A novel mechanism of RNase L inhibition: Theiler\u27s virus L* protein prevents 2-5A from binding to RNase L

<div><p>The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L <i>in vivo</i>. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.</p></div

Lineage A betacoronavirus NS2 proteins and the homologous torovirus Berne pp1a carboxy-terminal domain are phosphodiesterases that antagonize activation of RNase L

Viruses in the family Coronaviridae, within the order Nidovirales, are etiologic agents of a range of human and animal diseases, including both mild and severe respiratory diseases in humans. These viruses encode conserved replicase and structural proteins as well as more diverse accessory proteins, encoded in the 3′ ends of their genomes, that often act as host cell antagonists. We previously showed that 2′,5′-phosphodiesterases (2′,5′-PDEs) encoded by the prototypical Betacoronavirus, mouse hepatitis virus (MHV), and by Middle East respiratory syndrome-associated coronavirus antagonize the oligoadenylate-RNase L (OAS-RNase L) pathway. Here we report that additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses infecting both humans and animals, encode 2′,5′-PDEs capable of antagonizing RNase L. We used a chimeric MHV system (MHV(Mut)) in which exogenous PDEs were expressed from an MHV backbone lacking the gene for a functional NS2 protein, the endogenous RNase L antagonist. With this system, we found that 2′,5′-PDEs encoded by the human coronavirus HCoV-OC43 (OC43; an agent of the common cold), human enteric coronavirus (HECoV), equine coronavirus (ECoV), and equine torovirus Berne (BEV) are enzymatically active, rescue replication of MHV(Mut) in bone marrow-derived macrophages, and inhibit RNase L-mediated rRNA degradation in these cells. Additionally, PDEs encoded by OC43 and BEV rescue MHV(Mut) replication and restore pathogenesis in wild-type (WT) B6 mice. This finding expands the range of viruses known to encode antagonists of the potent OAS-RNase L antiviral pathway, highlighting its importance in a range of species as well as the selective pressures exerted on viruses to antagonize it. IMPORTANCE Viruses in the family Coronaviridae include important human and animal pathogens, including the recently emerged viruses severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome-associated coronavirus (MERS-CoV). We showed previously that two viruses within the genus Betacoronavirus, mouse hepatitis virus (MHV) and MERS-CoV, encode 2′,5′-phosphodiesterases (2′,5′-PDEs) that antagonize the OAS-RNase L pathway, and we report here that these proteins are furthermore conserved among additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses, suggesting that they may play critical roles in pathogenesis. As there are no licensed vaccines or effective antivirals against human coronaviruses and few against those infecting animals, identifying viral proteins contributing to virulence can inform therapeutic development. Thus, this work demonstrates that a potent antagonist of host antiviral defenses is encoded by multiple and diverse viruses within the family Coronaviridae, presenting a possible broad-spectrum therapeutic target

Synergistic effect on static and dynamic mechanical properties of carbon fiber-multiwalled carbon nanotube hybrid polycarbonate composites

Carbon fiber (CF) and multiwalled carbon nanotube (MWCNT)-reinforced hybrid micro-nanocomposites are prepared through melt mixing followed by injection moulding. The synergistic effect on both the static and dynamic mechanical properties with MWCNT/aMWCNT and CF reinforcement in a polycarbonate matrix is investigated by utilizing dynamic mechanical analysis, and flexural and tensile measurements. The enhancement in the flexural modulus and strength of the composite specimens as compared to pure PC for maximum loading of CF is 128.40% and 39.90%, respectively, which further improved to 142.94% and 42.60%, respectively, for CF-functionalized MWCNTs. Similarly, the storage modulus of the composite specimens reinforced with a maximum loading of CF and CF-functionalized MWCNTs show an increment of 176.57% and 203.33%, respectively over pure PC at 40 degrees C. Various types of parameter such as the coefficient C factor, degree of entanglement and adhesion factor have been calculated to analyze the interaction between fillers and the polymer matrix. Composite specimens containing 2 wt% of functionalized MWCNTs show a lower C value than the as-synthesized MWCNTs, which is indicative of a higher effectiveness of functionalized MWCNT-containing composite specimens. These results are well supported by optical microscopy and Raman spectroscopy by confirming the distribution of reinforcement

