Search Results (227)

Deoxynivalenol (DON), a trichothecene mycotoxin commonly found in cereal grains and their derived products, poses significant risks to human and animal health. In previous work, a fusion enzyme composed of the dehydrogenase DADH and the aldo-keto reductase AKR13B3 was engineered to convert DON into the non-toxic 3-epi-DON in a single step. However, the poor thermal stability of this fusion enzyme limited its industrial application. In this study, EVcouplings and the GRAPE-WEB platform were utilized to identify key amino acid residues governing the thermal stability of the fusion enzyme AKR13B3–DADH. Through single-point mutation screening and the combination of beneficial mutation sites, a triple mutant M361L/T508Y/Y603F (M1) was obtained. The half-life of M1 at 50 °C reached about 500 min, representing a 16.4-fold increase compared with the wild type, while its catalytic activity increased by 2.7-fold. The apparent melting temperature increased by approximately 4 °C. Molecular dynamics simulations verified that the improved thermostability results from reduced conformational flexibility in key regions, enhanced structural packing, and a strengthened hydrogen bond network. These results demonstrate the successful development of a thermostable DON-detoxifying fusion enzyme and provide a practical basis for its industrial application. Full article

Background/Objectives: With the advent of genome sequencing and its widespread use in the clinic, there is a great need to identify mutational biomarkers that predict therapeutic responses. DNA polymerase beta (Polβ) and the base excision repair (BER) pathway have been previously implicated as modulators of response to platinum-based chemotherapies and are mutated in as many as 30% of cancers. Methods: Here, we show in a triple-negative breast cancer (TNBC) model that two classes of mutations in Polβ, reduced catalytic activity (E295K and D256A mutations) and reduced fidelity (I260M), are sufficient to drive cisplatin and carboplatin-specific sensitivity. Cellular response to oxaliplatin in these Polβ mutant models is minimal relative to cisplatin and carboplatin. Results: We show that sensitivity is associated with reduced repair of both platinum-induced DNA intrastrand adducts and interstrand crosslinks (ICLs). Downregulation of the upstream BER factor uracil DNA glycosylase (UNG) reverses drug sensitivity, which is consistent with these Polβ mutations negatively impacting ICL DNA repair to drive drug sensitivity. In addition, the intrastrand adduct repair readout indicates that these lesions also play a role in the sensitivity observed in Polβ mutant models. In vivo studies demonstrate a significant effect on tumor growth delay with cisplatin treatment in tumor xenografts harboring Polβ mutations. Conclusions: These results support the potential for using Polβ mutations as predictive biomarkers for cisplatin and carboplatin therapies in the clinical setting. Full article

Background/Objectives: The EGFR T790M mutation drives lung cancer resistance by sterically hindering inhibitors and restoring ATP affinity. As C797S mutations render covalent inhibitors obsolete, novel non-covalent strategies are critical. This study identifies inhibitors that redefine the mutant methionine sulfur as a primary stabilizing anchor rather than a liability. Methods: A generative AI framework (DrugEx) sampled 100,000 molecules, prioritized via QSAR classification (ROC-AUC: 0.91 ± 0.01) and Applicability Domain (AD) mapping. The workflow was de-risked through retrospective benchmarking against the DUD-E database (35,590 molecules), achieving a 1% Enrichment Factor of 5.19. Lead candidates underwent 100 ns all-atom molecular dynamics (MD) simulations. Mechanistic stability was quantified via Free Energy Landscape (FEL) analysis and ensemble-averaged MM-GBSA binding free energy calculations. Results: Candidate 106 demonstrated exceptional mutation tolerance by redistributing interactions toward the Met790 sulfur atom. MD analysis confirmed potency is dictated by successful recruitment of the thioether environment, locking the complex within a narrow thermodynamic basin. Candidate 106 maintained stable binding (−11.0 kcal/mol) corroborated by an equipotent MM-GBSA ΔG_bind of −50.51 kcal/mol in the mutant system, driven by persistent π-sulfur contacts (85% occupancy). Conclusions: These results indicates that potential T790M resistance bypass is achievable by exploiting the gatekeeper methionine’s electronic environment. This modeled mutation-aware framework provides a candidate non-covalent strategy to be validated in future wet-lab campaigns. Full article

