SENSIBILIDAD DE LA GARRAPATA COMÚN DEL BOVINO Rhipicephalus (Boophilus) microplus A IVERMECTINA ESTIMADA CON EL MODELO SIGMOIDEO DE RESPUESTA MÁXIMA O DE HILL

The aim of this study was to obtain pharmacodynamics parameters to detect resistance or susceptibility of R. microplus strains to ivermectin (IVM). Two larvae samples; a susceptible strain (S) and field isolation (T) were treated with increasing concentrations of IVM using the larvae immersion technique the efficacy values measured at 24 hours were analysed with the sigmoidal maximum response so called Hill model as statistical analysis. The results obtained showed that the IVM have an all or nothing response represented by the Hill coefficient value >1 in both samples. Additionally, a low concentration effect was observed as E0 de 12.83% (S) and 9.91% (T). The field isolation larvae were susceptible to IVM in comparison with the susceptible strain by the resistance ratio (RR) which in one case was not significantly greater that one (RR50= 0.756 and RR90=1.009).

Generative adversarial networks for construction of virtual populations of mechanistic models: simulations to study Omecamtiv Mecarbil action

Biophysical models are increasingly used to gain mechanistic insights by fitting and reproducing experimental and clinical data. The inherent variability in the recorded datasets, however, presents a key challenge. In this study, we present a novel approach, which integrates mechanistic modeling and machine learning to analyze in vitro cardiac mechanics data and solve the inverse problem of model parameter inference. We designed a novel generative adversarial network (GAN) and employed it to construct virtual populations of cardiac ventricular myocyte models in order to study the action of Omecamtiv Mecarbil (OM), a positive cardiac inotrope. Populations of models were calibrated from mechanically unloaded myocyte shortening recordings obtained in experiments on rat myocytes in the presence and absence of OM. The GAN was able to infer model parameters while incorporating prior information about which model parameters OM targets. The generated populations of models reproduced variations in myocyte contraction recorded during in vitro experiments and provided improved understanding of OM’s mechanism of action. Inverse mapping of the experimental data using our approach suggests a novel action of OM, whereby it modifies interactions between myosin and tropomyosin proteins. To validate our approach, the inferred model parameters were used to replicate other in vitro experimental protocols, such as skinned preparations demonstrating an increase in calcium sensitivity and a decrease in the Hill coefficient of the force–calcium (F–Ca) curve under OM action. Our approach thereby facilitated the identification of the mechanistic underpinnings of experimental observations and the exploration of different hypotheses regarding variability in this complex biological system.

Dose- and Sex-Dependent Changes in Hemoglobin Oxygen Affinity by the Micronutrient 5-Hydroxymethylfurfural and α-Ketoglutaric Acid

5-Hydroxymethylfurfural (5-HMF) is known to increase hemoglobin oxygen affinity (Hb–O2 affinity) and to induce a left shift of the oxygen dissociation curve (ODC). It is under investigation as a therapeutic agent in sickle cell anemia and in conditions where pulmonary oxygen uptake is deteriorated or limited (e.g., various clinical conditions or altitude exposure). The combination of 5-HMF and α-ketoglutaric acid (αKG) is commercially available as a nutritional supplement. To further elucidate dose effects, ODCs were measured in vitro in venous whole blood samples of 20 healthy volunteers (10 female and 10 male) after the addition of three different doses of 5-HMF, αKG and the combination of both. Linear regression analysis revealed a strong dose-dependent increase in Hb–O2 affinity for 5-HMF (R2 = 0.887; p < 0.001) and the commercially available combination with αKG (R2 = 0.882; p < 0.001). αKG alone increased Hb–O2 affinity as well but to a lower extent. Both the combination (5-HMF + αKG) and 5-HMF alone exerted different P50 and Hill coefficient responses overall and between sexes, with more pronounced effects in females. With increasing Hb–O2 affinity, the sigmoidal shape of the ODC was better preserved by the combination of 5-HMF and αKG than by 5-HMF alone. Concerning the therapeutic effects of 5-HMF, this study emphasizes the importance of adequate dosing in various physiological and clinical conditions, where a left-shifted ODC might be beneficial. By preserving the sigmoidal shape of the ODC, the combination of 5-HMF and αKG at low (both sexes) and medium (males only) doses might be able to better maintain efficient oxygen transport, particularly by mitigating potentially deteriorated oxygen unloading in the tissue. However, expanding knowledge on the interaction between 5-HMF and Hb–O2 affinity in vitro necessitates further investigations in vivo to additionally assess pharmacokinetic mechanisms.

