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Measurement of cellular resting membrane potential (RMP) is important in understanding ion channels and their role in regulation of cell function across a wide range of cell types. However, methods available for the measurement of RMP (including patch clamp, microelectrodes, and potential-sensitive fluorophores) are expensive, slow, open to operator bias, and often result in cell destruction. We present non-contact, label-free membrane potential estimation which uses dielectrophoresis to determine the cytoplasm conductivity slope as a function of medium conductivity. By comparing this to patch clamp data available in the literature, we have demonstratet the accuracy of this approach using seven different cell types, including primary suspension cells (red blood cells, platelets), cultured suspension cells (THP-1), primary adherent cells (chondrocytes, human umbilical mesenchymal stem cells), and adherent (HeLa) and suspension (Jurkat) cancer cell lines. Analysis of the effect of ion channel inhibitors suggests the effects of pharmaceutical agents (TEA on HeLa; DMSO and neuraminidase on red blood cells) can also be measured. Comparison with published values of membrane potential suggest that the differences between our estimates and values recorded by patch clamp are accurate to within published margins of error. The method is low-cost, non-destructive, operator-independent and label-free, and has previously been shown to allow cells to be recovered after measurement.
Assuntos
Eletroforese , Potenciais da Membrana , Humanos , Potenciais da Membrana/fisiologia , Eletroforese/métodos , Células HeLa , Células Jurkat , Técnicas de Patch-Clamp/métodos , Eritrócitos/citologia , Eritrócitos/metabolismoRESUMO
This study examines the manufacturing, characterization, and biological evaluation of platinum nanoparticles, which were synthesized by Enterobacter cloacae and coated with Bovine Serum Albumin (BSA) and Resveratrol (RSV). The formation of PtNPs was confirmed with the change of color from dark yellow to black, which was due to the bioreduction of platinum chloride by E. cloacae. BSA and RSV functionalization enhanced these nanoparticles' biocompatibility and therapeutic potential. TGA, SEM, XRD, and FTIR were employed for characterization, where PtNPs and drug conjugation-related functional groups were studied by FTIR. XRD confirmed the crystalline nature of PtNPs and Pt-BSA-RSV NPs, while TGA and SEM showed thermal stability and post-drug coating morphological changes. Designed composite was also found to be biocompatible in nature in hemolytic testing, indicating their potential in Biomedical applications. After confirmation of PtNPs based nanocaompsite synthesis, they were examined for anti-bacterial, anti-oxidant, anti-inflammatory, and anti-cancer properties. Pt-BSA-RSV NPs showed higher concentration-dependent DPPH scavenging activity, which measured antioxidant capability. Enzyme inhibition tests demonstrated considerable anti-inflammatory activity against COX-2 and 15-LOX enzymes. In in vitro anticancer studies, Pt-BSA-RSV NPs effectively killed human ovarian cancer cells. This phenomenon was demonstrated to be facilitated by the acidic environment of cancer, as the drug release assay confirmed the release of RSV from the NP formulation in the acidic environment. Finally, Molecular docking also demonstrated that RSV has strong potential as an anti-oxidant, antibacterial, anti-inflammatory, and anticancer agent. Overall, in silico and in vitro investigations in the current study showed good medicinal applications for designed nanocomposites, however, further in-vivo experiments must be conducted to validate our findings.
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Nanopartículas Metálicas , Nanopartículas , Humanos , Soroalbumina Bovina/química , Nanopartículas Metálicas/química , Resveratrol/farmacologia , Platina/farmacologia , Platina/química , Antioxidantes/farmacologia , Simulação de Acoplamento Molecular , Nanopartículas/química , Anti-InflamatóriosRESUMO
Vaccinations can serve as an important preventive measure against the porcine epidemic diarrhea (PED) virus that currently threatens the swine industry. This study focuses on the development of a fusion protein vaccine, FliC99-mCOE, which combines the N-terminus of flagellin (FliC99) with a modified core neutralizing epitope (mCOE) of PEDV. In silico immunoinformatic analysis confirmed the construct's non-toxic, non-allergenic, and highly antigenic nature. Molecular docking and molecular dynamics (MD) simulations demonstrated FliC99-mCOE's strong binding to the TLR-5 immunological receptor. Repeated exposure simulations and immunological simulations suggested enhanced cell-mediated immunity. Both FliC99-mCOE and an inactivated PEDV vaccine were produced and tested in mice. The results from cell proliferation, ELISA, and neutralization assays indicated that FliC99-mCOE effectively stimulated cellular immunity and neutralized PEDV. We conclude that the FliC99-mCOE fusion protein may serve as a promising vaccine candidate against PEDV.
