The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.     

AT1R-CB₁R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II

The mechanism of G protein-coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero-oligomers facilitates signal integration of different receptor types, cross-talk between Gαi- and Gαq-coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi-coupled type I cannabinoid receptor (CB(1)R) and the Gαq-coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co-immunoprecipitation and resonance energy transfer assays, as well as receptor- and heteromer-selective antibodies, we show that AT1R and CB(1)R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol-administered rats in which CB(1)R is upregulated. We found a significant upregulation of AT1R-CB(1)R heteromers and enhancement of angiotensin II-mediated signalling, as compared with cells from control animals. Moreover, blocking CB(1)R activity prevented angiotensin II-mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer-dependent signal integration in pathology.     

Effects on sleep and dopamine levels of microdialysis perfusion of cannabidiol into the lateral hypothalamus of rats

AimsThe major non-psychoactive component of Cannabis sativa, cannabidiol (CBD), displays a plethora of actions including wakefulness. In the present study, we addressed whether perfusing CBD via microdialysis into lateral hypothalamus (LH) during the lights-on period would modify the sleep-wake cycle of rats as well as the contents of dopamine (DA) collected from nucleus accumbens (AcbC). Additionally, we tested whether perfusion of CBD into LH would block the sleep rebound after a sleep deprivation period.Main methodsElectroencephalogram and electromyogram electrodes were implanted in rats as well as a guide-cannula aimed to LH or AcbC. CBD perfusion was carried out via cannulae placed into LH whereas contents of DA were collected from AcbC and analyzed using HPLC means.Key findingsWe found that microdialysis perfusion of CBD (30, 60, or 90 nM) into LH of rat enhances alertness and suppresses sleep. This effect was accompanied with an increase in DA extracellular levels collected from the AcbC. Furthermore, perfusion of CBD into LH after total sleep deprivation prevented the sleep rebound.SignificanceThese findings enhance the investigation about the neurobiological properties of CBD on sleep modulation.     

Extended-spectrum Beta-lactamase gene sequences in gram-negative saprophytes on retail organic and nonorganic spinach

A substantial proportion of infections caused by drug-resistant Gram-negative bacteria (GNB) in community and health care settings are recognized to be caused by evolutionarily related GNB strains. Their global spread has been suggested to occur due to human activities, such as food trade and travel. These multidrug-resistant GNB pathogens often harbor mobile drug resistance genes that are highly conserved in their sequences. Because they appear across different GNB species, these genes may have origins other than human pathogens. We hypothesized that saprophytes in common human food products may serve as a reservoir for such genes. Between July 2007 and April 2008, we examined 25 batches of prepackaged retail spinach for cultivatable GNB population structure by 16S rRNA gene sequencing and for antimicrobial drug susceptibility testing and the presence of extended-spectrum beta-lactamase (ESBL) genes. We found 20 recognized GNB species among 165 (71%) of 231 randomly selected colonies cultured from spinach. Twelve strains suspected to express ESBLs based on resistance to cefotaxime and ceftazidime were further examined for bla(CTX-M) and bla(TEM) genes. We found a 712-bp sequence in Pseudomonas teessidea that was 100% identical to positions 10 to 722 of an 876-bp bla(CTX-M-15) gene of an E. coli strain. Additionally, we identified newly recognized ESBL bla(RAHN-2) sequences from Rahnella aquatilis. These observations demonstrate that saprophytes in common fresh produce can harbor drug resistance genes that are also found in internationally circulating strains of GNB pathogens; such a source may thus serve as a reservoir for drug resistance genes that ultimately enter pathogens to affect human health.     

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein–protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.     

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB1/2 receptors.     

Parahippocampal cortex: translating vision into space

Two recent imaging studies have shed new light on information representation in human parahippocampal cortex. Despite their different approaches, the two studies both support the view that this brain region represents space at an elementary level.     

pTransgenesis: a cross-species, modular transgenesis resource

As studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.