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The ability to introduce noncanonical amino acids as axial ligands in heme enzymes has provided a powerful experimental tool for studying the structure and reactivity of their FeIV=O ("ferryl") intermediates. Here, we show that a similar approach can be used to perturb the conserved Fe coordination environment of 2-oxoglutarate (2OG) dependent oxygenases, a versatile class of enzymes that employ highly-reactive ferryl intermediates to mediate challenging C-H functionalizations. Replacement of one of the cis-disposed histidine ligands in the oxygenase VioC with a less electron donating N δ-methyl-histidine (MeHis) preserves both catalytic function and reaction selectivity. Significantly, the key ferryl intermediate responsible for C-H activation can be accumulated in both the wildtype and the modified protein. In contrast to heme enzymes, where metal-oxo reactivity is extremely sensitive to the nature of the proximal ligand, the rates of C-H activation and the observed large kinetic isotope effects are only minimally affected by axial ligand replacement in VioC. This study showcases a powerful tool for modulating the coordination sphere of nonheme iron enzymes that will enhance our understanding of the factors governing their divergent activities.
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Multi-resistant Staphylococcus aureus (S. aureus) infection is a significant global health concern owing to its high mortality and morbidity rates. Coagulase (Coa), a key enzyme that activates prothrombin to initiate host coagulation, has emerged as a promising target for anti-infective therapeutic approaches. This study identified sinigrin as a potent Coa inhibitor that significantly inhibited S. aureus-induced coagulation at concentration as low as 32 mg/L. Additionally, at a higher concentration of 128 mg/L, sinigrin disrupted the self-protection mechanism of S. aureus. Thermal shift and fluorescence-quenching assays confirmed the direct binding of sinigrin to the Coa protein. Molecular docking analysis predicted specific binding sites for sinigrin in the Coa molecule, and point mutation experiments highlighted the importance of Arg-187 and Asp-222 as critical binding sites for both Coa and sinigrin. In vivo studies demonstrated that the combination of sinigrin with oxacillin exhibited greater antibacterial efficacy than oxacillin alone in the treatment of S. aureus-induced pneumonia in mice. Furthermore, sinigrin was shown to reduce bacterial counts and inflammatory cytokine levels in the lung tissues of S. aureus-infected mice. In summary, sinigrin was shown to directly target Coa, resulting in the attenuation of S. aureus virulence, which suggests the potential of sinigrin as an adjuvant for future antimicrobial therapies.
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Objective: Despite extensive research on the relationship between pulmonary tuberculosis (PTB) and inflammatory factors, more robust causal evidence has yet to emerge. Therefore, this study aims to screen for inflammatory proteins that may contribute to the susceptibility to PTB in different populations and to explain the diversity of inflammatory and immune mechanisms of PTB in different ethnicity. Methods: The inverse variance weighted (IVW) model of a two-sample Mendelian Randomization (MR) study was employed to conduct causal analysis on data from a genome-wide association study (GWAS). This cohort consisting PTB GWAS datasets from two European and two East Asian populations, as well as 91 human inflammatory proteins collected from 14,824 participants. Colocalization analysis aimed to determine whether the input inflammatory protein and PTB shared the same causal single nucleotide polymorphisms (SNPs) variation within the fixed region, thereby enhancing the robustness of the MR Analysis. Meta-analyses were utilized to evaluate the combined causal effects among different datasets. Results: In this study, we observed a significant negative correlation between tumor necrosis factor-beta levels (The alternative we employ is Lymphotoxin-alpha, commonly referred to as LT) (P < 0.05) and tumor necrosis factor receptor superfamily member 9 levels (TNFRSF9) (P < 0.05). These two inflammatory proteins were crucial protective factors against PTB. Additionally, there was a significant positive correlation found between interleukin-20 receptor subunit alpha levels (IL20Ra) (P < 0.05), which may elevate the risk of PTB. Colocalization analysis revealed that there was no overlap in the causal variation between LT and PTB SNPs. A meta-analysis further confirmed the significant combined effect of LT, TNFRSF9, and IL20Ra in East Asian populations (P < 0.05). Conclusions: Levels of specific inflammatory proteins may play a crucial role in triggering an immune response to PTB. Altered levels of LT and TNFRSF9 have the potential to serve as predictive markers for PTB development, necessitating further clinical validation in real-world settings to ascertain the impact of these inflammatory proteins on PTB.
