Applied Biochemistry and Biotechnology最新文献

Myocardial infarction (MI) is a coronary artery-related disease that seriously threatens human life and is the leading cause of sudden death worldwide, where a lack of nutrients and oxygen leads to an inflammatory response and death of cardiomyocytes. Ferroptosis is a form of non-apoptotic cell death associated with metabolic dysfunction, resulting in abnormal breakdown of glutamine and iron-dependent accumulation of reactive oxygen species (ROS) during metabolism. However, the molecular mechanism of ferroptosis in the pathogenesis of MI and the function of Klotho and KRAS on ferroptosis during MI remain unclear. The MI rat model was established by LAD ligation with a 6-0 suture. H9c2 cells were placed in glucose-deficient DMEM (Thermo) and cultured in an anaerobic environment (1% CO₂ and 5% CO) to establish an in vitro OGD cell model. The damage to rat heart tissue was detected by HE staining, and Klotho and KRAS were determined by RT-qPCR, Western Blot, and IHC. TUNEL staining was used to determine apoptosis in rat heart tissue samples. The interaction between Klotho and KRAS was verified by co-immunoprecipitation and Western Blot. The cardiomyocyte activity was measured by CCK-8 assay. LDH, CK-MB, cTnT, and Fe²⁺ markers were detected by the kits. For the assessment of ferroptosis, GSH and ROS in cardiomyocytes and serum were detected by kits, and PTSG was detected by Western Blot. IL-1β and IL-6 in cardiomyocytes and serum were determined by ELISA. Klotho was downregulated in MI. Downregulation of Klotho promoted myocardial injury; increased apoptosis of cardiomyocytes; promoted LDH, CK-MB, and cTnT concentrations; inhibited GSH; and promoted ROS levels, PTGS2 expression, and ferroptosis in rats. The same results were obtained in vitro. Klotho and KRAS had endogenous interactions. KRAS knockdown can reverse Klotho knockdown-mediated MI and ferroptosis. RAP1/ERK pathway was highly expressed in MI, and inhibiting RAP1/ERK pathway activation can reverse the promoting effect of overexpressed KRAS on MI progression and ferroptosis. Klotho interacts with KRAS and inhibits ferroptosis after MI by regulating the RAP1/ERK pathway.

Gymnostachyum febrifugum, a less-known ethnomedicinal plant from the Western Ghats of India, is used to treat various diseases and serves as an antioxidant and antibacterial herb. The present study aims to profile the cytotoxic phytochemicals in G. febrifugum roots using GC-MS/MS, in vitro confirmation of cytotoxic potential against breast cancer and an in silico study to understand the mechanism of action. Phytochemical profiling using GC-MS/MS showed the presence of eight cytotoxic molecules with lupeol in high abundance. A potent cytotoxic effect of G. febrifugum roots against breast cancer was also observed with antiproliferation, antimigration, inhibition in colony formation and death of breast cancer cells. Further, the cytotoxic potential of the plant was confirmed with the apoptosis of cells as observed in the flow cytometry. In silico network pharmacology, GO and KEGG analysis suggested the modulation of proteins of MAPK, PI3K-AKT and apoptosis pathways by lupeol to induce cytotoxicity in breast cancer. Further, dynamic simulation revealed MAPK and AKT as the major targets for lupeol. Our studies comprehensively elucidated the role of lupeol, a major phytochemical in G. febrifugum to induce cytotoxicity against breast cancer by targeting major cancer signaling pathways, providing a promising strategy and scientific basis to explore lupeol in targeted cancer therapy.

Antarctic organisms are known for producing unique secondary metabolites, and this study specifically focuses on the less-explored metabolites of the moss Warnstorfia fontinaliopsis. To evaluate their potential bioactivity, we extracted secondary metabolites using four different solvents and identified significant lipase inhibitory activity in the methanol extract. Non-targeted metabolomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on this extract predicted the presence of 12 compounds, including several not previously reported in mosses. To gain insights into their enzyme inhibitory activity, the binding affinities of these candidate compounds to lipase were evaluated through in silico molecular docking. Further validation by molecular dynamics (MD) simulations revealed that hyocholic acid and pheophorbide A form stable complexes with human pancreatic lipase (HPL). Based on these results, targeted fractionation experiments were performed, yielding eight fractions. Among these, Fractions 4 and 6, which are assumed to contain those compounds, exhibited higher lipase inhibitory activity compared to the crude extract. Additionally, pharmacokinetic properties of those compounds were analyzed using SwissADME and Molinspiration calculations, suggesting their potential as drug candidates. This study establishes a promising methodology for identifying rare bioactive compounds of low abundance in underexplored natural resources by combining LC-MS/MS analysis with molecular docking. These findings also provide new insights into the chemical ecology of Antarctic mosses and their potential applications in pharmaceutical development.

