Capsicum annuum L.). In current study, 16 putative chitin genes (CaChi) were retrieved from the latest pepper genome database, and were classified into four distinct classes  based on their sequence structure and domain architectures. Furthermore, the structure of gene, genome location, gene duplication and phylogenetic relationship were examined to clarify a comprehensive background of the CaChi genes in pepper. The tissue-specific expression analysis of the CaChi showed the highest transcript levels in seed followed by stem, flower, leaf and root, whereas the lowest transcript levels were noted in red-fruit. Phytophthora capsici post inoculation, most of the CaChi  were induced by both strains (PC and HX-9). Under abiotic and exogenous hormonal treatments, the CaChiIII2, CaChiIII7, CaChiVI1 and CaChiVI2 were upregulated by abiotic stress, while CaChiI1, CaChiIII7, CaChiIV1 and CaChiIV2 responded to hormonal treatments. Furthermore, CaChiIV1-silenced plants display weakened defense by reducing (60%) root activity and increase susceptibility to NaCl stress. Gene ontology (GO) enrichment analysis revealed that CaChi genes primarily contribute in response to biotic, abiotic stresses and metabolic/catabolic process within the biological process category. These results exposed that CaChi genes are involved in defense response and signal transduction, suggesting their vital roles in growth regulation as well as response to stresses in pepper plant. In conclusion, these finding provide basic insights for functional validation of the CaChi genes in different biotic and abiotic stresses.Chitin-binding proteins are pathogenesis-related gene family, which play a key role in the defense response of plants. However, thus far, little is known about the chitin-binding family genes in pepper ( PR-2 and PR-5) genes are involved in PTI and ETI, depending on the magnitude and time of the interacting signaling components [Plants being sessile organisms are exposed to a number of stresses. External environmental fluctuations, different insect pest and pathogen considerably affect the growth, development, yield and quality . To safemponents ,9.N-acetyl-d-neuraminic acid (NeuNAc) as building blocks. Thus far, the chitinase responsible genes have been classified into seven different classes (classes I–VII) as they belongs to the glycoside_hydrolase_families, thus signifying that the chitinase isozymes were encoded by a family of multi-genes [Arabidopsis thaliana [Triticum aestivum [Oryza sativa [Zea mays [Sorghum bicolor [Hordeum vulgare and has been shown antifungal activity [Nicotiana tobacum after the infection of tobacco mosaic virus (TMV) [Trichoderma harzianum showed higher resistance against phytopathogenic fungi in tobacco and apple [Chitin-binding proteins (CBP), encoded by chitin-gene family, are PR proteins, which enhance resistance to different stresses in several crop plants ,12,13,14ti-genes . Some meti-genes . Certainti-genes . CBPs arti-genes ,19. Up tthaliana , Triticuaestivum , Oryza sa sativa , Zea may bicolor . A classactivity . The patus (TMV)  while ennd apple ,25.Capsicum annuum L.) is one of the essential Solanaceous vegetable crop possessing great economic value throughout the world. Its growth, yield and quality are reduced by numerous biotic factors such as bacterial wilt, Phytophthora blight, viral infections, insect pests and abiotic stresses  [Phytophthora capsici infests pepper, eggplant, tomato, all cucurbits, and more recently snap and lima beans [Pepper  ,27. Thes metals) ,29. It hma beans ,31. To cma beans .cis-acting regulatory elements in the promoter regions and phylogenetic relationships of the pepper chitin-binding protein were elucidated. This study provides a base for future research regarding pepper chitin-binding protein. Furthermore, differential expression was recorded against biotic  and abiotic  stress and hormonal treatment  along with tissue specific expression in different plant parts. This study provides a foundation for further characterization of CaChi members in pepper and valuable information regarding function of this significant gene family in other important crops as well.The chitin-binding proteins are very important as they can enhance resistance against biotic and abiotic stress as well as in plant growth and development. The molecular function of chitin-binding protein genes in pepper plant are un-known. In the current study, sixteen chitin genes (CaChi) in pepper were mined through bioinformatics and their response to biotic and abiotic stresses and hormonal treatment were examined. Subsequently, the gene architecture, conserved domains, exon–intron structure, chromosomal location, gene duplication, gene ontology (GO) characterization, CaChiI1, CaChiI2, and CaChiI3), chitinase glycoside_hydrolase_19 (CaChiIV1 and CaChiIV2) and Barwin (CaChiVI2 and CaChi3) were also found in this gene family (CaChiVI1) to 996 bp (CaChiI1), whereas the deduced proteins had 85–331 amino acids. The predicted pI values ranged from 4.71 (CaChiIII7) to 9.19 (CaChiI2), MW ranged from 9.06 (CaChiVI1) to 35.49 (CaChiI1) kDa and the instability index varied from 18.45 (CaChiIV1) to 68.89 (CaChiIII4) (CaChiIII3 contains the most (36) sulfur elements while CaChiVI1 has the fewest (9) sulfur elements. All deduced proteins are shown in To comprehensively investigate and analyze the chitin-genes in pepper, the Hidden Markov Model (HMM) profile of the chitin-binding protein (Accession no. PF00187.17) was blast-searched in the pepper genome. As a result, 21 and 17 chitin genes were retrieved form CM334 and Zunla-1 databases, respectively. The gene sequences were aligned to avoid repetition and alternative splicing, and the longest sequences among them were chosen for further analysis. Among those 21 and 17 genes mined from the CM334 and Zunla databases, the genes having similar sequences with each other, were considered as a single gene. Consequently, we designed primer pairs  for the e family . In addiChiIII4) . The molAegilops tauschii, Arabidopsis thaliana, Artemisia annua, Brassica napus, Brassica rapa, Bromus inermis, Bupleurum kaoi, Capsicum annuum, Capsicum baccatum, Capsicum chinense, Carica papaya, Cryptomeria japonica, Dionaea muscipula, Drosera rotundifolia, Euonymus europaeus, Gossypium barbadense, Gossypium hirsutum, Glycine max, Hevea brasiliensis, Hippophae rhamnoides, Hordeum vulgare, Limonium bicolor, Linum usitatissimum, Lupinus albus, Malus domestica, Mikania micrantha, Momordica charantia, Nepenthes khasiana, Nepenthes maxima, Nicotiana attenuate, Nicotiana benthamiana, Olea europaea, Oryza sativa, Persea americana, Pisum sativum, Psophocarpus tetragonolobus, Rehmannia glutinosa, Saccharum officinarum, Secale cereal, Sesamum indicum, Solanum tuberosum, Sorghum bicolor, Triphyophyllum peltatum, Triticum aestivum, Urtica dioica, Vitis vinifera, Zea diploperennis, and Zea mays. The analysis shows that 16 CaChi were clearly classified into four distinct classes according to their sequence relatedness with previous research. Three CaChi  were clustered in class I, seven CaChi  in were clustered class III, two CaChi-genes (CaChiIV1 and CaChiIV2) were clustered in class IV, and four CaChi  were clustered in class VI . Motif 6 was present in 50% of the CaChi . Motifs 2 and 10 were present in five and four CaChi, respectively. Motifs 3, 4 and 9 each were found in three different sequence of CaChi while motifs 7 and 8 each existed in two CaChi , in which the HSE in the promoters of the CaChiIV1 and CaChiVI2 were highest (4) followed by CaChiIII2, CaChiIII6 and CaChiIV1 (each have 2). MeJA-responsiveness elements (CGTCA-motif) were found in the promoter region of 10 CaChi, where CaChiIII1 had the highest number (5) of elements followed by CaChiIII4 (3), while CaChiIII5 and CaChiIV2 each have two elements. Cis-acting elements involved in abscisic acid (ABA) responsiveness elements (ABRE), salicylic acid responsiveness (TCA-element) and ethylene-responsive element (ERE) were found in the promoter regions of six CaChi. The MYB binding site involved in drought-inducibility (MBS), resistance and stress responsiveness (TC-rich repeats), GA-responsive element (GARE-motif) and fungal elicitor-responsive element (W-box) were found in the promotor regions of 10, 8, 6 and 7 CaChi genes, respectively. The cis-acting element involved in low temperature sensitivity (LTR) was found in the promoter region of four CaChi. In addition, GA-responsive element (P box) and auxin-responsive elements (TGA-element and AuxRR-core) were also found in some of the CaChi promoter regions. All of the anticipated cis-elements were involved in response to signaling molecules and stresses.To examine the possible Gene ontology (GO) enrichment analysis of CaChi were predicted by the gene ontology slim analysis using Blast2GO tool. The analysis comprised three categories, i.e., biological process, molecular function, and cellular component same as mentioned by Di et al. (2018) . Our resPhytophthora capsici, pepper plants were inoculated with P. capsici via root drenching and their expression levels were analyzed by qRT-PCR. The results exposed that, post P. capsici inoculation, the CaChi were differentially expressed  (18.75%) were downregulated to both strains on maximum time points. Initially, CaChiI1 and CaChiVI4 exhibited downregulation after inoculation with virulent strain (HX-9), then CaChiI1 upregulated on 48 hpi and CaChiVI4 on 72 hpi, which were 1.18 and 1.45, respectively. However, CaChiIII2,CaChiIII6 and CaChiVI1 exhibited progressive upregulation at