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Glucose transporters are required to bring glucose into cells, where it is an essential energy source and precursor in protein and lipid synthesis. These transporters are involved in important common diseases such as cancer and diabetes. Here, we report the crystal structure of the Staphylococcus epidermidis glucose/H ⁺ symporter in an inward-facing conformation at 3.2-Å resolution. The Staphylococcus epidermidis glucose/H ⁺ symporter is homologous to human glucose transporters, is very specific and has high avidity for glucose, and is inhibited by the human glucose transport inhibitors cytochalasin B, phloretin, and forskolin. On the basis of the crystal structure in conjunction with mutagenesis and functional studies, we propose a mechanism for glucose/H ⁺ symport and discuss the symport mechanism versus facilitated diffusion.

Summary Background Despite inherent differences between the cytoskeletal networks of malignant and normal cells, and the clinical antineoplastic activity of microtubule-directed agents, there has yet to be a microfilament-directed agent approved for clinical use. One of the most studied microfilament-directed agents has been cytochalasin B, a mycogenic toxin known to disrupt the formation of actin polymers. Therefore, this study sought to expand on our previous work with the microfilament-directed agent, along with other less studied cytochalasin congeners. Materials and Methods We determined whether cytochalasin B exerted significant cytotoxic effects in vitro on adherent M109 lung carcinoma and B16BL6 and B16F10 murine melanomas, or on suspension P388/ADR murine leukemia cells. We also examined whether cytochalasin B, its reduced congener 21, 22-dihydrocytochalasin B (DiHCB), or cytochalasin D could synergize with doxorubicin (ADR) against ADR-resistant P388/ADR leukemia cells, and produce significant cytotoxicity in vitro. For in vivo characterization, cytochalasins B and D were administered intraperitoneally (i.p.) to Balb/c mice challenged with drug sensitive P388-S or multidrug resistant P388/ADR leukemias. Results Cytochalasin B demonstrated higher cytotoxicity against adherent lung carcinoma and melanoma cells than against suspension P388/ADR leukemia cells, as assessed by comparative effects on cell growth, and IC 50 and IC 80 values. Isobolographic analysis indicated that both cytochalasin B and DiHCB demonstrate considerable drug synergy with ADR against ADR-resistant P388/ADR leukemia, while cytochalasin D exhibits only additivity with ADR against the same cell line. In vivo, cytochalasins B and D substantially increased the life expectancy of mice challenged with P388/S and P388/ADR leukemias, and in some cases, produced long-term survival. Conclusion Taken together, it appears that cytochalasins have unique antineoplastic activity that could potentiate a novel class of chemotherapeutic agents.

Mesenchymal stem cells (MSCs) have a therapeutic potential in tissue repair because of capacity for multipotent differentiation and their ability to modulate the immune response. In this study, we examined the ability of human placental MSCs (pMSCs) to modify the differentiation of human monocytes into macrophages and assessed the influence of pMSCs on important macrophage functions. We used GM-CSF to stimulate the differentiation of monocytes into the M1 macrophage pathway and then co-cultured these cells with pMSCs in the early stages of macrophage differentiation. We then evaluated the effect on differentiation by microscopic examination and by quantification of molecules important in the differentiation and immune functions of macrophages using flow cytometry and ELISA. The mechanism by which pMSCs could mediate their effects on macrophage differentiation was also studied. The co-culture of pMSCs with monocytes stimulated to follow the inflammatory M1 macrophage differentiation pathway resulted in a shift to anti-inflammatory M2-like macrophage differentiation. This transition was characterized by morphological of changes typical of M2 macrophages, and by changes in cell surface marker expression including CD14, CD36, CD163, CD204, CD206, B7-H4 and CD11b, which are distinctive of M2 macrophages. Co-culture with pMSCs reduced the expression of the costimulatory molecules (CD40, CD80 and CD86) and increased the expression of co-inhibitory molecules (CD273, CD274 and B7-H4) as well as the surface expression of major histocompatibility complex (MHC-II) molecules. Furthermore, the secretion of IL-10 was increased while the secretion of IL-1β, IL-12 (p70) and MIP-1α was decreased; a profile typical of M2 macrophages. Finally, pMSCs induced the phagocytic activity and the phagocytosis of apoptotic cells associated with M2- like macrophages; again a profile typical of M2 macrophages. We found that the immunoregulatory effect of pMSCs on macrophage differentiation was mediated by soluble molecules acting partially via glucocorticoid and progesterone receptors. We have shown that pMSCs can transition macrophages from an inflammatory M1 into an anti-inflammatory M2 phenotype. Our findings suggest a new immunosuppressive property of pMSCs that may be employed in the resolution of inflammation associated with inflammatory diseases and in tissue repair.

Intercellular communication in the tumor microenvironment has a major impact on therapeutic outcomes. In particular, highly aggressive cancers such as glioblastoma benefit from efficient cellular communication by developing resistance to therapy or by infiltrating surrounding tissue to escape radiotherapy. Tiny membrane tunnels, such as tunneling nanotubes (TNTs), can serve as an effective communication tool for tumor cells. With these structures, the cells can build a large network that allows them to react and adapt to their environment as quickly as possible, thereby combating the therapy. In this study, we investigated the effects of chemotherapeutic agents on TNT networks in U87 MG cells to determine if they affect the number of TNTs and, consequently, possible therapeutic outcomes. The effect of drugs on TNT stability may be related to their effects on actin and tubulin, which are known stabilizers of membrane structures. Therefore, we also analyzed the cytoskeletal content of TNTs in U87 MG cells and examined the effect of the actin polymerization inhibitor cytochalasin B (CytoB) on TNT formation and networks. CytoB reduced the number of TNTs in this study, suggesting that actin, as a cytoskeletal component, is required for developing TNT networks. In contrast, the chemotherapeutic agents temozolomide (TMZ) and cytarabine (AraC) do not affect TNTs and actin content. As TNT networks are closely linked to increased therapy resistance of certain tumors, we believe that the lack of suppression of TNTs is a major problem in current glioblastoma therapy and should be a target for further research in the future.

