Cells In Medium Research Articles

A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis

AbstractOxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7β,9α,10β,13α‐hexaacetoxytaxa‐4(20),11(12)‐diene (1) as a substrate led to the production of a major oxetane derivative, 1β‐dehydroxybaccatin IV (1 a), and a minor 4β,20‐epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5‐deacetylated derivative of 1, yielded only 5α‐deacetylbaccatin I (2 b), a 4β,20‐epoxide. A possible reaction mechanism was proposed on the basis of substrate‐feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation‐acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4β,20‐epoxide might be a shunt product. In this process, the C5‐O‐acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)‐epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.

A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis.

Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7β,9α,10β,13α-hexaacetoxytaxa-4(20),11(12)-diene (1) as a substrate led to the production of a major oxetane derivative, 1β-dehydroxybaccatin IV (1 a), and a minor 4β,20-epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5-deacetylated derivative of 1, yielded only 5α-deacetylbaccatin I (2 b), a 4β,20-epoxide. A possible reaction mechanism was proposed on the basis of substrate-feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation-acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4β,20-epoxide might be a shunt product. In this process, the C5-O-acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)-epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.

Targeted therapies of inflammatory diseases with intracellularly gelated macrophages in mice and rats

Membrane-camouflaged nanomedicines often suffer from reduced efficacy caused by membrane protein disintegration and spatial disorder caused by separation and reassembly of membrane fragments during the coating process. Here we show that intracellularly gelated macrophages (GMs) preserve cell membrane structures, including protein content, integration and fluidity, as well as the membrane lipid order. Consequently, in our testing GMs act as cellular sponges to efficiently neutralize various inflammatory cytokines via receptor-ligand interactions, and serve as immune cell-like carriers to selectively bind inflammatory cells in culture medium, even under a flow condition. In a rat model of collagen-induced arthritis, GMs alleviate the joint injury, and suppress the overall arthritis severity. Upon intravenous injection, GMs efficiently accumulate in the inflammatory lungs of acute pneumonia mice for anti-inflammatory therapy. Conveniently, GMs are amenable to lyophilization and can be stored at ambient temperatures for at least 1 month without loss of integrity and bio-activity. This intracellular gelation technology provides a universal platform for targeted inflammation neutralization treatment.

Self-reporting intracellular delivery agent for singlet oxygen.

1,4-Dimethylphenazine endoperoxide releases singlet oxygen with a half-life of 89 hours at 37 °C. The thermal cycloreversion reaction is accompanied by a strong increase in the emission intensity with a peak at 490 nm, due to the formation of the phenazine core. The endoperoxide is effective against cancer cells in culture medium and tumor spheroids, with singlet oxygen-mediated cytotoxicity.

Label-free spatially maintained measurements of metabolic phenotypes in cells.

Metabolic reprogramming at a cellular level contributes to many diseases including cancer, yet few assays are capable of measuring metabolic pathway usage by individual cells within living samples. Here, autofluorescence lifetime imaging is combined with single-cell segmentation and machine-learning models to predict the metabolic pathway usage of cancer cells. The metabolic activities of MCF7 breast cancer cells and HepG2 liver cancer cells were controlled by growing the cells in culture media with specific substrates and metabolic inhibitors. Fluorescence lifetime images of two endogenous metabolic coenzymes, reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), were acquired by a multi-photon fluorescence lifetime microscope and analyzed at the cellular level. Quantitative changes of NADH and FAD lifetime components were observed for cells using glycolysis, oxidative phosphorylation, and glutaminolysis. Conventional machine learning models trained with the autofluorescence features classified cells as dependent on glycolytic or oxidative metabolism with 90%-92% accuracy. Furthermore, adapting convolutional neural networks to predict cancer cell metabolic perturbations from the autofluorescence lifetime images provided improved performance, 95% accuracy, over traditional models trained via extracted features. Additionally, the model trained with the lifetime features of cancer cells could be transferred to autofluorescence lifetime images of T cells, with a prediction that 80% of activated T cells were glycolytic, and 97% of quiescent T cells were oxidative. In summary, autofluorescence lifetime imaging combined with machine learning models can detect metabolic perturbations between glycolysis and oxidative metabolism of living samples at a cellular level, providing a label-free technology to study cellular metabolism and metabolic heterogeneity.

