Cell Culture Research Articles

Exploring the interactions between peritoneal dendritic cells and T cells: Implications for cancer metastasis

Abstract. Ovarian Cancer (OC) is a formidable challenge as a gynecological malignancy with ascites, the abnormal accumulation of fluid in the peritoneal cavity, serving as a clinical hallmark of advancement and metastasis. While OC immune analyses focus on solid tumors, little is known about malignant ascites inhabitants during peritoneal metastasis. Here, we focused on a single-cell landscape of the OC ecosystem in malignant ascites with comparison to tumor sites. Our data analysis reveals that compared to the immunosuppressed tumor, ascites contained less exhausted, more memory-like T cells, which correlated with prolonged survival in stages III/IV OC. Furthermore, we characterized the substantial interactions between dendritic cells and T cells. Lastly, an in vitro dendritic cell culture system from blood cells for potential novel cancer vaccine design was explored. Taken together, this study reveals T cell memory and effector potential amidst immunosuppressive pressures in ascites and provides valuable insight into the cross-talk between dendritic cells and T cells that potentially contributes to the unique T cell profile in the ascites ecosystem.

Cirsilineol improves anesthesia/surgery-induced postoperative cognitive dysfunction through attenuating oxidative stress and modulating microglia M1/M2 polarization.

Cirsilineol is a trimethoxy and dihydroxy flavonoid isolated from plant species such as Artemisia vestita and has a variety of pharmacological properties. This study analyzed whether cirsilineol could prevent postoperative cognitive dysfunction (POCD). A POCD mouse model induced by anesthesia/surgery induction and a cell model established with hydrogen peroxide (H2O2)-induced microglia BV-2 were employed to explore the efficacy of cirsilineol on POCD. The cognition function of the mice were assessed by carrying out behavioral tests (Morris water maze test and Y-maze test). We assessed the activation and polarization status of microglia using immunofluorescence analysis and detected the expression levels of CD86 and CD206 using the quantitative PCR (qPCR). Subsequently, cell viability was determined by CCK-8 assay and apoptosis was assessed using Calcein-AM/PI staining. Meanwhile, superoxide dismutase (SOD) and malondialdehyde (MDA) levels in plasma and cell culture medium were detected using chemiluminescence. Finally, the phosphorylation levels of JAK/STAT signaling pathway-related proteins were analyzed by Western blot. Cirsilineol reduced the escape latency and times of crossing island and increased spontaneous alternation (SA) rate, restoring the cognitive dysfunctions of POCD-modeled mice. Meanwhile, POCD elevated CD86 expression and malondialdehyde content and lowered the level of SOD; however, cirsilineol promoted CD206 expression and generation of SOD and inhibited malondialdehyde production. In H2O2-induced microglia BV-2, cirsilineol treatment increased SOD content and suppressed the generation of reactive oxygen species (ROS) and malondialdehyde, modulating microglia M1/M2 polarization and JAK/STAT pathway. Cirsilineol prevented against POCD by attenuating oxidative stress and modulating microglia M1/M2 polarization, providing novel insights for the management of POCD.

The Photocleavable Protein PhoCl-Based Dynamic Hydrogels.

Dynamic protein hydrogels have attracted increasing attention owing to their tunable physiochemical and mechanical properties, customized functionality, and biocompatibility. Among the different types of dynamic hydrogels, photoresponsive hydrogels are of particular interest. Here, we report the engineering of a photoresponsive protein hydrogel by using the photocleavable protein PhoCl. We employed the well-developed SpyTag and SpyCatcher chemistry to engineer PhoCl-containing covalently cross-linked hydrogels. In the hydrogel network, PhoCl, which can be cleaved into two fragments upon violet irradiation, is employed as a dynamic structural motif to regulate the cross-linking density of the hydrogel network. The resultant PhoCl-containing hydrogels showed photoresponsive viscoelastic properties. Upon violet irradiation, the PhoCl hydrogels soften, leading to an irreversible reduction in the storage moduli. However, no gel-sol transition was observed. Leveraging this light-induced stiffness change, we employed this hydrogel as a cell culture substrate to investigate the mechanobiological response of NIH-3T3 fibroblast cells. Our results showed that 3T3 cells can change their morphologies in response to the stiffness change of the PhoCl hydrogel substrate dynamically, rendering PhoCl-based hydrogels a useful substrate for other mechanobiological studies.

