Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development

A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine.     

Hepatoprotective mechanism of schisandrin B: role of mitochondrial glutathione antioxidant status and heat shock proteins

In this study, the time course of schisandrin B- (Sch B-) induced changes in hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (HSP) 25/70 induction was examined to study their differential roles in the hepatoprotection afforded by Sch B pretreatment against carbon tetrachloride (CCl(4)) toxicity in mice. Dimethyl diphenyl bicarboxylate (DDB), a nonhepatoprotective analog of Sch B, was also included for comparison. The results indicate that Sch B treatment (2 mmol/kg) produced maximum enhancement in hepatic mtGAS and increases in both hepatic HSP 25 and HSP 70 levels at 24 h after dosing. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl(4) was found to correlate inversely with the elapsed time postdosing, the protective effect was associated with the ability to sustain mtGAS and/or HSP 70 levels in a CCl(4)-intoxicated condition. On the other hand, DDB (2 mmol/kg) treatment, which did not sustain mtGAS and HSP 70 level, could not protect against CCl(4) toxicity. Abolition of the Sch B-mediated enhancement of mtGAS by buthionine sulfoximine/phorone did not completely abrogate the hepatoprotective action of Sch B. The results indicate that Sch B pretreatment independently enhances mtGAS and induces HSP 25/70 production, particularly under conditions of oxidative stress, thereby protecting against CCl(4) hepatotoxicity.     

Hebbian learning in parallel and modular memories

Many cognitive and sensorimotor functions in the brain involve parallel and modular memory subsystems that are adapted by activity-dependent Hebbian synaptic plasticity. This is in contrast to the multilayer perceptron model of supervised learning where sensory information is presumed to be integrated by a common pool of hidden units through backpropagation learning. Here we show that Hebbian learning in parallel and modular memories is more advantageous than backpropagation learning in lumped memories in two respects: it is computationally much more efficient and structurally much simpler to implement with biological neurons. Accordingly, we propose a more biologically relevant neural network model, called a tree-like perceptron, which is a simple modification of the multilayer perceptron model to account for the general neural architecture, neuronal specificity, and synaptic learning rule in the brain. The model features a parallel and modular architecture in which adaptation of the input-to-hidden connection follows either a Hebbian or anti-Hebbian rule depending on whether the hidden units are excitatory or inhibitory, respectively. The proposed parallel and modular architecture and implicit interplay between the types of synaptic plasticity and neuronal specificity are exhibited by some neocortical and cerebellar systems. Received: 13 October 1996 / Accepted in revised form: 16 October 1997     

Cyclin A2-Cyclin-Dependent Kinase 2 Cooperates with the PLK1-SCFβ-TrCP1-EMI1-Anaphase-Promoting Complex/Cyclosome Axis To Promote Genome Reduplication in the Absence of Mitosis

Limiting genome replication to once per cell cycle is vital for maintaining genome stability. Inhibition of cyclin-dependent kinase 1 (CDK1) with the specific inhibitor RO3306 is sufficient to trigger multiple rounds of genome reduplication. We demonstrated that although anaphase-promoting complex/cyclosome (APC/C) remained inactive during the initial G₂ arrest, it was activated upon prolonged inhibition of CDK1. Using cellular biosensors and live-cell imaging, we provide direct evidence that genome reduplication was associated with oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis at the single-cell level. Genome reduplication was abolished by ectopic expression of EMI1 or depletion of CDC20 or CDH1, suggesting the critical role of the EMI1-APC/C axis. In support of this, degradation of EMI1 itself and genome reduplication were delayed after downregulation of PLK1 and β-TrCP1. In the absence of CDK1 activity, activation of APC/C and genome reduplication was dependent on cyclin A2 and CDK2. Genome reduplication was then promoted by a combination of APC/C-dependent destruction of geminin (thus releasing CDT1), accumulation of cyclin E2-CDK2, and CDC6. Collectively, these results underscore the crucial role of cyclin A2-CDK2 in regulating the PLK1-SCF^β-TrCP1-EMI1-APC/C axis and CDC6 to trigger genome reduplication after the activity of CDK1 is suppressed.Limiting genome replication to once per cell cycle is critical for maintaining genome stability and suppressing tumorigenesis (reviewed in reference 18). DNA replication is a biphasic process consisting of origin licensing and origin firing. During late mitosis to early G₁ phase, origins are licensed by orderly loading of prereplicative complex components, including ORC, CDT1, CDC6, and MCM2-7 (reviewed in reference 3). Origin licensing occurs in a biochemical environment characterized by low activity of cyclin-cyclin-dependent kinase (CDK) and high activity of anaphase-promoting complex/cyclosome (APC/C) (reviewed in reference 51). Firing of the origins is coordinated by phosphorylation carried out by cyclin-CDK and DBF4-CDC7 and the binding of other replication factors, including CDC45, MCM10, RPA, and DNA polymerase (reviewed in reference 58).Critical roles in replication have been attributed to both cyclin A and cyclin E, but the distinct roles performed by the two cyclins remain incompletely understood. Cyclin A is especially interesting among the cyclins because of its association with multiple CDKs (CDK1 and CDK2) and its proposed functions in multiple points of the cell cycle (S phase and mitosis). In S phase, cyclin A is believed to be involved in the loading of CDC45 onto origins. Cyclin A is also involved in blocking the reloading of fired origins (reviewed in reference 43). CDT1 is targeted to degradation after phosphorylation by cyclin-CDK in SCF^SKP2- or CUL4-DDB1-mediated mechanisms (15). Geminin accumulates during S phase and inactivates the remaining CDT1 (32). Moreover, CDC6 is phosphorylated by cyclin-CDK and translocated out of the nucleus. Finally, ORC1 (the largest subunit of the ORC) is inactivated either by polyubiquitination by SCF^SKP2 and degradation by the proteasome (38) or by monoubiquitination and dissociation from the chromatin (29). Thus, the high cyclin-CDK and low APC/C activities during S phase prevent the formation of the prereplicative complex and reduplication. The system is reset during the next mitosis, when APC/C is activated and degrades cyclins and geminin, allowing the prereplicative complex to form again.Genome reduplication generates polyploid cells. A growing body of evidence indicates that polyploidization can initiate chromosomal instability and aneuploidy. A seminal study by Fujiwara et al. (16) indicates that tetraploids can be generated by transient blocking of cytokinesis in p53-null mouse mammary epithelial cells. Importantly, tetraploidization promotes aneuploidy and tumorigenesis (16). Another study reported that chromosome nondisjunction (both copies of a chromosome segregate to the same daughter cells) leads to binucleated tetraploids by promoting cleavage furrow regression; the tetraploid cells then become aneuploidy through further divisions (48). This and other studies provide strong evidence of the importance of tetraploidization as an early step in tumorigenesis (reviewed in reference 53).While rereplication is stringently prevented in the normal cell cycle, multiple rounds of genome reduplication, called endoreduplication, occur in cell types such as megakaryocytes, trophoblast giant cells, numerous plant cells (26), and in the salivary glands of Drosophila melanogaster (49). In yeasts, different levels of a single CDK are believed to allow origin licensing and firing and prevent relicensing and mitosis (reviewed in reference 45). In contrast, the complex interplay between different cyclin-CDK complexes and licensing factors to prevent genome reduplication in higher eukaryotes remains to be fully characterized.Cyclin E is required for the endoreduplication cycles in Drosophila cells (14, 31, 57), trophoblasts, and megakaryocytes (20, 40). In fact, ectopic expression of cyclin E can promote endoreduplication in megakaryocytes (19). In contrast, although a decrease in cyclin A promotes endoreduplication in plant cells (24, 62) and Drosophila cells (39), it does not appear to be the case for megakaryocytic cell lines (19, 65, 66). A decrease in cyclin B1, but not cyclin A2, has been reported to be required for endoreduplication in megakaryocytes (66). Precisely which cyclin-CDK complexes are involved in safeguarding rereplication remains largely unresolved. Rereplication induced by EMI1 depletion is correlated with a reduction of cyclin A2 and cyclin B1, which can be rescued with nondestructible cyclin A2 (34). However, rereplication in HeLa cells is induced only weakly by cyclin A2 depletion, but it occurs more efficiently after codepletion of geminin (34). Conversely, expression of cyclin A2 (but not cyclin E) potentiates the rereplication induced by CDC6 and CDT1 in mammalian cells (56).In contrast to the complexity and uncertainty about the different cyclins in DNA reduplication, the central role of inactivation of mitotic CDK1 is generally accepted. This has been observed in a wide range of endoreduplication cycles, including those in maize endosperm (21), Drosophila cells (49), trophoblasts (54), and megakaryocytes (65). Likewise, extensive DNA reduplication can be triggered by disruption of CDK1 in a mammalian cell line (7, 25, 28). The molecular basis of how CDK1 inactivation contributes to genome reduplication remains to be defined.The prevailing view is that APC/C plays a salient role in preventing rereplication. The orthologs of CDH1 in plant cells (Ccs52A) and in Drosophila cells (fzr) are both essential for endoreduplication (26, 49). Unscheduled activation of APC/C reduces the concentrations of mitotic cyclins and geminin, resulting in rereplication (12, 34). An extra level of regulation provided by the APC/C inhibitor EMI1 has been uncovered recently. EMI1 begins to accumulate at the G₁/S transition, thereby inactivating APC/C and allowing the accumulation of cyclins and geminin (46). Accordingly, depletion of EMI1 with RNA interference promotes unscheduled APC/C activation and rereplication (12, 34).In this study, we utilized a specific CDK1 chemical inhibitor to induce whole-genome reduplication in cancer cells. We found that genome reduplication was associated with spontaneous oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis. Moreover, the PLK1-SCF^β-TrCP1-EMI1 axis and cyclin A2-CDK2 were inextricably linked to the APC/C activation and genome reduplication. These data extended our understanding of the role of the different cyclin-CDK complexes in coordinating genome reduplication.     

