Search Results (2,818)

Hyperlipidemia is a major risk factor for vascular lesions in diabetes mellitus and other metabolic disorders, although its basis remains poorly understood. One of the key pathogenetic events in this condition is mitochondrial dysfunction associated with the opening of the mitochondrial permeability transition (MPT) pore, a drop in the membrane potential, and ROS overproduction. Here, we investigated the effects of bongkrekic acid and carboxyatractyloside, a potent blocker and activator of the MPT pore opening, respectively, acting through direct interaction with the adenine nucleotide translocator, on the progression of mitochondrial dysfunction in mouse primary lung endothelial cells exposed to elevated levels of palmitic acid. Palmitate treatment (0.75 mM palmitate/BSA for 6 days) resulted in an 80% decrease in the viability index of endothelial cells, which was accompanied by mitochondrial depolarization, ROS hyperproduction, and increased colocalization of mitochondria with lysosomes. Bongkrekic acid (25 µM) attenuated palmitate-induced lipotoxicity and all the signs of mitochondrial damage, including increased spontaneous formation of the MPT pore. In contrast, carboxyatractyloside (10 μM) stimulated cell death and failed to prevent the progression of mitochondrial dysfunction under hyperlipidemic stress conditions. Silencing of gene expression of the predominate isoform ANT2, similar to the action of carboxyatractyloside, led to increased ROS generation and cell death under conditions of palmitate-induced lipotoxicity in a stably transfected HEK293T cell line. Altogether, these results suggest that targeted manipulation of the permeability transition pore through inhibition of ANT may represent an alternative approach to alleviate mitochondrial dysfunction and cell death in cell culture models of fatty acid overload. Full article

Background: Earlier studies have established that culturing human ovarian tissue in a 3D system with a small amount of soluble Matrigel (a basement membrane protein) for 7 days in vitro increased gene fusion and alternative splicing events, cellular functions, and potentially impacted gene expression. However, this method was not suitable for in vitro culture of human testicular tissue. Objective: To test a new method for long-time in vitro culture of testicular fragments, thawed with two different regimes, with evaluation of transcriptomic differences by RNA sequencing. Methods: Testicular tissue samples were collected, cryopreserved (frozen and thawed), and evaluated immediately after thawing and following one week of in vitro culture. Before in vitro culture, tissue fragments were encapsulated in fibrin. Four experimental groups were formed. Group 1: tissue quickly thawed (in boiling water at 100 °C) and immediately evaluated. Group 2: tissue quickly thawed (in boiling water at 100 °C) and evaluated after one week of in vitro culture. Group 3: tissue slowly thawed (by a physiological temperature 37 °C) and immediately evaluated. Group 4: tissue slowly thawed (by a physiological temperature 37 °C) and evaluated after one week of in vitro culture. Results: There are the fewest differentially expressed genes in the comparison between Group 2 and Group 4. In this comparison, significantly up-regulated genes included C4B_2, LOC107987373, and GJA4, while significantly down-regulated genes included SULT1A4, FBLN2, and CCN2. Differential genes in cells of Group 2 were mainly enriched in KEGG: regulation of actin cytoskeleton, lysosome, proteoglycans in cancer, TGF-beta signaling pathway, focal adhesion, and endocytosis. These Group 2- genes were mainly enriched in GO: spermatogenesis, cilium movement, collagen fibril organization, cell differentiation, meiotic cell cycle, and flagellated spermatozoa motility. Conclusions: Encapsulation of testicular tissue in fibrin and long-time in vitro culture with constant stirring in a large volume of culture medium can reduce the impact of thawing methods on cryopreserved testicular tissue. Full article

Impaired tumor cell antigen presentation contributes significantly to immune evasion. This study identifies Berbamine hydrochloride (Ber), a compound derived from traditional Chinese medicine, as an effective inhibitor of autophagy that enhances antigen presentation in tumor cells. Ber increases MHC-I-mediated antigen presentation in melanoma cells, improving recognition and elimination by CD8⁺ T cells. Mutation of Atg4b, which blocks autophagy, also raises MHC-I levels on the cell surface, and further treatment with Ber under these conditions does not increase MHC-I, indicating Ber’s role in blocking autophagy to enhance MHC-I expression. Additionally, Ber treatment leads to the accumulation of autophagosomes, with elevated levels of LC3-II and p62, suggesting a disrupted autophagic flux. Fluorescence staining and co-localization analyses reveal that Ber likely inhibits lysosomal acidification without hindering autophagosome–lysosome fusion. Importantly, Ber treatment suppresses melanoma growth in mice and enhances CD8⁺ T cell infiltration, supporting its therapeutic potential. Our findings demonstrate that Ber disturbs late-stage autophagic flux through abnormal lysosomal acidification, enhancing MHC-I-mediated antigen presentation and curtailing tumor immune escape. Full article

