bluuebunny/crossref_metadata_2025 huggingface dataset:
Bronchopulmonary Dysplasia Patients Have Preserved CT‐Measured Central Airway Luminal Area
Authors: Guo Winston L. , Eli Ilhan M. , Ripoll Juan G. , Baker Sarah E , Shepherd John R.A. , Wiggins Chad C. , Joyner Michael J. , Dominelli Paolo B.
Published: April 2020

Objective

Bronchopulmonary dysplasia (BPD) is a condition of neonatal chronic lung disease due to disruption or dysregulation of pulmonary development. It is associated with decreased septation, alveolar hypoplasia, and abnormal disruption of pulmonary alveolar capillaries resulting in increased pulmonary resistance. However, the pathophysiology of BPD in the large conducting airways is not fully understood. The objective of our study was to determine if the luminal area of the central airways, measured by imaging, are altered in patients with a history of BPD. We hypothesized that compared to age‐ and sex‐matched controls, BPD patients would have a decreased luminal area of the central conducting airways and lung volume.

Methods

Fourteen BPD patients (8M/6F; Age=5.7±5.5 yrs) who had undergone a chest computed tomography (CT) scan were retrospectively identified. Age and sex matched controls (8M/6F, Age=5.6±5.3 yrs) who had undergone a CT scan for trauma were used for comparison. Measurement and analysis were performed by a single investigator using commercially available software (Aquarius, TeraRecon Inc.) that reconstructs the airways into 3D. Measurements of luminal airway area were conducted at three points based on anatomic bifurcations (corresponding to the beginning, middle and end of the airway) for each of the following structures: trachea, left main bronchus, left upper lobe, left lower lobe, right main bronchus, intermediate bronchus, and right upper lobe. Each airway’s luminal area was calculated based on the averages of the three measures obtained for each anatomic airway structure. Independent Student’s t‐test was used for statistical analysis.

Results

Airway luminal area was not different between BPD patients and matched controls (trachea: 89.8±57.1 vs 87.4±58.6 mm2; right main bronchus: 58.8±40.8 vs 64.0±44.5 mm2; intermediate bronchus: 33.6±21.2 vs 41.0±28.9 mm2; left main bronchus: 39.3±27.1 vs 46.6±36.5 mm2; left lower lobe: 22.7±14.6 vs 29.9±18.9 mm2; p>0.05 for all comparisons; BPD and controls, respectively). Total lung volume detected in the CT scans was not different between BPD patients and matched controls (3069±1073 vs 3215±984 cm3, p>0.05).

Conclusions

Our results suggest the luminal areas of the large conducting airways in patients with BPD are not different from matched controls. Given the degree of respiratory complications and sequelae associated with BPD, our findings suggest future research should focus on alveolar and vascular impaired physiological and anatomic developments versus the large conducting airways.

Support or Funding Information

National Institutes of Health Grants R35‐HL139854 (MJJ), F32 HL‐131151 (SEB), T32 DK‐007352‐39 (CCW) and Mayo Clinic Internal Funding and Natural Science and Engineering Council of Canada RGPIN‐2019‐04615 (PBD)
ACKR3 Regulates Endothelial Cell Function with Non‐canonical Integration of Gαi and β‐arrestin
Authors: Lee Claudia Y. , Decker Ann M. , Xiong Xinyu , Viswanathan Gayathri , Kohlmann Taylor , Smith Jeffrey S. , Inoue Asuka , Namjoshi Ojas A. , Rajagopal Sudarshan
Published: April 2020

Atypical Chemokine receptor 3 (ACKR3), also known as C‐X‐C chemokine receptor type 7 (CXCR7), is a G protein‐coupled receptor (GPCR) implicated in several physiological processes including leukocyte trafficking, cancer, and angiogenesis. Commonly co‐expressed with C‐X‐C chemokine receptor type 4 (CXCR4), ACKR3 has been shown to be unable to activate G protein pathways and appears to function as a “β‐arrestin‐biased” receptor. Furthermore, ACKR3’s ability to efficiently internalize its ligands, CXCL12 and CXCL11 (which are also ligands for the receptors CXCR4 and CXCR3, respectively), and degrade them, resulting in some of its functions through a “scavenger” or “decoy” mechanism. However, much is still unknown about the bona fide signaling mechanisms by ACKR3 and its implications on cellular physiology. Here we describe ongoing studies to characterize the mechanisms that underlie ACKR3 on endothelial cell function. We demonstrate in vitro and in vivo activity of two ACKR3 specific agonists, WW36 and WW38, that bind ACKR3 with nanomolar affinities, promote β‐arrestin recruitment, and promote proliferation and migration of human umbilical vein endothelial cells (HUVECs). Surprisingly, we found that ACKR3‐promoted proliferation and migration was sensitive to pertussis toxin, suggesting the involvement of Gαi/o proteins at a receptor which does not canonically signal via G proteins. We hypothesize that these results may in part be explained by the recently described coordination of Gαi and β‐arrestin to promote Gαi:βarr complex‐specific downstream signaling. Confirming this potential mechanism, we demonstrate that stimulation of ACKR3 promotes formation of a Gαi:βarr complex in a dose dependent manner. These studies reveal a potentially novel role for Gαi:βarr complexes in mediating ACKR3 function that contribute to endothelial cell biology. Additionally, these findings support an emerging paradigm in which G proteins and β‐arrestins coordinate their signaling downstream of GPCRs.

