Hitting two targets with one shot on pesticide genotoxicity assessment - Identifying risk while unveiling ex vivo approach as a throughput tool in gill-breathing animals.

Pesticides in the environment often compromise the ecosystem, thus requiring reliable approaches to assess their effects. Commonly used approaches, such as in vivo, come with several disadvantages, namely in the light of the 3 R's policy. Seeking for accurate and ethical approaches, this study intended to validate the ex vivo technique as an alternative, and to assess the genotoxicity of chemically-based pesticides and a biopesticide. The ex vivo approach was applied to gill cells of Procambarus clarkii for 2, 4 and 8 h. Cell viability and DNA integrity were evaluated to determine the applicability of this approach. Crayfish gill cells only showed to be suitable for exposures of 2 h. Accordingly, genotoxicity was evaluated in gill cells exposed, for 2 h, to environmentally relevant concentrations of the chemically-based pesticides dimethoate (20 µg L-1), imazalil (160 µg L-1) and penoxsulam (23 µg L-1), as well as to the bioinsecticide Turex® (25, 50, 100, 200 and 400 µg L-1). Every chemically-based pesticide demonstrated to be genotoxic, despite not inducing oxidative DNA damage. On the other hand, Turex® showed no genotoxic effects. Overall, the ex vivo approach demonstrated to be possible and practical to implement, improving the number of outcomes with a lower number of organisms. The findings from the screening test suggest that biological pesticides may pose a lower risk to non-target organisms compared to chemically-based pesticides.

Impact of CAD/CAM burs lifetime on surface properties of dental zirconia: Implications for biocompatibility of custom abutments.

OBJECTIVE: To evaluate the effect of the deterioration of computer aided design/computer aided manufacturing (CAD/CAM) burs during zirconia milling, on surface roughness, contact angle, and fibroblast viability. MATERIALS AND METHODS: Ceramic blocks were milled and 75 ceramic disks (8 × 1.5 mm) made and allocated into three groups (n = 25): G1-brand new 2L and 1L burs, G2-2L bur at the end of lifetime and brand new 1L bur and G3-both burs at the end of their lifetimes. Roughness (Ra, Rq, and Rz) was evaluated using a 3D optical profilometer, the contact angle by the sessile drop method and the cell viability of the mouse NIH/3T3 fibroblast, using the Alamar Blue assay at intervals of 24, 48, and 72 h (ISO 10993-5). Data were analyzed by one-way ANOVA and Kruskal-Wallis tests (p ≤ 0.05). RESULTS: Roughness increased as the burs deteriorated and G3 (0.27 ± 0.04) presented a higher value for Ra (p < 0.001). The highest contact angle was observed in G3 (86.2 ± 2.66) when compared with G1 (63.7 ± 12.49) and G2 (75.3 ± 6.36) (p < 0.001). Alamar Blue indicated an increase in cell proliferation, with no significant differences among the groups at 24 and 72 h (p > 0.05). CONCLUSIONS: The deterioration of the burs increased the surface roughness and decreased the wettability, but did not interfere in cell viability and proliferation. CLINICAL SIGNIFICANCE: The use of custom zirconia abutments represents an effective strategy for single crowns restorations. Our findings suggest that these abutments can be efficiently milled using CAD/CAM burs within their recommended lifetime.

m6A methylation of RNF43 inhibits the progression of endometriosis through regulating oxidative phosphorylation via NDUFS1.

