bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapeutic fasting mitigates metabolic and cardiovascular dysfunction in a prediabetic rat model: Possible role of adipose inflammation
Authors: Dwaib Haneen S. , Taher Maha F. , Mougharbil Nahed , Obeid Omar F. , El-Yazbi Ahmed F.
Published: April 2020

The risk of cardiovascular complications in type 2 diabetes increases as early as in the prediabetic stage. Our previous studies showed that perivascular adipose tissue inflammation contributes to vascular and cardiac autonomic dysfunction in prediabetic rats. Intermittent fasting has been extensively studied in the management of metabolic diseases. Here, we aim to examine the impact of therapeutic fasting (TF) on the metabolic and cardiovascular stress among prediabetic rats. Male SD rats (4–5weeks) were randomly allocated into 3 dietary groups; control diet (C), high‐calorie (HC) diet and HC‐diet with TF, for 24 weeks. Rats were fed ad libitum in the first 12 weeks. Afterwards, the TF group was subjected to daily fasting from 7.00pm – 7.00am (during the dark period) for 12 weeks with free access to water, and to HC‐diet during the light phase. Daily food intake, body weight (BW), blood glucose (fasting FBG and random RBG), body composition (BC) (using NMR), HbA1c, serum insulin levels, echocardiographic parameters, and noninvasive blood pressure were measured. At week 24; rats were catheterized for invasive hemodynamic examination. Cardiac autonomic neuropathy (CAN) was assessed by measuring baroreceptor sensitivity (BRS) using the vasoactive method. After sacrifice, aortic contractility and endothelial function were assessed using organ bath experiments. Daily calorie intake was higher in the HC group compared to control with no difference in body weight, blood glucose level, HbA1c, and glucose tolerance. Similar to our previous observations, HC feeding led to an increased fat/lean ratio, increased serum insulin level, insulin resistance, increased vascular reactivity, and reduced endothelium‐dependent relaxation. Despite a lack of effect on daily calorie intake, the previous parameters were reversed by TF. As well, SBP was higher in the HC‐fed rats (137.7 vs. 119 mm Hg), which was reversed in TF rats. This could be explained based on the observed increase in vascular sensitivity to phenylephrine‐induced contraction (pEC50 5.95 vs. 5.53) and a reduced acetylcholine mediated endothelium‐dependent relaxation in HC rats. Both observations were normalized in TF rats. Moreover, HC‐fed rats showed parasympathetic CAN manifesting as reduced BRS (DMAP vs. DHR slope decreased to −0.15 from −0.4) that was reversed in TF. Our results indicate that HC feeding induced vascular and cardiac autonomic dysfunction secondary to perivascular adipose inflammation. TF reverses signs of cardiovascular impairment. Future studies will be conducted to assess the effect of TF on PVAT inflammation.

Support or Funding Information

Funded by AUB‐FM MPP grant #320148
IL‐17 Signaling Stimulates Natural Killer Cell Activation and Causes Mitochondrial Dysfunction in Response to TH17 Cells Stimulated in Response to Placental Ischemia During Pregnancy
Authors: Fitzgerald Sarah Jayne , Deer Evangeline , Herrock Owen T. , Ibrahim Tarek , Amaral Lorena , Lamarca Babbette
Published: April 2020

Preeclampsia (PE) is a multisystem disorder characterized by new onset hypertension that is associated with placental ischemia, mitochondrial (mt) dysfunction, and an imbalance in T helper and Natural Killer (NK) cells during pregnancy. The reduced uterine perfusion pressure (RUPP) model is an established model for PE exhibiting hypertension in response to placental ischemia associated with mt dysfunction and mt reactive oxygen species (ROS), activation of T helper and NK cells. We have previously shown an important role for IL‐17 in the hypertension and activation of NK cells in the RUPP rat. However we don’t know the role for IL‐17 or TH17 cells in contributing to NK cell mediated mt dysfunction and ROS associated with placental ischemia. Therefore, we hypothesize that hypertension in response to placental ischemia stimulated TH17 cells causes mt ROS mediated through IL‐17 signaling to NK cells. On gestation day 12 (GD12) RUPP‐induced TH17s (splenic CD4+/CD25− cells) were injected into normal pregnant (NP) rats. Recombinant mouse IL‐17 receptor C (IL‐17RC) (100 pg/day) was administered from GD14‐19 via osmotic mini‐pump. On GD19, samples were collected and mean arterial pressure (MAP) was measured. Mt respiration and mt ROS data was measured in isolated mitochondria from renal and placental tissues using the Oxygraph 2K and fluorescent microplate reader, respectively. A one‐way ANOVA with Bonferroni post hoc test was used for statistical analysis. MAP was increased in NP+RUPP TH17 recipient rats (110 ± 0.94mmHg, n=10) compared to NP controls (91 ± 3.6mmHg, n=11) (p<0.05), and was lowered with IL‐17RC (100± 2.4 mmHg, n=4). Circulating activated NK cells were significantly increased with RUPP TH17 (4.74± 0.9 %, n=5) (p<0.0001) compared to NP controls (0.08± 0.06 %, n=8), and were attenuated with IL‐17RC (0.00± 0.00 %, n=3) (p<0.001). Placental activated NK cells were significantly increased with RUPP TH17 (2.5± 1.01 %, n=4) (p<0.05) compared to NP controls (0.03± 0.03 %, n=8) and were attenuated with IL‐17RC (0.21± 0.12 %, n=4) (p<0.05). Renal mtROS increased in NP RUPP TH17 recipients (593 ± 110%, n=9) compared to NP controls (289 ± 30%, n=6), and was attenuated by IL‐17RC (380± 103%, n=3). Placental mtROS significantly increased in NP RUPP TH17 recipients (3081 ± 720%, n=7) (P<0.0005) compared to NP controls (1287 ± 231.3%, n=6) (p<0.0001), and was decreased by administration of IL‐17RC (561.7± 105.5 %, n=3) (p<0.0001). These data demonstrate that IL‐17 signaling plays an important role in NK cell activation and tissue mt function in response to TH17 cells stimulated during placental ischemia of pregnancy.

