Cell Assay Research Articles

Evaluation of metabolite profiles and neuroprotective potential in four pomegranate cultivars for Alzheimer's disease prevention.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and memory loss, with oxidative stress and neuroinflammation playing crucial roles in its progression. Current treatments, such as acetylcholinesterase (AChE) inhibitors, offer limited symptomatic relief and can have side effects. With growing interest in functional foods that have fewer side effects, pomegranate (Punica granatum L.) has gained attention because of its rich antioxidant content. The present study aims to evaluate the antioxidant and neuroprotective effects of four pomegranate cultivars: 'Wonderful', 'Phoenicia', 'Azadi' and 'Eversweet'. Additionally, it investigates their metabolite profiles and predicts potential bioactive compounds that could help prevent AD. The 'Wonderful' cultivar demonstrated the highest levels of total phenolic content, total flavonoid content, total anthocyanin content and radical scavenging activities. The AChE and butyrylcholinesterase (BuChE) inhibition rates were highest in 'Phoenicia' and 'Wonderful' cultivars, suggesting potential neuroprotective effects. PC12 cell assays indicated that these cultivars significantly improved cell viability, reduced reactive oxygen species and malondialdehyde levels, and also enhanced catalase activity. Metabolomics analysis identified 141 metabolites, with the 'Wonderful' cultivar showing the highest metabolite abundance. Molecular docking studies indicated that some metabolites, such as taxifolin, demonstrated strong binding affinities for AChE and BuChE, suggesting potential for AD treatment. The 'Wonderful' and 'Phoenicia' cultivars demonstrated the most promising antioxidant and neuroprotective effects among the evaluated pomegranates, likely because of their high taxifolin content. These findings suggest that these cultivars could be valuable for developing functional foods aimed at AD prevention and treatment. © 2025 Society of Chemical Industry.

Potential of Newly Synthesized Sea Buckthorn Phytocarriers as Anti-Inflammatory Active Agents

Background: Phytocarriers are advanced drug delivery systems that use biocompatible and biodegradable materials to enhance the efficacy, stability, and bioavailability of natural products. The sea buckthorn (Hippophae rhamnoides L.) berry extract is rich in essential fatty acids and antioxidants, including vitamin C, vitamin E, and anthocyanins, which contribute to its wide-ranging health benefits. In this study, we assessed the morphology, intracellular delivery, and anti-inflammatory effect of sodium cholate (NaC) and sodium deoxycholate (NaDC)-based phytocarriers loaded with ethanolic extract from sea buckthorn berries (sea buckthorn carrier nanostructures, further defined as phytocarriers). Methods: Negative and electron cryo-microscopy were used to analyze hollow and loaded nanocarriers. The cyto-compatibility of nanocarriers was assessed by endpoint (LDH and MTS) and real-time cell assays, on both human fibroblasts (HS27) and human normal monocytes (SC). The anti-inflammatory effect of hollow and loaded nanocarriers was tested by multiplexing. Results: The negative and electron cryo-microscopy analyses showed that NaC-based phytocarriers were spherical, whilst NaDC-based phytocarriers were predominantly polymorphic. Moreover, the NaDC-based phytocarriers frequently formed large lipid networks or “plaques”. Although 24 h cytotoxicity testing showed both types of nanocarriers are biocompatible with human fibroblasts and monocytes, based on a long-term real-time assay, NaDC delayed fibroblast proliferation. NaC sea buckthorn phytocarriers did not impair fibroblast proliferation in the long term and they were uptaken by cells, as shown by hyperspectral microscopy. NaC nanocarriers and NaC sea buckthorn phytocarriers induced an anti-inflammatory effect, lowering IL-8 cytokine production in normal human monocytes as soon as 4 h of treatment lapsed. Conclusions: NaC-derived phytocarriers loaded with sea buckthorn alcoholic extract are a cell-compatible delivery system with anti-inflammatory properties.

Type IV PilD mutant stimulates the formation of persister cells in Pseudomonas aeruginosa.