Oxidative phosphorylation is becoming increasingly important in the induction and development of endometriosis. Recently, it has been reported that ring finger protein 43 (RNF43) is involved in the process of oxidative phosphorylation, but the mechanism remains unclear. Our investigation is to delve into the roles of RNF43 in endometriosis and elucidate the related mechanisms. We found RNF43 was downregulated in ectopic endometrial tissue and primary ectopic endometrial stromal cells (ECESCs). Knockdown of RNF43 enhanced cell viability and migration by activating oxidative phosphorylation in eutopic endometrial stromal cells (EUESCs), while overexpression of RNF43 led to the opposite results. Moreover, RNF43 reinforced the ubiquitination and degradation of NADH dehydrogenase Fe-S protein 1 (NDUFS1) by interacting with it. Likewise to RNF43 overexpression, NDUFS1 silencing inhibited cell viability, migration, and oxidative phosphorylation in ECESCs. NDUFS1 was a downstream target of RNF43, mediating its biological role in endometriosis. Interestingly, the expression and stability of RNF43 mRNA were regulated by the Methyltransferase-like 3 (METTL3)/IGF2BP2 m6A modification axis. The results of rat experiments showed decreased RNF43 expression and increased NDUFS1 expression in endometriosis rats, which was enhanced by METTL3 inhibition. Those observations indicated that m6A methylation-mediated RNF43 negatively affects viability and migration of endometrial stromal cells through regulating oxidative phosphorylation via NDUFS1. The discovery of METTL3/RNF43/NDUFS1 axis suggested promising therapeutic targets for endometriosis.

Establishment of Physalis alkekengi cell suspension culture: time-dependent behavior of genes related to the steroidal compounds, key enzymes, and physalins under static magnetic field.

Cell suspension culture has the potential to be a valuable source for the bioactive compound productions. In this study, an optimized procedure was established for callus and cell suspension culture of Physalis alkekengi for the first time, and the impact of static magnetic field (SMF, 6 mT) was studied on the high-value metabolic compounds through investigation of signaling molecules and gene expressions at the late log-to-stationary phase. Results showed that the growth regulators of 6-benzyl amino purine (BAP, 1.5 mg-1 L) and 1-naphthaleneacetic acid (NAA, 0.4 mg-1 L) induced the highest fresh weight, callus rate, callus index, and total withanolides. Cell suspension culture was established in the liquid MS medium supplied with BAP (1.5 mg-1 L) and NAA (0.1 mg-1 L). SMF application decreased slightly the cell growth and viability and enhanced the number of round-shaped cells. The hydrogen peroxide (H2O2) and nitric oxide (NO) levels increased at an all-time series after SMF exposure, and their maximum contents were observed after 12 h. A significant alteration of malondialdehyde content was also identified after 12 h of SMF exposure. The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxyD-xylulose 5-phosphate synthase (DXS), squalene synthase (SQS), sterol Δ7-reductase (DWF5), and C-7,8 sterol isomerase (HYD1) genes was upregulated significantly after 24 and 48 h. An increase in the total withanolides was related to more activity of HMGR and DXS enzymes in SMF-exposed cells and the maximum physalin A (12.8 mg g-1 DW) and physalin B (1.92 mg g-1 DW) obtained after 24 h compared to controls. Findings suggest that SMF can play a supportive factor in inducing steroidal compounds in P. alkekengi through modulating H2O2 and NO levels and the related-gene expressions.

Exploring the Biosafety Potential of Haberlea rhodopensis Friv. In Vitro Culture Total Ethanol Extract: A Comprehensive Assessment of Genotoxicity, Mitotoxicity, and Cytotoxicity for Therapeutic Applications.

The escalating elderly population worldwide has prompted a surge of interest in longevity medicine. Its goal is to interfere with the speed of ageing by slowing it down or even reversing its accompanying effects. As a field, it is rapidly growing and spreading into different branches. One of these is the use of nutraceuticals as anti-ageing drugs. This field is gaining massive popularity nowadays, as people are shifting towards a more natural approach to life and seeking to use natural products as a source of medicine. The present article focuses on the cellular effect of Haberlea rhodopensis Friv. in vitro culture total ethanol extract (HRT), produced by a sustainable biotechnological approach. The extract showed a similar phytochemical profile to plant leaf extract and was rich in primary bioactive ingredients-caffeoyl phenylethanoid glycosides, myconoside, and paucifloside. This study examined the biosafety potential, cytotoxicity, genotoxicity, and mitochondrial activity of the extract using in vitro cultures. The results showed high cell survival rates and minimal cytotoxic effects on Lep3 cells, with no induction of reactive oxygen species nor genotoxicity. Additionally, the extract positively influenced mitochondrial activity, indicating potential benefits for cellular health. The results are promising and show the beneficial effect of HRT without the observation of any adverse effects, which sets the foundation for its further testing and potential therapeutic applications.

