LysM-RLK genes in soybean. The majority of LysM-RLKs were highly conserved in Arabidopsis and soybean, while certain members of subclades III, VI, and VII are unique to soybean. The promoters of these LysM-RLKs contain specific cis-elements associated with plant development and responses to environmental factors. Notably, all LysM-RLK gene promoters feature nodule specificity elements, while 51.86% of them also possess NBS sites (NIN/NLP binding site). The expression profiles revealed that genes from subclade V in soybean roots were regulated by both rhizobia and nitrogen treatment. The expression levels of subclade V genes were then validated by real-time quantitative PCR, and it was observed that the level of GmLYK4a and GmLYK4c in roots was inhibited by rhizobia but induced via varying concentrations of nitrate. Consequently, our findings provide a comprehensive understanding of the soybean LysM-RLK gene family and emphasize the role of subclade V in coupling soybean symbiotic nitrogen fixation and nitrogen response.Lysin-Motif receptor-like kinase (LysM-RLK) proteins are widely distributed in plants and serve a critical role in defending against pathogens and establishing symbiotic relationships. However, there is a lack of comprehensive identification and analysis of LysM-RLK family members in the soybean genome. In this study, we discovered and named 27  Plants reside in a microbial-rich environment and must interact with a large number of pathogens and symbiotic bacteria . The iniArabidopsis thaliana , five Arabidopsis LysM-RLKs have been identified so far [Oryza sativa (O. sativa), OsCERK1, the orthologous of AtCERK1, encodes a LysM-RLK with a canonical intracellular kinase domain and is essential for chitin signal transduction. Although OsCERK1 does not directly bind to chitin, it interacts with OsCEBiP, which directly binds to chitin but lacks an intracellular domain, to transmit extracellular signals into the cell [Genetic evidence demonstrates that chitin perception is considered necessary in the process of pathogen infection to properly activate the defenses that limit pathogen invasion. In d so far ,10. AtLYd so far ,13,14,15the cell .Lotus japonicus (L. japonicus) and Medicago truncatula (M. truncatula), respectively, and the extracellular LysM domain of LjNFR1 and LjNFR5 can directly bind to the Nod Factors (lipochitooligosaccharide). Furthermore, the N-terminal LysM1 domain determines the specificity of receptor–ligand binding [GmNFR1α/GmNFR1β (orthologous to LjNFR1 and MtLYK3) encode LysM-RLKs with canonical kinases, while other Nod factor receptor genes GmNFR5α/GmNFR5β (orthologous to LjNFR5 and MtNFP) encode LysM-RLK with pseudo-kinases [M. truncatula LYK9 and O. sativa CERK1, as members of the LysM-RLK family, have been shown to function in regulating AMS [Apart from participating in plant immune responses, the LysM-RLK is also involved in the symbiotic establishment, including root nodule symbiosis (RNS) and arbuscular mycorrhizal symbiosis (AMS) . In 2003 binding ,20,21,22-kinases ,24. In cting AMS ,26.Soybean, a crucial legume crop, satisfies approximately 35% to 50% of the global population’s protein requirements via RNS under soil nitrogen deficiency status. Upon detecting low nitrogen signals, soybean triggers a series of molecular and metabolic responses. It releases flavonoids into the rhizosphere to attract rhizobia, where the rhizobia then secrete NFs, which will be recognized by GmNFR1α/β and GmNFR5α/β to initiate the NFs signaling pathway, while a high concentration of nitrogen inhibits RNS establishment ,27,28,29LysM-RLK genes  in subclade I might possess similar functions. Of note, only soybean LysM-RLKs were divided in subclades III, VI, and VII, suggesting that those LysM-RLK family members might have evolved novel functions in soybean.According to the reported role of AtLYK1 and GmNFR1α/β in recognition of chitin and NFs, respectively, we speculated that the remaining three genes  to 760 (GmLYK6) amino acids (aa), and the molecular weight (Mw) ranged from 57.071 kDa (GmNFR5β) to 84.586 kDa (GmLYK6). These LysM-RLKs also had different predicted protein isoelectric points (PI), where the PI values of GmLYK4e, GmLYK6, GmLYK7a, and GmLYK7c were greater than 8, while the PI values of other LysM-RLK proteins ranged from 5.21 to 7.48  to five (Chr 2). Notably, 22  of the 27 LysM-RLKs were localized close to the terminus region of the chromosome. In particular, the GmLYK8, GmLYK3a, GmLYK3d, GmLYK6, GmLYK3c, and GmLYK4d were closest to the end of the chromosome. In addition, the nod factor receptor gene GmNFR1α/β and GmNFR5α/β were distributed on four different chromosomes  of LysM-RLK proteins in soybean and Arabidopsis . As showWe then performed a conservative domain analysis to distinguish these LysM-RLKs B. All 27LysM-RLKs. Interestingly, the exon–intron structures of different subclades varied greatly, but genes within the same subclade had similar gene structures  or three (GmLYK7a) exons. In contrast, genes in subclades I, II, and VI contained nine to twelve exons. We also noticed that one soybean unique LysM-RLK gene (GmLYK7a) has no UTR. Through analyzing and comparing the orthologous gene structures of soybean and Arabidopsis, we discovered that AtLYK1 and AtLYK3 consisted of 12 and 11 exons, respectively. These numbers were similar to their corresponding soybean counterparts, GmLYK1 and GmLYK3, in subclades I and II. Similarly, AtLYK4 and AtLYK5 exhibited the presence of a single exon, just like their soybean counterparts. However, AtLYK2 differed from the genes in subclade IV of GmLYK2s as it contained two exons  sequence that is located upstream of the translation start codon (ATG) of all soybean ifferent . Among tcis-elements regulated by transcription factors. To investigate the expression profiles and potential functions of these soybean LysM-RLK genes, we analyzed cis-acting elements in the promoters (2 kb upstream of ATG) of LysM-RLK genes via the PlantCARE database [cis-elements with functional prediction were identified , plant growth and development-related , environment-related , and others. The findings implied that various LysM-RLK genes may exhibit diverse expression profiles during distinct developmental stages and in response to different biotic/abiotic stresses. In addition, we noted that genes from subclade I and subclade III, including GmNFR1α/β and GmNFR5α/β, contained more cis-elements in their promoter regions, indicating that the transcriptional regulation in these two subclades may be intricately complex.The expression of a gene is controlled by the promoter, which has various database . In addientified . These cGmNFR1α/β and GmNFR5α/β and its homologous genes may be regulated via nitrogen. To validate this hypothesis, we examined the cis-elements associated with plant response to nitrogen  and symbiotic nodulation  [LysM-RLK gene promoters contained nodule specificity elements, while 51.86% of the LysM-RLK gene promoters  had NBS  ,33,34. T had NBS B, suggesLysM-RLKs, we retrieved the expression data of 21 LysM-RLK genes (the remaining six members were not found in the database) via the eFP website and analyzed their expression profiles in response to rhizobia. The results demonstrated that the gene expression levels were particularly low in subclades II, IV VI, and VII, whereas a majority of the individuals in subclades I, III, and V had a substantially high expression (GmNFR5α had increased at 12 h after inoculation (HAI). Beside GmNFR5α, GmLYK4d was also up-regulated at 12 HAI, which indicates GmLYK4d may also