bluuebunny/crossref_metadata_2025 huggingface dataset:
The internal elastic lamina of resistance arteries facilitates vasodilation
Authors: Ruddiman Claire A. , Milosek Victoria , Peckham Richard , Mecham Robert P. , Wagenseil Jessica E. , Isakson Brant E.
Published: May 2022

In large conduit arteries, smooth muscle cell elastin produces elastic laminae, while in resistance arteries endothelial cell derived elastin is the major contributor to the internal elastic lamina (IEL). The IEL in resistance arteries separates smooth muscle and endothelium and contain distinct holes in the IEL (HIEL) where the two cell types communicate via a structure termed the myoendothelial junction (MEJ). The MEJ is an anatomical hallmark of resistance arteries that pierces through the IEL and facilitates vasodilation by movement of endothelial derived hyperpolarization (EDH) to smooth muscle. In contrast, large arteries dilate via nitric oxide (NO) and do not contain MEJs. To study the role of the IEL in resistance arteries in facilitating MEJ formation and EDH‐based vasodilation, we used a mouse with a Cdh5Cre to constitutively knockout elastin from endothelial cells (elastin ECKO). In en face preparations from third order mesenteric arteries, elastin ECKO mice had a 55.49% reduction in IEL surface area, and, importantly, completely lacked the distinct HIEL where MEJs could form. Regardless of this, en face claudin‐5 staining demonstrated a normal endothelial cell morphology. The lack of distinct HIEL, the canonical MEJ sites, led us to hypothesize vasodilatory mechanisms may be disrupted. To test this hypothesis, we first performed pressure myography on third order mesenteric arteries from elastin ECKO mice. Myogenic tone began to diverge from littermate controls starting at 80 mmHg, with preserved active, but not passive tone. For this reason, to test for changes in EDH between control and elastin ECKO, we used arteries pressurized to 60 mmHg and performed acetylcholine (Ach) dose‐response curves. We found elastin ECKO mice have decreased responses to Ach. Additionally, in the presence of 30 uM L‐NAME, the majority of dilation in elastin ECKO arteries was inhibited, compared to no inhibition with L‐NAME in the littermate controls. This indicated that NO‐mediated dilation was present in elastin ECKO mice and not littermate controls. En face immunohistochemistry demonstrated disruption of proteins localized to MEJs that regulate EDH in resistance arteries: both phosphatidylserine (See Abstract ID:R5515) and alpha globin, which is used to chelate NO. These results indicated that the disruption in the IEL, and thus canonical MEJ sites, affected the polarization of EDH‐associated proteins in the endothelium, supporting the observation of increased NO‐based signaling in elastin ECKO arteries. Overall, these data imply endothelial‐derived elastin is necessary for the functional formation of MEJs, the vasodilatory capacity of the endothelium, and the pathway of vasodilation in resistance arteries.
A theoretical model for flow regulation in the cerebral cortex: role of penetrating arterioles
Authors: Lee Grace V. , Secomb Timothy W.
Published: May 2022

Activation of a region of the brain results in a local increase in blood flow, a process known as neurovascular coupling (NVC). Here, a theoretical model of blood flow regulation in the cerebral cortex is utilized to investigate the potential role of penetrating arterioles as the primary mechanism for local control of blood flow. Penetrating arterioles connect pial arterioles with the capillary meshwork deeper in the cortex and have a typical length of approximately 600 μm. This is about one‐sixth of the length of corresponding small arterioles in skeletal muscle. The question arises whether these relatively short arterioles can achieve the range of flow modulation observed in the cerebral cortex. A previously developed compartmental model for flow regulation in skeletal muscle was modified to represent the structure of cerebral microvasculature. A compartment consisting of penetrating arterioles with a reference diameter of 14.8 μm and a length of 600 μm is assumed to provide active control of blood flow. Smooth muscle tone in these arterioles is assumed to depend on a combination of signals representing myogenic, shear‐dependent, and metabolic responses. The metabolic response is represented by a change in endothelial cell membrane potential, propagated upstream along arterioles by conducted responses. Sensitivity of diameter to membrane potential was determined based on experimental data. According to the model, a hyperpolarization of 40 mV can result in an increase in local blood flow by approximately 70%. Since increases in local blood flow of 50% with activation have been observed, these results imply that vasoconstriction and vasodilation by penetrating arterioles can provide typical levels of blood flow control seen in neurovascular coupling.
Systematic Integration of Epigenomic Landscapes in Human and Mouse Blood Cells to Predict Activity and Targets of Regulatory Elements
Authors: Hardison Ross C. , Xiang Guanjue , Jansen Camden , Keller Cheryl A. , Giardine Belinda M. , Cockburn April , He Xi , Sauria Michael E. , Weaver Kathryn , Li Qunhua , Zhang Yu
Published: May 2022

