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1.
Int J Mol Sci ; 25(12)2024 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-38928053

RESUMO

The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.


Assuntos
Doenças dos Peixes , Imunidade Inata , Filogenia , Piscirickettsia , Infecções por Piscirickettsiaceae , Renibacterium , Salmo salar , Animais , Piscirickettsia/genética , Imunidade Inata/genética , Salmo salar/microbiologia , Salmo salar/genética , Salmo salar/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/genética , Infecções por Piscirickettsiaceae/microbiologia , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/genética , Infecções por Piscirickettsiaceae/veterinária , Renibacterium/genética , Renibacterium/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteínas de Peixes/imunologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Evolução Molecular
2.
Microb Pathog ; 174: 105932, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36473669

RESUMO

Renibacterium salmoninarum is one of the oldest known fish bacterial pathogens. This Gram-positive bacterium is the causative agent of Bacterial Kidney Disease (BKD), a chronic infection that primarily infects salmonids at low temperatures. Externally, infected fish may show exophthalmos, skin blisters, ulcerations, and hemorrhages at the base of the fins and along the lateral line. Internally, the kidney, heart, spleen, and liver may show signs of inflammation. The best characterized virulence factor of R. salmoninarum is p57, a 57 kDa protein located on the bacterial cell surface and secreted into surrounding fish tissue. The p57 protein in fish is the main mediator in suppressing the immune system, reducing antibody production, and intervening in cytokine activity. In this review, we will discuss aspects such as single nucleotide polymorphisms (SNPs) that modify the DNA sequence, variants in the number of copies of MSA genes, physical-chemical properties of the signal peptides, and the limited iron conditions that can modify p57 expression and increase the virulence of R. salmoninarum.


Assuntos
Doenças dos Peixes , Infecções por Bactérias Gram-Positivas , Animais , Proteômica , Virulência/genética , Proteínas da Membrana Bacteriana Externa/genética , Genômica , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Infecções por Bactérias Gram-Positivas/microbiologia
3.
Microb Pathog ; 180: 106122, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37094756

RESUMO

Piscirickettsia salmonis is one of the main pathogens causing considerable economic losses in salmonid farming. The DNA gyrase of several pathogenic bacteria has been the target of choice for antibiotic design and discovery for years, due to its key function during DNA replication. In this study, we carried out a combined in silico and in vitro approach to antibiotic discovery targeting the GyrA subunit of Piscirickettsia salmonis. The in silico results of this work showed that flumequine (-6.6 kcal/mol), finafloxacin (-7.2 kcal/mol), rosoxacin (-6.6 kcal/mol), elvitegravir (-6.4 kcal/mol), sarafloxacin (-8.3 kcal/mol), orbifloxacin (-7.9 kcal/mol), and sparfloxacin (-7.2 kcal/mol) are docked with good affinities in the DNA binding domain of the Piscirickettsia salmonis GyrA subunit. In the in vitro inhibition assay, it was observed that most of these molecules inhibit the growth of Piscirickettsia salmonis, except for elvitegravir. We believe that this methodology could help to significantly reduce the time and cost of antibiotic discovery trials to combat Piscirickettsia salmonis within the salmonid farming industry.


Assuntos
Doenças dos Peixes , Piscirickettsia , Animais , Antibacterianos/farmacologia , Piscirickettsia/genética , DNA Girase/genética , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/microbiologia
4.
J Fish Biol ; 101(4): 1021-1032, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35838309

RESUMO

Piscirickettsia salmonis is the etiological agent of Piscirickettsiosis, a severe disease that affects Atlantic salmon (Salmo salar) farmed in Chile and many other areas (Norway, Scotland, Ireland, Canada and the USA). This study investigated the effects of low-dose P. salmonis infection (1 × 102 CFU/ml) on Atlantic salmon. In this study, we challenged fish with an isolated representative of the EM-90 genogroup via intraperitoneal injection for 42 days. Infected fish displayed decreased haematocrit and haemoglobin levels at day 13 post-infection, indicating erythropenia, haemolysis and haemodilution. Conversely, their white blood cell counts increased on days 13 and 21 post-infection. Additionally, their iron levels decreased from day 2 post-infection, indicating iron deficiency and an inability to retrieve stored iron before infection. Their magnesium levels also decreased at day 28 post-infection, possibly due to osmoregulatory problems. Also, we observed an increase in lactate dehydrogenase activity on days 5, 21, and 28 post-infection, suggesting early symptoms of hepatotoxicity. Later analyses determined a decrease in plasma glucose levels from day 2 post-infection. This may be attributed to the hypoxic conditions caused by P. salmonis, leading to an excess utilization of stored carbohydrates. Our results suggest that the blood parameters we studied are useful for monitoring the physiological status of Atlantic salmon infected with P. salmonis.


