Resistance Assays Research Articles

Abstract 2457: Brain derived endothelial cells as a mediator of cGAS-STING driven anti-tumor efficacy in glioblastoma

Abstract The median survival time for newly diagnosed patients with glioblastoma (GBM) is 15 months, highlighting the need for improved therapies. Current treatment for newly diagnosed and recurrent GBM consists of tumor resection and chemoradiation therapy. The immunosuppressive tumor microenvironment (TME) is a central obstacle in the treatment of GBM. Lack of NK cell and CD8+ T cell infiltration prevents efficacy with immunotherapies like immune checkpoint blockade (ICB).One approach our lab is pursuing to enhance tumor infiltrating lymphocytes is through harnessing the anti-viral cGAS-STING pathway. Upon cGAS activation by cytosolic double stranded DNA, the TBK1 kinase is phosphorylated, initiating a subsequent type I interferon (IFN) response and the recruitment of immune cells. This pathway is commonly altered in many cancer types to evade immune activation. Our lab has demonstrated survival benefits with STING agonist treatment in immunocompetent GBM mouse models. We have also shown strong STING localization in the vasculature, implicating endothelial cells as a potential treatment target for anti-tumor efficacy. Here we investigated the potential role of tumor vasculature in mediating the effects of STING agonists in GBM therapy.We have shown robust cGAS-STING activation in brain endothelial cell lines through enhanced secretion of CXCL10, IFN-β, and RANTES upon STING agonist treatment in vitro. Western blotting also shows pathway activation in response to treatment through increased phospho-TBK1 levels. Activation was further potentiated by manganese.To investigate the effects of STING activation in brain endothelial cells in detail, we performed RNA sequencing. This showed 262 differentially expressed genes in response to pharmacologic cGAS-STING activation, with 183 of these being upregulated. The top gene ontology terms enriched include type I interferon signaling pathway, defense response to virus, and antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-independent. In addition, gene expression of tight junction protein Claudin 5 was also enhanced. We have also shown increased resistance in response to activation, through transendothelial electrical resistance (TEER) assays. Taken together, these results suggest that cGAS-STING activation in endothelial cells leads to tight junction remodeling in addition to immunomodulatory changes, implicating roles for endothelial cells in barrier function and immune cell interactions respectively. Current research in our lab focuses on elucidating the mechanisms behind these changes. Our goal is to identify beneficial endothelial cell changes which promote anti-tumor immunity in order to inform future treatment strategies. We are currently performing studies aimed at demonstrating that cGAS-STING activation in endothelial cells is critical for the efficacy of STING agonist treatment. Citation Format: Andrea Schmidt, Sandra Remson, Philippa Vaughn-Beaucaire, Jorge L. Jimenez-Macias, Praveen Srinivasan, Maximillian Pinho-Schwermann, Sean Lawler. Brain derived endothelial cells as a mediator of cGAS-STING driven anti-tumor efficacy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2457.

Abstract 5110: Exploring the effects of Indirubins in pediatric diffuse high grade glioma models

Abstract Diffuse midline glioma (DMG) is an invasive pediatric brain tumor which grows in the pons of the brainstem. The current course of treatment is focal radiation however, DMG continues to have a very poor prognosis with a median survival of less than 10 months. A major barrier for chemotherapeutic treatment of DMG is the blood brain barrier (BBB) which tightly controls access of molecules to the brain. This limits the efficacy of drugs to treat brain cancer and other diseases of the central nervous system. Previous studies from the Lawler lab and others have shown that the indirubin derivative, 6-bromoindirubin-3′-acetoxime(BIO-acetoxime/BIA), has anti-invasive properties and can enhance survival in glioblastoma xenograft models. Here, we investigated the effects of BIA on pediatric glioma models using a range of relevant assays. In an in vitro collagen migration assay, BIA was observed to significantly slow cell migration at a concentration of 1µM in DIPG 36 cells, over 72 hours. Furthermore, we have also shown that BIA modulates the tumor vasculature, which could limit tumor growth, and improves drug delivery to tumors by targeting tight junctions in tumor associated endothelium. The effect on BBB drug penetration was assessed in a multicellular 3D in vitro BBB model and effects of BIA on endothelial barrier functions was determined using trans-endothelial resistance assays (TEER). BIA treatment increased dextran uptake into 3D BBB models, and also lowered endothelial cell permeability in vitro via a reduction in the expression of tight junction proteins as shown by staining and a decrease in TEER values. In order to assess potential synergistic interactions, a drug screen was performed on a panel of distinct pediatric glioma cell lines (DIPG 36 and KNS42). This identified several FDA approved drugs that combine well with BIA. From this screen, BIA was shown to synergize with DNA damaging chemotherapy to enhance toxicity and in combination with cisplatin promote DNA damage in pediatric glioma cell lines. Other drugs identified in the screen are currently under investigation and details will be reported. Thus, our hypothesis is that BIA may provide an effective approach for further development as a DMG therapeutic through targeting invasion and enhancing drug potency and delivery via modulation of endothelial cell tight junctions. Interrogating the mechanisms involved and developing drug formulations informed by the drug screen for in vivo studies to determine the translational potential of this approach is our focus for future studies. Citation Format: Jasmine S. Clark, Jorge Jimenez-Macias, Philippa Vaughn-Beaucaire, Shengliang Zhang, Wafik El-Deiry, Rishi Lulla, Sean Lawler. Exploring the effects of Indirubins in pediatric diffuse high grade glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5110.

