bluuebunny/crossref_metadata_2025 huggingface dataset:
Daidzein Attenuates Kidney Damage in Diabetic Rats
Authors: Laddha Ankit Pravin , Kulkarni Yogesh Anant
Published: April 2020

Diabetic nephropathy is one of the major complications of diabetes which is responsible for 40% of death among diabetes population. In persistent hyperglycaemia, nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) causes overproduction of reactive oxygen species in podocytes, endothelial cells and mesangial cells in glomeruli. It plays a key role in progression of diabetic nephropathy. Many natural products are widely been studied in management diabetic nephropathy. Daidzein is an isoflavone present in leguminous plants and has been reported for anti‐oxidant, anti‐inflammatory and anti‐apoptotic activity. Present study was designed to observe effect of daidzein in experimental model of diabetic nephropathy in rats. Streptozotocin was used for induction of diabetes in male Sprague Dawley rats at dose of 55 mg/kg, intraperitoneally. After 28 days of diabetic induction, animals were treated with daidzein at dose of 25, 50 and 100 mg/kg, orally for next 28 days. Biochemical parameters and urine parameters were assessed at the end of study. Kidney function parameters like glomerular filtration rate, creatinine clearance, urea clearance and oxidative stress marker in kidney tissue were evaluated. Histopathology of kidney tissue was studied using haematoxylin‐eosin staining. Diabetic animal showed significant increase in plasma biochemical parameters like creatinine and blood urea nitrogen (p<0.001). Treatment with daidzein at high dose (100 mg/kg) for 28 days showed significant reduction in elevated levels of creatinine and blood urea nitrogen (p<0.01 and p<0.001). Reduced level of total protein and albumin in diabetic animals was also significantly increased after daidzein treatment at high dose (p<0.01 and p<0.05). Urine creatinine and urine urea were significantly improved in daidzein treated animals as compared to diabetic control animals (p<0.01 and p<0.05). Kidney function parameters like urea clearance, creatinine clearance and glomerular filtration rate were significantly (p<0.001) reduced in diabetic control animals. Treatment with diadzein for 28 days significantly improved kidney function. Loss of anti‐oxidant enzymes like glutathione, catalase and superoxide dismutase was prevented and generation of malondialdehyde was inhibited in kidney tissue of diabetic animals treated with daidzein. Daidzein treatment also ameliorates histological changes in diabetic rats. From results, it can be concluded that daidzein attenuates progression of diabetic nephropathy and can be a considered as effective management option for diabetic nephropathy.
Comicronized PEA and Rutin reduces inflammation and oxidative stress in a mouse model of vascular injury
Authors: peritore Alessio Filippo , Impellizzeri Daniela , Gugliandolo Enrico , Siracusa Rosalba , Fusco Roberta , d’Amico Ramona , Cordaro Marika , Crupi Rosalia , Cuzzucrea Salvatore , di Paola Rosanna
Published: April 2020

