Search Results (51)

Bone defects from trauma, tumour resection, infection, and degenerative disease remain a major clinical burden, and autografts face limitations of supply and donor-site morbidity. Three-dimensional (3D) printing offers a route to patient-specific, architecturally defined bone scaffolds, while biopolymers from natural sources provide biodegradability, biocompatibility, and extracellular matrix-mimicking cues consistent with sustainable, green biomaterials science. This review synthesises recent progress in 3D printing of biopolymer-based scaffolds for bone tissue engineering. We first examine the principal feedstocks—alginate, gelatin and gelatin methacryloyl, collagen, chitosan, silk fibroin, cellulose, and microbial polyesters—and their preparation, crosslinking chemistry, and printability. We then compare extrusion, light-based, and indirect printing technologies and the process–property relationships governing resolution, mechanical competence, and cell viability. Composite and functionalisation strategies, including biopolymer–bioceramic hybrids and controlled delivery of growth factors and antimicrobial agents, are analysed as routes to osteoinduction, vascularisation, and infection control. Finally, we evaluate translational performance in preclinical models and outline central challenges of vascularisation, mechanical–degradation matching, scalability, and regulatory standardisation. Biopolymer 3D printing is positioned as a ve rsatile, sustainable platform whose clinical maturation depends on integrated material, structural, and biological design. Full article

Single-cell proteomics (SCP) is an exciting new field of study with developments in the areas of sample preparation, instrumentation and informatics. SCP has captured the imagination of biologists and clinicians and the critical interest of both academic and commercial mass-spectrometry groups. Currently (i.e., at the time this manuscript was written), SCP is still difficult and slow relative to competing single-cell technologies. What SCP may lose in relative throughput, it trades for direct analysis of protein and proteoforms, albeit with biases toward those of the highest relative concentration in each cell. These strengths may not make SCP the technology of choice for every study. This perspective is intended to identify current and future biological or clinical areas where SCP has or could have the greatest potential to advance human health and knowledge. I will also discuss applications where SCP would be less impactful than other technologies and where SCP, when mature, could play a true role in clinical diagnostics. Full article

Transmitochondrial cybrid technology is a key approach for elucidating the effects of mitochondrial DNA (mtDNA) mutations in defined nuclear genetic backgrounds and for studying nuclear–mitochondrial interactions. However, its application is limited by the availability of suitable recipient cell lines and by technically demanding enucleation procedures. We report three advances in cybrid technology: (1) enucleation using mitomycin C, a widely used agent for generating feeder layers in stem cell culture, which does not depend on cell attachment and provides a gentler alternative to actinomycin D; (2) selection of cybrids using mitochondrial uncouplers, which can reduce background survival of non-cybrid cells; and (3) cryopreservation of enucleated donor cells in liquid nitrogen, preserving fusion competence and increasing experimental flexibility. Additionally, we validate newly developed mtDNA-free (ρ⁰) derivatives of HCT116, HT1080, and U2OS cell lines as recipients for cybrid generation. These advances facilitated donor cell preparation, improved cybrid selection, and enhanced experimental flexibility, including the demonstration of preserved fusion competence of enucleated HeLa cells after 10 years of cryostorage. The ρ⁰ derivatives of HCT116, HT1080, and U2OS cells were confirmed as effective recipients. Together, these improvements enhance the efficiency and accessibility of transmitochondrial cybrid technology and are expected to facilitate its broader application. Full article