Impact of cellular ATP levels on cell viability in response to fluorouracil through lysophosphatidic acid (LPA) receptor-4 (LPA4) and LPA6 in colon cancer cells.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) mediates various aspects of cancer cell behaviors. This study aimed to investigate the variation in intracellular ATP levels and its impact on cell viability in response to fluorouracil (5-FU) through LPA4 and LPA6 in colon cancer DLD-1 cells. LPA4 and LPA6 are linked to Gs and Gi proteins. Gs protein stimulates the activity of adenylyl cyclase, which catalyzes the conversion of ATP to cAMP, whereas Gi protein inhibits this activity. In cell survival assay, cells were treated with 5-FU every 24 h for 3 days. The viability in response to 5-FU in DLD-1 cells was enhanced by LPA4 and LPA6 knockdowns. Furthermore, LPA4 and LPA6 knockdowns reduced the expression of cleaved-PARP1 protein when cells were treated with 5-FU. Since ethidium bromide (EtBr) reduces mitochondrial DNA level in cultured cells, EtBr-treated (DLD-EtBr) cells were generated from DLD-1 cells. The viability to 5-FU in DLD-EtBr cells was higher than that of DLD-1 cells. Additionally, culturing DLD-1 cells in a low glucose-containing medium led to increased viability to 5-FU. LPAR4 and LPAR6 expressions were reduced in both DLD-EtBr and low glucose-treated cells. The cellular ATP levels were significantly decreased in DLD-1 cells following EtBr treatment and exposure to low glucose conditions. Conversely, in the presence of LPA, LPA4 and LPA6 knockdowns resulted in a marked elevation of ATP levels. These results suggest that cell viability to 5-FU is negatively regulated via the activation of LPA4-and LPA6-Gs protein pathways in DLD-1 cells rather than Gi protein.

Bioinformatics comparison of hemolysin in different bacteria and experimental evaluation of anti-cancer properties of extracts of some hemolysin-producing bacteria.

Cancer is one of the main causes of death in the world. Resistance to anticancer treatments in patients with advanced solid tumors leads to new treatments. Therefore, more alternative anticancer methods have been found over time with greater specificity against tumor cells and with less or no adverse effects on normal cells. Bacterial spores of obligate anaerobes exclusively germinate in the hypoxic/necrotic areas and not in the well oxygenated areas of the body. This unique phenomenon has been exploited in using bacterial spores as a remedy for cancer. Bacterial toxins also play a significant role in either directly killing tumor cells or altering the cellular processes of the tumor cells which ultimately leads to the inhibition and regression of the solid tumor. In the microbial environment, pathogens such as Staphylococcus aureus, Bacillus cereus, or Streptococcus pyogenes produce hemolysin. This protein is used as an anti-cancer protein. To identify the production of hemolysin by bacteria, which can destroy cancer cells more effectively, different bacterial strains were first cultured in blood agar culture medium. The Strains that completely lysed red blood cells, creating transparent zones, were selected for further investigation. Then, to find out which strains have more ability to lyse red blood cells, the qualitative method of halo diameter measurement was used. Also, using quantitative methods, hemolysin strength in microtubes was determined compared to control samples. The results of the hemolysis in the microtube and the qualitative test results showed similar results. In the next step, the cell viability test was performed with the partially purified proteins. Then, bioinformatics studies such as secondary structure investigation, physicochemical properties, pseudo amino acid composition, and molecular docking were performed. The results of molecular docking showed that the hemolysin protein has the highest affinity for the cholesterol of the cytoplasmic membrane, respectively, of Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus bacteria which play a significant role in either directly killing tumor cells or altering the cellular processes of the tumor cells which ultimately leads to the inhibition and regression of the solid tumor.

Optimization of long-term incubation of precision-cut kidney slices.

