RESUMO
Blastocyst vitrification has significantly improved embryo transfer methods, leading to higher implantation success rates and better pregnancy outcomes in subsequent frozen embryo transfer cycles. This study aimed to simulate the transcriptional changes caused by vitrifying human blastocysts using mouse blastocysts as a model and to further investigate these changes' effects. Utilizing a human vitrification protocol, we implanted both vitrified and fresh embryos into mice. We observed the implantation success rates and performed transcriptomic analysis on the blastocysts. To validate the results from messenger RNA sequencing, we conducted reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of specific genes. Based on mRNA profiling, we predicted the microRNAs responsible for the regulation and used qPCR basic microRNA assays for validation. Our observations revealed a higher implantation success rate for vitrified embryos than fresh embryos. Transcriptomic analysis showed that vitrified-warmed blastocysts exhibited differentially expressed genes (DEGs) primarily associated with thermogenesis, chemical carcinogenesis-reactive oxygen species, oxidative phosphorylation, immune response, and MAPK-related signaling pathways. RT-qPCR confirmed increased expression of genes such as Cdk6 and Nfat2, and decreased expression of genes such as Dkk3 and Mapk10. Additionally, gene-microRNA interaction predictions and microRNA expression analysis identified twelve microRNAs with expression patterns consistent with the predicted results, suggesting potential roles in uterine epithelial cell adhesion, trophectoderm development, invasive capacity, and immune responses. Our findings suggest that vitrification induces transcriptomic changes in mouse blastocysts, and even small changes in gene expression can enhance implantation success. These results highlight the importance of understanding the molecular mechanisms underlying vitrification to optimize embryo transfer techniques and improve pregnancy outcomes.
Assuntos
Blastocisto , Criopreservação , Implantação do Embrião , Perfilação da Expressão Gênica , MicroRNAs , Vitrificação , Animais , Blastocisto/metabolismo , Camundongos , Implantação do Embrião/genética , Feminino , Criopreservação/métodos , Perfilação da Expressão Gênica/métodos , Gravidez , MicroRNAs/genética , Transcriptoma , Transferência Embrionária/métodos , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
OBJECTIVES: Hyperphenylalaninemia predicts poor outcomes in patients with cardiovascular disease. However, the prognostic value and factors associated with stress hyperphenylalaninemia (SHP) were unknown in critical patients in the cardiac ICU. DESIGN: Prospective observational study. SETTING: Single-center, cardiac ICU in Taiwan. PATIENTS: Patients over 20 years old with Acute Physiology And Chronic Health Evaluation II scores greater than or equal to 15 and/or ventilatory support in the cardiac ICU. INTERVENTIONS: We measured plasma phenylalanine levels serially during patients' stays in the ICU to investigate their prognostic value for 90-day mortality. Gene array was performed to identify genetic polymorphisms associated with SHP (phenylalanine level ≥ 11.2 µmol/dL) and to develop a Genetic Risk Score (GRS). We analyzed the associations between SHP and clinical factors and genetic variants and identified the correlation between pteridines and genetic variants. MEASUREMENTS AND MAIN RESULTS: The study enrolled 497 patients. Increased phenylalanine concentration was independently associated with increased mortality risk. Patients with SHP had a higher mortality risk compared with those without SHP (log rank = 41.13; p < 0.001). SHP was associated with hepatic and renal dysfunction and with genetic polymorphisms on the pathway of tetrahydrobiopterin (BH4) synthesis (CBR1 and AKR1C3) and recycling (PCBD2). Higher GRSs were associated with lower BH4 bioavailability in response to stress ( p < 0.05). In patients without SHP at baseline, those with GRSs gretaer than or equal to 2 had a higher frequency of developing SHP during the ICU stay (31.5% vs 16.1%; p = 0.001) and a higher mortality risk ( p = 0.004) compared with those with GRSs less than 2. In patients with SHP at baseline, genetic variants did not provide additional prognostic value. CONCLUSIONS: SHP in patients admitted to the ICU was associated with a worse prognosis. In patients without SHP, genetic polymorphisms associated with SHP measured using a GRS of greater than or equal to 2 was associated with the subsequent SHP and higher mortality risk.
