RNA localization involves cis-motifs that are recognized by RNA-binding proteins (RBP), which then mediate localization to specific sub-cellular compartments. RNA localization is critical for many different cell functions, e.g., in neuronal dendrites, localization is a critical step for long-lasting synaptic potentiation. However, there is little consensus regarding which RNAs are localized and the role of alternative isoforms in localization. A comprehensive catalog of localized RNA can help dissect RBP/RNA interactions and localization motifs. Here, we utilize a single cell sub-cellular RNA sequencing approach to profile differentially localized RNAs from individual cells across multiple single cells to help identify a consistent set of localized RNA in mouse neurons.Using independent RNA sequencing from soma and dendrites of the same neuron, we deeply profiled the sub-cellular transcriptomes to assess the extent and variability of dendritic RNA localization in individual hippocampal neurons, including an assessment of differential localization of alternative 3′UTR isoforms. We identified 2225 dendritic RNAs, including 298 cases of 3′UTR isoform-specific localization. We extensively analyzed the localized RNAs for potential localization motifs, finding that B1 and B2 SINE elements are up to 5.7 times more abundant in localized RNA 3′UTRs than non-localized, and also functionally characterized the localized RNAs using protein structure analysis.We integrate our list of localized RNAs with the literature to provide a comprehensive list of known dendritically localized RNAs as a resource. This catalog of transcripts, including differentially localized isoforms and computationally hypothesized localization motifs, will help investigators further dissect the genome-scale mechanism of RNA localization.The online version of this article (10.1186/s12915-019-0630-z) contains supplementary material, which is available to authorized users. RNA localization is critical to many inter-cellular processes. Neurons are an excellent system to study RNA localization because their extreme polar morphology (neurites of the neurons) creates clear spatial differentiation of the localized RNA and makes it relatively easy to isolate the localized RNA. In addition, RNA localization is critical to neuronal function in which neurons require local protein synthesis within the dendrites to produce long-lasting synaptic potentiation –3. In orThere are several major challenges in profiling the dendritic transcriptome: (1) cleanly separating the somatic and dendritic compartments so that they can be profiled separately, (2) differentiating transcript variation  in addition to localization, (3) accounting for single cell variation in both somatic expression and dendritic localization, and 4) distinguishing actively translocated RNA from randomly diffused RNA. Here, we approach these challenges by performing simultaneous RNA sequencing of the somatic and dendritic compartments of single neurons from primary cultures to allow for a direct contrast of the dendritic transcriptome with its parent soma and to enable the assessment of heterogeneity of localization across neurons. Approaching the problem by single-neuron-matched sub-cellular sequencing has two advantages. First, by matching the dendrite and soma samples, we are able to more clearly identify differentially expressed, and therefore likely to be actively translocated, RNA. Second, since randomly diffused RNA is likely to be different in different individual cells, by examining the variability and consistency across the individual cells, we are again likely to identify actively translocated RNA. Our approach also allows us to examine individual cell variability in localization as well as effects of isoform usage. Given that substantial gene expression heterogeneity has already been observed on the whole-neuron level , it woul distinguGrin1, Mtap2, and Neurod6, with little expression of other brain cell type markers   Fig. . NotablyActb, Bdnf, Calm1, Dlg4, Grin1, and Map2. To differentiate from the 387 differentially expressed genes described above, we refer to this set as the constitutive dendritic (consDend) RNAs, and the previous set as the differentially expressed dendritic (deDend) RNAs. The consDend RNAs covered many of the same ontology functions as the deDend RNAs, such as mitochondria and translation, but additionally were strongly enriched for a large number of synaptic and localization-related GO terms . Overall, the differences between the deDend and consDend lists suggest that at the single-cell level, RNAs with important dendritic and synaptic functions are often not localized to the point of having higher expression concentration in the dendrites relative to the soma, but are nonetheless consistently present in the dendrites at a lower level.Differential expression analysis may not identify all localized RNAs because not all localized RNAs are expected to have higher expression concentration in the dendrites than the soma. This may be particularly relevant when expression is profiled at the single-cell level, since factors such as bursting transcription and variable rates of localization can lead to high variability in the relative amounts of RNA in each compartment at the time of collection. Therefore, we additionally identified RNAs that were consistently present in the dendrites across the profiled cells, since these RNAs are likely to have important dendrite function even if they are not differentially at higher concentration in the dendrites compared to the soma. We found 1863 RNAs in at least 90% of the dendrite samples, which included well-characterized localized RNAs such as Serhl, Ptpn14, Liph, Mre11, Aox3, Casp4, Ddx58), most of which do not currently have a defined dendritic function, although mutations in Mre11 have been previously associated with Ataxia-telangiectasia-like disorder 1 {3,},’ which requires three consecutive matches to the pattern “three or more G’s followed by 1–7 of any nucleotide” and then ending with a fourth set of three or more G’s. The background set was the same as described in the previous section.De novo identification of enriched RNA secondary structures was performed using NoFold . SlidingGAGGCAGGCGGATTTCTGAGTTCGAGGCCAGCCTGGTCTACAGAGTGAGTTCCAGGACAGCCAGGGCTACACAGAGAAACCCTGTCTCB1 sequence:((((((((....(((((((((((..(((...(((((.((........))..)))))...))).)))))...))))))...))))))))B1 structure:GCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGCTCTTCCGAAGGTCAGGAGTTCAAATCCCAGCB2 sequence:(((((.((..((((((....((.(((((((......))))))))).........))).)))..)))))))B2 structure:Bitscore cutoffs for high-quality matches were set to 50 for B1 and 35 for B2 based on the length of the model. Enrichment was computed using Fisher’s exact test based on the number of high-quality matches in the localized set compared to the non-localized background (same background as above). Only one match was counted per gene for the purposes of enrichment testing.For each predicted dendritic RNA, we obtained the canonical protein sequence, if any, from UniProt . The canEach protein was split into domains based on DomainFinder Gene3D predictions , 52. If Additional file 1:GO annotations. Full list of enriched GO terms for differentially expressed (deDend and deSoma) and consistent dendrite (consDend) lists for biological processes (BP), molecular functions (MF), and cellular components (CC). Related to Fig. Additional file 2:Heatmap of subsampled variability of localization, related to Fig. Additional file 3:Re-analysis of differential localization of 3′UTR isoforms using 250 nt merge distance. (PDF 78 kb)Additional file 4:Table S1. Predicted transmembrane structures. Table S2. Predicted RNA-binding structures. Table S3. Predicted structures commonly found in synaptic proteins. (PDF 189 kb)Expanded structure analysis of potential locally translated proteins. Additional file 5:Full list of predicted protein structural folds for dendritic genes found in this study. (XLSX 506 kb)Additional file 6:Table S4. Full list of dendritic genes from current study and seven previous publications. Table S5. Full deDend gene list. Table S6. Full consDend gene list. Table S7. Full isoDend gene list. (XLSX 346 kb)Catalog of dendritic genes. Additional file 7:Overview of 3′UTR definition, quantification, selection of top two isoforms, and calculation of distal fraction. (PDF 275 kb)


TNF-α) is an important cytokine in acute inflammatory response to infectivefactors. Based on investigation in different populations, it is thought that this response increases in patients with endometrio-sis due to the presence of cytokines such as TNF-α. This study aimed to examine the association of four TNF-α polymor-phisms, namely -238G/A, -308G/A, -857C/T and -863C/A, with susceptibility to endometriosis in an Iranian population.Tumor necrosis factor-alpha .TNF-α -863C/A in the case and control groups showed a significant difference  but the result is not significant when Adjust-ing for multiple testing (P=0.188). No significant difference in the allele frequencies of -238G/A , -308G/A  and -857C/T  was observed. We adjusted all four polymorphism genotypes by age and body mass index (BMI),however, no significant difference was detected. There was an association between the case and control and BMI whenadjusting by age .The allele frequency of TNF-α gene withendometriosis has been conducted in women of Iranian origin. The present research reveals the -863 A allele may playa role in incidence of endometriosis among Iranian women. Development of endometriosis among those people with-863 A allele seems low. According to the results, the current study indicates that there might be a correlation betweenBMI and progression of endometriosis.For the first time the association of the four polymorphisms in the promoter region of the  Endometriosis is developed as a result of endometrial tissue exposing outside the uterine cavity. Studies have reported the pelvic and the peritoneum as the most common sites of replacement , 2. ThisTNF-α) is an important cytokine in acute inflammatory response to infective factors and is genetically variable. Based on multiple studies, TNF-α is thought to be a molecular indicator for gynecological-related diseases. It is suggested that the inflammatory response in endometriosis increases because of cytokines such as TNF-α  classification (stages I-IV of disease). The control group consisted of 150 women without endometriosis. Only women who underwent laparoscopy for non-endometriosis infertility and showed absence of endometriosis were included as controls. Stages I and II of endometriosis are commonly found in asymptomatic women . The excTNF-α was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Details on primers and restriction enzymes are presented in Blood was collected in tubes with 200 µl EDTA (0.5 M), as an anti-clotting factor, and stored at -20ºC until DNA extraction. Genomic DNA was extracted by salting out method from peripheral blood samples. Genotyping of the -238G/A (rs361525), -308G/A (rs1800629), -857C/T (rs1799724) and -863C/A (rs1800630) polymorphisms in the 5'-untranslated region of 2 , 0.4 µM of each dNTP , 5 pmol of each primer, 50 ng template DNA, 1 U Taq DNA polymerase  and sterile distilled water up to 25 µl. Amplification conditions start with an initial denaturation step of 5 minutes at 94ºC, followed by 30 cycles of 30 seconds denaturation (94ºC), 30 seconds annealing (63ºC) for -238G/A, -857C/T and -863C/A and 30 seconds annealing (66ºC) for -308G/A and 30 seconds extension (72ºC), ended by a final extension for 5 minutes (72ºC) and finally cooling to 4ºC.The PCR reactions carried out in final volume of 25 µl containing: 10X PCR Buffer , 1.5 mM MgClPolymerase chain reaction products were electrophoresed on a 1.5% agarose gel in 1X TAE and stained with ethidium bromide and visualized by ultraviolet light. After reviewing the PCR products, they were treated with restriction enzymes (Hpa II and NcoI at 37ºC and TaiI at 65ºC) overnight. The digestion products were subjected to 10% polyacrylamide gel electrophoresis and stained with silver nitrate .Results were analyzed by SPSS 24.0 software . The analysis of age and body mass index (BMI) in the study groups were performed using t test. The allele frequencies were compared using the Chi-squared test. Genotype distributions in the case and control groups were also analyzed. Age and BMI were considered as potential confounders. The analyses were performed and adjusted in terms of age and BMI using logistic regression. P<0.05 