The lack of disease-modifying therapies for Parkinson's disease (PD) places a severe burden on patients, their caregivers and aging societies. While the incomplete knowledge of underlying causes and pathophysiology remains a major bottleneck towards the development of rational therapies, there have been important advances in our understanding of neurodegenerative processes. Here, animal models have contributed substantially by allowing researchers to decipher PD-relevant pathology and the development of corresponding behavioral signs, which enabled development and refinement of treatment options. Without animal models, hypotheses of mechanisms underlying the development of motor symptoms after dopamine depletion, dyskinesia as side effects, the impact of diverse environmental neurotoxins, or the formation of alpha-synuclein pathology and spreading could not have been conclusively tested. Likewise, novel technologies are enabling the use of patient derived tissues for generating in vitro models that further enhance our understanding of mechanisms and of the effects of specific genetic modifications and their role in neurodegeneration. However, animal models are, as of yet, still required to model a fully functional central nervous system connected to the immune system and peripheral organs, which are impacted by the diversity of PD pathology and symptoms. Here, we provide an overview of key contributions of animal models, their ongoing role in PD research, and how this research can be enhanced by the concomitant use of advanced in vitro systems. Animal models and cell models to study Parkinson's diseaseThere is currently no treatment that can slow or stop the progression of Parkinson's disease (PD), which places a heavy burden on patients, caregivers, and aging societies. One major reason for this is that we still do not fully understand what causes PD. However, recent research has improved our understanding of the underlying disease processes. Animal models have played a crucial role in this progress. They allow scientists to study how PD-related loss of neurons lead to symptoms, and they have helped in developing and improving treatments. Without animal models, we would not be able to test important ideas about how dopamine loss leads to motor symptoms, why certain treatments cause side effects like dyskinesia, how environmental toxins contribute to the disease, or how abnormal proteins like alpha- synuclein spread in the brain. New models now let researchers study cells and tissues taken from patients. These tools make it easier to explore how specific genetic mutations contribute to brain cell damage. Still, animal models are currently the only way to study how Parkinson's affects the whole nervous system, including its connections to the immune system and other organs, all of which can be involved in the disease. This article reviews the important contributions of animal models to Parkinson's research, their ongoing value, and how combining them with advanced in vitro systems can improve our understanding of the disease and lead to better treatments.
The primary objective of this study is to evaluate whether anesthetic agents modulate the apoptotic activity and migration-inhibitory effects of chemotherapeutic agents, thereby informing the optimization of perioperative management strategies in oncologic interventions. SAOS-2 cells were treated with varying concentrations of thiopental, ketamine, and doxorubicin. Cell viability was assessed via MTT assay to determine IC₅₀ values. Thiopental exhibited greater antiproliferative activity than ketamine and was selected for further study. Its interaction with doxorubicin was analyzed using the Chou-Talalay method, yielding a combination index (CI) of 0.92608 at Fa = 0.5, indicating moderate synergism. The combination treatment also led to reduced IC₅₀ values. Cell migration was evaluated using a scratch wound healing assay. Gene expression changes related to apoptosis and metastasis were analyzed using qRT-PCR. In silico molecular docking simulations were performed to provide mechanistic insights into the observed biological effects. Thiopental and doxorubicin exhibited dose-dependent cytotoxicity, with the combination showing enhanced antiproliferative activity. Co-treatment reduced IC₅₀ values and suppressed SAOS-2 cell migration more effectively than either drug alone. Gene expression analysis revealed increased pro-apoptotic markers and decreased Bcl-2 expression. Metastasis-associated genes were significantly downregulated, despite upregulation of hypoxia-responsive genes. Molecular docking analyses identified interactions of thiopental and doxorubicin with HSP70, HSP90, and CYP450, providing complementary information for the interpretation of the observed cellular responses. The findings suggest that thiopental may influence the cellular response to doxorubicin in osteosarcoma through treatment-associated modulation of apoptosis- and migration-related molecular responses. The observed moderate synergistic interaction and dose-reduction potential warrant further investigation in more clinically relevant experimental models.
