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Acacia modesta (AM) and Opuntia monocantha (OM) are distributed in Pakistan, Afghanistan and India. Both of these plants have different pharmacological properties. This study was designed to evaluate anticancer potential of Acacia modesta (AM) and Opuntia monocantha (OM). Liver cancer cell line HepG2 was used for assessment of anticancer activity. For the evaluation of anti-proliferative effects, cell viability and cell death in all groups of cells were evaluated via MTT, crystal violet and trypan blue assays. For the evaluation of apoptosis ELISA of p53 performed. Furthermore, LDH assay to find out the ability of malignant cells to metabolize pyruvate to lactate and antioxidant enzymes activity (GSH, CAT and SOD) at the end HPLC was performed to find active compound of AM and OM. Cytotoxicity (MTT), Viability assays (trypan blue, crystal viability, MUSE analysis) showed more dead, less live cells in plant treated groups with increase of concentration. Scratch assay for the anti-migratory effect of these plants showed treated groups have not ability to heal scratch/wound. ELISA of p53 for cellular apoptosis showed more release of p53 in treated groups. Antioxidant assay via glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) showed less anti-oxidative potential in treated cancer groups. LDH assay showed more lactate dehydrogenase release in treated groups compared with untreated. HPLC analysis showed the presence of phytochemicals such as steroids, alkaloids, phenols, flavonoids, saponins, tannins, anthraquinone and amino acids in AM and OM plant extracts. Based on all these findings, it can be concluded that ethanolic extracts of Acacia modesta and Opuntia monocantha have promising anti-cancer potential. Resumo Acacia modesta (AM) e Opuntia monocantha (OM) estão distribuídas no Paquistão, Afeganistão e Índia. Ambas as plantas têm propriedades farmacológicas diferentes. Este estudo foi desenhado para avaliar o potencial anticancerígeno de Acacia modesta (AM) e Opuntia monocantha (OM). A linha celular de câncer de fígado HepG2 foi usada para avaliação da atividade anticâncer. Para a avaliação dos efeitos antiproliferativos, a viabilidade celular e a morte celular em todos os grupos de células foram avaliadas através dos ensaios de MTT, cristal violeta e azul de tripano. Para a avaliação da apoptose foi realizado ELISA de p53. Além disso, o ensaio de LDH para descobrir a capacidade das células malignas de metabolizar o piruvato em lactato e a atividade das enzimas antioxidantes (GSH, CAT e SOD) no final da HPLC foi realizado para encontrar o composto ativo de AM e OM. Citotoxicidade (MTT), ensaios de viabilidade (azul de tripano, viabilidade de cristal, análise MUSE) mostraram mais células mortas e menos células vivas nos grupos tratados com plantas com aumento da concentração. O ensaio de arranhão para o efeito anti-migratório dessas plantas mostrou que os grupos tratados não têm capacidade de cicatrizar arranhão/ferida. ELISA de p53 para apoptose celular mostrou maior liberação de p53 nos grupos tratados. Ensaio antioxidante via glutationa (GSH), superóxido dismutase (SOD), catalase (CAT) mostrou menor potencial antioxidante nos grupos de câncer tratados. O ensaio de LDH mostrou mais liberação de lactato desidrogenase nos grupos tratados em comparação com os não tratados. A análise de HPLC mostrou a presença de fitoquímicos como esteróides, alcalóides, fenóis, flavonóides, saponinas, taninos, antraquinona e aminoácidos nos extratos vegetais AM e OM. Com base em todos esses achados, pode-se concluir que os extratos etanólicos de Acacia modesta e Opuntia monocantha apresentam potencial anticancerígeno promissor.

This protocol describes a robust pipeline for simultaneously analyzing multiple samples by single-nucleus (sn)RNA-seq. cDNA obtained from each single sample are labeled with the same lipid-coupled oligonucleotide barcode (10X Genomics). Nuclei from as many as 12 individual samples can be pooled together and simultaneously processed for cDNA library construction and subsequent DNA sequencing. While previous protocols using lipid-coupled oligonucleotide barcodes were optimized for analysis of samples consisting of viable cells, this protocol is optimized for analyses of quick-frozen cell samples. The protocol ensures efficient recovery of nuclei both by incorporating high sucrose buffered solutions and by including a tracking dye (trypan blue) during nuclei isolation. The protocol also describes a procedure for removing single nuclei 'artifacts' by removing cell debris prior to single nuclear fractionation. This protocol informs the use of computational tools for filtering poorly labeled nuclei and assigning sample identity using barcode unique molecular identifier (UMI) read counts percentages. The computational pipeline is applicable to either cultured or primary, fresh or frozen cells, regardless of their cell types and species. Overall, this protocol reduces batch effects and experimental costs while enhancing sample comparison. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling cells with lipid oligo barcodes and generating multiplexed single-nucleus RNA-seq libraries Basic Protocol 2: Bioinformatic deconvolution of the multiplexed snRNAseq libraries.

Tissue engineering is an emerging method for replacing damaged tissues. In this study, the potential application of electrospun polycaprolactone/chitosan/ the internal layer of oak fruit (Jaft) as skin scaffolds was investigated. A combination of Polycaprolactone (PCL), chitosan (CH), and the internal layer of oak fruit (Jaft) was used to incorporate mechanical properties of synthetic polymers, biological properties of natural polymers, and antibacterial activity of Jaft. Physical and morphological characteristics of prepared scaffolds were investigated using a scanning electron microscope (SEM), mechanical analysis, swelling ratio, and contact angle. Moreover, chemical and biological properties were evaluated by Fourier-transform infrared spectroscopy (FTIR), chromatography, flow cytometry, DAPI staining, MTT assay, and trypan blue exclusion assay. Obtained results demonstrated that the fabricated scaffolds have good mechanical properties. Moreover, the addition of chitosan and Jaft to the PCL scaffolds improved their water absorption capacity as well as surface hydrophilicity. MTT results showed the fabricated nanofibrous scaffolds have adequate cell viability, which is higher than the cell culture plate at each time point of culture. Furthermore, SEM images of cultured scaffolds, trypan blue exclusion assay, and DAPI staining confirmed that fibroblast cells could be well-attached and proliferate on the PCL/CH/Jaft scaffolds. Results have proven that this novel bioactive scaffold has promising mechanical properties, suitable biocompatibility in vitro, and in vivo. Consequently, it could be a promising candidate for skin tissue engineering applications.

Understanding the interactions between drugs and lipid membranes is a prerequisite for finding the optimal way to deliver drugs into cells. Coadministration of statins and anticancer agents has been reported to have a positive effect on anticancer therapy. In this study, we elucidate the mechanism by which simvastatin (SIM) improves the efficiency of biological membrane penetration by the chemotherapeutic agent doxorubicin (DOX) in neutral and slightly acidic solutions. The incorporation of DOX, SIM, or a combination of them (DOX:SIM) into selected single-component lipid membranes, zwitterionic unsaturated 1-palmitoyl-2-oleoyl

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and β-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

Parathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found that in colorectal cancer (CRC) HCT116 cells, the binding of PTHrP to its receptor PTHR type 1 (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cell xenografts, PTHrP modulates the expression of molecular markers linked to tumor progression. Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.To elucidate the relationship among PTHR1, PTHrP, and Met in CRC models.ForBy Western blot analysis and using total Met antibody, we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 min (PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model. More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.

