Number Of Necrotic Cells Research Articles

Cold ischemia time alters cell-type specific senescence leading to loss of cellular integrity in mouse lungs

Purpose: Ischemia-reperfusion injury (IRI) is a major challenge in lung transplantation often causing graft dysfunction and chronic airway illnesses in recipients. To prevent potential transplant related complications, strict guidelines were put in place to choose viable donor lungs with minimal risk of IRI. These regulations deem most of the donor organs unfit for transplant which then are donated for research to understand the mechanisms of health and diseases in human. However, resected organs that are being transported undergo cold ischemia that can negatively affect the tissue architecture and other cellular functions under study. Thus, it is important to assess how cold ischemia time (CIT) affects the physiological mechanism. In this respect, we are interested in studying how CIT affects cellular senescence in normal aging and various pulmonary pathologies. We thus hypothesized that prolonged CIT exhibits cell-type specific changes in lung cellular senescence in mice. Methods: Lung lobes from C57BL/6J (n = 5–8) mice were harvested and stored in UW Belzer cold storage solution for 0, 4-, 9-, 12-, 24-, and 48-h CIT. Lung cellular senescence was determined using fluorescence (C12FdG) assay and co-immunolabelling was performed to identify changes in individual cell types. Results: We found a rapid decline in the overall lung cellular senescence after 4-h of CIT in our study. Co-immunolabelling revealed the endothelial cells to be most affected by cold ischemia, demonstrating significant decrease in the endothelial cell senescence immediately after harvest. Annexin V-PI staining further revealed a prominent increase in the number of necrotic cells at 4-h CIT, thus suggesting that most of the cells undergo cell death within a few hours of cold ischemic injury. Conclusions: We thus concluded that CIT significantly lowers the cellular senescence in lung tissues and must be considered as a confounding factor for mechanistic studies in the future.

Comparison the impact of mesenchymal stromal cells, their microvesicles and plasma enriched with soluble platelet factors on survival and apoptosis of rat spleen lymphocytes <i>in vitro</i>

BACKGROUND: The trophic function is one of the important but insufficiently studied features of mesenchymal stromal cells (MSCs), their microvesicles (MVs), and plasma enriched with soluble platelet factors (PRPs), which ensure the viability of target cells. AIM: To analyze the capability of MSCs, MVs, and PRP to affect the viability and spontaneous and activation-induced apoptosis of rat spleen lymphocytes during culturing in vitro. MATERIALS AND METHODS: MSCs were isolated from the mononuclear cell fraction of the femoral bone marrow of Wistar rats using the plastic adhesion method. MVs were obtained from the conditioned medium of MSCs by centrifugation at 14,500 g. PRP was prepared by freezing/thawing rat peripheral blood platelet concentrate. Rat spleen lymphocytes were isolated on a density gradient of 1.077 g/cm3. The viability and degree of lymphocyte apoptosis in vitro were assessed by flow cytometry for 7-AAD incorporation and binding to annexin V. RESULTS: The presence of MSCs at 10 and 20% concentrations caused an increase in the number of living intact and PMA-activated lymphocytes by the end of 3-day in vitro cultivation. Compared with controls, the amount of necrotic cells decreased 8.3–13.5 times, and the number of apoptotic cells decreased 2.3–4.0 times, mainly due to lymphocytes at the late stage of apoptosis. MVs of MSCs at the indicated concentrations did not significantly affect the viability of lymphocytes cultured in vitro but reduced the level of apoptosis of intact and PMA-activated lymphocytes by 3.6 (р=0.03) and 4.8–5.2 (р=0.048; р=0.03) times respectively. Studies have shown that 1.25% rat PRP has a growth-stimulatory activity against MSCs but not against lymphocytes cultured in vitro. In the culture of intact lymphocytes, PRPs did not significantly affect cell viability with a slight decrease (2.7–2.9 fold, p 0.05) in the number of necrotic cells. In the culture of PMA-activated lymphocytes, 1.25–2.50% PRP increased the number of living cells by 1.6–2.2 times (р=0.002; р=0.01) and the number of necrotic cells by two times (р=0.02). CONCLUSION: MSCs, MVs of MSCs, and PRP, in decreasing order, enhanced the viability of rat spleen lymphocytes and suppressed late apoptosis during in vitro cultivation. Lymphocytes activated by phorbol myristate acetate are more sensitive to their vital action compared with resting cells.

Streptococcus suis Induces Macrophage M1 Polarization and Pyroptosis.

