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PubMed2026

CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and zygotic genome activation during goat embryogenesis.

Timely and efficient degradation of maternal mRNA is essential for early embryonic development, which occurs from fertilization through the initiation of zygotic genome activation (ZGA). Yet, the regulatory mechanisms governing this process remain poorly characterized. In the present study, we investigated the function of CCR4-NOT transcription complex subunit 1 (CNOT1) during goat embryogenesis. We found that CNOT1 was upregulated during mammalian ZGA, and that its knockdown led to developmental arrest and a marked reduction in blastocyst formation. Moreover, CNOT1 knockdown impaired nascent RNA activity, resulting in 814 upregulated and 1014 downregulated genes, which were enriched for RNA splicing, regulation of chromosome organization, and RNA localization. RNA splicing analysis revealed differential splicing events in 2959 genes, of which 259 were downregulated following CNOT1 knockdown. Notably, CNOT1 was predicted to crosstalk with the m6A reader YTHDF2. Knockdown of YTHDF2 resulted in CNOT1 downregulation at the 8-cell stage in goats and increased transcription levels around polyadenylation sites during ZGA in mice. Together, these findings indicate that CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and ZGA during goat embryogenesis. Our work provides new insight into the complex regulatory landscape underlying ZGA and may inform strategies to improve the efficiency of goat embryogenesis.

Animals↗

PubMed2026

Simultaneously PYCR-1 and ALH-6 inhibition exacerbates 6-PPD quinone toxicity via disrupting proline and glutamate metabolisms and activating insulin signals in Caenorhabditis elegans.

Glutamate synthesized from the proline can serve as a precursor for key intermediate metabolites of citric acid cycle. Recently, we observed reduced glutamate content and expression of alh-6 controlling glutamate synthesis by 6-PPD quinone (6-PPDQ) in Caenorhabditis elegans. However, possible effect of 6-PPDQ on proline synthesis and the association with 6-PPDQ toxicity induction remain unclear. After 0.1-10 μg/L 6-PPDQ exposure, proline content was further reduced, and expression of pycr-1 governing proline biosynthesis was decreased. In 6-PPDQ exposed nematodes, RNA interference (RNAi) of pycr-1 decreased α-ketoglutarate content, enhanced mitochondrial dysfunction, reduced nicotinamide adenine dinucleotide (NADH) and reduced flavine adenine dinucleotide (FADH₂) contents, inhibited mitochondrial complex I/II activities, and decreased expressions of gas-1 and mev-1. Moreover, compared to single RNAi, double RNAi of pycr-1 and alh-6 exacerbated the 6-PPDQ toxicity in reducing α-ketoglutarate, NADH, and FADH₂ contents, and suppressing mitochondrial complex I/II activities and gas-1 and mev-1 expressions. Additionally, double RNAi of pycr-1 and alh-6 intensified toxicity of 6-PPDQ on longevity and caused upregulation of insulin ligand and receptor genes and downregulation of daf-16 and its targeted genes in 6-PPDQ exposed nematodes. Furthermore, after 6-PPDQ exposure, daf-16 RNAi suppressed pycr-1 and alh-6 expressions, suggesting formation of a regulatory feedback loop between pycr-1/alh-6 and daf-16. Our findings highlight involvement of disrupted proline and glutamate metabolisms in 6-PPDQ-induced mitochondrial dysfunction and reduced longevity.

Animals↗

PubMed2026

Protein sorting and proteostasis mechanisms in CFTR-related exocrine pancreas dysfunction: A systematic narrative review.

The pancreas consists of exocrine and endocrine compartments. In the exocrine pancreas, cystic fibrosis transmembrane conductance regulator (CFTR) functions mainly in ductal epithelial cells as a chloride and bicarbonate channel. Its activity depends on proper protein folding, trafficking, and localization to the apical membrane. This systematic narrative review aims to synthesize the available evidence on the role of protein sorting machinery in CFTR channelopathies and its contribution to exocrine pancreatic dysfunction. A thorough search was conducted using PRISMA criteria on PubMed, Wiley Online Library, and Scopus for studies published in English between January 2000 and November 2025. Twenty studies that met the inclusion criteria were included in this review. Pathogenic CFTR variants impair protein folding, endoplasmic reticulum (ER) exit, and endosomal recycling, resulting in reduced apical membrane expression and stability. These defects disrupt the localization of associated transporters and secretory proteins, impair ductal bicarbonate secretion, alter zymogen handling, and promote acinar injury, although these claims are supported mainly by indirect experimental models and therefore require clinical confirmation. CFTR channelopathies in the exocrine pancreas encompass both ion transport defects and broader disruptions of protein sorting machinery. CFTR may contribute to the assembly, stabilization, or localization of selected apical transport complexes, and its loss can secondarily alter epithelial organization. Therapeutic approaches targeting both channel correction and intracellular trafficking may improve pancreatic function and mitigate disease progression.

