Description: Nanomaterials have the characteristics associated with high surface-to-volume ratios and have been explored for their antiviral activity. Despite some success, cytotoxicity has been an issue in nanomaterial-based antiviral strategies. We previously developed a novel method to fully exfoliate montmorillonite clay to generate the most fundamental units of nanoscale silicate platelet (NSP). We further modified NSP by capping with various surfactants and found that the surfactant-modified NSP (NSQ) was less cytotoxic. In this study, we tested the antiviral potentials of a series of natural-clay-derived nanomaterials. Among the derivatives, NSP modified with anionic sodium dodecyl sulfate (NSQc), but not the pristine clay, unmodified NSP, a silver nanoparticle-NSP hybrid, NSP modified with cationic n -octadecanylamine hydrochloride salt, or NSP modified with nonionic Triton X-100, significantly suppressed the plaque-forming ability of Japanese encephalitis virus (JEV) at noncytotoxic concentrations. NSQc also blocked infection with dengue virus (DEN) and influenza A virus. Regarding the antiviral mechanism, NSQc interfered with viral binding through electrostatic interaction, since its antiviral activity can be neutralized by Polybrene, a cationic polymer. Furthermore, NSQc reduced the lethality of JEV and DEN infection in mouse challenge models. Thus, the surfactant-modified exfoliated nanoclay NSQc may be a novel nanomaterial with broad and potent antiviral activity. IMPORTANCE Nanomaterials have being investigated as antimicrobial agents, yet their antiviral potential is overshadowed by their cytotoxicity. By using a novel method, we fully exfoliated montmorillonite clay to generate the most fundamental units of nanoscale silicate platelet (NSP). Here, we show that the surfactant-modified NSP (NSQ) is less cytotoxic and that NSQc (NSP modified with sodium dodecyl sulfate) could potently block infection by dengue virus (DEN), Japanese encephalitis virus (JEV), and influenza A virus at noncytotoxic concentrations. For the antiviral mechanism, we find that the electrostatic interaction between the negatively charged NSQc and the positively charged virus particles blocks viral binding. Furthermore, we used mouse challenge models of JEV and DEN to demonstrate the in vivo antiviral potential of NSQc. Thus, NSQc may function as a potent and safe antiviral nanohybrid against several viruses, and our success in synthesizing surfactant-modified NSP with antiviral activity may shed some light on future antiviral development.

Description: CC chemokine ligand-2 (CCL2)/monocyte chemoattractant protein (MCP)-1 expression is upregulated during pulmonary inflammation, and the CCL2-CCR2 axis plays a critical role in leukocyte recruitment and promotion of host defense against infection. The role of CCL2 in mediating macrophage subpopulations in the pathobiology of noninfectious lung injury is unknown. The goal of this study was to examine the role of CCL2 in noninfectious acute lung injury. Our results show that lung-specific overexpression of CCL2 protected mice from bleomycin-induced lung injury, characterized by significantly reduced mortality, reduced neutrophil accumulation, and decreased accumulation of the inflammatory mediators IL-6, CXCL2 (macrophage inflammatory protein-2), and CXCL1 (keratinocyte-derived chemokine). There were dramatic increases in the recruitment of myosin heavy chain (MHC) II IA/IE int CD11c int cells, exudative macrophages, and dendritic cells in Ccl2 transgenic mouse lungs both at baseline and after bleomycin treatment compared with levels in wild-type mice. We further demonstrated that MHCII IA/IE int CD11c int cells engulfed apoptotic cells during acute lung injury. Our data suggested a previously undiscovered role for MHCII IA/IE int CD11c int cells in apoptotic cell clearance and inflammation resolution.

Description: The viral ribonucleoprotein (vRNP) complex of influenza A viruses (IAVs) contains an RNA-dependent RNA polymerase complex (RdRp) and nucleoprotein (NP) and is the functional unit for viral RNA transcription and replication. The vRNP complex is an important determinant of virus pathogenicity and host adaptation, implying that its function can be affected by host factors. In our study, we identified host protein Moloney leukemia virus 10 (MOV10) as an inhibitor of IAV replication, since depletion of MOV10 resulted in a significant increase in virus yield. MOV10 inhibited the polymerase activity in a minigenome system through RNA-mediated interaction with the NP subunit of vRNP complex. Importantly, we found that the interaction between MOV10 and NP prevented the binding of NP to importin-α, resulting in the retention of NP in the cytoplasm. Both the binding of MOV10 to NP and its inhibitory effect on polymerase activity were independent of its helicase activity. These results suggest that MOV10 acts as an anti-influenza virus factor through specifically inhibiting the nuclear transportation of NP and subsequently inhibiting the function of the vRNP complex. IMPORTANCE The interaction between the influenza virus vRNP complex and host factors is a major determinant of viral tropism and pathogenicity. Our study identified MOV10 as a novel host restriction factor for the influenza virus life cycle since it inhibited the viral growth rate. Conversely, importin-α has been shown as a determinant for influenza tropism and a positive regulator for viral polymerase activity in mammalian cells but not in avian cells. MOV10 disrupted the interaction between NP and importin-α, suggesting that MOV10 could also be an important host factor for influenza virus transmission and pathogenicity. Importantly, as an interferon (IFN)-inducible protein, MOV10 exerted a novel mechanism for IFNs to inhibit the replication of influenza viruses. Furthermore, our study potentially provides a new drug design strategy, the use of molecules that mimic the antiviral mechanism of MOV10.

