Engineering of zinc finger and MHC motifs to locked-in tertiary folds

The assembly of helical and -sheet peptide blocks containing reactive chain ends results inhighly branched chain architectures (locked-in folds) mimicking native tertiary structures.This molecular kit strategy allows to bypass the protein folding problem in protein de novodesign and gives access to protein mimetics of high thermodynamic stability. The validity ofthis concept is exemplified for the design and synthesis of locked-in folds mimicking the zincfinger and MHC folding motifs.     

Highly contrasted population genetic structures in a host–parasite pair in the Caribbean Sea

Evolution and population genetic structure of marine species across the Caribbean Sea are shaped by two complex factors: the geological history and the present pattern of marine currents. Characterizing and comparing the genetic structures of codistributed species, such as host–parasite associations, allow discriminating the relative importance of environmental factors and life history traits that influenced gene flow and demographic events. Using microsatellite and Cytochrome Oxidase I markers, we investigated if a host–parasite pair (the heart urchin Meoma ventricosa and its parasitic pea crab Dissodactylus primitivus) exhibits comparable population genetic structures in the Caribbean Sea and how the observed patterns match connectivity regions from predictive models and other taxa. Highly contrasting patterns were found: the host showed genetic homogeneity across the whole studied area, whereas the parasite displayed significant differentiation at regional and local scales. The genetic diversity of the parasitic crabs (both in microsatellites and COI) was distributed in two main groups, Panama–Jamaica–St Croix on the one hand, and the South‐Eastern Caribbean on the other. At a smaller geographical scale, Panamanian and Jamaican parasite populations were genetically more similar, while more genetic differentiation was found within the Lesser Antilles. Both species showed a signature of population expansion during the Quaternary. Some results match predictive models or data from previous studies (e.g., the Western‐Eastern dichotomy in the parasite) while others do not (e.g., genetic differentiation within the Lesser Antilles). The sharp dissimilarity of genetic structure of these codistributed species outlines the importance of population expansion events and/or contrasted patterns of gene flow. This might be linked to differences in several life history traits such as fecundity (higher for the host), swimming capacity of larval stages (higher for the parasite), and habitat availability (higher for the host).     

Toxoplasma gondii chromodomain protein 1 binds to heterochromatin and colocalises with centromeres and telomeres at the nuclear periphery

Background

Apicomplexan parasites are responsible for some of the most deadly parasitic diseases afflicting humans, including malaria and toxoplasmosis. These obligate intracellular parasites exhibit a complex life cycle and a coordinated cell cycle-dependant expression program. Their cell division is a coordinated multistep process. How this complex mechanism is organised remains poorly understood.

Methods and Findings

In this study, we provide evidence for a link between heterochromatin, cell division and the compartmentalisation of the nucleus in Toxoplasma gondii. We characterised a T. gondii chromodomain containing protein (named TgChromo1) that specifically binds to heterochromatin. Using ChIP-on-chip on a genome-wide scale, we report TgChromo1 enrichment at the peri-centromeric chromatin. In addition, we demonstrate that TgChromo1 is cell-cycle regulated and co-localised with markers of the centrocone. Through the loci-specific FISH technique for T. gondii, we confirmed that TgChromo1 occupies the same nuclear localisation as the peri-centromeric sequences.

Conclusion

We propose that TgChromo1 may play a role in the sequestration of chromosomes at the nuclear periphery and in the process of T. gondii cell division.     

Expression of caveolin‐1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor‐class B type I (SR‐BI) and cluster of differentiation‐36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin‐1. Our aim was to define the contribution of human CD36, SR‐BI, and caveolin‐1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)‐ and heavily (H)‐OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR‐BI, or caveolin‐1, we found that (1) CD36 increases M‐ and H‐OxLDL‐protein uptake; (2) SR‐BI drives M‐OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin‐1 increases M‐ and H‐OxLDL‐protein uptake and decreases CE selective uptake from M‐OxLDL. Also, incubation with M‐ or H‐OxLDL decreases the levels of SR‐BI and LDL‐receptor in control HepG2 cells which can be overcome by caveolin‐1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin‐1 in cells overexpressing this protein. Thus, hepatic caveolin‐1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels. J. Cell. Biochem. 108: 906–915, 2009. © 2009 Wiley‐Liss, Inc.     

