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Keywords = biophysical phenotyping

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20 pages, 3835 KiB  
Article
Host RhoA Signaling Controls Filamentous vs. Spherical Morphogenesis and Cell-to-Cell Spread of RSV via Lipid Raft Localization: Host-Directed Antiviral Target
by Manoj K. Pastey, Lewis H. McCurdy and Barney S. Graham
Microorganisms 2025, 13(7), 1599; https://doi.org/10.3390/microorganisms13071599 - 7 Jul 2025
Viewed by 351
Abstract
Respiratory syncytial virus (RSV) is a major human respiratory pathogen, particularly affecting infants, the elderly, and immunocompromised individuals. RSV exists in both spherical and filamentous forms, with the filamentous morphology associated with enhanced infectivity and cell-to-cell spread. Here, we demonstrate that RhoA, a [...] Read more.
Respiratory syncytial virus (RSV) is a major human respiratory pathogen, particularly affecting infants, the elderly, and immunocompromised individuals. RSV exists in both spherical and filamentous forms, with the filamentous morphology associated with enhanced infectivity and cell-to-cell spread. Here, we demonstrate that RhoA, a small GTPase involved in cytoskeletal regulation, is essential for filamentous RSV morphogenesis through its role in organizing lipid raft microdomains. Rhosin, a selective RhoA inhibitor developed through structure-guided screening, disrupts GEF–RhoA interactions to block RhoA activation. The pharmacological inhibition of RhoA with Rhosin significantly reduced filamentous virion formation, disrupted RSV fusion (F) protein colocalization with lipid rafts, and diminished cell-to-cell fusion, without affecting overall viral replication. Scanning electron microscopy revealed that Rhosin-treated infected HEp-2 cells exhibited fewer and shorter filamentous projections compared to the extensive filament formation seen in untreated cells. β-galactosidase-based fusion assays confirmed that reduced filamentation corresponded with decreased cell-to-cell fusion. The biophysical separation of RSV spherical and filamentous particles by sucrose gradient velocity sedimentation, coupled with fluorescence and transmission electron microscopy, showed that Rhosin treatment shifted virion morphology toward spherical forms. This suggests that RhoA activity is critical for filamentous virion assembly, which may enhance viral spread. Immunofluorescence microscopy using lipid raft-selective dyes (DiIC16) and fusion protein-specific antibodies revealed the strong co-localization of RSV proteins with lipid rafts. Importantly, the pharmacological inhibition of RhoA with Rhosin disrupted F protein partitioning into raft domains, underscoring the requirement for intact lipid rafts in assembly. These findings highlight a novel role for host RhoA signaling in regulating viral assembly through raft microdomain organization, offering a potential target for host-directed antiviral intervention aimed at altering RSV structural phenotypes and limiting pathogenesis. Full article
(This article belongs to the Special Issue Viral Diseases: Current Research and Future Directions)
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25 pages, 2451 KiB  
Article
Age-Related Increases in PDE11A4 Protein Expression Trigger Liquid–Liquid Phase Separation (LLPS) of the Enzyme That Can Be Reversed by PDE11A4 Small Molecule Inhibitors
by Elvis Amurrio, Janvi H. Patel, Marie Danaher, Madison Goodwin, Porschderek Kargbo, Eliska Klimentova, Sonia Lin and Michy P. Kelly
Cells 2025, 14(12), 897; https://doi.org/10.3390/cells14120897 - 13 Jun 2025
Viewed by 997
Abstract
PDE11A is a little-studied phosphodiesterase sub-family that breaks down cAMP/cGMP, with the PDE11A4 isoform enriched in the memory-related hippocampal formation. Age-related increases in PDE11A expression occur in human and rodent hippocampus and cause age-related cognitive decline of social memories. Interestingly, age-related increases in [...] Read more.
