bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cerebrovasculature Remodeling Accompanies Functional Impairment in Chronically Stressed Mice
Authors: Burrge Emily N. , Quintana Dominic D. , Coblentz Tyler , Childers Ryan , Price Christian , Bryner Randy W. , Frisbee Jefferson C. , Kelley Eric E. , Chantler Paul D. 
Published: April 2020

The cerebrovasculature system is inherently complex in both structure and function allowing it to dynamically regulate oxygen and nutrient flow within the brain. Chronic stress has been linked to various cerebrovasculature diseases, resulting from impaired function and altered architecture. Here we examined the effects of chronic stress on cerebrovasculature function, cerebral bloodflow, and angioarchitecture. Beginning at week 18, mice were exposed to the unpredictable chronic mild stress paradigm for 8 weeks (UCMS; 5 days/week for 7 hrs/day) or control conditions. Prior to euthanization, cerebral blood perfusion was examined using speckle‐pinpoint laser doppler flowmetry. Subsequently, mice were either subjected to vascular corrosion casting to provide a detailed 3D reconstruction of the complete cerebro‐microvasculature, or the middle cerebral arteries (MCA) were removed and positioned in a pressurize myobath and exposed to increasing concentrations of acetylcholine (ACh; 10 ‐9 M to 10 ‐4 M), phenylephrine (PE; 10 ‐9 M to 10 ‐4 M), and sodium nitroprusside (SNP; 10 ‐9 M to 10 ‐4 M). UCMS mice displayed a 25% (p<0.05) reduction in cerebral blood flow, and 18%(p<0.05) impairment in MCA dilation to ACh vs. control mice. MCA smooth muscle function was similar in UCMS and control mice, while UCMS mice displayed hypersensitivity to PE (20% increase vs. control, p<0.05). Cerebrovascular corrosion casting revealed increased tortuosity of microvasculature (49%), decreased cerebrovascular density, along with a 28% and 56% reduction capillaries and arteriole/veniole length in the overall brain (p<0.05). These data suggest that chronic stress results in significant changes to cerebrovascular function and angioarchitecture resulting in altered cerebral blood flow.Support or Funding InformationSupport: NIH Cobra Grant 5P20GM109098; NIH INBRE P20GM103434
Monoclonal Antibodies against Fentanyl and Fentanyl Analogs for Prevention of Opioid Overdose
Authors: Baehr Carly , Kelcher April Huseby , Robinson Christine , Crouse Bethany , Pravetoni Marco 
Published: April 2020

Anti‐opioid vaccines and antibodies have been proposed as a therapeutic agent to treat opioid use disorder and prevent opioid intoxication and overdose. In contrast to receptor‐targeting small molecule pharmacotherapy (e.g. naloxone) for opioid use disorder and overdose, antibody‐based therapeutics operate through sequestration of the target molecule in the serum, preventing access through the blood‐brain barrier. Monoclonal antibodies (mAb) offer several benefits as therapeutic agents, including long serum half‐life, high specificity for the target compound, and minimal side effects. Using a magnetic enrichment strategy to isolate opioid‐specific B cells, we have developed hybridomas expressing mAb against oxycodone, morphine, or fentanyl, that protect from opioid toxicity by preventing distribution of the target opioid to the brain. Passive immunization of mice with these mAbs significantly reduced both the antinociceptive effects and the concentration in the brain of each target opioid. Importantly, passive immunization of mice and rats with anti‐fentanyl mAb prevented fentanyl‐induced respiratory depression and bradycardia, two primary risk factors for opioid overdose‐related death. These anti‐fentanyl mAbs exhibited low nanomolar affinities in vitro, as measured by both competitive ELISA and biolayer interferometry. Finally, sequencing of opioid‐specific mAb identified recurring sequence motifs in antigen‐binding regions, which can inform rational design of mAbs to target structural variation in fentanyl analogs and related compounds. Overall, these results indicate passive immunization as a promising strategy to prevent toxic effects of opioids and other compounds of interest.Support or Funding InformationThis work is supported by the National Institutes of Health under grant T32DA007097 (CB) and National Institute of Drug Abuse under grant U01DA038876S1 (MP)
S‐Nitrosation and denitrosation of proteins by variation of superoxide/nitric oxide ratio – implications for prevention of sulfoxidation‐dependent enzyme inactivation during ischemia/reperfusion
Authors: Daiber Andreas , Oelze Matthias , Vujacic-Mirski Ksenija , Kalinovic Sanela , Jimenez Maria Teresa Bayo , Kuntic Marin , Ullrich Volker , Schildknecht Stefan 
Published: April 2020

