bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of FXR1 and Senescence in Vascular Biology and Intimal Hyperplasia
Authors: Corbett Cali , St. Paul Amanda , Leigh Tani , Okune Rachael , Preston Kyle , Kelemen Sheri , Autieri Michael
Published: May 2022

Introduction

Despite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle to success. Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis; therefore regulation of gene expression in VSMC represents a logical intervention point to attenuate this syndrome. FXR1 is a muscle‐enhanced RNA binding protein and expression is increased in injured human and mouse arteries. We have shown that modulation of FXR1 levels affects abundance and stability of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. This drives our hypothesis that FXR1 is involved in mitigating vascular disease and acts as a crucial regulator of inflammatory and proliferative mRNA in VSMC.

Approach/Results

Because FXR1 expression is increased in injured arteries, we have developed a novel VSMC‐specific conditional knockout mouse (FXR1VSMC/VSMC). In a carotid artery ligation model of intimal hyperplasia, FXR1VSMC/VSMC mice have significantly reduced neointima formation (p<0.001) post‐ligation compared to several controls. To determine the mechanism of these effects, we knocked down FXR1 in human VSMC by siRNA and observed decreased proliferation (p<0.05) as well as an increase in beta galactosidase (p<0.05) and gamma H2AX (p<0.01) staining compared with controls, indicative of senescence. Senescent cells exhibit phenotypic changes called the senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. RIP‐sequencing demonstrated that FXR1 interacts with transcripts involved in cell cycle control, and RNA stability of these transcripts is decreased with FXR1 KO. qPCR analysis from FXR1 KO mouse VSMC show an increase in transcripts associated with senescence (p21, p16, p53) as well as an increase in SASP‐associated mRNA compared with controls. Furthermore, wild‐type human VSMC cultured in conditioned media from cells transfected with FXR1 siRNA show an increase in SASP mRNA and an increase in proliferation compared with cells cultured in conditioned media from cells transfected with a scrambled control.

Summary & Conclusions

Our results are the first to suggest that in addition to destabilization of inflammatory transcripts, FXR1 may stabilize cell cycle related genes in VSMC, and absence of FXR1 leads to induction of a senescent phenotype, an increase in SASP gene expression, and reduction of intimal hyperplasia.
Two Pathways Diverge: Glycerophosphocholine Impacts Both Phosphate and Lipid Metabolism in Candida albicans
Authors: King William R. , Acosta‐Zaldivar Maikel , Qi Wanjun , Cooke Lauren , Cherico Nicholas , Köhler Julia R. , Patton‐Vogt Jana
Published: May 2022

Candida albicans (C. albicans)is a leading source of fungal infections in humans, resulting in over 250,000 deaths annually. C. albicanscolonizes multiple host niches and causes invasive disease, requiring its adaptation to wide variations in nutrient availability. Phosphate is an essential nutrient, and several genes associated with phosphate acquisition are differentially expressed during infection. In addition to inorganic phosphate (Pi), C. albicans utilizes the abundant lipid metabolite, glycerophosphocholine (GPC), as a phosphate source. GPC is transported into the cell via the Git3 and Git4 transporters. The Git transporters are required for full virulence in a mouse model of disseminated candidiasis, but the mechanism for that virulence defect is unknown. Here, we probe the importance of GPC uptake to cellular metabolism by examining two aspects of its metabolic fate: i) its catabolism to liberate Pi), and ii) its potential conversion to phosphatidylcholine (PC) via reacylation. To query its role in phosphate homeostasis, we employed a knockout strain of the high affinity phosphate transporter, Pho84. Cells lacking Pho84 display sensitivity to several host‐related stressors and decreased virulence. Our results show that provision of cells with GPC as phosphate source alleviates hypersensitivity of pho84∆/∆ mutants to osmotic, oxidative, and cell wall stress. The rate at which GPC and Pi are transported into the cell varies depending upon the total level of phosphate provided, with Pitransport exceeding GPC transport by a maximum of two‐fold at 500 µM total phosphate. The effect of exogenous GPC on induction of the PHO regulon, the cellular system of phosphate homeostasis, is complex and concentration dependent. Beyond its role in phosphate metabolism, GPC has other physiologically relevant metabolic fates, namely its reacylation to form a phosphatidylcholine (PC) molecule. In Saccharomyces cerevisiae, the first step of this novel reacylation pathway is catalyzed by the acyltransferase, Gpc1, which converts GPC to lyso‐PC. Initial in vivo labeling studies under phosphate replete conditions indicate that the C. albicans homolog of Gpc1 is a GPC acyltransferase and that the reacylation pathway is functional in C. albicans. In vitro assays in strains lacking or containing Gpc1 are underway. Taken together, our results indicate that C. albicans can utilize the abundant lipid metabolite, GPC, via at least two metabolic pathways to promote its growth and survival in the human host.
Effects of Prebiotic Fructooligosaccharides on Cytokine and Tight Junction Protein Expression in Cultured Intestinal Epithelial Cells
Authors: Miles Megan , Grantham Mackenzie , Walton Kristen L. W.
Published: May 2022

