Differential Gene Expression Profiles and Selected Cytokine Protein Analysis of Mediastinal Lymph Nodes of Horses with Chronic Recurrent Airway Obstruction (RAO) Support an Interleukin-17 Immune Response

Recurrent airway obstruction (RAO) is a pulmonary inflammatory condition that afflicts certain mature horses exposed to organic dust particulates in hay. Its clinical and pathological features, manifested by reversible bronchoconstriction, excessive mucus production and airway neutrophilia, resemble the pulmonary alterations that occur in agricultural workers with occupational asthma. The immunological basis of RAO remains uncertain although its chronicity, its localization to a mucosal surface and its domination by a neutrophilic, non-septic inflammatory response, suggest involvement of Interleukin-17 (IL-17). We examined global gene expression profiles in mediastinal (pulmonary-draining) lymph nodes isolated from RAO-affected and control horses. Differential expression of > 200 genes, coupled with network analysis, supports an IL-17 response centered about NF-κB. Immunohistochemical analysis of mediastinal lymph node sections demonstrated increased IL-17 staining intensity in diseased horses. This result, along with the finding of increased IL-17 concentrations in lymph node homogenates from RAO-affected horses (P = 0.1) and a down-regulation of IL-4 gene and protein expression, provides additional evidence of the involvement of IL-17 in the chronic stages of RAO. Additional investigations are needed to ascertain the cellular source of IL-17 in this equine model of occupational asthma. Understanding the immunopathogenesis of this disorder likely will enhance the development of therapeutic interventions beneficial to human and animal pulmonary health.     

A BLOC-1–AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers

Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1–dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3–BLOC-1 super-complex.     

Genomic divergence in sympatry indicates strong reproductive barriers and cryptic species within Eucalyptus salubris

Genetic studies are increasingly detecting cryptic taxa that likely represent a significant component of global biodiversity. However, cryptic taxa are often criticized because they are typically detected serendipitously and may not receive the follow‐up study required to verify their geographic or evolutionary limits. Here, we follow‐up a study of Eucalyptus salubris that unexpectedly detected two divergent lineages but was not sampled sufficiently to make clear interpretations. We undertook comprehensive sampling for an independent genomic analysis (3,605 SNPs) to investigate whether the two purported lineages remain discrete genetic entities or if they intergrade throughout the species’ range. We also assessed morphological and ecological traits, and sequenced chloroplast DNA. SNP results showed strong genome‐wide divergence (F _ST = 0.252) between two discrete lineages: one dominated the north and one the southern regions of the species’ range. Within lineages, gene flow was high, with low differentiation (mean F _ST = 0.056) spanning hundreds of kilometers. In the central region, the lineages were interspersed but maintained their genomic distinctiveness: an indirect demonstration of reproductive isolation. Populations of the southern lineage exhibited significantly lower specific leaf area and occurred on soils with lower phosphorus relative to the northern lineage. Finally, two major chloroplast haplotypes were associated with each lineage but were shared between lineages in the central distribution. Together, these results suggest that these lineages have non‐contemporary origins and that ecotypic adaptive processes strengthened their divergence more recently. We conclude that these lineages warrant taxonomic recognition as separate species and provide fascinating insight into eucalypt speciation.     

The helminth product,ES-62, protects against airway inflammation by resetting the Th cell phenotype

We previously demonstrated inhibition of ovalbumin-induced allergic airway hyper-responsiveness in the mouse using ES-62, a phosphorylcholine-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae. This inhibition correlated with ES-62-induced mast cell desensitisation, although the degree to which this reflected direct targeting of mast cells remained unclear as suppression of the Th2 phenotype of the inflammatory response, as measured by eosinophilia and IL-4 levels in the lungs, was also observed. We now show that inhibition of the lung Th2 phenotype is reflected in ex vivo analyses of draining lymph node recall cultures and accompanied by a decrease in the serum levels of total and ovalbumin-specific IgE. Moreover, ES-62 also suppresses the lung infiltration by neutrophils that is associated with severe asthma and is generally refractory to conventional anti-inflammatory therapies, including steroids. Protection against Th2-associated airway inflammation does not reflect induction of regulatory T cell responses (there is no increased IL-10 or Foxp3 expression) but rather a switch in polarisation towards increased Tbet expression and IFNγ production. This ES-62-driven switch in the Th1/Th2 balance is accompanied by decreased IL-17 responses, a finding in line with reports that IFNγ and IL-17 are counter-regulatory. Consistent with ES-62 mediating its effects via IFNγ-mediated suppression of pathogenic Th2/Th17 responses, we found that neutralising anti-IFNγ antibodies blocked protection against airway inflammation in terms of pro-inflammatory cell infiltration, particularly by neutrophils, and lung pathology. Collectively, these studies indicate that ES-62, or more likely small molecule analogues, could have therapeutic potential in asthma, in particular for those subtypes of patients (e.g. smokers, steroid-resistant) who are refractory to current treatments.     

