Partition decoupling for multi-gene analysis of gene expression profiling data

Background

Molecular dynamics (MD) simulations are powerful tools to investigate the conformational dynamics of proteins that is often a critical element of their function. Identification of functionally relevant conformations is generally done clustering the large ensemble of structures that are generated. Recently, Self-Organising Maps (SOMs) were reported performing more accurately and providing more consistent results than traditional clustering algorithms in various data mining problems. We present a novel strategy to analyse and compare conformational ensembles of protein domains using a two-level approach that combines SOMs and hierarchical clustering.

Results

The conformational dynamics of the α-spectrin SH3 protein domain and six single mutants were analysed by MD simulations. The Cα's Cartesian coordinates of conformations sampled in the essential space were used as input data vectors for SOM training, then complete linkage clustering was performed on the SOM prototype vectors. A specific protocol to optimize a SOM for structural ensembles was proposed: the optimal SOM was selected by means of a Taguchi experimental design plan applied to different data sets, and the optimal sampling rate of the MD trajectory was selected. The proposed two-level approach was applied to single trajectories of the SH3 domain independently as well as to groups of them at the same time. The results demonstrated the potential of this approach in the analysis of large ensembles of molecular structures: the possibility of producing a topological mapping of the conformational space in a simple 2D visualisation, as well as of effectively highlighting differences in the conformational dynamics directly related to biological functions.

Conclusions

The use of a two-level approach combining SOMs and hierarchical clustering for conformational analysis of structural ensembles of proteins was proposed. It can easily be extended to other study cases and to conformational ensembles from other sources.     

Investigation of Design Space for Freeze-Drying: Use of Modeling for Primary Drying Segment of a Freeze-Drying Cycle

In this work, we explore the idea of using mathematical models to build design space for the primary drying portion of freeze-drying process. We start by defining design space for freeze-drying, followed by defining critical quality attributes and critical process parameters. Then using mathematical model, we build an insilico design space. Input parameters to the model (heat transfer coefficient and mass transfer resistance) were obtained from separate experimental runs. Two lyophilization runs are conducted to verify the model predictions. This confirmation of the model predictions with experimental results added to the confidence in the insilico design space. This simple step-by-step approach allowed us to minimize the number of experimental runs (preliminary runs to calculate heat transfer coefficient and mass transfer resistance plus two additional experimental runs to verify model predictions) required to define the design space. The established design space can then be used to understand the influence of critical process parameters on the critical quality attributes for all future cycles.     

Phylogenetic relationships and evolution of pulmonate gastropods (Mollusca): new insights from increased taxon sampling

Phylogenetic relationships among higher clades of pulmonate gastropods are reconstructed based on a data set including one nuclear marker (complete ribosomal 18S) and two mitochondrial markers (partial ribosomal 16S and Cytochrome oxidase I) for a total of 96 species. Sequences for 66 of these species are new to science, with a special emphasis on sampling the Ellobiidae, Onchidiidae, and Veronicellidae. Important results include the monophyly of Systellommatophora (Onchidiidae and Veronicellidae) as well as the monophyly of Ellobiidae (including Trimusculus, Otina, and Smeagol). Relationships within Ellobiidae, Onchidiidae, and Veronicellidae are evaluated here for the first time using molecular data. Present results are compared with those from the recent literature, and the current knowledge of phylogenetic relationships among pulmonate gastropods is reviewed: despite many efforts, deep nodes are still uncertain. Identification uncertainties about early fossils of pulmonates are reviewed. Impacts of those phylogenetic and fossil record uncertainties on our understanding of the macro-evolutionary history of pulmonates, especially transitions between aquatic and terrestrial habitats, are discussed.     

Modulation of synaptic plasticity by stress hormone associates with plastic alteration of synaptic NMDA receptor in the adult hippocampus

Stress exerts a profound impact on learning and memory, in part, through the actions of adrenal corticosterone (CORT) on synaptic plasticity, a cellular model of learning and memory. Increasing findings suggest that CORT exerts its impact on synaptic plasticity by altering the functional properties of glutamate receptors, which include changes in the motility and function of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype of glutamate receptor (AMPAR) that are responsible for the expression of synaptic plasticity. Here we provide evidence that CORT could also regulate synaptic plasticity by modulating the function of synaptic N-methyl-D-aspartate receptors (NMDARs), which mediate the induction of synaptic plasticity. We found that stress level CORT applied to adult rat hippocampal slices potentiated evoked NMDAR-mediated synaptic responses within 30 min. Surprisingly, following this fast-onset change, we observed a slow-onset (>1 hour after termination of CORT exposure) increase in synaptic expression of GluN2A-containing NMDARs. To investigate the consequences of the distinct fast- and slow-onset modulation of NMDARs for synaptic plasticity, we examined the formation of long-term potentiation (LTP) and long-term depression (LTD) within relevant time windows. Paralleling the increased NMDAR function, both LTP and LTD were facilitated during CORT treatment. However, 1-2 hours after CORT treatment when synaptic expression of GluN2A-containing NMDARs is increased, bidirectional plasticity was no longer facilitated. Our findings reveal the remarkable plasticity of NMDARs in the adult hippocampus in response to CORT. CORT-mediated slow-onset increase in GluN2A in hippocampal synapses could be a homeostatic mechanism to normalize synaptic plasticity following fast-onset stress-induced facilitation.     

