ESKAPEEc contribute to a majority of bloodstream infections (BSIs) and their antibiogram have changed overtime, while data concerning about these alterations are lacking in China. Added that a paucity of studies referred to ESKAPEEc in pediatric BSIs, our study aimed to demonstrate the longitudinal alterations of ESKAPEEc distribution and antibiogram in adult and pediatric BSIs in Southwest China.Chi-square test or Fisher’s exact test was used to examine and compare temporal changes.A multicenter retrospective surveillance study was launched from 2012 to 2017. Data of China Antimicrobial Resistance Surveillance System (CARSS) was analyzed by Whonet 5.6 and Graphpad Prism 6 Software. Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) were the two predominant species. Carbapenem resistance was prevalent in 0.76, 4.60, 9.47,13.66, 59.47% of E. coli, K. pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii), respectively. The proportions of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREFM) were 28.91% and 2.20%, respectively. Between 2012-2014 and 2015–2017, E. coli and K. pneumonia showed significantly increased resistance rates to imipenem but decreased to ceftriaxone and ceftazidime, while A. baumannii exhibited reduced resistances to almost all the beta-lactams tested. The prevalence of antimicrobial resistance to most of agents against Gram-positive ESKAPEEc did not significantly varied during the same timeframe. In comparison with those from adult BSIs, K. pneumoniae from pediatric BSIs exhibited high resistance rates to all the beta-lactams tested, especially to carbapenems (12.79% vs 3.87%), while A. baumannii showed low resistance rates to all the agents.A total of 32,259 strains was isolated, with 17.4% from pediatric BSIs. ESKAPEEc contributed to 58.67%  of BSIs, with 65.3% of adult BSIs and 27.2% of pediatric BSIs. E. coli and K. pneumoniae call for continued surveillance. Carbapenems are still active against Gram-negative ESKAPEEc, except for A. baumannii and vancomycin or linezolid is still effective against Gram-positive ESKAPEEc. Carbapenem-resistant K. pneumoniae in children and carbapenem-resistant A. baumannii in adults necessitate effective antimicrobial strategies in consideration of age stratification.Ongoing burden of ESKAPEEc in BSIs and increasing trend of imipenem resistance in  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and Escherichia coli, have been reported to contribute 50–70% of all causative pathogens to BSIs [Sepsis has been recognized as a global health priority by world health organization (WHO) in 2017. Better assessment of antimicrobial resistance (AMR) of pathogens causing bloodstream infections (BSIs) is one of the most significant resolutions to stem the tide of BSIs and thus alleviate the burden of sepsis . ESKAPEE to BSIs –6. The s to BSIs . HoweverKlebsiella pneumoniae (10.6%) [Moreover, foregoing studies failed to discuss these alterations in pediatric populations. A longitudinal surveillance study in United States indicated that children witnessed an increased prevalence of carbapenem-resistant Enterobacteriaceae (CRE) in BSIs from 0.0% in 1999–2000 to 4.5% in 2011–2012 [To fill this gap, we launched a 6-year retrospective multicenter study from 2012 to 2017 to elucidate the longitudinal alterations of pathogen distributions and antibiogram of ESKAPEEc among pediatric and adult BSIs in Chongqing, Southwest China. Age distributions and AMR patterns were further compared between 2012–2014 and 2015–2017 two periods.This retrospective study was based on blood-culture proven BSIs and conducted in the first affiliated hospital of Chongqing Medical University, a branch of the China Antimicrobial Resistance Surveillance System (CARSS) in Southwest China, which included 54 microbiological laboratories from secondary or tertiary hospitals in Chongqing. Data of all the isolates were collected from the database of CARSS. Isolates were enrolled if specifically reported all the information including patients’ age, unique patient identification number and antibiotic susceptibilities with minimal inhibitory concentration (MIC) values. According to the Clinical Laboratory Standards Institute (CLSI) M39-A4, only the first isolate was evaluated in this present study, in consideration that inclusion of multiple isolates from an individual patient who has successive blood culture series, several episodes of different BSIs during hospitalization or poly-microbial BSIs may overestimate the risk of acquiring a resistant strain , 14.All participated laboratories followed standard procedures to fulfill blood culture, bacteria identification, and antimicrobial susceptibility testing on semi- or automated systems, according to the guidelines of CLSI. Antimicrobial susceptibilities of bacteria were interpreted by the recommendation of CLSI-M100 S27.Children was defined if patients were no elder than 14 years old, while adult was defined if patients were elder than 14 years old .Enterococcus faecium (E. faecium), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), Enterobacter cloacae (E. cloacae) and Escherichia coli (E. coli) [ESKAPEEc was a cluster of pathogens including E. coli) .K. pneumoniae, E. cloacae or E. coli strains was defined as resistance to at least one of carbapenems: ertapenem (MIC≥2), imipenem (MIC≥4) or meropenem (MIC≥4), while that of A. baumannii or P. aeruginosa strains was defined if resistance to either imipenem (MIC≥8) or meropenem (MIC≥8).Carbapenem resistance of Chi-square test or Fisher’s exact test. Statistical significance was confirmed if a two-tailed P value was no more than 0.05.Raw data was firstly processed by Whonet 5.6 software and then calculated on Graphpad prism 6 and SPSS v.21.0  software. Temporal changes in age distributions and AMR were further determined by E. coli (32.03%) was the most frequently isolated bacteria, followed by K. pneumoniae (11.10%), Staphylococcus epidermidis (10.23%) and S. aureus (6.05%). The total number of strains increased from 12,114 in 2012–2014 to 20,145 in 2015–2017 , largely due to the dramatical rise of E. coli (30.15% vs 33.16%) and K. pneumoniae (10.52% vs 11.46%) in isolation rate. During the same time frame, the percentages of A. baumannii (2.88% vs 1.99%) and P. aeruginosa (3.07% vs 2.59%) were decreased , whereas its resistance to meropenem, amikacin and piperacillin remained stable  was notably higher than that in adult BSIs (0.3%) .In contrast to SIs Fig. b. In briE. coli and K. pneumoniae, no significant difference in AMR profiles of E. cloacae was found between adult and pediatric BSIs , while isolates from children BSIs showed higher resistance rate to erythromycin . Similar trend was also observed in levofloxacin resistance . To our knowledge, this study presented the current available evidence of the time shifting of ESKAPEEc distributions and AMR patterns in children and adult BSIs from China. Our findings verified the increasing prevalence of ESKAPEEc in BSIs and their distinct AMR patterns in adult and pediatric BSIs.E. coli and K. pneumoniae in adult BSIs. Similar results were also concluded by a 9-year analysis of BSIs in Rome [ESKAPEEc contributed to 58.67% of BSIs, which was consistent with previous studies . Our obsA. baumannii and S. aureus. This drop may suggest that other successful species are prevalent in pediatric BSIs, but we noticed that CoNS was predominant in this present study and its predominance may override the prevalence of ESKAPEEc. Added that, data from Typhoid Surveillance in Africa Program of Sub-Saharan Africa had verified that children had significantly high odds of having a contaminated blood culture compared with adults [Interestingly, in accordance with an 11-year retrospective study in US , ESKAPEEh adults . Therefoh adults , 18.E. coli and K. pneumoniae. Similar trends were also observed by Lei Tian et al. [Unexpectedly, decreased resistance rates to ceftriaxone and ceftazidime but increased resistant rates to imipenem were observed among n et al. . The posn et al. –22.ISKpn27-blaKPC-2-ISKpn2 played an important role in CRKP transmission [blaNDM-1 among CRKP in Beijing, Shanghai and Shandong [blaNDM-1 among CRKP in Chongqing [Of note, our study alarmed high prevalence of CRKP (12.79%) among pediatric BSIs, which was higher than data reported by CHINET . Howeversmission . HoweverShandong –26. Our hongqing . TherefoE. coli was 0.76%, which was higher than results from an epicenter of CRE in the US with a ratio of 0.1% [The prevalence of carbapenem resistance among  of 0.1% . MoreoveA. baumannii in BSIs, reduced resistance rates to most of antibiotics were also observed, especially to carbapenems (From 63.61 to 55.86%). This rate of carbapenem resistance was lower than our previous study suggesting 71.19% of CRAB in BSIs [A. baumannii to all these antibiotics were 60% in adult BSIs and 25% in pediatric BSIs, respectively, hence, effective treatments to fight against A. baumannii BSIs are still in urgent need. Recently, Tigecycline Evaluation and Surveillance Trial (TEST) concerning global blood-borne pathogens reported 70.8% of A. baumannii were susceptible to minocycline and MIC90 of tigecycline against it was 2 μg/mL, suggesting the potential efficiencies of these two antibiotics to overcome A. baumannii BSIs [Accompanied with declined proportion of  in BSIs , but simnii BSIs .E. faecium, vancomycin or linezolid is still efficient to fight against Gram-positive ESKAPEEc in our settings.Moreover, TEST further reported high prevalence of MRSA and VREFM worldwide, with the proportions of 33.0 and 27.6%, respectively , while oSeveral limitations should be considered in our study. Firstly, although this study included almost all the microbiology laboratories in the secondary or tertiary hospitals in Chongqing, Southwest China, it excluded some laboratories which failed to perform antimicrobial susceptibility testing. Careful interpretation of our findings was advocated in different hospital settings. Secondly, this multicenter study was focused on laboratory based, blood-culture proven BSIs attributable to ESKAPEEc, it, therefore, was not designed to analyze blood culture contamination and to discuss hospital-onset or community-onset without sufficient clinical patient data. Thirdly, our study excluded the isolates with antimicrobial susceptibility results reported by Kirby-Bauer, which might lead to an overestimation of the incidences of antimicrobial resistance.E. coli and K. pneumoniae illustrated decreased resistances to ceftriaxone and ceftazidime but increased resistances to imipenem in the past 6 years. Carbapenems may be still efficient to fight against E. coli- and K. pneumoniae- BSIs, but it is noteworthy that high prevalence of CRKP in pediatric BSIs may wreck their efficiencies. Moreover, a decreasing tendency should not relax our vigilance of CRAB in consideration of their high resistance to all these first-line antibiotics and minocycline or tigecycline may be helpful to treat A. baumannii BSIs. AMR profiles of Gram-positive ESKAPEEc remained stable and vancomycin or linezolid remains active against them. Further stratified by age, most of ESKAPEEc from pediatric BSIs showed lower resistance rates to the first-line antimicrobials, except for E. coli and K. pneumoniae, which exhibited higher resistance rates to meropenem than isolates from adults, suggesting that AMR patterns of ESKAPEEc are associated with age. Taking into account age during the continuous surveillance and infection control of ESKAPEEc in BSIs was necessitated in Chongqing, Southwest China.Distributions and AMR patterns of ESKAPEEc had changed overtime and differed between adult and pediatric BSIs. We noted an ongoing burden of ESKAPEEc in BSIs, largely driven by an upward trend in adult BSIs. The two predominant species,