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Triple negative breast cancers (TNBC) are clinically heterogeneous but mostly aggressive malignancies devoid of expression of the estrogen, progesterone, and HER2 (ERBB2 or NEU) receptors. It accounts for 15-20% of all cases. Altered epigenetic regulation including DNA hypermethylation by DNA methyltransferase 1 (DNMT1) has been implicated as one of the causes of TNBC tumorigenesis. The antitumor effect of DNMT1 has also been explored in TNBC that currently lacks targeted therapies. However, the actual treatment for TNBC is yet to be discovered. This study is attributed to the identification of novel drug targets against TNBC. A comprehensive docking and simulation analysis was performed to optimize promising new compounds by estimating their binding affinity to the target protein. Molecular dynamics simulation of 500 ns well complemented the binding affinity of the compound and revealed strong stability of predicted compounds at the docked site. Calculation of binding free energies using MMPBSA and MMGBSA validated the strong binding affinity between compound and binding pockets of DNMT1. In a nutshell, our study uncovered that Beta-Mangostin, Gancaonin Z, 5-hydroxysophoranone, Sophoraflavanone L, and Dorsmanin H showed maximum binding affinity with the active sites of DNMT1. Furthermore, all of these compounds depict maximum drug-like properties. Therefore, the proposed compounds can be a potential candidate for patients with TNBC, but, experimental validation is needed to ensure their safety.Communicated by Ramaswamy H. Sarma.
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Simulação de Dinâmica Molecular , Neoplasias de Mama Triplo Negativas , Xantonas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Epigênese Genética , Detecção Precoce de Câncer , DNA , Simulação de Acoplamento MolecularRESUMO
Nanotechnology is a research hotspot that has gained considerable interest due to its potential inferences in the bioscience, medical, and engineering disciplines. The present study uses biomass from the Enterobacter hormaechei EAF63 strain to create bio-inspired metallic tin oxide nanoparticles (SnO2 NPs). The biosynthesized NPs were extensively analyzed using UV spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and Fourier transform infrared (FTIR) techniques. The identification of the crystalline phase was confirmed by XRD. The SEM technique elucidated the morphological characteristics and size of SnO2 NPs. SEM investigation revealed that the SnO2 NPs have a size of 10 nm with spherical morphology. The capping of NPs was confirmed by FTIR analysis that revealed the presence of different compounds found in the biomass of the E. hormaechei EAF63 strain. Later, EDX confirmed the elemental composition of NPs. Moreover, the synthesized SnO2 NPs were employed for important applications including anti-aging, anti-Alzheimer's, anti-inflammatory, anti-larvicidal, and antibacterial action against sinusitis pathogens. The highest value was observed for Streptococcus pyogenes (19.75 ± 0.46), followed by Moraxella catarrhalis (17.49 ± 0.82) and Haemophilus influenzae (15.31 ± 0.73), respectively. Among the used concentrations, the highest inhibition of 76.8 ± 0.93 for 15-lipoxygenase (15-LOX) was observed at 400 µg/mL, followed by 67.4 ± 0.91 for cyclooxygenase-1 (COX-1). So, as an outcome, E. hormaechei-mediated SnO2 NPs might be considered as the safe and effective nanoplatforms for multifunctional biological applications in the field of nanomedicine.
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BACKGROUND: Mentha arvensis has been utilized in diverse traditional medicines as an antidiabetic, anticarcinogenic, antiallergic, antifungal, and antibacterial agent. In this work, we have explored the phytochemical analyses and pharmacological potential of Mentha arvensis using both in silico and in vitro approaches for drug discovery. METHODS: To determine the extract with the highest potential for powerful bioactivity, ethanol was used as the solvent. The phytochemical components of the extracts were quantified using liquid chromatography-mass spectrometry analysis. The potential bioactivities of extracts and lead phytocompounds, including their antibacterial, cytotoxic, and anti-diabetic effects, were evaluated. RESULTS: The compounds oleanolic acid, rosmarinic acid, luteolin, isoorientin, and ursolic acid have been identified through liquid chromatography mass spectrometry analysis. Based on antimicrobial research, it has been found that the Mentha arvensis extract shows potential activity against K. pneumoniae which was 13.39 ± 0.16. Mentha arvensis has demonstrated a greater degree of efficacy in inhibiting α-glucosidase, with an inhibition rate of 58.36 ± 0.12, and in inhibiting α-amylase, with an inhibition rate of 42.18 ± 0.83. The growth of HepG2 cells was observed to be significantly suppressed upon treatment with extracts obtained from Mentha arvensis. Finally, In-silico methods demonstrated that the Luteolin and Rosmarinic acid exhibit acceptable drug-like characteristics. Furthermore, Molecular docking studies further demonstrated that both compounds have strong potential to inhibit the active sites of therapeutically relevant enzymes involved in Diabetes, Bacterial infections, and Cancer. CONCLUSIONS: The results of this study suggest that the Mentha arvensis extract possesses potent pharmacological potentials, particularly in terms of antibacterial, anti-diabetic, and cytotoxic effects. Particularly, Luteolin and Rosmarinic acid were identified as the top contenders for potential bioactivity with acceptable drug-like properties.
