Jove-Journal of Visualized Experiments最新文献

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during genome duplication can stall replisomes and convert replication forks into double-strand breaks. If left unrepaired, these toxic DNA breaks can trigger chromosomal rearrangements, leading to heightened genome instability and an increased likelihood of cellular transformation. Additionally, cancer cells exhibit persistent replication stress, making the targeting of replication fork vulnerabilities in tumor cells an attractive strategy for chemotherapy. A highly versatile and powerful technique to study DNA breaks during replication is the comet assay. This gel electrophoresis technique reliably detects the induction and repair of DNA breaks at the single-cell level. Herein, a protocol is outlined that allows investigators to measure the extent of DNA damage in mitotically dividing human cells using fork-stalling agents across multiple cell types. Coupling this with automated comet scoring facilitates rapid analysis and enhances the reliability in studying induction of DNA breaks.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or "nanodrug") administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert behavior with plant bioactive chemicals. This study explores the sustainable extraction of phenolic compounds and natural antioxidants from coffee silverskin using the microwave-assisted extraction (MAE) method with polyol-based solvents: glycerin, propylene glycol (PG), butylene glycol (BG), methylpropanediol (MPD), isopentyldiol (IPD), pentylene glycol, 1,2-hexanediol, and hexylene glycol (HG). A comparative analysis was conducted on conventional and non-conventional solvent extractions, focusing on their impact on the bioactive compounds of MAE, encompassing parameters such as total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities like the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH), the 2,2'-azino-bis(-3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging assay (ABTS), and the ferric reducing antioxidant power assay (FRAP). The highest values were observed for TPC with aqueous-1,2-hexanediol extraction (52.0 ± 3.0 mg GAE/g sample), TFC with aqueous-1,2-hexanediol extraction (20.0 ± 1.7 mg QE/g sample), DPPH with aqueous-HG extraction (13.6 ± 0.3 mg TE/g sample), ABTS with aqueous-pentylene glycol extraction (8.2 ± 0.1 mg TE/g sample), and FRAP with aqueous-HG extraction (21.1 ± 1.3 mg Fe (II) E/g sample). This research aims to advance eco-friendly extraction technology through natural plant components, promoting sustainability by minimizing hazardous chemical use while reducing time and energy consumption, with potential applications in cosmetics.

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron acceptor. This electron acceptor can be an electrode and electrons can be delivered indirectly via a redox-active mediator molecule. Here, we present a protocol to study mediated EET in Lactiplantibacillus plantarum, a probiotic lactic acid bacterium widely used in the food industry, using a bioelectrochemical system. We detail how to assemble a three-electrode, two-chambered bioelectrochemical system and provide guidance on characterizing EET in the presence of a soluble mediator using chronoamperometry and cyclic voltammetry techniques. We use representative data from 1,4-dihydroxy-2-naphthoic acid (DHNA)-mediated EET experiments with L. plantarum to demonstrate data analysis and interpretation. The techniques described in this protocol can open new opportunities for electro-fermentation and bioelectrocatalysis. Recent applications of this electrochemical technique with L. plantarum demonstrated an acceleration of metabolic flux towards producing fermentation end-products, which are critical flavor components in food fermentation. As such, this system has the potential to be further developed to alter flavors in food production or produce valuable chemicals.

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (<1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Microsurgical varicocelectomy is the most commonly used method for the treatment of varicocele (VC) in recent years. However, it is technically demanding with the risk of damaging the normal anatomical structure of the spermatic cord, such as the cremaster muscle, testicular artery, and vas deferens during the pampiniform plexus ligation. Also, traditional varicocelectomy hinders the drainage of the stagnant venous blood of the affected testicle, resulting in a persistent scrotal appearance of varicose veins and slower remission of swelling sensation in postoperative patients with grade III VC. Therefore, we developed a retroperitoneal varicocelectomy with a microscopical spermatic venous-superficial vein of the abdominal wall bypass procedure. The spermatic vein was transected and ligated proximally through the retroperitoneal space. Then, the distal spermatic vein was freed and passed through the internal ring; under the skin of the groin, a microscopic vascular anastomosis was performed to build the bypass of the distal spermatic vein and proximal inferior epigastric vein. The high ligation facilitates the protection of the normal anatomy of the spermatic cord, and the venous bypass allows rapid testicular blood drainage, which can effectively improve the degree of varicocele, testicular pain, and even spermatogenic function. In conclusion, the present protocol describes a promising way to reconstruct the spermatic return through high retroperitoneal ligation of the spermatic vein and anastomosis of the spermatic vein-inferior epigastric vein, which resulted in faster and more obvious improvement in symptoms and better prognosis of grade III VC.

