BackgroundSpidroins are well-known for their exceptional mechanical properties, which have inspired extensive research and applications across various fields. Large-scale production of spidroin continues to face major challenges due to the complexity involved in inducing host organisms to express full-length spidroins. This process requires advanced techniques and has issues such as plasmid instability and potential misfolding.ResultsIn this study, we developed a novel expression system by combining a fusion tag with a self-cleavage intein, enabling the convenient expression of three chimeric spidroins with varying numbers of repetitive units. After optimising expression conditions, NT2RepCT, NT4RepCT and NT6RepCT spidroins were obtained in soluble form, with yields of 266, 135 and 125 mg/L, respectively. All three spidroins exhibited an increased β-sheet content with increased numbers of repetitive units during transition from soluble to dry state. In terms of nanofibril morphologies, the self-assemblies of NT4RepCT and NT6RepCT closely resemble those of native silk proteins.ConclusionThis study can serve as a reference for preparation of high-performance spider silk materials and soluble expression of proteins, such as collagen, and as a foundation for preparation of other structurally complex polymer materials.

Bacteriophages are promising alternative antimicrobial agents due to their high specificity for host bacteria and minimal immunogenicity in humans. However, their therapeutic application is limited by their nature as biological entities, with potential evolutionary consequences. In this study, we address these challenges by repurposing only the structural components of bacteriophages as vesicles to deliver antibiotics directly into the cytoplasm of bacterial hosts. This approach is based on two key hypotheses: first, antibiotics such as β-lactams remain effective against resistant bacteria if injected directly into the cytoplasm, bypassing resistance mechanisms; second, phage structures can be synthesized and self-assembled in vitro using modular genomes and cell-free protein expression to carry small molecules as cargo. To test these hypotheses, we utilized T7 phages and penicillin-resistant Escherichia coli as a model system. First, we designed the T7 phage genome into a modular format containing only the genes encoding structural components and synthesized the gene fragments via de novo gene synthesis. These phage structures were then rebooted in vitro using cell-free protein expression in the presence of penicillin G, allowing the antibiotics to be incorporated as cargo during the self-assembly process. Finally, we tested the antimicrobial activity of these antibiotic-loaded phage syringes against penicillin-resistant E. coli. The results demonstrate that phage syringes effectively reduce the host population compared to negative controls, including free penicillin and water. This study highlights the potential of using phage structures as antibiotic delivery vehicles, offering a novel strategy to overcome both the limitations of small-molecule antibiotics and phage therapy.

Myocardial infarction, characterized by insufficient blood supply to the heart, leads to ischemia and hypoxia of myocardial tissues, causing injury and decreased cardiac function. Despite improvements in pharmaceutical and interventional therapies, it remains a leading cause of death worldwide. Human umbilical cord mesenchymal stem cells (hUC-MSCs) play an important role in the repair of infarcted myocardium by promoting angiogenesis, reducing inflammation, secreting growth factors and cytokines. However, the harsh hypoxic microenvironment of infarcted myocardial tissue poses a threat to the survival and function of transplanted hUC-MSCs. In this study, we modified the candidate gene promoter of hUC-MSCs under hypoxic conditions and created a promoter that can respond quickly under hypoxic conditions. We found that the modified promoter significantly promoted the transcription efficiency as hypoxia time increased. This indicates that the engineered hypoxia-response promoter can effectively drive gene expression in a hypoxic environment. Furthermore, the transcription efficiency of the modified promoter under normoxic conditions is lower than that of common promoters in eukaryotic organisms, suggesting that this effect can improve the efficacy and safety of hUC-MSC-based myocardial infarction treatment by ensuring that cells function effectively in the damaged hypoxic area.

