Search Results (4,392)

The short peptide AlaGluAspLeu (AEDL) stimulates shoot and root development in Nicotiana tabacum. Growing tobacco in the presence of AEDL was found to induce autophagy and programmed cell death, as demonstrated using immunodetection of the autophagy marker ATG8 and cytochrome c in the cytoplasm, as well as the detection of DNA breaks using the TUNEL assay. A detailed study of the ultrastructure of Nicotiana tabacum root cells grown in the presence of AEDL using transmission electron microscopy revealed fundamental structural differences from control cells. Control cells contained only lytic vacuoles, while in the presence of AEDL, tobacco root meristem cells contained predominantly protein-storing vacuoles and amyloplasts with numerous starch granules in the stroma. Characteristic types of phagophores were identified, forming numerous small autophagosomes with cytoplasmic regions, multivesicular bodies, or concentric membranes, possibly with cytoskeletal elements. Expression of autophagy protein genes revealed a decrease in TOR expression, which promoted autophagy activation and prevented ATG13 phosphorylation. ATG8 gene expression significantly increased in the presence of the AEDL peptide. Schematic diagrams of autophagy processes in root cells of control plants and those grown in the presence of AEDL are presented. Based on these data, it was concluded that stimulation of tobacco plant development in the presence of the AEDL peptide at a concentration of 10⁻⁷ M occurs due to the activation of metabolic processes and autophagy. Moreover, the synthesis of metabolites exceeds the required amount of nutrients, which accumulate in vacuoles and leucoplasts. Full article

In this study, for the first time, the genes encoding the mammalian steroidogenesis system—cytochrome P450scc (CYP11A1), and its native redox partners adrenodoxin and adrenodoxin reductase—were successfully expressed in the methylobacterium Methylorubrum extorquens. The advantage of using methylobacteria as an expression chassis is that they grow on inexpensive mineral media, use methanol as a carbon and energy source, and do not possess their own sterol catabolism systems. Using recombinant methylobacteria, the valuable steroid pregnenolone was obtained as a sole metabolite from cholesterol. The effect of media composition, bioconversion conditions such as methanol and N-sources content, modes of substrate addition, detergents, methyl-β-cyclodextrin, biomass, and aeration on pregnenolone accumulation was investigated. Under optimized conditions, its yield exceeded 100 mg/L. The results demonstrate a proof of concept relating to the use of bacteria lacking their own steroid degradation systems as microbial chassis for heterologous steroidogenesis systems, including mammalian cytochrome CYP11A1. Full article

Black soldier flies (Hermetia illucens) are widely used in organic waste bioconversion, and their adaptive capacity to region-specific food waste is critical for efficient application. This study aimed to explore the molecular mechanisms underlying the adaptation of black soldier fly larvae to long-term domestication on regional food waste from Kunming and Qujing. Integrated transcriptomic and metabolomic analyses were performed to identify differences in gene expression and metabolite profiles between the two groups of larvae. The results showed significant divergence in gene expression networks, with key differences in cytochrome P450 detoxification pathways, TOR nutrient-sensing pathways, and zf-C2H2 zinc finger transcription factor families. Metabolomic analysis revealed region-specific metabolic reprogramming, including enhanced branched-chain amino acid degradation in one group and activated sphingolipid signaling pathways with accumulated indole derivatives in the other. Additionally, 13.86% of unannotated metabolites in the metabolome exhibited high connectivity in metabolic networks, suggesting potential roles as “bridge metabolites” in adaptation. These findings demonstrate that long-term domestication on regional food waste drives adaptive differentiation in black soldier fly larvae through regulatory network remodeling, metabolic reprogramming, and activation of hidden metabolic reserves. The study provides a theoretical basis for optimizing the application of black soldier flies in waste treatment and strain breeding and establishes a “substrate-gene-metabolism” multi-omics framework for understanding insect adaptive evolution. Full article

