Tetronarce californica  is a classic preparation for biochemical studies of cholinergic neurotransmission. To broaden the usefulness of this preparation, we have performed a transcriptome assembly of the presynaptic component of the electric organ (the electric lobe). We combined our assembled transcriptome with a previous transcriptome of the postsynaptic electric organ, to define a MetaProteome containing pre- and post-synaptic components of the electric organ.The electric organ of De novo Trinity assembly was performed at Kmer 25 (default) and Kmers 27, 29, and 31. Trinity, generated around 103,000 transcripts, and 78,000 genes per assembly. Assemblies were evaluated based on the number of bases/transcripts assembled, RSEM-EVAL scores and informational content and completeness. We found that different assemblies scored differently according to the evaluation criteria used, and that while each individual assembly contained unique information, much of the assembly information was shared by all assemblies. To generate the presynaptic transcriptome (electric lobe), while capturing all information, assemblies were first clustered and then combined with postsynaptic transcripts (electric organ) downloaded from NCBI. The completness of the resulting clustered predicted MetaProteome was rigorously evaluated by comparing its information against the predicted proteomes from Homo sapiens, Callorhinchus milli, and the Transporter Classification Database (TCDB).Sequencing yielded 102 million paired-end 100 bp reads. T. californica MetaProteome and the proteomes of H. sapiens and C. milli, using the H. sapiens genome as a reference. This information was used to predict the position in human pathways of the conserved members of the T. californica MetaProteome. We found proteins not detected before in T. californica, corresponding to processes involved in synaptic vesicle biology. Finally, we identified 42 transporter proteins in TCDB that were detected by the T. californica MetaProteome (electric fish) and not selected by a control proteome consisting of the combined proteomes of 12 widely diverse non-electric fishes by Reverse-Blast-Hit Blast. Combined, the information provided here is not only a unique tool for the study of cholinergic neurotransmission, but it is also a starting point for understanding the evolution of early vertebrates.In summary, we obtained a MetaProteome containing 92%, 88.5%, and 66% of the expected set of ultra-conserved sequences , expected to be found for Eukaryotes, Metazoa, and Vertebrata, respectively. We cross-annotated the conserved set of proteins shared between the The online version of this article (doi:10.1186/s12864-017-3890-4) contains supplementary material, which is available to authorized users. Tetronarce californica, can provide over 1 kg of tissue highly enriched in both pre and postsynaptic proteins involved in cholinergic transmission.Electric rays have a long history of scientific inquiry, dating back to ancient times . The strT. californica has been studied , receptor subunit epsilon-3 (NR2C) [GABA(A) receptor subunit beta-1 [T. californica, we found two proteins that have been previously identified in the Marbled electric ray T. marmorata - Dispanin, which is a type of auxiliary transport protein, and the proton conducting portion of the vacuolar-ATPase, the V-type proton ATPase 16 kDa proteolipid subunit [We then asked if the ts Table . The majlutamate , and the (ClC-2) , which m(Cav1.2) , and ligt beta-1 . Finally subunit .T. californica-specific hits described here  were found despite the use of a combined 12-Fish proteome  composed of 12 largely evolutionarily diverse organisms ranging from bony vertebrates (Euteleostomi like Coelacanth (Latimeria), Spotted gar (Lepisosteus), Zebrafish (Danio), Blind Cave Fish (Astyanax), Atlantic Cod (Gadus), Southern Platyfish (Xiphophorus), Japanese Medaka (Oryzias), Nile Tilapia (Oreochromis), Fugu (Takifugu), Amazon molly (Poecilia), Three-spined Stickleback (Gasterosteus), and Spotted Green Pufferfish (Tetraodon). Many of these organisms , arguably are closer to H. sapiens than T. californica. Also, note that of the 42 transporters identified, only 22 of them were present in the set of proteins shared by C. milli, H. sapiens, and T. californica and 12 were present in the set shared by both H. sapiens and T. californica, and absent in C. milli. This suggests that these transporters are specific to T. californica (an electric fish) and are not just present in the Chondrichthyes. Finding Dispanin, a Torpedo protein, validated both the assembly and protein prediction strategy employed. The RBH-Blast strategy used here is very specific, as it establishes a one-to-one relationship between two datasets. RBH-Blast establishes that the hits observed are mutual, both from the query and from the subject (or database) point of view. Importantly, these results do not argue against nor discard the existence of homologous proteins present in the 12-Fish proteome. These results do establish, however, that in a RBH-Blast between the T. californica and current components of the TCDB-database, the T. californica transporter proteins selected, showed higher performance both at the level of Identity and Coverage, than the proteins of the combined 12-Fish-Proteome set used in this work. As a result, we were able to identify transporter proteins not identified by the other fish proteomes. In summary, we find that the set of transporters preferentially identified by the T. californica MetaProteome is highly enriched in proteins that play key roles in important neurological processes. These results are consistent with electric fish having a set of transporters that have been ’optimized’ to respond and recover quickly from a massive cellular depolarization. These results are also consistent with the idea that the genomes of organisms like H. sapiens and Mus musculus, in addition to other transporters, also posses the set of such ’electrically-optimized’ transporters or derivatives of them.The de novo transcriptome corresponding to the electric lobe of T. californica. We critically evaluated the quality of our assemblies using ‘Industry Standard’ methods. We found a high degree of variability between assemblies produced at different Kmers. Neither the number of transcripts assembled nor the value of the Detonate scores calculated gave us a definitive prediction for best assembly. In contrast, we observed that the only reliable parameter for assembly evaluation was related to the information content of the assembly in question, when compared to a standard database. We also generated a non-redundant set of transcripts by combining the transcriptome of the electric lobe with previous transcriptome of the electric organ, and determined those predicted proteins having high homology against the genomes of both H. sapiens and C. milli. Finally, we mapped and cross-annotated these highly-conserved predicted proteins against the well annotated Human biochemical and developmental pathways. We also identified transporter proteins present in the T. californica MetaProteome and in a MetaProteome set corresponding to the proteomes of 12-Fish genomes and identified a set of important transporters that were only detected by the T. californica MetaProteome. The combined information provides not only a unique tool for the study of cholinergic neurotransmission, but also a starting point for understanding the biology of early vertebrates, as well as, the biology of strongly electric fish, such as T. californica.We have assembled a Tetronarce californica was dissected from the central nervous system, and total RNA was isolated from the frozen tissue as described [+RNA was purified using Poly(A)Purist MAG Kit (Ambion) and further cleaned up with Turbo DNase (Ambion) and Terminator-5’-phosphate-dependent exonuclease (Epicentre) to remove any trace amount of DNA and rRNA. cDNA was synthesized from purified poly(A)+mRNA using random hexamer oligonucleotide or oligo dT as primer. Synthesized cDNA was then sheared into small pieces of 100 to 800 bp in length using a Biorupter. The fragmented cDNA was prepared for Illumina sequencing using TruSeq Sample Prep Kit (Illumina). Paired-end sequencing (100 bp) of the cDNA libraries was performed on a HiSeq 2500 instrument (Illumina).The electric lobe from a female marine ray escribed . RNA conescribed . Poly(A)The description of the software, version number and origin (when applicable) and representative commands and databases used are presented in Additional file Briefly, reads quality score was initially evaluated using FastQC. The first and last nucleotides of every read were trimmed using fastx_trimmer (FASTX-Toolkit). Similarly, sequencing artifacts were removed using fastx_artifacts_filter (FASTX-Toolkit). Sequencing adaptors were then removed using Cutadapt . Reads wAssembly was performed using Trinity at four different Kmers . All assemblies were performed with the ’jaccard_clip’ flag on Additional file 2Summary Of The Software And Commands Used In This Work. (PDF 158 kb)Additional file 3Supplemental Tables: File containing Table S01-to-S07. (PDF 128 kb)Additional file 4T. californica MetaTranscriptome. Transcripts IDs and their origin. (XLSX 2806 kb)Additional file 5T. cali MetaORFeome proteins. (XLSX 855 kb)Cross-Annotation and GO Analysis of the SwissProt proteins that have a coverage of 70% or higher to Additional file 6Proteome IDs of Blast Results and Reverse Blast Hit Results. (XLSX 2887 kb)Additional file 7T. californica Transcripts Present in C. milli. (XLSX 2201 kb)Cross-Annotated Additional file 8T. californica Transcripts Present in H. sapiens. (XLSX 3276 kb)Cross-Annotated Additional file 9H. sapiens and C. milli. (XLSX 3880 kb)Cross-Annotated Proteins Present in Additional file 10T. californica Transcripts Present in C. milli Not Present in H. sapiens. (XLSX 269 kb)Cross-Annotated Additional file 11T. californica Transcripts Present in H. sapiens Not Present in C. milli. (XLSX 1075 kb)Cross-Annotated Additional file 12H. sapiens and C. milli Not Present in T. californica. (XLSX 1976 kb)Cross-Annotated Proteins Present in Additional file 13T. californica Transcripts Present in H. sapiens and C. milli. (XLSX 2211 kb)Cross-Annotated Additional file 14H. sapiens, T. californica and C. milli that have a coverage of 100% between H. sapiens and C. Milli. (XLSX 99.2 kb)Cross-Annotated conserved SwissProt proteins shared between Additional file 15Set10 – GO Biological Processes Complete. (XLSX 47 KB)Additional file 16Set08 – GO Biological Processes Complete. (XLSX 24.6 kb)Additional file 17Set09 – GO Biological Processes Complete. (XLSX 41.2 kb)Additional file 18H. sapiens With T. californica Without C. milii (set 8). See Additional file KEGG Analysis. Intersection of Additional file 19H. sapiens With C. milli Without T. californica (set 9). See Additional file KEGG Analysis. Intersection of Additional file 20H. sapiens With T. californica (set 12). See Additional file KEGG Analysis. Intersection of Additional file 21H. sapiens With C. milli (set 13). See Additional file KEGG Analysis. Intersection of lli set 1. See AddAdditional file 22Selected KEGG Figures corresponding to pathways presented in Table Additional file 23T. californica. (XLSX 41.3 kb)Data Corresponding to the Eukaryotic Transporter Proteins Only Detected by Additional file 24Fasta File Containing Nucleotide Sequences Shorter Than 200 bp That Are Part Of The MetaProteome. (FA 791 kb)Additional file 25Fasta File Containing Predicted Peptide Sequences From The Nucleotide Sequences Shorter Than 200 bp That Are Part Of The MetaProteome. (FA 889 kb)


