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1.
J Transl Med ; 22(1): 272, 2024 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-38475878

RESUMO

BACKGROUND: In HBV-associated HCC, T cells often exhibit a state of functional exhaustion, which prevents the immune response from rejecting the tumor and allows HCC to progress. Moreover, polymerase-specific T cells exhibit more severe T-cell exhaustion compared to core-specific T cells. However, whether HBV DNA polymerase drives HBV-specific CD8+ T cell exhaustion in HBV-related HCC remains unclear. METHODS: We constructed a Huh7 cell line stably expressing HA-HBV-DNA-Pol and applied co-culture systems to clarify its effect on immune cell function. We also examined how HBV-DNA-Pol modulated PD-L1 expression in HCC cells. In addition, HBV-DNA-Pol transgenic mice were used to elucidate the underlying mechanism of HBV-DNA-Pol/PD-L1 axis-induced T cell exhaustion. RESULTS: Biochemical analysis showed that Huh7 cells overexpressing HBV-DNA-Pol inhibited the proliferation, activation, and cytokine secretion of Jurkat cells and that this effect was dependent on their direct contact. A similar inhibitory effect was observed in an HCC mouse model. PD-L1 was brought to our attention during screening. Our results showed that the overexpression of HBV-DNA-Pol upregulated PD-L1 mRNA and protein expression. PD-L1 antibody blockade reversed the inhibitory effect of Huh7 cells overexpressing HBV-DNA-Pol on Jurkat cells. Mechanistically, HBV-DNA-Pol interacts with PARP1, thereby inhibiting the nuclear translocation of PARP1 and further upregulating PD-L1 expression. CONCLUSIONS: Our findings suggest that HBV-DNA-Pol can act as a regulator of PD-L1 in HCC, thereby directing anti-cancer immune evasion, which further provides a new idea for the clinical treatment of liver cancer.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Vírus da Hepatite B/genética , DNA Viral , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos , DNA Polimerase Dirigida por DNA/metabolismo
2.
Pediatr Res ; 95(1): 227-232, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37580551

RESUMO

BACKGROUND: children who undergo CPB operations are at an elevated risk of infection due to immunosuppression. This study aims to investigate the association between lymphopenia following CPB and early postoperative infection in children. METHODS: A retrospective analysis including 41 children under 2 years old underwent CPB. Among them, 9 subjects had an early postoperative infection, and 32 subjects were period-matched without infection. Inflammatory cytokines, serum CRP and PCT values were measured in plasma, additionally, circulating total leucocyte and lymphocyte subpopulations were counted. RESULTS: Infected subjects exhibited significantly higher levels of inflammatory cytokines, including IL-6, IL-8, IL-10, IL-1ß and TNF-α, than non-infected subjects after CPB. Additionally, lower absolute number of lymphocyte and their subpopulations CD3+ T cells, CD4+ T-helper cells and CD8+cytotoxic T-cells, were observed in infected subjects. The impairment of T-cells Immune was found to be associated with higher levels of inflammatory cytokines IL-10. The ROC demonstrated that the absolute number of CD3+ T-cells <1934/ul, CD4+ T helper cells <1203/ul and CD8+cytotoxic T-cells <327/ul were associated with early postoperative infection. CONCLUSION: Higher levels of inflammatory cytokines resulted in T-cells lymphopenia after CPB, which significantly increasing the risk of postoperative infection in infants and young children. IMPACT: Infection complications after cardiopulmonary bypass (CPB) in pediatric CHD patients are serious issues, identifing the infection from after CPB remains a challenging. CPB can release numerous inflammatory cytokines associated with T cells lymphopenia, which increases the risk of postoperative infection after surgery. Monitoring T cells lymphopenia maybe more beneficial to predict early postoperative infection than C-reactive protein and procalcitonin.