Support or Funding Information

NIH (CYL, SR)
Measuring Cardiac Troponin I Phosphorylation in Viable Primary Cardiomyocytes
Authors: Jay Anthony G. , Han Young S. , Sieck Gary C.
Published: April 2020

Cardiac troponin I (cTnI) is a key regulator of cardiomyocyte contraction. Phosphorylation of cTnI at Ser22/23 is mediated by protein kinase A, C, and G, and is associated with reduced Ca2+ sensitivity of myocardial contraction. In previous in vivo and in vitro studies, we showed that hypothermia/rewarming (rewarming shock) reduces the Ca2+ sensitivity of myocardial contraction due to cTnI Ser22/23 phosphorylation. Rewarming shock was also found in isolated cardiomyocytes; however, demonstrating a relationship with cTnI phosphorylation is complicated by varying cardiomyocyte viability after isolation. Using a modified Langendorff technique, the cardiomyocyte viability gradually declines over a 4‐h period from ~80% to ~40%. This decline in cell viability is not accounted for in typical Western blot measurements and, therefore, assessments of cTnI phosphorylation are highly subject to misrepresenting actual physiological changes in cTnI phosphorylation. To address this issue, we developed a novel quantitative technique to measure cTnI phosphorylation at Ser22/23 in viable cardiomyocytes using confocal microscopy. We demonstrated that cardiomyocytes with a rod‐shaped morphology and uniform striation patterns display robust transient contractile and cytosolic Ca2+ responses to electrical stimulation. All of these rod‐shaped cardiomyocytes exclude trypan blue, while a disproportionate fraction of non‐rod‐shaped cardiomyocytes are non‐viable. Separately, using 2D gel electrophoresis, we validated the specificity of an antibody that targets phosphorylated cTnI. Using this antibody, we verified that the extent of cTnI phosphorylation in rod‐shaped cardiomyocytes increased with isoproterenol in a dose dependent fashion. This novel methodology allows a more accurate measurement of cTnI phosphorylation in isolated primary cardiomyocytes that is not affected as cell viability declines over time.

Support or Funding Information

This publication was made possible by Grant NumberT32 HL 105355 from the National Heart, Lung and Blood Institute (NHLBI), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIH.
Exogenous IL‐4 Promotes Myocardial Infarction Repair by Turning off Pro‐Inflammation in Neutrophils while Stimulating Anti‐Inflammation in Macrophages to Induce Neutrophil Phagocytosis
Authors: Tenkorang-Impraim Mavis Akosua Asantewaa , Ma Yonggang , Chalise Upendra , Daseke Michael J. , Konfrst Shelby R. , Garrett Michael R. , DeLeon-Pennell Kristine Y. , Lindsey Merry L.
Published: April 2020

Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While IL‐4 is an in vitro stimulus for anti‐inflammation, the MI myocardium does not express IL‐4. We hypothesized that continuous exogenous IL‐4 infusion starting 24 h after MI would promote a polarization switch in inflammatory cells towards a reparative phenotype. C57BL/6J male mice (3–6 months of age) were subcutaneously infused with either saline (n=17) or IL‐4 (20 ng/g/day; n=17) and evaluated at MI day 3. Macrophages and neutrophils were isolated ex vivo from the infarct region and examined for pro‐inflammatory genes (Ccl3, Ccl5, Il1b, Il12a, and Tnfa) and anti‐inflammatory genes (Arg1, Ym1, Mrc1, Il10, and Tgfb, Il6). Exogenous IL‐4 decreased pro‐inflammatory Ccl3, IL‐12a, Tnfa, and Tgfb1 in neutrophils while increasing anti‐inflammatory Arg1 and Ym1 in macrophages (all p<0.05); none of the other markers were different. Tissue clearance by IL‐4 treated neutrophils was not different, while phagocytosis of neutrophils, but not myocytes, doubled in IL‐4 treated macrophages (p<0.05). Because macrophage cell physiology was influenced by IL‐4, whole transcriptome analysis of macrophages isolated from the infarct regions of IL‐4 (n=10) or saline (n=11) treated mice was performed by RNA‐sequencing. Of the 24,341 genes sequenced, 2,042 genes were differentially expressed with IL‐4 stimulation (all p<0.05). Pdgfc gene expression was ranked first by p value, increasing from 1.8±0.3 in saline treated macrophages to 5.4±0.2 FPKM units in macrophages stimulated with IL‐4 (p=1×10−9). Importantly, changes in macrophage physiology and transcriptome occurred before differences in cardiac physiology evaluated by echocardiography, which allowed the isolation of effects in cell phenotype not induced by global myocardial effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF‐CC protein (10 μg/ml) or PDGF‐CC blocking antibody (10 μg/ml) revealed no effect on Arg1 or Ym1 expression, indicating the in vivo effect of IL‐4 to stimulate macrophage gene expression was independent of PDGF‐CC. Altogether, our results indicate that exogenous IL‐4 promotes inflammation resolution by turning off pro‐inflammation in neutrophils while stimulating anti‐inflammation in macrophages to induce neutrophil phagocytosis.