Pinus massoniana belonging to the genus Pinus (Pinaceae), which is a primary source of turpentine, was sequenced and analyzed in terms of gene rearrangements, ndh genes loss, and the contraction and expansion of short inverted repeats (IRs). P. massoniana CPG has a typical quadripartite structure that includes large single copy (LSC) , small single copy (SSC)  and two IRs . The 108 unique genes were identified, including 73 protein-coding genes, 31 tRNAs, and 4 rRNAs. Most of the 81 simple sequence repeats (SSRs) identified in CPG were mononucleotides motifs of A/T types and located in non-coding regions. Comparisons with related species revealed an inversion  in the LSC region; P. massoniana CPG lacks all 11 intact ndh genes . A pair of short IRs was found instead of large IRs, and size variations among pine species were observed, which resulted from short insertions or deletions and non-synchronized variations between “IRa” and “IRb”. The results of phylogenetic analyses based on whole CPG sequences of 16 conifers indicated that the whole CPG sequences could be used as a powerful tool in phylogenetic analyses.The chloroplast genome (CPG) of  It has trnaI-GAU, trnV-GAC and 16S rRNA) are located in the large IRs. However, there are some differences in the gene numbers among species because of the contraction or expansion of IRs ..P. massoP. massoniana organization with other conifers: P. taeda, Cedrus deodara, and Cryptomeria japonica. There were no structural variations between P. massoniana and P. taeda, while some inversions were observed among Pinus (P. massoniana and P. taeda), C. deodara, and C. japonica  changed to the inverted order in C. deodara   b. Large P. massoniana CPG, with minimum SSRs of eight, five, four, three, three, and three for mono-, di-, tri-, tetra-, penta- and hexa-nucleotides, respectively (P. massoniana CPG (ycf1 and ycf2) contained two or more SSRs motifs, while the other eight genes  contained one mono-SSR of the A/T type. Although gene sequences are strongly conserved in CPGs, some SSR variations had already been detected in CDSs [clpP and ndhA genes contain tri- and tetra-SSRs, respectively, in the P. edulis CPG [psbC, accD, cemA, petA, ycf2 and ycf1) were found that contained two or more SSRs in the Ananas comosus CPG [P. massoniana CPG [Microsatellites are SSRs with motifs of 1–6 bp that are distributed throughout the nuclear or plasmid genomes. In this study, 81 SSRs were detected in the ectively . The majectively a. Of theectively b. The siiana CPG c. Among  in CDSs . Some di in CDSs . The clposus CPG . These aiana CPG . In addiiana CPG . These andh genes are located in nuclear, mitochondrial, and CPGs that can encode the NAD(P)H-dehydrogenase-like (NDH). With the exception of some species in Pinaceae [ndh genes, including ndhA–K, were found in most land plant CPGs. In this study, the comparisons with CPGs of C. japonica [G. biloba [ndh genes. Among these ndh genes in the masson pine CPG, the four genes ndhA, ndhF, ndhG and ndhJ have been lost completely, the ndhC and ndhE genes remain as pseudogenes because of short insertions or short deletions, and the other five ndh genes  remain as truncated pseudogenes  lost their 3′ ends. ndhK lacks a short sequence from the 3′ end (79 bp), but it was not classified as a truncated pseudogene in a previous study of Pinus thunbergii [The Pinaceae , OrchidaPinaceae , and GnePinaceae , 11 ndh japonica  and G. b. biloba  indicateudogenes . As showunbergii .ndh gene loss have been observed. Some ndh genes are lost completely or transferred to nuclear genomes. For example, some non-functional plastid ndh gene fragments are found in the nuclear genome of Picea abies [ndh genes are retained as pseudogenes in CPG which are absent in the nuclear genome. For example, most of the ndh genes lost from CPGs are not located in the nuclear genomes of particular orchids [ndh genes is not necessary in some photoautotrophic plants [ndh genes  that were absent completely in the CPG of P. massoniana have been transferred to the nuclear genome. However, this still needs further verification.In previous studies, two phenomena of ea abies . Other n orchids . In addic plants ,16,17,18Large IRs play vital roles in maintaining the stability of the CPG ,7, and tPinus. The alignment results of “IRa” and “IRb” (screened using the reference sequences from P. taiwanensis) showed that the main causes of the variations are as follows: (1) Insertion or deletion of short sequences (variations in repeat numbers of microsatellite motifs). The IRs of P. massoniana are 10 bp shorter than those of P. taiwanensis because of the loss of a 5-bp microsatellite motif (AATGA and ACAAT) in two loci of IRs of P. massoniana  and an absence of large IRs. However, some variations in size and sequence were also found in short IRs among species in ssoniana a; (2) Noectively b. These  bp and a. biloba . Althoug. biloba , Taxodia. biloba  and LegumatK, rbcL and rpoB genes and non-CDSs of atpF-atpH, trnH-psbA and psbK-psbI have been used in phylogenetic studies [CPG sequences have been widely used in the phylogenetic analyses of land plants ,12,26. I studies ,20. HoweG. biloba as an outgroup. We obtained a congruent phylogenetic tree with different support values using maximum likelihood (ML) and Bayesian inference (BI) approaches . Total DNA was extracted using the Plant Genprep DNA Kit  and quantified using a NanoDrop 2000c . The entire CPG was amplified using PCR with 35 primer pairs [The needles of er pairs . AmplicoPinus taeda (KC427273.1).PCR products were evaluated using electrophoresis on agarose gels and purified using a Gel Mini purification Kit . All of the purified DNA products were sequenced using an ABI 3730 DNA sequencer. Sequence assemblies were performed using CAP3  and manuhttp://lowelab.ucsc.edu/tRNAscan-SE/) [http://ogdraw.mpimp-golm.mpg.de/) [The genome annotation was performed using a Dual Organellar GenoMe Annotator with default parameters , coupledcan-SE/) . The boumpg.de/) .http://pgrc.ipk-gatersleben.de/misa/misa.html) with the following parameters [The microsatellite motifs were identified using the MIcroSAtellite identification tool  . Specialng MAFFT  based onng MAUVE .Ginkgo biloba as the outgroup (The whole CPG sequences of 16 conifers were used in a phylogenetic analysis with outgroup . The phyoutgroup . The mul


Solenopsis invicta) encodes four hexamerin genes, hexamerin-like , hexamerin , arylphorin subunit alpha-like, and arylphorin subunit beta. In this study, a phylogenetic analysis of the S. invicta hexamerins determined that each predicted protein clustered with one of the orthologous Apis mellifera hexamerins. Gene expression analyses by RT-qPCR revealed differential expression of the hexamerins between queens and workers, and between specific task-allocated workers (nurses and foragers). Queens and nurses had significantly higher expression of all genes when compared to foragers. Hexamerin 1 was expressed at higher levels in queens, while hexamerin 2 and arylphorin subunit beta were expressed at significantly higher levels in nurses. Arylphorin subunit alpha-like showed no significant difference in expression between virgin queens and nurses. Additionally, we analyzed the relationship between the expression of hexamerin genes and S-hydroprene, a juvenile hormone analog. Significant changes in hexamerin expression were recorded in nurses, virgin queens, and foragers 12 h after application of the analog. Hexamerin 1 and arylphorin subunit alpha-like expression were significantly lower after analog application in virgin queens. In foragers, hexamerin 2 and arylphorin subunit beta were significantly lower after analog application, while in nurses expression of all genes were significantly lower after analog application. Our results suggest that in S. invicta hexamerin genes could be associated with reproductive division of labor and task-allocation of workers.The reproductive ground plan hypothesis proposes that gene networks regulating foraging behavior and reproductive female physiology in social insects emerged from ancestral gene and endocrine factor networks. Expression of storage proteins such as vitellogenins and hexamerins is an example of this co-option. Hexamerins, through their role modulating juvenile hormone availability, are involved in caste determination in termites. The genome of the fire ant ( Apis mellifera, Vg levels decrease as the workers transition from nursing to foraging tasks [Pogonomyrmex barbatus nurses express high levels of Vg transcript which decrease as the individual transitions into foraging tasks [Ectatomma tuberculatum, production of Vg is related to the worker age: workers produce high levels of Vg at the beginning of their adult life when they conduct tasks associated with the brood inside the nest, while older workers which perform tasks outside the nest do not produce Vg [The reproductive ground plan hypothesis proposes that the gene networks that regulate foraging behavior and reproductive female physiology in social insects emerged from ancestral networks of genes and endocrine factors ,2,3. Exang tasks , and silng tasks . Similarng tasks . Similaroduce Vg .A. mellifera [Pogonomyrmex californicus, Myrmicaria eumenoides and Harpegnathos saltator foragers have higher JH titers than the workers performing in-nest tasks [E. tuberculatum sterile workers following topical application of JH [In insects, juvenile hormone (JH) is a developmental hormone during the immature stages, and it can play a gonadotropic role promoting reproduction in adults . The behellifera ,23,24. Ist tasks ,26,27. Fon of JH .Polistes metricus, a social wasp, insects that will emerge to become gynes have higher Hexamerin 1 protein levels compared to those that will become workers [A. mellifera, two hexamerin genes are expressed in the adult fat body in a caste- and sex-specific manner, with higher expression in workers than in queens [In addition to Vg, hexamerins, a second family of storage proteins, have been also associated with social organization. Hexamerins, also known as larval storage proteins, are synthesized in the fat body, and are secreted and accumulate in the hemolymph of larvae. Later in development, they are reabsorbed into the fat body, broken down, and incorporated into adult tissues during the pupal molt . In term workers . Further workers ,34,35,36n queens . In antsn queens . In somen queens ,39.Solenopsis invicta is of particular interest because this species displays extreme reproductive division of labor between two basic caste forms. The worker caste is composed of polymorphic sterile females responsible for the tasks that support the growth and maintenance of the colony [S. invicta and we identified differences in the expression of Vg1 among workers performing different tasks [hexamerin-like  and arylphorin subunit alpha-like (LOC105192898), is significantly reduced in brains of mated queens [hexamerin  and arylphorin subunit beta (LOC105192897). Moreover, the topical application of the JH analog S-hydroprene resulted in decreased expression of hexamerin 1 in whole bodies of virgin queens [Investigating the expression and