Search Results (221)

Biofilms are the predominant lifeforms of microorganisms, contributing to over 80% of infections, including those caused by Candida species like C. albicans, C. parapsilosis and Candidozyma auris. These species form biofilms on medical devices, making infections challenging to treat, especially with the rise in drug-resistant strains. Candida infections, particularly hospital-acquired ones, are a significant health threat due to their resistance to antifungals and the risk of developing systemic infections (i.e., sepsis). We have previously shown that C14R reduces the viability of C. albicans and C. auris, but not of C. parapsilosis. Here, we show that C14R not only inhibits viability by pore formation, shown in a resazurin reduction assay, and in a C. parapsilosis and fluorescence-based permeabilization assay, but it also halts biofilm maturation and significantly reduces the biomass of preformed biofilms by over 70%. These findings suggest C14R could be an effective option for treating severe fungal infections, offering a potential new treatment approach for biofilm-related diseases. Further research is needed to fully understand its biofilm dispersal potential and to optimize its use for future applications as an antifungal in clinical settings. Full article

This article attempts to comprehensively assess plants from the Apiaceae family, such as Apium graveolens, Daucus carota or Petroselinum crispum, as raw plant materials with potential uses in cosmetic products with anti-inflammatory and antibacterial effects. The work compares the phytochemical profiles and activity of extracts and ferments from the roots of these plants obtained during fermentation using kombucha. The antioxidant properties of the tested extracts, the effect on the intracellular level of free radicals and their cytotoxicity towards skin cells were compared. Their anti-inflammatory and antibacterial properties were also assessed. The ABTS and DPPH tests indicated the highest antioxidant potential of the carrot ferments, achieving a 55.75% and 74.6% reduction of these radicals, respectively. The resazurin and Neutral Red assays indicated that in most cases, sample concentrations not exceeding 2.5% did not cause a cytotoxic effect, and in the case of a 20-day parsley ferment, they could increase viability by over 40%. The disk diffusion method indicated growth inhibition zones of over 20 mm for some bacteria. The minimum inhibitory concentrations for seven different bacterial strains ranged from 200 to 400 µg/mL. Anti-inflammatory properties were determined using the ELISA method, assessing the level of interleukins 1β, 6 and 10. The obtained results indicate a higher amount of phytochemicals, a lack of cytotoxic effect at lower concentrations of the tested samples and significantly stronger antioxidant, antibacterial and anti-inflammatory properties of the ferments compared to the extracts. This effect depends on the concentration and fermentation time used. Full article

Background/Objectives: Tuberculosis remains a major public health crisis, and it is imperative to search for new antimycobacterial drugs. Natural products, including medicinal macrofungi, have been used as sources for the discovery of pharmaceuticals; however, research on their antimycobacterial activity remains limited. This study aimed to isolate and identify the bioactive compound responsible for antimycobacterial activity, thereby expanding on the limited knowledge regarding the antimicrobial activity and bioactive compounds present in Gymnopilus junonius. Methods: Bioassay-guided fractionation using column chromatography and preparative thin-layer chromatography were employed to isolate the active compound. Antimycobacterial activity against Mycobacterium tuberculosis H37 was assessed using a resazurin microplate assay (REMA). The chemical structure was determined by ¹H nuclear magnetic resonance (NMR) spectroscopy, heteronuclear single quantum coherence (HSQC) spectroscopy, heteronuclear multiple bond correlation (HMBC) spectroscopy, and high-resolution electrospray ionization mass (HR-ESI-MS) spectrometry. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes in M. tuberculosis induced by the compound. Cytotoxicity was evaluated in African green monkey kidney cells (Vero), human liver cells (C3A), and zebrafish embryos/larvae. Results: Bioassay-guided fractionation led to the isolation of gymnopilene, which showed inhibitory activity against M. tuberculosis (MIC: 31.25 µg/mL). TEM analysis revealed that treatment with gymnopilene caused ultrastructural damage observed as the disruption and disintegration of the cell wall. While gymnopilene demonstrated cytotoxicity in Vero and C3A cells, no toxicity was observed in zebrafish embryos/larvae for the crude extract. Conclusions: This study highlights that macrofungi, such as G. junonius, could be a valuable resource of bioactive compounds. Full article

