Elements in the Canine Distemper Virus M 3′ UTR Contribute to Control of Replication Efficiency and Virulence

Canine distemper virus (CDV) is a negative-sense, single-stranded RNA virus within the genus Morbillivirus and the family Paramyxoviridae. The Morbillivirus genome is composed of six transcriptional units that are separated by untranslated regions (UTRs), which are relatively uniform in length, with the exception of the UTR between the matrix (M) and fusion (F) genes. This UTR is at least three times longer and in the case of CDV also highly variable. Exchange of the M-F region between different CDV strains did not affect virulence or disease phenotype, demonstrating that this region is functionally interchangeable. Viruses carrying the deletions in the M 3′ UTR replicated more efficiently, which correlated with a reduction of virulence, suggesting that overall length as well as specific sequence motifs distributed throughout the region contribute to virulence

Conserved motifs within the M 3′ UTR.

<p>(<b>A</b>) A pictogram, assembled as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031561#s4" target="_blank">Materials and Methods</a>, shows the sequence and frequency of the 410 nucleotides of the M 3′ UTR. A base height of 2 bits indicates 100% conservation of that nucleotide across the 26 CDV sequences. The numbering below the pictogram indicates the position in the UTR, with 1 being first nucleotide following the M stop codon and positions 408–410 being the intergenic triplet. (<b>B</b>) Conserved regions within the M 3′ UTR. The M 3′ UTR consists of 410 nucleotides and each is represented by a line. Black lines indicate 100% conservation across the 26 CDV sequences and grey lines represent variable nucleotides.</p

Characterization of M 3′ UTR mutants in ferrets.

<p>(<b>A</b>) Survival curve of ferrets infected with the different mutants. Animals were infected with 10⁵ TCID₅₀ intranasally. Death of an infected animal is indicated by a step down on the graph. (<b>B</b>) Course of cell-associated viremia displayed as the number of CDV-infected cells per million peripheral blood mononuclear cells (PBMC). (<b>C</b>) CDV-specific IgG response in serum samples over the course of the disease. An immunoperoxidase monolayer assay was performed and antibody titers are displayed as reciprocals of the highest antibody dilution at which viral antigen was observed. Error bars indicate the standard deviation.</p

Effects of the M 3′ UTR on early transcription and replication.

<p>Quantitative real-time RT-PCR analysis of viral genome and N, M and F mRNA. VerodogSLAMtag cells were infected with the parental and recombinant viruses at a MOI of 1. RNA was extracted after 0, 4, 16 and 24 h and subjected to reverse transcription. Primer pairs specific to the CDV trailer, N, M and F genes were used. The number of RNA molecules in the sample was quantified by plotting the Ct values against standard curves. Error bars indicate the standard deviation. *, P<0.05, **, P<0.01, and ***, P<0.001.</p

Effects of the M 3′ UTR on transcription and translation.

<p>(<b>A</b>) Northern blot analysis of viral mRNA with DNA probes. VerodogSLAMtag infected with the parental and recombinant viruses at an MOI of 1 were subjected to Northern blot analysis using DIG-labeled probes. Membranes were hybridized with DIG-labeled DNA probes for CDV N, P, M or F. Band intensities were determined by densitometry on non-saturated exposures using the Kodak Molecular Imaging software. The P, M and F expression were normalized by calculating the P/N, M/N and F/N ratios for each virus. The average mRNA ratios are shown beneath the appropriate bands and were calculated from at least four individual experiments. (<b>B</b>) Western blot analysis of viral N, M and F proteins. VerodogSLAMtag cells were infected with the parental and recombinant viruses at an MOI of 1 and overlaid with 0.5% methylcellulose. Cell lysates were extracted after 18 h and subjected to Western blot analysis. The membranes were probed with antibodies specific to the CDV N, M and F proteins. Intensities of N, M and F protein bands were determined by densitometry from non-saturated exposures using the Kodak Molecular Imaging Software. The M and F expression were normalized by calculating the M/N and F/N ratios for each virus and were calculated from at least four individual experiments.</p

Generation and characterization of recombinant CDVs with alterations in the M 3′ UTR.

