction, H+ attach themselves to acid-sensitive sites on ASICs, thereby diminishing Ca2+’s binding affinity towards ASICs. As a result, Ca2+ dissociates from the channel pore, facilitating ASIC opening and subsequent cellular entry [2+ flow in bone marrow mesenchymal stem cells (BMSCs). Lower pH levels activated ASIC1a, leading to the infiltration of Ca2+ into BMSCs. An acid-induced increase in Ca2+ influx leads to Ca2+-mediated activation of calpsin and calcineurin. The cytosolic phosphatase calcineurin, activated by calpsin, plays a crucial role in cellular apoptosis. Activation of calpsin by Ca2+ further stimulates calcineurin, thereby facilitating caspase-mediated apoptosis [2+ plays a role in acid-induced damage to NPCs through ASIC1a channels. The expression of ASIC1a was significantly upregulated in degraded NPCs compared to healthy ones. When NPCs were cultured under acidic conditions, there was a notable increase in intracellular Ca2+, which could be reduced by the presence of psalmotoxin-1 (PcTX1), an ASIC1a blocker, or the absence of extracellular Ca2+. ASIC1a may potentially contribute to intervertebral disc degeneration. Activation of ASIC1a leads to an elevation in intracellular Ca2+ flux in NPCs, thereby playing a crucial role in NPCs necrosis, apoptosis, and stress-induced premature aging during the process of intervertebral disc degeneration [ASIC1, ASIC2, ASIC3, and ASIC4). The expression levels of ECM-related genes  were downregulated, while the expression levels of stem cell-related genes  were also decreased. Sa12b is a wasp peptide that inhibits Ca2+ influx to counteract the effects caused by acidic conditions [2+ in NPCs also significantly increased following hypoxia treatment. Hypoxia treatment induces alterations in Ca2+ concentration and upregulates ASIC3 expression. Furthermore, hypoxia and ASIC3 overexpression exert inhibitory effects on NPC proliferation, arresting the cell cycle at the G1 phase, promoting apoptosis, initiating autophagy, and upregulating HIF-1α and LC3 expression [2+ plays a crucial role in regulating the microenvironment of NPCs and influencing their growth under hypoxic conditions.The Caar entry . When expoptosis . Previouneration . In addinditions . In addipression . The fin2+ in endoplasmic reticulum stress induced by mechanical load has been highlighted in several studies. After prolonged mechanical loading, the endoplasmic reticulum stress (ERS) pathway is activated, leading to the binding of endoplasmic reticulum and mitochondria. At the interface between these two organelles, an IP3R-GRP75-VDAC1 complex exists. This complex facilitates a significant upregulation of Ca2+ levels in NPCs, from the endoplasmic reticulum to mitochondria. The elevated Ca2+ levels then polarize mitochondria, resulting in a reduction of intracellular ATP levels. Additionally, this process leads to the release of lactate dehydrogenase (LDH) and high mobility group protein 1 (HMGB1) into extracellular mediators. The process of exchanging Ca2+ between the endoplasmic reticulum and mitochondria leads to cellular bioenergy depletion and triggers compression-induced programmed necrosis in NPCs [2+ mediates the activation of PARP and induces an increase in cleaved PARP (C-PARP) expression. Additionally, Ca2+ facilitates the translocation of AIF from mitochondria to cytoplasm and, subsequently, into the nucleus. By promoting the PARP-AIF pathway, Ca2+ plays a crucial role in mediating programmed necrosis of NPCs under stress conditions. Additionally, compression can induce reactive oxygen species (ROS) accumulation in NP cells, leading to enhanced endoplasmic reticulum stress and mitochondrial Ca2+ overload that ultimately triggers programmed cell necrosis [2+ and upregulated levels of intracellular ROS. These series of changes contribute to the down-regulation of collagen II and aggrecan expression, as well as an exacerbation of IVD degeneration [The involvement of Ca in NPCs . DNA rep in NPCs . Apoptos in NPCs . During necrosis . In anotneration .2+ concentration and a significant decrease in ER lumen Ca2+ concentration, as well as activation of downstream apoptotic pathways and an increased rate of cellular apoptosis [2+ levels, IP3R, RyR, and IP3 expression in severely degraded NP cells. Conversely, there is a notable decrease in endoplasmic reticulum Ca2+ levels and SERCA activity. In terms of regulating ER Ca2+ release, inhibiting IP3R proves more effective than inhibiting RyR activity to prevent subcellular calcium redistribution and subsequent ER stress-induced apoptosis. They hypothesized that inhibiting the IP3R-mediated release of calcium from the endoplasmic reticulum could further suppress the RyR-induced release of calcium (known as Ca2+-induced Ca2+ release or CICR) [2+ can serve as a protective mechanism against AGEs-induced IVD degeneration, while inhibiting the release of endoplasmic reticulum Ca2+ may ameliorate AGEs-induced IVD degeneration.In addition to mechanical loading, advanced glycation end products (AGEs) also impact calcium homeostasis. AGEs are proteins