Novel structural insights into rotavirus recognition of ganglioside glycan receptors

Rotaviruses ubiquitously infect children under the age of 5, being responsible for more than half a million diarrhoeal deaths each year worldwide. Host cell oligosaccharides containing sialic acid(s) are critical for attachment by rotaviruses. However, to date, no detailed three-dimensional atomic model showing the exact rotavirus interactions with these glycoconjugate receptors has been reported. Here, we present the first crystallographic structures of the rotavirus carbohydrate-recognizing protein VP8^? in complex with ganglioside G_M3 glycans. In combination with assessment of the inhibition of rotavirus infectivity by N-acetyl and N-glycolyl forms of this ganglioside, our results reveal key details of rotavirus-ganglioside G_M3 glycan recognition. In addition, they show a direct correlation between the carbohydrate specificities exhibited by VP8^? from porcine and by monkey rotaviruses and the respective infectious virus particles. These novel results also indicate the potential binding interactions of rotavirus VP8^? with other sialic acid-containing gangliosides.     

Circuit-Host Coupling Induces Multifaceted Behavioral Modulations of a Gene Switch

Quantitative modeling of gene circuits is fundamentally important to synthetic biology, as it offers the potential to transform circuit engineering from trial-and-error construction to rational design and, hence, facilitates the advance of the field. Currently, typical models regard gene circuits as isolated entities and focus only on the biochemical processes within the circuits. However, such a standard paradigm is getting challenged by increasing experimental evidence suggesting that circuits and their host are intimately connected, and their interactions can potentially impact circuit behaviors. Here we systematically examined the roles of circuit-host coupling in shaping circuit dynamics by using a self-activating gene switch as a model circuit. Through a combination of deterministic modeling, stochastic simulation, and Fokker-Planck equation formalism, we found that circuit-host coupling alters switch behaviors across multiple scales. At the single-cell level, it slows the switch dynamics in the high protein production regime and enlarges the difference between stable steady-state values. At the population level, it favors cells with low protein production through differential growth amplification. Together, the two-level coupling effects induce both quantitative and qualitative modulations of the switch, with the primary component of the effects determined by the circuit’s architectural parameters. This study illustrates the complexity and importance of circuit-host coupling in modulating circuit behaviors, demonstrating the need for a new paradigm—integrated modeling of the circuit-host system—for quantitative understanding of engineered gene networks.     

Spatiotemporal analysis of mycolactone distribution in vivo reveals partial diffusion in the central nervous system

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone’s diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin’s access to the brain.Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection.Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization.     

PHOTOSYNTHETIC RESPONSE OF NATURAL ASSEMBLAGES OF MARINE BENTHIC MICROALGAE TO SHORT-AND LONG-TERM VARIATIONS OF INCIDENT IRRADIANCE IN BAFFIN BAY,TEXAS1

This study was designed to understand the high variability characterizing primary production rates of microphytobenthos. The photosynthetic efficiency (α^B) and photosynthetic capacity (P^B_max) of the microphytobenthos were measured at different times of the day on two different dates (8 May and 7 July 1990). In July, unusually low light conditions were caused by the development of a brown tide (chrysophytes). Both light-limited and light-saturated photosynthesis changed at hourly and monthly scales. There was a linear relationship between α^B and P^B_max, suggesting a common response to environmental factors [α^B= 0.0075(±0.00063)·P^B_max+ 0.00097(±0.0071), R²= 0.94]. Incident irradiance at the sediment-water interface was the primary physical factor that explained variability of both α^B (84%) and P^B_max (92%). Temperature had a negative but minor effect that explained an extra 8% and 2% of the variance, respectively. There was no diel rhythm of α^B and P^B_max and incident irradiance was regulated by wind-induced currents. Therefore, microphytobenthos photosynthesis seemed to be primarily controlled by wind events in Baffin Bay.     

Synthesis and evaluation of alkenyl indazoles as selective Aurora kinase inhibitors

A series of alkenyl indazoles were synthesized and evaluated in Aurora kinase enzyme assays. Several promising leads were optimized for selectivity towards Aurora B. Excellent binding affinity and good selectivity were achieved with optimized compounds in isolated Aurora subfamily assays.     

Isolation and Purification of Colon Lamina Propria Dendritic Cells from Mice with Colitis

Dendritic cells are prime antigen presenting cells for stimulation of T cell immune responses. These cells are present in trace amounts in normal tissue. At sites of disease the increased frequency of these cells interacting with T cells may provide the basis for the release of pro-inflammatory mediators and contribute to localised cell and tissue damage. Studies on dendritic cells in the colon lamina propria of inflammatory bowel disease (IBD) mice have been limited due to the difficulties encountered in the isolation and purification of sufficient numbers of these cells. This is the first detailed, reproducible method provided in the literature for the isolation of colon lamina propria dendritic cells from mice with colitis, yielding optimum purity of cells and sufficient numbers to advance the study of dendritic cell function in the colons of mice. The most frequently used identification marker of murine DC is the CD11c surface antigen. We have adapted, combined, and improved procedures developed for the isolation of other cell types, to develop an efficient procedure for the isolation of dendritic cells from colon tissue. This protocol describes a step-by-step method for optimising the purity and recovery of lamina propria CD11c+ dendritic cells from mice colons.     

RP 67580, a potent and selective substance P non-peptide antagonist]

The pharmacological properties of 7,7-Diphenyl-2 [1-imino-2 (2-methoxy-phenyl)-ethyl] perhydroisoindol-4-one (3 aR, 7 aR) or RP67580 are described. This compound, derived from a novel chemical family, is a potent and selective substance P (SP) antagonist, in vitro and in vivo. In vitro, it inhibited in a competitive manner (IC50 = 10 nM) 3H-SP binding in rat brain (NK1 receptors). It did not interact with the two other tachykinin receptor sites (NK2 and NK3) nor the other receptor sites tested. Moreover, RP67580 competitively antagonized the contractile activity of SP on guinea-pig ileum (pA2 = 7.16); in contrast, it was inactive in rabbit pulmonary artery and in rat portal vein tissues which contain NK2 and NK3 receptors, respectively. In vivo, in the rat, RP67580 inhibited the plasmatic extravasation induced by administration of SP (ED50 = 0.04 mg/kg i.v.) as well as that induced by antidromic stimulation of a peripheral sensory nerve (ED50 = 0.15 mg/kg i.v.). In mice and rats, RP67580, like morphine, potently blocked the nociceptive effects of phenylbenzoquinone and formalin; its antinociceptive effect does not involve opiate receptors since it was not reversed by naloxone. These results indicate that RP67580 is a particularly valuable tool for investigating the physiological and pathological role of SP.     

Kinetic mechanism and nucleotide specificity of NADH peroxidase

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [14C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.