Background: The importance of the role of NF-κB is recognized in situations such as malignant transformation and metastasis of cancer, and it has been suggested that inhibiting this role can be one of the cancer treatment strategies. Gold preparations such as auranofin are known to have an indirect NF-κB inhibitory effect. Objective: We synthesized a novel gold complex [tiopronin monovalent gold-5-mercapto- 1-methyl tetrazole, abbreviated as TPN-Au(I)-MM4], with different physical properties and chemical structure from auranofin, and evaluated its cytotoxic activity and radiation sensitizing effect on human THP1 cells. Methods: The number of viable cells was counted by the trypan blue dye exclusion method. The cell death evaluation was performed by FITC-Annexin V+ and PI staining. In investigating the radiation sensitizing effect of TPN-Au(I)-MM4, this compound [10 or 25 μM] was added into the culture medium 1 h before X-ray irradiation. Results: In the cells treated with 25 μM TPN-Au(I)-MM4 for 72 h, a decrease in the proliferation of THP1 cells was observed [The relative values of viable cells in the control group and the 25 μM treatment group were approximately 6.8 and 4.2, respectively]. In the combination of 25 μM of the compound treatment and X-ray irradiation, an increase of approximately 3.0-fold was observed in 2 Gy irradiation and approximately 1.4-fold in 4 Gy irradiation as in comparison to the case of irradiation alone. Conclusion: These results suggest that TPN-Au(I)-MM4 reduces the proliferation of THP1 cells through the induction of cell death, and the combined use of TPN-Au(I)-MM4 and X-ray irradiation shows effective cytotoxicity against THP1 cells.

Purpose To quantify the damage to the corneal endothelium from the flow of balanced salt solution during phacoemulsification. Setting John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. Design Experimental Study. Methods Twelve New Zealand white rabbits received bilateral surgery. Six eyes served as controls with no irrigation (incision only). In 18 eyes, the Intrepid Balanced tip of the Centurion (Alcon) Ozil handpiece was inserted into the anterior chamber for continuous irrigation at 50mL/minute with either 250 mL or 500 mL of balanced salt solution (BSS). After euthanasia and enucleation, the corneas were removed, stained with trypan blue/alizarin red, and photographed in a standardized manner (X400 photographs from 5 specific areas, and 1 overview photograph from each corneal button). The ImageJ program was used to evaluate cell damage and loss in the photographs obtained from each cornea. Results Analysis of the X400 photographs showed no statistical differences between control, 250 mL, and 500 mL groups in the percentage of intact, damaged, or lost cells (P= 0.896, 0.851, and 0.972 respectively). For the overview photos, the differences in areas of intact and damaged cells among the groups were statistically significant, likely due to the peripheral areas of damage related to touches between the phaco tip and the endothelium during irrigation. Conclusion The volume of BSS flow alone does not appear to be a major contributing source of endothelial cell damage and loss, providing further insight on mechanisms of corneal endothelium damage during phacoemulsification.

Purpose To compare corneal endothelial damage from longitudinal and torsional ultrasound during phacoemulsification. Setting John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. Design Experimental Study. Methods Nine New Zealand white rabbits underwent bilateral surgery. After incision, the Intrepid Balanced tip of the Centurion (Alcon) Ozil handpiece was inserted into the anterior chamber and the following settings were used: 50 mL/minute flow, 70 mmHg intraocular pressure, 600 mmHg vacuum, and 60% longitudinal (one eye) or torsional (contralateral eye) ultrasound for 30 seconds. Cumulative dissipated energy (CDE) was noted. After euthanasia and enucleation, the corneas were removed, stained with trypan blue/alizarin red, and photographed (X400 photographs from 5 specific areas, and 1 overview photograph from each corneal button). The ImageJ program was used to evaluate cell damage and loss in the photographs obtained from each cornea. Results Cavitation bubbles around the phaco tip was generally observed in the longitudinal group. CDE was 17.4 +/- 0.58 and 6.93 +/- 0.15 in the longitudinal and torsional groups, respectively (P = 0.003). The percentage of intact cells was statistically higher in the torsional group, and the percentage of lost cells was statistically higher in the longitudinal group (P = 0.003). This was observed in the analysis of the X400 photographs, as well as the overview photographs. Conclusion This study suggests that torsional ultrasound was associated with significantly less corneal endothelial cell damage than classical longitudinal tip motion, providing further insight on mechanisms of corneal endothelial damage during phacoemulsification.

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200-300 μm) were randomly assigned into four groups. Group1 - Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2- FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2- FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (-196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

Chernobai N.A., Vozovik K.D., Kadnikova N.G. Comparative analysis of methods for assessing the safety of Dunaliella salina Teodoresco and Chlorococcum dissectum Korshikov (Chlorophyta) microalgae cultures after exposure to stress factors. Algologia 2021, 31(4): 353–364 https://doi.org/10.15407/alg31.04.353 The possibility of using various methods for determining the viability of cultures of microalgae Dunaliella salina and Chlorococcum dissectum before and after freezing-warming was investigated and analyzed. It has been established that the selection of an effective method should be carried out individually for each culture. For an integral assessment of the proliferative and metabolic activity of cells of both species of the studied microalgae, Alamar Blue-test and the ability to grow on liquid nutrient media can be used. The use of the Koch plate method, MTT-test and TTC staining is possible only for the microalga C. dissectum. Vital staining with trypan blue was found to be incorrect.

Piracanjunba (Brycon orbignyanus) is an endangered South American fish, and ovarian tissue cryopreservation is an alternative method for preserving maternal germplasm and genetic diversity. Therefore, our aim was to test a vitrification protocol for ovarian tissue containing primary growth (PG) oocytes of B. orbignyanus as a strategy to avoid the threat of extinction. Two vitrification solutions were evaluated (VS1: 1.5 M methanol + 4.5 M propylene glycol and VS2: 1.5 M methanol + 5.5 M Me2SO) and compared using control/fresh ovarian tissue. After vitrification, the following factors were analyzed: membrane integrity using trypan blue, morphology using a histological assessment, oxidative stress (total reactive antioxidant potential (TRAP) and reduced thiol [-SH]), mitochondrial activity using MTT, and DNA damage using a comet assay. The vitrified oocytes (VS1 = 24.3 ± 0.49% and VS2 = 24.8 ± 0.69%) showed higher DNA damage than the control group (control = 20.7 ± 1.03%) (P = 0.004). In contrast, in most evaluations (membrane integrity, membrane damage, oxidative stress, and mitochondrial activity), there were no discernible differences between the control group and the vitrified samples. In addition, oocyte (P = 0.883) and nuclear diameter (P = 0.118) did not change after vitrification. VS2 treatment resulted in higher nuclear damage (15.7 ± 1.45%) than in the control treatment (3.5 ± 1.19%); however, VS1 treatment did not result in significantly more damage (9.5 ± 3.01%) than in the control (P = 0.015). Therefore, the protocol for ovarian tissue vitrification tested in this study resulted in high maintenance of PG oocyte cell integrity, making it a promising alternative for B. orbignyanus maternal genome preservation.

The present study was designed to evaluate the possible protective effects of melatonin (MEL) and/or L-carnitine (L-CAR) against methotrexate (MTX)-induced toxicity in isolated rat hepatocytes. Hepatocytes were prepared using collagenase techniques of perfusion and digestion of rat liver. Trypan blue uptake, as well as, glutathione (GSH), lipid peroxidation (LPO), nitric oxide (NO), and tumor necrosis factor-alpha (TNF-α) levels were measured. Caspase-3 activity was also assessed. Pre-incubation of hepatocytes with MEL (1 mM) and/or L-CAR (10 mM) 30 min prior to intoxication with MTX, significantly protected hepatocytes against toxicity. In addition, LPO, NO, TNF-α levels, and caspase-3 activity were decreased in comparison to the MTX-intoxicated group. Furthermore, the two drugs increased the MTX-depleted GSH level. MEL and L-CAR prevented MTX-induced hepatocytotoxicity, at least partly, by their antioxidative, antiinflammatory, and antiapoptotic effects. Further studies are recommended on the clinical pharmacologic and toxicologic effects of MEL and L-CAR in patients receiving MTX.

