bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin 1‐7 reduces muscle atrophy and function loss following 16 week denervation
Authors: Renaud Jean-Marc , Albadrani Hind
Published: April 2020

Muscle atrophy is defined as a decrease in muscle mass that eventually leads to partial or complete muscle wasting. Muscle atrophy occurs in a variety of conditions including chronic diseases, limb immobilization and following a complete loss of muscle activity after spinal cord injury and peripheral nerve injury. A consequence of muscle atrophy is large force decrease associated with muscle mass loss as well as contractile components. The objective of this study was to determine whether angiotensin 1‐7 reduces the atrophy process and force loss following muscle denervation. Mouse extensor digitorum longus (EDL) and soleus were denervated by removing 3–4 mm of the sciatic nerve at the thigh level. Denervated mice were divided into 4 groups: i) denervated; ii) denervated and treated with angiotensin 1‐7; iii) denervated and treated with diminazene aceturate (DIZE), a ACE2 activator, to locally increase Ang 1‐7 levels, and iv) denervated and treated with sucrose. A 5th group of mice was used as control, i.e., normal innervated muscle. The results show that angiotensin 1‐7 significantly prevented muscle mass loss and fully prevented the loss of normalized force to cross sectional area that normally occurs over a 16 week denervation period. The maintenance of the normalized maximum force by angiotensin 1‐7/DIZE was in part due to a prevention of the loss of membrane excitability that occurs because denervated fibers are depolarized. This study is thus providing evidence that angiotensin1, 7 has therapeutic potential to prevent function loss following prolonged denervation.

Support or Funding Information

This project was supported by a grant from the National Science and Engineering Research Council of Canada (NSERC).
Chronic Stress Induces Persistent Hypertension and Increases NMDA Receptor Activity in the Paraventricular Nucleus in Borderline Hypertensive Rats
Authors: Zhou Jing-Jing , Shao Jian-ying , Chen Shao-Rui , Li De-Pei , Pan Hui-Lin
Published: April 2020

Stress activates sympathetic outflow and promotes hypertension development in humans with genetic predisposition. The enhanced N‐methyl‐D‐aspartate receptor (NMDAR) activity in the hypothalamus paraventricular nucleus (PVN) plays a critical role in controlling sympathetic output in hypertension. However, it is unclear how chronic stress affects NMDAR activity in the PVN in borderline hypertensive rats (BHR). BHR are the F1 male offspring from crossing female spontaneously hypertensive rats with male Wistars Kyoto rats (WKY). Arterial blood pressure (ABP) was monitored in free moving rats by using radiotelemetry. Using a 6‐week chronic unpredictable stress protocol, we found that stressed WKY rats had increased ABP during chronic stress but ABP returned to the normal level within a few days after stress. However, the stressed BHR showed a greater increase in ABP during stress and the elevated ABP was sustained for at least 2 weeks after discontinuing stress. Furthermore, whole‐cell voltage clamp recording in brain slices showed that the amplitude of evoked‐NMDAR currents in spinally projecting PVN neurons was significantly higher in stressed BHR than in non‐stressed BHR controls. Inhibiting α2δ‐1 with gabapentin normalized the increased synaptic NMDAR activity of spinally projecting PVN neurons in stressed BHR. In addition, the expression level of GluN1 and α2δ‐1 in the PVN was significantly greater in stressed BHR than in stressed WKY or non‐stressed BHR. These results suggest that chronic stress induces persistent hypertension in BHR, which is associated with α2δ‐1‐mediated NMDAR hyperactivity in PVN presympathetic neurons.

Support or Funding Information

Acknowledgments

Supported by NIH grants HL131161 and HL142133.
Anatomical Characterization Of Gαi2 Expressing Hypothalamic Paraventricular Nucleus Neurons
Authors: Chaudhary Parul , Wainford Richard D.
Published: April 2020

Background

Hypertension is a global health burden, and excess dietary salt is an established cause of hypertension. The hypothalamic paraventricular nucleus (PVN) is an essential component of the neuronal network that regulates sodium homeostasis and blood pressure. Previously, we have shown PVN specific GPCR‐coupled Gαi2 subunit proteins are essential to counter the development of salt‐sensitive hypertension by mediating the sympathoinhibitory and natriuretic responses to increased dietary sodium intake to maintain sodium homeostasis and normotension. However, the cellular localization and identity of PVN Gαi2 expressing neurons is currently unknown.

Objective

To determine the anatomical characterization of Gαi2 expressing PVN neurons.

Methodology

Three‐month‐old male (n=3) and female (n=3) Sprague Dawley rats were subjected to transcardial perfusion, and brains were post fixed in 4% paraformaldehyde and 30% sucrose. 20μm frozen brain slices were collected. Using in‐situ hybridization (ACD RNAscope kits) neuronal expression of Gαi2, vGLUT2, GAD67, corticotropin‐releasing hormone (CRH), tyrosine hydroxylase (TH), oxytocin, and arginine vasopressin (AVP) in hypothalamic PVN neurons was determined. The visualization and evaluation of co‐localization of Gαi2 expressing PVN parvocellular neurons with vGLUT2, GAD67, CRH, TH, oxytocin and AVP was done using a Keyence Bz‐9000e microscope and Image J respectively.

