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Purpose: To assess the antioxidant and antineoplastic effects of Hibiscus sabdariffa Linn. on oral squamous cell carcinoma cells. Materials and Methods: Human squamous cell carcinoma HSCC cells were tested for cytotoxicity by a methanol extract of Hibiscus sabdariffa (MEHSP). After 24, 48, and 72 h, the MTT assay and Trypan blue exclusion test were used to determine cell survival and death. 2, 2-diphenyl-1-picrylhydrazyl (DPPH), DNA Protection Assay (DPA), and ferric reducing antioxidant power assay (FRAPA) measured the antioxidant activity of MEHSP. Results: The antioxidant activity (%) ranged from 47.92-82.24 in the DPPH test, 11.61-73.65 in the DPA, and 4.97-52.09 in the FRAPA. The HSCC in-vitro cytotoxicity assay showed dose- and time-dependent cell viability. MEHSP at 5 μg/ml inhibited viable cells, while increasing MEHSP doses decreased cell viability. The Trypan blue exclusion test showed that MEHSP significantly reduced cell viability at 24, 48, and 72 h. Conclusion:Hibiscus sabdariffa contains antioxidant and HSCC-cytotoxic properties. [ABSTRACT FROM AUTHOR]

The lymphatic system is a crucial contributor to allograft rejection after corneal transplantation. However, no surgical procedures for the central pathway where conjunctival lymphatic vessels converge have been investigated. Therefore, we aimed to establish a murine model of lymphatic vessel ligation and evaluate its inhibitory effect on corneal allograft rejection. A tracer was used to visualise lymphatic vessels, and complications were evaluated. A surgical technique was developed to block the lymphatic vessels. Corneas from C57BL/6 mice were transplanted into BALB/c mice divided into two groups—one with and one without lymphatic vessel ligation, to evaluate their effects on allograft rejection. Graft opacity scores were evaluated for 8 weeks, and immunohistochemistry was used to quantify angiogenesis and lymphangiogenesis. 20% trypan blue used as a tracer showed clear inflow with no complications. The two sutures and cyanoacrylate glue combination demonstrated a blocking effect after 25 days and was thus used for lymphatic ligation. Three and nine out of fourteen eyes showed rejection at 8 weeks post-surgery in the lymphatic vessel ligation and control groups, respectively. Furthermore, neovascularisation and lymphangiogenesis significantly decreased in the lymphatic vessel ligation group. Overall, we present a novel therapeutic strategy for corneal transplantation. [ABSTRACT FROM AUTHOR]

This study investigated the bioactivity of both aerial (GNAR) and underground (GNUG) parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) (Orchidaceae). The obtained data proved interesting when the samples were tested in two adrenocortical cancer cell lines (SW13 and H295R). In particular, the GNAR 80% methanol extract distinctly inhibited their viability after 24 h at a concentration of 1 µg/µL by MTT assay and trypan blue dye exclusion method. Cell morphology evaluation by means Wright's staining also showed significant results, particularly in SW13 cells under the effect of both extracts. GNAR extract was able to scavenge the DPPH radical better than GNUG extract. It also was more active in albumin denaturation (a maximum % denaturation equal to 463.0 ± 8.3 vs 77.3 ± 13.3) and protease inhibition (a maximum % inhibition equal to 138.5 ± 7.0 vs 2.1 ± 2.0) tests. The results highlighted an important antitumor activity of G. nigra in vitro that deserves to be further studied. [ABSTRACT FROM AUTHOR]

The correction notice for the article "Intracellular Delivery of DNA and Protein by a Novel Cell-Permeable Peptide Derived from DOT1L" in Biomolecules addresses errors in Figure 2A, C, Figure 3A, C, and Figure S2C. The corrections involve the duplication of images and mismatched light and fluorescence images in the original publication. The corrected figures now accurately represent the data on Dot1l peptide penetration in cultured cells under different conditions. The scientific conclusions of the paper remain unaffected by these corrections. [Extracted from the article]

Atopic eczema patients exhibit high levels of Staphylococcus aureus (S. aureus) skin colonization. S. aureus can stimulate macrophages and the expression of proinflammatory cytokines. Berberine (BBR), an alkaloid, attenuates S. aureus toxin production. This study investigated if BBR suppressed bacterial growth and inflammatory response induced by eczema-patient-derived S. aureus using murine macrophage (RAW 264.7) and human monocyte cell lines (U937). RAW 264.7 and U937 were treated with BBR at different concentrations and stimulated with heat-killed S. aureus (ATCC #33591) or S. aureus derived from severe eczema patients (EC01–EC10), who were undergoing topical steroid withdrawal, for 24 h. TNF-α protein levels were determined by ELISA, gene expression by qRT-PCR, cell cytotoxicity by trypan blue excursion, and reactive oxygen species (ROS) levels by fluorometric assay. BBR showed a bacteriostatic effect in S. aureus (ATCC strain #33591 and clinical isolates (EC01–EC10) and suppressed TNF-α production in RAW 264.7 and U937 cells exposed to heat-killed S. aureus (ATCC and clinical isolates) dose-dependently without any cell cytotoxicity. BBR (20 µg/mL) suppressed >90% of TNF-α production (p < 0.001), downregulated genes involved in inflammatory pathways, and inhibited S. aureus ROS production in U937 and RAW 264.7 cells (p < 0.01). BBR suppresses S. aureus-induced inflammation via inhibition of TNF-α release, ROS production, and expression of key genes involved in the inflammatory pathway. [ABSTRACT FROM AUTHOR]

Background: The field of immunology has undoubtedly benefited from the in vitro use of cell lines for immunological studies; however, due to the "immortal" nature of many cell lines, they are not always the best model. Thus, direct collection and culture of primary cells from model organisms is a solution that many researchers utilize. To the best of our knowledge, there is not a singular protocol which encompasses the entire process of bone marrow cell collection through cryopreservation and resuscitation of cells from a murine model. Methods: Bone marrow cells were collected from mice with a C57BL6 genetic background. Cells were differentiated using L929 conditioned media. Cells were assessed using a combination of microscopy, differential staining, immunocytochemistry, and trypan blue. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. Conclusion: Crypopreserved and resuscitated primary murine BMDMs were viable and retained their pverall S. aureus clearance ability. [ABSTRACT FROM AUTHOR]

Objective: The Amazon has a rich biodiversity where many different plant species can be found. This diversity is an important source of bioactive substances, mainly due to the different structural components of their phytometabolites. Research for natural products is a strategy for the development of new agents in therapeutic applications, especially cosmetic applications, that have better pharmacological potential. Within this perspective, the objective of the study was to investigate the cosmetic application (anti‐aging potential) of the stem‐bark extract of Bertholletia excelsa H.B.K – (SBEBE), popularly known as the Brazil nut tree, here called SBEBE, a noble plant species of the Amazon that is rich in selenium. Methods: Enzymatic, glycation, proliferation, cell‐healing, collagen quantification, toxicity and genotoxicity assays were used. Results: Among the enzymes involved in the extracellular matrix of the skin, SBEBE was able to inhibit only elastase (62.67 ± 3.75) when compared to the standard sivelestat (89.04 ± 0.53), and the extract was also able to inhibit both the oxidative and the non‐oxidative pathway. When cell toxicity in fibroblasts (MRC‐5) and keratinocytes (HACAT) was evaluated, SBEBE did not present toxicity in 24 h of incubation. After this period, the extract showed average cytotoxicity in 48 and 72 h, but not enough to reach the concentration of 50% of MRC‐5 fibroblasts. In the trypan blue assay, the extract promoted fibroblast proliferation in 24, 48 and 72 h of incubation, which was evaluated through exponential cell growth, with emphasis mainly on the lowest concentration with results higher than the standard. When the cell healing capacity was evaluated, in 48 h of exposure to fibroblast, SBEBE was able to induce a cell carpet (cell film) in the cell monolayer scratch assay. Conclusions: SBEBE stimulated collagen production at all concentrations tested. In the alkaline comet assay, at the lowest concentration, the extract did not induce DNA damage when compared to the reference drug doxorubicin. This study proved that SBEBE extract can be considered an ally in the treatment of skin anti‐ageing as a possible biotechnological, phytocosmetic product. [ABSTRACT FROM AUTHOR]