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Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Tuberculose Pulmonar , Humanos , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Análise da Randomização Mendeliana , Fatores de Necrose Tumoral/genética , Povo Asiático/genética , MasculinoRESUMO
Background and Aims: Disease progression of chronic hepatitis B virus (HBV) infection is driven by the interactions between viral replication and the host immune response against the infection. This study aimed to clarify the relationship between HBV replication and hepatic inflammation during disease progression. Methods: Two cross-sectional, one validation cohort, and meta-analyses were used to explore the relationship between HBV replication and liver inflammation. Spearman analysis, multiple linear regression, and logistic regression were used to explore the relationship between variables. Results: In the cross-sectional cohorts A and B including 1,350 chronic hepatitis B patients, Spearman analysis revealed a negative relationship between HBV replication (such as HBV DNA) and liver inflammation (such as ALT) in HBeAg-positive patients with higher HBV DNA >2×106 IU/mL (rho=-0.160 and -0.042) which turned to be positive in HBeAg-positive patients with HBV DNA ≤2×106 IU/mL (rho=0.278 and 0.260) and HBeAg-negative patients (rho=0.450 and 0.363). After adjustment for sex, age, and anti-HBe, results from logistic regression and multiple linear regression showed the opposite relationship still existed in HBeAg-positive patients with different DNA levels; the opposite relationship in HBeAg-positive patients with different DNA levels was validated in a third cohort; the opposite relationship in patients with different HBeAg status was partially confirmed by meta-analysis (overall R: -0.004 vs 0.481). Conclusions: These results suggested a negative relationship between viral replication and liver inflammation in HBeAg-positive patients with high HBV DNA, which changed to a positive relationship for those HBeAg-positive patients with DNA less than 2×106 IU/mL and HBeAg-negative patients.
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BACKGROUND: In recent years, the prevalence of respiratory fungal diseases has increased. Polyene antifungal drugs play a pivotal role in the treatment of these conditions, with amphotericin B (AmB) being the most representative drug. This study aimed to evaluate the efficacy and safety of topical administration of AmB in the treatment of respiratory fungal infections. METHODS: We conducted a retrospective study on hospitalized patients treated with topical administered AmB for respiratory fungal infections from January 2014 to June 2023. RESULTS: Data from 36 patients with invasive pulmonary fungal infections treated with topical administration of AmB were collected and analyzed. Nebulization was administered to 27 patients. After the treatment, 17 patients evidenced improved conditions, whereas 10 patients did not respond and died in the hospital. One patient experienced an irritating cough as an adverse reaction. Seven patients underwent tracheoscopic instillation, and two received intrapleural irrigation; they achieved good clinical therapeutic efficacy without adverse effects. CONCLUSION: The combined application of systemic antifungal treatment and topical administration of AmB yielded good therapeutic efficacy and was well-tolerated by the patients. Close monitoring of routine blood tests, liver and kidney function, and levels of electrolytes, troponin, and B-type natriuretic peptide supported this conclusion.
