The structure of the O-antigen in the endotoxin of the emerging food pathogen Cronobacter (Enterobacter) muytjensii strain 3270

Strains of the Gram-negative bacterium Cronobacter (formerly known as Enterobacter) sakazakii have been identified as emerging opportunistic pathogens that can cause enterocolitis, bacteraemia, meningitis, and brain abscess, and they have been particularly associated with meningitis in neonates where infant milk formulae have been epidemiologically linked to the disease. A study of the lipopolysaccharides produced by clinical isolates using chemical, 2D ¹H and ¹³C NMR, and MS methods revealed that the O-polysaccharide produced by Cronobactermuytjensii strain 3270, isolated from powdered infant formula from Denmark, was a linear unbranched polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-d-galactose (d-GalNAc), 2-acetamido-2-deoxy-d-glucose (d-GlcNAc), 3-acetamido-3-deoxy-d-quinovose (d-Qui3NAc), l-rhamnose (l-Rha), and d-glucuronic acid (d-GlcA) in equimolar ratio, and has the structureThe specific structural characteristics of the O-polysaccharides of C.muytjensii may be of value in the identification and tracking of the bacterial pathogen.     

Mitochondrial Reactive Oxygen Species Mediate GPCR–induced TACE/ADAM17-dependent Transforming Growth Factor-α Shedding

Epidermal growth factor receptor (EGFR) activation by GPCRs regulates many important biological processes. ADAM metalloprotease activity has been implicated as a key step in transactivation, yet the regulatory mechanisms are not fully understood. Here, we investigate the regulation of transforming growth factor-α (TGF-α) shedding by reactive oxygen species (ROS) through the ATP-dependent activation of the P2Y family of GPCRs. We report that ATP stimulates TGF-α proteolysis with concomitant EGFR activation and that this process requires TACE/ADAM17 activity in both murine fibroblasts and CHO cells. ATP-induced TGF-α shedding required calcium and was independent of Src family kinases and PKC and MAPK signaling. Moreover, ATP-induced TGF-α shedding was completely inhibited by scavengers of ROS, whereas calcium-stimulated shedding was partially inhibited by ROS scavenging. Hydrogen peroxide restored TGF-α shedding after calcium chelation. Importantly, we also found that ATP-induced shedding was independent of the cytoplasmic NADPH oxidase complex. Instead, mitochondrial ROS production increased in response to ATP and mitochondrial oxidative complex activity was required to activate TACE-dependent shedding. These results reveal an essential role for mitochondrial ROS in regulating GPCR-induced growth factor shedding.     

Developmental Trajectories of Girls' BMI Across Childhood and Adolescence

This study describes qualitatively distinct trajectories of BMI change among girls participating in a longitudinal study of non‐Hispanic, white girls (n = 182) and their parents, assessed at daughters' ages 5, 7, 9, 11, 13, and 15 years. Height, weight, body fat, fasting blood glucose and lipids, blood pressure, waist circumference, and pubertal status were measured, and participants self‐reported dietary, physical activity, and television (TV) viewing patterns. Growth mixture models were used to model heterogeneity in girls' BMI trajectories over 10 years. Statistical support was strongest for four distinct BMI trajectories: (i) upward percentile crossing (UPC; n = 25, 14%); (ii) delayed downward percentile crossing (DDPC; n = 37, 20%); (iii) 60th percentile tracking (60PT; n = 52, 29%); and (iv) 50th percentile tracking (50PT; n = 68, 37%). Girls in the UPC group had more metabolic risk factors at age 15 years, even after adjusting for concurrent weight status. Girls in the UPC group had mothers with the highest BMIs at study entry and were breast‐fed for a shorter duration. This novel approach for examining differences in growth trajectories revealed four distinct BMI trajectories that predicted adolescent metabolic health outcomes in girls. The present study provides support for BMI monitoring in girls and for the potential utility of combining data on BMI tracking with data on familial characteristics for the early identification of girls at elevated risk for obesity and metabolic syndrome.     

Smoking Is Associated with Shortened Airway Cilia

Background

Whereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6–7 µm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers.

Methodology/Principal Findings

Cilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers.

Conclusions/Significance

Models predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease.     

Potencies of Cocaine Methiodide on Major Cocaine Targets in Mice

Cocaine methiodide (CM), a charged cocaine analog, cannot pass the blood brain barrier. It has been assumed the effects of systemic CM represent cocaine actions in peripheral tissues. However, the IC₅₀ values of CM have not been clearly determined for the major cocaine targets: dopamine, norepinephrine, and serotonin transporters, and sodium channels. Using cells transfected with individual transporters from mice and synaptosomes from mouse striatum tissues, we observed that the inhibition IC₅₀ values for monoamine uptake by CM were 31-fold to 184-fold higher compared to cocaine at each of the transporters. In dorsal root ganglion neurons, cocaine inhibited sodium channels with an apparent IC₅₀ of 75 µM, while CM showed no observable effect at concentrations up to 3 mM. These results indicate that an equal dose of CM will not produce an equivalent peripheral effect of cocaine.     

Replisome trafficking in growing vegetative hyphae of Streptomyces coelicolor A3(2)

We observed movies of replisome trafficking during Streptomyces coelicolor growth. A replisome(s) in the spore served as a replication center(s) until hyphae reached a certain length, when a tip-proximal replisome formed and moved at a fixed distance behind the tip at a speed equivalent to the extension rate of the tip.     

