Aconitum carmichaelii, Angelica gigas, and Zingiber officinale. Its anti-tumor effect has been reported; however, the efficacy of JI017 against oxaliplatin-induced allodynia has never been explored. Single oxaliplatin injection  induced both cold and mechanical allodynia, and oral administration of JI017 (500 mg/kg) alleviated cold but not mechanical allodynia in mice. Real-time polymerase chain reaction (PCR) analysis demonstrated that the upregulation of mRNA of spinal transient receptor potential vanilloid 1 (TRPV1) and astrocytes following oxaliplatin injection was downregulated after JI017 treatment. Moreover, TRPV1 expression and the activation of astrocytes were intensely increased in the superficial area of the spinal dorsal horn after oxaliplatin treatment, whereas JI017 suppressed both. The administration of TRPV1 antagonist  attenuated the activation of astrocytes in the dorsal horn, demonstrating that the functions of spinal TRPV1 and astrocytes are closely related in oxaliplatin-induced neuropathic pain. Altogether, these results suggest that JI017 may be a potent candidate for the management of oxaliplatin-induced neuropathy as it decreases pain, spinal TRPV1, and astrocyte activation.Oxaliplatin, a well-known chemotherapeutic agent, can induce severe neuropathic pain, which can seriously decrease the quality of life of patients. JI017 is an herb mixture composed of  Oxaliplatin has been widely used and recommended as a first-line treatment for metastatic colorectal cancer ,2. The cTRPV1 mRNA was downregulated with pain amelioration following oral administration of ginger extract and 6-shogaol [Trpv1 knockout (KO) mice affects pain behavior, as well the activation of spinal astrocytes [Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel that responds to physical or chemical stimuli, such as acid, capsaicin, or heat ,11. In t-shogaol . Moreovetrocytes ,17. In pSpinal astrocyte activation is a representative phenotype of various pain states, such as inflammatory pain, nerve injury-induced neuropathy, and chemotherapy-induced neuropathy ,20,21,22Aconitum carmichaelii, Angelica gigas, and Zingiber officinale. A recent study has revealed that JI017 has anti-tumor effects on prostate cancer cell lines [Aconitum carmichaelii has been reported to be potent against nerve injury-, oxaliplatin-, and paclitaxel-induced neuropathy [Angelica gigas showed anti-nociceptive properties by increasing the latency against heat stimuli [Zingiber officinale administration attenuated oxaliplatin-induced cold and mechanical allodynia [Despite its severity, there is no clear way to address oxaliplatin-induced neuropathic pain. Drugs, such as duloxetine or venlafaxine, are recommended; however, their use is limited because of adverse effects, such as somnolence, vertigo, and nausea ,31. Medill lines . Aconituuropathy ,39,40. A stimuli . In our llodynia . Based oIn this study, we investigated whether JI017 attenuates oxaliplatin-induced neuropathic pain. Furthermore, we studied the underlying mechanism of the effect of spinal TRPV1 alteration on the activation of astrocytes at the spinal dorsal horn in an oxaliplatin mouse model.In previous studies ,42,43, athe transient receptor potential cation channel subfamily V member 1 (Trpv1) and astrocytes glial fibrillary acidic protein (Gfap) was investigated. According to several studies of neuropathic pain, including oxaliplatin-induced neuropathy, upregulated GFAP is manifested as the activation of astrocytes [Trpv1 and Gfap were significantly upregulated compared with naïve mice (control vs. Oxa) , both the upregulated Trpv1 and Gfap expressions were significantly suppressed (Oxa vs. Oxa + JI017)  A,B. In c+ JI017) A,B. JI01TRPV1 and GFAP in the superficial dorsal horn.Immunohistochemistry was used to determine whether oxaliplatin treatment could affect the TRPV1 and GFAP levels in the superficial laminae of the spinal dorsal horn. As mentioned above, spinal cords from all groups of mice were collected on D4. First, we noted that the intensity of the TRPV1-positive signal in the superficial area of the dorsal horn was strengthened (control vs. Oxa) A,D. SecoTo investigate the role of TRPV1 in spinal astrocytic activation, capsazepine, a TRPV1 antagonist, was intrathecally injected into oxaliplatin-treated mice. Two hours after the injection, the spinal cords were acquired. Immunohistochemistry was conducted to determine whether capsazepine treatment altered oxaliplatin-induced spinal astrocytic activation. On D4, an overexpression of spinal TRPV1 and GFAP in the superficial area was observed in the oxaliplatin group compared with naïve mice . The intTo clarify whether JI017 could affect the activity of spinal TRPV1, levels of c-Fos and TRPV1 were evaluated in the L4/5 dorsal horn of treated mice. c-Fos expression in the spinal dorsal horn level has been used as an anatomical marker for neuronal activation related to pain . StudiesDespite the prominent anti-cancer effect of oxaliplatin, severe neuropathy, induced even after a single injection, can lower the quality of life of patients and may lead to discontinuation of the treatment. Moreover, the drugs used to decrease this pain also have adverse effects, which limits their use ,51,52. IIncreased TRPV1 expression in the superficial laminae I–II of the spinal dorsal horn has been reported in various types of pain, such as complete Freund’s adjuvant (CFA)-induced inflammatory pain, sciatic nerve injury, diabetes, and paclitaxel-induced neuropathic pain ,16,17,53Zingiber officinale downregulated mRNA levels of spinal TRPV1 and suppressed thermal allodynia in the same diabetic model [TRPV1 knockout (KO) mice that had pSNL [TRPV1 is expressed mostly in C fibers, which are known to be cold-sensitive fibers [In our study, JI017 only attenuated cold allodynia, while mechanical allodynia remained unchanged. Although the involvement of TRPV1 in mechanical pain has also been reported in some studies ,10, seveic model . Moreoveic model . Moreovehad pSNL ,17. It ie fibers ,19. Thise fibers , oral orTRPV1 KO lowered the activation of spinal astrocytes, and heat hypersensitivity was attenuated compared with the sham control [2+ influx and resulting in their activation [TRPV1 remains controversial. According to a study conducted on rats, 7% of the total TRPV1 is localized in spinal astrocytes [Along with spinal TRPV1, oxaliplatin also elicited increased activation of astrocytes in the spinal cord . Several control ,17. The  control . Several control ,57. In a control . It shoutivation ,60. Anottivation ,61. Howetrocytes . Our stuAconitum carmichaelii, Angelica gigas, and Zingiber officinale. In a previous study, JI017 showed anti-tumor potency in prostate cancer cell lines [Zingiber officinale in alleviating oxaliplatin-induced cold and mechanical allodynia in mice; 300 mg/kg of Zingiber officinale had higher efficacy than 100 mg/kg [Zingiber officinale only constitutes a quarter of JI017, in this study, it might have been in insufficient quantity to be efficacious against both cold and mechanical allodynia. Furthermore, HPLC data identified decursin, nodakenin, aconitine, and 6-gingerol as major phytochemicals of JI017 [Angelica gigas, while aconitine is present in Aconitum carmichaelii. 6-Gingerol is a key phytochemical of Zingiber officinale. Among the identified phytochemicals, it has been reported that decursin, aconitine, and 6-gingerol, can be delivered across the BBB [Angelica gigas and Aconitum carmichaelii, respectively, have shown potency in inflammatory and nerve injury-induced neuropathic pain by suppressing the activation of spinal astrocytes [Zingiber officinale, and ginger extract achieved pain relief by downregulating the mRNA of spinal TRPV1 [Medicinal herbs have been used to manage pain and treat various diseases since ancient times. Recently, they have attracted attention as alternative therapies because of their potency and safety . JI017 cll lines . Althougll lines ,39,41. W00 mg/kg . As Zingof JI017 . Decursi the BBB ,63,64. A the BBB , while a the BBB . The ana the BBB ,68,69. Itrocytes ,71. Moreal TRPV1 . HoweverTrpv1 and Gfap increased after single oxaliplatin treatment, whereas JI017 attenuated both. Moreover, our data showed that spinal TRPV1 inhibition by intrathecally injected TRPV1 antagonist blocked the activation of spinal astrocytes, suggesting that JI017 mediates its effect via spinal TRPV1 and astrocytes. However, more well-designed studies need to be conducted to clearly understand the relationship between TRPV1 and astrocytes in pain. Our study of JI017 would be a cornerstone for treating oxaliplatin-induced neuropathic pain by spinal TRPV1 and astrocyte regulation.In conclusion, we demonstrated that a single oral administration of JI017 could suppress cold but not mechanical allodynia in mice. Spinal expression of both Adult C57BL/6 mice (6 weeks old) were obtained from DBL  and housed in a specific pathogen-free (SPF) animal center. The animals were randomly distributed in cages. All these 96 mice were kept in a room with a temperature of 23 ± 2 °C, humidity of 65 ± 5%, fixed 12 h light/dark cycle, and with food and water ad libitum. All experimental protocols were approved by the Kyung Hee University Animal Care and Use Committee (KHUASP(SE)-20-448) on 15 November 2020 and were conducted in accordance with the guidelines of the International Association for the Study of Pain .Oxaliplatin  was dissolved in a 5% glucose solution at a concentration of 2 mg/mL, as previously described . A dose Angelica gigas and Zingiber officinale plants were purchased from GM PHARM , and the dried root of Aconitum carmichaelii plant was purchased from Shin Hung PHARM. . The roots were boiled for 3 h in distilled 70% ethanol. The extract was filtered twice through Whatman grade 2 filter paper  to remove insoluble materials. The filtered extract was lyophilized to a powder using a freeze dryer  and stored at 4 °C [The JI017  was obtained as a lyophilized powder. The dried roots of  at 4 °C . For admCapsazepine , a TRPV1 antagonist, was dissolved in 20% DMSO by vortexing. On D4 after oxaliplatin injection, capsazepine (10 μg) was intrathecally injected, and after 2 h, the mice were sacrificed.3) for 30 min before all behavioral tests. To assess the behavioral responses to cold stimuli, an acetone drop (10 μL) was applied to the mid-plantar hind paw of the mice. Behavioral responses (flicking and licking) against the stimulus were observed and counted for 30 s. Thus, the term “number of responses” mentioned in the figures represents the average number of responses to an acetone drop in 30 s.Behavioral tests were conducted to assess the degree of allodynia in mice as described in our previous study . All behTo measure mechanical allodynia, a series of von Frey filaments with bending forces of 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1, 1.4, and 2 g  was applied to the mid-plantar hind paws. The Dixon’s up-down method and Chaplan’s calculation method were used, and a withdrawal threshold of 15 g was applied as the cut-off ,74. The n = 6) and vehicle-  treated group (n = 6) or PBS  was orally administered. PBS was used as a control to JI017 , oxaliplatin-treated mice with PBS (Oxa), and JI017-on-oxaliplatin-treated mice (Oxa + JI017). Immunohistochemistry (IHC) was conducted three times. First, to observe the