Iguratimod Could Correct the Defective IL-35-Mediated Megakaryopoiesis in Immune Thrombocytopenia

Introduction: IL-35 is an inhibitory cytokine of the IL-12 family, and comprises an EBI3 subunit and a p35 subunit. Previous data indicated that serum levels of IL-35 were significantly lower in immune thrombocytopenia (ITP) patients than in healthy controls. Moreover, the concentration of plasma IL-35 in ITP patients achieving complete response returned to normal, but was not corrected in those who failed to achieve response. However, the underlying pathological function of IL-35 in impaired megakaryopoiesis in ITP remains unexplored. Our study aimed to evaluate the immunoregulatory role of IL-35 in megakaryocytes (MKs) differentiation. Iguratimod, a traditional therapy for inflammatory disorders, has been demonstrated to promote megakaryopoiesis. The effect of iguratimod on the treatment of ITP was retrospectively reviewed. Methods: Bone marrow samples were obtained from newly-diagnosed primary ITP patients and healthy donors. CD34+ cells from ITP patients and healthy donors were differentiated into MKs with and without IL-35 to determine the mechanism of IL-35 in megakaryopoiesis. Iguratimod was added to the culture medium of MKs to determine its function in MKs differentiation. Additionally, ITP mouse model was established to further validate the function of IL-35. Results: In comparison with healthy controls, ITP patients showed lower bone marrow levels of IL-35. IL-35 receptor expression was progressively induced on bone marrow MKs from healthy controls during the differentiation of CD34+ progenitors. In contrast, MKs from ITP patients exhibited defective expression of the IL-35 receptor. We then determined the effect of IL-35 on the megakaryocytic potential of CD34+ progenitor cells in culture medium containing TPO. IL-35 increased the number of medium-sized and large-sized colonies, and increased the total number of CD41+ cells. In IL-35-treated MKs, higher levels of Akt phosphorylation were observed. Pretreatment with LY294002, an inhibitor of Akt phosphorylation, blocked the differentiation of MKs induced by IL-35. LY294002 inhibited proplatelet formation and aggregation of β1-tubulin in MKs stimulated by IL-35. Flow cytometry analysis revealed that platelet production decreased in the LY294002 pretreatment group. These results demonstrated that IL-35 can enhance the proliferation and differentiation of MKs through Akt pathways. The decreased level of bone marrow IL-35 may partially explain the defective megakaryopoiesis in ITP. We then explored the potential causes of the decreased IL-35 levels in ITP. Flow cytometry analysis showed that the percentage of bone marrow iTr35 cells, the primary sources of IL-35, was significantly lower in ITP patients. Furthermore, we cocultured CD4+ T cells with MSCs from healthy controls and ITP patients, respectively. The percentage of iTr35 cells was lower when cultured with ITP-MSCs. These results showed that ITP-MSCs exhibited an impaired capability of inducing iTr35 cells, which might contribute to the reduced level of bone marrow IL-35 in ITP patients. We further investigated the effects of iguratimod on the differentiation of CD34+ progenitors into MKs. When CD34+ cells were cultured with TPO alone, the addition of iguratimod upregulated the expression of IL-35 receptors on MKs from ITP patients and a murine model of this disease, and consequently promoted the formation of MK colonies. In the retrospective study of iguratimod, a total of 68 adult patients with corticosteroid-resistant primary ITP were included. Initial response was achieved in 42/68 (62%) patients, and complete response was achieved in 25/68 (37%) patients. In patients who achieved initial response, the median time to response was 28 days. Sustained response at 6 months was seen in 35/68 (51%). Bleeding symptoms were reduced from 37/68 (54%) patients at baseline to 14/68 (21%) patients at 6 months. Most adverse events were mild (grade 1-2), and there were no grade 4 or worse adverse events in our patients. The most common adverse events were elevated ALT, gastrointestinal disorders, and nausea. Conclusions: ITP-MSCs exhibited an impaired capability of inducing iTr35 cells, which contributed to the reduced level of bone marrow IL-35 in ITP. IL-35 promoted MKs differentiation through the Akt pathway. Iguratimod promoted megakaryopoiesis by inducing the expression of IL-35 receptors on MKs. Iguratimod had good efficacy and safety in the treatment of ITP.

