Trypanosoma spp. , Leishmania spp. , trypanosomatids of insects, Phytomonas spp. of plants, and bodonids. Comparison of ACDps based on domain architecture, predicted tertiary structure, phylogeny and genome organization reveals a kinetoplastid evolutionarily conserved repertoire, which diversified prior to trypanosomatid adaptation to parasitic life. We identified 9 ACDp orthologs classified in 8 families of TryACD: four previously recognized , and four characterized for the first time in kinetoplastids . A single copy of each ortholog was identified in each genome alongside TryNudC1/TrypNudC2 homologs and, overall, ACDPs were under strong selection pressures at main phylogenetic lineages. Transcripts of all ACDPs were identified across the life stages of T. cruzi, T. brucei and Leishmania spp., but proteomic profiles suggested that most ACDPs may be species- and stage-regulated. Our findings establish the basis for functional studies, and provided evolutionary and structural support for an underestimated repertoire of ACDps in the kinetoplastids.Small Heat-Shock Proteins (sHSPs) and other proteins bearing alpha-crystallin domains (ACD) participate in defense against heat and oxidative stress and play important roles in cell cycle, cytoskeleton dynamics, and immunological and pathological mechanisms in eukaryotes. However, little is known about these proteins in early-diverging lineages of protists such as the kinetoplastids. Here, ACD-like proteins (ACDp) were investigated in genomes of 61 species of 12 kinetoplastid genera, including  Trypanosoma cruzi, T. brucei ssp. and Leishmania spp.), livestock  and plants (Phytomonas), but the majority are apparently harmless . Th. Th20]. T. brucei, SGT1 mRNA increases after heat shock in the presence of ZC3H11, a zinc finger protein that acts as a post-transcriptional regulator of chaperone [The SGT1 architecture includes three conserved domains: a TPR in the N-terminus, the central ACD-like, and a highly conserved SGT1-specific domain in the C-terminus. These domains are separated by V1 and V2 hypervariable regions. The TPR domain, present in many eukaryotic SGT1 proteins, is involved in chaperone machinery, cell-cycle and protein transport . This dohaperone .TrySGT1 genes encode a ~ 215 amino acid protein, lacking a signal peptide and showing ~ 65% amino acid identity among the orthologs of trypanosomatids. Notably, TrySGT1 exhibits a unique domain architecture comprising just two (SGT1-like and ACD-like) of the three domains present in human SGT1 archetype; i.e., TrySGT1 lacks the TPR domain plus the first hypervariable region in the N-terminus  SKP1-like protein. In a study with Toxoplasma gondii, SKP1 was involved in parasite detection of oxygen levels and genomic surveys suggested the existence of an ancestral SKP1 glycosylation pathway in other Apicomplexa species, and in protists in general [T. brucei SKP1 (Tb927.10.11610/Tb927.11.6130) and recovered a single SKP1-like gene from each genome examined, suggesting that a non-predicted interaction between SKP1/SGST1 may occur in the trypanosomatids. The genomic search revealed, in general, a single copy of TrySGT1. The exception was a frog trypanosome (Trypanosoma sp TCC878) with multiple nearly identical copies. MDS analyses clustered all TrySGT1 orthologs near to Tryp23B, TryDYX1C1 and TryACD-TPR clusters  proteins are conserved from fungi to mammals acting as chaperones and holdases that stabilize or enhance the folding of client proteins. Besides their multiple roles in the cell cycle, evidence has accumulated that NudC proteins have acquired new functions in metazoans, being implicated in many processes such as mitosis, neuronal differentiation and migration, regulation of inflammatory response, platelet production, cytoskeleton dynamics, and proliferation of tumor cells  22,50,5,52.TryNudC1 and TryNudC2 that clustered closest to each other but did not overlap in MDS analysis (TryNudC1 proteins (~315 amino acids) display domain architecture like the human homolog hNudC (NP_006591.1) with the NudC-N domain in the N-terminal extension, and a hypervariable region between residues 66–88 (TryNudC1 share ~70% amino acid identity and are quite conserved (~60% amino acid identity) to human NudC-N domain (NP_006591.1). Part of the amino acid alignment of TryNudC1 sequences is shown in the TryNudC1 orthologs from kinetoplastids and other eukaryotic lineages is shown in All members of the NudC family comprise one ACD domain. Conserved NudC homologs have been identified in a range of eukaryotes and three NudC genes, NudC, NudC-like (NudCL) and NudC-like 2 (NudCL2), have been identified exclusively in vertebrates . Our seaanalysis . TryNudCes 66–88 . NudC-N TryNudC1 homologs of human NudC, the kinetoplastids exhibited a NudC homolog hereafter referred to as TryNudC2 due to its similarity with NudCL2. Consistent with TryNudC2 being more related to NudCL2, both are small proteins lacking the NudC-N N-terminal domain (TryNudC2 (~170 amino acids) shared ~62% identity among trypanosomatid orthologs. In contrast to TryNudC1, TryNudC2 proteins are highly divergent from the NudCL2 of both human (~20% identity) and fungi (~30%). The alignment of TryNudC1 and TryNucC2 sequences is illustrated in the TryNudC1 and mammalian orthologs led us to investigate NudC-N domains (pfam14050) in other organisms. This analysis revealed NudC-N domains, similar to the TryNudC1 orthologs in trypanosomatids, protists of Alveolata, Plasmodium, Eimeria, Neospora and Toxoplasma, but not in those of Amebozoa, Fungi and green plants  of L. mexicana , and P. caudatus  amino acid identity with the smaller 420 amino acid human archetype. Size and amino acid polymorphisms among TryDYX1C1 orthologs are mainly due to differences in the two hypervariable regions between residues 170–218 and 533–550 , although the gene fragment and flanking genes were preserved in a locus syntenic with T. brucei (TryACDP was recovered from transcriptomes (T. vivax Y486 and TvLins). Present as single copy genes in almost all genomes, additional copies were so far identified in T. brucei, the frog trypanosome (TCC878) and P. caudatus , we recovered highly divergent sequences of proteins bearing ACD-like domain from pathogenic oomycetes Saprolegnia, Phytophthora, Aphanomyces, and Plasmopara . However, no clear inference about orthology of kinetoplastid and oomycetes genes was possible because of a very low sequence similarity through entire sequences including ACD-like domains. Therefore, TryACDP identified in the present study appears to form a kinetoplastid-specific family with an as yet unknown function. The analyses of transcriptomic and proteomic profiles , T. cruzi (CL Brener), and L. major (Friedlin). Synteny and gene neighborhood orientation indicate that ACDps belong to homologous genomic segments. The HSP20 and Tryp23B genes are in the same chromosomal segment in the alleles of both parentals (TcCh36-s and TcCh36-P) of the hybrid T. cruzi CL Brener. In T. brucei and L. major, HSP20 and Tryp23B orthologs are in different chromosomes. Tryp23A, TrySGT1, TryNudC1 and TryNudC2 are all present in syntenic genome segments, each in an individual chromosome. ATOM69, TryACDP and TryDYX1C1 are co-located in a chromosomal segment in T. brucei, but not in L. major and T. cruzi genomes , thus also suggesting the loss of duplicated ACD-like genes by most trypanosomatids. This feature is not conserved in trypanosomes from toads (TCC 339) or frogs (TCC2186 and TCC1307), which are very closely related to that from lizard, all belonging to the basal aquatic clade. More than one copy of ATOM69, Tryp23A and Tryp23B genes were detected in other trypanosomatids, whereas other genes such as HSP20, DYX1C1 and SGT1 rarely present more than one copy , divergences in numbers of ACD genes in a few species is likely the result of gene loss or duplication. Preliminary analyses suggested that the more basal bodonids tend to present additional families and duplicated copies of ACD-like orthologs, suggesting some reduction of copy number during the transition from free-living to parasitic lifestyles. Interestingly, a lizard trypanosome (TCC 878) that belongs to the most basal clade of one copy .TryHSP20, TryATOM69, TrySGT1 and TryDYX1C1  clustered in monophyletic assemblages. The best resolved phylogenetic relationships were obtained using ryDYX1C1 , in agre TryNudC .Trypanosoma, and the other harbored the species of subfamily Leishmaniinae comprising human infective and pathogenic species of Leishmania, and parasites predominantly of sloths of the genus Endotrypanum, all transmitted by sand flies, plus closely related insect trypanosomatids of the genera Zelonia, Crithidia, and Leptomonas. Relatives of Leishmaniinae are the species of Herpetomonas (monoxenous) and Phytomonas (plants and phytophagous hemipterans), and the more distantly related insect parasites of Angomonas and Strigomonas [Trypanosoma (the aquatic clade) comprises trypanosomes from fishes, anurans and lizards (T. brucei (T. cruzi (T. cruzi (pathogenic) and T. rangeli (non-pathogenic), trypanosomes restricted to bats, and some species parasitic in rodents, monkeys, carnivores and marsupials from both the New and Old Worlds. The species nested into the clade T. cruzi are transmitted by triatomine and cimicid bugs [T. theileri (cosmopolitan parasites of ruminants) and T. cyclops (Asian monkeys) [Comparison of phylogenies derived from ACDp, the SSU rRNA or gGAPDH  revealed remarkable congruence on branching patterns, with ACD-like genes supporting two major phylogenetic lineages. One lineage comprises igomonas ,67,68. Iigomonas , the mos lizards , transmi. brucei , constitT. cruzi , compriscid bugs ,73. In amonkeys) ,74, and monkeys) .TryHSP20 and TryNUDC2 acted in a few branches, whereas Tryp23A, Tryp23B, TryATOM69, TryNUDC1 and TryDYX1C1 genes diversified under selection pressure at many branches representing major lineages, genus, and species. Our analyses revealed positive selection (p>0.05) at the Trypanosoma branch in almost all ACDps: Tryp23B, TrySGT1, TryDYX1C1, TryNudC1, TryNudC2, ATOM69 and ACDP. The trypanosomes of the “aquatic” clade have been submitted to strong purifying selection on ATOM69 while this process on Tryp23B and TryACDp affected only the “terrestrial” clade. Therefore, different ACDp genes evolved under strong selective pressure on different branches in Trypanosoma. Species of the Leishmaniinae were affected by selection pressure on Tryp23B, TrySGT1, TryDYX1C1, TryNudC1, ATOM69 and TryACDP. Positive selection on TryNudC2 was exclusively inferred for trypanosomes , and Angomonas/Strigomonas . In contrast, Phytomonas spp. shows no evidence of episodic selection pressure in any ACDp.Trypanosomatid evolution is remarkable affected by events of host switching with life stages in different environments, and a range of vector-transmission strategies. These events lead to drastic changes in selective pressures, which are usually recorded in organism behavior, morphology, and genetics. To assess if specific clades evolved at different rates and could be under different selective pressures, we tested for significant positive diversifying selection on individual branches of the phylogenetic trees inferred for each ACDp. Most ACD-like genes were evidently influenced by positive selection, but some genes were more readily influenced than others. Positive selection of anosomes . HoweverTryp23A and TryNudC1 gene families during the transition from free living to the obligate parasitic lifestyle Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S1 Fig(DOC)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.


