Search Results (1,843)

Background: Stimuli-responsive hydrogels (SRHs) are smart polymeric materials that undergo reversible physicochemical changes in response to abiotic cues and externally applied fields, enabling applications in drug delivery, wound healing, and tissue engineering. However, they exhibit limited biological specificity and do not adequately reflect the dynamic, disease-relevant complexity of native tissue microenvironments. Microbe-colonized tissues display distinctive biochemical features driven, shaped by microbial metabolism, including localized pH gradients, short-chain fatty acid production, secretion of quorum-sensing molecules, biofilm formation, and expression of specialized enzymes. These endogenous, spatiotemporally regulated signals are closely linked to host physiology and pathology but remain underutilized in hydrogel design. This review aims to highlight microbiome-responsive hydrogels (MRHs) as a strategy to address this gap. Methods: This study summarizes current engineering approaches, key microbial stimuli, and emerging biomedical applications of MRHs, with emphasis on translational and regulatory challenges. Results: Microbiome-responsive hydrogels (MRHs) address this gap by leveraging microbial metabolic and biochemical cues to induce swelling, degradation, drug release, antibacterial activity, or structural transformation. By directly coupling to microbe-derived stimuli, MRHs offer improved physiological relevance, enhanced local specificity, and new opportunities for precision therapy targeting disease-associated microbial niches. Conclusions: Despite their promise, MRHs remain an early and fragmented field, lacking standardized biological triggers, material design frameworks, and performance evaluation strategies. This review summarizes current engineering approaches, key microbial stimuli, and emerging biomedical applications, with emphasis on translational and regulatory challenges, positioning MRHs as an underexplored platform for next-generation smart biomaterials. Full article

Soil contamination by per- and polyfluoroalkyl substances (PFAS) represents a pressing environmental and public health concern due to the exceptional persistence of carbon–fluorine bonds, which prevent natural attenuation and limit the effectiveness of conventional remediation. Agricultural and industrial soils serve as long-term sinks for PFAS, continuously releasing these pollutants into groundwater and facilitating their transfer through the food chain. Conventional chemical and physical remediation methods are often costly, energy-intensive, and yield incomplete removal, underscoring the need for sustainable and biologically driven alternatives. Microbial consortia have emerged as a promising solution due to their metabolic complementarities, cross-feeding interactions, and ecological resilience, which together enable PFAS transformation and partial defluorination under complex soil and subsurface conditions. Key enzymes such as oxygenases, reductive dehalogenases, and hydrolases are often operating within co-metabolic networks, which play central roles in these processes. Advances in metagenomics, CRISPR-based functional screening, and metabolic modelling are rapidly uncovering novel PFAS-degrading microbes and pathways. Integration of machine learning with multi-omics and environmental datasets further enables the prediction of degradation mechanisms, identification of keystone degraders, and rational design of synthetic consortia. Emerging sustainable strategies, including biochar- and nutrient-amended soil microcosms, plant–microbe partnerships for coupled soil–groundwater phytoremediation, and bioelectrochemical systems that offer new avenues for enhancing PFAS biodegradation in situ. This review synthesises recent research progress and provides critical perspectives on the mechanistic, ecological, and engineering dimensions of PFAS bioremediation, proposing an integrated conceptual framework linking microbial consortia dynamics, enzymatic pathways, and environmental engineering interventions to guide scalable field applications and sustainable management of PFAS-contaminated soil–groundwater ecosystems. Full article

Silphium perfoliatum is a promising economic plant rich in bioactive secondary metabolites, yet the molecular regulation of phenylpropanoid biosynthesis across development remains unclear. To elucidate the regulatory networks underlying these metabolic processes, we integrated metabolomic and transcriptomic analyses across six developmental stages, from cotyledon to flowering. LC–MS/MS identified 1964 metabolites, with phenylpropanoids representing the largest class (601 compounds). Differential accumulation analysis showed pronounced temporal dynamics in phenylpropanoid levels, especially chlorogenic acid and its derivatives, with many compounds peaking at the flowering stage. In parallel, RNA-seq revealed 31,624 differentially expressed genes (DEGs). Functional enrichment highlighted phenylpropanoid and flavonoid biosynthetic pathways as major metabolic hubs. Correlation analysis indicated that PAL, 4CL, HCT, F3H, FLS, and F3′H expression was tightly coordinated with the accumulation of phenolic acids and flavonoids, suggesting these gene encoded enzymes may represent rate-limiting steps. Furthermore, weighted gene co-expression network analysis (WGCNA) identified a “blue” module strongly associated with phenylpropanoid accumulation and significantly enriched in pathway-related genes. Together, these results provide a comprehensive regulatory framework for phenylpropanoid biosynthesis in S. perfoliatum and offer valuable genetic targets for metabolic engineering and molecular breeding to enhance bioactive compound production. Full article