Weaning stress predisposes piglets to intestinal barrier disruption and gut dysbiosis, which contribute to post-weaning diarrhea and poor feed efficiency. Chicory (Cichorium intybus L.) polysaccharide (CLP) is a fructan-rich prebiotic candidate; however, how CLP reshapes the microbiota-metabolite network to protect the colon remains unclear. In Exp. 1, 96 weaned piglets [Duroc × (Landrace × Yorkshire), 28 days old, 8.03 ± 0.2 kg] were fed a basal diet (CON group) or a 0.5% CLP supplemented diet (CLP group). In Exp. 2, fecal microbiota from piglets were transplanted into dextran sulfate sodium (DSS)-induced mice to confirm the causal role of the CLP-remodeled microbiota. Metagenomic and untargeted metabolomic analyses were employed to identify key microbial species and functional metabolites. In Exp. 3, Caco-2 cells were treated with varying concentrations of hyodeoxycholic acid (HDCA) for 24 h to functionally validate the regulatory effects on TGR5 and FXR expression levels. The results showed that dietary CLP significantly decreased the feed to gain ratio, diarrhea rate and histology index (P < 0.05), but increased goblet cell numbers (P < 0.05). Metagenomic sequencing revealed that CLP significantly increased microbial α-diversity and remodeled the community structure, specifically enriching beneficial microbes, such as Blautia sp., Eubacterium sp., and Ruminococcus sp. To test microbiota causality, fecal microbiota from CON or CLP piglets was transplanted into antibiotic treated mice followed by DSS challenge. The CLP modified microbiota alleviates DSS induced colitis, upregulated Occludin and ZO-1 expression, and reduced colonic IL-1β and TNF-α levels. Mechanistically, the CLP remodeled microbiota promoted the accumulation of HDCA, which functioned as a signaling ligand to activate the colonic TGR5 receptor. This activation subsequently suppressed the phosphorylation of Akt (P < 0.05), leading to the inhibition of the NF-κB signaling pathway through the reduced phosphorylation of IκBα and the p65 subunit (P < 0.05), thereby effectively abrogating the inflammatory response. Dietary CLP supplementation mitigates weaning induced intestinal injury and inflammation by remodeling the colonic microbiota, specifically enriching HDCA-producing species. The subsequent activation of the HDCA-TGR5-Akt signaling axis inhibits the NF-κB pathway, thereby improving host immune responses and intestinal barrier function.
Nanomaterials have gained huge importance in various fields, such as healthcare, electronics, and environmental management, due to their unique characteristics. However, the potent toxicity is a big concern regarding their utility. Toxicological assessment becomes an important step to ensure their safe applications. The significant hazards of nanomaterials have risen from their shape, size, charge, and makeup, which leads to genetic damage, cellular uptake, and internalization with oxidative stress (OS) and harm to specific organs. This review focuses on toxicity profiles and their possible interactions with cellular organelles leading to apoptosis (Apop) and long-term bioaccumulation.Size, surface area, and dissolving capability may affect the toxicity of nanomaterials. Different measures such as encapsulation methods, increasing surface area, and enhancing biodegradability are being considered to reduce toxicity from innovative nanomaterials. Knowledge gaps encompass uneven toxicity evaluations, inadequate chronic exposure data, and insufficient attention to individualized reactions. The integration of standardized models, computational predictions, mechanistic investigations, and regulatory compliance is crucial for the safer design of nanomaterials. The review highlights toxicological issues caused by nanomaterials such as nanoformulation, quantum dots, and nanoparticles. It also directs future strategies in nanotoxicology, highlighting increasing assessment models, strategies, and collaborative research efforts. Bringing together regulatory and research efforts in the context of nanomaterials is important for ensuring the safe application of nanomaterials across pharmacy and other industries. This review offers a comprehensive viewpoint connecting physicochemical factors, sophisticated in vitro models, and developing regulatory frameworks, emphasizing emerging trends including microphysiological systems and animal-free risk assessment methodologies.
Natural therapeutic alternatives are increasingly explored in endometriosis, a highly prevalent gynecological disorder with limited therapeutic options. Rosmarinus officinalis (rosemary) has attracted increasing scientific interest due to its biological activity. This study aimed to characterize a hydroethanolic rosemary extract (RE) and evaluate its effects on key cellular processes involved in endometriosis pathophysiology. Major phenolic compounds in RE were quantified by RP-HPLC, and antioxidant activity was assessed using DPPH, ABTS, and FRAP assays. After RE treatment, cell viability (WST-1), migration (wound healing assay), cell cycle distribution (DAPI staining), apoptosis (Annexin V/PI), p21 and cyclin A expression (Western blot), and intracellular ROS levels (DCFH-DA) were evaluated in endometrial stromal (t-HESC, St-T1b) and endometriotic epithelial (12-Z) cells. Phytochemical analysis revealed rosmarinic acid (RA) at 4.2%, while carnosic acid (CA) and carnosol (CS) together accounted for 23.7% of the extract. RE reduced cell viability and cell migration in 12-Z and t-HESC cells (p < 0.05). S-phase accumulation with a concomitant reduction in the G1 phase was observed across all evaluated cell lines (p < 0.05), along with increased p21 and cyclin A expression in stromal cells (p < 0.05). RE induced cell death in both 12-Z (p < 0.05) and St-T1b cells (p < 0.0001). In t-HESC cells, RE reduced both basal and H2O2-induced ROS levels (p < 0.01). These findings indicate that RE modulates key mechanisms involved in endometriosis pathophysiology, supporting its multi-target therapeutic potential as a nutraceutical approach for endometriosis management.
