Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease is described. The identified inhibitors bearing an amino nitrile warhead in P1 exhibit low nanomolar in vitro potency against cruzipain. Further SAR in P2 portion led to the identification of compounds, such as 26, that have a unique selectivity profile against other cysteine proteases and offering new opportunities for safer treatment of Chagas disease.     

Multivariate denoising methods combining wavelets and principal component analysis for mass spectrometry data

The identification of new diagnostic or prognostic biomarkers is one of the main aims of clinical cancer research. In recent years, there has been a growing interest in using mass spectrometry for the detection of such biomarkers. The MS signal resulting from MALDI‐TOF measurements is contaminated by different sources of technical variations that can be removed by a prior pre‐processing step. In particular, denoising makes it possible to remove the random noise contained in the signal. Wavelet methodology associated with thresholding is usually used for this purpose. In this study, we adapted two multivariate denoising methods that combine wavelets and PCA to MS data. The objective was to obtain better denoising of the data so as to extract the meaningful proteomic biological information from the raw spectra and reach meaningful clinical conclusions. The proposed methods were evaluated and compared with the classical soft thresholding denoising method using both real and simulated data sets. It was shown that taking into account common structures of the signals by adding a dimension reduction step on approximation coefficients through PCA provided more effective denoising when combined with soft thresholding on detail coefficients.     

Effects of glycosylation on the structure and function of the extracellular chaperone clusterin

Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation. Although carbohydrate comprises approximately 20-25% of the total mass of the mature molecule, its function is unknown. Results from the current study demonstrate that deglycosylation of human serum clusterin had little effect on its overall secondary structure content but produced a small increase in solvent-exposed hydrophobicity and enhanced the propensity of the molecule to aggregate in solution. These changes were associated with increased binding to a variety of ligands but did not substantially impact the ability of clusterin to inhibit heat-induced precipitation of citrate synthase. Evidence suggesting that the normally conjugated sugars are important in the interaction of secreted clusterin with a lectin-type receptor on liver cells is also presented. Bulk expression of fully processed, glycosylated clusterin in mammalian cells is difficult, often producing inappropriately disulfide-bonded high molecular weight aggregates; this has hampered previous studies aimed at identifying those regions of the molecule important in its chaperone action. The current results suggest that it may be possible in the future to study the structure and chaperone function of clusterin using recombinant protein (lacking sugars) conveniently bulk-expressed in bacteria.     

Coping with Stress: The Effectiveness of Different Types of Music

Listening to classical and self-selected relaxing music after exposure to a stressor should result in significant reductions in anxiety, anger, and sympathetic nervous system arousal, and increased relaxation compared to those who sit in silence or listen to heavy metal music. Fifty-six college students, 15 males and 41 females, were exposed to different types of music genres after experiencing a stressful test. Several 4 x 2 mixed design analyses of variance were conducted to determine the effects of music and silence conditions (heavy metal, classical, or self-selected music and silence) and time (pre-post music) on emotional state and physiological arousal. Results indicate listening to self-select or classical music, after exposure to a stressor, significantly reduces negative emotional states and physiological arousal compared to listening to heavy metal music or sitting in silence.     

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer.     

A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation

Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.