bluuebunny/crossref_metadata_2025 huggingface dataset:
High Intensity Exercise Improves Cardiorespiratory Capacity in a Mouse Model of Hypertrophic Cardiomyopathy
Authors: Herrera Jonathan , Szczesniak Danielle , Szczesniak Kate , Goddard Rose‐Carmel , Yob Jaime , Day Sharlene
Published: May 2022

Introduction

Cardiorespiratory capacity (pVO2) is an important prognostic indicator in patients with HCM. Individuals with HCM that have a lower pVO2 possess a greater risk for heart failure (HF) and mortality. High intensity exercise (HIE) can increase pVO2 to a greater degree compared to other training modalities, including when used for cardiac rehabilitation for in cardiac disease states (e.g., HF and Cardiovascular disease). The effects of HIE on HCM, however, are largely unknown. Our study sought to evaluate the effects of High Intensity Interval Training (HIIT) on pVO2, as well as cardiac structural and functional parameters, in a preclinical mouse model of HCM.

Methods

Female and male C57BL/6J non‐transgenic nTG (n=27) and TG (n=32) mice were subjected to HIIT protocol that mirrored methods used clinically in cardiac rehabilitation. A single treadmill training session included 4‐4 minute high intensity intervals (~80% preVO2 max speed) interspersed by 5‐3 minute recovery intervals (~50% preVO2 max speed) for 31 total minutes. Exercise training sessions were repeated 3 times per week for 6 weeks. Pre and post pVO2 and murine echocardiography were measured and analyzed by a blinded technician.

Results

Following 6 weeks of the HIIT, nTG female (+13.5 mL/kg/min ± 3.6, p<0.01), nTG male (+13.5 mL/kg/min ± 3.6, p<0.01) and TG female (+7.06 ± 3.3 mL/kg/min, p<0.05) mice demonstrated an average increase in pVO2. A change in pVO2 observed in TG male mice did not reach statistical significance (+4.34 ± p=0.12). Sedentary female (‐15.9 mL/kg/min ± 3.3, p<0.001) and male (‐13.1 mL/kg/min ± 3.9, p<0.01) TG control mice experienced a significant decrease in pVO2 following the same period, while no change was observed in nTG sedentary controls. Independent of sex, nTG and TG HIIT mice had a significantly greater change in pVO2 compared to sedentary counterparts. HIIT training did not result in changes to the following echocardiography measures in either nTG or TG mice: LV mass, LA volume, cardiac output, or stroke volume.

Conclusion

Our results indicate that a translationally derived HIIT protocol improved cardiorespiratory capacity in a preclinical HCM mouse model, absent of adverse events or worsening of organ level disease pathology.
Effect of TTfields interference MARK2 on proliferation, migration and invasion of glioma cells
Authors: Duan Hubin , Hao Chunyan , Xue Wen , Li Mingli , Zhang Jinbao , Qiao Binjun , Chen Xianghui , Zheng Juan , Qiao* Xiaohong
Published: May 2022

Objective

To study the effect of tumor‐treating fields intervention on proliferation, migration and invasion of glioma cells by microtubule affinity regulated kinase 2 (MARK2).

Methods

In this study, a set of tumor treatment field device developed and improved by us was used to dynamically observe the mitosis of U251 glioma cells, as well as the effect of tumor treatment fields on the proliferation, migration and invasion of tumor cells. The changes in cell activity of tumor cells were evaluated by CCK‐8 and EdU experiments. The proliferation ability of tumor cells was evaluated by colony formation experiment, the invasion and migration ability of tumor cells was evaluated by scratch healing experiment and Transwell experiment, and cell apoptosis was measured by flow cytometry. Westem blot assay was used to compare the difference of MARK2 protein expression between tumor cells in tumor treatment fields intervention group and control group.

Results

the results suggest that we developed a new device to dynamically observe the tumor cell mitosis, and also observed in tumor therapy fields affect tumor cell proliferation, migration and invasion, in addition to affect the mitosis, possibly by interfering with MARK2 expression, such as resist the tumor cell proliferation, migration, invasion, and promote the effect of tumor cell apoptosis.

Conclusion

Tumor treatment fields may inhibit tumor proliferation, migration, invasion and promote tumor cell apoptosis by affecting mitosis and interfering MARK2 expression.
Kinase Responsive to Stress B negatively regulates Rap1 in Dictyostelium discoideum
Authors: Flores Tiffany , Roberts Kelsey , Artemenko Yulia
Published: May 2022

Dynamic changes in cell adhesion to the substrate are essential for migration, although details of the pathways regulating adhesion, especially for amoeboid‐type migration are lacking. Social amoeba Dictyostelium discoideum is a commonly used model organism for the study of directed migration since its movement is very similar to that of other amoeboid cells, such as neutrophils and metastatic cancer cells. Rap1 is a small GTPase that regulates adhesion in Dictyostelium cells in part via its effects on myosin II and talin. Kinase responsive to stress B (KrsB), a homolog of mammalian tumor suppressor MST1/2 and DrosophilaHippo, also regulates cell adhesion and migration, although the molecular mechanism of KrsB action is not understood. Since KrsB has been shown to interact with active Rap1 by mass spectroscopy, we investigated the genetic interaction between Rap1 and KrsB. Cells lacking KrsB have increased contact with the substrate and are difficult to detach from the surface, which leads to reduced movement. Expression of constitutively active Rap1G12V increased cell adhesion, and inactive Rap1S17N reduced cell adhesion even in the absence of KrsB, suggesting that Rap1 does not require KrsB to mediate cell adhesion. However, Rap1G12V did not increase cell spreading when KrsB was overexpressed and Rap1‐mediated increase in spreading was dramatically enhanced in the absence of KrsB, suggesting that KrsB might negatively regulate Rap1. In addition, chemoattractant‐induced activation of Rap1, as assessed by transient cortical localization of the biosensor RalGDS, was impaired in krsB¯cells, possibly due to increased basal activity or membrane localization of Rap1. Since mammalian Rap1 localization can be regulated by phosphorylation, KrsB might phosphorylate Rap1, thereby disrupting its activation on the membrane. Overall, our study demonstrates that KrsB negatively regulates Rap1 signaling, and we are currently investigating the molecular mechanism of this regulation.
Distribution and Allelic Frequency of the Enamelin Gene Variant rs7671281 Associated to Amelogenesis Imperfecta and High‐risk Caries in Puerto Rico
Authors: Morales Morales Limary , Ramirez‐Aponte Edwin G. , Martinez‐Cruzado Juan C.
Published: May 2022

