bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neuroimmune Contributions to T‐lymphocyte‐Driven Inflammation in a Preclinical Model of Post‐traumatic Stress Disorder (PTSD)
Authors: Elkhatib Safwan K. , Moshfegh Cassandra M. , Watson Gabrielle F. , Case Adam J. 
Published: April 2020

Post‐traumatic stress disorder (PTSD) is a devastating psychological disorder with an idiopathic etiology. Interestingly, patients with PTSD have a >3 fold risk of developing comorbid autoimmune diseases compared to matched controls, yet, the exact mechanisms underlying this risk remain unclear. A hallmark of PTSD is increased sympathetic tone, and previous work from our lab has shown that immune cells, primarily T‐lymphocytes, display an autoreactive phenotype when exposed to elevated levels of norepinephrine (NE). Therefore, we hypothesized that the sympathoexcitation observed in PTSD may be causally linked to T‐lymphocyte‐driven inflammation and the predisposition to autoimmune diseases. To test this hypothesis, we adapted a preclinical mouse model of PTSD known as social defeat stress. After validating the PTSD‐like behavioral phenotype of our trauma‐exposed mice, we investigated the neuroimmune interactions of T‐lymphocytes in the spleen that may govern the increased predilection for inflammatory diseases in PTSD patients. The spleen is a primary site of adaptive immune system activation, and is innervated exclusively by the sympathetic efferent splenic nerve. Trauma‐exposed animals exhibited a 2.5 fold increase in splenic catecholamines and 4.1 fold increase in splenic tyrosine hydroxylase (TH) content (p<0.05) compared to controls. Additionally, purified splenic T‐lymphocytes from socially‐defeated mice showed elevations in the pro‐inflammatory cytokines interleukin 6 (IL‐6, 1.84 fold increase, p<0.0001) and interleukin 17A (IL‐17A, 5.5 fold increase, p=0.0032). Importantly, TH content was tightly correlated with T‐lymphocyte IL‐6 and IL‐17A expression (R=0.43, p=0.0001 and R=0.87, p<0.0001, respectively), suggesting a neuroimmune mechanism of pro‐inflammation. To test this, we performed targeted splenic denervation to deduce the contribution of the splenic nerve to T‐lymphocyte‐driven inflammation after psychological trauma. Denervation reduced splenic NE content by 80% (p<0.0001), and persisted to at least 30 days after denervation. While splenic denervation did not alter the PTSD‐like behavioral changes after social defeat, denervation ameliorated the characteristic increase in gene expression of splenic T‐lymphocyte IL‐6 and IL‐17A after social defeat stress. Overall, our data suggest that psychological trauma results in altered inflammation through sympathetic modulation of splenic T‐lymphocytes. Further elucidation of this neuroimmune interaction will provide possible new biomarkers or therapeutic strategies to reduce the inflammatory‐driven morbidity and mortality of PTSD patients.Support or Funding InformationNIH R00HL123471, T32NS105594
The HNF‐1a variant induces endogenous glucose production and Ketonuria in female mice
Authors: Sebastian Manuel , Morriseau Taylor , Dolinsky Vernon , Doucette Christine 
Published: April 2020

BACKGROUNDType 2 diabetes (T2D) diagnoses in the Anishininiiwuk (Oji‐Cree) linguistic group of central Canada are among the highest in the world. A genetic variant in the transcription factor hepatic nuclear factor‐1a (HNF‐1a), known as HNF‐1a G319S is present in this population which has been strongly associated with earlier onset of T2D. Historically, T2D was rare in Anishininiiwuk communities provided an active lifestyle required for land‐based food procurement, and potentially with prolonged periods of fasting. As a result of colonization, this lifestyle was abruptly altered leading to a decrease in hunter‐gatherer societies. In the liver, HNF‐1a plays an important role in glucose homeostasis and suppression of hepatic steatosis, but it is unclear if the G319S variant affects liver function. We
hypothesize that the G319S variant promotes greater hepatic fuel production and confers a metabolic advantage in the fasted state.METHODSCRISPR/Cas9 gene editing was used to knock‐in the HNF‐1a G319S G>A 955 single nucleotide substitution into C57BL6 mice. These mice express the G319S variant at endogenous levels and in appropriate tissues. Mice fed chow diet ad libitum were sacrificed at 6 months of age, and liver tissues were collected for gene expression analysis, glycogen content measurements, and for hematoxylin and eosin staining. In addition, pyruvate tolerance tests were performed, and urine ketone levels measured in mice fasted for 12 hours.RESULTSDifferences in hepatic steatosis and glycogen content were not observed between the wildtype (G/G), heterozygous (G/S), and homozygous (S/S) HNF‐1a mice. Female mice with two copies of the variant S‐allele (S/S) displayed elevated endogenous glucose production on a pyruvate tolerance test, as well as elevated ketones in the urine.MAIN FINDINGS/CONCLUSIONSIndependent of hepatic steatosis, female homozygous (S/S) HNF‐1a mice show greater endogenous glucose production, and higher ketone levels in their urine, indicative of a greater capacity to generate and mobilize endogenous fuels.Support or Funding InformationThis work was supported by a CIHR project grant awarded to CAD (PJT‐159633).
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

ObjectiveBronchopulmonary 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.MethodsFourteen 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.ResultsAirway 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).ConclusionsOur 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 InformationNational 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 InformationNIH (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 InformationThis 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 InformationNational 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 InformationFunding: 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 InformationBrigham Young University College Undergraduate Research AwardBryant 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 InformationThis project is funded by National Science Foundation Bridge to the Doctorate Award HRD‐1400868 and National Institutes of Health SC1GM127231.