or lipids that undergo glycation after exposure to sugar and are present in various cell types, affecting both extracellular and intracellular structure and function . Treatmepoptosis . Studiesor CICR) ,73. Treaor CICR) . In conc2+, Na+, and Mg2+ ions [2+ concentration and maintaining intracellular water balance, can be induced by mechanical stimulation, low osmotic pressure, as well as exposure to hot and acidic environments [bovine tail IVDs. In humans, degraded IVDs exhibit increased TRPV4 expression within the NP region compared to healthy discs, which is further promoted by decreased osmotic pressure. This heightened TRPV4 expression leads to an increase in intracellular calcium flux and interleukin-1β (IL-1β) production in IVD cells [2+ serving as the second messenger within the cell for further transmission of information. Notably, TRPV4 plays a pivotal role in regulating both calcium flux and matrix metabolism in AF cells.Transient receptor potential vanilloid type 4 (TRPV4) is a non-selective cation channel that responds to osmotic pressure and mechanical stimulation, allowing for the permeation of Cag2+ ions . The actronments . StudiesVD cells . In addiVD cells . After b2+ plays a crucial role in disc degeneration through its involvement in the Piezo1 channel. The study revealed that Piezo1 is an ion channel with seven transmembrane domains [FAM38A gene encodes Piezo1, a newly discovered mechanosensitive cation channel that facilitates the non-selective permeation of divalent ions such as Ca2+, Mg2+, and Mn2+ [2+ influx and thereby converting mechanical signals into chemical signals. This rise in intracellular calcium concentration triggers activation of the NF-κB pathway, leading to enhanced expression of NLRP3 and pro IL-1β. The Piezo1 inhibitor GsMTx4 attenuates the mechanically-induced upregulation of NLRP3 components, including NLRP3, ASC, caspase-1, and IL-1β, in nucleus pulposus cells [2+ serves as a mediator of the inflammatory response triggered by Piezo1 activation through mechanical stretching, ultimately leading to intervertebral disc degeneration. In addition to mechanical stretching, extracellular matrix stiffness can also act as a mechanical stimulus that activates Piezo1 channel opening and regulates Ca2+ influx. The latest research findings suggest that the activation of Piezo1 is triggered by extracellular matrix stiffness, resulting in an increase in intracellular calcium levels and ROS, which subsequently induces endoplasmic reticulum stress and ultimately leads to senescence and apoptosis of nucleus pulposus cells [Additionally, Ca domains . The FAMand Mn2+ . When thus cells . Ca2+ seus cells . A team us cells . The difrat models [The S100 protein family, a group of small acidic calcium-binding proteins, functions as either calcium sensors or extracellular factors to regulate diverse biological processes . It has t models ,87  and nuclear factor T (NFATs/TonEBP). Subsequently, CaN and NFATs promote CCN2 activity and expression. Thus, the extracellular signal is transmitted intracellularly through the Ca2+-CaN/NFAT-CCN2 signaling pathway to enhance CCN2 protein expression [Connective tissue growth factor (CTGF/CCN2) is a multifunctional protein belonging to the CCN family of stromal cell proteins, which governs cellular processes including proliferation, differentiation, adhesion, and various other biological phenomena. It actively participates in disease-associated pathways such as the Hippo pathway, p53 signaling, and NF-κB pathways, thus acting as a downstream effector that contributes to the development of inflammation, fibrosis, cancer progression, and other pathological conditions ,89,90,91pression .2+ concentration also contributes to the sensitization of dorsal root ganglion (DRG) neurons in a hypoxic and acidic environment. Hypoxia and acidosis can enhance the spontaneous calcium response in sheep DRG neurons. The direct exposure of DRGs to hypoxia and low pH resulted in a more pronounced increase in calcium flux compared to the indirect, simulated disc stimulation of DRGs. Transient elevations in cytoplasmic calcium concentrations reflect neuronal firing activity, while the proportion of cells exhibiting spontaneous calcium responses indicates the possibility of “spontaneous firing”. Notably, the growth spurt is more susceptible to alterations in calcium signaling than the neuronal cell body [2+ in nerve signal transduction. In summary, Ca2+ exerts a significant influence on regulating cellular physiological functions and forms a complex regulatory network. Precise regulation of Ca2+ is crucial for maintaining the stability of the microenvironment of nucleus pulposus cells  and H+ play pivotal roles in regulating these functions. At least seven subtypes of ASIC in mammals—ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4, and ASIC5—are reported to be encoded by five genes  [chicken ASIC1a channel reveals that a functional channel necessitates the presence of three subunits. The composition of these subunits within the functional channel can either be homomorphic, consisting of three identical ASIC subtypes, or heteromorphic, comprising different combinations of ASIC subtypes with distinct electrophysiological properties [2+ levels alteration, and high osmotic pressure [Maintaining acid-base balance is crucial for normal cellular physiology, yet various pathological conditions including ischemia, hypoxia, inflammation, and cancer can disrupt pH homeostasis . The H+ d Accn5) . Each suoperties . ASICs aoperties . Numerouoperties ,30,31,94pressure ,31.