Support or Funding Information

R01HD067541‐06, P20GM121334, AHA 19CDA34670055, T32HL105324
Dietary Creatine Supplementation Does Not Alter Trabecular Bone Morphology in Male and Female Rats
Authors: Murphy Jensen E. , Dunham Tyler , Yumol Jenalyn L. , Colonna Kaitlyn , Fajardo Val A. , Ward Wendy E. , Roy Brian D.
Published: April 2020

Background

Supplementation with dietary creatine has been reported to improve markers of bone structure in animals and humans. Trabecular bone is one of two types of bone responsible for strength and structural support in the body. It serves to support areas that experience mechanical stress. The purpose of this study was to investigate the effect of varying doses of dietary creatine on the trabecular bone morphology of male and female rats.

Methods

32 Sprague‐Dawley rats (16 male, 16 female) were randomized into one of four groups: 0g/L, 2.5g/L, 5g/L, or 10g/L of creatine monohydrate (CM) in drinking water with 1% sucrose (for palatability). Dietary interventions began at 12 weeks and lasted for 8 weeks. Ex vivo scanning and analysis was conducted on the proximal region of left femurs using micro‐computed tomography (mCT). Standard measures of trabecular bone morphology were measured at the proximal femur: trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), percent bone volume (BV/TV), connectivity (Conn) and connectivity density (Conn.Dn).

Results

There were no significant differences in Tb.Th, Tb.Sp, Tb.N, BV/TV, Conn, or Conn.Dn between creatine dose groups for males or females. Males had greater Tb.Th than females (p<0.01). Greater Tb.Sp was also observed in males as compared to females (p<0.01). Females demonstrated significantly higher BV/TV (p<0.01) as well as a greater Tb.N (p<0.01) than males. Conn was significantly different between sexes (p<0.05) with a higher value in females compared to males. Conn.Dn was also significantly higher in females (p<0.01) compared to males.

Conclusions

Eight weeks of different dietary intakes of creatine does not appear to have an effect on proximal femur Tb.Th, Tb.Sp, Tb.N, BV/TV, Conn, or Conn.Dn. However, differences in sex were observed for each dependent variable which is suggestive of a difference in the trabecular structure between males and females.

Support or Funding Information

Supported by NSERC (Canada).
A Light‐gated Integrator for Imaging Kinase Activity in Living Cells
Authors: Lin Wei , Phatarphekar Abhishek , Greenwald Eric C. , Zhang Jin
Published: April 2020

Kinase signaling pathways regulate many cellular processes and are often dysregulated in cancers. The intratumoral heterogeneity of kinase activity in the context of tumor microenvironment presents a challenge for kinase‐targeted cancer therapeutics. Therefore, characterizing kinase activity profiles in complex tumor environment can help address this challenge. Current genetically encoded fluorescent biosensors have been widely used in kinase signaling research. Even with the sensitive and dynamic readout of these biosensors, it is difficult to capture transient changes across a whole tumor section. Herein, we developed a light‐gated
kinase
activity
coupled
transcriptional (KINACT) integrator, which has the capability of “memorizing” differences in kinase activities over a certain period of time. The KINACT integrators have been applied for imaging PKA, PKC and Fyn activities in live cells, as well reporting subcellular changes in PKA activity. Moreover, we applied the PKA integrator to evaluate PKA response to a panel of G‐protein coupled receptor agonists and different kinase inhibitors, suggesting that KINACT integrator is a potential platform for screening various small molecule drugs. The KINACT integrator can also be applied to monitor PKA activity enhancement under pro‐inflammatory conditions.

Support or Funding Information

This work was supported by the National Institutes of Health (R35 CA197622 and R01 DK073368 to J.Z.)
The Mechanism of Actin Remodeling by the BAR Domain of ArfGAP ASAP1
Authors: Gasilina Anjelika , Luo Ruibai , Randazzo Paul
Published: April 2020

The actin cytoskeleton plays an important role in cellular processes. Normal processes, such as differentiation, use the actin cytoskeleton as a signaling and transport platform, while aberrant processes, such as cancer, repurpose actin for invasion. Actin remodeling involves assembly of globular actin into actin filaments and further assembly of filaments into bundles, which form higher order structures such as stress fibers, lamellipodia and filopodia. A given higher order actin structure relies on organized, timely and regulated mobilization of actin filaments and accessory actin binding and bundling proteins. For one of such structures, the actin stress fibers (SFs), assembly, maturation and maintenance depends on a carefully orchestrated crosstalk between actin filaments, actin binding proteins and the focal adhesion machinery. As a biological phenomenon, SFs provide a mechanotransduction platform, and as a model system, they provide a prototype of the mechanism and the regulation of actin filament assembly. The complexity and diversity of actin structures is, in part, dictated by the actin regulating proteins. Among these are well established actin remodeling families, such as actinin and fascin protein families. More recently a subset of BAR‐domain proteins, widely studied for inducing and sensing the membrane curvature, have been found to control actin filament assembly. However, the mechanism by which BAR domain proteins regulate of the actin cytoskeleton remains unclear. We recently showed that ASAP1, a BAR‐domain containing ArfGAP, remodels actin filaments. Depletion of ASAP1 led to reduction in overall levels of filamentous actin and the number of ECM‐interacting stress fibers. Our structure‐function studies using in vitro actin sedimentation, fluorescence microscopy, as well as cellular assays revealed that the BAR‐PH region of ASAP1 is sufficient and necessary to induce actin reorganization. During the study we observed that another member of the human ArfGAP family that also contains a BAR domain, ACAP1, does not induce the same phenotype as ASAP1. Our current work thus inquires whether structural differences between the BAR domains of ASAP and ACAP explain their disparate effects on actin organization. Molecular modeling‐mediated dissection of the mechanism of actin binding localizes the actin binding site to the 1st and 2nd helix of the BAR domain of ASAP1. Together these data highlight selectivity of the BAR domain proteins in actin remodeling and provide further insights into the different roles human ArfGAPs play in modulating the actin cytoskeleton.