CMTM6 is a membrane protein that acts as a regulator of PD-L1, maintaining its expression on the cell surface, and can prevent its lysosome-mediated degradation. It is unknown if CMTM6 is present in the plasma of patients with cervical cancer, and if it has non-canonical subcellular localizations in cell lines derived from cervical cancer. Our objective was to determine whether CMTM6 is found in plasma derived from cervical cancer patients and its subcellular localization in cell lines. Patient plasma was separated into exosome-enriched, exosome-free, and total plasma fractions. The levels of CMTM6 in each fraction were determined using ELISA and Western blot. Finally, for the cellular model, HeLa, SiHa, CaSki, and HaCaT were used; the subcellular locations of CMTM6 were determined using immunofluorescence and flow cytometry. Soluble CMTM6 was found to be elevated in plasma from patients with cervical cancer, with a nearly three-fold increase in patients (966.27 pg/mL in patients vs. 363.54 pg/mL in controls). CMTM6 was preferentially, but not exclusively, found in the exosome-enriched plasma fraction, and was positively correlated with exosomal PD-L1; CMTM6 was identified in the membrane, intracellular compartments, and culture supernatant of the cell lines. These results highlight that CMTM6, in its various presentations, may play an important role in the biology of tumor cells and in immune system evasion. Full article

GM1 gangliosidosis is a lysosomal storage disorder characterized by the accumulation of GM1 ganglioside, leading to severe neurodegeneration and early mortality. The disease primarily affects the central nervous system, causing progressive neurodegeneration, including widespread neuronal loss and gliosis. To gain a deeper understanding of the neuropathology associated with GM1 gangliosidosis, we employed single-nucleus RNA sequencing to analyze brain tissues from both GM1 gangliosidosis model mice and control mice. No significant changes in cell proportions were detected between the two groups of animals. Differential expression analysis revealed cell type-specific changes in gene expression in neuronal and glial cells. Functional analysis highlighted the neurodegenerative processes, oxidative phosphorylation, and neuroactive ligand–receptor interactions as the significantly affected pathways. The contribution of the impairment of neurotransmitter system disruption and neuronal circuitry disruption was more important than neuroinflammatory responses to GM1 pathology. In 16-week-old GM1 gangliosidosis mice, no microglial or astrocyte activation or increased expression of innate immunity genes was detected. This suggested that nerve degeneration did not induce the inflammatory response but rather promoted glial cell clearance. Our findings provide a crucial foundation for understanding the cellular and molecular mechanisms of GM1 gangliosidosis, potentially guiding future therapeutic strategies. Full article

Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays pivotal roles in both adaptive and innate immunities. GILT exhibits constitutive expression within antigen-presenting cells, whereas in other cell types, its expression is induced by interferon gamma (IFN-γ). Gaining insights into the precise molecular mechanism governing the induction of GILT protein by IFN-γ is of paramount importance for adaptive and innate immunities. In this study, we found that the 5′ segment of GILT mRNA inhibited GILT protein expression regardless of the presence of IFN-γ. Conversely, the 3′ segment of GILT mRNA suppressed GILT protein expression in the absence of IFN-γ, but it loses this inhibitory effect in its presence. Although the mTOR inhibitor rapamycin suppressed the induction of GILT protein expression by IFN-γ, the expression from luciferase sequence containing the 3′ segment of GILT mRNA was resistant to rapamycin in the presence of IFN-γ, but not in its absence. Collectively, this study elucidates the mechanism behind GILT induction by IFN-γ: in the absence of IFN-γ, GILT mRNA is constitutively transcribed, but the translation process is hindered by both the 5′ and 3′ segments. Upon exposure to IFN-γ, a translation inhibitor bound to the 3′ segment is liberated, and a translation activator interacts with the 3′ segment to trigger the initiation of GILT translation. Full article

ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER. Full article

Considering that the precise delivery of Celastrol (Cst) into mitochondria to induce mitochondrial dysfunction may be a potential approach to improve the therapeutic outcomes of Cst on TNBC, a novel tumor mitochondria dual-targeted mixed-micelle nano-system was fabricated via self-synthesized triphenylphosphonium-modified cholesterol (TPP-Chol) and hyaluronic acid (HA)-modified cholesterol (HA-Chol). The Cst-loaded mixed micelles (Cst@HA/TPP-M) exhibited the characteristics of a small particle size, negative surface potential, high drug loading of up to 22.8%, and sustained drug release behavior. Compared to Cst-loaded micelles assembled only by TPP-Chol (Cst@TPP-M), Cst@HA/TPP-M decreased the hemolysis rate and upgraded the in vivo stability and safety. In addition, a series of cell experiments using the triple-negative breast cancer cell line MDA-MB-231 as a cell model proved that Cst@HA/TPP-M effectively increased the cellular uptake of the drug through CD44-receptors-mediated endocytosis, and the uptake amount was three times that of the free Cst group. The confocal results demonstrated successful endo-lysosomal escape and effective mitochondrial transport triggered by the charge converse of Cst@HA/TPP-M after HA degradation in endo-lysosomes. Compared to the free Cst group, Cst@HA/TPP-M significantly elevated the ROS levels, reduced the mitochondrial membrane potential, and promoted tumor cell apoptosis, showing a better induction effect on mitochondrial dysfunction. In vivo imaging and antitumor experiments based on MDA-MB-231-tumor-bearing nude mice showed that Cst@HA/TPP-M facilitated drug enrichment at the tumor site, attenuated drug systemic distribution, and polished up the antitumor efficacy of Cst compared with free Cst. In general, as a target drug delivery system, mixed micelles co-constructed by TPP-Chol and HA-Chol might provide a promising strategy to ameliorate the therapeutic outcomes of Cst on TNBC. Full article

Combination therapy integrated with nanotechnology offers a promising alternative for breast cancer treatment. The inclusion of pequi oil, anacardic acid (AA), and docetaxel (DTX) in a nanoemulsion can amplify the antitumor effects of each molecule while reducing adverse effects. Therefore, the study aims to develop pequi oil-based nanoemulsions (PeNE) containing DTX (PDTX) or AA (PAA) and to evaluate their cytotoxicity against triple-negative breast cancer cells (4T1) in vitro. The PeNE without and with AA (PAA) and DTX (PDTX) were prepared by sonication and characterized by ZetaSizer^® and electronic transmission microscopy. Viability testing and combination index (CI) were determined by MTT and Chou-Talalay methods, respectively. Flow cytometry was employed to investigate the effects of the formulations on cell structures. PeNE, PDTX, and PAA showed hydrodynamic diameter < 200 nm and a polydispersity index (PdI) of 0.3. The association PDTX + PAA induced a greater decrease in cell viability (~70%, p < 0.0001) and additive effect (CI < 1). In parallel, an association of the DTX + AA molecules led to antagonism (CI > 1). Additionally, PDTX + PAA induced an expressive morphological change, a major change in lysosome membrane permeation and mitochondria membrane permeation, cell cycle blockage in G2/M, and phosphatidylserine exposure. The study highlights the successful use of pequi oil nanoemulsions as delivery systems for DTX and AA, which enhances their antitumor effects against breast cancer cells. This nanotechnological approach shows significant potential for the treatment of triple-negative breast cancer. Full article

The efficacy and potential toxicity of drug treatments depends on the drug concentration at its site of action, intricately linked to its distribution within diverse organelles of mammalian cells. These organelles, including the nucleus, endosome, lysosome, mitochondria, endoplasmic reticulum, Golgi apparatus, lipid droplets, exosomes, and membrane-less structures, create distinct sub-compartments within the cell, each with unique biological features. Certain structures within these sub-compartments possess the ability to selectively accumulate or exclude drugs based on their physicochemical attributes, directly impacting drug efficacy. Under pathological conditions, such as cancer, many cells undergo dynamic alterations in subcellular organelles, leading to changes in the active concentration of drugs. A mechanistic and quantitative understanding of how organelle characteristics and abundance alter drug partition coefficients is crucial. This review explores biological factors and physicochemical properties influencing subcellular drug distribution, alongside strategies for modulation to enhance efficacy. Additionally, we discuss physiologically based computational models for subcellular drug distribution, providing a quantifiable means to simulate and predict drug distribution at the subcellular level, with the potential to optimize drug development strategies. Full article

