Methylene Blue Assay Research Articles

Okanin Inhibits Cell Growth and Induces Apoptosis and Pyroptosis in Oral Cancer.

Okanin, a flavonoid compound derived from Bidens pilosa L., has garnered attention for its anti-inflammatory properties. Although Bidens pilosa is commonly used in healthcare products and functional foods, the anticancer potential of okanin, particularly in oral cancer, remains underexplored. This study aims to investigate the effects of okanin on oral cancer cell lines and its potential as a therapeutic agent. The study involved assessing the cytotoxic effects of okanin on oral cancer cell lines SAS, SCC25, HSC3, and OEC-M1. The IC50 values were determined using methylene blue assays, and the clonogenic capacity was evaluated through colony formation assays. Flow cytometry was used to analyze cell cycle progression and apoptosis. Caspase-3/7 activity assays and annexin V/7-AAD staining confirmed the induction of apoptosis and pyroptosis. In vivo efficacy was assessed using a SAS xenograft model, and immunohistochemical analysis of xenograft tissue was performed to examine pyroptosis-related markers. Okanin exhibited potent cytotoxic effects with IC50 values of 12.0 ± 0.8, 58.9 ± 18.7, 18.1 ± 5.3, and 43.2 ± 6.2 μM in SAS, SCC25, HSC3, and OEC-M1 cells, respectively. It caused dose- and time-dependent reductions in cell viability and significantly impaired clonogenic capacity. Flow cytometry revealed G2/M cell cycle arrest and increased sub-G1 population, indicating cell cycle disruption and death. Okanin induced both apoptosis and pyroptosis, as confirmed by caspase-3/7 activity and annexin V/7-AAD staining. In vivo, okanin reduced tumor growth and involved pyroptosis-related markers such as CASP1, GSDMC, GSDMD, and GSDME. Okanin demonstrates significant anticancer potential, particularly in oral cancer, by inducing both apoptosis and pyroptosis. Its efficacy in reducing tumor growth in vivo further supports its potential as a novel therapeutic option. Further mechanistic studies are needed to elucidate the pathways involved in okanin-mediated cell death and to explore its clinical applications.

SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis

Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.

Antifungal recombinant psoriasin of human origin effectively inhibits fungal growth on denture base.

To evaluate the efficacy of recombinant psoriasin as a novel treatment for oral candidiasis by eliminating Candida albicans growth on polymethyl methacrylate denture base. Recombinant psoriasin protein was expressed and purified from E. coli, and Candida growth was monitored invitro with varying concentrations of psoriasin. Subsequently, denture-base polymethyl methacrylate was immersed in psoriasin's solution or voriconazole, and fungal growth on the acrylic base and in the medium was examined by scanning electron microscopy and optical density, respectively. Cellular viability of HeLa and human gingival fibroblast cells treated with psoriasin was measured by methylene blue assay. The findings reveal an effective antifungal activity of psoriasin, completely inhibiting Candida albicans growth in RPMI at a protein concentration above 400 nM. Immersing the polymethyl methacrylate with 50 μM psoriasin completely eradicates fungal growth. Psoriasin has low cytotoxicity in HeLa cells at a concentration higher than 12 μM and no toxic effect on human gingival fibroblasts. This study marks psoriasin as an effective alternative to conventional antifungal treatments for denture stomatitis and a safe alternative to chemical antifungals in dental medicine and beyond.

Hydrogen sulfide mitigates ox‑LDL‑induced NLRP3/caspase‑1/GSDMD dependent macrophage pyroptosis by S‑sulfhydrating caspase‑1.