BACKGROUND The problem of resistance to antiparasitic drugs, associated with their side effects, suggest exploring other alternatives, including medicinal plants. Dracocephalum kotschyi (D. kotschyi), for example, from Lamiaceae family, is a plant widely used in Iran and in many countries, and to which interesting pharmacological properties have been attributed. This study aimed to investigate in vitro and in vivo anti-Toxoplasma activities of D. kotschyi extract in experimental models of acute toxoplasmosis. METHODS Anti-Toxoplasma activity of the extracts in vitro was performed on Vero Cell, using the MTT test. Vero cell were infected with (3 × 105 tachyzoites/well) following treatment with Dichloromethane (F1), dichloromethane: methanol (F2), methanol (F3), methanol: water (F4), and deionized water (F5) extracts of D. kotschyi, and pyrimethamine and sulfadiazine (positive control). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to measure cell viability. Effects of extracts on tachyzoites viability were evaluated by trypan blue exclusion method, followed by light microscopy. For in vivo test, 18 groups of 8-10-week-old Inbred Balb/c mice weighing 18-20 g, were intraperitoneally infected with 2 × 103 tachyzoites and then treated with sterile PBS (negative control), pyrimethamine (25 mg/kg) and sulfadiazine (500 mg/kg) as positive controls, and F1 to F5 extracts (at doses 50, 100 and 200 mg/kg). RESULTS The 50% Inhibitory Concentration of F1 extract, F2 extract, Sulfadiazine (Positive control) and Pyrimethamine (Positive control) were 8.77 µg, 7.1 µg 391.18 µg, and 84.20 µg, respectively, while the selectivity indices were 15.667, 30.197, 1.552 and 4.064, respectively. In vivo anti-Toxoplasma activity test showed that Methanol: water (F-4) 50 extract was more active than the positive control. CONCLUSIONS Indeed, the extract allowed a survival rate of 10% of the mice, compared to 0% for all the other groups. D. kotschyi has the potential to be valorized in the management of toxoplasmosis.

Sodium dodecyl sulfate (SDS) capped- Cu nanorods were prepared by using ascorbic acid under light limiting diode (LED) irradiation for the first time. The preliminary indications of CuNPs production were the appearance of yellow (Cu2O) and red-chocolate color (Cu0) in absence and presence of LED exposure at room temperature. The optical, spectroscopic and morphological properties of as-prepared NPs were characterized using UV–visible spectroscopy through the presence of a characteristic surface Plasmon resonance (SPR) band for Cu0 at 584 nm along with high absorbance at 800 nm for Cu2O. X-ray diffraction (XRD) shows that the red-chocolate color consist 90% metallic Cu0 and 9.7% Cu2O with face cubic center unit cell structure of CuNPs. Transmission electron microscope (TEM), energy dispersive X-ray (EDX), and scanning electron microscope (SEM) revealed that the CuNPs were nano-rods with an average length ranging from 300 to 350 nm. LED light assisted degradation of trypan blue (TB) was also investigated in presence of CuNPs and K2S2O8. The existence of sulfate radicals (SO4-•) and hydroxly radicals (HO•) generated during the degradation were identified by using different radical scavengers. Degradation follows first-order kinetics with respect to K2S2O8 concentrations. The oxidative dissolution of CuNPs was also observed along with the TB degradation. Based on the absorbance changes at 590 nm, the degradation and dissolution mechanisms were proposed and discussed.

Hepatocellular carcinoma is among the leading causes of cancer-related deaths worldwide and needs efficient and feasible approach of treatment. Present study focuses on exploring the anticancer activity of a secondary metabolite called siderophore of Aspergillus nidulans against hepatocellular carcinoma cell line HepG2. These small peptides are produced by microorganisms including fungi for scavenging iron from its surroundings. Fungi including Aspergillus spp. are known to produce siderophores under iron-limited conditions. Siderophores have high affinity towards iron and are classified into various types. In the present study, siderophore isolated and purified from fungal cultures was confirmed to be of hydroxamate type by chrome azurol sulfonate and Atkin's assay. HPLC analysis confirmed purity while LC-ESI-MS revealed that the siderophore is triacetyl fusigen. Cancerous cells, HepG2, grown under siderophore treatment showed inhibition in growth and proliferation in a dose- and time-dependent manner. Reduction in viability and metabolic activity was evident upon treatment as seen in trypan blue, MTT and WST assay. Fluorescent staining using PI and DAPI confirmed the same while DCFDA staining revealed increased reactive oxygen species production which might have led to cell death and deterioration. Such increase in ROS has been correlated with iron accumulation by assessing intracellular iron level through ICP-MS. To assess the effect of siderophore treatment on normal cells, WRL-68, same assays were carried out but the effect was mostly non-significant up to 48 h. Thus, present work suggests that an optimum dose of siderophore purified from A. nidulans culture might prove a useful anticancer agent.

Downstream processing is an expensive step for industrial production of recombinant proteins. Cell immobilization is known as one of the ideal solutions in regard to process intensification. In recent years, magnetic immobilization was introduced as a new technique for cell immobilization. This technique was successfully employed to harvest many bacterial and eukaryotic cells. But there are no data about the influence of magnetic immobilization on the eukaryotic inducted recombinant cells. In this study, impacts of magnetic immobilization on the growth and metabolic status of induced recombinant Pichia pastoris as a valuable eukaryotic model cells were investigated. Results based on colony-forming unit, OD600, and trypan blue assay indicated that magnetic immobilization had no adverse effect on the growth and viability of P. pastoris cells. Also, about 20-40% increase in metabolic activity was recorded in immobilized cells that were decorated with 0.5-2 mg/mL nanoparticles. Total protein and carbohydrate of the cells were also measured as main indicatives for cell function and no significant changes were observed in the immobilized cells. Current data show magnetic immobilization as a biocompatible technique for application in eukaryotic expression systems. Results can be considered for further developments in P. pastoris-based expression systems.

Background and Aim: The peripheral blood mononuclear cell (PBMC) is an excellent cell source for in vitro studies, particularly those involving immunology. The aim of this study was to determine the quality and quantity of chicken PBMCs isolated from freshly drawn blood as well as blood that had been chilled for 24 h. In addition, the survival of PBMCs cultured in medium was investigated. Materials and Methods: Blood samples were collected from 12 Betong and 12 Leghorn chickens. Hemograms were analyzed. Density gradient centrifugation was used to isolate PBMCs. PBMCs (2×106 cells/mL) were cultured in a culture medium and incubated in a CO2 incubator for 5 consecutive days. The number of viable cells was determined using the trypan blue dye exclusion method. Results: Blood samples were obtained from healthy chickens. There was no statistically significant difference in the total amount of PBMC between fresh and refrigerated blood samples from both chicken breeds. The viability of PBMCs isolated from fresh blood (95%) was significantly greater than blood refrigerated for 24 h (90-92%) in both breeds. Furthermore, the viability of PBMCs isolated from both blood samples decreased significantly over time, from 90-95% to 60-65%. Conclusion: The total number of PBMC in fresh and refrigerated blood was not significantly different. Fresh blood-derived PBMCs had significantly higher viability than 24 h refrigerated blood PBMCs. Furthermore, the viability of PBMCs decreased significantly over time.

