Search Results (374)

Serial block-face scanning electron microscopy (SBEM) is a powerful technique for three-dimensional ultrastructural analysis of biological samples, though its application to in vitro cultured human cells remains underutilized. In this study, we present an optimized SBEM sample preparation protocol using human dermal fibroblasts and induced pluripotent stem cells (iPSCs). The method includes key modifications to the original protocol, such as using only glutaraldehyde for fixation and substituting the toxic cacodylate buffer with a less hazardous phosphate buffer. These adaptations result in excellent preservation of cellular ultrastructure, with high contrast and clarity, as validated by transmission electron microscopy (TEM). The loss of natural cell morphology resulted from fixation during passage, when cells formed a precipitate, rather than from fixation directly within the culture medium. The protocol is time-efficient, safe, and broadly applicable to both stem cells and differentiated cells cultured under 2D conditions, providing a valuable tool for ultrastructural analysis in diverse biomedical research settings. Full article

Barium chloride (BaCl₂), a known environmental pollutant, induces organ-specific oxidative stress through disruption of redox homeostasis. This study evaluated the protective effects and safety profile of sodium copper chlorophyllin (SCC) and ascorbic acid (ASC) against BaCl₂-induced oxidative damage in the liver and brain of mice using a two-phase experimental protocol. Animals received either SCC (40 mg/kg), ASC (160 mg/kg), or their combination for 14 days prior to BaCl₂ exposure (150 mg/L in drinking water for 7 days), allowing evaluation of both preventive and therapeutic effects. Toxicological and behavioral assessments confirmed the absence of systemic toxicity or neurobehavioral alterations following supplementation. Body weight, liver and kidney indices, and biochemical markers (Aspartate Aminotransferase (ASAT), Alanine Aminotransferase (ALAT), creatinine) remained within physiological ranges, and no anxiogenic or locomotor effects were observed. In the brain, BaCl₂ exposure significantly increased SOD (+49%), CAT (+66%), GPx (+24%), and GSH (+26%) compared to controls, reflecting a robust compensatory antioxidant response. Although lipid peroxidation (MDA) showed a non-significant increase, SCC, ASC, and their combination reduced MDA levels by 42%, 37%, and 55%, respectively. These treatments normalized antioxidant enzyme activities and GSH, indicating an effective neuroprotective effect. In contrast, the liver exhibited a different oxidative profile. BaCl₂ exposure increased MDA levels by 80% and GSH by 34%, with no activation of SOD, CAT, or GPx. Histological analysis revealed extensive hepatocellular necrosis, vacuolization, and inflammatory infiltration. SCC significantly reduced hepatic MDA by 39% and preserved tissue architecture, while ASC alone or combined with SCC exacerbated inflammation and depleted hepatic GSH by 71% and 78%, respectively, relative to BaCl₂-exposed controls. Collectively, these results highlight a differential, organ-specific response to BaCl₂-induced oxidative stress and the therapeutic potential of SCC and ASC. SCC emerged as a safer and more effective agent, particularly in hepatic protection, while both antioxidants demonstrated neuroprotective effects when used individually or in combination. Full article