Streptococcus suis is an important bacterial pathogen that affects the global pig industry. The immunosuppressive nature of S. suis infection is recognized, and our previous research has confirmed thymus atrophy with a large number of necrotic cells. In this current work, we aimed to uncover the role of pyroptosis in cellular necrosis in thymic cells of S. suis-infected mice. Confocal microscopy revealed that S. suis activated the M1 phenotype and primed pyroptosis in the macrophages of atrophied thymus. Live cell imaging further confirmed that S. suis could induce porcine alveolar macrophage (PAM) pyroptosis in vitro, displaying cell swelling and forming large bubbles on the plasma membrane. Meanwhile, the levels of p-p38, p-extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) were increased, which indicated the mitogen-activated protein kinase (MAPK) and AKT pathways were also involved in the inflammation of S. suis-infected PAMs. Furthermore, RT-PCR revealed significant mRNA expression of pro-inflammatory mediators, including interleukin (IL)-1β, IL-6, IL-18, tumor necrosis factor (TNF)-α and chemokine CXCL8. The data indicated that the inflammation induced by S. suis was in parallel with pro-inflammatory activities of M1 macrophages, pyroptosis and MAPK and AKT pathways. Pyroptosis contributes to necrotic cells and thymocyte reduction in the atrophied thymus of mice.

Salt Tolerance Assessment in Triticum Aestivum and Triticum Durum.

Salt stress is a multicomponent phenomenon; it includes many processes that directly or indirectly affect the plant. Attempts have been made to comprehensively consider the processes of salt stress in plants Triticum aestivum (variety Orenburgskaya 22) and Triticum durum (variety Zolotaya). The study used methods of light and fluorescence microscopy, methods of immunofluorodetection, expression of DNA methyltransferase genes, genes of the ion transporter and superoxide dismutase families, as well as biochemical determination of total antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. According to morphometric indicators, the Orenburgskaya 22 variety showed greater tolerance to the action of 150 mM NaCl than the Zolotaya variety. The level of expression of genes of the HKT ion transporter family in the Orenburgskaya 22 variety is higher than in the Zolotaya variety. It was found that the expression of the DNA methyltransferase gene DRM2.1, which post-translationally methylates cytosine residues, is 22.3 times higher in Zolotaya compared to Orenburg 22 when exposed to salt. The accumulation of toxic ions is accompanied by an increase in reactive oxygen species (ROS) and increased damage to root tissue, especially in the Zolotaya variety. Using fluorescence microscopy using the Carboxy-H2DFF marker in the Orenburgskaya 22 variety at high NaCl concentrations, the highest fluorescence intensity was determined in the cap zone; in the Zolotaya variety-in the zones of the cap and root meristem. Excess ROS is more successfully removed in the Orenburgskaya 22 variety, which has a higher level of antioxidant activity (AOA), as well as the level of expression of the Cu/ZnSOD and MnSOD superoxide dismutase genes. Using programmed cell death (PCD) markers based on the release of cytochrome c from mitochondria into the cytoplasm, DNA breakage and the release of phosphatidylserine from mitochondria, the degree of damage to root cells was assessed in both wheat varieties. It has been proven that wheat cell death occurs through the mitochondrial pathway. It was noted that the salt-sensitive variety Zolotaya had a significant number of necrotic cells. Based on the data obtained, it was concluded that the Orenburgskaya 22 variety exhibits greater resistance to salinity than the Zolotaya variety. These data may be of practical importance for enhancing protective mechanisms under abiotic stress.

Abstract 5971: F3 targeted near-infrared photoimmunotherapy: The ideal therapeutic approach for numerous malignancies

Abstract Introduction: F3 is a cell surface protein that plays a role in blood clotting, but recent research has revealed its implication in cancer proliferation, metastasis, angiogenesis, and immune escape. TCGA cohort analysis has shown that elevated F3 is a common feature of many cancers, and generally correlates with poor patient survival. Therefore, the potential of F3 as a therapeutic target in cancer is worth exploring. Tisotsumab-vedotin, an antibody-drug conjugate (ADC) that targets F3, was approved by the FDA for cervical cancer and is undergoing clinical trials in some solid cancers. However, ADCs may give rise to concerns about drag resistance following cellular internalization. Herein, we developed and tested an F3 targeted near-infrared photoimmunotherapy (NIR-PIT) to overcome some of these resistance mechanisms. Methods: We created a conjugate of tisotumab as an F3 antibody and IR700. The efficacy of F3-targeted NIR-PIT was investigated using multiple cancer cell lines (A431; epidermoid carcinoma, HPAF-II; pancreatic adenocarcinoma, HSC-2; oral carcinoma, HT-1376luc; bladder carcinoma, MDAMB231; breast adenocarcinoma, and SKOV-3; ovarian serous cystadenocarcinoma) in vitro. In vivo, the efficacy of F3-targeted NIR-PIT was evaluated in HPAF-II tumor using xenograft mouse models. In addition, the pathological change after NIR-PIT was also evaluated in A431, HPAF-II, HSC-2, HT-1376luc, MDAMB231, and SKOV-3 tumors. Results: These cell lines showed F3 expression in vitro and in vivo. Additionally, F3 expression was specific to cancer cells in HPAF-II tumor model. In vitro, F3-targeted NIR-PIT damaged the multiple cell lines in an NIR light dose-dependent manner. In vivo, F3-targeted NIR-PIT suppressed tumor growth and improved survival rates compared to the untreated group. Pathologically, A431, HPAF-II, HSC-2, HT1376-luc, MDAMB231 and SKOV-3 tumors after NIR-PIT contain large numbers of necrotic cells that are shown typically less dark nuclei and eosinophilic cytoplasm with some vacuolar degeneration than untreated tumors. Conclusions: F3-targeted NIR-PIT holds the efficacy against multiple cancers expressing F3, regardless of the specific cancer type. Therefore, this modality exhibits significant promise as a potential NIR-PIT option. Citation Format: Seiichiro Takao, Aki Furusawa, Hiroshi Fukushima, Shuhei Okuyama, Makoto Kano, Hiroshi Yamamoto, Peter L. Choyke, Hisataka Kobayashi. F3 targeted near-infrared photoimmunotherapy: The ideal therapeutic approach for numerous malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5971.