Assuntos
Ponte Cardiopulmonar , Linfopenia , Lactente , Humanos , Criança , Pré-Escolar , Ponte Cardiopulmonar/efeitos adversos , Interleucina-10 , Estudos Retrospectivos , Citocinas , Linfócitos T , Linfopenia/etiologia
3.
Exp Cell Res ; 433(2): 113823, 2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-37890607

RESUMO

Breast carcinoma (BC) is one of the most common malignant cancers in females, and metastasis remains the leading cause of death in these patients. Chemotaxis plays an important role in cancer cell metastasis and the mechanism of breast cancer chemotaxis has become a central issue in contemporary research. PKCζ, a member of the atypical PKC family, has been reported to be an essential component of the EGF-stimulated chemotactic signaling pathway. However, the molecular mechanism through which PKCζ regulates chemotaxis remains unclear. Here, we used a proteomic approach to identify PKCζ-interacting proteins in breast cancer cells and identified VASP as a potential binding partner. Intriguingly, stimulation with EGF enhanced this interaction and induced the translocalization of PKCζ and VASP to the cell membrane. Further experiments showed that PKCζ catalyzes the phosphorylation of VASP at Ser157, which is critical for the biological function of VASP in regulating chemotaxis and actin polymerization in breast cancer cells. Furthermore, in PKCζ knockdown BC cells, the enrichment of VASP at the leading edge was reduced, and its interaction with profilin1 was attenuated, thereby reducing the chemotaxis and overall motility of breast cancer cells after EGF treatment. In functional assays, PKCζ promoted chemotaxis and motility of BC cells through VASP. Our findings demonstrate that PKCζ, a new kinase of VASP, plays an important role in promoting breast cancer metastasis and provides a theoretical basis for expanding new approaches to tumor biotherapy.


Assuntos
Neoplasias da Mama , Quimiotaxia , Proteína Quinase C , Feminino , Humanos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Quimiotaxia/genética , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteômica
4.
Int J Mol Sci ; 25(4)2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38396759

RESUMO

Caragana, a xerophytic shrub genus widely distributed in northern China, exhibits distinctive geographical substitution patterns and ecological adaptation diversity. This study employed transcriptome sequencing technology to investigate 12 Caragana species, aiming to explore genic-SSR variations in the Caragana transcriptome and identify their role as a driving force for environmental adaptation within the genus. A total of 3666 polymorphic genic-SSRs were identified across different species. The impact of these variations on the expression of related genes was analyzed, revealing a significant linear correlation (p < 0.05) between the length variation of 264 polymorphic genic-SSRs and the expression of associated genes. Additionally, 2424 polymorphic genic-SSRs were located in differentially expressed genes among Caragana species. Through weighted gene co-expression network analysis, the expressions of these genes were correlated with 19 climatic factors and 16 plant functional traits in various habitats. This approach facilitated the identification of biological processes associated with habitat adaptations in the studied Caragana species. Fifty-five core genes related to functional traits and climatic factors were identified, including various transcription factors such as MYB, TCP, ARF, and structural proteins like HSP90, elongation factor TS, and HECT. The roles of these genes in the ecological adaptation diversity of Caragana were discussed. Our study identified specific genomic components and genes in Caragana plants responsive to heterogeneous habitats. The results contribute to advancements in the molecular understanding of their ecological adaptation, lay a foundation for the conservation and development of Caragana germplasm resources, and provide a scientific basis for plant adaptation to global climate change.


Assuntos
Caragana , Caragana/genética , Perfilação da Expressão Gênica/métodos , Transcriptoma , Genes de Plantas , Fenótipo , Repetições de Microssatélites
5.
Echocardiography ; 40(3): 244-251, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36808139