change after JI017 treatment, animals were grouped into three groups: naïve mice (control), oxaliplatin-treated mice with PBS (Oxa), and JI017-on-oxaliplatin-treated mice (Oxa + JI017). Second, to analyze the effect of capsazepine, mice were divided into three groups: naïve mice (control), oxaliplatin-treated mice with 20% DMSO (Oxa), and capsazepine- on oxaliplatin-treated mice (Oxa + Capz). Third, to investigate the changes of c-Fos activity after JI017 treatment, animals were grouped into three groups: naïve mice (control), oxaliplatin-treated mice with PBS (Oxa), and JI017-on-oxaliplatin-treated mice (Oxa + JI017). .Trpv1) and GFAP (Gfap) were determined using SYBR Green qPCR Mastermix  in a CFX Connect Real-Time PCR system . Data were expressed as the ratio of targeted mRNA to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (relative mRNA expression). Because oxaliplatin-induced cold and mechanical allodynia peaked on D4 and behavioral tests were conducted on D4, the animals were perfused with PBS, and the lumbar segments of the spinal cord were collected on D4. The collected spinal cords were homogenized using easy Blue  solution, and RNA was extracted from the spinal cord according to the manufacturer’s protocol. The extracted RNA was qualified and quantified by NanoDrop  and converted to complementary DNA (cDNA) using a cDNA synthesis kit . The mRNA levels of TRPV1 . They were perfused with 0.1 M PBS, 4% paraformaldehyde (PFA)  by cardiac puncture. The spinal lumbar segments L4/5 were exposed by laminectomy. By tracing the projected dorsal roots from their respective dorsal root ganglia, the L4/5 segments were identified. The collected tissues were post-fixed in 4% PFA overnight at 4 °C and then soaked in 30% sucrose , dissolved in 0.1 M PBS, for 48 h at 4 °C. Using the optimal cutting temperature (OCT) compound , lumbar segment ice molds were prepared. These were sectioned into 20 μm thick slices using a cryostat . The slices were mounted onto glass slides  and dried for 3 h. These were then rinsed three times with PBS and then incubated for 45 min in 0.2% Triton X-100 in PBS at room temperature (RT). The blocking procedure was then performed using 3% bovine serum albumin  diluted in 0.05% phosphate buffered saline with Tween 20 (PBST) solution . The glass slides were again rinsed with PBS and incubated overnight at 4 °C with primary antibodies: rabbit anti-GFAP  and mouse anti-TRPV1 . For c-Fos staining, slides were incubated for 60 min in 5% bovine serum albumin in 0.3% Triton X-100 solution. After PBS rinsing, slides were incubated overnight at 4 °C with primary antibodies: rabbit anti-c-Fos  and mouse anti-TRPV1 . After rinsing again in PBS, secondary antibodies—anti-rabbit and anti-mouse immunoglobuling G (IgG) labeled with Alexa Fluor 488 and Alexa Fluor 594 , respectively—were embedded at RT, in the dark, for 2 h. Immunofluorescent images were acquired using confocal laser microscope  with a 10 × 0.45 numerical aperture (NA) objective lens. Quantitative analysis of GFAP- and TRPV1-positive cells in the spinal dorsal horn and co-localization of GFAP- and TRPV1-positive signals were performed using ImageJ software .The animals were deeply anesthetized with isoflurane inhalation (5% in 1:1 Np < 0.05 was considered to indicate significant differences.All data were presented as mean ± standard error of mean (SEM). Statistical analysis and graphic work were performed using Prism 7.0 . One-way and two-way analysis of variance (ANOVA) followed by Sidak’s or Tukey’s post-hoc test for multiple comparisons were used for statistical analyses. In all cases,


Brassica napus, B. rapa, and B. oleracea are utilized as valuable vegetables. Different biotic and abiotic stressors affect these crops, resulting in yield losses. Therefore, genome-wide analysis of the LysM-RLK gene family was conducted. From the genome of the examined species, 33 LysM-RLK have been found. The conserved domains of Brassica LysM-RLKs were divided into three groups: LYK, LYP, and LysMn. In the BrassicaLysM-RLK gene family, only segmental duplication has occurred. The Ka/Ks ratio for the duplicated pair of genes was less than one indicating that the genes’ function had not changed over time. The BrassicaLysM-RLKs contain 70 cis-elements, indicating that they are involved in stress response. 39 miRNA molecules were responsible for the post-transcriptional regulation of 12 Brassica LysM-RLKs. A total of 22 SSR loci were discovered in 16 Brassica LysM-RLKs. According to RNA-seq data, the highest expression in response to biotic stresses was related to BnLYP6. According to the docking simulations, several residues in the active sites of BnLYP6 are in direct contact with the docked chitin and could be useful in future studies to develop pathogen-resistant B. napus. This research reveals comprehensive information that could lead to the identification of potential genes for Brassica species genetic manipulation.The LysM receptor-like kinases (LysM-RLKs) play a crucial role in plant symbiosis and response to environmental stresses.  Because plants are stationary, they are subjected to a variety of biotic and abiotic stresses throughout their lives. Plants have been developed their immune strategies using signal transduction from the site of infection . Immune Catharanthus roseus like (CrRLK), the domain of unknown function 26 (DUF26), lectin (C-Lectin and L-Lectin), leucine-rich repeat (LRR), lysin motif (LysM), an extension like proline-rich extensin like (PERK), leaf rust kinase-like (LRK), thaumatin, self-incompatibility domain (S-domain), and unknown receptor kinase (URK) with various functions [Oryza sativa chitin elicitor-binding protein- OsCEBiP) was identified in rice which contains two extracellular LysM domains and a transmembrane LysM domain. Silencing of OsCEBiP prevented the plant from responding to chitin [OsCEBIP that are engaged in the plant defence system. The Mycosphaerella graminicola pathogen caused disease symptoms in wheat lines that were knocked down for TaCEBIP (Triticum aestivum chitin elicitor binding protein) using the virus-induced gene silencing (VIGS) approach [HvCEBIP (Hordeum vulgar chitin elicitor binding protein) via the VIGS, increased lesions owing to Magnaporthe oryzae infection were also observed [Oryza sativa chitin elicitor receptor kinase 1) is a LysM receptor-like protein kinase with three lysine motifs and a kinase domain that is required for signal transduction in rice. However, OsCEBiP is the main chitin-binding protein that uses OsCERK1 to activate the chitin-stimulated immune response [Arabidopsis thaliana chitin elicitor receptor kinase) has a dual role in biotic and abiotic stress signaling [Atlyk1 and Atlyk4 bind to chitin and transmit extracellular signals to the cell, activating downstream pathways of disease resistance [Botrytis cinerea and Alternaria brassicicola [Erysiphe necator resistance [PRRs are the main line of defense against infections. They are located on the cell membrane and belong to a receptor kinase family. They contain an intracellular kinase domain, a transmembrane domain, as well as an extracellular ligand-binding domain . The extunctions . Few RLKunctions . It was unctions  of a bacunctions ,14. LysMunctions ,16. LYKsunctions . The Lysunctions . LYKs arunctions . The firo chitin . Wheat aapproach . In the observed . Similarresponse ,23,24. Oresponse . The actignaling . In Arabsistance . AtLYM1 sistance . AtLYM2 sicicola ,31. VvLYsistance .PsLYK9 reduces levels of colonization by mycorrhizal fungi [In addition to the plant immune response, LysM-RLK genes are also involved in the plant and arbuscular mycorrhizal (AM) fungi interaction. Studies on chickpeas have shown that PsLYK9 is directly involved in the perception of long- and short-chain oligosaccharides in the hydrolyzed cell walls of the fungus and plays an important role in the immune response of chickpeas to fungal pathogens. PsLYK9, on the other hand, is involved in the symbiosis development of AM fungi, so that silencing al fungi . In tomaal fungi ,35.Brassica napus L. (2n = 4x = 38) is one of the most important allopolyploid oilseed crops derived from the hybridization of B. oleracea (2n = 2x = 18), and B. rapa (2n = 2x = 20). These species are used as important vegetables in the form of sauces, oil, and fodder, among many other items. Their crops are subjected to biotic and abiotic stressors throughout their life cycle, resulting in yield reductions. Alternaria spp., Fusarium oxysporum, Albuga candia, and Leptosphaeria maculans are the most prevalent fungal diseases that affect Brassica [Brassica crops are susceptible to bacterial rot disease as well [Brassica yield [Brassica can be developed through genetic engineering. In Arabidopsis, rice, lotus, sweet orange, potato, wheat, Chinese white pear, as well as other crops, the Lysin-Motif Receptor-Like Kinase (LysM-RLK) family has been explored [B. napus have been conducted to date. As a result, functional investigations of LysM-RLKs in Brassica have been investigated using bioinformatics techniques, given the importance of Brassica and the decrease of its products owing to biotic and abiotic stressors.Brassica . Brassic as well . Similarexplored ,42,43,44Brassica LysM-RLK proteins in the Ensembl Plants database to determine the LysM-RLK gene family in B. napus, B. oleracea, and B. rapa. Three domains including F-box-like (PF12937), protein tyrosine kinase (PF07714), and protein kinase domain (PF00069) were identified. The default parameters include significance E-values of 0.01 for sequence and 0.03 for hit matches, as well as reporting E-values of 1 for both sequences and hit. The ProtParam tool of the ExPASY bioinformatics resource portal [Brassica LysM-RLK. CELLO and DeepLoc were used to predict protein cellular localization [The HMM profile of the LysM domain (Pfam ID: PF01476) was retrieved from the Pfam database , and thelization ,49.LysM-RLK gene family, full-length protein sequence alignments of B. napus (Bn), B. oleracea (Bo), B. rapa (Br), Arabidopsis thaliana (At), Brachypodium distachyon (Bd), Oryza sativa (Os), and Vitis vinifera (Vv) were performed using ClustalX 2.0.8 software. The Neighbor-joining (NJ) method with 1000 bootstraps was used to generate a phylogenetic tree of LysM-RLK proteins using MEGA 7 [To study the evolutionary relationships of the g MEGA 7  and the g MEGA 7 .The coding sequence (CDS) of the examined genes was retrieved from the Ensemble Plants database using the biomart program  to invesLysM-RLK gene motifs [Brassica species was retrieved from the Ensemble Plants database and the appropriate analyses were done using TBtools software [The Multiple Em for Motif Elicitation (MEME 5.0.5) algorithm was used to find specific e motifs . Twenty e motifs . The GFFsoftware  to illusLysM-RLK genes from the Ensemble Plants database [BLysM-RLK-targeted miRNAs.PlantCare  was useddatabase . SSR mardatabase . CD sequBrassica LysM-RLK [CodonW 1.4.2 was used to analyze the sequences for frequency of optimal codons (FOP), codon adaptation index (CAI), GC content, effective codon number (ENC), GC content at the third site position of a codon (GC3s), and relative synonymous codon usage (RSCU) for LysM-RLK . The staBrassica napus reference genome using STAR [BnLysM-RLK genes in response to fungal infections such as Leptosphaeria maculans and