bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Chicken Model of Nonalcoholic Fatty Liver Disease Induced by High Cholesterol and Low Choline Diet
Authors: Huang Ting-Wei , Lin Chiao-Wei , Ding Shih-Torng
Published: April 2020

Non‐alcoholic fatty liver disease (NAFLD), a most common liver disease in human, is one of the primary causes of the laying hen death in chicken farming. It leads to a great commercial loss in agricultural sector. Therefore, reliable chicken models of NAFLD is required for the research of NAFLD etiology and prevention in chickens. To generate rapid chicken models of NAFLD, 7‐week‐old ISA chickens were fed with control diet (18% dietary protein, 4.72% fat, and 1300 mg/kg choline), low protein and high fat diet (LPHF, 13% dietary protein, 8.95% fat, and 1300 mg/kg choline), high cholesterol with low choline diet (CLC, 18% dietary protein, 4.72% fat, 2% cholesterol, and 800 mg/kg choline), or low protein, high fat, low choline, and high cholesterol diet (LPHFCLC, 13% CP, 8.95% fat, 2% cholesterol, and 800 mg/kg choline) for 4 weeks. Compared with control group, CLC and LPHFCLC treatments significantly elevated the level of plasma triglyceride and cholesterol. The appearance and the histological sections of the livers showed that CLC and LPHFCLC treatments significantly led to the accumulation of fat in the livers. The symptoms of steatohepatitis, hepatic ballooning and immune infiltration, were presented in the livers in CLC and LPHFCLC groups. In addition, the levels of dipeptidyl‐peptidase 4, a biomarker of fatty liver hemorrhagic syndrome in laying hens, were also increased in the plasma of CLC and LPHFCLC treatments. These data indicated that the diet with 2% cholesterol and 800 mg/kg choline was sufficient to induce hepatic steatosis and steatohepatitis. Moreover, the genes related to lipogenesis (PPARG, SREBP1C, and FASN) and pro‐inflammation cytokines (TNFA and IL1B) were elevated in the CLC group, further confirming the effects of CLC treatment on laying hens. Interestingly, CLC treatment also induced non‐alcoholic fatty livers in Plymouth Rock broiler chickens. It suggested that CLC treatment may induce non‐alcoholic fatty liver disease in different strains of chickens. Therefore, our data suggested that CLC treatment may induced rapid and reliable non‐alcoholic fatty liver disease in chickens. Accordingly, de novo lipogenesis is primary occurred in the livers both in chicken and human. This model can be expected to be utilized for the screening of functional components to prevent or improve fatty livers in chickens and human.
Diabetic metabolic dysregulation and chronic kidney disease induce specific perturbations in coronary microvascular function in swine
Authors: Sorop Oana , Van De Wouw Jens , Heinonen Ilkka , Merkus Daphne , Danser Jan A.H. , Duncker Dirk J.
Published: April 2020

Introduction

Comorbidities such as diabetes mellitus (DM), hypercholesterolemia (HC) and chronic kidney disease (CKD), are often associated with altered coronary microvascular function. However, the specific mechanisms affected by the various comorbidities have not been investigated in detail. Here we studied endothelium‐dependent and ‐independent vasodilation of isolated small coronary arteries, as well as vasoconstrictor responses to endothelin‐1 and their specific mechanisms of action in swine with multiple comorbidities.

Methods and Results

DM (streptozotocin, 150mg/kg i.v.) and hypercholesterolemia (high fat diet) were induced in 10 female swine, which were followed for 6 months (DM+HC). In a separate group of 9 female swine, CKD (by renal artery embolization) was added on top of the two risk factors and animals were followed for 6 months (DM+HC+CKD). 12 female healthy swine on normal pig‐chow served as controls. At sacrifice, coronary small arteries were isolated and vasomotor control was studied in vitro. Both DM+HC and DM+HC+CKD groups showed impaired endothelium‐dependent vasodilation to bradykinin, mainly via loss of EDHF‐dependent mechanism. No changes were seen in the response to the exogenous NO‐donor, SNAP, indicating preserved smooth muscle cell function. Coronary arteries from swine with DM+HC exhibited a blunted vasoconstriction to endothelin‐1, (ET‐1), due to increased ETB‐mediated vasodilation. In contrast, adding CKD to DM+HC resulted in enhanced ET‐1 vasoconstriction.

Conclusion

Diabetic metabolic dysregulation and chronic kidney disease induce comorbidityspecific perturbations in coronary microvascular function in swine.

Support or Funding Information

Supported by grants from the European Commission FP7‐Health‐2010 grant MEDIA‐261409, the Netherlands CardioVascular Research Initiative: an initiative with support of the Dutch Heart Foundation [CVON2014‐11(RECONNECT)] and The Academy of Finland 251272.
Phytochemical Characterization and Health Benefits of Omani Date Pollen
Authors: Waly Mostafa
Published: April 2020