Humans↗

PubMed2026

Olive leaf protein hydrolysates yield gastro-resistant peptides with antioxidant and anti-inflammatory potential: peptidomics, in vitro validation and molecular docking analyses.

Olive (Olea europaea L.) leaves are an abundant olive-oil by-product and a promising feedstock for sustainable valorisation. An olive leaf protein isolate (OLPI) from olive-leaf powder (OLP) was enzymatically hydrolysed to yield seven hydrolysates (OLPHs). All showed notable antioxidant activity as whole hydrolysate matrices (EC₅₀ = 0.11-0.28 mg mL-1); likely reflecting the combined contribution of released peptides and co-extracted phenolic compounds; the 15-min Alcalase product (OLPH15A) showed high activity with the shortest processing time. Its INFOGEST digest (dOLPH15A) attenuated LPS-induced inflammation in Caco-2 cells, down-regulating pro-inflammatory and up-regulating anti-inflammatory genes. Peptidomics identified 7037 peptides in OLPH15A and 534 in dOLPH15A, from which twenty gastro-resistant sequences were prioritised for in silico analysis. Multi-tool prediction and docking highlighted four peptides, GAAGGIGQPL, QSAYPGTGPL, GGGAGGGDGGIL and LDAQFPGVN, with favourable predicted affinity for the TLR4/MD2 complex, suggesting that they may contribute to the observed immunomodulatory response. These findings position olive leaves as a viable source of protein hydrolysate-based ingredients with antioxidant and anti-inflammatory potential, advancing the valorisation of olive-oil by-products.

Olea↗

PubMed2026

Dissemination of blaKPC-3-harbouring Klebsiella pneumoniae across ST48 and ST628 in multiple healthcare facilities in the Republic of Korea.

Klebsiella pneumoniae carbapenemase-3 (KPC-3) remains rare in South Korea, where KPC-2 is the dominant carbapenemase, making the repeated detection of a concentrated blaKPC-3 signal over five years notable. We performed genomic analyses of blaKPC-3-harbouring K. pneumoniae from a regional healthcare network. Two chromosomally distinct lineages with concordant capsule loci (ST628/KL15 and ST48/KL62) presented multidrug-resistant phenotypes, and the virulence-associated loci were confined to ST48. Single-nucleotide polymorphism (SNP) analyses revealed near-clonal relatedness within lineages, with 0-38 pairwise SNPs among ST628 isolates and 8 SNPs between the two ST48 isolates. Core-genome multilocus sequence typing (cgMLST) supported this structure, as ST628 isolates were assigned to complex type 19149 with 0-7 allelic differences, and ST48 isolates were assigned to complex type 19150 with 5 allelic differences. These patterns support vertical spread via clonal expansion across multiple facilities. Despite substantial chromosomal separation, most isolates carried the same IncFII(K) plasmid backbone and blaKPC-3, and they were nearly indistinguishable from a plasmid previously reported in South Korea. One isolate carried blaKPC-3 on a distinct multireplicon IncFIB(K)/IncFII(K) plasmid, indicating that the signal was not confined to a single plasmid backbone. In both plasmids, blaKPC-3 was embedded within Tn4401b. These findings indicate that a rare blaKPC-3 genotype can persist regionally through sustained clonal dissemination and that cross-lineage linkage is compatible with past horizontal transfer involving a conserved plasmid. These findings underscore the need for subtype-resolved, regionally coordinated genomic surveillance in connected healthcare networks to detect uncommon carbapenemase variants early.

Klebsiella pneumoniae↗

PubMed2026

Mitochondrial translocation of DNMT3L suppresses oxidative phosphorylation and restrains megakaryopoiesis.