Prebiotic molecules such as fructooligosaccharides (FOS) can promote the growth and metabolism of beneficial bacteria in the host intestine. There are several published studies suggesting that prebiotic supplementation can alleviate symptoms of colitis in animal models. In addition, a recent study showed evidence for direct effects of FOS on tight junction protein expression in cultured intestinal epithelial cells. This study aimed to assess effects of FOS on pro‐inflammatory cytokine and tight junction protein expression in CMT‐93 and Caco‐2 colonic epithelial cells. Cells were treated with 0.1 mg/ml, 1 mg/ml or 10 mg/ml FOS and expression of cyclooxygenase‐2 (COX‐2), pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐6, and tight junction proteins claudin and ZO‐1 were evaluated by real‐time qPCR. The number of viable cells was analyzed after overnight exposure to FOS using a cell survival assay. Expression of ZO‐1 and claudin and localization of nuclear factor kappa‐B (NFkB) were evaluated by immunofluorescent staining. Surprisingly, FOS‐treated CMT‐93 cells showed a slight reduction in claudin mRNA expression compared to untreated cells. ZO‐1 and COX‐2 mRNA expression were not altered by FOS treatment compared to untreated controls. Immunofluorescent staining showed no translocation of NFkB to the nucleus after 2 hours of exposure to FOS. In conclusion, prebiotic fructooligosaccharides had minimal to no effects on the mRNA expression of claudin, ZO‐1, and COX‐2. In addition, FOS did not activate the NFkB pathway at the time points studied. These data suggest that FOS may not cause inflammatory responses in intestinal epithelial cells.
Generation of an Adeno‐Associated Viral Model of Murine Obesity
Authors: Sellers Hunter , Padgett Caleb , Haigh Stephen , Bordan Zsuzsanna , Batori Robert , Fulton David
Published: May 2022

Cardiovascular disease (CVD) is a major contributor to US morbidity and mortality; significantly increasing chronic disabilities and shortening life expectancy. Obesity is a major driver of CVD and the rapid rise in the numbers of obese individuals portends an alarming societal burden. The etiology of CVD in obesity is incompletely understood and potential interventions to break the links between weight gain and vascular disease remain a critical barrier to treatment. Modeling complex disorders such as obesity in mice is an imperfect science but mouse models lacking leptin (ob/ob) or leptin receptors (db/db) mimic key aspects of human disease such as hyperphagia, overt obesity and vascular dysfunction. Limitations of these models include deficits in leptin signaling (not widely seen in humans), infertility which is a barrier to making compound genetic models and little to no temporal control of onset. To address these concerns, we hypothesized that delivery of AgRP, an orexigenic signaling molecule, to the brain of a C57BL6 mouse via a brain‐specific AAV would precipitate hyperphagia and obesity and recapitulate the vascular dysfunction exhibited by other hyperphagic models. Administration of this AAV elicits tightly restricted expression of AgRP in the brain and a significant (p=0.002) hyperphagic response on a standard chow diet. This leads to subsequent robust weight gain to a final weight of 63.1±2.1g, comparable to that of the db/db model. Pressure myography of mesenteric microvessels from lean control and obese AgRP AAV mice showed significant (p<0.001) impairment in endothelial dependent relaxation in the vessels from obese animals. Overall our findings with this model recapitulate the obesity and vascular dysfunction seen in other severely obese mouse models while providing increased flexibility and temporal control.
Remote Hind‐Limb Ischemia Mechanism of Preserved Ejection Fraction During Heart Failure
Authors: Tyagi Suresh C. , Homme Rubens P. , Zheng Yuting , Smolenkova Irina , Singh Mahavir
Published: May 2022

During acute heart failure (HF), remote ischemic conditioning (RIC) has proven to be beneficial; however, it is currently unclear whether it also extends benefits from chronic congestive, cardiopulmonary heart failure (CHF). Previous studies from our laboratory have shown three phases describing CHF viz. 1) HF with preserved ejection fraction (HFpEF), 2) HF with reduced EF (HFrEF), and 3) HF with reversed EF. Although reciprocal organ interaction, ablation of sympathetic, and calcium signaling genes are associated with HFpEF to HFrEF, the mechanism is unclear. The HFrEF ensues, in part, due to reduced angiogenesis, coronary reserve, and leakage of endocardial endothelial (EE) and finally breakdown of the blood‐heart barrier (BHB). In fact, our hypothesis states that a change in phenotype from compensatory HFpEF to decompensatory HFrEF is determined by a potential decrease in regenerative capacity, proangiogenic factors along with a concomitant increase in epigenetic memory, inflammation that combinedly cause oxidative, and proteolytic stress response. To test this hypothesis, we created CHF by aorta‐vena‐cava (AV) fistula in a group of mice that were subsequently treated with that of hind‐limb RIC. HFpEF vs. HFrEF transition was determined by serial or longitudinal echo measurements. Results revealed an increase in skeletal muscle musclin contents, bone‐marrow (CD71), and sympathetic activation (β2‐AR) by RIC. We also observed a decrease in vascular density, and attenuation of EE‐BHB function due to a corresponding increase in the activity of MMP‐2, VEGF, caspase, and calpain. This decrease was successfully mitigated by RIC‐released skeletal muscle derived exosomes that contain musclin; the myokine along with bone marrow, and sympathetic activation. In short, based on proteome (omics) analysis, ∼20 proteins that appear to be involved in the signaling pathway responsible for the synthesis, contraction, and relaxation of cardiac muscle were found to be the dominant features. Thus, our results support that the CHF phenotype causes dysfunction of cardiac metabolism affecting its contraction, and relaxation. Interestingly, RIC was able to mitigate many of the deleterious changes, as revealed by our multi‐omics findings.
In Arctic ground squirrels, diet and torpor alter skeletal muscle relaxation kinetics but not force development
Authors: Krishnan Jishnu K. , Rice Sarah , Mikes Monica , Sugiura M. Hoshi , Drew Kelly , Barati Zeinab , Oliver S. Ryan
Published: May 2022

