bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine‐regulation of myosin relaxed states in vertebrate muscle
Authors: Thomsen Elise M. , Sonne Alexander , Bertelsen Mads , Vermeulen Els , Ganswindt Andre , Wang Tobias , Ochala Julien
Published: May 2022

Vertebrate muscle myosin, the motor protein, has evolved over long periods of time. In the present study, we specifically aimed at identifying how this evolution has fine‐tuned myosin conformational states to the specific needs of organisms with different body masses. As myosin has two different relaxed states (super‐relaxed and disordered‐relaxed), we initially hypothesized that a linear positive correlation would exist between the amount of myosin molecules in the super‐relaxed conformation and the body weight of the organisms in which they are expressed. To verify this hypothesis, we extracted muscle fibres from multiple vertebrate species with body weights ranging from grams to tons. By using a loaded Mant‐ATP chase protocol, we observed that vertebrate species with body weights above 1000 kilograms have significantly higher proportions of super‐relaxed myosin proteins in their muscle fibres. As myosin heads in the super‐relaxed conformation consume five times less ATP than the ones in the disordered‐relaxed state, our finding suggest that heavy vertebrate animals have adapted their motor protein conformations to potentially avoid inappropriate and large energy consumption in their metabolically demanding muscles.
Alcohol exacerbates SARS‐CoV‐2 ‐induced ARDS in mice
Authors: Solopov Pavel A. , Colunga Biancatelli Ruben M. L. , Dimitropolou Christiana , Catravas John D.
Published: May 2022

SARS‐CoV‐2 – induced COVID‐19 causes acute respiratory distress syndrome (ARDS) and multiorgan failure. COVID‐19 can also cause psycho‐social problems, including increased alcohol consumption and consequent complications. Alcohol abuse is recognized as an independent factor that increases by three‐ to four‐fold the incidence of ARDS, a severe form of acute lung injury with a mortality rate of 40 to 50 percent. This translates to tens of thousands of excess deaths in the United States each year from alcohol‐associated lung injury. We developed a combined ARDS and alcohol abuse mouse model by intratracheally instilling the S1 subunit of SARS‐CoV‐2 Spike protein in K18‐hACE2 transgenic mice that express the human angiotensin‐converting enzyme 2 (ACE2) receptor for SARS‐CoV‐2 and which are kept on an ethanol diet. 72 hours after S1SP instillation, mice kept on the ethanol diet exhibited a strong decline in body weight, a dramatic increase in white blood cell content of bronchoalveolar lavage fluid (BALF), and an augmented “cytokine storm”, compared to S1SP treated mice on control diet. Histologic examination of lung tissue demonstrated abnormal recruitment of immune cells in the alveolar space, destructive effects on parenchymal architecture, and an overall worsening of the lung injury score (LIS) of S1SP‐ and alcohol‐treated animals. Along with the activation of pro‐inflammatory biomarkers (NF‐κB, STAT3, NLRP3 inflammasome), lung tissue homogenates from mice on alcohol diet, demonstrated overexpression of ACE2 compared to mice on control diet. In summary, K18‐hACE2 transgenic mice on an alcohol diet exhibit a more severe S1SP‐induced ARDS than corresponding mice on a control diet and may thus represent a useful model for the development of therapeutic interventions against alcohol‐exacerbated COVID‐19.
NVS‐HD1 preserves the mitochondria function and increase glucose uptake in C2C12 myoblasts exposed to lipopolysaccharide (LPS)‐induced inflammatory response
Authors: Wang Jianguo , Zhao Thomas , Slate‐Romano John , Zhao Ting C.
Published: May 2022

Background

Histone deacetylase plays a crucial role in modulating cellular survivals and metabolism. Specific HDAC isoform in the regulation of mitochondria function and glucose hemostasis in lipopolysaccharide (LPS)‐induced inflammatory response remains to be determined. We examined the effects of a newly synthesized HDAC4 inhibitor, NVS‐HD1 on cell survival and mitochondrial function in differentiated C2C12 cells.

Methods

C2C12 myoblasts were cultured in DMEM medium until 80% confluency before they were subjected to differentiation medium containing 2% horse serum for 3 consecutive days. C2C12 derived myotubes were then exposed to LPS at the concentration of 10 ng/L for 24 hours. NVS‐HD1 (10 nmol/L) and vehicle (Dimethylsulfoxide, DMSO) were treated in differentiated C2C12 cells with or without LPS to define the effect of HDAC4 inhibition on mitochondrial function and glucose uptake.

Results

Differentiated C2C12 cells decreased viability in response to LPS stress as evident by and lactate dehydrogenase leakage and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide MTT assay. LPS‐induced inflammatory attenuated glucose uptake as comparted to vehicle control. Furthermore, differentiated C2C12 cells displayed an impairment in mitochondrial function by decreasing mitochondrial membrane potential assessed by utilizing the red‐fluorescent probe TMRM (tetramethylrhodamine, methyl ester). As compared to DMSO treatment, NVS‐HD1 resulted in improvement in cell survival rate and increased glucose uptake, in association with the preservation of mitochondria function in LPS‐induced inflammatory response.