Assuntos
Mentha , Mentha/química , Luteolina , Hipoglicemiantes/farmacologia , Simulação de Acoplamento Molecular , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ácido RosmarínicoRESUMO
The current study aims to utilize the bacteria Paraclostridium benzoelyticum strain 5610 to synthesize bio-genic silver nanoparticles (AgNPs). Biogenic AgNPs were thoroughly examined using various characterization techniques such as UV-spectroscopy, XRD, FTIR, SEM, and EDX. Synthesis of AgNPs was confirmed by UV-vis analysis resulting in absorption peak at 448.31 nm wavelength. The SEM analysis indicated the morphological characteristics and size of AgNPs which was 25.29 nm. The face centered cubic (FCC) crystallographic structure was confirmed by XRD. Furthermore, FTIR study affirmed the capping of AgNPs by different compounds found in biomass of the Paraclostridium benzoelyticum strain 5610. Later, EDX was used to determine the elemental composition with respective concentration and distribution. Additionally, in the current study the antibacterial, anti-inflammatory, antioxidant, anti-aging, and anti-cancer ability of AgNPs was assessed. The antibacterial activity of AgNPs was tested against four distinct sinusitis pathogens: Haemophilus in-fluenza, Streptococcus pyogenes, Moraxella catarrhalis and Streptococcus pneumonia. AgNPs shows significant inhibition zone against Streptococcus pyogenes 16.64 ± 0.35 followed by 14.32 ± 071 for Moraxella catarrhalis. Similarly, the antioxidant potential was found maximum (68.37 ± 0.55%) at 400 µg/mL and decrease (5.48 ± 0.65%) at 25 µg/mL, hence the significant antioxidant ability was observed. Furthermore, anti-inflammatory activity of AgNPs shows the strongest inhibitory action (42.68 ± 0.62%) for 15-LOX with lowest inhibition activity for COX-2 (13.16 ± 0.46%). AgNPs have been shown to exhibit significant inhibitory actions against the enzyme elastases AGEs (66.25 ± 0.49%), which are followed by AGEs of visperlysine (63.27 ± 0.69%). Furthermore, the AgNPs show high toxicity against HepG2 cell line which shows 53.543% reduction in the cell viability after 24 h of treatment. The anti-inflammatory activity demonstrated a potent inhibitory effect of the bio-inspired AgNPs. Overall, the biogenic AgNPs have the ability to be served for the treatments of anti-aging and also due to their anti-cancer, antioxidant abilities NPs may be a useful therapy choice for a variety of disorders including cancer, bacterial infections and other inflammatory diseases. Moreover, further studies are required in the future to evaluate their in vivo biomedical applications. HIGHLIGHTS: Biogenic synthesis of AgNPs using Paraclostridium benzoelyticum Strain for the first time. FTIR analysis confirmed capping of potent biomolecules which are of great use in applied field especially Nanomedicines. Notable antimicrobial activity against sinusitis bacteria and cytotoxic potential of synthesized AgNPs on in vitro basis produce a new idea shifting us to treat cancerous cell lines.
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Nanopartículas Metálicas , Prata , Prata/farmacologia , Prata/química , Nanopartículas Metálicas/química , Antioxidantes/farmacologia , Bactérias , Extratos Vegetais/química , Antibacterianos/farmacologia , Antibacterianos/química , Produtos Finais de Glicação Avançada/farmacologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Clostridioides difficile is a gram-positive bacterium which is associated with different gastrointestinal related infections, and the numbers of cases related to it are continuously increasing in the past few years. Owing to high prevalence and development of resistance towards available antibiotics, it is required to develop new therapeutics to combat C. difficile infection. The current study was aimed to identify novel phytochemicals that could bind and inhibits the TcdB, an exotoxin which is required for the pathogenesis of bacteria, and hence can be considered as the future drug candidates against C. difficile. â¼2500 therapeutically important phyto-compounds were docked against the active sites of TcdB protein by using AutoDock-Vina software. The interactions between the ligands and the binding site of the top five docked complexes, based on the docking scores, were further elucidated by Molecular Dynamics Simulations of 500 ns, Molecular Mechanics Energies combined with the Poisson-Boltzmann and Surface Area (MMPBSA) or Generalized Born and Surface Area (MMGBSA), and WaterSwap Analysis. Findings of molecular docking suggested that natural compounds A183, A704, A1528, A2083, and A2129 with distinct chemical scaffolds are best docked in the binding site of TcdB and their bonding remained stable throughout the simulation studies of 500 ns. Compounds A2129 and A704 can be considered as prospective drug candidates against Clostridioides difficile, however, further wet lab experiments are needed to confirm our study.Communicated by Ramaswamy H. Sarma.