The Ednrb–Aim2–AKT axis regulates neural crest-derived melanoblast proliferation during early development

ABSTRACT Ednrb is specifically required to develop neural crest (NC) stem cell-derived lineages. However, it is still unknown why Ednrb signaling is only needed for the early development of melanoblasts in the skin and eye. We show that Ednrb is required for the proliferation of melanoblasts during early mouse development. To understand the mechanism of melanoblast proliferation, we found that the gene absent in melanoma 2 (Aim2) is upregulated in Ednrb-deficient NC cells by RNA-sequencing analysis. Consequently, the knockdown or knockout of Aim2 partially rescued the proliferation of Ednrb-deficient melanoblasts. Conversely, the overexpression of Aim2 in melanoblasts suppressed their proliferation. We further show that Ednrb signaling could act through the microRNA miR-196b to block the suppression of melanoblast proliferation by Aim2 in primary NC cell cultures. These results reveal the Ednrb–Aim2–AKT axis in regulating melanocyte development and suggest that Ednrb signaling functions as a negative regulator of Aim2, which inhibits the proliferation of melanoblasts in early development. These findings uncover a previously unreported role for Aim2 outside the inflammasome, showing that it is a significant regulator controlling NC stem cell-derived lineage development.

Single-cell analyses reveal that monocyte gene expression profiles influence HIV-1 reservoir size in acutely treated cohorts.

Elimination of latent HIV-1 is a major goal of AIDS research but the host factors determining the size of these reservoirs are poorly understood. Here, we investigated whether differences in host gene expression modulate the size of the HIV-1 reservoir during suppressive ART. Peripheral blood mononuclear cells (PBMC) from fourteen individuals initiating ART during acute infection who demonstrated effective viral suppression but varying magnitude of total HIV-1 DNA were characterized by single-cell RNA sequencing (scRNA-seq). Differentially expressed genes and enriched pathways demonstrated increased monocyte activity in participants with undetectable HIV-1 reservoirs. IL1B expression in CD14+ monocytes showed the greatest fold difference. The inverse association of IL1B with reservoir size was validated in an independent cohort comprised of 38 participants with different genetic backgrounds and HIV-1 subtype infections, and further confirmed with intact proviral DNA assay (IPDA ® ) measurements of intact HIV-1 proviruses in a subset of the samples. Modeling interactions with cell population frequencies showed that monocyte IL1B expression associated inversely with reservoir size in the context of higher frequencies of central memory CD4+ T cells, implicating an indirect effect of IL1B via the cell type well established to be a reservoir for persistent HIV-1. Signatures consisting of co-expressed genes including IL1B were highly enriched in the "TNFα signaling via NF-κB" geneset. Functional analyses in cell culture revealed that IL1B activates NF-κB, thereby promoting productive HIV-1 infection while simultaneously suppressing viral spread, suggesting a natural latency reversing activity to deplete the reservoir in ART treated individuals. Altogether, unbiased high throughput scRNA-seq analyses revealed that monocyte IL1B variation could decrease HIV-1 proviral reservoirs in individuals initiating ART during acute infection.

