Three-dimensional analysis of mitochondria in a patient-derived xenograft model of triple negative breast cancer reveals mitochondrial network remodeling following chemotherapy treatments.

Mitochondria are hubs of metabolism and signaling and play an important role in tumorigenesis, therapeutic resistance, and metastasis in many cancer types. Various laboratory models of cancer demonstrate the extraordinary dynamics of mitochondrial structure, but little is known about the role of mitochondrial structure in resistance to anticancer therapy. We previously demonstrated the importance of mitochondrial structure and oxidative phosphorylation in the survival of chemotherapy-refractory triple negative breast cancer (TNBC) cells. As TNBC is a highly aggressive breast cancer subtype with few targeted therapy options, conventional chemotherapies remain the backbone of early TNBC treatment. Unfortunately, approximately 45% of TNBC patients retain substantial residual tumor burden following chemotherapy, associated with abysmal prognoses. Using an orthotopic patient-derived xenograft mouse model of human TNBC, we compared mitochondrial structures between treatment-naïve tumors and residual tumors after conventional chemotherapeutics were administered singly or in combination. We reconstructed 1,750 mitochondria in three dimensions from serial block-face scanning electron micrographs, providing unprecedented insights into the complexity and intra-tumoral heterogeneity of mitochondria in TNBC. Following exposure to carboplatin or docetaxel given individually, residual tumor mitochondria exhibited significant increases in mitochondrial complexity index, area, volume, perimeter, width, and length relative to treatment-naïve tumor mitochondria. In contrast, residual tumors exposed to those chemotherapies given in combination exhibited diminished mitochondrial structure changes. Further, we document extensive intra-tumoral heterogeneity of mitochondrial structure, especially prior to chemotherapeutic exposure. These results highlight the potential for structure-based monitoring of chemotherapeutic responses and reveal potential molecular mechanisms that underlie chemotherapeutic resistance in TNBC.

A workshop to enrich physiological understanding through hands-on learning about mitochondria-endoplasmic reticulum contact sites.

Physiology is an important field for students to gain a better understanding of biological mechanisms. Yet, many students often find it difficult to learn from lectures, resulting in poor retention. Here, we utilize a learning workshop model to teach students at different levels ranging from middle school to undergraduate. We specifically designed a workshop to teach students about mitochondria-endoplasmic reticulum contact (MERC) sites. The workshop was implemented for middle school students in a laboratory setting that incorporated a pretest to gauge prior knowledge, instructional time, hands-on activities, interactive learning from experts, and a posttest. We observed that the students remained engaged during the session of interactive methods, teamed with their peers to complete tasks, and delighted in the experience. Implications for the design of future physiological workshops are further offered.NEW & NOTEWORTHY This manuscript offers a design for a workshop that utilizes blended learning to engage middle school, high school, and undergraduate students while teaching them about mitochondria-endoplasmic reticulum contact sites.

SOX6 expression and aneurysms of the thoracic and abdominal aorta.

Abdominal and thoracic aortic aneurysms (AAAs, TAAs) remain a major cause of deaths worldwide, in part due to the lack of reliable prognostic markers or early warning signs. Sox6 has been found to regulate renin controlling blood pressure. We hypothesized that Sox6 may serve as an important regulator of the mechanisms contributing to hypertension-induced aortic aneurysms. Phenotype and laboratory-wide association scans in a clinical cohort found that SOX6 gene expression is associated with aortic aneurysm in subjects of European ancestry. Sox6 and tumor necrosis factor alpha (TNF-α) expression were upregulated in aortic tissues from patients affected by either AAA or TAA. In Sox6 knockout mice with angiotensin-II-induced AAA, we found that Sox6 plays critical role in the development and progression of AAA. Our data support a regulatory role of SOX6 in the development of hypertension-induced AAA, suggesting that Sox6 may be a therapeutic target for the treatment of aortic aneurysms.

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. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and the proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Our analyses showed reductions in LD volume, area, and perimeter in aged samples in comparison to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for the mitochondria interacting with LDs. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology with mitochondrial function, metabolism, and bioactivity in aged BAT.

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes.

