Unveiling Contacts within Macromolecular Assemblies by Solving Minimum Weight Connectivity Inference (MWC) Problems*

Consider a set of oligomers listing the subunits involved in subcomplexes of a macromolecular assembly, obtained e.g. using native mass spectrometry or affinity purification. Given these oligomers, connectivity inference (CI) consists of finding the most plausible contacts between these subunits, and minimum connectivity inference (MCI) is the variant consisting of finding a set of contacts of smallest cardinality. MCI problems avoid speculating on the total number of contacts but yield a subset of all contacts and do not allow exploiting a priori information on the likelihood of individual contacts. In this context, we present two novel algorithms, MILP-W and MILP-W_B. The former solves the minimum weight connectivity inference (MWCI), an optimization problem whose criterion mixes the number of contacts and their likelihood. The latter uses the former in a bootstrap fashion to improve the sensitivity and the specificity of solution sets.Experiments on three systems (yeast exosome, yeast proteasome lid, human eIF3), for which reference contacts are known (crystal structure, cryo electron microscopy, cross-linking), show that our algorithms predict contacts with high specificity and sensitivity, yielding a very significant improvement over previous work, typically a twofold increase in sensitivity.The software accompanying this paper is made available and should prove of ubiquitous interest whenever connectivity inference from oligomers is faced.     

Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.     

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis

Therapeutic antibodies (Abs) are emerging as major drugs to treat respiratory diseases, and inhalation may provide substantial benefits for their delivery. Understanding the behavior of Abs after pulmonary deposition is critical for their development. We investigated the pharmacokinetics of a nebulized Ab by continuous sampling in lung parenchyma using microdialysis in non-human primates. We defined the optimal conditions for microdialysis of Ab and demonstrated that lung microdialysis of Ab is feasible over a period of several days. The concentration-profile indicated a two-phase non-linear elimination and/or distribution of inhaled mAbX. Lung exposition was higher than the systemic one over a period of 33 hours and above MabX affinity for its target. The microdialysis results were supported by an excellent relationship with dosages from lung extracts.     

PAT-12, a potential anti-nematode target, is a new spectraplakin partner essential for Caenorhabditis elegans hemidesmosome integrity and embryonic morphogenesis

Caenorhabditis elegans embryonic elongation depends on both epidermal and muscle cells. The hemidesmosome-like junctions, commonly called fibrous organelles (FOs), that attach the epidermis to the extracellular matrix ensure muscle anchoring to the cuticular exoskeleton and play an essential role during elongation.To further define how hemidesmosomes might control elongation, we searched for factors interacting with the core hemidesmosome component, the spectraplakin homolog VAB-10. Using the VAB-10 plakin domain as bait in a yeast two-hybrid screen, we identified the novel protein T17H7.4. We also identified T17H7.4 in an independent bioinformatic search for essential nematode-specific proteins that could define novel anti-nematode drug or vaccine targets. Interestingly, T17H7.4 corresponds to the C. elegans equivalent of the parasitic OvB20 antigen, and has a characteristic hemidesmosome distribution. We identified two mutations in T17H7.4, one of which defines the uncharacterized gene pat-12, previously identified in screens for genes required for muscle assembly. Using isoform-specific GFP constructs, we showed that one pat-12 isoform with a hemidesmosome distribution can rescue a pat-12 null allele. We further found that lack of pat-12 affects hemidesmosome integrity, with marked defects at the apical membrane. PAT-12 defines a novel component of C. elegans hemidesmosomes, which is required for maintaining their integrity. We suggest that PAT-12 helps maintaining VAB-10 attachment with matrix receptors.     

