Image_3_Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword.TIF

<p>Background: The beta-amyloid peptide (Aβ) involved in Alzheimer’s disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aβ in cultured hippocampal neurons.</p><p>Methods: To decrease or increase the levels of cholesterol in the membrane we used methyl-β-cyclodextrin (MβCD) and MβCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively.</p><p>Results: The results showed that cholesterol removal decreased the macroscopic association of Aβ to neuronal membranes (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.05) and induced a facilitation of the membrane perforation by Aβ with respect to control cells (half-time for maximal charge transferred: control = 7.2 vs. MβCD = 4.4). Under this condition, we found an increase in membrane fluidity (46 ± 3.3% decrease in GP value, p < 0.001). On the contrary, increasing cholesterol levels incremented membrane rigidity (38 ± 2.7% increase in GP value, p < 0.001) and enhanced the association and clustering of Aβ (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.01), but inhibited membrane disruption.</p><p>Conclusion: Our results strongly support the significance of plasma membrane organization in the toxic effects of Aβ in hippocampal neurons, since fluidity can regulate distribution and insertion of the Aβ peptide in the neuronal membrane.</p

Image_1_Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword.TIF

<p>Background: The beta-amyloid peptide (Aβ) involved in Alzheimer’s disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aβ in cultured hippocampal neurons.</p><p>Methods: To decrease or increase the levels of cholesterol in the membrane we used methyl-β-cyclodextrin (MβCD) and MβCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively.</p><p>Results: The results showed that cholesterol removal decreased the macroscopic association of Aβ to neuronal membranes (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.05) and induced a facilitation of the membrane perforation by Aβ with respect to control cells (half-time for maximal charge transferred: control = 7.2 vs. MβCD = 4.4). Under this condition, we found an increase in membrane fluidity (46 ± 3.3% decrease in GP value, p < 0.001). On the contrary, increasing cholesterol levels incremented membrane rigidity (38 ± 2.7% increase in GP value, p < 0.001) and enhanced the association and clustering of Aβ (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.01), but inhibited membrane disruption.</p><p>Conclusion: Our results strongly support the significance of plasma membrane organization in the toxic effects of Aβ in hippocampal neurons, since fluidity can regulate distribution and insertion of the Aβ peptide in the neuronal membrane.</p

DataSheet_1_γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids.docx

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.</p

Table_1_γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids.xlsx

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.</p

Image_2_Effect of Cholesterol on Membrane Fluidity and Association of Aβ Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword.tif

<p>Background: The beta-amyloid peptide (Aβ) involved in Alzheimer’s disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aβ in cultured hippocampal neurons.</p><p>Methods: To decrease or increase the levels of cholesterol in the membrane we used methyl-β-cyclodextrin (MβCD) and MβCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively.</p><p>Results: The results showed that cholesterol removal decreased the macroscopic association of Aβ to neuronal membranes (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.05) and induced a facilitation of the membrane perforation by Aβ with respect to control cells (half-time for maximal charge transferred: control = 7.2 vs. MβCD = 4.4). Under this condition, we found an increase in membrane fluidity (46 ± 3.3% decrease in GP value, p < 0.001). On the contrary, increasing cholesterol levels incremented membrane rigidity (38 ± 2.7% increase in GP value, p < 0.001) and enhanced the association and clustering of Aβ (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.01), but inhibited membrane disruption.</p><p>Conclusion: Our results strongly support the significance of plasma membrane organization in the toxic effects of Aβ in hippocampal neurons, since fluidity can regulate distribution and insertion of the Aβ peptide in the neuronal membrane.</p

Shape-Switching Microrobots for Medical Applications: The Influence of Shape in Drug Delivery and Locomotion

The effect of dynamic shape switching of hydrogel bilayers on the performance of self-folding microrobots is investigated for navigation in body orifices and drug release on demand. Tubular microrobots are fabricated by coupling a thermoresponsive hydrogel nanocomposite with a poly­(ethylene glycol)­diacrylate (PEGDA) layer, to achieve spontaneous and reversible folding from a planar rectangular structure. Graphene oxide (GO) or silica-coated superparamagnetic iron oxide nanoparticles are dispersed in the thermoresponsive hydrogel matrix to provide near-infrared (NIR) light sensitivity or magnetic actuation, respectively. The NIR light-responsive microstructures are fabricated for triggered drug delivery while magnetic nanocomposite-based microrobots are used to analyze the role of shape in locomotion. Experimental analysis and computational simulations of tubular structures show that drug release and motility can be optimized through controlled shape change. These concepts are finally applied to helical microrobots to show a possible way to achieve autonomous behavior

Tumor Selective Silencing Using an RNAi-Conjugated Polymeric Nanopharmaceutical

Small interfering RNA (siRNA) therapeutics have potential advantages over traditional small molecule drugs such as high specificity and the ability to inhibit otherwise “undruggable” targets. However, siRNAs have short plasma half-lives <i>in vivo</i>, can induce a cytokine response, and show poor cellular uptake. Formulating siRNA into nanoparticles offers two advantages: enhanced siRNA stability against nuclease degradation beyond what chemical modification alone can provide; and improved site-specific delivery that takes advantage of the enhanced permeability and retention (EPR) effect. Existing delivery systems generally suffer from poor delivery to tumors. Here we describe the formation and biological activity of polymeric nanopharmaceuticals (PNPs) based on biocompatible and biodegradable poly­(lactic-<i>co</i>-glycolic acid) (PLGA) conjugated to siRNA via an intracellular cleavable disulfide linker (PLGA–siRNA). Additionally, these PNPs contain (1) PLGA conjugated to polyethylene glycol (PEG) for enhanced pharmacokinetics of the nanocarrier; (2) a cation for complexation of siRNA and charge compensation to avoid high negative zeta potential; and (3) neutral poly­(vinyl alcohol) (PVA) to stabilize the PNPs and support the PEG shell to prevent particle aggregation and protein adsorption. The biological data demonstrate that these PNPs achieve prolonged circulation, tumor accumulation that is uniform throughout the tumor, and prolonged tumor-specific knockdown. PNPs employed in this study had no effect on body weight, blood cell count, serum chemistry, or cytokine response at doses >10 times the effective dose. PNPs, therefore, constitute a promising solution for achieving durable siRNA delivery and gene silencing in tumors