Diverse Experimental Protocols Research Articles

Probiotics Modulate Host Immune Response and Interact with the Gut Microbiota: Shaping Their Composition and Mediating Antibiotic Resistance.

The consortium of microbes inhabiting the human body, together with their encoded genes and secreted metabolites, is referred to as the "human microbiome." Several studies have established a link between the composition of the microbiome and its impact on human health. This impact spans local gastrointestinal inflammation to systemic autoimmune disorders and neurodegenerative diseases such as Alzheimer's and Autism. Some of these links have been validated by rigorous experiments that identify specific strains as mediators or drivers of a particular condition. Consequently, the development of probiotics to compensate for a missing beneficial microbe(s) has advanced and become popular, especially in the treatment of irritable bowel diseases and to restore disrupted gut flora after antibiotic administration. The widespread use of probiotics is often advocated as a natural ecological therapy. However, this perception is not always accurate, as there is a potential for unexpected interactions when administering live microbial cultures. Here, we designed this research to explore the intricate interactions among probiotics, the host, and microbes through a series of experiments. Our objectives included assessing their immunomodulatory effects, response to oral medications, impact on microbial population dynamics, and mediation of antibiotic resistance. To achieve these goals, we employed diverse experimental protocols, including cell-based enzyme -linked immunosorbent assay (ELISA), antibiotic susceptibility testing, antimicrobial activity assays, computational prediction of probiotic genes responsible for antibiotic resistance, polymerase chain reaction (PCR)-based validation of predicted genes, and survival assays of probiotics in the presence of selected oral medications. Our findings highlight that more than half of the tested probiotics trigger an inflammatory response in the Caco-2 cell line, are influenced by oral medications, exhibit antibacterial activity, and possess genes encoding antimicrobial resistance. These results underscore the necessity for a reevaluation of probiotic usage and emphasize the importance of establishing regulations to govern probiotic testing, approval, and administration.

The daily rhythms of working memory and their methodological constraints: a critical overview

ABSTRACT Working memory (WM) performance is not constant throughout the 24-hour cycle. Daily oscillations in WM performance are modulated by interactions between circadian and homeostatic factors. However, investigations that ascertained the circadian rhythm(s) of WM found contradictory results regarding the moments in which the lowest and highest peaks of WM performance occur across the light-dark cycle. This review analyses the findings and methodological approaches of past studies that assessed the circadian cycle(s) of WM and explores the main methodological constraints that may explain their mixed findings. Such differences may be explained by (1) the use of diverse experimental protocols; (2) the employment of different degrees of control of confounding exogenous variables; (3) the application of different types of WM paradigms; (4) the use of different conceptualizations and operationalizations to assess the daily rhythm(s) of WM. Some investigations that evaluated the daily pattern of WM performance considered this cognitive function as a single unit and used general/single measures of WM capacity to estimate the parameters of its circadian cycle, while others tried to disentangle the daily rhythms of the subcomponents of WM. Findings of structural and neurobehavioral studies suggest that the subcomponents of WM present independent circadian rhythms.

Pharmacological Conditioning of the Heart: An Update on Experimental Developments and Clinical Implications.

The aim of pharmacological conditioning is to protect the heart against myocardial ischemia-reperfusion (I/R) injury and its consequences. There is extensive literature that reports a multitude of different cardioprotective signaling molecules and mechanisms in diverse experimental protocols. Several pharmacological agents have been evaluated in terms of myocardial I/R injury. While results from experimental studies are immensely encouraging, translation into the clinical setting remains unsatisfactory. This narrative review wants to focus on two aspects: (1) give a comprehensive update on new developments of pharmacological conditioning in the experimental setting concentrating on recent literature of the last two years and (2) briefly summarize clinical evidence of these cardioprotective substances in the perioperative setting highlighting their clinical implications. By directly opposing each pharmacological agent regarding its recent experimental knowledge and most important available clinical data, a clear overview is given demonstrating the remaining gap between basic research and clinical practice. Finally, future perspectives are given on how we might overcome the limited translatability in the field of pharmacological conditioning.

Systematic Establishment of Robustness and Standards in Patient-Derived Xenograft Experiments and Analysis.

Patient-derived xenografts (PDX) are tumor-in-mouse models for cancer. PDX collections, such as the NCI PDXNet, are powerful resources for preclinical therapeutic testing. However, variations in experimental and analysis procedures have limited interpretability. To determine the robustness of PDX studies, the PDXNet tested temozolomide drug response for three prevalidated PDX models (sensitive, resistant, and intermediate) across four blinded PDX Development and Trial Centers using independently selected standard operating procedures. Each PDTC was able to correctly identify the sensitive, resistant, and intermediate models, and statistical evaluations were concordant across all groups. We also developed and benchmarked optimized PDX informatics pipelines, and these yielded robust assessments across xenograft biological replicates. These studies show that PDX drug responses and sequence results are reproducible across diverse experimental protocols. In addition, we share the range of experimental procedures that maintained robustness, as well as standardized cloud-based workflows for PDX exome-sequencing and RNA-sequencing analyses and for evaluating growth. SIGNIFICANCE: The PDXNet Consortium shows that PDX drug responses and sequencing results are reproducible across diverse experimental protocols, establishing the potential for multisite preclinical studies to translate into clinical trials.