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Toxinas Bacterianas , Clostridioides difficile , Fatores de Virulência , Simulação de Acoplamento Molecular , Clostridioides , Compostos Fitoquímicos/farmacologiaRESUMO
Hendra virus (HeV) belongs to the paramyxoviridae family of viruses which is associated with the respiratory distress, neurological illness, and potential fatality of the affected individuals. So far, no competitive approved therapeutic substance is available for HeV. For that reason, the current research work was conducted to propose some novel compounds, by adopting a Computer Aided Drug Discovery approach, which could be used to combat HeV. The G attachment Glycoprotein (Ggp) of HeV was selected to achieve the primary objective of this study, as this protein makes the entry of HeV possible in the host cells. Briefly, a library of 6000 antiviral compounds was screened for potential drug-like properties, followed by the molecular docking of short-listed compounds with the Protein Data Bank (PDB) structure of Ggp. Docked complexes of top two hits, having maximum binding affinities with the active sites of Ggp, were further considered for molecular dynamic simulations of 200 ns to elucidate the results of molecular docking analysis. MD simulations and Molecular Mechanics Energies combined with the Generalized Born and Surface Area (MMGBSA) or Poisson-Boltzmann and Surface Area (MMPBSA) revealed that both docked complexes are stable in nature. Furthermore, the same methodology was used between lead compounds and HeV Ggp in complex with its functional receptor in human, Ephrin-B2. Surprisingly, no major differences were found in the results, which demonstrates that our identified compounds can also perform their action even when the Ggp is attached to the Ephrin-B2 ligand. Therefore, in light of all of these results, we strongly suggest that compounds (S)-5-(benzylcarbamoyl)-1-(2-(4-methyl-2-phenylpiperazin-1-yl)-2-oxoethyl)-6-oxo-3,6-dihydropyridin-1-ium-3-ide and 5-(cyclohexylcarbamoyl)-1-(2-((2-(3-fluorophenyl)-2-methylpropyl)amino)-2-oxoethyl)-6-oxo-3,6-dihydropyridin-1-ium-3-ide could be considered as potential therapeutic agents against HeV; however, further in vitro and in vivo experiments are required to validate this study.
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Antivirais/química , Química Computacional/métodos , Proteínas Virais de Fusão/química , Antivirais/metabolismo , Efrina-B2/química , Efrina-B2/metabolismo , Vírus Hendra/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Virais/química , Receptores Virais/metabolismo , Bibliotecas de Moléculas Pequenas , Proteínas Virais de Fusão/antagonistas & inibidores , Proteínas Virais de Fusão/metabolismo , Água/químicaRESUMO
Human T-cell lymphotropic virus type 1 (HTLV-1) is an infectious virus that has been linked to adult T cell leukemia /lymphoma, aggressive CD4-T cell malignancy and many other immune-related medical illnesses. So far, no effective vaccine is known to combat HTLV-1, hence, the current research work was performed to design a potential multi-epitope-based subunit vaccine (MEBV) by adopting the latest methodology of reverse vaccinology. Briefly, three highly antigenic proteins (Glycoprotein, Accessory protein, and Tax protein) with no or minimal (<37%) similarity with human proteome were sorted out and potential B- and T-cell epitopes were forecasted from them. Highly antigenic, immunogenic, non-toxic, non-allergenic and overlapping epitopes were short-listed for vaccine development. The chosen T-cell epitopes displayed a strong binding affinity with their corresponding Human Leukocyte Antigen alleles and demonstrated 95.8% coverage of the world's population. Finally, nine Cytotoxic T Lymphocytes, six Helper T Lymphocytes and five Linear B Lymphocytes epitopes, joint through linkers and adjuvant, were exploited to design the final MEBV construct, comprising of 382 amino acids. The developed MEBV structure showed highly antigenic properties while being non-toxic, soluble, non-allergenic, and stable in nature. Moreover, disulphide engineering further enhanced the stability of the final vaccine protein. Additionally, Molecular docking analysis and Molecular Dynamics (MD) simulations confirmed the strong association between MEBV construct and human pathogenic immune receptor TLR-3. Repeated-exposure simulations and Immune simulations ensured the rapid antigen clearance and higher levels of cell-mediated immunity, respectively. Furthermore, MEBV codon optimization and in-silico cloning was carried out to confirm its augmented expression. Results of our experiments suggested that the proposed MEBV could be a potential immunogenic against HTLV-1; nevertheless, additional wet lab experiments are needed to elucidate our conclusion.