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1.
J Med Primatol ; 53(1): e12668, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37583034

RESUMO

Acute gastric dilatation (AGD) is one of the most prevalent and life-threatening diseases in nonhuman primates worldwide. However, the etiology of this syndrome has not been determined. Recently, sudden death occurred in a 7-year-old female cynomolgus monkey with a history of fecal microbiota transplantation using diarrheic stools. The monkey had undergone surgery previously. On necropsy, gastric dilatation and rupture demonstrated a tetrad arrangement on histopathologic examination. On 16S rRNA sequencing, a high population of Clostridium ventriculi was identified in the duodenum adjacent to stomach but not in the colon. This paper is the first report of Clostridium ventriculi infection in a cynomolgus macaque with acute gastric dilatation and rupture.


Assuntos
Clostridium , Dilatação Gástrica , Feminino , Animais , Macaca fascicularis , Dilatação Gástrica/veterinária , Dilatação Gástrica/patologia , RNA Ribossômico 16S
2.
J Med Virol ; 95(6): e28847, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37272485

RESUMO

Recently emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants are generally less pathogenic than previous strains. However, elucidating the molecular basis for pulmonary immune response alterations is challenging owing to the virus's heterogeneous distribution within complex tissue structure. Here, we revealed the spatial transcriptomic profiles of pulmonary microstructures at the SARS-CoV-2 infection site in the nine cynomolgus macaques upon inoculation with the Delta and Omicron variants. Delta- and Omicron-infected lungs had upregulation of genes involved in inflammation, cytokine response, complement, cell damage, proliferation, and differentiation pathways. Depending on the tissue microstructures (alveoli, bronchioles, and blood vessels), there were differences in the types of significantly upregulated genes in each pathway. Notably, a limited number of genes involved in cytokine and cell damage response were differentially expressed between bronchioles of the Delta- and Omicron-infection groups. These results indicated that despite a significant antigenic shift in SARS-CoV-2, the host immune response mechanisms induced by the variants were relatively consistent, with limited transcriptional alterations observed only in large airways. This study may aid in understanding the pathogenesis of SARS-CoV-2 and developing a clinical strategy for addressing immune dysregulation by identifying potential transcriptional biomarkers within pulmonary microstructures during infection with emerging variants.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , SARS-CoV-2/genética , Transcriptoma , COVID-19/genética , Alvéolos Pulmonares , Citocinas/genética , Macaca
3.
J Med Virol ; 95(12): e29309, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-38100632

RESUMO

The E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA-HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell-mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor-implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV-treated mice. Furthermore, tumor expansion was significantly reduced upon TC-1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV-related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine.


Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Vacinas contra Papillomavirus , Neoplasias do Colo do Útero , Humanos , Feminino , Animais , Camundongos , Papillomavirus Humano , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/prevenção & controle , RNA Mensageiro/genética , Proteínas E7 de Papillomavirus/genética , Camundongos Endogâmicos C57BL , Vacinação/métodos , Imunização , Neoplasias do Colo do Útero/prevenção & controle
4.
J Neurochem ; 162(2): 190-206, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35567753

RESUMO

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Astrócitos , Adesões Focais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Astrócitos/metabolismo , Proteínas de Transporte/genética , Adesões Focais/metabolismo , Camundongos
5.
Biochem Biophys Res Commun ; 637: 232-239, 2022 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-36410271

RESUMO

Enterococcus faecalis has recently shown signs of high antibiotic resistance. These bacteria can endure extremes of temperature and this may be due to the high thermostability of its proteins. E. faecalis has two acyl carrier proteins (ACPs), AcpA (EfAcpA), which is essential for de novo fatty acid synthesis (FAS), and EfAcpB, which plays an auxiliary role in the incorporation of exogenous fatty acids. Structural studies on EfAcpA and its interaction with FAS enzymes have not yet been reported. Here, we investigated the structures of EfAcpA using NMR spectroscopy, showing that EfAcpA consists of three α-helices with a long α2α3 loop, while the other ACPs have four α-helices. CD experiments showed that the melting temperature of EfAcpA is 76.3 °C and the Ala mutation for Ile10 reduced it dramatically by 29.5 °C. Highly conserved Ile10 of EfAcpA mediates compact intramolecular packing and promotes high thermostability. A docking simulation of EfAcpA and ß-ketoacyl-ACP synthase III (EfKAS III) showed that the α2α3 loop of EfAcpA contributes to specific protein-protein interactions (PPI) with EfKAS III. Unconserved charged residues, Lys52 and Glu54, in the α2α3 loop of EfAcpA formed specific electrostatic interactions with Asp 226 and Arg217 of EfKAS III, respectively. Binding interactions between EfAcpA and EfKASIII may provide insights for designing PPI inhibitors targeting FAS in E. faecalis to overcome its antibacterial resistance.