Support or Funding Information

National Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505
Nigella Sativa Seed Oil Effectively Attenuates Cyclophosphamide‐Induced Myelosuppression in Mice
Authors: Saad Shahad Ammar , Kathem Sarmed H.
Published: April 2020

Myelosuppression is one of the serious adverse effects of cancer chemotherapy that lead to life threatening febrile neutropenia and considered a limiting factor for successful therapy. Cyclophosphamide (Cyc), a widely used anticancer drug, induces severe myelosuppression by damaging hematopoietic stem cells. As cancer incidence expands globally, the demand for an effective myeloprotective therapy during cancer treatment is also increasing. Nigella sativa seed oil (NG oil), a well‐known plant extract that widely used for various health conditions. This study aims to evaluate the myeloprotective activity of NG oil in cyclophosphamide‐induced myelosuppression mice model. Myelosuppression induced by single IP injection of Cyc (200 mg/kg). Animals were divided into 4 groups each with 6 mice. First group served as negative control group received only normal saline. A second group served as experimental myelosuppression model group achieved by Cyc. Additional 2 groups were mice received NG oil (1ml/Kg/day) or (2ml/Kg/day) orally for 6 consecutive days starting day 1 with Cyc on day 3. On day 7, blood were collected from retro–orbital area for total and differential leukocytes counts. were collected from retro–orbital area for total and differential leukocytes counts. Compared to model group, we reported that NG oil (1ml/Kg) significantly increases total leukocytes count (1000±73 vs. 700±36 cell/μL), lymphocyte count (868±63 vs. 580±40 cell/μL), spleen index (1.60±0.11 vs. 1.25±0.025 mg/gm), bone marrow cells viability (50.17±5.36 vs. 9.5±0.76%) and bone marrow GM‐CSF (14.50±1.42 vs. 9.18±1.84 ng/l), while non significant change observed in neutrophil count and thymus index. Furthermore, increasing NG oil dose to (2ml/Kg) resulted in further improvement, were we reported a significant increase in total leukocytes count (1267±352 vs. 700±36 cell/μL), neutrophil count (279.67±90 vs. 95.5±8.4 cell/μL), lymphocyte count (1047±298 vs. 580±40 cell/μL), spleen index (2.35±0.49 vs. 1.25±0.025 mg/gm), thymus index (1.62±0.31 vs. 0.79±0.13 mg/gm), bone marrow cells viability (67.67±5.49 vs. 9.5±0.76%), and bone marrow GM‐CSF (16.18±1.50 vs. 9.18±1.84 ng/l). Bone marrow H&E histopathological sections confirm the myeloprotective effects of NG oil. In conclusion, we showed that NG oil has a promising strong myeoprotective and immunomodulatory effects against cyclophosphamide‐induced myelosuppression.

Support or Funding Information

Funding: University of Baghdad
γ‐T3 and α‐TEA reduce the amount of docetaxel required to decrease cell viability in human prostate cancer cells and enhance the efficacy of docetaxel in the treatment of drug‐resistant cells
Authors: Asay Spencer , Graham Andrew , Rankin Mason , Hollingsworth Sydney , Lindstrom Elijah , Burke Lexady , Barnes Bradley , Oblad Richard , Michaelis David , Kenealey Jason
Published: April 2020

Prostate cancer is the second most commonly diagnosed cancer in men, and metastatic prostate cancer is currently incurable. Prostate cancer frequently becomes resistant to standard of care treatments. Moreover, the administration of conventional chemotherapeutic drugs, such as docetaxel (DOC), poses the risk of debilitating toxic side effects. Combination chemotherapy, in which several compounds targeting multiple cellular pathways are administered jointly, is one tool that can be used to combat therapeutic resistance and drug toxicity. Vitamin E (VE) compounds and analogs have been proposed as potential non‐toxic chemotherapeutics. We modeled combination therapy using mixture design response surface methodology (MDRSM), a statistical technique designed to optimize mixture compositions, to determine whether combinations of three chemotherapeutic agents (γ‐tocotrienol (γ‐T3), α‐tocopherol ether acetate (α‐TEA), and DOC) would prove more effective than DOC alone in the treatment of PC‐3 and DU145 human prostate cancer cells. A response surface was generated for cell viability, and the optimal treatment combination for reducing PC‐3 cell viability was calculated. We found that a combination of 30 μM α‐TEA, 20 μM γ‐T3, and 25 nm DOC was most effective in the treatment of PC‐3 cells. We also found that combining γ‐T3 and α‐TEA with DOC decreased the dose of DOC required to significantly reduce cell viability in PC‐3 and DU145 cells. Further, combination treatment consisting of α‐TEA, γ‐T3, and DOC induced apoptosis in PC‐3 cells as evidenced by upregulation of caspases 3 and 7. Finally, we found that combining γ‐T3 and α‐TEA with DOC enhanced treatment efficacy in DOC‐resistant PC‐3 cells.

Support or Funding Information

Brigham Young University College Undergraduate Research Award

Bryant Adams
DNA binding specificity of cardiac transcription factor complex GATA4 and NKX2‐5
Authors: Rios Jessica Marie Rodriguez , Rodríguez Emili Patricia Rosado , Martínez José Arcadio Rodríguez
Published: April 2020

Transcription factors (TFs) are sequence‐specific DNA binding proteins responsible for cellular differentiation and development. Eukaryotic TFs are notorious for not working alone and they often bind DNA as multiprotein complexes to regulate gene expression, raising the possibility that complex formation might change their individual DNA binding specificities. Transcription factors GATA4 and NKX2‐5 are central components of the gene regulatory network that controls heart development. However, the DNA‐binding specificity of the cooperative complex between GATA4:NKX2‐5 remain undetermined. We want to identify the intrinsic DNA binding preferences and the emergent properties that result from this complex. In this study, we expressed full‐length GST‐Tagged GATA4 and NKX2‐5 using wheat germ cell‐free system. We validated DNA‐binding activity of GATA4 and NKX2‐5 through Electrophoretic Mobility Shift Assays (EMSA). We successfully determined the in vitro DNA‐binding specificity of GST‐GATA4, GST‐NKX2‐5 and GST‐GATA4:GST‐NKX2‐5 complex using Cognate Site Identification by High‐Throughput Systematic Evolution of Ligands by Exponential Enrichment (SELEX‐seq). DNA‐specificity models for GATA4, NKX2‐5 and GATA4:NKX2‐5 complex were generated using various computational tools. Specificity models will be used to make genome‐wide binding predictions. Findings of this study will help understand the spatial and temporal gene regulation rules involved in normal heart development.