Alopecia is a common disorder that can cause hair loss owing to various factors, including genetics, stress and diet. Androgenetic alopecia (AGA), also known as male pattern baldness, is the most common type of progressive hair loss. AGA symptoms can be alleviated by effective drugs, such as finasteride and minoxidil. Therefore, most patients with alopecia select drug therapy as the first-line treatment. However, their side effects and variable efficacy have increased the demand for alternative treatment options for these patients. Despite the high clinical demand, the development of novel therapeutic agents remains limited, primarily due to the lack of physiologically relevant drug testing models that accurately predict drug effects in humans. In this At a Glance article, we provide an overview of the currently available in vitro models for hair drug testing. First, we outline the limitations of traditional models, such as two-dimensional cultures and animal models, in reproducing hair follicle structures and multicellular signaling pathways. We then introduce three-dimensional culture platforms that partially overcome these drawbacks. Next, we discuss the differences and applications of various models in terms of functional readouts, reproducibility, high-throughput potential, standardization and cost. We highlight recent progress in the development of follicular skin model constructs and propose practical metrics for selecting the most appropriate model for the initial screening of potential hair drugs. Overall, this At a Glance article provides a roadmap towards a more translationally valuable screening system for the discovery and development of hair loss treatments.
Gestational diabetes mellitus (GDM) is a metabolic condition of pregnancy, characterized by hyperglycemia and glucose intolerance first diagnosed during the 2nd or 3rd trimester of pregnancy. In healthy pregnancies, pancreatic islets adapt by increasing beta-cell mass and function to maintain glucose homeostasis in the face of systemic insulin resistance, which develops with progression of pregnancy. However, in GDM, individuals experience hyperglycemia when beta-cells fail to compensate for this pregnancy-induced insulin resistance. GDM is a heterogeneous condition, with subtypes characterized by defects in insulin sensitivity, secretion, or both. Despite a substantial number of GDM cases being primarily the result of defects in insulin secretion, the majority of research concerns only insulin sensitivity in peripheral tissues, with less attention paid to insulin secretion and associated islet dysfunction, largely due to the absence of adequate models. Here, we review the pathophysiology of GDM at the islet and beta-cell levels. Numerous factors, including glucotoxicity and proinflammatory cytokines, can contribute to beta-cell dysfunction in GDM, similar to the metabolic dysfunction mechanisms of type 2 diabetes mellitus. Other potential contributors to beta-cell dysfunction in GDM are pregnancy-specific, including dysregulation of placental steroid hormones and lactogenic peptide hormones. Various models have been generated in an attempt to recapitulate the effects of GDM, with the use of modified diets and genetic alterations in animal models common in GDM research. However, there remains no well-characterized physiological animal model of GDM. In vitro models and analysis of human tissues offer promise, but human samples are difficult to access due to the rarity of cadaveric donors for this transient and survivable disease. Defining the molecular and cellular mechanisms driving GDM pathophysiology, specifically at the islet level, is fundamental to developing precise therapeutic strategies that prevent both gestational complications and long-term diabetic sequelae. Gestational diabetes mellitus (GDM) affects about 14% of all pregnancies. Whether left untreated or treated with current therapies, GDM has far-reaching impacts on both maternal health as well as future disease risk for babies born to pregnancies afflicted by the disease. We currently understand GDM to be caused by a combination of problems with two related systems: 1) the pregnant body’s inability to adequately respond to the hormone insulin, known as insulin resistance, and 2) an inability of the maternal insulin-producing cells, beta-cells, to secrete enough insulin to maintain healthy blood sugar levels. Despite insulin secretion by beta-cells being an extremely important aspect of GDM development, the vast majority of research on the disease concerns only insulin resistance. In this review, we dive into what is known about beta-cell failure in GDM, how nutrients, inflammation, and hormones direct the beta-cells toward function or dysfunction, and describe the challenges inherent in investigating these insulin-producing cells in the context of pregnancy.
