bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Infarct Border Zone Using Late Gadolinium Enhanced MRI in Rats
Authors: Mendiola Emilio , Merchant Samer , Xiang Qian , Neelakantan Sunder , Hsu Edward , Vanderslice Peter , Avazmohammadi Reza
Published: May 2022

Background

Following acute myocardial infarction (MI), cardiac myocyte death and the formation of a fibrotic scar in the left ventricular free wall (LVFW) alters the biomechanical behavior of the myocardium resulting in changes to organ‐level function. Despite significant advances in understanding LVFW remodeling in the setting of MI, the extent of tissue remodeling in the regions where infarct tissue transitions to healthy tissue, commonly called the border zone (BZ), is not well understood. Additionally, the effect of alterations in biomechanical behavior of the BZ on LV pump function remains understudied. We hypothesize that image‐based cardiac modeling can effectively address this gap. To this end, our objective was to develop an automated method to identify the infarct and BZ regions in rats using ex‐vivo late gadolinium‐enhanced magnetic resonance images (LGE‐MRI) and make use of these delineations to study regional structural remodeling in MI.

Methods

Anterobasal MI was induced in Wistar‐Kyoto rats (n=2) by ligating the left anterior descending artery. Prior to sacrifice, subjects in the infarct group were given gadolinium contrast agent for MRI enhancement. Animals were sacrificed at 4 weeks post‐infarct and cardiac MRI scans were performed at an isotropic 100 µm resolution. The mean value of the voxel signal‐intensity (SI) of the posterobasal region of the heart was calculated and is hereafter referred to as the baseline SI (BSI). The image voxels were then segmented in to three regions by using the following SIs as threshold value cutoffs: i) SI of greater than 125% of BSI was considered infarcted myocardium, ii) SI between 115% and 125% of BSI was considered BZ, iii) SI of less than 115% BSI was considered remote myocardium. The geometry and dimensions of the infarct region were confirmed with measurements from the excised heart of the animal.

Results

Increased SI was noted in the anterobasal region of the infarcted hearts (Fig 1), indicating myocyte necrosis and scar formation. Mean SI of the infarct region defined via this method was 1.6‐fold larger compared to that of the remote region. The infarct region resulting from the automated identification method closely matched the gemometry and dimensions of the infarct scar region of the excised hearts.

Conclusions

The extent of myocardial remodeling in LVFW during the post‐infarct healing process determines the contractile behavior in the LVFW and influences long‐term outcomes. Automated identification of infarct, BZ, and remote myocardium regions using LGE‐MRI is a step towards image‐based characterization of the heterogenous remodeling in the LVFW. Additionally, our algorithm enables the simple definition of a BZ of varied size and could allow us to better understand the influence of distinct regional remodeling events on organ‐level function as MI proceeds from early‐stage scarring to late‐stage remodeling.
MitoQ supplementation improves oxygen uptake kinetic by reduced reactive oxygen species levels and altered expression of miR‐155 and miR‐181b
Authors: Park Yoonjung , Aminizadeh Soheil , Lee Junghoon , Zarezadehmehrizi Aliasghar , Najafipour Hamid , Amiri‐Deh Ahmadi Maedeh , Moflehi Daruosh , Rashidzadeh Hamed
Published: May 2022

Background

Mitochondrial antioxidant (MitoQ) has been developed as a pharmaceutical for diseases. Few studies have investigated the role of MitoQ in enhancing athletic performance. Thus, the purpose of the study was to determine the effects of MitoQ supplementation to exercise training (EX) on the oxygen uptake kinetic, oxidative stress, and MicroRNAs (miRNAs) regulating vascular inflammation and reactive oxygen species (ROS) generation.

Methods

32 healthy young cyclists (25.6±3.8yr) were randomly divided into 4 groups (n=8); 1) Placebo, 2) EX (Cycle ergometer, moderate intensity), 3) MitoQ (20 mg/day, oral), and 4) EX+MitoQ. Oxygen uptake kinetics during low, moderate, and severe intensity cycling were measured before and after two weeks of interventions. Serum levels of ROS, glutathione peroxidase (GPx), and superoxide dismutases (SOD) were measured. miR‐19b, miR‐155, miR‐181b, and miRNA‐146a were measured by RT‐qPCR.

Results

EX+MitoQ accelerated the phase Ⅱ of oxygen uptake kinetic (moderate intensity) compared to EX (P<0.05). Both EX+MitoQ and MitoQ reduced ROS levels but there was no change in EX (P<0.05). GPx was increased in EX, MitoQ, and EX+MitoQ (P<0.05) whereas SOD levels in all groups were not different. miR‐155 and miR‐19b expressions were decreased in EX+MitoQ compared to EX (P<0.05). miR‐146a was increased in EX+MitoQ compared to EX only (P<0.05). There was no difference of miR‐181b between EX and Mito+Q. Placebo effects were not found.