Support or Funding Information

This project is funded by National Science Foundation Bridge to the Doctorate Award HRD‐1400868 and National Institutes of Health SC1GM127231.
Analysis of Platelet Aggregation, Secretion, Integrin Activation, and Calcium Release Stimulated by Different Agonists in Native Americans
Authors: Williquett Brady , Valen Grace E. , Goerger Krista , Clarke Santa , Abbott Karla , Larson Mark K.
Published: April 2020

Arterial thrombosis results from obstructive vascular blood clots that are largely initiated by platelet activation. As such, individuals whose platelets react more robustly to chemical signals generated by vascular damage or dysfunction are at a higher risk for morbidity and mortality caused by thrombosis. As a population, Native Americans have higher rates of obstructive arterial diseases, and previous research suggests African American populations have higher overall platelet reactivity that positively correlates to their higher risk for cardiovascular disease. Therefore, we set out to measure platelet reactivity in Native Americans and identify potential genetic alleles that correlate to the elevations in responsivity. Five platelet agonists were utilized to simulate vascular damage signals, followed by measurements of markers indicative of subsequent platelet activation: aggregation, secretion, integrin activation, and calcium ion mobilization. Preliminary results from 17 subjects showed that Native American platelets more rapidly aggregate in response to several agonists when compared with Caucasians, whereas other measures of platelet function are largely similar between the groups. These results suggest that Native Americans have more sensitive platelet aggregation responses, which could result in a higher risk for occlusive arterial diseases. Complete genomic sequences were also obtained from the highest responders for use in identifying alleles that correlate to this response.

Support or Funding Information

Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443
Cancerous Phenotypes Exhibited by NIH/3T3 Fibroblasts Overexpressing BST‐2
Authors: Alvarez Folnetti , Okeoma Chioma
Published: April 2020

Oncogenesis is a multistep process initiated by the accumulation of genetic and/or epigenetic changes that culminate in the transformation of normal cells into cancer cells. The genetic and/or epigenetic changes result in dysregulated gene expression signature that may involve key cancer driving genes such as Bone Marrow Stromal Antigen 2 (BST‐2). In this study, we sought to test the hypothesis that BST‐2, a type II transmembrane protein regulates early events of oncogenesis. BST‐2 is a multifaceted protein with both antiviral and proviral functions and has been implicated in promoting aggressive cancer cell behavior such as migration and invasion. Meta‐analysis of breast cancer datasets from The Cancer Genome Atlas (TCGA) show a statistically significant correlation between elevated BST‐2 expression and poor survival. In addition to its role in breast cancer progression, elevated BST‐2 expression correlates with poor prognosis in endometrial, brain, lung, and oral cavity cancers, further implicating elevated BST‐2 expression in cancer progression. However, the role of BST‐2 in regulating early oncogenic events is not well understood. Our approach consisted of stably overexpressing exogenous human BST‐2 in NIH/3T3 mouse embryonic fibroblasts that express very low endogenous mouse BST‐2 to generate 3T3BST‐2 cells. The control cells were stably transfected with pCDNA3.1 to generate 3T3pCDNA cells. The cells were tested for phenotypes associated with early oncogenic events under two‐dimensional (2D) and three‐dimensional (3D) culture conditions. In a confluent 2D monolayer, 3T3BST‐2 cells compared to 3T3pCDNA cells resisted contact inhibition and had enhanced growth and survival that is associated with increased phosphorylated protein kinase AKT (T308), increased phosphorylated mitogen‐activated protein kinase (ERK1/2), and decreased pro‐apoptotic factor Bak. In a 3D spheroids model, 3T3BST‐2 spheroids were larger in area and had higher ATP content, an indication of anchorage‐independent survival. Suspended as single cells in a gel, 3T3BST‐2 cells also had enhanced survival, especially when arginylglycylaspartic acid (RGD) adhesive peptide motif was present. Suppressed contact inhibition and enhanced anchorage‐independent survival suggests that overexpression of BST‐2 contributes to malignant transformation of normal cells. Discerning the mechanisms of BST‐2 mediated oncogenesis will expand our understanding of events that transforms a normal cell into an unwanted cancerous cell, which may potentially form the foundation for the development of BST‐2‐targeted cancer therapies.

Support or Funding Information

1. Molecular and Cellular Pharmacology PhD program 2. CIE’s IMSD‐MERGE scholars program 3. Scholars in Biomedical Sciences program (SBMS) 4. Stony Brook University Cancer Center
Canagliflozin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration
Authors: Durante Giovanna L. , Khanna Yash P. , Behnammanesh Ghazaleh , Peyton Kelly J. , Durante William
Published: April 2020