Effective treatment of skin wounds remains a clinical challenge owing to factors such as microbial infections, impaired fibroblast activity, disrupted angiogenesis, and collagen remodeling. In this study, we developed and evaluated novel curcumin–cyclodextrin hybrid nanoparticles (Cur/CD-HNPs) as a multifunctional platform for enhanced wound healing. Nanoparticles (NPs) were prepared via nanoprecipitation. Physicochemical and structural properties were systematically characterized by determining the encapsulation efficiency (EE), particle size, zeta potential, X-XRD, FTIR, SEM, in vitro release, and stability studies. The optimized Cur/CD-HNPs demonstrated a uniform particle size of 150.5 ± 2.8 nm, a surface charge of − 18.5 ± 0.59 mV, a PDI of 0.20 ± 0.03, and a high EE (90.2 ± 2.35%). Cur/CD-HNPs exhibited potent anti-inflammatory effects (97.93 ± 1.24% inhibition of protein denaturation), full antioxidant activity (100% ABTS radical scavenging, IC50 = 12.85 µg/mL), and broad-spectrum antibacterial efficacy. Cur/CD-HNPs exhibited a sustained biphasic release profile, with ~ 82% of Cur released over 24 h, supporting sustained delivery for wound healing applications. In vitro scratch assays revealed enhanced fibroblast proliferation and migration. For in vivo evaluation, the nanoparticles were incorporated into a hydrogel base and applied topically in a rat burn wound model, resulting in significantly accelerated wound closure (P < 0.05). Histopathological examination revealed improved epithelialization, collagen deposition, and tissue regeneration compared with the control groups. Our findings presented Cur/CD-HNPs as a promising therapeutic approach, offering Cur enhanced bioactivity, stability, and regenerative potential. This formulation addresses the key limitations of curcumin and presents a multifunctional and strong translational platform for clinical wound care.Graphical

In biogerontology, researchers are actively exploring the manipulation of sirtuin proteins, a family of enzymes that play a crucial role in regulating lifespan and age-related diseases through bioactive compounds found in the diet. This report explores potential new insights by leveraging the innovative intersection of natural bioactive compounds and sirtuin modulation through computational drug design. Using state-of-the-art in silico techniques, including high throughput virtual screening (HTVS) and molecular dynamics (MD) simulation, the study identifies natural compounds from various dietary sources for sirtuin proteins (SIRT 1, 3, 6, 7) related to the cellular process in aging. HTVS was automated with the AutoDock-VINA to pinpoint the novel candidate bioactive compounds for sirtuins, while the MD was conducted with GROMACS. The top hits, fagopyrine, emblicanin B, epicatechin-(4beta- > 8)-epicatechin-(2beta- > 7,4beta- > 8)-catechin (epicatechin), withanolide, and dulcisisoflavone, had superior docking scores ranging from − 12.7 to − 7.2 kcal/mol. The MD displayed steady interactions between target proteins and biocompounds throughout 100 ns without significant conformational shifts. This paper contributes significantly to anti-aging research by presenting a novel, computationally efficient approach to discovering and developing sirtuin-modulating compounds. Highlighting the untapped potential of natural bioactive substances in modulating key biological pathways associated with aging opens new avenues for future experimental exploration of diet-based therapeutic strategies that may support healthy aging.

BackgroundThe use of non-clonal CHO cell derived materials for preclinical studies has been found to be a valuable approach to accelerate the development of monoclonal antibodies (mAbs) for first-in-human (FIH) studies. In a comprehensive study, we assessed the culture performance, productivity, and product quality of non-clonal cell lines compared with clonal cell lines expressing various biologic modalities to determine if this approach can be applied to complex molecules.ResultsWe evaluated a multi-specific antibody, a cytokine-Fc fusion protein, and a mAb produced using the stable pool, the pool of top clones, and the lead clone utilizing transposase-mediated integration. The results indicated that the attributes were comparable regardless of the source of cells. Building upon these findings, the study progressed to the preclinical manufacturing of two multi-specific antibodies using both the pool of top clones and the lead clone. Subsequently, clinical manufacturing of these multi-specific antibodies was performed using the lead clone. The batches produced with the pool of clones and the lead clone demonstrated a high level of comparability in culture performance, productivity, and product quality.ConclusionsIn conclusion, non-clonal CHO cell derived materials can be effectively utilized for preclinical studies of complex molecules without compromising their quality, allowing for accelerated development for FIH studies.

This study investigates the effects of quercetin-conjugated nanocrystalline cellulose/cetyltrimethylammonium bromide/folic acid nanoparticles (NCC/CTAB/FA NPs) on AGS and A2780 cancer cell lines, focusing on their cytotoxicity and antioxidant capacity. Dynamic light scattering (DLS) analysis revealed an average particle size of 388.70 nm, suitable for cellular uptake and release kinetics. The NCC/CTAB/FA NPs exhibited a rod and spherical morphology and uniform distribution, as confirmed by field emission scanning electron microscopy (FESEM). Fourier-transform infrared (FTIR) spectroscopy confirmed the successful synthesis and functional group integration, supporting the NPs’ ability for drug delivery. The encapsulation efficiency value was 81.17%, demonstrating the effective incorporation of Quercetin. Cytotoxicity assays indicated significant reductions in cell viability for AGS and A2780 cells with IC50 values of 3.2 µg/mL and 16.04 µg/mL, respectively, while HDF cells exhibited higher viability. Flow cytometry analysis revealed a dose-dependent induction of apoptosis in AGS cells, supported by changes in gene expression related to apoptosis and inflammation. Furthermore, antioxidant capacity assays demonstrated practical free radical scavenging abilities, with IC50 values of 151.65 µg/mL for ABTS and 349.54 µg/mL for DPPH. NCC/CTAB/FA/Quercetin NPs exhibit promising characteristics for targeted cancer therapy and antioxidant applications.

BackgroundNanotechnology has been greatly examined for tumor medication, as nanoparticles (NPs) serve a crucial role in drug delivery mechanisms for cancer therapy. In contrast to traditional cancer therapies, NPs-based drug delivery offers several benefits, including increased stability and biocompatibility, improved retention capabilities and permeability, as well as precise targeting.AimThe objective of this study was to examine the tumor-targeting efficacy of Thymoquinone (TQ)–loaded gold NPs (AuNPs/TQ conjugate) or TQ–loaded silver NPs (AgNPs/TQ conjugate) in conjunction with the conventional chemotherapy agent cisplatin (CP) in Ehrlich ascites carcinoma (EAC)-bearing mice.MethodsThe loading capacity of synthesized conjugates was characterized by UV-Vis spectra and transmission electron microscope (TEM). We used CD-1 mice with a peritoneal EAC tumor xenograft model that received oral administration of TQ, AuNPs, AgNPs, AuNPs/TQ conjugate, and AgNPs/TQ conjugate.MethodsEAC-bearing mice received daily oral administration of one of the following treatments for six consecutive days: TQ, AuNPs, AgNPs, AuNPs/TQ, AgNPs/TQ, AuNPs/TQ + CP, or AgNPs/TQ + CP conjugates. Eleven days after EAC inoculations, assessments were conducted to evaluate the total number of tumor cells, splenocytes, white blood cells (WBCs), C-reactive protein (CRP) levels, flow cytometric analysis of apoptosis in EAC cells, as well as the functionality of the kidney and liver.ResultsEAC-bearing mice that received treatment with TQ, AuNPs, AgNPs, AuNPs/TQ, and AgNPs/TQ exhibited significantly enhanced anti-tumor activity and improved therapeutic efficacy. Our results further revealed that the combined synergistic approach of TQ’s anti-tumor properties, along with the efficient penetration abilities of AuNPs or AgNPs, led to a significant inhibition of the growth of tumor cells in EAC tumor-bearing mice. Moreover, the incorporation of CP into the AuNPs/TQ or AgNPs/TQ conjugates substantially augmented the anti-proliferative effects against EAC tumor cells, effectively overcoming resistance to chemotherapeutic agents. Furthermore, our data revealed that this combination resulted in an elevation of leukocyte counts, along with an increase in the absolute quantities of lymphocytes, neutrophils, and monocytes, thereby activating the immune system and reducing the inflammatory marker CRP. However, the restoration of splenocyte levels, which had been reduced due to EAC cell inoculation, required an extended period to return to baseline. Furthermore, the results indicated moderate alterations in the functionality of both the liver and kidney.ConclusionTo conclude, AuNPs, AgNPs, AuNPs/TQ, and AgNPs/TQ may hold great promise as potential nanoparticle-based therapies for cancer treatment. Additionally, provides numerous benefits compared to conventional cancer therapies, such as selectivity and minimal side effects. Additionally, AuNPs, AuNPs/TQ, AuNPs/TQ + CP, AgNPs, AgNPs/TQ, or AgNPs/TQ + CP can specifically target tumor tissues, suppress tumor growth, extend the lifespan of tumor-bearing mice, and minimize cytotoxic effects on normal tissues, relative to the administration of free CP alone. More research is needed to understand the mechanisms of these nanoparticle-based therapies in clinical and optimize their use as cancer therapies.

