Search Results (172)

Peripheral nerve injuries remain a major clinical problem, and cell-free therapies using stem cell-derived bioproducts have emerged as promising alternatives. This study evaluated the influence of neurogenic differentiation and passage number on the secretomic and exosomal profile of human dental pulp stem cells (hDPCSs). Conditioned media from undifferentiated and neurodifferentiated hDPSCs, and exosomes derived from undifferentiated hDPSCs at passages 4 and 7, were analyzed using multiplex immunoassays, RT-PCR, and scanning electron microscopy (SEM). Neurodifferentiated hDPSCs at early passages secreted higher levels of neurotrophic, angiogenic and immunomodulatory factors, including FGF-2, IL-6, IL-8, and PDGF-AA. Exosomes from early-passage undifferentiated cells showed a more abundant and relevant neuroregenerative mRNA cargo in comparison to the later passages. Both cell types and exosomes adhered to the Reaxon^® nerve guidance conduit, confirming the permissive nature of the materials regarding cells and cellular products, allowing adhesion and survival. Neurite outgrowth assays performed on neurodifferentiated hDPSCs confirmed functional neural behavior. In later passages, a decline in secretory and exosomal activity was noted. These results highlight the relevance of early-passage hDPSCs as a source of bioactive factors and support their application in cell-free approaches for peripheral nerve regeneration. Full article

An open question in radiobiology concerns whether low doses of radiation are harmful or if cells are able to tolerate such exposure with minimal or no disruption. This issue is relevant for evaluating public health risks associated with the increasing number of medical computed tomography (CT) diagnostic procedures. This study evaluated the impact of CT scan-level exposure on human adipose mesenchymal stem cells (hMSCs) by measuring DNA damage responses (γH2AX, 53BP1, pATM foci), proliferation (Ki-67), senescence (β-galactosidase), and multiple gene expressions. Responses to one or five CT exposures were compared to a 2 Gy X-ray dose at intervals from 1 h to 10 passages post-irradiation. It was shown that CT scan briefly increased DNA damage markers but showed no significant long-term effects. A high dose of 2 Gy X-ray exposure caused sustained DNA damage, decreased proliferation, increased senescence, and significant changes in hundreds of genes even after several cell generations. After a single CT exposure, gene expression changes were minimal, while high-dose exposure led to strong activation of DNA repair and stress response pathways. Five CT scans caused a slight activation of LIF and HSPA1B genes, but these effects were minor compared to the high-dose group. All detected effects from CT scans were not observed by ten cell passages, whereas high-dose effects persisted. In conclusion, typical CT scan exposures have only short-term, mild effects on hMSCs, while high-dose radiation causes lasting cellular and genetic changes. Full article

This study elucidates potential genetic determinants and mechanisms involved in the synergistic effects of daptomycin (DAP) + fosfomycin (FOF) combination therapy. Among 33 clinically derived DAP-susceptible (S)/DAP-resistant (R) isogenic strain pairs, mutations in the mprF gene occurred in 30/33 DAP-R strains, including polymorphisms of L826F (33%) or T345A/L/I (15%). Strain variants of DAP-S CB1483 serially passaged in vitro for 10 days in DAP +/− FOF identified a key non-synonymous mutation in mprF (L826F) only in the DAP monotherapy arm. Interestingly, passage in FOF alone or DAP + FOF prevented the emergence of this mprF mutation following 10-day passage. This L826F mprF polymorphism, associated with a “gain-in-function” phenotype, exhibited increased amounts of lysyl-phosphatidylglycerol (L-PG) in the cell membrane (CM). Transcriptomics revealed a relatively modest number (~10) of distinct genes that were significantly up- or downregulated (≥2 log fold) in both the DAP-S and DAP-R strain pairs upon DAP + FOF exposures (vs. DAP or FOF alone). Of note, DAP + FOF decreased expression of lrgAB and sdrE and increased the expression level of fosB. In a rabbit infective endocarditis (IE) model, the DAP-R CB185 strain treated with DAP +/− FOF showed significantly reduced lrgB expression in vegetations compared with DAP treatment alone. Overall, these findings indicate that DAP + FOF therapy impacts MRSA through multiple specific mechanisms, enhancing bacterial clearance. Full article