Dye-decolorizing peroxidases (DyPs) belong to a novel superfamily of heme peroxidases that can oxidize recalcitrant compounds. In the current study, the GlDyP2 gene from Ganoderma lucidum was heterologously expressed in Escherichia coli, and the enzymatic properties of the recombinant GlDyP2 protein were investigated. The GlDyP2 protein could oxidize not only the typical peroxidase substrate ABTS but also two lignin substrates, namely guaiacol and 2,6-dimethoxy phenol (DMP). For the ABTS substrate, the optimum pH and temperature of GlDyP2 were 4.0 and 35 °C, respectively. The pH stability and thermal stability of GlDyP2 were also measured; the results showed that GlDyP2 could function normally in the acidic environment, with a T50 value of 51 °C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg2+, Ni2+, Mn2+, and ethanol; 0.16 mM of Cu2+, Zn2+, methanol, isopropyl alcohol, and Na2EDTA·2H2O; and 0.016 mM of Fe2+ and SDS. The kinetic constants of recombinant GlDyP2 for oxidizing ABTS, Reactive Blue 19, guaiacol, and DMP were determined; the results showed that the recombination GlDyP2 exhibited the strongest affinity and the most remarkable catalytic efficiency towards guaiacol in the selected substrates. GlDyP2 also exhibited decolorization and detoxification capabilities towards several dyes, including Reactive Blue 19, Reactive Brilliant Blue X-BR, Reactive Black 5, Methyl Orange, Trypan Blue, and Malachite Green. In conclusion, GlDyP2 has good application potential for treating dye wastewater. [ABSTRACT FROM AUTHOR]

The repair process of periradicular tissues depends, among other factors, on the properties of endodontic cements. Macrophages are among the main cells involved in this process. Murine peritoneal macrophages obtained from C57BL/6 (MBL6) and BALB/c (MBalb) mice, respectively, were cultured with capillaries containing or not Endosequence BC Sealer (BC), Sealer Plus BC (MK), Bio-C Sealer (Ang), and mineral trioxide aggregate (MTA). Cell viability was measured by Trypan blue and MTT methods at 24, 48, and 72 hours. Cell adhesion, phagocytosis of Saccharomyces boulardii , production of reactive oxygen species (ROS), nitric oxide (NO), and the cytokines tumor necrosis factor-α and transforming growth factor (TGF)-β were also evaluated. The data were analyzed using the analysis of variance test (P <.05). Cell viability was similar between bioceramic sealers and MTA (P >.05). There was no statistical difference between both macrophages when adherence and phagocytose were assayed. The presence of inflammation stimulus significantly altered the production of ROS by MBL6 macrophages in contact with the cements. The production of TGF-β was similar for both lineages of macrophages. This study shows that the evaluated bioceramic cements do not interfere with MBL6 and MBalb macrophage adhesion, phagocytic capacity, or TGF-β production. The cements stimulated the production of ROS by MBL6 macrophages in response to induced inflammation, potentially favoring the elimination of residual pathogens. [ABSTRACT FROM AUTHOR]

Aging is a risk factor for various human disorders, including cancer. Current literature advocates that the primary principles of aging depend on the endogenous stress‐induced DNA damage caused by reactive oxygen species 50 Hz low‐frequency magnetic field was suggested to induce DNA damage and chromosomal instability. NF‐kB, activated by DNA damage, is upregulated in age‐related cancers and inhibition of NF‐kB results in aging‐related delayed pathologies. Metformin (Met), an NF‐kB inhibitor, significantly reduces both NF‐kB activation and expression in aging and cancer. This in vitro study, therefore, was set out to assess the effects of 5mT MF in 50 Hz frequency and Met treatment on the viability and proliferation of aged mouse NIH/3T3 fibroblasts and expression of RELA/p65, matrix metalloproteinases MMP2 and MMP9, and E‐cadherin (CDH1) genes. The trypan blue exclusion assay was used to determine cell viability and the BrdU incorporation assay to determine cell proliferation. The MMP‐2/9 protein analysis was carried out by immunocytochemistry, NF‐kB activity by ELISA and the expressions of targeted genes by qRT–PCR methods. Four doses of Met (500 uM, 1 mM, 2 mM and 10 mM) suppressed both the proliferation and viability of fibroblasts exposed to the MF in a dose‐dependent pattern, and the peak inhibition was recorded at the 10 mM dose. Met reduced the expression of NF‐kB, and MMP2/9, elevated CDH1 expression and suppressed NF‐kB activity. These findings suggest that Met treatment suppresses the carcinogenic potential of 50 Hz MFs in aged mouse fibroblasts, possibly through modulation of NF‐kB activation and epithelial‐mesenchymal transition modulation. [ABSTRACT FROM AUTHOR]

Chlorocresol has antibacterial and antifungal properties, yet its effectiveness in eradicating Acanthamoeba spp. remains unexplored. Acanthamoeba species trophozoites are usually sensitive to biocides, whereas cysts tend to be more resistant. This study aimed to evaluate the cysticidal activity of chlorocresol against Acanthamoeba polyphaga. Chlorocresol concentrations of 0.02, 0.04, and 0.08% were prepared and A. polyphaga cysts were incubated at room temperature (28–37 C) for 1, 24, 48, and 72 hr at each concentration. Cyst viability was evaluated using trypan blue staining and the percentage of nonviable cysts was calculated. For qualification assays, treated cysts were cultured on nonnutrient agar medium coated with Escherichia coli, incubated at 30 C, observed under a stereomicroscope for 30 days, and inoculated into peptone–yeast extract–glucose medium at 30 C for 72 hr. The results revealed that the A. polyphaga cysts were susceptible to 0.02, 0.04, and 0.08% chlorocresol. Chlorocresol made a significant difference in viability (P < 0.001) compared with the nontreated control for the same incubation time. This is the first study to examine the efficacy of chlorocresol against A. polyphaga cysts and it was highly effective. Chlorocresol could thus serve as an alternative chemical disinfectant for the eradication of A. polyphaga cysts as well as a prophylactic against transmission of other pathogenic microorganisms for which Acanthamoeba species can act as a carrier. [ABSTRACT FROM AUTHOR]

