High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Ventilatory support: a dynamical systems approach

Misunderstanding of the dynamical behavior of the ventilatory system, especially under assisted ventilation, may explain the problems encountered in ventilatory support monitoring. Proportional assist ventilation (PAV) that theoretically gives a breath by breath assistance presents instability with high levels of assistance. We have constructed a mathematical model of interactions between three objects: the central respiratory pattern generator modelled by a modified Van der Pol oscillator, the mechanical respiratory system which is the passive part of the system and a controlled ventilator that follows its own law. The dynamical study of our model shows the existence of two crucial behaviors, i.e. oscillations and damping, depending on only two parameters, namely the time constant of the mechanical respiratory system and a cumulative interaction index. The same result is observed in simulations of spontaneous breathing as well as of PAV. In this last case, increasing assistance leads first to an increase of the tidal volume (V_T), a further increase in assistance inducing a decrease in V_T, ending in damping of the whole system to an attractive fixed point. We conclude that instabilities observed in PAV may be explained by the different possible dynamical behaviors of the system rather than changes in mechanical characteristics of the respiratory system.     

Synthesis and evaluation of benzo[b]thiophene derivatives as inhibitors of alkaline phosphatases

Presence of basic calcium phosphate in knee joints of osteoarthritis patients could be prevented by inhibiting tissue non-specific alkaline phosphatase (TNAP) activity. Levamisole or the L stereoisomer of tetramisole (a known TNAP inhibitor) has been used as a treatment for curing rheumatoid arthritis but its therapeutical use is limited due to side effects. We report the synthesis and the TNAP inhibition property of benzo[b]thiophene derivatives, among which benzothiopheno-tetramisole and benzothiopheno-2,3-dehydrotetramisole, which could be involved in a drug therapy for osteoarthritis. Two water soluble racemic benzothiopheno-tetramisole and -2,3-dehydrotetramisole with apparent inhibition constants K_i = 85 ± 6 μM and 135 ± 3 μM (n = 3) comparable to that of enantiomeric levamisole 93 ± 4 μM were found. Several novel derivatives showed more pronounced inhibition properties towards intestinal alkaline phosphatase than TNAP.     

Using budding yeast to screen for anti-prion drugs

Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.     

Inhibition of Bcl-2-dependent cell survival by a caspase inhibitor: a possible new pathway for Bcl-2 to regulate cell death.

The REtsAF cell line expresses a temperature-sensitive mutant of the SV40 large tumor antigen. At restrictive temperature (39.5 degrees C), the cells undergo p53-mediated apoptosis, which can be inhibited by Bcl-2. Here, we show that Z-VAD-fmk, a caspase inhibitor, can suppress the Bcl-2-dependent cell survival at 39.5 degrees C. This result suggests that a caspase-like activity can act as an inhibitor of apoptosis in this model, downstream of Bcl-2. Our results also suggest that this activity may be up-regulated by Bcl-2 and may be responsible for cleavage of the tumor suppressor Rb protein.     

Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex_8–16GlcNAc₂ modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex₉GlcNAc₂ at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.     

Glucose Transporter (GLUT-4) Is Targeted to Secretory Granules in Rat Atrial Cardiomyocytes

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4–containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.     

Interleukin 2 production in iron-deficient children

The relationship between iron status and capacity for IL-2 production by lymphocytes was assessed in 81 children from 6 mo to 3 yr of age selected at random from a population with low socioeconomic status, undergoing free systematic examination in four children's health centers in the Paris area. Iron deficiency was defined by the existence of at least two abnormal values among the three indicators of iron status: serum ferritin level ≤12 μg/L, transferrin saturation <12%, and erythrocyte protoporphyrin concentration >3 μg/g hemoglobin. According to this definition, 53 children were classified as iron deficient and 28 as iron sufficient. No differences were observed between the iron-deficient and iron-sufficient groups in terms of the IL-2 concentration without stimulation by PHA. IL-2 production by lymphocytes stimulated with PHA, as well as the stimulation index (ratio of IL-2 concentration following stimulation by PHA to that of IL-2 concentration without stimulation by PHA) were significantly lower in iron-deficient children. The reduction in IL-2 production by activated lymphocytes observed in our study of iron-deficient children may be responsible for impairments in immunity found by other authors, particularly in cell-mediated immunity.