+ ions, due to the presence of negatively charged proteoglycans, leading to an acidic milieu. Furthermore, degenerated discs exhibit elevated lactate production as a consequence of pro-inflammatory cytokine activity [The intervertebral disc is the largest avascular tissue in the body . Researcactivity . Additioactivity ,99,100. activity . In the 2+ levels within NPCs, leading to the upregulation of ASIC3 expression and activation of the MAPK pathway. Consequently, this cascade results in cell cycle arrest at the G1 phase, inhibition of NPCs proliferation, initiation of apoptosis and autophagy processes, as well as upregulation of HIF-1α and LC3 expressions [MMP-3 and ADAMTS-4 gene expression, as well as a notable decrease in TIMP-2 expression [ASICs play a crucial role in modulating the microenvironment of nucleus pulposus cells and influencing their growth. Within the acidic milieu of the intervertebral disc, ASICs contribute to some extent to its degeneration. The expression levels of ASIC1 and ASIC3 gradually increase in degraded NP, while acidic conditions also induce upregulation of ASIC1 and ASIC3 expression in NP ,101. Morressions . The exppression . Similarpression . The acipression . Howeverpression ,101. Simpression .Oct4, Nanog, Jag1, and Notch1) [human NP-MSCs during intervertebral disc degeneration. Amiloride has been shown to protect NP-MSCs from acid-induced damage by blocking ASICs. Apoptosis also plays an important role in intervertebral disc degeneration [2+ levels, ASIC expression, LDH release, and cell damage in NPCs. Additionally, it stimulates apoptosis of NPCs by increasing the expression of Bax and cleaved caspase-3 proteins while decreasing the expression of Bcl-2. However, upon blocking the ASICs, the aforementioned process is reversed [2+ concentration and eventual apoptosis which can be halted by blocking ASIC1a channels [Furthermore, an acidic environment and upregulation of ASIC expression can induce inflammation and senescence in nucleus pulposus cells. The acidic microenvironment hinders the cell cycle progression of NP cells, leading to arrest in the G0/G1 phase. Consequently, there is a significant increase in the proportion of cells in the G1 phase along with elevated expression levels of p53, p21, and p16 proteins associated with aging. Additionally, IL-6 and IL-8 levels are also elevated, ultimately accelerating cellular aging ,25. More Notch1) . However Notch1) . After b Notch1) . Similar Notch1) . The acineration ,104. Acireversed ,8,101. Schannels  , decrease intracellular Ca2+ influx, and protect hNP-MSCs from proliferation inhibition and increased apoptosis caused by an acidic environment culture [2+ levels to suppress apoptosis [sheep dorsal root ganglion neurons, the combination of hypoxia and low pH led to a significantly enhanced spontaneous calcium response during neurite growth and augmented calcium response to bradykinin in DRG neurons compared to normoxic conditions and neutral pH [Sa12b, a  neurons . Additio culture . Some stpoptosis . In sheeutral pH . The aciASICs are susceptible to harmful factors such as an acidic microenvironment and lactic acid, which trigger subsequent processes that promote cellular adaptation to the harsh conditions of degenerated discs. In summary, an acidic environment can exacerbate intervertebral disc degeneration by increasing necrosis of NPCs, impairing proliferation leading to cell senescence, enhancing apoptosis, and augmenting extracellular matrix catabolism. Throughout this degenerative process, ASICs act as accomplices in disc degeneration caused by an acidic environment. Blocking ASIC receptors or inhibiting their expression could potentially delay the progression of disc degeneration. Acidic culture conditions can decrease the activity of NPCs, enhance cell matrix catabolism, and initiate cellular inflammation. Therefore, inhibiting ASICs may represent a promising approach for treating IVDD .rats revealed a gradual decrease in AQP-1 and AQP-3 expression with the progression of IVDD. In 2-month-old rats, AQP-1 and AQP-3 were predominantly localized in the inner layer of NP and AF, while weak immunostaining for AQP-1 and AQP-3 was observed in the outer region of AF. However, in 18-month-old rats, there was a significant reduction in the number of cells positive for both AQP-1 and AQP-3 within the IVD [rabbit lumbar NPs, with extracellular osmotic pressure and oxygen concentration exerting an influence on its regulation. Specifically, decreased osmotic pressure of the cellular environment resulted in down-regulation of AQP-1 