CpG promotes cross-presentation of dead cell-associated antigens by pre-CD8α+ dendritic cells [corrected

Cross-presentation of cell-associated Ags by dendritic cells (DC) plays an important role in immunity. DC in lymphoid tissues are short lived, being continuously replaced by precursors that proliferate and differentiate locally. Paradoxically, although TLR ligands promote immune responses and stimulate DC replenishment, they impair the cross-priming capacity of terminally differentiated splenic CD8α(+) DC, the major subset involved in cross-priming. In this study, we have investigated the cross-presentation capacity of newly generated murine DC and especially immediate precursors of CD8α(+) DC. We show that these DC do not cross-present Ag from dead cells unless stimulated by TLR ligands before Ag capture. TLR ligand CpG induced the expression of costimulatory molecules required for CD8 T cell activation but also regulated the intracellular mechanisms of cross-presentation such as Ag degradation rates without regulating Ag uptake. GM-CSF, an inflammatory cytokine associated with infections, also promoted cross-presentation acquisition by pre-CD8α(+) DC and synergized with TLR9 ligand. The concept that TLR ligands as well as inflammatory cytokines promote the acquisition of cross-presenting properties by pre-CD8α(+) DC has important implications during immune responses and when considering the use of these cells for vaccination.     

Insights into substrate gating in H. influenzae rhomboid

Rhomboids are a remarkable class of serine proteases that are embedded in lipid membranes. These membrane-bound enzymes play key roles in cellular signaling events, and disruptions in these events can result in numerous disease pathologies, including hereditary blindness, type 2 diabetes, Parkinson's disease, and epithelial cancers. Recent crystal structures of rhomboids from Escherichia coli have focused on how membrane-bound substrates gain access to a buried active site. In E. coli, it has been shown that movements of loop 5, with smaller movements in helix 5 and loop 4, act as substrate gate, facilitating inhibitor access to rhomboid catalytic residues. Herein we present a new structure of the Haemophilus influenzae rhomboid hiGlpG, which reveals disorder in loop 5, helix 5, and loop 4, indicating that, together, they represent mobile elements of the substrate gate. Substrate cleavage assays by hiGlpG with amino acid substitutions in these mobile regions demonstrate that the flexibilities of both loop 5 and helix 5 are important for access of the substrates to the catalytic residues. Mutagenesis indicates that less mobility by loop 4 is required for substrate cleavage. A reexamination of the reaction mechanism of rhomboid substrates, whereby cleavage of the scissile bond occurs on the si-face of the peptide bond, is discussed.     

Temporal dynamics of exchangeable K, Ca and Mg in acidic bulk soil and rhizosphere under Norway spruce (Picea abies Karst.) and beech (Fagus sylvatica L.) stands

Anthropogenic nitrogen (N) deposition significantly affects forest soil microbial biomass and extracellular enzymatic activities (EEA). However, the influence of mixed N fertilizations on soil microbial biomass and EEA remains unclear. In this work, NH₄NO₃ was chosen as inorganic N, while urea and glycine were chosen as organic N. They were used to fertilize subtropical forest soil monthly for 1 year with different ratios (inorganic N : organic N?=?10 : 0, 7 : 3, 3 : 7 and 1 : 9 respectively.) and N inputs were equivalent to 7.2 g?N?m^?2?y^?1. Soil samples were harvested every 2 months. Subsequently, soil microbial biomass and enzymatic activities were assayed. Multiple regression analysis (MRA) and principle components analysis (PCA) were utilized to illustrate the relationship between soil microbial biomass and EEA. Results showed that soil EEA displayed different changes in response to various mixed N fertilizations. Invertase, cellulase, cellobiohydrolase, alkaline phosphatase, and catalase activities under mixed N fertilization were higher than those of single inorganic N (NH₄NO₃) fertilization. Polyphenol oxidase activities were depressed after inorganic N fertilization and accelerated after mixed N fertilization. Acid phosphatase activities were accelerated in all N fertilization plots, while the influence of various mixed N fertilizations were not significant. Soil microbial biomass was enhanced by mixed N fertilization, while no significant changes were observed after inorganic N fertilization. The result revealed that although N fertilization may alleviate soil N-limitation, single inorganic N fertilization may disturb the balance of inorganic N and organic N, and depress the increases of soil enzymatic activities and microbial biomass in the end. Soil enzymes activities and microbial biomass showed the highest activities after medium organic N fertilization (inorganic : organic N?=?3 : 7), which might be the most suitable N fertilizer for soil microbes. Meanwhile, PCA showed that the alleviation of N-limited reached a maximum after medium organic N fertilization. All results indicated that soil EEA, microbial biomass, and their relationship are all affected by N type and inorganic to organic N ratio.