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The brain-derived neurotrophic factor (BDNF) involves stress regulation and psychiatric disorders. The Val66Met polymorphism in the BDNF gene has been linked to altered protein function and susceptibility to stress-related conditions. This in silico analysis aimed to predict and analyze the consequences of the Val66Met mutation in the BDNF gene of stressed individuals. Computational techniques, including ab initio, comparative, and I-TASSER modeling, were used to evaluate the functional and stability effects of the Val66Met mutation in BDNF. The accuracy and reliability of the models were validated. Sequence alignment and secondary structure analysis compared amino acid residues and structural components. The phylogenetic analysis assessed the conservation of the mutation site. Functional and stability prediction analyses provided mixed results, suggesting potential effects on protein function and stability. Structural models revealed the importance of BDNF in key biological processes. Sequence alignment analysis showed the conservation of amino acid residues across species. Secondary structure analysis indicated minor differences between the wild-type and mutant forms. Phylogenetic analysis supported the evolutionary conservation of the mutation site. This computational study suggests that the Val66Met mutation in BDNF may have implications for protein stability, structural conformation, and function. Further experimental validation is needed to confirm these findings and elucidate the precise effects of this mutation on stress-related disorders.

The industrial applications of the κ-carrageenases have been restricted by their poor thermostability. In this study, based on the folding free energy change (ΔΔG) and the flexibility analysis using molecular dynamics (MD) simulation for the alkaline κ-carrageenase KCgCD from Pseudoalteromonas porphyrae (WT), the mutant S190R was identified with improved thermostability. After incubation at 50 °C for 30 min, the residual activity of S190R was 63.7%, 25.7% higher than that of WT. The T_m values determined by differential scanning calorimetry were 66.2 °C and 64.4 °C for S190R and WT, respectively. The optimal temperature of S190R was 10 °C higher than that of WT. The κ-carrageenan hydrolysates produced by S190R showed higher xanthine oxidase inhibitory activity compared with the untreated κ-carrageenan. MD simulation analysis of S190R showed that the residues (V186-M194 and P196-G197) in F5 and the key residue R150 in F3 displayed the decreased flexibility, and residues of T169-N173 near the catalytic center displayed the increased flexibility. These changed flexibilities might be the reasons for the improved thermostability of mutant S190R. This study provides a useful rational design strategy of combination of ΔΔG calculation and MD simulation to improve the κ-carrageenase's thermostability for its better industrial applications.

The emergence of antibiotic resistance in pathogens is one of the major health concerns facing mankind as different bacterial strains have developed resistance to antibiotics over the period of time due to overuse and misuse of antibiotics. Besides this, ability to form biofilms is another major factor contributing to antibiotic resistance, which has necessitated the need for exploration for novel and effective compounds with ability to inhibit biofilm formation. Endophytic fungi are reported to exhibit antibacterial and anti-biofilm potential and could serve as a potent source of novel antibacterial compounds. Majority of the bioactivities have been reported from fungi belonging to phylum Ascomycota. Endophytic basidiomycetes, inspite of their profound ability to serve as a source of bioactive compounds have not been exploited extensively. In present study, an attempt was made to assess the antibacterial, anti-biofilm and biofilm dispersion potential of an endophytic basidiomycetous fungus Schizophyllum commune procured from the culture collection of our lab. Ethyl acetate extract of S. commune showed good antibacterial activity against Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica and Vibrio cholerae. Minimum inhibitory concentration and minimum bactericidal concentration of the extract were in the range of 1.25-10 mg/ml against the tested bacterial pathogens. The mode of action was determined to be bactericidal which was further confirmed by time kill studies. Good anti-biofilm activity of S. commune extract was recorded against K. pneumoniae and S. enterica, which was further validated by fluorescence microscopy. The present study highlights the importance of endophytic basidiomycetes as source of therapeutic compounds.

Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.