Thousands of genome‐wide epigenetic (epigenomic) datasets determined in the past decade have generated maps of chromatin accessibility, histone modifications, and binding by transcription factors and structural proteins in a large number of cell types in humans and model species. Such maps have revealed many informative associations between groups of epigenetic features and different classes of cis‐regulatory elements (CREs), and they enable a prediction of the locations of candidate CREs (cCREs) in many genomes. The actuation (opening of chromatin) and epigenetic state (combination of features such as histone modifications) acquired at specific cCREs has been associated with specific gene activation. However, despite the large number and rich variety of available epigenomic datasets, it is difficult for researchers to effectively utilize all the data relevant to their projects. Furthermore, comprehensive rules and catalogs have not been established for which CREs regulate specific target genes and how. Integrative analysis can help meet these needs, and our VISION project provides a ValIdated Systematic IntegratiONof epigenomic data in hematopoiesis in human and mouse blood cells. After careful curation and normalization of epigenomic data from many sources, we systematically integrated these data to generate a comprehensive view of the regulatory landscape (epigenetic states) and predicted cCREs in differentiating hematopoietic cell types jointly in both mouse and human. Multiple machine learning approaches reveal target genes predicted to be regulated by each element in different cell types. These approaches include predicting the regulatory impact of each element on targets by regression models, finding linkages between distinctive clusters of elements and potential target genes from self‐organizing maps, and visual overlays of chromatin interaction maps. Directed mutation and expression analysis around selected genes supports the utility of the assignments. These catalogs of highly annotated regulatory elements are useful for many applications, including interpretation of the phenotypic impact of genetic variants in noncoding genomic regions. Resources from the VISION project are available at our website usevision.org.
Nicotine Treatment Resulted in Decreased Protein Expression of Prorenin and Soluble Prorenin Receptor in 3T3‐L1 cells
Authors: Collins Katelyn B. , Nichols Kellea B. , Poupeau Audrey , Taylor Alexis , Yiannikouris Frederique
Published: May 2022

Over 36.5% of the United States population is affected by obesity, which is a major contributing factor to the rise of hypertension and cardiovascular disease. Furthermore, the CDC reports that over 16 million Americans suffer from nicotine‐related diseases. Nicotine consumption causes an increase in blood pressure and a decrease in adipogenesis. The prorenin receptor (PRR) and its soluble form (sPRR) are newly discovered components of a blood pressure regulatory pathway, the renin angiotensin system (RAS), and are upregulated during times of obesity. Currently, there is no research to understand the contrasting relationship between nicotine and PRR. Therefore, in this present study, we aimed to determine whether nicotine regulates blood pressure through an adipocyte PRR‐dependent.

3T3‐L1 cells were grown to confluence and differentiated over 8 days. On day 8, cells were treated with nicotine dose response treatments: vehicle (water), 0.01 µM nicotine, 1 µM nicotine, or 10 µM nicotine. After 24‐hour treatment, cells were collected to measure protein levels of PRR and it soluble form, sPRR. Additionally, gene expression levels of PRR, renin, 18S, furin, and S1P were measured. Media from the cells was collected after 2 hours, 4 hours, and 24 hours of incubation to measure the secretion levels of sPRR into the media.

Nicotine significantly decreased PRR protein expression at 1µM (n=2) compared to vehicle treatment (n=6) (vehicle: 1.00 0.37au; 1 µM: 0.46 0.09au; p <0.05). Nicotine did not affect the gene expression of PRR but significantly decreased PRR and sPRR protein levels in a dose response manner compared to vehicle treatment (vehicle: 1.00 0.42au, n=6; 0.01 µM: 0.47 0.18au, n=4, 1uM: 0.32 0.12au, n=2; p <0.05). Nicotine did not change the expression of S1P, and furin, two enzymes involved in PRR cleavage. Finally, renin and angiotensinogen (AGT) increased significantly in differentiated adipocytes treated with nicotine compared to those treated with vehicle. Together our data indicated that nicotine influences local renin angiotensin system by post‐transcriptionally down‐regulating PRR and sPRR protein and by upregulating renin and AGT. Together, these data suggest that nicotine could assert its effect on blood pressure and adipogenesis via an adipocyte‐PRR dependent mechanism.
Effects of a Recombinant Humanized Anti‐Cocaine Monoclonal Antibody on the Metabolism and Distribution of Cocaine In Vitro and in Mice
Authors: Zinani Dakota B. , Turner Mackenzie E. , Wetzel Hanna N. , Crutchfield Christopher A. , Norman Andrew B.
Published: May 2022

The anti‐cocaine monoclonal antibody (mAb), h2E2, is a candidate for treating cocaine‐use disorder. h2E2 binds to and sequesters cocaine in the plasma compartment, effectively decreasing cocaine concentrations in the brains of rats and mice. Despite the binding of cocaine to h2E2, plasma cocaine concentrations decline rapidly in rodents over time, but there was a drastic decrease in the urinary elimination of cocaine in the presence of h2E2. Since cocaine is not being renally excreted, the apparent disappearance of cocaine from the plasma must be explained by either metabolism or distribution. However, binding of cocaine to h2E2 may restrict the availability of cocaine for hydrolysis by endogenous esterases. Therefore, the antibody would be expected to extend the elimination half‐life of cocaine. In contrast, previous studies reported h2E2 as having no effect on the rate of cocaine clearance. It is important to examine the ultimate clearance of the cocaine to ascertain half‐life and potential for re‐intoxication. Therefore, we investigated the effects of h2E2 on cocaine hydrolysis in vitro and on cocaine metabolism and disposition in vivo over a six‐hour time course. The spontaneous and enzyme‐mediated in vitro hydrolysis of cocaine was drastically decreased in the presence of h2E2 in vitro. Additionally, in mice, h2E2 significantly increased the distribution and elimination half‐lives of cocaine relative to vehicle controls over an extended time course. Therefore, we concluded that h2E2 slowing the distribution and elimination of cocaine is the most appropriate explanation for the initial disappearance of cocaine from the plasma in vivo.
Phosphodiesterase 7B regulates prostanoid‐ but not ß‐adrenergic‐stimulated cAMP levels in primary human airway smooth muscle cells
Authors: McCuthcheon Dayanita P. , Fariborzi Mona , Cattani‐Cavalieri Isabella , Nuñez Francisco J. , Roosan Moom R. , Ostrom Rennolds S.
Published: May 2022

