Jove-Journal of Visualized Experiments最新文献

This paper presents a robot-based experimental program aimed at developing an efficient and fast colorimetric gas sensor. The program employs an automated Design-Build-Test-learning (DBTL) approach, which optimizes the search process iteratively while optimizing multiple recipes for different concentration intervals of the gas. In each iteration, the algorithm generates a batch of recipe suggestions based on various acquisition functions, and with the increase in the number of iterations, the values of weighted objective function for each concentration interval significantly improve. The DBTL method begins with parameter initialization, setting up the hardware and software environment. Baseline tests establish performance standards. Subsequently, the DBTL method designs the following round of optimization based on the proportion of recipes in each round and tests performance iteratively. Performance evaluation compares baseline data to assess the effectiveness of the DBTL method. If the performance improvement does not meet expectations, the method will be performed iteratively; if the objectives are achieved, the experiment concludes. The entire process maximizes system performance through the DBTL iterative optimization process. Compared to the traditional manual developing process, the DBTL method adopted by this experimental process uses multi-objective optimization and various machine learning algorithms. After defining the upper and lower limits of component volume, the DBTL method dynamically optimizes iterative experiments to obtain the optimal ratio with the best performance. This method greatly improves efficiency, reduces costs, and performs more efficiently within the multi-formulation variable space when finding the optimal recipe.

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.

Prenatal cannabinoid exposure (PCE) is becoming increasingly frequent as more states across the United States legalize recreational marijuana (cannabinoids). The consumption of cannabinoid products during pregnancy has been associated with various abnormal outcomes, although historical studies were conducted during a time when the potency of these products was approximately 300% lower than that of current products. Given the rising use of cannabinoids, it is essential to understand the potential impacts PCE may have on fetal neurodevelopment and subsequent infant and child development. Previous studies have demonstrated that PCE negatively affects learning and memory, behavioral skills, sleep, and attention in offspring. The aim of this study is to recapitulate PCE through voluntary ingestion of delta-9-tetrahydrocannabinol (THC), the psychoactive component of cannabinoid products, during pregnancy in a preclinical model. This article outlines the procedure for achieving moderate PCE throughout gestation. In this model, the control group consumes plain mini-chocolate/peanut butter cookies, while the PCE group consumes THC mixed into peanut butter paired with mini-chocolate cookies. This approach enables further investigation into the impact of PCE on developmental outcomes.

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of the specific role of individuals, defined or complex groups of microorganisms in host response. Traditionally bred and reared in flexible-film or semi-rigid isolators, germ-free mouse husbandry and experimental manipulation are costly and require numerous trained staff and a large space footprint in animal housing facilities. The IsoPositive caging system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages (isocages), reducing cost and enabling greater flexibility in experimental manipulations. Here, a protocol is described for transferring germ-free mice from breeding isolators to isocages and subsequent fecal transfer from human donor stool into mice to create stable long-term gut "humanized" mice for future studies. The materials and preparation needed for the utilization of the isocage system are described, including the use of chlorine-dioxide sterilant chemical sterilant to clean cages, supplies, equipment, and personal protective equipment. The methods for confirming the germ-free status of transferred mice and how to determine contamination in the caging system are discussed. The procedure for husbandry, including bedding, food, and water supply, is further discussed. The protocol for human fecal slurry preparation and gavage into germ-free mice to create gut "humanized" mice, along with stool collection to monitor the microbial community composition of these mice, are described. An experiment illustrates that two weeks post-human fecal transplant allows for stable colonization of donor microbiota in the murine hosts, enabling subsequent experimental usage. Furthermore, the collection of humanized mouse feces in viability preservation media, enabling use in further functional experiments, is described. Overall, these methods allow for the safe and effective establishment of humanized mouse communities in experimental gnotobiotic cages for further manipulation.

Compounds that form covalent bonds with specific target proteins offer a variety of advantages as chemical probes and therapeutic agents. Most commonly, mildly reactive, electrophilic small molecules are employed to form covalent bonds with select cysteine side chains in specific proteins. Electrophile-first approaches of ligand discovery, whereby a library of electrophilic small molecules are screened against a protein target, have become popular as they avoid the need for time-consuming downstream installation of an electrophilic warhead. Such screening is complicated, however, as electrophilic ligands can exhibit a wide range of different rates of spontaneous reaction with cysteines. Quantitative-irreversible tethering (qIT) offers a fluorescence-based method for hit identification and development that normalizes data for these differences in intrinsic compound reactivity. Rates of reaction of individual compounds with a target protein are determined and compared to compound reactivity with the unstructured tripeptide glutathione (this being a proxy for spontaneous compound reaction), enabling the identification of compounds that preferentially react with the protein of interest. This methodology has been successfully applied to identify selective covalent fragments against several drug targets, including SARS-CoV-2 main protease, cyclin-dependent kinase 2, and RAP27A. Here, we demonstrate the application of qIT to a target protein to generate a quantitative and robust data set, allowing prioritization of hit ligands for future development.

