Search Results (1,185)

Efficient and reproducible lentiviral vector production and T-cell transduction remain important technical challenges in CAR-T (Chimeric Antigen Receptor T-cell) cell manufacturing. In this study, we optimized HEK293T transfection and primary T-cell transduction parameters for lentiviral CAR constructs targeting BCMA (B-cell maturation antigen) and GPRC5D (G-protein coupled receptor family C group 5 member D). Lipofectamine 3000 and TurboFectin 8.0 were compared across different seeding densities and reagent-to-DNA ratios, with vector yields quantified by qPCR (Quantitative Polymerase Chain Reaction) and p24 ELISA (Enzyme-linked Immunosorbent Assay). Lipofectamine 3000 consistently generated higher viral titers and transduction efficiencies, as reflected by a greater proportion of GFP-positive (Green Fluorescent Protein) cells than TurboFectin 8.0, reaching peak titers of 9.65 × 10⁸ copies/mL for the anti-GPRC5D and 5.33 × 10⁸ copies/mL for the anti-BCMA vectors. Under optimized conditions, transduction efficiencies reached 43.8% GFP⁺ cells for BCMA-CAR and approximately 13–14% GFP-positive transduced cells for the GPRC5D construct within the tested TU/mL range. Co-transduction experiments yielded approximately 62–66% GFP⁺ cells with detectable BCMA-binding and presumptive GPRC5D-CAR-expressing subpopulations identified based on GFP reporter expression. Immunophenotypic analysis demonstrated a relatively stable CD4/CD8 distribution (~65/35), enrichment of effector memory CD8⁺ cells, and expression of activation-associated markers. Collectively, these findings describe an optimized lentiviral transfection and transduction workflow that may support the further development of dual-targeting BCMA/GPRC5D CAR-T manufacturing strategies in research and early translational settings. Full article

For over 3000 years, the perennial herb mugwort (Artemisia argyi) has served as a cornerstone of traditional Asian medicine. Its clinical efficacy is driven by a diverse array of specialized metabolites, most notably flavonoids and volatile oils. While B-box (BBX) transcription factors are known to dictate photomorphogenic development and secondary metabolic pathways in plants, this specific gene family has not yet been systematically analyzed in A. argyi. Leveraging a chromosome-level genomic assembly, we comprehensively identified and analyzed the complete repertoire of AarBBX genes, profiling their structural organization, physicochemical attributes, conserved motifs, promoter architecture, and spatial expression dynamics. The AarBBX family segregates into five distinct evolutionary clades and comprises 114 members, exceeding the gene counts in the diploid relatives Artemisia annua (27) and Arabidopsis thaliana (32), a numerical increase potentially attributable to the tetraploid genome architecture of A. argyi. Promoter scanning revealed a high density of cis-acting elements linked to light perception and environmental stress responses. Integrating RNA-seq transcriptomics with tissue-specific expression profiling, we identified prominent candidate light-responsive AarBBX genes that are highly active in green, photosynthetic tissues and acutely responsive to shifts in light conditions, providing a foundation for future exploration of their potential relationship with secondary metabolic pathways, including flavonoid and terpenoid biosynthesis. Furthermore, we validated the potential operational compartments and structural interactions of these proteins utilizing green fluorescent protein (GFP) subcellular localization and yeast two-hybrid (Y2H) screenings. Collectively, these findings provide new insights into the evolutionary trajectory and regulatory potential of the B-box (BBX) proteins in A. argyi, offering a prioritized candidate gene set for subsequent investigations into their potential roles in light-regulated secondary metabolism, including flavonoid and terpenoid pathways. Full article

Protein-based biosensors require controlled and site-selective functionalization strategies to enable stable and oriented immobilization without compromising protein structure and signal transduction efficiency. We evaluated a chemoselective linchpin-directed modification (LDM) approach targeting Lys–His pairs as a tool for site-specific labeling of the model fluorescent biosensor green fluorescent protein (GFP). LDM molecules with variable spacer lengths were prepared, and a structure-guided computational workflow was implemented to map Lys–His distances on the protein and identify candidate modification sites. Experimental validation by UV-Vis spectroscopy and mass spectrometry demonstrated efficient conjugation and a final degree of labeling close to unity, consistent with single-site modification, with all LDM molecules selectively targeting the same histidine residue (His181), independently of spacer length. Structural analysis revealed that this residue is located within an accessible internal cavity that favors productive interactions with the reactive group. Importantly, the modification preserves GFP fluorescence and pH response, confirming retention of sensing functionality. These results demonstrate that LDM enables selective modification not only of surface residues, but also of structurally guided, non-surface residues. This approach provides the proof of concept of a new, promising strategy for the controlled functionalization and immobilization of protein-based biosensors. Full article

