High-throughput screening assay for the identification of compounds regulating self-renewal and differentiation in human embryonic stem cells

High-throughput screening (HTS) of chemical libraries has become a critical tool in basic biology and drug discovery. However, its implementation and the adaptation of high-content assays to human embryonic stem cells (hESCs) have been hampered by multiple technical challenges. Here we present a strategy to adapt hESCs to HTS conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice during differentiation. Global gene expression analysis upon drug treatment defines known and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the repertoire of chemical compounds for manipulating hESC fate. The availability of high-content assays should accelerate progress in basic and translational hESC biology.     

Structural-dynamical properties of the transmembrane segment VI of the mitochondrial oxoglutarate carrier studied by site directed spin-labeling

Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidth analysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 to I298, indicating that it has an alpha-helical structure. Saturation behaviour, in presence of paramagnetic perturbants of different hydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect to the lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activity data indicate that the segment is made by an alpha helix, accommodated in an amphipathic environment, partially distorted in the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region of the segment is less restrained and more flexible than the N-terminus.     

Requirement of a plasmid-encoded catalase for survival of Rhizobium etli CFN42 in a polyphenol-rich environment

Nitrogen-fixing bacteria collectively called rhizobia are adapted to live in polyphenol-rich environments. The mechanisms that allow these bacteria to overcome toxic concentrations of plant polyphenols have not been clearly elucidated. We used a crude extract of polyphenols released from the seed coat of the black bean to simulate a polyphenol-rich environment and analyze the response of the bean-nodulating strain Rhizobium etli CFN42. Our results showed that the viability of the wild type as well as that of derivative strains cured of plasmids p42a, p42b, p42c, and p42d or lacking 200 kb of plasmid p42e was not affected in this environment. In contrast, survival of the mutant lacking plasmid p42f was severely diminished. Complementation analysis revealed that the katG gene located on this plasmid, encoding the only catalase present in this bacterium, restored full resistance to testa polyphenols. Our results indicate that oxidation of polyphenols due to interaction with bacterial cells results in the production of a high quantity of H(2)O(2), whose removal by the katG-encoded catalase plays a key role for cell survival in a polyphenol-rich environment.     

Variation in habitat preference and population structure among three species of the Lake Malawi cichlid genus Protomelas

Several studies have demonstrated strong population structuring over small distances in the rocky-shore mbuna cichlid fishes from Lake Malawi, suggesting the potential for allopatric speciation. However, many endemic Lake Malawi cichlids are neither mbuna, nor confined to rocky shores. Using microsatellites, we investigated the population structure in three species of the non-mbuna genus Protomelas. The rocky-shore P. taeniolatus showed high levels of population structure even over distances of less than 1 km, while the sandy-shore species P. similis showed no significant structure over distances up to 21 km. Protomelas fenestratus, which is generally found at the interface between rocks and sand, also showed low levels of population structure. Our results suggest that the model of allopatric speciation based on habitat fragmentation within the current lake basin may be equally applicable to rocky-shore non-mbuna as to mbuna, but that an alternative model is required to explain speciation among sandy-shore species as well as the deep-water and pelagic species.     

Profiling cancer

In the past couple of years, several very exciting studies have demonstrated the enormous power of gene-expression profiling for cancer classification and prediction of patient survival. In addition to promising a more accurate classification of cancer and therefore better treatment of patients, gene-expression profiling can result in the identification of novel potential targets for cancer therapy and a better understanding of the molecular mechanisms leading to cancer.     

Serum as a factor influencing adhesion of Enterococcus faecalis to glass and silicone

The purpose of this work was to analyze the effect of serum on the physicochemical surface properties and adhesion to glass and silicone of Enterococcus faecalis ATCC 29212 at 37 degrees C. As is presented using thermodynamics analysis, serum minimizes the interaction of cells with water, which correlates well with the increase in hydrophobicity and in bacterial adhesion to glass and silicone.     

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca²⁺ concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca²⁺-activated state of NCS proteins, little is known about the conformational dynamics of the Mg²⁺-bound and apo states, both of which are populated, at least transiently, at resting Ca²⁺ conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg²⁺ and in the absence of divalent ions. Under tension, the Mg²⁺-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca²⁺-bound state, the presence of Mg²⁺ allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.     

Efficacy and Safety of Non-Vitamin K Antagonist Oral Anticoagulants versus Vitamin K Antagonist Oral Anticoagulants in Patients Undergoing Radiofrequency Catheter Ablation of Atrial Fibrillation: A Meta-Analysis

BackgroundUse of the non-vitamin K antagonist oral anticoagulants (NOACs) is endorsed by current guidelines for stroke prevention in patients with atrial fibrillation (AF). However efficacy and safety of NOACs in patients undergoing catheter ablation (RFCA) of AF has not been well established yet.ObjectivesTo perform a meta-analysis of all studies comparing NOACs and vitamin K antagonist oral anticoagulants (VKAs) in patients undergoing RFCA.ResultsThere was no significant difference in thromboembolic complications (RR 1.39; p=0.13). Bleeding complications were significantly lower in the NOACs-treated arm as compared to VKAs (RR=0.67, p<0.001). Interestingly, a larger number of thromboembolic events was found in the VKAs-treated arm in those studies where VKAs had been interrupted during the periprocedural phase (RR=0.68; p=ns). In this same subgroup a significantly higher incidence of both minor (RR=0.54; p=0.002) and major bleeding (RR=0.41; p=0.01) events was recorded. Conversely, the incidence of thromboembolic events in the VKAs-treated arm was significantly lower in those studies with uninterrupted periprocedural anticoagulation treatment (RR=1.89; p=0.02).LimitationsAs with every meta-analysis, no patients-level data were available.

Conclusions and Implications

The use of NOACs in patients undergoing RFCA is safe, given the lower incidence of bleedings observed with NOACs. On the other side, periprocedural interruption of VKAs and bridging with heparin is associated with a higher bleeding rate with no significant benefit on onset of thromboembolic events.     

Characterization of tetranucleotide microsatellite loci in a Lake Victorian,haplochromine cichlid fish: a Pundamilia pundamilia × Pundamilia nyererei hybrid

The haplochromine cichlid fishes inhabiting Lake Victoria in East Africa are of great interest to evolutionary biologists. We have isolated and optimized six tetranucleotide and a single dinucleotide locus from a hybrid of the haplochromine cichlid fishes, Pundamilia pundamilia and P. nyererei. Characterization in 18 individuals of P. nyererei from a single wild population revealed between six and 18 alleles per locus, with expected heterozygosity ranging from 0.68 to 0.94. These loci will prove useful for investigations of population structure, and elucidating relationships between closely related species. An additional 26 unoptimized loci have been deposited with GenBank and the MEN database.