Culture Model Research Articles

Targeting KPNB1 with genkwadaphnin suppresses gastric cancer progression through the Nur77-mediated signaling pathway

Gastric cancer (GC) remains a global challenge due to the lack of early detection and precision therapies. Genkwadaphnin (DD1), a natural diterpene isolated from the bud of Flos GenkWa (Thymelaeaceae), serves as a Karyopherin β1 (KPNB1) inhibitor. In this study, we investigated the anti-tumor effect of DD1 in both cell culture and animal models. Our findings reveal that KPNB1, a protein involved in nuclear import, was highly expressed in GC tissues and associated with a poor prognosis in patients. We demonstrated that DD1, alongside the established KPNB1 inhibitor importazole (IPZ), inhibited GC cell proliferation and tumor growth by enhancing both genomic and non-genomic activity of Nur77. DD1 and IPZ reduced the interaction between KPNB1 and Nur77, resulting in Nur77 cytoplasmic accumulation and triggering mitochondrial apoptosis. The inhibitors also increased the expression of the Nur77 target apoptotic genes ATF3, RB1CC1 and PMAIP1, inducing apoptosis in GC cell. More importantly, loss of Nur77 effectively rescued the inhibitory effect of DD1 and IPZ on GC cells in both in vitro and in vivo experiments. In this study, we for the first time explored the relationship between KPNB1 and Nur77, and found KPNB1 inhibition could significantly increase the expression of Nur77. Moreover, we investigated the function of KPNB1 in GC for the first time, and the results suggested that KPNB1 could be a potential target for cancer therapy, and DD1 might be a prospective therapeutic candidate.

Single-domain antibody-based protein degrader for synucleinopathies

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Cultural Empowerment Models and Mechanisms in Rural Development: A Case Study in Zhang Village, China

Cultural Empowerment Models and Mechanisms in Rural Development: A Case Study in Zhang Village, China

Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

Circadian Regulation of Leishmania Parasite Internalisation in Macrophages and Downstream Cellular Events.

Leishmania spp. parasites use macrophages as a host cell during infection. As a result, macrophages have a dual role: clearing the parasite as well as acting as host cells. Recently, studies have shown that macrophages harbour circadian clocks, which affect many of their functions such as phagocytosis, receptor expression and cytokine release. Interestingly, Leishmania major infection in hosts was also shown to be under circadian control. Therefore, we decided to investigate what underlies the rhythms of L. major infection within macrophages. Using a culture model of infection of bone marrow-derived macrophages with L. major promastigotes, we show that the parasites are internalised into macrophages with a 24-h variation dependent on a functional circadian clock in the cells. This was associated with a variation in the number of parasites per macrophage. The cell surface expression of parasite receptors was not controlled by the cells' circadian clock. In contrast, the expression of the components of the endocytic pathway, EEA1 and LC3b, varied according to the time of infection. This was paralleled by variations in parasite-induced ROS production as well as cytokine tumour necrosis factor α. In summary, we have uncovered a time-dependent regulation of the internalisation of L. major promastigotes in macrophages, controlled by the circadian clock in these cells, as well as subsequent cellular events in the endocytic pathway, intracellular signalling and cytokine production.

Cryopreservation of human adult ventricular tissue for the preparation of viable myocardial slices

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): REGeRNA Myocardial slices are ultra-thin slices of living adult myocardium which can be cultured. As a multicellular 3-D culture model, myocardial slices offer a novel opportunity to investigate how adult cardiomyocytes (CMs) interact with other cardiac cell types and the extracellular matrix, while providing long-term preservation of CMs cytoarchitecture and electrical phenotype. Additionally, myocardial slices are a new tool in drug discovery by allowing to test chronic treatments, as opposed to isolated CMs which are limited to acute timepoints. Despite their benefits over other cardiac preparations, myocardial slices also have several limitations, of which one is the quality of the slices. The main determinant of myocardial slice quality is the procedure by which the cardiac tissue block is handled. Current methods depend on the preparation of slices directly after tissue excision, therefore limiting the moment when the experiment is performed. We developed a protocol for the cryopreservation of human adult ventricular tissue allowing preparation of viable myocardial slices afterwards. Fresh and cryopreserved cardiac tissue from donor hearts was processed and evaluated for viability, tissue integrity, and inflammatory markers using molecular biology and histological assays. The tissue samples retained viability, biomass, and tissue and vascular integrity following cryopreservation compared to fresh tissue. Additionally, we compared myocardial slices obtained from fresh or cryopreserved tissue and cultured for 24 hours. Histologically, normal cytomorphological features and gap junctions between CMs were preserved in all slices. Comparable retention of tissue viability and structural integrity post cryopreservation (4-6 weeks) and culture (up to 7 days) was observed in cardiac samples from 3 additional donors. In summary, cold storage extends the time-interval between sample collection and slice preparation, allowing for more long-term, complex endpoints. Moreover, efficient banking of cryopreserved cardiac samples from various donors and disease states is critical for regular use of myocardial tissue slices in drug discovery and development. Additional research on the effects of cryopreservation on different cardiac cell types will allow to improve donor organ management to extend the donor pool and increase utilization rate.