Precision-cut kidney slices (PCKS) provide a powerful model to close the gap between in vivo and in vitro research. Publications by various authors favor different incubation conditions, media and antibiotics, that have not yet been compared in a standardized manner. After preparation, rat-PCKS were incubated in a total of nine combinations of incubation media and antibiotics for 4 days. We found that a combination of DMEM/F-12 and gentamicin showed the highest levels of viability. Utilizing both qualitative and quantitative methods, we observed stable levels of cellular viability for ten days when incubated in the most suitable medium combination of DMEM and gentamicin. Additionally, a Calcein Acetoxymethyl/Ethidium Homodimer-1 based live/dead Staining, analysis of total protein content and lactate dehydrogenase (LDH) were explored to assess both short- and long-term tissue viability. PCKS showed a significant decrease in total protein content, leveling off at around 60% over the duration of 10 days. To be able to evaluate viability irrespective of decreases in total protein detected, we chose to utilize the alamarBlue Cell Viability Assay. Quantifying both intra- and extracellular activity of LDH, while using different concentrations of ethanol as a positive control, we explored enzyme content as a parameter for cell membrane damage and cytotoxicity in PCKS. Overall, we showed that PCKS are suitable for both short- and long-term observation by optimizing incubation parameters, with numerous possibilities for other assays and methods in future studies.

Buyang Huanwu Decoction suppresses ischemic stroke by suppressing glycolysis and cell apoptosis in rat brain microvascular endothelial cells.

BACKGROUND: Buyang Huanwu Decoction (BHD) is widely used in Chinese clinical practice for the treatment and prevention of ischemic cerebral vascular diseases. This study was designed to investigate the effects of BHD on ischemic stroke (IS) and its underlying mechanism. METHODS: The middle cerebral artery occlusion (MCAO) rat model and oxygen-glucose deprivation and reoxygenation (OGD/R) rat brain microvascular endothelial cell (RBMVEC) models were established. Brain infarction size and neurological score were calculated following MCAO surgery. Evans blue was used to measure blood brain barrier (BBB) permeability. Cell counting kit-8 (CCK-8) and TUNEL assays were performed to evaluate the cell viability and apoptosis of RBMVECs. Dual-luciferase reporter assay was used to analyze the transcriptional activities of apoptosis-related genes. RESULTS: Results showed that higher infarction volume, neurological scores, and BBB permeability in the MCAO group rats were reduced after BHD treatment. Drug serum (DS) treatment had no impact on the normal RBMVECs' cell viability and cell apoptosis. Besides, DS treatment decreased the lactate production, glucose uptake, and extracellular acidification rate in normal and OGD/R-induced RBMVECs. DS treatment downregulated the protein levels of pan-lysine lactylation (kla), histone H3 lysine 18 lactylation (H3K18la), and the transcriptional of apoptotic protease activating factor-1 (Apaf-1) in OGD/R-treated RBMVECs. In addition, Apaf-1 overexpression decreased cell viability and increased apoptosis and glycolysis activity of OGD/R-treated RBMVECs. CONCLUSION: In summary, BHD inhibited glycolysis and apoptosis via suppressing the pan-kla and H3K18la protein levels and the Apaf-1 transcriptional activity, thus restraining the progression of IS.

Synthesis of alga-coated copper oxide nanoparticles with potential applications in shrimp farming.

Copper (Cu) is a crucial element that plays a vital role in facilitating proper biological activities in living organisms. In this study, copper oxide nanoparticles (CuO NPs) were synthesized using a straightforward precipitation chemical method from a copper nitrate precursor at a temperature of 85 °C. Subsequently, these NPs were coated with the aqueous extract of Sargassum angustifolium algae. The size, morphology, and coating of the NPs were analyzed through various methods, revealing dimensions of approximately 50 nm, a multidimensional shaped structure, and successful algae coating. The antibacterial activity of both coated and uncoated CuO NPs against Vibrio harveyi, a significant pathogen in Litopenaeus vannamei, was investigated. Results indicated that the minimum inhibitory concentration (MIC) for uncoated CuO NPs was 1000 µg/mL, whereas for coated CuO NPs, it was 500 µg/mL. Moreover, the antioxidant activity of the synthesized NPs was assessed. Interestingly, uncoated CuO NPs exhibited superior antioxidant activity (IC50 ≥ 16 µg/mL). The study also explored the cytotoxicity of different concentrations (10-100 µg/mL) of both coated and uncoated CuO NPs. Following 48 h of incubation, cell viability assays on shrimp hemocytes and human lymphocytes were conducted. The findings indicated that CuO NPs coated with alga extract at a concentration of 10 µg/mL increased shrimp hemocyte viability. In contrast, uncoated CuO NPs at a concentration of 25 µg/mL and higher, as well as CuO NPs at a concentration of 50 µg/mL and higher, led to a decrease in shrimp hemocyte survival. Notably, this study represents the first quantitative assessment of the toxicity of CuO NPs on shrimp cells, allowing for a comparative analysis with human cells.