PDE11A is a little-studied phosphodiesterase sub-family that breaks down cAMP/cGMP, with the PDE11A4 isoform enriched in the memory-related hippocampal formation. Age-related increases in PDE11A expression occur in human and rodent hippocampus and cause age-related cognitive decline of social memories. Interestingly, age-related increases in PDE11A4 protein ectopically accumulate in spherical clusters that group together in the brain to form linear filamentous patterns termed “PDE11A4 ghost axons”. The biophysical/physiochemical mechanisms underlying this age-related clustering are not known. Here, we determine if age-related clustering of PDE11A4 reflects liquid–liquid phase separation (LLPS; biomolecular condensation), and if PDE11A inhibitors can reverse this LLPS. We show human and mouse PDE11A4 exhibit several LLPS-promoting sequence features, including intrinsically disordered regions, non-covalent pi–pi interactions, and prion-like domains that were particularly enriched in the N-terminal regulatory region. Further, multiple bioinformatic tools predict PDE11A4 undergoes LLPS. Consistent with these predictions, aging-like PDE11A4 clusters in HT22 hippocampal neuronal cells were membraneless spherical droplets that progressively fuse over time in a concentration-dependent manner. Deletion of the N-terminal intrinsically disordered region prevented PDE11A4 LLPS despite equal protein expression between WT and mutant constructs. 1,6-hexanediol, along with tadalafil and BC11-38 that inhibit PDE11A4, reversed PDE11A4 LLPS in HT22 hippocampal neuronal cells. Interestingly, PDE11A4 inhibitors reverse PDE11A4 LLPS independently of increasing cAMP/cGMP levels via catalytic inhibition. Importantly, orally dosed tadalafil reduced PDE11A4 ghost axons in old mouse ventral hippocampus by 50%. Thus, PDE11A4 exhibits the four defining criteria of LLPS, and PDE11A inhibitors reverse this age-related phenotype both in vitro and in vivo. Full article
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14 pages, 1916 KiB  
Article
Clinical Characteristics and Genetic Variants in Children with PAX2 Mutation-Associated Disorders
by Yanyan Jin, Na Li, Zipei Chen, Ke Zeng, Jingjing Wang, Aiqin Sheng, Haidong Fu, Lidan Hu and Jianhua Mao
Medicina 2025, 61(6), 959; https://doi.org/10.3390/medicina61060959 - 22 May 2025
Viewed by 469
Abstract
Background and Objectives: PAX2 serves as a critical transcription factor integral to the process of embryogenesis. Variations in the PAX2 gene could result in the aberrant development of numerous organs. Despite the identification of numerous mutations within the PAX2 gene, the correlation between [...] Read more.
Background and Objectives: PAX2 serves as a critical transcription factor integral to the process of embryogenesis. Variations in the PAX2 gene could result in the aberrant development of numerous organs. Despite the identification of numerous mutations within the PAX2 gene, the correlation between specific genotypes has yet to be fully clarified. The objective of this study was to examine the clinical phenotypes and genotypes associated with PAX2 mutation-induced disorders in pediatric patients of Chinese descent. The aim of our study was to forecast the pathogenic potential of these genetic mutations and to ascertain possible correlations between genotypic variations and the clinical manifestations of disorders linked to PAX2 mutations. Materials and Methods: We recruited 14 pediatric subjects with PAX2 mutations, meticulously examining the clinical characteristics and genetic alterations present in these individuals. Computational techniques were utilized to evaluate the pathogenicity, stability, and biophysical characteristics. A range of computational tools were employed for this assessment, including PredictSNP, MAGPIE, iStable, Align GVGD, ConSurf, and SNP effect. Results: The age at onset ranged from prenatal to 12 years. Five patients progressed to end-stage renal disease. Proteinuria and bilateral renal hypoplasia were observed in 92% of cases. Ocular and auditory abnormalities were also noted. We identified eleven different PAX2 mutations, including five novel variants not previously reported in the literature. We predicted that all mutations, with the exception of p.F27-L33 del and N188S, exhibited high pathogenicity scores. In particular, R117P and R140W are strongly associated with disease pathogenicity and are likely to cause more significant damage than other gene mutants. Conclusions: This study expands the mutational and phenotypic spectrum of PAX2-related disorders in the pediatric population. The identification of five novel variants enhances our understanding of the genetic basis of these conditions. Despite recurrent mutations, marked phenotypic heterogeneity persists, underscoring the need for further research. Full article
(This article belongs to the Section Pediatrics)
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21 pages, 3690 KiB  
Article
In-Season Predictions Using Chlorophyll a Fluorescence for Selecting Agronomic Traits in Maize
by Andrija Brkić, Sonja Vila, Domagoj Šimić, Antun Jambrović, Zvonimir Zdunić, Miroslav Salaić, Josip Brkić, Mirna Volenik and Vlatko Galić
Plants 2025, 14(8), 1216; https://doi.org/10.3390/plants14081216 - 15 Apr 2025
Viewed by 550
Abstract
Traditional maize (Zea mays L.) breeding approaches use directly measured phenotypic performance to make decisions for the next generation of crosses. Indirect assessment of cultivar performance can be utilized using various methods such as genomic predictions and remote sensing. However, some secondary [...] Read more.