Macrophage pyroptosis mediates vascular inflammation and atherosclerosis (AS). Hydrogen sulfide (H2S) exerts a protective role in preventing inflammation and AS. However, its molecular mechanisms of regulating the pyroptosis signaling pathway and inhibiting macrophage pyroptosis remain unexplored. The present study aimed to determine whether H2S mitigates macrophage pyroptosis by downregulating the pyroptosis signaling pathway and S‑sulfhydrating caspase‑1 under the stimulation of oxidized low‑density lipoprotein (ox‑LDL), a pro‑atherosclerotic factor. Macrophages derived from THP‑1 monocytes were pre‑treated using exogenous H2S donors sodium hydrosulfide (NaHS) and D,L‑propargylglycine (PAG), a pharmacological inhibitor of endogenous H2S‑producing enzymes, alone or in combination. Subsequently, cells were stimulated with ox‑LDL or the desulfhydration reagent dithiothreitol (DTT) in the presence or absence of NaHS and/or PAG. Following treatment, the levels of H2S in THP‑1 derived macrophages were measured by a methylene blue colorimetric assay. The pyroptotic phenotype of THP‑1 cells was observed and evaluated by light microscopy, Hoechst 33342/propidium iodide fluorescent staining and lactate dehydrogenase (LDH) release assay. Caspase‑1 activity in THP‑1 cells was assayed by caspase‑1 activity assay kit. Immunofluorescence staining was used to assess the accumulation of active caspase‑1. Western blotting and ELISA were performed to determine the expression of pyroptosis‑specific markers (NLRP3, pro‑caspase‑1, caspase‑1, GSDMD and GSDMD‑N) in cells and the secretion of pyroptosis‑related cytokines [interleukin (IL)‑1β and IL‑18] in the cell‑free media, respectively. The S‑sulfhydration of pro‑caspase‑1 in cells was assessed using a biotin switch assay. ox‑LDL significantly induced macrophage pyroptosis by activating the pyroptosis signaling pathway. Inhibition of endogenous H2S synthesis by PAG augmented the pro‑pyroptotic effects of ox‑LDL. Conversely, exogenous H2S (NaHS) ameliorated ox‑LDL‑and ox‑LDL + PAG‑induced macrophage pyroptosis by suppressing the activation of the pyroptosis signaling pathway. Mechanistically, ox‑LDL and the DTT increased caspase‑1 activity and downstream events (IL‑1β and IL‑18 secretion) of the caspase‑1‑dependent pyroptosis pathway by reducing S‑sulfhydration of pro‑caspase‑1. Conversely, NaHS increased S‑sulfhydration of pro‑caspase‑1, reducing caspase‑1 activity and caspase‑1‑dependent macrophage pyroptosis. The present study demonstrated the molecular mechanism by which H2S ameliorates macrophage pyroptosis by suppressing the pyroptosis signaling pathway and S‑sulfhydration of pro‑caspase‑1, thereby suppressing the generation of active caspase-1 and activity of caspase-1.

A methylene blue assay for the quantification of fucoidan – A specific application for seaweed extracts

Fucoidan is a fucose-rich sulfated polysaccharide found mainly in brown seaweeds. Fucoidan has recently gained much attention due to its bioactive and medicinal properties, hence its high potential to be utilized in the pharmaceutical industry. Therefore, an accurate, reliable, and simple method for the specific determination of fucoidan would be a useful tool when looking to recover this valuable polysaccharide. This research focused on developing an assay utilizing the thiazine dye, methylene blue, for the quantification of fucoidan, with a specific application for fucoidan found in algal extracts. Fucoidan could be specifically determined at pH 1, to maximum concentrations of 4 g L-1 in solution without the interference of alginate and other seaweed extract contaminants. At pH 7 and 11, quantification of fucoidan led to interference from alginate and gallic acid. A conversion factor of 1.8 allowed the quantification of fucoidan from Ecklonia maxima extracts using a commercial standard from Fucus vesiculosis. An Ecklonia maxima extract was found to contain 5.47 ± 0.061 g L-1 of fucoidan. Measuring the fucoidan and L-fucose concentration in crude seaweed extract through the methylene blue assay and modified Dische and Shettles J Biol Chem 175:595–603, (1948) method, respectively, confirmed the accuracy and specificity of the method. The limit of quantification of the methylene blue assay at pH 1 was 0.62 g L-1 of fucoidan.