DNMT3L, a catalytically inactive member of the DNA methyltransferase family, is identified here as a negative regulator of megakaryopoiesis. In K562 cells undergoing PMA-induced megakaryocytic differentiation, DNMT3L protein levels declined progressively, and shRNA-mediated depletion enhanced differentiation, whereas overexpression attenuated it. Consistent with these findings, Dnmt3l-knockout mice exhibited elevated peripheral blood platelet counts and expanded bone marrow megakaryocytes. Mechanistically, megakaryocytic differentiation triggered rapid mitochondrial translocation of DNMT3L within 6 h; mitochondrial DNMT3L suppressed oxidative phosphorylation (OXPHOS) capacity and ATP production and downregulated mitochondrial-encoded genes spanning Complex I, III, IV, and ATP synthase, without altering mitochondrial DNA copy number. This metabolic suppression was mediated through compartment-specific remodeling of DNMT3L-containing protein complexes: upon differentiation, DNMT3L selectively dissociated from DNMT1 and DNMT3B in mitochondria, relieving the repressive constraint on OXPHOS, whereas in the nucleus DNMT3L remained associated with DNMT3A, which concomitantly accumulated during differentiation. These findings reveal a previously unrecognized mechanism by which a catalytically inactive epigenetic co-regulator spatially redistributes to coordinate mitochondrial metabolic output with nuclear epigenetic control, thereby facilitating terminal megakaryocytic maturation.

Animals↗

PubMed2026

Viral replication through phase separation: Cytosolic and nuclear condensates.

Replication of many RNA and DNA viruses occurs within specialized intracellular hubs organized as membraneless biomolecular condensates (BCs) driven by liquid-liquid phase separation. As obligate intracellular parasites, viruses depend on the host cell machinery to complete their replication cycles and therefore actively remodel the intracellular environment to favor viral genome replication, transcription, and assembly. Cytosolic and nuclear phase-separated replication compartments (RC) provide concentrated and dynamic platforms that promote efficient interactions between viral genomes and viral or host proteins essential for infection. The formation of viral replication BCs is typically facilitated by viral proteins enriched in intrinsically disordered regions and low-complexity domains, which enable multivalent interactions with viral nucleic acids and cellular factors. These interactions are mediated by diverse biophysical forces, including hydrophobic and π interactions, hydrogen bonding, molecular crowding, and osmotic effects. Throughout infection, viral BCs remain highly dynamic, allowing continuous exchange of components and functional maturation of replication hubs. Their properties and activities are further regulated by post-translational modifications of viral and host proteins, such as phosphorylation, acetylation, and methylation. In this review, we summarize current evidence supporting liquid-liquid phase separation as a central organizing principle of viral RCs. We focus on representative RNA and DNA viruses that replicate in the cytosol or nucleus, highlighting virus-specific strategies, conserved mechanisms, and the consequences of BC formation for viral replication efficiency, host antiviral responses, and therapeutic intervention.

Phase Separation↗

PubMed2026

Bioprospecting microbial genomes to expand the biocatalytic toolbox of rubber oxygenases.

A set of rubber oxygenases was discovered through phylogenetic analysis and AI-based structural modeling of complexes of the putative enzymes with a substrate mimicking cis-1,4-polyisoprene. Sixteen candidate proteins were selected from thermophilic microorganisms, all sequence-related to the Latex clearing protein from Streptomyces sp. K30 (LcpK30). Sequence truncation and solubility tags were then evaluated to enhance protein expression, with the SUMO tag proving to be the most effective. Including LcpK30, nine heme-containing oxygenases were successfully expressed in E. coli NEB 10-beta cells, purified (35-157 mg L-1 yield) and characterized. Steady-state kinetics revealed significant rubber latex-degrading properties for six of them, with the truncated SUMO-fused LcpK30 (SUMO-LcpK30T) showing activity in agreement with literature. Notably, the catalytic efficiencies of all the expressed homologs lay within one order of magnitude and the oxygenase from Thermomonospora echinospora was found to be particularly promising in terms of activity, especially at high latex concentrations (more than 1% w/v). The analysis of reaction mixtures by both HPLC and HPLC-MS confirmed the oxidation of cis-1,4-polyisoprene to form the expected isoprenoid oligomers (n = 2-12), whose distribution was consistent with the usual endo-type cleavage pattern in all but one case. This bioprospecting effort afforded a platform of new rubber-degrading enzymes with diverse efficiencies and product profiles, capable of adapting to targeted applications.