Background: Cardiovascular diseases, particularly hypertension, and gastrointestinal disorders represent major public health concerns in Mexico. Although a range of pharmacological treatments exists, their use is associated with adverse effects, highlighting the need for safer therapeutic alternatives. Species of the Ipomoea genus are widely employed in Mexican traditional medicine (MTM) for their purgative, anti-inflammatory, analgesic, and sedative properties. Particularly, Ipomoea purpurea is traditionally used as a diuretic and purgative; its leaves and stems are applied topically for their anti-inflammatory and soothing effects. This study aimed to determine their phytochemical composition and to evaluate the associated vasodilatory activity, modulatory effects on intestinal smooth-muscle motility, and toxicological effects of the methanolic (ME-Ip) and dichloromethane (DE-Ip) extracts obtained from the aerial parts of I. purpurea. Methods: The phytochemical composition of the ME-Ip and DE-Ip extracts of I. purpurea was assessed using UPLC-QTOF-MS and GC-MS, respectively. For both extracts, the vasodilatory activity and effects on intestinal smooth muscle were investigated using ex vivo models incorporating isolated rat aorta and ileum, respectively, whereas acute toxicity was evaluated in vivo. Results: Phytochemical analysis revealed, for the first time, the presence of two glycosylated flavonoids within the Ipomoea genus; likewise, constituents with potential anti-inflammatory activity were detected. The identified compounds in I. purpurea extracts may contribute to the vasodilatory, biphasic, and purgative effects observed in this species. The EC₅₀ values for the vasodilatory effects of the methanolic (ME-Ip) and dichloromethane (DE-Ip) extracts were 0.80 and 0.72 mg/mL, respectively. In the initial phase of the experiments on isolated ileal tissues, both extracts induced a spasmodic (contractile) effect on basal motility, with ME-Ip exhibiting higher potency (EC₅₀ = 27.11 μg/mL) compared to DE-Ip (EC₅₀ = 1765 μg/mL). In contrast, during the final phase of the experiments, both extracts demonstrated a spasmolytic effect, with EC₅₀ values of 0.43 mg/mL for ME-Ip and 0.34 mg/mL for DE-Ip. In addition, both extracts exhibited low levels of acute toxicity. Conclusions: The phytochemical profile and the vasodilatory and biphasic effects of the I. purpurea extracts explain, in part, the use of I. purpurea in MTM. The absence of acute toxic effects constitutes a preliminary step in the toxicological safety assessment of I. purpurea extracts and demonstrates their potential for the development of phytopharmaceutic agents as adjuvants for the treatment of cardiovascular and gastrointestinal disorders. Full article

Vaccine adjuvants are non-immunogenic agents that enhance or modulate immune responses to co-administered antigens and are essential to modern vaccines. Despite their importance, few are approved for human use. The rise of new pathogens and limited efficacy of some existing vaccines underscore the need for more advanced and effective formulations, particularly for vulnerable populations. Aluminum-based adjuvants are commonly used in vaccines and effectively promote humoral immunity. However, they mainly induce a Th2-biased response, making them suboptimal for diseases requiring cell-mediated immunity. In contrast, saponin-based adjuvants from the Quillajaceae family elicit a more balanced Th1/Th2 response and generate antigen-specific cytotoxic T cells (CTL). Due to ecological damage and limited availability caused by overharvesting Quillaja saponaria Molina barks, efforts have intensified to identify alternative plant-derived saponins with enhanced efficacy and lower toxicity. Quillaja brasiliensis (A.St.-Hil. and Tul.) Mart. (syn. Quillaja lancifolia D.Don), a related species native to South America, is considered a promising renewable source of Quillajaceae saponins. In this review, we highlight recent advances in vaccine adjuvant research, with a particular focus on saponins extracted from Q. brasiliensis leaves as a sustainable alternative to Q. saponaria saponins. These saponin fractions are structurally and functionally comparable, exhibiting similar adjuvant activity when they were formulated with different viral antigens. An alternative application involves formulating saponins into nanoparticles known as ISCOMs (immune-stimulating complexes) or ISCOM-matrices. These formulations significantly reduce hemolytic activity while preserving strong immunoadjuvant properties. Therefore, research advances using saponin-based adjuvants (SBA) derived from Q. brasiliensis and their incorporation into new vaccine platforms may represent a viable and sustainable solution for the development of more less reactogenic, safer, and effective vaccines, especially for diseases that require a robust cellular immunity. Full article

Oral microbiota have been implicated in pancreatic ductal adenocarcinoma (PDAC) and may contribute to chemotherapy resistance. While previous studies attributed bacteria-induced resistance to indirect host modulation, recent findings suggest a direct mechanism. Escherichia coli expressing long-form cytidine deaminase (CDD_L) can degrade gemcitabine, a chemotherapeutic agent, into a non-toxic form, leading to resistance. In contrast, bacteria carrying short form (CDD_S) or lacking CDD did not induce resistance. This study investigates whether oral bacteria can cause gemcitabine resistance in PDAC cells through CDD-mediated degradation. Oral microbes associated with PDAC were selected based on CDD isoforms: Aggregatibacter actinomycetemcomitans carrying CDD_L, Enterococcus faecalis, Streptococcus mutans, Porphyromonas gingivalis, all carrying CDD_S, and Fusobacterium nucleatum lacking CDD. The selected microbes, along with wild-type and CDD-deficient E. coli, were co-incubated with gemcitabine to assess its degradation and PDAC cell proliferation. A. actinomycetemcomitans fully degraded gemcitabine and induced resistance. Surprisingly, CDD_S-expressing oral bacteria partially degraded gemcitabine in a strain-dependent manner. Expressing either CDD_L or CDD_S in CDD-deficient E. coli resulted in equivalent gemcitabine degradation and resistance, indicating that CDD function is independent of isoform length. These findings highlight the role of oral bacteria in gemcitabine resistance and the need for strategies to mitigate microbial-driven resistance in PDAC treatment. Full article

