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1.
Cardiovasc Drugs Ther ; 34(3): 345-356, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32236861

RESUMO

PURPOSE: Mitochondrial dysfunction plays a vital role in the pathophysiologic process of heart failure (HF). As a quality control system, mitochondrial fusion and fission are under control of mitochondrial fusion and fission-related proteins. The objective of this study was to investigate the effects of common variants in mitochondrial fusion and fission-related genes on the prognosis of HF. METHODS: We performed whole exome sequencing (WES) with 1000 HF patients; the statistically significant variant was further genotyped in the replicated population with 2324 HF patients. A series of function analysis including western blot, cell proliferation assay, and in vitro OMA1 activity assay were conducted to illuminate the underlying mechanism. RESULTS: We identified a missense variant rs17117699 associated with the prognosis of HF in group without ß-blocker use rather than with ß-blocker use in two-stage population: adjusted P = 0.79, HR = 0.88 (0.36-2.13) in group with ß-blocker use and adjusted P = 0.016, HR = 1.43 (1.07-1.91) in group without ß-blocker in first-stage population; adjusted P = 0.42, HR = 0.85 (0.56-1.28) in group with ß-blocker use and adjusted P = 0.015, HR = 1.39 (1.06-1.82) in group without ß-blocker in replicated stage. Functional analysis indicated that rs17117699-G allele increased the activity of OMA1 assessed by the ratio of S-OPA1 to L-OPA1 and suppressed cells proliferation under ISO treatment when compared with rs17117699-T allele. Furthermore, OMA1 functioned downstream of ß-adrenergic receptor signaling and ISO-induced OPA1 cleavage is dependent on OMA1. CONCLUSIONS: Our findings demonstrate that rs17117699T>G in OMA1 increases the risk of HF mortality via enhancing its OPA1 cleavage activity. It is a promising potential treatment target for HF. CLINICAL TRIAL REGISTRATION: NCT03461107. https://www.clinicaltrials.gov/ct2/show/NCT03461107?term=03461107&cond=Heart+Failure&cntry=CN&rank=1.


Assuntos
Insuficiência Cardíaca/genética , Metaloendopeptidases/genética , Mitocôndrias Cardíacas/genética , Dinâmica Mitocondrial/genética , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Idoso , Proliferação de Células , Feminino , GTP Fosfo-Hidrolases/metabolismo , Estudos de Associação Genética , Células HEK293 , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/mortalidade , Humanos , Masculino , Metaloendopeptidases/metabolismo , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Prognóstico , Sequenciamento Completo do Exoma
4.
Am J Physiol Renal Physiol ; 318(5): F1147-F1159, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174142

RESUMO

Meprin metalloproteases have been implicated in the progression of kidney injury. Previous work from our group has shown that meprins proteolytically process the catalytic subunit of protein kinase A (PKA-C), resulting in decreased PKA-C kinase activity. The goal of the present study was to determine the PKA-C isoforms impacted by meprin-ß and whether meprin-ß expression affects downstream mediators of the PKA signaling pathway in ischemia-reperfusion (IR)-induced kidney injury. IR was induced in 12-wk-old male wild-type (WT) and meprin-ß knockout (ßKO) mice. Madin-Darby canine kidney cells transfected with meprin-ß cDNA were also subjected to 2 h of hypoxia. Western blot analysis was used to evaluate levels of total PKA-C, PKA-Cα, PKA-Cß, phosphorylated (p-)PKA-C, and p-ERK1/2. Meprin-ß expression enhanced kidney injury as indicated by levels of neutrophil gelatinase-associated lipocalin and cystatin C. IR-associated decreases were observed in levels of p-PKA-C in kidney tissue from WT mice but not ßKO mice, suggesting that meprin-ß expression/activity is responsible for the in vivo reduction in kinase activity. Significant increases in levels of PKA-Cß were observed in kidney lysates for WT mice but not ßKO mice at 6 h post-IR. Proximal tubule PKA-Cß increases in WT but not ßKO kidneys were demonstrated by fluorescent microscopy. Furthermore, IR-induced injury was associated with significant increases in p-ERK levels for both genotypes. The present data demonstrate that meprin-ß enhances IR-induced kidney injury in part by modulating mediators of the PKA-Cß signaling pathway.