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Administração Tópica , Anfotericina B , Antifúngicos , Humanos , Anfotericina B/administração & dosagem , Anfotericina B/uso terapêutico , Anfotericina B/efeitos adversos , Masculino , Feminino , Estudos Retrospectivos , Pessoa de Meia-Idade , Antifúngicos/administração & dosagem , Antifúngicos/uso terapêutico , Antifúngicos/efeitos adversos , Idoso , Adulto , Resultado do Tratamento , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologia , Idoso de 80 Anos ou mais , Pneumopatias Fúngicas/tratamento farmacológico , Adulto JovemRESUMO
A 27-year-old female visited our hospital with a history of asthma, peripheral blood eosinophilia, increased total IgE, Aspergillus fumigatus specific IgE, reversible mild bronchiectasis, sinusitis, bronchial mucus plugs and cultivation of Aspergillus from BALF. Glucocorticoids therapy is effective. These results met the diagnostic criteria for both allergic bronchopulmonary aspergillosis (ABPM) and eosinophilic granulomatosis with polyangiitis (EGPA). Special attention should be paid to the possibility of both diseases coexisting in the disease process.
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Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.
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Cobre , Histidina , Oxigenases de Função Mista , Estresse Oxidativo , CatáliseRESUMO
The catalytic versatility of pentacoordinated iron is highlighted by the broad range of natural and engineered activities of heme enzymes such as cytochrome P450s, which position a porphyrin cofactor coordinating a central iron atom below an open substrate binding pocket. This catalytic prowess has inspired efforts to design de novo helical bundle scaffolds that bind porphyrin cofactors. However, such designs lack the large open substrate binding pocket of P450s, and hence, the range of chemical transformations accessible is limited. Here, with the goal of combining the advantages of the P450 catalytic site geometry with the almost unlimited customizability of de novo protein design, we design a high-affinity heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant coordination site for generating reactive intermediates, and a tunable distal pocket for substrate binding. A 1.6 Å X-ray crystal structure of dnHEM1 reveals excellent agreement to the design model with key features programmed as intended. The incorporation of distal pocket substitutions converted dnHEM1 into a proficient peroxidase with a stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned to generate enantiocomplementary carbene transferases for styrene cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.) by reconfiguring the distal pocket to accommodate calculated transition state models. Our approach now enables the custom design of enzymes containing cofactors adjacent to binding pockets with an almost unlimited variety of shapes and functionalities.
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Heme , Porfirinas , Heme/química , Metais , Sistema Enzimático do Citocromo P-450/metabolismo , Ferro/química , Porfirinas/química , Sítios de LigaçãoRESUMO
The development of bio-based polymer materials, such as polylactic acid (PLA) -based polymers, is an effective strategy to reduce dependence on petrochemical-based polymers. However, the preparation of bio-based polymers with high barrier properties is a major challenge. To overcome this challenge, a nacreous layer structure with a ' brick and mud ' pattern is mimicked to improve the overall performance of the material. In this paper, Poly (L -lactic acid) (PLLA) and Polypropylene Glycol (PPG) was combined to prepare bio-based polyurethane (PU-PLLA), which is used as the slurry structure of nacreous layer. The bio-based biomimetic composite membrane (PU-PLLA/BN) is then obtained by adding boron nitride (BN, brick structure of pearl layer) to it. The water vapor permeability test results show that the permeability of PU-PLLA material can be reduced by more than 50% by 5 wt.% BN, which is because the addition of BN can increase the length and tortuosity of the gas molecular diffusion path in the composite. Therefore, this pearl-inspired PU-PLLA/BN film has excellent moisture resistance, which opens up a broad road for the practical application of PLLA in flexible laminated packaging.
