The power of neuropeptide precursor sequences to reveal phylogenetic relationships in insects: A case study on Blattodea.

Recent state-of-the-art analyses in insect phylogeny have exclusively used very large datasets to elucidate higher-level phylogenies. We have tested an alternative and novel approach by evaluating the potential phylogenetic signals of identified and relatively short neuropeptide precursor sequences with highly conserved functional units. For that purpose, we examined available transcriptomes of 40 blattodean species for the translated amino acid sequences of 17 neuropeptide precursors. Recently proposed intra-ordinal relationships of Blattodea, based on the analysis of 2370 protein-coding nuclear single-copy genes (Evangelista et al., 2019), were corroborated with maximum support. The functionally different precursor units were analyzed separately for their phylogenetic information. Although the degree of information was different in the different sequence motifs, all precursor units contained phylogenetic informative data at the ordinal level, and their separate analysis did not reveal contradictory topologies. This study is the first comprehensive exploitation of complete neuropeptide precursor sequences of arthropods in such a context and demonstrates the applicability of these rather short but conserved sequences for an alternative, fast and simple analysis of phylogenetic relationships.

The ratio of N-terminal pro-B-type natriuretic peptide to troponin I for differentiating acute coronary syndrome.

INTRODUCTION: It is difficult to differentiate whether coronary or non-coronary causes in patients with elevated troponin I (TnI) in emergency department (ED). The aim of this study was to develop a clinical decision tool for differentiating a coronary cause in the patients with elevated TnI. METHODS: This was a retrospective observational study that enrolled consecutive ED patients. Patients were included in the study if they were ≥16 years of age, had admitted through ED with a medical illness, and TnI levels at initial evaluation in the ED were ≥0.2 ng/mL. Patients diagnosed with ST elevation myocardial infarction or congestive heart failure were excluded. Coronary angiography, electrocardiogram, laboratory results, echocardiography, and clinical characteristics were analyzed. RESULTS: Among the included 1441 patients, 603 and 838 patients were categorized into an acute coronary syndrome (ACS) group and non-acute coronary syndrome (non-ACS) group, respectively. The ratio of N-terminal pro-Btype natriuretic peptide (NT-proBNP) to TnI was significantly higher in the non-ACS group compared to the ACS group. The AUC of NT-proBNP/TnI (0.805, 95% CI, 0.784-0.826) was significantly superior to that of NT-proBNP/creatinine kinase-MB, TnI, and NT-proBNP. The patients of the non-ACS group with high levels of TnI and BNP showed more critically ill manifestation at the time of presentation and higher mortality. CONCLUSION: NT-proBNP/TnI may help to distinguish medical patients with elevated TnI whether the elevated TnIs were caused from ACSs or from conditions other than ACS.

Antimicrobial activities of a proline-rich proprotein from Spodoptera litura.

Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline-rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura. SlLebocin1 cDNA contains a 477-bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram-negative and two Gram-positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis, only the C-terminal fragment (124-158) showed strong inhibitory effects. For Staphylococcus aureus, all peptides showed partial inhibitions. None of them inhibited Serratia marcescens. Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms.

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide 'cocktails' is functionally important, but with plasticity over long evolutionary time scales.

Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.

Discovery of sea urchin NGFFFamide receptor unites a bilaterian neuropeptide family.

Neuropeptides are ancient regulators of physiology and behaviour, but reconstruction of neuropeptide evolution is often difficult owing to lack of sequence conservation. Here, we report that the receptor for the neuropeptide NGFFFamide in the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) is an orthologue of vertebrate neuropeptide-S (NPS) receptors and crustacean cardioactive peptide (CCAP) receptors. Importantly, this has facilitated reconstruction of the evolution of two bilaterian neuropeptide signalling systems. Genes encoding the precursor of a vasopressin/oxytocin-type neuropeptide and its receptor duplicated in a common ancestor of the Bilateria. One copy of the precursor retained ancestral features, as seen in highly conserved vasopressin/oxytocin-neurophysin-type precursors. The other copy diverged, but this took different courses in protostomes and deuterostomes. In protostomes, the occurrence of a disulfide bridge in neuropeptide product(s) of the precursor was retained, as in CCAP, but with loss of the neurophysin domain. In deuterostomes, we see the opposite scenario-the neuropeptides lost the disulfide bridge, and neurophysin was retained (as in the NGFFFamide precursor) but was subsequently lost in vertebrate NPS precursors. Thus, the sea urchin NGFFFamide precursor and receptor are 'missing links' in the evolutionary history of neuropeptides that control ecdysis in arthropods (CCAP) and regulate anxiety in humans (NPS).