Cellulase was effectively immobilized onto an epoxy-bound chitosan-modified zinc metal-organic framework (epoxy/ZIF-8/CS/cellulase) support, yielding a conjugation rate of 0.64 ± 0.02 mg/cm2 and retaining 80.01 ± 0.01% of its specific activity. The bare and cellulase-bound supports was characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, atomic force microscopy and energy-dispersive X-ray spectroscopy. The immobilized enzyme exhibited optimal activity at pH 5.5 and a temperature of 70 ℃. The efficiency, stability and reactivity of the enzyme improved after immobilization, as evidenced by a decrease in activation energy, enthalpy and Gibbs free energy along with an increase in entropy change. The epoxy-affixed ZIF-8/CS/cellulase strip was successfully employed for rice husk hydrolysis achieving an impressive conversion efficiency of 95%. The method demonstrated a linear range from 0.1 to 0.9% (0.1 × 10^-2 to 0.9 × 10^-2 mg/ml) and exhibited a strong correlation (R² = 0.998) with the widely adopted 3, 5-dinitrosalicylic acid method. The epoxy/ZIF-8/CS bound cellulase exhibited remarkable thermal stability, retaining 100% of its activity at 70 °C, in contrast to just 53% for the free enzyme and displayed a half-life of 21 days after storage at 4 °C compared to 9 days for the free enzyme. Furthermore, it retained over 95% activity after 12 h at pH levels of 4.5 and 5.5 and showcased excellent reusability, maintaining activity over 25 cycles. Overall, this method offers high conversion efficiency and selectivity under benign conditions, with no undesirable by-products, making it a cost-effective solution for the routine hydrolysis of lignocellulosic biomass feedstock.

Doxorubicin (DOX) is a commonly used chemotherapeutic medication for treating malignancies, although its cardiotoxicity limits its use. There is growing evidence that alteration of the mitochondrial fission/fusion dynamic processes accompanied by excessive reactive oxygen species (ROS) production and alteration of calcium Ca²⁺ homeostasis are potential underlying mechanisms of DOX-induced cardiotoxicity (DIC). Metformin (Met) is an AMP-activated protein kinase (AMPK) activator that has antioxidant properties and cardioprotective effects. The purpose of the study is to assess Met's possible cardioprotective benefits against DOX-induced cardiotoxicity. The study included 32 adult male rats. They were randomly divided into four groups: administered saline, DOX, Met, or DOX combined with Met respectively. Heart tissues were used for biochemical assays that measured oxidative stress markers, malondialdehyde (MDA), reduced glutathione (GSH), mitochondrial dynamics markers, optic atrophy-1(OPA-1) and dynamin-1-like protein (Drp1), calcineurin and caspase-3. Serum levels of myocardial injury markers, cardiac troponin I (cTn-I), and aspartate aminotransferase (AST), were also measured. The results revealed that DOX intoxication was associated with a significant increase in the levels of serum cTn-I and AST, increased cardiac MDA level, increased cardiac Drp1, calcineurin, and caspase-3 expressions, as well as reduced cardiac GSH level and cardiac OPA-1 expression. On the other hand, Met treatment significantly reduced DIC by decreasing oxidative stress, apoptosis, and improving mitochondrial and calcium balance. Finally, this study shows that Met may be able to protect the heart from damage caused by DOX by working as an antioxidant and anti-apoptotic agent and keeping the balance of calcium and mitochondria.

We report the first in situ reaction of the β-haloketones obtained from laccase catalysed oxidation of secondary alcohol 2-halo phenylethanol's in present study. To the best of our knowledge, this is the first ever fusion of laccase catalysed oxidation reaction with green organic synthetic reaction. The methodology employs molecular oxygen to oxidize secondary alcohol in biphasic medium by laccase from T. giganteum AGHP, to obtain β-haloketones. This research provides environmentally benign access to thiazole, β-ketosulfone and imidazopyridine derivatives in good to very good yields with wide functional group tolerance.

Caves are a unique ecosystem that harbor diverse microorganisms, and provide a challenging environment to the dwelling microbial communities, which may boost gene expression and can lead to the production of inimitable bioactive natural products. In this study, we obtained 59 actinobacteria from four different caves located in Bahadurkhel, District Karak, Pakistan. On the basis of taxonomic characteristics, 30 isolates were selected and screened for secondary metabolites production and bioactivity profiling. The extracts of all the isolates exhibited promising antibacterial activity against several pathogenic bacteria, with the best outcome seen in the extract of isolate SNK 21. The metabolomic analysis of the extracts by LC-MS/MS-based molecular networking and whole genome sequencing (WGS) followed by antiSMASH analysis revealed the presence of diverse secondary metabolites and biosynthetic gene clusters (BGCs) in SNK 21. Purification of compounds by manual chromatography, HPLC, and characterization by NMR, HR-MS, led to the identification of the active compounds, actinomycin D and its isomer. In addition, metabolomic analysis and genome mining of morphologically distinct isolates, SNK 202 and SNK 329, also showed diverse secondary metabolites and BGCs, underscoring the potential of actinobacteria from undisturbed caves in Pakistan as a new source of bioactive compounds.

