The dense packing of cells within tissues poses challenges for studying cellular properties. Sparse labeling, genetically targeting a small subset of densely distributed cells, provides a powerful approach to investigate cellular morphology, connectivity, dynamics, and functions, especially in neuroscience. However, current sparse labeling methods are generally restricted to fixed labeling densities and often exhibit a decline in sparsity over time. In this study, by maximizing the utility of commonly used recombinases and the relational recognition sites, we constructed a versatile sparse labeling system, termed Tri-M (Multi-recombinase, Multi-recognition site, and Multi-nested), built upon competitive recombination. The Tri-M system, which integrates transgenic mice with viral injection, enables tunable and graded sparse labeling of specific cell types within specific brain regions with long-term stability. This system significantly enhances the ability to track, analyze, and manipulate cells within tissues characterized by dense cellular packing, from single cells to populations.

Polycaprolactone (PCL) is a biodegradable polyester widely used in industry, but it degrades slowly in the environment. Enzymatic hydrolysis catalyzed by cutinases represents a promising approach for PCL waste utilization. Through screening, we selected the Aureobasidium pullulans VKM 1116 strain capable of degrading PCL. We amplified the ApCUT1 cutinase gene from the A. pullulans VKM 1116 genomic DNA. Heterologous expression in Komagataella phaffii yielded ~60 mg/L of the target protein. The recombinant enzyme exhibited maximum activity at pH 6.0-6.5 and 30-40°C. Using site-directed mutagenesis, we generated single mutants (Y58W and L186F) and a double mutant (Y58W/L186F). The double mutant demonstrated the highest catalytic efficiency toward the model substrate 4-nitrophenyl 16-methylsulfonyl hexadecanoate (4-NP-(16-MS-C16)), exhibiting a two-fold increase in the hydrolysis rate compared to the wild-type enzyme. During PCL hydrolysis, the Y58W mutant showed the highest activity, providing a 1.8-fold increase in the suspension degradation rate and complete polymer granule degradation at a rate of 1 mg/h per 1 mg of protein. Our molecular modeling suggests that the hydrophobic bridge at the entrance to the active site functions as a "molecular trap." For the low-molecular-weight substrate (4-NP-(16-MS-C16)), enzymatic activity is limited by the kinetics of the substrate retention step, which is consistent with the maximum catalytic efficiency of the Y58W/L186F mutant with a closing trap. Conversely, activity toward PCL is limited by the initial adsorption stage, for which an open conformation of the free enzyme - as observed in the Y58W variant - is critically important. These results demonstrate the potential of the ApCUT1_Y58W cutinase for developing advanced PCL recycling technologies.

Bombyx mori lipase-1 (Bmlipase-1) has strong anti-BmNPV activity, but the mechanism by which Bmlipase-1 resists BmNPV invasion has not been elucidated so far. In this paper, based on the construction of recombinant viruses overexpressing Bmlipase-1, we analyzed the effects of the recombinant viruses on the genes related to recombinant virus replication as well as viral replication and proliferation after the recombinant viruses infected BmN cells. In the presence of the virus, Bmlipase-1 localizes to the cell nucleus. Quantitative polymerase chain reaction (qPCR), northern blotting and western blot analyses showed that Bmlipase-1 inhibited the expression of BmNPV DNA-binding protein (dbp) as well as the replication and proliferation of the recombinant virus. DBP is a key factor for normal viral DNA replication and for the stabilization of nascent viral DNA. A biotin-labeled probe was synthesized around the c-Myb transcription factor binding site in the upstream promoter region of dbp and analyzed by EMSA with prokaryotically expressed and purified Bmlipase-1. It was found that Bmlipase-1 could bind to CACTTCAATT in the dbp promoter region, while the activity of the dbp promoter lacking CACTTCAATT was significantly reduced. In conclusion, Bmlipase-1 can bind to the CACTTCAATT motif on the dbp promoter in vitro, and this motif contributes positively to the activity of the dbp promoter. Overexpression of Bmlipase-1 significantly downregulates dbp expression and inhibits viral replication. Based on these findings, we propose the following hypothesis: Bmlipase-1 may inhibit viral replication by binding to the CACTTCAATT motif and interfering with the transcriptional regulation of dbp. However, this regulatory model requires further validation in the context of viral infection. This paper provides a new theoretical basis for the prevention and control of lepidopteran insect viruses, and also opens up a new way to study the antiviral mechanism of the silkworm, Bombyx mori.