Conclusions

A short‐term MitoQ supplementation to aerobic training enhanced oxygen utilization during a moderate performance in cyclists compared to training alone by reduced ROS levels and altered expression of miR‐155 and miR‐181b mediating vascular endothelial inflammation and ROS generation.
Modulation of GPCR Rhodopsin Function by Membrane Lipids and Water
Authors: Weerasinghe Nipuna , Fried Steven D. , Struts Andrey V. , Perera Suchithranga M. , Brown Michael F.
Published: May 2022

G protein‐coupled receptors (GPCRs) are responsible for the transduction of signals across lipid membranes and are the largest family of targets (~35%) for currently approved drugs. They exist as dynamic conformational ensembles with multiple inactive and active conformational substates described by an energy landscape model. Consequently, understanding the modulation of conformational dynamics of GPCRs by soft matter such as membrane lipids and cellular water in the receptor’s environment is critical to understanding GPCR efficacy and selectivity. Here we show how membrane lipids and cellular water play crucial roles in shaping the energy landscape of GPCR activation, thus acting as allosteric modulators. We investigated ways in which the receptor hydration level and lipid bilayer composition influence the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native and POPC recombinant membranes after light activation. Using different polyethylene glycol (PEG) solutions, the osmotic pressure on rhodopsin was varied, while shifting of the metarhodopsin equilibrium was probed using UV‐Visible spectroscopy. Our results show a flood of ~80‐100 water molecules into the rhodopsin interior during photoactivation, forming a solvent‐swollen Meta‐II active state. Dehydrating conditions favor Meta‐I through the efflux of water from the protein interior yet simultaneously increase bilayer thickness, which should favor Meta‐II. Because the osmotic effect on the protein is more significant than the effect of the lipid bilayer, the overall equilibrium is generally shifted to Meta‐I. However, small osmolytes favored the Meta‐II state because they can penetrate the protein core, decreasing the osmotic effect on the protein. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta‐I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C‐terminal peptide‐binding isotherms revealed that the G‐prottein binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta‐I state. By contrast, the native lipid membrane environment possesses a negative spontaneous curvature that favors the more expanded state of Meta‐II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment.
Biomechanical consequences of alveolar cleft defect
Authors: Smith Amanda L. , Nicholas Christina , Bakhsh Nora , Patankar Sayali , Ledogar Justin A. , Moreno‐Uribe Lina
Published: May 2022

Cleft palate is a common birth defect, requiring interventions that must be timed according to maxillary growth and patients often require multiple procedures to repair an alveolar cleft defect. Mechanical variables, such as pre‐surgical defect shape and size, have been suggested to affect the success of surgical repair, but the biomechanical consequences of these variables are not well understood. We hypothesize that mechanical performance of bony craniofacial structures will vary with cleft morphology.

3D surface models of alveolar cleft defects were created from cone‐bean (CB) CT scans of adolescent and young adult patients (n=36; IRB: 201601711). Surface semilandmarks were used to conduct a cluster analysis, identifying common axes of variation which broadly differentiated between defects that are mediolaterally wider from those that are superoinferiorly taller.

The centroid cleft defect shape from Cluster I was selected for preliminary biomechanical analysis. To isolate the mechanical effects of a defect of this shape/size, we used a previously published human cranial finite element model (FEM) as a baseline for model construction and comparison of performance. Patient CBCT was scaled and registered to the FEM and used to reconstruct the defect in the maxilla of the baseline model. Identical boundary conditions (material properties, muscle forces and joint and bite constraints) were applied to the cleft and non‐cleft FE models, to simulate biting on different teeth.

Our results reveal notable and unexpected differences in deformation and strain regimes of the cleft and non‐cleft models. Although both models show characteristic regions of elevated strain on the biting‐side, the cleft‐model experiences a marked reduction (50‐90%) in strain on the balancing (non‐biting) side, due to the cleft disrupting the transmission of internal forces across the maxilla and palate from the biting to the balancing side. Importantly, regions that experience diffuse low‐level shear strains in our baseline model drop to nearly undetectable levels in the cleft model.

We conclude that it is possible that the reduction of strain below this critical threshold could impede osteogenic mechanical signaling. This has important clinical implications for alveolar cleft defect repair and the development of novel scaffold‐based methods that both survive in vivo and restore functional orofacial load paths.
Identification and Characterization of Calcium Binding and Coiled Coil Domain 1 (Calcoco1) in Skeletal Muscle
Authors: Kajdic Amra , Gjoka Erisa , Waddell David
Published: May 2022

Skeletal muscle atrophy results in loss of muscle mass and reduced strength and is caused by disparate physiological conditions including aging, cancer, corticosteroid use, and denervation. To better understand the molecular genetic events that lead to muscle atrophy, a previous study isolated skeletal muscle from mice following 3 and 14 days of denervation. Gene expression was then analyzed by microarray and compared to control muscle to identify novel, atrophy‐induced genes. The microarray revealed that Calcium Binding and Coiled Coil Domain 1 (Calcoco1) is expressed in skeletal muscle and induced in response to denervation. To confirm that Calcoco1 is expressed in skeletal muscle, the cDNA was successfully amplified and cloned from cultured C2C12 cells. Moreover, quantitative PCR was conducted using RNA isolated from proliferating and differentiated muscle cells to determine the expression profile of Calcoco1 at the transcriptional level. We observed moderate activation of Calcoco1 through myoblast proliferation and early differentiation, followed by a robust increase in activation by the later stages of myotube differentiation. These results were mirrored at the protein level where it was observed that Calcoco1 is expressed during early proliferation and increases as muscle cell differentiation progresses. To elucidate a functional role for Calcoco1 in muscle, we transfected cultured muscle cells with a Calcoco1 expression plasmid and then harvested the cells at timepoints ranging from proliferation through late differentiation. The cell lysates were probed by Western blot for markers of muscle cell differentiation, including Myosin Heavy Chain (MyHC) and myogenin, which both show significant repression in response to Calcoco1 overexpression. The Calcoco1 protein is predicted to contain both a calcium binding domain and a coiled‐coiled domain, suggesting that it may play a role in protein‐protein interactions or as a putative transcription factor. To investigate these possibilities, we sought to determine the sub‐cellular localization of Calcoco1 in muscle cells by fusing Calcoco1 cDNA to Green Fluorescent Protein (GFP) and expressing it in muscle cells. Visualization by confocal microscopy revealed clear nuclear‐exclusion of Calcoco1 protein in unchallenged myoblast cells, suggesting that it may not participate directly in gene regulation. The discovery that Calcoco1 is expressed in skeletal muscle and is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of muscle wasting and may eventually contribute to the identification of new therapeutic targets for the treatment and of muscle atrophy.
The Effects of Metformin on Skeletal Muscle Differentiation and Satellite Cell Function in C2C12 Myocytes and Juvenile Lean and Obese Mice
Authors: Kinsey Stephen , Ochab Jess
Published: May 2022