expression, while decreased oxygen concentration led to upregulation. Notably, the expressions of collagen II and aggrecan were significantly reduced under conditions of decreased osmotic pressure or oxygen concentration; moreover, the combination of both factors led to a more pronounced reduction [human nucleus pulposus cells suggest a potential association between intervertebral disc degeneration and reduced expression of AQP-3. Inhibition of AQP-3 expression resulted in suppressed hNPCs proliferation and intensified extracellular matrix catabolism, while overexpression of AQP-3 inhibited the Wnt/β-catenin signaling pathway, significantly increased hNPCs proliferation, promoted aggrecan expression, and markedly decreased the expression of matrix-degrading enzymes ADAMTS-4 and ADAMTS-5 [Aquaporins (AQPs) play diverse roles in mammals, and their involvement in the transport of water and solutes is crucial for the proper functioning of various organ systems and cell types ,109. The the IVD . Studieseduction . AQP-1 eeduction . Therefoeduction . The finADAMTS-5 .mice but decreased in 22-month-old mice. Moreover, TonEBP-deficient mice exhibited fibrocartilage alterations and elevated levels of MMP-13, which are characteristic features of disc degeneration. Notably, TonEBP deficiency synergistically impairs disc health with aging [TonEBP, also known as activated T-cells nuclear Factor 5 (NFAT5), is implicated in the pathogenesis of various diseases including rheumatoid arthritis, atherosclerosis, diabetic nephropathy, acute kidney injury, hyperlipidemia and insulin resistance, autoimmune diseases such as type 1 diabetes and multiple sclerosis, salt-sensitive hypertension, and hepatocellular carcinoma ,113,114.th aging .rat NPCs. However, the knockdown of TonEBP expression reversed the hypertonicity-induced expression of both AQPs. Furthermore, fluorescence staining for AQP-1 and AQP-5 in the NP region was markedly reduced in TonEBP knockout mice [IL-1β and IL-6, indicating that TRPV4 signaling contributes to pro-inflammatory cytokine production in NPCs [Studies have demonstrated that exposure to a hypertonic environment significantly upregulates the expression of AQP-1 and AQP-5 in hNPCs, as well as AQP-1 in out mice . TonEBP out mice . It has  in NPCs . Aggreca in NPCs . The pro in NPCs .Notochordal cells (NCs) are the remnant cells of the notochords of all chordates originating in early embryogenesis and located in the center of the intervertebral disc ,118. TheHuman disc specimens consistently demonstrated the highest concentrations of iron, zinc, and copper, irrespective of degeneration status. Moreover, notable disparities in copper, magnesium, potassium, and calcium were observed between degraded discs and healthy ones [+ and K+ ions in the central region of the disc with lower concentrations at its boundaries. However, despite this disparity, the nucleus pulposus in the center of the disc contains fewer cells than the annulus fibrosus. The expression levels of HA and CS exhibit a positive correlation with sodium and potassium ion concentrations while cell density shows a negative correlation with them [Although the intervertebral disc is a relatively enclosed environment, its components exhibit complexity. thy ones . The conith them . These fhumans. It exhibits high expression levels in the brain, spinal cord, heart, kidney, ovary, and small intestine and is implicated in various physiological and pathological processes such as neuroprotection, epilepsy, depression, and pain perception [human intervertebral discs [The two-pore domain potassium channel TREK-1 is predominantly expressed in the central nervous system (CNS) of rception . Evidencal discs . Althoug2+ also plays a role in the degeneration of intervertebral discs. In nucleus pulposus cells, IL-1β can enhance the influx of Zn2+ into the cells, which subsequently leads to an increase in matrix metalloproteinases, indicating that elevated Zn2+ influx may contribute to the progression of IVDD [mice, accompanied by decreased glycosaminoglycan content and increased fibrosis. Tungsten also increases the expression of inflammatory cytokines IL-1β and TNF-α, as well as NGF and BDNF in intervertebral discs, while increasing pain marker calcitonin gene-related peptide (CGRP) expression [Additionally, several studies have suggested that Zn of IVDD . Recent pression , suggestpression . Howeverpression . The finPolE, as well as the catalytic subunit of DNA polymerase epsilon (Polε), along with two other Polε subunits, namely PolE2 and PolE3. Conversely, certain proteins involved in apoptosis, such as caspase-3 and caspase-8, exhibited a notable upregulation [human nucleus pulposus specimens revealed decreased expression of the iron transporter (FPN). Moreover, the down-regulation of FPN results in iron overload in human NPC cells, which is involved in TBHP-induced cell death and IVDD development [In a study of 217 patients with low back pain, it was discovered that serum ferritin levels can serve as an indicator for the severity of IVDD, with those having