Calculations of consciousness

Electroencephalography (EEG) was introduced into anesthesia practice in the 1990s as a tool to titrate anesthetic depth. However, limitations in current analysis techniques have called into question whether these techniques improve standard of care, or instead call for improved, more ubiquitously applicable measures to assess anesthetic transitions and depth. This review highlights emerging analytical approaches and techniques from neuroscience research that have the potential to better capture anesthetic transitions to provide better measurements of anesthetic depth. Since the introduction of electroencephalography, neuroscientists, engineers, mathematicians, and clinicians have all been developing new ways of analyzing continuous electrical signals. Collaborations between these fields have proliferated several analytical techniques that demonstrate how anesthetics affect brain dynamics and conscious transitions. Here, we review techniques in the following categories: network science, integration and information, nonlinear dynamics, and artificial intelligence. Up-and-coming techniques have the potential to better clinically define and characterize altered consciousness time points. Such new techniques used alongside traditional measures have the potential to improve depth of anesthesia measurements and enhance an understanding of how the brain is affected by anesthetic agents. However, new measures will be needed to be tested for robustness in real-world environments and on diverse experimental protocols.

β-adrenergic signaling broadly contributes to LTP induction.

Long-lasting forms of long-term potentiation (LTP) represent one of the major cellular mechanisms underlying learning and memory. One of the fundamental questions in the field of LTP is why different molecules are critical for long-lasting forms of LTP induced by diverse experimental protocols. Further complexity stems from spatial aspects of signaling networks, such that some molecules function in the dendrite and some are critical in the spine. We investigated whether the diverse experimental evidence can be unified by creating a spatial, mechanistic model of multiple signaling pathways in hippocampal CA1 neurons. Our results show that the combination of activity of several key kinases can predict the occurrence of long-lasting forms of LTP for multiple experimental protocols. Specifically Ca2+/calmodulin activated kinase II, protein kinase A and exchange protein activated by cAMP (Epac) together predict the occurrence of LTP in response to strong stimulation (multiple trains of 100 Hz) or weak stimulation augmented by isoproterenol. Furthermore, our analysis suggests that activation of the β-adrenergic receptor either via canonical (Gs-coupled) or non-canonical (Gi-coupled) pathways underpins most forms of long-lasting LTP. Simulations make the experimentally testable prediction that a complete antagonist of the β-adrenergic receptor will likely block long-lasting LTP in response to strong stimulation. Collectively these results suggest that converging molecular mechanisms allow CA1 neurons to flexibly utilize signaling mechanisms best tuned to temporal pattern of synaptic input to achieve long-lasting LTP and memory storage.

A Comprehensive Database and Analysis Framework To Incorporate Multiscale Data Types and Enable Integrated Analysis of Bioactive Polyphenols.

The development of a given botanical preparation for eventual clinical application requires extensive, detailed characterizations of the chemical composition, as well as the biological availability, biological activity, and safety profiles of the botanical. These issues are typically addressed using diverse experimental protocols and model systems. Based on this consideration, in this study we established a comprehensive database and analysis framework for the collection, collation, and integrative analysis of diverse, multiscale data sets. Using this framework, we conducted an integrative analysis of heterogeneous data from in vivo and in vitro investigation of a complex bioactive dietary polyphenol-rich preparation (BDPP) and built an integrated network linking data sets generated from this multitude of diverse experimental paradigms. We established a comprehensive database and analysis framework as well as a systematic and logical means to catalogue and collate the diverse array of information gathered, which is securely stored and added to in a standardized manner to enable fast query. We demonstrated the utility of the database in (1) a statistical ranking scheme to prioritize response to treatments and (2) in depth reconstruction of functionality studies. By examination of these data sets, the system allows analytical querying of heterogeneous data and the access of information related to interactions, mechanism of actions, functions, etc., which ultimately provide a global overview of complex biological responses. Collectively, we present an integrative analysis framework that leads to novel insights on the biological activities of a complex botanical such as BDPP that is based on data-driven characterizations of interactions between BDPP-derived phenolic metabolites and their mechanisms of action, as well as synergism and/or potential cancellation of biological functions. Out integrative analytical approach provides novel means for a systematic integrative analysis of heterogeneous data types in the development of complex botanicals such as polyphenols for eventual clinical and translational applications.

The two-process model of sleep regulation: a reappraisal.

