Limosilactobacillus fermentum NCDC400. Notably, this strain has demonstrated previous capabilities in enhancing dairy food texture and displaying in vivo hypocholesterolemic activity. Our investigation aimed to unveil EPS400′s potential biological roles, encompassing antioxidant, antibacterial, and immunomodulatory activities. The results underscore EPS400′s prowess in scavenging radicals, including the 2,2-diphenyl-1-picrylhydrazyl radical, 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) radical, superoxide radical, hydroxyl radical, and chelating activity targeting the ferrous ion. Furthermore, EPS400 displayed substantial antibacterial effectiveness against prevalent food spoilage bacteria such as Pseudomonas aeruginosa NCDC105 and Micrococcus luteus. Remarkably, EPS400 exhibited the ability to modulate cytokine production, downregulating pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and nitric oxide, while concurrently promoting the release of anti-inflammatory cytokine IL-10 within lipopolysaccharide-activated murine primary macrophages. Additionally, EPS400 significantly (p ≤ 0.05) enhanced the phagocytic potential of macrophages. Collectively, our findings spotlight EPS400 as a promising contender endowed with significant antioxidant, antibacterial, and immunomodulatory attributes. These characteristics propose EPS400 as a potential pharmaceutical or bioactive component, with potential applications in the realm of functional food development.Exopolysaccharides (EPS) are acknowledged for their diverse functional and technological properties. This study presents the characterization of EPS400, an acidic exopolysaccharide sourced from the native probiotic  Oxidative stress and inflammation play a pivotal role in the progression of a wide spectrum of metabolic disorders and chronic ailments in human beings. These conditions encompass diabetes mellitus, cancers, hypertension, coronary heart disease, and various other pathologies that also exacerbate unfavorable outcomes in cases of Coronavirus disease COVID-19) . . 2]. TheNatural bioactive compounds, exemplified by exopolysaccharides (EPSs), have emerged as a potential substitute for synthetic additives within the realms of both food and pharmaceuticals . These sHowever, it is important to note that a substantial portion of commercially available EPSs intended for food and pharmaceutical applications are derived from non-lactic-acid bacteria, exemplified by substances like xanthan gum, cellulose, and beta-glucan. This may be due to the lower yield of EPS derived from LAB . AlthougLimosilactobacillus fermentum holds an intriguing allure. Specifically, L. fermentum NCDC400, isolated from the traditional Indian dairy product known as “dahi,” has emerged as a lactic strain of notable technological significance. This strain not only produces EPS, but also enhances the rheological and sensory attributes of low-fat fermented dairy products .The capacity of EPS sourced from microbial origins to donate hydrogen has been identified as a prominent attribute contributing to specific antioxidant functions. This mechanism bears resemblance to the functioning of polysaccharides derived from alternative sources . A unifoPseudomonas aeruginosa NCDC105, Micrococcus luteus NCDC174, Escherichia coli, Staphylococcus aureus NCDC100, and Bacillus substilis NCDC70. The results showed that EPS400 generated very strong inhibition zones (≥15 mm) against Pseudomonas aeruginosa NCDC105 (18 ± 0.7 mm), and Micrococcus luteus NCDC174 (16 ± 0.57 mm), and a strong zone of inhibition (11–14 mm) against Escherichia coli NCDC135 (13 ± 0.57 mm), Staphylococcus aureus NCDC100 (11.3 ± 0.53 mm), and Bacillus substilis NCDC70 (11.3 ± 0.33 mm). However, no zone of inhibition against fungus, Rhodotorula glutinis NCDC51, Aspergillus niger NCDC55, Candida butyri NCDC280, and Penicillum camemberti NCDC56 was observed.EPS400 showed noteworthy antipathogenic activities against various food spoilage bacterial pathogens such as Lactobacillus sp. Ca6 strain against Micrococcus luteus and Salmonella enteric with 14 and 10 mm of inhibition zone, respectively, was observed by [E. coli and S. aureus by 2 to 3 logs, as reported by [Cronobacter sakazakii, Escherichia coli, Listeria monocytogenes, Staphyloccocus aureus, Candida albicans, Bacillus cereus, Salmonella typhimurium, and Shigella sonnei was reported by [Some studies have shown that EPS from different LAB has varying degrees of antimicrobial activity against a variety of pathogens in vitro and have proposed several possible antibacterial mechanisms of EPS, including (1) the chelation of metal, which causes the suppression of nutrients essential for microbial growth, (2) interaction with the ionic surface, which disrupts the cell wall and cytoplasmic membrane, and (3) the stimulation of different TLR-mediated signaling processes, which causes the inhibition of mRNA and proteins synthesis ,29,30,31It is well known that LPS can bind to Toll-like receptor 4 (TLR4) and activate the immune system by inducing cytokine release . Thus, Lp < 0.01) with the treatment of LPS for 24 or 48 h, up to 0.01 µg/mL, and decreased significantly (p < 0.01) from 1 µg/mL to 10 µg/mL, compared to the control (p < 0.001) in the production of IL-6 (p < 0.001) in the production of NO after 48 h of LPS treatment was found, while a non-significant difference was observed after 24 h compared to the control. The highest increase in NO was observed at a 1µg/mL concentration of LPS treatment after 24 h and 48 h, compared to the control, with no remarkable change between 24 h and 48 h (p < 0.001) in the production of NO, IL-6, and TNF-α, and a decrease in anti-inflammatory cytokine IL-10 (p < 0.01) were observed at 1 µg/mL concentration of LPS treatment after 24 h, compared to the control, whereas no significant difference between 1 µg/mL and 10 µg/mL concentrations of LPS treatment for 24 h and 48 h was observed. Therefore, the concentration of 1 µg/mL of LPS for 24 h was selected for the challenging the murine macrophage to evaluate the anti-inflammatory effect of EPS400.The production of the anti-inflammatory cytokine IL-10 increased significantly  in the viability of macrophage cells compared to the control. Contrarily, a noteworthy elevation in murine macrophage viability was evident at EPS400 concentrations of 12.5, 25, and 50 μg/mL relative to the control group (p < 0.05). This observed enhancement in cell viability could be attributed to the availability of vital nutrients, such as sugars, essential for cellular growth. This outcome resonates with the findings of [Lactiplantibacillus plantarum JLAU103 within the concentration range of 20–100 μg/mL. Moreover, ref. [Preceding the exploration of EPS400′s anti-inflammatory potential, an MTT assay was performed to assess its cytotoxicity towards murine macrophages A. As depdings of , whereiner, ref.  reportedp < 0.001). Treatment with different concentrations of EPS400 significantly decreased (p < 0.05) the pro-inflammatory cytokine, including IL-6 . EPS400 treatment resulted in 26.8%, 45.5%, and 60.1% decrease in NO production at the concentrations of 12.5, 25, and 50 μg/mL, compared to the LPS-alone-treated cell. In line with this result, polysaccharides derived from Cyclocarya paliurus have been reported to suppress the production of NO in a dose-dependent manner in LPS-treated macrophage cells [L. planetarium JLAU103 at concentrations of 40, 60, and 80 g/mL in comparison to the LPS-alone-treated cell group.LPS, an endotoxin of bacterial origin, triggers the release of pro-inflammatory cytokines along with other molecules such as nitric oxide (NO). Following LPS stimulation, the concentration of NO in the culture supernatant of macrophage cells exhibited a marked increase compared to untreated control cells, as illustrated in ge cells . Similarge cells  reportedThe immunomodulatory attributes exhibited by EPS can be attributed to factors encompassing its molecular weight, structure, and composition, which collectively influence the nature of the immune response generated. The interplay between EPS’s molecular weight and its potential to counteract inflammation has been comprehensively elucidated . NotablyL. plantarum JLAU103 [L. helveticus LZR-5 [Phagocytosis, as a fundamental cellular process, plays an important role in host defense. Phagocytic activity is a commonly used method for assessing the activities of macrophage function modulators. Hence, the biological effects of EPS400 on the phagocytic activity of macrophages were investigated and found to be positive, as shown in  JLAU103  and R-5-us LZR-5  have beeWhile the exact mechanism through which EPS influences the regulation of macrophage phagocytic activity remains elusive, it is evident that phagocytosis is triggered through various receptor–ligand interactions, leading to the elimination of pathogens and deceased cells within the host organism ,39,40,41L. fermentum NCDC400, has provided insights into its multifaceted potential encompassing antibacterial, antioxidant, and immunomodulatory activities. The findings presented herein underscore the noteworthy contributions of EPS400, notably its robust scavenging activity in targeting diverse radicals, as well as its remarkable antibacterial efficacy against prevalent food spoilage bacteria. Notably, EPS400 emerged as a regulator of cytokine release, effectively curbing the excessive secretion of TNF-α, IL-1β, IL-6, and NO, while concurrently fostering the release of the anti-inflammatory cytokine IL-10. Furthermore, EPS400 emerged as a potent enhancer of the phagocytic capability of macrophages. The cumulative outcomes of this investigation accentuate the promising prospects of EPS400 as a prospective bioactive constituent for integration into functional foods or medicinal formulations. However, it is prudent to acknowledge that further validation through in vivo research is imperative. This validation process should focus on corroborating the antioxidant, antibacterial, and immunomodulatory attributes of EPS400, particularly with an emphasis on unraveling its intricate structural features. In conclusion, this study lays the groundwork for potential applications of EPS400 in promoting health and combating ailments, while underscoring the necessity for continued research to realize its full therapeutic potential.In this study, an extensive exploration into the biological properties of EPS400, derived from