Cardiovascular disease is the major cause of morbidity and mortality in diabetes. Although numerous factors contribute to the development of diabetes‐associated cardiovascular disease, excessive proliferation and migration of vascular smooth muscle cells (SMCs) plays a critical role. Sodium‐glucose cotransporter 2 (SGLT2) inhibitors are the latest approved class of glucose lowering drugs. By blocking glucose uptake in the proximal tubule of the kidney, SGLT2 inhibitors induce glycosuria leading to decreases in fasting and postprandial glycemia. Notably, large multicenter clinical trials have demonstrated that SGLT2 inhibitors reduce cardiovascular disease and mortality in patients with type 2 diabetes. The mechanism underlying the cardiovascular benefits of SGLT2 inhibitors is not fully known; however, direct effects on vascular SMCs have not been considered. In the present study, we investigated the effect of three different SGLT2 inhibitors on vascular SMC function. Treatment of rat and human aortic SMCs with canagliflozin resulted in a potent, concentration‐dependent inhibition of cell proliferation and DNA synthesis. The anti‐proliferative effect of canagliflozin was observed at clinically relevant concentrations, and this was not seen with other SGLT2 inhibitors, including empagliflozin and dapagliflozin. The inhibition of SMC growth by canagliflozin occurred in the absence of cell death and was associated with the arrest of SMCs in the G0/G1 phase of the cell cycle. In addition, scratch‐wound assays found that vascular SMC migration was blocked by canagliflozin in a concentration‐dependent manner, whereas empagliflozin or dapagliflozin had no effect on SMC motility. In conclusion, the present study identified canagliflozin as a robust inhibitor of vascular SMC proliferation and migration. These inhibitory effects of canagliflozin are detected at pharmacologically relevant concentrations and are unique for this drug and not seen with other SGLT2 inhibitors. The ability of canagliflozin to exert these pleiotropic effects on vascular SMC function may contribute to the salutary cardiovascular actions of this drug in patients with type 2 diabetes.

Support or Funding Information

Supported by American Diabetes Association grant #1‐17‐IBS‐290.
A MAP kinase‐driven mechanical stress response mediates the hypertrophic response in skeletal muscle
Authors: Nordgaard Cathrine , Blasius Melanie , Vind Anna Constance , Reinert Marie Sofie , Snieckute Goda , Tiedje Christopher , Bekker-Jensen Simon
Published: April 2020

When cells are exposed to unfavorable conditions they cope by activating cellular stress responses. Such signaling depends on activation of the p38 and JNK MAP kinases and is crucial for both general tissue homeostasis and a range of physiological reactions. However, the mechanisms linking diverse stress stimuli to MAP kinase activation remain poorly understood.

Here we show how a specific MAP3 kinase mediates activation of p38 and JNK upon hyperosmotic‐ and mechanical stress. Employing fluorescence microscopy techniques, we show that this MAP3 kinase translocates from the cytoplasm to the nuclear lamina and other detergent‐resistant structures in the nucleus upon osmotic stress induction. Here, it co‐localizes with structures rich in DNA damage response factors such as 53BP1, but void of DNA strand breaks, which were shown to constitute nuclear sensor domains for mechanical stress.

Mechanical stress is experienced in skeletal muscle due to the repeated contraction of muscle fibers upon physical exercise. The signaling from these events underlie the hypertrophic response, where muscles respond to exercise by growing larger and stronger. We show that the above signaling pathway operates during exercise‐associated stimulus of C2C12 myotubes, where it is responsible for the production of a range of inflammatory transcripts and myokines (such as IL6). Consequently, in a mouse model deficient for the MAP3 kinase, we see clear signs of pathology associated with decreased muscle repair and adaptation to exercise.

In conclusion, our work provides novel insight into the mechanisms by which cells sense and react to mechanical stress stimuli and identify skeletal muscle as a major target organ upon deficiency in these responses.
TRPC3 is a critical contributor to the osmosensitivity and renal water handling in the mouse collecting duct
Authors: Tomilin Viktor N. , Mamenko Mykola , Zaika Oleg , Ren Guohui , Pochynyuk Oleh
Published: April 2020

Kidney plays a central role in control of systemic water balance depending on hydration status. Arginine vasopressin (AVP) augments water permeability of the collecting duct (CD) by facilitating aquaporin 2 (AQP2) translocation to the apical plasma membrane. Inability of CD to respond to AVP signal causes Nephrogenic Diabetes Insipidus (NDI) leading to polyuria, dehydration and thirst. AVP stimulates CD to reabsorb water, but this can only occur in presence of positive osmotic difference between the cytosol and luminal fluid. Extracellular hypotonicity increases [Ca2+]i and causes cell swelling due to AQP2‐driven water influx. The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remains unknown. TRPC3 is a Ca2+ permeable mechano‐activated channel abundantly expressed in the CD. Using ratiometric Fura 2‐based [Ca2+]i imaging, we simultaneously measured [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2‐dependent water reabsorption in freshly isolated split‐opened CDs of wild type and TRPC3 −/− mice and combined it with immunofluorescent detection of AVP‐induced AQP2 trafficking and assessment of systemic water balance. We found that TRPC3 inhibition or deletion precluded [Ca2+]i elevations induced hypotonicity and remarkably slowed the rate of cell swelling indicative of diminished water transport in the CD. TRPC3 −/− mice had comparable with WT serum and urine osmolarity in the presence of ad libitum water intake, but exhibited a significantly greater bodyweight loss, and larger urinary volume after 24 h water deprivation despite higher AVP levels when compared to WT. Furthermore, osmosensitive [Ca2+]i elevations were greatly increased in CDs from WT but not in TRPC3 −/− animals after 24 h water deprivation. The rate of cell swelling was greatly accelerated in WT, while was only modestly increased in TRPC3 −/− mice under this condition. Using immunofluorescent microscopy, we found that AQP2 translocated to the apical plasma membrane in WT, while remained largely cytosolic in TRPC3 −/− after 24 h water deprivation. Consistently, TRPC3 inhibition interferes with AVP‐induced AQP2 trafficking in cultured mpkCCDc14 cells. In summary, we demonstrate a significant role of TRPC3 in osmosensitivity and regulation of AVP‐dependent AQP2 trafficking in the CD. TRPC3 deletion compromises systemic water balance producing an NDI‐like phenotype.

