Search Results (65)

Michler’s Ketone (MK) is widely utilized as an additive in pigments, dyes, and other colorants, and has become a non-negligible environmental presence. Currently, environmental monitoring data and toxicity data for MK are extremely limited, and its specific mechanisms of neurotoxicity remain poorly characterized. A zebrafish model was employed to systematically delineate the neurotoxic mechanisms of MK through the integration of network toxicology predictions, transcriptomic profiling, and RT-qPCR validation. The results demonstrated that MK exposure was found to induce oxidative stress in zebrafish larvae, which subsequently disrupted the calcium signaling pathway and triggered apoptosis, ultimately leading to neurodevelopmental and locomotor behavioral impairments. This study provides a fundamental basis for elucidating MK’s developmental neurotoxicity mechanisms, while also holding significant value for its ecological risk assessment. Full article

The fluorescent dyes 9-aminoacridine (9-AA) and acridine orange (AO) are known mutagens that induce frameshift mutations in cells by intercalating between DNA bases. However, these chemicals can also affect other cellular components, such as proteins. In this study, we tested the ability of 9-AA and AO to induce heat shock in bacteria using the following methods: lux-biosensors based on Escherichia coli cells with the luxCDABE genes transcriptionally fused to heat shock-specific inducible promoters, RT-qPCR, and nanoDSF. We demonstrated that acridine dyes not only induce mutagenesis but also cause heat shock in bacterial cells. AO significantly reduced the melting temperature of proteins and strongly activated σ^E- and σ³²-dependent promoters, but not PluxC, which is activated by elevated temperatures via a different mechanism. In contrast, 9-AA weakly denatured the proteins and induced the σ^E-dependent promoter; however, it activated the σ³²-dependent promoters and PluxC, supporting the hypothesis that the σ³² heat shock response system is activated via hairpin RNA denaturation by 9-AA. The study on the application of lux-biosensors was hampered by the high general toxicity and luminescence shielding effect of AO, and RT-qPCR’s sensitivity was insufficient for detection of the response to 9-AA. Thus, methodologically, it is justified to conduct a comprehensive study of substances that cause heat shock or affect bioluminescence by both RT-qPCR and lux-biosensors. Full article

Ochratoxin A (OTA) is a widespread mycotoxin with documented nephrotoxic, hepatotoxic, immunotoxic, and carcinogenic effects, while its role in hematological malignancies and immune cells remains insufficiently defined. This study examined the cytotoxic and pro-apoptotic OTA activity in three human leukemia cell lines (CCRF-CEM, K-562, HL-60) and in peripheral blood mononuclear cells (PBMCs) from healthy donors. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue assays, mitochondrial membrane potential (ΔΨM) was assessed with JC-1 dye, caspase-3/7 activity was measured by flow cytometry, and the expression of apoptosis-related genes was analyzed by RT-qPCR. OTA did not significantly affect viability, mitochondrial function, or caspase activity in leukemia cell lines, suggesting relative resistance to OTA-induced apoptosis. In contrast, PBMCs exhibited clear dose- and time-dependent sensitivity, manifested by reduced viability, ΔΨM, caspase-3/7 activation, and transcriptional changes consistent with intrinsic apoptosis, including decreased BCL-2 (anti-apoptotic) and increased BAX (pro-apoptotic), APAF1 (apoptosome component), CASP3, and CASP9 (executioner and initiator caspases) expression. These findings demonstrate that OTA selectively targets healthy immune cells rather than leukemia cells, highlighting its pronounced immunotoxic risk and the importance of caution when considering its effect in a hematological context. Although limited to in vitro models, this study underscores the necessity of further research to clarify the molecular basis of differential OTA sensitivity and its contribution to immunosuppression and hematological disease. Full article