AMP activated protein kinase (AMPK) functions as a cellular energy sensor and it is a target for the treatment of obesity and type II diabetes. Activation of AMPK has well‐known benefits such as increased fuel oxidation and enhanced insulin sensitivity, however, it also may lead to compromised satellite cell function. This may be particularly detrimental to muscle development in juveniles, when satellite cell density and activity is high. In this study, the type II diabetic drug metformin was used to pharmacologically activate AMPK in C2C12 mouse skeletal muscle myoblasts and in juvenile lean and obese mice. In C2C12 cells 48 h of metformin treatment led to increased phosphorylation of AMPK and reduced mitochondrial respiration, as expected. Myocytes treated with metformin had a decreased expression of the myogenic markers, MyoD and myogenin, as well as a reduction in the satellite cell marker Pax7, and the myotubes were smaller in metformin treated cells. Further, nuclear recruitment was reduced in the metformin group, consistent with a reduction in satellite cell fusion with myotubes. In gastrocnemius from juvenile lean and obese mice, 6 weeks of metformin treatment had little effect on myogenic markers or on Pax7. However, nuclear recruitment was reduced in gastrocnemius from both lean and obese mice. These results support the hypothesis that AMPK activation with metformin in cultured muscle cells and juvenile lean and obese mice interferes with satellite cell fusion.
A Model of Repeated Opioid Use Producing Tolerance to Opioid Induced Respiratory Depression
Authors: Nwakudu Chinwendu , Szujewski Caroline , Ma Ellen , Garcia Alfredo J.
Published: May 2022

Repeated opioid users, such as those who suffer from opioid use disorder, are among the most vulnerable to overdose, overdose‐related death, and opioid‑related morbidities in breathing in the ongoing epidemic. Despite the significant progress made in defining the cellular and circuit basis by which opioid induced respiratory depression (OIRD) occurs, very little is known about the impact that repeated opioid use (ROU) has on the control of breathing. This ongoing work aims to define how ROU impacts the OIRD. We developed a mouse model of ROU that involved six consecutive days of fentanyl (0.7mg/kg via i.p. injection) administration. Breathing was measured via unrestrained whole‐body plethysmography prior to and immediately after fentanyl administration on Day 1 and Day 6. Prior to fentanyl administration, tidal volume (TV), respiratory rate (RR) and minute ventilation (Vmin) were similar on Day 1 and Day 6 (n= 7 mice). Peak depression of TV during fentanyl was similar on Day 1 and Day 6 (n=7, P=0.28); whereas, peak depression of RR (Day 1: 145.5±19.66­BPM vs. Day 6: 172.0 ± 18.89,P=0.005) and Vmin (Day 1: 142.0±30.23­microL•min‐1•g‐1 vs. Day 6: 210.1±21.96­microL•min‐1•g‐1, P=0.04) during fentanyl was greater on Day 1 (n= 6 of 7). Moreover, the change in body temperature during OIRD was not different between Day 1 and Day 6 (Day 1: 0.04±0.021oC vs. Day 6: 0.64 ± 0.021oC, P=0.62). Together these data suggest that tolerance to OIRD emerges following six days of ROU and does not involve significant changes in body temperature. The data from this ongoing study provides foundational knowledge to the impact of ROU on breathing and introduces a novel model of ROU that leads to tolerance to OIRD.
Clostridioides difficile surface layer protein triggered inflammasome activation is mediated by membrane lipid raft
Authors: Tsai Pei‐Jane , Tsai Yau‐Sheng
Published: May 2022

Clostridioides difficile is the leading cause of nosocomial infectious diarrhea, with clinical symptoms ranging from mild diarrhea to pseudomembranous colitis and toxic megacolon. Despite the use of vancomycin and fidaxomicin as standard drugs for the treatment of C. difficileinfection (CDI), clinical relapse rates remain high. Therefore, new alternative therapeutics to treat CDI are urgently required. Surface layer proteins (SLPs) are the most abundant proteins in the C. difficile cell wall, suggesting that they might involve in immune recognition. Here, we found SLPs as well as C. difficile induced inflammasome activation. In addition, the cholesterol‐rich microdomains, lipid rafts, on the cell membrane are thought to be crucial for bacterial adhesion and signal transduction. We demonstrated that lipid rafts participated in C. difficile SLPs binding to the cell membrane. Furthermore, both binding ability and inflammasome activation induced by SLP were abrogated with membrane cholesterol depletion by methyl‐β‐cyclodextrin (MβCD). The coalescence of SLPs in the cholesterol‐rich microdomains was confirmed in C. difficile‐infected cells. Our results demonstrate that SLPs recruit the lipid rafts, which may be a key step for C. difficile colonization and inducing inflammasome activation.
Do Adolescents With Insulin Resistance Demonstrate Impaired Heart Rate Recovery From Exercise?
Authors: Spaude Katie , Ward Aaron T. , Brubaker Justin M. , Zevin Erika L. , Livett Timothy A. , Carrel Aaron M. , Schrage William G.
Published: May 2022