Full-length cDNA cloning and structural characterization of preproinsulin in Alligator sinensis.

Insulin is an important endocrine hormone that plays a critical physiological role in regulating metabolism and glucostasis in vertebrates. In this study, the complete cDNA of Alligator sinensis preproinsulin gene was cloned for the first time by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods; the amino acid sequence encoded and protein structure were analyzed. The full-length of preproinsulin cDNA sequence consists of 528 base pairs (bp), comprising a 34-bp 5'-untranslated region, a 170-bp 3'-untranslated region and an open reading frame that is 324 bp in length. The open reading frame encodes a 107-amino acid preproinsulin with a molecular weight of approximately 12,153.8 Da, theoretical isoelectric point of 5.68, aliphatic index of 92.06, and grand average of hydropathicity of -0.157, from which a signal peptide, a B-chain, a C-peptide, and an A-chain are derived. Online analysis suggested that the deduced preproinsulin amino acid sequence contains a transmembrane region, and that it has a signal peptide whose cleavage site occurs between alanine 24 and alanine 25. Comparative analysis of preproinsulin amino acid sequences indicated that the A-chain and B-chain sequences of preproinsulins are highly conserved between reptiles and birds, and that the preproinsulin amino acid sequence of Alligator sinensis shares 89% similarity to that of Chelonia mydas, but low similarity of 48-63% to those of mammals and fishes. The phylogenetic tree constructed using the neighbor-joining method revealed that preproinsulin of Alligator sinensis had high homology with reptiles and birds, such as Chelonia mydas, Gallus gallus, and Columba livia.

NeuroPID: a classifier of neuropeptide precursors.

Neuropeptides (NPs) are short secreted peptides produced in neurons. NPs act by activating signaling cascades governing broad functions such as metabolism, sensation and behavior throughout the animal kingdom. NPs are the products of multistep processing of longer proteins, the NP precursors (NPPs). We present NeuroPID (Neuropeptide Precursor Identifier), an online machine-learning tool that identifies metazoan NPPs. NeuroPID was trained on 1418 NPPs annotated as such by UniProtKB. A large number of sequence-based features were extracted for each sequence with the goal of capturing the biophysical and informational-statistical properties that distinguish NPPs from other proteins. Training several machine-learning models, including support vector machines and ensemble decision trees, led to high accuracy (89-94%) and precision (90-93%) in cross-validation tests. For inputs of thousands of unseen sequences, the tool provides a ranked list of high quality predictions based on the results of four machine-learning classifiers. The output reveals many uncharacterized NPPs and secreted cell modulators that are rich in potential cleavage sites. NeuroPID is a discovery and a prediction tool that can be used to identify NPPs from unannotated transcriptomes and mass spectrometry experiments. NeuroPID predicted sequences are attractive targets for investigating behavior, physiology and cell modulation. The NeuroPID web tool is available at http:// neuropid.cs.huji.ac.il.

[Heterogeneity of hepatitis B virus and diagnostic potential of modern test systems for the detection of HBsAg].

AIM: Study heterogeneity ofhepatitis B virus in adult patients with chronic hepatitis B and determination of diagnostic potential of modern test systems with the detection of HBsAg with amino acid substitutions in the main hydrophilic region (MHR). MATERIALS AND METHODS: In 27 hepatitis B virus samples isolated from patients with chronic hepatitis B virus infection living in Vladimir, nucleotide sequence ofgenome region corresponding to preS1/preS2/S genes was determined. RESULTS: In all of the 27 isolates genotype D virus presented by 3 subgenotypes D1, D2, D3 was detected in 18%, 26% and 56% respectively. Based on the distribution of nucleotide substitutions in the compared functional regions of hepatitis B virus (virus entry into the cell coding site (2875 - 2991 n.b.), pre-S2/S promoter region (2994 - 3171 n.b.), 5'-end pre-S2 and S-genes sequences (3172 - 154 n.b. and 155-455 n.b.), MHR (455 - 635 n.b.) and 3'-end S-gene sequence (636 - 835 n.b.), substitutions are mostly concentrated in the promoter region of the S2/S-genes (30.8%). HBsAg serotypes were determined in 24 of 27 cases by using the predicted amino acid sequence, and in 17 cases HBsAg belonged to ayw2 (71%) serotype and in 7 cases - to ayw3 serotype (29%). Amino acid substitutions G145A, M133I, S132T localized in the main hydrophilic region and P217L, S207N, V184A localized in the C-end of the protein C that are connected with diagnostic and vaccine escape were identified in 5 isolates. CONCLUSION: Diagnostic potential of test systems with the detection of HBsAg with known amino acid sequence of the MHR region were studied. Approximately equal potential of 6 test systems to detect HBsAg with amino acid substitutions G145A, M133I and S132T localized in the MHR region were shown.

