Probing vocal fold fibroblast response to hyaluronan in 3D contexts

A number of treatments are being investigated for vocal fold (VF) scar, including designer implants. The aim of the present study was to validate a 3D model system for probing the effects of various bioactive moieties on VF fibroblast (VFF) behavior toward rational implant design. We selected poly(ethylene glycol) diacrylate (PEGDA) hydrogels as our base‐scaffold due to their broadly tunable material properties. However, since cells encapsulated in PEGDA hydrogels are generally forced to take on rounded/stellate morphologies, validation of PEGDA gels as a 3D VFF model system required that the present work directly parallel previous studies involving more permissive scaffolds. We therefore chose to focus on hyaluronan (HA), a polysaccharide that has been a particular focus of the VF community. Toward this end, porcine VFFs were encapsulated in PEGDA hydrogels containing consistent levels of high M _w HA (${\rm HA}_{{\rm H}{M}_{\rm W} } $ ), intermediate M_w HA (${\rm HA}_{{\rm I}{M}_{\rm W} } $ ), or the control polysaccharide, alginate, and cultured for 7 and 21 days. ${\rm HA}_{{\rm H}{M}_{\rm W} } $ promoted sustained increases in active ERK1/2 relative to ${\rm HA}_{{\rm I}{M}_{\rm W} } $ . Furthermore, VFFs in ${\rm HA}_{{\rm I}{M}_{\rm W} } $ gels displayed a more myofibroblast‐like phenotype, higher elastin production, and greater protein kinase C (PkC) levels at day 21 than VFFs in ${\rm HA}_{{\rm H}{M}_{\rm W} } $ and alginate gels. The present results are in agreement with a previous 3D study of VFF responses to ${\rm HA}_{{\rm I}{M}_{\rm W} } $ relative to alginate in collagen‐based scaffolds permissive of cell elongation, indicating that PEGDA hydrogels may serve as an effective 3D model system for probing at least certain aspects of VFF behavior. Biotechnol. Bioeng. 2009; 104: 821–831 © 2009 Wiley Periodicals, Inc.     

EVOLUTIONARY POTENTIAL OF A LARGE MARINE VERTEBRATE: QUANTITATIVE GENETIC PARAMETERS IN A WILD POPULATION

Estimating quantitative genetic parameters ideally takes place in natural populations, but relatively few studies have overcome the inherent logistical difficulties. For this reason, no estimates currently exist for the genetic basis of life-history traits in natural populations of large marine vertebrates. And yet such estimates are likely to be important given the exposure of this taxon to changing selection pressures, and the relevance of life-history traits to population productivity. We report such estimates from a long-term (1995–2007) study of lemon sharks ( Negaprion brevirostris ) conducted at Bimini, Bahamas. We obtained these estimates by genetically reconstructing a population pedigree (117 dams, 487 sires, and 1351 offspring) and then using an "animal model" approach to estimate quantitative genetic parameters. We find significant additive genetic (co)variance, and hence moderate heritability, for juvenile length and mass. We also find substantial maternal effects for these traits at age-0, but not age-1, confirming that genotype–phenotype interactions between mother and offspring are strongest at birth; although these effects could not be parsed into their genetic and nongenetic components. Our results suggest that human-imposed selection pressures (e.g., size-selective harvesting) might impose noteworthy evolutionary change even in large marine vertebrates. We therefore use our findings to explain how maternal effects may sometimes promote maladaptive juvenile traits, and how lemon sharks at different nursery sites may show "constrained local adaptation." We also show how single-generation pedigrees, and even simple marker-based regression methods, can provide accurate estimates of quantitative genetic parameters in at least some natural systems.     

System-level strategies for studying the metabolism of Mycobacterium tuberculosis

Despite decades of research many aspects of the biology of Mycobacterium tuberculosis remain unclear and this is reflected in the antiquated tools available to treat and prevent tuberculosis and consequently this disease remains a serious public health problem. Important discoveries linking M. tuberculosis's metabolism and pathogenesis have renewed interest in this area of research. Previous experimental studies were limited to the analysis of individual genes or enzymes whereas recent advances in computational systems biology and high throughput experimental technologies now allow metabolism to be studied on a genome scale. Here we discuss the progress being made in applying system level approaches to studying the metabolism of this important pathogen. The information from these studies will fundamentally change our approach to tuberculosis research and lead to new targets for therapeutic drugs and vaccines.     