all the time points in both strains, but CaChiIII2 was reached to peak (29.39) at 48 hpi in HX-9, while CaChiIII6 showed the highest transcription level after PC post inoculation (48 h), i.e., 26.17. Whereas CaChiVI2 peaked at 12 hpi in virulent (29.38) and avirulent (29.75), CaChiIV1 showed the highest expression compared with other CaChi, reaching a maximum at 38.52 (PC) and 45.51 (HX-9) and then slightly downregulated. Meanwhile, in the event of avirulent strain inoculation, CaChiI2 (2.25), CaChiIII1 (5.80) CaChiIII3 (2.30) and CaChiIII5 (2.59) were not predominantly upregulated but, at 72 hpi, showed slight expression. However, CaChiIII4 and CaChiVI2 were upregulated following the same pattern and reached a maximum 19.39 and 29.75, respectively, at 12 hpi. CaChiI3 and CaChiIV2 exhibited significant expression only to virulent (HX-9) strain. CaChiIII7 revealed upregulation for both virulent and avirulent strains and reached a peak (31.10 and 37.13 respectively) at 6 hpi, then downregulated, and subsequently upregulated. Six hours post inoculation, CaChiVI3 exhibited the highest transcription in PC strain and then later it was downregulated at every other time point; however, for HX-9 strain, its transcriptional level was raised.To examine the transcription levels of CaChi against the virulent (HX-9) and avirulent (PC) strain of xpressed . Twelve CaChiI3, CaChiIII2, CaChiIII4, CaChiIII6, CaChiIII7, CaChiIV1, CaChiIV2 and CaChiVI2) were selected from the CaChi, in which at least one gene was selected from each class on the basis of their cis-acting elements response and expression to P. capsici. Then, they were subjected to NaCl, mannitol and cold stresses , and then downregulated. CaChiIII2 showed a slight upregulation at 6 hpt in response to NaCl while exhibited concomitant up- and downregulation in response of mannitol stress. In the case of cold stress, no expression was recorded. In response to NaCl, CaChiIII4 initially exhibited no response, then upregulated at 6 hpt, reached a maximum at 12 hpt (16.84) and then showed a slight downregulation at 24 and 48 hpt, whereas no significant response was observed in response to cold and mannitol  in response to cold stress, where the expression was reduced in later hours. CaChiIII7 was gradually upregulated in response to NaCl, reached a maximum at 6 hpt (5.53) and then downregulated. In response to mannitol, a slight up regulation was noted at 12 hpt and then downregulated similarly. In cold stress, significant expression (7.32) was observed in all time points. The transcript level of CaChiIV1 was highly induced by mannitol, cold and NaCl at 12, 24 and 48 hpt, which were more than 12, 16 and 15 folds, respectively. CaChiIV2 was initially downregulated by NaCl and mannitol stress and then exhibited an abrupt upregulation in response to NaCl at 12 hpt (3.34) and again downregulated. In response to mannitol stress, no significant expression occurred. In cold stress, it shown initial abrupt upregulation and then smoothly downregulated  then smoothly downregulated. In response to cold and mannitol, it was upregulated and reached a maximum at 6 hpt (5.86) and 48 hpt (5.40), respectively.To examine the expression levels of the CaChi in response to abiotic stresses, eight representative genes  were significantly upregulated  at different time points, while CaChiIV1 and CaChiIV2 were gradually upregulated over time and maximum expressions at 48 (10 folds) and 12 hpt (two folds) were recorded in response to MeJA treatment. The CaChiIII4 was initially upregulated 1 hpt after ABA treatment (>5 folds), and then irregular changes were noticed, while, in response to MeJA and SA, the expression level was very low or even not obvious, except for SA where an abrupt upregulation (2.29) was noted at all given time points. CaChiIII7 responded to MeJA and reached a peak at 48 hpt (10.60), whereas the response to ABA was antagonistic-like initially where an upregulation and then smooth decline were observed. Expression was not induced by SA treatment. The transcript levels of CaChiIV1 gene were induced by MeJA, steadily increased and reached a peak (10.90) at 48 hpt. In the case of SA treatment, it was gradually upregulated, reached a peak (6.82) at 6 hpt, downregulated at 12 and 24 hpt, and again upregulated at 48 hpt. After ABA treatment, transcription level was high at 1 (7 folds) and 12 hpt (10 folds). The CaChiIV2 was upregulated by SA, reached to peak at 48 hpt (8.76). After ABA application, it abruptly upregulated at 1 and 12 hpt , while it showed no significant response to MeJA.Phyto-hormones and plant signaling molecules, such as MeJA, SA and ABA, are involved in various stress signaling pathways ,36,37,38CaChiI1, CaChiIII4, CaChiIII5, CaChiIV2 and CaChiVI3, while some of them had very low or no expression in all the tested tissues, i.e., CaChiI3, CaChiIII1, CaChiIII6 and CaChiVI1. (CaChiIV1 and CaChi13) are expressed only in particular tissue, mostly in seedling stage. Additionally, to further authenticate the CaChi expression level in various vegetative and reproductive tissues, we cultivated AA3 pepper variety in normal condition and different tissues were collected at different stages. Gene specific primers were used for qRT-PCR analysis , where the particular gene highly correspond to the development of red fruit, flower and leaf. Therefore, it can be assumed that the expression pattern implied by CaChiVI1 may function importantly in red fruit development. Moreover, CaChiI2, CaChiIII2, CaChiIII6, CaChiIII7 and CaChiIV2 did not show highly significant expression to any tested tissues excluding seed but CaChiIII6 having same results in silico. The integrated investigation of publicly available dataset revealed the ubiquitous expression of these genes and a number of CaChi exhibited a certain degree of tissue specificity. The noticeable differences in both expression level might be related to variation in biological materials, regulation of transcript, interpretation methodology and environmental fluctuation.To further elucidate the expression characteristics of CaChi genes in various vegetative and reproductive tissues , we carried out in silico analysis using public transcriptomic database of pepper ,40. The aChiVI1. A. Some ganalysis . As showCaChiIV1 under NaCl stress, the empty vector (used as control) and CaChiIV1-silenced plants were treated with NaCl (300 mM) solution. As shown in CaChiIV1 significantly compromised resistance to NaCl stress. A greater transcript level of CaChiIV1 was noted in the control (empty vector) plants than in the CaChiIV1-silenced plants in all time points, which is >3 folds. In addition, the transcript levels of other defense-related genes were also studied to see whether the silencing of CaChiIV1 changes their expression. It was noted that, with the passage of time after NaCl treatment, the expression of CaDEF1 (defensin) [CaSAR8.2A (systemic acquired resistance) [CaChiIV1-silenced plants (CaChiIV1-silenced plants (1.1) was less than TRV2:00 (2.7)   and CaSAistance)  were chad plants B,C. Addi00 (2.7) D.Arabidopsis thaliana, 37 in Oryza sativa and 17 chitin genes in Saccharum officinarum were reported [Chitin-binding protein (CBP) is an important biotic and abiotic resistance responsive multigene family in plant ,32,33,43reported ,32. CaChiIV1 contain two introns (CaChiVI1) to 331 aa (CaChiI1). The subcellular location of all the CaChi in pepper were predicted, and found that they exist in chloroplast, extracellular region, nucleus, cytoplasmic and vacuolar locations  distinct classes ,15,33. Tectively . Typicalectively , implyinCapsicum annuum, the highest number of CaChi genes (5) were found on chromosome 3 followed by 7 which had four CaChi. In the duplication and transposition analysis of CaChi, we obtained five clusters of segmental duplication and two genes tandemly duplicated on chromosome 8  of sugarcane [Plant diseases activated by fungal pathogens are one of the main concerns and promote defense to a plant pathogen is a difficult mechanism, which includes the triggering of several immune responses . Several bicolor ,56. Receugarcane . In addiugarcane ,49, and ugarcane .Musa spp. and Saccharum officinarum, respectively. In the current research work, during Phytophthora capsici inoculation (0–7 hpi), the transcription level of at least 11 CaChi  were highly induced by both strains  was exposed to be induced by S. scitamineum [CABPR1 gene in pepper was higher in the virulent strain interaction versus the avirulent interaction [CaSBPs was comparatively greater in the avirulent interaction than in the compatible interaction. Some other studies have revealed that the expression of oxysterol-binding protein gene (CanOBP) and a novel peroxidase gene (CanPOD) were higher in the incompatible interaction [CABGLU), disease-associated protein gene (CABPR1), and peroxidase gene (CAPO1) were expressed in a similar pattern, after the inoculation of virulent and avirulent strains of Phytophthora capsici on the roots of pepper as reported by Wang et al. (2013) [Phytophthora capsici strains, duration of infection, cultivar or the variation in their compatibility systems. The cellular predicted function of CaChi proteins indicated that they had mainly involved in defense response to fungi, bacterium infection, cell wall macromolecule catabolic process, chitin catabolic process and also in other stresses. The molecular functions of chitinase activities and chitin binding were predicted during GO analysis . Our res strains . Moreovencreased . An earl capsici , whereastamineum . The experaction . Howevereraction , it was eraction ,63 and t. (2013) . The difOsWRKY71 gene enhanced cold tolerance and mainly involved in metabolic as well as in regulatory pathways in rice, while Eroglu and Aksoy (2017) [CaChiI3, CaChiIII2, and CaChiIVI genes showed significant response to mannitol, while CaChiIII4, CaChiIII7, CaChiIVI CaChiIV2 and CaChiV12 exhibited greater response to cold stress. Therefore, we assumed that chilling induced intracellular Ca2+ overload may enhance the ROS production which is key component response to chilling stress. The CaChiIII6, CaChiIII7 and CaChiIVI genes expression was maximum at 6 hpt salt stress treatment. These results are supported by Yin et al. (2014) [CaAQP gene in pepper under salt stress and conform the highest expression at 4 h salt stress but in latter hours the expression was dramatically reduced. The reason for downregulation of permeability of membrane is it results in limitation of water loss from vacuole. These effects propose that the dissimilar pepper chitin-binding protein have separate functions in response to numerous environmental stresses. However, as discussed above, different CaChi exhibit strong spatiotemporal and tissue-specific expressions, demonstrating an obvious collaborative and/or divergence in both biological roles and evolutionary relationship of the chitin-binding protein family genes in pepper.Earlier studies have shown that chitins genes were expressed in different patterns in response to various abiotic stresses ,59,64. By (2017)  studied . (2014)  who studCaChiI1, CaChiIII2, CaChiIII6, CaChiIV1, CaChiIV2 and CaChiIV2 expression, CaChiIII7, CaChiIV1 and CaChiVI2 showed maximum expression level against MeJA and CaChiIII2, CaChiIII6, CaChiIII7 and CaChiV12 exhibit increased transcription by ABA application. It should be noticed that the expression profiles of the members of CaChi have distinct characteristics approach in response to these hormone treatments. For example, Guo et al. (2013) [Chitin-binding protein in plants are responsive for the certain level of abiotic  stresses and plant hormones ,58,59. P. (2013)  reportedCaChiIV1 gene initially, we searched the dataset of cis-regulatory elements in the promoter region and then we successfully knocked them down in pepper plant. Further, we performed an expression analysis under salt stress condition  as described in our previous study [http://peppergenome.snu.ac.kr/download.php) [http://peppersequence.genomics.cn/) [To identify the CaChi family members based on the conserved domain, accession No. “PF00187.17” was collected from Pfam database   and Zunlics.cn/)  databasehttps://itol.embl.de/) [pI), the amino acid sequences were blast in Expasy ProtoParam (Available online: http://web.expasy.org/protparam/) [http://www.genscript.com/wolf-psort.html) [http://www.cbs.dtu.dk/services/TargetP/) [Multiple sequence alignment of the pepper chitin-binding proteins were performed by ClustalW according to previous studies on plant chitinases ,32,33. Tmbl.de/)  using netparam/)  and WoLFrt.html) . The TarargetP/)  was usedhttp://meme-suite.org/tools/meme) as described by Guo et al. (2016) [http://www.ncbi.nlm.nih.gov/cdd/), SMART (Available online: http://smart.embl-heidelberg.de/), and EMBL-EBI (Available online: https://www.ebi.ac.uk/interpro/), and their diagrams were generated using online EXPASY server (Available online: https://prosite.expasy.org/mydomains/).The gene structures (exon–intron) of CaChi were obtained by aligning the CDS sequences with their corresponding genomic sequences, and their structures were shaped by using online Gene-Structure-Display Server 2.0 as described by Kang et al. (2016) . Conserv. (2016)  with maxcis-acting elements, 1500 bp upstream from the start codon (ATG) of the CaChi were obtained from the pepper genome database (PGD) and queried against the PlantCARE (Available online: http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [http://promotor.biosino.org/) online servers. The gene ontology analysis of CaChi protein sequences were obtained from Blast2GO (Available online: http://www.blast2go.com) program [To analyze the e/html/)  and Neur program .The amino acid sequences were blast in Blast2GO program and three groups of GO classification  were recovered.http://peppergenome.snu.ac.kr/) as described by Zhang et al. (2016) [n + 4.8L−0.32(1 + exp(−L/1000) otherwise, where n = 6 and L is the alignable length between the two protein sequences of the gene [Information about the chromosomal location of CaChi genes were obtained from Pepper Genome Platform (PGP) (Available online: . (2016) , and the. (2016) . Duplica. (2016)  further the gene ,89.http://peppergenome.snu.ac.kr/) which have been generated previously by Kim et al. (2014) [Publicly available transcriptomic data of root, stem, leaf, and for both pericarp and placenta at mature green (MG), breaker (B), 5 days post-breaker (5B), 10 days post-breaker, 6 days post anthesis (6DPA), 16 days post anthesis (16DPA) and 25 days post anthesis (25DPA) for pepper cultivar CM334 were retrieved from online server (Available online: . (2014) . The datP. capsici strains were obtained from the Laboratory of Vegetable Plant Biotechnology and Germplasm Innovation, Northwest A&F University-China. The method used for P. capsici inoculation was same as described by Khan et al. (2018) and Zhang et al. (2016) [P. capsici. For tissue specific expression, samples were collected from roots, stems, leaves, flower, green fruits, red fruits and seeds of untreated pepper plant for RNA extraction and qRT-PCR analysis [Pepper cultivar AA3, as well as . (2016) ,77. The analysis ,77.For hormonal treatments the plantlets were treated with 50 μM methyl jasmonate (MeJA), 5 mM salicylic acid (SA) and 0.57 mM ABA solution . Plantlehttp://www.premierbiosoft.com/primerdesign/index.html). The specificities of the primers were further confirmed through NCBI Primer BLAST (Available online: https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The pepper ubiquitin-conjugating protein gene (CaUbi3) was used as internal control [−ΔΔCt method [Total-RNA was extracted from different samples using Trizol reagent  following the instruction of manufacturer’s protocol. Further treatment of RNA was achieved with RNase-free DNaseI to eliminate DNA contamination. The cDNA was synthesis by using the Prime-Script TM RT Reagent Kit . The quality of cDNA was checked by nanodrop  and the required volume was calculated and adjusted the concentration up to 50 ng/µL. For qRT-PCR analysis, the gene-specific primers  were des control , with a t method .CaChiIV1 gene in the pepper plant cultivar AA3, and the VIGS assay was performed as described by Liu et al. (2016) [pTRV2:CaChiIV1, a 232-bp cDNA part of CaChiIV1 gene was PCR-amplified. Briefly, the CaChiIV1 gene was cloned into a pTRV2 vector to construct the recombinant plasmid pTRV2:CaChiIV1, which was further used in the subsequent research to confirm the exact silencing of CaChiIV1 (primer pairs used for vector construction are given in pTRV2:CaPDS (positive control) along with the combined vector pTRV2:CaChiIV1 into an Agrobacterium tumefaciens strain (GV3101). A. tumefaciens harboring pTRV1 was mixed at a 1:1 ratio with pTRV2, pTRV2-CaPDS and pTRV2-CaChiIV1. The agrobacterium inocula suspensions harboring pTRV1, pTRV2:00, pTRV2:CaPDS or pTRV2:CaChiIV1 (OD600 = 1.0) were infiltrated into the full extended cotyledons leaves of pepper plants using a 1.0 mL clean needleless syringe [CaChiIV1-silenced plants were collected to measure the silencing efficiency by RT-PCR. The triphenyltetrazolium chloride (TTC) method was used to measure the root activity [CaChiIV1-silenced (pTRV2:CaChiIV1) plants were collected at various time points after NaCl stress as described by Khan et al. (2018) [The VIGS approach was used for the knock-down of the . (2016) . For pTR syringe . Then, t syringe ,95. Fortactivity ,95. Befo. (2018) . These ep < 0.05) test was used to measure the significant differences among the given treatments. Data were presented in graphs and designed using GraphPad Prism 7.0 .The results were subjected to an analysis of variance (ANOVA) using SPSS software , and the analyzed data were expressed as means ± standard deviation (SD) of three replications in all measured parameters. The least significant difference (LSD; The findings of our research work briefly explain that the pepper chitin-binding protein gene family performs a vital role in the complex signaling networks system in response to numerous biotic and abiotic stresses and exogenous hormone applications. Every member of CaChi has its specific expression pattern and functional preference. Meanwhile, the particular molecular mechanism and function of pepper chitin-binding protein are still unclear. Additionally, the chitins–genes connections are also weakly discussed. The various transcription pattern of pepper chitin genes has been observed in tissues due to fluctuations in environmental circumstances, for example salt, drought and hormones, suggesting the diverse roles and inimitable transcription levels of chitin genes in the pepper plant growth, development and response to different stresses. Accordingly, different changes in transcription level in the same pepper chitin gene against several biotic and ambient alterations confirm the differences in their mechanism of regulation. These outcomes clarify the background for further experiments and provide the basic knowledge to explore the role and the possible cross-talk between pepper chitin-binding proteins in plants.