Alzheimer’s disease (AD) is characterized by cognitive decline and amyloid-β (Aβ)-related pathology. Non-rodent models that capture selected aspects of human AD remain limited. We established and characterized a human APP-driven, Aβ-related AD-like tree shrew model following AAV-mediated delivery of triple-mutant human amyloid precursor protein (hAPP-SLA) carrying the Swedish, Austrian, and London mutations by bilateral stereotaxic injection directed at CA1 coordinates. Adult tree shrews received bilateral AAV-hAPP-SLA injections directed at CA1 coordinates and were evaluated by bioluminescence imaging, behavioral testing, PCR, RT-qPCR, Western blotting, ELISA, and histopathology. Vector-associated reporter signals remained detectable for 6 months. The experimental group showed exogenous hAPP expression and reduced endogenous tsAPP expression, increased relative hippocampal Aβ₄₂ protein level, enhanced 4G8-reactive APP/Aβ-related signals, elevated total Aβ immunoreactivity, increased serum Aβ₄₂/Aβ₄₀ ratio, cytoarchitectural alterations, reduced Nissl staining, and Thioflavin S-reactive aggregate-associated signals. AT8 (Ser202/Thr205), GFAP, and Iba-1 immunoreactivity increased, whereas Synaptophysin and PSD-95 immunoreactivity was reduced. These changes were accompanied by reduced short-delay recognition-related performance and reduced social approach and social novelty preference. Aged tree shrews showed partly overlapping alterations. This model provides a non-rodent platform for studying human APP-driven Aβ-related pathology. Full article

Lytic polysaccharide monooxygenases (LPMOs) are promising enzymes for lignocellulose degradation; however, wild-type LPMOs often exhibit limited catalytic activity and stability. In this study, computer-aided virtual saturation mutagenesis was applied to AnLPMO15g from Aspergillus niger, and eight potentially beneficial mutants (S197H, S197F, E185V, E185L, E185M, E185I, Q108M, and A249P) were identified based on predicted changes in unfolding free energy (∆∆G). Six mutants demonstrated enhanced activity in a 2,6-dimethoxyphenol (2,6-DMP) oxidation assay, which serves as a proxy for peroxidase-like activity. The E185V mutant exhibited a 45% increase over the wild type. The triple mutant E185V/Q108M/A249P further increased the catalytic efficiency by 56%. Notably, when combined with cellulase, E185V/Q108M/A249P enabled a 202.5% increase in reducing sugars from wheat straw, achieving a synergy degree of 1.83, highlighting its potential to improve agricultural residue conversion. Molecular dynamics simulation suggested that the E185V/Q108M/A249P triple mutant induced flexible conformational changes in six residues, which may improve substrate binding affinity. This study presents an effective strategy for engineering AA9 family LPMOs to enhance catalytic performance, facilitating efficient and cost-effective degradation of lignocellulosic biomass with implications for sustainable agricultural waste management and circular bioeconomy. Full article