Background: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. The search for effective, new antineoplastic drugs with fewer side effects for the treatment of CRC continues, with marine-derived compounds emerging as promising candidates. Objectives: This study investigates the anticancer potential of Frondanol, a nutraceutical derived from the Atlantic Sea cucumber Cucumaria frondosa, known for its potent anti-inflammatory properties. Methods: Two human CRC cell lines, Caco-2 and HT-29, were used to test the effects of Frondanol using various in vitro approaches. Results: Frondanol significantly inhibited cell viability in a dose- and time-dependent manner. At a 1:10,000 dilution, viability decreased to around 30% in Caco-2 and 20% in HT-29 after 24 h, dropping to nearly 5% at 48 h. Furthermore, a clonogenic assay showed around 50% reduction in colony formation in both cell lines. Flow cytometry-based Annexin V staining revealed that Frondanol increased early apoptosis to ~5.2% in Caco-2 and ~9.4% in HT-29 cells, while cell cycle analysis showed accumulation of the sub G0 (apoptotic) phase increasing from 1.5% to 14.7% (Caco-2) and from 1.9% to 23.8% (HT-29). At the molecular level, Frondanol treatment significantly decreased anti-apoptotic protein B-cell lymphoma (Bcl)-2 expression while increasing the expression of the proapoptotic protein Bcl-2-associated X-protein. Additionally, Frondanol markedly induced cytochrome c release from the mitochondria and activated caspase-9, caspase-7, and caspase-3 after treatment, alongside cleavage of the caspase-3 substrate poly (ADP-ribose) polymerase. Frondanol inhibited 5-lipoxygenase activity, further contributing to its anticancer effects. Conclusions: In conclusion, Frondanol inhibits CRC cell proliferation and induces apoptosis through the mitochondrial pathway in vitro, suggesting that it is a potential nutraceutical for the prevention of human colorectal cancer or a valuable source of anticancer compounds. Full article

Background: The cytochrome P450 family 21 subfamily A member 2 gene (CYP21A2) encodes 21-hydroxylase, a key enzyme in adrenal steroid biosynthesis. Despite its physiological importance, the diversity of CYP21A2 transcript variants and their tissue-specific expression in domestic animals, including cattle, remains largely unexplored. This study aimed to characterize CYP21A2 transcription in adrenal glands and ovaries and assess the potential transcriptional activity of its pseudogene, CYP21A1P. Methods: CYP21A2 transcription was investigated in adrenal and ovarian tissues of 12 healthy cows using semi-quantitative PCR and Sanger sequencing. Real-time PCR was performed to confirm expression levels. Melting curve analysis and electrophoresis were used to validate distinct amplicons corresponding to different transcript variants. Extended amplicons were sequenced to identify transcripts corresponding to reference sequences and potential pseudogene products. Results: A single transcript variant (NM_001013596.1) was consistently detected in adrenal glands, whereas ovaries expressed two variants: NM_001013596.1 and XM_024983378.2. Semi-quantitative analysis showed significantly higher CYP21A2 expression in adrenal glands compared to ovaries (p < 0.01). In ovarian samples, the NM_001013596.1 variant was more abundant than the XM_024983378.2 (p < 0.01). Sanger sequencing revealed two products matching CYP21A2 reference transcripts and an additional, longer product containing sequence motifs specific to the pseudogene CYP21A1P, indicating its transcriptional activity. Conclusions: These results provide the first evidence of tissue-specific expression and differential abundance of CYP21A2 transcript variants in cattle and suggest the transcription of the CYP21A1P pseudogene. The findings reveal the complexity of CYP21A2 expression in steroidogenic tissues and suggest potential regulatory roles for transcript and pseudogene variants in bovine physiology. Full article

Background/Objectives: Molnupiravir (MOV) and nirmatrelvir (NMV) are antiviral drugs that were FDA-approved under the emergency use authorization (EUA) for coronavirus disease-2019 (COVID-19) treatment. MOV and NMV target the viral RNA-dependent RNA polymerase and main protease, respectively. Paxlovid is a combination of NMV and ritonavir (RTV), an inhibitor of the human cytochrome P450-3A4 (hCYP3A4). In this study, the structural consequences in the hCYP3A4 caused by MOV-induced mutations (MIM) were evaluated using in silico tools. Methods: MOV-induced mutations (MIM) were inserted into all the possible hotspots in the active site region of the hCYP3A4 gene, and mutant protein models were built. Structural changes in the heme-porphyrin ring of hCYP3A4 were analyzed in the presence and absence of substrates/inhibitors, including RTV. Molecular dynamics (MD) simulations were performed to analyze the effect of MIM-induced structural changes in hCYP3A4 on drug binding. Results: MD simulations confirm that MIMs, R375G and R440G in hCYP3A4 severely affect the heme-porphyrin ring stability by causing a tilt that in turn affects RTV binding, suggesting a possible inefficiency in the function of hCYP3A4. Similar results were seen for amlodipine, atorvastatin, sildenafil and warfarin, which are substrates of hCYP3A4. Conclusions: The current in silico studies indicate that hCYP3A4 containing MIMs can create complications in the treatment of COVID-19 patients, particularly with co-morbidities due to its functional inefficiency. Hence, clinicians must be vigilant when using MOV in combination with other drugs. Further in vitro studies focused on hCYP3A4 containing MIMs are currently in progress to support our current in silico findings. Full article