Lipid transfer proteins (LTPs) are allergens found in a wide range of plant-foods. Specifically, Pru p 3, the major allergen of peach, is commonly responsible for severe allergic reactions. The need for new alternatives to conventional food allergy treatments, like restrictive diets, suggests allergen immunotherapy as a promising option. It has been demonstrated that sublingual immunotherapy (SLIT) with synthetic glycodendropeptides, such as D1ManPrup3, containing mannose and Pru p 3 peptides induced tolerance in mice and that the persistence of this effect depends on treatment dose (2nM or 5nM). Moreover, it produces changes associated with differential gene expression and methylation profile of dendritic cells, as well as phenotypical changes in regulatory T cells (Treg). However, there are no works addressing the study of epigenetic changes in terms of methylation in the cell subsets that sustain tolerant responses, Treg. Therefore, in this work, DNA methylation changes in splenic-Treg from Pru p 3 anaphylactic mice were evaluated.It was performed by whole genome bisulphite sequencing comparing SLIT-D1ManPrup3 treated mice: tolerant (2nM D1ManPrup3), desensitized (5nM D1ManPrup3), and sensitized but not treated (antigen-only), with anaphylactic mice.Stat4, Stat5a, Stat5b, Foxp3, and Gata3. In fact, Foxp3 was observed exclusively as hypomethylated in tolerant group, whereas Gata3 was only hypomethylated in the desensitized mice.Most of the methylation changes were found in the gene promoters from both SLIT-treated groups, desensitized  and tolerant , followed by the antigen-only  group. Although tolerant and desensitized mice showed a similar number of methylation changes, only 445 genes were shared in both. Remarkably, interesting methylation changes were observed on the promoter regions of critical transcription factors for Treg function like In conclusion, diverse D1ManPrup3 doses induce different responses (tolerance or desensitization) in mice, which are reflected by differential methylation changes in Tregs. It consOne of the most common food allergens in adults is non-specific lipid-transfer proteins (nsLTPs), especially Pru p 3, which is the major allergen of peach. LTPs are widespread in multiples foods, although they are more relevant in plant-foods, where they display a defensive function . Their s4 with anti-inflammatory properties, and the change of the pro-inflammatory phenotype of dendritic cells towards a tolerogenic profile by increasing molecules as programmed cell death ligand-1 (PD-L1 or CD274)  containing these allergens is the only solution. Nevertheless, it worsens the quality of life of patients and an involuntary intake of allergen produces a high risk of suffering a serious allergic reaction, which also increases the patient’s fear and anxiety . Therefor CD274) .+ proliferative responses. Interestingly, mice treated with D1ManPrup3 showed a higher percentage of Cd4+/Cd25+/Foxp3+ cells, defined as regulatory T cells (Treg)  , which p+ T cells that expresses the antigen Cd25 (α-chain of IL-2 receptor), which can promote regulatory responses to mitigate immune and allergic diseases. Different subpopulations of Treg have been established, but the most important ones are those that express the forkhead box protein 3 (Foxp3) transcription factor, which has been considered a Treg cell maker essential to develop their functions . For this, the anaphylactic mice, which was used for developing the different comparison analysis, was considered as basal group. Differences found between desensitized and tolerant animals can be used to obtain prognostic biomarkers of long-term tolerance.22.1To develop the anaphylactic mouse model, female Balb/c mice of 4-5 weeks of age  were required. International standards of animal welfare were applied in experimental animal procedures. The Animal Experimentation Ethics Committee of Andalusian Centre for Nanomedicine and Biotechnology  approved all protocols related to animal manipulation.n = 7); 2) anaphylactic, non-treated (anaphylactic) (n = 7); 3) anaphylactic, treated with 2 nM D1ManPrup3 (tolerant) (n = 7); 4) anaphylactic, treated with 5 nM D1ManPrup3 (desensitized) (n = 7), according to previously described experimental procedures (Mice were separated into four groups: 1) sensitized, non-anaphylactic (antigen-only) , and rectal temperature and behavioral symptoms were evaluated 30-40 min after challenge, according to a scoring system . It is a2.21) were evaluated after Pru p 3-challenge by ELISA using Pru p 3 5 µg/mL for coating 96 wells plates . Then, sera were added and Pru p 3-specifc antibodies were revealed using biotinylated labelled rat anti-mouse IgE or IgG1 . ELISA results were expressed as 450nm optical density.Pru p 3-specifc serum antibody levels , following manufacturer’s protocol. +/Cd25+ population before and after isolation, as well as the proportion of obtained Foxp3+. Moreover, the percentage of Cd4+/Cd25+/Foxp3+ of each group of mice is represented. Isolated Cd4+/Cd25+ Treg were maintained in RLT Buffer with β-Mercaptoethanol and stored at -80°C until use.Treg from spleen were purified by positive magnetic immunoselection using CD42.4DNA of Treg cells from spleen was extracted using DNeasy Blood & Tissue Kit  according to manufacturer’s recommendations. DNA quality control, bisulphite conversion, library construction, and sequencing were performed by Novogene  .2.5To obtain the methylation ratio of each cytosine in CpG context, the following workflow was applied to the sequenced libraries. In first place, WGBS-seq libraries were processed by Trimmomatic , to trimq-value < 0.05 , and with at least 10 cytosines in the CpG context, were marked as differentially methylated, and those that did not overlap with gene promoters were filtered out. Thus, promoters were defined with a ChiPseeker annotatePeak function and covers 3 kb upstream and 3 kb downstream of the transcription starting site , REACTOME and KEGG pathways of the genes affected by DMPRs were conducted with clusterProfiler R package . Additio2.6post hoc test. Statistical software GraphPad Prism 8  were used for statistical analysis.Data is expressed as mean ± standard deviation (SD). Normality was analyzed using the Shapiro-Wilk test. Paired groups were compared by Mann-Whitney test, and multiple groups were contrasted by Kruskal-Wallis test with Dunn’s 33.1p < 0.001), whereas the rest of groups did not change their body temperatures after challenge and no further severe behavioral symptoms linked to anaphylactic reactions were observed , and desensitized vs anaphylactic   .p < 0.05), whereas the temperature of tolerant group was similar before and after challenge . Likewise, sIgE and sIgG1 levels were higher in desensitized mice in comparison to tolerant group . Therefore, these data confirm tolerant responses or temporary desensitization.Tolerant and desensitized responses were also assessed five weeks after the last dose of D1ManPrup3-SLIT, and tolerance was observed in mice treated with 2 nM D1ManPrup3, but not in mice treated with 5 nM D1ManPrup3. Only desensitized mice suffered a significant drop in the body temperature , confirming that the analyzed cells were Treg . Several types of mechanisms are involved in the regulation of gene expression, but cytosine methylation is one of the key elements in gene regulation . MoreoveAs we expected, and in line with our previous results, the dose of D1ManPrup3 administered in SLIT induces tolerance or desensitization, and indirectly changes in the methylation profile in a key cell type for tolerance acquisition: Treg cells . This faStat4 was found as differentially methylated in both treated-mouse groups and Stat4 ratio under Foxp3 defines effector Tregs by preventing Tregs identity destabilization by inflammatory conditions . Remarkably, we found that The data presented in the study are deposited in the Bioproject repository, accession number PRJNA937049.The animal study was reviewed and approved by Animal Experimentation Ethics Committee of Andalusian Centre for Nanomedicine and Biotechnology .Conceptualization, MT, JC and CM. Methodology, RN, MR, CL-M, MM-A, JR-S, JR and JC. Formal analysis, RN, MR, CL-M and JC. Data curation, RN, MR, CL-M, JC and CM. Original draft preparation, RN, CL-M, MM-A, JC and CM. Review and editing, MT, JC and CM. Supervision, JR, MT, JC and CM. Funding acquisition, MT, JR and CM. All authors contributed to the article and approved the submitted version.