Oryza sativa ssp. indica cv. Purpleputtu) is a unique rice landrace from southern India that exhibits predominantly purple color. This study reports the underlying genetic complexity of the trait, associated domestication and de-domestication processes during its coevolution with present day cultivars. Along-with genome level allelic variations in the entire gene repertoire associated with the purple, red coloration of grain and other plant parts. Comparative genomic analysis using ‘a panel of 108 rice lines’ revealed a total of 3,200,951 variants including 67,774 unique variations in Purpleputtu (PP) genome. Multiple sequence alignment uncovered a 14 bp deletion in Rc  locus of PP, a key regulatory gene of anthocyanin biosynthetic pathway. Interestingly, this deletion in Rc gene is a characteristic feature of the present-day white pericarped rice cultivars. Phylogenetic analysis of Rc locus revealed a distinct clade showing proximity to the progenitor species Oryza rufipogon and O. nivara. In addition, PP genome exhibits a well conserved 4.5 Mbp region on chromosome 5 that harbors several loci associated with domestication of rice. Further, PP showed 1,387 unique when SNPs compared to 3,023 lines of rice (SNP-Seek database). The results indicate that PP genome is rich in allelic diversity and can serve as an excellent resource for rice breeding for a variety of agronomically important traits such as disease resistance, enhanced nutritional values, stress tolerance, and protection from harmful UV-B rays.Purpleputtu ( O. sativa, namely indica and japonica, occupy more than 90% of the Asian rice crop acreage  is a colored landrace that exhibits purple color in all aerial parts including seeds except in nodes and pollen . It is as height . The gens height . Earlier pathway . Regulatciphered .japonica and indica rices provided an unprecedented opportunity to investigate the genome wide distribution of allelic variations that control complex pathways such as those that differentiate colored rices from white rice. Deep sequencing coupled with comparative genomic analysis using extensively sequenced diverse rice lines and SNP-Seek database provide a great opportunity to gain incisive insights into the genetic and molecular basis of a diverse array of traits. Further, genomic analysis of diverse genotypes such as wild progenitors, land races, cultivars and modern rices is expected to throw new light on domestication, selection sweeps of specific genomic regions and evolution of colorless grain phenotype. Present day colored rices, i.e., PP, had evolved from their colored progenitors such as O. rufipogon over thousands of years of cultivation, domestication and natural selection. It is interesting to investigate as to how such a complex trait governed by many genes across the genome has evolved and maintained even when selection is biased toward white grained rices in modern time. Interestingly, colored rices are known for traits associated with disease resistance and abiotic stress tolerance as demonstrated by numerous reports of introgression breeding via wide hybridization to essentially transfer useful genes from progenitors and wild relatives to present day cultivars, a form of de-domestication.Allelic variations at certain target loci of the anthocyanin pathway that lead to the formation of many diverse flavonoids and anthocyanins have been described . HoweverO. rufipogon, O. nivara; and the reference genome of Nipponbare rice  using BWA. Overall, 95.7% of the total reads were mapped covering 94% of the reference genome. The reads with mapping quality value ≥30 were retained for further analysis after removing duplicates. Out of total generated reads, 263,679,866 were aligned to the reference genome with an average of 49.5× read depth and 80.61% genome-wide coverage (Whole genome shotgun sequencing of PP genomic DNA (80× coverage) on Illumina HiSeq 2000 platform yielded 43.47 GB of raw data that include a total of 430,403,016 paired end reads of 100 bp. More than 81.7% of the data exceeded Q30 Phred quality score. The quality score of read 1 (forward) and read 2 (reverse) are shown in coverage . SupplemAssembled unmapped reads of PP was used for repeats masking using rice as reference model. A total of 2,124 genes were predicted, out of which 1,239 gene sequences were annotated based on UniProt, NCBI NR and Phytozome database . Of the Nipponbare reference genome  and Australian, aromatic and wild rices. This panel also includes other wild/progenitor species such as O. longistaminata, O. brachyantha, O. barthii, O. meridionalis, O. nivara, O. glaberrima, and O. rufipogon. A deeper analysis of the genome sequences with this panel allowed us to identify a set of unique alleles in PP genome. Out of 3,200,951 variants, 67,774 were found to be unique to PP  cutoff of <0.01. The dendrogram made with 3K dataset comparison shows that PP is close to IRIS-313-8921, IRIS-313-8498  assembly313-8498 . A total313-8498 . The ArmCaryophyllaceae); here it is worth noting that anthocyanins and betacyanins are mutually exclusive in flowering plants , anthocyanin biosynthesis [delphinidin 3-O-glucoside] (PWY-5153), proanthocyanidin biosynthesis from flavanols (PWY-641). Besides this, other biotic and abiotic stress responsive pathways were also found to harbor unique variants in PP (O-glucoside sub-pathway (PWY-5125) consists of 34 variations in which 9 are silent, 23 are missense, 1 each of frame-shift and in frame variations. In contrast, 24 variations were observed in an evolutionarily silenced delphinidin route of anthocyanin biosynthesis pathway (PWY-5153) of rice. Only one missense mutation was observed in enzymes responsible for proanthocyanidin pathway from flavanols (PWY-641) in rice. Trait ontology analysis for unique variants in PP was performed using Q-TARO database  family transcription factors which controls the far-red light signaling pathway by modulating phyA expression by activating FHY1 and FHL, thus maintaining homeostasis , which are involved in flavonoid/anthocyanin biosynthesis besides other biological processes such as wound and drought stress, light signaling and hormone signaling shows 672 variants  in rice including  and AP2 . The higeostasis . In contvariants . The bHLvariants . Interesvariants . The WRKesponses . A set oesponses .Bh4) and straw colored hulls; these are the phenotypic effects of Bh4 and straw hull 4 (Sh4) candidate gene expression , Ra (Chr4), Rc (Chr7), bHLH (Chr1), Rd (Chr1), MYB (Chr3), MYB (Chr6), MYBAS1 (Chr11), chalcone synthase (CHS) (Chr11), chalcone isomerase (CHI) (Chr3), leucoanthocyanidin dioxygenase (ANS) (Chr1), flavanone 3-hydroxylase (F3H) (Chr1), 3-O-glucosyltransferase (3GT) (Chr6), Bh4 (Chr4) and phenylalanine ammonia-lyase (PAL) (Chr2)  indicate divergence from other rice cultivars  on Chr7 exhibits an intronic variation responsible for deletion of ‘GAGA’ at position 138 that does not affect the functions of the gene. In addition, homology search for Rc genes against other rice genomes, did not provide any significant hit for any alternate functional gene. Notably, anthocyanin regulatory gene Ra (Pb/OSB1/LOC_Os04g47080) on Chr4 which is reported to be associated with purple color pericarp (∗) in the C terminus when compared to Nipponbare reference genome. bHLH (LOC_Os01g09900) on Chr1 shows interesting variations leading to amino acid changes . MYB gene (LOC_Os06g19980) on Chr3 shows a total of 14 variations in which 7, 3, 2, and 1 are downstream gene variant, in-frame deletion, intron variant and synonymous variant, respectively; remaining 1 variant belongs to missense variation and the changes in amino acid A263G possibly does not alter the secondary structure of the protein. MYB genes (LOC_Os06g19980) on Chr6 has 23 missense variations causing changes in amino acids, viz. S27R, M47L, R63L, R66L, D72G, L80N, I82S, A83P, I86V, Q112E, S116I, E147G, E148D, I151V, L163V, T202A, I232T, L296R, R298G, S446L, Y607H, and A639P; these changes possibly alter the structure and function of this MYB protein. The gene encoding PAL (LOC_Os02g41630) enzyme on Chr2 catalyzing the formation of 4-coumaroyl-CoA from phenylalanine has one synonymous, one intronic variation and only one missense variation changing amino acid A621V of the protein. CHS (Os11g0530600) on Chr11 has 1 SNP leading to a single amino acid change, N158S. F3H (LOC_Os01g50490) on Chr1, the gene encoding the enzyme involved in conversion of flavonone to dihydroflavonones has 49 unique variations in which 22 SNPs lead to protein structure variations. We found four SNPs each causing amino acid changes to proline and glutamic acid, viz. T3P, A79P, L99P, L474P, and Q80E, K407E, D410E, D414E, respectively. In addition, the remaining SNPs cause mutations of L6V, T15M, V34A, S55G, N77T, D103N, A110V, C389S, L392F, I393V, R418G, G420D, G424W, and T488A. Possibly, these mutations contribute toward hyper-accumulation of anthocyanins in PP. One silent variation was identified in ANS1 (LOC_Os01g27490) gene on Chr1, encoding the key enzyme involved in the conversion of leucoanthocyanidins to anthocyanidins, the penultimate step in anthocyanin biosynthesis. The last enzyme 3GT (LOC_Os06g18790) of this pathway was found on Chr6 of PP genome, converting anthocyanidins to anthocyanins and had three unique SNPs  that may have a role in accumulation of pigments in PP  (Chr2) . The var) (Chr2) . Of the ) (Chr2) . Comparace lines . Contrarultivars . MYBAS1 pericarp  shows onts in PP .Oryza sativa, an independently domesticated rice that has been in cultivation for more than 12,000 years, has become the predominant cereal staple for most of the ∼3 billion plus Asians. With an estimated 30% rise in population by 2050 and unpredictable climate changes, rice breeders must gear up for substantial increase in rice productivity across various agroclimatic regions. Modern rice breeding technologies are increasingly utilizing the genetic resources of progenitors and wild relatives by introgression of new genes associated with important agronomical traits into cultivars. These mainly include genes for biotic and abiotic stress tolerance, growth, and maturity traits. Though colored rices constitute a significant proportion of germplasm collections, they were not extensively used as genetic resources in breeding. Progenitors and domesticated colored rices were reported to be potential source of genes for resistance to diseases and pest, and tolerance to abiotic stresses. Besides, diverse colored pigments are recognized for their nutritional quality and anti-oxidant properties. Further, colored rices were found to be good subject material for understanding domestication and de-domestication processes in grasses in general and rice in particular  and all these genes are uncharacterized indicating the presence of novel genes associated with red/purple pigmentation. Further, comparative genomic analysis using ‘a panel of 108 rice lines’ spanning both indica and japonica, progenitors, land races uncovered 64,349 SNPs and 3,380 INDELs unique to PP genome. In all, we captured unique variants in one fifth of the total gene models of rice. Of the 3,200,951 polymorphic SNPs identified, about a third span across exonic regions and three fourth of them fall in coding sequences. Further, about 33% of the variants are mapped to repeat regions, retrotransposons and transposable elements, a finding that falls within the range reported for Oryza  deviating from the expected ratio of 0.5. Higher Ts/Tv ratios were reported in rice, maize, otus, medicago, diploid wheat, d barley . Due to d barley . Among td barley . Higher d barley . Generald barley , S5, S9.ca lines . It is we genome .Rc locus through the course of domestication and natural and artificial selection , 4-coumarate CoA ligase (4CL), CHS, CHI, F3H, dihydroxyflavonol reductase (DFR); leucoanthocyanidin dioxygenase (LDOX)/anthocyanidin synthase (ANS), GT/3GT, acyltransferase (AT), methyltransferase (MT)  was found in alpha helical region of the PAL enzyme that is positioned far away from the catalytic site, indicating unlikely effect on structural and functional role of this enzyme  indicated change of charged side change amino acid N to uncharged side chain amino acid S at a location near the catalytic residues in PP CHS. This change may be enhancing the catalytic activity of the enzyme  . We founs enzyme . Changese enzyme . The hydcapacity . Furthere family . Similarcyanidin , 2007.C-S-A gene model for rice hull pigmentation: C1 that encodes a MYB transcription factor and acts as a color-producing gene, and S1 that encodes a bHLH protein that functions in a tissue-specific manner. C1 interacts with S1 and activates expression of A1, which encodes a dihydroflavonol reductase . Qubit 2.0 fluorometer (Thermo Fisher) was used to quantify and NanoDrop 2000 (Thermo Fisher) for quality check of the isolated DNA. The DNA was fragmented to 300 bp size using a Covaris M220 focused ultrasonicator. The fragmented DNA was purified, and sequencing libraries were prepared using Illumina TruSeq DNA sample prep kit  as per manufacturer’s specifications. The quantity and size distribution of the libraries were carried out using a Bioanalyzer 2100 (Agilent Technologies). The quantified libraries were subjected for whole genome sequencing on Illumina HiSeq-2000 platform (Illumina Technologies) by paired-end sequencing to generate 90-base pair long, small reads with an insert size of 200–350 bp . Standar2 (BWA software (v0.7) was used for the mapping of high-quality filtered reads against reference genome reference genome  download from Rice Genome Annotation Project Database2 . Further2 .Unmapped reads of PP genome were assembled using MaSuRCA (v3.2.1) assembler with default options . The assMinimum variant frequency of ≥90%, average base quality of the SNP ≥30 and minimum read depth of 10 were the stringent criteria followed to filter the identified SNPs and INDELs. If three or more SNPs were present in any 10-bp window, the SNPs and INDELs were filtered. Genome wide distribution of DNA polymorphisms was analyzed by calculating their frequency at every 100 Kb interval on each rice chromosome. Circos was used to visualize the distribution of the SNPs and INDELs on rice chromosomes . Such diWe downloaded raw data of 108 selected rice cultivars  and alig3. Corresponding SNPs of PP were filtered as per SNP-seek database norms and compared using Bcftools to determine unique alleles. Identification of different transcripts associated with these variants were also performed. Variations in transcription factor genes were detected using PlantTFDB  using default parameters . QTLs/Gen Q-TARO .Bh4 gene for selected 108 rice lines against various public databases provides only 70 lines with bh4 genes sequences, while Rc gene was present for selected cultivars. Further multiple sequence alignments using ClustalW and phylogenetic tree construction using the Neighbor-Joining method and branch length is estimated using the bootstrap in MEGAv6 (Search of n MEGAv6 .PRJNA309223.Whole genome data of PP rice submitted in SRA database with BioProject ID: The datasets generated for this study can be found in data generated in this work was related to rice plant and submitted in SRA database (Acc No. PRJNA309223).in silico and in vitro experiments. RG, VL, GT, and AR analyzed the results. All authors contributed to writing the manuscript, discussed the results, and commented on the manuscript.GT, SS, and VL conceived the work and designed the experiments. RG, VL, BK, SL, AMK, NC, and LM performed VL, SL, BK, NC, AMK, AR, and GT were employed by AgriGenome Labs Pvt. Ltd., India, SS was employed by SciGenom Labs Pvt. Ltd., India, and RG and LM were employed by MedGenome Labs Ltd., India.


