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IntroductionSmall membrane particles called extracellular vesicles (EVs) transport biologically active cargo between cells, providing intercellular communication. The clinical application of EVs is limited due to the lack of scalable and cost-effective approaches for their production and purification, as well as effective loading strategies.MethodsHere we used EV mimetics produced by cell treatment with the actin-destabilizing agent cytochalasin B as an alternative to EVs for the delivery of therapeutic nucleic acids.ResultsCytochalasin-B-inducible nanovesicles (CINVs) delivered a fully modified N-(methanesulfonyl)- or mesyl (µ-) antisense oligonucleotide to B16 melanoma cells, selectively decreasing the level of target microRNA-21 with effectiveness comparable to that observed upon Lipofectamine 2000-mediated delivery. The efficiency of the CINV-mediated delivery of plasmid DNA encoding EGFP varied depending on the type of recipient cells. Surprisingly, under experimental conditions, CINVs were unable to deliver both modified and natural short RNA duplexes—small interfering RNA and immunostimulatory RNA—probably due to their poor loading into CINVs.DiscussionCINVs demonstrated unique properties for the delivery of therapeutic nucleic acids, especially for antisense oligonucleotide-based therapy.

Immunotherapy represents an innovative approach to cancer treatment, based on activating the body’s own immune system to combat tumor cells. Among various immunotherapy strategies, dendritic cell vaccines hold a special place due to their ability to activate T-lymphocytes, key players in cellular immunity, and direct them to tumor cells. In this study, the influence of dendritic cells processed with tumor-derived vesicles on the viability of melanoma cells in vitro was investigated. Dendritic cells were loaded with tumor-derived vesicles, after which they were used to activate T-cells. The study demonstrated that such modified T-cells exhibit high activity against melanoma cells, leading to a decrease in their viability. Our analysis highlights the potential efficacy of this approach in developing immunotherapy against melanoma. These results provide new prospects for further research and the development of antitumor strategies based on the mechanisms of T-lymphocyte activation using tumor-derived vesicles.

Triploid breeding with large individuals, high yield, and excellent disease resistance is widely used in aquaculture. However, the functional roles of triploid in the Zebra Calm II strain of Manila clam Ruditapes philippinarum has not been highlighted. In this study, the Zebra Clam II strain of the Manila clams were treated with cytochalasin B (CB) at a final concentration of 0.5 mg/L at 26 °C for 17 min to induce triploidity. The results showed that triploid induction rate of first-hatched D-larvae was 64.73 % and the triploid proportion of 180-day-old larvae was 72.5 %. Furthermore, the hatching rate in triploid (60.54 ± 2.19 %) was significantly lower than that in the control group (92.47 ± 0.58 %) (p 0.05). However, the daily relative growth rate of experimental group was significantly higher than that the control group at all daily ages except 360 days (p

Tumor-necrosis-factor-associated apoptosis-inducing ligand (TRAIL) is one of the most promising therapeutic cytokines that selectively induce apoptosis in tumor cells. It is known that membrane vesicles (MVs) can carry the surface markers of parental cells. Therefore, MVs are of interest as a tool for cell-free cancer therapy. In this study, membrane vesicles were isolated from TRAIL-overexpressing mesenchymal stem cells using cytochalasin B treatment (CIMVs). To evaluate the antitumor effect of CIMVs-TRAIL in vivo, a breast cancer mouse model was produced. The animals were intratumorally injected with 50 µg of native CIMVs or CIMVs-TRAIL for 12 days with an interval of two days. Then, tumor growth rate, tumor necrotic area, the expression of the apoptosis-related genes CASP8, BCL-2, and BAX and the level of CASP8 protein were analyzed. A 1.8-fold increase in the CAS8 gene mRNA and a 1.7-fold increase in the CASP8 protein level were observed in the tumors injected with CIMVs-TRAIL. The expression of the anti-apoptotic BCL-2 gene in the CIMV-TRAIL group remained unchanged, while the mRNA level of the pro-apoptotic BAX gene was increased by 1.4 times, which indicated apoptosis activation in the tumor tissue. Thus, CIMVs-TRAIL were able to activate the extrinsic apoptosis pathway and induce tumor cell death in the breast cancer mouse model.

To date, there are numerous protocols for the isolation of extracellular vesicles (EVs). Depending on the isolation method, it is possible to obtain vesicles with different characteristics, enriched with specific groups of proteins, DNA and RNA, which affect similar types of cells in the opposite way. Therefore, it is important to study and compare methods of vesicle isolation. Moreover, the differences between the EVs derived from tumor and mesenchymal stem cells are still poorly understood. This article compares EVs from human glioblastoma cells and mesenchymal stem cells (MSCs) obtained by two different methods, ultracentrifugation and cytochalasin B-mediated induction. The size of the vesicles, the presence of the main EV markers, the presence of nuclear and mitochondrial components, and the molecular composition of the vesicles were determined. It has been shown that EVs obtained by both ultracentrifugation and cytochalasin B treatment have similar features, contain particles of endogenous and membrane origin and can interact with monolayer cultures of tumor cells.

Multiple sclerosis (MS) is an incurable, progressive chronic autoimmune demyelinating disease. Therapy for MS is based on slowing down the processes of neurodegeneration and suppressing the immune system of patients. MS is accompanied by inflammation, axon-degeneration and neurogliosis in the central nervous system. One of the directions for a new effective treatment for MS is cellular, subcellular, as well as gene therapy. We investigated the therapeutic potential of adipose mesenchymal stem cell (ADMSC) derived, cytochalasin B induced artificial microvesicles (MVs) expressing nerve growth factor (NGF) on a mouse model of multiple sclerosis experimental autoimmune encephalomyelitis (EAE). These ADMSC-MVs-NGF were tested using histological, immunocytochemical and molecular genetic methods after being injected into the tail vein of animals on the 14th and 21st days post EAE induction. ADMSC-MVs-NGF contained the target protein inside the cytoplasm. Their injection into the caudal vein led to a significant decrease in neurogliosis at the 14th and 21st days post EAE induction. Artificial ADMSC-MVs-NGF stimulate axon regeneration and can modulate gliosis in the EAE model. [ABSTRACT FROM AUTHOR]

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Among perinatal stem cells of the umbilical cord, human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1–3 μM) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers. [ABSTRACT FROM AUTHOR]

The previously known cytotoxic cytochalasan, cytochalasin B (1), was isolated and purified from the ethyl acetate extract of the rice fermentation culture of the Alpine asphodel-associated Dothideomycetes fungus, Sparticola triseptata after iterative chromatographic purifications. The structure of 1 was established through extensive 1D and 2D-NMR spectroscopic experiments along with high-resolution electrospray ionization mass spectrometry (HRESIMS) and by comparison of its spectroscopic data with the literature. The potential of cytochalasin B (1) as antidiabetic and anti-obesity compound was screened using microplate, colorimetric enzyme inhibitory assays. Thus, 1 displayed significant in vitro α-glucosidase and porcine pancreatic lipase inhibitory activities with IC50 values of 5.46 µM and 8.43 µM, respectively when compared to the positive drug controls, acarbose and Orlistat®. Molecular simulation experiments using molecular docking showed moderately strong affinity of 1 onto the active sites of α-glucosidase and porcine pancreatic lipase with binding energies (BE) of -8.6 kcal/mol and -7.5 kcal/mol, respectively. [ABSTRACT FROM AUTHOR]