Assuntos
Proteína de Transporte de Acila , Enterococcus faecalis , Ácidos Graxos , Proteína de Transporte de Acila/química , Ácidos Graxos/biossíntese , Proteínas de Bactérias/química
6.
J Infect Dis ; 224(11): 1861-1872, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34718664

RESUMO

Germinal centers (GCs) elicit protective humoral immunity through a combination of antibody-secreting cells and memory B cells, following pathogen invasion or vaccination. However, the possibility of a GC response inducing protective immunity against reinfection following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown. We found GC activity was consistent with seroconversion observed in recovered macaques and humans. Rechallenge with a different clade of virus resulted in significant reduction in replicating virus titers in respiratory tracts in macaques with high GC activity. However, diffuse alveolar damage and increased fibrotic tissue were observed in lungs of reinfected macaques. Our study highlights the importance of GCs developed during natural SARS-CoV-2 infection in managing viral loads in subsequent infections. However, their ability to alleviate lung damage remains to be determined. These results may improve understanding of SARS-CoV-2-induced immune responses, resulting in better coronavirus disease 2019 (COVID-19) diagnosis, treatment, and vaccine development.


Assuntos
COVID-19 , Centro Germinativo , Imunidade Humoral , Reinfecção/imunologia , Animais , Anticorpos Antivirais , COVID-19/imunologia , Humanos , Pulmão/patologia , Pulmão/virologia , Macaca , Células B de Memória , Soroconversão
7.
Arch Virol ; 166(4): 1103-1112, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33575893

RESUMO

Dengue virus (DV) is a mosquito-borne virus that is endemic to many tropical and subtropical areas. Recently, the annual incidence of DV infection has increased worldwide, including in Korea, due to global warming and increased global travel. We therefore sought to characterize the molecular and evolutionary features of DV-1 and DV-4 isolated from Korean overseas travelers. We used phylogenetic analysis based on the full coding region to classify isolates of DV-1 in Korea into genotype I (43251, KP406802), genotype IV (KP406803), and genotype V (KP406801). In addition, we found that strains of DV-4 belonged to genotype I (KP406806) and genotype II (43257). Evidence of positive selection in DV-1 strains was identified in the C, prM, NS2A, and NS5 proteins, whereas DV-4 showed positive selection only in the non-structural proteins NS2A, NS3, and NS5. The substitution rates per site per year were 5.58 × 10-4 and 6.72 × 10-4 for DV-1 and DV-4, respectively, and the time of the most recent common ancestor was determined using the Bayesian skyline coalescent method. In this study, the molecular, phylogenetic, and evolutionary characteristics of Korean DV-1 and DV-4 isolates were evaluated for the first time.


Assuntos
Vírus da Dengue/genética , Dengue/virologia , Evolução Molecular , Viagem , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Dengue/epidemiologia , Vírus da Dengue/classificação , Vírus da Dengue/isolamento & purificação , Genótipo , Humanos , Filogenia , RNA Viral/genética , República da Coreia/epidemiologia , Seleção Genética , Sorogrupo , Proteínas Virais/genética
8.
Molecules ; 26(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916181

RESUMO

We have synthesized new magnetic resonance imaging (MRI) T1 contrast agents (CA1 and CA2) that permit the activatable recognition of the cellular vicinal thiol motifs of the protein thioredoxin. The contrast agents showed MR relaxivities typical of gadolinium complexes with a single water molecule coordinated to a Gd3+ center (i.e., ~4.54 mM-1s-1) for both CA1 and CA2 at 60 MHz. The contrast agent CA1 showed a ~140% relaxivity enhancement in the presence of thioredoxin, a finding attributed to a reduction in the flexibility of the molecule after binding to thioredoxin. Support for this rationale, as opposed to one based on preferential binding, came from 1H-15N-HSQC NMR spectral studies; these revealed that the binding affinities toward thioredoxin were almost the same for both CA1 and CA2. In the case of CA1, T1-weighted phantom images of cancer cells (MCF-7, A549) could be generated based on the expression of thioredoxin. We further confirmed thioredoxin expression-dependent changes in the T1-weighted contrast via knockdown of the expression of the thioredoxin using siRNA-transfected MCF-7 cells. The nontoxic nature of CA1, coupled with its relaxivity features, leads us to suggest that it constitutes a first-in-class MRI T1 contrast agent that allows for the facile and noninvasive monitoring of vicinal thiol protein motif expression in live cells.