This review also sums the crosstalk between these compounds. Besides, those signaling molecules can regulate the expressions of some genes during AR development, including cell division genes, auxin-related genes, and adventitious rooting-related genes. We conclude that these small-molecule compounds enhance adventitious rooting by regulating antioxidant, water balance, and photosynthetic systems as well as affecting transportation and distribution of auxin, and these compounds further conduct positive effects on horticultural plants under environmental stresses. Hence, the effect of these molecules in plant AR formation and development is definitely a hot issue to explore in the horticultural study now and in the future.Adventitious root (AR) is a kind of later root, which derives from stems and leaf petioles of plants. Many different kinds of small signaling molecules can transmit information between cells of multicellular organisms. It has been found that small molecules can be involved in many growth and development processes of plants, including stomatal movement, flowering, fruit ripening and developing, and AR formation. Therefore, this review focuses on discussing the functions and mechanisms of small signaling molecules in the adventitious rooting process. These compounds, such as nitric oxide (NO), hydrogen gas (H The root system of a plant is made up of the primary, lateral, and adventitious roots. The primary root originates during embryogenesis and will later elongate after germination. The AR is initiated and developed post-embryonically from differentiated cells; however, the later root is derived from pericycle cells, which is also defined anatomically as meristems ,2. AR is2S) were regarded as toxic molecule compounds in animals. Gradually, small signaling molecules have proven to be resistant to various abiotic stresses in plants, such as chilling, osmotic stress, drought stress, flooding, salt stress, heavy metal stress , and UV-A irradiation [2, CH4, ethylene (ETH), and H2O2 have been indicated to induce AR formation and development in plants [Many different kinds of molecules transmit information between the cells of multicellular organisms, including small-molecule compounds. These molecules are produced by signaling cells and subsequently bind to receptors in target cells, acting as ligands and chemical signals to coordinate responses and a variety of biological processes both in animals and plants ,9. Over adiation ,7,10. NOadiation ,12. Besiadiation ,13,14,15n plants ,17,18,19AR development is a complicated process and influenced by many abiotic factors such as mineral nutrition , light, temperature, and various biotic factors including ectomycorrhizas and agrobacterium rhizogenes ,20. In a2+, of plant signal transduction in the growth and development of plants, such as seed germination and dormancy, stomatal movement, and photosynthesis [NO plays a crucial role as a second messenger molecule, after Caynthesis . NO alsoynthesis . Tagetes erecta L. ‘Marvel’) [Cucumis sativus L.) [In plants, NO is synthesized through two synthetic pathways: enzymatic and nonenzymatic. The enzymatic pathway includes nitrate reductase (NR) and NO synthase (NOS)-like enzymes. Previous studies of NO showed that NR played crucial roles in the process of physiological activities in NO production . It was Marvel’)  and AR divus L.) , suggestivus L.) ,25. Addiivus L.) . Ample eivus L.) ,27. ThesDendranthema morifolium ‘Beiguozhicun’), and the explants of cucumber and marigold [It has been widely reported that NO may regulate the developmental process of adventitious rooting. Application of NO significantly enhanced AR length and number in the cuttings of ground-cover chrysanthemum  [NO can also regulate AR development through adjusting enzyme activities, such as peroxidase (POD), polyphenol oxidase (PPO), and indoleacetic acid oxidase (IAAO). Furthermore, NO accumulation significantly increased the activity of pro-oxidants including triphosphopyridine nucleotide (NADPH oxidase) and antioxidants such as superoxide dismutase (SOD), catalase (CAT), POD, ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) enzymes in AR development in mountain ginseng (. Meyer) . NO dono. Meyer) . Exogeno. Meyer) ,28. Thesw, SOD, CAT, and APX activities and chlorophyll content in cucumber explants [Oryza sativa L. cv. Komal) seedlings under arsenate (AsV) stress by increasing APX content [NO also regulates AR development under abiotic stresses. Exogenous NO enhanced the root number and root fresh weight in marigold explants under drought stress . And NO explants . Moreove content . Therefo2) is a colorless and tasteless molecule, and hydrogen is one of the most abundant elements in the universe, constituting nearly 75% of the mass of the universe [2 has come to the forefront in therapeutic medical gas research. Recently, the attention on the role of H2 has changed from animals to plants. It has been found that H2 could regulate the growth and development of plants, including seed germination, seedling growth, stomatal closure. and root elongation [2 enhances the tolerance of plants to abiotic stress conditions such as salt stress, osmotic stress, drought, cadmium toxicity, aluminum stress, mercury toxicity, and UV-A irradiation [Hydrogen gas  significantly induced adventitious rooting and enhanced NO content in a time-dependent manner, which reached a maximum at 24 h during the treatment. Also, the application of H2 triggered transition from G1 to S phase in plant cell cycle to enter a new cell cycle in a synchronous manner [Many studies have shown that Hs manner . And HRWs manner . Further2 exerted a positive effect on AR development under stress conditions. Fifty percent HRW significantly induced AR length and number and enhanced the RWC during rooting in cucumber explants under drought stress [2 dramatically increased leaf chlorophyll content, Fv/Fm, ɸPSII, and qP as well as the activities of SOD, POD, CAT, and APX enzymes during drought [2O2), superoxide radical (O2−), thiobarbituric acid reactive substances (TBARS), ascorbic acid (AsA), and reduced glutathione (GSH), as well as relative electrical conductivity (REC), lipoxygenase (LOX) activity, AsA/docosahexaenoic acid (DHA) ratio, and GSH/oxidized glutathione (GSSG) ratio, while increasing DHA and GSSG content. HRW + Cd treatment also significantly increased in the activity and related gene expressions of APX, DHAR, monodehydroascorbate reductase (MDHAR), and GR. Additionally, HRW + Cd treatment increased the contents of osmotic adjustment substances, as well as the activities of POD and PPO, while significantly decreasing IAAO activity [2 induces adventitious rooting under Cd stress by decreasing the oxidative damage, increasing osmotic adjustment substance content, and regulating rooting-related enzyme activity.Also, Ht stress . Also, e drought . Additio drought . Compareactivity . These r2S) is a naturally occurring, colorless, highly soluble, flammable, and toxic gas [2S has been found to be involved in abiotic stresses of plants such as salinity, drought, extreme temperatures, and heavy metals [2S regulated many aspects of plant development, like seed germination and adventitious root induction [2S, 10 μM sodium hydrosulfide (NaHS) significantly induced AR primordia in cucumber explants [Salix matsudana var. tortuosa Vilm) and soybean (Glycine max L.) seedlings [Hydrogen sulfide  in dose- and time-dependent manners [A number of studies showed that CO could trigger AR development. CO treatment induced adventitious rooting in the hypocotyl of mung bean , and ultraviolet radiation [4 on various plant developmental processes. Eighty percent methane-rich water (MRW) dramatically triggered the increase of AR length and number in cucumber [4-induced adventitious rooting in cucumber explants required γ-glutamyl cysteine synthetase (γ-ECS) [4 can be regarded as a crucial inducer during AR process.CH animals . CH4 alsstresses . It was adiation ,46. Howecucumber . CH4-ind (γ-ECS) ,47. ThesArabidopsis  [Arabidopsis mutant (max2 and max4) plants in a dose-dependent manner [ETH is a gaseous plant hormone and involved in many growth and development processes in plants, such as seed germination and dormancy, cell division and expansion, leaf senescence, and fruit ripening ,49. Zimmhaliana) . Howevert manner . The appt manner . 2O2 is viewed mainly as a type of ROS and a signaling messenger. A number of studies reveal that H2O2 is involved in many biological processes of plants, such as stomatal closure [H closure , floweri closure , leaf se closure , cellula closure , lateral closure ,59. 2O2 production, which showed that 3-O-C10-HL is likely to participate in auxin-prompted AR formation by H2O2-dependent cGMP signaling. Li et al. (2007) [2O2 could significantly increase the number of AR, while treatment with 10–50 μM H2O2 significantly increased the fresh weight of AR in cucumber. Moreover, in marigold, the treatment of 200 μM H2O2 significantly induced root length and root number, but its scavenger CAT alleviated its positive effects [2O2 and the synthetic phytohormone indole-3-butyric acid (IBA) may act synergistically to regulate adventitious rooting dependent on the auxin pathway in marigold explants [2O2, suggesting that H2O2 may be the downstream signal molecule in the auxin signaling cascade [2O2 promoted the AR length and number in marigold, and treatment with indole-3-butyric acid (IBA) enhanced the endogenous level of H2O2 in explants [2O2. In ground-cover chrysanthemum, 200 μM H2O2 was the most suitable dosage to promote AR development, at which this dosage increased the activities of PPO, IAAO, and WSC, as well as total nitrogen content, and decreased the total polyphenol content [2O2 could enhance AR development via increasing some enzyme activities, and carbohydrate and nitrogen contents, and inhibiting polyphenol production. According to Bai et al. (2012) , in mung. (2007)  found th effects . It has explants . Exogeno cascade . Also, 2explants , which s content . These r2O2 treatment significantly promoted root length and number in marigold, attenuated the destruction of the mesophyll cell ultrastructure, and increased leaf chlorophyll content, chlorophyll fluorescence parameters , and hypocotyl soluble carbohydrate and protein content, while decreasing starch content of marigold [2O2 on the ultra-microstructure of mesophyll cells improves the photosynthetic performance of blades and lightens the adverse impacts of drought on the build-up of carbohydrates and nitrogen in explants, boosting adventitious rooting. An overview of the effects and mechanisms in plant AR-related studies of small-molecule compounds under no stress and stress conditions are shown in Under drought stress, 600 μM Hmarigold . It implNO and CO are two toxic gases, but they function as second messenger molecules in decreasing vascular tone