Secretome analysis of breast cancer cells to identify potential target proteins of Ipomoea turpethum extract-loaded nanoparticles in the tumor microenvironment.

Background: Breast cancer is the leading cause of frequent malignancy and morbidity among women across the globe, with an increment of 0.5% incidences every year. The deleterious effects of traditional treatment on off-target surrounding cells make it difficult to win the battle against breast cancer. Hence, an advancement in the therapeutic approach is crucial. Nanotechnology is one of the emerging methods for precise, targeted, and efficient drug delivery in cells. The previous study has demonstrated the cytotoxic effect of Ipomoea turpethum extract on breast cancer cells delivered via NIPAAM-VP-AA nanoparticles (NVA-IT). Manipulating the tumor microenvironment (TME) to inhibit cancer progression, invasion, and metastasis seems to be very insightful for researchers these days. With the help of secretome analysis of breast cancer cells after treatment with NVA-IT, we have tried to find out the possible TME manipulation achieved to favor a better prognosis of the disease. Method: MCF-7 and MDA MB-231 cells were treated with the IC50 value of NVA-IT, and the medium was separated from the cells after 24h of the treatment. Nano LCMS/MS analysis was performed to identify the secretory proteins in the media. Further bioinformatics tools like GENT2, GSCA, GeneCodis 4, and STRING were used to identify the key proteins and their interactions. Result: From the nano LCMS/MS analysis, 70 differentially expressed secretory proteins in MCF-7 and 191 in MDA MB-231 were identified in the cell's media. Fifteen key target proteins were filtered using bioinformatics analysis, and the interaction of proteins involved in vesicular trafficking, cell cycle checkpoints, and oxidative stress-related proteins was prominent. Conclusion: This study concluded that I. turpethum extract-loaded NIPAAM-VP-AA nanoparticles alter the secretory proteins constituting the TME to cease cancer cell growth and metastasis.

Isolation and Characterization of Bacterial Gut Symbionts from Irradiated, Wild and Lab Reared Males of Melon Fly, Zeugodacus cucurbitae (Coquillett)

The melon fly, Bactrocera cucurbitae is of serious concern inflicting heavy losses to the horticultural industry. It attacks cucurbits right from the primordial stages of the crop up to harvest and causing yield loss of 30 to 100%. The Sterile Insect Technique is a species-specific and environmentally non-polluting method of insect control that relies on sterilization of males using gamma rays and systemic release of sterile males in wild environment. Male pupae of Zeugodacus cucurbitae were exposed to gamma radiation at 50 Gy using Cobalt-60 source. Bacterial gut symbionts from sterile males, wild males of field collected population and laboratory reared males of melon fly were isolated and characterized based on morphological characteristics (viz., colour, size, shape, opacity, margin, elevation and viscosity), gram staining, morphology and arrangement of bacterial cells in culture media. Ten adult flies from sterile, wild and laboratory reared males were used for isolation of gut symbionts. The most dominant phylum of bacteria found among the sterile, wild and lab reared male flies was Proteobacteria followed by the phylum Firmicutes. Different genera of bacteria isolated from sterile males were Enterobacter, Providencia and Bacillus. From wild males; Enterobacter, Providencia, Morganella, Klebsiella and Bacillus were identified. Bacterial genera obtained from lab reared males were Enterobacter, Providencia, Klebsiella and Bacillus. Among the entire bacterial genus, Enterobacter, Providencia and Bacillus were the common bacterial genera isolated from sterile, wild and lab reared male flies. Irradiation had resulted in loss of endosymbiotic bacteria in sterile males.

