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The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

Distinct mammalian RNA viruses trigger Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNA) and encode unrelated proteins to suppress vsiRNA biogenesis. However, the mechanism and function of the mammalian RNA interference (RNAi) response are poorly understood. Here, we characterized antiviral RNAi in a mouse model of infection with Nodamura virus (NoV), a mosquito-transmissible positive-strand RNA virus encoding a known double-stranded RNA (dsRNA)-binding viral suppressor of RNAi (VSR), the B2 protein. We show that inhibition of NoV RNA replication by antiviral RNAi in mouse embryonic fibroblasts (MEFs) requires Dicer-dependent vsiRNA biogenesis and Argonaute-2 slicer activity. We found that VSR-B2 of NoV enhances viral RNA replication in wild-type but not RNAi-defective MEFs such as Argonaute-2 catalytic-dead MEFs and Dicer or Argonaute-2 knockout MEFs, indicating that VSR-B2 acts mainly by suppressing antiviral RNAi in the differentiated murine cells. Consistently, VSR-B2 expression in MEFs has no detectable effect on the induction of interferon-stimulated genes or the activation of global RNA cleavages by RNase L. Moreover, we demonstrate that NoV infection of adult mice induces production of abundant vsiRNA active to guide RNA slicing by Argonaute-2. Notably, VSR-B2 suppresses the biogenesis of both vsiRNA and the slicing-competent vsiRNA-Argonaute-2 complex without detectable inhibition of Argonaute-2 slicing guided by endogenous microRNA, which dramatically enhances viral load and promotes lethal NoV infection in adult mice either intact or defective in the signaling by type I, II, and III interferons. Together, our findings suggest that the mouse RNAi response confers essential protective antiviral immunity in both the presence and absence of the interferon response.IMPORTANCE Innate immune sensing of viral nucleic acids in mammals triggers potent antiviral responses regulated by interferons known to antagonize the induction of RNA interference (RNAi) by synthetic long double-stranded RNA (dsRNA). Here, we show that Nodamura virus (NoV) infection in adult mice activates processing of the viral dsRNA replicative intermediates into small interfering RNAs (siRNAs) active to guide RNA slicing by Argonaute-2. Genetic studies demonstrate that NoV RNA replication in mouse embryonic fibroblasts is inhibited by the RNAi pathway and enhanced by the B2 viral RNAi suppressor only in RNAi-competent cells. When B2 is rendered nonexpressing or nonfunctional, the resulting mutant viruses become nonpathogenic and are cleared in adult mice either intact or defective in the signaling by type I, II, and III interferons. Our findings suggest that mouse antiviral RNAi is active and necessary for the in vivo defense against viral infection in both the presence and absence of the interferon response.

Intracellular diffusion underlies vital cellular processes. However, it remains difficult to elucidate how an unbound protein diffuses inside the cell with good spatial resolution and sensitivity. Here we introduce single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through local statistics of the instantaneous displacements of freely diffusing single molecules. We thus show that the diffusion of an average-sized protein in the mammalian cytoplasm and nucleus is spatially heterogeneous at the nanoscale, and that variations in local diffusivity correlate with the ultrastructure of the actin cytoskeleton and the organization of the genome, respectively. SMdM of differently charged proteins further unveils that the possession of positive, but not negative, net charges drastically impedes diffusion, and that the rate is determined by the specific subcellular environments. We thus unveil rich heterogeneities and charge effects in intracellular diffusion at the nanoscale.