play roles during the symbiosis establishment. The expression of GmNFR1α was suppressed by rhizobia at 12, 24, or 48 HAI, while the expression level of GmNFR1β remained very low, which was consistent with the previous findings [GmLYK4c, GmLYK4b, GmLYK5, and GmLYK4e) was slightly inhibited by rhizobia, while the expression of GmLYK4a was slightly upregulated at 12 HAI, and then reduced along with inoculation. Although GmLYK8 and GmLYK1b were expressed at high levels in the root hairs, their expression levels are basically unregulated by rhizobia.To further explore the potential functions of these pression . In partfindings . We alsoLysM-RLK genes under different nitrogen treatments based on the RNA-seq data from the JGI Plant Gene Atlas  and GmLYK1b from subclade I were induced via various nitrogen in roots, especially GmLYK4a and GmLYK4d. On the other hand, the expression of GmLYK4b was induced via ammonium and nitrate nitrogen but was inhibited by urea. In contrast, the expressions of GmLYK5, GmLYK3a, GmLYK3b were inhibited by various nitrogen in roots, and GmLYK7c was inhibited by nitrate or urea treatment  in roots were regulated by both rhizobia and nitrogen treatment, suggesting that genes from subclade V may function in coordinating the nodulation process and plant nitrogen response.Combined with the expression profiles under rhizobia treatment, we found that the genes from subclade V  under rhizobia and nitrate treatments in roots by performing quantitative PCR   A,C,F, anat 1 DPI D, which at 3 DPI E, while rhizobia B.GmLYK4a, GmLYK4b, GmLYK4c, and GmLYK4e were significantly induced via various concentrations of nitrate treatment at 1 day after treatments  and moderate levels of nitrate at 1 day after treatment and continuously decreased with prolonged low-nitrogen treatments but was induced via moderate and high levels of nitrate at 3 days after treatment  to six (subclades II and V), and different subclades exhibited distinct gene structures. Interestingly, genes in subclades III, IV, and V typically consisted of a single exon, except for GmLYK8 in subclade III which contained three exons. These genes encoded LysM-RLK with pseudo-kinase lacking in the G-rich motif. Therefore, based on the classification principle of Gust et al. [GmLYK7b and GmLYK7c) or three (GmLYK7a) exons, yet all GmLYK7 proteins with a canonical kinase domain belong to the LysM-RLK-I category. In contrast, members of subclades I, II, and VI comprised twelve genes containing nine to twelve exons, and the encoded LysM-RLK featured the classical kinase domain, categorizing them as LysM-RLK-I [In total, 27 soybean sM-RLK-I . AccordiLysM-RLKs. Almost all LysM-RLK promoters contained cis-elements associated with plant development and the responses to environmental factors, including phytohormones  and abiotic stresses . Notably, the number of cis-elements related to MeJA, abscisic acid, and gibberellin was significantly higher compared to others, as indicated in cis-elements in the promoter regions of soybean LysM-RLKs suggests their crucial role in facilitating the plant’s adaptation to environmental fluctuations. Moreover, previous studies have explored the responses of LysM genes to biotic and abiotic stresses in wheat and banana [LysM-RLK genes, identified via our bioinformatic analysis, in biological and abiotic stress remain uncertain, warranting further investigation and deliberation.The functional disparities of a gene are determined via the precise gene expression site and the magnitude of its promoter’s activity. Indeed, we observed substantial diversity in the promoter sequences of these d banana ,44. HoweLysM-RLK gene promoters contain nodule-specificity elements, while 51.86% of the LysM-RLK gene promoters  had NBS  were regulated by both rhizobia and nitrogen treatment in roots  genome, GFF3 file, and protein sequences were downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/ (accessed on 5 April 2023)). The hidden Markov model (HMM) file of the LysM domain (PF01476) and the Kinase domain (PF00069) were downloaded from the Pfam database (http://pfam.sanger.ac.uk/ (accessed on 10 April 2023)) [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 20 April 2023)) [Soybean (l 2023)) . Simple l 2023)) . The knol 2023)) .https://itol.embl.de/ (accessed on 20 April 2023)) [The amino acid sequences of selected LysM-RLKs were aligned via the MEGA-X software and the phylogenetic tree was constructed via the maximum likelihood method (ML) or the neighbor-joining method (NJ). Bootstrap analysis was calculated for 1000 replicates . The evol 2023)) .LysM-RLK genes was performed using TBtools software. The genomes of A. thaliana, L. japonicus, M. truncatula, and G. max were compared using One Step MCScanX-Super Fast with default parameters . TBtools was used to map the collinearity graphs and highlight the LysM-RLK genes on the graphs [A collinearity analysis of the e graphs .LysM-RLKs based on the GFF3 file and gene ID [TBtools software was utilized to display the gene density and chromosomal locations of soybean  gene ID .LysM-RLKs were obtained from the Phytozome database. The conserved motifs in LysM-RLK amino acid sequences and the promoter sequences of LysM-RLKs were analyzed via the MEME (https://meme-suite.org/meme/tools/meme (accessed on 15 April 2023)) online tool [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 20 April 2023)) [LysM-RLKs were visualized using the TBtools software based on the soybean GFF3 file, and TBtools software was also used to integrate the conserved motifs, protein conversed domains, and gene structures [The promoter sequences, CDSs, genomic sequences, and amino acid sequences of ine tool . The conl 2023)) . The exoructures .LysM-RLKs were obtained from the Phytozome database. The cis-elements in the promoter regions of 27 LysM-RLKs were analyzed using the PlantCARE online website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on accessed on 5 May 2023)). The data was visualized via TBtools [The promoters (2 kb upstream of ATG) of  TBtools .LysM-RLKs in response to rhizobia were downloaded from the eFP (Soybean eFP Browser (utoronto.ca)) website, and the LysM-RLKs’ gene expression data in response to different nitrogen treatments were obtained from the JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov/ (accessed on 5 May 2023)) [The expression data of y 2023)) ,50. Geney 2023)) .2/s, and 60% relative humidity) [LysM-RLKs’ expression pattern under rhizobia inoculation, 7-day-old plants were inoculated with Bradyrhizobium diazoefficiens USDA110  and roots were collected at 0 DPI, 1 DPI, and 3 DPI. For samples treated with various concentrations of nitrate, 7-day-old soybean plants were treated with 0 mM, 1 mM, 5 mM, and 10 mM of nitrate nitrogen, respectively. The roots were taken at 1 day and 3 days after nitrate treatment for subsequent RT-qPCR experiments.Seeds from soybean cultivar Williams 82 (Wm82) were sown in vermiculite soaked in a nitrogen-free BD nutrient solution, as described by Broughton and Dilworth, and cultured in a greenhouse under controlled conditions with a 16 h light/8 h dark at 26 °C, a light intensity of 200 μmol/mumidity) . To analLysM-RLKs, total RNA was extracted from different samples using a TRIpure reagent . cDNA was synthesized from the RNA via reverse transcription reagent kit ) and the Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus). Real-time quantitative PCR was performed using the Hieff qPCR SYBR Green Master Mix Kit (No Rox) (YEASEN). GmELF1b was used as an internal gene [To verify the expression of nal gene .