Pseudomonas aeruginosa clinical isolates that lack motility do not express type IV pilin, yet the biological roles of this absence in the infection process remain poorly understood. We asked whether the absence of motility in these bacteria is associated with increased antibiotic persistence. In this study, we analysed type IV PilD protein sequences in the database and conducted antibiotic-tolerant persister cell assays. We found that PilD variants were common in P. aeruginosa clinical isolates. Our results revealed that inactivation of PilD resulted in a significantly higher level of surviving persister cells following ciprofloxacin treatment. This PilD-mediated persistence did not involve previously described mechanisms, such as phenazine pyocyanin, biofilm or stringent response. Our findings connect the non-motility of clinical P. aeruginosa isolates with the survival of persister cells, highlighting the clinical significance for the development of strategies to eradicate P. aeruginosa infections.

Nonessential kinetochore proteins contribute to meiotic chromosome condensation through polo-like kinase.

Chromosome condensation plays a pivotal role during faithful chromosome segregation, hence, understanding the factors that drive condensation is crucial to get mechanistic insight into chromosome segregation. Previously, we showed that in budding yeast, the absence of the nonessential kinetochore proteins affects chromatin-condensin association in meiosis but not in mitosis. A differential organization of the kinetochores, that we and others observed earlier during mitosis and meiosis may contribute to the meiotic-specific role. Here, with our in-depth investigation using in vivo chromosome condensation assays in cells lacking a nonessential kinetochore protein, Ctf19, we establish that these proteins have roles in achieving a higher meiotic condensation without influencing much of the mitotic condensation. We further observed an accumulation of the polo-like kinase Cdc5 owing to its higher protein stability in ctf19Δ meiotic cells. High Cdc5 activity causes hyperphosphorylation of the condensin resulting in its reduced stability and concomitant decreased association with the chromatin. Overall, our findings highlight the role of Ctf19 in promoting meiotic chromosome condensation by influencing the activity of Cdc5 and thereby affecting the stability and association of condensin with the chromatin.

High-throughput screening to identify endocrine disruptors: Contribution of low-resolution tandem MS and high-resolution MS.

Considering the large diversity of chemicals present in the environment and the need to study their effects (alone or as mixtures), the development of high-throughput in vitro assays in line with the Replacement, Reduction, Refinement (3R) strategy is essential for chemical risk assessments. We developed a robust analytical workflow based on both low resolution tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry (HRMS) to quantify 13 steroids in NCI-H295R cell culture medium, human plasma and serum. The workflow was validated by screening media from the NCI-H295R cell line exposed in dose-response experiments to 5 endocrine disruptors (EDs) such as bisphenol A, prochloraz, ketoconazole, atrazine and forskolin. Absolute quantifications of the 13 steroids performed on a triple quadrupole (QqQ) MS/MS demonstrated that the performances obtained were in line with OECD recommendations. HRMS (MS1-HRMS) provided measurements nearly as sensitive and as reproducible as those obtained using multiple reaction monitoring (MRM) and ELISA. A bioinformatics workflow, using HRMS, was implemented to detect and annotate disrupted metabolites. HRMS allowed to detect disruptions in pathways associated to fatty acids, purines and amino acids metabolisms after exposure to the EDs tested, in addition to that linked to steroidogenesis. We developed a robust MS1-HRMS workflow, from sample preparation to compound quantification or annotation, compatible with absolute steroid quantification, to screen NCI-H295R cell media exposed to potential EDs. Using only 200μL of medium, the method integrates MS/MS and HRMS analyses, 96-well plate solid-phase extraction for high throughput, and automated pre-annotation for cost efficiency. This optimized workflow identifies EDs in cell assays by detecting disruptions in steroidogenesis and other biological pathways.

An Integrated Approach of Network Pharmacology, Bioinformatics, Molecular Docking, and Experimental Verification Uncovers Prunellae Spica as the potential Medicine of Prognosis Improvement for Oral Squamous Cell Carcinoma.

