Biochimie最新文献

Proteolytic enzymes from the bromeliad family are valuable biocatalysts with a broad range of biotechnological applications, thanks to their stability and broad substrate specificity. In this study, two novel cysteine proteases, AntB and AntC, were purified from ripe fruits of Bromelia antiacantha Bertol., using acetone precipitation and cation-exchange chromatography, thereby completing studies of the three main isoforms present in this plant. The functional characterization of both enzymes showed broad pH and temperature stability, with AntC retaining activity over pH 4.5-11 and both exhibiting optimal activity near 65 °C. Remarkably, AntB and AntC maintained activity in the presence of strong denaturants such as urea and guanidinium chloride, indicating high structural stability. Kinetic studies with synthetic substrates revealed differences in specificity and catalytic efficiency between the two enzymes and compared to stem bromelain, supporting their functional differences. Peptide mass fingerprinting further confirmed sequence homology with Fastuosain while highlighting structural divergences. Together, these findings establish AntB and AntC as robust and versatile proteases with promising potential for biotechnological, industrial, and biomedical applications and reinforce the value of underexploited bromeliad species as sustainable sources of biocatalysts.

The current immediate detection of influenza A (H1N1) virus faces the dual challenges of insufficient sensitivity and the risk of amplification contamination. Here, we developed a novel lateral flow nucleic acid biosensor using pre-amplified dsDNA tag (PADT). Linearized M13 phage was preamplified to generate a 3.5-kbp dsDNA product containing a 20-nt overhang probe and heavy biotin tags, followed by the formation of FAM probe/target RNA/PADT complexes by a sandwich-type detection strategy. RESULT: This technique achieves a 1 pM detection limit for H1N1-specific RNA, demonstrating 100-fold greater sensitivity than conventional lateral flow chromatography (p<0.01). This method eliminates nucleic acid amplification, preventing false positives from amplicon contamination. This method effectively prevents false positives induced by product contamination by avoiding direct amplification of the target nucleic acid. This stepwise strategy, marrying pre-amplification with point-of-need detection, achieves laboratory-grade accuracy while maintaining field applicability. The step-by-step strategy of pre-amplification and immediate detection combines laboratory precision with field applicability, and the design of interchangeable overhangs provides a platform solution for multi-virus detection.

Mogrosides are an important type of natural plant-derived sweeteners, which are used in foods and medicines. However, their sweetness and structure-activity relationship, especially those for the representative members mogrosides IV and V, have been rarely reported. In the present study, we characterized the sweet taste properties (threshold values, etc) of mogrosides IV and V with a cell-based calcium mobilization assay, showing that mogrosides represent a category of primitive sweet compounds which are edible by primates as well as other mammals. Furthermore, it was demonstrated that the sweetness of mogrosides can be inhibited by a classical sweet taste modulator, lactisole. Moreover, the binding site of mogroside IV in human Tas1R2 Venus flytrap domain was identified with the method of molecular docking, which was further validated by functional mutagenesis analysis. These results provide helpful guidelines for further exploring the structure-activity relationship and molecular design of mogrosides.

MicroRNAs play crucial roles in post-transcriptional regulation during environmental stress, yet their contribution to hypoxia adaptation in naturally hypoxia-tolerant species remains poorly understood. Here, we characterized the miRNA expression profile in kidneys of the naked mole-rat (Heterocephalus glaber), a subterranean rodent renowned for its exceptional hypoxia tolerance. Small RNA from naked-mole rat kidneys was sequenced under normoxic and hypoxic conditions to predict miRNA-mRNA interactions during low-oxygen stress. Bioinformatic analysis identified differentially expressed miRNAs and used pathway enrichment to predict regulatory mechanisms controlling kidney adaptation to hypoxia. Upregulated miRNAs, including let-7c-5p and miR-29a-3p target genes involved in cell cycle progression, extracellular matrix remodeling, and metabolic pathways, corresponding with negative enrichment of these processes. Conversely, downregulated miRNAs relieve inhibition of transcripts involved in chromatin remodeling, RNA processing, and immune signaling, aligning with positive enrichment of these adaptive pathways. Gene Ontology cellular component analysis suggested systematic subcellular reorganization, with suppression of extracellular and secretory compartments and enhancement of nuclear, RNA processing, and cytoskeletal structures. Notably, hypoxia induced upregulation of ribonucleoprotein complexes, spliceosomal machinery, and histone methyltransferase complexes, while downregulating extracellular matrix components and secretory pathway structures. This coordinated miRNA response appears to optimize energy utilization by suppressing non-essential pathways while selectively enhancing survival mechanisms through targeted post-transcriptional control; however further studies are required to confirm these findings. Our findings provide novel insights into the molecular mechanisms underlying the remarkable hypoxia tolerance of naked mole-rats and highlight miRNA-mediated regulation as a key adaptive strategy in mammalian hypoxic survival.