In the last decade, cholesterol level has been implicated in several types of cancer, including breast cancer. In the current study, we aimed to investigate the condition of lipid depletion, hypocholesterolemia or hypercholesterolemia reproduced in vitro to analyze the response of different human breast cancer cells. Thus, MCF7 as the luminal A model, MB453 as the HER2 model and MB231 as the triple-negative model were used. No effect on cell growth and viability was detected in MB453 and MB231 cells. In MCF7 cells, hypocholesterolemia (1) reduced cell growth, and Ki67 expression; (2) increased ER/PgR expression; (3) stimulated the 3-Hydroxy-3-Methylglutaryl-CoA reductase and neutral sphingomyelinase and; (4) stimulated the expression of CDKN1A gene coding cyclin-dependent kinase inhibitor 1A protein, GADD45A coding growth arrest and DNA-damage-inducible alpha protein and, PTEN gene coding phosphatase and tensin homolog. All these effects were exacerbated by the lipid-depleted condition and reversed by the hypercholesterolemic condition. The relationship between cholesterol level and sphingomyelin metabolism was demonstrated. In summary, our data suggest that cholesterol levels should be controlled in luminal A breast cancer. Scientific research aimed to study the pathogenetic mechanisms and clinical implications of cancer has always focused, and still is strongly concentrated on, breast cancer. Certainly, scientific progress has been remarkable in recent years, but, despite this, breast cancer remains the most common cause of cancer in women and affects increasingly younger women . MammogrAdditionally, a triple negative claudin-low and HER2  subtype was also described . GenomicCholesterol (Chol) mostly exerted opposite functions in physiological and pathological conditions as a source of hormones and vitamins, acting as atherogenic in the cardiovascular system, and acting as a pro-growth agent in cancer . HyperchChol has been shown to link sphingomyelin (SM) and form lipid rafts . In cancThe present study tested the effects of two different Chol conditions reproducing hypocholesterolemia (HypoChol) and hypercholesterolemia (HyperChol) in MCF7, MB453 and MB231 breast cancer cells. No differences were detected in MB453 and MB231 cells, while cell growth of MCF7 was influenced by Chol concentration. Therefore, the second part of the study aimed to investigate the possible metabolic mechanisms involved in the change in the Chol level. Notably, the effect of SM metabolism was investigated by studying neutral SMase 3 (nSMase3), encoded by the SMPD4 gene, which is deregulated in human cancer . . 8]. MCF7 appeared with an ovoid and incurved cell body with strong cell–cell adhesion, MB453 were round with small aggregates, and MB231 had a fusiform morphology and showed cellular dispersion . MB453 cWe analyzed the morphology, cell number and viability of MCF7, MB453 and MB231 breast cancer cells in our experimental model CTR, LD, HypoChol and HyperChol (see methods). After 48 h, cell culture differences in morphology and cell number were evident only in MCF7 cells a,b. IndeTo evaluate whether the endogenous Chol content was different in the three cell lines and whether the variation of the exogenous Chol might influence the endogenous one, the Chol dosage was performed in all samples. The results showed that MB453 and MB231 cells were richer in Chol content than MCF7. Moreover, the treatments of LD, HypoChol and HyperChol conditions affected the Chol content only in MCF7 cells .Since Chol level has emerged as a modulator of cell morphology and growth only in MCF7 cells without any effect in other cell lines, we aimed to explore whether this prediction translated into specific cellular activities. Thus, MCF7 cells in LD, HypoChol and HyperChol conditions were tested for Ki67, ER and PgR expression. LD dramatically reduced the Ki67 and strongly increased ER and PgR. HypoChol induced similar cell biological changes with a more moderated effect, and HyperChol had the opposite effect a,b. Of nNext, we attempted to establish whether the variation of exogenous Chol could influence its endogenous synthesis. To that end, we evaluated the key enzyme for Chol synthesis, the 3-Hydroxy-3-Methylglutaryl-CoA reductase (HMGCR). The gene expression was overexpressed in LD and HypoChol conditions and down-expressed in HyperChol conditions a. AccordCDKN1A gene coding cyclin-dependent kinase inhibitor 1A protein, GADD45A coding growth arrest and DNA-damage-inducible alpha protein and PTEN gene coding phosphatase and tensin homolog, an oncosuppressor protein. These genes were down-expressed in the HyperChol condition, which instead stimulated overexpression of CCND1 coding cyclin D1 . Cell culture medium , fetal bovine serum (FBS), penicillin G, streptomycin, glutamine, and sodium pyruvate were from GIBCO Invitrogen . FBS lipid-depleted was from Biowest . Human LDL was from Millipore . Anti-nSMase3, anti-β-tubulin, anti-HMGCR and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were from Abcam . ER antibody, clone 6F11, was from Leica Biosystems, Newcastle Upon Tyne, United Kingdom. PgR antibody, clone 16, was from Leica Biosystems, Newcastle Upon Tyne, United Kingdom. Ki-67 antibody clone MIB1 was from Agilent Dako, Glostrup, Denmark. 3--2,5-diphenyl-2-tetrazoliumbromide (MTT), Chol standard, nSMase and were from Sigma-Aldrich . TaqMan SNP Genotyping Assay and Reverse Transcription kit were purchased from Applied Biosystems . RNAqueous®-4PCR kit was from Ambion Inc. . SDS-PAGE molecular weight standards were purchased from Nzythech . Chemiluminescence kits were purchased from Amersham .MDA-MCF7 and MDA-MB231 cell lines were from Elabscience Biotechnology , and MDA-MD453 was from ATCC  and MB231 as triple-negative claudin-low  were used [2. To assess differences in cell growth of MCF7, MB435 and MB231 cell lines, the cells were counted at different times of culture .MCF7 as luminal A model , MB435 as HER2 basal model  and lipid-depleted (LD) FBS to induce a HypoChol and HyperChol condition. FBS contained 378 ± 3mg/dL of phospholipids (PLs) and 32 ± 4 mg/dL of Chol, corresponding to approximately 150 mg/dL of Chol in human serum, according to Haylett and Moore (2002) . In LD, 4 cells/well with DMEM complete culture medium. After 24 h, culture medium was replaced with respective culture medium with or without LDL, as reported above for each sample, and the cells were incubated for 24 h. Samples with 1% or 4% DMSO were added as controls due to their cytotoxic effect [MTT assay was used to test cell viability, as previously reported . MCF7, Mc effect ,47,48. TMCF7, MB435 and MB231 were cultured for 48 h, as reported above. The observations were performed by using inverted microscopy EUROMEX FE 2935  equipped with a CMEX 5000 camera system (20× or 40× magnification) and the morphometric analysis was performed by using ImageFocus software, as reported above .Lipids were extracted, and total phospholipids (PLs) and Chol were measured, as previously reported . Brieflyg for 10 min and treated as previously reported [® Automated Cell Block System  that rapidly creates a paraffin-embedded cell block was used, as previously reported. The inclusion was sectioned into 4 µm thick sections. Bond Dewax solution was used to remove paraffin from sections before rehydration and immunostaining on the Bond automated system . Immunostaining for ER, PgR and Ki-67 detection was performed using specific antibodies and Bond Polymer Refine Detection—Leica Biosystems . The observations were performed using inverted microscopy EUROMEX FE 2935  equipped with a CMEX 5000 camera system (40× magnification). The intensity of immunostaining was evaluated. The findings were classified as no reactive, low positive, medium positive, and strong positive cells. Only the strong positive cells were considered for quantification, as previously reported [MCF7 cells were cultured for 48 h, then centrifuged at 1000× reported . The pelreported .−ΔΔCt, comparing the results of the treated samples with control sample [Total RNA was extracted from MCF7 cells cultured for 24 h, as reported above, using RNAqueous-4PCR kit, as previously described . Samplesl sample .Protein concentration was measured as previously reported with modifications . Proteing for 10 min. The supernatants were used for nSMase assay. The enzyme activity was assayed in 60 µg proteins/10 µL Tris-MgCl2, pH 7.4, using Amplex Red Sphingomyelinase assay kit  according to the manufacturing instructions. The fluorescence was measured with FLUOstar Optima fluorimeter , using the filter set of 360 nm excitation and 460 nm emission.nSMase activity was assayed as previously reported . Cells wt-test was used for statistical analysis; each LD, HypoChol and HyperChol sample was versus the control sample (CTR). To compare the effect of HyperChol versus LD and/or HypoChol, ANOVA test was used.Three independent experiments performed in duplicate were carried out for each analysis. Data are expressed as mean ± SD; Student’s


Juglans sigillata L.  has a high economic value for nuts and wood and has been widely grown and eaten around the world. Light plays an important role in regulating the development of the walnut embryo and promoting nucleolus enlargement, which is one of the factors affecting the yield and quality of walnut. However, little is known about the effect of light on the growth and quality of walnuts. Studies have shown that far red prolonged hypocotyl 3 (FHY3) and far red damaged response (FAR1) play important roles in plant growth, light response, and resistance. Therefore, FHY3/FAR1 genes were identified in walnuts on a genome-wide basis during their growth and development to reveal the potential regulation mechanisms involved in walnut kernel growth and development.OF07056-RA, OF09665-RA, OF24282-RA, OF26012-RA, OF28029-RA, OF28030-RA, and OF08124-RA) were predicted to be associated with flavonoid biosynthetic gene regulation cis-acting elements in promoter sequences. RT-PCR was used to verify the expression levels of candidate genes during the development and color change of walnut kernels. In addition, light responsiveness and MeJA responsiveness are important promoter regulatory elements in the FHY3/FAR1 gene family, which are potentially involved in the light response, growth, and development of walnut plants.In the present study, a total of 61 FHY3/FAR1 gene family members in walnuts have been identified, ranging in length from 117 aa to 895 aa. These gene family members have FHY3 or FAR1 conserved domains, which are unevenly distributed on the 15 chromosomes (Chr) of the walnut (except for the Chr16). All 61 FHY3/FAR1 genes were divided into five subclasses  by phylogenetic tree analysis. The results indicated that FHY3/FAR1 genes in the same subclasses with similar structures might be involved in regulating the growth and development of walnut. The gene expression profiles were analyzed in different walnut kernel varieties . The result showed that some FHY3/FAR1 genes might be involved in the regulation of walnut kernel ripening and seed coat color formation. Seven genes (The results of this study provide a valuable reference for supplementing the genomic sequencing results of walnut, and pave the way for further research on the FHY3/FAR1 gene function of walnut.The online version contains supplementary material available at 10.1186/s12864-023-09629-2. The plants or crops are usually suffering from various biotic and abiotic stresses