RESUMO

OBJECTIVES: This study aimed to evaluate whether fetal echocardiographic parameters were predictive of the postnatal surgical treatment required for fetuses with Tetralogy of Fallot (TOF). METHODS: The fetal echocardiographic and postnatal clinical data of all cases of prenatal TOF at Xinhua Hospital from 2016 to 2020 were reviewed. Patients were categorized based on the operation type, and cardiac parameters were compared between groups. RESULTS: Of the 37 fetuses assessed, the development of the pulmonary valve annulus (PVA) was significantly poorer in the transannular patch group. Patients with a prenatal PVA z-score (Schneider's method) ≥ -2.645, a PVA z-score (Lee's method) ≥ -2.805, a PVA/aortic valve annulus diameter ratio ≥ .697, and a pulmonary annulus index ≥ .823 were more likely to undergo pulmonary valve-sparing surgery. There was a strong correlation between prenatal and postnatal PVA z-scores. The PVA growth potential was greater in the pulmonary valve-sparing surgery group. CONCLUSIONS: PVA-related parameters evaluated by fetal echocardiography can predict the type of surgical intervention required and are valuable in improving prenatal counseling in fetal cases of TOF.


Assuntos
Procedimentos Cirúrgicos Cardíacos , Valva Pulmonar , Tetralogia de Fallot , Feminino , Humanos , Gravidez , Tetralogia de Fallot/cirurgia , Estudos Retrospectivos , Valva Pulmonar/diagnóstico por imagem , Ecocardiografia , Resultado do Tratamento
6.
Arch Microbiol ; 204(9): 551, 2022 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-35951138

RESUMO

The enzymes are biological macromolecules that biocatalyze certain biochemical reactions without undergoing any modification or degradation at the end of the reaction. In this work, we constructed a recombinant novel Raoultella sp. NX-TZ-3-15 strain that produces heparinase with a maltose binding tag to enhance its production and activity. Additionally, MBP-heparinase was purified and its enzymatic capabilities are investigated to determine its industrial application. Moreover, the recombinant plasmid encoding the MBP-heparinase fusion protein was effectively generated and purified to a high purity. According to SDS-PAGE analysis, the MBP-heparinase has a molecular weight of around 70 kDa and the majority of it being soluble with a maximum activity of 5386 U/L. It has also been noted that the three ions of Ca2 + , Co2 + , and Mg2 + can have an effect on heparinase activities, with Mg2 + being the most noticeable, increasing by about 85%, while Cu2 + , Fe2 + , Zn2 + having an inhibitory effect on heparinase activities. Further investigations on the mechanistic action, structural features, and genomes of Raoultella sp. NX-TZ-3-15 heparinase synthesis are required for industrial-scale manufacturing.


Assuntos
Escherichia coli , Polissacarídeo-Liases , Enterobacteriaceae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Heparina Liase/química , Heparina Liase/genética , Heparina Liase/metabolismo , Plasmídeos/genética , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo
7.
Molecules ; 26(22)2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34833855

RESUMO

Ultrasound-assisted extraction (UAE) was used to extract carotenoids from the carrot pomace. To investigate the effect of independent variables on the UAE, the response surface methodology (RSM) with central-composite design (CCD) was employed. The study was conducted with three independent variables including extraction time (min), temperature (°C), and ethanol concentration (%). The results showed that the optimal conditions for UAE were achieved with an extraction time of 17 min, temperature of 32 °C, and ethanol concentration of 51% of total carotenoids (31.82 ± 0.55); extraction time of 16 min, temperature of 29 °C, and ethanol concentration of 59% for a combination of ß-carotene (14.89 ± 0.40), lutein (5.77 ± 0.19), and lycopene (2.65 ± 0.12). The non-significant (p > 0.05) correlation under optimal extraction conditions between predicted and experimental values suggested that UAE is the more productive process than conventional techniques for the extraction of carotenoids from the carrot pomace.


Assuntos
Carotenoides/isolamento & purificação , Daucus carota/química , Sonicação , Carotenoides/química
8.
Proc Biol Sci ; 287(1928): 20200675, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32486982

RESUMO

Biotic mechanisms associated with species diversity are expected to stabilize communities in theoretical and experimental studies but may be difficult to detect in natural communities exposed to large environmental variation. We investigated biotic stability mechanisms in a multi-site study across Inner Mongolian grassland characterized by large spatial variations in species richness and composition and temporal fluctuations in precipitation. We used a new additive-partitioning method to separate species synchrony and population dynamics within communities into different species-abundance groups. Community stability was independent of species richness but was regulated by species synchrony and population dynamics, especially of abundant species. Precipitation fluctuations synchronized population dynamics within communities, reducing their stability. Our results indicate generality of biotic stability mechanisms in natural ecosystems and suggest that for accurate predictions of community stability in changing environments uneven species composition should be considered by partitioning stabilizing mechanisms into different species-abundance groups.