Amelogenesis imperfecta (AI) is an inherited, rare dental disorder that affects only the formation of tooth enamel and shows both clinical and genetic heterogeneity. Similarly, the enamelin gene (ENAM) has a fundamental role in the mineralization and structural organization of enamel and any change in its sequence can affect the thickness of the enamel. The objective of this study is to be able to confirm the association between the genetic variant rs7671281 in the ENAM gene with AI and high‐risk caries subjects in the Puerto Rican population. This nonsense variant has been associated with the local, autosomal dominant hypoplastic form (type IB) of AI. Through Real‐time PCR, we determined the allelic frequency and geographic distribution of rs7671281 by genotyping 622 samples of healthy subjects from Puerto Rico. Due to its association with a reduction in enamel thickness, rs7671281 is also genotyped to check its role in the susceptibility to the risk of developing dental caries. The present study was carried out in the Puerto Rican population with phenotypes associated with amelogenesis imperfecta and subjects with a high to moderate prevalence of dental caries. In turn, subjects with a low to no prevalence of dental caries were considered as controls. Through the collection and analysis of saliva samples, the prevalence of the enamelin variant rs7671281 was confirmed in the Puerto Rican population. The island‐wide distribution pattern is non‐regionalized and influenced by coastal regions, but mostly predominated in the South and East area of the island. According to the 1000 Genomes Project, the allelic frequency of rs7671281 for the T allele was 0.885 (184) and for the C allele was 0.115 (24) based on a total of 208 samples. The present study shows a significant decrease in the allelic frequency of rs7671281 for the mutated allele C (0.0530) in comparison to the 1000 Genomes Project (0.115). Therefore, we concluded that the allelic frequencies of rs7671281 analyzed in this study are more accurate than the values given in the 1000 Genomes Project.
Analysis of Cortical Hemodynamic Responses in the Prefrontal Lobe during Chopsticks Usage
Authors: Acuña Stephan M. , Wagatsuma Mayumi , Carmona Christelli E. , Narasaki‐Jara Mai , Jung Aranne , Jung Taeyou
Published: May 2022

Fine motor skills, such as manipulating utensils, are found to be associated with increased brain activation in the prefrontal cortex (PFC). Limited previous research has examined PFC activation during chopsticks usage as compared to other fine motor tasks requiring a reliable hand‐eye coordination for object manipulations. The purpose of this study was to investigate the cortical hemodynamic responses in the PFC to chopsticks usage versus fork and hand use. Brain responses to chopsticks usage were further compared based on three subgroups categorized by performance proficiency, usage frequency, and chopsticks‐using cultural background. A total of 32 young adults (age 24.4 ± 2.36) participated in this cross‐sectional study and were instructed to transfer kidney beans from a source to a target plate at a comfortable pace using their dominant‐side hand. All participants completed the same task using 3 conditions: chopsticks, fork, and hand in a randomized order. A non‐invasive neuroimaging device, a functional near‐infrared spectroscopy (fNIRS) with 20‐channel configuration at the sampling rate of 7.81 Hz, was used to analyze and compare cortical hemodynamic responses by measuring oxyhemoglobin (HbO2) in the PFC among the three conditions. A repeated‐measures ANOVA revealed a significant difference between chopsticks and hand usage (p< 0.011). Chopsticks use resulted in a 36% greater increase of HbO2 when compared to hand use. However, there was no statistically significant difference between chopsticks and fork use. Subgroup analysis showed a systematic trend of showing decreased brain activation with lower HbO2 in the PFC when participants had better proficiency in using chopsticks, used chopstick more frequently, and had chopsticks‐using cultural background. Our results suggest that the use of chopsticks can increase brain activation in the PFC compared to fork or hand use. The findings also indicate that the brain activity during chopsticks usage can be influenced by other factors, such as a level of proficiency, frequency of use, and cultural background.
pQCT Analysis of Cortical Bone in Rats Exposed to High Levels of Cortisol in‐utero
Authors: Brown Sarah A.
Published: May 2022

Unperturbed fetal development is essential for future health of an individual. Previous studies have linked diseases of aging to harmful alterations that happen during fetal development. Given the significant long‐term impact that intrauterine environment has on an individual's life, it was hypothesized that maternal stress during pregnancy will have negative effects on the offspring's bone density and cross‐sectional area. To test this, twenty‐eight female and seven male Wistar rats (Rattus norvegicus) were purchased and bred to produce 176 offspring. During pregnancy, dams were randomly divided into four groups (n=7, per group) and immobilization stress induced as follows; Group 1 (GW1): immobilization stress on days 1–7 of pregnancy, Group 2 (GW2): on days 8–14, Group 3 (GW3): on days 15–21, Group 4 (Controls): left undisturbed. Maternal cortisol hormone, food intake, and weight gain were monitored during pregnancy. Pups were raised under normal laboratory conditions and sacrificed at ages: 4, 8, 12, and 16 weeks to determine the effect of prenatal stress. At necropsy, the femur was removed and preserved for image analysis.