Reporting multitargeted potency of Tiaprofenic acid against lung cancer: Molecular fingerprinting, MD simulation, and MTT-based cell viability assay studies.

Lung Cancer (LC) is among the most death-causing cancers, has caused the most destruction and is a gender-neutral cancer, and WHO has kept this cancer on its priority list to find the cure. We have used high-throughput virtual screening, standard precision docking, and extra precise docking for extensive screening of Drug Bank compounds, and the uniqueness of this study is that it considers multiple protein targets of prognosis and metastasis of LC. The docking and MM\GBSA calculation scores for the Tiaprofenic acid (DB01600) against all ten proteins range from -8.422 to -5.727 kcal/mol and - 47.43 to -25.72 kcal/mol, respectively. Also, molecular fingerprinting helped us to understand the interaction pattern of Tiaprofenic acid among all the proteins. Further, we extended our analysis to the molecular dynamic simulation in a neutralised SPC water medium for 100 ns. We analysed the root mean square deviation, fluctuations, and simulative interactions among the protein, ligand, water molecules, and protein-ligand complexes. Most complexes have shown a deviation of <2 Å as cumulative understanding. Also, the fluctuations were lesser, and only a few residues showed the fluctuation with a huge web of interaction between the protein and ligand, providing an edge that supports that the protein and ligand complexes were stable. In the MTT-based Cell Viability Assay, Tiaprofenic Acid exhibited concentration-dependent anti-cancer efficacy against A549 lung cancer cells, significantly reducing viability at 100 µg/mL. These findings highlight its potential as a therapeutic candidate, urging further exploration into the underlying molecular mechanisms for lung cancer treatment.

MiR-155 deficiency and hypoxia results in metabolism switch in the leukemic B-cells.

Hypoxia represents one of the key factors that stimulates the growth of leukemic cells in their niche. Leukemic cells in hypoxic conditions are forced to reprogram their original transcriptome, miRNome, and metabolome. How the coupling of microRNAs (miRNAs)/mRNAs helps to maintain or progress the leukemic status is still not fully described. MiRNAs regulate practically all biological processes within cells and play a crucial role in the development/progression of leukemia. In the present study, we aimed to uncover the impact of hsa-miR-155-5p (miR-155, MIR155HG) on the metabolism, proliferation, and mRNA/miRNA network of human chronic lymphocytic leukemia cells (CLL) in hypoxic conditions. As a model of CLL, we used the human MEC-1 cell line where we deleted mature miR-155 with CRISPR/Cas9. We determined that miR-155 deficiency in leukemic MEC-1 cells results in lower proliferation even in hypoxic conditions in comparison to MEC-1 control cells. Additionally, in MEC-1 miR-155 deficient cells we observed decreased number of populations of cells in S phase. The miR-155 deficiency under hypoxic conditions was accompanied by an increased apoptosis. We detected a stimulatory effect of miR-155 deficiency and hypoxia at the level of gene expression, seen in significant overexpression of EGLN1, GLUT1, GLUT3 in MEC-1 miR-155 deficient cells. MiR-155 deficiency and hypoxia resulted in increase of glucose and lactate uptake. Pyruvate, ETC and ATP were reduced. To conclude, miR-155 deficiency and hypoxia affects glucose and lactate metabolism by stimulating the expression of glucose transporters as GLUT1, GLUT3, and EGLN1 [Hypoxia-inducible factor prolyl hydroxylase 2 (HIF-PH2)] genes in the MEC-1 cells.

