Chrysanthemum morifolium is one of the most economically important and popular floricultural crops in the family Asteraceae. Chrysanthemum flowers vary considerably in terms of colors and shapes. However, the molecular mechanism controlling the development of chrysanthemum floral colors and shapes remains an enigma. We analyzed a cut-flower chrysanthemum variety that produces normal capitula composed of ray florets with normally developed pistils and purple corollas and mutant capitula comprising ray florets with green corollas and vegetative buds instead of pistils.Chrysanthemum morifolium ray florets with normally developed pistils and purple corollas with mutant ray florets with green corollas and vegetative buds. Additionally, the study findings will help to elucidate the molecular mechanisms underlying floral organ development and contribute to the development of techniques for studying the regulation of flower shape and color, which may enhance chrysanthemum breeding.We conducted a whole-transcriptome analysis of the differentially expressed genes (DEGs) in the mutant and normal capitula using third-generation and second-generation sequencing techniques. We identified the DEGs between the mutant and normal capitula to reveal important regulators underlying the differential development. Many transcription factors and genes related to the photoperiod and GA pathways, floral organ identity, and the anthocyanin biosynthesis pathway were differentially expressed between the normal and mutant capitula. A qualitative analysis of the pigments in the florets of normal and mutant capitula indicated anthocyanins were synthesized and accumulated in the florets of normal capitula, but not in the florets of mutant capitula. These results provide clues regarding the molecular basis of the replacement of C. morifolium capitula described herein indicates the anthocyanin deficiency of the mutant capitula may be related to the mutation that replaces ray floret pistils with vegetative buds. Moreover, pistils may be required for the anthocyanin biosynthesis in the corollas of chrysanthemum ray florets.The whole-transcriptome analysis of DEGs in mutant and normal The online version contains supplementary material available at 10.1186/s12863-021-00959-2. Chrysanthemum morifolium is one of the most economically important and popular floricultural crops in the family Asteraceae, and ranks second in the cut-flower industry after rose , leaving 11,508 (8.85%) unannotated unigenes. A total of 118,043, 101,048, 87,630, and 54,245 unigenes were annotated on the basis of searches of the nr, Swiss-Prot, KOG, and KEGG databases, respectively. Moreover, the Gene Ontology (GO) database was used for the functional annotation and analysis of genes, which were divided into the following three main categories: molecular function, cellular component, and biological process. Specifically, 36,144 unigenes were classified into 47 functional categories, including 19, 17, and 11 in the biological process, cellular component, and molecular function categories, respectively. The predominant biological process, molecular function, and cellular component GO terms among the genes were ‘metabolic process’ , ‘catalytic activity’ , and ‘cell’ , respectively. This implied that numerous metabolic activities were activated during the development of chrysanthemum capitula in a process regulated by the combined effects of the proteins encoded by these diverse genes. Additionally, a substantial proportion of the genes were annotated with the ‘cellular process’, ‘binding’, and ‘cell part’ GO terms, whereas ‘locomotion’, ‘transcription factor activity, protein binding’, and ‘extracellular matrix component’ were relatively uncommon GO terms  represented the largest group, followed by ‘signal transduction mechanisms’  and ‘posttranslational modification, protein turnover, chaperones’ . Conversely, the ‘defense mechanisms’ , ‘extracellular structures’ , and ‘cell motility’  clusters were the smallest groups . As a collection of manually drawn pathway maps, KEGG pathways present the networks of molecular interactions in cells and particular organisms. Of the 118,043 unigenes, 54,245 had significant matches with at least one KEGG pathway in the database and were assigned to 133 KEGG pathways in total  were identified between the normal and mutant capitula , ubiquitin-conjugating enzyme genes, zinc finger protein genes, and many unannotated genes. These genes may have important functions in developing chrysanthemum flowers, especially during the pistil determination and development stage. The production of normal capitula composed of ray florets with normally developed pistils and purple corollas and mutant capitula containing ray florets with green corollas and vegetative buds may be due to significant differences in the expression of these genes. Details regarding the annotation of the DEGs specifically expressed in the mutant and normal capitula are provided in Additional files A total of 131 DEGs were specifically expressed in the mutant capitula, including TCP1 and AP2/ERF domain-containing genes. Conversely, 2132 DEGs were specifically expressed in the normal capitula, including some important transcription factor genes  in the biological process category were all considerably lower in the mutant capitula than in the normal capitula. Of these genes, 11 were specifically expressed in the normal capitula, including WD40 and UBA1-like protein-encoding genes. These genes may play important roles in the regulatory pathways related to chrysanthemum reproduction , ARF (35 members), B3 (41 members), BBR-BPC (3 members), BES1 (8 members), bHLH (75 members), bZIP (22 members), C2H2 (84 members), C3H (51 members), CAMTA (2 members), CO-like (5 members), DBB (11 members), Dof (9 members), E2F/DP (4 members), ERF (98 members), FAR1 (15 members), G2-like (39 members), GATA (8 members), GeBP (1 member), GRAS (29 members), GRF (1 member), HB-other (6 members), HB-PHD (1 member), HD-ZIP (51 members), HSF (20 members), LBD (7 members), LSD (1 member), MIKC (12 members), M-type (9 members), MYB (80 members), NAC (25 members), NF-X1 (12 members), NF-YA (3 members), NF-YB (8 members), NF-YC (5 members), Nin-like (40 members), S1Fa-like (6 members), SBP (13 members), SRS (2 members), TALE (17 members), TCP (3 members), Trihelix (22 members), WRKY (56 members), YABBY (1 member), and ZF-HD (8 members). More specifically, the ERF, C2H2, MYB, bHLH, and WRKY transcription factor families respectively had 98, 84, 80, 75, and 56 members with expression levels that were extremely different between the normal and mutant capitula. Additionally, some important transcription factor genes were expressed only in the normal capitula, including 36 C2H2 genes, 6 bZIP genes, 5 bHLH genes, 5 MYB genes, 4 HB-other genes, 2 C3H genes, 2 E2F/DP genes, 2 GATA genes, 1 ERF gene, 1 HSF gene, 1 NF-X1 gene, 1 TALE gene, and 1 Trihelix gene.These results suggest that many transcription factors are important for floral development, but the functions of some transcription factors in developing flowers remain to be investigated. The important transcription factor genes substantially differentially expressed between the normal and mutant capitula may be crucial for the phenotypic variations between the normal and mutant capitula. These genes are presented in Additional file CRY1 and CRY2 homologs as well as homologs of PHYA, PHYB, FKF1, LHY, EFL1, EFL3, EFL4, TOC1, and GI. Moreover, homologs were detected for CONSTANS (CO), which is critical for the photoperiod response, and for FT (Flowering Locus T), which is targeted by CO. Many MADS-box genes are important for promoting floral meristem identity, including SHORT VEGETATIVE PHASE (SVP), SUPPRESSOR OF CONSTANS1 (SOC1), and APETALA1 (AP1). PISTILLATA (PI) is a floral organ identity gene that specifies petal and stamen identities in the A. thaliana flower , and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) . The MBWocyanins .MYB113, MYB114, MYB305, MYB46, Glabra 2 (GL2), Transparent Testa 12 (TT12), and TTG1. The expression levels of most of the R2R3 MYB genes were significantly down-regulated in the mutant capitula, with some genes not expressed at all  were also considerably down-regulated in the mutant capitula. Hua Li et al. suggested that MdMYB8 contributes to the regulation of flavonoid biosynthesis, with the overexpression of MdMYB8 promoting flavonol biosynthesis in crabapple  [The anthocyanin profiles in the normal and mutant capitula were analyzed by HPLC. A 1.0–10.0 g sample was ground into a fine powder in liquid N v/v/v)] . The ext v/v/v)] . Three bAdditional file 1 Chrysanthemum flowers. (A) Capitulum. (B) Disk floret. (C) Ray floret.Additional file 2. The DEGs specifically expressed in the mutant capitulaAdditional file 3. The DEGs specifically expressed in the normal capitulaAdditional file 4. The DEGs annotated with the “reproduction” GO term (GO:0000003)Additional file 5. The DEGs enriched in the biological process GO categoryAdditional file 6. The DEGs enriched in the molecular function GO categoryAdditional file 7. The DEGs enriched in the cellular component GO categoryAdditional file 8. The enriched KEGG pathways among the DEGs down-regulated in the mutant capitulaAdditional file 9. The enriched KEGG pathways among the DEGs up-regulated in the mutant capitulaAdditional file 10. Important transcription factor genes substantially differentially expressed between the normal and mutant capitulaAdditional file 11. Important photoperiod pathway genes in chrysanthemumAdditional file 12. The MADS-box genes identified in chrysanthemumAdditional file 13. The genes involved in the GA pathway in chrysanthemumAdditional file 14. Important anthocyanin biosynthesis pathway functional genes in chrysanthemumAdditional file 15 Primers used for the quantitative real-time PCR analysis of Chrysanthemum morifolium


EIL/EIN3) protein family can serve as a crucial factor for plant growth and development under diverse environmental conditions. EIL/EIN3 protein is a form of a localized nuclear protein with DNA-binding activity that potentially contributes to the intricate network of primary and secondary metabolic pathways of plants. In light of recent research advances, next-generation sequencing (NGS) and novel bioinformatics tools have provided significant breakthroughs in the study of the EIL/EIN3 protein family in cotton. In turn, this paved the way to identifying and characterizing the EIL/EIN3 protein family. Hence, the high-throughput, rapid, and cost-effective meta sequence analyses have led to a remarkable understanding of protein families in addition to the discovery of novel genes, enzymes, metabolites, and other biomolecules of the higher plants. Therefore, this work highlights the recent advance in the genomic-sequencing analysis of higher plants, which has provided a plethora of function profiles of the EIL/EIN3 protein family. The regulatory role and crosstalk of different metabolic pathways, which are apparently affected by these transcription factor proteins in one way or another, are also discussed. The ethylene hormone plays an important role in the regulation of reactive oxygen species in plants under various environmental stress circumstances. EIL/EIN3 proteins are the key ethylene-signaling regulators and play important roles in promoting cotton fiber developmental stages. However, the function of EIL/EIN3 during initiation and early elongation stages of cotton fiber development has not yet been fully understood. The results provided valuable information on cotton EIL/EIN3 proteins, as well as a new vision into the evolutionary relationships of this gene family in cotton species.The ethylene-insensitive3-like/ethylene-insensitive3 ( EIL/EIN3 genes also act as a foundation for ethylene connections with other signals, such as the crosstalk between ethylene and other hormones, light signaling, as well as various abiotic and biotic stress responses [EIN3) and EIN3-like (EIL) proteins are one of the main transcription factors [EIL/EIN3 proteins were indicated to be involved in the expression of the GCC-box-binding domain of Arabidopsis ethylene-response factors [EIN3/EIL protein family is a small group of transcription factors in higher plants. Several transcription factor genes of EIL/EIN3 proteins have been investigated in Arabidopsis [EIL/EIN3 proteins have highly conserved amino acid sequences at the N-termini, and are located in the nuclei compartment [EIL/EIN3 protein sequences, such as highly acidic N-terminus amino acid regions, 5-basic amino