Prunellae Spica (PS), the spike from Prunella vulgaris L., is a traditional Chinese medicine that can treat Oral Squamous Cell Carcinoma (OSCC), whereas its molecular mechanisms and effects on the prognosis of patients remain unclear. Our study aimed to identify potential anti-OSCC targets of PS and explore its mechanisms and effects on prognosis through network pharmacology, bioinformatics analysis, molecular docking, and in vitro cell assays. Sixty-two potential targets of 11 active anti-OSCC ingredients of PS were identified, with Quercetin, the core ingredient of PS, exhibiting the most significant number of OSCC-related targets. GO analysis indicated that the primary biological processes involved in OSCC treatment by PS were the cellular response to nitrogen compound, response to xenobiotic stimulus, and cellular response to organonitrogen compound. KEGG analysis revealed that Pathways in cancer were the top highly enriched signaling pathway in the treatment of OSCC by PS. DisGeNET analysis is mainly about Lip and Oral Cavity Carcinoma. More importantly, 6 of the 62 targets were markedly related to prognosis. Molecular docking revealed high affinities between the key component and the prognosis-related target proteins. Treatment of OSCC cell line SCC-25 with Quercetin could inhibit malignant biological behaviors, such as cell proliferation, colony formation, invasion, and migration, as well as affect the targets related to prognosis and promote autophagy. Overall, these results suggest that PS plays a significant role in treating and improving the prognosis of OSCC by directly influencing various processes in OSCC.

Synthesis and characterization of TiO2-ZnO composite thin films for biomedical applications.

Titania (TiO2) has superior biocompatibility, while zinc oxide (ZnO) is antibacterial. This investigation aimed to study the influence of TiO2-ZnO composite films on enhancing the biocompatibility of stainless steel (SS). Radio-frequency magnetron sputtering (RF-MS) technique is used to synthesize TiO2-ZnO composite thin films on 304-SS substrates from three sputtering targets with typical chemical compositions of 100% TiO2, 90%TiO2-10%ZnO, and 75%TiO2-25%ZnO, mixed by their respective weight percentages. The influence of surface chemistry, morphology, and wettability of TiO2-ZnO composite film on its osseointegration and antifouling characteristics was studied. The biocompatibility was assessed by protein adsorption kit, cytotoxicity assay, and cell adhesion of MG63 osteoblast cells, followed by S. aureus bacterial adhesion studies. All RF-MS films displayed hydrophobicity, minimal bacterial-cell adhesion, and higher cytocompatibility than the SS. RF-MS films deposited from the 75%TiO2-25%ZnO target exhibited the highest antifouling capability due to the least protein adsorption and the highest antibacterial ZnO concentration. However, increased ZnO concentration decreased MG63 cell viability. RF-MS films deposited from the 90%TiO2-10%ZnO target showed the highest mammalian cell viability of ≈88% and attachment. High plasma protein adsorption caused decreased mammalian cell viability and higher bacterial adhesion on 100% TiO2film and SS. Biocompatible and antifouling TiO2-ZnO composite thin films on SS substrates offer an alternative to conventional antibiotic coatings to combat antimicrobial resistance (AMR) and biofilm-related infections.

Rapid screening and identification of novel dipeptidyl peptidase IV inhibitory peptides from buffalo milk.

Peptidomics combined with molecular docking is an effective alternative method for rapid screening of novel bioactive peptides in food. Buffalo milk as a potential source of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides has been less studied. Peptidomics and molecular docking methods were employed to rapidly screen new DPP-IV inhibitory peptides from buffalo milk. The screened DPP-IV inhibitory peptides were further verified using an in vitro inhibition assay and a Caco-2 cell assay. The DPP-IV inhibition rate of buffalo milk was increased from 73.40 ± 6.01% to 97.23 ± 3.18% in an in vitro digestion assay, suggesting that buffalo milk could be a promising source of DPP-IV inhibitory peptides. Subsequently, two novel peptides (GPFPIIV and FPQYL) with potential DPP-IV inhibitory activity were screened using peptidomics, molecular docking and an in vitro inhibitory assay. The IC50 values for GPFPIIV and FPQYL were 0.2998 ± 0.03 and 0.1407 ± 0.01 mg mL-1, respectively. During simulated gastrointestinal digestion in vitro, FPQYL had an excellent digestive stability of 92.13 ± 1.03%, whereas that of GPFPIIV was 59.52 ± 2.56%. In addition, GPFPIIV and FPQYL (1.00 mg mL-1) showed significant DPP-IV inhibitory effects in a Caco-2 cell assay, with the inhibition rate increasing to 32% and 36%, respectively. In summary, two new DPP-IV inhibitory peptides were screened from buffalo milk through a combination of peptidomics and molecular docking, both of which exhibited significant DPP-IV inhibitory activities. The identified peptides, GPFPIIV and FPQYL, have promising applications in diabetes management. © 2025 Society of Chemical Industry.