Heart rate recovery (HRR) following maximal aerobic exercise can serve as a noninvasive method of analyzing the risk for cardiovascular diseases. Inability to recover heart rate to pre‐exercise values can indicate parasympathetic nervous system dysfunction. Poor HRR could also be an indicator of early mortality, as decreased ability to recover heart rate is linked to decreases in vagal tone. Insulin resistance (IR) has also been linked to diabetes, obesity, and parasympathic nervous system dysfunction in adults, through an increased ratio of sympathetic to parasympathetic nerve activity. However, it remains unknown if adolescents with IR exhibit similar parasympathetic dysfunction. Therefore, it was hypothesized that increased IR would be associated with decreased HRR in adolescents.

Data were collected from adolescents aged 12‐18. Insulin resistance was quantified as HOMA‐IR using fasting glucose and insulin. Subjects underwent maximal aerobic exercise testing on a cycle ergometer pedaling at 60‐70 RPM, starting at 25 W and increasing 25 W every two minutes. The test was terminated when subjects could no longer maintain the pedaling cadence for 5 consecutive seconds. Following cessation of exercise, subjects remained seated without movement for a two‐minute recovery period. Heart rate (HR) was collected via three lead ECG. Data was filtered and resampled to 1 Hz. A linear regression model was applied to the first 30s of HRR data for each subject, with the resulting slope used as the output variable in a subsequent linear regression model with HOMA‐IR as the predictor. Significance was set p<0.05.

Subjects were 15 ± 2 years old with a HOMA‐IR 2.08 ± 1.92. The average HRR slope was ‐0.63 ± 0.13 BPM (r=‐0.91). The slope of HRR vs. HOMA‐IR was ‐0.02 ± 0.02 (r=‐0.32, p=0.18). The slope of HRR vs. fasting glucose was ‐0.001 ± 0.003 (r=‐0.07, p=0.8). The slope of HRR vs. fasting insulin was ‐0.008 ± 0.004 (r=‐0.42, p=0.08).

Poor HRR has been linked to early mortality, and insulin resistance causes diabetes. However, IR and HRR were not found to have a statistically significant relationship. The relationships between HRR and fasting glucose and insulin levels were also not statistically significant. These data suggest there is no relationship between HRR and IR post exercise in adolescents.
Phosphorylation‐based regulation of mitochondrial metabolism
Authors: Niemi Nataile
Published: May 2022

Phosphorylation has long been appreciated to influence mitochondrial metabolism via the regulation of pyruvate dehydrogenase. However, the extent to which phosphorylation broadly influences mitochondrial function remains unclear, despite the presence of multiple protein phosphatases within the organelle. We recently demonstrated that deletion of the mitochondrial matrix phosphatase Pptc7 unexpectedly caused perinatal lethality in mice, suggesting that the regulation of mitochondrial phosphorylation is essential in mammalian development. Pptc7‐/‐ mice exhibit severe metabolic deficiencies, including hypoglycemia and lactic acidosis, and die within one day of birth. Biochemical and proteomic approaches revealed that Pptc7‐/‐ tissues have decreased mitochondrial function concomitant with a post‐transcriptional downregulation of mitochondrial proteins. Multiple elevated mitochondrial protein phosphorylation sites in Pptc7‐/‐tissues suggest novel functional connections between Pptc7‐mediated dephosphorylation and these observed metabolic consequences. Interestingly, these modifications occur on components of the import machinery of the mitochondria and within the mitochondrial targeting sequences of select nuclear‐encoded precursor proteins. Collectively, our data reveal an unappreciated role for a matrix‐localized phosphatase in the post‐translational regulation of the mitochondrial proteome and organismal metabolic homeostasis.
3D Bioprinted Artificial Breast Cancer Tumor Disks: A New Tool for Understanding Intra‐tumor Heterogeneity?
Authors: Lyden Timothy , Beethe Nadina , Hultgren Heather , Jaskens Randy , LeBeau Michelle , Thrun Lori
Published: May 2022