Sclerotinia sclerotiorum. Expression data related to the Leptosphaeria Maculans inoculation is available in the NCBI with the project ID number PRJNA311316. In total, they sequenced 36 samples (18 from the resistant (LepR1) genotype and 18 from the susceptible genotype (Westar)). Samples were collected 0, 3, 7, and 11 days post-inoculation in triplicate. RNA-seq data with accession number PRJNA274853 publicly available on the NCBI SRA database were mined and analyzed for expression patterns of the rapeseed LysM-RLK genes in response to S. sclerotiorum infection. The experiment consisted of 24 samples containing susceptible (J902) and resistant (J964) genotypes and was sampled at 24, 48, and 96 h after treatment with three biological replications.The transcript data for flower, leaf, root, silique, and stem tissues as well as dehydration stress at 1 and 8 h after treatment and ABA (25 M), cold (4 °C), and salinity (200 mM), stresses at 4 and 24 h after treatment were related to the study of Zhang et al.  with theing STAR . The couing STAR . ClusterIterative template-based fragment assembly simulations were used to create the full-length atomic structures of BnLYP6 proteins to forecast protein structures on the I-TASSER server . The topThe chitin ligand structure was retrieved from the PubChem database  and convLysM-RLK genes were discovered . The prefix Bn, Bo, and Br, as well as the protected domain discovered in each gene, were used to label the identified LysM-RLK genes. The chromosomal location of the genes has been used to estimate the gene number. They were divided into three groups based on their specific domains including LYK , LYP , and LysMn  (pI) ranging from 4.64 to 7.78  were acidic.In the current investigation, 33 leracea) . LYKs ar to 7.78 . Based oBrassica LysM-RLK proteins and their homologous in other plants. According to Brassica were highly similar to their counterparts in Arabidopsis (At), rice (Os), and grapevine (Vv). LysM-RLK proteins were divided into four subfamilies: LYK, LYP, LysMe, and LysMn. Except for LysMn, all subfamilies have been identified in the three Brassica species investigated. Based on previous studies in Arabidopsis, AtLYP1 and AtLYP3 recognize peptidoglycan while AtCERK1, AtLYK4, and AtLYK5 recognize chitin. Therefore, due to the existence of the BnLYP2, BrLYP2, BoLYP2, BnLYP3, BrLYP3, BoLYP3, BnLYP4, BrLYP4, BoLYP4, BnLYP5, BrLYP5, BoLYP5, BnLYP6, BoLYP7, BnLYP8, BnLYP9 in the clade of AtLYP1 and AtLYP3, they can recognize peptidoglycan while BnLYK1, BrLYK1, BnLYK5, and BoLYK2 formed a monophyletic cluster with AtLYK4 confirming their ability to recognize chitin. It seems that LYP and LYK subgroups can specifically identify peptidoglycan and chitin, respectively. However, some studies reported that some members of the LYP subfamily can bind to both chitin and peptidoglycan ligands such as LYP1, LYP4, LYP5, and LYP6 [A neighbor-joining phylogenetic dendrogram was constructed to establish the link between and LYP6 ,63,75,76Brassica LysM-RLK was unequal on chromosomes  was more than one, indicating positive selection, which resulted in their various functions as a result of mutations during their evolution.Except for Brassica LysM-RLK protein sequences  BoLYK1, BnLYK2, BrLYK2, BnLYK3, and BnLYK4 with all three splicing phases. The untranslated region was only found in 10 of the Brassica LysM-RLKs including BnLYP2-3, BnLYp4, BnLYP6, BnLYP8-10, BnLYK1-5, and BnLysMn2.The MEME tool was used to find conserved motifs in equences . Accordiequences A. The hial study B. IntronBrassica . Each suBrassica LysM-RLK start codon , MYC (100%), and ARE (100%), respectively. The lowest frequency of cis-regulatory elements was related to GC-motif (only in BoLYP2) AT-rich sequence (only in BnLYK4), CARE (only in BnLYK2), GTGGC-motif (only in BnLYR1), MSA-like (only in BoLYR2), and F-box (only in BnLYP5). Brassica LysM-RLK contained 218 stress-responsive elements, indicating that they may have a role in regulating the Brassica response to different environmental challenges. 168, 161, and 75 cis-acting elements associated with phytohormones, light, and different tissues were also detected. Therefore, Brassica LysM-RLKs have the potential to play a role in a variety of processes. 22 SSRs were identified in 16 out of 33 Brassica LysM-RLKs  (BnLYP5 (2 SSRs), BnLYP2, and BnLYP9 (4 SSRs each). The highest frequency was related to tetra-nucleotide repeats (9 SSRs) followed by di-nucleotide repeats (6 SSRs), tri-nucleotide repeats (4 SSRs), and pentanucleotide repeats (3 SSRs). 39 miRNAs for 12 Brassica LysM-RLKs targets have been detected  . Most gedetected . miRNAs B. napus is considered one of the plants that produce the healthiest oils. Due to its outstanding properties, such as rapid growth, this plant is also used as a useful species for genetic and molecular studies of development and adaptation to diverse conditions. Therefore, in the current study, the expression of LysM-RLK genes has been investigated in B.napus. RNA-seq data sets for B. napus at different developmental stages tissues have been studied in leaf, flower, root, seed, stem, and silique to discover the related LysM-RLKs , BnLYP2 (low expression in stem and leaf), BnLYP6 (low expression in seed and silique), BnLYP7 and BnLYP10 , BnLYP8 (low expression in flower and seed), and BnLYP9 ((low expression in leaf) and LYP4 low expression in flower. The highest expression in this subfamily was related to seed , leaf , silique , flower , and stem (BnLYP3). In the LYK subfamily, all BnLYKs demonstrated low expression except BnLYK1 and BnLYK5 (high level of transcripts in root and moderated expression in leaf), BnLYK2 (high level of transcripts in seed), and BnLYK3 (moderated expression in flower and silique). However, BnLYK5 showed no obvious expression in the flower. Based on RNA-seq data analysis of the BnLysMn subfamily, all members demonstrated moderate to high levels of transcripts in tissues. The expression patterns of BnLysM-RLK genes have been examined to predict their role in responding to abiotic stresses as well  > 1 implies that codons are used more frequently than other synonymous, an RSCU = 1 indicates that codons are not preferred, and an RSCU of 1 indicates that codons are rarely utilized by genes [BnLYP7, 22 BnLYP9, 23 codons in BoLYP3-5, BnLYP4-5, and BnLYK4, 24 codons in BrLYP4-5 and BnLYP2, 25 codons in BoLYK1-2, BoLYP1, BrLYP2, BrLYK1, BnLYP5, and BnLYK5, 26 codons in BnLYK3, 27 codons in BrLYP3, BrLysMn, and BnLYK1, 28 codons in BoLYP2, BnLYK2, BnLYP1, BnLysMn1, and BnLYP3, 29 codons in BrLYP1 and BnLYP6, and 30 codons in BoLysMn, BrLYK2, BnLysMn2, and BnLYP1 with RSCU > 1 indicating that these are the most desired codons for each gene. The higher RSCU value (the more frequent codons for each gene) is shown in red, while the lower RSCU value is shown in blue , cluster II , cluster III , and cluster IV . Each cluster had a similar preference for codons.The CAI (codon adaptation index), which was in the range of 0.221–0.262 in by genes . There a in blue . AccordiLysM-RLK genes were found among three Brassica species. In B. napus, 17 genes were detected, while only 8 genes were identified in each of B. oleracea, and B. rapa. Study of RGAs of 30 species of Brassicaceae showed that between 5 and 14 LysM-RLK genes are present in the different assembly versions of B. napus [Brassica juncea also showed that the number of LysM-RLK genes in this plant is low and in contrast, LRR-RLK genes have a high frequency [LysM-RLK genes identified in this study may be due to differences in detection criteria and differences in B. napus assembly versions. RLKs has been identified in many plants such as Arabidopsis thaliana, Oryza sativa, Brachypodium distachyon, Citrus sinensis, Triticum aestivum, Gossypium hirsutum, Pyrus bretschneideri, Malus domestica, Solanum tuberosum and B. juncea that containing 14, 20, 11, 9, 117, 60, 18, 21, 10 and 11 RLKs genes, respectively [LysM-RLK gene family is species-specific resulted from gene duplication events [LysM-RLKs, it may be concluded that there is no meaningful association between genome size and the number of genes in plants. For instance, Triticum aestivum and Gossypium hirsutum each have 117 and 60 LysM-RLK genes, while their genome sizes are 17 Gb and 2.5 Gb, respectively. The identified Brassica LysM-RLK were categorized into LYK, LYP, and LysMe groups. The distribution of LysM-RLK was uneven Brassica genome. Most, if not all, flowering plants had one or more genome duplication events in their evolution [LysM-RLK genes in Brassica.LysM-RLKs play an important role in the plant immune system against pathogens . In the requency .The obseectively ,78,81,82n events . Based ovolution . In the LysM-RLKs were less than 1 except for three duplicated gene pairs  with Ka/Ks more than 1 and two duplicated gene pairs (BnLYP7/BoLYP3 and BnLYP8/BoLYP4) with no Ka/Ks value due to the same sequence. It should be noted that during evolution, changes in the coding region of duplicated genes resulted in various functions due to amino acid substitution or exon-intron structural divergence [LysM-RLK gene family, the importance of the functional role of Brassica LysM-RLK genes has been determined. According to the phylogenetic tree, it was shown that Brassica LysM-RLKs have a close relationship with their counterparts due to their sequence conservation and similar function. The amino acid compositions of each cluster were similar, implying that the phylogenetic distribution of Brassica LysM-RLK proteins is associated with their motif contents. All members of the LYK cluster contained 10 common motifs 1, 3-4, 7-8, and 11-15. The difference between the members of this subfamily was related to motif 5 in the clade of BoLYK1, BnLYK2, BrLYK2, BnLYK3, and BnLYK4, while the clade of BnLYK1, BrLYK1, BoLYK2, and BnLYK5 contained motif 2. These results are completely consistent with the results of the phylogenetic tree. The LysMn subfamily had common motifs 1, 4, 8, 12, and 13. In the LYP subfamily, three groups were observed. The first group (Cluster I) consisted of BrLYP2, BnLYP2, BoLYP3, BoLYP5, BrLYP, BnLYP5, BnLYP7, and BnLYP9 proteins with common motifs 1-7 and 9-12. The second group (Cluster II), including BnLYP1, BoLYP1, BrLYP1, BnLYP10 with common motifs 1-7, 9-12, and 14 except for BoLYP1 with extra specific motif 13. The third group (Cluster III) consisted of BoLYP2, BrLYP3, BnLYP3, BnLYP4, BrLYP4, BoLYP4, BnLYP6, and BnLYP8 demonstrated 11 same motifs 1-7 and 10-13. The difference between clusters I and II was related to the existence of motifs 14 in the second cluster while cluster III was separated from the above two clusters due to the lack of motif 9. The structure of exons and introns, as well as the splicing phase, play crucial roles in the evolution of gene families [Brassica evolution. 