Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells.

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.

Creating Optimal Western Blot Conditions for OPA1 Isoforms in Skeletal Muscle Cells and Tissue.

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.

Quantitative assessment of morphological changes in lipid droplets and lipid-mito interactions with aging in brown adipose.

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LDs) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. For example, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids.

The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney.

The kidney filters nutrient waste and bodily fluids from the bloodstream, in addition to secondary functions of metabolism and hormone secretion, requiring an astonishing amount of energy to maintain its functions. In kidney cells, mitochondria produce adenosine triphosphate (ATP) and help maintain kidney function. Due to aging, the efficiency of kidney functions begins to decrease.

Quantitative Assessment of Morphological Changes in Lipid Droplets and Lipid-Mito Interactions with Aging in Brown Adipose.

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LD) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. Particularly, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids.

Methods to Utilize Pulse Wave Velocity to Measure Alterations in Cerebral and Cardiovascular Parameters.

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.

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The sorting and assembly machinery (SAM) Complex is responsible for assembling β-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity.

Recruiting and retaining autistic talent in STEMM.

Autistic adults (AA) have the highest unemployment rate relative to other groups, regardless of disability status. Systemic changes are needed to acquire and retain AA in science, technology, engineering, mathematics, and medicine (STEMM). Here, we discuss the unique challenges AA face in STEMM and possible solutions to overcome them.

A pilot study on our non-traditional, varied writing accountability group for historically excluded and underrepresented persons in STEMM.

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.

3D reconstruction of murine mitochondria reveals changes in structure during aging linked to the MICOS complex.

During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized.

Protocol for isolating mice skeletal muscle myoblasts and myotubes via differential antibody validation.

Isolation of skeletal muscles allows for the exploration of many complex diseases. Here, we present a protocol for isolating mice skeletal muscle myoblasts and myotubes that have been differentiated through antibody validation. We describe steps for collecting and preparing murine skeletal tissue, myoblast cell maintenance, plating, and cell differentiation.

Project Strengthen: An STEMM-focused career development workshop to prepare underrepresented minority students for graduate school.

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 to emulate some of the key aspects of MARC programs.

Three-dimensional mitochondria reconstructions of murine cardiac muscle changes in size across aging.

With sparse treatment options, cardiac disease remains a significant cause of death among humans. As a person ages, mitochondria breakdown and the heart becomes less efficient. Heart failure is linked to many mitochondria-associated processes, including endoplasmic reticulum stress, mitochondrial bioenergetics, insulin signaling, autophagy, and oxidative stress.

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue.

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells.

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue.

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells.

Components of Isolated Skeletal Muscle Differentiated Through Antibody Validation.

Isolation of skeletal muscles allows for the exploration of many complex diseases. Fibroblasts and myoblast play important roles in skeletal muscle morphology and function. However, skeletal muscles are complex and made up of many cellular populations and validation of these populations is highly important.

Ablation of is associated with fragmentation and alterations in metabolism in murine and human myotubes.

The Sorting and Assembly Machinery (SAM) Complex is responsible for assembling β-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system (MICOS) complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity.

Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells.

Proximity ligation assays (PLA) use specific antibodies to detect endogenous protein-protein interactions. PLA is a highly useful biochemical technique that allows two proteins within close proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of PLA in mouse skeletal muscle (SkM) is novel.

Creating Optimal Conditions for OPA1 Isoforms by Western Blot in Skeletal Muscle Cells and Tissue.

OPA1 is a dynamin-related GTPase that modulates various mitochondrial functions and is involved in mitochondrial morphology. There are eight different isoforms of OPA1 in humans and five different isoforms in mice that are expressed as short or long-form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial functions.

In the Age of Machine Learning Cryo-EM Research is Still Necessary: A Path toward Precision Medicine.

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.

Serial Block Face-Scanning Electron Microscopy as a Burgeoning Technology.

Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004. Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM.

Cardiovascular hemodynamics in mice with tumor necrosis factor receptor-associated factor 2 mediated cytoprotection in the heart.

Introduction: Many studies in mice have demonstrated that cardiac-specific innate immune signaling pathways can be reprogrammed to modulate inflammation in response to myocardial injury and improve outcomes. While the echocardiography standard parameters of left ventricular (LV) ejection fraction, fractional shortening, end-diastolic diameter, and others are used to assess cardiac function, their dependency on loading conditions somewhat limits their utility in completely reflecting the contractile function and global cardiovascular efficiency of the heart. A true measure of global cardiovascular efficiency should include the interaction between the ventricle and the aorta (ventricular-vascular coupling, VVC) as well as measures of aortic impedance and pulse wave velocity.

Using quotients as a mentor to facilitate the success of underrepresented students.

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.