Support or Funding Information

This work was supported by the Intramural Research Program of the National Cancer Institute, Department of Health and Human Services, project number BC007365
ShRNA‐mediated gene silencing of t‐PA prevents BBB disruption and elevation of MMP‐12 after ischemic stroke
Authors: Nalamolu Koteswara Rao , Challa Siva Reddy , Mohandass Adithya , Mussman Justin P. , Ilahi Sadia B. , Bedadala Mayurnath Reddy , Klopfenstein Jeffrey D. , Pinson David M. , Wang David Z. , Kalyanasundaram Ramaswamy , Vemuganti Raghu , Veeravalli Krishna Kumar
Published: April 2020

Post‐stroke induction of endogenous tissue‐type plasminogen activator (t‐PA) is known to promote BBB disruption after ischemic stroke, which contributes to edema, inflammation, hemorrhagic transformations and brain damage. We presently evaluated if this effect of t‐PA leads to the elevation of MMP‐12 in the ischemic brain. Cohorts of adult, male rats were administered with plasmids that express either a t‐PA shRNA or a scrambled control shRNA (1 mg/Kg; i.v.; n=9–13/group) formulated as nanoparticles at 5 min of reperfusion following transient focal middle cerebral artery occlusion. We also have sham‐operated control and untreated ischemia‐induced cohorts. As compared to control shRNA cohort, t‐PA shRNA treated cohort prevented the degradation of tight junction proteins claudin‐5 and ZO‐1, curtailed BBB disruption (studied by Evans blue extravasation into brain), and showed significantly reduced MMP‐12 expression. These results indicate that post‐stroke induced endogenous t‐PA mediated BBB disruption contributes to elevation of MMP‐12 in the ischemic brain.

Support or Funding Information

This work was supported by the NIH Grant 1R01NS102573‐01A1 to KKV.
Switching hypoxia tolerance strategies in a brainstem network of bullfrogs, Lithobates catesbeianus
Authors: Adams Sasha , Santin Joseph M.
Published: April 2020

Strategies to survive brain anoxia vary widely among vertebrates. Along a spectrum, some animals arrest neuronal activity to conserve ATP, while others can sustain neuronal functions during energetic stress. The neural circuit that produces breathing in the adult American bullfrog, Lithobates catesbeianus, is one example of a network that arrests activity during anoxia. This response is considered an energy saving arrest because 1) a hypoxia sensor that inhibits the network through noradrenergic signaling appears to contribute, and 2) activity recovers after anoxia. Unexpectedly, we found that this network becomes remarkably functionally tolerant to hypoxia after frogs emerge from 3–5 weeks of cold‐acclimation. Normally, control networks fail to produce rhythmic activity after 10.1±6.4 minutes of hypoxia (0% O2) at 22°C (n=9); however, networks at 22°C from cold‐acclimated frogs continue to produce coordinated respiratory bursts for about 16 times longer (159.1 ±42.2 minutes; n=5; p=0.001; Mann‐Whitney U test), representing a shift from an arrest strategy to functional tolerance. Thus, the transition may reflect a reduction in the hypoxia sensor (noradrenergic signaling) and/or a shift in energy use/metabolism. To begin to address these possibilities, we sought to identify if noradrenergic signaling is the sole cause of the arrest in control networks. To test this, we exposed in vitro brainstem preparations to norepinephrine and then hypoxia. We expected that pretreatment with norepinephrine would occlude the arrest during hypoxia if noradrenergic signaling was part of the oxygen sensor. However, this did not occur; preparations stopped activity over a similar time course during hypoxia when bathed with (n=6) or without (n=9) norepinephrine, implying the arrest does not involve noradrenergic signaling. We then hypothesized that metabolic deficits may contribute. To test this, we applied inhibitors of glycolysis [2‐deoxy‐D‐glucose substituted for D‐glucose (n=5) or 1 mM iodoacetate (n=4)] or mitochondrial respiration [1 mM sodium cyanide (n=5)] in baseline O2 tensions. Each inhibitor recapitulated the actions of hypoxia, demonstrating that deficits in ATP synthesis, regardless of oxygen tensions, produce an arrest. Cellular energy sensors, such as KATP channels and AMP‐dependent protein kinase, are known to reduce neuronal activity when ATP falls; however, activation of these mechanisms pharmacologically failed to arrest activity [KATP activator; 600 μM Diazoxide (n=5], AMPK activator; 1 mM AICAR (n=4)]. In sum, we hypothesize that deficits in ATP synthesis stop network output during hypoxia and that improvements in metabolic function or energy use contribute to the striking shift to functional hypoxia tolerance after cold‐acclimation.