Identification and classification of conopeptides using profile Hidden Markov Models.

Conopeptides are small toxins produced by predatory marine snails of the genus Conus. They are studied with increasing intensity due to their potential in neurosciences and pharmacology. The number of existing conopeptides is estimated to be 1 million, but only about 1000 have been described to date. Thanks to new high-throughput sequencing technologies the number of known conopeptides is likely to increase exponentially in the near future. There is therefore a need for a fast and accurate computational method for identification and classification of the novel conopeptides in large data sets. 62 profile Hidden Markov Models (pHMMs) were built for prediction and classification of all described conopeptide superfamilies and families, based on the different parts of the corresponding protein sequences. These models showed very high specificity in detection of new peptides. 56 out of 62 models do not give a single false positive in a test with the entire UniProtKB/Swiss-Prot protein sequence database. Our study demonstrates the usefulness of mature peptide models for automatic classification with accuracy of 96% for the mature peptide models and 100% for the pro- and signal peptide models. Our conopeptide profile HMMs can be used for finding and annotation of new conopeptides from large datasets generated by transcriptome or genome sequencing. To our knowledge this is the first time this kind of computational method has been applied to predict all known conopeptide superfamilies and some conopeptide families.

A novel class of peptide pheromone precursors in ascomycetous fungi.

Recently, sexual development in the heterothallic ascomycete Trichoderma reesei (anamorph of Hypocrea jecorina) has been achieved and thus initiated attempts to elucidate regulation and determinants of this process. While the α-type pheromone of this fungus fits the consensus known from other fungi, the assumed a-type peptide pheromone precursor shows remarkably unusual characteristics: it comprises three copies of the motif (LI)GC(TS)VM thus constituting a CAAX domain at the C-terminus and two Kex2-protease sites. This structure shares characteristics of both a- and α-type peptide pheromone precursors. Presence of hybrid-type peptide pheromone precursor 1 (hpp1) is essential for male fertility, thus indicating its functionality as a peptide pheromone precursor, while its phosphorylation site is not relevant for this process. However, sexual development in a female fertile background is not perturbed in the absence of hpp1, which rules out a higher order function in this process. Open reading frames encoding proteins with similar characteristics to HPP1 were also found in Fusarium spp., of which Fusarium solani still retains a putative a-factor-like protein, but so far in no other fungal genome available. We therefore propose the novel class of h-type (hybrid) peptide pheromone precursors with H. jecorina HPP1 as the first member of this class.

[Prediction of the efficacy of bevirimat used for the treatment of HIV infection in Russia].

The nucleotide sequences coding the CA-SP1 Pr55(gag) in 61 samples of HIV-1 subtype A variant IDU-A isolated in Russia were analyzed for bevirimat resistance mutations (CA-H226V, CA-L231F, CA-231M, SP1-A1V, SP1-A3T, SP1-A3V) and for polymorphisms in the GAG CA-SP1 cleavage site. None of the six bevirimat resistance mutations was found in the sequences analyzed. There were three polymorphisms CA-G225S, CA-R229K, CA-V2301 and a high variability in the C-terminus of SP1. The substitution SP-T8Q was observed in 98% of cases, which could probably affect the clinical efficacy of bevirimat. Therefore bevirimat can be potentially active in Russian patients infected with IDU-A variant, but strain-specific polymorphisms in combination with other virus genome mutations can influence the efficiency of bevirimat treatment.

Analysis and classification of circular proteins in CyBase.