Background: Toxoplasmosis, caused by Toxoplasma gondii , poses serious health issues for humans and animals. Individuals with impaired immune systems are more susceptible to severe toxoplasmosis. Pregnant women infected by T. gondii can face the possibility of birth defects and miscarriages. While pyrimethamine and sulfadiazine are commonly used drugs in clinical practice, concerns over their side effects and resistance are on the rise. A spider peptide XYP1 isolated from Lycosa coelestis had potent anti-T. gondii effects, but it had a high synthesis cost and strong cytotoxicity. Methods: This study intended to modify XYP1 for producing derived peptides via amino acid truncation and substitution. The anti-T. gondii effect was evaluated by trypan blue staining assay and killing experiment of RH strain tachyzoites. The CCK8 and hemolysis assays were used to compare their safeties. The morphological changes of T. gondii were observed by scanning electron microscope and transmission electron microscope. In addition, the mechanism of XYP1 against T. gondii through RNA-sequencing was further explored. Results: In vivo and in vitro experiments revealed that XYP1-18 and XYP1-18-1 had excellent anti-T. gondii activity with lower cytotoxicity and hemolysis activity than XYP1. XYP1, XYP1-18, and XYP1-18-1 were able to disrupt the surface membrane integrity of T. gondii tachyzoites, forming pores and causing the disruption of organelles. Furthermore, RNA-sequencing analysis indicated that XYP1 could stimulate the host immune response to effectively eliminate T. gondii and lessen the host's inflammatory reaction. Conclusions: XYP1-18 had lower cytotoxicity and hemolysis activity than XYP1, as well as significantly extending the survival time of the mice. XYP1 played a role in host inflammation and immune responses, revealing its potential mechanism. Our research provided valuable insights into the development and application of peptide-based drugs, offering novel strategies and directions for treating toxoplasmosis. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC), a major oral cancer, significantly challenges treatments and impacts patient quality of life; current therapies often cause severe side effects, highlighting the urgent need for gentler alternatives. Rice stands as one of the primary cereal grains providing the daily caloric intake for more than half of the global population. Extensive research has demonstrated the significant health benefits derived from rice, attributed to its abundance of bioactive compounds. This study endeavours to explore the potential cytotoxic effects of Bajong LN rice, a pigmented purple rice indigenous to Sarawak, on human squamous cell carcinoma, ORL-48 cells. Cells were cultured in complete DMEM/F-12 media and incubated under standard culture conditions. Upon reaching 80% confluency, the cells were treated to varied concentrations (ranging from 0 µg/ml to 2000 µg/ml) of Bajong LN rice methanol extract (BLN-ME) and cisplatin. Subsequently, the cells were incubated for 48 and 72 hours, and their cytotoxicity was assessed using the MTS assay. Results demonstrated that cisplatin inhibited ORL-48 cells with an IC50 of 7.483 µg/ml and 3.877 µg/ml; and an IC80 of 40.649 µg/ml; and 17.543 pg/ml for 48 and 72 hours, respectively. Correspondingly, BLN-ME exhibited a notable cytotoxic effect against ORL-48 cells at 48- and 72-hour intervals, with an IC50 of 354.4 µg/ml and 342.0 µg/ml; and an IC80 of 450.3 µg/ml and 423.63 µg/ml, respectively. The cytotoxic activity of BLN-ME against ORL-48 cells was observed in both a time and dose-dependent manner. Morphological analysis and the Trypan blue exclusion assay corroborated the MTS assay's findings. Our preliminary findings provide the first scientific evidence of the cytotoxic effect of BLN-ME specifically against human squamous cell carcinoma, ORL-48 cells. This study suggests the potential of BLN-ME as a promising anti-cancer agent, presenting opportunities for further investigation into its underlying cytotoxic mechanisms. [ABSTRACT FROM AUTHOR]

Background: Transfusion‐related acute lung injury (TRALI) remains a major contributor to transfusion‐associated mortality. While the pathogenesis of TRALI remains unclear, there is evidence of a role for blood components. We therefore investigated the potential effects of fresh frozen plasma (FFP), cryoprecipitate, and extracellular vesicles (EVs) derived from these blood components, on the viability of human lung microvascular endothelial cells (HLMVECs) in vitro. Methods: EVs were isolated from FFP and cryoprecipitate using size‐exclusion chromatography and characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscopy. The potential effects of these blood components and their EVs on HLMVEC viability (determined by trypan blue exclusion) were examined in the presence and absence of neutrophils, either with or without prior treatment of HLMVECs with LPS. Results: EVs isolated from FFP and cryoprecipitate displayed morphological and biochemical properties conforming to latest international criteria. While FFP, cryoprecipitate, and EVs derived from FFP, each reduced HLMVEC viability, no effect was observed for EVs derived from cryoprecipitate. Conclusion: Our findings demonstrate clear differences in the effects of FFP, cryoprecipitate, and their respective EVs on HLMVEC viability in vitro. Examination of the mechanisms underlying these differences may lead to an improved understanding of the factors that promote development of TRALI. [ABSTRACT FROM AUTHOR]

Cervical and ovarian cancers account for the majority of deaths among women. Due to the failure of conventional therapies to completely cure these diseases, research has shifted focus to traditional and plant-based medicines. This study aimed to assess the potential anticancer activity of tamarind leaf extract on ovarian cancer PA-1 and cervical cancer HeLa cell lines. The extract was prepared using Soxhlet equipment with ethanol as the solvent and fractionated using Thin Layer Chromatography (TLC) under bioassay guidance. Anticancer activity was determined through in vitro tests, including MTT assay, fluorescence microscopy, trypan blue assay, LDH activity assay, caspase-9 activity assay, analysis of DNA fragmentation, Annexin V/PI staining and flow cytometry. Bioactive compound characterization was performed using gas chromatography-mass spectrometry (GC-MS) followed by NIST library search, to understand the underlying principles behind the anticancer attributes of tamarind leaf. The results revealed that treatment with tamarind leaf ethanol extract drastically reduced HeLa and PA-1 cell viabilities to 26.8% and 11.9%, respectively, after 72 hours. The yellow fraction (TLYF) of the extract was identified as the bioactive component through bioassay-guided fractionation. Trypan blue assay results showed cell concentrations reduced to 1.3x105 and 1.2x105 cells/mL compared to about 3.6x105 cells/mL in control HeLa and PA-1 cells. Cell cycle analysis demonstrated that TLYF reduced overall cell count, with a significant decrease in S and G2/M phases, and increased percentage of cells (50.9 and 18.8%) in early and late apoptotic stages as per annexin V/PI staining. Caspase assay confirmed apoptosis initiation through caspase-9 activation in cancer cells induced by TLYF. According to GC-MS data, apigenin, an anticancer compound reported in several fruits, was found in tamarind leaf. It can be assumed that apigenin in tamarind leaf might be the reason for its profound antiproliferative effects against cancer cells. [ABSTRACT FROM AUTHOR]

Sangre de drago, the sap of Croton lechleri Müll. Arg. tree, has been used for centuries in traditional medicine owing to its diverse biological activities. Extracts derived from different parts of the species contain a multitude of phytochemicals with varied applications. Twigs, however, are among the least studied parts of the plant. This study unveils new biological activities of Croton lechleri twig extracts recovered by applying Soxhlet and advanced green techniques. For all extracts, total phenolic content and antioxidant activity were determined. Subsequently, four were selected, and their cytotoxic effects were assessed on both normal (HaCat) and malignant melanoma (A375) skin cell lines using the MTT assay and trypan blue exclusion assay. All showed dose-dependent cytotoxicity, with the Soxhlet ethanol extract demonstrating the highest selectivity towards A375 cells over HaCat cells. The extracts induced apoptosis and necrosis, as confirmed by Annexin V/PI dual-labeling and flow cytometry, highlighting their ability to trigger programmed cell death in cancer cells. The selective inhibition of cell cycle progression in A375 compared to HaCat observed both for Soxhlet ethanol and pressurized ethanol extracts induces cell cycle arrest at multiple points, primarily in the G1 and G2/M phases, and significantly reduces DNA synthesis as evidenced by the decrease in the S-phase population, confirmed by the EdU assay. Consequently, the Soxhlet extract composition was analyzed using LC-MS, which revealed their richness in polyphenolic compounds, particularly flavonoids from the flavonol subclass. [ABSTRACT FROM AUTHOR]

Bladder cancer (BCa) is one of the most lethal genitourinary malignancies owing to its propensity for recurrence and poor survival. The biochemical pathway, signal transducer and activator of transcription 3 (STAT3), has gained significance as a molecular pathway that promotes proliferation, invasion, and chemoresistance. In this study, we explored the targeting of STAT3 with TTI-101 and SH5-07 in BCa and elucidated the mechanisms in three-dimensional (3D) spheroid and organoid models. We optimized the growth of spheroids from human, rat, and mouse BCa cell lines (J82, NBT-II, and MB49 respectively) and organoids from BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine)-induced rat bladder tumors. Cell viability was assessed using MTT and trypan blue assays. Intracellular ATP production, ROS production, and calcium AM (CA)/EtBr staining were used to measure the spheroid and organoid inhibition and mitochondrial function. Western blot analysis was performed to evaluate the pharmacodynamic markers involved in cell proliferation, apoptosis, cancer stem cells (CSCs), and STAT3 signaling in BCa. We found that targeting STAT3 (using TTI-101 and SH5-07) significantly reduced the proliferation of BCa spheroids and organoids, which was accompanied by decreased expression of pSTAT3, Cyclin D1, and PCNA. Our data also demonstrated that treatment with STAT3 inhibitors induced ROS production and cell death in BCa spheroids and organoids. STAT3 inhibition-induced cell death was associated with the activation of caspase 3/7 and PARP cleavage. Moreover, TTI-101 and SH5-07 target cancer stem cells by downregulating the expression of CD44 and CD133 in 3D models. This study provides the first evidence for the prevention of BCa with small-molecule inhibitors TTI-101 and SH5-07 via suppression of CSCs and STAT3 signaling. [ABSTRACT FROM AUTHOR]