Conclusion

Our finding is the first demonstration that specific HDAC4 plays a pivotal role in preserving cellular survival, glucose uptake and mitochondrial function in LPS induced inflammatory response.
Potential Sex Differences in Bone Strength: A Cadaveric Study
Authors: Chapin Madison M. , Vranish Jennifer R. , Luckett Brittany S.
Published: May 2022

This study was conducted to determine if there was a difference in bone strength between men and post‐menopausal women. Prior research suggests that women are four times more likely to be affected by osteoporosis and experience fractures twice as often as men. Bone mass loss found in osteoporosis increases in women after menopause due to marked estrogen deficiency. However, it is unclear whether this bone mass difference between men and women contributes to a reduction in bone strength. Therefore, the hypothesis of this study was that men will have a higher bone density than women and will therefore have stronger bones when tested to failure. To test this hypothesis, eight cadavers (four men, four women) from the Wayne State University Willed Body Program were used (age: 81±16, range: 51‐104). The bone density of the ulna was calculated via a volume displacement test and its mass. The ulna was then secured and weight was added incrementally until failure. There was no significant difference found in ulna bone density between men and women (p=0.776); however, the weight required to break the ulnas in men was greater (p=0.0001). Once normalized by the mass of the ulna this effect was reduced (p>0.05). These data suggest potential sex differences in bone strength, independent of bone density, indicating future investigation is needed at different locations in the body.
Smooth Muscle At1a Receptor Mediates Perivascular Fibrosis in Angiotensin II‐infused Mice
Authors: Okuno Keisuke , Torimoto Keiichi , Cicalese Stephanie , Sparks Matthew , Eguchi Satoru
Published: May 2022

Angiotensin II (Ang II) signaling via AT1 receptor has been shown to play a critical role in the pathogenesis of hypertension, cardiovascular hypertrophy and fibrosis. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates EGF receptor activation and promotes cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. In a recent study, deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affects hypertensive fibrosis in vasculature. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis during Ang II stimulation using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the endogenous sm22α promoter (Kock‐In, KIsm22α‐Cre). Male AT1a flox/flox KIsm22α‐Cre+/‐ (SMKO) and control (AT1a flox/flox KIsm22α‐Cre‐/‐) mice were infused with Ang II (1 µg/kg/min) for 2 weeks via osmotic mini‐pump. In control mice, Ang II infusion for 2 weeks induced cardiac hypertrophy indicated by heart‐to‐body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart‐to‐body weight ratios were significantly increased in control mice compared with AT1a SMKO mice (6.04 versus 4.89, respectively, p=0.032). Cardiac wall hypertrophy was seen in control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice (n=5) showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 µm vs. 56.4 µm, respectively, p<0.0001). In conclusion, AT1a receptors expressed in VSMC could mediate Ang II‐induced cardiovascular hypertrophy and perivascular fibrosis. Whether the data can be fully explained by the prevention of hypertension remains to be determined. Our data also contrast with the past manuscript showing a protective effect in AT1a flox/flox S100A4‐Cre+/‐ (fibroblast silencing) but not in AT1a flox/flox sm22α‐Cre+/‐ mice with Ang II infusion.
FXR ligand structure impact on FXR‐DNA binding
Authors: Meinert Emily , Yu Tracy
Published: May 2022

The farnesoid X‐receptor (FXR), a nuclear receptor, is a ligand‐regulated transcription factor that regulates bile acid, lipid, and glucose metabolism. FXR consists of a ligand‐binding domain (LBD), that bind small lipophilic ligands, and a DNA binding domain (DBD) that targets specific areas of DNA called FXR response elements (FXRE). When ligands bind to the LBD, allosteric structural changes occur in the DBD and affect the DNA binding preferences. Once bound to FXREs, ligand‐bound FXR regulates the transcription of specific genes. While the natural ligands of FXR are bile acids, many synthetic ligands have also been developed to regulate FXR. Previous studies have shown that structurally similar FXR ligands activate FXR in a gene‐specific fashion. However, it is not clear how these similar FXR ligands achieve such varying effects on DNA binding preferences. My project focuses on a structurally related group of synthetic FXR ligands (fexaramine, fexarine and fexarene) to understand how ligand structure impacts FXR‐DNA binding. To investigate how modifications in the ligand scaffold regulate FXR transcriptional activity, I am performing dual‐luciferase assays to measure the transcription in a large set of FXRE‐driven promoters.
The Stress Hormone ACTH Causes a Prolonged Reduction in Heart Rate Variability
Authors: Hamner J.W. , Taylor J. Andrew , Bonyhay Istvan , Adler Gail K. , Freeman Roy
Published: May 2022

Hypoglycemia is a complex stressor that activates multiple hormonal systems and the sympathetic nervous system. Hypoglycemia also impairs autonomic control of blood pressure. It is possible that stress hormones produced during hypoglycemia (such as ACTH) play a role in the attenuation of autonomic function after recurrent hypoglycemia known as “cardiovascular hypoglycemia‐associated autonomic failure”. Prior work from this placebo‐controlled cross‐over study has shown that ACTH infusion reduces Modified Oxford baroreflex gain and that the reduction persists to the following day. The purpose of this analysis was to determine whether measures of heart rate variability (HRV) demonstrate a similar pattern. In the original study, men and women aged 18 – 55 years (n = 22) completed 2 study visits (ACTH or placebo in random order) with a 1–3‐month washout period between. A study visit consisted of a 3‐day/2‐night inpatient stay with the infusion occurring on day 2. Standard HRV measures from 3 minutes of 0.1 Hz breathing (6 breaths/minute) were collected on each of the 3 days. Overall HRV (time or frequency domain), as well as low frequency HRV, were reduced on the infusion day and the day after by ACTH, with no effect on blood pressure variability. Low‐frequency transfer function gain was similarly reduced, but only on day 3. In addition, these frequency domain gains were significantly correlated with the Modified Oxford derived gains. These results suggests that the ACTH impairment of autonomic control of blood pressure is reflected in HRV measures.
Nuclear Entry of DNA Tumor Viruses: Finding the LINC in Nuclear Transport
Authors: Spriggs Chelsey C. , Tsai Billy
Published: May 2022

