Targeted delivery of chemotherapy agents using a liver cancer-specific aptamer

Background

Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.

Methodology/Principal Findings

In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.

Conclusions/Significance

Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general.     

Topological characterization of neuronal arbor morphology via sequence representation: II - global alignment

Background

The increasing abundance of neuromorphological data provides both the opportunity and the challenge to compare massive numbers of neurons from a wide diversity of sources efficiently and effectively. We implemented a modified global alignment algorithm representing axonal and dendritic bifurcations as strings of characters. Sequence alignment quantifies neuronal similarity by identifying branch-level correspondences between trees.

Results

The space generated from pairwise similarities is capable of classifying neuronal arbor types as well as, or better than, traditional topological metrics. Unsupervised cluster analysis produces groups that significantly correspond with known cell classes for axons, dendrites, and pyramidal apical dendrites. Furthermore, the distinguishing consensus topology generated by multiple sequence alignment of a group of neurons reveals their shared branching blueprint. Interestingly, the axons of dendritic-targeting interneurons in the rodent cortex associates with pyramidal axons but apart from the (more topologically symmetric) axons of perisomatic-targeting interneurons.

Conclusions

Global pairwise and multiple sequence alignment of neurite topologies enables detailed comparison of neurites and identification of conserved topological features in alignment-defined clusters. The methods presented also provide a framework for incorporation of additional branch-level morphological features. Moreover, comparison of multiple alignment with motif analysis shows that the two techniques provide complementary information respectively revealing global and local features.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0605-1) contains supplementary material, which is available to authorized users.     

The Role of Spatial Configuration in Multiple Identity Tracking

Background

The simultaneous tracking and identification of multiple moving objects encountered in everyday life requires one to correctly bind identities to objects. In the present study, we investigated the role of spatial configuration made by multiple targets when observers are asked to track multiple moving objects with distinct identities.

Methodology/Principal Findings

The overall spatial configuration made by the targets was manipulated: In the constant condition, the configuration remained as a virtual convex polygon throughout the tracking, and in the collapsed condition, one of the moving targets (critical target) crossed over an edge of the virtual polygon during tracking, destroying it. Identification performance was higher when the configuration remained intact than when it collapsed (Experiments 1a, 1b, and 2). Moreover, destroying the configuration affected the allocation of dynamic attention: the critical target captured more attention than did the other targets. However, observers were worse at identifying the critical target and were more likely to confuse it with the targets that formed the virtual crossed edge (Experiments 3–5). Experiment 6 further showed that the visual system constructs an overall configuration only by using the targets (and not the distractors); identification performance was not affected by whether the distractor violated the spatial configuration.

Conclusions/Significance

In sum, these results suggest that the visual system may integrate targets (but not distractors) into a spatial configuration during multiple identity tracking, which affects the distribution of dynamic attention and the updating of identity-location binding.     

Axons Amplify Somatic Incomplete Spikes into Uniform Amplitudes in Mouse Cortical Pyramidal Neurons

Background

Action potentials are the essential unit of neuronal encoding. Somatic sequential spikes in the central nervous system appear various in amplitudes. To be effective neuronal codes, these spikes should be propagated to axonal terminals where they activate the synapses and drive postsynaptic neurons. It remains unclear whether these effective neuronal codes are based on spike timing orders and/or amplitudes.

Methodology/Principal Findings

We investigated this fundamental issue by simultaneously recording the axon versus soma of identical neurons and presynaptic vs. postsynaptic neurons in the cortical slices. The axons enable somatic spikes in low amplitude be enlarged, which activate synaptic transmission in consistent patterns. This facilitation in the propagation of sequential spikes through the axons is mechanistically founded by the short refractory periods, large currents and high opening probability of axonal voltage-gated sodium channels.

Conclusion/Significance

An amplification of somatic incomplete spikes into axonal complete ones makes sequential spikes to activate consistent synaptic transmission. Therefore, neuronal encoding is likely based on spike timing order, instead of graded analogues.     

Wax boluses and accuracy of EBT and RTQA radiochromic film detectors in radiotherapy with the JINR Phasotron proton beam

Aim

To present the results obtained using radiochromic films EBT and RTQA 1010P for the reconstruction the dose distributions for targets irradiated by proton beam and modified by wax boluses.

