Search Results (4,516)

Insects present extraordinary potential for obtaining recombinant proteins, both in terms of the quantity and quality of the synthesized product. This work proposes the use of artificial diets including pDNA as an oral transfection system for the Lepidoptera Spodoptera exigua. It is hypothesized that oral transfection can lead to the effective expression of the reporter genes carried in plasmids. Prior to their incorporation into the artificial diet, plasmids (pCMVβ and pEGFP-N2) were protected from inactivation in the digestive tract by chitosan nanoparticulation. The survival of plasmids and their oral uptake by larvae was evaluated, as well as the persistence of pDNA in larvae throughout their ontogeny. The results confirmed that transfection occurred and that pDNA persisted during the ontogeny, even after discontinuing plasmid administration. The transcription of reporter genes was quantified by qRT-PCR, and the results indicate a dose-dependent synthesis of mRNA as the inclusion level of pDNA in diets increased. Moreover, the measurement of the biological activity of the recombinant proteins (β-galactosidase activity and green fluorescence) paralleled the results obtained for gene transcription, also dose-dependently. Therefore, effective oral transfection is feasible in S. exigua, provided that pDNA is protected against gut inactivation prior to its incorporation in artificial diets. Full article

Background: Medulloblastoma is an aggressive pediatric brain tumor characterized by marked molecular heterogeneity, which significantly impacts prognosis. The low frequency of genomic mutations in medulloblastoma suggests that alternative mechanisms, such as epigenetic regulation, may play a critical role in its pathogenesis. Methods: Using the EpiFactors database, we investigated the expression of epigenetic regulators in two independent RNA sequencing cohorts [Pediatric Brain Tumor Atlas (PBTA) and Williamson], stratified by molecular subgroups and clinical outcomes. We further analyzed expression heterogeneity at the single-cell level in malignant medulloblastoma cells using single-cell RNA sequencing. Results: Members of the SWI/SNF superfamily were dysregulated across all four molecular subtypes of medulloblastoma. Subtype-specific alterations were also observed: the acetyltransferase complex was shared between Group 3 (with SMARCD3 as a potential marker) and Group 4 (with RBM24 as a potential marker); SWR1, β-catenin/TCF, and protein–DNA complexes were specifically enriched in SHH-MB (with EYA1 and SATB2 as SHH markers); and RSC-type, PRC1, DNA polymerase complexes, and X-chromosome-related factors were enriched in WNT-MB (with FOXA1 and PIWIL4 as WNT markers). An epigenetic score (epi-score), linked to RNA metabolism and S-adenosyl-L-methionine pathways, was developed and identified as an independent adverse prognostic factor. High epi-scores were associated with proliferative, stem-like SHH malignant cells (characterized by G2/M phase, low pseudotime, and high entropy), exhibiting alterations in RNA splicing, DNA recombination, and nuclear division. Conclusions: Expression heterogeneity of epigenetic regulators is closely associated with molecular subgroups and clinical outcomes in medulloblastoma. These findings highlight the role of epigenetic dysregulation in RNA metabolism and tumor progression, particularly in SHH-driven proliferative cells. Full article

The rise in diabetes and obesity worldwide has created an urgent demand for low-sugar, nutrient-dense foods with appealing flavors. This study established an endogenous and “rapid validation–stable production” platform to enhance the flavor of edible tomato fruits by integrating two key technologies in the MicroTom cherry tomato: (1) TRV viral vector-mediated transient expression and (2) Agrobacterium-mediated stable genetic transformation. We employed the human sweet taste receptor TAS1R2 for in vitro functional validation and objectively demonstrated that tomato-derived recombinant thaumatin II exhibits receptor-binding activity equivalent to that of the native protein, overcoming the limitations of traditional sensory evaluation. Non-targeted metabolomic analysis (covering 1236 metabolites) confirmed that thaumatin II expression did not significantly alter the profiles of sugars, organic acids, or key flavor compounds in tomato fruits. This provides safety data supporting the development of “ready-to-eat sugar-substitute fruits.” Our strategy offers a solution and theoretical technical support for the development of low-sugar, high-nutrient foods. Full article