Multiple myeloma (MM) is an incurable malignancy of plasma cells that accounts for 10% of all haematological malignancies diagnosed worldwide. The poor outcome of patients with MM highlights the ongoing need for novel treatment strategies. Ferroptosis is a recently characterised form of non-apoptotic programmed cell death. Phospholipids (PLs) containing polyunsaturated fatty acids (PUFAs) play a crucial role as ferroptosis substrates when oxidised to form toxic lipid reactive oxygen species (ROS). Using a range of scientific techniques, we demonstrate a strong correlation between the PL profile of MM and diffuse large B cell lymphoma (DLBCL) cells with their sensitivity to ferroptosis. Using this PL profiling, we manufacture liposomes that are themselves composed of PL-PUFA ferroptosis substrates relatively deficient in MM cells, with and without the GPX4 inhibitor, RSL3, for investigation of their ferroptosis-inducing potential. PL-PUFAs were more abundant in DLBCL than MM cell lines, consistent with greater ferroptosis sensitivity. In contrast, MM cells generally contained a significantly higher proportion of PLs containing monounsaturated fatty acids. Altering the lipid composition of MM cells through exogenous supplementation with PL-PUFAs induced ferroptosis-mediated cell death and further sensitised these cells to RSL3. Liposomes predominantly comprising PL-PUFAs were subsequently manufactured and loaded with RSL3. Uptake, cytotoxicity and lipid ROS studies demonstrated that these novel liposomes were readily taken up by MM cells. Those containing RSL3 were more effective at inducing ferroptosis than empty liposomes or free RSL3, resulting in IC₅₀ values an average 7.1-fold to 14.5-fold lower than those for free RSL3, from the micromolar to nanomolar range. We provide a better understanding of the mechanisms associated with ferroptosis resistance of MM cells and suggest that strategies such as liposomal delivery of relatively deficient ferroptosis-inducing PL-PUFAs together with other targeted agents could harness ferroptosis for the personalised treatment of MM and other cancers. Full article

p-coumaric acid (p-CA) is one of the main allelochemicals of cultivable Morchella mushrooms. However, its toxicity mechanism has not been elucidated. Therefore, we used physiological and comparative transcriptomic analyses to reveal its toxicity mechanism. The results suggest that the mycelial growth and sclerotial production of M. importuna were promoted under treatment with a low dosage of p-CA (10 μg/mL). The treatment induced moderate reactive oxygen species (ROS) accumulation, with an upregulation of genes associated with antioxidant regulation, energy supply and damage repair. In contrast, oxidative stress induced under treatment with a high dosage of p-CA (50 μg/mL) led to strain ageing. The contents of ROS were significantly increased, along with decreased peroxidase and catalase activity. Moreover, the genes associated with H₂O₂ synthesis were upregulated, while those responsible for H₂O₂ decomposition, non-enzymatic antioxidant components and damage repair were downregulated. Meanwhile, the carbohydrate and lipid metabolic pathways, and the signal transduction and cell division pathways, were impaired. Taken together, moderate stress induced under a low concentration of p-CA promotes the mycelial growth and sclerotial metamorphosis of M. importuna. This study provides new insights into the potential mechanisms of continuous cropping obstacles in the cultivation of morel mushrooms, which is of great importance for the practical aspects of mushroom cultivation. Full article