Results

We observed no differences among male and female rats, therefore, all data is presented as n=6. Our data show that neurons containing Gαi2 mRNA are localized in parvocellular but not magnocellular region of hypothalamic PVN. In the parvocellular region Gαi2 mRNA co‐localized with 87.5 ± 3.0% of GABA expressing neurons and 25.1 ± 2.7 % of vGLUT2 neurons, signifying that the Gαi2 mRNA is highly co‐localized with GABAergic neurons. Additionally, Gαi2 co‐localized with 76.3 ± 5.2% of CRH neurons and 33.5 ± 1.8% of TH expressing parvocellular neurons. We observed no localization of Gαi2 neurons with oxytocin or AVP expressing magnocellular neurons.

Conclusion

Given 1) our prior data demonstrate PVN Gαi2 proteins play a critical role in mediating sympathoinhibition and, 2) that the GABAB receptor signals predominantly through a Gαi2 pathway we speculate, given the high co‐localization of Gαi2 mRNA with GABAergic neurons, that Gαi2 proteins modulate GABAergic signaling to impact sympathetic outflow and blood pressure.

Support or Funding Information

T32 HL007224 to PC

R01 HL139867 to RDW

R01 HL141406 to RDW

R56 AG057687 to RDW
Interrogating the role of eIF3 on the ribosome and across the transcriptome
Authors: Aitken Colin Echeverría , Yoo Kyungyoon , Stanciu Andrei , Luo Juncheng
Published: April 2020

Translation initiation is the rate‐limiting and most‐regulated phase of translation. Misregulation of translation initiation has been implicated in myriad maladies, including cancer and neurodegenerative disease. In eukaryotes, initiation is a complex pathway requiring the participation of at least twelve initiation factors (eIFs). Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these factors and appears to participate in events throughout the pathway.

In particular, eIF3 is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC.

These two arms appear to play distinct roles during translation initiation. The exit channel arm has been shown to interact with mRNA sequences 5′ of the AUG and to stabilize mRNA binding at the exit channel of the PIC. While mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA, mutations in the entry channel have the most pronounced effects.

eIF3 also appears to collaborate with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within the ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. The eIF3i and eIF3g components of the entry‐channel arm also appear to contribute to the PIC‐dependent enhancement of eIF4A ATPase activity. Recent work suggests that eIF4A may function to remodel the mRNA entry channel and thereby promote mRNA recruitment or scanning.

Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches. In particular, we are exploring the transcriptome‐wide translational effects of depleting or disrupting the eIF3 complex, using ribosome profiling. We observe dramatic decreases in global translation for both eIF3 variants, as well as specific mRNAs whose translation is hypersensitive to each. In both cases, these mRNAs possess longer and more structured 5′ UTRs, consistent with the role of eIF3 in mRNA recruitment and scanning. In parallel, we are investigating the effects of mutations to the 40S protein Rps2 on the PIC:mRNA interaction, as well as how mutations to Rps2 and Rps3 affect the sensitivity of this interaction to mutations within the eIF3 complex.
Identifying the compositional features facilitating recruitment of prion‐like domains to stress granules
Authors: Baer Matthew H. , Ross Eric D.
Published: April 2020

Stress granules are highly dynamic nonmembranous cytoplasmic RNA‐protein assemblies that form via interactions between mRNAs stalled in translation initiation and RNA‐binding proteins. Many of the RNA‐binding proteins in stress granules contain prion‐like domains (PrLDs). PrLDs are intrinsically disordered protein domains that compositionally resemble yeast prion domains; some PrLDs have been identified as playing a key role in stress granule formation. PrLDs are thought to be recruited to stress granules in part by liquid‐liquid phase separation (LLPS). Mutations in various RNA binding proteins containing PrLDs are associated with degenerative diseases, including Amyotrophic Lateral Sclerosis. These mutations are associated with the formation of cytoplasmic inclusions that share common components with stress granules, suggesting that the mutations perturb stress granule dynamics. However, the sequence features that drive recruitment of PrLDs into stress granules have yet to be completely defined. We recently demonstrated that many PrLDs are sufficient for stress granule recruitment in yeast, and that this recruitment is driven largely by amino acid composition. Here, we utilize synthetic prion‐like domains to rigorously examine the compositional features driving stress granule recruitment. We show that based solely on amino acid composition, we can rationally design synthetic PrLDs that are recruited to stress‐induced assemblies. Surprisingly, although aromatic amino acids are widely believed to play a key role in recruitment of PrLDs to stress granules, we find that aliphatic amino acids can functionally replace aromatic amino acids in supporting recruitment.
Morphometric Characterization of Extracellular Vesicles in Adults with Obesity
Authors: Alvarado Herminia Mendivil , Munguia Bibiana Chavez , Millan Elizabeth Carvajal , Hernandez Manuel Hernandez , Teros Veronica Lopez , Moreno Bertha Pacheco , Duran Lucila Rascon , Garcia Humberto Astiazaran
Published: April 2020