Acute and chronic inflammation responses are important risk factors for vascular remodeling processes such as in atherosclerosis, arteriosclerosis and restenosis. Inflammation and oxidative stress in the intimal region after a vascular damage are key events in the development of neo intimal hyperplasia. Sustained failure to counteract the excessive production of reactive oxygen species (ROS) and dysregulation of the antioxidant defence system in the endothelium elicits cellular damage and dysfunction. Palmitoylethanolamide (PEA) an endogenous fatty acid amide belonging to the N‐acylethanolamine family, has anti‐inflammatory and neuroprotective effects. However, PEA lacks direct capacity to prevent formation of free radicals. Rutin is a kind of flavonoid glycoside known as Vitamin P and has antiplatelet, antiviral and antihypertensive properties, as well as strengthing the capillaries of blood vessels, which are the results of its high radical scavenging activity and antioxidant capacity. Thus, the combination of PEA with rutin could have beneficial effects on inflammatory process and oxidative stress. The aim of this study was to investigate the protective effects of a new comicronized compound Palmitoylethanolamide with antioxidant Rutin (Rutamid) in a ratio 1:1 at dose of 10mg/kg (daily oral adminstration) in an experimental model of vascular damage, which involves the complete ligature of the left carotid artery for 14 days. This model showed that 14 days after carotid artery ligation there was a significant structural change within the vessel, and an important involvement of inflammatory pathways in the progression of this disease. Rutamid treatment reduces vessel damage, adhesion molecules expression such as intercellular adhesion molecules‐1(ICAM‐1) and vascular cell adhesion molecules‐1(V‐CAM), proinflammatory cytokines production (Tumor Necrosis Factor alpha (TNF‐) and Interleukin 1 beta(IL‐1), the inducible nitricoxide synthase(iNOS) and Poly(ADP‐ribose) polymerase (PAR), formation. Nuclear factor kappa‐B and NRF‐2 pathways activation and apoptosis were evaluated. Our results clearly demonstrated that treatment with Rutamid was able to reduce vascular damage and attenuates the inflammatory process and oxidative stress. Rutamid could represent a new therapeutic strategy in the treatment of conditions associated to vascular damage.
Inhibition of Epstein‐Barr Virus by Serotonin‐Dopamine Antagonists
Authors: Gorres Kelly , Hoffman Nate
Published: April 2020

Epstein‐Barr Virus (EBV), a herpesvirus family member, was the first human cancer‐associated virus discovered. EBV maintains a lifelong latent infection within the host B cells. During periodic reactivation into the lytic phase, the virus replicates and spreads among cells. The virus is reactivated into the lytic phase in response to environmental stress conditions. In cultured EBV‐positive cells, the lytic switch can be initiated by the addition of a variety of small‐molecule inducing agents. Viral reactivation is inhibited by diverse natural products and pharmaceuticals. Viral reactivation can be induced, or blocked, through a number of different mechanisms. Since one viral inhibitor is also a drug used to treat neurological conditions, we investigated the response of the virus to atypical antipsychotic drugs. Previously, we discovered clozapine is an inhibitor of EBV reactivation. Here we screened a panel of atypical antipsychotic drugs for effects on the expression of the viral lytic gene BZLF1. We found antagonists of serotonin and dopamine receptors inhibited the EBV lytic cycle in Burkitt lymphoma cells. The inhibitors may be functioning through their associated receptors or through any of their off target signaling pathways. These results highlight the lesser known roles of serotonin and dopamine signaling outside the central nervous system. Inhibition of EBV through novel mechanisms will be useful in elucidating the natural pathways of viral reactivation and in the treatment of EBV‐associated diseases.

Support or Funding Information

This work was funded by the UWL College of Science and Health and UWL Faculty Research Grants to KLG.
A Comparison of Spherical Harmonic and Sliding Semilandmark Analyses as Methods for Three‐Dimensional Shape Evaluation
Authors: Goldstein Deanna M. , Harper Christine M. , Sylvester Adam D.
Published: April 2020

The ability to quantify morphological variation is integral to biological research. The use of sliding semilandmarks in geometric morphometric (GM) analyses allows for the evaluation of whole bone morphology in three dimensions. A sliding semilandmark analysis, however, is difficult to complete when structures lack obvious homologous features for landmark placement. Spherical harmonics (SPHARM) offers an alternative method for shape analysis in such cases. Here we evaluate the SPHARM method by comparing results to those from a sliding semilandmark analysis of the calcaneus in African apes and modern humans.

Modern human (n=20), chimpanzee (n=20), and gorilla (n=20) calcanei were surface scanned. For the GM analysis, 1007 sliding semilandmarks were evenly distributed across the external surface of each calcaneus. Semilandmarks were slid to minimize the bending energy of the thin plate spline interpolation function relative to an updated Procrustes average. Final landmark configurations were then aligned using Generalized Procrustes Analysis. For the SPHARM analysis, surface models were mapped onto the surface of a sphere (parameterization), and then decomposed into a weighted sum of spherical harmonic functions. Because the spherical harmonics provide a standardized basis for quantifying shape, coefficients of the harmonic functions can be used as shape descriptors. Calcaneal shape variation was summarized using a principal components (PC) analysis of the Procrustes coordinates and SPHARM coefficients. Analyses were compared through calculation of the root mean square (RMS) and maximum distance between surface models of the average specimen from each species, as well as surface models of PC warps created two standard deviations from the average along PCs 1–3.