P-57. Durability of SARS-CoV-2 specific Cellular Immune Response after COVID-19 Vaccination in PLWH

Abstract Background People living with HIV (PLWH) face the dual challenge of human immunodeficiency virus(HIV) infection and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. It is associated with severe outcomes and a higher risk of mortality. In January 2021, the vaccination campaign against SARS-CoV-2 was launched in India and started mass administration of two types of vaccines. The two vaccines used on a large scale were, a recombinant, replication-deficient chimpanzee adenovirus vectored vaccine/ ChAdOx1 nCoV-19 (Covishield) and whole virion inactivated vaccine/ BBV152 (Covaxin).The study of cellular immune responses to COVID-19 vaccination in PLWH was vital for safeguarding this vulnerable population. Methods Study design: Prospective observational study Study population: The individuals recruited in the study were vaccinated with at least 2 doses and/or booster dose of the COVID-19 vaccine (COVISHIELD/COVAXIN). The individuals were segregated into two groups (PLWH and non-HIV-infected healthy individuals). Testing Method: Peripheral blood mononuclear cell (PBMC) were cultured to test the SARS-CoV-2-specific T cell immune response using activation induced marker/ intracellular cytokine staining (AIM/ICS) assay. Distribution of CD4 T lymphocytes count in HIV patients with mean CD4 count 470 cell/µl. Results We conducted did this study in two phases. The first phase was conducted between August 2022 and December 2022, while the second phase, or the follow-up, took place from January 2023 to December 2023. In the first phase/ first visit (V1), we recruited 50 adult PLWH and 40 adult healthy controls. Subsequently, in phase 2/follow-up/ second visit (V2), we achieved a follow-up rate of 100% in each arm. SARS-CoV2-specific cellular immune response after COVID-19 vaccination was comparable and durable IN PLWH as compared to healthy controls. Conclusion Comparable and durable immune response was observed in PLWH population on regular highly active antiretroviral therapy (HAART), with stable viral suppression and good CD4+ T cell counts. This was consistent over time across 1 year gap in both phases of the study and across different vaccine types. This outcome indicates that the cellular immune responses in our PLWH cohort is comparable and durable to that of the general population, which is a reassuring finding for the public health. SARS-CoV-2-specific CD8 T-cell immune response (scatter plots displaying the results of CD8 T cell assays, specifically evaluating the production of various cytokines (IFNγ (Interferon-gamma), TNFα (Tumor Necrosis Factor-alpha), and IL-2 (Interleukin 2), Granzyme B (Gran-B) at two different time points V1 and V2 (visit 1 and visit 2) with p values mentioned at the top. Disclosures All Authors: No reported disclosures

ZNF169 promotes thyroid cancer progression via upregulating FBXW10

BackgroundZinc finger protein 169 (ZNF169) plays a key role in cancer development. However, the specific role of ZNF169 in the tumorigenesis of thyroid carcinoma (THCA) remains poorly understood.MethodsThe expression of ZNF169 was measured using immunohistochemistry, RT-qPCR, and western blot. Cell proliferation was detected using CCK-8 assay and cell colony formation assays, while cell migration was determined by Transwell assay. Flow cytometry was used to detect cell apoptosis and cell cycle distribution. The interaction of ZNF169 and its downstream gene was studied using luciferase assay and CHIP-PCR. Recovery assay in cells and animals were also performed to demonstrate the mechanism.ResultsZNF169 was highly expressed in THCA tissues and cells lines compared with matched adjacent non-cancerous thyroid tissues or normal thyroid epithelial cell. Moreover, thyroid cancer cell proliferation and migration were suppressed following ZNF169 knockdown, while were potentiated by ZNF169 overexpression. ZNF169 also regulate THCA cell apoptosis and cell cycle progression. Mechanically, ZNF169 enhanced the transcription activity and expression of F-box/WD repeat-containing protein 10 (FBXW10) via the binding to its promoter. There was a positive correlation between ZNF169 and FBXW10 in THCA patients. In addition, knockdown of FBXW10 suppressed the proliferation of THCA cells. Recovery assays in vitro and in vivo demonstrated that FBXW10 knockdown reversed the effects of ZNF169 overexpression on THCA cell proliferation and tumor growth.ConclusionsIn summary, ZNF169 promotes THCA progression via upregulation of FBXW10, which may provide a novel theoretical basis for the development of clinical therapies for THCA.