during their growth, which can severely affect their growth and yield, such as drought, light, temperature, salinity, waterlogging, mycosis, virus, and bacteria stress. Among them, light is the most important environmental factor for plant growth and development . TherefoThe FHY3/FAR1 transcription factor family plays an important role in regulating plant growth, light response, and resistance. FHY3/FAR1 is derived from an ancient mutation-like transposase, which is a typical transcription factor that binds the FHY3/FAR1 binding site (FBS) cis-acting elements on the promoter in the form of homologous dimers or heterodimers to regulate downstream gene expression . A molecCamellia sinensis), Eucalyptus , and peanuts (Arachis hypogaea) had 12, 25, 33, and 246 FHY3/FAR1 family members, respectively [At present, the FHY3/FAR1 transcription factor family has been analyzed and reported in many plant species. In Arabidopsis, tea plants . According to the Food and Agriculture Organization of the United Nations (2017), China’s walnut production is 1.925 million tons, accounting for 47% of the world’s total walnut production. Walnuts have a high economic value in the food industry because of their rich nutrient content, such as fatty acids, polyphenols, flavonoids, steroids, carbohydrates, protein, dietary fiber, heart glycosides, melatonin, folic acid, and vitamins [Walnut (2n = 32), as the oldest cultivated economic tree, is widely grown and eaten around the world due to its valuable nuts and wood, which originated in Central Asia and Southeast Europe . Juglansvitamins . With thations 207, China’In order to obtain sufficient genetic resources, revealing the growth and development of walnut and its response mechanisms to environmental stress such as light is extremely important. Therefore, it is necessary to identify the FHY3/FAR1 genes during the growth and development of walnut and to reveal the mechanism of growth and resistance regulation, which can provide insights for further research on the FHY3/FAR1 gene function of walnut response to light and quality breeding.Three walnut varieties were grown under the same environmental conditions and used for transcriptome sequencing in this study. These three walnut varieties have different colored pellicle, which includes the varieties of shajiwuren, hongguowuren, and songguowuren renamed F, Q, and T, respectively . Wuren whttp://www.arabidopsis.org/). Then, these Arabidopsis FHY3/FAR1 protein sequences were used for online tool BLASTP queries  in the walnut whole genome to identify homologous FHY3/FAR1 protein sequences in walnut. The whole genome information of walnuts was obtained from the Research Institute of Forestry, Chinese Academy of Forestry [https://www.ebi.ac.uk/Tools/hmmer/) corresponding to Pfam (http://pfam.xfam.org/) scanning to predict the protein walnut genome. In addition, with the fake members eliminated, these real walnut FHY3/FAR1 gene members were renamed in the present study as JsFAR1s. The gene transcript length and chromosome localization of the FHY3/FAR1 genes from walnut were calculated according to the method of Liu et al. (2023) [https://web.expasy.org/compute_pi/) website.All known FHY3/FAR1 protein sequences in Arabidopsis were obtained from the TAIR (Arabidopsis Information Resource) database  in walnut. The NCBI  Conservative Domain Database (https://www.ncbi.nlm.nih.gov/cdd/) was used to confirm the results [The exon or intron structure information was obtained from the  members . The conhttps://www.arabidopsis.org/) database. MEGA  version 7.0 software was used to perform multiple sequence alignments on the obtained FHY3/FAR1 protein sequences. Based on the results of multiple sequence alignments, the Neighbor-Joining algorithm is used to construct a phylogenetic tree, and the Replications of Bootstrap are set to 1,000 [Phylogenetic analysis was conducted on the newly identified 61 FHY3/FAR1 proteins of walnut and Arabidopsis. The FHY3/FAR1 protein sequences of Arabidopsis were obtained from the TAIR (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Secondary structure analysis of FHY3/FAR1 proteins was conducted using the online prediction software of DETAIBIO (http://www.detaibio.com/tools/chou-fasman-forecast.html).The 2 kb (non-coding region sequence) upstream region before the coding sequence (CDS) of members of the FHY3/FAR1 gene family was predicted [The total RNA of walnuts was isolated from approximately 100 mg of frozen pericarp samples using the RNeasy plant mini kit (Qiagen) according to the manufacturer’s instructions. The RIN algorithm was determined by the Agilent Bio analyzer 2100 system  for 18 cDNA library constructions (RNA integrity number > 7) on an Illumina HiSeq 2000 platform. After removing poly-N and low-quality reads, the clean reads were assembled, and the Q30 and GC contents were calculated by using Trinity . Based o−ΔΔCt method [To verify the usability of the gene expression data and FHY3/FAR1 gene function for walnut growth, the relative transcript levels of eight candidate genes were determined using quantitative real-time reverse-transcription PCR (qRT-PCR). Gene-specific primer pairs were designed using Primer 4.0. Revert Aid First Strand cDNA Synthesis Kit  was used to reverse-transcribe total RNA in walnuts to obtain first-strand cDNA according to the manufacturer’s instructions. qRT-PCR was performed using the SYBR1 Green PCR Kit . Plant actin was used as the internal reference to normalize the cDNA content. The mRNA expression level of genes was calculated using the 2t method . All genp < 0.05. All data had three biological repeats. Differentially expressed genes were defined as genes with an FDR < 0.001 and a fold change > 2-fold [Data from three biological repeats were analyzed using GraphPad Prism 5, Excel 2013, and SPSS 20.0 and rendered as the means ± SD. A one-way Analysis of Variance (ANOVA) was performed, followed by Tukey’s significant difference test at > 2-fold . A p-val> 2-fold .In this study, amino acid sequences of the FHY3/FAR1 gene family in Arabidopsis thaliana and peanuts were used to BLAST  the walnut genome. After deleting the fake gene members, a total of 61 FHY3/FAR1 gene family members of walnuts were obtained in this study , Songhe Wuren (T), and Sachijiwuren (F), were selected for transcriptome sequencing at two stages of kernel development (immature and mature stages). The expression profile and regulation mechanism of the FHY3/FAR1 gene in walnut during maturation were analyzed. Q walnut seed coat is red after maturity, T walnut seed coat is yellow after maturity, and F walnut seed coat is pink after maturity Fig. .In the Q variety, most of the FHY3/FAR1 gene family members in walnuts showed up-regulated expression in Q2 (maturity stage) compared with Q1 Fig. A. In T, The potential cis-acting elements were predicted in the 2 kb sequence (non-coding region sequence) before the CDS sequence (start codon) of the FHY3/FAR1 gene family. A total of 19 main cis-acting elements were distributed in the 2 kb promoter regions of FHY3/FAR1 gene family members, including abscisic acid responsiveness (142), gibberellin responsiveness (59), light responsiveness (608), methyl jasmonate (MeJA) responsiveness (164), low-temperature responsiveness (40), flavonoid biosynthetic genes regulation (7), salicylic acid responsiveness (18), seed-specific regulation (6), auxin responsiveness (62), defense and stress responsiveness (26), zein metabolism regulation (47), drought-inducibility (41), 60 K protein binding site, endosperm expression (16), anaerobic induction (151), cell cycle regulation (4), circadian control (9), wound-responsive element (3), and meristem expression (27) . Among them, light responsiveness is the most cis-acting component, followed by MeJA responsiveness, anaerobic induction, and abscisic acid responsiveness. Among them, seven FHY3/FAR1 gene family members possess flavonoid biosynthetic gene regulation cis-acting elements. This might be related to the seed coat color formation during the ripening process of Q, T, and F walnut varieties, which include OF07056-RA, OF09665-RA, OF24282-RA, OF26012-RA, OF28029-RA, OF28030-RA, and OF08124-RA.p < 0.05) .In order to verify the accuracy and reliability of the RNA-Seq detection results in this experiment. The previously screened candidate FHY3/FAR1 genes were detected by RT-PCR. These candidate genes include JsFAR1-1, JsFAR1-2, JsFAR1-3, JsFAR1-7, JsFAR1-10, JsFAR1-13, JsFAR1-15, and JsFAR1-21. The results showed that the expression levels and trends of these genes in the RNA-Seq profile were consistent with those in the RT-PCR detection results (05) Fig. . A correWalnuts are among the oldest foods known today, and they are grown throughout temperate zones of the world for nut production. Fresh walnuts are delicious, crispy, and more nutrient-denser than dried walnut products ; therefoSeveral previous studies suggested that FHY3/FAR1 have multiple functions in different physiological and developmental processes of plants, such as abiotic stress responses , flowering, reactive oxygen species (ROS) homeostasis, chlorophyll biosynthesis, UV-B signaling, circadian clock entrainment, and abscisic acid (ABA) signaling . HoweverPrevious studies have reported that sequences of FHY3/FAR1 gene family members are homologous and conserved among land plants, which are considered the founding members of the FRS and FRS-RELATED FACTOR gene families, and the proteins encoded by these genes might be involved in different signaling processes in response to various biological and abiotic stresses , 26–28. Due to morphological limitations, plants often use their complex sensory systems to monitor their environment and adapt to growth. As a result, plants develop a range of defense and perception systems, such as light. Light plays an essential role in the growth and development of plants throughout their lives for seed germination, seedling and stem elongation, phototropism, chloroplast movement, circadian rhythms, and flowering , 26, 27.Arabidopsis Thaliana [Arabidopsis Thaliana, encoding different phytochromes that sense red (R) and FR light and regulate various growth and development processes in plants, including seed germination, stomatal membrane opening, chlorophyll synthesis, low-quality growth, and flower initiation [In recent years, studies have shown that FHY3/FAR1 appears to constitute a key transcriptional signaling factor that coordinates the expression of downstream genes to ensure optimal plant growth, development, and immunity to various internal and external signals. In Arabidopsis thaliana and rice plants, the FHY3/FAR1 gene controls the accumulation of photosensitive pigments through FHY1 and is associated with the photosensitive pigment signaling pathway , 30. StuThaliana . Similaritiation , 33. TheThe phyA is primarily responsible for very low flux responses and FR light dependent high irradiance responses, including inhibition of hypocotyl elongation, opening of the apical hook, cotyledon expansion, anthocyanin accumulation, and green blocking of FR preconditioning . In thisBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Ustilago maydis to identify the gene network required for cell survival in the absence of telomerase. Mutations in either of the telomerase core subunits (trt1 and ter1) of the dimorphic fungus U. maydis cause deficiencies in teliospore formation. We report the global transcriptome analysis of two ter1Δ survivor strains of U. maydis, revealing