During the hibernation season, Arctic ground squirrels (AGS) experience extreme temperature fluctuations (body temperature, Tb, as low as ‐3°C), during which they are mostly physically inactive. Once Tb reaches ~15°C during interbout arousals, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to reach Tb of ~35°C. Polyunsaturated fatty acids (PUFA) in the diet are known to influence SkM function and metabolism. Recent studies in the cardiac muscle of hibernators have revealed that increased levels of ω‐6 and the ω‐6:ω‐3 PUFA ratio correlates with sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and hibernation status. Muscular activity is characterized by contraction and relaxation mediated by calcium ions cycling released from SR stores. Ryanodine receptors (RyR1) on SR membranes release calcium ions into the cytosol, facilitating muscle contraction via actin‐myosin cross‐bridging. Whereas SERCA catalyzes Ca2+ reuptake back into the SR, releasing Ca2+ from actin/myosin filaments, completing the calcium cycle, and causing muscle relaxation. We hypothesized that diet (increased ω‐6:ω‐3 PUFA ratio) and torpor status are important in the regulation of the SERCA pump and that this may improve SkM performance during hibernation. Ex‐vivo functional assays were used to characterize performance changes in SkM (diaphragm) from AGS fed the following diets. 1) Standard rodent chow with an ω‐6:ω‐3 ratio of 5:1, and 2) a balanced diet with an ω‐6:ω‐3 ratio of 1:1 that roughly mimics wild diet. We collected diaphragms at three different stages of hibernation (early torpor, late torpor, and arousal) and evaluated muscle function under hypothermic temperature stress at 4°C, 15°C, 25°C, and 37°C to determine functional resilience. Our data show that torpid animals fed standard rodent chow appear to have an increase in SERCA activity, resulting in a significant increase in calcium mobilization from the sarcoplasm to the sarcoplasmic reticulum by the SERCA pump, allowing faster SkM relaxation when compared to the balanced diet animals. Furthermore, we discovered that standard rodent chow AGS during torpor has higher SkM relaxation kinetics, but this effect of torpor is eliminated in balanced diet AGS. Interestingly, neither diet nor torpor influenced the rate of force development (rate of calcium release). This is the first study to show that increasing the dietary ω‐6:ω‐3 PUFA ratio improves skeletal muscle performance during hypothermic stress in a hibernating animal. This evidence supports the interpretation that diet can change some functional properties of the SkM, presumably through membrane lipid composition, ambient temperature, and torpor interaction, with an impact on SkM performance.
Biochemical Mechanisms that Control the Effects of RhoA Small GTPase Signaling on Synaptic Stability and Cognition
Authors: Bjornson Kathryn , Cahill Michael
Published: May 2022

Dendritic spines are the site of most excitatory connections in the brain. Small GTPases act as binary switches that link cell surface receptors to persistent modifications in the morphogenesis of dendritic spines. Small GTPases are active when bound to GTP and inactive when GDP bound. The direct activation of small GTPases is accomplished by guanine nucleotide exchange factors (GEFs). The small GTPase RhoA, is enriched in dendritic spines and its activation can destabilize the actin cytoskeleton within spines reducing dendritic spine stability. Rodent studies suggest that excessive RhoA signaling causes cognitive impairment in part via its ability to reduce dendritic spine density in forebrain neurons. Despite the broad therapeutic potential of targeting the RhoA pathway as a means of enhancing cognition, the specific biochemical mechanisms that control its activity in neurons remain largely unknown. PDZ‐RhoGEF is a RhoA‐specific guanine nucleotide exchange factor that shows an unusually potent ability to activate RhoA. We show that PDZ‐RhoGEF destabilizes dendritic spines in cortical neurons and causes cortical‐dependent cognitive impairment. Ongoing studies are extending this analysis to determine if PDZ‐RhoGEF is similarly important for activity‐mediated remodeling of dendritic spines after NMDA receptor activation. Despite the importance of PDZ‐RhoGEF to synaptic and cognitive phenotypes, owing to a lack of studies, minimal is known about the biochemical mechanisms that regulate PDZ‐RhoGEF. Our recent findings indicate that PDZ‐RhoGEF activity and cellular localization is controlled by its interaction with the DISC1 scaffolding‐like protein, such that DISC1 helps localize PDZ‐RhoGEF to cellular membranes yet inhibits PDZ‐RhoGEF’s ability to activate RhoA. Experiments utilizing structured illumination microscopy are being conducted to further assess the functional consequences of the interaction between PDZ‐RhoGEF and DISC1 in neurons, and in particular, within dendritic spines. We theorize that the knockdown of DISC1 in neurons will result in the improper synaptic localization of PDZ‐RhoGEF with a concomitant increase in PDZ‐RhoGEF‐mediated RhoA activity. Collectively, these findings identify PDZ‐RhoGEF as a potent regulator of synaptic stability and cognition and highlight a previously unrecognized involvement for DISC1 in controlling RhoA activity via PDZ‐RhoGEF.
The Analysis and Experimentation of Aspergillus flavus Colonizing Corn in West Tennessee
Authors: Adams Valeanna , McLaughlin Autumn , Kelley Heather M.
Published: May 2022