and inhibiting platelet aggregation by simultaneously inducing cGMP content in animals . The bioIn plants, NO and CO are able to act as two typical downstream signals in auxin-induced adventitious rooting. In mung bean seedlings, addition of the CO donor hematin induced AR formation in dose- and time-dependent manners, similarly with the phenotype induced by NO donor SNP. Furthermore, the NO-specific scavenger cPTIO suppressed the entire NO induced by CO, and the root numbers of the mung bean hypocotyls were decreased by NOS inhibitor L-NAME and hematin treatments when compared to that under hematin treatment alone. However, the inhibitor of HO-1 zinc protoporphyrin IX (ZnPPIX) could not affect the SNP action, suggesting that the CO-induced adventitious rooting process is mediated by the NO/NOS pathway, and NO may act downstream of CO during AR formation in mung bean hypocotyls .heme oxygenase1 (CsHO1) gene was cloned and proved to exhibit HO activity, which was the inducible isoform of heme oxygenase, and it participated in the heme degradation pathway while concomitantly releasing CO. The nitric oxide donor SNP, as well as other AR inducers, could increase the level of the CsHO1 transcript and the corresponding protein, which further suggested the crosstalk between endogenous HO-1/CO and NO during adventitious rooting and their signal transduction pathway [In cucumber, the  pathway . Subsequent research found that application of CO and hemin to cucumber explants induced the production of NO and the NOS-like enzyme inhibitors, and the NO scavenger blocked NO content and AR formation stimulated by CO, but not the NR inhibitors. Simultaneously, researchers found that NO might act as a downstream regulator of CO during adventitious rooting in a cGMP-dependent manner .2 in AR development in cucumber and marigold. The NO scavenger cPTIO, NOS inhibitor NG-nitro-L-Arg methyl ester hydrochloride (l-NAME) or NR inhibitor sodium azide (NaN3), and tungstate could partly reduce the effect of H2-triggered AR in cucumber, which indicated that NO acts as the downstream signaling molecule, and that NOS and NR might be responsible for NO generation during H2-induced AR formation [2 induced AR development through regulating the cell cycle and the activity of root organogenesis-related enzymes POD, IAAO, and PPO [It was reported that NO had a close relation with Hormation . Moreove and PPO .3, suggesting that the positive role of ETH on AR formation was partially NO-dependent [The marigold and cucumber explants treated with ETH and NO together exhibited a better phenotype of AR formation than those treated with ETH or NO alone. These effects of ETH were restrained by cPTIO, L-NAME and NaNependent . It was ependent ,27. In mependent . Togethe4 in cucumber (C. sativus ‘Lufeng’) explants triggered generation of NO and was followed by adventitious rooting, which was similar to the effects of the NO donor SNP and NONOate, suggesting the involvement of NO in CH4-elicited root organogenesis. Further studies illustrated that CH4 induced endogenous NO production through NOS-like enzymes and diamine oxidases (DAOs), and it triggered AR development via controlling expressions of NO-targeted genes [The applied CHed genes . Ipomoea batatas L.), application of the H2S donor NaHS induced ARs in a dose-dependent manner. Treatment of NaHS increased endogenous H2S, IAA, and NO contents, and H2S mediated AR, which was blocked by NPA and cPTIO, This indicated that IAA and NO participate in H2S-induced adventitious rooting, and this regulation mechanism could also be applicable to root organogenesis in willow (S. matsudana var. tortuosa Vilm) and soybean (G. max L.) stem cuttings [In sweet potato , researchers further discovered that CO may be involved in H2-triggered adventitious root formation through adjusting RWC, photosynthesis, and metabolic constituent content and alleviating oxidative damage under drought stress conditions [In alfalfa seedlings, Hstresses . In cucustresses . In cucunditions .2S donor NaHS induced adventitious rooting, and the expression of CsHO-1 as well, and ZnPPIX significantly suppressed the effects induced by NaHS. Furthermore, NaHS regulated similar target genes of HO-1/CO in AR formation, but the H2S scavenger hypotaurine (HT) could not influence HO-1/CO AR formation, which suggested that HO-1/CO acts downstream of H2S during AR formation [In cucumber, the Hormation .2+ pathway in the MRW-induced AR process was certified. Together, CH4 could stimulate adventitious rooting through partially adjusting HO-1/CO and Ca2+ pathways [In cucumber, MRW significantly induced AR formation in IAA-depleted explants, and the inducible affect was still effective in soybean and mung bean explants. Meanwhile, auxin signaling-related genes and cell cycle regulatory genes exerted by MRW during AR formation were blocked by ZnPPIX and further reversed by CO, and the involvement of the Capathways .2S and CH4 mediated adventitious rooting in cucumber explants. Further study demonstrated that CH4 induced endogenous generation of H2S during adventitious rooting, and the H2S scavenger HT restrained the development of adventitious root primordia and gene expressions as well as S-sulfhydration level elicited by CH4, indicating the important role of endogenous H2S in participating in CH4-induced cucumber adventitious root development [Previous results showed that Helopment .2 increased adventitious root mass, ginsenoside contents, and polysaccharides, while CO2 and C2H4 were found to operate opposite impacts as O2 on secondary metabolism in plant cell cultures [During the adventitious root bioreactor culture of ginseng, the enrichment of Ocultures . 2, NO, CO, ETH, and other small-molecule compounds work together by regulating antioxidant, water balance, and photosynthetic systems, and they affect the transportation and distribution of auxin as well as expedite plant adventitious rooting. The mechanisms of the cross-talk between NO and other small signal molecules during plant adventitious rooting are summarized in In summary, Hcyclins  and two CDK (CDKA and CDKB) genes are found to analyze the effect of cell division on AR formation in plants. In cucumber, NO and H2 treatment up-regulated the expression of the cell-cycle regulatory genes CycA, CycB, CDKA, and CDKB, which participate in mediating cell division in internode growth and root meristem induction to boost the process of AR development. This result suggests that both NO and H2 take part in regulating the cell cycle progress to the mitotic phase [CsCDPK1 and CsCDPK5 genes in cucumber explants [4 could also up-regulate cell cycle regulatory genes in cucumber, including CsCDC6, CsCDPK1, CsCDPK5, and CsDNAJ-1 [It is well known that cell cycle regulation genes are often related to the regulation of cell division in plant internode growth. Two ic phase . Also, Nexplants . The appCsDNAJ-1 ,47.CsDNAJ-1 and CsCDPK1/5 genes. However, when NO and ETH were applied alone, the relative expressions of CsDNAJ-1, CsCDPK1, and CsCDPK5 genes were significantly higher than that of any other treatments. The CsDNAJ-1 and CsCDPK1/5 target genes were up-regulated by the application of H2 and HO-1/CO during AR development in cucumber [2S donor NaHS triggered up-regulation of target genes responsible for HO-1/CO-induced cucumber adventitious root formation, including CsDNAJ-1 and CsCDPK1/5 [CsDNAJ-1 and CsCDPK1/5 genes were the possible target genes in the MRW-induced HO-1/CO-mediated cucumber adventitious development [CsHO1 can inhibit antioxidant processes in some abiotic stresses. CsHO1 was found to be involved in cucumber adventitious rooting via NO, H2S, and CO treatments [CsmiR160 and CsmiR167 were increased or decreased, respectively, by the application of MRW, indicating that CH4 may play a positive effect on AR development in cucumber [2, CO, H2S, and CH4 are able to induce high expressions of the CsDNAJ-1, CsCDPK1/5, and CsHO1 genes to mediate adventitious rooting.Furthermore, molecular evidence illustrates that the corresponding genes of DNAJ-like proteins and calcium-dependent protein kinases (CDPKs) are related to the initiation and development of adventitious roots. Xu et al. 2017)  found thcucumber . The H2SsCDPK1/5 . CsDNAJ-elopment . Also, t  found teatments . In addicucumber . TherefoARF6 and ARF8, which were targeted by miR167, had important effects on the regulation of AR development by adding ETH in Arabidopsis; however, ARF17, a target of miR160, was a negative regulator [CsAUX22B-like and CsAUX22D-like were target genes to HRW-induced AR formation [CsAux22D-like and CsAux22B-like in cucumber explants [4 could induce two auxin-signaling genes, such as CsAux22D-like and CsAux22B-like, which are responsible for AR formation in cucumber [Besides, some auxin-response genes are associated with AR formation. Previous studies showed that the auxin response factors ormation . Recentlexplants . Also, Ccucumber . Table 32S, were previously considered as toxic. They recently were assumed to be the gaseous substances to induce AR development. This review is based on a number of studies to show that small-molecule compounds such as NO, H2, H2S, CO, CH4, and ETH can be necessary to trigger adventitious rooting. However, the mechanisms of these compounds in adventitious rooting should still be illustrated and consummated. Many results have reported that some small molecules exert interactions with each other. For instance, H2 is involved in CO-induced AR development, but the concrete pathway is still unclear. Therefore, the combined effects of these compounds can mediate the induction of AR development. Besides, some target genes responsible for adventitious rooting, including CsDNAJ-1 and CsCDPK1/5, could also be regulated by these small molecules.In recent years, there have been increasing studies about the effects on the small signaling molecules of AR formation and development. Some molecules, like CO and HAbove all, these small-molecule compounds are involved in AR development through modifying antioxidant defenses, water balance, photosynthesis, and auxin transportation. In addition, the small compound molecules are widely and easily applied in various plant species and regulate many processes in plants, and they are more efficient and cheaper than some chemicals. Therefore, it is very meaningful to study the influence of small molecules on AR development and formation. However, the mechanism of the promotion of AR development by these small molecules has not been clear. Thus, further research is required to investigate their mechanisms. Finally, the question of whether there any other signaling compounds that can be used to promote AR formation and development should be considered. In the future, related work should be done to improve our knowledge in AR promotion and the future application of signaling molecules to horticultural products.