the deregulation of telomerase-deleted responses (TDR) genes, such as DNA-damage response, stress response, cell cycle, subtelomeric, and proximal telomere genes. Other differentially expressed genes (DEGs) found in the ter1Δ survivor strains were related to pathogenic lifestyle factors, plant–pathogen crosstalk, iron uptake, meiosis, and melanin synthesis. The two ter1Δ survivors were phenotypically comparable, yet DEGs were identified when comparing these strains. Our findings suggest that teliospore formation in U. maydis is controlled by key pathogenic lifestyle and meiosis genes.We analyzed the global expression patterns of telomerase-negative mutants from haploid cells of  Telomerase does this using its unique and essential reverse-transcriptase activity ,2. The ced motif . Telomered motif ; in the advances . Howeveradvances ,6.Saccharomyces cerevisiae, telomerase activity is expressed at the late S-phase during an undetermined number of replication rounds [tlc1 cells, the telomeres shorten and senescence sets in.In n rounds . This isn rounds ,9. When When a critical telomere-shortening threshold is reached, most of the cells stop growing and die; at any point after this, in the post-senescent culture, survivor cells may arise ,11. In ttlc1 mutants of S. cerevisiae along the cell progression  have been achieved to identify the network of genes involved in cell surviving in the absence of telomerase [Schizosaccharomyces pombe [S. cerevisiae and have been named telomerase-deleted signature (TDS) genes [Transcriptome analyses of lomerase ,16. In tlomerase . Among tes pombe . Additioes pombe ,19, but TDR includes genes from carbohydrate metabolism, protein synthesis, folding, membrane structure, membrane trafficking, and genes harbored in subtelomeric sequences. The wider number and deepest response of TDR genes are attained when chromosomes reach the shortest telomere length; then, most of the genes return to a middle stage of deregulation in the survivor phase of the cells ; more reU. maydis, which has reported the telomere structure and drafted its organization [Multiple research groups have successfully addressed the efforts to elucidate the details of telomere metabolism in the basidiomycetous model organism nization ,22,23,24nization ; in addinization .U. maydis telomerase is located on chr 8 as an overlapping sense lncRNA spanning the last exon of UMAG_03168 and the proximal intergenic sequence ter1 of 1626 bp (GenBank TPA: BK059259). The length of the gene has been calculated as at least 1280 bp; it transcribes more than one isoform, but the characterization of a full set of transcript products remains to be completed [The TER subunit of the U. maydis and S. cerevisiae prompted us to study the transcriptome of two ter1-disrupted U. maydis survivor strains. These strains lack the essential template domain. We set out to describe the variance in gene expression between ter1Δ and WT strains. This objective was chosen for two reasons: (1) TER (telomerase RNA) is the limiting subunit dictating telomerase functionality in yeast; and considering (2) in U. maydis, additional genetic constituents facilitate and regulate the transition from a yeast-like to a mycelial form.The shared telomere-maintenance pathways between ter1Δ strains of U. maydis. Many of the identified TDRs are shared with other TER-disrupted fungi, but we also identify deregulated genes involved in meiosis, pathogenic development, and pathogen–plant crosstalk. Although a similar pattern of differentially expressed genes (DEGs) was found in both ter1Δ strains, they were not identical. In both strains, some genes involved in the pathogenic-mycelium lifestyle were deregulated in sporidia, suggesting an early interference with their ability to cross-fertilize with compatible strains and complete the life cycle.Here, we report the global transcriptome analysis of two U. maydis 518 (a2b2) strain was previously donated by WK. Holloman , the telomerase-negative derivative strains ter1-02 and ter1-24  were constructed and characterized as previously described in [The ribed in . The parRNA extraction was performed as previously described . For thehttps://ftp.ensemblgenomes.ebi.ac.uk/pub/fungi/release-56/fasta/ustilago_maydis/cds/Ustilago_maydis.Umaydis521_2.0.cds.all.fa accessed on 30 June 2021) using Bowtie2 v2.4.2 [http://www.uusmb.unam.mx/ideamex/ accessed on 30 June 2021; [The quality of the RNA-Seq reads was evaluated with the FastQC v0.11.7 software. Trimming of adapters and remotion of poor-quality reads was performed with Trimmomatic v0.36 , keeping2 v2.4.2 . Next, tne 2021;  to use Dne 2021; , NOISeq ne 2021; , Limma-Vne 2021; , and Edgne 2021; , the cut−5. Next, protein domains were sought in the outcomes employing the hmmer v3.3.1 software of Trinotate suite and the database Pfam-A. Likewise, SignalIP v4.1 and TMHMM v2.0c programs were used to seek the cleavage sites of signal peptides and to predict transmembrane helix [https://www.biobam.com/omicsbox accessed on 30 June 2021), the GO identifiers of DEGs were filtered by taxonomy (fungi) and classified by functional annotation.The annotation of the resulting transcripts and products was carried out using the Trinotate v3.2.2 pipeline ; the seqne helix ,39,40. DThe global expression profile of TER-deleted mutants at the surviving stage was analyzed using RNA-seq libraries of the parental strain 518 and its telomerase-negative derivatives ter1-02 and ter1-24. The assays were elaborated in independent triplicate repeats and sequenced using the Illumina NextSeq 500 platform , yielding a total of 126,587,419 paired reads from nine libraries. After raw read processing, a total of 110,227,520 paired reads were preserved . The aliter1Δ mutants, the reads mapping to the reference transcripts were used for the analysis of differential expression on the IDEAMEX platform, setting up cutoff points of: logFC ≥ 2 and FDR ≤ 0.01, and only the genes appearing at the intersection of DESeq2, NOISeq, Limma-Voom, and EdgeR were considered differentially expressed. Likewise, the changes in the expression profile between ter1-02 vs. ter1-24 mutants were compared to find the differences exhibited by both mutants [To identify the DEGs between the parental strain and  mutants .Concurrently with the differential expression analysis, the input data were analyzed to detect sample variations. In U. maydis, deletion of TER causes the deregulation of a core of 90 genes shared by the two TER-mutants, hence named core genes; this represents 1.32% of the total coding genes. Eight of those genes showed clear differences in transcriptional expression among the mutants. When the search for unique DEGs was carried out for each pair of transcriptomes analyzed, 57 genes were identified for ter1-02, and 124 genes were identified for ter1-24. When the differential analysis was conducted against wild-type, 47 unique DEGs were identified when comparing ter1-24 with ter1-02  and metabolic processes  followed by genes involved in localization and response to stimuli . In the case of downregulated DEGs from ter1-24, GO analysis showed 23 downregulated DEGs, which are involved in cellular processes, 15 DEGs in metabolic processes, and ten DEGs in cellular localization as the most representative categories; remarkably, there also were downregulated genes from multi-organism processes and stimuli response .To determine which of the cellular functions were affected by TER deletion, DEGs were grouped into the three categories of the GO analysis: biological process (BP), molecular function (MF), and cellular component (CC). Regarding the MF category of the GO analysis, the main enrichment of upregulated genes in ter1-02 occurred in the categories of catalytic activity (95 DEGs), binding capability (69 DEGs), and transporter activity (22 DEGs); likewise, in ter1-24, overexpressed genes also were from catalytic activity (99 DEGs), binding capability (81 DEGs), and transporter activity (22 DEGs). Downregulated DEGs in the same MF categories were found in the ter1-24 strain with catalytic activity (15 DEGs), binding capability (13 DEGs), and transporter activity (10 DEGs). Of the overexpressed genes in ter1-02, it was interesting to observe that 41.05% (39) of those assigned in the status of “catalytic activity” have hydrolase activity, and 38.84% (35 genes) possess oxidoreductase activity; a similar enrichment of these two activities was found in the ter1-24 DEGs reaching 31.31% (31 genes with hydrolase activity) and 38.83% (38 genes with oxidoreductase activity).In the CC category of upregulated genes, the most enriched DEGs of ter1-02 mutant were intracellular anatomical structure (66 DEGs), organelle (49 DEGs), and cytoplasm (47 DEGs). In the ter1-24 strain, the most enriched DEGs also were intracellular anatomical structure (81 DEGs), organelle (66 DEGs), and cytoplasm (64 DEGs). Likewise, in ter1-24, there were also downregulated DEGs in the same categories; the intracellular anatomical structure was enriched with a count of (13 DEGs), organelle (ten DEGs), and cytoplasm (nine DEGs). Finally, from the DEG analysis for the BP category in both mutants, the most abundant deregulated genes included those from the cellular process, metabolic process, localization, and response to stimuli. Agreeing with the above results, those DEGs were assigned to the catalytic activity group, genes that regulate molecular function, binding and transporter activity in the MF category. Interestingly, genes that regulate molecular function were among the most enriched DEGs (26.54%) obtained from the analysis of both mutant strains. For the CC category, the main enriched DEGs were assigned to the intracellular anatomical structure group, and the number of DEGs assigned to those located in the membrane group increased, as well as those assigned to organelles and intrinsic components of membranes .UMAG_02709 was repressed (logFC = −2.26), a locus that codes for the histone replication-coupled H3.2; repression of other core histone complex components was also noted (H2B (UMAG_01505), H2A (UMAG_01504), and H4 (UMAG_02710)) and were also found with logFC values of −1.98, −1.93, and −1.74, respectively, in addition to the putative H1 (UMAG_10447) with logFC value −1.83. However, although repression of the same genes was observed in the ter1-02 mutant, such changes were not significant at the cutoff point used, ranging between −0.93 (UMAG_01504) and −0.62 (UMAG_10447). Additionally, in both mutants, there were no significant changes in the expression of H2A.Z (UMAG_00469) and histone replication-independent H3.1 (UMAG_03916). Regarding other components involved in chromatin remodeling, in ter1-24, the changes were subtle, below the threshold in the genes UMAG_02567 (logFC = −1.51) and UMAG_06201 (logFC = −1.19) encoding for the catalytic subunit of histone acetyltransferase type B and RuvB-like helicase1 respectively. However, they need to be further explored.Within the DEGs in ter1-24, we found that U. maydis has multiple predicted telomere-linked helicase (TLH) isoforms. These include RecQ-like sequences UT5 (AF030886.2) and UT6 (AF030887.1) [UTASa and UTASb, respectively. UTASa is adjacent to telomere repeats and possess the same helicase domain as the RecQ helicases subfamily. UTASb is near telomere repeats or 5’ upstream of UTASa. UTASb may be a truncated segment generated from the homing endonuclease gene HEG [UTASb consists of a highly conserved “core” sequence, an OrsD domain, or