Background/Objectives: The low permeability of the blood-brain barrier (BBB) represents a significant challenge to effective systemic chemotherapy for primary and metastatic brain cancers. Kinin receptors play a crucial role in modulating BBB permeability, and their agonist analogs have been explored in preclinical animal models to enhance drug delivery to the brain. In this study, we investigated whether des-Arg⁹-bradykinin (DBK), a physiological agonist of kinin B₁ receptor (B1R), acts as a brain drug delivery adjuvant by promoting the transient opening of the BBB. Methods: Human brain microvascular endothelial cells (HBMECs) were treated with DBK in the culture medium and in conditioned media from glioblastoma cell lines, namely T98G (CMT98G) and U87MG (CMU87). Immunofluorescence, RT-qPCR, in-cell Western assay, and proximity ligation assay (PLA) were performed to analyze BBB components, kinin receptors and TLR4, a receptor associated with the kinin pathway and inflammation. The effect of DBK on enhancing paracellular molecule transport was evaluated using Evans blue dye (EB) quantification in a cell culture insert assay and in an in vivo model, where mice with and without brain tumors were treated with DBK. To assess the functional impact of the transient BBB opening induced by DBK, the chemotherapeutic drug doxorubicin (DOX) was administered. Results: Treatment with DBK facilitates the presence of EB in the brain parenchyma by transiently disrupting the BBB, as further evidenced by the increased paracellular passage of the dye in an in vitro assay. B1R activation by DBK induces transient BBB opening lasting less than 48 h, enhancing the bioavailability of the DOX within the brain parenchyma and glioma tumor mass. The interaction between B1R and TLR4 is disrupted by the secreted factors released by glioblastoma cells, as conditioned media from T98G and U87 reduce TLR4 staining in endothelial cells without affecting B1R expression. Conclusions: These results further support the potential of B1R activation as a strategy to enhance targeted drug delivery to the brain. Full article

Feline infectious peritonitis (FIP), a devastating disease with near-complete mortality, is caused by the feline coronavirus (FCoV) and affects domestic cats worldwide. Herein, we report the development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay incorporating xylenol orange (XO) as a visual indicator for FCoV detection. The assay employed six oligonucleotide primers targeting regions of the nucleocapsid (N) gene. Under optimized conditions (65 °C, 60 min), amplification products were detected through pH-dependent colour changes in the XO dye. The RT-LAMP-XO assay exhibited high specificity for FCoV, with no cross-reactivity against other common feline viral pathogens. While the detection limit (1.7 × 10¹ copies/µL) was an order of magnitude higher than that of qPCR, the method offered advantages in simplicity and speed compared to existing diagnostic approaches. Although less sensitive than qPCR, the RT-LAMP-XO assay may serve as a rapid screening tool when used in combination with additional primer sets. These findings demonstrate the potential utility of XO-based RT-LAMP as a simple, visual detection method for FCoV infection. Full article

(1) Background: There are many cases of human disease caused by the hepatitis A virus contamination of aquatic products, so the development of the rapid detection of hepatitis A virus in aquatic products is crucial. (2) Methods: In this study, we developed three visual loop-mediated isothermal amplification methods for the rapid and intuitive detection of hepatitis A virus in aquatic products. New specific LAMP primers were designed for the HAV-specific VP1 protein shell. (1) HNB dye was added to the LAMP reaction system. After the reaction, the color of the reaction mixture changed from violet to sky blue, showing a positive result. (2) Cresol red dye was added to the LAMP reaction system, and a positive result was indicated by orange, while a negative result was indicated by purple. (3) By labeling FIP with biotin and LF with 6-FAM, the amplified product simultaneously contained biotin and 6-FAM, which bound to the anti-biotin antibody on the gold nanoparticles on the lateral flow dipstick (LFD). Subsequently, biotin was further combined with the anti-fam antibody on the T-line of the test strip to form a positive test result. (3) Results: The three visual LAMP methods were highly specific for HAV. The sensitivity of the visual assay was 2.59 × 10⁰ copies/μL. The positive detection ratio for 155 bivalve shellfish samples was 8.39%, which was the same as that for RT-qPCR. The three visual LAMP methods established in our work have better sensitivity than the international gold standard, and their operation is simple and requires less time. (4) Conclusions: The results can be obtained by eye color comparison and lateral flow dipsticks. Without the use of large-scale instrumentation, the sensitivity is the same as that of RT-qPCR. The test strips are lightweight, small in size, and easy to carry; they are suitable for emergency detection, on-site monitoring, field sampling, or remote farms and other non-laboratory environments for rapid identification. Full article