acid groups, and proline-rich regions [EIN3/EIL protein family has multiple functions in plant growth and development. In Arabidopsis, EIL3 acts as the central transcriptional regulator of plant sulfur response and metabolism process [EIL3 restores the sulfur limitation responseless morphologies of Arabidopsis mutants (slim1 mutants) [EIN3 significantly associates with the expression of chlorophyll biosynthesis genes [Arabidopsis mutants found several EIN3-regulated genes that were coregulated by the other transcription factors such as PIFs  and RHD6 (root hair development) [EIL/EIN3-activated transcription by certain developmental processes and environmental conditions. In tomato, overexpression of EIN3-binding F-box protein2 gene prompted elongated fruit shape and delayed fruit development and ripening [EIL/EIN3 gene family have been reported [EIL/EIN3 gene family in cotton. Fiber mutants are powerful tools for investigating the molecular mechanism and physiological process of fiber cell developmental stages. Mutant cotton analysis has accelerated the identification and characterization of interest genes or molecular elements associated with various stages of cotton fiber development [EIL/EIN3 genes from cotton species, including Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii were analyzed, according to their tree topology and sequence similarity. Moreover, the expression levels of upland cotton EIL/EIN3 genes were studied on a variety of fiber-cell developmental stages including 0, 3, 5, 8, and 10 days post anthesis (DPA). The results of this study provide fundamental evidence into the role of EIL/EIN3 genes in cotton fiber development and will support future functional examination of this vital gene family. In cotton, the EIL/EIN3 transcription factor plays an important role in the ethylene signaling pathway, which is involved in regulating the posttranscriptional level, whereas transcriptional regulations of EIL/EIN3 are not yet fully known.The gaseous ethylene phytohormone plays an important role in the regulation of different biological processes such as plant growth, plant development, and stress responses ,4,5,6,7.esponses ,10. In cesponses . Ethylen factors ,13,14. T factors . The EINbidopsis ,17, tobabidopsis , tomato bidopsis , cucumbebidopsis ,21,22, abidopsis . Plant spartment . Some im process . Overexpmutants) . In addiis genes . Transcrlopment) , which iripening . In cottripening . The genripening . Recentlreported ,29,30. Helopment ,32,33,34EIL/EIN3 transcription factor family in three cotton genome protein sequences  were downloaded from cottongen database resources (https://www.cottongen.org/). To identify EIL/EIN3 transcription factors in the cotton genome a BLASTP search was conducted using Arabidopsis, P. trichocarpa, maize, sorghum, and rice. EIL/EIN3 protein sequences were identified in the Phytozome database . The Hidden Markov Model profile of the EIL/EIN3 protein domain (PF04873) was downloaded from the Pfam database resources (http://pfam.xfam.org) and used as query sequences to identify EIL/EIN3 genes with an E-value < 10−10. To confirm the presence of the EIL/EIN3 protein domain in the three cotton genome protein sequences, all putative protein sequences were scanned by the InterPro online tool (http://www.ebi.ac.uk/interpro/) and SMART database (http://smart.embl-heidelberg.de/) to validate the EIL/EIN3 domain. Additionally, only the protein sequences with the EIN3/EIL domain were kept for further investigation. The physiochemical features (isoelectric point and molecular mass) of all the identified EIL/EIN3 protein domains were examined by the ExPASy Server tool. Moreover, the prediction of subcellular location was done by WoLF PSORT (https://wolfpsort.hgc.jp/) and confirmed by Protein Prowler Subcellular Localization Predictor version 1.2 (http://bioinf.scmb.uq.edu.au:8080/pprowler_webapp_1-2/) and TargetP1.1 Server11 (http://www.cbs.dtu.dk/services/TargetP/). The physical locations of EIN3/EIL genes were determined through blastN queries against the three cotton genomes. The MapChart tool (https://mapchart.net/) was used to locate EIN3/EIL genes on the specific chromosome.The identification of the EIL/EIN3 genes from G. hirsutum, G. arboreum, G. raimondii, Arabidopsis thaliana, P. trichocarpa, maize, sorghum, and rice. Moreover, a neighbor-joining (NJ) phylogeny method was conducted using MEGA 6.0 software (https://www.megasoftware.net/) with the following parameters: A Poisson model and 1000 bootstraps. The EIL/EIN3 gene organizations (exon/intron) were created by Gene Structure Displayer Server 2.0 (http://gsds.cbi.pku.edu.cn/) according to their genomic DNA sequences [EIN3/EIL proteins were examined by the MEME program (http://meme-suite.org/).The ClustalW method was used to align full amino acid sequences of equences . The 10 cis-acting regulatory elements in each promoter sequence of EIN3/EIL genes (2 kb upstream of the translation starting site) were investigated using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The plant small RNA target analysis server (http://plantgrn.noble.org/psRNATarget/) [G. hirsutum EIL/EIN3 genes.The Target/)  was usedEIL/EIN3 genes involvement in three ways: biological processes, cellular components, and molecular function. GO annotation analysis of cotton EIN3/EIL genes was done based on an OmicsBox/Blast2Go (https://www.biobam.com/omicsbox/). The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway was also used for a bioinformatics investigation that sought to examine the organismal and cellular functions of the EIL/EIN3 genes. To visualize the KEGG-pathway-enrichment of cotton EIL/EIN3 genes, Arabidopsis gene homologs for the identified EIL/EIN3 proteins in cotton were utilized as input data for the David database (http://david.abcc.ncifcrf.gov/).Gene Ontology (GO) was used to get information about the EIN3/EIL genes in biological involvement through protein–protein interaction, the STRING online database (https://string-db.org/) was used to conduct the protein interaction networks.The investigation of protein–protein interactions simplifies the understanding of plant gene functions . ConsequG. hirsutum EIN3/EIL genes. The raw reads were mapped to the upland reference genome [G. hirsutum EIL/EIN3 genes associated with cotton fiber developmental stages, 0, 3, 5, 8, and 10 DPA. The SYBER premix ExTaq kit  and Applied Biosystems 7500 Real-Time PCR system  were used to conduct RT-qPCR experiments. The G. hirsutum constitutive β-actin gene was applied as a reference gene and special EIL/EIN3 gene primers were employed for RT-qPCR. The following thermal cycle circumstances were applied: 95 °C for 2 min, 40 cycles of 95 °C for 5 s, and 60 °C for 34 s. The expression profiles for each gene were measured as the mean signal intensity across the three biological replicates. The Ct was used for the relative calculation of the input target number.Upland cotton, Ligonlintless-1 mutant, and wild-type genotypes were planted in the experimental field at the ICR, Institute of Cotton Research, CAAS  under normal conditions. For self-pollination, cotton flowers before the anthesis day were tied and labeled. All cotton samples were collected with biological triplicates for each sample from both the Ligonlintless-1 mutant and wild-type at the following stages of cotton fiber development: 0, 3, 5, 8, and 10 DPA and cotton leaf. The collected cotton samples were quickly frozen in liquid nitrogen and kept at −80 °C until further analysis. RNA-seq data for upland cotton, Ligonlintless-1, and wild-type from cotton fiber developmental stages at 0, 3, and 8 DPA, and cotton leaf were used to identify the spatio-temporal expression levels of e genome . The trae genome . The FPKEIL/EIN3 transcription factor family in G. hirsutum, G. raimondii, and G. arboreum, two methods of the blast  were used to search against the tree cotton genomes for E-values ≤ 10−10. Multiple sequence alignment was implemented to remove the redundant sequences of EIL/EIN3 genes. Pfam (http://pfam.xfam.org/) and SMART (http://smart.embl-heidelberg.de/) databases were used to confirm the presence of the EIL/EIN3 domain in cotton protein sequences. Finally, the redundant protein sequences were removed, resulting in 37 genes, including, 18, 10, and 9 EIL/EIN3 genes in G. hirsutum, G. raimondii, and G. arboreum, respectively. The results of this study revealed that the EIN3/EIL transcription factor family is significantly smaller than other transcription factor families [EIN3 protein domain in each protein sequence of the EIL/EIN3 gene members was found and localized in the nucleus, and various EIL/EIN3 genes were found in higher plant genomes [EIL/EIN3 gene family may be involved in regulating various functions in different species. Additionally, the predicted nuclear positions may prove that the EIL/EIN3 genes act as transcription factors. To obtain more insight into the possible role of the proteins encoded by the EIL/EIN3 gene under examination, it is necessary to understand their physiochemical properties, for instance, the proteins can be divided based on their molecular weight and isoelectric point properties [EIL/EIN3 genes significantly varied in size and were divergent in physicochemical characteristics (EIL/EIN3 proteins varied in length from 109 to 690 amino acids. Cotton EIL/EIN3 proteins had a huge divergence in the isoelectric point (pI), ranging from 4.78 to 9.35. The molecular weight of the cotton EIN3/EIL proteins ranged from 13052.13 kDa to 77907.98 kDa . Two EIL/EIN3 genes were mapped to chromosomes Ah05, Ah13, Dh03, and Dh13, and one EIL/EIN3 gene was localized on chromosomes Ah02, Ah03, Ah06, Ah08, Dh05, Dh06, Dh07, Dh08, and Dh12  did not have introns, while 12 genes contained an intron. Of the 12 EIL/EIN3 genes with introns, ten genes had one intron, one contained two introns, and the other contained four introns  domain, which was found to be located in the first half of the amino acid sequences . In previous identification of conserved motifs in higher plants, it was found that motifs 1, 3, 4, 5, and 6 were related to the domain of EIN3/EILs [EIL/EIN3 gene was found to contain highly homologous sequences, which were associated with the domain of EIN3 [EIL/EIN3.In order to obtain further insight into the conserved motifs, 10 motifs in cotton  program . A set oIN3/EILs ,14,29,30 of EIN3 . This wa of EIN3 . Additio of EIN3 . MoreoveEIL/EIN3 protein sequences from 18 G. hirsutum, 9 G. arboreum, 10 G. raimondii, 6 Arabidopsis, 7 P. trichocarpa, 6 maize, 3 sorghum, 3 T. cacao, and 12 soybean EIL/EIN3 proteins , GARE-motif (cis-acting regulatory element involved in the gibberellin-responsive element), TGA-element (cis-acting regulatory element involved in auxin responsiveness), TCA-element , and ABRE (cis-acting regulatory element involved in abscisic acid responsiveness)  . In cottEIL/EIN3 proteins. Cotton EIL/EIN3 genes were classified into two groups by GO annotation analysis according to their categories: biological processes and cellular components. The GO annotation results assigned the EIL/EIN3 genes into 14 clusters of biological processes; a great number of EIL/EIN3 genes were involved in the ethylene-activated signaling pathway, cellular macromolecule metabolic process, nitrogen compound metabolic process, primary metabolic process, and cellular response to iron ions . Ethylene plays a key role in promoting cotton fiber elongation by increasing the expression of tubulin, sucrose synthase, and expansin genes [The GO annotations and KEGG pathway analyses were conducted using the OmicsBox/Blast2Go tool and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway, and they presented the possible functions of ron ions A. In terin genes . The ethin genes .EIL/EIN3 genes in regulating cotton fiber developmental processes, the STRING database was used to generate the protein–protein interaction networks , and E3 ubiquitin ligase complexes (EBF1 and EBF2). Serine/threonine protein kinase plays an important role in a multiplicity of developmental pathways including cell migration, cell proliferation, and cytoskeleton regulation [Arabidopsis as a result of its connections with ethylene receptors [EIL/EIN3 protein family might form functional transcriptional factor complexes, mediating the expression of ethylene-insensitive3-like/ethylene-insensitive3 (EIL/EIN3) protein genes in cotton fiber developmental stages.Protein interaction networks are significantly involved in regulation of various cellular functions, including metabolic pathways, signal transduction, and