Genetic studies of human embryonic morphogenesis are constrained by ethical and practical challenges, restricting insights into developmental mechanisms and disorders. Human pluripotent stem cell (hPSC)-derived organoids provide a powerful alternative for the study of embryonic morphogenesis. However, screening for genetic drivers of morphogenesis in vitro has been infeasible due to organoid variability and the high costs of performing scaled tissue-wide single-gene perturbations. By overcoming both these limitations, we developed a platform that integrates reproducible organoid morphogenesis with uniform single-gene perturbations, enabling high-throughput arrayed CRISPR interference screening in hPSC-derived organoids. To demonstrate the power of this platform, we screened 77 transcription factors in an organoid model of anterior neurulation to identify ZIC2, SOX11, and ZNF521 as essential regulators of neural tube closure. We discovered that ZIC2 and SOX11 are required for closure, while ZNF521 prevents ectopic closure points. Single-cell transcriptomic analysis of perturbed organoids revealed co-regulated gene targets of ZIC2 and SOX11 and an opposing role for ZNF521, suggesting that these transcription factors jointly govern a gene regulatory program driving neural tube closure in the anterior forebrain region. Our single-gene perturbation platform enables high-throughput genetic screening of in vitro models of human embryonic morphogenesis. Proper organ morphology is a critical outcome of embryonic development. However, the genetic basis of human developmental disorders affecting tissue morphogenesis, such as neural tube closure (a process essential for the formation of the brain and spinal cord), has proven difficult to study because of ethical limitations on human embryo research. Three-dimensional stem cell-derived models of human embryogenesis and organogenesis provide a promising alternative for investigating the genetics of morphogenesis. Nevertheless, high-throughput genetic screens in organoid systems typically rely on ‘pooled perturbation approaches’, in which only a small fraction of cells within each organoid carries a given genetic perturbation. While these methods have advanced our understanding of gene function at the single-cell level, they are not ideal for studying how individual genes regulate multicellular processes such as morphogenesis. Huang et al. sought to identify genes required for neural tube closure in the developing human forebrain using a stem cell-derived organoid model. The researchers first aimed to establish a more reproducible three-dimensional model of human neural tube closure. They then sought to develop a high-throughput method for targeting individual genes across micropatterned organoids, enabling parallel assessment of gene-specific contributions to neural tube closure. Huang et al. developed an approach for uniform gene knockdown across entire human pluripotent stem cell (hPSC)-derived organoids. They used this method to identify three transcriptional regulators of neural tube closure in the developing human forebrain. They found that arranging organoids in a hexagonal pattern promoted highly symmetrical breaking of ectodermal tissues, resulting in a reproducible three-dimensional model of human neural tube closure. They next developed a high-throughput method for targeting single genes broadly and uniformly across each organoid, by generating and applying a high density of lentiviruses carrying gene-editing tools (CRISPRi dual-guide RNAs). This allowed them to examine the roles of individual genes in the tissue-wide process of morphogenesis. Guided by neural gene expression analyses, 77 transcription factor candidates were screened, and three genes (ZIC2, SOX11, and ZNF521) were identified as essential for proper neural tube closure. The platform developed by Huang et al. is broadly applicable to genetic investigations of morphogenesis in human stem cell-derived models, enabling high-throughput analyses of genes that regulate developmental processes. Future studies could evaluate the roles of ZIC2, SOX11 and ZNF521 in human neural tube defects by assessing their sequence and expression in patients affected by such conditions. In addition, the relationship between these genes and other known genetic contributors to neural tube defects remains an important area for future investigation.
The gut microbiota is a complex microbial community that plays a crucial role in host health. Environmental and biological factors within the ecosystem influence the dynamic interactions among its members. Although dietary and host-derived nutrient availability play a key role in shaping microbial ecology and interaction patterns, the dynamics of these interactions within the mucus layer remain poorly understood. In this study, we analyzed a synthetic community comprised of six species with variable abilities to utilize mucin. We performed in vitro growth analyses and monitored the interactions among community members in monoculture, co-culture, and community batch culture under different nutrient conditions. Our results showed that positive interactions were prevalent among bacteria when mucin served as the sole carbon source. In contrast, the addition of glucose or high nutrient availability significantly increased inter-bacterial competition. These findings suggest that mucin mitigates competitive antagonism and potentially promotes community diversity. Further in vivo studies supported the role of mucin in increasing community diversity and modulating bacterial metabolic patterns. Deciphering these intricate relationships is essential for understanding how gut microbiota stability is maintained, and what factors might disrupt this delicate balance.IMPORTANCEThe gut microbiota is essential for host health, yet microbial interactions within the intestinal mucus layer remain poorly understood. Current understanding of gut microbial ecology is largely based on nutrient-rich media that do not accurately reflect the mucosal environment. Here, we demonstrate that when bacteria rely solely on mucin as a carbon source, cooperative interactions predominate. In contrast, the introduction of simple sugars shifts the balance toward intensified interbacterial competition. Mucin mitigates competitive antagonism, promotes resource utilization, and enhances community diversity. By demonstrating that mucus actively shapes microbial interaction patterns, this study provides a mechanistic framework for understanding gut ecosystem resilience. Furthermore, these findings support the development of more physiologically relevant in vitro models for predicting gut microbial dynamics and may guide microbiome-based therapies.
