In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes

Background  

Serum paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme involved in organophosphate (OP) degradation and prevention of atherosclerosis. PON1 comprises a potential candidate for in vivo therapeutics, as an anti-atherogenic agent, and for detoxification of pesticides and nerve agents. Because human PON1 exhibits limited stability, engineered, recombinant PON1 (rePON1) variants that were designed for higher reactivity, solubility, stability, and bacterial expression, are candidates for treatment. This work addresses the feasibility of in vivo administration of rePON1, and its HDL complex, as a potentially therapeutic agent dubbed BL-3050.     

Using genome-wide measurements for computational prediction of SH2–peptide interactions

Peptide-recognition modules (PRMs) are used throughout biology to mediate protein–protein interactions, and many PRMs are members of large protein domain families. Recent genome-wide measurements describe networks of peptide–PRM interactions. In these networks, very similar PRMs recognize distinct sets of peptides, raising the question of how peptide-recognition specificity is achieved using similar protein domains. The analysis of individual protein complex structures often gives answers that are not easily applicable to other members of the same PRM family. Bioinformatics-based approaches, one the other hand, may be difficult to interpret physically. Here we integrate structural information with a large, quantitative data set of SH2 domain–peptide interactions to study the physical origin of domain–peptide specificity. We develop an energy model, inspired by protein folding, based on interactions between the amino-acid positions in the domain and peptide. We use this model to successfully predict which SH2 domains and peptides interact and uncover the positions in each that are important for specificity. The energy model is general enough that it can be applied to other members of the SH2 family or to new peptides, and the cross-validation results suggest that these energy calculations will be useful for predicting binding interactions. It can also be adapted to study other PRM families, predict optimal peptides for a given SH2 domain, or study other biological interactions, e.g. protein–DNA interactions.     

Expression Quantitative Trait Loci Are Highly Sensitive to Cellular Differentiation State

Genetical genomics is a strategy for mapping gene expression variation to expression quantitative trait loci (eQTLs). We performed a genetical genomics experiment in four functionally distinct but developmentally closely related hematopoietic cell populations isolated from the BXD panel of recombinant inbred mouse strains. This analysis allowed us to analyze eQTL robustness/sensitivity across different cellular differentiation states. Although we identified a large number (365) of “static” eQTLs that were consistently active in all four cell types, we found a much larger number (1,283) of “dynamic” eQTLs showing cell-type–dependence. Of these, 140, 45, 531, and 295 were preferentially active in stem, progenitor, erythroid, and myeloid cells, respectively. A detailed investigation of those dynamic eQTLs showed that in many cases the eQTL specificity was associated with expression changes in the target gene. We found no evidence for target genes that were regulated by distinct eQTLs in different cell types, suggesting that large-scale changes within functional regulatory networks are uncommon. Our results demonstrate that heritable differences in gene expression are highly sensitive to the developmental stage of the cell population under study. Therefore, future genetical genomics studies should aim at studying multiple well-defined and highly purified cell types in order to construct as comprehensive a picture of the changing functional regulatory relationships as possible.     

α‐MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV‐induced DNA damage in human melanocytes

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of α‐melanocortin (α‐MSH) that were more potent and stable than the physiological α‐MSH, and mimicked its photoprotective effects against UV‐induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified α‐MSH core His⁶‐d ‐Phe⁷‐Arg⁸, which contained different N‐capping groups, C‐terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C‐terminal modifications. The most effective C‐terminal tripeptide mimicked α‐MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non‐functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.     

Cholinergic modulation of activation sequence in the atrial myocardium of non-mammalian vertebrates

Cholinergic changes of electric activity were studied in isolated atrium preparations from fishes (cod and carp), amphibians (frog) and reptilians (lizard) using the microelectrode technique and high-resolution optical mapping. Perfusion of isolated atrium with acetylcholine (10^? 6–5 · 10^? 5 M) caused gradual suppression of action potential generation and, eventually, completely blocked the excitation in a part of the preparation. Other regions of atrium, situated close to the sinoatrial and atrioventricular junctions, remained excitable. Such cholinergic suppression of electric activity was observed in the atrial myocardium of frog and in both fish species, but not in reptilians. Ba²⁺ (10^? 4 M), which blocks the acetylcholine-dependent potassium current (I_KACh), prevented cholinergic reduction of action potential amplitude. In several preparations of frog atrium, cholinergic suppression of excitation coincided with episodes of atrial fibrillation. We conclude that the phenomenon of cholinergic suppression of electric activity is typical for atria of fishes and amphibians. It is likely to be caused by I_KACh activation and may be important for initiation of atrial arrhythmias.     