Multiple molecular events determine stochastic cell fate switching in a eukaryotic bistable system

Eukaryotic transcriptional networks are often large and contain several levels of feedback regulation. Many of these networks exhibit bistability, the ability to generate and stably maintain two distinct transcriptional states and to switch between them. In certain instances, switching between cell states is stochastic, occurring in a small subset of cells of an isogenic population in a seemingly homogenous environment. Given the scarcity and unpredictability of switching in these cases, investigating the determining molecular events is challenging. White-opaque switching in the fungal species Candida albicans is a complex bistable eukaryotic system and can serve as an experimentally accessible model system to study characteristics important for bistability and stochastic cell fate switching. In standard lab media, switching is rare, and genetically identical cells maintain their cellular identity (either “white” or “opaque”) through thousands of cell divisions. By isolating populations of white or opaque cells and measuring switching frequencies, previous studies have elucidated the many differences between the two cell states and identified a set of transcriptional regulators needed for cell type switching. Yet little is known about the molecular events that determine the rare, stochastic switching events that occur in single cells. We use microfluidics combined with fluorescent reporters to directly observe rare switching events between the white and opaque states. We investigate the stochastic nature of switching by beginning with white cells and monitoring the activation of Wor1, a master regulator and marker for the opaque state, in single cells and throughout cell pedigrees. Our results indicate that switching requires two stochastic steps; first an event occurs that predisposes a lineage of cells to switch. In the second step, some but not all, of those predisposed cells rapidly express high levels of Wor1 and commit to the opaque state. To further understand the rapid rise in Wor1, we used a synthetic inducible system in Saccharomyces cerevisiae into which a controllable C. albicans Wor1 and a reporter for its transcriptional control region have been introduced. We document that Wor1 positive autoregulation is highly cooperative (Hill coefficient > 3), leading to rapid activation and producing an “all or none” rather than a graded response. Taken together, our results suggest that reaching a threshold level of a master regulator is sufficient to drive cell type switching in single cells and that an earlier molecular event increases the probability of reaching that threshold in certain small lineages of cells. Quantitative molecular analysis of bistability in the white-opaque circuit can serve as a model for the general understanding of complex circuits.

Critical Re-evaluation of the Slope Factor of the Operational Model of Agonism Short Title: Slope Factor of the Operational Model of Agonism

Although being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves. Functional analysis of OMA demonstrates that the slope factor implemented by Black et al. affects relationships among parameters of the OMA. Fitting of the OMA with Black et al. slope factor to concentration-response curves of experimental as well as theoretical data (homotropic allosteric modulation, substrate inhibition and non-competitive auto-inhibition) resulted in wrong estimates of operational efficacy and affinity. In contrast, fitting of the OMA modified by the Hill coefficient to the same data resulted in correct estimates of operational efficacy and affinity. Therefore OMA modified by the Hill coefficient should be preferred over Black et al. equation for ranking of agonism and subsequent analysis, like quantification of signalling bias, when concentration response curves differ in the slope factor.

TBP and SNAP50 transcription factors bind specifically to the Pr77 promoter sequence from trypanosomatid non-LTR retrotransposons

Background Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5′ ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported. Methods TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence. Results This study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein–protein interactions through other trypanosomatid nuclear proteins. Conclusions Identification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite. Graphic abstract

On slope factor of the operational model of agonism

Although being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1995, Black et al. introduced slope factor to the OMA that makes the OMA more flexible and allows for fitting steep as well as flat concentration-response curves. Here I opinion drawbacks of the slope factor implemented by Black et al. that affects relationships among parameters of the OMA. Instead, I propose the implementation of the Hill coefficient in the OMA that does not affect observed parameters. The OMA modified by the Hill coefficient is more practical in the determination of operational efficacies for agonism ranking and subsequent analysis, like quantification of signalling bias.