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into various lineages. They have also the potential to protect themselves against harmful stimuli to maintain their functional integrity. Drug resistance-related transporters such as ABCB1 (P-glycoprotein; P-gp), ABCC1 (MRP1; multidrug resistance-related Protein 1), and LRP (lung resistance protein) may protect MSCs against toxic substances such as chemotherapeutic agents. This study evaluated ABCB1, ABCC1, and LRP before and after the differentiation of MSCs derived from human amniotic fluid (AF) and bone marrow (BM). P-gp expression in both AFMSCs and BMMSCs was analyzed by immunocytochemistry, and pump function was analyzed by cell viability assay with doxorubicin (DOX) and Rhodamine 123 (Rh 123) dye exclusion. ABCB1, ABCC1, and LRP gene expression was determined by RT-PCR both before and after osteogenic and adipogenic differentiation. The MES-SA/DX5 cell line was used as a model of resistance to DOX and the overexpression of P-gp. Both AFMSCs and BMMSCs displayed a high P-gp expression, although lower than MES-SA/DX5 control cells. It was shown that both, undifferentiated AFMSCs and BMMSCs, have high cell viability in response to DOX, similar to the MES-SA/DX5 lineage. ABCB1 was less expressed in BM than in AFMSCs in undifferentiated samples, while no differences were observed in the expression of ABCC1 and LRP. AFMSCs showed an increase in ABCB1 after osteogenic differentiation, whereas BMMSCs exhibited lower ABCB1 and ABCC1 expression after osteogenic and adipogenic differentiation. The findings suggest that ABCB1, ABCC1, and LRP gene expression in AFMSCs and BMMSCs is influenced by differentiation processes and further support the concept that these transporters modulate MSC differentiation in a cell source-dependent way. Full article

Background: Acute myeloid leukemia (AML) is a common and aggressive form of leukemia, yet current treatment strategies remain insufficient. Venetoclax, a BH3-mimetic approved for AML treatment, induces Bcl-2-dependent apoptosis, though its therapeutic efficacy is still limited. Therefore, new strategies to enhance the effect of venetoclax are highly sought. Valproic acid (VPA), commonly used for epilepsy, has also been studied for potential applications in AML treatment. Methods: AML cells were treated with venetoclax, with or without VPA. Cell viability was assessed using the trypan blue dye exclusion assay, while cell cycle progression was analyzed by flow cytometry. The expression of pro-apoptotic proteins Bax and Bak was measured by RT-qPCR. Results: Venetoclax and VPA individually had only mild effects on AML cell proliferation. However, their combination significantly inhibited cell growth and triggered pronounced cell death. This combination also led to the cleavage of poly (ADP-ribose) polymerase (PARP), a substrate of caspases, indicating activation of apoptosis. VPA treatment upregulated the expression of Bax and Bak, further supporting apoptosis induction. The cell death induced by the venetoclax–VPA combination was predominantly apoptotic, as confirmed by the near-complete blockade of cell death by a pan-caspase inhibitor. Conclusions: Our study demonstrates that VPA enhances venetoclax-induced apoptosis in AML cell lines, providing a novel role for VPA and suggesting a promising combinatory strategy for AML treatment. These findings offer valuable insights into potential clinical applications of venetoclax and VPA in AML management. Full article

Chalcones are phenolic compounds with biological properties. This study had the aim to evaluate the effects of topical administration of a new synthetic chalcone, Chalcone T4, in an animal model of periodontitis induced by ligature. Forty rats were distributed in the following experimental groups: negative control (without periodontitis and topical application of distilled water), positive control (periodontitis and topical application of distilled water), chalcone I and II (periodontitis and topical application of 0.6 mg/mL and 1.8 mg/mL, respectively). Chalcone or distilled water was administered into the gingival sulcus of the first molars daily for 10 days, starting with the ligature installation. The following outcomes were evaluated: alveolar bone loss (µCT and methylene blue dye staining), quantification of osteoclasts (histomorphometry), cell infiltrate and collagen content (stereometry), gene expression of mediators (Nfact11, Tnf-α, Mmp-13, iNos, Sod and Nrf2) by (RT-qPCR); expression of BCL-2 and Caspase-1 (immunohistochemistry). Chalcone T4 inhibited bone resorption and prevented collagen matrix degradation. Reduction in the expression of inflammatory markers (Nfact11, Tnf-α, Mmp-13, and Caspase-1), attenuation of oxidative stress (iNOS reduction, and increase in Sod), and pro-apoptotic effect of the compound (BCL-2 reduction), were associated its effects on periodontal tissues. Topical application of Chalcone T4 prevented bone resorption and inflammation, demonstrating potential in the adjunctive treatment of periodontitis. Full article