Assuntos
Lesão Renal Aguda/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Rim/enzimologia , Metaloendopeptidases/metabolismo , Traumatismo por Reperfusão/enzimologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Hipóxia Celular , Modelos Animais de Doenças , Cães , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Rim/patologia , Células Madin Darby de Rim Canino , Masculino , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais
5.
Mol Med Rep ; 21(3): 1285-1295, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32016477

RESUMO

Of the different types of lung cancer, lung squamous cell cancer (LUSC) has the second highest rates of morbidity and mortality, which have been increasing in recent years. Epigenetic abnormalities may serve as potential biomarkers and diagnostic and/or therapeutic targets, which may help to monitor and improve the prognosis of patients with cancer. In the present study, data were obtained from The Cancer Genome Atlas database and survival and joint survival analyses were conducted using the R MethylMix package. Peptidase, mitochondrial processing a subunit pseudogene 1 (PMPCAP1), sosondowah ankyrin repeat domain family member C (SOWAHC) and zinc finger protein (ZNF) 454 were identified as independent prognosis­related hub methylation­driven genes (MDGs). Of these three genes, PMPCAP1 and SOWAHC, characterized by hypomethylation and high expression levels, were associated with poor prognosis in patients with LUSC, whilst ZNF454 was associated with an improved prognosis. In addition, pathway enrichment analysis suggested that PMPCAP1, SOWAHC and ZNF454 were primarily involved in gene expression or transcription pathways. Furthermore, 5, 1 and 10 key methylation sites of PMPCAP1, SOWAHC and ZNF454, respectively, were confirmed to be significantly relevant to gene expression, establishing a basis for further investigation into the mechanisms and more precise targets of these 3 genes. In conclusion, the MDGs PMPCAP1, SOWAHC and ZNF454 may be potential prognostic biomarkers of LUSC for guiding diagnosis and therapy options, as well as providing a theoretical basis for further investigation.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/diagnóstico , Metaloendopeptidases/metabolismo , Proteínas/metabolismo , Algoritmos , Biomarcadores/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Metilação de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metaloendopeptidases/genética , Prognóstico , Proteínas/genética , Análise de Sobrevida , Dedos de Zinco
6.
J Vet Diagn Invest ; 32(2): 259-267, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31924132

RESUMO

Two putative zinc metalloproteases encoded by Clostridium perfringens have been implicated in the pathogenesis of necrotic enteritis, an economically significant poultry disease that is caused by this anaerobic bacterium. These proteases have ~64% amino acid identity and are encoded by the zmpA and zmpB genes. We screened 83 C. perfringens isolates by PCR for the presence of these genes. The first gene, zmpB, is chromosomally located and was present in all screened strains of C. perfringens, regardless of their origin and virulence. The second gene, zmpA, is plasmid-borne and was only found in isolates derived from chickens with necrotic enteritis. We describe the generation of insertionally inactivated mutants of both zmpA and zmpB in a virulent C. perfringens isolate. For each mutant, a significant (p < 0.001) reduction in virulence was observed in a chicken necrotic enteritis disease model. Examples of each mutant strain were characterized by whole genome sequencing, which showed that there were a few off-site mutations with the potential to affect the virulence of these strains. To confirm the importance of these genes, independently derived zmpA and zmpB mutants were constructed in different virulent C. perfringens isolates and shown to have reduced virulence in the experimental disease induction model. A zmpA-zmpB double mutant also was generated and shown to have significantly reduced virulence, to the same extent as the respective single mutants. Our results provide evidence that both putative zinc metalloproteases play an important role in disease pathogenesis.