Extracellular vesicles (EVs) are an extracellular communication system. Recently, they have more studied for their function as intercelular communication vehicles and their participation in physiological processes and specific health conditions. However, little is know about the influence of excess weight on their morphometric characteristics. The aim was to characterize populations of extracellular vesicles in adults with obesity and normal weight. Study design was cross‐sectional and subjects were assigned to: group A (n=10) which included participants with obesity (Body Mass Index (BMI)>30 kg/m2), and group B (n=10) included subjects with normal weight (BMI 18 – <25 kg/m2). Subjects were free of co‐morbidities. Weight, height, electrical bioimpedance, and serum lipids were evaluated. EVs were isolated from blood plasma using Invitrogen total exosome isolation reagent. Isolation was later corroborated by detecting the expression of the marker ALIX, an EV‐associated protein by Western blot (WB), and the particle size was assessed using Zetasizer Nano ZS. The isolated EVs were also analyzed by transmission electron microscopy (TEM). We identified two sizes of EVs, that were later classified as exosomes and microvesicles. The characterization of EVs was confirmed with the expression of ALIX and the images of TEM. In subjects from group A, EVs size (65.9 ± 16.1nm) was larger than in those subjects without obesity (42.3 ± 10.7nm), but this was not statistically significant. Body weight and tryglycerides concentration were positively associated (p<0.05) with EVs’ size. The variability in the size of EVs between groups could reflect different composition or maturation level of EVs. The size of EVs is related with state of obesity, also its content could reflect higher risk of developing comorbidities associated with excess weight and nutritional status.

Support or Funding Information

The project was funded by CIAD and MAH received a scholarship from CONACYT.
A Morphometric Analysis of Early Eocene Euprimate Calcanei from Gujarat, India
Authors: Llera Catherine J. , Rose Kenneth D. , Sylvester Adam D.
Published: April 2020

Postcrania dating back to the early Eocene from the Cambay Shale Formation of Gujarat, India, provide a unique window for understanding early primate behavior. Previous metric analyses of postcrania assigned to Asiadapinae indicate that these primates were generalized arboreal quadrupeds. Examination of long bone cross‐sectional properties, however, complicates the initial behavioral interpretations because it shows that these fossils overlap with primates of differing locomotor behaviors. The purpose of this study is to re‐examine calcaneal morphology of the Indian material in order to refine the locomotor reconstructions of these fossils.

Surface models of fossil calcanei (n=6) were generated from micro‐computed tomography scans while those (n=73) representing 23 extant primate species were downloaded from MorphoSource. A weighted spherical harmonics analysis was carried out on the total sample. This morphological analysis represents genus‐zero surfaces using a common set of spherical harmonic functions. The harmonic coefficients associated with each calcaneus serve as shape descriptors that can be summarized via principal components (PC) analysis. Bivariate plots and models representing plus and minus two standard deviations of each PC were created to visualize morphological differences.

The first three PCs explain 52.4%, 9.7%, and 9.3% of the morphological variation in the sample respectively (71.4% combined). The first PC differentiates primates with elongated calcanei that are more likely to engage in vertical clinging and leaping, from more quadrupedal primates. The second PC distinguishes calcanei of slow climbers from primates that engage in other forms of locomotion (e.g., leaping and quadrupedalism). Extant primate morphology seems to follow a continuum in which relative anteroposterior calcaneal elongation negatively correlates with slow climber calcaneal morphology. As expected, the calcaneal morphology of Marcgodinotius indicus aligns with that of extant lemuriforms, and most closely resembles that of a generalized arboreal quadruped with some leaping ability. There are two fossils tentatively attributed to Asiadapis cambayensis included in the analysis. One groups with other generalized arboreal quadrupeds, while the other falls outside the range of calcaneal variation observed in extant primates. The outlier does not follow the general pattern of calcaneal morphology observed in extant primates; instead it is characterized by some aspects of slow climber morphology, but with a relative anteroposterior calcaneal elongation. These results may reflect the generalized morphology of this fossil, and consequently the behavior of this primitive euprimate.

Support or Funding Information

Supported by NSF DGE‐1746891, National Geographic Society, Leakey Foundation
Translational Study Tracking Dynamic Changes in Cardiac Pattern Variability and Systemic Inflammation in Critically Ill Patients
Authors: Tackett Shiloh , Guo Fei , Clifford Caitlyn , Campanaro Cara , Nethery David , Horton Kofe-Kermit , Fletcher David , Kaffashi Farhad , Hsieh Yee-Hsee , Bonfield Tracey , Loparo Kenneth , Dick Thomas , Jacono Frank
Published: April 2020

Sepsis is a dynamic complex condition with high patient mortality and health‐care system cost for which the understanding of underlying pathophysiology is still evolving. We hypothesize that pattern variability of centrally controlled physiological rhythms, like cardiac and respiratory rhythms, decreases with an increase in systemic inflammation, such as sepsis. In our lab, we have previously demonstrated in an animal sepsis model that respiratory pattern predictability will increase with disease onset and progression. We describe a complementary IRB approved clinical study, in which physiological waveform data were recorded continuously from consented adult patients (n=43 to date) admitted to the medical intensive care unit (MICU). Serum was collected at intervals determined clinically and subsequently quantified using Luminex assays. The electrocardiogram data was analyzed for heart rate variability (HRV) indices, including Coefficient of Variation, Poincare Plots and Power Spectral Density (high frequency power (HFP)) for the hour preceding and succeeding each serum sampling. Of the 15 patients analyzed, we identified 18 intervals between successive blood draws with a significant change in IL‐6 serum concentration, defined as a 20% relative change accompanied by >20 pg/mL absolute change. In each of the 6 time intervals where IL‐6 was significantly increased there was an associated decrease in HRV and/or HFP. In 12 time intervals IL‐6 decreased; 7 of these were associated with an increase in HRV and/or HFP. Previous studies and our own studies on humans and rats conclude that decreases in the variability, or increases in the predictability, of physiological rhythms correlate to diseased state. Our working hypothesis is that neural inflammation in the brainstem alters central regulation of cardio‐respiratory rhythms and their coupling. As sepsis has had consistent and widespread negative impact on patients, the heath‐care system, and healthcare economy, it is imperative to identify and describe biometrics that serve as early warnings to clinicians.

