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Virus-like particles (VLPs) is one of the most favourable subjects of study, especially in the field of nanobiotechnology and vaccine development because they possess good immunogenicity and self-adjuvant properties. Conventionally, VLPs can be tagged and purified using affinity chromatography or density gradient ultracentrifugation which is costly and time-consuming. Turnip yellow mosaic virus (TYMV) is a plant virus, where expression of the viral coat protein (TYMVc) in Escherichia coli (E. coli) has been shown to form VLP. In this study, we report a non-chromatographic method for VLP purification using C-terminally His-tagged TYMVc (TYMVcHis6) as a protein model. Firstly, the TYMVcHis6 was cloned and expressed in E. coli. Upon clarification of cell lysate, nickel (II) chloride [NiCl2; 15 µM or equivalent to 0.0000194% (w/v)] was added to precipitate TYMVcHis6. Following centrifugation, the pellet was resuspended in buffer containing 1 mM EDTA to chelate Ni2+, which is then removed via dialysis. A total of 50% of TYMVcHis6 was successfully recovered with purity above 0.90. Later, the purified TYMVcHis6 was analysed with sucrose density ultracentrifugation, dynamic light scattering (DLS), and transmission electron microscopy (TEM) to confirm VLP formation, which is comparable to TYMVcHis6 purified using the standard immobilized metal affinity chromatography (IMAC) column. As the current method omitted the need for IMAC column and beads while significantly reducing the time needed for column washing, nickel affinity precipitation represents a novel method for the purification of VLPs displaying poly-histidine tags (His-tags).
Assuntos
Brassica napus , Tymovirus , Humanos , Níquel/química , Níquel/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cromatografia de Afinidade/métodosRESUMO
MicroRNAs (miRNAs) are a family of small, single-stranded, and non-protein coding RNAs about 19 to 22 nucleotides in length, that have been reported to have important roles in the control of bone development. MiRNAs have a strong influence on osteoblast differentiation through stages of lineage commitment and maturation, as well as via controlling the activities of osteogenic signal transduction pathways. Generally, miRNAs may modulate cell stemness, proliferation, differentiation, and apoptosis by binding the 3'-untranslated regions (3'-UTRs) of the target genes, which then can subsequently undergo messenger RNA (mRNA) degradation or protein translational repression. MiRNAs manage the gene expression in osteogenic differentiation by regulating multiple signalling cascades and essential transcription factors, including the transforming growth factor-beta (TGF-ß)/bone morphogenic protein (BMP), Wingless/Int-1(Wnt)/ß-catenin, Notch, and Hedgehog signalling pathways; the Runt-related transcription factor 2 (RUNX2); and osterix (Osx). This shows that miRNAs are essential in regulating diverse osteoblast cell functions. TGF-ßs and BMPs transduce signals and exert diverse functions in osteoblastogenesis, skeletal development and bone formation, bone homeostasis, and diseases. Herein, we highlighted the current state of in vitro and in vivo research describing miRNA regulation on the canonical TGF-ß/BMP signalling, their effects on osteoblast linage, and understand their mechanism of action for the development of possible therapeutics. In this review, particular attention and comprehensive database searches are focused on related works published between the years 2000 to 2022, using the resources from PubMed, Google Scholar, Scopus, and Web of Science.
