Search Results (112)

Background/Objective: This study aimed to use multiple disk domain receptor 1 (DDR1), transforming growth factor β1 (TGFβ-1), tumor-associated calcium signal transduction protein 2 (TACSTD2), and polyligand proteoglycan 1 (SDC1) siRNA to treat pancreatic cancer with the goals of high specificity, significant therapeutic efficacy, and relatively low toxicity. Methods: (1) A microfluidic method was used to prepare siRNA-LNPs with different formulations. (2) Quantitative PCR (qPCR) and Western blot assays were used to detect the inhibitory effect of different-prescription siRNA-LNP formulations on mRNA and protein expression levels of related genes in PaTu 8988 pancreatic cells. (3) The anti-pancreatic cancer effect of multiple siRNAs combined with LNPs in vivo was evaluated using the BALB/c nude mouse model with subcutaneous pancreatic cancer xenografts. Results: (1) Three siRNA-LNP formulations, DMG, CE 1.5, and CE 0.75, were successfully prepared, exhibiting small particle sizes and uniform distribution. (2) qPCR and Western blot results indicated that DDR1, TGFβ-1, TACSTD2, and SDC1 siRNA-LNP significantly inhibited related genes’ mRNA and protein expression in pancreatic cancer PaTu 8988 cells. (3) Efficacy studies in animals indicated that multiple siRNA combined with LNPs in each group exhibited significant antitumor effects on pancreatic cancer tumor-bearing nude mice. The therapeutic efficacy of the combined siRNAs was superior to that of single siRNA treatments, indicating a clear combined effect, especially with three- and four-siRNA combinations. Conclusions: The prepared DDR1/TGFβ-1/TACSTD2/SDC1 siRNA-loaded LNP demonstrated a small particle size, high gene inhibition efficiency, and a significant therapeutic effect in treating pancreatic cancer. Its safety is generally acceptable, but attention should be paid to the toxicity caused by LNP excipients, especially cationic lipids. Full article

Potyvirus rapae (turnip mosaic virus, TuMV) is widely used as a model system in plant–virus interaction studies. The TuMV RNA genome encodes 11 proteins, some of which remain poorly characterized, while the functions of others are well defined. Studying individual proteins in isolation may not recapitulate native expression levels, subcellular localization, and interaction with host factors during virus replication and movement. An alternative approach is to tag individual viral proteins in the context of an infectious clone. Epitope tags may alter protein functions and affect viral replication, movement, or a combination of essential steps, thus leading to changes in pathogenicity. Because they have central roles in viral infection, here we measured the effect of individually tagging the helper component proteinase (HC-Pro) and nuclear inclusion protein b (NIb) with a 6His-3xFLAG tag. Epitope tags were placed at the N-terminus of HC-Pro and the N- and C-termini of NIb within a TuMV infectious clone carrying coding sequences for the green fluorescent protein (TuMV-GFP). Constructs carrying a tagged HC-Pro displayed pathogenicity similar to that observed for TuMV-GFP in Nicotiana benthamiana and Arabidopsis thaliana plants. In contrast, infectivity of NIb-tagged clones became temperature sensitive and, even at the permissive temperature, showed reduced pathogenicity compared to TuMV-GFP. Providing a silencing suppressor in trans did not restore infection efficiency, suggesting reduced viral fitness due to structural or functional disruption caused by the epitope tags. Structural models generated using AlphaFold2 showed no effect of the tag on HC-Pro. In contrast, structural models illustrated tag interference with the NIb catalytic site. AlphaFold2 was further used to predict the structural impact of several tags on NIb and to predict the effect of a 6HIS-3xFlag tag on all other TuMV proteins. This study provides a broadly applicable framework for selecting suitable epitope tags to mark viral proteins and maintain function in the context of virus infection. Full article