Assuntos
Rastreamento de Células/métodos , Meios de Contraste , Espectroscopia de Ressonância Magnética , Compostos de Sulfidrila , Tiorredoxinas , Linhagem Celular Tumoral , Meios de Contraste/síntese química , Meios de Contraste/química , Humanos , Imagens de Fantasmas , Compostos de Sulfidrila/metabolismo , Tiorredoxinas/metabolismo
9.
J Infect Dis ; 222(10): 1596-1600, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32745172

RESUMO

Using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we exposed macaques to high titers of SARS-CoV-2 via combined transmission routes. We observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. All macaques had a significant loss of total lymphocytes during infection, which were restored over time. These data show that SARS-CoV-2 causes a coronavirus disease 2019 (COVID-19)-like disease in macaques. This new model could investigate the interaction between SARS-CoV-2 and the immune system to test therapeutic strategies.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/complicações , Modelos Animais de Doenças , Doenças Pulmonares Intersticiais/complicações , Linfopenia/complicações , Doenças dos Macacos/virologia , Pneumonia Viral/complicações , Animais , COVID-19 , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Doenças Pulmonares Intersticiais/patologia , Linfopenia/patologia , Macaca fascicularis , Macaca mulatta , Masculino , Doenças dos Macacos/patologia , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2
10.
J Med Primatol ; 49(1): 56-59, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31642533

RESUMO

Chronic inflammatory enteric diseases occur commonly in humans and animals, especially in captive bred macaques. However, information about the etiology of idiopathic chronic inflammatory diarrhea in cynomolgus monkeys is limited. In this paper, we reported the unusual case of idiopathic chronic diarrhea in a captive cynomolgus monkey based on microbial, imaging, and microbiome examinations.


Assuntos
Diarreia/veterinária , Disbiose/veterinária , Macaca fascicularis , Doenças dos Macacos/etiologia , Animais , Doença Crônica/veterinária , Diarreia/complicações , Diarreia/etiologia , Diarreia/imunologia , Disbiose/complicações , Disbiose/etiologia , Disbiose/imunologia , Feminino , Doenças dos Macacos/imunologia
11.
Arch Virol ; 165(8): 1739-1748, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32409874

RESUMO

In Korea, dengue infection has been frequently reported in travelers to tropical and subtropical countries. Global warming increases the probability of autochthonous dengue outbreaks in Korea. In this report, the molecular and evolutionary properties of four dengue virus (DENV) type 2 isolates from Korean overseas travelers were examined. Three of these isolates were classified as Cosmopolitan genotypes and further divided into sublineages 1 (43,253, 43,254) and 2 (43,248), while the other isolate (KBPV-VR29) was related to American genotypes. The variable amino acid motifs related to virulence and replication were identified in the structural and non-structural proteins. A negative selection mechanism was clearly verified in all of the DENV proteins. Potential recombination events were identified in the NS5 protein of the XSBN10 strain. The substitution rate (5.32 × 10-4 substitutions per site) and the time of the most recent common ancestor (TMRCA) for each evolutionary group were determined by the Bayesian skyline coalescent method. This study shows that DENV type 2 strains with distinct phylogenetic, evolutionary, and virulence characteristics have been introduced into Korea by overseas travelers and have the potential to trigger autochthonous dengue outbreaks.


Assuntos
Vírus da Dengue/genética , Vírus da Dengue/isolamento & purificação , Substituição de Aminoácidos/genética , Dengue/virologia , Surtos de Doenças , Evolução Molecular , Genoma Viral/genética , Genótipo , Humanos , Filogenia , RNA Viral/genética , República da Coreia , Sorogrupo , Proteínas Virais/genética , Virulência/genética , Replicação Viral/genética
12.
Anaerobe ; 64: 102236, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32623046

RESUMO

Clostridium perfringens is ubiquitous in the environment and the gastrointestinal tract of warm-blooded animals. While part of the gut microbiome, abnormal growth of C. perfringens causes histotoxic, neurologic, and enteric diseases in a variety of animal species, including humans, due to the production of toxins. There is extremely limited information on C. perfringens infection in non-human primates. Presently, 10 strains were successfully isolated from 126 monkeys and confirmed by molecular and biochemical analyses. All isolates were genotype A based on molecular analysis. Alpha toxin was identified in all isolates. Beta 2 toxin was detected in only three isolates. No other toxins, including enterotoxin, beta, iota, epsilon, and net B toxin, were identified in any isolate. All isolates were highly susceptible to ß-lactam antibiotics. Double hemolysis and lecithinase activity were commonly observed in all strains. Biofilm formation, which can increase antibiotic resistance, was identified in 90% of the isolates. The data are the first report the prevalence and characteristics of C. perfringens isolated from captive cynomolgus monkeys.