The liver, the largest internal organ and a metabolic hub, undergoes significant declines due to aging, affecting mitochondrial function and increasing the risk of systemic liver diseases. How the mitochondrial three-dimensional (3D) structure changes in the liver across aging, and the biological mechanisms regulating such changes confers remain unclear. In this study, we employed Serial Block Face-Scanning Electron Microscopy (SBF-SEM) to achieve high-resolution 3D reconstructions of murine liver mitochondria to observe diverse phenotypes and structural alterations that occur with age, marked by a reduction in size and complexity. We also show concomitant metabolomic and lipidomic changes in aged samples. Aged human samples reflected altered disease risk. To find potential regulators of this change, we examined the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex, which plays a crucial role in maintaining mitochondrial architecture. We observe that the MICOS complex is lost during aging, but not Sam50. Sam50 is a component of the sorting and assembly machinery (SAM) complex that acts in tandem with the MICOS complex to modulate cristae morphology. In murine models subjected to a high-fat diet, there is a marked depletion of the mitochondrial protein SAM50. This reduction in Sam50 expression may heighten the susceptibility to liver disease, as our human biobank studies corroborate that Sam50 plays a genetically regulated role in the predisposition to multiple liver diseases. We further show that changes in mitochondrial calcium dysregulation and oxidative stress accompany the disruption of the MICOS complex. Together, we establish that a decrease in mitochondrial complexity and dysregulated metabolism occur with murine liver aging. While these changes are partially be regulated by age-related loss of the MICOS complex, the confluence of a murine high-fat diet can also cause loss of Sam50, which contributes to liver diseases. In summary, our study reveals potential regulators that affect age-related changes in mitochondrial structure and metabolism, which can be targeted in future therapeutic techniques.

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. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Analysis showed reductions in LD volume, area, and perimeter in aged samples compared to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for mitochondria interacting with LD lipids. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology and mitochondrial functionality, metabolism, and bioactivity in aged BAT.

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. Dysfunction in mitochondria and cristae, the inner folds of mitochondria, is a hallmark of aging. Therefore, age-related kidney function decline could be due to changes in mitochondrial ultrastructure, increased reactive oxygen species (ROS), and subsequent alterations in metabolism and lipid composition. We sought to understand if there is altered mitochondrial ultrastructure, as marked by 3D morphological changes, across time in tubular kidney cells. Serial block facing-scanning electron microscope (SBF-SEM) and manual segmentation using the Amira software were used to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented, compared to the 3-month, with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we show that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney due to aging. Disruption of the MICOS complex shows altered mitochondrial calcium uptake and calcium retention capacity, as well as generation of oxidative stress. We found significant, detrimental structural changes to aged kidney tubule mitochondria suggesting a potential mechanism underlying why kidney diseases occur more readily with age. We hypothesize that disruption in the MICOS complex further exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, thus impacting kidney health.

Myeloid Cell Glucocorticoid, Not Mineralocorticoid Receptor Signaling, Contributes to Salt-Sensitive Hypertension in Humans via Cortisol.

BACKGROUND: Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular morbidity and mortality, yet the etiology is poorly understood. We previously found that serum/glucocorticoid-regulated kinase 1 (SGK1) and epoxyeicosatrienoic acids (EETs) regulate epithelial sodium channel (ENaC)-dependent sodium entry into monocyte-derived antigen-presenting cells (APCs) and activation of NADPH oxidase, leading to the formation of isolevuglandins (IsoLGs) in SSBP. Whereas aldosterone via the mineralocorticoid receptor (MR) activates SGK1 leading to hypertension, our past findings indicate that levels of plasma aldosterone do not correlate with SSBP, and there is little to no MR expression in APCs. Thus, we hypothesized that cortisol acting via the glucocorticoid receptor (GR), not the MR in APCs mediates SGK1 actions to induce SSBP. METHODS: We performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) analysis on peripheral blood mononuclear cells of humans rigorously phenotyped for SSBP using an inpatient salt loading/depletion protocol to determine expression of MR, GR, and SGK1 in immune cells. In additional experiments, we performed bulk transcriptomic analysis on isolated human monocytes following in vitro treatment with high salt from a separate cohort. We then measured urine and plasma cortisol, cortisone, renin, and aldosterone. Subsequently, we measured the association of these hormones with changes in systolic, diastolic, mean arterial pressure and pulse pressure as well as immune cell activation via IsoLG formation. RESULTS: We found that myeloid APCs predominantly express the GR and SGK1 with no expression of the MR. Expression of the GR in APCs increased after salt loading and decreased with salt depletion in salt-sensitive but not salt-resistant people and was associated with increased expression of SGK1. Moreover, we found that plasma and urine cortisol/cortisone but not aldosterone/renin correlated with SSBP and APCs activation via IsoLGs. We also found that cortisol negatively correlates with EETs. CONCLUSION: Our findings suggest that renal cortisol signaling via the GR but not the MR in APCs contributes to SSBP via cortisol. Urine and plasma cortisol may provide an important currently unavailable feasible diagnostic tool for SSBP. Moreover, cortisol-GR-SGK1-ENaC signaling pathway may provide treatment options for SSBP.