KIF1A (kinesin family member 1A) gene have been associated with hereditary spastic paraplegia (HSP) type 30 (SPG30), encopassing autosomal dominant and recessive, pure and complicated forms.Pathogenic variants in KIF1A gene.We report the long-term follow-up of a 19 years-old boy first evaluated at 18 months of age because of toe walking and unstable gait with frequent falls. He developed speech delay, mild intellectual disability, a slowly progressive pyramidal syndrome, microcephaly, bilateral optic subatrophy and a sensory axonal polyneuropathy. Brain MRI showed cerebellar atrophy, stable along serial evaluations (last performed at 18 years of age). Targeted NGS sequencing disclosed the de novo c.914C > T missense, likely pathogenic variant on KIF1A variant associated with slowly progressive complicated SPG30 and stable cerebellar atrophy on long-term follow-up, adding to current knowledge on this HSP subtype.We report on a previously unpublished de novo heterozygous likely pathogenic  Hereditary spastic paraplegias (HSP) are a group of clinically and genetically heterogeneous neurodegenerative disorders featuring progressive loss of corticospinal motor tract function, resulting in spasticity predominantly in the lower limbs, which can be isolated (pure forms) or associated with additional neurological or non-neurological features (complex forms) , 2.KIF1A were first associated with two neurodegenerative diseases in 2011: hereditary sensory and autonomic neuropathy (HSAN) type 2 [KIF1A pathogenic variants cause SPG30 [KIF1A pathogenic variants. A third phenotype  (OMIM #614255), initially thought to represent a distinct disease, has been later demonstrated to be also caused by KIF1A pathogenic variants.Pathogenic variants in ) type 2  and a fo) type 2 . One yease SPG30 , and in se SPG30  and AD [se SPG30 , 8, purese SPG30 , 9 formsDescription of long-term follow-up data on patients with rare neurogenetic conditions can be crucial to increase our knowledge on the natural history of the disease and to correctly disentangle it from phenotypic heterogeneity .We present the clinical, neuroimaging and genetic data on a patient recently diagnosed with SPG30 with long-term neurologic follow-up, ranging from infancy to early adulthood.A male patient, now aged 19 years, was born at term after uneventful pregnancy and delivery , from healthy non-consanguineous Caucasian parents. He has an older sister reportedly toe walking in the first 2 years of life. His early developmental milestones were normally achieved . He made his first steps at 14 months, however, concerns arose regarding toe walking and unstable gait with frequent falls, and he was first referred to child neurology evaluation at 18 months of age. Pyramidal signs at his lower limbs were detected. Subsequently, he developed speech delay, learning difficulties, and mild intellectual disability. During follow-up, microcephaly, bilateral optic subatrophy, hyperlordosis and asymmetric equino-valgo-pronated feet also became evident. Moreover, his spastic paraparesis has shown a slowly progressive course, with increasing difficulties in independent walking and a worsening in his hypertonus in the lower limbs. No quantitative scale was administered during follow-up.At the last follow-up, he is slightly microcephalic . He shows toe walking with a broad-based gait and valgo-pronated feet.During this long follow-up, he underwent extended diagnostic investigations. Brain MRI, first performed at 18 months of age, documented superior vermis atrophy, and reduced volume of the optic nerves and optic chiasm, with no significant progression (last examined at 18 years of age) .LMLN gene) and arr4q22.1 x3mat (including the GRID2 gene).Negative neurometabolic and neuromuscular investigations include alpha-fetoprotein, amino acids, beta-exosaminidase, muscle biopsy with immuno-hystochemistry, and respiratory chain enzimes activity. Neurogenetically-wise, direct sequencing of POLG, Twinkle, PLA2G6, OPA1, OPA3, SPG20, and SCA5 retrieved negative results, while two known polymorphisms were detected on the exons 11 and 15 of the SPG7 gene. Array-CGH detected two partial duplications, both of maternal origin: arr3q29 x3mat  on The variant we identified is a previously unpublished in the literature, missense, non-tolerated variant, absent in controls. It has been previously reported in ClinVar in four cases, in two in association with mental retardation, autosomal dominant 9 (OMIM #614255), as a de novo variant, interepreted as likely pathogenic in the first patient and as pathogenic in the second one. Of note, the third and fourth cases report it as pathogenic, and in one the associated phenotype is characterized by developmental delay, cerebellar atrophy, ataxia, and eye movement abnormalities (patient tested at GeneDx). The variant is located inside the motor domain of kinesin 1A, in the N terminus of the protein (aa 1–365), where the vast majority of pathogenic variants associated with both AR and AD SPG30 are clustered. This domain is necessary for kinesin 1A movement along neurites and for binding with ATP and microtubules . The p.PThe variant is classified as likely pathogenic according to ACMG criteria : PM1: LoThe clinical picture associated with complicated forms of SPG30 typically features intellectual disability, along with cerebellar atrophy and spastic paraplegia, with very wide range of onset. In some cases, optic nerve atrophy, thinning of the corpus callosum, periventricular white matter lesions, epilepsy and microcephaly (in two literature patients as well as the present one) , have alClinically, in contrast with the first descriptions of heterozygous cases, initially reported to be associated to both younger age at onset and more severe phenotypes , our patIn summary, with long-term follow-up, this case adds to current knowledge on phenotypic and genotypic variability in complex AD SPG30.


Fatigue is a common, distressing, and persistent symptom for patients with malignant tumor including colorectal cancer (CRC). Although studies of cancer-related fatigue (CRF) have sprung out in recent years, the pathophysiological mechanisms that induce CRF remain unclear, and effective therapeutic interventions have yet to be established. To investigate the effect of the traditional Chinese medicine YangZheng XiaoJi (YZXJ) on CRF, we constructed orthotopic colon cancer mice, randomly divided into YZXJ group and control (NS) group. Physical or mental fatigue was respectively assessed by swimming exhaustion time or suspension tail resting time. At the end of the experiment, serum was collected to measure the expression level of inflammatory factors by ELISA and feces to microbiota changes by 16s rDNA, and hepatic glycogen content was detected via the anthrone method. P < 0.05) s, respectively; the suspension tail resting time of YZXJ group was shorter than that of the control group (49.85 ± 4.56) s and (68.83 ± 7.26) s, P < 0.05)). Serum levels of IL-1β and IL-6 in YZXJ group were significantly lower than the control group (P < 0.05). Liver glycogen in YZXJ group was (5.18 ± 3.11) mg/g liver tissue, which was significantly higher than that in control group (2.95 ± 2.06) mg/g liver tissue (P < 0.05). At phylum level, increased abundance of Bacteroidetes, Verrucomicrobia, Actinobacteria, and Cyanobacteria and decreased Proteobacteria in YZXJ group emerged as the top differences between the two groups, and the Firmicutes/Bacteroidetes ratio was decreased in YZXJ group compared to the control group. At genus level, the abundance of Parabacteroides, unidentified Saprospiraceae, and Elizabethkingia which all belong to phylum Bacteroidetes were increased, while Arcobacter, Marinobacter, Alkanindiges, Sulfuricurvum, Haliangium, and Thiobacillus in phylum Proteobacteria were decreased after YZXJ intervention. YZXJ can also increase Pirellula, Microbacterium, and Alpinimonas and decrease Rubrobacter and Iamia. The nutritional status of the YZXJ group was better than that of the control group, and there was no statistical difference in tumor weight. The swimming exhaustion times of YZXJ group and control group were (162.80 ± 14.67) s and (117.60 ± 13.42, YZXJ may reduce the physical and mental fatigue caused by colorectal cancer by inhibiting inflammatory reaction, promoting hepatic glycogen synthesis, and changing the composition of intestinal microbiota. Cancer-related fatigue (CRF) is one of the common, suffering and persistent symptoms of cancer patients, often accompanied by the entire course of malignancy. The prevalence of CRF may vary with an estimate of 60%–96% in malignant tumor patients who are undergoing treatment and experiencing fatigue , 2. Unliβ in peripheral blood activate the vagus nerve, which in turn leads to fatigue . This suggested that the energy and mental state of mice with orthotopic colon cancer were better in those taking YZXJ.α/β, IL-2, IL-6, IL-10, TNF-α, IFN-γ. In expectation, fatigue-related inflammatory factors in YZXJ group were generally lower than NS group, particularly IL-1β and IL-6. The level of IL-1β in YZXJ group was significantly lower than NS group . The IL-6 level in YZXJ group was (92.40 ± 71.43) pg/ml, and NS group was (229.44 ± 117.54) pg/ml (P = 0.002)  , Table 1P = 0.04, To investigate the effect of YZXJ on hepatic glucose metabolism, we detected liver glycogen. The results showed that the level of hepatic glycogen in NS group was (2.95 ± 2.06) mg/g, which increased to (5.18 ± 3.11) mg/g in YZXJ group  triggering metabolic disorders, as a result of insufficient energy supply to cells; (2) increasing energy consumption of immune system, resulting in impaired immune function; and (3) causing neuroendocrine dysfunction, neurotransmitter metabolism disorder, or abnormal neuronal formation [At present, the most accepted view on the pathogenesis of CRF is inflammatory response. We found that YZXJ can reduce various inflammatory factors in mice blood, with the most significant decrease in IL-1ormation , 38. Stuormation . Certainormation , 41. Howβ induced by LPS and P-JNK signaling pathway activation [κB pathway that reduces the generation of proinflammatory cytokines like IL-1β and IL-6 [α or IL-1β through NF-κB, ERK, JNK, and other pathways to regulate immune function and reduce intestinal inflammation [YZXJ contains ingredients with heat-clearing and detoxifying properties such as Scutellaria barbata, Bittersweet herb, and Herba artemisiae scopariae. Scutellaria barbata, a traditional anticancer Chinese medicine, has been found to have the additional effect of anti-inflammation and anti-infection in recent years. Diterpenoid alkaloids and 6-methoxynaringenin in the Scutellaria barbata extracts had the strongest effect on downregulating the secretion of NO, PE2, IL-6, IL-1tivation –44. Moreand IL-6 –47. Otheammation , 49. In ammation . Furtherammation .Except for the inflammation, oxidative stress, energy metabolism, and abnormal hormone levels are also important mechanisms for the development of CRF, especially physical fatigue. Emerging evidence demonstrated that persistent fatigue occurs when the body kinetic energy consumption exceeds the cells energy supply capacity. Physical fatigue is almost accompanied by metabolic disorders, mainly liver glycogen and muscle glycogen metabolism . In fact Fusobacterium, Bacteroides fragilis (Prevotella), Shigella, Helicobacter, etc. can promote inflammation, conversely, Clostridium, especially Faecalibacterium prausnitzii, can inhibit the inflammatory response. Meanwhile, the proinflammatory intestinal flora mentioned above can cause fatigue, whereas Clostridium, Bifidobacterium, and Faecalibacterium can alleviate fatigue. In view of these above, people are trying to treat CRF from the perspective of regulating intestinal microbiota.Flora imbalance is involved in the development of CRC by affecting inflammatory response or immune status; on the other hand, it can induce fatigue through the inflammatory or metabolic pathways. Among the numerous bacteria associated with CRC, Firmicutes/Bacteroidetes ratio, which was identical with those reported in literature. Studies have shown that the ratio of Firmicutes/Bacteroidetes is related to fatigue, and the higher the ratio, the severer the fatigue, and vice versa [ Parabacteroides, unidentified Saprospiraceae, and Elizabethkingia in Bacteroidetes were increased and Arcobacter, Marinobacter, Alkanindiges, Sulfuricurvum, Haliangium, and Thiobacillus belonging to Proteobacteria were decreased in YZXJ group. A large amount of data confirmed that Bacteroides can promote the metabolism of ginsenoside that indirectly improves the state of physical fatigue [ Proteobacteria mainly include Helicobacter and Shigella, both of them can promote the occurrence of CRC by inducing inflammatory reaction and also play an important role in causing CRF [ Proteobacteria can affect the deglycosylated metabolism of panax notoginseng saponins via regulating the activities of glycosidases, while upregulation of Bacteroidetes may promote the redox metabolism of this drug through improving related enzymes activities in intestine [In order to study the effect of YZXJ on intestinal microbiota, we used a method of colon cancer orthotopic transplantation in BALB/c mice to restore the growth environment of colon cancer as much as possible. We found that the YZXJ group had a decrease in thece versa –63. At g fatigue , 65. Prosing CRF –68. Accontestine . Streptococcus [ Streptococcus [There is still lack of systematic study in depth on the elevated effects of YZXJ on certain beneficial intestinal bacteria. However, it can be seen that some components of YZXJ have an antimicrobial effect. Oleanolic acid and ursolic acid, the main components of YZXJ, extracted from Fructus ligustri lucidi, can inhibit the transcription of genes related to peptidoglycan biosynthesis, thereby preventing bacterial growth which manifested a strong lethality ontococcus . Other itococcus . The acttococcus . ChangesThis study did not perform metabolomics analysis and in vitro experiments that could not fully elucidate the exact mechanism of YZXJ on alleviating CRF. Nevertheless, this study has clarified that YZXJ was obviously effective in improving physical and mental fatigue in mice with orthotopic colorectal cancer, from which we almost can speculate that this effect is exerted by altering intestinal microbiota, reducing inflammation, and improving metabolism, which hence shows good prospects for application.


Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate  The highly divergent outer membrane translocase (TOM) from the Trichomonas hydrogenosome (an organelle related to mitochondria) is composed of conserved core and lineage-specific subunits, and has an unconventional skull-like triplet-pore structure. Trichomonas vaginalis, is composed of highly divergent core subunits and lineage-specific peripheral subunits. Despite the evolutionary distance, the T. vaginalis TOM (TvTOM) complex has a conserved triplet-pore structure but with a unique skull-like shape suggesting that the TOM in the early mitochondrion could have formed three pores. Our results contribute to a better understanding of the evolution and adaptation of protein import machinery in anaerobic forms of mitochondria.Mitochondria carry out many vital functions in the eukaryotic cells, from energy metabolism to programmed cell death. These organelles descended from bacterial endosymbionts, and during their evolution, the cell established a mechanism to transport nuclear-encoded proteins into mitochondria. Embedded in the mitochondrial outer membrane is a molecular machine, known as the translocase of the outer membrane (TOM) complex, that plays a key role in protein import and biogenesis of the organelle. Here, we provide evidence that the TOM complex of hydrogenosomes, a metabolically specialised anaerobic form of mitochondria in  Mitochondria originated from proteobacterial endosymbionts , and oveTrypanosoma brucei, the TOM complex (named the archaic translocase of the outer membrane [ATOM]) has only two orthologues, a highly divergent Tom40 (ATOM40) and a Tom22-like protein (ATOM14) )  . To subsle media . Such anle media . The groeratures . Thus, iTo identify interaction partners for TvTom40-2, we performed co-immunoprecipitations (coIPs) of HA-tagged TvTom40-2 under crosslinking and native conditions, and the eluted proteins were analysed using label-free quantitative MS (LFQ-MS). CoIPs using anti-HA antibody were performed with hydrogenosomes isolated from both the strain expressing HA-tagged TvTom40-2 and the wild-type (WT) strain, used as a negative control. The analysis revealed that 50 and 36 proteins were enriched with HA-tagged TvTom40-2 under crosslinking and native conditions, respectively . As TOM T. brucei -methionine. Under standard in vitro import conditions, using hydrogenosomes isolated from the double-transfected TvTom40-2-HA/Tom36-V5 strain, TvFdx1-DHFR was imported into hydrogenosomes, which was confirmed by a protease protection assay. The autoradiograph showed a time-dependent import of TvFdx1-DHFR , which has an NTS fused to dx1-DHFR . Next, idx1-DHFR . Finallydx1-DHFR . The twodx1-DHFR . No subsdx1-DHFR . These rE. coli BL21 (DE3) strain, respectively, and coupled with Ni-nitrilotriacetic acid (Ni-NTA) agarose beads  synthetase (αSCS), with the latter fused to DHFR at the C-terminus (αSCS-DHFR), were incubated with Tom36cd-His or Tom46cd-His coupled with or mock-treated beads for 1 hour. Then, the His-tagged proteins with the bound substrates were eluted with imidazole. The eluate from the Tom36cd-His and Tom46cd-His binding assay showed the presence of two radiolabeled proteins, frataxin and αSCS-DHFR  and Tom46  were expressed with a C-terminal polyhistidine (His) tag in se beads . Beads pT. vaginalis expressing TvTom40-2-HA under native conditions. The isolated hydrogenosomes were solubilised with digitonin to release the complex, and then the TvTOM complex was purified by IP using α-HA antibody coupled to Dynabeads and negatively stained for electron microscopy. The identity of the HA-tagged TvTom40-2 in the IP eluate was verified by immunoblotting and silver staining  and 40% , respectively, while TvTOM with three pores accounted for 25% (n = 860). The single-pore particles were oval, 70 × 125 Å in size with an eccentric pore placement. Two-pore particles were oval or triangular and 140 × 100 Å in size. The particles with three pores were skull-shaped and measured 150 × 175 Å in size, although a fourth spot of stain accumulation with a low contrast was observed in one of the class averages , in kinetoplastids, and interestingly, in a fungus Neocallimastix californiae  suggest a peculiar evolutionary history for TvTOM complex. Therefore, we searched for orthologues of TOM components using a local HMM in selected genomes across different eukaryotic supergroups, with a focus on Excavata to estimate the conservation, gain, and loss of components . For our. brucei ,35, althmponents   nonredundant protein database regardless of the domain composition that resulted in a data set of 299 eukaryotic, 810 bacterial, and 5 archaeal sequences that were analysed using CLuster ANalysis of Sequences (CLANS) algorithm  Fig 11A. Tom36 amologues .S. cerevisiae and N. crassa , 50 mM potassium acetate, 10% glycerol, and EDTA-free cOmplete protease inhibitor cocktail [Roche]) containing 1% digitonin (Merck Millipore), and the clarified extract was incubated with Dynabeads (Thermo Fisher Scientific) coupled with α-HA antibody for 90 minutes on an overhead rotator at room temperature. The beads were washed thrice before elution with either SDS-PAGE buffer for crosslinking coIPs or elution buffer  for native coIPs. The coupling of α-HA antibody to the Dynabeads was performed according to the manufacturer’s instructions.T. vaginalis database , the quantifications were performed with the label-free algorithms, and the data analysis was performed using Perseus 1.5.2.4 software. The MS data have been deposited to the ProteomeXchange consortium via the PRIDE  methionine (MGP spol sro) according to the manufacturer’s instructions (NEB PURExpress in vitro protein synthesis kit). Cytoplasmic extract was prepared from the T. vaginalis strain T1 as described elsewhere  methionine as described earlier. The recombinant His-tagged proteins were expressed in E. coli BL21 (DE3) strain at 37 °C for 3 hours following the induction with 0.5 mM IPTG. The cells from a 10 mL culture of E. coli (negative control) and strains expressing His-tagged proteins were harvested, resuspended in 4.5 mL lysis buffer , incubated on ice for 45 minutes, and lysed using QSonica sonicator. The homogenised extract was clarified at 9,000 rcf for 30 minutes at 4 °C. Aliquots of supernatant and pellet were used for immunoblotting to test the solubility of the proteins. The supernatant obtained was split into three equal parts and was incubated with 50 μL of Ni-NTA agarose resin (Qiagen) on an overhead rotator for 2 hours at room temperature. The resin collected was washed five times using 10 volumes of wash buffer . To block, the beads were washed thrice with wash buffer II . To the mock-treated beads or beads bound with His-tagged protein, binding buffer , 50 μL of Trichomonas cytosolic extract, and 10 μL of radiolabeled precursors were added and incubated for 1 hour at 37 °C with gentle shaking. The beads were washed three times with the binding buffer, and the proteins were eluted with the elution buffer . The samples were electrophoresed, and the gel was vacuum dried. The gel was exposed for 4 to 5 days prior to phosphorimaging with Typhoon TLA 7000. The oligonucleotides used for cloning are listed in The gene encoding for the cytosolic domain of Tom36 and Tom46 (Tom36cd and Tom46cd) were cloned into pET42b vector tagged to polyhistidine at the C-terminus. The genes encoding for cytochrome b5 (TVAG_063210), frataxin (TVAG_182610), and αSCS  were subcloned into NEB PURExpress control plasmid, and the radiolabeled precursors were synthesised in the presence of L-[E. coli BL21 (DE3) strain following an induction with 1 mM IPTG, and the His-tagged Sam50 was purified using affinity chromatography under denaturing conditions. The purified antigen was separated via SDS-PAGE, and the Coomassie-stained band was used to generate polyclonal antibody in rat.The gene encoding Sam50 was cloned into pET42b fused to a C-terminal His tag. The protein was expressed in S1 FigT. vaginalis TOM; VDAC, voltage-dependent anion channel.The conserved residues of the β-motif, PxGxxHxH, are highlighted: P is polar (fluorescent green), x is any amino acid, G is glycine (fluorescent yellow), and H is hydrophobic (turquoise). All TvTom40 isoforms have the conserved β-motif except TvTom40-3, where the last hydrophobic residue has been replaced by serine. TOM, translocase of the outer membrane; TvTOM, (PDF)Click here for additional data file.S2 FigS. cerevisiae. The selected 21 sequences were chosen out of the multiple alignment of 140 sequences to demonstrate the potential conservation of key residues and to highlight the differences between VDAC and Tom40 proteins. TOM, translocase of the outer membrane; TvTOM, T. vaginalis TOM; VDAC, voltage-dependent anion channel.Highlighted residues mark anchoring positions for possible interactions between the Tom40 β-barrel and essential subunits of the TOM complex in (PDF)Click here for additional data file.S3 FigT. vaginalis, S. cerevisiae, Arabidopsis thaliana, Rattus rattus, Mus musculus, Homo sapiens, and Bos taurus. The TMD is marked by a box, and the conserved residues are highlighted—tryptophan (yellow), hydroxylated residues (turquoise), and proline (green). TMD, transmembrane domain; Tom, translocase of the outer membrane.Names of the organisms are as follows: (PDF)Click here for additional data file.S4 FigE. coli strain expressing Tom36cd-His (panel A) and Tom46cd-His (panel B), respectively, before (Control) and 1 hour and 3 hours after induction with 0.5 mM IPTG. IPTG, Isopropyl β-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulphate-PAGE; Tom, translocase of the outer membrane. Expression of Tom36cd-His and Tom46cd-His. SDS-PAGE gels stained with Coomassie and immunoblots probed with α-His antibody for the whole cell lysate from a 250 μL culture of (TIF)Click here for additional data file.S5 FigTrichomonas strain expressing HA-tagged TvTom40-2. Two bands marked were identified by MS as TvTom40-2. The common contaminant was identified as Cpn60. (C) Purified TvTOM complexes were applied on EM grids and negatively stained with phosphotungstic acid. Electron micrograph of negatively stained TvTOM complexes recorded at a magnification of 78,000×. Scale bar, 40 nm. Bottom panel: magnified view of selected particles with three, two, and one pore(s) (left to right). Scale bar, 10 nm. EM, electron microscopy; HA, human influenza hemagglutinin; In, Input; IP, immunoprecipitation; MS, mass spectrometry; TOM, translocase of the outer membrane; TvTOM, T. vaginalis TOM; WT, wild-type. Preparation of purified TvTOM for EM analysis. (A) Immunoblot of digitonin-lysed extract of hydrogenosomes  and the eluate  from TvTom40-2-HA IP under native conditions using α-HA antibody. (B) Silver stained-gel showing the α-HA IP eluates from TvT1 WT strain and (PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S1 TableT. vaginalis TOM.NCBI, National Center for Biotechnology Information; TOM, translocase of the outer membrane; TvTom, (PDF)Click here for additional data file.S2 TableT. vaginalis TOM.HMM, hidden Markov model; PDB, Protein Data Bank; TOM, translocase of the outer membrane; TvTOM, (PDF)Click here for additional data file.S3 TableTOM, translocase of the outer membrane.(XLSX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 DataHMM, hidden Markov model; TOM, translocase of the outer membrane.(TXT)Click here for additional data file.S2 DataThe data sets shown were obtained were four independent coIP experiments indicated by columns A, B, C, and D. A protein was considered enriched either if the protein was present only in the test sample and absent in the control or if the protein was enriched by a fold change of >1 in the test sample. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource.(XLSX)Click here for additional data file.S3 DataTOM, translocase of the outer membrane.(FASTA)Click here for additional data file.S4 DataATOM, archaic translocase of the outer membrane; CLANS, cluster analysis of sequences; TOM, translocase of the outer membrane.(FASTA)Click here for additional data file.S5 DataCLANS, cluster analysis of sequences; TPR, tetratricopeptide repeat.(FASTA)Click here for additional data file.S6 DataHMM, hidden Markov model; Tom, translocase of the outer membrane.(TXT)Click here for additional data file.S7 DataHMM, hidden Markov model; Tom, translocase of the outer membrane.(TXT)Click here for additional data file.S8 DataEntamoeba sp., and Tom36 homologues from parabasalids using MAFFT; Tom40 and VDAC (Porin_3) homologues, fungal Tom5, metazoan Tom5, plant Tom5, metazoan Tom6, fungal Tom6, Tom7, Tom20, plant Tom20, and Tom22 homologues from the Pfam database; plant Tom6 homologues from the Eggnog database; and Tom70 homologues from the COG database. ATOM, archaic TOM; COG, clusters of orthologous groups; MAFFT, multiple sequence alignment based on fast Fourier transform; Pfam, Protein families; Tom, translocase of the outer membrane.Alignments of ATOM11, ATOM12, ATOM46, and ATOM69 homologues from kinetoplastids, Tom60 homologues from (TXT)Click here for additional data file.S9 DataT. vaginalis TOM; VDAC, voltage-dependent anion channel.Tom, translocase of the outer membrane; TvTom, (TXT)Click here for additional data file.