Both sliding semilandmark and SPHARM analyses separate species along the first two PCs. For both analyses, African apes and modern humans separate along PC1. Average RMS between species averages was 0.25 mm. Maximum surface distances between average surface models from the two analyses were all less than 0.23 mm. Average RMS between all respective PC warps was 0.82 mm. Maximum surface distances between PC warps were all less than 1 mm. Although there were minor differences between PC plots produced from the two analyses, the same general morphological distinctions between species were identified. The primary differences between SPHARM and sliding semilandmark‐based surface models of PC warps and species averages occur around sharp edges, which are less defined using the SPHARM analysis, although these differences are small. Overall, SPHARM provides the same shape representation as a sliding semilandmark analysis, making it a comparable method for the evaluation of shape in three dimensions.

Support or Funding Information

This project was supported by NSF grant # BCS ‐ 1824630.
Reducing Turbulent Airflow Lowers Healthy Females Work Of Breathing During Exercise To A Level Similar To Males
Authors: Granger Emily A. , Mann Leah M. , Chan Jason S. , Yu Annie , Dominelli Paolo
Published: April 2020

Objective

Imaging studies have shown that healthy females have smaller conducting airways than height matched males. The smaller conducting airways would increase the propensity towards turbulent airflow and this is thought to be the underlying mechanism behind greater resistive and total work of breathing in females. Heliox (21% O2: balance He) is considerably less dense than room air and has a greater propensity towards laminar flow. As such, respiring heliox should produce a physiologically similar effect as increasing airway size and allows for experimental manipulation of work of breathing between the sexes. The objective of the study was to determine if turbulent flow is responsible for the greater work of breathing in healthy females.

Methods

Six healthy men (Age=24±4 years) and five healthy women (Age=22±2 years) completed two step‐wise maximal exercise tests on separate days. On one day subjects respired compressed room air and on the other a compressed helium‐oxygen mixture (~21% O2: balance He). Room air and heliox were humidified and delivered in an identical manner. Esophageal pressure was measured with a balloon catheter and allowed for the determination of the work of breathing via integration of transpulmonary pressure‐volume loops. The work of breathing‐ventilation relationship was modelled using a previously described equation consisting of two constants that describe the mechanical work to overcome i) viscous resistance and laminar flow and ii) turbulent resistance.

Results

End‐exercise ventilation was significantly greater for females during the heliox trial (82±9 vs. 94±11 L min−1, P=0.02) but there was no difference in work of breathing (122±38 vs. 116±42 J min−1, P=0.45), for room air and heliox respectively. Using the modeled work of breathing data, compared to males, females had a significantly greater work of breathing at ~70 L min−1 during the room air trial and this was due to greater work to overcome turbulent resistance. When comparing the heliox trials between sexes, there was no difference in the work of breathing. Similarly, after ~60 L min−1 compared to the room air trial, the work of breathing among females had significantly lowered during the heliox trial and this was due to a lower contribution of turbulent resistance.

Conclusion

Our results suggest that increased turbulent flow, as a result of smaller conducting airways, is responsible for the greater work of breathing in healthy young females during exercise.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada RGPIN‐2019‐04615 (PBD). Canada Foundation for Innovation #38432 (PBD).
Cerebrovascular Impairment Due to Maternal Electronic Cigarette Usage During Pregnancy is not Dose Dependent
Authors: Burrage Emily N. , Aboaziza Eiman , Reppert Sarah A. , O’Reilly Juliana , Hare Lane A. , Chantler Paul D. , Olfert I. Mark
Published: April 2020

