Search Results (544)

Pseudomonas aeruginosa and Scedosporium/Lomentospora often coexist in the lungs of cystic fibrosis patients, where their interaction can affect disease outcomes. Our group has recently demonstrated that P. aeruginosa suppresses the growth of Scedosporium/Lomentospora species partly through mechanisms involving iron sequestration. In this study, we have investigated how molecules secreted by P. aeruginosa under high (36 µM) and low (3.6 µM) iron conditions affect the planktonic growth and biofilm formation by S. apiospermum, S. minutisporum, S. aurantiacum and L. prolificans. Although P. aeruginosa exhibited enhanced proliferation under high-iron conditions, spectrophotometric analyses revealed a marked increase in phenazine and pyoverdine production under low-iron conditions, with siderophore activity confirmed by Chrome Azurol S assays. Supporting these findings, supernatants from P. aeruginosa cells grown under iron limitation markedly inhibited fungal growth (≈30%) and biofilm formation (≈70%), whereas those from high-iron cultures were less effective. Notably, low-iron bacterial-free supernatants exhibited pronounced cytotoxic effects on mammalian cells, reducing metabolic activity by an average of 20% in A549 lung epithelial cells and 40% in THP-1 macrophages, and significantly compromising survival in the Tenebrio molitor infection model, resulting in 100% larval mortality within 7 days. Collectively, these results indicate that the antifungal activity of P. aeruginosa is closely coupled with increased host toxicity. Moreover, the results demonstrate that environmental iron availability plays a critical role in modulating both antifungal activity and toxicity, thereby shaping P. aeruginosa interactions with Scedosporium/Lomentospora species. Such iron-dependent dynamics may influence the progression and severity of respiratory co-infections, with important implications for patient management and therapeutic interventions. Full article

The post-implantation phase of mammalian development is crucial yet challenging to study due to ethical and technical constraints, particularly in humans. Recent revolutionary advances in extended in vitro culture systems for mammalian embryos now offer unprecedented windows into this developmental “black box”. This review synthesizes how these platforms, alongside stem cell-derived embryo models, are transforming our ability to model early human development in a dish. We detail the technological evolution from two-dimensional (2D) to three-dimensional (3D) cultures that support mouse, non-human primate, and human embryos through key stages of implantation and gastrulation, recapitulating events like lineage specification and axial patterning. Furthermore, we explore how these models serve as powerful tools for investigating the etiology of early pregnancy failure, screening for developmental toxicity of pharmaceuticals, and deciphering the molecular pathogenesis of birth defects. By bridging fundamental embryology with clinical and pharmacological applications, these innovative models herald a new era in biomedical research, holding significant promise for advancing reproductive medicine and regenerative strategies. Full article

Rabies is a neglected tropical zoonotic disease caused by rabies-virus (RV) infection and is responsible for almost 60,000 annual deaths globally, largely affecting the socio-economically disadvantaged population. Although fatality is preventable by immunization either before or after exposure with therapeutic antibodies, the high cost of prophylaxis or treatment limits their accessibility for the affected population. However, due to the almost 100% fatality rate in symptomatic individuals, almost 29 million annual vaccinations are performed, imposing high financial burden. Human transmission occurs principally through bites from infected dogs and although multiple mammalian species are permissive to RV, transmission from them or from symptomatic humans is rare. To overcome the limitations posed by the requirement of biosafety level-3 (BSL-3) containment for live virus culture, we established a replication-deficient vesicular stomatitis virus (VSV) pseudovirus expressing the Rabies-G (RV-G) protein and a multiplexed Luminex immunoassay for quantifying anti-rabies antibodies in equine sera. The purified pseudovirus exhibited robust luciferase activity and was able to infect multiple mammalian cell lines, although with variable efficiency. Using hyper-immunized equine serum, we observed a strong correlation (ρ > 0.9, p < 0.001) between binding antibody titers measured by the Luminex assay with neutralizing antibody titers determined using the pseudovirus-based neutralization assay. These assays provide a safe, quantitative, and BSL-2-compatible platform for rabies serological evaluation and vaccine testing. Full article

