Aspiration of THP1 into a micropipette. Mechanical deformation of monocytic THP-1 cells: occurrence of two sequential phases with differential sensitivity to metabolic inhibitors

Blood leukocytes can exhibit extensive morphological changes during their passage through small capillary vessels. The human monocytic THP-1 cell line was used to explore the metabolic dependence of these changes in shape. Cells were aspirated into micropipettes for determination of the rate of protrusion formation. They were then released and the kinetics of morphological recovery was studied. Results were consistent with Evans’ model (Blood 64:1028, 1984) of a viscous liquid droplet surrounded by a tensile membrane. The estimated values of cytoplasmic viscosity and membrane tension were 162 Pa.s and 0.0142 mN/m respectively. The influence of metabolic inhibitors on cell mechanical behavior was then studied: results strongly suggested that deformation involved two sequential phases. The cell elongation rate measured during the first 30 s following the onset of aspiration was unaffected by azide, an inhibitor of energy production, and it was about doubled by cytochalasin D, a microfilament inhibitor, and colchicine, a microtubule inhibitor. However, during the following 2 min, deformation was almost abolished in cells treated with azide and cytochalasin D, whereas the protrusion of control cells exhibited an approximately threefold increase in length. It is concluded that, although cells seemed to deform as passive objects, active metabolic processes were required to allow extensive morphological changes triggered by external forces.     

Brachypodium distachyon grain: characterization of endosperm cell walls

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ～52% (w/w) of the endosperm in comparison with 2-7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1-3) (1-4)-β-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1-3) (1-4)-β-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell-cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains.     

O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1

The human epigenetic cell-cycle regulator HCF-1 undergoes an unusual proteolytic maturation process resulting in stably associated HCF-1(N) and HCF-1(C) subunits that regulate different aspects of the cell cycle. Proteolysis occurs at six centrally located HCF-1(PRO)-repeat sequences and is important for activation of HCF-1(C)-subunit functions in M phase progression. We show here that the HCF-1(PRO) repeat is recognized by O-linked β-N-acetylglucosamine transferase (OGT), which both O-GlcNAcylates the HCF-1(N) subunit and directly cleaves the HCF-1(PRO) repeat. Replacement of the HCF-1(PRO) repeats by a heterologous proteolytic cleavage signal promotes HCF-1 proteolysis but fails to activate HCF-1(C)-subunit M phase functions. These results reveal an unexpected role of OGT in HCF-1 proteolytic maturation and an unforeseen nexus between OGT-directed O-GlcNAcylation and proteolytic maturation in HCF-1 cell-cycle regulation.     

Unsuspected diversity of arsenite-oxidizing bacteria as revealed by widespread distribution of the aoxB gene in prokaryotes

In this study, new strains were isolated from an environment with elevated arsenic levels, Sainte-Marie-aux-Mines (France), and the diversity of aoxB genes encoding the arsenite oxidase large subunit was investigated. The distribution of bacterial aoxB genes is wider than what was previously thought. AoxB subfamilies characterized by specific signatures were identified. An exhaustive analysis of AoxB sequences from this study and from public databases shows that horizontal gene transfer has likely played a role in the spreading of aoxB in prokaryotic communities.     

Tuberculosis drug resistance testing by molecular methods: opportunities and challenges in resource limited settings

One of the greatest threats to global tuberculosis (TB) control is the growing prevalence of drug-resistant bacilli. Correctly diagnosing drug-resistant TB patients is more problematic in resource-limited settings as there is no or limited infrastructure for drug susceptibility testing (DST) of TB bacilli. The conventional phenotypic DST method for TB takes weeks before declaring the results and initiating proper anti-TB treatment. Molecular DST offers advantages over the phenotypic methods mainly because of the short turnaround time. This review summarizes the different molecular DST methods for TB and discusses challenges and opportunities in implementing them in resource-limited settings.     