Support or Funding Information

This work was supported by start‐up funds to JS provided by UNC‐Greensboro.
The ERG1 Potassium Channel is Abundant in Cachectic Human Skeletal Muscle
Authors: Pond Amber Lynn , Zampieri Sandra , Sandri Marco , Cheatwood Joseph , Kohli Punit , Balaraman Rajesh , Anderson Luke Brian , Latour Chase Doyne , Hockerman Gregory H. , Kern Helmut , Sartori Roberta , Merigiano Stefano , Da Dalt G , Davie Judy K. , Carraro Ugo
Published: April 2020

Introduction

A heteromultimer of the ERG1a/1b potassium channel is known to contribute to repolarization of the cardiac action potential. A homomultimer of the ERG1a subunit has been detected in the atrophying skeletal muscle of mice experiencing muscle disuse and cancer cachexia and has been shown to contribute to muscle atrophy by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1 has not been reported in human skeletal muscle.

Methods and Results

Here, using immunohistochemistry we detect ERG1 immunofluorescence at low levels in Rectus abdominis muscle of young adult humans and show that it trends toward greater levels (10.6%) in the same muscle of healthy aged adults. Further, we detect ERG1 immunofluorescence at a statistically greater level (53.6%; p<0.05) in the Rectus abdominis muscle of older people having cancer cachexia than in age‐matched adults. Additionally, we observe ERG1 immunofluorescence in skeletal muscle sarcolemma and detect that its fluorescent pattern is consistent with I‐band localization.

Discussion

The data suggest that ERG1 may be related to muscle loss in humans and may be located in t‐tubules where it could influence calcium handling.

Support or Funding Information

This work was supported in part by Southern Illinois University School of Medicine (Research Seed Grant to ALP). The work was also supported in part by the Italian Society for Cancer Research (AIRC grant number 17388 to M Sandri) and by Austrian national co‐financing of the Austrian Federal Ministry of Science and Research; Ludwig Boltzmann Society (Vienna, Austria) to H Kern. PK would like to acknowledge National Science Foundation (CHE 0748676), NIH (GM 106364) for partial financial support of this research. The authors wish to thank Dr. Don Caspary who graciously provided FBN rat muscles. Human Pectoralis minor muscle samples were provided by the SIU School of Medicine Tissue Bank, a Simmons Cancer Institute funded program. Other investigators may have received samples from these same tissue specimens. UC thanks the IRCCS Fondazione Ospedale San Camillo, Venezia, Italy and the A&C M‐C Foundation for Translational Research for scientific support.
Identification of PDZ‐RhoGEF as a Novel Target of Gαi Signaling
Authors: Chandan Naincy R. , Abraham Saji , Smrcka Alan V.
Published: April 2020

Cell‐migration and adhesion are fundamental biological processes regulated by chemokine G‐protein‐coupled receptors (GPCRs). GPCRs bind to heterotrimeric G proteins, which are composed of Gα and Gβγ subunits. Chemokine receptors couple to the PTX‐sensitive Gi/o family of Gα (Gαi) proteins. The role of Gβγ subunits released from Gi heterotrimers in chemokine signaling has been well characterized and is thought to be the major transducer of these processes. In contrast, Gαi has been concluded to have no signaling role other than to regulate the release of Gβγ. However, this conclusion is complicated by the fact that tools to inactivate Gαi signaling also inactivates obligatory Gβγ signaling. Previous reports from our laboratory have identified that Gαi‐GTP directly regulates cell adhesion but the direct effector of Gαi has not been identified. To identify the novel effectors of Gαi, we adopted a proximity‐based labeling technique. BioID2 (promiscuous biotin ligase enzyme) generates reactive Biotin‐AMP from biotin. Bio‐AMP then diffuses away from the enzyme’s active site and labels proteins covalently with biotin that are in ∽10 nm radius. HT1080 fibrosarcoma cells were transfected with Gαi1‐WT‐BioID2 (inactive Gαi), Gαi1‐QL‐BioID2 (constitutively active Gαi) or BioID2‐CaaX (membrane‐targeted control samples). Cell extracts were prepared, and biotinylated proteins were selectively isolated by biotin‐streptavidin affinity capture. Tandem mass spectrometry was then performed using quantitative tandem mass tag (TMT) labeling. Known binding partners for Gαi subunits including, GPCRs, Gβ and Gγ, adenylate cyclase, Ric8A, and others were identified as enriched in the Gαi‐WT‐BioID2 or Gαi‐QL‐BioID2 samples. One candidate Gαi effector, significantly enriched in Gαi1‐QL‐BioID2 as compared to Gαi1‐WT‐BioID2 samples, was PDZ‐RhoGEF (ARHGEF11), a Rho guanine nucleotide exchange factor. PDZ‐RhoGEF is a known major regulator of cell adhesion in neutrophils downstream of Gi‐coupled chemoattractant receptors. To determine if PDZ‐RhoGEF is a true effector of Gαi; we co‐transfected PDZ‐RhoGEF with Gαi subunits and an SRE luciferase reporter construct responsive to Rho activation in A293 cells. Gαi1‐QL but not Gαi1‐WT strongly activated SRE luciferase only when co‐expressed with PDZ‐RhoGEF. The activation of RhoA by Gαi1‐QL was confirmed with a Rhotekin‐GST pulldown assay. Activation is specific to the Gαi subfamily of G proteins and requires the RGS domain within PDZ‐RhoGEF. Since PDZ‐RhoGEF is known to be involved in cell migration and adhesion, this protein is likely a key downstream target of Gαi that regulates cell migration. Overall, identification of the relatively ubiquitously expressed PDZ‐RhoGEF as a new Gαi‐GTP target has implications for regulation of downstream signaling by Gi‐coupled GPCRs in multiple biological systems.
Microglia regulate neuronal network formation and synaptogenesis
Authors: Ruß Tamara Heike , Spittau Björn , Piecha Robin
Published: April 2020

Microglia are the resident immune cells of the central nervous system (CNS) and play an essential role under physiologic and pathologic conditions. Next to their well‐established role in the progression in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), recent reports underline the involvement of microglia during postnatal CNS maturation. The main objective of the present study was to examine the influence of microglia on neuronal network formation and synaptogenesis in cortical/midbrain neuron cultures.