Photocatalytic Activity and Antibacterial Properties of Mixed-Phase Oxides on Titanium Implant Alloy Substrates

Titanium alloys are commonly used for implants, but the naturally forming oxides are bioinert and not ideal for bacterial resistance or osseointegration. Anodization processes are a modification technique that can crystallize the oxides, alter oxide surface topography, and introduce beneficial chemistries. Crystalline titanium oxides are known to exhibit photocatalytic activity (PCA) under UVA light. Anodization was used to create mixed-phase oxides on six titanium alloys including commercially pure titanium (CPTi), Ti-6Al-4V (TAV), Ti-6Al-7Nb (TAN), two forms of Ti-15Mo (TiMo-β and TiMo-αβ), and Ti-35Nb-7Zr-5Ta (TNZT). Combined EDS and XPS analyses showed uptake of the electrolyte and substrate alloying elements into the oxides. The relative oxide PCA was measured using methylene blue degradation assays. CPTi and TAN oxides exhibited increased PCA compared to other alloys. Combined XRD and EBSD oxide phase analyses revealed an unfavorable arrangement of anatase and rutile phases near the outermost surfaces, which may have reduced PCA for other oxides. The relative Staphylococcus aureus attachment to each oxide was also assessed. The CPTi and TiMo-αβ oxides showed significantly reduced S. aureus attachment after 1 h of UVA compared to un-anodized CPTi. Cell culture results verified that the UVA irradiation did not negatively influence the MC3T3-E1 attachment or proliferation on the mixed-phase oxides.

The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1β, Transforming Growth Factor (TGF β1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene β-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

Material properties in regenerating axolotl limbs using inverse finite element analysis

BackgroundThe extracellular mechanical environment plays an important role in the skeletal development process. Characterization of the material properties of regenerating tissues that recapitulate development, provides insights into the mechanical environment experienced by the cells and the maturation of the matrix. In this study, we estimated the viscoelastic material properties of regenerating forelimbs in the axolotl (Ambystoma mexicanum) at three different regeneration stages: 27 days post-amputation (mid-late bud) and 41 days post-amputation (palette stage), and fully-grown time points. A stress-relaxation indentation test followed by two-term Prony series viscoelastic inverse finite element analysis was used to obtain material parameters. Glycosaminoglycan (GAG) content was estimated using a 1,9- dimethyl methylene blue assay. ResultsThe instantaneous and equilibrium shear moduli significantly increased with regeneration while the short-term stress relaxation time significantly decreased with limb regeneration. The long-term stress relaxation time in the fully-grown time point was significantly lower than 27 and 41 DPA groups. The GAG content was not significantly different between 27 and 41 DPA but the GAG content of cartilage in the fully-grown group was significantly greater than in 27 and 41 DPA. ConclusionsThe mechanical environment of the proliferating cells changes drastically during limb regeneration. Understanding how the tissue's mechanical properties change during limb regeneration is critical for linking molecular-level matrix production of the cells to tissue-level behavior and mechanical signals.

Chelators as Antineuroblastomas Agents.

Neuroblastoma represents 8-10 % of all malignant tumors in childhood and is responsible for 15 % of cancer deaths in the pediatric population. Aggressive neuroblastomas are often resistant to chemotherapy. Canonically, neuroblastomas can be classified according to the MYCN (N-myc proto-oncogene protein) gene amplification, a common marker of tumor aggressiveness and poor prognosis. It has been found that certain compounds with chelating properties may show anticancer activity, but there is little evidence for the effect of chelators on neuroblastoma. The effect of new chelators characterized by the same functional group, designated as HLZ (1-hydrazino phthalazine), on proliferation (WST-1 and methylene blue assay), cell cycle (flow cytometry), apoptosis (proliferation assay after use of specific pharmacological inhibitors and western blot analysis) and ROS production (fluorometric assay based on dichlorofluorescein diacetate metabolism) was studied in three neuroblastoma cell lines with different levels of MYCN amplification. The molecules were effective only on MYCN-non-amplified cells in which they arrested the cell cycle in the G0/G1 phase. We investigated the mechanism of action and identified the activation of cell signaling that involves protein kinase C.