Cytopathology induced by methamphetamine (METH) is reminiscent of degenerative disorders such as Parkinson’s disease, and it is characterized by membrane organelles arranged in tubulo-vesicular structures. These areas, appearing as clusters of vesicles, have never been defined concerning the presence of specific organelles. Therefore, the present study aimed to identify the relative and absolute area of specific membrane-bound organelles following a moderate dose (100 µM) of METH administered to catecholamine-containing PC12 cells. Organelles and antigens were detected by immunofluorescence, and they were further quantified by plain electron microscopy and in situ stoichiometry. This analysis indicated an increase in autophagosomes and damaged mitochondria along with a decrease in lysosomes and healthy mitochondria. Following METH, a severe dissipation of hallmark proteins from their own vesicles was measured. In fact, the amounts of LC3 and p62 were reduced within autophagy vacuoles compared with the whole cytosol. Similarly, LAMP1 and Cathepsin-D within lysosomes were reduced. These findings suggest a loss of compartmentalization and confirm a decrease in the competence of cell clearing organelles during catecholamine degeneration. Such cell entropy is consistent with a loss of energy stores, which routinely govern appropriate subcellular compartmentalization. Full article

Marburg hemorrhagic fever (MHF) is a fatal infectious disease caused by Marburg virus (MARV) infection, and MARV has been identified as a priority pathogen for vaccine development by the WHO. The glycoprotein (GP) of MARV mediates viral adhesion and invasion of host cells and therefore can be used as an effective target for vaccine development. Moreover, DNA vaccines have unique advantages, such as simple construction processes, low production costs, and few adverse reactions, but their immunogenicity may decrease due to the poor absorption rate of plasmids. Lysosome-associated membrane protein 1 (LAMP1) can direct antigens to lysosomes and endosomes and has great potential for improving the immunogenicity of nucleic acid vaccines. Therefore, we constructed a DNA vaccine based on a codon-optimized MARV GP (ID MF939097.1) fused with LAMP1 and explored the effect of a LAMP targeting strategy on improving the immunogenicity of the MARV DNA vaccine. ELISA, ELISpot, and flow cytometry revealed that the introduction of LAMP1 into the MARV DNA candidate vaccine improved the humoral and cellular immune response, enhanced the secretion of cytokines, and established long-term immune protection. Transcriptome analysis revealed that the LAMP targeting strategy significantly enriched antigen processing and presentation-related pathways, especially the MHC class II-related pathway, in the candidate vaccine. Our study broadens the strategic vision for enhanced DNA vaccine design and provides a promising candidate vaccine for MHF prevention. Full article

Several years ago, dozens of cases were described in patients with symptoms very similar to mucopolysaccharidosis (MPS). This new disease entity was described as mucopolysaccharidosis-plus syndrome (MPSPS). The name of the disease indicates that in addition to the typical symptoms of conventional MPS, patients develop other features such as congenital heart defects and kidney and hematopoietic system disorders. The symptoms are highly advanced, and patients usually do not survive past the second year of life. MPSPS is inherited in an autosomal recessive manner and is caused by a homozygous-specific mutation in the gene encoding the VPS33A protein. To date, it has been described in 41 patients. Patients with MPSPS exhibited excessive excretion of glycosaminoglycans (GAGs) in the urine and exceptionally high levels of heparan sulfate in the plasma, but the accumulation of substrates is not caused by a decrease in the activity of any lysosomal enzymes. Here, we discuss the pathomechanisms and symptoms of MPSPS, comparing them to those of MPS. Moreover, we asked the question whether MPSPS should be classified as a type of MPS or a separate disease, as contrary to ‘classical’ MPS types, despite GAG accumulation, no defects in lysosomal enzymes responsible for degradation of these compounds could be detected in MPSPS. The molecular mechanism of the appearance of GAG accumulation in MPSPS is suggested on the basis of results available in the literature. Full article