FgF23 and soluble klotho effects on the myocardium: an ex vivo study

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Center for Cardiovascular research Background Fibroblast Growth Factor 23 (FGF23) and soluble Klotho (sKL) play a critical role in mineral homeostasis in the body by acting on the kidney to regulate phosphate levels. Circulating FGF23 levels rise in patients with kidney disease and dysregulation of the FGF23 –sKL- axis is a common phenomenon in uremic cardiomyopathy. Studies have shown that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4), while sKL mitigates these effects and may also exert an independent protective effect on the heart(1,2). Purpose We aimed to study the effects of FGF23 and sKL on myocardial contractility, calcium handling and molecular signaling pathways in the human heart using an ex vivo model of cultured contracting myocardial slices. Methods We obtained human heart slices from explanted hearts from patients undergoing cardiac transplantation at the LMU Klinikum Munich, Germany. The 300 µm thick slices were cultured under continuous stimulation (1 Hz, 3 ms pulse width) with 1 mN preload, and contractile function were recorded continuously. Slices with vehicle, recombinant FGF23 protein (100ug/ml), recombinant sKL protein (200ug/ml) and both FGF23((100ug/ml)+sKL (200ug/ml) were continuously monitored over 10 days. We checked for mRNA expression using RT PCR. Results Treatment of human left ventricular tissue with recombinant FGF23 protein resulted in enhanced mRNA expression of the FGFR4. sKL treatment alone induced downregulation of the FGFR4 gene and mitigated upregulation of FGFR4 in FGF23+ sKL treated slices. Post pause potentiation (PPP), assessed by pausing the stimulation of the slices for 120s and then measuring the relative height of the first beat after resumption of pacing, is an indirect measure of sarcoplasmic reticulum Ca2+ handling capacity. PPP was reduced in FGF23 treated slices and heightened in the slices with sKL compared to vehicle treated ones, indicating that FGF23 and sKL play a role in intracellular Ca2+ handling. Conclusions sKL and FGF23 have opposite effects on human cardiac muscle with sKL mitigating FGF23-induced upregulation of FGFR4. Furthermore, sKL improves, while FGF23 impairs myocardial calcium handling . As altered calcium handling may also be involved in hypertrophic signaling altered calcium handling may provide a means by which FGF23 promotes and sKL inhibits myocardial hypertrophy. sKL is advocated as a promising candidate in exploring it therapeutic role in FGF23 mediated cardiomyopathy. We intend to advance our understanding of sKL-FGF23signaling mechanisms by combining transcriptomic analysis with functional data in this model of myocardial slice culture.

Uniform sized cancer spheroids production using hydrogel-based droplet microfluidics: a review.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

Single-cell RNA sequencing analysis of the human fetal epicardium reveals several new genes involved in regulation of epicardial activation