Vanadium promoted ZnO films: effects on optical and photocatalytic properties

ABSTRACT The effect of electrodeposition temperature, vanadium doping, and oxidation state of vanadium on the photocatalytic activity of ZnO thin films over ITO substrates were investigated. NH4VO3 aqueous solution was used as precursor to dope ZnO thin film samples with V5+ by dip coating method and oxalic acid was used as reducing agent to synthesize V+4 doped samples. UV-Vis spectroscopy was used to determine the direct and indirect band gap values of thin film samples. Methylene blue (MB) degradation assays were used to evaluate the photocatalytic activity. XPS analysis was used to determine the analyse oxidation state of vanadium over the samples. Photocatalytic activity studies revealed that ZnO samples electrodeposited at 70°C have higher band gap and MB degradation activity under artificial solar irradiation. The presence of vanadium also enhances the photocatalytic activity of ZnO and samples doped with V+4 have higher photocatalytic activity than the samples doped with V+5 sites.

Advances and Opportunities in H2S Measurement in Chemical Biology.

Hydrogen sulfide (H2S) is an important biological mediator across all kingdoms of life and plays intertwined roles in various disciplines, ranging from geochemical cycles to industrial processes. A common need across these broad disciplines is the ability to detect and measure H2S in complex sample environments. This Perspective focuses on key advances and opportunities for H2S detection and quantification that are relevant to chemical biology. Specifically, we focus on methods for H2S detection and quantification most commonly used in biological samples, including activity-based H2S probes, the methylene blue assay, the monobromobimane assay, and H2S-sensitive electrode measurements. Our goal is to help simplify what at first may seem to be an overwhelming array of detection and measurement choices, to articulate the strengths and limitations of individual techniques, and to highlight key unmet needs and opportunities in the field.

Synergistic effects of Thymus vulgaris essential oil in combination with antifungal agents and inhibition of virulence factors of Candida albicans

BackgroundCombination antifungal therapy has become a prominent approach in medical practice as it takes advantage of synergistic interactions by interfering with multiple targets of the pathogen, broadening the spectrum of activity, reducing the development of resistance, and improving treatment outcomes. Therefore, combining conventional antifungal drugs with natural products can increase antifungal activity, reduce side effects, and optimize therapeutic effects. PurposeThe present study aimed to evaluate the antifungal activity of thyme essential oil (ThyEO) in combination with amphotericin B, caspofungin, fluconazole, itraconazole, and posaconazole (PSZ), as well as its inhibitory effect on Candida albicans virulence factors. Materials and MethodsThe microbroth dilution assay was employed to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Interactions were assessed using the microdilution checkerboard assay. Ergosterol and methylene blue assays were used to detect effects on fungal membrane, while the sorbitol assay was used to detect effects on fungal cell wall. Inhibition of yeast virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation, secretion of hydrolytic enzymes, and biofilm formation) was assessed with previously reported methods. ResultsAlmost all combinations showed additivism against C. albicans, except ThyEO/PSZ (31.25/0.0039 µg/ml), which showed partial synergism. Furthermore, all mixtures were fungicidal against C. albicans strains. ThyEO/PSZ, its components alone, and thymol were shown to disrupt the fungal cytoplasmic membrane, increasing its permeability. ThyEO/PSZ, at sub-inhibitory concentrations, significantly decreased the ability of C. albicans to adhere to buccal epithelial cells. ThyEO/PSZ, ThyEO, and PSZ were able to reduce the pseudomycelium production of C. albicans while thymol completely inhibited its formation. ThyEO/PSZ, each combination component on its own, and thymol inhibited biofilm formation and preformed biofilm of C. albicans. Notably, ThyEO/PSZ showed synergistic and fungicidal activity against a resistant strain of C. albicans, reducing the PSZ dose by 4-fold. ConclusionThese findings make ThyEO and ThyEO/PSZ mixture valuable candidates for the development of alternative antifungals with a lower incidence of adverse effects.