9.09% of Brassica LysM-RLK genes were intronless.The Ka/Ks ratios of the most duplicated vergence . Becausefamilies . The higfamilies ,88. The Brassica LysM-RLK genes. In response to environmental stresses, transcription factors play a significant role. They bind to the target genes’ promoters, regulating their expression [LysM-RLKs have a role in the plant’s response to a variety of biological processes. Several cis-elements associated to plant resistance against biotic and abiotic stresses were identified based on the promoter analysis, including ARE, DRE, GC-motif, LTR, MBS, MYB, MYC, STRE, AP1, S-box, W-box, WUN-motif, and WRE3. The TGACG and CGTCA motifs are found on methyl jasmonate-responsive genes [LysM-RLK genes are active in a variety of stress responses in Brassica species. However, the existence of specific regulatory elements is not sufficient evidence for these genes’ responses to specific hormones or stresses, requiring the use of laboratory procedures to precisely determine their function. SSRs are 1-6 nucleotide tandem repeats that have been shown to play a crucial function in gene regulation [LysM-RLK may be examined in different cultivars in the future, and they may be useful for marker-assisted selection (MAS) development in Brassica genetic improvement to choose genotypes with higher resistance to various stresses. MicroRNAs (miRNA) are non-coding small RNAs with a length of 19-24 bp. They are crucial in the regulation of post-transcriptional modifications. Plants, animals, and viruses all have miRNAs. Plant development and responses to environmental stressors are also influenced by them [Brassica miRNAs targeted 6, 5, and 1 transcript in the LYK , LYP (BnLYP2-3 and BrLYP2-3), and LysMn (BnLysMn2) subfamilies, respectively. No LysM-RLK-targeted miRNA was found in LysMn and LYP subfamilies. miR156 is required for the vegetative phase transition of a plant from a juvenile to an adult [BnLYK1 and BnLYK3-5 are likely to play a role in root development. miR396 with reduced activity has been demonstrated to give widespread resistance to necrotrophic and hemibiotrophic fungal infections in Arabidopsis [B.napus defense against fungal infections. miR397 has been reported that target laccase family genes through transcript cleavage in Arabidopsis and rice [BnLYK4 in this study. miR5717 regulates genes involved in lipid metabolism and pollen tube growth [BrLYP3 is likely to have a role in reproductive development. It was hypothesized that miR5721 may target genes that encode biotinyl-lipoyl-containing proteins [B. napus, miR2111 plays a significant role in the response to phosphorus deficiency [B. napus seeds [The study of promoter regions is necessary to understand the function of pression . The preve genes . Senesceve genes . In respgulation . In the gulation . SSR pol by them . Brassican adult . Under nan adult . Therefobidopsis , thus, Band rice . As a reand rice . In Araband rice ,100,101 e growth . Therefoproteins . In B. nficiency . Finallyus seeds .RLKs are thought to play a crucial role in stress responses [BnLysM-RLK genes with moderate to high expression was observed in seeds (76.92%) followed by roots (76.47%), and silique (52.94%) while the lowest number of moderate to highly expressed genes was related to stem (35.39%) preceded by leaf and flower (41.17% each). BnLYK5 was considered not expressed in flower tissue. The highest expression in root and flower tissues was related to BnLYP6 and BnLYP2, respectively, while in stem and leaves the highest expression was related to BnLYP3 and in seed and silique was related to BnLYP9. Most of the low-expression and high-expression genes were related to LYK and LYP subfamilies, respectively. The results demonstrated that the expression patterns of genes belonging to the same subfamily can differ significantly. For instance, the BnLYP3 and BnLYP8 of the LYP subfamily are consistently expressed at high levels while other LYP genes demonstrated a minimum expression except for BnLYP4-6 and BnLYP9 with moderate to high expressions. The results reinforced the hypothesis of divergence that the duplicated genes may be the result of one of two processes: 1) subfunctionalization, and 2) neofunctionalization. In the subfunctionalization process, some of the characteristics of new genes vary from the parental genes [Arabidopsis receptor dead kinase 1 (RDK1) plays an important role in drought stress response. The Arabidopsis rdk1 mutants were hypersensitive to drought stress due to the down-regulation of ABA-responsive genes [LysM-RLK genes in response to abiotic stresses varies depending on the stress type and duration. Thus, BnLYP3 and BnLYP8 genes were up-regulated by salt after 4 h of treatment, while they were down-regulated after 24 h under salinity condition. The highest transcript level under dehydration conditions after 1 and 8 h was related to BnLYP3 and BnLysMN2, respectively. Interestingly, in all treatments including salt (after 4 h), ABA (after 4 h), and cold (after 4 and 24 h) BnLYP3 showed the highest expression while the transcript levels of BnLYP9 and BnLysMn2 was higher than other LysM-RLKs in response to salinity and ABA treatments after 24 h, respectively. These findings suggested that the BnLYP3 gene may play a critical role in B. napus response to abiotic stresses, which can be utilized to improve the resistance of B. napus cultivars in future researches. We can also suggest this gene as a marker of abiotic stresses in B. napus. Pathogens and pests are believed to be capable of causing 50–60% losses in Brassica crop yield and quality, resulting in significant economic losses [B. napus, caused by S. sclerotiorum. The highest expression in response to S. sclerotiorum was related to BnLYP6, followed by BnLYP4, BnLYP3, BnLYP8, and BnLYP5 in both susceptible and resistant cultivars except in resistant cultivar after 96 h that the highest expression was related to BnLYP3, followed by BnLYP6, BnLYP8, BnlYP9, and BnLYP4. Based on the results of Brotman et al. (2012), CERK1 (LysM-RLK1) receptor is required for chitinase-induced salt and heavy metal tolerance in plants. Likewise, they suggested that ectopic chitinases are largely involved in inducing plant immune response against pathogens mediated by the CERK1 receptor [Gossypium barbadense. According to their findings, the majority of the up-regulated genes associated with disease resistance were chitin responsive, implying that the transgenic Arabidopsis showed improved resistance against Verticillium dahlia by modulating the chitin response signaling pathway [L. maculans, is a serious production limitation in B. napus. It has been observed in all canola-growing regions except China and causes yearly yield losses of 10–20% [BnLysM-RLKs is suppressed after L. maculans infection, except in BnLYP5 and BnLYP9 that their expression was slightly increased after pathogen infection. Taken collectively, all members of the gene family are expressed in B. napus.The expression profile of genes provides important information about the function of the genes that have been found. According to recent studies, esponses ,107. Theal genes , whereasal genes . Droughtal genes . As an Ave genes . Considec losses . Sclerotreceptor . June et pathway . Blacklef 10–20% . ExpressLysM-RLK gene family in other plants also shows the response of these genes to fungal and bacterial pathogens. A study of transcriptome data has shown that the expression of wheat LysM-RLK genes is induced in response to Flg22 and chitin. Therefore, these genes are involved in wheat resistance to fungal and bacterial pathogens [Citrus sinensis, the expression of LYK genes have increased in response to Xanthomonas citri, the Citrus bacterial canker (CBC) causing plant bacterial pathogen, and the salicylic acid (SA), methyl jasmonate (MeJA), and abscisic acid (ABA) hormones. Accordingly, there is a link between the LYK genes, the ABA, SA, and MeJA signaling pathways, and CBC resistance [Fusarium graminearum (Fg), the causative agent of Fusarium head blight (FHB), induces the expression of BdLYK2, BdLYK3, and BdLYK4 genes in Brachypodium distachyon. On the other hand, the expression of BdLYP1 and BdLYP4 genes has decreased in response to this pathogen. The function of these genes seems to be similar to the Arabidopsis AtLYP2 and AtLYP3 genes, which are involved in responding to bacterial pathogens [LYP genes to bacterial PGN, in rice, LYP4 and LYP6 genes are dual-functional and can respond simultaneously to fungal chitin and PGN [BnLYP6 gene expression is induced in response to fungal pathogens. On the other hand, molecular docking analysis showed that BnLYP6 has a high affinity for chitin, which indicates the role of this gene in responding to fungal pathogens in B. napus. These results together indicate the different functions of LysM-RLK genes in the response of plants to bacterial and fungal pathogens as well as abiotic stresses [The study of the athogens . In Citrsistance . Fusariuathogens . Althoug and PGN . The prestresses .Brassica LysM-RLKs ranged from 48.97 to 59.64, indicating that the codons of this family are not affected by strong codon bias and there are various synonymous codons [the BnLysM-RLKs is almost low. Although the codon preference of highly expressed genes is stronger with a higher CAI and lower NC values, low-expression genes have more rare codons, resulting in a lower CAI and a higher NC. For instance, BnLYP6 showed increased expression in response to biotic stresses with almost larger CAI and relatively lower NC. The optimal codon frequency is represented by the FOP and CBI indices, which range from 0 to 1 and -1 to 1, respectively. Based on the results of the FOP and CBI, the frequency of optimum codons in this gene family was low. The majority of Brassica LysM-RLKs showed a GC content of more than 0.5, implying that Brassica LysM-RLKs have obvious preference for GC. 69.69% of Brassica LysM-RLKs demonstrated a GC3s value greater than 0.5, indicating that G/C end codons are preferred.CUB can represent the origin of a gene and can be utilized as a theoretical model for analyzing gene evolution and function . The amos codons . The CAIs codons . AccordiLysM-RLK genes with significant structural diversity in three Brassica species. Based on the phylogenetic analysis, Brassica LysM-RLK genes were divided into three groups including LYK, LYP, and LysMn. Only segmental duplication was found during the investigation of the mechanism of gene family expansion. The function of most duplicated Brassica LysM-RLK genes has been conserved over evolution due to negative selection. During promoter analysis, several elements in the Brassica LysM-RLK promoters were found, showing that they play a role in stress response and plant growth. 22 SSR and 39 miRNA were detected which can be employed in MAS and genetic transformation, respectively. The functional involvement of LysM-RLK genes in Brassica tissues in response to environmental stressors was revealed by their expression patterns in diverse tissues. Due to the high expression of BnLYP3 genes in response to Sclerotinia stem rot infection and BnLYP3 in response to abiotic stresses, these genes can be exploited in the production of B. napus plants resistant to biotic and abiotic stresses. The discovery of these residues might be important in future investigations to improve the efficiency of the LYP6 enzymes and generate pathogen-resistant B. napus by site-directed mutagenesis. This research has given fundamental information on the LysM-RLK genes, which will be useful in future investigations aimed at improving Brassica quality.Bioinformatic analyses were performed in this work to discover 33