Traditional and folk medicines of different countries have great potential for introducing new natural remedies for various human diseases. One of these remedies is date pollen, which is an edible component of the date palm tree, and has been used in Oman and other countries as a therapeutic agent for male infertility. There is a need to identify the bioactive/phytochemical compounds in the Omani date palm pollen which might attribute for its therapeutic effects on infertility and other human diseases. Date palm pollen is rich in bioactive compounds with phytochemicals and nutraceutical properties. There is no information about the efficacy of the Omani date pollen bioactive compounds and their structure. This study is focusing on optimizing the extraction conditions of different Omani date palm pollen and to compare their bioavailability as well as their therapeutic properties. The project was carried out in two phases. In the first phase: Extraction of date pollen from different date palm cultivars and the characterization of their phytochemicals profile. In the second phase: Novel therapeutic (antioxidant and anticancer activities) properties of the date pollen extracts were investigated. The results of this study support the use of Omani date palm pollen extract as a functional food by the pharmaceutical and nutraceutical industries.
G protein βγ translocation to the Golgi activates the mitogen‐activated protein kinases via phosphoinositide 3‐kinase γ
Authors: Khater Mostafa Kamel , Wei Zhe , Huang Wei , Lokeshwar Bal L. , Lambert Nevin A. , Wu Guangyu
Published: April 2020

The activation of G protein‐coupled receptors (GPCRs) induces the translocation of G protein βγ complex from the plasma membrane (PM). However, its functions are not well known. Here we demonstrated that among the entire twelve Gγ family members, Gγ9 is the most translocatable Gγ subunit. More than 80% of Gγ9, in complex with Gβ1, relocated from the PM to the Golgi in response to CXCR4 activation by stromal cell‐derived factor 1α (SDF1α) and the half time of the translocation was about 5 seconds. To study the function of Gβγ translocation to the Golgi, we focused on the activation of the mitogen‐activated protein kinases (MAPK) ERK1/2. CRISPR‐Cas9‐mediated knockout of Gγ9 abolished ERK1/2 activation by SDF1α, whereas knockout of the least‐translocatable Gγ3 had no effect. To investigate if Gβγ‐mediated ERK1/2 activation indeed occurred on the Golgi, we employed an inducible system to specifically express Gβγ onto the Golgi. Interestingly, the targeted expression of Gγ2, Gγ3 and Gγ9, in complex with Gβ1, onto the Golgi each induced remarkable ERK1/2 activation. Such constitutive ERK1/2 activation was blocked by the expression of Gα subunits, the targeted expression of the Gβγ blocker GRK2ct at the Golgi, and chemical treatments to disrupt the Golgi structure. By contrast, the expression of individual Gγ subunits at the PM did not activate ERK1/2. To define the downstream effectors of Gβγ, we determined the effect of selective pharmacological inhibition of phosphoinositide 3‐kinase (PI3K), phospholipase C, PKC and PKD on ERK1/2 activation. This strategy identified PI3Kγ and PI3Kδ to be essential for Gβγ‐mediated ERK1/2 activation. Indeed, knockout of PI3Kγ by CRISPR‐Cas9 almost abolished ERK1/2 activation induced by both SDF1α and Gγ9 targeting to the Golgi. Similar to CXCR4, α2‐adrenergic receptor‐mediated ERK1/2 activation was also dramatically inhibited by Gγ9 and PI3Kγ knockout. We further showed that knockout of Gγ9 and PI3Kγ markedly suppressed the migration and invasion of prostate cancer PC3 cells in response to SDF1α stimulation in vitro. Gγ9 and PI3Kγ knockout also significantly reduced the bone metastasis of PC3 tumors following intra‐cardiac injection of PC3‐Luc cells in athymic nude mice. Overall, our data demonstrate a novel function for Gβγ translocation to the Golgi to spatiotemporally control the activation of the MAPK pathway by GPCRs and tumor progression.

Support or Funding Information

NIH R01 GM118915
Compensatory Plasticity of Breathing in Patients with Neuromuscular Disease
Authors: Smith Barbara K
Published: April 2020

Neuromuscular diseases are comprised of variable, often inherited conditions and characterized by muscular (e.g. Duchenne muscular dystrophy), neural (e.g. amyotrophic lateral sclerosis) or a mixed neuromuscular (e.g. Pompe disease) dysfunction. While individual neuromuscular disorders differ in the underlying mechanism and exhibit a unique disease presentation, the consistently leading cause of morbidity and mortality is respiratory failure. Loss of breathing function is literally a “life‐or‐death" issue for patients. Clinical management of breathing relies upon pulmonary function tests to estimate the degree of respiratory involvement in patients, but standard pulmonary function test results can be biased by learning, cognition, or poor effort. This presentation will discuss compensatory neuromuscular adaptations to preserve breathing in the face of worsening disease, including changes in neural drive, neuromuscular junction plasticity, and a greater reliance on less‐affected respiratory motor pools. Detection of these adaptations is needed for prescribing supportive therapies with greater precision and identifying clinically meaningful changes in response to treatments. Citing natural history studies from our team and others, this presentation will feature clinical approaches to objectively evaluate respiratory motor control and gain mechanistic insights into neuromuscular disease. The control of breathing will then be discussed in the context of clinical trials, with an emphasis on current and emerging treatments directed toward improvements in ventilation. Examples will highlight basic‐clinical collaborations that promote new therapeutic frontiers for patients with neuromuscular disease.

Support or Funding Information

This work is supported by National Institutes of Health awards R21 HD090752 and U01 HL121842.
Role of TMAO, a Gut Metabolite, on Proline‐Rich Proteins
Authors: Kumari Kritika , Singh Laishram Rajendrakumar
Published: April 2020

Osmoprotectants, a group of low molecular weight compounds, are accumulated in various cells to protect organism from various stresses. Most of the osmoprotectants (osmolytes) are known to stabilize proteins by assisting them in proper folding into the native conformation. However, Trimethylamine N‐oxide (TMAO), a human gut metabolite, is one such osmolyte that is believed to have both stabilizing and destabilizing effect on different proteins. Till date the destabilizing effect of TMAO has been confined only to the fast‐folding proteins. We, therefore, investigated the destabilizing effect of TMAO on folding pattern of various slow‐folding proteins.