Primary mouse E14 cortical/midbrain neuron cultures were used to determine the development of functional synapses in the presence and absence of microglia. Microglia depletion was performed using the Csf1r inhibitor BLZ945. Neuronal network formation and maturation of synapses were analyzed using the Microelectrode Array Technology (MEA) and immunocytochemistry labeling of synaptic markers such as Synapsin and PSD95.

Electrophysiology using MEAs indicated that microglia regulate the abundance of functional synapses during a time of 28 days in vivo (DIV). Using a Csf1r inhibitor, we were able to show that the efficient depletion of microglia abrogated the inhibitory effects on synaptogenesis.

Taken together, our data demonstrate that microglia are effective regulators of neuronal network formation and synaptic connectivity further broadening the portfolio of versatile functions of these resident immune cells of the central nervous system.
Identification of Residues Controlling dUTPase Enzyme Activity from Epstein‐Barr Virus
Authors: Robbins Alex M. , Gorres Kelly
Published: April 2020

Epstein Barr‐Virus (EBV) is primarily known for causing infectious mononucleosis, but also increases the incidence of lymphomas and autoimmune disorders. There is currently no vaccine or treatment once infected by EBV. The EBV genome encodes a dUTPase enzyme. This enzyme catalyzes the reaction of dUTP to dUMP which is a precursor of the thymidine nucleotide. The enzyme activity regulates the dUTP/dTTP ratio and decreases misincorporation of uracil into newly synthesized DNA. A dUTPase knockout is lethal in mice, E. coli., and S. cerevisiae. The Murine gammaherpesvirus lacking its viral dUTPase has reduced viral replication. It is hypothesized that inhibition of EBV dUTPase would result in uracil misincorporation, double stranded DNA breaks, and a reduction in replication of the viral genome. Characterization of residues required for enzyme activity in EBV dUTPase was performed using sequence alignments and structural data. Mutagenesis and comparative enzyme kinetics on these residues identified aspartate 76 as being critical for enzyme activity. Analysis of structural data identified a histidine residue hypothesized to regulate substrate specificity. A small molecule binding site was identified using computational mapping of two EBV dUTPase 3D structures. We hypothesis that this allosteric binding site contains a serine that post transcriptionally regulates dUTPase activity. This research will guide the development of inhibitors that bind to critical dUTPase residues offering a potential treatment for EBV.

Support or Funding Information

This work was supported by, UWL College of Science and Health, UWL Research and Creativity, and Toce Deans Distinguished Fellowship
Possible Role of Cannabinoid Receptors in Modulating Parkinsonian‐Like Behaviour
Authors: Nassar Noha N. , Ghaith Wessam Z. , Mobarez Elham A. , Abdallah Dalaal M.
Published: April 2020

Cannabinoid‐receptors are one of the most abundant G‐protein coupled receptors in the brain which raises interest regarding their role in the brain physiology and pathology. To this end, this study aimed to investigate the possible role of cannabinoid‐receptors signalling modulation in Parkinsonian Disease (PD) using a model of reserpine‐induced PD‐like symptoms. Adult male Sprague‐Dawley rats were divided into 5 groups, the 1st served as a control that received the vehicle. Animals in the 4 other groups were subcutaneously injected with reserpine (3mg/kg). Rats in the PD group were left untreated, whereas those in the other 3 group were given WIN55,212 (0.1mg/kg I.P.; group 3), rimonabant (0.1mg/kg I.P.; group 4) or a combination of both (group 5) 24 h after reserpine. Microscopical examination of the substantia nigra revealed necrosis and pyknosis in PD rats that was prevented by rimonabant and to a lesser extent by its combination with WIN55,212. Similarly, substantia nigral tyrosine hydroxylase immunostaining, as well as striatal dopamine content was reduced in PD rat, but preserved with rimonabant. Notably, rimonabant was able to hinder in part reserpine‐dependent increments in pT180/Y182 mitogen‐activated protein kinase p38, pS473‐AKT and pS536‐nuclear factor‐κB p65, as well as its down‐stream effector TNF‐α. Besides, rimonabant was able to restore the oxidant/antioxidant pool designated by increased glutathione versus decreased MDA. Notably, WIN55,212 per se intensified some of the reserpine effects and when used in combination with rimonabant, it partially antagonized the effect of the latter on almost all the aforementioned assessments. In all groups, the ATP/ADP/AMP and SIRT1 were decreased without changing mitochondrial transcription factor A (TFAM) and peroxisome proliferator‐activated receptor gamma coactivator (PGC)1α. Nonetheless, Nrf1 and mtDNA were increased with reserpine but decreased by rimonabant or combined treatment. Therefore, the study nominates a pivotal role for cannabinoid receptor activation in combating neuroinflammation instigated by PD with reduced mitochondrial biogenesis.
Investigating Plasma Membrane Monoamine Transporter (PMAT) as a Target for Development of Novel Antidepressant Treatments for Juveniles
Authors: Bowman Melodi Ann , Vitela Melissa , Gilman T. Lee , Daws Lynette C.
Published: April 2020

