Biotechnology and applied biochemistry最新文献

This study emphasizes to investigate the modulatory activity of trans-ferulic acid (TFA) on anti-inflammatory activity of etoricoxib (ETO) and underlying mechanisms via formalin-induced licking and paw edema model and in silico study. Inflammation was induced by injecting formalin (50 µL) into the right hind paw of mice. The animals were treated with different doses of TFA (25, 50, and 75 mg/kg, p.o.). The vehicle and ETO (35 mg/kg, p.o.) were provided as positive and negative control, respectively. ETO also served combined with TFA to evaluate the modulatory activity. The licking behavior was counted for the early and late phases, whereas the paw edema diameter was measured by using a slide caliper. All treatment was continued for 7 days until the edema was totally minimized to determine the inflammation's recovery capability for a specific group. Different computed and web tools were used to estimate molecular binding affinity, binding interactions, and pharmacokinetics. The findings demonstrated that TFA significantly (p < 0.05) enhanced the onset of licking and reduced the number of licks compared to vehicle group. TFA also showed a significant (p < 0.05) diminished in paw edema and complete recovered of the edema after 5 days of treatment indicating the anti-inflammatory effects. However, TFA with ETO notably diminished the anti-inflammatory effects of ETO by enhancing paw edema diameter and licking number. TFA also expressed elevated binding affinity of -7.5 and -6.5 kcal/mol toward nitric oxide (NO) synthase and COX-1, respectively. In conclusion, TFA exerted anti-inflammatory effects and reduces anti-inflammatory capability of ETO.

As a significant cause of global mortality, the cancer has also economic impacts. In the era of cancer therapy, mitigating side effects and costs and overcoming drug resistance is crucial. Microbial species can grow inside the tumor microenvironment and inhibit cancer growth through direct killing of tumor cells and immunoregulatory effects. Although microbiota or their products have demonstrated anticancer effects, the possibility of acting as pathogens and exerting side effects in certain individuals is a risk. Hence, several genetically modified/engineered bacteria (GEB) have been developed to this aim with ability of diagnosing and selective targeting and destruction of cancers. Additionally, GEB are expected to be considerably more efficient, safer, more permeable, less costly, and less invasive theranostic approaches compared to wild types. Potential GEB strains such as Escherichia coli (Nissle 1917, and MG1655), Salmonella typhimurium YB1 SL7207 (aroA gene deletion), VNP20009 (∆msbB/∆purI) and ΔppGpp (P_Tet and P_BAD), and Listeria monocytogenes Lm^at-LLO have been developed to combat cancer cells. When used in tandem with conventional treatments, GEB substantially improve the efficacy of anticancer therapy outcomes. In addition, public acceptance, optimal timing (s), duration (s), dose (s), and strains identification, interactions with other strains and the host cells, efficacy, safety and quality, and potential risks and ethical dilemmas include major challenges.

The role of C-X-C chemokine receptor type 4 (CXR-4) in chemotherapy resistance remains crucial in promoting proliferation, invasion, and progression in acute myeloid leukemia (AML) cells. This study aims to screen and investigate a potential lead candidate as a therapeutic agent targeting CXCR-4 in AML cells. Diversity-based virtual screening process using AutoDock-Vina was employed to screen approximately 850,000 compounds from the ChemBridge-small molecule database. The binding stability and dynamics were investigated through GROMACS-based molecular dynamics simulations and root mean square deviation (RMSD). AML cells (THP-1, HL-60, and SKM-1 cell lines) were used to assess proliferation CXCR-4 expression, and apoptosis induction was measured using flow cytometry and trans-endothelial migration was assessed using calorimetric method in AML cells. The absorption, distribution, metabolism, and excretion (ADME) properties were predicted using SwissADME server. The computational evaluations revealed SSB-2548 as a lead candidate that binds stably to CXCR-4. Molecular dynamics simulations provided detailed insights into the conformational changes of the SSB-2548/CXCR-4 complex. The compound inhibited the THP-1, HL-60, and SKM-1 cell proliferations with GI₅₀ values of 84.57, 41.30, and 120.50 nM, respectively. SSB-2548 decreased the trans-endothelial migration and CXCR-4 expression in while inducing early and late phase apoptosis in all three AML cell types. ADME predictions indicated a favorable lead-likeness, gastrointestinal absorption, and lack of notable toxicity. Computational assessments identified SSB-2548 as a novel CXCR-4 inhibitor. In vitro evaluations proved this lead compound effective against AML cells. These findings lay the groundwork for future, investigations positioning SSB-2548 as a candidate for the development of targeted therapies against AML.

