Search Results (320)

Background/Objectives: Subunit vaccines composed of purified proteins and adjuvants offer excellent safety, but often generate short-lived immunity due to rapid antigen clearance and limited antigen-presenting cell engagement. Sustained, localized delivery of antigen and adjuvant may improve the magnitude and durability of the immune response without compromising safety. This study evaluated an in-situ polymerizing type I oligomeric collagen (Oligomer) scaffold to localize antigen/adjuvant at the injection site and prolong antigen presentation. Methods: Mice were immunized intramuscularly with ovalbumin (OVA) and CpG oligonucleotide adjuvant delivered alone or co-formulated with Oligomer. Antibody response and inflammation at the injection site were assessed post-booster at early (Day 32) and late (Day 68) time points. Antigen retention and dendritic cell trafficking to draining lymph nodes were evaluated using fluorescently labeled OVA. Results: The Oligomer scaffold retained vaccine antigen at the injection site without eliciting a material-mediated foreign body response. Co-delivery of OVA and CpG within the scaffold enhanced germinal center activity, increased follicular helper T cells and germinal center B cells, and skewed CD4⁺ T cells toward a Th1 phenotype. Humoral responses were greater and more durable, with higher OVA-specific IgG, IgG1, and IgG2a titers and an increased number of bone marrow antibody-secreting cells persisting through Day 68. Antigen-positive dendritic cells, including both resident and migratory subsets, were elevated in draining lymph nodes, indicating enhanced antigen transport. No anti-mouse collagen I antibodies were detected, confirming the maintenance of collagen self-tolerance. Conclusions: The Oligomer delivery platform functioned as a localized, immunotolerant vaccine depot, sustaining antigen availability and immune cell engagement. This spatiotemporal control enhanced germinal center responses and generated a more robust, durable humoral immune response, supporting its potential to improve subunit vaccine efficacy while maintaining an excellent safety profile. Full article

Total internal reflection fluorescence (TIRF) microscopy excites fluorophores within a few hundred nanometers of the sample–substrate interface, enabling high-contrast imaging near the cell membrane. When cultured cells differentiate, the membrane in contact with the coverslip generally acquires basal characteristics, while the opposite membrane develops apical features. Consequently, conventional TIRF microscopy is limited to imaging the basal surface. We developed an immersed-prism TIRF (IP-TIRF) microscope, in which a prism immersed in the culture medium generates TIR at the cell/medium–prism interface, illuminating the apical membrane and reducing cytosolic background. In proof-of-principle experiments, we imaged fluorescent beads and 3xmNeonGreen-tagged intraflagellar transport (IFT) particles in cilia, and compared the performance with confocal microscopy. In cellular regions where both methods can be applied (such as the IFT base pool), on average, IP-TIRF achieved approximately 1.8 times the contrast-to-noise ratio (CNR~31) compared to confocal microscopy. Furthermore, IFT-particle motion was detected in IP-TIRF image sequences and Kymographs of cilia, with adequate spatial resolution. Kymograph analysis revealed an average anterograde IFT velocity of 0.156 ± 0.071 µm/s and an average retrograde velocity of 0.020 ± 0.007 µm/s, approximately one-quarter and one-twentieth, respectively, of the values reported for mammalian primary cilia, which we attribute to acquisition at room temperature rather than physiological conditions. Therefore, these velocity measurements should be regarded as proof-of-principle demonstrations obtained at room temperature, not as validated physiological transport rates. Our IP-TIRF method provides a high-resolution, cost-effective, and broadly accessible approach for imaging the apical membrane in live cells. Full article