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Poliésteres , Polímeros , Poliésteres/química , Polímeros/química , Embalagem de Produtos , PermeabilidadeRESUMO
Cases of empyema associated with Histoplasma infection are rarely reported. Here, we discuss a case of Histoplasma-associated empyema successfully treated with amphotericin B intravenous and pleural infusion therapy and multiple medical thoracoscopies. A 57-year-old Chinese woman with preexisting diabetes mellitus and gastric cancer had massive left-sided pleural effusion diagnosed by chest computed tomography. Her pleural effusion was purulent through pleural catheter drainage, and the culture of the pleural fluid showed Escherichia coli and Streptococcus constellation. Histopathology of the thoracoscopic pleural biopsy after hexamine silver and PAS staining supported Histoplasma infection. The patient was treated with intravenous injection and local thoracic irrigation of amphotericin B and continuous oral administration of itraconazole. At the same time, the patient received thoracic cannulation, daily thoracic lavage and thoracoscopy for purulent and necrotic tissue removal three times during hospitalization. The patient's pleural effusion and necrotic tissue in the pleural cavity were significantly reduced in a short time, and the clinical symptoms were significantly improved. After discharge, the patient recovered well and had no obvious complications or sequelae. Intravenous injection and local thoracic irrigation of amphotericin B are safe and effective drug therapies to treat fungal-associated empyema such as Histoplasma. Medical thoracoscopy effectively shortens the recovery time of empyema, improving the prognosis and reducing complications.
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Empiema Pleural , Histoplasmose , Derrame Pleural , Anfotericina B/uso terapêutico , Empiema Pleural/tratamento farmacológico , Empiema Pleural/etiologia , Feminino , Histoplasma , Histoplasmose/complicações , Histoplasmose/tratamento farmacológico , Humanos , Injeções Intravenosas , Pessoa de Meia-Idade , Derrame Pleural/complicações , Derrame Pleural/tratamento farmacológico , Irrigação Terapêutica/efeitos adversos , Toracoscopia/efeitos adversos , Toracoscopia/métodosRESUMO
Early and effective identification of severe coronavirus disease 2019 (COVID-19) may allow us to improve the outcomes of associated severe acute respiratory illness with fever and respiratory symptoms. This study analyzed plasma concentrations of heat shock protein gp96 in nonsevere (including mild and typical) and severe (including severe and critical) patients with COVID-19 to evaluate its potential as a predictive and prognostic biomarker for disease severity. Plasma gp96 levels that were positively correlated with interleukin-6 (IL-6) levels were significantly elevated in COVID-19 patients admitted to the hospital but not in non-COVID-19 patients with less severe respiratory impairment. Meanwhile, significantly higher gp96 levels were observed in severe than nonsevere patients. Moreover, the continuous decline of plasma gp96 levels predicted disease remission and recovery, whereas its persistently high levels indicated poor prognosis in COVID-19 patients during hospitalization. Finally, monocytes were identified as the major IL-6 producers under exogenous gp96 stimulation. Our results demonstrate that plasma gp96 may be a useful predictive and prognostic biomarker for disease severity and outcome of COVID-19. IMPORTANCE Early and effective identification of severe COVID-19 may allow us to improve the outcomes of associated severe acute respiratory illness with fever and respiratory symptoms. Some heat shock proteins (Hsps) are released during oxidative stress, cytotoxic injury, and viral infection and behave as danger-associated molecular patterns (DAMPs). This study analyzed plasma concentrations of Hsp gp96 in nonsevere and severe patients with COVID-19. Significantly higher plasma gp96 levels were observed in severe than those in nonsevere patients, and its persistently high levels indicated poor prognosis in COVID-19 patients. The results demonstrate that plasma gp96 may be a useful predictive and prognostic biomarker for disease severity and outcome of COVID-19.