Two metabolic stages of SRB strain Desulfovibrio bizertensis affecting corrosion mechanism of carbon steel Q235

Desulfovibrio bizertensis (D. bizertensis), an SRB isolated and cultured from the rust layer of the South China Sea, is widespread in the corrosive microbial communities on steel surface. Although the corrosion mechanism of SRB was widely studied, the mechanism of different metabolic stages inducing corrosion was still unclear. In this work, D. bizertensis was used to study the corrosion process of carbon steel Q235 at different metabolic stages by surface analysis and electrochemical methods. Combined with the metabolic process of SRB, corrosion pits of carbon steel could be influenced by two stages. In the first stage, the rapid growth of planktonic D. bizertensis cells in culture medium was accompanied by quick consumption of the electron donor (lactate) till complete consumption. D. bizertensis cells began to adhere to Q235 surface and promote anodic dissolution of iron. In the second stage, the growth of planktonic cells was suppressed, thus the colonized D. bizertensis cell in the pitting could obtain energy directly from Fe(0) through DET, which would further aggravate pitting corrosion and the maximum depth of the pits approached 15.7 µm. Sulfides produced by D. bizertensis metabolism would accumulate on steel coupon and form FeS coverage. Thus D. bizertensis could survive for a long time in this conductive FeS particles network. Electrochemical results showed the promotion effect of D. bizertensis on cathodic reaction, and the values of Rct in the SRB medium were much lower than that in the sterile medium.

Glutamine deprivation induces ferroptosis in pancreatic cancer cells.

Ferroptosis is a type of programmed cell death closely related to amino acid metabolism. Pancreatic cancer cells have a strong dependence on glutamine, which serves as a carbon and nitrogen substrate to sustain rapid growth. Glutamine also aids in self-protection mechanisms. However, the effect of glutamine on ferroptosis in pancreatic cancer remains largely unknown. Here, we aim to explore the association between ferroptosis and glutamine deprivation in pancreatic cancer. The growth of pancreatic cancer cells in culture media with or without glutamine is evaluated using Cell Counting Kit-8. Reactive oxygen species (ROS) are measured by 2',7'-dichlorodihydrofluorescein diacetate staining. Ferroptosis is assessed by BODIPY-C11 dye using confocal microscopy and flow cytometry. Amino acid concentrations are measured using ultrahigh-performance liquid chromatography-tandem mass spectrometry. Isotope-labelled metabolic flux analysis is performed to track the metabolic flow of glutamine. Additionally, RNA sequencing is performed to analyse the genetic alterations. Glutamine deprivation inhibits pancreatic cancer growth and induces ferroptosis both in vitro and in vivo. Additionally, glutamine decreases ROS formation via glutathione production in pancreatic cancer cells. Interestingly, glutamine inhibitors (diazooxonorleucine and azaserine) promotes ROS formation and ferroptosis in pancreatic cancer cells. Furthermore, ferrostatin, a ferroptosis inhibitor, rescues ferroptosis in pancreatic cancer cells. Glutamine deprivation leads to changes in molecular pathways, including cytokine-cytokine receptor interaction pathways ( CCL5, CCR4, LTA, CXCR4, IL-6R, and IL-7R). Thus, exogenous glutamine is required for the detoxification of ROS in pancreatic cancer cells, thereby preventing ferroptosis.