Aspergillus flavus is an opportunistic fungus that affects many crops including corn. A. flavus colonizes the ear of the corn, causing a disease known as Aspergillus ear rot (AER). AER contributes to yield loss in two ways; through fungal infection, which results in decreased kernel weight, and through contamination of the grain with aflatoxins. A. flavus has the ability to produce toxic secondary metabolites called aflatoxins which are harmful to humans and animals when consumed. Contamination with aflatoxin can result in reduced price, or rejection of grain depending on FDA regulatory limits. Due to their ability to compete with and displace toxigenic strains, atoxigenic (non‐toxin producing) strains of A. flavus have been used as a biological control, such as the Syngenta product Afla‐Guard®. The evaluation of local populations of A. flavus strains enables researchers to gather information about what is present in the field (atoxigenic or toxigenic) and can help make more informed management recommendations. Therefore, the purpose of this research was to evaluate the characteristics of an unknown isolate of A. flavus isolated from corn in West Tennessee in comparison to known atoxigenic (1 isolate from Afla‐Guard®) and toxigenic isolates (2 isolates). Each isolate was grown on barley seed and plated onto MDRB media three times (i.e. 3 replications) along with a negative control (autoclaved barley) (total of 15 plates). Radial growth and colony morphology of each isolate were evaluated at 3‐ and 5‐days after plating in a laboratory setting. Based on observations of the activity of the TN field collected isolate, it was predicted to grow slower than the Afla‐Guard®/atoxigenic isolate. Preliminary results demonstrate that the Afla‐Guard®/atoxigenic isolate did outgrow the TN strain as hypothesized. Additional research is required to determine the toxicity of the TN strain, as well as evaluation of other naturally occurring strains in TN in relation to potential use as biological controls.
A 14‐3‐3‐mediated mechanism of regulation for the ubiquitin‐sensing kinase TNK1
Authors: Egbert Christina M. , Chan Tsz‐Yin , Lopez‐Palacios Tania , Ashworth Spencer , McCormack Katherine , Vaughan Alec , Andersen Joshua
Published: May 2022

Thirty ‐eight negative kinase 1 (TNK1) is a poorly characterized member of the ACK family of non‐receptor tyrosine kinases. Previous studies suggest a role for TNK1 in oncogenic transformation; however, the biological function, regulation and substrates of TNK1 remain unknown. We previously discovered that the phospho‐binding protein 14‐3‐3 binds to a phospho‐serine (S502) within a C‐terminal proline‐rich domain of TNK1, sequestering TNK1 in the cytosol and inhibiting its kinase activity. Conversely, the release of TNK1 from 14‐3‐3 activates TNK1 and increases its oncogenic activity1. However, how 14‐3‐3 controls TNK1 localization and activity is still not understood. We recently found that the release of TNK1 from 14‐3‐3 allows TNK1 to interact with ubiquitin‐rich cytosolic condensates, where TNK1 is fully active. Furthermore, we found that the interaction between TNK1 and ubiquitin‐rich condensates requires an unusual ubiquitin association (UBA) domain on the TNK1 C‐terminus that binds with high affinity to K63‐linked poly‐ubiquitin chains. Thus, 14‐3‐3 acts as a toggle to turn on and off the ubiquitin‐binding ability of TNK1, which in turn controls TNK1 oncogenic activation. Our current data suggest that this occurs through a two‐step mechanism. First, the release of TNK1 from 14‐3‐3 activates the TNK1 kinase domain, which leads to an autophosphorylation within the TNK1 UBA domain. Second, this autophosphorylation enhances the affinity of the UBA domain for poly‐ubiquitin, which promotes the clustering of active TNK1 at ubiquitin‐rich condensates where TNK1 phosphorylates downstream substrates.

1. Chan and Egbert et al. TNK1 is a ubiquitin‐binding and 14‐3‐3 regulated kinase that can be targeted to block tumor growth. Nat Commun 12, 5337 (2021).
Contractile response of avian ductus arteriosus to chronic elevated oxygen levels
Authors: Rippamonti Jessica , Dzialowski Edward M.
Published: May 2022

The ductus arteriosus (DA) is an oxygen sensitive embryonic blood vessel present in all developing amniotes. The DA connects the pulmonary artery to the aorta and shunts blood away from nonfunctioning lungs and towards the fetal respiratory organ. In birds at hatching, a rise in arterial PO2 initiates DA smooth muscle cell constriction to initiate closure. As the avian embryo approaches hatching, the DA contraction becomes more sensitive to oxygen. In this study we asked how 3‐hr chronic exposure to elevated oxygen levels of 5, 25, or 73 kPa oxygen influenced the oxygen stimulated contraction of the late term chicken DA. During the 3‐hr exposure, the greatest increase in contraction was seen in response to 73 kPa oxygen with smaller change occurring at 5 kPa oxygen exposure. Exposure to all three levels of oxygen resulted in resetting the baseline tension when oxygen was returned to 2.5 kPa. These changes in response to chronic elevated oxygen disappeared in the presence of cytochalasin B. These findings suggest that DA exposure to chronic elevated oxygen results in cellular changes in the contractile properties of the vessel allowing it to respond with greater contractile strength in response to a rise in oxygen.
A Low‐carbohydrate, Ketogenic Diet Enhances Hippocampal Mitochondrial Bioenergetics and Efficiency
Authors: Saito Erin R. , Warren Cali E. , Campbell Rachel J. , Miller Gavin , du Randt Johannes D. , Cannon Mio E. , Saito Jeremy Y. , Hanegan Cameron M. , Kemberling Colin M. , Edwards Jeffrey G. , Bikman Benjamin T.
Published: May 2022