Human airway smooth muscle (HASM) cells contain G‐protein coupled receptor (GPCR) signal transduction pathways that are organized in the plasma membrane and form cAMP signaling compartments. One such compartment contains ß2‐adrenergic receptors (ß2AR) coupled to adenylyl cyclase (AC)6 and is principally localized in lipid rafts. Another compartment expresses E prostanoid receptors 2 and 4 (EP2/4) coupled to AC2 and is restricted to non‐raft regions of the plasma membrane. Various phosphodiesterase (PDE) isozymes modulate cyclic AMP (cAMP) levels in response to these pathways. Non‐specific or PDE‐family specific PDE inhibitors have been used to treat asthma and chronic obstructive pulmonary disease with limited success, in part due to expression of these PDE isoforms throughout the body. Drugs that inhibit specific PDE isozymes might produce therapeutic effects in airways with fewer off‐target effects. RNA‐sequencing data in HASM cells indicates that PDE7B is the third most highly expressed PDE isoform and includes expression of both the PDE7B‐201 and PDE7B‐203 splice variants. We sought to characterize the role of PDE7B in the lipid raft and non‐raft cAMP signaling compartments. We used siRNA to knock down both PDE7B splice variants, confirmed protein reduction via Western Blot analysis, and then observed live cells’ real‐time cAMP levels in response to either isoproterenol (ßAR agonist) or PGE2 (EP agonist). The cADDis‐FMP biosensor, which localizes in lipid raft regions, did not report altered cAMP dynamics in response to ß2AR or EPR agonists following PDE7B knockdown. However, the cADDis‐FS15 biosensor, which reports cAMP levels in non‐raft plasma membrane regions, reported significant increases in cAMP levels stimulated by EPR agonists but no change in cAMP levels stimulated by ß2AR agonist. PDE7B may play a unique role in regulating non‐raft associated EP2/4 signaling without altering cAMP levels in other areas of the cell, thereby presenting a novel therapeutic target for pulmonary diseases.
Global DNA Methylation Status in Relation to Resistance Training with High vs Low Loads to Failure
Authors: Sexton Casey , Godwin Joshua , Ruple Bradley , Mcintosh Mason , Osburn Shelby , Hollingsworth Blake , Agostinelli Philip , Kavazis Andreas , Zeigenfuss Timothy , Lopez Hector , Smith Ryan , Young Kaelin , Dwaraka Varun , Mobley Christopher , Sharples Adam , Roberts Michael
Published: May 2022

Objective

Our objective was to determine how global DNA methylation status of skeletal muscle differs with resistance training (RT) to failure in trained participants using either 80% of their estimated one‐repetition maximum (Est. 1‐RM) (80FAIL) or 30% of their Est. 1‐RM (30FAIL).

Hypotheses

We hypothesize there will be a significant decrease in global DNA methylation with both RT conditions, but that 30FAIL RT will elicit a greater magnitude of hypomethylation than 80FAIL RT.

Methods

Previously trained college‐aged males (n= 11, age 23 ± 4 years, percent fat 11.4 ± 6.4%, training experience 4 ± 3 years, squat strength relative to body weight 1.7 ± 0.3) voluntarily underwent two bouts of RT. Participants completed both the 80FAIL and 30FAIL conditions (separated by one week). For each bout of RT, participants completed 4 sets of back squats and 4 sets leg extensions with either the 80FAIL or 30FAIL training conditions. Muscle biopsies were collected from the vastus lateralis before (PRE), 3 hours (3hPOST), and 6 hours (6hrPOST) after each RT bout. DNA was then batch‐isolated from muscle tissue and submitted for analysis using the Illumina MethylationEPIC array.

Results

Total number of repetitions performed were significantly higher for 30FAIL training vs 80FAIL training (p< .001), however total training volume (sets x reps x load) was not significantly different between conditions (p= 0.571). Global methylation changes between PRE, 3hPOST, and 6hPOST are presented herein. In addition, significant differences between conditions are emphasized for each post‐exercise time point.

Conclusions

With this study we expand our understanding of how the manipulation of RT variables affect epigenetic modifications in skeletal muscle.
Loss of Myosin Vb leads to dysregulation of colonic goblet cell structure and function
Authors: Digrazia Jessica , Engevik Melinda , Engevik Amy C.
Published: May 2022

Background

Intestinal goblet cells secrete a protective mucus layer preventing bacteria and harmful ingested substances from contacting the columnar epithelial cells that line the intestine. Single cell RNAseq data has revealed that goblet cells have high expression of Myosin 5b (Myo5b), a molecular motor that regulates vesicle trafficking in epithelial cells. Myo5b is most widely studied in the small intestine, but it’s role in the colonic epithelium and particularly goblet cells is unknown. A recent study found that Myo5b expression was reduced in inflammatory bowel disease (IBD), a condition that is characterized by dysfunctional mucus; potentially providing a link between Myo5b and goblet cell function. We hypothesized that loss of Myo5b would perturb goblet cell mucus production.

Methods

Adult control and intestinal specific tamoxifen inducible VillinCreERT2;Myo5bflox/flox(Myo5b KO) mice were injected intraperitoneally with 2 mg of tamoxifen to deplete intestinal Myo5b. After four days colonic tissue was collected and analyzed.