Mycotoxins (MTs), produced by filamentous fungi, represent a severe hazard to the health of humans and food safety, affecting the quality of various agricultural products. They can contaminate a wide range of foods, during any processing phase before or after harvest. Animals and humans who consume MTs-contaminated food or feed may experience acute or chronic poisoning, which may result in serious pathological consequences. Accordingly, developing rapid, easy, and accurate methods of MTs detection in food becomes highly urgent and critical as a quality control and to guarantee food safety and lower health hazards. In this review, we highlighted and discussed innovative approaches like biosensors, fluorescent polarization, capillary electrophoresis, infrared spectroscopy, and electronic noses for MT identification pointing out current challenges and future directions. The limitations, current challenges, and future directions of conventional detection methods versus innovative methods have also been highlighted and discussed.

The use of attenuated bacteria for oral delivery of DNA vaccines is a recent innovation. We designed and constructed the naked plasmid DNA vaccine pcDNA-CCOL2A1, which effectively prevented and treated a rheumatoid arthritis model by inducing immunotolerance. We aimed to ensure a reliable, controllable dosage of this oral DNA vaccine preparation and establish its stability. We transformed pcDNA-CCOL2A1 via electroporation into attenuated Salmonella typhimurium SL7207. A resistant plate assay confirmed the successful construction of the transformed strain of the SL7207/pcDNA-CCOL2A1 oral DNA vaccine. We verified its identification and stability in vitro and in vivo. Significant differences were observed in the characteristics of the transformed and blank SL7207 strains. No electrophoretic restriction patterns or direct sequencing signals were observed in the original extract of the transformed strain. However, target gene bands and sequence signals were successfully detected after PCR amplification. CCOL2A1 expression was detected in the ilea of BALB/c mice that were orally administered SL7207/pcDNA-CCOL2A1. The pcDNA-CCOL2A1 plasmid of the transformed strain was retained under the resistant condition, and the transformed strain remained stable at 4 °C for 100 days. The concentration of the strain harboring the pcDNA-CCOL2A1 plasmid was stable at 10⁹ CFU/mL after 6-8 h of incubation. The results demonstrated that the transformed strain SL7207/pcDNA-CCOL2A1 can be expressed in vivo, has good stability, and may be used to prepare the oral DNA vaccine pcDNA-CCOL2A1 with a stable, controllable dosage and the capacity to provide oral immunization. This vehicle can effectively combine both oral immunotolerance and DNA vaccination.

Staphylococcus aureus is a major threat in infectious diseases due to its varied infection types and increased resistance. S. aureus could form persister cells under certain condition and could also attach on medical apparatus to form biofilms, which exhibited extremely high resistance to antibiotics. 3-Acetyl-11-keto-beta-boswellic acid (AKBA) is a well-studied anti-tumor and antioxidant drug. This study is aimed to determine the antimicrobial effects of AKBA against S. aureus and its persister cells and biofilms. The in vitro antimicrobial susceptibility of AKBA was assessed by micro-dilution assay, disc diffusion assay and time-killing assay. Drug combination between AKBA and conventional antibiotics was detected by checkerboard assay. And the antibiofilm effects of AKBA against S. aureus were explored by crystal violet staining combined with SYTO/PI probes staining. Next, RBC lysis activity and CCK-8 kit were used to determine the cytotoxicity of AKBA. In addition, murine subcutaneous abscess model was used to assess the antimicrobial effects of AKBA in vivo. Our results revealed that AKBA was found to show effective antimicrobial activity against methicillin-resistant S. aureus (MRSA) with the minimal inhibitory concentration of 4-8 µg/mL with undetectable cytotoxicity. And no resistant mutation was induced by AKBA after 20 days of consecutive passage. Further, we found that AKBA could be synergy with gentamycin or amikacin against S. aureus and its clinical isolates. By crystal violet and SYTO9/PI staining, AKBA exhibited strong biofilm inhibitory and eradication effects at the concentration of 1 ~ 4 µg/mL. In addition, the effective antimicrobial effect was verified in vivo in a mouse model. And no detectable in vivo toxicity was found. These results indicated that AKBA has great potential to development as an alternative treatment for the refractory S. aureus infections.