Differences among groups were determined by T‐test or analysis of variance (ANOVA). P values ≤ 0.05 were considered significant. Cortisol hormone levels in controls were lower than those of stressed animals. Stressed dams consumed 12.5% less food per day compared to controls. Animals in GW1 and GW2 gained less weight during pregnancy but had larger litters than did GW3 or the control group. Offspring born to GW3 were heavier compared to all other groups. The femurs were scanned using the XCT Research SA+ peripheral Quantitative Computed Tomography (pQCT). Preliminary analysis of cortical bone shows the GW1 males as having a significantly lower cortical thickness compared to the controls at the age of 8, 12 and 16 weeks. The GW1 males also had a significantly lower periosteal perimeter and endosteal perimeter compared to controls at the age of 8 weeks. There was no significant difference in cortical thickness, periosteal perimeter and endosteal perimeter of females. This preliminary analysis shows that bone development in males was significantly impacted when fetuses in this study were exposed to high levels of cortisol in‐utero during the first week of gestation. Analysis of the remaining data is ongoing.
Inhibition of Hsp90 regulates fluconazole resistance in the pathogenic fungus Cryptococcus neoformans
Authors: Darrah Beka , Shah Dhriti , Chandrasekaran Srikripa
Published: May 2022

Cryptococcus neoformans is an encapsulated yeast, found in the lungs of immunocompromised hosts. In the human host, Cryptococcusis subject to multiple forms of environmental stress, including heat stress. Heat shock protein 90 (Hsp90) is an essential protein that has been shown to protect fungal cells against extreme temperature in the human host and to assist in proper cell division. Hsp90 has also been implicated in resistance to the anti‐fungal drug fluconazole. To better characterize the role of Hsp90 in fluconazole resistance in Cryptococcuswe addressed two questions: 1. Does concomitant partial inhibition of Hsp90 and treatment with fluconazole lead to increased sensitivity to fluconazole and 2. Does prolonged partial inhibition of Hsp90 prior to treatment with fluconazole lead to subsequent increase in development of fluconazole resistance. Using growth assays, disk assays, and a standardized e‐test to determine the Minimum Inhibitory Concentration (MIC) of fluconazole, we found that indeed, pharmacological partial inhibition of Hsp90 with radicicol, concomitant with fluconazole treatment leads to increased sensitivity to fluconazole. However, initial pre‐treatment of Cryptococcuswith sub‐inhibitory concentration of radicicol followed by exposure to fluconazole leads to development of fluconazole resistant Cryptococcuscolonies. We characterized the resistant colonies, which show a lower MIC to fluconazole compared to cells that were never exposed to radicicol and fluconazole. Cells derived from the resistant colonies exhibit slower growth on drug‐free media, and lose resistance to the drug after passaging in drug‐free media.

Based on the fact that resistance to fluconazole is lost when fluconazole is removed from the environment, we hypothesize that compromising Hsp90 function during the pre‐treatment with radicicol triggers aneuploidy in Cryptococcus neoformans. Furthermore, we hypothesize that the resistant colonies are aneuploids that possess an altered chromosome number to maintain drug resistance in response to an environmental insult, such as presence of fluconazole.
MKP‐2 Deficiency Improves Insulin Sensitivity and Protects Against Fatty Liver Disease
Authors: Lawan Ahmed
Published: May 2022

The function of MAPK phosphatase‐2 (MKP‐2), a type 1 dual‐specific phosphatase (DUSP) in metabolic regulation is largely unknown. Here we demonstrate that MKP‐2 expression was upregulated in liver tissue in humans with obesity and fatty liver disease, and in insulin‐responsive tissues in mice with obesity. MKP‐2 deficient mice have enhanced p38 MAPK, JNK and ERK activities in insulin‐responsive tissues compared with wild type mice. MKP‐2 deficiency in mouse protects against diet‐induced obesity and hepatic steatosis and was accompanied with improved glucose homeostasis and insulin sensitivity. This was associated with enhanced circulating insulin‐like growth factor‐1 (IGF‐1) and stromal cell‐derived factor 1 (SDF‐1) levels in Mkp‐2‐/‐ mice. PTEN, a negative regulator of Akt, was upregulated in livers of Mkp‐2‐/‐ mice, resulting in enhanced Akt activity consistent with increased insulin sensitivity. Pancreatic islet analysis demonstrated that MKP‐2 deficiency altered islet composition and this has the potential to regulate in b‐cell physiology. These studies demonstrate for the first time that MKP‐2 is essential in the regulation of metabolic homeostasis and pathophysiology of obesity‐induced insulin resistance and fatty liver disease.
A Cellular & Network Level Investigation of Thalamocortical Neuron Oscillations & the Role of the Transcription Factor, Shox2
Authors: Febbo Isabella G. , Martinez Luis A. , Warkins Valerie , Vargas Nadia , Anderson Anne E. , Schrader Laura A.
Published: May 2022