The objectives of this study were to examine cytotoxic and genotoxic damage in human BJ fibroblasts caused by three pesticides used worldwide by trypan blue dye exclusion assays and to measure the relative level of phosphorylated histone H2A.X by flow cytometry at different concentrations. Captan-based fungicide and methyl thiophanate-based fungicide (100 and 1000 µΜ) showed immediate cytotoxic effects; furthermore, after 24 h, captan-based fungicide, chlorothalonil-based fungicide and methyl thiophanate-based fungicide caused cytotoxic effects in the concentration ranges of 40-100 µM, 30-100 µM and 150-1000 µM, respectively. All fungicides generated DNA damage in the treated cells by activating ATM and H2A.X sensor proteins. The three fungicides tested generated DNA double-stranded breaks and showed cytotoxicity at concentrations 33, 34, and 5 times lower (captan, chlorothalonil and thiophanate-methyl respectively) than those used in the field, as recommended by the manufacturers.

Simple staining of cells is a widely used method in basic medical diagnostics, education, and research laboratories. The stains are low-cost, but the extensive consumption results in excessive toxic waste generation. Thus, to decrease the amount of toxic waste resulting from the cell staining procedure is a need. In this study, we developed a magnetically driven and compartmentalized passive microfluidic chip to perform simple staining of human eukaryotic cells, K562 cells, and lymphocyte cells derived from patients. We demonstrated simple staining on cells with trypan blue, methylene blue, crystal violet, and safranin for high, medium, and low cell densities. The stained cells were imaged using a bright field optical microscope and a cell phone to count cells on the focal plane. The staining improved the color signal of the cell by 25-135-pixel intensity changes for the microscopic images. The validity of the protocol was determined using Jurkat and MDA-MB-231 cell lines as negative controls. In order to demonstrate the practicality of the system, lymphocyte cells derived from human blood samples were stained with trypan blue. The color intensity changes in the first and last compartments were analyzed to evaluate the performance of the chip. The developed method is ultra-low cost, significantly reduces the waste generated, and can be integrated with mobile imaging devices in terms of portability. By combining microfabrication technology with cell staining, this study reported a novel contribution to the field of microfluidic biosensors. In the future, we expect to demonstrate the detection of pathogens using this method. This research has been supported by Erciyes University Scientific Research Projects Coordination Unit under grant number FDK-2022-11974.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 µm cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 µm and 40 µm cell strainers. The contents retained in the 40 µm strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

We aimed to quantitatively analyze the corneal endothelial cell damage by measuring the area stained with trypan blue dye, and to confirm the degree of corneal endothelial cell damage resulting from enucleation, corneal buttoning, and storage in donor corneas intended for use in human corneal transplantation. This study was a retrospective analysis of medical records and videos recorded during keratoplasty. Twenty-one corneal buttons of 21 donors that underwent endothelial cell staining using trypan blue for the donor preparation during DALK or DMEK were included in the study. The percentage of stained area in entire corneal endothelia and the percentage of the stained area in the 8-mm diameter circle were quantitatively analyzed using Adobe Photoshop. The mean percentage of the stained area in the entire corneal endothelia in 13 corneas was 8.1 ± 13.3% (range, 0.0-56.1%), and the mean percentage of the stained area in a circle with a diameter of 8 mm in 21 corneas was 3.4 ± 5.2% (range, 0.0-18.9%). The correlations between the death-to-preservation time, the training duration of the residents who performed donor corneal buttoning, and the percentage of the stained area in the 8-mm diameter circle were not significant(P = .441, P = .495, respectively). Cornea thickness and endothelial cell density did not differ between 10 eyes in the group with the percentage of the stained area in a circle with a diameter of 8 mm5% and 5 eyes in the group with the percentage more than 5% damage (P = .854, P = .358). The corneal endothelial cell damage could be quantitatively analyzed using trypan blue staining before keratoplasty in donor cornea. The amount of corneal endothelial cell damage in the central 8-mm circle was mostly acceptable, but some cases showed significantly severe endothelial cell damage. The corneal thickness and endothelial cell density did not differ between 10 eyes in the group with the percentage of the stained area in a circle with a diameter of 8 mm5% and 5 eyes in the group with the percentage more than 5% damage. Therefore, pachymetry and specular microscopy are not sufficient for evaluating donor corneas before keratoplasty.

BACKGROUND Intraoperative tumour spillage can be concerning during cancer excisions, given it can lead to tumour-cell re-implantation and local recurrence. Examples include bladder tumour recurrences post-transurethral resection, or peritoneal spillage during laparotomy/laparoscopy for bowel and ovarian cancers. One approach to reducing implantation is mechanical wash out of free-floating tumour cells. Irrigation with water may have additional effectiveness compared to iso-osmotic irrigants (e.g. saline) by causing osmotic cytolysis, but this is not well-characterised. This in vitro study aimed to ascertain the time-course of osmotic effects of water on various cancer cell lines to provide guidance for clinical usage. METHODS Assays were conducted on six cancer cell lines (bladder [HT1197, HT1376], colon [KM12, LIM2405], kidney [SKRC52], and ovarian [COV434]). Cells were exposed to water or 0.9% saline and cell counts were performed using a haemocytometer at 10, 20, 40, 60, 120 and 180 min. Cell viability was determined using Trypan Blue exclusion. RESULTS In all cell lines, exposure to water led to 100% cell lysis within a median time of 40 min (range 10-180 min), while exposure to saline led to a gradual decline in cell viability (median 50.2%, range 6.7%-100.0%) over 3 h, and did not result in complete cell lysis. An increase in osmotic gradient equivalent to a concentration of 5% NaCl was sufficient to impede the effects of water-mediated cell lysis. CONCLUSION Our studies suggest that water has a rapid osmolytic effect on cancer cells. The required exposure time to reach 0% cell viability varied between individual cell lines.

Intraoral fixed appliances remain in the potentially corrosive environment of the mouth for an average of two years. Over time, corrosion causes the release of metal ions, such as nickel and chromium. These metals can become allergenic and cytotoxic, causing different conditions in the human body. The aim of this study therefore is to carry out a systematic review of the available scientific evidence on the accumulation of metal ions, and the genotoxic and cytotoxic effects in oral mucosa cells deriving from short- and long-term exposure to them. The systematic review is reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The primary outcome (quantification of metal ion deposits and assessment of their genotoxic and/or cytotoxic effects) and secondary outcome (complementary analysis of cytotoxic and genotoxic effects) were examined. The Cochrane Collaboration tool and Toxicological data Reliability Assessment Tool (ToxRTool) were used for quality assessment. Once the search was performed, a total of seven articles met the inclusion criteria and were included in this study. Two main techniques were used to assess genotoxic effects: alkaline comet assay (6/7) and micronucleus method (1/7). Cytotoxicity was evaluated (4/7) using the trypan blue dye test. Accumulations of nickel (7/7), chromium (5/7), and other metals (zinc, cobalt, iron, manganese, molybdenum, titanium) were also quantified. The results allowed us to conclude that release of metal ions and acute cell and DNA damage in oral mucosa cells takes place in the early stages of treatment. However, more long-term studies are needed to evaluate chronic exposure to metals and DNA damage, as well as cellular capacity to recover DNA integrity.