Lentiviral vectors (LVs) are indispensable tools in cell and gene therapy. Rising demand has created a global shortage of LVs, driving the development of novel packaging approaches. We report a novel vector packaging approach using autonomously replicating cytoplasmic RNAs (replicons) to express packaging proteins. Yellow fever virus (YFV) was used as a source of replicons encoding the HIV-1 Gag–Pol polyprotein together with reporter or selectable markers. YFV replicons were able to establish chronic infection in HEK293FT cells. Replicons expressing HIV-1 Gag–Pol containing the wild-type HIV-1 protease caused strong cytotoxicity, which prevented the selection of polyclonal cell pools harboring the replicon. In contrast, a replicon carrying the T26S mutation in the HIV-1 protease gene showed no measurable cytotoxic effects, enabling the generation of stable replicon-containing cell pools. The replicon cell pools were established using antibiotic selection and maintained Gag-Pol expression for at least ten passages under selection pressure. Using these first-generation replicon cell pools as packaging cells, LV production required only transient transfection of a transfer vector, a Tat/Rev plasmid, and an envelope plasmid. Yields reached ~10⁶ TU/mL prior to concentration and ~10⁹ TU from multilayer cell stacks, which fall within the range typically reported for conventional transient transfection systems under similar culture conditions. The resulting vectors efficiently transduced target cells, and no replication-competent lentivirus (RCL) was detected using a two-phase RCL assay with p24 ELISA detection. This demonstrator platform utilizing replicon cell pools represents a novel approach for LV packaging. Full article

Producing single-cell protein (SCP) from syngas-derived ethanol and acetate offers a sustainable solution to global protein shortages, yet microbial utilization mechanisms for these mixtures remain underexplored. This study establishes a systematic bioconversion strategy using Cyberlindnera jadinii TU389. To mitigate acetaldehyde accumulation during ethanol metabolism, we engineered the strain TU546 to overexpress acylating acetaldehyde dehydrogenase (ADA6). TU546 achieved a maximum biomass of 46.7 g/L and a protein yield of 21.69 g/L, representing enhancements of 28.16% and 23.02% over the wild-type, respectively. Transcriptomic analysis revealed extensive metabolic reprogramming. In the C2 assimilation pathway, upregulated aldehyde dehydrogenase and acetyl-CoA Synthetase 1 accelerated acetate conversion to acetyl-CoA, while downregulated pyruvate decarboxylase and alcohol dehydrogenase minimized carbon flux loss. The upregulation of tricarboxylic acid cycle enzymes, the glyoxylate shunt, and acyl-coA oxidase improved carbon skeleton retention. Moreover, the upregulation of transaminases and N-acetylglutamate synthase, synergized with intensified cell proliferation signaling, redirected amino acid metabolism toward a synthesis-enhanced and degradation-controlled paradigm. This synergistic regulatory network drives the high-efficiency bioconversion of ethanol and acetate into SCP, establishing a molecular mechanistic foundation for the valorization of syngas-derived C2 substrates in biological macromolecule production. Full article

Background: The root of Schisandra propinqua (Wall.) Baill. var. sinensis Oliv is a traditional ethnomedicine in China; it was widely used to treat abscesses, sores, carbuncles, rheumatism, and so on. The purpose of this study was to elucidate the bacteriostatic mechanism of the ethyl acetate extract from the root of Schisandra propinqua (Wall.) Baill. var. Sinensis Oliv (Xiao Xue Teng) against Staphylococcus aureus ATCC 25923 (S. aureus ATCC 25923). Methods: Bioactive bacteriostatic constituents in Xiao Xue Teng were identified through Hybrid Quadrupole-TOF LC/MS/MS. The minimum inhibitory concentration (MIC) of Xiao Xue Teng against S. aureus ATCC 25923 was determined using the microbroth dilution method. A time–kill curve analysis was used to evaluate the bacteriostatic effects. SDS-PAGE coupled with nano-liquid NanoLC-ESI-MS/MS, real-time PCR, and scanning electron microscopy (SEM) was used to study the bacteriostatic mechanism of Xiao Xue Teng against S. aureus ATCC 25923. Results: The MIC of Xiao Xue Teng against S. aureus ATCC 25923 was determined to be 15.625 µg/mL. The translation initiation factor (IF-2) and elongation factor (EF-Tu) were significantly decreased in S. aureus ATCC 25923 after treatment with Xiao Xue Teng, while the proteins SodA and AhpC were obviously increased. The intracellular levels of total reactive oxygen species (ROS) and hydrogen peroxide (H₂O₂) were significantly increased (p < 0.01) after the treatment with Xiao Xue Teng. Concurrently, the activities of SOD, CAT and GSH-Px were significantly increased (p < 0.01). Moreover, cellular swelling and shrinkage were observed using SEM. Conclusions: The bacteriostatic mechanism of Xiao Xue Teng against S. aureus ATCC 25923 was related to eliciting oxidative stress, inhibiting protein synthesis and enhancing cytoplasmic membrane permeability. Full article