Background/Objectives: Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. Human ccRCCs have increased glycolytic metabolism and decreased mitochondrial oxidative metabolism relative to normal kidneys. Our research using human RCC4 ccRCC cells and a murine model of ccRCC, TRACK (TRAnsgenic model/Cancer/Kidney), in which a triple-mutant (P402A, P564A, N803A) human HIF1α is selectively expressed in proximal tubule cells (PTCs), revealed highly induced ATF4, a stress-responsive transcription factor. We then investigated the role of ATF4 in the metabolic changes in ccRCC. Methods: We performed comprehensive analysis of the ccRCC Cancer Genomics Atlas (TCGA) data. We deleted ATF4 in PTCs of TRACK mice and human RCC4 cells. We conducted genome-wide transcriptomic and untargeted metabolomic studies of cortices of WT and CGERA∆T (TRACK mice with PTC-specific ATF4-knockout (KO)) mice and performed glucose isotopologue tracing in parental and ATF4 KO RCC4 cells. Results: Analysis of TCGA data showed increased mRNAs of enzymes in glycolysis and reduced mRNAs of enzymes in the TCA cycle. Transcriptomic and metabolomic studies demonstrated that ATF4 deletion suppressed glycolysis and enhanced TCA cycle metabolism in CGERA∆T versus WT cortices. Glucose isotopologue tracing showed that ATF4 deletion altered glycolysis pathway metabolite levels and shifted glucose metabolism towards the TCA cycle, evidenced by increased intracellular [¹³C₂]citrate in RCC4-ATF4 KO cells. Using the Seahorse XFe96 analyzer we also showed reduced glycolytic capacity and reserve in RCC4-ATF4 KO cells. Conclusions: Collectively, our results demonstrate that ATF4 regulates glycolysis in ccRCC, supporting ATF4 as a therapeutic target. Full article

Cellulose is the strongest cell wall polymer defining plant cell shape and growth, and the most abundant biopolymer on the Earth. Its synthesis by the plasma membrane (PM)-localized cellulose synthase complexes (CSCs) depends on surrounding lipids that establish the membrane microenvironment in which CSCs work and form vesicles delivering and removing CSCs to and from the PM by exo- and endocytosis. The role of exact lipid molecular species in these processes is poorly understood. In the present work we used hypocotyls of etiolated wild-type Col-0 and mutant ixr1-1 Arabidopsis thaliana seedlings grown with or without isoxaben, a specific cellulose synthesis inhibitor, as a model to reveal lipid molecular species associated with cellulose biosynthesis. Different lipid classes were separated by thin-layer chromatography (TLC) and their molecular species were analyzed by liquid chromatography–triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). A total of 250 lipid molecular species were identified. Col-0 plants maintained stable levels of membrane glycerophospholipids but displayed significant remodeling of their acyl chains. In the presence of isoxaben, they accumulated three times more phosphatidic acids, a hallmark of a stress response. The isoxaben-resistant mutant ixr1-1 was characterized by a higher relative content of phosphatidylethanolamines, potentially rendering membranes more labile, as well as plastid galactolipids, which accumulated under isoxaben treatment. The multifaceted effects of isoxaben, including its impact on endomembrane lipids, suggest that it has additional binding sites beyond CSC. Full article

Background: Type 2 diabetes is projected to affect millions of people annually as the number of cases rises year on year. This includes children. Treating diabetes and its related comorbidities has a huge economic impact and puts pressure on healthcare providers. Understanding the disease at a molecular level is key for developing better therapeutics. The protein Islet Amyloid Polypeptide (IAPP) or amylin is important for glucose regulation; however, it is also instrumental in type 2 diabetes pathology. Human IAPP can misfold into oligomers and amyloid fibrillar aggregates within pancreatic islets, promoting β-cell dysfunction and death, contributing to progressive insulin deficiency and worsening hyperglycaemia. Methods: Based on previous studies on mutations at residues 18, 28 and 31,we have designed three novel IAPP analogues (two double and one triple mutant) to assess whether the combined amino acid substitutions impact fibril formation, solubility and toxicity. Results: All three of our analogues show a reduced propensity to aggregate and are more soluble than wild type IAPP. Compared with pramlintide, a clinically prescribed synthetic analogue of human amylin, all of our analogues appeared to have similarly reduced toxicity and improved solubility relative to human IAPP. Additionally, two of our analogues exhibited a markedly slower rate of fibril formation. Conclusions: Our results highlight the importance of targeting multiple residues as a promising strategy for developing improved diabetes therapeutics in the future. Full article