Recombinant human granulocyte colony-stimulating factor (rh-G-CSF) is useful in tissue regeneration due to its angiogenic and anti-inflammatory actions. The current study focused on the downstream process of A7G mutant rh-G-CSF (m-rh-G-CSF) and the effects of m-rh-G-CSF on human fibroblasts. The m-rh-G-CSF was purified and had 97.24% purity, a final protein concentration of 0.814 g/L and a molecular weight of 18,784 Da. The addition of m-rh-G-CSF significantly elevated the ATP level in the treated cells in a concentration-dependent manner. The maximal ATP level was achieved at a concentration of 2.5 μg/mL and was 89% higher than in the control cells and 13% higher than in reference rh-G-CSF. In the scratch-wound assay, 92.67% and 98.69% wound closure were observed at 24 and 48 h after the addition of m-rh-G-CSF, respectively, compared with 49.93% in control cells at 48 h. These findings support the potential of m-rh-G-CSF as a therapeutic candidate for fibroblast-mediated wound healing.

Bacteria can convert CO2 into acetate and ethanol via the Wood-Ljungdahl pathway with H2 as an electron donor, providing a green route for renewable energy production. However, the low solubility of H2 in water limits conversion efficiency. Although in semi-artificial biohybrid systems such as InP-based systems, photogenerated electrons can replace H2 as an alternative driving force, rapid electron-hole recombination limits carbon fixation efficiency. In this work, CQDs were introduced to construct a CQDs/InP composite to facilitate carrier separation and interfacial electron transfer, forming a CQDs/InP@bacteria biohybrid system. Compared with pure InP, CQDs/InP showed a 78.3% reduction in interfacial charge transfer resistance and a 1.95-fold increase in photocurrent. Metabolomic analysis further revealed that key intermediates involved in intracellular respiration and ATP synthesis, such as succinic acid and fructose 1,6-bisphosphate, were downregulated by 23% and 21.35%, respectively, indicating that the input of external electrons enabled the biohybrid system to effectively reduce the bacterial dependence on intracellular catabolic energy production. Consequently, the CQDs/InP@bacteria biohybrid system under 450 nm blue light achieved acetate and ethanol production of 0.70 and 0.24 g·L-1, respectively, representing increases of 114.46% and 126.98% over the pure bacterial system, with an apparent quantum yield of 1.76%, indicating improved light-to-chemical energy conversion efficiency. This work effectively enhances interfacial electron transfer in InP-based systems and promotes light-driven CO2 fixation and chemical production in non-photosynthetic bacteria, providing a pathway for constructing efficient semi-artificial photosynthetic microbial CO2 conversion systems.

The gold standard for SARS-CoV-2 neutralization assays involves wild-type virus, which requires Biosafety Level 3 (BSL-3) containment. To improve safety and accessibility, pseudovirus-based neutralization assays utilizing non-replicating particles like Vesicular Stomatitis Virus (VSV) expressing the SARS-CoV-2 spike protein can be conducted under BSL-2 conditions. This study aimed to perform the analytical validation of a VSV-based pseudovirus system for SARS-CoV-2 using recombinant monoclonal antibody. Pseudo-VSV carrying SARS-CoV-2 Spike proteins were produced using LentiX-293T cells. The assay system was optimized for Multiplicity of Infection (MOI) and assessed for specificity, limit of quantification (LOQ), linearity, accuracy, and precision using the neutralizing mAb BD-604. The system was optimized at an MOI of 0.25. The assay proved highly specific, as mAb BD-604 showed clear neutralizing activity while mAb 1A9 did not. The limit of quantification (LOQ) was determined to be 125 ng of mAb BD-604, with a linear range of 125 - 1000 ng. The relative accuracy remained within the 80-120% range, and the precision (%CV) ranged from 1.92% to 13.57%. Additionally, the system successfully characterized variant-specific neutralization, revealing that BD-604 was effective against the Wuhan, Delta, and Omicron BA.1/BA.2 strains but lacked activity against the Omicron XBB.1.5 variant. This validated pseudo-VSV based SARS-CoV-2 neutralization assay is a valuable bioassay for evaluating neutralizing antibody potency against various SARS-CoV-2 strains in a BSL-2 environment, thus making it useful for vaccine and therapeutic development.