both [UMAG_12032 and UMAG_12076, while portions of UT6 are conserved in UMAG_06476, UMAG_06474, UMAG_11065, UMAG_04094, and UMAG_04308. UMAG_12032 was upregulated in both mutant strains ter1-02 (logFC = 3.16) and ter1-24 (logFC = 5.20)  . UT5 andgene HEG . UTASb c or both . UT5 exh = 5.20) . The othith TLH1 . The disith TLH1   were differentially expressed, while in ter1-24, 10.27% of telomere-proximal genes were among DEGs (5.43% of DEGs), and from these only UMAG_06259 (peptidyl-prolyl cis-trans isomerase D) was repressed. Seven genes were overexpressed uniquely in ter1-02  with logFC greater than two, without being classified as differentially expressed in ter1-24 at the same cutoff point used. From the sets of DEGs located at the chromosomal ends, four belong to the core genes of the ter1Δ strains; among those are UMAG_12032 and UMAG_04695 that encodes a choline transporter and UMAG_06146 and UMAG_12031 that code for hypothetical proteins.Transcriptional expression of genes arranged on the 20 kb adjacent to each of the 46 chromosome termini of UMAG_06490, which encodes a predicted inorganic phosphate transporter, was differentially expressed in ter1-24 (logFC = −2.21); in NW_011929456.1, the UMAG_06503, which encodes a DEG similar to the uncharacterized protein gene C1198.03c, had a logFC = 2.88; finally, placed on NW_011929457.1, the loci UMAG_06504 (encoding a protein of unknown function) and UMAG_12076 (encoding a protein related to ATP-dependent DNA Q5/DEAD/DEAH box helicase) were upregulated in ter1-02. The UMAG_06504 fitted in the core of DEGs, and UMAG_12076, another RecQ-like helicase, was upregulated in ter1-02 with logFC = 4.93 but showed no change in ter1-24 (logFC = 0.22).In addition to the DEGs from subtelomeric regions , we founUMAG_03122 , located within the first 20 kb of chromosomal ends. Most of these genes could be analogous to stress-induced responses and cellular senescence of S. cerevisiae [UMAG_01937, UMAG_02224, and UMAG_03728. In addition, UMAG_04553  was differentially expressed in ter1-24 because of a slight downregulation in ter1-02, whereas UMAG_11483 (predicted protein-L-isoaspartate (D-aspartate) O-methyltransferase) and UMAG_01262 (predicted β-DNA-polymerase) were uniquely upregulated at logFC > 2 in ter1-24 but induced in ter1-02 at logFC = 1.15 and 1.83, respectively.DEGs involved in stress and DNA-damage responses are listed in revisiae . The groUMAG_12336) and MutS5/Msh5 (UMAG_12155) homologs were annotated as members of the DEGs core genes  homolog also occurred in both mutants  and Mlh1 homolog (UMAG_05208) were upregulated more than two-fold in ter1-02. However, no such expression changes of UMAG_05208 occur in ter1-24 , UMAG_05917 , and UMAG_01262 , which did not reach significance. Further study is now needed.wAn increase in expression of Mer3  in the ter1-02 strain, whereas in ter1-24, overexpression of the same gene reaches only a logFC = 0.76. Also, we detected a slight upregulation of the genes encoding the RPA-heterotrimer: RFA1 , RFA2  and RFA3 .Regarding the homologs of the yeast U. maydis. The putative rad7 (UMAG_00657) showed an upregulation of nearly three times in the ter1-02 strain and of nearly two times in ter1-24 (rad16 homolog (UMAG_03263) did not exhibit expression changes. Finally, in ter1-24, the repression of some genes encoding DDR constituents at values above the logFC threshold of −2 was considered in this report because the interaction of their protein products with those of each other suggests alterations in DNA replication and/or repair among repressed DEGS: PCNA , DNA Polε catalytic subunit A , and DNA ligase1 . Regarding components of the heteropentameric RFC homologs in U. maydis, putative RFC5 (UMAG_00920) was downregulated marginally by logFC = −1.45; RFC4 (UMAG_00729) had a logFC = −1.05, and MCM10 (UMAG_10135) had a logFC = −1.25. On the other hand, the putative MCM2-7 complex exhibited logFC values between −0.62 and −1.01, except MCM5 (UMAG_05064) logFC = −0.10, which could also be considered an ambiguous value. Finally, the following genes encoding the homologous subunits of the DNA-directed RNA polymerase II complex were slightly downregulated: RPB11 , RPABC1 , RPABC2 , and RPABC3   . FurtherUMAG_02326; Rap1, UMAG_04676; Tpp1, UMAG_11538; Pot1, UMAG_05117) [UMAG_11687; Ten1, UMAG_11842) [UMAG_04704; Rad50, UMAG_01085) [Except for Tpp1, which showed near to 50% repression in the ter1-24 mutant, no significant changes were found in the expression of genes encoding the components of the shelterin complex , SMD2 , and SMD3  were slightly downregulated; a marginal change was found in SMB , but no expression changes were found in SME (UMAG_10312), SMF (UMAG_12130), and SMG (UMAG_10805) in the ter1-24 mutant. The same strain also showed a slight downregulation  of components of the predicted Dyskerin complex: Dyskerin/CBF5 , NPH2 , GAR1 ; and NAF1 ; no expression changes were predicted for NOP10 (UMAG_03354).A highly conserved motif has been found at the 3′ end of the putative TER of all the members of Ustilaginales studied in , which sUTASa and UTASb harboring ORFs encoding helicase-like sequences in telomere maintenance has been proposed by some authors [UMAG_12032 locus was upregulated; this could be associated with a possible gene de-repression of subtelomeric sequences after telomere-repeats loss or a possible role of this RecQ-like helicase in the primary DNA metabolism in the absence of telomerase [UMAG_12032 in ter1-24 is sufficiently high compared to ter1-02 to be considered as a DEG (logFC = 2.06) and is the only helicase-related gene overexpressed in this strain in contrast to the seven genes observed in ter-02  to pathogenic mycelium (hyphae); this lifestyle change requires the reprogramming of gene expression [The dimorphic fungus pression ,53,54. Gpression ,55,56.ter1Δ mutants, there were no expression changes detected in the master regulator cdk1, nor in the cdk5 involved in cell differentiation [pcl12 [pcl12 and rbf1 are controlled by the heterodimeric master regulator bE/bW; however, its expression pattern suggests an independent mechanism of control [In the ntiation ,58, yet n [pcl12  reached  control  (see Tabter1Δ mutants [chk1 (logFC = 1.40) and wee1 kinase (logFC = 1.18) in ter1-02 and ter1-24, respectively, although both were below the logFC threshold of 2.0. Notably, no alterations were detected in the predicted apical kinase atr1 expression within the ter1-02 and ter1-24 mutants (logFC = 0.51 and 0.49). This finding weakens the likelihood of a link between the apical members of DDR and the changes described above [The analysis revealed a significant downregulation of Rad21 (logFC = −1.21). This Rad21 downregulation in ter1-24 went near half in the wild-type cell expression. The genes responsible for encoding the postulated cohesin proteins, Smc1 and Smc3, also exhibited substantial deregulation, with their normal expression levels decreasing by nearly half at logFC = −1.15 and logFC = −0.94, respectively. This downregulation could be correlated with the changes in cell morphology and nuclear irregularities observed in  mutants , possibled above ,70. Simied above ,72,73.Since the third portion of the DEGs in mutant strains lacks an assigned function or a defined role, a database of genes involved in pathogenic development was constructed, which included fungal secreted proteins, cell-wall-degrading enzymes, plant component-degrading enzymes, and proteins involved in the organization of the fungal cell wall, to further learn about the influence of chromosome architecture and fungal development ,66,68,74egl1 and egl3 endoglucanases, xin1 endoxylanase, which is engaged in the fungal cell wall organization, and lac1 and lac2 involved in melanin synthesis [About 10% of the DEGs were involved in pathogenic development in both mutants. Those genes were included in the group of core genes and included some encoding plant cell wall-degrading enzymes such as ynthesis ,64.eff1) located on the chr 5; those genes whose products have a role in virulence were upregulated [fer3, the downregulation of the fer gene cluster, located on the telomere-proximal right end of the chr 2, also occurred in ter1-24 strain of U. maydis  and UMAG_01438 (predicted Acyl-CoA-dependent acyltransferase gene) loci, which separate fer9-11 from the remaining genes of the fer cluster, were upregulated in ter1-24; whereas strong upregulation (logFC = 7.35) of the UMAG_11874 gene, encoding a hypothetical protein of unknown function, was detected in ter1-02. Moreover, UMAG_11874 is the last gene at the end of chromosome 2 , lipases (UMAG_06274), and proteases (UMAG_12330) [Other DEGs-encoding proteins of unknown function located within clusters 2A, 10A, 19A, and 22A  were deres UMAG_074, and pter1Δ mutants, some were associated with pathogenic development, such as hdp1, which was differentially expressed in both the ter1-02 and ter1-24 mutant strains. The hdp1 is required for filamentous growth and cell cycle arrest [hdp2 (UMAG_04928), which plays a role in appressorium formation [fox1 gene, involved in suppressing plant defenses [hdp1, hdp2, and fox1) are activated by the Rbf1 master regulator (UMAG_03172), which in turn is activated by the bE/bW heterodimer in a dependent manner and also participates in the activation of biz1 and MAP kinase Kpp6, which are involved in appressorium formation [The DEGs-encoding transcriptional factors (TFs) are listed in e arrest ; likewisormation , was uprdefenses , was higormation ,79.kpp6 was identified as differentially expressed in ter1-24, as was rbf1. Those results in sporidia, which lack the functional bE/bW heterodimer because they are haploid cells, together with the absence of deregulation, or with a slight increase in ter1-24, in the expression of factors upstream of Pfr1 suggest that other unidentified components or possibly Ter1 could be involved in the regulation of rbf1; and hence, some of the target genes of Rbf1 (ter1Δ mutants as core genes were: the homolog of Yap (UMAG_3296) bZIP protein from the AP-1 family, involved in response to oxidative stress and plant–host interaction  were deregulated in either mutant, suggesting the cooperation of other unidentified factors in the regulation of these genes.Also,  of Rbf1  as occur meiosis . IntriguUMAG_01456, UMAG_06308, UMAG_02835, and UMAG_04778, which in natural circumstances are upregulated during pathogenic development [UMAG_01025, which is progressively repressed during early infection and responds to unfolded proteins [UMAG_12304 and UMAG_04101 that encode Mtf1, a transcription regulator of the gene cluster located at the end of chromosome 12 involved in melanin production [pks5, pks4, vbs1, orf4, pks3, omt1, pmo1, and cyp4) were also identified as DEGs, while orf1, orf5, aox1, and deh1 do not exhibit changes in transcriptional expression, nor mtf2 or its target orf2  situated 5′ upstream from the intergenic region housing the conserved domains of the ter1 gene [Here, we present a transcriptomic DEGs analysis of three strains: the wild-type eviously . This reer1 gene . NeverthUMAG_03169 gene is at the 3′ end of ter1 and encodes a putative ornithine-oxo-acid transaminase. UMAG_03169 was also upregulated in the mutants (logFC = 1.74 in ter1-02 and logFC = 3.0 in ter1-24). This upregulation in these two genes cannot be solely attributed to the transcriptional disruption caused by ter1. Instead, it may be linked to the Telomere Damage Response (TDR). This would be consistent with