Gliomas are one of the most prevalent types of solid tumors in the brain. Imbalances in mitochondrial metabolism have been implicated in the pathological progression of gliomas. Etomidate, an agonist of the γ-aminobutyric acid type A (GABAA) receptor, is widely used in clinical settings. In this study, we report a novel pharmacological function of etomidate in regulating mitochondrial metabolism in glioma cancer cells. U87 glioma tumor cells were treated with etomidate (0.5, 1.0, and 2.0 µg/mL) for 24 h. Quantitative real-time PCR, western blot analysis, mtDNA/nDNA ratio, MitoTracker Red staining, Complex I and IV activity, intracellular ATP levels, and mitochondrial respiration were assessed. First, etomidate exposure inhibited the expression of PGC-1α in U87 glioma tumor cells. Further investigation revealed that etomidate suppressed the expression of Nrf1 and TFAM, the two key executors of mitochondrial biogenesis. Etomidate treatment led to damage in mitochondrial biogenesis by decreasing the mtDNA/nDNA ratio, reducing the protein expression of cytochrome B, and lowering mitochondrial mass. These changes suggest impaired mitochondrial replication and function. Correspondingly, etomidate exposure induced a "loss of mitochondrial function" by diminishing the activities of Complex I and Complex IV, the mitochondrial respiratory rate (MRR), and ATP generation. These effects highlight the detrimental impact of etomidate on the energy metabolism of glioma cells. Mechanistically, etomidate inactivated the transcription factor CREB by reducing its phosphorylation at Ser133. Activation of CREB with the second messenger cAMP restored the expression of PGC-1α, the mtDNA/nDNA ratio, Complex IV activity, summarized mitochondrial respiratory rate (MRR), and ATP production. This suggests that CREB activation may serve as a potential therapeutic strategy to counteract etomidate's inhibitory effects on mitochondrial function in glioma cells. Our results suggest that damage to mitochondrial biogenesis is a key step in the anticancer properties of etomidate in gliomas, and the decrease in PGC-1α and its downstream molecules may be the critical mechanism behind this effect.

Grifola frondosa antioxidant peptides (GFAP) were prepared through trypsin enzymolysis and characterized. This study conducted a comprehensive assessment of clinical symptoms, colon pathological injuries, levels of inflammatory factors, expression of inflammation-related proteins, and alterations in gut microbiota composition in mice with ulcerative colitis (UC). The findings demonstrated that GFAP effectively mitigated UC, alleviated mucosal damage, and reduced inflammatory infiltration. Specifically, GFAP administration resulted in significant reductions in pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, while enhancing the expression levels of tight junction proteins such as Occludin and ZO-1. Additionally, GFAP treatment led to decreased levels of Toll-like receptor 4 (TLR-4), inducible nitric oxide synthase (iNOS), and TNF-α. Noteworthy, GFAP also influenced the gut microbiota by decreasing the abundance of Proteobacteria and increasing Bacteroidetes and Firmicutes. Moreover, specific bacteria like Bacteroides uniformis and Alistipes exhibited elevated abundances following GFAP treatment. In summary, GFAP exhibited preventive and protective effects against UC in mice by effectively alleviating clinical symptoms and modulating gut microbiota composition.

Lymphatic filariasis (LF) is a mosquito-borne disease caused by parasitic nematodes Brugia malayi, Brugia timori, and Wuchereria bancrofti. The drugs available are effective in several cases, and the absence of vaccination is the crucial factor hindering the elimination of LF. The UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase (MurE) plays an important role in the peptidoglycan biosynthesis of Wolbachia endosymbiont B. malayi, which are reported to be a vital drug target for bacterial and endosymbiotic hosts. Thus, we selected the ATP binding cavity of MurE as the potential site to screen inhibitors. The MurE structure was modeled using AlphaFold due to the absence of an experimental structure. Structure-based screening identified five potent phytochemicals targeting the ATP binding site with higher Glide scores and affinity. The top five phytochemicals CID 311, CID 445713, CID 441626, CID 39077, and CID 10814 showed a docking score of -16.812, -16.117, -15.668, -15.324, and -13.442 kcal/mol, respectively. Further, the molecular dynamics simulations depicted the binding stability of the phytochemical inhibitors bound to the MurE complex. Moreover, ADME assessment and Density Functional Theory analyses of the predicted compounds have shown acceptable pharmacokinetic properties and high reactivity with the drug target of MurE.

Colorectal cancer (CRC) progression involves complex genetic changes. This study examines circRNA expression in CRC to identify biomarkers for improved diagnosis and staging. The objective of this study is to explore the role of circular RNA (circRNA) in CRC progression and identify specific circRNA biomarkers. Using high-throughput circRNA chip technology, cancerous and adjacent tissues from three CRC patients (staged as T1-3N0M0) were analyzed to identify differentially expressed circRNAs. Bioinformatics analyses, including co-expression network construction, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, were performed to evaluate circRNA function and pathways. A total of 404 differentially expressed circRNAs were identified, with significant variations between cancerous and adjacent tissues. Trend analysis revealed that circRNA expression decreased progressively with CRC advancement. Co-expression network analysis highlighted eight key circRNAs, including hsa_circ_0000007, associated with CRC progression. GO and KEGG analyses indicated these circRNAs are involved in ribosome biogenesis, metabolism, and the regulation of G1-S phase transcription through the RB1 gene. The expression of hsa_circ_0000007, hsa_circ_0023608, hsa_circ_0026694, and hsa_circ_0029903 decreased as CRC progressed, suggesting their potential as biomarkers for CRC diagnosis and staging. These findings offer insights into the molecular mechanisms underlying CRC progression.