The role of HCN channels in pain has been widely investigated as targets for the development of new therapeutic drugs, but the comprehensive design of HCN channel modulators has been restricted due to the lack of crystallographic data. The three-dimensional structure of the human HCN1 channel was recently reported, opening new possibilities for the rational design of highly-selective HCN modulators. In this review, we discuss the structural and functional properties of HCN channels, their pharmacological inhibitors, and the potential strategies for designing new drugs to block the HCN channel function associated with pain perception.Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are highly regulated proteins which respond to different cellular stimuli. The HCN currents (I V10–KV12 channels [h is similar for both cyclic nucleotides, with the same efficacy at least in mammalians, the apparent affinities of Ih are 10–100 fold higher for cAMP than for cGMP [Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated pore loop channel superfamily ,2,3, andchannels   located at C-terminal. This binding leads to accelerated activation kinetics and to a shift of the conductance voltage curve toward positive voltages up to 20 mV) ,2,3,5. A0 mV ,2,3Moreover, in HCN channels the voltage dependence goes in opposite directions to the classical voltage-dependent ion channels, which opens with a depolarized stimulus. Hyperpolarization-activated cyclic nucleotide-gated channels are closed to a depolarized stimulus and opened to the membrane hyperpolarization ,2,3.In mammals, four HCN isoforms have been identified to encode for the subunits HCN1 to HCN4 . To form+ ions observed in K+ channels [+/K+ permeability ratio in HCN channels is ~1/4 and also displays a small but significant permeability to Ca2+ ions [2+, the Ca2+ current in the native HCN current (Ih) as well as in the expression system, where channels HCN2 and HCN4 are expressed, is about 0.5% [2+ current of HCN channels is relatively small (0.47% in HCN2 and 0.6% in HCN4 channels) compared with the fractional Ca2+ currents in other ion channels, such as the nicotinic acetylcholine receptor (2.5%) [N-methyl-d-aspartic acid) [2+ current through HCN channels may be enough to modulate Ca2+-dependent cellular functions [+ binding sites (denoted as 1–4 from the extracellular to the intracellular side), adopts a non-canonical configuration [+ ion occupies the 3 and 4 sites (+-selective filter (HCN1 channel) and a K+-selective filter  where all sites are occupied by K+  motif that confers the high selectivity for Kchannels . Howevera2+ ions . For insout 0.5% ,24. Thisr (2.5%) , glutamaic acid) , AMPA, αic acid) , CNG chaic acid) , and calic acid) . Howeverunctions ,24. Lee  4 sites C, left. ed by K+ C, right.A cyclic nucleotide-binding domain (CNBD) is found at the intracellular C-terminus of HCN channels. The CNBD, connected to the S6 transmembrane domain by a C-linker, regulates the HCN channel activity by facilitating the cyclic adenosine monophosphate (cAMP) binding ,31 FiguB,D promoh)—a Na+/K+ current when the membrane potential is hyperpolarized, producing rhythmic electrical activity in specialized neurons of the brain [h currents, including the determination of resting membrane potential (RMP), action potential (AP) firing rate, dendritic integration, and synaptic transmission [The HCN channels are widely expressed in peripheral sensory neurons, neurons in the central nervous system , and carhe brain ,41,42 anhe brain . Diversesmission .Hyperpolarization-activated cyclic nucleotide-gated channel activity plays important roles in behavior and physiological process such as sleep and arousal, learning and memory, and anesthesia ,45,46,47The cyclic nucleotide adenosine-3′,5′-cyclic monophosphate regulates the voltage dependence of HCN subunits ,10. In c+, and Cl− ions [2+/calmodulin-dependent protein kinase II) [In addition to cAMP, HCN channels are also allosterically regulated by other molecules, such as phosphatidylinositol 4,5-biphosphate, cholesterol, HCl− ions , and modnase II) ,55,56,57nase II) ,59. Alsonase II) ,61,62. NAs previously mentioned, cholesterol within lipid rafts can disrupt the HCN channel function since the HCN1, HCN2, and HCN4 subunits expression were downregulated after cholesterol depletion .h) and can inhibit the passive propagation of excitatory post-synaptic potentials [f) or pacemaker currents in cardiac cells [All HCN members (HCN1–4) have been found in central and peripheral nervous systems, where they are associated with synaptic integration, neuronal excitability, and the formation of resting membrane potentials . Neuronstentials ,65,66,67ac cells ,41,68,69ac cells ,41,42,68ac cells ,42. The ac cells . In the ac cells ,42. In cac cells , whereasac cells ,42.Immunochemistry studies have shown the localization of various HCN channels in different tissues ,71. The Although the hyperpolarization of the membrane potential activates HCN channels expressed in the central nervous system tissues, a few channels are activated by resting membrane potentials (between −50 and −60 mV). The activation of HCN channels, by resting membrane potentials, brings them closer to the threshold which regulates the action potential ,72. Giveh can induce the repetitive firing of primary nociceptive neurons [Pain affects millions of individuals worldwide . Chronic neurons ,84,85. I neurons ,77,83, a neurons ,81,82. I neurons . AdditioDiabetic patients develop a painful diabetic neuropathy (PDN)—a chronic pain condition induced by nerve damage . The mol+ channel mRNA in mouse DRGs, with an increase in HCN1 subunit and a down regulation of TREK1, TREK2 (TWIK-Related K+ Channel), TRAAK (TWIK-related arachidonic acid-stimulated K+ channel), and KV1.1 in nociceptors [V1.2, KV11.3, Kir3.1, and HCN1 channels along with reductions in Kir1.1, Kir3.4, and K2P1.1 channels [Even though distinct chemotherapeutic drugs have different modes of action, several drugs can also cause chronic pain. Three of this main chemotherapeutic classes, taxanes, platinum-based agents, and vinca alkaloids are commonly associated with chemotherapy-induced peripheral neuropathy (CIPN) . Althougiceptors ,90. Resticeptors  recentlyiceptors , abolishiceptors ,89. Studchannels . These cchannels . HCN1 chchannels .Given the importance of HCN channels in the pain process ,82,84,94h current were also observed in the periaquaductal gray of CCI rats, whereas infusion of ZD-7288 into this brain region attenuated neuropathic pain [Nociceptive pain involves the transduction, conduction, transmission, modulation, and perception of noxious signals to the brain , which ahic pain ,105. Morhic pain  or CCI [hic pain . ZD-7288hic pain . Thus, t50 of ~4 µM for HCN1, HCN2, and HCN4 subunits [Additionally, further studies where the HCN channel inhibitors ivabradine and gabapentin were used , was tested in a spared nerve injury (SNI) model [12m, for treatment of neuropathic pain were displayed [Other inhibitors have a greater affinity for specific HCN channels over others. The structural analogs related to zatebradine EC18 and MEL57A displayed selectivity for homomeric HCN channel isoforms ,113. AmoI) model . The conisplayed .Amino acid residues involved in the binding of drugs to HCN channels may also be different. Unlike ivabradine, which is predicted to bind to amino acid residues Y386, F389, and V390 of the HCN1 subunit, ZD-7288 binds to residues A425 and I432 located in the S6 transmembrane domain of the HCN2 subunit . NeverthN-methyl-d-aspartic acid, glycine receptors, and K2P and HCN channels [2P channels as well as gamma-aminobutyric acid (GABA), NMDA and glycine receptors. On the other hand, a wide range of volatile anesthetics  potentiation, and general anesthesia. Thus, 2,6- and 2,4-di-tert-butylphenol  were shown to be more potent HCN1 antagonists than propofol while 2,6- and 2,4-di-sec-butylphenol  were less potent [sec-butylphenols), but not DTBPs (di-tert-butylphenols), enhance GABAA receptor activity and are general anesthetics. Also, in a neuropathic pain model, 2,6-DTBP and subhypnotic propofol were antihyperalgesic [A receptor, and suggest that central HCN1 channel antagonism may be of limited importance to general anesthesia [Regarding anesthetic drugs, there is compelling evidence supporting the involvement of ion channels and receptors, such as type A gamma-aminobutyric acid, channels ,127,128.alothane ,132,133,alothane ), intravalothane , ketaminalothane ,137, andalothane ,47,138) alothane ,140 and alothane ,142,143)alothane , and thealothane ,138. Conalothane  hypothess potent . In contralgesic . These festhesia . Alkylphesthesia . Thus, testhesia .h current as well as the current generated by HCN1 and HCN2 channels heterologously expressed [−/− mice and HCN1f/f,cre mice (with a HCN1 deletion restricted to the forebrain). These tests confirm a role for volatile anesthetic-mediated inhibition of HCN1 when contributing to hypnosis, and likely amnesia but not immobility [The volatile anesthetics  have previously been shown to inhibit the Ixpressed ,132,133.xpressed  tested tmobility .h in hippocampal pyramidal neurons and cortical pyramidal neurons [For instance, the intravenous anesthetic drug propofol  inhibits neurons  by block neurons  subunit. neurons .Ketamine, a drug with anesthetic, analgesic, and psychotropic effects, also inhibits the HCN1 subunit in cortical pyramidal neurons , and itsh in cortical neurons mediated by monomeric and heteromeric HCN2 and HCN1 channels [h in motoneurons and cortical pyramidal neurons [Volatile anesthetic drugs, such as the commonly used isoflurane and halothane, decrease Ichannels ,133. IsoRational drug design targeting ion channels with specific properties is a constantly evolving research field. The design offers revolutionary advantages for the development of new therapeutic drugs through the use of newly developed methods such as computational tools and structure-based approaches, where the structural, physical-chemical, and pharmacological properties of the ligands and targets are critical to gain high potency and binding affinity.Nowadays, for drugs whose molecular target is an ion channel with a known 3D structure, experimental and theoretical approaches are used to study protein-ligand interactions. Structure-based drug design (SBDD) is most powerful when it is a part of an entire drug discovery process. This is a handy and versatile computational tool that examine the 3D structure of a given target  spectroscopy, cryoelectron microscopy, or any other molecular modeling technique ), followThe cDNA cloning and partial characterization of different HCN subunits have set the pace for their extensive study using biochemical, biophysical, genetic, and cellular approaches. HCN subunits are highly regulated proteins which respond to different stimuli.Hyperpolarization-activated cyclic nucleotide-gated channels are widely distributed in both excitable (and non-excitable) cells of the nervous system and the heart. Their expression patterns in different tissue and cells are affected by different pathological conditions. These channels transmit electrical signals in excitable cells such as neurons; for instance, they control heart rate and also inhibit pain ,110. DesTherefore, the 3D structure of the human HCN1 subunit  has open