The rice-field eel (Monopterus albus) is an important economic aquaculture species in China. Aeromonas veronii is one of the most common pathogens, which has a high infection and mortality rate in rice-field eel culture. In this study, a strain of A. veronii (AV-MA), isolated from the intestine of a diseased rice-field eel, was labelled with green fluorescent protein (GFP) using mini-Tn7-based systems. The growth curve and virulence of the GFP-labelled AV-MA (AV-MA^GFP) strain showed no significant differences compared to AV-MA. After immersion infection with AV-MA^GFP at 2.5 × 10⁹ CFU/mL, bacterial loads in the liver, stomach, intestine, spleen, kidney, and heart tissues peaked within 1–6 h, and then declined gradually over time. AV-MA^GFP infection remarkably increased intestinal malondialdehyde (MDA) content, superoxide dismutase (SOD) and catalase (CAT) activities. Levels of interleukin-8 (IL-8), interleukin-10 (IL-10), Tumor necrosis factor-α (TNF-α) and nuclear factor kappa-B repressing factor (nkrf) mRNAs fluctuated in the intestine after infection. In conclusion, this study demonstrated that GFP DNA was inserted into the chromosome of A. veronii using mini-Tn7 system for tracking its invasion pathway in vivo of rice-field eels, which provides a useful tool for further studying the pathogen–host interaction mechanisms between A. veronii and M. albus. Full article

A comprehensive analysis of the microcin J25 (MccJ25) biosynthetic gene cluster revealed a previously uncharacterized 96-base pair overlapping gene, designated mcjX. This gene features a +1 reading frame shift relative to the primary sequence and encodes a 31-amino acid peptide. Notably, 53 nucleotides overlap with the 3′ terminus of the structural gene mcjA. Such significant overlaps are rare features in the Escherichia coli genome, highlighting the hidden complexity of microbial operon architectures. In this study, we demonstrate that mcjX is actively translated. Functional assays, including green fluorescent protein reporter systems, suggest that McjX acts as a negative regulator of the mcjA promoter, modulating MccJ25 expression. This discovery represents the first report of a regulatory mechanism mediated by an overlapping gene within a lasso peptide operon, providing new perspectives on how microbial genomes fine-tune the production of antimicrobial peptides through compact genetic organization. Full article

Antigen detection provides rapid and convenient diagnosis of respiratory infections. This study develops an innovative dual-fluorescence aptasensing method based on polydopamine-functionalized MXene (PDA-MXene) for the simultaneous detection of spike protein and hemagglutinin protein. The method employs green- and red-emitting quantum dot (QD) probes as fluorescence reporters, and the PDA-MXene as an effective adsorption and separation substrate. Coupled with a centrifugation-assisted separation strategy, this design method reduces background interference and enhances detection reliability. The method demonstrates good analytical performance, with detection limits of 0.82 ng/mL for spike protein and 2.11 ng/mL for hemagglutinin protein in single-channel mode. The dual-channel mode enables reliable and simultaneous quantification of both target proteins with minimal spectral cross-talk. Furthermore, this method exhibits high specificity against interferents including ions, proteins, and toxins. Artificial saliva, chosen as real sample, is spiked with target proteins to investigate the practical applicability of the method, showing recovery rates for both target proteins between 100 and 114 sensing strategy is simple to operate and allows the detection of new targets by simply replacing the azide-modified aptamer lyophilized powder. It therefore holds promising application for the simultaneous detection of multiple proteins in point-of-care testing and health monitoring fields. Full article