Results

We found that loss of Myo5b in vivo results in reduced number of goblet cells in the colon by H&E staining. Muc2 immunostaining and alcian blue staining similarly demonstrated a reduction of mucin within goblet cells. Mucin proteins are heavily o‐glycosylated which makes up 80% of the molecular weight. Normally, mucin structures are terminated by sialic acid which provides a negative charge that protects the mucus protein from bacterial degradation. Staining for sialic acid with the lectin SNA revealed a decrease in sialic acid levels in the mucus of Myo5b KO mice compared to controls. Once sialic acid is removed from mucins, the underlying glycan structures are exposed. Using the lectin WGA to identify the internal glycan oligosaccharide n‐acetyl glucosamine, we observed increased exposure of n‐acetyl glucosamine in Myo5b KO mice compared to control mice; which was consistent with our sialic acid findings. Intestinal goblet cells also secrete wound healing factors including the peptide Trefoil factor 3 (TFF3). Similar to the pattern we observed for Muc2 and sialic acid content, we found decreased levels of TFF3 in the mucosa of Myo5b KO mice.

Conclusions

Our data shows that loss of functional Myo5b in the colonic epithelium results in decreased goblet cell numbers and alterations in mucin composition. These data suggest that Myo5b may play an important role in goblet cell mucus production and that mutations affecting Myo5b function may contribute to IBD.
CYP1A1 and CYP1A2 Form Distinct Protein Complexes with NADPH‐cytochrome P450 Reductase, Cytochrome b5, and Heme Oxygenase‐1
Authors: Connick Patrick , Reed James R. , Backes Wayne L.
Published: May 2022

The cytosolic face of the endoplasmic reticulum (ER) membrane contains a variety of enzymes involved in oxidative metabolism. Among these, NADPH‐cytochrome P450 reductase (POR) and cytochrome b5 (CYB5) provide reducing electrons necessary for cytochrome P450 (P450) and heme oxygenase‐1 (HO‐1) activity. HO‐1 catalyzes the first and rate‐limiting step in free heme catabolism while the P450s are responsible for the metabolism of endogenous and exogenous compounds, including a majority of prescribed drugs. P450s have been shown to form heteromeric and homomeric complexes with other P450s and these complexes have been associated with alterations in P450 activity. Additionally, the P450 catalytic cycle requires 2 electrons—while POR is able to provide both, CYB5 can provide the second electron in some cases. However, binding of POR and CYB5 to the same P450 has been reported to be mutually exclusive, raising the question of how CYB5 can stimulate P450 activity if POR must dissociate prior to CYB5 binding.

In order to shed light on this question, we set out to collect more information on the protein‐protein interaction network that the ER‐membrane proteins participate in. Using bioluminescence resonance energy transfer (BRET), we are able to detect interactions between a pair of proteins and, further, to measure the stability of that pair in the presence of a third protein. To detect protein‐protein interactions, HEK293T cells were co‐transfected with vectors encoding two potentially interacting proteins tagged with either Renilla luciferase (Rluc) or GFP2.

While human CYP1A2 showed evidence of a specific complex, with analysis of BRET data showing the presence of homodimers, human CYP1A1 did not form a specific homomeric complex. CYP1A1 and CYP1A2 also differed in how they interacted with other ER membrane proteins. Both proteins showed evidence of heteromeric complex formation with HO‐1, but CYP1A1 only did so in the presence of excess POR. POR and CYB5 do not appear to interact with each other and in the presence of CYP1A2, HO‐1, and several other P450s, a complex between POR and CYB5 was not detected. However, a complex including the two proteins was detected in the presence of CYP1A1 which indicated the presence of a ternary CYB5‐POR‐CYP1A1 complex. The presence of a ternary complex could explain the stimulation of P450 activity by CYB5, though the specific mechanism remains to be determined.
Gut Dysbiosis: A Review of the Effects of a High‐Fat Diet on Iron Regulation and Brain‐Derived Neurotrophic Factor (BDNF) Expression in the Brain‐Gut Axis
Authors: Ty Darren , Kania Barbara , Sotelo Alexis , Wisco Jonathan
Published: May 2022

Introduction

The human microbiome in relation to the ‘gut‐brain’ bi‐directional axis is becoming increasingly relevant in our study to understand the effects of gastro‐intestinal microbiota on brain development and neurological diseases such as Alzheimer’s disease. One particular protein of interest is called brain‐derived neurotrophic factor (BDNF), which is a protein found in the brain that is important for learning and memory through stabilization of long‐term potentiation (LTP). Through past research, its expression levels have been found to be greatly affected by diet and exercise. Furthermore, in events such as aging, decreased levels of BDNF is associated with cognitive decline and increased susceptibility to neurodegenerative diseases such as Alzheimer’s disease (AD). Our study aims to look at how the human microbiome, particularly that of a Western diet, affects BDNF expression levels and modulation in the ‘gut‐brain’ bi‐directional axis.

Methods

In order to explore our research topic further, we conducted a literature review of about 80 papers using keywords: Small intestines, brain, liver, hepcidin, TMAO, BDNF, iron, astrocytes, Western Diet, microbiota, gut microbiome, gut dysbiosis, iron, inflammation, oxidative stress, IL‐6, vagus nerve.

Results

After reviewing papers, it was concluded that a Western diet decreases brain BDNF expression. However, the exact mechanism of how gut microbiota modulates this expression and function in the brain is unclear. Some papers mentioned BDNF in the gut and the periphery, but not much else other than that they existed.

