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PubMed2026

Third-generation whole-genome sequencing reveals the role of CNTNAP2 as a tumor suppressor gene in high-risk neuroblastomas.

BACKGROUND: Neuroblastoma is a common and aggressive pediatric sympathetic nervous system tumor. Genomic structural variants (SVs) contribute substantially to neuroblastoma, yet remain under-characterized in high-risk neuroblastomas. We aimed to elucidate neuroblastoma pathogenesis using third-generation whole-genome sequence high-risk cases to identify driver aberrations and explore potential therapeutic strategies. METHODS: We analyzed third-generation whole-genome sequencing data of 20 high-risk neuroblastoma samples and combined the findings with those obtained from the analysis of clinical samples, in vitro models, and public datasets. RESULTS: The contactin-associated protein-like 2 (CNTNAP2) gene was observed to be frequently aberrated because of structural variants in high-risk neuroblastoma samples. CNTNAP2 expression was significantly correlated with favorable histology and could be used to predict prognosis using clinical samples and neuroblastoma datasets. Overexpression and knockdown experiments and transcriptomic analysis revealed that CNTNAP2 was primarily involved in neuronal differentiation and axon guidance pathways; moreover, CNTNAP2 was required for neuroblastoma differentiation and affected cancer stemness. Immunoprecipitation and mass spectrometry revealed that CNTNAP2 interacted with cytoskeletal proteins like drebrin 1 (DBN1) and myosin-heavy chain 9 (MYH9). CNTNAP2 dynamically reorganises actin and microtubules for DBN1-mediated neuronal differentiation. CNTNAP2 also reduces CTNNB1 transcription and β-catenin pathway activation by inhibiting MYH9 nuclear translocation. CNTNAP2 overexpression in neuroblastoma cell lines resulted in cell cycle arrest, decreased cell proliferation and metastasis. CONCLUSIONS: The recurrent loss of CNTNAP2 in neuroblastoma contributes to an aggressive phenotype by impairing neuronal differentiation and increasing cancer stemness. These findings may serve as a foundation for developing therapeutic strategies to overcome barriers to differentiation.

Humans↗
PubMed2025

ves1α genes expression is the major determinant of Babesia bovis-infected erythrocytes cytoadhesion to endothelial cells.

Babesia bovis causes the most pathogenic form of babesiosis in cattle, resulting in high mortality in naive adults. This parasite invades red blood cells (RBCs) within the bovine hosts where they multiply and produce clinical disease. Babesia bovis exports numerous proteins into invaded RBCs changing its properties. Thus, the infected RBCs (iRBCs) are capable to cytoadhere in the microvasculature of internal organs and brain, leading to respiratory distress, neurologic signs, and mortality. Variant Erythrocyte Surface Antigen 1 (VESA1) is one of those exported proteins by B. bovis which represents a major virulence factor due to its central role in immune evasion by antigenic variation and intravascular parasite sequestration. VESA1 is a heterodimer protein encoded by ves1α and ves1β multigene family and localized on the ridges, the focal point for cytoadhesion. To gain further insights into the molecular mechanisms of cytoadhesion of B. bovis, we panned the parasites with bovine brain microvasculature endothelial cells, which resulted in obtaining several clones with different cytoadherence abilities. The transcriptome analysis of 2 high and 2 low cytoadherent clones revealed that ves1α sequences were diversified, likely resulting from genomic recombination. On the other hand, ves1β sequences were almost identical among these 4 clones. Insertion and expression of ves1α of a clone with high binding into ef-1α locus of a low binding clone increased cytoadherence confirming the role of ves1α suggested by our transcriptome data. Whole genome sequencing of cytoadherent clones revealed active locus of ves1 on chromosome 2. These results suggest that VESA1a proteins encoded by ves1α genes determine the cytoadherence strength of B. bovis and they are in the active site for recombination.

Animals↗
PubMed2026

Comparative metabolomic and transcriptomic profiling of flavonoid diversity and antioxidant capacity in three Isatis species.