FliZ Is a Global Regulatory Protein Affecting the Expression of Flagellar and Virulence Genes in Individual Xenorhabdus nematophila Bacterial Cells

Heterogeneity in the expression of various bacterial genes has been shown to result in the presence of individuals with different phenotypes within clonal bacterial populations. The genes specifying motility and flagellar functions are coordinately regulated and form a complex regulon, the flagellar regulon. Complex interplay has recently been demonstrated in the regulation of flagellar and virulence gene expression in many bacterial pathogens. We show here that FliZ, a DNA-binding protein, plays a key role in the insect pathogen, Xenorhabdus nematophila, affecting not only hemolysin production and virulence in insects, but efficient swimming motility. RNA-Seq analysis identified FliZ as a global regulatory protein controlling the expression of 278 Xenorhabdus genes either directly or indirectly. FliZ is required for the efficient expression of all flagellar genes, probably through its positive feedback loop, which controls expression of the flhDC operon, the master regulator of the flagellar circuit. FliZ also up- or downregulates the expression of numerous genes encoding non-flagellar proteins potentially involved in key steps of the Xenorhabdus lifecycle. Single-cell analysis revealed the bimodal expression of six identified markers of the FliZ regulon during exponential growth of the bacterial population. In addition, a combination of fluorescence-activated cell sorting and RT-qPCR quantification showed that this bimodality generated a mixed population of cells either expressing (“ON state”) or not expressing (“OFF state”) FliZ-dependent genes. Moreover, studies of a bacterial population exposed to a graded series of FliZ concentrations showed that FliZ functioned as a rheostat, controlling the rate of transition between the “OFF” and “ON” states in individuals. FliZ thus plays a key role in cell fate decisions, by transiently creating individuals with different potentials for motility and host interactions.     

Resting State fMRI Reveals Diminished Functional Connectivity in a Mouse Model of Amyloidosis

Introduction

Functional connectivity (FC) studies have gained immense popularity in the evaluation of several neurological disorders, such as Alzheimer’s disease (AD). AD is a complex disorder, characterised by several pathological features. The problem with FC studies in patients is that it is not straightforward to focus on a specific aspect of pathology. In the current study, resting state functional magnetic resonance imaging (rsfMRI) is applied in a mouse model of amyloidosis to assess the effects of amyloid pathology on FC in the mouse brain.

Methods

Nine APP/PS1 transgenic and nine wild-type mice (average age 18.9 months) were imaged on a 7T MRI system. The mice were anesthetized with medetomidine and rsfMRI data were acquired using a gradient echo EPI sequence. The data were analysed using a whole brain seed correlation analysis and interhemispheric FC was evaluated using a pairwise seed analysis. Qualitative histological analyses were performed to assess amyloid pathology, inflammation and synaptic deficits.

Results

The whole brain seed analysis revealed an overall decrease in FC in the brains of transgenic mice compared to wild-type mice. The results showed that interhemispheric FC was relatively preserved in the motor cortex of the transgenic mice, but decreased in the somatosensory cortex and the hippocampus when compared to the wild-type mice. The pairwise seed analysis confirmed these results. Histological analyses confirmed the presence of amyloid pathology, inflammation and synaptic deficits in the transgenic mice.

Conclusions

In the current study, rsfMRI demonstrated decreased FC in APP/PS1 transgenic mice compared to wild-type mice in several brain regions. The APP/PS1 transgenic mice had advanced amyloid pathology across the brain, as well as inflammation and synaptic deficits surrounding the amyloid plaques. Future studies should longitudinally evaluate APP/PS1 transgenic mice and correlate the rsfMRI findings to specific stages of amyloid pathology.     