Toxicological studies of pesticides in animal models provide critical insights into their mechanisms of action, while adsorption strategies offer potential solutions for decontaminating polluted waters. We evaluated toxicity induced by tetraethyl pyrophosphate (TEPP), an organophosphate pesticide and AChE inhibitor, on zebrafish (Danio rerio) development and behavior, alongside the efficacy of wine cork granules as a natural adsorbent. TEPP exposure reduced embryo viability following an inverted U-shaped dose–response curve, suggesting non-monotonic neurodevelopmental effects, but did not alter developmental timing or morphology in survivors. In juveniles, TEPP increased preference for dark environments (33% vs. controls) and enhanced swimming endurance approximately 3-fold, indicating disrupted phototaxis and stress responses. Most strikingly, water treated with cork granules retained toxicity, increasing mortality, delaying embryogenesis, and altering behavior. This directly contradicts in vitro adsorption studies that suggested cork’s efficacy. These results demonstrate the high sensitivity of zebrafish to TEPP at nanomolar concentrations, which contrasts with in vitro models that require doses approximately 1000 times higher. Our findings not only highlight TEPP’s ecological risks but also reveal unexpected limitations of cork granules for environmental remediation, urging caution in their application. Full article

Alzheimer’s disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder characterized by memory loss and cognitive decline. In addition to its two major pathological hallmarks, extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles (NFTs), recent evidence highlights the critical roles of mitochondrial dysfunction and neuroinflammation in disease progression. Aβ impairs mitochondrial function, which, in part, can subsequently trigger inflammatory cascades, creating a vicious cycle of neuronal damage. Estrogen receptors (ERs) are widely expressed throughout the brain, and the sex hormone 17β-estradiol (E2) exerts neuroprotection through both anti-inflammatory and mitochondrial mechanisms. While E2 exhibits neuroprotective properties, its mechanisms against Aβ toxicity remain incompletely understood. In this study, we investigated the neuroprotective effects of E2 against Aβ-induced mitochondrial dysfunction and neuroinflammation in primary cortical neurons, with a particular focus on the role of AMP-activated protein kinase (AMPK). We found that E2 treatment significantly increased phosphorylated AMPK and upregulated the expression of mitochondrial biogenesis regulator peroxisome proliferator-activated receptor gamma coactivator-1 α (PGC-1α), leading to improved mitochondrial respiration. In contrast, Aβ suppressed AMPK and PGC-1α signaling, impaired mitochondrial function, activated the pro-inflammatory nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), and reduced neuronal viability. E2 pretreatment also rescued Aβ-induced mitochondrial dysfunction, suppressed NF-κB activation, and, importantly, prevented the decline in neuronal viability. However, the pharmacological inhibition of AMPK using Compound C (CC) abolished these protective effects, resulting in mitochondrial collapse, elevated inflammation, and cell death, highlighting AMPK’s critical role in mediating E2’s actions. Interestingly, while NF-κB inhibition using BAY 11-7082 partially restored mitochondrial respiration, it failed to prevent Aβ-induced cytotoxicity, suggesting that E2’s full neuroprotective effects rely on broader AMPK-dependent mechanisms beyond NF-κB suppression alone. Together, these findings establish AMPK as a key mediator of E2’s protective effects against Aβ-driven mitochondrial dysfunction and neuroinflammation, providing new insights into estrogen-based therapeutic strategies for AD. Full article

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality globally, with metabolic-dysfunction-associated steatohepatitis (MASH) and alcohol-related liver disease (ALD) emerging as major etiologies. This review explores the epidemiological trends, pathogenesis, and clinical management of HCC arising from MASH and ALD, highlighting both the shared and distinct mechanisms. MASH-HCC is driven by metabolic dysregulation, including obesity, insulin resistance, and lipotoxicity, with genetic polymorphisms such as PNPLA3 and TM6SF2 playing critical roles in disease progression. ALD-HCC, in contrast, is propelled by the toxic byproducts of ethanol metabolism, including acetaldehyde and reactive oxygen species, which induce chronic inflammation, and fibrosis. Both conditions also involve immune dysregulation, gut dysbiosis, and increased intestinal permeability, contributing to hepatic carcinogenesis. The review emphasizes that, while there is consensus regarding the screening of HCC in cirrhosis patients, there is lack of consensus on screening strategies for non-cirrhotic MASH patients who are also at risk for HCC. This underscores the importance of the early detection of cirrhosis using advanced diagnostic tools such as transient elastography and fibrosis scores. Current therapeutic approaches, ranging from surgical resection, liver transplantation, and locoregional therapies to systemic therapies like immune checkpoint inhibitors, are discussed, with an emphasis on the need for personalized treatment strategies. Finally, the review highlights future research priorities, including the development of novel biomarkers, exploration of the gut–liver axis, and deeper investigation of the interplay between genetic predisposition and environmental factors. By synthesizing these insights, the review aims to inform multidisciplinary approaches to reduce the global burden of MASH- and ALD-related HCC and improve patient outcomes. Full article

Kynurenic acid (KYNA), a putative neuroprotective agent, modulates glutamatergic pathways in schizophrenia and Parkinson’s disease but is limited by acute motor activity impairments (e.g., ataxia). Research leveraging animal disease models explores its structure–activity relationship to enhance therapeutic efficacy while mitigating adverse effects, addressing global neuropsychiatric disorders affecting over 1 billion people. Structural analogs of KYNA (SZR-72, SZR-73, and SZR-81) were designed to uncouple therapeutic benefits from motor toxicity; yet, systematic comparisons of their acute behavioral profiles remain unexplored. Here, we assess the motor safety, time-dependent effects, and therapeutic potential of these analogs in mice. Using acute intracerebroventricular dosing, we evaluated motor coordination (rotarod), locomotor activity (open-field), and stereotypic behaviors. KYNA induced significant ataxia and stereotypic behaviors at 15 min, resolving by 45 min. In contrast, all analogs avoided acute motor deficits, with SZR-73 maintaining baseline rotarod performance and eliciting a delayed decrease in ambulation and inquisitiveness in open-field assays. These findings demonstrate that the structural optimization of KYNA successfully mitigates motor toxicity while retaining neuromodulatory activity. Here, we show that SZR-73 emerges as a lead candidate, combining transient therapeutic effects with preserved motor coordination. This study advances the development of safer neuroactive compounds, bridging a critical gap between preclinical innovation and clinical translation. Future work must validate chronic efficacy, disease relevance, and mechanistic targets to harness the full potential of KYNA analogs in treating complex neuropsychiatric disorders. Full article

The life expectancy of patients with psychotic disorders is significantly shorter than that of the general population; antipsychotic-induced metabolic disorders play a significant role in reducing life expectancy. Both metabolic syndrome (MetS) and schizophrenia are multifactorial conditions. One area where the two conditions overlap is oxidative stress, which is present in both diseases. The glutathione-S-transferase (GST) system is a major line of defense against exogenous toxicants and oxidative damage to cells. The aim of our study was to perform an association analysis of gene polymorphisms with metabolic disorders in patients with schizophrenia treated with antipsychotic therapy. Methods: A total of 639 white patients with schizophrenia (ICD-10) from Siberia (Russia) were included in the study. Genotyping was carried out using real-time polymerase chain reaction for two single-nucleotide polymorphisms (SNPs) in the GSTP1 (rs614080 and rs1695) and one SNP in the GSTO1 (rs49252). Results: We found that rs1695*GG genotype of GSTP1 is a risk factor for the development of overweight (OR 2.36; 95% CI: 1.3–4.29; p = 0.0054). In the subgroup of patients receiving first-generation antipsychotics as basic therapy, the risk of overweight was associated with carriage of the rs1695*GG (OR 5.43; 95% CI: 2.24–13.16; p < 0.001) genotype of GSTP1 in a recessive model of inheritance. In contrast, an association of rs1695*G GSTP1 with obesity (OR: 0.42; 95% CI: 0.20–0.87; p = 0.018) was shown in the dominant model of inheritance in patients receiving second-generation antipsychotics. Conclusions: The pilot results obtained confirm the hypothesis of a violation of the antioxidant status, in particular the involvement of GSTP1, in the development of antipsychotic-induced metabolic disorders in schizophrenia. Further studies with larger samples and different ethnic groups are needed to confirm the obtained results. Full article

