Molecular Biotechnology最新文献

Since December 2019, the SARS-CoV-2 virus has caused the global COVID-19 pandemic. Antiviral and anti-inflammatory treatments have had limited success, positioning vaccine development as a key strategy for public health. This study constructed a chimeric S1 protein fused to a human Fc domain using the Pichia pastoris expression system. Yeast expression system was selected for its low-cost and relatively easier process comparing mammalian and insect. In addition, two human commercial vaccines including human Hepatitis B virus and human papilloma virus are produced currently in yeast system. The chimeric protein named S1Fc was codon-optimized and expressed via the pPICZaA vector as pPICZaA-S1Fc construct. This construct consists of 918 amino acids: 673 amino acids of the S1 protein (N-terminal) linked to 227 amino acids from the human IgG1 Fc region (C-terminal) via 18 amino acids linker. Two yeast strains, a standard glycosylating strain and a mammalian-like GlycoSwitch strain, were selected for expression. SDS-PAGE and western blot analyses indicated successful S1Fc expression in both strains, with a molecular weight of approximately 130 kDa. The GlycoSwitch strain demonstrated enhanced antigenicity in ELISA, indicating a glycosylation pattern more similar to the native viral S1 protein. Purification was achieved using a protein G chromatography column, yielding 14.6 µg/ml in the GlycoSwitch strain and 18.9 µg/ml in the standard strain. These findings highlight the Pichia pastoris expression system as a cost-effective platform for S1Fc protein production, meriting further study as a potential vaccine antigen.

Microorganisms can produce various amphiphilic compounds known as biosurfactants, with diverse applications in distinct industries. This study was focused on the biosurfactant production by Limosilactobacillus fermentum HBUAS62516 for the synthesis of silver nanoparticles. The biosurfactant obtained was characterized as glycolipid using FTIR which showed prominent peaks at 2932.3, 1116.3, and 1084.4 cm^-1, indicating major functional groups which was further confirmed using techniques, such as EDS, NMR, and HPLC. Biosurfactant was utilized as the reducing agent for the biosynthesis of silver nanoparticles which was confirmed using UV-Vis spectral measurements that showed maximum absorbance at 421 nm and FTIR revealed peaks at 109 and 665 cm^-1, indicating silver nanoparticle formation. EDS confirmed the presence of silver nanoparticles with a mass percentage of 100.00 ± 4.56%. Dynamic light scattering (DLS) and zeta potential were 87.93 nm and - 21 mV, respectively, indicating stability. The nanoparticles showed significant antibiofilm and antioxidant activity (90.1%). The synergistic antibacterial effect of the biosurfactant and nanoparticles was studied against Staphylococcus aureus and Pseudomonas putida, as well as their antifungal activity against Aspergillus niger, with a MIC value of 12.5 μg/mL. Nanoparticles synthesized using biosurfactants obtained from probiotic bacteria can act as alternative therapeutics to treat infections caused by the biofilm-forming bacteria.

Pediatric sepsis remains one of the leading causes of mortality in children worldwide. Despite advances in medical care, the prognosis of pediatric sepsis is still poor, necessitating the need for more precise diagnostic and therapeutic strategies. Recently, metabolic reprogramming, particularly glycolysis, has been implicated in the pathogenesis of sepsis, offering new avenues for biomarker discovery and targeted therapy. We applied the GSVA algorithm to the GSE26440 dataset to score glycolysis pathways and identified key glycolysis-related genes (GRGs) using LASSO and logistic regression. We then constructed a predictive nomogram with these GRGs and used consensus clustering to define new molecular subgroups, followed by analyzing their metabolic and immune characteristics. The signature genes were validated by animal experiments. We found increased glycolysis pathway activity in sepsis patients. Through the application of LASSO and logistic regression, GNPDA2, PRKACB, and TGFBI emerged as potential glycolysis-based diagnostic markers. The nomogram showed significant diagnostic accuracy in both the original (GSE26440) and the separate validation datasets (GSE13904 and GSE26378). We distinguished two sepsis subtypes, with the C2 subtype exhibiting higher GRGs, glucose metabolism, and inflammation. Immune infiltration and checkpoint gene expression also varied between the subtypes. Our research identifies glycolysis-based diagnostic markers and molecular subtypes in sepsis, enhancing our understanding and potentially leading to better diagnosis and treatment strategies, including immunotherapy.