Background. Experimental studies suggest that mechanical cell washing to remove pro-inflammatory components that accumulate in the supernatant of stored donor red blood cells (RBCs) might reduce inflammation and organ injury in transfused patients.Methods. Cardiac surgery patients at increased risk of large-volume RBC transfusion were eligible. Participants were randomized to receive either mechanically washed allogenic RBCs or standard care RBCs. The primary outcome was serum interleukin-8 measured at baseline and at four postsurgery time points. A mechanism substudy evaluated the effects of washing on stored RBCs in vitro and on markers of platelet, leucocyte, and endothelial activation in trial subjects.Results. Sixty adult cardiac surgery patients at three UK cardiac centres were enrolled between September 2013 and March 2015. Subjects received a median of 3.5 (interquartile range 2–5.5) RBC units, stored for a mean of 21 (sd 5.2) days, within 48 h of surgery. Mechanical washing reduced concentrations of RBC-derived microvesicles but increased cell-free haemoglobin concentrations in RBC supernatant relative to standard care RBC supernatant. There was no difference between groups with respect to perioperative serum interleukin-8 values  or concentrations of plasma RBC microvesicles, platelet and leucocyte activation, plasma cell-free haemoglobin, endothelial activation, or biomarkers of heart, lung, or kidney injury.Conclusions. These results do not support a hypothesis that allogenic red blood cell washing has clinical benefits in cardiac surgery.Clinical trial registration. ISRCTN 27076315. Editor’s key pointsExperimental evidence supports a benefit to red blood cell (RBC) washing to reduce inflammatory factors before transfusion.In a randomized trial of washed and standard unwashed RBCs in high-risk cardiac surgery patients, the experimental benefit was not replicated.Owing to the limited power of this trial, larger studies are necessary to test the hypothesis that RBC washing is beneficial.Organ injury associated with red blood cell (RBC) transfusion has been attributed to a ‘storage lesion’; a progressive disruption of erythrocyte homeostasis associated with depletion of high-energy phosphates during storage that results in accumulation of microparticles and other inflammatory substances in the supernatant of RBCs.vs standard care (no washing). The trial had ethical approval (REC Reference 12/EM/0475) and was registered (ISRCTN 27076315). The trial protocol has been published;The Red Cell Washing for the Attenuation of Organ Injury Following Cardiac Surgery (REDWASH) trial was a multicentre, single-blinded, parallel-group, randomized controlled trial of washing of allogeneic RBCs before transfusion Adults (≥16 yr of age) undergoing cardiac surgery with blood cardioplegia identified as representing a high-risk group for large-volume blood transfusion (LVBT) using a modified risk scoreSubjects were randomly assigned with concealed allocation using an Internet-based randomization system  recognized facility). Randomization was stratified by study site and type of procedure. Outcome assessors were blinded to allocation.g force centrifugation with this device minimizes RBC trauma.Eligible subjects who consented to participate were randomly allocated, in a 1:1 ratio, to receive either standard care  or washed red blood cells, between the commencement of surgery and 48 h after surgery. Allogeneic saline–adenine–glucose–mannitol (SAGM) stored RBC units used in the trial were issued by National Health Service Blood and Transplant (NHSBT) as per standard care. The Continuous AutoTransfusion System  was used on the basis that low The primary outcome was severity of the systemic inflammatory response as indicated by serum interleukin (IL)-8 measured at baseline and at four postsurgery time points. We have previously shown that IL-8 is increased in transfused patients.sd) for normally distributed data, median  for non-normally distributed data, or n (%). Means for continuous outcomes for clinical and experimental data  were compared using mixed effects models, adjusting for baseline values where available. Treatment estimates were reported as effect sizes with 95% confidence intervals (CIs). A statistical analysis plan was written before database lock  is reported here. Secondary outcomes are reported in the online -1. 1.73m-2) in the washing group [68 (24) vs 90 (30)] in the standard care arm. The types of procedures and the bypass and cross-clamp times were well matched between groups  of RBC transfusions was similar between groups  in the standard care arm compared with 4 (2–6) in the washing arm P=0.04; . In the n groups . The meaP=0.078; . There wP=0.078; , serial P=0.078; , or expoP=0.078; .vs washing, 0.239 , P=0.318; P=0.039; There was no difference between groups for the primary outcome, serum IL-8 [adjusted mean difference, standard urements . Serial P=0.039; . HoweverP=0.039; . ClinicaMicroparticles positive for CD235a  and annexin V (AV), which labels phosphatidylserine and oxidized lipids, accumulated in the supernatant of RBC units throughout storage . MechaniThere was no difference between groups with respect to serial measures of total, AV-positive, or RBC-derived (CD235a positive) microparticles in plasma up to 48 h postsurgery . There wPlasma cell-free Hgb concentrations were increased significantly in both washing and standard care groups immediately postsurgery . HoweverThe greater part of plasma free Hgb immediately postsurgery was probably not altered by RBC washing but was most probably attributable to lysis of recipient RBCs by the cardiopulmonary bypass (CPB) circuit. However, free Hgb released after washing of stored RBCs is more reactive than that released by lysed fresh RBCs, as generated by the CPB circuit , implyinRed blood cell-derived (CD235a-positive) microparticles accumulate progressively in RBC units from the start of storage and are effectively removed by mechanical washing. In the REDWASH trial, however, transfusion of washed RBCs did not reduce plasma concentrations of CD235a-positve microparticles or levels of platelet or leucocyte activation in recipients compared with standard RBCs. Cell-free Hgb also increases in the supernatant of RBC units during storage and is increased by washing. However, in the REDWASH trial there was no difference in plasma free Hgb concentrations or markers of haem/iron metabolism, endothelial activation, or oxidative stress between groups.This is the first randomized trial to evaluate the risks and benefits of RBC washing in adults. We recruited a high-risk population of cardiac surgery patients that should in principle have benefited from safer RBC transfusion. Patients in both groups received significant volumes of RBCs, 1000 ml, within 48 h of surgery and experienced significant morbidity; 86% of patients experienced a composite clinical outcome of death, sepsis, or organ failure. Subjects in the washing group received more RBC units despite similar perioperative levels of blood loss and serial serum haemoglobin concentrations. This is consistent with previous clinical and experimental studies that report loss of 10–25% of red cells during mechanical washing.in vitro. Furthermore, we have shown that mechanical RBC washing in a swine model effectively removes microparticles and attenuates platelet and leucocyte sequestration in lungs and the clinical and histological correlates of transfusion-associated lung injury . Likewise, we have shown that cell-free Hgb activates endothelial cells by a pathway previously described only in human aortic endothelial cells in the presence of minimally modified low-density lipoprotein.Experimental work has documented both potential risks and benefits of RBC washing. We have shown that RBC-derived microparticles activate platelets and leucocytes −1. In the standard care patients in the REDWASH trial, the peak IL-8 concentration was 220 pg ml−1. We speculate that the effects of washing were superseded by greater platelet and leucocyte activation, free Hgb release, and endothelial injury attributable to surgery and CPB.Our experimental results were not replicated in the REDWASH trial. This could be attributable to differences between species, differences in the time period over which RBCs were transfused, or both, in porcine experiments and trial subjects. Typically, 4 units were transfused during 2 h in swine, whereas a similar volume, on average, was transfused during 2 days in the clinical trial. The porcine experiments were also conducted without CPB. These factors might have diminished the clinical benefits of washing; neither microparticle depletion nor the release of free Hgb observed after washing was reflected by differences in plasma microparticle concentrations or cell-free Hgb in subjects or by differences in inflammation and organ injury. Moreover, the peak mean IL-8 concentration in our porcine experiments after transfusion with older stored RBCs was 10 pg mlIn conclusion, the results of the mechanism substudy of the REDWASH trial did not support our hypothesis that mechanical washing of transfused RBCs would attenuate platelet and leucocyte activation and organ injury in cardiac surgery patients.Conceived the trial and wrote the application for funding (with others): G.J.M.Designed the trial: G.J.M., M.J.W., A.H.G.Managed the conduct of the trial: W.D., S.K.B., N.B., T.K.Managed the data during the trial: W.D., T.K., T.M. Laboratory analyses: M.J.W., R.C., M.W., N.S.Statistical analyses: T.M., M.N.Drafted the report: M.J.W., G.J.M., A.H.G.All authors reviewed the report for important intellectual content and approved the final version. All authors, external and internal, had full access to all of the data  and can take responsibility for the integrity of the data and the accuracy of the data analysis.British Journal of Anaesthesia online.Supplementary DataClick here for additional data file.