The Enhanced Metastatic Potential of Hepatocellular Carcinoma (HCC) Cells with Sorafenib Resistance

Acquired resistance towards sorafenib treatment was found in HCC patients, which results in poor prognosis. To investigate the enhanced metastatic potential of sorafenib resistance cells, sorafenib-resistant (SorR) cell lines were established by long-term exposure of the HCC cells to the maximum tolerated dose of sorafenib. Cell proliferation assay and qPCR of ABC transporter genes (ABCC1-3) were first performed to confirm the resistance of cells. Migration and invasion assays, and immunoblotting analysis on the expression of epithelial to mesenchymal transition (EMT) regulatory proteins were performed to study the metastatic potential of SorR cells. The expression of CD44 and CD133 were studied by flow cytometry and the gene expressions of pluripotency factors were studied by qPCR to demonstrate the enrichment of cancer stem cells (CSCs) in SorR cells. Control (CTL) and SorR cells were also injected orthotopically to the livers of NOD-SCID mice to investigate the development of lung metastasis. Increased expressions of ABCC1-3 were found in SorR cells. Enhanced migratory and invasive abilities of SorR cells were observed. The changes in expression of EMT regulatory proteins demonstrated an activation of the EMT process in SorR cells. Enriched proportion of CD44⁺ and CD44⁺CD133⁺ cells were also observed in SorR cells. All (8/8) mice injected with SorR cells demonstrated lung metastasis whereas only 1/8 mouse injected with CTL cells showed lung metastasis. HCC cells with sorafenib resistance demonstrated a higher metastatic potential, which may be due to the activated EMT process. Enriched CSCs were also demonstrated in the sorafenib resistant cells. This study suggests that advanced HCC patients with acquired sorafenib resistance may have enhanced tumor growth or distant metastasis, which raises the concern of long-term sorafenib treatment in advanced HCC patients who have developed resistance of sorafenib.     

The extracellular chaperone clusterin potently inhibits human lysozyme amyloid formation by interacting with prefibrillar species

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment.     

Numerical investigations of the haemodynamic changes associated with stent malapposition in an idealised coronary artery

The deployment of a coronary stent near complex lesions can sometimes lead to incomplete stent apposition (ISA), an undesirable side effect of coronary stent implantation. Three-dimensional computational fluid dynamics (CFD) calculations are performed on simplified stent models (with either square or circular cross-section struts) inside an idealised coronary artery to analyse the effect of different levels of ISA to the change in haemodynamics inside the artery. The clinical significance of ISA is reported using haemodynamic metrics like wall shear stress (WSS) and wall shear stress gradient (WSSG). A coronary stent with square cross-sectional strut shows different levels of reverse flow for malapposition distance (MD) between 0 mm and 0.12 mm. Chaotic blood flow is usually observed at late diastole and early systole for MD=0 mm and 0.12 mm but are suppressed for MD=0.06 mm. The struts with circular cross section delay the flow chaotic process as compared to square cross-sectional struts at the same MD and also reduce the level of fluctuations found in the flow field. However, further increase in MD can lead to chaotic flow not only at late diastole and early systole, but it also leads to chaotic flow at the end of systole. In all cases, WSS increases above the threshold value (0.5 Pa) as MD increases due to the diminishing reverse flow near the artery wall. Increasing MD also results in an elevated WSSG as flow becomes more chaotic, except for square struts at MD=0.06 mm.     

miR-200a Regulates Epithelial-Mesenchymal to Stem-like Transition via ZEB2 and β-Catenin Signaling

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133⁺ side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.     

Host defense peptide LL-37 selectively reduces proinflammatory macrophage responses

The human cathelicidin peptide, LL-37, is a host defense peptide with a wide range of immunomodulatory activities and modest direct antimicrobial properties. LL-37 can exert both pro- and anti-inflammatory effects and can modulate the proinflammatory responses of human peripheral blood monocytes and epithelial cells. In this study, we evaluated the effect of LL-37 on mouse bone marrow-derived macrophages (BMDM) and tissue macrophages in vitro and in vivo. LL-37 dramatically reduced TNF-α and NO levels produced by LPS and IFN-γ-polarized M1-BMDM and slightly reduced reactive oxygen species production by these cells. LL-37 did not affect the ability of IL-4-polarized M2-BMDM to upregulate arginase activity, although it did inhibit LPS-induced TNF-α secretion in these cells. LL-37 did not compromise the ability of M1-polarized BMDM to phagocytose and kill bacteria and did not affect the uptake of apoptotic neutrophils by M2-polarized BMDM. However, LL-37-treated M1-BMDM were more efficient at suppressing tumor growth in vitro. LL-37 significantly reduced LPS-induced TNF-α secretion in ex vivo alveolar macrophages, whereas its effect on peritoneal macrophages was much less dramatic. Effective inhibition of LPS-induced TNF-α secretion by alveolar macrophages also occurred in vivo when LL-37 was administered by intratracheal injection. This demonstrates a selective ability of LL-37 to decrease M1-BMDM, M2-BMDM, and tissue macrophage production of the proinflammatory cytokine TNF-α in response to LPS while leaving other crucial anti-inflammatory M1 and M2 macrophage functions unaltered.