cell cycle development . In animnetworks . The resgulation ,62. Actigulation . CTR1 aseceptors . The funeceptors . Many E3eceptors ,67,68. Teceptors . The resEIL/EIN3 genes in cotton fiber developmental stages and their potential functions in the early termination of the fiber elongation process, RNA-seq data, including three stages of fiber cell development  and cotton leaf, from Ligonlintless-1 and its wild-type were generated by Illumina sequencing. It was shown that all EIL/EIN3 genes were expressed at three stages  of cotton fiber development and leaf using the FPKM value  were differentially expressed in cotton fiber development during initiation and elongation stages  and the great number of them are linked to cotton fiber developmental stages [EIL/EIN3 genes were targeted by more than one microRNA; for example, Gh_A05G0871, Gh_D05G3883, and Gh_D08G2099 genes were targeted by 4 microRNAs, and the Gh_A08G1750 gene was targeted by 2 microRNAs . The interactions between EIL/EIN3 genes and microRNAs provide fundamental information on the regulation of gene expression.Fiber developmental stages rely on the multicomplex regulation networks of numerous genes. The miRNAs play an important role in the regulation of cotton fiber development. The miRNAs are defined as part of the noncoding RNAs, which broadly play an important role in regulating various aspects of plant growth and development . In cottNATarget  for inven stages . These nicroRNAs . In cottEIL/EIN3 transcription factor family in cotton, a representative cash crop. These results show that some EIL/EIN3 genes were directly targeted by miRNAs related to cotton fiber developmental stages. Further examination with functional studies of EIL/EIN3 transcription factors is needed to understand the regulation and interaction of various molecular pathways in enhancing cotton fiber development during the initiation and elongation stages. The findings of this study provided fundamental evidence into the role of EIL/EIN3 genes, which will support future functional examination of various important biological molecular mechanisms of this vital gene family in cotton.The results of this work revealed insights into the


Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enabling primitive eukaryotic cells to survive and reproduce. In animals, cilia have undergone functional and structural speciation giving raise to typical motile cilia, motile nodal cilia, and sensory immotile cilia. In contrast to other cilia types, typical motile cilia are able to beat in complex, two-phase movements. Moreover, they contain many additional structures, including central apparatus, composed of two single microtubules connected by a bridge-like structure and assembling numerous complexes called projections. A growing body of evidence supports the important role of the central apparatus in the generation and regulation of the motile cilia movement. Here we review data concerning the central apparatus structure, protein composition, and the significance of its components in ciliary beating regulation. Typical motile cilia and flagella (CFs) are several to several dozen micrometers long, microtubule-based organelles formed at the surface of eukaryotic cells, from protists to humans. These two homologous structures are early evolutionarily inventions, proposed to be present in the last eukaryotic common ancestor (LECA) ,2. DurinThe functional specialization of cilia correlates with the differences in their ultrastructure. In all CF types, the skeleton (an axoneme) is composed of nine peripherally positioned microtubular doublets, but only in motile cilia, both typical and nodal; these microtubules are docking sites for multiprotein complexes ,13,14. IIt is postulated that signals initiated at the CA are transmitted through mechano-chemical interactions between CA projections and RS heads to the RSs, and next to N-DRC and dyneins . In consDysfunction of motile CFs, resulting in altered beating pattern and beating frequency, causes rare human genetic disorders, including primary ciliary dyskinesia (PCD), normal pressure hydrocephalus (NPH), and different forms of male infertility  ,20,21.Although numerous data suggest that CA is essential for the generation and regulation of motile CF beating, our knowledge of its composition and functioning is fragmentary. Here, we present a systematic review on CA, including the newest data concerning CA proteomics, analysis of its functioning, and human disorders caused by the mutations in genes encoding CA proteins.In typical motile cilia, the central apparatus consists of two thirteen-protofilament microtubules, called C1 and C2, which are docking sites for numerous protein complexes called projections   [In contrast to outer doublets, CA microtubules do not originate from the basal body, but above it, in the distal region of the transition zone. Most frequently, the minus-end of one of two CA microtubules is capped  and embeylinder) ,31  . Little ylinder) ,33.Chlamydomonas  [Tetrahymena [Drosophila sperm cells [The process of CA assembly is poorly characterized. Up to now, it was shown that lack of katanin, the heterodimeric microtubule severing complex, composed of the catalytic p60 and regulatory p80 subunits, is indispensable for the formation of the central apparatus in f15, p80 , pf19, p mutants ) and in rahymena . Lack ofrahymena ,38. Howee below) ,40 and ie below) ,41,42,43rahymena  and Drosrm cells , CA was Chlamydomonas [Chlamydomonas central tube [Trypanosoma, the RNAi-dependent depletion of γ-tubulin or γ-tubulin-small-complex proteins, GCP2 or GCP3, resulted in the formation of CA-less flagellum [Trypanosoma-specific component of the basal plate, was also shown to be necessary for the formation of this structure and CA [In growing CFs, the CA assembly is initiated at the transition zone region and progresses toward the CF distal part, as was shown in ydomonas ,48, suggral tube , while ilagellum ,51. Moree and CA , furtherInterestingly, CA formation was initiated in the full-length flagella of katanin CA-less mutant once they were mated with wild-type cells. In fused cells (dikaryons) and in the presence of katanin (from wild-type partner), the nucleation of the CA structure is initiated in sub-distal parts of the CFs , but theParamecium [Chlamydomonas [Chlamydomonas [Chlamydomonas) a convey (inside) side of the bend [Tetrahymena, based on the analysis of cilia cross-sections of neighboring oral ciliary rows, which are in different phases of metachronal wave [In animals and parasitic protozoa, CA microtubules were shown to have a fixed position with respect to the outer doublets and, in consequence, to the plane of the bend, with both CA microtubules facing the bend ,54,55,56ramecium  and Chlaydomonas , it was ydomonas ) faces athe bend ,58  or one (27%) central pair microtubules , have also been observed in isolated wild-type CFs) [Trypanosoma mutants with a reduced level of PF20 (RNAi) seems to support this supposition. Flagella of PF20 mutant cells are nearly paralyzed, but surprisingly, 80% of them have both CA microtubules (but frequently misoriented), and the remaining 20% miss only one CA microtubule [Trypanosoma-specific flagellar structure contributing to flagella stability, or fixation method used .The protein composition of the so-called bridge-like structure extending between C1 and C2 microtubules remains largely unknown. Up to now, PF20/SPAG16 is the only protein shown to localize between two central microtubules in g bridge . PF20 isg bridge . Chlamydype CFs) ,40. Thusrotubule . The staPF20/Spag16 gene encodes two proteins, a 71 kDa Spag16L (large) and a 35 kDa Spag16S . The latter corresponds to the C-terminal part of Spag16L, containing seven WD-repeats. The WD-repeat containing fragment can bind to another CA protein, PF16/Spag6 [In mammals, 16/Spag6 , indicatSpag16L is expressed in motile CF-forming organs, including testis, oviduct, and brain ,94. HoweSpag16S is expressed exclusively in testes , where iThe targeted disruption of exon 11, and thus elimination of both Spag16 variants, in embryonic cells subsequently injected into blastocysts, yield infertile chimeric male mice . The matThese results suggest that Spag16 plays an important role during germ cell development and maturation. No abnormalities were observed in chimera bronchial cilia, indicating that CF-related Spag16 functions are most likely restricted to sperm cells. Importantly, the presence of Spag16S in the nucleus and its interactions with a nuclear protein, MEIG1, led to the hypothesis that Spag16 proteins play the additional role(s) in the process of spermatogenesis besides its structural function in the axoneme ,93.Chlamydomonas CFs caused by PF20/SPAG16 deletion. Because Chlamydomonas flagella lack additional supporting structures, such as paraflagellar rod in Trypanosoma, one could speculate that the lack of CA in Chlamydomonas pf20 mutants could be a secondary effect caused by forces generated during flagellum bending. It was also proposed that the differences in ciliary waveform  could explain higher resistance of CA in sperm cells flagella assembled by spag16 mutant [The experiments conducted in mice revealed surprising differences in the phenotypic and structural alterations in respiratory cilia and sperm cells flagella as well as between mammals and 6 mutant . On the SPAG16 allele did not cause PCD symptoms or infertility [In humans, the mutation in a single ertility , while iC1a projection of the estimated size of 1.1–1.7 MDa, C1c (1.9–2 MDa), and C1e projection (0.2–0.5 MDa) , previouChlamydomonas PF6, together with evolutionarily conserved proteins, calmodulin, two orthologous FAP114/CCDC189 and FAP119/CCDC189, and two nonconserved proteins, were reported to form a C1a projection [In ojection  , one or even both central pair microtubules are missing [Spag17, an ortholog of rm cells  and playrm cells . Mice wi missing .Deletion of Spag17 in mice also affected the formation of sperm cells. Surprisingly, the majority of studied axonemes showed normal ultrastructure. Instead, affected males produced a low number of sperm cells, and mature sperm cells had abnormal head shape and reduced tail length, indicating that Spag17 is involved in spermatogenesis . It was Spag17-null mutant [Intriguingly, Spag17 also regulates the formation of the primary cilia, at least in a subset of cell types. Osteoblasts and chondrocytes isolated from Spag17-deficient mice form shorter primary cilia and with lower frequency. This phenotype could explain defects in bone formation and mineralization in l mutant .Chlamydomonas pf16 mutant lack the entire C1a-c-e supercomplex [PF16/SPAG6, an approximately 60 kDa armadillo repeats-containing protein , plays arcomplex  and are rcomplex .Spag6 causes symptoms typical for PCD, but ciliary defects are less severe than those observed in Chlamydomonas. Respiratory CFs in mutant mice seem to have normal ultrastructure and only beat with reduced frequency [Xenopus embryos with morpholino-induced knockdown of Spag6 assemble a normal number of CF. Yet, similar to mice, they show some ultrastructural defects and their motility and orientation are affected [In mice, knockout of requency . Howeverrequency , suggestaffected .Spag6-deficiency also causes male infertility. Mostly, the sperm cells produced by Spag6 knockout males are hardly motile, and nearly half of them have structural abnormalities  .When overexpressed, Spag6 was shown to colocalize with cytoplasmic microtubules in non-ciliated and primary cilia-forming cells ,104, sugChlamydomonas pf16 flagella and mouse PF16/Spag6 knockout epididymal sperm cells, the level of PF6/Spag17 is reduced  [Chlamydomonas, in mice also the level of PF20/Spag16 is lowered in PF16/Spag6-null sperm cells [Biochemical studies suggest that PF16/Spag6, PF6/Spag17, and the bridge protein, PF20/Spag16, interact and likely localize interdependently. PF16/Spag6 interacts with PF6/Spag17 via the C-terminal part of PF6/Spag17  and withof mice) ,104. Howrm cells , suggestFAP81/Dlec1 in mouse causes male infertility, but due to aberrant spermatogenesis [Chlamydomonas fap81 mutant lack C1c and e projections and beat with reduced beating frequency, altered waveform and asynchrony [The knowledge regarding functions of the remaining components of the C1a-c-e supercomplex is sparse. Bulls with a homozygous null-mutation in an ~30–40 kDa, coiled-coil domain-containing protein, CCDC189, produce sperm cells with flagella of normal ultrastructure but nearly immotile . Furtherogenesis  manifestogenesis . In contynchrony . Interesynchrony .Finally, some data suggest that MOT17/SPATA17, a small ~30 kDa protein, can also be involved in the process of spermatogenesis, as overexpression of human SPATA17 in mouse testes increased germ cell apoptosis . SPATA17Chlamydomonas C1b-less mutant  lack both C1b and C1f projections [Chlamydomonas flagella showed that C1b/C1f is likely composed of several evolutionarily conserved proteins, CPC1/SPEF2, FAP69/CFAP69, enolase (ENO), and HSP70 [C1b (estimated MW 1.3–1.9 MDa) is located at the opposite side of the C1a and near C1f (1.1–1.6 MDa) , earlierjections . Thus, Cnd HSP70 ,69. Addind HSP70 . Of notend HSP70 .Chlamydomonas cpc1 mutant exhibit normal waveform, but their beating frequency is only half of that observed in wild-type cells [CPC1 and its mammalian ortholog SPEF2 are large, ~200–250 kDa proteins, containing CH  domain, adenylate kinase domain, and EF-hand motif . Flagellpe cells . Ultrastpe cells .Spef2 causes PCD symptoms and male infertility. The ciliary defects in murine Spef2 mutants are similar but not identical to those observed in Chlamydomonas. Although the beating frequency of the respiratory cilia is slightly (~20%) reduced, the cilia ultrastructure was described as normal [Spef2 mouse mutants. A lack of CA was observed at the early-stage of spermatogenesis, while in maturing sperm cells, all flagellar structures were highly disorganized [SPEF2 mutation.In mice ,82 and hs normal ,82. Howerganized ,82. Simirganized  and humarganized ,118,119 SPEF2 mutation, the respiratory cilia did not show apparent ultrastructural abnormalities  than to axoneme itself . During Chlamydomonas, it was shown that HSP70 participates in the regulation of flagellar microtubule assembly, probably playing a role in protein folding, transport, and repair as in other systems [Although the role of HSP70 was broadly studied, little is known about its function in motile CFs. In  systems ,124,125.FAP69/CFAP69 is an ~100–120 kDa armadillo repeats-containing protein . The knoCFAP69 or in Cfap69 mutant mice [CFAP69 was shown to be located exclusively in the proximal part of sperm flagella (midpiece) and to be required for flagellar localization of Spef2 ,127. Knoant mice ,127.Chlamydomonas, lack of the ~120 kDa FAP246 slightly reduces cells swimming rate [In ing rate . The maming rate ,128. A sing rate . Accordiing rate .Chlamydomonas indicate that evolutionarily conserved proteins, FAP46, FAP54, FAP74, FAP221, FAP297/WDR93, calmodulin, and likely FAP99 [C1d is the largest CA projection (estimated MW of 2.4–2.6 MDa), although it does not extend as close to the radial spokes as C1a and C1b . The stuly FAP99 ,65,71 buSimilar to C1a projection, mutations in mammalian C1d subunits are associated with male infertility caused by sperm cell malformations. However, mammalian C1d-mutants also develop PCD, indicating that aberration in this projection strongly affects the functioning of motile CFs assembled by different cell types.CFAP221 was also identified in humans; in the affected individual, the respiratory cilia beat with normal frequency but abnormal waveform, forming rotatory-like rather than planar movement [FAP221 and its mammalian ortholog, CFAP221/Pcdp1 (primary ciliary dyskinesia protein 1), are ~100–110 kDa proteins containing a calmodulin-binding domain. Thus, FAP221 orthologs could be involved in calcium-dependent regulation of ciliary functions. In mice, lack of Cfap221/Pcdp1 causes PCD and male infertility without any obvious ultrastructural abnormalities in the respiratory cilia . A PCD-cmovement .FAP54/CFAP54, an ~300 kDa protein, can bind calmodulin . In miceChlamydomonas. Flagella of fap46 mutant lack C1d projection, beat asynchronously, with reduced frequency and changed waveform [FAP46, an ~300 kDa protein, and FAP74, an approximately 200 kDa protein, were analyzed only in waveform .fap46 mutant but with even lower frequency [Flagella of FAP74 knockdown are depleted of both C1d and f projections. In consequence, cells with FAP74 knockdown swim with flagella beating patterns similar to requency . Thus, arequency .Chlamydomonas and mouse mutants, it appears that approximately 540 kDa protein, hydin , is the main subunit of C2b. Recent proteomic studies suggest that another ciliary protein, FAP47/CFAP47, could also form a part of C2b projection [The C2b projection is facing the C1b projection but is much smaller (approximately 1–1.1 MDa ). Based ojection .Chlamydomonas, knockdown of HYD results in lack (about 30–40% of cells) or assembly of shorter (40% of the wild-type length) CFs, with residual hydin present most frequently in the proximal part of only one of them. Mutant CFs lack C2b and part of C2c projection, suggesting that besides C2b, hydin either builds a part of C2c or is required for its stabilization. Less frequently, also other abnormalities, such as a loss of one or both CA microtubules or outer doublet abnormalities, were observed [Chlamydomonas CA mutants , CFs point away from the cell body , in approximately 80% of Chlamydomonas hyd cells, one or less frequently, both CFs are positioned alongside the cell body , indicating probable defects with the initiation of the recovery stroke [Chlamydomonas hyd mutants, flagella could twitch (mostly the ones in “hands-up position”), with occasional very quick power and recovery strokes [In observed . These uy stroke . In some strokes , likely Trypanosoma, similar to Chlamydomonas, depletion of hydin causes cell immotility, with some flagella lacking one or both CA microtubules. Regretfully, it was not described if, in CA-containing CFs, C2b and C2c projections are normally formed. Interestingly, in Trypanosoma hydin knockdown, the CA was frequently misoriented [In oriented .In mammals, mutations in hydin cause PCD-related phenotype. Hydin-deficient mice develop hydrocephalus ,132,133,Chlamydomonas, murine and human hydin-depleted epithelial CFs were able to beat, although with lowered amplitude (and frequency in mouse) [In contrast to n mouse) ,89. On tn mouse) .hydin mutant mice, similar as in PF16/Spag6 mutant, the basal bodies in ependymal ciliated cells are frequently misaligned [In saligned , and indsaligned ,89.In silico study suggests that hydin has four ASH domains. The biological significance of ASH is unknown, but it was hypothesized that the ASH domain mediates the targeting of CF proteins to the CA ,134. AccChlamydomonas Klp1/Kif9 [Identified in lp1/Kif9  is, so flp1/Kif9 . Klp1/Kilp1/Kif9 ,135,136.lp1/Kif9 , but theChlamydomonas causes a decrease of CF beating frequency related to the loss of a part of C2c and reduction or displacement of C2b projection. In contrast, in Trypanosoma knockdown of KIF9A, an ortholog of KLP1 reduced cell swimming rate, but the apparent ultrastructural changes in mutant flagella were not detected [The knockdown of KLP1 in detected .Until now, there is no evidence that mutations in Kif9 cause PCD in humans or mice. However, mouse males with Kif9 mutation have lower fertility, likely due to reduced sperm cell motility and abnormal flagellar waveform .Drosophila melanogaster Fu is a part of the intracellular multiprotein hedgehog signaling complex (HSC) that mediates hedgehog (Hh) signaling (reviewed in [Fu (Fused)/STK36 is an ~140 kDa serine-threonine kinase. In iewed in ). In mamiewed in . Similariewed in . Moreoveiewed in ,41 and Ciewed in  further iewed in . Thus, Siewed in  or its dIn mammals, mutations of Fu/STK36 cause PCD symptoms ,42. In SAs in the case of depletion of other CA components, in both human patients and knockout mice, the basal bodies and CFs in respiratory epithelial cells are disoriented, and thus very likely, the ciliary synchronization is compromised ,42.Chlamydomonas flagellar and CA proteomes [Chlamydomonas genome. However, it cannot be excluded that STK36 is present at a very low-level in Chlamydomonas CFs and therefore, it is difficult to detect.Surprisingly, Fu/STK36 ortholog was not identified in the roteomes ,71,139, Spef1 is an evolutionarily conserved protein with microtubule binding and bundling properties . The vasTrypanosoma, a Spef1 ortholog, TbSpef1, was shown to colocalize with a bundle of MTs supporting CFs (between the kinetoplast and flagellum attachment zone), but not within the CF itself [The immunofluorescence analysis showed that Spef1 likely localizes to the CA in ependymal cilia . SurprisF itself . Thus, iChlamydomonas mutants  are paralyzed [The analysis of the CA-less mutants indicates that CA plays an important role in CF beating generation and regulation. Interestingly, the phenotypic outcome of the lack of CA can vary depending on the organism or even tissue type within the same organism. For example, CFs of CA-less aralyzed  while muaralyzed ,41.Chlamydomonas mutants lacking specific projection suggests that each of them could have its own specific, not interchangeable, functions in the regulation of CF beating. For example, lack of the whole C1a-c-e supercomplex (pf16 mutant) results in nearly total CF paralysis  [hyd mutant) also causes nearly total CF paralysis, but frequently one or two CFs extend along the cell surface , suggesting problems in recovery stroke initiation and/or execution [The comparison of the phenotype of osition)  suggestixecution . Thus, ipf6 mutant), the biggest part of the C1a-c-e supercomplex, results in “twitchy” CFs, moving with very low frequency and slightly abnormal waveform [fap81 mutant) causes asynchronous beating, likely due to different frequencies and slightly modified waveform. CF with the elimination of C1e projection (fap76 mutant) beat asynchronously, but with only slightly reduced frequency and normal waveform (see Video 3 in [cpc1 mutant) only decreases CF beating without affecting flagellar waveform [Loss of other projections is less severe. Lack of C1a . Loss owaveform , while Cwaveform ,65.pf6 mutant), but not in C1b-lacking flagella (cpc1 mutant) [How CA regulates CF beating? The presence of proteins with functional domains and enzymes in different projections could suggest that particular projections are involved in specific signaling pathways. For example, C1a contains calmodulin, suggesting its involvement in calcium signaling . Accordi mutant) . On the  mutant) ,71, two  mutant) . In fact mutant) .Chlamydomonas pf6 mutant lacking C1a projection could be partially suppressed by expression of RSP3, RSP4, or RSP6 with a tag attached to their C-terminal end. Such a tag is exposed toward the CA. The RSP fusion proteins were unable to rescue the slow-motility of cpc1 mutant lacking C1b projection [It is proposed that signals produced at the CA are transmitted to radial spokes by mechano-chemical pathways . Howeverojection . These rChlamydomonas mutant cells with the negatively charged amino acids replaced by uncharged ones swam in spiral-like rather than straight paths, and CF beating frequency was reduced compared to wild-type.A recent study from the Vale group  suggestsDespite the recent significant progress, we are only at the beginning of the road to answer questions regarding structure, composition and role(s) played by the CA in the intricate process of cilia beating regulation.Chlamydomonas as a model [Most information regarding the protein composition of the CA complex and its functions was obtained using  a model ,71. SurpThe structural CA proteins can form a scaffold for enzymes that could be loosely or transiently associated with CA projections. Such enzymes could locally regulate the level of signaling molecules, energy sources  or modify CA subunits . It was already shown that enolase , calmoduAlthough it is widely accepted that the signals regulating cilia beating originate at the CA and are transmitted via radial spokes to dynein arms, the nature of these signals is still unclear. Available data suggest that mechanical or mechanochemical signals can be transmitted by transient physical interactions between radial spokes and C1a projection . This raChlamydomonas), each outer doublet axonemal unit contains three radial spokes that slightly differ in their architecture [With few exceptions (e.g., itecture  and, thuFinally, the mechanism behind the CA assembly and the role of the IFT particles in this process are unclear. In addition, we can only speculate about the significance of the microtubule severing protein, katanin, STK36 kinase, SPEF1 or tubulin modification in this process.Likely, further analysis and use of sophisticated experimental tools will help researchers to answer these and many other questions and dissect the molecular mechanisms behind this ciliary kickstarter activity.