Innate lymphoid cells (ILCs) comprise a recently identified subset of innate immune cells that are mainly localized to mucosa-associated tissues. Although they have not yet been fully characterized, they can generally be divided into ILC1s, ILC2s, and ILC3s. ILCs and their corresponding cytokines act as important mediators of the early stages of the immune response during inflammation, tissue repair, and the maintenance of epithelial integrity. Consequently, the dysregulation of ILC subsets might promote inflammation and cancer. Numerous studies have demonstrated that these cells play an important role in maintaining the microecological balance of the small intestine; however, their specific roles in mediating inflammation in this tissue and tumorigenesis remain unclear and controversial. In this review, we focus on recent progress that has helped to gain a better understanding of the role of ILCs in intestinal homeostasis, chronic inflammation, and cancer. Further focused research on the regulation and role of ILCs in intestinal homeostasis and pathology will help to reveal valuable diagnostic and therapeutic targets for the treatment of intestinal diseases. Intestinal epithelial cells (IECs) cover the luminal surface of both the small and large intestines of the gastrointestinal tract. As part of the intestinal mucosa layer, IECs are single-layer, columnar cells organized with tight junctions that form a contiguous and relative impermeable membrane . The priα4β7 in the bone marrow, which interacts with the chemokine receptor CXCR6 and adhesion molecule MadCAM-1 when cells migrate to the intestine [Innate lymphoid cells (ILCs) are recently identified mucosal immune cells considered the gatekeeper of mucosa-associated tissues such as the gut. Their function is regulated by IEC-secreted cytokines in response to physiological and pathological processes including immune defense, tissue remodeling, inflammation, and cancer , 4, 5. Intestine . Unlike ntestine .γ and lack the production of T helper 2- (Th2-) and Th17-associated cytokines. ILC1s express high levels of the transcription factor T-bet and low levels of the transcription factor retinoid-related orphan receptor γt (RORγt) but lack expression of CD117 [α in mice [γt for their development and function and produce IL-17A and/or IL-22 [+/− ILC3s.Based on their phenotypic and functional characteristics, ILCs can be generally divided into cytolytic and noncytolytic ILCs , 10. Cytof CD117 . Group 2of CD117 , IL-33, of CD117 . The dev in mice , 18, 19.or IL-22 , 20. ThiRM) cells owing to some common features [In addition, recent studies have revealed a regulatory subpopulation of ILCs  and memory ILCs, which include circulating memory NK cells and tissue-resident memory ILCs –23. ILCrfeatures .β, ILC2s and ILC3s can transdifferentiate into ILC1s, which can in turn transdifferentiate back into ILC2s and ILC3s in the presence of IL-4 and IL-23, respectively [ILCs display certain levels of functional diversity and plasticity. Under the influence of IL-12, IL-18, and IL-1ectively –27. Thus+ ILCs accounting for 5% and 2% of total lymphocytes in the human and mouse small intestine, respectively [The intestinal epithelium is the largest barrier that isolates an organ system from the immediate environment. ILC subsets have been found to play a role in gut homeostasis in both humans and mice , 28. In ectively , and ILCectively . Given t+ and IFN-γ-producing subsets, respectively. However, although they share many characteristics, based on recent studies, NK cells and ILC1s develop from different bone marrow progenitors, and therefore, they are substantially different in their tissue tropism, migratory capacity, and effector functions [γt and NKp44 in humans and NKp46 in mice [+ ILC3s produce IL-22 upon interaction with the transcription factor aryl hydrocarbon receptor ligand, which can be derived from the diet and microflora [Cytolytic ILCs (NK cells) and noncytolytic ILC1s are known as T-betunctions , 32. ILCunctions . In the  in mice . NCR+ ILcroflora . AlthougLactobacillus plantarum to attenuate enterotoxigenic Escherichia coli- (ETEC-) induced epithelial damage. Moreover, defense against ETEC is considered to involve the stimulation of NK cells to enhance IL-22 production [The functional activity of ILCs requires exposure to the gut microbiota, as shown by a mouse model study in which germ-free or antibiotic-treated mice displayed impaired NK cell activity . Intestioduction   α [γ to limit the early viral burden [γ by ILC1s, which enhances the expression of chemokines CXCL9, CXCL10, and CXCL11 in IECs, which recruit CXCR3+ leukocytes including Th1, ILC1s, and NKp46+ ILC3 cells [The absence of ILC1s in T-betfections . ILC1s m(TNF-) α , 39. Siml burden . IL-15 pILC2s express the signature transcription factor GATA-3, as well as CD90, CD127, CD25, IL-25R, and IL-33R, and are distributed throughout the intestinal lamina propria , 42. ILCC. difficile infection, ILC1s provide immune protection, whereas ILC2s are activated by IL-33 as an essential pathway for in recovery from C. difficile infection-associated colitis [β) production by intestinal goblet cells, which help to limit parasitic infections [During  colitis . Importafections , 50, 51.fections , 53  including ulcerative colitis (UC) and Crohn's disease (CD) –65. Steewith IBD  and Takaoduction   [However, NK cells might not always act as potent antitumor effectors, as the cytotoxic function of these cells in the tumor microenvironment can be dampened, and NK cells can even have a tumor-promoting effect under certain conditions . The expof IFN-γ . A possiN-γ [β1) –94. In aN-γ [β1)  (Figure in vivo [Owing to the limited quantity of cytotoxic tumor-infiltrating NK cells and their markedly impaired cytotoxic function in colorectal cancer, the adoptive transfer of activated NK cells has emerged as a potential strategy for clinical therapy. A phase I clinical trial with colorectal cancer patients showed that patients who had previously undergone IgG1-based chemotherapy could tolerate the autologous transfer of NK cells well and could successfully induce type-1 immune responses in vivo .γ and TNF-α are released, and these cytokines promote chronic gut inflammation. IFN-γ and TNF-α are involved in antitumor immunity [γ plays a role in cell-mediated tumor cell lysis, which inhibits tumor cell proliferation, reduces neoangiogenesis, and suppresses tumor progression [α can facilitate tumor cell apoptosis and macrophage and dendritic cell infiltration into the local tumor, suggesting direct and indirect antitumor immune responses [Nevertheless, the detailed role of ILC1s in colorectal tumors remains unclear. However, their well-established function in intestinal chronic inflammation could provide a clue based on their potential to create a suitable environment for subsequent malignant transformation . Upon thimmunity  (Figure gression . In turnesponses , 99. HowCRTH2, GATA3, and RORα, in the peripheral blood of gastric cancer patients [+ T cells [Further, the function of ILC2s during intestinal carcinogenesis has been well researched in both mouse models and humans. Bie's group reported increased numbers of ILC2s, as well as the transcription of ILC2-related genes including patients . The frepatients –103. ILCpatients  (Figure patients , 106 and T cells . Thus, f−/− mouse tumor model induced by Helicobacter hepaticus oral infection, indicating that ILCs and IL-22 are essential for the formation of colonic tumors. ILC3s are the main producers of IL-22, which generally has a tumor-promoting effect, but its condition-dependent nature has been proposed with different roles observed in different cancer microenvironments [An increased frequency of ILC3s has also been found in colorectal cancer tissues in a mouse model, and this might play a role in tumor progression , 108. Asronments –114. As ronments , 116 (Fironments . Collect+ cNK cells were found to effectively control B16 tumor development in a mouse model [RM cells, they are considered to have potential benefits in long-term tumor control and vaccination, with promising clinical value for tumor immunotherapies and vaccine-development strategies [Finally, monobenzone-induced memory CD49bse model . Althougrategies .Mounting evidence has now demonstrated that ILCs are critical regulators of intestinal homeostasis, inflammation, and cancer. Although tremendous progress has been made in understanding the detailed roles of ILCs in the restoration of epithelial barrier integrity and protection against infiltrating pathogens, and intestinal inflammation and cancer, this research has also revealed that ILCs represent a highly heterogeneous group of cells. The different groups and subsets of ILCs and their corresponding cytokines have now emerged as important mediators of various pathological conditions, and even the same subtype might have diverse roles in different contexts or at different stages of disease. Moreover, ILCs also play roles in metabolic homeostasis and contribute to the pathogenesis of graft-versus-host disease. Thus, further studies focused on exploring the regulation and pathophysiology of ILCs might reveal potential targets for future therapeutic interventions.


