Immunotoxins are widely applied for cancer therapy. However, bacterial expression of immunotoxins usually leads to the formation of insoluble and non-functional recombinant proteins. This study was aimed to improve soluble expression of a novel anti-HER2 immunotoxin under the regulation of the trc promoter in Escherichia coli by optimization of the cultivation conditions using response surface methodology (RSM). To conduct RSM, four cultivation variables (i.e., inducer concentration, post-induction time, post-induction temperature, and medium recipe), were selected for statistical characterization and optimization using the Box-Behnken design and Design Expert software. Based on the developed model using the Box-Behnken design, the optimal cultivation conditions for soluble expression of anti-HER2 immunotoxin were determined to be 0.1 mM IPTG for induction in the LB medium at 33 °C for 18 h. The expressed immunotoxin was successfully purified using affinity chromatography with more than 90% purity and its bioactivity was confirmed using cell-based ELISA. Technical approach developed in this study can be generally applied to enhance the production yield and quality of recombinant proteins using E. coli as the gene expression system.
A bacterial consortium was isolated from a soil in Noblejas (Toledo, Spain) with a long history of mixed hydrocarbons pollution, by enrichment cultivation. Serial cultures of hydrocarbons polluted soil samples were grown in a minimal medium using diesel (1 mL/L) as the sole carbon and energy source. The bacterial composition of the Noblejas Consortium (NC) was determined by sequencing 16S rRNA gene amplicon libraries. The consortium contained around 50 amplicon sequence variants (ASVs) and the major populations belonged to the genera Pseudomonas, Enterobacter, Delftia, Stenotrophomonas, Achromobacter, Acinetobacter, Novosphingobium, Allorhizobium-Neorhizobium-Rhizobium, Ochrobactrum and Luteibacter. All other genera were below 1%. Metagenomic analysis of NC has shown a high abundance of genes encoding enzymes implicated in aliphatic and (poly) aromatic hydrocarbons degradation, and almost all pathways for hydrocarbon degradation are represented. Metagenomic analysis has also allowed the construction of metagenome assembled genomes (MAGs) for the major players of NC. Metatranscriptomic analysis has shown that several of the ASVs are implicated in hydrocarbon degradation, being Pseudomonas, Acinetobacter and Delftia the most active populations.
Pseudomonas aeruginosa biofilms shield the bacteria from antibiotics and the body's defenses, often leading to chronic infections that are challenging to treat. This study aimed to assess the impact of sub-lethal doses of antimicrobial photodynamic inactivation (sAPDI) utilizing carbon dots (CDs) derived from gentamicin and imipenem on biofilm formation and the expression of genes (pelA and pslA) associated with P. aeruginosa biofilm formation.The anti-biofilm effects of sAPDI were evaluated by exposing P. aeruginosa to sub-minimum biofilm inhibitory concentrations (sub-MBIC) of CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN, combined with sub-lethal UVA light irradiation. Biofilm formation ability was assessed by crystal violet (CV) assay and enumeration method. Additionally, the impact of sAPDI on the expression of pelF and pslA genes was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR).Compared to the control group, the sAPDI treatment with CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN resulted in a significant reduction in biofilm activity of P. aeruginosa ATCC 27853 (P < 0.0001). The CV assay method demonstrated reductions in optical density of 83.70%, 81.08%, 89.33%, and 75.71%, while the CFU counting method showed reductions of 4.03, 3.76, 4.39, and 3.21 Log10 CFU/mL. qRT-PCR analysis revealed decreased expression of the pelA and pslA genes in P. aeruginosa ATCC 27853 following sAPDI treatment compared to the control group (P < 0.05).The results indicate that sAPDI using CDs derived from gentamicin and imipenem can decrease the biofilm formation of P. aeruginosa and the expression of the pelA and pslA genes associated with its biofilm formation.
Developing a potent antiviral agent to combat Coronavirus Disease-19 (COVID-19) is of critical importance as we may be at risk of the emergence of new virus strains or another pandemic recurrence. The interaction between the SARS-CoV-2 spike protein and Angiotensin Converting Enzyme 2 (ACE2) is the main protein-protein interaction (PPI) implicated in the virus entry into the host cells. Spike-ACE2 PPI represents a major target for drug intervention. We have repurposed a previously described protein-protein interaction detection method to be utilized as a drug screening assay. The assay was standardized using Chitosan nanoparticles (CNPs) as the drug and SARS-CoV-2 spike-ACE2 interaction as the PPI model. The assay was then used to screen four natural bioactive compounds: Curcumin (Cur), Gallic acid (GA), Quercetin (Q), and Silymarin (Sil), and their cytotoxicity was evaluated in vitro. Production of the spike protein and the evaluation of its activity in comparison to a standard commercial protein was part of our work as well. Here we describe a novel simple immunofluorescent screening assay to identify potential SARS-CoV-2 inhibitors that could assess the inhibitory effect of any ligand against any PPI.