Chrysemys picta) genome assembly and use de novo fluorescent in situ hybridization (FISH) of bacterial artificial chromosome (BAC) clones, BAC-FISH, to physically map the genomes of the painted and slider turtles (Trachemys scripta elegans). Optical mapping increased C. picta’s N50 by ~242% compared to the previous assembly. Physical mapping permitted anchoring ~45% of the genome assembly, spanning 5544 genes (including 20 genes related to the sex determination network of turtles and vertebrates). BAC-FISH data revealed assembly errors in C. picta and T. s. elegans assemblies, highlighting the importance of molecular cytogenetic data to complement bioinformatic approaches. We also compared C. picta’s anchored scaffolds to the genomes of other chelonians, chicken, lizards, and snake. Results revealed a mostly one-to-one correspondence between chromosomes of painted and slider turtles, and high homology among large syntenic blocks shared with other turtles and sauropsids. Yet, numerous chromosomal rearrangements were also evident across chelonians, between turtles and squamates, and between avian and non-avian reptiles.Recent sequencing and software enhancements have advanced our understanding of the evolution of genomic structure and function, especially addressing novel evolutionary biology questions. Yet fragmentary turtle genome assemblies remain a challenge to fully decipher the genetic architecture of adaptive evolution. Here, we use optical mapping to improve the contiguity of the painted turtle ( The evolution of genomic structure and function is an important area of inquiry in evolutionary biology. The rapid advent of whole-genome sequencing technologies has permitted the assembly of a growing number of complex genomes, including those of non-model organisms. However, many of these new genome assemblies are fragmentary, preventing the full discovery of organizational changes that accompanied diversification across the tree of life. This problem is alleviated by the continual development of longer-read and longer-range linkage sequencing methods, software enhancements, and molecular cytogenetics ,2,3, allChrysemys picta) [Trachemys scripta elegans) [Pelodiscus sinensis) [Chelonia mydas) [Gopherus agassizii) [Platysternon megacephalum) [Chelonoidis abingdonii and Aldabrachelys gigantea) [C. picta [Among reptiles, turtles are an exemplar system to study biological responses to natural environmental variation due to overwintering, anoxia, thermal variation affecting sexual development, and environmental xenobiotics that can disrupt these and other fundamental processes . Karys picta) ,12, the elegans) , the Chiinensis) , the grea mydas) , the Agaassizii) , the bigephalum) , and twoigantea) . Yet, geigantea)  and fluoC. picta ,12.Trachemys scripta elegans, which helped establish the homology and evolution between the chromosomes of painted and slider turtles which split from each other ~29 Mya (www.timetree.org). We then compared our improved painted turtle genome assembly to those of some other turtles and sauropsid vertebrates, and documented shared homologous syntenic blocks and numerous chromosomal rearrangements that occurred between these vertebrate genomes, thus advancing our understanding of turtle chromosome organization and vertebrate karyotypic evolution.The painted turtle is an emerging model for ecology, evolution, and human health . It is aC. picta hatchling who was produced by egg incubation at 31 °C, digested with two rare cutter restriction enzymes (BssSI and BspQI), and labeled with a fluorophore at the restriction sites. The linearized DNA was loaded into micro-channels where it was photographed. These optical maps were converted into molecules bioinformatically and assembled de novo to obtain consensus genomic maps, which were aligned to the CPI 3.0.3 reference genome [C. picta assembly was enhanced and refined by alignment with the optical maps to produce hybrid scaffolds that ordered and oriented previous sequences. This improved assembly was uploaded to the National Center for Biotechnology Information , and will be referred to hereafter as CPI 3.0.4.The BioNano Genomics (BNG) platform uses a technical approach that images native DNA molecules to produce an optical map of the genome sequences which can be used to scaffold DNA sequences obtained by other Next Generation Sequencing (NGS) methods . For oure genome . OpticalC. picta and T. scripta cell cultures established previously using our standard protocols [2 supplementation, and 10 µg/mL colcemid  was added four hours prior to harvesting. Metaphase chromosomes were harvested after hypotonic exposure and fixed in 3:1 methanol:acetic acid. Cell suspensions were dropped onto glass slides and air-dried. The slides were digested with pepsin and fixed with formaldehyde. High quality metaphase chromosomes were imaged and used for FISH. Because the subspecies of the slider turtle individual used for cytogenetics was not confirmed, we will use T. scripta or TSC to refer to cytogenetic data related to this individual, and T. s. elegans or TSE to refer to data from the recently published genome assembly [All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Iowa State University. Metaphase chromosome preparations were obtained from rotocols ,21,26. Bassembly .The coordinates of the VMRC CHY3 BAC library (produced by the Joint Genome Institute) in the CPI 3.0.3 genome were downloaded from the NCBI Clone database (accession number GCF_000241765.2.101). The complete sequences of unique and concordant BACs were extracted and mapped to the BioNano hybrid scaffolds using BWA-MEM . Out of C. picta cot-1 DNA and human cot-1 DNA, which was shown empirically to successfully suppress repetitive DNA sequences in turtles [T. scripta metaphase chromosomes as described earlier [BAC clones were grown by standard protocols in autoclaved Luria Borth (LB) media with 25 mg/µL chloramphenicol. Cultures were grown in a 37 °C incubator shaker overnight. BAC DNA was extracted using the ZR BAC DNA Miniprep Kit  following the manufacturer’s instruction. A total of ~1 µg DNA was labeled by standard nick-translation  using biotin-16-dUTP, digoxigenin-11-dUTP, or fluorescein-12-dUTP  and co-precipitated with a combination of unlabeled  turtles ,12,26. B turtles ,26. Brie earlier .® cytogenetic analysis system (Leica Microsystems). A minimum of 10 complete metaphase spreads were examined and analyzed for each specimen. The FISH signal and hybridization pattern revealed the BAC sequence relative position within chromosomes, and chromosomes were distinguished according to their morphology, size, shape, and DAPI-banding [Images were taken with a Leica DFC365 FX camera  attached to an Olympus BX41 fluorescent microscope , equipped with DAPI, FITC (fluorescein isothiocyanate), and TRITC (tetramethylrhodamine) filter cubes, and analyzed using a CytoVision-banding .C. picta BAC sequences were aligned to the genome for chromosomal assignment of scaffolds within the C. picta (CPI 3.0.4) and T. s. elegans genome assemblies. The BAC sequences were split into sliding windows of 150 bp length with a step size of 50 bp. These windows were mapped to each genome using BWA-MEM [C. picta scaffolds assigned to a particular chromosome (and unplaced scaffolds) were also mapped to corresponding chromosomes in T. s. elegans to produce synteny plots using nucmer/mummerplot [ BWA-MEM . A custommerplot  for visuC. picta  and seven sauropsid chromosome-level genome assemblies available in NCBI, including slider turtle T. s. elegans , Goode’s thornscrub tortoise Gopherus evgoodei , leatherback sea turtle Dermochelys coriacea , green anole Anolis carolinensis , sand lizard Lacerta agilis , western terrestrial garter snake Thamnophis elegans , and chicken Gallus gallus .Pairwise genome alignments were performed to compare the synteny between the genomes of C. picta genome was built, suitable for comparison between less-similar sequences, keeping the region masked in the genome assembly and additionally masking simple repeats (lastdb -uMAM4 -R11). Then, substitution and gap frequencies were determined for each CPI and query species comparison (last-train) and local alignments  were performed between CPI and each query sequence. To visualize the pairwise synteny between genomes, a filter was set on minimum size blocks , and chord diagrams were generated using Circos [The pairwise genome alignments were performed using LAST ,31, follg Circos .Optical maps were obtained for DNA digested with BssSI and BspQI restriction enzymes. The initial hybrid scaffolding incorporated 1117 CPI 3.0.3 scaffolds larger than 150 kb into the analysis. A large number of conflicts were detected between the original CPI 3.0.3 scaffolds and this first BioNano assembly (which used the BssSI data) due to the genetic difference between the specimen used for BioNano and the individual sequenced to generate the original assembly [Optical mapping alone increased the proportion of the genome that had been physically mapped to the CPI 3.0.3 genome assembly in previous studies ,12 via FWe also collected de novo BAC-FISH data from the 64 newly selected BACs, discarding from further analysis any BAC that failed to produce clean or consistent signals . Thus, our final new dataset includes results from 26 BAC clones with ~150 kb inserts and containing sequences corresponding to 21 additional hybrid scaffolds. These new data permitted assigning these 26 BAC sequences to 13 chromosomes, representing the physical mapping of an additional 260,371,119 bp  of the CPI 3.0.4 genome assembly . Thus, aImportantly, we detected a few discrepancies between the BioNano assembly and BAC-FISH physical mapping, revealing what appear to be chimeric BioNano scaffolds , thus hiC. picta chromosomes by our BAC-FISH data  co-localized in chromosome 1 of both C. picta and chicken, but their relative order differs (Fhl2-Dax1-Fgf9 in C. picta and Dax1-Fhl2-Fgf9 in chicken). Three other genes  map to CPI-3, but in chicken (Esr1 and Gata4) map to GGA-3 in the same gene order as in turtle, whereas Sox9 is localized in a chicken microchromosome (GGA-18). Two additional genes (Emx2 and Gata2) found in a single turtle chromosome (CPI-5) map to two separate chromosomes in chicken (GGA-6 and GGA-12). In another case, four genes  map to CPI-7 and GGA-Z, with Dmrt1-Dmrt2-Rps6 retaining the same gene order in both species, while Aco1 changes from first to last position in the cluster . Finally, two genes (Lhx9 and Dmrtb1) detected in chromosome 8 of C. picta and chicken retained the same gene order , Goode’s thornscrub tortoise (Gopherus evgoodei), leatherback sea turtle (Dermochelys coriacea), green anole (Anolis carolinensis), sand lizard (Lacerta agilis), Western terrestrial garter snake (Thamnophis elegans), and chicken , revealed numerous chromosomal rearrangements interspersed within large homologous blocks of shared synteny  follow a similar pattern. First, the homology between C. picta and T. s. elegans macrochromosomes revealed by our physical mapping data extends to G. evgoodei. Specifically, 11 macrochromosomes in C. picta shared a high degree of homology with the 11 macrochromosomes in these three turtle species, albeit smaller-scale chromosomal rearrangements were evident. Namely, C. picta’s three largest chromosomes  remained highly conserved in all three turtle species, with a large block of conserved synteny identified in the three largest chromosomes in these species. These three chromosomal homologies were also observed in chicken. Seven of the 11 macrochromosomes display one-to-one correspondence, whereas CPI-4, CPI-6 and CPI-7 share homology with several chromosomes in other turtles: TSE-4 (GEV-4), TSE-5 (GEV-5), and TSE-6 (GEV-6) of T. s. elegans and G. evgoodei, respectively, and to DCO-6, DCO-4, and DCO-5 of D. coriacea, respectively. CPI-5 is