function of storage proteins in e colony , while tnt tasks . Furthernt tasks . In a did queens . In the n queens . HoweverS. invicta has not yet been investigated. In particular, knowledge gaps exist regarding the relationship between hexamerin expression and JH, a key regulator of social organization. In this species, while JH has retained its gonadotropic role in the queen [S. invicta has not been investigated. Thus, in this study, we evaluated the expression of the four hexamerin genes in S. invicta workers and queens, and investigated whether those genes were regulated by a JH analog in a caste- or task-specific manner.Despite the knowledge about the involvement of storage proteins in caste determination in social insects, the expression and roles of hexamerin genes in he queen , it is sS. invicta were collected in College Station, Brazos County, Texas, from July to September of 2017 and maintained in the Department of Entomology at Texas A&M University, College Station, Texas, as laboratory colonies in plastic containers (27 x 40 x 9 cm). The inside of these containers was coated with Fluon to prevent ant escape . The colonies were kept at 27± 2°C in a 12:12 hour dark-light photoperiod. Colonies were provided with half-filled water tubes, capped with cotton as a damp nesting area, and fed daily with both 20% honey solution and crickets (Acheta domestica) as a protein source. Water was provided ad libitum. Colonies contained mated queens , virgin queens , drones, polymorphic workers and brood . Mated queens for gene expression analyses were collected immediately after a mating flight in College Station, Texas in May 2018. These mated queens were kept as previously described in a container with half-filled water tubes, capped with cotton as a damp nesting area for approximately one week after the first batch of eggs were laid.Polygyne colonies of Apis mellifera, Nasonia vitripennis, Camponotus floridanus, Harpegnathos saltator, Acromyrmex echinatior, Atta colombica, and Lasius niger hexamerin proteins were identified using Blastp searches or mining the respective genomes. The protein sequences were downloaded in fasta format and aligned using MAFFT v.7 [http://tree.bio.ed.ac.uk/software/figtree/) and rooted using the termite Reticulitermes flavipes hexamerin proteins as the outgroup.AFFT v.7  using thAFFT v.7  and the AFFT v.7  on the CAFFT v.7  with fouVirgin queens  were selected based on the presence of wings. Mated queens  were selected if they had laid eggs. The workers were classified based upon their head width , and forGene expression analyses were conducted using pools of ten individual workers or of five individual queens to obtain sufficient RNA. Individuals in each pool were from the same colony; each pool was obtained from a different colony. Workers were pooled according to the task performed (nursing or foraging). Pooled alate virgin and mated queens were of unknown age. Pools were flash-frozen in liquid nitrogen upon collection and stored at -80°C until gene expression analysis.Insects were ground in liquid nitrogen to a fine powder using a mortar and pestle. Total RNA extractions were conducted using Trizol reagent  following the manufacturer’s protocol. Purification of RNA was performed using the Direct-zol microprep kit  for clean-up of the samples. RNA was resuspended in 20 μl of nuclease-free water. Genomic DNA was eliminated using the Turbo DNAse kit  following the manufacturer’s protocol. Total RNA was quantified using the Infinite 200 PRO NanoQuant , and its integrity was assessed using gel electrophoresis, visualized on a 2% agarose gel stained with ethidium bromide.rp18 which is stable among different S. invicta castes [For gene expression analyses, RT-qPCR reactions were performed using the SensiFAST SYBR Hi-ROX One Step kit  following the manufacturer’s protocol. All reactions contained 50 ng of total RNA, 250 nM of forward and 250 nM of reverse primers , 2X Sensa castes . For eacAlate virgin queens and medium-size workers (nurses and foragers) were collected and treated topically with 1 μl of the JH analog, S-hydroprene  (25 ng/μl dissolved in acetone) with a pipette on the dorsal surface of the abdomen, or with 1 μl of acetone on the abdomen once during the assay. The chosen S-hydroprene dose was used in previous experiments on ants ,41. FresTen medium-size foragers, medium-size nurses, or alate virgin queens from each treatment group were collected twelve hours after treatment, pooled by treatment, flash-frozen, and stored at -80°C until gene expression analyses. Total RNA extractions from each pool and RT-qPCR analyses were conducted as described before.p-values were considered significant when below the 0.05 threshold.Data are reported as the mean ± the standard error of the mean (SEM). Data were log transformed and the normality of the log CT was verified after transformation. Statistical analyses were conducted using the one-way ANOVA test and Tukey-Kramer Post Hoc with JMP Version 13  and the estimated S. invicta hexamerins with other hymenopteran hexamerins , N. vitripennis NP_001164356.1 and N. vitripennis XP_001599110.3. Similar to the proteins from these other species, S. invicta Hexamerin 2 is characterized by a high glutamic acid and glutamine (Glx) content (25.4%)  . S. inviS. invicta hexamerins, which are adjacent in the S. invicta genome on the same contig (NW_020521759.1), show a higher degree of similarity among themselves (S. invicta Hexamerin 1 clustered with A. mellifera NP_001011600.1 (Hex 70b). Interestingly, N. vitripennisXP_001605292.3 and XP_001600430.1, which had previously clustered with A. mellifera Hex 70b [S. invicta Arylphorin subunit alpha-like clustered with A. mellifera NP_001092187.1 (Hex 70c), and two N. vitripennis proteins, NP_001153417.1 and XP_016839327.1. Finally, S. invicta Arylphorin subunit beta clustered with A. mellifera NP_001104234.1 (Hex 70a) and N. vitripennis NP_001164332.1. Each of these S. invicta proteins were more similar to the ant hexamerins than to the A. mellifera or N. vitripennis hexamerins. Both S. invicta arylphorin proteins have high aromatic amino acid content (S. invicta hexamerins are leucine-rich (>10% leucine) or methionine-rich (> 4% methionine) proteins [The other three emselves . S. invi Hex 70b  were not content . Hexamer content . None ofproteins .hexamerin 1, hexamerin 2, arylphorin subunit alpha-like and arylphorin subunit beta transcripts were obtained for alate virgin queens, dealate mated queens, medium-size foragers, and medium-size nurses. Significant differences were found in the expression of all genes. Foragers had the lowest expression of all tested genes (hexamerin 1 (p < 0.0001). Alate virgin queens had the highest level of expression, significantly higher than the other three groups, while dealate mated queens had significantly higher levels of expression compared to nurses and foragers, and nurses had significantly higher levels than foragers. Similar expression profiles were obtained for hexamerin 2 (p < 0.0001) and arylphorin subunit beta (p < 0.0001). Nurses had the highest levels of expression. Alate virgin and dealate mated queens had similar levels of expression, which was significantly higher than foragers. For arylphorin subunit alpha-like (p < 0.0001). No significant difference in expression was found between nurses and alate virgin queens. Dealate mated queens had significantly lower expression than the nurses and alate virgin queens, but significantly higher than foragers.The patterns of expression of ed genes . For hexamerin 1 , signifiamerin 2  . However, significant differences in expression were identified for hexamerin 2 , while differences were found for hexamerin 1 . Based on the phylogenetic analysis, they correspond to each of the four hexamerin types found in A. mellifera . This is consistent with previous analyses of hymenopteran genomes which typically harbor at least one copy of each of the four hexamerins commonly identified in A. mellifera [hex 70c in the leaf-cutting ants A. colombica and in A. echinatior which could be linked with the mutualism established between these species and fungi [hex 70c in the aforementioned ants, a homolog of hex 110 was not found in the current annotation of L. niger genome.In holometabolous insects, hexamerin proteins accumulate during larval development  and servS. invicta the four hexamerin genes were expressed in queens and in workers. The gene expression of all hexamerins was lower in foragers than in nurses or in queens. Hexamerin 2 and arylphorin subunit beta were expressed at higher levels in nurses than in queens, while arylphorin subunit alpha-like was expressed at higher levels in nurses than in mated queens, but not virgin queens. In ants, expression of hexamerin genes in adults, and differences of expression between the worker and queen castes were recorded in Formica exsecta [Camponotus festinatus has been associated with claustral colony founding [A. mellifera in which the expression of hex 70a and hex 110 genes in queens is relatively low and occurs in the ovaries of egg-laying queens [A. mellifera adult worker fat body [In  exsecta . In this exsecta ). Moreovfounding . This isg queens ,54,55. Ffat body .S. invicta hexamerins was down-regulated in foragers when compared to nurses. This finding is in accordance with transcriptomic results comparing gene expression between S. invicta nurses and foragers . Similaforaging . This ch ovaries ,58. S. iignaling ,41. For f larvae .hexamerin 1 expression in S. invicta virgin queens was down-regulated following topical application of a JH analog [S. invicta hexamerin genes following topical application of the JH analog to virgin queens, nurses, and foragers. The four genes were down-regulated in S. invicta nurses after application of the JH analog. However, only hexamerin 2 and arylphorin subunit beta were down-regulated in foragers, while hexamerin 1 and arylphorin subunit alpha-like were down-regulated in virgin queens following the topical application of the JH analog. Therefore, it is possible that JH regulates hexamerins differentially in the reproductive caste than in worker caste. The reduced expression of all hexamerins in foragers compared to nurses, and the reduction of hexamerin expression following the topical application of the JH analog to nurses are consistent with the existence of a low JH titer in nurses and a high titer in foragers as shown in other ant species.Previously, we had shown that H analog . Here, wS. invicta in a caste-specific manner  Materials used for the cage are a 15 mL tube, fine netting cut into a square, parafilm to hold down the netting. (B) Constructed treatment cage. (C) After treatment alate queens were put into tubes and lid fastened. (D) Cages were put back into the colony. (E) 100% of alate queens from the S-hydroprene treatment dealated.(TIFF)Click here for additional data file.S2 Fig indicates non conservative mutations, (*) indicates conserved regions, (:) indicates conservative replacement mutations and (.) indicates semi conservative mutations.