In Vitro Anthelmintic Effect of Ferula assa-foetida Hydroalcoholic Extract Against Flukes of Fasciola hepatica and Dicrocoelium dendriticum

Background: Dicrocoeliasis and fascioliasis are foodborne parasitic diseases of the biliary tract, resulting from Dicrocoelium dendriticum and Fasciola hepatica causing extensive financial losses and serious health problems in ruminants. Due to low-performance medications, drug delivery is a tremendous project to improve interventions available for these diseases. Objectives: This study aimed to determine the anthelmintic properties of Ferula assa-foetida extract against F. hepatica and D. dendriticum using in vitro assay. Methods: The effects of diverse concentrations of F. assa-foetida extract (400 - 1000 µg/mL) for 12-24 hours were examined for the treatment of D. dendriticum and F. hepatica. The anthelmintic efficacies were evaluated using scanning electron microscopy (SEM). The MTT assay was carried out to evaluate the cell viability of all cells in culture media. Results: The SEM images of treated worms by F. assa-foetida extract (200 µg/mL) confirmed excessive damage, which included an entire lack of sensory papillae and destruction of distinguished network structures and tegument vesicles. Variables of duration and concentration presented a considerable effect on both the mortality rate and the anthelmintic properties of F. assa-foetida; accordingly, as the time and concentration increased, the mortality rate became higher. Based on the MTT assay, the toxicity of F. assa-foetida at 800 µg/mL concentration was 8.7%. Therefore, it can be argued that F. assa-foetida had anthelmintic properties. Conclusions: This is the first study that evaluated the impact of F. assa-foetida on liver flukes of D. dendriticum and F. hepatica. Therefore, it paved the way for further studies on the control of those trematodes. It is recommended to document and look into the indigenous understanding of viable medicinal plants to provide evidence for their use.

Eukaryotic CRFK Cells Motion Characterized with Atomic Force Microscopy.

We performed a time-lapse imaging with atomic force microscopy (AFM) of the motion of eukaryotic CRFK (Crandell-Rees Feline Kidney) cells adhered onto a glass surface and anchored to other cells in culture medium at 37 °C. The main finding is a gradient in the spring constant of the actomyosin cortex along the cells axis. The rigidity increases at the rear of the cells during motion. This observation as well as a dramatic decrease of the volume suggests that cells may organize a dissymmetry in the skeleton network to expulse water and drive actively the rear edge.

The Epigenetic Regulation in Plant Specialized Metabolism: DNA Methylation Limits Paclitaxel in vitro Biotechnological Production.

Environmental conditions are key factors in the modulation of the epigenetic mechanisms regulating gene expression in plants. Specifically, the maintenance of cell cultures in optimal in vitro conditions alters methylation patterns and, consequently, their genetic transcription and metabolism. Paclitaxel production in Taxus x media cell cultures is reduced during its maintenance in in vitro conditions, compromising the biotechnological production of this valuable anticancer agent. To understand how DNA methylation influences taxane production, the promoters of three genes (GGPPS, TXS, and DBTNBT) involved in taxane biosynthesis have been studied, comparing the methylation patterns between a new line and one of ~14 years old. Our work revealed that while the central promoter of the GGPPS gene is protected from cytosine methylation accumulation, TXS and DBTNBT promoters accumulate methylation at different levels. The DBTNBT promoter of the old line is the most affected, showing a 200 bp regulatory region where all the cytosines were methylated. This evidence the existence of specific epigenetic regulatory mechanisms affecting the last steps of the pathway, such as the DBTNBT promoter. Interestingly, the GGPPS promoter, a regulatory sequence of a non-specific taxane biosynthetic gene, was not affected by this mechanism. In addition, the relationship between the detected methylation points and the predicted transcription factor binding sites (TFBS) showed that the action of TFs would be compromised in the old line, giving a further explanation for the production reduction in in vitro cell cultures. This knowledge could help in designing novel strategies to enhance the biotechnological production of taxanes over time.

Review on Human Placental Proteomics

The human placenta plays a crucial part during conception of another human life. Through this time the placenta forms and grows along with the fetus and removed after the birth of the child. The human placenta is rich in proteins and other nutrients required for the growth of the fetus is now discarded. The proteins present in the placenta is studied by extracting proteins from the spongy tissue or by growing the cells in culture media. Use of appropriate technique is also essential in case of diagnosis of pathological condition or to use these proteins as potent drug. This paper reviews the basic components of the placenta and proteomic techniques used in various studies for normal and pathological conditions in the human placenta.