CyBase is a database dedicated to the study of the sequences and three-dimensional structures of ribosomally synthesized, backbone cyclized proteins, and their synthetic variants. This article describes CyBase data and tools that are useful in the analysis of circular proteins. Circular proteins have now been discovered in organisms from all kingdoms of life, and given the current rate of discovery they could soon number in the thousands. Presently CyBase manages 427 protein sequences, 106 nucleic acid sequences, and 49 protein three-dimensional structures from 44 different species. Circular proteins are grouped into distinct classes according to their origin and sequence similarities. These classes include trypsin inhibitors, bacterial proteins, mushroom toxins, cyclotides, and cyclic defensins from primates. Several protein classification types are used in CyBase to designate proteins extracted from natural resources (wild type and precursor) or engineered (modified wild type, grafted, mutant, cyclic permutant, and acyclic permutant). CyBase has tools for the analysis of mass spectrum fingerprints of cyclic peptides, and assists in the discovery of new circular proteins. Some of the developments detailed here have been made specifically for the largest class of circular proteins, the cyclotides, but could be adapted for other classes of cyclic proteins. The cyclotide-specific tools include two-dimensional representations of domains and alternative displays of alignments for precursor sequences. This alignment prompted us to propose a revision of the cydclotide precursor organization, in which the repeated regions now include a small C-terminal region, which appears to have a significant role in the biosynthesis of mature cyclotides.

Novel alpha D-conopeptides and their precursors identified by cDNA cloning define the D-conotoxin superfamily.

AlphaD-conotoxins are peptide inhibitors of nicotinic acetylcholine receptors (nAChRs) first described from Conus vexillum (alphaD-VxXIIA-C and renamed here to alphaD-VxXXA, alphaD-VxXXB, and alphaD-VxXXC). In this study, we report cDNA sequences encoding D-superfamily conopeptides identified in the Clade XII Conidae Conus vexillum, Conus capitaneus, Conus mustelinus, and Conus miles, together with partial sequences of corresponding peptides from this family. The D-superfamily signal peptide sequences display greater heterogeneity than reported for other conotoxin superfamilies. Phylogenetic analysis of the relationships among alphaD-conotoxin precursors reveals two distinct groups containing either an EMM or AVV signal peptide sequence motif. Homodimer and heterodimer combinations of predicted mature toxin sequences likely account for the partial amino acid sequences and mass values observed for several of the native dimeric peptide components identified in C. capitaneus, C. miles, and C. mustelinus venom. The discovery of the precursors and several novel conotoxins from different species defines this large conotoxin family and expands our understanding of sequence diversification mechanisms in Conus species.

Novel brevinins from Chinese piebald odorous frog (Huia schmackeri) skin deduced from cloned biosynthetic precursors.

Antimicrobial peptides represent the most characterized and diverse class of peptides within the defensive skin secretions of anuran amphibians. With an ever expanding database of primary structures, the current accepted rules for nomenclature have become increasingly difficult to apply to peptides whose primary structural attributes are either unique or that fall between those that define existing groups. An additional factor that adds to the confusion is the regular re-classification or revision of existing taxa. In the present study, we have identified five new antimicrobial peptide homologs in the defensive skin secretion of the Chinese piebald odorous frog, Huia schmackeri (formerly Rana (Odorrana) schmackeri), by cloning of their respective biosynthetic precursors. As these peptides are obvious homologs of the brevinin-1 and brevinin-2 families we have named these in accordance: (1) brevinin-1HS1, (2) brevinin-2HS1, (3) brevinin-2HS2, (4) brevinin-2HS3 and (5) brevinin-1HS2. The reasons for adopting these names are discussed. It is clear that with an ever-increasing number of amphibian skin antimicrobial peptides appearing in the literature that a consistent nomenclature scheme needs to be established.

Differential expression of duplicated genes for prothymosin alpha during zebrafish development.

We show that ptma, a single copy gene found in all organisms investigated so far, is duplicated in zebrafish. The two genes, ptmaa and ptmab, are individually controlled as indicated by their different expression patterns during embryonic development. Only the ptmab transcript is observed at 4 and 8 hpf of development in all embryonic cells, whereas both genes are expressed at later stages as revealed by in situ hybridization studies. In most cases, the two genes are expressed in the same territories, but only the ptmaa transcript was found in the trigeminal ganglion and in endodermal pouches. In the eye, at 72 hpf, the ptmaa and ptmab transcripts were found in amacrine cells, whereas only the ptmab transcript appeared in horizontal cells. The existence of two prothymosin genes indicates that their function in cell proliferation and differentiation is more complex in fishes than in mammals.