Preclinical Toxicological Assessment of 2-(Benzoxazol-2-yl)[(2-hydroxynaphthyl)diazenyl]phenol Derivatives for Blue Light and UV Radiation Photoprotection Applications.

The widespread use of electronic devices has led to increased blue light exposure, highlighting the need for effective radiation blockers with blue light protection. Two synthetic 2-(2'-hydroxyphenyl)benzoxazole derivatives named azo-4'-benzoxazole and azo-5'-benzoxazole have shown an unprecedented blue light absorption capacity but had not been subjected to a safety evaluation. This study aimed to evaluate the cytotoxic, genotoxic, and mutagenic activities of these compounds. The cytotoxic and genotoxic activities were evaluated using MTT assay and comet assay in L929 fibroblast cells. Salmonella/microsome assay and micronucleus test were performed to detect gene and chromosomal mutations. The IC50 was 87.9 μg/mL for azo-4'-benzoxazole and 79.5 μg/mL for azo-5'-benzoxazole. In the Salmonella/microsome assay, the azo-5'-benzoxazole compound induced frameshift mutations in the TA97a strain in the presence of metabolic activation (S9 mix), while azo-4'-benzoxazole did not show mutagenic activities in all five strains tested. The azo-5'-benzoxazole showed genotoxic and mutagenic effects in L929 cells that were probably associated to the cleavage of azo-5' into its analogs 2-(4'-amino-2'-hydroxyphenyl)benzoxazole and 2-amino-1-naphthol. In conclusion, the azo-substituted group at the 5' position of the phenyl ring appears to have greater toxicological risks than substituents at the 4' position of 2-(phenyl)benzoxazole. The findings warrant further preclinical studies to ensure the safety of these compounds for use as blue light filters.

Effective eradication of acute myeloid leukemia stem cells with FLT3-directed antibody-drug conjugates.

Refractory disease and relapse are major challenges in acute myeloid leukemia (AML) therapy attributed to survival of leukemic stem cells (LSC). To target LSCs, antibody-drug conjugates (ADCs) provide an elegant solution, combining the specificity of antibodies with highly potent payloads. We aimed to investigate if FLT3-20D9h3-ADCs delivering either the DNA-alkylator duocarmycin (DUBA) or the microtubule-toxin monomethyl auristatin F (MMAF) can eradicate quiescent LSCs. We show here that DUBA more potently kills cell-cycle arrested AML cells compared to microtubule-targeting auristatins. Due to limited stability of 20D9h3-DUBA ADC in vivo, we analyzed both ADCs in advanced in vitro stem cell assays. 20D9h3-DUBA successfully eliminated leukemic progenitors in vitro in colony-forming unit and long-term culture initiating cell assays, both in patient cells and in patient-derived xenograft (PDX) cells. Further, it completely prevented engraftment of AML PDX leukemia-initiating cells in NSG mice. 20D9h3-MMAF had a similar effect in engraftment assays, but a less prominent effect in colony assays. Both ADCs did not affect healthy stem and progenitor cells at comparable doses providing the rationale for FLT3 as therapeutic LSC target. Collectively, we show that FLT3-directed ADCs with DUBA or MMAF have potent activity against AML LSCs and represent promising candidates for further clinical development.

Modeling the response to interleukin-21 to inform natural killer cell immunotherapy.

Natural killer (NK) cells are emerging agents for cancer therapy. Several different cytokines are used to generate NK cells for adoptive immunotherapy including interleukin (IL)-2, IL-12, IL-15 and IL-18 in solution, and membrane-bound IL-21. These cytokines drive NK cell activation through the integration of signal transducers and activators of transcription (STAT) and nuclear factor-kappa B (NF-κB) pathways, which overlap and synergize, making it challenging to predict optimal cytokine combinations for both proliferation and cytotoxicity. We integrated functional assays for NK cells cultured in a variety of cytokine combinations with mathematical modeling using feature selection and mechanistic regression models. Our regression model successfully predicts NK cell proliferation for different cytokine combinations and indicates synergy of activated STATs and NF-κB transcription factors between priming and post-priming phases. The use of IL-21 in solution in the priming of NK cell culture resulted in an improved NK cell proliferation, without compromising cytotoxicity potential or interferon gamma secretion against hepatocellular carcinoma cell lines. Our work provides an integrative framework for interrogating NK cell proliferation and activation for cancer immunotherapy.