Assuntos
Doenças dos Peixes , Salmo salar , Animais , Glicemia , Magnésio , Doenças dos Peixes/microbiologia , Ferro , Lactato Desidrogenases , Hemoglobinas
5.
Int J Mol Sci ; 23(3)2022 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-35163678

RESUMO

CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.


Assuntos
Técnicas e Procedimentos Diagnósticos/tendências , Doença/genética , Edição de Genes/métodos , COVID-19/genética , Sistemas CRISPR-Cas/genética , DNA/genética , Diagnóstico , Humanos , Reprodutibilidade dos Testes , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade
6.
J Cell Physiol ; 235(1): 166-175, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31180589

RESUMO

The pancreatic islets of Langerhans, mainly formed by glucagon-producing α-cells and insulin-producing ß-cells, are critical for glucose homeostasis. Insulin and glucagon oppositely modulate blood glucose levels in health, but a combined decline in insulin secretion together with increased glucagon secretion contribute to hyperglycemia in diabetes. Despite this bi-hormonal dysregulation, most studies have focused on insulin secretion and much less is known about glucagon secretion. Therefore, a deeper understanding of α-cell metabolism and glucagon secretion is of great interest. Here, we show that phosphoenolpyruvate carboxykinase (PCK1), an essential cataplerotic enzyme involved in metabolism and long considered to be absent from the pancreatic islet, is expressed in pancreatic α-cells of both murine and human. Furthermore, PCK1 transcription is induced by fasting and diabetes in rat pancreas, which indicates that the PCK1 activity is required for α-cell adaptation to different metabolic states. To our knowledge, this is the first evidence implicating PCK1 expression in α-cell metabolism.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Células Secretoras de Glucagon/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Pâncreas/enzimologia , Pâncreas/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Ratos
7.
BMC Musculoskelet Disord ; 20(1): 60, 2019 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-30736762

RESUMO

BACKGROUND: The purpose of this study was to compare the biomechanical behaviour of two bioabsorbable interference screws with different geometries. METHODS: Two different pitch (2.5 and 5 mm) bioabsorbable interference screws, both 9 × 30 mm, were tested. Tests were performed with forty bovine digital extensor tendons and skeletally mature porcine tibiae. Two protocols of cyclic tests at 1 Hz were performed: 1000 cycles from 50 to 250 N, and 5000 cycles from 100 to 300 N (n = 10 for each type of test and screw). After the cyclic loading, a final ramp displacement until failure at 0.5 mm/s was applied. RESULTS: The stiffness after the cyclic phase of the tests was not statistically different between the two screws (1000th cycle: 2.5 mm pitch 280.3 ± 56.4 N/mm, 5 mm pitch 275.2 ± 65.0 N/mm, P = .965; 5000th cycle: 2.5 mm pitch 281.3 ± 66.4 N/mm, 5 mm pitch 286.1 ± 79.4 N/mm, P = .814). The yield load was not significantly different between the screws (1000 cycle tests: 2.5 mm pitch 482.2 ± 120.2 N, 5 mm pitch 495.9 ± 131.3 N, P = .508; 5000 cycle tests: 2.5 mm pitch 476.4 ± 65.3 N, 5 mm pitch 494.3 ± 39.2 N, P = .391). No correlation was found between the insertion torque and yield load (1000 cycle tests, R2 = 0.013; 5000 cycle tests, R2 = 0.006). CONCLUSIONS: The pitch of bioabsorbable interference screws does not seem to affect fixation strength. Also, the authors recommend not to use insertion torque alone to estimate the fixation strength.