Weintraub et al. Cold Spring Harb Perspect Med 2012doi:10.1101/cshperspect.a006171.Dr. Weintraub et al. has written a comprehensive review of cognitive profiles amongaging and dementias, comparing them to the neuropsychological deficits seen inAlzheimer's disease (AD). The authors described the neuropsychological findingsregarding episodic memory, language and semantic knowledge, executive functions,working memory and attention; and visuospatial abilities. The article highlightedthat although memory impairment is a hallmark of AD, it may also occur in otherneurodegenerative dementias. This represents an excellent review of topics thatcenters on only neuropsychological findings, providing a rapid overview ofunderlying pathophysiological mechanisms.Deramecourt et al. Neurology 2012;78:1043-1050.Vascular cognitive impairment is a relatively new concept that encompasses mildcognitive impairment without dementia to vascular dementia and mixed dementia. Thecondition is a clinical diagnosis. There are presently no consensual pathologiccriteria for the various degrees of cognitive decline. The authors analyzed 135brains with varying degrees of cerebrovascular lesions (CVL) and Deramecourt et al.attempted to conceptualize the natural history of CVL. The most common lesions werevessel wall modifications ,followed by perivascular modifications , myelin loss and infarcts (microinfarcts and large infarcts). Thesefindings were present in all cerebral regions. After descriptions, authors proposeda global vascular score. Brains previously considered with vascular or mixeddementia had a vascular score ≥ 10 in 98.9% whereas those considered withpure degenerative dementia had a score <10 in 92.5%.Baborie et al. Arch Neurol 2012. Doi: 10.1001/archneurol.2011.3323.Frontotemporal lobar degeneration (FTLD) has been considered a presenile dementia,being the second leading cause after Alzheimer's disease during this period. Thismanuscript shows differences in patients diagnosed with FTLD dividing them intoelderly and presenile-onset FTLD. This was a retrospective study over a 25-yearperiod (1974-2004) with 11 cases of FTLD in elderly patients and 19 cases ofpresenile-onset FTLD, all of which were neuropathologically identified. All elderlyFTLD patients except for one had behavioral changes. Memory loss was one thepresenting symptoms in 7 out of 11 the elderly patients. Subsequently, a severedegree of memory loss was present in 10 out of the 11 patients. Atrophy of temporaland frontal lobes was present in all elderly patients with FTLD but proved lesssevere than in patients with presenile-onset FTLD. Temporal lobe atrophy wassignificantly less in elderly patients in whom parietal and occipital atrophypredominated. Nine out of the 11 elderly patients with FTLD had selective loss ofpyramidal neurons in the hippocampus; characteristic of hippocampal sclerosis (82%)while 37% of presenile-onset had hippocampal sclerosis. This study provides somepointers for diagnosing possibly under-recognized elderly FTLD cases.Jung et al. Arch Neurol 2012 doi: 10.1001/archneurol.2011.2874.The role of cholinesterase inhibitors in vascular dementia (VaD) treatment isunclear, due to heterogeneity of lesions among patients in clinical trials. Jung etal. hypothesized that in subcortical VaD, white matter lesions could lead toretrograde degeneration of the nucleus basalis (NB) neurons. Patients withsubcortical ischemic vascular disease (SIVD)(n=16), AD (n=20), and mixed AD and SIVD(n=10), plus healthy controls matched for age and educational level, were comparedregarding number of neurons in the NB. Loss of neurons was observed in AD and mixedgroups, but not in SIVD and healthy groups. There was preservation of NB neurons inSIVD patients compared to controls. A negative correlation was observed between NBneurons and CDR scores in the AD group. These findings pointed to an absence ofprimary loss of cholinergic neurons in SIVD patients.Smith et al. Arch Neurol 2012 doi: 10.1001/archneurol.2012.590.Last year a phase I study was published by the same group using deep brainstimulation (DBS) on fornix in six AD patients with encouraging results. In thisonline published study, the group investigated changes in cerebral glucosemetabolism (PET) and clinical outcomes in 5 mild AD patients. After one year of DBS,two networks showed increased metabolism: afrontal-temporal-parietal-striatal-thalamic network and afrontal-temporal-parietal-occipital-hippocampal network. In similar cortical areas,higher metabolism prior to DBS was correlated with better outcomes while increasedmetabolism after 1 year of DBS showed same correlation with cognitive and functionalmeasures.Yoon et al. Age Ageing 2012. Doi: 10.1093/ageing/afs018.2) –odds ratio 2.61, and classification in the top tertile of visceral adiposity area –odds ratio 2.58, were associated with poor cognitive performance in subjects youngerthan 70 years. Adiposity is related to many mechanisms that link it to brainfunction.High body mass index (BMI) is regarded by some studies as a risk factor for cognitivedecline and dementia. Yoon et al. measured abdominal adiposity by computedtomography  and cognitive function withthe Mini-Mental State Examination, in addition to BMI and waist circumference in 188subjects <70 years of age and 62 subjects aged 70 years or older. On themultivariate logistic regression analyses, obesity (BMI >25 kg/m


Dictyostelium discoideum, is a popular model system for cell biology and developmental biology with key signalling pathways controlling cell-specialization being resolved recently. For the most dominant pathways, evolutionary origins were retraced to a stress response in the unicellular ancestor, while modifications in the ancestral pathway were associated with acquisition of multicellular complexity. This review summarizes our current understanding of developmental signalling in D. discoideum and its evolution.Dictyostelia represent a tractable system to resolve the evolution of cell-type specialization, with some taxa differentiating into spores only, and other taxa with additionally one or up to four somatic cell types. One of the latter forms,  Current Opinion in Genetics & Development 2016, 39:29–34Developmental mechanisms, patterning and evolutionThis review comes from a themed issue on Detlev Arendt and Cassandra ExtavourEdited by Issue and the EditorialFor a complete overview see the Available online 16th June 2016http://dx.doi.org/10.1016/j.gde.2016.05.0140959-437X/© 2016 Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).The Dictyostelid social amoebas evolved multicellularity 0.6 billion years ago as a strategy to survive starvation as aerially borne spores, supported by dead stalk cells . Their lDictyostelium discoideum, a robust laboratory model that uses cAMP as a chemoattractant for aggregation. The development of genetic transformation, gene knock-out, targeted mutagenesis and high resolution imaging techniques in the '80 and '90s, make it the organism of choice for research into fundamental problems in cell biology and developmental biology . D. . D. 6]. ividuals . The enzividuals , 35. Knotellanii , 35. Comactivity , 37. In carA or pdsA in P. pallidum disrupts fruiting body morphogenesis, but not aggregation [carA in P. pallidum also prevents cAMP-induced prespore differentiation, causing cysts to differentiate in the ‘spore’ head [Dictyostelium cells secrete most of their cAMP [The enzymes AcaA and PdsA and the cAMP receptor CarA, which are important for extracellular cAMP signalling, are conserved in Dictyostelia, but, apart from PdsA, not in unicellular Amoebozoa. CarA is only expressed after aggregation in groups 1–3, and deletion of regation , 39. Theeir cAMP . While sD. discoideum aggregation, favouring spiral over concentric waves. Spiral waves organize larger territories and hence give rise to large aggregates and robust fruiting bodies [D. discoideum PdsA, and only partially restore aggregation of a D. discoideum pdsA null mutant [carA gene expression.The extracellular cAMP phosphodiesterase PdsA and its secreted inhibitor PdiA affect the kinetics of cAMP wave propagation during g bodies , 42. Pdil mutant . The usein vitro [StlB, dmtA and chlA genes are conserved throughout Dictyostelia, but a group 2 dmtA does not complement the D. discoideum dmta-mutant [DIF-1 is a secreted chlorinated polyketide, that induces differentiation of stalk-like cells in vitro . Howeverin vitro , 19, 20.a-mutant , and a sa-mutant . The DIFa-mutant . These dThe comparative studies allow reconstruction of a tentative narrative about the evolutionary origins of developmental signalling in Dictyostelia, and innovations that occurred at successive stages of their evolution . This na• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


AbstractPolyommatus (Agrodiaetus) morgani  from the region of its type locality in the southern Zagros Mountains in Iran, provide first chromosomal evidence for P. (A.) antidolus  in Iran and demonstrate that these two species can be easily identified through analysis of their karyotypes whereas they share their mitochondrial barcodes.DNA barcoding has been suggested as a universal tool for molecular species identification; however, it cannot be applied in cases when morphologically similar species share their DNA barcodes due to the common ancestry or mitochondrial introgression. Here we analyze the karyotype of  COI and the non-coding nuclear sequence, internal transcribed spacer 2 (ITS2), is sometimes insufficient to distinguish between evolutionarily young sister species, either because they can be weakly differentiated regarding these markers or because they are too polymorphic  antidolus , P. (A.) kurdistanicus  and P. (A.) morgani , a complex of three closely related allopatric species distributed in east Turkey as well as in west and central Iran  morgani, n=39-42 in P. (A.) antidolus and n=61-62 in P. (A.) kurdistanicus (P. (A.) morgani  , in the vicinity of Khalifen (15 km W of Mahabad)  and in Seir (near Urmia)   kurdistanicus and P. (A.) antidolus specimens available from GenBank , in the vicinity of Darman  morgani, we first searched for it in its exact type locality in “Deh Tcheshma”   antidolus - P. (A.) kurdistanicus - P. (A.) morgani complex. In our opinion, P. (A.) morgani is extinct in its type locality, probably due to PageBreakclimate change and aridification during the last 100 years. Fortunately, we were able to find typical P. (A.) morgani in a small, relatively humid mountain valley between Fereydunshahr and Sibak, 90 km NW of Farsan   and a group of smaller bivalents (from 17 to 21 in different cells). The number of bivalents that were classified as “larger” and “smaller” was variable, most likely depending on the bivalent orientation. However, in some metaphase plates, the distinction between the larger and smaller bivalents was unclear, and the bivalents gradually decreased in size, with the largest bivalent approximately 10 times larger than the smallest one. Thus, the results obtained confirm the previous taxonomic interpretations (P. (A.) morgani.In order to investigate the topotypical population of etations  that conP. (A.) antidolus - P. (A.) kurdistanicus - P. (A.) morgani complex from the vicinity of Mahabad (West Azerbaijan Province)  antidolus; however, they did not provide any chromosomal data to confirm this conclusion. We analyzed seven specimens from two localities close to Mahabad  antidolus. The number of elements within the karyotype was unstable, varying from n=27 to n=29, most likely due to the presence of two chromosomal fusions/fissions  morgani described above. The chromosome numbers n=28 and n=29 were not previously reported for P. (A.) morgani  morgani.ons Figs , 4a, b.  morgani , 2015b. P. (A.) antidolus in the neighboring Province Hakkari in south-east Turkey, thus providing first chromosomal evidence for P. (A.) antidolus in Iran.Finally, in three specimens collected in Seir (near Urmia, locality 5 in Fig.


