Kenneth O. Willeford

Prediction of Cell Penetrating Peptides by Support Vector Machines

Cell penetrating peptides (CPPs) are those peptides that can transverse cell membranes to enter cells. Once inside the cell, different CPPs can localize to different cellular components and perform different roles. Some generate pore-forming complexes resulting in the destruction of cells while others localize to various organelles. Use of machine learning methods to predict potential new CPPs will enable more rapid screening for applications such as drug delivery. We have investigated the influence of the composition of training datasets on the ability to classify peptides as cell penetrating using support vector machines (SVMs). We identified 111 known CPPs and 34 known non-penetrating peptides from the literature and commercial vendors and used several approaches to build training data sets for the classifiers. Features were calculated from the datasets using a set of basic biochemical properties combined with features from the literature determined to be relevant in the prediction of CPPs. Our results using different training datasets confirm the importance of a balanced training set with approximately equal number of positive and negative examples. The SVM based classifiers have greater classification accuracy than previously reported methods for the prediction of CPPs, and because they use primary biochemical properties of the peptides as features, these classifiers provide insight into the properties needed for cell-penetration. To confirm our SVM classifications, a subset of peptides classified as either penetrating or non-penetrating was selected for synthesis and experimental validation. Of the synthesized peptides predicted to be CPPs, 100% of these peptides were shown to be penetrating.

Reducing Mortality in Salmonella enterica Serovar Typhimurium-Infected Mice with a Tripeptidic Serum Fraction

ABSTRACT Salmonellosis-induced mortality in female Swiss Webster mice decreased significantly when tripeptidic immunostimulant (TPI) was administered prophylactically. Prophylactic benefits developed in a dose-dependent manner wherein 15 mg of TPI given 1 day before challenge reduced mortality by 70%.

Interleukin-6 expression in response to innate immune regulatory factor stimulation

Innate immune regulatory factor (IIRF) is a serum-derived biological response modifier, or agent that modifies/bolsters the pathogenic immune response. IIRF has been shown to provide protection (e.g., increase host survival) against a variety of pathogenic challenges including Salmonella typhimurium and Pasteurella multocida. In this report, we analyzed cytokine production in mice, whole blood and cultured cells in response to IIRF challenge. When IIRF was administered to mice, it stimulated the release of pro-inflammatory cytokines. Both IL-6 and IL-10 were temporally stimulated when IIRF was introduced. Serum IL-6 peaked at 3h (3957pg/ml) before returning to baseline by 8h, while IL-10 began to appear at 3h, peaked at 8h (734.5pg/ml), and returned to baseline by 36h. IIRF challenge also increased the expression of two positive acute phase proteins: haptoglobin increased 61-fold, while serum amyloid A levels increased ∼3500-fold in mice. A whole blood immunoassay indicated the effect of IIRF on production of IL-6 was dose- and time-dependent. Anti-asialo GM 1.1 provided to partially (>95%) eliminate functional natural killer cells elevated IL-6 in whole blood. IIRF was also able to induce the production of IL-6 in cultured human monocytic THP-1 cells. Based upon this study and previously reported data, we propose that IIRF activates the innate immune response via induction of IL-6 production.

Comparison of inhibition kinetics of several organophosphates, including some nerve agent surrogates, using human erythrocyte and rat and mouse brain acetylcholinesterase.

Because testing of nerve agents is limited to only authorized facilities, our laboratory developed several surrogates that resemble nerve agents because they phosphylate the acetylcholinesterase (AChE) with the same moiety as the actual nerve agents. The inhibition kinetic parameters were determined for AChE by surrogates of cyclosarin (NCMP), sarin (NIMP, PIMP and TIMP) and VX (NEMP and TEMP) and other organophosphorus compounds derived from insecticides. All compounds were tested with rat brain and a subset was tested with mouse brain and purified human erythrocyte AChE. Within the compounds tested on all AChE sources, chlorpyrifos-oxon had the highest molecular rate constant followed by NCMP and NEMP. This was followed by NIMP then paraoxon and DFP with rat and mouse brain AChE but DFP was a more potent inhibitor than NIMP and paraoxon with human AChE. With the additional compounds tested only in rat brain, TEMP was slightly less potent than NEMP but more potent than PIMP which was more potent than NIMP. Methyl paraoxon was slightly less potent than paraoxon but more potent than TIMP which was more potent than DFP. Overall, this study validates that the pattern of inhibitory potencies of our surrogates is comparable to the pattern of inhibitory potencies of actual nerve agents (i.e., cyclosarin>VX>sarin), and that these are more potent than insecticidal organophosphates.

Serum Derived Transfer Factor Stimulates the Innate Immune System to Improve Survival Traits in High Risk Pathogen Scenarios

Preclinical Research

Quantification and comparison of baseline cortisol levels between aqueous and plasma from healthy anesthetized hound dogs utilizing mass spectrometry

OBJECTIVE The purpose of this study is to quantify and compare the resting cortisol levels between aqueous (Aq) and plasma of anesthetized hound dogs utilizing mass spectrometry. ANIMALS Nine hound breed dogs weighing between 20.8 and 29.2 kg (x = 26.3 kg, SD ± 2.6 kg) were utilized from a previous project. PROCEDURES All dogs underwent two anesthesia sessions to harvest Aq from each eye respectively. A paired blood sample was taken immediately after aqueous centesis. The Aq and plasma were analyzed for cortisol levels using mass spectrometry. Correlation of cortisol levels in dog serum and ocular fluid was determined with Sigma Stat using Pearsons correlation analysis. The level of significance for correlation analysis was set at P < 0.05. RESULTS The plasma resting cortisol levels in the dog ranged from 3.59 to 89.35 nm (x = 31.68 nm, SD ± 28.53 nm), while the Aq cortisol levels ranged from 0.82 to 5.62 nm (x = 2.73 nm, SD ± 1.94 nm). The correlation of cortisol levels in Aq compared to plasma was significant (P = 0.008) with a correlation coefficient of r = 0.779. Based on these results, 61% (r = 0.779(2) ) of the variation in Aq cortisol levels is explained by the variations in plasma cortisol levels. CONCLUSION AND CLINICAL RELEVANCE This study identified significant levels of cortisol in the Aq of the canine eye and positively correlated them with plasma cortisol levels. The dog may offer a representative model for further studies to help clarify the overall role of cortisol in the anterior chamber.

Phosphorylation of phosphoenolpyruvate carboxylase from Crassula argentea

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) was purified to electrophoretic homogeneity from Crassula argentea leaves according to established methods incorporating DEAE, hydroxylapatite, and Mono Q chromatography. The purified enzyme had a specific activity of 20-25 units/ mg of protein. Purified PEPC was further processed by blue agarose affinity chromatography and gel filtration to help ensure the removal of potential contaminating kinases. Autoradiography revealed that phosphorylation of PEPC occurred when the purified enzyme was incubated with [γ- 32 P]ATP-Mg 2+ . Radiolabel was not incorporated when [α- 32 P]ATP-Mg 2+ was utilized as substrate. Phosphorylation of the PEPC culminated in its activation: the K i for L-malate increased 2.5-fold while the maximum inhibition dropped from 73 to 39% and the K m for magnesium phosphoenolpyruvate dropped from 69 to 53 μM. These data are consistent with phosphorylation sensitive PEPC from C. argentea possessing an auto-kinase function or, alternatively, the copurification of a kinase that possesses a high specific activity and tightly associates with PEPC.