Assuntos
Implantes Absorvíveis , Reconstrução do Ligamento Cruzado Anterior/instrumentação , Ligamento Cruzado Anterior/cirurgia , Parafusos Ósseos , Tíbia/cirurgia , Animais , Ligamento Cruzado Anterior/fisiopatologia , Reconstrução do Ligamento Cruzado Anterior/efeitos adversos , Fenômenos Biomecânicos , Bovinos , Análise de Falha de Equipamento , Desenho de Prótese , Falha de Prótese , Sus scrofa , Tíbia/fisiopatologia , Torque
8.
J Fish Dis ; 42(9): 1283-1291, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31241770

RESUMO

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron-acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT-qPCR to assess the effects of normal and iron-limited conditions on iron-uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H-2 and the type strain DSM20767T . Further evaluated was the in vitro immune-related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron-limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H-2 than DSM20767T . These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF-α, IL-1ß, TLR1 and INF-γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron-limited conditions, especially H-2. These observations indicate that iron-acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron-uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.


Assuntos
Infecções por Actinomycetales/veterinária , Doenças dos Peixes/imunologia , Ferro/metabolismo , Micrococcaceae/imunologia , Micrococcaceae/patogenicidade , Salmo salar , Infecções por Actinomycetales/imunologia , Infecções por Actinomycetales/metabolismo , Infecções por Actinomycetales/microbiologia , Animais , Linhagem Celular , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia , Imunidade Inata , Micrococcaceae/metabolismo , Renibacterium , Virulência
9.
J Fish Dis ; 42(12): 1645-1655, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31591746

RESUMO

The aetiological agent of Piscirickettsiosis is Piscirickettsia salmonis, a Gram-negative intracellular pathogen, and high doses of antibiotics have regularly been employed to treat this infection. Seven florfenicol and/or oxytetracycline resistance genes (tet pump, tetE, Tclor/flor, Tbcr, TfloR, ompF and mdtN) were identified in strains by in silico genome analyses. Later, the number of single nucleotide polymorphisms (SNPs) and its relationship with the resistance to these antibiotics were identified and analysed, using the original LF-89 strain as reference. Trials to determine and compare the minimum inhibitory concentration (MIC) of oxytetracycline and florfenicol in each strain, as well as to quantify the gPCR transcripts levels in the selected genes, were performed. Therefore, variations in the resistance to both antibiotics were observed, where the strain with fewer SNPs showed the highest susceptibility. Consistently, the in silico 3D analyses of proteins encoded by the selected genes revealed structural changes, evident in the sequences with the highest number of SNPs. These results showed that the bacterial resistance to oxytetracycline was mainly linked to the presence of SNPs in relevant sites, antibiotic resistance genes and an OmpF porin, leading to important changes in the protein structure.


Assuntos
Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Piscirickettsia/genética , Polimorfismo de Nucleotídeo Único , Animais , Doenças dos Peixes/microbiologia , Testes de Sensibilidade Microbiana , Oxitetraciclina , Piscirickettsia/efeitos dos fármacos , Infecções por Piscirickettsiaceae/microbiologia , Infecções por Piscirickettsiaceae/veterinária , Tianfenicol/análogos & derivados
10.
J Fish Dis ; 42(5): 721-737, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30851000

RESUMO

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic-resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty-two Chilean P. salmonisisolates, as well as the type strain LF-89T , were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF-89T and EM-90 strains, as well as a seven-isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.


Assuntos
Doenças dos Peixes/microbiologia , Variação Genética , Genótipo , Tipagem de Sequências Multilocus/métodos , Piscirickettsia/genética , Infecções por Piscirickettsiaceae/veterinária , Salmonidae , Animais , Aquicultura , Sequência de Bases , Chile , Oncorhynchus kisutch , Oncorhynchus mykiss , Filogenia , Infecções por Piscirickettsiaceae/microbiologia , Salmo salar , Alinhamento de Sequência/veterinária
11.
J Cell Physiol ; 232(2): 355-362, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27186953