Methodology

Folding pattern of chosen proteins in absence and presence of TMAO were studied using enzyme kinetic assays. Different spectroscopic probes were further used to measure effect on native and refolded state structure. Light Scattering measurements and electron micrographs were obtained using Dynamic light scattering (DLS) and Transmission electron microscopy (TEM) respectively to measure aggregation status of different protein species.

Results

Protein stability and enzyme activity studies revealed TMAO acts as a denaturant. Among all osmolytes this effect was found to be unique to TMAO only. No recovery of the enzyme activity of refolded protein even after prolonged incubation without formation of aggregates confirms TMAO interfere with refolding of proteins with high number of proline residues. Structural studies revealed refolding of protein to a compact denatured state. Changed behavior of protein from denatured to native state was observed upon addition of Peptidyl‐Prolyl Isomerase (PPIase).

Conclusion

We discovered that TMAO fails to stabilize or refold the slow folding proteins. Furthermore, we showed that in proteins with high number of proline residues, proline cis‐trans isomerization becomes the rate limiting step in protein folding process, thus resulting into slow folding of proteins. Recovery of native like activity of protein in presence of PPIase indicate that TMAO might have interfered cis‐trans conversion generating a non‐native protein species. This study highlights the potential role of TMAO in protein folding and associated pathological consequences.
To investigate whether metastasis‐related mechanism are the same in different organs through the in vivo selection of colorectal cancer cell lines from liver and lung metastases
Authors: Hsu Tai-I , Hsiao Micheal
Published: April 2020

The mortality and incidence rate of colorectal cancer is currently one of the top three cancers in Taiwan. In the United States the mortality and incident rate of colorectal cancer also the one of top three cancers. The colorectal cancer can use the stool occult blood examination as the early screening tool to achieve the early prevention. Although, the non‐invasive and highly effective method of early prevention, there still have some patients who cannot be screened or who are false negatives may have had micrometastasis or some treatment difficulties when the disease is discovered. The current therapeutic strategies are surgery, radiation, chemotherapy, chemotherapy concurrent radiotherapy and target therapy. The survival rate of patients is improved within the advanced medical technology and drug. However, for metastatic colorectal cancer, it is still unable to effectively improve the survival rate. The metastasis is also the majority cause of death in cancer. In our proposal, the genetic profile of metastatic colorectal cancer will be analyzed and the drug efficacy will be verified in metastatic cancer cells. The xerography model had been established. The cancer cells can be injected on the subserosal of cecum wall. In the in vivo tracking, the mice used the IVIS to tracking the immunofluorescence signal. After 4 weeks the signal can be detected in the other organ such as liver and lung. To confirm the signal from the metastasis organs, the mice were sacrificed and collected the organ to confirm the cells metastatic to liver and lung. However the cells in the transwell assay seem can not show the migration or metastatic function. The IHC staining for human mitochondria cells verified the cells with large ratio of nucleus and substance. The optical observation did not show the difference. The experimental assay showed the control group did not show highly growth rate. The cell growth rate in lung group is increased compared with the other groups. In the cancer progression theory, the cancer stemness theory has been mentioned. The western blotting assay related of stemness theory showed that lung group with dominates notch1 level decreased. Therefore the non‐coding RNA array was a tool for us to screen the candidate non‐coding RNA included 4 up‐regulation and 1 down‐regulation miRNAs. In the future, the miRNAs will be the target to investigated.

Support or Funding Information

This research was supported by Academia Sinica [AS‐SUMMIT‐108].
Molecular and cellular analysis of intestinal lactase‐phlorizin hydrolase gene variants unravel a heterogeneous pathogenic pattern of congenital lactase deficiency
Authors: Marten Lara M. , Wanes Dalanda , Santer René , Naim Hassan Y.
Published: April 2020

Congenital lactase deficiency (CLD) is a rare autosomal recessive genetic disorder, leading to a dysfunctional lactase‐phlorizin hydrolase (LPH). LPH is a ß‐galactosidase located in the intestinal brush border membrane and indispensable for lactose digestion. Impaired LPH‐function results in severe symptoms within a few days after birth with the onset of nursing. The symptoms include watery diarrhea and consequently dehydration, leading to potential life‐threatening disease of the newborn. Several mutations in the coding region of LPH have been characterized, occurring in homozygote or compound heterozygote patterns of inheritance. The basis of the genotype‐phenotype relationship in CLD on a molecular and cellular level has not been studied in details. Here we analyze the influence of the mutations p.S688P, p.E1612X, p.S1121L, that have been identified in patients from different ethnic groups, on the structure, trafficking and function of the LPH protein. For this purpose, LPH‐mutants were generated by site‐directed mutagenesis and transiently expressed in COS‐1 cells. Additionally we concluded studies regarding the interaction of these mutants with the wild‐type‐LPH protein. The results reveal phenotypic variations in the protein trafficking of the mutants between the ER and the Golgi. Additionally, the LPH‐mutant harboring the mutation p.S688P showed residual lactose digesting capacity. These variations implicate possible clinical correlation as for example different course and severity of disease. These data are relevant for the further understanding of CLD, potential impact for heterozygote mutation carriers and possible invention of treatment strategies, as for example protein stabilization through chaperones.