As an isoflavone metabolite with diverse physiological activities, the development of efficient and sustainable manufacturing technologies for (S)-equol holds significant importance. This study focuses on the semi-rational design of daidzein reductase (DZNR), the first key enzyme in the (S)-equol biotransformation pathway. Through multiple sequence alignment and three-dimensional structural analysis, two critical residues, Gly30 and Ala105, were identified in DZNR. A library of single and combinatorial mutants was constructed and screened, yielding the double variant DZNR30S+105S with substantially enhanced catalytic performance. In a whole-cell biocatalytic system, the recombinant E. coli (Escherichia coli) strain harboring this combinatorial mutant achieved a yield of 238.3 mg/L (S)-equol at a substrate concentration of 1 mM daidzein, demonstrating markedly improved catalytic efficiency. Upon increasing the daidzein concentration to 2 mM, the reaction reached equilibrium within 5 h, producing 384.6 mg/L (S)-equol, which highlights the mutant’s excellent potential for high-substrate-concentration applications. This study not only provides novel mechanistic insights into DZNR catalysis but also successfully establishes a DZNR variant with enhanced activity, offering an efficient biocatalytic component for the industrial-scale biomanufacturing of (S)-equol and thereby advancing the development of green biosynthesis technologies for this valuable compound. Full article

The ability of enzymes to recognize and process structurally diverse substrates is fundamental to metabolic flexibility and biological regulation. In melanin biosynthesis, human tyrosinase (Tyr) catalyzes the oxidation of several chemically distinct intermediates, including L-tyrosine, L-DOPA, DHICA, and DHI. Although its catalytic chemistry is well established, the structural basis of substrate selectivity and how it is altered by disease-associated mutations remains unclear. Using molecular docking and molecular dynamics simulations, we mapped the Tyr active site and identified 23 evolutionarily conserved residues that mediate multi-substrate recognition and binding. Across all substrates, binding induces coordinated conformational responses, particularly within an anchoring region (334–347) that provides electrostatic and hydrophobic steering, and a flexible gating loop (374–386) that modulates access and stabilizes bound intermediates. The OCA1B-associated P406L mutation, although distant from the catalytic core, disrupts long-range dynamic coupling and impairs loop flexibility, while 25 ClinVar-listed genetic variants at substrate-interacting residues weaken active-site organization, underscoring the sensitivity of Tyr’s dynamic network to perturbation. Integrating these findings, we propose an ordered multi-substrate binding mechanism in which substrates are first guided by the anchoring region, then aligned by the universal triad, and finally refined through loop-mediated, substrate-specific contacts. Our work suggests a dynamic framework that could be useful for understanding human tyrosinase catalysis, genetic mutation impact, and future engineering strategies. Full article

Ochratoxin A (OTA) is a globally distributed mycotoxin that poses serious threats to food safety and human health due to its nephrotoxic, hepatotoxic, and carcinogenic properties. Previous enzymatic detoxification strategies for OTA have been constrained by low degradation efficiency or poor soluble expression of highly active enzymes. In this study, a bacterial strain with strong OTA-degrading activity was isolated and identified as Acinetobacter kookii AK4, which degraded 95.44% of 1 μg/mL OTA within 6 h. The predominant OTA-degrading activity was derived from intracellular enzymes. Through genome mining and experimental validation, gene2102 was identified as encoding an amidohydrolase. The enzyme was designated AMH2102 and was heterologously expressed in Escherichia coli. Codon optimization combined with fusion of an N-terminal SUMO tag increased the soluble expression of AMH2102 by 14.81-fold, enabling complete (100%) OTA degradation within 3 min. Overall, this study achieved the identification of an efficient OTA-degrading strain and enzyme and explored strategies for improving enzyme expression, yielding effective outcomes that provide useful references for future studies on strain mining and enzyme engineering. Full article