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Background The outside mesothelial layer of the heart, the epicardium, is essential for cardiac development. Cells from this layer undergo epithelial-to-mesenchymal transition (EMT), migrate into the tissue and partake in cardiogenesis by contributing cells and by secreting growth factors. In the adult heart, the epicardium is quiescent but upon ischemic injury it is activated and conveys a partial regenerative response. This activation step is important for repair, but suboptimal compared to the cardiogenic potential during development. Understanding the processes and cells that underlie developmental epicardial activation could contribute to enhancing the adult post-injury response, but little is known about the human developing epicardium. Purpose to determine the cellular composition of the human fetal epicardial layer and to identify the processes that regulate the activation of the epicardium. Methods An epicardial-cell enriched dataset was obtained by specifically dissecting the epicardial layers from 4 fetal human hearts (14-15 weeks gestation, obtained under informed consent and according to local ethical approval). Single live cells were sorted into 384-wells plates and sequenced. Data analysis was performed on 2073 cells using R-packages RaceID3 and StemID2. Findings were validated using qPCR and immunohistochemistry on tissue sections and cell culture models. Results Sequencing analysis revealed a distinct clustering of epicardial (epithelial, pre-EMT) and epicardial-derived mesenchymal (post-EMT) cell populations. Besides the identification of novel markers for epicardial cell states, the regulation of epicardial cell activation and EMT was investigated by applying pseudo-time analysis. We selected genes that were highly regulated (>2 fold change) in this trajectory for further analysis in vitro. We identified several genes including CRIP1, SULF1 and others that are expressed in epithelial cells in vitro and in vivo and are rapidly downregulated upon EMT. The expression profiles positively correlated with epithelial cell related genes and negatively with mesenchymal cell genes. Based on GO-terms, and protein expression within the epicardial layers in fetal and adult epicardium, we anticipated a role for these genes in maintaining the epithelial-state. Indeed, siRNA experiments in vitro with cultured primary human epicardial cells confirmed an essential role in EMT regulation. We continue to evaluate other genes identified in our sequencing dataset, resulting in several other potent and previously unknown pathways involved in regulation of epicardial activation. Conclusions We shed light on the composition of the human fetal epicardium using a highly enriched scRNA dataset. We identify specific markers for different cell-states, and reveal novel regulators of activation that in the future may be influenced to optimize the post-injury response.

A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer.

The aim of the present study was to assess the cytocompatibility of epoxy resin-based AH Plus Jet (Dentsply De Trey, Konstanz, Germany), Sealer Plus (MK Life, Porto Alegre, Brazil), calcium silicate-based Bio-C Sealer (Angelus, Londrina, PR, Brazil), Sealer Plus BC (MK Life) and AH Plus BC (Dentsply) through a tridimensional (3D) culture model of human osteoblast-like cells. Spheroids of MG-63 cells were produced and exposed to fresh root canal sealers extracts by 24h, and the cytotoxicity was assessed by the Lactate Dehydrogenase assay (LDH). The distribution of dead cells within the microtissue was assessed by fluorescence microscopy, and morphological effects were investigated by histological analysis. The secreted inflammatory mediators were detected in cell supernatants through flow luminometry (XMap Luminex). Cells incubated with AH Plus Jet, AH Plus BC, Sealer Plus BC and Bio-C Sealer extracts showed high rates of cell viability, while the Sealer Plus induced a significant reduction of cell viability, causing reduction on the spheroid structure. Sealer Plus and Seaker Plus BC caused alterations on 3D microtissue morphology. The AH Plus BC extract was associated with the downregulation of secretion of pro-inflammatory cytokines IL-5, IL-7, IP-10 and RANTES. The new AH Plus BC calcium silicate-based endodontic sealer did not reduce cell viability in vitro, while led to the downregulation of pro-inflammatory cytokines. Choosing the appropriate endodontic sealer is a crucial step. AH Plus BC demonstrated high cell viability and downregulation of pro-inflammatory cytokines, appearing reliable for clinical use, while Sealer Plus presented lower cytocompatibility.

Small Peptide Derived from SFRP5 Suppresses Melanogenesis by Inhibiting Wnt Activity.

Melanocytes, located in the epidermis' basal layer, are responsible for melanin pigment production, crucial for skin coloration and protection against UV radiation-induced damage. Melanin synthesis is intricately regulated by various factors, including the Wnt signaling pathway, particularly mediated by the microphthalmia-associated transcription factor (MITF). While MITF is recognized as a key regulator of pigmentation, its regulation by the Wnt pathway remains poorly understood. This study investigates the role of Sfrp5pepD, a peptide antagonist of the Wnt signaling pathway, in modulating melanogenesis and its potential therapeutic implications for pigmentary disorders. To tackle this issue, we investigated smaller peptides frequently utilized in cosmetics or pharmaceuticals. Nevertheless, there is a significant scarcity of reports on peptides associated with melanin-related signal modulation or inhibiting melanin production. Results indicate that Sfrp5pepD effectively inhibits Wnt signaling by disrupting the interaction between Axin-1 and β-catenin, thus impeding downstream melanogenic processes. Additionally, Sfrp5pepD suppresses the interaction between MITF and β-catenin, inhibiting their nuclear translocation and downregulating melanogenic enzyme expression, ultimately reducing melanin production. These inhibitory effects are validated in cell culture models suggesting potential clinical applications for hyperpigmentation disorders. Overall, this study elucidates the intricate interplay between Wnt signaling and melanogenesis, highlighting Sfrp5pepD as a promising therapeutic agent for pigmentary disorders. Sfrp5pepD, with a molecular weight of less than 500 Da, is anticipated to penetrate the skin unlike SFRPs. This suggests a strong potential for their use as cosmetics or transdermal absorption agents. Additional investigation into its mechanisms and clinical significance is necessary to enhance its effectiveness in addressing melanin-related skin conditions.