Despite advances with novel targeted agents (e.g., BCL-2 or IDH inhibitors) combined with chemotherapy for acute myeloid leukemia (AML), drug resistance persists. We investigated whether blocking Na⁺/H⁺ exchanger 1 (NHE1) could enhance AML cell sensitivity to the BCL-2 inhibitor venetoclax and sought to determine the molecular mechanisms. Our results demonstrated that co-treatment with venetoclax and the NHE1 inhibitor 5-(N,N-hexamethylene) amiloride (HMA) synergistically induced apoptosis in both venetoclax-sensitive and -resistant leukemic cell lines. Specifically, the combination significantly increased apoptosis in venetoclax-resistant THP-1 cells to 72.28% (17.79% with 100 nM venetoclax and 10.15% with 10 μM HMA alone; p < 0.001). Conversely, another venetoclax-resistant line, U-937, showed no significant apoptotic response to the combination. In THP-1 cells, this synergy was mediated via a caspase-dependent programmed cell death pathway, evidenced by an increased BAX/BCL-2 ratio, mitochondrial cytochrome c release, and subsequent caspase-9 and caspase-3 activation. Furthermore, co-treatment downregulated the anti-apoptotic protein MCL-1 and reduced PI3K and Akt phosphorylation, suggesting that inhibition of these survival pathways also contributed to the synergistic effect. Inhibition of NHE1 may substantially enhance venetoclax sensitivity in certain AML models, particularly in venetoclax-resistant THP-1 cells but not in U-937, highlighting biological diversity and the probable involvement of alternative survival pathways. Full article

Multi-Wall Carbon Nanotubes (MWCNTs) are under investigation for their use in biomedical applications, especially in neurological diseases, due to their electrochemical properties. Nevertheless, conflicting results have cast doubt on their safety. To advance their translational potential, we evaluated the cytotoxicity of two MWCNT samples in vivo in both vertebrate and invertebrate animal models. Pristine MWCNTs were, in part, used as prepared (MWCNTs), and, in part, annealed at 2400 °C (a-MWCNTs). The two samples differ in their electrochemical properties: MWCNTs are not electro-conductive, while a-MWCNTs are electro-conductive and negatively charged on their surface. We evaluated the effects of both intranasally delivered MWCNTs on several key markers of cell viability in the olfactory bulbs and hippocampus from healthy adult Wistar rats, as well as their impact on lifespan, genotoxicity, oxidative stress, and aging-related functional markers in the nematode Caenorhabditis elegans. Neither of the two MWCNT samples was cytotoxic towards neuronal cells in the hippocampus. In olfactory bulbs, only electro-conductive a-MWCNTs interacted with two positively charged mitochondrial proteins: Translocase of Outer Mitochondrial Membrane 20 (TOM20) and Cytochrome C (CytC). In C. elegans, neither type of MWCNT affected lifespan or brood size, and cytosolic ROS levels remained unchanged. Notably, treated worms exhibited a significantly delayed aging phenotype. Metallic MWCNTs are biocompatible in living organisms and possess the potential to modulate neural cells functioning in vivo. Full article

Researchers strive to exploit kinetic potentials of multistep reactions by positioning enzymes in a regulated fashion. Therein, the proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus is a promising biomolecular tool due to its extraordinary architecture. PCNA is a circular DNA sliding clamp, which can bind and move along DNA and thus, be applied for the immobilization and transport of biomolecules on versatile DNA scaffolds. Additionally, its heterotrimeric character facilitates the colocalization of enzyme cascades with defined stoichiometry. This study provides insights into the in vitro binding behavior of PCNA and its potential as protein scaffold for DNA functionalization and controlled biocatalysis: (1) PCNA was capable of binding circular DNA and wireframe DNA nanostructures. (2) DNA binding was predominantly mediated by the PCNA1 subunit. (3) PCNA assembly around DNA was compromised when cysteines were introduced at the PCNA–PCNA interfaces to stabilize the ring via disulfide bonds. (4) A two-enzyme cascade, comprising a pseudo-monomeric cytochrome P450 BM3 monooxygenase and a monomeric alcohol dehydrogenase (ADH), was successfully fused to PCNA, retaining catalytic activity. (5) When immobilized on DNA, the cascade performance was not assessable, due to nearly complete loss of ADH activity in proximity to DNA. Full article