Support or Funding Information

American Heart Association Career Development Award for Viktor Tomilin (19CDA34660148)

NIH RO1 awards for Oleh Pochynyuk (DK117865 and DK119170)
Integration of Gross Anatomy Laboratory sessions into Medical Physics Curriculum using Segmentation and Augmented‐Reality
Authors: Noel Geoffroy P.J.C. , Kang Esther S.H. , Popovic Marija
Published: April 2020

Anatomy teaching to Medical Physics (MP) trainees is recognized by the Commission on Accreditation of Medical Physics Education Programs, and yet anatomy laboratory sessions have not been integrated in MP training curricula. This study outlines the development of anatomy laboratory sessions for MP trainees and serves as the initial step to developing an anatomical curriculum guideline for MP. The two objectives of this qualitative study are: (a) to explore the educational potential of integrated anatomy laboratory sessions in the MP curriculum and (b) to evaluate the benefits of interprofessional education activities between MP trainees and Radiation Oncology residents.

Participants included 17 MP graduate students, 2 MP residents and 8 Radiation Oncology residents, over two years. Two 2‐hour anatomy laboratory sessions per year were organized following the respective in‐class lectures on the pelvis, head and neck and thorax. After the two laboratory sessions, participants were asked to fill out a survey reflecting on their experience in the laboratory.

Both MP students and radiation oncology residents voiced an appreciation for the anatomy laboratory sessions for the consolidation and application of knowledge. Most mentioned that these sessions helped to “visualize” and “have a better understanding” of the anatomy taught in class and made them more “confident”. In terms of interprofessionalism, both the MP and the radiation oncology trainees expressed a better understanding of one another’s level of knowledge in anatomy. Both groups suggested to have more interprofessional sessions. They also suggested to build a stronger link between cadaveric anatomy and imaging anatomy through augmented reality technology.

The integration of anatomy laboratory sessions into the anatomy curriculum for MP enhances trainees’ understanding of human anatomy and its pivotal role in radiation therapy treatment. Furthermore, interprofessional activities prior to clinical placement of MP students reinforce technical and professional communication.

Support or Funding Information

The authors would like to thank the support provided by the Centre for Medical Education Innovation and Research Seed Fund (to GPJCN).
Dendritic Cell Specific Jak2 Expression plays a role in in Salt‐Sensitive Hypertension through an ENaC‐dependent Mechanism
Authors: Aden Luul A. , Pitzer Ashley A. , Grimes Samantha , Ray Evan , Kleyman Thomas , Kirabo Annet
Published: April 2020

Salt‐sensitivity of blood pressure affects 50% of hypertensive and 25% of normotensive individuals and is an independent predictor of death due to cardiovascular disease. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase‐dependent formation of immunogenic isolevuglandin (IsoLG)‐protein adducts leading to inflammation and salt‐sensitive hypertension. We hypothesized that Jak2 expression in dendritic cells regulates expression of ENaCγ and promotes salt‐sensitive hypertension. Mice lacking Jak2 in CD11c+ cells developed blunted hypertension (123.8±4.7) during the high salt feeding phase of the N‐Nitro‐L‐arginine methyl ester hydrochloride (LNAME)/high salt model of salt‐sensitive hypertension compared to their wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention (69.55±5.8) in response to high salt‐feeding when compared to the wildtype litter mate controls (57.89±9.5). We also found that deletion of Jak2 in dendritic cells reduced the salt‐induced expression of ENaCγ in CD11c+ cells. Following high salt feeding, mice lacking Jak2 in DCs exhibited less aortic infiltration of CD11c+ cells with less expression of CD86 and production of IsoLGs. These results indicate that dendritic cell Jak2 plays a role in salt‐sensitive hypertension through an ENaC‐dependent mechanism.

Support or Funding Information

K01HL130497R01HL147818T32HL144446
Pharmacokinetic studies of moracin C in mice using various blood sampling methods
Authors: Choi Young-Hee , You Byung-Hoon , Chin Young-Won
Published: April 2020

Mice have been increasingly used in pharmacokinetic evaluation due to using less amount of compound and availability of various disease models and blood sampling methods in mice have produced the distinct pharmacokinetic profiles even in the same compounds. In this study, three blood sampling methods [i.e., automated dosing/blood sampling (ABS), tail artery collection (TA) and heart puncture (HP)] were applied to investigate the pharmacokinetics of moracin C in mice. Depending on blood sampling sites, significant differences were observed in plasma concentrations as well as pharmacokinetic parameters such as AUC (area under the plasma‐time concentration curve). The AUC values in ABS and TA groups were smaller than that in HP group. Considering that moracin C was greatly distributed to liver and gastrointestinal tracts (e.g., stomach, small intestine, and large intestine) and was extensively metabolized via glucuronidation in liver and small intestines, the AUC differences could be mainly caused by the stronger stress from blood sampling via heart puncture, which might interfere the elimination (i.e., hepatic and/or intestinal first‐pass effects). The remaining amounts of moracin C in gastrointestinal tract at 24 h after its oral administration were similar among three groups with different blood sampling methods, indicating that the absorption of moracin C was not affected during the blood sampling stress of HP. These results indicate that the ABS or TA method can be utilized to obtain better reliable and accurate pharmacokinetic parameters in mice.

