The convergence of cell-free protein synthesis (CFPS) and vesicle-based delivery platforms presents a promising avenue for therapeutic development. The open environment of CFPS offers precise control over protein synthesis by enabling the modulation of synthetic conditions. Additionally, vesicle-based platforms provide enhanced stability, bioavailability, and targeted delivery. This synergy facilitates the efficient production of complex proteins—including membrane proteins, antibody fragments, and proteins requiring post-translational modifications (PTMs)—and supports novel drug delivery strategies. While existing reviews have covered synthetic cells and biomanufacturing broadly, a dedicated analysis of CFPS system-containing vesicles (CFVs) for therapeutic applications remains absent from the literature. This review addresses this knowledge gap by providing a comprehensive examination of CFVs, highlighting their potential as programmable drug delivery platforms through the integration of genetic circuits. It emphasizes the advantages of CFPS over traditional cell-based approaches and explores the synergistic benefits of combining CFPS with various vesicle systems. These systems offer dynamic control over therapeutic protein production and targeted delivery, enabling precise responses to specific signals in complex environments. Although challenges such as low protein yield and imperfect targeting remain, potential optimization strategies are discussed. This analysis highlights the significant potential of integrating CFPS and vesicle-based delivery to advance biomanufacturing, therapeutic development, and synthetic cell systems, thereby opening new avenues in medicine and healthcare.

Polycystic Ovary Syndrome (PCOS) is a hormonal disorder affecting women of reproductive age, often associated with oxidative stress and inflammation. This study explores the therapeutic potential of Arctium lappa phenolic-rich fraction encapsulated nanofiber (ALPRF-NF) in a testosterone-induced PCOS mouse model. All experiments were performed in triplicate and Duncan’s Multiple Range Test was used to assess significant differences between means, with significance determined at p < 0.05. The ALPRF-NF formulation demonstrated favorable physicochemical properties, including a ribbon-like structure (216.9 nm), a zeta potential of -19.3 mV, and a high encapsulation efficiency (93.1%). In vivo findings showed that ALPRF-NF significantly improved body weight, feed intake, and liver enzyme profiles in PCOS-induced mice (p ≤ 0.05). It also enhanced the antioxidant defense system by elevating levels of glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT). Mechanistically, ALPRF-NF reduced oxidative stress and inflammation by delivering phenolic compounds that scavenge reactive oxygen species (ROS) and modulate gene expression in ovarian tissue. This included downregulation of inducible nitric oxide synthase (iNOS) and upregulation of SOD expression. These results suggest that ALPRF-NF effectively mitigates testosterone-induced ovarian oxidative damage and inflammation, offering a targeted, nanotechnology-based therapeutic approach for PCOS. The study provides valuable insights into novel strategies for improving women’s reproductive health through bioactive compound delivery.

BackgroundSynthesis of organic@inorganic hNFs is achieved by the coordination of organic compounds containing amine, amide, and diol groups with bivalent metals. The use of bio-extracts containing these functional groups instead of expensive organic inputs such as DNA, enzymes, and protein creates advantages in terms of cost and applicability. In this study, the application potentials (antioxidant, antibacterial, anticancer, guaiacol, anionic, and cationic dye degradation) of hybrid (organic@inorganic) nanoflowers (hNFs) synthesized with Cu+2 and snakeskin (SSS) were proposed.ResultsMorphology, presence, and composition of elements of Cu and SSS-coordinated hNFs (Cu@SSS hNFs) were shown through FE-SEM–EDX spectroscopy. According to FE-SEM findings, hNFs synthesized with 0.5 ml and 1 ml extract have diameters of 12.81 and 3 µm, respectively. Diffraction peaks of hNFs determined by XRD were consistent with JCPDS Card 00–022 –0548. Cu@SSS NFs showed antioxidant properties depending on time through DPPH scavenging behavior (ability (R2: 0.5612, IC50: 2.07 mg/ml). Cu@SSS hNFs synthesized coordination of SSS and Cu degraded (75%) methylene blue at the highest pH 9 condition. However, hNFs highest degraded (68%) brilliant blue in an acidic PBS medium. hNFs oxidized guaiacol depending on exposure time. Cu@SSS hNFs demonstrated antibacterial properties towards Gram (-/ +) pathogen strains (MIC: 60 µg/ml). The catalytic and antimicrobial properties of hNFs were mentioned by the Fenton reaction. The cytotoxicity of Cu@SSS hNFs on the lung carcinoma (A549) cell line was shown to be concentration-dependent by the MTT test assay (IC50: 56.4 µg/ml).ConclusionAs a result, Cu-based hNFs synthesized by using an organic waste (SSS) might be improved for environmental and biomedical applications.