The identification of genetic biomarkers that play a crucial role in cancer survival is challenging, but requires attention. The present study aimed to identify common biomarkers that affect the survival of patients with cancer. For this purpose, The Cancer Genome Atlas datasets of liver, lung, cervical and pancreatic cancers were used to identify differentially expressed genes (DEGs) that have an impact on cancer survival. The STRING database and ComplexHeatmap package were used to develop a protein-protein interaction network and heatmaps of the DEGs, respectively. The inhibitors of DEGs were identified using CMap software. Molecular docking and molecular dynamics simulations were performed using PyRx and NAMD software. Of note, two common genes [human NDC80 kinetochore complex component (NDC80) and human disks large-associated protein 5] in liver and lung cancers and five common genes [human minichromosome maintenance complex component (MCM)2, origin recognition complex subunit 1, cell division cycle 45, MCM3 and human DNA topoisomerase II alpha] in cervical and liver cancers were found to significantly affect overall survival. Several inhibitors of these seven common genes were identified and it was found that cytochalasin (NDC80 inhibitor) demonstrated the highest binding affinity. The results of molecular dynamics suggested that cytochalasin B can be used as a therapeutic drug in cancers with a high expression of NDC80. In addition, the present study identified unique biomarkers present in specific types of cancer and these data were validated using the c-BioPortal database. The c-BioPortal data analysis demonstrated that the expression of the cytochrome P450 family 2 subfamily C member 9 and acyl-CoA dehydrogenase short chain genes, together with the American Joint Committee on Cancer Pathological Staging, can be used as prognostic markers of liver cancer recurrence (AUC value=0.71). No such data were obtained for other cancers. On the whole the present study highlights that the selection of DEGs as potential treatment targets should be based on their effects on cancer survival along with tumor stages. [ABSTRACT FROM AUTHOR]

Tunneling nanotubes (TNTs) are F‐actin‐based open‐ended tubular extensions that form following stresses, such as nutritional deprivation and oxidative stress. The chemotherapy agent 5‐fluorouracil (5‐FU) represents a significant stressor to cancer cells and induces thymidine deficiency, a state similar to nutritional deprivation. However, the ability of 5‐FU to induce TNT formation in cancer cells and potentially enhance survival has not been explored. In this study, we examined whether 5‐FU can induce TNT formation in MCF‐7 breast cancer cells. Cytotoxic doses of 5‐FU (150–350 μm) were observed to significantly induce TNT formation beginning at 24 h after exposure. TNTs formed following 5‐FU treatment probably originated as extensions of gap junctions as MCF‐7 cells detach from cell clusters. TNTs act as conduits for exchange of cellular components and we observed mitochondrial exchange through TNTs following 5‐FU treatment. 5‐FU‐induced TNT formation was inhibited by over 80% following treatment with the F‐actin‐depolymerizing agent, cytochalasin B (cytoB). The inhibition of TNTs by cytoB corresponded with increased 5‐FU‐induced cytotoxicity by 30–62% starting at 48 h, suggesting TNT formation aides in MCF‐7 cell survival against 5‐FU. Two other widely used chemotherapy agents, docetaxel and doxorubicin induced TNT formation at much lower levels than 5‐FU. Our work suggests that the therapeutic targeting of TNTs may increase 5‐FU chemotherapy efficacy and decrease drug resistance in cancer cells, and these findings merits further investigation.

Introduction: Small membrane particles called extracellular vesicles (EVs) transport biologically active cargo between cells, providing intercellular communication. The clinical application of EVs is limited due to the lack of scalable and cost-effective approaches for their production and purification, as well as effective loading strategies., Methods: Here we used EV mimetics produced by cell treatment with the actin-destabilizing agent cytochalasin B as an alternative to EVs for the delivery of therapeutic nucleic acids., Results: Cytochalasin-B-inducible nanovesicles (CINVs) delivered a fully modified N-(methanesulfonyl)- or mesyl (µ-) antisense oligonucleotide to B16 melanoma cells, selectively decreasing the level of target microRNA-21 with effectiveness comparable to that observed upon Lipofectamine 2000-mediated delivery. The efficiency of the CINV-mediated delivery of plasmid DNA encoding EGFP varied depending on the type of recipient cells. Surprisingly, under experimental conditions, CINVs were unable to deliver both modified and natural short RNA duplexes-small interfering RNA and immunostimulatory RNA-probably due to their poor loading into CINVs., Discussion: CINVs demonstrated unique properties for the delivery of therapeutic nucleic acids, especially for antisense oligonucleotide-based therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Oshchepkova, Chernikov, Miroshnichenko, Patutina, Markov, Savin, Staroseletz, Meschaninova, Puchkov, Zhukov, Kupryushkin, Maslov, Sen’kova, Vlassov, Chernolovskaya and Zenkova.)

Despite significant advances in modern medicine, effective therapeutics for Alzheimer's disease (AD) treatment have not yet been found. Currently, the use of exosomes and microvesicles derived from mesenchymal stem cells (MSCs) has begun to be considered as one of the promising approaches to the therapy of this disease. Exosomes and microvesicles in comparison with MSCs have significant benefits such as nanometric size, inability to divide, lack of risks of immunogenicity, malignant transformation, and thrombosis. In this work, cytochalasin B–induced membrane vesicles (CIMVs) from MSCs isolated from the adipose tissue were obtained and characterized, and the possibility of their penetration into the brain after intranasal administration was studied. Preliminarily, MSCs were transduced with the recombinant replication-deficient lentivirus containing RFP gene. The study was carried out on a model of sporadic AD in olfactory bulbectomized (OBX) mice, demonstrating behavioral, biochemical, and morphological signs of neurodegenerative process of the Alzheimer's type. Four hours after intranasal administration, presence of vesicles containing the fluorescent protein RFP was observed in the hippocampus and neocortex of OBX mice, i.e., in the brain areas most vulnerable to AD. It is assumed that microvesicles administrated intranasally can act as nanocontainers for target delivery of biologically active compounds present in MSCs, or even drugs to the brain regions affected by neurodegeneration, and are a promising therapeutic agent for the treatment of AD. [ABSTRACT FROM AUTHOR]