Copper and nickel ions play pivotal, albeit distinct, roles as essential trace elements in living systems, and primarily serve as co-factors for a range of enzymes. However, as with all trace metal ions, excessive concentrations can exert adverse toxicological properties. Interestingly, the incorporation of these in cell culture media can establish novel chemical interactions, with their speciation status markedly influencing characteristics, including cell maturation, and cellular uptake mechanisms. Thus, the primary objective of this study was to investigate and determine the speciation status (i.e., complexation) of nickel(II) and copper(II) ions by biomolecules present in RPMI 1640 mammalian cell culture medium using virtually non-invasive high-resolution proton NMR analysis, an investigation of much relevance to now routine studies of their toxicological actions towards cultured cells. Samples of the above aqueous culture medium were ¹H NMR-titrated with increasing added concentrations of 71–670 µmol/L Ni(II)(aq.), and 0.71–6.7, 7.1–67 and 71–670 µmol/L Cu(II)(aq.), in duplicate or triplicate. ¹H NMR spectra were acquired on a JEOL ECZ-600 spectrometer at 298 K. Results demonstrated that addition of increasing concentrations of Ni(II) and Cu(II) ions to the culture medium led to the selective broadening of a series of biomolecule resonances, results demonstrating their complexation by these agents. The most important complexants for Ni(II) were histidine > glutamine > acetate ≈ methionine ≈ lysine ≈ threonine ≈ branched-chain amino acids (BCAAs) > asparagine ≈ aspartate > tyrosine ≈ tryptophan, whereas for Cu(II) they were found to be histidine > glutamine > phenylalanine ≈ tyrosine ≈ nearly all remaining aliphatic metabolites (particularly the wealth of amino acids detectable) > 4-hydroxyphenylacetate (trace culture medium contaminant), in these orders. However, Cu(II) had the ability to influence the linewidths of these signals at much lower added levels (≤7 µmol/L) than that of Ni(II), the broadening effects of the latter occurring at concentrations which were approximately 10-fold greater. Virtually all of these added metal ion-induced resonance modifications were, as expected, reversible on addition of equivalent or excess levels of the chelator EDTA. From this study, changes in the co-ordination sphere of metal ions in physiological environments can give rise to marked modifications in their physicochemical properties (e.g., redox potentials, electronic charges, the potential catalytic generation of reactive oxygen species (ROS), and cell membrane passages). Moreover, given that the above metabolites may also function as potent hydroxyl radical (^●OH) scavengers, these findings suggest that generation of this aggressively reactive oxidant directly from Cu(II) and Ni(II) ions in physiologically-relevant complexes may be scavenged in a ‘site-dependent’ manner. This study is of further relevance to trace metal ion research in general since it enhances our understanding of the nature of their interactions with culture medium biomolecules, and therefore provides valuable information regarding their overall chemical and biological activities, and toxicities. Full article

Early mammalian embryo development is a temporally regulated process initially governed by maternal factors during the first few cleavage divisions. In porcine embryos, the transition from oocyte to embryonic control occurs around the 4-cell stage. This developmental progression depends on embryonic genome activation (EGA), epigenetic reprogramming, metabolic cues, and extracellular signaling pathways. While fundamental aspects of early development are conserved across mammals, porcine embryos exhibit distinct molecular features, including unique EGA timing, altered regulatory gene expression, and a pronounced reliance on lipid metabolism. This review provides a comprehensive overview of recent advances in understanding the molecular mechanisms underlying early porcine embryo development, from fertilization to blastocyst formation. It summarizes molecular changes associated with the maternal regulation of initial embryonic divisions, genome activation, chromatin remodeling, and the role of transcription factors and metabolic pathways. Additionally, the review examines the impact of in vitro culture conditions on these molecular processes. A thorough understanding of these mechanisms is critical for optimizing embryo culture systems, improving developmental outcomes, and advancing agricultural biotechnology. Full article

The development of new alternative models is essential to overcome the limitations of traditional two-dimensional (2D) cell cultures and animal models. Three-dimensional (3D) models, such as organoids, better mimic the structural and functional complexity of mammalian organs, thereby reducing the ethical and economic issues related to animal experimentation. These systems provide more physiologically relevant environments, improving the accuracy of disease modeling and drug response prediction. In this context, we have developed mouse hepatic organoids from livers of adult BALB/c mice and characterized them by microscopy and transcriptional analysis. This model was applied to a robust and reproducible high-throughput screening (HTS) platform for testing cytotoxicity at the preclinical stage of drug discovery. In addition, mouse hepatic organoids were co-cultured with amastigotes of Leishmania donovani parasites to establish a model of host–parasite interaction, which was characterized by RNA-seq linked to differential expression analysis and cytokine production by the hepatic organoids. The findings provided in this work establish mouse hepatic organoids as an alternative model for drug discovery and pathogenesis studies. Full article

