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Abstract Proximity ligation assays (PLAs) use specific antibodies to detect endogenous protein‐protein interactions. PLAs are a highly useful biochemical technique that allow two proteins within proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of a PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein‐protein interactions within mitochondria‐endoplasmic reticulum contact sites (MERCs). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Proximity ligation assay for skeletal muscle tissue and myoblast for MERC proteins 

Abstract OPA1 is a dynamin‐related GTPase that modulates mitochondrial dynamics and cristae integrity. Humans carry eight different isoforms of OPA1 and mice carry five, all of which are expressed as short‐ or long‐form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance. However, western blot isolation of all long and short isoforms of OPA1 can be difficult. To address this issue, we developed an optimized western blot protocol based on improving running time to isolate five different isoforms of OPA1 in mouse cells and tissues. This protocol can be applied to study changes in mitochondrial structure and function. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Western Blot Protocol for Isolating OPA1 Isoforms in Mouse Primary Skeletal Muscle Cells 

Abstract While some established undergraduate summer programs are effective across many institutions, these programs may only be available to some principal investigators or may not fully address the diverse needs of incoming undergraduates. This article outlines a 10‐week science, technology, engineering, mathematics, and medicine (STEMM) education program designed to prepare undergraduate students for graduate school through a unique model incorporating mentoring dyads and triads, cultural exchanges, and diverse activities while emphasizing critical thinking, research skills, and cultural sensitivity. Specifically, we offer a straightforward and adaptable guide that we have used for mentoring undergraduate students in a laboratory focused on mitochondria and microscopy, but which may be customized for other disciplines. Key components include self‐guided projects, journal clubs, various weekly activities such as mindfulness training and laboratory techniques, and a focus on individual and cultural expression. Beyond this unique format, this 10‐week program also seeks to offer an intensive research program that emulates graduate‐level experiences, offering an immersive environment for personal and professional development, which has led to numerous achievements for past students, including publications and award‐winning posters. 

Abstract Machine learning has proven useful in analyzing complex biological data and has greatly influenced the course of research in structural biology and precision medicine. Deep neural network models oftentimes fail to predict the structure of complex proteins and are heavily dependent on experimentally determined structures for their training and validation. Single‐particle cryogenic electron microscopy (cryoEM) is also advancing the understanding of biology and will be needed to complement these models by continuously supplying high‐quality experimentally validated structures for improvements in prediction quality. In this perspective, the significance of structure prediction methods is highlighted, but the authors also ask, what if these programs cannot accurately predict a protein structure important for preventing disease? The role of cryoEM is discussed to help fill the gaps left by artificial intelligence predictive models in resolving targetable proteins and protein complexes that will pave the way for personalized therapeutics. 

Abstract Alzheimer’s Disease (AD) is a global health issue, affecting over 6 million in the United States, with that number expected to increase as the aging population grows. As a neurodegenerative disorder that affects memory and cognitive functions, it is well established that AD is associated with cardiovascular risk factors beyond only cerebral decline. However, the study of cerebrovascular techniques for AD is still evolving. Here, we provide reproducible methods to measure impedance-based pulse wave velocity (PWV), a marker of arterial stiffness, in the systemic vascular (aortic PWV) and in the cerebral vascular (cerebral PWV) systems. Using aortic impedance and this relatively novel technique of cerebral impedance to comprehensively describe the systemic vascular and the cerebral vascular systems, we examined the sex-dependent differences in 5x transgenic mice (5XFAD) with AD under normal and high-fat diet, and in wild-type mice under a normal diet. Additionally, we validated our method for measuring cerebrovascular impedance in a model of induced stress in 5XFAD. Together, our results show that sex and diet differences in wildtype and 5XFAD mice account for very minimal differences in cerebral impedance. Interestingly, 5XFAD, and not wildtype, male mice on a chow diet show higher cerebral impedance, suggesting pathological differences. Opposingly, when we subjected 5XFAD mice to stress, we found that females showed elevated cerebral impedance. Using this validated method of measuring impedance-based aortic and cerebral PWV, future research may explore the effects of modifying factors including age, chronic diet, and acute stress, which may mediate cardiovascular risk in AD. New and NoteworthyHere, we presented a new technique which is an application of the concept of aortic impedance to determining cerebral impedance. While aortic PWV is typically utilized to study aortic stiffness, we also developed a technique of cerebral PWV to study cerebral vascular stiffness. This method may be useful in improving the rigor of studies that seek to have a dual focus on cardiovascular and cerebral function. 

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