Assuntos
MicroRNAs , Fator de Crescimento Transformador beta , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Proteínas Hedgehog/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/genética , Transdução de Sinais , Osteoblastos/metabolismoRESUMO
The current influenza vaccines only confer protection against the circulating influenza subtypes, therefore universal vaccines are needed to prevent upcoming influenza outbreaks caused by emerging influenza subtypes. The extracellular domain of influenza A M2 protein (M2e) is highly conserved among different subtypes of influenza A viruses, and it is able to elicit protective immunity against the viruses. The influenza nucleoprotein (NP) was used to display the M2e in this study due to its promising T-cell response and adjuvanticity. The M2e gene was fused to the 5'-end of the NP gene and then cloned into pRSET B vector. The DNA sequencing analysis revealed six point mutations in the M2e-NP fusion gene, including one mutation in the M2e peptide and five mutations in the NP. The mutations were reverted using PCR site-directed mutagenesis. The recombinant plasmids (pRSET B-M2e-NP and pRSET B-mM2e-NP) were introduced into Escherichia coli (E. coli) BL21 (DE3) for protein expression. The mutated and non-mutated proteins were subsequently expressed and named mM2e-NP and M2e-NP, respectively. The expression of mM2e-NP and M2e-NP was not affected by the mutations. The binding of anti-M2e antibody to the purified native mM2e-NP and M2e-NP also remained active. However, when the anti-NP antibody was tested, the signal produced by mM2e-NP was very weak. The results implied that the amino acid changes in the NP had adversely impacted on the conformation of mM2e-NP and subsequently affected the antibody binding. In light of the remarkable antibody binding to the M2e-NP fusion protein, this study highly recommends the potential of M2e-NP as a universal influenza vaccine candidate.
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Discovery of conserved antigens for universal influenza vaccines warrants solutions to a number of concerns pertinent to the currently licensed influenza vaccines, such as annual reformulation and mismatching with the circulating subtypes. The latter causes low vaccine efficacies, and hence leads to severe disease complications and high hospitalization rates among susceptible and immunocompromised individuals. A universal influenza vaccine ensures cross-protection against all influenza subtypes due to the presence of conserved epitopes that are found in the majority of, if not all, influenza types and subtypes, e.g., influenza matrix protein 2 ectodomain (M2e) and nucleoprotein (NP). Despite its relatively low immunogenicity, influenza M2e has been proven to induce humoral responses in human recipients. Influenza NP, on the other hand, promotes remarkable anti-influenza T-cell responses. Additionally, NP subunits are able to assemble into particles which can be further exploited as an adjuvant carrier for M2e peptide. Practically, the T-cell immunodominance of NP can be transferred to M2e when it is fused and expressed as a chimeric protein in heterologous hosts such as Escherichia coli without compromising the antigenicity. Given the ability of NP-M2e fusion protein in inducing cross-protective anti-influenza cell-mediated and humoral immunity, its potential as a universal influenza vaccine is therefore worth further exploration.
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The present study aimed to investigate the antioxidant and anti-inflammatory properties of defatted kenaf seed meal (DKSM) and its phenolic-saponin-rich extract (PSRE) in hypercholesterolemic rats. Hypercholesterolemia was induced using atherogenic diet feeding, and dietary interventions were conducted by incorporating DKSM (15% and 30%) or PSRE (at 2.3% and 4.6%, resp., equivalent to the total content of DKSM-phenolics and saponins in the DKSM groups) into the atherogenic diets. After ten weeks of intervention, serum total antioxidant capacities of hypercholesterolemic rats were significantly enhanced by DKSM and PSRE supplementation (p < 0.05). Similarly, DKSM and PSRE supplementation upregulated the hepatic mRNA expression of antioxidant genes (Nrf2, Sod1, Sod2, Gsr, and Gpx1) of hypercholesterolemic rats (p < 0.05), except for Gpx1 in the DKSM groups. The levels of circulating oxidized LDL and proinflammatory biomarkers were also markedly suppressed by DKSM and PSRE supplementation (p < 0.05). In aggregate, DKSM and PSRE attenuated the hypercholesterolemia-associated oxidative stress and systemic inflammation in rats, potentially by enhancement of hepatic endogenous antioxidant defense via activation of the Nrf2-ARE pathway, which may be contributed by the rich content of phenolics and saponins in DKSM and PSRE. Hence, DKSM and PSRE are prospective functional food ingredients for the potential mitigation of atherogenic risks in hypercholesterolemic individuals.