Innovative hydroxyapatite/submicron mesoporous SiO2/HA particles composite coatings for enhanced osteogenic activity of NiTi bone implants: A comprehensive investigation of materials and biological interactions

In this study, hydroxyapatite (HA) incorporated in mesoporous SiO2 particles (SiO2/HA) was initially synthesized using a soft template method. The resulting structure owned a high surface area (796.62 m2 g-1) and a substantial pore volume (0.757 cm3 g-1). The TEM and FESEM images indicated that the synthesized mesoporous particles possessed a spherical morphology, with a diameter of approximately 200 nm, and pores of slit-shape. The mission was to improve the surface characteristics of NiTi implants by composite coatings. This investigation emphasized specifically the impact of different concentrations of mesoporous SiO2/HA particles within the electrolyte on the properties of HA/mesoporous composite coatings. The surface characteristics of the coatings were examined using ATR-FTIR, FESEM, EDS, AFM, and hydrophilicity measurements. Furthermore, the osteogenic activity of the coatings was evaluated by culturing the osteosarcoma cell line SAOS-2 on the coated samples. The introduction of mesoporous particles resulted in a decrease in the coatings’ surface roughness along with an improvement in their wettability. Moreover, there was an enhancement in the cell viability (99% after 7 days), proliferation, adhesion, and growth of SAOS-2 cells at the optimized concentration of mesoporous SiO2/HA particles (1000 mg/L). DAPI images illustrated that the mesoporous particles did not cause any adverse impact on the cellular nuclei. Finally, in the apoptosis test, the composite coating displayed a reduced number of necrotic cells compared to the pure HA coating.

Selective Apoptotic Effects of Resilon and Epiphany Se: In Vitro Investigation in Cell Cultures

Objective: The aim of this investigation is to examine the programmed cell death mechanism using Resilon, a root canal filling material, and Epiphany SE, a root canal sealant, in a microenvironment of cultured epithelial-like cells. Methods: Vero cells were distributed into a 24-well plate at a density of 2 x 105 cells/ml per well. Fol- lowing an incubation period of 18 hours, Resilon was applied to the cell monolayer. The monolayer was combined with Resilon and covered with a pre-warmed medium before being incubated at 37 0 C in a 5% CO2 incubator. Following this, the wells were treated with a 50% diluted eluate of Epiphany SE. Resilon and Epiphany SE were then left to incubate for 0.5, 1, 3, 6, 12, 18 and 24 hours at 37 0C. Data were analyzed in IBM SPSS V23. Statistical analysis involved determining the differences in means of the specimens using a one-way ANOVA, followed by Duncan’s multiple range test to as- sess specific statistical differences across various time periods. Results: It was found that between 0 and 6 hours, it was unclear whether Resilon and Epiphany SE induce apoptosis in Vero cells. Significant differences were observed 12 hours after examination comparing both the Resilon and Epiphany SE groups to the examination time prior to 12 hours (p < 0.05). These cells decreased at 18 and 24 hours. However, necrotic cells increased at each stage of the study period. The increase in the number of necrotic cells was statistically significant at 18 and 24 hours (p < 0.05). Conclusion: It is inferred that necrosis of fibroblastic cells is caused by the cytotoxic effects of both Resilon and Epiphany SE. Hence, it can cause problems with root canal over obturation.

Ex Vivo Treatment By Mitapivat, an Allosteric Pyruvate Kinase Activator, Reduced Oxidative Stress and Promoted Terminal Erythropoiesis in a Severe Hemolytic Anemia Patient Due to Krϋppel-like Factor 1 Mutations