Laparoscopic caudate lobectomy (LCL) is one of the most challenging types of laparoscopic liver resections. The main difficulty lies in the deep anatomical position of the caudate lobe, which is closely adjacent to the first and second hepatic ports and the inferior vena cava, increasing the risk of major bleeding during surgery. In addition to a thorough understanding of the anatomy of the caudate lobe tumor, comprehensive imaging assessment, and three-dimensional reconstruction, the flexible choice of surgical approach is also key to reducing surgical difficulty and improving safety. We perform laparoscopic caudate lobectomy using a left-sided approach, especially when the tumor is located in the area of the caudate lobe close to the inferior vena cava. This method avoids the step of splitting the liver substance to expose the field of view required by the traditional anterior approach, with the advantages of larger operating space and shorter operation time. At the same time, combined with preoperative three-dimensional reconstruction technology, we have significantly reduced the risk of damaging important blood vessels and increased the success rate of resection of tumors in the caudate lobe.

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic strains and genetically engineered cells. Here, an alternative method to detect biochemical interaction between the macrophage microbial sensor SLAMF1 and Mycobacterium tuberculosis is described. Two technical approaches employing flow cytometry and fluorescence microscopy were developed. Total cell protein extracts from human macrophages were generated, then incubated with whole cells of M. tuberculosis (WCMtb) or M. tuberculosis antigens (Mtb Ags) overnight at 4 °C and finally cross-linked using formaldehyde/glycine/ethylene glycol bis (succinimidyl succinate) treatment. SLAMF1 interaction with WCMtb by flow cytometry was detected with a PE-specific anti-SLAMF1 antibody. The existence of interaction by fluorescence microscopy was performed by attaching Rhodamine-PE stained Mtb Ags to poly-D-lysine coated slides, which were incubated with the total protein extract from monocyte-derived macrophages. After cross-linking treatment, SLAMF1 was visualized using primary (anti-SLAMF1) and secondary (Alexa Fluor 488) antibodies. The assays provided a strong biochemical tool to measure pathogen-immunoreceptor interactions, overcoming the difficulties associated with transgenic cell lines and protein gene expression modulation experiments.

Step width is a critical factor influencing lower limb biomechanics during running, significantly affecting stability, performance, and injury risk. Understanding these effects is essential for optimizing running performance and minimizing injury risk. This study evaluated the effects of varying step widths on lower limb biomechanics at different running speeds. Thirteen healthy Chinese males (aged 20-24) participated in the study, running at speeds of 3.0 m/s and 3.7 m/s using six distinct step widths: the preferred step width and five variations (reductions of 13% and 6.5%, and increases of 6.5%, 13%, and 25%, based on leg length). Data were collected using a motion capture system and force plates and analyzed through repeated measures ANOVA and correlation tests. The results indicated that wider step widths significantly reduced peak knee abduction moments and hip adduction angles, whereas narrower step widths increased knee joint loading. These findings have important implications for clinicians and runners, suggesting that careful step width selection can help reduce injury risk and enhance running efficiency. This study contributes a new dataset that lays the foundation for future research into the relationship between step width and running biomechanics and serves as a reference for training and rehabilitation practices.

Choledochal cysts (CCs), known as congenital choledochal dilatations, are more prevalent in Asia. The majority of patients with abdominal symptoms are diagnosed and treated during early childhood, which results in a lower prevalence of CCs in adults. The treatment of choice for CCs is complete cyst excision followed by choledochojejunostomy. Laparoscopic surgery is now more widely accepted than traditional open surgery due to its smaller incisions, faster recovery, and less postoperative pain. However, there are few reports on laparoscopic excision of CCs in adults. This article presents a protocol that describes and demonstrates the complete procedure for laparoscopic excision of a choledochal cyst and Roux-en-Y choledochojejunostomy. A 32-year-old woman diagnosed with a 2.5 cm by 3 cm CC underwent surgery using a laparoscopic approach with post-colonic anastomosis. The procedure lasted 290 min with an estimated blood loss of approximately 100 mL. A follow-up abdominal CT scan on the sixth postoperative day showed a satisfactory recovery, leading to her discharge on the ninth day. This study aims to demonstrate the feasibility and safety of laparoscopically assisted excision of CCs in adults. This procedure is expected to become the preferred surgical option for CCs in adults due to its minimal surgical trauma and rapid postoperative recovery.

Sprouting angiogenesis is the formation of new blood vessels from pre-existing vasculature and is of great importance for physiological such as tissue growth and repair and pathological processes, including cancer and metastasis. The multistep process of sprouting angiogenesis is a molecularly and mechanically driven process. It consists of induction of cellular sprout by vascular endothelial growth factor, leader/follower cell selection through Notch signaling, directed migration of endothelial cells, and vessel fusion and stabilization. A variety of sprouting angiogenesis models have been developed over the years to better understand the underlying mechanisms of cellular sprouting. Despite advancements in understanding the molecular drivers of sprouting angiogenesis, the role of mechanical cues and the mechanical driver of sprouting angiogenesis remains underexplored due to limitations in existing models. In this study, we designed a 2.5D ex vivo model that enables us to mechanically characterize cellular sprouting from a porcine carotid artery using traction force microscopy. The model identifies distinct force patterns within the sprout, where leader cells exert pulling forces and follower cells exert pushing forces on the matrix. The model's versatility allows for the manipulation of both chemical and mechanical cues, such as matrix stiffness, enhancing its relevance to various microenvironments. Here, we demonstrate that the onset of sprouting angiogenesis is stiffness-dependent. The presented 2.5D model for quantifying cellular traction forces in sprouting angiogenesis offers a simplified yet physiologically relevant method, enhancing our understanding of cellular responses to mechanical cues, which could advance tissue engineering and therapeutic strategies against tumor angiogenesis.