Leishmania amazonensis, a cause of cutaneous leishmaniasis in Brazil, is a neglected disease with toxic and inconsistently effective treatments. The parasite’s survival depends on managing oxidative stress, making redox-regulating enzymes potential therapeutic targets. Geopropolis, a resinous product from native stingless bees, shows promising antiparasitic effects. This study aims to evaluate the anti-L. amazonensis activity of geopropolis produced by Melipona bicolor, M. marginara, M. mondury, and M. quadrifasciata (two samples), targeting enzymes responsible for the parasite’s redox balance. Ethanol extracts of geopropolis produced by each bee (BCRL, MRGT, MNDY, MNDA(1), and MNDA(2), respectively) were analyzed for total phenolics and flavonoids. Promastigotes and axenic amastigotes were treated with various extract concentrations, and parasite viability was assessed using the resazurin reduction method. Cytotoxicity was tested on peritoneal macrophages, RAW 264.7, VERO cell lines (MTT assay), and erythrocytes (hemolysis assay). Additionally, mitochondrial dehydrogenase activity, reactive oxygen species (ROS) production, the inhibition of recombinant arginase, and autophagic activity were also evaluated in treated parasites. MRGT showed the highest levels of phenolics (762 mg GAE/g) and flavonoids (345 mg QE/g). MDRY was more effective against promastigote and axenic amastigote forms (IC₅₀ = 168 and 19.7 µg/mL, respectively). MRGT showed lower cytotoxicity against RAW 264.7 and VERO (CC₅₀ = 654 µg/mL and 981 µg/mL, respectively). Erythrocytes exhibited reduced sensitivity to MNDA(2) (HC₅₀ = 710 µg/mL). The activity of dehydrogenases and LiARG was reduced by treating the parasites with the extracts following the induction of ROS and autophagic activity. These results highlight geopropolis extracts as a source of substances with anti-L. amazonensis activity capable of inducing oxidative stress on the parasite. Full article

Background/Objectives: Natural cytokinins are a promising group of anti-tumor agents. In this work, we hypothesized that modification of the ethylenediurea moiety with alkyl and oxygen-containing groups could be a way to enhance the anti-proliferative properties of the molecule. Methods: Ten new analogs of ethylenediurea with these substitutions were tested for anti-proliferative activity in the human cancer cell lines MDA-MB-231 (breast cancer), A-375 (melanoma), and U-87 MG (glioblastoma) during 72 h of incubation using resazurin test and evaluated the substances receptor using molecular docking. Results: The compound with the carbamate link and ethylene substituent on the phenyl ring inhibited proliferation in these models by 70–90% without cytotoxic effects. The compound did not affect the viability of the immortalized fibroblast cell line Bj-5ta. The compound was also able to enhance the action of doxorubicin and temozolomide by about 20%. According to the molecular modeling data, the probable receptor target for the synthesized compound was the A2AR adenosine receptor. Conclusions: The results obtained on the ethylenediurea analogs with ethyl substituent in the aromatic ring are promising for the development of novel anticancer therapeutics. Full article

2-arachnadoyl glycerol (2-AG) is one of the most common endocannabinoid molecules with anti-proliferative, cytotoxic, and pro-proliferative effects on different types of tumors. Typically, it induces cell death via cannabinoid receptor 1/2 (CB1/CB2)-linked ceramide production. In breast cancer, ceramide is counterbalanced by the sphingosine-1-phosphate, and thus the mechanisms of 2-AG influence on proliferation are poorly understood. We evaluated the mechanism of the anti-proliferative action by 2-AG and the influence of lysophaosphatidylinositol (LPI) on it in six human breast cancer cell lines of different tumor degree (MCF-10A, MCF-7, BT-474, BT-20, SK-BR-3, and MDA-MB-231) using resazurin test, inhibitor, blocker, and anti-oxidant analysis, and siRNA interference. To avoid acyl migration in 2-AG, we replaced it with the analog 2-arachidonoyl-1,3-difluoropropanol (2-ADFP) newly synthesized by us. Using a molecular docking approach, we showed that at the CB2 receptor, 2-ADFP and 2-AG were very close to each other. However, 2-ADFP demonstrated a stronger affinity towards CB1 in the antagonist-bound conformation. 2-ADFP was anti-proliferative in all the cell lines tested. The toxicity of 2-ADFP was enhanced by LPI. 2-ADFP activity was reduced or prevented by the CB2 and vanilloid receptor 1 (TRPV1) blockers, inositol triphosphate receptor, CREB, and cyclooxygenase 2 inhibitor, and by anti-oxidant addition. Together with the literature data, these results indicate CB2- and TRPV1-dependent COX-2 induction with concomitant cell death induction by the oxidized molecule’s metabolites. Full article