Actinobacillus succinogenes is a ruminal microorganism of biotechnological importance due to its capacity to produce succinic acid at high yields. Despite the scientific interest in this organism, molecular vehicles for the transfer and expression of genetic material are limited compared to the existing demand. To facilitate gene cloning and expression in A. succinogenes, we report the generation and characterization of two novel shuttle expression vectors containing the chloramphenicol acetyltransferase gene (catA) as a selection marker and replication origins from A. succinogenes, other members of the Pasteurellaceae family, and Escherichia coli. Vector pAVP_trc includes the following features from the E. coli expression vector pTrc99A2: lacI^q gene, an IPTG-inducible trc promoter (P_trc), the lacZ ribosome-binding site, a multiple cloning site, and the rrnB transcription terminator. The second novel vector pAVP_mdh contains 200 bp of the promoter region (P_mdh) from the A. succinogenes malate dehydrogenase (MDH) gene Asuc_1612 (mdh). Gene mdh was cloned in the two novel vectors to generate pAVP_trcmdh and pAVP_mdhmdh. Promoter activity in these vectors was determined by measuring transcript levels with RT-qPCR analysis and MDH specific activity. In cultures with A. succinogenes/pAVP_trcmdh with 2 mM IPTG, a two-fold increase in MDH specific activity and a 22-fold increase in mdh transcript level were observed. In the case of pAVP_mdhmdh, a four-fold increase in MDH-specific activity and a 19-fold increase in mdh transcript level were observed. Analysis by qPCR showed plasmids pAVP_trc, pAVP_trcmdh, pAVP_mdh, and pAVP_mdhmdh to be present in a range of seven to nine copies per cell.

This article delves into the application of design of experiments methodologies for the integration of a second-order definitive screening design (DSD) and artificial neural network (ANN) to comprehensively assess and predict nine operational variables aimed at increasing the yield of α-amylase from Bacillus velezensis species. By utilizing environmentally friendly and cost-effective agro-solid substrates such as moong husk and soya bean cake, the physical and chemical parameters influencing α-amylase biosynthesis from B. velezensis species were optimized, gathering early data from experiments conducted in shake flasks through the standard one-factor-at-a-time (OFAT) technique. In the realm of response surface methodology (RSM) utilizing the DSD model, nine process variables were taken into account, including pH, temperature, carbon source, nitrogen source, K₂PO₄, MgSO₄, NaCl, fructose, and NaNO₃. Furthermore, optimization based on ANN modeling was employed to enhance the enzyme yield further. Experiments were then executed under the optimal conditions as defined by RSM and ANN to corroborate the predicted optimized enzyme activity. As a result, B. velezensis species exhibited enzyme activity of 1092.49 U/mL under the optimal process variables identified by both RSM and ANN optimization methods, which included pH 5.48, temperature (34.28°C), carbon source (4.09%), nitrogen source (2.02%), K₂PO₄ (0.34%), MgSO₄ (0.14%), NaCl (0.23%), fructose (1.54%), and NaNO₃ (0.53%). To encapsulate, compared to the OFAT technique, where the enzyme activity was 418.25 U/mL, a 2.6-fold increase in enzyme activity was achieved by integrating DSD and ANN optimization, considering only nine significant process parameters for the proliferation of B. velezensis species and the maximization of α-amylase activity. The α-amylase enzyme from the B. velezensis species was further purified and characterized. The purification process achieved a 71.77-fold increase in specific activity, with the purified enzyme exhibiting optimal activity at pH 5.5 and 55°C. The enzyme displayed high thermal stability, with minimal activity loss up to 4°C. Kinetic analysis revealed a K_M of 0.85 mg/mL and a V_max of 250 U/mg/min. The enzyme was found to be metal-independent, with inhibition observed for certain metal ions.

Dengue fever, a mosquito-borne viral infection, poses a significant global health challenge, particularly in tropical and subtropical regions. The absence of non-effective vaccines and specific treatments underscores the need for advanced diagnostic tools for early detection and management. This study presents a novel biosensor for detecting dengue virus type 4 (DENV-4) by combining carbonyldiimidazole nanoflower (CDI-NF) with Mn₃O₄ on laser-scribed graphene (LSG). Material characterization techniques, including Raman spectroscopy, TEM, XRD, XPS, and FTIR, were employed to confirm the successful integration of Mn₃O₄ and CDI-NF, resulting in a unique 3D flower-like structure. In order to verify the sensing efficiency, a selective DNA sample captured on LSG/Mn₃O₄-CDI-NF was investigated for specifc binding with Aedes aegypti target DNA through selective hybridization and mismatch analysis. Electrochemical impedance studies further confirmed sensitive detection of up to 1 fM, where the sensitivity was confirmed by large transfer resistance (R_ct) before and after hybridization with a regression coefficient 0.97373. EIS results demonstrated successful surface modifications and the biosensor's specificity in distinguishing between complementary, mismatched, and non-complementary target sequences. The biosensor's ability to differentiate between these sequences highlights its potential for accurate and targeted DENV-4 detection, offering a promising avenue for advancing dengue diagnostics.