Abstract I009: Leveraging engineered virus-like particles for protein and RNA delivery

Abstract Precision gene editing agents enable the manipulation of genomic DNA in living organisms and raise the possibility of treating the root cause of many genetic diseases. However, realizing the full promise of therapeutic gene editing requires the ability to safely and efficiently deliver gene editing agents into relevant tissues and cell types. We developed engineered virus-like particles (eVLPs) that efficiently package and deliver gene editing proteins or RNAs into cells in culture and in mice1. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient gene editing in several primary mouse and human cell types. Single injections of eVLPs into mice supported therapeutic levels of gene editing in multiple tissues, including liver and retina. Off-target gene editing from eVLPs was virtually undetected, an improvement over canonical viral delivery methods. Recently, we further improved the delivery potency of eVLPs by applying a novel directed evolution platform to identify eVLP capsid mutants that exhibit improved properties. Collectively, these results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and non-viral delivery. 1. S. Banskota*; A. Raguram*; S. Suh; S. W. Du; J. R. Davis; E. H. Choi; X. Wang; S. C. Nielsen; G. A. Newby; P. B. Randolph; M. J. Osborn; K. Musunuru; K. Palczewski; D. R. Liu. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 185, 250–265 (2022). Citation Format: Aditya Raguram. Leveraging engineered virus-like particles for protein and RNA delivery [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I009.

Metal-Free CO Prodrugs Activated by Molecular Oxygen Protect against Doxorubicin-Induced Cardiomyopathy in Mice.

Carbon monoxide has been extensively studied for its various therapeutic activities in cell cultures and animal models. Great efforts have been made to develop noninhalational approaches for easy and controlled CO delivery. Herein, we introduce a novel metal-free CO prodrug approach that releases CO under near-physiological conditions. CO from the quinone-derived CO prodrugs is initiated by general acid/base-catalyzed tautomerization followed by oxidation by molecular oxygen to form the key norbornadienone intermediate, leading to cheletropic CO release only in an aerobic environment. Representative CO prodrug analog QCO-105 showed marked anti-inflammatory effects and HO-1 induction activity in RAW264.7 macrophages. In a mouse model of doxorubicin-induced cardiomyopathy, we show for the first time that the CO prodrug QCO-105 prevented cardiomyocyte injury, consistent with the known organ-protective effects of HO-1 and CO. Overall, such a new CO prodrug design serves as the starting point for developing CO-based therapy in attenuating the cardiotoxicity of doxorubicin.

Multi-well plate-based versatile platform for online fabricating alginate hydrogel microspheres and in-situ 3D cell culture

BackgroundHydrogel microspheres with monodisperse and homogeneous dimensions have potential application in the field of three-dimensional (3D) cell culture due to its ability to provide a similar microenvironment. Currently, alginate hydrogel microspheres (AHMs) have received much attention due to the favorable properties of alginate such as biocompatibility, inexpensiveness, nontoxicity, and biodegradability. The fabrication methods of AHMs mainly include extrusion, electrostatic dripping and microfluidic chip techniques. These current methods suffer trade-offs between operational complexity, fabrication cost and practical application. ResultsWe proposed a novel and versatile multi-well plate-based platform for online fabricating AHMs and in-situ 3D cell culture. The AHMs could be easily fabricated based on gravity-driven gelation combined with our recently developed bent-capillary-centrifugal-driven (BCCD) system. Ca-EDTA complex was used as Ca2+ source for crosslinking reaction of the alginate chains. The whole preparation process of AHMs included four steps: emulsification, pre-gelation, spontaneous demulsification and further solidification. The gravity-driven hydrogel microsphere gelation could produce the AHMs with good sphericity (Φ = 0.96) and monodispersity (PDI% = 0.94 %). The rapid drug susceptibility testing and single-cell encapsulation in the AHMs were well demonstrated. It also provided a novel in-situ 3D cell culture strategy, which demonstrated more than 85 % cell viability in practice. SignificanceThe proposed platform avoided the complex and laborious microfabrication. Moreover, cell-encapsulated AHMs could be directly produced in the multi-well plate, which could facilitate the subsequent cultivation and observation. It is expected to be a versatile in-situ 3D cell culture tool in the fields of biomedicine and tissue engineering.