Micro- and nanoplastic particles (MNPLs) present in the environment have recently become a potential health hazard factor due to the ability to penetrate living organisms, their organs, and cells. MNPLs interact with and absorb chemicals and elements, including metals, such as iron, copper, and zinc, and transport them into the cells. The cells subsequently respond with the altered gene expression profiles. In this study, we applied freely accessible online bioinformatic tools to draw out the sets of genes modulated by the metal ions and MNPLs. We focused on the gene interactome as revealed by The Comparative Toxicogenomics Database (CTD). To achieve a deeper insight into the biological processes that are potentially modulated, the retrieved CTD lists of genes, whose expression was influenced by MNPLs and metals, were subsequently analyzed using online tools: Metascape and String database. The genes from the revealed networks were arranged into functional clusters, annotated mainly as inflammation and immune system activity, regulation of apoptosis, oxidative stress response, Wingless-related Integration Site (WNT) signaling and ferroptosis. The complexity of the interactions between the gene sets altered by MNPLs and metal ions illustrates their pleiotropic effects on living systems. Full article

Histone deacetylase inhibitors (HDACis) induce the expression of multidrug resistance (MDR) pumps and can even display the MDR phenotype in cell lines. This is the first report to include the profiles of ATP-binding cassette (ABC) transporters in intrinsically expressed HeLa cells as well as those acquired due to a 5 mM valproic acid (VPA) treatment. Expression of ABC transporters related to the MDR phenotype was analyzed by RT-PCR in untreated HeLa cells and HeLa cells treated with 5 mM VPA. The ABCB5 protein was identified in HeLa cells by immunocytochemistry. HeLa cell treatment with 5 mM VPA increased ABCB1 expression and triggered the de novo expression of ABCB5 in mRNA and protein. Despite the expression of ABCB5 and the overexpression of ABCB1, VPA reduced the growth rate by 20%, delayed doubling time by 25%, and decreased the number of living cells per well to 50% after 72 h. Pretreatment with VPA for 24 h followed by cotreatment with doxorubicin (DOX) sensitized HeLa cells to DOX. However, for the de novo expression of ABCB5, HeLa cells did not acquire the MDR phenotype from the 5 mM VPA treatment. The ABCB5 isoform induced by VPA treatment probably lacks MDR activity. Full article

Anesthesia has emerged as a critical strategy for maintaining fish viability during transport, with natural anesthetics gaining increasing attention in recent research. The active ingredients in Laurus nobilis L. have antioxidant effects and reduce cell apoptosis. Studies have shown that they can upregulate expression of Nrf2 in mitochondrial biosynthetic factors. This study aimed to investigate the effects of laurel (Laurus nobilis) essential oil on oxidative stress and apoptosis mechanisms during the live transport of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂). The addition of laurel essential oil during transport activated the Nrf2-Keap1 antioxidant pathway, resulting in up-regulated expression of catalase (cat) and superoxide dismutase (sod) genes. This led to increased enzymatic activity and reduced levels of oxidative stress markers. The mitigation of oxidative stress contributed to physiological stability by downregulating apoptotic gene expression (Bax, Caspase 8), reducing gill and liver tissue damage, and lowering the activity of hepatocyte damage markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Metabolomic analysis revealed several key metabolic pathways affected during transport, with the FoxO signaling pathway demonstrating the most significant impact. Within this pathway, reduced glutamate levels appeared to inhibit apoptosis, while decreased ADP and AMP levels potentially influenced antioxidant capacity. The addition of laurel essential oil to transport water proved beneficial in reducing biochemical markers of stress responses in hybrid grouper during keep live transport. Full article

Intercellular pathways of viral infection in host cells offer advantages, such as efficiency of viral spread and immune surveillance evasion, compared to cell-free viral infection. Therefore, some enveloped viruses present both cell-to-cell and cell-free forms of infection in the host organisms. In this study, we investigated the occurrence of Ebola virus (EBOV) and Marburg virus (MARV) nucleocapsid exchange in vitro between interconnected Huh7 cells using live-cell imaging methods. Moreover, through plasmid transfection methods, we demonstrated that nucleocapsid-like structures (NCLSs) formed with EBOV NP, VP35, VP24, and VP30 proteins can also be transported intercellularly to non-transfected cells through cell-to-cell contact regions in a process involving interaction with the host cell actin cytoskeleton. Our results provide further evidence of cell-to-cell transport as a mechanism of filovirus spread and support the need for further research in this field to develop new intervention methods targeting this transmission pathway. Full article