Mechanisms of vascular inflammaging: TNF-alpha mediates endothelial CEACAM1 expression by NF-KB and beta-Catenin in a biphasic manner

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Research Foundation Introduction Chronic inflammation with progressive age, also called inflammaging, is a hallmark of the pathogenesis of cardiovascular diseases. Previously we have shown increased expression of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) with progressive age in the vasculature of mice and humans that leads to a chronic pro-inflammatory milieu via mutual upregulation with TNF-α. Furthermore, CEACAM1 critically promotes vascular aging processes like collagen deposition, endothelial permeability as well as enhanced oxidative stress. Therefore, CEACAM1 is a main mediator of vascular inflammaging that may facilitate progress into cardiovascular diseases. In this study we identified the mechanisms that underly the upregulation of endothelial CEACAM1 expression by TNF-α in detail. Methods Wildtype (WT) and TNF-α knockout (Tnf-/-) mice of different age were used to determine the impact of TNF-α on age-dependent CEACAM1 expression in vivo. Underlying mechanisms of TNF-α-dependent CEACAM1 upregulation were further analyzed in an endothelial cell culture model using pharmacological inhibition and siRNA-mediated knockdown of signaling pathways. Activation of signaling pathways was confirmed by determining the phosphorylation status of signaling components via Western Blot and analysis of nuclear translocation of NF-κB and β-Catenin using subcellular fractionation. Results Immunohistochemical analyses of aortic sections from WT and Tnf-/- mice of different age reveal that TNF-α critically contributes to the aging-related upregulation of endothelial CEACAM1 expression in vivo. Mechanistic analyses in vitro showed that TNF-α upregulated CEACAM1 expression in a time-dependent manner. In pharmacological experiments we identified an early NF-κB- and a delayed β-Catenin-mediated response. This finding was supported by time-matched nuclear translocation of NF-κB and β-Catenin. Furthermore, siRNA-mediated knockdown of the β-Catenin-targeted transcription factor TCF4 reduced TNF-α-mediated CEACAM1 upregulation. Co-immunoprecipitation analyses show that β-Catenin is released by TNF-α-mediated adherens junctions disassembly. Furthermore, TNF-α enhanced activating phosphorylation of Akt kinase at Ser473. Akt kinase in turn increased phosphorylation of β-Catenin at Ser552, a modification reported to augment its transcriptional activity. Additionally, Akt kinase facilitated β-Catenin signaling by inhibition of its degradation via phosphorylation of GSK3β at Ser9. Conclusion Taken together, this study identified the signaling mechanisms that underly the vascular upregulation of CEACAM1 with age. Our results directly link endothelial barrier impairment, an initial step in the pathogenesis of atherosclerosis, to CEACAM1 expression. Since CEACAM1 critically promotes vascular inflammaging and age-dependent vascular alterations this further emphasizes the potential of CEACAM1 as a novel target to prevent cardiovascular diseases.

Novel ex-vivo calcification model for human aortic valves reveals patient-specific effect of compound