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Biomarcadores/sangue , Teste para COVID-19/métodos , COVID-19/diagnóstico , Glicoproteínas de Membrana/sangue , Índice de Gravidade de Doença , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Citocinas/sangue , Feminino , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Monócitos , SARS-CoV-2/isolamento & purificação , Adulto JovemRESUMO
Objective: This study aimed to evaluate the factors associated with death in patients with coronavirus disease 2019 by clarifying the clinical characteristics and immune responses. Methods: The clinical characteristics and laboratory findings, including cytokine and lymphocyte subsets, were obtained from the electronic medical records of patients in Wuhan Tongji Hospital. Results: This study included 836 patients with confirmed COVID-19. In total, 699 (83.6%) were cured and discharged, and 137 (16.4%) died. Our analysis revealed that age ≥ 65 years, male sex, malignancy, chronic obstructive pulmonary disease, dyspnea, dizziness, respiratory rate > 20 bpm, heart rate > 100 bpm, systolic blood pressure < 90 mmHg, neutrophils > 6.3×109/L, lymphopenia, thrombocytopenia, D-dimer ≥ 0.5 mg/L, lactate dehydrogenase > 250 U/L, aspartate aminotransferase > 40 U/L, total bilirubin > 26 µmol/L, albumin < 35 g/L, blood urea nitrogen > 9.5 mmol/L, estimated glomerular filtration rate < 90 ml/min/1.73, elevated cardiac troponin I, N-terminal pro-brain natriuretic peptide ≥ 900 pg/ml, C-reactive protein ≥ 25 mg/L, procalcitonin ≥ 0.05 ng/ml and ferritin > 400 µg/L were associated with death in patients with COVID-19. The multivariate logistic regression analysis revealed that an estimated glomerular filtration rate < 90 ml/min/1.73, elevated cardiac troponin I, C-reactive protein ≥ 25 mg/L and procalcitonin ≥ 0.05 ng/ml were predictive of mortality. Regarding immune responses, IL-2R, IL-6, IL-8, IL-10, and TNFα were remarkably higher in the deceased group at admission, and the levels of IL-2R, IL-6, IL-8, IL-10, and TNFα in the deceased group showed a rapid increase; the dynamics of these cytokines were highly consistent with disease deterioration. Lymphocyte subset analysis revealed that the deceased patients showed significant decreases in lymphocyte counts, especially helper T cells, suppressor T cells and NK cells. Conclusions: This study identified that an estimated glomerular filtration rate < 90 ml/min/1.73, elevated cardiac troponin I, C-reactive protein ≥ 25 mg/L and procalcitonin ≥ 0.05 ng/ml were predictors of mortality in COVID-19 patients. Elevated cytokine levels and a continued increasing trend, including in IL-2R, IL-6, IL-8, IL-10 and TNFα, and a decrease in lymphocyte subsets, especially helper T cells, suppressor T cells and NK cells, were associated with a poor prognosis.
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COVID-19/epidemiologia , Pandemias , SARS-CoV-2 , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína C-Reativa/análise , COVID-19/sangue , COVID-19/imunologia , COVID-19/mortalidade , Doenças Cardiovasculares/epidemiologia , Comorbidade , Citocinas/sangue , Feminino , Humanos , Subpopulações de Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Neoplasias/epidemiologia , Prognóstico , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento , Adulto Jovem , Tratamento Farmacológico da COVID-19RESUMO
Recent studies have identified the curative effects of traditional Chinese medicine for constipation. The mechanism of action of Guiren Runchang granules (GRGs) in the treatment of slow transit constipation (STC) was evaluated in this study. Here, we assessed the efficacy of GRG by comparing the differences in fecal characteristics, stool weight, and intestinal transit rate (ITR) among 6 groups (n = 12/group), which were administered three concentrations of GRG, mosapride, and saline. The influence of GRG on the SCF/c-kit pathway, AQP4, and serum motilin of mice was assessed through ELISA, western blot, and immunohistochemical analysis. The dry weight of mouse feces at 24 hr and ITR in the MD (medium-dose GRG; 9.44 g/kg/d) and HD (high-dose GRG; 18.88 g/kg/d) groups was higher than that in the MC (model control) group. The serum motilin of morphine-induced mice level was lower in the MC group than in the NC (normal control) group, and this condition was improved in the HD group. The HD group expressed significantly higher levels of SCF and c-kit protein but lower levels of AQP4 and simultaneously presented more SCF-positive and c-kit-positive cells. However, no differences in the serum SCF level were found among the six groups. Certain concentrations of GRG are effective in STC mice, the potential mechanism of which may be associated with repairing the SCF/c-kit pathway and reducing the expression of AQP4 in the colon. GRG improved the serum motilin level but had no influence on the serum SCF level.