Aims: To investigate the effects of the selective serotonin reuptake inhibitors (SSRIs) sertraline and paroxetine at therapeutically relevant concentrations on beta‐cell mass and function. Methods: Viability was quantified in mouse insulinoma (MIN6) beta cells and mouse islets after 48‐h exposure to sertraline (1–10 μM) or paroxetine (0.01–1 μM) using the Trypan blue exclusion test. The effects of therapeutic concentrations of these SSRIs on insulin secretion were determined by static incubation and perifusion experiments, while islet apoptosis was investigated by Caspase‐Glo 3/7 assay, TUNEL staining and quantitative PCR analysis. Finally, proliferation of MIN6 and mouse islet beta cells was assessed by bromodeoxyuridine (BrdU) enzyme‐linked immunosorbent assay and immunofluorescence. Results: Sertraline (0.1–1 μM) and paroxetine (0.01–0.1 μM) were well tolerated by MIN6 beta cells and islets, whereas 10 μM sertraline and 1 μM paroxetine were cytotoxic. Exposure to 1 μM sertraline and 0.1 μM paroxetine significantly potentiated glucose‐stimulated insulin secretion from mouse and human islets. Moreover, they showed protective effects against cytokine‐ and palmitate‐induced apoptosis of islets, they downregulated cytokine‐induced Stat1 and Traf1 mRNA expression, and they significantly increased proliferation of mouse beta cells. Conclusions: Our data demonstrate that sertraline and paroxetine act directly on beta cells to enhance glucose‐stimulated insulin secretion and stimulate beta‐cell mass expansion by increasing proliferation and decreasing apoptosis. These drugs are therefore likely to be appropriate for treating depression in people with type 2 diabetes. [ABSTRACT FROM AUTHOR]

Reliable monitoring of mammalian cells in bioreactors is essential to biopharmaceutical production. Trypan blue exclusion is a method of determining cell density and viability that has been used for over one hundred years to monitor cells in culture and is the current standard method in biomanufacturing. This method has many disadvantages however and there is a growing demand for more detailed and in‐line measurements of cell growth in bioreactors. This article assesses a novel dynamic imaging system for single cell analysis. This data shows that comparable total cell density, viable cell density and percentage viability data shown here, generated by the imaging system, aligned well with conventional trypan blue counting methods for an industrially relevant Chinese Hamster Ovary (CHO) cell line. Furthermore, detailed statistical analysis shows that the classification system used by the PharmaFlow system can reveal trends of interest in monitoring the health of mammalian cells over a 6‐day bioreactor culture. The system is also capable of sampling at‐line, removing the necessity for taking samples off‐line and enabling real time monitoring of cells in a bioreactor culture. [ABSTRACT FROM AUTHOR]

Introduction: The purpose of this study was to evaluate whether vital blue dyes could enhance the contrast of intraoperative optical coherence tomography (OCT) during macular surgery. Methods: Consecutive patients undergoing elective pars plana vitrectomy for vitreomacular interface disorders were enrolled. Intraoperative OCT was performed with the Artevo 800 microscope (Carl Zeiss Meditec AG, Jena, Germany) before and after injection of 0.2 mL of Trypan Blue and Brilliant Blue G Ophthalmic Solution. The OCT contrast ratio was measured with ImageJ, while the overall scan quality was subjectively classified using a 4-point scale. Results: Ten eyes of 10 patients were enrolled in the study. The OCT contrast ratio was 9.39 ± 5.35 without blue dye and significantly improved to 14.31 ± 10.50 after blue dye injection (p = 0.027). The percentage of patients with a grade 4 scan quality also significantly improved (from 40% without blue dye to 90% with blue dye injection; p = 0.012). Conclusion: The use of blue dyes during intraoperative OCT is an effective strategy for improving contrast and scan quality without affecting the surgical time and workflow. [ABSTRACT FROM AUTHOR]

Aims: Multiple myeloma (MM) remains a challenging condition to cure, with persistent drug resistance negating the benefits of treatment advancements. The unraveling complexities in programmed cell death (PCD), inclusive of apoptosis, autophagy, and ferroptosis, have highlighted novel therapeutic avenues. Our study focuses on deciphering how adapalene (ADA), a small molecule compound, accelerates the demise of MM cells via targeting their compensatory survival mechanisms. Methods: To assess the impact of ADA on MM, we employed flow cytometry and trypan blue exclusion assays to determine cell viabilities across MM cell lines and primary patient samples post-treatment. To delineate ADA's therapeutic targets and mechanisms, we conducted RNA sequencing (RNA-seq), gene set enrichment analysis (GSEA), molecular docking, and molecular dynamics simulations. We further designed pre-clinical trials emphasizing MM, exploring the efficacy of ADA as a standalone and in combination with bortezomib (BTZ). Results: ADA elicited a dose-responsive induction of MM cell death. Building upon ADA's anti-MM capabilities as a single agent, we proposed that ADA-BTZ co-treatment might amplify this lethality. Indeed, ADA and BTZ together greatly potentiated MM cell death. ADA proved beneficial in restoring BTZ susceptibility in BTZ-resistant relapsed or refractory MM (RRMM) patient cells. Molecular simulations highlighted ADA's high affinity (-9.17 kcal/mol) for CD138, with MM-GBSA revealing a binding free energy of -27.39 kcal/mol. Detailed interaction analyses indicated hydrogen-bonding of ADA with CD138 at the Asp35 and Gln34 residues. Additionally, ADA emerged as a versatile instigator of both ferroptosis and apoptosis in MM cells. Furthermore, ADA disrupted activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway triggered by BTZ, fostering cell death in BTZ-resistant MM subsets. Conclusion: ADA demonstrates a comprehensive capability to orchestrate MM cell death, exerting pronounced anti-MM activity while disrupting NF-κB-related drug resistance. ADA sensitization of MM cells to BTZ unravels its potential as a novel therapeutic drug for MM management. [ABSTRACT FROM AUTHOR]

Introduction: Human trophoblastic cell lines, such as BeWo, are commonly used in 2Dmodels to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. Methods: We developed a 3D culturemodel using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81)models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring b-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-a gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Results: Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 mm observed at 5 days. Functionality, assessed through b-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. Discussion: This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment. [ABSTRACT FROM AUTHOR]

Tracking cell death in vivo can enable a better understanding of the biological mechanisms underlying tissue homeostasis and disease. Unfortunately, existing cell death labeling methods lack compatibility with in vivo applications or suffer from low sensitivity, poor tissue penetration, and limited temporal resolution. Here, we fluorescently labeled dead cells in vivo with Trypan Blue (TBlue) to detect single scattered dead cells or to generate whole-mount three-dimensional maps of large areas of necrotic tissue during organ regeneration. TBlue effectively marked different types of cell death, including necrosis induced by CCl4 intoxication in the liver, necrosis caused by ischemia-reperfusion in the skin, and apoptosis triggered by BAX overexpression in hepatocytes. Moreover, due to its short circulating lifespan in blood, TBlue labeling allowed in vivo "pulse and chase" tracking of two temporally spaced populations of dying hepatocytes in regenerating mouse livers. Additionally, upon treatment with cisplatin, TBlue labeled dead cancer cells in livers with cholangiocarcinoma and dead thymocytes due to chemotherapy-induced toxicity, showcasing its utility in assessing anticancer therapies in preclinical models. Thus, TBlue is a sensitive and selective cell death marker for in vivo applications, facilitating the understanding of the fundamental role of cell death in normal biological processes and its implications in disease. [ABSTRACT FROM AUTHOR]