Recombinantly produced monoclonal antibodies (mabs) belong to the fastest growing class of biotherapeutics. In humans, antibodies are classified into five different classes: IgA, IgD, IgE, IgG and IgM. Most of the therapeutic mabs used in the clinic belong to the IgG class, albeit other antibody classes, e.g., IgM, have been evaluated in clinical stages. Antibodies are composed of heavy chains paired with a light chain. In IgM and IgA, an additional chain, the J-chain, is present. Two types of light chains exist in humans: the κ-light chain and the λ-light chain. The κ-light chain predominates in humans and is used in the vast majority of therapeutic IgG. The reason for the preference of the κ-light chain in humans is not known. Our study investigates whether light-chain selection influences the productivity of the clinically validated mabs adalimumab and trastuzumab. Both mabs were expressed as IgG and IgM with a κ- or a λ-light chain in HEK293 cells. Besides comparing the expression levels of the different mabs, we also evaluated whether the passage number of the cell line has an impact on product yield. In addition, the expressions of adalimumab, trastuzumab, an anti-CD38 and an anti-PD-L1-antibody were analyzed in HEK293 and CHO cells when both the κ- and λ-light chains are present. In summary, IgG outperformed IgM variants in expression efficacy, while light-chain selection had minimal impact on the overall expression levels. The yields of all mab variants were higher in fresh cells, despite cell cultures with a high cell passage number having higher cell densities and cell numbers at the time of harvest. The incorporation of a particular light chain occurred at similar rates in HEK293 and CHO cells. Full article

Background: The marine bacterium Vibrio campbellii has been a model system for the study of bacterial quorum sensing and is increasingly recognized as a formidable aquatic animal pathogen. While genetically tractable, the study of this species in basic and applied research still relies upon laborious and time-consuming conjugation methods for plasmid DNA transformation. Methods: In this study, we developed an electroporation protocol using the most studied strain of this species, V. campbellii ATCC BAA-1116. An electroporation efficiency of up to 3 × 10⁴ CFU/μg DNA was demonstrated using derived parameters (10 kV/cm, 400 Ω, 25 μF), which took cell growth phase at harvest, plasmid DNA amount, and recovery conditions into account. The electroporation protocol was tested using several different plasmids and with additional strains of V. campbellii and sister species V. harveyi. Results: Interestingly, of the eight other V. campbellii strains tested, only three others, which also happened to be the three most recent environmental isolates with the fewest number of laboratory passages, were amenable to electroporation-mediated transformation. Conclusions: This electroporation protocol expands the tool set for studying V. campbellii and provides interesting insights into DNA transformation and uptake in this and related bacterial species. Full article

Nanoplastic (NP) pollution has emerged as a growing concern due to its potential impact on human health, although its adverse effects on different organ systems are not yet fully understood. This systematic scoping review, conducted in accordance with international guidelines, aimed to map the current evidence on the biological effects of NPs. In vitro animal studies assessing cellular damage caused by exposure to any type of NP were searched on PubMed, Web of Science, and Scopus. Data on primary outcomes related to genotoxicity and cytotoxicity (cell viability, oxidative stress, inflammation, DNA and cytoplasmic damage, apoptosis) were extracted from the included studies, and overall reporting quality was assessed. A total of 108 articles published between 2018 and 2024, mostly by China (54%), Spain (14%), and Italy (9%), were included. Polystyrene (PS) was the most frequently studied polymer (85%). NP sizes in solution ranged from 15 to 531 nm, with a higher prevalence in the 40–100 nm range (38%). The overall quality of studies was rated as moderate (60%), with many lacking essential details about cell culture conditions (e.g., pH of the medium, passage number, substances used). A higher frequency of negative effects from NP exposure was observed in respiratory cell lines, while immune, digestive, and hepatic cell lines showed greater resistance. Nervous, urinary, and connective tissue systems were impacted by NPs. Positively charged and smaller PS particles were consistently associated with higher toxicity across all systems. In summary, this review highlights the multifactorial nature of NP toxicity, influenced by size, surface charge, and polymer type. It also reveals a significant knowledge gap, stemming from the predominant use of immortalized monocultures exposed to commercially available PS NPs, the limited use of environmentally relevant particles, and the underutilization of advanced experimental models (e.g., organ-on-chip systems) that better mimic physiological conditions. Full article