Flavonoids are key bioactive compounds in plants with significant health benefits. This study employs an integrated multi-omics approach to investigate flavonoid diversity and antioxidant capacity across three Isatis species: I. oblongata, I. tinctoria, and I. indigotica. Metabolomic profiling identified 200 flavonoids, with glycosides being the most abundant class. I. tinctoria exhibited the highest total flavonoid content and antioxidant activity, strongly correlated with the accumulation of 53 core differential flavonoid metabolites, most of which were glycosylated derivatives. Transcriptomic analysis revealed coordinated upregulation of phenylpropanoid pathway genes and specific UDP-glycosyltransferases (UGTs) in I. tinctoria, providing a genetic basis for its enhanced glycoside production. The study establishes a clear genotype-metabolite-phenotype linkage, highlighting glycosylation as a key mechanism underlying flavonoid-driven antioxidant superiority in Isatis. Although the current evidence is primarily correlative, the consistent and strong associations across independent transcriptomic, metabolomic, and antioxidant datasets provide a robust foundation for this conclusion. These findings offer new insights into the metabolic evolution and regulatory networks of flavonoids, with implications for breeding and metabolic engineering of high-value medicinal plants.

Flavonoids↗
PubMed2026

Metabolome-driven rhizosphere microbiome assembly determining the health of medicinal herb (Angelica sinensis) against root rot.

BACKGROUND: The rhizosphere-associated microbiota plays a crucial role in plant responses to disease stress. Plant secondary metabolites are recognized as crucial mediators in the assembly of rhizosphere microbial communities, particularly by enhancing the colonization of beneficial microorganisms. Despite this recognized importance, a deeper understanding of how such metabolome-driven microbiome assembly specifically determines plant resistance against soil-borne diseases is still lacking. RESULTS: Here, we focused on the widely planted medicinal plant Angelica sinensis and demonstrated that root rot-diseased rhizosphere soils (DRS) exhibited a higher relative abundance of Fusarium and a lower relative abundance of Streptomyces compared to healthy rhizosphere soils (HRS). Shotgun metagenomic sequencing revealed that metabolism-associated genes, particularly those related to steroid degradation, are significantly enriched in HRS samples. Subsequent genome and functional gene analysis of Streptomyces revealed that the steroid degradation-related genes are associated with rhizosphere colonization in hosts. Rhizosphere Streptomyces S15 directly antagonized Fusarium and enhanced the root resistance of A. sinensis. Comparative metabolomics showed that A. sinensis plants from HRS secreted more lipid and lipid-like molecules than those from DRS, especially sterol lipids and long-chain fatty acids, which promoted the growth of Streptomyces S15 isolates. Transcriptome analysis validated that the lipid hormones are essential for sporulation, biofilm formation, and streptomycin biosynthesis of S15 strain. Finally, exogenous application of synbiotics (lipid prebiotics and S15) to A. sinensis resulted in the enrichment of S15-homologous Streptomyces amplicon sequence variant (ASV), further establishing beneficial bacterial communities in Fusarium-stressed rhizospheres. CONCLUSIONS: Our study proposes that A. sinensis recruits steroid-metabolizing Streptomyces species by exuding key lipid compounds (i.e., methyl jasmonate and brassinolide) to combat Fusarium root rot. This study provides novel insights into using functional synbiotics as a promising strategy for manipulating plant-microbiome interactions to promote sustainable agriculture. Video Abstract.

Rhizosphere↗
PubMed2026

Cis-regulatory variation in the MdCKX6 promoter is associated with allele-specific expression and fruit size in apple.