Elevated Serum Hydrogen Sulfide in Age Related Macular Degeneration

Introduction: Age related macular degeneration is one of the major causes of global blindness and vision impairment. The study was done to determine the levels of hydrogen sulfide (H2S) in AMD subjects in the study cohort. Methods: Twenty-six blood samples were collected, 12 control and 14 AMD subjects, the serum obtained from these samples were used for the measurement of H2S by Methylene blue (MB) assay and IL6 using ELISA. Results: We observed that both the levels of H2S and IL6 to be elevated in AMD subjects when compared to control and H2S and IL6 showed a positive correlation. Conclusion: We propose H2S to be a marker in both dry and wet AMD

Poster 152: Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics

Objectives: Chlorhexidine gluconate (CHG) is commonly used in antiseptic irrigation solutions for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG to articular cartilage has been reported, CHG irrigation is utilized by some surgeons to limit surgical site infection and to salvage grafts after accidental contamination during joint surgery. The full extent of CHG irrigation-related chondrotoxicity, as well as its effects on articular cartilage extracellular matrix and mechanical properties, are unknown. The purpose of this study was to determine the in vitro effects of a single one-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. Methods: Articular cartilage explants (n=6 per group) were harvested from the femoral condyles of the porcine stifle and sectioned at the tidemark. Explants were bathed in Irrisept® chlorhexidine solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) to stimulate surgical irrigation for 1 minute, followed by complete phosphate-buffered saline wash and culture. After seven days of culture in chondrogenic medium, the explants were analyzed for viability (live/dead assay); collagen content (hydroxyproline assay); glycosaminoglycan (GAG) content (dimethyl methylene blue assay); and compressive mechanical testing (creep indentation testing). Statistical analyses were performed using a one-way ANOVA with post hoc Tukey test. Results: Seven days after CHG exposure, a dose-dependent decrease in mean chondrocyte viability was observed in the CHG groups (p<0.001), with <3% viability observed in the 0.05% CHG group (Figure 1). There was no significant difference in mean collagen content per wet weight among groups. Conversely, 0.05% CHG exposure led to a decrease in mean GAG per wet weight compared to negative control (p=0.029) and 0.01% CHG (p=0.046) (Figure 2). These changes in GAG content corresponded to observed changes in the compressive mechanical properties of the explants. A dose-dependent decrease in mean aggregate modulus and shear modulus was observed after CHG exposure (p<0.029), indicating weakening of the cartilage matrix and increased deformation in response to compressive loads (Figure 3). Conclusions: One-minute CHG exposure to articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. These detrimental effects were observed for both 0.05% and 0.01% CHG concentrations. There is a paucity of literature examining the effects of CHG on articular cartilage matrix and mechanics, and the findings of this study suggest a lack of matrix maintenance following cell death, raising concern for risk of mechanical failure of the cartilage tissue. Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Evaluation of cancer drug infusion devices prior to the implementation of a compounding robot.

Compounding robots are increasingly being implemented in hospital pharmacies. In our hospital, the recent acquisition of a robot (RIVATM, ARxIUM) for intravenous cancer drug compounding obliged us to replace the previously used infusion devices. The objective of the present study was to assess and qualify the new intravenous sets prior to their use in our hospital and prior to the implementation of the compounding robot. The ChemoLockTM (ICU Medical) was compared with the devices used previously for compounding (BD PhaSealTM, Becton-Dickinson) and infusion (Connect-ZTM, Codan Medical). The connection/disconnection of infusion devices to/from 50 mL infusion bags was tested with a dynamometer (Multitest-i, Mecmesin). Leakage contamination was visualized by a methylene blue assay and was quantified in simulated pump infusions with 20 mg/mL quinine sulfate (N = 36/group); after the analytical assay had been validated, quinine was detected by UV-spectrophotometry at 280 and 330 nm. Groups were compared using chi-squared or Mann-Whitney U tests. The connection/disconnection test showed that although all the devices complied with the current standard, there was a statistically significant difference in the mean ± standard deviation compression force (51.5 ± 11.6 for the Connect-ZTM vs. 60.3 ± 11.7 for the ChemoLockTM; p = 0.0005). Leaks were detected in 32 (29.1%) of the 110 tests of the ChemoLockTM. The contamination rates were also significantly different: 13.9% for the BD PhaSealTM versus 75.0% for the ChemoLockTM; p < 0.0001). Our results showed that the new infusion device complied with current standards. However, the presence of contamination emphasizes the need for operators to use the recommended personal protective equipment. Further studies of contamination with cancer drugs are required.