Cyclophosphamide (CTX) is a commonly used immunosuppressant and chemotherapeutic drug; however, its clinical application is often restricted by drug-induced liver injury (DILI). Pterostilbene (PTS), a dietary polyphenol abundant in blueberries and other berries, is known for its antioxidant and anti-inflammatory properties; however, its hepatoprotective effects and underlying mechanisms on CTX-induced liver injury remain largely unexplored. This study adopted an integrated approach combining network pharmacology, in vivo and in vitro models, untargeted metabolomics, and gut microbiota 16S rRNA sequencing to investigate the protective effects of PTS on CTX-induced liver injury and explore the underlying multi-dimensional mechanisms. PTS significantly ameliorated CTX-induced liver injury by regulating inflammation and oxidative stress. Network pharmacology corroborated these findings, revealing that the targets of PTS against DILI were enriched in pathways closely related to inflammation and oxidative stress, such as reactive oxygen species and MAPK signaling, with key molecules including EGFR, PIK3CA, and RELA. Experimental validation confirmed that PTS inhibited the overactivation of EGFR/MAPK, PI3K/AKT, and NF-κB pathways and activated the Nrf2/HO-1 antioxidant pathway. Metabolomics analysis revealed that PTS ameliorates CTX-induced hepatic metabolic perturbations by regulating key metabolites, including lipids (e.g., PGP), amino acids (e.g., taurine), vitamins (e.g., tocotrienol), and oxidative stress-related markers (e.g., ophthalmate). Gut microbiota profiling showed that PTS restores CTX-induced dysbiosis by increasing the abundance of beneficial genera (e.g., Bacteroides) and reducing that of pathogenic taxa (e.g., Proteobacteria). Multi-omics correlation analysis further uncovered a robust network linking gut microbial composition, hepatic metabolites, and markers of inflammation and oxidative stress. In conclusion, PTS protects against CTX-induced liver injury via multiple targets and pathways, involving the gut-liver axis, inflammatory-oxidative regulation, and metabolic homeostasis. This study provides a theoretical basis for the potential application of PTS in the prevention and treatment of DILI and offers novel insights into liver-protective strategies targeting intestinal microecology.
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global liver disorder with a rising incidence. Early-to-middle-stage MASLD remains amenable to clinical intervention; without timely management, it may progress to fibrosis and cirrhosis. Therefore, identifying novel targets through integrated multi-technical approaches is critical for preventing and treating MASLD. We developed an integrative multi-omics and spatial proteomic strategy combined with in vitro validation. CyTOF was applied to high-dimensional immunophenotyping of the MASLD liver microenvironment. Key cell populations were then sorted accordingly for mass spectrometry to uncover their core signaling. Focusing on identified key amino acid metabolic enzymes, we performed IMC for spatial single-cell proteomic profiling to localize metabolic aberrant subsets. Finally, functional phenotypes and mechanisms were validated in cell models. CyTOF revealed significant expansion of myeloid-derived cells. Thus, by targeting myeloid-derived cells, proteomic and phosphoproteomic analyses identified two severely impaired amino acid metabolic pathways: the glycine metabolic pathway regulated by AGXT2 and the proline metabolic pathway regulated by PYCR3. We then focused on AGXT2 and PYCR3 for spatial exploration via IMC and ultimately identified a macrophage subset negative for both AGXT2 and PYCR3. These macrophages were significantly elevated within MASLD and exhibited high spatial colocalization with inflammatory cells and fibrotic cells. Compared with dysregulated M1 subsets (M1-C3), dysregulated M2 subsets (M2-C1) indicated stronger pro-inflammatory and pro-fibrotic potential. In vitro models using human and murine cells confirmed that AGXT2⁻PYCR3⁻ macrophages exhibited enhanced proliferation and migration, secreted higher levels of inflammatory cytokines, chemokines, and classic fibrotic proteins, and exerted strong inductive effects on hepatic fibrotic cells, with downregulation of intercellular GSH. Mechanistically, the NF-κB and MAPK/AP-1 pathways were involved in inflammatory responses, while the p-SMAD3(T8)/TGFβ pathway contributed to the fibrotic phenotype. However, the abnormal biological functions could be effectively rescued by supplementation with corresponding amino acids, accompanied by the reversal of aberrant signaling pathway activation. In summary, our study explores an initial link between amino acid metabolism and early-to-middle-stage MASLD. AGXT2⁻PYCR3⁻ macrophages are significantly enriched in MASLD with pro-inflammatory and pro-fibrotic potential, and amino acid supplementation can rescue their aberrant phenotypes in vitro, warranting further validation of their treatment potential.