Introduction: Human erythroid Krüppel-like Factor 1 (EKLF or KLF1), a hematopoietic-specific transcription factor encoded from KLF1 plays roles as the master regulator of erythroid development, including globin switching and expression, pyruvate kinase ( PKLR) function, heme synthesis, structural membrane, minor blood group, and cell cycle regulation. Most patients carrying KLF1 mutations present with severe chronic hemolytic anemia with low pyruvate kinase-red blood cell (PKR) activity and require regular transfusion. Previously, our group described the effects of ex vivo mitapivat (AG-348), an allosteric activator of PKR, treatment on the peripheral RBCs of two non-regular transfused chronic hemolytic anemia patients due to KLF1 mutations by increased PKR activity and ATP production of treated RBCs (Suksangpleng et al, Blood 2019; 134 (Supplement_1): 2220). We are currently studying the erythropoietic characteristics and investigating the effects of treatment with mitapivat to improve the severe anemia observed in these KLF1 patients. Objective: To evaluate the effects of ex vivo mitapivat treatment on the terminal erythropoiesis characteristics, including cell viability, oxidative stress, necrosis, and erythroid differentiation, of one severe hemolytic anemia patient due to KLF1 mutations. Methods: The 20-mL EDTA whole blood sample was collected from one regular transfused severe hemolytic anemia patient who was identified with KLF1 alleles mutations. The CD34 +-peripheral blood mononuclear cells (PBMCs) were separated by the specific anti-CD34 antibody-labelled magnetic beads and cultured by 3 phases of primary erythroid culture system, including phase 1: erythroblast transformation, phase 2: erythroblast expansion, and phase 3: erythroid differentiation. Cell viability was monitored in all 3 phases. The erythroid differentiation was monitored by stage-specific erythroid surface marker antibodies using flow cytometry. The ex vivo treatment was performed in phase 3 by incubating the cultured erythroid cells with the completed tissue cultured medium containing 40 nM mitapivat (Agios Pharmaceuticals, Inc.). Intracellular reactive oxygen species (ROS) were measured by fluorescent detection and necrotic cells were stained with Annexin V/PI and numbered by flow cytometry. Results: One index patient with regular transfused chronic hemolytic anemia showed low level of Hb (< 11 g/dL), high reticulocyte count (> 2%), high Hb F level (> 2%), and no PKLR mutation identified. The allele genotypes of KLF1 were identified as compound heterozygous mutation including unusual amino acid (Q58X) and missense mutation (A298P) on allele 1 and 2, respectively (Table 1). Proerythroblasts were transformed from the separated CD34 +-PBMCs in the Phase 1 culture period (Day 1 - Day 6) and highly expanded in Phase 2 (Day 7 - Day 13). After being induced to erythroid differentiation in Phase 3, the viability of erythroblasts gradually decreased while increased intracellular ROS was found. These phenomena corresponded to the increased numbers of necrotic cells during erythroid stage differentiation. Interestingly, the ex vivo treatment with mitapivat decreased intracellular ROS and necrotic cell numbers of the stage-differentiated erythroblasts, with more viable cell counts compared with the untreated condition in terminal erythropoiesis (Figure 1A, 1B, 1C). Moreover, the ex vivo treatment with mitapivat supported the effective stage differentiation shown by higher FITC- and lower PE-mean fluorescent intensity (MFI) that represented two stage-specific erythroid markers, including CD235a (glycophorin A) and CD71 (transferrin receptor), respectively, than those of untreated differentiated erythroblasts (Figure 1D). Conclusion: This study showed the effects of ex vivo treatment with mitapivat which could reduce oxidative stress, promote cell viability and stage differentiation in terminal erythropoiesis of one regular transfused hemolytic anemia patient due to KLF1 mutations. Mitapivat is being studied in clinical trials in PK deficiency (NCT03853798, NCT05144256, and NCT05175105), thalassemia (NCT04770753 and NCT04770779) and sickle cell disease (NCT05031780). Further evaluation of mitapivat in patients with severe hemolytic anemia due to KLF1 mutations is warranted as a potential therapeutic option for patients with KLF1 disease.

Interaction between mesenchymal stromal cells and tuberculous mycobacteria in vitro

The objective: in an in vitro experiment, we compared phagocytic parameters of mesenchymal stromal cells (MSCs) and macrophages to tuberculous mycobacteria, assessed the ability of MSCs and macrophages to lyse mycobacteria or maintain their intracellular growth, their effect on formation of phenotypic drug resistance of mycobacteria, as well as the effect of tuberculous mycobacteria on the type of MSCs cell death.Subjects and Methods. Balb/c male mice, aged 6 to 8 weeks, were used in the experiment. Bone marrow MSCs were obtained from femurs and tibias by further cultivation, peritoneal macrophages were elicited with 4% alpha-glucan. The intracellular content of mycobacteria was counted using a confocal microscope with x 400 magnification. Susceptibility of mycobacteria to isoniazid and development of phenotypic drug resistance after culturing MSCs and macrophages with MTB on Lowenstein–Jensen medium was assessed by counting CFU. In 5 days after the infection, the number of apoptotic and necrotic MSCs and macrophages was determined by a flow cytometer.Results. On Day 1, the total number of phagocytosed MTB, as well as the number of phagocytic-active macrophages, exceeds the corresponding figures for MSCs more than twice. MSCs phagocytize tuberculous mycobacteria in a smaller amount, but MTB reproduces in them more actively: the number of CFU after 7 days of cell cultivation with MTB exceeded the corresponding parameter by almost 50 times after 24 hours of cultivation. In cultures of infected MSCs cultivated for 7 days, regardless of the presence of isoniazid, there was a rapid growth of tuberculous mycobacteria. On Day 5 after infection of macrophage culture with tuberculous mycobacteria, the number of necrotic cells was 2.7 times greater than that of uninfected necrotic macrophages, but the number of apototic cells in these groups differed slightly. In the culture of MSCs, there were 8.5 times more infected nectrotic cells versus uninfected necrotic MSCs, and the number of necrotic MSCs was 4.5 times higher than the number of MSCs with apoptosis, while in the culture of infected macrophages, the number of necrotic cells was the same as number of apoptotic cells. Unlike macrophages, treatment of MSCs with isoniazid did not inhibit the intracellular proliferation of MTB.Conclusion. MSCs have the ability to phagocytose mycobacteria, but they do it less actively than macrophages and, unlike macrophages, they are not able to restrain the reproduction of tuberculous mycobacteria. Mycobacteria have phenotypic drug resistance in MSCs. In MSCs, when infected with tuberculous mycobacteria, there is a pronounced shift towards necrosis in the type of cell death, which can lead to dissemination of MTB and development of local destructive changes.