Reversible resistance induced by FLT3 inhibition: a novel resistance mechanism in mutant FLT3-expressing cells

Objectives

Clinical responses achieved with FLT3 kinase inhibitors in acute myeloid leukemia (AML) are typically transient and partial. Thus, there is a need for identification of molecular mechanisms of clinical resistance to these drugs. In response, we characterized MOLM13 AML cell lines made resistant to two structurally-independent FLT3 inhibitors.

Methods

MOLM13 cells were made drug resistant via prolonged exposure to midostaurin and HG-7-85-01, respectively. Cell proliferation was determined by Trypan blue exclusion. Protein expression was assessed by immunoblotting, immunoprecipitation, and flow cytometry. Cycloheximide was used to determine protein half-life. RT-PCR was performed to determine FLT3 mRNA levels, and FISH analysis was performed to determine FLT3 gene expression.

Results and Conclusions

We found that MOLM13 cells readily developed cross-resistance when exposed to either midostaurin or HG-7-85-01. Resistance in both lines was associated with dramatically elevated levels of cell surface FLT3 and elevated levels of phosphor-MAPK, but not phospho-STAT5. The increase in FLT3-ITD expression was at least in part due to reduced turnover of the receptor, with prolonged half-life. Importantly, the drug-resistant phenotype could be rapidly reversed upon withdrawal of either inhibitor. Consistent with this phenotype, no significant evidence of FLT3 gene amplification, kinase domain mutations, or elevated levels of mRNA was observed, suggesting that protein turnover may be part of an auto-regulatory pathway initiated by FLT3 kinase activity. Interestingly, FLT3 inhibitor resistance also correlated with resistance to cytosine arabinoside. Over-expression of FLT3 protein in response to kinase inhibitors may be part of a novel mechanism that could contribute to clinical resistance.     

Phylogenetic biogeography of the leafy liverwort Herbertus (Jungermanniales, Herbertaceae) based on nuclear and chloroplast DNA sequence data: correlation between genetic variation and geographical distribution

Aim The cosmopolitan genus Herbertus is notorious for having a difficult taxonomy and for the fact that there is limited knowledge of species ranges and relationships. Topologies generated from variable molecular markers are used to discuss biogeographical patterns in Herbertus and to compare them with the geological history of continents and outcomes reported for other land plants. Location Africa, Asia, Azores, Europe, southern South America, northern South America, North America, New Zealand. Methods Phylogenetic analyses of nuclear ribosomal internal transcribed spacer and chloroplast (cp) trnL–trnF sequences of 66 accessions of Herbertus and the outgroup species Triandrophyllum subtrifidum and Mastigophora diclados were used to investigate biogeographical patterns in Herbertus. Areas of putative endemism were defined based on the distribution of species included in the analyses. Maximum parsimony analyses were undertaken to reconstruct ancestral areas and intraspecies migration routes. Results The analyses reveal species‐level cladograms with a correlation between genetic variation and the geographical distribution of the related accessions. The southern South American Herbertus runcinatus is sister to the remainder of the genus, which is split into two main clades. One contains the Neotropical–African Herbertus juniperoideus and the New Zealand/Tasmanian Herbertus oldfieldianus. An African accession of H. juniperoideus is nested within Neotropical accessions. The second main clade includes species that inhabit Asia, the Holarctic, Africa, and northern South America. Maximum parsimony analyses indicate that this clade arose in Asia. Herbertus sendtneri originated in Asia and subsequently colonized the Holarctic and northern South America. An Asian origin and colonization into Africa is indicated for H. dicranus. Main conclusions The current distribution of Herbertus cannot be explained by Gondwanan vicariance. A more feasible explanation of the range is a combination of short‐distance dispersal, rare long‐distance dispersal events (especially into regions that faced floral displacements as a result of climatic changes) extinction, recolonization, and diversification. The African Herbertus flora is a mixture of Asian and Neotropical elements. Southern South America harbours an isolated species. The molecular data indicate partial decoupling of molecular and morphological variation in Herbertus. Biogeographical patterns in Herbertus are not dissimilar to those of other groups of bryophytes, but elucidation of the geographical ranges requires a molecular approach. Some patterns could be the result of maintenance of Herbertus in the inner Tropics during glacial maxima, and dispersal into temperate regions in warm phases.