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Helicobacter pylori infection is the principal cause of peptic ulcer disease, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma. Recent studies have shown that it may interfere with many biological processes and determine or influence the occurrence of many diseases outside the stomach. Currently, the role of H. pylori in idiopathic thrombocytopenic purpura and iron deficiency anemia is well documented. Emerging evidence suggests that it may also contribute to vitamin B12 deficiency, insulin resistance, metabolic syndrome, diabetes mellitus and non-alcoholic liver disease. Additionally, it may increase the risk of acute coronary syndrome, cerebrovascular disease, neurodegenerative disease and other miscellaneous disorders. Different pathogenic mechanisms have been hypothesized, including the occurrence of molecular mimicry and the induction of a low-grade inflammation. This review summarizes the results of the most relevant studies on the extra-gastroduodenal manifestations of H. pylori infection. Helicobacter pylori infection is the principal cause of chronic gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma ) ) . Polyzossubjects . Howevernd NAFLD , 64. Recnfection . NonetheH. pylori infection and NAFLD remains contradictory.In summary, the association between H. pylori and CAD in 1994 [H. pylori were associated with atherosclerosis [H. pylori plays an important role in the development of CAD by altering the lipid profile and enhancement of chronic inflammation [H. pylori were associated with high serum levels of interleukin-6 and B-type natriuretic peptide in patients with CAD [H. pylori infection increased the risk of acute coronary syndrome [H. pylori infection and the risk of myocardial infarction  [H. pylori and CAD [H. pylori eradication in decreasing the incidence of CAD.Mendall et al. first showed a link between  in 1994 . Severalclerosis –69. Al-Gammation . Figura with CAD . A natiosyndrome . In addi1.8–2.5) . However and CAD , 75. AddH. pylori infection and CAD. H. pylori has been detected in human carotid atherosclerotic plaques [H. pylori infection with systemic inflammation and endothelial dysfunction in healthy male subjects [H. pylori infection may cause atherogenesis through persistent low-grade inflammation. Recently, molecular mimicry between CagA antigen of H. pylori and atherosclerotic plaque peptides has also been proposed as a possible mechanism [There are several proposed mechanisms underlying the association between  plaques . Oshima subjects . They prechanism .H. pylori infection and CAD. No adequate interventional trials demonstrating a lower incidence of CAD as a result of anti-H. pylori therapy exit.In conclusion, there is controversial evidence linking H. pylori infection and stroke in 1996  [H. pylori predict incident stroke in fully adjusted models  [H. pylori infection and non-cardioembolic stroke [H. pylori infection and stroke mortality, and this reverse association was stronger for H. pylori cagA positivity [H. pylori infection and cerebrovascular disease.Wincup et al. first reported a link between to 2.60) . A Mexic to 2.3) . Recentlc stroke . Howeversitivity . In summH. pylori with dementia and Alzheimer’s disease (AD) [H. pylori infection in patients with AD than in the control group [H. pylori infection and non-Alzheimer’s dementia (non-AD) using a nationwide population-based dataset in Taiwan, and found that patients with H. pylori infection were 1.6-fold more likely to develop non-AD than those without infection [H. pylori was associated with a decreased progression of dementia as compared to no eradication of H. pylori in AD patients with peptic ulcers [Some studies also disclosed the relationship of ase (AD) , 84. A sol group . Hung etnfection . A retroc ulcers . HoweverH. pylori infection and allergic asthma has been reported. A meta-analysis by Zhou et al... in 2013 found lower prevalence rate of H. pylori infection in patients with allergic asthma [H. pylori infection has been found in cirrhotic patients with hepatoencephalopathy than in those without hepatoencephalopathy [H. pylori infection with elevated blood ammonia levels in cirrhotic patients [H. pylori infection increases the risk of colon adenocarcinoma and adenoma [H. pylori infection and chronic spontaneous urticaria has been reported but remains controversial. Fukuda et al. demonstrated a significant improvement of chronic spontaneous urticaria by anti-H. pylori therapy in Japanese patients [H. pylori infection and chronic spontaneous urticaria [The inverse relationship between c asthma . Higher alopathy . Jaing epatients . Several adenoma –92. Recepatients . This wopatients . Howeverrticaria .H. pylori infection and aforementioned miscellaneous disorders. Adequate interventional trials are needed to clarify these associations.In summary, there are still controversial evidences linking H. pylori may interfere with many biological processes and determine or influence the occurrence of many diseases outside the stomach (Table H. pylori infection is a perfect model for the study of interplay between human beings and bacteria. Further studies are mandatory to clarify the pathogenesis of extragastroduodenal diseases induced by H. pylori infection.Recent studies have shown that ch Table . Current


Entamoeba histolytica, Leishmania species (sp.), Trypanosoma brucei, T. cruzi, Cryptosporidium sp., Plasmodium sp., and Toxoplasma gondii. Clan CA proteases from three organisms  are well characterized as druggable targets based on in vitro and in vivo models. A number of candidate inhibitors are under development. CPs from these organisms and from other protozoan parasites should be further characterized to improve our understanding of their biological functions and identify novel targets for chemotherapy.Cysteine proteases (CPs) play key roles in the pathogenesis of protozoan parasites, including cell/tissue penetration, hydrolysis of host or parasite proteins, autophagy, and evasion or modulation of the host immune response, making them attractive chemotherapeutic and vaccine targets. This review highlights current knowledge on clan CA cysteine proteases, the best-characterized group of cysteine proteases, from 7 protozoan organisms causing human diseases with significant impact:  Proteases are enzymes that catalyze the hydrolysis of peptide bonds and are important in a number of biological activities, including digestion of peptides, activation of other enzymes, modulation of the immune system, participation in the cell cycle, and differentiation and autophagy. There are at least 6 classes of proteases classified according to the nucleophilic group responsible for the first step in the proteolysis: serine, cysteine, metallo, aspartate, glutamate, and threonine proteases. Cysteine proteases (CPs) are categorized into 72 families, but not all are represented in protozoan parasites . The mosDespite the essentiality of CPs for parasite survival, there are, as yet, no approved drugs targeting this group of enzymes. The most advanced drug in development against a parasite CP is the vinyl sulfone inhibitor K11777, with in vivo efficacy in an animal model of Chagas disease , 3; K117E. histolytica causes amebiasis and is responsible for about 70,000 deaths per year  deaths estimated in 2015 using different modeling approaches. Plasmodium falciparum and P. vivax are the most common species infecting humans, with P. falciparum responsible for nearly all deaths. The parasites express multiple CPs, some of which are the subjects of recent reviews [P. falciparum, the genome sequence predicts 33 CP-like proteins, although a number of these are probably not active enzymes. The best characterized are 4 falcipains and 3 dipeptidyl peptidases, all clan CA proteases [Malaria is by far the deadliest parasitic disease of humans, with 446,000 or 631,000 , 95 deat reviews –98. For roteases . in a cooperative process involving enzymes of multiple catalytic classes, including CPs, aspartic proteases, and metalloproteases. Incubating parasites with broadly active CP inhibitors causes the food vacuole to swell and fill with undegraded hemoglobin, suggesting CP essentiality in hemoglobin processing. CPs also contribute indirectly to hemoglobin hydrolysis via the processing of plasmepsins to active enzymes [P. falciparum trophozoites to fill with undegraded hemoglobin, but this abnormality resolved later in the life cycle, presumably due to expression of falcipain-3 [, the functional CP SERA6 [ and dipeptidyl aminopeptidase 3 [P. falciparum development , 111, and antibodies against the endogenous P. falciparum CP inhibitor falstatin blocked invasion, suggesting that inhibiting falcipain-like CP activity facilitates invasion [P. falciparum CP appears to be required for the development of erythrocytic parasites [P. falciparum otubain-like CP was recently shown to localize to the apicoplast organelle and to be required for normal apicoplast and parasite development via inhibition of the predicted role of P. falciparum Atg8 in protein import to the apicoplast [The functions of plasmodial CPs have been characterized using selective CP inhibitors and in some cases by gene knockout. Erythrocytic malaria parasites import erythrocyte cytosol and degrade hemoglobin in an acidic food vacuole as a source of amino acids  enzymes . The CPscipain-3 . In contcipain-3 . Some stcipain-3 . CP inhiCP SERA6 , and dipCP SERA6 , 106, alCP SERA6 , 107. ReCP SERA6 . Some sttidase 3 . Howeverinvasion . Considearasites , 114. Alicoplast .P. berghei, the orthologue of falcipain-1 appears to be critical for invasion of erythrocytes by hepatocyte-derived merozoites [.CPs appear to play additional roles in nonerythrocytic plasmodial stages. Considering liver stages, an unidentified plasmodial CP cleaves the circumsporozoite protein, which coats the sporozoites injected by mosquitoes, to enable invasion of hepatocytes . In the rozoites . Considerozoites , 118. AlP. berghei and P. vinckei; differences in specificity between falcipain-2 and falcipain-3 were less pronounced [; and mediation of binding to the native substrate, hemoglobin, by a small insertion near the C-terminus of the catalytic domain.Our understanding of the roles of CPs in the plasmodial life cycle suggests numerous potential drug targets. Falcipain-2 and falcipain-3 have low pH optima, consistent with activity in the acidic food vacuole, and both enzymes were localized to the food vacuole . Falcipaonounced . Both enonounced . The falP. falciparum development is accompanied by a specific block in hemoglobin hydrolysis, marked by the appearance of swollen, hemoglobin-filled food vacuoles, and antiparasitic effects correlated with the degree of inhibition of falcipain-2 and falcipain-3. Drug discovery directed against falcipains is facilitated by the available structures of falcipain-2 and falcipain-3 complexed with small-molecule and protein inhibitors .Multiple studies have demonstrated that CP inhibitors have potent antimalarial effects . With thPeptidyl falcipain inhibitors with nanomolar antimalarial activity have included fluoromethyl ketones, vinyl sulfones, and aldehydes; in some cases, in vivo activity against murine malaria has also been demonstrated . Promisi; thus, falcipain inhibitors should probably not be combined with artemisinins. In contrast, inhibitors of CPs and aspartic proteases showed synergistic antimalarial effects, consistent with a complementary role for these two classes of enzymes and suggesting the potential for synergistic combination antimalarial therapy [Concerning the potential for resistance, parasites were selected for resistance to a vinyl sulfone falcipain inhibitor, but the selection was slow and the mechanism of resistance complex, without mutations in target enzymes, suggesting that resistance to falcipain inhibitors may develop slowly, especially with combination therapy . Conside therapy .Toxoplasma gondii is a foodborne pathogen with seroprevalence ranging from 10%–30% in North America and northern Europe to more than 80% in areas of Latin America and Africa [T. gondii CPs have been shown to be important for invasion, digestion of host proteins for nutrition [Toxoplasma genome project revealed that the redundancy of CP genes is lower in T. gondii than in most other studied parasites. For example, E. histolytica has more than 50 genes encoding CPs with similar structure and specificity [T. gondii has genes encoding only one cathepsin B (TgCPB), one cathepsin L (TgCPL), and three cathepsin Cs , potentially making them more amenable drug targets. Active recombinant proteases have been expressed for all the T. gondii CPs, simplifying structure-based drug design.d Africa . Most ind Africa . T. gondutrition , 137, anutrition . The Toxcificity . In cont and key adhesins, the microneme proteins MIC2-associated protein (M2AP) and MIC3. TgCPL knockouts were attenuated in virulence in acute infection in mice [TgCPB and TgCPL have been linked to host cell invasion. Targeting TgCPB with a peptidyl cathepsin B inhibitor or antisense RNA inhibited host cell invasion and in vitro growth and blocked infection in a chick embryo model of toxoplasmosis . TgCPL a in mice . Both Tg in mice . In TgCPThe most developed peptidyl inhibitors target TgCPB and/or TgCPL. The crystal structure of TgCPL with morpholinurea-leucyl-homophenyl-vinyl sulfone has been determined, and the inhibitor can block host cell invasion  and cystT. gondii cathepsin Cs are exopeptidases that are also potential drug targets. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites; TgCPC3 was only identified in oocysts [The  oocysts . Both Tg oocysts .T. gondii tachyzoites to survive extracellular stress and for bradyzoites during latent infection [T. gondii genome, and there are no classic lysosomes in this organism, autophagosome-like bodies are formed [Autophagy is a key process in all eukaryotic cells to remove and recycle misfolded proteins and damaged organelles. Autophagy is likely to be important in nfection . Althouge formed , 145. TgT. gondii have been identified as potential vaccine candidates. DNA vaccines containing TgCPB or TgCPL gene sequences individually or together produced both humoral and cellular immune responses in BALB/c mice. Following immunization, survival was prolonged following intraperitoneal challenge with tachyzoites, with the most significant effect from the combined TgCPB/TgCPL vaccine [The clan CA cysteine proteinases of  vaccine . Similar vaccine .Trypanosoma and Plasmodium) have at least one well-characterized clan CA CP: cruzain in T. cruzi, TbCatL in T. brucei, and falcipain-2 and falcipain-3 in the malaria parasite. These enzymes were validated as drug targets using a variety of inhibitor chemistries.From the 7 protozoan parasites causing human disease that are of interest in this review, only 2 genera :833–7. PubMed PMID: 10480720. Sijwali PS, Rosenthal PJ. Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum. Proc Natl Acad Sci U S A. 2004;101(13):4384–9. PubMed PMID: 15070727.Sajid M, McKerrow JH. Cysteine proteases of parasitic organisms. Mol Biochem Parasitol. 2002;120(1):1–21. PubMed PMID: 11849701.10.1146/annurev.pathol.1.110304.100151. PubMed PMID: 18039124 McKerrow JH, Caffrey C, Kelly B, Loke P, Sajid M. Proteases in parasitic diseases. Annu Rev Pathol. 2006;1:497–536. doi:


PERKs) are an important class of receptor kinases in plants. Receptor kinases comprise large gene families in many plant species, including the 15 PERK genes in Arabidopsis. At present, there is no comprehensive published study of PERK genes in G. hirsutum. Our study identified 33 PERK genes in G. hirsutum. Phylogenetic analysis of conserved PERK protein sequences from 15 plant species grouped them into four well defined clades. The GhPERK gene family is an evolutionarily advanced gene family that lost its introns over time. Several cis-elements were identified in the promoter regions of the GhPERK genes that are important in regulating growth, development, light responses and the response to several stresses. In addition, we found evidence for gene loss or addition through segmental or whole genome duplication in cotton. Gene duplication and synteny analysis identified 149 orthologous/paralogous gene pairs. Ka/Ks values show that most GhPERK genes experienced strong purifying selection during the rapid evolution of the gene family. GhPERK genes showed high expression levels in leaves and during ovule development. Furthermore, the expression of GhPERK genes can be regulated by abiotic stresses and phytohormone treatments. Additionally, PERK genes could be involved in several molecular, biological and physiological processes that might be the result of functional divergence.Proline-rich extensin-like receptor kinases ( Arabidopsis. The Arabidopsis receptor kinase family contains approximately 600 members and their homologs have been investigated in ~20 different species [Receptor kinases comprise large gene families in many plant species, including rice and  species ,2,3. The species ,5. RecepArabidopsis receptor kinases, including BRI1 and BAK1, which are involved in brassinosteroid (BR) perception [CLV1 and ERECTA receptor kinases are evidence that functional redundancy exists [PERKs) are an important class of receptor kinases. The Arabidopsis PERK family is similar to Brassica napus PERK1 and shares maximum sequence identity. In the Arabidopsis genome, a total of 15 PERK genes have been identified; however, their biological functions are largely unknown [PERK1 is localized to the plasma membrane as predicted for a receptor kinase and the gene is induced rapidly in response to wounding in Arabidopsis [PERK4 is a novel regulator of Ca2+ signaling that also mediates the early events leading up to the abscisic acid response in root tip growth [Previously, receptor kinases were classified into many distinct classes on the basis of motifs in their extracellular domains ,6. For erception ,8,9. Amorception ; for exay exists ,12,13. P unknown ,15. PERKbidopsis . Furtherp growth ,17.PERK genes in 15 different plant species, including 33 PERK genes in G. hirsutum. Next, sequence logos, phylogenetic analysis, biophysical properties, exon/intron and protein motif distribution, promoter cis-element analysis, chromosomal distribution, gene duplication, synteny analysis and Ka/Ks values were also determined. Furthermore, tissue-specific expression patterns, abiotic stress responses and functional roles with respect to BR signaling and other phytohormones were investigated for the G. hirsutum PERK genes. The present study will enable a detailed understanding of the molecular and biological functions of PERK genes in cotton.Plants face continuously changing environmental conditions during growth and development. Among these environmental challenges, abiotic and hormonal stresses are the crucial factors that affect plant growth and biological yield. Cotton is one of the leading fiber crops cultivated worldwide . Variousin silico approaches to identify a total of 207 PERK gene family members in 15 different plant species following confirmation with different tools such as PROSITE (http://prosite.expasy.org/), SMART (http://smart.embl-heidelberg.de/) and InterProscan 63.0 (http://www.ebi.ac.uk/interpro/)  of PERK genes and P. patens having only five genes, indicating that PERK genes were subjected to a large scale expansion in higher plants. Because our main interest is in upland cotton, G. hirsutum, we compared the retrieved sequences of two different sequenced genomes of upland cotton including BJI and NAU and found no differences among the candidate genes. For the following analyses we used the gene sequences retrieved from the NAU genome sequence database. Here, allotetraploid upland cotton had more than double the number of PERK genes compared to diploids cotton species such as G. arboreum and G. raimondii, illustrating the effect of polyploidy on the PERK gene family in G. hirsutum which is derived from the hybridization of two progenitor species resembling the diploids G. arboreum and G. raimondii.In this study, we used various terpro/) . The PERGhPERK family members including locus ID, the corresponding chromosome, start and end points, strand polarity, gene length (bp), CDS length (bp), protein length (aa), predicted protein molecular weights (MW) and isoelectric points (pl), predicted masses and predicted cellular localization. We found that 14 GhPERK genes originated from the At sub-genome, 15 from the Dt sub-genome and four were from scaffolds of the G. hirsutum genome. Predicted protein lengths range from 178-884 aa, with molecular weights (MW) of 19,755.66–93,594.32 Da for GhPERK7 and GhPERK14, respectively. Moreover, the cellular localizations of all GhPERK proteins were related to the plasma membrane. All other estimated biophysical properties are shown in We next determined the biophysical properties of the Arabidopsis, rice and upland cotton G. hirsutum , an earliest plant lineage (PERK genes were present in A. comosus (angiosperm), P. taeda (gymnosperm), P. patens (bryophytes), S. moellendorffii (lycophytes), dicots  and monocots . These results indicated that PERK genes were originated from early land plants phylostratum and potential orthologous genes of PERK are present throughout pant kingdom. Next to construct the evolutionary relationships among the 207 PERK genes from 15 different plant species including A. thaliana, G. arboreum, G. hirsutum, G. raimondii, G. max, P. trichocarpa, T. cacao, O. sativa, S. bicolor, Z. mays, A. comosus, C. reinhardtii, P. patens, S. moellendorffii and P. taeda, a Maximum likelihood (ML) tree was generated. Phylogenetic analysis grouped all PERK proteins into four clades (PERKa-d) on the basis of their sequence homologies , P. taeda (gymnosperm), A. comosus (angiosperm), dicots and monocots except C. reinhardtii (chlorophyte) and P. patens (bryophytes), indicated the evolution of these PERK genes after the split of lycophytes, chlorophyte and bryophytes. While PERK-b clade contains PERK genes from all plant species except P. taeda. Similarly, PERK-c clade lacks the PERK family members from S. moellendorffii, C. reinhardtii and P. patens. While PERK-d clade lacks the PERK genes from S. moellendorffii, C. reinhardtii, P. patens and P. taeda. Surprisingly, PERK genes from C. reinhardtii were only present in clade PERK-b. Interestingly, PERK genes from dicot and monocot plant species were randomly distributed to all four clades. Moreover, PERK genes from G. hirsutum were present in almost all clades with PERK-c having highest number (11) of GhPERK genes and PERK-b having only three GhPERK genes. These results demonstrated that PERK-b clade is an ancient group of PERK genes having PERK gene family members from almost all plant species except P. taeda. Further, the phylogenetic analysis indicated that G. hirsutum experienced significant gene family expansion, because it has almost double the number of PERK genes compared to the other plant species except Z. mays. At least eight pairs or triplets of GhPERK proteins cluster together in the phylogenetic tree, which can be interpreted as evidence of gene duplication. All species except for G. raimondii and S. bicolor had gene pairs derived from the same node, demonstrating that the PERK genes in all species included in