The lipopolysaccharide (LPS)-induced inflammation model is widely used in mammalian studies but remains poorly investigated in a variety of fish species. We reproduce this model in rainbow trout, Oncorhynchus mykiss, a species of high economic value, to validate specific inflammatory biomarkers for reliably assessing the health and immune status of farmed fish. The inflammatory process, together with the effect of preconditioning, was modeled using two consecutive intraperitoneal injections of 300 and 600 µg LPS. We assessed innate immunity biomarkers, including the expression of inflammation-related genes (il1ß, il8), peripheral blood leukocyte profiles, serum bactericidal activity (SBA), and various serum and hepatic biochemical parameters. These parameters comprised the concentration of C-reactive protein (CRP, an acute phase protein), and the activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione-S-transferase) measured at 24 and 96 h post-injection. In trout, LPS-induced effects involved the overexpression of pro-inflammatory interleukins (il1ß, il8), a left shift in white blood cells (characterized by a prevalence of immature neutrophils), and enhanced SBA. In contrast to warm-blooded animals, LPS challenge in trout did not appear to significantly elevate CRP levels or antioxidant enzyme activity. Further investigations in other fish species are needed to determine whether these are traits specific to trout or common to bony fish. Our findings provide a foundation for developing a biomarker panel suitable for the routine assessment of welfare, early detection of infection-associated inflammation in cultured fish, and the screening of the anti-inflammatory and immunostimulant activities of drugs and feed additives. Full article

Advanced cell-based therapies, including immunotherapy, regenerative medicine, and other biotechnological applications, require large quantities of viable mammalian cells for research and clinical use. Conventional enzymatic harvesting methods, such as trypsini-zation, can compromise cell integrity and reduce viability. This study investigates an al-ternative temperature-responsive approach using alginate beads incorporated with poly(N-isopropylacrylamide) (PNIPAAm), a polymer exhibiting a lower critical solution temperature (LCST) of approximately 32 °C. This system enables temperature-controlled cell detachment while preserving cellular structure and extracellular matrix components, thereby potentially improving post-harvest viability compared to trypsin treatment. Ho-mogeneous alginate hydrogel beads were synthesized using a standard infusion pump and ionically crosslinked with calcium cations. The beads were characterized by scanning electron microscopy (SEM) for morphology and by Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and micro-computed tomography (µ-CT) for compositional and thermal analysis. Mouse fibroblast cells (L929 cell line) were cultured on the beads, and their proliferation and viability were assessed using CCK-8 and Live/Dead assays, demonstrating significant cell growth over seven days. The results suggest that PNIPAAm-modified alginate beads provide a promising, enzyme-free platform for efficient mammalian cell harvesting and delivery, with potential applications across advanced cell manufacturing and therapeutic technologies. Full article

The evaluation of the use of extracellular vesicles (derived from different cellular sources and mammalian fluids) in regenerative medicine has produced interesting results. This includes their great potential for the treatment of chronic skin ulcers, which is related to their effects on migration, proliferation, inflammation and angiogenesis, among other processes. However, large-scale production of mammalian extracellular vesicles may be limited by the need to maintain cell cultures continuously, without losing their ability to secrete extracellular vesicles with regenerative capacity. This may require complex and expensive infrastructures. It is therefore necessary to identify other possible, more efficient alternatives that can be easily transferred to clinical practice. Among these substitutes are plant-derived extracellular vesicles (PDEVs). Fortunately, they resemble those of mammals, playing a role in cell communications. As expected, their compositions depend on source tissues and the physiological conditions of the plants. They may carry numerous molecules with high biological activity. Interestingly, PDEVs are easy to obtain on a large scale, have good stability and are less immunogenic than mammalian-derived EVs. Numerous preclinical studies indicate that they can enhance chronic-wound healing through their immunomodulatory and angiogenic effects, among others. Thus, this review aims to describe the current state of knowledge on the potential therapeutic use of PDEVs in wound healing. It also describes the methods of obtaining and applying them, as well as regenerative processes in which they may intervene. The information provided shows the need to continue advancing knowledge about the production, isolation and mechanisms of action of PDEVs. This will allow new effective therapeutic strategies for the treatment of chronic cutaneous ulcers to be developed. Full article

Research on mammalian embryogenesis has been revolutionised by rapid technological and methodological advancements. This review seeks to synthesise historical and contemporary studies on mouse embryos to provide comprehensive conceptual frameworks of early embryogenesis. Key research findings from live-cell imaging, fluorescence staining, molecular, and sequencing analyses were reassessed, mainly focusing on in vivo and in vitro mouse embryo models. Through critical reassessment, this review first presents historical progress in mammalian models and culture systems for the study of embryogenesis. Subsequently, it elucidates the spatiotemporal progression of morphological events from pre- to peri-implantation across tissue, cellular, and molecular scales. The critical analysis in this review highlights that prevailing and alternative models for pre-implantation cell lineage specification are not mutually exclusive but, rather, describe complementary aspects of embryogenesis. While multiple factors in peri-implantation cell lineage specification are discussed, establishing integrative theoretical models requires further investigation into the crosstalk between different factors during lineage decision-making. Furthermore, this work identifies that precise spatiotemporal dynamics and mechanisms, particularly those governing the initiation, progression, and maintenance of morphological and lineage decisions, remain major knowledge gaps. Lastly, this review provides critical thinking frameworks for assessing current models and defining specific experimental pathways to address enduring unresolved knowledge gaps regarding early embryogenesis. Full article