The Yes-associated protein (YAP) is a key regulator in the pathogenesis of gastric cancer (GC), yet its role in modulating autophagy remains unclear. This study investigated the effects of YAP modulation on autophagy and tumor progression using the SNU-484 and MKN-74 gastric cancer cell lines. YAP overexpression led to increased B-cell lymphoma-2 (BCL-2) transcription, reducing autophagy and enhancing cell survival, while YAP knockdown resulted in elevated autophagic activity. In vivo experiments with nude mice confirmed that YAP overexpression promotes tumor growth, whereas YAP silencing inhibits it. Further analysis revealed that YAP directly binds to the BCL-2 promoter, driving its transcription and thereby inhibiting autophagy-induced cell death. Importantly, silencing BCL-2 mitigated the autophagy inhibition caused by YAP without affecting YAP expression itself. These findings indicate that YAP, by upregulating BCL-2, suppresses autophagy and contributes to gastric cancer progression, suggesting a potential therapeutic strategy targeting the YAP-BCL-2 axis in GC treatment.

Glioblastoma demands the designing of potential drugs as there is no specific treatment available. In this study, we employed computational screening techniques to identify potential modulators of the c-Met receptor from a library of 273 Chrysopogon zizanioides derived compounds which can pass blood brain barrier (BBB) due to their low molecular weight and BBB permeability. Through rigorous molecular docking simulations utilizing Auto Dock Vina plugin integrated with Chimera software, Ketone (C₂₉H₅₆O) (IMPHY012701) emerged as a standout candidate, exhibiting a lower binding energy compared to the reference molecule, AMG 337 which was used as a control compound. The optimal orientation of Ketone (C₂₉H₅₆O) (IMPHY012701) within the c-Met receptor's active site was elucidated, indicating favourable molecular interactions conducive to stable binding. Ketone (C₂₉H₅₆O) (IMPHY012701) shows equilibrium state during 50 ns simulation with least root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values. Notably, Ketone (C₂₉H₅₆O) (IMPHY012701) demonstrated superior binding affinity relative to the control compound, underscoring its potential as a lead for further investigation. This study underscores the utility of computational approaches in drug discovery from natural sources and highlights Ketone (C₂₉H₅₆O) (IMPHY012701) as a promising candidate for the modulation of c-Met-mediated signalling pathways, warranting further experimental validation and exploration of its pharmacological properties.

Polymerase chain reaction (PCR), a revolutionary molecular tool, has transformed genetic studies by facilitating rapid DNA amplification. The PCR process relies on several key components: a DNA template or cDNA, two primers, Taq polymerase, nucleotides, and a buffer. These elements collectively facilitate the amplification process, which comprises three stages: denaturation, annealing, and extension. These stages are repeated in cycles to exponentially amplify the target DNA sequence. Furthermore, the power of PCR lies in its ability to generate exponential copies of target DNA in a remarkably short period. Moreover, various PCR techniques are available, encompassing traditional approaches like quantitative PCR, reverse transcription PCR, and nested PCR, as well as innovative methods such as extreme PCR, inverse PCR, and touchdown PCR. These techniques are extensively utilized in molecular, biological, and medical research laboratories for both research and diagnostic applications. This review explores a comprehensive overview of PCR, covering its history, underlying principles, and diverse applications in diagnostics, research, and drug development.

Naegleria fowleri causes primary amoebic meningoencephalitis (PAM), a lethal disease with a mortality rate of 97%. Current treatment options are limited and often ineffective, highlighting the urgent need for novel therapeutic agents. This study aimed to identify potential inhibitors of the S-adenosyl-L-homocysteine hydrolase (SAHH) enzyme from N. fowleri using an in-silico approach including Molecular Docking, Density Functional Theory (DFT), Molecular Dynamics (MD) simulation, and Molecular Mechanics Generalized Born Surface Area (MMGBSA) analysis. This study included compounds capable of crossing the blood-brain barrier after screening the Asinex Library of 261120 compounds. After molecular docking, ligands had binding energies ranging from - 5.3 to - 11.4 kcal/mol. Only one ligand 2-[(3-Chlorobenzoyl)amino]-N-(2,3-dihydro-1H-inden-5-yl)-4-methyl-1,3-thiazole-5-carboxamide had a better binding energy of - 11.4 kcal/mol as compared to the reference compound adenosine (- 10.1 kcal/mol). DFT calculations revealed HOMO-LUMO energy gaps of 0.14301 eV (α-spin) and 0.07565 eV (β-spin). MD simulations conducted throughout 100 ns confirmed the stable binding and interaction of the ligand with key active site residues, including Asp130, His232, Phe150, Leu200, and Lys68. Stable root-mean-square deviation (RMSD) and continuous interactions between the ligand and critical active site residues were observed. MMGBSA analysis confirmed the ligand's strong binding affinity, indicated by a negative binding energy with substantial lipophilic and Coulombic contributions. The selected ligand demonstrated significant binding affinity, stability, and inhibitory potential against NfSAHH, making it a promising candidate for further development as a therapeutic agent against PAM. The findings reveal novel binding interactions and structural insights into the binding mechanism of NfSAHH inhibitors. By employing a strategic in silico approach, this study provides a robust foundation for identifying and prioritizing potential inhibitors, optimizing resources for experimental validation, and streamlining the drug discovery process.