Autism spectrum disorder (ASD) is sexually dimorphic in brain structure, genetics, and behaviors. In studies of brain tissue, the age of the population is clearly a factor in interpreting study outcome, yet sex is rarely considered. To begin to address this issue, we extend our previously published microarray analyses to examine expression of small noncoding RNAs (sncRNAs), including microRNAs (miRNAs), in ASD and in the control temporal cortex in males and females. Predicted miRNA targets were identified as well as the pathways they overpopulate.After considering age, sexual dimorphism in ASD sncRNA expression persists in the temporal cortex and in the patterning that distinguishes regions. Among the sexually dimorphic miRNAs are miR-219 and miR-338, which promote oligodendrocyte differentiation, miR-125, implicated in neuronal differentiation, and miR-488, implicated in anxiety. Putative miRNA targets are significantly over-represented in immune and nervous system pathways in both sexes, consistent with previous mRNA studies. Even for common pathways, the specific target mRNAs are often sexually dimorphic. For example, both male and female target genes significantly populate the Axonal Guidance Signaling pathway, yet less than a third of the targets are common to both sexes.Our findings of sexual dimorphism in sncRNA levels underscore the importance of considering sex, in addition to age, when interpreting molecular findings on ASD brain.The online version of this article (doi:10.1186/s13229-017-0117-0) contains supplementary material, which is available to authorized users. Autism spectrum disorder (ASD) is one of a number of neurodevelopmental disorders that display sexual dimorphism, occurring more frequently in males, which affect brain structure, gene expression, pathways, function, and ultimately behaviors that will require individualized treatments –3. ExtenFew molecular studies of ASD brain tissue to date have considered potential sexual dimorphism often because of limited tissue availability of female cases. In typical brain development, male-biased gene expression changes are enriched for extracellular matrix, immune response, chromatin, and cell cytoskeleton pathways that have been implicated in ASD . Sex difHere, we extend our analyses of previously published microarray data , 18 to eThe methods used in the current study are similar to our recent publications , 18 , which is suitable for unbalanced designs . REML optimizes the parameter estimates for the effects in the model. We used repeated measures to account for multiple regions measured within the same subjects (STS and PAC), and we made the SubjectID a random effect. Considering that our sample size was unbalanced between males and females, some identified differences may be due to this limitation. Statistical significance was defined as P < 0.05 was used for determining significance of pathway enrichment. The sexually dimorphic predicted mRNA target genes and pathways were compared to three other data sets: (1) autism risk genes from SFARI using the hypergeometric probability function phyper in R with population size set to all protein-coding human genes of 20,687 ,, 26,3, 2There are a number of sexually dimorphic miRNAs from our study that have been associated with ASD-relevant diseases and processes Table . For exaIn STS of ASD female compared to control female, miR-219 and miR-338 showed the highest level of downregulation. Both miRNAs are involved in regulating oligodendrocyte differentiation and likely myelin production . This wamiR-219 and miR-338 both promote oligodendrocyte differentiation –41. HoweIt is important to note that sex differences in the miRNAome are prominent in health and disease –47. SeveAs we have reported previously , there aspecific miRNA regulated in ASD and control brains in this study and the miRNA described by Ziats and Rennert for typically developing human brain [In general, there is little overlap of the an brain . Howeveran brain  and a tran brain  and our There are important limitations to consider when interpreting this data that are common to studies of postmortem human brains, including small sample size and variation in cause of death, postmortem interval, age at death, agonal state, postmortem RNA integrity, and tissue preparation at different brain banks. In addition, the changes in miRNA and predicted targets and pathways studied here could lead to some aspects of ASD, but they could just as well be a consequence of the condition and even represent compensatory mechanisms. Future studies will need to assess both miRNA and mRNA in the same samples, so that one can determine if specific miRNA target mRNA are present and are regulated in an inverse direction as occurs for most miRNA-mRNA interactions. Small sample size is also limitation of this report and findings need to be confirmed in larger cohorts and with alternative gene expression profiling techniques such as qRT-PCR. However, this study clearly indicates that sex needs to be considered when interpreting data on postmortem human brains in ASD.Our own molecular studies, and many others, have demonstrated clear age-related changes in the brains of both typically developing and autistic individuals , 24, 56.Additional file 1:Supplementary Data Tables. Table S1. sncRNA differentially expressed in ASD-STS vs CTRL-STS. Table S2. sncRNA differentially expressed in ASD-PAC vs CTRL-PAC. Table S3. Female (F) ASD Regional Analysis. F_ASD-STS vs F_ASD-PAC. Table S4. Female (F) controls (CTRL) regional analysis. F_CTRL-STS vs F_CTRL-PAC. Table S5. Male (M) ASD regional analysis. M_ASD-STS vs M_ASD-PAC. Table S6. Male (M) controls (CTRL) regional analysis. M_CTRL-STS vs M_CTRL-PAC. Table S7. Summary of putative target analyses. Table S8. Pathway analysis of putative targets of the mature miRNA from the PAC analysis in females. Table S9. Pathway analysis of putative targets of the mature miRNA from the PAC analysis in males. Table S10. Pathway analysis of putative targets of the mature miRNA from the STS analysis in females. Table S11. 227 common targets between female and male regional analyses. Table S12. Pathway analysis of putative targets of the mature miRNA from the female regional analysis. Table S13. Pathway analysis of putative targets of the mature miRNA from the male regional analysis. Table S14. Pathway analysis of putative targets of the mature miRNA from the female regional analysis: immune pathways. Table S15. Pathway analysis of putative targets of the mature miRNA from the male regional analysis: immune pathways. Table S16. Pathway analysis of putative targets of the mature miRNA from the female regional analysis: Neurotransmitter and other nervous system signaling pathways. Table S17. Pathway analysis of putative targets of the mature miRNA from the male regional analysis: neurotransmitter and other nervous system signaling pathways. Table S18. Pathway overlapping between our male regional analysis and the male-enriched pathways from Ziats and Rennert (Ref 15). Table S19. Seventy-four ASD-implicated genes amongst the 1382 putative targets of the miRNAs from the female regional analysis. Table S20. Canonical pathway enriched in the 74 ASD-implicated genes from the predicted targets in the female regional analysis Table S21. Targets overlapping between our analysis and the sexually dimorphic mRNA from Werling et al, Nature Communications, 2016; Supplementary Material: ncomms10717-s2. Table S22. Pathway overlapping between our male and female regional analysis and pathways were derived based on combined sexually dimorphic mRNAs from Werling et al, Nature Communications, 2016; Supplementary Material: ncomms10717-s2. (XLSX 130 kb)Additional file 2:P of overlap = 0.0034. B Overlap between all our sexually dimorphic pathways (290 pathways) and Ziats and Rennert, Mol. Psychiatry, 2014, sexually dimorphic pathways (204 pathways); P of overlap < 0.00001. C Overlap between all our sexually dimorphic pathways (287 pathways) and Werling et al, Nat. Communications, 2016, sexually dimorphic pathways (14 pathways). P of overlap between Werling et al, 2016 and our regional analysis = 0.098, P of overlap between Werling et al, 2016, and our PAC analysis = 0.092. D Overlap between the predicted targets from our female regional analysis (STS vs PAC) (1382 targets) and SFARI ASD-implicated genes (768). P of overlap < 0.001. (TIF 48 kb)Supplementary Figure. Overlap with relevant studies. A Overlap between our male regional analysis (118 pathways) and Ziats and Rennert, Mol. Autism, 2013, male-enriched pathways (10 pathways);


Trypanosoma cruzi. It is an important public health problem affecting around seven to eight million people in the Americas. A large number of hematophagous triatomine insect species, occupying diverse natural and human-modified ecological niches transmit this disease. Triatomines are long-living hemipterans that have evolved to explode different habitats to associate with their vertebrate hosts. Understanding the molecular basis of the extreme physiological conditions including starvation tolerance and longevity could provide insights for developing novel control strategies. We describe the normalized cDNA, full body transcriptome analysis of three main vectors in North, Central and South America, Triatoma pallidipennis, T. dimidiata and T. infestans.Chagas disease is a parasitic infection caused by Rhodnius prolixus genome and proteome. A Triatoma expansion of the calycin family and two types of protease inhibitors, pacifastins and cystatins were identified. A high number of transcriptionally active class I transposable elements was documented in T. infestans, compared with T. dimidiata and T. pallidipennis. Sequence identity in Triatoma-R. prolixus 1:1 orthologs revealed high sequence divergence in four enzymes participating in gluconeogenesis, glycogen synthesis and the pentose phosphate pathway, indicating high evolutionary rates of these genes. Also, molecular evidence suggesting positive selection was found for several genes of the oxidative phosphorylation I, III and V complexes.Two-thirds of the de novo assembled transcriptomes map to the Protease inhibitors and calycin-coding gene expansions provide insights into rapidly evolving processes of protease regulation and haematophagy. Higher evolutionary rates in enzymes that exert metabolic flux control towards anabolism and evidence for positive selection in oxidative phosphorylation complexes might represent genetic adaptations, possibly related to prolonged starvation, oxidative stress tolerance, longevity, and hematophagy and flight reduction. Overall, this work generated novel hypothesis related to biological adaptations to extreme physiological conditions and diverse ecological niches that sustain Chagas disease transmission.The online version of this article (10.1186/s12864-018-4696-8) contains supplementary material, which is available to authorized users. Trypanosoma cruzi, transmitted to humans by hematophagous insects of the Reduviidae family. Among the Reduviidae, the Triatominae subfamily conforms one of 21 subfamilies. Triatomines are obligatory blood feeders evolved from predatory ancestors. The Rhodniini and Triatomini tribes contain most species within the subfamily. The Rhodniini tribe is an accepted monophyletic group [Chagas disease is an important neglected tropical disease in Central and South America. The causative agent is the protozoan ic group .Rhodnius species mostly dwell in palm tree canopies were they feed on birds, while Triatoma species are land dwellers, living in crevices and burrows were they feed on birds and mammals [T. cruzi result in Chagas disease endemism in Latin America, spanning from Northern México to Southern Argentina with an estimated seven to eight million people infected [With few exceptions, triatomine species occurs in the Americas occupying a large variety of ecological niches. In this complex mosaic of natural and human-modified ecotopes, triatomines have evolved to explode different habitats associated with their vertebrate hosts.  