RESUMO

Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti-diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type-2-diabetic (D-RPTEC) and non-diabetic (N-RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development, and repair, all processes that are dysregulated during DKD. In basal conditions, D-RPTEC cells secreted higher levels of prototypical pro-inflammatory IL-6, IL-8, and MCP-1 than N-RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL-6, IL-8, and MCP-1 secretion in both N- and D-RPTEC, together with lower levels of IL-1 RA, IL-4, IL-10, and GM-CSF, suggesting that it may contribute to the extent of renal damage/repair during DKD. Besides, NaW induced the accumulation of IκBα, the main inhibitor protein of one major pathway involved in cytokine production, suggesting further anti-inflammatory effect in the long-term. A better understanding of the mechanisms involved in the interplay between the anti-diabetic and immunomodulatory properties of NaW will facilitate future studies about its clinical relevance. J. Cell. Physiol. 232: 355-362, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Citocinas/metabolismo , Hipoglicemiantes/farmacologia , Túbulos Renais Proximais/citologia , Compostos de Tungstênio/farmacologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP) , Fosforilação/efeitos dos fármacos , Fatores de Tempo
12.
Microb Pathog ; 110: 586-593, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28789875

RESUMO

Piscirickettsia salmonis is an intracellular bacterium and the causative agent of Piscirickettsiosis, a disease responsible for considerable mortalities in the Chilean salmon farming industry. Currently, P. salmonis protein translocation across the membrane and the mechanisms by which virulence factors are delivered to host cells are poorly understood. However, it is known that Gram-negative bacteria possess several mechanisms that transport proteins to the periplasmic and extracellular compartments. The aim of this study was to evaluate the expressional changes of several genes in the P. salmonis Sec-dependent pathway and type 4B secretion system during in vitro infection. Genes homologous and the main proteins belonging to Sec-dependent pathway and Type 4 Dot/Icm secretion system were found in the genome and proteome of P. salmonis AUSTRAL-005 strain. Additionally, several genes of these protein transport mechanisms were overexpressed during in vitro P. salmonis infection in SHK-1 cell line. The obtained data indicate that the Sec-dependent pathway and Type 4B secretion system are biologically active during P. salmonis infection. These mechanisms could contribute to the recycling of proteins into the inner and outer bacterial membrane and in translocate virulence factors to infected cell, which would favor the structural integrity and virulence of this bacterium.


Assuntos
Perfilação da Expressão Gênica , Piscirickettsia/crescimento & desenvolvimento , Piscirickettsia/genética , Sistemas de Secreção Tipo IV/biossíntese , Sistemas de Secreção Tipo IV/genética , Animais , Linhagem Celular , Células Epiteliais/microbiologia , Genômica , Proteômica , Salmão
13.
Microb Pathog ; 107: 436-441, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28438636

RESUMO

Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, which, as the main systemic disease in the Chilean salmon industry, causes significant economic losses. This bacterium can produce biofilm as a persistence and survival strategy in adverse conditions. In other bacteria, cheA is a key gene for modulating the onset of bacterial chemotaxis, as well as having a secondary role in biofilm production. Notwithstanding this association, the potential relationships between biofilm formation and genes involved in P. salmonis chemotaxis are poorly understood. This study aimed to determine P. salmonis cheA gene expression when grown in different culture media known to induce biofilm production. Piscirickettsia salmonis AUSTRAL-005 produced moderate/high biofilm levels after 144 h of incubation in the AUSTRAL-SRS and marine broths. In contrast, LF-89 biofilm production was weak/nonexistent in the aforementioned broths. Both assessed P. salmonis strains contained the cheYZA operon. Additionally, AUSTRAL-005 cheA transcripts increased in both culture media. In conclusion, these results suggest potential relationships between biofilm formation and genes related to chemotaxis in the fish pathogen P. salmonis.


Assuntos
Quimiotaxia/genética , Regulação Bacteriana da Expressão Gênica/genética , Óperon/genética , Piscirickettsia/genética , Animais , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Quimiotaxia/fisiologia , Meios de Cultura/química , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Genes Bacterianos/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Proteínas Quimiotáticas Aceptoras de Metil/fisiologia , Microscopia Eletrônica de Varredura , Piscirickettsia/crescimento & desenvolvimento , Piscirickettsia/patogenicidade , Infecções por Piscirickettsiaceae/microbiologia , Virulência/genética , Virulência/fisiologia
14.
Virol J ; 14(1): 17, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28143585

RESUMO

BACKGROUND: The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level. METHODS: In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S). RESULTS: Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates. CONCLUSIONS: This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.