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Carcinoma de Células em Anel de Sinete/diagnóstico , Neoplasias Gástricas/diagnóstico , Microangiopatias Trombóticas/diagnóstico , Adulto , Biomarcadores , Biópsia , Carcinoma de Células em Anel de Sinete/etiologia , Carcinoma de Células em Anel de Sinete/metabolismo , Diagnóstico Diferencial , Feminino , Humanos , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/metabolismo , Tomografia Computadorizada por Raios XRESUMO
Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technology, will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.
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Aminoácidos/química , Enzimas/química , Enzimas/metabolismo , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/metabolismo , Biocatálise , Domínio Catalítico , Compostos de Diazônio/química , Código Genético , Conformação Molecular , Oxirredução , Processos Fotoquímicos , Engenharia de Proteínas , beta-Alanina/análogos & derivados , beta-Alanina/químicaRESUMO
BACKGROUND: Irritable bowel syndrome (IBS) is a chronic functional bowel disease characterized by abdominal pain and changes in bowel habits in the absence of organic disease. Electroacupuncture (EA) has been shown to alleviate visceral hypersensitivity (VH) in IBS rat models by inhibiting the activation of astrocytes in the spinal cord. However, the underlying molecular mechanisms mediated by P2Y1 receptor of this effect of electroacupuncture remain unclear. AIM: To explore whether EA inhibits the activity of astrocytes in the spinal cord dorsal horn of rat with visceral hypersensitivity by inhibiting P2Y1 receptor and its downstream mitogen activated protein kinase/extracellular regulated kinase 1 (MAPK/ERK) pathway. METHODS: Ten-day-old Sprague-Dawley (SD) male rats were given an intracolonic injection of 0.2 ml of 0.5% acetic acid (AA) to establish a visceral hypersensitivity model. EA was performed at Zusanli (ST 36) and Shangjuxu (ST 37) at 100 Hz for 1.05 s and 2 Hz for 2.85 s alternately, pulse width for 0.1 ms, 1 mA, 30 min/d, once a day, for 1 week. Cytokines IL-6, IL-1ß, and TNF-α were analyzed by ELISA. The expressions of the P2Y1 receptor and pERK1/2 were analyzed by Western Blot and real-time PCR in the model and EA treated animals to explore the molecular mechanism of EA in inhibiting the activity of spinal cord dorsal horn (L6-S2 segment) astrocytes in rats with IBS visceral hypersensitivity. RESULTS: EA significantly reduced the behavioral abdominal withdrawal reflex score (AWRs) of IBS rats with visceral hypersensitivity induced by AA. For comparison, intrathecal injection of astrocytes activity inhibitor fluorocitrate (FCA) also reduced visceral hypersensitivity in IBS rats. EA at Zusanli and Shangjuxu inhibited the mRNA and protein expression of the glial fibrillary acidic protein (GFAP) and in rat spinal cord and reduced the release of inflammatory cytokines IL-6, IL-1, and TNF-α were analyzed by ELISA. The expressions of the P2Y1 receptor and pERK1/2 were analyzed by Western Blot and real-time PCR in the model and EA treated animals to explore the molecular mechanism of EA in inhibiting the activity of spinal cord dorsal horn (L6-S2 segment) astrocytes in rats with IBS visceral hypersensitivity. ß, and TNF-µg, 10 µg, 10 . CONCLUSION: EA inhibited astrocyte activity in the spinal cord dorsal horn of rat with IBS visceral hypersensitivity by inhibiting the P2Y1 receptor and its downstream, PKC, and MAPK/ERK1/2 pathways.
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The biotin-streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17â¯000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.