Support or Funding Information

Intramural funds, University of Veterinary Medicine Hannover
Glucocorticoid Receptor Maintains Vasopressin‐Regulated Water Reabsorption Pathway in the Kidney Collecting Duct Cells
Authors: Su Shih-Han , Ho Cheng-Hsuan , Yu Ming-Jiun
Published: April 2020

Aquaporin‐2 (AQP2) is a transmembrane protein expressed in the principal cells of the kidney collecting ducts, which mediate osmotic water reabsorption. AQP2 is regulated by the antidiuretic peptide hormone arginine vasopressin (AVP). When AVP binds to the vasopressin type 2 receptor (V2R) in the principal cells, it initiates a signaling network that results in 1) AQP2 trafficking to the apical plasma membrane for water transport and 2) AQP2 gene expression. In a previous study, we found that dexamethasone, a glucocorticoid receptor (GR) agonist, enhanced AVP‐induced AQP2 gene expression in the collecting duct cell model mpkCCD. Here, we showed that GR knockdown drastically decreased AVP‐induced AQP2 mRNA and protein levels, indicative of a role of GR in AVP‐induced AQP2 gene expression. We further showed that the 1,000bp region upstream to the AQP2 transcription start site was sufficient to mediate GR‐enhanced AVP‐induced promoter activity and that GR bound to the above AQP2 promoter region. RNA‐seq was used to profile the transcriptome of the GR knockdown vs. control cells. A total of 93,308 transcripts belonging to 22,025 genes were quantified. GR knockdown decreased expression of 5,266 genes and increased expression of 3,709 genes. These two gene groups respectively represent positive and negative regulators for the AVP responses. Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis showed 62 pathways associated with the positive regulators and 48 pathways associated with the negative regulators. The AVP‐regulated water reabsorption pathway was among the 62 positive pathways whose gene expression was reduced upon GR knockdown. Quantitative RT‐PCR analysis showed about 60% reduction in the V2R mRNA level upon GR knockdown. Taken together, our results implicated that GR in one hand potentiates AVP‐induced AQP2 gene expression and in the other hand maintains expression of genes involved in the AVP‐regulated water reabsorption pathway in the kidney collecting duct cells.

Support or Funding Information

This work was supported by the Ministry of Science and Technology, Taiwan (107‐2320‐B‐002‐057‐MY3).
Anti‐Biofilm Properties of Flax, Chia, and Hemp Seed Oil Extracts
Authors: Menjivar Jessica Liseth , Bendaoud Meriem
Published: April 2020

Biofilm formation is an important virulence factor and a crucial step in the pathogenesis of bacterial infection for certain microorganisms. Bacteria attaching to surfaces and forming biofilms are resistant to antibiotic treatment and antimicrobial agents, which makes them difficult to eradicate. Worldwide, an increasing number of pathogenic bacteria and yeast have been gaining antibiotic and anti‐fungal resistance. This is a critical problem for public health as millions of people acquire infections from pathogenic antibiotic resistant microorganisms such as Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Candida albicans. For many years now, scientists have been looking for natural alternatives like herbs, oils, and seed extracts to control the rise of antibiotic resistant microbial infections and support better health. Chia, Flax, and Hemp seed oil extracts have been found to support skin health and improve immunity and cardiovascular function. However, very little is known about their effects on the growth of pathogenic microorganisms. The purpose of this study is to test these oil‐seed‐extracts for anti‐biofilm properties. Using the biofilm assay we were able to show that Chia seed oil extract and Hemp seed oil extract had the most effective anti‐biofilm properties against P. aeruginosa and S. epidermidis. Preliminary chemical characterization of these oils was initiated to determine the active fraction displaying anti‐biofilm properties. Additional testing will be done to further characterize the active compound in each of these oils.

Support or Funding Information

This project received support from US Education Department Title III Part F HSI grant # P031C160155
Some Believe, Not All Achieve: The Role of Active Learning Practices in Anxiety and Academic Self‐Efficacy in First Generation College Students
Authors: Hull Kerry , Hood Suzanne , Page Olivia , Birdsong-Farr Melaney , Barrickman Nancy , Djerdjian Nancy , Roychowdhury Hiranya , Stowe Skye , Gerrits Ron , Ross Kyla , Ott Betsy , Jensen Murray
Published: April 2020

Although active learning (AL) techniques improve student performance and persistence in STEM programs of study, AL may provoke anxiety in certain student populations. Given that high anxiety impairs academic performance, there is a need to understand how psychological variables in students interact with AL. As part of a larger study examining the adoption of AL in human anatomy and physiology courses in community colleges, we asked students to rate various teaching strategies in terms of their perceived educational value and their capacity to invoke anxiety. We also examined the relationship of these scores to two psychological constructs: self‐reported social anxiety (psychological distress relating to the fear of negative evaluation by others) and academic self‐efficacy (confidence in one’s ability to overcome academic challenges). In general, the degree of anxiety induced by a teaching strategy was inversely related to its perceived educational value such that the most anxiety‐inducing (e.g., cold calling) were reported as having the least educational value (N=330). Compared to students low in social anxiety, socially anxious students rated all techniques as more anxiety‐inducing and of lower educational value, and anticipated a lower grade in the course. Notably, individual differences in academic self‐efficacy mediated the relationship between social anxiety and anticipated grade. Consistent with this, low‐efficacy students reported more anxiety in response to AL techniques involving a social component (e.g., non‐graded group activities) than did high‐efficacy students. Since mastery experiences (such as those provided by active learning strategies) can significantly improve academic self‐efficiency, our results suggest that anxious students may not benefit from the beneficial and reciprocal relationship between active learning strategies and academic self‐efficacy to the same extent as less anxious students. Other documented strategies that improve academic self‐efficacy, such as the use of trained peer facilitators, may thus facilitate the introduction of AL in the classroom.