Characterization of Novel Plantaricin-Derived Antiviral Peptides Against Flaviviruses

Flaviviruses, including West Nile virus, Zika virus, and Dengue virus, pose global health challenges due to their distribution, pathogenicity, and lack of effective treatments or vaccines. This study investigated the antiviral activity of novel truncated peptides derived from the two-peptide plantaricins PLNC8 αβ, PlnEF, PlnJK, and PlnA. The antiviral potential was predicted using machine learning tools, followed by in vitro evaluation against the Kunjin virus using plaque reduction assays in Vero cells. Molecular docking assessed peptide interactions with KUNV and ZIKV. Full-length and truncated peptides from PlnA, PlnE, PlnF, PlnJ, and PlnK demonstrated limited antiviral efficacy against KUNV in vitro, despite in silico predictions suggesting antiviral potential for PlnA, PlnE, and PlnJ. Large discrepancies were observed between the predicted and experimentally determined activities. However, complementary two-peptide plantaricins PlnEF and PlnJK exhibited significant synergistic effects. Furthermore, the truncated peptides PLNC8 α1-15 and PLNC8 β1-20 reduced KUNV viral load by over 90%, outperforming their full-length counterparts. Molecular docking revealed interactions of PLNC8 α and PLNC8 β, and their truncated variants, with KUNV and ZIKV, suggesting a mechanism involving viral envelope disruption. These findings highlight the potential of plantaricin-derived peptides as promising antiviral candidates against flaviviruses, warranting further investigation into their mechanisms and applications.

Evaluation of the Anti-Mycobacterial and Anti-Inflammatory Activities of the New Cardiotonic Steroid γ-Benzylidene Digoxin-15 in Macrophage Models of Infection

Cardiotonic steroids modulate various aspects of the inflammatory response. The synthetic cardiotonic steroid γ-benzylidene digoxin 15 (BD-15), a digoxin derivative, has emerged as a promising candidate with potential immunomodulatory effects. However, its biological activity remains largely unexplored. This study investigated the anti-mycobacterial and anti-inflammatory effects of BD-15 in an in vitro macrophage infection model with Mycobacterium spp. Unlike digoxin, which showed significant toxicity at higher concentrations, BD-15 exhibited no cytotoxicity in RAW 264.7 cells (a murine macrophage cell line). Both compounds were evaluated in Mycobacterium smegmatis-infected RAW 264.7 cells, reducing bacterial burden without direct bactericidal activity. Additionally, both modulated pro-inflammatory cytokine levels, notably by decreasing tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) levels. BD-15 specifically reduced NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome expression and increased interleukin-10 (IL-10) production. Notably, BD-15 reduced colony-forming unit (CFU) counts in Mycobacterium tuberculosis-infected RAW 264.7 cells. Toxicity assays in HepG2 cells (a human liver cancer cell line) showed that BD-15 had minimal hepatotoxicity compared to digoxin, and both demonstrated negligible acute toxicity in an Artemia salina bioassay. These findings revealed the immunomodulatory effects of cardiotonic steroids in a bacterial infection model and highlighted BD-15 as a safer alternative to digoxin for therapeutic applications.

Theoretical and practical considerations for validating antigen-specific B cell ImmunoSpot assays.

Owing to their ability to reliably detect even very rare antigen-specific B cells in cellular isolates such as peripheral blood mononuclear cells (PBMC), and doing so robustly in a high throughput-compatible manner, B cell ELISPOT/FluoroSpot (collectively "B cell ImmunoSpot") tests have become increasingly attractive for immune monitoring in regulated settings. Presently, there are no guidelines for the qualification and validation of B cell ImmunoSpot assay results. Here, we propose such guidelines, building on the experience acquired from T cell ImmunoSpot testing in an environment adhering to the requirements of regulatory bodies yet taking the unique features of B cell assays into account. A streamlined protocol is proposed that permits the performance of all tests needed for the formal validation of an antigen-specific B cell ImmunoSpot assay in only three experiments, utilizing 2.2 × 107 PBMC per donor. Subsequently, utilizing only 1-2 × 106 PBMC per sample (obtainable from 1 to 2 mL of blood), a validated multiplexed assay enables accurate quantification of the frequency of antigen-specific memory B cell-derived blasts secreting IgM, IgG, IgA or IgE antibodies. Collectively, such multiplexed B cell ImmunoSpot assays offer immense value for B cell immune monitoring programs due to their ease of implementation, scalability, applicability to essentially any antigenic system, economy of PBMC utilization, and last but not least, the high content information gained.