Chlorhexidine and cetrimide are often used as antiseptics and disinfectants. While their individual activities are well-documented, their synergism has rarely been evaluated. Here, we attempted to evaluate the antimicrobial and antibiofilm effects of the combination of these two antimicrobial agents against two environment isolates, viz., P. aeruginosa and S. aureus. The synergism was assayed by determining the fractional inhibitory concentrations, while the antibiofilm effects were determined using crystal violet staining and the resazurin assay. Further, the effects on the biofilms were visualized using brightfield and confocal laser scanning microscopy. Our results show that the combination of these antimicrobials resulted in synergistic inhibition of P. aeruginosa growth. When tested at concentrations below the individual MICs (one-quarter of the MICs), the combination was able to significantly reduce the adherence of S. aureus biofilms to a polystyrene surface, while no effect was observed for P. aeruginosa. The combination was also able to significantly reduce the viability of pre-formed biofilms of both bacteria, thereby showing its antibiofilm potential. Next, we evaluated the performance of this combination against a wide array of micro-organisms. This fixed-dose combination formulation exhibited a significant reduction in the viability of an array of clinically relevant micro-organisms, including ESKAPE pathogens, Mycobacterium sp., MRSA, Leptospira, Candida sp., norovirus and adenovirus. Overall, it can be inferred that the combination of chlorhexidine and cetrimide is a potential biocide that continues to be relevant for use in antisepsis and disinfection against infection-causing pathogens. Full article

Background/Objectives: Côa Valley, located in the northeast of Portugal, harbors more than 500 medicinal plant species. Among them, four species stand out due to their traditional uses: Equisetum ramosissimum Desf. (hemorrhages, urethritis, hepatitis), Rumex scutatus L. subsp. induratus (Boiss. and Reut.) Malag. (inflammation, constipation), Geranium purpureum Vill., and Geranium lucidum L. (pain relief, gastric issues). Given their rich ethnomedicinal history, we evaluated their protective effects on an in vitro model of metabolic dysfunction-associated steatotic liver disease (MASLD). Methods: Decoction (D) and hydroalcoholic (EtOH80%) extracts were prepared and chemically characterized. Their safety profile and effects on lipid accumulation were assessed in palmitic acid (PA)-treated HepG2 cells using resazurin, sulforhodamine B, and Nile Red assays. Results: Chemical analysis revealed diverse phenolic compounds, particularly kaempferol derivatives in E. ramosissimum. All extracts showed minimal cytotoxicity at 25–50 µg/mL. At 100 µg/mL, only E. ramosissimum extracts maintained high cell viability. In the lipotoxicity model, E. ramosissimum decoction demonstrated the most potent effect, significantly reducing PA-induced neutral lipid accumulation in a dose-dependent manner, while other extracts showed varying degrees of activity. Conclusions: These findings highlight E. ramosissimum’s decoction, rich in kaempferol derivatives, as particularly effective in reducing lipid accumulation in this MASLD cell model while also providing a comprehensive characterization of traditionally used plants from the Côa Valley region. Full article

Oxidative stress (OS) is a common feature of many inflammatory diseases, oral pathologies, and aging processes. The impact of OS on periodontal ligament cells (PDLCs) in relation to oral pathologies, including periodontal diseases, has been investigated in different studies. However, its impact on orthodontic tooth movement (OTM) remains poorly understood. This study used an in vitro model with human PDLCs previously exposed to H₂O₂ to investigate the effects of OS under a static compressive force which simulated the conditions of OTM. Human PDLCs were treated with varying concentrations of H₂O₂ to identify sub-lethal doses that affected viability minimally. To mimic compromised conditions resembling OTM under OS, the cells were pretreated with the selected H₂O₂ concentrations for 24 h. Using an in vitro loading model, a static compressive force (2 g/cm²) was applied for an additional 24 h. The cell viability, proliferation, and cytotoxicity were evaluated using live/dead and resazurin assays. Apoptosis induction was assessed based on caspase-3/7 activity. The gene expression related to bone remodeling (RUNX2, TNFRSF11B/OPG, BGLAP), inflammation (IL6, CXCL8/IL8, PTGS2/COX2), apoptosis (CASP3, CASP8), and autophagy (MAP1LC3A/LC3, BECN1) was analyzed using RT-qPCR. This study suggests an altering effect of previous OS exposure on static-compression-related mechanosensing. Further research is needed to fully elucidate these mechanisms. Full article

