Experimental Considerations Research Articles

The Mediator complex as a master regulator of transcription by RNA polymerase II.

The Mediator complex, which in humans is 1.4 MDa in size and includes 26 subunits, controls many aspects of RNA polymerase II (Pol II) function. Apart from its size, a defining feature of Mediator is its intrinsic disorder and conformational flexibility, which contributes to its ability to undergo phase separation and to interact with a myriad of regulatory factors. In this Review, we discuss Mediator structure and function, with emphasis on recent cryogenic electron microscopy data of the 4.0-MDa transcription preinitiation complex. We further discuss how Mediator and sequence-specific DNA-binding transcription factors enable enhancer-dependent regulation of Pol II function at distal gene promoters, through the formation of molecular condensates (or transcription hubs) and chromatin loops. Mediator regulation of Pol II reinitiation is also discussed, in the context of transcription bursting. We propose a working model for Mediator function that combines experimental results and theoretical considerations related to enhancer–promoter interactions, which reconciles contradictory data regarding whether enhancer–promoter communication is direct or indirect. We conclude with a discussion of Mediator’s potential as a therapeutic target and of future research directions.

Considerations on the use of spectroelectrochemistry in reflection mode for quantitative analysis: Study of the Fe(III)/Fe(II) – orthophenanthroline system

Theoretical and experimental considerations are made about possible analytical applications of spectroelectrochemistry (SEC) in reflection mode using screen-printed electrodes. The system Fe(III)/Fe(II) – orthophenanthroline is taken as a model of electrochemical generation and consumption of a substance that is strongly absorbing: the Fe(II) – orthophenanthroline complex. Different possibilities of quantification are discussed, including cathodic and anodic peaks in cyclic voltammetry and both electrochemical and optical signals acquired in chronoamperometric experiments. Among these, the evolution of absorbance with the square root of time at a constant potential has shown interesting possibilities for the determination of Fe(III)-ion at micromolar level (LOD = 2 10-6 mol L-1). Moreover, this method appears to be resistant to the presence of other electroactive species such as Cu(II)-ions.

Spectroscopic ellipsometry from 10 to 700 K

Abstract The temperature dependence of the optical constants of materials (refractive index, absorption and extinction coefficients, and dielectric function) can be determined with spectroscopic ellipsometry over a broad range of temperatures and photon energies or wavelengths. Such results have practical value, for example for applications of optical materials at cryogenic or elevated temperatures. The temperature dependence of optical gaps and their broadenings also provides insight into the scattering of electrons and holes with other quasiparticles, such as phonons or magnons. This review presents a detailed discussion of the experimental considerations for temperature-dependent ellipsometry and selected results for insulators, semiconductors, and metals in the infrared to ultraviolet spectral regions.

Modification of FAU zeolite as an active heterogeneous catalyst for biodiesel production and theoretical considerations for kinetic modeling

In this work, a high purity FAU-type zeolite catalyst was prepared from shale rock and modified as a heterogeneous efficient catalyst for biodiesel production from sunflower oil. The characterization properties for both of the prepared catalysts were determined using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDAX), Brunauer–Emmett–Teller (BET), and Fourier-transform infrared spectroscopy (FTIR). The incipient wetness impregnation method was adopted for loading the catalyst with three base precursors: NaOH, KOH, and Ca(OH)3. Different factors affecting transesterification reaction onto modified Na-K-Ca-FAU zeolite were investigated such as; temperature (35, 45, 55, and 65 °C), catalyst concentrations (2, 3,4, 5, and 6 wt%) and the molar ratio of methanol to sunflower oil (3:1, 6:1, 9:1 and 12:1). The optimum conditions of transesterification reactions were obtained for reaction time (4 h) and agitation rate (700 rpm) in a batch reactor at 65 °C reaction temperature, 5% catalyst concentration, and a 9:1 M ratio of methanol to oil. The experimental results showed that the conversion of triglyceride in sunflower oil to fatty acid methyl ester (FIME) increased from 48.62 to 91.6% when the FAU zeolite was loaded with 15 wt% of the three bases. The properties of the produced biodiesel were evaluated within the standard performance ASTM D-6751. This study shows that the three base precursors (i.e., NaOH, KOH, and Ca(OH)3) were successfully loaded onto support FAU zeolite and functioned as excellent catalysts for biodiesel production. Theoretical considerations for kinetic modeling in the heterogeneous transesterification reaction were investigated using MATLAB programming. The experimental and theoretical considerations for kinetic modeling were fitted well.