Traditional maize (Zea mays L.) breeding approaches use directly measured phenotypic performance to make decisions for the next generation of crosses. Indirect assessment of cultivar performance can be utilized using various methods such as genomic predictions and remote sensing. However, some secondary traits might expand the breeder’s ability to make informed decisions within a single season, facilitating an increase in breeding speed. We hypothesized that assessment of photosynthetic performance with chlorophyll a fluorescence (ChlF) might be efficient for in-season predictions of yield and grain moisture. The experiment was set with 16 maize hybrids over three consecutive years (2017–2019). ChlF was measured on dark-adapted leaves in the morning during anthesis. Partial least squares models were fitted and the efficiency of indirect selection was assessed. The results showed variability in the traits used in this study. Genetic correlations among all traits were mainly very weak and negative. Heritability estimates for all traits were moderately high to high. The model with 10 latent variables showed a higher predictive ability for grain yield (GY) than other models. The efficiency of the indirect selection for GY using biophysical parameters was lower than direct selection efficiency, while the indirect selection efficiency for grain moisture using biophysical parameters was relatively high. The results of this study highlight the significance and applicability of the ChlF transients in maize breeding programs. Full article
(This article belongs to the Section Plant Genetics, Genomics and Biotechnology)
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23 pages, 8139 KiB  
Article
Efficient Fabrication of Human Corneal Stromal Cell Spheroids and Promoting Cell Stemness Based on 3D-Printed Derived PDMS Microwell Platform
by Yuexi Chen, Jianing Gu, Zekai Cui, Xihao Sun, Yuqin Liang, Chunwen Duan, Xiaoxue Li, Zhanyu Su, Bo Zhang, Jiansu Chen and Zheng Wang
Biomolecules 2025, 15(3), 438; https://doi.org/10.3390/biom15030438 - 19 Mar 2025
Cited by 1 | Viewed by 778
Abstract
Spherical culture could promote the plasticity and stemness of human corneal stromal cells (hCSCs). Here, we introduce a novel three-dimensional (3D) cell culture system based on a polydimethylsiloxane (PDMS) microwell platform composed of many V-bottom microcavities to generate human corneal stromal cell spheroids [...] Read more.
Spherical culture could promote the plasticity and stemness of human corneal stromal cells (hCSCs). Here, we introduce a novel three-dimensional (3D) cell culture system based on a polydimethylsiloxane (PDMS) microwell platform composed of many V-bottom microcavities to generate human corneal stromal cell spheroids and promote cell stemness. We isolated hCSCs from SMILE-derived lenticules and maintained their physiological phenotype by culturing them in a medium supplemented with human corneal stromal extract (hCSE). Utilizing a PDMS microwell platform fabricated through 3D printing technology, we successfully generated 3D corneal stromal cell spheroids (3D-CSC) with uniform size and stable structure, exhibiting increased expression of pluripotency factors, including OCT4, NANOG, SOX2, KLF4, and PAX6. Furthermore, the iPS supernatant of E8-conditioned medium (E8-CM) significantly enhanced the stemness properties of these cells. RNA sequencing and proteomics analyses revealed that 3D-CSCs exhibited superior proliferation, differentiation, cell adhesion, migration, and neurogenesis compared to traditional monolayer cultures, underscoring the role of biophysical cues in promoting hCSCs stemness. In summary, this study presents an effective 3D cell culture platform that mimics the in vivo microenvironment, facilitating the enhancement of stemness properties and providing valuable insights into corneal tissue engineering and regenerative medicine, particularly for treating corneal opacities and diseases. Full article
(This article belongs to the Section Cellular Biochemistry)
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23 pages, 25297 KiB  
Article
Bacterial Adhesion on Soft Surfaces: The Dual Role of Substrate Stiffness and Bacterial Growth Stage
by René Riedel, Garima Rani and Anupam Sengupta
Microorganisms 2025, 13(3), 637; https://doi.org/10.3390/microorganisms13030637 - 11 Mar 2025
Cited by 1 | Viewed by 1717
Abstract
The surface adhesion and stiffness of underlying substrates mediate the geometry, mechanics, and self-organization of expanding bacterial colonies. Recent studies have qualitatively indicted that stiffness may impact bacterial attachment and accumulation, yet the variation in the cell-to-surface adhesion with substrate stiffness remains to [...] Read more.