Support or Funding Information

NIH U01 EB021960‐01A1 and VA Research Service Merit Award I01BX004197‐01A1
Oxycodone‐induced Gene adaptations in the brain reward center in a murine Model of Neuropathic pain
Authors: Pryce Kerri D. , Kronman Hope , Ramakrishnan Aarthi , Torres-Berrio Angelica , Polizu Claire D. , Pena Catherine , Shen Li D. , Nestler Eric , Zachariou Venetia
Published: April 2020

The long‐term use of opioids for the treatment of chronic pain can lead to physical dependence and transition to addiction. Recent studies from our group revealed that chronic neuropathic pain promotes several adaptations in the brain reward regions (Descalzi et al., Sci. Signaling, 2017). Here, we are using Next Generation RNA sequencing and upstream pathway analysis to gain insight on gene expression adaptations triggered by persistent exposure to oxycodone in the presence and in the absence of pain. To this extent, we employed the spared nerve injury model (SNI) of neuropathic pain in adult C57Bl/6 mice and assessed peripheral neuropathy symptoms for 9 weeks. We then applied an oxycodone misuse paradigm, where mice were treated daily with oxycodone for two weeks, and gene expression was monitored three weeks after oxycodone withdrawal. Mice were also assessed for emotional and pain‐related behaviors during the three‐week drug withdrawal period. Oxycodone treatment‐induced thermal hyperalgesia regardless of pain states, while withdrawal produced significant mechanical allodynia and deficits in social interaction. Using RNA Sequencing, we monitored changes in gene expression in the medial prefrontal cortex, the nucleus accumbens, and the ventral tegmental area. Although oxycodone treatment promotes mostly unique transcriptome profiles across brain regions, we observed similar downstream effectors and transcription factors to be affected in pain states when compared with non‐pain states. Furthermore, our findings suggest that chronic pain states exacerbate the behavioral and transcriptomic signatures of oxycodone withdrawal. Overall this work will provide new insights on the influence of long term pain in oxycodone‐induced plasticity in the brain reward center.

Support or Funding Information

5T32DA007135‐34, NIDA, R01 NS086444, R56 NS111351 NINDS, and P01 DA008227, NIDA
A cellular model of human mesothelium to study the physiology of water transport during peritoneal dialysis and the biocompatibility of innovative glucose‐sparing solutions.
Authors: Procino Giuseppe , Piccapane Francesca , Arduini Arduino , Corciulo Roberto , Carmosino Monica , Gerbino Andrea , Caroppo Rosa , Svelto Maria
Published: April 2020

The three pore model postulates that the endothelium of peritoneal capillaries is the major limiting barrier regulating water transport across peritoneal membrane during peritoneal dialysis (PD).

We hypothesize that the mesothelium may represent an additional selective barrier to water diffusion in PD.

We characterized an immortalized cell line of human mesothelium (HMC) to study the functional role of the water channel AQP1 expressed in vivo by mesothelial cells and also to test the biocompatibility of glucose‐sparing PD solutions containing xylitol and carnitine as the main osmotic agents.

Transepithelial water transport was measured by TEA+‐sensitive microelectrodes. The biocompatibility of conventional versus innovative PD solutions was evaluated by MTT‐test, measurement of transepithelial electrical resistance (TEER) and production of pro‐inflammatory cytokines.

HMCs showed polarized expression of Na+/K+‐ATPase and tight junctions markers but no endogenous expression of AQP1.

HMC showed a low TEER compared to renal cells not expressing AQP1. However, the transepithelial water transport was comparable between the two cell types.

Experiments in HMCs transfected with AQP1 cDNA, suggest that the water permeability of HMC is strictly dependent of the presence of AQP1.

Biocompatibility assays indicate that conventional dialysis solutions with high glucose significantly reduce both cell viability and TEER, and increase pro‐inflammatory cytokines production.

Interestingly, innovative PD solutions minimize these effects. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD. Importantly, we also demonstrated that the formulation of glucose‐sparing PD solutions containing xylitol and carnitine better preserve mesothelial cells viability and may represent a useful means to prolong the dialysis life of patients undergoing peritoneal dialysis.

Support or Funding Information

Apulian Region NATURe project Code: XUANRO4
Bringing 23andMe Into the Human Genetics Classroom
Authors: Smith Emmett M.
Published: April 2020

Research has shown that using a direct‐to‐consumer (DTC) genetic test in a genetics course enhances student performance (Adams 2016, Linderman 2018, Weber 2015, Weitzel 2016). I incorporated the 23andMe Health+Ancestry kit into my upper‐class undergraduate BIOL351 Human Genetics and Genomics course at Earlham College in 2018. Students opted in and were given informational forms, consent documentation and a list of local genetic counselors (approved by the college IRB, Parker and Grubs 2014). Students were never required to share their personal results in the classroom. Throughout the semester, I identified 23andMe tests that aligned with the disorders we studied. Students were asked to complete a scaffolded assignment about DTC genetic testing companies, beginning with an understanding of 23andMe’s privacy policy, extending to analysis of 11 DTC genetic testing companies’ validity (ranging from
Ancestry.com to Vinome), culminating in the creation and presentation of infographics to the general public in collaboration with our local Joseph Moore Museum. Students responded favorably in course evaluations. Of the 7 evaluations submitted (9 total students), 4 relayed that 23andMe was the most positive part of the course, stating “Seeing my own results … helped broaden my understanding of the subject;” “the 23andMe opportunity was very positive;” “23andMe … helped me to relate more to the material.” Here I present materials and projects that will help you incorporate DTC genetic testing into your own genetics course.