Protective Effect of Vitis gracilis Wall (Vitaceae) Leaf Decoction on Sexual Vitality and Testis of Alloxan-Induced Diabetic Mice

Diabetes mellitus is profoundly associated with various detrimental outcomes including sexual dysfunction and infertility in males. On the other hand, a medicinal plant namely Vitis gracilis Wall (Vitaceae) has been used as a traditional medicine to enhance vitality. This present study aimed to investigate the protective effect of V. gracilis leaf decoction against diabetes-induced sexual dysfunction and testicle histopathology in adult male mice. The experiment was composed of five different groups namely the control (non-diabetic) group, the diabetes group (without any treatments), and the diabetes treated with V. gracilis decoction at the doses of 25, 50, and 100 g/L, respectively. In addition, the phytochemical constituents of leaf decoction were determined by using Ultra performance-liquid chromatography-mass spectroscopy (UPLC-MS). Our data demonstrated that, despite failing to improve blood glucose profile and body weight, V. gracilis leaf decoction sustained intense sexual behaviors including face and genital kissing, genital licking, and mount toward estrous females. Moreover, lower doses of decoction (25 and 50 g/L) attenuated the diabetes-induced reduction of testis weight and precluded malondialdehyde accumulation in the testicle tissue. The decoction at the lower doses also ameliorated histopathological alterations in the testis, particularly the wall thickness of tubulus seminiferous and the number of necrotic cells. V. gracilis decoction also improved hematological values including hemoglobin, red blood cell count and hematocrit level. In addition, UPLC-MS analysis revealed a total of 26 phytochemical compounds with seven predominant substances. In conclusion, leaf decoction of V. gracilis, particularly at lower doses but not at a higher dose, exerted a protective effect on sexual vitality, testicle tissue, and hematological value under diabetic condition. The beneficial effects of V. gracilis decoction might be associated with its various bioactive compounds. Therefore, V. gracilis leaves may be a future candidate as a potent natural drug for male sexual vitality and testicle protection against diabetes. 

Chitin Extracted from the Shell of Blue Swimming Crabs (Portunus pelagicus Linn.) Inhibits NF-kappaB p65 in Ethanol-Induced Gastric Ulcerative Wistar Rats.

Peptic ulcer disease is generated by the activation of NF-kappaB activity. A recent clinical study reported a significant increase in NF-kappaB2 gene expression in 79 samples of peptic ulcer patients compared to the control group. Moreover, the deacetylated chitin could alter the translocation of NF-kappaB p65 to the nucleus. Considering this, our work aims to explore the effect of chitin extracted from the shell of blue swimming crabs (Portunus pelagicus Linn.) towards NF-kappaB p65 levels in ethanol-induced gastric ulcerative Wistar rats. The shells are found abundantly as the waste of seafood processing in the northern part of West Java, Indonesia. In this study, chitin extraction was carried out using the microwave-assisted extraction method by employing choline chloride (C5H14ClNO) and DL-malic acid (C₄H₆O₅) as the solvents. The inhibitory activity assay of chitin on the expression of NF-kappaB p65 was performed by using Western blot. The extraction yielded a good quality of chitin with a deacetylation degree of 30.8026%, molecular weight of 3.35 × 105 Da, and a negligible heavy metals level. Moreover, chitin extract at doses of 150, 300, and 600 mg/kg BW significantly reduced the percentage of gastric ulcer index compared to the negative control group. Meanwhile, chitin extract at doses of 300 and 600 mg/kg BW significantly inhibited NF-kappaB expression compared to the negative control group. Histopathological examination demonstrated a decrease in the number of necrotic cells and fat degeneration in the gastric mucosa and an increase in normal cells. Taken together, chitin extract obtained from the shells of blue swimming crabs may be able to prevent gastric ulcers induced by ethanol via the inhibition of NF-kappaB p65; however, further studies are needed to verify its anti-ulcerative properties.