German coasts harbor non-O1/non-O139 Vibrio cholerae with clinical virulence gene profiles

Non-O1/non-O139 Vibrio cholerae (NOVC) are ubiquitous in aquatic ecosystems. In rare cases, they can cause intestinal and extra-intestinal infections in human. This ability is associated with various virulence factors. The presence of NOVC in German North Sea and Baltic Sea was observed in previous studies. However, data on virulence characteristics are still scarce. Therefore, this work aimed to investigating the virulence potential of NOVC isolated in these two regions. In total, 31 NOVC strains were collected and subjected to whole genome sequencing. In silico analysis of the pathogenic potential was performed based on the detection of genes involved in colonization and virulence. Phenotypic assays, including biofilm formation, mobility and human serum resistance assays were applied for validation. Associated toxin genes (hlyA, rtxA, chxA and stn), pathogenicity islands (Vibrio pathogenicity island 2 (VPI-II) and Vibrio seventh pathogenicity island 2 (VSP-II)) and secretion systems (Type II, III and VI secretion system) were observed. A maximum likelihood analysis from shared core genes revealed a close relationship between clinical NOVCs published in NCBI and environmental strains from this study. NOVC strains are more mobile at 37 °C than at 25 °C, and 68% of the NOVC strains could form strong biofilms at both temperatures. All tested strains were able to lyse erythrocytes from both human and sheep blood. Additionally, one strain could survive up to 60% and seven strains up to 40% human serum at 37 °C. Overall, the genetic virulence profile as well as the phenotypic virulence characteristics of the investigated NOVC from the German North Sea and Baltic Sea suggest potential human pathogenicity.

Sulforaphane Regulates Macrophage M1/M2 Polarization to Attenuate Macrophage-induced Caco-2 Cell Injury in an Inflammatory Environment.

The imbalance between M1 and M2 macrophage activation is closely associated with the pathogenesis of inflammatory bowel diseases (IBDs). Sulforaphane (SFN) plays an important role in the treatment of inflammatory diseases. To investigate the effect of SFN on macrophage polarization and its underlying regulatory mechanism. Mouse bone marrow-derived macrophages (BMDMs) were treated with SFN and an Nrf2 inhibitor, Brusatol. M1 macrophages were induced by LPS and IFN-γ stimulation, whereas M2 macrophages were induced by stimulation with IL-4 and IL-13. LPS-stimulated BMDMs were co-cultured with Caco-2 cells. Flow cytometry, qRT-PCR, and Western blot were performed to assess macrophage polarization. Cell function was assessed using CCK8 assay, transepithelial electrical resistance (TEER) assay, and biochemical analysis. Higher concentrations of SFN resulted in better intervention effects, with an optimal concentration of 10 μM. SFN decreased the levels of IL-12, IL-6, and TNF-α, as well as the percentages of CD16/32 in M1 BMDMs. At the same time, SFN increased the levels of YM1, Fizz1, and Arg1 as well as the percentages of CD206+ cells in M2 BMDMs. In addition, SFN enhanced the accumulation of Nrf2, NQO1, and HO-1 in M1 BMDMs, and the downregulation of Nrf2 reversed the regulatory effect of SFN on M1/M2 macrophages. LPS-stimulated BMDMs induced Caco-2 cell damage, which was partially alleviated by SFN. Our findings indicate that SFN may act as an Nrf2 agonist to regulate macrophage polarization from M1 to M2. Furthermore, SFN may represent a potential protective ingredient against IBD.