Comparative Evaluation of the Differentiation and Proliferation Potential of Dental Pulp Stem Cells on Hydroxyapatite/Beta-Tricalcium Bone Graft and Bovine Bone Graft: An In Vitro Study.

BACKGROUND: Stem cells of mesenchymal origin have good proliferative capacity when compared to other stem cell types. Dental pulp stem cells (DPSCs) are a variety of mesenchymal cells obtained from the pulpal tissue of teeth and are abundantly available and easy to obtain. DPSCs facilitate and improve the formation of new bone using different bone graft scaffolds. This present study aims to evaluate and compare the osteogenic potential of DPSCs on alloplastic and xenogeneic bone grafts. MATERIALS AND METHODS: Hydroxyapatite and beta-tricalcium bone graft and bovine bone graft were used in a triplicate manner in the laboratory. DPSCs were obtained from the pulpal tissue of extracted third molars in the laboratory. The cytotoxicity, osteogenic potential, and difference in the rate of proliferation of mesenchymal cells on the biomaterials were assessed. RESULTS: Darker purple staining was seen in the case of hydroxyapatite/beta-tricalcium bone graft on MTT colorimetric assay stating that there was an increase in cell viability in hydroxyapatite/beta-tricalcium bone graft as compared to the bovine bone graft. Hydroxyapatite/beta-tricalcium bone graft showed more osteogenic potential as compared to the bovine bone graft as a higher degree of red staining was seen in Alizarin staining. CONCLUSION: Higher cell viability and higher osteogenic proliferation and differentiation were seen on the hydroxyapatite/beta-tricalcium bone graft compared to the bovine bone scaffold.

Coenzyme Q10 and Vitamin E Regulate the Bioactivity of Human Corneal Fibroblast Cells.

Purpose: Corneal fibroblasts are involved in the wound healing of the cornea with proliferation, migration, and differentiation processes. Coenzyme Q10 (CoQ10) and vitamin E can enhance corneal wound healing when applied after a corneal lesion as an eye drop. Thus, this study was performed to determine the potential efficiency of a CoQ10 ophthalmical solution containing a CoQ10 and vitamin E D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-derived formulation in human corneal fibroblasts (HCFs) in vitro. Methods: Primary HCFs were obtained from cadaveric corneal tissue, and cell viability was determined using MTT assay at 24 and 72 h. Cell migration was evaluated using an in vitro wound healing assay, and mRNA expressions of collagen type I (COL-I), collagen type III (COL-III), lumican, hyaluronan, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, tissue inhibitors of MMP (TIMP)-1, TIMP-2, interleukin (IL)-1ß, IL-6, IL-8, and IL-10 were assessed using reverse transcription polymerase chain reaction at 24 and 72 h. Results: At various concentrations of CoQ10 ophthalmical solution (CoQ10-os), cell viability and wound healing rates of HCFs increased compared with the control group. The expressions of COL-I, COL-III, lumican, and hyaluronan were increased by CoQ10-os, whereas those of MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were not affected by CoQ10-os at 24 and 72 h. In treating HCFs with a CoQ10-os medium, IL-1ß, IL-6, and IL-8 decreased, whereas IL-10 was significantly increased in a time- and dose-dependent manner. Conclusions: The findings indicate that CoQ10 and vitamin E-TPGS are potent regulators of the bioactivity of HCFs, thus supporting their potential application as ophthalmical solutions in therapies aimed at the fast regeneration of damaged cornea tissues.

Impact of Spirulina platensis biomass on the viability of Lactobacillus delbrueckii subsp. bulgaricus strain during the freeze-drying process.