Developing of functional foods is a promising strategy to reduce the increasing burden of colorectal cancer worldwide. Fruit pomace, particularly polyphenol and anthocyanin-rich chokeberry and blueberry, is a valuable ingredient for functional foods and nutraceuticals. Our study aimed to evaluate the anti-inflammatory and antiproliferative effects of chokeberry and blueberry pomace extracts on C2BBe1 colorectal carcinoma cells and explore the underlying signaling pathways. We analyzed both pomace extracts for total polyphenols and anthocyanins using Folin-Ciocalteu method and ultra-performance liquid chromatography, while antioxidative activity was assessed via the 2,2-diphenyl-1-picrylhydrazyl radical scavenging method. We evaluated the in vitro anti-inflammatory and antiproliferative effects using trypan blue exclusion, MTT and LDH assays, and assessed protein levels of p-Erk1/2, Akt-1, STAT1, STAT3, occludin, oxidized proteins, and MDA-protein adducts through western blotting, as well as analysis of a 37-plex panel of inflammatory markers. Chokeberry extracts exhibited higher total polyphenol content, anthocyanin levels, and antioxidative activity compared to blueberry extracts, however, blueberry extracts effects on cell viability and proliferation in C2BBe1 cells were stronger. Both fruit pomaces induced non-inflammatory cell death characterized by membrane integrity loss, beneficial in cancer therapy. Our data suggests chokeberry's cytotoxicity may be mediated by Erk signaling and Akt-1 inhibition, while blueberry uniquely decreased occludin levels. These berries pomaces' potential to mitigate cancer risks and enhance treatment efficacy is promising, warranting further investigation for functional foods development. [ABSTRACT FROM AUTHOR]

For the first time, this study shows the nanoarchitectonic process to obtain an acetogenin-enriched nanosystem (AuNPs-Ac) using an aqueous extract from Annona cherimola Mill (ACM) composed of gold nanoparticles embedded in an organic matrix that acts as stabilizing agent and presents anti-inflammatory activity and cytotoxical effect against HepG2 cell line, promoting apoptosis. The synthesis of AuNPs-Ac was confirmed by x-ray diffraction analysis, showing metallic gold as the only phase, and the scanning transmission microscope showed an organic cap covering the AuNPs-Ac. Fourier-transformed infrared suggests that the organic cap comprises a combination of different annonaceous acetogenins, alkaloids, and phenols by the presence of bands corresponding to aromatic rings and hydroxyl groups. High-Performance Liquid Chromatography has demonstrated the presence of annonacin, a potent acetogenin, in the extract of ACM. An in vitro anti-inflammatory activity of the extract of ACM and the AuNPs-Ac was performed using the albumin denaturation method, showing a nonlinear response, which is better than sodium diclofenac salt in a wide range of concentrations that goes from 200 to 400 μ g ml−1 with both samples. The viability assay was studied using trypan blue, treating IMR90 and HepG2 at different concentrations of AuNPs-Ac. The results defined a median lethal dose of 800 μ g ml−1 against HepG2 through apoptosis according to the ratio of caspase-cleaved 9/alpha-tubulin evaluated. It was also demonstrated that the nanosystem presents a higher cytotoxic effect on the HepG2 cell line than in IMR90, suggesting a targeted mechanism. In addition, the nanosystem performs better than using only the extract of ACM in the anti-inflammatory or antiproliferative test, attributed to their higher surface area. [ABSTRACT FROM AUTHOR]

N,N-dibenzyl dithiocarbamate silver(I) was synthesized and characterized by spectroscopic techniques and single crystal X-ray crystallography. The complex crystallizes in the trigonal space group P31 to form a one-dimensional, chain-like polymeric structure connected by Ag-Ag argentophilic and Ag-S interactions. The complex was used as single source precursor and thermolyzed at 120, 160 and 200 °C in 1-dodecanethiol to prepare silver sulfide nanoparticles (AgS-120, AgS-160 and AgS-200). TEM images showed quasi-spherical nanoparticles with average particle sizes of 4.0, 5.4 and 5.5 nm for nanoparticles prepared at 120, 160 and 200 °C, respectively, with optical band gaps of 3.76, 3.81 and 3.71 eV. The as-prepared silver sulfide nanoparticles potential as photocatalysts were evaluated using rhodamine 6G and trypan blue under irradiation. The photocatalytic degradation efficiency of 42.4% (k = 3.03 × 10−3), 41.9% (k = 2.73 × 10−3) and 56.7% (k = 4.16 × 10−3) for rhodamine 6G dye and 35.4% (2.94 × 10−3), 49.7% (3.87 × 10−3) and 16.9% (9.0 × 10−4) for trypan blue dye were obtained in the presence of AgS-120, AgS-160 and AgS-200, respectively. The kinetics and mechanism of photocatalytic reaction show that the dye degradation in the presence of the as-prepared nanoparticles follow pseudo-first order kinetics. [ABSTRACT FROM AUTHOR]

The synthesis of silver nanoparticles (AgNPs) using environmentally friendly methods has become increasingly important due to its sustainability and cost-effectiveness. This study investigates the green synthesis of AgNPs using gall extracts from the plant Ficus recemosa, known for its high phytochemical content. The formation of AgNPs was verified through multiple analytical techniques, including UV-Vis spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), zeta potential analysis, and dynamic light scattering (DLS). The UV-Vis spectroscopy results displayed a distinct surface plasmon resonance peak indicative of AgNP formation. FTIR analysis revealed specific interactions between silver ions and phytochemicals in the gall extract, while TEM images confirmed the nanoscale morphology and size of the synthesized particles. Zeta potential and DLS analyses provided insights into the stability and size distribution of the AgNPs, demonstrating good colloidal stability. Biological properties of the AgNPs were assessed through various assays. Antimicrobial activity was tested using the disc diffusion method against Escherichia coli and Staphylococcus aureus, showing significant inhibitory effects. The anticancer potential was evaluated using the trypan blue exclusion assay on Dalton's Lymphoma Ascites (DLA) cells, revealing considerable cytotoxicity. Additionally, antimitotic activity was studied in the dividing root cells of Allium cepa, where the AgNPs significantly inhibited cell division. This research highlights the effective use of F. recemosa gall extracts for the green synthesis of AgNPs, presenting an eco-friendly approach to producing nanoparticles with strong antimicrobial, anticancer, and antimitotic properties. The promising results suggest potential applications of these biogenic AgNPs in medical and agricultural sectors, paving the way for further exploration and utilization. [ABSTRACT FROM AUTHOR]

Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss. [ABSTRACT FROM AUTHOR]

To evaluate the clinical implications of the different trypan blue dyeing techniques used during liquid bubble (LBT) and manual peel (MPT) DMEK lenticule preparation techniques. This study retrospectively compared the degree to which endothelial cells are preserved using selective Descemet Membrane (DM) staining (LBT) versus bath-staining (MPT) when performed by a single surgeon, sourced from a single eye bank. Endothelial cell density measured after the 3-month follow-up was 1805 and 1916 cells/mm2 respectively, differing significantly (p = 0.012). A double-scroll graft formation was found and maintained until implantation in 94% of preparations with bath staining and 50% of preparations using selective DM staining. Preoperative visual acuity was comparable between preparation techniques at 0.4 logMAR as well as postoperatively, at an average of 0.1 logMAR. Reducing chemical stress on the endothelium by avoiding any contact with trypan blue allows for a significantly higher degree of cell preservation. However, achieving the often-desired double-scroll graft formation was possible less frequently. It remains unclear which factors define the differences graft scrolling behavior observed between LBT and MPT. [ABSTRACT FROM AUTHOR]

