Presynaptic to postsynaptic relationships of the neuromuscular junction are held constant across age and muscle fiber type

The neuromuscular junction (NMJ) displays considerable morphological plasticity as a result of differences in activity level, as well as aging. This is true of both presynaptic and postsynaptic components of the NMJ. Yet, despite these variations in NMJ structure, proper presynaptic to postsynaptic coupling must be maintained in order for effective cell‐to‐cell communication to occur. Here, we examined the NMJs of muscles with different activity profiles (soleus and EDL), on both slow‐ and fast‐twitch fibers in those muscles, and among young adult and aged animals. We used immunofluorescent techniques to stain nerve terminal branching, presynaptic vesicles, postsynaptic receptors, as well as fast/slow myosin heavy chain. Confocal microscopy was used to capture images of NMJs for later quantitative analysis. Data were subjected to a two‐way ANOVA (main effects for myofiber type and age), and in the event of a significant (p < 0.05) F ratio, a post hoc analysis was performed to identify pairwise differences. Results showed that the NMJs of different myofiber types routinely displayed differences in presynaptic and postsynaptic morphology (although the effect on NMJ size was reversed in the soleus and the EDL), but presynaptic to postsynaptic relationships were tightly maintained. Moreover, the ratio of presynaptic vesicles relative to nerve terminal branch length also was similar despite differences in muscles, their fiber type, and age. Thus, in the face of considerable overall structural differences of the NMJ, presynaptic to postsynaptic coupling remains constant, as does the relationship between presynaptic vesicles and the nerve terminal branches that support them. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 744–753, 2013     

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.     

Model-Based Assessment of Estuary Ecosystem Health Using the Latent Health Factor Index,with Application to the Richibucto Estuary

The ability to quantitatively assess ecological health is of great interest to those tasked with monitoring and conserving ecosystems. For decades, biomonitoring research and policies have relied on multimetric health indices of various forms. Although indices are numbers, many are constructed based on qualitative procedures, thus limiting the quantitative rigor of the practical interpretations of such indices. The statistical modeling approach to construct the latent health factor index (LHFI) was recently developed. With ecological data that otherwise are used to construct conventional multimetric indices, the LHFI framework expresses such data in a rigorous quantitative model, integrating qualitative features of ecosystem health and preconceived ecological relationships among such features. This hierarchical modeling approach allows unified statistical inference of health for observed sites (along with prediction of health for partially observed sites, if desired) and of the relevance of ecological drivers, all accompanied by formal uncertainty statements from a single, integrated analysis. Thus far, the LHFI approach has been demonstrated and validated in a freshwater context. We adapt this approach to modeling estuarine health, and illustrate it on the previously unassessed system in Richibucto in New Brunswick, Canada, where active oyster farming is a potential stressor through its effects on sediment properties. Field data correspond to health metrics that constitute the popular AZTI marine biotic index and the infaunal trophic index, as well as abiotic predictors preconceived to influence biota. Our paper is the first to construct a scientifically sensible model that rigorously identifies the collective explanatory capacity of salinity, distance downstream, channel depth, and silt–clay content–all regarded a priori as qualitatively important abiotic drivers–towards site health in the Richibucto ecosystem. This suggests the potential effectiveness of the LHFI approach for assessing not only freshwater systems but aquatic ecosystems in general.     

KCa3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma

Background

The Ca²⁺-activated K⁺ channel K_Ca3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K_Ca3.1 modifies experimental asthma in sheep.

Methodology and Principal Findings

Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K_Ca3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.

Conclusions

These findings suggest that K_Ca3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K_Ca3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.     

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.     

Erectile Dysfunction Severity as a Risk Marker for Cardiovascular Disease Hospitalisation and All-Cause Mortality: A Prospective Cohort Study

Background

Erectile dysfunction is an emerging risk marker for future cardiovascular disease (CVD) events; however, evidence on dose response and specific CVD outcomes is limited. This study investigates the relationship between severity of erectile dysfunction and specific CVD outcomes.

Methods and Findings

We conducted a prospective population-based Australian study (the 45 and Up Study) linking questionnaire data from 2006–2009 with hospitalisation and death data to 30 June and 31 Dec 2010 respectively for 95,038 men aged ≥45 y. Cox proportional hazards models were used to examine the relationship of reported severity of erectile dysfunction to all-cause mortality and first CVD-related hospitalisation since baseline in men with and without previous CVD, adjusting for age, smoking, alcohol consumption, marital status, income, education, physical activity, body mass index, diabetes, and hypertension and/or hypercholesterolaemia treatment. There were 7,855 incident admissions for CVD and 2,304 deaths during follow-up (mean time from recruitment, 2.2 y for CVD admission and 2.8 y for mortality). Risks of CVD and death increased steadily with severity of erectile dysfunction. Among men without previous CVD, those with severe versus no erectile dysfunction had significantly increased risks of ischaemic heart disease (adjusted relative risk [RR] = 1.60, 95% CI 1.31–1.95), heart failure (8.00, 2.64–24.2), peripheral vascular disease (1.92, 1.12–3.29), “other” CVD (1.26, 1.05–1.51), all CVD combined (1.35, 1.19–1.53), and all-cause mortality (1.93, 1.52–2.44). For men with previous CVD, corresponding RRs (95% CI) were 1.70 (1.46–1.98), 4.40 (2.64–7.33), 2.46 (1.63–3.70), 1.40 (1.21–1.63), 1.64 (1.48–1.81), and 2.37 (1.87–3.01), respectively. Among men without previous CVD, RRs of more specific CVDs increased significantly with severe versus no erectile dysfunction, including acute myocardial infarction (1.66, 1.22–2.26), atrioventricular and left bundle branch block (6.62, 1.86–23.56), and (peripheral) atherosclerosis (2.47, 1.18–5.15), with no significant difference in risk for conditions such as primary hypertension (0.61, 0.16–2.35) and intracerebral haemorrhage (0.78, 0.20–2.97).

Conclusions

These findings give support for CVD risk assessment in men with erectile dysfunction who have not already undergone assessment. The utility of erectile dysfunction as a clinical risk prediction tool requires specific testing. Please see later in the article for the Editors'' Summary     

Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3⁺ bacteria were present within single-membrane phagosomes that are LAMP1⁺. Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3⁺ bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.