Hypoxia-induced circRNAs encoded by PPARA are highly expressed in human cardiomyocytes and are potential clinical biomarkers of acute myocardial infarction

BackgroundAcute myocardial infarction (AMI) is a serious cardiovascular disease that adversely affects human health. Circular RNAs (circRNAs) are involved in the pathological and physiological processes of AMI, but the biological mechanism of their involvement and their clinical significance remain unknown. We aimed to identify circRNAs that are significantly associated with morbidity in the peripheral blood of patients with AMI and evaluate their diagnostic utility.MethodsHigh-throughput sequencing was used to screen for differentially expressed circRNAs in peripheral blood samples obtained from five patients with AMI and five sex- and age-matched healthy controls. A series of bioinformatics tools and databases were used to determine the biological functional classification and pathway enrichment of the circRNAs based on data obtained from sequencing. A hypoxia model was established and used to evaluate the effect of hypoxia on circRNA expression in human cardiomyocytes. A cytoplasmic separation assay and enzyme resistance assay were employed to identify the biological characteristics of circRNA. Polymerase chain reaction validity testing and receiver operating characteristic (ROC) curve analysis were used to evaluate the utility of circRNA assessments in the diagnosis of AMI.ResultsA large number of circRNAs were found to be differentially expressed in the peripheral blood of patients with AMI, and significantly more of these circRNAs were highly expressed than lowly expressed. The genes encoding these circRNAs have a wide range of effects on various functions in the body. A hypoxic environment promoted the upregulation of circRNA expression in human cardiomyocytes, and hsa_circ_0116795 encoded by PPARA was highly expressed in the peripheral blood of the patients with AMI. In terms of biological characteristics, under physiological conditions, hsa_circ_0116795 (circ_PPARA) was mainly located in the cytoplasm of cardiomyocytes and found to be resistant to exonuclease. The ROC curve analysis showed that the expression levels of circ_PPARA in the peripheral blood of patients with AMI were significantly different from those in the peripheral blood of healthy controls.ConclusionA large number of abnormally expressed circRNAs are detectable in the peripheral blood of patients with AMI. In particular, circ_PPARA is highly expressed in human myocardial cells under hypoxic conditions, and its biological characteristics indicate that it could be employed as a biomarker for the early diagnosis of AMI.

Woolly mutation with the Get02 locus overcomes the polygenic nature of trichome-based pest resistance in tomato.

Type-IV glandular trichomes, which only occur in the juvenile developmental phase of the cultivated tomato (Solanum lycopersicum), produce acylsugars that broadly protect against arthropod herbivory. Previously, we introgressed the capacity to retain type-IV trichomes in the adult phase from the wild tomato, Solanum galapagense, into the cultivated species cv. Micro-Tom (MT). The resulting MT-Galapagos enhanced trichome (MT-Get) introgression line contained 5 loci associated with enhancing the density of type-IV trichomes in adult plants. We genetically dissected MT-Get and obtained a subline containing only the locus on Chromosome 2 (MT-Get02). This genotype displayed about half the density of type-IV trichomes compared to the wild progenitor. However, when we stacked the gain-of-function allele of WOOLLY, which encodes a homeodomain leucine zipper IV transcription factor, Get02/Wo exhibited double the number of type-IV trichomes compared to S. galapagense. This discovery corroborates previous reports positioning WOOLLY as a master regulator of trichome development. Acylsugar levels in Get02/Wo were comparable to the wild progenitor, although the composition of acylsugar types differed, especially regarding fewer types with medium-length acyl chains. Agronomical parameters of Get02/Wo, including yield, were comparable to MT. Pest resistance assays showed enhanced protection against silverleaf whitefly (Bemisia tabaci), tobacco hornworm (Manduca sexta), and the fungus Septoria lycopersici. However, resistance levels did not reach those of the wild progenitor, suggesting the specificity of acylsugar types in the pest resistance mechanism. Our findings in trichome-mediated resistance advance the development of robust, naturally resistant tomato varieties, harnessing the potential of natural genetic variation. Moreover, by manipulating only 2 loci, we achieved exceptional results for a highly complex, polygenic trait, such as herbivory resistance in tomato.

Comparison of gradient diffusion and molecular methods using Allplex™ NG&DR assay (Seegene®) for macrolide and fluoroquinolone screening resistance in Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae (NG) is increasing worldwide. Second-line treatments with macrolides or fluoroquinolones are an option for NG infections in some cases following the STI guideline recommendations. In our study, we compared the gradient diffusion test using EUCAST 2024 breakpoints with a new molecular method using the Allplex™ NG&DR assay (Seegene®) including A2059G/C2611 mutations (23S rRNA) associated with high/moderate-level macrolide resistance and S91F mutation (gyrA) relationship with fluoroquinolone resistance in NG isolates (n = 100). We calculated the sensitivity, specificity, and correlation of the molecular test for fluoroquinolone using the gradient diffusion as the reference method. In twenty-three strains was not detected any mutation associated with macrolides or fluoroquinolone resistance. No A2059G/C2611T mutations were detected, and the S91F mutations were detected in 77 out of the 100 isolates screened. Twenty-three NG isolates were reported to be resistant to azithromycin (ECOFF: >1mg/L), and 78 NG isolates were resistant to ciprofloxacin (MIC: >0.06mg/L). The molecular method showed a sensitivity of 96.1% and, a specificity of 90.9% for fluoroquinolone susceptibility, but the statistical analysis between the molecular test and gradient diffusion test was not statistically significant for fluoroquinolone resistance (p = 1). Statistical analysis was not performed for macrolides because of the absence of positive RT-PCR results. According to our data, Allplex™ assay cannot replace the gradient diffusion test for macrolide resistance. However, the assay could be used to test fluoroquinolone resistance in NG isolates as a replacement for phenotypic methods.