Recently the UWRF Tissue and Cellular Innovation Center and the Century College Biofabrication Program have engaged in a series of new collaborative research projects using bioprinting technology and methods to model and study in‐vitro 3D artificial tumor and stromal tissues. In this presentation, we report on results obtained from efforts to bioprint cancer disks using the human breast adenocarcinoma cell line MCF‐7. This breast cancer cell line is considered to be one of the “gold standard” cultures to study this type tumor. In extensive previous studies in our lab using other 3D modeling methods, we have established a robust library of experience and data with these cells and artificial tissue constructs. In these new studies, bioprinting methods were applied to establish tumor structures which initially contained specific cell numbers per unit volume as well as relatively mono‐cellular distribution patterns. Both of these characteristics had been very difficult to predictably generate and control in earlier studies using precast or molded hydrogels made of chitosan and hyaluronic acid or decellularized natural marine sponge ECM. In this project, two distinct bioinks were tested including one called GelMa, a gelatin/methacrylate hydrogel and another called GelMa‐C which is basically the same, but with nanocellulose added to enhance the structural stability of constructs during printing. While both bioinks generated structures and tissues, the GelMa bioink specifically generated relatively transparent tumor structures that provided a unique window into the cell interactions and eventual early development of these artificial tumor tissues. Using a Cellink “Inkcredible” model printer loaned to the TCIC by Century College for the duration of this project, we printed tumor disks with a diameter of 5 mm and thickness of 2.5 mm and then cultured these in 6 well plates for up to 3 months. These breast cancer disks displayed features and tissue progression similar to that seen with the other methods used in our earlier work, while also providing new and unique insights into the interior artificial tumor tissue development in real‐time. These new observations also demonstrated a unique cell/tissue behavior in the resulting constructs that we hypothesize may directly address the origins of intra‐tumor heterogeneity of various cell subpopulations and their relative contributions to the overall tumor development. Future studies are planned to further evaluate and qualify these observations and to apply this new 3D artificial tissue tool to further elucidate cancer biology as a whole.
Mitochondrial PKD Activates Mitochondrial Fission and Proliferative Signaling in Cardiac Fibroblasts
Authors: Zhou Xiaoxu , Adhikari Neeta , Cypress Michael W. , Polina Iuliia , Landherr Maria , Chaput Isabel , Suckow Mark A. , Choudhary Gaurav , O‐Uchi Jin , Jhun Bong Sook
Published: May 2022

Introduction

Cardiac fibrosis, characterized by wall stiffening, reduced contractility, and impaired overall heart performance, is a self‐reinforcing process in response to injury (e.g., myocardial infarction) and pressure overload (e.g., systemic hypertension and pulmonary arterial hypertension [PAH]). The severity of cardiac fibrosis is associated with cardiac dysfunction, increased risk of arrhythmia, and mortality. However, there is a lack of effective therapies designed to inhibit or reverse cardiac fibrosis. Hyperproliferation of cardiac fibroblasts (CFs) and their differentiation into myofibroblasts are the key contributors to cardiac fibrosis. We previously showed that mitochondrial reactive oxygen species (mROS) are one of the regulators for activating proliferative signaling in rat neonatal CFs. Moreover, we reported that phosphorylation of dynamin‐related protein 1 (DRP1) by a stress‐responsive protein kinase D (PKD) at the outer mitochondrial membrane (OMM) promotes mROS generation via increased mitochondrial fission in H9c2 cardiac myoblasts.

Hypothesis

Mitochondrial PKD‐DRP1 signaling increases mitochondrial fission leading to increased mROS levels, which activates proliferative signaling.

Methods

Primary adult CFs from human ventricles and the right ventricles (RVs) of a SU5415/hypoxia‐induced rat PAH model were used. Mitochondrial morphology and mROS levels were measured by confocal microscopy.

Results

Overexpression of PKD1 increased DRP1 phosphorylation at Ser637, and activates proliferative signaling pathways including ERK1/2 and p38 in human CFs, but did not promote their differentiation into myofibroblasts, assessed by the amount of a‐smooth muscle actin. To specifically identify the involvement of OMM‐localized PKD activity in these processes, we generated an OMM‐targeted dominant‐negative PKD1 (termed mt‐PKD‐DN) by adding an OMM‐target sequence (amino acid 1‐33 from human TOM20) at the N‐terminus of PKD1‐K612W. Importantly, mt‐PKD‐DN was able to inhibit mitochondrial fission, decrease mROS, hyperpolarize mitochondrial membrane potential, and ultimately decrease proliferation in human CFs. To further understand the role of PKD‐DRP1 signaling in vivo, we employed a preclinical PAH animal model. Immunofluorescence staining of RVs from PAH rats revealed that PKD activity and DRP1 phosphorylation increased only in CFs, but not in ventricular myocytes. RV‐CFs isolated from PAH rats exhibited increased mitochondrial fission, mROS levels, and proliferative signaling compared to RV‐CFs from control rats, and these changes were abolished by adenoviral expression of mt‐PKD‐DN.

Conclusion

Mitochondrial mROS elevation via PKD‐DRP1‐dependent mitochondrial fission promotes CF proliferation. Targeting mitochondrial PKD may be a potential therapeutic strategy to reduce cardiac fibrosis under pathological conditions such as PAH.
Palmitoylation Targets the Calcineurin Phosphatase to the Phosphatidylinositol 4‐kinase Complex at the Plasma Membrane
Authors: Cyert Martha , Ulengin‐Talkish Idil , Parson Matthew A. , Jenkins Meredith L. , Roy Jagoree , Shih Alexis Z. , St‐Denis Nicole , Gulyas Gergo , Balla Tamas , Gingras Anne‐Claude , Várnai Péter , Conibear Elizabeth , Burke John E.
Published: May 2022