Phthonandria atrilineata, also known as the mulberry looper, is a major defoliator of mulberry trees. This feeding behavior directly affects the growth of the trees and reduces the quality and yield of mulberry leaves for its use in sericulture. Despite its importance the molecular basis of its resistance to insecticides remains poorly understood. Therefore, this study aimed to comprehensively characterize the cytochrome P450 monooxygenases (P450s) gene family in P. atrilineata and identify key effectors responsible for responses to diverse chemical stressors. We integrated genome-wide re-annotation, phylogenetic analysis, and comparative transcriptomics following exposure to five chemically distinct insecticides. We identified a high-confidence set of 70 P450 genes, dominated by the CYP6 and CYP4 families, whose expansion was driven by tandem gene duplication. Transcriptomic analysis revealed a powerful yet highly selective “elite-driven” response, wherein a small subset of P450s was strongly induced by multiple insecticides. Random Forest and Support Vector Machine (SVM) models converged with differential expression data to pinpoint a core trio of P450s as primary drivers of detoxification: two generalists, CYP6(09521) and CYP6(04876), responsive to all compounds, and one potent specialist, CYP4(04803), exhibiting massive induction to a specific subset of insecticides. Our findings uncover a complex, energy-efficient metabolic strategy in P. atrilineata and identify pivotal P450 genes for broad-spectrum detoxification. These genes represent high-priority targets for developing molecular diagnostic tools for resistance monitoring and informing scientifically guided insecticide rotation strategies. Full article

Background/Objectives: Honokiol (HK), a bioactive phenolic compound, exhibits significant anti-cancer properties. This study aimed to investigate the anti-cancer effects of HK in colorectal cancer (CRC) cells by focusing on its direct interaction with heat shock protein 27 (Hsp27) as a molecular target, and to elucidate the underlying mechanisms involved. Methods: HK was isolated via silica/ODS chromatography. Anchorage-independent growth of CRC cells was quantified using a soft agar assay with increasing HK concentrations. Apoptosis and cell cycle were analyzed by flow cytometry, and cell viability by MTS assay. Hsp27 binding to HK was validated by pull-down assay with HK-conjugated Sepharose 4B beads. Hsp27 knockdown was performed using lentiviral shRNA in CRC cells. Molecular docking of HK-Hsp27 interaction employed Schrödinger Suite 2016. Protein expressions, including chaperone and apoptotic proteins, were evaluated by Western blotting. Results: HK dose-dependently suppressed anchorage-independent growth of CRC cells and induced G₀/G₁ arrest. It triggered apoptosis through cytochrome c release, PARP cleavage, and Bcl-2 downregulation. HK directly bound to the α-crystallin domain of Hsp27 at Asn102 and His103 residues, confirmed by computational molecular docking and site-directed mutagenesis. Hsp27 knockdown in CRC cells dramatically reduced anchorage-independent growth. HK markedly decreased Hsp27 protein levels while having less effect on other heat shock proteins in CRC cells. Conclusions: HK exerts anti-cancer effects in CRC cells, associated with Hsp27 inhibition, resulting in suppressed cell growth and increased apoptosis. This interaction between HK and Hsp27 may support a mechanistic foundation supporting the potential utility of HK as a natural therapeutic agent for CRC. Full article

The Red-legged Partridge (Alectoris rufa, Phasianidae) is a non-migrant gamebird endemic to southwestern Europe that was introduced into Mediterranean and Atlantic islands in historical times. This is the case for Madeira, Portugal, where a population morphologically assigned to A. r. hispanica has been present since the XV century. We assessed its genetic identity using 2248 (Cytochrome-b, Cyt-b + Control Region, CR) and 297 bp-long (CR) mitochondrial DNA sequences obtained from modern and archival (1900–1964, including Caccabis rufa maderensis syntypes) partridges, respectively. These sequences were compared against an already published dataset covering the entire Iberian A. rufa range. We found that all the haplotypes of modern birds from Madeira were private to this island. The putative subspecies was confirmed, and northern Portugal with northwestern Spain turned out to host the closest mainland populations. This result was in line with the origin of the first human settlers of Madeira from, among other historical provinces, Douro Litoral and Minho, the latter neighboring Galicia. Despite relatively recent A. rufa importations from continental Europe, we did not find any significant change over time in the haplotypic pattern of Madeiran partridges as well as any evidence for maternal introgression from species such as the congeneric Chukar Partridge (A. chukar). Studies relying on genome-wide markers and including the only captive-bred population of Madeira are needed to gain more comprehensive information for the management of the local A. rufa. Full article

