Development of Mass Spectrometry Imaging on skeletal muscle to characterize the local pro-inflammatory and pro-resolution lipid responses in a vaccination context.

Vaccine reactogenicity is well documented at the clinical level but the mechanism involved at the local or systemic level are still poorly understood. Muscular tissue where most vaccines are administered is the first place of interaction between the vaccine formulation and the host's immune cells. So far, this site of vaccine administration is not well documented from a mechanistic standpoint. The study of early molecular events at the injection site is crucial to understand the local response to vaccines. In this paper, we report a standardized workflow, from the injection of vaccine formulations in rabbit muscle, to the analysis by desorption electrospray ionization and histology staining to understand the role of lipids involved in the inflammation and its resolution on striated muscular tissue. The analysis of lipid mediators was optimized at the site of needle insertion to allow the spatial comparison of cellular infiltrates at the injection site. We showed that lipids were distributed across the spatial tissue morphology in a time-dependent manner. The MS imaging applied to vaccinology could pave the way to a better understanding of vaccine reactogenicity and mechanism of action.

Deep longitudinal multi-omics analysis of Bordetella pertussis cultivated in bioreactors highlights medium starvations and transitory metabolisms, associated to vaccine antigen biosynthesis variations and global virulence regulation.

Bordetella pertussis is the bacterial causative agent of whooping cough, a serious respiratory illness. An extensive knowledge on its virulence regulation and metabolism is a key factor to ensure pertussis vaccine manufacturing process robustness. The aim of this study was to refine our comprehension of B. pertussis physiology during in vitro cultures in bioreactors. A longitudinal multi-omics analysis was carried out over 26 h small-scale cultures of B. pertussis. Cultures were performed in batch mode and under culture conditions intending to mimic industrial processes. Putative cysteine and proline starvations were, respectively, observed at the beginning of the exponential phase (from 4 to 8 h) and during the exponential phase (18 h 45 min). As revealed by multi-omics analyses, the proline starvation induced major molecular changes, including a transient metabolism with internal stock consumption. In the meantime, growth and specific total PT, PRN, and Fim2 antigen productions were negatively affected. Interestingly, the master virulence-regulating two-component system of B. pertussis (BvgASR) was not evidenced as the sole virulence regulator in this in vitro growth condition. Indeed, novel intermediate regulators were identified as putatively involved in the expression of some virulence-activated genes (vags). Such longitudinal multi-omics analysis applied to B. pertussis culture process emerges as a powerful tool for characterization and incremental optimization of vaccine antigen production.

Compaction of RNA Hairpins and Their Kissing Complexes in Native Electrospray Mass Spectrometry.

When electrosprayed from typical native MS solution conditions, RNA hairpins and kissing complexes acquire charge states at which they get significantly more compact in the gas phase than their initial structure in solution. Here, we also show the limits of using force field molecular dynamics to interpret the structures of nucleic acid complexes in the gas phase, as the predicted CCS distributions do not fully match the experimental ones. We suggest that higher level calculation levels should be used in the future.

Electrostatics Explains the Position-Dependent Effect of G⋠U Wobble Base Pairs on the Affinity of RNA Kissing Complexes.

In the RNA realm, non-Watson-Crick base pairs are abundant and can affect both the RNA 3D structure and its function. Here, we investigated the formation of RNA kissing complexes in which the loop-loop interaction is modulated by non-Watson-Crick pairs. Mass spectrometry, surface plasmon resonance, and UV-melting experiments show that the G⋅U wobble base pair favors kissing complex formation only when placed at specific positions. We tried to rationalize this effect by molecular modeling, including molecular mechanics Poisson-Boltzmann surface area (MMPBSA) thermodynamics calculations and PBSA calculations of the electrostatic potential surfaces. Modeling reveals that the G⋅U stabilization is due to a specific electrostatic environment defined by the base pairs of the entire loop-loop region. The loop is not symmetric, and therefore the identity and position of each base pair matters. Predicting and visualizing the electrostatic environment created by a given sequence can help to design specific kissing complexes with high affinity, for potential therapeutic, nanotechnology or analytical applications.

Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity.

Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.

Insights into the preferential order of strand exchange in the Cre/loxP recombinase system: impact of the DNA spacer flanking sequence and flexibility.

The Cre/loxP system is widely used as a genetic tool to manipulate DNA. Cre recombinase catalyzes site-specific recombination between 34 bp loxP sites. Each loxP site is recognized by two Cre molecules assuming a cleaving (CreC) and non-cleaving (CreNC) activity. Despite the symmetry in the sequences of the arms of loxP, available biochemical data show strong evidence that the recombination reaction is asymmetric with a preferred strand exchange order. The asymmetry comes from the spacer separating the two sets of palindromic arms of the loxP sequence. However, it remains to be understood how this preferential order is established. We apply computational structure-based methods and perform a thorough detailed analysis of available structural and biochemical information on the Cre/loxP system in order to investigate such asymmetry in the recombination, and we propose a rationale to explain the determinants favoring the strand exchange order. We show that the structural properties of the DNA flanking sequence of the spacer guide the recombination, and we establish the role of residues R118, R121 and K122 from CreC, which contact the spacer region and by clamping the DNA inhibit the cleavage on the second arm of loxP. Our studies give an atomistic insight on the synapsis state of the recombination process in the Cre/loxP system and highlight the importance of the flexibility and other intrinsic properties of the flanking regions of the DNA spacer to establish a preferential strand exchange order.

Universal Tre (uTre) recombinase specifically targets the majority of HIV-1 isolates.

Current drugs against HIV can suppress the progression to AIDS but cannot clear the patient from the virus. Because of potential side effects of these drugs and the possible development of drug resistance, finding a cure for HIV infection remains a high priority of HIV/AIDS research. We recently generated a recombinase (termed Tre) tailored to efficiently eradicate the provirus from the host genome of HIV-1 infected cells by specifically targeting a sequence that is present in the long terminal repeats (LTRs) of the viral DNA [1]. In vivo analyses in HIV-infected humanized mice demonstrated highly significant antiviral effects of Tre recombinase [2]. However, the fact that Tre recognizes a particular HIV-1 subtype A strain may limit its broad therapeutic application. To advance our Tre-based strategy towards a universally efficient cure, we have engineered a new, universal recombinase (uTre) applicable to the majority of HIV-1 infections by the various virus strains and subtypes. We employed the search tool SeLOX [3] in order to find a well-conserved HIV-1 proviral sequence that could serve as target site for a universal Tre from sequences compiled in the Los Alamos HIV Sequence Database. We selected a candidate (termed loxLTRu) with a mean conservation rate of 94% throughout the major HIV-1 subtype groups A, B and C. We applied loxLTRu as substrate in our established substrate-linked protein evolution (SLiPE) process [4] and evolved the uTre recombinase in 142 evolution cycles. Highly specific enzymatic activity on loxLTRu is demonstrated for uTre in both Escherichia coli and human cells. Naturally occurring viral variants with single mutations within the loxLTRu sequence are also shown to be efficiently targeted by uTre, further increasing the range of applicability of the recombinase. Potential off-target sites in the human genome are not recombined by uTre. Furthermore, uTre expression in primary human T cells shows no obvious Tre-related cytopathic or genotoxic effects. Finally, uTre expressing mice show no undesired phenotypes during their normal lifespan. We have developed a broad-range HIV-1 LTR specific recombinase that has the potential to be effective against the vast majority of HIV-1 strains and to cure HIV-1 infected cells from the infection. These results strongly encouraged us in our confidence that a Tre recombinase-mediated HIV eradication strategy may become a valuable component of a future therapy for HIV-infected patients.

Nucleic acid ion structures in the gas phase.

