Suppressor analysis links trans-translation and ribosomal protein uS7 to RluD function in Escherichia coli.

The pseudouridine (ψ) synthase, RluD is responsible for three ψ modifications in the helix 69 (H69) of bacterial 23S rRNA. While normally dispensable, rluD becomes critical for rapid cell growth in bacteria that are defective in translation-termination. In slow-growing rluD- bacteria, suppressors affecting termination factors RF2 and RF3 arise frequently and restore normal termination and rapid cell growth. Here we describe two weaker suppressors, affecting rpsG, encoding ribosomal protein uS7 and ssrA, encoding tmRNA. In K-12 strains of E. coli, rpsG terminates at a TGA codon. In the suppressor strain, alteration of an upstream CAG to a TAG stop codon results in a shortened uS7 and partial alleviation of slow growth, likely by replacing an inefficient TGA stop codon with the more efficient TAG. Inefficient termination events, such as occurs in some rluD- strains, are targeted by trans-translation. Inactivation of the ssrA gene in slow-growing, termination-defective mutants lacking RluD and RF3, also partially restores robust growth, most probably by preventing destruction of completed polypeptides on ribosomes at slow-terminating stop codons. Finally, an additional role for RluD has been proposed, independent of its pseudouridine synthase activity. This is based on the observation that plasmids expressing catalytically dead (D139N or D139T) RluD proteins could nonetheless restore robust growth to an E. coli K-12 rluD- mutant. However, newly constructed D139N and D139T rluD plasmids do not have any growth-restoring activity and the original observations were likely due to the appearance of suppressors.

Musculoskeletal ultrasound for predicting remission in patients with rheumatoid arthritis: results from a 1-year prospective study.

To assess the role of musculoskeletal ultrasound as a predictor for the achievement of DAS28 remission in patients with rheumatoid arthritis (RA). One hundred and forty-one patients underwent physical and ultrasound examination at five visits (at baseline and after 1, 3, 6 and 12 months). Patients were divided into two groups according to the type of treatment, which involved synthetic (sDMARDs) and biologic (bDMARDs) disease-modifying antirheumatic drugs. Ultrasound assessment of the wrist, second and third metacarpophalangeal, second and third proximal interphalangeal joint, second and fifth metatarsophalangeal joint (the German US7 score) was performed on gray scale (GS) and on power Doppler ultrasound (PDUS). The rate of clinical remission and clinical and sonographic predictors for the achievement of DAS28 remission at month 12 were assessed. In the sDMARDs group at month 12, 43.6% of the patients achieved DAS28 remission, 5.1%-SDAI, 3.8%-CDAI, and 3.8%-Boolean remission. In the bDMARDs group, 49.2% achieved DAS28 remission, 6.3%-SDAI, 4.8%-CDAI, and 4.8%-Boolean remission. Predictors for DAS28 clinical remission in the sDMARDs group were low baseline DAS28 (p = 0.002), short disease duration (p = 0.007) and lower baseline PDUS score (p = 0.038). In the bDMARDs group low baseline DAS28 (p < 0.001) and PDUS score (p = 0.035) predicted DAS28 remission. Shorter disease duration, lower baseline DAS28 and PDUS scores are associated with a higher probability of achieving DAS28 remission at month 12 in patients with RA. Musculoskeletal ultrasound and in particular the German US7-scoring system may be used as a predictor for the achievement of clinical remission in RA patients.

Relation among anti-rheumatic drug therapy, CD14(+)CD16(+) blood monocytes and disease activity markers (DAS28 and US7 scores) in rheumatoid arthritis: A pilot study.