Bovine chymosin is key for cheese production, yet its traditional sourcing is unsustainable. While microbial and plant-based alternatives exist, they often cause non-specific proteolysis, leading to bitter flavors in cheese. This study aims to develop a high-yield, methanol-independent platform for recombinant bovine chymosin production by engineering the expression system of Komagataella phaffii through multi-factorial optimization. Initially, the native bovine prochymosin gene (pcw) was codon-optimized (pcm14) and cloned, along with an mCherry-tag construct (clpcm14), into inducible vector pPIC9 for expression in Komagataella phaffii GS115. Screening identified the fusion-tagged strain clp2-91 as the highest producer. Subsequently, the inducible AOX1 promoter in the previously selected clp2-91 strain was replaced with a constitutive GAP promoter, yielding engineered strain GH1. Cultivated in a 3L fermenter, GH1 exhibited a volumetric productivity of 105.03 SU/(mL·h), twice that of inducible strain clp2-91 (53.59 SU/(mL·h)). The further optimization of fermentation conditions (pH 4.0, glucose as carbon source, fed-batch mode) boosted the enzyme activity of GH1 to 12,000 SU/mL. The recombinant chymosin exhibited enzymatic properties similar to those of the native enzyme and, importantly, demonstrated a broader pH stability (pH 2.0–6.0). This study demonstrates an efficient strategy for chymosin expression in K. phaffii, offering insights that may support the future development and optimization of heterologous protein production in this yeast. Full article

RNA editing is a way to diversify, regulate expression, and expand the cell transcriptome. The most common RNA editing is the reversible conversion of adenosine (A) to inosine (I) driven by double-stranded RNA-binding adenosine deaminases (ADARs). As inosine is recognized as guanosine (G) during translation, the RNA editing may result in non-synonymous codon changes. For this reason, ADARs have gained attention as promising enzymes to rewrite mRNA. Many efforts were undertaken to engineer a precise, effective, and controllable ADAR-based system to target certain Adenines on RNA to repair pathological mutations. This review summarizes the advances in ADAR-mediated RNA editing, evolving from systems using antisense oligonucleotides as guide RNA to recruit endogenous or overexpressed ADARs, through more complex setups additionally expressing other RNA-binding proteins, to rational designs harnessing ADARs to convert other nucleotides and amplify the low initial signal. Increasing the specificity and yield of RNA editing, expanding the number of targetable sites, and reducing off-target and bystander activity remain key challenges for these technologies. Improving delivery efficiency across a broad range of cell types, as well as optimizing delivery routes in in vivo studies are also critical to harness them as advantageous tools for both research and therapy. Full article

Advances in affordable genetic engineering have accelerated the creation and large-scale environmental release of genetically modified microorganisms (GMMs). While beneficial applications exist, GMMs may present unique, long-term risks to human and environmental health. Unlike static chemicals, GMMs are biologically active, self-replicating entities capable of rapid mutation and global dispersal. Current regulatory frameworks place responsibility on each country to regulate GMMs, without a clear, coordinated international policy. This review details critical risk scenarios, including horizontal gene transfer to native species and the possible disruption of vital human microbiomes (gut, oral, and infant), which could increase resistance to degradation, promote traits that expand a microbe’s range of hosts or ecological niches, and enhance the production of novel metabolites with unexpected biological activity. In soil, GMMs may support the emergence of “super bugs” or destabilize carbon sequestration cycles, potentially impacting climate resilience. Engineered microbial enzymes in the food supply may also act as environmental drivers of autoimmunity. Given the limited understanding of microbial ecology, we propose a decision-based biosafety workflow emphasizing pre-release risk assessment and continuous post-release monitoring. We urge national and international regulators to adopt the precautionary principle to better protect human health and the environment from the potential negative outcomes of GMMs. Full article

Hepatitis C virus (HCV) chronically infects over 50 million people worldwide and poses a significant risk to global health. The HCV NS3/4A complex, a bifunctional enzyme comprising a protease and a helicase domain, is indispensable for viral replication and immune evasion, making it a pivotal target for direct-acting antiviral agents (DAAs). Here, we summarize its structural features, functional mechanisms, and implications in drug design and protein engineering (e.g., nanopore sequencing applications). The NS3 protease domain is activated by the NS4A cofactor, which mediates viral polyprotein processing and relies on a zinc-binding site for structural stability. The C-terminal helicase domain catalyzes ATP-dependent 3′→5′ unwinding, and allosteric crosstalk between the protease and helicase domains dynamically modulates the enzymatic activity, balancing unwinding velocity and processivity. Beyond supporting viral replication, NS3/4A cleaves MAVS to abolish RIG-I/MDA5 signaling but spares TRIF, leaving TLR3-mediated immunity intact; it also modulates host lipid and iron metabolism, contributing to HCV pathogenesis. Notably, structural and functional studies of NS3/4A lay a solid theoretical foundation for developing novel therapeutic strategies. Currently, DAAs targeting NS3/4A have achieved high sustained virologic response rates; however, resistance-associated substitutions remain a major clinical challenge, particularly in genotype 3 infections. Emerging therapeutic strategies targeting NS3/4A include allosteric inhibition and proteolysis-targeting chimeras (PROTACs)-mediated degradation. Full article

