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Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.

Abstract Choosing a mentor requires a certain level of introspection for both the mentor and the mentee. The dynamics of mentorship may change depending on the academic status of the mentee. Regardless, mentors should help their trainees grow both academically and professionally. The success of an individual in the fields of science, technology, engineering, mathematics, and medicine (STEMM) depends on more than intellectual capacity; a holistic view encompassing all factors that contribute to scientific achievement is all-important. Specifically, one new method scientists can adopt is quotients, which are scales and techniques that can be used to measure aptitude in a specific area. In this paper, we focus on these factors and how to grow one’s adversity quotient (AQ), social quotient (SQ), and personal growth initiative scale (PGIS). We also look at how mentors can better understand the biases of their trainees. In addressing this, mentors can help trainees become more visible and encourage other trainees to become allies through reducing biases. Quotients that contribute to success are discussed, and suggestions in the context of COVID-19 are provided to facilitate student success in science, technology, engineering, mathematics, and medicine (STEMM).

Abstract Disability remains an underacknowledged and underdiscussed topic in science, technology, engineering, mathematics, and medicine (STEMM). Social stigma and fear of negative outcomes have resulted in a consistent lack of disclosure. Disabilities cause social and professional difficulties for those that have them. While some faculty can be allies, past literature shows that steps must be taken to make disabilities visible in STEMM at both student and faculty levels. Here, we offer suggestions to better support faculty and students in enhancing the outcomes of individuals who have invisible disabilities. Critically, techniques such as abolishing stigma, universal learning, and better mentoring may improve the challenges faced by those who self-identify as an individual with a disability. We discuss disability in science, technology, engineering, mathematics, and medicine, why faculty and students with a disability remain underresourced, and potential solutions to increase universal design and representation of individuals with a disability.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

N-type calcium (CaV2.2) channels are widely expressed in the brain and the peripheral nervous system, where they play important roles in the regulation of transmitter release. Although CaV2.2 channel expression levels are precisely regulated, presently little is known regarding the molecules that mediate its synthesis and degradation. Previously, by using a combination of biochemical and functional analyses, we showed that the complex formed by the light chain 1 of the microtubule-associated protein 1B (LC1-MAP1B) and the ubiquitin-proteasome system (UPS) E2 enzyme UBE2L3, may interact with the CaV2.2 channels promoting ubiquitin-mediated degradation. The present report aims to gain further insights into the possible mechanism of degradation of the neuronal CaV2.2 channel by the UPS. First, we identified the enzymes UBE3A and Parkin, members of the UPS E3 ubiquitin ligase family, as novel CaV2.2 channel binding partners, although evidence to support a direct protein-protein interaction is not yet available. Immunoprecipitation assays confirmed the interaction between UBE3A and Parkin with CaV2.2 channels heterologously expressed in HEK-293 cells and in neural tissues. Parkin, but not UBE3A, overexpression led to a reduced CaV2.2 protein level and decreased current density. Electrophysiological recordings performed in the presence of MG132 prevented the actions of Parkin suggesting enhanced channel proteasomal degradation. Together these results unveil a novel functional coupling between Parkin and the CaV2.2 channels and provide a novel insight into the basic mechanisms of CaV channels protein quality control and functional expression.