Support or Funding Information

Emmett M Smith

Earlham College, Richmond IN

Smithem3@earlham.edu
Fluvastatin Decreases Endothelial Nitric Oxide Synthase O‐Glcnacylation by Decreasing Glucose Uptake
Authors: Bohlman Samantha , Clyne Alisa Morss
Published: April 2020

Proper function of the endothelium, which lines all blood contacting surfaces, is essential in cardiovascular health. Endothelial nitric oxide (NO) production, which is regulated by endothelial nitric oxide synthase (eNOS) phosphorylation, is considered the gold standard measure of endothelial function. eNOS is O‐GlcNAcylated through the hexosamine biosynthetic pathway (HBP), which may inhibit eNOS phosphorylation. Statins, a cholesterol lowering drug class, increase NO production. This study aimed to determine if statins impact NO production through eNOS O‐GlcNAcylation. We hypothesized statins decrease eNOS O‐GlcNAcylation through a concomitant decrease in glucose metabolism and thereby increase NO production. To investigate this hypothesis, human umbilical vein endothelial cells (HUVEC) were treated with a physiologically relevant fluvastatin dose. Total, phosphorylated, and O‐GlcNAcylated eNOS were measured by Western blot. Metabolites were measured by YSI biochemical analyzer or mass spectrometry using U‐13C6 glucose. Fluvastatin increased eNOS protein in addition to decreasing eNOS O‐GlcNAcylation 24 hours post treatment. To determine whether fluvastatin directly decreased O‐GlcNAcylation via the catalytic enzyme O‐GlcNAc transferase (OGT), endothelial cells were treated with statins along with glucosamine, an O‐GlcNAc substrate which feeds directly into the HBP. Glucosamine reversed fluvastatin effects on eNOS O‐GlcNAcylation, suggesting that fluvastatin does not directly affect catalytic addition of O‐GlcNAc to protein targets. Further, OGlcNAcase (OGA) activity, which would catalyze O‐GlcNAc removal, did not increase with statin treatment. Instead, endothelial cell glucose consumption and lactate production decreased 24 and 48 hours after statin treatment. Since the ratio of glucose consumption to lactate production was similar across all samples, statin treatment did not change the preferential use of glucose for anaerobic glycolysis. Metabolic mass spectrometry showed that statin treated HUVEC had markedly reduced glycolytic metabolites after fructose‐1,6‐bisphosphate, suggesting that fluvastatin likely decreases O‐GlcNAcylation by acting at or below phosphofructokinase to decrease glucose metabolism. These studies show that statins may have additional beneficial cardiovascular effects through lowered endothelial glucose metabolism.
Endocannabinoid hydrolases are differentially distributed in human blood fractions and differentially influenced by thrombin
Authors: Alexander Stephen , O’Sullivan Saoirse , Anajirih Nuha
Published: April 2020

Plasma levels of the endocannabinoids anandamide and 2‐arachidonoylglycerol have been reported in a number of studies using both humans and laboratory animals, with typically much higher levels of 2‐arachidonoylglycerol than anandamide. In this investigation, we sought to characterize activities of the two primary endocannabinoid hydrolases, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) responsible for anandamide and 2‐arachidonoylglycerol hydrolysis, respectively, in different fractions from human blood.

Following approval from the School of Life Sciences Ethics Committee, University of Nottingham, overnight fasting blood samples were taken in the morning from otherwise healthy volunteers. Blood fractions were isolated, including platelet‐rich plasma, packed erythrocytes (RBC), washed platelets, plasma and serum. Thrombin was employed to trigger clot formation, particularly with RBC and platelet‐rich plasma. Following the generation of a clot, the supernatant layer from a low speed centrifugation, equivalent to serum, was harvested and assayed for endocannabinoid hydrolase activity. The activities of FAAH and MAGL were quantified using both radiometric and ABPP assays.

Of the blood fractions examined, MAGL activity was highest in platelets (92 % of the MAGL activity in whole blood), while FAAH activity was highest in RBC. Intriguingly, expressing the FAAH activity of various fractions relative to whole blood summated to 854 %, with RBC constituting 824 % of the whole blood activity. There were no differences between male and female volunteers in the levels and pattern of enzyme activities in the different blood fractions. Assessing enzyme activities from individual donors in these fractions on three visits separated by two week intervals showed no significant differences. The level of FAAH and MAGL activities was relatively stable in washed platelet and RBC samples after being stored in liquid nitrogen, while more than 50 % of their activities were lost after 3 days of storage at −80 °C.

Interestingly, FAAH activity was low but detectable, while MAGL was essentially undetectable in serum. Thrombin addition to a washed platelet or RBC fraction caused clot formation within 5 min. FAAH activity in the supernatant fraction following thrombin exposure was much higher from RBC relative to platelets, in accordance with the activities in those fractions. In contrast, the activity of MAGL was significantly reduced in RBC and platelet fractions induced by thrombin compared to nonactivated erythrocytes and platelets, with negligible activities present in the releasate.