was considered statistically significant. The P value corrected using Bonferroni method for the multiple testing. Logistic regression was used to predict the odds of developing a given disease based on observed characteristics of the patients. In our study, the criterion variable was the logistic regression of disease and no-disease. To perform the statistical analysis using SPSS, we considered the two case and control groups as dependent variables. Age and BMI were considered as covariants and genotype selected as the basis of categorical covariant. http://string-db.org/) was used to acquire the network of protein-protein interactions for TNF-α.For interaction analysis, the STRING online server (2) in the case and control groups were 25.2 (SD=3.7) and 26.2 (SD=4) respectively. Genotypes of the TNF-α -238G/A, -308G/A, -857C/T and -863C/A polymorphisms were obtained in 150, 150, 148, 150 patients and 149, 150, 143, 150 control samples respectively. Genotype frequencies of the TNF-α -238G/A, -308G/A, -857C/T and -863C/A polymorphisms in the case and control groups were in Hardy-Weinberg equilibrium. Genotype and allele frequencies for the TNF-α -238G/A, -308G/A, -857C/T and-863C/A are shown in Table 2According to the analysis of descriptive variables, the age range was from 19 to 50 years  in the patients, and from 19 to 44 years  in the control group. The mean BMI  but the result is not significant when adjusting for multiple testing (P=0.188). However, no significant difference was observed in the allele frequencies of the -238G/A (P=0.862), -308G/A (P=0.438) and -857C/T (P=0.878) polymorphisms in TNF-α between the case and control groups. We adjusted all four polymorphism genotypes by age and BMI but according to the results, no significant difference was discovered between the groups. But there was an association between the case and control and BMI when adjusting by age .The TNF-α interacts with 10 other proteins according to STRING (TAB2), ii. Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1), iii. TNF receptor-associated factor 2 (TRAF2), iv. TNFRSF1A-associated via death domain (TRADD), v. Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB), vi. Receptor interacting serine-threonine kinase 1 (RIPK1), vii. Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), viii. Baculoviral IAP repeat containing 2 (BIRC2), ix. Tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A), and x. Tumor necrosis factor receptor superfamily, member 1B; (TNFRSF1B). STRING . In specTNF-α polymorphisms, -238G/A, -308G/A, -857C/T and -863C/A with endometriosis in an Iranian sample.Endometriosis is a multifactorial disease with both genetic and environmental components . StudiesTNF-α polymorphisms with augmented endometriosis risk r et al.  in Austr et al.  found a  et al.  discoverTNF-α -238G>A has been inspected in three studies, -308G>A in five articles, -857C>T in three articles and -863C>A in four articles, all of which showed compatible findings where no significant association was reported between these four polymorphisms and endometriosis in any of the models. Our study reported an association between the -863C/A polymorphism in the promoter region of TNF-α and endometriosis. We also observed a direct relationship between the case and control and BMI when adjusting by age  is essential for mediating most of the metabolic efficacy of TNF-α . TNF-α -863 A allele was significantly lower in women with endometriosis than controls, suggesting that the -863 A allele may play a role in incidence of endometriosis among Iranian women. Development of endometriosis among those people with -863 A allele seems low although it should be noted that the calculations show is not significant when adjusting for multiple testing. According to the results, the current study indicates that there might be a direct relationship between BMI and progression of endometriosis.We investigated the association of four polymorphisms in the promoter region of


Leishmania species , Trypanosoma cruzi and three Trypanosoma brucei subspecies . The P-type ATPase complement in these trypanosomatids includes the P1B , P2A , P2B , P2D (Na+ pumps), P3A (H+ pumps), P4 (aminophospholipid translocators), and P5B (no assigned specificity) subfamilies. These subfamilies represent the P-type ATPase transport functions necessary for survival in the Trypanosomatidae as P-type ATPases for each of these seven subfamilies are found in all Leishmania and Trypanosoma species included in this analysis. These P-type ATPase subfamilies are correlated with current molecular and biochemical knowledge of their function in trypanosomatid growth, adaptation, infectivity, and survival.P-type ATPases are critical to the maintenance and regulation of cellular ion homeostasis and membrane lipid asymmetry due to their ability to move ions and phospholipids against a concentration gradient by utilizing the energy of ATP hydrolysis. P-type ATPases are particularly relevant in human pathogenic trypanosomatids which are exposed to abrupt and dramatic changes in their external environment during their life cycles. This review describes the complete inventory of ion-motive, P-type ATPase genes in the human pathogenic Trypanosomatidae; eight  Leishmania and Trypanosoma) is a significant public health problem with widespread disease and limited therapeutic options. Leishmaniasis is endemic in 97 countries, putting 350 million people at risk, and there are an estimated 12 million individuals currently infected, with 700,000–1,000,000 new cases and 26,000–65,000 deaths worldwide each year [Leishmania-HIV co-infection is a serious and growing problem in many areas as HIV dramatically increases the risk of fulminant VL, and VL promotes the clinical progression of HIV [Trypanosoma cruzi, is a major illness in Latin America with 6–7 million infected individuals, 25 million at risk of infection, and an annual death toll of over 10,000 [T. cruzi trypomastigotes circulating in the bloodstream can be mild to severe with fatalities resulting from myocardial damage. Chronic Chagas disease occurs after trypomastigotes enter cells, particularly myocardial cells, to grow as intracellular amastigotes. Clinical manifestations can appear decades later and include chronic chagasic heart disease , due to destruction of cardiac innervation and myocardial cells, and chronic gastrointestinal disease (megaesophagus and megacolon) caused by impaired autonomous neuronal regulation. Trypanosoma brucei rhodesiense and T. b. gambiense are causative agents of human African trypanosomiasis (HAT) or “sleeping sickness” and if untreated the disease is generally fatal. As recently as 2006, HAT infected 50,000–70,000 people annually, but through the sustained efforts of the World Health Organization and public health officials in affected countries, the annual burden of disease was reduced to less than 3000 cases in 2015 [T. b. rhodesiense is rapidly fatal (weeks) with early CNS involvement and recurrent waves of high parasitemia. Trypanosoma brucei gambiense infection produces a chronic disease with low blood parasitemia and late CNS involvement that ends fatally years later. Each of these trypanosomatid infections can be associated with serious medical complications and fatal outcomes, even when treatment is administered. Chemotherapeutic interventions for these diseases are inadequate due to toxic side effects and drug resistance to the current treatment regimens, and there is an urgent need for improved therapeutic alternatives.Human infection by insect-borne parasites of the family Trypanosomatidae  and non-dividing (stumpy) trypomastigote forms in the mammalian host.These organisms have a complex digenetic life cycle with different morphologic forms in the human host and within the insect vectors of the disease; sandflies for Adaptation of the trypanosomatids to these differing environs, and the abrupt transitions that occur, present a challenge for the parasites to adjust to the changing ionic environments and to the structural modifications required for their morphologic changes. The maintenance of intracellular ion homeostasis is critical to growth and survival in all organisms. Proper ionic balance is required for a wide array of cellular processes including regulating osmolarity and cell volume, maintaining pH homeostasis, controlling levels of toxic ions such as heavy metals, providing co-factors for protein function and cellular signaling pathways, and establishing membrane potentials to energize secondary transport systems. To counter the different ionic environments they encounter, cells have evolved a diverse array of proteins to regulate and move ions across both internal and external cellular membranes. These include passive systems such as ion exchangers, ion symporters, and ion channels, as well as active transport systems which require energy for ion movement. The crucial players in this regulation of cellular ion homeostasis are the P-type ATPases which are the only ion transport proteins capable of moving ions against a concentration gradient and thus can facilitate and coordinate the activity of other ion motive mechanisms. This study examines the complete genomes of the human pathogenic Trypanosomatidae in order to identify their complement of P-type ATPases and to correlate current molecular and biochemical knowledge of their function with trypanosomatid adaptation and survival.+, H+, Na+, Ca2+, and Mg2+ ions, and are capable of translocating large phospholipid molecules across membranes. P-type ATPases are typically inhibited by orthovanadate. P-type ATPases are multi-domain membrane proteins with molecular masses ranging from 70 kDa to 150 kDa and share conserved sequence motifs and a common structural organization. All of the P-type ATPases have a similar tertiary structure in their catalytic subunits and a common reaction cycle with two conformational states (E1 and E2), characterized by the reversible phosphorylation of a conserved aspartate residue, which is part of the phosphorylation site motif DKTGT. The P-type ATPases contain 6–13 hydrophobic transmembrane domains (TMs), typically with cytoplasmic exposure for the amino and carboxyl termini of the proteins, which may contain regulatory elements. P1A-ATPases contain 7 TMs, P1B-ATPases have 6–8 TMs, P2, P3 and P4-ATPases each have 10 TMs, and P5-ATPases have 11–13 TMs [P-type ATPases are ubiquitous in nature and have been described in archaebacteria, eubacteria, protozoa, fungi, plants, invertebrates and vertebrates. Proteins of the P-type ATPase family utilize the energy of ATP hydrolysis to transport ions across plasma and organellar membranes and to generate membrane lipid asymmetry , 159. P-1–13 TMs . The B-d1–13 TMs , 106.1–P5) with P1, P2, P3, and P5-ATPases further subdivided into eleven additional classes. Each of these subfamilies and classes is characterized by unique substrate specificity and group-specific sequence motifs [1–P5-ATPase classification of Axelsen and Palmgren [http://www.tcdb.org). The P1A-ATPases (3.A.3.7) are bacterial membrane transporters of K+ ions. The P1B-ATPases (3.A.3.5 and 3.A.3.6) transport heavy metals such as copper, cadmium, lead, and zinc. The P2 subfamily transports non-heavy metal cations; P2A- and P2B-ATPases (3.A.3.2) transport Ca2+ ions and are located in internal organelles, particularly the sarcoplasmic and endoplasmic reticulum (SERCA), and in the plasma membrane (PMCA) respectively, P2C-ATPases (3.A.3.1) are Na+/K+- and H+/K+-ATPases, and P2D- and P2E-ATPases (3.A.3.9) function as either efflux or influx proteins, respectively in the transport of K+ and Na+ ions. The P3-ATPases translocate H+  and Mg2+  ions. The P4-ATPases (3.A.3.8) are phospholipid “flippases” which maintain membrane lipid asymmetry by imparting selectivity and directionality to lipid bilayer movement. The final subfamily, consisting of P5A and P5B-ATPases (3.A.3.10–3.A.3.22), does not have an identified substrate specificity or biological function although their involvement in protein maturation, protein secretion, and in anion transport has been proposed.Based on shared sequence homologies, a phylogenetic analysis of P-type ATPases has identified five major evolutionary subfamilies , to identify the complete inventory of P-type ATPases in these organisms [+-ATP binding site DGVND, the actuator domain LTGES, and other highly conserved P-type ATPase motifs, DPPR, KGAP and MLTGD. The annotation for predicted protein sequences was also queried for the terms ATPase, P-type ATPase, ion translocation, cation transport, and phospholipid