Depression is a psychiatric illness that affects individuals of all ages, yet only two antidepressants are approved to treat depression in children – both belong to the selective serotonin reuptake inhibitor (SSRI) class. Moreover, children are less effectively treated by SSRIs than adults. SSRIs block reuptake of serotonin via the high‐affinity, low‐capacity serotonin transporter (SERT). The resulting increase in extracellular serotonin is thought to initiate a cascade of downstream effects, which underlie the therapeutic utility of SSRIs. However, other transporters also clear serotonin from extracellular fluid, including the low‐affinity, high‐capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT). Our lab has shown that, in adults, decynium‐22 (D22), an inhibitor of OCTs and PMAT, produces antidepressant‐like effects when SERT function is either genetically or pharmacologically impaired. However, it is unknown whether OCTs and/or PMAT may be useful targets for therapeutic interventions in juveniles. In contrast to adults, our preliminary studies show that D22 has antidepressant‐like effects in juvenile (postnatal day 21 (P21)) wild‐type mice, suggesting that OCTs and/or PMAT may be functionally upregulated in juvenile mice. Since PMAT has a higher affinity for serotonin compared to OCTs, we investigated the antidepressant‐like effects of D22 in male and female P21 PMAT wild‐type (+/+) and constitutive PMAT knockout (−/−) mice and found the antidepressant‐like effects of D22 were lost in P21 PMAT−/− mice. Using in vivo chronoamperometry, we examined serotonin clearance kinetics in CA3 region of hippocampus in P21 PMAT+/+ and PMAT−/− mice. Across serotonin concentrations ranging from 0.25 to 1.5 micromolar, we found no difference in serotonin clearance rate between genotypes. These data suggest that either a) these concentrations of serotonin are insufficient to engage PMAT, and/or b) SERT, or other OCT isoform, is upregulated in constitutive PMAT−/− mice. In either case, detection of PMAT‐dependent serotonin clearance would be obscured. Ongoing studies are investigating the effect of D22 on serotonin clearance in juveniles and adults over a range of serotonin concentrations, as well as evaluating PMAT expression in brain as a function of age and PMAT genotype. Together, these studies will expand our understanding of differences in serotonin transport mechanisms between juvenile and adult brain, and will aid in discovery of novel targets for the development of antidepressants with improved therapeutic efficacy for children suffering from depression.

Support or Funding Information

This work was supported by the National Institutes of Health R01 MH106978 to LCD
A Proposed Proton Relay Mechanism in Methylenetetrahydrofolate Reductase
Authors: Pan Yuwei , Tetrick Maxwell , Li Richard , Seng John , Trimmer Elizabeth Eloise
Published: April 2020

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10‐methylenetetrahydrofolate (CH2‐H4folate) to 5‐methyltetrahydrofolate (CH3‐H4folate) by NAD(P)H. This reaction can be divided into two half‐reactions: the NAD(P)H reductive halfreaction and the folate oxidative half‐reaction. The X‐ray crystal structure of the E. coli enzyme has revealed a hydrogen‐bonded network of three active site amino acids, Glu28, Ser26 and His273. Previous studies with a Glu28Gln mutant indicated that Glu28, located near N10 of CH3‐H4folate, is an essential amino acid. Since Ser26 and His273 form hydrogen bonds to Glu28, we propose that Ser26, His273 and Glu28 are involved in a proton relay mechanism which donates a proton to CH2‐H4folate in the folate oxidative half‐reaction. In order to elucidate the role of Ser26 in this mechanism, we have characterized Ser26Thr and Ser26Ala mutants using the steady‐state CH3‐H4folate‐menadione oxidoreductase assay, in which the folate oxidative half‐reaction is rate limiting. We have hypothesized that the Ser26Thr mutant will have activity comparable to the wild‐type enzyme and the Ser26Ala mutant will have reduced activity due to its inability to hydrogen bond. Contrary to our hypothesis, we show that both mutants catalyze the folate reaction similar to the wild‐type enzyme. We also demonstrate that the Ser26Ala mutant shows competitive substrate inhibition in the CH3‐H4folate‐menadione assay, which is not observed for the wild‐type enzyme. Examination of the mutants in the NADH‐menadione assay shows kinetic parameters similar to those of the wild‐type enzyme. Taken together, our results suggest that Ser26 may not play an important role in either folate or NADH catalysis of MTHFR. However, we propose that in the case of the folate reaction, a water molecule inserted between the alanine and His273 might function like a serine in the proton relay mechanism, explaining the lack of a defect in the Ser26Ala mutant. To investigate the proton relay mechanism and the role of Ser26 further, we are studying the pH‐dependency of MTHFR in the CH3‐H4folate‐menadione assay.
Bioelectrical Impedance Analysis in Breastfeeding Women After an Individualized Dietary Intervention
Authors: Trecierras Larisa , Limon-Miro Ana T. , Valencia Mauro E. , Plata Trinidad Quizan , Slater Christine , Guzman-Leon Alan E. , Padilla Andrea Garcia , Valenzuela Jose C. , Lopez-Teros Veronica
Published: April 2020

Body composition assessment by bioelectrical impedance analysis (BIA) in breastfeeding women can provide an insight on the changes in body tissues and the state of hydration. Our general aim was to assess the changes of BIA parameters in overweight or obese breastfeeding women after an individualized nutritional intervention. After an informed consent was signed, breastfeeding women (2–8 weeks postpartum) were randomly assigned by simple allocation in blocks (1:1) to either the intervention (In) or control (Cn) group. These two groups received general recommendations on healthy eating during the lactation period; additionally, the In group received a dynamic menu with meals based on equivalent interchangeable foods every 2 weeks for 3 months. Body composition was evaluated using anthropometry and multi‐frequency bioelectrical impedance (Impedimed™) at baseline and 3 months after the intervention. Out of 345 women invited, 37 were eligible but only twenty (n = 10/group) completed the intervention program. At baseline, there were no differences in age and body mass index (BMI) and 60% of both groups of women declared exclusive breastfeeding. At the end of the program, the In group showed differences of −2.9 kg of fat mass [IQR −7.1– −0.9 (p<0.05)], −2.5 cm in waist [(IQR −5.0 – 0.2 (p=0.06)] and −3.2 cm in hip circumferences [IQR −4.7– −1.7 (p<0.05)]. Change in resistance standardized by height was ‐ 27 ohms [IQR −63–0.7 (p<0.05)]. Fat free mass and total body water remained unchanged with medians of +1.4 kg [IQR −0.15–2.6 (p=0.4)] and +0.95 kg [IQR −1.5– 3.2 (p=0.1)], respectively. This individualized nutrition intervention program designed for breastfeeding women was effective in improving the mother’s body composition. Multi‐frequency BIA can be a useful tool to monitor these changes during postpartum subjects in clinical practice. This trial was registered at clinicaltrials.gov NCT03640104.