Plant defensin-like proteins, as the core effector molecules of innate immunity, play an important role in resistance against fungi and bacteria. However, their function in plant antiviral resistance remains unclear. Here, NbDLP, a defensin-like protein from Nicotiana benthamiana (N. benthamiana), is identified through transcriptome analysis. NbDLP is upregulated upon viral infection of Tobacco Vein Mottling Virus (TVMV). Then, we cloned NbDLP into plant expression vector by gateway recombination to obtain pEAQ-NbDLP. We found that the transient expression of NbDLP in N. benthamiana could significantly inhibit TVMV, TuMV and TMV infection. Further, silencing NbDLP contributed to TuMV and TMV infection. In conclusion, the results indicate that NbDLP participates in the plant antiviral resistance against plant viral infection and might be used for defining antiviral strategies in application points. Full article

The expanding insect farming industry generates up to 67,000 tons of frass per year. Its potential use as fertilizer is promising, but has not yet been widely studied. This study aimed to characterize the chemical composition, organic matter structure, ecotoxicity, and phytotoxicity of frass from four insect species in order to evaluate its potential as a fertilizer. We compared four types of insect frass (IF) (Tenebrio molitor, Galleria mellonella, Hermetia illucens, and Acheta domesticus) to Eisenia fetida vermicompost (EFV). We used physicochemical analyses (pH, electrical conductivity (EC), macro-micronutrients and dissolved organic carbon (DOC), spectroscopy (solid-state ¹³C nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FTIR)) and thermogravimetry/differential scanning calorimetry (TGA/DSC: R₁, R₂, Tmax), together with phytotoxicity (germination index, %GI) and ecotoxicity (toxicity units, TU) bioassays. Composition was species-dependent: A. domesticus showed the highest levels of nitrogen (N), phosphorus (P), and potassium (K); the concentration of DOC was higher in insect frass (IF) than in EFV, with the highest concentration found in IF of T. molitor. ¹³C NMR/FTIR profiles distinguished between frass (carbohydrates/proteins and chitin signals) and EFV (humified, oxidized matrix). Thermal stability followed: G. mellonella (R₁ ≈ 0.88) ≥ A. domesticus (0.79) > H. illucens (0.73) > EFV (0.67) > T. molitor (0.50). In bioassays, T. molitor and A. domesticus exhibited phytotoxicity (%GI < 30), whereas G. mellonella and H. illucens did not. EFV exhibited the highest %GI. Dilution increased %GI in all materials, especially in T. molitor and A. domesticus, and reduced acute risk (TU). Frass is not a uniform input: its agronomic performance emerges from the interaction between EC (ionic stress), the availability of labile C (DOC, C/N and low-temperature exotherms), and structural stability (R₁/R₂ and aromaticity). In terms of formulation, IF can provide nutrients that mineralize rapidly, whereas EFV contributes stability. Controlling the inclusion and dilution of materials (e.g., limiting the amount of T. molitor in blends) and considering the mixing matrix helps to manage phytotoxicity and ecotoxicity, and realize the fertilizer value of the product. Full article