At present, there is an increasing interest in the potential role of extracellular vesicles (EVs), acting as multi-signal messengers of the tumor stroma, in the development and progression of tumor. Tumor cell-derived EVs are considered a potential vector for the targeted delivery of antitumor agents due to the ability to fuse with parental cells through endocytosis and release their contents into the cytoplasm of the recipient cell. Tumor cell-derived EVs could be also used for priming immune cells and therapeutic vaccine development. It is also known that mesenchymal stem cells (MSCs) have a tropism toward tumor niches. It is believed that MSC migration to the tumor is due to its inflammatory signaling. Presumably, with the accumulation of MSCs at tumor sites, these cells differentiate into pericytes or tumor-associated fibroblasts, thereby forming a supporting tumor growth microenvironment. However, besides the ability to promote tumor progression, MSCs can also suppress its growth by inhibiting proliferation and cell cycle progression, and angiogenesis. Thus, the further studies of the MSC role in TME and MSC interaction with other cells of the tumor stroma, including through EVs, are of particular interest. To increase the yield of vesicles the isolation method based on pharmacological disorganization of the actin cytoskeleton induced by treating with cytochalasin B was used in this study. In this investigation the interaction of SH-SY5Y neuroblastoma cell-derived membrane vesicles, obtained using cytochalasin B (CIMVs), with human bone marrow-derived MSCs was analyzed using imaging flow cytometry. Using transmission electron microscopy, it was shown that CIMVs have a size similar to that of natural microvesicles, which is 100–1000 nm. Using imaging flow cytometry, it was shown that after 24 h of co-cultivation 6% of the MSCs contained a large number of CIMVs, and 42% of the MSCs contained a small amount of CIMVs. Cultivation of MSCs with SH-SY5Y cell-derived CIMVs also induced dose-dependent decrease in the expression of CD markers typical for MSCs. Thus, the internalization of SH-SY5Y cell-derived CIMVs within MSCs and the ability of the CIMVs to modulate immunophenotype of the recipient cells were shown. However, further studies are required to determine the effect of CIMVs on pro- or antioncogenic phenotype and function of MSCs. [ABSTRACT FROM AUTHOR]

Cytoskeletal proteins provide architectural and signaling cues within cells. They are able to reorganize themselves in response to mechanical forces, converting the stimuli received into specific cellular responses. Thus, the cytoskeleton influences cell shape, proliferation, and even differentiation. In particular, the cytoskeleton affects the fate of mesenchymal stem cells (MSCs), which are highly attractive candidates for cell therapy approaches due to their capacity for self-renewal and multi-lineage differentiation. Cytochalasin B (CB), a cyto-permeable mycotoxin, is able to inhibit the formation of actin microfilaments, resulting in direct effects on cell biological properties. Here, we investigated for the first time the effects of different concentrations of CB (0.1–10 μM) on human adipose-derived stem cells (hASCs) both after 24 h (h) of CB treatment and 24 h after CB wash-out. CB influenced the metabolism, proliferation, and morphology of hASCs in a dose-dependent manner, in association with progressive disorganization of actin microfilaments. Furthermore, the removal of CB highlighted the ability of cells to restore their cytoskeletal organization. Finally, atomic force microscopy (AFM) revealed that cytoskeletal changes induced by CB modulated the viscoelastic properties of hASCs, influencing their stiffness and viscosity, thereby affecting adipogenic fate. [ABSTRACT FROM AUTHOR]

Tunneling nanotubes (TNTs) are F‐actin‐based open‐ended tubular extensions that form following stresses, such as nutritional deprivation and oxidative stress. The chemotherapy agent 5‐fluorouracil (5‐FU) represents a significant stressor to cancer cells and induces thymidine deficiency, a state similar to nutritional deprivation. However, the ability of 5‐FU to induce TNT formation in cancer cells and potentially enhance survival has not been explored. In this study, we examined whether 5‐FU can induce TNT formation in MCF‐7 breast cancer cells. Cytotoxic doses of 5‐FU (150–350 μm) were observed to significantly induce TNT formation beginning at 24 h after exposure. TNTs formed following 5‐FU treatment probably originated as extensions of gap junctions as MCF‐7 cells detach from cell clusters. TNTs act as conduits for exchange of cellular components and we observed mitochondrial exchange through TNTs following 5‐FU treatment. 5‐FU‐induced TNT formation was inhibited by over 80% following treatment with the F‐actin‐depolymerizing agent, cytochalasin B (cytoB). The inhibition of TNTs by cytoB corresponded with increased 5‐FU‐induced cytotoxicity by 30–62% starting at 48 h, suggesting TNT formation aides in MCF‐7 cell survival against 5‐FU. Two other widely used chemotherapy agents, docetaxel and doxorubicin induced TNT formation at much lower levels than 5‐FU. Our work suggests that the therapeutic targeting of TNTs may increase 5‐FU chemotherapy efficacy and decrease drug resistance in cancer cells, and these findings merits further investigation. [ABSTRACT FROM AUTHOR]

Among perinatal stem cells of the umbilical cord, human Wharton’s jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1–3 μM) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers.

Owing to their self-renewal and multi-lineage differentiation capability, mesenchymal stem cells (MSCs) hold enormous potential in regenerative medicine. A prerequisite for a successful MSC therapy is the rigorous investigation of their function after in vitro cultivation. Damages introduced to mitochondria during cultivation adversely affect MSCs function and can determine their fate. While it has been shown that microtubules and vimentin intermediate filaments are important for mitochondrial dynamics and active mitochondrial transport within the cytoplasm of MSCs, the role of filamentous actin in this process has not been fully understood yet. To gain a deeper understanding of the interdependence between mitochondrial function and the cytoskeleton, we applied cytochalasin B to disturb the filamentous actin-based cytoskeleton of MSCs. In this study we combined conventional functional assays with a state-of-the-art oxygen sensor-integrated microfluidic device to investigate mitochondrial function. We demonstrated that cytochalasin B treatment at a dose of 16 μM led to a decrease in cell viability with high mitochondrial membrane potential, increased oxygen consumption rate, disturbed fusion and fission balance, nuclear extrusion and perinuclear accumulation of mitochondria. Treatment of MSCs for 48 h ultimately led to nuclear fragmentation, and activation of the intrinsic pathway of apoptotic cell death. Importantly, we could show that mitochondrial function of MSCs can efficiently recover from the damage to the filamentous actin-based cytoskeleton over a period of 24 h. As a result of our study, a causative connection between the filamentous actin-based cytoskeleton and mitochondrial dynamics was demonstrated. [ABSTRACT FROM AUTHOR]

Abstract Circulating tumor cells (CTCs) are receiving a great amount of scientific interest as a diagnostic biomarker for various types of cancer. Despite the recent progress in the development of highly sensitive CTC isolation devices, post-capture analysis of CTCs is still hindered by technical challenges associated with their rarity. Herein, we present a multi-modal CTC screening platform which is capable to analyze CTCs and CTC-derived extracellular vesicles (EVs), simultaneously from a single sample. Cytochalasin B (CB) treatment promotes cells to release large number of EVs from their surface, as demonstrated by CB-treated cells (5 µg/mL for 3 h) secreting 3.5-fold more EVs, compared to the non-treated cells. CB further generates 1.7-fold more EVs from the cells captured on our CTC filtration device (the fabric filter), compared to those from the cell culture flasks, owing to its multiple pore structure design which reduces the non-specific binding of EVs. Both CB-treated cancer cells and CB-induced EVs are found to overexpress tumor-associated markers, demonstrating a potential for the development of CTC dual-screening platform. Collectively, the results presented in this study reveal that our multi-modal cancer screening platform can synergistically improve the reliability and efficacy of the current CTC analysis systems.