A simple method for the efficient high-throughput transformation of hybrid poplar (Populus tremula x alba clone 717 1B) is described. Factors considered in developing the method included the ease and efficiency of preparing large numbers of explants for transformation, and selection of culture media that enhanced cell and tissue growth while promoting shoot regeneration competence. We found that petiole explants from in vitro-cultured plantlets can be easily collected and prepared for transformation and regenerate shoots comparable to stem or leaf explants. Culturing petiole explants on Driver Kuniyuki Walnut (DKW) medium resulted in significantly greater tissue growth compared to Murashige Skoog (MS) medium. Moreover, the inclusion of low concentrations of thidiazuron (TDZ) in callus-inducing media (CIM) significantly enhanced shoot regeneration competence of cultured petiole explants. As a consequence, the combination of petiole explants cultured on DKW medium along with 2.2 ug/L TDZ during the callus induction phase resulted in rapid and efficient transformation of this hybrid poplar genotype. When applied to genomic approaches such as activation tagging or CRISPR-Cas9 and Cas12a gene editing, we obtained transformation efficiencies ranging between 70% and 90%. The described protocol provides a simple and efficient method that is easily scalable for high-throughput approaches, which could facilitate genome-wide methods for the rapid and efficient production of transformed hybrid poplars. Full article

Background/Objectives: Recombinant adeno-associated virus (rAAV) has become a leading vector in gene therapy. However, manufacturing limitations may result in replication-competent AAV (rcAAV) contamination of clinical rAAV products, posing safety risks. Rigorous testing is therefore essential, and the use of accurately calibrated rcAAV reference standard materials is critical for ensuring assay stability and reliability. A disadvantage of the widely used Tissue Culture Infectious Dose 50 (TCID₅₀) assay is its high variability. This study introduces an optimized TCID₅₀ assay for the precise quantification of infectious rcAAV particles. Methods: We developed a TCID₅₀ assay tailored to rep2-based rcAAV, optimizing key aspects such as viral infection conditions, qPCR reaction systems, and standard curve preparation. We employed an innovative strategy to prepare the standard curve using serial dilutions of rcAAV in cell lysate, ensuring alignment with the test sample matrices. Results: The rcAAV-derived standard curve demonstrated exceptional linearity (R² > 0.99), sensitivity (LOQ ≈ 38 copies), and reproducibility, enabling robust endpoint qPCR analysis. The optimized assay significantly improved the precision of the TCID₅₀ assay, as an inter-assay coefficient of variation (CV) of 11.4% was achieved. Conclusions: This refined TCID₅₀ assay is a reliable method for calibrating infectious titers of rcAAV reference standard materials, thereby enabling the standardization of rcAAV testing. Full article

Objectives: The epidermal growth factor receptor (EGFR) is a clinically relevant membrane receptor that is frequently overexpressed or dysregulated in multiple types of cancer, making it an important target for antibody-based strategies. Nanobodies, derived from camelid heavy-chain antibodies, possess favorable properties such as small size, high stability, and strong antigen-binding capacity. This study aimed to generate EGFR-specific nanobodies and to systematically characterize their binding properties and initial functional activity. Methodology: Bactrian camels were immunized with a whole-cell antigen prepared from 293F cells transiently transfected to express full-length human EGFR. A high-diversity phage display nanobody library was constructed from peripheral blood lymphocytes. After two rounds of biopanning against EGFR, positive clones were screened and selected. The identified nanobodies were recombinantly expressed in Escherichia coli and purified. Binding specificity, epitope relationships, and kinetic parameters were evaluated using high-performance liquid chromatography (HPLC), bio-layer interferometry (Octet), and flow cytometry. The effect of selected nanobodies on EGF-induced cell proliferation was evaluated using a CCK-8 assay. Results: Two EGFR-specific nanobodies, Nb2H4 and Nb2B6, were successfully isolated. Both nanobodies exhibited specific binding to EGFR and recognized distinct, non-competing epitopes. Kinetic analyses revealed favorable binding affinities, and flow cytometry confirmed their ability to recognize EGFR in its native cellular context. In addition, Nb2H4 significantly suppressed EGF-induced proliferation in an EGFR-overexpression cell model, indicating preliminary functional activity. Conclusions: This study reports on the successful generation and in vitro characterization of EGFR-targeting nanobodies based on the extracellular domain of EGFR. The identified nanobodies provide useful molecular tools for epitope mapping, structural studies, and the further exploration of EGFR-directed antibody engineering strategies. Full article

