- The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - mansoni elicits significant changes in neuropeptide dynamics in the B. - As ion channels are the fundamental units of excitabil- ity and synaptic transmission in the nervous system, characterization and identification of ion channels is enormously important to the field of neuroscience.. - Mutations in the genes encoding ion channels, or encod- ing proteins that regulate ion channels, have dramatic effects on normal functioning, resulting in a wide range of debilitating diseases [17, 18]. - This refrain of invertebrate channels being conserved in mammals is repeated in the mouse homologues of Drosophila Shaker, Shal, Shab, and Shaw K + channels, and in humans as well, with the Drosophila K+ channels all having been shown to be expressed in human cardiac tissue [31]. - 1 Workflow of quality control, assembly and prediction of protein-coding transcripts in the L. - With the aid of a preliminary, recently sequenced and Table 1 BLAST hit of the top 20 expressed transcripts in the L. - Table 2 BUSCO analyses of protein-coding sequences identified in the published EST library and de novo assembly, the current assembly and combined set. - stagnalis protein-coding transcripts identified in the current assembly with those identified in the previously published EST library [49] and de novo assembly [50]. - stagnalis CNS transcriptome To compare the number and quality of predicted protein-coding transcripts uncovered in our current as- sembly with those in the previously published expressed sequence tags (EST) library [49] and de novo assembly [50], these two earlier sets of sequences were processed as described above to identify potential protein coding transcripts. - We found 1946 sequences (out of 10,375 in total) that contained at least one protein domain and/or were annotated as a signaling peptide by both Phobius and SignalP in the EST library, and 11,742 such se- quences (out of 116,355 in total) in the de novo assem- bled library. - BUSCO analysis showed that the predicted protein-coding transcripts identified in the current as- sembly contained 92.7% of single-copy ortholog genes present in >. - current: 13,109 aa) sequence lengths in the current assembly were all higher than those in the ESTs library and de novo assembly. - Comparison of unique Nr hits in the three sets of predicted protein- coding sequences showed that 7198 Nr hits were found in all three, with and 316 hits found only in the current assembly, de novo assembly, and ESTs li- brary, respectively (Fig. - We next examined this comprehensive set of sequences to characterize gene expression in the L. - 3 Comparison of KOG annotations of protein-coding transcripts expressed in the CNS of key vertebrate and invertebrate neuroscience model organisms (E-value <1E-5). - To this end, we analyzed publicly deposited RNA-seq librar- ies to characterize gene expression in the CNS of adult Mus musculus (mouse) [53], Xenopus tropicalis [53], Da- nio rerio (zebrafish) [54], Drosophila melanogaster (fruit fly) (BioProject: PRJNA320764), and Caenorhabditis elegans [55] (Table S1). - An- notation of the top 20 expressed transcripts in each of these five species (Tables S7, S8, S9, S10 and S11), in the same manner as done for L. - “Transcription”, and “Cytoskeleton” than in the three in- vertebrate species. - Conversely, in the three invertebrate species, more transcripts were identified to be function- ally involved in “Carbohydrate transport and metabol- ism”, “Lipid transport and metabolism”, and. - “Translation, ribosomal structure and biosynthesis” than those in the three vertebrate species.. - We further clustered CNS protein-coding transcripts in the six species into orthologous groups to identify. - 4 Membership of species in the orthogroups identified amongst protein-coding transcripts expressed in the CNS of M. - The number of orthogroups that each species is present in is shown in the inset bar graph. - Furthermore, we identified 222 transcription factors whose binding motifs were enriched in the promoter region of this set of mouse genes (Table S15), identifying both well-characterized neuronal tran- scription factors such as CREB1 and AP2, but also novel proteins that may provide targets of future studies. - We also performed similar enrichment analyses of genes in the 2154 vertebrate-only and 88 invertebrate-only orthogroups, in order to gain insights into differential functional