Fruit size is a key determinant of apple fruit quality and market value and is strongly influenced by phytohormone-regulated cell proliferation and expansion during early fruit development. Cytokinin oxidase/dehydrogenase (CKX) enzymes regulate cytokinin homeostasis by irreversibly degrading active cytokinins, but the contribution of natural variation in CKX genes to fruit size remains poorly understood. Here, we identified MdCKX6 as a candidate regulator of fruit growth in apple (Malus domestica). MdCKX6 exhibited pronounced allele-specific expression during fruit development in the cultivar 'Royal Gala'. Sequence analysis identified a promoter SNP associated with differential promoter activity and allele-specific expression. Genotyping of diverse apple cultivars and wild Malus accessions revealed a significant association between MdCKX6 promoter genotype and fruit size. Cultivars carrying low-expression alleles produced larger fruits, whereas high-expression alleles were associated with smaller fruits. To investigate gene function, MdCKX6 was overexpressed in tomato, resulting in reduced fruit size. Histological analyses of the transgenic tomato fruit revealed smaller pericarp cells. Transcriptome analysis of transgenic fruits revealed widespread changes in genes associated with cell-cycle regulation, cell wall modification, hormone-related processes, and transcriptional regulation. Together, these results identify MdCKX6 as a potential negative regulator of apple fruit growth and reveal an association between cis-regulatory variants, gene expression, and fruit size. This study provides new insights into the role of cytokinin metabolism in fruit development and highlights regulatory variation in MdCKX6 as a potential target for apple breeding.

Malus↗
PubMed2026

De Novo Assembly of the Trypanosoma congolense Genome Reveals an Organization Influenced by Antigenic Variation but Distinct from Trypanosoma brucei.

Antigenic variation allows pathogens to evade mammalian adaptive immunity through the continuous change in exposed antigens. In African trypanosomes, antigenic variation involves changes in expressed Variant Surface Glycoproteins (VSGs). Understanding of VSG expression control and change amongst African trypanosomes is most advanced in Trypanosoma brucei. In the important animal trypanosome, Trypanosoma congolense, incomplete genome assembly has held back understanding of the mechanics of antigenic variation. Here, we have used long-read DNA sequencing and Hi-C DNA interaction analysis to provide a telomere-to-telomere assembly of the T. congolense genome. This assembly reveals a genome comprising 12 diploid chromosomes, one tetraploid chromosome, and more than 100 small chromosomes. With this assembly we reveal several features of VSG organization and expression that differ from T. brucei. The majority of the T. congolense VSG archive, estimated at ∼1,500 genes, localizes to subtelomeres in 12 of the 13 large chromosomes, but these loci are notably smaller than are found in T. brucei. Furthermore, transcriptome analysis suggests expression of VSGs across the T. congolense subtelomeres, which are not separated within the nucleus from non-VSG chromosome regions, suggesting that there is no dedicated VSG expression site. Strikingly, one chromosome contains approximately 40% of the VSG archive and is largely transcriptionally silent, potentially acting as the major reservoir of new VSG variants. Finally, we show that VSG expression can be detected from multiple small chromosomes. In summary, the new genome assembly provides a platform for understanding a potentially unusual operation of VSG expression and switching in T. congolense.

Trypanosoma congolense↗
PubMed2025

Exploring Hox Genes and Their Temporal Expression in an Embryonic Model of Freshwater Crustaceans.

Hox genes have been investigated in various Arthropod species, resulting in the identification of ten Hox genes, organized in a colinear arrangement within the genome. Among arthropods, crustaceans exhibit a remarkable diversity of body shapes, which are associated with a variety of egg types, embryonic development patterns, and importantly, with the modulation of Hox genes to specify the identity of body segments along the antero-posterior axis of the embryo. Although there are more than 52,000 species of crustaceans described, their genomic resources are relatively limited, making it challenging to employ several molecular tools for studying embryonic development. In this regard, we present a protocol for identifying Hox genes in a freshwater prawn using degenerate primers and transcriptome analysis. This method enables the study of specific functions of Hox genes, thereby contributing to the evolutionary understanding of the diversity of body shapes in crustaceans.

Animals↗
PubMed2022

Lipid hydroperoxides and oxylipins are mediators of denervation induced muscle atrophy.