Background: Cystic echinococcosis (CE) is a globally distributed zoonosis triggered by the larval stage of Echinococcus granulosus (E. granulosus), impacting humans and an extensive array of mammalian intermediate hosts. EGR-01664 is the major egg antigen of E. granulosus, but almost nothing is currently known about the function of EGR-01664 from E. granulosus. Methods: This study aimed to investigate the E. granulosus EGR-01664 gene (GenBank ID: 36337379), and the recombinant EGR-01664 protein was expressed successfully. Next, the transcription of the EGR-01664 gene across various developmental stages of E. granulosus was analyzed. Its spatial expression patterns in adult worms and protoscoleces were characterized using both quantitative PCR (qPCR) and immunofluorescence assays. Furthermore, the immunomodulatory effects of rEGR-01664 on cell proliferation, nitric oxide production, and cytokine secretion were examined by co-culturing the recombinant protein with canine PBMCs. Results: The rEGR-01664 could be recognized by sera from dogs infected with E. granulosus. Immunofluorescence assay (IFA) localization revealed the protein’s presence in the epidermis of protoscoleces, the adult epidermis, and some parenchymal tissues. qPCR revealed that EGR-01664 mRNA levels were significantly higher in protoscoleces compared to adults (p < 0.0001). At a concentration of 20 μg/mL, rEGR-01664 could significantly activate the transcription and expression of IL-10, TGF-β1, IL-17A, and Bax in canine PBMCs. However, with an increase in concentration, it inhibited the expression of IFN-γ, Bcl-2, GSDMD, IL-18, and IL-1β. These results suggest that the EGR-01664 gene plays a crucial role in the development, parasitism, and reproduction of E. granulosus. In vitro studies have shown that rEGR-01664 protein regulates the immune regulation function of canine PBMCs, suggesting its potential as a vaccine adjuvant or immunotherapy target. Conclusions: EGR-01664 may modulate canine PBMC functions to regulate host immune responses, thereby facilitating our understanding of how E. granulosus EGR-01664 contributes to the mechanism of parasitic immune evasion. Full article

Background. Dystroglycanopathies (DGPs) constitute a set of recessive, neuromuscular congenital dystrophies that result from impaired glycosylation of dystroglycan (DG). These disorders typically course with CNS alterations, which, alongside gradual muscular dystrophy, may include brain malformations, intellectual disability and a panoply of ocular defects. In this process, the protein products of 22 genes, collectively dubbed DGP-associated genes, directly or indirectly participate sequentially along a complex, branched biosynthetic pathway. POMGNT2 and POMGNT1 are two enzymes whose catalytic activity consists of transferring the same substrate, a molecule of N-acetylglucosamine (GlcNAc) to a common substrate, the O-mannosylated α subunit of DG. Despite their presumptive role in retinal homeostasis, there are currently no reports describing their expression pattern or function in this tissue. Purpose. This work focuses on POMGNT2 and POMGNT1 expression in the mammalian retina, and on the characterization of their distribution across retinal layers, and in the 661W photoreceptor cell line. Methods. The expression of POMGNT2 protein in different mammalian species’ retinas, including those of mice, rats, cows and monkeys, was assessed by immunoblotting. Additionally, POMGNT2 and POMGNT1 distribution profiles were analyzed using immunofluorescence confocal microscopy in retinal sections of monkeys and mice, and in 661W cultured cells. Results. Expression of POMGNT2 was detected in the neural retina of all species studied, being present in both cytoplasmic and nuclear fractions of the monkey and mouse, and in 661W cells. In the cytoplasm, POMGNT2 was concentrated in the endoplasmic reticulum (ER) and/or Golgi complex, depending on the species and cell type, whereas POMGNT1 accumulated only in the Golgi complex in both monkey and mouse retinas. Additionally, both proteins were present in the nucleus of the 661W cells, concentrating in the euchromatin and heterochromatin, as well as in nuclear PML and Cajal bodies, and nuclear speckles. Conclusions. Our results are indicative that POMGNT2 and POMGNT1 participate in the synthesis of O-mannosyl glycans added to α-dystroglycan in the ER and/or Golgi complex in the cytoplasm of mammalian retinal cells. Also, they could play a role in the modulation of gene expression at the mRNA level, which remains to be established, in a number of nuclear compartments in transformed retinal neurons. Full article