DNA viruses that replicate in the nuclei of infected cells must transport to and deliver their viral genomes into the nucleus to cause infection. How polyomavirus (PyV), a tumor virus that causes debilitating disease in humans, accomplishes this essential step in infection is not well‐understood. During entry, PyV sorts from the cell surface to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol. From there, the virus is transported to the nucleus for entry through the narrow nuclear pore complex (NPC). The incoming viral genome is harbored within an icosahedral capsid that is approximately 45 nm in diameter and larger than the NPC cargo limit. Using the archetype PyV, simian virus 40 (SV40), which shares both structural and genetic organization with human PyVs as well as the same infectious life cycle, we recently reported that the virus is disassembled in the cytosol by the bicaudal D2 (BICD2) dynein cargo adaptor to generate a subviral particle competent for transport through the NPC. How this disassembled particle is subsequently targeted to the nuclear membrane as well as the exact mechanism of nuclear import is unclear. In mammalian cells, intracellular transport to the nucleus is facilitated largely by the cytoplasmic dynein motor, which moves cargo along microtubules towards the center of the cell. Interestingly, BICD2 was shown to mediate the recruitment of dynein to the nuclear membrane through its association with either the linker of nucleoskeleton and cytoskeleton (LINC) complex component Nesprin‐2 or the NPC cytoplasmic filament RanBP2. Our preliminary experiments reveal that loss of Nesprin‐2 (and not RanBP2) blocks nuclear entry, suggesting that SV40 is recruited to the nucleus by a dynein‐BICD2‐Nesprin‐2 nexus. Moreover, SV40 associates with Nesprin‐2 and this interaction appears to mediate a second, previously unreported, disassembly step at the nuclear membrane. We are currently investigating the role of BICD2/Nesprin‐2 in nuclear arrival of SV40 as well as the functional relationship between Nesprin‐2 and the NPC during nuclear import. Our studies will clarify the mechanism responsible for driving these final steps of viral entry, and more broadly, further our understanding of host cellular nucleocytoplasmic transport.
Direct Observation of Na,K‐ATPase Oligomers in The Plasma Membrane of Living Cells by FRET‐FCS
Authors: Nordahl Linnea , Wennmalm Stefan , Jönsson Jakob , Elsässer Simon , Akkuratov Evgeny E. , Brismar Hjalmar
Published: May 2022

The sodium pump, Na,K‐ATPase, is an integral plasma membrane protein, expressed in all eukaryotic cells. Na,K‐ATPase transforms chemical energy from ATP into a gradient of Na+ and K+ over the plasma membrane by actively exporting three Na+ ions and importing two K+ ions for each hydrolyzed ATP. It is responsible for maintenance of the transmembrane Na+ gradient and is the major determinant of the membrane potential. It provides the driving force for all Na+‐coupled transport processes, thereby controlling essential functions in the cell. Na,K‐ATPase is formed by three subunits alpha/beta/FXYD, where alpha is the catalytic ion‐transporting subunit, beta is a regulatory subunit and FXYD is accessory.

Self‐interaction and oligomerization of the Na,K‐ATPase alpha/beta heterodimer in cell membranes has been proposed and discussed for a long time but is still an open question.

Here we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET‐FCS, in order to detect oligomers of Na,K‐ATPase. Compared to conventional cross‐correlation FCCS, FRET‐FCS is one to two orders of magnitude more sensitive when detecting oligomers. Moreover, FRET‐FCS is inherently insensitive to unbalanced labeling, which is a great advantage during live cell measurements.

We hypothesized that Na,K‐ATPase can exist in a higher order oligomeric state and demonstrate the use of FRET‐FCS to test this hypothesis

We have introduced fluorescent labels by using expression of non‐canonical amino acid modified beta subunits. The FRET pair Alexa488 and Alexa647 was directly conjugated to the beta subunits using selective click chemistry. Conventional FCS measurements of labeled cells revealed the absolute density of labeled and unlabeled Na,K‐ATPase. With FRET‐FCS we could observe FRET signals and FCS curves demonstrating the existence of oligomers. Positive controls for the FRET‐FCS measurements were constructed by labeling alpha subunits with Alexa488 and beta subunits with Alexa647.

Furthermore, we performed Monte Carlo simulations of Na,K‐ATPase, as monomer and as oligomer of increasing order, together with its ligands in a picket and fence diffusion model of the plasma membrane. The simulations suggest that oligomerization can have an impact on the net efficiency of the Na,K‐ATPase measured as ATP turnover.