The surface adhesion and stiffness of underlying substrates mediate the geometry, mechanics, and self-organization of expanding bacterial colonies. Recent studies have qualitatively indicted that stiffness may impact bacterial attachment and accumulation, yet the variation in the cell-to-surface adhesion with substrate stiffness remains to be quantified. Here, by developing a cell-level force–distance spectroscopy (FDS) technique based on atomic force microscopy (AFM), we simultaneously quantify the cell–surface adhesion and stiffness of the underlying substrates to reveal the stiffness-dependent adhesion of the phototrophic bacterium Chromatium okenii. As the stiffness of the soft substrate, modeled using a low-melting-point (LMP) agarose pad, was varied between 20 kPa and 120 kPa by changing the agarose concentrations, we observed a progressive increase in the mean adhesion force by over an order of magnitude, from 0.21±0.10 nN to 2.42±1.16 nN. In contrast, passive polystyrene (PS) microparticles of comparable dimensions showed no perceptible change in their surface adhesion, confirming that the stiffness-dependent adhesive interaction of C. okenii is of a biological origin. Furthermore, for Escherichia coli, the cell–surface adhesion varied between 0.29±0.17 nN and 0.39±0.20 nN, showing a weak dependence on the substrate stiffness, thus suggesting that stiffness-modulated adhesion is a species-specific trait. Finally, by quantifying the adhesion of the C. okenii population across different timescales, we reported the emergent co-existence of weak and strongly adherent sub-populations, demonstrating diversification of the adherent phenotypes over the growth stages. Taken together, these findings suggest that bacteria, depending on the species and their physiological stage, may actively modulate cell-to-surface adhesion in response to the stiffness of soft surfaces. While the surface properties, for instance, hydrophobicity (or hydrophilicity), play a key role in mediating bacterial attachment, this work introduces substrate stiffness as a biophysical parameter that could reinforce or suppress effective surface interactions. Our results suggest how bacteria could leverage stiffness-dependent adhesion and the diversity therein as functional traits to modulate their initial attachment to, colonization of, and proliferation on soft substrates during the early stages of biofilm development. Full article
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16 pages, 3074 KiB  
Article
Breast Cancer Tissues and Organoids BioBank: Constitution, Research Activities and Samples Access
by Lucia Miranda, Luigi Mandrich, Simona Massa, Teresa Nutile, Clotilde Crovella, Ilaria De Rosa, Raffaella Lucci, Filippo De Rosa, Pasquale Somma, Vincenzo Mercadante, Ciro Abate, Salvatore Arbucci, Luigi Panico and Emilia Caputo
Organoids 2025, 4(1), 5; https://doi.org/10.3390/organoids4010005 - 3 Mar 2025
Viewed by 1066
Abstract
In 2023, at the Center for Biological Resources (CRB) at the Institute of Genetics and Biophysics (IGB, Naples, Italy) of the National Research Council (CNR), the Breast Cancer Tissues and Organoids Biobank (BCTO BioBank) was founded. This is a new generation Biobank, dedicated [...] Read more.
In 2023, at the Center for Biological Resources (CRB) at the Institute of Genetics and Biophysics (IGB, Naples, Italy) of the National Research Council (CNR), the Breast Cancer Tissues and Organoids Biobank (BCTO BioBank) was founded. This is a new generation Biobank, dedicated to the collection, characterization, storage, and distribution of tissues and their 3D ‘organoid’ patients-derived. Tumor and healthy tissues from breast cancer patients have been collected from surgeons at Monaldi Hospital (Naples, Italy) and used to generate the corresponding tumor and healthy organoids from the same patient. After their establishment in culture, both organoids were characterized for their receptor status on a microfluidic 2-lane OrganoPlate, by immunofluorescence. The resulting data were compared with the expression profile obtained by immunohistochemistry on respective parental tissues. These data allowed us to phenotypically validate the generated organoids and classify them in a dedicated database, where also the clinical data of the corresponding patients were collected. During the six months of activities, we collected and characterized 27 samples. The continuous BCTO BioBank activity is fundamental to generating a high number of samples, for a broader and efficiently elaborated patient stratification at molecular level, biomarker discovery investigations, and for tailored treatment protocols design. Full article
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15 pages, 2842 KiB  
Article
NanoBioAnalytical (NBA) Platform to Decipher Extracellular Vesicles Secreted by Microvascular Endothelial Cells Under Benzo[a]pyrene Exposure
by Geetika Raizada, Joan Guillouzouic, Alain Rouleau, Eric Lesniewska, Eric Le Ferrec, Céline Elie-Caille and Wilfrid Boireau
Biosensors 2025, 15(2), 103; https://doi.org/10.3390/bios15020103 - 11 Feb 2025
Viewed by 1119
Abstract
Recent advances in the clinical extracellular vesicles (EVs) field highlight their potential as biomarkers for diverse diseases and therapeutic applications. This study provides an in-depth characterization of 10k EVs from human microvascular endothelial cells (HMEC-1) exposed to benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon [...] Read more.
