The fluorescence viewing of mitochondria is commonly performed by MitoTracker, a lipophilic cationic dye that is taken up by the mitochondria. In this forum, we highlight several issues that may ...occur with MitoTracker, including staining of other organelles. Our aim is to offer alternative dyes and discuss their advantages and disadvantages. We also offer options for software with alternatives to MitoTracker to expedite future experimental design.
This review provides an overview of the current methods for quantifying mitochondrial ultrastructure, including cristae morphology, mitochondrial contact sites, and recycling machinery and a guide to ...utilizing electron microscopy to effectively measure these organelles. Quantitative analysis of mitochondrial ultrastructure is essential for understanding mitochondrial biology and developing therapeutic strategies for mitochondrial-related diseases. Techniques such as transmission electron microscopy (TEM) and serial block face-scanning electron microscopy, as well as how they can be combined with other techniques including confocal microscopy, super-resolution microscopy, and correlative light and electron microscopy are discussed. Beyond their limitations and challenges, we also offer specific magnifications that may be best suited for TEM analysis of mitochondrial, endoplasmic reticulum, and recycling machinery. Finally, perspectives on future quantification methods are offered.
•Suggestions for transmission electron microscopy measurement of mitochondria, their contact sites, and recycling machinery.•Measurement techniques for mitochondria, their contact sites, recycling machinery, endoplasmic reticulum, and Golgi Apparatus.•Critique of the current literature regarding TEM quantification and considerations for future 3D analysis.•TEM from our laboratory across a range of tissues demonstrating the functional impact of these literature-backed suggestions.
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
, 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.
Mitochondria contain connexins, a family of proteins that is known to form gap junction channels. Connexins are synthesized in the endoplasmic reticulum and oligomerized in the Golgi to form ...hemichannels. Hemichannels from adjacent cells dock with one another to form gap junction channels that aggregate into plaques and allow cell-cell communication. Cell-cell communication was once thought to be the only function of connexins and their gap junction channels. In the mitochondria, however, connexins have been identified as monomers and assembled into hemichannels, thus questioning their role solely as cell-cell communication channels. Accordingly, mitochondrial connexins have been suggested to play critical roles in the regulation of mitochondrial functions, including potassium fluxes and respiration. However, while much is known about plasma membrane gap junction channel connexins, the presence and function of mitochondrial connexins remain poorly understood. In this review, the presence and role of mitochondrial connexins and mitochondrial/connexin-containing structure contact sites will be discussed. An understanding of the significance of mitochondrial connexins and their connexin contact sites is essential to our knowledge of connexins' functions in normal and pathological conditions, and this information may aid in the development of therapeutic interventions in diseases linked to mitochondria.
Hypertension is the leading remediable risk factor for cardiovascular morbidity and mortality in the United States. Excess dietary salt consumption, which is a catalyst of hypertension, initiates an ...inflammatory cascade via activation of antigen-presenting cells (APCs). This pro-inflammatory response is driven primarily by sodium ions (Na
) transporting into APCs by the epithelial sodium channel (ENaC) and subsequent NADPH oxidase activation, leading to high levels of oxidative stress. Oxidative stress, a well-known catalyst for hypertension-related illness development, disturbs redox homeostasis, which ultimately promotes lipid peroxidation, isolevuglandin production and an inflammatory response. Natural medicinal compounds derived from organic materials that are characterized by their anti-inflammatory, anti-oxidative, and anti-mutagenic properties have recently gained traction amongst the pharmacology community due to their therapeutic effects. Flavonoids, a natural phenolic compound, have these therapeutic benefits and can potentially serve as anti-hypertensives. Flavones are a type of flavonoid that have increased anti-inflammatory effects that may allow them to act as therapeutic agents for hypertension, including diosmetin, which is able to induce significant arterial vasodilation in several different animal models. This review will focus on the activity of flavones to illuminate potential preventative and potential therapeutic mechanisms against hypertension.
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.
It is well-understood that the science, technology, engineering, and mathematics (STEM) fields have unique challenges that discourage recruiting and retaining underrepresented minorities. Research ...programs aimed at undergraduates have arisen as a critical mechanism for fostering innovation and addressing the challenges faced by underrepresented minorities. Here, we review various undergraduate research programs designed to provide exposure to undergraduates, with a focus on underrepresented minorities in STEM disciplines. We provide insight into selected programs' objectives, key features, potential limitations, and outcomes. We also offer recommendations for future improvements of each research program, particularly in the context of mentorship. These programs range from broad-reaching initiatives (e.g., Leadership Alliance) to more specific programs targeting underrepresented students. By offering a nuanced understanding of each program’s structure, we seek to provide a brief overview of the landscape of diversity-focused STEM initiatives and a guide on how to run a research program effectively.