Assuntos
Unidades de Terapia Intensiva , Pteridinas , APACHE , Adulto , Humanos , Fenilalanina/genética , Prognóstico , Estudos Prospectivos , Adulto JovemRESUMO
Viruses are obligate intracellular parasites relying on host cells to obtain biosynthetic precursors and energy to successfully infect the host. The metabolic profile of the host cell is known to be altered in response to viral infection to satisfy the resources demanded during viral replication. Previous data of ours showed that white spot syndrome virus (WSSV) elicited in a crustacean host (Procambarus clarkii) a rapid and long-lasting release of crustacean hyperglycemic hormone (CHH), a well-known carbohydrate-regulating and stress response-mediating endocrine hormone. Therefore, the WSSV-enhanced release of CHH could be responsible at least in part for the metabolic alterations in the WSSV-challenged host. To investigate the possible metabolic roles of CHH in the host-parasite interaction, we studied whether silencing CHH gene expression could inhibit WSSV propagation in tissues and reduce the mortality of the WSSV-infected animals. Data presented in this study showed that CHH gene silencing indeed resists the WSSV infection. Injection of CHH dsRNA at the dosage of 140 µg/g BW caused significant decreases of viral copy number in tissues of WSSV-infected host, particularly showing a pronounced effect in the endodermal tissues (including hepatopancreas and gastrolith disk). Furthermore, results from the cumulative mortality showed that the treatment of CHH dsRNA delayed death from WSSV. Injection of CHH dsRNA at the dosages of 70, 17, and 10 µg/ g BW significantly extended the mean survival time. Together, this study concludes that the silencing of the CHH gene does have an inhibitory effect on the replication of the white spot syndrome virus and can assist the host to mitigate the invasion of WSSV, through attenuating CHH-mediated stress responses.
Assuntos
Penaeidae , Viroses , Vírus da Síndrome da Mancha Branca 1 , Animais , Astacoidea , Hepatopâncreas , RNA de Cadeia Dupla/metabolismo , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/genéticaRESUMO
A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9-Asn28; α3, His34-Gly38; and α5, Glu62-Arg72), and a π-helix (π4, Cys43-Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7-Cys43, Cys23-Cys39, and Cys26-Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand-receptor binding specificity.
Assuntos
Proteínas de Artrópodes , Braquiúros , Hormônios de Invertebrado , Proteínas do Tecido Nervoso , Neuropeptídeos , Receptores de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Braquiúros/genética , Braquiúros/metabolismo , Hormônios de Invertebrado/química , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Modelos Moleculares , Família Multigênica , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/química , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Pericárdio/metabolismo , Ligação Proteica , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
To identify endogenous peptides using MS/MS analysis and searching against a polypeptide sequence database, a non-enzyme specific (NES) search considering all of the possible proteolytic cleavages is required. However, the use of a NES search generates more false positive hits than an enzyme specific search, and therefore shows lower identification performance. In this study, the use of the sub-ranked matches for improving the identification performance of the Mascot NES search was investigated and a new scoring method was developed that considered the contribution of all sub-ranked random match probabilities, named the contribution score (CS). The CS showed the highest identification sensitivity using the Mascot NES search with a full protein database when compared to the use of the Mascot first ranked score and the delta score (DS). The confident peptides identified by DS and CS were shown to be complementary. When applied to plant endogenous peptide identification, the identification numbers of tomato endogenous peptides using DS and CS were 176.3% and 184.2%, respectively, higher than the use of the first ranked score of Mascot. The combination of DS and CS identified 200.0% and 8.6% more tomato endogenous peptides compared to the use of Mascot and DS, respectively. This method by combining the CS and DS can significantly improve the identification performance of endogenous peptides without complex computational steps and is also able to improve the identification performance of the enzyme specific search. In addition to the application in the plant peptidomics analysis, this method may be applied to the improvement of peptidomics studies in different species. A web interface for calculating the DS and CS based on Mascot search results was developed herein.
Assuntos
Algoritmos , Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Cromatografia Líquida/métodos , Bases de Dados de Proteínas , Escherichia coli , Humanos , Solanum lycopersicum/química , Proteínas de Plantas/análise , Coelhos , Saccharomyces cerevisiae , Ferramenta de BuscaRESUMO
Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (Solanum lycopersicum) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in Arabidopsis thaliana, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.