Salmonella Infantis Adhesion to Various Surfaces and In Vitro Antimicrobial Efficacy of Commercial Disinfectants.

Salmonella Infantis poses a significant challenge in poultry production due to its persistence and resistance to disinfectants. This study investigated the survival of the S. Infantis strain on different surfaces and evaluated the efficacy of disinfectants in both preventing and treating biofilms. The survival of the tested S. Infantis strain was assessed on plastic and stainless steel surfaces after 24 and 48 h. The minimum inhibitory concentrations (MICs) of five disinfectants were determined, and their antiadhesion effectiveness was evaluated using crystal violet. The efficacy of biofilm treatment was evaluated by cell culturability. The results showed that the adhesion of S. Infantis was significantly higher on the plastic surface. The disinfectants were effective at reducing biofilm formation only within the first 24 h. Fresh solutions of disinfectants based on quaternary ammonium compounds exhibited the highest antimicrobial efficacy, while chlorocresol was the most effective for both the prevention and treatment of biofilms. The study results suggest that the presence of plastic surfaces may contribute to the dissemination of Salmonella. Additionally, the effectiveness of disinfectants varied based on storage conditions and contact time, while biofilms demonstrated reduced susceptibility compared to planktonic cells. However, given the laboratory scale of this study, further validation on a commercial scale is necessary to confirm these findings.

Euonymus maximowiczianus aril-derived long-term suspension-cultured cells: Light and methyl jasmonate impact in the anthocyanin and VLCFA accumulation

The genus Euonymus (L.) consists of shrubs and woody plants, distributed mainly in the Northern Hemisphere. Several hundred of secondary metabolites have been isolated from Euonymus spp. In addition, fatty oil was found in the fruits of some Euonymus spp., which accumulates not only in the seeds but also in the arils. This study presents the research of unique over ten-year-old suspension cell cultures of the endemic plant Euonymus maximoviczianus Prokh., obtained from the aril tissue of unripe capsules. The suspension cells retain the ability to form oil droplets containing neutral lipids. Both cells growing in the dark (Em-D culture) and cells growing in the light (Em-L culture) can synthesize very-long-chain fatty acids (VLCFAs) as well as cyanidin-3-O-hexoside, delphinidin-3-O-hexoside, and peonidin-3-O-hexoside. Here, we researched the VLCFA and anthocyanin accumulation dynamics during subcultivation, as well as the influence of methyl jasmonate (MeJA) and light on these processes. In the darkness, the formation of VLCFAs was more intense, while the biosynthesis of anthocyanins was significantly activated in the light. In Em-L cells, more than 76% of anthocyanins were represented by cyanidin-3-O-hexoside, and in Em-D cells delphinidin-3-O-hexoside was more actively synthesized (45%). MeJA substantially enhanced the accumulation of anthocyanins (especially in the light) and, surprisingly, the formation of VLCFAs in both Em-L and Em-D cell cultures. The possible competition between the biosynthetic pathways of VLCFAs and anthocyanins is discussed in connection with the commonality of the cytosolic pool of their precursor, malonyl-CoA.

Synthesis and effect of 4-acetylphenylamine-based imidazole derivatives on migration and growth of 3D cultures of breast, prostate and brain cancer cells

In this study, we have synthesized novel 4-acetophenone moiety-bearing functionalized imidazole derivatives containing S-, and N-ethyl substituents and evaluated their anticancer activity. Their anticancer activity was studied against human breast carcinoma (MDA-MB-231), human prostate carcinoma (PPC-1), and human glioblastoma (U-87). Compounds 4, 9, 14, and 22 were identified as the most promising anticancer agents from a series of imidazole derivatives. They showed the highest cytotoxicity by MTT assay against MDA-MB-231, PPC-1 and U-87 cell lines. Compounds 14 and 22 were most selective against PPC-1 and U-87 cell lines, and their EC50 values against these cell lines ranged from 3.1 to 47.2 µM. Most tested compounds showed lower activity against the triple-negative breast cancer MDA-MB-231 cell line. None of the imidazole derivatives possessed an inhibiting effect on the migration of PPC-1 and U-87 cells by ‘wound’ healing assay. In spheroid assay, the most promising were compounds 14 and 22, especially in PPC-1 3D cultures. They efficiently reduced both the size and the viability of PPC-1 spheroid cells.