The incidence and mortality rate of cancer has been quickly increasing in the past decades. At present, cancer has become the leading cause of death worldwide. Most of the cancers cannot be effectively diagnosed at the early stage. Although there are multiple therapeutic treatments, including surgery, radiotherapy, chemotherapy, and targeted drugs, their effectiveness is still limited. The overall survival rate of malignant cancers is still low. It is necessary to further study the mechanisms for malignant cancers, and explore new biomarkers and targets that are more sensitive and effective for early diagnosis, treatment, and prognosis of cancers than traditional biomarkers and methods. Long non-coding RNAs (lncRNAs) are a class of RNA transcripts with a length greater than 200 nucleotides. Generally, lncRNAs are not capable of encoding proteins or peptides. LncRNAs exert diverse biological functions by regulating gene expressions and functions at transcriptional, translational, and post-translational levels. In the past decade, it has been demonstrated that the dysregulated lncRNA profile is widely involved in the pathogenesis of many diseases, including cancer, metabolic disorders, and cardiovascular diseases. In particular, lncRNAs have been revealed to play an important role in tumor growth and metastasis. Many lncRNAs have been shown to be potential biomarkers and targets for the diagnosis and treatment of cancers. This review aims to briefly discuss the latest findings regarding the roles and mechanisms of some important lncRNAs in the pathogenesis of certain malignant cancers, including lung, breast, liver, and colorectal cancers, as well as hematological malignancies and neuroblastoma. It was estimated that there were about 18.1 million newly diagnosed cancer cases and about 9.6 million cancer-related deaths worldwide in 2018 . Becausehttp://cpc2.cbi.pku.edu.cn [http://www.bioinfo.org/software/cnci), CPAT (http://lilab.research.bcm.edu/cpat/index.php), ESTScan (http://estscan.sourceforge.net/), PLEK (https://sourceforge.net/projects/plek/files/), PORTRAIT (http://bioinformatics.cenargen.embrapa.br/portrait), FEELnc (https://github.com/tderrien/FEELnc), TransDecoder (http://trinityrnaseq.sf.net), and GeneMarkS-T (http://topaz.gatech.edu/GeneMark/license_download.cgi); CPAT and ESTScan can also provide a web server [http://www.cuilab.cn/gic/) [Long non-coding RNAs (lncRNAs) are a class of RNA transcripts with a length larger than 200 nucleotides. Generally, lncRNAs do not encode proteins or peptides. In addition to the size of other classes of non-coding RNAs , lncRNAs also have secondary and three-dimentional structures which enable them to have both RNA- and protein-like functions . LncRNAsu.edu.cn , and canb server . Moreovecn/gic/) .cis-regulation or trans-regulation [It has been shown that the majority of lncRNAs are localized in the nucleus , but somgulation . It has gulation . So far,gulation ,15, and gulation ,18,19,20LncRNAs are a class of RNA transcripts with a length greater than 200 bp, characterized by more spatial and temporal specificity and lower interspecific conservation when compared with mRNAs . LncRNAsFirstly, lncRNAs play a crucial role in regulating gene expression though chromatin modification and remodeling, histone modification, and nucleosome localization changes . Among tFurthermore, microRNAs (miRNAs) inhibit the translation efficiency and/or induce mRNA degradation of the target gene by complementary pairing with the target RNA, regulating gene expression at the post-transcriptional level. There is a type of competitive endogenous RNA (ceRNA) that can indirectly regulate the expression of target genes via competitively binding with miRNAs; this competitive binding of miRNAs is also called microRNA sponges ,38. ManyLC is the leading cause of cancer-related deaths, accounting for 18.4% of total cancer-related deaths and taking the first place with an incidence rate of 11.6% among males and females . LC is pMALAT1, also known as nuclear-enriched abundant transcript 2 (NEAT2), is an 8.7 kb intergenic lncRNA located on chromosome 11q13 , and is Several studies have suggested that MALAT1 could serve as a potential prognostic biomarker and therapeutic target for patients with early-stage LC due to its specificity and stability ,49,50,51Functionally, MALAT1 is considered as an oncogenic lncRNA because of its role in promoting tumor differentiation, proliferation, migration, invasion, EMT, and chemoresistance ,41. SileH19 is a 2.3 kb intergenic and maternally-expressed lncRNA located on chromosome 11p15.5, which is also one of the first identified imprinting lncRNA ,41,46,63Studies have revealed that the expression of H19 is significantly increased in LC tissues when compared with that in adjacent tissues ,66. H19 H19 plays an important role in promoting cell proliferation and differentiation, cell migration and invasion, and EMT . Zhou etTUG1 is a 7.1 kb intergenic lncRNA located on chromosome 22q12 ,79,80. TTUG1 is upregulated in the majority of the above cancer tissues but downregulated in LSCC and LAD tissues ,82,83,84TUG1 expression is induced by the wild-type p53 in three NSCLC cell lines , and TUG1 knockdown increases cell proliferation in vitro and in vivo ,84. AddiMEG3 is 6.9 kb in length and acts as a tumor suppressor gene located on chromosome 14q32.2 within the DLK1-MEG3 locus ,88,89. MMEG3 is significantly downregulated in LC cell lines A549 and HCC823 . UpregulThe drug resistance to chemotherapy drug vincristine is negatively associated with the expression of MEG3 in vitro . UpregulAFAP1-AS1 is a 6.8 kb antisense lncRNA, which is located on chromosome 4p16.1. AFAP1-AS1 is transcribed from AFAP1 in the antisense direction and can also affect the expression of AFAP1 . AFAP1-AAFAP1-AS1 expression is positively correlated with tumor pathological grade, TNM staging, smoking history, infiltration degree, distant metastasis, and clinical outcomes in NSCLC patients ,100,101.AFAP1-AS1 is upregulated in NSCLC tissues and cell lines ,106,107.According to the Global Cancer Statistics 2018, the incidence of BC in both sexes is 11.6%, which is almost as much as that of LC . HerceptHOTAIR is located on human chromosome 12q13.13, and is a 2158 bp intergenic lncRNA situated inside the Homeobox C (HOXC) gene. HOTAIR is among the first lncRNAs reported to be involved in the development of cancer . HOTAIR The expression of HOTAIR is important for BC cell growth, and loss of HOTAIR induces apoptosis in MCF-7 cells . The proHOTAIR plays a critical role in regulating BC cell apoptosis, proliferation, migration, and invasion. Knockdown of HOTAIR suppresses cell proliferation and promotes apoptosis in several BC cell lines ,130. MorIn the MDA-MB-231 BC cell line, overexpression of HOTAIR promotes the expression of stemness-related genes such as SOX1, SOX10, aldehyde dehydrogenase 2 family member (ALDH2), and CD44 . When coGAS5 is localized on chromosome 1q25.1, and was originally isolated from the NIH 3T3 cell line . AlthougMiR-21 is an important oncogene in various malignant tumors ,152,153.GAS5 is associated with drug and chemotherapy resistance in BC. Trastuzumab administration is a therapy method in clinic to treat HER2-positive, early-stage, and metastatic BC, but the effectiveness is limited . The levThe expression of H19 is remarkably increased in BC tissues ,160 and Abolished H19 expression suppresses cell survival and blocks estrogen-induced cell growth in BC cells . KnockdoH19 has been also reported to be involved in chemoresistance in BC. In paclitaxel-resistant TNBC cells, the expression of level H19 is significantly upregulated, and knockdown of lncRNA H19 enhances paclitaxel sensitivity and promotes apoptosis by suppressing the Akt signaling pathway in TNBC cells . The expUCA1 is a 2314 bp lncRNA composed of three exons and two introns located in human chromosome 19p13.12 ,179. UCAP27 (Kip1) is a tumor suppresser, and its level is decreased in BC tissues . UCA1 coTamoxifen is usually used to treat ER-positive BC patients, but the resistance of tamoxifen is still a question to be resolved . When coLincRNA-ROR is a 2.6 kb lncRNA, which is located at 18q21.31 ,198. The+/CD44+/CD24− MCF10DCIS stem cell population to regulate self-renewal of CSCs in BC [In TNBC tissues ,203 and Cs in BC .Silencing of lincRNA-ROR inhibits BC cell proliferation, increases sensitivity to tamoxifen, and represses the activation of the PI3K/Akt/mTOR signaling pathway in MDA-MB-231 cell . InhibitLiver cancer is the sixth most common malignancy worldwide, and the mortality rate is strikingly high. In 2018, there were approximately 782,000 deaths worldwide, which accounted for 8.2% of all cancer deaths . AlthougHOTAIR has been considered to be a risk factor of HCC ,208. In Inhibition of HOTAIR represses the proliferation, migration, and invasion of HCC cells ,211,213.HULC is located on chromosome 6p24.3, which is firstly characterized as a novel mRNA-like lncRNA, and HULC is shown to be significantly upregulated in human HCC . A high Knockdown of HULC in HCC cell lines represses cell proliferation, invasion, and migration, and promotes cell apoptosis , whereasHepatitis B Virus X protein (HBx) plays vital roles in the development of HCC. The expression levels of HULC are positively related with HBx in HCC patients, and HBx can induce the expression of HULC . AnotherNEAT1 is located on chromosome 11q13.1. NEAT1 has two transcription variants, which are termed as NEAT1_1 and NEAT1_2, respectively. Both of them are located in nuclear paraspeckles and constitute an important structure of paraspeckles . When coIn one study, it was reported that although cell growth is not affected after knockdown of NEAT1 in HepG2, cell invasion is significantly decreased . Ling et+T cell apoptosis and the cytolysis activity in HCC patients [The balance between NEAT1_1 and NEAT1_2 may take part in the process of HCC cell malignant growth and metastasis . Silencipatients . CollectSNHG1 is located in the 11q12.3 region of the chromosome. Both public microarray data and a cohort study discovered that SNHG1 levels are significantly increased in HCC tissues, and the high expression of SNHG1 is positively associated with tumor size ,254. A mIn HepG2 cells, the expression level of SNHG1 is remarkably increased, and the high level of SNHG1 downregulates miR-195 to promote HCC cell proliferation, invasion, and migration . AnotherP21 is a canonical transcriptional target of p53 and plays a critical role in cell cycle and tumorigenensis . lincRNAIn different tumor cell lines, lincRNA-p21 is induced by p53. lincRNA-p21 can interact with hnRNP-K to be involved in the apoptosis regulated by p53 . UpregulCRC is one of the most common gastrointestinal tumors, with the second highest incidence among females (9.5%) and the third among males 10.9%) [0.9% 1,2. About 1CCAT1 is a newly discovered oncogenic lncRNA, 2628 bp in length, which is located on chromosome 8q24.21 ,273. CCAMechanistically, CCAT1 expression is considered to be associated with cell cycle, proliferation, apoptosis, migration, invasion, and EMT process in multiple cells . OverexpCCAT2 is a 1752 bp lncRNA located on chromosomal region 8q24.21 ,289,290.Overexpression of CCAT2 in CRC cells could promote tumor growth, migration, and invasion in vitro and vivo and also induce chromosomal instability in vitro . There iCRNDE is located on chromosome 16, and contains six exons: E1–E6 . CRNDE iCRNDE is significantly upregulated in both CRC tissues and cell lines compared with that in adjacent tissues and normal colon cell line . CRNDE kA research clarified that the level of UCA1 in CRC tissues is positively correlated with CRC tumor size and advanced tumor stages . A meta-UCA1 is demonstrated to be involved in the process of CRC tumorigenesis and progression, including cell proliferation, cell cycle, autophagy, apoptosis, and drug resistance ,310. UCAHematological malignancies are one of the most common types of malignant tumors that are genetically divided into acute leukemia (AL), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), chronic lymphocytic leukemia (CLL), and lymphoma . AccordiPVT1 is an oncogenic lncRNA located at chromosome 8q24.2 downstream of c-Myc, and this region is thought to be cancer-related because of its function with c-Myc and rearrangement ,317,318.Zeng et al. reported that PVT1 level in peripheral blood samples from patients with acute promyelocytic leukemia (APL) is significantly upregulated when compared with healthy donors, which indicates that PVT1 may act as a novel biomarker for APL diagnosis . IzadifaLncRNA DLEU1 and DLEU2 are located in the region of chromosome 13q14.3 ,333, andLee et al. found that knockdown of DLEU1 in the human Burkitt lymphoma (BL) cell line results in a significant increase in cell proliferation and a reduction in apoptosis when compared to WT cells. Conversely, overexpression of DLEU1 in Raji cells causes a significant decrease in cell proliferation and an increase in apoptosis compared to control cells. The authors further showed that DLEU1 knockdown leads to a significant upregulation of anti-apoptotic genes and a downregulation of pro-apoptotic genes . AdditioResearches have shown that DLEU1, DLEU2, and embedded miRNA cluster miR-15a/16-1 are frequently deleted in CLL, MM, mantle cell lymphoma (MCL), and some types of solid tumors ,343,344.MEG3 is significantly downregulated in AML patients’ tissue samples and cell lines when compared to normal controls, particularly in the Wilms’ tumor 1 (WT1) or ten–eleven translocation 2 (TET2) mutant AML patients ,345,346.Huang et al. reported that the expression of MALAT1 in patients with AML-M5 is significantly increased when compared with healthy controls. The OS of AML-M5 patients with high MALAT1 expression is significantly decreased compared with that of AML-M5 patients with low MALAT1 expression . UpregulThe expression of MALAT-1 in patients with MM and MM cell lines is significantly higher than that in normal controls ,353,354.The expression of MALAT-1 is significantly higher in diffuse large B cell lymphoma (DLBCL) cell lines than that in normal controls . MALAT1 The expression of HOTAIR is significantly upregulated in DLBCL tissues and cell lines when compared with normal controls. DLBCL patients with higher expression of HOTAIR have poorer OS . KnockdoHOTAIR expression level is tested to be significantly upregulated in BM/peripheral white blood cell (PB) samples from AML patients when compared with AML-complete remission (CR) patients and healthy controls. Its level is markedly downregulated in AML patients after treatment ,360,361.HOTAIR can interact with miR-143, regulating CML cell proliferation, knockdown of HOTAIR, or overexpression of miR-143, inhibiting the phosphorylation of the PI3K/AKT/mTOR signaling pathway . MoreoveNB is the most common pediatric extracranial solid tumor, which derives from primitive sympathetic neural precursors, and accounts for more than 7–10% of all pediatric cancers and 15% of all pediatric cancer deaths ,367,368.Under hypoxic conditions, the expression of MALAT1 in NB cells promotes endothelial cell migration, invasion, and vasculature formation . MALAT1 The expression of SNHG7 is markedly higher in NB tissues than that in non-tumor tissues . Kaplan–Cancer susceptibility 15 (CASC15) and neuroblastoma associated transcript 1 (NBAT1) are tumor suppressors that are located at the NB risk-associated 6p22.3 locus ,380. CASLncRNA Paupar is transcribed from 8.5 kb upstream of the paired box 6 (Pax6). Reports have shown that Paupar knockdown can regulate the expression of multiple genes related to cell cycle, DNA replication, and mitosis, induce elevation of cell count in S and G2 phases, and increase neurite outgrowth in N2A cells . Paupar LINC01105 and hyperpolarization activated cyclic nucleotide gated potassium channel 3 (HCN3) are significantly upregulated, whereas MEG3 is downregulated in NB tissues when compared to control tissues . It has Kaplan–Meier curves analyses have shown that NB patients with high expression of Ets-1 promoter–associated noncoding RNA (pancEts-1) and hnRNPK are associated with poor OS and EFS. Significant higher pancEts-1 and hnRNPK transcription levels can be observed in primary NB tumors than that in normal dorsal ganglia tissues, and upregulation of pancEts-1 and hnRNPK is also correlated with NB cases with poor differentiation and advanced International Neuroblastoma Staging System (INSS) stage. Mechanistically, pancEts-1 can promote NB cell growth, invasion, and metastasis in vitro and in vivo through β-catenin transactivation and other mechanisms .Zhang et al. found that X inactive specific transcript (XIST) is markedly upregulated in NB tissues when compared with that in control tissues. XIST can stimulate NB cell proliferation, migration, and invasion by epigenetically downregulating dickkopf WNT signaling pathway inhibitor 1 (DKK1) expression through induction of H3 histone methylation via EZH2 .Knockdown of lncRNA myocardial infarction associated transcript (MIAT) reduces cell growth and migration, and enhances apoptosis in NB cell line when compared with that in control . AtmadibOverall, multiple lncRNAs have been shown to be involved in the pathogenesis of NB. Further study is needed to explore the distinct roles and mechanisms of lncRNAs in the development of NB. Moreover, whether lncRNAs could serve as plasma biomarkers for predicting the risk of NB also awaits exploration. LncRNAs play an important role in the pathogenesis of various malignant cancers by promoting cell proliferation, migration, and invasion at transcriptional, translational, and post-translational levels. In particular, several lncRNAs play important roles in the pathogenesis of various cancers, suggesting that they represent a new class of targets for treating different cancers , Table 2MALAT1 plays an important role in the development of various cancers by modulating cell proliferation, migration and apoptosis. The symbol “↑” means activation, and symbol “⊥” represents repression.HOTAIR plays an important role in the pathogenesis of various cancers by multiple signaling pathways. Targeting HOTAIR represents a novel strategy for treating various cancers. The symbol “↑” means activation, and symbol “⊥” represents repression.


Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are key transmembrane proteins leading to reactive oxygen species (ROS) overproduction. However, the detailed roles of NOXs in retinal pigment epithelial (RPE) cell metabolic stress induced by Earle's balanced salt solution (EBSS) through starvation remain unclear. In this study, we investigated what roles NOXs play in regard to calpain activity, endoplasmic stress (ER), autophagy, and apoptosis during metabolic stress in ARPE-19 cells. We first found that EBSS induced an increase in NOX2, NOX4, p22phox, and NOX5 compared to NOX1. Secondly, suppression of NOXs resulted in reduced ER stress and autophagy, decreased ROS generation, and alleviated cell apoptosis. Thirdly, silencing of NOX4, NOX5, and p22phox resulted in reduced levels of cell damage. However, silencing of NOX1 was unaffected. Finally, taurine critically mediated NOXs in response to EBSS stress. In conclusion, this study demonstrated for the first time that NOX oxidases are the upstream regulators of calpain-2, ER stress, autophagy, and apoptosis. Furthermore, the protective effect of taurine is mediated by the reduction of NOX-derived ROS, leading to sequential suppression of calpain induction, ER stress, autophagy, and apoptosis. Reactive oxygen species (ROS) are signaling molecules that result in metabolic stress, changes in mitochondrial membrane permeability, DNA damage, and cell apoptosis –3. ROS a2+ [Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is another key source of ROS besides mitochondria , 12. NOX2+ –27. The 2+ .2+ disturbances [There is growing evidence showing that NOXs are important sources of ROS during ER stress . The incurbances , 31. Numurbances . Oxidatiurbances , 34. ROSurbances . It is kurbances . In addiurbances , 38.RPE cells are highly metabolically active cells that are located in the retina and play a vital role in maintaining normal visual function. Hence, RPE cells are vulnerable to oxidative stress , 39. In In our previous study, we demonstrated that taurine inhibited starvation-triggered cell damage in ARPE-19 cells . Howeverμg/mL). Cells were maintained at 37°C and 5% CO2.ARPE-19 cells were procured from Shanghai GuanDao Biotech Co. Ltd. Cells were subcultured in Dulbecco's modified Eagle's medium (DMEM)/F-12  containing 10% FBS , penicillin (100 IU/mL), and streptomycin  were obtained from GenePharma . The siRNAs (two short sequences of siRNA specific for each one) were transfected into ARPE-19 cells using lipofectamine 2000 reagent  for 12 h. Next, cells were incubated for another 48 h in normal culture conditions. Protein expression was detected using Western blot, and the better siRNAs were chosen for subsequent experiments. The scrambled (nontargeting) siRNAs were used as the negative control.Human The protocol of the Western blot analysis is described in detail elsewhere . AntibodμM) in PBS for 30 min. Fluorescence was measured with a FACSCalibur flow cytometer . The protocol of the annexin V/PI staining is described in detail elsewhere [Cellular ROS levels were monitored using the fluorescent probe (DCFH-DA). Cells were seeded in 6-well plates overnight and treated with EBSS for 24 h with or without inhibitors/siRNA/taurine pretreatment. Next, cells were loaded with DCFH-DA  for 30 min. Nuclei were stained with Hoechst for 20 min. The protocol of image analysis is described in detail elsewhere [Cells were cocultured with DCFH-DA  was considered significant.Data was expressed as the mean ± standard error of the mean (SEM). Statistics were analyzed by SPSS 21.0 statistics program . One-way ANOVA and Dunnett's posttest were used to determine the statistical significance. A μM) as a positive control for 6 h. The expression of NOX proteins was examined by Western blot. The results show that the protein expression of NOX2, NOX4, p22phox, and NOX5 was dramatically increased from 6 h until 24 h after EBSS treatment. However, the expression of NOX1 was unchanged . We observed that the fluorescence intensity of the peaks shifted immediately after 3 h until 24 h post-EBSS treatment. Furthermore, EBSS induced the loss of calcium homeostasis in ARPE-19 cells  or with tunicamycin , or the NOX inhibitor, apocynin , and then cultured with EBSS (6 or 12 h). We found that pretreatment with NAC or Apo strongly and significantly reduced the expression of NOX2, NOX4, p22phox, and NOX5 Figures , suggestTo further explore the possible roles of NOXs and ROS in EBSS-induced autophagy and apoptosis in ARPE-19 cells, we examined the expression of autophagy-related proteins and mitochondrial apoptotic proteins by Western blot. Our results suggest that both inhibitors, NAC and Apo, caused a significant decrease in the expression of beclin-1, LC3, Bax, and cleaved caspase-3. Meanwhile, levels of P62 and Bcl-2 were noticeably increased Figures , indicatNOX4, p22phox, and NOX5 silencing resulted in lower levels of calpain-2 and ER stress-related proteins. However, induction of calpain-1 was unaffected  species leading to a marked decrease of NOX protein levels Figures . To demo Figures . Our finNOX4, p22phox, and NOX5 silencing downregulated the expression of beclin-1, LC3, Bax, and cleaved caspase-3. Meanwhile, the levels of P62 and Bcl-2 were increased .We also investigated the effects of taurine (30 mM) on NOXs and ROS. Our results suggest that the expression of NOX2, NOX4, p22phox, and NOX5 in the EBSS group was increased compared with the control group. The level of proteins was decreased after intervention with taurine. The expression of NOX1 levels was not influenced in models (EBSS treatment) and taurine + EBSS groups Figures . OxidatiOur study demonstrates the involvement of NOX-derived ROS in EBSS-induced RPE metabolic disorders. NOXs are upstream regulators of calpain-2, ER stress, autophagy, and apoptosis. Furthermore, NOXs are involved in calpain-2, ER stress, autophagy, and apoptosis regulation by NOX4, p22phox, and NOX5 but not by NOX1 or NOX2. Taurine alleviated cell injuries through regulation of NOX levels .During metabolic stress (starvation), the balance of ROS level changes. NOXs are activated and result in excessive production of ROS (termed oxidative stress) . Further2+ also leads to ROS accumulation [The interaction of ROS and calcium signaling can be considered bidirectional; ROS can regulate cellular calcium signaling, while calcium signaling is crucial for ROS generation , 47, 48.mulation , 57. Ovemulation –60. Indumulation , 28, 50.mulation , 61. Bclmulation , 63. We 2+ sensitive, because calpain-2 induction was also inhibited. Hence, NOX influence generation of ROS also regulates ER stress, autophagy, and apoptosis. We showed that NOX4, p22phox, and NOX5 are critical for damage and cell death of ARPE-19 cells. In addition, we found for the first time that NOX5 induction is required for calpain-2 induction, ER stress, autophagy, and apoptosis in ARPE-19 cells (Figures To elucidate the functional significance of NOXs, we examined ROS generation, ER stress, autophagy, and apoptosis in which NOX2/NOX4/p22phox/NOX5 was knocked down with siRNA. Although NOX4/p22phox/NOX5 was not completely knocked down, induction of NADPH oxidase was inhibited. The exact reasons for this are complex and not well-understood but relate to the fact that NOX4/p22phox/NOX5 downregulation influences other NADPH oxidase subunits that may have an impact on the induction of oxidases , 64. It The antioxidation of taurine may prevent disease progression . The antIn conclusion, our data demonstrate that ROS generated via the NADPH oxidase system is the major contributing factor in RPE dysfunction induced by EBSS. NOX silencing or suppression partly protects cells from cell damage. NOX4, p22phox, and NOX5 play key roles in the production of ROS, ER stress, autophagy, and apoptosis. Our observations support the hypothesis that oxidative stress is the main causative for cell injury, and taurine attenuates ER stress, autophagy, and apoptosis in ARPE-19 cells via a suppression of the NADPH oxidase-derived reactive oxygen species-mediated calpain induction pathway. Our data provides novel insights into ocular diseases and mechanisms of taurine.