Glycation, or non-enzymatic glycosylation, has recently attracted increasing interest in the context of its impact on aging. Advanced glycation end products (AGEs) contribute to various age-related pathological conditions such as inflammation, fibrosis, and vascular calcification. However, the molecular mechanisms underlying glycation-induced disruption of cell–matrix interactions during cellular senescence are not fully understood. The aim of this study was to investigate transcriptomic changes in young and senescent dermal fibroblasts (HdFbs) cultured in 3D post-glycated collagen type I matrices after 10 and 17 days. Our findings indicate that D-ribose-mediated glycation increases the accumulation of fluorescent AGEs and the stiffness of matrices in a dose-dependent manner. The transcriptome alterations in cells encompassed the modulation of age-related genes and signaling pathways, including activation of genes related to senescence-associated secretory phenotype (SASP). Notably, the alterations in the transcriptome profiles due to glycation were more pronounced (in terms of both the number of genes and their fold changes) after 10 days of culture compared to day 17 in both passages. These findings suggest that cellular responses to glycation and resulting stiffness depend on both the concentration of reducing sugar and the time spent under those conditions. Full article

Mesenchymal stem cells (MSCs) are multipotent progenitors capable of self-renewal and differentiation into various cell lineages, making them essential for tissue repair and regenerative medicine. However, their regenerative potential is constrained by replicative senescence, an irreversible growth arrest that occurs after a finite number of cell divisions. In this study, we serially passaged human bone marrow-derived MSCs (bMSCs) and compared young, pre-senescent, and senescent cells. The onset of senescence was accompanied by progressive alterations in mitochondrial dynamics, leading to a decline in mitochondrial membrane potential, and increased reactive oxygen species (ROS) production, alongside a diminished cellular antioxidant capacity. These mitochondrial defects play a role in metabolic reprogramming in senescent bMSCs. Our findings underscore the intricate interplay between ROS, mitochondrial dysfunction, and replicative senescence, offering valuable insights to guide the development of therapeutic strategies for preserving MSC functionality in aging and MSC-based therapies. Full article

Human mesenchymal stem cells (hMSCs) are widely used in regenerative medicine, but large-scale in vitro expansion alters their function, impacting proliferation and differentiation potential. Currently, a predictive marker to assess these changes is lacking. Here, we used dielectrophoresis (DEP) to characterize the electrical phenotype of hMSCs derived from bone marrow (BM), adipose tissue (AT), and umbilical cord (UC) as they aged in vitro from passage 4 (P4) to passage 9 (P9). The electrical phenotype was defined by the DEP spectra, membrane capacitance, and cytoplasm conductivity. Cell morphology and size, growth characteristics, adipogenic differentiation potential, and osteogenic differentiation potential were assessed alongside label-free biomarker membrane capacitance and cytoplasm conductivity. Differentiation was confirmed by histological staining and RT-qPCR. All hMSCs exhibited typical morphology, though cell size varied, with UC-hMSCs displaying the largest variability across all size metrics. Growth analysis revealed that UC-hMSCs proliferated the fastest. The electrical phenotype varied with cell source and in vitro age, with high passage hMSCs showing noticeable shifts in DEP spectra, membrane capacitance, and cytoplasm conductivity. Correlation analysis revealed that population doubling level (PDL) correlated with membrane capacitance and cytoplasm conductivity, indicating PDL as a more precise marker of in vitro aging than passage number. Additionally, we demonstrate that membrane capacitance correlates with the osteogenic marker COL1A1 and that cytoplasm conductivity correlates with the adipogenic markers ADIPOQ and FABP4, suggesting that DEP-derived electrical properties serve as label-free biomarkers of differentiation potential. While DEP has previously been applied to BM-hMSCs and AT-hMSCs, and more recently to UC-hMSCs, few studies have provided a direct comparison across all three sources or tracked changes across continuous expansion. These findings underscore the utility of DEP as a label-free approach for assessing hMSC aging and function, offering practical applications for optimizing stem cell expansion and stem cell banking in clinical settings. Full article

Background/Objectives: Breast cancer is the most prevalent cancer in adult women. Currently, new therapies and protein determinations with prognostic value are under development. Fascin (encoded by the FSCN1 gene) is an actin-binding protein that is critical for the development of cytoplasmic projections that are essential for tumor invasion. DNA topoisomerase 2-alpha (TOP2A) is a nuclear protein crucial for ATP-dependent breakage, passage, and rejoining of double-stranded DNA and cell division. Both proteins are associated with higher proliferation rates and worse prognosis in breast cancer and together can provide comprehensive information on prognosis and treatment response. Methods: We simultaneously assessed fascin expression and TOP2A/CEP17 DNA copy number ratios in various histological and molecular subtypes. Additionally, these markers were analyzed along with previously established diagnostic markers and other relevant clinical data. Results: Our series included 265 patients, four of whom were male, and all of which were diagnosed with breast carcinoma. Of the 265 patients initially included, sufficient material for analysis was available for 175 cases, as some samples were excluded because of insufficient tissue quantity, poor preservation, or lack of hybridization in certain assays. Immunohistochemical (IHC) expression of fascin, both in its aggregated form and by category, showed no association with the TOP2A gene alteration ratio. Fascin expression was significantly associated with histological subtype (p < 0.001), molecular subtype (p < 0.001), hormone receptor (HR) (p < 0.001), BCL2 (p = 0.003), Ki67 (p = 0.002), and histological grade (p < 0.001). TOP2A was significantly associated with molecular subtype (p = 0.041), Ki67 (p = 0.048), and histological grade (p = 0.033). In our study, molecular subtype (p = 0.037) emerged as an independent variable for the complete histological response to neoadjuvant treatment. Multivariate analysis linked pathological stage (p = 0.002) and estrogen receptor (ER) expression (p = 0.004) to overall survival (OS) and disease-free survival (DFS). Conclusions: No statistical relationship was evident between fascin expression (IHC) and the TOP2A copy ratio. The results of this study suggested that the mechanisms of increased cell proliferation associated with alterations in fascin and TOP2A are independent. Full article