Assuntos
Pradaria , Animais , Biodiversidade , China , Ecossistema , Gerbillinae , Dinâmica Populacional
9.
Crit Rev Biotechnol ; 40(3): 365-379, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948287

RESUMO

Chitosan, obtained as a result of the deacetylation of chitin, one of the most important naturally occurring polymers, has antimicrobial properties against fungi, and bacteria. It is also useful in other fields, including: food, biomedicine, biotechnology, agriculture, and the pharmaceutical industries. A literature survey shows that its antimicrobial activity depends upon several factors such as: the pH, temperature, molecular weight, ability to chelate metals, degree of deacetylation, source of chitosan, and the type of microorganism involved. This review will focus on the in vitro and in vivo antimicrobial properties of chitosan and its derivatives, along with a discussion on its mechanism of action during the treatment of infectious animal diseases, as well as its importance in food safety. We conclude with a summary of the challenges associated with the uses of chitosan and its derivatives.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Quitina/química , Quitosana/química , Quitosana/farmacologia , Doenças dos Animais/tratamento farmacológico , Animais , Bactérias/efeitos dos fármacos , Biotecnologia , Bovinos , Terapia por Quelação , Indústria Alimentícia , Inocuidade dos Alimentos , Fungos , Concentração de Íons de Hidrogênio , Ostreidae/efeitos dos fármacos , Temperatura , Indústria Têxtil
10.
Appl Microbiol Biotechnol ; 103(4): 1557-1571, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30607489

RESUMO

Chitosan is a naturally occurring biodegradable as well as a non-toxic polymer generated from chitin through alkaline deacetylation reaction, and it is insoluble in organic/inorganic solvents and water. Furthermore, chitosan is one of the most plentiful cationic polymers in natural surroundings. Due to its non-toxicity and biocompatibility, chitosan is extensively employed in industrial, biomedical, food, pharmaceutical, environmental, and agricultural industry. Chitosan-based biomaterials exhibit great potential in various biotechnological applications, such as anti-hypertensive therapy, anti-oxidant, anti-microbial, anti-allergic, immunostimulant, cancer therapy, delivery of genetic materials, delivery of bone morphogenetic type-2, wound healing, treatment of wastewater, hypocholesterolemic, and bio-imaging. Therefore, this review mainly focuses on the biotechnological potential of chitosan and its derivatives as well as presents the potential of chitosan-based biomaterial/pharmaceutical for the prevention of various life-threating chronic disorders.


Assuntos
Anticolesterolemiantes/metabolismo , Materiais Biocompatíveis/metabolismo , Biotecnologia/métodos , Quitosana/metabolismo , Tecnologia Farmacêutica/métodos
11.
Prenat Diagn ; 39(13): 1198-1203, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31600411

RESUMO

OBJECTIVES: Isolated prenatal ventricular disproportion with a dominant right ventricle represents a challenge in decision-making for both physicians and pregnant women. In the current study, we sought to delineate the postnatal outcomes of these cases. METHODS: This retrospective analysis included consecutive cases of isolated ventricular disproportion identified using complete fetal echocardiography at the Fetal Heart Center of Xinhua Hospital from January 2014 to October 2017. Postnatal cardiac outcome was examined using transthoracic echocardiography within the first 6 months after birth. RESULTS: A total of 90 fetuses were included in the final analysis. The median gestational age (GA) at diagnosis was 29 weeks (range 24 to 36). At postnatal examination, cardiac malformations were detected in 39 cases (43.3%), including 25 (27.8%) cases of congenital cardiac septal defects, eight (8.9%) of persistent left superior vena cava, four (4.4%) of left-sided obstructive diseases, and one (1.1%) case of coronary fistula. Nineteen cases (21.1%) with fetal cardiac malformations had significant lower GA at diagnosis (P = .01) and greater right to left ventricle ratio (1.38 vs 1.30, P = .02). Neonatal surgical intervention was not required in any of the cases. CONCLUSIONS: Isolated prenatal ventricular disproportion with a dominant right ventricle comprises minor postnatal cardiac malformations and doesn't require neonatal intervention.