Recent advances in the clinical extracellular vesicles (EVs) field highlight their potential as biomarkers for diverse diseases and therapeutic applications. This study provides an in-depth characterization of 10k EVs from human microvascular endothelial cells (HMEC-1) exposed to benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon found in food and smoke. Given EVs’ complexity, with numerous surface and cargo proteins, phenotyping remains challenging. Here, we introduce a multiplex biosensor, in µarray format, for profiling EVs from distinct cellular conditions, employing a multimodal approach that combines surface plasmon resonance imaging (SPRi) and in situ atomic force microscopy (AFM) to decipher EVs’ biochemical and biophysical properties. SPRi experiments showed notable EV capture differences on ligands such as Anti-CD36, Anti-CD81, and Anti-ApoA between treated and control conditions, likely due to B[a]P exposure. A complementary AFM study and statistical analyses revealed size differences between EVs from treated and control samples, with ligands like Annexin-V, Anti-CD36, and Anti-VEGFR1 emerging as ligands specific to potential cytotoxicity biomarkers. Our findings suggest that B[a]P exposure may increase EV size and alter marker expression, indicating phenotypic shifts in EVs under cytotoxic stress. The original combination of SPRi and AFM reveals valuable data on the phenotypical and morphological heterogeneities of EV subsets linked to cytotoxic stresses and highlights the potential of EVs as specific toxicological markers. Full article
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21 pages, 6651 KiB  
Article
Electrospun Aligned Gelatin/Chitosan Nanofibrous Membranes for a Better Culture of Mesothelial Cells
by Hao-Hsi Kao, Darshan Tagadur Govindaraju, Banendu Sunder Dash and Jyh-Ping Chen
J. Compos. Sci. 2025, 9(1), 31; https://doi.org/10.3390/jcs9010031 - 10 Jan 2025
Viewed by 1264
Abstract
The delivery of mesothelial cells by nanofibrous membranes (NFMs) can repair a damaged peritoneal mesothelium and enhance peritoneal healing in patients with chronic renal failure. On the other hand, the orientation of the nanofibers in NFMs may affect cell attachment, proliferation, and the [...] Read more.
The delivery of mesothelial cells by nanofibrous membranes (NFMs) can repair a damaged peritoneal mesothelium and enhance peritoneal healing in patients with chronic renal failure. On the other hand, the orientation of the nanofibers in NFMs may affect cell attachment, proliferation, and the phenotype of mesothelial cells in the nanostructured scaffold. We prepare composite gelatin/chitosan NFMs with aligned or random fiber orientations by electrospinning. We cross-link the nanofibers to maintain the fiber orientation during in vitro cell culture. We then study the cellular response of attached mesothelial cells to fiber orientation in the scaffold. From in vitro cell culture with rat mesothelial cells, the prepared NFMs show high biocompatibility to support cellular growth, regardless of fiber orientation. However, the alignment of electrospun nanofibers in a well-defined geometry can promote cell adhesion and proliferation rates with directional cell organization. The anisotropic arrangement of mesothelial cells in the aligned NFM also coincides with the phenotypic maintenance of the attached mesothelial cells, with biophysical cues provided by the aligned nanofibers. The aligned NFMs may find applications in tissue engineering of a damaged mesothelium layer or in other regenerative therapies where cellular alignment is critical for neo-tissue regeneration. Full article
(This article belongs to the Special Issue Feature Papers in Journal of Composites Science in 2024)
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17 pages, 4231 KiB  
Article
A Spike-Based mRNA Vaccine Encapsulated in Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine Containing Lipid Nanoparticles Induced Potent B- and T-Cell Responses Associated with Protection Against SARS-CoV-2 Infection and COVID-19-like Symptoms in Hamsters
by Afshana Quadiri, Swayam Prakash, Latifa Zayou, Nisha Rajeswari Dhanushkodi, Amruth Chilukuri, Gemma Ryan, Kelly Wang, Hawa Vahed, Aziz A. Chentoufi and Lbachir BenMohamed
Vaccines 2025, 13(1), 47; https://doi.org/10.3390/vaccines13010047 - 8 Jan 2025
Cited by 1 | Viewed by 2433
Abstract
Background: Nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) have emerged as a promising vaccine strategy, especially for COVID-19. While the LNPs protect mRNA from degradation and efficiently deliver the mRNA to antigen-presenting cells, the effect of lipid composition on the immunogenicity and protective [...] Read more.