specializations of the vertebrate and inverte- brate CNS. - Enrichment of mouse genes in the vertebrate-only orthogroups showed a constellation of GO (Fig. - Enrichment of fruit fly genes in the invertebrate-only orthogroups found terms relating to response to chemical stimuli, such as “Sensory perception of chemical stimulus” (GO:. - Identification of ion channels and ionotropic receptors expressed in the L. - Ion channels and ionotropic neurotransmitter receptors play critical roles in regulating neuronal excitability and synaptic transmission in the CNS. - stagnalis CNS is widely employed to study the fundamental mechanisms of neuronal func- tion and circuit function we next sought to identify ion channels and ionotropic receptors expressed in the L. - We identified 211 protein- coding transcripts that contained both 1) ion channel or ionotropic receptor protein domains and 2) transmem- brane helices to characterize the complement of ion channels and ionotropic receptors expressed in the L.. - A proportion of sequences in this set were substantially shorter or longer than their best matched sequence in the Nr database. - stagnalis sequence in the Nr database, indicating that majority of the sequences were identified here in this species for the first time.. - 5 Enriched GO terms of genes encoded by transcripts in the 3729 orthogroups shared amongst M. - Using the REVIGO tool, GO terms in the “ Biological process ” (a. - 6 Enriched GO terms of genes encoded by transcripts in the 2154 orthogroups shared amongst only M. - GO terms in the “ Biological process ” (a. - Cellular components ” (b) and “ Molecular function ” (c) classes are clustered and coloured in the same manner as described in Fig. - Firstly, we per- formed strand-specific paired-end RNA-seq that pro- duced 150 bp long reads in this study, as compared to the unstranded 100 bp single-end reads used in the pre- viously published de novo assembly [50]. - 7 Enriched GO terms of genes encoded by transcripts in the 88 orthogroups shared amongst only L. - 8 Ionotropic neurotransmitter receptor families identified in the L. - The tree is rooted at the mid-point and is drawn to scale, with branch lengths measured in the number of substitutions per site (see scale in bottom left). - There were a total of 133 positions in the final dataset. - Then, we characterized the functional distribu- tion of protein-coding transcripts expressed in the L.. - and 14, Tables S15, S16, S17, S18, S19, S20, S21, S22 and S23), greatly expanding our understanding of the molecular determinants of neural excitability and transmission in the L. - 9 K + channel subtypes and families identified in the L. - 8, where the analysis involved 36 amino acid sequences and yielded a total of 75 positions in the final dataset, resulting in a tree with highest log likelihood of − 4511.88. - stagnalis show the highest degrees of sequence similarity with their homologs in the other five species (Fig. - 10 Ca 2+ channel subtypes and families identified in the L. - 8, where the analysis involved 24 amino acid sequences and yielded a total of 4018 positions in the final dataset, resulting in a tree with highest log likelihood of . - 11 Na + channel subtypes and families identified in the L. - 8, where the analysis involved 12 amino acid sequences and yielded a total of 345 positions in the final dataset, resulting in a tree with highest log likelihood of − 6272.64. - In the case of cyto- skeleton dynamics, this is consistent with known differ- ences in multidirectional polarity and absence of dendritic spines in invertebrate neurons as compared to their vertebrate counterparts [87]. - Finally, we clustered all protein-coding transcripts expressed in the CNS of these six species into orthogroups, each of which was predicted based on se- quence similarities to be descended from a common gene in the last common ancestor of these six species.. - 12 Cl − channel subtypes and families identified in the L. - 8, where the analysis involved 18 amino acid sequences and yielded a total of 1233 positions in the final dataset, resulting in a tree with highest log likelihood of . - Similarly, most invertebrates lack dendritic spines, plasticity of which relies on actin remodeling [95], and differential conser- vation of actin dynamics between vertebrates and inver- tebrates identified in the current study is consistent with this. - Finally, differences in enrichment of genes related to GPCR signaling is expected