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.

Call to action to properly utilize electron microscopy to measure organelles to monitor disease.

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.

A workshop on mitochondria for students to improve understanding of science and hypothesis forming.

There remains a clear deficiency in recruiting middle school students in science, technology, engineering, mathematics, and medicine fields, especially for those students entering physiology from underrepresented backgrounds. A large part of this may be arising from a disconnect between how science is typically practiced at a collegiate and K-12 level. Here, we have envisioned mitochondria and their diverse subcellular structures as an involver for middle school students. We present the framework for a workshop that familiarizes students with mitochondria, employing three-dimensional visual-spatial learning and real-time critical thinking and hypothesis forming. This workshop had the goal of familiarizing middle school students with the unique challenges the field currently faces and better understanding the actuality of being a scientist through critical analysis including hypothesis forming. Findings show that middle school students responded positively to the program and felt as though they had a better understanding of mitochondria. Future implications for hands-on programs to involve underrepresented students in science are discussed, as well as potential considerations to adapt it for high school and undergraduate students.NEW & NOTEWORTHY Here we employ a workshop that utilizes blended and tactile learning to teach middle schoolers about mitochondrial structure. By creating an approachable and fun workshop that can be utilized for middle school students, we seek to encourage them to join a career in physiology.

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells.

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery.

Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer-scale micrographs of cellular degradation components in a fixed sample. Serial block face-scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

The SWELL1-LRRC8 complex regulates endothelial AKT-eNOS signaling and vascular function.

The endothelium responds to numerous chemical and mechanical factors in regulating vascular tone, blood pressure, and blood flow. The endothelial volume-regulated anion channel (VRAC) has been proposed to be mechanosensitive and thereby sense fluid flow and hydrostatic pressure to regulate vascular function. Here, we show that the leucine-rich repeat-containing protein 8a, LRRC8A (SWELL1), is required for VRAC in human umbilical vein endothelial cells (HUVECs). Endothelial LRRC8A regulates AKT-endothelial nitric oxide synthase (eNOS) signaling under basal, stretch, and shear-flow stimulation, forms a GRB2-Cav1-eNOS signaling complex, and is required for endothelial cell alignment to laminar shear flow. Endothelium-restricted Lrrc8a KO mice develop hypertension in response to chronic angiotensin-II infusion and exhibit impaired retinal blood flow with both diffuse and focal blood vessel narrowing in the setting of type 2 diabetes (T2D). These data demonstrate that LRRC8A regulates AKT-eNOS in endothelium and is required for maintaining vascular function, particularly in the setting of T2D.

CD28 Superagonistic Activation of T Cells Induces a Tumor Cell-Like Metabolic Program.

CD28 superagonist (CD28SA), a therapeutic immunomodulatory monoclonal antibody triggered rapid and exaggerated activation of CD4+ effector memory T cells (TEMs) in humans with unwanted serious adverse effects. It is well known that distinct metabolic programs determine the fate and responses of immune cells. In this study, we show that human CD4+ TEMs stimulated with CD28SA adopt a metabolic program similar to those of tumor cells with enhanced glucose utilization, lipid biosynthesis, and proliferation in hypoxic conditions. Identification of metabolic profiles underlying hyperactive T cell activation would provide a platform to test safety of immunostimulatory antibodies.

Metabolic Syndrome, Dyslipidemia and Regulation of Lipoprotein Metabolism.

BACKGROUND: Metabolic syndrome is associated with increased risk for both type 2 diabetes and cardiovascular disease. Development of these pathologies is associated with the disorders of lipid and lipoprotein metabolism. Dyslipidemia leads to the overproduction of potentially atherogenic lipid and lipoproteins. Furthermore, there is a decrease in the levels of high-density lipoproteins and an increase in the levels of remnant and small dense LDL particles. CONCLUSION: In the current review, we have discussed the pathophysiology of lipoprotein biosynthesis and metabolism in the metabolic syndrome. Finally, we describe regulation of lipoprotein metabolism which may be used as a potential target for treating dyslipidemia in metabolic syndrome.

RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

Brusatol provokes a rapid and transient inhibition of Nrf2 signaling and sensitizes mammalian cells to chemical toxicity-implications for therapeutic targeting of Nrf2.

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs.

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist.

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.