Assuntos
Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/genética , Farmacorresistência Bacteriana Múltipla , Macaca fascicularis/microbiologia , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Infecções por Clostridium/microbiologia , Infecções por Clostridium/veterinária , Clostridium perfringens/isolamento & purificação , DNA Bacteriano/genética , Fezes/microbiologia , Feminino , Genótipo , Masculino , Filogenia , Prevalência , RNA Ribossômico 16S/genética , beta-Lactamas/farmacologia
14.
Biochem Biophys Res Commun ; 495(1): 145-150, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29101034

RESUMO

MeCP2 is a chromatin associated protein which is highly expressed in brain and relevant with Rett syndrome (RTT). There are AT-hook motifs in MeCP2 which can bind with AT-rich DNA, suggesting a role in chromatin binding. Here, we report the identification and characterization of another AT-rich DNA binding motif (residues 295 to 313) from the C-terminal transcription repression domain of MeCP2 by nuclear magnetic resonance (NMR) and isothermal calorimetry (ITC). This motif shows a micromolar affinity to AT-rich DNA, and it binds to the minor groove of DNA like AT-hook motifs. Together with the previous studies, our results provide an insight into a critical role of this motif in chromatin structure and function.


Assuntos
DNA/metabolismo , Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , DNA/química , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Síndrome de Rett/metabolismo
15.
Biophys J ; 112(10): 2089-2098, 2017 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-28538146

RESUMO

The periplasmic domain of OmpA from Acinetobacter baumannii (AbOmpA-PD) binds to diaminopimelate and anchors the outer membrane to the peptidoglycan layer in the cell wall. Although the crystal structure of AbOmpA-PD with its ligands has been reported, the mechanism of ligand-mediated folding of AbOmpA remains elusive. Here, we report that in vitro refolded apo-AbOmpA-PD in the absence of ligand exists as a mixture of two partially folded forms in solution: mostly unfolded (apo-state I) and hololike (apo-state II) states. Binding of the diaminopimelate or glycine ligand induced complete folding of AbOmpA-PD. The apo-state I was highly flexible and contained some secondary structural elements, whereas the apo-state II closely resembled the holo-state in terms of both structure and backbone dynamics, except for the ligand-binding region. 15N-relaxation-dispersion analyses for apo-state II revealed substantial motion on a millisecond timescale of residues in the H3 helix near the ligand-binding site, with this motion disappearing upon ligand binding. These results provide an insight into the ligand-mediated folding mechanism of AbOmpA-PD in solution.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Acinetobacter baumannii , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Cromatografia em Gel , Dicroísmo Circular , Escherichia coli , Fluorometria , Glicina/química , Glicina/metabolismo , Simulação de Dinâmica Molecular , Método de Monte Carlo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Soluções
16.
Biochem Biophys Res Commun ; 493(1): 28-33, 2017 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-28935372

RESUMO

Enoyl-ACP reductase (ENR, also known as FabI) has received considerable interest as an anti-bacterial target due to its essentiality in fatty acid synthesis. All the FabI structures reported to date, regardless of the organism, are composed of homo-tetramers, except for two structures: Bacillus cereus and Staphylococcus aureus FabI (bcFabI and saFabI, respectively), which have been reported as dimers. However, the reason for the existence of the dimeric form in these organisms and the biological meaning of dimeric and tetrameric forms of FabI are ambiguous. Herein, we report the high-resolution crystal structure of a dimeric form of Bacillus anthracis FabI (baFabI) and the crystal structures of tetrameric forms of baFabI in the apo state and in complex with NAD+ and with NAD+-triclosan, at 1.7 Å, 1.85 Å, 1.96 Å, and 1.95 Å, respectively. Interestingly, we found that baFabI with a His6-tag at its C-terminus exists as a dimer, whereas untagged-baFabI exists as a tetramer. The His6-tag may block the dimer-tetramer transition, since baFabI has relatively short-length amino acids (255LG256) after the 310-helix η7 compared to those of FabI of other organisms. The dimeric form of baFabI is catalytically inactive, because the α-helix α5 occupies the NADH-binding site. During the process of dimer-tetramer transition, this α5 helix rotates about 55° toward the tetramer interface and the active site is established. Therefore, tetramerization of baFabI is required for cofactor binding and catalytic activity.