Thalamocortical neurons (TCNs) densely innervate the cortex and rhythmically burst fire in synchrony with cortical electrical activity that generates thalamus‐driven sleep spindles. Proper development of thalamocortical neuron cortical connectivity and firing activity underlie these sleep spindles—which are critical for memory consolidation—yet a transcriptional regulator that promotes these critical TCN characteristics has not been identified. Previously, we have shown that the transcriptional factor, Shox2, is important for the expression of key ion channels that generate TCN burst firing. Here, we have developed a targeted model using stereotactic injections of Cre virus into Shox2fl/flmice to isolate the role of Shox2 function in a single thalamic nucleus, the ventrobasal (VB) nucleus, upon TCN burst firing, cortical connectivity and organization, sleep spindles, and ultimately memory consolidation. We utilize patch clamp electrophysiology in slices to analyze burst firing of TCNs, cytochrome oxidase and VGluT2 staining of VB cortical targets (cortical barrels), in vivo electrophysiology analysis of sleep spindles, and novel object recognition to assess the effects of Shox2 KO in VB TCNs. Our findings indicate that the area under the burst curve and number of action potentials is significantly reduced, cortical barrel organization is disrupted in a P6 Shox2 KO model, sleep spindle occurrence is significantly reduced, and novel object recognition is impaired. These results show that Shox2 contributes to regulation of TCN cell properties and overall thalamic function.
Uncovering the mechanistic basis of MAGEA5/A10 driven resistance to chemotherapeutic drugs
Authors: Bagsby Clarke N. , Colemon Ashley , Ramanathan Saumya
Published: May 2022

Melanoma Antigen Genes (MAGEs) are defined as cancer‐testis antigens due to their unique expression pattern. There are two families of MAGEs, Type I and Type II. Type I MAGEs are expressed in the testis and in no other normal somatic tissue and then re‐expressed in many cancers. Type II MAGEs on the other hand, are ubiquitous in their expression.. The MAGE‐A family are members of the type 1 family located on the X chromosome. Type I MAGEs, considered to be true cancer‐testis antigens are all located on the X chromosome. Previous studies show that reversible DNA methylation of CpG sites upstream of MAGE‐A promoters regulates their expression. Many members of the MAGE1 subfamily cause increase in proliferative phenotypes. However, MAGEA5 and MAGEA10 expressing cells, demonstrated resistance to high doses of chemotherapeutic agent 5‐flurouracil. We aim to understand the mechanism behind this resistance. We will perform RNA‐Sequencing on cells expressing MAGEA5/A10 and control cells and determine protein binding partners of MAGEA5/A10 using IP‐Mass Spectrometry. In addition, we will test the hypothesis that in the heterogenous tumor cell population, MAGEA5/A10 is expressed in the cancer “stem cells” and that is responsible for driving chemo‐resistance. We will also test resistance of these cells to other chemotherapeutic agents such as taxols.
Understanding the Structural Basis of Epha1 and Epha2 Homo‐Dimerization, Membrane Proximal Domain Interactions and its Implications for Cancer
Authors: Buck Matthias , Sahoo Amita
Published: May 2022

EphA1 and EphA2 are receptor tyrosine kinases which play a critical role in cellular growth, differentiation and cell motility. In line with the mRNA expression in multiple tissues and organs, the overexpression of these receptors has been reported in several different cancer types, and even in cancer‐derived cell lines. This makes them key therapeutic targets for treatment of several cancers. In Eph receptors, ligand binding regulates the monomer‐dimer equilibrium through stabilization of the dimeric state by inducing a conformational change in the extracellular region (ECR). Thus, dimerization is a key regulatory step in the activity and signaling process of these receptors. However, the involvement of the transmembrane (TM), the immediate two extracellular FibronectinIII domains (FN1&2) and the juxtamembrane (JM) region in the dimerization process is not clearly defined [1]. Moreover, it is clear that the FN domains and the JM region also functions in synergy with the TM domain for signal transduction. The JM domain has several basic residues (K/R) that are closely positioned to the membrane surface, while the surface of the EphA2 2nd FN domain has also been reported to interact with the lipid bilayer. Therefore, studying the structural mechanisms of EphA1 and EphA2 dimerization and membrane interactions will help us better understand the activity and signaling of these receptors in different cancer types. Importantly, working with membrane proteins is extremely challenging. However, the recent developments in molecular dynamics simulation have provided a powerful tool to study membrane proteins and their interactions. Here, we modeled the relevant domains of the EphA1 and EphA2 receptors and studied their structural mechanism of activation by using the coarse‐grain (CG) molecular dynamics simulations using the recently published Martini3 potential function [2]. We show that the Fibronectin domains, TM and JM domains all differ in their interactions and in their interactions with the membrane between EphA1 and A2. Furthermore, the FN domains constrain the TM region of EphA2 to be non‐dimeric in many of the simulations, whereas FN domain dimerization and membrane orientation is more compatible with TM helix dimers in EphA1. The underlying differences in the primary sequence which are likely responsible for these observations as well as future directions of the project are discussed.

References:

[1] Sahoo AR, Buck M. Structural and Functional Insights into the Transmembrane Domain Association of Eph Receptors. Int J Mol Sci. 2021 Aug 10;22(16):8593.