Loss of innervation is a key driver of age associated muscle atrophy and weakness (sarcopenia). Our laboratory has previously shown that denervation induced atrophy is associated with the generation of mitochondrial hydroperoxides and lipid mediators produced downstream of cPLA2 and 12/15 lipoxygenase (12/15-LOX). To define the pathological impact of lipid hydroperoxides generated in denervation-induced atrophy in vivo, we treated mice with liproxstatin-1, a lipid hydroperoxide scavenger. We treated adult male mice with 5 mg/kg liproxstain-1 or vehicle one day prior to sciatic nerve transection and daily for 7 days post-denervation before tissue analysis. Liproxstatin-1 treatment protected gastrocnemius mass and fiber cross sectional area (∼40% less atrophy post-denervation in treated versus untreated mice). Mitochondrial hydroperoxide generation was reduced 80% in vitro and by over 65% in vivo by liproxstatin-1 treatment in denervated permeabilized muscle fibers and decreased the content of 4-HNE by ∼25% post-denervation. Lipidomic analysis revealed detectable levels of 25 oxylipins in denervated gastrocnemius muscle and significantly increased levels for eight oxylipins that are generated by metabolism of fatty acids through 12/15-LOX. Liproxstatin-1 treatment reduced the level of three of the eight denervation-induced oxylipins, specifically 15-HEPE, 13-HOTrE and 17-HDOHE. Denervation elevated protein degradation rates in muscle and treatment with liproxstatin-1 reduced rates of protein breakdown in denervated muscle. In contrast, protein synthesis rates were unchanged by denervation. Targeted proteomics revealed a number of proteins with altered expression after denervation but no effect of liproxstain-1. Transcriptomic analysis revealed 203 differentially expressed genes in denervated muscle from vehicle or liproxstatin-1 treated mice, including ER stress, nitric oxide signaling, Gαi signaling, glucocorticoid receptor signaling, and other pathways. Overall, these data suggest lipid hydroperoxides and oxylipins are key drivers of increased protein breakdown and muscle loss associated with denervation induced atrophy and a potential target for sarcopenia intervention.

Male↗
PubMed2026

Disruption of the ubiquitin-mediated proteolysis pathway: a study of seed aging in Saposhnikovia divaricata caused by UBC1 gene family suppression.

BACKGROUND: Saposhnikovia divaricata (Turcz.) Schischk. is a perennial herb whose seed aging during storage significantly reduces germination rates, limiting industrial-scale production. Reactive oxygen species (ROS)-induced oxidative damage is a key driver of seed aging, but the underlying mechanisms in Saposhnikovia divaricata remain unclear. RESULTS: Suppression of the UBC1 gene family reduces the activity of ubiquitin-conjugating enzymes, leading to dysfunction of the ubiquitin-mediated proteolysis pathway, which in turn decreases protein degradation efficiency and causes the accumulation of damaged proteins. Transcriptome analysis revealed predominant downregulation of genes crucial for seed physiological maintenance. By the fourth year of storage, germination dropped sharply to 30.67%, accompanied by embryo cavitation. Downregulation of ribosome pathway genes hindered ribosome assembly and protein synthesis, while suppression of endoplasmic reticulum protein processing genes led to unfolded/misfolded protein accumulation and intensified cellular stress, accelerating aging. Proteomic analysis showed increased total differential and antioxidant-related proteins. ROS content fluctuated with storage time: peroxyl radicals peaked in year two (5.68 RFU/mg), whereas hydroxyl radicals and hydrogen peroxide were highest in year four (0.0655 pg/mL and 0.0946 pg/mL, respectively), with significant differences across periods. Elevated membrane-related proteins, increased electrical conductivity, and malondialdehyde content (maximum 54.30 nmol/g at year four) confirmed oxidative membrane damage. ROS-induced stress promotes protein misfolding, and reduced UBC1 expression is associated with impaired clearance of misfolded proteins by the ubiquitin-mediated proteolysis pathway. CONCLUSIONS: This study provides the first integrated transcriptomic and proteomic insight into UBC1 deficiency-mediated seed aging in Saposhnikovia divaricata. The findings enhance molecular understanding of seed aging and offer new directions for improving seed storage and viability.

Ubiquitin-Conjugating Enzymes↗
PubMed2026

Progressive salinity drives flavonoid branch reprogramming in Anoectochilus roxburghii.