Calcineurin (CN), the serine/threonine protein phosphatase and the target of immunosuppressants, is a critical regulator of Ca2+ signaling. To discover novel CN‐regulated processes, we examined an understudied isoform, CNAb1, that is highly conserved across vertebrates and expressed in every human tissue. CN is a heterodimer of regulatory and catalytic subunits whose functions and regulation by Ca2+ and calmodulin are well understood for canonical CN isozymes. The few studies of CNAb1, demonstrate its unique physiological and biochemical properties, however, mechanisms that confer these properties and CNAb1‐ specific substrates remain unknown. Here, we show that the non‐canonical C‐terminus of CNAb1, generated by alternative 3’ pre‐mRNA processing, confers unique intracellular localization, enzymatic regulation and substrate specificity for this isoform. In vitro, CNAb1 displays distinct enzymatic properties, where instead of the auto‐inhibitory domain that blocks the active site of canonical CN isoforms under non‐signaling conditions, CNAβ1 is autoinhibited by a sequence motif at its C tail that blocks substrate binding. In cells, unlike canonical cytosolic CN, CNAb1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C‐terminal tail. This palmitoylation, which is dynamically regulated by the ABHD17A depalmitoylase, targets CNAb1 to distinct set of membrane‐associated interactors including the phosphatidylinositol 4‐kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. This complex recruits the cytosolic PI4KIIIA to the PM where it synthesizes phosphatidylinositol‐4‐phosphate (PI4P), a precursor of the critical signaling phospholipid, PI(4,5)P2, required for sustained Ca2+signaling through GPCRs. Using hydrogen‐deuterium exchange, we reveal multiple CN‐PI4KA complex contacts, including a calcineurin‐binding peptide motif in the disordered tail of FAM126A which we further establish as a novel calcineurin substrate. Using BRET‐based detection of phosphoinositides in live cells, we show that CN inhibitors decrease PM PI4P production during Gq‐coupled muscarinic receptor signaling, suggesting that CN dephosphorylates and promotes PI4KA complex activity. Together, this work not only discovers dynamic palmitoylation as a novel mechanism that confers unique localization, substrate specificity and regulation to CNAb1 but also uncovers a novel Ca2+/CN‐mediated, feedback loop, that promotes PI4P replenishment at the PM during GPCR signaling.
Enhancing RNA aggregation through temperature cycling and intrinsically disordered RNA binding peptides
Authors: Tenaglia Brandon M. , Finnegan Casey , Sokoloski Joshua E.
Published: May 2022

RNA‐protein aggregates akin to membraneless organelles in modern biology offer a potential model for protocells and precursors to protocells in the chemistry of early Earth. In this project, we explore the aggregation properties of nucleic acids and their nucleotide monomers under a variety of conditions using a fluorescence dye‐based assay for aggregation. The building blocks of RNA – guanosine monophosphate and adenosine monophosphate showed intrinsic concentration‐dependent aggregation. Cyclic incubation between high and low temperatures, mimicking for example conditions around a hydrothermal vent, increased aggregation. This trend was enhanced when experiments were repeated with total RNA extracted from bacterial and fungal sources. RNA aggregation was further enhanced by the incorporation of peptides featuring an RNA binding sequence combined with an intrinsically disordered region. Our results show that RNA aggregation, a key first step on the pathway to protocells, can be enhanced through temperature cycling and the binding of short disordered peptides.
A Workflow for Digitizing Anatomical Specimens by Combining Photogrammetry and 3D Scanning
Authors: Dixit Ishan , Dunne Connor , Logan Curtis J. , Fejtek Monika , Blumer Paige , Prakitpong Ratthamnoon , Krebs Claudia
Published: May 2022

The 3D visualization of anatomical specimens relies on two main technologies: 3D laser surface scanning and photogrammetry (PGM). While 3D laser scanning provides the most accurate 3D model, photogrammetry provides the best, photorealistic surface texture. Typically, a specimen would be assessed to see which technology is best to capture it ‐ in this study we set out to investigate whether it is feasible to combine both methods, incorporating the advantages of both. Artec Space Spider scanner was used to capture accurate geometry of specimens and create a mesh with high polygon count and topological accuracy in Artec Studio 15, and Reality Capture was used to capture photographs around the specimen at regular 5 degree increments until the entire surface was captured to produce a high‐resolution textured mesh. The meshes were combined in Artec Studio 15 by matching their geometric centers in 3D space and ensuring that all polygons are precisely overlapped ‐ even a minor amount of deviation would result in blurry reprojection of texture. This is followed by mapping and reprojecting the PGM texture on the 3D scanned mesh (now aligned and scaled) in Reality Capture and cleaning up the produced texture map in Adobe Photoshop to ensure that any artifacts are cleaned up. Through this combination, models that gather ‘best of both worlds’ qualities from 3D scanning and photogrammetry ‐ laser accurate geometry and photorealism. We determined that 3D scanning is sufficient for specimens with simple texture, and photogrammetry is effective for specimens with complex texture and simple geometry; the combination method shines where great detail and high resolution texture is required to meet the learning objectives, such as organs and musculoskeletal specimens and other specimens with key topological details and colorations. Gathering both PGM and 3D scanning data allows for error correction, especially when performed in collaboration with a content expert and a 3D artist. Availability of these methods to produce photorealistic and accurate scans of specimens lends to accessibility of high quality online anatomy educational materials for students. This technology may also be useful in museum settings and to scan rare collections where to date a single method was not able to capture the complexity of the specimens.
Rapid Onset Vasodilation: Impact of Cardiorespiratory Fitness
Authors: Sherman Sara R. , Hibner Brooks A. , DeJonge Sydney R. , Rosenberg Alexander J. , Lima Natalia S. , Baynard Tracy , Fernhall Bo
Published: May 2022

Introduction

Higher cardiorespiratory fitness is associated with greater vascular function that leads to improved regulation of skeletal muscle blood flow during exercise. Rapid onset vasodilation (ROV) describes the immediate increase in blood flow following a single contraction, which is integral to support greater blood flow during exercise. As such, blood flow regulation during exercise hyperemia is important. However, it is unclear if cardiorespiratory fitness plays a role in ROV of small muscle mass in young, healthy individuals.