Assuntos
Infecções por Birnaviridae/veterinária , Variação Genética , Vírus da Necrose Pancreática Infecciosa/genética , Vírus da Necrose Pancreática Infecciosa/isolamento & purificação , Oncorhynchus kisutch/virologia , Oncorhynchus mykiss/virologia , Salmo salar/virologia , Animais , Aquicultura , Infecções por Birnaviridae/virologia , Chile , Genótipo , Vírus da Necrose Pancreática Infecciosa/classificação , Análise de Sequência de DNA , Proteínas Estruturais Virais/genética
15.
Fish Shellfish Immunol ; 67: 189-198, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28600194

RESUMO

Infections caused by the facultative intracellular bacterial pathogen Piscirickettsia salmonis remains an unsolved problem for the aquaculture as no efficient treatments have been developed. As a result, substantial amounts of antibiotic have been used to limit salmonid rickettsial septicemia (SRS) disease outbreaks. The antibiotic usage has not reduced the occurrence, but lead to an increase in resistant strains, underlining the need for new treatment strategies. P. salmonis produce membrane vesicles (MVs); small spherical structures know to contain a variety of bacterial components, including proteins, lipopolysaccharides (LPS), DNA and RNA. MVs mimics' in many aspects their mother cell, and has been reported as alternative vaccine candidates. Here, MVs from P. salmonis was isolated and evaluated as a vaccine candidate against SRS in an adult zebrafish infection model. When zebrafish was immunized with MVs they were protected from subsequent challenge with a lethal dose of P. salmonis. Histological analysis showed a reduced bacterial load upon challenge in the MV immunized group, and the mRNA expression levels of several immune related genes altered, including mpeg1.1, tnfα, il1b, il10 and il6. The MVs induced the secretion of IgM upon immunization, indicating an immunogenic effect of the vesicles. Taken together, the data demonstrate a vaccine potential of MVs against P. salmonis.


Assuntos
Vacinas Bacterianas/imunologia , Vesículas Citoplasmáticas/metabolismo , Doenças dos Peixes/prevenção & controle , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/veterinária , Sepse/veterinária , Peixe-Zebra , Animais , Carga Bacteriana , Vesículas Citoplasmáticas/imunologia , Feminino , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Expressão Gênica , Imunidade Inata , Masculino , Modelos Animais , Piscirickettsia/metabolismo , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/prevenção & controle , RNA Mensageiro/genética , Sepse/imunologia , Sepse/prevenção & controle
16.
J Cell Physiol ; 231(8): 1635-7, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26661279

RESUMO

At present, diabetes mellitus is the main cause of end-stage renal disease. Effective glycaemic management is the most powerful tool to delay the establishment of diabetic complications, such as diabetic kidney disease. Together with reducing blood glucose levels, new anti-diabetic agents are expected not only to control the progression but also to restore known defects of the diabetic kidney. Sodium-glucose co-transporter 2 (SGLT2) inhibitors are promising anti-diabetic agents that reduce hyperglycaemia by impairing glucose reabsorption in proximal tubule of the kidney and increasing glucosuria. SGLT2 inhibitors have shown to reduce glucotoxicity in isolated proximal tubule cells and also to attenuate expression of markers of overall kidney damage in experimental animal models of diabetes, but the actual renoprotective effect for downstream nephron segments is still unknown and deserves further attention. Here, we briefly discuss possible undesired effects of enhanced glucosuria and albuminuria in nephron segments beyond the proximal tubule after SGLT2 inhibitor treatment, offering new lines of research to further understand the renoprotective action of these anti-diabetic agents. Strategies blocking glucose reabsorption by renal proximal tubule epithelial cells (RPTEC) may be protective for RPTEC, but downstream nephron segments will still be exposed to high glucose and albumin levels through the luminal face. The actual effect of constant enhanced glucosuria over distal nephron segments remains to be established. J. Cell. Physiol. 231: 1635-1637, 2016. © 2015 Wiley Periodicals, Inc.