Development of a rapid detection assay for acetolactate synthase inhibitors resistance in three Amaranthus weed species through loop-mediated isothermal amplification.

The early detection of herbicide resistance in weeds is a key factor to avoid herbicide waste and improve agriculture sustainability. The present study aimed to develop and validate an allele-specific loop-mediated isothermal amplification (AS-LAMP) assay for the quick on-site detection of the resistance-endowing point mutation Trp-574-Leu in the acetolactate synthase (ALS) gene in three widely diffused Amaranthus weed species: Amaranthus retroflexus, Amaranthus hybridus and Amaranthus tuberculatus. The AS-LAMP protocol was developed on wild-type and ALS-mutant plants of the three species and revealed that the amplification approach with only the primer set specific for the mutant allele (574-Leu) was the most promising. The validation and estimation of the AS-LAMP performance evaluated by comparing the results with those of the molecular marker (cleaved amplified polymorphic sequences) indicated that, although the sensitivity and specificity were relatively high in all species (overall 100 and > 65%, respectively), precision was high for A. hybridus L. and A. retroflexus L. (75 and 79%, respectively), but quite low for A. tuberculatus (Moq.) J. D. Sauer (59%). The LAMP assay was also effective on crude genomic DNA extraction, allowing the quick detection of mutant plants in field situation (on site resistance detection). The proposed AS-LAMP method has proven to be a promising technique for rapid detection of resistance as a result of Trp-574-Leu on the two monoecious weedy Amaranthus species but resulted less effective in the genetically variable dioecious species A. tuberculatus. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development of tight junction-strengthening compounds using a high-throughput screening system to evaluate cell surface-localized claudin-1 in keratinocytes

Tight junctions (TJs) are important factors constituting the physical barriers of the skin, and their suppression has been described in various conditions, such as aged skin and atopic dermatitis lesions. However, the methods for improving skin TJ function remain insufficient. Therefore, to obtain compounds that can improve TJ function, we developed a novel high-throughput screening system termed live-cell immunostaining to evaluate cell surface-localized claudin-1 (CLDN1) with high selectivity using normal human epidermal keratinocytes (NHEKs). Heparinoid and phospho-pyridoxal (p-Pyr), a metabolite of pyridoxine, were identified as hit compounds. In addition, heparinoid was strongly suggested to increase CLDN1 expression by inhibiting epidermal growth factor receptor signaling. By contrast, p-Pyr did not enhance CLDN1 expression, but it accelerated the translocation of CLDN1 to the cell surface. Finally, we confirmed that heparinoid and p-Pyr improved barrier function in NHEKs in a transepithelial electrical resistance assay. In conclusion, heparinoid and p-Pyr could potentially ameliorate skin conditions by improving TJ function.

Boosting Solanum tuberosum resistance to Alternaria solani through green synthesized ferric oxide (Fe2O3) nanoparticles

Potato (Solanum tuberosum) is the third crucial global crop facing threats from Alternaria solani, a necrotrophic fungal pathogen causing early blight disease. Beyond crop impact, it leads to substantial production reduction and economic losses worldwide. This study introduces a green synthesis method for producing Ferric Oxide nanoparticles (FNPs) using dried Guava (Psidium guajava) leaves. Guava leaf extract acts as a reducing agent, with iron (III) chloride hexahydrate (FeCl3·6H2O) as the oxidizing agent. This study employed various characterization techniques for Ferric Oxide nanoparticles (FNPs). Fourier Transform Infrared Spectroscopy (FTIR) revealed peaks at 877 cm−1, 1180 cm−1, 1630 cm−1, 1833 cm−1, 2344 cm−1, and 3614 cm−1, associated with Maghemite vibrations, polyphenol compounds, and amino acids. UV–Vis spectroscopy exhibited a characteristic absorbance peak at 252 nm for FNPs. Scanning Electron Microscope (SEM) images illustrated particle sizes of 29-41 nm, and Energy Dispersive Spectroscopy (EDS) indicated elemental composition. X-ray diffraction (XRD) confirmed crystalline FNPs with peaks at 26.78, 30.64, 36.06, 38.21, 43.64, 53.52, 57.42, 63.14 and 78.32. Disease resistance assays demonstrated FNPs’ effectiveness against A. solani, reducing disease incidence and severity. In the leaf detach assay, concentrations of 15, 10 and 5 mg/L showed a dose-dependent reduction in disease severity and incidence. The Greenhouse Assay confirmed FNPs’ concentration-dependent effect on disease incidence and severity. The study also explored FNPs’ potential as biocontrol agents showing no adverse effects on overall plant development. Additionally, the study highlighted the agronomic potential of FNPs in enhancing plant growth and development emphasizing their role as micronutrients in biofortification. The findings suggest the promising application of FNPs in plant protection and biofortification strategies.