Oxygenases↗

PubMed2026

An overview of the use of proteomics and peptidomics to characterize alternative protein foods.

The global protein transition is accelerating the development of alternative protein foods, mainly derived from plants, insects, algae, fungi, and cellular agriculture. Ensuring the authenticity, safety, and nutritional adequacy of these emerging protein matrices requires molecular-level characterization beyond traditional compositional analyses. Proteomics and peptidomics have emerged as transformative analytical platforms capable of decoding the molecular signatures that define protein origin, structural integrity, digestibility, functionality, and health potential. The review comprehensively examines the application of proteomics, and peptidomics for profiling alternative protein foods. Further, the source authentication strategies based on species-specific protein and peptide biomarkers, detection of adulteration in complex matrices, and allergenicity assessment is discussed. Special attention is also given to nutritional proteomics with protein digestibility, gastrointestinal peptide release, and identification of bioactive sequences. SIGNIFICANCE: The importance of this review is that proteomics and peptidomics are becoming central in the management of the fast-growing environment of alternative protein foods, such as plant-based, insect, algal, fungal, and cultured meat products. It provides an explanation of the application of mass spectrometry-based processes to decode molecular signatures defining the origin of proteins, their structural integrity, digestibility, allergenicity, and bioactive properties, and thus directly contribute to safety, nutritional analysis, and authenticity of the product. Presentation of the article includes the integration of the knowledge of traditional muscle foods with alternative systems of proteins, where validated protein and peptide biomarkers are used in authentication, fraud detection, and allergy risk assessment in a wide variety of matrices. It also indicates the role of nutritional proteomics and peptidomics in informing the formulation strategy to promote digestibility and release of health-promoting peptides. In general, this review will guide scientists, the food industry, and regulatory bodies to use modern proteomic technologies in quality assurance, and decision-making, for the advancementof sustainable protein-based foods.

Proteomics↗

PubMed2026

Engineering bubble structures as Cas12a activators for highly sensitive monitoring of WRN helicase function.

The Werner syndrome helicase (WRN) is a critical synthetic lethal target in microsatellite instability cancers, essential for resolving complex genomic structures like replication bubbles and R-loops. However, strategies to simultaneously discriminate WRN activity on DNA versus DNA-RNA substrates in living cells are lacking. Here, we developed a structure-specific CRISPR/Cas12a biosensing strategy to visualize WRN functional activity by engineering bubble-structure probes. These probes were rationally designed to structurally mimic DNA replication bubbles and R-loop associated DNA-RNA hybrids. Upon specific unwinding by WRN, the probes release a sequestered activator strand that triggers Cas12a trans-cleavage, effectively converting the unwinding event into an amplified fluorescent signal. This assay achieves low picomolar sensitivity (LODs: 5.6-6.0 pM) and exceptional selectivity against homologous RecQ helicases. Uniquely, this strategy enables the parallel quantification of WRN activity on both substrate types, providing insights into distinct WRN-mediated pathways for resolving genomic stress. We further demonstrated the strategy's utility by visualizing endogenous WRN dynamics in living cells and profiling the efficacy of small-molecule inhibitors. This work offers a powerful molecular toolkit for dissecting WRN biology and facilitating high-throughput drug screening in targeted cancer therapy.

Werner Syndrome Helicase↗

PubMed2026

Cre-loaded integrase-defective lentiviral vectors for targeted cassette exchange in CHO cells.

Genome-modifying enzymes, such as recombinases and CRISPR-associated nucleases, enable targeted gene insertion when delivered transiently to minimize off-target effects. Precise genome engineering requires controlled enzyme activity, as well as efficient donor DNA transfer. Integrase-defective lentiviral vectors (IDLVs) provide a promising platform for transient episomal DNA transfer; however, their integration efficiency depends on complementary genome-targeting strategies. Here, we engineered Cre-loaded IDLVs (Cre-IDLVs) that co-package lentiviral vector genomes together with bioactive Cre recombinase. Cre was inserted into the Gag region of an integrase-defective gag-pol construct, allowing for efficient encapsidation and protease-mediated release during virion maturation without compromising the viral titer. The resulting particles carried donor cassettes flanked by heterospecific loxP sites. When applied to CHO founder cells harboring compatible genomic loxP landing pads, Cre-IDLVs efficiently mediated recombination-mediated cassette exchange, producing the highest number of G418-resistant colonies among the plasmid ratios tested. Genomic PCR and sequencing confirmed precise locus-specific insertion without detectable random integration in the analyzed clones. These findings establish Cre-IDLVs as a streamlined dual-delivery platform that couples transient recombinase activity with episomal donor DNA transfer. This hybrid lentiviral strategy provides a programmable approach for controlled and site-specific genome modification in mammalian cells.