Background: The accurate determination of cell confluence is a critical step for generating reasonable results of designed experiments in cell biological studies. However, the cell confluence of the same culture may be diversely predicted by individual researchers. Herein, we designed a systematic quantification scheme implemented on the Matlab platform, the so-called "Confluence-Viewer" program, to assist cell biologists to better determine the cell confluence. Methods: Human normal oral fibroblasts (hOFs) seeded in 10 cm culture dishes were visualized under an inverted microscope for the acquisition of cell images. The images were subjected to the cell segmentation algorithm with top-hat transformation and the Otsu thresholding technique. A regression model was built using a quadratic model and shape-preserving piecewise cubic model. Results: The cell segmentation algorithm generated a regression curve that was highly correlated with the cell confluence determined by experienced researchers. However, the correlation was low when compared to the cell confluence determined by novice students. Interestingly, the cell confluence determined by experienced researchers became more diverse when they checked the same images without a time limitation (up to 1 min). Conclusion: This tool could prevent unnecessary human-made mistakes and meaningless repeats for novice researchers working on cell-based studies in health care or cancer research. [ABSTRACT FROM AUTHOR]

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an antioxidant enzyme which is associated with poor prognosis in human breast, colon, lung and liver cancers. However, the molecular mechanisms underlying the pro-tumorigenic function of NQO1 remains unclear. This study investigated the function of NQO1 in the context of hepatocellular carcinoma (HCC) development. We found that NQO1 was frequently up-regulated in human liver cancer, and its high expression level was correlated with the tumor stage and low survival rate of HCC patients. Loss-of-function of NQO1 inhibited growth in HCC cells with increased apoptosis in vitro, and suppressed orthotopic tumorigenicity in vivo. Mechanistically, high level of NQO1 in HCC cells enhanced protein stability of X-linked inhibitor of apoptosis protein (XIAP) by increasing its phosphorylation at Ser 87. Reintroduction of wile type XIAP and the phospho-mimic mutants XIAPS87D significantly reversed NQO1 knock-down/out induced growth inhibition and apoptosis. In mouse model with orthotopically implanted hepatocarcinoma, NQO1 suppression and NQO1 inhibitor suppressed tumor growth and induced apoptosis. NQO1 plays an important role in sustaining HCC cell proliferation and may thus act as a potential therapeutic target in HCC treatment. [ABSTRACT FROM AUTHOR]

Abstract Fine particulate matter (PM 2.5) exposure could cause many acute and chronic respiratory diseases. In this study the protective effects of polysaccharide from Morchella esculenta (FMP-1) and its derivatives against PM 2.5 -induced inflammation were evaluated. By flow cytometry and ELISA analysis, sulfated polysaccharide SFMP-1 showed the best protective effect in reducing PM 2.5 -induced cell death, cell apoptosis and production of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), which was accompanied by a diminished level in reactive oxygen species (ROS) formation caused by PM 2.5 in rat alveolar macrophage NR8383 cells. Furthermore, the mechanism was studied by immunofluorescence, qRT-PCR and western blotting. SFMP-1 could down-regulate the expression of inducible NO synthesis (iNOS) and cyclooxygenase-2 (COX-2) at both mRNA and protein levels in PM 2.5 -treated cells. The PM 2.5 -induced phosphorylation of nuclear factor-kappa B (NF-κB) was also reduced through suppressing nuclear translation of the NF-κB and inhibiting the degradation and phosphorylation of IκBα. These results indicated that SFMP-1 could protect NR8383 cells from PM 2.5 -induced inflammation by inhibiting NF-κB activation. Highlights • Polysaccharide from Morchella esculenta (FMP-1) was chemically modified to obtain sulfated and carboxymethylated derivatives. • Protection of FMP-1 and its derivatives against PM 2.5 -induced inflammation were comparatively investigated. • Sulfated polysaccharide SFMP-1 showed the best protective effect in PM 2.5 -treated alveolar macrophage NR8383 cells. • SFMP-1 could protect NR8383 cells from PM 2.5 -induced inflammation by inhibiting NF-κB activation. [ABSTRACT FROM AUTHOR]