Support or Funding Information

Funding from the National Science Foundation (Award #1829157)
Investigating EWS‐FLI1 Induced Replication Stress in Ewing Sarcoma Pathogenesis
Authors: Halbkat Halle , Ghosal Gargi
Published: April 2020

Ewing sarcoma (EWS) is the second most common malignant bone cancer affecting young children and adolescents. Although most patients achieve long‐lasting remission, patients with metastatic or recurrent disease have very poor outcomes and chemo‐resistance is a significant problem. The majority of Ewing sarcomas are driven by a chromosomal translocation resulting in the EWS‐FLI1 fusion gene. EWS‐FLI1 is an oncogenic transcription factor that plays roles in transcriptional regulation, DNA damage response, cell cycle checkpoint control, and alternative splicing. However, the exact molecular mechanisms behind these functions are not understood fully. Although EWS‐FLI1 is specific to EWS cells and required for tumorigenesis, successfully targeting EWS‐FLI1 has proved challenging. Therefore, other therapeutic targets must be identified to improve outcomes for patients with metastatic or recurrent disease.

Using a quantitative PCR array, we identified USP1, a deubiquitinase that functions in both the DNA damage and replication stress responses, as being overexpressed in EWS cell lines compared to its cell of origin, the human mesenchymal stem cell. Importantly, we found USP1 to be essential for EWS cell line proliferation, as growth of these cell lines is arrested with knockdown of USP1. USP1 downregulation has been linked to oncogene‐induced cellular senescence dependent on functional p53. However, because p53 is mutated in EWS, the growth arrest we observe following USP1 downregulation must occur via a p53‐independent mechanism. Therefore, we hypothesize that USP1 overexpression functions to bypass cellular senescence in a p53‐independent manner in response to EWS‐FLI1 induced replication stress, resulting in tumorigenesis. Our studies aim to test this hypothesis in EWS and explore the potential to target USP1 alone or in combination with the current chemotherapy regimen for treating EWS.

Support or Funding Information

This work was supported by Pediatric Cancer pilot funding UNMC and UNMC startup funds to Gargi Ghosal and UNMC Graduate Assistantship Fellowship to Halle Halbkat
Pattern of sympathetic vasomotor activity induced by GABAergic inhibition in the brain and spinal cord
Authors: Campos Ruy R. , Milanez Maycon I. , Silva Adilson M. , Perry Juliana C. , Faber Jean , Nishi Erika E. , Bergamaschi Cassia
Published: April 2020

Background

The knowledge of the central areas involved in the control of sympathetic vasomotor activity has been advanced in the last few decades. The γ‐aminobutyric acid (GABA) is the mainly inhibitory neurotransmitter in the mammal central nervous system and bicuculline, an antagonist of GABA type A receptors (GABA‐A), microinjected into the paraventricular nucleus of the hypothalamus (PVN) alters the pattern of sympathetic vasomotor activity to the renal, splanchnic and lumbar territories. However, studies are needed to clarify the role of GABAergic inputs in other central areas involved in the control of sympathetic vasomotor activity. Thus, the present work studied the cardiovascular effects evoked by GABAergic inhibition in the PVN, RVLM and the spinal cord.

Methods and Results

All experimental approach applied in this present study agreed to the guidelines recommended by the National Institute of Health and were approved by the Ethics in Research Committee of the Escola Paulista de Medicina – Universidade Federal de São Paulo (8724270715/15). In urethane (1.4 g/kg, iv) Wistar male anesthetized rats, Bicuculline microinjection (400 pMol in 100 nL) into the PVN and rostral ventrolateral medulla (RVLM), as well its intrathecal administration (1.6 nmol in 2 μL) evoked an increase in blood pressure, heart rate, renal and splanchnic sympathetic nerve activity (rSNA and sSNA, respectively) inducing a higher coherence of rSNA and sSNA pattern. However, some of these responses were more intense when the GABA‐A antagonism was performed into the RVLM.

Conclusions

Administration of bicuculline into the RVLM, PVN and SC induced a similar pattern of renal and splanchnic sympathetic vasomotor burst discharge, characterized by a low frequency (0.5 Hz) and high amplitude pattern despite different blood pressure responses. Thus, the 0.5 Hz recorded in sympathetic nerves to different targets is dependent in part of tonic GABAergic inputs in the brain (RVLM and PVN) and spinal cord. The origin of the GABAergic projections is unknown.

Support or Funding Information

CAPES ‐ Finance Code 001; FAPESP ‐ 18/02671‐3 and CNPq 309020/2017‐4.
Anomalous Origin of Obturator Artery from Inferior Epigastric Artery
Authors: Walter Brianna Leigh , Suhrawardy Ameen , Ahmadi Payaum , Walton Hayley , Mrowczynski Stephanie , Khatter Neil , Mielke Nicholas , Armstrong Zachary , Barremkala Malli
Published: April 2020