Purpose

To investigate the relationship of cardiorespiratory fitness on ROV following a single handgrip (HG) contraction in young, healthy adults.

Methods

Cardiorespiratory fitness (VO2peak) was assessed during a maximal cycle ergometer test in 14 volunteers (M/F, 6/8; 28 ± 5yrs; 24.8 ± 4.1kg/m2). Brachial velocity and diameter were measured using duplex Doppler ultrasound for 10 cardiac cycles prior to and 20 cardiac cycles following the first cardiac cycle after a single 30% of maximal voluntary handgrip contraction. Brachial artery blood flow (FBF) was calculated for each cardiac cycle: (diameter2/2)*π*velocity*60. Peak forearm (brachial) blood flow (FBFpeak) was determined as the maximal FBF from baseline following the first cardiac cycle after the single HG contraction. Time to peak blood flow response was determined as the time to reach FBFpeak and heart rate was measured using a three‐lead ECG. The relationship between FBFpeak and time to peak blood flow was tested using a multivariate regression analysis with cardiorespiratory fitness and sex as independent variables.

Results

Cardiorespiratory fitness (VO2peak: 30.6 ± 8.0mL/kg/min); β for VO2peak= 0.162 [0.003, 0.327]) was the only predictor of time to peak blood flow response, F(1,12)= 4.602, p= 0.05, R2= 0.217, r= 0.526, while sex (β for time to peak= ‐106.5 [‐156.9, ‐56.2]) was the only predictor of FBFpeak, F(1,12)= 21.245, p<0.001, R2= 0.609, r= 0.799, indicating that females had a lower FBFpeakoverall.

Conclusion

Our data suggests blood flow responsiveness (e.g., time to peak) is positively associated with cardiorespiratory fitness and these data contribute to our understanding of potential mechanisms between improved blood flow regulation commonly associated with greater cardiorespiratory fitness. More research is necessary to fully elucidate the role biological sex has on these relationships, but our data suggest there may be sex differences in certain blood flow responses.
Essential role for epithelial HIF‐xenophagy axis in control of Salmonella infection and dissemination
Authors: Dowdell Alexander S. , Kitzenberg David A. , Kostelecky Rachael E. , Mahjoob Omemh , Welch Nichole , Glover Louise E. , Colgan Sean P.
Published: May 2022

The intestinal mucosa exists in a state of “physiologic hypoxia,” in which oxygen tensions of the epithelium and lumen are markedly lower than those encountered in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain mucosal homeostasis in this austere environment in part through the actions of dedicated oxygen‐sensitive transcription factors, including hypoxia‐inducible factors (HIFs). HIFs are composed of a labile α‐subunit that becomes stabilized most notably in hypoxic environments and a constitutive β‐subunit. Using an unbiased chromatin immunoprecipitation (ChIP) promoter array screen for HIF‐1 targets in IEC, we identified autophagy as a major pathway induced by hypoxia and HIF in cultured IECs. Autophagy is a conserved eukaryotic system for degradation of intracellular cargo through the lysosome. Validation of the ChIP screen revealed that HIF promotes expression of multiple autophagy genes in IEC cell lines and further validated in primary murine organoids. One important function of autophagy is to defend against intracellular pathogens, a process termed xenophagy, a crucial innate immune mechanism in the mucosa that serves to control invasion of intracellular bacteria in vivo and to limit extraintestinal dissemination of enteric pathogens. Loss of function analysis revealed that HIF is a central regulator of autophagic flux and that in vitro infection of IECs with SalmonellaTyphimurium, a model intracellular bacterial pathogen, results in stabilization of HIF with the subsequent increase in HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Studies in vivo revealed that Salmonella­ drives increases in HIF activity using HIF luciferase reporter mice (HIF∆ODD) in combination with streptomycin‐pretreatment model of murine colitis. Further studies in vivo demonstrated that intestinal epithelial‐specific deletion of HIF (Hif1b∆IEC) compromised IEC xenophagy and exacerbated bacterial dissemination to multiple extraintestinal organs, implicating xenophagy‐assisted clearance of Salmonella in mucosal innate immunity.
The characteristics of metformin nanoliposomes
Authors: Fan Baochan , Zu Yujiao
Published: May 2022

Objectives

Metformin is an FDA‐approved anti‐diabetic drug. The oral bioavailability of metformin is low. After oral intake of metformin at 0.5‐3 g/day, blood metformin levels are less than 40 μM in human subjects. We successfully synthesized biocompatible and biodegradable metformin nanoliposomes. The objectives of this study are to measure the characteristics of metformin nanoliposomes and to determine their stability at three different temperatures.

Methods

The particle size and polydispersity index of metformin nanoliposomes were measured using a dynamic light scattering technique, and their surface charges were measured using a zeta potential analyzer. The metformin nanoliposomes were stored at 4°C, 22°C and 37°C for 5 days, and their stability was measured every day.

Results

The mean particle size of metformin nanoliposomes was around 100 nm. The polydispersity index value was less than 0.3. Moreover, the zeta potential of metformin nanoliposomes was around −20 mV. The particle size, polydispersity index and zeta potential of metformin nanoliposomes were stable at 4°C for 24 hours, but were increased at 22°C and 37°C after 24 hours.

