Discovery and Structure Activity Relationship of Small Molecule Inhibitors of Toxic β-Amyloid-42 Fibril Formation

Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity.     

CaV1.3 as pacemaker channels in adrenal chromaffin cells: specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca (2+) -dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (CaV1.2 and CaV1.3), CaV1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using CaV1 .3 (-/-) KO mice and the AP-clamp it has been possible to resolve the time course of CaV1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells CaV1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca (2+) currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca (2+) loading during long interspike intervals and, possibly, to control the Ca (2+) -dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.     

Dissection of reverse gyrase activities: insight into the evolution of a thermostable molecular machine

Reverse gyrase is a peculiar DNA topoisomerase, specific of thermophilic microorganisms, which induces positive supercoiling into DNA molecules in an ATP-dependent reaction. It is a modular enzyme and comprises an N-terminal helicase-like module fused to a C-terminal topoisomerase IA-like domain. The exact molecular mechanism of this unique reaction is not understood, and a fundamental mechanistic question is how its distinct steps are coordinated. We studied the cross-talk between the components of this molecular motor and probed communication between the DNA-binding sites and the different activities (DNA relaxation, ATP hydrolysis and positive supercoiling). We show that the isolated ATPase and topoisomerase domains of reverse gyrase form specific physical interactions, retain their own DNA binding and enzymatic activities, and when combined cooperate to achieve the unique ATP-dependent positive supercoiling activity. Our results indicate a mutual effect of both domains on all individual steps of the reaction. The C-terminal domain shows ATP-independent topoisomerase activity, which is repressed by the N-terminal domain in the full-length enzyme; experiments with the isolated domains showed that the C-terminal domain has stimulatory influence on the ATPase activity of the N-terminal domain. In addition, the two domains showed a striking reciprocal thermostabilization effect.     

Locomotor muscle fatigue increases cardiorespiratory responses and reduces performance during intense cycling exercise independently from metabolic stress

Locomotor muscle fatigue, defined as an exercise-induced reduction in maximal voluntary force, occurs during prolonged exercise, but its effects on cardiorespiratory responses and exercise performance are unknown. In this investigation, a significant reduction in locomotor muscle force (-18%, P < 0.05) was isolated from the metabolic stress usually associated with fatiguing exercise using a 100-drop-jumps protocol consisting of one jump every 20 s from a 40-cm-high platform. The effect of this treatment on time to exhaustion during high-intensity constant-power cycling was measured in study 1 (n = 10). In study 2 (n = 14), test duration (871 +/- 280 s) was matched between fatigue and control condition (rest). In study 1, locomotor muscle fatigue caused a significant curtailment in time to exhaustion (636 +/- 278 s) compared with control (750 +/- 281 s) (P = 0.003) and increased cardiac output. Breathing frequency was significantly higher in the fatigue condition in both studies despite similar oxygen consumption and blood lactate accumulation. In study 2, high-intensity cycling did not induce further fatigue to eccentrically-fatigued locomotor muscles. In both studies, there was a significant increase in heart rate in the fatigue condition, and perceived exertion was significantly increased in study 2 compared with control. These results suggest that locomotor muscle fatigue has a significant influence on cardiorespiratory responses and exercise performance during high-intensity cycling independently from metabolic stress. These effects seem to be mediated by the increased central motor command and perception of effort required to exercise with weaker locomotor muscles.     

Role of Capsular Colanic Acid in Adhesion of Uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Two types of binding sites for hexokinase, designated as Type A or Type B sites, have been shown to coexist on brain mitochondria. The ratio of these sites varies between species.HK1 attaches by reversibly binding to the voltage dependent anion channel (VDAC). Regarding the nature of hexokinase binding sites, we investigated if it was linked to distinct VDAC interactomes. We approached this question by 2D BN/SDS-PAGE of mitochondria, followed by mass spectrometry.Our results are consistent with the possibility that the ratio of Type A/Type B sites is due to differential VDAC interactions in bovine and rat neuronal cells.     

Insular Organization of Gene Space in Grass Genomes

Wheat and maize genes were hypothesized to be clustered into islands but the hypothesis was not statistically tested. The hypothesis is statistically tested here in four grass species differing in genome size, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, and Aegilops tauschii. Density functions obtained under a model where gene locations follow a homogeneous Poisson process and thus are not clustered are compared with a model-free situation quantified through a non-parametric density estimate. A simple homogeneous Poisson model for gene locations is not rejected for the small O. sativa and B. distachyon genomes, indicating that genes are distributed largely uniformly in those species, but is rejected for the larger S. bicolor and Ae. tauschii genomes, providing evidence for clustering of genes into islands. It is proposed to call the gene islands “gene insulae” to distinguish them from other types of gene clustering that have been proposed. An average S. bicolor and Ae. tauschii insula is estimated to contain 3.7 and 3.9 genes with an average intergenic distance within an insula of 2.1 and 16.5 kb, respectively. Inter-insular distances are greater than 8 and 81 kb and average 15.1 and 205 kb, in S. bicolor and Ae. tauschii, respectively. A greater gene density observed in the distal regions of the Ae. tauschii chromosomes is shown to be primarily caused by shortening of inter-insular distances. The comparison of the four grass genomes suggests that gene locations are largely a function of a homogeneous Poisson process in small genomes. Nonrandom insertions of LTR retroelements during genome expansion creates gene insulae, which become less dense and further apart with the increase in genome size. High concordance in relative lengths of orthologous intergenic distances among the investigated genomes including the maize genome suggests functional constraints on gene distribution in the grass genomes.     

Decision-Making in Multiple Sclerosis Consultations in Italy: Third Observer and Patient Assessments

Objective

To assess decision-making in multiple sclerosis (MS) from third observer and patient perspectives.

Method

Audio recordings of first-ever consultations with a participating physician (88 outpatients, 10 physicians) at four tertiary MS care clinics in Italy, were rated by a third observer using the Observing Patient Involvement in Shared Decision Making (OPTION) and by patients using the Perceived Involvement in Care Scale (PICS).

Results

Mean patient age was 37.5, 66% were women, 72% had MS, and 28% had possible MS or other disease. Mean PICS subscale scores (range 0 poor, 100 best possible) were 71.9 (SD 24.3) for "physician facilitation" (PICS-F); 74.6 (SD 22.9) for "patient information exchange" (PICS-I); and only 22.5 (SD 16.2) for "patient decision making" (PICS-DM). Mean OPTION total score (0 poor, 100 best possible) was 29.6 (SD 10.3). Poorest OPTION scores were found for items assessing “preferred patient approach to receiving information” and “preferred patient level of involvement.” Highest scores were for “clinician drawing attention to identified problem”, “indicating need for decision making,” and “need to review the decision.” Consultation time, woman physician, patient-physician gender concordance and PICS-F were associated with higher OPTION total score; older physician and second opinion consultation were associated with lower OPTION score.

Conclusions

In line with findings in other settings, our third observer findings indicated limited patient involvement abilities of MS physicians during first consultations. Patient perceptions of physician skills were better than third observers’, although they correlated. Consultations with women physicians, and younger physicians, were associated with higher third observer and patient-based scores. Our findings reveal a need to empower Italian MS physicians with better communication and shared decision-making skills, and show in particular that attention to MS patient preferences for reception of information and involvement in health decisions, need to be improved.