Developmental harm to offspring from maternal exposure to electronic cigarettes (E‐cig) during pregnancy is still poorly understood. Similar to cigarettes, we hypothesized the even low‐level E‐cig exposure would produce similar evidence of cerebral vascular dysfunction compared to a higher exposure dose. We examined the effects of maternal E‐cig exposure (Joyetech eGrip OLED using 5‐sec puffs @17.5 W) on cerebrovascular function in offspring (n=2–4 from each dam) from Sprague‐Dawley rat dams exposed to air (Control), E‐cig with 18 mg/ml nicotine (E‐cig18) and without nicotine (E‐cig0). Dams were exposed to low (20 puffs) or high (60 puffs) dose for 1‐hour each day, 5 days/week, starting on gestational day 2 and continued until pups were weaned. Pups themselves we never directly exposed to E‐cig aerosol. Middle cerebral arteries (MCA) were obtained from pups at 3‐months of age, isolated and positioned in a pressurized myobath, and exposed to increasing concentrations of acetylcholine (ACh; 10 ‐9 M to 10 ‐4 M), serotonin (5‐ HT;10 ‐9 M to 10 ‐4 M), and sodium nitroprusside (SNP; 10 ‐9 M to 10 ‐4 M), and in the presence or absence of Tempol (a superoxide dismutase mimetic). At the lower dose, MCA dilation to ACh was impaired by 60±1% and 50±0.1% in E‐cig0 and E‐cig18 groups, respectively, compared to controls (p<0.05). At the higher dose, MCA dilation to ACh was similarly impaired by 63±1% and 60±0.2% in E‐cig0 and E‐cig18 groups, respectively (p<0.05). Incubation with tempol reversed the cerebrovascular dysfunction seen in both E‐cig groups at both doses, suggesting the superoxide pathway is involved in the impairment observed in offspring. At both low and high doses, the vasocontrictory response to 5‐HT, and dilation response to SNP (non‐endothelial‐dependent mechanism), were similar across all groups. These data show that maternal E‐cig usage results in similar cerebrovasculature impairment with either 20 or 60 puffs/day during pregnancy and lactation in adolescent offspring with prior in utero exposure from maternal vaping at suggesting that even low‐levels of maternal E‐cig use confers significant post‐natal vascular health risks for the offspring.

Support or Funding Information

WVU Cancer Institute Philip R Dino Innovative Research Grant (IMO); APS STRIDE Fellowship (JO); NIHGMS 5U54GM104942‐03 (PDC)
Psoas Quintus: A Novel Muscle Variant of the Posterior Abdominal Wall
Authors: Jones Shiloh , Fenster Trenten , Kamande Stanley , Maini Aneel , Marcelle Madeline , McConathy Emily , Suarez-Quian Carlos A.
Published: April 2020

Anatomical variations of the psoas muscles are rare, with only a few examples reported in the literature. Typical musculature consists of two psoas muscles on the posterior abdominal wall. These include the psoas major, which is universally present in the human population, and the psoas minor, an inconsistent, but commonly occurring muscle. Other rarely reported psoas variants include the psoas tertius and psoas quartus muscles. Both are predicted to play a role in femoral nerve compression due to their proximity to the femoral nerve and their location on the posterior abdominal wall. Here we report the presence of both psoas quartus and psoas tertius muscles, as well as the discovery of a previously unreported psoas variant, which we have named psoas quintus. The muscle was discovered during a routine dissection of the left posterior abdominal wall of a 57‐year‐old Caucasian male. The psoas quintus had a proximal attachment via a slender tendon to the body of the L3 vertebrae and spread to the area of the anterior superior iliac spine by a relatively broad tendon. Its muscles fibers were oriented distinctly from those of the iliacus muscle below and the psoas quartus medially.
Inflammation and oxidative damage as risk factors of neurodegeneration after Traumatic Brain Injury in young and aged mice
Authors: Cordaro Marika , Remigante Alessia , Crupi Rosalia , Morabito Rossana , Marino Angela
Published: April 2020