During routine cadaver dissection at Oakland University William Beaumont School of Medicine, an aberrant Obturator artery (OBA) was observed originating from the inferior epigastric artery (IEA). Typically, OBA originates from the anterior division of the internal iliac artery (IIA). When it originates from the IEA, it passes over the pelvic brim and places the individual at risk for hemorrhage during surgery in that region or after pelvic trauma. This phenomenon is called ‘corona mortis.’ The objective of this study is therefore to characterize how many OBAs arise from IIA and how many arise from aberrant branching patterns, with a focus on those which arise from IEA. The origin of the OBA was studied in 24 formalin fixed cadavers (13 female, 11 male, average age 80) which had been dissected by first year medical students. To confirm identification, the obturator nerve was first identified passing into the obturator canal in each cadaver, and then the main arterial contribution passing into the obturator canal was identified. The OBA was found to originate from the anterior division of the IIA in 28 pelvic halves (58%). In 12 (25%) cases , OBA was found to arise from the IEA. In 7 (15%) cases, it arose from other locations such as the posterior division of the internal iliac artery (6) or as a branch of the superior gluteal artery (1). There was one hemisected pelvis in which the accessory OBA arising from the IEA was the same size as the OBA contribution from the anterior division of IIA. We found that 60% of the aberrant OBAs were on the right and 40% on the left. There were 13 aberrant OBAs in the female cadavers in contrast to 6 found in males. Literature review by the authors reveals widely varying estimates for the origin of OBAs.
Jakubowicz et al (1996) describe incidence of OBA from IEA as only 2.6%,
Mahato (2009) reports incidence at 8%, and
Pai et al (2009) report incidence of 14%. Additionally, Pai et al found that this aberrance was more common in males than females, the opposite of the present findings. Furthermore, they demonstrated a similar pattern of variances of OBA arising from external iliac artery and IEA on the right (26.5%) vs left (16.3%).
Urinary Angiotensinogen is Essential for Renal Potassium Handling
Authors: Wang Bangchen , Wang-France Jun , Sansom Steven
Published: April 2020

Apart from the well‐known systemic renin‐angiotensin system (RAS), the urinary RAS acts as a local autocrine/paracrine system along the nephron. The regulation of renal K+ handling by systemic vs urinary RAS remains unclear. Liver‐specific (Alb‐Cre) angiotensinogen knockout (LKO) and liver + proximal tubule (Alb‐Cre + KAP‐Cre) angiotensinogen knockout (DKO) were utilized in this study. WT, LKO, and DKO mice were kept on either a regular diet (RD) or a high K diet (HK; 5% K+) for 10 days. Mice were acclimated to metabolic cages twice before collecting urine for 24 hours. Mice were then euthanized to collect blood and kidneys. Plasma and urinary [Na+] and [K+] were measured by flame photometry. Plasma and urinary angiotensin II (Ang II) concentrations were measured by ELISA (Enzo Life Sciences). Plasma and urinary aldosterone (Aldo) concentrations were measured by ELISA (DRG Diagnostics). Urinary creatinine (Cr) concentrations were measured by colorimetric assays (Cayman Chemical). Plasma [Ang II] was significantly lower in LKO, compared to WT, and the value in DKO was not significantly different from zero. Urinary [Ang II] was significantly lower in DKO compared to WT and LKO. Plasma [K+] was significantly higher in DKO but not LKO compared to WT on HK. Urinary K:Cr ratio was significantly lower in DKO compared to WT on HK. However, plasma and urinary [Aldo] were not different between DKO and WT on HK. Overall, our results showed that urinary RAS but not systemic RAS plays an essential role in renal K handling.

Support or Funding Information

This project was funded by F30 DK108456.
Is skeletal muscle a functional immunosensory organ of pathogens? A murine model of polymicrobial sepsis.
Authors: Robinson Gerard Patrick , Iwaniec John , Alzahrani Jamal , Sheikh Laila , de Carvalho Lucas , Garcia Christian , Murray Kevin , Morse Deborah , Laitano Orlando , Clanton Thomas
Published: April 2020

The body’s ability to sense lipopolysaccharide (LPS) is critical in the inflammatory response to bacterial infection. LPS is a ligand for toll‐like receptors (TLRs), expressed on immune and many other cell types. When activated, they lead to secretion of cytokines such as IL‐6 and IL‐1ß. These cytokines, in turn, promote the activity and trafficking of inflammatory cells to the site of infection. We hypothesize that skeletal muscle (skm), which also expresses TLRs, plays a role in directing inflammatory cells via cytokine secretion during sepsis. In order to test this, we developed an inducible skeletal muscle‐specific knockdown of MYD88 (an adaptor protein necessary for TLR function) using the Mer‐CRE‐Mer system.

METHODS

Male and female mice on skmCRE background were injected with raloxifene to induce skmMYD88 knockdown vs. vehicle control (no CRE induction). Three weeks after CRE induction, sepsis was induced via intraperitoneal injection of a calibrated dose of cecal slurry. The animals were sacrificed 6 and 12 hours post‐injection. Plasma and peritoneal lavage were collected at the time of sacrifice, and analyzed for the presence of various inflammatory cells via a HESKA hematological analyzer.

RESULTS

skmMYD88−/− females showed a significant increase in the %neutrophils in the lavage at both the 6h (P<0.05) and 12h time points (P<0.0001) compared to skmMYD88+/+. The % of lavage basophils were elevated in female skmMYD88−/− at 12h (P< 0.01), as were %eosinophils (P<0.0001). Female lavage %monocytes increased at 12h in skmMYD88−/−. There were no differences in lavage %lymphocytes. No differences between the male skmMYD88−/− were observed for any cell types in the peritoneal lavage. In the blood, the only significant differences were found in males, in which the skmMYD88−/− group displayed increased %monocytes and decreased %lymphocytes at 12h.