Purpose Methods Results Conclusions To evaluate the use of vital dyes and light microscopy for assessing canine corneal endothelial morphology ex vivo.The corneas of 40 canine eyes (n = 20 dogs) enucleated <24 h following euthanasia or death were isolated and flat‐mounted on a slide. Corneal endothelium was stained via 0.25% trypan blue followed by 0.5% alizarin red (pH 4.2), photographed, then the following morphological features were calculated using ImageJ: mean cell density (MCD), mean cell area (MCA), polymegathism (coefficient of variation of cell area), and pleomorphism (% hexagonality).Mean ± standard deviation (range) outcomes were: MCD, 2544 ± 541 cells/mm2 (1750–3922 cells/mm2); MCA, 431 ± 97 μm2 (251–626 μm2); polymegathism, 17 ± 2% (14%–22%); pleomorphism, 84 ± 3% (80%–90%). No significant differences (p ≥ .122) were noted for any outcome between male versus female or brachycephalic versus non‐brachycephalic dogs. Young dogs (<10 years) had lower MCA (p = .044), lower pleomorphism (p = .003), and higher MCD (p = .035) when compared to older dogs (≥10 years). Age was significantly (p ≤ .049) correlated with MCA (r = 0.467), MCD (r = −0.476), polymegathism (r = 0.444), and pleomorphism (r = 0.609).The combination of vital dyes and light microscopy allowed for clear visualization and evaluation of the corneal endothelium in canine eyes ex vivo. Our findings can be used in future studies to deepen our understanding of the corneal endothelium in health and disease. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to evaluate the efficacy and safety of McCarey-Kaufman (MK) medium supplemented with Colistin and amphotericin B in inhibiting the growth of multidrug-resistant Pseudomonas (P.) aeruginosa, using an ex vivo experimental model with human donor corneas. Methods: Cadaveric human corneas deemed unsuitable for corneal transplantation were obtained, and MK media were supplemented with colistin and amphotericin B. Multidrug-resistant P. aeruginosa was cultured and used to infect the human donor corneas ex vivo. Infected corneas were placed in the MK media with additional antibiotics (colistin and amphotericin B) and the standard MK media, which served as the control arm for comparison. Corneal opacity due to infiltration and quantitative analysis of colony-forming units (CFUs) were assessed. The viability of the corneal endothelium was assessed using trypan blue staining. Results: Corneas incubated in MK media supplemented with additional antibiotics showed less corneal opacification compared with those in standard MK media at both 48- and 96-hour (hr) time points. Quantitative analysis revealed a lower bacterial load and a significant reduction in CFU in the corneas incubated in MK media with additional antibiotics compared with the control group. At 48 hrs, there was 84% (P value = 0.024) reduction in bacterial load, and at 96 hr, a 53% (P value = 0.016) reduction was observed in comparison with those placed in standard MK media. The trypan blue staining tests revealed that the extent of endothelial cell loss in corneas incubated in supplemented MK media was comparable to the ones in standard MK media. Conclusion: The addition of colistin and amphotericin B to MK media demonstrated efficacy in inhibiting the growth of multidrug-resistant P. aeruginosa in an ex vivo cornea infection model. The supplemented media had no detrimental effect on the corneal endothelium. The findings suggest that supplementing the MK media with these broad-spectrum antimicrobial agents may help mitigate the risk of postoperative donor-related infection in the recipients by reducing and containing the load of microbial contamination in donor corneas. [ABSTRACT FROM AUTHOR]

Purpose: We describe an alternative automated technique that consists of simultaneous anterior capsule puncture and decompression of the capsular bag by using an insulin needle attached to the aspiration tubing of the phacoemulsification device to prevent the occurrence of the "Argentinian Flag sign" during capsulorhexis in intumescent cataract. Setting: Instituto de Olhos de Assis and Center of Specialties Hoftalmed, located in the state of São Paulo, Brazil. Design: Prospective interventional study. Methods: Eighty-eight eyes of 88 patients with white or intumescent cataracts were included in this study. Routine clear cornea incision, capsule staining with trypan blue, intracameral anesthesia, and ophthalmic viscoelastic device were used before the procedure. A 26-gauge needle was connected to the phacoemulsification aspiration tubing using a double male Luer connector for irrigation, and aspiration was inserted into the anterior chamber through a new paracentesis incision with the bevel facing down. Immediately after insertion, automated aspiration of the liquefied cortex was performed to remove anterior intralenticular material and achieve capsular decompression. Compression of the nucleus with the needle tip was performed to remove any liquefied material trapped between the posterior surface of the nucleus and the posterior capsule. All surgeries were performed using the same phacoemulsification and parameters. The rate of complete continuous capsulorhexis was observed and noted. Results: No complications were observed in any of the cases. A single-stage, continuous, and well-centered capsulorhexis was achieved in 100% of cases. Conclusion: We conclude that a simultaneous puncture and decompression of the capsular bag using an insulin needle attached to the aspiration tubing of the phacoemulsification machine effectively avoided the "Argentinian Flag sign" in intumescent cataract surgery. [ABSTRACT FROM AUTHOR]

Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 µM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy. NEW & NOTEWORTHY: Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells. [ABSTRACT FROM AUTHOR]

The high objectives to this study were to determine and evaluate incidence of Sarcocystis fusiform infection in slaughtered buffaloes in Assiut abattoir, and additionally study the effect of chilling on their viability and infectivity. Tissue specimens were collected from three hundred and fifty-six buffaloes (337 male buffaloes and 19 female buffaloes) slaughtered at Assiut abattoir during the period from 2/2022 to 1/2023. Samples including esophagus, tongue, and masseter muscles were examined macroscopically and histopathologically. The total prevalence of macroscopic Sarcocystis (S. fusiform) in examined buffaloes was 8.43%, the infection rate in males (Less than 2 years) and in females buffaloes (4 to 7 years old) were (8.3%) and (10.53%), respectively. The predilection seats of S. fusiform in examined buffaloes were; esophagus (90%), followed by tongue (23.3%), throat muscles (20%) and the lowest one skeletal muscle (total body) (3.3%). Concerning seasonal variation the high infection rate was happened and detected in spring (13.9%) while the lowest infection was happened in the autumn season (5.4%). The effect of chilling on the viability of S. fusiform cysts was investigated using a vital stain (0.4% trypan blue). Heavily infected muscles were chilled at 4°C for 24, 48 and 72h and was carried out on cats. The reduction rate of S. fusiformis bradyzoites after chilling was (0%), (20%) and (54.6%), respectively. However, the parasite lost its infectivity after chilling for three days, where heavily infected muscles cooling at 4°C were rendered non-infective to cats. Conclusion: The present study concluded that S. fusiformis infection in buffaloes constitutes one of the main causes of economic losses in Assiut slaughterhouses. The incidence of S. fusiform was higher in aged female buffaloes and esophagus was the main predilection seat. Additionally, our results showed that cooling heavily infected muscles with S. fusiformis at 4 °C leads to a significant reduction that touched as a fact in parasite viability and trypan blue stain effectively measured the viability of Sarcocystis. Also, the infectivity of the parasite to cats was lost after chilling for three days. [ABSTRACT FROM AUTHOR]

Background: Sarcocystis is a food‐borne zoonotic protozoan whose final hosts are humans, dogs, cats, and other carnivores and intermediate hosts are birds and mammals, especially humans and herbivores. Humans become infected by eating raw and undercooked meat contaminated with bradyzoites or by consuming water or food contaminated with the sporocyst stage of the parasite. Objectives: The aim of this study was to investigate the effects of gamma radiation and electron beam on the survival rate of Sarcocystis bradyzoites in infected beef and to determine the effective dose. Methods: Three replicates of 100 g of infected meat were treated with different doses (0.5, 1, 1.5 and 2 kGy). As a control, 20 g of contaminated meat was stored separately at 4°C. The viability of the bradyzoites after digestion in pepsin solution was assessed, stained (trypan blue) and unstained, under a stereomicroscope. To assess survival of the bradyzoites, the irradiated meat samples were fed to 30 dogs. After 10 days, faecal samples were examined for sporocysts. Results: The results showed that the highest and lowest mortality rate of Sarcocystis bradyzoites in infected organs using electron beam at a dose of 2 kGy were 92.5% and 100%, respectively, and the lowest mortality rate at a dose of 0.5 kGy were 2.5% and 7.89%, respectively. Conclusion: The results of statistical analysis showed that the mortality rate of Sarcocystis bradyzoites was significant between different doses of gamma ray and electron beam, so that gamma rays were better compared to electron beam in destroying Sarcocystis bradyzoites. [ABSTRACT FROM AUTHOR]