Lagenaria siceraria (Molina) Standl.]) is a relatively drought-tolerant cucurbit due to the high composition of unique biochemical compositions, including cucurbitacin. The objective of this study was to determine the concentrations of cucurbitacins in bottle gourd and their relationship to drought tolerance. The study assessed 12 bottle gourd accessions grown under two moisture levels  and drought-stressed (DS)) and three drought stress intensities  using a 12 × 2 × 3 factorial experiment designed in a randomized complete block design with three replications. Control studies were undertaken under glasshouse conditions. The content of cucurbitacins B, E, and I were quantified in leaves and roots using high-performance liquid Cchromatography–mass spectrometry (HPLC-MS). The free radical scavenging activities of pure cucurbitacins B, E, and I were quantified using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and a ferrulic acid power assay (FRAP). Results revealed that cucurbitacins B and I were present in accessions BG-48, BG-58, BG-70, BG-78, BG-79, BG-81, BG-52, and GC in leaves and roots under DS condition. The contents of cucurbitacins B and I were enhanced under increased drought intensity for accessions BG-48, BG-81, and GC. In all the leaf and root samples, cucurbitacin E was not detectable. Based on the DPPH test, pure cucurbitacins I, B, and E reduced free radicals at maximum values of 78, 60, and 66%, respectively. Based on the FRAP assay, pure cucurbitacins I, B, and E had maximum ferric-reducing powers of 67, 62, and 48%. Additionally, cucurbitacin I recorded the highest antioxidant activity compared to cucurbitacins B and E. Increased cucurbitacin accumulation and antioxidant properties indicate their role in minimising cell damage caused by oxidative stress under drought-stressed environments. The present study revealed that cucurbitacins B and I serve as novel biochemical markers for screening drought tolerance in bottle gourd or related cucurbits.Bottle gourd [ Lagenaria siceraria (Molina) Standl.] is an important cucurbit crop in Sub-Saharan Africa (SSA) and is widely cultivated by small-holder growers for its nutritious fruit. Young and succulent leaves are consumed as leafy vegetables after cooking and are often used for medicinal purposes to treat headaches [Fusarium and Verticilium wilts [Bottle gourd [eadaches ,2. Bottleadaches ,4,5,6. Seadaches . The frueadaches ,9,10. Theadaches ,11,12,13eadaches . The seeeadaches . The mateadaches . Bottle um wilts ,17,18,19Cucurbita maxima) and cucumber (Cucurbita pepo) [Bottle gourd shows considerable drought tolerance when compared to other cucurbits, including pumpkin (ta pepo) . In SSA,ta pepo) ,21,22. Tta pepo) ,24,25,26Cucurbitaceae crops and genotypes of the same species. For example, cucurbitacin B is found in melon [Hemsleya macrosperma [Cucurbit crops, including bottle gourd, produce secondary metabolites, including cucurbitacins ,25,26,27in melon ,39, C inin melon , E and Iin melon , E in wain melon , and IIarosperma . Cucurbirosperma , anti-inrosperma , anti-turosperma , hepatoprosperma , hypoglyrosperma , and antrosperma , thus marosperma . Other prosperma .2, OH¯, H2O2, and 1O2) [Cucurbitacins are produced in cucurbits as a defense strategy against insect pests and diseases . They arand 1O2) ,52. In aand 1O2) ,47,53. Rand 1O2) . Mashiloand 1O2)  detectedv/v) throughout the experiment. Under DS conditions, a steady decrease in the soil water moisture was recorded across the different accessions. At 7 days, the soil moisture level had reached approximately 25 to 30% for most accessions. At 14 days of withholding water, the soil moisture content decreased to approximately 7 to 14% for most accessions, except for BG-81, which was at approximately 21%. At 21 days, which corresponded to the severe drought stress, the soil moisture content reached approximately 0 to 5% across the different accessions.Significant differences were observed in soil water content across the twelve accessions grown under DS conditions as presented in The contents of cucurbitacins B and I in bottle gourd leaves and roots grown under DS and NS conditions are shown in Cucurbitacin I was not detected in the leaves of any of the accessions under mild drought stress. However, under moderate and severe drought stress cucurbitacin I was detected for the accessions BG-48, BG-81, and GC. In the leaf samples of the accessions BG-48, BG-81, and GC, drought stress did not induce significant changes in the accumulation of cucurbitacin I. Cucurbitacin I was not detected in leaf samples under NS conditions. Root cucurbitacin I content increased with increasing drought stress intensity for accessions BG-48 and GC, whereas no significant changes were observed for accessions BG-70, BG-79, and BG-81. Under the NS condition, cucurbitacin I was not detected in the root samples.Antioxidant potential of cucurbitacins B, E, and I based on the DDPH assay is presented in 3+) to Fe2+ is presented in Antioxidative activity of cucurbitacin B, E, and I as monitored by their ability to convert potassium ferricyanide  [QBt.1 and QBt.2 could probably enhance the concentration of cucurbitacins B and I in the leaves and roots of bottle gourd [The biosynthesis of cucurbitacin B and I is conditioned by various genes . The exptadienol ,54,57. Ctadienol . The acttadienol . In bottermelon) . Therefole gourd  subjecteCmBi, Cm710, and CmAC showed varied expression to regulate cucurbitacin B contents in melon (Cucumis melo) [Citrullus lanantus var. lanantus), genotypic-specific accumulation of cucurbitacins B and E has been reported [In the present study, cucurbitacin B and I did not accumulate in most of the bottle gourd accessions, suggesting accession-specific accumulation of the cucurbitacins. This is associated with a preferential selection pressure for the non-bitter types in bottle gourd for various uses, including food. For example, the tender leaves of bottle gourd are cooked as leafy vegetables by the Venda tribe of South Africa . The conis melo) . In watereported . Accessireported , perhaps+) to this radical causes a color change from violet to pale yellow in a solution [3+)— to form Fe2+. The potassium ferrocyanide then reacts with ferric chloride to form a ferric-ferrous complex, a reaction monitored through the color change from yellow to green [Cucumis melo L.), cucurbitacin B accumulation has been associated with increased antioxidative activity that enhanced tolerance to biological stress [Blastania cerasiformis (Stocks) A. Meeuse and B. garcinii (Burm.f.) Cogn., were associated with high antioxidant activity [Citrullus lanatus var. citroides), cucurbitacin E was reported to be an inefficient scavenger of reactive oxygen species [To determine the possible roles of cucurbitacins as antioxidant molecules, pure cucurbitacin samples were evaluated for their antioxidant activity using DPPH and FRAP assays . DPPH rasolution . The yelsolution ,65. The to green . To deteto green ,68. Drouto green . In melol stress . Cucurbiactivity . In citr species ,71,72. IThe study used 12 selected accessions of bottle gourd assigned the following codes by the Limpopo Department of Agriculture and Rural Development (LDARD) namely: BG-27, BG-31, BG-48, BG-52, BG-58, BG-67, BG-70, BG-78, BG-79, BG-80, BG-81, and GC. BG stands for botte (B) gourd (G), whereas the numbers denote the accession entry number based on the collection mission. These accessions possess varying drought tolerance levels as reported in our previous work ,41. Furtv/v field capacity. One plant was maintained per pot upon germination and three weeks after emergence. The plants were watered with tap water until a sixth fully expanded leaf was developed approximately 18 to 22 days after planting. Water was then withheld for DS treatment for the entire duration of the experiment, whereas the NS plants were supplied with water. The experimental units were without water on days 7, 14, and 21, corresponding to mild, moderate, and severe drought stress, in that order. Three fully expanded leaves were collected from each plant in DS and NS conditions. The same plants used for leaf sampling were carefully exhumed to expose the roots. The roots were gently washed with tap water to remove soil debris. The leaf and root samples were placed in 50 mL centrifuging tubes and kept at −80 °C in a −86 °C ultralow freezer until analysis.The experiment was conducted during the 2021 season under a growth chamber environment at the University of Limpopo , South Africa. The study used the 12 bottle gourd accessions grown under two moisture levels  and drought-stressed (DS)) and three drought stress intensities  using a randomized complete block design with 3 replications. Seeds of the accessions were grown in a 2 L capacity polyethylene plastic pot containing approximately 2 kg of loamy soil collected from the University of Limpopo, Syferskuil Experimental farm . Three seeds were sown per accession and were watered daily to maintain soil moisture content at approximately 40% g, 15 min at 4 °C. The supernatant was removed and filtered through hydrophilic polypropylene membrane (13 mm × 0.2 μm) and the cucurbitacin-containing extract was stored at −20 °C in a freezer.Cucurbitacin extraction was carried out as previously outlined ,73 with Luffa operculate (L.) Cohn, and isolation was conducted using a column chromatography. Molecular identification was performed with nuclear magnetic resonance. Cucurbitacins B, E, and I were quantified using an external calibration curve on a dry weight basis  and 0.1% of formic acid in methanol (B). Gradient elution was as follows: 0–0.5 min 20% B, 0.5–10 min 90% B, 10–12.50 min 90% B, 12.50–12.60 min 20%, 12.60–15 min 20% B (run time 15 min). Flow rate was maintained at 0.4 mL/min at 40 °C. Cucurbitacin identification was performed via the comparison of the retention times of the cucurbitacin B, E, and I standards detected at 230 nm. Pure cucurbitacin B, E, and I standards were purchased from Sigma-Aldrich . The samples were extracted from ht basis . Figure ® 730 Life Science UV/Vis spectrophotometer . The absorbance reading was averaged for each of the samples, and the percentage antioxidant potential (percentage inhibition) was calculated using the following formula [Ac = Absorbance of control (negative) solutionAs = Absorbance of cucurbitacin solutionThe free radical scavenging activities of pure cucurbitacins B, E, and I were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay as previously outlined  with som formula :(1)% Inhw/v) in distilled water in test tubes. After adding each solution, the mixtures were vortexed for approximately 10 s. This was followed by incubation at 50 °C for 20 min. After that, 2 mL of trichloroacetic acid  (10% w/v) in distilled water was added to each test tube. The tubes were left to stand at room temperature for approximately 20 min; during this time, the sediments settled at the bottom of the tube and the yellow top parts of the mixtures were transferred to new, clean test tubes. To the yellow solution in a new test tube, 5 mL of distilled water and 1 mL ferric chloride  in distilled water were added, and the mixture was vortexed. The same procedure was followed for the preparation of the negative control; instead of adding cucurbitacin, distilled water was added. The color change for all the mixtures from yellow to green or blue was monitored and measured at a wavelength of 700 nm using DU® 730 Life Science UV/Vis spectrophotometer. The percentage increase of the potassium ferrocyanide (Fe2+), which is directly proportional to the antioxidative power of the compound, was calculated using the following formula [Ac = Absorbance of control (negative) solutionAs = Absorbance of cucurbitacin solutionEvaluation of the ferric-reducing power of cucurbitacins B, E, and I was carried out as previously outlined  with som formula :(2)% incThe plant population of bottle gourd was relatively low due to larger within- and between-row planting space, limiting genotype evaluation under variable drought tolerance and multiple sampling. The collection of variable age leaf and root samples  could affect the accumulation patterns and estimation of cucurbitacin contents. Furthermore, data presented in one season could limit the repeatability of the experiment and an overestimation of measurements needing multiple sampling across environments.The present study determined the potential roles of cucurbitacins in conferring drought tolerance in bottle gourd. Accumulation of cucurbitacins B and I were identified in leaves and roots of some bottle gourd accessions subjected to drought stress. Based on the DDPH test and FRAP assays, pure cucurbitacins B, E, and I reduced free radicals, indicating their ability as antioxidant molecules in reducing cell damage caused by reactive oxygen species in drought-exposed bottle gourd crops. The present study revealed that cucurbitacins B and I are novel biochemical markers for screening drought tolerance in bottle gourd or related cucurbits.