The Supplementary Motor Area Exerts a Tonic Excitatory Influence on Corticospinal Projections to Phrenic Motoneurons in Awake Humans

Introduction

In humans, cortical mechanisms can interfere with autonomic breathing. Respiratory-related activation of the supplementary motor area (SMA) has been documented during voluntary breathing and in response to inspiratory constraints. The SMA could therefore participate in the increased resting state of the respiratory motor system during wake (i.e. "wakefulness drive to breathe").

Methods

The SMA was conditioned by continuous theta burst magnetic stimulation (cTBS, inhibitory) and 5 Hz conventional rTMS (5 Hz, excitatory). The ensuing effects were described in terms of the diaphragm motor evoked response (DiMEPs) to single-pulse transcranial magnetic stimulation over the motor cortex. DiMEPs were recorded at baseline, and at 3 time-points ("post1", "post2", "post3") up to 15 minutes following conditioning of the SMA.

Results

cTBS reduced the amplitude of DiMEPs from 327.5±159.8 µV at baseline to 243.3±118.7 µV, 217.8±102.9 µV and 240.6±123.9 µV at post 1, post 2 and post 3, respectively (F = 6.341, p = 0.002). 5 Hz conditioning increased the amplitude of DiMEPs from 184.7±96.5 µV at baseline to 270.7±135.4 µV at post 3 (F = 4.844, p = 0.009).

Conclusions

The corticospinal pathway to the diaphragm can be modulated in both directions by conditioning the SMA. This suggests that the baseline respiratory activity of the SMA represents an equipoise from which it is possible to move in either direction. The resting corticofugal outflow from the SMA to phrenic motoneurones that this study evidences could putatively contribute to the wakefulness drive to breathe.     

Nonesterified cholesterol content of lysosomes modulates susceptibility to oxidant-induced permeabilization

Reactive oxygen species (ROS) can induce lysosomal membrane permeabilization (LMP). Photoirradiation of murine hepatoma 1c1c7 cultures preloaded with the photosensitizer NPe6 generates singlet oxygen within acidic organelles and causes LMP and the activation of procaspases. Treatment with the cationic amphiphilic drugs (CADs) U18666A, imipramine, and clozapine stimulated the accumulation of filipin-stainable nonesterified cholesterol/sterols in late endosomes/lysosomes, but not in mitochondria. Concentration-response studies demonstrated an inverse relationship between lysosomal nonesterified cholesterol/sterol contents and susceptibility to NPe6 photoirradiation-induced intracellular membrane oxidation, LMP, and activation of procaspase-9 and -3. Similarly, the kinetics of restoration of NPe6 photoirradiation-induced LMP paralleled the losses of lysosomal cholesterol that occurred upon replating U18666A-treated cultures in CAD-free medium. Consistent with the oxidation of lysosomal cholesterol, filipin staining in U18666A-treated cultures progressively decreased with increasing photoirradiating light dose. U18666A also suppressed the induction of LMP and procaspase activation by exogenously added hydrogen peroxide. However, neither U18666A nor imipramine suppressed the induction of apoptosis by agents that did not directly induce LMP. These studies indicate that lysosomal nonesterified cholesterol/sterol content modulates susceptibility to ROS-induced LMP and possibly does so by being an alternative target for oxidants and lowering the probability of damage to other lysosomal membrane lipids and/or proteins.     

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.     

Size matters in quantitative radar monitoring of animal migration: estimating monitored volume from wingbeat frequency

Quantitative radar studies are an important component of studying the movements of birds. Whether a bird, at a certain distance from the radar, is detected or not depends on its size. The volume monitored by the radar is therefore different for birds of different sizes. Consequently, an accurate quantification of bird movements recorded by small‐scale radar requires an accurate determination of the monitored volume for the objects in question, although this has tended to be ignored. Here, we demonstrate the importance of sensitivity settings for echo detection on the estimated movement intensities of birds of different sizes. The amount of energy reflected from a bird and detected by the radar receiver (echo power) depends not only on the bird's size and on the distance from the radar antenna, but also on the beam shape and the bird's position within this beam. We propose a method to estimate the size of a bird based on the wingbeat frequency, retrieved from the echo‐signal, independent of the absolute echo power. The estimated bird‐size allows calculation of size‐specific monitored volumes, allowing accurate quantification of movement intensities. We further investigate the importance of applying size‐specific monitored volumes to quantify avian movements instead of using echo counts. We also highlight the importance of accounting for size‐specific monitored volume of small scale radar systems, and the necessity of reporting technical information on radar parameters. Applying this framework will increase the quality and validity of quantitative radar monitoring.