Ophiocordyceps xuefengensis is an important medicinal fungus with considerable pharmaceutical and economic value. However, its industrial and scientific utilization has been severely limited by the lack of an efficient genetic transformation system, largely due to limited genomic information and wild growth. In this study, we established an efficient and stable plasmid transformation system within O. xuefengensis protoplasts mediated by PEG. To overcome low protoplast yield and transformation efficiency, key factors influencing protoplast preparation including enzyme composition and concentration, fungal age, and digestion conditions were systematically optimized. The optimal protocol involved digesting 4-day-old mycelia with a mixture of 1.5% lywallzyme 1 and 1.5% snailase at 34 °C and 130 rpm for 3.5 h, yielding at least 9.42 × 10⁷ CFU/mL protoplasts. Protoplast regeneration was significantly enhanced in PY medium supplemented with 0.6 M mannitol. Under these optimized conditions, a transformation efficiency of 45.5% was achieved, with stable plasmid integration confirmed over four successive generations. Furthermore, the transformation system was successfully applied to functional gene characterization by driving exogenous gene expression using the endogenous gpd1 promoter. This study provides a foundational platform for functional gene analysis and paves the way for further elucidation of growth and development mechanisms and metabolic engineering in O. xuefengensis. Full article

Plant-parasitic nematodes (PPNs) cause big crop losses globally. Safe/reliable methods for their durable management strategies can harness various beneficial relationships among the plant immune system and related microbiomes. Molecular mechanisms basic to these relations reveal wide arrays of significant roles for plant-healthy growth. This review focuses on such relations of microbiomes to prime and immunize plants against PPNs. It also highlights molecular issues facing PPN-resistant varieties with possible solutions such as genetic breeding/engineering, grafting, PPN-antagonistic root exudates, and novel resistant cultivars. These issues call for optimal uses of various widespread groups of microbiomes. Related plant signaling hormones and transcription factors that regulate gene expression and modulate nematode-responsive genes to ease positive/negative adaptation are presented. Exploring PPN-resistance genes, their activation mechanisms, and signaling networks offers a holistic grasp of plant defense related to biotic/abiotic factors. Such factors relevant to systemic acquired resistance (SAR) via plant–microbe interactions to manage PPNs are stressed. The microbiomes can be added as inoculants and/or steering the indigenous rhizosphere ones. Consequently, SAR is mediated by the accumulation of salicylic acid and the subsequent expression of pathogenesis-related genes. To activate SAR, adequate priming and induction of plant defense against PPNs would rely on closely linked factors. They mainly include the engaged microbiome species/strains, plant genotypes, existing fauna/flora, compatibility with other involved biologicals, and methods/rates of the inoculants. To operationalize improved plant resistance and the microbiome’s usage, novel actionable insights for research and field applications are necessary. Synthesis of adequate screening techniques in plant breeding would better use multiple parameters (molecular and classical ones)-based ratings for PPN-host suitability designation. Sound statistical analyses and interpretation approaches can better identify genotypes with high-level, stable resistance to PPNs than the commonly used ones. Linking molecular mechanisms to consistent field relevance can be progressed via dissemination of many advanced techniques. The CRISPR/Cas9 system has been effective in knocking out both the OsHPP04 gene in rice to confer resistance against Meloidogyne graminicola and the GhiMLO3 gene in cotton to minimize the Rotylenchulus reniformis reproduction. Its genetic modifications in crops synthesized “transgene-free” PPN-resistant plants without decreased growth/yield. Characterizing microbiome species/strains needed to prime and immunize plants requires better molecular tools for fine-scale taxonomic resolution than the common ones used. The former can distinguish closely related ones that exhibit divergent phenotypes for key attributes like stability and production of enzymes and secondary metabolites. As PPN-control strategies via tritrophic interactions are more sensitive to the relevant settings than chemical nematicides, it is suggested herein to test these settings on a case-by-case basis to avoid erratic/contradictory results. Moreover, expanding the use of automated systems to expedite detection/count processes of PPN and related microbes with objectivity/accuracy is discussed. When PPNs and their related microbial distribution patterns were modeled, more aspects of their field distributions were discovered in order to optimize their integrated management. Hence, the feasibility of site-specific microbiome application in PPN–hotspot infections can be evaluated. The main technical challenges and controversies in the field are also addressed herein. Their conceptual revision based on harnessing novel techniques/tools is direly needed for future clear trends. This review also engages raising growers’ awareness to leverage such strategies for enhancing plant resistance and advancing the microbiome role. Microbiomes enjoy wide spectrum efficacy, low fitness cost, and inheritance to next generations in durable agriculture. Full article