Although blood–brain barrier (BBB) models are of great value in investigating neurological diseases, the structural complexity and intricate function based on cell–cell interactions of the BBB bring various limitations to the applications of existing models. In this study, a novel BBB micro-organoid model was established by culturing neurovascular unit (NVU) cells on a decellularized squid mantle scaffold (DSMS) film to reconstitute a more authentic and reliable NVU microenvironment for in vitro research. The DSMS applied was obtained from squid mantle scaffolds via decellularization, followed by defatting, and showed good biocompatibility with no cytotoxicity. The DSMS film was finally prepared by lyophilization. The lyophilized film exhibited a void ratio and pore size suitable for the adhesion and growth of endothelial cells (hCMEC/D3) and astrocytes (hACs), which led to the formation of a BBB-like spatial structure. The BBB micro-organoid model exhibited functional barrier properties, including an effective transendothelial electrical resistance (TEER) of approximately 230 Ω/cm², restricted permeability to macromolecules—with apparent permeability coefficients (Papp) of 6.3 × 10⁻⁷ cm/s for 10 kDa and 2.7 × 10⁻⁷ cm/s for 70 kDa FITC–dextran—and expression of tight junctional complex (TJC) proteins such as vascular endothelial cadherin (VE-cad) and Zonula Occludens-1 (ZO-1). Furthermore, low-density lipoprotein receptor-related protein 1 (LRP1), a key receptor stably expressed in these two NVU cell types, was utilized as a critical indicator to assess the integrity of the BBB micro-organ model and its responsiveness to pathophysiological stimuli, particularly under thrombotic conditions. This study not only validates the feasibility of constructing a functionally competent BBB micro-organ model using DSMS films integrated with NVU cells but also provides a promising in vitro platform for subsequent studies on the BBB-related pathological mechanisms and the evaluation of drug permeability across the BBB. Full article

Synergetic therapeutic study using multifunctional nanoplatforms has been developed as an innovative modality for effective cancer treatment to improve the clinical efficiency of anticancer drugs and reduce severe off-target side effects. Herein, an artificial nanoplatform (denoted as PPy-RhB-PDA-CD-LA) was prepared by grafting β-cyclodextrin (β-CD) derivatives and lactobionic acid (LA) on the surface of rhodamine B (RhB)-doped polypyrrole nanoparticles (PPy-RhB NPs) using polydopamine (PDA) as the intermediate linker. Doxorubicin (DOX) was selected and successfully loaded onto the nanoplatforms with a high loading content of 327 mg/g. Furthermore, significant NIR light-triggered release of DOX was observed in a weak acidic tumor microenvironment. The nanoplatform exhibited superior photostability with a high photothermal effect of 51.7% under irradiation by a 808 nm laser and a competent temperature sensitivity (SR is 1.44% °C⁻¹) under a single wavelength excitation. MTT assay against SMMC-7721 cells clearly illustrated that the nanoplatform had low cytotoxicity at a high level (200 μg/mL) after 24 h and high therapeutic efficacy of chemo-phototherapy. Thus, it is highly promising for use in biomedical applications. Full article

Background/Objectives: 5-Fluorouracil (5-FU) and Irinotecan (IRT) are two of the most used chemotherapeutic agents in CRC treatment. However, achieving treatment goals has been hampered by poor drug delivery to tumor sites and associated toxicity from off-target binding to healthy cells. Though the synergism of 5-FU-IRT has provided incremental improvements in clinical outcomes, the short elimination half-life and off-target binding to healthy cells remain significant challenges. We postulated that nanoencapsulation of a combination of 5-FU and IRT in niosomes would prolong the drugs’ half-lives, while over-encapsulation lyophilized powder in Targit^® oral capsules would passively the CRC microenvironment and avoid extensive systemic distribution. Methods: Ranges of formulation and process variables were input into design of experiment (DOE Fusion One) software, to generate screening experiments. Niosomes were prepared using the thin-film hydration method and characterized by size, the polydispersity index (PDI), morphology and intrastructure, and drug loading. Blank niosomes ranged in size from 215 nm to 257 nm. Results: After loading with the 5-FU-IRT combination, the niosomes averaged 251 ± 2.20 nm with a mean PDI of 0.293 ± 0.01. The surfactant-to-cholesterol ratio significantly influenced the niosome size and the PDI. The hydrophilic 5-FU exhibited superior loading compared to the lipophilic IRT molecules, which probably competed with other lipophilic niosome components in niosomes’ palisade layers. In vitro dissolution in biorelevant media showed delayed release until lower intestinal region (IRT) or colonic region (5-FU). Conclusions: Thus, co-nanoencapsulation of 5-FU/IRT in niosomes, lyophilization, and over-encapsulation of powder in colon-specific capsules could passively target the CRC cells in the colonic microenvironment. Full article

Ulcerous lesions can arise in primary skin cancers and upon infiltration of the skin by malignant cells originating from other organs. These malignant fungating wounds are difficult to treat, and they cause pain, itching and malodor. Distressing malodor imposes a major burden on patients. The carrion odor of decaying tissue is—at least in part—due to the bacterial breakdown products cadaverine and putrescine. Here, we examined the binding of cadaverine, histamine, putrescine, spermidine and spermine to the preparation of micronized purified clinoptilolite-tuff (PCT) by relying on three radiolabeled tracers ([³H]cadaverine, [³H]histamine and [³H]spermidine). Binding was rapid, stable and of high capacity. The binding affinities were in the low µM range. Displacement experiments indicated that the binding sites were non-equivalent. These three properties combined to support effective binding for any given ligand in the presence of the expected, submillimolar concentrations of competing ligands. This was further verified by measuring the binding of [³H]cadaverine in the presence of wound drainage fluids. [³H]Cadaverine was effectively adsorbed by a wound dressing, into which purified clinoptilolite-tuff had been incorporated: the observed binding capacity of this wound dressing was consistent with its content of purified clinoptilolite-tuff. Based on these findings, we propose that purified clinoptilolite-tuff be further investigated as a means to control malodor emanating from chronic wounds. Full article

Gonadotropins and progestins are the primary regulators of follicle maturation and ovulation in fish, and they require complex communication among the oocyte and somatic cells of the follicle. The major progestin and the maturation-inducing hormone in salmonids is 17α,20β-dihdroxy-4-pregnen-3-one (17,20βP), and traditional nuclear receptors and membrane steroid receptors for the progestin have been identified within the follicle. Herein, RNA-seq was used to conduct a comprehensive survey of changes in gene expression throughout the intact follicle in response to in vitro treatment with these hormones to provide a foundation for understanding the coordination of their actions in regulating follicle maturation and preparation for ovulation. A total of 5292 differentially expressed genes were identified from our transcriptome sequencing datasets comparing four treatments: fresh tissue; untreated control; 17,20βP-treated; and salmon pituitary homogenate-treated follicles. Extensive overlap in affected genes suggests many gonadotropin actions leading to the acquisition of maturational and ovulatory competence are mediated in part by gonadotropin induction of 17,20βP synthesis. KEGG analysis identified signaling pathways, including MAPK, TGFβ, FoxO, and Wnt signaling pathways, among the most significantly enriched pathways altered by 17,20βP treatment, suggesting pervasive influences of 17,20βP on actions of other endocrine and paracrine factors in the follicle complex. Full article

Thinking and reasoning competencies are crucial for the success of future healthcare professionals and are noted as pre-professional competencies for medical school admissions. At Agnes Scott College, our graduate-level Medical Cell Biology class focuses on cellular structure and function in human disease. In this course, students complete assignments meant to foster critical thinking competencies, wherein they analyze primary articles on the cellular pathogenesis of disease and relevant drug therapies. To assess student perspectives on these assignments, we developed a survey to gauge student attitudes toward the effectiveness of these assignments in supporting their learning and preparing them as applicants to various health professions programs. Attitudinal data shows that these assignments have helped students think critically when evaluating scientific literature and bolstered their understanding of cell biology in the progression and treatment of human pathologies, better preparing them for their future careers in the health professions. Full article