Assuntos
Proteínas de Bactérias/genética , Infecções por Clostridium/veterinária , Clostridium perfringens/fisiologia , Clostridium perfringens/patogenicidade , Enterocolite Necrosante/veterinária , Metaloendopeptidases/genética , Doenças das Aves Domésticas/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Infecções por Clostridium/microbiologia , Clostridium perfringens/enzimologia , Enterocolite Necrosante/microbiologia , Metaloendopeptidases/metabolismo , Virulência
7.
PLoS One ; 14(12): e0225666, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31805094

RESUMO

The hatching enzymes or choriolysins are key proteases in fish life cycle controlling the release of larvae to surrounding environment that have been suggested as target for novel biotechnological uses. Due to the large amounts of eggs released by the flatfish Solea senegalensis, during the spawning season, the hatching liquid properties and choriolysin-encoding genes were investigated in this species. A genomic analysis identified four putative genes referred to as SseHCEa, SseHCEb, SseLCE and SseHE. The phylogenetic analysis classified these paralogs into two clades, the clade I containing SseHCE paralogs and the clade II containing two well-supported subclades named as HE and LCE. The two SseHCE paralogs were intron-less and both genes were tandemly arrayed very close in the genome. The synteny and gene rearrangement identified in the flatfish lineage indicated that the duplication of these two paralogs occurred recently and they are under divergent evolution. The genes SseHE and SseLCE were structured in 8 exons and 7 introns and the synteny was conserved in teleosts. Expression studies confirmed that the four genes were expressed in the hatching gland cells and they migrate co-ordinately from the head to around the yolk sac close to the hatch with specific temporal and intensity expression profiles. Although the mRNA levels of the four genes peaked in the hours previous to larval hatching, the SseHCE and SseLCE paralogs kept a longer expression than SseHE after hatching. These expression patterns were consistent even when larvae were incubated at different temperatures that modified hatching times. The analysis of hatching-liquid using SDS-PAGE and zymography analyses of hatching liquid identified a major band of expected choriolysin size. The optimal pH for protease activity was 8.5 and inhibition assays using EDTA demonstrated that most of the activity in the hatching liquid was due to metalloproteases with Ca2+ ions acting as the most effective metal to restore the activity. All these data provide new clues about the choriolysin evolution and function in flatfish with impact in the aquaculture and the blue cosmetic industry.


Assuntos
Evolução Molecular , Linguados/metabolismo , Metaloendopeptidases , Animais , Linguados/genética , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Metaloendopeptidases/classificação , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Filogenia , RNA Mensageiro/genética
8.
Ital J Pediatr ; 45(1): 155, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796088

RESUMO

BACKGROUND: Pathogenic variants in KIF1A (kinesin family member 1A) gene have been associated with hereditary spastic paraplegia (HSP) type 30 (SPG30), encopassing autosomal dominant and recessive, pure and complicated forms. CASE PRESENTATION: We report the long-term follow-up of a 19 years-old boy first evaluated at 18 months of age because of toe walking and unstable gait with frequent falls. He developed speech delay, mild intellectual disability, a slowly progressive pyramidal syndrome, microcephaly, bilateral optic subatrophy and a sensory axonal polyneuropathy. Brain MRI showed cerebellar atrophy, stable along serial evaluations (last performed at 18 years of age). Targeted NGS sequencing disclosed the de novo c.914C > T missense, likely pathogenic variant on KIF1A gene. CONCLUSIONS: We report on a previously unpublished de novo heterozygous likely pathogenic KIF1A variant associated with slowly progressive complicated SPG30 and stable cerebellar atrophy on long-term follow-up, adding to current knowledge on this HSP subtype.