The starch content of lotus (Nelumbo nucifera) rhizomes is a key determinant of their taste and overall quality. In our previous work, a candidate transcription factor, NnAP2, was identified and its coding-region single-nucleotide polymorphisms (SNPs) were significantly associated with rhizome enlargement and carbohydrate-related traits. Owing to limitations in stable genetic transformation systems in lotus, potato (Solanum tuberosum) was employed as a heterologous model to investigate the regulatory role of NnAP2 in starch and soluble sugar metabolism. Overexpression of two allelic variants of the NnAP2 transcription factor (CC and TT) in potato resulted in pronounced differences between CC- and TT-overexpressing lines (NnAP2^CC-OE and NnAP2^TT-OE) in microtuber carbohydrate composition and proteome dynamics, accompanied by divergence in transgene copy number and substantial variation in transgene expression levels among independent lines. Six months after planting transgenic lines NnAP2^CC-OE and NnAP2^TT-OE, the NnAP2^CC-OE micro-tubers exhibited significantly higher starch content and lower soluble sugar levels compared with NnAP2^TT-OE. To uncover the underlying molecular basis, profiling of proteoforms was conducted on leaves, stems and tubers of both genotypes through a label-free proteomic strategy. A total of 51,299 peptides matched to 7292 proteins. Principal component analysis demonstrated clear separation of treatment groups, indicating robust differential accumulation of proteoforms. In total, 1715 differentially expressed proteins (DEPs) were identified across tissues (fold change ≥ 1.5 or ≤0.67, p  <  0.05), of which 1516 (88.4%) were tissue-specific. GO and KEGG enrichment analyses revealed that in leaves, DEPs were enriched for amino sugar metabolism, protein transporter activity and cell-wall macromolecule modification; in stems, enrichment included response to biotic stimulus, defense response and transporter activity; in tubers, DEPs were strongly enriched for carbohydrate metabolic processes, starch and sucrose metabolism, the TCA cycle and nucleotide sugar biosynthesis. Key starch-biosynthetic enzymes (e.g., ADP-glucose pyrophosphorylase, UDP-glucose-4-epimerase) were up-regulated in NnAP2^CC-OE tubers relative to NnAP2^TT-OE, while soluble sugar synthesis pathways (e.g., trehalose-6-phosphate synthase) were down-regulated. Together, these data suggest that elevated NnAP2^CC expression in transgenic potato is associated with allele-dependent shifts in central carbon allocation between starch and soluble sugar pathways, as revealed by comparative analyses between NnAP2^CC-OE and NnAP2^TT-OE. This study provides a comprehensive proteoform framework for allelic variation in an AP2 transcription factor involved in source–sink carbon partitioning and tuber starch accumulation in potato. Full article

Enzyme performance parameters, including the turnover number and specificity constant, exhibit remarkable diversity due to biological evolution and natural selection. In some bacterial and human enzymes, catalytic efficiencies approach fundamental physical limits, underscoring the importance of physical constraints on enzymatic function. A deeper understanding of these constraints, particularly in far-from-equilibrium irreversible processes, is therefore essential for rational enzyme engineering. Such constraints are most naturally addressed within the frameworks of nanothermodynamics and stochastic thermodynamics, which remain relatively unfamiliar to much of the molecular biology community. Recent theoretical and experimental advances indicate that classical enzyme kinetic parameters are not independent, but are systematically linked to energetic dissipation. In particular, enzymes appear to occupy a characteristic dissipation plane defined by entropy production, reflecting the coupled influence of thermodynamic principles and evolutionary selection. In this review, we synthesize evidence across diverse enzyme families demonstrating correlated increases in housekeeping dissipation, evolutionary divergence, and enzymatic performance. Together, these findings support dissipation as a physically grounded parameter that connects enzyme kinetics, biological evolution, and nonequilibrium thermodynamics. Full article