Assuntos
Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Solanum lycopersicum/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Cromatografia Líquida , Ciclopentanos/farmacologia , Resistência à Doença/efeitos dos fármacos , Resistência à Doença/genética , Resistência à Doença/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Oxilipinas/farmacologia , Peptídeos/genética , Peptídeos/farmacologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteoma/genética , Proteoma/farmacologia , Proteômica , Pseudomonas syringae/imunologia , Pseudomonas syringae/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Espectrometria de Massas em Tandem , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Transcriptoma/imunologiaRESUMO
As part of the study of the resilience of Antarctic crustaceans to global warming, the shrimp Chorismus antarcticus was subjected to an analysis of global approach using the Next Generation Sequencing Illumina Hi-Seq platform. With this data a detailed study into the principal neuropeptides and neurohormones of this species have been undertaken. Total RNAs from whole animals were enriched with eyestalk extracts to ensure maximum sequencing depth of the different neurohormones and neuropeptides mainly expressed into the X organ-sinus gland complex, which is a major endocrine organ of their synthesis. Apart from the information that can provide the availability of the transcriptome of a polar crustacean, the study of neuropeptides of a caridean shrimp will partially fill the limited data available for this taxon. Illumina sequencing was used to produce a transcriptome of the polar shrimp. Analysis of the Trinity assembled contigs produced 55 pre-pro-peptides, coding for 111 neuropeptides belonging to the following families: adipokinetic-corazonin-like peptide, Allatostatins (A, B et C), Bursicon (α), CCHamide, Crustacean Hyperglycemic Hormones (CHH), Crustacean Cardioactive Peptide (CCAP), Corazonin, Crustacean Female Sex Hormone (CSFH), Diuretic Hormones 31 and 45 (DH), Eclosion Hormone (EH), FLRFamide, GSEFLamide, Intocin, Ion Transport Peptide-like (ITP-like), Leucokinin, Molt-inhibiting Hormone, Myosuppresin, Neuroparsin, Neuropeptide F (NPF), Orcokinin, Orcomyotropin, Pigment Dispersing Hormone (PDH), Pyrokinin, Red Pigment Concentrating Hormone (RPCH), SIFamide, small Neuropeptide F (sNPF), Sulfakinin and finally Tachykinin Related peptides. Among the new peptides highlighted in this study, the focus was placed on the peptides of the CHH family and more particularly on a new ITP-like in order to confirm its belonging to a new group of peptides of the family. A phylogeny made from more than 200 sequences of peptides, included new sequences from new species besides Chorismus antarcticus, confirms the peculiarity of this new set of peptides gathered under the name ITP-like.
Assuntos
Decápodes/metabolismo , Neuropeptídeos/metabolismo , Oceanos e Mares , Proteoma/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Animais , Regiões Antárticas , Neuropeptídeos/química , Filogenia , Alinhamento de Sequência , Análise de Sequência de RNARESUMO
Pathophysiological studies of rhizocephalan infections are rare. We describe differences in the levels of tissue and hemolymph metabolites between Polyascus plana-parasitized and unparasitized individuals of Metopograpsus thukuhar. Crabs were assigned to either a parasitized (carrying at least 1 externa, i.e. a protruding reproductive body) or an unparasitized (not carrying externae and determined to be rootlet-free by a barnacle 18S rRNA-based polymerase chain reaction) group. Quantification of metabolites showed that muscle glycogen levels were significantly lower and hepatopancreas levels were significantly higher in parasitized crabs compared to unparasitized crabs; hepatopancreas triacylglycerol levels were significantly higher and hemolymph levels significantly lower in parasitized hosts, and there was no significant difference in muscle triacylglycerol levels between unparasitized and parasitized animals. Glucose levels in the hepatopancreas, muscle, and hemolymph were all significantly higher in parasitized hosts. Significant levels of glucose, triacylglycerol, and glycogen were present in the barnacle externae. In addition, levels of crustacean hyperglycemic hormone in the sinus glands were not significantly different between unparasitized and parasitized animals. Glucose mobilized from the muscle is likely converted to glycogen and triacylglycerol in the rootlet-infiltrated hepatopancreas of parasitized hosts, and the eyestalk neuroendocrine system appears not to be significantly impaired, in terms of hormone production and storage, by parasitization.