translocation. The P-type ATPase sequences identified were utilized to search for additional homologous genes in these trypanosomatid genomes. As P-type ATPases were found, they were sequentially numbered using the designations of LA, LB, LD, LI, LM, LMX, LP, LT, TB, TBG, and TC for Leishmania aethiopica, L. braziliensis, L. donovani, L. infantum, L. major, L. mexicana, L. panamensis, L. tropica, Trypanosoma brucei, T. b. gambiense, and T. cruzi ATPases, respectively. The P-type ATPases identified in these searches were subjected to a phylogenetic analysis based on the method of Axelsen and Palmgren [This review examines the genome sequences of rganisms , 139. ThPalmgren  which exPalmgren , 51. ThiL. major (strain Friedlin), 12 in T. cruzi (strain CL Brener) and 14 in T. brucei TREU927 (strain 927/4 GUTat10.1) [Leishmania species and two more Trypanosoma species have been reported and are included in this analysis [Leishmania and Trypanosoma species were either from non-human pathogens or were incomplete as judged by the absence of functional genes for some essential P-type ATPase subfamilies and were not analyzed. The T. cruzi CL Brener stain used for genome sequencing is a hybrid of two distantly related T. cruzi lineages, designated as Esmeraldo-like and Non-Esmeraldo-like, which initially complicated assignment of chromosome specificity [T. cruzi ATPases, each represented as nearly identical copies (>97%) on separate contigs , and these duplications are only included once each in the P-type ATPase count for T. cruzi strain CL-Brenner. However, the true complement of P-type ATPases in T. cruzi is likely 14–15, as partial sequences for several homologues of H+-ATPase TC8 were present in different chromosomal locations in the CL-Brener genome sequence and other investigators have reported tandemly linked arrays of three H+-ATPase genes in T. cruzi Y strain and four H+-ATPase genes in the Sylvio X10/7 strain [L. major and the two Trypanosoma species are due to the absence of homologues for calcium motive ATPase LM6 in T. brucei and T. cruzi and for proton motive ATPase LM10 in T. brucei, as well as variable numbers of repeated genes in the additional calcium and proton ATPase loci. The P-type ATPases of the trypanosomatid species were found to be highly syntenic, showing considerable conservation in the surrounding gene order as was true for most of their genome sequence.A total of 42 complete P-type ATPase protein sequences, listed in Tat10.1) , 46, 67.analysis , 139. Avcificity , 173. As7 strain , 101. ThLeishmania and four Trypanosoma strains, respectively. All of the prior P-type ATPases reported were detected in the trypanosomatid genomes, indicating the reliability of the current search of the trypanosomatid genomes. The trypanosomatid proteins are organized in 1B , P2A (SERCA), P2B (PMCA), P2D (Na+ pumps), P3A (H+ pumps), P4 (aminophospholipid translocators), and P5B (no assigned specificity) subfamilies. These represent the P-type ATPase transport functions necessary for survival in the Trypanosomatidae as P-type ATPases for each of these seven subfamilies are found in all Leishmania and Trypanosoma species included in this analysis. Although H+/K+-ATPase activity has been described in L. donovani and Na+/K+-ATPase activity reported for L. mexicana, L. amazonensis, T. brucei, and T. cruzi, no P2C-ATPases which might account for these findings were found in the parasite genomes [1A-ATPases, K+ transporters or P3B-ATPases, Mg++ transporters, both found exclusively in prokaryotes, were identified.P-type ATPases previously characterized are listed in  genomes , 95, 1111B , P2A (SERCA), P2D (Na+ pumps) and P5B (no assigned specificity) subfamilies. Several of the P2B (PMCA) ATPase genes are also single copy and each of the five different genes found in the P4 aminophospholipid subfamily are also present in a single copy with the exception of a duplication of the LB15 (LB15–LB17) gene in L. braziliensis. The only additional P-type ATPases present in linked, repeated assemblies in these parasite genomes are P2B ATPases (PMCA) of Trypanosoma brucei (TB3–TB4–TB4b), Leishmania major (LM3–LM4) and L. mexicana (LMx3–LMx4) each separated by a single gene, and P3A ATPases (H+ pumps) of L. major (LM8–LM9), L. infantum (LI8–LI9) and T. brucei (TB8–TB9). Other Leishmania and Trypanosoma species have only a single gene in these two loci, although in many cases gene fragments and pseudogenes are present, indicating the ancestral existence of duplicate genes in these loci. As shown in subsequent sections, differences in the presence and extent of tandem arrays in these P-type ATPase subfamilies as well the presence of loci containing additional P2B ATPase (PMCA) and P3A (H+ pumps) genes in Leishmania species results in a variable content of P-type ATPases among the different Trypanosomatidae species.Although tandemly linked repeated genes are common in the Trypanosomatidae, most of the P-type ATPases are present as single copy genes , 3, 4. ALeishmania including those causing visceral disease , Old World cutaneous sores , New World cutaneous lesions (L. mexicana), and New World mucocutaneous leishmaniasis . The L. panamensis and L. braziliensis sequences belong to the Viannia subgenus of Leishmania. Alignments of the Leishmania ATPases within each subfamily show them to be highly homologous, generally >90% identical in their core sequences, which exclude amino and carboxyl sequences outside the first and last membrane spanning domains. P-type ATPases from Old World species L. major and L. infantum were usually most closely related in each subfamily and L. panamensis and L. braziliensis proteins were the least homologous as compared to the other species. The differences in protein sizes within each ATPase family were principally due to alterations in the length of the amino and carboxyl ends of the protein, shorter or longer than the consensus length although in several cases internal sequence was missing. The Leishmania ATPases were highly syntenic with identical chromosomal locations, except differences attributable to breakage/fusion rearrangements in chromosomes 8/29 and 20/36 in L. mexicana and chromosomes 20/34 in L. braziliensis and L. panamensis [Leishmania appears to be 16. However, rearrangements, deletions, and duplications have altered this complement as only L. major has 16 functional P-type ATPase genes. The other Leishmania species have reduced numbers of P2B (PMCA), P3A (proton pumps) or P4 (aminophospholipid translocators), with only fragments of the original gene remaining, and Leishmania braziliensis has an additional gene copy (LB17) in the P4-ATPase family, arising from an apparent duplication of LB15.namensis , 121. ThTrypanosoma species; Trypanosoma brucei TREU927, T. brucei Lister strain 427, T. brucei gambiense DAL972, and T. cruzi CL Brener. There are 14 P-type ATPases in T. brucei TREU927 and Lister strain 427, 11 in T. brucei gambiense, and 12 in T. cruzi. Trypanosoma brucei TREU927 and Lister strain have repeated genes in the P2B ATPase (PMCA) and P3A (H+ pumps) subfamilies, TB/TBL3–TB/TBL4–TB/TBL4b and TB/TBL8–TB/TBL9 respectively, as compared to T. b. gambiense and T. cruzi which have a single gene in these loci. The three Trypanosoma brucei strains also lack a homologue for TC12, one of the five P4 (aminophospholipid translocators) present in T. cruzi. Homologues for TC12 are present in all eight Leishmania species  to 8  transmembrane segments (TMs) flanking the large cytoplasmic loop that contains the ATP binding and phosphorylation sites, and the presence of metal binding signature sequences in TM4, TM5 and TM6 which determine ion selectivity. P1B-ATPases also possess a diverse array of regulatory cytoplasmic N-terminal and C-terminal metal binding domains that control the enzyme turnover rate, but do not affect metal ion binding to the transmembrane transport sites. Seven P1B-ATPase subgroups have been identified and the metal selectivity identified for the first four; P1B-1: Cu+/Ag+; P1B-2: Zn2+/Pb2+/Cd2+, P1B-3: Cu2+; P1B-4: Co2+, with substrate specificity as yet to be definitively defined for P1B-5, P1B-6, and P1B-7 [1B-1-ATPases (3.A.3.5) are the most widely represented, present in archaea, prokaryotes and eukaryotes. The P1B-2, P1B-3, and P1B-4 subgroups (3.A.3.6) which transport divalent metal ions are restricted to archaea, prokaryotes and plants where they are responsible for metal biotolerance and also function in the hyper-accumulation of divalent metals in some plant species.All organisms require trace amounts of metals ions, principally as cofactors in biological catalysis; over 40% of enzymes classified by EC (Enzyme Commission) number, whose three-dimensional structures have been deposited in the Protein Bank Database, are metal dependent . Howevernd P1B-7 . Members1B-ATPase is present in each of the Leishmania and Trypanosoma species described in this report. Those in the Leishmania species are 175–292 amino acids longer than their Trypanosoma counterparts. This size differential is primarily related to longer amino terminal sequences proximal to the first transmembrane segment present in the Leishmania P1B-ATPases. However, excluding the cytoplasmic amino and carboxyl terminal portions of the enzymes the Leishmania and Trypanosoma P1B-ATPases are highly homologous, sharing 72–83% identity in this region. The trypanosomatid proteins each exhibit a structural organization characteristic of the Cu+ transporting P1B-1 subgroup of P1B-ATPases; a long N-terminal domain, eight predicted transmembrane domains  with the large cytoplasmic loop located between TM4 and TM5, and a relatively short C-terminal domain. The trypanosomatid P1B-ATPases also contain the transmembrane signature motifs present in the Cu+ transporting P1B-1 subgroup; CPCALGLATP in TM4, NX6YNX4P in TM5, and PX6MX2SSX5N in TM6 [1B-ATPase subgroups with other metal ion specificities were absent in the trypanosomatid proteins. P1B-1-ATPases also have an N-terminal consensus regulatory metal binding domain, GMXCXXC, which is present in 2–3 copies in the Leishmania species and as a single copy in the Trypanosoma species. The trypanosomatid P1B-1-ATPases do not possess the Cys or His-rich or (HX)n regulatory metal binding domains associated with other P1B-ATPase subgroups but the eight Leishmania species each do have an N-terminal CC dipeptide motif that has been associated with metal binding in other subgroups. A phylogenetic comparison with 52 additional P1B-ATPases from organisms ranging from archaea and prokaryotes thru vertebrates shows that the trypanosomatid ATPases segregate with the P1B-1 Cu+ pumps from fungi, plants, vertebrates and invertebrates, and are most closely related to the yeast  and plant (Sorghum) representatives (data not shown).A single P1B-ATPases function primarily as efflux pumps, conferring resistance to high levels of heavy metals in the environment. Non-photosynthetic eukaryotes appear to lack P1B-ATPases specific for divalent metal ions but their P1B-1-ATPases have a dual function; they can localize to the plasma membrane and function in copper efflux, conferring metal resistance, or be localized to organellar membranes and supply copper to intracellular compartments to meet metabolic requirements. The Saccharomyces copper transporting P1B-1-ATPase, CCC2, is located in the membrane of the post-Golgi network where it transfers copper from the copper chaperone Atx1p into the lumen of the Golgi network for insertion into secreted copper-dependent enzymes [1B-1-ATPases, ATP7A and ATP7B, localize to the trans-Golgi network and undergo copper-dependent trafficking, but can also be relocated to the plasma membrane or to vesicles proximal to the plasma membrane in various tissues [In archaea and prokaryotes, P enzymes , 184. In tissues . Defects1B-1-ATPases in their life cycle remains speculative. However, a role similar to CCC2 or ATP7A/B in the transport of copper into the trans-Golgi network or other intracellular organelles for incorporation into metalloproteins seems likely. For instance, a Cu/Zn-type superoxide dismutase is present in Leishmania glycosomes [Leishmania species and T. cruzi to survive within phagolysosomal vacuoles of macrophages highlight the need for the trypanosomatid P1B-1-ATPases to participate in active copper efflux. Macrophages introduce Cu+ ions into the lumen of phagolysosomal vacuoles, via the ATP7A copper pump, as part of the toxic antimicrobial milieu present in this compartment [1B-1-ATPases is required for virulence and survival from macrophage-mediated bacterial clearance in