In this work, we evaluated the protective capacity of Spirulina platensis biomass in preserving Lactobacillus delbrueckii subsp. bulgaricus WDCM 00102. The L. bulgaricus strain was freeze-dried in the presence of S. platensis biomass and the freeze-dried samples were then stored at 5 and 25°C for varying periods. Subsequently, the samples were rehydrated and bacterial plate counts were determined. The results indicate that a concentration of 12% S. platensis biomass was highly effective in preserving L. bulgaricus. Commercial products with higher S. platensis biomass content exhibited greater protective capacity. While S. platensis biomass is well-known for its prebiotic properties, its protective role has not been previously reported or thoroughly explored. This study demonstrates the protective capacity of S. platensis biomass in preserving L. bulgaricus, a strain particularly sensitive to preservation processes.

Modified human skin cell isolation protocol and its influence on keratinocyte and melanocyte culture.

Introduction: With the increasing emphasis on the use of nonanimal ingredients in clinical care, studies have proposed the use of TrypLE™ as an alternative to trypsin. However, previous research has reported insufficient cell yield and viability when using TrypLE to isolate skin cells compared to the dispase/trypsin-EDTA method. This study aimed to propose an improved method for increasing the yield and viability of cells isolated by TrypLE and to evaluate isolated keratinocytes and melanocytes. Methods: Foreskin tissues were isolated to keratinocytes and melanocytes using the trypsin-EDTA protocol and our modified TrypLE protocol. The yield and viability of freshly isolated cells were compared, the epidermal residue after cell suspension filtration was analyzed histologically, and the expression of cytokeratin 14 (CK14) and Melan-A was detected by flow cytometry. After cultivation, keratinocytes and melanocytes were further examined for marker expression and proliferation. A coculture model of melanocytes and HaCaT cells was used to evaluate melanin transfer. Results: The yield, viability of total cells and expression of the keratinocyte marker CK14 were similar for freshly isolated cells from both protocols. No differences were observed in the histologic analysis of epidermal residues. Moreover, no differences in keratinocyte marker expression or melanocyte melanin transfer function were observed after culture. However, melanocytes generated using the TrypLE protocol exhibited increased Melan-A expression and proliferation in culture. Conclusion: Our TrypLE protocol not only solved the problems of insufficient cell yield and viability in previous studies but also preserved normal cell morphology and function, which enables the clinical treatment of depigmentation diseases.

Year-long and seasonal differences of PM2.5 chemical characteristics and their role in the viability of human lung epithelial cells (A549).

Fine particulate matters-PM2.5 in the air can have considerable negative effects on human health and the environment. Various human cell-based studies examined the effect of PM2.5 on human health in different cities of the world using various chemical parameters. Unfortunately, limited information is available regarding the relationship between toxicity and chemical characteristics of PM2.5 collected in Istanbul, Türkiye, located in one of the most populated cities in the world. To investigate the chemical characteristics and cytotoxicity of PM2.5 in Istanbul, samples were collected for 12 months, then potentially toxic metals, oxidative potential, and particle indicators (e.g., functional groups and elements) were determined, and the cytotoxicity of PM2.5 on human A549 lung alveolar epithelial cells was examined. The mean PM2.5 mass concentration was 24.0 ± 17.4 µg m-3 and higher in cold months compared to other seasons. Moreover, the results of the metals, elemental, and functional groups indicated that seasonal and monthly characteristics were influenced by the regional anthropogenic sources and photochemistry input. The cytotoxicity results also showed that the viability of A549 cells was reduced with the exposure of PM2.5 (30-53%) and higher cytotoxicity was obtained in summer compared to the other seasons due to the impact of the metals, elements, and oxidative characteristics of PM2.5.

Production of recombinant human epidermal growth factor fused with HaloTag protein and characterisation of its biological functions.