Snake venom is an ecologically critical functional trait, primarily applied for foraging and accordingly shaped by selective pressures. Recent insights underpinned the high variability of snake venoms down to the intraspecific level, with regional, ontogenetic, and seasonal variation being mostly investigated. In contrast, sex-based venom variation has received considerably less attention so far, and its influence on venom compositions is poorly described. Here, we compare venom profiles and bioactivity from pooled male and female samples of Central European adders (Vipera berus) to provide insights into potential sex-based venom variation in this species. Proteomics, paired with SDS-PAGE and RP-HPLC, revealed highly similar venom profiles. Likewise, phospholipases A₂ and proteases activity profiling, as well as bioassays targeting the effects of venom on the coagulation cascade and the viability of different mammalian cell lines revealed similar activity spectra. Our results do not suggest a noteworthy extent of sex-based intraspecific venom variation in V. berus. We further discuss our data in light of the species' venom composition at larger geographic scales and its clinical relevance. This work contributes to a clearer framework for understanding venom biology in the world's most widespread medically relevant venomous snake.

Orf virus, a member of the Parapoxvirus genus within the Poxvirus family, is the causative agent of Contagious Ecthyma, a zoonotic infection primarily affecting goats and sheep. The immune response against Orf virus is short-lived, enabling the virus to repeatedly infect animals, regardless of their vaccination status. Several virulence factors, including the OH1 tyrosine phosphatase, are responsible for the modulation of the host immune response. Here, we report the direct interaction between the viral OH1 and the human transcription factor STAT1 identified as a physiological OH1 substrate. Indeed, our results demonstrate that OH1 dephosphorylates STAT1 at pTyr701, resulting in a subsequent impairing of its nuclear import. By employing protein-protein docking and molecular dynamics simulations, we modelled the STAT1-OH1 complex, phosphorylated or not, and dissected the structural basis of its mutual recognition. Also, we identified additional potential substrates of OH1 that show to be involved in cell trafficking, thereby expanding our understanding of the host-triggered immune responses elicited by Poxviruses. Overall, this study provides insights into the molecular mechanisms underlying immune evasion in Orf virus, extending our knowledge towards new therapeutic strategies against this pathogen.

Ischemic brain damage is characterized by mitochondrial dysfunction and oxidative stress. Mitochondrial reactive oxygen species induce cellular damage during reperfusion. Dysregulated mitochondrial calcium influx triggers the opening of the mitochondrial permeability transition pore (MPTP), disrupting mitochondrial function and causing cell death. Dioclea violacea lectin (DVL) is a plant lectin which exhibits a diverse range of biological activities. Here, we aimed to investigate and characterize the effects of in vivo treatment with DVL in a rat model of ischemic stroke. DVL (0.5 μg) or saline were administrated by stereotaxic intracerebroventricular injection (volume of 1 μl) 15 mins prior to ischemia. Global cerebral ischemia was induced by bilateral carotid occlusion for 30 min. After 24 hours reperfusion we evaluated locomotor activity, oxidative stress markers (nitrite, thiobarbituric acid reactive substances - TBARS, mitochondrial H₂O₂ levels, and superoxide dismutase (SOD) activity), mitochondrial oxygen consumption and ADP/O (using a clark-type electrode). MPTP opening was determined by Ca²⁺-induced swelling. DVL (prior to ischemia) induced neuroprotective effects in rats` cerebral tissue. It restored the rats` exploratory behavior and mitigated depression. DVL-treated rats had improved brain mitochondrial oxygen consumption rates and ADP/O ratio. Additionally, DVL reduced oxidative stress (mitochondrial H₂O₂ production, TBARS, and nitrate levels) and preserved SOD activity. Finally, mitochondria isolated from DVL-treated rats had lower susceptibility to Ca²⁺-induced MPTP opening. Mechanistically, we found that DVL binds glutamate - an excitotoxic neurotransmitter highly released after ischemic insults. This study reveals a novel neuroprotective mechanism of a plant-derived lectin acting through mitochondrial preservation and oxidative stress reduction.