Support or Funding Information

This study was supported by National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (NRF‐2016R1C1B2010849, NRF‐2019R1A2C2009053 and NRF‐2018R15A2023217).
Biomechanical and biophysical properties as diagnostic markers in breast cancer progression in diabetic conditions
Authors: Ortega Jose , Schmidt Roman , Capellen Caleb , Dutta Diganta , Chandra Surabhi
Published: April 2020

Cancer is the uncontrollable and continuous proliferation of cells. Hyperglycemia has been known to be a source for increasing cancer cell proliferation and furthering metastasis through a phenomenon known as Warburg effect. It is understood that mechanical properties may influence a cell’s overall functionality. Cancer cells have been shown to have altered cytoskeletal structure compared to their normal cell counterparts. It is hypothesized that diabetic conditions affect the cellular mechanics of breast epithelial cancer cells. Mechanical properties of cells involve mobility, invasiveness, deformability, and cytoskeletal filament organization. Cell lines used in this study include MCF‐7 (early‐stage breast cancer), MDA‐MB‐231 (late‐stage breast cancer cells) and MCF‐10A (normal breast epithelial cells). Cells were treated with low glucose (LG, 5mM) or high glucose (HG, 25mM) for varying time periods (24–72 h). The mechanical properties were assayed using single‐cell atomic force microscopy (AFM). The elastic modulus quantification of breast cancer cells based on results from AFM showed a distinct physical difference versus the normal breast epithelial cells. Differences in cytoskeletal organization will be further determined by visualization of actin filaments under various treatments. Cell mobility will be assayed using microfluidic devices fabricated to apply an alternating current flow to exploit the electrophoretic and electroosmotic properties under hyperglycemic conditions. Results of cellular biomechanics and cellular mobility under applied voltage in cells treated with glucose can thus potentially serve as a basis to prepare a diagnostic device for efficient, economical, and rapid diagnosis and prognosis of cancer.
A nigro‐vagal pathway controls colonic motility and may be impaired in a model of environmental Parkinson’s disease
Authors: Nanni Giorgia , Travagli R. Alberto
Published: April 2020

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor deficits caused by the loss of substantia nigra pars compacta (SNpc) dopaminergic neurons. It is well known that the majority of PD patients experience gastrointestinal (GI) dysfunctions such as gastroparesis, impaired swallowing and severe constipation. Upper GI tract functions are modulated by vagal reflexes, the central neurocircuit of which are located in the dorsal vagal complex (DVC, i.e. dorsal motor nucleus of the vagus (DMV), nucleus tractus solitarius and area postrema). We discovered a monosynaptic nigro‐vagal pathway recently that modulates gastric tone and motility and is impaired in an environmental PD model induced by oral administration of subthreshold doses of paraquat (P) and lectin (L). It is not known, however, whether this pathway also modulates colonic motility and whether the P+L model of PD decreases intestinal transit.

Proximal colonic motility and tone were recorded with a strain gauge aligned with the colonic circular smooth muscle, approximately 3 cm distal to the ileal‐cecal valve. Microinjections of the ionotropic glutamate selective agonist, NMDA, were made at the level of the SNpc (5nmoles/210nL) and the effects on colonic motility and tone assessed. Microinjection of NMDA, but not vehicle, increased colonic tone (472±54mg) and motility (+358±166% of baseline; N=2).

Gastro‐cecal transit, an indicator of intestinal motility, was measured using the 13C lactose ureide‐ breath test. Female rats had a slower gastro‐cecal transit time (178±24 min, N=4) than male rats (138±1.5 minutes, N=3). Treatment with P+L delayed gastro‐cecal transit time to 272±18 minutes (N=3).

These data suggest that the nigro‐vagal pathway also modulates colonic motility and the P+L model of PD delayes intestinal transit and thus may be a useful model to study PD‐related constipation.

Support or Funding Information

supported by NIH grant DK‐55530 to R. Alberto Travagli
Nox4‐mTORC1 connection in salt‐induced hypertension and kidney injury in Dahl S rats
Authors: Kumar Vikash , Geurts Aron , Kurth Theresa , Zheleznova Nadezhda N. , Yang Chun , Cowley Allen W.
Published: April 2020

Chronic treatment of Dahl salt‐sensitive (SS) rats with rapamycin which specifically inhibits mTORC1 (mammalian target of rapamycin complex 1) was found to blunt the chronic phase of hypertension, kidney injury, immune cell infiltration, renal hypertrophy, and cell proliferation in high‐salt (4.0% NaCl ) fed SS rats. Many of the upstream regulators of mTORC1 are known including amino acids, insulin/insulin‐like growth factor, and cellular energy. In SS rats, high‐salt intake is known to increase the renal production of reactive oxygen species (ROS) from the mitochondria and from NADPH oxidases (NOXs), especially NOX4 which is the most abundant NOX isoform in the kidney. NOX4 exclusively releases H2O2 and we recently reported that a chronic 3‐day renal interstitial infusion of H2O2 activates mTORC1 in vivo as evidenced by phosphorylation and activation of the ribosomal protein S6 (pS6S235/236/S6). In the present study, we hypothesized that activation of NOX4 and elevations of H2O2 in the high‐salt fed SS rat stimulate mTORC1 leading to renal injury. Western blot analysis found that mTORC1 activity was significantly higher in SS rats fed a high‐salt diet which also have high levels of H2O2 compared to SS‐NOX4 knock out rats (SSNox4−/−). SSNox4−/− rats with reduced H2O2 exhibited reduced level of mTORC1 activity (e.g., reduced phosphorylation of ribosomal protein S6 kinase 1, ribosomal protein S6) and mean arterial pressure (MAP) compared to SS rats fed a 4.0% NaCl diet for 21 days. It was also found in vitro that H2O2 activation of mTORC1 was direct and independent of phosphoinositide 3‐kinase (PI3K). Rapamycin treatment of SSNox4−/− rats fed a 4.0% NaCl diet for 21 days somewhat further reduced the already low levels of mTORC1 activity and immune cell infiltration (CD68, CD3), although no further reduction of MAP or renal injury was observed compared to vehicle treated SSNox4−/− rats. We conclude that H2O2 produced by NOX4 is a major upstream regulator of mTORC1 independent of PI3K activity and contributes importantly to blood pressure salt‐sensitivity and renal injury in SS rats.