Coleus aromaticus plant is having potential medicinal properties. In this study, we have purified a protein and characterized it by investigating the active protein was purified by G-50 column chromatography and its homogeneity was confirmed with SDS PAGE and RP HPLC. The protein molecular weight was confirmed with MS MALDI. The Phosphomolybdenum assay was used to evaluate the antioxidant capacity, where α- tocopherol was used as standard. The protective effect of purified Coleus aromaticus leaves protein CALP against t-BOOH induced DNA damage was studied using submarine gel electrophoresis, where ascorbic acid is used as the positive control. The trypan blue dye exclusion method was used to find the protective and non toxic nature of protein, where antioxidants like Butylated Hydroxylnisole (BHA) and Ascorbic acid were used as the positive control. The results showed that the molecular weight of Coleus aromaticus plant leaves protein (CALP) is 36 kDa. The purified CALP antioxidant capacity is compared to α- tocopheroland showed maximum protection at a concentration of 25ug.The cell viability study showed that lymphocytes treated with t-BOOH in presence of CALP (25µg) exhibited 69 ± 1% viability, whereas BHA (400μM) had 70 ± 2% viability and ascorbic acid (400µM) had 75 ± 2 % viability when compared to untreated lymphocytes (81 ± 3%) as they are not affected by any external factor like t-BOOH. The purified 36kDa CALP is a nontoxic antioxidant which provides protection against DNA damage induced by t-BOOH.

In the present clinical study, it was aimed to investigate the possible effects of Trypan blue (TB) use on the corneal endothelium during cataract surgery in eyes with pseudoexfoliation syndrome (PEX) during a three-month follow-up period using the contralateral eye control design.This prospective, randomised controlled, individual cohort study included 92 eyes of 46 patients with bilateral PEX and cataracts. While 1% TB was applied to one eye of the patients before capsulorhexis (study group), it was not applied to the other eye (control group). Both groups were compared preoperatively and postoperatively in terms of endothelial cell density (ECD), endothelial cell loss (%), pleomorphism, polymegathism and central corneal thickness (CCT) using specular microscopy.Preoperative corneal ECD was measured as 2362.56 ± 253.27 in the study group, 2380.84 ± 220.54 in the control group, and 2145.58 ± 221.71 in the study group and 2184.97 ± 200.94 cells/mmThis study demonstrated that the injection of 1% TB into the anterior chamber for staining the anterior capsule during cataract surgery did not cause significant corneal endothelial changes at postoperative 3

Single crystals of pure and trypan blue (TB; C34H28N6O14S4) stained (0.01 mol%) potassium hydrogen phthalate (KAP; C8H5KO4) were grown by slow evaporation solution method. Experimentally obtained crystal habit of KAP matched well with the theoretical morphology predicted using BFDH laws. Eight planes ({111} & { 11 1 ¯ } ) out of total fourteen planes got selectively coloured by TB dye molecules. Affinity of TB dye towards specific growth sectors can help various scientists to understand the selective nature of dye molecules and thereby will help in proposing novel tailor-made stained photonic crystals. UV–Vis-NIR spectra for TB stained KAP crystals displayed a strong absorption peak at 563 nm, which was blue shifted for TB dye in KAP matrix relative to neutral aqueous solution of TB dye. This shift in the absorption maxima towards lower wavelength confirms the successful colour insertion into selective growth sectors of stained KAP crystals. The effect of TB staining on various optical parameters namely optical conductivity, optical band gap, refractive index and extinction coefficient of host KAP matrix was studied. The structural conformation and the recognition of different functional groups present in as grown KAP crystals were approved by powder XRD and FTIR studies, respectively. Dielectric constant increased and dielectric loss decreased as a result of TB dye staining in KAP material. The piezoelectric conversion efficiency got increased from 1.22 pC/N for un-dyed KAP single crystal to 1.53 pC/N for TB stained KAP single crystals. In Vickers micro-hardness studies, TB stained KAP crystals displayed better mechanical strength.

Trypan blue staining is a classic way of visualizing leaf disease and wound responses in plants, but it involves working with toxic chemicals and is time-consuming (2–3 days). Here, the investigators established near-infrared scanning with standard lab equipment as a fast and nondestructive method for the analysis of leaf injuries compared with trypan blue staining. Pathogen-inoculated and wounded leaves from potato, tomato, spinach, strawberry, and arabidopsis plants were used for proof of concept. The results showed that this newly developed protocol with near-infrared scanning gave the same results as trypan blue staining. Furthermore, a macro in FIJI was made to quantify the leaf damage. The new protocol was time-efficient, nondestructive, chemical-free and may be used for high-throughput studies.

Arbuscular mycorrhizal (AM) fungi form mutualistic symbiosis with root of host plant. Staining technique to detect AM fungi usually used hazardous chemical. The ink stain and vinegar were used as an alternative technique to replace trypan blue and lactic acid in root staining method. This study aimed to determine time for clearing root cell contents and ink stain type to visualize the best AM fungal structures within the root observed under light microscope. Pueraria phaseoloides var. javanica roots colonized by AM fungi were cut into 1 cm long, cleared in KOH solution and stained. Four clearing time were done vis 5, 10, 15 and 20 minutes, and four stains were used namely Shaeffer black ink, Parker Quink blue ink, blue stamp ink, and trypan blue. Twenty stained roots were taken randomly from each tratment, and observed. Root clearing process for 20 minutes showed the best result. Only Shaeffer black ink and trypan blue produced clear structure of external hyphae, internal hyphae, vesicles and arbuscules. Arbuscular structure stained only by Shaeffer black ink and trypan blue. This indicated that Shaeffer black ink could be used as an alternative stain to detect AM fungi within the root of host plant

Trypan blue dye exclusion-based cell viability measurements are highly dependent upon image quality and consistency. In order to make measurements repeatable, one must be able to reliably capture images at a consistent focal plane, and with signal-to-noise ratio within appropriate limits to support proper execution of image analysis routines. Imaging chambers and imaging systems used for trypan blue analysis can be inconsistent or can drift over time, leading to a need to assure the acquisition of images prior to automated image analysis. Although cell-based autofocus techniques can be applied, the heterogeneity and complexity of the cell samples can make it difficult to assure the effectiveness, repeatability and accuracy of the routine for each measurement. Instead of auto-focusing on cells in our images, we add control beads to the images, and use them to repeatedly return to a reference focal plane. We use bead image features that have stable profiles across a wide range of focal values and exposure levels. We created a predictive model based on image quality features computed over reference datasets. Because the beads have little variation, we can determine the reference plane from bead image features computed over a single-shot image and can reproducibly return to that reference plane with each sample. The achieved accuracy (over 95%) is within the limits of the actuator repeatability. We demonstrate that a small number of beads (less than 3 beads per image) is needed to achieve this accuracy. We have also developed an open-source Graphical User Interface called Bead Benchmarking-Focus And Intensity Tool (BB-FAIT) to implement these methods for a semi-automated cell viability analyser.It is critical for the manufacturing and release of living cell-based therapies to determine the viability, the ratio of living cells to the total number of cells (live and dead), in the therapy. Dead cells can be a safety concern for the patient, and dosing is often based on the number of living cells which are the active ingredient of the drug product. Currently, the most common approach to evaluating cell viability is based on the staining of cell samples with the trypan blue marker of cell membrane integrity: a loss in cell membrane integrity with cell death allows the dye into the cell, which can be seen using brightfield microscopy. To classify cells as live/dead, the brightness of the cells is evaluated and cells with bright centres are considered live, while those with dark centres are considered dead. Unfortunately, this approach of staining, imaging and classification is very sensitive to image acquisition settings, including image focus and brightness. This paper introduces a method to establish the required image quality for image viability analysis, providing a tool to return to image acquisition settings that will ensure image quality even when there is variability from sample to sample. In this method, polymeric beads are added to each cell sample prior to cell viability analysis. Using image processing, we extract key features from the beads in the image such as sharpness of the edges of the beads. The image features of the cells can vary significantly from sample to sample and under different cell conditions, but image features of beads have proved to be consistent across samples. We are thus able to collect reference datasets quantifying bead features over a wide range of image acquisition settings (brightness and focus), allowing us to establish a reference focal plan for image acquisition for any cell sample based on bead features. We show that with as few as three beads per image, the reference focal plane can be found from a single acquisition of beads image data over a wide range of image focuses and brightness, allowing users to consistently acquire images for cell viability that meet pre-defined quality requirements.