serum ferritin levels below 170.5 ng/mL exhibiting more severe IVDD . The samgulation . In addielopment . Other selopment . Thereforabbit model of disc degeneration induced by fine needle puncture, it was observed that the cartilage endplate underwent remodeling, the vasculature changed with disc degeneration, and there was reduced transport of small molecules into the degenerative disc [2O2 in cartilage endplate cells enhances the activity of alkaline phosphatase (ALP) and promotes the expression of RUNt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and collagen I, which are involved in the maturation and vascularization of cartilage. Furthermore, H2O2 treatment reduces levels of inorganic pyrophosphate (PPi) while increasing levels of inorganic phosphate (Pi). PPi effectively inhibits calcium/phosphate crystal nucleation and propagation, whereas Pi promotes mineralization [The hardening of the cartilage endplate and decreased permeability are considered significant factors contributing to disc degeneration . In a raive disc . Calciumive disc . The expive disc . Treatmelization . These flization . In concIn this article, we present a summary of the changes in the microenvironment of the nucleus pulposus during intervertebral disc degeneration. The microenvironmental homeostasis of the nucleus pulposus plays a crucial role in protecting the disc, as the disc is subjected to daily internal and external pressures. Environmental and internal pressures can affect various factors such as aquaporin (AQP), calcium and acid-sensitive ion channels (ASIC), Piezo1, transient receptor potential vanilloid type 4 (TRPV4), tension response enhancer binding protein (TonEBP), and metallic elements that influence the microenvironment where the nucleus pulposus resides. Various environmental factors, such as increased levels of reactive oxygen species or hypoxia, elevated calcium concentrations, accumulation of cellular metabolites, altered osmotic pressure, and the presence of metallic elements, are more or less present in the microenvironment surrounding the nucleus pulposus. Alterations in the microenvironment of the nucleus pulposus can induce cellular aging, apoptosis, inflammation, extracellular matrix degradation, endoplasmic reticulum stress, ferroptosis, and other pathological changes within nucleus pulposus cells. Furthermore, this may lead to cartilage endplate calcification and exacerbate the already compromised microenvironment of the nucleus pulposus, thereby establishing a vicious cycle. Disc degeneration, an age-related disease, has become increasingly prevalent. While there are numerous treatments available for this condition, the key to successful treatment lies in reducing adverse factors within the disc environment while maintaining a normal internal environment.2O2-induced intervertebral disc degeneration, apoptosis of nucleus pulposus cells, and degradation of extracellular matrix [The management of age-related disc degeneration involves various strategies aimed at reducing or mitigating the progression of disc degeneration. Hypertension, inadequate physical activity, and gout have been identified as potential factors contributing to disc degeneration, according to a study . Furtherr matrix . Simultar matrix . Furtherr matrix . The bacr matrix . Furtherr matrix . Therefor matrix ,151,152.r matrix ,155,156.


LEF1 and TCF3 are over-expressed in breasttumors regarding non-tumor samples, while TCF4 andTCF7 are down-expressed, with the gene’s methylation statusbeing associated with its expression dysregulation. All four transcriptionfactors presented significance at the diagnostic and prognostic levels.LEF1, TCF4, and TCF7presented a significant correlation with immune cell infiltration, beingassociated with the immune subtypes of less favorable outcomes. Altogether, thisresearch contributes to a more accurate understanding of the expression andclinical and biomarker significance of the LEF1/TCF transcription factors inbreast cancer.The LEF1/TCF transcription factor family is related to the development of diversetissue types, including the mammary tissue, and dysregulation of its expressionand function has been described to favor breast tumorigenesis. However, theclinical and biological relevance of this gene family in breast cancer is stillpoorly understood. Here, we used bioinformatics approaches aiming to reduce thisgap. We investigated its expression patterns in molecular and immune breastcancer subtypes; its correlation with immune cell infiltration, and itsprognostic values in predicting outcomes. Also, through regulons construction,we determined the genes whose expression is influenced by these transcriptionfactors, and the pathways in which they are involved. We found that LEF1(TCF1α), TCF3 (TCF7L1),TCF4 (TCF7L2), and TCF7(TCF1) represent the LEF1/TCF family, a group of nuclearDNA-binding transcription factors. These proteins regulate the expression of a largesum of targets through their multiple binding sites and splicing variants . This analysis only included cancer types with at least