Escherichia coli, Pseudomonas, pneumococcus, and Salmonella infections [Trypanosoma cruzi P1B-1-ATPases are upregulated over three-fold in the intracellular amastigote stage present in host cells versus the extracellular epimastigote in the insect vector, emphasizing its importance in an intracellular environment for T. cruzi [Trypanosoma brucei, which lacks an intracellular stage in the human host, there is little difference in P1B-1-ATPase transcript and protein levels between insect and human stages [L. mexicana and L. major, the presence of multiple regulatory copper-binding domains in the amino terminal segments of the Leishmania P1B-1-ATPases may provide an alternative mechanism for increasing efflux activity under the high copper conditions encountered within macrophage phagolysosomes [In the absence of biochemical characterization and subcellular localization data, the precise role of the trypanosomatid Pycosomes . In addipartment , 65, 174fections , 174. TrT. cruzi . Howevern stages . Althougysosomes , 79.Leishmania and T. cruzi must contend with the low free calcium content of host cells. As a consequence, the trypanosomatid parasites have an additional calcium storage compartment, an acidic, calcium and phosphorous rich organelle known as an acidocalcisome [2+-ATPases participate in regulation of intracellular calcium levels; the P2A-ATPases, also known as the sarcoplasmic-endoplasmic reticulum calcium ATPases (SERCA), and P2B-ATPases known as plasma membrane calcium ATPases (PMCA), though PMCA pumps can also be found in intracellular organelles such as Golgi vesicles, vacuoles, and acidocalcisomes. SERCA pumps are found within intracellular organelles, notably the sarcoplasmic and endoplasmic reticulum, where they mediate Ca2+ sequestration, maintaining low intracellular Ca2+, and providing a recruitable Ca2+ store for intracellular signaling. The P2A-ATPases in most organisms have a high affinity for calcium and are inhibited by sub-micromolar concentrations of the sesquiterpene lactone thapsigargin. In trypanosomatids, calcium similarly functions as a second messenger to control intracellular signaling but also mediates several events unique to these organisms [Trypanosoma brucei maintains a low intracellular concentration of free Ca2+ and increases in calcium concentration are implicated in release of variant surface glycoprotein (VSG) from the plasma membrane [T. cruzi trypomastigotes and L. amazonensis is also dependent on increased cytosolic calcium levels [The maintenance of intracellular calcium homeostasis is critical to living cells. Calcium is an integral component in signaling pathways that control many aspects of cellular function, including transcription, motility, cell division, apoptosis, metabolism, and signal transduction. Free calcium level within cells is maintained at micro-molar concentrations more than three orders of magnitude lower than intracellular calcium storage sources or the extracellular environment. Vertebrate cells utilize two sources of calcium for signaling, the sarcoplasmic reticulum and the extracellular space but intracellular parasites such as alcisome . Two clarganisms , 129. Trmembrane , 141. Thm levels , 182.2+-ATPases, including a single representative of the P2A-SERCA pumps and 2–4 members of the P2B-PMCA pumps. The SERCA pumps in the three trypanosomatid genomes initially sequenced  are high affinity calcium pumps that participate in the maintenance of low intracellular Ca2+ levels characteristic of eukaryotic cells by exporting Ca2+ from the cytosol to the extracellular environment. Vertebrates possess multiple PMCA genes and each transcript can be alternatively spliced to produce multiple variants that are developmentally regulated in a tissue- and cell-specific manner [Plasma membrane Cac manner . In morec manner , 142. Noc manner .Leishmania, two copies on chromosome 7, although only partial sequences or gene fragments are noted for some Leishmania species at this locus, and in T. brucei, three copies on chromosome 8  and TbPMC2 (TB3) can complement yeast mutants whose vacuolar calcium ATPase and Ca2+/H+ antiporter are deleted, and suppress their calcium hypersensitivity [Trypanosoma cruzi Tca1  LM3-4, TB3-4-4b, Tc3s/3p, (ii) LM5, TB5, Tc5p, and (iii) LM6, each group presumably with different physiological roles. The first group exists as tandemly linked copies in mosome 8  and 4. T90–100%,  to TB3, sitivity . A partisitivity . TrypanoTC3s/3p,  co-localstigotes . Althougmexicana –14. In Lcterized . No gene+ ion participation in many metabolic activities, as well as the toxic effects of a high intracellular Na+ content, requires active transport mechanisms to regulate K+ and Na+ ion levels and maintain high K+ and low Na+ within cells. In lower eukaryotes, fungi, and bryophytes, a combination of inward K+ ion channels, K+ uptake proteins, and P-type ATPase efflux and import pumps capable of transporting either Na+ or K+ has evolved to serve this function [2D-ATPases, function to confer tolerance to high concentrations of Na+ and K+ in the environment by exchanging Na+ (or K+) for H+. This efflux can be either across the plasma membrane or into intracellular organelles to sequester sodium. In fungi and lower eukaryotes, this will also necessitate efflux of the imported H+ ions via a proton pump (see P3A-ATPases). In invertebrates and vertebrates Na+ and K+ homeostasis is regulated by a counter-transporting P-type Na+/K+-ATPase which couples the export of three Na+ ions with the import of two K+ ions. The Na+/K+-ATPase thus requires the presence of both Na+ and K+ for activity [+/K+-ATPase, classified as a type P2C-ATPase, is inhibited by ouabain and insensitive to furosemide, whereas the yeast P2D Na+ pumps are typically insensitive to ouabain and sensitive to furosemide. In animal cells, the electrogenic nature of Na+/K+-ATPase activity also produces a large transmembrane electrochemical gradient of sodium ions that is harnessed to drive the secondary transport of nutrients and other substrates [The physiologic dependence of living cells on Kfunction . These Aactivity . The Na+bstrates .+ and K+ ion homeostasis. In the human host, high serum Na+ (135–150 mM) and low serum K+ concentrations (3.6–5.6 mM) are encountered during growth in blood and low Na+ (5–15 mM) and high K+ (140–155 mM) concentrations are present in the intracellular environments seen by T. cruzi and Leishmania amastigotes. While information on the ionic environment in tsetse flies and sandflies, vectors for T. brucei and Leishmania, is lacking, it is reasonable to expect that Na+ and K+ concentrations in their insect hosts during the digestion of the parasite acquired blood meal will be similar to those seen in human serum, as has been shown in the gut of reduviid bug vectors of T. cruzi after feeding [+ and K+ ions in the trypanosomatids during the life cycle has been controversial; both P2C-ATPases with properties similar to vertebrate Na+/K+-ATPases and P2D ATPases similar to fungal and plant Na+ ATPases have been reported [+/K+-ATPase inhibitor, as distinguishing criteria, resulting in the discrepancies in interpretation between different investigators. Ouabain does not have absolute specificity for the Na+/K+-ATPase and can inhibit additional cellular functions as well as other P-type ATPases, albeit to a lesser extent and at higher concentrations. Cystamine transport is inhibited by 60% in Saccharomyces by 200 μM ouabain although S. cerevisiae lacks an Na+/K+-ATPase [+/K+-ATPase [Streptococcus sanguis and divalent cation stimulated ATPase activity in Staphylococcus aureus are also slightly inhibited by micromolar concentrations of ouabain [During their life cycle, the trypanosomatid parasites are exposed to differing challenges in regulating Na feeding . Howeverrum Na+ 1–150 mM areported , 165. Th+-ATPase . Ouabain+-ATPase . Ecto-AT ouabain , 93. TheT. brucei, T. cruzi and Leishmania protein sequences show 65–73% identity and 77–82% homology within the region between the first and last trans-membrane segments, excluding amino and carboxyl terminal amino acids. Homologues for each of these genes have also been cloned and partially characterized  that form the cation pore of Na+/K+-ATPases are conserved in each of the trypanosomatid sodium pumps [2D-ATPases as well, and simply reflect a common evolutionary origin of the ability of P2C- and P2D-ATPases to transport Na+ and K+. There is strong similarity in the trypanosomatid P2D-ATPase sequences (matching at 6–9 amino acids) to a conserved motif, MIEALHRRKK, located in ATPase conserved region g, that is present in fungal P2D-ATPase ENA pumps but absent in Na+/K+ P2C-ATPases [Zygosaccharomyces rouxii ENA1 ATPase, D852 in TM7 and E981 in TM10, are also conserved in the trypanosomatid P2D-ATPases, but are not present in Ca2+-, Na+/K+-, or H+-ATPases [A single sodium pump is present in each of the trypanosomatid genomes  and these are highly homologous: pairwise alignments of the cterized . Examinas report . Althougescribed . None ofarasites . A DSYGQum pumps , 29. In um pumps . However-ATPases . Two res-ATPases .+- and K+-ATPase activities are also most consistent with a classification as P2D-ATPases. In yeast, the primary function of their P2D-ATPases is sodium or potassium efflux in conditions of high concentrations of these ions. A similar role is likely for the trypanosomatid sodium pumps. ATPase activity in Trypanosoma cruzi epimastigote membranes is stimulated by addition of Na+ ions and does not require the presence K+ for activity, as do Na+/K+-ATPases, and this stimulation is ouabain-insensitive and furosemide-sensitive [T. cruzi Tulahuen strain, TcENA  and it also localizes to the plasma membrane and is upregulated in trypomastigote stages [Leishmania amazonensis ATPase activity in promastigote plasma membranes is also stimulated by Na+ ions, does not require the presence K+ for activity, and is ouabain-insensitive and furosemide-sensitive [+ would also be encountered, modulates this (Na+ + K+) ATPase by increasing intracellular H2O2 production which activates a protein kinase C signaling pathway to increase (Na+ + K+) ATPase activity [+ efflux through the plasma membrane the trypanosomatid P2D-ATPases may also function in cyclic AMP-protein kinase A (PKA) signaling and other intracellular signaling pathways. The PKA regulatory subunit immunoprecipitates with multiple P-type ATPases  in trypomastigote membranes and P-type ATPases may play a role in anchoring PKA to the plasma membrane [+/K+-ATPases also serve as an anchor for a signalosome, containing specific binding motifs for proteins such as caveolin, phosphoinositide 3′ kinase, ankyrin, and AP2 adaptor complex. When bound to ouabain, the Na+/K+-ATPases signalosome can activate Src family tyrosine kinases and transduce signals via multiple pathways independent of its pumping function [+ pumps.Biochemical properties of the trypanosomatid Naensitive . The chan, TcENA , provideum pumps . The ATPe stages . Leishmaensitive . Heme, aactivity , 37, 135membrane . In humafunction , 181. A +-ATPases are found in plants, fungi, protozoa and archaebacteria. They are a major determinate in the maintenance of cytosolic pH and by pumping H+ ions out of the cell they generate and maintain an electrochemical H+ gradient across the plasma membrane , which is used to energize secondary transport of solutes [+ ions and is controlled by P2C Na+/K+-ATPases. P3A-ATPases are characterized by the presence of an auto-inhibitory domain which regulates protein activity. In fungi, this domain is located in the C-terminal cytoplasmic portion of the protein and interacts intra-molecularly to lock the pump in a low activity state. In plants, both the C-terminal and N-terminal domains are involved in controlling H+-ATPase activity [+-ATPases or glucose to fungal H+-ATPases neutralizes this interaction and moves the pump to a high activity state. This regulation is principally dependent on the phosphorylation of serine and threonine residues in the C-terminal auto-inhibitory domain of both fungi and plants [+-ATPase mediates glucose-dependent activation of Pma1 [Plasma membrane H solutes . In animactivity . Bindingd plants , 118. Ta of Pma1 .3A-ATPases are present in the trypanosomatid species. Leishmania has H+-ATPase genes in two chromosomal locations; a multi-copy gene locus on chromosome 18 and a single copy H+-ATPase gene on chromosome 4  or two  complete H+-ATPases gene sequences found in the genomes of the other Leishmania species  in both procyclic and bloodstream forms of the parasite as shown by plasma membrane