<p>(<b>A</b>) Schematic drawing of recombinant viruses produced. The recombinant 58ΔF₁₀₆ genome is drawn to scale. Black and white boxes represent open reading frames and UTRs, respectively. The genes are indicated by their respective abbreviation. The M-F region of the recombinant viruses is expanded below. The UTRs are indicated by a thinner box, and the signal peptide coding region of the F gene (Fsp) is shaded. (<b>B</b> and <b>C</b>) Growth curves of the parental 58ΔF₁₀₆ and the three recombinant viruses in VerodogSLAMtag cells expressed as titers of released (<b>B</b>) and cell-associated virus (<b>C</b>). Cells were infected with an MOI of 0.01, and samples were harvested daily for five days. Titers are expressed as TCID₅₀ and error bars indicate the standard deviation. **, P<0.01, and ***, P<0.001. (<b>D</b>) Syncytia formation in VerodogSLAMtag cells. Cells were infected with a MOI of 0.01 and overlaid with 0.5% methylcellulose. Photographs show a representative syncytia of one replicate taken 48 h post infection using phase contrast at 100× magnification.</p

Evolution of the Therapeutic Management of Giant Cell Arteritis: Analysis of Real-Life Practices over Two Timeframes (2014–2017 and 2018–2020)

Objectives: To determine how therapeutic strategies for giant cell arteritis (GCA), especially glucocorticoid (GC) management, evolved between 2014 and 2020. Patients and Methods: Consecutive GCA patients followed for at least 24 months in a single tertiary center were enrolled and separated into two groups: those diagnosed from 2014 to 2017 and those diagnosed from 2018 to 2020. GC doses (mg/kg/day) were analyzed at onset, at Month 3 (M3) and, if continued, at M6, M12, M18 and M24. Physicians’ practices were also individually analyzed. Results: Among the 180 patients included, 96 (53%) were diagnosed in 2014–2017 and 84 (47%) in 2018–2020. All patients received GC at diagnosis without a difference in the initial dose between the two groups (p = 0.07). At M3, the daily dose was lower in patients treated after 2017 (p = 0.002). In patients who still received GC at M6 (p = 0.0008), M12 (p = 0.01) and M24 (p = 0.02), the daily GC dose was still lower in patients treated after 2017. The proportion of patients who definitively discontinued GC use before M18 (42% versus 21%, p = 0.003) was higher in those treated after 2017. The rates of immunosuppressant use were not different between the two time periods (31% versus 38%, p = 0.34), but tocilizumab replaced methotrexate. Significant differences were observed among practitioners regarding the GC doses at M6 (p = 0.04) and M12 (p = 0.04), the total GC duration (p = 0.02) and the ability to stop GC before M18 (p = 0.007). Conclusions: This real-life study showed a global change in GC management over time for GCA patients, with important variability among physicians’ practices

Apelin treatment increases complete Fatty Acid oxidation, mitochondrial oxidative capacity, and biogenesis in muscle of insulin-resistant mice.

International audienceBoth acute and chronic apelin treatment have been shown to improve insulin sensitivity in mice. However, the effects of apelin on fatty acid oxidation (FAO) during obesity-related insulin resistance have not yet been addressed. Thus, the aim of the current study was to determine the impact of chronic treatment on lipid use, especially in skeletal muscles. High-fat diet (HFD)-induced obese and insulin-resistant mice treated by an apelin injection (0.1 μmol/kg/day i.p.) during 4 weeks had decreased fat mass, glycemia, and plasma levels of triglycerides and were protected from hyperinsulinemia compared with HFD PBS-treated mice. Indirect calorimetry experiments showed that apelin-treated mice had a better use of lipids. The complete FAO, the oxidative capacity, and mitochondrial biogenesis were increased in soleus of apelin-treated mice. The action of apelin was AMP-activated protein kinase (AMPK) dependent since all the effects studied were abrogated in HFD apelin-treated mice with muscle-specific inactive AMPK. Finally, the apelin-stimulated improvement of oxidative capacity led to decreased levels of acylcarnitines and enhanced insulin-stimulated glucose uptake in soleus. Thus, by promoting complete lipid use in muscle of insulin-resistant mice through mitochondrial biogenesis and tighter matching between FAO and the tricarboxylic acid cycle, apelin treatment could contribute to insulin sensitivity improvement

Quantification of HER Expression and Dimerization in Patients’ Tumor Samples Using Time-Resolved Förster Resonance Energy Transfer

<div><p>Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with <em>EGFR</em> mRNA copy number (<em>P</em><0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients’ treatment in oncology.</p> </div