tennon-tumor samples available. The analysis of variance (ANOVA) was performed toaccess the differential expression in the comparisons . The same statistical approach was performed to examine theexpression of LEF1, TCF3,TCF4, and TCF7 in the breast cancermolecular subtypes, first applying a T x NT comparison to each subtypeseparately, and after a comparison between the tumor samples of each subtype (Tx T). Also, using the GEPIA2 database, we investigated the mRNA levels ofLEF1, TCF3, TCF4, andTCF7 across different breast tumor stages .GEPIA2  is a webLEF1, TCF3, TCF4, andTCF7 to discriminate tumor breast samples from non-tumorsamples. The performance of each gene was obtained by receiver operatingcharacteristic curves , andquantified by the area under de curve (AUC).Using the TCGA mRNA data and the binary regression model implemented in the IBMSPSS Statistics (v.26) software, we tested the potential ofLEF1 (Antibody:CAB019405), TCF3 (Antibody: CAB018351), TCF4(Antibody: CAB020722), and TCF7 (Antibody: CAB019402) proteinsin tumor and non-tumor breast samples. The protein expression levels weredefined based on the staining intensity . Weselected the tumor samples with both stronger and weaker staining for comparisonwith the non-tumor samples. The Human Protein Atlas (HPA)  is an onLEF1, TCF3, TCF4, andTCF7 and the breast cancer prognostic factors ER (ER+/ER-),PR (PR+/PR-), HER2 (HER2+/HER2-), nodal status (negative/positive) and patientsage (≤51 and >51). TP53 status (mutated/wild-type), PAM50/TNBC status, Nottingham Prognostic Index (NPI) and ScarffBloom & Richardson grade (SBR) were also evaluated . Next,Kaplan-Meier survival analyses were performed to evaluate the associations ofLEF1, TCF3, TCF4, andTCF7 with overall survival (OS), distant metastasis-freesurvival (DMFS), and disease-free survival (DFS) . Groups ofhigh and low expression were defined using the median value. ThroughBc-GenExMiner, we also investigated the expression of these genes accordingly tothe histological subtypes of breast cancer .Bc-GenExMiner (v.4.5) is a statistical tool for mining transcriptomic breastcancer data from DNA microarrays and TCGA samples . Using tLEF1,TCF3, TCF4, and TCF7genes was determined through UALCAN online tool . Following to the database analysis pipeline, all thecorrelations were adjusted by tumor purity. The Tumor Immune Estimation Resource (TIMER) database  is an onLEF1, TCF3,TCF4, and TCF7 were investigated acrossthe immune subtypes of breast cancer, using the data and subtype classificationfrom TCGA .In the TISIDB web portal , the expLEF1,TCF3, TCF4, and TCF7.Firstly, we calculated the mutual information (MI) between each TF and allpotential targets. Afterward, we applied the MI-based algorithm of theReconstruction of Accurate Cellular Networks (ARACNe) method (RTN is an R package available in the Bioconductor open-source software  that tes) method  to remov2FC ±0.58; P-value <0.05).The entire process resulted in consensus regulatory networks, which include a MIvalue for each TF- gene association combined with a sign (“+” or “-”) thatrepresents the direction of Pearson’s correlation between the pair. Theparameters used in the network construction were 1000 permutations, a P-valuecutoff of 0.01, and 100 bootstraps. The input data comprised a gene expressionmatrix originated from the TCGA-BRCA data, containing only the differentiallyexpressed genes identified by GEPIA2 .The molecular signatures database (MSigDB)  is a webResultsLEF1, TCF3, and TCF7 werefrequently over-expressed in several cancer types, while TCF4was commonly down-expressed. More detailed gene expression data are displayed inWe used the GEPIA2 database to explore the mRNA levels of the LEF1/TCFtranscription factor family members, comparing the differences in theirexpression between tumor and non-tumor tissue samples of 16 cancer types. Thesegenes were found deregulated in cancer, with expression levels at least 1.5folds altered in tumor tissues .LEF1, TLEF1 and TCF3 are over-expressed in tumorsamples, and TCF4  and TCF7  are down-expressed (TCF7 (AUC = 0.844) have the mostpromising discriminative potential do differentiate tumor from non-tumor breastsamples, followed by TCF4 (AUC = 0.636), TCF3(AUC = 0.539) and LEF1 (AUC = 0.515). In the T x NT breast cancer comparisons, GEPIA2 shows that xpressed . To deteTCF3 and down-expression of TCF4 andTCF7 in breast tumors at levels consistent with that ofmRNA. Controversially, LEF1 showed stronger staining innon-tumor than in the tumor tissue.Also, to determine if the mRNA expression matches the protein levels, we used theHPA database to analyze the immunohistochemical staining of breast tumor andnon-tumor tissues . We founLEF1 was over-expressed in tumors of all the subtypes;controversially, its promoter region was found hypermethylated in basal andluminal tumors. TCF3 was hypomethylated in basal and HER2enriched tumors, but not in luminal tumors. Concordantly, TCF3showed no significant differential expression in both luminal subtypes but wasover-expressed in basal and HER2 enriched tumors. TCF4presented