The paper discusses the transport functions of the main classes of blood plasma lipoproteins (LP) that are not associated with the metabolism of lipids that make up their composition. The aim of the study was to study the ability of various plasma LP fractions (very low (VLDL), low (LDL) and high density (HDL)) to interact with certain hydrophilic and hydrophobic compounds and show the role of LP as transport forms of xenobiotics in the organs and tissues of the body. Material and methods. The studies were performed with tritium-labeled cytochalasin B, benzylpenicillin, benzanthracene, benzo(a)pyrene, ultracentrifugation of human plasma LP fractions, column chromatography; in vivo experiments with intravenous injection of LP complexes with tritium-labeled benzanthracene were conducted. Results. The ability of various classes of LP to form complexes with hydrophilic (cytochalasin B, benzylpenicillin) and hydrophobic (benzanthracene, benzo(a)pyrene) compounds is shown by the method of ultracentrifugation. More than 50 % of the radioactivity of hydrophilic compounds in human blood plasma was represented in the composition of the LDL and HDL fractions, and in the composition of the VLDL fractions it was minimal – 6.3 and 5.1 %, respectively. A significant part of cytochalasin and benzylpenicillin was also present in the protein infranatant – 43.6 and 40.9 %, respectively. The distribution in blood plasma for hydrophobic (benzanthracene, benzo(a)pyrene) compounds was different. More than 80 % of the radioactivity was represented in the composition of the LP fractions. The polar protein infranatant contained 16.1 % of the radioactivity of benzantracene and 13.6 % of benzo(a)pyrene. The features of the lipophilic xenobiotics uptake by organs and tissues were shown in vivo experiments with intravenous injection of complexes of LP with tritium-labeled benzanthracene to rats. The highest specific radioactivity was found in the liver and adrenal glands after the intravenous injection of 3H-benzanthracene in the composition of VLDL and LDL. Twice less uptake of the labeled drug was observed in the testis and kidneys. Radioactivity decreased in the series: lungs,adipose tissue, thymus, heart, and spleen. A feature of the use of HDL as a platform for 3H-enzanthracene is the intense accumulation of lipophilic xenobiotics in steroid-producing organs: the adrenal glands and testis. Conclusion. The results obtained allow us to consider the real possibility of using blood plasma PL as transport platforms for hydrophilic and hydrophobic compounds into the cells of organs and tissues.

Fertilized Zhikong scallop (Chlamys farreri) eggs were treated with cytochalasin B (CB 0.5 mg/L) at 14–15 min postfertilization to inhibit first polar body formation. The eggs were then stained with fluorescein isothiocyanate (FITC) -anti-α-tubulin and propidium iodide (PI) to examine their microtubule patterns and chromosome, respectively. Fluorescent microscope observations of treated eggs sampled every 2–3 min during meiotic maturation revealed meiotic apparatus assembly and correlated chromosome segregation. In CB-treated groups, meiosis I proceeded normally and produced two groups of dyads, with 19 in each group. Both dyad groups were retained in the eggs as they entered meiosis II. Two, three, or four asters (centrosome with microtubules around it) in meiosis II rearranged the spindle in several patterns: bipolar [24.0 ± 4.1 μm (long axis) × 18.3 ± 4.1 μm (diameter: metaphase plate)], tripolar (18.6 ± 3.9 μm × 9.9 ± 1.3 μm), separated bipolar (18.3 ± 2.8 μm × 11.2 ± 1.8 μm), and other unclassified spindle patterns. Corresponding chromosome segregation, including bipolar (18.9%), tripolar (38.9%), double bipolar (16.5%), and unclassified (25.6%), was observed during meiosis II in CB-treated eggs. The data indicated that chromosome segregation patterns determined by spindle patterns were critically influenced by the number of centrosomes in meiosis II eggs following inhibition of polar body 1 (PB1) formation with CB.

In order to obtain suitable conditions for tetraploid induction of Crassostrea gigas "Haida No. 2", the effects of cytochalasin B (CB) concentration, initial induction time and induction duration on tetraploid rate and D larvae rate were estimated. The ploidy of induced spat was tested to verify the effectiveness of the induction method. The result showed that the linear effects of CB concentration and initial induction time on tetraploid rate were significantly (P<0.05), but induction duration had no significant effect on tetraploid rate (P>0.05). The interactive effect of CB concentration, initial induction time and induction duration influenced tetraploid rate significantly (P<0.05). The linear effects of CB concentration, initial induction time and induction duration on D larvae rate were significant (P<0.05), but the interactive effect of CB concentration, initial induction time and induction duration had no significant effect on D larvae rate (P>0.05). The maximum point of tetraploid rate was found at CB concentration of 0.5 mg/L, initial induction time of 15min and induction duration of 20min. Under these conditions, the tetraploid rate reached to (37.82±4.75)%. On day 180, tetraploid spat of C. gigas "Haida No. 2" existed in two of the treatment groups, and the tetraploid rates were 2.22% and 3.03%, respectively. [ABSTRACT FROM AUTHOR]

Cytoskeletal proteins provide architectural and signaling cues within cells. They are able to reorganize themselves in response to mechanical forces, converting the stimuli received into specific cellular responses. Thus, the cytoskeleton influences cell shape, proliferation, and even differentiation. In particular, the cytoskeleton affects the fate of mesenchymal stem cells (MSCs), which are highly attractive candidates for cell therapy approaches due to their capacity for self-renewal and multi-lineage differentiation. Cytochalasin B (CB), a cyto-permeable mycotoxin, is able to inhibit the formation of actin microfilaments, resulting in direct effects on cell biological properties. Here, we investigated for the first time the effects of different concentrations of CB (0.1–10 μM) on human adipose-derived stem cells (hASCs) both after 24 h (h) of CB treatment and 24 h after CB wash-out. CB influenced the metabolism, proliferation, and morphology of hASCs in a dose-dependent manner, in association with progressive disorganization of actin microfilaments. Furthermore, the removal of CB highlighted the ability of cells to restore their cytoskeletal organization. Finally, atomic force microscopy (AFM) revealed that cytoskeletal changes induced by CB modulated the viscoelastic properties of hASCs, influencing their stiffness and viscosity, thereby affecting adipogenic fate.

The factors affecting intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high-resolution cryo-imaging technology, the broad bean intracellular ice formation and growth processes were successfully captured during freezing. Cytochalasin B（CB）was used to solubilize the cytoskeleton. Images of IIF were compared between cells with and without cytoskeleton. The behavior of intracellular ice crystal formation in plant tissues with or without CB was evaluated using changes of cell areas, the probability of crystallization, and growing rate of intracellular ice crystal. Moreover, light intensity figures were used to determine cell damage. This study showed that the cytoskeleton was involved in ice crystal nucleation mechanism during freezing responses of the plant cells.