In conclusion we find that Na,K‐ATPase can be found in the plasma membrane as oligomers. Further we discuss the consequences of oligomerization and propose that it can have a regulatory effect for the Na,K‐ATPase net efficiency.
Metallated Anticancer Peptides: An Expanded Mechanism that Encompasses Physical and Chemical Bilayer Disruption
Authors: Mihailescu Ella , Comert Fatih , Heinrich Frank , Chowdhury Ananda , Schoeneck Mason , Darling Caitlin , Anderson Kyle , Libardo M. Daben , Angeles‐Boza Alfredo , Silin Vitalii , Cotten Myriam
Published: May 2022

In the search for novel broad‐spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically‐active conformations and minimum motifs for function represents a requisite step to developing them into efficacious and safe therapeutics. Here, we demonstrate that metallating HDPs is an effective chemical strategy to improve their cytotoxicity on cancer cells. Mechanistically, we find that the metallated peptides not only physically but also chemically damage lipid membranes. Our testing ground features piscidins 1 and 3 (P1/3), two amphipathic, histidine‐rich, membrane‐interacting, and cell‐penetrating HDPs that are α‐helical bound to membranes. To investigate their membrane location, permeabilization effects, and lipid‐oxidation capability, we employ neutron reflectometry, impedance spectroscopy, neutron diffraction, and UV spectroscopy. While P1‐apo is more potent than P3‐apo, metallation boosts their cytotoxicities by up to two‐ and seven‐fold, respectively. Remarkably, P3 is particularly effective at inserting its metallated motif in bilayers, causing water crevices in the hydrocarbon region and placing Cu2+ near the double bonds of the acyl chains, as needed to oxidize them. This study points at a new paradigm where metallating HDPs to expand their mechanistic reach could be explored to design more potent peptide‐based anticancer therapeutics.
Effect of Short‐Term Estradiol Exposure on Endothelin‐B Receptor‐Mediated Vasodilation in Postmenopausal Women
Authors: Welti Laura M. , Haigh Katherine M. , McGinty Shane J. , Del Vecchio Angelica R. , Edwards David G. , Wenner Megan M.
Published: May 2022

Endothelin (ET)‐1 contributes to endothelial dysfunction in postmenopausal women (PMW) via the ETB receptor. We have previously shown that the ETB receptor mediates vasodilation in young women, an effect that is lost in PMW. However, it is unclear if ETB receptor‐mediated dilation can be restored in PMW exposed to E2.

PURPOSE

To test the hypothesis that E2 exposure restores ETB receptor‐mediated vasodilation in PMW.

METHODS

We tested 9 PMW (55±2 yrs, 24±3 kg/m2, 91±5 mmHg) who self‐reported natural menopause and were at least 1 year without menses. We measured vasodilation in the cutaneous microcirculation via local heating using laser Doppler flowmetry coupled with intradermal microdialysis during perfusions of lactated Ringer’s (control), ETB receptor antagonist (BQ‐788, 300nM) and ETA receptor antagonist (BQ‐123, 500nM). Cutaneous vascular conductance (CVC) was calculated during the plateau phase of local heating (42°C), and normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C. All women were tested at baseline (BL) and after 7 days of E2 (Vivelle‐Dot, 0.1mg/day, transdermal patch) administration (+E2). A two‐way repeated measures ANOVA was performed to compare %CVCmax in PMW before and after E2 exposure. Data presented are mean ± standard deviation.

RESULTS

E2 administration increased serum estradiol (BL: 45±29 vs +E2: 71±42 pg/mL, P<0.01) and reduced follicle stimulating hormone (BL: 71±13 vs +E2: 57±17 pg/mL, P<0.01), but there were no changes in serum progesterone (BL: 0.57±0.93 vs +E2: 0.53±0.88 pg/mL, P=0.14) nor plasma ET‐1 (BL: 1.62±0.66 vs +E2: 1.54±0.54 pg/mL, P=0.42). E2 administration had no effect on cutaneous microvascular responses (Time P=0.41, Drug P=0.17, Interaction P=0.71). At BL, vasodilatory responses to local heating were not different during control and ETB receptor antagonism (control: 83±8 vs. BQ‐788: 88±6 %CVCmax). Similarly, vasodilation was not altered at BL during ETA receptor antagonism in a subset of women (n=6, control: 83±8 vs. BQ‐123: 87±8 %CVCmax). E2 administration did not improve vasodilation in the control site (BL: 83±8 vs. +E2 85±13 %CVCmax). Furthermore, there was no effect of E2 exposure on cutaneous vasodilatory responses during perfusion of ETB (BL: 88±6 vs. +E2: 89±6 %CVCmax) nor ETA (n=6, BL: 87±8 vs +E2: 92±7 %CVCmax) receptor antagonists.

CONCLUSION

These preliminary data suggest that short‐term E2 exposure does not modulate ETB receptor function in PMW. These findings are in contrast to our observations in young women demonstrating that ETB receptors mediate vasodilation in the presence of E2. The role of alternative therapies to modulate ETB receptor function should be considered in future studies.
Lysine Deacetylases Have Distinct Preferences for Multiply Acetylated Substrates
Authors: Bezue Jada A. , Mohammed Gipson Hajjia S. , Nguyen Trang T. , Toro Tasha B. , Watt Terry J.
Published: May 2022