mammals . Most trinfected .Rhodnius prolixus, Triatoma dimidiata, T. pallidipennis, and T. infestans. R. prolixus, a model organism for insect physiology, is a main vector in Venezuela, Colombia, Peru and whole Central America [Triatoma dimidiata, consist of several variants occupying sylvatic, peri-domestic and domestic habitats, making a main Chagas disease vector in Central America and Mexico [. Triatoma pallidipennis stands out by showing the capacity to colonize domestic environments in at least 11 Mexican States with a T. cruzi infection prevalence up to 90% [T. infestans, largely adapted to domiciliary habitats, but with sylvatic populations that repopulate the human environment. This is one of the reasons for failure of the indoor insecticide spraying strategy deployed in South America endemic countries [Four species are relevant vectors of Chagas disease in the Americas:  America , 7. Triad Mexico –10. Triap to 90% . The mosountries .R. prolixus has been sequenced [Triatoma genus is still limited to organ-specific transcriptomes or selected gene families [New strategies for Chagas control are urgently needed; information on the genetic basis of their biological adaptations to extreme physiological conditions such as prolonged starvation periods, their extended life span and their capability to adapt to diverse ecological niches could provide insights for novel control interventions. However, despite of their medical significance, detailed genetic information in triatomines is still scarce. The genome of equenced , but genfamilies –21. ThusT. infestans, T. dimidiata and T. pallidipennis cDNA libraries from all stages of their life cycle and compared them with the genome of R. prolixus. This allowed the identification of a large set of shared genes, gene expansions and higher sequence divergence in energy metabolism-related genes, possibly related to adaptations to their life styles, providing the basis for a better understanding of triatomine biology and insights for development of novel control strategies.Transcriptome analysis of organisms without sequenced genomes provides a preliminary catalogue, useful for gene discovery, including specific molecules and their putative functions for the comparative analysis among related organisms, as well as a means for gene prediction validation in future genome projects. We generated transcriptomes derived from normalized T. infestans, T. dimidiata and T. pallidipennis, respectively, which resulted in a cDNA assembly of 3904, 4847 and 5148 isogroups (unigenes) containing from 35% to 69% of assembled reads using the Newbler assembler .Totals of 164.6, 112.8 and 202.6 megabases of raw sequence data were generated for T. dimidiata mitochondrial genome [T. dimidiata coverage of the mitochondrial genome was near complete, whereas for T. pallidipennis and T. infestans 86% and 46% of the mitochondrial genome was covered, respectively  that covered 100% of the reported Triatoma virus genome sequence , as revealed by transcriptome mapping to the l genome . The T. ly Table . NeverthTriatoma transcripts that best-matched to the R. prolixus predicted peptide dataset were similar among the three species (~ 7272 to 8002), which corresponds to 48–53% of the R. prolixus predicted proteome  in the Triatoma transcriptomes. Transcriptome completeness assessment by searching the Core Eukaryotic Genome Dataset (CEGMA) [Triatoma and R. prolixus revealed very high coverage values. The BUSCO coverage was 82.2% in T. infestans, 85.8% in T. dimidiata and 86.9% in T. pallidipennis, while these coverage values were higher than 90% for the CEGMA in the three species (Table The number of  (CEGMA)  and the  (CEGMA)  for the Triatoma transcriptome covers between 20 to 55% of the proteome of other insects  among the three - R. prolixus predicted proteome. Despite library normalization, the Triatoma transcriptomes were consistently enriched for transcripts involved in core cellular functions, such as protein translation, folding and sorting, generation of precursor metabolites and energy, mitochondrial and ribosomal organization and assembly , as well as for technical variables . To assess transcriptional representation bias, we tested Gene Ontology annotated transcriptomes for enrichment compared to the ly Table . In contTriatoma transcriptomes. In the T. infestans transcriptome, we found overrepresentation of the GO term “symbiosis, encompassing mutualism through parasitism” (GO:0044403). No significant enrichment was found within the T. dimidiata and T. pallidipennis transcriptomes. The biased transcriptional representation found in the Triatoma datasets, using the R. prolixus as reference dataset, may result from transcriptional abundance of core cellular processes and the underrepresentation of certain organs/tissues, such as nervous system and sensory organs during library preparation, and may not reflect accurately gene content. Moreover, the lack of significant enrichment within Triatoma datasets indicates that the source of bias may be predominantly due to transcriptional abundance, and in a lesser extent to differences in gene content, with little participation of technical variation during library preparation.We next asked if there was biased transcriptional representation according to GO within the three - T. infestans, T. dimidiata, T. pallidipennis non-redundant dataset, and 59.6% of the R. prolixus transcript dataset had at least one InterPro annotation. A Fisher’s exact test was performed to identify enrichment of InterPro entries in the test transcriptome isotig dataset  compared with the R. prolixus transcript dataset as reference. In some cases, significant enrichment  was not congruent with absolute gene number enrichment. To account for confounding effects due to transcriptome expression bias, emphasis was put on those InterPro entries in which absolute transcript counts were greater than in R. prolixus in at least one Triatoma species. Enrichment results are summarized in Table To assess potential gene expansions, transcriptomes were annotated using InterProScan  through T. dimidiata and T. pallidipennis) and cystatin (the three species) families, was identified (Table T. infestans and R. prolixus had nine transcripts/genes encoding pacifastin domains (IPR008037), which corresponded to a statistical enrichment in T. infestans (FDR 3.0− 2) . Domain architecture analysis revealed novel domain configurations in T. pallidipennis absent in R. prolixus predicted proteome such as a pacifastin with von Willebrand factor, type C (IPR001007) and D domains (IPR001846), as well as a membrane-anchored two pacifastin-repeat coding transcript, and a six pacifastin domain transcript encoding a putative secreted product  Table , althougnization . PacifasR. prolixus, but cystatins were more abundant in T. dimidiata (n = 9), T. pallidipennis (n = 6), and T. infestans (n = 4).Transcripts encoding for InterPro domains belonging to protease inhibitors other than pacifastins were also more abundant. Cystatins are important regulators of cathepsin-mediated intracellular proteolysis , 29. TheT. infestans, regarding T. dimidiata (FDR = 1.0− 4) and T. pallidipennis (FDR = 2.0− 3).Serine-type protease gene expansions are common in hematophagous arthropods , 30. EnrTriatoma, and function as anti-hemostatic factors [Triatoma species (Table T. infestans (n = 79), compared to R. prolixus (n = 72) (FDR = 1.0− 30). This calycin enrichment can be attributed to a significant triabin/procalin (IPR005657) expansion in the three species, but again higher in T. infestans (n = 70 versus 40 in R. prolixus. FDR = 4.0− 32). As expected, the calycin fold was also enriched in T. infestans over T. pallidipennis  and T. dimidiata . Calycins are classified according to the GO biological process “symbiosis, encompassing mutualism through parasitism” (GO:0044403), explaining the corresponding GO term enrichment in Triatoma sp., particularly in T. infestans , triatins (clade II), salivary lipocalins (clade IV), triafestins (clade V), collagen-induced platelet aggregation calycins (clade VI), and lipocalins/palidipin-like proteins (clade VII) [T. infestans, and triabins - clade III calicyns in our Triatoma transcriptomes  . Howevermes Fig. .Fig. 3ClT. infestans, T. dimidiata and T. pallidipennis contigs sharing high sequence identity with nitrophorins, salivary heme-containing proteins, which participate in nitric oxide (NO) transport and storage [R. prolixus nitrophorins (RPRC000537 and RPRC000163)  highly conserved in insects and vertebrates , compared with T. dimidiata (n = 13) and T. pallidipennis (n = 23) , mainly CR1, Jockey, Nimb and Loa-type; followed by DNA TEs (36%), mainly Mariner-like. There was at least a five-fold increase in the number of LTRs in T. infestans , mainly ERV-type. In T. pallidipennis, the majority were DNA TEs (69%), being Mariner-like the most common  were found in database  revealed286 were T. infestans best matched with R. prolixus TEs, a large proportion (45%) best matched to bird TEs  with their corresponding bird best match was significantly higher than those best-matching R. prolixus non-LTRs    Fig.  and DNA oa) Fig.  , OBPs and CSPs were the most abundant families. As for pacifastins in T. infestans, validation of the OBP family expansion awaits the availability of the full genome sequence of the corresponding triatomine.A statistically significant enrichment of odorant binding proteins (OBPs) (IPR006170) was found in ns Table . Becausens Table . Taking Triatoma species or in a particular species . Other protein families were enriched only in statistical terms. The thioredoxin-like domain (IPR012336) was enriched in the three species; however, absolute numbers were lower than in R. prolixus. In T. dimidiata, there were 15 tubulin (IPR000217) transcripts compated to 19 in R. prolixus (FDR = 2.3− 2). Finally, statistical enrichment regarding R. prolixus of insect cuticle protein (IPR000618) (FDR = 2.0− 4) and chitin-binding domains (IPR002557) (FDR = 7.0− 3) was found in T. infestans, which might be implicated in cuticle formation, melanization and perimicrovilliar membrane formation [An additional number of protein families/domains were enriched in the three -es Table . The numormation –39. As pTriatoma and R. prolixus 1:1 orthologs. To compare protein sequence conservation within Triatoma species, Z values (number of standard deviations from the mean % identity) were used. The majority of GO classes were distributed around Z = 0 (i.e. average divergence), but few classes had negative Z values (more conserved than the average), while fewer classes had positive Z values (more divergent than the average)  Fig. .Fig. 5Stp < 0.05). A high proportion of orthologs belonging to this GO term participate in protein synthesis and different components of the translation machinery and cell signaling. In contrast, orthologs annotated as “single-organism metabolic processes” (GO:0044710) were significantly less conserved in the three - Triatoma species . Only in T. infestans, orthologs annotated as “interspecies interaction between organisms” (GO:0044419) were significantly less conserved , “heterocyclic compound binding” (GO:1901363), and “localization in the ribonucleoprotein complex” (GO:1990904) were significantly conserved at the protein sequence level. In contrast, significant higher protein sequence divergence was observed in “oxidoreductases” (GO:0016491) in the three species  according to biological process Fig.  and cellny. Fig. .T. infestans , and “peptidase inhibitor activity”, GO:0030414) in T. pallidipennis. Importantly, biological processes such as carbohydrate, ketone and lipid metabolism were enriched in the most divergent group, suggesting that the whole energy generation and storage pathway may be evolving at higher rates. Additionally, “serine-type endopeptidase activity” (GO:0004252) was enriched in the most divergent group only in T. dimidiata and T. pallidipennis, indicating that, in addition to a numerical expansion  and the whole ranked dataset was subdivided into equal terciles or groups  [R. prolixus genes, orthologs for 36 were found in the three - Triatoma species, and the majority of these belonged to the intermediate or most conserved group. Only five orthologs involved in carbohydrate metabolism with coverage higher than 40% belonged to the most divergent group. A putative phosphoglucomutase 1 (Pgm1) ortholog, involved in glycolysis and glycogen synthesis was found in T. pallidipennis, but absent in T. dimidiata and T. infestans  of the glycolytic pathway, fructose-1,6-bisphosphatase (Fbp) involved in gluconeogenesis was identified in the three – Triatoma species. Interestingly, only Fbp in T. dimidiata and T. pallidipennis belonged to the most divergent group, whereas the corresponding T. infestans ortholog belonged to the most conserved group  , as wellR. prolixus genes encoding for PPP enzymes, only glucose-6-phosphate dehydrogenase (G6pd) was not found in the three - Triatoma species. A putative phosphoglucomutase/phosphopentomutase 2 (Pgm2), involved in purine and pyrimidine biosynthesis was found in T. pallidipennis and T. infestans, but only in the later belonged to the most divergent group  orthologs, linking the PPP to glycolysis, also belonged to the most diverse group , succinate dehydrogenase complex (II), cytochrome B complex (III), cytochrome c oxidase complex (IV) and ATP synthase complex (V) were identified in Triatoma species and R. prolixus. All the mitochondria-encoded subunits, two and seven subunits of the α and β subcomplex, respectively belonged to the most divergent group , an α subcomplex composed by 13 nuclear-encoded subunits, and a β subcomplex composed by 11 nuclear-encoded subunits. We identified 11 core subunits, 10 α subcomplex subunits, and all β subcomplex subunits in the three - Triatoma and R. prolixus. Only SdhD were in the most divergent group  are transferred to oxygen to form water, linked to the translocation of additional protons. Complex IV is composed by 20 subunits. Cox1, Cox2 and Cox3 are mitochondria-encoded. We identified 12 subunit orthologs, five of which were in the most divergent group in the three - O subcomplex, which constitutes the proton pore embedded in the inner mitochondrial membrane, and a F1 subcomplex, which mediates ATP synthesis. The FO subcomplex is composed of at least eight subunits, two of which are mitochondria-encoded (ATP6 and ATP8). The F1 subcomplex is composed of five nuclear-encoded subunits . Orthologs for the eight FO subunits and all F1 were identified in the three - Triatoma and R. prolixus. All the components of the FO subunit, but subunit C (pore-forming subunit), belonged to the most divergent group, while all F1 subunits were in the most or intermediately conserved groups  and Proline dehydrogenase (ProDH), which may play an important role in insect vectors physiology , 41. Thep > 0.05). Furthermore, we used the HyPhy package to estimate selection per codon, based on the difference of non-synonymous (dN) and synonymous (dS) substitutions maximum likelihood [A, showed biased dN-dS distribution towards positive values  selection in 23 orthologs of the NADH dehydrogenase complex (I), six of the cytochrome B complex (III), 12 of the cytochrome C complex (IV) and 13 of the ATP synthase complex (V) using MEGA7 . No evidence of positive selection (dN > dS) was found using a codon-based Z-test of positive selection  towards the identification of genetic signatures that could suggest adaptations to their particular and diverse life styles. From this approach, we expected to generate novel hypothesis regarding triatomine biology, rather than providing exhaustive characterization and hypothesis testing of genes involved in different biological processes.We conducted a transcriptomic analysis of normalized cDNA libraries that cover the entire life cycle of three triatomine species relevant for Chagas disease transmission from Mexico to Argentina, R. prolixus genome and proteome, and three quarters of the R. prolixus proteome had a homologous match in the three - Triatoma datasets. Despite the relatively low throughput of the 454 sequencing technology compared with other sequencing technologies, library normalization contributed to the high coverage of the core eukaryotic genome (CEG) [Triatoma species will be required for definitive genome content comparisons among reduviids.Almost two-thirds of our de novo assembled datasets map to the me (CEG)  and the me (CEG)  (Table 2me (CEG) ; howeverme (CEG) . TherefoR. prolixus as reference , particularly in membrane bound pacifastins  inhibitors in plants, prokaryotes and animals . Cystatid so far , 47, 51.nfestans , thus thR. prolixus [Triatoma species, suggesting a gene family expansion that occurred in Triatomini after divergence from Rhodniini. Calycins form a large protein superfamily divided into lipocalins, fatty acid binding proteins, triabin, and thrombin inhibitors [A calycin/lipocalin lineage specific expansion was described in prolixus . We founhibitors . Of theshibitors , 47, 51.T. infestans, where salivary lipocalins (clade IV), triafestin-like (clade V) and lipocalin/palidipin-like (clade VII) calycins groups doubled the numbers in T. pallidipennis and T. dimidiata  of transcriptionally active transposable elements in T. infestans, particularly non-LTR retrotransposons (8 and 12-fold increase)  . Although we cannot rule out that non-LTR transcripts could derive from chicken blood used for feeding, these were not found in T. dimidiata and T. pallidipennis transcriptomes, which were fed from the same blood source under the same conditions. Further research and the availability of the T. infestans genome will contribute to clarify this issue.An important difference among the three - se) Fig. . This maeduviids ; and geneduviids . Accordieduviids  and basepe) Fig. . Horizonprolixus . Non-LTRprolixus –60. AlsoA. pisum  and R. p genomes . Taken tTriatoma-R. prolixus 1:1 orthologs as an indicator of differences in evolutionary rates. Clustering of divergent proteins in a biological pathway, a function or a biological process is suggestive of high evolutionary rates of that particular process or function, and could guide the search for molecular adaptation. To document adaptation, a directed test for selection is required. In our work, an important limitation of using these approaches was that in some cases, the full ORF for each pair was unavailable. Nevertheless, the analysis of protein sequence conservation based on Z score comparisons and enrichment in the most divergent group suggest high evolutionary rates in oxidoreductase-mediated central metabolism, protease inhibitors, serine proteases in the three Triatomini, and in the calycin family at least in T. infestans, , glycogen synthesis (Pgm1) and the PPP ; and one enzyme that participates in glycolysis (Pyk)  Fig. . Fbp genclimates , 66. MolPgm1 is a critical enzyme catalyzing the interconversion of glucose 1-phosphate to glucose 6-phosphate, a critical step in the initiation of glycolysis and glycogen synthesis. Glycogen synthesis and storage is required to cope with different metabolic demands in insects. In Drosophila, adaptive mutations in the Pgm1 locus show latitudinal clinal variation correlating with increased glycogen storage in temperate regions [R. prolixus, is required for oogenesis and is completely depleted after prolonged starvation periods [Pgm1 locus in reduviids, associated with an optimization in glycogen metabolic flux to cope with prolonged starvation and to achieve reproductive success. regions , 68. Gly periods –72  overexpression, increases the levels of NADPH and extends the fly lifespan [Triatoma PPP enzymes, Pgm2 and Tald belonged to the most divergent group, both mediate reactions in the non-oxidative phase. Ribose-5-phosphate isomerase (Rpi) generates the substrate for Pgm2, which in turn promotes ribonucleotide synthesis  required for fatty acid synthesis and reactive oxygen species (ROS) scavenging via reduced glutathione. In its non-oxidative phase, it generates precursors for the synthesis of ribonucleotides. In lifespan . Among tife span . Tald de elegans . This opPyk mediates the last step of glycolysis, catalyzing the bidirectional inter-conversion between phosphoenolpyruvate and pyruvate  subunits , NDUFB4, NDUFB8, NDUFB9 (β subcomplex of NADH dehydrogenase) and Cox7A2 mammalian homologs are implicated in the formation of respiratory chain “supercomplexes”, which may be required for adequate mitochondrial function . Moreoveosophila , 79. Alsosophila .p values in the context of biological plausibility , a coherent effect in protein divergence in certain functionally related genes, and an equally coherent biased distribution of dN-dS of certain, but not all OxPhos sub-complexes  to suggest, although not prove, the likelihood of selection in complexes I, IV and V. These observations suggest that triatomines underwent molecular adaptations in oxidative phosphorylation which may have resulted in optimization of energy metabolism and ROS generation balance. This optimization may allow these insects, on the one hand, to cope with prolonged starvation periods and on the other, the production the high amounts of energy demanded