Glycogen storage disorders (GSDs) are a group of inherited metabolic disorders characterized by defects in enzymes involved in glycogen metabolism. Deficiencies in enzymes responsible for glycogen breakdown and synthesis can impair mitochondrial function. For instance, in GSD type II (Pompe disease), acid alpha-glucosidase deficiency leads to lysosomal glycogen accumulation, which secondarily impacts mitochondrial function through dysfunctional mitophagy, which disrupts mitochondrial quality control, generating oxidative stress. In GSD type III (Cori disease), the lack of the debranching enzyme causes glycogen accumulation and affects mitochondrial dynamics and biogenesis by disrupting the integrity of muscle fibers. Malfunctional glycogen metabolism can disrupt various cascades, thus causing mitochondrial and cell metabolic dysfunction through various mechanisms. These dysfunctions include altered mitochondrial morphology, impaired oxidative phosphorylation, increased production of reactive oxygen species (ROS), and defective mitophagy. The oxidative burden typical of GSDs compromises mitochondrial integrity and exacerbates the metabolic derangements observed in GSDs. The intertwining of mitochondrial dysfunction and GSDs underscores the complexity of these disorders and has significant clinical implications. GSD patients often present with multisystem manifestations, including hepatomegaly, hypoglycemia, and muscle weakness, which can be exacerbated by mitochondrial impairment. Moreover, mitochondrial dysfunction may contribute to the progression of GSD-related complications, such as cardiomyopathy and neurocognitive deficits. Targeting mitochondrial dysfunction thus represents a promising therapeutic avenue in GSDs. Potential strategies include antioxidants to mitigate oxidative stress, compounds that enhance mitochondrial biogenesis, and gene therapy to correct the underlying mitochondrial enzyme deficiencies. Mitochondrial dysfunction plays a critical role in the pathophysiology of GSDs. Recognizing and addressing this aspect can lead to more comprehensive and effective treatments, improving the quality of life of GSD patients. This review aims to elaborate on the intricate relationship between mitochondrial dysfunction and various types of GSDs. The review presents challenges and treatment options for several GSDs. Full article

Rhodamine 6G (R6G) and 4-nitro-2,1,3-benzoxadiazole (NBD) linked through a spacer molecule spermidine (spd), R6G-spd-NBD, produces a fluorescent probe with pH-sensitive FRET (Förster (fluorescence) resonance energy transfer) effect that can be useful in a variety of diagnostic applications. Specifically, cancer cells can be spotted due to a local decrease in pH (Warburg effect). In this research, we applied this approach to intracellular infectious diseases—namely, leishmaniasis, brucellosis, and tuberculosis, difficult to treat because of their localization inside macrophages. R6G-spd-NBD offers an opportunity to detect such bacteria and potentially deliver therapeutic targets to treat them. The nanogel formulation of the R6G-spd-NBD probe (nanoparticles based on chitosan or heparin grafted with lipoic acid residues, Chit-LA and Hep-LA) was obtained to improve the pH sensitivity in the desired pH range (5.5–7.5), providing selective visualization and targeting of bacterial cells, thereby enhancing the capabilities of CLSM (confocal laser scanning microscopy) imaging. According to AFM (atomic force microscopy) data, nanogel particles containing R6G-spd-NBD of compact structure and spherical shape are formed, with a diameter of 70–100 nm. The nanogel formulation of the R6G-spd-NBD further improves absorption and penetration into bacteria, including those located inside macrophages. Due to the negative charge of the bacteria surface, the absorption of positively charged R6G-spd-NBD, and even more so in the chitosan derivatives’ nanogel particles, is pronounced. Additionally, with a pH-sensitive R6G-spd-NBD fluorescent probe, the macrophages’ lysosomes can be easily distinguished due to their acidic pH environment. CLSM was used to visualize samples of macrophage cells containing absorbed bacteria. The created nanoparticles showed a significant selectivity to model E. coli vs. Lactobacillus bacterial cells, and the R6G-spd-NBD agent, being a mild bactericide, cleared over 50% E.coli in conditions where Lactobacillus remained almost unaffected. Taken together, our data indicate that R6G-spd-NBD, as well as similar compounds, can have value not only for diagnostic, but also for theranostic applications. Full article