Tetracenomycins are anticancer polyketides that arrest cancer cell proliferation via binding to the large mammalian ribosomal subunit near the polypeptide exit channel. The tetracenomycins are natural products that many members of the actinomycete family produce. The first goal of this study was to improve the biosynthesis of tetracenomycin analogs via metabolic engineering. The second goal was to probe more deeply into the antiproliferative activity of tetracenomycin aglycones. The tetracenomycins were assessed via several assays, including cell viability assays, clonogenic assays, and flow cytometry apoptosis assays. The data suggest that tetracenomycins C and X inhibit cell proliferation and arrest cell growth, supporting their cytostatic action mechanism. In addition, tetracenomycins C and X induced degeneration of 3D spheroid cultures and exhibited concentration-dependent inhibition of cell survival and colony formation in clonogenic assays. This work demonstrates that tetracenomycins act mainly as cytostatic rather than apoptotic agents. Full article

Background: Borrelia burgdorferi, the causative agent of Lyme disease, releases outer membrane vesicles (OMVs) that may contribute to infection and modulate the host immune response. Although interest in OMVs is growing, few studies have systematically compared methods for isolating OMVs from B. burgdorferi. Methods: In this study, we evaluated two OMV isolation techniques—standard ultracentrifugation and an ion-exchange chromatography-based ExoBacteria™ kit—and examined how serum supplements (rabbit serum vs. exosome-depleted fetal bovine serum, ED-FBS) influence Bb-OMV yield and composition. Gene expression profiles were assessed using RT-PCR, and specific protein content was identified by Western blot analyses. To assess the ability of Bb-OMVs to interact with host cells, Bb-OMVs were co-cultured with MDA-MB-231 triple-negative breast cancer cells. Results: Transmission electron microscopy confirmed that both methods produced spherical Bb-OMVs with intact membrane bilayers. Ultracentrifugation generated larger vesicles (15–180 nm), while the ExoBacteria™ kit yielded smaller vesicles (<50 nm) with a higher double-stranded DNA (dsDNA) content, and protein levels were similar across samples. Cultures grown with rabbit serum produced more Bb-OMVs and had cleaner backgrounds in the TEM images than those grown with ED-FBS. All Bb-OMV samples lacked intracellular markers (DnaK and 16S rRNA) and consistently expressed the outer surface protein OspA, confirming high purity. All isolated Bb-OMVs were taken up by the cells, as indicated by OspA expression, without detectable 16S rRNA, confirming vesicle internalization without bacterial contamination. Conclusions: These findings indicate that isolated OMVs are biologically active and capable of interacting with mammalian cells, highlighting their potential role in host–pathogen interactions and the broader relevance of OMVs in studying bacterial modulation of mammalian cell behavior. Overall, both isolation methods produced high-quality OMVs, with ultracentrifugation yielding slightly more pure vesicles, emphasizing the importance of selecting appropriate isolation methods and culture conditions for functional OMV studies. Full article

The naphthazarins shikonin and alkannan are strongly chromogenic, dark red enantiomers, each of which has biological activity, that are found primarily in the plant family Boraginaceae. These compounds and their many chemical metabolites, derivatives, oligomers, and analogs (“shikonoids”) are an important group of phytochemicals, utilized since antiquity as components of dyes, traditional medicines, and food and cosmetics. They are now recognized for their potent anti-inflammatory and regulatory activity on a variety of molecular signaling pathways in humans. Since many Boraginaceae species are overly exploited or endangered, we developed a pilot-scale in vitro shikonoid production system using Plagiobothrys arizonicus (Gray) Greene ex A.Gray, the Arizona popcorn flower, native to the southwestern USA and the Sonoran floristic province in the Madrean region of Mexico. Aseptic root cultures were initiated from fresh leaf tissue and stimulated to continuously produce shikonoids in liquid shake cultures layered under paraffin oil from which the shikonoids were extracted and concentrated. The crude, red extracellular product from these rapidly expanding root masses was also fractionated by Centrifugal Counter-Current Chromatography (CCC) into its component shikonin derivatives. A number of these shikonoids profoundly up-regulated detoxification and antioxidant proteins (phase 2 enzymes) and inhibited inflammation in mammalian cell bioassay systems. This prototype shikonoid production methodology can be readily scaled to either batch or chemostat culture. Full article