Jove-Journal of Visualized Experiments最新文献

Functional Near-Infrared Spectroscopy (fNIRS) hyperscanning is an innovative technique that enables real-time monitoring of brain activity among multiple individuals engaged in social interactions. Researchers in this field quantify concurrent brain activities through the index of inter-brain synchrony (IBS). In psychological counseling research, the use of fNIRS to measure IBS has garnered attention for its potential to illuminate the dynamics of counselor-client interactions. Nevertheless, the field currently lacks a standardized protocol for precisely measuring IBS between counselors and clients, which would facilitate the revelation of real-time interaction patterns during counseling sessions. To address this need, this paper proposes a detailed standardized protocol, outlining the procedural steps for conducting fNIRS hyperscanning in psychological counseling settings, focusing on the acquisition of brain signals, calculation of IBS between counselors and clients, and analysis of lead-lag patterns of IBS throughout counseling sessions. Implementing this standardized fNIRS hyperscanning pipeline not only enhances the reproducibility and reliability of IBS measurements in psychological counseling research but also facilitates deeper insights into the neural mechanisms underlying working alliance. By integrating fNIRS hyperscanning into naturalistic counseling environments, researchers can advance understanding of how IBS correlates with counseling outcomes, potentially informing personalized approaches to mental health treatment.

Elastase, a serine protease, plays an essential role in elastin degradation. Elastin is an extracellular protein that helps maintain tissue elasticity in the lungs, skin, and blood vessels. Tight regulation of elastase activity is crucial for tissue homeostasis, as dysregulation can contribute to pathologies such as emphysema, wrinkles, and atherosclerosis. Some compounds, such as naturally occurring phytochemicals, have shown potential for therapeutic intervention and have attracted significant interest. Elucidating the modulatory effects of different compounds on elastase, whether inhibitory or stimulatory, is crucial for developing novel therapeutic and cosmetic strategies targeting elastase-associated disorders. A widely accepted method for measuring elastase activity is the colorimetric elastase assay. In this assay, a specific substrate is used to break down elastase, releasing a detectable yellow compound, p-nitroaniline (pNA). The amount of pNA produced reflects elastase activity in the sample and can be measured by colorimetry. This assay offers several benefits, including simplicity, high sensitivity, rapid results, and adaptability to various research needs. The colorimetric elastase assay remains a valuable tool for studying how compounds impact elastase activity. Due to its ease of use and effectiveness, this assay is a cornerstone of research in this field.

This study aimed to assess the impact of various fabrication techniques and polishing procedures on the surface roughness (Ra) of resin-based materials used in the fabrication of complete dentures. A total of 90 specimens were produced from three different resin materials: heat-polymerized polymethyl methacrylate (PMMA) resin, CAD-CAM milled PMMA resin, and 3D-printed resin (n = 30). Each specimen measured 10 mm in diameter and 2 mm in height. The surface roughness (Ra) values of the specimens were initially determined using a contact profilometer following fabrication. Subsequently, each group of specimens was polished with 600-, 800-, and 1000-grit silicon carbide abrasive papers under running water. A second measurement of the surface roughness (Ra) values was then performed. The data were analyzed statistically using the Kruskal-Wallis test, Mann-Whitney U test, Wilcoxon signed-rank test, and paired samples t-test (p = 0.05). A statistically significant difference was identified between the groups in terms of surface roughness (Ra) prior to the polishing process (p < 0.001). However, no statistically significant difference was observed between the milled and heat-polymerized PMMA base materials following the polishing process. The 3D-printed specimens showed the most notable improvement in surface roughness due to the polishing process. Nevertheless, their surface roughness remained statistically significantly higher compared to the other samples, both before and after polishing (p < 0.001). The fabrication method of complete denture base materials was observed to influence surface roughness. The surface roughness values of the base materials fabricated using the 3D printing method were higher compared to those fabricated with milled and heat-polymerized PMMA resin, both before and after polishing.

