Search Results (441)

Ehrlichia chaffeensis is an obligate intracellular bacterium that proliferates within monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening zoonosis. Doxycycline is the choice of treatment for HME, yet it has prominent side effects. Host cells lack the lysine biosynthetic pathway; thus, the enzymes in this pathway are essential for bacterial growth and recognized as potential targets for the development of novel antibiotics. Here, we demonstrated that inhibitors targeting DapE, which is a key enzyme in the lysine biosynthetic pathway, especially disulfiram, effectively inhibit E. chaffeensis infection and intracellular growth. Through complementation experiments and peptide nucleic acid-mediated dapE knockdown, we showed that DapE in E. chaffeensis is functional and essential for bacterial intracellular growth. Using purified recombinant protein, we found that DapE induces IL-8 expression in host cells. Finally, we identified that NtrX, the response regulator of the two-component system NtrY/NtrX, regulates dapE expression using an electrophoretic mobility shift assay and a reporter assay. Our findings deepen the understanding of E. chaffeensis pathogenesis as well as illustrate that DapE in E. chaffeensis is a potential therapeutic target for the development of novel HME treatments. Full article

Lepidolite flotation was investigated using Armeen C, a conventional primary amine, and a cationic Gemini surfactant (hexanediyl-α,ω-bis(dimethyldodecylammonium bromide), 12-6-12) (HBDB), as collectors. Microflotation tests, electrophoretic mobility measurements, adsorption isotherms, and dynamic foamability experiments were conducted to compare their performance and interaction mechanisms. HBDB produced high lepidolite recoveries at relatively low concentrations of 25–75 mg/L, particularly between pH 5 and 9, whereas Armeen C required higher dosages of 100–300 mg/L to achieve comparable recoveries over a broader pH range. Electrophoretic mobility and adsorption results confirmed the interaction of both collectors with the lepidolite surface, although Armeen C showed stronger charge reversal and higher adsorption density. In contrast, HBDB exhibited substantially stronger foamability, with DFI values ~7.2–7.8 higher than those of Armeen C. These results indicate that HBDB can enhance lepidolite flotation through efficient surface hydrophobization and strong froth stabilization. Full article

Listeria monocytogenes is a foodborne pathogen capable of persisting under acid, osmotic, oxidative, thermal, and other environmental stresses. The glutamate decarboxylase (GAD) system is a major determinant of survival under acidic conditions. Previous work showed that the two-component system LiaSR negatively regulates the gadT2/gadD2 locus in L. monocytogenes 10403S, but the intervening regulator was unknown. Here, we combined transcription-factor mutant screening, promoter-reporter assays, RT-qPCR, Western blotting, acid-survival assays, and electrophoretic mobility shift assays to define this regulatory relationship. Deletion of mntR reduced P_gadT2-gfp activity, gadD2 transcript abundance, and GadD2 protein levels at pH 4.5, 7, and 9, whereas complementation restored expression toward the wild-type level. The ΔmntR strain also showed reduced survival under inorganic and organic acid stresses. LiaSR deletion increased P_mntR-gfp activity, and LiaR bound directly to P_mntR. Conversely, mntR deletion increased LiaR abundance, although MntR did not bind to P_liaSR, indicating indirect feedback regulation. The liaSR/mntR double mutant showed reduced gadT2/gadD2 expression under acidic conditions and impaired acid survival. Together, these findings support a LiaSR-MntR-gadT2/gadD2 pathway in which MntR promotes GAD-mediated acid resistance. Full article

Approximately 30% of tumors contain an activating mutation in the oncogene KRAS, leading to increased cell proliferation that often promotes non-small cell lung cancers, colorectal adenocarcinomas, pancreatic ductal adenocarcinomas (PDAC), and other cancers. Among the most common point mutations in KRAS is G12D, an example of an oncogenic sequence present in tumor cells but not normal cells. We developed peptide nucleic acid (PNA) oligomers that selectively bind KRAS G12D sequences and fused them with novel cell-penetrating peptide flanking regions (CPP-PNA-G12D) then evaluated them. Electrophoretic mobility shift assays demonstrated in vitro binding to and selectivity for KRAS G12D over wild-type KRAS and KRAS G12C. Cells and nuclei were able to uptake CPP-PNA-G12D at high efficiency as shown by fluorescent microscopy and flow cytometry. Cell viability assays showed a striking dose-response effect in on-target cells expressing KRAS G12D, while relatively sparing off-target cells expressing KRAS G12C. CPP-PNA-G12D constructs were effective against a panel of PDAC cell lines and in female Balb/c mice bearing patient-derived xenografts. These results show promise for an enhanced PNA-delivery peptide conjugate strategy as a potential therapeutic strategy to selectively target KRAS mutant cancer cells, with the potential to expand this technology to additional cancer-derived mutant oncogenes. Full article