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host's microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples. Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

Personal identity, possible worlds, and medical ethics

Thought experiments that concoct bizarre possible world modalities are standard fare in debates on personal identity. Appealing to intuitions raised by such evocations is often taken to settle differences between conflicting theoretical views that, albeit, have practical implications for ethical controversies of personal identity in health care. Employing thought experiments that way is inadequate, I argue, since personhood is intrinsically linked to constraining facts about the actual world. I defend a moderate modal skepticism according to which intuiting across conceptually incongruent worlds constitutes ‘invalid intuition-inferences’—i.e., carrying over intuitions gathered from facts about possible worlds that are at odds with facts about the actual world, for the purpose of making claims about real-life persons and their identity, leads to conceptual incongruences. Such a methodological fallout precludes accurate, informative judgments about personal identity in the actual world, calling into question the adequacy of thought experimental considerations for potential real-world applications in medical ethics.

Leveraging Isothermal Titration Calorimetry to Obtain Thermodynamic Insights into the Binding Behavior and Formation of Metal-Organic Frameworks.

Isothermal titration calorimetry (ITC) is a technique which directly measures the thermodynamic parameters of binding events. Although historically it has been used to investigate interactions in biological macromolecules and the kinetics of enzyme-catalyzed reactions, ITC has also been demonstrated to provide relevant thermodynamic information about interactions in synthetic systems, such as those in metal-organic frameworks (MOFs). MOFs are a family of crystalline porous materials that have been widely studied as supports for molecules ranging from gases to biomolecules through physisorption and chemisorption. Herein, we offer a perspective on the current applications of ITC in MOFs, including the mechanism of small molecule adsorption and the formation of MOF-based composite materials through noncovalent interactions. Experimental considerations specific to running ITC experiments in MOF systems are reviewed on the basis of existing reports. We conclude by discussing underexplored, but promising, MOF-related research directions which could be elucidated by ITC.

A trace formula for metric graphs with piecewise constant potentials and multi-mode graphs

We generalize the scattering approach to quantum graphs to quantum graphs with piecewise constant potentials and multiple excitation modes. The free single-mode case is well-known and leads to the trace formulas of Roth (1983 C. R. Acad. Sci., Paris I 296 793–5), Kottos and Smilansky (1997 Phys. Rev. Lett. 79 4794). By introducing an effective reduced scattering picture we are able to propose new exact trace formulas in the more general settings. The latter are derived and discussed in details with some numerical examples for illustration. Our generalization is motivated by both experimental applications and fundamental theoretical considerations. The free single-mode quantum graphs are an extreme idealization of reality that, due to the simplicity of the model allows to understand a large number of generic or universal phenomena. We lift some of this idealization by considering the influence of evanescent modes that only open above threshold energies. How to do this theoretically in a closed model in general is a challenging question of fundamental theoretical interest and we achieve this here for quantum graphs.

Electrochemical etching of p-type GaN using a tunnel junction for efficient hole injection

In this work electrochemical etching (ECE) of p-type GaN under constant bias without an external light source is demonstrated for the first time. A tunnel junction (TJ) was used as a cap to ensure stable and controllable hole injection to the p-type semiconductor. GaN and In0.02Ga0.98N layers with two different Mg dopant concentrations (5.6∙1018 and 2∙1019cm−3, respectively) were investigated. Structuring of the samples into double-step stripes allowed examination of the etching mechanism. The etch rate and threshold etching voltage were determined. Finally, a model of the band structure of the etched samples is presented. Based on experimental results and theoretical considerations this work provides a method for controllable ECE of p-type GaN using TJ for efficient hole injection.

Experimental investigation and design considerations on cold-formed steel built-up​ I-section columns subjected to interactive buckling modes

An experimental investigation into the behavior of cold-formed steel (CFS) built-up column assembly compressed between fixed ends is presented in this paper. The built-up column assembly is designed to be doubly symmetric and locally slender to study the interactive buckling mode of failures. A total of forty-one columns were tested including different cross-section dimensions, lengths, intermediate connection spacing, and slendernesses. It is experimentally shown that the back-to-back connected doubly symmetric built-up cross-section assembly columns fail predominantly in interactive local and flexural–torsional buckling due to the shift in the line of action of the internal force caused by local buckling deformations. The influence of intermediate fastener spacing was also prevalent in the failure modes of the doubly symmetric CFS built-up columns. The appropriateness of the AISI’s maximum intermediate connection spacing limitation is verified to prevent global instability failures. The test and design results comparison indicated that the current AISI’s DSM design curve for interactive buckling is unconservative for the CFS built-up columns with predominant interactive local–global failure mode vulnerability. Therefore, a modified design curve for interactive buckling is suggested based on the test results with reliability analysis.