due to differences in the ensemble of GPCRs expressed in invertebrates compared to vertebrates. - 13 Cation channel subtypes and families identified in the L. - 8, where the analysis involved 17 amino acid sequences and yielded a total of 8 positions in the final dataset, resulting in a tree with highest log likelihood of − 339.65. - This phenomenon occurs due to a thermolabile hidden break in the molecule, as described in various in- vertebrates, which leads to its cleavage into two sub- parts of size similar to that of 18S RNA [100]. - 14 Transient receptor potential (TRP) channel subtypes and families identified in the L. - 8, where the analysis involved 33 amino acid sequences and yielded a total of 133 positions in the final dataset, resulting in a tree with highest log likelihood of − 7343.03. - Workflow for assembling and identifying protein-coding transcripts in the L. - blob/master/FilterUncorrectabledPEfastq.py), and processed and assembled in the same manner, except using a modified custom script to remove unfixable single-end reads after rCorrector analysis. - stagnalis CNS were obtained from the European Nucleotide Archive and processed in the same manner as described above to predict protein-coding sequences. - Each bitscore is standardized by the average and standard deviation of all bitscores in the matrix. - Quality of reads was visualized using FastQC, and erroneous bases were corrected and fil- tered in the same manner as described above. - Pfam IDs used to identify transcripts encoding ion channels and ionotropic receptors in the L. - Proportion of transcripts containing complete and fragmented ORFs as identified by the Evigene pipeline in the “ okay ” and “ okalt ” sequence sets. - Top 20 expressed transcripts in the adult mouse brain. - Top 20 expressed transcripts in the adult X.. - Top 20 expressed transcripts in the adult zebra- fish brain. - Top 20 expressed transcripts in the adult fruitfly brain. - Transcript factors whose binding motifs are enriched in the set of mouse genes in orthogroups shared amongst all the species examined.. - Transcripts encoding ionotropic acetylcholine receptors in the L. - Transcripts encoding ionotropic GABA or glycine receptors in the L. - Transcripts encoding ionotropic glutamate receptors in the L.. - Transcripts encoding K+ channels in the L.. - Transcripts encoding Ca2+ channels in the L.. - Transcripts encoding Na + channels in the L.. - Transcripts encoding Cl- channels in the L.. - Transcripts encoding cation channels in the L.. - Transcripts encoding TRP channels in the L.. - ZZ and YW participated in the bioinformatic analysis. - Changes in the activity of a CpG neuron after the reinforcement of an operantly conditioned behavior in Lymnaea. - Excitatory actions of GABA mediate severe-hypoxia-induced depression of neuronal activity in the pond snail (Lymnaea stagnalis). - Effects of chronic waterborne nickel exposure on growth, ion homeostasis, acid-base balance, and nickel uptake in the freshwater pulmonate snail, Lymnaea stagnalis. - Pyrosequencing-based transcriptomic resources in the pond snail Lymnaea stagnalis, with a focus on genes involved in molecular response to diquat-induced stress. - Changes in the neuropeptide content of Biomphalaria ganglia nervous system following Schistosoma infection. - Ligand-gated ion channels in the brain: the amino acid receptor superfamily. - Cloning and Characterization of a P2X Receptor Expressed in the Central Nervous System of Lymnaea stagnalis. - Distinguishing protein-coding and noncoding genes in the human genome. - The participation of NMDA receptors, PKC, and MAPK in the formation of memory following operant conditioning in Lymnaea. - Combining Transcriptome assemblies from multiple de novo assemblers in the Allo-Tetraploid plant Nicotiana benthamiana. - Discovery of leucokinin-like neuropeptides that modulate a specific parameter of feeding motor programs in the molluscan model, Aplysia. - Gene splicing of an invertebrate beta subunit (LCav β ) in the N-terminal and HOOK domains and its regulation of LCav1 and LCav2 calcium channels. - Identification and functional expression of a family of nicotinic acetylcholine receptor subunits in the central nervous system of the mollusc Lymnaea stagnalis. - Characterization of the transcriptome, nucleotide sequence polymorphism, and natural selection in the desert adapted mouse Peromyscus eremicus
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