during hematophagy [Trypanosoma cruzi in the vector [We have based our interpretation of position-based selection test atophagy . Their ee vector .Overall, our results documented a set of shared gene expansions in triatomines possibly related to biological adaptations to their lifestyles and diverse ecological niches. Protease inhibitor and calycin-coding gene expansions points at rapidly evolving processes of protease regulation and hematophagy, which require further research for a better understanding of their role in vectorial capacity. Higher evolutionary rates in critical enzymes that exert metabolic flux control towards glycogen synthesis and the PPP, in oxidative phosphorylation complexes, and the expansion of the thioredoxin fold-containing proteins could represent genetic adaptations favoring hallmarks of triatomine life styles. Particularly, glycogen storage to cope with prolonged starvation and reproductive success, antioxidant mechanisms to cope with the oxidative stress favoring longevity, and optimization of aerobic metabolism to meet the energetic demands of hematophagy. Overall, this work represents a source of novel hypotheses related to triatomine biology to guide experimental testing.T. dimidiata , T. infestans  and T. pallidipennis (colony 0230 from Mexico) established in Centro Nacional de Chagas, Córdoba, Argentina were reared in the Centro Regional de Estudios Genómicos (CREG), Universidad Nacional de La Plata (UNLP) and the Centro de Bioinvestigaciones, Universidad Nacional del Noroeste de Buenos Aires (UNNOBA), at 28 °C and a partial humidity of 70% with a 12 h light/dark schedule. Insects were regularly fed using an artificial feeder and chicken blood. Insect handling was performed in accordance to the World Health Organization protocol [Colonies of T. infestans, T. dimidiata and T. pallidipennis was used to generate a normalized cDNA library for each species as described [T. infestans (SRX2600754), T. dimidiata (SRX2600753) and T. pallidipennis (SRX2600752).A mixture of RNA derived from different life-cycle stages of escribed . LibrariR. prolixus genome (Rhodnius-prolixus-CDC_SCAFFOLDS_RproC3.fa) using BLASTN and proteome (Rhodnius-prolixus-CDC_PEPTIDES_RproC3.2.fa) using BLASTX. Non-redundant mapped reads to either database were included as singletons in to the assembled dataset (full_dataset).Raw reads were analyzed with PRINSEQ  and filtfull_dataset in the assessment of statistical representation of GO and InterPro annotations, two additional sequence databases for each species were built. A non-redundant database (nr_dataset) discards alternative isotigs belonging to the same isogroup or unigene, by keeping the largest isotig (transcript) per isogroup. The third database includes only non-redundant isotigs (isotig_dataset). An additional transcriptome database was created for each Triatoma species, by mapping raw reads to the R. prolixus predicted transcript dataset (Rhodnius-prolixus-CDC_TRANSCRIPTS_RproC3.2.fa) with the GS (Newbler) Mapper v.2.8. This dataset (RproC3.2_mapped) was used only for the identification of metabolic pathways enzyme-coding transcripts  [Triatoma virus genome (Genbank accession: NC_003783.1)  . The samhmmscan command and the -T 40 and --domT 40 filters, as described in [The assembled dataset for each species was used to identify the proportion of the core eukaryotic genome coverage, as described . We usedribed in . Followiribed in  was usedR. prolixus predicted proteome [R. prolixus and the three - Triatoma transcriptomes was done using the BLAST best reciprocal hit strategy as described [To estimate the proportion of each transcriptome database that is homologous to the proteome , we usedescribed .full_dataset transcriptome assemblies were used to find homolog proteins using BLASTX (cut-off –e 1.0E-05) in the following arthropod proteome databases  downloaded from VectorBase [D. melanogaster [Ac. pisum [The ctorBase , as wellnogaster  and Ac. c. pisum .e value of 1.0E-6, was used. Once annotated, Gene Ontology term enrichment using as input the isotig_dataset was performed by means of Fisher’s exact test followed by a P value adjustment to correct for multiple testing with the Benjamini-Hochberg method (cut-off FDR < 0.05).To classify transcripts according to their putative biological process, molecular function and structural relationships (protein conserved domains), these were analyzed according to Gene Ontology (GO) “terms”  and Inteisotig_dataset was used to estimate enrichment, using the R. prolixus InterPro annotations as reference. To avoid spurious enrichments, repeated InterPro ID’s in the same transcripts/gene were counted only once. A Fisher Exact Test, a 2 X 2 contingency table was built between the number of annotated and the non-annotated transcripts for a given IPR entry for each Triatoma species (test), as well as for R. prolixus (reference). The H0 is that there is no difference in the probability distribution between species and belonging to a given IPR entry. Given that transcript underrepresentation in transcriptome analysis in meaningless due the lack of certainty of completeness, emphasis was put in enrichment of IPR terms in Triatoma (Ha) by using a one-sided Fisher’s test with the R function fisher.test , which indicates an enrichment in the test dataset. The function fisher.test  was also used to complement the enrichment analysis within Triatoma species, which indicates depletion in the reference dataset. P value adjustment with the Benjamini-Hochberg method was performed to correct for multiple testing using the R function p.adjust). A False Discovery Rate (FDR) < 0.05 was considered as a significant enrichment [The absolute count of InterPro annotations in the non-redundant richment . Individrichment  or HMMERrichment .e value of 1.0E-6 against the RepBase database, Version 21.08 [Analysis of expressed transposable elements (TEs) was performed with local NCBI-BLASTN, with a cut-off on 21.08 . MatchesP-value cutoff of 1e − 37, repuls = 2 and attract = 2.To analyze the global phylogenetic relationships of our calycin/lipocalin ortholog predictions, we performed a clustering analysis with CLANS software . CLANS iR. prolixus - Triatoma ortholog pair identified by BLAST – best reciprocal hit strategy (isotig_dataset) was grouped according to Gene Ontology biological process, molecular function and cellular component categories (Level 3). Median % identity for each category was used to calculate Z values (number of standard deviations from the % identity mean) by subtracting the overall median % identity . Statistical significance (p < 0.05) was determined with Kruskal-Wallis one-way ANOVA test with Dunn’s correction for multiple comparisons. Z values were hierarchically clustered using Cluster 3.0 [http://jtreeview.sourceforge.net).The sequence identity (%) for each 1:1 ster 3.0  accordinisotig-dataset) was subdivided into equal terciles according to % identity (BLASTx)   and nonsynonymous (dN) substitutions per site was computed using the Nei-Gojobori method . In the Additional file 1: InterProScan analysis of Pacifastin domain containing proteins in Reduviids (IPR008037). Data for Fig. Additional file 2: Triatoma. Data for Fig. Analysis of the calycin family in Additional file 3: Triatoma. BLASTn analysis search in the RepBase database. Data for Fig. Transcriptionally active transposable elements in Additional file 4: Triatoma. (XLS 78 kb)Transcripts coding for odorant receptors and odorant binding proteins in Additional file 5: Protein sequence divergence in putative 1:1 orthologs according to Gene Ontology terms (Level 3). Isotig dataset. Data for Fig. Additional file 6: R. prolixus-Triatoma 1:1 putative orthologs divided into equal terciles or groups. (PDF 450 kb)Sequence identity distribution for Additional file 7: Gene ontology term enrichment in divergent vs. conserved group putative 1:1 orthologs (isotig dataset). (XLS 43 kb)Additional file 8: Identification of Glycolysis, gluconeogenesis and pentose-phosphate putative orthologs and classification according to protein sequence conservation (RproC3.2_mapped dataset. Data for Fig. Additional file 9: nr-dataset and mitochondrial_mapped datasets. Data for Fig. Oxidative phosphorylation. Identification of respiratory complex subunit putative orthologs and classification according to protein sequence conservation (


The functional studies of GPAT in plants demonstrated its importance in controlling storage and membrane lipid. Identifying genes encoding GPAT in a variety of plant species is crucial to understand their involvement in different metabolic pathways and physiological functions. Here, we performed genome-wide and evolutionary analyses of GPATs in plants. GPAT genes were identified in all algae and plants studied. The phylogenetic analysis showed that these genes group into three main clades. While clades I (GPAT9) and II (soluble GPAT) include GPATs from algae and plants, clade III (GPAT1-8) includes GPATs specific from plants that are involved in the biosynthesis of cutin or suberin. Gene organization and the expression pattern of GPATs in plants corroborate with clade formation in the phylogeny, suggesting that the evolutionary patterns is reflected in their functionality. Overall, our results provide important insights into the evolution of the plant GPATs and allowed us to explore the evolutionary mechanism underlying the functional diversification among these genes. GPAT catalyzes the first step in the synthesis of almost all membrane phospholipids. GPAT transfers an acyl group from acyl-CoA or acyl-ACP at the sn-1 or -2 position of a glycerol 3-phosphate generating lysophosphatidic acids (LPAs)  . LPA is Other enzymes involved in triacylglycerol (TAG) biosynthesis have also been studied. Diacylglycerol acyltransferase  was demonstrated to be crucial for enhancing the control of seed oil content through bioengineering . EvolutiGPATs were first characterized biochemically over 60 years ago from animal and plant tissues . The reaArabidopsis thaliana genome, named GPAT1-9 and Soluble GPAT . GPAT9 plays an essential role in plant membrane and storage lipid biosynthesis  there are multiple copies of GPAT genes in plant genomes, (ii) different GPAT gene paralogs can encode enzymes with different glycerolipid synthesizing ability, and (3) GPATs may be involved in many different metabolic and physiologic pathways. All these findings shed new light on glycerolipid biosynthetic pathway in plants and emphasize the need for a deeper understanding of the complexity of plant GPATs. In this study, we performed a genome-wide comparative analysis, including a phylogenetic approach, gene structure comparison and gene expression analyses to provide further insights into the present-day diversity and ortholog/paralog relationship of plant GPATs.A. thaliana. Then, we retrieved the A. thaliana GPAT sequences using BLAST and keyword searches in the http://www.phytozome.net/). The A. thaliana GPATs sequences  were used as queries to perform BLASTp and TBLASTx searches in the Phytozome database. BLAST searches were conducted against 39 