[2] Sahoo, AR, Souza, PCT, Meng, Z, Buck M. Predicting Transmembrane (TM) Domain Dimer Structures using Martini 3. BioRxiv doi: https://doi.org/10.1101/2021.09.10.459840.
The β‐galactoside binding lectin Galectin regulates metabolism in Drosophila
Authors: Luo Liping , Robinson Brian
Published: May 2022

The β‐galactoside binding lectins, termed galectins, have garnered immense scientific interest for their ability to regulate diverse biologic processes, from adaptive immune responses to epithelial cell biology. Yet, an understanding of the role of galectins in regulating developmental processes has been limited by the functional redundancies across galectins which exist in vertebrates. Thus, we utilized the model organism Drosophila, which contains a single galectin ortholog, to ask what are the developmental requirements for galectins in metazoans. Using CRISPR/Cas9, we generated deletions in the galectin locus which efficiently eliminated galectin protein (Dmgal) across tissues. Intriguing, loss of galectin is not essential in Drosophila as animals ecclose with relatively minimal phenotypes. Among these, dmgal‐deficient animals are significantly smaller than their wild‐type counterparts. Moreover, when challenged to nutrient‐deprivation or Paraquat‐toxicity, dmgal‐deficient animals show marked sensitivity compared to their wild‐type animals. Transcriptomics analysis reveals that dmgal‐deficient animals show significantly different profiles than their wild‐type counterparts, with enrichment in metabolic pathways. Similarly, metabolomics shows distinct profiles from dmgal‐deficient and wild‐type animals with significant alterations in amino acid and nutrient‐sensing pathways. Finally, analysis of the Drosophila fat body reveals alterations in fat content, and tissue specific deletion of galectin in this organ renders animals sensitive to nutrient loss. Taken together, these findings uncover a novel role for dmgal in regulating Drosophila metabolism in the Drosophila fat body, and may provide clues into the role of galectins in regulating insulin sensitivity and NASH/NAFLD in vertebrates.
Large‐scale profiling of physiologically relevant naturally occurring rare GPCR variants using the bioSensAll® technology
Authors: Cao Frank , Lau Jenny , Gadzinski Adeline , Normand Claire , Semache Meriem , Mancini Arturo , Miller Melissa R. , Guan Shunjie , Yan Dong , Hyde Craig L. , Fortin Jean‐Philippe , Fauman Eric B. , Leroy Xavier , Sabbagh Laurent
Published: May 2022

G protein‐coupled receptors (GPCRs) represent the largest class of membrane‐bound proteins which regulate diverse physiological processes by engaging multiple signaling pathways. GPCR signaling can be differentially modulated by specific ligands to selectively promote the engagement of different subsets of signaling pathways, an outcome known as biased signaling. Although dysregulation of GPCR activity has been linked to pathological consequences, the involvement of their signaling pathways toward specific clinical repercussions remain unresolved. Thus, linking the role of GPCR signaling pathways to distinct physiological processes is not only important for understanding GPCR biology, but also essential for successfully targeting these receptors as therapeutic avenues. Here, to exploit the potential of naturally occurring GPCR variants emerging from large scale exome and genome sequencing efforts, we assessed the signaling profiles of rare variants across different GPCRs found in human populations with documented clinical phenotypes. Signaling profiles were assessed through an automated platform using our bioSensAll® technology, which measures the activity of various signaling pathways using a panel of 16 selective bioluminescence resonance energy transfer (BRET)‐based biosensors that monitor the activation of heterotrimeric G proteins (Gαs, Gαi1, Gαi2, GαoA, GαoB, Gαz, Gαq, Gα11, Gα14, Gα15/16, Gα12, Gα13) or recruitment of β‐arrestins (β‐arrestin 1 and β‐arrestin 2) to the plasma membrane upon agonist stimulation. To achieve the large‐scale screen in a high throughput system, bioSensAll® assays were miniaturized and adapted to a 384‐well format. HEK293 cells were co‐transfected with receptors and biosensors, and a scheduling software was used to coordinate the interaction between various instruments via a robotic microplate mover. Analysis was performed through an automated tool to generate and compile signaling profiles for each variant normalized to wild‐type receptor, revealing gain‐ or loss‐of‐function properties induced by variants across different signaling pathways, thus also allowing us to identify genetic alterations that impart biased signaling profiles. By pairing the multiparametric signaling profiles of variants found in human populations generated through our automated bioSensAll® platform with information on their associated clinical phenotypes, we can uncover associations between altered receptor pharmacology and clinical outcomes to accelerate the discovery of disease‐relevant targets and improve translation from bench to clinic.
Increased HDAC1 Expression Increases Mitochondrial Dysfunction in Endothelial Cells
Authors: Jones Bobby , Pati Paramita , Pollock Jennifer
Published: May 2022

Endothelial dysfunction is a critical determinant of many chronic diseases including cardiovascular and kidney disease. Endothelial dysfunction is defined by a loss of NO bioavailability and increased reactive oxygen species (ROS). Previous reports show that mitochondrial‐derived ROS (mtROS) is a major source of ROS in endothelial cells. Endothelial senescence and apoptosis are regulated by mtROS and changes in mitochondrial gene expression. Histone deacetylases (HDACs) remove acetyl groups from histones epigenetically regulating gene transcription via deacetylation of histone proteins or post‐translation deacetylation of non‐histone proteins. We previously presented that increased HDAC1, a class I HDAC, expression reduces NO production and increases maximum respiratory oxygen consumption rate in endothelial cells. Reports also show HDACs function as gene silencers or activators. We hypothesized that increased HDAC1 expression in endothelial cells leads to increased mtROS and mitochondrial dysfunction via increased expression of key mitochondrial genes. Bovine aortic endothelial cells (BAECs) were transiently transfected with HDAC1. BAECs were then evaluated for mtROS by mitoSox fluorescence and mRNA expression of key mitochondrial regulatory genes, NRF2, TFAM, and PGC1a, by RT‐PCR. MitoSox fluorescence was increased in cells with HDAC1 OE as compared to vehicle transfections. Gene expression of TFAM was significantly increased (p=0.0367; n=5); NRF2 expression trended up (p=0.0746; n=5); and, PGC1a expression remained unchanged (p=0.9952; n=5) as compared to vehicle transfections (n=6). These results suggest increased HDAC1 in endothelial cells may lead to increased mtROS and alterations in mitochondrial function.
Autonomic Testing in Collegiate Athletes Following SARS‐Cov‐2 Infection
Authors: Luck J. C. , Blaha Cheryl , Cauffman Aimee , Arnold Amy C. , Gao Zhaohui , Hamaoka Takuto , Sinoway Lawrence I. , Cui Jian
Published: May 2022