Background: Ocean acts as a wealthy resource of biological diversity producing prospective novel metabolites from the organisms present in the environment. Novel bioactive compounds from Echinoderms mainly from sea stars have been extensively studied procuring rich diverse compounds which exhibits in vitro cytotoxicity comparable to or better than those of the potent anticancer drug. These sea stars which reside in the benthic region serves as their habitat and they are therefore predators and persistently being attacked by various organisms subsequently they acclimatize the survival approach to defend themselves from the external pathogens. Since these metabolites are obscured as a metabolic product during their endurance an initial evaluation of these compounds is necessary to assess human risk. Objectives: The present study focuses in vitro toxicity evaluation of isolated sterol like isolate from echinoderm sea star Stellaster equestris by incorporating hemolytic, chromosomal aberration assay and the cell viability tryphan blue exclusion assay against human peripheral blood. Methods: The in vitro toxicity evaluation was studied against the human peripheral blood collected from the healthy donors with the defined concentration of the sterol like compound from the sea star Stellaster equestris. The hemolytic, cell viability trypan blue exclusion method and chromosomal aberration assay were performed to check the hemolytic, mutagenic and genotoxic effect against the lymphocytes and the red blood cells. Results and Discussion: The result suggested that the hemolytic assay and the cell viability assay even in a dose dependent manner were non-hemolytic and percentage of the viability was not affected due to the exposure of the compound. The absence of genotoxicity was evident for the chromosomal aberration assay indicated that the isolated compound from the sea star Stellaster equestris might be considered as effective novel compound.

This work aimed to evaluate antiviral, antibacterial and anti-quorum sensing effects of a lyophilized aqueous pine resin extract. The pine resin toxicity was evaluated by a crystal violet assay at 492 nm for Hep-2 cells and the trypan blue method for P3HR1. The cytotoxicity expressed in CC50 was 12.5 mg/ml and 0.23 mg/ml on Hep-2 and P3HR1 cells, respectively. The antiviral effect was obtained at 4 mg/ml against coxsackievirus B4 (CVB4). Using the diffusion method discs for the antibacterial activity, the extract was more active against Gram+ than Gram−. Inhibition zones of 22 mm were obtained at a 275 mg/ml concentration. Inhibition of violacein production by pine resin extract was tested using Chomobacterium violaceum 026 (CV026) in a medium supplemented with acyl-homoserine lactone (AHL); cytotoxicity was evaluated toward Artemia and its protecting effect against Vibrio harveyi BB120 and then AHL degradation was studied. Results showed that pine resin inhibited violacein production by CV026, was not toxic for Artemia and protected it against Vibrio harveyi BB120; AHL was degraded with inhibition areas from 9.6, 11.03 and 10.26 to 12.34 mm at the contact times of the mixtures “resin-AHL” respectively at 0 h, 4 h 24 h and 72 h. This study constitutes a confirmation of aqueous extract pine resin as antibacterial, by anti-quorum sensing pathway and antiviral efficacy.

Background: Over-expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1 is associated with resistance to chemotherapeutic agents such as fludarabine. Moreover, an inverse relationship between miRNA-15a levels with Bcl-2 and Mcl-1 expression has been observed in CLL patients. In this study, the effect of miRNA-15a on apoptosis and sensitivity of the CLL cells to fludarabine was investigated. Methods: After treatments, the Mcl-1 and Bcl-2 expression levels were quantified by RT-qPCR. Trypan blue assay was used to explore the effects of miRNA-15a and fludarabine on cell proliferation. The cytotoxicity was measured using MTT assay and combination index analysis. Cell death was determined using cell death detection ELISA assay and caspase-3 activity assay Kits. Results: Results showed that miRNA-15a clearly decreased the mRNA levels of Bcl-2 and Mcl-1 in a time dependent manner, which led to CLL-II cell proliferation inhibition and enhancement of apoptosis (p < 0.05, relative to control). Transfection of the miRNA-15a synergistically reduced the cell survival rate and lowered the IC50 value of fludarabine. Furthermore, miRNA-15a significantly enhanced the apoptotic effect of fludarabine. Conclusions: Our data propose that suppression of Bcl-2 and Mcl-1 by miRNA-15a can effectively inhibit the cell proliferation and sensitize CLL cells to fludarabine. Therefore, miRNA-15a can be considered as a potential therapeutic target in CLL resistant patients.

Cell lines have been established to preserve the genetic material of endangered animals. This study aims to establish, characterize and authenticate fibroblast cells derived from the kidney tissue of a Sumatran rhinoceros’ carcass. The primary cultures were obtained using the mixed enzymatic-explant method, supplemented with complete media and maintained at 37oC with 5% CO2 in an incubator. Following routine trypsinization, viability and growth curves were generated through the Trypan Blue counting method. Cellular senescence was quantified by Sa-β-gal staining assay and G-banding for karyotyping. As a result, the cell derivation had generated 81 frozen stocks. The viability of cells at P5 and P10 showed reasonable recovery after six months. Cell population doubling time at P5 was 20.45 hours, while it was 22.35 hours at P10 and P15. The senescence level significantly increased from P5 to P10, and was especially significant at P15. Genetic stabilities were considered stable at P5 and P10, with frequency of over 70 %. In conclusion, this study was able to derive a primary fibroblast culture from the preserved tissue of a Sumatran rhinoceros, with certain changes in morphology, senescence level, growth curves and cell viability as the number of passages increased.

Introduction. Boron neutron capture therapy (bnct) is a promising method for treating tumors, in particular, infiltrative malignant tumors, due to the selective destruction of tumor cells without damaging the surrounding normal tissues. This type of therapy is based on nuclear reaction of neutron capture by stable 10b isotope. For the successful implementation of bnct, boron delivery drugs that must be selectively accumulated in malignant cells in a sufficient amount, and a neutron source with the energy required for the neutron capture reaction are needed. At the budker institute of nuclear physics, the accelerator-based neutron source was designed with flux parameters allowing studies on bnct to be conducted.Objective: to assess the effect of bnct on tumor and normal cell lines using borphenylalanine (bpa), borcaptate (bsh) and liposomal borcaptat as boron delivery drugs.Materials and methods. Human cell cultures: glioblastoma (u87), colorectal human adenocarcinoma (sw-620), human melanoma (sk-mel28) and primary embryonic cell lines were irradiated with a neutron flux at the presence of bpa, bsh and liposomal bsh with a concentration of 10b 40 μg/ml. The short-term cytotoxic effect of irradiation was evaluated using trypan blue. Cell survival 96 hours after irradiation was determined using mtt test, and survival fraction was evaluated using the clonogenic test.Results. Early cytotoxic effects of irradiation were not observed for all 4 cell lines. According to mtt and clonogenic tests, the most pronounced effect of bnct was noticed for sw-620 and u87 lines, regardless of boron delivery drug used. For sk-mel28 line, the best effect was achieved after irradiation with liposomal borocaptate. For the primary transplanted embryonic line, high toxicity was revealed when bnct was performed with borphenylalanine and borcaptate.Conclusion. The data obtained indicate that the accelerator-based bnct using boron delivery drugs, such as borphenylalanine, borcaptate and liposomal borcaptat, has a positive effect on tumor lines of glioblastoma, colorectal adenocarcinoma and melanoma.