In the last three decades the two-process model of sleep regulation has served as a major conceptual framework in sleep research. It has been applied widely in studies on fatigue and performance and to dissect individual differences in sleep regulation. The model posits that a homeostatic process (Process S) interacts with a process controlled by the circadian pacemaker (Process C), with time-courses derived from physiological and behavioural variables. The model simulates successfully the timing and intensity of sleep in diverse experimental protocols. Electrophysiological recordings from the suprachiasmatic nuclei (SCN) suggest that S and C interact continuously. Oscillators outside the SCN that are linked to energy metabolism are evident in SCN-lesioned arrhythmic animals subjected to restricted feeding or methamphetamine administration, as well as in human subjects during internal desynchronization. In intact animals these peripheral oscillators may dissociate from the central pacemaker rhythm. A sleep/fast and wake/feed phase segregate antagonistic anabolic and catabolic metabolic processes in peripheral tissues. A deficiency of Process S was proposed to account for both depressive sleep disturbances and the antidepressant effect of sleep deprivation. The model supported the development of novel non-pharmacological treatment paradigms in psychiatry, based on manipulating circadian phase, sleep and light exposure. In conclusion, the model remains conceptually useful for promoting the integration of sleep and circadian rhythm research. Sleep appears to have not only a short-term, use-dependent function; it also serves to enforce rest and fasting, thereby supporting the optimization of metabolic processes at the appropriate phase of the 24-h cycle.

QTreds: a flexible LIMS for omics laboratories

QTreds: a flexible LIMS for omics laboratories

ChemInform Abstract: Recent Efforts Directed to the Development of More Sustainable Asymmetric Organocatalysis

AbstractReview: 211 refs.

Recent efforts directed to the development of more sustainable asymmetric organocatalysis

In line with the principles of "green" chemistry, organocatalysis seeks to reduce energy consumption and to optimize the use of the available resources, aiming to become a sustainable strategy in chemical transformations. Nevertheless, during the last decade diverse experimental protocols have made organocatalysis an even "greener" alternative by the use of friendlier reaction conditions, or via the application of solvent-free methodologies, or through the design and synthesis of more selective catalysts, or via the development of multicomponent one-pot organocatalytic reactions, or by the recycling and reuse of organocatalysts, or by means of the application of more energy-efficient activation techniques, among other approaches. In this feature article we review some of the remarkable advancements that have made it possible to develop even more sustainable asymmetric organocatalyzed methodologies.

Comparative effects of fatty acids on endothelial inflammatory gene expression

Endothelial dysfunction may be related to adverse effects of some dietary fatty acids (FAs). Although in vitro studies have failed to show consistent findings, this may reflect the diverse experimental protocols employed and the limited range of FAs and end points studied. To investigate the effect of dietary FA type (saturated, monounsaturated, n-6 and n-3 polyunsaturated fatty acids), concentration, incubation time and cell stimulation state, on a broad spectrum of endothelial inflammatory gene expression. Using human umbilical vein endothelial cells, with and without stimulation (+/-10 ng/ml TNFalpha), the effects of arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA), linoleic (LA), oleic (OA) and palmitic acids (PA) (10, 25 and 100 microM), on the expression of genes encoding a number of inflammatory proteins and transcription factors were assessed by quantitative real time RT-PCR. Individual FAs differentially affect endothelial inflammatory gene expression in a gene-specific manner. EPA, LA and OA significantly up-regulated MCP-1 gene expression compared to AA (p = 0.001, 0.013, 0.008, respectively) and DHA (p < 0.0005, = 0.004, 0.002, respectively). Furthermore, cell stimulation state and FA incubation time significantly influenced reported FA effects on gene expression. The comparative effects of saturated, monounsaturated, n-6 and n-3 polyunsaturated FAs on endothelial gene expression depend on the specific FA investigated, its length of incubation, cell stimulation state and the gene investigated. These findings may explain existing disparity in the literature.

The nature of the heterozygous Trp53 knockout model for identification of mutagenic carcinogens.

The heterozygous Trp53 null allele C57BL/6 (N5) mouse is susceptible to the rapid development of neoplasia by mutagenic carcinogens relative to control strains. This mouse model of chemical carcinogenesis demonstrates 1) dose-related rapid induction of tumors (26 wks), 2) multiple sites of carcinogen-specific tissue susceptibility, and 3) carcinogen-induced loss of heterozygosity involving the Trp53 wild-type allele or a p53 haploinsufficiency permitting mutation of other critical protooncogenes and/or inactivation of tumor suppressor genes driving tumorigenesis. Demonstration of mutation or loss of heterozygosity involving the Trp53 locus is consistent with a common finding in human cancers and supports extrapolation between rodents and humans. Using diverse experimental protocols, almost all mutagenic rodent carcinogens (including all mutagens that are carcinogenic to humans), but not nonmutagenic rodent carcinogens, induce tumors within 26 weeks of continuous exposure. These characteristics and results indicate that the mouse heterozygous for the Trp53 null allele may be of significant use for the prospective identification of mutagenic carcinogens of potential risk to human health.