Traumatic Brain Injury (TBI) causes transitory or permanent structural and functional impairments and may increase the risk of neurodegeneration. As age correlates with development of neurodegenerative disease after TBI, the aim of the present study was to verify the possible onset of neurodegeneration after TBI and the related pathways, namely associated to oxidative stress and neuroinflammation in both young (3 months) and aged mice (12 months). TBI was induced in mice by controlled cortical impact and behavioral tests (open field, elevated plus maze, novel object recognition, Morris water maze) performed after 30 days and 3 months of trauma. Thereafter, the animals were sacrificed and the brains processed for Western blot and immunohistochemical analyses. In particular, IL‐6, IL‐8 and TNF‐a levels accounting for inflammation and malonildialedhyde (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) levels accounting for oxidative damage have been monitored. With regard to behavioural anaylis at both time intervals after TBI, aged animals exhibited a more pronounced anxiety‐like behavior along with impaired memory than what estimated in young animals. Moreover, a strong increase in neuroinflammation, shown by increased levels of cytokines, and oxidative damage, attested by increased MDA and GSH along with decreased SOD quantity suggested that neuroinflammation and oxidative stress damage were more significantly exhibited by aged than young mice.

The present study suggests that aging may exacerbate the damage deriving from TBI and increases the risk of neurodegeneration due to oxidative stress and inflammation response, with specific regard to anxiety‐like behavior. Further studies may be useful to monitor risk factors of neurodegenerative diseases over a longer time lapse after TBI, namely in aged animal, in order to add more knowledge about development of neurodegeneration.

Considering the complex multifactorial nature of neurodegenerative diseases, a preventive strategy able to simultaneously target multiple risk factors and disease mechanisms at an early stage is most likely to be effective to slow/halt the progression of neurodegenerative diseases.
The Impact of Chromatin Structure on the Formation of Double Strand Breaks by CRISPR‐CAS9 in the Yeast Genome
Authors: Heitmiller Courtney Anne , Burgess Rebecca C.
Published: April 2020

DNA is persistently at risk for being damaged by endogenous and exogenous factors. One type of DNA damage is a double‐strand break (DSB), which occurs when both strands of the helix become severed. DSBs are important because upon repair, they could lead to mutations or genomic alteration. The packaging of the DNA into chromatin may affect the susceptibility of an area to DSBs and thus could make the area more prone to mutation. The purpose of this study is to look at how the chromatin environment affects the susceptibility of genes to DSBs in Saccharomyces cerevisiae. A new system, tetracycline‐inducible CRISPR‐Cas9, was used in this experiment to induce DSBs at different sites in the yeast genome Histone modification levels were measured at each site to correlate these chromatin marks with DSB formation frequency. DSB frequency at each genomic locus was measured using quantitative PCR and directly compared to those same sites in unchromatinized DNA to see how chromatin environment and the surrounding histone modifications affects susceptibility to DSBs.

To measure the frequency of DSBs at different genomic sites using qPCR, the break site was amplified. DSBs would be indicated through a decrease in product when compared to an uncut control site NFS1 on Chromosome III. While correlation analysis of histone modifications and DSB frequency is still ongoing, preliminary data suggests that repressed genes have higher levels of cutting than active genes after three hours of DSB induction. Of the repressed genes investigated, PPE1 had 82.06% DSB formation and RRT8 had 66.67% after three hours of induction. Active genes RPL22A, TDH2, and TDH3 had on average about 15%, 9.5%, and 56% DSB formation after three hours of DSB induction, respectively. The level of DSB formation in these sites will be directly compared to DSB formation of the same sites on an unchromatinized plasmid, which is currently underway.

This research is important because it shows which areas of the genome may be at higher risk for DSBs and whether or that is affected by chromatin structure. Understanding this could help deepen the knowledge of how the genome evolves through spontaneous DSB formation and repair.
Phosphorylation and function of OATP1B1 with tyrosine kinase inhibitors.
Authors: Hayden Elizabeth R.
Published: April 2020

Introduction

The organic anion transporting polypeptide OATP1B1 regulates hepatic uptake of many therapeutics. As a result, drug‐drug interactions (DDI’s) are commonly mediated by OATP1B1 activity. Reduced drug elimination via this transporter can lead to increased rates of drug‐associated adverse reactions, such as statin‐induced rhabdomyolysis. Surprisingly, numerous tyrosine kinase inhibitors (TKIs) that are either weak or non‐substrates of OATP1B1 increase systemic concentrations of various known OATP1B1 substrates by an unknown mechanism. We hypothesize that OATP1B1 function is dependent on tyrosine phosphorylation, which can be reduced by specific TKIs.