The sulfo-Embden–Meyerhof–Parnas (sulfo-EMP) pathway enables Escherichia coli to utilize sulfoquinovose, (SQ) a sulfonated sugar derived from plant sulfolipids, as a carbon source. This pathway is encoded by the yih gene cassette. However, structural and functional diversity of this cassette across E. coli strains has not been fully characterized. We identified two structural variants of the yih cassette across E. coli and Shigella strains: a long form (ompL-yihOPQRSTUVW) and a truncated short form (yihTUVW). Both forms occupy the same genomic location but differ in orientation and are scattered across the phylogenetic tree, suggesting frequent recombination events. Transcriptome analyses revealed that only the long cassette, as found in E. coli K-12 MG1655, is transcriptionally induced during growth on sulfoquinovose. The short cassette, found in E. coli Nissle 1917 and other host-adapted strains, showed no differential expression. Despite this, both strains grew comparably on sulfoquinovose, indicating different metabolic adaptation strategies. Gene expression profiling revealed shared stress responses but distinct central metabolic patterns. Electrophoretic mobility shift assays further demonstrated that the transcription factor YihW from Nissle 1917 binds its DNA targets with lower affinity than the homolog from K-12 and shows weaker sulfoquinovose-dependent dissociation. Full article

Fusarium head blight caused by Fusarium graminearum is a serious fungal disease on wheat and maize worldwide, resulting in a significant economic loss. DUG3-mediated glutathione utilization has been revealed to play important roles in fungal differentiation, metabolism, stress adaptation, and plant infection. However, functional roles of the DUG3 homolog in F. graminearum remain uncharacterized. In the present study, FgDUG3 was knocked-out via homologous recombination to investigate functions of this gene. The deletion mutant (ΔFgDUG3) was normal in mycelial growth, but showed impairments in conidiation, conidial germination, and plant infection, compared to the wild-type strain. The defects of ΔFgDUG3 were recovered in the complemented strain (ΔFgDUG3-C). Transcriptomic analysis revealed that deletion of FgDUG3 caused significantly differential expression of genes, mainly related to metabolism, catabolism, cellular structure organization, and signal transduction. Taken together, these results suggest that FgDUG3 plays important roles in the differentiation and pathogenicity of F. graminearum. Full article

Background: Fascioliasis, caused by Fasciola species, is a significant public health concern affecting over 250 million people globally and causing annual economic losses exceeding USD 6 billion. The sole FDA-approved treatment, triclabendazole (TCZ), faces increasing resistance due to extensive use, highlighting the urgent need for alternative therapeutic targets. A promising candidate is thioredoxin glutathione reductase (TGR), a multifunctional enzyme unique to platyhelminths, essential for redox balance and parasite survival. Methods: This study investigated the antioxidant and enzymatic activities of recombinant Fasciola gigantica TGR (FgTGR), its localization within the parasite, and its inhibition by furoxan derivatives. FgTGRsec (FgTGR containing selenocysteine) was expressed and purified, and its enzymatic activities, including thioredoxin reductase (TrxR), glutathione reductase (GR), and glutaredoxin (Grx), were characterized. Results: Immunolocalization studies revealed FgTGR’s presence in critical tissues, underscoring its functional significance. Antioxidant assays demonstrated the protein’s role in protecting against oxidative damage. Inhibition assays with furoxan derivatives identified potential inhibitors targeting TGR activity. Sequence and phylogenetic analyses showed FgTGR’s evolutionary conservation among trematodes, confirming its potential as a drug target. Conclusions: The study’s findings establish FgTGR as a critical enzyme for parasite survival and a promising target for developing novel therapeutics. These results pave the way for the further screening and optimization of TGR inhibitors, offering a strategic approach to overcoming TCZ resistance and improving fascioliasis control. Full article