Nucleic acids are diverse polymeric macromolecules that are essential for all life forms. These biomolecules possess a functional three-dimensional structure under aqueous physiological conditions. Mass spectrometry-based approaches have on the other hand opened the possibility to gain structural information on nucleic acids from gas-phase measurements. To correlate gas-phase structural probing results with solution structures, it is therefore important to grasp the extent to which nucleic acid structures are preserved, or altered, when transferred from the solution to a fully anhydrous environment. We will review here experimental and theoretical approaches available to characterize the structure of nucleic acids in the gas phase (with a focus on oligonucleotides and higher-order structures), and will summarize the structural features of nucleic acids that can be preserved in the gas phase on the experiment time scale.

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach.

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre's enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.

Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system.

Targeted genome engineering has become an important research area for diverse disciplines, with site-specific recombinases (SSRs) being among the most popular genome engineering tools. Their ability to trigger excision, integration, inversion and translocation has made SSRs an invaluable tool to manipulate DNA in vitro and in vivo. However, sophisticated strategies that combine different SSR systems are ever increasing. Hence, the demand for additional precise and efficient recombinases is dictated by the increasing complexity of the genetic studies. Here, we describe a novel site-specific recombination system designated Vika/vox. Vika originates from a degenerate bacteriophage of Vibrio coralliilyticus and shares low sequence similarity to other tyrosine recombinases, but functionally carries out a similar type of reaction. We demonstrate that Vika is highly specific in catalyzing vox recombination without recombining target sites from other SSR systems. We also compare the recombination activity of Vika/vox with other SSR systems, providing a guideline for deciding on the most suitable enzyme for a particular application and demonstrate that Vika expression does not cause cytotoxicity in mammalian cells. Our results show that Vika/vox is a novel powerful and safe instrument in the 'genetic toolbox' that can be used alone or in combination with other SSRs in heterologous hosts.

How cations can assist DNase I in DNA binding and hydrolysis.

DNase I requires Ca²+ and Mg²+ for hydrolyzing double-stranded DNA. However, the number and the location of DNase I ion-binding sites remain unclear, as well as the role of these counter-ions. Using molecular dynamics simulations, we show that bovine pancreatic (bp) DNase I contains four ion-binding pockets. Two of them strongly bind Ca²+ while the other two sites coordinate Mg²+. These theoretical results are strongly supported by revisiting crystallographic structures that contain bpDNase I. One Ca²+ stabilizes the functional DNase I structure. The presence of Mg²+ in close vicinity to the catalytic pocket of bpDNase I reinforces the idea of a cation-assisted hydrolytic mechanism. Importantly, Poisson-Boltzmann-type electrostatic potential calculations demonstrate that the divalent cations collectively control the electrostatic fit between bpDNase I and DNA. These results improve our understanding of the essential role of cations in the biological function of bpDNase I. The high degree of conservation of the amino acids involved in the identified cation-binding sites across DNase I and DNase I-like proteins from various species suggests that our findings generally apply to all DNase I-DNA interactions.

DNA structures from phosphate chemical shifts.

For B-DNA, the strong linear correlation observed by nuclear magnetic resonance (NMR) between the (31)P chemical shifts (deltaP) and three recurrent internucleotide distances demonstrates the tight coupling between phosphate motions and helicoidal parameters. It allows to translate deltaP into distance restraints directly exploitable in structural refinement. It even provides a new method for refining DNA oligomers with restraints exclusively inferred from deltaP. Combined with molecular dynamics in explicit solvent, these restraints lead to a structural and dynamical view of the DNA as detailed as that obtained with conventional and more extensive restraints. Tests with the Jun-Fos oligomer show that this deltaP-based strategy can provide a simple and straightforward method to capture DNA properties in solution, from routine NMR experiments on unlabeled samples.

Sequence-dependent DNA flexibility mediates DNase I cleavage.

Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3' phosphate linkage flexibility, monitored by (31)P chemical shifts. Examining NMR-refined DNA structures highlighted that sequence-dependent flexible phosphates were associated with large minor groove variations that may promote the affinity of DNase I, according to relevant DNA-protein complexes. In sum, this work demonstrates that specificity in DNA-DNase I interaction is mediated by DNA flexibility, which influences the induced-fit transitions required to form productive complexes.