PIK3CD and PIK3R1 cause combined immunodeficiencies that can also present as CSR/HIGM defects. These defects, their pathophysiology and derived clinical manifestations are described in depth. Previously reported forms of CSR/HIGM syndromes are briefly reviewed and compared to the phosphoinositide 3-kinase (PI3K) pathway defects. Diseases involving the PI3K pathway represent a distinctive subset of CSR/HIGM syndromes, presenting with their own characteristic clinical and laboratory attributes as well as individual therapeutic approaches.Antibody production and function represent an essential part of the immune response, particularly in fighting bacterial and viral infections. Multiple immunological phenotypes can result in dysregulation of the immune system humoral compartment, including class-switch recombination (CSR) defects associated with hyper-IgM (HIGM) syndromes. The CSR/HIGM syndromes are defined by the presence of normal or elevated plasma IgM levels in the context of low levels of switched IgG, IgA, and IgE isotypes. Recently described autosomal dominant gain-of-function (GOF) mutations in  Effective humoral immunity relies on the ability of B cells to recognize a wide variety of antigens and respond properly. A diverse BCR repertoire is established by V(D)J recombination in early stages of B-cell development, prior to antigen encounter. Functional commitment of antibodies takes place at a later stage, allowing responses to be tailored upon antigen recognition in a process called class-switch recombination (CSR) , 2.Defective CSR results in an abnormal humoral pattern known as hyper-IgM syndrome (HIGM), characterized by normal or elevated serum levels of IgM in the context of low levels of switched IgG, IgA, and IgE isotypes. A heterogeneous group of primary immunodeficiencies (PID) underlies HIGM, collectively known as CSR defects , 3–8.PIK3CD and PIK3R1, inducing hyperactivation of the enzyme phosphoinositide 3-kinase δ (PI3Kδ)  mutations in  PI3Kδ) –12, can  (PI3Kδ) , 14. ThiKδ –12, c (PI3Kδ) , a deepeIn this review, antibody maturation steps as well as previously known forms of CSR/HIGM syndromes are briefly described, with special emphasis on the more recently reported PI3K pathway activating mutations. The pathophysiology and clinical manifestations associated with CSR/HIGM syndrome in PIK3CD and PIK3R1 defects are described and analyzed in more detail.B-cell development occurs in the bone marrow. Upon completion, naïve B cells express unique B-cell receptors (BCRs) in the form of membrane-bound IgD and IgM. Naïve B cells circulate throughout the blood and lymphatics to secondary lymphoid organs where activation occurs upon ligation of the BCR with its cognate antigen. Subsequently, and mostly with the help of T cells, two critical steps for antibody maturation occur predominantly inside germinal centers: CSR and somatic hypermutation (SHM) , 8, 15.+ T cells, including T follicular helper cells (Tfh), binds to CD40, a receptor constitutively expressed on B lymphocytes (and monocytes). The CD40L/CD40 engagement in the germinal center, in the presence of the appropriate cytokine milieu, promotes B cells to undergo proliferation, CSR and SHM, through activation of transcription factors, such as nuclear factor-κB (NF-κB). Although in a less effective way, CSR can also be induced in a T-cell-independent manner, via concurrent engagement of BCR or TACI and Toll-like receptors (TLRs) , which is expressed on activated CD4s (TLRs) , 15, 16.The CSR process involves genomic recombination of the IgM-defining constant (C)μ region in the Ig locus for a downstream Cα, Cγ, or Cε region, coding for the constant regions of IgA, IgG, or IgE isotypes, respectively. Constant regions are flanked by switch (S) regions and CSR occurs when two S regions upstream of a C region undergo switch-region recombination, excising the preceding C regions from the Ig locus. CSR involves a sequential, multi-step process. First, there is a requirement for transcriptional access to the S regions, which allows for activation-induced cytidine deaminase (AID) to catalyze the conversion of cytidine nucleotides to uracil. Next, uracil removal by uracil N-glycosylase (UNG), which, along with endonucleases and the mismatch repair (MMR) machinery, facilitates the generation of double-stranded DNA breaks. Finally, the removal of the intervening DNA as excision circles and repair of the DNA double-stranded breaks, primarily by non-homologous end joining (NHEJ), complete the main steps involved in CSR. Thus, the CSR machinery preserves the Ig variable (V) locus region, thereby maintaining antigen specificity , 8, 15.The SHM process randomly introduces mutations into the Ig V regions, altering the affinity for antigens while maintaining the same Ig C isotype. Uracil residues are introduced by AID and can be further modified by UNG. These transitions and transversions generated at the Ig V region are perpetuated during DNA replication, the MMR process, and error-prone polymerase enzymatic activities. SHM additionally allows for affinity maturation by providing a higher diversity of antibodies from which clones with the highest affinity to foreign antigens can be selected. While all HIGM syndromes are determined by CSR defects, SHM abnormalities are not always involved in this group of diseases , 8, 15.Deficiencies in CD40L, CD40, AID, UNG, NEMO, IkBα, ATM, and PMS2 represent the previously identified forms of CSR/HIGM syndromes. While the clinical manifestations can be variable, the HIGM phenotype can be divided into those disorders that directly affect the CD40/CD40L pathway (B- and T-cells are impacted), and those like AID, UNG, NEMO, IkBα, ATM, and PMS2 where mainly B cells are affected in the CSR/HIGM defect , 5–7. ThCD40L (Pneumocystis jirovecii pneumonia (PJP), or Cryptosporidium-induced diarrhea and sclerosing cholangitis, which may predispose patients to tumors of the liver, pancreas or biliary tree . These iewed in .ATM) gene, which encodes an enzyme that contributes to DNA mismatch repair. Clinical symptoms include cerebellar ataxia, oculomotor apraxia, telangiectasias and sinopulmonary infections. Because the ATM gene is involved in DNA double-stranded repair, patients are also sensitive to ionizing radiation and susceptible to malignancy. Laboratory manifestations can also be variable with some patients presenting with a HIGM phenotype, but most other patients having only reduced levels of IgG2 and IgA. The SHM process is not affected in these patients  result in recurrent severe infections, café-au-lait spots, and a HIGM humoral phenotype. While CSR is defective as in other CSR/HIGM syndromes, SHM is only mildly affected  or PIK3R1 (APDS2/PASLI-R1) that induce hyperactivation of the enzyme PI3Kδ  is caused by heterozygous GOF mutations in me PI3Kδ –12, 42.PIK3CD) is restricted to leukocytes, and it is activated by antigen receptors, co-stimulatory receptors, cytokine receptors and growth factor receptors. Briefly, class IA PI3Ks catalyze the phosphorylation of phosphatidylinositol--bisphosphate (PIP2) to generate phosphatidylinositol--triphosphate (PIP3) at the plasma membrane of lymphocytes upon receptor activation and leading to downstream Akt-mTOR signaling  that phosphorylate the inositol ring of phosphatidylinositol lipids in the plasma membrane Figure . There aignaling –45. Phosignaling , 46, 47 ignaling .+ T cells, the Akt-mTOR pathway is critical for regulating whether naïve cells differentiate into effector or memory cells by controlling the transition from oxidative phosphorylation, a lower energy yielding process, to aerobic glycolysis, better suited for the rapid growth and proliferation needed by effector T cells. Akt also suppresses the transcription factor FOXO1 function via phosphorylation and through PI3Kδ-dependent downregulation of IL-7Rα and CD62L , ICOS and IL-2R stimulation. Particularly in CD8nd CD62L , 47, 48.RAG, IKAROS, CD62L and AICDA genes, which as a group are critical for B-cell development, as well as CSR and SHM , and the same E1021K mutation reported by Angulo et al.  cells. In vitro, patients' B cells underwent normal proliferation in response to stimuli, but were unable to secrete class-switched immunoglobulin isotypes , they were predominantly effector memory terminal cells (CCR7−/CD45RA−) also showing signs of increased activity (by CD38 staining). Persistent Akt hyperactivation was hypothesized as the driver for the increased CD8 T-cell proliferation, that in turn determined higher terminally differentiated CD8 T-effector cells, increased senescent CD8 T cells and decreased long-lived memory CD8 T cells, altogether resulting in impaired control of EBV- and CMV-infected cells . Other features consistently seen in patients with SHORT syndrome include mild intrauterine growth restriction, characteristic facies, lipodystrophy and insulin resistance in adolescence progressing to diabetes mellitus in early adulthood. These patients do not typically present with a HIGM humoral phenotype. Immunoblot analysis has shown that levels of p85α were ~50% lower in SHORT syndrome patients than in controls  patients . Growth controls . Normal controls . Interescontrols , 61.PIK3R1 leading to complete absence of p85α. This patient had colitis and history of Campylobacter infection, in the context of absent B cells with resulting agammaglobulinemia. Early in life, erythema nodosum and arthritis were also observed  and T-dependent in vivo stimulation. Of note, Pi3kcdE1020K GOF B cells behaved similarly to WT cells regarding proliferation, germinal center formation, SHM and affinity maturation. Induction of AICDA mRNA in B cells following stimulation with anti-CD40/IL-4 was significantly reduced in mutants compared to WT and positively correlated with the percentage of IgG1+ B cells generated after activation. In vitro treatment with the p110δ inhibitor leniolisib , that are shown to be reduced in PI3K defects   , 14, 42. in APDS , are mar in APDS .Germinal centers' architecture also helps to distinguish between different CSR/HIGM syndromes. While generally absent in CD40/CD40L deficiencies and giant in AID deficiency , 79, theTreatment for APDS patients includes prophylactic measures, such as antibiotics, antivirals, and antifungals, as well as immunoglobulin replacement. Most patients undergo immunosuppressive therapy aiming to control lymphoproliferation and/or autoimmunity. Rapamycin was a frequently used immunossupressor in the ESID-APDS-registry cohort, showing positive results in controlling benign lymphoproliferation, but not as effective for gastrointestinal manifestations and cytopenias . RituximTherapeutics created for the treatment of leukemia and lymphoma, such as idelalisib, duvelisib, or ibrutinib block PI3Kδ either directly or indirectly, making them exciting potential options for treating patients with APDS . LenioliWhen focused on curative treatment, Nademi et al. reported an 81% survival among 11 APDS patients who underwent HSCT in seven pediatric centers. Acute GvHD was a common complication (81%), 2 patients presented low chimerism, and 2/3 of the surviving patients are off immunosuppressive therapy and immunoglobulin replacement .PIK3CD and PIK3R1 GOF mutations, have recently been highlighted as forms of combined immunodeficiency compromising both the T- and B-cell compartments. Common clinical features include recurrent bacterial respiratory tract infections, EBV/CMV viremia, T-cell lymphopenia, memory B-cell deficiency, increased transitional Bcells and elevated IgM levels accompanied by low IgG and IgA levels. Due to elevated IgM levels and low IgG, IgA, and IgE levels, PI3K diseases ultimately fit under the umbrella of the CSR/HIGM syndromes.Diseases involving the PI3K pathway due to AD-AICDA, UNG, NEMO, NFKBIA, ATM, and PMS2, but shown to be variably affected in patients carrying PI3K-associated diseases, although more consistently normal in animal models (PIK3CD and PIK3R1 mutations appear to be intermediate between the above-mentioned defects in terms of opportunistic infections susceptibility. Over time, as with many other immunodeficiencies, autoimmunity has become a more relevant feature demonstrating overall immune dysregulation within HIGM syndromes in general and PI3K defects in particular.While all the diseases presenting with HIGM humoral phenotypes described in this review involve by definition, CSR defects, this mechanism is not univocally disrupted in all these defects. Expression and function of CD40L/CD40 and AID are crucial to this process, and when either of them is altered, they markedly affect its outcome. In PI3K-related defects, evidence points at AID expression and function through FOXO1 regulation playing a central role in CSR integrity. In terms of SHM, this process is generally preserved when defects involve l models , 53, 68.In conclusion, the CSR/HIGM syndromes encompass a wide variety of diseases, each with their own defining features. Diseases involving the PI3K pathway are indeed combined immunodeficiencies, as well as a subset of CSR/HIGM syndromes, presenting with their own characteristic clinical and laboratory attributes as well as individual therapeutic approaches.RDJ wrote the first draft. RDJ, CJN-S, JB and SDR contributed and reviewed all the data presented. JB and SDR supervised the project.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.