Deciphering the RNA universe in sperm in its role as a vertical information carrier.

The inheritance of neurophysiologic and neuropsychologic complex diseases can only partly be explained by the Mendelian concept of genetic inheritance. Previous research showed that both psychological disorders like post-traumatic stress disorder and metabolic diseases are more prevalent in the progeny of affected parents. This could suggest an epigenetic mode of transmission. Human studies give first insight into the scope of intergenerational influence of stressors but are limited in exploring the underlying mechanisms. Animal models have elucidated the mechanistic underpinnings of epigenetic transmission. In this review, we summarize progress on the mechanisms of paternal intergenerational transmission by means of sperm RNA in mouse models. We discuss relevant details for the modelling of RNA-mediated transmission, point towards currently unanswered questions and propose experimental considerations for tackling these questions.

Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic mastectomy is the most effective primary prevention strategy. Prophylactic mastectomy is an aggressive surgical procedure that completely removes the mammary epithelial cells from which breast cancer arises along with the surrounding tissue. We seek to develop a minimally invasive intraductal procedure as an alternative to prophylactic mastectomy to locally ablate the mammary epithelial cells before they can become malignant. We and others have developed an intraductal delivery procedure to reach and treat these epithelial cells in rodent models of breast cancer. While the mouse mammary gland with a single non-anastomosed ductal tree opening at the nipple has a much less complex and tortuous architecture than the human breast, chemically induced and genetically engineered mouse models of breast cancer are valuable to produce proof-of-concept studies of new preventative strategies. Here, we describe a procedure for intraductal delivery of an ethanol-based ablative solution containing micro-CT/X-ray tantalum-based contrast agent within the mouse mammary ductal tree for the therapeutic purpose of primary prevention of breast cancer. Intraductal delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) has been previously described in mouse models. Thus, we focus our protocol description on methodological modifications and unique experimental considerations for optimizing delivery of ethanol, for minimizing local and systemic side effects of ethanol administration, and for in vivo visualization of ductal tree filling via micro-CT/fluoroscopy imaging. Visualization of the ductal tree immediately after injection of a contrast-containing solution allows for confirmation of complete filling or unsuccessful outcomes such as underfilling or overfilling. This procedure can be applied for delivery and imaging of other ablative compounds aimed at either preventing tumor formation or locally treating early-stage tumors accessible via the ductal tree.

Electron pair emission from surfaces: Some general experimental considerations

We discuss some experimental facets of electron pair emission from surfaces using two different experimental approaches. In the first case the instrument consists of a pair of hemispherical analyzers which are operated with continuous primary beams of electrons or photons. The second instrument employs a pair of time-of-flight spectrometers which require a pulsed excitation source. A key experimental quantity is the ratio of ‘true’ to ‘random’ coincidences which can be determined in different ways. Regardless of the type of instrument the primary flux has to adopt a much smaller value than in single electron spectroscopy. We describe different approaches to obtain the relevant count rates, in particular the concept of operating with a delayed coincidence circuit. We also address the question on how to compare the two types of spectrometer in terms of their performance.

Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models.

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic mastectomy is the most effective primary prevention strategy. Prophylactic mastectomy is an aggressive surgical procedure that completely removes the mammary epithelial cells from which breast cancer arises along with the surrounding tissue. We seek to develop a minimally invasive intraductal procedure as an alternative to prophylactic mastectomy to locally ablate the mammary epithelial cells before they can become malignant. We and others have developed an intraductal delivery procedure to reach and treat these epithelial cells in rodent models of breast cancer. While the mouse mammary gland with a single non-anastomosed ductal tree opening at the nipple has a much less complex and tortuous architecture than the human breast, chemically induced and genetically engineered mouse models of breast cancer are valuable to produce proof-of-concept studies of new preventative strategies. Here, we describe a procedure for intraductal delivery of an ethanol-based ablative solution containing micro-CT/X-ray tantalum-based contrast agent within the mouse mammary ductal tree for the therapeutic purpose of primary prevention of breast cancer. Intraductal delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) has been previously described in mouse models. Thus, we focus our protocol description on methodological modifications and unique experimental considerations for optimizing delivery of ethanol, for minimizing local and systemic side effects of ethanol administration, and for in vivo visualization of ductal tree filling via micro-CT/fluoroscopy imaging. Visualization of the ductal tree immediately after injection of a contrast-containing solution allows for confirmation of complete filling or unsuccessful outcomes such as underfilling or overfilling. This procedure can be applied for delivery and imaging of other ablative compounds aimed at either preventing tumor formation or locally treating early-stage tumors accessible via the ductal tree.