Assuntos
Braquiúros/parasitologia , Thoracica/fisiologia , Animais , Interações Hospedeiro-ParasitaRESUMO
The glutamic acid at position 100 (E(100)) in the capsid protein (CP) of Odontoglossum ringspot virus (ORSV) plays an important role in long-distance viral movement in Nicotiana benthamiana. The ORSV(E100A) mutant, which has a glutamic acid to alanine substitution, shows a loss of systemic infectivity in N. benthamiana. Transmission electron microscopy and size-exclusion chromatography assays showed that E(100) is essential for CP-CP interaction and viral particle assembly. To identify the ORSV triggering or response genes and CP-interacting proteins (CP-IP), an integrated omics approach based on next-generation sequencing and proteomics profiling was used in this study. The whole-transcriptomes of healthy and ORSV-infected leaves of N. benthamiana were analyzed, and the gene information was used to create a N. benthamiana protein database that was used for protein identification following mass spectrometry analysis. The integrated omics approach identified several putative host proteins that interact with ORSV CP(WT) and were categorized as photosystem subunits, defense-associated proteins, and cell division components. The expression pattern and CP interaction of these CP-IP were examined by semiquantitative reverse transcription polymerase chain reaction and an in vitro binding assay, respectively, to verify the in silico data. Among these proteins, a proteinase inhibitor of N. benthamiana (NbPI2) was highly associated with CP(E100A) as compared with CP(WT), and NbPI1 and NbPI2 were highly induced in ORSV-infected plants. NbPI1- and NbPI2-silenced plants (via a Tobacco rattle virus-induced gene-silencing system) did not exhibit a difference in ORSV infection. Thus, whether NbPI1 and NbPI2 play a role in plant immunity requires further investigation. In summary, the integrated omics approach provides massive and valuable information to identify the ORSV CP-IP and these CP-IP will help us to understand the movement of this virus and plant-virus interaction.
Assuntos
Proteínas do Capsídeo/metabolismo , Biologia Computacional , Nicotiana/genética , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Tobamovirus/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Genômica , Ácido Glutâmico , Modelos Moleculares , Dados de Sequência Molecular , Imunidade Vegetal , Folhas de Planta/virologia , Proteínas de Plantas/genética , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Análise de Sequência de DNA , Nicotiana/metabolismo , Nicotiana/virologia , Tobamovirus/genética , TranscriptomaRESUMO
PURPOSE: Copper peptide (GHK-Cu) plays an important role in skin regeneration and wound healing. However, its skin absorption remains challenging due to its hydrophilicity. Here we use polymeric microneedle array to pre-treat skin to enhance GHK-Cu skin penetration. METHODS: Two in vitro skin models were used to assess the capability of microneedles in facilitating skin delivery of GHK-Cu. Histological assay and confocal laser scanning microscopy were performed to characterize and quantify the microconduits created by the microneedles inside skin. Cellular and porcine models were used to evaluate the safety of microneedle-assisted copper peptide delivery. RESULTS: The depth and percentage of microneedle penetration were correlated with application forces, which in turn influenced the extent of enhancement in the skin permeability of GHK-Cu. In 9 h, 134 ± 12 nanomoles of peptide and 705 ± 84 nanomoles of copper permeated though the microneedle treated human skin, while almost no peptide or copper permeated through intact human skin. No obvious signs of skin irritation were observed with the use of GHK-Cu after microneedle pretreatment. CONCLUSIONS: It is effective and safe to enhance the skin permeation of GHK-Cu by using microneedles. This approach may be useful to deliver similar peptides or minerals through skin.