Coronavirus disease 2019 (COVID-19) is an infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The clinical and epidemiological analysis reported the association between SARS-CoV-2 and neurological diseases. Among neurological diseases, Alzheimer’s disease (AD) has developed as a crucial comorbidity of SARS-CoV-2. This study aimed to understand the common transcriptional signatures between SARS-CoV-2 and AD.System biology approaches were used to compare the datasets of AD and COVID-19 to identify the genetic association. For this, we have integrated three human whole transcriptomic datasets for COVID-19 and five microarray datasets for AD. We have identified differentially expressed genes for all the datasets and constructed a protein–protein interaction (PPI) network. Hub genes were identified from the PPI network, and hub genes-associated regulatory molecules (transcription factors and miRNAs) were identified for further validation.AKT1, ALB, BDNF, CD4, CDH1, DLG4, EGF, EGFR, FN1, GAPDH, INS, ITGB1, ACTB, SRC, TP53, CDC42, RUNX2, HSPA8, PSMD2, GFAP, VAMP2, MAPK8, CAV1, GNB1, RBX1, and ITGA2B. Specific miRNA targets associated with Alzheimer’s disease and COVID-19 were identified through miRNA target prediction. In addition, we found hub genes-transcription factor and hub genes-drugs interaction. We also performed pathway analysis for the hub genes and found that several cell signaling pathways are enriched, such as PI3K-AKT, Neurotrophin, Rap1, Ras, and JAK–STAT.A total of 9,500 differentially expressed genes (DEGs) were identified for AD and 7,000 DEGs for COVID-19. Gene ontology analysis resulted in 37 molecular functions, 79 cellular components, and 129 biological processes were found to be commonly enriched in AD and COVID-19. We identified 26 hub genes which includes Our results suggest that the identified hub genes could be diagnostic biomarkers and potential therapeutic drug targets for COVID-19 patients with AD comorbidity. COVID-1Alzheimer’s Disease is a neurodegenerative disorder more than 50 m people are affected worldwide and this count is expected 150 m in 2050 . The maj2.2.1.We have used gene expression datasets such as transcriptome datasets and microarray datasets to find the differentially expressed genes. This collection of datasets was extracted from gene expression omnibus (GEO) at the National Center for Biotechnology InformationFor our analysis, we used the following inclusion criteria:Dataset which contains samples from the disease group and the control group in original experimental studies.Expression profiling by array used for AD with GEO2R tool support.Expression profiling by high throughput sequencing with raw counts data used for COVID-19.Only homo-sapiens datasets were included.A dataset containing at least eight samples included.The keywords used for AD include “Alzheimer’s Disease” and further the results were filtered by the term “homo-sapiens,” and we selected the study type “expression profiling by array” which resulted in five datasets for AD. Among the five datasets, three of them were associated with peripheral blood mononuclear cells (PBMCs), and two of them were brain tissue-based. For COVID-19 we used the keywords “SARS-CoV-2” to narrow down the results and further filtered them by “homo-sapiens,” and “expression profiling by high-throughput sequencing.” We retrieved three datasets related to COVID-19, including two PBMC datasets and one brain tissue dataset. Both control (non-diseased) and diseased samples are included in all the datasets 2.2.p value  in RStudio version 2022. The transcriptome profile of COVID-19 tissues and blood samples was compared with control tissues and blood samples. DESeq2 is a statistical model designed to identify differentially expressed genes between two or more conditions, it is often used in the analysis of RNA-Seq data, to identify the genes which change in expression between different biological samples or conditions , 44. Thep value and log fold changes are then used to rank the genes based on their level of differential expression  was constructed using the common differentially expressed genes between the four groups and on other hand, the PPI network (module 2) was constructed using the genes with common GO terms. The protein interactions were constructed using STRING version 11.52.5.in vitro assessment is possible of some of the mechanistic functions of candidate miRNAs prior to conducting preclinical animal tests (The interaction between hub genes-transcription factors (TFs) and hub genes-microRNAs (miRNA) has been conducted. Transcription factors play a crucial role, it binds with specific genes and regulates the rate of transcription of genetic information. Bioinformatically and/or al tests . Cytoscaal tests . Therefoal tests . The ideal tests , 56.2.6.The drug-gene interaction was identified using Drug Gene Interaction Database (DGIdb) . DGIdb i2.7.Cluster Profiler (Version 4.1.0) Bioconductor package in R was used for creating Gene ontology of the hub genes . The top3.3.1.p-values based on the moderated t-statistic (adj P) <0.05 along with an absolute value of logFC (log foldchange) of >1. The logFC ≥1 was considered as upregulated genes and logFC ≤ −1 was considered as downregulated genes.DEGs were identified for each data set by using adjusted 3.2.The enrichment analysis of the gene ontology terms was confirmed using the “cluster Profiler” package, the analysis was performed separately for each comparison with applied hypergeometric statistical test, through the below equation,p-values were adjusted for multiple comparisons, and q-values were also calculated for FDR control as well. p-values <0.05 were considered to be significantly enriched terms . The modified Fisher exact p-value (EASE score) ≤ 0.05 and FDR < 0.05 are considered strongly enriched  and indirect  associations. PPI networks can be constructed by calculating the distance ‘D’ between pairs of proteins ,STRING tool provides four thresholds as a default including low (0.15), medium (0.40), high (0.70), and highest (0.90) and, we created a network using a medium threshold value .4.4.1.We retrieved five microarray datasets for AD and three transcriptome datasets for COVID-19 which includes disease and healthy samples. The AD microarray datasets were GSE4226, GSE4229, GSE18309, GSE97760, and GSE36980 analyzed through GEO2R. The transcriptome-based COVID-19 datasets GSE152418, GSE166190, and GSE174745 were analyzed through the DESeq2 Bioconductor package in R software. The datasets were analyzed individually and identified the DEGs , S2. The4.2.HST6, POLR3G, SLC6A20, ITGA2B, HOMER3, GMPR, AGBL1, CRABP2, OLFML2B) genes have been found to be shared between AD-PBMC, AD-Tissue, COVID-19-PBMC, and COVID-19-Tissue. We identified the genes which were present in at least 3 groups and tabulated them  protein interaction network construction. 4.4.via Cytoscape software. The edges represent the interactions between the genes, and the nodes represent the genes. The STRING database was used to construct the protein interaction network then visualized 4.5.ACTB, CDC42, RUNX2, HSPA8, PSMD2, GFAP, VAMP2, MAPK8, CAV1, GNB1, RBX1, ITGA2B) obtained from common genes (module 1) PPI network. A group of 17  overlapping genes was obtained through gene ontology (module 2) PPI network  has been associated with aging in both senescent cells and aged tissues in vitro and in vivo. In murine embryonic fibroblasts, Cav-1 knockout accelerates premature senescence, while loss of Cav-1 accelerates neurodegeneration and aging. In most cell types, ACTB (Actin-Beta) is abundantly and stably expressed and is commonly used to normalize gene expression as an internal control  and transcription factors (TFs) are involved in the development and progression of COVID-19 and its comorbid conditions. Based on the analysis of the hub genes-miRNA and hub genes-Transcription factors, we have obtained a clear network of interactions. The results revealed that the miRNAs regulate 26 hub genes, which could be a possible target of the comorbidity. All the hub genes have targeted a total of 839 miRNAs of which 27 miRNAs were targeted in more than three hub genes .Also, we have identified the hub miRNAs using four ranking methods  of the CytoHubba plugin in Cytoscape. We extracted the top 40 nodes from each ranking method and the overlapped miRNAs were identified using a Venn diagram . The miR4.7.iRegulon predicted 85 TFs for the hub genes and importantly four TFs