Bitter gourd (Momordica charantia L.) is widely consumed worldwide due to its unique flavor and medicinal value. In subtropical regions, low spring temperatures limit bitter gourd growth, leading to plant mortality and yield loss. Thus, elucidating the mechanisms of cold tolerance in bitter gourd could facilitate the development of cold-resistant cultivars via genetic engineering or molecular breeding. In this study, a cold-tolerant (CT) and a cold-sensitive (CS) inbred line of bitter gourd were used to investigate proteomic differences under cold stress. Before cold stress, 504 differentially accumulated proteins (DAPs) were identified, with 123 up-accumulated in CT plants compared to CS plants. Upon exposure to cold stress, these numbers changed to 388 DAPs (259 up-accumulated in CT) at 6 h and further to 649 DAPs (415 up-accumulated in CT) at 24 h. K-means cluster analysis identified 65 cold-stress response proteins that may contribute to cold tolerance in CT plants, including evm.TU.chr4.3733 (Proline dehydrogenase 1), evm.TU.chr10.115 (Delta(1)-pyrroline-2-carboxylate reductase), and evm.TU.chr10.815 (Calcium-dependent protein kinase 3). Glucose and starch levels remained stable in both CS and CT plants during cold stress, and the baseline concentration of glucose was consistently and significantly higher in CT plants than in CS plants. Before cold stress, proline content was similar in both CT and CS plants. Following 6 h of cold stress, CS plants accumulated significantly higher proline levels than CT plants. This trend, however, reversed after 24 h, with proline content becoming significantly lower in CS plants. Differential protein accumulation between CT and CS plants under cold stress reflects their distinct responses, with core DAPs serving as key functional determinants of enhanced cold tolerance in the CT genotype. This study revealed important proteomic data underlying the cold stress response in bitter gourd. Full article

Objective: To investigate the mechanism by which sphingosine-1-phosphatase 2 (SGPP2) modulates endoplasmic reticulum stress (ERS) through the SIRT1/AMPK pathway to improve ischemic cardiomyopathy-induced chronic heart failure (IHF). Methods: Key genes of IHF and ERS were identified through bioinformatics analysis, and significantly associated pathways of the key genes were obtained via single-gene enrichment analysis. In vivo, IHF was induced in Sprague–Dawley (male) rats via ligation of the left anterior descending coronary artery, with cardiac function examined through echocardiography. Myocardial tissue injury and fibrosis were evaluated utilizing hematoxylin-eosin, Masson, and TUNEL staining. Serum levels of NT-proBNP and cTnT were measured via ELISA. SGPP2 protein expression was assessed via immunohistochemistry and Western blotting (WB). In vitro, neonatal rat cardiomyocytes (NRCMs) were isolated and underwent oxygen-glucose deprivation (OGD) to establish an IHF model. SGPP2-overexpressing NRCMs were constructed and treated with the ERS inducer tunicamycin (Tu) or the SIRT1 inhibitor EX527. Cell injury was evaluated using Cell Counting Kit-8 and lactate dehydrogenase release assays, as well as flow cytometry. Endoplasmic reticulum structure was examined by transmission electron microscopy. The endoplasmic reticulum was labeled with the ER-Tracker Red molecular probe. WB was utilized to detect the expression of apoptosis- and ERS-linked proteins, and the activity of the SIRT1/AMPK signaling pathway. Results: Six key genes (CTSK, FURIN, SLC2A1, RSAD2, SGPP2, and STAT3) were identified through bioinformatics analysis, with SGPP2 showing the most significant differential expression. Additionally, SGPP2 was found to be downregulated in IHF. Single-gene enrichment analysis showed that SGPP2 exhibited a significant association with the AMPK signaling pathway. Animal experiments demonstrated that rats with IHF exhibited significantly impaired cardiac function, marked myocardial tissue injury and fibrosis, ERS in myocardial tissue, lowered SGPP2 expression, and decreased SIRT1/AMPK signaling pathway activity. In vitro experiments confirmed that SGPP2 overexpression alleviated OGD-induced cardiomyocyte injury by inhibiting ERS and simultaneously activating the SIRT1/AMPK signaling pathway. Rescue experiments further demonstrated that both Tu and EX527 significantly promoted ERS and cellular injury, thereby counteracting the protective effects of SGPP2. Conclusions: SGPP2 alleviates IHF by inhibiting ERS modulated by the SIRT1/AMPK pathway. Full article