Practical identifiability of parametrised models: A review of benefits and limitations of various approaches

This systematic review of practical identifiability (PI) explores the challenging issue of how parameter identification of models is affected by both experimental considerations and model structure. Structural identifiability (SI) analyses that yield binary assessment of parameter uniqueness have been historically dominant in the field. However, recent developments in the less explored PI domain have facilitated more nuanced estimates of identified model parameter trade-off and variance. As PI acknowledges variation in parameter estimates due to real-world limitations in data quality and quantity, it can both explore how parameters may trade-off, and guide more informative experimental design.In this review, PI analysis methodologies used across various fields of study are compared, and their role in aiding experimental design is discussed. The methods presented show that the choice of PI approach requires careful consideration based on the modelling context and desired research outcomes. Illustrative examples are included for common methodologies, and some ongoing research is briefly reviewed. Overall, the concept of PI brings value to model-based analyses across a broad range of disciplines.

Topical bioequivalence: Experimental and regulatory considerations following formulation complexity

Documenting topical bioequivalence can be an extremely complex process, which is intrinsically dependent on the formulation technological features. According to EMA guideline, for simple formulations, BE may be demonstrated by documenting the qualitative (Q1), quantitative (Q2), microstructure (Q3) and performance (Q4) equivalence. Nevertheless, when addressing complex semisolids, equivalence regarding local availability should also be demonstrated. The purpose of this study is to pursue this strategy using two opposite scenarios: a simple dimetindene maleate 1 mg/g gel formulation and a diclofenac diethylammonium 23.2 mg/g emulgel, representing a complex formulation. For both formulations, Q1/Q2 test (TP) and reference products (RP) were used. Rheology, in vitro release (IVRT) and in vitro permeation methods (IVPT) were developed and validated for both products.For the dimetindene formulation, equivalence pertaining to Q4 was established. However, high variability was observed for some rheology endpoints, especially for the different RP batches. Therefore, equivalence could not be established for Q3 as per EMA requirements. Can some rheology endpoints be waived? Can we establish reasonable criteria that are overall feasible for generic manufacturers and at the same time safe for the patient? An attempt was made to propose a wider acceptance range based on the inter-batch variability of the RP. For that, the rationale presented in the EMA guideline on bioequivalence for highly variable products was used.For the diclofenac formulation, Q3 equivalence was likewise not established. Q4 equivalence was only found for some batch combinations and when applying a wider acceptance criterion (75–133%). Furthermore, IVPT equivalence also failed to be demonstrated. Nevertheless, since the TP displays an equivalent pharmacokinetic profile compared to the RP, the observed Q3, Q4 and local availability differences are not expected to be clinically significant. This study draws attention to an effective search to determine the most appropriate strategy for assessing topical bioequivalence on a case-by-case basis.

Bias‐driven marketing that instigates pledging to a crowdfunding campaign: An experimental consideration of behavioral anomalies

