Analysis of the Trajectory of Drosophila melanogaster in a Circular Open Field Arena

BACKGROUND: Obtaining a complete phenotypic characterization of a freely moving organism is a difficult task, yet such a description is desired in many neuroethological studies. Many metrics currently used in the literature to describe locomotor and exploratory behavior are typically based on average quantities or subjectively chosen spatial and temporal thresholds. All of these measures are relatively coarse-grained in the time domain. It is advantageous, however, to employ metrics based on the entire trajectory that an organism takes while exploring its environment. METHODOLOGY/PRINCIPAL FINDINGS: To characterize the locomotor behavior of Drosophila melanogaster, we used a video tracking system to record the trajectory of a single fly walking in a circular open field arena. The fly was tracked for two hours. Here, we present techniques with which to analyze the motion of the fly in this paradigm, and we discuss the methods of calculation. The measures we introduce are based on spatial and temporal probability distributions and utilize the entire time-series trajectory of the fly, thus emphasizing the dynamic nature of locomotor behavior. Marginal and joint probability distributions of speed, position, segment duration, path curvature, and reorientation angle are examined and related to the observed behavior. CONCLUSIONS/SIGNIFICANCE: The measures discussed in this paper provide a detailed profile of the behavior of a single fly and highlight the interaction of the fly with the environment. Such measures may serve as useful tools in any behavioral study in which the movement of a fly is an important variable and can be incorporated easily into many setups, facilitating high-throughput phenotypic characterization

Knots: Attractive Places with High Path Tortuosity in Mouse Open Field Exploration

When introduced into a novel environment, mammals establish in it a preferred place marked by the highest number of visits and highest cumulative time spent in it. Examination of exploratory behavior in reference to this “home base” highlights important features of its organization. It might therefore be fruitful to search for other types of marked places in mouse exploratory behavior and examine their influence on overall behavior

Behavioral Sequence Analysis Reveals a Novel Role for ß2* Nicotinic Receptors in Exploration

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system and modulate neuronal function in most mammalian brain structures. The contribution of defined nAChR subunits to a specific behavior is thus difficult to assess. Mice deleted for ß2-containing nAChRs (ß2−/−) have been shown to be hyperactive in an open-field paradigm, without determining the origin of this hyperactivity. We here develop a quantitative description of mouse behavior in the open field based upon first order Markov and variable length Markov chain analysis focusing on the time-organized sequence that behaviors are composed of. This description reveals that this hyperactivity is the consequence of the absence of specific inactive states or “stops”. These stops are associated with a scanning of the environment in wild-type mice (WT), and they affect the way that animals organize their sequence of behaviors when compared with stops without scanning. They characterize a specific “decision moment” that is reduced in ß2−/− mutant mice, suggesting an important role of ß2-nAChRs in the strategy used by animals to explore an environment and collect information in order to organize their behavior. This integrated analysis of the displacement of an animal in a simple environment offers new insights, specifically into the contribution of nAChRs to higher brain functions and more generally into the principles that organize sequences of behaviors in animals

Modulation of the peripheral blood transcriptome by the ingestion of probiotic yoghurt and acidified milk in healthy, young men

The metabolic health benefits of fermented milks have already been investigated using clinical biomarkers but the development of transcriptomic analytics in blood offers an alternative approach that may help to sensitively characterise such effects. We aimed to assess the effects of probiotic yoghurt intake, compared to non-fermented, acidified milk intake, on clinical biomarkers and gene expression in peripheral blood. To this end, a randomised, crossover study was conducted in fourteen healthy, young men to test the two dairy products. For a subset of seven subjects, RNA sequencing was used to measure gene expression in blood collected during postprandial tests and after two weeks daily intake. We found that the postprandial response in insulin was different for probiotic yoghurt as compared to that of acidified milk. Moreover changes in several clinical biomarkers were associated with changes in the expression of genes representing six metabolic genesets. Assessment of the postprandial effects of each dairy product on gene expression by geneset enrichment analysis revealed significant, similar modulation of inflammatory and glycolytic genes after both probiotic yoghurt and acidified milk intake, although distinct kinetic characteristics of the modulation differentiated the dairy products. The aryl hydrocarbon receptor was a major contributor to the down-regulation of the inflammatory genesets and was also positively associated with changes in circulating insulin at 2h after yoghurt intake (p = 0.05). Daily intake of the dairy products showed little effect on the fasting blood transcriptome. Probiotic yoghurt and acidified milk appear to affect similar gene pathways during the postprandial phase but differences in the timing and the extent of this modulation may lead to different physiological consequences. The functional relevance of these differences in gene expression is supported by their associations with circulating biomarkers