Classical fungal mutant strains obtained by mutagenesis have helped to elucidate fundamental metabolic pathways in the past. In the filamentous fungus Neurospora crassa, the gluc-1 strain was isolated long ago and characterized by its low level of β-glucosidase activity, which is essential for the degradation of cellulose, the most abundant biopolymer on Earth and the main polymeric component of the plant cell wall. Based on genomic resequencing, we hypothesized that the causative mutation resides in the β-glucosidase gene gh3-3 (bgl6, NCU08755). In this work, growth patterns, enzymatic activities and sugar utilization rates were analyzed in several mutant and overexpression strains related to gluc-1 and gh3-3. In addition, different mutants affected in the degradation and transport of cellobiose were analyzed. While overexpression of gh3-3 led to the recovery of β-glucosidase activity in the gluc-1 mutant, as well as normal utilization of cellobiose, the full gene deletion strain Δgh3-3 was found to behave differently than gluc-1 with lower secreted β-glucosidase activity, indicating a dominant role of the amino acid substitution in the point mutated gh3-3 gene of gluc-1. Our results furthermore confirm that GH3-3 is the major extracellular β-glucosidase in N. crassa and demonstrate that the two cellodextrin transporters CDT-1 and CDT-2 are essential for growth on cellobiose when the three main N. crassa β-glucosidases are absent. Overall, these findings provide valuable insight into the mechanisms of cellulose utilization in filamentous fungi, being an essential step in the efficient production of biorefinable sugars from agricultural and forestry plant biomass.

Giardiasis, which is caused by Giardia duodenalis, has clinical symptoms such as steatorrhea and can be very dangerous in children. In addition, some documents reported that this parasite is present inside the tissue of patients with cancer. In this study, we analyzed the gene expression profiles of some main genes important to apoptosis and anti-apoptosis in humans.Expression profile arrays of Genomic Spatial Event (GSE) 113666, GSE113667, and GSE113679 obtained from Gene Expression Omnibus were used for meta-analysis using R commands. Cytoscape and STRING databases used the protein-protein Interaction network. Then, the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis was performed. Similar genes in Homo sapiens were identified using Basic Local Alignment Search Tool analysis. The validation was performed on eight people using real-time Polymerase chain reaction. In addition to the candidate genes, the gene expression of some other genes, including Serine/Threonine Kinase 1 (AKT1), Cyclin Dependent Kinase Inhibitor 2A (CDKN2A), Kirsten Rat Sarcoma (KRAS), and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) were also examined. Analysis of the expression of serum amyloid A1 (SAA1), Regenerating Islet-Derived 3 Gamma (REG3G), and REG3A genes did not show any difference between the two groups of healthy and diseased people. Examining the mean expression of the four genes AKT1, CDKN2A, KRAS, and PIK3CA showed that three genes of AKT1, CDKN2A, and KRAS had increased expression in people with a history of giardiasis compared to healthy people. We showed that the gene expression pattern differs in apoptosis and anti-apoptosis signaling in people with a history of giardiasis. Giardia duodenalis seems to induce post-non-infectious symptoms with stimulation of human gene expression.

Leishmaniasis is a vector-borne disease, one of the most important neglected tropical diseases. Existing anti-leishmanial treatments are not effective for a long time and associated with toxic side effects so searching for a new, effective and safe alternative treatments against infectious diseases is greatly needed. This study is aimed to assess the leishmaniacidal effects of methanolic extracts of Eryngium planum (E. planum) and Ecbilliun elaterum (E. elaterum) on Leishmania major (L. major), In vitro. The selected plants were collected from northern areas of Iran. The methanolic extract from the aerial parts of plants were prepared using maceration methods. GC- Mass analysis was used to determine the compounds of the plants. Promastigotes of L. major was cultured in RPMI-1640 medium and the anti-leishmanial and cytotoxicity effects of extracts at concentrations of 100, 200, 400 and 800 µg/ml were assessed using MTT assay. The data obtained from gas chromatography revealed that α-Pinene, Caryophyllene oxide, β-Caryophyllene, Bicyclogermacrene and α-Bisabolol are the main compounds extracted from E. planum and α-Pinene, Germacrene D, Caryophyllene oxide, γ-Eudesmol and α-Bisabolol are the main components of E. elaterum. The results of MTT Assay revealed that E. planum at concentrations of 800 µg/ml after 24 h at 400 µg/ml after 48 h and the E. elaterium at concentrations of 800 µg/ml after 48 h at 400 µg/ml after 72 h had similar anti-leishmanial effects to the positive control. These results indicated that E. planum and E. elaterum are the potential sources for the discovery of novel anti-leishmanial treatments.