We report a workflow and a software description for digital image colorimetry aimed at obtaining a quantitative dose–response curve and the minimal inhibitory concentration in the Resazurin Microtiter Assay (REMA) test of the activity of antimycobacterial drugs. The principle of this analysis is based on the newly established correspondence between the intensity of the ${\mathrm{a}}^{*}$ channel of the CIE L*a*b* colour space and the concentration of resorufin produced in the course of this test. The whole procedure can be carried out using free software. It has sufficiently mild requirements for the quality of colour images, which can be taken by a typical smartphone camera. Thus, the approach does not impose additional costs on the medical examination points and is widely accessible. Its efficiency is verified by applying it to the case of two representatives of substituted 2-(quinolin-4-yl) imidazolines. The direct comparison with the data on the indicator’s fluorescence obtained using a commercial microplate reader argues that the proposed approach provides results of the same range of accuracy on the quantitative level. As a result, it would be possible to apply the strategy not only for new low-cost studies but also for expanding databases on drug candidates by quantitatively reprocessing existing data, which were earlier documented by images of microplates but analysed only qualitatively. Full article

The A549 cell line has become a cornerstone in biomedical research, particularly in cancer studies and serves as a critical tool in cytotoxicity studies and drug screening where it is used to evaluate the impact of pharmaceutical compounds on cellular viability. One of the most widely adopted methods for viability assessment, which is also used in evaluating drug cytotoxicity, is the resazurin-based assay. This assay exploits the ability of living cells to convert resazurin into fluorescent resorufin, providing a reliable indicator of metabolic activity. By measuring this conversion, cell viability can be estimated. Resazurin assay is extensively used for evaluating cytotoxic effects on various cell lines, including A549 cells, thereby bridging the gap between in vitro experimentation and drug development. However, frequent data inconsistencies in pre-clinical drug screening highlight the critical need for standardization to ensure reliability and reproducibility. This manuscript addresses these challenges by describing the optimization of resazurin-based viability assays for A549 cells in both 2D cultures and 3D fibrin gel models. By optimizing this test, the study aims to enhance the reliability of cytotoxicity results and introduces a new standard operating procedure, thus providing consistent results with minimal measurement uncertainty. This standardization is crucial for advancing drug screening and ensuring robust research findings. Full article

Background/Objectives: The development of innovative materials for disease diagnostics and therapeutics is a fast-growing area of scientific research. In this work, we report the development of innovative hydrogels incorporating carbon dots (Cdots) for bioimaging purposes. Methods: The Cdots were prepared using a sustainable and low-cost process, starting with an underused fiber from the Brazilian semiarid region. Spectroscopy analysis (Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, UV-visible spectroscopy), X-ray diffraction, photoluminescence, zeta potential, scanning electron microscopy, and transmission electron microscopy were used to characterize these hydrogels. In addition, biocompatibility using the resazurin assay and cellular uptake by confocal microscopy were evaluated. Results: Our results showed that the Cdots changed the structure and crystallinity of hydrogels, mainly due to heat treatment. In addition, hydrogels’ chemical groups suffer red and blue shifts following the Cdots incorporation. Moreover, the Cdots were homogeneously incorporated into the hydrogel matrix. Importantly, the cytotoxicity levels were maintained above 90% (p < 0.01), and cellular uptake studies using HeLa cells demonstrated intracellular fluorescence of both the Cdots and hydrogels after incubation. Additionally, the concentration of Cdots within hydrogels significantly affected fluorescence intensity, even compared with pure Cdots. Conclusions: These results showcase the potential for these hydrogels to be further developed as biomarkers and therapeutic biomaterials for women’s health. Full article

