Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody

Elicitation of potent and broadly neutralizing antibodies is an important goal in designing an effective human immunodeficiency virus-1 (HIV-1) vaccine. The HIV-1 gp41 inner-core trimer represents a functionally and structurally conserved target for therapeutics. Here we report the 2.0-A-resolution crystal structure of the complex between the antigen-binding fragment of D5, an HIV-1 cross-neutralizing antibody, and 5-helix, a gp41 inner-core mimetic. Both binding and neutralization depend on residues in the D5 CDR H2 loop protruding into the conserved gp41 hydrophobic pocket, as well as a large pocket in D5 surrounding core gp41 residues. Kinetic analysis of D5 mutants with perturbed D5-gp41 interactions suggests that D5 persistence at the fusion intermediate is crucial for neutralization. Thus, our data validate the gp41 N-peptide trimer fusion intermediate as a target for neutralizing antibodies and provide a template for identification of more potent and broadly neutralizing molecules.     

Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study

The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259-264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance.     

Evidence for Sub-Haplogroup H5 of Mitochondrial DNA as a Risk Factor for Late Onset Alzheimer's Disease

Background

Alzheimer''s Disease (AD) is the most common neurodegenerative disease and the leading cause of dementia among senile subjects. It has been proposed that AD can be caused by defects in mitochondrial oxidative phosphorylation. Given the fundamental contribution of the mitochondrial genome (mtDNA) for the respiratory chain, there have been a number of studies investigating the association between mtDNA inherited variants and multifactorial diseases, however no general consensus has been reached yet on the correlation between mtDNA haplogroups and AD.

Methodology/Principal Findings

We applied for the first time a high resolution analysis (sequencing of displacement loop and restriction analysis of specific markers in the coding region of mtDNA) to investigate the possible association between mtDNA-inherited sequence variation and AD in 936 AD patients and 776 cognitively assessed normal controls from central and northern Italy. Among over 40 mtDNA sub-haplogroups analysed, we found that sub-haplogroup H5 is a risk factor for AD (OR = 1.85, 95% CI:1.04–3.23) in particular for females (OR = 2.19, 95% CI:1.06–4.51) and independently from the APOE genotype. Multivariate logistic regression revealed an interaction between H5 and age. When the whole sample is considered, the H5a subgroup of molecules, harboring the 4336 transition in the tRNA^Gln gene, already associated to AD in early studies, was about threefold more represented in AD patients than in controls (2.0% vs 0.8%; p = 0.031), and it might account for the increased frequency of H5 in AD patients (4.2% vs 2.3%). The complete re-sequencing of the 56 mtDNAs belonging to H5 revealed that AD patients showed a trend towards a higher number (p = 0.052) of sporadic mutations in tRNA and rRNA genes when compared with controls.

Conclusions

Our results indicate that high resolution analysis of inherited mtDNA sequence variation can help in identifying both ancient polymorphisms defining sub-haplogroups and the accumulation of sporadic mutations associated with complex traits such as AD.     

Tracing the evolutionary history of the mole,Talpa europaea,through mitochondrial DNA phylogeography and species distribution modelling

Our understanding of the effect of Pleistocene climatic changes on the biodiversity of European mammals mostly comes from phylogeographical studies of non‐subterranean mammals, whereas the influence of glaciation cycles on subterranean mammals has received little attention. The lack of data raises the question of how and to what extent the current amount and distribution of genetic variation in subterranean mammals is the result of Pleistocene range contractions/expansions. The common mole (Talpa europaea) is a strictly subterranean mammal, widespread across Europe, and represents one of the best candidates for studying the influence of Quaternary climatic oscillation on subterranean mammals. Cytochrome b sequences, as obtained from a sampling covering the majority of the distribution area, were used to evaluate whether Pleistocene climate change influenced the evolution of T. europaea and left a trace in the genetic diversity comparable to that observed in non‐subterranean small mammals. Subsequently, we investigated the occurrence of glacial refugia by comparing the results of phylogeographical analysis with species distribution modelling. We found three differentiated mitochondrial DNA lineages: two restricted to Spain and Italy and a third that was widespread across Europe. Phylogenetic inferences and the molecular clock suggest that the Spanish moles represent a highly divergent and ancient lineage, highlighting for the first time the paraphyly of T. europaea. Furthermore, our analyses suggest that the genetic break between the Italian and the European lineages predates the last glacial phase. Historical demography and spatial principal component analysis further suggest that the Last Glacial Maximum left a signature both in the Italian and in the European lineages. Genetic data combined with species distribution models support the presence of at least three putative glacial refugia in southern Europe (France, Balkan Peninsula and Black Sea) during thelast glacial maximum that likely contributed to post‐glacial recolonization of Europe. By contrast, the Italian lineage remained trapped in the Italian peninsula and, according to the pattern observed in other subterranean mammals, did not contribute to the recolonization of northern latitudes. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 495–512.     

Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells

Background

The elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases.

Methods

Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance.

Results

We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction.

Conclusion

Our findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma.     

Mitogenome sequence comparison in the endangered congeneric Pinna nobilis and Pinna rudis bivalves

Molecular Biology Reports - The pen shells Pinna nobilis and Pinna rudis are large wedge-shaped bivalve molluscs. Both species are threatened by different anthropogenic pressures. In the last few...