HAND2, GATA1, GATA2, and GATA6 interacted with 23 hub genes . The hea4.8.We investigated the drug interactions of hub genes using the DGIdb. A total of 26 hub genes were explored through the drug-gene interactions network. The network result shows that a total of 106 were interacting with the hub genes . Some of4.9.p value <0.05, as illustrated in Functional enrichment analysis results showed that hub genes are involved in several biological functions. We identified hub genes related gene ontology using cluster profiler package in r, and we plotted the significantly enriched terms based on adjusted 5.HST6, POLR3G, SLC6A20, ITGA2B, HOMER3, GMPR, AGBL1, CRABP2, and OLFML2B) were commonly expressed between these groups. In addition, we performed Gene Set Enrichment Analysis for the DEGs of Alzheimer’s disease and SARS-CoV-2 DEGs, then we retrieved the genes with common gene ontology terms for constructing a PPI network (module 2).High-throughput sequencing technologies, bioinformatics, and systems biology analysis methods could identify and reveals the changes in the expression level of genes and also assists to identify the potential biomarkers for several diseases importantly neurodegenerative diseases. In this study, the focus is on understanding how AD and COVID-19 disease are related through pathogenetic processes and molecular crosstalks. We followed systems biology approaches including DEGs identification, PPI network construction, hub genes identification, gene set enrichment analysis, and pathway analysis. Also, we explored and identified the regulatory network and drug-genes interaction of the hub genes. To investigate the relationship between AD and COVID-19 we performed gene set enrichment analysis using AD and COVID-19 DEGs discretely. The datasets were further classified into four different groups such as AD-PBMC, AD-Tissue, COVID-19-PBMC, and COVID-19-Tissue. We collected the common DEGs from among the four groups for constructing a Protein–Protein interaction network (module 1). While only 9 DEGs  is the olfactomedin domain protein photomedin-2, with an important role in neural crest development and neurogenesis, cell–cell adhesion, and cell cycle regulation. The OLFML2B gene may contribute to the treatment of bladder cancer in the future based on individual prognostic markers (via the AMPK (AMP-activated protein kinase) and adenosine receptor pathways (via an increase in glycine levels in the brain and N-Methyl-D-Aspartate receptors (NMDAR) activity . Also selecting overlapping DEGs from separate analyses of tissues and blood samples may not completely eliminate the confounding effect of sample variation. Additionally, the large number of DEGs identified in the study may have caused a potential for false positive results. While we attempted to address these issues by performing additional analyses including hub genes and pathway analysis.6.AKT1, ALB, BDNF, CAV1, CD4, CDC42, CDH1, DLG4, EGF, EGFR, FN1, GAPDH, INS, ITGB1, ACTB, SRC, TP53, RUNX2, HSPA8, PSMD2, GFAP, VAMP2, MAPK8, GNB1, RBX1, ITGA2B) hub genes were identified. Transcription factor network analyses revealed that several TFs play a crucial role in post-transcriptional and transcriptional regulators of the differentially expressed genes. The identified shared pathways between AD and COVID-19 provide there are several similar underlying mechanisms play in both diseases. Our findings could lead to identifying a potential biomarker to predict the highest risk of neurological complications with COVID-19. Also, the identified transcription factor might be a potential therapeutic drug target for both diseases.The present study aims to understand the molecular crosstalk between COVID-19 and Alzheimer’s Disease, including discovering the gene expression signatures, TFs, Drug-gene interaction, miRNAs associations, and dysregulated molecular pathways. As a result of integrated analyses of microarrays and transcriptomics of PBMC cells and tissue cells, we were able to identify AD and COVID-19 DEGs. Through PPI network analysis twenty-three (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/TP analyzed the data and wrote the manuscript. SS conceptualized and designed the work, revised, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TP53 or its regulator MDM4 may cause short telomeres and alter hematopoiesis, but also revealed features of Diamond–Blackfan anemia (DBA) or Fanconi anemia (FA), two BMFSs, respectively, caused by defects in ribosomal function or DNA repair. p53 downregulates several genes mutated in DC, either by binding to promoter sequences (DKC1) or indirectly via the DREAM repressor complex , and the p53-DREAM pathway represses 22 additional telomere-related genes. Interestingly, mutations in any DC-causal gene will cause telomere dysfunction and subsequent p53 activation to further promote the repression of p53-DREAM targets. Similarly, ribosomal dysfunction and DNA lesions cause p53 activation, and p53-DREAM targets include the DBA-causal gene TSR2, at least 9 FA-causal genes, and 38 other genes affecting ribosomes or the FA pathway. Furthermore, patients with BMFSs may exhibit brain abnormalities, and p53-DREAM represses 16 genes mutated in microcephaly or cerebellar hypoplasia. In sum, positive feedback loops and the repertoire of p53-DREAM targets likely contribute to partial phenotypic overlaps between BMFSs of distinct molecular origins.Mice with a constitutive increase in p53 activity exhibited features of dyskeratosis congenita (DC), a bone marrow failure syndrome (BMFS) caused by defective telomere maintenance. Further studies confirmed, in humans and mice, that germline mutations affecting  TP53, the gene encoding tumor suppressor p53, is the most frequently mutated gene in human sporadic tumors [TP53 mutations [TP53 mutations in cancers affect the core DNA-binding domain of the protein and alter its capacity to bind specific DNA sequences within target gene promoters [Trp53 locus, which created an allele encoding p53Δ31, a mutant p53 that lacks the last 31 amino acid residues [Δ31/Δ31 mouse embryonic fibroblasts (MEFs), the p53Δ31 protein appeared more stable than wild-type (WT) p53 and correlated with increased activity, as exemplified by a stronger transactivation of p53 target genes , a more efficient cell cycle arrest response to γ-irradiation, and accelerated senescence [Δ31/Δ31 thymocytes [ΔCTD mutant mouse, another model characterized by a smaller deletion of 24 C-terminal residues [c tumors , and Li–utations . Most TPromoters , and posresidues . In p53Δnescence . Furthernescence , and an ymocytes . These rresidues .+/Δ31 mice, p53Δ31/Δ31 mice were born in Mendelian proportions, but they were much smaller than their WT or p53+/Δ31 littermates, exhibited darker footpads and tails, and most died before weaning. Upon dissection, p53Δ31/Δ31 mice exhibited cerebellar hypoplasia, hypogonadism in males, and hypertrophic hearts typical of anemic animals. Accordingly, sternum sections from p53Δ31/Δ31 mice revealed bone marrow hypocellularity, hematopoietic progenitors were very scarce, and hemograms revealed severe pancytopenia, establishing bone marrow failure as a likely cause of premature death [Δ31/Δ31 mice. However, p53Δ31/Δ31 mice exhibited an additional trait not previously reported in this type of mutant: pulmonary fibrosis. In humans, the association of bone marrow failure with pulmonary fibrosis had been proposed to predict germline defects in telomerase [Δ31/Δ31 cells. Indeed, p53Δ31/Δ31 bone marrow cells exhibited a two-fold decrease in average telomere length compared with WT cells, and p53Δ31/Δ31 MEFs were also shown to exhibit short and dysfunctional telomeres [Δ31/Δ31 mice recapitulated the pathological features  and the molecular diagnostic feature (short telomeres) of dyskeratosis congenita (DC), the archetypal syndrome of defective telomere maintenance, caused by mutations in genes encoding subunits of the telomerase or shelterin complexes or telomerase regulators [Δ31/Δ31 mice exhibited skin hyperpigmentation and oral leukoplakia, whereas nail dystrophy was observable but rare. Furthermore, short stature, heart hypertrophy, testicular atrophy, and cerebellar hypoplasia are features observed in patients with Hoyeraal–Hreidarsson syndrome (HHS), a severe variant of DC, and 30% of the p53Δ31/Δ31 mice had a hypoplastic cerebellum, 63% a short stature, and 100% a hypertrophic heart, and 91% of male mice exhibited testicular hypoplasia. Interestingly, 10% of the heterozygous