The cytochalasin B-induced membrane vesicles (CIMVs) are suggested to be used as a vehicle for the delivery of therapeutics. However, the angiogenic activity and therapeutic potential of human mesenchymal stem/stromal cells (MSCs) derived CIMVs (CIMVs-MSCs) remains unknown. Objectives: The objectives of this study were to analyze the morphology, size distribution, molecular composition, and angiogenic properties of CIMVs-MSCs. Methods: The morphology of CIMVs-MSC was analyzed by scanning electron microscopy. The proteomic analysis, multiplex analysis, and immunostaining were used to characterize the molecular composition of the CIMVs-MSCs. The transfer of surface proteins from a donor to a recipient cell mediated by CIMVs-MSCs was demonstrated using immunostaining and confocal microscopy. The angiogenic potential of CIMVs-MSCs was evaluated using an in vivo approach of subcutaneous implantation of CIMVs-MSCs in mixture with Matrigel matrix. Results: Human CIMVs-MSCs retain parental MSCs content, such as growth factors, cytokines, and chemokines: EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFN-γ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-15, sCD40L, IL-17A, IL-1RA, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP_1a, MIP-1b, TNF-α, TNF-β, VEGF. CIMVs-MSCs also have the expression of surface receptors similar to those in parental human MSCs (CD90+, CD29+, CD44+, CD73+). Additionally, CIMVs-MSCs could transfer membrane receptors to the surfaces of target cells in vitro. Finally, CIMVs-MSCs can induce angiogenesis in vivo after subcutaneous injection into adult rats. Conclusions: Human CIMVs-MSCs have similar content, immunophenotype, and angiogenic activity to those of the parental MSCs. Therefore, we believe that human CIMVs-MSCs could be used for cell free therapy of degenerative diseases. [ABSTRACT FROM AUTHOR]

BackgroundAirway narrowing associated with chronic asthma results in the transmission of injurious compressive forces to the bronchial epithelium and promotes the release of pro-inflammatory mediators and the denudation of the bronchial epithelium. While the individual effects of compression or denudation are well characterized, there is no data to elucidate how these cells respond to the application of mechanical compression in the presence of a compromised epithelial layer.MethodsAccordingly, differentiated normal human bronchial epithelial cells were exposed to one of four conditions: 1) unperturbed control cells, 2) single scrape wound only, 3) static compression (6 hours of 30 cmH2O), and 4) 6 hours of static compression after a scrape wound. Following treatment, wound closure rate was recorded, media was assayed for mediator content and the cytoskeletal network was fluorescently labeled.ResultsWe found that mechanical compression and scrape injury increase TGF-beta2 and endothelin-1 secretion, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound augmented these observations, and also decreased PGE2 media content. Compression stimulated depolymerization of the actin cytoskeleton and significantly attenuated wound healing. Closure rate was partially restored with the addition of exogenous PGE2, but not EGF.ConclusionOur results suggest that mechanical compression reduces the capacity of the bronchial epithelium to close wounds, and is, in part, mediated by PGE2 and a compromised cytoskeleton.

The main advantage of extracellular vesicles (EVs) as a drug carrier system is their low immunogenicity and internalization by mammalian cells. EVs are often considered a cell-specific delivery system, but the production of preparative amounts of EVs for therapeutic applications is challenging due to their laborious isolation and purification procedures. Alternatively, mimetic vesicles prepared from the cellular plasma membrane can be used in the same way as natural EVs. For example, a cytoskeleton-destabilizing agent, such as cytochalasin B, allows the preparation of membrane vesicles by a series of centrifugations. Here, we prepared cytochalasin-B-inducible nanovesicles (CINVs) of various cellular origins and studied their tropism in different mammalian cells. We observed that CINVs derived from human endometrial mesenchymal stem cells exhibited an enhanced affinity to epithelial cancer cells compared to myeloid, lymphoid or neuroblastoma cancer cells. The dendritic cell-derived CINVs were taken up by all studied cell lines with a similar efficiency that differed from the behavior of DC-derived EVs. The ability of cancer cells to internalize CINVs was mainly determined by the properties of recipient cells, and the cellular origin of CINVs was less important. In addition, receptor-mediated interactions were shown to be necessary for the efficient uptake of CINVs. We found that CINVs, derived from late apoptotic/necrotic cells (aCINVs) are internalized by in myelogenous (K562) 10-fold more efficiently than CINVs, and interact much less efficiently with melanocytic (B16) or epithelial (KB-3-1) cancer cells. Finally, we found that CINVs caused a temporal and reversible drop of the rate of cell division, which restored to the level of control cells with a 24 h delay.

Intracellular bacterial pathogens employ a variety of strategies to invade their eukaryotic host cells. From an ultrastructural standpoint, the processes that bacteria employ to invade their host cells include conventional phagocytosis, coiling phagocytosis, and ruffling/triggered macropinocytosis. In this paper, we describe a novel process by which Francisella tularensis, the agent of tularemia, enters host macrophages. F. tularensis is a remarkably infectious facultative intracellular bacterial parasite--as few as 10 bacteria can cause life-threatening disease in humans. However, the ultrastructure of its uptake and the receptor mechanisms that mediate its uptake have not been reported previously. We have used fluorescence microscopy and electron microscopy to examine the adherence and uptake of a virulent recent clinical isolate of F. tularensis, subspecies tularensis, and the live vaccine strain (LVS), subspecies holarctica, by human macrophages. We show here that both strains of F. tularensis enter human macrophages by a novel process of engulfment within asymmetric, spacious pseudopod loops, a process that differs ultrastructurally from all previously described uptake mechanisms. We demonstrate also that adherence and uptake of F. tularensis by macrophages is strongly dependent upon complement receptors and upon serum with intact complement factor C3 and that uptake requires actin microfilaments. These findings have significant implications for understanding the intracellular biology and virulence of this extremely infectious pathogen.