Toxoplasma gondii is a globally distributed protozoan parasite, and reliable serodiagnosis is essential for effective management of toxoplasmosis. Conventional assays rely on tachyzoite lysate antigen (TLA), which suffers from limited standardization and reproducibility. In this study, immunodominant fragments of six dense granule proteins—GRA29, GRA35, GRA36, GRA45, GRA54, and GRA64—were expressed in Escherichia coli, purified, and evaluated as candidate antigens in IgG ELISAs using human sera. This study represents the first assessment of their diagnostic utility. Initial screening identified GRA29, GRA45, and GRA54 as promising candidates, with AUC values of 0.9983, 0.8507, and 0.9323, respectively, while GRA35-, GRA36-, and GRA64-based ELISA showed poor discrimination between seropositive and seronegative samples. Extended evaluation of GRA29-based assay with a larger serum panel (n = 286) confirmed excellent diagnostic performance, yielding an AUC of 0.9942 and higher sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) compared with TLA-ELISA. A comparative analysis revealed that GRA29 produced stronger reactivity in positive sera and lower background in negatives. These findings highlight GRA29 as a promising recombinant antigen for the serodiagnosis of human toxoplasmosis and a potential standardized alternative to TLA. Full article

In the past decade, research on recombinant oncolytic viral agents in the treatment of solid tumors has evolved from the initial stage of simple genetic engineering to the current stage of multiple pipelines of parallel clinical application and combination therapy. Compared with T-VEC, the classical therapeutic agent that only expresses GM-CSF, which was approved in 2015, most new oncolytic virus designs include diverse gene constructs to reduce toxic effects, enhance multiple antitumor immunity, avoid immune clearance, or enhance tumor targeting. The single route of administration that activates the inflammatory tumor immune microenvironment by intratumoral injection is no longer sufficient to meet the treatment needs of refractory solid tumors. In this review, we illustrated the construction patterns of typical recombinant oncolytic viral agents and their latest clinical trial progress. Secondly, we summarized the underlying mechanisms of the combined application of antiviral and antitumor immunity in the field of solid tumor immunotherapy. Finally, we explored the feasibility of the intravenous application of oncolytic viruses and their future development directions. We believe that the diversified treatment design of oncolytic viruses will bring more surprises to the immunotherapy of refractory tumors. Full article

The focus of this study was to explore phage display systems employing bacteriophage lambda (λ) gene fusions to its capsid decoration protein gpD as reagent tools for tackling disease. The biological activity of gpD-fusions was examined by testing for the retained antimicrobial toxicity of cathelicidins or defensins fused to gpD. Our previous finding that only COOH fusions of either cathelicidins or defensins to gpD were toxigenic was expanded to show that only the reduced form of fused defensin antimicrobial polypeptides was found to be toxigenic. Compared in review are gene-fusion lytic display systems (where the fusion-display gene is integrated within the viral genome) with a surrogate system, employed herein, that exogenously provides the fusion-display protein for addition to phage capsid. It is easily possible to produce fully coated lambda display particles (LDP) serving as single epitope vaccines (SEV), or antimicrobials, or to produce partially coated LDP without any complex bacteriophage genetic engineering, making the system available to all. The potential to build vaccine vector phage particles (LDNAP) comprising essentially sheathed DNA vaccines encapsulated within an environmentally protective capsid is described. LDNAP are produced by introducing a cassette into the phage genome either by phage–plasmid recombination or cloning. The cassette carries a high-level eukaryotic expression promoter driving transcription of the vaccine candidate gene and is devoid of plasmid resistance elements. Full article