Methods

Primary human hepatocytes and HEK293 cells transfected with OATP1B1, or tyrosine (Y) to phenylalanine (F) mutant variants of OATP1B1, were used to assess the uptake of 8‐[2‐[Fluoresceinyl]‐aminoethylthio)‐adenosine‐3′,5′‐cyclic‐monophosphate (5–40 μM) or estradiol‐b‐glucuronide (2 μM) in the presence or absence of FDA approved TKIs. A biotinylation labeling assay was utilized to measure surface expression of OATP1B1 treated with and without nilotinib (0–20 μM). Phosphorylation of OATP1B1 was assessed by Western blot and mass spectrometry‐based proteomic analysis. OATP1B1 protein was purified by immunoprecipitation from overexpressing cell lines in the presence or absence of nilotinib. siRNA was utilized to identify kinases that potentially mediate OATP1B1 function. Statistical analysis was performed with Graph‐pad Prism, and each experiment was repeated in triplicate.

Results

Nine of 46 FDA approved TKIs inhibit OATP1B1 function over 80%, compared to untreated controls. Nilotinib, the most potent TKI among assessed, does not decrease OATP1B1 expression or membrane localization. Instead, it elicits inhibition of OATP1B1 through a non‐ or mixed‐competitive and reversible mechanism. Nilotinib also reduces OATP1B1‐mediated uptake in primary human hepatocytes (P < 0.001). Mass spectrometry analysis of OATP1B1 revealed a variety of tyrosine phosphorylation sites, among them, phosphorylation at position 625 is greatly reduced in the presence of nilotinib. Moreover, mutation of Y625F results in significantly reduced transport activity compared to the wild‐type OATP1B1 activity (P < 0.001). Finally, we confirmed that SiRNA knocking down of LYN kinase activity, a Src tyrosine kinase, significantly reduces OATP1B1 activity (P < 0.001).

Conclusion

We found that OATP1B1 is tyrosine phosphorylated at numerous sites and that position 625 is highly sensitive to nilotinib exposure. The Y625F mutation of OATP1B1 results in decreased transport activity and similarly, loss of LYN expression also leads to lower OATP1B1 transport. These findings indicate that clinically relevant TKIs may promote drug‐drug interactions by interfering with a post‐translational event that is required for OATP1B1‐mediated transport.

Support or Funding Information

University at Buffalo School of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Seed Support Program
STING Activation and its Role in IFN Production
Authors: Link Tina , Rajput Aashita , Kona Abhinav , Lee Chase , Sheth Heli , Vemana Riya , Wright Aryn
Published: April 2020