depolarization by H+-ATPase inhibitors [T. brucei H+-ATPase also regulates intracellular pH in procyclic trypomastigotes and bloodstream trypomastigotes [+-ATPases  and slowed recovery of pHi after acidification [T. cruzi epimastigotes, trypomastigotes and amastigotes is similarly dependent on plasma membrane H+-ATPase activity and participation of a P-type H+-ATPase in the acidification of internal organelles has been shown [+-ATPases, TcHA1 and TcHA2  unable to support yeast growth at pH 3 and LDH1B (with LDH1A tail) now able to support growth at pH 3. Replacement of the COOH-terminal eight amino acids of LDH1B with those from LDH1A also confers the ability to support growth at pH 3 [Leishmania H+-ATPases. Multiple Ser and Thr residues are present in all of the proton pumps for the additional Leishmania species described here, including a Ser-Thr pair in each, as well as NxS and FS motifs in most of the Leishmania species. The COOH-terminal tails of the Trypanosoma proton pumps also contain multiple Ser and Thr residues and NxS/FS motifs but no Ser-Thr pairs. However, the T. cruzi proton pump, TcHA1, is apparently not regulated via a COOH-terminal auto-inhibitory domain as COOH-terminal truncation of TcHA1 did not affect its ability to complement yeast deficient in H+-ATPase activity [The nd LDH1B  are diff and NxS . Functio at pH 3 . Potentiactivity .4-ATPases is common. A unique feature of eukaryotes is the asymmetrical nature of their membranes and aminophospholipid translocases are responsible for maintaining the asymmetrical distribution of lipids in the plasma membrane, trans-Golgi network and endosomal system membranes by their ability to “flip” phospholipids such as phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) between bilayer leaflets to increase PS and PE exposure on the cytosolic leaflet and PC and sphingolipid exposure on the extracellular leaflet [4-ATPases work in synergy with other ATP-dependent and ATP-independent protein families, such as ATP binding cassette (ABC) transporters and flippases respectively, to effect this non-random phospholipid distribution in eukaryotic membranes. This asymmetric lipid arrangement is critical to cellular function as it confers different biophysical properties and physiological roles to the two sides of the bilayer. P4-ATPases are also essential to membrane biogenesis by generating lipid asymmetry in the new membrane as it transverses from its origin in the endoplasmic reticulum through the Golgi network. In addition to their role in vesicular trafficking, P4-ATPases have also been implicated in cell signaling events, apoptosis, blood clotting, phagocytosis, cell polarity and growth, adhesion, migration, lysophospholipid transport, and apical barrier function [The aminophospholipid translocases are a large and diverse group of P-type ATPases that appear to be ubiquitous in eukaryotes where the expression of multiple P leaflet , 82, 134function .4-ATPases can differentiate phospholipids based on their head groups and lipid backbones. Yeast possesses five P4-ATPases; Drs2p transports PS with a weaker affinity for PE, Dnf1p, Dnf2p, and Dnf3p transport PC and PE, and the transport specificity of Neo1p appears to be PE with a weaker activity towards PS [4-ATPases also exhibit specificity in their intracellular location; yeast Dnf1p and Dnf2p localize to the plasma membrane and early endosomes, Dnf3p and Drs2p are located in the trans-Golgi network, and Neo1p is present in the trans-Golgi network and late endosomes [4-ATPases require at least three accessory (β subunit) molecules, which form heterodimeric complexes with the P4-ATPases in membranes and are required for their stability, proper intracellular targeting, and transport activity and specificity. In yeast, Cdc50p complexes with Drs2p and Neo1p, Lem3p/Ros3p complexes with Dnf1p and Dnf2p, and Crf1p binds Dnf3p. Five classes of P4-ATPase ATPases , analogous to those in yeast, with different substrate specificities, a similar diverse array of cellular membrane localizations, and accompanied by accessory chaperone proteins,  are also present in humans [Different members of the Pwards PS , 158. Yendosomes . The P4-4-ATPases are present in the trypanosomatid genomes initially sequenced  LM11, TC11s/11p, and TB11; (ii) LM12 and TC12s; (iii) LM13, TB13 and TC13s; (iv) LM14, TB14, and TC14p; and (v) LM15, TB15, and TC15s/15p. The first two groups show higher homology to each other than to other groups, a finding also true for the last two groups. This result is confirmed by phylogenetic analysis with 42 additional P4-ATPases (not shown) wherein the trypanosomatid sequences from the first two groups (LM11-TB11-TC11s/11p and LM12-TB12) segregate on a separate branch of the phylogram, as do the last two groups (LM14-TB14-TC14p and LM15-TB15-TC15s/15p), while LM13, TB13 and TC13s segregate together on a branch that includes yeast Neo1p. Homologous proteins in each of these five P4-ATPase groups are present in all the other Leishmania and Trypanosoma species analyzed as well, with the exception of a homologue for LM12 and TC12 which is missing in all of the Trypanosoma brucei strains , localizes to the plasma membrane and mediates uptake of glycerophospholipids and the lysophospholipid analogue miltefosine (hexadecylphosphocholine), a highly effective oral anti-leishmanial drug [L. donovani promastigotes and amastigotes to miltefosine is characterized by mutations to LdMT, which decrease internalization of miltefosine and other phospholipid analogues [T. cruzi and to a lesser extent versus T. brucei, suggest a similar functional role for their LM15 homologues, TC15s/15p  and TB 15  [Except for the Pficities , 71, 140ial drug , 123. Renalogues . Resistanalogues , 150. Thto LM15) , 146.L. donovani (strain MHOM/ET/67/HU3) reveals a similar requirement for accessory β subunits in trypanosomatid P4-ATPase function [Leishmania and Trypanosoma genomes described in this report. The LdRos protein homologue in L. major genome is 93% identical to LdRos and homologous proteins in T. brucei and T. cruzi are 53% and 55% identical to LdRos.The characterization of a functional homologue, LdRos, of the Lem3/CDC50 family in function . LdRos i4-ATPases participate in entry and intracellular survival of Leishmania. Phagocytosis of Leishmania amastigotes and infective stationary phase promastigotes is enhanced by increased exposure of phosphatidylserine on the outer membrane [Leishmania exploit this process by mimicking the apoptotic programmed cell death phenotype to gain entry into target cells and evade immune responses [Leishmania has not been identified, the dependence of PS exposure on an ATP driven process in Leishmania has been demonstrated, an observation consistent with inhibition or reduction of membrane P4-ATPase PS translocating activity [Pmembrane . The preesponses , 171. Alactivity .5-ATPases are the least well understood subfamily of the P-type ATPases, which is surprising as they appear to be ubiquitous among eukaryotes. P5-ATPases appear to be specific for eukaryotes as no P5-ATPases have been identified in bacterial or archaeal species to date. P5-ATPases group into two subfamilies, P5A and P5B, based on the composition of a characteristic signature sequence present in their fourth transmembrane domain, which corresponds to the PEGLP sequence of P2A-ATPases that participates in forming a Ca2+ binding site [5A-ATPases this motif contains the sequence PP(D/E)LPxE and in P5B-ATPases the two negative charged residues have been replaced with hydrophobic residues, PP(A/V)LPAx, with x denoting a hydrophobic residue [5A- and P5B-ATPases may differ in their ion specificities. The P5A-ATPases are present in all eukaryotic lineages but P5B-ATPases are not found in three eukaryotic lineages; excavates, which includes Leishmania and Trypanosoma, entamoebas, and land plants.The Ping site . In P5A- residue . The los5A-ATPases to the endoplasmic reticulum (ER), early Golgi network, vacuoles, and secretory vesicles [5-ATPases and loss of function mutants in both yeast and plants exhibit pleiotropic effects with diverse phenotypes [5-ATPases in protein folding, maturation, degradation, and secretion through maintenance of ER homeostasis. The Saccharomyces Cod1 mutant is deficient in the regulation of hydroxymethylglutaryl-coenzyme A degradation, an integral ER protein, and disruption of this P5A-ATPase  yields a glycosylation defective phenotype, perturbs cellular calcium homeostasis, and increases expression of Kar2p, an ER stress chaperone, and calcineurin-activated genes, indicating secretory calcium store depletion [2+ and microsomes from Spf1P overexpressing yeast have increased Mn2+ as compared to wild type [Schizosaccharomyces pombe P5A-ATPase, SPAC29A4.19c, are hypersensitive to elevated calcium in the absence of the vacuolar Ca2+-ATPase, Pmc1p, a condition that can be suppressed by overexpression of another Ca2+-ATPase, Pmr1p. Double deletion mutants of SPAC29A4.19c and Pmr1p are hypersensitive to Mn2+ depletion in the culture medium. Schizosaccharomyces SPAC29A4.19c deletion mutants are also hypersensitive to the antiarrhythmic drug amiodarone, which disrupts Ca2+ homeostasis [5A-ATPase of Arabidopsis thaliana, MIA, exhibit abnormal morphology, altered cell wall structure, imbalances in cation homeostasis, and changes in the expression of secretory proteins [Multiple studies associate Pvesicles , 175. Ioenotypes . Availabepletion , 156. Spepletion . Microsoild type . Deletioeostasis . Mutantsproteins .5B-ATPase, are linked to the lysosomal storage disorder neuronal ceroid lipofuscinosis and neurodegenerative diseases such as Kufor-Rakeb syndrome, Parkinson’s disease, and hereditary spastic paraplegia, and are characterized by lysosomal and mitochondrial dysfunction [Dictyostelium amoeba, phagocytic bacterial predators found in soil, deletion of kil2, a P5B-ATPase present in phagolysosomal membranes, prevents killing and growth on Klebsiella bacteria and this defect is rescued by addition of Mg2+ ions, suggesting that kil2 is a magnesium pump [Caenorhabditis elegans on the presence of the P5B-ATPase plasma membrane transporter, CATP-5, demonstrates the existence of an additional potential function for the P5B-ATPases [Mutations in ATP13A2, a human neuronal Pfunction , 47, 130ium pump . However-ATPases .2+, Mg2+, and Mn2+ homeostasis seen in many studies has led to speculation that P5-ATPases transport one or more of these ions. An alternative hypothesis is that the P5-ATPases function to move lipids or organic molecules across membranes to assist in the generation of membrane vesicles, participate in lipid recycling and/or lipid distribution in membranes, or to move glycolipid precursors, needed for synthesis of N-linked oligosaccharides or GPI-anchors, from the cytosol to the lumen of the ER [5-ATPases are most closely related to the P4-aminophospholipid translocating ATPases among the P-type ATPases [5A- or P5B-ATPases is lacking at this time.The perturbations in Caf the ER –145. Sup ATPases . Unfortu5-ATPase genes are present in the all of the Trypanosomatidae examined  to inhibit the human gastric H+/K+-ATPase and decrease stomach acidity, cardiotonic steroids  for the treatment of congestive heart failure and heart arrhythmia by specific inhibition of the heart Na+/K+-ATPase, artemisinin compounds for the treatment of malaria via inhibition of the parasite sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and clotrimazole, another SERCA inhibitor, as an anti-mycotic agent [Leishmania and Trypanosoma parasites; artemisinins have anti-trypanosomal activity, omeprazole inhibits L. donovani, thapsigargin inhibits the SERCA pump of T. brucei, the Na+-ATPase of L. amazonensis is sensitive to the diuretic furosemide (Lasix), pentamidine inhibits the plasma membrane Ca2+-ATPase of T. brucei, the aminophospholipid translocator, LdMT, in L. donovani is required for miltefosine transport and miltefosine inhibits the Na+-ATPase of T. cruzi [Leishmania and trypanosome infections should be a priority.Chemotherapeutic interventions for ic agent . The antic agent . There aT. cruzi , 146. MoThe identification of the genes responsible for ATP-dependent ion movements should facilitate future studies of their structure and function. Importantly, the exploitation of P-type ATPase genes as drug targets should be advanced as their presence offers the possibility of exploiting differences in their ion pumps for the biologically based design of new therapeutic interventions for these infections.The author declares that he has no conflict of interest.