AbstractBias‐driven marketing leverages the behavioral biases of investors for greater marketing gains. To transform cognitively biased individuals from antagonists into allies, the marketers need to decipher the critical crowdfunding attributes, which ultimately motivate behaviorally biased crowdfunders to invest in crowdfunding campaigns. Hence, this study seeks to investigate the influential factors concerning four behavioral biases that accelerate crowdfunding adoption. A conceptual model, parameterized using conjoint analysis, is developed and administered in an agent‐based behavior model to effectively determine the influential crowdfunding attributes, which will induce the biased investors to adopt crowdfunding. The results revealed that status quo biased individuals adopted the crowdfunding campaign when launched by well‐known and reputed fund raisers. Whereas, hyperbolic discounting bias accelerated the adoption when the campaign reached more than 75% of subscription and duration was lesser than 30 days. The results also indicated that framing bias enhanced the adoption when the risk inherent in the project is disclosed. Finally, the results also claimed that individuals liable to loss aversion bias expected a known funder, who posts frequent updates and preferred the All‐or‐Nothing crowdfunding model. The experimental results of the study provide insights to the marketers that crowdfunding campaigns can be successfully strategized through the exploration of underlying cognitive biases and the corresponding motivational cues that accentuate the pledging behavior of the funders. Given the importance of an effective marketing strategy in crowdfunding adoption, this study offers one such recommendation to the crowdfunding campaigners by demonstrating how bias driven marketing strategy enhances the adoption rate of the campaigns. With the strong pieces of evidence obtained from this study, it is anticipated that behavioral biases, drawn from the neuromarketing theory, will no longer be viewed as cognitive errors from the investor's perspective. Rather, it will be viewed as a boon from the marketer's perspective as the bias‐based marketing strategies have the potential to achieve a fully subscribed crowdfunding campaign.

Structurally characterized salamo-based mononuclear Cu(II) complex fluorogenic sensor with high selectivity for CN− and Cys-Cys

A highly selective “off-on” fluorogenic sensor (L-Cu2+), as an aldehyde-exposed salamo-based mononuclear Cu(II) complex, was designed and used as an effective sensor for the double pronged sensing of cyanide ion (CN−) and Cystine (Cys-Cys). By combining and calculating with experimental data, it can be fully characterized by a variety of spectroscopy techniques. The LODs of CN− and Cys-Cys were calculated to be 3.3792 × 10−7 and 1.2757 × 10−7 M. Consequently, the developed sensor showed good sensitivity and selectivity for CN− and Cys-Cys in DMF/water (v/v, 9:1) solution. Meanwhile, the designed complex sensor can discriminate and detect CN− ion but not other multiple anions. No interference from other ions was observed during the detection of CN− and Cys-Cys. Finally, we made known the rationality of fluorogenic “turn on” response phenomenon of L-Cu2+ to CN− and Cys-Cys by means of ESI-mass spectrometry, FT-IR spectra analyses to further understand experimental consideration results observed.

The Power of Single-Cell RNA Sequencing in eQTL Discovery.

Genome-wide association studies have successfully mapped thousands of loci associated with complex traits. During the last decade, functional genomics approaches combining genotype information with bulk RNA-sequencing data have identified genes regulated by GWAS loci through expression quantitative trait locus (eQTL) analysis. Single-cell RNA-Sequencing (scRNA-Seq) technologies have created new exciting opportunities for spatiotemporal assessment of changes in gene expression at the single-cell level in complex and inherited conditions. A growing number of studies have demonstrated the power of scRNA-Seq in eQTL mapping across different cell types, developmental stages and stimuli that could be obscured when using bulk RNA-Seq methods. In this review, we outline the methodological principles, advantages, limitations and the future experimental and analytical considerations of single-cell eQTL studies. We look forward to the explosion of single-cell eQTL studies applied to large-scale population genetics to take us one step closer to understanding the molecular mechanisms of disease.

Nonlinear quantum behavior of ultrashort-pulse optical parametric oscillators

The quantum features of ultrashort-pulse optical parametric oscillators (OPOs) are investigated theoretically in the nonlinear regime near and above threshold. Viewing the pulsed OPO as a multimode open quantum system, we rigorously derive a general input-output model that features nonlinear coupling among many cavity (i.e., system) signal modes and a broadband single-pass (i.e., reservoir) pump field. Under appropriate assumptions, our model produces a Lindblad master equation with multimode nonlinear Lindblad operators describing two-photon dissipation and a multimode four-wave-mixing Hamiltonian describing a broadband, dispersive optical cascade, which we show is required to preserve causality. To simplify the multimode complexity of the model, we employ a supermode decomposition to perform numerical simulations in the regime where the pulsed supermodes experience strong single-photon nonlinearity. We find that the quantum nonlinear dynamics induces pump depletion as well as corrections to the below-threshold squeezing spectrum predicted by linearized models. We also observe the formation of non-Gaussian states with Wigner-function negativity and show that the multimode interactions with the pump, both dissipative and dispersive, can act as effective decoherence channels. Finally, we briefly discuss some experimental considerations for potentially observing such quantum nonlinear phenomena with ultrashort-pulse OPOs on nonlinear nanophotonic platforms.