Conclusions

Metformin can be encapsulated into biocompatible and biodegradable nanoliposomes. They are stable at 4°C for 24 hours.
Three‐dimensional functional capacity of the intrinsic hand musculature in humans and chimpanzees
Authors: Fields Mara G. , Mackereth Eden M. , Sullivan Spiro P. , McGechie Faye R. , Middleton Kevin M. , Holliday Casey A. , Ward Carol V.
Published: May 2022

Accurately interpreting the functional significance of variation in hand morphology has been central to discussions of the evolution of in hominoids, especially humans. Although there is an extensive literature on bony morphology, muscle size and strength, kinematics, and use of the hand, to date it has not been possible to visualize and quantify the mechanics of the of the hand in situ.

This study compared the intrinsic hand muscles of Homo sapiens and Pan troglodytes in 3D. Each specimen was fixed, stained with Lugol’s iodine, and microCT scanned [<61 µm resolution]. Bones, muscles and the flexor retinaculum were segmented using Avizo. Avizo Xfiber was used to visualize and quantify fascicle orientation within muscles, which permits quantification of both whole muscle and within muscle fascicle orientations. Fascicle length, pennation angles and muscle volume were used to calculate PCSA and estimate force output of each muscle. Together, these data are used to compare functional capability of muscles individually and in aggregate.

Data reveal differences in force‐generating capacity and force orientations of muscles, and with finer‐scale variation of within‐muscle fascicle orientations. The most significant differences were found in the adductor pollicis and opponens pollicis, which are larger and more transversely oriented in humans than in chimpanzees. Data also reveal differences in other thenar and hypothenar muscles, affecting the capacity to effect different grips.

Our approach and results highlight the importance of evaluating of the musculoskeletal system of the hand in 3D which can then be used to compare to bone form across hominoids.
SIRT1 Mutation Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine
Authors: Reed John , Thamba Aish , Strobel John , Byrd James , Alloosh Mouhamad , Coutts Alex , Sturek Michael
Published: May 2022

Background

Metabolic syndrome (MetS) impairs sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) activity in coronary smooth muscle (CSM) and is implicated in coronary atherosclerosis in Ossabaw miniature swine. Sirtuin 1 (SIRT1) is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 impairment exacerbates MetS and vascular disease, including calcification. After Ca2+ is increased in CSM by Ca2+ influx or release from the sarcoplasmic reticulum (SR), the Ca2+ is then buffered by either Ca2+ efflux from the cell by the plasmalemmal Ca2+ ATPase or sequestration back into the SR via SERCA. SIRT1 inhibition hyper‐acetylates SERCA and decreases activity, thereby impairing the Ca2+ buffering capacity of the cell.

Hypothesis

MetS and SIRT1 mutation will impair Ca2+ buffering in CSM.

Methods

Using CRISPR/Cas9 methodology a point mutation (SIRT1L100P) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation of Ca2+transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, and SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. CSM cells were enzymatically dispersed and Ca2+ was measured with fura‐2. The main Ca2+ regulation protocol was the maximal release of Ca2+ from the SR by 5 mM caffeine and then assessment of Ca2+ efflux and sequestration. Time to half recovery of Ca2+ to baseline during caffeine exposure was measured to assess Ca2+ efflux (rapid phase Ca2+buffering) and deviation from baseline was measured to assess SERCA function (slow phase Ca2+buffering).

Results

There was no effect of MetS or SIRT1 mutation on Ca2+ efflux. Two‐way ANOVA showed SIRT1 mutation alone inhibits SERCA buffering of Ca2+ in CSM (p=0.0005). There was no effect of MetS diet (p=0.184) and no interactions of SIRT1 genotype and MetS diet (p=0.113) on SERCA activity.

Conclusion

SIRT1L100P mutation is likely to contribute to coronary atherosclerosis and SIRT1 activators may be effective therapies.
Metabolomic Signatures of Ocean Acidification Stress in the Coral Acropora millepora
Authors: Patel Sajin U. , Hauser Paul , Ronan Jehnna
Published: May 2022

The rapid acidification of seawater poses a significant threat to the persistence of coral reefs. However, taxa‐specific, mechanistic understandings of holobiont responses to ocean acidification (OA) stress remain largely unknown. Understanding these mechanisms is crucial to uncovering predictive markers of OA stress. This could subsequently be used to assist field‐based conservation efforts. We hypothesized that exposure to elevated pCO2 levels would decrease the abundance of primary metabolites such as amino acids and carbohydrates and increase the production of secondary metabolites, specifically those involved in cnidarian‐symbiodinium cell signaling. In this study, we applied untargeted capillary electrophoresis time‐of‐flight mass spectrometry (CE‐TOFMS) to profile changes in the intracellular polar and semi‐polar metabolite composition of the coral holobiont after exposure to elevated pCO2 concentrations. Nubbins of Acropora millepora were exposed to ambient (410 µatm) or elevated (805 µatm) pCO2 levels under controlled conditions over a period of 10 days. Measurement of treatment‐induced bleaching was performed by quantification of symbiodinium cell density and chlorophyll a concentration. We hope to identify molecules in the metabolite profiles associated with the individual cellular responses of holobiont members to osmotic stress. Additionally, we hope to provide further insight into unknown roles of secondary and tertiary metabolite pools in cellular homeostasis and acclimation to thermal stress in the coral holobiont, and specifically the cnidarian‐symbiodinium symbiosis. Our goal is for these findings to assist conservation efforts, specifically in the development of rapid tests for field‐based conservation efforts.