Bovine herpesvirus type 1 (BoHV-1) causes severe respiratory and reproductive diseases in cattle, leading to significant economic losses worldwide. Current inactivated vaccines in China fail to induce robust cellular immunity and cannot differentiate infected from vaccinated animals (DIVA). To address these limitations, we constructed two triple-gene-deleted BoHV-1 vaccine candidates, ∆T3 (∆TK-∆gE/gI) and ∆g3 (∆gG-∆gE/gI), using Red/ET two-step recombination technology based on an infectious BAC clone. Their safety and immunogenicity were evaluated in a rabbit model. Both deletion mutants exhibited growth kinetics similar to the wild-type strain but with significantly reduced viral titers and plaque areas. The triple-gene deletion mutants showed superior safety compared with single-gene deletion mutants, with minimal clinical signs and low viral shedding. Immunization with ∆T3 and ∆g3 induced robust gB-specific and neutralizing antibody responses. Following challenge with the virulent BoHV-1 BC01 strain, vaccinated rabbits maintained normal body temperatures, showed significantly reduced viral shedding (approximately 100-fold), and exhibited milder pulmonary lesions. These findings demonstrate that ∆T3 high-dose and ∆g3 low-dose regimens are promising DIVA-compatible vaccine candidates, offering an effective strategy for IBR control and eradication programs. Full article

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited targeted therapeutic options, underscoring the urgent need for novel treatment strategies. Feilike (FLK), a Traditional Chinese Medicine formula with heat-clearing and detoxifying properties, aligns with key pathological features implicated in breast cancer progression. In addition, several of its components have demonstrated anti-tumor activity, positioning FLK as a potential therapeutic candidate for TNBC. In this study, we employed an integrated approach combining network pharmacology, transcriptomic analysis, and experimental validation to investigate the anti-TNBC effects of FLK. Our results demonstrate that FLK significantly inhibits the proliferation of TNBC cell lines and patient-derived organoids and induces typical pyroptotic features, including cell swelling and increased lactate dehydrogenase (LDH) release. Mechanistically, FLK triggers a mutant p53 signaling cascade involving calcium dysregulation, endoplasmic reticulum stress (ERS) activation, mitochondrial dysfunction, and reactive oxygen species (ROS) accumulation, which collectively activate the P38/JNK–Caspase-3/GSDME pathway to induce pyroptosis. In vivo, FLK markedly suppresses tumor growth in a 4T1 orthotopic mouse model and enhances the anti-tumor efficacy of Cyclophosphamide. Furthermore, Licochalcone B (LCB) is identified as a key bioactive constituent that recapitulates the pyroptosis-inducing effects of FLK. Collectively, our findings uncover a previously unrecognized mutant p53–ERS–ROS–MAPK signaling axis underlying FLK-induced pyroptosis and provide mechanistic insight and experimental evidence supporting the repurposing of FLK as a potential therapeutic strategy for TNBC. Full article

Targeted inhibition of the MAPK pathway with BRAF and MEK inhibitors (BRAFi/MEKi) produces rapid tumor regressions in BRAF V600-mutant melanoma, yet most patients ultimately develop acquired resistance. Resistance is not solely a tumor-cell-intrinsic phenomenon; it is accompanied by time-dependent remodeling of the tumor immune microenvironment (TIME) that can shape sensitivity to immune checkpoint inhibitors (ICIs) and inform rational combination or sequencing strategies. Early during MAPK inhibition, melanomas often display increased melanoma antigen expression and enhanced CD8⁺ T-cell infiltration, along with reduced immunosuppressive cytokines, suggesting a transient “immune-permissive” window. However, the same period can show induction of PD-L1 and T-cell exhaustion markers, foreshadowing adaptive immune resistance. At progression, immune-favorable features may diminish and immune evasion mechanisms, such as impaired antigen presentation and MHC-I downregulation, can become prominent and associate with resistance to immunotherapy. Here we review the temporal dynamics of TIME under MAPK inhibition, mechanistic links between resistance programs and immune remodeling, including signaling adaptation, focal adhesion/FAK signaling, dendritic cell dysfunction, antigen-presentation defects, and lymphatic/perilymphatic adipose remodeling, and practical biomarker opportunities across baseline, on-treatment, and progression timepoints. We also summarize emerging therapeutic strategies for post-resistance disease, including optimized ICI combinations, triple therapy concepts, and novel approaches such as combining FAK inhibition with RAF-MEK “clamp” therapy. Finally, we highlight key gaps and propose a framework for longitudinal sampling, standardized multi-omics integration, and TIME-informed trial design. The key distinguishing feature of this review is its time-resolved perspective on TIME remodeling, which links baseline immune contexture, early treatment-induced immune permissiveness, and the immune-evasive state that emerges during acquired resistance. Full article