Conclusions

This study sparked further questions: Does gut microbiota affect brain BDNF directly through the gut‐brain axis? Or is it indirect through an intermediate such as gut BDNF? Is there a relationship between gut BDNF and brain BDNF? It is known that brain BDNF is associated with learning and memory and is important for preserving cognitive abilities, however little is known about how gut microbiota affects it through the gut‐brain axis or even its relation to other peripheral BDNF. Looking forward, we plan on measuring different protein levels, including that of BDNF, in the brains and guts across different cohorts of mice. The cohorts will differ in diet, age, and having the APP gene. From this analysis we hope to find more insight on the unknown mechanism that bridges gut microbiota to BDNF in the brain, which may ultimately help in finding ways to prevent cognitive decline seen in many neurological diseases.
Antinociceptive Effects of Morphine:Ketamine Mixtures in Rats
Authors: Strumberger Conor , Minervini Vanessa
Published: May 2022

Chronic pain is a serious public health concern with an economic burden of $600 million annually and personal burden for approximately 100 million Americans. Prescription opioids (mu opioid receptor agonists) are the “gold standard” for treating moderate to severe pain despite their well‐documented adverse effects (dependence, respiratory depression, constipation). Of all prescription medications, opioids are abused most widely, and fatal overdoses have reached epidemic levels. Thus, there is a dire need for safer, more effective treatments for pain. One strategy for improving the margin of safety of opioids is combining them with other analgesic drugs to decrease the opioid dose needed for pain relief, thereby avoiding adverse effects that occur with larger doses. The NMDA receptor antagonist ketamine has been used safely and effectively to treat pain, but only under a narrow range of conditions (in emergency departments, post‐operative recovery, and combat casualty). The current studies used a model of acute pain (warm water tail withdrawal) and a model of chronic inflammatory pain (Von Frey paw withdrawal) to determine the antinociceptive effects of morphine and ketamine alone and in mixtures (in 3:1, 1:1, and 1:3 ratios) in 16 male Sprague Dawley rats. Given alone, both morphine (1‐10 mg/kg) and ketamine (3.2‐32 mg/kg) dose‐dependently increased tail withdrawal latency, with morphine having greater potency and efficacy as compared to ketamine. Similarly, given alone, both morphine (0.56‐5.6 mg/kg) and ketamine (3.2‐32 mg/kg) dose‐dependency increased force required to elicit a paw withdrawal response, with morphine having greater potency as compared to ketamine. ED50 values were used to determine the doses for mixtures. In mixtures, the potency of morphine or ketamine to produce antinociception was enhanced by 2‐3 fold as compared to either drug given alone. Dose‐equivalence and dose‐additivity analyses showed that the effects of morphine:ketamine mixtures were additive. Furthermore, morphine:ketamine mixtures that relieved pain also were tested in an assay of constipation in a separate group of 8 rats. While both morphine (1‐10 mg/kg) alone and ketamine (3.2‐32 mg/kg) alone dose‐dependently decreased fecal output, morphine:ketamine mixtures did not enhance constipation. Therefore, morphine:ketamine mixtures appear to selectively enhance antinociception. It remains unknown whether morphine:ketamine mixtures have other adverse effects (abuse, physical dependence, respiratory depression) and whether interactions between morphine and ketamine on these outcomes might be related to the ratio of each drug in mixtures. Morphine:ketamine mixtures might have greater therapeutic potential than mu opioids alone for treating moderate to severe pain, but only if adverse effects of each drug are not enhanced.
Exercise Capacity Mediated by the Gut Microbiome
Authors: Campbell Sara C. , Oydanich Marko , Zhang Jie , Longoria Candace R. , Vatner Dorothy E. , Siderovski David P. , Vatner Stephen F.
Published: May 2022

Female C57BL/6J wild‐type (WT) mice (5 months old) exhibited greater maximal exercise capacity for running distance (489 ± 15 meters; n=6, p<0.05) than age‐matched male WT mice (307 ± 17 m; n=8), as well as a 21% greater capacity for work to exhaustion. We hypothesized that these observed differences may be mediated by gut microbiota, as the sexes are known to have distinctly different microbiota. Predominant species in the male mice were found to be Faecalibacterium prausnitzii, Roseburia intestinalis and Allobaculum spp. while female mice had predominantly Parabacteroides goldstenii and Eubacterium coprostanoligenes. To determine if microbiota contribute to exercise tolerance, we administered antibiotic therapy (ABX) in drinking water to eliminate gut microbes. ABX eliminated the enhanced exercise tolerance of WT females. We then examined a model of enhanced exercise capacity ‐‐ Regulator of G Protein Signaling 14 (RGS14) knockout (KO) mice ‐‐ which exhibit greater maximal running distance (648 ± 38 m vs. 405 ± 21 m; p<0.05) than age matched WT mice, as well as a 53% increase in work to exhaustion (n=10/group). There is no difference in the enhanced exercise capacity of RGS14 KO mice between males and females. The greater exercise capacity in the RGS14 KO mice was also abolished by ABX. In conclusion, the gut microbiome appears to be a key factor in mediating exercise performance, since eliminating its action with chronic antibiotic therapy also eliminates the enhanced exercise performance in female vs male WT mice and in RGS14 KO mice of either sex.
Functional Modularization in the Neck Muscles of the Gerenuk (Litocranius walleri)
Authors: Manfredi Kevin R. , Georgi Justin A. , Townsend K.E. B.
Published: May 2022