Assuntos
Cinesina/genética , Mutação de Sentido Incorreto , Paraplegia Espástica Hereditária/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Atrofia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Éxons , Humanos , Imagem por Ressonância Magnética , Masculino , Metaloendopeptidases/genética , Polimorfismo Genético , Adulto Jovem
9.
Orphanet J Rare Dis ; 14(1): 294, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856865

RESUMO

BACKGROUND: Mandibuloacral Dysplasia with type B lipodystrophy (MADB) is a rare premature aging disorder with an autosomal recessive inheritance pattern. MADB is characterized by brittle hair, mottled, atrophic skin, generalized lipodystrophy, insulin resistance, metabolic complications and skeletal features like stunted growth, mandibular and clavicular hypoplasia and acro-osteolysis of the distal phalanges. MADB is caused by reduced activity of the enzyme zinc metalloprotease ZMPSTE24 resulting from compound heterozygous or homozygous mutations in ZMPSTE24. METHODS: In 2012, and again in 2018, eight related patients from the remote tropical rainforest of inland Suriname were analysed for dysmorphic features. DNA analysis was performed and clinical features were documented. We also analysed all previously reported genetically confirmed MADB patients from literature (n = 12) for their clinical features. Based on the features of all cases (n = 20) we defined major criteria as those present in 85-100% of all MADB patients and minor criteria as those present in 70-84% of patients. RESULTS: All the Surinamese patients are of African descent and share the same homozygous c.1196A > G, p.(Tyr399Cys) missense variant in the ZMPSTE24 gene, confirming MADB. Major criteria were found to be: short stature, clavicular hypoplasia, delayed closure of cranial sutures, high palate, mandibular hypoplasia, dental crowding, acro-osteolysis of the distal phalanges, hypoplastic nails, brittle and/or sparse hair, mottled pigmentation, atrophic and sclerodermic skin, and calcified skin nodules. Minor criteria were (generalized or partial) lipoatrophy of the extremities, joint contractures and shortened phalanges. Based on our detailed clinical observations, and a review of previously described cases, we propose that the clinical diagnosis of MADB is highly likely if a patient exhibits ≥4 major clinical criteria OR ≥ 3 major clinical criteria and ≥ 2 minor clinical criteria. CONCLUSIONS: We report on eight related Surinamese patients with MADB due to a homozygous founder mutation in ZMPSTE24. In low-income countries laboratory facilities for molecular genetic testing are scarce or lacking. However, because diagnosing MADB is essential for guiding clinical management and for family counselling, we defined clinical diagnostic criteria and suggest management guidelines.


Assuntos
Anormalidades Craniofaciais/genética , Lipodistrofia/genética , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Homozigoto , Humanos , Masculino , Mutação/genética , Linhagem , Fenótipo , Suriname , Adulto Jovem
10.
Nature ; 575(7782): 361-365, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695197

RESUMO

Reprogramming of mitochondria provides cells with the metabolic flexibility required to adapt to various developmental transitions such as stem cell activation or immune cell reprogramming, and to respond to environmental challenges such as those encountered under hypoxic conditions or during tumorigenesis1-3. Here we show that the i-AAA protease YME1L rewires the proteome of pre-existing mitochondria in response to hypoxia or nutrient starvation. Inhibition of mTORC1 induces a lipid signalling cascade via the phosphatidic acid phosphatase LIPIN1, which decreases phosphatidylethanolamine levels in mitochondrial membranes and promotes proteolysis. YME1L degrades mitochondrial protein translocases, lipid transfer proteins and metabolic enzymes to acutely limit mitochondrial biogenesis and support cell growth. YME1L-mediated mitochondrial reshaping supports the growth of pancreatic ductal adenocarcinoma (PDAC) cells as spheroids or xenografts. Similar changes to the mitochondrial proteome occur in the tumour tissues of patients with PDAC, suggesting that YME1L is relevant to the pathophysiology of these tumours. Our results identify the mTORC1-LIPIN1-YME1L axis as a post-translational regulator of mitochondrial proteostasis at the interface between metabolism and mitochondrial dynamics.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Metabolismo dos Lipídeos , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Hipóxia Celular , Linhagem Celular , Proliferação de Células , Humanos , Lipídeos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , Proteólise
11.
Clin Lab ; 65(10)2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31625357