Flavonoids play critical roles in plant adaptation to abiotic stress; however, how salt stress modulates metabolic flux distribution within flavonoid branches remains poorly understood, particularly in non-model medicinal plants. Here, we integrated targeted metabolomics, transcriptomics, and proteomics to examine flavonoid regulation in Anoectochilus roxburghii under 0, 50, 100, and 200 mmol·L- 1 NaCl. Metabolite profiling showed that salinity reshaped flavonoid composition rather than uniformly increasing flavonoid abundance. A metabolite-derived branch bias index (MI), representing the balance between reductive branch metabolites and flavonol products, increased under salt treatment, peaked at 100 mmol·L- 1 NaCl, and declined at 200 mmol·L- 1, indicating maximal branch bias under moderate stress followed by partial rebalancing under severe stress. Transcriptomic analysis showed induction of upstream phenylpropanoid and flavonoid entry genes, including PAL, 4CL, and CHS, whereas F3H was suppressed and FLS showed no induction. Furthermore, several short-chain dehydrogenase/reductase homologs (IFR-like SDR homologs) were upregulated, and the transcript-derived reductive branch index (EI) increased progressively across the salt gradient. EI was positively associated with MI, although the relationship was not strictly proportional under severe stress (200 mmol·L- 1 NaCl). Proteomic profiling further provided supportive evidence for sustained activation of upstream flavonoid biosynthesis, such as salt-induced accumulation of chalcone synthase (CHS) protein, complementing the transcriptomic and metabolomic datasets. Together, these results indicate that salt stress reorganizes flavonoid metabolism in A. roxburghii through persistent upstream activation and branch-specific regulation, favoring the reductive branch under moderate salinity.

Orchidaceae↗
PubMed2026

Oncogenic PIK3CA reprograms glutamine metabolism to drive bladder cancer progression.

BACKGROUND: Genomic analysis has revealed that approximately 40% of bladder cancer (BLCA) tumors harbor alterations in the PI3K/AKT pathway, with PIK3CA mutations occurring in 15-25% of cases. PIK3CA, which encodes the catalytic p110α subunit of PI3K, plays a critical role in regulating cell survival, proliferation, and metabolism. However, the metabolic and functional consequences of PIK3CA mutations in BLCA remain poorly defined. METHODS: To investigate the role of PIK3CA mutations in BLCA, we performed targeted sequencing on tumors from patients, identifying recurrent alterations. Using CRISPR/Cas9 knock-in models in SCaBER and UM-UC-3 cell lines, we introduced the PIK3CA E545K mutation to study its effects. We conducted transcriptomic profiling, targeted metabolomics, and stable isotope tracing to assess metabolic reprogramming. Functional assays measured proliferation, mitochondrial complex I activity, and glutaminolysis. Orthotopic xenografts in mice were used to evaluate in vivo tumor growth and metabolism. RESULTS: PIK3CA mutations were present in 20% of cases, consistent with TCGA data. The E545K and E545Q hotspots accounted for 70% of these mutations. PIK3CA E545K strongly activated PI3K/AKT signaling. Transcriptomic analysis revealed enrichment of OXPHOS, fatty acid metabolism, and mTORC1 signaling. Metabolomics indicated changes in TCA cycle metabolites and enhanced reductive carboxylation of glutamine to citrate, driving fatty acid synthesis. Mutant cells showed increased expression of GLS1 and FASN, higher proliferation rates, and elevated mitochondrial complex I activity. In vivo, PIK3CA-mutant xenografts displayed significantly increased tumor growth. CONCLUSION: PIK3CA mutations are frequent drivers of metabolic reprogramming in BLCA, leading to increased glutamine flux, elevated OXPHOS activity, and enhanced fatty acid synthesis, all of which contribute to tumor progression. These findings provide the first comprehensive evidence that PIK3CA-driven metabolic alterations are both biomarkers of aggressive disease and actionable therapeutic targets. The efficacy of PI3Kα inhibition in combination with metabolic targets may support its potential in precision medicine for PIK3CA-mutant BLCA and highlights the value of integrating metabolic biomarkers into treatment strategies for advanced BLCA.

Journal Article↗
PubMed2025

Genetic effect of the Ph1 locus on transcriptome atlas of anther development-related genes, meiotic chromosome behavior and agronomic traits in bread wheat.