Background: Nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) have emerged as a promising vaccine strategy, especially for COVID-19. While the LNPs protect mRNA from degradation and efficiently deliver the mRNA to antigen-presenting cells, the effect of lipid composition on the immunogenicity and protective efficacy of mRNA/LNP vaccines is not well characterized. Studies on using the mRNA/LNP platform for vaccines have largely focused on the nucleic acid cargo with less attention paid to the LNP vehicle. Whether the composition and biophysical properties of LNPs impact vaccine performance remains to be fully elucidated. Methods: In the present study, we used SARS-CoV-2 Spike-mRNA as a prototype vaccine to study the effect of four different LNPs with various lipid compositions. Results: We demonstrate that when the same Spike-mRNA was delivered in the LNP4 formulation based on phospholipid 1,2-dioleoyl-sn-glycero-3-Phosphoethanolamine, it outperformed other LNPs (LNP1, LNP2, and LNP3) that are based on different lipids. Compared to the other three LNPs, LNP4 (i) enhanced the phenotypic and functional maturation of dendritic cells; (ii) induced strong T-cell responses; (iii) increased the secretion of proinflammatory cytokines and pro-follicular T helper (Tfh) cell cytokines; (iv) induced higher neutralization IgG titers; and (v) provided better protection against SARS-CoV-2 infection and COVID-19-like symptoms in the hamster model. Furthermore, we compared LNP-4 with the commercially available LNPs and found it to provide better T-cell immunity against COVID-19 in hamsters. Conclusion: This study suggests mRNA vaccines encapsulated in Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine containing LNPs induced Potent B- and T cell immunity. The mechanisms by which Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine-based LNPs may activate protective B and T cells are discussed. Full article
(This article belongs to the Special Issue Role of Next Generation Vaccines in Immunotherapeutics)
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19 pages, 6330 KiB  
Article
Characterisation of Castration-Resistant Cell-Derived Exosomes and Their Effect on the Metastatic Phenotype
by Jorge Recio-Aldavero, Lorena Parra-Gutiérrez, Laura Muñoz-Moreno, Irene D. Román, María Isabel Arenas and Ana M. Bajo
Cancers 2025, 17(1), 141; https://doi.org/10.3390/cancers17010141 - 4 Jan 2025
Viewed by 1298
Abstract
Background/Objectives: Prostate cancer (PCa) is characterised by its progression to a metastatic and castration-resistant phase. Prostate tumour cells release small extracellular vesicles or exosomes which are taken up by target cells and can potentially facilitate tumour growth and metastasis. The present work studies [...] Read more.
Background/Objectives: Prostate cancer (PCa) is characterised by its progression to a metastatic and castration-resistant phase. Prostate tumour cells release small extracellular vesicles or exosomes which are taken up by target cells and can potentially facilitate tumour growth and metastasis. The present work studies the effect of exosomes from cell lines that are representative of the different stages of the disease on the tumoral phenotype of PC3 cells. Methods: Exosomes were isolated by ultracentrifugation from human prostate epithelial cells (RWPE-1) and androgen-dependent PCa cells (LNCaP) and castration-resistant PCa cells (CRPC) with moderate (DU145) or high (PC3) metastatic capacity. The biophysical and biochemical properties of the exosomes were characterised as well as their effects on PC3 cell viability and migration. Results: The study of the exosomes of prostate cell lines shows heterogeneity in their size, presenting in some of them two types of populations; in both populations, a larger size in those derived from PC3 cells and a smaller size in those derived from non-tumourigenic prostate cells were detected. Differences were found in the physical properties of those derived from healthy and PCa cells, as well as between cells representative of the most aggressive stages of the disease. The highest gamma-glutamyl transferase (GGT) activity was observed in androgen-dependent cells and differences in the pro-metalloproteinases (MMP) activity were detected in healthy cells and in castration-resistant cells with moderate metastatic capacity with respect to PC3 cells. The treatment of PC3 cells with their own exosomes increased PC3 cell viability and migration. Conclusion: Exosomes represent a promising field of research in the diagnosis, prognosis, and treatment of prostate cancer. Full article
(This article belongs to the Special Issue Exosomes in Cancer Metastasis)
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22 pages, 5227 KiB  
Article
BDNF Differentially Affects Low- and High-Frequency Neurons in a Primary Nucleus of the Chicken Auditory Brainstem
by Kristine McLellan, Sima Sabbagh, Momoko Takahashi, Hui Hong, Yuan Wang and Jason Tait Sanchez
Biology 2024, 13(11), 877; https://doi.org/10.3390/biology13110877 - 29 Oct 2024
Viewed by 1558
Abstract
Neurotrophins are proteins that mediate neuronal development using spatiotemporal signaling gradients. The chicken nucleus magnocellularis (NM), an analogous structure to the mammalian anteroventral cochlear nucleus, provides a model system in which signaling between the brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B [...] Read more.