Assuntos
Bacillus anthracis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , NAD/química , NAD/ultraestrutura , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
17.
Cytokine ; 77: 127-34, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26579632

RESUMO

Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-ß (TRIF), one of the two major TLRs-adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-ß in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células Epiteliais/metabolismo , Imunidade Inata/imunologia , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Western Blotting , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Escherichia coli/fisiologia , Expressão Gênica/efeitos dos fármacos , Imunidade Inata/genética , Interferon beta/genética , Interferon beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Peritônio/citologia , Peritônio/metabolismo , Poli I-C/farmacologia , Pseudomonas aeruginosa/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/genética
18.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 7): 1944-53, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25004971

RESUMO

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1-RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1-RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432-Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1-RASSF5 complex showed a longer helical structure (Ser438-Lys480) than that in the MST1 homodimer (Val441-Lys480). Moreover, extensive polar and nonpolar contacts in the MST1-RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST-RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1-RASSF5 SARAH domain in apoptosis signalling.


Assuntos
MAP Quinase Quinase Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Via de Sinalização Hippo , Humanos , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
19.
EJNMMI Radiopharm Chem ; 9(1): 12, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38358577

RESUMO

BACKGROUND: Nanoparticles exhibit distinct behaviours within the body, depending on their physicochemical properties and administration routes. However, in vivo behaviour of poly(lactic-co-glycolic acid) (PLGA) nanoparticles, especially when administered nasally, remains unexplored; furthermore, there is a lack of comparative analysis of uptake efficiency among different administration routes. Therefore, here, we aimed to comprehensively investigate the real-time in vivo behaviour of PLGA nanoparticles across various administration routes. PLGA-NH2 nanoparticles of three sizes were synthesised using an oil-in-water single-emulsion method. We assessed their uptake by murine macrophage RAW264.7 cells using fluorescence microscopy. To enable real-time tracking, we conjugated p-SCN-Bn-deferoxamine to PLGA-NH2 nanoparticles and further radiolabelled them with 89Zr-oxalate before administration to mice via different routes. Nanoparticle internalisation by lung immune cells was monitored using fluorescence-activated cell sorting analysis. RESULTS: The nanoparticle sizes were 294 ± 2.1 (small), 522.5 ± 5.58 (intermediate), and 850 ± 18.52 nm (large). Fluorescent labelling did not significantly alter the nanoparticle size and charge. The level of uptake of small and large nanoparticles by RAW264.7 cells was similar, with phagocytosis inhibition primarily reducing the internalisation of large particles. Positron emission tomography revealed that intranasal delivery resulted in the highest and most targeted pulmonary uptake, whereas intravenous administration led to accumulation mainly in the liver and spleen. Nasal delivery of large nanoparticles resulted in enhanced uptake by myeloid immune cells relative to lymphoid cells, whereas dendritic cell uptake initially peaked but declined over time. CONCLUSIONS: Our study provides valuable insights into advancing nanomedicine and drug delivery, with the potential for expanding the clinical applications of nanoparticles.

20.
Heliyon ; 10(9): e30222, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38737246

RESUMO

After the first outbreak, SARS-CoV-2 infection continues to occur due to the emergence of new variants. There is limited information available on the comparative evaluation of evolutionary characteristics of SARS-CoV-2 among different countries over time, and its relatedness to epidemiological and socio-environmental factors within those countries. We assessed comparative Bayesian evolutionary characteristics for SARS-CoV-2 in eight countries from 2020 to 2022 using BEAST version 2.6.7. Additionally, the relatedness between virus evolution factors and both epidemiological and socio-environmental factors was analyzed using Pearson's correlation coefficient. The estimated substitution rates in the gene encoding S protein of SARS-CoV-2 exhibited a continuous increase from 2020 to 2022 and were divided into two distinct groups in 2022 (p value < 0.05). Effective population size (Ne) generally showed decreased patterns by time. Notably, the change rates of the substitution rates were negatively correlated with the cumulative vaccination rates in 2021. A strict and rapid vaccination policy in the United Arab Emirates dramatically reduced the evolution of the virus, compared to other countries. Also, the average yearly temperature in countries were negatively correlated with the substitution rates. The changes of six epitopes in SARS-CoV-2 were related to various socio-environmental factors. We figured out comparative virus evolutionary traits and the association of epidemiological and socio-environmental factors especially cumulative vaccination rates and average temperature.

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