Dye is a common pollutant present in many chemical industrial waste water. Advanced oxidation processes are widely used for dye degradation. In this study, the degradation of trypan blue dye was examined by Fenton process. Neutralized red mud catalyst was used as a source of ferrous ion for Fenton’s process. The dye degradation performance has been analyzed by using circulating fluidized-bed reactor. The influence of some key parameters such as pH, initial dye concentration, catalyst dosage and hydrogen peroxide concentration on the degradation of dye has been investigated. All the experiments were performed for 90 min. The initial dye concentration was taken as 1.56 × 10−5 and 2.60 × 10−5 mol L−1 and the amount of catalyst was varied from 0.5 to 0.7 gm/L. The hydrogen peroxide was taken in the ratio of 1:20 with the catalyst. The effect of pH was studied in the varying range from 3–5. It was found that the more the acidic pH, more will be the rate of degradation. The increase in pH results in the lower degradation rate. As the amount of catalyst was increased, the degradation rate got increased. The optimized results were obtained at pH 3, catalyst dosage of 0.7 gm/L and dye concentration of 1.56 × 10−5 mol L−1. Subsequently, the reaction kinetics of Fluidized-bed reactor was also studied.

BACKGROUND/AIMS: Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. METHODS: We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. RESULTS: We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. CONCLUSION: In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.

ObjectivesTo develop a new dye formulation for vitreoretinal surgery, which shows increased transparency for better intraoperative handling with better parameters important for use.Methods and analysisA new blue dye, DDG, was synthesised and tested for toxicity and staining. Diglycerol as new density-increasing additive was identified, and its toxicity and lack of influence of the staining with trypan blue (TB) on a collagen membrane as model for the epiretinal membrane was determined. Transparency of the dye solution was evaluated.ResultsDDG is as alternative to Brilliant Blue G (BBG), with good staining properties for interna limitans models, and a good safety profile. Diglycerol is a new non-toxic additive replacing PEG3350, with reduced viscosity and no reduction in staining, allowing the reduction of TB to achieve the same staining level of the collagen membrane by 40%, with greater transparency of the dye solution and reduced viscosity. Both factors should facilitate a safe removal during surgery.ConclusionA new dye preparation with improved performance in comparison to marketed combinations of BBG and TB was developed. Its reduced TB concentration and viscosity with maintained density allow better tolerance and handling.

The use of femtosecond laser to create sub-microscopic transient pores on the cell membrane allowing exogenous material into mammalian cells has become a very efficient optical delivery method over the past decade. This study focuses on laser-enabled delivery of antiretroviral (ARV) drugs into HIV-1 infected TZM-bl cells in vitro. A 1 kHz femtosecond laser emitting at a wavelength of 800 nm was used to photoporate cells at 6.5 μW. Trypan blue was used for characterisation and its uptake was quantified using Matlab software. Cell membrane damage was assessed using the lactate dehydrogenase (LDH) assay while HIV-1 infection was assessed using luciferase assay. Our results showed successful delivery of ARVs into HIV-1 infected cells without compromising their cell membranes, subsequently reducing the level of infection. The LDH assay showed no significant cell membrane damage of laser-treated cells, and the luciferase assay demonstrated significant reduction in the level of HIV-1 infection.

The anti-cancer effects of vitamin D are of fundamental interest. Cholecalciferol is sequentially hydroxylated endogenously to calcidiol and calcitriol. Here, SiHa epidermoid cervical cancer cells were treated with cholecalciferol (10–2600 nmol/L). Cell count and viability were assayed using Crystal Violet and Trypan Blue, respectively. Apoptosis was assessed using flow cytometry for early and late biomarkers along with brightfield microscopy and transmission electron microscopy. Autocrine vitamin D metabolism was analysed by reverse transcription-quantitative PCR and immunoblotting for activating enzymes: 25-hydroxylases (CYP2R1 and CYP27A1) and 1α-hydroxylase (CYP27B1), the catabolic 24-hydroxylase (CYP24A1), and the vitamin D receptor (VDR). Data were analysed using one-way ANOVA and Bonferroni post-hoc test, and p

Purpose: To evaluate the influence of DMEM extract of Turkish propolis (TP) on the morphology of metastatic MDA-MB-231 cells. Methods: The cells were incubated with DMEM extract of TP (collected from Trabzon in Turkey) at a dose of 2.5 mg/mL for 72 h. The effect of DMEM extract on proliferation and cytotoxicity of the cells was determined using 3-[4,5-dimethyltiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assay. MDA-MB-231 cells incubated with or without extracts were randomly photographed with a camera-coupled inverted microscope. Treated and control MDA-MB-231 cells were classified as monopolar, bipolar or multipolar, and their dimensions measured with an electronic caliper. Results: Although the extract reduced the proliferation of the cells, the effect was not statistically significant (p < 0.05). Moreover, no cytotoxic effect was observed. Field diameters, process length and cell body diameters of the treated cells were increased by DMEM extract treatment in bipolar and multipolar cell types, but these parameters were decreased in monopolar cell type, although insignificantly (p < 0.05). In addition, the process thickness of treated MDA-MB-231 cells increased insignificantly in all cell types (p < 0.05). Conclusion: These findings indicate that DMEM extract of TP at a dose of 2.5 mg/mL morphologically suppresses monopolar MDA-MB-231 cells. Future studies would examine the morphological effects of different concentrations of the propolis extract in anti-proliferation, cytotoxicity and morphological investigations in MDA-MB-231 cells.

Purpose: To investigate the neuroprotective effect of lidocaine in Neuro2A cells Methods: Differentiated N2a cells were used in this study. Cell viability and neuroprotection were assessed using dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays, while Bax/Bcl-2 expression was assayed by western blotting. Mitochondrial membrane potential, reactive oxygen species and calcium levels were measured using flow cytometry. Results: Lidocaine protected differentiated N2a cells against cadmium-induced toxicity, and also attenuated cadmium toxicity-induced changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and calcium (Ca2+) levels. Furthermore, Bax/Bcl-2 ratio, which was disrupted by cadmium, and cadmium-induced apoptosis, were reversed by lidocaine. Conclusion: Lidocaine protects differentiated N2a cells against cadmium-induced toxicity by reversing apoptosis. Thus, lidocaine is a potential neuroprotective agent.

