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An oxetane intermediate during a direct aldol reaction was trapped with an internal aryl group to yield trans -tetraline products. The contribution of the oxetane intermediate was confirmed by 18 O-isotope labeling experiments.

Unlabelled: A functional immune response is crucial to prevent and limit infections with Streptococcus pneumoniae. Dendritic cells (DCs) play a central role in orchestrating the adaptive and innate immune responses by communicating with other cell types via antigen presentation and secretion of cytokines. In this study, we set out to understand how pneumococci activate human monocyte-derived DCs to produce interleukin-12 (IL-12) p70, an important cytokine during pneumococcal infections. We show that IL-12p70 production requires uptake of bacteria as well as the presence of the adaptor molecule TRIF, which is known to transfer signals of Toll-like receptor 3 (TLR3) or TLR4 from the endosome into the cell. While TLR4 is redundant for IL-12p70 production in DCs, we found that TLR3 is required to induce full IL-12p70 secretion. Influenza A virus (IAV) infection of DCs did not induce IL-12p70 but markedly upregulated TLR3 expression that during coinfection with S. pneumoniae significantly enhanced IL-12p70 secretion. Finally, we show that pneumococcal RNA can act as a bacterial stimulus for TLR3 and that it is a key signal to induce IL-12p70 production during challenge of DCs with pneumococci., Importance: Streptococcus pneumoniae, a common colonizer of the nose, is the causative agent of severe and deadly diseases. A well-orchestrated immune response is vital to prevent and limit these diseases. Dendritic cells (DCs) reside in the mucosal linings of the lungs and sample antigens. They are activated by pathogens to present antigens and secrete cytokines. While many studies focus on murine models, we focused our work on human monocyte-derived DCs. We found that pneumococcal RNA is an important stimulus in DCs to activate the endosomal receptor TLR3, a receptor previously not identified to sense pneumococci, and its adaptor molecule TRIF. This leads to secretion of the cytokine interleukin-12 (IL-12). Severe pneumococcal pneumonia occurs closely after influenza A virus (IAV) infection. We show that IAV infection upregulates TLR3 in DCs, which sensitizes the cells to endosomal pneumococcal RNA. This new insight contributes to unlock the interplay between pneumococci, IAV, and humans., (Copyright © 2016 Spelmink et al.)

We propose a new optical coherent detection scheme with a "two-tone" local light, which is also in principle insensitive to the laser phase fluctuation and is assisted by the digital signal processing technique for radio-over-fiber (RoF) systems. The main feature of our proposal is that the frequency separation of the two-tone local light is different from that of the RoF signal, which is called "offset-frequency-spaced" in this paper. First, we explain the principle of our new proposal and experimentally demonstrate the data recovery. Then, the influence of the frequency detuning between photo-detected modulated and unmodulated signals is discussed. Moreover, the transmission performance with an error vector magnitude (EVM) is evaluated for the optical coherent detection of a 10-Gbaud quadrature-phase-shift-keying RoF signal after a 20-km-long standard single-mode fiber transmission.

Secondary infections with Streptococcus pneumoniae (SP) are frequently observed following influenza A virus (IAV) infection and have a substantial impact on global health. Despite this, the basis for the disease progression is incompletely understood. To investigate the effect of co-infection on human monocyte-derived dendritic cells (MDDCs) we analysed the expression of clinically important pro-inflammatory and immune-modulatory cytokines. IAV infection or treatment with supernatants from IAV-infected cell cultures resulted in priming of the DCs which subsequently influenced the production of IL-12p70, as well as IL-6, following SP infection. Co-infection of the same cell was not required but this effect was dependent on the time, dose and duration of the infections, as well as pathogen viability, bacterial uptake and endosome acidification. Bacterially infected cells were characterized as the main producers of IL-12p70. Finally, we showed that type I interferons were primarily responsible for the priming of IL-12p70 that was observed by infection with IAV. These results provide a probable mechanism for the elevated levels of particular cytokines observed in IAV and SP co-infected cell cultures with implications for the pathogenic outcome observed during in vivo infection., (© 2013 John Wiley & Sons Ltd.)

Multi-input multi-output (MIMO) transmission of two millimeter-wave radio signals seamlessly converted from polarization-division-multiplexed quadrature-phase-shift-keying optical signals is successfully demonstrated, where a radio access unit basically consisting of only optical-to-electrical converters and a radio receiver performs total signal equalization of both the optical and the radio paths and demodulation with digital signal processing (DSP). Orthogonally polarized optical components that are directly converted to two-channel radio components can be demultiplexed and demodulated with high-speed DSP as in optical digital coherent detection. 20-Gbaud optical and radio seamless MIMO transmission provides a total capacity of 74.4 Gb/s with a forward error correction overhead of 7%.

Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.

The generation of a 40-Gb/s 16-QAM radio-over-fiber (RoF) signal and its demodulation of the wireless signal transmitted over free space of 30 mm in W-band (75-110 GHz) is demonstrated. The 16-QAM signal is generated by a coherent polarization synthesis method using a dual-polarization QPSK modulator. A combination of the simple RoF generation and the versatile digital receiver technique is suitable for the proposed coherent optical/wireless seamless network., (© 2011 Optical Society of America)

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Several plus-strand RNA viruses encode proteins containing macrodomains. These domains possess ADP-ribose-1″-phosphatase (ADRP) activity and/or bind poly(ADP-ribose), poly(A) or poly(G). The relevance of these activities in the viral life cycle has not yet been resolved. Here, we report that genetically engineered mutants of severe acute respiratory syndrome coronavirus (SARS-CoV) and human coronavirus 229E (HCoV-229E) expressing ADRP-deficient macrodomains displayed an increased sensitivity to the antiviral effect of alpha interferon compared with their wild-type counterparts. The data suggest that macrodomain-associated ADRP activities may have a role in viral escape from the innate immune responses of the host.

The 2'-5' oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, both in vitro and in vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.

SARS coronavirus (SARS-CoV), the causative agent of severe acute respiratory syndrome, is a versatile pathogen armed with a host of factors countering the antiviral type I interferon (IFN) system. Hence, tissue cells infected with SARS-CoV are unable to launch an IFN response. Plasmacytoid dendritic cells, however, produce high levels of IFN after infection. We recently demonstrated that minute amounts of IFN applied before infection (IFN priming) can ameliorate the IFN response of tissue cells to SARS-CoV. IFN priming of SARS-CoV-infected cells activated genes for IFN transcription, IFN signaling, antiviral effector proteins, ubiquitinylation and ISGylation, antigen presentation, and other cytokines and chemokines, whereas IFN treatment or infection alone had no major effect. Thus, the IFN which is produced by plasmacytoid dendritic cells could enable tissue cells to at least partially overturn the SARS-CoV-induced block in innate immune activation.

Background: Studies of the host response to infection often require quantitative measurement of the antiviral type I interferons (IFN-alpha/beta) in biological samples. The amount of IFN is either determined via its ability to suppress a sensitive indicator virus, by an IFN-responding reporter cell line, or by ELISA. These assays however are either time-consuming and lack convenient readouts, or they are rather insensitive and restricted to IFN from a particular host species., Results: An IFN-sensitive, Renilla luciferase-expressing Rift Valley fever virus (RVFV-Ren) was generated using reverse genetics. Human, murine and avian cells were tested for their susceptibility to RVFV-Ren after treatment with species-specific IFNs. RVFV-Ren was able to infect cells of all three species, and IFN-mediated inhibition of viral reporter activity occurred in a dose-dependent manner. The sensitivity limit was found to be 1 U/ml IFN, and comparison with a standard curve allowed to determine the activity of an unknown sample., Conclusions: RVFV-Ren replicates in cells of several species and is highly sensitive to pre-treatment with IFN. These properties allowed the development of a rapid, sensitive, and species-independent antiviral assay with a convenient luciferase-based readout.

The human coronaviruses (CoVs) severe acute respiratory syndrome (SARS)-CoV and NL63 employ angiotensin-converting enzyme 2 (ACE2) for cell entry. It was shown that recombinant SARS-CoV spike protein (SARS-S) downregulates ACE2 expression and thereby promotes lung injury. Whether NL63-S exerts a similar activity is yet unknown. We found that recombinant SARS-S bound to ACE2 and induced ACE2 shedding with higher efficiency than NL63-S. Shedding most likely accounted for the previously observed ACE2 downregulation but was dispensable for viral replication. Finally, SARS-CoV but not NL63 replicated efficiently in ACE2-positive Vero cells and reduced ACE2 expression, indicating robust receptor interference in the context of SARS-CoV but not NL63 infection.

SARS coronavirus (SARS-CoV) is known to efficiently suppress the induction of antiviral type I interferons (IFN-alpha/beta) in non-lymphatic cells through inhibition of the transcription factor IRF-3. Plasmacytoid dendritic cells, in contrast, respond to infection with production of high levels of IFNs. Here, we show that pretreatment of non-lymphatic cells with small amounts of IFN-alpha (IFN priming) partially overturns the block in IFN induction imposed by SARS-CoV. IFN priming combined with SARS-CoV infection substantially induced genes for IFN induction, IFN signalling, antiviral effector proteins, ubiquitination and ISGylation, antigen presentation and other cytokines and chemokines, whereas each individual treatment had no major effect. Curiously, however, despite this typical IFN response, neither IRF-3 nor IRF-7 was transported to the nucleus as a sign of activation. Taken together, our results suggest that (i) IFN, as it is produced by plasmacytoid dendritic cells, could enable tissue cells to launch a host response to SARS-CoV, (ii) IRF-3 and IRF-7 may be active at subdetectable levels, and (iii) SARS-CoV does not activate IRF-7.

Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.

In this study, we analyzed the replication and budding sites of severe acute respiratory syndrome coronavirus (SARS-CoV) at early time points of infection. We detected cytoplasmic accumulations containing the viral nucleocapsid protein, viral RNA and the non-structural protein nsp3. Using EM techniques, we found that these putative viral replication sites were associated with characteristic membrane tubules and double membrane vesicles that most probably originated from ER cisternae. In addition to its presence at the replication sites, N also accumulated in the Golgi region and colocalized with the viral spike protein. Immuno-EM revealed that budding occurred at membranes of the ERGIC (ER-Golgi intermediate compartment) and the Golgi region as early as 3 h post infection, demonstrating that SARS-CoV replicates surprisingly fast. Our data suggest that SARS-CoV establishes replication complexes at ER-derived membranes. Later on, viral nucleocapsids have to be transported to the budding sites in the Golgi region where the viral glycoproteins accumulate and particle formation occurs.

A novel method of optical phase-shift detection using differential-phase-to-intensity conversion (DPIC) based on injection locking of a semiconductor laser is proposed. We predict DPIC numerically and verify it experimentally. We then demonstrate detection of OC-48 (2.48832-Gbit/s) phase-modulated signals by use of DPIC.