It is well-understood that science, technology, engineering, and mathematics (STEM) fields have unique challenges that discourage recruiting and retaining underrepresented minorities. Research programs aimed at undergraduates have arisen as a critical mechanism for fostering innovation and addressing the challenges faced by underrepresented minorities. Here, we review various undergraduate research programs designed to provide exposure to undergraduates, with a focus on underrepresented minorities in STEM disciplines. We provide insight into the selected program objectives, key features, potential limitations, and outcomes. We also offer recommendations for future improvements of each research program, particularly in the context of mentorship. These programs range from broad-reaching initiatives like the Leadership Alliance to more specific programs targeting underrepresented students. By offering a nuanced understanding of each program’s structure, we seek to provide a brief overview of the landscape of diversity-focused STEM initiatives and a guide on how to run a research program effectively.
Underrepresented faculty have higher burnout rates and lower grant attainment rates when compared with their non-minority counterparts. Many in science, technology, engineering, mathematics, and ...medicine (STEMM) disciplines, including underrepresented individuals, often have difficulty dedicating time to the writing process, with trainees often being relegated to laboratory tasks in their training years, resulting in a lack of practice in academic writing. Notably, past studies have shown that grant attainment rates of underrepresented individuals are lower than their majority counterparts. Here, we sought to consider a mechanism targeted to underrepresented individuals, although applicable to everyone, to help overcome traditional barriers to writing in STEMM. The authors have hosted a writing accountability group (WAG) that uniquely provides a format focused on physical activity and different forms of writing to strengthen both career development and award/funding attainment. Our objectives were to evaluate this unique format, thus creating a resource for individuals and institutions to learn about WAGs and expand upon the framework to formulate their own WAG. To do this, we performed a small pilot study (n = 21) to investigate attitudes towards the WAG. We present the results of a survey conducted among underrepresented WAG participants, which spanned different career stages and was highly diverse demographically. Our results show that following attendance of our WAG, individuals did not note a significant change in scales pertaining to John Henryism (high-effort coping), resilience, sense of belonging, or grit. However, significant increases were noted in the self-perceived ability to handle stress, confidence in applying for awards, appreciation for mentoring, and satisfaction of WAGs. Taken together, the results of this study suggest that our unique WAG format can have some positive results as a career and writing development opportunity and may be able to support underrepresented individuals in attaining funding at higher education institutions.
Maximizing Access to Research Careers (MARC) programs are aimed to increase diversity in science, technology, engineering, math, and medicine (STEMM) fields. However, limited programs and eligibility ...requirements limit the students who may apply to similar programs. At Winston-Salem State University, we piloted a series of workshops, collectively termed Project Strengthen, to emulate some of the key aspects of MARC programs. Following the workshop, Project Strengthen students showed a significant increase in their understanding of essential educational development skills, such as writing personal statements, applying to graduate school, studying for the GRE, and seeking summer internships. This suggests Project Strengthen may be a potential lower cost comparable option than MARC to make up for current deficiencies in preparedness for graduate school. We also provide educational materials from Project Strengthen, including a clear framework for this seminar series, six ready-made PowerPoints to share with trainees that have been demonstrated to be effective.
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•Project Strengthen improves minority graduate school preparedness at a low cost•Project Strengthen is similar to the Maximizing Access to Research Careers program•Need for other low-cost programs to increase minority student retention•All workshops included in Project Strengthen are available for future use here
Social sciences; Education
Diversity, equity, and inclusion (DEI) initiatives are critical for fostering growth, innovation, and collaboration in science, technology, engineering, mathematics, and medicine (STEMM). This ...article focuses on four key topics that have impacted many Black individuals in STEMM: know-your-place aggression, environmental microaggressions, peer mediocrity, and code-switching. We provide a comprehensive background on these issues, discuss current statistics, and provide references that support their existence, as well as offer solutions to recognize and address these problems in the STEMM which can be expanded to all historically underrepresented individuals.
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