Elongation Factor P is Dispensable in Escherichia coli and Pseudomonas aeruginosa

3-Hydroxypropionic acid (3-HP) is a commercially important platform chemical from which a panel of chemicals can be generated. Klebsiella pneumoniae has been regarded as a promising host strain in glycerol-based 3-HP production for its exceptional ability to metabolize glycerol. Since the glycerol dissimilation mechanism governs the carbon flux distribution from glycerol, inducible strong promoters were usually employed to enhance the glycerol consumption and 3-HP production. Here, we report an alternative strategy that the native promoter of dhaB gene was applied to enhance 3-HP production in K. pneumoniae. The key enzyme genes (ald4 and dhaB) for 3-HP biosynthesis were co-expressed under this promoter. Metabolic analysis revealed that the 3-HP formation was partially coupled with cell metabolism. To optimize the production of 3-HP, the effects of glucose as energy source assistant were investigated based on the analysis of fermentation process kinetics. The highest 3-HP yield (3.77 g/L in flask) was observed upon optimized conditions. Since there were no additional inducers needed, the strategy of employing native promoter seems more feasible to industrial application. More importantly, the employment of constitutive promoter demonstrated an effective approach for decoupling the natural correlation between respiratory metabolism and glycerol dissimilation in K. pneumoniae.     

Macrophage Glucose-6-Phosphate Dehydrogenase Stimulates Proinflammatory Responses with Oxidative Stress

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that regulates cellular redox potential. In this study, we demonstrate that macrophage G6PD plays an important role in the modulation of proinflammatory responses and oxidative stress. The G6PD levels in macrophages in the adipose tissue of obese animals were elevated, and G6PD mRNA levels positively correlated with those of proinflammatory genes. Lipopolysaccharide (LPS) and free fatty acids, which initiate proinflammatory signals, stimulated macrophage G6PD. Overexpression of macrophage G6PD potentiated the expression of proinflammatory and pro-oxidative genes responsible for the aggravation of insulin sensitivity in adipocytes. In contrast, when macrophage G6PD was inhibited or suppressed via chemical inhibitors or small interfering RNA (siRNA), respectively, basal and LPS-induced proinflammatory gene expression was attenuated. Furthermore, macrophage G6PD increased activation of the p38 mitogen-activated protein kinase (MAPK) and NF-κB pathways, which may lead to a vicious cycle of oxidative stress and proinflammatory cascade. Together, these data suggest that an abnormal increase of G6PD in macrophages promotes oxidative stress and inflammatory responses in the adipose tissue of obese animals.     

Cancer screening behaviors among smokers and non-smokers

Objective: We explored whether smoking is associated with cancer screening behaviors. Methods: We used data from the 2007 Florida Behavioral Risk Factor Surveillance System and the Florida Tobacco Callback Survey to examine screening behaviors related to four cancer types (breast, cervical, prostate, and colorectal). Using multiple logistic regression analyses, we examined the association between smoking status and health screening behaviors. Results: For 10 of the 11 cancer screening variables, being a current smoker was significantly associated with being less likely to ever have been screened and also less likely to be compliant with screening guidelines. For breast and cervical cancer, level of nicotine dependence was also significantly related to compliance with screening recommendations; women with higher levels of dependence were less likely to be compliant. Conclusions: Our results support the notion that individuals’ actions related to their health are consistent across different types of behaviors. We found that smokers were less likely to engage in cancer screening behaviors. In addition, among smokers, individuals with greater nicotine dependence had lower compliance with some screening tests. Physicians should ensure that their patients who smoke are receiving appropriate and adequate screening for cancer.     

Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes

A mutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype "chocolate" (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38(G19V) is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles.     

Glial regulation of the axonal membrane at nodes of Ranvier

Action potential conduction in myelinated nerve fibers depends on a polarized axonal membrane. Voltage-gated Na(+) and K(+) channels are clustered at nodes of Ranvier and mediate the transmembrane currents necessary for rapid saltatory conduction. Paranodal junctions flank nodes and function as attachment sites for myelin and as paracellular and membrane protein diffusion barriers. Common molecular mechanisms, directed by myelinating glia, are used to establish these axonal membrane domains. Initially, heterophilic interactions between glial and axonal cell adhesion molecules define the locations where nodes or paranodes form. Subsequently, within each domain, axonal cell adhesion molecules are stabilized and retained through interactions with cytoskeletal and scaffolding proteins, including ankyrins and spectrins.