homologous to chromosome 7 of all three turtles. In addition, chromosomes 8, 9, 10, and 11 are retained in all four turtle species, whereas some of the smaller microchromosomes CPI-15, CPI-18, and CPI-19 shared homology with the same chromosomes in T. s. elegans, G. evgoodei, and D. coriacea . Two other noticeable rearrangements were revealed by the homology between CPI-8 and chromosomes 8 and 9 of all other turtles, and between CPI-9 and chromosomes 9 and 10 of all other turtles, both of which indicate a likely remodeling of these medium size chromosomes in C. picta.Overall, the chromosomal homology and synteny blocks identified between C. picta , and 26 of the 39 chicken chromosome pairs . Painted turtle (C. picta) chromosomes exhibited homology with at least one and up to 14 chicken chromosomes. Specifically, chicken and painted turtle macrochromosomes 1, 2, and 3 contain large conserved gene blocks, as well as regions orthologous to other chromosomes. Namely, C. picta chromosome 1 (CPI-1) shared homology with 11 chicken chromosomes, with a large block of conserved synteny identified in GGA-1. Similarly, CPI-2 contains gene blocks homologous to GGA-2 and also partial homology to 13 other chicken chromosomes, and CPI-3 shows homology to a large block of GGA-3 plus six other chicken chromosomes. Other turtle chromosomes that contain relatively large conserved syntenic blocks with chicken include CPI-4 to GGA-5 and GGA-26, CPI-5 to GGA-12, CPI-6 to GGA-4, and CPI-7 to GGA-Z. Homology to two chicken chromosomes was identified in C. picta chromosome CPI-10, and homology to three chicken chromosomes was detected in CPI-6, CPI-7, CPI-13, CPI-15, CPI-17, and CPI-18  by the use of optical mapping and de novo BAC-FISH data. This improved assembly adds to the genomic resources available for the painted turtle [C. picta as an emerging system to study the genomic architecture of adaptive traits within species and across vertebrates. In silico alignment of the complete CPI and TSE genome assemblies showed that CPI scaffolds map to almost all regions of the TSE assembly  using DNA from BAC clones as probes can help validate and correct genome assemblies , by deteC. picta and T. scripta, two turtles that have evolved independently for ~29 My (www.timetree.org), retained a largely conserved synteny, yet karyotypic evolution is still visible in changes in the shape, size, and gene arrangements of homologous chromosomes (C. picta macrochromosomes display relatively larger p-arm (short arm) than their homologous chromosomes in T. scripta, perhaps due to intrachromosomal inversions. These chromosomes include sub-metacentric CPI-4, CPI-8, and CPI-13 , as well as metacentric CPI-9 and CPI-10 . Furthermore, shape differences between the telocentric CPI-12 and its homologous acrocentric TSC-12 may be due to a pericentric inversion in C. picta or in T. scripta. We observed that omosomes . In partImportantly, despite the karyotypic changes mentioned above, most of the chromosome assignments from the genome assembly are consistent with our physical mapping data for both species, such that no major chromosomal fusion or fission events were detected between painted and slider turtles based on BAC-FISH. This highly one-to-one correspondence between chromosomes  could beT. s. elegans genome assembly [Indeed, we found some discrepancies in the chromosomal assignment between the genome assembly  and our assembly . For insassembly  are harbC. picta [C. picta as the reference and re-naming several scaffolds in the T. s. elegans’s genome assembly (accession number GCA_013100865.1). Specifically, we recommend renaming TSE-13 as TSE-15, TSE-14 as TSE-13, TSE-19 as TSE-24, and TSE-20 as TSE-18  physical mapping for turtles was carried out on C. picta , we recos TSE-18 . We poinectively ) indicatMore broadly, our molecular cytogenetics data contributed to growing efforts to illuminate chromosome evolution across distantly related taxa ,21,37,42C. picta CPI-7 is homologous to red-eared slider (T. scripta) chromosome 6 [Staurotypus triporcatus) XY sex chromosomes [Apalone spinifera and Pelodiscus sinensis [Aco1, Dmrt1, Dmrt2, Rps6. Dmrt1 is a key regulator of male sexual development across a variety of species [Aco1 between CPI-7 and GGA-Z , revealed an intrachromosomal rearrangement between painted turtle and chicken. A similar gene order to painted turtle (Aco1-Dmrt1-Rps6) was reported in S. triporcatus XY sex chromosomes [Gpn3) localized in CPI-17, a chromosome that shares homology to GGA-15, which is homologous to the Z chromosome of the softshell turtle family Trionychidae (A. spinifera and P. sinensis) [Our data expanded the physical mapping of genes involved in sexual development by detecting 20 genes out of 32 core genes in the vertebrate sex determination/differentiation cascade, including 10 genes previously reported . Our finmosome 6   .Fhl2, Dax1, and Fgf9 differed in CPI-1 and GGA-1 , whereas the order observed in C. picta is shared by the distantly related softshell family Trionychidae [C. picta and Trionychidae yielding a convergent gene order.Besides genes harbored in sex chromosomes, we found that while the relative position of some sexual development genes remained conserved between turtles and birds, others were captured in chromosomal rearrangements. Namely, the relative position of nd GGA-1 . Interesnychidae ,12. ThusSox9), and another involving Gata2 and Emx2, were detected between turtles and birds. Namely, Sox9 is found in the third largest chromosome across TSD and GSD turtles from both suborders Cryptodira and Pleurodira (which is homologous to the largest chromosome of Emydura macquarii where Sox9 is located) [Sox9 co-localizes with Esr1 and Gata4 in CPI-3, a chromosome sharing high homology with GGA-3 where Esr1 and Gata4 map, Sox9 is found in a microchromosome in chicken (GGA-18), indicating that an evolutionary transition took place between turtles and birds. On the other hand, Gata2 and Emx2 are linked to CPI-5, but map to two separate chicken chromosomes. All these examples underscore the occurrence of gene transposition or inversion events during the evolution of these vertebrate lineages.A rearrangement involving a key regulator of male differentiation (located) , and thuC. picta, T. s. elegans, G. evgoodei, and D. coriacea turtles in 11 macrochromosomes. In particular, C. picta’s three largest chromosomes  remained conserved across all turtles and chicken, indicating that large syntenic blocks of these chromosomes are likely ancestral to archelosaurs (turtles plus archosaurs)  A. Likewire level . Furtherre level B expand re level , as wellre level B, whereaC. picta microchromosomes are syntenic to portions of chicken macrochromosomes, whereas others are conserved as microchromosomes in chicken.Numerous chromosome fusions and fissions were proposed to have occurred after the split of turtle and birds ~250 Mya, leading to changes in chromosome number ,35,42. AC. picta chromosomes shared synteny with the six largest chromosomes of A. carolinensis. Similarly, CPI-4 and CPI-5 contain gene blocks homologous to parts of chromosome 1 and 2 (respectively) of anolis, sand lizard, and snake, indicating that this genomic arrangement is unique to squamates. Furthermore, the Z sex chromosomes of chicken, sand lizard, and garter snake share homology with three different C. picta chromosomes , highlighting their distinct evolutionary origin [Outside archosaurs, eight y origin ,50,52,53In conclusion, our study adds valuable information on chromosome evolution in vertebrates, uncovering homologies among turtles, and contributing to improve turtle genomic resources. This growing body of knowledge is essential to fully discern the role of chromosome repatterning on chromatin organization and transcription, and its ultimate influence on phenotype diversity and adaptation to environmental variation such as climate change . Future


The activation of succinate receptor 1 (SUCNR1) by extracellular succinate has been found to regulate immune cell function. However, the clinical significance of SUCNR1 in ovarian cancer and its correlation with tumor-infiltrating lymphocytes remain unclear.The genetic alteration and expression patterns of SUCNR1 were analyzed by using cBioPortal and Gene Expression Omnibus (GEO) datasets. Kaplan-Meier Plotter was used to assess the prognostic value of SUCNR1 in patients with ovarian cancer. The correlations between SUCNR1 expression and immune infiltration, gene markers of immune cells, cytokines, chemokines, or T cell exhaustion were explored by using TIMER and TISIDB platforms. We also performed Gene Set Enrichment Analysis (GSEA) to reveal biological function of SUCNR1 in ovarian cancer.p = 0.0073, HR = 1.49, 95% CI = 1.11–2).The expression of SUCNR1 was closely related to tumor infiltrating lymphocytes, multiple gene markers of immune cells, and T cell exhaustion in ovarian cancer. The expression of SUCNR1 was also associated with the expression of cytokine- or chemokine-related genes. Moreover, GSEA revealed that various immune-related pathways might be regulated by SUCNR1. In addition, we found that SUCNR1 was amplified in ovarian cancer, and the high expression of SUCNR1 predicted worse progression-free survival (These results highlight the role of SUCNR1 in regulating tumor immunity in ovarian cancer. Accumulating evidences indicate that tumor microenvironment (TME) and immune cell infiltration play critical roles in the development and treatment of cancers . The metSucnr1–/– mice also showed impaired migration of dendritic cells (Extracellular succinate function is achieved by binding its specific G protein-coupled receptor succinate receptor 1 (SUCNR1/GPR91) . SUCNR1 ic cells , a reducic cells , and weaic cells . HoweverOvarian cancer is one of the most malignant cancers in the female reproductive system. It is estimated that there are more than 290,000 new cases and 180,000 deaths each year worldwide . Nowaday1 was used to analyze the correlation of SUCNR1 expression with tumor-infiltrating lymphocytes, including activated CD8+ T cells, effector memory CD8+ T cells, activated CD4+ T cells, effector memory CD4+ T cells, regulatory T cells, natural killer cells, neutrophils, macrophages, activated dendritic cells, activated B cells, and myeloid-derived suppressor cells (MDSC) (The TISIDB platforms (MDSC) . We also2 and TIMER2.03 platforms were used to investigate the association between SUCNR1 expression and the infiltration level of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells , and to explore the differential gene expression in visceral adipose tissue from Sucnr1flox/flox and LysM-Sucnr1–/– mice (GSE120121). We used GSE9891 (including 285 ovarian cancer samples) to analyze the correlation of SUCNR1 expression with markers of immune cells, exhausted T cells, cytokines and chemokines in ovarian cancer. We also used GSE39204 to analyze the expression of SUCNR1 in 30 serous ovarian cancer samples, 13 endometrioid ovarian cancer samples and 16 clear cell cancer samples.Gene Expression Omnibus (GEO) datasets6 was used to assess the prognostic value of SUCNR1 in patients with ovarian cancer  to explore the biological functions and immune-related pathways potentially regulated by SUCNR1 in ovarian cancer  and presented as mean ± SD. p-values <0.05 were considered statistically significant.The correlation of SUCNR1 expression with immune infiltration level was determined by the TIMER and TISIDB platforms using Spearman’s correlation analysis. The correlation of gene expressions were analyzed by using Spearman’s correlation analysis. The association between the expressions of succinate-related regulators and molecular subtypes in ovarian cancer was assessed by the TISIDB platform using the Kruskal-Wallis Test. Survival rates were assessed using Kaplan-Meier curves and the log-rank test. Student’s + T cells , effector memory CD8+ T cells , activated CD4+ T cells , effector memory CD4+ T cells , regulatory T cells , natural killer cells , neutrophils , macrophages , activated dendritic cells , activated B cells , and MDSC , in ovarian cancer , neutrophil markers , natural killer cell markers , tumor-associated macrophage (TAM) markers  and M2 macrophage markers  showed significant correlation with the expression of SUCNR1 (p < 0.0001), T-helper 1 cells (Th1) , and T-helper 2 cells (Th2)   . Moreove 0.0001) . Togethep = 4.48e-25), CCL3 , ENTPD1 , CTLA4 , EOMES , LAG3 , PD1 , TIGIT , and HAVCR2 , were significantly correlated with the expression of SUCNR1 (p = 5.9e-14), PD-L2 , CD48 , CD80 , CD86 , and TNFRSF14  (The exhaustion of T cells is often associated with inefficient control of tumor growth and metastasis . We assef SUCNR1 . Moreove 5.1e-9) . Moreove 5.1e-9) . These fp = 2.86e-18), IL7 , IL16 , and IL18 , in ovarian cancer (p < 0.0001), interferon receptors , and even interferon regulatory factors , in ovarian cancer (p < 0.0001), C-X-C motif chemokine ligands  and C-C motif chemokine receptors  had a strong correlation with SUCNR1 expression  were downregulated in Sucnr1–/– tissues (GSE120121). Besides, MHC molecules, including HLA-B , HLA-DMA , HLA-DPA1 , HLA-DQA1 , and HLA-E , were significantly correlated with the expression of SUCNR1 in ovarian cancer , but not with OS , in ovarian cancer patients  , whereas high expression of SUCNR1 plus a high infiltration level of M1 macrophages predicted a better survival rate  in ovarian cancer  . Addition cancer .To further understand the molecular mechanism of SUCNR1 in ovarian cancer immunology, we performed a GSEA based on the Cancer Genome Atlas (TCGA) ovarian cancer RNAseq data. Gene Ontology (GO) analysis showed that SUCNR1 might be involved in adaptive immune response, lymphocyte mediated immunity, the regulation of cytokine production, regulation of the immune system process, regulation of the immune effector process, and the immune response-regulation signaling pathway . AntigenThe membrane receptor SUCNR1 is a ligand of succinate and its biological function is related to metabolic indications and the inflammatory program. However, the role of SUCNR1 in regulating tumor immune cell infiltration is unclear. In the current study, we reported that SUCNR1 was amplified and predicted a poor prognosis in ovarian cancer. The expression of SUCNR1 was closely correlated with immune infiltrates, diverse immune markers, and T cell exhaustion. The upregulation of SUCNR1 could affect the expression of cytokine- or chemokine-related genes. Furthermore, GSEA also revealed that various immune-related pathways might be regulated by SUCNR1. Therefore, our study highlights the immunoregulatory role of SUCNR1 in ovarian cancer.Succinate-triggered SUCNR1 signaling affects cellular metabolism and has been documented in multiple pathological processes, including ischemia-reperfusion, renin-induced hypertension, cardiac hypertrophy, and diabetic nephropathy and inflammation . InteresThe biological roles of SUCNR1 in ovarian cancer were identified by GSEA. It is worth noting that the high expression of SUCNR1 was closely related to a variety of immune-related processes and pathways, including cytokine and MHC protein binding, regulation of cytokine production, regulation of immune effector process, lymphocyte mediated immunity, T cell activation, interleukin signaling pathway, natural killer cell mediated cytotoxicity, and interferon alpha/beta signaling. This indicates that SUCNR1 has an immunoregulatory role in ovarian cancer.+ T cells, CD4+ T cells, macrophages, B cells, neutrophils, dendritic cells, and MDSC. In addition, the expression of SUCNR1 is closely related to the gene markers of various immune cells, including Treg (FOXP3 and CCR8), Th1 (TBX21 and CXCR3), Th2 (GATA3 and STAT5A), natural killer cells , M2 macrophages , and even TAM . To our knowledge, different immune cells are recruited by different chemokines and inflammatory cytokines (+ T cells and natural killer cells (+ Treg cells and macrophages (Although SUCNR1 has been found to regulate the function of immune cells , it remaytokines . For exaer cells . CCL5 prrophages . Here, wT cell dysfunction and exhaustion can lead to cancer immune evasion . In the in vivo experiments to confirm the immunoregulatory role of SUCNR1 in ovarian cancer. On the other hand, the detailed mechanisms of SUCNR1 in regulating immune cell infiltration needs further study.There are still some limitations in our study. We draw our conclusions through correlation analysis based on several public databases, and did not conduct In summary, our bioinformatics analysis revealed that increased SUCNR1 expression is associated with a high level of immune infiltration. Furthermore, the expression of SUCNR1 was closely associated with T cell exhaustion, gene markers of various immune cells, cytokines, and chemokines. The prognostic value of SUCNR1 was also demonstrated in patients with ovarian cancer. These findings identify SUCNR1 as a novel immune regulator and could be a valuable target for ovarian cancer immunotherapy.The original contributions presented in the study are included in the article/QW and YY conceived the project. JZ and QZ analyzed the data. JZ, QZ, YY, and QW contributed toward the interpretation of the data. All authors wrote and approved the final manuscript.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.


Trypanosoma cruzi has a high genetic diversity, and each group of strains may elicit specific pathological responses in the host. Conflicting results have been reported in studies using various combinations of mammalian host—T. cruzi strains. We previously profiled the transcriptomic signatures resulting from infection of L6E9 rat myoblasts with four reference strains of T. cruzi . The four strains induced similar overall gene expression alterations in the myoblasts, although only 21 genes were equally affected by all strains. Cardiotrophin-like cytokine factor 1 (Clcf1) was one of the genes found to be consistently upregulated by the infection with all four strains of T. cruzi. This cytokine is a member of the interleukin-6 family that binds to glycoprotein 130 receptor and activates the JAK/STAT signaling pathway, which may lead to muscle cell hypertrophy. Another commonly upregulated gene was tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta , present in the Cell Cycle Pathway. In the present work, we reanalyzed our previous microarray dataset, aiming at understanding in more details the transcriptomic impact that each strain has on JAK/STAT signaling and Cell Cycle pathways. Using Pearson correlation analysis between the expression levels of gene pairs in biological replicas from each pathway, we determined the coordination between such pairs in each experimental condition and the predicted protein interactions between the significantly altered genes by each strain. We found that although these highlighted genes were similarly affected by all four strains, the downstream genes or their interaction partners were not necessarily equally affected, thus reinforcing the idea of the role of parasite background on host cell transcriptome. These new analyses provide further evidence to the mechanistic understanding of how distinct T. cruzi strains lead to diverse remodeling of host cell transcriptome.Chagas disease is responsible for more than 10,000 deaths per year and about 6 to 7 million infected people worldwide. In its chronic stage, patients can develop mega-colon, mega-esophagus, and cardiomyopathy. Differences in clinical outcomes may be determined, in part, by the genetic background of the parasite that causes Chagas disease.  Trypanosoma cruzi and affects about 6 to 7 million people worldwide   is caused by the protozoan ide WHO, . The caride WHO,  is the mide WHO, . These dT. cruzi   by all four strains of T. cruzi. Clcf1 belongs to the interleukin (IL)-6 family of cytokines that have the glycoprotein gp130 as a common signal-transducing receptor and is involved in cell differentiation, survival, apoptosis, and proliferation through activation of Janus kinase (JAK). JAKs in turn, activate signal transducer and activator of transcription (STAT) factors  revealed cardiac overexpression of CLCF1 and gp130  infected with four different strains of STAT proteins include STAT1–4, –5a, –5b, and –6 and have been shown to play an important role in cytokine signaling. These proteins are tyrosine phosphorylated by JAKs following the binding of cytokine to its receptor. Upon tyrosine phosphorylation, STAT proteins form homodimers or heterodimers and rapidly translocate to the nucleus and induce gene expression. Recent evidence has demonstrated the necessity of STAT3 in cell growth and transformation , and there were different levels of apoptosis induction by each strain. Moreover, T. cruzi infection provoked variable apoptosis rates in distinct host cell types  .In this context, transcriptomic analyses may be expected to elucidate associations between the DTUs and prediction of the pathogenesis of Trypanosoma cruzi were isolated from supernatants of infected Vero cells and used at a multiplicity of infection (MOI) of 10. Twenty-four hours after infection, cultures were washed twice with Ringer's saline solution and fresh supplemented medium was added. Medium was replaced daily and cultures were kept up to 72 h post infection.For microarrays analyses, rat skeletal myoblast L6E9 were used as described in Adesse et al. . Before T. cruzi   was >0.05. Experimental details and raw and processed expression data have been deposited and are publically available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18175.Microarray data were obtained from our previous publication  expression levels of each pair of pathway genes in the biological replicas. Two genes were considered as synergistically expressed if their expression levels increased and decreased together (positive covariance) in a set of similar samples or as antagonistically expressed (negative covariance) when they manifest opposite tendencies and as independently expressed when their expressions are not correlated (close to zero covariance). In the case of four biological replicas, the (p < 0.05) cut-off for synergism is a pairwise Pearson correlation coefficient ρ > 0.90, for antagonism ρ < −0.90, and for independence |ρ| < 0.05. To illustrate, Antxr1), antagonistically (Dus3l), and independently (Golim4) expressed genes with Clcf1 in control L6E9 rat myoblasts.As previously described : a) JAK/STAT Signaling Pathway  and b) Cell Cycle Pathway .On the basis of our initial analyses of genes whose expression was altered by infection with different Trypanosoma cruzi. We now listed the 30 genes most downregulated and upregulated genes by each strain  networks and subsequently applied K-means algorithm to determine clusters of genes with a similar expression profile . The cluT. cruzi induced upregulation of the Clcf1 transcript in infected rat myoblasts by 2.2-, 2.3-, 1.8-, and 2.3-fold by the Brazil, CL, Tulahuen, and Y strains, respectively. Because this cytokine is one of the known activators of the JAK/STAT signaling pathway, we investigated whether other genes in such pathway might be altered by all four strains, which would validate JAK/STAT activation as a hallmark of T. cruzi infection. Using KEGG pathway database, we highlighted which genes were significantly upregulated or downregulated by each parasite strain , IL-21 (9.4-fold), and colony stimulating factor 3 receptor (2.3-fold). The Y strain also led to increased expression of IL-21 transcript (4.4-fold). Concerning the membrane receptors that trigger JAK/STAT signaling, we found that the Brazil and Tulahuen strains both induced upregulation of prolactin receptor . Cells infected with the CL strain showed increased expression of IL-3 (1.9-fold) and IL-21 (3.0-fold) receptors, as well as IFN-α receptor 1 . The Y strain also increased the expression of IL-21 receptor (2.5-fold). Other constituents of this pathway were altered by the Y and CL strains: PIAS4 was upregulated by 1. 