The gerenuk (Litocranius walleri) is an African ruminant that feeds on high growing foliage by standing erect on its hindlimbs and extending its long neck. We hypothesize that extensive myological adaptation would be present in the muscles that facilitate head stability and movement, as well as muscles that anchor the neck. The vertebral musculature from the atlas to the thoracic vertebrae of a gerenuk specimen was dissected and compared to that of other ruminants. Several muscles were found to have narrowed or shifted caudally along the vertebral column, resulting in their cranial‐most attachment point being the fifth cervical vertebrae (C5). Similarly, two muscles were found to have shifted cranially, resulting in their caudal‐most attachment point also being C5. Caudally shifted muscles include m. serratus ventralis cervicis, the scalene muscles, m. rhomboideus cervicis, and the cervical part of the trapezius. Cranially shifted muscles include m. splenius cervicis and m. longissimus capitis. These shifts indicate a significant degree of modularization occurring between head movement muscles and neck stability muscles, with C5 being the boundary. Further adaptation that was not part of modularization around C5 was found in the segmental muscles attaching between vertebral processes. Segments of m. intertransversarii and m. multifidi have elongated and undergone specialization at the cranial attachments. The segments of m. spinalis cervicis were found to have a complex configuration that interdigitates with the multifidi. Additionally, m. levator claviculae and m. iliocostalis cervicis were present, neither of which have been previously described in ruminants. Previously published work reported that osteological adaptation in the gerenuk neck is especially pronounced in C1 – C4, which is consistent with our findings of myological modularization occurring with C5 as the boundary.
A human prefrontal cortex tissue microarray to study Alzheimer’s disease
Authors: Nielsen Rikke S. , Hawkinson Tara R. , Clarke Harrison A. , Young Lyndsay E. , Nelson Peter , Gentry Matthew S. , Sun Ramon C.
Published: May 2022

Alzheimer’s disease (AD) is characterized by proteinopathy, glucose hypometabolism, progressive neurodegeneration and eventually death from organ failure. One of the disease characteristics is glucose hypometabolism in the brain that can lead to perturbed cerebral glycan and glycogen metabolism. A key driver of disrupted glucose metabolism could be linked to glycans, which is a co‐translational modification of proteins involved in various biological functions of the cell. Prior research has demonstrated profound glycosylation differences in AD tissue compared to normal tissue, however, these differences have yet to be linked to disease progression. In collaboration with AD biobank at the University of Kentucky led by Dr. Peter Nelson, human AD brain tissue samples from over 90 deceased patients with and without known cognitive impairment were collected, in addition to their corresponding metadata. To analyze the N‐linked glycan content of the samples a Tissue Microarray (TMA) was produced using cores punched from the patients’ cortex tissue, and run through an optimized, enzyme‐assisted matrix‐assisted laser desorption/ionization‐mass spectrometer imager (MALDI‐MSI) workflow. Our data not only revealed interesting correlations between glycosylation and disease pathology of AD but also interesting differences in the glycan profiles between male and female patients. Further indepth analyses will be required to further determine specific markers for the correlation between glycosylation and disease progression of AD.
ATG9A‐mediated turnover of p62 condensates requires ubiquitin and occurs independently of the LC3‐lipidation machinery
Authors: McEwan Colten M. , Broadbent David G. , Poole Daniel M. , Naylor Brad C. , Price John C. , Schmidt Jens C. , Andersen Josh L.
Published: May 2022

Autophagy is a tightly controlled cellular recycling process that requires a host of autophagy machinery to form a double membraned vesicle called the autophagosome. This process is most understood in the context of stress‐induced autophagy, with little known about autophagosome biogenesis in basal (nutrient replete) conditions. To understand the regulation of basal autophagy, our work has focused on the poorly understood protein ATG9A, a multi‐pass transmembrane lipid scramblase that is essential for basal autophagy. To broadly understand the role ATG9A plays in basal autophagy, we utilized a quantitative proteome‐level MS/MS approach to measure how ATG9A affects protein flux. We show that loss of ATG9A in basal conditions impairs the degradation of autophagy adaptors, particularly p62/SQSTM1. Using a panel of ATG knock‐out cells, we demonstrate that the lipid transferase proteins ATG2A, ATG2B, and ATG9A promote the basal autophagic turnover of p62 and TAX1BP1 over other autophagy adaptors and do so independently of the LC3‐lipidation machinery. Furthermore, we demonstrate that ATG2A and ATG9A lipid transferase activity regulates the rate of p62 condensate degradation. Finally, we show in CRISPR knock‐in cell lines that ubiquitin is required for recruiting ATG9A to p62 condensates. Taken together, our data suggest that the lipid transferase activity of ATG9A and ATG2A is vital to basal autophagic regulation of protein homeostasis, and that ubiquitination is an apical signal that initiates recruitment of ATG9A to p62 condensates.
Effects of Sustained Hypobaric Hypoxia on Amplitude of Forced Hemodynamic Oscillations During Central Hypovolemia
Authors: Anderson Garen K. , Rosenberg Alexander , Barnes Haley , Bird Jordan , Pentz Brandon , Byman Britta R. , Jendzjowsky Nicholas , Wilson Richard , Day Trevor , Rickards Caroline
Published: May 2022

Introduction

Forcing oscillations in arterial pressure and cerebral blood flow at 0.1 Hz during simulated hemorrhage protects cerebral oxygenation at both low and high altitude. Arterial pressure oscillations at 0.1 Hz are endogenously driven by rhythmic fluctuations in sympathetic nerve activity. As hypobaric hypoxia increases basal sympathetic activity, we hypothesize that the amplitude of forced oscillations in arterial pressure and cerebral blood flow during simulated hemorrhage will be greater at high altitude compared to low altitude.