Ehrlichia chaffeensis is an obligate intracellular Gram-negative bacterium that infects human monocytes and macrophages, causing human monocytic ehrlichiosis (HME). HME is an emerging and highly threatening tick-borne infectious disease. CtrA functions as the response regulator of the CckA/CtrA two-component regulatory system, which exhibits high sequence conservation among α-proteobacteria. As a global transcriptional regulator, CtrA regulates the expression of genes associated with E. chaffeensis infection and intracellular survival. Here, we identified consensus CtrA-binding motifs in the promoter regions of genes encoding the phosphate-specific transport (Pst) system and twin-arginine translocation (Tat) system. Using electrophoretic mobility shift assay and reporter assay, our experimental evidence demonstrated that CtrA directly binds to the promoter regions of these target genes and further activates the expression of the corresponding genes. Through complementation experiments, we found that the E. chaffeensis Pst and Tat systems are functional and play critical roles in bacterial swimming motility, biofilm formation, and oxidative stress resistance. Using peptide nucleic acid-mediated knockdown, we further confirmed that the Pst and Tat systems are essential for E. chaffeensis intracellular survival. Collectively, our data advance the current understanding of the function and regulation of the Pst and Tat systems, which are essential for E. chaffeensis intracellular survival, and E. chaffeensis pathogenesis and host adaptation. Full article

Inorganic polyphosphate (polyP) is a ubiquitous polymer increasingly recognized as a regulator of protein function through lysine polyphosphate modification (KPM), a reversible interaction with lysine-rich protein regions. Although numerous KPM targets have been identified, the functional consequences of polyP binding remain poorly understood. N-myristoyltransferase 2 (NMT2), an essential enzyme that catalyzes protein N-myristoylation and regulates membrane-associated signaling pathways, was previously identified as a candidate KPM target. Here, we investigated the molecular and functional relationship between polyP and NMT2. Using a fluorescence-based coenzyme A release assay, we found that polyP directly down-regulated NMT2 enzymatic activity in a dose-dependent manner, with long-chain polyP (polyP₇₀₀) producing significantly greater inhibition than medium-chain polyP (polyP₁₀₀). In cells, both exogenous polyP treatment and induction of endogenous polyP synthesis reduced phosphorylation of Src, a downstream signaling protein whose activation depends on N-myristoylation, supporting inhibition of NMT2 function in vivo. PolyP also decreased the viability of NMT2-overexpressing HeLa cells in a dose-dependent manner, suggesting functional consequences for cellular fitness. Biochemical analysis further revealed that polyP induced a pronounced electrophoretic mobility shift in NMT2 that was completely reversed by a 25-residue lysine-rich competitor peptide, consistent with a specific and reversible interaction mediated through lysine-enriched regions of the enzyme. These findings identify NMT2 as a functional target of polyP and demonstrate that polyP negatively regulates NMT2 activity, downstream Src signaling, and cellular viability. Our results expand the emerging concept of lysine polyphosphate modification and establish polyP as a previously unrecognized regulator of protein lipidation-dependent signaling pathways. Full article

Coconut (Cocos nucifera L.) is an important tropical horticultural crop valued for its edible endosperm with high oil content. However, the molecular mechanisms governing carbon partitioning and the regulation of glycolytic genes during endosperm development remain largely unclear. Fructose-1,6-bisphosphate aldolase (FBA), a key enzyme in glycolysis, plays a central role in carbohydrate metabolism, yet its transcriptional regulatory mechanisms in coconut have not been elucidated. In this study, the CoFBA promoter (proFBA) was isolated and used for yeast one-hybrid screening, leading to the identification of a GATA transcription factor, CoGATA2. Subcellular localization analysis confirmed that CoGATA2 is localized in the nucleus. Yeast one-hybrid assays, electrophoretic mobility shift assays (EMSA), and transient expression in coconut protoplasts demonstrated that CoGATA2 directly binds to a proFBA fragment containing the predicted GATA motif and represses CoFBA expression. Moreover, virus-induced gene silencing (VIGS) of CoGATA2 in coconut callus resulted in significant upregulation of CoFBA expression and increased fructose-1,6-bisphosphate (FBP) levels. Collectively, these findings demonstrate that CoGATA2 functions as a transcriptional repressor of CoFBA and that this regulation correlates with altered FBP levels in coconut callus. However, the precise mechanism by which FBP accumulation occurs and its relationship to glycolytic flux require further investigation. This study provides new insights into the transcriptional regulation of a key glycolytic gene in coconut and offers a foundation for future efforts to manipulate carbon partitioning in this important horticultural crop. Full article