Neurotrophins are proteins that mediate neuronal development using spatiotemporal signaling gradients. The chicken nucleus magnocellularis (NM), an analogous structure to the mammalian anteroventral cochlear nucleus, provides a model system in which signaling between the brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) is temporally regulated. In the NM, TrkB expression is high early in development (embryonic [E] day 9) and is downregulated until maturity (E18–21). It is currently unknown how BDNF–TrkB signaling affects neuronal properties throughout development and across a spatial (i.e., frequency) axis. To investigate this, we exogenously applied BDNF onto NM neurons ex vivo and studied intrinsic properties using whole-cell patch clamp electrophysiology. Early in development (E13), when TrkB expression is detectable with immunohistochemistry, BDNF application slowed the firing of high-frequency NM neurons, resembling an immature phenotype. Current measurements and biophysical modeling revealed that this was mediated by a decreased conductance of the voltage-dependent potassium channels. Interestingly, this effect was seen only in high-frequency neurons and not in low-frequency neurons. BDNF–TrkB signaling induced minimal changes in late-developing NM neurons (E20–21) of high and low frequencies. Our results indicate that normal developmental downregulation of BDNF–TrkB signaling promotes neuronal maturation tonotopically in the auditory brainstem, encouraging the appropriate development of neuronal properties. Full article
(This article belongs to the Special Issue Roles and Functions of Neurotrophins and Their Receptors in the Brain)
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24 pages, 3367 KiB  
Article
Mechanobiological Strategies to Enhance Ovine (Ovis aries) Adipose-Derived Stem Cells Tendon Plasticity for Regenerative Medicine and Tissue Engineering Applications
by Arlette A. Haidar-Montes, Annunziata Mauro, Mohammad El Khatib, Giuseppe Prencipe, Laura Pierdomenico, Umberto Tosi, Guy Wouters, Adrián Cerveró-Varona, Paolo Berardinelli, Valentina Russo and Barbara Barboni
Animals 2024, 14(15), 2233; https://doi.org/10.3390/ani14152233 - 31 Jul 2024
Viewed by 1904
Abstract
Adipose-derived stem cells (ADSCs) hold promise for tendon repair, even if their tenogenic plasticity and underlying mechanisms remain only partially understood, particularly in cells derived from the ovine animal model. This study aimed to characterize oADSCs during in vitro expansion to validate their [...] Read more.
Adipose-derived stem cells (ADSCs) hold promise for tendon repair, even if their tenogenic plasticity and underlying mechanisms remain only partially understood, particularly in cells derived from the ovine animal model. This study aimed to characterize oADSCs during in vitro expansion to validate their phenotypic properties pre-transplantation. Moreover, their tenogenic potential was assessed using two in vitro-validated approaches: (1) teno-inductive conditioned media (CM) derived from a co-culture between ovine amniotic stem cells and fetal tendon explants, and (2) short- (48 h) and long-term (14 days) seeding on highly aligned PLGA (ha-PLGA) electrospun scaffold. Our findings indicate that oADSCs can be expanded without senescence and can maintain the expression of stemness (Sox2, Oct4, Nanog) and mesenchymal (CD29, CD166, CD44, CD90) markers while remaining negative for hematopoietic (CD31, CD45) and MHC-II antigens. Of note, oADSCs’ tendon differentiation potential greatly depended on the in vitro strategy. oADSCs exposed to CM significantly upregulated tendon-related genes (COL1, TNMD, THBS4) but failed to accumulate TNMD protein at 14 days of culture. Conversely, oADSCs seeded on ha-PLGA fleeces quickly upregulated the tendon-related genes (48 h) and in 14 days accumulated high levels of the TNMD protein into the cytoplasm of ADSCs, displaying a tenocyte-like morphology. This mechano-sensing cellular response involved a complete SOX9 downregulation accompanied by YAP activation, highlighting the efficacy of biophysical stimuli in promoting tenogenic differentiation. These findings underscore oADSCs’ long-term self-renewal and tendon differentiative potential, thus opening their use in a preclinical setting to develop innovative stem cell-based and tissue engineering protocols for tendon regeneration, applied to the veterinary field. Full article
(This article belongs to the Section Veterinary Clinical Studies)
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14 pages, 3760 KiB  
Article
The Biallelic Inheritance of Two Novel SCN1A Variants Results in Developmental and Epileptic Encephalopathy Responsive to Levetiracetam
by Giorgia Dinoi, Elena Conte, Orazio Palumbo, Mario Benvenuto, Maria Antonietta Coppola, Pietro Palumbo, Patrizia Lastella, Brigida Boccanegra, Ester Di Muro, Marco Castori, Massimo Carella, Vittorio Sciruicchio, Marina de Tommaso, Antonella Liantonio, Annamaria De Luca, Angela La Neve and Paola Imbrici
Biomedicines 2024, 12(8), 1698; https://doi.org/10.3390/biomedicines12081698 - 31 Jul 2024
Cited by 3 | Viewed by 1830
Abstract
Loss-, gain-of-function and mixed variants in SCN1A (Nav1.1 voltage-gated sodium channel) have been associated with a spectrum of neurologic disorders with different severity and drug-responsiveness. Most SCN1A variants are heterozygous changes occurring de novo or dominantly inherited; recessive inheritance has been reported in [...] Read more.