Cathepsins represent a crucial group of protein enzymes involved in insect metabolism. Within the Hemiptera order, comprising a diverse array of predatory, blood-feeding, and herbivorous species, the understanding of cathepsin types and their roles as venom components in predatory bugs remains limited. This investigation systematically identified cathepsin genes present in Hemiptera genomes, highlighting a prevalence of cathepsin B and L, with cathepsin D exhibiting a higher gene count in the Heteroptera suborder. Examining the predatory assassin bug Sycanus bifidus, eight cathepsin genes were notably expressed in its venom glands, with the SbCAB2 gene from the cathepsin B subfamily demonstrating the highest expression in the posterior main gland, indicating its significance as a venom component. Subsequent expression and purification of the recombinant SbCAB2 protein revealed heightened hydrolytic activity (0.91 U/mg protein) compared to extracts from the anterior main gland, accessory gland, and gut. Functional assays demonstrated that SbCAB2, at lower doses (0.625–2.5 μg), can impede phenoloxidase activity in Tenebrio molitor pupal hemolymph, with a 2.5 μg dose inhibiting 86.5% of this activity, thereby preventing hemolymph melanization. Conversely, a higher dose of 10 μg led to effects akin to human placental cathepsin B, promoting melanization in T. molitor pupal hemolymph. These findings lay the foundation for further exploration of the adaptive evolution of cathepsin genes in Hemiptera and offer crucial insights into the functional role of venomous cathepsins in predatory bugs. Full article

Type II collagen (Col2), a crucial structural protein in hyaline cartilage, is essential for cartilage integrity and facilitating injury repair. However, research on recombinant type II collagen still faces many challenges, such as structure and yield, which limit the application of recombinant Col2 in biomedical fields. In this study, we achieved high-yield expression of full-length human Col2 (rhCol2) in CHO cells. The physical and chemical properties of rhCol2 were very close to native Col2, including molecular weight, triple helix structure, thermal stability and self-assembly capacity. Functional assays of primary chondrocytes have demonstrated that rhCol2 can effectively promote chondrocyte proliferation and increase the expression levels of cartilage-specific genes (Col2a1, Aggrecan, and Sox-9). Moreover, a cartilage defect model was surgically created in SD rats demonstrated that rhCol2 significantly enhanced cartilage repair, and the severity of the defect was assessed through histological and micro-CT analyses. Human chondrocytes were utilized to compare the effects of different collagens and verified through a series of functional experiments. In conclusion, these findings indicate that rhCol2 is an effective biomaterial and is expected to promote the application of recombinant collagen in the field of cartilage repair. Full article

Post-translational modifications (PTMs) are critically important for protein structure and function, with glycosylation being one of the most common forms of PTM. The gastric pathogen Helicobacter pylori has a general glycosylation system, which performs complex glycosylation of lipopolysaccharide, flagella proteins, and outer membrane proteins (OMPs). One of the best-described OMPs of H. pylori is the blood group binding adhesin (BabA), which interacts with the Lewis histo-blood group antigen, Lewis b. The 3D structure for BabA has been determined, and the ligand specifically described. Although BabA is reported to be a glycoprotein, there are limited data examining the effects of glycosylation on the structure and function of this protein. This study examined the folding and thermostability of non-glycosylated recombinant BabA and used computational approaches to predict the effect of glycosylation on the protein, with a focus on its possible heterologous expression in mammalian cells. Three potential O-linked and three potential N-linked glycosylation sites were predicted. Furthermore, the effect of glycan shielding on the solvent-accessible surface area of BabA was examined. Molecular dynamics simulations highlighted local indicators, including root mean square fluctuation and the number of protein-glycan contacts that were affected by glycosylation. Taken together, the findings support a role of glycans in surface shielding and promoting local stabilization in specific areas of the BabA protein. This study helps to strengthen the understanding of the importance of glycosylation and the role it plays in the structure, function, and stability of H. pylori proteins. Full article