This study investigates the phylogenetic relationships, genetic diversity, and biogeographic structure of Pipistrellus species in Türkiye using mitochondrial cytochrome b (Cytb) sequences from 156 specimens collected across 26 localities. Our primary aim was to clarify taxonomic boundaries of morphologically cryptic species and elucidate the evolutionary role of Anatolia in the Western Palearctic. Analyses strongly confirmed that molecular data are mandatory for defining taxonomic boundaries. Crucially, all individuals morphologically identified as P. pygmaeus were genetically determined to be P. pipistrellus, highlighting the inadequacy of external traits for cryptic species. We resolved deep intraspecific divergence across the genus. In P. pipistrellus, two major lineages (Eastern and Western) were identified, partially separated by the Anatolian Diagonal. Their co-occurrence in multiple localities confirms Anatolia’s function as a secondary contact zone. Similarly, P. kuhlii populations represent a transition zone where two distinct lineages, one of Asiatic origin (P. k. lepidus) and one Mediterranean-Levantine (P. k. kuhlii), meet. Furthermore, while P. nathusii is largely associated with migratory European lineages; a genetically distinct, potentially resident lineage was revealed in southwestern Anatolia. Divergence time estimations indicate that this diversification was shaped by major climatic events from the Miocene to the Pleistocene. This study demonstrates that Anatolia is more than just a geographic bridge; it is a dynamic center of evolution, functioning critically as both a glacial refugium and a secondary contact zone for Palearctic bat fauna. Full article

The unique cytochrome P450 BM3 from Priestia megaterium (syn. Bacillus megaterium) is renowned for its versatile high catalytic activity. The cyp102A1-LG23 gene encoding its CYP102A1-LG23 mutant variant was expressed in Escherichia coli and Mycolicibacterium smegmatis. The in vivo activity of the heterologous enzyme was assessed with respect to androstenedione (AD), androstadienedione (ADD), testosterone (TS) and dehydroepiandrosterone (DHEA). Alongside 7β-hydroxylation, the heterologous enzyme catalyzed the mono- and dihydroxylation of C19 steroids. For the first time, the formation of 7β-, 6β- and 11α-hydroxylated derivatives of ADD using a bacterial enzyme, as well as the hydroxylation of DHEA at the C7α and C7β positions, and its dihydroxylation with the formation of the 7α,15α-dihydroxylated derivative using the mutant cytochrome P450 BM3 were demonstrated. The steroid structures were confirmed using mass spectrometry and ¹H NMR spectroscopy. The advantages of using mycolicibacteria as a bacterial chassis for gene expression were also shown. The results demonstrate the unusual properties of the mutant cytochrome P450 BM3-LG23 and open up prospects for its application in the biotechnological production of valuable hydroxysteroids. Full article

Background/Objectives: The ¹³C-Methacetin Breath Test (MBT) is a non-invasive tool to evaluate hepatic microsomal function via exhaled ¹³CO₂, reflecting cytochrome P450 1A2 (CYP1A2)-mediated metabolism. Despite decades of evidence demonstrating its utility in diagnosing cirrhosis, stratifying liver disease severity, and predicting outcomes, MBT adoption remains limited due to methodological inconsistencies and variable diagnostic thresholds. This review aimed to summarize MBT data in adults and assess its diagnostic and prognostic performance. Methods: A literature review was conducted using PubMed, Web of Science, and Scopus. Eligible studies included those applying oral or intravenous methacetin with defined reference values or diagnostic cutoffs. Outcomes of interest were percent dose recovery (PDR), cumulative PDR (cPDR), and LiMAx^® values. Due to heterogeneity in protocols, units, and endpoints, results were synthesized narratively. Results: Healthy individuals typically demonstrated rapid metabolism (e.g., cPDR30 10–15%), whereas cirrhotic patients showed significantly reduced values (e.g., cPDR30 ≈ 1%). Diagnostic cutoffs varied widely (<0.35% to <8%), reflecting methodological and population differences. MBT reliably identified advanced liver disease but showed inconsistent sensitivity for early-stage fibrosis and metabolic dysfunction-associated steatotic liver disease. Variability in dosing, timing, measurement duration, and analytic technique limited cross-study comparability. Conclusions: MBT is a validated, dynamic marker of liver function with both diagnostic and prognostic relevance. However, inconsistent protocols and thresholds hinder its clinical implementation. Standardization of MBT procedures, reference ranges, and reporting metrics is essential. A harmonized protocol (“MBT-60”), supported by multicenter validation, demographic stratification, and direct comparison with structural and serologic liver tests, is recommended to facilitate MBT integration into routine hepatology practice. Full article