With the current spread of clinically relevant multidrug-resistant (MDR) pathogens, insufficient unearthing of new anti-infectives, and the high cost required for approval of new antimicrobial agents, a strong need for getting these agents via more economic and other alternative routes has emerged. With the discovery of the biosynthetic pathways of various antibiotics pointing out the role of each gene/protein in their antibiotic-producing strains, it became apparent that the biosynthetic gene clusters can be manipulated to produce modified antibiotics. This new approach is known as the combinatorial biosynthesis of new antibiotics which can be employed for obtaining novel derivatives of these valuable antibiotics using genetically modified antibiotic-producing strains (pathway engineering). In this review and based on the available biosynthetic gene clusters of the major aminoglycoside antibiotics (AGAs), the possible alterations or modifications that could be done by co-expression of certain gene(s) previously known to be involved in unique biosynthetic steps have been discussed. In this review defined novel examples of modified AGA using this approach were described and the information provided will act as a platform of researchers to get and develop new antibiotics by the antibiotic-producing bacterial strains such as Streptomyces, Micromonospora,…etc. This way, novel antibiotics with new biological activities could be isolated and used in the treatment of infectious diseases conferring resistance to existing antibiotics.
In this paper we investigate the influence of cold plasma as novel method on the external otitis treatment which is a frequent cause of earache. 24 infected external auditory canals in 24 rats were categorized in four experimental groups including control, plasma exposed, ciprofloxacin drug and mixed of plasma-ciprofloxacin groups. In plasma group, dielectric barrier discharge was employed as the source of cold plasma in 5 days. All rats were observed with otoscope daily and a scoring system was used to evaluate swelling and effusion of the ear canal. Number of colonies in microbiological culture were counted in each group during the first 5 days after treatment. For the multiple group comparisons of swelling and effusion measured in the external auditory canal, Kruskal–Wallis analysis was applied and one-way anova and Kruskal–Wallis analysis was used for the statistical analysis of the results of the cultures in different days. Also, Tukey and Mann–Whitney tests was applied for multiple comparisons. Our findings show that swelling and effusion were obviously reduced in plasma group compared to control group (P < 0.01). Number of colonies in control group was statistically different from those in drug, plasma, and mixed group on the second to fifth day (p < 0.001). According to the results cold plasma can be introduced as an impressive method for external otitis treatment. Moreover, when cold plasma joined to antibiotic method, it leads to a superior performance respecting plasma or antibiotic method alone.
Non-alcoholic fatty liver disease (NAFLD) is becoming a significant global public health threat. Seabuckthorn (Hippophae rhamnoides L.) has been used in traditional Chinese medicine (TCM). The hypolipidemic effects of Seabuckthorn polysaccharides (SP) against high-fat diets (HFD)-induced NAFLD were systematically explored and compared with that of Bifidobacterium lactis V9 (B. Lactis V9). Results showed that HFD-induced alanine transaminase (ALT) and aspartate aminotransferase (AST) levels decreased by 2.8-fold and 4.5-fold, respectively, after SP supplementation. Moreover, the alleviating effect on hepatic lipid accumulation is better than that of B. Lactis V9. The ACC and FASN mRNA levels were significantly reduced by 1.8 fold (P < 0.05) and 2.3 folds (P < 0.05), respectively, while the CPT1α and PPARα mRNA levels was significantly increased by 2.3 fold (P < 0.05) and 1.6 fold (P < 0.05), respectively, after SP administration. SP activated phosphorylated-AMPK and inhibited PPARγ protein expression, improved serum oxidative stress and inflammation (P < 0.05). SP supplementation leads to increased hepatic expression of nuclear factor erythroid 2-related factor 2 (Nrf-2), heme oxygenase-1 (HO-1) and Superoxide dismutase-2 (SOD-2). Furthermore, SP treatment improved HFD-induced intestinal dysbiosis. Lentisphaerae, Firmicutes, Tenericutes and Peptococcus sp., RC9_gut_group sp., and Parabacteroides sp. of the gut microbiota were significantly associated with hepatic steatosis and indicators related to oxidative stress and inflammation. Therefore, SP can mitigate hepatic lipid accumulation by regulating Nrf-2/HO-1 signaling pathways and gut microbiota. This study offers new evidence supporting the use of SP as a prebiotic treatment for NAFLD.
Cystic fibrosis transmembrane conductance regulator (CFTR) protein is an ion channel found in numerous epithelia and controls the flow of water and salt across the epithelium. The aim of our study to find natural compounds that can improve lung function for people with cystic fibrosis (CF) caused by the p.Gly628Arg (rs397508316) mutation of CFTR protein. The sequence of CFTR protein as a target structure was retrieved from UniProt and PDB database. The ligands that included Armepavine, Osthole, Curcumin, Plumbagine, Quercetin, and one Trikafta (R*) reference drug were screened out from PubChem database. Autodock vina software carried out docking, and binding energies between the drug and the target were included using docking-score. The following tools examined binding energy, interaction, stability, toxicity, and visualize protein-ligand complexes. The compounds having binding energies of -6.4, -5.1, -6.6, -5.1, and - 6.5 kcal/mol for Armepavine, Osthole, Curcumin, Plumbagine, Quercetin, and R*-drug, respectively with mutated CFTR (Gly628Arg) structure were chosen as the most promising ligands. The ligands bind to the mutated CFTR protein structure active sites in hydrophobic bonds, hydrogen bonds, and electrostatic interactions. According to ADMET analyses, the ligands Armepavine and Quercetin also displayed good pharmacokinetic and toxicity characteristics. An MD simulation for 200 ns was also established to ensure that Armepavine and Quercetin ligands attached to the target protein favorably and dynamically, and that protein-ligand complex stability was maintained. It is concluded that Armepavine and Quercetin have stronger capacity to inhibit the effect of mutated CFTR protein through improved trafficking and restoration of original function.
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