Nanobodies are characterized by their small size, high specificity, and strong affinity, making them promising antiviral agents. In this study, a dual-plasmid yeast surface display (YSD) system based on the Saccharomyces cerevisiae a-agglutinin (Aga1p-Aga2p) platform was evaluated for the functional presentation of H5-specific nanobody. To investigate the influence of fusion design on display performance, enhanced green fluorescent protein (EGFP) was fused to Aga2p in two different orientations. Both configurations enabled successful surface display, while the EGFP-AGA2 orientation showed significantly higher display efficiency than AGA2-EGFP (p < 0.001). This optimized configuration was subsequently used to display Nb10, a broadly neutralizing nanobody targeting the hemagglutinin (HA) protein of H5 influenza viruses. Indirect ELISA, immunofluorescence, and confocal microscopy confirmed successful surface localization of Nb10, while flow cytometry revealed 22.10% positive cells compared with 0.30% in the negative control (p < 0.001). In hemagglutination inhibition (HI) assays, the YSD-Nb10 strain exhibited an HI titer of 3log2, whereas no detectable HI activity was observed in the control strain. Collectively, these results demonstrate the feasibility of displaying a functional H5-specific nanobody using a dual-plasmid YSD system and highlight the importance of fusion orientation for efficient surface presentation, providing preliminary practical guidance for optimization of YSD applications. 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

Protein–protein interactions (PPIs) are fundamental to viral replication, regulating processes such as assembly, genome packaging, and virion maturation. Despite their biological importance, these interactions remain challenging to study and are relatively underexploited as therapeutic targets. Split reporter systems, based on protein-fragment complementation, provide quantitative platforms to measure PPIs by reconstituting reporter activity when interacting protein partners are brought into proximity. These systems can be applied in vitro and in live cells which enables detection of dynamic and multimeric interactions in physiologically relevant contexts. Major classes of split reporter systems include β-lactamase, alkaline phosphatase, luciferase-based platforms, green fluorescent protein, and horseradish peroxidase. Assay performance depends on factors such as fusion protein stability, expression levels, and reporter kinetics, which influence sensitivity, dynamic range, and reliability. These approaches have been applied to study viral protein interactions across diverse systems, including HIV-1 matrix and nucleocapsid proteins, flaviviral capsid proteins, hepatitis B virus core protein, and chikungunya virus capsid. Split reporter assays also enable high-throughput screening for small-molecule inhibitors that disrupt viral PPIs and multimerization. This provides a functional readout linked to viral replication. Despite the challenges that exist in assay optimization and protein stability, the sensitivity and versatility of these systems provide a framework to interrogate viral protein interactions and support the development of antiviral therapeutics.: Full article

Chlorophylls are the major light-harvesting pigments in photosynthetic organisms. Their biosynthesis requires the coordinated supply of metabolic intermediates from two independent upstream branches: the methylerythritol 4-phosphate (MEP)-derived terpenoid pathway, which supplies the phytyl side chain via geranylgeranyl diphosphate (GGPP), and the tetrapyrrole biosynthesis pathway (TBP), which provides the porphyrin ring. How flux through these two branches is coordinated remains poorly understood. In this study, we report the identification of a direct protein–protein interaction between δ-aminolevulinic acid dehydratase (ALAD), the second enzyme of the TBP, positioned immediately upstream of the first metabolic branch point, and the Type II small subunit of GGPP synthase (SSUII), a key regulator of terpenoid flux toward chlorophyll biosynthesis. ALAD was identified as a candidate SSUII-interacting protein by co-immunoprecipitation coupled with LC-MS analysis of rice leaf tissue, with a sequence coverage of 57.04%. The interactions between OsALAD1 and OsSSUII in rice, and between AtALAD1 and AtSSUII in Arabidopsis thaliana, were validated by yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts. BiFC imaging demonstrated that the interaction is localized to the chloroplast. Sequence analysis revealed that plant ALAD proteins are highly conserved, with 92% similarity between OsALAD1 and AtALAD1, and 76.9% similarity between OsALAD1 and the green alga Chlamydomonas reinhardtii CrALAD1, indicating cross-species conservation of the ALAD–SSUII interaction. In vitro enzyme activity assays showed that AtSSUII does not directly alter AtALAD1 catalytic activity, suggesting the interaction operates through post-translational rather than direct catalytic mechanisms. Overexpression of AtALAD1 caused severe chlorosis and seedling lethality, while AtSSUII overexpression produced no distinct phenotype; neither transgene altered the transcript level of the other. Together, our results reveal a conserved cross-pathway protein–protein interaction linking the terpenoid regulatory machinery to the early TBP, suggesting a molecular possibility for the coordinated regulation of chlorophyll biosynthesis. Full article