Assuntos
Antioxidantes/metabolismo , Hibiscus/química , Hipercolesterolemia/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Biomarcadores/sangue , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Hibiscus/metabolismo , Hipercolesterolemia/patologia , Inflamação/prevenção & controle , Lipoproteínas LDL/sangue , Fígado/metabolismo , Masculino , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fenóis/química , Fenóis/farmacologia , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Saponinas/química , Saponinas/farmacologia , Sementes/química , Sementes/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Mesenchymal stem cells (MSCs) are involved in bone formation in the embryo, bone repair and remodeling. The differentiation of these cells is a complex multistep pathway that involves discrete cellular transitions and is similar to that which occurs during hematopoiesis. MSCs have self-renewal capacity without differentiation in long-term culture. In the present study, MSCs were isolated from human bone marrow and characterized by the presence of cluster of differentiation 105 marker using the labeled streptavidin biotin method. The MSCs were cultured in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, ascorbic acid, ß-glycerol phosphate and dexamethasone to differentiate into osteoblasts. Biological in vitro analysis showed the rapid proliferation of the MSCs. Further evaluation of specific osteogenic markers using von Kossa staining and the alkaline phosphate assay demonstrated that the MSCs were stimulated to differentiate into osteoblast-lineage cells. This mesengenic potential indicated that the bone marrow-derived cells were multipotent MSCs. The findings of this study show that bone marrow can be a legitimate source of MSCs for the production of osteoblasts for utilization in bone replacement therapy.
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AIM: Synovial fibroblast has emerged as a potential cellular target in progressive joint destruction in rheumatoid arthritis development. In this study, BDMC33 (2,6-bis[2,5-dimethoxybenzylidene]cyclohexanone), a curcumin analogue with enhanced anti-inflammatory activity has been synthesized and the potency of BDMC33 on molecular and cellular basis of synovial fibroblasts (SF) were evaluated in vitro. METHODS: Synovial fibroblast cells (HIG-82) were cultured in vitro and induced by phorbol-12-myristate acetate (PMA) to stimulate the expression of matrix metalloproteinase (MMPs) and pro-inflammatory cytokines. The protective effects of BDMC33 were evaluated toward MMP activities, pro-inflammatory cytokine expression and nuclear factor kappa-B (NF-κB) activation by using various bioassay methods, including zymography, Western blotting, reverse transcription polymerase chain reaction, immunofluorescense microscopy and electrophoretic mobility shift assay. RESULTS: The results showed that BDMC33 significantly inhibited the pro-gelatinase B (pro-MMP-9) and collagenase activities via suppression of MMP-1 in activated SF. In addition, BDMC33 strongly suppressed MMP-3 gene expression as well as inhibited COX-2 and IL-6 pro-inflammatory gene expression. We also demonstrated that BDMC33 abolished the p65 NF-κB nuclear translocation and NF-κB DNA binding activity in PMA-stimulated SF. CONCLUSIONS: BDMC33 represents an effective chemopreventive agent and could be used as a promising lead compound for further development of rheumatoid arthritis therapeutic intervention.
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Antirreumáticos/farmacologia , Compostos de Benzilideno/farmacologia , Cicloexanonas/farmacologia , Fibroblastos/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Mediadores da Inflamação/metabolismo , Metaloproteinases da Matriz/metabolismo , Coelhos , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1+pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP+pDis/IL-18 inoculated groups. The pDis/N1+pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P<0.05). The flow cytometry results from both trials demonstrated that the pDis/N1+pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P<0.05). Meanwhile, pDis/N1+pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P<0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P>0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1+pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/imunologia , Interleucina-15/farmacologia , Interleucina-18/farmacologia , Linfócitos T/imunologia , Animais , Western Blotting/veterinária , Galinhas/imunologia , Galinhas/virologia , Chlorocebus aethiops , DNA Viral/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico , Células Vero/virologiaRESUMO
Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00 ± 0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00 ± 0.52 µg/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug.