Crystal Structure of the C-Terminal Cytoplasmic Domain of Non-Structural Protein 4 from Mouse Hepatitis Virus A59

BACKGROUND:The replication of coronaviruses takes place on cytoplasmic double membrane vesicles (DMVs) originating in the endoplasmic reticulum (ER). Three trans-membrane non-structural proteins, nsp3, nsp4 and nsp6, are understood to be membrane anchors of the coronavirus replication complex. Nsp4 is localized to the ER membrane when expressed alone but is recruited into the replication complex in infected cells. It is revealed to contain four trans-membrane regions and its N- and C-termini are exposed to the cytosol. METHODOLOGY/PRINCIPAL FINDINGS:We have determined the crystal structures of the C-terminal hydrophilic domain of nsp4 (nsp4C) from MHV strain A59 and a C425S site-directed mutant. The highly conserved 89 amino acid region from T408 to Q496 is shown to possess a new fold. The wild-type (WT) structure features two monomers linked by a Cys425-Cys425 disulfide bond in one asymmetric unit. The monomers are arranged with their N- and C-termini in opposite orientations to form an "open" conformation. Mutation of Cys425 to Ser did not affect the monomer structure, although the mutant dimer adopts strikingly different conformations by crystal packing, with the cross-linked C-termini and parallel N-termini of two monomers forming a "closed" conformation. The WT nsp4C exists as a dimer in solution and can dissociate easily into monomers in a reducing environment. CONCLUSIONS/SIGNIFICANCE:As nsp4C is exposed in the reducing cytosol, the monomer of nsp4C should be physiological. This structure may serve as a basis for further functional studies of nsp4

RTA Promoter Demethylation and Histone Acetylation Regulation of Murine Gammaherpesvirus 68 Reactivation

Gammaherpesviruses have a common biological characteristic, latency and lytic replication. The balance between these two phases in murine gammaherpesvirus 68 (MHV-68) is controlled by the replication and transcription activator (RTA) gene. In this report, we investigated the effect of DNA demethylation and histone acetylation on MHV-68 replication. We showed that distinctive methylation patterns were associated with MHV-68 at the RTA promoter during latency or lytic replication. Treatment of MHV-68 latently-infected S11E cells with a DNA methyltransferases (DNMTs) inhibitor 5-azacytidine (5-AzaC), only weakly reactivated MHV-68, despite resulted in demethylation of the viral RTA promoter. In contrast, treatment with a histone deacetylase (HDAC) inhibitor trichostatin A (TSA) strongly reactivated MHV-68 from latency, and this was associated with significant change in histone H3 and H4 acetylation levels at the RTA promoter. We further showed that HDAC3 was recruited to the RTA promoter and inhibited RTA transcription during viral latency. However, TSA treatment caused rapid removal of HDAC3 and also induced passive demethylation at the RTA promoter. In vivo, we found that the RTA promoter was hypomethylated during lytic infection in the lung and that methylation level increased with virus latent infection in the spleen. Collectively, our data showed that histone acetylation, but not DNA demethylation, is sufficient for effective reactivation of MHV-68 from latency in S11E cells

Access to Digital Financial Services and Green Technology Advances: Regional Evidence from China

Using data of 265 Chinese cities from 2010 to 2017, we studied the impact of access to digital financial services on green technology advances in the context of regional competition. We found that access to digital financial services significantly promotes green technology advances within the region but inhibits those in other regions. We also found that modest regional competition can promote green technology advances, whereas excessive competition impairs the positive relationship between access to digital financial services and green technology advances. We identified a significantly positive spatial spillover effect for green technology advances

A path forward for the chlamydial virulence factor CPAF

CPAF is a conserved and secreted protease from obligate intracellular bacteria of the order Chlamydiales. Recently, it was demonstrated that most of its host targets are an artifact of inaccurate methods. This review aims to summarize key features of CPAF and propose new approaches for evaluating its role in chlamydial pathogenesis

Metal-nitrogen-doped carbon materials as highly efficient catalysts: Progress and rational design