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dekker grant Background Aortic stenosis (AS) is a narrowing of the aortic valve opening due to calcification and valve thickening. There are currently no pharmacological treatments for AS, and surgical interventions are the only therapies. To understand the molecular mechanisms underlying AS and test new pharmacological agents, advanced calcification models are required. These models need to closely approach native tissue response to stimuli by maintaining the valvular interstitial cells within their native environment. We have recently developed an ex vivo culture model that fulfills this requirement and allows the induction of calcification in slices of human aortic valves. Purpose Our goal is to evaluate the applicability of our ex-vivo calcification model as a pre-clinical test system for potential therapies. Methods Diseased aortic valves were dissected, embedded in agarose, then cut into 300-500 um slices using a vibratome. The use of multiple slices from a single patient ensured proper internal controls. To induce calcification, slices were cultured with 3mM phosphate(n=12), and control slices without phosphate (n=11). To investigate molecular modulation of induced calcification, dexamethasone was added to phosphate cultures(n=9). The slices were assessed for calcification using Alizarin Red staining. ImageJ was used to quantify the percentage of alizarin red-positive area. The average thickness was determined using 3DHISTECH software. Results In the presence of phosphate, a significant increase in the calcification percentage was observed in cultured valve slices compared with cultures without phosphate (p<0.05). Interestingly, within the phosphate-treated group, two sub-groups could be distinguished, one sub-group exhibiting extensive calcification, while the other showed virtually no sign of calcification. Paired comparisons revealed strong effect of dexamethasone depending on the extent of calcification-inducing capability of phosphate in each patient-sample. More precisely, dexamethasone exhibited potent inhibitory effect on calcification in valvular slices from patients where calcification was stimulated in the presence of phosphate. Conversely, dexamethasone exhibited a stimulatory effect on calcification in slices that did not show a stimulation of calcification in the presence of phosphate. This differential effect was strongly correlated with valve thickness.(r=-0.884,p< 0.01). Conclusions Our novel ex-vivo calcification model for human aortic valves revealed potential presence of patient-specific effects of pharmacological agents. Dexamethasone can have both stimulatory and inhibitory effects on calcification levels, influenced by phosphate response and valve thickness. As thickness might be indicative of the disease stage, this correlation suggests that the direction of compound’s effect might depend on disease stage. Hence, proper understanding of potential opposite effects of compounds is crucial in developing novel therapies.

Multimodal Mass Spectrometry Imaging of an Osteosarcoma Multicellular Tumour Spheroid Model to Investigate Drug-Induced Response.

A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

A systematic review on the culture methods and applications of 3D tumoroids for cancer research and personalized medicine.

Cancer is a highly heterogeneous disease, and thus treatment responses vary greatly between patients. To improve therapy efficacy and outcome for cancer patients, more representative and patient-specific preclinical models are needed. Organoids and tumoroids are 3D cell culture models that typically retain the genetic and epigenetic characteristics, as well as the morphology, of their tissue of origin. Thus, they can be used to understand the underlying mechanisms of cancer initiation, progression, and metastasis in a more physiological setting. Additionally, co-culture methods of tumoroids and cancer-associated cells can help to understand the interplay between a tumor and its tumor microenvironment. In recent years, tumoroids have already helped to refine treatments and to identify new targets for cancer therapy. Advanced culturing systems such as chip-based fluidic devices and bioprinting methods in combination with tumoroids have been used for high-throughput applications for personalized medicine. Even though organoid and tumoroid models are complex in vitro systems, validation of results in vivo is still the common practice. Here, we describe how both animal- and human-derived tumoroids have helped to identify novel vulnerabilities for cancer treatment in recent years, and how they are currently used for precision medicine.

Model of Culture in The Context of Modern Arts and Humanities Approaches

Model of Culture in The Context of Modern Arts and Humanities Approaches

GM1 Ameliorates Neuronal Injury in Rats after Cerebral Ischemia and Reperfusion: Potential Contribution of Effects on SPTBN1-mediated Signaling

Monosialoganglioside GM1 (GM1) has long been used as a therapeutic agent for neurological diseases in the clinical treatment of ischemic stroke. However, the mechanism underlying the neuroprotective function of GM1 is still obscure until now. In this study, we investigated the effects of GM1 in ischemia and reperfusion (I/R) brain injury models. Middle cerebral artery occlusion and reperfusion (MCAO/R) rats were treated with GM1 (60 mg·kg−1·d−1, tail vein injection) for 2 weeks. The results showed that GM1 substantially attenuated the MCAO/R-induced neurological dysfunction and inhibited the inflammatory responses and cell apoptosis in ischemic parietal cortex. We further revealed that GM1 inhibited the activation of NFκB/MAPK signaling pathway induced by MCAO/R injury. To explore its underlying mechanism of the neuroprotective effect, transcriptome sequencing was introduced to screen the differentially expressed genes (DEGs). By function enrichment and PPI network analyses, Sptbn1 was identified as a node gene in the network regulated by GM1 treatment. In the MCAO/R model of rats and oxygen-glucose deprivation and reperfusion (OGD/R) model of primary culture of rat cortical neurons, we first found that SPTBN1 was involved in the attenuation of I/R induced neuronal injury after GM1 administration. In SPTBN1-knockdown SH-SY5Y cells, the treatment with GM1 (20 μM) significantly increased SPTBN1 level. Moreover, OGD/R decreased SPTBN1 level in SPTBN1-overexpressed SH-SY5Y cells. These results indicated that GM1 might achieve its potent neuroprotective effects by regulating inflammatory response, cell apoptosis, and cytomembrane and cytoskeleton signals through SPTBN1. Therefore, SPTBN1 may be a potential target for the treatment of ischemic stroke.