Furthermore, the JEV mutants with G1 glycosylation showed enhanced DC-SIGN binding, which may then lead to reduced brain invasion and explain the reason why WT JEV is devoid of this G1 site of glycosylation. Overall, the patterns of N-linked glycosylation on JEV E proteins may affect viral interaction with cellular lectins and contribute to viral replication and pathogenesis.The major structural envelope (E) protein of Japanese encephalitis virus (JEV) facilitates cellular binding/entry and is the primary target of neutralizing antibodies. JEV E protein has one N-linked glycosylation site at N154 (G2 site), but the related dengue virus E protein has two glycosylation sites at N67 (G1 site) and N153 (G2 site). We generated three recombinant JEVs with different glycosylation patterns on the E protein. As compared with wild-type (WT) JEV with G2 glycosylation, viral growth in culture cells as well as neurovirulence and neuroinvasiveness in challenged mice were reduced when infected with the G1 mutant (E-D67N/N154A) with glycosylation shifted to G1 site, and the G0 mutant (E-N154A) with non-glycosylation. The G1G2 mutant (E-D67N), with E-glycosylation on both G1 and G2 sites, showed potent  The flavivirus genus contains many important human pathogens such as Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus, West Nile virus (WNV), and Zika virus (ZIKV). Flaviviral virion is enveloped and contains a positive-sense, single-stranded RNA genome. The viral RNA translates a polyprotein that is proteolytically processed into structural proteins  and seven nonstructural proteins   lectins are transmembrane proteins that primarily display on antigen-presenting cells for recognition of specific carbohydrates on glycoconjugates. Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin  and its homolog DC-specific ICAM-3-grabbing nonintegrin-related protein  can be attachment factors tethering glycosylated virions onto the cell surface  and N153 (G2 site), whereas JEV E proteins have only one N-linked glycosylation at residue N154 (G2 site). Cryo-electron microscopy (EM) study demonstrated that the carbohydrate recognition domain of DC-SIGN binds preferentially to a subset of glycans present at N67 (G1 site) on DENV particles  and these mutants, we determined the contribution of N-glycans on E proteins to JEV infection.To address the precise role, especially the location and number of N-linked glycosylation on JEV E proteins, we generated three JEV mutants—glycosylation at N67 (G1), N67 plus N154 (G1G2), or no glycosylation (G0) on E proteins, respectively. By comparing the Animal studies were conducted according to the guidelines outlined by the Council of Agriculture, Executive Yuan, Republic of China. The animal protocol was approved by the Academia Sinica Institutional Animal Care and Utilization Committee (Protocol ID 13-08-570). All surgery was performed under Isoflurane anesthesia to minimize suffering. For biosafety, we followed the “Guidelines for Research Involving Recombination DNA Molecules” issued by the Ministry of Science and Technology, Republic of China NSC, , in whicBaby hamster kidney BHK-21 fibroblast cells  were grown in RPMI 1640 medium containing 5% fetal bovine serum (FBS) and 2 mM L-glutamine. Human embryonic kidney 293T cells  and the human microglial cell line (CHME3)  and E-D67N (5′-GTTACTGTTATCATGCTTCAGTCACTAATATCTCGACGGTGGCTCG-3′) (mutation underlined), respectively. For infectious virus production, plasmids carrying WT, or mutated JEV cDNA were transfected into BHK-21 cells, and culture supernatants were collected after incubation for 4 days. Viruses were amplified in mosquito C6/36 cells  for further study  was used as a template to generate glycosylation-mutated viruses by single primer mutagenesis as described  containing RPMI-1640 with 2% FBS for 4 days, then fixed with 10% formaldehyde and stained with 0.5% crystal violet.To obtain extracellular E proteins, culture supernatant from JEV WT- or mutant-infected C6/36 cells was concentrated and purified by ultracentrifugation. To obtain intracellular E proteins, C6/36 cell lysates were collected. E glycoproteins were digested with N-glycosidase F (Roche) and endoglycosidase H (Roche) under reducing conditions for 16 h at 37°C according to the manufacturer's instructions.−/−) mice  of each virus was calculated by the Reed and Muench method  of a lethal JEV strain RP-9  or i.c. route  as described previously  for 4 h were transfected with Flag-tagged DC-SIGN by use of Lipofectamine 2000 (Invitrogen) for 24 h. Cells were rinsed with phosphate-buffered saline (PBS) and lysed with protein lysis buffer  containing a cocktail of protease inhibitors (Roche). Cell lysates were immunoprecipitated with anti-Flag M2 affinity gel  overnight at 4°C. The immunocomplex was washed with cold protein lysis buffer at 4°C and suspended in sample buffer with 2-mercaptoethanol. Proteins were separated with 4 to 12% NuPage (Invitrogen) and probed with anti-E antibody by western blot analysis.CHME3 cells with DC-SIGN or eGFP overexpression were established by transduction with lentivirus expressing DC-SIGN or eGFP. Cells were adsorbed with JEV WT or mutants at 4°C for 2 h, and then washed extensively with cold serum-free RPMI three times. Total RNA was extracted by using the RNeasy kit (QIAGEN) and analyzed by real-time RT-PCR. The cDNA was reverse transcribed from 1 μg of RNA with random hexamers by using the ThermoScript RT kit (Invitrogen). PCR involved the LightCycler FastStart DNA Master PLUS SYBR Green I kit (Roche) with the primer sequences JEV nt 10603–10619, forward (5′-AAGTTGAAGGACCAACG-3′) and JEV nt 10789–10772, reverse (5′-GCATGTTGTTGTTTCCAC-3′), or β-actin nt 811–831, forward (5′-TCCTGTGGCATCCACGAAACT-3′) plus β-actin nt 1125–1105, reverse (5′-GAAGCATTTGCGGTGGACGAT-3′). For relative quantification, JEV RNA was measured relative to actin RNA in each sample and the concentration was calculated from a standard curve. Melting curves were used to verify the specificities of products.SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) with Prism (GraphPad Software) and two-tailed Student t-test. Determination of the median survival time (T50) and P-values by the log-rank test, involved use of Prism (GraphPad Software).Data are presented as mean ± standard deviation , which removes all N-linked carbohydrates, and endoglycosidase H (Endo-H), which only removes high mannose and some hybrid types of N-linked carbohydrates. G0 has no N-linked carbohydrates, so the gel mobility of E proteins was similar with or without enzyme treatment  and peripheral injection , respectively. G1 and G0 mutants showed reduced neurovirulence  as compared with the G1G2 mutant and WT JEV   was also noted for the attenuated G1 and G0 mutants (Table 50 (>106 PFU for G1G2 vs. ~104 PFU for WT) and longer T50  Table −/− mice via subcutaneous route. Lower neuroinvasiveness was noted for G1G2 with higher survival rate (40% for G1G2 vs. 0% for WT)  (Table To study the ts Table . Further) Figure  and long1, 102 and 103 PFU per mouse), then challenged with a virulent JEV strain 2 weeks later. A single vaccination with 103 PFU of G1G2 or G0 completely protected the mice against JEV challenge, whereas degrees of mortality were noted in groups vaccinated with lower doses of JEVs or the PBS control , but not for the glycosylation-null G0 mutant, as compared to the GFP control cells Figure . FurtherWe then tested the ability of WT and mutant JEVs to bind with DC-SIGN by using two binding assays. The first was immunoprecipitation-western blot assay of HEK293T cells infected with JEVs and co-transfected with Flag-tagged DC-SIGN or vector control by using anti-Flag antibody-conjugated beads and anti-JEV E antibody. Interaction of DC-SIGN with JEV E protein was readily seen in G1G2 and G1 mutants but not much in G2 and G0 mutants Figure . Next, win vivo neurovirulence as the WT JEV, but its neuroinvasiveness was reduced. Thus, the neurotropic flaviviral E proteins with only one glycosylation at N153 might have a selection advantage for crossing the blood-brain barrier during evolution. This finding also supports that glycans on flaviviral E proteins play important roles in viral infection and affect viral tropism.Mature flaviviral particles have 180 copies of E glycoproteins and M proteins embedded in the viral membrane (Kuhn et al., Glycan diversity on the virion surface should be related to differential usage of lectin receptors by these flaviviruses (Sirohi and Kuhn, in vitro, have an attenuation phenotype in animals and lead to vaccine potential (Lee et al., Single amino acid replacements in structural proteins have been found to affect receptor usage, tropism and pathogenesis for many viruses (Baranowski et al., JEV activity has been reported in most of Southeast Asia (Fan et al., J-JL and Y-LL designed research, analyzed data, and wrote the paper. J-JL and M-WC performed research and animal study. M-WC provided technical assistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Overall, we show that TMEM39A plays a positive regulatory role in EMCV proliferation and that TMEM39A expression is dependent on the autophagy pathway.Encephalomyocarditis virus (EMCV) causes encephalitis, myocarditis, neuropathy, reproductive disorders, and diabetes in animals. EMCV is known to induce cell autophagy; however, the molecular mechanisms underlying this remain unclear. Here, we show that the type III-transmembrane protein, transmembrane protein 39A (TMEM39A), plays a critical role in EMCV replication. We showed that EMCV GS01 strain infection upregulated TMEM39A expression. Importantly, EMCV induced autophagy in a range of host cells. The autophagy chemical inhibitor, 3-MA, inhibited EMCV replication and reduced TMEM39A expression. This is the first study demonstrating TMEM39A promoting the replication of EMCV  Picornaviridae family  is a positive sense single-stranded RNA virus belonging to the e family . EMCV ise family . EMCV cae family . EMCV ine family . EMCV cae family . Therefoe family . EMCV lie family . Howevere family . TMEM39Ae family . Furthere family .via an interaction with ATG3 and ATG7  supplemented with 10% (v/v) newborn bovine serum  in a 37°C incubator. We used the EMCV GS01 strain in this study and was isolated as previously described . pET28a,Anti-HA antibody (A02040) was purchased from Abbkine. Antibodies against ACTB (ab6276), 6 × His tag (ab18184), GST (ab92), and TMEM39A (ab175618) were purchased from Abcam. Anti-LC3B antibody (14600-1-AP) was purchased from Proteintech. Anti-ATG7 antibody (AA820) was purchased from Beyotime. Anti-SQSTM1/p62 antibody (WH098631) was purchased from Abclonal. Peroxidase AffiniPure goat anti-rabbit IgG (H + L) (111-035-003) and anti-mouse IgG (H + L) (115-035-003) were purchased from Jackson ImmunoResearch Laboratories. RIPA (P0013K), NP40 (P0013F) and PMSF (ST506-2) were purchased from Beyotime. Lipofectamine™ 2000 (11668019), Pierce™ GST protein interaction pull-down kit (21516), and protein G dynabeads™ (10004D) were purchased from ThermoFisher. 