COMPARISON OF CELL DEATH RATE IN VARIOUS CHICKEN LYMPHOCYTES POPULATIONS IN A RESPONSE TO THREE TEMPERATURE TREATMENTS

The goal of the experiment was to evaluate susceptibility of lymphocytes populations derived from bursa of Fabricius, spleen, thymus, and blood to temperature treatments in vitro. Temperatures used in study was 37 °C (thermoneutral), 41 °C (mild heat stress) and 45 °C (severe heat stress). Cells were incubated for 8 hours and after 3 and 8 hours were measured numbers of apoptotic, necrotic and CellROX positive cells by flow cytometry. Representation of live lymphocyte phenotypes (CD3, CD4, CD8, BU1) in cell populations were also measured. At all temperature treatments, thymus lymphocytes population had the lowest (p < 0.05) percentage of apoptotic cells. On the other hand, significantly highest numbers (p < 0.05) of apoptotic cells were found in bursal lymphocytes after 8 hours of incubation. Differences in numbers of necrotic cells were more pronounced at 45 °C, where after 3 and 8 hours of incubation were significantly highest numbers (p < 0.05) of necrotic cells in spleen. Increasing temperature induce production of reactive oxygen species (ROS) in cells and highest proportion (p < 0.05) of ROS-positive cells were found in bursal and spleen cell population after 8 hours of incubation. Transmission electron microscopy showed loss of mitochondrial matrix and disrupted mitochondrial membrane in mitochondria of lymphocytes at 45 °C as initial damage caused by ROS. From individual cell phenotypes, there were significantly decreased (p < 0.05) B lymphocytes in bursa of Fabricius, CD4+ and CD8+ lymphocytes in thymus and whole T lymphocytes population in spleen at 45 °C. Various susceptibility to temperature can be predictive factor of worse function in some organs in stress conditions where body temperature increase.

RIP3/MLKL regulates necroptosis via activating 4EBP1-eIF4E pathway.

Necroptosis is a cell death type mediated by receptor interacting protein 3 (RIP3)/mixed lineage kinase domain-like protein (MLKL). It has been reported that mammalian target of rapamycin plays a regulatory role in necroptosis. Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1)-eukaryotic initiation factor 4E (eIF4E) pathway is a key down streamer of mammalian target of rapamycin. However, whether 4EBP1-eIF4E pathway is involved in necroptosis is still unknown. This study aims to investigate the changes of 4EBP1-eIF4E pathway in necroptosis. TNF-α/SM-164/Z-VAD-FMK (TSZ), a necroptosis inducer, was used to induce necroptosis in murine fibroblastoid cell line L929. Cell necrosis was observed under an optical microscope. Then, TSZ was added to L929 cells with RIP3 and MLKL gene knockout. Propidium iodide (PI) staining was used to observe cell necrosis. Real-time fluorescence quantitative PCR and Western blotting were used to determine the mRNA and protein expression of 4EBP1 and eIF4E, respectively. After treating L929 cells with TSZ, the number of necrotic cells was increased, the mRNA and protein expression levels of 4EBP1 were significantly downregulated, and the ratio of phosphorylated 4EBP1 (p-4EBP1) to 4EBP1 was increased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly upregulated, and the ratio of phosphorylated eIF4E (p-eIF4E) to eIF4E was increased (both P<0.01). After knocking out RIP3 and MLKL in L929 cells, PI positive necrotic cells were significantly reduced, the mRNA and protein expression levels of 4EBP1 were significantly upregulated, and the ratio of p-4EBP1 to 4EBP1 was decreased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly downregulated, and the ratio of p-eIF4E to eIF4E was decreased (both P<0.01). 4EBP1-eIF4E pathway is activated in the RIP3/MLKL mediated-necroptosis.

Melatonin blunted the angiogenic activity in 3D colon cancer tumoroids by the reduction of endocan

BackgroundComplexity and heterogeneity of the tumor niche are closely associated with the failure of therapeutic protocols. Unfortunately, most data have been obtained from conventional 2D culture systems which are not completely comparable to in vivo microenvironments. Reconstructed 3D cultures composed of multiple cells are valid cell-based tumor models to recapitulate in vivo-like interaction between the cancer cells and stromal cells and the oncostatic properties of therapeutics. Here, we aimed to assess the tumoricidal properties of melatonin on close-to-real colon cancer tumoroids in in vitro conditions.MethodsUsing the hanging drop method, colon cancer tumoroids composed of three cell lines, including adenocarcinoma HT-29 cells, fibroblasts (HFFF2), and endothelial cells (HUVECs) at a ratio of 2: 1: 1, respectively were developed using 2.5% methylcellulose. Tumoroids were exposed to different concentrations of melatonin, from 0.005 to 0.8 mM and 4 to 10 mM, for 48 h. The survival rate was measured by MTT and LDH leakage assays. Protein levels of endocan and VEGF were assessed using western blotting. Using histological examination (H & E) staining, the integrity of cells within the tumoroid parenchyma was monitored.ResultsDespite the reduction of viability rate in lower doses, the structure of tumoroids remained unchanged. In contrast, treatment of tumoroids with higher doses of melatonin, 4 and 10 mM, led to disaggregation of cells and reduction of tumoroid diameter compared to the non-treated control tumoroids (p < 0.05). By increasing melatonin concentration from 4 to 10 mM, the number of necrotic cells increased. Data showed the significant suppression of endocan in melatonin-treated tumoroids related to the non-treated controls (p < 0.05). According to our data, melatonin in higher doses did not alter protein levels of VEGF (p > 0.05).ConclusionsMelatonin can exert its tumoricidal properties on colon cancer tumoroids via the reduction of tumor cell viability and inhibition of the specific pro-angiogenesis factor.