SOS2 modulates the threshold of EGFR signaling to regulate osimertinib efficacy and resistance in lung adenocarcinoma.

Son of sevenless 1 and 2 (SOS1 and SOS2) are RAS guanine nucleotide exchange factors (RasGEFs) that mediate physiologic and pathologic receptor tyrosine kinase (RTK)-dependent RAS activation. Here, we show that SOS2 modulates the threshold of epidermal growth factor receptor (EGFR) signaling to regulate the efficacy of and resistance to the EGFR tyrosine kinase inhibitor (EGFR-TKI) osimertinib in lung adenocarcinoma (LUAD). SOS2 deletion (SOS2KO ) sensitized EGFR-mutated cells to perturbations in EGFR signaling caused by reduced serum and/or osimertinib treatment to inhibit phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation, oncogenic transformation, and survival. Bypassing RTK reactivation of PI3K/AKT signaling represents a common resistance mechanism to EGFR-TKIs; SOS2KO reduced PI3K/AKT reactivation to limit osimertinib resistance. In a forced HGF/MET-driven bypass model, SOS2KO inhibited hepatocyte growth factor (HGF)-stimulated PI3K signaling to block HGF-driven osimertinib resistance. Using a long-term in situ resistance assay, most osimertinib-resistant cultures exhibited a hybrid epithelial/mesenchymal phenotype associated with reactivated RTK/AKT signaling. In contrast, RTK/AKT-dependent osimertinib resistance was markedly reduced by SOS2 deletion; the few SOS2KO cultures that became osimertinib resistant primarily underwent non-RTK-dependent epithelial-mesenchymal transition (EMT). Since bypassing RTK reactivation and/or tertiary EGFR mutations represent most osimertinib-resistant cancers, these data suggest that targeting proximal RTK signaling, here exemplified by SOS2 deletion, has the potential to delay the development osimertinib resistance and enhance overall clinical responses for patients with EGFR-mutated LUAD.

An eco-friendly and ultrasensitive micro-orifice resistance assay for the detection of Listeria monocytogenes based on calcium carbonate signal probes harvested from discarded eggshells

The development of eco-friendly analytical methods for the detection of foodborne pathogens is of great importance for protecting human health and alleviating environmental pressure. Here, we demonstrate that a material (CaCO3 microparticles) prepared from discarded eggshells can be used as signal probes for micro-orifice resistance assay (MORA) and can be combined with ligation-mediated branching hybridization chain reaction (LB-HCR)-based signal-amplification technique (e-LMORA) for high sensitivity detection of Listeria monocytogenes. This strategy used LB-HCR-induced aggregation of streptavidin-modified CaCO3 microparticles, which were recognized by the particle counter for quantitative analysis of the target DNA. Compared with traditional MORA using polystyrene microspheres as signal probes, CaCO3 microparticles prepared from waste eggshells are more environmentally friendly. The combination of DNA-recognition probe, signal probes, and enzyme-free signal-amplification strategy not only reduces the cost synergistically, but also enables the biosensor to attain high sensitivity (limit of detection = 21 CFU/mL) due to its efficient signal amplification.

Mechanism of Circ_HECW2 regulating osteoblast apoptosis in osteoporosis by attenuating the maturation of miR-1224-5p