• Validation of MN assay in mouse fibroblasts cells L929. • Verification of phenobarbital/5,6-benzoflavone as S9 inducer vs. Aroclor 1254. • Verification of the best L929 cells staining method to visualize MNs. The MNa (in vitro the micronucleus assay) is recommended for studying genotoxicity of chemicals. However, no protocol is currently available for experiments with mouse fibroblast L929 cells. The aim of this study was to improve the scope of CBMNb (cytokinesis-block micronucleus) test. Optimization consisted of: selection of a non-cytotoxic concentration of cytokinesis blocker - cytoBc (cytochalasin B) and type and definition of the positive controls, verification of the efficacy of phenobarbital/5,6-benzoflavone as an S9 enzyme inducer as well as the identification of an optimal staining method. The compounds were tested in three exposure regimens: 6 h exposure with S9 activation followed by a 24 h recovery period, 6 h exposure followed by a 24 h recovery without metabolic activation of S9 and 30 h continuous exposure without S9. Different parameters, such as internal and interlaboratory reproducibility were investigated and criteria for test correctness were proposed. Higher MN rates were achieved using 1 μg/mL cytoBc as a cytokinesis blocker, and MMSd (methyl methanesulfonate), (250 μM), Cole (colchicine), (0.5 μM) and CPf (cyclophosphamide), (30 μM) as positive controls. In regard to the recommended S9 inducer, phenobarbital/5,6-benzoflavone was more effective as Aroclor 1254. Giemsa and acridine orange stains were optimal for the evaluation of MN formation. The protocol described in this study with L929 cells produced the reliable results and is suitable for performing the CBMNb experiments according to the current OECD Guideline #487. [ABSTRACT FROM AUTHOR]

This study aimed to examine the evidence of direct interaction among actin, myosin and phosphatidylinositol 3‐kinase (PI3K) in the polarisation and formation of the tetraspore germ tube of Gelidium floridanum.After release, tetraspores were exposed to cytochalasin B, latrunculin B, LY294002 and BDM for a period of 6 h.In control samples, formation of the germ tube occurred after the experimental period, with cellulose formation and elongated chloroplasts moving through the tube region in the presence of F‐actin. In the presence of cytochalasin B, an inhibitor of F‐actin, latrunculin B, an inhibitor of G‐actin, and BDM, a myosin inhibitor, tetraspores showed no formation of the germ tube or cellulose. Spherical‐shaped chloroplasts were observed in the central region with a few F‐actin filaments in the periphery of the cytoplasm. Tetraspores treated with LY294002, a PI3K inhibitor, showed no formation of the tube at the highest concentrations. Polarisation of cytoplasmic contents did not occur, only cellulose formation.It was concluded that F‐actin directs the cell wall components and contributes to the maintenance of chloroplast shape and elongation during germ tube formation. PI3K plays a fundamental role in signalling for the asymmetric polarisation of F‐actin. Thus, F‐actin regulates the polarisation and germination processes of tetraspores of G. floridanum. [ABSTRACT FROM AUTHOR]

During early gestation, hypoxic condition is critically maintained by optimal glucose metabolism and transporter activities. Glucose is readily available energy nutrient required for placentation. However, limited data are available on glucose uptake and its transporters during first trimester placentation processes. To this end, effects of glucose and the roles of glucose transporters (GLUTs) were investigated during hypoxia on trophoblast migration and placental angiogenesis processes using early gestation-derived trophoblast cells, HTR8/SVneo, and first trimester human placental explant tissues. Exogenously added glucose (25 mM) significantly increased tube formation (in vitro angiogenesis) in HTR8/SVneo cells with concomitant activation of AKT-PI3K pathway and increased expression of vascular cell adhesion molecule 1 (VCAM1) compared with those in the presence of 11 mM glucose. Cobalt chloride (CoCl2)-induced hypoxia also significantly increased glucose uptake and GLUT1 expression along with tube formation and migration of HTR8/SVneo cells. During hypoxia, addition of glucose further stimulated HIF1α expression than by hypoxia alone. Cytochalasin B (cyt-B) inhibited the glucose uptake both in the presence of 11 mM and 25 mM glucose. Insulin (1 ng/ml) stimulated GLUT1 expression and tube formation and up-regulated the expression of VEGFR2 in HTR8/SVneo cells. Insulin and glucose-stimulated tube formation was inhibited by cyt-B but had no effect on hypoxia-induced tube formation. Silencing of GLUT1 inhibited the glucose and insulin-stimulated tube formation as well as glucose uptake. However, fatty acid-stimulated tube formation was not affected in GLUT1 knockdown cells. All these data suggest that glucose uptake, glucose-stimulated tube formation, and insulin-stimulated glucose uptake of the first trimester trophoblast cells, HTR8/SVneo, are mediated in part via GLUT1. [ABSTRACT FROM AUTHOR]

Interleukin 2 (IL2) was one of the first cytokines used for cancer treatment due to its ability to stimulate anti-cancer immunity. However, recombinant IL2-based therapy is associated with high systemic toxicity and activation of regulatory T-cells, which are associated with the pro-tumor immune response. One of the current trends for the delivery of anticancer agents is the use of extracellular vesicles (EVs), which can carry and transfer biologically active cargos into cells. The use of EVs can increase the efficacy of IL2-based anti-tumor therapy whilst reducing systemic toxicity. In this study, human adipose tissue-derived mesenchymal stem cells (hADSCs) were transduced with lentivirus encoding IL2 (hADSCs-IL2). Membrane vesicles were isolated from hADSCs-IL2 using cytochalasin B (CIMVs-IL2). The effect of hADSCs-IL2 and CIMVs-IL2 on the activation and proliferation of human peripheral blood mononuclear cells (PBMCs) as well as the cytotoxicity of activated PBMCs against human triple negative cancer MDA-MB-231 and MDA-MB-436 cells were evaluated. The effect of CIMVs-IL2 on murine PBMCs was also evaluated in vivo. CIMVs-IL2 failed to suppress the proliferation of human PBMCs as opposed to hADSCs-IL2. However, CIMVs-IL2 were able to activate human CD8+ T-killers, which in turn, killed MDA-MB-231 cells more effectively than hADSCs-IL2-activated CD8+ T-killers. This immunomodulating effect of CIMVs-IL2 appears specific to human CD8+ T-killer cells, as the same effect was not observed on murine CD8+ T-cells. In conclusion, the use of CIMVs-IL2 has the potential to provide a more effective anti-cancer therapy. This compelling evidence supports further studies to evaluate CIMVs-IL2 effectiveness, using cancer mouse models with a reconstituted human immune system.

Electric cell–substrate impedance sensing (ECIS) is an attractive method for monitoring cell behaviors in tissue culture in real time. The time series impedance fluctuations of the cell-covered electrodes measured by ECIS are the phenomena accompanying cellular micromotion as cells continually rearrange their cell–cell and cell–substrate adhesion sites. Accurate assessment of these fluctuations to extract useful information from raw data is important for both scientific and practical purposes. In this study, we apply discrete wavelet transform (DWT) to analyze the concentration-dependent effect of cytochalasin B on human umbilical vein endothelial cells (HUVECs). The sampling rate of the impedance time series is 1 Hz and each data set consists of 2048 points. Our results demonstrate that, in the Daubechies (db) wavelet family, db1 is the optimal mother wavelet function for DWT-based analysis to assess the effect of cytochalasin B on HUVEC micromotion. By calculating the energy, standard deviation, variance, and signal magnitude area of DWT detail coefficients at level 1, we are able to significantly distinguish cytotoxic concentrations of cytochalasin B as low as 0.1 μM, and in a concentration-dependent manner. Furthermore, DWT-based analysis indicates the possibility to decrease the sampling rate of the micromotion measurement from 1 Hz to 1/16 Hz without decreasing the discerning power. The statistical measures of DWT detail coefficients are effective methods for determining both the sampling rate and the number of individual samples for ECIS-based micromotion assays.