Lysine deacetylases (KDACs) are enzymes that remove an acetyl group from a lysine, thereby regulating the post‐translational modification of lysine. KDACs are important in many different biological processes and are associated with various mechanisms of diseases in the human body. However, factors contributing to substrate specificity of the KDACs are poorly understood. Moreover, many potential substrate proteins contain adjacent or nearby lysine residues that can be independently acetylated and deacetylated. We hypothesized that KDACs will demonstrate preferences for a particular acetyllysine when reacting with multiply acetylated substrates. We additionally hypothesized that KDAC activity would exhibit a dependence on the acetylation status of nearby lysine residues. We identified several proteins reported as putative substrates of KDACs and which contain multiple nearby lysines, each of which has been reported as acetylated. Using peptides derived from these proteins, we measured activity of the putative substrates with each KDAC via a fluorescence‐based assay. The acetyllysine(s) targeted by each specific KDAC was determined by mass spectrometry. Molecular dynamics was used to model the interactions of the peptides with the KDACs. Our results demonstrate that nearby acetylation does influence the ability of individual KDACs to deacetylate a particular lysine residue, in a manner specific to each KDAC. We propose distinct contacts in the KDACs that may contribute to this selectivity. Future plans include evaluating the enzymatic activity of the KDACs with the full‐length putative substrates in cells to more firmly establish biological relevance.
Normalization of the α‐1Na/K‐ATPase Signalosome Rescinds Epigenetic Modifications in NASH‐Related Hepatocellular Carcinoma
Authors: Rajan Pradeep K. , Udoh Utibe‐ Abasi S. , Sanabria Juan D. , Nakafuku Yuto , Sodhi Komal , Pierre Sandrine V. , Xie Zijian , Shapiro Joseph I. , Sanabria Juan R.
Published: May 2022

OBJECTIVE

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers and is characterized by an acquisition of multiple abnormal phenotypes driven by genetic and epigenetic alterations. Epigenetic changes including DNA methylation and histone modification are associated with uncontrolled cell growth and proliferation, and even initiation and progression of HCC from chronic inflammation with or without fibrosis in the liver. Among others, non‐alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are two major risk factors as both of them may develop cirrhosis and hepatocellular carcinoma (HCC) if left untreated. Most of the existing clinical and experimental reports provide only a snapshot of abnormal histone modifications in HCC rather than their dynamic changes. This makes it difficult to elucidate the significance of these changes in the development of HCC. Therefore, the objective of the present study is to investigate the role of histone acetylation/methylation mediated alterations using in vitro cell line and high fat diet animal model of non‐alcoholic steatohepatitis (NASH)‐derived liver carcinogenesis.

Hypothesis

Most of the existing clinical and experimental reports provide only a snapshot of abnormal histone modifications in HCC rather than their dynamic changes. Hence, the present study hypothesised that the histone acetylation/methylation disturbances mediated by the α‐1Na/K‐ATPase signalosome may help to elucidate the significance of these changes in the development of HCC.

METHODS

In vitro studies were done using two human HCC cell lines, complemented with our in vivo murine models of NASH and NASH‐HCC. Experimental groups were exposed to pNaKtide, a selective α‐1 subunit signalling inhibitor. Acetylated/tri methylated H3K9 in cell lysates and liver homogenates were measured by ELISA, and their expression by confocal microscopy on immuno‐stained livers. Significant differences among groups were established at p<0.05 using ANOVA/t‐test.

RESULTS

Epigenetic changes in cell lines and liver malignancies correlated with morphological changes in NASH progression to malignancy. Acetylated and tri‐methylated H3K9 were significantly increased in untreated cells and livers when compared with pNaKtide treated cells and animals (p<0.05). Quantitative analysis of H3acetylK9 and H3tri‐methyl K9 proteins on confocal images from cells and livers confirmed our findings.

CONCLUSIONS

Normalization of the α‐1Na/K‐ATPase signalosome in human HCC cell lines and murine NASH‐HCC results epigenomic reprogramming.
Anti‐cancer Effects of Coumestrol in Triple‐Negative Inflammatory Breast Cancer 3D Models: Opportunities for In Vivo Studies
Authors: Colon‐Ortiz Ayeisha N. , Rodriguez‐Martir Keishla , Negron‐Figueroa Dalissa , Peterson‐Peguero Esther A.
Published: May 2022

Inflammatory breast cancer (IBC) is the most aggressive subtype of breast cancer. Each molecular subtype is characterized by the presence or absence of two hormonal steroid receptors, estrogen receptor (ERα‐66) and progesterone receptor (PR), and a growth factor receptor called HER2. Particularly, a subtype of breast cancer that lacks these three biomarkers is known as triple‐negative breast cancer (TNBC). However, previous studies showed that TNBC can express alternate estrogen receptors known as ERα‐36 and GPR30, mediating a non‐genomic signaling upon estrogen (E2) treatment. Today, effective targeted therapeutics for triple‐negative inflammatory breast cancer (TN‐IBC) patients, accounting 20‐40% of TN‐IBC cases, is limited. Coumestrol (Cou), a plant derived compound structurally similar to estrogen has shown to induce a cytotoxic effect in TNBC. Till this day, there is no known study of the effects of Cou in three‐dimensional culture models using Matrigel, a solubilized membrane matrix that mimics the tissue microenvironment. Additionally, we want to test Cou’s inhibitory effect on tumor emboli formation, a distinct hallmark of IBC. This tumor cell clusters are known to infiltrate and block the breast lymphatics of patients, presenting a barrier for successful IBC treatment (Arora Etl. 2017). We hypothesize that Cou inhibits cell viability and tumor emboli formation in TN‐IBC cells. First, we performed 3D dose response curves to determine the half‐maximal inhibitory concentration (IC50) of Cou (50µM). Next, 3D proliferationassays were performed using 50µM of Cou. After 10 days, E2 showed a significant increase in colony size in comparison to vehicle control (DMSO). Also, proliferation decreased more than a half after treating the cells with Cou alone and in combination of both treatments. When testing the tumor emboli formation after 72 hours, Cou did not inhibit the formation of emboli, but it decreased the tumor emboli growth rate shown by the decreased in tumor emboli size in both culture conditions. In comparison, E2 promoted a significant increase in the size of the tumor emboli. Lastly, we used Western Blot analysis to validate the decrease in phosphorylation levels of ERK1/2 and AKT, two kinases involved in estrogen non‐genomic signaling, upon Cou treatment. Together, these data demonstrated that Cou decreases cell viability in 3D proliferation assays and decreased growth rate of tumor emboli in TN‐IBC cells. In summary, the anticancer effects of Coumestrol in IBC three‐dimensional models provide the basis for future in vivo studies using animal models.
Neuregulin‐1β promotes myoblast differentiation and is anti‐cachexic in respiratory muscles of post‐infarcted swine
Authors: Hill Braxton , Nguyen Van Thuan , Cleator John H. , Sawyer Douglas B. , Galindo Cristi L.
Published: May 2022