The effect of cerium dioxide nanoparticles on the viability of hippocampal neurons in Alzheimer's disease modeling.

The possibilities of using nanoparticle materials based on cerium dioxide (CNPs) are exciting since they are low toxic and have specific redox, antiradical properties. It can be supposed that CNPs' biomedical use is also relevant in neurodegenerative diseases, especially Alzheimer's disease (AD). AD is known as the pathologies leading to progressive dementia in the elderly. The factor that provokes nerve cell death and cognitive impairment in AD is the pathological accumulation of beta-amyloid peptide (Aβ) in the brain tissue. In our studies, we examined the impact of Aβ 1-42 on neuronal death and evaluated the potential neuroprotective properties of CNPs during AD modeling in cell culture. Our findings show that, under AD modeling conditions, the number of necrotic neurons increased from 9.4% in the control to 42.7% when Aβ 1-42 was used. In contrast, CNPs alone showed low toxicity, with no significant increase in the number of necrotic cells compared to control conditions. We further explored the potential of CNPs as a neuroprotective agent against Aβ-induced neuronal death. We found that introducing CNPs 24 h after Aβ 1-42 incubation or prophylactically incubating hippocampal cells with CNPs 24 h before amyloid administration significantly reduced the percentage of necrotic cells to 17.8 and 13.3%, respectively. Our results suggest that CNPs in the cultural media can significantly reduce the number of dead hippocampal neurons in the presence of Aβ, highlighting their neuroprotective properties. These findings suggest that CNPs may hold promise for developing new treatments for AD based on their neuroprotective properties.

Development of hyaluronic acid-anchored polycaprolactone nanoparticles for efficient delivery of PLK1 siRNA to breast cancer.

PlK1 has a significant role in the development of breast cancer. Thus, silencing of PlK1 gene may arrest the growth of breast cancer. However, the in vivo stability of PlK1 siRNA after injection remains a challenge to target the specific site. The delivery of siPlK1 RNA via viral vector and amine group-terminated dendrimer is associated with immune reaction and cellular cytotoxicity. Thus, in the present study, hyaluronic acid-functionalized and -thiolated polycaprolactone nanoparticles (SH-HPP NPs) were developed for enhancing the targeting capabilities of siRNA towards human breast cancer cells. NPs displayed size in the range of 180-217nm, and with sustain and pH-dependent release of siRNA up to 120h. The in vitro treatments with siRNA-containing NPs showed the high number of necrotic cells and the cell cycle arrest at the G2/M phase. The gene expression analysis depicts the decrease of endogenous PLK1 siRNA expression on MCF-7 cells upon PLK1 NPs treatment. In vitro cytotoxicity experiments demonstrated effective anticancer properties against MCF-7. Finally, in vivo results showed that substantial tumor inhibition was achieved with PLK1 siRNA-containing SH-HPP NPs in comparison of the control group. Hence, HPP NPs have enormous potential for the selective delivery of siRNA, i.e., breast cancer cells.

High Levels of Extracellular Potassium Can Delay Myxoma Virus Replication by Preventing Release of Virions from the Endosomes.

Potassium (K+) is one of the most abundant cations in the human body. Under normal conditions, the vast majority of K+ is found within cells, and the extracellular [K+] is tightly regulated to within 3.0 to 5.0 mM. However, it has recently been shown that high levels of localized necrosis can increase the extracellular concentration of K+ to above 50 mM. This raises the possibility that elevated extracellular K+ might influence a variety of biological processes that occur within regions of necrotic tissue. For example, K+ has been shown to play a central role in the replication cycles of numerous viral families, and in cases of lytic infection, localized regions containing large numbers of necrotic cells can be formed. Here, we show that the replication of the model poxvirus myxoma virus (MYXV) is delayed by elevated levels of extracellular K+. These increased K+ concentrations alter the cellular endocytic pathway, leading to increased phagocytosis but a loss of endosomal/lysosomal segregation. This slows the release of myxoma virus particles from the endosomes, resulting in delays in genome synthesis and infectious particle formation as well as reduced viral spread. Additionally, mathematical modeling predicts that the extracellular K+ concentrations required to impact myxoma virus replication can be reached in viral lesions under a variety of conditions. Taken together, these data suggest that the extracellular [K+] plays a role in determining the outcomes of myxoma infection and that this effect could be physiologically relevant during pathogenic infection. IMPORTANCE Intracellular K+ homeostasis has been shown to play a major role in the replication of numerous viral families. However, the potential impact of altered extracellular K+ concentrations is less well understood. Our work demonstrates that increased concentrations of extracellular K+ can delay the replication cycle of the model poxvirus MYXV by inhibiting virion release from the endosomes. Additionally, mathematical modeling predicts that the levels of extracellular K+ required to impact MYXV replication can likely be reached during pathogenic infection. These results suggest that localized viral infection can alter K+ homeostasis and that these alterations might directly affect viral pathogenesis.