The immune system’s ability to respond to foreign DNA is a highly conserved mechanism. One crucial part of this mechanism is Stimulator of Interferon Genes (STING), a central innate immune protein located in the endoplasmic reticulum. It is a pattern recognition receptor that is activated in response to intracellular infection and, ultimately, mediates type 1 interferon (IFN) production. Encoded by the gene TMEM173, STING is expressed in T‐cells, monocytes, natural killer cells, and dermal fibroblasts.. It is composed of four transmembrane helices and a cytoplasmic ligand binding and signaling domain, which form a domain‐swapped dimer assembly. The STING pathway is initiated when double‐stranded DNA (dsDNA) is sensed, causing cyclic‐GMP‐AMP‐synthase (cGAS) to cyclize GTP and ATP and produce 2′3′‐cyclic‐GMP‐AMP (cGAMP), the main ligand that activates STING. Upon STING activation, the ligand‐binding domain closes, inducing a 180 degree rotation of the ligand‐binding domain relative to the transmembrane domain. The C‐terminal tail of STING has been found to autoinhibit the protein by blocking the polymerization interface of inactive STING, preventing auto activation. The conformational change made in response to cGAMP releases the CTT, exposing the polymer interface and allowing disulfide link formation. Alternatively, STING can be activated by bacterial cyclic‐di‐GMP in a cooperative manner, with cyclic‐di‐GMP serving as a partial antagonist of the cGAMP signaling pathway. Structural and biochemical studies have provided elegant insight into the mechanism of differential of STING by cGAMP and cDi‐GMP. STING activates tank binding kinase 1 (TBK1) which phosphorylates transcription factor IRF‐3 and causes it to translocate to the nucleus. Gain‐of‐function mutations of TMEM173 causing elevated transcription of IFNs have been proven to cause autoimmune syndromes such as systemic lupus erythematosus and, most recently, three mutations of TMEM173 have been found to directly cause STING‐associated vasculopathy with onset in infants (SAVI). New research proposes a therapeutic strategy that uses molecules that induces a different STING conformation than cGAMP‐bound STING as a STING inhibitor. This method would prevent constitutive STING activation in autoimmune syndromes while being less immunosuppressive than previously proposed therapies involving STING inhibition. The Walton High School MSOE Center for Biomolecular Modeling SMART Team has designed a 3‐D model of STING using a recently published cryo‐electron microscopy structure that reveals the transmembrane domain structure to further investigate and examine the structure‐function relationship of STING.

Support or Funding Information

MSOE Center for Biomolecular Modeling
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 Information

Support: 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 Information

This work is supported by the National Institutes of Health under grant T32DA007097 (CB) and National Institute of Drug Abuse under grant U01DA038876S1 (MP)
Integrating 3D Animated Videos in Case‐Based Study of Cranial Nerve Pathology
Authors: Ayoub Amara , Afifi Linda , Zumwalt Ann
Published: April 2020

The structures and functions of cranial nerves are essential topics in the gross anatomical study of the head and neck. Cranial nerves with complicated pathways carry multiple modalities and exhibit several symptoms when injured. Conventionally, the course and branching patterns of these nerves are taught using two‐dimensional resources with limited perspective and depth. Three‐dimensional anatomical applications allow us to map the pathways of cranial nerves toward their peripheral targets to reinforce understanding of complex spatial relationships. We created a series of animated videos through the 3D4Medical Complete Anatomy application to demonstrate the courses and modalities of three challenging cranial nerves (CN), VII, IX and X. These videos cater to flipped learning, a student‐centered pedagogical approach. Here we describe the methodology and effectiveness of integrating 3D‐animated videos into a flipped classroom paradigm to enhance understanding of cranial nerve pathways, functions, and pathologies. Forty students participated in two flipped sessions (20 students per session) administered as a series of clinical cases studies. Students were expected to watch the cranial nerve animation videos prior to the session. Class time was devoted to small‐group discussion and application of knowledge through case‐based learning and formative assessments. Each of the answer choices were reviewed following discussion of the case. Individual student performances (n = 34) were determined using pre‐ and post‐test scores. Pre‐ and post‐test scores were compared using the non‐parametric Wilcoxon signed rank test, as differences do not follow a normal distribution. Our results indicate an overall significant improvement in performance between pre‐ and post‐assessment scores (p = 0.036, one‐sided). Students (n = 31) completed a post‐session survey exploring the effectiveness of instruction, pre‐work material and case‐based approach. Based on a modified Likert scale of 1–5, students found the 3D‐animation videos effective (score of 5, 64.5%; score of 4, 12.9%) in preparing for the session and future exam. Students found the case‐based approach, discussion and instruction to be highly effective (score of 5, 96.8%; score of 4, 3.2%), which strongly supports the use of this pedagogical method. We conclude that flipped, case‐based sessions integrating 3D‐animated videos are an effective method for enhancing understanding of cranial nerve structure, function and pathology.
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 Information

The 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 Information

JRC 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 Information

Supported 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 Information

Supported 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 Information

This 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).