Drug resistance of sorafenib seriously affects the treatment effect of late-stage hepatocellular carcinoma (HCC) patients. However, the precise mechanism of resistance to sorafenib remains unclear. Therefore, to obtain a deep understand of sorafenib resistance mechanisms and find potential therapeutic targets are very important for improving the clinical prognosis of HCC patients. In this study, a label-free quantitative proteomics method was performed to investigate the proteins differentially expressed between HepG2 and the sorafenib-acquired resistance HepG2 (HepG2-R) cells. In total, 84 differential expressed proteins were identified between the two cell lines. Bioinformatics analysis results demonstrated the dysregulated metabolic processes have a significant impact on the drug resistance of HepG2-R cells. Among them, the expression of Microsomal glutathione S-transferase 1 (MGST1) in two cell lines was further confirmed by western blot method. Moreover, colony formation assay and trypan blue dye assay results revealed that MGST1 is closely connected with the sorafenib resistance of HepG2-R cells, and the knockdown of MGST1 increased the sensitivity of sorafenib resistance HepG2-R cells to sorafenib treatment. In conclusion, these results lay a foundation for deciphering the mechanism for HCC sorafenib resistance and present a possibility of MGST1 serving as a therapeutic target for the treatment of sorafenib resistance HCC.

Background: Toxoplasmosis is a disease that results from infection with an obligate intracellular T. gondii parasite, one of the world's most common parasites. Considering the complications of chemical drugs and the need for an appropriate drug combination for treatment of toxoplasmosis and also considering the antimicrobial potential of chitosan, as a natural source, this study was aimed to evaluate in vitro activity of commercial chitosan (CC) on T. gondii. Methods: In this experimental study, the tachyzoites of T. gondii was collected from the peritoneal exudates from infected Balb/c mice. The tachyzoites were diluted in phosphate buffer saline (PBS). Chitosan with low molecular weight was commercially purchased. Then, at concentrations of 10, 50, 100 and 200 µg/mL and after 30, 60, 120 and 180 minutes the viability of tachyzoites were determined by using trypan blue 0.1%. Anti-T.gondii activity of CC in all concentration was significantly higher than pyrimethamine as control group (P=0.05). Results: The concentration of 200 µg/mL of CC had the highest effects and killed 30.5, 52, 59 and 81.5% of tachyzoites after 30, 60, 120 and 180 minutes. Moreover, IC50 values of CC were 515, 171, 12.5 and Conclusion: Our results indicate chitosan in low molecular weight had potent activity against T. gondii tachyzoites and could be an appropriate candidate for treatment of at least acute toxoplasmosis, certainly, after complementary in vivo experiments.

Background and objective. Glass-Ionomer (G.I.) restoratives are used to manage cervical tooth defects; however certain chemicals could leach out of the material and affect the adjacent gingival tissue. Therefore, this study aimed to assess the viability and attachment of human gingival epithelial cells (HGECs) on both resin-coated and uncoated Equia fil G.I. restorative materials. Materials and Methods. Two groups of G.I. disc specimens (n= 34 each) were constructed from Equia fil G.I. restorative by mixing the content of G.I. capsules, then injection into silicone molds, the material was left for setting before cutting the excess. Surfaces of group I specimens were coated with the accompanied protective Equia resin, while those in group II were left uncoated. HGECs were isolated and incubated with the constructed specimens. The cell viability was then assessed using both Methyl Tetrazolium (MTT) and Trypan blue dye exclusion (TBDE) assays. The micromorphology and the attaching ability of the isolated cells to specimens’ surfaces were assessed using SEM. The surface roughness was measured and the chemical analysis of G.I. surfaces was also analyzed by energy disperse x-ray. Results. Reduction in HGECs viability was noticed in contact with G.I. of both test groups. However, HGECs showed better viability and attaching ability to the resin-coated G.I. surface than were in presence of uncoated restorative. Conclusion. Resin Coating preserves the cell viability and improves the attachment of HGECs on Equia fil G.I restorative surfaces. This finding, therefore, offers sound evidence to select such material in treating sub-gingival cervical tooth defects.

The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay is one of the most commonly used tests of cell proliferation. Hydralazine has been reported to interfere with the performance of the MTS assay when used on adherent cells. This study aimed to investigate whether hydralazine interferes with the performance of the MTS assay on suspended cells. THP-1 (a monocytic leukemia cell line) cells were cultured in the presence or absence of hydralazine (0, 10, 50, 100, and 500 μM) for 2 or 24 h. Cell numbers were analyzed using the MTS, trypan blue exclusion, or microscopic assays. A modified version of the standard MTS assay was established by centrifuging the cells and replacing the test medium with fresh culture medium immediately before the addition of the MTS reagent. Culture of THP-1 cells with hydralazine at concentrations of 50, 100, and 500 μM for 2 h increased absorbance (p

Estimation of realized fecundity (Freal, number of viable oocytes produced) is an essential, yet seldom-achieved element in the understanding of marine animal production and population dynamics. We used the Neutral Red (NR) vital stain to determine oocyte viability in spawns and gonad strippings of four species of commercially-important bivalves: Cerastoderma edule (L), Crassostrea gigas Thunberg, Mytilus edulis L, and Tapes philippinarum (Adams and Reeve). The utility of Trypan Blue as a complementary mortal stain was also assessed, and found to be unnecessary. Normal, live oocytes stained with NR, and were either spherical (mature oocytes) or pedunculated (immature oocytes); atresic/abnormal oocytes also stained with NR, allowing their ready recognition due to their abnormal shapes and cytoplasmic retraction. Dead oocytes did not stain with NR. Across the species studied, a considerable and highly variable proportion of spawned or stripped oocytes was either dead or non-viable; quantitative counts were performed for Cerastoderma edule, the only species for which > 5 spawns occurred. The high level and variability (34–85%) of dead or non-viable oocytes is consistent with a reproductive Red Queen dilemma, in which greater oocyte numbers do not translate to commensurately greater real fecundities, and also with a Sweepstakes Reproductive Success strategy, in which a large range of Freal confronts the considerable variability of intertidal environmental conditions. Neutral Red vital staining is a promising tool for the elucidation and optimization of crucial yet previously intractable aspects of bivalve hatchery production, genetic improvement, restocking, stock management, and conservation.

Plants possess unique bioactive principles justifying their multifunctional properties. In this work silver nanoparticles (AgNPs) were prepared via a photo-mediated green approach for enhanced antimicrobial and anticancer activity. The aqueous rhizome extract of Curcuma zanthorrhiza Roxb. (Cz) was employed as a source of reducing and capping agent for the synthesis of colloidal Cz-AgNPs. The materialization of Cz-AgNPs was identified from the surface plasmon resonance observed at 416 nm using UV–Visible spectrophotometer. The FTIR analysis of both Cz-AgNPs and plant rhizome extract revealed the as-synthesized Cz-AgNPs were capped with plant derived phytoconstituents. The crystallinity, surface morphology, hydrodynamic size and surface area of Cz-AgNPs were characterized by PXRD, HR-TEM, FESEM, DLS and BET analysis. The as-synthesized Cz-AgNPs exhibited strong antimicrobial activities against bacterial strains and plant pathogenic fungi in a dose-dependent manner. The antimicrobial activity of Cz-AgNPs was assessed using standard disc diffusion, broth microdilution assay for bacterial strains and poisoned food technique for fungal strains. The Cz-AgNPs were inhibitory to all the microbes even at the lowest concentration. The biocompatible nature of Cz-AgNPs was analyzed for hemolytic properties with human erythrocytes and promising results were observed. In addition, the cytotoxicity activity (Trypan Blue Exclusion Method) on the cell lines of Dalton’s Lymphoma Ascites (DLA) and Ehrlich Ascites Carcinoma (EAC) showed Cz-AgNPs exhibited potential anticancer activity with concentration as low as 1.67 µg/mL and 1.84 µg/mL respectively (IC50 value). This work attempted at optimizing a safe dosage of silver nanoparticles using hemolysis assay and thus for application in therapy.