Abstract B004: Ovarian cancer long intergenic noncoding RNA 1 is a potential therapeutic target in advanced ovarian cancer by promoting glycolytic pathway and hypoxic response

Abstract Ovarian cancer (OvC) is often diagnosed as an advanced disease because symptoms do not develop at earlier stages. Besides current treatments, alternative efficient therapies for OvC need to be established. We have previously identified that a novel long intergenic noncoding RNA (lincRNA) originated from chromosome 10q21 was predominantly expressed in clinical ovarian cancer tissues, designated as ovarian cancer lincRNA 1 (OIN1). Silencing of OIN1 in OvC cell lines substantially exhibited the inhibition of in vitro and in vivo cell proliferation, by upregulating apoptosis-related genes. In the present study, we aimed to evaluate whether OIN1 can be a therapeutic target in advanced OvC and how OIN1 drives the progression of OvC. We established long-term culturable spheroid cultures of OvC patient-derived cells (OvC-PDCs) from ascites of OvC patients as an advanced OvC model. The OvC-PDC spheroid cultures abundantly expressed OIN1 and stemness-related genes. OIN1-targeted siRNAs could suppress the spheroid growth and upregulate apoptosis-related genes such as RASSF5 and ADORA1 in OvC-PDC cultures. In a bloody ascites-generating xenograft in vivo model of mice intraperitoneally injected with OvC-PDC, OIN1-targeted siRNA treatment engineered by drug delivery system alleviated ascites production and prolonged the mice survival time. Based on transcriptomic analysis of OIN1 silencing in OvC-PDC cultures, we found that OIN1 inhibition exerts the downregulation of genes involved in glycolytic pathway and hypoxic response. We further demonstrated that OIN1 modulates HIF1a-mediated transcriptional regulation in OvC- PDCs. The present findings provide promising evidence that OIN1 can be a potential therapeutic target for advanced OvC. Citation Format: Kuniko Horie, Wataru Sato, Kazuhiro Ikeda, Satoshi Inoue. Ovarian cancer long intergenic noncoding RNA 1 is a potential therapeutic target in advanced ovarian cancer by promoting glycolytic pathway and hypoxic response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B004.

Human Supplementation with AM3, Spermidine, and Hesperidin Enhances Immune Function, Decreases Biological Age, and Improves Oxidative-Inflammatory State: A Randomized Controlled Trial.

The positive effect of AM3, spermidine, and hesperidin, which have antioxidant and anti-inflammatory properties, on immunity is known, but their effect on the rate of aging, known as biological age (BA), is unclear. This work aims to test if the intake of a blend of AM3 (150 mg), spermidine (0.6 mg), and hesperidin (50 mg) for 2 months could decrease BA and improve immunity, redox, and inflammatory states. For this, 41 participants (30-63 years) were randomly divided into placebo and supplement groups. The supplement group took two capsules daily with AM3, spermidine, and hesperidin for two months, while the placebo group took capsules containing only calcium phosphate and talcum powder. Before and after the treatment, peripheral blood was collected. Immune function was assessed in leukocytes, redox state in whole-blood cells, erythrocytes, and plasma, and cytokine concentration in both mononuclear cell cultures and plasma. Finally, the Immunity Clock model was applied to determine BA. The results show that the intake of this blend improves the immune functions that constitute the Immunity Clock, decreasing BA by 11 years and reducing the oxidative-inflammatory state of the participants. Therefore, this supplement can be proposed as a strategy to rejuvenate BA and achieve healthy aging.