In summary, we have identified a differential distribution of endocannabinoid hydrolases across different fractions of human blood, and identified the potential for FAAH, but not MAGL, release from RBC and platelets during thrombus formation.

Support or Funding Information

Funded by the Saudi Cultural Bureau
Systemic blockade of adenylyl cyclase type 1 (AC1) prevents the recall of extinction memory
Authors: Gonzalez Alexandra , Brust Tarsis , Santini Edwin
Published: April 2020

Previous transgenic studies have shown that the expression of adenylyl cyclases is necessary for memory consolidation. Studies using genetically modified mice have shown that the expression of the type 1 adenylyl cyclase (AC1) is required for hippocampal‐dependent long‐term memories, including the acquisition of contextual fear conditioning. However, to our knowledge, no pharmacological study has been done to examine the role of AC1 in auditory fear conditioning and extinction. To test the effects of pharmacological inhibition of AC1 during fear extinction, rats were fear conditioned and divided into the control (n=12) and experimental groups (n=12) on day 1. Groups were matched based on equivalent levels of conditioned freezing (p > 0.05). On day 2, rats were injected with vehicle control or the selective AC1 inhibitor, ST034307, prior to extinction training. On day 3, both groups were tested for recall of extinction memory. During extinction training, ST034307‐injected rats showed similar rates of extinction within a session compared to vehicle controls (p > 0.05). However, 24‐hrs later at test, ST034307‐injected rats showed significantly higher levels of conditioned freezing compared to vehicle‐injected rats (p < 0.01). Together these data suggest that AC1 plays a critical role in memory consolation and long‐term recall of fear extinction memory. Based on clinical data, it has been proposed that patients suffering from anxiety disorders, such as post‐traumatic disorder (PTSD) might be deficient in extinction‐related mechanisms. A better understating the role of AC1 might lead to the identification of novel targets for drug pharmacotherapy aimed for the treatment of PTSD.

Support or Funding Information

Palm Beach Atlantic University Quality Initiative (QI) Grant to ES; Intergra Connect Faculty Research Grant at Palm Beach Atlantic University to ES; New Investigator Award, American Association of Colleges of Pharmacy to TB
Structural and Functional Insights into TAZ2‐mediated CBP/p300 Recruitment by the Melanogenic Transcription Factor MITF
Authors: Brown Alexandra , Vergunst Kathleen , Branch Makenzie , Baillie George , Dupre Denis , Langelaan David
Published: April 2020

The microphthalmia‐associated transcription factor (MITF) is a master regulator of development and differentiation within the melanocyte lineage. However, aberrant MITF activity can lead to multiple malignancies such as skin cancer, where it plays a key role in modulating the proliferation and invasiveness of melanoma. MITF is a basic helix‐loop‐helix leucine zipper transcription factor that binds to specific gene promoters via a central DNA‐binding domain. MITF also recruits transcriptional co‐activators, such as the histone acetyltransferase CREB‐binding protein and its homologue p300 (CBP/p300) through an N‐terminal acidic transactivation domain (TAD), however the details of these interactions are not yet fully understood. In order to gain insight into the mechanisms of gene regulation by MITF, we investigated the structure and functional interaction between MITF‐TAD and the transcription adapter putative zinc finger (TAZ2) domain of CBP/p300. In mammalian‐one‐hybrid assays MITF transcriptional activity was enhanced in the presence of co‐transfected CBP/p300 and abolished upon deletion of residues within the MITF‐TAD. Peptide microarrays indicated that no one residue of MITF is essential for TAZ2 binding, however, deletion of multiple residues in MITF‐TAD ablated its ability to bind TAZ2. NMR‐based chemical shift mapping experiments determined that MITF‐TAD interacts with the same surface of TAZ2 as the adenoviral protein E1A, which has been shown to inhibit MITF function. We determined that E1A and MITF‐TAD directly compete for CBP/p300 through the TAZ2 domain using NMR‐based titrations, pulldown, and mammalian‐hybrid assays. Furthermore, we used qPCR to measure the effect of disrupting these interactions on the transcription of MITF‐specific target genes in melanoma‐derived cell lines. Understanding mechanistic details regarding the interaction between MITF and its co‐activators is fundamental to our understanding of gene regulation by MITF and may outline a potential new strategy to inhibit MITF function.

Support or Funding Information

This research was funded by the Beatrice Hunter Cancer Research Institute, Nova Scotia Heath Research Foundation Scotia Scholars Award, a Canadian Institutes of Health Research studentship, Dalhousie Nova Scotia Graduate Scholarship, and Izaak Walton Killam Predoctoral Scholarship.
Actin Destabilization Enhances Thermally‐induced Morphological Abnormalities in Erythrocytes
Authors: Weber-Fishkin Samantha , Eligulashvili Anna , Frame Lesley , Frame Mary
Published: April 2020