Support or Funding Information

LT, AGP, JCV recieved a fellowship from The National Research Council (CONACYT)
Protein Arginine Methyltransferase 7 (PRMT7): A Human Enzyme Often Overexpressed in Cancer is Most Active Under Non‐physiological Conditions
Authors: Lowe Troy L. , Vernon Annabel , Clarke Steven G.
Published: April 2020

Arginine methylation is a ubiquitous eukaryotic posttranslational modification that can modulate transcriptional activation and suppression. Protein arginine methyltransferase 7 (PRMT7), one of the nine mammalian PRMT family members, catalyzes only monomethylation of a terminal guanidino nitrogen atom on its substrates. In an effort to understand the role of PRMT7 in the cell, we characterized a recombinant human PRMT7 expressed as a GST‐fusion protein in bacterial cells. Using S‐adenosyl‐L‐[methyl‐3H]methionine (3H‐AdoMet) as a substrate, we measured the rate of methylation of a synthetic peptide containing residues 23–37 of human histone H2B. Although the physiological methyl‐accepting substrates of PRMT7 are not known, this peptide contains major in vitro monomethylation sites at arginine residues 29, 31, and 33. A P81 phosphocellulose binding assay was performed that separates the 3H‐methylated product from unused 3H‐AdoMet. We confirmed the unusual temperature dependence of PRMT7, where optimal activity was found at about 20 °C with only about 30% of this activity at 37 °C. We found that the Km of PRMT7 for the H2B‐derived peptide was about 10 μM at 20 °C and about 20 μM at 37 °C. The Km for AdoMet was about 0.5 μM at both temperatures. Additionally we showed that physiological salt conditions can significantly decrease the activity. At 150 mM concentrations, we found only about 10% activity with NaCl or KCl, but about 80–90% activity with potassium acetate or ammonium acetate. We also detected a loss of activity with high ionic strength. These results suggest that there may be a specific inhibition by chloride ion as well as by ionic strength. Finally we show that PRMT7 activity is optimal in alkaline environments. The activity of PRMT7 at the near‐physiological pH of 7.5 is only about 20% that at pH 8.5. Taken together, these results open the possibility that PRMT7 can respond to cells undergoing temperature, salt, or pH stress. These findings demonstrate a potential mechanism for the in vivo regulation of arginine methylation by PRMT7 and provide clues to why PRMT7 has pleiotropic effects involved with transcriptional regulation and translation in the cell.

Support or Funding Information

This work was supported by National Science Foundation Grant MCB‐1714569 (to S.G.C), by a predoctoral Ruth L. Kirschstein National Service Award GM007185 (to T.L.L), and by an Amgen Scholars Award (to A.V.).
SPARC ‐ The Functional Anatomy of Murine Myelinated Renal Afferents
Authors: Schramm Lawrence P. , Tyshynsky Roman S. , Van Helden Dusty A. , Osborn John W.
Published: April 2020

Perhaps because they are a small minority of the total population of renal afferents, myelinated renal afferents (MRA ) have been the least studied. Yet, they are promising targets for neuromodulation because of their low thresholds for electrical stimulation. We are studying the calibers, branching patterns, terminal morphologies, and intrarenal targets of MRA. We identify the myelin sheaths and axons of MRA using antibodies to myelin protein zero and heavy‐chain neurofilament, respectively. The diameters of a majority of MRA range between 1 and 4 u. The diameters of a small minority range between 6 and 10u. Thus, the diameters of MRA are distinctly bimodal. Unmyelinated terminal segments of MRA arise from the myelinated segments of axons at two sites. First, an unmyelinated segment of axon projects from the end of each MRA. Second, unmyelinated segments arise as axon collaterals at nodes of Ranvier along the course of the myelinated axon. Most unmyelinated segments end without any terminal specialization. However, some terminate in small “bulbs, that are approximately twice the diameter of the axon. We have not observed receptor‐related, connective tissue specializations surrounding either type of termination. The distal myelinated portions of MRA are frequently tortuous, sometimes forming hairpin turns and loops. These complex features result in spatial concentrations of nodes of Ranvier, concentrations of unmyelinated collaterals, and concentrations of receptors. A marked narrowing at the origin of each unmyelinated segment suggests the presence of a spike‐initiation zone. The majority of MRA terminate deep in the renal cortex, most often in the extracellular space and frequently within bundles of connective tissue. Some terminations closely appose, but never penetrate, the renal tubular system. However, a small number of MRA appear to penetrate the walls of small arteries and venules. Terminations of MRA show no consistent relationship to glomeruli. The functions of MRA are, thus far, uncertain. Their terminations in the extracellular space would permit them to respond to changes in the composition of the extracellular fluid. However, their associations with connective tissue in the extracellular space could also provide them with mechanoreceptor properties. Their presence in the walls of the vasculature could mediate either chemoreceptor or mechanoreceptor functions. The distinctly bimodal distribution of the diameters of MRA suggests that they may participate in more than one physiological function. From a clinical perspective, the differences in stimulation thresholds afforded by those differences in axon caliber could facilitate selective neuromodulation of multiple physiological processes.