Immortalized mammosphere-derived epithelial cells retain a bioactive secretome with antimicrobial, regenerative, and immunomodulatory properties

BackgroundThe secretome of primary bovine mammosphere-derived epithelial cells (MDECs) has been shown to exert antimicrobial, regenerative, and immunomodulatory properties in vitro, which warrants its study as a potential biologic treatment with the potential to be translated to human medicine. Currently, the use of the MDEC secretome as a therapy is constrained by the limited life span of primary cell cultures and the decrease of secretome potency over cell passages.MethodsTo address these limitations, early-passage bovine MDECs were immortalized using hTERT, a human telomerase reverse transcriptase. The primary and immortal MDECs were compared morphologically, transcriptomically, and phenotypically. The functional properties and proteomic profiles of the secretome of both cell lines were evaluated and compared. All experiments were performed with both low and high passage cell cultures.ResultsWe confirmed through in vitro experiments that the secretome of immortalized MDECs, unlike that of primary cells, maintained antimicrobial and pro-migratory properties over passages, while pro-angiogenic effects of the secretome from both primary and immortalized MDECs were lost when the cells reached high passage. The secretome from primary and immortalized MDECs, at low and high passages exerted immunomodulatory effects on neutrophils in vitro.ConclusionsHigh passage immortalized MDECs retain a bioactive secretome with antimicrobial, regenerative, and immunomodulatory properties, suggesting they may serve as a consistent cell source for therapeutic use.

Unveiling tryptophan dynamics and functions across model organisms via quantitative imaging

BackgroundTryptophan is an essential amino acid involved in critical cellular processes in vertebrates, serving as a precursor for serotonin and kynurenine, which are key neuromodulators to influence neural and immune functions. Systematic and quantitative measurement of tryptophan is vital to understanding these processes.ResultsHere, we utilized a robust and highly responsive green ratiometric indicator for tryptophan (GRIT) to quantitatively measure tryptophan dynamics in bacteria, mitochondria of mammalian cell cultures, human serum, and intact zebrafish. At the cellular scale, these quantitative analyses uncovered differences in tryptophan dynamics across cell types and organelles. At the whole-organism scale, we revealed that inflammation-induced tryptophan concentration increases in zebrafish brain led to elevated serotonin and kynurenine levels, prolonged sleep duration, suggesting a novel metabolic connection between immune response and behavior. Moreover, GRIT’s application in detecting reduced serum tryptophan levels in patients with inflammation symptoms suggests its potential as a high-throughput diagnostic tool.ConclusionsIn summary, this study introduces GRIT as a powerful method for studying tryptophan metabolism and its broader physiological implications, paving the way for new insights into the metabolic regulation of health and disease across multiple biological scales.

Augmenting antitumor efficacy of Th17-derived Th1 cells through IFN-γ-induced type I interferon response network via IRF7

The importance of CD4+ T cells in cancer immunotherapy has gained increasing recognition. Particularly, a specific subset of CD4+ T cells coexpressing the T helper type 1 (Th1) and Th17 markers has demonstrated remarkable antitumor potential. However, the underlying mechanisms governing the differentiation of these cells and their subsequent antitumor responses remain incompletely understood. Single-cell RNA sequencing (scRNA-seq) data reanalysis demonstrated the presence of Th171 cells within tumors. Subsequent trajectory analysis found that these Th171 cells are initially primed under Th17 conditions and then converted into IFN-γ-producing cells. Following the in vivo differentiation trajectory of Th171 cells, we successfully established in vitro Th171 cell culture. Transcriptomic profiling has unveiled a substantial resemblance between in vitro-generated Th171 cells and their tumor-infiltrating counterparts. Th171 cells exhibit more potent antitumor responses than Th1 or Th17 cells. Additionally, Th171chimeric antigen receptor T (CAR-T) cells eradicate solid tumors more efficiently. Importantly, Th171 cells display an early exhaustion phenotype while retaining stemness. Mechanistically, Th171 cells migrate faster and accumulate more in tumors in an extracellular matrix protein 1 (ECM1)-dependent manner. Furthermore, we show that IFN-γ up-regulated IRF7 to promote the type I interferon response network and ECM1 expression but decreased the exhaustion status in Th171 cells. Taken together, our findings position Th171 cells as a great candidate for improving targeted immunotherapies in solid malignancies.