As a typical class of single-atom catalysts (SACs) possessing prominent advantages of high reactivity, high selectivity, high stability, and maximized atomic utilization, emerging metal-nitrogen-doped carbon (M-N-C) materials, wherein dispersive metal atoms are coordinated to nitrogen atoms doped in carbon nanomaterials, have presented a high promise to replace the conventional metal or metal oxides-based catalysts. In this work, recent progress in M-N-C-based materials achieved in both theoretical and experimental investigations is summarized and general principles for novel catalysts design from electronic structure modulating are provided. Firstly, the applications and mechanisms on the advantages and challenges of M-N-C-based materials for a variety of sustainable fuel generation and bioinspired reactions, including the oxygen reduction reaction (ORR), oxygen evolution reaction (OER), hydrogen evolution reaction (HER), carbon dioxide reduction reaction (CO2RR), nitrogen reduction reaction (NRR), and nanozyme reactions are reviewed. Then, strategies toward enhancing the catalytic performance by engineering the nature of metal ion centers, coordinative environment of active centers, carbon support, and their synergistic cooperation, are proposed. Finally, prospects for the rational design of next generation high-performance M-N-C-based catalysts are outlined. It is expected that this work will provide insights into high-performance catalysts innovation for sustainable and environmental technologies.</p

Inclusive Green Growth and Regional Disparities: Evidence from China

It is determined that inclusive green growth comprises processes of economic development and inclusiveness as a system of inclusions, taking into account the anthropogenic burden on the ecosystem, as well as the relational nature of socio-economic transformations. This article is an evaluation of this issue in the context of a contemporary Chinese society beset by regional inequalities that uses the Yangtze River basin as a case study. An index system has been constructed for inclusive green growth measurement, and kernel density and the Dagum Gini coefficient are used to analyze and describe characteristics regarding the distribution and spatial disparities within and between city clusters. The article then concludes that all city clusters are developing towards an inclusive green economy. There are still significant inequalities in inclusive growth among city clusters. Most city clusters are converging so slow that it will take a long time for weaker cites to catch up with stronger cites. City clusters also suffer major inner imbalances and gaps are widening. This paper argues that the profession needs to be more proactive in promoting strategic and targeted policies within such an unequal growth context

Transformation of sexually transmitted infection-causing serovars of chlamydia trachomatis using Blasticidin for selection.

Plasmid-free Chlamydia trachomatis serovar L2 organisms have been transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT using β-lactamase as a selectable marker. However, the recommendation of amoxicillin, a β-lactam antibiotics, as one of the choices for treating pregnant women with cervicitis due to C. trachomatis infection has made the existing shuttle vectors unsuitable for transforming sexually transmitted infection (STI)-causing serovars of C. trachomatis. Thus, in the current study, we modified the pGFP::SW2 plasmid by fusing a blasticidin S deaminase gene to the GFP gene to establish blasticidin resistance as a selectable marker and replacing the β-lactamase gene with the Sh ble gene to eliminate the penicillin resistance. The new vector termed pGFPBSD/Z::SW2 was used for transforming plasmid-free C. trachomatis serovar D organisms. Using blasticidin for selection, stable transformants were obtained. The GFP-BSD fusion protein was detected in cultures infected with the pGFPBSD/Z::SW2-trasnformed serovar D organisms. The transformation restored the plasmid property to the plasmid-free serovar D organisms. Thus, we have successfully modified the pGFP::SW2 transformation system for studying the biology and pathogenesis of other STI-causing serovars of C. trachomatis

TSA and 5-AzaC did not act synergistically to induce MHV-68 reactivation.

<p>(A) RT-PCR analysis of MHV-68 RTA mRNA expression after 5-AzaC and/or TSA treatment. S11E cells were induced with 5-AzaC (10 µM) and/or TSA (200 ng/ml) for 24 hrs and then total RNA were isolated for RT-PCR, GAPDH mRNA was amplified as a control. (B) Western blotting analysis of MHV-68 lytic protein expression after 5-AzaC and/or TSA treatment. TPA (25 ng/ml) plus NaB (4 mM) treatment served as a positive control.</p

A working model for DNA demethylation and histone acetylation regulation of MHV-68.

<p>(A) In S11E cells or latent infection <i>in vivo</i>, the RTA promoter is methylated and HDAC3 complex is recruited to the RTA promoter to suppress transcription. (B) When treated with TSA, the HDAC3 complex is rapidly removed, the RTA promoter demethylated through a passive mechanism, and RTA transcription is turned on. (C) However, when treated with 5-AzaC, though the RTA promoter becomes demethylated, HDAC3 stays at the promoter and the RTA promoter remains suppressed. M: methylation; A: acetylation.</p