Molecular mechanisms creating bistable switches at cell cycle transitions

Progression through the eukaryotic cell cycle is characterized by specific transitions, where cells move irreversibly from stage i−1 of the cycle into stage i. These irreversible cell cycle transitions are regulated by underlying bistable switches, which share some common features. An inhibitory protein stalls progression, and an activatory protein promotes progression. The inhibitor and activator are locked in a double-negative feedback loop, creating a one-way toggle switch that guarantees an irreversible commitment to move forward through the cell cycle, and it opposes regression from stage i to stage i−1. In many cases, the activator is an enzyme that modifies the inhibitor in multiple steps, whereas the hypo-modified inhibitor binds strongly to the activator and resists its enzymatic activity. These interactions are the basis of a reaction motif that provides a simple and generic account of many characteristic properties of cell cycle transitions. To demonstrate this assertion, we apply the motif in detail to the G1/S transition in budding yeast and to the mitotic checkpoint in mammalian cells. Variations of the motif might support irreversible cellular decision-making in other contexts.     

Hydrophilic compounds in liquids of enzymatic hydrolyzed spruce and pine biomass

This study was focused on organic acids and metals in biofluids of wood. Without seasoning, fresh woods from spruce and bark, phloem, and heartwood from pine were used as materials, which were degraded with either microbes of oyster mushroom, baker’s yeast, or lactic acid bacteria. Due to neutral pH of the fluids, ambient temperature, atmospheric pressure, and short reaction time, native wood microbe populations were supposed to be present. The water content of the fresh woods was 4 to 20%. The study showed that process methodology and experimental conditions affected the generation of lactic, citric, succinic, and adipic acids, which are considered as source chemicals in the biopolymer industry. In addition to the organic acids and metals, the process produced monosaccharides, polysaccharides, and phenolic acids such as benzoic, salicylic, cinnamic, vanillic, tannic, and conifer (ferulic) acids. Concentrations of total acids and acetic and succinic acids in pine fluids from bark, phloem, and heartwood were 58.4 g/kg and 3.5 to 6.9 g/kg, respectively. In spruce, the most dominant acids were l-lactic and l-malic acids. As for metals, Ag and Cr were detected at 0.01-g/kg quantities in pine bark. Alkali metals K, Mg, Sr, and Ca were detected at 10, 8, 1.3, and 4 g/kg, respectively.     

Validation of Walking Trails for the Urban TrainingTM of Chronic Obstructive Pulmonary Disease Patients

Purpose

Accessible interventions to train patients with chronic obstructive pulmonary disease (COPD) are needed. We designed urban trails of different intensities (low, moderate and high) in different types of public spaces (boulevard, beach and park). We aimed to validate the trails’ design by assessing the physiological response to unsupervised walking trails of: (1) different intensities in COPD patients, and (2) same intensity from different public spaces in healthy adults.

Methods

On different days and under standardized conditions, 10 COPD patients walked the three intensity trails designed in a boulevard space, and 10 healthy subjects walked the three intensity trails in three different spaces. We measured physiological response and energy expenditure using a gas analyzer. We compared outcomes across trails intensity and/or spaces using mixed-effects linear regression.

Results

In COPD patients, physiological response and energy expenditure increased significantly according to the trails intensity: mean (SD) peak V˙O₂ 15.9 (3.5), 17.4 (4.7), and 17.7 (4.4) mL/min/kg (p-trend = 0.02), and MET-min 60 (23), 64 (26), 72 (31) (p-trend<0.01) in low, moderate and high intensity trails, respectively. In healthy subjects there were no differences in physiological response to walking trails of the same intensity across different spaces.

Conclusions

We validated the trails design for the training of COPD patients by showing that the physiological response to and energy expenditure on unsupervised walking these trails increased according to the predefined trails’ intensity and did not change across trails of the same intensity in different public space. Walkable public spaces allow the design of trails that could be used for the training of COPD patients in the community.     

An Improved Method for P2X7R Antagonist Screening

ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimer''s disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC₅₀ value of the known agonist ATP and the IC_s50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 mM and 173-285 μM for ATP, BBG and OATP, respectively). The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist     

Role of P2 Receptors as Modulators of Rat Eosinophil Recruitment in Allergic Inflammation

ATP and other nucleotides are released from cells through regulated pathways or following the loss of plasma membrane integrity. Once outside the cell, these compounds can activate P2 receptors: P2X ionotropic receptors and G protein-coupled P2Y receptors. Eosinophils represent major effector cells in the allergic inflammatory response and they are, in fact, associated with several physiological and pathological processes. Here we investigate the expression of P2 receptors and roles of those receptors in murine eosinophils. In this context, our first step was to investigate the expression and functionality of the P2X receptors by patch clamping, our results showed a potency ranking order of ATP>ATPγS> 2meSATP> ADP> αβmeATP> βγmeATP>BzATP> UTP> UDP>cAMP. This data suggest the presence of P2X1, P2X2 and P2X7. Next we evaluate by microfluorimetry the expression of P2Y receptors, our results based in the ranking order of potency (UTP>ATPγS> ATP > UDP> ADP >2meSATP > αβmeATP) suggests the presence of P2Y2, P2Y4, P2Y6 and P2Y11. Moreover, we confirmed our findings by immunofluorescence assays. We also did chemotaxis assays to verify whether nucleotides could induce migration. After 1 or 2 hours of incubation, ATP increased migration of eosinophils, as well as ATPγS, a less hydrolysable analogue of ATP, while suramin a P2 blocker abolished migration. In keeping with this idea, we tested whether these receptors are implicated in the migration of eosinophils to an inflammation site in vivo, using a model of rat allergic pleurisy. In fact, migration of eosinophils has increased when ATP or ATPγS were applied in the pleural cavity, and once more suramin blocked this effect. We have demonstrated that rat eosinophils express P2X and P2Y receptors. In addition, the activation of P2 receptors can increase migration of eosinophils in vitro and in vivo, an effect blocked by suramin.