Camellia oleifera oil (CO oil) extracted from C. oleifera seeds has a 2300-year consumption history in China. However, there is relatively little research regarding its non-edible uses. This study determined the physicochemical properties of CO oil extracted via direct pressing, identified its main components using GC-MS, and evaluated its antioxidant, moisturizing, and anti-inflammatory activities. The results revealed that CO oil’s acid, peroxide, iodine, and saponification values were 1.06 ± 0.031 mg/g, 0.24 ± 0.01 g/100 g, 65.14 ± 8.22 g/100 g, and 180.41 ± 5.60 mg/g, respectively. CO oil’s tocopherol, polyphenol, and squalene contents were 82.21 ± 9.07 mg/kg, 181.37 ± 3.76 mg/kg, and 53.39 ± 6.58 mg/kg, respectively; its unsaturated fatty acid (UFA) content was 87.44%, and its saturated fatty acid (SFA) content was 12.56%. CO oil also demonstrated excellent moisture retention properties, anti-inflammatory effects, and certain free radical scavenging. A highly stable CO oil emulsion with competent microbiological detection was developed using formulation optimization. Using CO oil in the emulsion significantly improved the formulation’s antioxidant and moisturizing properties compared with those of the emulsion formulation that did not include CO oil. The prepared emulsion was not cytotoxic to cells and could reduce cells’ NO content; therefore, it may have potential nutritional value in medicine and cosmetics. Full article

Extracellular vesicles (EVs) such as exosomes have been shown to play physiological roles in cell-to-cell communication by delivering various proteins and nucleic acids. In addition, several studies revealed that the EVs derived from the cells that are infected with certain viruses could transfer the full-length viral genomes, resulting in EVs-mediated virus propagation. However, the possibility cannot be excluded that the prepared EVs were contaminated with infectious viral particles. In this study, the cells that harbor subgenomic replicon derived from the Japanese encephalitis virus and dengue virus without producing any replication-competent viruses were employed as the EV donor. It was demonstrated that the EVs in the culture supernatants of those cells were able to transfer the replicon genome to other cells of various types. It was also shown that the EVs were incorporated by the recipient cells primarily through macropinocytosis after interaction with CD33 and Tim-1/Tim-4 on HeLa and K562 cells, respectively. Since the methods used in this study are free from contamination with infectious viral particles, it is unequivocally indicated that the flavivirus genome can be transferred by EVs from cell to cell, suggesting that this pathway, in addition to the classical receptor-mediated infection, may play some roles in the viral propagation and pathogenesis. Full article

The introduction of plasmids into Agrobacterium cells is one of the key steps in the Agrobacterium-mediated transformation of plants for gene editing applications. Depending on chromosomal background, some Agrobacterium strains exhibit a very low transformation efficiency, which results in a low number of colonies for subsequent screening and thus limits the potential for automated high-throughput transformation processes, especially with low copy or large plasmids. This study demonstrates improvements of transformation frequency by modifying the competent cell preparation process and optimizing electroporation parameters for two Agrobacterium strains. The competent cell preparation process was modified by prolonging bacterial growth in the log phase and optimizing the endpoint cell density for cell harvest which resulted in a significant cell yield increase and transformation frequency improvement. Optimization of electroporation by fine-tuning the parameters not only resulted in a 30-fold transformation frequency increase but also revealed a strain-dependent requirement for field strength and electric pulse length. To further improve transformation of a recalcitrant strain, different concentrations of dimethyl sulfoxide (DMSO) in recovery medium were examined. The study revealed an important role of DMSO in transformed cell recovery, with 5% DMSO resulting in the highest transformation frequency. The significant improvements in Agrobacterium transformation frequency addressed a critical bottleneck towards establishing a high throughput process. Full article