observations for several other genes.The U. maydis. This finding is consistent with previous studies addressing other telomerase-negative mutants [rad51 was upregulated (logFC = 2). The rad51 gene is involved in the ALT pathways in S. cerevisiae and in DNA repair through homologous recombination. Conversely, the gene encoding its associated protein Brh2  showed UMAG_05721. This gene encodes the srbA homolog in U. maydis, termed srb1, which plays a role in hypoxia adaptation and is a potential co-regulator of tumor formation [UMAG_05721 is repressed, whereas srb1 is upregulated in ter1-24 (The chromatin modification pattern of telomere-associated sequences (TAS) shifts as telomeres shorten. This shift influences the condensation status of both the TAS chromatin and neighboring genes. Reducing chromatin compaction could lead to a variegated gene expression pattern, impacting genes close to telomeres like ormation . Interes ter1-24 .terΔ mutants exhibited divergent transcriptional behaviors. However, as these strains share the same mutation, induce similar infection symptoms, and cannot produce tumors in planta, it is likely that a set of “core genes” orchestrates control over these strains during the early stages of their biotrophic development [ter1Δ mutation or the telomere-position effect (TPE), as previously discussed by [U. maydis.Other genes within the two elopment . Additioussed by . The TPEussed by . The pheussed by , playingussed by . SimilarS. pombe and various eukaryotes, transcripts originating from subtelomeric sequences have been observed. Notable examples include TERRA, ARRET, and α-ARRET, as well as transcripts like ARIA, that initiate from the telomere itself [S. pombe and humans, TERRA and ARIA transcripts, which incorporate telomere repeats, display a lesser degree of polyadenylation compared to ARRET and α-ARRET, representing antisense and sense transcripts from subtelomeric regions [Due to the TPE, it was previously believed that the ends of chromosomes were transcriptionally silent. However, recent findings challenge this notion. In organisms like e itself . In both regions .ter1Δ mutants studied here, the poly(A) fraction was extracted from total RNA. Notably, a strand-specific approach was not employed for sequencing, presenting transcripts as homologous to subtelomeric sequences. Previously, in U. maydis, sense and antisense transcripts derived from chromosome ends were amplified using RT-PCR. Although sequences analogous to TERRA, ARRET, and α-ARRET were investigated by our group, their characterization remains incomplete, including determination of polyadenylation extent and percentage. Given that UTASa primarily localizes adjacent to telomere repeats, it is conceivable that their transcripts may lack polyadenylation, akin to the case of TERRA. This absence of poly(A) tail in transcripts could impede their detection in polyadenylated transcriptomes, thereby accounting for their underrepresentation in the analysis. Consequently, this could explain the prevailing abundance of UTASb sequences.For the transcriptomes of the UMAG_12032 and UMAG_12076 are two upregulated UTASa sequences containing RecQ-like motifs. These UTASa sequences lack adjacency to telomeres. This suggests the possibility of these sequences undergoing transcription in a manner akin to ARRET or α-ARRET sequences. Also, UMAG_11065 experiences an approximately 100-fold increase in expression within the ter1-02 strain. Both this sequence and UMAG_06474 share homologous traits with UTASb and are notably detached from telomere repeats.Moreover, the significance of TERRA extends to its role in telomere lengthening in scenarios where telomerase is absent. This is attributed to TERRA’s involvement in the formation of telomeric R-loops, which represent RNA/DNA hybrids. These structures facilitate recombination-based alternative mechanisms (ALT) that amplify chromosome ends. This phenomenon, in turn, ensures the survival of cells even in the absence of functional telomerase .Non-telomeric functions have also been attributed to TERRA, shelterin proteins, constituents of the CST complex, and TERT, collectively referred to as telomere-associated factors (TAFs). These proteins have been found to intricately modulate the transcriptional activity of critical genes dispersed along the length of the human genome . AdditiohTERP [Consequently, it has come to light that the TER component engages in various non-telomeric activities. For instance, it plays a pivotal role in responding to DNA damage , offerinhTERP ,102. FurhTERP ,105,106.hTERP .ter1Δ mutants, the deregulation of at least 12 pivotal developmental TFs has been identified  appear to exert divergent and substantial impacts on the fer3 to fer8 genes within the strain. This effect is particularly prominent in the ter1-24 strain  gene cluster at the terminal end of chromosome 12 B encompa cluster . ConsequbW gene is also influenced by the disruption of ter1. bW and bE play a pivotal role in governing dimorphism and the pathogenic transition in U. maydis. These genes produce proteins that form a heterodimeric TF named bE/bW; both partners are needed for functionality [b locus, these key components are transcribed from divergent promoters. Intriguingly, in the ter1-02 strain, solo expression of bW2 was observed. This occurrence echoes previous reports wherein solo bW and other genes exclusive to the pathogenic lifestyle were de-repressed in sporidia lacking the histone deacetylase hda1Δ [bW reinforces the notion of epigenetic factors influencing the development of the pathogenic lifestyle within haploid sporidia mutants. However, beyond epigenetic influence, the atypical transcription at the b locus raises the intriguing question of how its target genes become de-repressed. An unconventional possibility could be the formation of functional homodimers by bW under exceptional circumstances in U. maydis, analogous to the situation in the distantly related basidiomycetous fungus Cystofilobasidium capitatum [The ionality . Encodedse hda1Δ . The derapitatum .rbf1, a transcription factor that governs the filamentous and pathogenic transition in U. maydis. In the ter1-02 strain, rbf1 experiences a slight upregulation. Whereas in ter1-24, rbf1 demonstrates more pronounced upregulation . Within both mutants, Hdp1 displays elevated expression levels. Removal of hdp1 produces filament elongation and G2 cell cycle arrest in wild-type cells [ter1Δ mutants. Conversely, Fox1 regulates genes responsible for suppressing plant defenses through secreted effectors, some of which are harbored in clusters 10 and 19, such as UMAG_03751, UMAG_05308, and UMAG_05314. These genes were upregulated in the ter1Δ mutants, but it has been reported that deletion of Fox1 causes their downregulation [One of the prime targets of the bE/bW complex is gulation . Additiope cells  akin to gulation . Other mU. maydis’s lifestyle and their contributions to the phenotypic changes and DEGs uncovered in this study. Notably, other factors not explicitly depicted, such as unh1 , were unse UPR; , were up meiosis – and sevrevisiae , suggesttrt1Δ mutants, ter1Δ mutants failed to induce tumor galls when mated with wild-type strains. The discovery of this asymmetric effect on teliospore formation following trt1 or ter1 disruption [ter1 subunit of telomerase can undergo regulation during the dimorphic transition in this fungus. Future work should also determine whether core genes are subject to additional telomerase-independent activities within U. maydis.Equally noteworthy, in contrast to the sruption ,25 is a UMAG_03168 in the initial stages of biotrophic development [UMAG_03168 encodes the 5′ end of the bicistronic ter1 transcript [Data supporting the notion of telomerase deregulation during the dimorphic transition are drawn from , whereinelopment . Intriguanscript . It willU. maydis elicits the upregulation of genes associated with ALT, environmental stress response (ESR), and pathogenesis. The concurrent telomere shortening and loss of TPE may account for the observed ALT and ESR patterns. However, the mechanisms underlying the upregulation of genes located outside subtelomeric regions warrant further investigation. The findings of this study underscore the involvement of TER during the dimorphic transition, potentially suggesting a negative regulatory relationship between Ter1 and the master regulator Rbf1 or other factors that influence its functionality. A comparative analysis of the transcriptomes of ter1Δ and trt1Δ mutants could shed additional light on potential non-telomeric roles played by the core telomerase subunits, as well as by genes neighboring ter1, such as UMAG_03168, UMAG_03169, and rbf1.The absence of the RNA component of telomerase in


Nonsegmental vitiligo is a depigmented skin disorder showing acquired, progressive, and depigmented lesions of the skin, mucosa, and hair. It is believed to be caused mainly by the autoimmune loss of melanocytes from the involved areas. It is frequently associated with other autoimmune diseases, particularly autoimmune thyroid diseases including Hashimoto's thyroiditis and Graves' disease, rheumatoid arthritis, type 1 diabetes, psoriasis, pernicious anemia, systemic lupus erythematosus, Addison's disease, and alopecia areata. This indicates the presence of genetically determined susceptibility to not only vitiligo but also to other autoimmune disorders. Here, we summarize current understanding of autoimmune pathogenesis in non-segmental vitiligo. Nonsegmental vitiligo is acquired depigmented skin lesions mainly caused by autoimmune loss of melanocytes. Genetic and environmental factors are involved in the development. Recent genetic studies identified predisposed genes involving the development of nonsegmental vitiligo –5. CurrePTPN22, LPP, IL2RA, UBASH3A, C1QTNF6, RERE, GZMB, TYR, FOXP1, CCR6, TSLP, XBP1, and FOXP3 [RNASET2, FGFR1OP, and CCR6 [Genome-wide association studies are applied for identifying the candidate genes in multifactor-associated disorders such as nonsegmental vitiligo –5. In 20nd FOXP3 . Anotherand CCR6 –91. Thesin vitro and trigger apoptosis [Nonsegmental vitiligo can be caused by an immunologically complex mechanism. A variety of melanocytes-expressing proteins have been identified as autoantigens. Cui et al. showed that 24 (83%) of 29 vitiligo patients had autoantibody to melanocytes-associated autoantigen versus 2 (7%) of 28 healthy controls . Until npoptosis . Howeverpoptosis . Histopathological studies demonstrated the increased dendritic cells , Th17 ceβ and IL-18, inducing apoptosis [β level in the vitiligo lesion and the expression of NALP-1 in the activated epidermal Langerhans cells and dermal dendritic cells [β is the essential cytokine to develop Th17 cells [β produced by activated inflammasome may involve the development of nonsegmental vitiligo.The activation of inflammasome constructed by NOD-like receptors such as NALP-1 overproduces proinflammatory cytokines of IL-1poptosis . Interesic cells . As IL-117 cells , IL-1β pConsiderable progress is being made towards understanding the pathogenesis of nonsegmental vitiligo. Although a number of genes have been implicated by well-designed genome-wide association studies, we do not have good genotype-phenotype correlations. In the future, we can anticipate further advancement regarding specific interactions between disease-susceptible genes and gene-environment interactions.The authors declared that there is no conflict of interests.