6- and 2.0-fold, respectively. Conversely, the Tulahuen strain downregulated expression of protein inhibitor of STAT1 and STAT3 (PIAS1 and PIAS3) by 1.7- and 2.5-fold, respectively  was one of the few genes found to be upregulated by all four strains studied , a major player in the cell cycle pathway, as has also been previously reported  and 27% (15/55 spots) altered genes, respectively. The Brazil strain (TcI) and Y strain  had an impact on these two pathways, although to a lesser extent, affecting eight and five of the 55 measured genes, respectively. Specific Cell Cycle Pathway genes whose expression was altered following infection are described below.cyclin-dependent kinase inhibitor 1B ; ATM serine/threonine kinase ; checkpoint kinase 1 homolog , and MAD2 -like 1 . Downregulated genes includes SMAD family member 2  and cell division cycle 7 . Whereas the theta polypeptide of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (Ywhaq) was upregulated (2.9-fold), the zeta polypeptide (Ywhaz) was downregulated (−1.9-fold)  .Cyclin D1 , histone deacetylase 1 , cyclin dependent kinase 2 , proliferating cell nuclear antigen , growth arrest and DNA-damage-inducible beta , Checkpoint kinase 1 homolog , protein kinase, membrane associated tyrosine/threonine 1 , MAD2 -like 1 (yeast) , anaphase promoting complex subunit 2 , and cell division cycle 20 . Regarding the members of the 14-3-3 complex, CL infection upregulated the theta polypeptide   .S-phase kinase-associated protein 1 , CyclinD1 , cyclin-dependent kinase inhibitor 1B , minichromosome maintenance deficient 2 mitotin , fizzy/cell division cycle 20 related 1 , cell division cycle 20 homolog , proliferating cell nuclear antigen . Downregulated genes included MAD homolog 2 , MAD homolog 4 , V-abl Abelson murine leukemia viral oncogene homolog 1 , protein kinase, DNA activated, catalytic polypeptide , and tumor protein p53 . Three members of the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein were affected by the Tulahuen strain infection: downregulated the eta  and zeta , and upregulated the theta   .glycogen synthase kinase 3 beta (Gsk3b 1.5-fold), cyclin-dependent kinase inhibitor 2B , Ywhaq (2.6-fold), and downregulation of anaphase promoting complex subunit 4   .The pathway expression analysis shown in From these measurements, we determined the number of gene pairs with significant pairwise Pearson correlation coefficients, with synergistic correlations shown in blue and antagonistic in red in In the non-infected control group, the JAK/STAT signaling pathway had 16 synergistic and six antagonistic coordination. Samples obtained for each of the four strains exhibited higher numbers of synergisms ; antagonistic correlations for the Brazil, Talahuen, and Y strains were similar to those of control (six antagonisms each) . The CL T. cruzi infection by all strains resulted in fewer synergisms , the coordination changed to a neutral profile.Additionally, we determined whether infection by each of the JAK/STAT  and CellJAK/STAT  with allTrypanosoma cruzi infection results in CD that has different clinical forms that include asymptomatic, cardiac, digestive, and neurological features  replication and dissemination in STAT1 knockout mice  had such interactions among the downregulated genes. Regarding the pairwise coordination profiling of genes belonging to the cell cycle pathway, we verified an overall reduction of synergistic interactions induced by all four strains. Accordingly, infection of L6E9 myoblasts by the Brazil strain led to no significant alteration of cyclin D1 promotor activity or cyclin D1 protein stability in infected cultures , 14-3-3γ (YWHAG), 14-3-3ε (YWHAE), 14-3-3ζ (YWHAZ), 14-3-3η (YWHAH), 14-3-3θ (YWHAQ), and 14-3-3σ also known as stratifin (SFN) (MacKay et al., T. cruzi genetic background on host cell transcriptome, as a good predictor of biological outcomes. Although Clcf-1 and Ywhaq were equally altered in the infected L6E9 cells, their network ensemble was in fact composed of different transcripts, which may lead to variations in the degrees of activations in these molecular pathways. These observations are important to deepen the understanding of how CD can present multiple pathologies, according to parasite background, combined with host diversity and environmental aspects. Such variability should be taken in consideration when proposing chemotherapeutic or immunomodulatory approaches to control of this disease.In summary, the bioinformatic tools used in this work allowed the further description of the differential impact of The raw datasets generated in this study can be found in NCBI, accession number GSE18175.DA, LG, DS, HT, and DI conceptualized the study. DA, DI, and DS contributed to methodology, resources, and funding acquisition. PCV, PHV, LN, TM, and LC helped with the validation. SI, DI, and TM did the formal analysis. DA, LN, PCV, PHV, LC, and TM carried out the investigation. DA and LN contributed to data curation and writing the original draft. DA, LG, DS, and DI wrote, reviewed, and edited the manuscript. DA was responsible for the visualization and project administration. DA, DI, DS, and HT supervised the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Proteins associated with the yeast plasma membrane often accumulate asymmetrically within the plane of the membrane. Asymmetric accumulation is thought to underlie diverse processes, including polarized growth, stress sensing, and aging. Here, we review our evolving understanding of how cells achieve asymmetric distributions of membrane proteins despite the anticipated dissipative effects of diffusion, and highlight recent findings suggesting that differential diffusion is exploited to create, rather than dissipate, asymmetry. We also highlight open questions about diffusion in yeast plasma membranes that remain unsolved. Saccharomyces cerevisiae, involves the creation of a new cell (the bud) connected to a mother cell. Bud formation involves sequential changes in the pattern of secretion during the cell cycle, changing from uniform secretion in the mother during G1 phase, to polarized secretion into the bud during S/G2/M phases, to directed secretion towards the mother-bud neck during septation  [Yeast cells are small, with diameters of 4–6 µm . Over su2/s) ,6,7 woul2/s) ,9. Howev2/s) .2/s, while multipass transmembrane proteins diffuse with D < 0.0005 µm2/s [2/s) [Fluorescence Recovery After Photobleaching (FRAP) studies showed that single-pass transmembrane proteins in the yeast plasma membrane diffuse with D < 0.0025 µm05 µm2/s ,12. Beca/s [2/s) . Consist/s [2/s) ,14,15. TInterestingly, several long-lived membrane proteins are enriched in the mother plasma membrane, and this has been linked to the process of aging in yeast ,16,17. TGiven the very restricted lateral mobility of plasma membrane proteins in yeast, one way to obtain mother-enriched protein localization would be to restrict protein synthesis to a specific phase of the cell cycle, as discussed for cell wall proteins B. IndeedSlow diffusion also has implications for the distribution of short-lived proteins. The yeast pheromone (α-factor) receptor, Ste2, undergoes endocytosis and vacuolar degradation ,19. In cEndocytosis and recycling can also change the distribution of long-lived membrane proteins. In a series of elegant experiments, the polarized distribution of v-SNAREs on the yeast plasma membrane was shown to arise from the combination of polarized delivery, slow diffusion, and rapid endocytosis that recycles v-SNAREs before they diffuse too far from their site of deposition  Figure A. SimilaIn aggregate, these studies highlight two key aspects of yeast cell biology that enable asymmetric accumulation of integral membrane proteins. First, secretion is directed towards different sites in a stereotypical manner during the cell cycle, allowing targeted delivery of proteins depending on their time of synthesis. Second, slow diffusion in the yeast plasma membrane can allow for asymmetries to persist, either passively for long-lived almost immobile multipass membrane proteins, or via endocytic recycling for more mobile single-pass membrane proteins.2/s) [Unlike integral membrane proteins, peripheral membrane proteins can detach from the membrane to the cytoplasm, where diffusion is much more rapid  ,7. For aCdc42 localization changes dramatically through the cell cycle B. Initia2/s: [2/s: [Cdc42 is prenylated, which promotes its attachment to membranes. An early hypothesis to explain Cdc42 polarization was that, as for the v-SNAREs discussed above, directed secretion combined with endocytosis could mediate asymmetric accumulation of Cdc42  Figure A. Howeve2/s: ) were fas: [2/s: ). With ss: [2/s: ,29. A prs: [2/s:  also seeAt the membrane, Cdc42 switches between GTP- and GDP-bound states in a manner assisted by GDP exchange factors (GEFs) and GTPase-activating proteins (GAPs) C. The CdThe first report to consider differential diffusion of Cdc42 suggested that Cdc42 could diffuse more slowly in one part of the plasma membrane than another, perhaps due to membrane domains of different lipid composition . Such loEvidence for such location-dependent diffusion came from an unexpected observation . GFP-Cdc2/s (fast zones) and 0.0061 µm2/s (slow zones). The authors speculated that fast and slow diffusion zones might represent patches of membrane with different lipid composition, reminiscent of lipid rafts, although current models for lipid rafts consider much smaller and more transient entities.The authors proposed that the patches represented zones in which Cdc42 mobility was altered, so that bright patches were slow-mobility zones and dark patches (as well as areas outside the polarity site) were high-mobility zones. In this view, location-dependent diffusion explained the GFP-Cdc42 localization pattern C. FittinAn alternative, and in our view more likely, interpretation of the bright and dark patches is based on the observation that fluorescent markers of endocytic sites at the membrane showed a pattern that was anticorrelated with the GFP-Cdc42 signal . We suggOther studies proposed a distinct scenario in which diffusion of any given Cdc42 molecule is similar at all locations, but different forms of Cdc42 (GTP-Cdc42 vs. GDP-Cdc42) have inherently different mobility ,38. Cons2/s), while GTP-Cdc42 diffusion was at least 10-fold slower [2/s for GTP-Cdc42 and 0.023 µm2/s for GDP-Cdc42) [Convincing evidence for a stark GDP/GTP-dependent difference in Cdc42 diffusion came from studies in fission yeast, where (unlike in budding yeast) Cdc42 mobility was largely independent of the GDI. GDP-Cdc42 was estimated to diffuse rapidly in the membrane  . These fDiffusion is best known as a dissipative process that acts to reduce concentration gradients in cells and membranes. Nevertheless, many proteins in yeast plasma membranes develop markedly asymmetric patterns of accumulation. The studies discussed above suggest that this is enabled by two features. First, protein diffusion in the yeast plasma membrane is unusually slow, which reduces the dissipative effects of diffusion. Second, some proteins, including Cdc42, may diffuse at different rates depending on their conformation. These studies raise several unsolved questions, a few of which are highlighted below.Mutations that perturb the normal lipid composition of the plasma membrane can modestly increase protein diffusion ,15. ThusWith the exception of the bud-site selection landmarks  and perhThe structures of GDP- and GTP-bound Cdc42 differ in well-appreciated ways , but it