Photocatalytic and Antimicrobial Activity of TiO2 Films Deposited on Fiber-Cement Surfaces

In this study, TiO2 films were deposited via the doctor blade technique on fiber-cement surfaces. Two types of nanoparticles (TiO2-P25 from Degussa and TiO2-PC105 from Tronox) were used to produce films. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images revealed films with homogeneous and nanoparticulated morphology. The TiO2 PC105 film presented a lower roughness parameter (RMS) in relation to that of the TiO2 P25-based film. Both films exhibited high hydrophilicity when exposed to UV-A radiation (contact angle θ &lt; 6°). The photocatalytic activity of the films was evaluated by standardized methylene blue dye degradation assays under UV-A irradiation (1.0 mW/cm2). The TiO2-PC105 film showed a photonic efficiency of ξ = 0.1%, while for the films obtained with TiO2-P25, ξ = 0.08%. The cement surface modified with the PC105 film was evaluated for antimicrobial activity through the use of multiple pathogens commonly found in hospitals. A considerably high efficiency was measured with visible light. Growth inhibition rates of 99.0% ± 0.2, 99.1% ± 0.2, 99.1% ± 0.2, 97.5% ± 0.5, 98.0% ± 0.5 and 98.0% ± 0.5 were found for Staphylococcus aureus, Klebsiella sp., Escherichia coli, Rhizobium sp., Fusarium sp. and Penicillium sp., respectively. The results show the self-cleaning ability and their potential use for protection, by preventing contamination of the fiber-cement surface and opening new possibilities for the use of this building material.

Anticancer and antioxidant activities of ethanolic extract and semi-purified fractions from guava and mango seeds

Mango (Mangifera indica) and guava (Psidium guajava) seeds have several pharmaceutical applications and biological activities because as they have been recognized with different bioactive molecules (phenolic compounds) such as flavonoids, phenolic acids, and catechins, so they have antioxidant and anticancer activities. The aim of the present study was to assess in vitro antioxidant and anticancer activities of successive extracts and semi-purified fractions from mango seeds. In this work, mango and guava seeds were collected and extracted using two solvents (ethanol 70% and ethyl acetate) followed by phytochemical screening and determination of biological activities such as antioxidant activity using five assays (DPPH, ABTS, KMnO4, Methylene blue and H2O2) additionally the antiradical activity and hybrid reaction for ethanolic extract of mango seeds as promising extract. The total phenolic, flavonoid, and catechin compounds were determined for all successive extracts, and finally, the anticancer activity of extracts was evaluated using MTT assay against HepG2 cell line and phenolic compounds were identified by HPLC. The phytochemical screening and TLC showed the primary investigation for phenolic compounds of ethanol extracts of both kind of seeds and only ethyl acetate of guava extract as promising extracts. However, HPLC determination of these three extracts showed high amount of gallic acid, naringenin, ellagic acid, and ferulic acid as they have anticancer and antioxidant activities. The antioxidant tests showed that the ethanolic mango extract is the highest antioxidant extract against DPPH by 84.0%, but recorded 82.0% with methylene blue and ABTS assays when compared with ascorbic acid. The ethyl acetate of guava extract showed strong cytotoxic effect with IC50 75.5 μg/mL against HepG2 cell line in all tested concentrations. From the obtained results, it could be concluded that mango ethanolic extract and its fractions are the most promising as antioxidants and ethyl acetate of guava extract the most promising in the anticancer activity.