Listeria monocytogenes (L. monocytogenes) is a zoonotic foodborne pathogen that triggers life-threatening invasive illnesses. MarR-type transcriptional regulators widely control bacterial stress adaptation, antibiotic tolerance and virulence, playing crucial roles in the pathogenesis of diverse bacterial pathogens. Lmo0840 is a conserved member of this family; however, its physiological functions and regulatory networks in L. monocytogenes remain completely unexplored. To this end, we constructed an lmo0840 deletion mutant and its complemented strain in the L. monocytogenes EGD-e background and systematically characterized the biological functions of Lmo0840 using phenotypic assays, transcriptomic profiling, and electrophoretic mobility shift assays (EMSA). Phenotypic characterization revealed that loss of lmo0840 impaired growth rate under hyperosmotic, oxidative, and iron-limitation stresses, while unexpectedly promoting growth rate at 30 °C and under tetracycline stress. Consistently, Disk diffusion assays further showed that the Δlmo0840 mutant exhibited significantly reduced inhibition zone diameters against tetracycline antibiotics compared to the EGD-e strain. Moreover, the Δlmo0840 mutant exhibited reduced early biofilm formation and attenuated macrophage adhesion, yet showed enhanced cellular invasion and decreased virulence in a murine model. EMSA further demonstrated that Lmo0840 directly binds to the promoter region of lmo0839 and represses its transcription, thereby contributing to bacterial adaptation to tetracycline stress. Collectively, these findings establish Lmo0840 as a pleiotropic regulator involved in environmental stress responses, tetracycline adaptation, and virulence in L. monocytogenes. Notably, this study identifies for the first time an Lmo0840–Lmo0839 regulatory cascade underlying tetracycline adaptation. These insights not only deepen our understanding of the molecular mechanisms governing Lm environmental adaptation and pathogenicity but also provide a potential target for the prevention and control of listeriosis. Full article

Low-temperature stress severely inhibits the growth and development of sweetpotato, restricts its planting geographical range, and frequently causes seedling death and yield reduction, posing a major threat to sweetpotato sustainable production. Nevertheless, the molecular regulatory mechanisms by which sweetpotato perceives and adapts to low-temperature stress have not been fully elucidated and deserve further in-depth investigation. In this study, a basic helix–loop–helix (bHLH) transcription factor IbbHLH129 was cloned from a cold-tolerant sweetpotato variety Xs33. The IbbHLH129 protein is localized in the nucleus. IbbHLH129 is most highly expressed in sweetpotato leaves and upregulated by low temperature, GA3, MeJA, and IAA, while repressed by ABA. Overexpressing IbbHLH129 enhanced cold tolerance in sweetpotato seedlings under short-term cold stress through enhancing antioxidant capability and regulating hormone-related pathways. Dual-luciferase and electrophoretic mobility shift assays demonstrated that IbbHLH129 binds to the IbYUCCA2 and IbGID1 promoters to activate their expression. These findings suggest that IbbHLH129 enhances cold tolerance in sweetpotato seedlings under short-term cold stress by activating IAA and GA signaling pathways. Our study provides a novel gene resource with promising applications for reducing low-temperature injury to sweetpotato seedlings cultivated in cold growing areas. Full article