Tomato brown rugose fruit virus (ToBRFV) poses a major threat to global tomato (Solanum lycopersicum) production, as it can overcome conventional resistance genes that are effective against tobamoviruses. In this study, a multiplex CRISPR/Cas9 system was developed to target the SlTOM1 susceptibility gene family (SlTOM1a–d), which encodes host factors essential for tobamovirus replication. Six guide RNAs (gRNAs), designed following 12 off-target analyses, were assembled into a multiplex CRISPR/Cas9 construct using a Golden Gate cloning strategy and introduced into tomato genotypes through an Agrobacterium-based tissue culture transformation procedure. Although primary T₀ transformants exhibited chimeric mutation patterns, stable inheritance and segregation of edited alleles were confirmed in the T₁ generation. Sequence analyses identified diverse indel mutations across target loci, with SlTOM1d exhibiting the highest editing efficiency. Multiplex genome editing successfully generated single-, double-, and triple-mutant combinations, with higher-order mutants displaying the strongest tolerance phenotypes. Following mechanical ToBRFV inoculation, edited T₁ plants exhibited markedly reduced symptom severity, low viral accumulation, and improved fruit health compared to wild-type controls. RT-qPCR analysis further confirmed significantly reduced viral RNA levels, supporting a host-factor-mediated tolerance mechanism. Importantly, edited lines maintained normal growth and agronomic performance. Collectively, these findings demonstrate that multiplex CRISPR/Cas9-mediated targeting of SlTOM1 homologs represents a promising and practical strategy for improving ToBRFV tolerance in tomato breeding programs. Full article

Background/Objectives: Capivasertib is a selective pan-AKT inhibitor recently approved in combination with fulvestrant for the treatment of hormone receptor-positive (HR+)/HER2- breast cancer with alterations in the PI3K/AKT pathway. The PI3K/AKT/mTOR signaling cascade represents a critical indication of endocrine resistance and tumor progression in this subtype of breast cancer. The present review summarizes current clinical data regarding the efficacy of capivasertib, either as monotherapy or in combination with other therapeutic agents and discusses emerging biomarkers and mechanisms of resistance. Methods: A literature search of the PubMed database was conducted to identify clinical trials evaluating capivasertib in breast cancer. Studies on capivasertib as monotherapy or in combination with fulvestrant, paclitaxel, or olaparib were included. Results: Findings from phase I–III clinical trials indicate that capivasertib in combination with fulvestrant significantly prolongs progression-free survival in patients with HR+/HER2- advanced breast cancer, particularly in tumors containing PIK3CA, AKT1, or PTEN alterations. Drug combination approaches with paclitaxel or olaparib have demonstrated additive or synergistic effects in triple-negative and DNA repair-deficient contexts, respectively. Monotherapy studies confirm effective pathway inhibition with modest clinical benefit, primarily in AKT1-mutant tumors. Translational analyses suggest that persistent mTORC1-mediated protein synthesis and compensatory signaling activation contribute to acquired resistance. Conclusions: Capivasertib constitutes a clinically validated therapeutic approach for the inhibition of AKT signaling in breast cancer. Its efficacy is most evident when combined with endocrine therapy; however, optimization of patient selection and rational combination strategies remains necessary to overcome resistance associated with mTORC1 activation and signaling redundancy. Full article