BackgroundOsteoporosis (OP) poses a significant clinical challenge with escalating morbidity. This study explores Circ_HECW2 expression in OP patients and its regulatory role in lipopolysaccharide (LPS)-induced osteoblast apoptosis.MethodsCirc_HECW2 expression in OP patient serum and healthy controls was quantified using RT-qPCR. Diagnostic value of Circ_HECW2 for OP was assessed via ROC curve. Pearson’s correlation model examined associations between indicators. Human osteoblasts HFOB1.19, treated with LPS, were analyzed for Circ_HECW2, pre-miR-1224, miR-1224-5p, and PDK2 mRNA levels. TUNEL assay determined cell apoptosis and Western blot assessed cleaved-caspase-3 protein levels. RNase R resistance assay and actinomycin D assay confirmed Circ_HECW2’s cyclic structure. RNA pull-down and dual-luciferase reporter assay verified binding relationships between Circ_HECW2 and miR-1224 and between miR-1224-5p and PDK2.ResultsCirc_HECW2 exhibited elevated expression in OP patients with diagnostic significance and a negative correlation with lumbar T-score. LPS co-culture increased Circ_HECW2 expression in HFOB1.19 cells, significantly elevating apoptosis index and cleaved-caspase-3. Circ_HECW2 downregulation inhibited HFOB1.19 apoptosis, reduced pre-miR-1224 expression, and elevated mature miR-1224-5p. Circ_HECW2 bound to pre-miR-1224, and inhibiting miR-1224-5p reversed the effect of Circ_HECW2 downregulation on osteoblast apoptosis. miR-1224-5p targeted PDK2 transcription.ConclusionCirc_HECW2, highly expressed in OP, holds diagnostic significance and reflects disease severity. Circ_HECW2 reduces mature miR-1224-5p by binding to pre-miR-1224, upregulating PDK2, and facilitating LPS-induced osteoblast apoptosis.Graphic

Liquid-liquid phase separation-related lncRNA prognostic signature and ZNF32-AS2 as a novel biomarker in hepatocellular carcinoma

BackgroundLiquid-liquid phase separation (LLPS) enhances oncogenic signaling pathways and advances cancer progression, and has been proposed as a promising cancer biomarker and intervention target. Nevertheless, doubts remain about the prognostic importance of LLPS-related long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC). Methods: An LLPS-related lncRNA prognostic signature was generated by drivers and regulators of LLPS, and was validated in external datasets. The underlying genetic changes and functional enrichment of the signature were assessed. The drug sensitivity and response to immunotherapy were predicted in patients categorized as high-risk and low-risk. Clinical samples, phase separation agonist, and dispersant were used to identify lncRNAs with the most significant expression change. Cancer cells with ZNF32-AS2 expression regulation were subjected to colony formation assay, scratch test assay, migration and invasion assay, sorafenib resistance assay, and xenograft tumor model. Results: The signature of LLPS-related hub lncRNAs identified through Weighted Gene Co-Expression Network Analysis showed outstanding performance in training and external validation cohorts consistently, and the molecular characteristics varied between different risk groups. Potential drugs for high-risk individuals were identified, and low-risk individuals demonstrated a more favorable reaction to immunotherapy. ZNF32-AS2 showed the most significant expression change in phase separation agonist and dispersant treatment. ZNF32-AS2 promoted the proliferation, mobility, and sorafenib resistance of liver cancer cells. Conclusions: The LLPS-related lncRNA signature may help assess prognosis and predict treatment efficacy in clinical settings. LLPS-related ZNF32-AS2 promoted the proliferation, mobility, and sorafenib resistance of liver cancer cells, and may be a novel potential biomarker in hepatocellular carcinoma.

In vitro study of the blood-brain barrier transport of bioactives from Mediterranean foods.

The Mediterranean diet (MD), characterized by olive oil, olives, fruits, vegetables, and wine intake, is associated with a reduced risk of dementia. These foods are rich in bioactives with neuroprotective and antioxidant properties, including hydroxytyrosol (HT), tyrosol (TYRS), serotonin (SER) and protocatechuic acid (PCA), a phenolic acid metabolite of anthocyanins. It remains to be established if these molecules cross the blood-brain barrier (BBB), a complex interface that strictly controls the entrance of molecules into the brain. We aimed to assess the ability of tyrosine (TYR), HT, TYRS, PCA and SER to pass through the BBB without disrupting its properties. Using Human Brain Microvascular Endothelial Cells as an in vitro model of the BBB, we assessed its integrity by transendothelial electrical resistance, paracellular permeability and immunocytochemical assays of the adherens junction protein β-catenin. The transport across the BBB was evaluated by ultra-high-performance liquid chromatography high resolution mass spectrometry. Results show that tested bioactives did not impair BBB integrity regardless of the concentration evaluated. Additionally, all of them cross the BBB, with the following percentages: HT (∼70%), TYR (∼50%), TYRS (∼30%), SER (∼30%) and PCA (∼9%). These results provide a basis for the MD neuroprotective role.

Red raspberry (Rubus idaeus) preserves intestinal barrier integrity and reduces oxidative stress in Caco-2 cells exposed to a proinflammatory stimulus.