The cytochalasin B-induced membrane vesicles (CIMVs) are suggested to be used as a vehicle for the delivery of therapeutics. However, the angiogenic activity and therapeutic potential of human mesenchymal stem/stromal cells (MSCs) derived CIMVs (CIMVs-MSCs) remains unknown. Objectives: The objectives of this study were to analyze the morphology, size distribution, molecular composition, and angiogenic properties of CIMVs-MSCs. Methods: The morphology of CIMVs-MSC was analyzed by scanning electron microscopy. The proteomic analysis, multiplex analysis, and immunostaining were used to characterize the molecular composition of the CIMVs-MSCs. The transfer of surface proteins from a donor to a recipient cell mediated by CIMVs-MSCs was demonstrated using immunostaining and confocal microscopy. The angiogenic potential of CIMVs-MSCs was evaluated using an in vivo approach of subcutaneous implantation of CIMVs-MSCs in mixture with Matrigel matrix. Results: Human CIMVs-MSCs retain parental MSCs content, such as growth factors, cytokines, and chemokines: EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFN-γ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-15, sCD40L, IL-17A, IL-1RA, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP_1a, MIP-1b, TNF-α, TNF-β, VEGF. CIMVs-MSCs also have the expression of surface receptors similar to those in parental human MSCs (CD90+, CD29+, CD44+, CD73+). Additionally, CIMVs-MSCs could transfer membrane receptors to the surfaces of target cells in vitro. Finally, CIMVs-MSCs can induce angiogenesis in vivo after subcutaneous injection into adult rats. Conclusions: Human CIMVs-MSCs have similar content, immunophenotype, and angiogenic activity to those of the parental MSCs. Therefore, we believe that human CIMVs-MSCs could be used for cell free therapy of degenerative diseases.

Methodology was developed to measure osmotic water permeability in monolayer cultured cells and applied to examine the proposed role of glucose transporters in the water pathway (1989. Proc. Natl. Acad. Sci. USA. 86:8397-8401). J774 macrophages were grown on glass coverslips and mounted in a channel-type perfusion chamber for rapid fluid exchange without cell detachment. Relative cell volume was measured by 45 degrees light scattering using an inverted microscope; measurement accuracy was validated by confocal imaging microscopy. The time required for greater than 90% fluid exchange was less than 1 s. In response to a decrease in perfusate osmolality from 300 to 210 mosM, cells swelled without lag at an initial rate of 4.5%/s, corresponding to a water permeability coefficient of (6.3 +/- 0.4) x 10(-3) cm/s (SE, n = 20, 23 degrees C), assuming a cell surface-to-volume ratio of 4,400 cm-1. The initial rate of cell swelling was proportional to osmotic gradient size, independent of perfusate viscosity, and increased by amphotericin B (25 micrograms/ml), and had an activation energy of 10.0 +/- 1 kcal/mol (12-39 degrees C). The compounds phloretin (20 microM) and cytochalasin B (2.5 micrograms/ml) inhibited glucose transport by greater than 85% but did not influence Pf in paired experiments in which Pf was measured before and after inhibitor addition. The mercurials HgCl2 (0.1 mM) and p-chloromercuribenzoate (1 mM) did not inhibit Pf. A stopped-flow light scattering technique was used to measure Pf independently in J774 macrophages grown in suspension culture. Pf in suspended cells was (4.4 +/- 0.3) x 10(-3) cm/s (assuming a surface-to-volume ratio of 8,800 cm-1), increased more than threefold by amphotericin B, and not inhibited by phloretin and cytochalasin B under conditions of strong inhibition of glucose transport. The glucose reflection coefficient was 0.98 +/- 0.03 as measured by induced osmosis, assuming a unity reflection coefficient for sucrose. These results establish a quantitative method for measurement of osmotic water transport in adherent cultured cells and provide evidence that glucose transporters are not involved in the water transporting pathway.

Immunotherapy represents an innovative approach to cancer treatment, based on activating the body's own immune system to combat tumor cells. Among various immunotherapy strategies, dendritic cell vaccines hold a special place due to their ability to activate T-lymphocytes, key players in cellular immunity, and direct them to tumor cells. In this study, the influence of dendritic cells processed with tumor-derived vesicles on the viability of melanoma cells in vitro was investigated. Dendritic cells were loaded with tumor-derived vesicles, after which they were used to activate T-cells. The study demonstrated that such modified T-cells exhibit high activity against melanoma cells, leading to a decrease in their viability. Our analysis highlights the potential efficacy of this approach in developing immunotherapy against melanoma. These results provide new prospects for further research and the development of antitumor strategies based on the mechanisms of T-lymphocyte activation using tumor-derived vesicles.

Quercetin, a common dietary flavone, is a competitive inhibitor of glucose uptake and is also thought to be transported into cells by GLUT1. In this study, we confirm that quercetin is a competitive inhibitor of GLUT1 and also demonstrate that newly synthesized compounds, WZB-117 and BAY-876 are robust inhibitors of GLUT1 in L929 cells. To measure quercetin interaction with L929 cells, we develop a new fluorescent assay using flow cytometry. The binding of quercetin and its inhibitory effects on 2-deoxyglucose (2DG) uptake showed nearly identical dose dependent effects, with both having maximum effects between 50 and 100 μM and similar half maximum effects at 8.9 and 8.5 μM respectively. The interaction of quercetin was rapid with t 1/2 of 54 s and the onset and loss of its inhibitory effects on 2DG uptake were equally fast. This suggests that either quercetin is simply binding to surface GLUT1 or its transport in and out of the cell reaches equilibrium very quickly. If quercetin is transported, the co-incubation of quercetin with other glucose inhibitors should block quercetin uptake. However, we observed that WZB-117, an exofacial binding inhibitor of GLUT1 reduced quercetin interaction, while cytochalasin B, an endofacial binding inhibitor, enhanced quercetin interaction, and BAY-876 had no effect on quercetin interaction. Taken together, these data are more consistent with quercetin simply binding to GLUT1, but not actually being transported into L929 cells via the glucose channel in GLUT1. [ABSTRACT FROM AUTHOR]

In this study we examined the effects of JNJ-7706621, a cyclin-dependent kinase inhibitor, on the in vitro growth of pig embryos that had been produced either by parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT). A significantly higher percentage of PA embryos reached the blastocyst stage by Day 7 after exposure to 10 mM JNJ-7706621 for 4 h compared with embryos exposed to 5 μgmL-1 cytochalasin B for 4 h (P,0.05). Similarly, the rate of Tyr15 phosphorylation of the complex of cyclin and p34cdc2 (CDK1) was significantly elevated in the JNJ-7706621- treated embryos compared with embryos exposed to cytochalasin B or non-treated controls (P,0.05). In contrast, Thr161 phosphorylation of CDK1 was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated as well as the non-treated group (P,0.05). Similarly, the level of M-phase-promoting factor (MPF) in embryos was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated and non-treated groups (P,0.05). In addition, more SCNT embryos reached the blastocyst stage after treatment with JNJ-7706621 than following exposure to cytochalasin B (P,0.05). In conclusion, these results reveal that exposure to 10 mMJNJ-7706621 for 4 h improves early development of PA and SCNT porcine embryos by suppressing the activity of CDK1 and a concomitant reduction in the level of MPF. [ABSTRACT FROM AUTHOR]