Contributions of A2A and A2B adenosine receptors in coronary flow responses in relation to the KATP channel using A2B and A2A/2B double-knockout mice

Adenosine plays a role in physiological and pathological conditions, and A(2) adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A(2B)AR and its relationship to the A(2A)AR in coronary flow (CF) changes using A(2B) single-knockout (KO) and A(2A/2B) double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A(2A)KO and A(2B)KO and A(2A/2B)DKO mice. BAY 60-6583 (a selective A(2B) agonist) had no effect on CF in A(2B)KO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min(-1)·g(-1)). 5'-N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A(2B)KO mice (maximum of 34.6 ± 4.7 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Also, CGS-21680 (a selective A(2A) agonist) increased CF in A(2B)KO mice (maximum of 29 ± 1.9 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min(-1)·g(-1)). SCH-58261 (an A(2A)-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A(2B)KO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min(-1)·g(-1)) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min(-1)·g(-1)). NECA did not induce any increase in CF in A(2A/2B)DKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Furthermore, the mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker 5-hydroxydecanoate had no effect on the NECA-induced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min(-1)·g(-1)), A(2A)KO (12.5 ± 2.3 ml·min(-1)·g(-1)), and A(2B)KO (16.2 ± 0.8 ml·min(-1)·g(-1)) mice was significantly blunted by the K(ATP) channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min(-1)·g(-1), respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min(-1)·g(-1)) and BAY 60-6583-induced (16.4 ± 1.60 ml·min(-1)·g(-1)) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min(-1)·g(-1), respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A(2A)ARs in A(2B)KO mice and demonstrates that both A(2A)ARs and A(2B)ARs induce CF changes through K(ATP) channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders.     

EphrinA/EphA‐induced ectodomain shedding of neural cell adhesion molecule regulates growth cone repulsion through ADAM10 metalloprotease

EphrinA/EphA‐dependent axon repulsion is crucial for synaptic targeting in developing neurons but downstream molecular mechanisms remain obscure. Here, it is shown that ephrinA5/EphA3 triggers proteolysis of the neural cell adhesion molecule (NCAM) by the metalloprotease a disintegrin and metalloprotease (ADAM)10 to promote growth cone collapse in neurons from mouse neocortex. EphrinA5 induced ADAM10 activity to promote ectodomain shedding of polysialic acid‐NCAM in cortical neuron cultures, releasing a ~ 250 kDa soluble fragment consisting of most of its extracellular region. NCAM shedding was dependent on ADAM10 and EphA3 kinase activity as shown in HEK293T cells transfected with dominant negative ADAM10 and kinase‐inactive EphA3 (K653R) mutants. Purified ADAM10 cleaved NCAM at a sequence within the E‐F loop of the second fibronectin type III domain (Leu⁶⁷¹‐Lys⁶⁷²/Ser⁶⁷³‐Leu⁶⁷⁴) identified by mass spectrometry. Mutations of NCAM within the ADAM10 cleavage sequence prevented EphA3‐induced shedding of NCAM in HEK293T cells. EphrinA5‐induced growth cone collapse was dependent on ADAM10 activity, was inhibited in cortical cultures from NCAM null mice, and was rescued by WT but not ADAM10 cleavage site mutants of NCAM. Regulated proteolysis of NCAM through the ephrin5/EphA3/ADAM10 mechanism likely impacts synapse development, and may lead to excess NCAM shedding when disrupted, as implicated in neurodevelopmental disorders such as schizophrenia.

     

Flow‐mediated plasticity in the expression of stickleback nesting glue genes

Nest construction is an essential component of the reproductive behavior of many species, and attributes of nests – including their location and structure – have implications for both their functional capacity as incubators for developing offspring, and their attractiveness to potential mates. To maximize reproductive success, nests must therefore be suited to local environmental conditions. Male three‐spined sticklebacks (Gasterosteus aculeatus) build nests from collected materials and use an endogenous, glue‐like multimeric protein – “spiggin” – as an adhesive. Spiggin is encoded by a multigene family, and differential expression of spiggin genes potentially allows plasticity in nest construction in response to variable environments. Here, we show that the expression of spiggin genes is affected significantly by both the flow regime experienced by a fish and its nesting status. Further, we show the effects of flow on expression patterns are gene‐specific. Nest‐building fish exhibited consistently higher expression levels of the three genes under investigation (Spg‐a, Spg‐1, and Spg‐2) than non‐nesting controls, irrespective of rearing flow treatment. Fish reared under flowing‐water conditions showed significantly increased levels of spiggin gene expression compared to those reared in still water, but this effect was far stronger for Spg‐a than for Spg‐1 or Spg‐2. The strong effect of flowing water on Spg‐a expression, even among non‐nesters, suggests that the increased production of spiggin – or of spiggin rich in the component contributed by Spg‐a – may allow more rapid and/or effective nest construction under challenging high flow conditions.     

Both CP15 and CP25 are left as trails behind gliding sporozoites of Cryptosporidium parvum (Apicomplexa)

Abstract Sporozoites of Cryptosporidium parvum were examined after gliding upon glass microscope slides using monoclonal antibodies to the 15 and 25 kDa surface molecules and immunogold-silver enhancement. Both antibodies bound to surface antigen deposited as trails behind parasites, suggesting that both surface molecules are involved in substrate attachment.