Mitochondrial abnormalities underscore a variety of neurologic injuries and diseases and are well-studied in adult populations. Clinical studies identify critical roles of mitochondria in a wide range of developmental brain injuries, but models that capture mitochondrial abnormalities in systems representative of the neonatal brain environment are lacking. Here, we develop an organotypic whole-hemisphere (OWH) brain slice model of mitochondrial dysfunction in the neonatal brain. We extended the utility of complex I inhibitor rotenone (ROT), canonically used in models of adult neurodegenerative diseases, to inflict mitochondrial damage in OWH slices from term-equivalent rats. We quantified whole-slice health over 6 days of exposure for a range of doses represented in ROT literature. We identified 50 nM ROT as a suitable exposure level for OWH slices to inflict injury without compromising viability. At the selected exposure level, we confirmed exposure- and time-dependent mitochondrial responses showing differences in mitochondrial fluorescence and nuclear localization using MitoTracker imaging in live OWH slices and dysregulated mitochondrial markers via RT-qPCR screening. We leveraged the regional structures present in OWH slices to quantify cell density and cell death in the cortex and the midbrain regions, observing higher susceptibilities to damage in the midbrain as a function of exposure and culture time. We supplemented these findings with analysis of microglia and mature neurons showing time-, region-, and exposure-dependent differences in microglial responses. We demonstrated changes in tissue microstructure as a function of region, culture time, and exposure level using live-video epifluorescence microscopy of extracellularly diffusing nanoparticle probes in live OWH slices. Our results highlight severity-, time-, and region-dependent responses and establish a complimentary model system of mitochondrial abnormalities for high-throughput or live-tissue experimental needs.

Adenosine triphosphate (ATP) assays have a faster turnaround time and higher sensitivity than traditional cultivation methods for microbial monitoring. Challenges implementing ATP testing include incompatibility with chlorine quenching agents and hold time sensitivity, which are not well-studied. Chlorinated distribution system samples were collected from two Canadian utilities, Metro Vancouver (n = 40 samples) and Halifax Water (n = 283). No significant correlations were observed between heterotrophic cell count (HPC) and cellular ATP, suggesting these do not correlate well in waters with low biological activity (median HPC < 2 CFU/mL). However, interpretation of HPC and cATP results (based on the HPC guideline of 100 CFU/mL and cATP of 10 pg/mL) yielded the same conclusion for 95% of samples, suggesting a potential decision-making framework to replace HPC with cATP. Moreover, cATP correlates better with free chlorine (p < 0.04) compared with HPC for one of the studied systems. Importantly, adding chlorine quench (10% sodium thiosulfate) did not produce significantly different cATP results, nor did analyzing at various hold times of 4-, 6-, and 24-h. This study supports the integration of ATP testing into existing sampling procedures for water utilities, as a sensitive, fast, and reliable monitoring method.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s13036-024-00446-z.

BackgroundDe-novo-designed synthetic transcriptional regulators have great potential as the genetic parts for constructing complex multilayered gene circuits. The design flexibility afforded by advanced nucleic acid sequence design tools vastly expands the repertoire of regulatory elements for circuit design. In principle, the design space of synthetic regulators should allow for the construction of regulatory circuits of arbitrary complexity; still, the orthogonality and robustness of such components have not been fully elucidated, thereby limiting the depth and width of synthetic circuits.ResultsIn this work, we systematically explored the design strategy of synthetic transcriptional regulators, termed switchable transcription terminators. Specifically, by redesigning key sequence domains, we created a high-performance switchable transcription terminator with a maximum fold change of 283.11 upon activation by its cognate input RNA. Further, an automated design algorithm was developed for these elements to improve orthogonality for a complex multi-layered circuit construction. The resulting orthogonal switchable transcription terminators could be used to construct a three-layer cascade circuit and a two-input three-layer OR gate.ConclusionsWe demonstrated a practical strategy for designing standardized regulatory elements and assembling modular gene circuits, ultimately laying the foundation for the streamlined construction of complex synthetic gene circuits.

The production of value-added bio-compounds from rejuvenated sources and their recruitment for healthcare services are paramount objectives in the agenda of white biotechnology. Hereupon, the current study focused on economic production of single cell oils (SCOs) from oleaginous fungi Alternaria sp. (A-OS) and Drechslera sp. (D-OS) using cheese whey waste stream, followed by their evaluation as antibiofilm and anticancer agents, for the first time. As a sole substrate for growth, the whey aided in lipid accumulation by 3.22 and 4.33 g/L, which representing 45.3 and 48.2% lipid content in Drechslera sp. (D-OS) and Alternaria sp. (A-OS), respectively. Meanwhile, a higher unsaturation degree was detected in A-OS by 62.18% comparing to 53.15% of D-OS, with advantageous presence of omega-6 poly unsaturated fatty acid by 22.67% and 15.04% for A-OS and D-OD, respectively, as revealed by GC-MS and FTIR characterization analysis. Interestingly, an eminent and significant (P ≤ 0.05) antibiofilm potency was observed in a dose-dependent modality upon employing both SCOs as antibiofilm agents. Whereas, 100 µg/mL of A-OS recorded superior inhibition of P. aeruginosa, S. aureus and C. albicans biofilms development by 84.10 ± 0.445, 90.37 ± 0.065 and 94.96 ± 0.21%, respectively. Whereas, D-OS (100 µg/mL) thwarted the biofilms of P. aeruginosa, S. aureus and C. albicans by 47.41 ± 2.83, 62.63 ± 5.82 and 78.67 ± 0.23%, correspondingly. Besides, the metabolic performance of cells within biofilm matrix, protein, carbohydrate contents and hydrophobicity of examined biofilms were also curtailed in a significant correlation with biofilm biomass (r ≥ 0.9). Further, as anticancer agents, D-OS recorded higher potency against A549 and CaCo-2 cell lines with IC50 values of 2.55 and 3.425% and SI values of 10.1 and 7.5, respectively. However, A-OS recorded 8.275% and 2.88 for IC50 and SI of Caco-2 cells, respectively. Additionally, A-OS activated caspase 3 by 64.23 ± 1.18% and 53.77 ± 0.995% more than D-OS (52.09 ± 0.222% and 49.72 ± 0.952%) in A549 and Caco-2 cells, respectively. Furthermore, the enzymes, which associated with cancer invasion, metastasis, and angiogenesis (i.e., MMP2 and MMP9) were strongly inhibited by A-OS with 18.58% and 8.295%, respectively as IC50 values; while D-OS results recorded 23.61% and 13.16%, respectively, which could be ascribed to the higher ω-6/ω-3 contents of A-OS. The promising results of the current study opens up the vision to employ SCOs as anti-infective nutraceuticals and in complementary/alternative therapy and prophylactic programs as well.

Type 2 diabetes mellitus (T2DM) is a major public health concern with significant cardiovascular complications (CVD). Despite extensive epidemiological data, the molecular mechanisms relating hyperglycemia to CVD remain incompletely understood. We here investigated the impact of chronic hyperglycemia on human aortic smooth muscle cells (HASMCs) cultured under varying glucose conditions in vitro, mimicking normal (5 mmol/L), pre-diabetic (10 mmol/L), and diabetic (20 mmol/L) conditions, respectively. Normal HASMC cultures served as baseline controls, and patient-derived T2DM-SMCs served as disease controls. Results showed significant increases in cellular proliferation, area, perimeter, and F-actin expression with increasing glucose concentration (p < 0.01), albeit not exceeding the levels in T2DM cells. Atomic force microscopy analysis revealed significant decreases in Young’s moduli, membrane tether forces, membrane tension, and surface adhesion in SMCs at higher glucose levels (p < 0.001), with T2DM-SMCs being the lowest among all the cases (p < 0.001). T2DM-SMCs exhibited elevated levels of selected pro-inflammatory markers (e.g., ILs-6, 8, 23; MCP-1; M-CSF; MMPs-1, 2, 3) compared to glucose-treated SMCs (p < 0.01). Conversely, growth factors (e.g., VEGF-A, PDGF-AA, TGF-β1) were higher in SMCs exposed to high glucose levels but lower in T2DM-SMCs (p < 0.01). Pathway enrichment analysis showed significant increases in the expression of inflammatory cytokine-associated pathways, especially involving IL-10, IL-4 and IL-13 signaling in genes that are up-regulated by elevated glucose levels. Differentially regulated gene analysis showed that compared to SMCs receiving normal glucose, 513 genes were upregulated and 590 genes were downregulated in T2DM-SMCs; fewer genes were differentially expressed in SMCs receiving higher glucose levels. Finally, the altered levels in genes involved in ECM organization, elastic fiber synthesis and formation, laminin interactions, and ECM proteoglycans were identified. Growing literature suggests that phenotypic switching in SMCs lead to arterial wall remodeling (e.g., change in stiffness, calcific deposits formation), with direct implications in the onset of CVD complications. Our results suggest that chronic hyperglycemia is one such factor that leads to morphological, biomechanical, and functional alterations in vascular SMCs, potentially contributing to the pathogenesis of T2DM-associated arterial remodeling. The observed differences in gene expression patterns between in vitro hyperglycemic models and patient-derived T2DM-SMCs highlight the complexity of T2DM pathophysiology and underline the need for further studies.

BackgroundMesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation- relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs.ResultsUsing an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-α /IFN-γ stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism.ConclusionThis study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphometric approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.