Characterization of Potency of the P2Y13 Receptor Agonists: A Meta-Analysis

P2Y13 is an ADP-stimulated G-protein coupled receptor implicated in many physiological processes, including neurotransmission, metabolism, pain, and bone homeostasis. Quantitative understanding of P2Y13 activation dynamics is important for translational studies. We systematically identified PubMed annotated studies that characterized concentration-dependence of P2Y13 responses to natural and synthetic agonists. Since the comparison of the efficacy (maximum response) is difficult for studies performed in different systems, we normalized the data and conducted a meta-analysis of EC50 (concentration at half-maximum response) and Hill coefficient (slope) of P2Y13-mediated responses to different agonists. For signaling events induced by heterologously expressed P2Y13, EC50 of ADP-like agonists was 17.2 nM (95% CI: 7.7–38.5), with Hills coefficient of 4.4 (95% CI: 3.3–5.4), while ATP-like agonists had EC50 of 0.45 μM (95% CI: 0.06–3.15). For functional responses of endogenously expressed P2Y13, EC50 of ADP-like agonists was 1.76 μM (95% CI: 0.3–10.06). The EC50 of ADP-like agonists was lower for the brain P2Y13 than the blood P2Y13. ADP-like agonists were also more potent for human P2Y13 compared to rodent P2Y13. Thus, P2Y13 appears to be the most ADP-sensitive receptor characterized to date. The detailed understanding of tissue- and species-related differences in the P2Y13 response to ADP will improve the selectivity and specificity of future pharmacological compounds.

Cytochrome c adsorption in a continuous flow system by using Cu(II)-chelated magnetic affinity particles

Background and methodology: In the current study magnetic poly(ethylene glycoldimethacrylate-N-methacryloyl-(L)-histidine methyl ester) poly(Egdma–Mah)) was used as ametal chelated affinity particles. Cu2+ ions loaded directly to MAH ligand of magnetic particlesfor the adsorption of cytochrome c (Cyt c) in a continuous flow system. Results: The maximumCyt c adsorption capacity on the magnetic particles and Cu2+-chelated magnetic particles were42 mg/g and 197 mg/g in phosphate buffer (pH 8.0), respectively. Cu2+ loading increased theCyt c adsorption capacity, significantly. Cyt c adsorption capacity decreased with increasedtemperature and with increasing magnetic field. According to reusability studies Cyt c moleculescould be reversibly adsorbed and desorbed five times. The binding isotherm was determined byscatchard analysis followed by application of Hill equation to the data obtained, then bindingconstant and nH Hill coefficient were calculated. Bangladesh Journal of Medical Science Vol.20(1) 2021 p.159-163

Entangled gene regulatory networks with cooperative expression endow robust adaptive responses to unforeseen environmental changes

Living organisms must respond to environmental changes. Generally, accurate and rapid responses are provided by simple, unidirectional networks that connect inputs with outputs. Besides accuracy and speed, however, biological responses should also be robust to environmental or intracellular noise and mutations. Furthermore, cells must also respond to unforeseen environmental changes that have not previously been experienced, to avoid extinction prior to the evolutionary rewiring of their networks, which takes numerous generations. To address the question how cells can make robust adaptation even to unforeseen challenges, we have investigated gene regulatory networks that mutually activate or inhibit, and have demonstrated that complex entangled networks can make appropriate input-output relationships that satisfy such adaptive responses. Such entangled networks function when the expression of each gene shows sloppy and unreliable responses with low Hill coefficient reactions. To compensate for such sloppiness, several detours in the regulatory network exist. By taking advantage of the averaging over such detours, the network shows a higher robustness to environmental and intracellular noise as well as to mutations in the network, when compared to simple unidirectional circuits. Furthermore, it is demonstrated that the appropriate response to unforeseen environmental changes, allowing for functional outputs, is achieved as many genes exhibit similar dynamic expression responses, irrespective of inputs including unforeseen inputs. The similarity of the responses is statistically confirmed by applying dynamic time warping and dynamic mode decomposition methods. As complex entangled networks are commonly observed in the data in gene regulatory networks whereas global gene expression responses are measured in transcriptome analysis in microbial experiments, the present results give an answer how cells make adaptive responses and also provide a novel design principle for cellular networks.Author summaryRecent experimental advances have demonstrated that cells often have appropriate, robust responses to environmental changes, including those that have not previously been experienced. It is known that accurate and rapid responses can be achieved by simple unidirectional networks that connect straightforwardly input and outputs. However, such responses were not robust to perturbations. Here we have made numerical evolution of gene regulatory networks with mutual activation and inhibitions, and uncovered that complex entangled networks including many feedforward and feedback paths can make robust input-output responses, when each gene expression is not accurate. Remarkably, they make appropriate responses even to unforeseen environmental changes, as are supported by global, correlated responses across genes that are similar for all input signals. The results explain why cells adopt complex gene regulatory networks and exhibit global expression changes, even though they may not be advantageous in terms of their energy cost or response speed. The present results are consistent with the recent experiments on microbial gene expression changes and network analyses. This investigation provides insights into how cells survive fluctuating and unforeseen unpredictable environmental changes, and gives a universal conceptual framework to go beyond the standard picture based on a combination of network motifs.