In Plasmodium species, Ca2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca2+ reservoirs. Several Ca2+-binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca2+-dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca2+-mediated signaling correlated to the developmental stages of the malaria parasite life cycle.The calcium ion (Ca The control of intracellular Ca2+ signals is dynamic. Overall, fluctuations in Ca2+ concentrations are modulated by an influx and/or efflux promoted by membrane channels, such as store-operated calcium channels (SOCEs), plasma membrane Ca2+-ATPase (PMCA) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps. Generally, this orchestration in the Ca2+ concentration follows a signaling pathway that obeys the order: stimuli, G-protein coupled receptor, phospholipase C (PLC) activation, mobilization of phosphatidylinositol 4,5-biphosphate P2), production of inositol 1,4,5-phosphate (IP3), IP3 recognition by IP3-sensitive receptor channels (IP3Rs) in the endoplasmic reticulum (ER) and downstream Ca2+ cascade , Ca2+ has to cross several barriers to reach the parasite, which include the red blood cell membrane (RBCM) and parasitophorous vacuole membrane (PVM) (Cane (PVM) .2+ fluctuations in Plasmodium species are very complex and demand the support of intracellular Ca2+ storage. For example, gametocytes or schizont fractions from Plasmodium chabaudi infected RBCs (iRBCs) present 10-20 times more Ca2+ than uninfected RBCs. Moreover, it has been observed that this ion concentrates in parasite storage compartments -sensitive channel ‐mediated protein export across the PV and out into the RBC cytosol by conditional knockdown approach, significantly reduced Ca2+ permeability in iRBCs  was reported in the parasitophorous vacuole (PV) required for proper parasite development (2+ sensor yellow cameleon (YC)-Nano, dynamic measurement of intracellular Ca2+ in different life stages of P. falciparum shows significant fluctuations throughout the parasite development: ring (~370 nM), trophozoite (~30 nM), schizont (~310 nM), merozoite (~950 nM), and gametocyte  stages  stages .2+ fluctuations have now begun to be identified, although it is still a subject under debate. For example, cytoplasmic Ca2+ increase may be related to potassium (K+) availability, especially when parasites are faced with an abrupt change from high to low K+ concentration. Exposition of P. falciparum merozoites to an ionic environment with a low K+ concentration  increases the levels of cytosolic Ca2+  pathway in ecretion . Nonetheecretion . Moreovesurvival .2+ levels. For example, melatonin, which appears as a critical signal controlling synchronous maturation of Plasmodium in vivo, triggers an increase in Ca2+ cytoplasmic concentration through Ca2+ release from intracellular stores by an IP3-dependent pathway activation  and dihydroartemisinin (dART) was ~2.3-fold weaker than those observed between human SERCA and dART, indicating that dART do not inhibit PfSERCA pump activity, refuting the initial conclusion , which stock Ca2+ in an acidic environment  and vacuolar-H+-ATPase (V-ATPase), can use PPi and ATP, respectively, to pump protons toward the lumen of acidocalcisomes, providing acidification of the structures, supporting Ca2+-storage maintenance in these organelles , causing an increase in cytosolic Ca2+ levels , present in the food vacuole membrane (FVM), is apparently very important to balance these processes  in the parasite  and a vacuolar-type H+-pyrophosphatase (TgVP1) are localized in these organelles  was also revealed, highlighting evidence of an acidic environment for Ca2+ maintenance  from P. falciparum in the Xenopus laevis oocystes has been shown to cause Ca2+ uptake after the alkalinization of the intracellular environment, suggesting that the out- or inward-directed Ca2+ proton movement is pH-dependent , essential to ookinete forms and parasite transmission to the mosquito in vivo, but not essential to the erythrocytic stages of P. berghei. A pbcax disrupted strain revealed a stage-specific role of this transporter for Plasmodium survival  identified 103 potential proteins with EF-hand motifs. Nonetheless, this number is undoubtedly overestimated due to the divergence of the EF-hand motif and some rifins. Without rifins, 43 proteins containing EF-hands were recorded in P. falciparum (2+-dependent protein kinases (CDPK) family  family .P. falciparum (PfCaM) is localized diffusely in the cytoplasm during mature stages of the intraerythrocytic cycle and at the apical pole end of merozoites within the ductule of rhoptries , that contain four EF-hand motifs. This group of proteins are involved in parasite cell division at centrosome-like structures, probably in a Ca2+-dependent manner  are known in mammals. P. falciparum protein kinase 2 (PfPK2) is the unique homolog of human functional CaMK that phosphorylates its substrate in a Ca2+- and CaM-dependent manner, and it is expressed during invasion , which are not present in humans. Canonical CDPKs have four EF-hand Ca2+-binding domains attached to the C-terminus of a catalytic kinase domain that shows high homology with CaMK. While CaMKs can self-inhibit through a C-terminal helix, CDPKs are regulated by their Ca2+-binding domains. In these cases, CDPKs undergo structural and conformational changes, promoting the regulation of other proteins by phosphorylation , with correspondent orthologs in plants, but not in animals or fungi . The PfCDPK1 inhibitor, purfalcamine, blocked SERA5 phosphorylation, leading to the blockage of merozoite egress  showed prominently reduced activity compared to wild-type parasites. This lower activity seems to be compensated by PKG, influencing the up-regulation of transcription levels of CDPK5 and CDPK6 in the CDPK1 T145M mutant parasites , suggestarasites . Some rearasites . PfCDPK1 by cAMP . Additioe egress . Despitee egress . Neverthe egress .Plasmodium. In all rodent and some other malaria species, the cdpk2 gene is lacking , such as hepatocytes, involving the induction of the circumsporozoite protein (CSP) cleavage upon contact with hepatocytes . As prev PfCDPK1 . Still, rphology . DespiteP. falciparum genome by in silico analysis , also known as STPP 2B or PP3, is a heterodimeric complex containing a catalytic subunit (CNA) and a regulatory subunit (CNB) . CN is cs target .Plasmodium spp. is required for host cell attachment and invasion in a receptor-dependent pathway distinct from the AMA1-RON2  system but with some degree of functional overlap  contains EF-hands and IQ (the first two amino acids of the motif: Ile and Gln) calmodulin-binding motifs but, in contrast, is monomeric. The CaM-binding motif was found to inhibit phosphatase activity in psis PP7 . PP7 is psis PP7 . These opsis PP7 .2+ signaling and phosphatases are focused on calcineurin. However, other phosphatases without Ca2+-binding motifs could be affected by Ca2+ signaling through their protein partners possessing these motifs. According to STRING prediction and Gene Ontology analyses, amongst the 67 candidate phosphatases identified in P. falciparum, ten potentially interact with proteins involved in Ca2+ signaling: putative acid phosphatase (PF3D7_0918000), putative protein phosphatase 2C (PF3D7_0829100), putative 4-nitrophenylphosphatase (PF3D7_0715000), putative protein phosphatase (PF3D7_0802800), putative RNA lariat debranching enzyme (PF3D7_1340600), putative acid phosphatase (PF3D7_1403900), hypothetical protein (PF3D7_1464600), hypothetical protein (PF3D7_1469200), protein phosphatase 2C (PF3D7_0410300) and putative phosphoesterase (PF3D7_1206000)  . Howeveralarials .P. falciparum reticulocyte binding-like protein 1 (PfRh1) performs a role in initial sensing of Ca2+ followed by signal transduction, causing erythrocyte binding antigen-175 (EBA-175) release from microneme and allowing tight junction formation  has been proposed as a protein to potentially connect this motor complex to the IMC membrane. It is also involved in gliding and invasion processes in a Ca2+-dependent manner. PfIMC1l interacts directly with Ca2+ and its interaction with actin is enhanced in the presence of this ion  and the CDPK4 substrate SOC6 (PBANKA_070770), involved in IMC biogenesis  and P. falciparum reticulocyte binding-like protein 2b (PfRh2b) (P. berghei rhoptry neck protein 11 (PbRON11) in sporozoites reduced attachment and motility, leading to the impairment of the infection in the mosquito salivary gland and hepatocyte cells. This protein contains putative EF-hand domains and might act by controlling rhoptry protein secretion in a Ca2+-dependent manner  results in a cytoplasmic lowering of Ca(PfRh2b) . Rhoptry(PfRh2b) . The rept manner .2+ with parasite egress was previously reported . Conditional knockdown of ICM1 revealed an essential role in Ca2+ mobilization to initiate both Plasmodium gametogenesis and merozoite egress  and PKG activation, essential for the protein discharge of secretory organelles, known to support this process . Correlareported . Events  by Ca2+ . More ree egress . Additiol stores .P. falciparum phosphodiesterase β (PfPDEβ) leads to a dramatic reduction in schizont cAMP and cGMP hydrolytic activity, resulting in elevated cAMP levels and inappropriate cAMP-induced increased phosphorylation of PKA substrates. In addition, PKA seem to assume a compensatory role with PKG, in order to phosphorylate P. falciparum myosin A (PfMyoA), an important component of the so-called glideosome, a parasite complex involved in host cell invasion, in PfPDEβ mutants, bypassing the need for PKG activity by elevated cAMP levels upon Ca2+ signaling, possibly by PKA action   and an ionophore, ionomycin, did not change the amounts of cytosolic Ca2+ in knockdown parasites bearing a glucosamine-inducible ribozyme gene (PfERC-glmS) from ER or neutral Ca2+ storages, suggesting that the availability of Ca2+ from different sources does not change upon knockdown of PfERC. Moreover, PfERC is required for the complete maturation of the aspartic protease plasmepsin X (PMX) in a Ca2+-dependent manner, which is required to cleave the subtilisin-like protease (SUB1) , has been remarkably underexplored, considering that proteins from this family are widely found from protozoans to mammals . A membeprotein) . This pre (SUB1) .2+ importance for SUB1 discharge and proteolytic cascade events have been reported. Chelation of intracellular Ca2+ in P. falciparum schizonts blocks the SUB1 discharge from merozoite exonemes into PV, resulting in a decrease of SERA5 proteolytic cleavage and harming PVM rupture and merozoite egress  in a temperature-dependent process  in hepatocytes and intracellular Ca2+ elevation, essential to EEF development (Ca process . It has e stages . Sporozoelopment . The intPlasmodium species contain distinctive features when compared to other eukaryotes. Such characteristics define its phylum or genus, in which attachment to the host cell, motility, invasion and egress are essential for survival and dissemination. Since Ca2+ signaling regulates important and specific Plasmodium cellular processes such as microneme secretion, attachment, gliding motility, invasion and egress, actors involved in these pathways, which are regulated by this ion, could be considered potential drug targets. Striking progress to achieve a broader understanding of Ca2+ signaling in Plasmodium has been made, including the potential involvement of host compounds in Ca2+ uptake, such as K+, Na+, ionic strength and melatonin (elatonin .2+ uptaking by iRBCs, together with transport through PVM and the parasite cellular membrane. Moreover, IP3R or alternative functional protein identification in Plasmodium would be an important breakthrough to explore Ca2+ mobilization and storage, as well as backstage actors which support those processes as promising therapeutic targets. Actually, a plethora of Plasmodium proteins with standard and unusual Ca2+ binding motifs, which are known or suspected to be involved in Ca2+ signaling, could be explored to this end. This also includes proteins without Ca2+-binding motifs acting as indirect effectors.However, several gaps in understanding remain in these organisms, covering mechanisms involved in increased Cain vivo protein-partners, such as BioID and APEX-2 proximity-labelling techniques (2+ ratiometric techniques coupled to imaging reporters (2+ in the backstage of malaria parasite biology and drug screening assay design.The association of classical techniques employed to study permeability, protein channels and pumps, together with more recent high-throughput approaches is a promise to fulfill these gaps. Mass spectrometry-based proteomics , includichniques , metabolchniques  and new eporters  are examAll 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.