Mucin 21 confers resistance to apoptosis in an O-glycosylation-dependent manner

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.

Label-Free Digital Holographic Microscopy for In Vitro Cytotoxic Effect Quantification of Organic Nanoparticles.

Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots® nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.

Alkaline intracellular pH (pHi) activates AMPK–mTORC2 signaling to promote cell survival during growth factor limitation

The mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) signaling controls cell metabolism, promotes cell survival, and contributes to tumorigenesis, yet its upstream regulation remains poorly defined. Although considerable evidence supports the prevailing view that amino acids activate mTOR complex 1 but not mTORC2, several studies reported paradoxical activation of mTORC2 signaling by amino acids. We noted that after amino acid starvation of cells in culture, addition of an amino acid solution increased mTORC2 signaling. Interestingly, we found the pH of the amino acid solution to be alkaline, ∼pH 10. These observations led us to discover and demonstrate here that alkaline intracellular pH (pHi) represents a previously unknown activator of mTORC2. Using a fluorescent pH-sensitive dye (cSNARF1-AM) coupled with live-cell imaging, we demonstrate that culturing cells in media at an alkaline pH induces a rapid rise in the pHi, which increases mTORC2 catalytic activity and downstream signaling to the pro-growth and pro-survival kinase Akt. Alkaline pHi also activates AMPK, a canonical sensor of energetic stress. Functionally, alkaline pHi activates AMPK-mTOR signaling, which attenuates apoptosis caused by growth factor withdrawal. Collectively, these findings reveal that alkaline pHi increases mTORC2- and AMPK-mediated signaling to promote cell survival during conditions of growth factor limitation, analogous to the demonstrated ability of energetic stress to activate AMPK–mTORC2 and promote cell survival. As an elevated pHi represents an underappreciated hallmark of cancer cells, we propose that the alkaline pHi stress sensing by AMPK–mTORC2 may contribute to tumorigenesis by enabling cancer cells at the core of a growing tumor to evade apoptosis and survive.

Investigating the cytotoxicity of four types of freshly prepared root canal sealers using human gingival fibroblasts

Introduction: One of the most important characteristics of endodontic treatment seals is their biocompatibility. The aim of present study was to investigate the cytotoxicity of four types of freshly prepared root canal sealers using human gingival fibroblasts. Materials and Methods: In this experimental laboratory study, freshly prepared sealers were investigated. To investigate the cytotoxicity, each sample was extracted separately in RPMI-1640 (Sigma Aldrich-USA). Then, the resulting extracts were exposed to C165 fibroblast cells in culture medium for 1, 3 and 7 days. Then, the level of cytotoxicity was assessed by MTT colorimetric method based on the level of light absorption. In the present study, multivariate analysis of variance was used to analyze the data. Results: The results showed that the cytotoxicity of AH 26 and tgsealer sealers decreased and MTA Fillapex sealer increased over time. Also, Adseal sealer did not follow a logical trend but, in general, its cytotoxicity increased over time. In addition, based on mean of all data obtained from each sealer, MTA Fillapex had the lowest cytotoxicity and Adseal had the highest cytotoxicity, and AH 26 cytotoxicity was higher than that of tgsealer. Conclusion: According to the results of the present study, the MTA-based sealer of MTA Fillapex has the lowest cytotoxicity. Keywords: Root canal sealers, fibroblasts, freshly prepared roots

CLL Cell-Derived Exosomes Promote Bone Marrow Fibrosis and Inhibit Normal Hematopoietic Colony Proliferation

CLL Cell-Derived Exosomes Promote Bone Marrow Fibrosis and Inhibit Normal Hematopoietic Colony Proliferation

Genotoxic Effects of Radiofrequency-Electromagnetic Fields

Genotoxic Effects of Radiofrequency-Electromagnetic Fields