To date, more than 46 million people in the United States have contracted the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), which causes COVID‐19. Over 700 thousand total cases have been linked to American colleges and universities. Post‐acute sequelae of SARS‐CoV‐2 infection may affect varied systems. It was reported that sympathetic nerve activity following SARS‐CoV‐2 infection was elevated in young individuals. Cardiovascular autonomic function post‐acute SARS‐CoV‐2 infection in other populations such as athletes, however, has not been thoroughly investigated. Collegiate athletes are a unique population given that they might have a high level of physical fitness before the infection. The present study examined 16 collegiate athletes (12 men and 4 women, age: 20±1 yrs, height: 181±10 cm, weight: 81±18 kg) who were evaluated ~2 weeks after testing positive for SARS‐CoV‐2 by polymerase chain reaction assay. These subjects were asymptomatic or with mild/moderate symptoms during infection. None of the subjects had been hospitalized for COVID‐19 and all subjects were asymptomatic at the time of the study. Beat‐by‐beat blood pressure (BP) with Finometer, and heart rate (HR) from ECG were recorded during 10 minutes of supine rest, Valsalva maneuver, slow breathing (6 breaths/min), handgrip exercise, and 10 minutes of standing. BP, HR, heart rate variability (HRV), and cardiac baroreflex sensitivity (CBRS), and venous blood aldosterone and renin levels were analyzed. The vagal tone index from HRV in 3 of 16 athletes was lower than normative values. The responses in BP to Valsalva maneuver during phases IIa (i.e., “early”) and IIb (i.e., “late”) were mildly abnormal in 4 athletes and moderately abnormal in one athlete. The Valsalva ratio was below the normal range in 6 athletes. In response to slow breathing, the respiratory sinus arrhythmia amplitude was below the normal range in 3 athletes. In response to handgrip exercise, one athlete had an atypical response where BP decreased while HR was increased from baseline. Three of the subjects were unable to tolerate an orthostatic challenge for 10 minutes. Four out of 16 athletes had abnormally elevated renin activity and 1 out 16 elevated aldosterone levels. It is important to note that while several athletes had abnormal responses to the various autonomic tests, these abnormalities did not correlate with symptoms seen during the infection. Our data suggest that SARS‐CoV‐2 infection may reduce parasympathetic and increase sympathetic tone and may contribute to the autonomic disfunction associated in a subset of collegiate athletes even after recovering from COVID‐19. This study provides further evidence for potential long‐term cardiovascular autonomic effects of Covid‐19 infection even in healthy young trained athletes. Further studies with a larger patient population as well as suitable healthy control subjects are warranted.
Intestinal Dmt1 is Required for Iron Loading in Mice Modeling β‐Thalassemia Intermedia
Authors: Yu Yang , Yang Chunhua , Shine Jacob S. , Lee Jennifer K. , Ebea Pearl , Zhu Sean , Merlin Didier , Collins James F.
Published: May 2022

β‐thalassemia intermedia (βTI) is characterized by chronic anemia, ineffective erythropoiesis, and excessive intestinal iron absorption leading to systemic iron overload. Divalent metal‐ion transporter 1 (DMT1), the main intestinal iron transporter, may mediate excessive iron absorption in Th3/+ mice, which model βTI.

Objective

To determine if intestinal DMT1 is required for iron‐loading in beta‐thalassemia, and if so, to utilize a ginger nanoparticle‐derived lipid vector (GDLVs) siRNA delivery system to normalize iron absorption in Th3/+ mice.

Hypothesis

DMT1 mediates excessive iron absorption in βTI mice and decreasing DMT1 expression by GDLVs‐siRNA will prevent iron accumulation in these mice.

Methods

Th3/+ mice were crossed with mice lacking intestinal DMT1 generating double KOs. Also, weanling mice were gavaged daily for 16 days with GDLVs carrying functional DMT1 siRNA, and iron absorption and iron loading were assessed.

Results

Liver, spleen, kidney, heart, and duodenum non‐heme iron levels were decreased in Th3/+ mice lacking intestinal DMT1. Serum non‐heme iron content, serum ferritin levels, and transferrin saturation also decreased in Th3/+ mice lacking intestinal DMT1. Splenomegaly was alleviated in the double KO mice. Oral gavage of GDLVs carrying DMT1 siRNA blunted intestinal DMT1 expression and mitigated hepatic and splenic iron loading. Serum non‐heme iron levels, serum ferritin, and transferrin saturation also decreased in Th3/+ mice receiving GDLVs with DMT1 siRNA.