Proper spatiotemporal expression of meiosis-related genes (MRGs) and other male-microsporogenesis/microgametogenesis-related genes (MMRGs) is crucial for normal anther development, yet their expression patterns remain largely unknown in wheat. The Ph1 locus in wheat is known to contain the Ph1 gene that plays a dual role in promoting pairing between homologous chromosomes but repressing pairing between homoeologous chromosomes, but its genetic function is still unclear. Here, we investigated these issues by conducting a comprehensive transcriptome analysis during wheat anther development in Chinese Spring (CS) and its ph1b deletion mutant under greenhouse and field conditions. Our results revealed that MRGs and MMRGs are predominantly expressed during pre-meiosis stages, with MMRGs also being highly expressed in meiotic-II. Gene co-expression analysis showed that C2H2 and B3 transcriptional factors (TFs) are associated with MRGs, and MYB regulators interacted mainly with MMRGs during microgametogenesis. Deletion of genes within the Ph1 locus failed to induce compensatory transcriptional activation of their homoeologous counterparts, while genes outside the Ph1 locus showed environmental-specific responses, especially during meiotic-II and mature pollen stages. Notably, early disjunction of bivalent chromosomes is a primary factor leading to defective meiocytes during metaphase I. Furthermore, the ph1b deletion mutant exhibited a substantially delayed heading date, potentially contributing to environment-stable and environment-specific alterations in fertility and grain-related traits. Our study highlights the significant impact of the Ph1 locus on the transcriptome during anther development, and a previously unheeded effect on meiotic chromosome pairing and agronomic traits, suggesting potential for genetic manipulations within the Ph1 locus for wheat improvement.

Triticum↗
PubMed2026

Longitudinal multiorgan transcriptomic atlas of salt-induced hypertension.

High dietary salt intake elevates blood pressure and drives multiorgan damage. However, the molecular programs underlying progressive organ injury remain poorly defined. Here, we present a longitudinal multiorgan transcriptomic atlas of salt-induced hypertensive injury. We profiled kidney cortex, kidney medulla, heart, and liver across 4 stages, spanning early hypertension to advanced pathology in Dahl salt-sensitive rats. We identified dynamic and tissue-specific molecular trajectories, including a shared early proliferative response that converges on proinflammatory and fibrotic remodeling. Notably, we uncovered compartment-specific renal responses, showing that the cortex and medulla, despite their proximity, follow distinct molecular trajectories during disease progression. We further identified 79 stage- and tissue-specific transcription factors that drive gene expression dynamics in salt-induced hypertensive injury. Integration with human genome-wide association studies revealed conserved pathways in endocrine signaling, ion transport, lipid metabolism, and detoxification, establishing cross-species relevance and highlighting mechanistic targets of clinical importance. Compound-transcriptome analysis revealed stage- and organ-specific therapeutic opportunities, prioritizing kinase and epigenetic modulators as candidates to rebalance maladaptive gene programs. Overall, this study provides a resource for understanding molecular mechanisms from early salt-induced hypertension to tissue-specific injury and underscores the need for precision interventions.

Animals↗
PubMed2026

Comprehensive identification and evolutionary analysis of the Wnt gene family in bivalves: Insights into the larval development of the noble scallop Chlamys nobilis.

The Wnt gene family regulates fundamental developmental processes in metazoans, but its evolutionary composition and developmental deployment in bivalves remain largely unresolved. Here, we performed a comparative genomic analysis of Wnt genes in 19 bivalve species and examined developmental expression profiles in the noble scallop Chlamys nobilis, with Crassostrea gigas and Chlamys farreri used for cross-species comparison. A total of 235 Wnt genes were identified and assigned to 12 subfamilies. No reliable Wnt3 ortholog was detected in any analyzed bivalve, supporting the view that Wnt3 loss occurred early during lophotrochozoan evolution rather than representing a lineage-specific absence. Most Wnt proteins retained the conserved WNT domain, indicating strong structural conservation, whereas lineage-specific copy-number variation and gene loss were observed among species. C. farreri and C. gigas each retained 12 Wnt genes and lacked Wnt3, whereas C. nobilis lacked Wnt3, Wnt7, and Wnt16. Developmental transcriptome analysis and RT-qPCR revealed clear stage-specific expression patterns. In C. gigas, Wnt2/10/A were highly expressed during earlydevelopment and peaked around the D-shaped larval stage, while Wnt8 and Wnt11 showed distinct stage-specific peaks. By contrast, Wnt1/5/6/9 were more active during later larval development or juvenile formation. These results provide a comparative framework for bivalve Wnt evolution and identify candidate Wnt genes potentially involved in larval development and aquaculture-relevant developmental transitions.