Growing evidence showed the capacity of (poly)phenols to exert a protective role on intestinal health. Nevertheless, the existing findings are still heterogeneous and the underlying mechanisms remain unclear. This study investigated the potential benefits of a red raspberry (Rubus idaeus) powder on the integrity of the intestinal barrier, focusing on its ability to mitigate the effects of tumor necrosis factor-α (TNF-α)-induced intestinal permeability. Human colorectal adenocarcinoma cells (i.e., Caco-2 cells) were used as a model to assess the impact of red raspberry on intestinal permeability, tight junction expression, and oxidative stress. The Caco-2 cells were differentiated into polarized monolayers and treated with interferon-γ (IFN-γ) (10 ng mL-1) for 24 hours, followed by exposure to TNF-α (10 ng mL-1) in the presence or absence of red raspberry extract (1-5 mg mL-1). The integrity of the intestinal monolayer was evaluated using transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-D) efflux assay. Markers of intestinal permeability (claudin-1, occludin, and zonula occludens-1 (ZO-1)) and oxidative stress (8-hydroxy-2-deoxyguanosine (8-OHdG) and protein carbonyl) were assessed using ELISA kits. Treatment with red raspberry resulted in a significant counteraction of TEER value loss (41%; p < 0.01) and a notable reduction in the efflux of FITC-D (-2.5 times; p < 0.01). Additionally, red raspberry attenuated the levels of 8-OHdG (-48.8%; p < 0.01), mitigating the detrimental effects induced by TNF-α. Moreover, red raspberry positively influenced the expression of the integral membrane protein claudin-1 (+18%; p < 0.01), an essential component of tight junctions. These findings contribute to the growing understanding of the beneficial effects of red raspberry in the context of the intestinal barrier. The effect of red raspberry against TNF-α-induced intestinal permeability observed in our in vitro model suggests, for the first time, its potential as a dietary strategy to promote gastrointestinal health.

Bacillus halotolerans attenuates inflammation induced by enterotoxigenic Escherichia coli infection in vivo and in vitro based on its metabolite soyasaponin I regulating the p105-Tpl2-ERK pathway.

Soyasaponins, recognized for their anti-inflammatory and antioxidant effects, have not yet been fully explored for their role in combating enterotoxigenic Escherichia coli (ETEC) infections. Recent findings identified them in small-molecule metabolites of Bacillus, suggesting their broader biological relevance. This research screened 88 strains of B. halotolerans, identifying the strain BH M20221856 as significantly inhibitory against ETEC growth in vitro. It also reduced cellular damage and inflammatory response in IPEC-J2 cells. The antimicrobial activity of BH M20221856 was attributed to its small-molecule metabolites rather than secretory proteins. A total of 69 small molecules were identified from the metabolites of BH M20221856 using liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS). Among these, soyasaponin I (SoSa I) represented the largest multiple change in the enrichment analysis of differential metabolites and exhibited potent anti-ETEC effects in vivo. It significantly reduced the bacterial load of E. coli in mouse intestines, decreased serum endotoxin, D-lactic acid, and oxidative stress levels and alleviated intestinal pathological damage and inflammation. SoSa I enhanced immune regulation by mediating the p105-Tpl2-ERK signaling pathway. Further evaluations using transepithelial electrical resistance (TEER) and cell permeability assays showed that SoSa I alleviated ETEC-induced damage to epithelial barrier function. These results suggest that BH M20221856 and SoSa I may serve as preventative biologics against ETEC infections, providing new insights for developing strategies to prevent and control this disease.

Bacillus paralicheniformis 809 and Bacillus subtilis 810 support in vitro intestinal integrity under hydrogen peroxide and deoxynivalenol challenges.

We designed and conducted two in vitro experiments to evaluate the effects of two Bacillus spp. probiotics on gut barrier integrity using the transepithelial electrical resistance (TEER) assay under two different challenge models. In Exp. 1, intestinal epithelial cells received or not (CON) B. paralicheniformis 809 (BLI) or B. subtilis 810 (BSU) at a rate of 1 × 108 colony forming units (CFU)/transwell. Two hours after treatment application (CON, BLI, or BSU), 5mM of the reactive oxygen species hydrogen peroxide, mimicking mucosal oxidative stress, was added alone (HYP) or with each of the Bacillus spp. (HYP + BLI or HYP + BSU). In Exp. 2, cells were assigned to the same treatments as in Exp. 1 (CON, BLI, and BSU), or mycotoxin deoxynivalenol (DON), which was added alone or in combination with BLI or BSU, resulting in another two treatments (DON + BLI and DON + BSU). Transepithelial electrical resistance was measured for 14h postchallenge. In Exp. 1, a treatment × hour interaction was observed for TEER (P < 0.0001). Adding BLI and BSU resulted in greater TEER values vs. CON for most of the experimental period (P < 0.02), whereas HYP reduced mean TEER and area under the curve (AUC), while increasing the amount of sugar that translocated through the monolayer cells (P < 0.001). A treatment × hour interaction was also observed in Exp. 2 (P < 0.0001), as DON led to an immediate and acute drop in TEER that lasted until the end of the experimental period (P < 0.0001). Both BLI and BSU alleviated the DON-induced damaging effects on the integrity of intestinal epithelial cells, whereas both Bacillus spp. alleviated the damage caused by DON alone and the proportion of sugar that translocated through the monolayer cells was not different between CON and DON + BLI (P = 0.14) and DON + BLI and DON + BSU (P = 0.62). In summary, both Bacillus spp. strains (B. paralicheniformis 809 and B. subtilis 810) were able to counteract the damaging effects of the challenge agents, hydrogen peroxide and deoxynivalenol, on gut barrier integrity.