Mitochondrial dysfunction and cognitive impairment are common symptoms in many neurologic and psychiatric disorders, as well as in nonpathological aging. Ketones have been suggested as therapeutic for their relevance in epilepsy as well as other neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Here we explored whether a low‐carbohydrate, ketogenic diet (KD) alters recognition memory, hippocampal mitochondrial bioenergetics, and expression of proteins involved in mitochondrial dynamics. Mature‐to‐middle‐aged adult male and female mice were placed on a lard‐based KD supplemented with an exogenous ketone ester for eight weeks. Changes in behavioral recognition memory were measured in a two‐object novel object recognition test. Hippocampal mitochondrial physiology was assessed using high‐resolution respirometry to measure changes in oxygen flux, biochemical assays to quantify ATP production, and western blot to measure changes in Drp1 expression. Here we demonstrate that the KD reduces hippocampal oxygen consumption (p=0.013), but does not alter ATP production (p=0.42) or Drp1 expression (p=0.51). These results indicate an enhancement of mitochondrial coupling and efficiency independent of mitochondrial dynamics, as changes in oxygen flux occurred without changes in Drp1 expression. Together, these findings add to growing support for the use of ketones and KDs in pathological brain states in which mitochondrial function is compromised, especially within the hippocampus.
Acute Intermittent Hypoxia Preconditioning Elicits Age and Sex‐Dependent Changes in Molecules Known to Regulate Phrenic Motor Plasticity
Authors: Nair Jayakrishnan , Marciante Alexandria B. , Seven Yasin B. , Kelly Mia N. , Lurk Carter , Oberto Jose R. , Mitchell Gordon S.
Published: May 2022

Acute intermittent hypoxia (AIH) elicits a form of respiratory motor plasticity known as phrenic long‐term facilitation (pLTF); preconditioning with daily AIH (dAIH) enhances pLTF, demonstrating metaplasticity. Two additional factors impacting pLTF expression are age and sex: whereas moderate AIH‐induced pLTF decreases with age in males, it increases in middle aged females. Unfortunately, little is known concerning mechanisms of dAIH preconditioning or age‐dependent sexual dimorphism in pLTF. Moderate AIH elicits 2 distinct signaling cascades in phrenic motor neurons initiated by 5HT2A/2B receptors (Q pathway) versus adenosine 2A or 5HT7 receptors (S pathway), respectively. The Q and S pathways interact via mutual cross‐talk inhibition, a powerful regulator of pLTF expression. To begin exploring the hypotheses that dAIH preconditioning and age/sex effects on pLTF can be accounted for by shifts in molecules regulating the Q and S pathways, we assessed mRNA of key molecules known to regulate pLTF in ventral cervical spinal homogenates from segments containing the phrenic motor nucleus from young (3 month) and middle‐aged (12 month) male and female Sprague‐Dawley rats. Rats (n=8/group) were exposed to sham or dAIH for 14 days (15, 1 min hypoxic episodes/day), and sacrificed 24 hours after the last AIH exposure. Tissues were harvested, preserved in RNA stabilizing solution (<10 min), and stored (‐80°C) until mRNA extraction. mRNA levels of molecules that regulate pLTF were assessed via RT PCR, including: serotonin 2A (Htr2a) & 2B (Htr2b); adenosine 2A (Adora2a); exchange protein activated by cAMP (Epac); PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (Cx3cl1); phosphodiesterase type 4 (Pde4b); Gp91 and p47(NOX genes); and the Pkcδisoform. We also quantified protein expression in phrenic motor neurons (via immunofluorescence) for BDNF and adenosine‐1A & ‐2A receptors in rats fixed with 4% paraformaldehyde (n=4/group; analysis via MATLAB codes to quantify optical density in labeled motor neurons). We found significantly more Pde4b, Adora2a, and Pkcδ mRNA in young and older females versus age matched males; Epacwas also elevated, but only in young females (p<0.001). p47 was increased in older rats of both sexes (p<0.01). p47, Cx3cl1, Adora2a and Pkcδ mRNA were reduced by dAIH in older females (p<0.05), but not other groups. There were age‐dependent increases in adenosine 1A (p <0.0001) and 2A (p <0.0001) receptors, and BDNF (p<0.001) protein post‐AIH in both sexes. Thus, there are age, sex and dAIH preconditioning effects on molecules known to regulate the Q and S pathways to phrenic motor facilitation. These novel findings advance our understanding of pLTF, and inform translational research concerning the therapeutic potential of dAIH to treat breathing deficits in individuals of different ages or sex.
Skeletal Muscle Injury is Sufficient for Angiogenesis but Myofiber Regeneration is Required for Restoring Microvascular Integrity
Authors: Jacobsen Nicole L. , Cornelison D.D.W. , Segal Steven S.
Published: May 2022