Near Scale-Free Dynamics in Neural Population Activity of Waking/Sleeping Rats Revealed by Multiscale Analysis

A neuron embedded in an intact brain, unlike an isolated neuron, participates in network activity at various spatial resolutions. Such multiple scale spatial dynamics is potentially reflected in multiple time scales of temporal dynamics. We identify such multiple dynamical time scales of the inter-spike interval (ISI) fluctuations of neurons of waking/sleeping rats by means of multiscale analysis. The time scale of large non-Gaussianity in the ISI fluctuations, measured with the Castaing method, ranges up to several minutes, markedly escaping the low-pass filtering characteristics of neurons. A comparison between neural activity during waking and sleeping reveals that non-Gaussianity is stronger during waking than sleeping throughout the entire range of scales observed. We find a remarkable property of near scale independence of the magnitude correlations as the primary cause of persistent non-Gaussianity. Such scale-invariance of correlations is characteristic of multiplicative cascade processes and raises the possibility of the existence of a scale independent memory preserving mechanism.     

Polymorphisms of the ITGAM gene confer higher risk of discoid cutaneous than of systemic lupus erythematosus

Background

Lupus erythematosus (LE) is a heterogeneous disease ranging from mainly skin-restricted manifestations (discoid LE [DLE] and subacute cutaneous LE) to a progressive multisystem disease (systemic LE [SLE]). Genetic association studies have recently identified several strong susceptibility genes for SLE, including integrin alpha M (ITGAM), also known as CD11b, whereas the genetic background of DLE is less clear.

Principal Findings

To specifically investigate whether ITGAM is a susceptibility gene not only for SLE, but also for cutaneous DLE, we genotyped 177 patients with DLE, 85 patients with sporadic SLE, 190 index cases from SLE families and 395 population control individuals from Finland for nine genetic markers at the ITGAM locus. SLE patients were further subdivided by the presence or absence of discoid rash and renal involvement. In addition, 235 Finnish and Swedish patients positive for Ro/SSA-autoantibodies were included in a subphenotype analysis. Analysis of the ITGAM coding variant rs1143679 showed highly significant association to DLE in patients without signs of systemic disease (P-value  = 4.73×10⁻¹¹, OR  = 3.20, 95% CI  = 2.23–4.57). Significant association was also detected to SLE patients (P-value  = 8.29×10⁻⁶, OR  = 2.14, 95% CI  = 1.52–3.00), and even stronger association was found when stratifying SLE patients by presence of discoid rash (P-value  = 3.59×10⁻⁸, OR  = 3.76, 95% CI  = 2.29–6.18).

Significance

We propose ITGAM as a novel susceptibility gene for cutaneous DLE. The risk effect is independent of systemic involvement and has an even stronger genetic influence on the risk of DLE than of SLE.     

Genetic variants in toll-like receptors are not associated with rheumatoid arthritis susceptibility or anti-tumour necrosis factor treatment outcome

Background

Several studies point to a role of Toll-like receptors (TLRs) in the development of rheumatoid arthritis (RA). We investigated if genetic variants in TLR genes are associated with RA and response to tumour necrosis factor blocking (anti-TNF) medication.

Methodology and Principal Findings

22 single nucleotide polymorphisms (SNPs) in seven TLR genes were genotyped in a Dutch cohort consisting of 378 RA patients and 294 controls. Significantly associated variants were investigated in replication cohorts from The Netherlands, United Kingdom and Sweden (2877 RA patients and 2025 controls). 182 of the Dutch patients were treated with anti-TNF medication. Using these patients and a replication cohort (269 Swedish patients) we analysed if genetic variants in TLR genes were associated with anti-TNF outcome. In the discovery phase of the study we found a significant association of SNPs rs2072493 in TLR5 and rs3853839 in TLR7 with RA disease susceptibility. Meta-analysis of discovery and replication cohorts did not confirm these findings. SNP rs2072493 in TLR5 was associated with anti-TNF outcome in the Dutch but not in the Swedish population.

Conclusion

We conclude that genetic variants in TLRs do not play a major role in susceptibility for developing RA nor in anti-TNF treatment outcome in a Caucasian population.     

Abnormal compartmentalization of norepinephrine in mouse dentate gyrus in alpha-synuclein knockout and A30P transgenic mice

In the dentate gyrus of the mouse hippocampus, presynaptic recruitment of norepinephrine in response to repeated-burst stimulation can be described in terms of an interaction between storage and readily releasable pools. The dynamics of this interaction depends on neuronal activity (bursting), so that the higher the demand for norepinephrine, the faster it is delivered from the storage pool. We also found that alpha-synuclein, a presynaptic protein that plays a crucial role in dopamine compartmentalization in the striatum, is also involved in the compartmentalization of norepinephrine in the dentate gyrus. Experiments in transgenic mice with modified or absent alpha-synuclein revealed that the familial Parkinson's disease-linked alpha-synuclein mutation A30P can cause selective changes in the function of noradrenergic terminals. Addition of mutated human alpha-synuclein abolished the normal norepinephrine mobilization. There were no compensatory mechanisms available in the norepinephrine presynaptic terminals. In contrast, deletion of mouse alpha-synuclein is compensated for by increased vesicle transport from the storage pool. The effects are essentially the same as previously reported for dopaminergic terminals in the striatum, indicating that the important role of alpha-synuclein in neurotransmitter mobilization is not limited to dopaminergic terminals.