Malformation of cortical development is an important cause of drug-resistant epilepsy in young children. Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) has been added to the last focal cortical dysplasia (FCD) classification and commonly involves the frontal lobe. The semiology at the onset of epilepsy is dominated by non-lateralizing infantile spasm; the boundaries of the malformation are usually difficult to determine by magnetic resonance imaging (MRI) and positron emission tomography (PET), and electroencephalography (EEG) findings are often widespread. Therefore, the traditional concept and strategy of preoperative evaluation to determine the extent of the epileptogenic zone by comprehensive anatomo-electro-clinical methods are difficult to implement. Frontal disconnection is an effective surgical method for the treatment of epilepsy, but there are few related reports. A total of 8 children with histo-pathologically confirmed MOGHE were retrospectively studied. MOGHE was located in the frontal lobe in all patients, and frontal disconnection was performed. The periinsular approach was used in the disconnective procedures, divided into several surgical steps: the partial inferior frontal gyrus resection, the frontobasal and intrafrontal disconnection, and the anterior corpus callosotomy. One patient presented with a short-term postoperative speech disorder, while another patient exhibited transient postoperative limb weakness. No long-term postoperative complications were observed. At 2 years after surgery, 75% of patients were seizure-free, with cognitive improvement in half of them. This finding suggested that frontal disconnection is an effective and safe surgical procedure for the treatment of MOGHE instead of extensive resection in the frontal lobe.

The commercial wasabi pastes commonly used for food preparation contain a homologous compound of chemosensory isothiocyanates (ITCs) that elicit an irritating sensation upon consumption. The impact of sniffing dietary alcoholic beverages on the sensation of wasabi spiciness has never been studied. While most sensory evaluation studies focus on individual food and beverages separately, there is a lack of research on the olfactory study of sniffing liquor while consuming wasabi. Here, a methodology is developed that combines the use of an animal behavioral study and a convolutional neural network to analyze the facial expressions of mice when they simultaneously sniff liquor and consume wasabi. The results demonstrate that the trained and validated deep learning model recognizes 29% of the images depicting co-treatment of wasabi and alcohol belonging to the class of the wasabi-negative liquor-positive group without the need for prior training materials filtering. Statistical analysis of mouse grimace scale scores obtained from the selected video frame images reveals a significant difference (P < 0.01) between the presence and absence of liquor. This finding suggests that dietary alcoholic beverages might have a diminishing effect on the wasabi-elicited reactions in mice. This combinatory methodology holds potential for individual ITC compound screening and sensory analyses of spirit components in the future. However, further study is required to investigate the underlying mechanism of alcohol-induced suppression of wasabi pungency.

Zebrafish and their mutant lines have been extensively used in biomedical investigations, cardiovascular studies, and drug screening. In the current study, the commercial version of the novel system, Zebra II, is presented. The protocol demonstrates electrocardiogram (ECG) acquisition and analysis from multiple zebrafish within controllable working environments. The device is composed of an external and independent perfusion system, a 4-point electrode, temperature sensors, and an embedded electronic system. In previous studies, the device prototype underwent validation against the established iWORX system through several tests, demonstrating similar data quality and ECG response to drug interventions. Following this, the study delved into examining the impact of anesthetic drugs and temperature fluctuations on zebrafish ECG, necessitating instant data evaluation. Thanks to the apparatus's capacity for consistent delivery of anesthetics and drugs, it was possible to extend ECG data collection up to 1 h, markedly longer than the 5 min duration supported by current systems. This paper introduces a pioneering, cloud-based, automated analysis utilizing data from four zebrafish, offering an efficient method for conducting combination experiments and significantly reducing time and effort. The system proved effective in capturing and analyzing ECG, especially in detecting drug-induced arrhythmias in wild-type zebrafish. Additionally, the capability to gather data across multiple channels facilitated the execution of randomized controlled trials with zebrafish models. The developed ECG system overcomes existing limitations, showing the potential to greatly expedite drug discovery and cardiovascular research involving zebrafish.

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.