Acute injury of skeletal muscle damages myofibers, microvessels, and supporting tissue components. To restore the structural and functional integrity of intact muscle, each tissue component must regenerate. In adult skeletal muscle, resident muscle stem cells (satellite cells) efficiently regenerate myofibers within 10 days post injury (dpi) and microvascular integrity recovers by 21 dpi. However, the requirement for crosstalk between regenerating myofibers and microvessels is poorly understood. We tested the hypothesis that myofiber regeneration is necessary for revascularization and restoration of microvascular integrity. To ablate satellite cells and impair myofiber regeneration, we studied male and female mice (age, ~4 mo) in which tamoxifen‐dependent Cre recombination induced the expression of diphtheria toxin in quiescent satellite cells, killing them prior to injury. For acute muscle injury, the gluteus maximus muscle (GM) was injected with BaCl2 (1.2%, 75 µL). Microvascular perfusion and permeability were evaluated by injecting fluorescent dextrans (70 & 155 kDa) into the circulation during intravital imaging of the GM at defined timepoints following injury. Despite the absence of myofiber regeneration, sprouting angiogenesis was robust and capillary perfusion was restored within 7 dpi, as observed in wild‐type mice. However, the permeability barrier of nascent microvessels was compromised as shown by profuse leakage of both fluorescent dextrans. At 21 dpi, the absence of regenerated myofibers was accompanied by sustained inflammation and myosteatosis. Areas of ectopic adipocyte accumulation were hypervascular and localized around venules. Adjacent areas contained empty myofiber “sheaths” which were underperfused; residual microvascular networks exhibited pruning. These findings suggest that, following acute injury of adult skeletal muscle, revascularization occurs in the absence of myogenesis but is defective and nascent vessels are unstable. We conclude that myofiber regeneration is essential to recovering a functionally intact microcirculation following muscle injury.
Identifying Mucus‐Degrading Microbes Within the Human Gut Microbiota
Authors: Glover Janiece S. , Ticer Taylor , Engevik Melinda
Published: May 2022

Introduction

Mucus provides a critical barrier for the colonic epithelium by excluding microbes and antigens from direct interaction with epithelial cells, providing a lubricant for stool, and limiting the diffusion of harmful compounds. This barrier function is due in part to the extensive glycan structures found in mucus. Mucin protein is heavily O‐glycosylated with core structures containing α‐ and β‐ linked N‐acetyl‐galactosamine, N‐acetyl‐glucosamine, and galactose. These core structures are elongated and commonly modified by α‐linked fucose, sialic acid and sulfate residues. The sialic acid and sulfate residues protect the underlying mucin glycans from degradation. However, some gut microbes harbor glycosyl hydrolases (GHs) that enzymatically degrade mucin glycans. The released oligosaccharides can then be used as a primary carbohydrate source for the mucus‐associated microbiota. Despite the growing number of bacterial genome sequences available, our knowledge of the mucin‐degrading capacity of human gut commensal microbes remains fragmented. The aim of the present study was to systematically examine the CAZyme mucin‐degrading profiles of the human gut microbiota.

Methods & Results

Using the CAZy database, we identified 13,156 genomes harboring at least one gene copy of GH families involved in mucin degradation. Commensal microbes were identified using the Human Microbiome Project consortium in the Joint Genomes Institute Integrated Microbial Genomes database, resulting in 4,385 genomes for downstream analysis. Within the Verrucomicrobia phlyum, all Akkermansia glycaniphila and muciniphila genomes harbored gene copies of sialidases (GH33), fucosidases (GH29, GH95), endo‐β‐1,4‐galactosidases (GH2, 35), mucin core GHs (GH84 and GH89), endo‐acting O‐glycanases (GH16), and sulfatases (GH20, GH2, GH42); consistent with their known ability to degrade mucus. Interestingly, the only representative of the Lentisphaerae phylum, Victivallales, harbored a GH profile that closely mirrored Akkermanisa. In the Actinobacteria phlyum, we found that several Actinomadura, Actinomyces, Bifidobacteria, Streptacidiphilus and Streptomyces species contained gene copies of mucin‐degrading GHs. Within the Bacteroidetes phylum, we identified mucin degrading GHs in Alistipes, Alloprevotella, Bacteroides, Fermenitomonas Parabacteroides, Prevotella and Phocaeicola species. Firmicutes contained Abiotrophia, Blautia, Enterococcus, Paenibacillus, Ruminococcus, Streptococcus, and Viridibacillus species with mucin‐degrading GHs. Far fewer mucin‐degrading GHs were observed in the Proteobacteria phylum, with only 3‐4 GHs families found in Klebsiella, Mixta and Enterobacter. We confirmed the mucin‐degrading capability of 26 representative gut microbes by growing these bacteria in a chemically defined media lacking glucose supplemented with porcine intestinal mucus.

Conclusions

These data greatly expand our knowledge of mucin degradation within the human gut microbiota.
Towards Bacterial Expression of Unstable Mutants of a Mitochondrial Enzyme
Authors: Randa Lora , Beyene Eledon , Gjertson Max , Chihade Joseph
Published: May 2022

Mutations in the human AARS2 gene, which codes for the mitochondrial alanyl‐tRNA synthetase, are linked with severe disease, including infantile cardiomyopathy and leukodystrophy. Our laboratory is interested in understanding, on a molecular level, why different mutations result in different disease phenotypes. Attempts to produce some mutants of AARS2 using a bacterial expression system have resulted in poor expression of proteins contaminated with proteolytic fragments. To improve expression of these mutants, we have used solubility tags to facilitate the purification of full‐length mutant protein. Addition of an MBP solubility tag greatly improved expression and purification of the wildtype and mutant enzymes. We are currently investigating strategies for solubility tag removal and characterization of wildtype and mutant thermal stability.
A novel HDAC1/2 inhibitor alleviates physical and emotional symptoms associated with spontaneous oxycodone withdrawal in neuropathic pain mice
Authors: Pryce Kerri D. , Ramakrishnan Aarthi , Kronnman Hope , Nicolais Andrew , Polizu Claire , Ruiz Anne , Serafini Randal , Gaspari Sevasti , Pena Catherine J. , Torres‐Berrio Angelica , Mitsi Vassiliki , Darpe Matthew , Shen LI , Nestler Eric J. , Zachariou Venetia
Published: May 2022