no differential expression in luminal A tumors but was down-expressedin the other three subtypes. Regarding methylation, TCF4 washypermethylated in all tumor subtypes. TCF7 presenteddown-expression in tumors of all subtypes and was hypermethylated in luminal andHER2 enriched tumors. Moreover, The T vs. NT comparisons were further performed by verifying the methylationstatus in the gene promoter region  -D, and LEF1, TCF3, TCF4, andTCF7 presented lightly high expression in invasive lobularcarcinoma (ILC) type, while TCF4 had a lower expression inmucinous type    -D. LEF1LEF1, TCF3,TCF4, and TCF7 were evaluated according tothe five classical breast cancer prognostic factors - ER, PR, and HER2 status,age, and nodal status; the TP53 status and PAM50/TNBC statuswere also included in the analysis . The mRNAexpression levels of analysis .LEF1 was significantly associated withpositive ER/PR status and HER2 negative status (P < 0.0001), andTCF7 had its lower expression associated with ER/PRpositive and HER2 negative tumors . In contrast, lowexpression of TCF4 was related to negative ER/PR status (P <0.0001), and higher levels of TCF3 were associated withnegative ER/PR status and HER2 positive status .Concordantly, LEF1 and TCF4 were positivelyassociated with Non-basal-like/Non-TNBC tumors , whileTCF3 and TCF7 were positively associatedto basal-like/TNBC tumors . The high expression of TP53 status and nodal status were alsoanalyzed, highlighting that LEF1 had a positive correlationwith wild type TP53 tumors ;TCF3 presented higher levels in ≤ 51 years patients (P <0.0001) and a positive relation with mutated TP53 tumors; TCF4 showed a lower expression in > 51years patients  and in TP53 mutated tumors, and TCF7 presented lower expression in >50 years patients , and TP53 wild type tumors.The parameters age, LEF1, TCF3, TCF4, andTCF7 was accessed using bc-GenExMiner (v.4.5), searchingfor associations between their expression levels and overall survival (OS),disease-free survival (DFS), and distant metastasis-free survival (DMFS).Considering its associations with clinicopathological and molecular parameters ofthe disease, together with the possibility that its deregulated expression inbreast cancer may impact tumorigenesis, we investigated the potential value ofthe LEF1/TCF transcription factors as prognostic markers. The prognostic valueof LEF1 was associated with a better OS considering all thesamples  using the TIMER database. Inaddition, we observed their expression pattern in the immunologic subtypes ofbreast cancer using the TISIDB web source. We evaluated the correlation between LEF1 was related to the infiltration of immune cells, showing anegative association with tumor purity , andsignificant-positive associations with five cell markers , except for B-cell infiltrations   and, except forB-cells infiltration , presented positivecorrelations with the other five tumor-infiltrating immune cell markers  , and positively with tumor infiltration by fiveimmune cells , but not with macrophages  (< 0.05) .TCF4, a < 0.05) . TCF7 wa= 0.165) . In thise <0.05) .LEF1, TCF3,TCF4, and TCF7 according to differentimmune subtypes of breast cancer are displayed in LEF1 and TCF4 weremostly expressed in the inflammatory and TGF-beta dominant subtypes. Incontrast, TCF3 was expressed highly in wound healing andIFN-gamma dominant, and TCF7 in IFN-gamma in dominant andinflammatory subtypes.The expression levels of LEF1, TCF3, TCF4, andTCF7 associations with 5269 breast cancer differentiallyexpressed target genes . These genes potentially have itsexpression influenced by the LEF1/TCF transcription factors. The regulonspredicted for LEF1 and TCF3, bothover-expressed in breast tumor samples, included 640 and 2421 genesrespectively, while the down-expressed TCF4 andTCF7 presented 3109 and 2284 genes in its regulonsrespectively. To retain only the most significant associations for theenrichment analysis, 5% of the most positive and 5% of the negative associations were filtered and maintained. Thefinal regulons included 801 differentially expressed target genes:LEF1 filtered regulon was composed of 64 genes; theTCF3 filtered regulon presented 242 genes;TCF4 retained 311 and TCF7 228 genes composed of28 genes.The MSigDB analysis showed that the genes present in the regulons weresignificantly enriched in pathways and biological functions associated withcarcinogenesis  . Figure LEF1 regulon was mainly associated with cell cycleregulation, RHO GTPase signaling, chromosome maintenance, and processes relatedto the CCT/TriC chaperonins functions , including degradation and organization of ECM,collagen degradation and trimerization, and MET signaling ,however, varied greatly between cancer types, indicating a possible tissue-dependenttumorigenic action. Regarding the biomarker potential in breast cancer, our resultssuggest that LEF1, TCF3, TCF4, and specially TCF7, have significant diagnostic valueto distinguish breast cancer patients from healthy individuals and a role insubtyping insight. A pan-cancer view revealed that LEF1 with the