A New Method for the Preparation of Minicells for Physiological Studies

A procedure has been developed for preparing minicells that does not rely on sucrose gradients in a rate-zone centrifuge. In the presence of low levels (10 units/ml) of penicillin, the contaminating bacteria present in minicell cultures after low-speed differential centrifugation are turned into long filamentous cells and can be killed by sonic treatment. An additional low-speed centrifugation (2, 000 g for 5 min) yields purified minicells with less than one contaminating cell per 10 minicells.     

A Computational Model of Lipopolysaccharide-Induced Nuclear Factor Kappa B Activation: A Key Signalling Pathway in Infection-Induced Preterm Labour

Preterm birth is the single biggest cause of significant neonatal morbidity and mortality, and the incidence is rising. Development of new therapies to treat and prevent preterm labour is seriously hampered by incomplete understanding of the molecular mechanisms that initiate labour at term and preterm. Computational modelling provides a new opportunity to improve this understanding. It is a useful tool in (i) identifying gaps in knowledge and informing future research, and (ii) providing the basis for an in silico model of parturition in which novel drugs to prevent or treat preterm labour can be “tested”. Despite their merits, computational models are rarely used to study the molecular events initiating labour. Here, we present the first attempt to generate a dynamic kinetic model that has relevance to the molecular mechanisms of preterm labour. Using published data, we model an important candidate signalling pathway in infection-induced preterm labour: that of lipopolysaccharide (LPS) -induced activation of Nuclear Factor kappa B. This is the first model of this pathway to explicitly include molecular interactions upstream of Nuclear Factor kappa B activation. We produced a formalised graphical depiction of the pathway and built a kinetic model based on ordinary differential equations. The kinetic model accurately reproduced published in vitro time course plots of Lipopolysaccharide-induced Nuclear Factor kappa B activation in mouse embryo fibroblasts. In this preliminary work we have provided proof of concept that it is possible to build computational models of signalling pathways that are relevant to the regulation of labour, and suggest that models that are validated with wet-lab experiments have the potential to greatly benefit the field.     

Automated analysis of courtship suppression learning and memory in Drosophila melanogaster

Study of the fruit fly, Drosophila melanogaster, has yielded important insights into the underlying molecular mechanisms of learning and memory. Courtship conditioning is a well-established behavioral assay used to study Drosophila learning and memory. Here, we describe the development of software to analyze courtship suppression assay data that correctly identifies normal or abnormal learning and memory traits of individual flies. Development of this automated analysis software will significantly enhance our ability to use this assay in large-scale genetic screens and disease modeling. The software increases the consistency, objectivity, and types of data generated.     

What Are the Health Benefits of Active Travel? A Systematic Review of Trials and Cohort Studies

Background

Increasing active travel (primarily walking and cycling) has been widely advocated for reducing obesity levels and achieving other population health benefits. However, the strength of evidence underpinning this strategy is unclear. This study aimed to assess the evidence that active travel has significant health benefits.

Methods

The study design was a systematic review of (i) non-randomised and randomised controlled trials, and (ii) prospective observational studies examining either (a) the effects of interventions to promote active travel or (b) the association between active travel and health outcomes. Reports of studies were identified by searching 11 electronic databases, websites, reference lists and papers identified by experts in the field. Prospective observational and intervention studies measuring any health outcome of active travel in the general population were included. Studies of patient groups were excluded.

Results

Twenty-four studies from 12 countries were included, of which six were studies conducted with children. Five studies evaluated active travel interventions. Nineteen were prospective cohort studies which did not evaluate the impact of a specific intervention. No studies were identified with obesity as an outcome in adults; one of five prospective cohort studies in children found an association between obesity and active travel. Small positive effects on other health outcomes were found in five intervention studies, but these were all at risk of selection bias. Modest benefits for other health outcomes were identified in five prospective studies. There is suggestive evidence that active travel may have a positive effect on diabetes prevention, which may be an important area for future research.

Conclusions

Active travel may have positive effects on health outcomes, but there is little robust evidence to date of the effectiveness of active transport interventions for reducing obesity. Future evaluations of such interventions should include an assessment of their impacts on obesity and other health outcomes.     

Weight-Bearing Locomotion in the Developing Opossum,Monodelphis domestica following Spinal Transection: Remodeling of Neuronal Circuits Caudal to Lesion

Complete spinal transection in the mature nervous system is typically followed by minimal axonal repair, extensive motor paralysis and loss of sensory functions caudal to the injury. In contrast, the immature nervous system has greater capacity for repair, a phenomenon sometimes called the infant lesion effect. This study investigates spinal injuries early in development using the marsupial opossum Monodelphis domestica whose young are born very immature, allowing access to developmental stages only accessible in utero in eutherian mammals. Spinal cords of Monodelphis pups were completely transected in the lower thoracic region, T10, on postnatal-day (P)7 or P28 and the animals grew to adulthood. In P7-injured animals regrown supraspinal and propriospinal axons through the injury site were demonstrated using retrograde axonal labelling. These animals recovered near-normal coordinated overground locomotion, but with altered gait characteristics including foot placement phase lags. In P28-injured animals no axonal regrowth through the injury site could be demonstrated yet they were able to perform weight-supporting hindlimb stepping overground and on the treadmill. When placed in an environment of reduced sensory feedback (swimming) P7-injured animals swam using their hindlimbs, suggesting that the axons that grew across the lesion made functional connections; P28-injured animals swam using their forelimbs only, suggesting that their overground hindlimb movements were reflex-dependent and thus likely to be generated locally in the lumbar spinal cord. Modifications to propriospinal circuitry in P7- and P28-injured opossums were demonstrated by changes in the number of fluorescently labelled neurons detected in the lumbar cord following tracer studies and changes in the balance of excitatory, inhibitory and neuromodulatory neurotransmitter receptors’ gene expression shown by qRT-PCR. These results are discussed in the context of studies indicating that although following injury the isolated segment of the spinal cord retains some capability of rhythmic movement the mechanisms involved in weight-bearing locomotion are distinct.     

Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial–mesenchymal transition

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague–Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial–mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.     

The dark shadow of white Australia: Racial anxieties in Australia in World War II

Although World War II was perceived as a race war, historical attention has almost exclusively been focused upon the Axis powers’ racial conceptualizations. Little scholarly research has been directed towards investigating the Allies’ internal racial attitudes and policies. Building upon long‐standing formulations that emerged from the process of colonial conquest, the rhetoric of racism in Australia was recast and expanded in order to address the unprecedented crisis of the war in the Pacific. Aboriginal people were thus portrayed as potential collaborators who would aid the invading Japanese; enemy aliens, most particularly the Italians, the Japanese and the Germans become sinister fifth columnists. Lastly, the introduction of large numbers of black GIs among the American forces challenged notions of racial and sexual purity. Australians were ultimately fighting to preserve a white British‐derived nation. The ‘race war’ on the Pacific frontline intensified racial awareness and allowed hysterical overreaction that had tragic consequences for its victims.     

Characterization of functionally distinct mitochondrial subpopulations

Mitochondrial stress results in changes in mitochondrial function, morphology and homeostasis (biogenesis, fission/fusion, mitophagy) and may lead to changes in mitochondrial subpopulations. While flow cytometric techniques have been developed to quantify features of individual mitochondria related to volume, Ca²⁺ concentration, mtDNA content, respiratory capacity and oxidative damage, less information is available concerning the identification and characterization of mitochondrial subpopulations, particularly in epithelial cells. Mitochondria from rabbit kidneys were stained with molecular probes for cardiolipin content (nonyl acridine orange, NAO) and membrane potential (tetramethylrhodamine, TMRM) and analyzed using flow cytometry. We validated that side scatter was a better indicator of volume and that as side scatter (SSC) decreased mitochondrial volume increased. Furthermore, those mitochondria with the highest NAO content had greater side scattering and were most highly charged. Mitochondria with average NAO content were of average side scattering and maintained an intermediate charge. Those mitochondria with low NAO content had minimal side scattering and exhibited minimal charge. Upon titration with the uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), it was found that the high NAO content subpopulations were more resistant to uncoupling than lower NAO content populations. Ca²⁺-induced swelling of mitochondria was evaluated using probability binning (PB) analyses of SSC. Interestingly, only 30 % of the mitochondria showed changes in response to Ca²⁺, which was blocked by cyclosporine A. In addition, the small, high NAO content mitochondria swelled differentially in response to Ca²⁺ over time. Our results demonstrate that flow cytometry can be used to identify mitochondrial subpopulations based on high, mid and low NAO content and/or volume/complexity. These subpopulations showed differences in membrane potential, volume, and responses to uncoupling and Ca²⁺-induced swelling.     

Neutralizing Nanobodies Targeting Diverse Chemokines Effectively Inhibit Chemokine Function

Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.     

Phylogenetic analyses of anatomical and nuclear SSU rDNA sequence data indicate that the Dasyaceae and Delesseriaceae (Ceramiales,Rhodophyta) are polyphyletic

The aim of the current investigation was to test the general convention that the Dasyaceae, Delesseriaceae and Rhodomelaceae are all monophyletic families of the red algal order Ceramiales. Phylogenetic relationships among 45 ceramialean taxa were determined, including eight ceramiacean, 18 dasyacean, nine delesseriacean and eight rhodomelacean species, plus two of uncertain ceramialean affinities, based on 34 anatomical characters and nuclear small subunit (SSU) rDNA sequences. Results from our ‘total-evidence’ approach were consistent with the notion that the Dasyaceae, Delesseriaceae and Rhodomelaceae have evolved from a common ancestor within a paraphyletic Ceramiaceae. Our data indicate, however, that the Rhodomelaceae alone is monophyletic at the family level, with both the Dasyaceae and Delesseriaceae polyphyletic. In particular we resolved two independent and well-supported lineages for the included Dasyaceae, viz., a Dasya group and a Heterosiphonia group, which were as distinct from one another as they were from the Rhodomelaceae and the various lineages of Delesseriaceae. The molecular data alone were equivocal on the issue of monophyly of the Dasyaceae. We therefore advocate conservative taxonomic revisions as an interim step towards eventual resolution of familial-level taxonomy in the Ceramiales. Based on our results, the Heterosiphonioideae H.-G. Choi, Kraft, I.K. Lee et G.W. Saunders subfam. nov. is proposed for Heterosiphonia and five closely related genera, and the Dasyoideae Schmitz et Falkenberg is emended for the remaining taxa. Although the Dasyoideae is a natural group, it is in need of a thorough systematic reinvestigation at the generic level. Our analyses indicate that the genus Dasya is polyphyletic or paraphyletic in excluding Dasysiphonia, Eupogodon and Rhodoptilum and that Heterosiphonia japonica also has affinities to this group, taxonomic issues that will be addressed in detail elsewhere.