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) has been circulating in China for several years, causing substantial economic losses to the local pig industry. Current commercial vaccines have failed to provide complete protection against NADC34-like PRRSV infection. Additionally, the poor adaptation of NADC34-like strains to Marc-145 cells presents a considerable challenge for developing effective vaccines against these strains. This study addresses these challenges by developing a novel vaccine candidate against NADC34-like PRRSV. We engineered a recombinant PRRSV, rNADC34-CHSps, by replacing the structural protein region of the JS2021NADC34 strain with that of the CHR6 strain to improve its adaptation to Marc-145 cells. The rescued strain could proliferate well in Marc-145 cells, maintaining high titers and stable growth kinetics even at high passage numbers. Piglets were vaccinated with rNADC34-CHSps at passage 80 and then challenged with the virulent NADC34-like PRRSV strain, JS2021NADC34, at 28 days post-vaccination. All vaccinated piglets developed specific antibodies against PRRSV at 14 dpv and showed no significant clinical symptoms, even after exposure to PRRSV JS2021NADC34. Furthermore, the vaccinated piglets gained significantly more weight, displayed much less severe pathological lesions, and reduced viremia compared to the challenge control piglets. These results indicate that rNADC34-CHSps is a promising vaccine candidate against NADC34-like PRRSV infection, highlighting the potential of targeted genomic modifications to enhance vaccine efficacy. Full article

Burn patients treated with tissue-engineered skin substitutes (TESs) often experience pigmentation irregularities, including hypopigmentation and pigmentation spots. These issues are thought to stem from the reduced presence of melanocytes through dilution during TES manufacturing. To address this, we hypothesized that supplementing epithelial cell cultures—primarily composed of keratinocytes but also containing melanocytes—with Fibroblast Growth Factor-2 (FGF-2), a known promoter of melanocyte proliferation, could enhance melanocyte growth. This would potentially increase their numbers in TESs and improve pigmentation outcomes. Our findings indicate that FGF-2, at an optimal dose of 0.2 nM, effectively maintains melanocyte numbers in 2D cultures and epithelial cell cultures through the first passage. Importantly, this treatment does not interfere with keratinocyte proliferation or differentiation, nor does it affect TES integrity. However, FGF-2 supplementation alone did not increase the proportion of melanocytes in epithelial cultures beyond the first passage or in TESs. In summary, while FGF-2 supports melanocyte growth in culture, its addition alone was insufficient to significantly improve TES pigmentation. Full article