Bacterial cellulose (BC) is reported as an extracellular polysaccharide distinguished by its exceptional purity, mechanical features, and biocompatibility, making it an attractive material for applications in food, plant tissue culture, and Biomedicine. Traditionally, BC production relies on chemically defined synthetic media under either static or agitated fermentation conditions. However, the high production cost remains a critical barrier, limiting its large-scale utilization and broader adoption. To address this challenge, recent advances in sustainable strategies, such as employing agro-industrial byproducts and low-cost carbon sources, have significantly reduced costs while improving yield and functional properties. In particular, diverse environmental waste streams, including agricultural residues, industrial wastes, and food processing byproducts, have been explored as renewable substrates for BC synthesis. BC can be obtained through static fermentation, yielding gelatinous films (pellicles), or agitated fermentation, generating suspended fibers or pellets, each with unique structural features. Moreover, innovative functionalization approaches, including in situ and ex situ modifications, incorporation of bioactive agents, and the development of BC-based nanocomposites, have further expanded its biomedical potential. This review emphasizes sustainable strategies to overcome the cost limitations of BC production, while also highlighting recent advances in functionalization techniques and their pivotal role in advancing medical applications, including cartilage engineering, bone regeneration, wound healing, and dentistry.

The importance of lysine 2-hydroxyisobutyrylation (Khib) in regulating biological processes has become increasingly apparent, but its functional significance in prokaryotes, especially in the Lactobacillus paracasei PC-01, is poorly understood. In this study, the regulatory mechanism of Khib in high-density fermentation of L. paracasei PC-01 was investigated using mass spectrometry proteomics. In total, 6783 Khib sites on 1361 proteins were identified, with 73.48% proteins containing multiple sites. GO and KEGG enrichment analysis showed Khib affected cellular functions and metabolic pathways, especially those involved in ribosomal activity and protein biosynthesis. Subsequently, a Khib-mediated regulatory network of L. paracasei PC-01 revealed most of the significantly upregulated enzymes in the glycolysis, TCA cycle, purine metabolism, amino acid metabolism, and fatty acid biosynthesis pathways were modified by Khib. This study elucidated the biological function of Khib modification in L. paracasei during high-density cultures and provides theoretical basis for the preparation of highly active probiotic preparations.

Chronic lead exposure poses severe threats to human health, which demands a rapid detection strategy beyond conventional instrumentation-dependent approaches. While CRISPR/Cas12a systems offer promising alternatives through trans-cleavage activity, conventional Pb²⁺ biosensors relying on DNAzyme-generated intact activators suffer from high background signals due to interference from uncleaved substrates. To address this limitation, we developed a steric-hindrance-controlled activation strategy by employing a chimeric DNAzyme substrate (Sub) that prevents Cas12a binding until Pb²⁺-dependent cleavage occurs. This DNAzyme-mediated splitting releases two fragments (A1/A2) that rearrange into split activators, triggering the CRISPR/Cas12a trans-cleavage of a quenched reporter (6-FAM/BHQ1). Under the optimal condition, the sensor achieved a linear detection range of 2.5-25 μM (R² = 0.998) with 2.18 μM LOD and high selectivity against interferents. Validation in tap water matrices demonstrated 98.6%-102.6% recovery (RSD 3.0%-7.5%), which showed robustness in real samples. This split-activator design paradigm eliminates background from uncleaved substrates without additional pretreatment steps to provide a versatile template for converting metal ions into CRISPR-detectable signals.