Thermal burns are a significant clinical problem with limited treatment options. Burn area progresses radially over several days, blocking oxygenated flow to a progressively increasing region. By 4 hours post‐burn, red blood cell (RBC) aggregates form at the burn margin, without evidence of fibrin clots, indicating a potential for early intervention (Clark, 2013). The present study focuses on understanding the early aggregation process. The RBC’s biconcave shape is due to the cytoskeletal structure that is tethered to the membrane; many pathologies occur when this is disrupted. The RBC cytoskeleton includes spectrin‐actin meshwork that affects membrane curvature, mechanics, and microdomain formation. Our specific hypothesis is that elevated temperatures in arterial oxygen conditions (10%) contributes to the actin cytoskeleton destabilization, thus permitting membrane deformation. Washed human RBCs (BioIVT, Westbury, NY) were suspended in saline (PBS) containing 1% DMSO, 3.7μM Jasplakinolide (JASPA, polymerizes F‐actin), 26.14μM Latrunculin A (LatA, depolymerizes F‐actin), 0.5μM Cytochalasin D (CytoD, disrupts all cytoskeletal integrity), 2μM 4,4′‐Diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS, binds to band 3, untethering the cytoskeleton from the membrane) (1% DMSO) or control (PBS and platelet poor plasma, PPP). All conditions were exposed to five temperatures (37, 41, 43, 45, or 49°C) and three oxygen levels (0, 5, or 10% O2) for 10 minutes. Suspensions were fixed with 4% paraformaldehyde (PFA) at the test temperature/oxygen and imaged using a 60X water immersion objective. Singlet RBCs were scored by their morphology, in accordance with clinical standards, and identified by degree of echinocyte (I, II, and III), type of non‐echinocyte abnormal shape, or as discocytes. With increasing temperature, the population shifted from the discocyte shape for all control conditions. Increasing oxygen (in plasma) likewise shifted RBC morphology to pathological shapes, but with a less pronounced effect. CytoD, DIDS, JASPA and LatA each eliminated the discocyte populations at all temperatures and oxygen levels. Cells were also prepared for SEM imaging using 1.5% glutaraldehyde and 1.5% PFA and serial dehydration. RBC shapes obtained from brightfield images (current clinical practice) were compared to shapes obtained from SEM images. Population distribution of shapes from these two image pools were not different. Overall, we show that heat and increasing oxygen promotes pathological RBC morphologies. Deliberate disruption of the actin cytoskeleton induces these morphologies, which is exacerbated by increasing temperature. Further, LatA and JASPA cause similar population distributions of shapes, thereby indicating any imbalance of the equilibrium between filamentous and globular actin triggers morphological abnormalities.
"Effects of the Human SULT1A1 Polymorphisms on the Sulfation of 4‐hydroxytamoxifen and Endoxifen"
Authors: Rasool Mohammed Ibrahim , Liu Ming-Cheh
Published: April 2020

Background

Human sulfotransferase 1A1 (SULT1A1) catalyzes the sulfation of a wide variety of substances including 4‐hydroxytamoxifen (4‐HT) and endoxifen, the active metabolites of tamoxifen. Polymorphisms were reported in SULT1A1 gene. A functional single nucleotide polymorphism (SNP) in the SULT1A1 gene (R213H) showed lower catalytic activity and thermal enzyme stability. Previous data have suggested that this particular genotype may account for a portion of individual variation in responding to tamoxifen therapy. The current study was performed to investigate the effects of SNPs of human SULT1A1 genes on the enzymatic characteristics of the sulfation of 4‐HT and endoxifen by SULT1A1 allozymes.

Methods

Online databases were searched for SULT1A1 nonsynonymous coding SNPs. Nine SULT1A1 allozymes coded by missense SNPs were selected based on the predicted importance of the amino acid variations, were generated, bacterially expressed and purified by glutathione‐Sepharose affinity chromatography. Purified SULT1A1 allozymes were analyzed for sulfating activity using an established assay procedure.

Results

Specific activity obtained showed the differential sulfating activities of SULT1A1 allozymes toward both antiestrogenic compounds.

Conclusion

The activity data obtained for the SULT1A1 allozymes analyzed indicated the importance of the amino acid residues in the proper functioning of the SULT1A1 enzyme, which may affirm the variability in the metabolism of tamoxifen that could affect its efficacy in individuals with different SULT1A1 genotypes.

Support or Funding Information

This work was supported in part by a grant from National Institutes of Health (Grant #R03HD071146).
Kv7.1 Channels Contribute to Neonatal Renal Blood Flow Autoregulation Induced by a Step Decrease in Arterial Pressure
Authors: Peixoto-Neves Dieniffer , Soni Hitesh , Adebiyi Adebowale
Published: April 2020

Kv7 channels, the voltage‐gated potassium channels encoded by KCNQ genes, are expressed in a wide variety of blood vessels, including coronary, mesenteric, cerebral, and renal arteries. In adult rodents, Kv7 channels mediate heterogeneous basal and agonist‐induced vascular reactivity. Although postnatal changes in functional expression of mammalian Kv7 channels have been reported, the physiological significance of these channels in neonatal renal microvessels is unknown. The objective of this study was to determine whether Kv7 channels regulate neonatal renal microcirculation. Polymerase chain reaction indicated that out of the five members of KCNQ genes, only KCNQ1 (Kv7.1) is expressed in smooth muscle cells (SMCs) isolated from preglomerular microvessels of neonatal pigs. Confocal microscopy demonstrated that Kv7.1 is expressed in the plasma membrane of renal vascular SMCs. Kv7.1 channel activators induced whole‐cell currents in neonatal pig renal vascular SMCs, which were inhibited by HMR1556 (HMR), a selective Kv7.1 inhibitor. However, HMR did not cause a significant change in steady‐state diameters of neonatal pig distal interlobular arteries that were pressurized to physiological renal arterial pressure. Also, intrarenal artery infusion of HMR did not alter the mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance in the pigs. An approximately 20 mmHg reduction in the MAP evoked effective autoregulation of the RBF in neonatal pigs, which was inhibited by HMR. Together, these findings suggest that renal vascular SMC Kv7.1 does not control basal renal vascular tone in neonatal pigs, but contribute to RBF autoregulation induced by a step decrease in arterial pressure.