Feline infectious peritonitis virus (FIPV) remains as one of the leading causes of morbidity and mortality in young cats from shelters and catteries worldwide. Since little is known about the molecular characteristics of currently circulating FIPV strains in Long Island, New York, samples from two shelter cats submitted to the Pathology Diagnostic Services of the Long Island University College of Veterinary Medicine, with gross and microscopic lesions consistent with those of FIP were processed for virus isolation, molecular characterization and full-length genome decoding. The younger shelter cat, a 1-year-old male (A15) was found dead without previous signs of illness. Postmortem examination revealed gross and microscopic lesions characterized by vasculitis, necrosis, hemorrhage, and pyogranulomatous inflammation confined to the colon and associated lymph nodes. The second cat, a 7-year-old spayed female (A37) had an identical clinical history and similar but widespread lesions, including fibrinous peritoneal effusion, cecal, colonic, renal, and hepatic involvement. The gross and microscopic diagnosis of FIP in these cats was confirmed by immunohistochemistry (IHC) demonstration of feline coronavirus antigen using mouse anti-FIPV3-70 monoclonal antibody. Virus isolation from saved frozen kidney and colon tissue was performed through several subsequent blind passages in MDCK and Vero cell lines. Confirmation of the FIPV isolation was done through qRT-PCR, IFA, western blot using N protein antibodies, and NGS of the full-length genome sequencing. The full-length genome sequences of the virus isolate from the two cats were decoded using next-generation sequencing (NGS) and deposited in the GenBank as accession numbers PQ192636 and PQ202302. The genome size of these isolates was (29355 and 29321) nucleotides (nt) in length, respectively. While their genome organization was consistent with other FIPV genomes as follows (5’UTR-ORF1ab-S-3abc-M-E-7b-3’UTR-3’), marked differential mutations were observed in the ORF1a/b, S, 3Abc, and 7b protein genes of the two FIPV isolates. One notable deletion of 34 nucleotides was observed in the 7b genes of one of these isolates but was absent in the other. We confirmed the potential recombination events during the evolution of those two FIPV field isolates with the potential parent virus as FECoV-US isolated in 1970 and the potential minor parent as the Canine coronavirus. Our results provide a comprehensive molecular analysis of two novel FIPV isolates causing fatal disease in shelter cats from Long Island. Diagnostic surveillance with molecular characterization and sequencing analysis of circulating FIPV strains within animal shelters may help early detect unique emerging clinical and pathological manifestations of the disease and develop more targeted prophylactic and therapeutic approaches to control it. Full article

Background: The Vero cell rabies vaccine is currently the most widely used human rabies vaccine. However, owing to the presence of residual host cell DNA (HCD) in the final product and the potential tumorigenicity of the DNA of high-passage Vero cells, the WHO not only sets a limit on the number of times cells used in production can be passaged, but also imposes strict requirements on the amount of residual HCD in the final vaccine product. Objectives: To systematically reduce the HCD level in the final vaccine product, multiple purification steps are included in the vaccine production process. This study investigated the effectiveness of key production steps in antigen recovery and DNA removal. Methods: The residual HCD fragment content and size distribution were detected using fluorescence quantitative PCR (qPCR) and capillary gel electrophoresis (CGE), and the rabies virus glycoprotein antigen content was detected using enzyme-linked immunosorbent assay (ELISA). The antigen recovery rate and HCD removal rate in each key process were calculated to evaluate the scientific basis and effectiveness of each production step. Additionally, the stability of the process was studied using multiple commercial batches of the product. Results: The results revealed that the total antigen recovery rate in the production process described in this report was no less than 8.5%, and the effective removal rate of residual HCD was not lower than 99.99%. Moreover, the amount of residual HCD in the final product was far below the quality standard of 2 ng/dose, and most of the residual HCD fragments were smaller than 200 bp. The results of the process stability studies on multiple commercial batches showed that the bulk human rabies vaccine produced by this process had excellent safety and efficacy and that the production process was stable and thus suitable for large-scale batch production. Conclusions: The production process described in this study achieved effective recovery of viral antigens and efficient removal of residual HCD, and the process was stable and controllable, enabling the continuous and stable production of vaccine products that meet WHO recommendations and the relevant requirements of the current edition of the Chinese Pharmacopeia. In addition, this study provides theoretical guidance for optimizing the vaccine production process. Full article

Rat dental pulp stem cells (DPSCs) can be used to elucidate mesenchymal stem cell (MSC) applications in regenerative medicine. However, information on rat DPSCs during long-term passage, which could lead to replicative senescence, is limited. In this study, we investigated the phenotypic changes in DPSCs after 3–26 passages (3P–26P). The results show that cell morphology and nuclear size increase proportionally with passage number. The phosphorylated histone H2A.X (γ-H2A.X) positive cells (indicating DNA damage) increased significantly earlier than the 4-Hydroxynonenal (4-HNE) stained cells (indicating an abundance of intracellular reactive oxygen species). Compared to the cells subjected to 3P and 5P, the cells subjected to 15P showed reduced proliferation despite being positive for Ki67. Furthermore, cell growth was completely arrested after 26P. The senescence markers, senescence-associated β-galactosidase (SA-β-gal) and p16, exhibited similar expression patterns that were not correlated with those of p21 and urokinase-type plasminogen activator receptor (uPAR). Nearly all cells expressed SA-β-gal and p16 after 26P, whereas only half expressed p21 and uPAR. These results will contribute to understanding the characteristics of DPSCs toward replicative senescence, which are applicable to elucidate mechanisms related to regenerative medicine and stem cell aging. Full article