Background and Objectives: Expansion of white adipose tissue causes systemic inflammation and increased risk of metabolic diseases due to its endocrine function. Resveratrol was suggested to be able to prevent obesity-related disorders by mimicking caloric restriction; however, its structure–activity relationships and molecular targets are still unknown. We aimed to compare the effects of resveratrol and its analogues on adipocyte metabolism and lipid accumulation in vitro. Methods: Mouse embryonic fibroblasts were differentiated to adipocytes in the absence or presence of resveratrol or its derivatives (oxyresveratrol, monomethylated resveratrol, or trimethylated resveratrol). Intracellular lipid content was assessed by Oil Red O staining. Glucose uptake and its response to insulin were estimated by 2-NBDG, and mitochondrial activity was assayed via resazurin reduction. Involvement of potential molecular pathways was investigated by concurrent treatment with their inhibitors. Results: Although lipid accumulation was significantly reduced by all analogues without altering protein content, oxyresveratrol was the most potent (IC50 = 4.2 μM), while the lowest potency was observed with trimethylated resveratrol (IC50 = 27.4 μM). Increased insulin-stimulated glucose uptake was restored by each analogue with comparable efficiency. The enhanced mitochondrial activity was normalized by resveratrol and its methylated derivatives, while oxyresveratrol had a minor impact on it. Among the examined pathways, inhibition of SIRT1, PGC-1α, and JNK diminished the lipid-reducing effect of the compounds. Autophagy appeared to play a key role in the effect of all compounds but oxyresveratrol. Conclusions: Resveratrol and its analogues can mimic caloric restriction with complex mechanisms, including activation of SIRT1, PGC-1α, and JNK, making them possible drug candidates to treat obesity-related diseases. Full article

Bioengineered livers are currently an acceptable alternative to orthotopic liver transplants to overcome the scarcity of donors. However, the challenge of using a bioengineered liver is the lack of an intact endothelial layer in the vascular network leading to thrombosis. Heparin-modified surfaces have been demonstrated to decrease thrombogenicity in earlier research. However, in our study, we aimed to apply heparin immobilization to enhance the hemocompatibility, endothelial cell (EC) adhesion, and angiogenesis of rat decellularized liver scaffolds (DLS). Heparin was immobilized on the DLS by the end-point attachment technique. The scaffold’s hemocompatibility was assessed using ex vivo blood perfusion and platelet adhesion studies. The heparinized scaffold (HEP-DLS) showed a significantly reduced thrombogenicity and platelet aggregation. HEP-DLS was recellularized with EA.hy926 cells via the portal vein and maintained in the bioreactor for 7 days, showing increased EC adhesion and coverage within the blood vessels. The Resazurin reduction assay confirmed the presence of actively proliferating cells in the HEP-DLS. The scaffolds were implanted subcutaneously into the dorsum of mice for 21 days to evaluate cell migration and angiogenesis. The results showed significant increases in the number of blood vessels in the HEP-DLS group. Our results demonstrated that heparin immobilization reduces thrombosis, promotes re-endothelialization, and enhances angiogenesis in DLS. The research provides insight into the potential use of heparin in the formation of a functioning vasculature. Full article

Ovarian cancer (OC) remains one of the leading causes of cancer-related mortality among women. Targeting the insulin-like growth factor 1 (IGF-1) signaling pathway has emerged as a promising therapeutic strategy. Linsitinib, an IGF-1 receptor (IGF-1R) inhibitor, has shown potential in disrupting this pathway. Additionally, metformin, commonly used in the treatment of type 2 diabetes, has been studied for its anti-cancer properties due to its ability to inhibit metabolic pathways that intersect with IGF-1 signaling, making it a candidate for combination therapy in cancer treatments. This study explores the anti-cancer effects of linsitinib and metformin on OVCAR3 cells by the suppression of the IGF-1 signaling pathway by siRNA-mediated IGF-1 gene silencing. The goal is to evaluate their efficacy as therapeutic agents and to emphasize the critical role of this pathway in OC cell proliferation. Cellular viability was evaluated by resazurin-based assay, and apoptosis was assessed by flux cytometry. The results of this study indicate that the combination of linsitinib and metformin exhibits an antagonistic effect (obtained by SynergyFinder 2.0 Software), reducing their anti-neoplastic efficacy in OC cell lines. Statistical analyses were performed using ordinary one-way or two-way ANOVA, followed by Tukey’s or Šídák’s multiple comparison tests. While linsitinib shows promise as a therapeutic option for OC, further research is needed to identify agents that could synergize with it to enhance its therapeutic efficacy, like the combination with standard chemotherapy in OC (carboplatin and paclitaxel). Full article