our study have experienced gene duplication events that resulted in PERK gene family expansion. However, gene duplication among the different groups varies for the different species. These results indicate that gene duplication is the main contributor toward PERK gene family expansion in all plant species. These results could be considered as evidence for lineage-specific expansion of the PERK genes after the divergence of dicots and monocots. Further, a NJ tree was constructed for the cotton PERK proteins in order to investigate the relationship between the common ancestors of diploid (G. arboreum and G. raimondii) and allotetraploid cottons (G. hirsutum)  . The treGhPERK genes lack introns and that similar genes were clustered close to one another in the same groups (GhPERK genes showed conserved patterns of gene structure (exon/intron) and protein motif distribution for the At and Dt sub-genomes even though gene duplication occurred long ago during evolution.It has been previously reported that the exon/intron distribution pattern in a gene is related to its biological function. Exon/intron analysis along with phylogenetic tree results showed that all e groups A. Furthee groups B. In faccis-elements located in upstream regions, cis-element analysis is important to understanding gene regulation and function [cis-elements in the promoters of the GhPERK genes and categorized them based on their functional relevance (cis-elements were classified with respect to their functions in growth and development and light and stress responses. All of the GhPERK genes had various cis-elements for different responses such as the GCN4 and Skn-1 motifs (endosperm expression), the as-2-box (shoot-specific expression), the 3-AF1 binding site, AC-II, the CCGTCC-box, Sp1 and circadian for growth and development, ACE, AE-box, ATCT-motif, Box 4, Box I, Box II, G-box, GA-motif, GAG-motif, GATA-motif, GC-motif, GT1-motif, I-box, L-box, Sp1, TCT-motif and chs-CMA1a for light response and MBS and TC-rich repeats (For stress and defense), LTR (for low temperature), Box-W1 , ARE, CGTCA-motif, GARE-motif, HSE, TCA-element, TCCACCT-motif, TGACG-motif, TGA-element and TCCC-motif for different stress responses. Additional cis-elements that regulate responses to different hormones such as auxin (TGA-element and AuxRE-core), salicylic acid (TCA-element and SARE), gibberellin , MeJA (CGTCA-motif and TGACG-motif) and ethylene (ERE-box) were also identified in the promoter regions of many GhPERK genes.Because gene expression is regulated by promoters and by the binding of transcription factors to function ,40. We ielevance . The ideGhPERK genes were mapped to their corresponding chromosomal loci. The results showed that 14 genes mapped to At sub-genome chromosomes, 15 to Dt sub-genome chromosomes and four to scaffolds . Upland cotton is an allotetraploid crop that is a model species for the study of natural polyploidy [G. arboreum and G. raimondii [G. arboreum) or D (G. raimondii) genomes and the observation that the genes from any one from these four genomes  have the corresponding orthologous from the other three genomes provides evidence that cotton PERK genes may not have experienced genomic arrangements during polyploidization. A total of 149 orthologous/paralogous gene pairs were identified, of which 10 pairs were attributed to segmental duplication to form paralogous gene pairs within the At or Dt sub-genomes  for 22 different tissues including roots, stems and leaves, reproductive tissues such as torus, petals, stamens, pistils, calyces and ovules sampled at -3, -1, 0, 1, 3, 5, 10, 20, 25 and 35 days post-anthesis (DPA) and fibers sampled at 5, 10, 20 and 25 DPA. Heat mapping of gene expression indicated that genes with similar expression levels clustered close together and that the expression patterns for many of the genes differed considerably in the different tissues (GhPERK genes in previously published RNA-seq data from two lintless/fuzzless mutants named M1l and M2l [GhPERK1) was up-regulated and two genes (GhPERK13 and GhPERK26) were down-regulated in the M1l mutant, while the three genes display the reverse expression patterns in the M2l mutant. However, the relative expression of 10 genes was the same in both mutants. Overall, 13 GhPERK genes were down- while seven GhPERK genes were up-regulated for both mutants, indicating both positive and negative roles in cotton fiber formation  and fibers . Interestingly, the results were generally similar to analyses of previously published transcriptomic and RNA-seq data, as all 12 genes were highly expressed in leaves and also during some stages of ovule development. However, all estimated me genes C. TogethGhPERK gene expression in response to various abiotic stresses. First, we analyzed the relative expression of GhPERK genes in publicly-available transcriptomic data for changes related to abiotic stresses including cold, heat, salt and PEG (drought) at several time points [GhPERK genes was up- or down-regulated under different treatments at different time points , first we analyzed the tissue-specific expression levels of selected genes in the cotton brassinosteroid mutant Pag1 [GhPERK genes did not show high expression levels during fiber development and in the other tested tissues in the WT plants because Pag1 mutant plants display short stature with no obvious fiber development. The GhPERK genes could be responsible for this altered morphology through brassinosteroid catabolism but a more extensive investigation may be needed to explore the complex gene regulation. These results demonstrate the negative regulation of GhPERK genes in the brassinosteroid pathway.To investigate the roles of ant Pag1  comparedGhPERK gene expression can be regulated by brassinosteroids, we used qRT-PCR to examine the expression levels of GhPERK genes in cotton seedlings treated with the active form of brassinosteroid BL (brassinolide) and two brassinosteroid biosynthesis inhibitors, BRZ (brassinozole) and PCZ (propiconazole), for 0.5, 1, 3 and 5 hours  during root tip growth [cis-elements identified in this study, we evaluated the GhPERK genes for their responses to phytohormones including GA, IAA, SA and MeJA after 0.5, 1, 3 and 5 hours of treatment , P. taeda (gymnosperm), A. comosus (angiosperm), C. reinhardtii (chlorophyte), P. patens (bryophytes), seven dicots  and three monocots  into four phylogenetic clades. The phylogenetic analysis showed that the PERK proteins from S. moellendorffii, A. comosus andP. taeda divided the PERK proteins into these four clades. The phylostratum analysis of PERK gene family identified the earliest plant lineage as PERK genes were present in C. reinhardtii (chlorophyte), indicated that PERK genes were originated from early land plants phylostratum and potential orthologous genes of PERK are present throughout pant kingdom. Phylogenetic tree showed that PERK-b clade is an ancient group having PERK gene family members from all selected plant species except P. taeda. While, PERK-d clade could be more advance than others, lacking PERK genes from S. moellendorffii, P. taeda, C. reinhardtii and P. patens. The presence of PERK genes in each selected plant species, with 33 PERK genes from G. hirsutum and only five PERK genes from P. patens, demonstrated that PERK genes are evolutionary conserved and experienced a large scale expansion in higher plants. Moreover, cotton and cacao PERK genes depicted close relationship during phylogenetic analysis strengthening the previous finding that cotton and cacao share a common ancestor [Arabidopsis, rice and G. hirsutum at both the N and C termini and there was no compositional bias in any conserved amino acid residues indicating that the PERK genes are evolutionarily conserved which will help to identify the pattern of PERK protein sequence conservation in other plant species. The WOX and YABBY gene families were also found to be evolutionarily conserved and exhibit similar conserved sequence logos [In the present study, we classified PERK protein family members from ancestor . Sequencce logos ,45.GhPERK genes contain various predicted cis-elements that are specific for plant growth, development and light and stress responses. Transcription is regulated by the binding of transcription factors to upstream cis-acting regulatory elements. Many studies have shown the important role that light plays in the processes of plant growth and development [cis-elements such as the TCA and CGTCA elements for MeJA and SA stress, respectively [GhPERK gene promoter regions. In our study, most of GhPERK genes have features typical of genes involved in growth and the responses to various stresses. For example, AtPERK4 is involved in ABA-responsive root-tip growth in Arabidopsis [cis-elements in their promoters and were shown to be functionally relevant [The promoter sequences of the elopment . A numbeectively ,47, dehyectively , abscisiectively , heat stectively , and theectively  were prebidopsis ,17. Prevrelevant ,53.GhPERK genes on the At and Dt chromosomes of G. hirsutum indicates possible gene loss or addition through segmental or whole genome duplication events and incomplete genome sequencing could be an issue here as well. In addition, all GhPERK genes have no introns and the proteins have similar patterns of protein motif distribution. Previous studies have demonstrated the importance of introns in the evolution of various plant genes [GhPERK genes might have evolved relatively rapidly. Furthermore, the number of introns present early in gene family evolution were reduced over time, resulting in gene families with fewer or no introns [In our results, the uneven distribution of nt genes . Many gent genes ,52,54. Tnt genes . Introns introns  and thes introns , while gGhPERK gene family is an advanced gene family in which the members lost their introns over time but underwent expansion in a rapid phase of evolution.It has been established that tandem duplication might cause an increase in intron numbers and create new genes . ConsistG. hirsutum is an allotetraploid cotton species that is cultivated globally and accounts for 95% of global production [G. arboreum and G. raimondii, respectively) experienced whole genome duplication and, as the result of hybridization, the allotetraploid AtDt genome species G. hirsutum emerged between 5 and 10 mya (million years ago) [G. hirsutum is one of the best model species in which to study polyploidy in plants [oduction . The A- ars ago) . As allon plants . Gene dun plants . Gene dun plants . Duplican plants . Two dupn plants . In segmn plants . Approxin plants ,63. Durin plants .GhPERK genes, double the number of PERK genes found in any other plant species, which demonstrates the effect of polyploidy on the PERK gene family in G. hirsutum. Polyploidy is a feature of genome evolution in G. hirsutum that consequently doubled the size of the PERK family as a result of segmental and whole genome duplication (WGD). As previously reported, polyploidy enabled many plant species to adapt to new environments [GhPERK gene family can be evaluated by examining the PERK genes in the A- (G. arboreum) and D-genome (G. raimondii) cottons. Gene loss is an essential process that occurs after the hybridization event during genomic arrangements and chromosome doubling [PERK gene family in plants could be the result of segmental and whole genome duplication. Moreover, the G. hirsutum genome experienced fewer arrangements compared to paleopolyploid maize and