Iron overload is a common pathophysiological state underlying many diseases that has a detrimental influence on cells. The protective effects of Dexmedetomidine (Dex), a high selective alpha-2-adrenoceptor agonist, have been revealed through many experimental models, whereas no study reports its effects on an iron overload model. To elucidate these effects, we used FeCl2 with or without Dex to treat SH-SY5Y cells for 24 h and then detected indicators of oxidative stress, inflammation and apoptosis and investigated possible mechanisms further. After treatment with FeCl2 for 24 h, cell viability decreased in a dose dependent manner, and Dex promoted cell survival in FeCl2 incubation, also in a dose-dependent manner. Compared with the FeCl2 group, 20 µM Dex significantly attenuated ROS accumulation, reduced pro-inflammatory cytokine expression, and inhibited apoptosis. Furthermore, 20 µM concentration of Dex remarkably downregulated the expression of pro-apoptotic protein and activation of caspase 3 while increasing anti-apoptotic protein expression. Additionally, Dex also effectively suppressed the expression of NF-κB and its activation. In conclusion, Dex exerted anti-oxidative stress, anti-inflammation, and anti-apoptosis effects on FeCl2-treated SH-SY5Y cells, possibly by inhibiting NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

The incidence of cancer is increasing at an alarming rate despite recent advances in prevention strategies, diagnostics and therapeutics for various types of cancer. The identification of novel biomarkers to aid in prognosis and treatment for cancer is urgently required. Uncontrolled proliferation and dysregulated apoptosis are characteristics exhibited by cancer cells in the initiation of various types of cancer. Notably, aberrant expression of crucial oncogenes or cancer suppressors is a defining event in cancer occurrence. Research has demonstrated that SAD1/UNC84 domain protein-2 (SUN2) serves a suppressive role in breast cancer, atypical teratoid/rhabdoid tumors and lung cancer progression. Furthermore, SUN2 inhibits cancer cell proliferation, migration and promotes apoptosis. Recent reports have also shown that SUN2 serves prominent roles in resistance to the excessive DNA damage that destabilizes the genome and promotes cancer development, and these functions of SUN2 are critical for evading initiation of cancer. Additionally, increasing evidence has demonstrated that SUN2 is involved in maintaining cell nuclear structure and appears to be a central component for organizing the natural nuclear architecture in cancer cells. The focus of the present review is to provide an overview on the pharmacological functions of SUN2 in cancers. These findings suggest that SUN2 may serve as a promising therapeutic target and novel predictive marker in various types of cancer. [ABSTRACT FROM AUTHOR]

Abstract: The capability of MSCs to differentiate into neurons has been proven by many studies. Recently, other studies have cast doubt on MSCs neurogenic differentiation with non-physiological chemical inducing agents in vitro. This present study was designed to use conditioned medium to investigate whether signals from pathological condition of ALS were competent to induce a program of neurogenic differentiation in expanded cultures of hMSCs. Incubation of hMSCs with conditioned medium prepared from CNS extracts of ALS mice (SOD1-G93A ALS mice) resulted in a time-dependent morphological change from fibroblast-like into neuron-like, concomitant with increase in the expression of Nestin and subsequent β-tubulin III, NSE and GAP43. Moreover, signals in pathological CNS extracts of ALS mice were more effective in promoting hMSCs neurogenic differentiation than those in physiological extracts of normal adult mice. These results show that pathological condition of ALS is endowed with capacity to induce hMSCs neurogenic differentiation and hMSCs have shown a potential candidate in cellular therapy for ALS. [Copyright &y& Elsevier]

RNA silencing is a sequence-specific RNA degradation mechanism that is operational in plants and animals. Here, we show that flock house virus (FHV) is both an initiator and a target of RNA silencing in Drosophila host cells and that FHV infection requires suppression of RNA silencing by an FHV-encoded protein, B2. These findings establish RNA silencing as an adaptive antiviral defense in animal cells. B2 also inhibits RNA silencing in transgenic plants, providing evidence for a conserved RNA silencing pathway in the plant and animal kingdoms. [ABSTRACT FROM AUTHOR]