Abstract 386: Metastatic models of resistance to next generation tyrosine kinase inhibitors (TKIs) in HER2-positive breast cancer (BC)

Abstract HER2+ BC is an aggressive subtype with increased metastatic tropism towards brain. Small molecule anti-HER TKIs are still a key treatment choice in this setting owing to their competency to cross the blood brain barrier. Despite the efficacy, resistance to HER2-targeted agents often arises either due to compensatory signaling from other HER receptors or due to dysregulation in the downstream signaling pathway. We have previously shown that the mechanism of resistance to each TKI differs based on its HER receptor specificity profile. Here, we sought to evaluate the aggressiveness and metastatic ability of our HER2+ cell models with acquired resistance to different clinically relevant anti-HER TKIs. Dual GFP-Luc tagged HER2+ BT474 naïve parental cells and their acquired lapatinib (LapR), neratinib (NeraR), and tucatinib (TucaR) resistant (Res) derivatives harboring acquired HER2 L755S, HER2 L755S and a pathogenic PIK3CA mutation, or EGFR amplification, respectively were used. We have previously shown that while the LapR and NeraR cells are cross-Res to Tuca, the LapR and TucaR cells remain sensitive to Nera. Changes in cell growth and migration were evaluated by methylene blue or Incucyte wound healing assays, respectively. The metastatic phenotype of parental and the acquired Res models was assessed by intracardiac injection in NSG mice and metastasis was monitored by bioluminescence imaging. Metastatic lesions from different organs/sites were harvested to establish GFP-positive in vitro cell cultures. Our recent data showed that the LapR, NeraR, and TucaR derivatives are significantly more migratory compared to naïve parental cells suggesting their enhanced metastatic potential. Indeed, our in vivo studies revealed that all the Res derivatives and parental cells were highly metastatic to the brain, proving the well-established notion that HER2+ BC cells are brain tropic. Further, the involvement of ovary, lung, and liver, among others, were observed as additional common metastatic sites. We have now successfully established in vitro cultures of harvested metastatic lesions from different sites for each cell model. Growth assays using the cells established from LapR ovary metastatic lesions (LapR/OvMet) revealed that they are indeed Res to Lap, while importantly, consistent with the drug sensitivity profile of the original LapR 2D cells, the LapR/OvMet line was highly sensitive to Nera but cross-Res to Tuca. Additional studies to investigate the organ-specific metastatic tropism of each acquired resistant model are currently ongoing. Molecular and functional characterization of our established metastatic cultures will guide future in vivo studies, including testing the efficacy of new targeted treatment regimens to treat organ-specific metastasis, and expanding such studies to additional relevant cell and patient-derived xenograft/organoid models. Citation Format: Fu-Tien Liao, Lanfang Qin, Martin J. Shea, Igor Bado, Yi-Hsuan Wu, Ling Wu, Yang Gao, Sarmistha Nanda, Tia Gordon, Chia Chia Liu, Xiang H.-F. Zhang, C. Kent Osborne, Mothaffar F. Rimawi, Jamunarani Veeraraghavan, Rachel Schiff. Metastatic models of resistance to next generation tyrosine kinase inhibitors (TKIs) in HER2-positive breast cancer (BC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 386.

Abstract P1-13-17: Hyperactivation of the EGFR pathway is associated with resistance to tucatinib in HER2-positive breast cancer models