Support or Funding Information

American Heart Association (AHA), National Institutes of Health (NIH).
19‐(S/R)Hydroxyeicosatetraenoic Acid is a Novel Endogenous Inhibitor of Cytochrome P450 1B1 in Enantioselective Manner
Authors: Shoieb Sherif , El-Sherbeni Ahmed A. , El-Kadi Ayman O. S.
Published: April 2020

Purpose

CYP1B1 is known to contribute to the pathogenesis of several diseases such as cancer and cardiac hypertrophy by forming the toxic metabolites. We recently reported that the arachidonic acid metabolite, 19(S/R)hydroxyeicosatetraenoic (HETE) acid, confers cardioprotection against cardiac hypertrophy by inhibiting the formation of cardiotoxic metabolites, midchain HETEs, known to be formed by CYP1B1. This raised the question whether 19(S/R)‐HETE can directly inhibit the CYP1B1.

Methods

Human ventricular cardiomyocytes, RL‐14 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 20 μM 19(R)‐HETE or 19(S)‐HETE for 24 h. Gene and protein expression were measured using real‐time PCR and Western blot analysis, respectively. The level of mid‐chain HETEs was determined using liquid chromatography–mass spectrometry (LC/MS). The O‐dealkylation rate of 7‐ethoxyresorufin (EROD) by recombinant human CYP1B1 was measured in the absence and presence of 19(R)‐HETE or 19(S)‐HETE.

Results

The results showed that both 19(R)‐HETE and 19(S)‐HETE significantly decreased the metabolite formation rate of midchain HETEs, namely 8‐, 9‐, 12‐ and 15‐HETE compared to control group. Nonlinear regression analysis and comparisons showed that the mode of inhibition for 19(R)‐HETE and 19(S)‐HETE is non‐competitive inhibition of CYP1B1 enzyme. Dixon plots showed that 19(R)‐HETE and 19(S)‐HETE have Ki values of 89.1 and 37.3 nM, respectively. The Ki values of both enantiomers showed that the S‐enantiomer is more potent than the R‐enantiomer by approximately 2.4 fold.

Conclusion

The current study suggests that 19(R)‐HETE and 19(S)‐HETE could be considered a novel therapeutic modality in the treatment of cardiac hypertrophy, and cancer. Moreover, given that a non‐competitive inhibitor might bind to the enzyme regulatory region, 19(R)‐HETE and 19(S)‐HETE could be the focus for unraveling the obscure mechanisms of CYP1B1 enzymatic reaction.

Support or Funding Information

This work was supported by a grant from CIHR to A.O.S.E. S.M.S. is the recipient of Alberta Innovates Graduate Student Scholarship.
Effects of Wastewater Treatment Plant Effluents on the Microbial Biomass and Community Structure of the Big Walnut Creek
Authors: Atkinson Sophia , Luchmun Hashleyna , Akinwole Philips
Published: April 2020

Excessive organic matter input and nutrient loading are considered a major ongoing threat to water quality. Communal wastewater treatment is a process whereby sewage water is treated to remove contaminants before being discharged back to the environment. As most Wastewater Treatment Plants (WWTPs) do not remove all contaminants from sewage waters, we examined the impact of WWTPs effluents on microbial biomass and community structure of the Big Walnut Creek, Greencastle, IN. We took triplicate sediment samples along a 6,000 meter transect at five different locations, two upstream and three downstream of the outfall in June, September, and November of 2019. We quantified microbial community structure as community level physiological profiling (CLPP) using Biolog EcoPlates as well as microbial biomass using phospholipid phosphate analysis. The samples in June right above and below the outfall had comparably lower carbon to nitrogen (C/N) ratios (39.63 and 32.44), and therefore more available nutrients for microbial uptake. The samples further downstream from the outfall (1,452 m and 4,677 m) had high C/N ratios of 65.8 and 88.09 respectively, while samples farther upstream from the outfall (1,600 m) had a C/N ratio of 93.53. Microbial biomass increased from June to November for all five sites due to seasonal allochthonous material inputs. We noticed that the C/N ratio correlated to the increase in microbial biomass observed at the outfall for the June samples. Interestingly, the further away from the outfall, the lower the biomass measured and the higher the C/N ratios. We did not observe any significant difference in microbial biomass across sites during the months of September and November. Increased inputs of allochthonous materials during these months (falling leaves and algae) might have obscured the effects of the WWTP effluent. Principal component analysis (PCA) of the CLPP showed two distinct microbial communities for samples collected at the outfall across seasons and separately grouped together microbial communities from non‐outfall samples. PC 1 explained 28.04% variation observed in microbial community and the most dominant microbial groups on the positive component of PC 1 includes Corynebacteriaceae, Acetobacteraceae, and Lactobacillaceae. Overall, this study showed that WWTP effluent impacts the immediate surroundings of its input, but can be obscured by allochthonous materials or biological transformation of nitrogen downstream. Further research should look into antibiotic resistant genes and resistant bacteria at these separate sites to see if WWTP effluents might have further health impacts on the stream ecology of the Big Walnut Creek.

Support or Funding Information

Support from the Biology Department and the Science Research Fellows program at DePauw University.