Neuregulin‐1β (NRG‐1β) is a growth and differentiation factor with pleiotropic systemic effects. Because NRG‐1β has therapeutic potential for heart failure and known growth effects in skeletal muscle, we hypothesized that it might affect heart failure‐associated cachexia, a severe co‐morbidity characterized by a loss of muscle mass. We therefore assessed NRG‐1β’s effect on skeletal muscle gene expression in a swine model of heart failure using recombinant Glial Growth Factor 2 (USAN ‐ cimaglermin alfa), a version of NRG‐1β currently being tested in humans with systolic heart failure. Animals received one of two intravenous doses (0.67 or 2 mg/kg) of NRG‐1β bi‐weekly for 4 weeks, beginning one week after infarct. Skeletal muscle was collected at the time of euthanasia from the intercostal space and paired‐end RNA sequencing performed. NRG‐1β treatment altered expression of 583 transcripts including 242 transcripts altered at both doses. These included genes required for myofiber growth, maintenance and survival such as MYH3, MYHC, MYL6B, KY and HES1. Importantly, NRG‐1β altered the directionality of at least 85 genes associated with cachexia, including myostatin, which negatively regulates myoblast differentiation by down‐regulating MyoD expression. Consistent with this, MyoD was increased in NRG‐1β treated animals. In vitro experiments with myoblast cell lines confirmed that NRG‐1β induces skeletal muscle differentiation with an absolute requirement for ERBB signaling on myoblast differentiation. These findings suggest a NRG‐1β‐mediated anti‐atrophic, anti‐cachexia effect that may provide additional benefits to this potential therapy in heart failure.
Mice Deficient in Endothelial Cell‐Selective Adhesion Molecule Develop Vascular Endothelial and Left Ventricle Diastolic Dysfunction
Authors: Buncha Vadym , Tian Yanna , Fopiano Katie Anne , Lang Liwei , Bagi Zsolt
Published: May 2022

The Endothelial Cell‐Selective Adhesion Molecule (ESAM) mediates inflammatory cell transmigration and also involved in pathological angiogenesis. Here we investigated the role of ESAM in affecting vasodilator and cardiac contractile function. To that end ESAM knockout and wild type (WT) mice were employed. Using transthoracic echocardiography it was found that ESAM knockout mice displayed left ventricle (LV) diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT) and E wave deceleration time, with no change in the ejection fraction. The systolic blood pressure, as measured by tail‐cuff plethysmography, was not different between ESAM knockout and WT mice, whereas the heart to body weight ratio is increased in ESAM knockout mice. Using an unbiased automated tracing of the microvasculature we found a decreased vascularization (total vascular length) of the myocardium in ESAM knockout mice. In addition, vasodilator function of the pulmonary artery and aorta was assessed using wire myography. We found that the endothelium‐dependent, acetylcholine‐induced relaxation of the pulmonary artery was significantly reduced in ESAM knockout mice, whereas endothelium‐independent, sodium nitroprusside‐induced relaxation was similar in two groups. Thus, we conclude that ESAM deficiency causes vasodilator dysfunction, impaired angiogenesis underlying the development LV diastolic dysfunction.
Opioid Analgesic Use in Patients with Sleep Disorders: A Retrospective Cohort Study Using Electronic Health Records
Authors: Jedlicka Charles R. , Bhagwat Siddharth , Tomasson Michael H. , Bates Melissa L.
Published: May 2022

The objective of this study was to utilize electronic health records to determine the incidence of opiate prescription following diagnosis of a sleep related disorder. Opiate use disrupts sleep architecture triggering sleep disturbances, and neurobiological mechanisms exist through which sleep disorders modify opiate reward pathways. We hypothesized patients with sleep disorders will have increased opiate prescriptions following diagnosis. We used data from the TriNetX electronic health records network which includes over 80 million patients from 57 health care organizations across the world. We compared adult patients with the most common sleep disorders against adults with negative polysomnography. Patients with a sleep disorder were more likely to use opioids than those without a sleep disorder (RR 1.39, 95% CI 1.35, 1.44). Patients with a sleep disorder also had significantly more unique opiate uses in their medical record (2.56, sd 12.18, p < 0.001) compared to individuals without a sleep disorder (1.101, sd 5.57). Sleep disorder patients also used an opiate closer to the date of their sleep assessment compared to patients without a sleep disorder (median 2078 and 3345 days, respectively). These results support our hypothesis of increased opiate use by patients with a sleep disorder. Sleep disorder patients are more likely to receive opiates, receive a higher quantity, and receive them closer to their sleep assessment. These results support that the association between opiates and nighttime breathing disorders is bidirectional.
Structural Basis of Nanobody Induced ACKR3 Inhibition
Authors: Schlimgen Roman , Peterson Francis , Heukers Raimond , Smit Martine , McCorvy John , Volkman Brian
Published: May 2022