Human pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of diseases associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate time points of retinal pigment epithelium (RPE) cultures that reflect native counterparts, we characterised the transcriptomic profiles of the hPSC-derived RPE cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single-cell RNA-sequencing of a total of 16,576 cells to assess the molecular changes of the RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial–mesenchymal transition, and with the maturing populations of the RPE observed with time in culture. Assessment of Gene Ontology pathways revealed that as the cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirms a maturing transcriptional profile of RPE cells in culture. These results suggest that long-term in vitro culture of RPE cells allows the modelling of specific phenotypes observed in native mature tissues. Our work highlights the transcriptional landscape of hPSC-derived RPE cells as they age in culture, which provides a reference for native and patient samples to be benchmarked against. The retinal pigment epithelium (RPE) is a monolayer of post-mitotic, pigmented polarised cells that is key to the health and function of photoreceptors and underlying vasculature. In particular, the RPE protects the retina against photo-oxidation and phagocytoses photoreceptor outer segments. The RPE is also essential to the immune privilege of the eye, as it physically contributes to the blood–retina barrier and also expresses molecules repressing the migration of immune cells into the retina in vitro and give rise to any cell types in the body, including cells that form the retina. Various protocols have been described to differentiate hPSCs into RPE cells in vitro. Here, we used single-cell RNA-seq (scRNA-seq) of hPSC-derived RPE cells maintained in culture for 1 month or 12 months to assess the impact of time on the RPE transcriptome and whether genetic hallmarks of maturation can be observed over time. A short-time differentiation (1 month) was chosen as it represents a time point routinely used in in vitro assays of RPE Human pluripotent stem cells (hPSCs) have the ability to propagate indefinitely The human embryonic stem cell (hESC) line H9 was differentiated to RPE cells following the protocol described in the Materials and methods section. To generate a transcriptional map of the RPE cells reflecting time in culture, RPE cells from the same culture and original passaging were isolated after 1 month or 12 months of differentiation, dissociated to single cells, and processed to generate libraries for scRNA-seq analysis A. The cain vitro, by analysing differential expression. A range of RPE markers were observed as conserved between both time points , melanin biosynthesis , lipid biosynthesis (PTGDS and INPP5K), visual cycle , and secretion (SERPINF1) were expressed at both time points (We compared variations in the transcriptomic profiles between the 1-month-old and 12-month-old samples B and C te points D. In pare points D.BMP7 and SOX4) and canonical RPE genes INPP5K and PTGDS), visual cycle , melanin biosynthesis ; secretion , phagocytic activity (GULP1), and extracellular structure organisation . Most canonical RPE genes were expressed across most populations, although many were found at lower levels in the 1-month-old cells, confirming the purity of the RPE cell cultures over time  in each sample . After iver time B. As theMITF, PMEL, TTR, TYR, TYRP1, and DCT), extracellular structure organisation , secretion (SERPINF1 and VEGFA), visual cycle , tight junctions (TJP1), phagocytic activity (GULP1), and lipid biosynthesis  were conserved in all or some of the subpopulations  and 11-cis-retinol (RPE65) for phototransduction is expressed at low levels in the 1-month-old RPE samples but becomes more comprehensive as cells age in culture  were clustered into six Common subpopulations that intersect the 1-month-old and 12-month-old cell cultures B and C, ulations . Expresselopment B. More hd stable D and 2B. culture C and D.  culture A–C. Gene samples B. SimilaCRISPLD1 C, which P < 0.74) , as well as DCT, PMEL, MITF, TYR, and TYRP1 (genes involved in pigment/melanin biosynthesis). This subpopulation also expressed genes involved in early retinal development including of the RPE and eye morphogenesis  and in the retinoid cycle (RPE65 and RLBP1). In addition, 79 ribosomal genes (32 RPS and 47 RPL) and 11 mitochondrially-encoded genes were identified. Expression of ribosomal genes and mitochondrially-encoded genes has been correlated with development and maturation The common subpopulation 1  was characterised by 891 conserved markers identified ( < 0.74) . The mosCST3 is abundantly produced by RPE cells DCT is expressed in the developing retina The subpopulation Common 2 (2204 cells) identified 1077 conserved markers. The 15 most conserved markers in this subpopulation were all known RPE markers. Many of the expressed markers are involved in the generation of RPE cells or in maturation and homeostasis of these cells. For instance, cystatin C encoded by DCT, TTR, CST3, AQP1, FTH1BEST1), with some markers, such as TFPI2, known to promote survival and maintenance of RPE cells e.g., GNGT1 that encodes a protein found in rod outer segments, suggesting that cells are not yet fully committed. Together, these markers indicate cellular functions suggestive of functional and maturing RPE cells.The subpopulation Common 3 (1811 cells) identified 1226 conserved markers. The most conserved markers in this subpopulation were mostly known to be expressed in the RPE  identified 1421 conserved markers. Many of the most conserved markers of this subpopulation are not specifically linked to the RPE. For instance, SERPINE2SFRP5AQP1, CST3, PTGDS, SERPINF1, BEST1, and SMOC2) was downregulated when compared to that in other populations. Expression patterns of other genes indicate an immaturity/ differentiation or proliferation of cells, such as GAP43, DAAM1, CD44DUSP4. Together, these data describe a subpopulation comprised of cells in early differentiation to RPE.The subpopulation Common 5  was characterised by expression of conserved RPE markers  or found at low levels in the RPE  was downregulated. These data thus suggest a subpopulation of immature cells.The subpopulation Common 6  was characterised by markers associated with retinal cell types other than RPE Finally, the analysis of the PANTHER GO slim biological processes conserved within the Common subpopulations identified pathways that are predominantly involved in mitochondrial, metabolic, and ribosomal processes, as well as purine biosynthesis, nucleotide metabolism, protein biogenesis, localisation, and transport . AltogetPTGDS), visual cycle , melanin biosynthesis , phagocytic activity (GULP1), secretion , and extracellular structure organisation .Around one third of all cells (5625 cells) were clustered into six Young subpopulations . Common SERPINF1, CST3, DCT, TSC22D4, IGFBP5, and RNASE1. Expression of most of these genes has been reported in the retina  IGFBP5The subpopulation Young 0  comprises singleton cells that expressed CTNNB1, NOG, ATP1B1, GSTP1, CD63, and HNRNPH1. All these genes play fundamental roles in the homeostasis and functions of the RPE. ATP1B1 encodes an apical Na+/K+ ATPase, whose expression reduces with age and in age-related macular degeneration (AMD) CTNNB1 are linked to abnormalities in RPE development and pigmentation GSTP1 is a survival factor for RPE cells, whose expression increases as cells mature CD63 is a late endosome/exosome marker known to be released by RPE cells HNRNPH1 levels are associated with improved survival of RPE cells in culture SERPINF1, RLBP1, TTR, PMEL, and CRYAB, were expressed at lower levels in this subpopulation than in all other subpopulations. GO biological process analysis highlights that this population is highly metabolically active  was characterised by the expression of y active . These dCCL2, SFRP1, and B2M, downregulation of CTSV and TMSB4X, as well as expression of DCT, which is known to be expressed during RPE development The subpopulation Young 2  was characterised by the specific upregulation of genes including TOP2A, PCLAF, PTTG1, ANLN, MKI67, RRM2, TPX2, and PBK. Although all genes are found to be expressed in the retina TOP2A, PCLAF, PTTG1, MKI67, RRM2, TPX2, and PBK) and cellular rearrangements (ANLN), which have been described as characteristics of immature RPE cells ANLN is reported to promote maturity of intercellular adhesions (tight junctions and adherens junctions) in epithelial cells PCLAF, PTTG1, and MKI67, suggests a proliferating cell population. Low expression of RPE markers  further demonstrates the RPE identity of the developing retinal cell subpopulation. Altogether, this expression profile specifies an immature differentiating cell population.The subpopulation Young 3  was characterised by the expression of genes such as CRYAB (downregulated), CRX, FTH1, TFPI2  suggests a differentiation to RPE, yet the presence of the photoreceptor-specific GNGT1 expression could also indicate an early differentiation step where cells are not yet fully committed.The subpopulation Young 4  was characterised by expression of genes that are not traditionally associated with the RPE identity. The expression of BEST1, VEGFA, ENPP2, TIMP3, and TYRP1, as well as genes involved in early retinal development including of the RPE and eye morphogenesis  Interestingly, the subpopulation Young 5  displayed a signature comprising mitochondrial and ribosomal transcripts with 9 mitochondrial genes (MT-) and 14 ribosomal genes (RPS- or RPL-). These genes are ubiquitous and have not been specifically correlated to the retina or the RPE, however they are known to facilitate fundamental biological processes, including electron transfer, energy provision, ribosome biogenesis, and protein synthesis. PANTHER GO-slim biological process and GO biological process analyses confirmed that the conserved pathways within this subpopulation are mostly related to the mitochondrial energetic metabolism and nucleotide metabolism . This