Circulating human monocytes, a functionally and phenotypically heterogeneous population, are emerging as fundamental cell types in rheumatoid arthritis (RA). The aim of this pilot study was to assess the correlation, if any, among anti-rheumatic drug therapy, circulating CD14(+)CD16(+) monocytes and validated clinical scales (e.g., DAS28 score and ultrasonography US7 score) of disease severity in RA. Thirty consecutive RA patients, either naïve or under disease-modifying anti-rheumatic drugs (DMARDs) or biological therapy, and 10 age-matched healthy volunteers, were enrolled. Monocytes were prepared from heparinized blood samples; surface expression of CD14 and CD16 was determined by flow cytometry. RA patients presented a significantly higher percentage of CD14(+)CD16(+) monocytes, as compared to healthy subjects. There was a good correlation between DAS28 clinical score and the ultrasound composite score US7 (r=0.66), as well as between both scores and the percentage of CD14(+)CD16(+) monocytes (r=0.43 and 0.47, respectively). Naïve RA patients had the highest expression (19.2±3.2%) of CD14(+)CD16(+) monocytes and elevated DAS28 score; patients on DMARDs presented a 7-fold increased expression of CD14(+)CD16(+) monocytes, relatively to healthy volunteers (2.1±1.4%), and an intermediate disease severity. The RA patients treated with biological therapy had a low percentage of CD14(+)CD16(+) monocytes (5.1±3.6%; p<0.01 vs naïve and DMARDs groups), similar to the one detected in healthy controls, and reduced US7 and DAS28 scores. Interestingly, for the same DAS28 score, monocytes isolated from RA patients on biological therapy had a lower CD16 expression than patients on DMARDs. Therefore, CD14(+)CD16(+) circulating blood monocytes may represent an appropriate biomarker to assess RA disease activity along with DAS28 and US7 scores. Together, these three parameters may represent a better indicator for evaluating therapy efficacy.

Ultrasound and magnetic resonance imaging of hands in systemic sclerosis: A cross-sectional analytical study of prevalence of inflammatory changes in patients with subclinical arthropathy.

Objectives: Prevalence of synovitis, tenosynovitis, erosions, acro-osteolysis and bone marrow edema in systemic sclerosis is not extensively reported. We aimed to estimate the prevalence of changes in individual joints of hands in systemic sclerosis patients. Method: A cross-sectional analytical study consisting of 34 adults (females, n = 32) with systemic sclerosis. Patients with clinical synovitis were excluded. All patients underwent ultrasound (US) and magnetic resonance imaging of bilateral hands. Results: On US, synovitis, tenosynovitis, erosions, and acro-osteolysis were detected in 97%, 94%, 97%, and 29% patients. Grade I synovitis observed in 67% joints-first carpometacarpal joint (55%), first metacarpophalangeal joint (54%), distal radioulnar joint (50%), and intercarpal joints (47%) were commonly affected. Erosions were common in distal phalanges (first DP72% to fifth DP39%). On magnetic resonance imaging, synovitis, tenosynovitis, erosions, and bone edema were observed in 91%, 85%, 97%, and 85% patients. Grade I synovitis was seen in 70% joints, affecting intercarpal joint (70.6%) and third metacarpophalangeal joint (52.9%) commonly. Grade I erosions were seen in 61%, affecting distal phalanges (55.8%), capitate (60.3%), and lunate (55.8%). Grade I edema was commonly affecting lunate (39%) and capitate (26%). On magnetic resonance imaging, acro-osteolysis was present in 28% (97/340) distal phalanges. Fair agreement (0.21-0.40) was noted between US and magnetic resonance imaging for synovitis and erosions. Conclusion: High prevalence of low-grade inflammation is found in systemic sclerosis patients on US and magnetic resonance imaging. Distal joint assessment in addition to proximal joints improves accurate estimation of prevalence of early arthropathy.

Construction of a US7/US8/UL23/US3-deleted recombinant pseudorabies virus and evaluation of its pathogenicity in dogs.

