Rhinovirus species and clinical characteristics in the first wheezing episode in children

The clinical data on the first wheezing episodes induced by different rhinovirus (RV) species are still limited. We aimed to investigate the prevalence of RV genotypes, sensitization status, and clinical characteristics of patients having a respiratory infection caused by either different RV species or other respiratory viruses. The study enrolled 111 patients (aged 3–23 months, 79% hospitalized, 76% with RV infection) with the first wheezing episode. RV‐specific sequences were identified by partial sequencing of VP4/VP2 and 5′ non‐coding regions with 80% success rate. The investigated clinical and laboratory variables included atopic characteristics and illness severity, parental atopic illnesses, and parental smoking. Of the study children, 56% percent had > 1 atopic characteristic (atopy, eczema and/or blood eosinophil count > 0.4 × 10⁹/L) and 23% were sensitised to allergens. RV‐C was detected in 58% of RV positive samples, followed by RV‐A (20%) and RV‐B (1.2%). Children with RV‐A and RV‐C induced wheezing were older (P = 0.014) and had more atopic characteristics (P = 0.001) than those with non‐RV. RV‐A and RV‐C illnesses had shorter duration of preadmission symptoms and required more bronchodilator use at the ward than non‐RV illnesses (both P < 0.05, respectively). RV‐C is the most common cause of severe early wheezing. Atopic and illness severity features are associated with children having RV‐A or RV‐C induced first wheezing episode rather than with children having a non‐RV induced wheezing. J. Med. Virol. 88:2059–2068, 2016. © 2016 Wiley Periodicals, Inc.

     

Physical mechanisms of amyloid nucleation on fluid membranes

Biological membranes can dramatically accelerate the aggregation of normally soluble protein molecules into amyloid fibrils and alter the fibril morphologies, yet the molecular mechanisms through which this accelerated nucleation takes place are not yet understood. Here, we develop a coarse-grained model to systematically explore the effect that the structural properties of the lipid membrane and the nature of protein–membrane interactions have on the nucleation rates of amyloid fibrils. We identify two physically distinct nucleation pathways—protein-rich and lipid-rich—and quantify how the membrane fluidity and protein–membrane affinity control the relative importance of those molecular pathways. We find that the membrane’s susceptibility to reshaping and being incorporated into the fibrillar aggregates is a key determinant of its ability to promote protein aggregation. We then characterize the rates and the free-energy profile associated with this heterogeneous nucleation process, in which the surface itself participates in the aggregate structure. Finally, we compare quantitatively our data to experiments on membrane-catalyzed amyloid aggregation of α-synuclein, a protein implicated in Parkinson’s disease that predominately nucleates on membranes. More generally, our results provide a framework for understanding macromolecular aggregation on lipid membranes in a broad biological and biotechnological context.

The aggregation of normally soluble proteins into β-sheet-rich amyloid fibrils is a common form of protein assembly that has broad implications across biomedical and biotechnological sciences, in contexts as diverse as the molecular origins of neurodegenerative disorders to the production of functional materials (1, 2). The presence of surfaces and interfaces can strongly influence amyloid aggregation, either catalyzing or inhibiting it, depending on the nature of the surface. This effect has been studied for the cases of amyloid nucleation on nanoparticles (3–5), on flat surfaces (6–10), and on the surface of amyloid fibrils themselves (11, 12).Lipid bilayers are a unique type of surface, which is ubiquitous in biology and is the main contributor to the large surface-to-volume ratio characteristic of biological systems. They are highly dynamic, self-assembled structures that can induce structural changes in the proteins bound to them (13, 14) and markedly affect protein-aggregation propensities (15, 16). While nucleation on the surfaces of lipid membranes can influence fibril formation dramatically, alternative surfactant-driven fibrillation pathways in solution have been proposed at surfactant concentrations where formation of bilayer structures is not observed (17).Increasing experimental evidence supports the principle that the interaction between amyloidogenic proteins and the lipid cell membrane catalyzes in vivo amyloid nucleation, which is involved in debilitating pathologies. Remarkably, through surface-driven catalysis, lipid bilayers can enhance the kinetics of α-synuclein aggregation, the protein involved in Parkinson’s disease, by over three orders of magnitude with respect to nucleation in solution (18).Bilayer membranes can exist in different structural phases and can undergo local and global phase changes. A large body of work has focused on exploring how the membrane’s dynamical properties, such as its fluidity, relate to amyloid aggregation of bound proteins (19–26).For instance, fluid membranes, constituted of short and saturated lipid chains, were found to most effectively catalyze the nucleation of α-synuclein (19), while less-fluid membranes composed of long lipid chains had less catalytic power. Furthermore, the addition of cholesterol to lipid membranes was found to alter its fluidity and govern the nucleation rate of Aβ42 (25), a peptide implicated in Alzheimer’s disease. In these cases, the physical properties of the membrane are controlled through variations in its composition, and decoupling the role of the membrane’s physical properties from its chemical specificity is extremely challenging.The question we focus on here is how the microscopic steps that drive amyloid nucleation at the membrane surface are altered by the inherently dynamic nature of lipid bilayers.Computer simulations can be of great help in this case, enabling us to systematically investigate the role of the physical and chemical properties of lipid membranes independently from one another, thus helping to identify key players behind membrane-driven amyloid nucleation.In this work, we develop a coarse-grained Monte Carlo model for studying the nucleation of amyloidogenic proteins on lipid membranes. We use it to identify the microscopic mechanisms which connect the membrane fluidity, the rate of amyloid nucleation, and the morphology of amyloid aggregates. We find that the membrane most efficiently catalyzes amyloid nucleation by donating its lipids to the nucleating fibril, which depends 1) on the lipid solubility and often correlates with membrane fluidity, and 2) the affinity of proteins to the membrane. This interdependence controls both the morphology of the resulting aggregates, which can range from protein-rich to lipid-rich, and the rate of fibril formation. We then discuss how our results provide a mechanistic explanation for a number of recent experimental observations regarding accelerated nucleation kinetics on fluid membranes (19), lipid–protein coaggregation (27), and altered aggregate morphology (23). Furthermore, the framework developed here offers a platform for studying strategies for bypassing amyloid nucleation in a cellular context.     