Brucellosis remains one of the most significant zoonotic diseases, posing a serious threat to both human health and livestock. This issue is particularly relevant for Kazakhstan, which is among the countries endemic for brucellosis with a high incidence rate. Such circumstances highlight the urgent need for the development and implementation of effective preventive measures, including modern vaccine platforms capable of providing reliable protection for the population and reducing the economic impact on the agricultural sector. Recombinant capripoxviruses are considered promising vector platforms for vaccine development, as they ensure high expression of target antigens, elicit strong immune responses, and are safe for humans. In this study, the replication of recombinant capripoxviruses expressing Brucella antigens (SPPV (TK-) OMP19/SODC and SPPV (TK-) OMP25) was evaluated in Vero cells using the BelloStage™-3000 bioreactor system in combination with BioNOC II^® macrocarriers. Application of the bioreactor resulted in nearly a 100-fold increase in Vero cell density compared with static cultures and provided optimal conditions for cell adhesion, growth, and metabolic activity. Consequently, a significant increase in viral titers was observed: for SPPV (TK-) OMP19/SODC, mean titers reached 7.50 log₁₀ TCID₅₀/mL versus 4.50 in static culture (p < 0.0001), while SPPV (TK-) OMP25 achieved 7.08 log₁₀ TCID₅₀/mL versus 4.33 (p < 0.001). These findings confirm the reliability, reproducibility, and scalability of this bioreactor-based approach, demonstrating clear advantages over conventional cultivation methods. Overall, the study highlights the high potential of the BelloStage™-3000 system with BioNOC II^® macrocarriers for the industrial production of recombinant capripoxvirus-based vaccines against brucellosis and for the broader development of other recombinant viral vaccines. Full article

Rational design of capripoxvirus-based vaccine vectors can be achieved by knockout of immunomodulatory genes. In this study, the effect of knockout of the immunomodulatory genes LSDV005, LSDV008 and LSDV066 on the replication of Lumpy skin disease virus in cell cultures and the immune response to an integrated foreign antigen were assessed. The knockout of genes was performed by homologous recombination under conditions of temporary dominant selection. It was found that single knockout of the LSDV005 gene and the LSDV008 gene did not affect the replicative activity of recombinant viruses in vitro (Atyrau-5 and Atyrau-B). Both single knockout of the LSDV066 gene and in combination with knockout of LSDV005 or LSDV008 led to a decrease in the replicative activity of recombinant LSDVs. The recombinant Atyrau-5J(IL18) with LSDV005 gene knockout induced production of antibodies to the integrated antigen in mice. Prime-boost vaccination with all studied recombinants increased the level of interferon-γ. In addition, during immunization with the recombinant Atyrau-5J(IL18) secretion of interleukin-2 was significantly increased. The study of the functions of immunomodulatory genes and their effect on the expression of inserted sequences of foreign antigens is promising for the creation of highly effective polyvalent vector vaccines for animals. Full article

Human granulocyte colony-stimulating factor (hG-CSF) is primarily used to treat neutropenia induced by cancer chemotherapy and bone marrow transplantation. The current identification test for hG-CSF relies on Western blot (WB), a labor-intensive and technically demanding method. This study aimed to screen and prepare an anti-hG-CSF nanobody to identify and quantify hG-CSF, with the ultimate goal of developing colloidal gold-labeled nanobody test strips for rapid identification. An alpaca was immunized with hG-CSF, and the VHH gene sequence encoding the anti-hG-CSF nanobody was obtained through sequencing following phage display library construction and multiple rounds of biopanning. The nanobody C68, obtained from screening, was expressed by E. coli, and its physicochemical properties such as molecular weight, isoelectric point, and affinity were characterized after purification. WB analysis demonstrated excellent performance of the nanobody in identification tests in terms of specificity, limit of detection (LOD), applicability with products from various manufacturers, and thermal stability. Additionally, we established an ELISA method for hG-CSF quantification utilizing the nanobody C68 and conducted methodological validation. Finally, colloidal gold-based test strips were constructed using the nanobody C68, with a LOD of 30 μg/mL, achieving rapid identification for hG-CSF. This study represents a novel application of nanobodies in pharmaceutical testing and offers valuable insights for developing identification tests for other recombinant protein drugs. Full article