Comprehensive property combination for biomedical application achieved in a Ti35Zr35Nb15Mo5Fe5Cr5 complex concentrated alloy

A novel Ti35Zr35Nb15Mo5Fe5Cr5 complex concentrated alloy (CCA) with potential for biomedical application was developed by vacuum arc melting and its microstructural evolution, mechanical properties, corrosion and wear behavior, and cytocompatibility were systematically evaluated. The alloy has an experimentally measured density of 6.37 g/cm³, suitable to fulfill the requirement of light weight. XRD analysis revealed that the as-cast and annealed specimens contain two BCC solid solution phases and a TiCr2 type Laves phase. The average microhardness (H) and elastic modulus (E) of the as-cast CCA are 618.39 ± 9.26 HV and 97.32 ± 3.58 GPa, respectively. Moreover, the yield strength (YS) of the as-cast alloy, estimated from elastoplastic analysis of the microindentation data, is 1203.94 ± 30.28 MPa. However, the CCA annealed at 1100˚C exhibits a microhardness of 833.08 ± 7.58 HV and a YS of 1669.65 ± 24.79 MPa, with the elastoplastic stress-strain response revealing no significant loss of plasticity due to the increased hard Laves phase. Corrosion and wear tests conducted in simulated body fluid confirmed the alloy's excellent corrosion and wear resistance. Cell culture experiments with MG-63 and HEK-293 cells demonstrated superior cell viability and proliferation compared to CP-Ti and 316 L SS. Moreover, confocal fluorescence images of MG-63 cells stained with AO/EtBr, Rh-123, and DCFH-DA revealed that the present CCA exhibits good biocompatibility. Thus, a comprehensive combination of low density, compatible elastic modulus, good strength with adequate plasticity, and sufficient corrosion resistance and biocompatibility were successfully achieved, unraveling significant potential of the alloy for biomedical applications.

Anti-Inflammatory Effects of Cordyceps Cs-HK1 Fungus Exopolysaccharide on Lipopolysaccharide-Stimulated Macrophages via the TLR4/MyD88/NF-κB Pathway.

Chronic inflammation is a common factor in the pathological processes of multiple human diseases. EPS-LM, an exopolysaccharide (EPS) from the Cordyceps sinensis fungus Cs-HK1, has shown notable anti-inflammatory activities in previous studies. This study aimed to investigate the major signaling events mediating the anti-inflammatory effects of EPS-LM in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cell culture. EPS-LM treatment significantly reduced LPS-induced production of pro-inflammatory mediators, including nitric oxide (NO) and reactive oxygen species (ROS). It also suppressed the expression levels of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), subsequently delaying the translocation of nuclear factor-kappa B (NF-κB) to the nucleus. Additionally, co-immunoprecipitation (Co-IP) experiments demonstrated that EPS-LM inhibited the binding of TLR4 to MyD88. The ability of EPS-LM to inhibit the TLR4/MyD88/NF-κB pathway, coupled with its capacity to reduce oxidative stress, underscores its multifaceted anti-inflammatory effects. These effects render EPS-LM as a promising candidate for the comprehensive management of various inflammatory and oxidative stress-related conditions, protecting against cell damage.

Paclitaxel-Loaded, Pegylated Carboxylic Graphene Oxide with High Colloidal Stability, Sustained, pH-Responsive Release and Strong Anticancer Effects on Lung Cancer A549 Cell Line.