Animals↗
PubMed2026

The dirigent protein MsDIR6 functions in drought tolerance and modulates reactive oxygen species scavenging and secondary metabolite biosynthesis in alfalfa.

Alfalfa (Medicago sativa L.) is a globally significant forage crop essential for ensuring global food security. However, soil water deficit leads to a substantial decline in its yield, posing a severe threat to sustainable forage production. Dirigent (DIR) proteins play important roles in lignan biosynthesis and plant stress responses. Here, we identified 52 MsDIR genes in alfalfa through a genome-wide analysis, and screened MsDIR6 as a key candidate gene associated with drought tolerance. The results of qRT-PCR showed that MsDIR6 transcription was significantly induced by drought stress in alfalfa. MsDIR6 was preferentially expressed in roots and leaves, and its protein was localized in the nucleus and plasma membrane. Heterologous expression of MsDIR6 in yeast improved tolerance to mannitol-triggered osmotic stress. Heterologous overexpression of MsDIR6 in Arabidopsis significantly increased seed germination rate, seedling survival rate, and antioxidant capacity under drought stress, while improving leaf water-holding capacity by regulating stomatal movement. In transgenic alfalfa hairy roots, MsDIR6 alleviated drought-induced growth inhibition and enhanced reactive oxygen species (ROS) scavenging mediated by the antioxidant defense system under drought stress. Transcriptomic analysis revealed that MsDIR6 activated key genes in the phenylpropanoid and flavonoid biosynthesis pathways, which are crucial for ROS scavenging during drought adaptation. Additionally, we observed elevated flavonoid and lignin contents in MsDIR6-overexpressing alfalfa. Collectively, our findings offer novel insights into alfalfa's drought tolerance mechanisms and identify MsDIR6 as a promising genetic resource for molecular breeding strategies to improve this vital forage crop.

Alfalfa↗
PubMed2026

An Instrumental Optimization of a Label-Free Proteomic Method for Trace Protein Input.

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics of trace-level samples, such as tens of cells or spatially resolved tissue regions, offers unique biological insights but is often constrained by the requirement for specialized, costly instrumentation. In this study, we developed a scalable workflow for the deep proteomic analysis of low- to ultralow-input samples by systematically optimizing a widely adopted Orbitrap and UHPLC platform to maximize sensitivity, precision, and throughput. This optimized workflow identified over 5600 proteins from 5 ng of peptides and 3400 proteins from 20 sorted cells, achieving a throughput of 30 analyses per day while maintaining deep proteome coverage and high quantitative reproducibility. Furthermore, by applying this method to spatially resolved proteomics, we identified over 6100 proteins from microscale regions of interest (ROIs) within a formalin-fixed, paraffin-embedded (FFPE) tissue. A data-driven normalization strategy was employed to correct for variable cellularity across tissue regions, effectively revealing intratumor heterogeneity and distinct molecular and functional signatures, including pathway activations not apparent in parallel spatial transcriptomic analysis. Ultimately, this accessible, high-performance method substantially lowers the instrumentation barrier for the deep proteomic profiling of trace-level biological samples.

Proteomics↗
PubMed2026

Identification and characterization of non-canonical azole antifungal resistance pathways in Aspergillus fumigatus.