This study focused on the inhibitory effects of wheat bran feruloyl oligosaccharides (FOs) on the formation of AGEs in three bovine serum albumin (BSA)-based non-enzymatic glycation models, namely BSA-fructose, BSA-methylglyoxal (MGO), and BSA-glyoxal (GO). In the BSA-fructose model, FOs at 0.25 mg/mL achieved a 62% inhibition rate of fructosamine, equivalent to approximately 78% of the activity of the positive control aminoguanidine (AG), and reduced fluorescent AGEs by over 50% on day 12. Additionally, FOs suppressed the accumulation of α-dicarbonyl compounds, key intermediates in the glycation pathway. In the BSA-MGO and BSA-GO system, the decreased fluorescence intensity of tryptophan residues indicated that FOs bound to BSA, inducing conformational changes in the protein microenvironment; this binding also inhibited protein carbonyl formation and the loss of thiol groups, thereby modulating the protein glycation process. Compared with their precursors (ferulic acid, FA; xylooligosaccharides, XOS), FOs exhibited comparable or even superior inhibitory activity against specific AGE subtypes, suggesting a synergistic effect between the feruloyl and oligosaccharide moieties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that FOs reduced the band intensity of 90 kDa AGEs in the glycation system, indicating the inhibition of protein-fructose cross-linking. Fluorescence spectroscopy confirmed that FOs dynamically quenched BSA with a single binding site, and thermodynamic calculations demonstrated that the binding was spontaneous (ΔG < 0), primarily driven by hydrogen bonds and van der Waals forces (ΔH < 0, ΔS < 0). This study systematically investigated the anti-glycation activities of FOs and their precursors. The findings demonstrate that FOs are promising natural glycation inhibitors and provide important theoretical and experimental support for related research. Furthermore, this study establish a basis for the green and high-value utilization of agricultural by-products like wheat bran. Full article

(1) Background: Currently, there is a problem of prompt determination of fat and protein content in the milk–air mixture of milking machines. (2) Methods: A design of a sensor prototype is proposed, combining measurements of light scattering (scatterometry) and fluorescence (fluorometry) to determine the component composition of the milk–air mixture formed during milking. (3) Results: An optical and electronic circuit of a flow sensor has been developed, using four sources of optical radiation: blue, green and red semiconductor lasers (light scattering in milk) and a UV LED (milk fluorescence), as well as an axial photodiode array for recording the light scattering indicatrix and the fluorescence intensity of the milk–air mixture. The use of three laser sources in the scatterometric circuit allows for the determination of the fat content in milk with an error of 0.05%, which is better than all currently known analogs. The developed sensor enables the detection of counterfeit milk containing palm oil instead of milk fat. It operates reliably in a temperature range of 5–35 °C and at milk flow rates of up to 100 mL/sec. (4) Conclusions: The sensor is capable of transmitting real-time data on the fat and protein content of milk to an RS-232 serial port, enabling integration into milking robots and automated milking systems. Full article

Class B scavenger receptor BI splice variants (SR-BI) and BII (SR-BII) internalize lipoproteins but also bind and internalize bacteria. Their individual roles in sepsis are unknown. We overexpressed human SR-BI or BII in transgenic mice, primarily in the liver, but also in the kidney and in bone marrow-derived macrophages, and then performed cecal ligation and puncture (CLP) surgery. SR-BI and BII transgenic mice had significantly worse survival compared to WT mice. Twenty-four hours after CLP, liver injury markers and histological damage were elevated in both SR-BI and BII transgenic mice, whereas kidney damage was similar. Systemic inflammatory cytokines were markedly increased in SR-BI and BII transgenic mice; parallel increases were seen in liver mRNA expression, but not in the kidney. The highest degree of neutrophil infiltration was observed in the liver of SR-BI. Human SR-BI and BII dramatically decreased bacterial accumulation in the liver. Green fluorescent protein-labeled E. coli were efficiently phagocytosed in hepatic macrophages of SR-BI and BII transgenic mice; phagocytosis was more prominent in SR-BII transgenic mice. Finally, human SR-BI overexpression reduced systemic HDL-C levels, eliminated adrenal cortex lipid droplets, and dampened the systemic increase of corticosterone after CLP. Supplementation with glucocorticoid and mineralocorticoid improved survival in SR-BI but not in SR-BII transgenic mice after CLP. In summary, our findings suggest human SR-BI and BII overexpression contributes to higher mortality after CLP by different mechanisms: excessive inflammatory response due to adrenal insufficiency (SR-BI) or hyperactive phagocytosis (SR-BII) in the liver. Full article