Background: Graphene Oxide (GO) has shown great potential in biomedical applications for cancer therapeutics. The biosafety and stability issues of GO in biological media have been addressed by functionalization with polyethylene glycol (PEG). Methods: In this work, carboxylated, nanosized GO (nCGO) was evaluated as a potential carrier of paclitaxel (PCT). The effect of PEG characteristics on particle size and surface charge, colloidal stability, drug, and release, and the hemolytic potential of nCGO, was investigated. Optimum PEG-nCGO/PCT formulations based on the above properties were evaluated for their anticancer activity (cytotoxicity and apoptosis induction) in the A549 lung cancer cell line. Results: An increase in the length of linear PEG chains and the use of branched (4-arm) instead of linear PEG resulted in a decrease in hydrodynamic diameter and an increase in ζ potential of the pegylated nCGO particles. Pegylated nCGO exhibited high colloidal stability in phosphate-buffered saline and in cell culture media and low hemolytic effect, even at a relatively high concentration of 1 mg/mL. The molecular weight of PEG and branching adversely affected PCT loading. An increased rate of PCT release at an acidic pH of 6.0 compared to the physiological pH of 7.4 was observed with all types of pegylated nCGO/PCT. Pegylated nCGO exhibited lower cytotoxicity and apoptotic activity than non-pegylated nCGO. Cellular uptake of pegylated nCGO increased with incubation time with cells leading to increased cytotoxicity of PEG-nCGO/PCT with incubation time, which became higher than that of free PCT at 24 and 48 h of incubation. Conclusions: The increased biocompatibility of the pegylated nCGO and the enhanced anticancer activity of PEG-nCGO/PCT compared to free PCT are desirable properties with regard to the potential clinical application of PEG-nCGO/PCT as an anticancer nanomedicine.

IN VIVO INOCULATION OF GBM CELLS IN RATS AND TREATMENT WITH RUXOLITINIB

GlioStat (patent pending) denotes an invention that addresses the current deficiencies of glioblastoma (GBM) chemotherapy by developing a molecularly imprinted drug reservoir that is specifically designed for post-surgical recovery. The goal of our research was to guarantee the long-term release of the antitumor drug ruxolitinib (RUX), which is specifically designed to target residual infiltrative cancer cells, while simultaneously reducing the risk of adverse effects. In order to achieve this goal, we have successfully developed and characterized four distinctive molecularly imprinted polymers (MIPs), with one of them advancing to in vivo experimentation. The selection of one MIP for further in vivo investigations was guided by the Alamar Blue viability assay on C6 GBM cell cultures, which took into account both the efficacy and the potential toxicity of residual monomers. Over the course of 96 hours, MIP 2 demonstrated the most favorable risk-benefit profile, providing superior efficacy against GBM C6 cells. In contrast, its non-imprinted counterpart (NIP 2) exhibited minimal toxicity. The protocol involved anesthetising the male Wistar rats, immobilising them on a corkboard, without the use of a stereotactic headholder. After shaving the head and local disinfection with iodine solution, we made a linear sagittal incision and exposed the parietal bones. A 3x3 mm burr hole was made with a pneumatic drill in the right parietal bone, revealing the dura mater. Subsequently, we injected 20,000 C6 GBM cells suspended in 5 microliters of solution in the cerebral parenchyma at a depth of 3 mm. Clinical and imaging control was performed. For the animals that developed tumors, a second therapeutic intervention was performed. The rats were anesthetised, disinfected, and readied in the same manner as before. The burr hole was exposed, with the tumor being partially resected via a “microscooping” technique. Next, 5-10 microliters of MIP2 solution (4 mg/mL) were injected into the parenchyma respective of the tumor bed. Five microliters of thrombin solution were then added (100 UI/mL) to form the fibrin network. In comparison to the untreated controls and animals treated with free RUX, animals treated with MIP2 exhibited a substantial increase in survival time during the in vivo evaluation, extending from 20 to 50 days. As such, our research had yielded a potentially life-changing drug delivery system in GBM, with the capacity to significantly increase postoperative survival. More in vivo tests should be conducted to validate our findings.