Methods

8 healthy human participants (4 M, 24.7 ± 4.1 y; 4 F, 34.3 ± 8.3 y) underwent a hypovolemic oscillatory lower body negative pressure (OLBNP) protocol, where chamber pressure reduced to ‐60 mmHg then oscillated every 5‐s between ‐30 mmHg and ‐90 mmHg over 10‐min (0.1 Hz). This protocol was performed at both low altitude (LA; Calgary, Alberta, Canada; 1045 m) and high altitude (HA; White Mountain, California, USA; 3800 m). Mean arterial pressure (MAP), mean middle cerebral artery velocity (MCAv), and cerebral tissue oxygenation (ScO2) were recorded continuously. Frequency analysis (via continuous wavelet transform) was used to quantify oscillations in MAP and mean MCAv at ~0.1 Hz. Data were analyzed with linear mixed‐models and paired t‐tests. All data are represented as mean ± SD.

Results

Baseline amplitude of oscillations were similar between HA and LA for MAP (1.9 ± 0.6 mmHg vs. 1.2 ± 0.5 mmHg; P = 0.47) and mean MCAv (0.9 ± 0.4 cm/s vs. 1.1 ± 0.3 cm/s; P = 0.91). Oscillatory amplitudes increased with 0.1 Hz OLBNP and altitude for MAP (ANOVA main effect, OLBNP: P < 0.001, Altitude: P = 0.007) and mean MCAv (ANOVA main effect, OLBNP: P = 0.002, Altitude: P = 0.008). Amplitude of oscillations during OLBNP were greater at HA for both MAP (4.0 ± 2.1 mmHgvs. 2.6 ± 1.4 mmHg, P = 0.05) and mean MCAv (2.4 ± 1.1 cm/svs. 0.9 ± 0.4 cm/s; P = 0.01). The relative (%∆) decrease in ScO2 was not different between HA and LA (‐0.63 ± 0.92 % vs. ‐2.56 ± 2.61 %, P = 0.11).

Conclusions

Oscillatory amplitudes at 0.1 Hz in both MAP and mean MCAv increased during OLBNP at high altitude. This effect may be due, in part, to the sympathoexcitatory stimulus of hypobaric hypoxia, and does not alter the protection of cerebral tissue oxygenation in this environment.
Research Handling Effects on Stress Hormones, Blood Parameters, and Heart Rate in Juvenile Northern Elephant Seals (Mirounga angustirostris)
Authors: Cooley Lauren A. , Costa Daniel P. , Hannah Shawn M. , Hindle Allyson G. , Holser Rachel R. , Horning Markus , Klinck Holger , Ponganis Paul J. , Williams Cassondra L. , McDonald Birgitte I.
Published: May 2022

Wildlife researchers must balance the need to safely capture and handle their study animals while simultaneously ensuring study results are not confounded by stress artifacts caused by handling. To determine the physiological effects of research activities including chemical immobilization, transport, instrumentation with biologgers, and overnight holding on a model marine mammal species, we collected hormone, blood parameter, and heart rate data from 19 juvenile northern elephant seals (Mirounga angustirostris) throughout a translocation experiment. Blood samples were collected during the initial capture (baseline), the instrumentation procedure conducted under isoflurane gas anesthesia, the post‐procedure recovery period, and the recapture (2‐10 days post‐release). Continuous heart rate data was collected using electrocardiogram biologgers during the procedure, recovery, transport, and release. Serum cortisol levels increased from 6.6 ± 5.3 μg/dL at capture to a maximum of 29.2 ± 23.7 μg/dL during recovery. Similarly, serum aldosterone increased from baseline levels of 69.7 ± 63.1 pg/mL to 217.8 ± 101.2 pg/mL during the procedure. Cortisol was positively associated with aldosterone, which supports growing evidence that aldosterone is a component of the acute stress response in marine mammals. Blood glucose and hematocrit both varied throughout the experiment, while there were no significant changes in β‐hydroxybutyrate blood ketones or erythrocyte sedimentation rate. Seals exhibited bradycardia (mean heart rate = 49.7 ± 3.7 bpm) while anesthetized, followed by a gradual increase in mean heart rate during the recovery period to 73.5 ± 7.9 bpm. Despite these short‐term changes, by the time seals were recaptured after several days at sea, all hormonal and hematological parameters had returned to baseline levels and 95% of study animals were resighted in the wild up to two years post‐translocation. Together these findings suggest that while northern elephant seals exhibit mild physiological stress responses to capture and handling activities, they recover rapidly and show no long‐term deleterious effects, making them a robust species for ecological and physiological research.
Regulatory Modulations and Dendritic Arborization in the Mouse Hippocampus Following Gulf War Toxicant Exposure
Authors: Murray Kathleen E. , Ratliff Whitney A. , Delic Vedad , Citron Bruce A.
Published: May 2022