PurposeTo study the potential of a candidate probiotic strain belonging to the Enterococcus durans species in alleviating hypercholesterolemia and improving the microbial milieu of rat gut.MethodsA previously isolated and characterized E. durans strain NPL 1334 was further screened in vitro for its bile salt hydrolyzation and cholesterol assimilation ability. An in vivo trial using diet-induced hypercholesterolemic rats was conducted to evaluate the effects of the administered test probiotic strain on the animal’s blood biochemical parameters such as total cholesterol (TC), high-density lipopolysaccharides (HDL), low-density lipopolysaccharides (LDL), triglycerides (TG), on body weight, oxidative stress markers, and its impact on intestinal and fecal microbiota as well as a histopathological examination of the test animal’s livers.ResultsE. durans strain showed good bile salt hydrolyzing ability and ample cholesterol assimilation in vitro. Probiotic-fed hypercholesterolemic rats showed significantly lowered cholesterol, triglyceride and LDL levels. The body weight of probiotic-fed rats was reduced as compared to the control. E. durans also stimulated the growth of beneficial LAB in the intestine of experimental rats and did not harm the liver of the experimental rats.ConclusionE. durans can be a natural therapeutic alternative to manage diet-induced hypercholesterolemia and may eventually enhance anti-cholesterolemic therapies.

BackgroundFalse flax, or gold-of-pleasure (Camelina sativa) is an oilseed that has received renewed research interest as a promising vegetable oil feedstock for liquid biofuel production and other non-food uses. This species has also emerged as a model for oilseed biotechnology research that aims to enhance seed oil content and fatty acid quality. To date, a number of genetic engineering and gene editing studies on C. sativa have been reported. Among the most common targets for this research are genes, encoding fatty acid desaturases, elongases, and diacylglycerol acyltransferases. However, the majority of these genes in C. sativa are present in multiple copies due to the allohexaploid nature of the species. Therefore, genetic manipulations require a comprehensive understanding of the diversity of such gene targets.ResultsHere we report the detailed analysis of FAD2, FAD3 and FAE1 gene diversity in five Camelina species, including hexaploid C. sativa and four diploids, namely C. neglecta, C. laxa, C. hispida var. hispida and var. grandiflora. It was established that FAD2, FAD3 and FAE1 homeologs in C. sativa retain very high conservancy, despite their allohexaploid inheritance. High sequence conservancy of the identified genes along with their different expression patterns in C. sativa suggest that subfunctionalization of these homeologs is mainly grounded on the transcriptional balancing between subgenomes. Finally, fatty acid composition of seed lipids in different Camelina species was characterized, suggesting potential variability in the activity of fatty acid elongation/desaturation pathways may vary among these taxa.ConclusionIt was shown that the FAD2, FAD3 and FAE1 genes retain high conservation, even in allohexaploid C. sativa after polyploidzation, in which the subfunctionalization of the described homeologs is mainly grounded on the expressional differences. The major differences in FA accumulation patterns within the seeds of different species were identified as well. These results provide a foundation for future precise gene editing, which would be based on targeting of particular FAD2, FAD3 and FAE1 gene copies in C. sativa that allow regulating the dosage of the mentioned genes, thus shaping the desired FA composition in cultivated false flax.