Cbln and C1q family proteins: new transneuronal cytokines.

The C1q family is characterized by a C-terminal conserved global C1q domain, which is structurally very similar to the tumor necrosis factor homology domain. Although some C1q family members are expressed in the central nervous system, their functions have not been well characterized. Cbln1, a member of the Cbln subfamily of the C1q family, is predominantly expressed in cerebellar granule cells. Interestingly, Cbln1 was recently shown to play two unique roles at excitatory synapses formed between cerebellar granule cells and Purkinje cells: the formation and stabilization of synaptic contact, and the control of functional synaptic plasticity by regulating the postsynaptic endocytosis pathway. Since other Cbln subfamily members, Cbln2-Cbln4, are expressed in various regions of developing and mature brains, Cbln subfamily proteins may generally serve as a new class of transneuronal regulators of synapse development and synaptic plasticity in various brain regions.

[Molecular forms of PSA]. / Les formes moléculaires du PSA.

Molecular forms of serum PSA (prostate specific antigen) have been developped to improve total PSA sensitivity and specificity in prostate cancer diagnosis and staging. Total PSA is measured in bound (complexed PSA) and unbound (free PSA) molecular forms. Their levels in absolute values and in relation to total PSA (f/t PSA and c/t PSA) have been evaluated. The percentage of free PSA is more specific but less sensitive than tPSA and it is not recommended as a first line diagnostic test. It may be useful as a second-line test, prescribed by the urologist after a first series of negative biopsies. There is general agreement that at high sensitivity, cPSA provides higher specificity compared with tPSA in the gray zone (2-10 ng/ml). Nevertheless the widespread use of tPSA an the small benefit in terms of specificity explains why cPSA is not generally recommanded. Molecular derivates of free PSA have been identified: proPSA (precursor inactive form of PSA), intact PSA (an additionnal form of proPSA that is found intact and inactive), human Kallikrein 2 and BPSA (for benign PSA wich is associated to BPH) have been evaluated. Preliminary studies did not have demonstrate their ability to discriminate between cancer and BPH, and did not define cutoff values.

Molecular characterization of prepro-orexin in Atlantic cod (Gadus morhua): cloning, localization, developmental profile and role in food intake regulation.

A full-length cDNA encoding for prepro-orexin (prepro-OX) was cloned from Atlantic cod (Gadus morhua) hypothalamus using reverse transcription and rapid amplification of cDNA ends (RACE). The 143 amino acids (aa) prepro-OX contains a 38 aa signal peptide, a 50 aa orexin-A peptide and a 29 aa orexin-B peptide. Semi-quantitative RT-PCR shows that prepro-OX mRNA is present in brain and pituitary and in peripheral tissues, including gill, spleen, stomach and gut. Within the brain, high expression levels are seen in the hypothalamus. During development, prepro-OX is expressed from the cleavage stage up to the hatched larvae. Slot blot analysis shows that prepro-OX expression levels are higher in fish fed low (0.2% BW) and medium (0.8% BW) rations than in fish fed high rations (1.5% BW). Fish fed low and medium rations also display periprandial changes in prepro-OX expression, with higher expression levels at meal time (0 h) compared to 2h before and 2h after feeding. Our results suggest that orexins might be involved in development and feeding regulation in Atlantic cod.

Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii revealed from a Duvernoy's (venom) gland transcriptome.

We investigated the putative toxins of Philodryas olfersii (Colubridae), a representative of a family of snakes neglected in venom studies despite their growing medical importance. Transcriptomic data of the venom gland complemented by proteomic analysis of the gland secretion revealed the presence of major toxin classes from the Viperidae family, including serine proteases, metalloproteases, C-type lectins, Crisps, and a C-type natriuretic peptide (CNP). Interestingly, the phylogenetic analysis of the CNP precursor showed it as a linker between two related precursors found in Viperidae and Elapidae snakes. We suggest that these precursors constitute a monophyletic group derived from the vertebrate CNPs.