When bound to their small, secreted chemotactic cytokine ligands (chemokines), the G protein‐coupled receptor (GPCR) subfamily known as chemokine receptors (CKRs) can promote cell proliferation, survival, and migration towards increasing chemokine concentration gradients. Their strong association with these phenotypic hallmarks of cancer and their upregulation in cancer cells has made these receptors a topic of extensive research in the cancer field. Recent findings show upregulation of the atypical chemokine receptor 3 (ACKR3) in a wide variety of cancers including various breast, lung, and brain cancers, suggesting the involvement of ACKR3 in cancer pathogenesis. Acting as a scavenger receptor for a narrow range of chemokine ligands (CXCL12/CXCL11), binding of ACKR3 to a cognate ligand induces non‐canonical β‐arrestin dependent signaling, which can lead to receptor‐ligand internalization, and cause a unique signaling cascade to directly promote cell proliferation, cell growth, and angiogenesis. Moreover, silencing of ACKR3 in murine models has been shown to significantly decrease the growth and invasiveness of breast, lung, and endothelial cancer. The development of therapeutics capable of selectively inhibiting ACKR3 has now become an area of active interest. Despite this drive, the characteristic promiscuity of the CKR network and a lack of understanding in ACKR3’s mechanism of inhibition has left all ACKR3 antagonists to date with varying degrees of partial agonism, low specificity, or low stability. Here we identify and characterize a new antagonist of ACKR3, turning to recombinant variable domain monomers from the unique heavy‐chain only camelid antibodies or “nanobodies”. These 13 kDa nanobodies have highly conserved structures with the exception of three structural loops which encode the nanobody complimentary‐determining regions (CDRs) involved in antigen recognition and protein binding. This intricate binding epitope allows nanobodies to uniquely target complex GPCR epitopes, offering increased receptor specificity and selectivity versus small molecules and traditional antibody therapeutics. Using NMR spectroscopy, we have resolved the solution structure of the nanobody VUN701, an ACKR3 antagonist capable of completely ablating ACKR3 signaling at nanomolar concentrations. The structure of this inhibitor contains an uncharacteristically well‐ordered extended beta‐hairpin in the CDR3 region. In combination with cell‐based assays and molecular modeling, this analysis has allowed us to develop an atomic view of the VUN701‐ACKR3 interface. These findings shed light on the mechanism behind ACKR3 inhibition and drive forward the development of highly specific lead compounds for cancer therapy.
Network mapping of asynchronous student interactions in online discussion boards
Authors: Downey Ryan
Published: May 2022

The recent movement towards asynchronous learning activities in higher education has transformed student and educator interactions. However, as the efficacy of such approaches are unknown, many educators must rely on personal experience and peer anecdotes regarding student engagement without underlying data to inform assessment. As educators transition from pandemic‐driven pedagogy to sustainable models of course design, the use of course‐based analytics can help instructors understand the ways in which students are interacting with asynchronous activities and each other.

To develop fluency in reading and understanding scientific journal articles and to provide an interactive forum for discussions between students, weekly discussion boards were assigned in a graduate‐level neuroscience course. Based on an assigned article, each student was required to post an original response to the reading and reply to two posts made by their peers. These discussion board interactions were analyzed to form a social network map of asynchronous student interactions. Of the 27 students initially enrolled in the course, 24 completed it. The 3 students that did not complete the course were included as nodes and edges but excluded from statistical analysis.

During the 9‐week semester (9 original posts, 18 required peer replies), students responded well to instructions about ensuring that the majority of posts received peer interaction, with 91.5% of all original posts receiving at least one reply and 28.7% of posts receiving 3 or more replies. Prestige or indegrees, measured as the average number of replies each student received, was 17.96 (SD 6.39). Centrality or outdegrees, measured as the average number of replies each student made, was 17.92 (SD 1.64). Students connected to an average of 45% (SD 0.07) of their peers. There was no correlation between student quiz performance and either prestige or centrality. Students that tended to post earlier saw a non‐significant trend to receive more peer replies than those who tended to post later. This trend became significant (p < 0.001) when analyzing the correlation between absolute post order and number of replies rather than the average post order of each student.