Radiation therapy continues to be the cornerstone treatment for malignant brain tumors, the majority of which express wild-type p53. Therefore, the identification of drugs that promote the ionizing radiation (IR)-induced activation of p53 is expected to increase the efficacy of radiation therapy for these tumors. The growth inhibitory effects of CEP-1347, a known inhibitor of MDM4 expression, on malignant brain tumor cell lines expressing wild-type p53 were examined, alone or in combination with IR, by dye exclusion and/or colony formation assays. The effects of CEP-1347 on the p53 pathway, alone or in combination with IR, were examined by RT-PCR and Western blot analyses. The combination of CEP-1347 and IR activated p53 in malignant brain tumor cells and inhibited their growth more effectively than either alone. Mechanistically, CEP-1347 and IR each reduced MDM4 expression, while their combination did not result in further decreases. CEP-1347 promoted IR-induced Chk2 phosphorylation and increased p53 expression in concert with IR in a Chk2-dependent manner. The present results show, for the first time, that CEP-1347 is capable of promoting Chk2-mediated p53 activation by IR in addition to inhibiting the expression of MDM4 and, thus, CEP-1347 has potential as a radiosensitizer for malignant brain tumors expressing wild-type p53. Full article

In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca²⁺ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca²⁺ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca²⁺ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca²⁺ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk. Full article

As a toxic xenobiotic compound, the anthraquinone dye Remazol Brilliant Blue R (RBBR) poses a serious threat to aquatic ecosystems. In the present study, the ability of Trametes hirsuta to remove RBBR from the medium was investigated, and the role of adsorption by fungal mycelium and biodegradation by fungal enzymes was evaluated. It was shown that the whole fungal culture was able to remove up to 97% of the dye within the first four hours of incubation. Based on enzymatic activities in the culture broth, laccases were proposed to be the main enzymes contributing to RBBR degradation, and RT-qPCR measurements demonstrated an increase in transcription for the two laccase genes—lacA and lacB. Composite mycelial pellets of T. hirsuta with improved adsorption ability were prepared by adding activated carbon to the growth medium, and the induction of laccase activity by carbon was shown. For composite pellets, the RBBR decolorization degree was about 1.9 times higher at 1 h of incubation compared to carbon-free pellets. Hence, it was shown that using fungal mycelium pellets containing activated carbon can be an effective and economical method of dye removal. Full article

The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method. Full article

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e.g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 μM and 100 μM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 μM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed −0.52–1.15, 0.9–1.51, and 0.31–1.69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater. Full article

Organophosphorus pesticides (OPs) are important factors in the etiology of many diseases, including obesity and type 2 diabetes mellitus. The aim of this study was to investigate the effect of a representative of OPs, chlorpyrifos (CPF), on viability, proliferation, differentiation, and fatty acid uptake in 3T3-L1 cells. The effect of CPF exposure on preadipocyte proliferation was examined by the MTT, NR, and BrdU assays. The impact of CPF exposure on the differentiation of preadipocytes into mature adipocytes was evaluated by Oil Red O staining and RT-qPCR. The effect of CPF on free fatty acid uptake in adipocytes was assessed with the fluorescent dye BODIPY. Our experiments demonstrated that exposure to CPF decreased the viability of 3T3-L1 cells; however, it was increased when the cells were exposed to low concentrations of the pesticide. Exposure to CPF inhibited the proliferation and differentiation of 3T3-L1 preadipocytes. CPF exposure resulted in decreased lipid accumulation, accompanied by down-regulation of the two key transcription factors in adipogenesis: C/EBPα and PPARγ. Exposure to CPF increased basal free fatty acid uptake in fully differentiated adipocytes but decreased this uptake when CPF was added during the differentiation process. Increased free fatty acid accumulation in fully differentiated adipocytes may suggest that CPF leads to adipocyte hypertrophy, one of the mechanisms leading to obesity, particularly in adults. It can therefore be concluded that CPF may disturb the activity of preadipocytes and adipocytes, although the role of this pesticide in the development of obesity requires further research. Full article