RESUMO

BACKGROUND: The reagent red blood cells used to screen and identify antibodies have to include K+ cells in all batch productions. The data of K/k phenotypes among differing Thai blood donor populations remains unknown; hence, mass screening for uncommon K+ donors by serological test has some limitations. Implementing K/k genotyping may be useful to predict uncommon K+ donors to overcome this challenge. This study aimed to establish an in-house K/k genotyping technique and to report KEL*01 and KEL*02 allele frequencies among three Thai blood donor populations to increase the selection of K+ donors in rare blood group databases. METHODS: A total of 2,239 DNA samples obtained from 1,512 central, 427 southern, and 300 northern Thai blood donors were included. The KEL*01 and KEL*02 genotyping using PCR with sequence-specific primers (PCR-SSP) was developed and validated. All samples were genotyped using developed PCR-SSP. Moreover, the possibility of finding group O and predicted K+ phenotypes among Thai blood donor populations was calculated. RESULTS: The DNA controls were validated using two sets of primer combinations and the results of KEL*01 and KEL*02 genotyping were in agreement. The KEL*01 allele frequencies were 0.0007, 0.0047, and 0.0000, and KEL*02 allele frequencies were 0.9993, 0.9953, and 1.0000 among central, southern, and northern Thai donors, respectively. In addition, mass screening among 3,795 and 566 donors in central and southern Thai populations was required to find at least one group O and predicted K+ phenotypes. CONCLUSIONS: The in-house PCR-SSP for KEL*01 and KEL*02 genotyping provided reproducible and accurate results with cost effectiveness. Our results confirmed the low KEL*01 allele frequencies among Thais. PCR-SSP could be used as an alternative technique to simply increase the number of uncommon predicted K+ phenotypes for reagent red blood cell recruitments.


Assuntos
Doadores de Sangue , Eritrócitos/metabolismo , Técnicas de Genotipagem/métodos , Sistema do Grupo Sanguíneo de Kell/genética , Grupo com Ancestrais do Continente Asiático/genética , Sequência de Bases , DNA/análise , DNA/genética , Primers do DNA/genética , Frequência do Gene , Genótipo , Humanos , Glicoproteínas de Membrana/genética , Metaloendopeptidases/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Tailândia
13.
Biochimie ; 167: 81-92, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31476328

RESUMO

Loxosceles spiders are found in almost all countries of South America. In Peru, Loxosceles laeta species is the main responsible for the accidents caused by poisonous animals, being known as "killer spiders", due to the large number of fatal accidents observed. Astacin-like metalloproteases, named LALPs (Loxosceles astacin-like metalloproteases) are highly expressed in Loxosceles spiders venom gland. These proteases may be involved in hemorrhage and venom spreading, being relevant to the envenoming proccess. Thus, the aim of this work was to analyze Peruvian L. laeta venom gland transcripts using bioinformatics tools, focusing on LALPs. A cDNA library from Peruvian L. laeta venom glands was constructed and sequenced by MiSeq (Illumina) sequencer. After assembly, the resulting sequences were annotated, seeking out for similarity with previously described LALPs. Nine possible LALPs isoforms from Peruvian L. laeta venom were identified and the results were validated by in silico and in vitro experiments. This study contributes to a better understanding of the molecular diversity of Loxosceles venom and provide insights about the action of LALPs.


Assuntos
Isoenzimas , Metaloendopeptidases , Diester Fosfórico Hidrolases , Venenos de Aranha , Aranhas/genética , Animais , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Isoenzimas/genética , Isoenzimas/toxicidade , Metaloendopeptidases/genética , Metaloendopeptidases/toxicidade , Peru , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/toxicidade , Coelhos , Venenos de Aranha/genética , Venenos de Aranha/toxicidade
14.
BMC Cancer ; 19(1): 879, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488085