TC2N maintains stem cell-like characteristics to accelerate lung carcinogenesis by blockade of dual specificity protein phosphatase 3

BackgroundTandem C2 domains, nuclear (TC2N) is a protein that has been characterized to contain C2A domain, C2B domain, and a short C-terminus with a WHXL motif. In previous studies, we have uncovered the oncogenic role and mechanisms of TC2N in lung cancer: TC2N achieves this by inhibiting the p53 signaling pathway and activating the NF-kappaB signaling pathway. Beyond that, its precise function in tumorigenesis is not fully understood.MethodsTC2N-engineered mice model was used to assess the effect of TC2N knockout on normal lung and urethane-induced carcinogenesis. Tumor tissues of 395 lung cancer patients were subjected to tissue microarray and further assessed the associations of TC2N expression with tumor differentiation degree. The protein levels of TC2N and stem cell markers in cell lines and tissue specimens were monitored by WB and immunohistochemistry. In vitro cell assays were performed to assess the effect of TC2N ectopic expression on the stem cell-like characteristics of lung cancer cells. The downstream signaling pathway or target molecule of TC2N was mined using a combination of transcriptomics and proteomics, and the underlying mechanism was explored by WB and co-IP assays.ResultsHerein, TC2N appeared to have a strong effect in promoting lung tumorigenesis caused by urethane, whereas it seemed to lose its function in the normal lung. Meanwhile, we found that the functional differences of TC2N between lung tumor and normal lung were linked to its potential role in cancer cell stemness. Function-wise, TC2N overexpression maintained stem-like properties of lung cancer cell. Mechanism-wise, TC2N upregulated the phosphorylation of EGFR, ERK, STAT3 and FAK1 to activate these signaling pathways by the inhibition of DUSP3 phosphatase via a dual mechanism. Firstly, TC2N competes with EGFR, ERK, STAT3 and FAK1 for binding to DUSP3. This competition prevents these signaling molecules from being dephosphorylated by DUSP3, resulting in their sustained activation. Secondly, TC2N bind to DUSP3 and restrict the enzyme’s ability to dephosphorylate the signaling molecules.ConclusionsOverall, this study revealed a previously unknown role and mechanism of TC2N in the regulation of tumorigenesis and stemness in lung cancer cells.

OP22 NR1D1 Deficiency Induces CD47 Upregulation Impairing Macrophage Efferocytosis and Exacerbating Intestinal Inflammation in Ulcerative Colitis

Abstract Background Ulcerative colitis (UC) is marked by excessive apoptosis of colonic epithelial cells, driven by intrinsic epithelial defects and impaired macrophage efferocytosis. Inefficient clearance of apoptotic cells perpetuates inflammation and obstructs tissue resolution. NR1D1, a nuclear receptor involved in cellular homeostasis and inflammation, has been implicated in immune regulation, but its role in UC pathogenesis, particularly in macrophage efferocytosis, remains poorly understood. Methods A clinical cohort of 300 individuals (200 UC patients and 100 healthy controls) was analyzed to assess the relationship between NR1D1 expression and disease severity, using the Mayo score as a measure of clinical progression. In parallel, dextran sulfate sodium (DSS) was used to induce acute colitis in wild-type and NR1D1-knockout mice. Proteomic profiling was employed to elucidate the effects of NR1D1 deficiency on intestinal inflammation. Mechanistic insights into NR1D1 regulation of CD47 expression were obtained using CUT&Tag, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays in colonic epithelial cells from UC patients and healthy controls. The impact of NR1D1 deficiency on macrophage efferocytosis was explored in vitro using co-cultures of macrophages and organoids. Therapeutic potential was evaluated by administering a hydrogel loaded with SR9009, an NR1D1 agonist, to DSS-treated mice, followed by single-cell RNA sequencing (scRNA-seq) to assess cellular and molecular changes. Results NR1D1 expression was significantly reduced in UC patients and inversely correlated with disease severity, as reflected by the Mayo score. Proteomic analysis in DSS-treated mice confirmed that NR1D1 deficiency aggravated intestinal inflammation. CUT&Tag and ChIP assays identified CD47 as a direct transcriptional target negatively regulated by NR1D1. Loss of NR1D1 led to elevated CD47 expression in epithelial cells, which impaired macrophage efferocytosis and disrupted the resolution of inflammation. Administration of SR9009 via a hydrogel delivery system restored NR1D1 activity, reduced CD47 expression, and improved efferocytosis. scRNA-seq of colonic tissues from treated mice revealed a shift toward anti-inflammatory cellular states, with enhanced macrophage functionality and improved epithelial integrity. Conclusion NR1D1 deficiency exacerbates epithelial apoptosis and inflammation in UC by upregulating CD47, thereby impairing macrophage efferocytosis. Restoring NR1D1 activity with SR9009 ameliorates inflammation and promotes resolution through improved apoptotic cell clearance. Targeting the NR1D1-CD47 axis represents a promising therapeutic strategy for UC.