Organic acids mitigate Streptococcus agalactiae virulence in Tilapia fish gut primary cells and in a gut infection model

BackgroundStreptococcus agalactiae, a Gram-positive bacterium, has emerged as an important pathogen for the aquaculture industry worldwide, due to its increased induced mortality rates in cultured fish. Developing interventions to cure or prevent infections based on natural alternatives to antibiotics has become a priority, however, given the absence of scientific evidence regarding their mode of action progress has been slow.MethodsIn this study we aimed to investigate the effect of a mixture of organic acids (natural antimicrobials), AuraAqua (Aq), on the virulence of S. agalactiae using Tilapia gut primary epithelial cells and an in vitro Tilapia gut culture model. Our results show that Aq was able to reduce significantly, in vitro, the S. agalactiae levels of infection in Tilapia gut primary epithelial cells (TGP) when the MIC concentration of 0.125% was tested.Results and discussionAt bacterial level, Aq was able to downregulate bacterial capsule polysaccharide (CPS) gene expression, capC, resulting in a significant decrease in bacterial surface capsule production. The decrease in CPS production was also associated with a reduction in the pro-inflammatory IFNγ, IL1β, TNFα, SOD and CAT gene expression and H2O2 production in the presence of 0.125% Aq (P < 0.0001). The antimicrobial mixture also reduced the levels of S. agalactiae infection in an in vitro gut culture model and significantly reduced the IFNγ, IL1β, TNFα, SOD, CAT gene expression and H2O2 production in infected tissue. Moreover, genes involved in Tilapia resistance to S. agalactiae induced disease, MCP-8 and Duo-1, were also downregulated by Aq, as a consequence of reduced bacterial levels of infection.ConclusionConclusively, our study shows that mixtures of organic acids can be considered as potential alternative treatments to antibiotics and prevent S. agalactiae infection and inflammation in the Tilapia fish digestive tract.

The Nolan Index: An R Program for Calculating List Resemblances

The Nolan Index (NI) is a normed, quantitative measure for comparing the degree of resemblance (similarity or dissimilarity) between free listings with an Excel program for calculating it. This article enhances that effort with the addition of an R program and additional applications. Free-list resemblance measures have been used to investigate and compare historical and cultural connections, ecological relationships, cultural item utilities, practices, knowledge, and values both within and between various groups. Their utility for cultural domain analysis and the theory of cognitive cultural models is also considered.

3D-Bioprinted Hepar-on-a-Chip Implanted in Graphene-Based Plasmonic Sensors.

Precise three-dimensional (3D) bioprinting designs enable the fabrication of unique structures for 3D-cell culture models. There is still an absence of real-time detection tools to effectively track in situ 3D-cell performance, hindering a comprehensive understanding of disease progression and drug efficacy assessment. While numerous bioinks have been developed, few are equipped with internal sensors capable of accurate detection. This study addresses these challenges by constructing a 3D-bioprinted hepar-on-a-chip embedded with graphene quantum dot-capped gold nanoparticle-based plasmonic sensors, featuring strong surface-enhanced Raman scattering (SERS) enhancement, biostability, and signal consistency. Such an integrated hepar-on-a-chip demonstrates excellent capability in the secretion of liver function-related proteins and the expression of drug metabolism and transport-related genes. Furthermore, the on-site detection of cell-secreted biomarker glutathione transferase α (GST-α) was successfully achieved using the plasmonic probe, with a dynamic linear detection range of 20-500 ng/mL, showcasing high anti-interference and specificity for GST-α. Ultimately, this integrated hepar-on-a-chip system offers a high-quality platform for monitoring liver injury.