Structural Diversity, LC-MS-MS Analysis and Potential Biological Activities of Brevibacillus laterosporus Extract.

Lake Mariout is Egypt's degraded coastal marine habitat that encompasses a variety of wastes. The biodiversity and hard environmental conditions allow the co-existence of organisms with high resistance and rich metabolism, making them potential candidates for screening and isolating novel microbial strains. A bacterial isolate (BF202) cultured from the marine sediments of Alexandria's Mariout Lake (Egypt) was tested for its antimicrobial and anticancer potential. The phylogenetic analysis of the isolated strain's 16S rDNA and gyrB revealed that BF202 belongs to Brevibacillus laterosporus (B. laterosporus). Antibiosis of B. laterosporus was confirmed against microbial pathogens including Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Staphylococcus aureus. The highest antibacterial activity was detected on glucose peptone medium after 18 h of incubation at 35 °C, and at pH of 7.0 in the presence of mannose and ammonium carbonate as carbon and nitrogen sources, respectively. The cytotoxicity of the methanolic extract against breast cancer (MCF-7) and normal Vero cell lines, using the MTT test, revealed IC50 values of 7.93 and 23.79 µg/mL, respectively. To identify apoptotic and necrotic cells, a flow cytometric analysis using annexin V-FITC/PI dual-labeling was utilized and recorded a higher number of necrotic cells compared to apoptotic ones. Similarly, the cell cycle S-phase arrest was reported. The LC-MS-MS investigation of B. laterosporus extract and the molecular networking database analysis demonstrated five strategic diketopiperazine compounds with antimicrobial and anticancer activities. Taken together, this research shows that the crude extract of B. laterosporus might be an effective agent against drug-resistant bacteria and malignant disorders due to its richness in diketopiperazines.

Effect of Coated Silver Nanoparticles on Cancerous vs. Healthy Cells.

Unique properties of silver nanoparticles (NPs) ensure their wide applications, in biomedicine; for this reason, it is very important carefully to study the toxicity of such NPs. The influence of silver nanoparticles coated with natural resin (Ag NPs) on the morphological and functional features of healthy BHK-21 and cancerous Hep-2 cells were studied using fluorescence microscopy, MTT, and neutral red assays. Ag NPs induced morphological changes in both cell cultures. The modifications were dose-dependent and more pronounced with an increase in NPs concentration. The IC50 value of Ag NPs for Hep-2 cells was found to be 2.19 ± 0.22 µg/mL, whereas for BHK-21 cells it was significantly (5x) higher at 10.92 ± 2.48 µg/mL. The use of NPs at a concentration close to IC50 leads to significant increase (up to 40%) in the number of necrotic cells in cancerous cell population and a decrease in the number of mitotic cells (up to 1.3%). In noncancerous cells the cellular parameters were similar to the control cells. These data suggest that the silver nanoparticles coated with natural resin can be potentially used in cancer therapy.

Enhanced Cytotoxic Effects of Arenite in Combination with Active Bufadienolide Compounds against Human Glioblastoma Cell Line U-87.

The cytotoxicity of a trivalent arsenic derivative (arsenite, AsIII) combined with arenobufagin or gamabufotalin was evaluated in human U-87 glioblastoma cells. Synergistic cytotoxicity with upregulated intracellular arsenic levels was observed, when treated with AsIII combined with arenobufagin instead of gamabufotalin. Apoptosis and the activation of caspase-9/-8/-3 were induced by AsIII and further strengthened by arenobufagin. The magnitude of increase in the activities of caspase-9/-3 was much greater than that of caspase-8, suggesting that the intrinsic pathway played a much more important role in the apoptosis. An increase in the number of necrotic cells, enhanced LDH leakage, and intensified G2/M phase arrest were observed. A remarkable increase in the expression level of γH2AX, a DNA damage marker, was induced by AsIII+arenobufagin. Concomitantly, the activation of autophagy was observed, suggesting that autophagic cell death associated with DNA damage was partially attributed to the cytotoxicity of AsIII+arenobufagin. Suppression of Notch signaling was confirmed in the combined regimen-treated cells, suggesting that inactivation of Jagged1/Notch signaling would probably contribute to the synergistic cytotoxic effect of AsIII+arenobufagin. Given that both AsIII and arenobufagin are capable of penetrating into the blood–brain barrier, our findings may provide fundamental insight into the clinical application of the combined regimen for glioblastoma.