The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na + ) channels. Paradoxically, the results of patch-clamp studies suggest that the Na + channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na + imaging, together with modeling, which indicate that the Na + channel density at the AIS of cortical pyramidal neurons is ∼50 times that in the proximal dendrites. Anchoring of Na + channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na + current measured in patches from the AIS. Computational models required a high Na + channel density (∼2,500 pS μm −2 ) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na + channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.

Cow mastitis is a major challenge in dairy farming, significantly affecting both milk quality and cow health. Cytochalasin B (CB) is a fungal toxin and an actin cytoskeleton depolymerizing agent that exhibits anti-inflammatory and antitumor properties; however, its mechanism in cow mastitis remains unclear. In this study, we systematically evaluated the effects of CB on mastitis using an LPS-induced inflammation model in bovine mammary epithelial cells (MAC-T) and a mouse mastitis model. The techniques employed included Real-time quantitative PCR detecting system (qPCR), Western blot, HE staining, immunofluorescence (IF), and immunohistochemistry (IHC). The results demonstrated that CB significantly alleviated LPS-induced mastitis by downregulating the expression of pro-inflammatory factors IL-1β, TNF-α, and the NLRP3 inflammasome while also reducing cell apoptosis. Further mechanistic investigations revealed that CB mitigates the inflammatory response by inhibiting the expression of ARPC3, ARPC4, and HSP70, thereby disrupting cytoskeletal rearrangement and the activation of the NLRP3 inflammasome. Overall, this study reveals the potential therapeutic role of CB in cow mastitis and provides a theoretical foundation for developing novel intervention strategies.

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5-6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase-deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

Triploids are widely used to rapidly achieve genetic improvements of organisms due to their fast growth and enhanced environmental adaptability. Artificially induced triploids are generally considered to be infertile owing to the obvious inhibition of gonadal development. Recently, some fertile individuals with reduced advantages have been found in triploid bivalves, which is a notable deviation from the original intention of artificially inducing triploids. This study utilized dwarf surf clams ( Mulinia lateralis ), a promising model organism of bivalves, to develop a model for exploring the potential mechanism of triploid reproduction. The results showed that the optimal induction condition for triploid M. lateralis , determined by orthogonal experiments, was 0.5 mg/L cytochalasin B (CB) to inhibit PB2 for 20 min, resulting in a triploidy rate of 95.57% and a hatching rate of 60.25%. By tracking the development of M. lateralis , we found that the induced triploids could develop normally to maturity and exhibited significant growth and survival advantages post-metamorphosis. Although the triploidy rate exhibited a slight decline overtime, it remained high, with a ratio of 90.63% at 120 dpf. Histological observation confirmed that the gonadal development pattern of triploid M. laterali was similar to that of diploids, but it also showed characteristics such as developmental retardation, few mature gametes, and gamete gigantism. The dynamic expression of genes related to gonadal development provided further molecular evidence for this phenomenon. Additionally, 82.6% of triploid M. laterali exhibited normal spawning behavior, produced fewer but larger viable gametes, and could generate offspring with full developmental potential. Flow cytometry analysis revealed that sperm of triploid M. laterali was aneuploid, with a DNA content of about 1.5 times that of diploid sperm, and the ploidy levels of mating offspring were 2N (DD, diploid female × diploid male), 2.5N (DT, diploid female × triploid male), 2.5N (TD, triploid female × diploid male), and 3N (TT, triploid female × triploid male), respectively. Overall, the artificially induced triploid M. laterali has been confirmed to be bisexually fertile, which will provide a unique model for exploring the underlying mechanisms of advantageous trait formation and fertility regulation in triploids, and offer a valuable platform for the study of ploidy control and polyploidization in bivalves. Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary. Yes, i have checked and it is OK.

By preventing G-actin accumulation, Rho-GTPase promotes the transcriptional activity of myocardin-related transcription factors (MRTFs), known co-factors of serum response factor (SRF). Rho-dependent MRTF expression is required for injected metastatic cell lines to colonize the lung. Rho GTPases control cytoskeletal dynamics through cytoplasmic effectors and regulate transcriptional activation through myocardin-related transcription factors (MRTFs), which are co-activators for serum response factor (SRF). We used RNA interference to investigate the contribution of the MRTF–SRF pathway to cytoskeletal dynamics in MDA-MB-231 breast carcinoma and B16F2 melanoma cells, in which basal MRTF–SRF activity is Rho-dependent. Depletion of MRTFs or SRF reduced cell adhesion, spreading, invasion and motility in culture, without affecting proliferation or inducing apoptosis. MRTF-depleted tumour cell xenografts showed reduced cell motility but proliferated normally. Tumour cells depleted of MRTF or SRF failed to colonize the lung from the bloodstream, being unable to persist after their arrival in the lung. Only a few genes show MRTF-dependent expression in both cell lines. Two of these, MYH9 (NMHCIIa) and MYL9 (MLC2), are also required for invasion and lung colonization. Conversely, expression of activated MAL/MRTF-A increases lung colonization by poorly metastatic B16F0 cells. Actin-based cell behaviour and experimental metastasis thus require Rho-dependent nuclear signalling through the MRTF–SRF network.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4′′-O-methylligstroside (1), (8E)-4′′-O-methyldemethylligstroside (2), and 3′′,4′′-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4–26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4′′-O-methylligstroside (1), (8E)-4′′-O-methyldemethylligstroside (2), 3′′,4′′-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 μg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 μg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.