Assuntos
Cobre/administração & dosagem , Oligopeptídeos/administração & dosagem , Administração Cutânea , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Cultura em Câmaras de Difusão , Sistemas de Liberação de Medicamentos , Humanos , Técnicas In Vitro , Irritantes , Queratinócitos/efeitos dos fármacos , Agulhas , Oligopeptídeos/química , Ratos , Pele/patologia , Absorção Cutânea , SuínosRESUMO
Recent developments in high resolution mass spectrometry (HR-MS) technology have ushered proteomics into a new era. However, the importance of using a common, open data platform for signal processing of HR-MS spectra has not been sufficiently addressed. In this study, a MS signal processor was developed to facilitate data integration from different instruments and different proteomics approaches into a unified platform without compromising protein identification and quantitation performance. This processor supports parallel processing capability which allows full utilization of computing resources to speed up signal processing performance to >1 gigabytes/min. The storage space occupied by the processed MS data can be reduced to ~10%, which helps the analysis and management of large quantities of data from comprehensive proteomics studies. For quantitation at the MS level, processing accuracy is improved and processing time for ASAPRatio is reduced to ~50%. For quantitation at the MS/MS level, accurate reporter ion ratios from different instruments can be directly determined by the processed MS/MS spectra and reported in the Mascot search result directly without using specialized iTRAQ software.
Assuntos
Proteínas/análise , Proteômica , Software , Células Cultivadas , Humanos , Células Jurkat , Espectrometria de MassasRESUMO
It was demonstrated in a previous study (Wu et al., 2012b) that crustacean hyperglycemic hormone (CHH) gene is expressed in the hemocyte of Procambarus clarkii. In the present study, 2 additional cDNAs (CHH2-L and tCHH2) from the hemocyte and a CHH gene (CHH2) from the abdominal muscle of the same species were cloned. Analyses of the cDNA and genomic sequences suggested that, similar to other previously reported CHH genes, 2 precursor transcripts (CHH2 and CHH2-L) would be derived from CHH2 gene through a process of RNA alternative splicing, and CHH2 and CHH2-L each encode a precursor containing a signal peptide, a CHH precursor-related peptide, and a mature peptide. Further, tCHH2 sequence consists of exon I, exon II, and a truncated segment of intron II of CHH2 gene, followed by a previously unknown 3'sequence. It is suggested that, because the truncation disrupts the highly conserved RNA splice acceptor site, the truncated segment is retained within tCHH2, resulting in encoding a precursor containing the typical precursor components except the mature peptide is truncated with only 40 residues. In addition, unlike 2 other previously identified transcripts (referred to as CHH1 and CHH1-L), CHH2-L, CHH2, tCHH2 contain in the 3'-UTRs 3-5 AU-rich elements (AREs). The data showed that multiple CHH genes are expressed in crayfish hemocytes. Novel sequence characteristics of the transcripts result in an RNA splicing pattern that yields a transcript (tCHH2) encoding a precursor with an atypical truncated mature peptide and possibly leads to a different expression dynamics of the precursors encoded by the ARE-containing transcripts.
Assuntos
Processamento Alternativo/genética , Proteínas de Artrópodes/genética , Astacoidea/genética , Hemócitos/metabolismo , Hormônios de Invertebrado/genética , Proteínas do Tecido Nervoso/genética , Animais , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologiaRESUMO
The objectives of the present study were to characterize the changes in crustacean hyperglycemic hormone (CHH) transcript and peptide levels in response to infection of white spot syndrome virus (WSSV) in a crustacean, Procambarus clarkii. After viral challenge, significant increase in virus load began at 24 h post injection (hpi) and the increase was much more substantial at 48 and 72 hpi. The hemolymph CHH levels rapidly increased after viral challenge; the increase started as early as 3 hpi and lasted for at least 2 d after the challenge. In contrast, the hemolymph glucose levels did not significantly changed over a 2 d period in the WSSV-infected animals. The CHH transcript and peptide levels in tissues were also determined. The CHH transcript levels in the eyestalk ganglia (the major site of CHH synthesis) of the virus-infected animals did not significantly change over a 2 d period and those in 2 extra-eyestalk tissues (the thoracic ganglia and cerebral ganglia) significantly increased at 24 and 48 hpi. The CHH peptide levels in the eyestalk ganglia of the virus-infected animals significantly decreased at 24 and 48 hpi and those in the thoracic ganglia and cerebral ganglia remained unchanged over a 2 d period. These data demonstrated a WSSV-induced increase in the release of CHH into hemolymph that is rapid in onset and lasting in duration. Changes in the CHH transcript and peptide levels implied that the WSSV-induced increase in hemolymph CHH levels primarily resulted from an enhanced release from the eyestalk ganglia, but the contribution of the 2 extra-eyestalk tissues to hemolymph pool of CHH increased as viral infection progressed. The combined patterns of change in the hemolymph glucose and CHH levels further suggest that the virus-enhanced CHH release would lead to higher glycolytic activity and elevated glucose mobilization presumably favorable for viral replication.
Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea , Infecções , Hormônios de Invertebrado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vírus da Síndrome da Mancha Branca 1 , Animais , Astacoidea/metabolismo , Astacoidea/virologia , Glucose/metabolismo , Hemolinfa/metabolismo , Hemolinfa/virologia , Infecções/metabolismo , Infecções/patologia , Infecções/virologia , Sistemas Neurossecretores/metabolismo , Sistemas Neurossecretores/patologia , Vírus da Síndrome da Mancha Branca 1/metabolismo , Vírus da Síndrome da Mancha Branca 1/patogenicidadeRESUMO
Crustacean hyperglycemic hormone (CHH) was originally identified in a neuroendocrine system-the X-organ/sinus gland complex. In this study, a cDNA (Prc-CHH) encoding CHH precursor was cloned from the hemocyte of the crayfish Procambarus clarkii. Analysis of tissues by a CHH-specific enzyme-linked immunosorbent assay (ELISA) confirmed the presence of CHH in hemocytes, the levels of which were much lower than those in the sinus gland, but 2 to 10 times higher than those in the thoracic and cerebral ganglia. Total hemocytes were separated by density gradient centrifugation into layers of hyaline cell (HC), semi-granular cell (SGC), and granular cell (GC). Analysis of extracts of each layer using ELISA revealed that CHH is present in GCs (202.8±86.7 fmol/mg protein) and SGCs (497.8±49.4 fmol/mg protein), but not in HCs. Finally, CHH stimulated the membrane-bound guanylyl cyclase (GC) activity of hemocytes in a dose-dependent manner. These data for the first time confirm that a crustacean neuropeptide-encoding gene is expressed in cells essential for immunity and its expression in hemocytes is cell type-specific. Effect of CHH on the membrane-bound GC activity of hemocyte suggests that hemocyte is a target site of CHH. Possible functions of the hemocyte-derived CHH are discussed.
Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/metabolismo , Hemócitos/metabolismo , Hormônios de Invertebrado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Astacoidea/genética , Centrifugação com Gradiente de Concentração , Clonagem Molecular , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Feminino , Guanilato Ciclase/metabolismo , Hormônios de Invertebrado/genética , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Precursores de Proteínas/genéticaRESUMO
The new generation, i.e., second- and third-generation, drug-eluting stents (DESs) remain a risk of in-stent restenosis (ISR). We evaluated the power of a genetic risk score (GRS) model to identify high-risk populations for new generation DES ISR. We enrolled patients with coronary artery disease (CAD) treated with new generations DESs by a single-center cohort study in Taiwan and evaluated their genetic profile. After propensity score matching, there were 343 patients and 153 patients in the derivation and validation cohorts, respectively. Five selected single-nucleotide polymorphisms (SNPs), i.e., SNPs in CAMLG, GALNT2, C11orf84, THOC5, and SAMD11, were included to calculate the GRS for new generation DES ISR. In the derivation and the validation cohorts, patients with a GRS greater than or equal to 3 had significantly higher new generation DES ISR rates. We provide biological information for interventional cardiologists prior to percutaneous coronary intervention by specific five SNP-derived GRS.