Structure of the Human Angiotensin II Type 1 (AT1) Receptor Bound to Angiotensin II from Multiple Chemoselective Photoprobe Contacts Reveals a Unique Peptide Binding Mode

Breakthroughs in G protein-coupled receptor structure determination based on crystallography have been mainly obtained from receptors occupied in their transmembrane domain core by low molecular weight ligands, and we have only recently begun to elucidate how the extracellular surface of G protein-coupled receptors (GPCRs) allows for the binding of larger peptide molecules. In the present study, we used a unique chemoselective photoaffinity labeling strategy, the methionine proximity assay, to directly identify at physiological conditions a total of 38 discrete ligand/receptor contact residues that form the extracellular peptide-binding site of an activated GPCR, the angiotensin II type 1 receptor. This experimental data set was used in homology modeling to guide the positioning of the angiotensin II (AngII) peptide within several GPCR crystal structure templates. We found that the CXC chemokine receptor type 4 accommodated the results better than the other templates evaluated; ligand/receptor contact residues were spatially grouped into defined interaction clusters with AngII. In the resulting receptor structure, a β-hairpin fold in extracellular loop 2 in conjunction with two extracellular disulfide bridges appeared to open and shape the entrance of the ligand-binding site. The bound AngII adopted a somewhat vertical binding mode, allowing concomitant contacts across the extracellular surface and deep within the transmembrane domain core of the receptor. We propose that such a dualistic nature of GPCR interaction could be well suited for diffusible linear peptide ligands and a common feature of other peptidergic class A GPCRs.     

Ecological immunology in a fluctuating environment: an integrative analysis of tree swallow nestling immune defense

Evolutionary ecologists have long been interested by the link between different immune defenses and fitness. Given the importance of a proper immune defense for survival, it is important to understand how its numerous components are affected by environmental heterogeneity. Previous studies targeting this question have rarely considered more than two immune markers. In this study, we measured seven immune markers (response to phytohemagglutinin (PHA), hemolysis capacity, hemagglutination capacity, plasma bactericidal capacity, percentage of lymphocytes, percentage of heterophils, and percentage of eosinophils) in tree swallow (Tachycineta bicolor) nestlings raised in two types of agro‐ecosystems of contrasted quality and over 2 years. First, we assessed the effect of environmental heterogeneity (spatial and temporal) on the strength and direction of correlations between immune measures. Second, we investigated the effect of an immune score integrating information from several immune markers on individual performance (including growth, mass at fledging and parasite burden). Both a multivariate and a pair‐wise approach showed variation in relationships between immune measures across years and habitats. We also found a weak association between the integrated score of nestling immune function and individual performance, but only under certain environmental conditions. We conclude that the ecological context can strongly affect the interpretation of immune defenses in the wild. Given that spatiotemporal variations are likely to affect individual immune defenses, great caution should be used when generalizing conclusions to other study systems.     

New and revised maimetshid wasps from Cretaceous ambers (Hymenoptera, Maimetshidae)

New material of the wasp family Maimetshidae (Apocrita) is presented from four Cretaceous amber deposits - the Neocomian of Lebanon, the Early Albian of Spain, the latest Albian/earliest Cenomanian of France, and the Campanian of Canada. The new record from Canadian Cretaceous amber extends the temporal and paleogeographical range of the family. New material from France is assignable to Guyotemaimetsha enigmatica Perrichot et al. including the first females for the species, while a series of males and females from Spain are described and figured as Iberomaimetsha Ortega-Blanco, Perrichot & Engel, gen. n., with the two new species Iberomaimetsha rasnitsyni Ortega-Blanco, Perrichot & Engel, sp. n. and Iberomaimetsha nihtmara Ortega-Blanco, Delclòs & Engel, sp. n.; a single female from Lebanon is described and figured as Ahiromaimetsha najlae Perrichot, Azar, Nel & Engel, gen. et sp. n., and a single male from Canada is described and figured as Ahstemiam cellula McKellar & Engel, gen. et sp. n. The taxa are compared with other maimetshids, a key to genera and species is given, and brief comments made on the family.     

¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using 13C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with 13C labeled glycerol or sodium bicarbonate. Through measurements of the 13C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO? into biomass. As the human host is abundant in CO? this finding requires further investigation in vivo as CO? fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using 13C-MFA.