p53+/Δ31 mutant mice died within a year with a hypertrophic heart, suggesting that they might be mildly affected. Consistent with this, when we analyzed heterozygous p53+/Δ31 mutants in a genetic context hemizygous for the p53 negative regulator Mdm4, 70% of the p53+/Δ31 Mdm4+/− mice died in less than 3 months, exhibiting bone marrow hypocellularity and short telomeres [Importantly, following intercrosses of p53re death . Similarre death ,11,12,13lomerase , which lelomeres . Thus, pgulators . Interesgulators . All p53Δ31/Δ31 data [Δ31/Δ31 mice recapitulated all the phenotypic traits of DC was also surprising, given the previous phenotypes of mouse models of DC. Indeed, laboratory mice have much longer telomeres than humans, and mice lacking telomerase were reported to exhibit short telomeres only after three or four generations of intracrosses [Δ31/Δ31 mice [Δ31/Δ31 mice more closely resembled mice with a telomerase haploinsufficiency and a deficient shelterin complex, reported to develop some DC features in one generation—although neither oral leukoplakia nor pulmonary fibrosis [Δ31 might exert pleiotropic effects on telomeres, possibly by impacting both telomerase and shelterin complexes. We thus analyzed if genes mutated in dyskeratosis congenita or implicated in aplastic anemia might be regulated by p53. Ten genes corresponding to these criteria were tested, and four were found downregulated by p53: Dkc1, Rtel1, Tinf2, and Terf1 [Δ31/Δ31 mice in our study were of mixed genetic backgrounds (C57Bl/6J and 129S2/SvPas), which made it possible to correlate decreased Rtel1 gene expression with decreased p53Δ31/Δ31 mouse survival, further indicating that an abnormal p53-mediated regulation of Rtel1 might have pathological consequences [DKC1 and RTEL1 expression in human cells. Furthermore, the phenotypes resulting from additional nonsense Trp53 mutations targeted in mouse cells, when compared with the distribution of TP53 nonsense mutations in human cancers, suggested that p53 nonsense mutations might have similar consequences in humans and mice [Together, these data revealed that a germline increase in p53 activity could cause defects in telomere maintenance in mice, a rather paradoxical finding given the well-accepted notion of “p53, guardian of the genome” . In factΔ31 data , suggestΔ31 data . The facacrosses ,21, wherΔ31 mice . In thatfibrosis ,23. Thisnd Terf1 . InteresΔ31/Δ31 mice phenocopied syndromes of defective telomere maintenance in a single mouse generation, which suggested that p53 had pleiotropic effects on telomeres, and consistent with this, p53 was found to downregulate three genes mutated in dyskeratosis congenita and one gene implicated in aplastic anemia. However, we initially tested only 10 genes associated with dyskeratosis congenita or aplastic anemia, but several tens of proteins were known to impact telomere maintenance [Tpp1, Parn) [Fancd2, Recql4) [Gar1, Terf2) [Blm, Dek, Fancd2, Fen1, Gar1, Recql4, and Timeless [Gar1 encodes a subunit of the telomerase complex, the finding that it is downregulated by p53 provided further evidence that p53 regulates telomere maintenance. However, the gene most dramatically downregulated upon p53 activation was Fancd2. This was unexpected, because patients with a FANCD2 mutation are diagnosed with Fanconi anemia (FA), a bone marrow failure syndrome caused by defective DNA repair, rather than telomere dysfunction. Intriguingly, Blm encodes a helicase associated with Fanconi proteins in a multienzyme complex [Fen1 encodes an endonuclease stimulated by Fanca [Rtel1 encodes a Fancj-like helicase [Fanca, Fancb, Fancm, Ube2t), the pivotal ID2 complex , and downstream effector proteins  [Δ31/Δ31 cells exhibited a typical feature of cells from FA patients: the decreased capacity to repair DNA interstrand crosslinks induced by mitomycin C. Importantly, out of the 12 FA genes downregulated by p53 in mouse cells, 9 were also downregulated by p53 in human fibroblasts, and an increased expression of FA genes could be used as a marker of human tumor progression [Δ31/Δ31 mice exhibited all the typical features of DC, but their cells also phenocopied cells from patients with FA [p53ntenance  and 30–4ntenance . This suntenance . The 42 1, Parn) ,28, gene Recql4) ,30, gene, Terf2) , or otheTimeless . Becauseby Fanca , and Rtehelicase , which l Rad51c) . Accordigression . Furthergression , and p53gression . Thus, a with FA ,26.Δ31/Δ31 mice, Tummala et al. reported a mutation in PARN, encoding a polyadenylate-specific ribonuclease, in three families with severe DC [Δ31 mouse model, because PARN was proposed to regulate p53 mRNA stability [DKC1, RTEL1, and TERF1 [Δ31/Δ31 mice [The phenotypes associated with human mutations causing germline p53 activation also led to a more complex picture. Following the description of p53evere DC . This aptability  and becand TERF1 , three gΔ31 mice . HoweverΔ31 mice . AccordiΔ31 mice , and TERΔ31 mice , raisingTP53 mutations in two patients with an atypical bone marrow failure syndrome [TP53 mutations were found which both led to a hyperactive mutant p53 protein truncated of its 32 C-terminal residues (p53Δ32). Both patients exhibited pure red cell aplasia, growth delays, hypogammaglobulinemia, microcephaly, and telomere length between the 1st and 10th percentile, and one patient also presented reticular skin pigmentation, hypogonadism, and tooth anomalies. These phenotypes were suggestive of a DBA-like syndrome with additional features more common in DC [TP53 mutations were later confirmed to present DBA-like features, but could exhibit normal telomere length [Toki et al. next reported de novo germline syndrome . In bothon in DC . Additioe length ,40.MDM4. In this family, carriers of the T454MMDM4 mutation exhibited considerable heterogeneity in their phenotypes, with features suggestive of DC, e.g., bone marrow hypocellularity, short telomeres, tongue squamous cell carcinoma, and acute myeloid leukemia [Mdm4 missense mutation (p.T454M) and found that Mdm4T454M/T454M MEFs exhibited decreased Mdm4 protein levels, increased p53 activity, and short telomeres. Mdm4T454M/T454M mice were born in Mendelian proportions but were smaller than their WT and Mdm4+/T454M littermates, and they died immediately after birth from neonatal respiratory failure. Importantly however, 80% of the Mdm4T454M/T454M p53+/− mice died from bone marrow failure in 2–6 months, which indicated that the Mdm4T454M mutant is extremely sensitive to a decrease in p53 signaling. Conversely, most Mdm4+/T454M heterozygous mice remained alive after 6 months and appeared similar to WT mice, except for a very subtle increase in skin pigmentation in 30% of animals. By contrast, all the Mdm4+/T454M p53+/Δ31 compound heterozygotes died in less than 3 months and exhibited intense skin hyperpigmentation, short stature, cardiac hypertrophy, testicular hypoplasia, bone marrow failure, and short telomeres, again indicating that the Mdm4T454M mutant is extremely sensitive to variations in p53 signaling [+/T454M human family relatives. Consistent with this, the most affected family member, with a lymphocyte telomere length within or below the first percentile of age-matched participants and a tongue squamous cell carcinoma at age 27, exhibited a WT MDM4 allele with three single-nucleotide polymorphisms (SNPs) associated with increased p53 activity in addition to the T454MMDM4 allele. Opposite this, less severely affected family members presented SNPs in MDM4 or TP53 that might antagonize the effects of the MDM4T454M mutation [MDM4 were reported in two additional studies: all presented bone marrow abnormalities, and two exhibited very short telomeres, thus confirming the impact of MDM4 on telomere maintenance [In collaboration with Sharon Savage, we identified a family with a heterozygous germline missense mutation in leukemia . We creaignaling . These omutation . Importantenance ,43.MDM2 was found in a patient affected by a Werner-like segmental progeroid syndrome, with clinical traits notably including short stature, prematurely gray hair, and testicular hypoplasia [TP53, MDM4, or MDM2 were found in a few patients, but were not shown to cause disease phenotypes [MDM2 may cause bone marrow failure syndromes remains uncertain at this time.Germline mutations of MDM2, another p53 negative regulator, were also reported. A homozygous germline antiterminating mutation in poplasia . Althougpoplasia , the potenotypes . Thus, wAltogether, the studies in mice and humans indicate that mutations leading to a germline activation of the p53 pathway may cause bone marrow failure, with variable phenotypic features that resemble DC, DBA, or FA.Trp53/TP53 [MDM4 genes [Rtel1 expression, and increased risk of bone marrow failure in p53Δ31/Δ31 mice [Δ31/Δ31 cells exhibited a decreased capacity to repair DNA interstrand crosslinks, a distinctive feature best explained by their increased repression of multiple genes in the Fanconi anemia DNA repair pathway [TP53 mutation also revealed features of DBA [Fbl, encoding a rRNA methyl-transferase [Genes whose mutations cause DC, FA, or DBA will lead to distinct molecular outcomes, i.e., defective telomeres, unrepaired DNA interstrand crosslinks, or dysfunctional ribosomes, respectively . Importap53/TP53 ,38,40 orM4 genes ,42,43, aM4 genes ,53,54,55M4 genes ,57,58,59M4 genes ,26, whic pathway ,61. In f pathway ,62,63, a pathway ,65. Thuss of DBA ,40, a bos of DBA ,68,69,70nsferase . TogetheDkc1 and Fbl are among those genes [dimerization partner, RB-like, E2F4/5, and MuvB) [Rtel1 and 12 genes of the FA DNA repair pathway, and identified functionally DREAM-binding sites for 3 FA genes  [Cenpa, Hjurp) in a later study [p53 was shown to repress transcription over 30 years ago ,73, and se genes ,71. Howend MuvB) ,81,82,83, Rad51)  as well er study . Thus, sTo search for genes downregulated upon p53 activation, we exploited transcriptomic changes associated with the in vitro differentiation of bone marrow cells (BMCs). The system we analyzed relies on the conditional expression of Hoxa9 , a transRTEL1, DCLRE1B), Fanconi anemia , and Diamond–Blackfan anemia (TSR2). These results suggest a model of molecular circuitry that may account for the partial clinical overlap between bone marrow failure syndromes of distinct molecular origins: independently of the initial causal mutation, the resulting molecular defect will lead to p53 activation, and this will lead to the repression of a set of genes implicated in telomere maintenance, DNA repair, and ribosome function  or cerebellar hypoplasia , which might account for the brain-related phenotypes often observed in patients with DC [DREAM binding sites were notably identified for genes mutated in dyskeratosis congenita (function . Due to ntenance , but DKCfunction , whereasA repair ,99. LikeA repair , but FANfunction , FANCD2 ntenance ,102, andfunction ,104. Imp with DC , FA [100 with DC  (Figure BRCA1 and BRCA2 appear as very good candidate p53-DREAM targets [NUF2 and MTHFD1 [It should be noted that the molecular circuitry represented in  targets ,88 for wd MTHFD1 ,108. ThuTP53 or MDM4 genes, which might account for the variable expressivity of disease symptoms among family members with the same syndrome-causing mutation. Evidence for this was already obtained in a familial syndrome of defective telomere maintenance caused by a MDM4 mutation [Ten years after finding that mice with a germline activating p53 mutation exhibit features of a telomere disorder syndrome, we now know that p53 downregulates the expression of many genes implicated in telomere maintenance, DNA repair, and ribosome biology. This may contribute to explaining why p53 activation plays a central role in bone marrow failure processes, and account for the partial clinical overlap between inherited bone marrow failure syndromes. Interestingly, these studies revealed positive feedback loops that may vary in strength because of SNPs affecting the mutation , and it mutation  or even


Biological aging can be described as accumulative, prolonged metabolic stress and is the major risk factor for cognitive decline and Alzheimer’s disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes overexpressed with age, and it is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that, in human cells, genetic removal of QR2 produced a shift in the proteome opposing that found in AD brains while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2is) to enable evaluation of chronic QR2 inhibition as a means to reduce biological age–related metabolic stress and cognitive decline. QR2is replicated results obtained by genetic removal of QR2, while local QR2i microinjection improved hippocampal and cortical-dependent learning in rats and mice. Continuous consumption of QR2is in drinking water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity and pathway function in the healthy and neurodegenerative brain and what we believe to be the great therapeutic potential of QR2is as first-in-class drugs.  Accumulating metabolic dysfunction in the ageing brain creates chronic stress, which disrupts homeostasis and results in a wide spectrum of pathologies that risk the occurrence of dementia, such as Alzheimer’s disease (AD) , 2. ConsQR2 inhibitors (QR2is) have previously been made, in efforts to develop anti-malarial drugs, research melatonin, cancer, and basic biochemistry –24. HoweWe therefore created small molecule QR2is that enable safe and effective evaluation of chronic QR2 inhibition in vitro and in vivo as a way to reduce biological age–related metabolic stress and cognitive decline. We carried out a high throughput screen (HTS) to assay approximately 200,000 compounds against QR2 activity. Following concentration-response hit validation using standard and orthogonal assays against QR2 and QR1, a series of diverse sulfonamides were identified and selected as a lead series. Structure-activity-relationship (SAR) studies of the sulfonamide series’ effective chemical domains resulted in a family of specific QR2is, with potential for further expansion. These inhibitors possess high selectivity and potency against QR2, while showing extremely low toxicity. A water-soluble version of the most potent QR2i was used to obtain a crystal structure, providing insight into the unique interactions these inhibitors make with QR2. By using these QR2is, we hypothesize that (a) cells expressing QR2, such as neural or HCT116 cells, will display lowered metabolic stress, (b) rodents will demonstrate better cognitive abilities and (c) a profound AD mouse model (5XFAD) , at an ahttps://doi.org/10.1172/JCI162120DS1). Proteomic analysis  was carried out, using leading QR2is YB’s-537, 800, and 808. Following a 1 hour preincubation with 5 μM of the QR2is or vehicle, thermal aggregation curves (Taggs) of QR2 expressed in HEK293T were measured in increasing temperatures. All QR2is increased QR2 thermal stability compared with vehicle . Based o50 . This shd YB-537 . ROS levd YB-537 , indicatd YB-537 , C and Dd YB-537 ; howeverd YB-537 , similarQR2 elimination or inhibition has been shown to improve rodent memory in several tasks , 16, 17,To make QR2-ligand crystallization amenable, enable oral administration of the inhibitors, and eliminate undesirable formulations, HCl was conjugated to YB-537 . This reTo estimate the evolutionary conservation of amino acids in hQR2 and related proteins, we used the ConSurf server . The serWe next aimed to test the effect of QR2 inhibition in AD model mice using YB-537, taking advantage of its extremely high specificity, solubility, and lack of toxicity. First, we determined YB-537 pharmacokinetics (PK) and oral bioavailability and assessed any acute observable toxicity in mice. It was found that YB-537 was well tolerated at 50 mg/kg per os (p.o.) or 10 mg/kg i.v. with no discernable adverse symptoms at any time up to 24 hours following administration , which was much more tightly centered around the mean compared with controls , with all animals receiving YB-537 showing measurements centered around the mean , while controls showed far greater variability , and compared cognitive performance to male 5xFAD and WT control mice of the same age not being treated with YB-537, using DFC in double-blind experiments. We found that, while all male mouse groups showed similar learning curves during conditioning , 5xFAD mFollowing the behavioral results obtained with 4-month YB-537 treatment in the male 5xFAD mice, we evaluated AD-related brain pathologies using IHC, similar to the 1-month treatment experiment . The marAge-related cognitive decline and prolonged metabolic stress in the brain represent major medical challenges in the modern era . A centrHere, we found that removing such chronically high levels of QR2 results in a proteome that is functionally opposed to that seen in AD brains , where hTo date, a number of QR2is have been developed , providiSAR studies of QR2-inhibiting molecules discovered by HTS demonstrated