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Biotina/química , Engenharia Química/métodos , Metaloproteínas/química , Compostos Organometálicos/química , Estreptavidina/química , Sítios de Ligação , Biotina/genética , Biotinilação , Catálise , Cromatografia de Afinidade , Escherichia coli/genética , Hidrogenação , Irídio/química , Cinética , Metaloproteínas/genética , Estereoisomerismo , Estreptavidina/genéticaRESUMO
OBJECTIVES: This study was aimed to develop small molecule inhibitors of the P. aeruginosa heme oxygenase (pa-HemO) as potential treatment of infections caused by P. aeruginosa. METHODS: New compounds were designed based on the crystal structure of pa-HemO. The binding affinities (KD) were determined using intrinsic fluorescence quenching assays. The anti-microbial effects of the new compounds was evaluated by minimal inhibitory concentration 50% (MIC50). RESULTS: Eleven compounds were synthesized as potential pa-HemO inhibitors. New compounds demonstrated KD values ranging from 1.5 to 180⯵M, and MIC50 values ranging from 26 to 260⯵g/mL. The compounds had good affinity with HemO and promising anti-microbial effects on P. aeruginosa. CONCLUSIONS: The new inhibitors described herein can inhibit the growth of P. aeruginosa via the inhibition of pa-HemO. There may be broad prospects for HemO inhibitors to treat P. aeruginosa related infections.
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Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Antibacterianos/síntese química , Antibacterianos/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Pseudomonas aeruginosa/enzimologiaRESUMO
Macrophages, whether M1 or M2 subtype, have been found to be implicated in the pathogenesis of osteoarthritis (OA). However, no study regarding the status of M1 and M2 macrophages has been reported in knee OA. To investigate the status of M1 and M2 macrophages in knee OA, synovial fluid as well as peripheral blood were collected from 80 patients with knee OA and 80 healthy controls. Reverse transcription-quantitative PCR was used to quantitatively detect the expression of CD11c as a marker for M1 macrophages and CD206 as a marker for M2 macrophages from synovial fluids. As confirmation, flow cytometry was employed to count the number of monocytes from whole blood using the CD86 (M1) and CD163 (M2) markers on monocytes. The ratio of M1 to M2 macrophages was shown to be markedly higher in knee OA than that of control and that the ratio was significantly positively correlated with level of Kellgren-Lawrence grade in knee OA, that is, the higher the ratio the more severe the knee OA seems to be. Thus, our study presented direct evidence for the involvement of macrophages in the pathogenesis of knee OA.
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BACKGROUND: Cystic fibrosis (CF) is a disease characterized by chronic airway infection with a high incidence and poor prognosis. Pseudomonas aeruginosa and Aspergillus fumigatus are pathogens commonly found in CF patients. Clinically, these two microorganisms often coexist in the airway of CF patients. Combined infection with P. aeruginosa and A. fumigatus results in worsening lung function and clinical condition. METHODS: In this review, we focus on the mutual inhibition and promotion mechanisms of P. aeruginosa and A. fumigatus in CF patients. We also summarized the mechanisms of the interaction between these pathogenic microorganisms. RESULTS: P. aeruginosa inhibits A. fumigatus growth through the effects of phenazines, the quorum sensing system, iron competition, bacteriophages, and small colony variants. P. aeruginosa induces A. fumigatus growth through volatile organic compounds and subbacteriostatic concentrations of phenazines. A. fumigatus interferes with P. aeruginosa, affecting its metabolic growth via phenazine metabolic transformation, gliotoxin production, and reduced antibiotic sensitivity. DISCUSSION: Coexistence of P. aeruginosa and A. fumigatus can lead to both mutual inhibition and promotion. In different stages of CF disease, the interaction between these two pathogenic microorganisms may shift between promotion and inhibition. A discussion of the mechanisms of P. aeruginosa and A. fumigatus interaction can be beneficial for further treatment of CF patients and for improving the prognosis of the disease.