Crohn's disease (CD), a complex gastrointestinal disorder, can be attributed to a combination of genetic factors, immune system dysfunction, and environmental triggers. Aquaporin 9 (AQP9) has been implicated in immunoregulation and inflammation in various conditions, yet its function in CD remains unclear. Herein, we investigated the contribution of AQP9 to CD pathogenesis and its impact on inflammation and pyroptosis. Bioinformatic analysis showed a significant increase in AQP9 expression (above 2.5-fold change) in CD patients compared to controls. In vitro experiments using human colonic epithelial cells (HT-29) demonstrated that AQP9 inhibition attenuated lipopolysaccharide (LPS)-induced cell damage, inflammatory cytokine secretion, and pyroptosis. Mechanistically, AQP9 silencing suppressed NLRP3 inflammasome activation, suggesting a role in regulating pyroptosis. AQP9 silencing inhibited p38 MAPK phosphorylation, indicating a direct involvement in modulating this inflammatory pathway. Furthermore, our findings indicate that AQP9 exacerbates inflammation and pyroptosis via activating the p38 MAPK signaling pathway, known to contribute to CD pathogenesis. In vivo studies using a murine model of CD-like colitis revealed that AQP9 inhibition led to about 45% reduction in colitis severity scores and about 30% decrease in the production of inflammatory cytokine by inactivating NLRP3 inflammasome and the p38 MAPK signaling. To sum up, our study highlights the involvement of AQP9 in CD pathogenesis through modulation of inflammation and pyroptosis via the NLRP3 inflammasome and p38 MAPK signaling pathway. Targeting AQP9 may offer a promising therapeutic approach for CD by suppressing inflammatory responses and preventing tissue damage.

Myocardial DNA damage plays a critical role in the pathogenesis of cardiovascular diseases, frequently leading to adverse outcomes such as myocardial infarction and heart failure. This study elucidated the protective effects of sodium alginate (SA) against myocardial DNA damage and explored the underlying molecular mechanisms involved. Hydrogen peroxide (H₂O₂) -stimulated AC16 cells were employed as an in vitro model to induce myocardial DNA damage, and CCK-8 assays established that SA exhibited no cytotoxicity at concentrations up to 800 µM. The protective effects of SA on myocardial DNA damage were shown to be mediated by VSNL1 using immunofluorescence, western blotting and qPCR analyses. To further substantiate this mechanism, lentiviral transduction was utilized to achieve VSNL1 overexpression, whereas targeted siRNA silencing was employed for VSNL1 knockdown. Following VSNL1 overexpression, a reduction in γ-H2AX protein expression was observed, accompanied by increased levels of CNP and NPR-B proteins on the cell membrane, as well as a decrease in intracellular calcium ion concentrations. Conversely, knockdown of VSNL1 reduced the protective effects of SA, highlighting its critical role in the mediation of cardioprotective mechanisms. Taken together, these findings suggest that SA exerts a potential protective effect against myocardial DNA damage through upregulating VSNL1, activating the CNP/NPR-B signaling pathway, and decreasing intracellular calcium ion accumulation. These results underscore that SA is a promising therapeutic candidate for the attenuation of myocardial injury.

This study investigated the microbial community composition and structure in healthy and diseased rhubarb (Rheum rhabarbarum) root systems, examining both root tissue and rhizosphere environments. Alpha diversity analysis revealed significantly higher microbial abundance in the rhizosphere compared to root tissues, with notable differences between healthy and diseased plants. Principal coordinate analysis demonstrated that bacterial community composition was primarily influenced by ecological niches (47.5% variation explained), whereas fungal communities segregated based on plant health status. Network analysis revealed increased bacterial community complexity in diseased plants rhizosphere (579 nodes, 13,016 edges) compared to healthy plants (542 nodes, 8700 edges), while fungal networks showed opposite trends with significant reduction in diseased conditions (147 nodes, 30 edges vs. 205 nodes, 418 edges). Correlation analysis identified significant associations between specific microbial taxa and soil properties, with notable positive correlations between certain bacteria (Oscillospirales) and fungi (Barnettozyma, Mortierella) with soil organic matter and nutrient availability. Pathogenic taxa, including Fusarium and members of Burkholderiales, showed negative correlations with beneficial microorganisms, suggesting potential antagonistic relationships. These findings provide crucial insights into the complex interactions within the rhubarb root microbiome and their implications for plant health, contributing to our understanding of root rot disease dynamics and potential management strategies.