Over the past several decades, veno-arterial extracorporeal membrane oxygenation (V-A ECMO) has become a critical tool in the management of patients with severe cardiogenic shock and cardiopulmonary failure. Due to the inherent instability of these patients, their transport away from intensive care units is fraught with risk. As a result, bedside diagnostic tools are essential for their daily care. One such tool is point-of-care ultrasound (POCUS) of the heart, which can non-invasively assess several parameters: left ventricular (LV) performance (size, systolic function, stroke volume, aortic valve opening), right ventricular (RV) performance (size, systolic function), and the presence of intracardiac thrombus. Additionally, POCUS can assist in evaluating readiness for V-A ECMO weaning and eventual decannulation. Despite its potential, the use of POCUS in the context of V-A ECMO remains inconsistent due to variability in provider training. This study aims to address this gap by detailing POCUS image acquisition in V-A ECMO, particularly in the absence of LV venting. It covers key aspects such as patient positioning, transducer selection, probe placement, acquisition sequence, and image optimization.

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular persistence contributes to chronic and recurrent infections, complicating treatment and prolonging the disease. Consequently, there is a critical need for an intracellular infection model to better understand, prevent, and treat infections caused by S. aureus. This study indicated that antibiotics effectively eliminated extracellular bacteria but could not eradicate those that had entered the cells. Thus, a stable intracellular infection in vitro was established by RAW264.7 infected with S. aureus and co-culturing them with antibiotics. Subsequently, an intracellular infection model in mice was established by injecting peritoneal macrophages containing the intracellular infection. Vancomycin effectively cleared bacterial loads in mice challenged with planktonic S. aureus; however, it was ineffective against mice infected with equal or lower levels of intracellular bacteria within the peritoneal macrophages. This indicates that the intracellular infection model of S. aureus was successfully established, offering potential insights for the prevention and treatment of intracellular infections.

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

The glucose concentration is a key indicator of cellular metabolism and could indicate the total metabolism rate, an aberration in glucose metabolism, and, in some cases, how cells couple glucose metabolism to energetic metabolism. In addition, intracellular and extracellular glucose levels are indicative of the cellular metabolic status. Enzymatic techniques, such as Bergmeyer glucose quantification, are more accurate and sensitive than other techniques, such as dinitrosalicylic acid or fluorescence methods, which are usually utilized in microbiology. Although mainly used in the clinical area, Bergmeyer glucose quantification can also be applied to any cell but has not been reported in detail for bacteria, fungi, yeasts, or other microorganisms. Herein, we present a methodology to quantify glucose from bacteria and yeast samples using the enzymatic Bergmeyer glucose quantification method. The procedure involved the enzymes glucose oxidase, peroxidase, and o-dianisidine dihydrochloride incubated at 37 °C for 20 min, followed by the addition of sulfuric acid. The absorbance is then measured at 545 nm. It is important to highlight that although this technique presents difficulties in measuring high concentrations of glucose (above 60 g/L), it is possible to measure concentrations below 50 g/L using dilution factors. This enzymatic approach is valuable for research and analysis in microbiology and other scientific areas. The precision and sensitivity of the method make it helpful for detecting even low concentrations of glucose in microbiological samples.

This study aimed to determine whether fusing pre- and post-operative computed tomography (CT) images could help in assessing electrode placement and center frequency (CF) in cochlear implant (CI) recipients. A secondary objective was to compare automatic fusion with manual methods for measuring cochlear parameters. The study included twenty ears with CIs that underwent both pre- and post-operative CT scans. Manual measurements of cochlear parameters were initially taken from the post-operative CT images, followed by automatic detection using fused pre- and post-operative CT images with otological software (OTOPLAN). Angular insertion depth (AID) and CF of each electrode contact were calculated using both methods, and error differences were assessed. The analysis showed significant differences between the two methods for cochlear width (B-value) and cochlear duct length (CDL); however, these differences were not clinically significant. Furthermore, there was no statistically significant difference in cochlear diameter (A-value). The mean differences were 0.04 mm for the A-value, 0.21 mm for the B-value, and 0.73 mm for the CDL. The comparison of AID and CF revealed non-significant differences between manual and automatic fusion methods across all electrode contacts, except for electrode number five. According to this study, fusing pre- and post-operative CT images can be used to determine electrode positions in CI recipients. Automatic fused images can potentially measure cochlear parameters, AID, and CF with reduced human interference. Therefore, this method may serve as another basis for creating anatomy-based fitting.