3--2,5-diphenyltetrazolium bromide  and 3-methyladenine  were purchased from Sigma-Aldrich.Total RNA was extracted from cells and supernatant using the RNAiso Plus kit  according to the manufacturer’s protocol, and RNA was reverse transcribed. RT-qPCR was done using the PowerUp™ SYBR Green Master Mix  or the Premix Ex Taq Probe qPCR . The expression level of each target gene was calculated by normalization to ACTB using the ΔΔCT method, and the ratio of the two genes in the control group was normalized to 1. All RT-qPCR primer sequences are listed in Cell monolayers were incubated on ice with RIPA lysis buffer  containing PMSF . Proteins were separated on 15% SDS-PAGE gels and subsequently transferred to PVDF membranes  using a Trans-Blot Turbo™ RTA Mini PVDF Transfer Kit . The membranes were then sequentially incubated with primary antibodies and peroxidase affinipure goat anti-rabbit or mouse IgG (H + L). Specific protein bands were analyzed using an ECL kit .E.coli BL21 strain. GST alone or GST-TMEM39A protein was mixed with the glutathione agarose resin  and incubated overnight at 4°C. The following day, His-labeled proteins were added separately to the above mixture. After conjugation, the unbound proteins were washed away and subjected to immunoblotting.Protein G dynabeads™  were mixed with the corresponding antibody overnight at 4°C. For Co-IP assays, cells were incubated in NP40 lysis buffer  containing PMSF . The following day cell lysates containing the antigen was added to the Protein G mixture. The bead-Ab-Ag complexes were washed five times by gentle pipetting and subjected to immunoblotting. For the pull-down assays, His-labeled VP1, VP2, and VP3, pGEX-6P-1, and GST-TMEM39A were expressed in Cells were seeded in six-well plates and allowed to reach 50% confluency. Cells were transfected with 150 nM specific siRNA using Lipofectamine™ 2000  according to the manufacturer’s instructions. siRNAs targeting TMEM39A (GGACCCTCGTCAATCTCTT) and non-targeting control (NC) (ACGTGACACGTTCGGAGAA) were purchased from RiboBio. At 48 h post-transfection, knockdown efficiency of endogenous TMEM39A was confirmed by RT-qPCR and immunoblotting.50 assay (Reed-Muench method).HEK293, C2C12 cells, and BHK-21 cells were infected with EMCV at a multiplicity of infection (MOI) of 0.0001 and cultured in DMEM/3%NBS for a further 24 h post-infection (hpi). Cells and another group of the mixture of cells and supernatants after repeated freezing and thawing for three times were harvested separately for RT-qPCR analysis and the TCIDCell viability was detected by the MTT assay as described previously .t-test. p <0.05 was considered a statistically significant difference between two groups .Results were displayed as the mean ± SD. Statistical analysis was performed using the Student’s via the yeast two-hybrid assay. To verify this, we performed in vitro Co-IP and pull-down assays. Since HEK293 cells endogenously express TMEM39A, these cells were transfected with HA-labeled VP1 or VP2. As expected, overexpressed HA-labeled VP1 and VP2 co-precipitated with endogenous TMEM39A  and VP2 (HUVEC cell cDNA library)  TMEM39A . Similar TMEM39A . These rTo further investigate the role of TMEM39A in EMCV infection, we overexpressed TMEM39A in HEK293 cells. TMEM39A mRNA and protein expression levels were significantly increased 48 h post-transfection ,B. CompaPrevious work has shown that EMCV HB10 infection can induce autophagy in BHK-21 cells and that autophagy promotes the replication of EMCV . We wantTo investigate the effect of EMCV infection on TMEM39A expression, we evaluated the expression of TMEM39A in different cells by immunoblotting. We found that the TMEM39A was markedly upregulated in EMCV-infected HEK293 and C2C12 cells at 24 hpi ,B. Since3-Methyladenine (3-MA) is a widely used autophagy inhibitor, which can impede the activity of class III PI3K . 3-MA alTMEMs are not only used as “carrier terminals” or channels to selectively allow the transport of substances into cells or on biofilms but also as “carriers” to transport substances out of cells . At presvia activating the EIF2S1-ATF4 pathway -inducible gene. However, GWS studies have implicated TMEM39A in lupus, an autoimmune disease characterized with dysfunctional IFN responses. We have included these references in our manuscript , coxsackie virus B (CVB), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), and classical swine fever virus (CSFV) can induce autophagy in host cells, while promoting viral replication using autophagy mechanisms . Subsequ pathway . One stu pathway . In 2014 pathway . The nucnuscript . Whethernuscript , screeniIn the present study, we showed that TMEM39A directly interacted with EMCV VP1 and VP2 and played a positive regulatory role in the proliferation of EMCV. This is the first study to clarify the role of TMEM39A in viral infection and provide evidence of a relationship with autophagy pathway. Our findings provide novel ideas for clarifying the role of TMEM39A in the process of viral infection and clinical therapy of certain autoimmune diseases and cancers related to TMEM39A.The original data supporting the conclusions of this manuscript will be provided by the authors to any qualified researcher without reservation.XL wrote the draft manuscript. XL and RF designed the experiment. XL, RM, SL, and QL performed experiments and processed the data. HZ, JX, JB, and RF revised and proofread the draft manuscript. AI provided valuable suggestions and comments and was actively involved in the final editing and proofreading of this manuscript. RF supervised the entire process.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C2H2-zinc finger protein family is commonly found in the plant, and it is known as the key actors in the regulation of transcription and vital component of chromatin structure. A large number of the C2H2-zinc finger gene members have not been well characterized based on their functions and structure in cotton. However, in other plants, only a few C2H2-zinc finger genes have been studied.Gossypium hirsutum (upland cotton), Gossypium arboreum and Gossypium raimondii, respectively. Phylogenetic tree analysis of the C2H2-zinc finger proteins encoding the C2H2-zinc finger genes were classified into seven (7) subgroups. Moreover, the C2H2-zinc finger gene members were distributed in all cotton chromosomes though with asymmetrical distribution patterns. All the orthologous genes were detected between tetraploid and the diploid cotton, with 154 orthologous genes pair detected between upland cotton and Gossypium arboreum while 165 orthologous genes were found between upland cotton and Gossypium raimondii. Synonymous (Ks) and non-synonymous (Ka) nucleotide substitution rates (Ka/Ks) analysis indicated that the cotton C2H2-zinc finger genes were highly influenced mainly by negative selection, which maintained their protein levels after the duplication events. RNA-seq data and RT-qPCR validation of the RNA seq result revealed differential expression pattern of some the C2H2-zinc finger genes at different stages of cotton fiber development, an indication that the C2H2-zinc finger genes play an important role in initiating and regulating fiber development in cotton.In this work, we performed a comprehensive analysis and identified 386, 196 and 195 C2H2-zinc finger genes in This study provides a strong foundation for future practical genome research on C2H2-zinc finger genes in upland cotton. The expression levels of C2H2-zinc finger genes family is a pointer of their involvement in various biochemical and physiological functions which are directly related to cotton fiber development during initiation and elongation stages. This work not only provides a basis for determining the nominal role of the C2H2-zinc finger genes in fiber development but also provide valuable information for characterization of potential candidate genes involved in regulation of cotton fiber development.The online version of this article (10.1186/s12870-019-2003-8) contains supplementary material, which is available to authorized users. Furtherchocarpa  and 211 chocarpa . C2H2-zichocarpa . They plchocarpa , floral chocarpa , seed gechocarpa . In ricechocarpa –18. It hm annuum . Moreove tobacco . In cott) mutant . Recentl) mutant . Cotton ) mutant  in more ) mutant . Advance) mutant  Fiber in) mutant . Elongat) mutant  Cotton f) mutant . TherefoG. hirsutum (upland cotton) genome sequence [G. arboreum [G.raimondii [V. vinifera, Arabidopsis and P. trichocarpa. G. raimondii and G. arboreum underwent whole genome duplication events about 16.6 million years ago (Mya), and upland cotton  emerged from hybridizations of A or D diploid ancestral species nearly 1.5 Mya, which produce high quality of fiber as compare to their diploid relatives. To understand if at all increase or decrease of the number of the Zinc figer genes could have resulted into any changes in the dynamics of cotton fiber development. Moreover, upland cotton is a polyploid species in which its genome contains both maternal genome (G. arboreum like A-genome) and paternal genome (G. raimondii like D genome) [G. hirsutum, G. arboreum and G. raimondii, and their chromosomal positions, duplicated gene events, phylogenetic relationships, gene structures, conserved motifs and expression profiles at different developmental stages were analyzed.In addition, the current published sequence , G. arboarboreum  and G.raaimondii , provide genome) . Minimal genome) . In high genome) . Here wehttp://pfam.sanger.ac.uk/) and used to query against entire cotton proteome sequence. We identified 420, 236 and 230 C2H2-zinc finger candidate genes in upland cotton, G. arboreum and G. raimondii, respectively. Additionally, the retrieved sequences were physically checked by SMART (http://smart.emblheidelberg.de/) to examine the presence or absence of the C2H2-zinc finger protein domain. Finally, 386, 196 and 195 C2H2-zinc finger genes were identified in upland cotton, G. arboreum and G. raimondii, respectively and contained one or more C2H2-zinc finger protein domains  [G. hirsutum. The difference could be due to improved gene annotation. The lengths of all upland cotton C2H2-zinc finger proteins had a range of 101 to 1614 amino acids with an average of 374 amino acids. In order to understand the possible function of the proteins encoded by the gene under investigation, understanding their physiochemical properties is inevitable, for example the proteins can be separated according to their molecular mass (size) and isoelectric point (charge) properties and their abundance then determined subsequently [G. hirsutum is a polyploid, having emerged through whole genome duplication (WGD) [In the identification of all the C2H2- zinc finger gene members, in the whole cotton genome, Hidden Markov Model (HMM) profile of C2H2-zinc finger protein domain PF00096), obtained from Pfam database (edu.cn/)  in whichequently . The isoequently . Moreoveequently . The mol, obtaineon (WGD) .GhZF 1 to GhZF 378, while only 8 GhZF genes were not mapped to any chromosome as referred to scaffolds and designated as GhZF 379 to GhZF 386. One hundred and ninety-six (196) G. arboreum (A2) and 195 G. raimondii (D5) C2H2-zinc finger genes were all mapped to chromosomes and named based on their chromosomal positions. The distribution of C2H2-zinc finger genes on different chromosomes was not uniform  upland cotton C2H2-zinc finger genes were mapped across in all chromosomes and names assigned as per their chromosomal locations as 5, with tandem duplication  with an average of 1.05 mya which is consistent to whole genome duplication of upland cotton [2 and D5 genome, mostly arose from whole genome duplication events during their evolution.Gene duplication, tandem and segmental duplication events have been demonstrated to be the two main mechanisms initiating gene family expression in plants . The dupon Table . To expld cotton , 37. TheG. arboreum, 195 G. raimondii, 97 T. cacao, 179 Z. mays, 64 V. vinifera, 118 P. trichocarpa and 60 A. thaliana were used to construct phylogenetic tree, by employing the neighbor-joining (NJ) method of the MEGA 6 software. Based on the sequence similarity and tree topology, the C2H2-zinc finger proteins were divided into 3 main groups, designated as A, B and C  and G. hirsutum (At sub genome), as well as those between