Do Experience and Body Size Play a Role in Responses of Larval Ringed Salamanders,Ambystoma annulatum,to Predator Kairomones? Laboratory and Field Assays

Prey may experience ontogenetic changes in vulnerability to some predators, either because of changes in morphology or experience. If prey match their level of antipredator behavior to the level of predatory threat, prey responses to predators should reflect the appropriate level of threat for their stage of development. For larval salamanders, responses to predators may change with body size because larger larvae are less vulnerable to predation by gape‐limited predators or because fleeing responses by large salamanders may be more effective than for smaller salamanders. In a field experiment, small larval ringed salamanders, Ambystoma annulatum, responded to chemical stimuli (‘kairomones’) from predatory newts, Notophthalmus viridescens, with an antipredator response (decreased activity). Laboratory‐reared larvae decreased their activity following exposure to newt kairomones, indicating that larval ringed salamanders do not require experience with newts to recognize them as predators. In both experiments, larvae distinguished between chemical stimuli from newts and stimuli from tadpoles (non‐predators) and a blank control. In a third experiment, field‐caught (experienced) larvae showed a graded response to newt kairomones based on their body size: small larvae tended to decrease their activity while larger larvae showed no change or an increase in activity. This graded response was not observed for neutral stimuli, indicating that it is predator‐specific. Therefore, ringed salamander larvae exhibit threat‐sensitive ontogenetic changes in their response to chemical stimuli from predatory newts.     

Stroke Damage Is Exacerbated by Nano-Size Particulate Matter in a Mouse Model

This study examines the effects of nano-size particulate matter (nPM) exposure in the setting of murine reperfused stroke. Particulate matter is a potent source of inflammation and oxidative stress. These processes are known to influence stroke progression through recruitment of marginally viable penumbral tissue into the ischemic core. nPM was collected in an urban area in central Los Angeles, impacted primarily by traffic emissions. Re-aerosolized nPM or filtered air was then administered to mice through whole body exposure chambers for forty-five cumulative hours. Exposed mice then underwent middle cerebral artery occlusion/ reperfusion. Following cerebral ischemia/ reperfusion, mice exposed to nPM exhibited significantly larger infarct volumes and less favorable neurological deficit scores when compared to mice exposed to filtered air. Mice exposed to nPM also demonstrated increases in markers of inflammation and oxidative stress in the region of the ischemic core. The findings suggest a detrimental effect of urban airborne particulate matter exposure in the setting of acute ischemic stroke.     

Binding and internalization of heparin by vascular smooth muscle cells

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.     

Linkage analysis of a completely ascertained sample of familial schizophrenics and bipolars from Palau, Micronesia

We report on linkage analysis of a completely ascertained population of familial psychosis derived from the oceanic nation of Palau. Palau, an archipelago of islands in the Southern Pacific, currently has a population of approximately 23,000 individuals. The peoples of Palau populated these islands recently in human history, approximately 2,000 years ago. As both historical and genetic evidence suggest, the population is far more homogeneous than most other populations undergoing genetic studies, and should therefore prove quite useful for mapping genetic variants having a meaningful impact on susceptibility to psychotic disorders. Moreover, for our study, essentially all on-island schizophrenics (150) and individuals with other psychotic disorders (25) participated. By analysis of narrow (only schizophrenia) and broad (all psychosis) diagnostic schemes, two-point linkage analyses suggest that two regions of the genome harbor genetic variants affecting liability in most families, 3q28 (LOD=3.03) and 17q32.2 (LOD=2.80). Results from individual pedigrees also support 2q37.2, 2p14, and 17p13 as potentially harboring important genetic variants. Most of these regions have been implicated in other genetic studies of psychosis in populations physically quite distant from this Oceanic population, although some (e.g., 3q28) appear to be novel results for schizophrenia linkage analyses.     

Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.     

Accurate Distinction of Pathogenic from Benign CNVs in Mental Retardation

Copy number variants (CNVs) have recently been recognized as a common form of genomic variation in humans. Hundreds of CNVs can be detected in any individual genome using genomic microarrays or whole genome sequencing technology, but their phenotypic consequences are still poorly understood. Rare CNVs have been reported as a frequent cause of neurological disorders such as mental retardation (MR), schizophrenia and autism, prompting widespread implementation of CNV screening in diagnostics. In previous studies we have shown that, in contrast to benign CNVs, MR-associated CNVs are significantly enriched in genes whose mouse orthologues, when disrupted, result in a nervous system phenotype. In this study we developed and validated a novel computational method for differentiating between benign and MR-associated CNVs using structural and functional genomic features to annotate each CNV. In total 13 genomic features were included in the final version of a Naïve Bayesian Tree classifier, with LINE density and mouse knock-out phenotypes contributing most to the classifier''s accuracy. After demonstrating that our method (called GECCO) perfectly classifies CNVs causing known MR-associated syndromes, we show that it achieves high accuracy (94%) and negative predictive value (99%) on a blinded test set of more than 1,200 CNVs from a large cohort of individuals with MR. These results indicate that this classification method will be of value for objectively prioritizing CNVs in clinical research and diagnostics.     