Integrases↗

PubMed2026

The composition of the periostracum in the razor clam Sinonovacula constricta and the mantle's response to sulfide.

The razor clam Sinonovacula constricta inhabits sulfide-rich intertidal sediments and exhibits remarkable tolerance to this toxicant, yet the role of its periostracum in sulfide adaptation remains poorly understood. In this study, we investigated the composition and structure of the periostracum proteins, and the response of the mantle to sulfide stress. Scanning electron microscopy and energy-dispersive X-ray spectroscopy revealed that the periostracum is approximately 10 μm thick and contains 1.43 wt% sulfur, and proteomic analysis further confirmed the presence of organic sulfur (Cys/Met-rich proteins), suggesting its involvement in sulfur deposition. Using LC-MS/MS, we identified 77 high-confidence proteins from the periostracum, which were classified into six functional categories: enzymes, framework proteins, immune-related proteins, calcium ion-related proteins, other proteins, and proteins with unknown functions. Phylogenetic analyses of representative proteins revealed bivalve-specific evolutionary patterns, with several proteins exclusively present in Bivalvia, such as Unknown protein 2 and 7, which possess signal peptides and low-complexity domains. For the sulfide exposure experiment, razor clams were subjected to three Na2S concentrations (0, 10, and 100 μM). qPCR analysis showed that, compared with the control group, Chitin-binding protein 3 and Tyrosinase were significantly upregulated in the mantle, peaking in the 100 μM group at 48 h (5677.84-fold and 157.20-fold, respectively), whereas Collagen and Cadherin 3 were generally suppressed. This study represents one of the most comprehensive proteomic profiles of the razor clam periostracum and highlights the mantle's potential role in sulfide tolerance, offering insights for sulfur-tolerant aquaculture breeding and bioremediation applications.

Animals↗

PubMed2026

Whole-Genome Deep Learning Predicts Chemotherapy Response in Colorectal Cancer.

Chemotherapy response in colorectal cancer (CRC) exhibits significant heterogeneity, with current clinical predictors failing to capture complex genomic determinants of resistance. We developed a hybrid deep learning framework integrating convolutional neural networks (CNNs) and bidirectional long short-term memory (BiLSTM) networks to analyze whole-genome somatic mutations, evolutionary conservation, chromatin accessibility, and 3D genome architecture in 2,546 TCGA patients. An attention mechanism identified predictive genomic regions. The model achieved an AUC of 0.92 (95% CI: 0.89-0.94) in cross-validation and 0.88 (95% CI: 0.85-0.91) in independent validation, outperforming clinical models (ΔAUC = +0.18, p < 0.001). Key predictors included non-coding variants in TP53, KRAS, and PIK3CA regulatory regions. Triple-positive patients (mutations in all 3 regions) had significantly worse progression-free survival (HR = 4.7, p < 0.001). Our framework enables accurate chemotherapy response prediction and reveals novel non-coding resistance mechanisms, advancing precision oncology in CRC.

Humans↗

PubMed2026

Performance comparison of rapid and native barcoding methods for Oxford Nanopore sequencing of Poliovirus Viral Protein 1 (VP1) amplicons.

Accurate and timely sequencing of poliovirus is critical for global eradication efforts, particularly for molecular epidemiology based on the typing region of the genome, viral protein 1 (VP1). While Oxford Nanopore Technologies (ONT) sequencing has expanded capabilities for poliovirus surveillance, the relative performance of different ONT library preparation methods, including ligation-based (Native Barcoding) and transposase-based (Rapid Barcoding) approaches, has not been systematically evaluated. In this study, we compared rapid barcoding and native barcoding workflows for sequencing VP1 amplicons from 17 type 2 poliovirus-positive samples, each processed in triplicate. Native barcoding generated significantly more sequencing output, producing approximately 2.3-fold greater total read yield than rapid barcoding, and demonstrated higher run-to-run reproducibility (R2 = 0.979-0.998 vs. 0.847-0.929, respectively; p < 0.001). In addition, native barcoding generated 80% of the total yield achieved by rapid barcoding within approximately 7 h, whereas rapid barcoding required approximately 40 h to reach the same output. Despite these differences, both methods produced identical VP1 consensus sequences across all samples, with comparable read quality (median per-base Q-scores of approximately Q17-Q18). Rapid barcoding provided substantial practical advantages, reducing hands-on library preparation time (55 vs. 200 min) and per-sample cost ($12.82 vs. $16.54), while simplifying workflow and reducing technical complexity. These findings indicate that sequencing yield may not be a determinant of downstream analytical outcomes for poliovirus VP1 ONT sequencing. Rapid barcoding therefore represents a cost-effective and efficient approach for routine poliovirus surveillance, whereas native barcoding remains advantageous in applications requiring rapid data generation or maximal sequencing depth.