Brassica [In the present we study identified 33 ronments . The dradoubling . Even thBrassica ,67.GhPERK genes. Segmental duplication is the main impetus during evolution and it has occurred in many plant genomes that contain many duplicated chromosomal blocks [Arabidopsis gene families experienced coherent evolutionary dynamics that led to gene family expansion [WOX, YABBY, GRAS, RH2FE3, MIKC-Type and MADS-Box, soybean WRKY and sesame heat shock proteins experienced expansion as the result of segmental and whole genome duplication [GhPERK genes clustered into pairs, which indicates an ancient genome duplication event. The two copies of a gene can be subjected to shuffling and rearrangements that create potential diversity. Four possible fates have been described for duplicated genes [PERK genes in the G. hirsutum genome may be important for the plant’s normal growth and development and in responding to phytohormone and abiotic stresses. In our study, we found that the GhPERK genes experienced strong purifying selection.Generally, polyploidy is associated with gene duplication and in our study we found that segmental and whole genome duplication were the main factors responsible for doubling the number of l blocks . For exaxpansion ,69. Furtlication ,70,71,72ed genes . One, onArabidopsis experienced whole genome duplication twice in the Brassicaceae lineage [PERK genes share a common ancestor, while D-genome and Dt sub-genome PERK genes belong to the same ancestor. Our investigations into cotton PERK gene family expansion provides useful information on chromosomal interaction and polyploidization in addition to differential rates of evolution and information transfer through inter-genomic hereditary. Taking all of our data together, we conclude that the cotton PERK gene family experienced segmental and whole genome duplication, which subsequently caused expansion of the GhPERK gene family. lineage . In Addi lineage . OrtholoArabidopsis, PERK1 was shown to be induced rapidly in response to wounding [PERK4 a novel regulator of Ca2+ signaling, mediates the early events of the abscisic acid response in root tip growth [A limited number of studies have been conducted to investigate the molecular and physiological functions of PERK proteins in plants. Previously, it has been reported that receptor-like protein kinases (RLKs) are likely to respond to external challenges in an-ever changing environment and are involved in hormonal response pathways, plant growth and development, self-incompatibility, cell differentiation, symbiosis and pathogen recognition . In Arabwounding . PERK4 ap growth ,17. HoweGhPERK genes are highly expressed in leaves and some stages of ovule development, demonstrating the positive roles that these genes may play in cotton leaf morphology with no obvious contribution to fiber development, implying a negative role in fiber development. Also, the expression of GhPERK genes can be significantly regulated (positively or negatively) by abiotic stress treatments such as cold, heat, salt, and PEG because all of the GhPERK genes displayed ubiquitous expression patterns that did not follow any particular fashion. Our qRT-PCR results agreed with previously published RNA-seq and transcriptomic data. Coupled with these findings, we conclude that GhPERK genes may provide important genetic strategies for breeding cotton lines that are tolerant to several abiotic stresses.The results of our study show that all predicted GhPERK genes could play a role in negative regulation of the brassinosteroid signaling pathway; the expression of all genes was up-regulated in all examined tissues in the cotton brassinosteroid mutant Pag1 compared to WT plants. Moreover, the expression of all tested GhPERK genes could be either up- or down-regulated by the active form of brassinosteroid, BL and by treatment with the two brassinosteroid biosynthesis inhibitors BRZ and PCZ. These findings show that further investigations are needed to explore the functions of GhPERK genes in the brassinosteroid signaling pathway. Additionally, all GhPERK genes showed significant responses upon exposure to several phytohormones including GA, IAA, SA and MeJA; the expression of GhPERK8, GhPERK9, GhPERK12, GhPERK23, GhPERK27 and GhPERK29 was up-regulated with a few exceptions, while GhPERK5 was down-regulated when the plants were exposed to all four plant hormones at all time points from 0.5 to 5 hours, supporting our hypothesis that GhPERK genes play essential roles in certain hormone signaling pathways. We can also conclude that the GhPERK genes experienced strong functional divergence following gene duplication during the evolution of the GhPERK gene family in cotton.Further experiments indicated that the PERKs) are an important class of receptor kinases. These are associated with multiple molecular and cellular mechanisms affecting developmental, signaling and physiological processes in plants [PERK genes were co-expressed with one or more other G. hirsutum genes. Gene Ontology (GO) showed that these genes were related to enzyme inhibitor activity (GO:0004857), polygalacturonase activity (GO:0004650), glycoprotein binding (GO:0005515), calcium ion binding (GO:0005509), actin binding (GO:0003779), zinc ion binding (GO:0008270) and defense response (GO:0006952). Additionally, a KEGG enrichment analysis revealed that these two PERK genes could be involved in plant-pathogen interaction (K02183), pentose and glucuronate interconversions (K01051), regulation of actin cytoskeleton (K05765), nitrogen metabolism (K01674), monoterpenoid biosynthesis (K07385) and microbial metabolism in diverse environments (K00860). These results indicated that PERK genes could be involved in several molecular, biological and physiological processes that might be the result of PERK genes functional divergence during long evolutionary history of cotton.Proline-rich extensin-like receptor kinases , G. hirsutum , G. raimondii , G. max (version 10), P. trichocarpa (version 2.0), T. cacao (version 10), O. sativa (version 10), Z. mays (version 10), S. bicolor (version 10), A. comosus (version 3.0), C. reinhardtii (version 5.5), P. patens (version 3.3), S. moellendorffii (version 1.0) and P. taeda (version 1.0). Moreover, the definition of the PERK (PF07714) domain was downloaded from Pfam: http://pfam.janelia.org/ [PERKs from other species. The G. arboreum database was downloaded from (ftp://bioinfo.ayit.edu.cn/downloads/) while G. raimondii and G. hirsutum were downloaded from COTTONGEN (https://www.cottongen.org/). Also, Arabidopsis database was downloaded from TAIR 10 (http://www.Arabidopsis.org), while sequence databases for the other plant species were downloaded from online Phytozome v11 . All retrieved sequences were verified by SMART (http://smart.embl-heidelberg.de/) [http://www.ebi.ac.uk/InterProScan/) [http://us.expasy.org/tools/protparam.html) and subcellular localization was predicted using softberry (www.softberry.com).The RK genes  and encolia.org/  and thenerg.de/)  and Inte PERK PF014 domainSequence logos for conserved amino acid residues and phylogenetic analysis were performed as described previously . In briehttp://gsds.cbi.pku.edu.cn/index.php) [http://meme-suite.org/tools/meme) [cis-element analysis in promoter regions, 2-kb upstream sequences were analyzed in PlantCARE [To determine exon/intron structure, bed-files obtained from databases were analyzed using GSDS 2.0 (dex.php)  and the ls/meme)  as descrls/meme) . For cislantCARE  and the GhPERK genes, extracted gff3-files of cotton genome annotation data (ftp://ftp.bioinfo.wsu.edu/species/Gossypium_hirsutum/ NAU-NBI_G) were used to map the genes to the chromosomes using MapInspect (http://www.plantbreeding.wur.nl/UK/software_mapinspect.html) [http://www.bork.embl.de/pal2nal/) [To determine the chromosomal distribution of ct.html) . Gene duct.html) . In shorct.html)  was usedal2nal/)  and the al2nal/) .G. hirsutum variety CRI24 and the cotton brassinosteroid mutant Pag1 were used for gene expression analysis. To determine the tissue-specific expression patterns, all sampled tissues were obtained from cotton plants grown under field conditions with standard cultural practices. Abiotic stress treatments were conducted as described previously [In our study, the eviously ,54. For GhHis3 gene (GenBank accession no. AF024716) was used as the internal control for normalization of gene expression and the qRT-PCR assays were performed with SYBR Green on a LightCycler 480 system . All the primers used in this study are given in GhPERK genes was downloaded from online tool ccNET [http://structuralbiology.cau.edu.cn/gossypium/cytoscape/network.list.php) and finally, we visualized co-expression network using Cytoscape (version 3.4.0) program [http://www.cottonfgd.org/analyze/).Total RNA was extracted from cotton samples using the RNAprep Pure Plant Kit  and cDNA was synthesized from 1 μg total RNA using the Prime-Script® RT reagent kit . The ol ccNET  (http:// program . AdditioPERK gene family members in 15 different plant species. Among these genes, we identified 33 GhPERK genes from the allotetraploid cotton species G. hirsutum. Sequence analysis of conserved amino acid residues among the PERK proteins from Arabidopsis, rice and G. hirsutum showed that PERK genes are highly conserved in plants. Phylogenetic analysis grouped all PERK proteins into four clades on the basis of sequence homology. GhPERK genes do not have introns and the protein motif distribution pattern is conserved across all proteins. Furthermore, the predicted cis-elements in the GhPERK gene promoter regions indicate their functional relevance to growth, development and the response to various stresses. Uneven distribution of the GhPERK genes on the At and Dt chromosomes of G. hirsutum may be due to gene loss or addition through segmental or whole genome duplication during evolution, and/or incomplete genome sequencing. Also, gene duplication and synteny analysis indicates that the cotton PERK genes experienced segmental and whole genome duplication during evolution that subsequently resulted in a major expansion of the GhPERK gene family. Additionally, our data shows that the GhPERK genes experienced functional divergence during evolution and their high levels of expression in leaves can be regulated by abiotic and hormonal stresses, indicating that members of this gene family could play crucial roles in hormone signaling pathways, especially for the brassinosteroids. Moreover, PERK genes could be involved in several molecular, biological and physiological processes that might be the result of PERK genes functional divergence during long evolutionary history of cotton.The present study identified 207