UNLABELLED: Human fungal infections, especially those caused by Aspergillus fumigatus, pose a significant global health threat, particularly in immunocompromised individuals. Azole antifungals are the primary treatment for this pathogen; however, the prevalence of azole-resistant A. fumigatus strains is steadily increasing. Mutations in cyp51A, which encodes an enzyme involved in ergosterol biosynthesis and the molecular target of the azoles, are well established to confer resistance in this fungal species. However, additional mechanisms governing resistance to this antifungal class remain understudied and poorly characterized, despite growing recognition of their importance in clinical resistance. In this study, we investigated the genetic basis of azole resistance in A. fumigatus isolates from clinical settings worldwide, with a particular focus on mechanisms independent of cyp51A (non-canonical). Using a combination of genomic and functional approaches, including whole-genome sequencing and transcriptomic analysis, we identified novel genetic variants and characterized population structure, advancing our understanding of the genetic diversity and evolutionary dynamics of resistance in A. fumigatus. By expanding our understanding of the complex genetic and molecular factors underlying azole resistance in this important human fungal pathogen, this research is poised to inform the development of novel antifungal strategies and contribute to global efforts to combat fungal infections. IMPORTANCE: Azole antifungals are the frontline therapy for infections caused by the opportunistic mold Aspergillus fumigatus, yet resistance to these drugs is rapidly increasing worldwide. Most studies have focused on mutations in cyp51A, the canonical target of azoles; however, a growing proportion of resistant clinical isolates lack these mutations, indicating that alternative resistance mechanisms are emerging. Here, we integrate population genomics, transcriptomics, and functional analyses across a global collection of isolates to define the architecture of cyp51-independent (non-canonical) azole resistance. We show that this resistance phenotype is strongly associated with a distinct population lineage and is driven by a highly polygenic network of metabolic, mitochondrial, and regulatory adaptations rather than single target site mutations. These isolates exhibit extensive transcriptional rewiring and metabolic remodeling under azole stress, suggesting distinct survival strategies beyond canonical resistance. Our findings reveal that azole resistance in A. fumigatus can evolve through diverse evolutionary routes and emphasize the need to monitor and therapeutically target non-canonical pathways that may increasingly contribute to antifungal treatment failure.

Aspergillus fumigatus↗
PubMed2026

An XRE-type regulator in Streptococcus mutans plays an important role in brpA expression and oxidative stress tolerance response.

This study used a functional genomics approach to explore the role of a xenobiotic response element (XRE)-type regulator (SMU.405c) in Streptococcus mutans physiology, including the expression of biofilm regulatory protein BrpA. Results showed that deletional mutation of xre significantly reduced the ability of the deficient mutant to grow in the presence of methyl viologen, a commonly used oxidative stressor (P < 0.001). When challenged in a hydrogen peroxide killing assay, the survival rate of the &#x2206;xre mutant was >2-log less than the parent strain after 60 min (P < 0.001). Luciferase reporter fusion assays showed that xre deficiency had no significant effect on luciferase expression when it was under the control of the intact brpA promoter, but the reporter activity increased by >6-fold (P < 0.001) when the reporter gene was fused to a brpA promoter derivative with deletion of a putative XRE-binding box. Electrophoretic mobility shift assay (EMSA) showed that recombinant XRE interacted with the brpA promoter, resulting in an electrophoretic shift of the promoter probes. In vitro transcription assay also showed that inclusion of XRE caused transcription to fall off, significantly reducing full-length brpA transcripts. RNA-seq analysis revealed that deficiency of XRE led to altered expression of >102 genes by >2-fold (P < 0.05), including 28 with increased expression, and 74 with decreased expression. Among the down-regulated were genes for DNA repair and oxidative stress tolerance response. These results suggest that XRE (SMU.405c) in S. mutans plays an important role in brpA expression and oxidative stress tolerance response.IMPORTANCEStreptococcus mutans, a keystone pathogen in human dental caries, primarily lives in the highly diverse microbiota on tooth surfaces, where the conditions are often harsh and fluctuate frequently. Locus SMU.405c was annotated to encode a xenobiotic response element (XRE)-like transcriptional regulator, but no information is available concerning the role of this protein in S. mutans pathophysiology. This study used a functional genomics approach along with molecular and transcriptomic analysis to characterize a deletional xre mutant, and the results showed that xre deficiency in S. mutans resulted in weakened oxidative stress tolerance response and alterations in transcription of >102 genes, including those known to play an important role in cell envelope biogenesis and stress tolerance response. Reporter fusion assay, electrophoretic mobility shift assay (EMSA), and in vitro transcription further demonstrated that the XRE-like regulator encoded by SMU.405c is a repressor of brpA expression and plays an important role in oxidative stress tolerance response.

Streptococcus mutans↗