Poliovirus↗

PubMed2026

Differential contributions of mt-Tr and Cs variants to developmental cochlear defects and mitochondrial dysfunction in A/J mice.

A/J mice exhibit early-onset hearing loss linked to Cdh23, mitochondrial tRNA-Arg (mt-Tr), and citrate synthase (Cs) variants. Although developmental cochlear defects have been identified in juvenile A/J mice, the hierarchical contributions of mt-Tr versus Cs remain unclear. Using reciprocal intercross-derived strains to decouple mitochondrial haplotypes from nuclear factors, we demonstrate that the mitochondrial background is the primary determinant of auditory dysfunction. Mice with A/J mtDNA (AXB strains) displayed significantly higher ABR thresholds, accelerated hair cell attrition, and severe stereocilia dysmorphology compared to those with B6 mtDNA (BXA strains), occurring largely independently of the Cs genotype. While the Cs mutation exacerbated hearing loss, its impact was secondary to that of the dominant mitochondrial background. Systemic behavioral assessments and mitochondrial assays confirmed that A/J mitochondria exert a more profound metabolic impact than the Cs mutation. Our findings establish that the mitochondrial genomic background, with the mt-Tr locus as a prominent candidate variant, serves as the principal driver of developmental cochlear defects and early-onset hearing loss in A/J mice, while the nuclear Cs mutation acts as a synergistic modifier. This study underscores the critical role of mitonuclear crosstalk in inner ear maturation and provides new insights into the etiology of hereditary hearing loss.

Animals↗

PubMed2026

Early hepatic protein responses to dietary restriction-refeeding in Japanese quail: A proteomic investigation.

Feed intake and refeeding after nutrient scarcity induce rapid metabolic adaptations in the poultry liver; however, hepatic proteomic recovery pathways in the early hours post-refeeding remain poorly defined. This study aimed to characterize early liver protein signatures in Japanese quail (Coturnix japonica) recovering from nutritional stress under two refeeding conditions. Eighteen 12-week-old male quails (245.20 ± 0.213 g) were assigned to three groups (n = 6): control fed ad libitum (12.13 MJ/kg), 24 h feed deprivation followed by 6 h refeeding, and 24 h low metabolizable energy (6.30 MJ/kg) diet followed by 6 h refeeding. In total, 854 proteins were identified, of which 515 met the filtering criteria. The low metabolizable energy refeeding showed higher abundance of proteins linked to ATP binding and carbohydrate/carboxylic acid metabolism, alongside detoxification-related proteins, while suppressing translation/RNA-binding machinery and antioxidant pathways. Feed-deprived refeeding enriched in oxidative phosphorylation and mitochondrial complex I assembly with reduced cytoplasmic translation, NMD-related components, and sulfur compound metabolism. A direct comparison indicated divergent recovery strategies: low metabolizable energy refeeding mainly reflected oxidoreductase activity and translation initiation, whereas feed-deprived refeeding potentially enriched mitochondrial ATP production and glutathione-based defenses. Our analysis indicate that 6 h of refeeding initiates an early, incomplete recovery toward hepatic homeostasis, with the severity of prior nutritional restriction dictating distinct liver metabolic priorities. Collectively, these findings might provide a preliminary understanding of the hepatic mechanisms involved in recovery from nutrient deprivation and may help in the development of feeding strategies for managing metabolic recovery in poultry. However, these findings should be considered hypothesis-generating pending further validation.

Animals↗

PubMed2026

Insights into the mechanism of enhanced tetramethylpyrazine production in dehulled adlay fermented by Bacillus subtilis BJ3-2.