MeLEAs distributed in 10 chromosomes and 11 MeLEA paralogues were subjected to purifying selection. Transcriptomic analysis showed the expression profiles of MeLEAs in different tissues of stem, leaves, and storage roots of three accessions. Comparative transcriptomic analysis revealed that the function of MeLEAs in response to drought may be differentiated in different accessions. Compared with the wild subspecies W14, more MeLEA genes were activated in cultivated varieties Arg7 and SC124 after drought treatment. Several MeLEA genes showed induction under various stresses and related signaling treatments. Taken together, this study demonstrates the transcriptional control of MeLEAs in tissue development and the responses to abiotic stress in cassava and identifies candidate genes for improving crop resistance to abiotic stress.Late embryogenesis abundant (LEA) proteins, as a highly diverse group of polypeptides, play an important role in plant adaptation to abiotic stress; however, LEAs from cassava have not been studied in cassava. In this study, 26 LEA members were genome-wide identified from cassava, which were clustered into seven subfamily according to evolutionary relationship, protein motif, and gene structure analyses. Chromosomal location and duplication event analyses suggested that 26  Arabidopsis, maize, barley, wheat, sunflower bean, and Brassica napus [Late embryogenesis abundant (LEA) protein was first found to accumulate during the late embryogenesis in cotton seed . Subsequca napus ,5,6,7,8.ca napus ,10. LEA ca napus ,12. Baseca napus .Most of the LEA protein families have a unique subcellular localization, which is involved in protecting the stability of cells under stress conditions ,14. AccuOsLEA3-2 was able to enhance salt and drought tolerance in transgenic rice plants [ZmLEA3 could increase the transgenic tobacco tolerance to low temperature, osmotic and oxidative stresses [CuCOR19 in tobacco increased cold tolerance by inhibiting lipid peroxidation [WCOR410 improved freezing resistance in transgenic strawberry leaves [SiLEA14 conferred salt and osmotic tolerance in transgenic Arabidopsis and foxtail millet [JcLEA in transgenic Arabidopsis plants improved resistance to dehydration and salinity [B. napus enhanced salt and drought tolerance [Moreover, genetic evidences also support the function of LEA proteins on abiotic stress response, including dehydration, osmotic, drought, salt, and chilling. Overexpression of the e plants . ZmLEA3 stresses ,21. Overxidation . Overexpy leaves . SiLEA14l millet . Overexpsalinity . Transgeolerance . TogetheLEA genes have been genome-wide identified in various plant species, such as 51 LEAs in Arabidopsis [LEAs in rice [LEAs in Brassica napus [LEAs in Moso Bamboo [LEAs in Brachypodium distachyon [LEAs in Populus trichocarpa [To date, bidopsis , 39 LEAs in rice , 108 LEAo Bamboo , 36 LEAsstachyon , and 53 chocarpa . Howeverchocarpa ,31. Casschocarpa . Transcrchocarpa . Some gechocarpa . lncRNAschocarpa . In addichocarpa ,38,39. T2O2 treatments. This systematic study should increase our understanding of LEA proteins related to abiotic stress response and lay a foundation for genetic improvement of crops.In this study, an effort was made to identify MeLEAs from the cassava genome and investigate their phylogenetic relationship, conserved motif, gene structure, chromosomal location, duplication event, and expression profiles in distinct tissues and in response to drought, salinity, osmotic, cold, ABA, and HArabidopsis, rice, and cassava , which evidenced that segmental duplications had an important role in MeLEAs expansion in the cassava genome (To study the expansion of the a genome .Ka) and synonymous (Ks) values were calculated for the duplicated MeLEA genes based on nucleotide sequences. The pairwise comparison data revealed that the Ka/Ks ratios in all the paralogous genes were between 0.2 and 0.45, showing the purifying selection of these genes  were uncovered in the transcriptome dataset of different tissues of Arg7 and W14. These 7 MeLEA genes may have no, or low, expression levels in different tissues of cassava. In these 19 MeLEA genes, eight (42%), seven (37%), and five (26%) MeLEAs had high expression levels  in storage roots, stems and leaves of Arg7, respectively. The MeLEAs with high expression levels  in storage roots, stems and leaves of W14 were seven (37%), six (32%), and six (32%), respectively. Notably, MeLEA1 in the LEA_5 subgroup, MeLEA15 in the LEA_2 subgroup, and MeLEA-6, -7, -21 in the LEA_3 subgroup showed high expression abundance  in all the tested tissues of Arg7 and W14. These LEA genes may be beneficial for cassava tissue development and function. To examine the transcriptional levels of the analysis . NineteeMeLEAs under drought stress, water withholding was applied to a wild subspecies W14 and two cultivated varieties Arg7 and SC124. Then, total RNA was collected from roots and leaves for transcriptomic analyses  were not covered in the transcriptomic datasets. In W14, three (14%) and two (10%) MeLEA genes were transcriptionally up-regulated (log2-based fold change > 1) by drought stress in roots and leaves, respectively. In Arg7 variety, three (14%) and seven (33%) MeLEA genes were induced (log2-based fold change > 1) after drought stress in roots and leaves, respectively. In SC124 variety, four (19%) and six (29%) MeLEA genes showed up-regulation (log2-based fold change > 1) by drought stress in roots and leaves, respectively. Additionally, MeLEA5 and MeLEA26 were induced (log2 based fold change > 1) by drought in the two cultivated varieties. Together, these results revealed that the number of LEA genes induced by drought was more in Arg7 and SC124 than that in W14.To further investigate the possible role of analyses . AccordiMeLEA-2, -4, -5, -6, -7, -15, -16, -20, -26) were induced by drought stress in different cassava accessions, which were chosen for further examination of their transcriptional patterns after salt, osmotic, cold, and H2O2 and ABA treatments , in which AtM was unique to the Brassicaceae [LEA genes (25/26) harbored less introns (0–2) in cassava  and low/no expression in different tissues. In contrast, MeLEA15 in LEA_2 subfamily and MeLEA-6, -7, -21 in the LEA_3 subfamily did not contain intron, but had high expression levels in various tissues  were identified from cassava, suggesting that segmental duplication may be the main expansion mechanism for MeLEAs , whereas distinct expression profiles in different tissues; MeLEA11/MeLEA12 showed different exon-intron organization, but commonly low/no expression in different tissues; MeLEA13/MeLEA22 and MeLEA19/MeLEA24 commonly had three exons and low/no expression in different tissues; and MeLEA6/MeLEA7/MeLEA21 commonly displayed 1 exons and high expression in different tissues. Additionally, these paralogous gene pairs exhibited different expression patterns after drought treatment in different cassava accessions  by drought in different cassava accessions. Additionally, we observed that the total number of MeLEAs up-regulated by drought was greater in Arg7 and SC124 than that in W14, indicating the comprehensive activation of LEAs in Arg7 and SC124 (LEAs-mediated drought responsive mechanism are differentiated between wild subspecies and cultivated varieties. Due to the differences for the genetic background between wild species (W14) and cultivated varieties (Arg7 and SC124) [MeLEA genes to drought tolerance of cassava is elucidated.Genome-wide expression analysis have demonstrated the significant response of t orange ,3,27,43.nd SC124 C. This id SC124) , the conMeLEA-2, -4, -5, -6, -7, -15, -16, -20, -26) under salt, osmotic, cold, and H2O2 and ABA treatments. The results showed that almost all the MeLEA genes could be induced at least by one of the treatment  is a wild subspecies of cultivated cassava. Arg7 is a variety containing elite agronomic traits, including a certain level of growth under moderate drought stress. SC124 is a widely planted cassava cultivar in China and can survive in prolonged severe drought stress. All the cassava plants were cultured with soil and vermiculite (1:1) under the glass house in the CATAS . To analyze transcripts of MeLEAs in distinct tissues at the early development stage, stems (90 days old), leaves (90 days old), and storage roots (150 days old) were gathered from Arg7 (cultivated variety) and W14 (wild subspecies) under normal conditions. To examine the transcriptional levels of MeLEAs under drought stress, cassava plants (90 days old) of W14, Arg7, and SC124 were subjected to water withholding for 12 days, and the leaves and roots were collected for RNA sequencing. To detect the expression levels of MeLEAs under abiotic stress, leaves were collected from Arg7 (60 days old) for quantitative real-time PCR (qRT-PCR) detection. For salt and osmotic treatments, cassava seedlings grew in soil irrigated with 300 mM NaCl or 200 mM mannitol for 14 days. For cold treatment, cassava seedlings were cultured in an incubator with low temperature (4 °C) for 48 h. For ABA and H2O2 treatments, cassava seedlings grew in soil irrigated with 100 μM ABA or 10% H2O2 for 24 h.The characteristics of W14, SC124, and Arg7 cassava accessions were described in previous studies ,46. W14 Arabidopsis and rice were obtained from the Arabidopsis information resource (TAIR) and rice genome annotation project (RGAP) databases, respectively [Arabidopsis, and rice by ClustalX 2.0  and MEGA 6.0 software packages  bootstrap values for 1000 replicates) [The whole LEA protein sequences in ectively ,48. The ectively . To confectively ,51,52. Tlicates) ,54. MeLEAs were identified by the Gene Structure Display Server (GSDS) database [The ExPASy database  and DNASTAR software packages  were used to predict the molecular weight and isoelectric points of MeLEA proteins ,56. The database .MeLEA genes was identified according to the data information obtained from the Phytozome 12.0 cassava database. Paralogous LEA genes were selected according to calculation of sequence identity and the phylogenetic relationship. Gene duplication events of paralogous LEA genes in cassava were identified based on the following two rules: (1) the alignment covered >80% of the longer gene; (2) the aligned region had an identity >80% [Ks (synonymous substitution) and Ka (non-synonymous substitution) were calculated by DnaSP 5.0 software  [Ka/Ks ratio < 1 indicates a purifying selection, a Ka/Ks ratio = 1 indicates a neutral selection, and a Ka/Ks ratio > 1 indicates a positive selection [The chromosomal location of ity >80% ,61. The , Spain) . A Ka/Kselection ,64.Samples were gathered to extract total RNA with plant RNA extraction kit . The total RNA of each sample was used to construct cDNA libraries. Then, Illumina GAII platform  was used to perform high throughput sequencing according to manufacturer’s RNA-seq protocol. Adapter sequences were cleaned by FASTX-toolkit in the raw reads . To obtaMeLEAs in response to various stimuli were determined by quantitative real-time PCR (qRT-PCR) on Stratagene Mx3000P instrument . AlleleID6.0 was used to design gene-specific primer pairs that were further validated by melting curve and agarose gel electrophoresis . The PCR conditions were implemented as follows: 5 min at 95 °C; 40 cycles of 10 s at 95 °C, 15 s at 55 °C and 20 s at 72 °C; 60 s at 95 °C, 30 s at 55 °C and 30 s at 95 °C. The Tubulin gene was used to normalize as a reference. The 2Ct−ΔΔ approach was carried out to calculate the relative expression of MeLEA genes. The relative expression levels of MeLEA genes in each time point was calculated according to the control and treated samples. Each sample has three replicates and three biological experiments were performed.Expression of phoresis . Each reMeLEAs were distributed in 10 chromosomes and 11 LEAs were paralogues which were subjected to purifying selection. Transcriptomic analyses revealed the tissue expression and drought response diversity of MeLEAs in wild species and cultivated varieties. Several MeLEA genes were observed to respond to multiple abiotic stresses, and H2O2 and ABA signaling. This systematic research will provide a solid foundation for understanding the function of LEA genes and LEAs-mediated abiotic stress response in cassava.In conclusion, this study identified 26 LEAs from cassava and studied their classification by phylogenetic, protein motif, and gene structure analyses. Chromosomal location and duplication event analysis revealed that 26