Conotoxin Prediction: New Features to Increase Prediction Accuracy.

Conotoxins are toxic, disulfide-bond-rich peptides from cone snail venom that target a wide range of receptors and ion channels with multiple pathophysiological effects. Conotoxins have extraordinary potential for medical therapeutics that include cancer, microbial infections, epilepsy, autoimmune diseases, neurological conditions, and cardiovascular disorders. Despite the potential for these compounds in novel therapeutic treatment development, the process of identifying and characterizing the toxicities of conotoxins is difficult, costly, and time-consuming. This challenge requires a series of diverse, complex, and labor-intensive biological, toxicological, and analytical techniques for effective characterization. While recent attempts, using machine learning based solely on primary amino acid sequences to predict biological toxins (e.g., conotoxins and animal venoms), have improved toxin identification, these methods are limited due to peptide conformational flexibility and the high frequency of cysteines present in toxin sequences. This results in an enumerable set of disulfide-bridged foldamers with different conformations of the same primary amino acid sequence that affect function and toxicity levels. Consequently, a given peptide may be toxic when its cysteine residues form a particular disulfide-bond pattern, while alternative bonding patterns (isoforms) or its reduced form (free cysteines with no disulfide bridges) may have little or no toxicological effects. Similarly, the same disulfide-bond pattern may be possible for other peptide sequences and result in different conformations that all exhibit varying toxicities to the same receptor or to different receptors. We present here new features, when combined with primary sequence features to train machine learning algorithms to predict conotoxins, that significantly increase prediction accuracy.

Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum.

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

Endometrial microstructure after long-term use of a 91-day extended-cycle oral contraceptive regimen.

OBJECTIVE: To assess the effect on the endometrial microstructure of an extended-cycle oral contraceptive (OC) regimen containing ethinyl estradiol (EE) and levonorgestrel (LNG). METHODOLOGY: Subjects received up to four cycles of a 91-day extended-cycle OC regimen (84 consecutive days of monophasic 30 microg EE/150 microg LNG followed by 7 days of placebo). Endometrial biopsies were performed prior to the initiation and at the completion of therapy. All endometrial samples were processed centrally and reviewed by three independent pathologists blinded to treatment groups. RESULTS: Endometrial biopsies were performed in 50 women. In general, samples taken after completion of therapy with no further hormonal exposure demonstrated rapid return to normal endometrial cycling. In contrast, the majority of subjects still on active extended hormonal OC therapy at the time of biopsy had inactive or atrophic endometrium. No intravascular blood clots were observed in any of the specimens. CONCLUSION: The endometrial findings observed in this cohort of women treated with a 91-day extended-cycle OC regimen for up to 1 year showed no significant pathology. Additionally, the endometrium reverted quickly to normal cyclic changes in those subjects who, after completing therapy, elected not to continue with hormonal contraception.