RESUMO
Sco-CHH and Sco-CHH-L (CHH-like peptide), two structural variants of the crustacean hyperglycemic hormone family identified in the mud crab (Scylla olivacea), are presumably alternatively spliced gene products. In this study, Sco-CHH and Sco-CHH-L were isolated from the tissues using high performance liquid chromatography. Identity of the native peptides was confirmed using mass spectrometric (MS) analyses of purified materials and of trypsin-digested peptide fragments. Additionally, characterizations using circular dichroism (CD) spectrometry revealed that the 2 peptides have similar CD spectral profiles, showing they are composed mainly of alpha-helices, and are similarly thermo-stable with a melting temperature of 74-75 degrees C. Results of bioassays indicated that Sco-CHH exerted hyperglycemic and molt-inhibiting activity, whereas Sco-CHH-L did not. Further, recombinant Sco-CHH-Gly (rSco-CHH-Gly, a glycine extended Sco-CHH) and Sco-CHH-L (rSco-CHH-L) were produced using an Escherichia coli expression system, refolded, and purified. rSco-CHH-Gly was further alpha-amidated at the C-terminal end to produce rSco-CHH. MS analyses of enzyme-digested peptide fragments of rSco-CHH-Gly and rSco-CHH-L showed that the two peptides share a common disulfide bond pattern: C7-C43, C23-C39, and C26-C52. Circular dichroism analyses and hyperglycemic assay revealed that rSco-CHH and rSco-CHH-L resemble their native counterparts, in terms of CD spectral profiles, melting curve profiles, and biological activity. rSco-CHH-Gly has a lower alpha-helical content (32%) than rSco-CHH (47%), a structural deviation that may be responsible for the significant decrease in the biological activity of rSco-CHH-Gly. Finally, modeled structure of Sco-CHH and Sco-CHH-L indicated that they are similarly folded, each with an N-terminal tail region and 4 alpha-helices. Putative surface residues located in corresponding positions of Sco-CHH and Sco-CHH-L but with side chains of different properties were identified. The combined results support the notion that Sco-CHH and Sco-CHH-L are functionally different, but resemble each other at higher-level structures. Functional diversity between the 2 peptides is probably due to critical residues located in the C-terminus. The availability of large amounts of recombinant proteins will permit additional functional and structural studies of these CHH family peptides.
Assuntos
Braquiúros/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Proteínas de Artrópodes , Dicroísmo Circular , Hormônios de Invertebrado , Espectrometria de Massas , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Peptídeos/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Early studies recognizing the importance of the decapod eyestalk in the endocrine regulation of crustacean physiology-molting, metabolism, reproduction, osmotic balance, etc.-helped found the field of crustacean endocrinology. Characterization of putative factors in the eyestalk using distinct functional bioassays ultimately led to the discovery of a group of structurally related and functionally diverse neuropeptides, crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). These peptides, along with the first insect member (ion transport peptide, ITP), constitute the original arthropod members of the crustacean hyperglycemic hormone (CHH) superfamily. The presence of genes encoding the CHH-superfamily peptides across representative ecdysozoan taxa has been established. The objective of this review is to, aside from providing a general framework, highlight the progress made during the past decade or so. The progress includes the widespread identification of the CHH-superfamily peptides, in particular in non-crustaceans, which has reshaped the phylogenetic profile of the superfamily. Novel functions have been attributed to some of the newly identified members, providing exceptional opportunities for understanding the structure-function relationships of these peptides. Functional studies are challenging, especially for the peptides of crustacean and insect species, where they are widely expressed in various tissues and usually pleiotropic. Progress has been made in deciphering the roles of CHH, ITP, and their alternatively spliced counterparts (CHH-L, ITP-L) in the regulation of metabolism and ionic/osmotic hemostasis under (eco)physiological, developmental, or pathological contexts, and of MIH in the stimulation of ovarian maturation, which implicates it as a regulator for coordinating growth (molt) and reproduction. In addition, experimental elucidation of the steric structure and structure-function relationships have given better understanding of the structural basis of the functional diversification and overlapping among these peptides. Finally, an important finding was the first-ever identification of the receptors for this superfamily of peptides, specifically the receptors for ITPs of the silkworm, which will surely give great impetus to the functional study of these peptides for years to come. Studies regarding recent progress are presented and synthesized, and prospective developments remarked upon.