Support or Funding Information

HL‐116264
G Protein‐coupled Estrogen Receptor Protects Against Aging‐Induced Vascular Dysfunction in Females but Not Males.
Authors: Zimmerman Margaret , Ogola Benard , Lindsey Sarah
Published: April 2020

G protein‐coupled estrogen receptor (GPER) promotes the vasoprotective effects of estrogen, and we previously published that resistance vessels from middle‐aged female rodents are less responsive to selective GPER activation compared with adult vessels. Therefore, we hypothesized that aging or global GPER deletion would induce similar dysfunction in the vasculature. Mesenteric resistance arteries were isolated from adult (21–25 weeks old) and middle‐aged (52–57 weeks old) male and female wildtype (WT) and GPER knockout (KO) mice. Vessel reactivity was measured via wire myography in response to increasing doses of vasoconstrictors phenylephrine (PE) and prostaglandin F2α (PGF2α) and vasodilators acetylcholine (Ach) and sodium nitroprusside (SNP). PGF2α‐induced constriction was significantly decreased with aging in WT males (446 ± 15% vs. 378 ± 14%; P=0.02) but not in vessels from KO males (434 ± 24% vs. 416 ± 30%; ns). PE‐induced constriction was comparable between male middle‐aged and adult WT (401 ± 19% vs. 416 ± 22%; ns) and KO vessels (416 ± 22% vs. 398 ± 19%; ns). Relaxation to SNP in male animals was comparable between age groups and unaltered by GPER deletion (WT Middle‐Aged: 26 ± 6% vs. Adult: 15 ± 4%; ns; KO Middle‐Aged: 14 ± 3% vs. Adult: 11 ± 2%; ns). However, GPER deletion enhanced relaxation to Ach in middle‐aged males (WT: 42 ± 7% vs. KO: 18 ± 5%; P<0.01) but not adult males (WT: 37 ± 9% vs. KO: 28 ± 12%; ns). In females, PGF2α‐induced vasoconstriction was significantly greater in middle‐aged versus adult WT vessels (446 ± 18% vs. 388 ± 32%; ns) and abolished with GPER deletion (463 ± 12% vs. 413 ± 25%; ns). PE‐induced constriction was comparable between female middle‐aged and adult WT vessels (454 ± 17% vs. 387 ± 18%; ns) and unaltered by GPER deletion (474 ± 25% vs. 412 ± 26%; ns). In females, relaxation to SNP was not altered by aging or GPER deletion (WT Middle‐Aged: 8 ± 1% vs. Adult: 11 ± 3%; ns; KO Middle‐Aged: 13 ± 3% vs. Adult: 20 ± 6%; ns). However, relaxation to Ach was impaired by GPER deletion in adult female vessels (WT: 7 ± 1% vs. KO: 26 ± 10%; P=0.01) but not in middle‐aged vessels (WT: 6 ± 1% vs. KO: 20 ± 5%; ns). The current study demonstrates that the role of GPER in resistance artery function is dependent on sex and age. In males, GPER deletion enhanced vasoconstriction in adult animals but improved endothelial‐dependent vasodilation in middle‐age. In females, GPER deletion was detrimental for endothelial function in adulthood and enhanced vasoconstriction in middle‐age. Therefore, GPER function may contribute to vascular protection in aging women but not men.

Support or Funding Information

This study was funded by NIH grants HL133619 and U54 GM104940.
Identification of potential agonist of human and mouse FFAR2 by homology modeling and molecular docking study approach
Authors: Karunakar Prashantha , Mishra Sidharth P. , Razazan Atefeh P. , Sharma Shailesh , Yadav Hariom
Published: April 2020

The contribution of gut microbiome and its metabolites like short chain fatty acids (SCFAs; such as acetate, propionate, butyrate) became apparent to impact human health. SCFAs not only feed the intestinal cells, but also function as gut microbiome sensors on distinct cell types through activating G‐couple protein receptors like free fatty acid receptor 2 and 3 (FFAR2&3). FFAR2 is widely expressed in intestine, immune cells, adipose tissues, pancreas and others, and plays important role in several human diseases like diabetes, obesity and immune disorders. FFAR2 plays an important role in the regulation of homeostasis and in the intestine immunity. SCFAs are strong agonists of FFAR2, but they are often found decreased in the gut of diseased host, therefore, developing agonists that can enhance FFAR2 activity could of therapeutic importance of several human diseases. In the present study, we screened 125,000 natural compound library with human FFAR2 docking patterns, and selected 14 compounds. These selected compounds were further docked with the homology modeled human and mouse FFAR2 to identify the potential agonist and antagonist using molecular docking approach. The interaction of the ligands with the key residues of human FFAR2 namely His242, Arg180 and Arg255 found to be the determining molecules to identify the agonist and antagonist nature of molecules. Using AutoDock Vina and hydrogen bonding scores, ZINC00388198 showed a potential agonist probability against human FFAR2 and mouse FFAR2. Compound ZINC00968099 and ZINC00403588 showed a potential antagonist probability against human FFAR2 and mouse FFAR2, respectively. Further studies are underway to will demonstrate the direct activation of FFAR2 signaling in cell culture and mouse models of selected molecules, and their biological functions such as anti‐aging and anti‐diabetic effects in C. elegans and mice. Results of these studies will uncover novel natural compounds that can activate FFAR2 to exhibit beneficial functions against several human diseases, associated with microbiota dysbiosis and decreased SCFAs levels.

Support or Funding Information

We thank for the Department of Defense, the National Institutes of Health and resources from Wake Forest School of Medicine.