Combinatorial effects of amoxicillin and metronidazole on selected periodontal bacteria and whole plaque samples

Objective

The aim of the present study was to analyze in vitro the combinatorial effects of the antibiotic combination of amoxicillin plus metronidazole on subgingival bacterial isolates.

Design

Aggregatibacter (Actinobacillus) actinomycetemcomitans, Prevotella intermedia/nigrescens, Fusobacterium nucleatum and Eikenella corrodens from our strain collection and subgingival bacteria isolated from patients with periodontitis were tested for their susceptibility to amoxicillin and metronidazole using the Etest. The fractional inhibitory concentration index (FICI), which is commonly used to describe drug interactions, was calculated.

Results

Synergy, i.e. FICI values ≤ 0.5, between amoxicillin and metronidazole was shown for two A. actinomycetemcomitans (FICI: 0.3), two F. nucleatum (FICI: 0.3 and 0.5, respectively) and one E. corrodens (FICI: 0.4) isolates. Indifference, i.e. FIC indices of >0.5 but ≤4, occurred for other isolates and the 14 P. intermedia/nigrescens strains tested. Microorganisms resistant to either amoxicillin or metronidazole were detected in all samples by Etest.

Conclusion

Combinatorial effects occur between amoxicillin and metronidazole on some strains of A. actinomycetemcomitans, F. nucleatum and E. corrodens. Synergy was shown for a few strains only.     

Efficacy of various side-to-side toothbrushes for noncontact biofilm removal

Objectives

The aim of this study was to evaluate the efficacy of four different powered toothbrushes with side-to-side action for noncontact biofilm removal in vitro.

Materials and methods

A three-species biofilm was formed in vitro on protein-coated titanium disks using a flow chamber combined with a static biofilm growth model. Subsequently, the biofilm-coated substrates were exposed to four different side-to-side toothbrushes (A, B, C, and D) with various brushing times (2, 4, and 6 s) and brushing (bristle-to-disk) distances (0, 2, and 4 mm). The biofilm volumes were measured using volumetric analyses with confocal laser scanning microscope images and Imaris version 7.5.2 software.

Results

The median percentages of biofilm reduction by the analyzed toothbrushes ranged from 9 % to 80 %. The abilities of the tested toothbrushes to remove the in vitro biofilm differed significantly (p?<?0.05). Two of the tested toothbrushes (C and D) were capable of significant biofilm reduction by noncontact brushing.

Conclusions

It was possible to reduce a three-species in vitro biofilm by noncontact brushing with two out of four side-to-side toothbrushes.

Clinical relevance

Toothbrushes C and D show in vitro a high efficacy in biofilm removal without bristle contact.     

H-ras oncogene point mutations in arthritic synovium

Objective. To examine mutational activation of ras proto-oncogenes in synovial tissue from patients with rheumatoid arthritis (RA) compared with synovial specimens from patients with osteoarthritis (OA) or other arthropathies. Synovial samples from cadavers, without any signs of joint disease, were used as control material. Methods. Using a combination of polymerase chain reaction (PCR) and automated sequencing of the amplified PCR product, regions around codons 12, 13, and 61 of the H-, K-, and N-ras proto-oncogenes were analyzed. Confirmation of mutations was based on restriction fragment length polymorphism analysis and/or oligonucleotide hybridization. Results. Four (6%) of 72 patients with RA, 2 (13%) of 16 with OA, and 1 (8%) of 12 with other arthropathies harbored mutant H-ras proto-oncogenes, and were heterozygous at codon 13 for the GGT→GAT (Gly→Asp) change. An unexpected mutation was found in the H-ras gene, in which a heterozygous GTG→ATG (Val→Met) mutation was observed over codon 14. The incidence for this mutation was 39% (28 of 72) in RA patients, 94% (15 of 16) in OA patients, and 42% (5 of 12) in patients with other arthropathies. All samples carrying the codon 13 mutation of H-ras were also codon 14-mutated, i.e., double mutations existed. Identical point mutations were also detected in a few synovial specimens obtained from cadavers (n = 8), including a single case of double mutation. All specimens showed normal K- and N-ras loci. Conclusion. Activation of proto-oncogene H-ras by point mutation in codons 13 and 14 occurred in the synovial tissue of patients with RA, OA, or other arthropathies, as well as, to some extent, in the control synovia, indicating that the phenomenon is not specific for RA. In codon 14, incidence of the H-ras point mutation was highest in OA tissue. The possible significance of this codon 14-mutated H-ras gene needs to be clarified.