Streptococcus suis is an important zoonotic pathogen responsible for severe infections in pigs and humans. Its capacity to survive within phagocytic cells is considered a key virulence mechanism that contributes to dissemination and persistence in host tissues. This study employed comparative proteomic profiling to investigate intracellular adaptation of S. suis serotypes 2 (SS2) and 14 (SS14) during infection of human U937 macrophages. Five isolates originating from humans and pigs were analyzed using gel electrophoresis with liquid chromatography–tandem mass spectrometry (GeLC–MS/MS), revealing 118 differentially expressed proteins grouped into 11 functional categories. Translation-related proteins represented the largest group (48%), including upregulated ribosomal subunits (30S: S2, S5, S7, S8, S12, S15; 50S: L1, L5, L18, L22, L24, L33, L35) and translation factors such as GidA/TrmFO and RimP. Enrichment of carbohydrate metabolism and DNA replication proteins, including phosphoenolpyruvate carboxylase (PEP), UDP-N-acetylglucosamine pyrophosphorylase (GlmU), and ATP-dependent DNA helicase RuvB, indicated metabolic reprogramming and stress adaptation under intracellular conditions. Stress-response proteins such as molecular chaperone DnaK were also induced, supporting their multifunctional, “moonlighting” roles in virulence and host interaction. Comparative analysis showed that SS2 expressed a broader range of adaptive proteins than SS14, consistent with its higher virulence potential. These findings reveal conserved intracellular responses centered on translation, energy metabolism, and stress tolerance, which enable S. suis to survive within human macrophages. Integration of these intracellular proteomic signatures with previous exoproteomic, peptidomic, and network-based studies highlights translational and metabolic proteins—particularly DnaK, enolase, elongation factor EF-Tu, and GlmU—as multifunctional candidates linking survival and immunogenicity. This work establishes a comparative proteomic foundation for understanding S. suis intracellular adaptation and highlights potential targets for future vaccine or therapeutic development against this zoonotic pathogen. Full article

Microtubule minus-end binding proteins (−TIPs) are critical regulators of microtubule dynamics and stability, whose dysfunctions are increasingly associated with tumorigenesis and cancer progression. This review systematically consolidates current research advances on the molecular characteristics, oncogenic mechanisms, and therapeutic potential of −TIPs in cancer. By integrating preclinical studies, multi-omics data, and clinical evidence, it was found that calmodulin-regulated spectrin-associated proteins (CAMSAPs) and abnormal spindle microtubule assembly (ASPM) primarily exhibit oncogenic properties, whereas CAMSAP3 acts as a tumor suppressor by negatively regulating tumor cell migration. Studies also demonstrate that pharmacological inhibition of the γ-tubulin ring complex (γ-TuRC) effectively attenuates the centrosomal hyper-clustering capacity of malignant cells, thereby suppressing invasive phenotypes. This result underscores the therapeutic value of targeting −TIPs. In summary, −TIPs play critical and complex roles in cancer progression and hold significant potential as prognostic biomarkers and therapeutic targets. Intervention strategies focusing on specific −TIPs, such as γ-TuRC, offer promising strategies for precision cancer therapy; however, the context-dependent functions of these proteins require further investigation to facilitate clinical translation. Full article

Background: Ethylene is one of the most important plant hormones. ACC oxidase (ACO) plays a vital role in ethylene synthesis and responses to biotic and abiotic stresses in plants. However, its function in Triticum urartu remains unclear. This study aims to systematically identify the members of the TuACO gene family to elucidate its response characteristics and functions under biotic and abiotic stresses. Methods: Through homologous alignment, phylogenetic evolution analysis, and investigations of gene structure and promoter cis-elements, a total of eight TuACO genes were identified in the T. urartu genome based on their homology to OsACO and AtACO protein sequences. Results: These genes were classified into five ACO subfamilies and distributed across chromosomes 1A, 4A, 5A, 6A, and 7A. TuACO gene families contained 0–3 introns and 1–4 exons. The protein sequence contains 10 different conservative motifs. QRT-PCR expression analysis revealed that the transcript levels of TuACO5a, TuACO5b, and TuACO3a were significantly upregulated at 6 and 24 h after infection with powdery mildew, a biotic stress. Under boron deficiency, an abiotic stress, the expression of TuACO6 and TuACO1b increased, whereas the expression of TuACO5b and TuACO3b was markedly induced under high-boron conditions. Conclusions: These results demonstrate that TuACO genes exhibit functional diversification in response to biotic and abiotic stresses, which lays the foundation for elucidating their gene functions. Full article

Ubiquitin-conjugating enzyme (UBC) plays a significant role in plant hormone signal transduction. In this study, we observed that TuMV infection markedly upregulates UBC mRNA expression, suggesting a close association between UBC and viral infection. Using Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to downregulate UBC expression in Nicotiana benthamiana, we found that UBC-silenced plants exhibited enhanced susceptibility to TuMV compared with control plants. Conversely, transient overexpression of UBC protein suppressed viral propagation. Further analysis by reverse transcription quantitative PCR (RT-qPCR) revealed a substantial downregulation in the expression of SA and JA biosynthetic genes in UBC-silenced plants. Accordingly, liquid chromatography–tandem mass spectrometry (LC-MS/MS) confirmed a marked decrease in the accumulation of the corresponding hormones. Exogenous application of SA or JA partially restored antiviral resistance in UBC-silenced plants, indicating that hormonal deficiency contributes to enhanced viral susceptibility. Collectively, our results demonstrate that UBC positively regulates SA and JA biosynthesis. UBC silencing impairs both SA- and JA-mediated defense pathways, thereby facilitating viral infection in N. benthamiana. Full article

Chloroplasts, which are essential for plant defense and phytohormone signaling, contain ribosomal proteins that play key roles in viral infection processes. Plastid-specific ribosomal proteins (PSRPs), unique to chloroplasts, remain unexplored in their mechanistic roles during plant-virus interactions. In this study, we identified two PSRPs from non-heading Chinese cabbage (Brassica campestris ssp. chinensis) as interacting with turnip mosaic virus (TuMV, Potyvirus rapae). Subcellular localization revealed that BcPSRP2/4 is targeted to chloroplasts, while BiFC, Y2H, and LCAs confirmed their interaction with TuMV VPg (virus protein, genome-linked). Intriguingly, VPg altered the subcellular localization of BcPSRP2/4, suggesting an important role for BcPSRP2/4 in TuMV infection. Strikingly, overexpression of BcPSRP2/4 enhanced TuMV cell-to-cell movement, while psrp2 knockdown mutants in Arabidopsis exhibited a significant reduction in viral accumulation, highlighting their proviral roles. Furthermore, virus-induced gene silencing (VIGS)-mediated suppression of BcPSRP2/4 in non-heading Chinese cabbage resulted in milder symptoms upon TuMV infection without compromising plant growth: a distinct advantage over conventional resistance genes that incur fitness costs. These findings highlight PSRP2/4 as pivotal molecular hinges in chloroplast-virus interplay, offering novel targets for engineering sustainable antiviral strategies in cruciferous crops. Full article

Reprogramming is a hallmark of cancer, enabling tumour cells to sustain rapid proliferation, resist cell death, and adapt to hostile microenvironments. This review explores the expression profiles of key metabolic enzymes and transporters involved in glucose, amino acid, and lipid metabolism across the five most deadly cancers worldwide: lung, breast, colorectal, liver, and gastric cancers. Through a comparative analysis, we identify consistent upregulation of glycolytic enzymes such as LDHA, PKM2, and HK2, as well as nutrient transporters like GLUT1, ASCT2, and LAT1, which contribute to cancer progression, metastasis, and therapy resistance. The role of enzymes involved in glutaminolysis (e.g., GLS1, GDH), one-carbon metabolism (e.g., SHMT2, PHGDH), and fatty acid synthesis (e.g., FASN, ACLY) is also examined, with emphasis on their emerging relevance as diagnostic, prognostic, and predictive biomarkers. While several metabolic proteins show strong potential for clinical translation, only a few, such as tumour M2-pyruvate kinase (TuM2-PK) and serum LDH measurement, have progressed into clinical use or trials. This review addresses some of the challenges in biomarker development. Ultimately, our findings underscore the importance of metabolic proteins not only as functional drivers of malignancy but also as promising candidates for biomarker discovery. Advancing their clinical implementation could significantly enhance early detection, treatment stratification, and personalized oncology. Full article