Drug-induced liver injury (DILI) is one of the most serious adverse drug reactions in clinical practice and a major concern in toxicity assessment during drug development. In particular, DILI caused by antifungals can become life-threatening in severe cases; therefore, early detection and appropriate intervention are essential. Here, potential early biomarkers of DILI induced by azole antifungals were explored by performing metabolomic analysis of the supernatants from spheroid cultures of primary human hepatocytes. Primary human hepatocytes obtained from 2 different donors were independently cultured under 3-dimensional spheroid conditions and exposed to 9 antifungal agents. Culture supernatants were collected and analyzed using untargeted metabolomics. The sampling time point was determined based on longitudinal evaluation of cell viability, and the latest time point at which concentration-dependent cytotoxicity was observed was selected for analysis. In parallel, the levels of conventional liver injury markers, aspartate aminotransferase and alanine aminotransferase, were also evaluated in the supernatants. Cell viability was assessed at the time of supernatant collection. Several candidate biomarkers associated with hepatotoxicity were identified, whereas conventional liver injury markers, including aspartate aminotransferase and alanine aminotransferase, showed no clear concentration-dependent changes. Among these, one compound was identified as glycocholic acid through comparison with an authentic standard. Another metabolite detected in positive ion mode could not be conclusively identified and requires further characterization. These findings support the development of novel biomarkers for the early detection of DILI and highlight their potential applications in toxicity screening during drug development and in clinical biomarker research. SIGNIFICANCE STATEMENT: This study identifies novel metabolite-based biomarkers for azole antifungal-induced liver injury using 3-dimensional-cultured primary human hepatocytes and untargeted metabolomics. Glycocholic acid and an additional unidentified metabolite showed stronger associations with hepatotoxicity than conventional markers (aspartate aminotransferase and alanine aminotransferase). These findings highlight the potential of advanced in vitro models and metabolomics to improve early detection of drug-induced liver injury in drug development and clinical settings.
Excessive mechanical stress is a main cause of intervertebral disc degeneration (IDD). However, the specific mechanism remains unclear. We established in vivo and in vitro models to investigate the role of cytoskeletal proteins in excessive mechanical stress-induced NP cell pyroptosis and IDD. The expression level of Vimentin was decreased in degenerated NP cells induced by excessive mechanical stress. Knockdown of Vimentin promoted NP cell pyroptosis and IDD in rats, whereas Vimentin overexpression significantly alleviated excessive mechanical stress-induced NP cell pyroptosis and degeneration. Further mechanistic studies revealed that Vimentin ameliorated mitochondrial dysfunction triggered by excessive mechanical stress through PINK1-Parkin-dependent mitophagy, thereby attenuating NP cell pyroptosis and degeneration. Co-immunoprecipitation-mass spectrometry analysis suggested an interaction between Itgb1 and Vimentin, which was validated by Co-immunoprecipitation assays. Itgb1 enhanced Vimentin protein stability via the ubiquitin-proteasome pathway and inhibited mechanical stress-mediated Vimentin degradation. Subsequent experiments confirmed that Itgb1 reduced Vimentin ubiquitination and degradation by blocking the binding of MNAT1 to Vimentin. Itgb1 ameliorated excessive mechanical stress-induced NP cell pyroptosis and degeneration via Vimentin. Restoring Vimentin function through gene overexpression effectively inhibited NP cell pyroptosis and delayed the progression of IDD in rats. In summary, this study reveals a mechanotransduction pathway from mechanical stress sensing to cellular functional regulation in IDD, providing novel insights into the pathological mechanisms underlying IDD. Moreover, this study demonstrates that Vimentin exerts a significant protective effect against excessive mechanical stress-induced NP cell pyroptosis and IDD, offering a potential therapeutic target for the clinical management of IDD.
Tetrabromobisphenol A (TBBPA) and polystyrene nanoparticles (PSNPs) are contaminants of emerging concern frequently detected in environmental matrices, with dietary exposure representing a major route. Despite their co-occurrence, data on combined toxicity remain limited. This study evaluated the effects of TBBPA and PSNPs individual and combined exposure on human intestinal barrier integrity, differentiation and function using differentiated Caco-2 cells and Caco-2/HT29-MTX co-cultures as validated in vitro models of increasing complexity. Cells were exposed to TBBPA (1-150 µM) and PSNPs (0.1-50 µg/mL) individually or in combination, either for 24 h in fully differentiated monolayers or continuously during the 21-day differentiation period. In differentiated Caco-2 cells, combined exposure altered cytoskeletal organization, with PSNPs being internalized and accumulating as large intracellular aggregates that persisted for at least 144 h post-exposure. Ultrastructural analysis revealed microvilli alterations and multilamellar bodies formation, although tight junction architecture remained intact and cellular adhesion structures recovered progressively following contaminant removal, suggesting retained barrier repair capacity. During the differentiation period, TBBPA was the primary cytotoxic agent and PSNPs alone showed no cytotoxicity at the concentrations tested, as confirmed by benchmark dose analysis, which yielded low relative potency factor values for PSNPs across all tested combinations. Genotoxicity assessment revealed oxidative DNA damage in selected TBBPA and PSNPs combinations using the FPG-modified comet assay, with gene expression analysis indicating activation of DNA damage response and oxidative stress pathways. Co-exposure modulated the expression of intestinal differentiation markers, including ALPI, suggesting interference with epithelial maturation processes. The complexity of the dose-response relationships observed underscores the importance of comprehensive dose-range testing and the use of physiologically relevant differentiated models in the risk assessment of contaminant mixtures.