RESUMO

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) is an enterotoxin-producing bacterium that possibily has a role in the occurrence and progression of colorectal cancer (CRC) by modulating the mucosal immune response and inducing epithelial cell changes. The aim of this study was to investigate the frequency of ETBF in stool samples of CRC patients and healthy volunteers. METHODS: A total of 60 stool samples from confirmed CRC patients and 60 stool samples from healthy volunteers with no personal or familial history or diagnosis of colorectal disease were collected. Stool samples were screened for direct detection of B. fragilis using PCR targeting the marker genes of neu and bft. Enterotoxin isotypes bft-1, bft-2 and bft-3 were also detected in B. fragilis positive samples. RESULTS: The frequency of B. fragilis among CRC and control cases was 58.3 and 26.6%, respectively (P < 0.05). The rate of bft gene in CRC cases was significantly higher than in controls (P < 0.05). Also, the presence of bft gene in CRC patients stage III was significantly higher than stages I and II (P < 0.05). Enterotoxin isotype bft-2 was detected with higher frequency among CRC patients than healthy control (P < 0.05). CONCLUSION: Our results show the association between fecal ETBF and CRC, and we suggest that detection of ETBF may be a potential marker for colorectal cancer diagnosis. However, additional investigations on tumor and paired normal tissue samples are required to substantiate this possible correlation.


Assuntos
Infecções por Bacteroides/microbiologia , Bacteroides fragilis/genética , Neoplasias Colorretais/microbiologia , Enterotoxinas/genética , Fezes/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Toxinas Bacterianas/genética , Bacteroides fragilis/isolamento & purificação , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Feminino , Microbioma Gastrointestinal , Voluntários Saudáveis , Humanos , Irã (Geográfico) , Masculino , Metaloendopeptidases/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase
15.
Mar Drugs ; 17(9)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461929

RESUMO

Quorum quenching (QQ) is a promising alternative infection-control strategy to antibiotics that controls quorum-regulated virulence without killing the pathogens. Aeromonas hydrophila is an opportunistic gram-negative pathogen living in freshwater and marine environments. A. hydrophila possesses an N-acyl homoserine lactone (AHL)-based quorum-sensing (QS) system that regulates virulence, so quorum signal-inactivation (i.e., QQ) may represent a new way to combat A. hydrophila infection. In this study, an AHL lactonase gene, aiiA was cloned from Bacillus sp. strain QSI-1 and expressed in Escherichia coli strain BL21(DE3). The A. hydrophila hexanoyl homoserine lactone (C6-HSL) QS signal molecule was degraded by AiiAQSI-1, which resulted in a decrease of bacterial swimming motility, reduction of extracellular protease and hemolysin virulence factors, and inhibited the biofilm formation of A. hydrophila YJ-1 in a microtiter assay. In cell culture studies, AiiAQSI-1 decreased the ability of A. hydrophila adherence to and internalization by Epithelioma papulosum cyprini (EPC) cells. During in vivo studies, oral administration of AiiAQSI-1 via feed supplementation attenuated A. hydrophila infection in Crucian Carp. Results from this work indicate that feed supplementation with AiiAQSI-1 protein has potential to control A. hydrophila aquaculture disease via QQ.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/farmacologia , Doenças dos Peixes/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/veterinária , Metaloendopeptidases/farmacologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/antagonistas & inibidores , Administração Oral , Aeromonas hydrophila/patogenicidade , Ração Animal , Animais , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biofilmes/efeitos dos fármacos , Carpas/microbiologia , Linhagem Celular , Clonagem Molecular , Doenças dos Peixes/microbiologia , Pesqueiros , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Metaloendopeptidases/genética , Metaloendopeptidases/isolamento & purificação , Percepção de Quorum/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fatores de Virulência/antagonistas & inibidores
16.
N Z Vet J ; 67(6): 329-332, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31378159