Characteristic of KPC-12, a KPC Variant Conferring Resistance to Ceftazidime-Avibactam in the Carbapenem-Resistant Klebsiella pneumoniae ST11-KL47 Clone Background.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a great threat to public health worldwide. Ceftazidime-avibactam (CZA) is an effective β-lactam/β-lactamase inhibitors against CRKP. However, reports of resistance to CZA, mainly caused by Klebsiella pneumoniae carbapenemase (KPC) variants, have increased in recent years. In this study, we aimed to describe the resistance characteristics of KPC-12, a novel KPC variant identified from a CZA resistant K. pneumoniae. The K. pneumoniae YFKP-97 collected from a patient with respiratory tract infection was performed whole-genome sequencing (WGS) on the Illumina NovaSeq 6000 platform. Genomic characteristics were analyzed using bioinformatics methods. Antimicrobial susceptibility testing was conducted by the broth microdilution method. Induction of resistant strain was carried out in vitro as previously described. The G. mellonella killing assay was used to evaluate the pathogenicity of strains, and the conjugation experiment was performed to evaluate plasmid transfer ability. Strain YFKP-97 was a multidrug-resistant clinical ST11-KL47 K. pneumoniae confers high-level resistance to CZA (16/4 μg/mL). WGS revealed that a KPC variant, KPC-12, was carried by the IncFII (pHN7A8) plasmids (pYFKP-97_a and pYFKP-97_b) and showed significantly decreased activity against carbapenems. In addition, there was a dose-dependent effect of bla KPC-12 on its activity against ceftazidime. In vitro inducible resistance assay results demonstrated that the KPC-12 variant was more likely to confer resistance to CZA than the KPC-2 and KPC-3 variants. Our study revealed that patients who was not treated with CZA are also possible to be infected with CZA-resistant strains harbored a novel KPC variant. Given that the transformant carrying bla KPC-12 was more likely to exhibit a CZA-resistance phenotype. Therefore, it is important to accurately identify the KPC variants as early as possible.

Plant growth promoting potential of Arthrobacter spp., an early colonizer on the volcanic deposits of Mt. Merapi, Indonesia, as a restorative agent for degraded land

Exploration of novel and effective plant growth-promoting bacteria (PGPB) from unexplored environments is an intriguing option for restoring degraded land in Indonesia caused by mining activity. Due to their ability to develop in the absence of carbon, PGPB derived from volcanic environments may be a sustainable alternative. Previous research on the volcanic deposits of Mt. Merapi, Indonesia and Miyake-Jima, Japan, revealed that Arthrobacter spp. dominated the pioneer bacterial communities. Consequently, this research aimed to investigate the biochemical and physiological characteristics of volcanic Arthrobacter groups and assess their potential as PGPB. Using the conventional API system (20NE, 50CH, Zym), biochemical characteristics were evaluated. In vitro evaluations of plant growth-promoting activities such as siderophore production, phosphate solubilization, growth in media devoid of nitrogen, heavy metal resistance and weathering activity were conducted. The results showed broad substrate utilization and enzyme activity profiles of the Mt. Merapi isolates (BRU 37, BRU I.13, and BRU I.45). During 15 days of incubation on a minimal medium containing potassium feldspar, glucose, and NH4Cl, all of the isolates grew and produced acids. In nitrogen-free media, all of the isolates grew. These isolates showed positive reaction for the phosphate solubilization assay using Pikovskaya plates. After one day of incubation, all isolates produced yellow zones on the blue-green pigmented media (CAS-overlay media) and were therefore classified as potent siderophore producers. The heavy metal resistance assay showed that BRU37 grew in the heavy metal media. These results indicate the potential of selected indigenous Mt. Merapi Arthrobacter to enhance plant growth. Further characterization of plant growth-promoting traits is now in progress.