Digital Twins (DTs) are transforming manufacturing by bridging the physical and digital worlds, enabling real-time insights, predictive analytics, and enhanced decision making. In Industry 4.0, DTs facilitate automation and data integration, while Industry 5.0 emphasizes human-centric, resilient, and sustainable production. However, implementing DTs in robotic metal additive manufacturing (AM) remains challenging because of the complexity of the wire-based laser metal deposition (LMD) process, the need for real-time monitoring, and the demand for advanced defect detection to ensure high-quality prints. This work proposes a structured DT architecture for a robotic wire-based LMD cell, following a standard framework. Three DT implementations were developed. First, a real-time 3D simulation in RoboDK, integrated with a 2D Node-RED dashboard, enabled motion validation and live process monitoring via MQTT (message queuing telemetry transport) telemetry, minimizing toolpath errors and collisions. Second, an Industrial IoT-based system using KUKA iiQoT (Industrial Internet of Things Quality of Things) facilitated predictive maintenance by analyzing motor loads, joint temperatures, and energy consumption, allowing early anomaly detection and reducing unplanned downtime. Third, the Meltio dashboard provided real-time insights into the laser temperature, wire tension, and deposition accuracy, ensuring adaptive control based on live telemetry. Additionally, a prescriptive analytics layer leveraging historical data in FireStore was integrated to optimize the process performance, enabling data-driven decision making. Full article

The transport of intensity equation enables quantitative phase imaging from only two axially displaced intensity images, facilitating the characterization of low-contrast samples like cells and microorganisms. However, the rapid selection of the correct defocused planes, crucial for real-time phase imaging of dynamic events, remains challenging. Additionally, the different images are normally acquired sequentially, further limiting phase-reconstruction speed. Here, we report on a system that addresses these issues and enables user-tuned defocusing with snapshot phase retrieval. Our approach is based on combining multi-color pulsed illumination with acousto-optic defocusing for microsecond-scale chromatic aberration control. By illuminating each plane with a different color and using a color camera, the information to reconstruct a phase map can be gathered in a single acquisition. We detail the fundamentals of our method, characterize its performance, and demonstrate live phase imaging of a freely moving microorganism at speeds of 150 phase reconstructions per second, limited only by the camera’s frame rate. Full article

In eukaryotic cells, vesicle-mediated transport interconnects the endomembrane system. These vesicles are formed by coat proteins via deformation of donor membranes. Here, we constructed a set of fluorescent protein-based markers for major coat protein complexes in the yeast model system, and examined their subcellular localization patterns. Our markers covered COPII, COPI, AP-1, AP-2, AP-3, and retromer complexes. Our live cell imaging demonstrates that COPII puncta were primarily associated with the endoplasmic reticulum (ER), and occasionally with early Golgi. COPI was present on both early Golgi and late Golgi/early endosomes. AP-1 puncta were present on late Golgi/early endosomes. AP-2 was present on plasma membrane (PM)-associated puncta, and around the bud neck. AP-3 puncta were present on late Golgi/early endosomes and on the surface of vacuoles. Retromer was present on the surface of vacuoles, late endosomes, and other perivacuolar puncta. Notably, more than half of AP-1 puncta and AP-3 puncta were not associated with the donor compartments where they are thought to be generated, implying that these were coated transport vesicles. This work provides a convenient tool set for the investigation of vesicular transport in yeast and live cell imaging evidence for the presence of certain coated transport vesicles. Full article