Base excision repair (BER) and nucleotide excision repair (NER) are traditionally regarded as independent pathways; however, accumulating evidence indicates that ultraviolet (UV)-damaged DNA-binding protein (UV-DDB), a core NER factor, stimulates BER DNA glycosylases, including alkyladenine DNA glycosylase (AAG). Despite this functional link, the molecular basis of the UV-DDB/AAG interaction and its regulation by DNA remain unclear. This study investigated the direct interaction between AAG and UV-DDB using electrophoretic mobility shift assays (EMSA), surface plasmon resonance (SPR), biolayer interferometry (BLI) and AlphaFold3-based structural modeling under DNA-free and DNA-bound conditions. SPR analysis revealed that AAG and UV-DDB form a high-affinity complex in the absence of DNA ($K_D$ ≈ 17.5 nM), which is maintained but reduced approximately 2.6-fold upon binding to apurinic/apyrimidinic site (AP site)-containing dsDNA ($K_D$ ≈ 46.2 nM). BLI analysis independently confirmed this interaction under both DNA-free and DNA-bound conditions, with inter-platform differences consistent with previously reported BLI/SPR variability. EMSA showed UV-DDB-mediated ternary complex formation accompanied by redistribution of binary AAG/DNA species. AlphaFold3 modeling predicted that AAG associates with DDB1 in the DNA-free state, whereas under DNA-bound conditions, DDB2 recognizes the AP site while AAG repositions toward the lesion with multiple active site residues placed in close proximity. These findings support a model in which DNA binding acts as a molecular switch that reconfigures the UV-DDB/AAG interaction, potentially enabling UV-DDB to function as a recruitment platform that facilitates directional progression of AAG through the BER cycle, and providing a structural basis for coordinated integration of BER and NER. Full article

Cadmium (Cd) toxicity poses a significant threat to crop production and food safety. Although proline is known to enhance plant tolerance to Cd, the molecular mechanisms regulating Cd detoxification through proline accumulation remain unclear. This study identifies the proline transporter TaPROT2 as a crucial positive regulator of Cd tolerance in wheat. We demonstrate that overexpression of TaPROT2 directly promotes proline accumulation in transgenic wheat while simultaneously activating the antioxidant enzyme system, thereby reducing both Cd accumulation and translocation. Using electrophoretic mobility shift assays (EMSA), yeast one-hybrid (Y1H) assays, and luciferase reporter assays, we confirmed that TaERF3 directly binds to the GCC-box element in the TaPROT2 promoter, thereby activating its transcription. Furthermore, overexpression of TaERF3 enhances the expression of TaPROT2, leading to increased proline accumulation and decreased Cd content. In summary, our study reveals a novel TaERF3-TaPROT2 module that promotes proline accumulation, reduces Cd accumulation, and enhances Cd tolerance, providing a promising target for breeding low-Cd wheat. Full article

The seeds of Impatiens balsamina L. have been traditionally used in East Asian medicine and are known to contain bioactive compounds with antioxidant properties. However, studies focusing on seed-derived callus remain limited. This study aimed to comparatively evaluate the antioxidant activities and lipid accumulation-inhibitory effects of 70% ethanol extracts from seeds (IB) and seed-derived callus (IBC) of I. balsamina. Callus was induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Antioxidant activities were evaluated using DPPH radical scavenging, superoxide anion scavenging, deoxyribose-based hydroxyl radical scavenging, DNA nicking, lipid peroxidation, and relative electrophoretic mobility (REM) assays, along with the determination of total phenolic, flavonoid, and tannin contents. Cell viability and lipid accumulation were assessed in FFA-treated HepG2 cells. In silico network and transcription factor (TF) enrichment analyses were performed to explore underlying mechanisms. Callus induction was most effective at 1 mg/L 2,4-D. Both IB and IBC exhibited antioxidant activities across all assays, with IB showing higher activity and greater phytochemical content than IBC. Both extracts reduced lipid accumulation in FFA-treated HepG2 cells at non-cytotoxic concentrations. Network analysis identified enrichment in pathways related to oxidative stress, inflammation, and lipid metabolism, and TF enrichment analysis identified NFKB1 and ATF3 as major upstream regulators. Both IB and IBC exhibited antioxidant activities across multiple in vitro assays, with IB showing higher activity attributable to its more complex phytochemical content. The lipid accumulation-inhibitory effects observed in FFA-treated HepG2 cells suggest a potential association between antioxidant capacity and lipid regulation, although the underlying mechanisms remain to be experimentally validated. Seed-derived callus may serve as a useful in vitro model for studying plant-derived bioactive compounds, pending further optimization. Full article