Assuntos
Proteínas de Artrópodes/metabolismo , Crustáceos/metabolismo , Hormônios de Invertebrado/metabolismo , Família Multigênica , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteínas de Artrópodes/genética , Crustáceos/genética , Hormônios de Invertebrado/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
The Antarctic krill, Euphausia superba, is a Southern Ocean endemic species of proven ecological importance to the region. In the context of predicted global warming, it is particularly important to understand how classic biomarkers of heat stress function in this species. In this respect, Hsp70s are acknowledged as good candidates. However, previous studies of expression kinetics have not been able to demonstrate significant upregulation of these genes in response to heat shocks at 3 °C and 6 °C for 3 and 6 h. The current work complements these previous results and broadens the prospects for the use of Hsp70s as a relevant marker of thermal shock in this krill species. New experiments demonstrate that induction of Hsp70 isoforms was not detected during exposure to heat shock, but increased expression was observed after several hours of recovery. To complete the analysis of the expression kinetics of the different isoforms, experiments were carried out over short time scales (1 and 2 h at 3 °C and 6 °C) as well as at higher temperatures (9 °C, 12 °C, and 15 °C for 3 h), without any significant response. A 6-week monitoring of animals at 3 °C showed that the time factor is decisive in the establishment of the response. CTmax experiments with incremental times of 1 °C per day or 1 °C every 3 days have shown a particularly high resilience of the animals. The demonstration of the abundance of Hsp70s present before thermal stress in various species of krill, as well as in specimens of E. superba of various origins, showed that the delay in the response in expression could be related to the high constitutive levels of Hsp70 available before the stress experiments. The alternative labelling of the two main isoforms of Hsp70 according to the origin of the animals allowed hypotheses to be put forward on the functioning of thermoregulation in Antarctic krill as well as ice krill.
Assuntos
Euphausiacea/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Termotolerância , Animais , Regiões Antárticas , Aquecimento Global , Oceanos e MaresRESUMO
In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.
Assuntos
Crustáceos/metabolismo , Hormônios de Invertebrado/metabolismo , Muda , Transdução de Sinais , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Crustáceos/genética , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Regulação para Baixo , Ecdisteroides/metabolismo , Glândulas Endócrinas/metabolismo , Olho/metabolismo , Hemolinfa/metabolismo , Hormônios de Invertebrado/sangue , Hormônios de Invertebrado/química , Hormônios de Invertebrado/genética , Dados de Sequência Molecular , Neuropeptídeos/metabolismo , Filogenia , Conformação Proteica , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
To comprehensively characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), metabolites in two CHH target tissues of the crayfish Procambarus clarkii, whose levels were significantly different between CHH knockdown and control (saline-treated) animals, were analyzed using bioinformatics tools provided by an on-line analysis suite (MetaboAnalyst). Analysis with Metabolic Pathway Analysis (MetPA) indicated that in the muscle Glyoxylate and dicarboxylate metabolism, Nicotinate and nicotinamide metabolism, Alanine, aspartate and glutamate metabolism, Pyruvate metabolism, and Nitrogen metabolism were significantly affected by silencing of CHH gene expression at 24 hours post injection (hpi), while only Nicotinate and nicotinamide metabolism remained significantly affected at 48 hpi. In the hepatopancreas, silencing of CHH gene expression significantly impacted, at 24 hpi, Pyruvate metabolism and Glycolysis or gluconeogenesis, and at 48 hpi, Glycine, serine and threonine metabolism. Moreover, analysis using Metabolite Set Enrichment Analysis (MSEA) showed that many metabolite sets were significantly affected in the muscle at 24hpi, including Ammonia recycling, Nicotinate and nicotinamide metabolism, Pyruvate metabolism, Purine metabolism, Warburg effect, Citric acid cycle, and metabolism of several amino acids, and at 48 hpi only Nicotinate and nicotinamide metabolism, Glycine and serine metabolism, and Ammonia recycling remained significantly affected. In the hepatopancreas, MSEA analysis showed that Fatty acid biosynthesis was significantly impacted at 24 hpi. Finally, in the muscle, levels of several amino acids decreased significantly, while those of 5 other amino acids or related compounds significantly increased in response to CHH gene silencing. Levels of metabolites related to nucleotide metabolism significantly decreased across the board at both time points. In the hepatopancreas, the effects were comparatively minor with only levels of thymine and urea being significantly decreased at 24 hpi. The combined results showed that the metabolic effects of silencing CHH gene expression were far more diverse than suggested by previous studies that emphasized on carbohydrate and energy metabolism. Based on the results, metabolic roles of CHH on the muscle and hepatopancreas are suggested: CHH promotes carbohydrate utilization in the hepatopancreas via stimulating glycolysis and lipolysis, while its stimulatory effect on nicotinate and nicotinamide metabolism plays a central role in coordinating metabolic activity in the muscle with diverse and wide-ranging consequences, including enhancing the fluxes of glycolysis, TCA cycle, and pentose phosphate pathway, leading to increased ATP supply and elevated protein and nucleic acid turnovers.