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 ß-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. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

A review of undergraduate research programs aimed at underrepresented students.

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.

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. 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.

Strategies for inducing and validating zinc deficiency and zinc repletion.

Given the growing interest in the role of zinc in the onset and progression of diseases, there is a crucial demand for reliable methods to modulate zinc homeostasis. Using a dietary approach, we provide validated strategies to alter whole-body zinc in mice, applicable across species. For confirmation of zinc status, animal growth rates as well as plasma and urine zinc levels were evaluated. The accessible and cost-effective methodology outlined will increase scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of diseases.NEW & NOTEWORTHY This methods paper details dietary approaches to alter zinc homeostasis in rodents and qualitative and quantitative methods to ensure the zinc status of experimental animals. The outlined accessible and cost-effective protocol will elevate scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of a multitude of health conditions and diseases.

Running a successful STEMM summer program: A week-by-week guide.

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.

Strategies for Inducing and Validating Zinc Deficiency and Zinc Repletion.

Given the growing interest in the role of zinc in the onset and progression of diseases, there is a crucial demand for reliable methods to modulate zinc homeostasis. Using a dietary approach, we provide validated strategies to alter whole-body zinc in mice, applicable across species. For confirmation of zinc status, animal growth rates as well as plasma and urine zinc levels were evaluated. The accessible and cost-effective methodology outlined will increase scientific rigor, ensuring reproducibility in studies exploring the impact of zinc deficiency and repletion on the onset and progression of diseases.

Considerations for developing mitochondrial transplantation techniques for individualized medicine.

Tweetable abstract Mitochondrial transplantation has been used to treat various diseases associated with mitochondrial dysfunction. Here, we highlight the considerations in quality control mechanisms that should be considered in the context of mitochondrial transplantation.

A workshop to showcase the diversity of scientists to middle school students.

Identity matters in science, technology, engineering, mathematics, and medicine (STEMM) because it can affect an individual's long-term sense of belonging, which may in turn affect their persistence in STEMM. Early K-12 science classes often teach students about the foundational discoveries of the field, which have been predominately made, or at least published, by White men. This homogeneity can leave underrepresented individuals in STEMM feeling isolated, and underrepresented K-12 students may feel as though they cannot enter STEMM fields. This study aimed to examine these feelings of inclusivity in STEMM through an interactive workshop that asked middle schoolers to identify scientists from images of individuals with various racial and gender identities. We found that a plurality of students had a positive experience discussing diversity in science and recognizing underrepresented individuals as scientists.NEW & NOTEWORTHY We observed positive sentiments from middle school students following a workshop that showcased diversity in science. This workshop uniquely encourages students to recognize that physiologists and scientists today are much more diverse than textbooks typically demonstrate and can be adapted for middle schoolers, high schoolers, and college students.

3D Mitochondrial Structure in Aging Human Skeletal Muscle: Insights into MFN-2 Mediated Changes.

Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 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 we 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.

Disparities in funding for Nobel Prize awards in medicine and physiology across nationalities, races, and gender.

Since 1901, the Nobel Prize in Physiology and Medicine has been awarded to numerous individuals for their outstanding contributions. This article presents a comprehensive analysis of the Nobel Prize recipients, focusing on gender, race, and nationality. We observe that an alarming disparity emerges when we examine the underrepresentation of Black scientists among Nobel laureates. Furthermore, trends in nationalities show how Americans make up the majority of Nobel Prize winners, while there is a noticeable lack of gender and racial minority winners of the Nobel Prize in Physiology and Medicine. Together, this highlights the importance of diversity and inclusion in scientific achievement. We offer suggestions and techniques, including funding opportunities and expanding nominators, to improve the gender, racial, and geographical diversity of Nobel Prizes.

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. Therefore, it is hypothesized that significant morphological changes in BAT mitochondria and cristae will be present with aging. A quantitative 3D electron microscopy approach is developed to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, the 3D morphology of mitochondrial cristae is investigated in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, an increase is found in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging.