bluuebunny/crossref_metadata_2025 huggingface dataset:
Mass Spectrometry Based Identification of Novel HLA Class I Restricted Peptides in Merkel Cell Carcinoma
Authors: Rachimi Suzanna , Klaeger Susan , Lee Patrick C. , Le Phuong M. , Chea Vipheaviny A. , Carulli Isabel P. , Ananthapadmanabhan Varsha , Tarren Anna , Sarkizova Siranush , Apffel Annie , Clauser Karl R. , DeCaprio James A. , Carr Steven A. , Wu Catherine J. , Keskin Derin B.
Published: May 2022

Highly polymorphic class I human leukocyte antigen (HLA‐I) molecules present endogenous peptides to CD8+ T cells, an essential part of immune regulation. This mechanism allows for the presentation of thousands of epitopes per allele and individual. Liquid chromatography‐mass spectrometry (LC‐MS/MS) is a high throughput method that can be utilized to identify endogenously processed and presented peptides. Merkel cell carcinoma (MCC) is a rare and aggressive form of neuroendocrine skin cancer. A major subtype of this cancer is caused by the Merkel cell Polyomavirus (MCPyV), so MCPyV+ MCC is an informative model system to explore virally derived cancer‐specific epitopes. MCC, however, often downregulates or eliminates HLA‐I antigen presentation to evade recognition by the immune system. We hypothesized that reversing the mechanisms of immune evasion will allow us to detect novel epitopes. Here, we used optimized LC‐MS/MS methods to characterize the immunopeptidome of MCC in a native and perturbed state. First, we compared the immunopeptidome of primary tumor samples and tumor‐derived MCC cell lines. Peptide identities presented on primary tumors and tumor‐derived cell lines overlapped by 50% or more, indicating that the tumor‐derived cell lines retain their innate tumor properties and are a useful model to study immune presentation in MCC. Next, we investigated mechanisms to restore HLA‐I presentation in MCC, such as IFNγ treatment and knockout or inhibition of components of the Polycomb repressive complex (PRC1.1). We found that IFNγ treatment increased peptide yield up to 8‐fold across seven cell lines while also increasing presentation on HLA‐B alleles. Using tandem mass tag labeling, we observed relative quantitative changes in the immunopeptidome of an MCPyV+ cell line when PRC1.1 components were knocked down. We discovered increased HLA‐I presentation upon USP7 inhibitor treatment in comparison to the control. In contrast to IFNγ treatment, USP7 inhibition did not alter the fraction of observed peptides assigned to alleles. Finally, use of High Field Asymmetric Ion Mobility Spectrometry (FAIMS) to analyze samples with reinstated HLA presentation enabled identification of a number of novel antigens, including three MCPyV viral epitopes. The immunogenicity of these viral peptides was determined by ELISpot assays with peripheral blood mononuclear cells (PBMCs). These findings can be translated into medical treatments for patients diagnosed with this cancer.
Resting Sympathetic Transduction in Young Healthy non‐Hispanic Black Women: Potential Race and Sex Differences
Authors: Stephens Brandi Y. , Young Benjamin E. , Nandadeva Damsara , Skow Rachel J. , Grotle Ann‐Katrin , Vranish Jennifer R. , Greaney Jody L. , Brothers R. M. , Fadel Paul J.
Published: May 2022

Background

The prevalence of hypertension (HTN) is highest in the non‐Hispanic Black (BL) population. Notably, BL women have an equal or greater HTN prevalence compared to BL men; however, limited studies have included women. Heightened sympathetic nervous system reactivity has been proposed as a potential mediator of elevated HTN risk in the BL population. In this regard, our laboratory previously demonstrated exaggerated sympathetic transduction at rest in young normotensive BL men compared to their non‐Hispanic white (WH) counterparts. Whether similar responses are present in BL women is not known. Furthermore, whether there are sex differences in sympathetic transduction in young BL adults remains unclear.

Hypothesis

We hypothesized that BL women would exhibit elevated resting sympathetic transduction compared to WH women, and that sympathetic transduction would be comparable between BL women and men.

Methods

Six young healthy BL women (age: 20 ± 1 years), six WH women (age: 21 ± 1 years) and six BL men (age: 21 ± 1 years) were studied. Heart rate (ECG), muscle sympathetic nerve activity (MSNA; peroneal microneurography) and beat‐to‐beat blood pressure (BP; Finometer) were continuously measured under resting conditions for 19 ± 1 min. Sympathetic transduction was quantified as peak changes in mean arterial pressure (MAP) and the nadir of total vascular conductance (TVC) over 10 cardiac cycles following spontaneous bursts of MSNA using signal‐averaging. All data are expressed as mean ± SEM.

Results

Resting BP and heart rate were not different between groups (all P > 0.05). Likewise, BL women had similar MSNA burst frequency (BL women: 12 ± 2 vs. WH women: 14 ± 1 bursts/min; P = 0.475) and burst incidence (BL women: 21 ± 2 vs. WH women: 24 ± 2 bursts/100 cardiac cycles; P = 0.477) compared to WH women, and BL men (13 ± 1 bursts/min, 21 ± 1 bursts/100 cardiac cycles; P = 0.539). For peak increases in MAP following MSNA bursts, there were no differences between BL and WH women (BL women: Δ+2.3 ± 0.4 vs. WH women: Δ+2.1 ± 0.2 mmHg; P = 0.741). Peak reductions in TVC were also not different in women (BL women: Δ‐2.1 ± 0.3 vs. WH women Δ‐2.4 ± 0.6 mL/min/mmHg; P = 0.966). However, in comparison to BL men, BL women had lower MAP (women: Δ+2.3 ± 0.4 vs. men: Δ+5.5 ± 1.1 mmHg; P = 0.011) and TVC (P = 0.036) responses.

Conclusion

Contrary to our hypothesis, these preliminary data suggest similar beat‐to‐beat sympathetic transduction at rest in BL and WH women. Interestingly, sympathetic transduction at rest was lower in BL women compared to BL men highlighting a potential differential effect of sex on BP regulation in young BL adults. Collectively, these findings suggest that augmented sympathetic transduction is not likely an early contributor to the greater prevalence of HTN in BL women.
Development of Tumor‐selective Transmembrane Peptides to Promote the Activity of the Receptor Protein Tyrosine Phosphatase J by Disrupting its Homodimerization
Authors: Rizzo Sophie , Sikorski Eden , Hart Will , Gerristen Jacqueline , Lazzarra Matthew , White Forest , Thévenin Damien
Published: May 2022

Receptor Protein Tyrosine Phosphatases (RPTPs) are one of the most important regulators of receptor tyrosine kinases (RTKs) and therefore play a crucial role in mammalian signal transduction. But our still‐incomplete understanding of the structural aspects of their regulation and the lack of selective agonists have hampered efforts to understand their roles in downstream signaling regulation, and to pursue them as potential therapeutic targets. However, the reported ability of RPTP homodimerization to antagonize their catalytic activity presents potential opportunities to develop unique strategies to modulate the activity of RPTPs.

We recently reported (Block et al., JBC, 2019; doi:10.1074/jbc.RA119.010229) that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ; also known as DEP1) is regulated by specific transmembrane (TM) residues, and that these interactions are essential in regulating its enzymatic activity and substrate access in cells.

Building on these new insights and in response to the lack of selective agonists, we present here the design and testing of a novel class of tumor‐selective peptide capable of binding to the TM domain of PTPRJ and disrupting its homodimerization. By doing so, it promotes PTPRJ TM‐mediated access to EGFR, reduces the phosphorylation of EGFR (a known substrate) and other downstream signaling effectors, and antagonizes EGFR‐driven cancer cell phenotypes.

This class of peptide represents a novel allosteric and a possibly orthogonal way to target the activity of PTPRJ and RTKs phosphorylation. It could therefore be used not only as probes to tease out PTPRJ regulating mechanisms but also for therapeutic purposes via the attenuation of signaling by dysregulated RTKs.
Investigating the Role of PKC‐delta in Voltage‐Independent Contractile Pathways in Mouse Resistance Arteries
Authors: Haghbin Nadia , Richter David M. , Kim Michelle S. , Welsh Donald G.
Published: May 2022

Vascular smooth muscle cells (VSMCs) tune blood flow delivery through G protein‐coupled receptor (GPCR) associated signaling pathways and two general contractile mechanisms. The first, electromechanical coupling ties membrane potential (VM) to cytosolic [Ca2+] via voltage‐sensitive L‐type Ca2+ channels. The second—independent of voltage—centers on mechanisms of Ca2+ sensitization via Rho kinase and Protein Kinase C (PKC). While untested, agonists presumably activate both, with their relative contribution remaining static across concentration range. We hypothesize that voltage‐dependent mechanisms proceed voltage‐independent, and this order is contingent on the mode of agonist application.

Mouse mesenteric/cerebral arteries mounted in a pressure myograph (60 mmHg) were globally or focally exposed to GPCR agonists (U46619 and Phenylephrine) and vasomotor responses, intercellular [Ca2+] levels, and VM monitored. Global application of either agonists increased arterial tone in a concentration dependent manner. Subsequent Nifedipine (L‐type Ca2+ channel blocker) application attenuated constrictive responses, particularly at the lower agonist concentrations. The remaining voltage‐independent constriction was blocked by Calphostin C (PKC inhibitor). While these findings highlight a hierarchical arrangement (voltage‐dependent preceding voltage‐independent), this order is not static. Stimulating a reduced number of VSMCs through focal agonist application, induces localized vasomotor responses that do not conduct across the arteriole or alter intercellular [Ca2+] or VM. Such focal responses could bypass electromechanical coupling owing to insufficient charge to change VM. Thus, this response is heavily dependent on Ca2+ sensitization mechanisms. Western blot analysis indicates that PKCα and PKCδ are likely the key regulatory isoforms in agonist‐induced constriction. Ongoing work is focused on translocation of PKCα and PKCδ and phosphorylation state of their key downstream targets including MYPT1, CPI‐17, Caldesmon, and HSP27.

In summary, our findings reveal a hierarchical but malleable contractile arrangement in VSMCs whereby voltage‐dependent signaling precedes voltage‐independent, as long as sufficient smooth muscle cells are activated. These findings are of particular translational value to the pathobiology/management of cerebral vasospasm.
Inducible Smooth Muscle‐Specific Knockout of Acid Sensing Ion Channel 1 Protects Against Pulmonary Hypertension
Authors: Garcia Selina M. , Yellowhair Tracylyn R. , Ahmadian Rosstin , Herbert Lindsay M. , Gonzalez Bosc Laura V. , Resta Thomas C. , Jernigan Nikki L.
Published: May 2022

Acid‐sensing ion channel 1 (ASIC1) is a proton‐gated cation channel that conducts both Na+ and Ca2+. ASIC1 is expressed in multiple cell types including vascular smooth muscle cells (SMCs) and endothelial cells (ECs). Our laboratory has previously shown that ASIC1 contributes to pulmonary arterial constriction and the development of chronic hypoxia (CH)‐induced pulmonary hypertension. However, the contribution of SMC versus EC ASIC1 to CH‐induced pulmonary hypertension is unknown. We tested the hypothesis that SMC expression of ASIC1 contributes to pulmonary hypertension and vascular remodeling by examining the development of CH‐induced pulmonary hypertension (barometric pressure of 380 mmHg for 6 weeks) in inducible SMC‐specific ASIC1 knockout (SMC‐Asic1‐/‐) and EC‐specific ASIC1 knockout (EC‐Asic1‐/‐) mice. The SMC‐Asic1‐/‐animals were used to examine the prevention and reversal of pulmonary hypertension by either inducing Asic1 knockout before CH exposure or following the establishment of pulmonary hypertension (3 weeks post‐CH). Pulmonary hypertension was assessed by direct cardiac puncture in anesthetized mice to measure right ventricular systolic pressure. Similar to global ASIC1 knockout (Asic1‐/‐) mice, SMC‐Asic1‐/‐ mice were protected from the development of CH‐induced pulmonary hypertension. In contrast, EC‐Asic1‐/‐ mice developed pulmonary hypertension comparable to wildtype Asic1+/+ mice (Figure 1). Furthermore, CH‐induced pulmonary hypertension was reversed by induction of SMC‐specific ASIC1 knockout following established pulmonary hypertension. CH‐induced vascular remodeling was assessed using smooth muscle α‐actin immunofluorescence to determine the medial thickness of small pulmonary arteries. The degree of arterial muscularization was significantly attenuated and/or reversed in SMC‐Asic1‐/‐ arteries (P<0.05). These findings were additionally supported by decreased CH‐induced proliferation (P<0.05) and migration (P<0.05) of pulmonary arterial SMC from Asic1‐/‐ mice, assessed by flow cytometry of BrdU positive cells and transwell migration assay, respectively. Together these data demonstrate that SMC, but not EC, ASIC1 contributes to CH‐induced pulmonary hypertension and vascular remodeling. Furthermore, these studies provide evidence for the therapeutic potential of ASIC1 inhibition to reverse pulmonary hypertension.
Structural and Functional Characterization of PLCβ3
Authors: Outlaw Kennedy , Fisher Isaac J. , Muralidharan Kaushik , Lyon Angeline
Published: May 2022

Phospholipase C β (PLCβ) plays an important role in cardiovascular diseases and opioid analgesia. PLCβ catalyzes the hydrolysis of the inner membrane lipid phosphatidylinositol‐4,5‐bisphosphate (PIP2) to inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control responses to extracellular signals. PLCβ is a downstream effector of G‐protein coupled receptors (GPCRs) and is activated by both the Gαq and Gβγ subunits. Activation by Gα q requires a unique 400 amino acid C‐terminal region of the lipase, which is subdivided into the proximal and distal C‐terminal domains (CTDs). These domains are required for allosteric activation by Gα q and for membrane binding. Our lab and others have reported that PLCβ is more flexible in solution, as compared to crystal structures of the protein. In small angle X‐ray scattering (SAXS) experiments, the solution structure of PLCβ had additional density that cannot be accounted by the crystal structure. Additionally, crosslinking the PH and EF hand domains decreased this density and the maximum particle diameter. We propose that this additional density corresponds to an open conformation, wherein the PH and EF hand domains are extended from the core of the lipase, and that this conformation of PLCβ represents an autoinhibited state. To test this hypothesis, we mutated residues in PLCβ3 at the interface between the PH and EF hand domains to disrupt their interaction, and then measured changes in PLCβ basal activity and Gβγ‐stimulated activity. These mutations had no significant impact on basal activity, however in many of these mutants, there was a decrease in apparent Gβγ activation. Since these mutations should stabilize an open conformation of PLCβ, this suggests the open conformation has less ability to bind to or be activated by Gβγ. We are currently working to solve the structure of PLCβ3 in solution using single particle cryo‐electron microscopy (cryo‐EM), which will allow us to determine a solution structure of PLCβ to a higher resolution than SAXS studies. We hypothesize that PLCβ3 will exist in the open, autoinhibited conformation, and will also shed light on possible allosteric interactions between the distal CTD and the core.
Single Particle Cryo‐EM Reconstruction of an Activated Gβγ‐PLCβ Complex
Authors: Fisher Isaac J. , Muralidharan Kaushik , Outlaw Kennedy , Garland‐Kuntz Elisabeth , Lyon Angeline
Published: May 2022

G protein‐coupled receptors (GPCRs) regulate diverse physiological processes in health and disease through activation of the heterotrimeric G protein subunits, Gα and Gβγ. G proteins in turn activate effector enzymes, such as phospholipase C β (PLCβ), to produce second messengers. The PLCβ subfamily has modest basal activity which is robustly increased by direct binding of Gαq. PLCβ1‐3 are also directly activated by Gβγ. PLCβ hydrolyzes phosphatidylinositol‐4,5‐bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol‐1,4,5 trisphosphate (IP3), activating protein kinase C (PKC) and increasing intracellular Ca2+. Inhibition of G protein‐dependent activation of PLCβ has been shown to have therapeutic potential in multiple pathologies, including inflammation, cardiac hypertrophy, opioid analgesia, and cancer. Much has been learned about how Gαq binds to and activates PLCβ from structural and functional studies, but there is no consensus as to the binding site for Gβγ on PLCβ, or its mechanism of activation. Gαq and Gβγ synergistically activate PLCβ, suggesting the G proteins bind to nonoverlapping sites on the lipase and activate through independent mechanisms. Atomic resolution structures of the Gβγ‐PLCβ complex are needed to fully understand the mechanism of Gβγ activation of PLCβ. We have isolated a stable, active Gβγ–PLCβ3 complex and are using single particle cryo‐electron microscopy (cryo‐EM) to investigate its structure. We have so far identified two particle populations using heterogeneous refinement, and generated reconstructions at 4 and 7 Å resolution. The structures reveal strong density for Gβγ binding to PLCβ through multivalent interactions with its pleckstrin homology (PH) domain, EF hands, and C2 domain, dispelling controversies about the Gβγ binding site that have persisted for over three decades. The greatest difference between structures is the position of Gβγ with respect to the PH‐EF hand interface. Surprisingly, one conformation of the Gβγ–PLCβ complex is compatible with simultaneous binding of Gαi, which has not been reported in any other Gβγ–effector enzyme structure. This suggests that Gβγ may also function as a scaffold for Gαi and PLCβ, in which a pre‐activated complex is maintained at the membrane. Dissociation of activated Gαi would allow rotation of Gβγ, leading to full engagement of PLCβ and maximum activation. Further investigation into the scaffolding and activating roles of Gβγ in PLCβ activation will be essential in developing chemical probes to selectively target this pathway.
Determining Biomarkers of Myosteatosis for Sarcopenia and Cachexia Using MRI and Ultrasound
Authors: Rush Benjamin , Lortie Jevin , Osterbauer Katie , Garlapati Sujay , Campbell Toby , Traynor Anne , Leal Ticiana , Lee Kenneth , Reeder Scott B. , Kuchnia Adam
Published: May 2022

Background

Lung cancer patients have low survival rates resulting in over 131,000 deaths in the US in 2021. Muscle health decline from sarcopenia and cachexia increases the risk of death among patients with lung cancer. Though muscle mass is often used for sarcopenia diagnosis and non‐tissue specific weight loss is typically used for cachexia diagnosis, both conditions have no consensus definitions. Therefore, the need for quantitative biomarkers that reflect muscle health changes due to aging and weight loss is critical to mitigate sarcopenia and cachexia. We propose using highly accurate MRI‐based proton density fat fraction (PDFF) as a reference measure of muscle health to develop bedside ultrasound measures as biomarkers of muscle health. We hypothesized that PDFF is associated with ultrasound‐based echointensity (EI) and shear wave elastography (SWE), and that all three modalities are sensitive to muscle health differences between healthy young, healthy older, and older adults undergoing treatment for lung cancer.

Methods

We compared muscle health operationalized as myosteatosis in the mid‐thigh of the rectus femoris using ultrasound‐based brightness‐mode for EI, ultrasound‐based SWE for tissue elasticity, and MRI‐based PDFF for percent fat, which was the reference measure. We compared the 3 measures among young healthy (n=10), older healthy (n=10), and older adults with non‐small cell lung cancer (n=10) in a cross‐sectional pilot study. We also compared non‐myosteatosis measures including subcutaneous adipose tissue thickness and rectus femoris thickness via ultrasound, whole‐leg cross‐sectional area via MRI, and BMI between groups and between myosteatosis measures. We used ANOVAs with Tukey post‐hoc comparisons to assess muscle health between groups and Pearson correlations to determine muscle health measure associations between imaging modalities.

Results

We found young healthy adults had significantly lower PDFF than older healthy adults and older adults with lung cancer (0.33% vs 2.83% vs 2.93%, respectively; p=0.001). Young adults also had significantly lower EI than older healthy adults, but not older adults with cancer (48.58 vs 81.81 vs 75.35; p=0.008). When comparing between measures, PDFF was highly associated with EI (r=0.62, p<0.001) and moderately inversely associated with SWE (r=‐0.41, p=0.025) but not BMI, rectus femoris thickness, or rectus femoris cross‐sectional area.

Conclusion

PDFF was sensitive to myosteatosis differences between young and both older adult groups. EI was less sensitive to myosteatosis differences between groups, yet EI was highly associated with PDFF unlike BMI. Our results suggest that the ultrasound measures could serve to determine muscle health at the bedside and are more sensitive to muscle health differences than BMI, which could improve interventions for cachexia and patient outcomes.
A Reduction in Tafazzin Decreases Mitochondrial Function in C2C12 Myotubes
Authors: Richards Brandon J. , Oliviera Ashley N. , Hood David A.
Published: May 2022

Mitochondria, which are often regarded as the “powerhouse of the cell”, are labile organelles that regulate cellular metabolism, determine cell fate, and act as important signaling hubs. A phospholipid that is exclusively found within mitochondria that serves numerous roles is cardiolipin (CL). In healthy mitochondria, CL is predominately localized in the inner mitochondrial membrane (IMM) and binds to several membrane proteins including those in the electron transport chain (ETC) and in the protein import machinery (PIM). Similarly, CL has also been shown to regulate cell death and mitophagy in dysfunctional mitochondria. Following the biosynthesis of nascent CL in the inner membrane, it is remodeled into its mature form by a transacylase, tafazzin (Taz). The absence of this enzyme is associated with Barth Syndrome, a disease characterized by cardiomyopathy and skeletal myopathy. Interestingly, little is known about the role of mature CL in skeletal muscle. Therefore, the objective of this study was to identify whether Taz deficiency diminishes mitochondrial function in the presence or absence of changes in organelle volume and function. We hypothesized that a reduction in Taz will attenuate mitochondrial function, while lysosomal and mitochondrial content will be elevated. To test this hypothesis, C2C12 myotubes were transfected on day 3 of differentiation with either a scrambled or Taz siRNA vector. Cells were harvested for analysis on day 7 of differentiation. Organelle volume was assessed using immunohistochemistry and Western blot techniques, whereas Seahorse technology was used to measure mitochondrial respiration. In comparison to C2C12 myotubes that were treated with a scrambled vector, Taz protein content was reduced by 85% (P<0.05) in siRNA‐treated cells. Maximal oxygen consumption and ATP production were attenuated in Taz‐deficient cells by 18% and 14%, respectively, while basal respiration remained unaffected. Lysosome and mitochondrial content were increased 20% and 24%, respectively, in Taz‐deficient cells. Similarly, myotubes lacking Taz exhibited a 7% increase (P=0.056) in Complex IV protein content, but lysosomal‐associated membrane protein 1 (LAMP1) decreased 33% (P<0.05). Myosin heavy chain (MHC) IIX was also elevated by 32% in mature CL depleted myotubes. CL‐deficient myotubes were 32% smaller in diameter and 10% longer when compared to scrambled treated myotubes. These data suggest that a reduction in mature CL impairs skeletal muscle mitochondrial function. Moreover, although lysosomal content was elevated, lysosomes appear to have a reduced capacity to fuse with autophagosomes, which may in part explain why there is an increase in dysfunctional mitochondria. Future research should evaluate mitophagy flux and mitochondrial membrane potential in the presence of reduced mature CL in C2C12 myotubes, and whether exercise has the ability to rescue mitochondrial function.
Integrated Streaming of Anatomy Dissection during Lecture Hours
Authors: Nonabur Venkatesh , Weitzel Sarah E. , Rarey Kyle E.
Published: May 2022

Streaming of live faculty anatomy dissections was utilized to teach clinically relevant gross anatomy to first‐year Physician Assistant students during the summer of 2021 to correlate lecture with relevant dissection part. The online anatomy dissections by faculty were integrated with PowerPoint presentations before onsite dissection laboratory periods by the students.

MATERIAL AND METHODS

The PA students viewed the dissections via zoom sessions during lecture hours coordinated by lecture presenter. During their six weeks of study, each session was scheduled for one hour lecture, followed by 3 hours of dissections. At the beginning of each session, ten quiz questions relative to prior sessions were asked to enhance students’ retention and recall. Following the daily online practical quizzes, students watched step by step dissection and power point lecture integrated with relevant slides and/or cross‐sections. During three hour laboratory time of each daily session, students were divided into small groups to dissect anatomy region for that day assisted by faculty member. Sixty four percent of the PA Class of 2023 completed an anonymous electronic survey at the conclusion of the course.

The online survey of the enrolled PA students (n=40) in their first‐year training identified that the online dissections and the teaching format were extremely beneficial to their learning. Ninety seven percent of the students strongly agreed or agreed that the streaming of anatomy dissections was helpful in learning the assigned anatomy. Ninety five percent of the students strongly agreed or agreed that integrated power point presentations were beneficial, and Ninety five percent of the students strongly agreed or agreed that daily integrated quizzes (self‐assessment modules) were beneficial for their retention and recall. Although setting up online dissections and integrating the streaming dissections with relevant power points were time‐intensive, the teaching faculty viewed the online teaching experiences as effective means to further develop students’ knowledge base of anatomy education.
Probing Local Protein Environments in Adenylate Kinase with 4‐Cyano‐L‐Phenylalanine
Authors: Mishra Pratiksha , Camilo Angelica , Phillips‐Piro Christine M. , Brewer Scott H.
Published: May 2022

Unnatural amino acids (UAAs) containing vibrational reporters, such as nitrile, azide, and nitro groups, can be utilized to effectively probe local protein environments. Specifically, the nitrile symmetric stretching frequency of 4‐cyano‐L‐phenylalanine (pCNF) is sensitive to local protein hydration and occurs in a relatively clear region of the infrared spectrum. In addition, the magnitude of change in this vibrational frequency over a given temperature range is dependent on the local protein environment of the nitrile group of pCNF. Finally, the UAA pCNF can also be site‐specifically incorporated into proteins with high efficiency and fidelity using the Amber codon suppression methodology. Here, pCNF was incorporated into several different local protein environments in the enzyme Adenylate Kinase (AK). The successful incorporation was verified by time‐of‐flight mass spectrometry. These results in addition to the linear temperature‐dependent IR analysis of these constructs to extract the hydration state of the nitrile group of pCNF in each of these AK constructs will be presented.
Role of NOX2 in Ceramide‐Induced Human Microvascular Endothelial Dysfunction
Authors: Katunaric Boran , SenthilKumar Gopika , Schulz Mary E. , Freed Julie K.
Published: May 2022

Endothelial dysfunction, or the loss of nitric oxide (NO)‐mediated dilation to shear stress (flow‐induced dilation; FID), is observed in human adipose arterioles from patients with coronary artery disease (CAD). FID is maintained during CAD by release of the mitochondrial‐derived, pro‐inflammatory dilator, H2O2. Ceramide, a sphingolipid that when elevated in plasma is an independent risk factor for future cardiac events, also induces this transition in human arterioles (10µM, 16‐20hrs). The initial events leading to ceramide‐induced endothelial dysfunction are unknown. Here we hypothesize that activation of NADPH oxidase 2 (NOX2), an endothelial membrane enzyme activated by acute stress, occurs prior to the formation of mitochondrial H2O2 in arterioles exposed to chronic ceramide. Human arterioles (100‐250µm in diameter) were dissected from discarded surgical adipose tissues and prepared for videomicroscopy. Following preconstriction with endothelin‐1, internal diameters were measured in response to increased flow. To determine the minimum exposure time necessary to convert to H2O2‐dependent FID in arterioles exposed to ceramide, microvessels from healthy nonCAD adults were incubated with C2 ceramide for 30 min, 2 hrs, and 4 hrs. FID remained mediated by NO at 30min and 2hrs, however, following a 4hr incubation, FID was significantly impaired in the presence of PEG‐catalase (44.6% of maximal dilator capacity ±13.8 (SEM), n=8, p<0.05, one‐way ANOVA*) compared to vehicle control (83.5%±5.0, n=8). The source of H2O2 generated during FID in arterioles treated for 4hr with ceramide was not mitochondrial as increases in fluorescent intensity of mito peroxy yellow 1 (mitoPY1) were not observed during maximal flow. To test whether NOX2 is the initial source of H2O2 during FID in arterioles exposed to 4hr ceramide, nonCAD arterioles were first treated with the NOX2 inhibitor GSK2795039 (NOX2i, 10‐6 M) or underwent intraluminal administration of siRNA to decrease NOX2 expression prior to treatment with ceramide. FID was reduced in vessels treated with both ceramide and the NOX2 inhibitor in the presence of cPTIO (NO scavenger) (50.2%±30.1, n=4* vs. NOX2 inhibitor and ceramide alone 80.8%±6.4, n=7). L‐NAME also impaired FID in vessels with reduced expression of NOX2 and subsequently treated with ceramide (10.8%±38.9, n=3* compared to siNOX2 and ceramide alone 85.5%±5.8, n=3). This suggests that activation of NOX2 may be the initial event in the conversion of NO‐ to H2O2‐dependent FID due to ceramide and may serve as a potential therapeutic target in those at risk for developing cardiovascular disease due to increased plasma ceramide.
A predictive model for estimating protection against CKD and CVD with SGLT2 inhibition in patients with diabetes
Authors: Sadria Mehrshad , Layton Anita
Published: May 2022

Diabetes affects >450 million people worldwide. Elevated blood glucose caused by diabetes can lead to chronic kidney disease (CKD), which increases the risk of end‐stage kidney disease (ESKD) requiring dialysis or a kidney transplant. In type 2 diabetes (T2D), medications called sodium‐glucose cotransporter‐2 (SGLT2) inhibitors have become part of standard of the care for improving glycemic control by promoting urine glucose excretion. These drugs reduce urinary albumin excretion (quantified as urinary albumin‐to‐creatinine ratio, “UACR”, an effect linked with kidney protection), slow CKD progression and delay ESKD, with similar benefits in males and females. In people with T2D, SGLT2 inhibitors also reduce heart failure and cardiovascular events by 20‐40%. But in people with type 1 diabetes (T1D), there are no studies evaluating SGLT2 inhibitors in patients at the highest risk of ESKD or cardiovascular disease (CVD). Building on the benefits of SGLT2 inhibitors in people with T2D, we seek to evaluate the efficacy and mechanisms in people with T1D and CKD. We hypothesize that SGLT2 inhibition will slow loss of kidney function (glomerular filtration rate, or “GFR”) in people with T1D and CKD. To test that hypothesis, we analyze clinical data for a cohort of patients with diabetes and kidney disease, and identify key features that determine the rate of progression of CKD. We then develop personalized assessment tools that yield estimates for protection against CKD and CVD with SGLT2 inhibition in patients with T1D or T2D.
Role of TFE3 in Mitochondrial Adaptations to Skeletal Muscle Disuse
Authors: Oliveira Ashley N. , Memme Jonathan M. , Wong Jenna , Hood David A.
Published: May 2022

Prolonged muscle disuse is accompanied by a phenotypic shift characterized by declines in mitochondrial content and function within skeletal muscle. This loss in mitochondrial content can be partially attributed to elevations in various catabolic processes that occur with atrophy. One of these is termed mitophagy, a selective form of cellular recycling (autophagy) whereby dysfunctional mitochondria are degraded via lysosomes. TFEB and TFE3 are key transcription factors that regulate lysosomes by activating the expression of various lysosomal genes. Loss of TFE3 has been associated with depressed levels of autophagy and with mitochondrial dysfunction. It is speculated that these functional impairments are due to diminished clearance via the lysosomes, however to date this has not been examined. We hypothesized that the loss of TFE3 would amplify the mitochondrial dysfunction associated with muscle disuse, while paradoxically preserving muscle mass and mitochondrial content. Using a severe model of muscle disuse, sciatic denervation, we observed a 10% loss in hindlimb muscle mass within 7 days of denervation. In the absence of TFE3, a trend for muscle preservation was observed, as these animals lost 20% less muscle mass than WT counterparts. Reduced rates of respiration supported by complex I and II were observed irrespective of genotype, concurrent with elevated ROS emissions, suggesting an impaired oxidative capacity following 7 days of denervation. Surprisingly, TFE3 KO animals did exhibit lower levels of oxidative stress basally, but this too increased following 7 days of denervation, and taken relative to baseline the fold induction of ROS emission was 3 times greater than WT animals. Higher levels of mitophagy flux were observed in the absence of TFE3 basally, which supports the observed 35% decline in mitochondrial content as measured by COX activity, as well as the lower levels of ROS emissions. Following only 1 day of denervation, increases in mitophagy flux were observed in WT animals, while the response in KO animals was clearly attenuated. In the WT animals, denervation led to a 30% reduction in mitochondrial content, however no change was observed in the absence of TFE3. Finally, increases in a number of autophagy‐related markers such Beclin‐1 and ATG7 were observed following denervation irrespective of genotype. However, the mature form of Cathepsin B, a hydrolytic enzyme, was markedly reduced by 55% in the absence of TFE3 and did not increase to the same extent as WT following 7 days of denervation suggesting an impairment in lysosomal function. Together, our results suggest that TFE3 exerts multiple roles in skeletal muscle plasticity, as a partial mediator of muscle mass, and in the control of lysosomal function and mitophagy in response to the acute stress of muscle disuse.
Measurement of Betaine‐Homocysteine Methyltransferase Activity via Fluorescent Detection of Free Thiols
Authors: Spiegelberg Bryan D. , Robinson Zaria
Published: May 2022

High concentrations of homocysteine correlate with early onset heart disease and other disorders. The enzyme betaine‐homocysteine methyltransferase (BHMT) consumes homocysteine by transferring a methyl group from betaine to the free thiol group of homocysteine to form methionine. BHMT activity in turn can cause an imbalance of betaine, leading to other health issues like fatty liver and digestive disorders. Clearly, then, understanding the activity and regulation of BHMT can lead to insights into several key health problems. Unfortunately, currently available in vitro BHMT enzyme assays include the use of NMR or HPLC and thus are either slow or not readily scalable.

Our goals are to create a new high throughput in vitro assay to measure BHMT activity and to implement this assay to better understand the enzyme’s regulation. This new assay utilizes the dye monobromobimane (mBBr), which becomes fluorescent upon conjugation to a free thiol group. The free thiol of BHMT substate homocysteine is consumed upon methylation; mBBr fluorescence, then, serves as a quantitative reporter of the progress of the BHMT reaction. We have confirmed that mBBr detects the model thiol, L‐cysteine, in a 96‐well plate format, and we can detect sub‐nanomolar amounts of homocysteine deprotected from commercially available DL‐homocysteine thiolactone. This assay has been used to follow the progress of BHMT activity and to investigate the effects of several potential inhibitors. The development of a simple fluorescent assay will enhance the speed of study of this and related enzymes and could spur high throughput searches for small molecule modulators.
Comprehensive Characterization of Biological Properties of Human Urine‐Derived Stem Cells
Authors: Danisovic Lubos , Culenova Martina , Novakova Zuzana V. , Nicodemou Andreas , Debreova Michaela , Smolinksa Veronika , Bernatova Sona , Varga Ivan , Ziaran Stanislav
Published: May 2022

Mesenchymal (stromal) stem cells (MSCs) represent a unique tool in the field of regenerative medicine. However, their harvesting often requires invasive medical procedures. Urine‐derived stem cells (UDSCs) display similar properties to MSCs, and their sampling and further processing is non‐invasive for the donors. Here, we offer a comprehensive analysis of their biological properties. The goal of this study was to analyze their morphology, stemness, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm2, they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed that two distinct types of the cell populations have been present. The first type had a rice‐grain shape and the second one was characterized by a polyhedral shape. All examined UDSCs expressed typical MSC‐like surface markers, CD73, CD90 and CD105. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL‐8, IL‐6 and chemokines MCP‐1 and GM‐CSF. We have also successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Our findings indicate that UDSCs might have immense potential in the regeneration of the damaged tissues.
Understanding the Role of Gβγ in the Cellular Regulation of Mutationally Activated Gαq/11
Authors: Aumiller Jenna , Inoue Asuka , Wedegaertner Philip
Published: May 2022

Uveal melanoma (UM) is the most common intraocular malignancy in adults. UM metastasizes to the liver in 50% of patients, and there are currently no effective therapies for metastatic UM. Approximately 90% of UM cases harbor mutually exclusive mutations in the GNAQ and GNA11 genes, which code for the heterotrimeric G protein subunits Gαq and Gα11, respectively. The most common mutations within these genes are within residues Q209 and R183, resulting in Q209L, Q209P, or R183C mutations in UM patients. These mutations prevent GTP hydrolysis and render the proteins constitutively active (CA) to promote oncogenic signaling. The Ras/mitogen‐activated protein kinase (MAPK) and Hippo/YAP pathways are commonly stimulated via CA Gαq/11. Although it is generally thought that active, GTP‐bound Gα subunits signal independently of Gβγ, we have found that these CA mutants of Gαq/11 retain some level of binding to Gβγ and that this interaction is necessary for oncogenic signaling. The main objective of this project is to understand the role of Gβγ in the regulation of CA Gαq/11. We hypothesized that disrupting the interaction between CA Gαq/11 and Gβγ can ultimately inhibit oncogenic signaling in UM. To test this hypothesis, we introduced a single point mutation within the N‐terminus of the CA Gαqmutants, which has been shown previously to disrupt the interaction between wild‐type Gαqand Gβγ and prevent agonist‐dependent signaling of Gαq.We also overexpressed another Gα subunit, Gαo, to bind endogenous Gβγ and thereby prevent Gβγ binding by CA Gαqmutants. We found that these methods disrupted binding of CA Gαq to Gβγ and further disrupted oncogenic signaling via the Hippo/YAP and MAPK pathways. Interestingly, we found that the GαqQ209L mutant binds more strongly to Gβγ, and signaling by the GαqQ209P and GαqR183C mutants are more sensitive to disruption of Gβγ binding. This was further confirmed within UM cell lines where siRNA knockdown of Gβ1 and Gβ2 had a more profound effect on inhibition of signaling in cells with the GαqQ209P mutation compared to cell lines with the GαqQ209L mutation. In conclusion, our study challenges the idea that CA Gαq/11signals independently of Gβγ. Our work also suggests a novel difference in sensitivity between the GαqQ209L, GαqQ209P, and GαqR183C mutants. We propose that disrupting the interaction between CA Gαq/11and Gβγ can be a novel therapeutic target in UM.
Determining the role of ATF4 in the regulation of mitochondrial remodeling during myotube differentiation and contractile activity
Authors: Memme Jonathan M. , Sanfrancesco Victoria C. , Hood David A.
Published: May 2022

ATF4 is a transcriptional regulator that is selectively induced in response to cellular stress conditions, such as exercise, through the activation of the integrated stress response (ISR). Specifically, in the context of mitochondrial‐specific stress, ATF4 is induced as a key component of the mitochondrial unfolded protein response (UPRmt) and is suggested to upregulate various organellar chaperones and proteases that both preserve, and promote, mitochondrial function. In response to the stress brought about by contractile activity, ATF4 has been implicated in regulating skeletal muscle health by mediating the various signaling events associated with mitochondrial quality control (MQC), including i) mitochondrial biogenesis (expansion), ii) the mitophagy‐lysosomal clearance of damaged and thus potentially harmful organelles, or iii) by activating the mitochondrial unfolded protein response (UPRmt) as an intermediate response to acute cellular stress. However, it remains to be determined whether ATF4 is necessary for mitochondrial adaptations in skeletal muscle. Therefore, our aim was to determine whether ATF4 is required for the maintenance of mitochondrial function and adaptation following an acute 3h bout of contractile activity (ACA), or after repeated bouts (4 days; CCA) in C2C12 myotubes in which ATF4 was either overexpressed (OE) or knocked down (KD) via lentiviral transduction of plasmids containing the ATF4 open reading frame, or siRNA, respectively. Knockdown of ATF4 promoted elongated myotube formation following 5 days of differentiation, whereas ATF4 OE contributed to the opposite effect in which shorter myotubes were observed relative to the control condition. Induction of PGC‐1α mRNA following ACA in ATF4 KD myotubes was attenuated, suggesting diminished drive for mitochondrial biogenesis in the absence of ATF4. The mRNA expression of ATF5, a downstream target of ATF4, was induced both in response to ACA as well as with ATF4 OE, and was reduced in the absence of ATF4. Mitophagy flux, measured by mitochondrial‐localized LC3‐II, was upregulated in ATF4 OE and KD myotubes basally, and was augmented in both control and ATF4 OE cells following ACA, but not in the absence of ATF4. Furthermore, ATF4 OE revealed decrements in mitochondrial content indicated by 20%, and 40%, reductions in VDAC and COX I protein expression relative to control, while 1.2‐1.5‐fold increases in these markers were observed when ATF4 was knocked down. However, following CCA, COX I and VDAC protein content were increased 3‐4‐fold in ATF4 OE cells, while there was no observable increase in mitochondrial content in ATF4 KD myotubes. Together, these data highlight a potential role of ATF4 in regulating basal mitochondrial content and further suggest that ATF4 may be required for contractile activity‐induced increases in mitochondrial content.
The Discovery of Antivirals and Targets for SARS‐CoV‐2 and EV‐A71
Authors: Haddad Christina , Tolbert Blanton S. , Hargrove Amanda E. , Brewer Gary , Li Mei‐Ling
Published: May 2022

Positive Strand RNA (PSR) viruses, such as coronaviruses and enteroviruses, cause serious health and economic threats worldwide, as currently seen with the COVID‐19 pandemic. This has drawn attention to the importance of identifying new antivirals and molecular targets in RNA viruses. The multifunctionality of PSR genomes make them desirable targets for therapeutic intervention. Here, we present a class of antivirals that can inhibit SARS‐CoV‐2 replication in vitro by targeting conserved viral RNA structures at the 5’‐end. Specifically, stem loops 1, 4, 5a, and 6 of the viral 5’‐region have shown a degree of binding with these small molecules as determined by NMR structural analysis. These results open the door to potentially develop specific small molecules against SARS‐CoV‐2 and related coronaviruses. Additionally, Enterovirus A71 (EV‐A71), which is the etiological agent of the hand, foot, and mouth disease, has caused severe morbidity and high mortality rates in children for decades. Thus, understanding the mechanisms by which EV‐A71 replicates within the cellular environment can bring to light efficient drug targets for viral inhibition. The multifunctional viral protein, 3C protease (3Cpro), is essential for viral protein and RNA synthesis. Here, we investigate how RNA binding allosterically modulates the enzymatic activity of 3Cpro. We identify an overlooked dimerization surface on 3Cpro that is proximal to its active site and distal to its RNA binding domain. Our data show that RNA binding is allosterically coupled to 3Cpro dimerization, and we posit that this is a novel mechanism to regulate its enzymatic function. To that point, single, double, and triple point mutations in the 3Cpro dimerization domain attenuates viral growth and kinetics. Taken together, we present compelling data that demonstrates novel targeting surfaces on 3Cpro that can be pursued as antiviral targets.