DISCUSSION

The data suggest that skeletal muscle is capable of selectively governing the direction and presence of immune cells at the site infection, i.e. the peritoneum. The cause of this influence appears to be, in part, due to skeletal muscle TLR activation. While total leukocytes remain unchanged in the peritoneal lavage, knocking down skmMYD88 resulted an altered immune cell profile during the time‐course of infection. In the case of neutrophils, skeletal muscle appears to play a major suppressive regulatory role. Overall, these results implicate skeletal muscle as a significant player in pathogen recognition and response.

Support or Funding Information

Supported by NIH 1R01GM118895 (TLC) and the BK Betty Stevens Endowment
Morphology of the Semicircular Canals and Locomotion of Zionodon satanus
Authors: Dunn Rachel Heather , Penkrot Tonya , Zack Shawn
Published: April 2020

Zionodon satanus is a relatively large “insectivore” from the Uinta Formation of Utah, known from associated fragmentary dental, cranial, and postcranial elements. Features of the postcrania, particularly the knee and ankle joints, suggest that this species primarily engaged in swift terrestrial locomotion, but with some climbing and digging capability. Among the cranial fragments of Z. satanus are two petrosal bones preserving portions of the bony labyrinth, particularly the semicircular canals, which are known to vary among extant mammals according to locomotor agility, with more agile animals having semicircular canals with a larger radius of curvature than slower ones. Thus, semicircular canal dimensions can provide insight into locomotion independent of postcranial morphology. Here we compare measurements of the semicircular canals of Z. satanus to other extant and extinct mammals to assess the degree of locomotor agility of this species and compare those results to those obtained by functional analysis of the postcrania.

The petrosal bones of the type specimen of Z. satanus were microCT scanned and virtual models of the semicircular canals were extracted with AMIRA 6.5.0 software. One petrosal includes a complete posterior and partial lateral semicircular canal, whereas the other preserves a complete lateral, damaged but complete posterior, and most of the anterior semicircular canal. The height and width of each semicircular canal was measured from the extracted surface models and the radius of curvature was calculated from height and width measurements. The average radius of curvature of the semicircular canals of Z. satanus overlaps with extant mammals of similar body size of “fast” or “medium” agility and lies well outside the range of slow‐moving small mammals. Compared to other fossil “insectivores”, Z. satanus has relatively smaller semicircular canals than Leptictidium from the middle Eocene of Messel, Germany, which has been reconstructed as a bipedal runner and saltator, but larger ones than the North American leptictids Paleictops and Leptictis. These results corroborate observations from the postcranial skeleton, suggesting that Z. satanus likely engaged in frequent, rapid locomotion.
CYP11B and CYP17A1 in the Biosynthesis and Metabolism of C11‐oxy Steroids
Authors: Swart Amanda Cecilia , van Rooyen Desmaré , Gent Rachelle , du Toit Therina
Published: April 2020

Cytochrome P450 (CYP11B1) and cytochrome 17α‐hydroxylase (CYP17A1) play pivotal roles in the biosynthesis of adrenal glucocorticoids and androgens. It is generally accepted that CYP11B1 catalyses only the production of cortisol and corticosterone while CYP17A1 catalyses the hydroxylation of pregnenolone and progesterone at C17 and, in humans, the 17,20‐lyase of 17‐hydroxypregnenolone to produce DHEA. CYP11B1, however, also catalyses the biosynthesis of C11‐oxy C21 steroids, 11β‐hydroxyandrostenedione, 11β‐hydroxyprogesterone (11OHPROG) and 21‐deoxycortisol (21dF) in the adrenal
[1,
2].

We investigated the potential role of CYP17A1 in the metabolism of these C11‐oxy C21steroids leading to the production of 11keto‐dihydrotestosterone since 17‐hydroxyprogesterone has been shown to lead to the production of dihydrotestosterone in the backdoor pathway.

The 17α‐hydroxylase/17,20‐lyase activity of CYP17A1 towards 11OHPROG and 21dF was assayed as well as the catalytic activity of the enzyme towards the downstream C11‐oxy C21steroid products. In vitro conversions were carried out with 1 μM steroid substrate in HEK293 and in LNCaP cells transiently expressing CYP17A1 and metabolites analysed using UPC2‐MS/MS. Both 11OHPROG and 11keto‐progesterone (11KPROG) were hydroxylated at C17 producing 21dF, 0.27 μM and 21‐deoxycortisone, 0.65 μM, respectively while no lyase activity was detected. It was only once reduced by 5α‐reductase and 3α‐hydroxysteroid dehydrogenase that the aforementioned steroids were also lyased to produce the C11‐oxy C19steroids, 11‐hydroxyandrosterone, 0.6 μM and 11‐ketoandrosterone, 0.43 μM. In LnCaP cells 11OHPROG and 11KPROG were metabolised to C11‐oxy C19steroids indicative of the C11‐oxy backdoor pathway. In addition, in HEK293 cells transiently transfected with 11β‐hydroxysteroid dehydrogenase (11βHSD) type 1 and 11βHSD2 the isozymes readily catalysed the interconversion of all the C11‐keto C21steroid intermediates produced in the pathway with the production of the keto intermediates being favoured.

Although the C11‐oxy C21steroids produced in the adrenal may serve as ligands for the progesterone receptor they also potentially add to the pool of potent androgens via the C11‐oxy backdoor pathway. These C11‐oxy steroids would thus further contribute to the androgen pool in adrenal disorders and diseases characterised by androgen excess in which these steroids have been identified. We recently reported 11OHPROG, 11keto‐dihydroprogesterone and pregnanetriol in circulation in BPH patients
[3]. The C11‐oxy C21steroids and C11‐oxy C19steroids, however, remain to be fully explored –not only in pathophysiology but also in sexual differentiation and development.