Eugenol, the main constituent of clove oil possesses a number of medicinal activities. To enhance the medicinal property, structural modification is required. On the other hand, bismuth nitrate pentahydrate has been established as an excellent eco-friendly nitrating agent for several classes of organic compounds.Bismuth nitrate pentahydrate-induced nitration of eugenol has been investigated very thoroughly. Twenty five different conditions have been studied. The microwave-induced solvent-free reaction has been identified as the best condition.Spectral analyses confirm that 5-nitroeugenol is the sole product in all the cases. No oxidized or isomerized product could be detected. Syzygium aromaticum L., popularly known as clove, belongs to the plant family Myrtaceae, and has been used in folk medicine and dental treatment. Eugenol , the main component of clove oil, is an allyl chain-substituted guaiacol in the biosynthesized phenylpropanoid compound class derived from Syzygium aromaticum L. and widely used in medicine[ medicine. It is w medicine. Clove o medicine. It is s medicine. Moreove medicine.3/H2SO4[3/Et2O[In order to study the biological activities of eugenol derivatives, nitration by conventional nitric acid-sulfuric acid or a nitronium tetrafluoborate method were performed. These reactions require a mixture of concentrated or fuming nitric acid with sulfuric acid leading to excessive use of hazardous chemicals. Nitrati3/H2SO4 or by HNO4[3/Et2O. In addiOn the other hand, the usefulness of clay-mediated organic synthesis has been documented in a large number of publications which includes Michael addition , regioseWe have been studying metal/metal salts-mediated reactions with the aim of developing several biologically active compounds; including anticancer polyaromatic compounds  and antiIn previous work we reported the nitration of estrone with different types of metal salts in the presence of solid surfaces under various conditions. It has 3)2, Ca(NO3)2, LaNO3, NaNO3, and Cu(NO3)2 were also studied elsewhere. Bismuth nitrate pentahydrate was confirmed as the best nitrating agent among all others. Dry conditions and solvent-free methods along with commercial solvents without any purification were investigated in order to identify the best conditions for this reaction. Reactions were performed at high temperature using Dean-Stark water separator, traditional reflux, and conventional kitchen microwave-induced methods. Solid surfaces such as florisil, silica gel, molecular sieves, KSF clay, and neutral alumina were used as solid support in the reaction. It was discovered that silica gel is the best solid surface. In some cases , the reaction gave 100% yield of the product . Eugenol and bismuth nitrate along with KSF clay as solid support, under the conventional microwave and solvent-free condition produced 100% yield (entry 19). Quantitative yield was also observed under reflux in benzene with bismuth nitrate in presence of silica gel (entry 22). No reaction was observed when Bi(NO3)3 was used at room temperature even after 24 h (entries 11-15).Bismuth nitrate pentahydrate is the metal nitrate used in this experimentation process, although the effect of many others such as CAN, Zn has been observed in variable yields. The exploratory results described herein confirm that bismuth nitrate pentahydrate is the reagent of choice in the absence of any solvent under microwave-irradiation condition (entry 19). Importantly, in contrast with nitric acid-mediated method, these reactions mediated by bismuth nitrate have several important characteristics. For example, no isomerization of the alkene moiety has been observed, regioselectivity remains identical irrespective of the solid supports and conditions, no oxidation of the alkene/aromatic systems has been observed, and phenolic hydroxyl group has no influence on the regioselectivity of the reactions. On the basis of these important and selective observations, this method will find very useful applications in synthetic chemistry of electrophilic aromatic nitration reaction.1H-NMR (600 MHz) and 13C-NMR (125 MHz) spectra were obtained at room temperature with Bruker-600 equipment using TMS as internal standard and CDCl3 as solvent. Analytical grade chemicals (Sigma-Aldrich Corporation) were used throughout the project. Deionized water was used for the preparation of all aqueous solutions.FT-IR spectra were registered on a Bruker IFS 55 Equinox FTIR spectrophotometer as KBr discs. In general, eugenol 1 mmol) and bismuth nitrate pentahydrate (1 eqv.) were mixed and the mixture was studied under different conditions varying the method, solid support and/or solvent as mentioned in Table  mmol and-1): 2369, 1522, 1457, 1243, 1136, 1061, 941, 810 and 712; 1H NMR  δ: 10.67 , 7.45 , 6.84 , 5.89 , 5.05 , 3.83 , 3.27 . 13C NMR  δ: 149.86, 144.88, 135.94, 133.64, 132.46, 128.63, 127.43, 125.07, 56.70, 36.74.sticky mass; IR (KBr disc, cmThe authors declare that they have no competing interests.


Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are the most common human adult-onset neurodegenerative diseases. They are characterized by prominent age-related neurodegeneration in selectively vulnerable neural systems. Some forms of AD, PD, and ALS are inherited, and genes causing these diseases have been identified. Nevertheless, the mechanisms of the neuronal cell death are unresolved. Morphological, biochemical, genetic, as well as cell and animal model studies reveal that mitochondria could have roles in this neurodegeneration. The functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations, triggering neurodegeneration according to a cell death matrix theory. In AD, alterations in enzymes involved in oxidative phosphorylation, oxidative damage, and mitochondrial binding of Aβ and amyloid precursor protein have been reported. In PD, mutations in putative mitochondrial proteins have been identified and mitochondrial DNA mutations have been found in neurons in the substantia nigra. In ALS, changes occur in mitochondrial respiratory chain enzymes and mitochondrial cell death proteins. Transgenic mouse models of human neurodegenerative disease are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria and the mitochondrial permeability transition pore. This review summarizes how mitochondrial pathobiology might contribute to neuronal death in AD, PD, and ALS and could serve as a target for drug therapy. Pathologists conceived the concept of cell death as a mechanism of disease to aid in diagnosis and therapy . PatholoAn exciting new understanding of mitochondrial biology has emerged over the past two decades from multiple disciplines that is likely to be very relevant to adult-onset neurodegenerative disorders of the central nervous system (CNS). . Mitocho2+ homeostasis, synthesis of steroids, heme and iron-sulfur clusters, and programmed cell death (PCD) . H2O. H2O2+ h2O2 in the presence of reduced transitional metals (Fe2+) can be converted to hydroxyl radical (•OH) or hydroxyl-like intermediates . ON. ON2O2 is in DNA . Cu/ZnSOchondria  and has sylation .2 and substrate delivery to the brain and bioenergetic failure as seen in tissue ischemia and toxic exposures .,40.39,40collapse ,42,43,44l damage . The morphology and some biochemical features of classic necrosis in neurons are distinctive  24]. Th. Th24]. Recent work has shown that cell necrosis might not be as chaotic, random, and incomprehensible as envisioned originally but can involve the activation of specific signaling pathways to eventuate in cell death ,47,48. T2+ overload, excessive oxidative stress, and decreased electrochemical gradient (Δψm), ADP, and ATP can favor mPT. This alter condition of mitochondria involves the mitochondrial permeability transition pore (mPTP) that functions as a voltage, thiol, and Ca2+ sensor  re re262] reostasis ,267, defeostasis ,271,272,eostasis ,274, andeostasis ,276. There are possible disease links between intraneuronal Aβ and mitochondria suggesting an intracellular toxicity of Aβ ,278,279.ppif) in human mutant APP tg mice (J-20 line) protects neurons from Aβ- and oxidative stress-induced cell death . T. T317]. The degeneration of pigmented SNc neurons is characterized by chromatolysis A, nucleaThe molecular pathogenesis of PD is still not understood. At least 2 forms of PD exist: idiopathic (sporadic) and heritable  . The majα-syn gene (PARK1) result in amino acid substitutions Ala-53→Thr, Ala-30→Pro, Glu-46→Lys; in addition, duplication and triplication mutations in the α-syn gene (PARK4) have been found . Cy. Cy81]. e manner . Oxidatie manner . Some SOe manner ,465, ande manner ,491. Toghigh-mSOD1 mice without CyPD show markedly delayed disease onset and lived significantly longer than tg mice with CyPD. The effect of CyPD deletion was much more prominent in females than in males [Ppif gene ablation in tg mice with much lower levels of human mSOD1 expression and a slower disease progression (G93Alow-mSOD1 mice) also show significantly delayed disease onset and lived significantly longer than tg mice with CyPD [The role of CyPD in the process of motor neuron disease in ALS mice has been examined through gene-ablation . G93Ahigin males . Remarkaith CyPD . Thus, s2+ (2 mM) can still lead to mPTP activation without CyPD and that cell deaths caused by Bid, Bax, DNA damage and TNF still occur without CyPD [Nevertheless, most G93A-mSOD1 mice without CyPD develop eventually motor neuron disease and die. Other work on CypD null mice has shown that high concentrations of Caout CyPD . The effout CyPD .Mitochondria have diverse functions and properties and could be critically important for the development of human adult-onset neurodegenerative disorders, including AD, PD, and ALS . Structu