RESUMO

Aims: To determine if presence of the Bacteroides fragilis toxin (bft) gene, a molecular marker of colonic carriage of entertoxigenic Bacteroides fragilis (ETBF) in humans, was associated with a finding of small intestinal adenocarcinomas (SIA) in sheep in New Zealand. Methods: Samples of jejunal tissue were collected from the site of tumours and from grossly normal adjacent tissue in 20 sheep, in different consignments, diagnosed with SIA based on gross examination of viscera following slaughter. Two jejunal samples were also collected from a control sheep in the same consignment that had no gross evidence of SIA. A PCR assay was used to detect the presence of the bft gene in the samples. Results: Of the sheep with SIA, the bft gene was amplified from one or both samples from 7/20 (35%) sheep, and in sheep that had no gross evidence of SIA the bft gene was amplified from at least one sample in 11/20 (55%) sheep (RR 0.61; 95% CI = 0.30-1.25; p = 0.34). Of 11 positive samples analysed, ETBF subtype bft-1 was detected in one, bft-2 was detected in 10, and none were bft-3. Conclusions and Clinical Relevance: There was a high prevalence of detection of the bft gene in both SIA-affected and non-affected sheep, but there was no apparent association between carriage of ETBF, evidenced by detection of the bft gene, and the presence of SIA. ETBF are increasingly implicated in the aetiology of human colorectal cancer, raising the possibility that sheep may provide a zoonotic reservoir of this potentially carcinogenic bacterium. Abbreviation: Bft: Bacteroides fragilis toxin; ETBF: Enterotoxigenic Bacteroides fragilis; SIA: Small intestinal adenocarcinoma.


Assuntos
Adenocarcinoma/veterinária , Toxinas Bacterianas/genética , Neoplasias Intestinais/veterinária , Metaloendopeptidases/genética , Doenças dos Ovinos/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/microbiologia , Animais , Toxinas Bacterianas/isolamento & purificação , DNA Bacteriano/análise , Genes Bacterianos , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/microbiologia , Metaloendopeptidases/isolamento & purificação , Ovinos , Doenças dos Ovinos/microbiologia
17.
mBio ; 10(4)2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409684

RESUMO

Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools.IMPORTANCE We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.


Assuntos
Bacteroidetes/genética , Microbioma Gastrointestinal , Engenharia Genética/métodos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Bacteroides/genética , Bacteroides/metabolismo , Bacteroidetes/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Marcadores Genéticos , Vetores Genéticos , Humanos , Inulina/genética , Inulina/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mutação , Regiões Promotoras Genéticas , Ramnose/metabolismo
18.
Am J Physiol Renal Physiol ; 317(4): F1034-F1046, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411076

RESUMO

Meprin metalloproteases have been implicated in the pathophysiology of diabetic kidney disease (DKD). Single-nucleotide polymorphisms in the meprin-ß gene have been associated with DKD in Pima Indians, a Native American ethnic group with an extremely high prevalence of DKD. In African American men with diabetes, urinary meprin excretion positively correlated with the severity of kidney injury. In mice, meprin activity decreased at the onset of diabetic kidney injury. Several studies have identified meprin targets in the kidney. However, it is not known how proteolytic processing of the targets by meprins impacts the metabolite milieu in kidneys. In the present study, global metabolomics analysis identified differentiating metabolites in kidney tissues from wild-type and meprin-ß knockout mice with streptozotocin (STZ)-induced type 1 diabetes. Kidney tissues were harvested at 8 wk post-STZ and analyzed by hydrophilic interaction liquid chromatography ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Principal component analysis identified >200 peaks associated with diabetes. Meprin expression-associated metabolites with strong variable importance of projection scores were indoxyl sulfate, N-γ-l-glutamyl-l-aspartic acid, N-methyl-4-pyridone-3-carboxamide, inosine, and cis-5-decenedioic acid. N-methyl-4-pyridone-3-carboxamide has been previously implicated in kidney injury, and its isomers, 4-PY and 2-PY, are markers of peroxisome proliferation and inflammation that correlate with creatinine clearance and glucose tolerance. Meprin deficiency-associated differentiating metabolites with high variable importance of projection scores were cortisol, hydroxymethoxyphenylcarboxylic acid-O-sulfate, and isovaleryalanine. The data suggest that meprin-ß activity enhances diabetic kidney injury in part by altering the metabolite balance in kidneys, favoring high levels of uremic toxins such as indoxyl sulfate and N-methyl-pyridone-carboxamide.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Rim/metabolismo , Metabolômica/métodos , Metaloendopeptidases/genética , Animais , Biomarcadores/urina , Cromatografia Líquida , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/patologia , Rim/patologia , Masculino , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proliferadores de Peroxissomos , Espectrometria de Massas em Tandem
19.
Med Hypotheses ; 131: 109309, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31443781

RESUMO

Stroke and traumatic brain injury (TBI) are significant clinical problems characterized by high rate of mortality and long-lasting disabilities, and an unmet need for new treatments. Current experimental stroke and TBI research are evolving to focus more on understanding the brain's self-protective mechanisms to meet the critical need of developing new therapies for these disorders. In this hypothesis-based manuscript, I provide several lines of evidence that peptidase neurolysin (Nln) is one of the brain's potent, self-protective mechanisms promoting preservation and recovery of the brain after acute injury. Based on published experimental observations and ongoing studies in our laboratory, I posit that Nln is a compensatory and cerebroprotective mechanism in the post-stroke/TBI brain that functions to process a diverse group of extracellular neuropeptides and by that to reduce excitotoxicity, oxidative stress, edema formation, blood brain barrier hyper-permeability, and neuroinflammation. If this hypothesis is correct, Nln could potentially serve as a single therapeutic target to modulate the function of multiple targets, the involved neuropeptide systems, critically involved in various mechanisms of brain injury and cerebroprotection/restoration. Such multi-pathway target would be highly desired for pharmacotherapy of stroke and TBI, because targeting one pathophysiological pathway has proven to be ineffective for such complex disorders.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Metaloendopeptidases/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuropeptídeos/metabolismo , Acidente Vascular Cerebral/metabolismo , Doença Aguda , Animais , Lesões Encefálicas Traumáticas/fisiopatologia , Morte Celular , Hipóxia Celular , Células Cultivadas , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Terapia Genética , Humanos , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurotoxinas/farmacologia , Bulbo Olfatório/metabolismo , Estresse Oxidativo , Oxigênio/farmacologia , Ratos , Proteínas Recombinantes/metabolismo , Sistema Renina-Angiotensina/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapia , Regulação para Cima
20.
Mol Biol Evol ; 36(10): 2127-2142, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251352

RESUMO

Introgression among parasite species has the potential to transfer traits of biomedical importance across species boundaries. The parasitic blood fluke Schistosoma haematobium causes urogenital schistosomiasis in humans across sub-Saharan Africa. Hybridization with other schistosome species is assumed to occur commonly, because genetic crosses between S. haematobium and livestock schistosomes, including S. bovis, can be staged in the laboratory, and sequencing of mtDNA and rDNA amplified from microscopic miracidia larvae frequently reveals markers from different species. However, the frequency, direction, age, and genomic consequences of hybridization are unknown. We hatched miracidia from eggs and sequenced the exomes from 96 individual S. haematobium miracidia from infected patients from Niger and the Zanzibar archipelago. These data revealed no evidence for contemporary hybridization between S. bovis and S. haematobium in our samples. However, all Nigerien S. haematobium genomes sampled show hybrid ancestry, with 3.3-8.2% of their nuclear genomes derived from S. bovis, providing evidence of an ancient introgression event that occurred at least 108-613 generations ago. Some S. bovis-derived alleles have spread to high frequency or reached fixation and show strong signatures of directional selection; the strongest signal spans a single gene in the invadolysin gene family (Chr. 4). Our results suggest that S. bovis/S. haematobium hybridization occurs rarely but demonstrate profound consequences of ancient introgression from a livestock parasite into the genome of S. haematobium, the most prevalent schistosome species infecting humans.


Assuntos
Introgressão Genética , Proteínas de Helminto/genética , Hibridização Genética , Metaloendopeptidases/genética , Schistosoma/genética , Animais , Variação Genética , Genoma Mitocondrial , Sequenciamento Completo do Exoma
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