Assuntos
Albuminúria/induzido quimicamente , Glicemia/efeitos dos fármacos , Diabetes Mellitus/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Glicosúria/induzido quimicamente , Hipoglicemiantes/efeitos adversos , Neoplasias Renais/induzido quimicamente , Túbulos Renais Proximais/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Transformação Celular Neoplásica/induzido quimicamente , Diabetes Mellitus/sangue , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/fisiopatologia , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/fisiopatologia , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiopatologia , Terapia de Alvo Molecular , Medição de Risco , Fatores de Risco , Transportador 2 de Glucose-Sódio/metabolismo
17.
Fish Shellfish Immunol ; 51: 97-103, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26876354

RESUMO

Streptococcus phocae subsp. salmonis is a Gram-positive bacterium that causes mortality only in Atlantic salmon (Salmo salar) farmed in Chile, even when this species is co-cultured with rainbow trout (Oncorhynchus mykiss). This susceptibility could be determined by innate immune response components and their responses to bacterial infection. This fish pathogen shares subspecies status with Streptococcus phocae subsp. phocae isolated from seals. The present study compared innate immune system mechanisms in Atlantic salmon and rainbow trout when challenged with different S. phocae, including two isolates from Atlantic salmon (LM-08-Sp and LM-13-Sp) and two from seal (ATCC 51973(T) and P23). Streptococcus phocae growth was evaluated in the mucus and serum of both species, with rainbow trout samples evidencing inhibitory effects. Lysozyme activity supported this observation, with significantly higher (p < 0.01) expression in rainbow trout serum and mucus as compared to Atlantic salmon. No differences were found in phagocytic capacity between fish species when stimulated with ATCC 51973(T) and P23. Against all S. phocae strains, rainbow trout and Atlantic salmon showed up to two-fold increased bactericidal activity, and rainbow trout demonstrated up to three-fold greater reactive oxygen species production in macrophages. In conclusion, the non-specific humoral and cellular barriers of Atlantic salmon were immunologically insufficient against S. phocae subsp. salmonis, thereby facilitating streptococcosis. Moreover, the more robust response of rainbow trout to S. phocae could not be attributed to any specific component of the innate immune system, but was rather the consequence of a combined response by the evaluated components.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata , Oncorhynchus mykiss , Salmo salar , Infecções Estreptocócicas/veterinária , Streptococcus/fisiologia , Animais , Chile , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/veterinária , Macrófagos/imunologia , Muco/imunologia , Infecções Estreptocócicas/imunologia
18.
Fish Shellfish Immunol ; 58: 259-265, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27640334

RESUMO

The NOD-like receptors (NLRs) were recently identified as an intracellular pathogen recognition receptor family in vertebrates. While the immune system participation of NLRs has been characterized and analyzed in various mammalian models, few studies have considered NLRs in teleost species. Therefore, this study analyzed the Atlantic salmon (Salmo salar) NLRC5. Structurally, Atlantic salmon NLRC5 presented leucine-rich repeat subfamily genes. Phylogenetically, NLRC5 was moderately conserved between S. salar and other species. Real-time quantitative PCR revealed NLRC5 expression in almost all analyzed organs, with greatest expressions in the head kidney, spleen, and hindgut. Furthermore, NLRC5 gene expression decreased during smolt stage. These data suggest that NLRC5 participates in the Atlantic salmon immune response and is regulated, at least partly, by the smoltification process, suggesting that there is a depression of immune system from parr at smolt stage. This is the first report on the NLRC5 gene in salmonid smolts.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica , Expressão Gênica , Inflamassomos/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Salmo salar/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Imunidade Inata/genética , Inflamassomos/química , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Salmo salar/classificação , Salmo salar/imunologia , Homologia de Sequência de Aminoácidos
19.
Biochem J ; 472(2): 225-37, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26417114

RESUMO

Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K(+). We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes.


Assuntos
Metabolismo Energético , Frutose-Bifosfatase/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Gluconeogênese , Glicólise , Hepatócitos/metabolismo , Modelos Biológicos , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Frutose-Bifosfatase/química , Frutose-Bifosfatase/genética , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/genética , Células HeLa , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Microscopia Confocal , Transporte Proteico , Ratos Wistar , Proteínas Recombinantes de Fusão/metabolismo
20.
Biochim Biophys Acta ; 1840(6): 1798-807, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24444799

RESUMO

BACKGROUND: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure. METHODS: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. RESULTS: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. CONCLUSIONS: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. GENERAL SIGNIFICANCE: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.


Assuntos
Biocatálise , Frutose-Bifosfatase/química , Rim/enzimologia , Animais , Sequência de Bases , Sítios de Ligação , Frutose-Bifosfatase/antagonistas & inibidores , Frutose-Bifosfatase/metabolismo , Frutosedifosfatos/química , Dados de Sequência Molecular , Subunidades Proteicas , Especificidade por Substrato , Suínos
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