This research examined host metabolic processes associated with gingivitis and potential impacts of interventions. First, gingival brush samples from a previously reported clinical study in which participants used an intervention (stannous fluoride (SnF2) toothpaste/cetylpyridinium chloride rinse/oscillating-rotating electric toothbrush) or control (standard fluoride toothpaste/manual toothbrush) were analyzed with Metabolon metabolomic assessment. Next, hypotheses were generated about mechanisms of metabolic observations and tested in two in vitro models. Human primary blood cells were treated with endotoxins and outer membrane vesicles (OMVs) in the presence of SnF2. In clinical brush samples, 46 metabolites decreased with intervention at Week 3, including 9- and 13-hydroxyoctadecadienoic acid, citrulline, adenine, and hypoxanthine. Seven metabolites related to nitrogen metabolism and citrulline production were selected for targeted quantification. They were low in health and increased in gingivitis. Targeted quantification demonstrated reduced citrulline, succinate, ornithine and deoxycarnitine (p ≤ 0.001 for all) between baseline and Week 6 with intervention; reductions were greater for intervention vs. control for citrulline, succinate and deoxycarnitine at Week 6 (p ≤ 0.019). In vitro, endotoxin and OMVs increased citrulline and nitric oxide (NO). SnF2 inhibited elevation of both. Citrulline, deoxycarnitine, succinate, and ornithine were identified as potential novel markers of gingivitis. Elevated NO production is a potential driver of oxidative stress, reduced with intervention.
The study of aroma release is essential in food science to better understand flavour perception. Analytical techniques such as APCI-MS and PTR-MS allow real-time monitoring of volatile compounds, including isoamyl acetate and diacetyl, directly in food systems. In vitro models are particularly useful because they enable controlled studies of parameters like temperature or container volume, which are difficult to isolate in real consumption conditions. In parallel, aroma modelling has been developed to predict release kinetics under different conditions. Early mechanistic approaches focused on describing mass transfer processes governing aroma behaviour after a disturbance, such as opening a container. These models have since been extended to more complex food matrices to better simulate real systems. This paper presents data on the kinetics of in vitro aroma release for two volatile compounds, in aqueous solution or incorporated into gelatin discs, measured using PTR-ToF-MS. A total of 84 experiments were conducted under varying conditions, including temperature, initial concentration or duration of preliminary equilibrium. The dataset includes raw PTR-ToF-MS time-series data in .csv format, together with associated metadata describing the experimental conditions, analysed products, target compounds, and instrumental parameters required for data interpretation and reuse. This dataset can be used for: - documenting PTR-ToF-MS measurements under multiple conditions initiating a database for modelling aroma release, - relating signal intensity and real concentration, - tracking ion and fragment relationships.
Preeclampsia affects 2-4% of pregnancies worldwide and remains a leading contributor to maternal and perinatal morbidity and mortality. The prevailing framework, anchored in defective second-trimester spiral-artery remodelling, placental hypoxia, and antiangiogenic imbalance, continues to guide screening, prediction, and prevention. Evidence accumulated over the past 5 years, however, indicates that several pillars of this framework may require revision. This narrative review synthesises six inter-related paradigm shifts emerging principally from the research programme of the Huppertz group at the Medical University of Graz between 2020 and 2026, and situates them against contemporaneous mainstream formulations. The six shifts addressed are: first-trimester villous origins rather than second-trimester deep-placentation failure; intervillous hyperoxia rather than placental hypoxia in early-onset disease; metabolic and glycocalyx-based pathogenesis rather than pure angiogenic imbalance; the placenta as an endogenous exposome via extracellular vesicles; steroid imbalance coupled with alternative renin-angiotensin-leptin signalling; and reduced immune tolerance together with dynamic in vitro models that challenge inferences drawn from static explants. Collectively, these shifts reframe preeclampsia as a first-trimester syndrome of villous trophoblast dysregulation that propagates to maternal endothelial injury through multiple, partly redundant pathways. They suggest that maternal-foetal medicine may benefit from earlier, multimodal risk assessment, from biomarker panels that capture senescence, metabolic and extracellular vesicle signatures, and from a more cautious mechanistic interpretation of the soluble fms-like tyrosine kinase 1 to placental growth factor ratio, whilst acknowledging its established short-term clinical utility for triage. Several of these propositions, particularly those concerning intervillous hyperoxia and alternative renin-angiotensin-leptin signalling, still require independent replication in cohorts beyond the originating research environment before clinical translation. The six shifts do not individually overturn the two-stage framework; collectively they relocate the initiating lesion, broaden the signalling vocabulary, and argue for translation into first-trimester multimodal panels that extend beyond current angiogenic and Doppler measures.
