Outside-binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1.

The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as a wild-type structure. After selecting the target NA sequences, their three-dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM-PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature.

Plasma let-7i, miR-16, and miR-221 levels as candidate biomarkers for the assessment of ankylosing spondylitis in Mexican patients naïve to anti-TNF therapy.

Novel biomarkers are needed for the diagnosis and clinical assessment of ankylosing spondylitis (AS). Our objective was to investigate plasma microRNAs differentially expressed between AS patients and controls and to evaluate their potential as biomarkers in Mexican subjects. A cross-sectional study including 15 AS patients naïve to anti-TNF therapy and 13 controls was performed. Disease activity, physical function, and global well-being were evaluated by the AS Disease Activity Score (ASDAS-CRP), the Bath AS Disease Activity Index (BASDAI), the Bath AS Functional Index (BASFI), and the AS Quality of Life Questionnaire (ASQoL), respectively. Total RNA was isolated from plasma of participants and relative expression of let-7i, miR-16, and miR-221 was evaluated. Serum levels of C-reactive protein (CRP), matrix metalloproteinase-1 (MMP-1), MMP-9, and intercellular adhesion molecule-1 (ICAM-1) were also measured. Expression of let-7i was higher in patients than in controls (1.8, 0.9 to 3.4 versus 0.6, 0.4 to 1.2; P = 0.033) with an AUC/ROC of 0.74 (0.54 to 0.93; P = 0.032). Levels of miR-16 and miR-221 were unable to discriminate between patients and controls. Notably, plasma miR-16 levels were inversely correlated with the ASDAS-CRP score (rho - 64, - 0.87 to - 0.18; P = 0.011), the BASFI score (rho - 0.78, - 0.93 to - 0.43; P = 0.001), and serum MMP-1 levels (rho - 0.59, - 0.85 to - 0.09; P = 0.022). No other associations were found. Plasma let-7i levels may serve as a biomarker for the diagnosis of AS in Mexican individuals. Additionally, plasma miR-16 levels are associated with disease activity and physical functionality in patients naïve to anti-TNF therapy.

HPV vaccination syndrome. A questionnaire-based study.

Isolated cases and small series have described the development of complex regional pain syndrome, postural orthostatic tachycardia, and fibromyalgia after human papillomavirus (HPV) vaccination. These illnesses are difficult to diagnose and have overlapping clinical features. Small fiber neuropathy and dysautonomia may play a major role in the pathogenesis of these entities. We used the following validated questionnaires to appraise the chronic illness that might appear after HPV vaccination: The 2010 American College of Rheumatology Fibromyalgia Diagnostic Criteria, COMPASS 31 dysautonomia questionnaire, and S-LANSS neuropathic pain form. These questionnaires and a "present illness" survey were e-mailed to persons who had the onset of a chronic ailment soon after HPV vaccination. Forty-five filled questionnaires from individuals living in 13 different countries were collected in a month's period. Mean (±SD) age at vaccination time was 14 ± 5 years. Twenty-nine percent of the cases had immediate (within 24 h) post-vaccination illness onset. The most common presenting complaints were musculoskeletal pain (66%), fatigue (57%), headache (57%), dizziness/vertigo (43%), and paresthesias/allodynia (36%). Fifty-three percent of affected individuals fulfill the fibromyalgia criteria. COMPASS-31 score was 43 ± 21, implying advanced autonomic dysfunction. Eighty-three percent of the patients who had ongoing pain displayed S-LANSS values >12, suggesting a neuropathic component in their pain experience. After a mean period of 4.2 ± 2.5 years post-vaccination, 93% of patients continue to have incapacitating symptoms and remain unable to attend school or work. In conclusion, a disabling syndrome of chronic neuropathic pain, fatigue, and autonomic dysfunction may appear after HPV vaccination.

Molecular distribution of amino acid substitutions on neuraminidase from the 2009 (H1N1) human influenza pandemic virus.

The pandemic influenza AH1N1 (2009) caused an outbreak of human infection that spread to the world. Neuraminidase (NA) is an antigenic surface glycoprotein, which is essential to the influenza infection process, and is the target of anti-flu drugs oseltamivir and zanamivir. Currently, NA inhibitors are the pillar pharmacological strategy against seasonal and global influenza. Although mutations observed after NA-inhibitor treatment are characterized by changes in conserved amino acids of the enzyme catalytic site, it is possible that specific amino acid substitutions (AASs) distant from the active site such as H274Y, could confer oseltamivir or zanamivir resistance. To better understand the molecular distribution pattern of NA AASs, we analyzed NA AASs from all available reported pandemic AH1N1 NA sequences, including those reported from America, Africa, Asia, Europe, Oceania, and specifically from Mexico. The molecular distributions of the AASs were obtained at the secondary structure domain level for both the active and catalytic sites, and compared between geographic regions. Our results showed that NA AASs from America, Asia, Europe, Oceania and Mexico followed similar molecular distribution patterns. The compiled data of this study showed that highly conserved amino acids from the NA active site and catalytic site are indeed being affected by mutations. The reported NA AASs follow a similar molecular distribution pattern worldwide. Although most AASs are distributed distantly from the active site, this study shows the emergence of mutations affecting the previously conserved active and catalytic site. A significant number of unique AASs were reported simultaneously on different continents.