Loss-, gain-of-function and mixed variants in SCN1A (Nav1.1 voltage-gated sodium channel) have been associated with a spectrum of neurologic disorders with different severity and drug-responsiveness. Most SCN1A variants are heterozygous changes occurring de novo or dominantly inherited; recessive inheritance has been reported in a few cases. Here, we report a family in which the biallelic inheritance of two novel SCN1A variants, N935Y and H1393Q, occurs in two siblings presenting with drug-responsive developmental and epileptic encephalopathy and born to heterozygous asymptomatic parents. To assess the genotype–phenotype correlation and support the treatment choice, HEK 293 cells were transfected with different combinations of the SCN1A WT and mutant cDNAs, and the resulting sodium currents were recorded through whole-cell patch-clamp. Functional studies showed that the N935Y and H1393Q channels and their combinations with the WT (WT + N935Y and WT + H1393Q) had current densities and biophysical properties comparable with those of their respective control conditions. This explains the asymptomatic condition of the probands’ parents. The co-expression of the N935Y + H1393Q channels, mimicking the recessive inheritance of the two variants in siblings, showed ~20% reduced current amplitude compared with WT and with parental channels. This mild loss of Nav1.1 function may contribute in part to the disease pathogenesis, although other mechanisms may be involved. Full article
(This article belongs to the Special Issue Epilepsy: From Mechanisms to Therapeutic Approaches)
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29 pages, 1624 KiB  
Review
Are Aminoglycoside Antibiotics TRPing Your Metabolic Switches?
by Alfredo Franco-Obregón and Yee Kit Tai
Cells 2024, 13(15), 1273; https://doi.org/10.3390/cells13151273 - 29 Jul 2024
Cited by 1 | Viewed by 3145
Abstract
Transient receptor potential (TRP) channels are broadly implicated in the developmental programs of most tissues. Amongst these tissues, skeletal muscle and adipose are noteworthy for being essential in establishing systemic metabolic balance. TRP channels respond to environmental stimuli by supplying intracellular calcium that [...] Read more.
Transient receptor potential (TRP) channels are broadly implicated in the developmental programs of most tissues. Amongst these tissues, skeletal muscle and adipose are noteworthy for being essential in establishing systemic metabolic balance. TRP channels respond to environmental stimuli by supplying intracellular calcium that instigates enzymatic cascades of developmental consequence and often impinge on mitochondrial function and biogenesis. Critically, aminoglycoside antibiotics (AGAs) have been shown to block the capacity of TRP channels to conduct calcium entry into the cell in response to a wide range of developmental stimuli of a biophysical nature, including mechanical, electromagnetic, thermal, and chemical. Paradoxically, in vitro paradigms commonly used to understand organismal muscle and adipose development may have been led astray by the conventional use of streptomycin, an AGA, to help prevent bacterial contamination. Accordingly, streptomycin has been shown to disrupt both in vitro and in vivo myogenesis, as well as the phenotypic switch of white adipose into beige thermogenic status. In vivo, streptomycin has been shown to disrupt TRP-mediated calcium-dependent exercise adaptations of importance to systemic metabolism. Alternatively, streptomycin has also been used to curb detrimental levels of calcium leakage into dystrophic skeletal muscle through aberrantly gated TRPC1 channels that have been shown to be involved in the etiology of X-linked muscular dystrophies. TRP channels susceptible to AGA antagonism are critically involved in modulating the development of muscle and adipose tissues that, if administered to behaving animals, may translate to systemwide metabolic disruption. Regenerative medicine and clinical communities need to be made aware of this caveat of AGA usage and seek viable alternatives, to prevent contamination or infection in in vitro and in vivo paradigms, respectively. Full article
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