Klebsiella pneumoniae (K. pneumoniae) is a critical pathogen responsible for a wide range of severe infections. Its escalating resistance to frontline antimicrobials, particularly carbapenems, severely compromises existing therapeutic options and undermines current treatment strategies. This clinical challenge is further exacerbated by the emergence of multidrug-resistant (MDR) strains and hypervirulent lineages, which substantially complicate infection management and worsen patient outcomes. Ultimately, this alarming convergence of resistance and virulence presents a severe public health threat that necessitates the urgent development of novel therapeutic approaches; in this regard, phage therapy is emerging as a promising strategy. Bacteriophages and their derivatives have garnered increasing recognition as targeted antibacterial agents, demonstrating potent activity against carbapenemase producing and MDR K. pneumoniae in vitro models. They effectively lyse MDR strains, disrupt biofilm formation, and have shown promising therapeutic efficacy across various in vivo models. To ensure safety in clinical applications, genome sequencing is essential to identify and exclude undesirable genes, thereby determining therapeutic suitability. Here, we have sequenced and analyzed the genomic characteristics of seven lytic bacteriophages targeting carbapenemase-producing MDR K. pneumoniae. Genomic evaluation confirmed that these phages are strictly lytic and revealed the presence of distinct lysis modules across the genomes. Phylogenetic analysis showed that these phages cluster within four genera of Caudoviricetes: Taipeivirus, Drulisvirus, Przondovirus, and Webervirus. The phage genomes ranged from 15,773 to 166,437 base pairs (bp) in length and encoded core structural and replication modules. Furthermore, distinct lytic modules predicted to have function in bacterial capsule degradation were identified across the genomes. Importantly, the absence of known virulence or toxin genes guarantees therapeutic safety and satisfies rigorous genomic criteria for clinical translation. These findings underscore the potential of these phages as a promising alternative for combating MDR and hypervirulent K. pneumoniae infections. Future in vivo investigations and clinical trials are essential to validate their therapeutic efficacy, safety profile, and optimal treatment protocols.
Digestion in the small intestine is a crucial step in the breakdown and absorption of nutrients from human milk. In vitro models of intestinal digestion are commonly employed to investigate the digestion behaviour of breast milk nutrients. Accurate replication of the infant small intestine requires a detailed understanding of its biochemical environment. This review summarises current knowledge on small intestinal digestion in infants, integrating evidence from both human studies and in vitro intestinal digestion models. Key challenges in simulating the infant small intestinal conditions, including motility, intestinal fluid dynamics and composition, enzymatic activity, and absorption are discussed. This literature review also provides recommendations for enhancing the physiological relevance of in vitro small intestinal digestion systems for studying intestinal digestion in early infancy.
Sepsis-associated acute kidney injury (SA-AKI) is a common complication in septic patients and is associated with high mortality and progression to chronic kidney disease (CKD). Previous studies have demonstrated that thymosin β4 (Tβ4) attenuates lipopolysaccharide (LPS)-induced liver injury and ischemic AKI by suppressing inflammatory responses. Here, we aimed to investigate the effects of Tβ4 on SA-AKI and elucidate its underlying mechanisms. In vivo and in vitro models of SA-AKI were established using LPS. Tβ4 was administered 30 min after LPS exposure. Transcriptome sequencing was performed to explore the underlying molecular mechanisms, followed by validation using western blotting and qRT-PCR. In SA-AKI mice, serum Tβ4 levels were significantly decreased. Administration of Tβ4 ameliorated renal dysfunction and morphological injury in LPS-treated mice, accompanied by reduced renal inflammation and apoptosis. Transcriptome-sequencing analysis indicated that the MAPK signaling pathway was involved in both SA-AKI pathogenesis and the protective effect of Tβ4. Western blotting of the renal tissues confirmed that LPS enhanced the phosphorylation of JNK1/2, p38 MAPK and ERK1/2, while Tβ4 significantly suppressed their activation. In HK-2 cells, pharmacological activation of the MAPK pathway with anisomycin restored MAPK phosphorylation and partially reversed the inhibitory effects of Tβ4 on the mRNA expression of the pro-inflammatory cytokines IL-6, IL-1β and IL-18. In conclusion, Tβ4 alleviates renal inflammation and apoptosis in SA-AKI by suppressing the MAPK signaling pathway. Tβ4 may represent a potential therapeutic strategy for early intervention in SA-AKI.