Assuntos
Coração Fetal/diagnóstico por imagem , Cardiopatias Congênitas/diagnóstico por imagem , Adulto , Ecocardiografia , Feminino , Ventrículos do Coração/diagnóstico por imagem , Humanos , Gravidez , Resultado da Gravidez , Estudos Retrospectivos , Ultrassonografia Pré-Natal
12.
Artigo em Inglês | MEDLINE | ID: mdl-30150473

RESUMO

ß-Lactam antibiotics are the mainstay for the treatment of bacterial infections. However, elevated resistance to these antibiotics mediated by metallo-ß-lactamases (MBLs) has become a global concern. New Delhi metallo-ß-lactamase-1 (NDM-1), a newly added member of the MBL family that can hydrolyze almost all ß-lactam antibiotics, has rapidly spread all over the world and poses serious clinical threats. Broad-spectrum and mechanism-based inhibitors against all MBLs are highly desired, but the differential mechanisms of MBLs toward different antibiotics pose a great challenge. To facilitate the design of mechanism-based inhibitors, we investigated the active-site conformational changes of NDM-1 through the determination of a series of 15 high-resolution crystal structures in native form and in complex with products and by using biochemical and biophysical studies, site-directed mutagenesis, and molecular dynamics computation. The structural studies reveal the consistency of the active-site conformations in NDM-1/product complexes and the fluctuation in native NDM-1 structures. The enzymatic measurements indicate a correlation between enzymatic activity and the active-site fluctuation, with more fluctuation favoring higher activity. This correlation is further validated by structural and enzymatic studies of the Q123G mutant. Our combinational studies suggest that active-site conformational fluctuation promotes the enzymatic activity of NDM-1, which may guide further mechanism studies and inhibitor design.


Assuntos
beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Escherichia coli/metabolismo , Humanos , Conformação Proteica/efeitos dos fármacos
13.
Microb Pathog ; 125: 12-19, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30201590

RESUMO

Edwardsiella tarda poses a threat to human health and has resulted in enormous economic losses in aquaculture. Low temperatures are usually applied to contain the growth of this microorganism. In this study, stable isotope labelling by amino acids in cell culture (SILAC) was used to conduct comparative proteomic quantitation of E. tarda ATCC 15947 under cold stress for two weeks. We identified 1391 proteins, of which 898 were quantifiable. Of these, 72 proteins were upregulated and 164 were downregulated in response to cold stress. Even though E. tarda ATCC 15947 is not a psychrophile, several key proteins related to DNA synthesis and transcription were significantly upregulated. Additionally, proteins related to haemolytic activities and gluconeogenesis were upregulated, even though E. tarda ATCC 15497 is considered non-virulent in aquaculture. This study therefore delineated the specific proteomic response of this E. tarda ATCC 15947 to prolonged cold stress.


Assuntos
Aminoácidos/metabolismo , Resposta ao Choque Frio , Edwardsiella tarda/fisiologia , Edwardsiella tarda/efeitos da radiação , Marcação por Isótopo/métodos , Proteômica/métodos , Técnicas de Cultura de Células/métodos
14.
Appl Microbiol Biotechnol ; 102(2): 703-712, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29184989