plant species genomes and proteomes available in Phytozome, including algae ; the lycophyte Selaginella moellendorffii, the mosses Physcomitrella patens and Sphagnum fallax; the single living representative of the sister lineage to all other extant flowering plants Amborella trichopoda; monocots ; and eudicots . The cDNA, genomic DNA, and amino acid sequences corresponding to each GPAT or putative GPAT were downloaded from the Phytozome database. All taxa were indicated by three-letter acronyms in which the first letter is the first letter of the genus and the next two letters are the first two letters of the species name (e.g. Osa corresponds to O. sativa). The sequences were identified in all analyses using the acronym followed by the protein accession number . The names for the previously reported A. thaliana GPATs were added before the acronym, and the accession number . A detailed description of the sequences used in this study, including their corresponding accession numbers, protein length, presence of protein domain and intron numbers is provided in Supplementary Table S1.To identify GPAT genes and their homologs, we first performed a literature survey to find GPAT genes that have already been characterized in the model plant http://beast.bio.ed.ac.uk/Tracer) was used to verify the convergence of the Markov chains and the adequate effective sample sizes (> 200). The trees were visualized and edited using http://tree.bio.ed.ac.uk/software/figtree).The nucleotide and protein sequences were aligned using MUSCLE  implemenA. thaliana, G. max, O. sativa, B. distachyon, S. moellendorffii, P. patens and V. carteri) to GSDraw and retrieved the gene structures with conserved protein motifs and phylogenetic trees. We also performed searches for each GPAT in all species deposited in the Piece database and retrieved gene structure organization and intron phase for all these species. The online Gene Structure Display Server (http://gsds.cbi.pku.edu.cn) was also used to analyze the intron/exon distribution and intron phase patterns along with the phylogenetic tree for the seven species cited above.In order to determine the intron/exon distribution in the GPAT genes of plants and understand the rules and possible consequences of gene structure and organization on protein functionality and evolutionary changes among species (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and in A. thaliana, O. sativa, S. moellendorffii, P. patens and V. carteri). Potential functional motifs of GPAT proteins were identified using the multiple expectation maximization for motif elicitation (MEME) utility program (http://meme.sdsc.edu) (http://weblogo.berkeley.edu/logo.cgi)  ; soluble GPAT, located in the plastid; and GPAT1-8, of which GPAT1-3 are localized in mitochondria and GPAT4-8 in ER. Using nucleotide and amino acid sequences from these 10 A. thaliana GPATs as queries, we conducted a broad survey of fully sequenced genomes for the presence of GPAT homologs genes in 39 species  (Tables S1 and S2). Candidate GPAT genes were found in all examined plant genomes. Interestingly, the BLAST searches against A. thaliana returned 11 putative GPAT sequences, among them 10 are known GPATs . One of these, AT3G11325, is annotated as a member of the phospholipid/glycerol acyltransferase protein family in the Phytozome database and is more similar to Arabidopsis GPAT5 and GPAT7 .Currently, there are 10 genes annotated as GPATs in the O. lucimarinus that presented three. The mosses P. patens and S. fallax presented nine and 12 putative GPAT genes, respectively. The lycophyte S. moellendorffii presented 12, while A. trichopoda has seven putative GPAT genes. Among the monocot species, B. distachyon, O. sativa and P. hallii presented 18 putative GPAT genes, while S. italica, S. viridis, S. bicolor and Z. mays presented 20, 19, 16 and 17 putative GPAT genes, respectively. In the eudicot species, the number of genes ranged from eight (C. sativus) to 28 (G. max). C. sinensis presented nine and C. clementina, A. lyrata, E. salsugineum, S. lycopersicum, P. trichocarpa, R. comunis, presented 10 putative GPAT genes. M. esculenta, A. thaliana, C. grandiflora and C. rubella presented 11 putative GPAT genes. E. grandis, T. cacao, M. truncatula and P. vulgaris presented 12, while M. guttatus and S. tuberosum presented 13 putative GPAT genes. A. coerulea presented 15 putative GPAT genes. G. raimondii and B. rapa presented 17 putative GPAT genes. To verify the reliability of the BLAST results, the 450 protein sequences retrieved were subjected to InterPro and Pfam analyses (Table S1), and most of them were classified into the acyltransferase family (Pfam: PF01553). This family contains acyltransferases involved in phospholipid biosynthesis and proteins of unknown function.GPAT genes were ubiquitously found in all algae and land plants studied. In total, we retrieved 450 sequences from 39 species . The algTo investigate the evolutionary relationships among the plant GPATs, we reconstructed phylogenetic trees using the protein sequences of putative GPATs identified by homology searches in 39 species. In G. max, G. raimondii, B. rapa, M. truncatula, E. grandis and Z. mays, these possibly presenting gene duplication events.Within clade I (GPAT9)  and cladO. sativa. The GPATs from P. patens and S. fallax (moss) and S. moellendorffii (lycophyte), two basal lineages of land plants, are phylogenetically more related with GPAT4-8 (subclade IIIa) and GPAT6 (subclade IIIb), implying that GPAT4-6-8 are the most ancient forms of GPATs exclusive of land plants (GPAT1-8). Within subclade IIIa, most of the species presented only one sequence. The species that presented more than one sequence are G. max, M. gutattus, B. rapa, A. lyrata, A. thaliana (well characterized GPAT 4 and GPAT8) and E. salsugineum. This indicates that duplication events that originated GPAT 4 and GPAT8 were independent, lineage specific events. Subclade IIIb (GPAT6) is closely related with subclade IIIa suggesting that GPAT4, GPAT8 and GPAT6 have a common ancestral gene and diverged from duplication events. GPAT5 and GPAT7 within subclade IIId are also likely resulted from independent and lineage-specific duplication events. GPAT1, GPAT2 and GPAT3 (subclade IIIe) are closely related and may have originated by duplication events in vascular plants. The A. thaliana AT3G11325 gene retrieved in BLAST searches and annotated as Phospholipid/glycerol acyltransferase family protein in the Phytozome database is placed in subclade IIId, close to GPAT5 and GPAT7. This sequence also presents an acyltransferase domain.Clade III (GPAT1-8)  and 5 isTable S1 and A. thaliana GPAT 4 and 8 that have three introns . Conserved motifs in the representative proteins from plant and algae species are depicted in Table S3, Figure S1), while no TrM was identified for plastid GAPT, indicating that GPAT1-9 proteins are associated with membrane systems and that plastid GPAT is a soluble form.After the examination of gene structure, we continued our analysis with a focus on the protein properties of 450 putative GPATs, including protein length, presence of putative transmembrane domains, and conserved motifs. Overall, the length of the GPAT amino acid sequences ranged from 237 to 621 residues  and G. max  and the monocots O. sativa  and Z. mays . We found probes for nine GPATs in A. thaliana . For G. max, eight out of 28 genes identified in our BLAST searches presented available probes . For the monocots, we found 17 available probes for 17 putative GPATs for Z. mays  and 17 probes for O. sativa . We analyzed 105 anatomical parts and 10 developmental stages from A. thaliana, 68 anatomical parts and five developmental stages from G. max, 85 anatomical parts and 7 developmental stages from Z. may, and 38 anatomical parts and 9 developmental stages from O. sativa.The available plant expression data from GENEVESTIGATOR was used to obtain information about potential functional roles of each GPAT. We analyzed the temporal and spatial expression patterns of the GPAT genes in plant tissues, using public microarray expression data of the eudicots In silico analyses of the expression profiles showed that all plant GPAT genes present some expression level in developmental stages and anatomical parts. However, different expression patterns across different tissues and plant developmental stages were found across different GPATs within each species  is more expressed in radicle, maturation zone and root hair, while Glyma.14G028300  and Glyma.02G249300 (included in subclade IIId – GPAT5 and 7) are more expressed in flower cluster (raceme), flower, androecium, stamen, anther and pollen. O. sativa LOC_Os01g63580 included in the GPAT6 group (subclade IIIb) is more expressed in inflorescence, panicle, spikelet, coleoptile, anther and pistil. O. sativa LOC_Os01g44069 included in the GPAT1 group (subclade IIIe) is highly expressed in stigma. O. sativa LOC_Os11g45400 grouped into subclade IIId is more expressed in seedling, leaf, inflorescence, anther and pistil. Z. mays GRMZM2G070304 included in subclade IIIe (GPAT1-3) is highly expressed in spikelet cell, floret cell, stamen cell, anther cell and meyocite. species . For exaGlycerolipids play crucial role in plant biology, since they serve as major components of cellular membranes, storage lipids in developing seeds, and the protective hydrophobic barrier on the cuticular surface of plant organs . In addiA. trichopoda and P. trichocarpa. Our phylogenetic analysis showed that GPAT9 is a very divergent clade. It has already been reported that A. thaliana GPAT9 is more closely related to the mammalian ER-localized GPAT3 and GPAT4 compared to other members of the A. thaliana GPAT family (GPAT1–8), suggesting that the divergence of the GPAT9 gene from the GPAT1–8 of this species occurred prior to the evolutionary split between plants and mammals  presents higher expression compared to other GPATs in endosperm tissue, suggesting that it is likely important in castor oil synthesis   were included in the clade III and are present only in Embryophyta lineages. It was shown for invasion . The expinvasion  is assocIn conclusion, our study provides a comprehensive genomic analysis of GPAT genes in plants, covering phylogenetic, gene structure, protein properties, and gene expression analysis. These results can improve our understanding of the evolutionary history of GPAT genes in plants and shed light on their function. Phylogenetic analysis indicates that plant GPATs can be grouped into three distinct clades, which is further supported by their conservation and variation in gene structure, protein properties, motif occurrences and gene expression patterns. Our study, together with previous studies, suggests that the presence of several genes encoding GPATs in land plants may be related to their adaptation to a terrestrial environment. Current knowledge regarding the functions of plant GPATs is limited to few species. To obtain a more thorough understanding of the function of GPATs in plants, the functional characterization of GPATs in more species will be necessary.