Epidermal growth factor (EGF) protein is a crucial biomolecule involved in regulating cell growth, proliferation, migration and differentiation, which is used in various therapeutic applications, such as wound healing and tissue regeneration. The production of recombinant EGF is essential for studying its biological function and for its clinical translation. However, EGF protein expressed in prokaryotic cells often occurs in inclusion bodies, and co-expression with soluble tag protein is an effective method to prepare recombinant EGF. In this study, we expressed recombinant human EGF (rhEGF) fused to a HaloTag (Halo-rhEGF) and a large portion of Halo-rhEGF was found in the soluble fraction. Cell growth assay showed that the purified Halo-rhEGF protein could promote the proliferation of fibroblasts (NIH 3T3) and epithelial cells (HaCaT), and significantly increased their viability. Phosphorylation of the intracellular signaling proteins, ERK1/2 and c-Jun, was stimulated by treatment with Halo-rhEGF and the expression levels of proteins regulating cell proliferation were significantly increased. RNA sequencing analysis revealed that rhEGF could increase the transcription of genes enriched in ribosome generation and cell proliferation. Moreover, Halo-rhEGF can be labelled by HaloTag ligand for fluorescence imaging and can be slowly released in tissue repair by binding to anion biomaterials. In conclusion, HaloTag is an efficient fusion tag for rhEGF protein expression, purification and controlled release, and Halo-rhEGF can promote the proliferation and viability of epithelial and fibroblast cells.

Carbon nanotubes induce cytotoxicity and apoptosis through increasing protein levels of Bax and ROS in mouse skin fibroblasts.

Background and purpose: Carbon nanotubes (CNTs) are a significant discovery in nanotechnology, with widespread applications in modern technology. However, there are concerns about their potential toxicity, particularly in skin cells. This study aimed to investigate the mechanisms by which CNTs induced cytotoxicity and apoptosis in mouse skin fibroblasts. Experimental approach: The mice skin fibroblasts were isolated and exposed to two types of CNTs at various concentrations and then analyzed for changes in viability, reactive oxygen species (ROS) production, the levels of Bcl-2-associated X protein (Bax), and lactate production. Findings/Results: The results demonstrated that CNTs reduced cell viability and increased ROS production in a dose-dependent manner. Additionally, the current study found that CNTs increased the protein levels of Bax, a pro-apoptotic protein, in mouse skin fibroblasts. Furthermore, it was observed a significant decrease in lactate production in cells exposed to CNTs. Conclusion and implications: The findings concluded that CNTs have the potential to be toxic substances for skin fibroblasts, which serve as the body's first line of defense. This is evidenced by their ability to increase the production of ROS and the protein levels of Bax, as well as reduce lactic acid levels. As lactic acid has been reported to have beneficial effects on skin collagen production, further studies are needed to fully understand the impact of carbon nanotube exposure on human skin health.

The protective effects of alpha-pinene on high glucose-induced oxidative stress and inflammation in HepG2 cells.

Objectives: Hyperglycemia, a prevalent metabolic condition observed in diabetes, leads to oxidative damage, inflammatory responses, and other consequences. Natural compounds alleviate the adverse impacts of diabetes. We aimed to explore the effects of alpha-pinene (AP) as a monoterpene on oxidative damage and inflammation caused by high glucose (HG) in the human hepatocellular liver carcinoma (HepG2) cell line. Materials and Methods: The HepG2 cells were subjected to non or HG concentration (50 mM) and treated with or without AP (8, 16, and 32 µg/ml) for 48 hr. The effect of treatments on cellular viability, malondialdehyde (MDA), glutathione (GSH), and activity of anti-oxidant enzymes, including glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), was determined. The gene expression levels of nuclear factor-κß (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and dipeptidyl peptidase-4 (DPP-4) were estimated using quantitative real-time polymerase chain reaction (qRT-PCR). Results: HG exposure significantly increased cell death, MDA formation, and depletion of GSH content and GPx, CAT, and SOD activity (P<0.05). We have also seen a significant induction in NF-κB, TNF-α, IL-6, and DPP-4 gene expression in hepatocytes under HG conditions (P<0.05). Interestingly, co-treatment with AP in a dose-dependent manner improved cell death and altered levels of MDA and GSH, and activity of GPx and CAT (P<0.05). AP could also modulate the gene expression of NF-κB and inflammatory biomarkers dose-dependently (P<0.05). Conclusion: Our findings suggested the protective effect of AP on hepatocytes under HG conditions through attenuating oxidative stress markers and suppression of inflammatory pathways.