These findings provide evidence regarding student engagement behaviors as they pertain to online discussion boards. Students generally did not exceed the mandated minimums for full points, and in some cases, self‐reported through end‐of‐semester feedback that they strategically engaged either to maximize earned points or to minimize overall effort. Collectively, in their feedback, the students provided deep and nuanced feedback on the activity experience. Overall, while many students noted the time required and initial difficulty in regularly reading scientific literature, 92% of responses indicated that the student found value and utility in reading and discussing the scientific papers in this format, supporting the use of this activity in future versions of this course.
Molecular landscape of canine soft tissue sarcoma
Authors: Das Sunetra , Idate Rupa , Regan Daniel P. , Lana Susan E. , Duval Dawn L.
Published: May 2022

Canine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features including hemangiosarcoma, lymphangiosarcoma, histiocytic sarcoma, synovial sarcoma, leiomyosarcoma, and rhabdomyosarcoma. Histologically, canine STS sub‐types are difficult to discern at the light microscopic level due to their overlapping microscopic features. Thus, genomic, and transcriptomic profiling of canine STS may prove more valuable in differentiating the diverse sub‐types of mesenchymal neoplasms within this grouping. To this purpose we sought to characterize the mutation and expression profiles of canine STS. In this study we used whole exome capture (Agilent Sure Select Canine V2) to sequence 29 tumors, grade I‐III, from non‐visceral locations, and matched normal samples, along with RNAseq of tumor samples. For the WES data, 40‐170 million 150 bp paired‐end reads were obtained from Illumina sequencing which were mapped against CanFam3.1 genome via BWA and short variants were called and annotated Mutect2 and VEP tools, respectively. The median depth of sequencing for normals and tumors were 97X (range: 35X–128X) and 110X (range: 22X–133X), respectively. Additionally, RNAseq reads were sequenced from 29 tumors and mapped against CanFam3.1 genome via STAR and gene count data was generated by HTSeq‐count tool. The total number of somatic variants identified across 29 samples ranged from 275 to 4,196, of which, 6% to 31% were annotated as cDNA variants. The protein coding mutations per callable megabase ranged from 0.3 to 37.3. Known cancer genes (COSMIC database) with recurrent mutations were KMT2D(21% of the samples), TP53 (21%), CNTNAP2(14%) genes. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B (PDGFB) and collagen genes. These gene fusions were confirmed through Sanger sequencing of amplified cDNA. To delineate the transcriptomic sub‐types, we used hierarchical and K‐means clustering methods. This resulted in 4 groups with similar expression profiles comprising of 11, 4, 8 and 6 samples in each. Further, genes set variation analysis (GSVA) was used to profile the distinct pathways across all 4 clusters. Preliminary data indicates that the 11‐sample and 6‐sample clusters had up‐regulated non‐homologous end joining pathway and immune response pathways, respectively. Further study of genes and pathways that are enriched in sample clusters will help in categorizing the canine STS tumors for diagnosis and treatment.
Interactions of a high‐fat Western diet and crystalline silica inhalation on airway epithelial ion transport and airway reactivity
Authors: Thompson Janet A. , Kashon Michael L. , McKinney Walter S. , Fedan Jeffrey S.
Published: May 2022

Silicosis, an irreversible occupational lung disease caused by crystalline silica inhalation, is a serious health risk for silica‐exposed workers. NIOSH reports that Appalachian coal miners have higher rates of obesity and metabolic dysfunction (MetDys) compared to the general U.S. adult population. MetDys is a risk factor for lung function impairment, pulmonary hypertension, and asthma. Consumption of a high‐fat Western diet (HFWD) is associated with obesity and MetDys. In this study, we investigated the effects of, and determine interactions between, HFWD‐consumption and silica‐exposure on airway epithelial ion transport and smooth muscle reactivity in the F344 rat. Six‐week‐old male F344 rats were fed either a HFWD [40.6% fat (19.5% lard), 40.6% total carbohydrate (20% sucrose), 14.8 % protein] or standard rat chow (STD) [6.2 % fat, 44.2 % carbohydrate (grain sources), 18.6 % protein] for the duration of the study. Following 16 weeks of diet‐consumption, inhalation exposure to respirable crystalline silica (Min‐U‐Sil 5®, 15 mg/m3, 6 h/d, 5 d/wk, for 39 d) or filtered air began, with endpoint experiments conducted at 0, 4, and 8 wk post‐exposure. Airway epithelial ion transport maintains airway surface liquid osmolarity and depth required for effective cilia motility and clearance of xenogens. Changes in ion transport were determined ex vivo by measurement of transepithelial potential difference (Vt), short‐circuit current (ISC) and transepithelial resistance (Rt) in rat tracheal segments mounted in Ussing chambers, and administered the ion transport inhibitors amiloride (Na+ channel blocker; apical), 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB; Clˉ channel blocker; apical), and ouabain (Na+, K+‐pump blocker; basolateral). Airway hyperresponsiveness is associated with obesity and pulmonary diseases such as asthma and COPD; thus, the isolated perfused trachea apparatus was employed to ascertain whether silica or HFWD altered airway smooth muscle reactivity to serosal or mucosal applied methacholine (MCh). HFWD‐consumption had no effect on basal Vt. Silica exposure increased Na+ transport at 0 wk, decreased basal ISC at 4 wk, and reduced Cl‐ channel and Na+, K+‐pump activity at both 4 wk and 8 wk compared to STD+AIR controls. HFWD‐consumption caused a reduction in Cl‐ transport and Na+, K+‐pump activity at 4 wk, while increasing Rt in response to ouabain at 0 wk and NPPB at 8 wk compared to STD+AIR. HFWD+SIL increased basal ISC at 0 and 4 wk, caused reduction in Cl‐ transport and Na+, K+‐pump activity at 4 wk, while reducing Rtin response to ouabain at 4 wk compared to STD+SIL. No significant changes in tracheal reactivity to MCh were observed. In conclusion, HFWD and silica altered epithelial ion transport, but the combined effects of HFWD+SIL were not synergistic.