RNA viruses exhibit high mutation rates, necessitating antivirals targeting conserved infection mechanisms. In this study, viral hemorrhagic septicemia virus (VHSV), a non-human pathogenic negative-sense RNA virus, was used as a surrogate model to enable high-throughput antiviral screening under reduced biosafety conditions. A recombinant VHSV expressing enhanced green fluorescent protein was used to screen 17,265 compounds, 2000 plant extracts, and 100 marine extracts. Among the candidates, the leaf extract of Phellodendron amurense Rupr. (PL extract) exhibited antiviral activity with low cytotoxicity (selectivity index ≈ 10). The extract reduced viral infectivity in a dose-dependent manner and showed cross-activity against snakehead rhabdovirus. Mechanistic analyses indicated that the PL extract acts primarily at early stages of infection. Virucidal assays demonstrated direct, time-dependent inactivation of viral particles, while pre-treatment reduced host cell susceptibility. Time-of-addition experiments confirmed that antiviral activity was restricted to early infection, suggesting interference with viral attachment or entry rather than intracellular replication. Fractionation revealed that activity was associated with the non-polar n-hexane fraction, implicating lipophilic compounds that may disrupt viral envelope integrity or membrane interactions. These findings suggest that P. amurense leaf extract is a promising candidate for broad-spectrum antivirals targeting conserved entry processes in enveloped RNA viruses. Full article

Rhoptroceros cyatheae (Hymenoptera: Selandriidae) is a dominant herbivorous pest of Alsophila spinulosa in southwestern China, including Guizhou and Sichuan provinces. Infestation by this pest impairs spore reproduction of A. spinulosa and reduces the photosynthetic capacity of host plants. However, the chemosensory genes of R. cyatheae have not been reported, and the molecular basis of antennal detection of host volatile organic compounds (VOCs) is poorly understood. This study aims to screen and identify bioactive VOCs potentially involved in host searching behavior of R. cyatheae, analyze antennal VOC detection patterns, and explore the in vitro binding characteristics of an odorant-binding protein (OBP) involved in olfactory recognition, thereby providing a preliminary theoretical basis for the green management of R. cyatheae. Dynamic headspace sampling, gas chromatography-mass spectrometry (GC-MS), and gas chromatography-electroantennography (GC-EAD) were used to measure antennal electrophysiological responses of R. cyatheae to volatiles from its host A. spinulosa. Y-tube olfactometer assays were conducted to evaluate behavioral responses. For RcyaOBP7, fluorescence competitive binding assays, structural modeling, and molecular docking were integrated to investigate its in vitro binding characteristics with nine selected bioactive VOCs. Nine A. spinulosa volatiles were identified that elicited antennal electrophysiological responses in R. cyatheae, and the sawfly showed behavioral orientation to these VOCs, confirming that its antennae can detect host VOCs. In vitro binding assays showed that RcyaOBP7 exhibited strong binding affinity to p-ethylacetophenone, suggesting its potential involvement in antennal olfactory recognition of this volatile. Specific VOCs released by A. spinulosa are among the signaling molecules detected by the antennae of R. cyatheae. In vitro findings indicate that RcyaOBP7 binds specifically to p-ethylacetophenone, suggesting a possible role in antennal olfactory recognition and behaviors such as host location. However, in vivo functional validation and field trials under ecologically relevant conditions are needed to confirm these roles. This study characterizes the in vitro binding properties of RcyaOBP7 and provides a basis for further research on green management strategies for R. cyatheae based on antennal olfactory signals. Full article