Ricin is a highly potent toxin that causes severe lung injury upon inhalation by initiating a complex cascade of cellular responses that ultimately leads to cell death. The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in various physiological processes, including ricin-mediated toxicity. This study explores the role of LRP1 shedding in the development of ricin-induced lung injury. Analysis of bronchoalveolar lavage fluid (BALF) from ricin-intoxicated mice and swine showed a significant increase in soluble LRP1 (sLRP1) levels, whereas serum LRP1 levels remained largely unchanged, suggesting the lungs are the primary source of sLRP1 release. In vitro assays demonstrated the formation of ricin-sLRP1 complexes, indicating that sLRP1 in BALF retained ricin-binding capability. Flow cytometric analysis of lung cells revealed a reduction in both the percentage and total number of LRP1-expressing cells following ricin exposure. Further investigation of specific lung cell populations showed that alveolar epithelial type II (AT-II) cells, despite experiencing significant injury, exhibited minimal LRP1 shedding. No shedding of LRP1 occurred in neutrophils. In contrast, fibroblasts, which were resistant to ricin-induced cell death, exhibited increased shedding of LRP1 and a corresponding decrease in membrane-bound LRP1 expression. This shedding of the LRP1 ectodomain was mediated by metalloproteinases. Immunohistochemical staining further confirmed decreased LRP1 expression in fibroblasts from ricin-exposed mice. Macrophages also showed substantial LRP1 shedding, despite undergoing significant depletion. These findings highlight the complex cell-specific nature of LRP1 shedding in response to ricin intoxication and suggests the potential role of LRP1 in modulation of cellular susceptibility and resistance to ricin-induced lung injury. Full article

Antibiotics are indispensable in medical patient care, yet they may elicit off-target effects, particularly by affecting mitochondrial function. This study investigates three commonly used antibiotics, gentamicin, ciprofloxacin, and amoxicillin, for their direct effects on mitochondrial respiration and membrane potential. Using high-resolution respirometry, we show that gentamicin and ciprofloxacin markedly increase mitochondrial leak respiration in permeabilized human embryonic kidney cells, suggesting alterations in the mitochondrial inner membrane. This is supported by a gentamicin-induced decrease in mitochondrial membrane potential. Especially gentamicin, but also ciprofloxacin, dose- and time-dependently inhibit oxidative phosphorylation and the mitochondrial electron transfer capacity, pronouncedly in the NADH-linked but also in the succinate-linked pathway. Furthermore, gentamicin decreases Complex IV (CIV) activity in a time-dependent fashion. In contrast, amoxicillin has no significant effect on mitochondrial respiration. These findings emphasize the importance of evaluating the potential direct toxicity of antibiotics on mitochondria, as they are most critical off-target sites. High-resolution respirometry provides a powerful approach to characterize such effects early in the drug development process. Full article

Cervical cancer remains a major threat to women’s health, with advanced cases often exhibiting recurrence and metastasis due to cancer stem cells driving therapy resistance. This study evaluated fenbendazole (FBZ), a repurposed veterinary anthelmintic, for its antitumor activity dual targeting cervical cancer cells (CCCs) and cervical cancer stem cells (CCSCs). CD133⁺CD44⁺ CCSCs were isolated from HeLa and C-33 A cell lines via immunomagnetic sorting and validated for stemness. Cell proliferation, cell cycle and apoptosis, and protein expression were detected by MST assay, flow cytometry, and Western blot analysis, respectively. FBZ dose-dependently inhibited proliferation, induced G2/M arrest, and triggered apoptosis in both CCCs and CCSCs. Mechanistically, FBZ upregulated cyclin B1 and phosphorylation of cdc25C-Ser198, while downregulating Wee1, phosphorylation of CDK1, and phosphorylation of cdc25C-Ser216, collectively enforcing G2/M blockade. In vivo, FBZ (100 mg/kg) significantly suppressed tumor growth in xenograft models without weight loss, contrasting with cisplatin-induced toxicity. Survival analysis revealed 100% survival in FBZ-treated mice versus 40% in cisplatin and 0% in untreated controls. These findings demonstrate FBZ’s unique ability to simultaneously target bulk tumor cells and therapy-resistant CCSCs via cell cycle disruption, supported by its preclinical safety and efficacy, positioning it as a promising therapeutic candidate for cervical cancer. Full article