Oxidative stress plays a key role for the development of cardiovascular, metabolic and neurodegenerative disease. This concept was never widely proven by the therapeutic use of classical antioxidants in large scale clinical trials. However, we know from numerous (pre)clinical studies that the formation of reactive oxygen and nitrogen species (RONS) as well as stable oxidative stress markers are a hallmark of most forms of diseases and higher oxidative stress markers are frequently associated with higher disease risk and mortality. Besides the detrimental role of RONS, they are also involved in cellular functions via redox signalling. We have previously identified an efficient mechanism of S‐nitrosation by low levels of nitric oxide and superoxide (3:1 ratio) with potential formation of N2O2 [Daiber A, Schildknecht S, Müller J, Kamuf J, Bachschmid MM, Ullrich V. Chemical model systems for cellular nitros(yl)ation reactions. Free Radic Biol Med. 2009 Aug 15;47(4):458–67.]. Here we, elucidated whether S‐nitrosation in the presence of higher nitric oxide than superoxide concentrations (as observed under hypoxic conditions) could prevent sulfoxidation and thereby oxidative inactivation of enzymes in the presence of higher superoxide than nitric oxide concentrations (as observed during reoxygenation). We found that increasing concentrations of xanthine oxidase/hypoxanthine caused conversion of S‐nitrosoglutathione (GSNO) to reduced glutathione (GSH) up to a certain concentration of xanthine oxidase, indicating that superoxide can induce denitrosation of GSNO. This finding was unexpected since in the presence of excess superoxide one would not expect regeneration of reduced GSH from GSNO. This was even more surprising since we observed substantial dihydrorhodamine oxidation that was prevented by uric acid and tyrosine nitration of albumin during denitrosation of GSNO by superoxide, all of which points to intermediary formation of peroxynitrite. In summary, we propose that S‐nitrosation of (mitochondrial) proteins during ischemia represents a protective mechanism to prevent irreversible overoxidation of thiols during the reperfusion phase and to re‐establish reduced thiol state in (mitochondrial) key enzymes of energy metabolism and cell survival. Wide‐spread mitochondrial protein S‐nitrosation may represent a central feature of the protective preconditioning effects of nitric oxide.Support or Funding InformationThe present work was supported by the European Cooperation in Science and Technology (COST Action BM1203/EU‐ROS and COST Action CA16225/EU‐CARDIOPROTECTION).
Investigation of the Conformational State of the 26S Proteasome Using FRET
Authors: Cresti Julianna Rose , Kraut Daniel Adam 
Published: April 2020

The 26S proteasome is a multiprotein complex in eukaryotic organisms that mechanically degrades misfolded, mutated, and obsolete proteins. As a result, proteasome function, or the lack thereof, has implications for neurodegenerative diseases. As it unfolds and degrades protein substrates, the proteasome undergoes a series of conformational changes that have been seen in CryoEM structures, proceeding from the s1, or substrate‐accepting state, to the s3 and s3‐like substrate processing and translocating states. In this research, the 19S particle of yeast proteasome was tagged with the fluorescent proteins (FPs) mTurquoise and mNeonGreen. The distance‐dependent spectroscopic technique Förster Resonance Energy Transfer (FRET) was employed to track the conformational changes associated with proteasomal function. The non‐hydrolyzable ATP analog ATPγS increased the observed FRET signal relative to ATP, consistent with the known ability of ATPγS to bias the proteasome’s conformation toward the s3 or s3‐like states.Support or Funding InformationJRC is a Beckman Scholar. This material is based upon work supported by the National Science Foundation under Grant No. 1515229 to DAK.
The Evolution of the Malate Dehydrogenase CUREs Community
Authors: Bell Jessica , Provost Joseph , Bell Ellis 
Published: April 2020

The Malate Dehydrogenase CUREs Community, MCC, was founded in 2017, supported by NSF IUSE Funding (NSF 1726932 EHR‐IUSE). The founding member schools (12 diverse institutions including PUI, Research Intensive, Comprehensive, and Community College) met in Summer 2017 to design and implement the first round of MCC CUREs which were taught in Spring 2018. Since then the community has grown and now consists of 23 institutions, (7 research intensive or comprehensive, 4 community college and 12 PUI) teaching MCC CURE classes to approximately 600 students per semester. As the community has grown we have learned that three key aspects of developing and sustaining such a community depend on not only the annual meeting of the group (in person or virtually) but on the availability of a network of colleagues willing to act as a support system for new members who are developing MCC CUREs for their classrooms. First, faculty interactions involve not only sharing of syllabi and written resources but more importantly the willingness to act as a sounding board and troubleshooting mechanism for new faculty participants who are in similar institutional settings. A significant strength of MCC is the breadth and balance of institution types involved. Second, the development of clusters of themed MDH CUREs focusing on specific scientific questions and supported by the development of appropriate biological resources and written background information has proved important for increased faculty and student engagement‐ this year one group of participating classes are engaged in exploration of the role of the flexible loop of Malate Dehydrogenase while another group is focusing on Protein‐Protein Interactions in metabolons and the regulation of these interactions by post translational modifications. Finally, the use of virtual meetings, of classes, and of participating faculty appears to energize students enhancing their ownership of the projects as well as providing a further resource for new faculty in the consortium. The faculty, like our students, progress from novice to expert on aspects of the project that they may be less familiar with. For example, the ability of the community to hold virtual PyMol tutorials significantly lowers the barrier for faculty less familiar with molecular visualization. In summary, what we learned during the initial phases of the project has contributed significantly to both the expansion of MCC and the sustainability of our classroom activities helping to provide CURE experiences for an increasingly diverse pool of students.Support or Funding InformationSupported by NSF 1726932 EHR‐IUSE
Sex complicates the effect and treatment of heat stress
Authors: Rudolph Tori , Lozano Johana Mayorga , Rhoads Robert P. , Baumgard Lance H. , Selsby Joshua T. 
Published: April 2020

Global temperature is continuing to rise, and resultant heat stress (HS) poses a major threat to human health. Using large animal models, we have shown that 12 and 24 h, and 7 d of heat exposure induced oxidative stress and impaired autophagy in oxidative skeletal muscle but not glycolytic muscle. We and others have implicated mitochondrial dysfunction in this process. We hypothesized that treatment with a mitochondrially targeted antioxidant, MitoQ, would decrease oxidative stress and alleviate cellular dysfunction during HS. To address this hypothesis, 3 mo old castrated male pigs (59 ± 1.0m kg; n=8/group) were administered either placebo or MitoQ (40 mg/day) divided into two daily doses every 12 h beginning 48 h prior to environmental treatment and continuing through environmental exposure. Half of the placebo‐ and half of the MitoQ‐treated pigs were assigned to thermoneutral (TN; 20°C) or HS (35°C) conditions for 24 h. The red portion of the semitendinosus (STR) was collected for subsequent analysis. Unexpectedly, malondialdehyde (MDA) concentration, a marker of oxidative stress, was unaffected by treatment or environment. Additionally, LC3A/B‐I was decreased 0.5‐fold (p<0.05) during HS, LC3A/B‐II was similar, and the ratio of LC3A/B‐II/I was increased 1.5‐fold (p<0.05) in HS; p62 was similar between treatment groups. These data suggest increased degradation of autophagosomes in HS males, which may explain maintenance of redox balance but conflicts with previous findings in female pigs; a scenario which raises the possibility of a sex effect on the muscular response to HS. To probe this idea, we compared STR from these HS males to archived HS STR from female pigs treated with a similar experimental design and environmental protocol described above. We confirmed our previous findings of oxidative stress and dysfunctional autophagy in females, and maintenance of redox balance with increased degradation of autophagosomes in males. In total, these data suggest that muscle from female pigs is more susceptible to detrimental effects of environment‐induced hyperthermia than muscle from male pigs.Support or Funding InformationSupported by USDA grant 2017‐05931
Approaches to Targeting Adenylyl Cyclase 1 for Novel Pain Therapeutics
Authors: O’Brien Joseph , Hayes Michael , Watts Val , Roman David 
Published: April 2020

Adenylyl cyclases (AC) catalyze the formation of cyclic AMP (cAMP) from ATP and are involved in a number of disease states, making them attractive potential drug targets. Recent preclinical studies have identified neuronal adenylyl cyclase type 1 (AC1) as a novel target for treating chronic pain. AC1 is highly expressed in neuronal tissues associated with pain processing and neuronal plasticity, and studies using AC1 knockout mice provide direct evidence linking AC1 to chronic inflammatory pain conditions. Furthermore, AC1 inhibitors would lack the side effects associated with other agents (e.g. opioids) used to treat chronic inflammatory pain. We have designed our studies to target NOT the conserved P‐site or forskolin‐binding site, but rather a novel approach, targeting the unique protein‐protein interaction of AC1 and calmodulin (CaM). AC1 and AC8 are both activated by CaM; however, the CaM binding domains are unique in structure and location providing an unprecedented opportunity to achieve AC1 selectivity. We hypothesize that developing a small molecule inhibitor of AC1 will allow us to mimic the AC1 knockout phenotype and provide a new avenue for the treatment of chronic inflammatory pain. Through the development and implementation of a novel biochemical high‐throughput screening paradigm we will interrogate a library of 100,000 compounds to identify inhibitors of the AC1/CaM protein‐protein interaction. Hit compounds will be validated and chemically optimized to lead molecules using cellular assays focused on selectivity and potency to guide medicinal chemistry efforts. To date, we have screened 14,080 compounds and identified 18 compounds capable of disrupting the AC1/CaM protein‐protein interaction, a hit rate of 0.13%. We anticipate the identification of selective AC1 inhibitors that will ultimately be improved and applied in models of chronic inflammatory pain.Support or Funding InformationThis work was supported by The National Institutes of Health (1R61NS111070 DLR & VJW), The University of Iowa Center for Biocatalysis and Bioprocessing and the NIH‐sponsored Predoctoral Training Program in Biotechnology (5T32GM008365 JBO).
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 InformationFunded 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 InformationR01HD067541‐06, 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

BackgroundSupplementation 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.Methods32 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).ResultsThere 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.ConclusionsEight 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 InformationSupported by NSERC (Canada).