suTaken together, our results demonstrate that all Young subpopulations are immature cells developing to RPE cells.INPP5K), visual cycle (RLBP1), melanin biosynthesis (TTR and DCT), secretion (SERPINF1 and VEGFA), and extracellular structure organisation (CST3 and CRISPLD1) were conserved in some of the “Aged” subpopulations  are clustered into the “Aged” category, which comprises six subpopulations . All sixulations .CRISPLD1, PCCA, WFDC1, TTR, SH3BGRL3, and TMSB4X, but at lower average levels per cell than those in all other subpopulations. Some of these genes have been found to be expressed in the RPE  and encode for proteins involved in more universal cellular events. For instance, mitochondrial protein PCCA plays roles in death/survival, SH3BGRL3 is involved in oxidoreduction, whilst TMSB4X encodes proteins of the cytoskeleton. Likewise, the subpopulation Aged 1  presented more cells expressing HSD17B2, TPM1, MYL9, NDUFA4L2, BNIP3, CALD1, TTR, DCT, MT-CYB, FTH1, CRYAB, and TMSB4X but at lower average levels per cell than in all other subpopulations. Nine out of the twelve genes are known to be expressed in the RPE . Some of these markers are associated with late RPE development  with PCCA, WFDC1, SERPINE2, TMSB4X, and TTR being expressed in more cells with lower average levels than all other subpopulations. In addition, SPON2, which encodes ECM proteins important for cell adhesion, was expressed in more cells but at higher levels. The close similarities of these three Aged subpopulations point to a late RPE phenotype, with higher levels of maturation, towards regionalisation of cells.The subpopulation Aged 0  is composed of singleton cells. These cells expressed ment TTR, whilst DCT , CALD1 , TTR , SOX9 , RBP1 , SERPINF1 , VEGFA , TMSB4X , and CRYAB (downregulated in Aged 2 and Aged 3) confirms their RPE identity.Similarly, the subpopulations Aged 2 , Aged 3 , and Aged 4  showed close similarities in terms of gene expression profiles. The presence of known RPE markers, such as MT1E, MT1F, MT1G, DCT, MT2A), as well as in oxidoreduction (DCT), were significantly differentially expressed between the “Aged” subpopulations compared to all others, with an overall increased expression per cell as cells age  and PANTHER GO biological process analyses  were performed. Subpopulations Aged 2 and 4 were similar, with high significance in pathways associated with response to metal ions ; response to stress, chemicals, and toxins; and neural crest fate commitment. Among them, neural crest fate commitment is the most significantly identified biological process in Aged 3, with a 92.9-fold enrichment. Interestingly, GO analysis indicates that nuclear genes encoding metallothioneins involved in metal binding  was examined to assess whether some progenitor populations remain with proliferative potentials, and whether these vary over time in culture. Our data indicate that only a small portion of cells remained proliferative over time in culture. In particular, MKI67 was almost uniquely expressed in the immature subpopulation Young 3, together with other cell proliferation markers  and RPE genes  were performed in all subpopulations to measure gene expression trajectories . CCL2 was more uniformly expressed in the 12-month-old sample, suggesting more homogeneity in its expression as cells mature.RPE cells can modulate immune responses and differentiation of other retinal cell types, in part via paracrine signalling e points A. CCL2 w Young 2  and was  Young 2 A. UnsurpSFRP 1, 2, and 5; FZD 1 and 8; as well as WNT 2B, 3, 4, and 5A, with more expression homogeneity in the 12-month-old samples for many of them , reflective of a dynamic expression pattern of SFRP genes by RPE cells in culture  are secreted ligands for receptors Frizzled (FZD1–10), which are also receptors for WNT proteins (WNTs). Therefore, SFRPs are considered WNT antagonists and can modulate WNT signalling. Cells in culture from both time points expressed genes involved in the WNT signalling pathways, in particular  of them B–J. SFRP Young 2 . SFRP2 wulations . It is i culture . Interestectable E–J. The PE cells B–J. ThisSERPINF1, which is highly expressed by RPE cells and was found as a marker of subpopulations within the Young, Common, and Aged subpopulations  was detected in the samples, again indicating that the RPE-secreted VEGF likely acts on neighbouring cells to maintain homeostasis (Pigment epithelium-derived factor (PEDF) is another factor key to various retinal cell differentiation, maturation, and survival, including RPE and photoreceptors ulations . Howevereostasis . These eAPOE was highly expressed in cells across both time points in culture  are proteins involved in lipid metabolism including cholesterol and are also regulated with complement activation in the RPE  culture L. APOE wulations . The lev3, and 6 L. Intere culture . The opp culture . Express culture M–P. Inte culture M–P. ThesC1s, C1r, and C1QBP, the genes encoding complement regulators that form the C1 complex, were conserved markers in a few Young and Common subpopulations  were not identified as markers of any subpopulation  were characterised in any examined subpopulation , whilst other genes, including TYRP1, were found at similar expression levels in 1-month-old and 12-month-old cultures, as well as in Young, Common, and Aged populations . Media was changed every second day, with the first sample of cells harvested after 30 days (D30) and the second sample harvested on day 367 (D367) for scRNA-seq analysis (The hESC line H9 (Wicell) was maintained on vitronectin-coated plates using StemFlex , with medium changed every second day analysis A.RPE cells were dissociated to single cells using 0.25% trypsin-EDTA for 8 min and inactivated with RPEM. Cells were centrifuged at 300 g for 1 min to pellet and resuspended in a small volume of RPEM containing 0.1% v/v propidium iodide  to exclude non-viable cells. Single cell suspensions were passed through a 35-µm filter prior to sorting. A minimum of 60,000 live cells (PI-negative) were sorted on a BD FACSAria IIu  into culture medium. Cells were centrifuged at 300 g for 5 min and resuspended in PBS containing 0.04% BSA to a concentration of ~800–1000 cells/µl. Approximately 17,400 cells were loaded onto a 10X chip for a target recovery of 10,000 cells. Cultures at the two time points were captured separately to prepare two separate 10X reactions.To generate single-cell gel beads in emulsion, single cell suspensions were loaded onto 10X Genomics Single Cell 3′ Chips together with the reverse transcription master mix following the manufacturer's protocol for the Chromium Single Cell 3′ v2 Library . For each sample, sequencing libraries were generated with unique sample indices (SI), assessed by gel electrophoresis  and quantified with qPCR . Following pooling and normalisation to 4 nM, libraries were denatured and diluted to 1.6 pM for loading onto the sequencer. Libraries were sequenced on an Illumina NextSeq 500  using NextSeq 500/550 High Output Kit v2.5 (150 Cycles)  as follows: 26 bp (Read 1), 8 bp (i7 Index), and 98 bp (Read 2).cellranger mkfastq and cellranger count pipelines from the Cell Ranger Single Cell Software Suite (version 3.0.2) by 10x Genomics (http://10xgenomics.com) for initial quality control, sample demultiplexing, mapping, and quantification of raw sequencing data. The cellranger count pipeline was run with the following argument: “--expect-cells = 10,000”, and reads were mapped to the Homo sapiens reference genome . Filtered count matrices were then used for downstream analyses in R.We used the SCTransform function from Seurat Using Seurat v3.1.3 Data dimensionality was reduced with principal component analysis (PCA). Subsequently, the 30 most statistically significant PCs were reduced to two dimensions using the Uniform Manifold Approximation and Projection (UMAP) and used to construct a shared nearest neighbour (SNN) graph for each cell. The Louvain method for community detection was then used to identify clusters in each dataset with resolutions ranging from 0 to 1.5. The results for all resolutions were plotted using clustree Homo sapiens genome (PANTHER version 14.1 released 2019-03-12). For some clusters, insufficient gene markers are available for GO analysis . Canonical RPE markers, gene expression profiles, and their associated GO terms specific to each cluster are provided in Network analysis was performed on differentially expressed genes (DEGs) using Reactome functional interaction analysis Monocle 3 v0.2.4 graph_test” function using the following arguments: neighbor_graph = “principal_graph”, reduction_method = “UMAP”, and expression_family = “quasiposson”. DEGs with FDR ≤ 0.05 were clustered into co-expression modules using the “find_gene_modules” function, and the resulting protein interactions were characterised with STRING.Trajectory analysis was performed with The experimental work was approved by the Human Research Ethics Committee of the University of Melbourne (1545484), with the requirements of the National Health and Medical Research Council (NHMRC) of Australia in accordance with the Declaration of Helsinki.https://github.com/powellgenomicslab/RPE_scRNA_AgedStudy. This repository consists of code used to process raw sequencing data in FASTQ format to cell-gene expression tables via the Cell Ranger pipeline, and code used to perform the following analyses: quality control, normalisation, dimensionality reduction, clustering, differential expression, and integration.Code and usage notes are available at: Sequencing data are available at ArrayExpress (ArrayExpress: E-MTAB-8511). Files are raw FASTQ files, and a tab separated matrix of UMIs per gene for each cell passing quality control filtering. BAM files can be generated using the supplied repository to process the FASTQ files via Cell Ranger.Grace E. Lidgerwood: Conceptualization, Data curation, Formal analysis, Visualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing - original draft, Writing - review & editing. Anne Senabouth: Conceptualization, Data curation, Formal analysis, Visualization, Methodology, Investigation, Software, Writing - original draft, Writing - review & editing. Casey J.A. Smith-Anttila: Conceptualization, Methodology, Investigation, Writing - review & editing. Vikkitharan Gnanasambandapillai: Conceptualization, Methodology, Investigation, Writing - review & editing. Dominik C. Kaczorowski: Conceptualization, Methodology, Investigation, Writing - review & editing. Daniela Amann-Zalcenstein: Conceptualization, Methodology, Investigation, Writing - review & editing. Erica L. Fletcher: Conceptualization, Methodology, Funding acquisition, Writing - review & editing. Shalin H. Naik: Conceptualization, Methodology, Funding acquisition, Writing - review & editing. Alex W. Hewitt: Conceptualization, Funding acquisition, Methodology, Resources, Writing - review & editing, Supervision. Joseph E. Powell: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Software, Writing - original draft, Writing - review & editing, Supervision. Alice Pébay: Conceptualization, Formal analysis, Visualization, Funding acquisition, Methodology, Project administration, Resources, Writing - original draft, Writing - review & editing, Supervision. All authors read and approved the final manuscript.The authors have declared no competing interests.