Feline infectious peritonitis virus (FIPV) is a lethal feline pathogen with no widely effective prophylactic vaccine available. To address this unmet need, we explored the feasibility of using Toxoplasma gondii (whose definitive host is felids) as a live delivery platform to develop a bivalent vector vaccine. We generated a transgenic T. gondii strain engineered to express and secrete the FIPV spike protein S1 subunit into the parasitophorous vacuole. Immunization in mice confirmed the immunogenicity of this recombinant parasite, which induced specific antibody responses targeting both the T. gondii vector and the FIPV S1 antigen. In vitro neutralization assays revealed limited FIPV-neutralizing capacity in immune sera, with only low-level inhibitory activity observed at the lowest serum dilution, which was markedly inferior to the neutralization potency induced by recombinant S1 protein vaccination. Collectively, these data preliminarily verify the potential of T. gondii as a multivalent antigen delivery vector. This work provides a proof-of-concept framework for a dual-target vaccination strategy intended to mitigate the epidemiological burden of both FIPV and T. gondii in cats, and underpins integrated One Health-oriented disease prevention and control efforts.

Dengue virus (DENV) nonstructural protein 3 (NS3) is essential for viral replication and host immune evasion. While its protease domain (NS3pro) has been linked to T-cell anergy, its role in inducing regulatory T cells (Tregs) requires further exploration. To analyze the induction of diverse regulatory T-cell populations in BALB/c mice immunized with recombinant rNS3pro proteins from the four DENV serotypes. Mice were immunized with rNS3pro, and splenocytes were stimulated in vitro with homologous proteins. Cytokine expression and T-cell phenotypes-including markers for activation, exhaustion, and cytotoxicity-were analyzed via RT-PCR, ELISA, and flow cytometry. rNS3pro immunization induced a predominantly regulatory-associated response characterized by significant IL-10 expression. A marked expansion of CD4+CD25+ T cells (23.5% vs. 1.05% in controls) was observed, encompassing CD25+FoxP3+ regulatory-like T cells, alongside FoxP3-independent subsets expressing CD25+IL-10+ and CD25+TGF-β+. These cells co-expressed cytotoxicity-associated (FoxP3+Perforin+/GzB+) and co-inhibitory (CTLA-4+, PD-1+) markers. Effector CD8+ T-cell proliferation was significantly inhibited and could not be restored by exogenous IL-2. In conclusion, DENV rNS3pro immunization drives a robust expansion of multifaceted regulatory-like T-cell phenotypes. This immunomodulatory response correlates with compromised CD8+ effector proliferation, identifying the NS3 protease domain as a potential player in DENV immune evasion strategies.

Metal-organic frameworks (MOFs) have attracted tremendous interest in the photocatalytic hydrogen evolution reaction (HER). Nevertheless, their practical catalytic performance is limited by the rapid recombination of photogenerated charge carriers and sluggish interfacial reaction kinetics. Herein, a hierarchically structured UiO-66-NH2(Hf) / ZnIn2S4 (denoted UN-66/ZIS) heterostructure was rationally fabricated via a solvothermal strategy to simultaneously regulate charge separation efficiency and optimize interfacial catalytic kinetics. Benefiting from the intimate heterointerface and built-in electric field, the optimized UN-66/ZIS sample exhibits remarkably improved visible-light absorption capability and charge transport efficiency, achieving a photocatalytic hydrogen production of 6052 ± 206 μmol·g-1, which greatly outperforms pristine UN-66 and ZIS. More importantly, in-situ X-ray photoelectron spectroscopy combined with density functional theory calculations directly unveils the mechanism of interfacial charge redistribution and directional electron transfer responsible for the enhanced carrier separation efficiency, offering mechanistic insights into the improved catalytic activity. Furthermore, loading of Pt cocatalyst effectively accelerates the surface hydrogen evolution kinetics. The corresponding hydrogen evolution rate increases to 14,403 ± 287 μmol·g-1·h-1, approximately seven times that of the Pt-free heterostructure. This work proposes a synergistic strategy integrating heterojunction engineering and cocatalyst modulation to simultaneously optimize light harvesting, charge separation and surface reaction kinetics, which provides new guidelines for the rational design of high-performance MOF/sulfide photocatalysts toward solar hydrogen production.

Inspired by natural photosynthetic systems, the rational design of artificial photocatalysts featuring asymmetrically coordinated dual-centers and a tailored microenvironment offers promising opportunities to overcome the intrinsic limitations in selective chemical reactions. Here, we present an asymmetric coordination catalyst by immobilizing porphyrins within a defect-engineered metal-organic framework. By leveraging the matched size of guest molecules in heterogeneous pores, the electron-hole recombination obstacle was significantly overcome. Across the library of multivariate zirconia metal-organic frameworks, the optimal member stands out for its photocatalytic properties, delivering an excellent apparent quantum yield (11.31%) for ultra-fast degradation of sulfaclozine (SCL) with a 10-minute reaction time. Consistent with the extended X-ray absorption fine structure (EXAFS) and theoretical calculations, the confined groups in microporous environments remarkably increased the charge separation ability of the Zr-O nano-capsules in biomimetic porous PSI and PSII systems. The turnover frequency is 32.1 and 36.9 times higher than that of the pristine framework and the commercial TiO2 photocatalyst, respectively. This work establishes a broadly applicable strategy for multivariate zirconia metal-organic frameworks and provides a platform for tailoring active-site configuration for photocatalytic reactions.

Global marine fisheries face increasing pressures from overexploitation and environmental degradation, thereby necessitating sustainable alternatives to conventional seafood production. Cellular aquaculture emerges as a promising approach to separate fish protein supply from ecological constraints. This review aims to synthesize current knowledge on the efficient production of cell-cultured fish meat, consolidating fragmented advances across disciplines. This review systematically examines three core concepts: marine fish stem cell biology, serum-free production technologies, and sensory quality reconstruction. The key findings indicate that marine fish cells exhibit distinct evolutionary traits compared to mammalian cells, including unique telomerase-mediated immortalization potential and cold-tolerance mechanisms. Furthermore, recent breakthroughs in serum-free media using plant hydrolysates and recombinant proteins have substantially reduced costs, while edible microcarriers and suspension cultures provide viable pathways for scale-up. However, challenges remain in replicating the lipid profiles, specifically n-3 polyunsaturated fatty acids, and the textural hierarchy of native fish fillets. This review concludes that future industrial success depends on the development of species-specific bioprocesses and strictly food-grade culture systems. This work provides a foundational framework for the transition of cultivated fish from laboratory prototypes to commercial production.

Inclusion bodies are insoluble nanostructured recombinant proteins that have emerged as innovative biomaterials for disease control in animal production. This systematic literature review aimed to explore the use of inclusion bodies for disease control in aquaculture and livestock. We conducted a literature search in four databases: PubMed, Web of Science, ScienceDirect and Scopus. Of the 3139 research articles retrieved, only 16 met our eligibility criteria: 6 for aquaculture, including studies in rainbow trout, senegalese sole, and zebrafish, and 10 for livestock, including studies in cattle, sheep, swine, and mouse models. The proteins produced in these studies were mainly host immune system proteins and pathogen antigenic proteins. The diseases or pathogens potentially controlled by inclusion bodies in aquaculture were Pseudomonas aeruginosa, Mycobacterium marinum, Spring viremia of carp virus and Viral nervous necrosis virus. Conversely, the diseases or pathogens evaluated to be controlled by inclusion bodies in livestock were mastitis, Fasciola hepatica, Lawsonia intracellularis, and Vibrio sp. The administration strategies varied greatly, including intra-mammary gland infusion, nasal suspension, gastric injection, esophageal intubation, intramuscular injection, and oral administration. Regardless of administration route, these biomaterials consistently activated immune responses through two mechanisms: modulation of the host immune system via immunostimulatory proteins, and activation of adaptive immunity via antigenic proteins. Although promising, widespread adoption remains limited by technological challenges including purification standardization, batch variability, and lack of studies in commercially relevant species. These findings establish a robust evidence base to inform the development of IB-based interventions in animal health and guide future research directions.

Four-and-a-half LIM domains protein 2 (FHL2) is a modular adaptor composed entirely of LIM domains that mediate protein-protein interactions linking the cytoskeleton to transcriptional regulation. Here, we report a streamlined preparation of recombinant tag-free full-length FHL2 in bacteria and describe its biophysical characterization using size-exclusion chromatography coupled to static light scattering (SEC-SLS), small-angle X-ray scattering (SEC-SAXS), and cross-linking coupled with mass spectrometry (XL-MS). Soluble expression was enhanced by zinc supplementation and low-temperature induction. Purified FHL2 eluted from SEC-SLS as a monomeric species consistent with a flexible, elongated multidomain architecture. Integrative modeling based on SAXS and XL-MS confirmed that FHL2 exists in an extended and a compact bent conformation in solution. SEC-SLS and Isothermal titration calorimetry (ITC) measurements confirmed a direct 2:1 interaction between FHL2 and β-catenin with submicromolar affinity, supporting FHL2's scaffolding role within the Wnt/β-catenin signaling pathway. These results establish the groundwork for structural and functional analyses of FHL2 and related LIM-domain proteins.