Abstract: The in vitro micronucleus (MN) assay is a well‐established test for evaluating genotoxicity and cytotoxicity. The use of manual microscopy to perform the assay can be laborious and often suffers from user subjectivity and interscorer variability. Automated methods including slide‐scanning microscopy and conventional flow cytometry have been developed to eliminate scorer bias and improve throughput. However, these methods possess several limitations such as lack of cytoplasmic visualization using slide‐scanning microscopy and the inability to visually confirm the legitimacy of MN or storage of image data for re‐evaluation using flow cytometry. The ImageStreamX® MK II (ISX) imaging flow cytometer has been demonstrated to overcome all of these limitations. The ISX combines the speed, statistical robustness, and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery of microscopy and possesses the ability to store all collected image data. This paper details the methodology developed to perform the in vitro MN assay in human lymphoblastoid TK6 cells on the ISX. High resolution images of micronucleated mono‐ and bi‐nucleated cells as well as polynucleated cells can be acquired at a high rate of capture. All images can then be automatically identified, categorized and enumerated in the data analysis software that accompanies the ImageStream, allowing for the scoring of both genotoxicity and cytotoxicity. The results demonstrate that statistically significant increases in MN frequency when compared with solvent controls can be detected at varying levels of cytotoxicity following exposure to well‐known aneugens and clastogens. This work demonstrates a fully automated method for performing the in vitro micronucleus assay on the ISX imaging flow cytometry platform. © 2018 The Author. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC. [ABSTRACT FROM AUTHOR]

Argopecten nucleus is a small scallop from the Caribbean Sea and a relatively new species for aquaculture. One of the key challenges to develop the farming operations for this species from the current pilot scale to commercial level is to improve its harvest size. In this study, we tested three different methods for triploidy induction. Additionally, the effect of these protocols on survival, developmental rate and size of larvae and post-larvae were assessed. Three different mechanisms to stimulate the inhibition of the release of the second polar body were tested; (1) cold shock (18°C); (2) 6-dimethylaminopurine (6-DMAP); (3) cytochalasin B (CB) and (4) dimethylsulphoxide (DMSO). The treatment with 6-DMAP yielded the highest percentage of triploid larvae (39%). The survival and development rate, however, were higher in non-treated larvae (control) than in the treatment groups. Interestingly, larvae from CB and the DMSO control groups exhibited lower growth rates in length than those from control and the other two treatments. No influence of the triploidy induction treatments was observed on post-larvae survival, but the size of post-larvae was larger for the cold shock treatment and DMSO control group. Our results indicate that the use of 6-DMAP has the greatest potential to produce triploid larvae of A. nucleus without affecting negatively growth and survival of post-larvae. [ABSTRACT FROM AUTHOR]

Two types of triploid hard clams Mercenaria mercenaria were produced by inhibiting polar body I ( PB1) or polar body II ( PB2) with cytochalasin B. Treatments were applied at 22-23°C, with PB1 inhibition starting at 4-7 min postfertilization and ending when PB2 was first observed in control groups, and with PB2 inhibition starting at 17-23 min postfertilization and ending when 80% of control eggs released PB2. Triploid induction success was evaluated by chromosome counting in 2-4 cell embryos and by flow cytometry at larval and juvenile stages. PB2 inhibition produced more triploids (82%-100%) than PB1 inhibition (71%-83%), although the difference was not significant ( p ≥ .088). Triploid percentages in PB1- or PB2-inhibited groups showed a small but insignificant decline during the first 6 months. At month 3, PB1 and PB2 triploids were not different from their within-group diploids, but significantly larger than control diploids; PB1 triploids were significantly larger than PB2 triploids ( p ≤ .003). At month 6, PB1 triploids were not different from either within-group or control-group diploids, while PB2 triploids were significant larger than both within-group and control diploid; PB1 triploids were smaller than PB2 triploids. At month 16, PB1 and PB2 triploids in one remaining replicate were not different from their within-group diploids. Overall, this study shows that triploids can be efficiently produced by PB1 or PB2 inhibition, and their growth performance relative to diploids is variable depending on age and replicates or parental genotype. [ABSTRACT FROM AUTHOR]

The factors affecting intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high-resolution cryo-imaging technology, the broad bean intracellular ice formation and growth processes were successfully captured during freezing. Cytochalasin B（CB）was used to solubilize the cytoskeleton. Images of IIF were compared between cells with and without cytoskeleton. The behavior of intracellular ice crystal formation in plant tissues with or without CB was evaluated using changes of cell areas, the probability of crystallization, and growing rate of intracellular ice crystal. Moreover, light intensity figures were used to determine cell damage. This study showed that the cytoskeleton was involved in ice crystal nucleation mechanism during freezing responses of the plant cells. [ABSTRACT FROM AUTHOR]

This study was conducted to optimize the efficiency of cloning and to produce cloned mice. The majority of cloned mammals derived by nuclear transfer (NT) die during gestation and have enlarged and dysfunctional placentas. In this study, the optimized conditions were established to produce clone mice. The parthenogenetic oocytes were activated after 6 h regardless of cytochalasin B (CB) concentration. CB treatment (2 μg/ml) was found second polar body. Lower concentration of CB was decreased the activation rate, but the second polar body was the best highly increased during 6 h incubation. The small fragments were exhibited in the 5 μg/ml treatment of CB, but it was not found in lower concentration groups (> 2.5 μg/ml). To examine effects of SrCl2 on the adult cumulus cells, somatic cell NT oocytes were exposed during 0.5, 1 and 6 hrs. The second polar body was significantly greater in 0.5 h exposure group (6.6%) than 1, 6 hrs. Developmental rate from 2-cell to 4-cell was the lowest in 7.5 mM Strontium chloride (SrCl2) groups (84.1% and 64.3%) than 5, 10 m MSrCl2. The implantation rate was not significantly difference among 5, 7.5 and 10 m MSrCl2 group. Three live fetuses were produced by SCNT. SCNT placentas were remarkably heavier than IVF group (8 fetuses) (0.34, 0.34, 0.33 vs 0.14 g) compared with the placenta weight of IVF and SCNT clones.

Incorporation of rhodamine- and fluorescein-isothiocyanate labeled bovine serum albumin (BSA-TRITC, BSA-FITC) was examined in different root zones of the 3-day-old seedlings in Melandrium noctiflorum, Allium cepa and Zea mays. The uptake of fluorescent-labeled BSA was found: (1) species-specific, (2) cell-type dependent, and (3) cytochalasin B-sensitive. The characteristic punctute distribution of vesicles within the cytoplasm suggests the internalization of labeled proteins by endocytosis.