Trichomonas vaginalis is an organism of interest for understanding eukaryotic evolution. Despite having an unusually large genome and a rich gene repertoire among protists, spliceosomal introns in T. vaginalis appear rare: only 62 putative introns have been annotated in this genome, and little or no experimental evidence exists to back up these predictions.The human protozoan parasite T. vaginalis derived from the genome sequencing plus previous publications. After experimental validation and a new genome-wide search, we confirmed the presence of introns in 32 genes and 18 others were concluded to be intronless. Sequence analyses classified the validated introns into two types, based on distinctive features such as length and conservation of splice site motifs.This study revisited the 62 annotated introns of T. vaginalis. Our findings suggests the existence of two intron ‘families’ spread among T. vaginalis protein-coding genes. Additional studies are needed to understand the functional separation of these two classes of introns and to assess the existence of further introns in the T. vaginalis genome.Our study provides an updated list of intron-containing genes in the genome of The online version of this article (10.1186/s13071-018-3196-7) contains supplementary material, which is available to authorized users. Introns, intervening non-coding sequences in genes of eukaryotes, are precisely removed from pre-mRNA by splicing, yielding mature mRNA. Splicing is achieved by a ribonucleoprotein complex (i.e. the spliceosome) through recognition of sequence elements on the introns such as the 5' and 3' splice sites (SS) and the branch site (BS) . PhylogeTrichomonas vaginalis. This is a protozoan parasite of the human urogenital tract and the causative agent of trichomoniasis, the most prevalent non-viral sexually transmitted infection worldwide [T. vaginalis has experienced a recent genome expansion as a result of gene duplications, transposon activities and lateral gene transfer [T. vaginalis (~170 Mbp) harbours a protein-coding capacity of ~60,000 genes [T. vaginalis genes that contain spliceosomal introns is predicted to be very low (~0.001 introns/gene) [Kipferlia bialata, also a member of the Excavata, exhibits an average of ~7 introns/gene which is similar to the most intron-rich eukaryotic genomes [One species where our understanding of introns remains particularly patchy is orldwide . As a meorldwide , 10, T. transfer , 12. The00 genes , and the00 genes , 14. Desns/gene) , 15, 16.ginalis ~0 Mbp harns/gene) . Kipferl genomes .T. vaginalis genome, it is not clear whether this is a result of natural variation or limited data. It is also worth noting that only a very few introns have been assigned to protein-coding genes of T. vaginalis experimentally [T. vaginalis. A 35-nt intron, modified from Giardia lamblia [T. vaginalis [T. vaginalis protein-coding genes [Giardia-derived intron [Rab1a of T. vaginalis where the splicing motifs did not match to the strict motif used by the previous report [T. vaginalis, released between the aforementioned studies, included the annotation of 62 introns among the ~60,000 predicted protein-coding genes [While intron density appears low in mentally , 16. Vanmentally  were the lamblia , was shoaginalis . Mutagenng genes . Howeverd intron , suggests report . The drang genes .T. vaginalis has an intron-poor genome and short introns with conserved SS and BS, or whether other introns may be present in the genome. As a first step towards addressing this question, we revisited all T. vaginalis introns that were predicted from the genome annotation and described in previous publications [T. vaginalis experimentally. Splicing activity was confirmed by sequencing the exon-exon boundaries of the spliced products. Our results support the existence of two types of introns in T. vaginalis with distinctive length, SS and BS features.Given the limited data currently available, it is therefore difficult to ascertain whether these observations indicate that ications , 15, 16.T. vaginalis strain G3 was cultured in Diamond’s media [6 cells/ml) were washed and resuspended in 0.9 ml of phosphate-saline buffer. Cells were lysed by adding 0.1 ml of lysis buffer . Lysis was followed by phenol and chloroform extractions and the DNA/RNA from the aqueous phase was precipitated with 0.6 volumes of isopropanol. After washing with 70% ethanol and air-drying, the pellet was resuspended in 0.5 ml of TE buffer  followed by digestion with RNase A (5 μg/ml) and stored for use. Total RNA was extracted using TRIzol Reagent  and treated with DNase I  to remove gDNA contamination.’s media  suppleme’s media . BrieflyT. vaginalis protein-coding genes with exon count ≥ 2 were downloaded from trichdb.org . The available features of the 42 intron-containing sequences described by the two early studies  were extracted from the original publications [T. vaginalis strain G3. The old gene IDs given by the early publications were replaced with the TrichDB ID if the intron fragment was located in a protein-coding gene according to the current genome annotation.Sequences of the 61 ications , 16 and Splicing activity was assessed by reverse transcription and PCR (RT-PCR). The first-strand, complementary DNA (cDNA) was synthesised from the purified total RNA using SuperScript III Reverse Transcriptase (RT) and oligo dT primer as recommended . gDNA and cDNA were used as templates for polymerase chain reaction (PCR) using specific primers that target the flanking regions of the putative intronic sequences  were downloaded from the Sequence Read Archive at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/sra). These datasets contain expressed sequences of T. vaginalis strains G3 and B7RC2, respectively. Over 180 and 160 million sequencing reads of 125 bp long from these 2 datasets were imported to Geneious 11.1.2 separately and mapped to the 13 genes carrying the undetermined introns. The genes TVAG_416520 and TVAG_337250, containing a functional and a non-functional intron, were chosen as positive and negative controls, respectively. The number of sequencing reads from the RNA-Seq data covering each nucleotide position was determined for each gene. A marked drop in sequencing depth on the intron regions as compared to their respective flanking exons, and in reference to controls above, was considered as evidence of splicing activity.A strategy based on the available RNA sequencing (RNA-Seq) data was used to evaluate the splicing activity of 13 introns that could not be determined experimentally, as described above (i.e. undetermined introns). The two largest and most recent T. vaginalis were updated, according to the previous studies on T. vaginalis introns [de novo for matches on T. vaginalis genome segments using the DNA motif pattern search tool at TrichDB (http://trichdb.org/trichdb/showQuestion.do?questionFullName=SpanQuestions.DynSpansByMotifSearch). Potential hits found by this search were subjected to the same RT-PCR validation, as described above.Sequence properties of the newly discovered introns in this study were described using a Python script which reads FASTA sequences one by one and retrieves all intron-related information as presented here. Introns were classified here into two different families, based on sequence characteristics and putative splicing motifs. The consensus sequences of the two intron families in  introns , 16. TheT. vaginalis . The second dataset consists of 42 gene sequences, each carrying a single putative intron that were described in earlier publications [T. vaginalis genome. Nineteen of the 41 putative intron fragments predicted by Vanacova et al. [TvRab1a, the only intron-containing gene confirmed by Deng et al. [In this study, we interrogated two datasets of intron-containing genes in lis Fig. . The firications , 16. Theications , 16, wera et al.  were locg et al.  did not Primers flanking all 62 putative introns were designed to assess splicing activity using RT-PCR, gel electrophoresis and DNA sequencing  functional; (B) non-functional or (C) undetermined  of the protein-coding genes as one intron per gene. Finally, 18 introns in 17 protein-coding genes were assigned as non-functional as they were certainly transcribed but the predicted intronic sequences were not removed  instead of A or G as in type A introns (RCTAAC). In contrast to type A introns, a space flexibility between the branch adenosine and the 3' SS is apparently allowed for type B introns. The type B intron found in the gene TVAG_416520 shows that this distance can be either 7 or 8 nt . As this intron had not been experimentally validated  since cDNA samples indicate expression, but only yielded full-length PCR products, consistent with the absence of splicing activity. We could not ascertain the splicing status for the remaining 13 putative introns experimentally. However, by mapping available T. vaginalis transcriptome reads to these genes in silico, we observed that all 13 genes are transcribed and that five of them show read mapping patterns consistent with functional introns . Introns of type A conform to those previously reported by Vanacova et al. , except observed , i.e. siobserved . DespiteT. vaginalis [G. lamblia [T. vaginalis.We confirmed the original observation of the positional conservation of the branch adenosine, precisely 7 nt away from the 3' SS , among taginalis , 16 and  lamblia . HoweverIn addition to the short length of type B introns, the low conservation of the 5' SS is another feature that separates them from type A introns according to the previous mutagenesis study . Three oT. vaginalis transcript. All other introns were found in CDS of T. vaginalis protein-coding genes. The 5' UTRs of T. vaginalis mRNAs are known to be short in length, based on the close proximity between conserved core promoter elements and the ATG start codon [T. vaginalis protein-coding genes [T. vaginalis, further investigation is necessary to confirm the functional differences on sequence conservation of their splicing motifs.The type B intron found in TVAG_269270 after new genome search was the only one found in the UTR of a rt codon . These cng genes . The 5' T. vaginalis. This study is by no means an exhaustive screen: however, it indicates that more introns might be present in this genome. As expected, standard intron-prediction models potentially fail when applied to genetically divergent eukaryotes such as T. vaginalis. Transcriptomic-based surveys, such as the one recently conducted for G. lamblia and Spironucleus salmonicida [T. vaginalis. These studies together should help close our gaps of understanding in the evolution of splicing, a signature of eukaryotic life.In summary, we have examined evidence for splicing for a number of intron-containing genes in monicida , will beT. vaginalis genome. A large number of misannotated introns indicates the inaccuracy of intron prediction algorithms used by genome projects when applied to a non-model eukaryotic organism. From this updated list, we were able to identify two potential intron families carrying distinctive features. A new intron consensus allowed us to discover one additional intron-containing gene, suggesting that further studies may expand the list of introns in the genome of this evolutionarily divergent eukaryote.Our study provided an updated list of intron-containing genes in the Additional file 1:Table S1. Primers used in this study to validate the putative introns by RT-PCR. Nucleotides that target 5' and 3' UTRs were indicated with lower-case letters. Underlined bases are located outside the transcribed region of these genes. (PDF 36 kb)Additional file 2:Table S2. An update on the gene ID of the 42 introns previously reported, with references as indicated [ndicated , 16.  functional, (b) non-functional or (c) undetermined. (PDF 256 kb)Additional file 4:Table S3.T. vaginalis genome segments that match the consensus sequence of type A intron . (PDF 84 kb)Additional file 5:Table S4.T. vaginalis genome segments that match the consensus sequence of type B intron . (PDF 85 kb)Additional file 6:Figure S2. Evidence of gene transcription and mRNA splicing for the 13 putative introns that could not be experimentally determined in this study. (PDF 1422 kb)