Introduction: Post-ovulatory aging is a time-dependent deterioration of ovulated oocytes and a major limiting factor reducing the fitness of offspring. This process may lead to the activation of cell death pathways like apoptosis in oocytes. Methodology: We evaluated oocyte membrane integrity, egg developmental competency, and mRNA abundance of apoptosis-related genes by RT-qPCR. Oocytes from zebrafish Danio rerio were retained in vivo at 28.5°C for 24 h post-ovulation (HPO). Viability was assessed using trypan blue (TB) staining. The consequences of in vivo oocyte aging on the developmental competence of progeny were determined by the embryo survival at 24 h post fertilization, hatching, and larval malformation rates. Results: The fertilization, oocyte viability, and hatching rates were 91, 97, and 65% at 0 HPO and dropped to 62, 90, and 22% at 4 HPO, respectively. The fertilizing ability was reduced to 2% at 8 HPO, while 72% of oocytes had still intact plasma membranes. Among the apoptotic genes bcl-2 (b-cell lymphoma 2), bada (bcl2- associated agonist of cell death a), cathepsin D, cathepsin Z, caspase 6a, caspase 7, caspase 8, caspase 9, apaf1, tp53 (tumor protein p53), cdk1 (cyclin-dependent kinase 1) studied, mRNA abundance of anti-apoptotic bcl-2 decreased and proapoptotic cathepsin D increased at 24 HPO. Furthermore, tp53 and cdk1 mRNA transcripts decreased at 24 HPO compared to 0 HPO. Discussion: Thus, TB staining did not detect the loss of oocyte competency if caused by aging. TB staining, however, could be used as a simple and rapid method to evaluate the quality of zebrafish oocytes before fertilization. Taken together, our results indicate the activation of cell death pathways in the advanced stages of oocyte aging in zebrafish. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N‐methyltransferase (NNMT) catalyses the N‐methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. Methods: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. Results: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti‐cancer therapy. [ABSTRACT FROM AUTHOR]

Complex nutrient media are used for the germination of orchids in the laboratory. These substrates are also suitable for the growth of microbes and thus the sterilization of both the medium and the seeds prior to sowing is mandatory. The surface disinfection of orchid seeds with chemical substances eliminates possible infections and enhances germination in several cases, by chemically scarifying their seed coats. However, long durations of chemical treatments may result in viability loss, and consequently, precisely determining the optimal treatment duration is essential for developing effective germination protocols. In this study, the double staining technique with Tetrazolium Chloride and Trypan Blue is tested as a proxy method to determine the optimal chemical treatment duration for 10 orchid taxa of Greece. Various durations of chemical treatment with 5% Ca(OCl)2 were carried out on independent seed samples which were subsequently tested, pairwise, for both germination and viability. The results in the corresponding experiments are extraordinarily similar and we conclude that the double viability staining is a quick and accurate technique in determining the permeability and optimal duration for chemical scarification prior to germination experiments. The storability potential of the taxa studied is discussed in comparison with previous data of the NKUA Seed Bank. [ABSTRACT FROM AUTHOR]

On a global basis, colorectal cancer is one of the foremost causes of cancer-related death rate. The present research encompasses the fabrication of CuO nanoparticles employing leaf extract of Coleus amboinicus (CA–CuO NPs) and evaluating the anti-cancerous capability against HCT 116 cells (Colon cancer). The fabricated CA–CuO NPs were assessed by UV–Vis. spectrophotometer, FT-IR, SEM, EDAX and DLS. The cytotoxic action upon the HCT 116 cells was evaluated using MTT, trypan blue exclusion assay, phase contrast microscopy, DAPI, and MMP Staining. The oxidative stress markers such as nitric oxide (NO), lipid peroxidation (LPO), and reactive oxygen species (ROS) levels were analyzed. The antioxidant markers including catalase (CAT) and superoxide dismutase (SOD) were further analyzed. Using RT-PCR, the expression levels of genes including Bax, p53, and Bcl-2 were analyzed. The peak at 225 nm in the UV spectrum disclosed the formation of CA–CuO NPs. The possible existence of phytoconstituents responsible for reducing CuO ions was confirmed by FT-IR results. The SEM images showed the formation of irregular rice-shaped and elongated tetragonal-shaped CuO nanoparticles. The EDAX results confirm the existence of C, Cu, O and S peaks. The DLS findings revealed average particle size of CA–CuO NPs was 118.6 nm. The MTT assay demonstrated a dose-dependent, substantial suppression of HCT 116 cell growth, with an IC50 of 12.5 µg/mL indicating 49.71% inhibition. The proportion of viable cells in the trypan blue exclusion assay steadily dropped as treated cells suffered morphological deformity as observed by phase contrast microscopy. The DAPI and MMP staining revealed an increase in dead cells in a dose-wise manner. Oxidative stress indicators including NO, LPO, and ROS levels were considerably higher in the treated groups than in the control. The antioxidant markers such as CAT and SOD declined significantly in treated groups. The endpoints of the gene expression study revealed the overexpression of tumor suppression genes Bax and p53 and anti-apoptotic gene, Bcl-2 was unexpressed. The biogenically synthesized CA–CuO NPs exhibited exceptional anti-cancerous activity towards HCT 116 cell line. [ABSTRACT FROM AUTHOR]

The aim of the present study was to develop potential drug carrier system formulations, namely PLAROsomes for the delivery of Proanthocyanidin (PACY), isolated from the extract of dried grape seeds. The PLAROsomes loaded with PACY were prepared using the thin film hydration method. The influence of various process parameters and material attributes was investigated using the design of experiments (DoE). The PACY‐loaded PLAROsomes were characterized using hyphenated tools. The in‐vitro anticancer activities of PACY‐loaded PLAROsomes were confirmed using the Trypan blue method, MTT method, and flow cytometric analysis on MCF‐7 cells. The polymer‐to‐lipid ratio, among various process parameters and material attributes, significantly influenced the average particle size. Additionally, it played a crucial role in determining the percentage of PACY released from PACY‐loaded PLAROsomes. The size of the PACY‐loaded PLAROsomes ranged from 40 to 300 nm, and the optimized batch demonstrated a drug entrapment efficiency of 86.38±0.22%. PACY‐loaded PLAROsomes exhibited improved in vitro anticancer activities against MCF‐7 breast cancer cell lines compared to PACY. PACY‐loaded PLAROsomes showed greater activity at lower concentrations in cytotoxicity studies, as supported by apoptosis analysis. Therefore, PLAROsomes could present a promising drug carrier system for delivering PACY in breast cancer treatment, offering a sustained release effect. [ABSTRACT FROM AUTHOR]

Starting compound 1 was synthesized according to reference.1 Benzylidene thiazole pyrimidin-3(5H)-ones were synthesized reactions of 1 with bromoacetic acid and various aryl-aldehydes in the same vessel via one-step, unlike studies in the literature. Quantum chemical parameters and full geometry optimizations for all compounds were computed using DFT based on B3LYP. Cytotoxic action potential of synthesized compounds was evaluated using trypan blue dye exclusion and MTT assays in different cell lines including adenocarcinoma alveolar basal epithelial-like adherent A549 cells, the colon adenocarcinoma HT-29 cells, prostate adenocarcinoma DU-145 cells, and diploid ARPE-19 retinal pigment epithelial cells. Embryotoxicity and genotoxicity assessments were performed on pluripotent human embryonal carcinoma NT2 and human lymphocyte cells, respectively. Compound A1 exhibited good anticancer activity on A549 and DU-145 cell lines, and the compounds including A3, 4, 6, and 9 induced cytotoxicity on A549 cells. The compounds A1-10 also showed a good biosafety profile at relatively lower concentrations. [ABSTRACT FROM AUTHOR]