that, at either end of a central sulfonamide linker, several amines and heterocycles strongly inhibited QR2 and could be added in various combinations and modules and allow potential development of hundreds of inhibitor variants. The medicinal chemistry carried out initially, based on this approach, resulted in a family of highly effective, QR2 selective, nontoxic inhibitors, shown to directly bind QR2 in human cells. Of these, some of the most promising were tested for water solubility and in vivo efficacy, the latter by way of measuring the known and previously demonstrated memory-enhancing effect of QR2 inhibition in rodent models , 16, 17.Taking advantage of this, we found that by providing YB-537 in the drinking water of 5xFAD mice for 1 month, a significant reduction in brain pathologies and improvement in memory was observed, though this was unexpectedly mainly seen in females. While male and female 5xFAD mice tended to show quicker learning in the MWM, only females demonstrated significant improvement in NOR and DFC paradigms compared with controls. The more potent effect of QR2i in female mice following 1 month of treatment was also noted when measuring brain pathologies. In the hippocampus, both p-tau and amyloid β were significantly diminished following QR2i in the female mouse hippocampus, and microglia activation was also significantly reduced in female mice only. Microglia also showed reduced activation in the female cortex. Interestingly, average 4-HNE levels remained unchanged, but, in females, the number of animals with higher or lower levels of 4-HNE was significantly reduced. This revealed higher homogeneity in 4-HNE levels within the QR2i-group females, compared with much higher heterogeneity in oxidative stress in the control females. Males, in comparison, showed a reduction in astrocyte activation in the cortex, with no other significant changes in any of the other markers, either in the hippocampus or cortex. When assessing amyloid β42 in the whole brain, an insignificant reduction was seen in the soluble but not insoluble fraction in both males and females. Since males showed a slower, more muted response to QR2i than females, we carried out an additional 4 month treatment regimen with male mice and compared them to 5xFAD controls and WT mice, to test whether a longer treatment period would show increased effect in the male population. The longer QR2i treatment did result in much more significant effect, as male 5xFAD mice ingesting YB-537 for 4 months showed similar contextual memory to WT mice, and brain pathologies showed far greater reduction across brain areas. This included significantly reduced 4-HNE in the cortex, with 4 months of QR2i lowering the oxidative stress marker in the 5xFAD mice to levels measured in WT mice. Amyloid β tended to show a reduction in the hippocampus and showed significant reduction in the cortex, while astroglia activation markers GFAP and Iba1 were both similarly reduced in the 5xFAD mice, following the 4 month treatment period. YB-537 therefore reduces brain pathologies and reverses cognitive deficits in the 5xFAD mouse model at an advanced age, with sex-dependent variation in results and a more rapid effect in females. Why females respond to QR2is so much faster than males will be the subject of future studies and may be related to estrogen metabolism , 48, thoWith the recent description of the QR2 pathway in the brain and its association with cognitive dysfunction and metabolic pathologies with age, we believe that an exceptional opportunity to tackle neurodegenerative diseases like AD has been found. The approach of QR2 inhibition for AD treatment may provide direct or combinatory therapeutic efficacy, along with other disease modifying drugs, such as ISRIB , PKR 4)4), and Phttps://www.stat.ubc.ca/~rollin/stats/ssize/n2.html).In this study we created orally available QR2is with the aim to replicate genetic QR2 removal phenotypes, such as reduction of metabolic stress and memory enhancement. Inhibitor specificity for QR2 against QR1 was measured using standardized fluorescent- and orthogonal-absorption activity assays. QR2is were evaluated for in vitro activity and toxicity in QR2-expressing HEK293FT  and THLE-2  cells, and occlusion experiments in QR2-KO cells . QR2i-ta2DCFDA ROS assay was carried out according to established protocols, with some adjustment (Hjustment  6). Cel. Cel2DCFjustment  with somjustment , using TRats were taught to drink from 2 pipettes each filled with 10 mL of water during a 20 minute period, over 3 days. On the fourth day, they were given a novel, palatable taste (2 pipettes each filled with 10 mL of 0.3% NaCl), 30 minutes after a local microinfusion of YB-808 or vehicle. On the fifth day, rats were once again given water in the pipettes. On the sixth day, the rats were presented with a choice test, in which they were given 2 pipettes of water and 2 pipettes of NaCl (10 mL in each pipette). The memory for the novel taste was then assessed after 20 minutes of liquid consumption by calculating a preference index as follows: (novel taste consumed/[novel taste consumed+water consumed]) × 100 , 56.DFC was done as previously described . BrieflyMWM was carried out as previously described . Mice weFollowing a 10 minute exploration of a 50 × 50 cm open-field arena, mice were returned to the arena the next day, which contained 2 identical objects. Mice were allowed to explore the objects and the arena 3 times for 10 minutes, with an inter-trial interval of 10 minutes. The following day, one of the objects was replaced with a novel object and the mice were returned to the arena and allowed to explore for 10 minutes. Mouse movement, exploration, and nuzzling was automatically recorded with an Ikegami ICD-49E camera with EthoVision 14 (Noldus Information Technology). Discrimination of the novel object was assessed by calculating (time exploring novel object – time exploring familiar object)/(time exploring novel object + time exploring familiar object).Perfused and fixed brain slices were blocked with 10% normal donkey serum (DNS) and 0.2% triton (Sigma-Aldrich) in PBS. Antibodies, including 4-HNE , Iba1 , AT-8 , Amyloid β , diluted in PBS with 10% DNS, were incubated at 4°C overnight. Secondary antibodies including donkey-anti-goat Alexa Fluor 568 , donkey-anti-mouse Cy 5 , and donkey-anti-rabbit DyLight 488  all diluted 1:500 in PBS with 1% BSA, were applied to the slices at RT for 2 hours the next day. Slices were mounted onto glass slides using DAPI containing Vectashield (H-1200). Images were taken using a confocal microscope (Olympus IX83). Three slices were used per mouse per antibody. Tiling images of the dorsal CA1 and cortex (Bregma: –1.58mm to –2.30mm) were taken using ×20 objective, acquiring a Z-stack of the whole section. Analysis of the images was done blind by an experimenter unaware of the experimental conditions or details, using Imaris (Bitplane) software. A surface reconstruction module was used to extract the data as volumes of the used markers . Marker volume and intensity were normalized to the corresponding brain volume. The normalized value was averaged for each triplicate and presented here.t tests, 1-way ANOVA followed by Tukey’s or Šidák’s posthoc analysis) and for data not normally distributed, nonparametric tests . Data are presented as means ± SEM. All statistical analysis were done using GraphPad Prism 7 software, unless stated otherwise. P values less than 0.05 were considered statistically significant.Detailed statistics for the manuscript are available in the Supplemental Materials in All animal experiments were approved by the University of Haifa Animal Care and Use Committee committee  and in compliance with the National Institutes of Health guidelines for the ethical treatment of animals.https://neurosenblum.haifa.ac.il/ hQR2 crystal structure is available at https://www.rcsb.org/structure/7O4D The proteomic data set is available upon request from the corresponding author.Data presented is available in the NLG led the project. NLG and KR designed the research and KR supervised the research. NLG, GRS, SC, KS, HK, EE, AE, O David, MF, DT, TP, VS, MW, MM, O Dym, SA, YP, NG, IB, HS, MK, M Lalzar, M Lévesque, and GL performed the research. NLG, GRS, SC, AE, MF, DT, TP, O. Dym, M Lalzar, MA, KS, NH, BL, CS, B Shilton, B Shklyar, VS, GA, and HB analyzed the data. NLG, GRS, O. Dym, KS, SC, BL, M Lalzar, and KR wrote the paper. All authors reviewed the manuscript.