Anisakis pegreffii impacts differentiation and function of human dendritic cells

Human dendritic cells (DCs) show remarkably phenotypic changes when matured in presence of helminth-derived products. These modifications frequently elicited a polarization towards Th2 cells and regulatory T cells thus contributing to immunological tolerance against these pathogens. In this study, the interaction between DCs and larvae of the zoonotic anisakid nematode Anisakis pegreffii was investigated. A. pegreffii larvae were collected from fish hosts and monocyte derived DCs were co-cultured in the presence of the live larvae (L) or its crude extracts (CE). In both experimental conditions A. pegreffii impacted DC viability, hampered DC maturation by reducing the expression of molecules involved in antigen presentation and migration (i.e. HLA-DR, CD86, CD83 and CCR7), increased the phagosomal ROS levels and modulated the phosphorylation of ERK1,2 pathway. These biological changes were accompanied by the impairment of DCs to activate a T cell mediated IFN. Interestingly, live larva appeared to differently modulate DC secretion of cytokines and chemokines as compared to CE. These results demonstrate for the first time the immunomodulatory role of A. pegreffi on DCs biology and functions. In addition, they suggest a dynamic contribution of DCs to the induction and maintenance of the inflammatory response against A. pegreffi

Influence of mechanical, thermal, and electrical perturbations on the dielectric behaviour of guest-encapsulated HKUST-1 crystals

Metal–organic frameworks (MOFs) are emerging low-k dielectric materials for application in next-generation microelectronics and telecommunication devices. MOF dielectrics can function as smart sensors with high sensitivity and chemical selectivity, by leveraging the ubiquitous dielectric response of MOFs and overcoming the limitations of DC conductivity and fluorescence approaches. Herein we study the effects of material synthesis, applied mechanical stress (37–520 MPa), varying temperature (20–100 °C), and guest encapsulation on the frequency-dependent dielectric response (4 Hz to 1 MHz) and AC conductivity of the HKUST-1 MOF. Particularly, we show that the confinement of the triethylamine (NEt3) guest molecules in HKUST-1 (host) yields a NEt3@HKUST-1 system that is tuneable via mechanical, thermal and electrical perturbations. Within the frequency range of 10 kHz to 1 MHz, at 20 °C, we show that the dielectric constant (ε′) of the guest-encapsulated system could be tuned to attain a value in the range of 2.8 to 7.2; at 100 °C, an even greater value of ε′ in the range of 3.1 to 9.5 could be achieved. Conversely, we found that the dielectric tuneability of the porous (guest-free) HKUST-1 is relatively more limited (ε′ = 2.8 to 4.9) whilst employing the same operational parameters. Furthermore, the confinement of guest molecules in HKUST-1 enhances the mechanical resilience and yield strength of the powders subject to a compressive pelleting stress. Together, the results elucidate the new potential for exploiting host–guest interactions in MOFs, coupled with electrical, thermal, and mechanical stimuli to regulate the precise dielectric response of a designer low-k material

Identification of rare cell types in lung cancer and investigation of their use in prognosis

Cancer is a complex, dynamic and heterogeneous disease. Identification of tumor heterogeneity holds a significant promise not only on tumor classification but also on predicting the prognosis and choosing the right treatment. Therefore, it is essential to investigate tumor heterogeneity in a regular diagnosis procedure. Tumors contain variety of cell types such as malignant cells, non-malignant cells, immune cells, and rare cells (i.e., dormant cells (DRM) and cancer stem cells (CSC)). [1] All these cell types contribute to the disease progression. For example, dormant cells are major cause of recurrence of cancer because they disseminate from the tumors and enter into a state of cellular dormancy (G0/G1 arrest), and they may start dividing again after a prolonged period of dormancy. [2]The arrival of single cell RNA sequencing (scRNAseq) provides new opportunities for exploring gene expression profile at the single-cell level. scRNA technique has become favorable for studying the heterogeneous diseases such as cancer. [3]In this study, rare cell types from single-cell RNA sequencing data [1]were identified by using the Seurat tool[4]in R programing language and single-cell references were determined to analyze bulk data. Bulk data from TCGA[5]were analyzed to find cell type proportions by using the MuSiC tool [6]and survival models were generated to discover whether these rare cell types contribute to the survival time and if they can be used for prognosis. DRM cells negatively affect survival probability of LUADand LUSC patients over time. These results may indicate that cells in dormant state cannot be detected and eliminated in any treatment applied, and that the disease relapses after a whil

Structural flexibility of multifunctional HABP1 may be important for regulating its binding to different ligands

Hyaluronan-binding protein 1 (HABP1)/p32/gC1qR was characterized as a highly acidic and oligomeric protein, which binds to different ligands like hyaluronan, C1q, and mannosylated albumin. It exists as trimer in high ionic and reducing conditions as shown by crystal structure. In the present study, we have examined the structural changes of HABP1 under a wide range of ionic environments. HABP1 exhibits structural plasticity, which is influenced by the ionic environment under in vitro conditions near physiological pH. At low ionic strength HABP1 exists in a highly expanded and loosely held trimeric structure, similar to that of the molten globule-like state, whereas the presence of salt stabilizes the trimeric structure in a more compact fashion. It is likely that the combination of the high net charge asymmetrically distributed along the faces of the molecule and the relatively low intrinsic hydrophobicity of HABP1 result in its expanded structure at neutral pH. Thus, the addition of counter ions in the molecular environment minimizes the intramolecular electrostatic repulsion in HABP1 leading to its stable and compact conformations, which reflect in its differential binding toward different ligands. Whereas the binding of HABP1 toward HA is enhanced on increasing the ionic strength, no significant effect was observed with the two other ligands, C1q and mannosylated albumin. Thus, although HA interacts only with compact HABP1, C1q and mannosylated albumin can bind to loosely held oligomeric HABP1 as well. In other words, structural changes in HABP1 mediated by changes in the ionic environment are responsible for recognizing different ligands

Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan-binding protein 1 (HABP1)/p32/gC1qR

Hyaluronan-binding protein 1 (HABP1), a ubiquitous multifunctional protein, interacts with hyaluronan, globular head of complement component 1q (gC1q), and clustered mannose and has been shown to be involved in cell signalling. In vitro, this recombinant protein isolated from human fibroblast exists in different oligomeric forms, as is evident from the results of various independent techniques in near-physiological conditions. As shown by size-exclusion chromatography under various conditions and glutaraldehyde cross-linking, HABP1 exists as a noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers, i.e. a hexamer. The formation of a covalently-linked hexamer of HABP1 through Cys186 as a dimer of trimers is achieved by thiol group oxidation, which can be blocked by modification of Cys186. The gradual structural transition caused by cysteine-mediated disulfide linkage is evident as the fluorescence intensity increases with increasing Hg2+ concentration until all the HABP1 trimer is converted into hexamer. In order to understand the functional implication of these transitions, we examined the affinity of the hexamer for different ligands. The hexamer shows enhanced affinity for hyaluronan, gC1q, and mannosylated BSA compared with the trimeric form. Our data, analyzed with reference to the HABP1/p32 crystal structure, suggest that the oligomerization state and the compactness of its structure are factors that regulate its function

Pengaruh kinerja keuangan daerah terhadap tingkat kemiskinan melalui pertumbuhan ekonomi: studi pada Kabupaten/Kota Provinsi Nusa Tenggara Timur Periode 2017-2019

This study aims to analyze the effect of financial performance as measured by the ratio of regional independence, effectiveness of PAD, efficiency of PAD, and compatibility of capital expenditures on poverty through economic growth in districts and cities in East Nusa Tenggara Province for the period 2017-2019. The type of data used in this research is quantitative data and the source of the data used in this research is secondary data. The samples used in this study were 21 districts and 1 city in the province of East Nusa Tenggara. Data collection techniques in research by means of documentation, namely looking at the realization report and budget at the East Nusa Tenggara Central Statistics Agency (BPS) and the Director General of Fiscal Balance (DJPK) for the 2017-2019 period. The analytical method used in this research is path analysis. The results of this study indicate that in the sub-structural equation I together the ratio of regional independence, effectiveness of PAD, efficiency of PAD, and compatibility of capital spending have an effect on economic growth. Whereas in the sub-structural equation II the ratio of regional independence, effectiveness of PAD, efficiency of PAD, compatibility of capital expenditures, and economic growth together have an effect on poverty. In the substructural partial test I, the ratio of independence and efficiency has a positive and significant effect on economic growth. The ratio of effectiveness and compatibility of capital expenditures has no effect on economic growth. In the sub-structural equation II, the independence ratio has a negative and significant effect on poverty, the balance ratio of capital expenditures and economic growth has a positive and significant effect on poverty. Meanwhile, the ratio of effectiveness and efficiency has no effect on poverty. The ratio of independence, effectiveness, efficiency does not affect poverty through economic growth. Meanwhile, the ratio of harmony bel capital anja indirectly no effect on poverty through economic growth

Rola infekcji wirusem Epstein-Barr’a w rozwoju autoimmunologicznych chorób tarczycy

Introduction: Autoimmune thyroid diseases, including Graves’ and Hashimoto’s thyroiditis, are the most frequent autoimmune disorders. Viral infection, including Epstein-Barr virus (EBV), is one of the most frequently considered environmental factors involved in autoimmunity. Its role in the development of AITD has not been confirmed so far.Material and methods: Surgical specimens of Graves’ and Hashimoto’s diseases and nodular goitres were included in the study. The expression of EBV latent membrane protein 1 (LMP1) was analysed by immunohistochemistry, with the parallel detection of virus-encoded small nuclear non-polyadenylated RNAs (EBER) by in situ hybridisation.Results: In none of the Graves’ disease specimens but in 34.5% of Hashimoto’s thyroiditis cases the cytoplasmic expression of LMP1 was detected in follicular epithelial cells and in infiltrating lymphocytes. EBER nuclear expression was detected in 80.7% of Hashimoto’s thyroiditis cases and 62.5% of Graves´ disease cases, with positive correlation between LMP1 and EBER positivity in all Hashimoto’s thyroiditis LMP1-positive cases.Conclusions: We assume that high prevalence of EBV infection in cases of Hashimoto’s and Graves’ diseases imply a potential aetiological role of EBV in autoimmune thyroiditis. The initiation of autoimmune thyroiditis could start with EBV latency type III infection of follicular epithelium characterised by LMP1 expression involving the production of inflammatory mediators leading to recruitment of lymphocytes. The EBV positivity of the infiltrating lymphocytes could be only the presentation of a carrier state, but in cases with EBER+/ LMP1+ lymphocytes (transforming latent infection) it could represent a negative prognostic marker pointing to a higher risk of primary thyroid lymphoma development. (Endokrynol Pol 2015; 66 (2): 132–136)Wstęp: Autoimmunologiczne choroby taczycy, w tym zapalenia tarczycy Graves’a i Hashimoto, są najczęstszymi zaburzeniami autoimmunologicznymi. Infekcja wirusowa, w tym wirusem Epstein-Barr’a (EBV), jest jednym z najczęściej rozważanych czynników środowiskowych łączonych z autoimmunologią. Jego rola w rozwoju autoimmunologicznyh chorób taczycy (AITD) nie została do tej pory potwierdzona.Materiały i metody: Zbadano chirurgicznie pobrane preparaty od chorych na chorobę Gravesa, chorobę Hashimoto i wole guzowate. Ekspresja latentnego białka błonowego 1 (LMP1) EBV była zanalizowana metodami immunohistochemicznymi, z równoczesnym wykrywaniem kodowanych wirusem, małych, jądrzastych, niespoliadenylowanych RNA (EBER) poprzez hybrydazcję in situ.Wyniki: W żadnym z przypadków choroby Gravesa nie stwierdzono w komórkach pęcherzykowych i naciekających limfocytach ekspresji LMP1w cytoplazmie, ale wykryto ją w 34,5% przypadkach choroby Hashimoto. Jądrowa ekspresja EBER została wykryta w 80,7% przypadków zapalenia tarczycy Hashimoto i w 62,5% choroby Gravesa. Zauważono również dodatnią korelację pomiędzy LMP1 i występowaniem EBER we wszystkich przypadkach choroby Hashimoto LMP1+.Wnioski: Autorzy uwazają, że powszechne występowanie infekcji EBV w chorobach Hashimoto i Gravesa sugeruje potencjalną rolą etiologiczną EBV w rozwoju autoimmunologicznych zapaleń tarczycy. Zapoczątkowaniem autoimmunologicznych zapaleń tarczycy może być utajona infekcja EBV typu III, charakteryzująca się ekspresją LMP1 w komórkach pęcherzykowych i związana z produkcją mediatorów zapalnych prowadzącą do migracji limfocytów. Obecność EBV w naciekach limfocytarnych może być jedynie charakterystyczna dla nosiciela wirusa ale w przypadku obecności limfocytów EBER+/LMP1+ (przekształcajcych infekcję utajoną) może być negatywnym markerem prognostycznym wskazując podwyższone ryzyko rozwoju pierwotnego chłoniaka tarczycy. (Endokrynol Pol 2015; 66 (2): 132–136