Objectives: Diabetes mellitus is a complex heterogenous metabolic disease that significantly affects the world population. Although many treatments exist, including medications such as metformin, sulfonylureas, and glucagon‐like peptide‐1 (GLP) receptor agonist, there is growing interest in finding alternative methods to noninvasively treat this disease. It has been previously shown that low‐intensity ultrasound stimulation of pancreatic β‐cells in mice can elicit insulin secretion as a potential treatment for this disease. This is desirable as therapeutic ultrasound has the ability to induce bioeffects while selectively focusing deep within tissues, allowing for modulation of hormone secretion in the pancreas to mitigate insufficient levels of insulin. Methods: Exactly 800 kHz ultrasound with intensity 0.5 W/cm2 was administered 5 minutes continuously, that is, 100% duty cycle, to donor pancreatic human islets, followed by 1 hour incubation and RT‐qPCR to assess the effect of ultrasound stimulation on gene expression. The genes were insulin (INS), glucagon (Glu), amylin (Amy), and binding immunoglobulin protein (BiP). Nine donor pancreatic human islets were used to assess insulin and glucagon secretion, while eight samples were used for amylin and BiP. Fold change (FC) was calculated to analyze the effect of ultrasound stimulation on the gene expression of the donor islet cells. High‐glucose and thapsigargin‐treated islets were utilized as positive controls. Cell viability testing was done using a Trypan Blue Exclusion Test. Results: Ultrasound stimulation did not cause a statistically significant upregulation in any of the tested genes (INS FC = 1.15, P‐value =.5692; Glu FC = 1.60, P‐value =.2231; Amy FC, P‐value =.2863; BiP FC = 2.68, P‐value =.3907). Conclusions: The results of this study show that the proposed ultrasound treatment parameters do not appear to significantly affect gene expression of any gene tested. [ABSTRACT FROM AUTHOR]

The estimation of acrosomal integrity is crucial for predicting the fertility of spermatozoa. The semen quality analysis demands assessment techniques to be reliable, quick and easy to perform for routine application in semen station. A study was conducted to assess the acrosomal integrity of Kangayam cattle and Salem Black goat using Giemsa stain and modified Trypan Blue-Giemsa stain. For this procedure, 70 numbers of frozen semen straws of Kangayam bull and Salem Black bucks were stained. The staining quality and acrosomal integrity were assessed at the interval of 3, 5 and 24 hours interval up to 5 days, and evaluated as acrosomal intact and non-intact spermatozoa. In Kangayam bull semen, the quality of the staining was good and better in trypan blue – Giemsa stain than Giemsa alone but for Salem Black buck semen Giemsa showed better staining quality than trypan blue – Giemsa combination. However, the working solution of Giemsa once prepared can be used for 4 days but the trypan blue-Giemsa stain can be used for only 24 hours once prepared. Hence, stain is required to be prepared on daily basis for routine semen evaluation in semen stations. As a conclusion trypan blue – Giemsa staining method is recommended for better staining of acrosome in bull semen and Giemsa alone in Buck semen. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "The Auto-Regulation of ATL2 E3 Ubiquitin Ligase Plays an Important Role in the Immune Response against Alternaria brassicicola in Arabidopsis thaliana." The correction addresses a mistake in Figure 4 of the original publication, where an image was mistakenly duplicated. The corrected figure is provided, and the authors state that this correction does not affect the scientific conclusions. The document also includes additional figures and data related to the disease response to A. brassicicola in Arabidopsis plants. [Extracted from the article]

Abstract Brilliant blue 0.05% and trypan blue 0.1% were mixed in a proportion of 1:1 in a 1-mL syringe. This combination produced a waterfall effect with the fast sinking of the dye to the posterior pole and little diffusion through the vitreous cavity. Therefore, it can effectively stain the internal limiting membrane and the epiretinal membrane with a good contrast during surgeries for a macular hole, myopic foveoschisis, and macular pucker.

Exposure to indoor air pollutants (IAP) has increased recently, with people spending more time indoors (i.e. homes, offices, schools and transportation). Increased exposures of IAP on a healthy population are poorly understood, and those with allergic respiratory conditions even less so. The objective of this study, therefore, was to implement a well-characterised in vitro model of the human alveolar epithelial barrier (A549 + PMA differentiated THP-1 incubated with and without IL-13, IL-5 and IL-4) to determine the effects of a standardised indoor particulate (NIST 2583) on both a healthy lung model and one modelling a type-II (stimulated with IL-13, IL-5 and IL-4) inflammatory response (such as asthma). Using concentrations from the literature, and an environmentally appropriate exposure we investigated 232, 464 and 608ng/cm2 of NIST 2583 respectively. Membrane integrity (blue dextran), viability (trypan blue), genotoxicity (micronucleus (Mn) assay) and (pro-)/(anti-)inflammatory effects (IL-6, IL-8, IL-33, IL-10) were then assessed 24 h post exposure to both models. Models were exposed using a physiologically relevant aerosolisation method (VitroCell Cloud 12 exposure system). No changes in Mn frequency or membrane integrity in either model were noted when exposed to any of the tested concentrations of NIST 2583. A significant decrease (p < 0.05) in cell viability at the highest concentration was observed in the healthy model. Whilst cell viability in the "inflamed" model was decreased at the lower concentrations (significantly (p < 0.05) after 464ng/cm2). A significant reduction (p < 0.05) in IL-10 and a significant increase in IL-33 was seen after 24 h exposure to NIST 2583 (464, 608ng/cm2) in the "inflamed" model. Collectively, the results indicate the potential for IAP to cause the onset of a type II response as well as exacerbating pre-existing allergic conditions. Furthermore, the data imposes the importance of considering unhealthy individuals when investigating the potential health effects of IAP. It also highlights that even in a healthy population these particles have the potential to induce this type II response and initiate an immune response following exposure to IAP. [ABSTRACT FROM AUTHOR]

Purpose: To compare 3 capsulotomy centration methods. Setting: Private clinic, Zlin, Czech Republic. Design: Prospective, consecutive case series. Methods: 180 eyes undergoing cataract surgery had anterior capsule staining with microfiltered 0.4% trypan blue solution before selective laser capsulotomy. The first 60 eyes (Group 1) had mydriatic dilated pupil centered capsulotomies. The next 60 eyes (Group 2) were centered on the trypan blue central landmark (TCL). The final 60 capsulotomies (Group 3) were centered on the patient fixated coaxial Purkinje reflex (CPR). Measurements between key anatomical landmarks and the TCL, CPR capsuloto- mies, and implanted intraocular lens (IOL) center were made. Results: The TCL, observed in >94% of eyes in the study, coincided with the CPR with a displacement of <0.1 ± 0.1 mm. Group 1 capsulotomies were noticeably decentered on the IOLs by 0.3 ± 0.2 mm. The Group 2 symmetrical IOL relationship was maintained with a decentration of 0.15 ± 0.1 mm. Group 3 had a similar decentration with the IOLs with 0.15 ± 0.1 mm. Verification with IOLMaster 700 data and CALLISTO Eye System showed that the CPR and the TCL were coincident with the measured visual axis. Conclusions: The clearly visible TCL served as an alternate landmark to the patient fixated CPR, and being on the anterior capsule was not sensitive to tilt. Further patient compliance was not required. Both were superior to dilated pupil centration, to achieve symmetric IOL coverage. This has application for both capsulotomies and capsulorhexes. [ABSTRACT FROM AUTHOR]