Description: n -Alkanes are ubiquitous in nature and are widely used by microorganisms as carbon sources. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of n -alkanes, which plays important roles in natural alkane attenuation and is used in industrial and environmental applications. The alkane oxidation operon, alkW1-alkX , in the alkane-degrading strain Dietzia sp. strain DQ12-45-1b is negatively autoregulated by the TetR family repressor AlkX via a product positive feedback mechanism. To predict the gene regulation mechanism, we determined the 3.1-Å crystal structure of an AlkX homodimer in a non-DNA-bound state. The structure showed traceable long electron density deep inside a hydrophobic cavity of each monomer along the long axis of the helix bundle, and further gas chromatography-mass spectrometry analysis of AlkX revealed that it contained the Escherichia coli -derived long-chain fatty acid molecules as a ligand. Moreover, an unusual structural feature of AlkX is an extra helix, α6', forming a lid-like structure with α6 covering the inducer-binding pocket and occupying the space between the two symmetrical DNA-binding motifs in one dimer, indicating a distinct conformational transition mode in modulating DNA binding. Sequence alignment of AlkX homologs from Dietzia strains showed that the residues involved in DNA and inducer binding are highly conserved, suggesting that the regulation mechanisms of n -alkane hydroxylation are possibly a common characteristic of Dietzia strains. IMPORTANCE With n -alkanes being ubiquitous in nature, many bacteria from terrestrial and aquatic environments have evolved n -alkane oxidation functions. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of n -alkanes, which plays important roles in natural alkane attenuation and petroleum-contaminating environment bioremediation. The gene regulation of the most common alkane hydroxylase, AlkB, has been studied widely in Gram-negative bacteria but has been less explored in Gram-positive bacteria. Our previous study showed that the TetR family regulator (TFR) AlkX negatively autoregulated the alkane oxidation operon, alkW1-alkX , in the Gram-positive strain Dietzia sp. strain DQ12-45-1b. Although TFRs are one of the most common transcriptional regulator families in bacteria, the TFR involved in n -alkane metabolism has been reported only recently. In this study, we determined the crystal structure of AlkX, which implies a distinct DNA/ligand binding mode. Our results shed light upon the regulation mechanism of the common alkane degradation process in nature.

Description: Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-B or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs. IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ~200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ~10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

Description: The alveolar epithelium is composed of type I cells covering most of the gas-blood exchange surface and type II cells secreting surfactant that lowers surface tension of alveoli to prevent alveolar collapse. Here, we have identified a subgroup of type II cells expressing a higher level of cell surface molecule CD44 (CD44 high type II cells) that composed ~3% of total type II cells in 5–10-wk-old mice. These cells were preferentially apposed to lung capillaries. They displayed a higher proliferation rate and augmented differentiation capacity into type I cells and the ability to form alveolar organoids compared with CD44 low type II cells. Moreover, in aged mice, 18–24 mo old, the percentage of CD44 high type II cells among all type II cells was increased, but these cells showed decreased progenitor properties. Thus CD44 high type II cells likely represent a type II cell subpopulation important for constitutive regulation of alveolar homeostasis.

Description: Japanese encephalitis virus (JEV) is an enveloped flavivirus with a single-stranded, positive-sense RNA genome encoding three structural and seven nonstructural proteins. To date, the role of JEV nonstructural protein 2A (NS2A) in the viral life cycle is largely unknown. The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) phosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α) after sensing viral RNA and results in global translation arrest as an important host antiviral defense response. In this study, we found that JEV NS2A could antagonize PKR-mediated growth inhibition in a galactose-inducible PKR-expressing yeast system. In human cells, PKR activation, eIF2α phosphorylation, and the subsequent translational inhibition and cell death triggered by dsRNA and IFN-α were also repressed by JEV NS2A. Moreover, among the four eIF2α kinases, NS2A specifically blocked the eIF2α phosphorylation mediated by PKR and attenuated the PKR-promoted cell death induced by the chemotherapeutic drug doxorubicin. A single point mutation of NS2A residue 33 from Thr to Ile (T33I) abolished the anti-PKR potential of JEV NS2A. The recombinant JEV mutant carrying the NS2A-T33I mutation showed reduced in vitro growth and in vivo virulence phenotypes. Thus, JEV NS2A has a novel function in blocking the host antiviral response of PKR during JEV infection.

Description: High-precision measurements of size changes of individual bacterial spores based on ellipse fitting to bright-field images recorded with a digital camera were employed to monitor the germination of Bacillus spores with a precision of ~5 nm. To characterize the germination of individual spores, we recorded bright-field and phase-contrast images and found that the timing of changes in their normalized intensities coincided, so the bright-field images can be used to characterize spore size and refractility changes during germination. The major conclusions from this work were as follows. (i) The sizes of germinating B. cereus spores were nearly unchanged until T release , the time of the completion of CaDPA (a 1:1 chelate of Ca 2+ and dipicolinic acid [DPA]) release after addition of nutrient germinants. (ii) The minor axis of germinating B. cereus spores rapidly increased by ~50 nm in a few seconds right after T release , while the major axis was slightly decreased or unchanged. Both the minor and major axes remained unchanged for a further 30 to 45 s and then increased by 100 to 200 nm by T lys , the time of completion of cortex lysis. (iii) Individual spores in a population showed significant heterogeneity in the timing of germination events, such as T release and T lys , but also variation in size changes during germination. (iv) Bacillus subtilis wild-type spores, B. subtilis spores lacking the cortex-lytic enzyme CwlJ, and wild-type Bacillus megaterium spores showed similar kinetics of size changes during nutrient germination. The size increases in germinating spores probably result from uptake of water and cortex lysis after completion of CaDPA release.

Description: There is a global epidemic of obesity, and obesity is known to inhibit AMP-activated protein kinase (AMPK) activity and impairs myogenesis. Myogenin mediates the fusion of myoblasts into myotubes, a critical step in myogenesis. We observed that inhibition of AMPKα1 downregulates myogenin expression and myogenesis, but the underlying mechanisms are unclear. We postulated that AMPK regulates myogenin expression through phosphorlytion of histone deacetylase 5 (HDAC5). In C2C12 cells, HDAC5 knockdown increased while HDAC5 stablization by MC1568 reduced myogenin expression. Consistently, using luciferase assay, we observed that myogenin promoter activity was negatively regulated by HDAC5. Using RNA interference and primary myoblasts prepared from wild-type and AMPKα1 knockout mice, we further demonstrate that AMPKα1 regulates HDAC5 phosphorylation at Ser 259 and 498. Mutation of these two Ser to Ala in HDAC5 abolished the regulatory role of AMPKα1 on myogenin expression, clearly showing the necessity of these phosphorylation sites in mediating myogenin expression. In aggregate, these data show that AMPK inhibition downregulates myogenin transcription and myogenesis through phosphorylation of HDAC5, mediated mainly by AMPKα1. These data demonstrate that AMPK is a key molecular target for promoting myogenesis and muscular regeneration. Because drugs activating AMPK activity, such as metformin, are widely available, our finding has critical clinical implications to ensure proper muscle development and regeneration in obese subjects and under other pathophysiological conditions where AMPK activity is attenuated.

Description: Brucella abortus and Yersinia enterocolitica serotype O:9 serologically cross-react in the immune response with the host; therefore, our aim was to compare the immune responses to these two pathogens. We selected typical B. abortus and Y. enterocolitica O:9 strains to study the cytokine immune response and the histopathological changes in livers and spleens of BALB/c mice. The data showed the cytokine responses to the two strains of pathogens were different, where the average levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-α) were higher with B. abortus infections than with Y. enterocolitica O:9 infections, especially for IFN-, while the IL-10 level was lower and the levels of IL-1β, IL-4, IL-5, and IL-6 were similar. The histopathological effects in the livers and spleens of the BALB/c mice with B. abortus and Y. enterocolitica O:9 infections were similar; however, the pathological changes in the liver were greater with B. abortus infections, while damage in the spleen was greater with Y. enterocolitica O:9 infections. These observations show that different cytokine responses and histopathological changes occur with B. abortus and Y. enterocolitica O:9 infections.