Anthracyclines have been clinically well established in cancer chemotherapy for decades. The main limitations of this drug class are the development of resistance and severe side effects. In the present investigation, we analyzed 30 anthracyclines in a panel of 59 cell lines of the National Cancer Institute, USA. The log₁₀IC₅₀ values varied from −10.49 M (3′-deamino-3′-(4″-(3″-cyano)morpholinyl)-doxorubicin, 1) to −4.93 M (N,N-dibenzyldaunorubicin hydrochloride, 30). Multidrug-resistant NCI-ADR-Res ovarian cancer cells revealed a high degree of resistance to established anthracyclines (between 18-fold to idarubicin (4) and 166-fold to doxorubicin (13) compared to parental, drug-sensitive OVCAR8 cells). The resistant cells displayed only low degrees of resistance (1- to 5-fold) to four other anthracyclines (7, 18, 28, 30) and were even hypersensitive (collaterally sensitive) to two compounds (1, 26). Live cell time-lapse microscopy proved the cross-resistance of the three chosen anthracyclines (4, 7, 9) on sensitive CCRF/CEM and multidrug-resistant CEM/ADR5000 cells. Structure–activity relationships showed that the presence of tertiary amino functions is helpful in avoiding resistance, while primary amines rather increased resistance development. An α-aminonitrile function as in compound 1 was favorable. Investigating the mRNA expression of 49 ATP-binding cassette (ABC) transporter genes showed that ABCB1/MDR1 encoding P-glycoprotein was the most important one for acquired and inherent resistance to anthracyclines. Molecular docking demonstrated that all anthracyclines bound to the same binding domain at the inner efflux channel side of P-glycoprotein with high binding affinities. Kaplan–Meier statistics of RNA sequencing data of more than 8000 tumor biopsies of TCGA database revealed that out of 23 tumor entities high ABCB1 expression was significantly correlated with worse survival times for acute myeloid leukemia, multiple myeloma, and hepatocellular carcinoma patients. This indicates that ABCB1 may serve as a prognostic marker in anthracycline-based chemotherapy regimens in these tumor types and a target for the development of novel anthracycline derivatives. Full article

One of the most significant factors shaping the formation of new urban structures is climate change—including global warming and the associated emerging issues—heatwaves, storms, hurricanes, floods, droughts, fires and others. In recent times, new threats have emerged, including war risks, radiation, pandemics and other potential factors, whose devastating consequences are no less severe than those of climate change. Concerning these and other potential threats, this work aims to develop a new, sustainable urban structure element—a territorial unit or complex to be used in creating a new city planning framework. The formation of this sustainable urban unit or complex is based on three fundamental sustainability principles—social, ecological and economic—the harmonious interaction of which can enable the creation of a safe, healthy and convenient urban environment for living, working and leisure. Such a structural urban complex would consist of a group of neighbourhoods with various building densities, enclosed by public transport streets that integrate the complex into the city’s overall spatial structure. To support the complex’s functioning, a structural element—a green core—is planned at its centre, serving as a space for residents’ recreation, protection from various threats and social interaction. Given that this technical, structural and urban territorial unit, in terms of its autonomous functionality, structure, composition, significance and other characteristics, is identical to a natural cell, it is proposed (based on the principles of bionics) to name this structural urban territorial unit an ‘urban cell’ or ‘urbocell’ for semantic clarity. Full article

Disorders of vesicular trafficking and genetic defects in autophagy play a critical role in the development of metabolic and neurometabolic diseases. These processes govern intracellular transport and lysosomal degradation, thereby maintaining cellular homeostasis. In this article, we present two siblings with a novel homozygous variant in VPS51 (Vacuolar protein sorting 51) gene (c.1511C>T; p.Thr504Met), exhibiting developmental delay, a thin corpus callosum, severe intellectual disability, epilepsy, microcephaly, hearing loss, and dysphagia. This study aimed to investigate the effects of the novel VPS51 gene variation at the RNA and protein level in fibroblasts derived from patients. A comparative proteomic analysis, which has not been previously elucidated, was performed to identify uncharacterized proteins associated with vesicular trafficking. Furthermore, the impact of disrupted pathways on mitochondria–lysosome contact sites was assessed, offering a thorough pathophysiological evaluation of GARP/EARP (Golgi Associated Retrograde Protein / Endosome Associated Retrograde Protein) complex dysfunction. An analysis of mRNA expression indicated decreased levels of the VPS51 gene, alongside modifications in the expression of autophagy-related genes (LC3B, p62, RAB7A, TBC1D15). Western blotting demonstrated a reduction in VPS51 and autophagy-related protein levels. Proteomic profiling revealed 585 differentially expressed proteins, indicating disruptions in vesicular trafficking, lysosomal function, and mitochondrial metabolism. Proteins involved in mitochondrial β-oxidation and oxidative phosphorylation exhibited downregulation, whereas pathways related to glycolysis and lipid synthesis showed upregulation. Live-cell confocal microscopy revealed a notable increase in mitochondria–lysosome contact sites in patient fibroblasts, suggesting that VPS51 protein dysfunction contributes to impaired organelle communication. The findings indicate that the novel VPS51 gene variation influences intracellular transport, autophagy, and metabolic pathways, offering new insights into its involvement in neurometabolic disorders. Full article