Dynamic causal models and physiological inference: a validation study using isoflurane anaesthesia in rodents

Generative models of neuroimaging and electrophysiological data present new opportunities for accessing hidden or latent brain states. Dynamic causal modeling (DCM) uses Bayesian model inversion and selection to infer the synaptic mechanisms underlying empirically observed brain responses. DCM for electrophysiological data, in particular, aims to estimate the relative strength of synaptic transmission at different cell types and via specific neurotransmitters. Here, we report a DCM validation study concerning inference on excitatory and inhibitory synaptic transmission, using different doses of a volatile anaesthetic agent (isoflurane) to parametrically modify excitatory and inhibitory synaptic processing while recording local field potentials (LFPs) from primary auditory cortex (A1) and the posterior auditory field (PAF) in the auditory belt region in rodents. We test whether DCM can infer, from the LFP measurements, the expected drug-induced changes in synaptic transmission mediated via fast ionotropic receptors; i.e., excitatory (glutamatergic) AMPA and inhibitory GABA(A) receptors. Cross- and auto-spectra from the two regions were used to optimise three DCMs based on biologically plausible neural mass models and specific network architectures. Consistent with known extrinsic connectivity patterns in sensory hierarchies, we found that a model comprising forward connections from A1 to PAF and backward connections from PAF to A1 outperformed a model with forward connections from PAF to A1 and backward connections from A1 to PAF and a model with reciprocal lateral connections. The parameter estimates from the most plausible model indicated that the amplitude of fast glutamatergic excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) behaved as predicted by previous neurophysiological studies. Specifically, with increasing levels of anaesthesia, glutamatergic EPSPs decreased linearly, whereas fast GABAergic IPSPs displayed a nonlinear (saturating) increase. The consistency of our model-based in vivo results with experimental in vitro results lends further validity to the capacity of DCM to infer on synaptic processes using macroscopic neurophysiological data.     

Prebiotic effects of wheat arabinoxylan related to the increase in bifidobacteria, Roseburia and Bacteroides/Prevotella in diet-induced obese mice

Background

Alterations in the composition of gut microbiota - known as dysbiosis - has been proposed to contribute to the development of obesity, thereby supporting the potential interest of nutrients targeting the gut with beneficial effect for host adiposity. We test the ability of a specific concentrate of water-extractable high molecular weight arabinoxylans (AX) from wheat to modulate both the gut microbiota and lipid metabolism in high-fat (HF) diet-induced obese mice.

Methodology/Principal Findings

Mice were fed either a control diet (CT) or a HF diet, or a HF diet supplemented with AX (10% w/w) during 4 weeks. AX supplementation restored the number of bacteria that were decreased upon HF feeding, i.e. Bacteroides-Prevotella spp. and Roseburia spp. Importantly, AX treatment markedly increased caecal bifidobacteria content, in particular Bifidobacterium animalis lactis. This effect was accompanied by improvement of gut barrier function and by a lower circulating inflammatory marker. Interestingly, rumenic acid (C18:2 c9,t11) was increased in white adipose tissue due to AX treatment, suggesting the influence of gut bacterial metabolism on host tissue. In parallel, AX treatment decreased adipocyte size and HF diet-induced expression of genes mediating differentiation, fatty acid uptake, fatty acid oxidation and inflammation, and decreased a key lipogenic enzyme activity in the subcutaneous adipose tissue. Furthermore, AX treatment significantly decreased HF-induced adiposity, body weight gain, serum and hepatic cholesterol accumulation and insulin resistance. Correlation analysis reveals that Roseburia spp. and Bacteroides/Prevotella levels inversely correlate with these host metabolic parameters.

Conclusions/Significance

Supplementation of a concentrate of water-extractable high molecular weight AX in the diet counteracted HF-induced gut dysbiosis together with an improvement of obesity and lipid-lowering effects. We postulate that hypocholesterolemic, anti-inflammatory and anti-obesity effects are related to changes in gut microbiota. These data support a role for wheat AX as interesting nutrients with prebiotic properties related to obesity prevention.