Cell surface and substrate distribution of the 67-kDa laminin-binding protein determined by using a ligand photoaffinity probe.

Peptide 11, a nine-amino acid sequence from the beta1 chain of laminin-1, has been reported to inhibit tumor cell invasion of basement membranes, and to reduce tumor lung colonization (Iwamoto et al.: Science 238:1132-1134, 1987; Landowski et al.: Clin Exp Metastasis 13:357-372, 1995). The peptide is a ligand for the 32/67-kDa laminin-binding protein (LBP); however, the mechanism by which the 67-kDa LBP promotes invasion is unknown. We have synthesized a highly specific probe for the 67-kDa LBP by adding a biotinylated residue, and replacing the required tyrosine in peptide 11 with the photoactivatable bezophenone crosslinker, 4-benzoyl-L-phenylalanine. This probe was used to follow the distribution of the 67-kDa LBP by gel electrophoresis, fluorescence-activated cell scanning, and confocal microscopy techniques. A single crosslinked protein, consistent with the high molecular weight form of the LBP, was found on Western blots of membrane detergent extracts from cells treated with the ligand probe. A CHO cell line, manipulated to overexpress the laminin-specific alpha6beta1 integrin, exhibited increased invasiveness, and expressed more cell surface 67-kDa LBP. Membrane-associated 67-kDa LBP was found in the vicinity of focal adhesion plaques and also associated with the matrix substrate. Studies on conditioned medium indicated that the matrix-associated LBP derived from material that was shed from the cells, with more being shed from the more invasive CHO variants. These results demonstrate the utility of this novel probe in diverse experimental protocols, and suggest that shedding of the 67-kDa LBP may have a role in promoting tumor cell invasion.

Experiment manager software for an automated chemistry workstation, including a scheduler for parallel experimentation

An automation system with the ability to work relentlessly, precisely, strategically, adn autonomously in pursuit of scientific goals will provide a powerful tool for tackling difficult scientific problems. We now present our work over the past seven years aimed at developing the hardware and software architecture for such an automated chemistry workstation. The workstation is designed for microscale experimentation in relatively clean domains of synthetic chemistry. This paper has two themes. First, the software system is presented in a comprehensive manner. An experiment manager software package has been developed that provides for composition of experimental plans, controls all aspects of automated experimentation, and manages the data. Experiment manager software is comprised of open-loop and closed-loop experiment planners bundled together with supporting features for timing, scheduling, material databases, resource management, automated start-up procedures, running display, data handling, configuration options, and maintenance operations. The experiment planners provide a menu-driven user interface, editing features, and a modular set of procedural events with which diverse experimental protocols can be composed. An overarching objective has been to achieve versatility in experiment planning yet still maintain access to the power that parallelism can confer in experimentation. Accordingly, the second theme in this paper is a description of approaches for performing experiments in parallel. Parallelism originates chiefly through simultaneous processing of samples at semi-autonomous hardware modules, at the user interface, and through the use of a scheduler. The scheduler takes as input a set of experimental protocols, establishes a sequence of the protocols, then interleaves the experimental protocols without altering the relative times of the procedures within each protocol. Experimental throughput can be increased by up to ten-fold by this approach. The straightforward planning and implementation of concurrent experimental protocols should yield major increases in research productivity.

Experiment planner for strategic experimentation with an automated chemistry workstation

An automation system capable of modifying its actions based on experimental data will serve as a useful assistant to the scientist. In order to achieve flexibility in experimentation, we have developed a software system for stating experimental protocols that involve decision-making based on experimental data. These decision-making capabilities enable strategic experimentation, where experiments are altered or terminated based on prior or newly acquired data. An experiment planner provides for experimentation in an automated chemistry workstation using the simplex algorithm or a decision-tree programming approach. The composite modified simplex algorithm is used for optimization experiments. Simplex experiments are planned through a series of menus. The simplex algorithm can be altered in sophistication by turning features on or off in software. The simplex algorithm is well-suited for optimizations but provides no general decision-making capabilities. A general decision-tree programming module enables the composition of diverse experimental protocols which capture the logic of experimentation. Experimental protocols written with a text editor are converted by an interpreter to a set of procedural events. Experimental protocols can be composed that execute incisive actions based on data from ongoing experiments. Protocols have been written for automatically matching samples with analytical instruments and performing an automated dye titration. Resource consumption cannot be predicted in advance in closed-loop experimentation, thus a static resource manager is used with each simplex cycle and a dynamic resource manager is used in decision-tree experimentation. The resource managers and strategic decision-making features empower the workstation to function autonomously in pursuit of the research objectives stated by the scientist.