Solution structure of NOD1 CARD and mutational analysis of its interaction with the CARD of downstream kinase RICK

NOD1 is a cytosolic signalling host pattern-recognition receptor composed of a caspase-activating and recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NOD) and leucine-rich repeats. It plays a crucial role in innate immunity by activating the NF-kappaB pathway via its downstream effector the kinase RICK (RIP2) following the recognition of a specific bacterial ligand. RICK is recruited by NOD1 through interaction of their respective CARDs. Here we present the high resolution NMR structure of the NOD1 CARD. It is generally similar to other CARDs of known structure, consisting of six tightly packed helices, although the length and orientation of the last helix is unusual. Mutations in both the NOD1 and RICK CARD domains were assayed by immuno-precipitation of cell lysates and in vivo NF-kappaB activation in order to define residues important for CARD-CARD interaction and downstream signalling. The results show that the interaction is critically dependent on three acidic residues on NOD1 CARD and three basic residues on RICK CARD and thus is likely to have a strong electrostatic component, similar to other characterised CARD-CARD interactions.     

Trogocytosis-based generation of suppressive NK cells

Trogocytosis is a fast uptake of membranes and associated molecules from one cell by another. Trogocytosis between natural killer (NK) cells and tumors is already described, but the functional relevance of NK-tumor targets material exchange is unclear. We investigated whether the immunosuppressive molecule HLA-G that is commonly expressed by tumors in vivo and known to block NK cytolytic function, could be transferred from tumor cells to NK cells, and if this transfer had functional consequences. We show that activated NK cells acquire HLA-G1 from tumor cells, and that upon this acquisition, NK cells stop proliferating, are no longer cytotoxic, and behave as suppressor cells. Such cells can inhibit other NK cells' cytotoxic function and protect NK-sensitive tumor cells from cytolysis. These data are the first demonstration that trogocytosis of HLA-G1 can be a major mechanism of immune escape that acts through effector cells made to act as suppressor cells locally, temporarily, but efficiently. The broader consequences of membrane sharing between immune and non-immune cells on the function of effectors and the outcome of immune responses are discussed.     

Pandemic influenza due to pH1N1/2009 virus: estimation of infection burden in Reunion Island through a prospective serosurvey, austral winter 2009

Background

To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season.

Methodology/Principal Findings

A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive.

Conclusions

Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.     

A simple cryogenic holder for tensile testing of soft biological tissues

To overcome the difficulty of gripping soft biological materials for tensile test, a simple inexpensive cryogenic holder was developed which allows rapid (3 min) preparation of samples. It is made of 6 parts, built in a bakelite cloth, which is an excellent thermal isolant, and is used with rectangular (8x10(-2)x10(-2)x10(-2)m) samples. The holder with the sample inside is completely immersed in liquid nitrogen for 50 s. This duration allows the freezing of the sample ends on a 10(-2)m length and gives a very flat freezing surface throughout the sample cross section. The 6x10(-2)m central part of the sample remained at ambient temperature. Two parts of the holder help maintain the sample until its ends are vertically gripped in the tensile machine thus avoiding any sample deformation during this step. No pressure was applied on the frozen part of the sample by grips of the tensile machine and this avoids breaks in this region. The sample is fixed by adhesion forces (>1 kN) between its frozen parts and 2 pieces of the holder. The procedure has been successfully tested with bovine and salmon muscle samples and results show tensile breaks randomly distributed in the unfrozen region of the samples. Particular attention has been paid to obtain a very flat freezing surface so that the axial strain is equal throughout the sample and therefore any strain-related mechanical parameters can be accurately determined. The dimensions of the holder can be easily modified to fit other sample geometries and can be used with other biological materials.     

Formation of isodialuric acid lesion within DNA oligomers via one-electron oxidation of 5-hydroxyuracil: characterization, stability and excision repair

5-Hydroxyuracil is a major oxidized nucleobase that can be generated by the action of ^•OH radical and one-electron oxidants. The latter modified base that exhibits a low ionization potential is highly susceptible to further degradation upon exposure to various oxidants. Emphasis was placed in thiswork on the formation and characterization of one-electron oxidation products of 5-hydroxyuracil within DNA fragments of defined sequence. For this purpose, 5-hydroxyuracil containing single- and double-stranded oligonucleotides of various lengths were synthesized and then exposed to the oxidizing action of iridium salts. Isodialuric acid was found to be formed almost quantitatively by a one-electron oxidation mechanism for which relevant information was inferred from a freeze-quenched ESR study. Information on the stability of isodialuric acid thus formed and its conversion products in aqueous solutions was also gained from experiments performed at acidic, neutral and alkali pH’s. Moreover, biochemical features dealing with the substrate specificity of several bacterial and yeast base excision repair enzymes to remove isodialuric acid from site-specifically modified DNA fragments were determined.     

Aerodynamics of cyclist posture, bicycle and helmet characteristics in time trial stage

The present work is focused on the aerodynamic study of different parameters, including both the posture of a cyclist's upper limbs and the saddle position, in time trial (TT) stages. The aerodynamic influence of a TT helmet large visor is also quantified as a function of the helmet inclination. Experiments conducted in a wind tunnel on nine professional cyclists provided drag force and frontal area measurements to determine the drag force coefficient. Data statistical analysis clearly shows that the hands positioning on shifters and the elbows joined together are significantly reducing the cyclist drag force. Concerning the saddle position, the drag force is shown to be significantly increased (about 3%) when the saddle is raised. The usual helmet inclination appears to be the inclination value minimizing the drag force. Moreover, the addition of a large visor on the helmet is shown to provide a drag coefficient reduction as a function of the helmet inclination. Present results indicate that variations in the TT cyclist posture, the saddle position and the helmet visor can produce a significant gain in time (up to 2.2%) during stages.     

Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.     

A structural analysis of the catalytic mechanism of methionine sulfoxide reductase A from Neisseria meningitidis

The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product—methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.