Support or Funding Information

The South African National Research Foundation (IFR170125217588, CSUR160414162143)
Cigarette smoke extract increases expression and plasma membrane trafficking of α2C ‐ adrenoceptors in human microvascular smooth muscle cells
Authors: Maaliki Dina Salem , Eid Ali H.
Published: April 2020

Vasoconstriction of cutaneous arterioles in response to cold temperatures or emotion is a normal adaptive process in which the body diverts blood flow from the superficial circulation to internal organs to prevent heat loss. This is mediated by increased sympathetic activation as well as increased sensitivity of the digital arteriolar vascular smooth muscle to vasoconstrictors. When cold‐induced constriction is exacerbated, Raynaud’s phenomenon (RP) is precipitated. Remarkably, the entirety of this augmented cold‐induced vasoconstriction is mediated by α2Cadrenoceptors (α2C‐ARs) . We have previously established that cooling induces translocation of α2C‐ARs from the Golgi compartment, where they are trapped, to the plasma membrane of microvascular smooth muscle cells.

Epidemiological studies show that cigarette smoking is a significant risk factor for RP. Here, we provide exciting evidence which shows that cigarette smoke extract (CSE) increases α2C‐AR protein expression in a concentration and time‐dependent manner in human arteriolar smooth muscle cells. This expression appears to be due to increased transcriptional activity of the α2C‐AR promoter since pretreatment with actinomcyin D, a transcription inhibitor, abolished CSE‐induced α2C‐AR protein expression. Moreover, CSE increased transcriptional activity of α2C‐AR promoter: reporter construct. Interestingly, apocynin, a ROS scavenger, abolished CSE‐induced α2C‐AR protein expression, indicative of a ROS‐mediated pathway. CSE also increased RhoA activation, a pathway that we have previously established to drive trafficking of α2C‐ARs to the membrane. Indeed, CSE evoked mobilization of α2C‐ARs to the cell membrane. Taken together, these results may explain the underlying mechanism of CSE‐exacerbated cold‐induced vasoconstriction and provide an alternative avenue for treatment of patients who have RP and are smokers.

Support or Funding Information

This publication was made possible by an MPP fund (#320133) from the American University of Beirut to Ali Eid
Urinary Concentrating Defect Contributes to Polyuria in a Mouse Model of Cystinosis
Authors: Amlal Hassane , Dong Fei , Kao Winston
Published: April 2020

Cystinosis is a rare inherited disorder caused by mutations in the CTNS gene, which encodes the lysosomal cystine transporter, cystinosin. Both humans and cystinosin deficient mice exhibit significant polyuria, which is attributed to proximal tubule fluid reabsorption defect. However, whether cystinosis is also associated with impaired urinary concentrating ability of the kidney remains elusive.

We have developed a new mouse model of cystinosis using an insertional mutation generated by CRISPR gene editing techniques of fertilized eggs of C57/BL6 mice. Homozygous mutant (Cyst−/−) mice exhibited cysteine crystals in the cornea within 4 weeks of age, as compared to Wild‐type (WT) mice. Wild‐type, Cyst−/− and heterozygous (Cyst+/−) mice were placed in metabolic cages with free access to rodent chow and distilled water and tested for renal phenotype at 5, 12 and 20 months. To assay the activity of the proximal tubule, the animals were given a single injection of acetazolamide (ACTZ) in a 24hour period, or subjected to metabolic acidosis using 280 mM NH4Cl loading for 5 days. The diuretic effect of ACTZ and the adaptation to chronic metabolic acidosis in terms of urinary ammonium (NH4+) excretion level were similar between all three genotypes at 5 and 12 months of age. Further, the baseline data for water balance and urine osmolality were also similar among the three genotypes at 5 and 12 months of age. However, at 20 months, Cyst−/− mice exhibited a steady‐state significant polyuria, polydipsia and reduced urine osmolality, as compared to WT or Cyst+/− mice. Immunoblotting and immunofluorescence studies showed that the impaired water balance correlated with a significant reduction in AQP2 protein abundance throughout the kidney regions in Cyst−/− vs. WT or Cyst+/− mice. AQP1 and NKCC2 expression was not altered, whereas AQP4 protein was rather significantly upregulated with a shift in the molecular size of the protein in Cyst−/− vs. WT or Cyst+/− mice. When the animals were treated with a vasopressin V2 receptor specific analog (dDAVP), urine osmolality increased sharply in WT and Cyst+/− but remained unchanged in Cyst−/− mice. Blood Chemistry analysis showed a ~3‐fold increase in BUN levels, hyperkalemia, metabolic acidosis, and lower hematocrit and hemoglobin in Cyst−/− vs. WT or Cyst+/− mice, indicating the onset on renal disease in mutant homozygous mice.

In conclusion, Cyst−/− mice exhibited urinary concentrating defect, which is associated with vasopressin resistance of the collecting duct system, and manifested by the reduction in the protein abundance of the apical water‐absorbing channel AQP2. We propose that the impaired water balance and the proximal tubulopathy both contribute to massive volume depletion, which progressively lead to renal injury in cystinosis disease.

Support or Funding Information

Dialysis Clinic, INC. and NIDDK RO1‐DK‐083582 for H. Amlal and Cystinosis Research Foundation for W. Kao.