Air exposure stress during live transport and subsequent reoxygenation are factors in the development of molecular/pathological and compensatory/adaptive responses. They affect the physiological functions and survival of economically important invertebrate species, in particular, crustaceans. In this study, we consider the effects of anoxia and subsequent reoxygenation on the physiological responses, signaling pathways involved in stress, and cell apoptosis in the central nervous system (CNS) of the horsehair crab, Erimacrus isenbeckii. The results showed that 1 day of air exposure stress and 1 subsequent day of reoxygenation cause the immunoreactivity of tyrosine hydroxylase (TH) and neuropeptide Y (NPY) to change, suggesting that these changes may be associated with adaptive responses, which are presumably employed to avoid oxidative damage and provide the initial mechanism for survival. Caspase-3 immunoreactive neurons increased eight-fold in the brain and 7.2-fold in the VNC after 1 day of reoxygenation, and the TUNEL-positive cell percentage rose from 0% (control) to 8.4% in the brain and from 1.7% (control) to 13% in the VNC. The results of our study provide evidence that anoxia and reoxygenation can activate caspase-3 and facilitate apoptosis in the CNS of crabs. These results provide evidence that even short-term air exposure stress followed by reoxygenation can trigger significant apoptotic cell death in crustacean neural tissue, which is important for developing better live transport practices. Full article

Pollen allergy is rated as a major public health problem, causing significant morbidity and adversely affecting the quality of people’s lives. The airway epithelium serves as the first line of defense in the respiratory system, playing a crucial role in orchestrating immune responses to allergens. In this work, we studied the important transport steps in the major alder pollen allergen Aln g 1 through the human airway epithelium in comparison with those of the birch pollen allergen Bet v 1. Using fluorescence spectroscopy, we showed that both allergens can destroy liposomes with a composition modeling the adult human pulmonary surfactant. Using a polarized Calu-3 monolayer, we showed similar efficiencies of Aln g 1 and Bet v 1 transport through the artificial epithelial barrier. Using qPCR, we showed that Aln g 1 upregulates the expression of IL-33, TSLP, IL-1β, CXCL8 in epithelial cells, playing an important role in the sensitization process. The obtained results may improve our understanding of the primary sensitization mechanisms with the involvement of the PR-10 family of lipid-binding allergens. Full article

Perfusion offers unique benefits to tissue-engineered systems, enhancing oxygen and nutrient transport, which improves tissue formation and growth. In this study, we present a novel and integrated portable perfusion system. Weighing < 10 lbs, the system can maintain continuous flow in a standard incubation environment (37 °C, 5% CO₂), effectively functioning as a portable perfusion and tissue culturing system. To characterize the perfusion system’s flow parameters, we measured the volumetric flow rate across a range of pressures and found that the system could achieve flow velocities between 1.69 to 4.6 $\mathsf{\mu }$m/s, which is similar to in vivo interstitial flow. Computational fluid dynamics revealed fully developed laminar flow within the sample-containing region of the perfusion system, helping ensure even fluid and nutrient distribution. To study the system’s compatibility with live tissues, bioengineered tissue patches were created and perfused. After 24 h of perfusion, no significant difference in cell viability was observed between the perfused samples and static controls, indicating no adverse effects on cell health. Perfusion also facilitated enhanced spatial organization within tissue patches, reducing the inter-spheroids distance. Furthermore, perfusion strengthened the tissue matrix and reduced the degradation rate of the hydrogel scaffold. Complemented by its ability to provide mobile perfusion and incubation, this novel integrated portable perfusion system holds promise for promoting tissue maturation and advancing tissue bioengineering studies. Full article