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Age‐related skeletal muscle atrophy, known as sarcopenia, is characterized by loss of muscle mass, strength, endurance, and oxidative capacity. Although exercise has been shown to mitigate sarcopenia, the underlying governing mechanisms are poorly understood. Mitochondrial dysfunction is implicated in aging and sarcopenia; however, few studies explore how mitochondrial structure contributes to this dysfunction. In this study, we sought to understand how aging impacts mitochondrial three‐dimensional (3D) structure and its regulators in skeletal muscle. We hypothesized that aging leads to remodeling of mitochondrial 3D architecture permissive to dysfunction and is ameliorated by exercise. Using serial block‐face scanning electron microscopy (SBF‐SEM) and Amira software, mitochondrial 3D reconstructions from patient biopsies were generated and analyzed. Across five human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria are less spherical and more complex, indicating age‐related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age‐related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved, as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age‐related structural changes in mitochondria and further suggest that exercise may mitigate age‐related structural decline through modulation of mitofusin 2. Changes in mitochondrial structure and dynamics during aging provide a mechanism for the development of age‐related sarcopenia, a condition characterized by muscle mass loss. Through the creation of three‐dimensional models of mitochondria from quadriceps muscle tissue taken from old and young humans, a loss in mitochondrial complexity was observed to occur during aging. A decrease in the expression of mitochondrial fusion protein mitofusin 2 (MFN‐2) in older populations may drive these mitochondrial structural changes. A Drosophila model with the MFN‐2 ortholog knocked down demonstrated a loss of mitochondrial complexity and lower quality cristae, which parallel changes in mitochondria observed in older humans. The use of an in vitro cell exercise model showed that the mechanism by which exercise counteracts the effects of sarcopenia, age‐related disease may be due to increased expression of MFN‐2 during exercise.

Mitochondrial contact site and cristae organizing system (MICOS) complexes are critical for maintaining the mitochondrial architecture, cristae integrity, and organelle communication in neurons. MICOS disruption has been implicated in neurodegenerative disorders, including Alzheimer's disease (AD), yet the spatiotemporal dynamics of MICOS-associated neuronal alterations during aging remain unclear. Using three-dimensional reconstructions of hypothalamic and cortical neurons, we observed age-dependent fragmentation of mitochondrial cristae, reduced intermitochondrial connectivity, and compartment-specific changes in mitochondrial size and morphology. Notably, these structural deficits were most pronounced in neurons vulnerable to AD-related pathology, suggesting a mechanistic link between MICOS disruption and the early mitochondrial dysfunction observed in patients with AD. Our findings indicate that the loss of MICOS integrity is a progressive feature of neuronal aging, contributing to impaired bioenergetics and reduced resilience to metabolic stress and potentially facilitating neurodegenerative processes. MICOS disruption reduced neuronal firing and synaptic responsiveness, with miclxin treatment decreasing mitochondrial connectivity and inducing cristae disorganization. These changes link MICOS structural deficits directly to impaired neuronal excitability, highlighting vulnerability to AD-related neurodegeneration. These results underscore the importance of MICOS as a critical determinant of neuronal mitochondrial health and as a potential target for interventions aimed at mitigating AD-related mitochondrial dysfunction.

ABSTRACT Mentoring is a developmental experience intended to increase the willingness to learn and establish credibility while building positive relationships through networking. In this commentary, we focus on intentional mentoring for underrepresented mentees, including individuals that belong to minority racial, ethnic and gender identity groups in Science, Technology, Engineering, Mathematics and Medicine (STEMM) fields. Intentional mentoring is the superpower action necessary for developing harmony and comprehending the purpose and value of the mentor/mentee relationship. Regardless of a mentor's career stage, we believe the strategies discussed may be used to create a supportive and constructive mentorship environment; thereby improving the retention rates of underrepresented mentees within the scientific community. This article discusses how to be an intentional mentor in the 21st Century.

ABSTRACT While it is commonly thought that microaggressions are isolated incidents, microaggressions are ingrained throughout the academic research institution (Young, Anderson and Stewart 2015; Lee et al. 2020). Persons Excluded from science because of Ethnicity and Race (PEERs) frequently experience microaggressions from various academicians, including graduate students, postdocs and faculty (Asai 2020; Lee et al. 2020). Here, we elaborate on a rationale for concrete actions to cope with and diminish acts of microaggressions that may otherwise hinder the inclusion of PEERs. We encourage Science, Technology, Engineering and Mathematics (STEM) departments and leadership to affirm PEER scholar identities and promote allyship by infusing sensitivity, responsiveness and anti-bias awareness. This article focuses on how mentors can be allies against microaggressions in STEM.