Meeting Report: Moving Upstream—Evaluating Adverse Upstream End Points for Improved Risk Assessment and Decision-Making

Background Assessing adverse effects from environmental chemical exposure is integral to public health policies. Toxicology assays identifying early biological changes from chemical exposure are increasing our ability to evaluate links between early biological disturbances and subsequent overt downstream effects. A workshop was held to consider how the resulting data inform consideration of an “adverse effect” in the context of hazard identification and risk assessment. Objectives Our objective here is to review what is known about the relationships between chemical exposure, early biological effects (upstream events), and later overt effects (downstream events) through three case studies (thyroid hormone disruption, antiandrogen effects, immune system disruption) and to consider how to evaluate hazard and risk when early biological effect data are available. Discussion Each case study presents data on the toxicity pathways linking early biological perturbations with downstream overt effects. Case studies also emphasize several factors that can influence risk of overt disease as a result from early biological perturbations, including background chemical exposures, underlying individual biological processes, and disease susceptibility. Certain effects resulting from exposure during periods of sensitivity may be irreversible. A chemical can act through multiple modes of action, resulting in similar or different overt effects. Conclusions For certain classes of early perturbations, sufficient information on the disease process is known, so hazard and quantitative risk assessment can proceed using information on upstream biological perturbations. Upstream data will support improved approaches for considering developmental stage, background exposures, disease status, and other factors important to assessing hazard and risk for the whole population

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

Sequence specific nonenzymatic ligation of single- and double-stranded DNA by triple helix formation

Phosphodiesters link the units of chemical information within nucleic acids. The formation of a phosphodiester linkage by condensation of phosphate and hydroxyl termini of DNA requires activation of the phosphate toward nucleophilic displacement and proper placement of the hydroxyl for nucleophilic attack on the activated phosphate in competition with water. Sequence information is transferred when a nucleic acid template promotes this condensation reaction by the sequence specific formation of a complex in which condensing functionalities are juxtaposed. This thesis describes investigations of the sequence specific formation of phosphodiester linkages by the assembly of triple-helical complexes. In the first part of Chapter I, sequence specific recognition of double stranded DNA by triple helix formation is reviewed. Structural features of nucleic acid triple helices, the sequence specificity of their formation, and functions associated with them are considered in this review. In the second part of Chapter I, literature regarding template-directed formation of phosphodiesters in aqueous solution is reviewed. Chapter 2 describes investigations of the sequence specific formation of a phosphodiester linkage between pyrimidine oligodeoxyribonucleotides using a double-stranded DNA template to juxtapose their termini in a triple helix. Several approaches to activation of the condensing phosphate were explored. The most effective of these was activation in situ with the condensing agent N -cyanoimidazole with which condensation yields greater than 80% could be obtained. The reaction was directed by the double stranded template and could be shown to form a 3',5' phosphodiester linkage between the two oligodeoxyribonucleotides. A single mismatch in one of the condensing oligodeoxyribonucleotides at the condensing terminus resulted in at least a 25 to 60 fold decrease in the rate of the reaction. The relevance of this reaction to the detection of sequences, nucleic acid catalysts, and the evolution of template-directed information transfer is discussed. Chapter 3 describes the nonenzymatic sequence specific ligation of blunt-ended duplex DNA by triple helix formation. Using a pyrimidine oligodeoxyribonucleotide as a template and N-cyanoimidazole as a condensing agent, a double-stranded plasmid with homopurine tracts at one 3' terminus and one 5' terminus could be covalently circularized In yields exceeding 50%. Ligation on both strands was demonstrated in some of the circularized product. Ligation of duplexes with homopurine tracts at their 3' termini was directed by a pyrimidine oligodeoxyribonucleotide of two segments joined 3' to 3' through an abasic linker, creating a duplex of the sequence type 5'-(purine)_m(pyrimidine)_n-3'. The linkages formed in the ligation reaction were demonstrated to be substrates for a restriction endonuclease, identifying them as phosphodiesters. The sequence specificity of these reactions is not accessible by enzymatic ligation of double-stranded DNA

NMR characterization of an oligonucleotide model of the miR-21 pre-element.

We have used NMR spectroscopy to characterize an oligonucleotide stem loop structure based on the pre-element of an oncogenic microRNA, miR-21. This predicted stem-loop structure is cleaved from the precursor of miR-21 (pre-miR-21) by the nuclease Dicer. It is also a critical feature recognized by the protein complex that converts the primary transcript (pri-miR-21) into the pre-miRNA. The secondary structure of the native sequence is poorly defined by NMR due to rapid exchange of imino protons with solvent; however, replacement of two adjacent putative G•U base pairs with G•C base pairs retains the conformation of the hairpin observed by chemical probing and stabilizes it sufficiently to observe most of the imino proton resonances of the molecule. The observed resonances are consistent with the predicted secondary structure. In addition, a peak due to a loop uridine suggests an interaction between it and a bulged uridine in the stem. Assignment of non-exchangeable proton resonances and characterization of NOEs and coupling constants allows inference of the following features of the structure: extrahelicity of a bulged adenosine, deviation from A-form geometry in a base-paired stem, and consecutive stacking of the adenosines in the 5' side of the loop, the guanosine of the closing base pair, and a cross-strand adenosine. Modeling of the structure by restrained molecular dynamics suggests a basis for the interaction between the loop uridine, the bulged uridine in the stem, and an A•U base pair in the stem