Background

In Medico-Technical Complex at the Joint Institute for Nuclear Research in Dubna implemented technology of wax boluses.

Materials and methods

Wax boluses are easier to make and they give better dose distributions than boluses made from modeling clay previously used at our center. We irradiated two imaginary targets, one shaped as a cylinder and the other one as two cuboids. The evaluated calibration curve was used for calculation of the dose distributions measured by the EBT and RTQA radiochromic film. In both cases, the measured dose distributions were compared to the dose distributions calculated by the treatment planning system (TPS). We also compared dose distributions using three different conformity indices at a 95% isodose.

Results

Better target coverage and better compliance of measurements (semiconductor detectors and radiochromic films) with calculated doses was obtained for cylindrical target than for cuboidal target. The 95% isodose covered well the tumor for both target shapes, while for cuboidal target larger volume around the target received therapeutic dose, due to the complicated target shape. The use wax boluses provided to be effective tool in modifying proton beam to achieve appropriate shape of isodose distribution.

Conclusion

EBT film yielded the best visual matching. Both EBT and RTQA films confirmed good conformity between calculated and measured doses, thus confirming that wax boluses used to modify the proton beam resulted in good dose distributions.     

MicroRNA expression profiling of the fifth-instar posterior silk gland of Bombyx mori

Background

The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production.

Results

Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, of the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the targets into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing.

Conclusion

The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs and their targets in silk production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-410) contains supplementary material, which is available to authorized users.     

High-Content,Image-Based Screening for Drug Targets in Yeast

Background

Drug discovery and development are predicated on elucidation of the potential mechanisms of action and cellular targets of candidate chemical compounds. Recent advances in high-content imaging techniques allow simultaneous analysis of a range of cellular events. In this study, we propose a novel strategy to identify drug targets by combining genetic screening and high-content imaging in yeast.

Methodology

In this approach, we infer the cellular functions affected by candidate drugs by comparing morphologic changes induced by the compounds with the phenotypes of yeast mutants.

Conclusions

Using this method and four well-characterized reagents, we successfully identified previously known target genes of the compounds as well as other genes involved with functionally related cellular pathways. This is the first demonstration of a genetic high-content assay that can be used to identify drug targets based on morphologic phenotypes of a reference mutant panel.     

Cre-dependent expression of multiple transgenes in isolated neurons of the adult forebrain

Background

Transgenic mice with mosaic, Golgi-staining-like expression of enhanced green fluorescent protein (EGFP) have been very useful in studying the dynamics of neuronal structure and function. In order to further investigate the molecular events regulating structural plasticity, it would be useful to express multiple proteins in the same sparse neurons, allowing co-expression of functional proteins or co-labeling of subcellular compartments with other fluorescent proteins. However, it has been difficult to obtain reproducible expression in the same subset of neurons for direct comparison of neurons expressing different functional proteins.

Principal Findings

Here we describe a Cre-transgenic line that allows reproducible expression of transgenic proteins of choice in a small number of neurons of the adult cortex, hippocampus, striatum, olfactory bulb, subiculum, hypothalamus, superior colliculus and amygdala. We show that using these Cre-transgenic mice, multiple Cre-dependent transgenes can be expressed together in the same isolated neurons. We also describe a Cre-dependent transgenic line expressing a membrane associated EGFP (EGFP-F). Crossed with the Cre-transgenic line, EGFP-F expression starts in the adolescent forebrain, is present in dendrites, dendritic protrusions, axons and boutons and is strong enough for acute or chronic in vivo imaging.

Significance

This triple transgenic approach will aid the morphological and functional characterization of neurons in various Cre-dependent transgenic mice.     

Scaling up towards international targets for AIDS, tuberculosis, and malaria: contribution of global fund-supported programs in 2011-2015

Objective

The paper projects the contribution to 2011–2015 international targets of three major pandemics by programs in 140 countries funded by the Global Fund to Fight AIDS, Tuberculosis and Malaria, the largest external financier of tuberculosis and malaria programs and a major external funder of HIV programs in low and middle income countries.