Toxigenic Klebsiella oxytoca strains linked to antibiotic-associated hemorrhagic colitis produce the cytotoxins tilimycin and tilivalline, which contribute to intestinal epithelial damage during infection. Tilimycin and tilivalline are synthesized by enzymes encoded within the nonribosomal peptide synthetase (NRPS) operon, yet the regulatory mechanisms controlling operon expression remain poorly understood. SdiA, an orphan LuxR-type quorum-sensing regulator, detects exogenous N-acyl homoserine lactones (AHLs) produced by neighboring bacterial species and modulates gene expression in response to interspecies communication. Although SdiA has been implicated in virulence regulation in several enteric pathogens, its role in K. oxytoca remains unclear. This study demonstrates that SdiA positively regulates npsA, the first gene in the NRPS operon, and that this regulatory effect is enhanced in the presence of exogenous AHL. Electrophoretic mobility shift assays indicate that SdiA directly binds to the upstream regulatory region of npsA, supporting a direct interaction consistent with positive transcriptional regulation. Furthermore, deletion of sdiA significantly reduces cytotoxicity toward HeLa cells under the conditions tested. Collectively, these findings identify SdiA as a quorum-sensing-responsive activator of npsA expression and support its role in modulating cytotoxicity in toxigenic K. oxytoca strains. These results provide new insight into the influence of interspecies quorum-sensing signals on virulence-associated regulatory pathways in K. oxytoca. Full article

N⁶-methyladenosine (m⁶A) is an important epigenetic modification of eukaryotic RNA, playing a significant role in various biological processes. Metasequoia glyptostroboides (M. glyptostroboides) is an ancient tree species in China, with a long history and excellent genetic characteristics. In this study, we identified six MgYTH genes in the genome of M. glyptostroboides, elucidating their phylogenetic relationships, conserved domains, gene structures, conserved motifs, chromosome locations, and prediction of LLPS. The analysis of the cis-regulatory elements in the promoter region suggested that MgYTH genes are associated with drought and the ABA-responsive expression patterns signaling pathway, which was further supported by expression pattern analysis. In addition, to directly evaluate the m⁶A binding ability of MgYTH proteins, we selected MgYTH5 as the representative for homology modeling analysis and electrophoretic mobility shift assay (EMSA). The results demonstrated that MgYTH5 has the ability to bind m⁶A in vitro, thereby providing biochemical evidence that MgYTH5 can bind m⁶A-modified RNA in vitro mRNAs. The subcellular localization results showed that MgYTH5 is located in the cytoplasm. These findings provide new insights into the epigenetic regulation mechanisms in gymnosperms and provide a resource for future functional studies in this species. Full article

In this study, we investigate two RpoN homologs in Bradyrhizobium sp. DOA9—chromosomal RpoNc and megaplasmid-borne RpoNp—and their roles in free-living conditions and nitrogen fixation. Phylogenetic analysis showed that RpoNc clusters with RpoN proteins from symbiotic nitrogen-fixing strains, whereas RpoNp forms a distinct clade, consistent with a function in stress responses. RpoNc proved essential for free-living conditions: ΔrpoNc mutants displayed severe growth defects that RpoNp could not compensate for. Transcriptomic comparisons between wild type and mutant RpoN identified 541 differentially expressed genes (DEGs) grouped into three clusters: 100 downregulated, 175 upregulated, and 254 moderately downregulated (with a fold change > 2, and a q-value (FDR, padj) < 0.05). Affected pathways involved nitrogen metabolism, motility, and environmental adaptation. RpoNc controlled major nitrogen fixation genes (nif and fix) along with core growth and stress response functions, while RpoNp mainly influenced stress-adaptation pathways. Genome-wide promoter motif analysis predicted 68 putative RpoNc targets, mainly associated with nitrogen fixation and metabolism, compared with only 22 predicted RpoNp targets, indicating a more restricted regulon. Electrophoretic mobility shift assays (EMSAs) further confirmed that both RpoN proteins directly bind σ⁵⁴-dependent promoters identified from transcriptomic data, supporting their regulatory roles under free-living conditions. Two mutants (ΔrpoNc and ΔrpoNp::ΩrpoNc) showed broad transcriptional disruption across nitrogen fixation, metabolism, and stress responses, underscoring complementary regulation. Overall, RpoNc is the dominant regulator of nitrogen fixation and core metabolism during free-living conditions, whereas RpoNp fine-tunes stress responses, revealing new regulatory insights for DOA9 adaptation. These results clarify how RpoN systems optimize survival across fluctuating conditions. Full article