Support or Funding Information

Supported by SPARC Award 1U01DK116320‐02, JW Osborn, Jr., PI
Cerebrovascular Function is Impaired in Offspring from a Pre‐Clinical Rat Model of Preeclampsia that Exhibits Sex‐Dependent Changes in Blood Pressure
Authors: Cunningham Mark W , Pabbidi Mallikarjuna R , Williams Jan , Duncan Jeremy W
Published: April 2020

Background

Adult offspring (AOS) born from hypertensive mothers have an increased risk of developing hypertension, stroke, and neurological complications later in life. Women with preeclampsia (PE) and the pre‐clinical RUPP (reduced uterine perfusion pressure) rat model of PE have an increase in blood pressure, impaired cerebral blood flow (CBF) autoregulation, and cerebrovascular dysfunction during pregnancy. Studies have shown that blood pressure is elevated early in male AOS and later in female AOS born from RUPP dams. However, no study has examined the cerebrovascular function in AOS from RUPP dams.

Hypothesis

We hypothesize that AOS from RUPP dams will exhibit impaired cerebral vascular myogenic responses and CBF autoregulation.

Methods

Male and female Sprague Dawley rats were born from normal pregnant (NP) and RUPP dams. After 3 weeks of weening, OS rats were separated by sex and groups determined by their mother. OS rats were aged to 17 weeks and mean arterial pressure (MAP) and body weight (BW) was recorded. At 22–23 weeks of age, myogenic responses were determined by changes in vascular diameter of the middle cerebral artery in response to increases in intraluminal pressure. At 29–30 weeks, CBF autoregulation was measured by laser doppler flowmetry through a 4 mm x 4 mm cranial window under anesthesia. MAP was elevated step‐wise from 100–190 mmHg by infusion of phenylephrine to determine changes in CBF.

Results

At 17 weeks, BW was smaller in male AOS born from RUPP vs NP (381±10 vs 414±3g, p<0.05). No change was observed in female AOS BWs. MAP was elevated in male AOS RUPP vs NP (143±2 vs 133±3 mmHg, p<0.05) at 17 weeks, with no change in female AOS. At 22–23 weeks, the myogenic response was impaired and tone was increased in male AOS RUPP vs NP (p<0.05). At 29–30 weeks, CBF autoregulation was impaired in male OS RUPP vs NP rats at 120 mmHg (129±12% vs 103±4), at 140 mmHg (149±21 vs 107±12%), and at 160 mmHg (172±31 vs 108±15%) respectively. Interestingly, CBF autoregulation was also impaired in female AOS RUPP vs NP rats at 140 mmHg (130±14 vs 110±18%) and 160 mmHg (175±2 vs 99±8%, p<0.05) in the absence of hypertension.

Conclusion

A decrease in BW, increase in blood pressure, and impairments of CBF autoregulation was observed in male RUPP AOS. In contrast, female RUPP AOS exhibited no changes in body weight or blood pressure, but showed a marked impairment in CBF autoregulation, suggesting that elevated MAP is not a prerequisite or required for impaired CBF autoregulation in females. Future studies are warranted to examine the mechanisms underlying CBF autoregulation impairment and their associated sex differences in AOS born from hypertensive pregnancies associated with placental ischemia.

Support or Funding Information

Supported: AHA18CDA34110264
Topical Analgesic and Local Anesthetic Agents for Pain Associated with Chronic Leg Ulcers: A Systematic Review
Authors: Purcell Anne , Buckley Thomas , King Jennie , Moyle Wendy , Marshall Andrea P.
Published: April 2020

GENERAL PURPOSE

To provide information about the effectiveness of topical analgesic and local anesthetic agents for reducing pain associated with chronic leg ulcers.

TARGET AUDIENCE

This continuing education activity is intended for physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care.

LEARNING OBJECTIVES/OUTCOMES

After participating in this educational activity, the participant will:

1. Distinguish adverse reactions to topical analgesics and local anesthetic agents.

2. Evaluate the effectiveness of topical analgesics and local anesthetic agents for pain associated with chronic leg ulcers.

3. Identify substances used as topical analgesics and local anesthetic agents and the application of those agents.

OBJECTIVE

To examine the evidence related to the effectiveness of topical analgesic and topical local anesthetic agents for reducing pain associated with chronic leg ulcers.

METHODS

A systematic search and review of the literature were undertaken using key search terms such as leg ulcers, topical anesthetics, topical analgesics, and pain. Six databases were electronically searched for articles published between January 1990 and August 2019.

RESULTS

A total of 23 articles were identified that met the inclusion criteria. Data were extracted using content analysis. Most of the included studies were randomized controlled trials; however, the reported methodology for most of studies was poor, so the validity and reliability of the evidence are uncertain. Lidocaine/prilocaine cream, ibuprofen foam, and morphine gel were the most examined topical agents. Lidocaine/prilocaine cream significantly improved wound-related pain compared with all other studied agents. For topical analgesic agents, ibuprofen foam reduced chronic leg ulcer pain significantly, whereas morphine gel was ineffective.

CONCLUSIONS

Lidocaine/prilocaine cream and ibuprofen foam are effective agents for reducing wound-related pain associated with chronic leg ulcers. Effective use of topical agents could reduce the need for systemic pain relief agents, mitigating potential adverse effects, while giving clinicians another treatment option to manage wound-related pain associated with chronic leg ulcers.