Abstract Background: The HER2-specific tyrosine kinase inhibitor (TKI) tucatinib (Tuca) recently approved for advanced HER2+ breast cancer is making a move towards the early setting. Given its growing use, resistance is inevitable, as observed in the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on Tuca. Driven by the prevailing lack of knowledge about the mechanisms of resistance, in this study, we sought to define these mechanisms and identify treatment strategies to overcome them. We previously reported (SABCS 2021) that our BT474 TucaR models acquired EGFR amplification and showed elevated levels of phosphorylated (p) and total (t) EGFR, pHER2, pHER3, and downstream pAKT and pS6. Since the HER pathway is activated by ligands, here we aim to assess if hyperactivation of EGFR via high levels of its ligands is an alternative mechanism of Tuca resistance. Materials and Methods: Our recently developed HER2+ BT474 (ATCC and AZ) cell models with acquired resistance to Tuca (TucaR) developed through long-term exposure to gradually increasing doses of Tuca and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (western blot) characterization were performed. Changes in cell growth and migration were assessed by methylene blue and Incucyte wound healing assays, respectively. Results: RNA-seq analysis demonstrated that the levels of TGFα was significantly higher in our BT474 TucaR models compared to P cells. Our results now demonstrate that exogenous supplementation of EGF to BT474-P cells rescues the Tuca-mediated inhibition of pEGFR, pHER2, and the downstream pAKT, pERK, and pS6 levels. Exogenous EGF was also found to reduce the levels of apoptosis, as assessed by cleaved PARP, mitigating the Tuca-induced cell death. Exogenous EGF or TGFα rendered naïve BT474 and SKBR3 cells resistant to Tuca while neratinib, a pan-HER TKI, effectively inhibited this ligand-driven cell growth. We previously showed that the HER signaling reactivation observed in our EGFR-amplified TucaR cells was inhibited by the EGFR-specific TKI gefitinib (Gef) (SABCS 2021) and that the TucaR cells displayed enhanced migratory capabilities (AACR 2022). Here, we demonstrate that in addition to curbing the growth of TucaR cells, Gef, either alone or together with Tuca, also markedly reverts the migration of the TucaR cells. Knockdown (KD) of EGFR but not HER2 selectively and substantially inhibited the migration of the TucaR cells. KD of EGFR also had a marked cell killing effect on only the TucaR cells, whereas HER2 KD inhibited the growth of P but not TucaR cells. Our findings are consistent with the notion that while the P cells are functionally dependent on HER2, in TucaR cells the survival dependence could be rewired to rely primarily on the hyperactive EGFR signaling. Genomic analysis further revealed that in addition to EGFR amplification, the AZ TucaR cells also acquired a gain of YES1, a src family receptor tyrosine kinase implicated in cancer cell growth, invasion, and metastasis. Functional studies using 2 siRNAs, however, showed that YES1 KD had no effect on the growth of TucaR cells, and the migration of both TucaR and P cells was equally affected by YES1 KD, precluding the potential role of YES1 in driving the resistant and enhanced migratory phenotypes. Conclusions: Hyperactivation of the EGFR pathway via amplification of EGFR or increased expression of its ligands confers resistance to Tuca, which may be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Given the rapidly evolving treatment landscape of HER2+ breast cancer and biomarkers of resistance, our novel findings have potentially crucial therapeutic implications and suggest that rationally sequencing the currently available TKIs may be clinically important. Citation Format: Fu-Tien Liao, Tia Gordon, Chia Chia Liu, Pier Selenica, Yingjie Zhu, Juber Patel, Sarmistha Nanda, Lanfang Qin, Xiaoyong Fu, Andrea Gazzo, Antonio Marra, Juan Blanco-Heredia, Britta Weigelt, Jorge Reis-Filho, C. Kent Osborne, Mothaffar Rimawi, Rachel Schiff, Jamunarani Veeraraghavan. Hyperactivation of the EGFR pathway is associated with resistance to tucatinib in HER2-positive breast cancer models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-17.

Paper sensor-based method using a portable 3D-printed platform and smartphone-assisted colorimetric detection for ammonia and sulfide monitoring in anaerobic digesters and wastewater

A simple and low-cost field-deployable method using paper sensors in combination with a 3D-printed portable platform to simultaneous determination of ammonia and sulfide has been developed. Ammonia and sulfide are harmful pollutants for the environment and important toxicants inhibiting anaerobic digestion, therefore, are key monitoring parameters during wastewater treatment. The sample preparation system consisted of a well microplate and sensor paper discs placed inside the microplate sealing system. Gas pervaporation and diffusion of ammonia and hydrogen sulfide across a Teflon membrane onto a paper discs, impregnated with bromothymol blue and methylene blue assay reagents, allowed the ammonia and sulfide determination, respectively. The detection system consisted in a 3D-printed photographic lightbox with LED plates and a smartphone. A code was developed in ImageJ for a fast colorimetric measurement of the digital images. Under optimal conditions, limits of detection of 1.3 and 1.7 mg L-1 and linear concentration ranges between 5 and 50 mg L-1 and 5–45 mg L-1, were obtained for ammonia and sulfide, respectively. The analysis of an anaerobic digester and wastewater samples revealed good precisions (RSDs < 10 %) and recoveries (91–105 %). This methodology shows advantages such as a high precision, a simple paper discs preparation and a reusable system.