Cigarette smoke impairs NK cell-dependent tumor immune surveillance

In this study, we investigated the impact of cigarette smoke on tumor immune surveillance and its consequences to lung tumor burden in a murine lung metastasis model. Cigarette smoke exposure significantly increased the numbers of lung metastases following B16-MO5 melanoma challenge. This effect was reversible; we observed significantly fewer tumor nodules following smoking cessation. Using RAG2(-/-) and RAG2(-/-)gamma(c)(-/-) mice, we provide strong evidence that increased tumor incidence was NK cell dependent. Furthermore, we show that cigarette smoke suppressed NK activation and attenuated NK CTL activity, without apparent effect on activating or inhibitory receptor expression. Finally, activation of NK cells through bone marrow-derived dendritic cells conferred protection against lung metastases in smoke-exposed mice; however, protection was not as efficacious as in sham-exposed mice. To our knowledge, this is the first experimental evidence showing that cigarette smoke impairs NK cell-dependent tumor immune surveillance and that altered immunity is associated with increased tumor burden. Our findings suggest that altered innate immunity may contribute to the increased risk of cancer in smokers.     

Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.     

Multivalent binding and facilitated diffusion account for the formation of the Grb2-Sos1 signaling complex in a cooperative manner

Despite its key role in driving cellular growth and proliferation through receptor tyrosine kinase (RTK) signaling, the Grb2-Sos1 macromolecular interaction remains poorly understood in mechanistic terms. Herein, using an array of biophysical methods, we provide evidence that although the Grb2 adaptor can potentially bind to all four PXψPXR motifs (designated herein S1-S4) located within the Sos1 guanine nucleotide exchange factor, the formation of the Grb2-Sos1 signaling complex occurs with a 2:1 stoichiometry. Strikingly, such bivalent binding appears to be driven by the association of the Grb2 homodimer to only two of four potential PXψPXR motifs within Sos1 at any one time. Of particular interest is the observation that of a possible six pairwise combinations in which S1-S4 motifs may act in concert for the docking of the Grb2 homodimer through bivalent binding, only S1 and S3, S1 and S4, S2 and S4, and S3 and S4 do so, while pairwise combinations of sites S1 and S2 and sites S2 and S3 appear to afford only monovalent binding. This salient observation implicates the role of local physical constraints in fine-tuning the conformational heterogeneity of the Grb2-Sos1 signaling complex. Importantly, the presence of multiple binding sites within Sos1 appears to provide a physical route for Grb2 to hop in a flip-flop manner from one site to the next through facilitated diffusion, and such rapid exchange forms the basis of positive cooperativity driving the bivalent binding of Grb2 to Sos1 with high affinity. Collectively, our study sheds new light on the assembly of a key macromolecular signaling complex central to cellular machinery in health and disease.     

Novel device to sample the esophageal microbiome-the esophageal string test

A growing number of studies implicate the microbiome in the pathogenesis of intestinal inflammation. Previous work has shown that adults with esophagitis related to gastroesophageal reflux disease have altered esophageal microbiota compared to those who do not have esophagitis. In these studies, sampling of the esophageal microbiome was accomplished by isolating DNA from esophageal biopsies obtained at the time of upper endoscopy. The aim of the current study was to identify the esophageal microbiome in pediatric individuals with normal esophageal mucosa using a minimally invasive, capsule-based string technology, the Enterotest?. We used the proximal segment of the Enterotest string to sample the esophagus, and term this the "Esophageal String Test" (EST). We hypothesized that the less invasive EST would capture mucosal adherent bacteria present in the esophagus in a similar fashion as mucosal biopsy. EST samples and mucosal biopsies were collected from children with no esophageal inflammation (n?=?15) and their microbiome composition determined by 16S rRNA gene sequencing. Microbiota from esophageal biopsies and ESTs produced nearly identical profiles of bacterial genera and were different from the bacterial contents of samples collected from the nasal and oral cavity. We conclude that the minimally invasive EST can serve as a useful device for study of the esophageal microbiome.