Hans Hebert

The gut microbiota influences blood-brain barrier permeability in mice

The intestinal microbiota helps to maintain the integrity of the blood-brain barrier in fetal and adult mice. The Gut Microbiota and the Blood-Brain Barrier The blood-brain barrier is an important gateway that controls the passage of molecules and nutrients in and out of the brain. An intact blood-brain barrier is a crucial checkpoint for appropriate development and function of the brain. Braniste et al. now show that germ-free pregnant dams, devoid of maternal microbes, have offspring that show increased permeability of the blood-brain barrier. This elevated permeability was also observed in adult germ-free mice. However, fecal transplants from mice exposed to bacteria into adult germ-free mice reduced blood-brain barrier permeability, possibly through the regulation of tight junction proteins. These findings suggest that crosstalk between the gut microbiota and the brain, initiated during the intrauterine period, is perpetuated throughout life. Pivotal to brain development and function is an intact blood-brain barrier (BBB), which acts as a gatekeeper to control the passage and exchange of molecules and nutrients between the circulatory system and the brain parenchyma. The BBB also ensures homeostasis of the central nervous system (CNS). We report that germ-free mice, beginning with intrauterine life, displayed increased BBB permeability compared to pathogen-free mice with a normal gut flora. The increased BBB permeability was maintained in germ-free mice after birth and during adulthood and was associated with reduced expression of the tight junction proteins occludin and claudin-5, which are known to regulate barrier function in endothelial tissues. Exposure of germ-free adult mice to a pathogen-free gut microbiota decreased BBB permeability and up-regulated the expression of tight junction proteins. Our results suggest that gut microbiota–BBB communication is initiated during gestation and propagated throughout life.

Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype

Ileal mucus in CftrΔ508 mice is more adherent, denser, and less penetrable than that of WT mice, but addition of bicarbonate normalizes the properties of CftrΔ508 mucus.

Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin

MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D′D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. The MUC2-N aggregates were dissolved by removing Ca2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

Structural basis for induced formation of the inflammatory mediator prostaglandin E2

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH2 into PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH2 to PGE2 after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.

The projection structure of Perfringolysin O (Clostridium perfringens θ‐toxin)

The cytolysin Perfringolysin O was applied to lipid layers and the obtained ring‐shaped oligomers analyzed by electron microscopy and image processing. The final result shows the periodic repeat of 2.4 nm along the outer rim of the ring. The asymmetric protein unit, corresponding to one monomer, spans the ring from the convex to the concave surface. It shows a clear protein peak close to the outer radius and less density in the middle of the oligomer. The number of monomers in the average ring is 50, and the inner radius of the aggregate is approximately 15 nm.

Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-1

Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E2. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E2 production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substrate PGH2, or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.

Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support Multimodal Imaging

Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.

Two-dimensional crystals of membrane-bound gastric H,K-ATPase

H,K‐ATPase protomer. Through stain‐deficient contact regions four adjacent protein units were connected forming a tetrameric structure.

Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure

The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus–cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N- and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.