Background: Acquired resistance to the third-generation EGFR tyrosine kinase inhibitor osimertinib, often mediated by EGFR triple mutations, poses a major clinical challenge in non-small cell lung cancer (NSCLC) treatment. Among these, some rare mutations, such as L858R/T790M/L792H and L858R/T790M/G796R, create steric hindrance that directly interferes with osimertinib binding, yet effective targeted therapeutic strategies for these specific mutations remain lacking. Cudratricusxanthone A (CTXA), a natural xanthone derivative isolated from Cudrania tricuspidata Bur., has demonstrated various pharmacological activities, but its effects against EGFR triple-mutant NSCLC have not been systematically investigated. Methods: Stable Ba/F3 and NIH/3T3 cell lines expressing EGFR L858R/T790M/L792H or L858R/T790M/G796R triple mutations were generated via electroporation. The antiproliferative effects of CTXA were evaluated by MTT/MTS assays, colony formation, and wound healing assays. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Protein expression of EGFR signaling pathway components (p-EGFR, p-ERK, p-AKT, p-STAT3) and cell cycle regulators (Cyclin D1, CDK4) were examined by Western blotting. Molecular docking and 200 ns molecular dynamics simulations were performed to investigate the stability and binding modes of CTXA to the mutant EGFR kinase domains. Results: The successfully established triple-mutant cell lines exhibited high EGFR expression, IL-3-independent growth, and significant resistance to osimertinib. CTXA inhibited the proliferation of all triple-mutant cell lines in a time- and concentration-dependent manner, with 48 h IC₅₀ values ranging from 0.362 to 2.488 μM. Mechanistically, CTXA suppressed EGFR autophosphorylation and downregulated downstream p-ERK, p-AKT, and p-STAT3. CTXA induced G1 phase cell cycle arrest by downregulating Cyclin D1 and CDK4, significantly promoted apoptosis, and inhibited cell migration. Molecular docking revealed that while osimertinib binding was blocked by steric hindrance from His-792 or Arg-796, CTXA adapted to the mutated ATP-binding pockets through multiple hydrogen bonds and extensive hydrophobic interactions. Molecular dynamics simulations confirmed the stable binding of CTXA to both mutant EGFR proteins over the 200 ns simulations. Conclusions: This study demonstrates for the first time that the natural compound CTXA possesses antitumor efficacy against EGFR L858R/T790M/L792H and L858R/T790M/G796R mutants by regulating EGFR-ERK/AKT/STAT3 signaling. Our findings position CTXA as a promising lead compound for tackling this challenging form of acquired resistance and highlight the value of natural products in multi-target antitumor drug discovery. Full article

Listeria monocytogenes (Lm) is an important zoonotic foodborne pathogen that causes severe rhombencephalitis in ruminants. The trigeminal ganglion is a critical node for Lm invasion of the central nervous system via neural pathways. However, the roles of key virulence factors InlA, InlB, and LLO from ovine-derived Lm in trigeminal ganglion neuron infection remain unclear. In this study, LM90SB2, an ovine-derived Lm strain isolated from a sheep with encephalitis in Xinjiang, China, was used as the wild type, and its ΔInlAB double-gene deletion and ΔInlABO triple-gene deletion mutants were constructed. Primary mouse trigeminal ganglion cells (TGCs) were infected with these strains, and cell-association and invasion assays, bacterial colonization analysis, cell scratch tests, Western blotting, and qRT-PCR were performed to explore the effects of InlA, InlB, and LLO on Lm infection of TGCs and their regulatory roles in host adhesion molecules N-cadherin and NCAM1. The results showed that the wild-type LM90SB2 had significantly stronger cell-association, invasion, and colonization abilities in TGCs than the ΔInlAB and ΔInlABO mutants (p < 0.01 or p < 0.0001). LM90SB2 infection significantly upregulated the mRNA and protein expression levels of N-cadherin and NCAM1 in TGCs and enhanced TGC migration, while these effects were gradually attenuated with the sequential deletion of InlA, InlB and LLO. This study clarifies the synergistic roles of InlA, InlB, and LLO in mediating the infection of trigeminal ganglion neurons by ovine-derived Lm and reveals the molecular mechanism by which Lm promotes neural invasion by regulating the expression of host cell adhesion molecules. Our findings provide important experimental data for elucidating the neural invasion pathway of Lm in ruminants and lay a theoretical foundation for the development of targeted prevention and control strategies for ruminant listeriosis in veterinary clinical practices. Full article