Self-association of model transmembrane alpha-helices is modulated by lipid structure

We have developed a fluorescence quenching method using peptides containing 3,5-dibromotryrosine to measure oligomerization of model transmembrane alpha-helices in lipid bilayers. Peptides of the type Ac-LysLysGlyLeu(m)XLeu(n)LysLysAla-amide where X is tryptophan or 3,5-dibromotyrosine were found to form heterodimers in bilayers of phosphatidylcholine in the liquid-crystalline phase. The free energy of dimer formation changed little with increasing number of Leu residues from 16 to 22 but increased with increasing phospholipid fatty acyl chain length, with a slope of about 0.5 kJ mol(-1) per fatty acyl chain carbon. Peptides were excluded from lipid in the gel phase, resulting in increased levels of oligomerization. Addition of cholesterol to form the liquid-ordered state led to increased dimerization but without phase separation. The presence of phosphatidylethanolamine had little effect on dimerization.     

In vivo modification of porin activity conferring antibiotic resistance to Enterobacter aerogenes

Cephalosporins are widely used in chemotherapy of bacterial infections and resistance mechanisms seriously impair their antibacterial activity. Several resistant strains of Enterobacter aerogenes, a frequently isolated nosocomial pathogen, were analyzed. One isolate exhibited a strong modification of the porin antigenic pattern, especially with an immunological probe directed against an epitope located inside the pore lumen. A strong decrease of cefepime uptake was evidenced for this isolate, similarly to ones observed for porin-deficient strains: these kinetics show a serious alteration of the channel properties which may support cephalosporin resistance. This is the first E. aerogenes isolate using such adaptive response which defines an original enterobacterial answer to cephalosporin.     

Membrane permeability modifications are involved in antibiotic resistance in Klebsiella pneumoniae

Two Klebsiella pneumoniae strains selected according to their high cross-resistance pattern to cephalosporins were characterized by (i) outer membrane protein content such as OmpA or nonspecific porins, (ii) MICs of various cephalosporins and unrelated antibiotics, (iii) beta-lactamase production, and (iv) active efflux of fluoroquinolones. An association of porin deficiency and beta-lactamase production induced a noticeable cephalosporin resistance. In addition to these mechanisms, the presence of an active efflux participating in high-level fluoroquinolone resistance was identified in one strain. The decrease of antibiotic uptake associated with efflux explains the Klebsiella adaptation against the drugs present in the environment.     

Characterization of a class III peroxidase from Artemisia annua: relevance to artemisinin metabolism and beyond

Key message

A class III peroxidase from Artemisia annua has been shown to indicate the possibility of cellular localization-based role diversity, which may have implications in artemisinin catabolism as well as lignification.

Abstract

Artemisia annua derives its importance from the antimalarial artemisinin. The –O–O– linkage in artemisinin makes peroxidases relevant to its metabolism. Earlier, we identified three peroxidase-coding genes from A. annua, whereby Aa547 showed higher expression in the low-artemisinin plant stage whereas Aa528 and Aa540 showed higher expression in the artemisinin-rich plant stage. Here we carried out tertiary structure homology modelling of the peroxidases for docking studies. Maximum binding affinity for artemisinin was shown by Aa547. Further, Aa547 showed greater binding affinity for post-artemisinin metabolite, deoxyartemisinin, as compared to pre-artemisinin metabolites (dihydroartemisinic hydroperoxide, artemisinic acid, dihydroartemisinic acid). It also showed significant binding affinity for the monolignol, coniferyl alcohol. Moreover, Aa547 expression was related inversely to artemisinin content and directly to total lignin content as indicated by its transient silencing and overexpression in A. annua. Artemisinin reduction assay also indicated inverse relationship between Aa547 expression and artemisinin content. Subcellular localization using GFP fusion suggested that Aa547 is peroxisomal. Nevertheless, dual localization (intracellular/extracellular) of Aa547 could not be ruled out due to its effect on both, artemisinin and lignin. Taken together, this indicates possibility of localization-based role diversity for Aa547, which may have implications in artemisinin catabolism as well as lignification in A. annua.

     

Encapsulation of moss buds: an efficient method for the in vitro conservation and regeneration of the endangered moss <Emphasis Type="Italic">Splachnum ampullaceum</Emphasis>

In vitro culture techniques are usually employed for ex situ conservation of endangered plant species. However, encapsulation to preserve threatened bryophytes is scarcely used, and only as a pretreatment prior to cryopreservation. In our study, two different methods of germplasm conservation, involving calcium-alginate encapsulation of moss material, were assessed. The plant material used was gametophyte buds (gametophores) of Splachnum ampullaceum Hedw., a rare species of moss. Moss regeneration was evaluated at different periods of time to examine the efficacy of the technique for moss germplasm conservation. The effects of encapsulation and cold storage on developmental parameters such as protonematal colony diameter, bud length, and number of buds were also studied. The results suggest that moss encapsulation with no prior treatment may be a suitable method for germplasm conservation during long periods of time. With our method survival rates as high as 50% could be reached after 2.5 years of cold storage versus 0% of 24-h cryopreserved beads. This technique together with cold storage, avoiding freezing, may be especially important in desiccation intolerant mosses.     

Airway Surface Dehydration Aggravates Cigarette Smoke-Induced Hallmarks of COPD in Mice

Introduction

Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown.

Objective

We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the β-subunit of the epithelial Na⁺ channel (βENaC).

Methods

βENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured.

Results

Airway surface dehydration in βENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in βENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements.

Conclusions

We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD.     

Comparison of Microbiomes from Different Niches of Upper and Lower Airways in Children and Adolescents with Cystic Fibrosis

Changes in the airway microbiome may be important in the pathophysiology of chronic lung disease in patients with cystic fibrosis. However, little is known about the microbiome in early cystic fibrosis lung disease and the relationship between the microbiomes from different niches in the upper and lower airways. Therefore, in this cross-sectional study, we examined the relationship between the microbiome in the upper (nose and throat) and lower (sputum) airways from children with cystic fibrosis using next generation sequencing. Our results demonstrate a significant difference in both α and β-diversity between the nose and the two other sampling sites. The nasal microbiome was characterized by a polymicrobial community while the throat and sputum communities were less diverse and dominated by a few operational taxonomic units. Moreover, sputum and throat microbiomes were closely related especially in patients with clinically stable lung disease. There was a high inter-individual variability in sputum samples primarily due to a decrease in evenness linked to increased abundance of potential respiratory pathogens such as Pseudomonas aeruginosa. Patients with chronic Pseudomonas aeruginosa infection exhibited a less diverse sputum microbiome. A high concordance was found between pediatric and adult sputum microbiomes except that Burkholderia was only observed in the adult cohort. These results indicate that an adult-like lower airways microbiome is established early in life and that throat swabs may be a good surrogate in clinically stable children with cystic fibrosis without chronic Pseudomonas aeruginosa infection in whom sputum sampling is often not feasible.