RESUMO

Avermectins, produced by Streptomyces avermitilis, are important antiparasitic agents. The use of traditional microbial breeding methods for this organism has been limited by the low-throughput shake flask-based screening process. The unique growth cycle of actinomycetes makes the establishment of a reliable high-throughput screening (HTS) process difficult. To enhance the efficiency of screening strains with high yields of avermectin, a HTS process aided by fluorescence-activated cell sorting (FACS) was established. Four different spore solutions were investigated for maintaining a relatively high viability of spores. Propidium iodide (PI) and fluorescein diacetate (FDA) were used to discriminate between dead and live spores using the FACS system. Spores stained with 7-µg/mL PI and 15-µg/mL FDA at 4 °C in the dark for 30 min resulted in optimum sorting. Spores were treated by atmospheric and room temperature plasma (ARTP). Single live spores were sorted and sprayed into 96-well microtiter plates containing 50 µL of solid agar culture medium. Solid-liquid combinatorial microculture was used for high-throughput avermectin culture. A high-titer avermectin producer (G9) was obtained from 5760 mutants after mutagenesis and HTS. Compared with the original strain, the titer was improved by 18.9% on flask culture and 20.6% on fermenter, respectively. The HTS process established in this study could easily be transferred to other similar target products produced by actinomycetes.


Assuntos
Antiprotozoários/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Ivermectina/análogos & derivados , Streptomyces/metabolismo , Reatores Biológicos , Citometria de Fluxo , Corantes Fluorescentes , Regulação Bacteriana da Expressão Gênica , Ivermectina/metabolismo , Mutagênese , Esporos , Streptomyces/genética
15.
Appl Microbiol Biotechnol ; 102(19): 8275-8289, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30066188

RESUMO

The age-related changes in the diversity and composition of the gut microbiota are well described in recent studies. These changes have been suggested to be influenced by age-associated weakening of the immune system and low-grade chronic inflammation, resulting in numerous age-associated pathological conditions. Gut microbiota homeostasis is important throughout the life of the host by providing vital functions to regulate various immunological functions and homeostasis. Based on published results, we summarize the relationship between the gut microbiota and aging-related diseases, especially Parkinson's disease, immunosenescence, rheumatoid arthritis, bone loss, and metabolic syndrome. The change in composition of the gut microbiota and gut ecosystem during life and its influence on the host immunologic and metabolic phenotype are also analyzed to determine factors that affect aging-related diseases. Approaches to maintain host health and prevent or cure geriatric diseases are also discussed.


Assuntos
Envelhecimento/patologia , Microbioma Gastrointestinal/fisiologia , Animais , Artrite Reumatoide/microbiologia , Artrite Reumatoide/patologia , Homeostase/fisiologia , Humanos , Inflamação/microbiologia , Inflamação/patologia
16.
Curr Microbiol ; 75(1): 20-26, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28831596

RESUMO

Vibrio parahaemolyticus is a kind of food-borne pathogenic bacterium, which can seriously infect food, especially seafood causing gastroenteritis and other disease. We studied the global proteome responses of V. parahaemolyticus under cold stress by nano-liquid chromatography-tandem mass spectrometry to improve the present understanding of V. parahaemolyticus proteomics events under cold stress. A total of 1151 proteins were identified and 101 proteins were differentially expressed, of which 69 were significantly up-regulated and 32 were downregulated. Functional categorization of these proteins revealed distinct differences between cold-stressed and control cells. These proteins were grouped into 21 functional categories by the clusters of orthologous groups (COG) analysis. The most of up-regulated proteins were functionally categorized as nucleotide transport and metabolism, transcription, function unknown, and defense mechanisms. These up-regulated proteins play an important role under cold stress.


Assuntos
Proteínas de Bactérias/química , Vibrio parahaemolyticus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Perfilação da Expressão Gênica , Humanos , Espectrometria de Massas , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Proteômica , Vibrioses/microbiologia , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificação
17.
Immunology ; 151(1): 43-55, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28092402

RESUMO

Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation-induced hepatitis is unknown. In this study, using HBV transgenic (HBs-Tg) and HBs-Tg T-cell receptor-δ-deficient (TCR-δ-/- ) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon-γ (IFN-γ) levels and more extensive necrosis. γδ T-cell deficiency resulted in elevated IFN-γ in CD4+ T cells but not in natural killer or natural killer T cells. The depletion of CD4+ T cells and neutralization of IFN-γ reduced liver damage in HBs-Tg and HBs-Tg-TCR-δ-/- mice to a similar extent. Further investigation revealed that HBs-Tg mice showed an enhanced interleukin-17 (IL-17) signature. The administration of exogenous IL-23 enhanced IL-17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs-Tg mice, but not in HBs-Tg-TCR-δ-/- mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A-induced hepatitis by inhibiting IFN-γ production from CD4+ T cells and are indispensable for IL-23-mediated protection against Con A-induced hepatitis in HBs-Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation-induced liver damage.


Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatite B/imunologia , Fígado/patologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Concanavalina A/imunologia , Hepatite B/terapia , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-23/uso terapêutico , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Necrose , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/virologia
18.
Biotechnol Lett ; 39(4): 567-575, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28044225

RESUMO

OBJECTIVE: To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. RESULTS: A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). CONCLUSION: The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.


Assuntos
Aminoácidos/química , Códon , Mutagênese Sítio-Dirigida/métodos , Mutagênese , Engenharia de Proteínas/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Mutação , Oligonucleotídeos/química , Plasmídeos/genética , Reação em Cadeia da Polimerase , Transformação Genética
19.
BMC Biotechnol ; 16(1): 49, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27255274

RESUMO

BACKGROUND: Microalgae have been recognized as a good food source of natural biologically active ingredients. Among them, the green microalga Euglena is a very promising food and nutritional supplements, providing high value-added poly-unsaturated fatty acids, paramylon and proteins. Different culture conditions could affect the chemical composition and food quality of microalgal cells. However, little information is available for distinguishing the different cellular changes especially the active ingredients including poly-saturated fatty acids and other metabolites under different culture conditions, such as light and dark. RESULTS: In this study, together with fatty acid profiling, we applied a gas chromatography-mass spectrometry (GC-MS)-based metabolomics to differentiate hetrotrophic and mixotrophic culture conditions. CONCLUSIONS: This study suggests metabolomics can shed light on understanding metabolomic changes under different culture conditions and provides a theoretical basis for industrial applications of microalgae, as food with better high-quality active ingredients.


Assuntos
Reatores Biológicos/microbiologia , Suplementos Nutricionais/microbiologia , Euglena/metabolismo , Ácidos Graxos/metabolismo , Metaboloma/fisiologia , Microalgas/metabolismo , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Euglena/classificação , Análise do Fluxo Metabólico/métodos , Microalgas/classificação , Especificidade da Espécie
20.
J Immunol ; 192(12): 5599-609, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24842756

RESUMO

The differentiation and function of IL-17-producing Th17 cells are tightly regulated by specific transcription factors and cytokines, which are the key participants in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Although specific miRNAs have been shown to be involved in the development of MS and EAE, the potential role of miRNAs in the context of Th17-driven autoimmunity is just beginning to be clarified. miR-20b has been reported as a downregulated miRNA in blood cells of MS patients. In this report, it was further studied in greater detail because we found it was significantly downregulated during EAE, and, in the in vitro differentiation model, Th17 cells had lower expression of miR-20b than did Th1, Th2, or inducible T regulatory cells. Ectopic expression of miR-20b repressed Th17 differentiation in vitro. Using lentiviral vectors for miR-20b overexpression in vivo, we demonstrated that overexpression of miR-20b led to decreased Th17 cells and reduced severity of EAE. Furthermore, we also identified both RAR-related orphan receptor γt and STAT3 as potential targets of miR-20b. Finally, we confirmed that the mild disease severity and low number of Th17 cells in LV-miR-20b-infected mice were largely reversed by coinfection of these mice with lentivirus-expressing RAR-related orphan receptor γt or STAT3 3'-untranslated regions. Taken together, our results contribute to the importance of miRNAs in Th17 differentiation and pathogenesis of MS and EAE.


Assuntos
Diferenciação Celular/imunologia , Encefalomielite Autoimune Experimental/imunologia , MicroRNAs/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Fator de Transcrição STAT3/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Fator de Transcrição STAT3/genética , Células Th17/patologia
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