The development of physical dependence and addiction disorders due to misuse of opioid analgesics is a major concern with pain therapeutics. In this study, we developed a mouse model of oxycodone misuse in order to gain insight into genes and molecular pathways in reward‐related brain regions that are affected by prolonged exposure to oxycodone and subsequent withdrawal in the presence or absence of chronic neuropathic pain. RNA‐sequencing (RNA‐seq) and bioinformatic analyses revealed that oxycodone withdrawal alone triggers robust gene expression adaptations in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and ventral tegmental area (VTA), with numerous genes and pathways selectively affected by oxycodone withdrawal under peripheral nerve injury states. Our pathway analysis predicted that histone deacetylase 1 (HDAC1), an epigenetic modifier with a prominent role in striatal plasticity, is a top upstream regulator in opioid withdrawal in both the NAc and mPFC. Indeed, treatment with the novel HDAC1/2 inhibitor RCY1305 attenuated behavioral manifestations of oxycodone withdrawal, with the drug being more efficacious under states of neuropathic pain. Our studies also suggest that RCY1305 treated mice did not develop conditional place preference. Since RCY1305 displays antiallodynic actions with no rewarding effects in models of neuropathic pain, inhibition of HDAC1/2 may provide an avenue for chronic pain patients dependent on opioids to transition to non‐opioid analgesics. Overall, our study highlights transcriptomic events in components of the reward circuitry associated with oxycodone withdrawal under pain‐naïve and prolonged neuropathic pain states, thereby providing information on possible new targets for the treatment of physical dependence to opioids and transitioning individuals to non‐opioid medications for chronic pain management.
Higher Levels of Shear Stress Downregulate Endothelin‐1 mRNA Expression
Authors: Rodriguez Carmen , Garcia Mario , Cabral Shelsea , Conde Daniel , Gurovich Alvaro N.
Published: May 2022

Background

Physical inactivity has been shown to be a risk factor in the development of endothelial dysfunction. Endothelin‐1 (EDN‐1), a potent vasoconstrictor contributes to the pathogenesis of cardiovascular disease. Exercise‐induced endothelial shear stress (ESS) is a mechanical stimulus caused by alterations in blood flow patterns that regulates vascular function. Although it has been reported that higher levels of ESS may act as a protective mechanism to prevent vascular dysfunction, the role of pulsatile exercise‐induced ESS with varying exercise intensities has not been explored. Therefore, the purpose of this study was to determine the effects of pulsatile exercise‐induced ESS based on in vivo exercise intensity on EDN‐1 expression.

Methods

Commercially available human umbilical vein endothelial cells (HUVEC; Sigma‐Aldrich, St Louis, MO) were cultured until 90–100% confluence. Exercise‐induced ESS in vivo was obtained from our previous reports. Cultured HUVEC cells were exposed to pulsatile resting ESS (18 dynes/cm2) for 2 hours, followed by 1 hour at resting ESS, low‐intensity exercise‐induced ESS (35 dynes/cm2), moderate exercise‐induced ESS, or high‐intensity exercise‐induced ESS (75 dynes/cm2) on a closed circuit pump and channeled slide (Ibidi pump system (PumpControl Software 1.5.4) and slides, Ibidi Inc., Fitchburg, WI). A set of cells were exposed to physiologically low shear stress levels (10 dynes/cm2) for 3 hours as a control. Total RNA was extracted and reverse transcribed followed by measurement of EDN‐1 mRNA expression via qRT‐PCR. qRT‐PCRs were performed in duplicates and changes in gene expression were calculated using the ΔΔ‐CT method with GAPDH used as the normalizing control gene. Statistical analysis was performed using graphpad prism 8 software (Graphpad Software.). Significance was considered at p<0.05.

Results

EDN‐1 mRNA expression was significantly downregulated following low and high intensity exercise‐induced ESS compared to resting conditions (p<0.05). Moderate intensity exercise induced ESS showed a trend downregulation in EDN‐1 mRNA expression compared to resting conditions (p<0.10).

Conclusions

Our data suggests that higher levels of pulsatile exercise‐induced ESS are needed to suppress mRNA expression of EDN‐1. Although moderate intensity exercise induced ESS showed only a trend difference in mRNA expression compared to resting conditions, we have previously reported that moderate exercise‐induced ESS was shown to upregulate endothelial nitric oxide gene expression, a key vasodilatory enzyme that may prevent vascular dysfunction.
Endothelial dysfunction drives aneurysm development in Marfan syndrome
Authors: Cantalupo Anna , Asano Keiichi , Hansen Jens , Halabi Carmen , Mecham Robert , Iyengar Ravi , Ramirez Francesco
Published: May 2022

Thoracic aortic aneurysm (TAA) is the major morbidity/mortality factor in Marfan syndrome (MFS), a connective tissue disease caused by mutations in fibrillin‐1. We examined the impact of fibrillin‐1 (Fbn1) deficiency on the integrity and function of the tunica intima by using the Cdh5‐Cre transgene to selectively inactivate the Fbn1 gene in endothelial cells (ECs). The resulting Fbn1Cdh5‐/‐mice had a normal life span and displayed no overt signs of arterial disease. However, ultrastructural analyses of the ascending aorta of Fbn1Cdh5‐/‐mice at P18 and P45 revealed severe fragmentation of the internal elastic lamina (IEL) with biosynthetically active sub‐endothelial cells (SMCs) breaching into the intima. Additional analyses related loss of fibrillin‐1 deposition in the IEL with increased endothelial permeability, impaired EC‐dependent vascular relaxation and dysregulated gene expression in mural cells. The first two abnormalities were found in common with MFS mice that are hypomorphic for fibrillin‐1 throughout the entire vessel (Fbn1mgR/mgR mice). As these MFS mice also displayed aortic hypercontractility and a more abnormal aortic transcriptome, we concluded that Fbn1Cdh5‐/‐mice replicate the early EC‐dependent events that eventually trigger full blown TAA pathology in MFS. Preliminary analyses of single cell RNASeq data indicate that loss of intimal fibrillin‐1 altered the profile of aortic subclusters, particularly those corresponding to immune cells and ECs.
Ameliorative Effect of Morinda Lucida on L‐nitro Arginine Methyl Ester (L‐name) Induced Hypertension in Male Wistar Rats
Authors: Ogunpolu Blessing S. , Omobowale Temidayo , Rauff Barakat , Oyagbemi Ademola , Falayi Funke , Hassan Fasilat
Published: May 2022

Hypertension is a chronic cardiovascular disease of extreme and important global concern. It has been established as a leading cause of death especially in humans. The aim of this study was to evaluate the possible antihypertensive effect of a common locally available plant Morinda lucidaagainst L‐Nitro‐Arginine‐Methyl‐Ester (L‐NAME)‐induced hypertension in male Wistar rats.

Forty‐eight rats were used for the experiment. They were divided into six groups labelled A‐F, each containing eight animals. The animals were dosed for six weeks with group A being the control group (normotensive) given feed and water ad libitun; Group B animals were administered L‐NAME only, 40 mg/kg (hypertensive); Groups C, D and E were simultaneously treated with L‐NAME (40 mg/kg) and Morinda lucida extract in increasing doses of 100 mg/kg, 200 mg/kg and 400 mg/kg, respectively, while Group F were given lisinopril at 20 mg/kg for a period of 6 weeks. From the result, there was significant increase in systolic, diastolic, and mean arterial blood pressure in the hypertensive group compared to the normotensive group and hypertensive treated groups. Hypertension caused significant (p<0.05) increases in oxidative stress markers, including malondialdehyde (MDA), protein carbonyl (PC), hydrogen peroxide (H2O2), while reducing the activities of superoxide dismutase (SOD), and serum nitric oxide (NO) levels. Also, serum myeloperoxidase (MPO), blood urea nitrogen and creatinine were increased with high expression of cardiac injury biomarkers such as cardiac troponin and angiotensin 2 type 1 receptor. These indicated oxidative stress, cardiac damage because of hypertension, most especially in the untreated hypertensive group. The co‐treatment of hypertensive rats with Morinda lucida extract caused reduction in markers of oxidative stress and inflammation and normalized blood pressure parameters. The findings from this study suggest the possibility of using Morinda lucida extract as a novel antihypertensive agent.
TNK1 negatively regulates TBK1 at ubiquitin condensates
Authors: Lopez‐Palacios Tania , Egbert Christina M. , Chan Tsz‐Yin , Ashworth Spencer , Vaughan Alec , Andersen Joshua
Published: May 2022

Thirty‐eight‐negative kinase 1 (TNK1) is a poorly understood member of the ACK family of non‐receptor tyrosine kinases. TNK1 has been linked to oncogenic activity. However, the biological function of TNK1 remains unclear. Recently, we discovered the presence of a functional ubiquitin association (UBA) domain on the C‐terminus of TNK1, which is an unusual feature on a kinase. Previously, we demonstrated that the TNK1 UBA domain binds to poly‐ubiquitin with high affinity and with no preference for length or linkage type (1). Interestingly, deletion of the UBA domain (TNK1 ΔUBA) affects TNK1 intracellular localization and decreases its phospho‐substrates network. Based on these data, we hypothesized that the UBA domain homes TNK1 to substrates. To test this hypothesis, we used quantitative phospho‐tyrosine proteomics in pro‐B cells transformed with either TNK1 or TNK1 ΔUBA to identify UBA‐dependent TNK1 substrates. Among the putative UBA‐dependent substrates, we identified phospho‐Y235 of TANK‐binding kinase 1 (TBK1). TBK1 is an important serine/threonine protein kinase involved in the regulation of inflammation, autophagy, and NF‐kB signaling. Through confocal microscopy, we demonstrated a colocalization between TNK1 and TBK1 at p62‐positive phase‐separated clusters of ubiquitin—referred to here as ubiquitin condensates. We found that TNK1 localizes to ubiquitin condensates in a UBA‐dependent manner. Furthermore, our data suggest that TBK1 is inhibited by phosphorylation at Y235, as phospho‐null mutation at Y235 (Y235F) significantly increases phosphorylation of the TBK1 substrates p62 and OPTN. We also found that CRISPR/Cas9 deletion of TNK1 increased TBK1 activity and phosphorylation of p62 and OPTN. These data suggest that TNK1 negatively regulates TBK1 activity. Intriguingly, we also identified an enrichment of other putative UBA‐dependent TNK1 substrates in the NF‐kB signaling pathway, suggesting that the UBA domain tethers TNK1 to multiple substrates in this pathway, perhaps as a means to dampen NF‐kB signaling. Our current work focuses on the biological impact of these phosphorylations on NF‐kB activity, inflammatory signaling, and autophagy.

1. Chan, TY., Egbert, C.M. et al. TNK1 is a ubiquitin‐binding and 14‐3‐3‐regulated kinase that can be targeted to block tumor growth. Nat Commun 12, 5337 (2021).