CSF1R encodes the colony-stimulating factor 1 receptor which regulates the proliferation, differentiation, and biological activity of monocyte/macrophage lineages. Pathogenic variants in CSF1R could lead to autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia or autosomal recessive skeletal dysplasia. In this study, we identified three heterozygous deleterious rare variants in CSF1R from a congenital vertebral malformation (CVM) cohort. All of the three variants are located within the carboxy-terminal region of CSF1R protein and could lead to an increased stability of the protein. Therefore, we established a zebrafish model overexpressing CSF1R. The zebrafish model exhibits CVM phenotypes such as hemivertebral and vertebral fusion. Furthermore, overexpression of the mutated CSF1R mRNA depleted of the carboxy-terminus led to a higher proportion of zebrafish with vertebral malformations than wild-type CSF1R mRNA did (p = 0.03452), implicating a gain-of-function effect of the C-terminal variant. In conclusion, variants affecting the C-terminal of CSF1R could cause CVM though a potential gain-of-function mechanism. CSF1R is also expressed in Paneth cells, renal proximal tubule epithelial cells, and placental trophoblasts, indicating a pleiotropic role of CSF1R protein in embryonic development   regulates the proliferation, differentiation and biological activity of monocyte/macrophage (Mϕ) lineages , which is characterized by progressive neurologic deterioration and sclerotic bone dysplasia , which has been partially attributed to genetic defects previously . Detailed phenotypic data was recorded. X-ray, computed tomography (CT), and magnetic resonance imaging (MRI) were also taken. Exome sequencing and bioinformatic analysis were conducted as described previously . Rare variants in CSF1R were extracted and filtered with the following criteria: (1) truncating  and minor allele frequency (MAF) ≤ 0.001 or (2) missense variants absent from public databases in the general population.Five hundred and eighty-three patients diagnosed with CVM were consecutively enrolled and collected in the cohort between 2009 and 2018 at Peking Union Medical College Hospital, as a part of the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) study . Sanger sequencing was then performed on an ABI3730XL instrument.Candidate variants of CSF1R and mutated CSF1R  plasmids. All plasmids were verified by DNA sequencing.The pEGFP-C1-based vector was used for the construction of wild-type Cos-7 cells were maintained in Dulbecco's Modified Eagle's medium  supplemented with fetal bovine serum (Gibco), penicillin (50 U/ml), and streptomycin (50 μg/ml) in six-well plates. The full-length wild-type or mutant CSF1R constructs (plasmid pEGFP-C1 1 μg) was transfected into the cells, respectively. After 48 h, western blotting was used to assess the protein expression level in the cell lysates with the following antibodies: rabbit anti-human CSF1R monoclonal antibody  and mouse anti-GAPDH monoclonal antibody .Tg (Ola.Sp7:nlsGFP) transgenic zebrafish, where GFP expression was driven by an Sp7 promoter in osteoblasts, was utilized for animal model establishment. The zebrafish were kept at 28°C and fed with brine shrimp twice a day. From the 2–4-cell stage to 3 days post fertilization (dpf), zebrafish were exposed to methylene blue which inhibited fungal contamination.CSF1R DNA sequence was cloned into the PCS2+ plasmid, constructing a human wild-type CSF1R plasmid. Then, an indel variant (c.2749_2758delGACAGGAGAG) was constructed to generate a human-mutated CSF1R plasmid containing a mutated gene sequence. After the linearization of plasmids, transcription was performed with mMESSAGE mMACHINE™ SP6 Ultra Transcription Kit (Ambion) to obtain corresponding mRNA.A human wild-type CSF1R mRNA (50 pg for each), i.e., the wild-type mRNA or the mutated mRNA, was dissolved into distilled water (2 nl for each) and injected into embryos at the 1–2-cell stage, respectively. An equal amount of water was injected into the embryos, which were fertilized as the control group. All the individuals in the experimental groups and control group were maintained for phenotype evaluation.Human Fluorescence images were collected at 14 dpf by fluorescent microscopy. The vertebral morphology was observed and recorded for phenotypic evaluation and statistical analysis.IL34, CSF1, SOS, GRB2, INPP5D, INPPL1, PIK3R1, PIK3R2, SOCS1, SOCS3, CBL, FYN, GRAP2, LYN, RASA1, SHC11, THOC5, TES1, CBL, PLCG2, SLA2) were selected for analysis according to public databases including Pubmed (https://pubmed.ncbi.nlm.nih.gov/) and Online Mendelian inheritance in Man . Further, variant filtration of those genes was performed according to the same interpretation method as we did for CSF1R.Twenty genes which are biologically linked to CSF1R , which was then confirmed by Sanger sequencing (CSF1R. The mRNA harboring this mutation is predicted to escape from non-sense-mediated mRNA decay (NMD) by the NMD Esc Predictor . Besides, a structure disorder of T3–T7 was also observed in CT scan and three-dimensional reconstruction . A misseCSF1R, which has been reported in the East Asian population with low frequency (0.0005)  . This deCSF1R variants identified in our cohort were located within or near the carboxy-terminal region downstream of the PTK domain  which is depleted of all the carboxy-terminal regions downstream of the PTK domain in the zebrafish model. This mutated mRNA-deleted carboxy-terminal region is referred to as CSF1RCΔ mRNA in the following text.Therefore, we proposed that the heterozygous deleterious CSF1R and CSF1RCΔ mRNAs were injected into Tg (Ola.Sp7:nlsGFP) zebrafish embryos, respectively. Fluorescence images of the spine were collected at 21 dpf by fluorescent microscopy. The zebrafish of the CSF1R mRNA overexpression group exhibited vertebral malformations including vertebral fusion, hemivertebra, and fused vertebral arches   . These rIL34, CSF1, SOS, GRB2, INPP5D, INPPL1, PIK3R1, PIK3R2, SOCS1, SOCS3, CBL, FYN, GRAP2, LYN, RASA1, SHC11, THOC5, TES1, CBL, PLCG2, SLA2) and performed a genetic spectrum analysis. Twelve potential deleterious rare variants were identified in different patients, including four truncating variants and eight missense variants , which offer binding sites for c-Cbl and participate in the degradation of CSF1R  zebrafish also exhibited neurological abnormalities primarily, along with skeletal phenotypes like osteopetrosis  identified in our cohort was related to higher protein expression level and led to more severe clinical phenotypes as compared with that of the other two deleterious variants. This variant (NM_005211.3: c.2749_2758delGACAGGAGAG) deletes the entire carboxy-terminus region sequence (p.Asp917SerfsTer32), which is important for the binding of c-Cbl and thus the ubiquitination-mediated protein degradation. However, the other two CSF1R variants only lead to minor structural disturbance of the protein carboxy terminus and consequently confer milder effects on the protein expression and human orthopedic manifestations. This phenomenon indicates a genotype–phenotype correlation in CSF1R-related CVM.Furthermore, we focused on the detailed structural and biological effects of the variants and hypothesized how these variants affected the biological function of CSF1R. One of the CSF1R-related CVM, we also focused on this protein and identified two deleterious rare CBL variants in our cohort. It is demonstrated that c-Cbl binds to activated CSF1R and mediates its degradation through the tyrosine-kinase binding (TKB) domain  leads to amino acid change (p.Gly933Cys), for which glycine was replaced by cystine. Glycine is a relatively stable amino acid with low molecule weight. The amino acid is replaced by cystine, which is much more active and easily oxidizable. The oxidized cystine tends to form a disulfide bond with each other, potentially promoting the formation and stabilization of the CSF1R homodimer. The biological activity of protein could be enhanced by such structure disorder, which contributes to vertebral malformation. All the hypothesized mechanisms mentioned before remains to be explored further.CSF1R in CVM. The variants affect the biological function of the CSF1R product through potential gain-of-function effects.In conclusion, our study reveals the pathogenicity of heterozygous carboxy-terminal variants of https://data.mendeley.com/datasets/wzjxyz99st/1.The datasets generated during the current study are available in the Mendeley repository, The studies involving human participants were reviewed and approved by ethics committee at Peking Union Medical College Hospital (JS-098). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. The animal study was reviewed and approved by ethics committee at Peking Union Medical College Hospital (JS-098). Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.in vivo experiments. BL, LZ, and XL performed the in vitro experiments. BL, SZ, and NW wrote the first draft of the manuscript and critically revised the work for important intellectual content. All authors contributed to the article and approved the submitted version.NW, ZW, TZ, BL, SZ, and GQ conceived of the project and designed the study. NW, BL, SZ, ZY, SW, and YN recruited the patients and collected and interpreted the data. BL and SZ conducted the statistical analysis and bioinformatic analyses. BL and JL performed the 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.