Tetramethylpyrazine (TTMP) is a vital bioactive alkaloid and characteristic flavor compound in fermented foods. Our previous study found that fermentation of adlay by Bacillus subtilis BJ3-2 efficiently accumulates TTMP, whereas the underlying high-yield mechanism remains unclear. This study investigated the fermentation characteristics, gene transcription and protein expression of B. subtilis BJ3-2 in dehulled adlay (BDA) and soybean (BSB), respectively, and elucidated the mechanism responsible for high-yield TTMP production. The results showed that glutamate, leucine and phenylalanine were major free amino acids in BDA. The TTMP yield in BDA at 48 h (6.11 mg/g dry weight) was 360-fold higher than that in BSB. Transcriptomic and proteomic analysis demonstrated that compared with the soybean substrate, dehulled adlay substrate significantly up-regulated the expression of alsSD and ilvBH genes and their encoding proteins in B. subtilis BJ3-2, which were involved in C5-branched dibasic acid metabolism, 2-oxocarboxylic acid metabolism, and valine, leucine and isoleucine biosynthesis. Meanwhile, acetoin degradation was inhibited by down-regulating acetoin dehydrogenase complex (acoABCL) in citrate cycle, glycolysis/gluconeogenesis and carbon metabolism. Additionally, nitrogen metabolism pathway was transcriptionally enhanced to guarantee sufficient ammonium supply. Notably, protein-protein interaction and molecular docking analyses revealed that acetohydroxyacid synthase (ilvBH) interacted tightly with α-acetolactate decarboxylase (alsD), potentially forming a metabolic channel for acetoin synthesis. In conclusion, the efficient synthesis of TTMP in BDA was primarily attributed to the high synthesis and low degradation of acetoin, and the moderate synthesis of ammonium/ammonia. This study provided a theoretical basis for the targeted and efficient biosynthesis of TTMP.

Bacillus subtilis↗

PubMed2026

A STORM-based protocol for nanoscale imaging and quantitative analysis of protein-associated and phospholipid-associated structures in natural rubber.

Stochastic Optical Reconstruction Microscopy (STORM) enables nanoscale mapping of molecular components beyond the diffraction limit; however, its reproducible implementation in hydrophobic polymer matrices remains challenging because fluorescence-labeling specificity, fluorophore photoswitching, three-dimensional localization, chromatic registration, and quantitative image analysis must be carefully controlled. This protocol presents a standardized experimental workflow for dual-color labeling, astigmatism-based three-dimensional STORM acquisition, and quantitative analysis of protein-associated and phospholipid-associated structures in natural rubber (NR). The workflow covers sample pretreatment, Cy5 NHS ester labeling of protein-associated primary amines, DiI labeling of phospholipid-rich domains, STORM imaging-buffer preparation, three-dimensional single-molecule localization, dual-channel registration, generation of standardized xy projections, aggregate-size analysis, and projected lateral spatial correlation assessment. Reproducibility is supported by defined acquisition and localization criteria, three independent sample preparations with at least five fields of view analyzed per condition, and unlabeled, single-color, dye-only matrix, and processing-associated Cy5 controls. Mean lateral localization precisions of 11.8 ± 2.3 nm for Cy5 and 13.5 ± 2.9 nm for DiI were obtained, while two-dimensional Fourier ring correlation analysis of the xy projections yielded effective lateral image resolutions of approximately 25 and 28 nm, respectively. Image-based particle segmentation and localization-coordinate-based density-based spatial clustering of applications with noise (DBSCAN) were applied to standardized xy projections as complementary quantitative approaches. Application of the protocol to untreated, centrifuged, and protease-treated NR samples demonstrated treatment-associated changes in the detected abundance and projected size distributions of protein- and phospholipid-associated aggregates, together with a non-monotonic change in their projected lateral spatial correlation. These observations describe alterations in nanoscale organization but do not, by themselves, establish stable protein-phospholipid complex formation. Unlike previous studies that primarily demonstrated the feasibility of STORM imaging in rubber materials, the principal contribution of this work is an end-to-end, step-by-step protocol incorporating defined controls, three-dimensional localization, image-quality metrics, chromatic-registration procedures, and complementary quantitative-analysis pipelines for non-expert users. The workflow may be adaptable to other hydrophobic polymers and soft-material systems after appropriate optimization and validation.

Rubber↗