Enhanced antitumor efficacy of nanostructured lipid carrier co-loaded with docetaxel and 5-fluorouracil for targeted gastric cancer therapy.

This study presents nanostructured lipid carrier (NLC) co-loaded with Docetaxel (DCT) and 5-Fluorouracil (5-FU) as a targeted therapeutic approach for gastric cancer (GC). Using nanoprecipitation, NLC-DCT/5-FU were synthesized and exhibited an average particle size of 215.3 ± 10.4nm, a polydispersity index (PDI) of 0.29, and a zeta potential of -17.1mV. Encapsulation efficiency reached 95.9% for DCT and 5-FU, with a loading efficiency of 11.2%. In vitro release studies demonstrated a biphasic release profile, with an initial burst and sustained release, achieving 85.6% DCT and 75.8% 5-FU release over 72h. Cytotoxicity assays in MKN45 cells showed a significantly lower half-maximal inhibitory concentration (IC50) for NLC-DCT/5-FU (0.3µM) compared to free DCT (3.9µM) and free 5-FU (19.5µM), indicating enhanced efficacy. In vivo evaluation in a GC mouse model confirmed substantial tumor volume reduction to 213 mm3 with NLC-DCT/5-FU treatment, compared to 432mm3 with the free-drug combination. Systemic safety assessment showed minimal adverse effects, suggesting the nanoparticles' enhanced therapeutic index. These results demonstrate that NLC-based co-delivery systems could substantially improve the clinical outcomes of GC therapy.

Development of curcumin-loaded ultra deformable lipid vesicles for enhanced anti-melanoma activity: in vitro, ex-vivo, and cell line studies

This research aims to formulate and characterize transfersomes containing curcumin (CUR-TFS) for the enhanced melanoma activity. Thin film hydration was used to prepare the CUR-TFS with Phospholipon® 90 G and sodium cholate. The formulated transfersomes were studied for their size, zeta potential (ZP), entrapment efficiency (EE), skin penetration and ex-vivo skin permeation. Differential scanning calorimetry (DSC) and x-ray diffraction (XRD) were used to determine the physical state of the drug in the optimized curcumin transfersomes (OPT-CUR-TFS). In vitro cytotoxicity, cellular uptake, live and dead cell assay, soft agar colony assay, and apoptosis analysis were carried out using the A375 melanoma cell line. The size and EE of the OPT-CUR-TFS were 191 ± 5.25 nm and 91.72 ± 0.36%, respectively. When compared to conventional liposomes, transfersomes showed significantly increased drug permeation and deposition in the skin layers. The penetration of transfersomes into the deeper skin layers was illustrated through fluorescence microscopy. The DSC and XRD analysis suggested that CUR in transfersomes was amorphous, demonstrating its successful encapsulation inside the transfersomes. The OPT-CUR-TFS was found to significantly decrease the cell viability in A375 cell lines due to their higher cellular uptake. In addition, the OPT-CUR-TFS showed enhanced antiproliferative action by suppressing the clonogenic potential of A375 cells. Furthermore, the OPT-CUR-TFS induced apoptosis-mediated cell death in A375 cells. These findings could offer a powerful justification for CUR transfersomes as a viable treatment strategy for the management of melanoma.