Gulf War illness (GWI) is a chronic multi‐symptom syndrome which affects approximately 30% of the nearly 700,000 Veterans who were deployed to the Persian Gulf from 1990 to 1991. These Veterans have reported experiencing a variety of symptoms including difficulties with learning and memory, depression and anxiety, and increased incidence of neurodegenerative diseases. Evidence suggests that combined exposure to both reversible and irreversible acetylcholinesterase (AChE) inhibitors is a likely risk factor. We modeled Gulf War exposure in male C57Bl/6J mice with three AChE inhibitors that have been implicated as causative agents for GWI: pyridostigmine bromide (PB), the anti‐sarin prophylactic; chlorpyrifos (CPF), an organophosphate insecticide; and N,N‐diethyl‐m‐toluamide (DEET), a common insect repellent. Previously, we reported acute hippocampal gene expression changes following 10 d of toxicant exposure, including significant downregulation of several neuronal immediate early genes (IEGs) such as Arc, Egr1, and Nr4a1, as well as hippocampal‐dependent memory impairment in a Y‐maze task. Arc is predominantly expressed in cortical and hippocampal neurons and is critical for long‐term potentiation (LTP) and stabilization of synaptic plasticity. IEGs such as Arc and Egr1 have also been suggested to play a role in determining the risk of developing major depressive disorder, which is often comorbid with GWI. Here, we quantified Arc protein expression in granule cells of the dentate gyrus with IHC at 2‐4 h post‐exposure and examined the effects of treatment with a neuroprotective Nrf2 activator, tert‐butylhydroquinone (tBHQ), at 14 weeks post‐exposure. Additionally, mice were either placed in a novel enriched environment for 5 min or kept in their home cage to evaluate induced Arc expression in granule cells at 4 h post‐exposure. We also assessed dendritic arborization, important in connectivity, in granule cells by measuring dendritic lengths and spine densities with Golgi staining at 12 weeks post‐exposure. We hypothesized that toxicant‐exposed mice would have fewer Arc+ granule cells in the dentate gyrus and show reductions in both basal and induced Arc expression at 4 h post‐exposure. We also hypothesized that exposed mice would display dendritic arbor reduction and loss of spines in granule cells at the chronic timepoint, and that this effect would be reversed by antioxidant treatment. Preliminary results indicate that Arc protein expression is decreased in granule cells of exposed mice at the acute but not chronic timepoint. Dendritic arbor complexity was significantly reduced at the chronic phase in exposed mice but improved when treated with 1% tBHQ formulated food pellets. Our results suggest that IEGs may only be dysregulated immediately following exposure but could contribute to long‐term detrimental effects on hippocampal neuroplasticity, which may be improved with antioxidant treatment.
Serum and Glucocorticoid‐Inducible Kinase 1 (SGK1): An Important Contributor to Diarrhea and Malabsorption in Microvillus Inclusion Disease (MVID)
Authors: Ahsan Md Kaimul , dos Reis Diego Carlos , Barbieri Andrea , Sumigray Kaelyn , Nottoli Timothy , Salas Pedro J. , Ameen Nadia A.
Published: May 2022

Microvillus Inclusion Disease (MVID) is a lethal congenital diarrheal disease resulting from loss of function mutations in the actin motor myosin VB (MYO5B) that regulates apical traffic. MVID remains without treatment to reverse the severe diarrhea that leads to death. MVID diarrhea results from both increased fluid secretion and malabsorption of ions and carbohydrates in the small intestine. Serum and Glucocorticoid‐inducible kinase 1 (SGK1), is a potent regulator of ion transporters including cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine. Since SGK1 upregulates CFTR function in the intestine, we hypothesized that loss of SGK1 could potentially reduce MVID diarrhea by decreasing CFTR fluid secretion.

Using CRISPR‐Cas 9 approaches, we first generated MYO5B‐floxed mice and to generate conditional MYO5B‐KO (R26creER;MYO5Bf/f) mice, we cross bred with R26creER mice. Tamoxifen‐treated R26creER;MYO5Bf/fmice resulted in characteristic features of human MVID including severe diarrhea, Microvillus Inclusions (MIs), defective apical traffic, depolarization of proteins, and ion transporters including CFTR, NHE3 and DRA. MYO5B‐KO mice also showed increased phosphorylation of SGK1 and PDK1 in the intestine. SGK1‐floxed (SGK1f/f)mice were crossed with R26creER;MYO5Bf/fmice to generate conditional MYO5B/SGK1 DcKO (R26creER;MYO5Bf/f;SGK1f/f) upon Tamoxifen induction. Surprisingly, tamoxifen treatment (3 day) of MYO5B/SGK1 DcKO mice resulted in more severe diarrhea compared to MYO5B (R26creER;MYO5Bf/f) mice. Immunoblots of intestinal lysates revealed decreased CFTR, increased alpha‐ and beta‐ENaC, increased phosphorylation of PDK1, Nedd4‐2 and pPKCιThr563 in MYO5B/SGK1 DcKO vs. MYO5B ScKO mice. Finally, fecal glucose loss was higher with reduced SGLT1 and GLUT2 by immunoblot in MYO5B/SGK1 DcKO vs MYO5B ScKO mice. We conclude that activation of SGK1 pathway and CFTR contribute to worsening of diarrhea and carbohydrate malabsorption in MVID.
Horseshoe Kidney with Surplus Renal Arteries, Veins, and Roots of the Ureters
Authors: Dixon Jared , Choobchian Paoula , Perozich Garrett , Raciti Catherine , Barremkala Malli
Published: May 2022

We report the anatomical findings of a kidney discovered in a 92‐year‐old male during a routine dissection of the abdominal viscera performed by medical students at the Oakland University William Beaumont School of Medicine. The lower poles of the kidney are fused together, forming an isthmus anterior to the aorta and inferior vena cava at the L3‐L4 vertebral level. The right and left kidneys ascend past the isthmus, forming a “U” shape, the defining feature of a horseshoe kidney. The left kidney stops prematurely, approximately 3.2 cm below the inferior mesenteric artery at the level of the left inferior renal artery. The right kidney drains into superior, inferior, and middle renal arteries which have three, two, and one branches respectively. Due to the size and position of the hilum, five separate roots extend outside the kidney into the right ureter. The left side is supplied by a superior and inferior renal artery, each dividing into two branches. The left inferior renal artery branches off the ventral face of the descending aorta. The left renal vein notably has three prominent tributaries from the kidney and an additional branch of the left adrenal vein. Six roots extend from the left hilum and drain into the left ureter. In total, the kidney has nine veins, six arteries, and eleven ureteric branches draining into the left and right ureters. These findings will be useful in regard to research and surgery on other horseshoe kidneys.