Optimisation of induction conditions for a bacterial strain producing proinsulin aspart

Diabetes poses a serious threat to the health of people around the world. Therefore, in 2021, the World Health Organisation launched the Global Diabetes Compact, an initiative aimed at improving the management and prevention of diabetes. The rapid growth in the number of diabetic patients has increased the need for insulin. Rapid-acting human insulin analogues, including insulin aspart, improve the efficacy of insulin therapy. Methods for insulin aspart production include its biosynthesis in the proinsulin form in Escherichia coli. However, the yield of the recombinant protein largely depends on the optimisation of the production process.The aim of the study was to optimise the induction conditions for an E. coli strain expressing recombinant proinsulin aspart through applying the Design of Experiment (DoE) approach to enhance bacterial cell productivity.Materials and methods. The study focused on a strain of E. coli producing proinsulin aspart. The authors planned the experiment using MODDE software and the reduced face-centred central composite design (CCF) enabling the assessment of factor interactions and the creation of design spaces. The authors carried out fermentations of the producing strain in a 5 L Biostat® B bioreactor and measured proinsulin aspart concentrations by capillary gel electrophoresis. The results were analysed using GraphPad Prism 6.Results. Using the DoE approach, the authors optimised the conditions for the growth of the producer strain and the biosynthesis of proinsulin aspart. Based on data from response surface plots for wet biomass concentration, specific productivity, and volumetric productivity, as well as plotted models, the authors established design spaces for the induction of proinsulin aspart expression in E. coli. The plotted models demonstrated high predictive power and high reproducibility of the results. The authors successfully validated the induction process for the synthesis of proinsulin aspart in a bioreactor under optimised conditions. The volumetric productivity of the strain producing proinsulin aspart increased from 3.06±0.16 g/L (conventional conditions) to 4.93±0.80 g/L (optimised conditions).Conclusions. The authors achieved a 60% increase in the volumetric yield of proinsulin aspart. The study results may be used to intensify the industrial production of insulin aspart

Mouse Cognition-Related Behavior in the Open-Field: Emergence of Places of Attraction

Spatial memory is often studied in the Morris Water Maze, where the animal's spatial orientation has been shown to be mainly shaped by distal visual cues. Cognition-related behavior has also been described along “well-trodden paths”—spatial habits established by animals in the wild and in captivity reflecting a form of spatial memory. In the present study we combine the study of Open Field behavior with the study of behavior on well-trodden paths, revealing a form of locational memory that appears to correlate with spatial memory. The tracked path of the mouse is used to examine the dynamics of visiting behavior to locations. A visit is defined as either progressing through a location or stopping there, where progressing and stopping are computationally defined. We then estimate the probability of stopping at a location as a function of the number of previous visits to that location, i.e., we measure the effect of visiting history to a location on stopping in it. This can be regarded as an estimate of the familiarity of the mouse with locations. The recently wild-derived inbred strain CZECHII shows the highest effect of visiting history on stopping, C57 inbred mice show a lower effect, and DBA mice show no effect. We employ a rarely used, bottom-to-top computational approach, starting from simple kinematics of movement and gradually building our way up until we end with (emergent) locational memory. The effect of visiting history to a location on stopping in it can be regarded as an estimate of the familiarity of the mouse with locations, implying memory of these locations. We show that the magnitude of this estimate is strain-specific, implying a genetic influence. The dynamics of this process reveal that locations along the mouse's trodden path gradually become places of attraction, where the mouse stops habitually

Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma

Introduction. Romiplostim is an analogue of the fusion protein peptide of thrombopoietin (TPO), which increases platelet count by binding and activating the human thrombopoietin receptor (TPO-R). It is used to treat thrombocytopenia associated with chronic immune thrombocytopenia. For romiplostim, one of the possible adverse reactions from the immune system is immunogenicity: the production of anti-drug antibodies to the medicinal product, including neutralising antibodies, which may affect the efficacy and safety profile of the medicinal product.Aim. Validate the procedure for determining neutralising antibodies to romiplostim in human plasma for further clinical studies of immunogenicity.Materials and methods. The study used rabbit polyclonal antibodies to romiplostim, Nplate® produced by Amgen Europe as a standard sample; a placebo produced by LLC "GEROPHARM", a cell line 32D-hTPOR clone 63 with stable expression of human TPO receptor and a chemiluminescence assay kit CellTiter-Glo® Luminescent Cell Viability Assay produced by Promega to assess specificity. The experiment was carried out on cell line 32D-hTPOR clone 63, which was seeded on the first day and the neutralizing antibody concentrations were titrated with a constant concentration of romiplostim, then the chemiluminescence was detected on the second day. Statistical processing of the results was carried out using Prism 9 software.Result and discussion. The specificity of the procedure was demonstrated; at maximum concentration, the medicinal product differs from placebo by 309 times in the residual level of cell viability. The linearity of the procedure in terms of the coefficient determination is 0.9969. The precision of the procedure was determined: the repeatability was 1–9 %, the intermediate precision was 3–18 %. The coefficient of variation in selectivity of the procedure was 22 %. For the accuracy parameter, the values for recovery/spike were determined as 90–101 %. It was proven that there was no matrix effect.Conclusion. It can be stated that the procedure is linear, specific, highly precise, correct, selective and with a proven absence of matrix effect, which allows it to be used to determine the immunogenicity of romiplostim medicinal products in clinical studies

Physiology, pharmacology and prospects for dipeptidilpeptidase-4 inhibitors use

Modern requirements for the treatment of type 2 diabetes mellitus (DM2) include not only achieving a glycemic control, but also reducing the risk of developing cardiovascular complications. Dipeptidyl peptidase 4 (DPP-4) inhibitors are inferior in the effectiveness to some other actively developing groups of hypoglycemic drugs (SGLT2 inhibitors and GLP-1 receptor agonists); however, they seem relevant at the present time.The aim of the study is to analyze the literature data on the therapeutic potential and results of the of DPP-4 inhibitors research.Materials and methods. When searching for the review article materials, the abstracting databases of PubMed, Google Scholar and e-Library were used. The search was carried out on the publications for the period from 2006 to 2022, using the following keywords: DPP-4 inhibitors; glucagonlike peptide-1 (GLP-1); glucose-dependent insulinotropic peptide (GIP); sitagliptin, and other drugs.Results. DPP-4 belongs to the serine proteases family and is involved in the degradation of various chemokines and peptide hormones, including incretins secreted by intestinal L- and K-cells – GLP-1 and GIP. They regulate a postprandial insulin secretion and a β-cell function, modulate a fasting and postprandial glucagon secretion, regulate the eating behavior and have many pleiotropic (immunomodulatory, anti-inflammatory, antifibrotic, etc.) effects. DPP-4 inhibitors reduce an enzyme activity by 70–90%, increasing plasma incretin levels by 2–4 times and have been used to treat DM2 since 2006. Now there are 13 DPP-4 inhibitors on the market in different countries, differing primarily in pharmacokinetic parameters. They are actively used in the combination therapy for type 2 diabetes, increasing the glycemic control effectiveness without increasing the risk of hypoglycemia. The evidence is emerging about the therapeutic potential of DPP-4 inhibitors in COVID-19.Conclusion. A peroral form, an ability to create effective combinations with other hypoglycemic drugs without increasing the risk of hypoglycemia, the pleiotropic effects of DPP-4 inhibitors, make this group relevant at the present time