G. raimondii (D5) and G. hirsutum (Dt sub genome). The value of Ka/Ks ratio is an indicator of selective pressure acting on a protein coding gene during evolutionary time , which varied in number from 4 to 105 genes  of C2H2-zinc finger transcription factors were intronless and 48.4% (187) of C2H2-zinc finger genes had introns, which varied from 1 to 10. Moreover, most C2H2-zinc finger genes with the intron, 101 contained either 1 or 2 introns while the rest were disrupted by more than two introns  and cytokinin signaling. [Gh_A05G1815, Gh_D06G2303, Gh_A01G0984, Gh_D01G1033, Gh_A02G0836, Gh_D13G0451, Gh_A13G2112 among others. Some of these genes had differential expression during fiber development, implying that they could be involved in the regulation mechanism at a different stage of fiber development in cotton. High numbers of genes containing ALGGH motifs were found to be members of subgroup IV, which includes, Gh_A09G0743, Gh_D09G0744, Gh_D10G0401 and Gh_Sca004883G01  between Li1 mutant as compared to the wild-type during initiation (0 DPA) and elongation (8 DPA) stages. The result showed that 63 and 96 C2H2-zinc finger genes were differentially expressed between Li1 mutant and wild-type at 0 and 8 DPA, respectively  and 8 down-regulated genes , which were differentially expressed at initiation and elongation stages of fiber development. RT-qPCR was used to validate the expression levels of the C2H2-zinc finger gene family in Li1 mutant and wild-type at different stages of cotton fiber, 0, 5, 8 and 10 DPA , but not in wild-type. Gh_A03G1611 was expressed highly expressed in Ligon-lintless-1 at 5, 8 and 10 DPA but showed low expression level at 0 DPA in Li1 mutant as compared to wild-type. The outcome of this result was consistent with RNA-seq analysis which all these genes were up-regulated in Li1 mutant as compared to its wild-type, indicating these genes could be having role in inhibiting normal processes in fiber initiation and development. In contrast, 8 down-regulated C2H2-zinc finger genes such as Gh_A10G1157, Gh_Sca004794G01, Gh_D08G1830 and Gh_A01G2114 were expressed highly in wild-type had lower expression levels in the Ligon-lintless-1 mutant at various stages of cotton fiber development, which indicate that these are important for normal fiber development in mutant cotton. Other groups of C2H2-zinc finger genes such as Gh_A07G2108, Gh_D01G1033, Gh_A09G2473 and Gh_D05G0849 were expressed at different levels in the wild-type and Ligon-lintless-1 mutant, which could be an indication of functional divergence of C2H2-zinc finger genes during initiation and elongation stages of cotton fiber development.To examine whether these differentially expressed C2H2-zinc finger genes had a role in cotton fiber development during initiation and elongation stages, 16 C2H2-zinc finger genes were selected based on their expression patterns at initiation and elongation stages of cotton fiber development, 8 up-regulated genes  which is homolog to Gh_D09G0619 involved in regulating trichome development (initiation) through integrating cytokinin and gibberellin signaling pathways in Arabidopsis. These two Arabidopsis genes and their homologous genes from upland cotton showed low expression levels in diverse stages of fiber development, suggesting that these genes may play a negative role in controlling fiber development by integrating plant hormones or plant pathways. AT2G41940 (ZFP8) and ZFP5 interaction, facilitates the regulation of epidermal cell differentiation [Gh_A05G2741, Gh_D05G3769, Gh_A04G0449 and Gh_Sca045498G01 (homologs to AT2G41940) were significantly expressed at initiation stages (0 and 3 DPA) but not in elongation stage of cotton fiber. C2H2-zinc finger genes were differentially expressed in Li1 mutant and wild-type during fiber development. A total of 27 and 45 C2H2-zinc finger genes were down-regulated at 0 and 8 DPA, respectively, while 36 and 51 C2H2-zinc finger genes were up-regulated at the same period of 0 and 8 DPA, respectively, which provide a stronger evidence of the involvement of these genes either positively or negatively in initiation and elongation stages of cotton fiber development of Li1 mutant and the wild-type  genome sequence were extracted from the Cotton Gen database (http://www.cottongen.org); G. arboreum (A2) genome sequence was downloaded from the Cotton Genome Project (http://cgp.genomics.org.cn/page/species/download). The Hidden Markov Model (HMM) profile of the C2H2-zinc finger domain (PF00096) was downloaded from Pfam database (http://pfam.sanger.ac.uk/) and was used to identify the C2H2-zinc finger genes in the cotton genome (proteome sequence) using HMMER 3.0 software [− 10. Moreover, the corresponding protein sequences of C2H2-zinc finger proteins were downloaded from Arabidopsis database , T. cacao, Z. mays, V. vinifera and P. trichocarpa protein sequences were obtained from the plant transcription factor database (http://planttfdb.cbi.edu.cn/). Hence, they were utilized as query sequences to identify all the cotton C2H2-zinc finger proteins encoded by searching also against cotton proteome sequences, followed by removal of the same sequences from all the search results. Furthermore, to confirm the protein sequences derived from the selected cotton C2H2-zinc finger, candidate genes were examined using the domain analysis SMART (http://smart.emblheidelberg.de/). Only the protein sequences with C2H2-zinc finger domains were taken for further analyses. All redundant sequences were manually removed, resulting in 386 protein sequences in G. hirsutum contained C2H2-zinc finger domain. Moreover, the isoelectric points (pI) and molecular weights of upland cotton C2H2-zinc finger proteins were estimated by ExPASy Server tool (http://web.expasy.org/compute_pi/). In addition, WoLFPSORT (http://wolfpsort.org/) was used to predict the subcellular localization of the upland cotton C2H2-zinc finger proteins.The upland cotton and software  with E vG. arboreum, G. raimondii, T. cacao, Z. mays, V. vinifera, P. trichocarpa and A. thaliana C2H2-zinc finger proteins were performed using ClustalW . Phylogenetic tree was constructed with MEGA 6.0 software (http://www.megasoftware.net/) using the neighbor-joining (NJ) algorithm with 1000 bootstrap repetitions. The tree was constructed with the following parameters: Substitution, Poisson Model; data subset to use, the p-distance, pairwise deletion; replication, bootstrap analysis with 1000 replicates. In addition, minimum evolution method was also used to validate the result of the NJ method. A separate phylogenetic tree was constructed with all the upland cotton C2H2-zinc finger proteins for further analysis.Multiple sequence alignments of upland cotton, http://gsds.cbi.pku.edu.cn/index.php) [http://meme.nbcr.net/meme/) [The Gene structure display server   was usedt/meme/)  was used− 6 (Mya) synonymous substitution rate per 10− 8 years for cotton [C2H2-zinc finger genes were located on cotton chromosomes based on their location data retrieved from the cotton genome sequences. The distribution of genes on the cotton chromosomes was mapped by Map Chart software. The duplicated genes events were identified based on the criteria defined in the previous study, which stated that the aligned region of two sequences covers > 80% of the longer sequence and the similarity of the aligned region is > 70% . The Dnar cotton .G. hirsutum L.), were used in this study, Ligon-lintless-1 (Li1) which is a mutant form and its wild-type (TM-1) were planted in the experimental field at the Institute of Cotton Research, Chinese Academy of Agricultural Sciences  under normal field conditions. The Ligon-lintless-1 (Li1) is a mutant upland cotton, with abnormal morphological characteristics such as distorted leaves, stems and significantly reduced or short lint fiber length approximately 4 to 6 mm on the mature seed [G. hirsutum β-actin gene (GenBank accession no: AY305733) was used as the reference gene and primers specific to 16 C2H2-zinc finger genes were used for RT-qPCR analysis based on their expression levels at initiation and elongation stages of cotton fiber development. The detailed information of RT-qPCR procedure and gene expression analysis was done as described by Salih et al. 2016 [Two Upland cotton  were downloaded from the database in National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/) under accession number (PRJNA248163) [http://cufflinks.cbcb.umd.edu/). Heat maps were generated and hierarchical clustering was performed using MeV_4_9_0 software [G. hirsutum (TM-1) [P-values were adjusted using Benjamini’s and Hochberg’s method to control the false rate [P-value ≤0.05 found using DESeq were categorized as differentially expressed.RNA-sequencing (RNA-seq) data from various tissues of wild-type (TM-1) (A248163) . To estiA248163)  (http://software . In addim (TM-1)  using Tom (TM-1) . Cufflinm (TM-1)  was usedm (TM-1) . The reslse rate . Only geAdditional file 1:Figure S1. Distribution of C2H2-zinc finger genes on cotton chromosomes. The chromosomal position of each C2H2-zinc finger gene was located to the G. hirsutum genome. (PDF 96 kb)Additional file 2:Figure S2. Phylogenetic tree analysis, gene structure and conserved motifs of upland cotton C2H2-zinc finger genes were performed. (A) An unrooted tree is constructed by MEGA6.0 software using the full length amino acid sequences of the 386 upland cotton C2H2-zinc finger proteins by the Neighbor-Joining method, with 1000 bootstrap replicates. Based on phylogenetic tree, upland cotton C2H2-zinc finger divided into seven main subgroups (group I to VII) and each subgroup indicated with different colors and (B) Exon-intron structure of upland cotton C2H2-zinc finger genes. The yellow boxes represent exons, black lines represent introns and blue boxes represent the upstream/downstream (UTRs). The sizes of exons and introns can be estimated using the scale at the bottom. (C) Expression patterns of upland cotton C2H2-zinc finger members in different tissues  and different of fiber developmental stages . The expression levels are represented by the color bar. (D) Distribution of conserved motifs in upland cotton C2H2-zinc finger members and different motif boxes present by different colors (motifs 1 to 20). (TIFF 6685 kb)Additional file 3:Figure S3. The neighbor-joining (NJ) and minimum evolution methods of C2H2-zinc finger proteins family in G. hirsutum. (TIFF 3125 kb)Additional file 4:Figure S4. Motif analysis of C2H2-zinc finger gene family in upland cotton. A total of 20 putative conserved motifs of upland cotton TPS proteins were identified using the MEME online program. (PDF 300 kb)Additional file 5:Table S1. Location, annotation and protein domains of C2H2-zinc finger genes in cotton genomes. (XLSX 54 kb)Additional file 6:Table S2. List of 386 C2HC2-zinc finger genes in upland cotton and their proteins sequence features including, proteins length, molecular weight and subcellular positions. (XLSX 23 kb)Additional file 7:Table S3. Synonymous substitution rates (Ks) and non-synonymous substitution rates (Ka) are represented for each orthologous gene pairs between upland cotton and their ancestral diploid cotton, G. arboreum (A2) and G. raimondii (D5). (XLSX 35 kb)Additional file 8:Table S4. Multiple sequence alignment of upland cotton C2H2-zinc finger proteins for each group. (PDF 494 kb)Additional file 9:Table S5. Expression patterns of C2H2-zinc finger genes at various developmental stages in G. hirsutum (TM-1) were measured by RNA-seq. (XLSX 49 kb)Additional file 10:Table S6. Expression patterns of C2H2-zinc finger genes at two stages of cotton fiber development in Ligon-lintless-1 and wild-type (WT) were measured by RNA-seq. (XLSX 38 kb)