Design

Estimates, using past trends, for the period 2011–2015 of the number of persons receiving antiretroviral (ARV) treatment, tuberculosis case detection using the internationally approved DOTS strategy, and insecticide-treated nets (ITNs) to be delivered by programs in low and middle income countries supported by the Global Fund compared to international targets established by UNAIDS, Stop TB Partnership, Roll Back Malaria Partnership and the World Health Organisation.

Results

Global Fund-supported programs are projected to provide ARV treatment to 5.5–5.8 million people, providing 30%–31% of the 2015 international target. Investments in tuberculosis and malaria control will enable reaching in 2015 60%–63% of the international target for tuberculosis case detection and 30%–35% of the ITN distribution target in sub-Saharan Africa.

Conclusion

Global Fund investments will substantially contribute to the achievement by 2015 of international targets for HIV, TB and malaria. However, additional large scale international and domestic financing is needed if these targets are to be reached by 2015.     

In the Absence of Frazzled Over-Expression of Abelson Tyrosine Kinase Disrupts Commissure Formation and Causes Axons to Leave the Embryonic CNS

Background

In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl''s role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures.

Methodology/Principal Findings

The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS.

Conclusion/Significance

Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border.     

Brain deletion of insulin receptor substrate 2 disrupts hippocampal synaptic plasticity and metaplasticity

Objective

Diabetes mellitus is associated with cognitive deficits and an increased risk of dementia, particularly in the elderly. These deficits and the corresponding neurophysiological structural and functional alterations are linked to both metabolic and vascular changes, related to chronic hyperglycaemia, but probably also defects in insulin action in the brain. To elucidate the specific role of brain insulin signalling in neuronal functions that are relevant for cognitive processes we have investigated the behaviour of neurons and synaptic plasticity in the hippocampus of mice lacking the insulin receptor substrate protein 2 (IRS-2).

Research Design and Methods

To study neuronal function and synaptic plasticity in the absence of confounding factors such as hyperglycaemia, we used a mouse model with a central nervous system- (CNS)-restricted deletion of IRS-2 (NesCreIrs2KO).

Results

We report a deficit in NMDA receptor-dependent synaptic plasticity in the hippocampus of NesCreIrs2KO mice, with a concomitant loss of metaplasticity, the modulation of synaptic plasticity by the previous activity of a synapse. These plasticity changes are associated with reduced basal phosphorylation of the NMDA receptor subunit NR1 and of downstream targets of the PI3K pathway, the protein kinases Akt and GSK-3β.

Conclusions

These findings reveal molecular and cellular mechanisms that might underlie cognitive deficits linked to specific defects of neuronal insulin signalling.     

Gogo Receptor Contributes to Retinotopic Map Formation and Prevents R1-6 Photoreceptor Axon Bundling

Background

Topographic maps form the basis of neural processing in sensory systems of both vertebrate and invertebrate species. In the Drosophila visual system, neighboring R1–R6 photoreceptor axons innervate adjacent positions in the first optic ganglion, the lamina, and thereby represent visual space as a continuous map in the brain. The mechanisms responsible for the establishment of retinotopic maps remain incompletely understood.

Results

Here, we show that the receptor Golden goal (Gogo) is required for R axon lamina targeting and cartridge elongation in a partially redundant fashion with local guidance cues provided by neighboring axons. Loss of function of Gogo in large clones of R axons results in aberrant R1–R6 fascicle spacing. Gogo affects target cartridge selection only indirectly as a consequence of the disordered lamina map. Interestingly, small clones of gogo deficient R axons perfectly integrate into a proper retinotopic map suggesting that surrounding R axons of the same or neighboring fascicles provide complementary spatial guidance. Using single photoreceptor type rescue, we show that Gogo expression exclusively in R8 cells is sufficient to mediate targeting of all photoreceptor types in the lamina. Upon lamina targeting and cartridge selection, R axons elongate within their individual cartridges. Interestingly, here Gogo prevents bundling of extending R1-6 axons.

Conclusion

Taken together, we propose that Gogo contributes to retinotopic map formation in the Drosophila lamina by controlling the distribution of R1–R6 axon fascicles. In a later developmental step, the regular position of R1–R6 axons along the lamina plexus is crucial for target cartridge selection. During cartridge elongation, Gogo allows R1–R6 axons to extend centrally in the lamina cartridge.