Conclusion

Intestinal DMT1 is required for iron‐loading in Th3/+ mice, and in vivo DMT1 knock down is an effective approach to blunt iron loading in mice modeling βTI.
Sex Differences in Cocaine Self‐Administration Behavior in Rats
Authors: Dean Calista R. , Zinani Dakota B. , Norman Andrew B.
Published: May 2022

Inter‐injection intervals during maintained cocaine self‐administration behavior in male rats is regular and increases with higher unit doses due to the longer time required for the higher cocaine concentrations to decline to the minimum maintained concentration, the satiety threshold. In addition, lever pressing behavior is induced by cocaine when it is below the satiety threshold and above the priming threshold, termed the compulsion zone. This paradigm provides a framework for the development of medications to treat cocaine use disorder. However, this paradigm has never been tested in female rats. It is a regulatory requirement to test potential medications in both males and females. Female Sprague‐Dawley rats also acquired iv cocaine self‐administration behavior. The inter‐injection intervals were regular and increased as a function of unit dose, indicating the underlying pharmacokinetic/pharmacodynamic cocaine induced behavior in female rats is consistent with the observed self‐administration behavior in male rats. However, compared to age‐matched male rats the inter‐injection intervals in females were shorter across all unit doses. It is not yet clear whether this is due to a higher satiety threshold, faster cocaine elimination, or both in female rats. Interestingly, there was no detectable cyclicity of inter‐injection intervals over several estrous cycles, implying that the faster rate of self‐administration was not related to estrous hormone levels. The compulsion zone theory applies to female rats and females should be included in pre‐clinical medication screening protocols.
Does Metabolism Regulate Cardiac Repair in the Regeneration Competent Axolotl Salamander?
Authors: Dittrich Anita , Andersson Sofie A. , Busk Morten , Nielsen Rasmus J. , Lauridsen Henrik
Published: May 2022

Adult human hearts form permanent scar tissue after a myocardial infarction, leaving millions of survivors in western societies with ongoing heart complications and increased risk of heart failure. Unlike, humans and other mammals, axolotl salamanders have an extraordinary ability to repair similar cardiac injuries. After forming an initial collagenous scar in response to a cryo‐infarction, axolotls completely repair their heart, with existing cardiomyocytes dedifferentiating and proliferating, with the transient scar tissue eventually completely replaced by new functioning myocardium. The mechanisms leading to this immense discrepancy in capacity for repair within vertebrates is not fully understood, although the proliferative capacity of cardiomyocytes are a perquisite for regeneration. In recent years, research across species has demonstrated that metabolism may be instrumental in regulating cardiomyocyte proliferation and regeneration. In both adult and neonatal mice as well as zebrafish, studies have shown that a pro‐glycolytic metabolic profile is favorable to regeneration, although the mechanisms involved are unclear. We hypothesize that metabolic adaptations are an instrumental part of the regenerative response in the axolotl and that thereby altering metabolism can both promote and inhibit cardiac regeneration in this animal model. In this currently ongoing study, we are employing a range of methods to uncover the interplay between metabolism and regeneration in the axolotl, combining whole‐animal and high resolution cardiac respirometry as well as autoradiography to assess glucose and acetate consumption, metabolite analysis, immunochemistry and enzyme histochemistry. We are currently exploring if specific metabolic adaptations occur after injury both in the animal as a whole and locally in the different regions of the heart, which could help facilitate cardiac regeneration. In addition, we are looking at how altering axolotl metabolism can support or inhibit regeneration. For this purpose, axolotls are given the same type of cryoinjury and housed at different rearing temperatures (10 ◦C, 20 ◦C or 30 ◦C), or kept in isosmotic water, or fasted for the duration of regeneration which takes 60 ‐ 90 days. By augmenting metabolism in this way in a regenerative animal model, we hope to uncover key insights about how metabolism affects cardiac regenerative ability.
The Regulation of Basal Autophagic Flux by an ATG13‐ATG101 Complex
Authors: Youngs Joshua C. , Poole Daniel , Kannangara Ashari , Lewis Riley J. , Andersen Joshua L.
Published: May 2022

Autophagy is an essential cellular process by which cellular debris is collected and broken down in the lysosome. Cancer cells rely on autophagy for survival during chemotherapy treatment and periods of hypoxia within solid tumors. In this project, we investigate the ATG13‐ATG101 protein complex, a sub‐complex of the ULK1 initiatory complex whose regulatory role in autophagy is not completely understood. Our recent data demonstrate that an ULK1‐independent ATG13‐ATG101 complex is essential for the basal autophagic degradation of protein aggregates—a process known as aggrephagy. Furthermore, we found that the ULK1‐independent ATG13‐ATG101 complex traffics to condensates of poly‐ubiquitinated protein aggregates where it colocalizes with the autophagy regulator ATG9A.1 Our current data suggest that this ATG13‐ATG101 complex cooperates with ATG9A in the degradation of these protein aggregates. To further elucidate the function of the ATG13‐ATG101 complex, we have developed a protein‐fragment complementation assay using a novel split TurboID (BioID) system in which complementary halves of TurboID are fused to ATG13 and ATG101. Using this split BioID system, together with quantitative LC‐MS/MS and mutants of ATG13 that cannot bind ULK1, we are identifying interactors that are unique to the ULK1‐independent ATG13‐ATG101 complex. Based on previous data, we hypothesize that the ATG13‐ATG101 sub‐complex participates in ATG9A trafficking pathways and recruitment of autophagy regulators to protein aggregate condensates. Thus, our project characterizes the first proximity interactome for the ATG13‐ATG101 complex.

1. Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L., BioID reveals an ATG9A interaction with ATG13‐ATG101 in the degradation of p62/SQSTM1‐ubiquitin clusters. EMBO Rep2021, e51136.
Calcium Assimilation for Regenerative Purposes in Axolotls
Authors: Braagaard‐Sørensen Alexander , Lauridsen Henrik
Published: May 2022

Background

The Mexican axolotl (Ambystoma mexicanum (Shaw and Nodder, 1798)) is an urodele amphibian with intrinsic regenerative capacity in a range of organs and tissues e.g. heart, spinal cord and appendages.

Urodeles represents one of few tetrapods that can recreate the embryonic/undifferentiated environment of cells to regenerate once differentiation has already occurred. When trauma occurs, the wound is sealed off by wound epithelium which stems from migrating epithelial cells. Dedifferentiated local cells are activated in the stump accumulate at the tip and form the blastema. The blastema cells proliferate and control the pattern formation so the limb reforms in the right way. This includes the position of cartilage cells, and it takes around 2‐3 months to have a fully differentiated limb. Most studies although only look at the first stages of the regenerated limb (until cartilage formation) whereas calcification may take even longer.

The route of calcium uptake during limb regeneration in the axolotl is not known. However, there is anecdotal evidence for the observation that age matched axolotls develop mineralized limbs at different rates in different laboratories with varying water supply and food, suggesting an effect of the of ambient calcium level.

Our hypothesis is that calcium uptake during limb regeneration is maintained by ambient available calcium dissolved in either the food or aquatic environment, and low levels of ambient calcium results in lack of mineralization in the regenerated appendage and/or results in transient/permanent osteopenia in the remaining skeleton.

Materials and methods

Axolotls (n=24) is divided into four groups respectively: Control (n=6), calcium enriched food (n=6), Calcium free water and enriched food (n=6), High calcium water (0,61 g/l CaCl2) and food(n=6). Calcium enriched pellets was soaked in a saturated CaCl2 solution (850g/l) for 1h and dried for 24h at 21oC.

To assess mineral density the animals was scanned by non‐invase microCT prior to amputation. It is planned to be done again 3 months post amputation, 4,5 months and before euthanization.

Bone mineral content will subsequently be validated by ashing and some animals will sacrificed at different stages of mineralization for histological staining, but this has yet to be done.

Results

To ascertain the amount of calcium uptake trough eating we found the calcium enriched pellets to have mean factor of 4.67 times more calcium in them than the normal pellets (Ca enriched mean: 56ppm and normal pellets mean: 12ppm).

Conclusion

With the initial assessment of regeneration between the different groups. We expect to see a difference in mineralization.
Circulating Exosomal Proteins are linked to Neuropathogenesis in SIV‐infected Rhesus Macaque: A Proteomic Approach
Authors: Chandra Partha K. , Braun Stephen E. , Baddoo Melody C. , Kim Hogyoung , Castorena‐Gonzalez Jorge A. , Cikic Sinisa , Rutkai Ibolya , Guidry Jessie J. , Worthylake David K. , Flemington Erik K. , Abdel‐Mageed Asim B. , Busija David W.
Published: May 2022

Background

Combined antiretroviral therapy suppresses HIV replication, but 30‐60% of patients suffer from HIV‐1 associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in HIV CNS infection. Using proteomics, we investigated possible circulating exosomal protein links to neuropathogenesis in SIV‐infected rhesus macaque (RM).

Methods

Exosomes were isolated from sera of SIV‐infected (SIV‐Exo) and uninfected (CTL‐Exo) RM (N = 3/group) by QIAGEN exoEasy kit and characterized by the qNano‐IZON system. Proteomic analysis of the isolated exosomes was performed using liquid chromatography/mass spectrometry (LC‐MS/MS).

Results

qNano‐IZON analysis indicated that isolated EVs were predominantly exosomes (particle size < 150 nm). In the LC‐MS/MS study, 5,654 proteins were quantified, with 236 proteins (~ 4%) significantly differentially expressed (DE) between CTL‐Exo and SIV‐Exo. Two or more unique peptides were detected in 85% (4777/5654) of quantified proteins, and in 89% (211/236) of significant DE proteins, indicating the depth of analysis. We quantified most of the exosome‐associated proteins (tetraspanins, enzymes, lipid rafts, cytoskeletal, and endosome‐specific proteins) reported in previous studies. The heat‐maps and hierarchical clustering indicated that proteins involved in latent viral reactivation (heat shock transcription factor 1), inflammation (complement factor H, antioxidants, glycoproteins), unfolded protein response (UPR) (proteasome activators, cochaperones), neuropathology (amyloid beta [Aβ] precursor, chromogranin‐A and ‐B), and signaling (cytoskeleton regulators, cyclin‐H, mTOR complex 2, CD74) were expressed at significantly higher levels in SIV‐Exo than CTL‐Exo. However, proteins involved in mitochondrial (Mt) fission (Mt‐fission 1, ‐fission factor, and fission regulator 1), and ATP production (Mt Complex‐I, ‐IV, and ‐V), that play a critical role in the brain energy supply, were significantly decreased in SIV‐Exo. Moreover, exosomal‐proteins involved in autophagy‐mediated degradation (autophagy related 9A, ‐2B, lysosomal associated membrane protein 2), endosomal recycling (sorting nexin 4) and exocytosis (synaptogyrin), sprouting angiogenesis (jumonij domain‐containing 6), and cytoskeleton organization (calponin) were also expressed at significantly lower levels in SIV‐Exo than CTL‐Exo.

Conclusions

Our novel findings suggest that circulating exosomal proteins are associated with viral reactivation, inflammation, UPR, mitochondrial dysfunction, defective autophagy, and Aβ and Tau pathology that may elucidate the etiology of HAND, and possibly provide novel therapeutic targets.