expression of ER/PR and activation of the Wntpathway in luminal subtypes, as well as a negative correlation betweenLEF1 and HER2 expression, indicating that LEF1tends to mediate tumor cell invasion mainly in tumors positives to ER/PR and lackingHER2 over-expression  tumors. TCF3 also appears over-expressed inbreast tumor tissues, but when subgrouping tumors by subtypes, TCF3showed higher expression only in basal and HER2 enriched subtypes, corroboratingprevious observations of over-expression of TCF3 in ER- tumors andits association with basal-like tumors , while TCF4 and TCF7 weremainly down-expressed in the lymphocyte depleted subtype, a subtype with mixedsignatures (LEF1 in augmentation of the levels of immune infiltrating cellsin breast microenvironment, and low expression of TCF4 andTCF7 to ablation of immune cells infiltration.TCF3 was highly expressed in wound healing and IFN-gammadominant subtypes, but with a non-significant correlation with tumor purity.Together, these results suggest a relevant role of LEF1,TCF4, and TCF7 in the immune tumormicroenvironment of breast cancer and support their application as prognosismarkers. Immune cells can significantly influence the tumor microenvironment and growththrough anti-tumor immunity, cell-mediated cytotoxicity, inflammation, and secretionof cytokines and growth factors . In breagnatures . AnegatLEF1 wasmainly associated with pathways related to cell cycle regulation, Rho GTPasessignaling, and metastasis induction through CCT/TriC chaperonins. These findingssupport previous reports on the LEF1 function in cancer malignancy:In colon cancer, for example, knockdown of LEF1 reduced cellviability, invasion capacity, and proliferation through cell cycle stabilization(LEF1 is involved in cell cycle regulation, proliferation, andmetastasis (LEF1 acts in metastatic processes (LEF1 leads to deregulation of several pathways, contributing totumorigenic processes. However, as a prognosis marker, it is low expression ofLEF1 that is associated with poor prognosis in breast cancer:This conflict may be the result of the interaction patterns or changes in the tumormicroenvironment that are yet to be unraveled.Finally, we investigated the potential role of these transcription factors on breasttumorigenesis by determining its regulons, and the processes and pathways in whichthey are involved. Our analysis showed that the regulon of lization. In prostastasis , and intastasis . In brearocesses  and is orocesses . ReporteTCF3 over-expression is associated withtumorigenic processes. In colorectal and gastric cancer, TCF3 isrelated to proliferation stimulation and metastasis (TCF3 knockdown decreased tumor growth andaggressiveness (TCF3 is linked with tumor growth and initiation (TCF3 was related to proliferation,migration, and apoptosis (TCF3 regulon in cellcycle regulation, Rho GTPases cycle, adaptive immune system, RET signaling, PI3K/AKTsignaling, besides signal transduction by growth factor receptors andtyrosine-kinase receptors.In several cancer types, tastasis , and insiveness . In breaitiation , and in poptosis .Our resTCF4 is known as a tumor suppressor in some cancer types: In coloncancer, loss of TCF4 leads to tumorigenesis via dysregulation ofproliferation (TCF4 suppressed cellproliferation and growth (TCF4 is also suggested to playa role in tumor suppression (TCF4 being related to chemoresistance inbreast cancer xenograft models via cell cycle deregulation (TCF4 regulon mainly with metastasis-related processes, likeextracellular matrix organization, degradation and proteoglycans, cell surfaceintegrin interactions, and collagen biosynthesis and degradation via regulation ofcollagen genes. Altogether, our results reinforce that low expression ofTCF4 contributes to breast cancer malignancy. feration  andmetaferation , and ind growth . In breapression ,with logulation  and to mgulation . Our enrTCF7 regulon was mainly enriched in processes involving the immunesystem, cytokine signaling, chemokine receptors, and PI3K/AKT signaling. Thedown-expression of TCF7 is rarely related to cancer, however, ithas been demonstrated that depletion of TCF7 can impact immunesystem regulation and immunotherapy response (TCF7 also participates inchemokine signaling in several cancer types (response . TCF7 aler types , highligLEF1, TCF3,TCF4, and TCF7 have the potential to bebiomarkers in breast cancer clinics. Our study appoints these transcription factorsas differentially expressed in breast tumor samples, and that its expression can berelated to outcome prediction, immunological subtypes, and immune infiltration inthe breast tumor microenvironment. Regarding biological significance, our analysisshowed that these transcription factors and their targets are involved in breasttumorigenesis, mainly through cell cycle regulation, metastatic processes, andimmune system regulation. This study contributes with relevant data in biomarkerdiscovery and diagnosis/prognosis refinement, suggesting biomarkers that cancomplement the classic breast cancer prognostic factors. In summary, we suggest that