Nelumbo nucifera, an aquatic crop cultivated throughout Asian countries, belongs to the Nelumbonaceae family and has been widely used in traditional medicines with key pharmacological activities such as anti-viral, antipyretic, antioxidant, anti-steroid, anti-inflammatory, anti-arrhythmia, anti-obesity, and anti-aging properties. The present study aims to explore and assess the phytochemical composition, GC-MS profiling, antioxidant efficacy, and the major phytoconstituent phytol subjected to theoretical spectroscopic characterization using the DFT method. The phytochemical profiling of N. nucifera reveals the presence of alkaloids, carbohydrates, saponin, phenol, and flavonoids. The antioxidant efficacy of N. nucifera extract against DPPH and ABTS radicals increased in a concentration-dependent manner, with an IC₅₀ value of 222.84 µg and 52.67 µg, respectively. The simulated structural parameters of phytol exhibited strong concordance with experimental values. The simulated wavenumbers identified characteristic peaks corresponding to hydroxyl (OH), methylene (CH₂), and methyl (CH₃) groups. The simulated electronic spectrum of phytol exhibits a prominent absorption peak at 174 nm, predominantly attributed to the transitions H-1 → L (58%) and H → L (36%). NBO analysis reveals significant stabilization energy (7.09 kJ/mol) due to the donation of electrons from the C₂₀-H₅₈ bonding orbital to the anti-bonding orbital of C₁₈-C₁₉ via a σ → σ* transition. In Mulliken charge distribution, compared to other hydrogen, hydrogen H₆₁ in the hydroxyl (O-H) group exhibits a higher positive potential due to the influence of the oxygen atom. In addition, molecular docking was performed against breast cancer SMAD proteins to confirm its antagonist property, with binding energies of - 3.64 kcal/mol (6OM2), - 5.49 kcal/mol (1U7F), - 5.05 kcal/mol (1U7V), and - 3.73 kcal/mol (6FX4).

The presence of antibiotics in wastewater discharges significantly affects the environment, mainly due to the generation of bacterial populations with multiple antibiotic resistances. The cometabolic capacity of nitrifying sludge to simultaneously remove ammonium (NH₄⁺) and emerging organic contaminants (EOCs), including antibiotics, has been reported. In the present study, the removal capacity of 50 mg ampicillin (AMP)/L by nitrifying cultures associated with biosorption and biotransformation processes was evaluated in a sequencing batch reactor (SBR) system. The contribution of nitrifying enzymes (ammonium monooxygenase (AMO) and nitrite oxidoreductase (NOR)) and β-lactamases in AMP biodegradation was evaluated using specific inhibitors in batch cultures. AMP was 100% eliminated after 5 h since the first cycle of operation. The sludge maintained its ammonium oxidizing capacity with the total consumption of 102.0 ± 2.5 mg NH₄⁺-N/L in 9 h, however, the addition of AMP altered the nitrite-oxidizing process of nitrification, recovering 30 cycles later at both physiological and kinetic level. The kinetic activity of the nitrifying sludge improved along the operating cycles for both AMP removal and nitrification processes. The elimination of 24% AMP was attributed to the biosorption process and 76% to biotransformation, wherein the AMO enzyme contributed 95% to its biodegradation. Finally, the repeated exposure of the sludge to AMP for 72 operating cycles (36 days) was not sufficient to detect β-lactamase activity. The cometabolic ability of ammonium-oxidizing bacteria for biodegrading AMP could be employed for bioremediation of wastewater.

As a novel protein post-translational modification, lysine succinylation is widely involved in metabolism regulation. To describe succinylated lysine's physiological functions and distribution patterns in Saccharopolyspora erythraea, a large and global protein succinylome was identified in a hypersuccinylated strain E3ΔsucC, using high-resolution 4D label-free mass spectrometry. Bioinformatic analysis was conducted to examine the succinylated proteins further in this study. The results showed that succinylated proteins were identified to be predominantly involved in protein synthesis, central carbon and nitrogen metabolism, and secondary metabolism. The process of lysine succinylation was found intricately regulated by a delicate interplay of factors, such as the relative abundance of lysine within the protein, the strategic positioning of polar amino acids flanking the succinylated sites, and the degree to which lysine residues are exposed to the solvent, thereby shaping the landscape of post-translational modifications. This systematic analysis has represented the global analysis of lysine succinylation in S. erythraea and has provided an important resource for exploring the function and regulation of lysine succinylation in S. erythraea and likely in all actinomycetes.