The effects of microRNAs on human neural stem cell differentiation in two- and three-dimensional cultures

INTRODUCTION: Stem cells have the ability to self-renew or to differentiate into numerous cell types; however, our understanding of how to control and exploit this potential is currently limited. An emerging hypothesis is that microRNAs (miRNAs) play a central role in controlling stem cell-fate determination. Herein, we have characterized the effects of miRNAs in differentiated human neural stem cells (hNSCs) by using a cell line currently being tested in clinical trials for stroke disability (NCT01151124, Clinicaltrials.gov). METHODS: HNSCs were differentiated on 2- (2D) and 3-dimensional (3D) cultures for 1 and 3 weeks. Quantification of hNSC differentiation was measured with real-time PCR and axon outgrowth. The miRNA PCR arrays were implemented to investigate differential expression profiles in differentiated hNSCs. Evaluation of miRNA effects on hNSCs was performed by using transfection of miRNA mimics, real-time PCR, Western blot, and immunocytochemistry. RESULTS: The 3D substrate promoted enhanced hNSC differentiation coupled with a loss of cell proliferation. Differentiated hNSCs exhibited a similar miRNA profiling. However, in 3D samples, the degree and timing of regulation were significantly different in miRNA members of cluster mi-R17 and miR-96-182, and hsa-miR-302a. Overall, hNSC 3D cultures demonstrated differential regulation of miRNAs involved in hNSC stemness, cell proliferation, and differentiation. The miRNA mimic analysis of hsa-miR-146b-5p and hsa-miR-99a confirmed induction of lineage-committed progenitors. Downregulated miRNAs were more abundant; those most significantly downregulated were selected, and their putative target mRNAs analyzed with the aim of unraveling their functionality. In differentiated hNSCs, downregulated hsa-miR-96 correlated with SOX5 upregulation of gene and protein expression; similar results were obtained for hsa-miR-302a, hsa-miR-182, hsa-miR-7, hsa-miR-20a/b, and hsa-miR-17 and their target NR4A3. Moreover, SOX5 was identified as a direct target gene of hsa-miR-96, and NR43A, a direct target of hsa-miR-7 and hsa-mir-17 by luciferase reporter assays. Therefore, the regulatory role of these miRNAs may occur through targeting NR4A3 and SOX5, both reported as modulators of cell-cycle progression and axon length. CONCLUSIONS: The results provide new insight into the identification of specific miRNAs implicated in hNSC differentiation. These strategies may be exploited to optimize in vitro hNSC differentiation potential for use in preclinical studies and future clinical applications

Spatial chaos of an extensible conducting rod in a uniform magnetic field

The equilibrium equations for the isotropic Kirchhoff rod are known to form an integrable system. It is also known that the effects of extensibility and shearability of the rod do not break the integrable structure. Nor, as we have shown in a previous paper does the effect of a magnetic field on a conducting rod. Here we show, by means of Mel'nikov analysis, that, remarkably, the combined effects do destroy integrability; that is, the governing equations for an extensible current-carrying rod in a uniform magnetic field are nonintegrable. This result has implications for possible configurations of electrodynamic space tethers and may be relevant for electromechanical devices

Dynamic phase transition in the conversion of B-DNA to Z-DNA

The long time dynamics of the conformational transition from B-DNA to Z-DNA is shown to undergo a dynamic phase transition. We obtained the dynamic phase diagram for the stability of the front separating B and Z. The instability in this front results in two split fronts moving with different velocities. Hence, depending on the system parameters a denatured state may develop dynamically eventhough it is thermodynamically forbidden. This resolves the current controversies on the transition mechanism of the B-DNA to Z-DNA.Comment: 5 pages, 4 figures. New version with correction of typos, new references, minor modifications in Fig 2, 3. To appear in EP

Lead clinical and pre-clinical antimalarial drugs can significantly reduce sporozoite transmission to vertebrate populations

To achieve malarial elimination we must employ interventions that reduce the exposure of human populations to infectious mosquitoes. To this end, numerous anti-malarial drugs are under assessment in a variety of transmission-blocking assays which fail to measure the single crucial criteria of a successful intervention; namely impact on case incidence within a vertebrate population (reduction in Ro/effect size). Consequently, any reduction in new infections due to drug treatment (and how this may be influenced by differing transmission settings) is not currently examined, limiting the translation of any findings. We describe the use of a laboratory population model to assess how individual anti-malarial drugs can impact the number of secondary P.berghei infections over a cycle of transmission. We examine the impact of multiple clinical and pre-clinical drugs on both insect and vertebrate populations at multiple transmission settings. Both primaquine (>6mg/kg) and NITD609 (8.1mg/kg) have significant impact across multiple transmission settings, but artemether/lumefantrine (57/11.8mg/kg), OZ439 (6.5mg/kg) and primaquine (<1.25mg/kg) demonstrated potent efficacy only at lower transmission settings. While directly demonstrating the impact of drug treatment on anti-malarial drug treatment on vertebrate populations, we additionally calculate effect size for each treatment, allowing for head-to-head comparison of the potential impact of individual drugs within epidemiologically relevant settings, supporting their usage within elimination campaigns

PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

Published onlineComparative StudyEvaluation StudiesJournal ArticleResearch Support, Non-U.S. Gov'tBACKGROUND: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared with three previously described PCR techniques. METHODS: A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium species and discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the new assay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in a blind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. The performance of the assays was then compared using more than 450 field-collected specimens that had been stored on silica gel, in ethanol or in isopropanol. RESULTS: The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template. Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitive of the four methods followed by the 'gold standard' nested PCR approach and the results generated using these two methods were in good concordance. The sensitivity of the other two methods and their agreement with the nested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of the methods (including the nested protocol) showed a high degree of non-specific amplification when using DNA derived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when using the same samples. CONCLUSION: This study describes a new high-throughput TaqMan assay that very effectively detects the four Plasmodium species that cause malaria in humans and discriminates the most deadly species, P. falciparum, from the others. This method is at least as sensitive and specific as the gold standard nested PCR approach and because it has no requirement for post-PCR processing is cheaper, simpler and more rapid to run. In addition this method is not inhibited by the storage of mosquito specimens by drying or in ethanol or isopropanol.BBSRCInnovative Vector Control Consortiu

A length-dynamic Tonks gas theory of histone isotherms

We find exact solutions to a new one-dimensional (1D) interacting particle theory and apply the results to the adsorption and wrapping of polymers (such as DNA) around protein particles (such as histones). Each adsorbed protein is represented by a Tonks gas particle. The length of each particle is a degree of freedom that represents the degree of DNA wrapping around each histone. Thermodynamic quantities are computed as functions of wrapping energy, adsorbed histone density, and bulk histone concentration (or chemical potential); their experimental signatures are also discussed. Histone density is found to undergo a two-stage adsorption process as a function of chemical potential, while the mean coverage by high affinity proteins exhibits a maximum as a function of the chemical potential. However, {\it fluctuations} in the coverage are concurrently maximal. Histone-histone correlation functions are also computed and exhibit rich two length scale behavior.Comment: 5 pp, 3 fig

Integrability of a conducting elastic rod in a magnetic field

We consider the equilibrium equations for a conducting elastic rod placed in a uniform magnetic field, motivated by the problem of electrodynamic space tethers. When expressed in body coordinates the equations are found to sit in a hierarchy of non-canonical Hamiltonian systems involving an increasing number of vector fields. These systems, which include the classical Euler and Kirchhoff rods, are shown to be completely integrable in the case of a transversely isotropic rod; they are in fact generated by a Lax pair. For the magnetic rod this gives a physical interpretation to a previously proposed abstract nine-dimensional integrable system. We use the conserved quantities to reduce the equations to a four-dimensional canonical Hamiltonian system, allowing the geometry of the phase space to be investigated through Poincar\'e sections. In the special case where the force in the rod is aligned with the magnetic field the system turns out to be superintegrable, meaning that the phase space breaks down completely into periodic orbits, corresponding to straight twisted rods.Comment: 19 pages, 1 figur

Integrability, localisation and bifurcation of an elastic conducting rod in a uniform magnetic field

The classical problem of the buckling of an elastic rod in a magnetic ¯eld is investigated using modern techniques from dynamical systems theory. The Kirchhoff equations, which describe the static equilibrium equations of a geometrically exact rod under end tension and moment are extended by incorporating the evolution of a fixed external vector (in the direction of the magnetic field) that interacts with the rod via a Lorentz force. The static equilibrium equations (in body cordinates) are found to be noncanonical Hamiltonian equations. The Poisson bracket is generalised and the equilibrium equations found to sit, as the third member, in a family of rod equations in generalised magnetic fields. When the rod is linearly elastic, isotropic, inextensible and unshearable the equations are completely integrable and can be generated by a Lax pair. The isotropic system is reduced using the Casimirs, via the Euler angles, to a four-dimensional canonical system with a first integral provided the magnetic field is not aligned with the force within the rod at any point as the system losses rank. An energy surface is specified, defning three-dimensional flows. Poincare sections then show closed curves. Through Mel'nikov analysis it is shown that for an extensible rod the presence of a magnetic field leads to the transverse intersection of the stable and unstable manifolds and the loss of complete integrability. Consequently, the system admits spatially chaotic solutions and a multiplicity of multimodal homoclinic solutions exist. Poincare sections associated with the loss of integrability are displayed. Homoclinic solutions are computed and post-buckling paths found using continutaion methods. The rods buckle in a Hamiltonian-Hopf bifurcation about a periodic solution. A codimension-two point, which describes a double Hamiltonian-Hopf bifurcation, determines whether straight rods buckle into localised configurations at either two critical values of the magnetic field, a single critical value or do not buckle at all. The codimension-two point is found to be an organising centre for primary and multimodal solutions

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls

Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studie

Phase Behavior of Columnar DNA Assemblies

The pair interaction between two stiff parallel linear DNA molecules depends not only on the distance between their axes but on their azimuthal orientation. The positional and orientational order in columnar B-DNA assemblies in solution is investigated, based on the DNA-DNA electrostatic pair potential that takes into account DNA helical symmetry and the amount and distribution of adsorbed counterions. A phase diagram obtained by lattice sum calculations predicts a variety of positionally and azimuthally ordered phases and bundling transitions strongly depending on the counterion adsorption patterns.Comment: 4 pages, 3 figures, submitted to PR