Since 2011, to control the spread of pseudorabies (PR), US7/US8/UL23-deleted recombinant PRV (rPRV) vaccines based on current variants have been developed. The vaccines can provide effective immune protection to pigs, but fur-bearing animals, such as dogs, foxes, and minks, are increasingly infected by PRV due to consuming contaminated raw meat or offal from immunized pigs. It is suspected that the attenuated PRV vaccine strain is not safe for these fur-bearing animals. To confirm this, we construct a US7/US8/UL23-deleted and a US7/US8/UL23/US3-deleted rPRV based on PRV GL isolated from fox using the CRISPR/Cas9 method. Growth kinetics in vitro and pathogenicity in dogs were compared between the wild type and both rPRVs. The results showed that the growth kinetics of wild-type PRV and US7/US8/UL23-deleted rPRV were faster than those of US7/US8/UL23/US3-deleted recombinant PRV from 24 h to 48 h post infection. Moreover, PRV GL- and rPRVdelUS7/US8/UL23-infected cells formed cell-cell fusion, but the rPRVdelUS7/US8/UL23/US3-infected cells did not. Dogs challenged with wild-type PRV or US7/US8/UL23-deleted rPRV showed obvious nervous symptoms, and all the dogs died, but the group challenged with the US7/US8/UL23/US3-deleted rPRV did not show any nervous symptoms, and all the dogs survived for the duration of the experiment. Tissue viral load analyses also showed that the virulence of the US7/US8/UL23/US3-deleted rPRV was significantly reduced in dogs. This study provides evidence that the US7/US8/UL23-deleted rPRV variant still exhibits high virulence for dogs and also highlights the role of the US3 gene in the pathogenicity of PRV in dogs and provides a strategy for developing a safer vaccine.

Relationship Between Rheumatoid Arthritis Disease Activity Assessed with the US7 Score and Quality of Life Measured with Questionnaires (HAQ, EQ-5D, WPAI).

BACKGROUND: Rheumatoid arthritis is a chronic disease considerably affecting the quality of life of patients, their functional abilities and capability to work. In everyday clinical practice, the condition is commonly monitored by assessing the disease activity using a joint count and laboratory markers of inflammation with calculating the DAS28 or other systems for scoring the disease activity. OBJECTIVES: This study is aimed to evaluate the benefit of ultrasound examination with a standardized instrument and correlate its results with the quality of life of patients and their functional abilities as assessed by standardized questionnaire methods. METHODS: 98 rheumatoid arthritis patients were assessed clinically for disease activity and ultrasound examination with assessment of German US 7 score was performed. The data from patientreported outcomes (Stanford Health Assessment Questionnaire and the EQ-5D instrument) were collected. RESULTS: The results of ultrasound examination were correlated with both disease activity and patient- assessed quality of life and limitations to daily activities. A very good predictor of impaired quality of life mainly appears to be the presence of synovitis detected by grayscale (GS) ultrasonography, shown to be significantly negatively correlated with patient mobility (r=-0.268, p=0.017), self-care (r-0.349, p= 0.002) and usual activities (r=-0.264, p= 0.019) as well as with patients' global health assessment (r=-0.243, p= 0.031). CONCLUSIONS: The study results showed a direct relationship between rheumatoid arthritis activity assessed with the US7 and patients' impaired functional abilities, mobility, self-care and usual activities.

Interface between 40S exit channel protein uS7/Rps5 and eIF2α modulates start codon recognition in vivo.

The eukaryotic pre-initiation complex (PIC) bearing the eIF2·GTP·Met-tRNAiMet ternary complex (TC) scans the mRNA for an AUG codon in favorable context. AUG recognition evokes rearrangement of the PIC from an open, scanning to a closed, arrested conformation. Cryo-EM reconstructions of yeast PICs suggest remodeling of the interface between 40S protein Rps5/uS7 and eIF2α between open and closed states; however, its importance was unknown. uS7 substitutions disrupting eIF2α contacts favored in the open complex increase initiation at suboptimal sites, and uS7-S223D stabilizes TC binding to PICs reconstituted with a UUG start codon, indicating inappropriate rearrangement to the closed state. Conversely, uS7-D215 substitutions, perturbing uS7-eIF2α interaction in the closed state, confer the opposite phenotypes of hyperaccuracy and (for D215L) accelerated TC dissociation from reconstituted PICs. Thus, remodeling of the uS7/eIF2α interface appears to stabilize first the open, and then the closed state of the PIC to promote accurate AUG selection in vivo.

The ß-hairpin of 40S exit channel protein Rps5/uS7 promotes efficient and accurate translation initiation in vivo.

The eukaryotic 43S pre-initiation complex bearing tRNAi(Met) scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the ß-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2∙GTP∙Met-tRNAi ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in ß-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred 'leaky-scanning' of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the ß-strand-1 substitution greatly destabilized the 'PIN' conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in ß-hairpin loop residues increased initiation fidelity and destabilized PIN at UUG, but not AUG start codons. We conclude that the Rps5 ß-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition.