bluuebunny/crossref_metadata_2025 huggingface dataset:
5‐Aminosalicylic Acid Treatment Affects the Rate of Caloric Intake in Fattening Ground Squirrels
Authors: Witman Nathan , Bradley Rachel , Roberts Emily , Kovalenko Ivanna , Duddleston Khrystyne , Kurtz Courtney
Published: April 2020

Obesity is the second leading cause of preventable death in the United States. Though prevalent, very little is known about the biological mechanisms that lead to obesity. A connection between increased gut permeability and the consequent immune response in systemic tissues exists in obese humans and mice. Systemic inflammation brought on by this immune response has been shown to decrease tissue efficacy and lead to adverse effects, such as insulin resistance. Recently, it was shown in mice on a high‐fat diet that 5‐aminosalicylic acid (5‐ASA, mesalazine), a gut‐specific anti‐inflammatory drug, attenuated insulin resistance and metabolic inflammation. We used a natural model of rapid adiposity, fattening 13‐lined ground squirrels (Ictidomys tridecemlineatus) during their active season, to examine the effects of 5‐ASA on caloric intake, body mass, glucose tolerance and inflammation. These squirrels normally double their adipose mass during their active season in preparation for hibernation. We found that the rate of caloric intake was significantly affected by 5‐ASA treatment and that these squirrels tended to eat more while gaining equal or less mass during the same period. Adipose depot mass was not different between groups but did differ between males and females. Despite a trend in the early weeks of 5‐ASA treatment, glucose tolerance did not differ between treated and control squirrels. Examination of pro‐ and anti‐inflammatory cytokines in white adipose depots and gut tissues revealed trends toward increased IL‐4 and TNF in visceral adipose of treated squirrels. Our results suggest that 5‐ASA treatment has different effects on development of metabolic inflammation in ground squirrels versus mice. Further studies are needed to determine if a higher dose would prove more effective in these seasonal hibernators.

Support or Funding Information

Supported by NIH award 1R15GM124586‐01A1
Post‐Exercise Central Hypotension is Associated with Changes in Forward Pulse Wave Amplitude and Reflected Pulse Wave Timing in Young, Healthy Individuals
Authors: Patik Jordan C. , Stock Joseph M. , Chouramanis Nicholas V. , Mehrer Justin D. , Chirinos Julio A. , Edwards David G.
Published: April 2020

In the hours following moderate intensity aerobic exercise (AE), persistent dilation of the arterial circulation supplying previously exercised muscle decreases total peripheral resistance (TPR) and contributes to post‐exercise hypotension. In addition to the effect of vasodilation on TPR, the diameter and tone of arterial vessels also influences central blood pressures via changes in the amplitude of forward (Pf) and backward (Pb) pulse waves, as well as the apparent wave reflection return timing (i.e. reflected wave transit time, RWTT). Prior research has assessed these parameters over a limited timeframe (e.g. 15 min) and relied on synthesized aortic flow waves rather than measured left ventricular outflow. As such, the prolonged post exercise effects of AE on pulse wave characteristics and central blood pressure are unclear.

Purpose

This study aimed to characterize changes in pulsatile hemodynamics in the two hours following AE. We hypothesized that Pf and Pb would be reduced, and RWTT would be increased post‐AE relative to baseline (BL).

Methods

Twelve young, recreationally active participants (8M/4F, 26±3 years, VO2max: 50.8±14.3 ml·kg−1·min−1) were studied at BL and then every 20 min after completing 60 min of upright cycling at 60% VO2max. At each timepoint, central pressure waveforms were derived from radial artery applanation tonometry and aortic inflow waves were acquired via echocardiographic pulsed‐Doppler assessment of blood velocity in the left ventricular outflow tract. Pressure and flow waves were averaged over 10 cardiac cycles and pressure‐flow relations were determined offline via wave separation analysis.

Results

As expected, brachial systolic blood pressure (SBP) was decreased across the 120 min period post‐AE relative to BL (time effect P<0.001) while TPR was only reduced at 20 min (Δ−15±5%, P<0.05 vs BL) and cardiac output (CO) was maintained at all timepoints (time effect P=0.12). The nadir in central SBP occurred at 60 min post‐AE (Δ−11±2%, P<0.001 vs BL). At 60 min post‐AE, Pf and Pb were reduced by Δ−14.3±2.5% and Δ−24.2±3.3%, respectively (each P<0.001 vs BL) and RWTT was increased (Δ19.5±5.3% P<0.02 vs BL). The percent change in central SBP from BL to 60 min was related to changes in RWTT (r=−0.79, P=0.02) and Pf (r=0.65, P=0.02) but not Pb (r=0.38, P=0.22), CO (r=0.27, P=0.40) or TPR (r=0.14, P=0.66).

Conclusions

These data highlight the contribution of changes in pulsatile hemodynamic parameters, in contrast to changes in non‐pulsatile parameters (CO and TPR), to reductions in central BP following moderate intensity AE.

Support or Funding Information

Supported by NIH grants R01‐HL104106 and P20‐GM113125
Comparative Neuromorphology of Purkinje Neurons Across Species
Authors: Liu Yi Fan , Bradley Stefanie , Bailey Craig D.C. , Vickaryous Matthew K.
Published: April 2020

Neuromorphology of cells vary both across and within species, the differences of which contribute to a variety of cellular functions. Purkinje cells (PCs) in the cerebellum play a key role in the fine tuning of motor control and movement, and are characterized by their large soma and distinctive pattern of dendritic branching. However, outside of few selected taxa, the neuromorphology of PCs have not been well characterized. Here, we investigate PC neuromorphology in a representative reptile (leopard gecko), mammal (laboratory mouse), and bird (domestic chicken). Using a modified Golgi‐Cox protocol, differences in PC neuromorphology were quantified using Sholl and branched structure analyses. While PCs in all species demonstrated elaborate patterns of dendritic arborization, we quantified species‐specific differences in both cell size and branching complexity. The average dendritic length of PCs in geckos and mice were comparable, while those of chickens were almost twice as long. Chicken PCs are also characterized by a greater dendritic diameter and dendritic volume. Mouse and chicken PCs were significantly more complex than those of geckos, based on the number of dendritic terminals. Taken together, our findings demonstrate that even within the same cell type, there is considerable neuromorphological variation between species, potentially related to aspects of phylogeny, ecology, and functional morphology.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants 400358 to MKV and 2019‐04989 to CDCB
Relation of the Marginal Mandibular Nerve to the Inferior Border of the Mandible in Fresh and Embalmed Cadavers
Authors: Marolt Clayton , Freed Blair , Johnson Kenneth , Rowe Joanna , Coker Charles , Steele Robert , Stephens Robert , Surek Chris C.
Published: April 2020

The most frequent cause of damage and paralysis to the facial nerve is iatrogenic injury during operations in the parotid or submandibular regions, and the marginal mandibular branch (MMBr) is the least likely to recover due to its infrequent anastomosis with other branches. Literature has stated that the MMBr courses superior to the inferior border of the mandible more often than in the submandibular region. However, various studies report conflicting results, and variations in location have been seen between embalmed and fresh specimens. The purpose of this study was to determine the location of the MMBr in relation to the inferior border of the mandible in embalmed specimens and compare those findings with fresh specimens. In both embalmed and fresh cadavers, the superficial fascial planes were dissected to reveal the MMBr and its anatomical relationships and distances. Perpendicular distance between the most inferior branch of the MMBr and the antegonial notch were measured on both sides to the nearest 0.01mm using a caliper. The most inferior position of the MMBr between the antegonial notch and gonion was also measured. A negative value was used to denote branches superior to the inferior border of the mandible, and positive values for those below. This study found that the MMBr was located below of the border of the mandible more often than above. There was also no significant difference in MMBr location between fresh and embalmed cadavers. These results indicated that the MMBr postion was more inferior than initially stated, and that there was no significant change in the location of the nerve due to the embalming process.
Structural details of GEF‐mediated Sar1 activation
Authors: Joiner Aaron , Fromme Chris
Published: April 2020

The first trafficking step of the eukaryotic secretory pathway is the packaging of cargo proteins into COPII‐coated vesicles that exit the endoplasmic reticulum (ER). COPII vesicle coat proteins are recruited to the ER membrane via direct interaction with the activated (GTP‐bound) form of the Sar1 GTPase. Sar1 is activated on the surface of the ER by Sec12, a membrane‐anchored GEF (guanine nucleotide exchange factor). Here we report the crystal structure of the complex between Sar1 and the cytoplasmic domain of Sec12, representing a snapshot of the activation reaction. This structure provides detailed insight into the mechanism of Sar1 activation. The structure reveals that despite structural homology to the RCC1 GEF, a distinct surface of the beta‐propeller fold of Sec12 interacts with Sar1 to displace nucleotide. The structural results suggest that the cytoplasmic domain of Sec12 may adopt an unexpected orientation relative to the membrane surface. This orientation would position Sar1 on the ER membrane so that binding to GTP and engagement of its amphipathic N‐terminal helix with the lipid leaflet can occur simultaneously. Together with accompanying functional data, our results provide a structural explanation for the critical regulatory step of COPII vesicle biogenesis.

Support or Funding Information

This work was funded by the National Institutes of Health grant
R01GM116942 to J.C.F.

Alfred P. Sloan Foundation Fellowship and NSF GRFP DGE‐1650441 to A.J.
The Impact of Bacteriophage on Complex Bacterial Populations in Urban Waterways
Authors: Lammers Abigail , Enz Alicia , Carlson Clayton D.
Published: April 2020

The purpose of this research project is to investigate the population and activity of bacteriophage in Chicagoland waterways with the ultimate goal of treating bacteria‐polluted water with their respective phage counterparts. The focus waterway in this study is the Tinley Creek: a small waterway that flows through residential areas, forest preserves, and industrial areas, eventually flowing into a major channel that carries treated wastewater. In our preliminary research, we have used phage amplification, absorbance readings, and metagenomic analysis to show that the presence of phage in a population greatly shapes the growth patterns of bacteria in that population. Our particular bacteria of focus has been Klebsiella oxytoca, a member of the family Enterobacteriaceae and an opportunistic human pathogen. K. oxytoca is present in the Tinley Creek. Our treatment of wild bacteria populations has been focused on using K. oxytoca phage as well as a mixed population of wild phage from the Tinley Creek. We are scaling up our tests to larger replications of the ecosystem to examine how microbial populations respond to phage treatment. Our research suggests that treating polluted waterways with phage may be a viable means to improve water in urban waterways.
c‐Fos Responses Produced by the Central Microinjection of Salvinorin B in Conscious Rats
Authors: Ali Aboozar B. , Gonzalez Christopher , Lee Eunice , Rockey Anna , Hall Allison , Franklin Cynthia , Rangel Yolanda , Helmut Gottlieb B.
Published: April 2020

Intracerebroventricular (ICV) injection of kappa opioid agonists produces a marked diuresis in rats via inhibition of vasopressin secretion. Salvinorin B (Sal B) is a plant‐derived high‐affinity ligand for the kappa opioid receptor (KOR). Previous studies have proposed that Sal B has similar potency and efficacy as other synthetic kappa agonists in mediating the CNS effects of KOR activation. The present study examines the effects of Sal B on renal function and the expression of the proto‐oncogene c‐Fos in several hindbrain regions. Sal B effects were compared to either acetonitrile or saline vehicle treated animals. Sal B is a highly lipophilic drug and past studies utilized drug dissolving vehicles (e.g., DMSO) that produced significant cardiovascular and renal effects. In this study, we dissolved Sal B in acetonitrile/saline (1:9 dilution) to minimize solvent viscosity and cardiovascular effects.

Methods

Animals were habituated to a metabolic cage and injected with either Sal B, acetonitrile or saline. Urine samples were collected during the habituation period and used as an additional control. On the day of the experiment, urine was collected during two 15 min control periods followed by six 15 min periods beginning 15 min after the ICV injection into the lateral ventricle. After 90 minutes, animals were perfused with phosphate buffer and PFA. Brains were removed and cut on a cryostat (40 μm thick). Brain sections were processed for c‐Fos using a commercially available antibody. Sections were double labeled with dopamine beta‐hydroxylase (DBH; CY3 Anti‐mouse) to define anatomically specific hindbrain regions.

Results

ICV Sal B failed to produce any significant changes to renal function (urine volume, sodium and potassium excretion rate) as compared to control vehicle. Sal B decreased c‐Fos expression in the medial nucleus of the solitary tract (NTS‐m) and rostral ventrolateral medulla (RVLM), and noradrenergic cell group (A5).

Conclusion

Previous studies suggested that Sal B has affinity for the KOR; however, our results indicate that Sal B lacks efficacy and does not produce diuretic or anti‐natriuretic effects associated to synthetic kappa opioid agonists. In addition, KOR agonists increase renal sympathetic nerve activity via changes to cardiovascular controlling centers in the brain. Sal B decreased c‐Fos expression in certain hindbrain regions, but further studies are necessary to determine the role Sal B plays in these regions.
Effects of the Gut Microbiome on Aged‐Related Cognitive Decline and Inflammation
Authors: Lee Juneyoung , Venna Venugopal R. , Durgan David J. , McCullough Louise D. , Bryan Robert M.
Published: April 2020

Introduction

Aging is associated with low level inflammation and cognitive decline. We tested the hypothesis that the gut microbiome has a major role in these age‐related changes and that this age‐related decline is transferrable through the gut microbiome in mice.

Methods

For this study we gavaged a suspension of fresh fecal contents from aged (~20 months) or young (~3 months) BL/6 mice into germ‐free BL/6 mice (N=10 per group). The absence of gut bacteria in germ‐free mice reduced competition and enhanced the colonization of the aged or young microbiomes. Gavages began in the germ‐free mice at 12 weeks of age and were repeated monthly for 3 months. Behavioral testing and collection of fecal samples occurred over the study period. After 3 months, mice were euthanized, and the brain and colon were harvested for assessing the inflammatory state using flow cytometry. Bacterial community structures and relative abundances were measured in fecal samples by sequencing the bacterial 16S rRNA gene. Short‐chain fatty acids (SCFA) were measured in feces using LC‐MS.

Results

Mice with an aged microbiome demonstrated depressive‐like behavior, impaired short‐term memory, and impaired spatial memory for at least 3 months after the initial gavage using the tail‐suspension (P=0.008), the Novel Object Recognition (P<0.001), and the Barnes Maze (P=0.019) tests respectively. The aged microbiome increased inflammation as revealed by flow cytometry studies. In brain γΔ T cells were increased (P=0.017) with an aged microbiome. In the gut, leukocytes were increased (P<0.001) and Tregs were decreased (P=0.029) with an aged microbiome. Mice with aged and young microbiomes showed clear differences in bacterial α diversity (Simpsons and Fisher, P<0.001 for both) and β diversity (weighted and unweighted PCoA, P=0.001 for both). At 3 months after the initial gavage, the bacteria families, Defluviitaleaceae and Lactobacillaceae, were significantly decreased in mice with aged microbiomes (P<0.001 and P=0.01 respectively). Fecal SCFAs, including acetate, propionate, and butyrate (P<0.01 for each), were decreased in mice with an aged microbiome. These 3 SCFAs are gut protective and anti‐inflammatory. Of note, Lactobacillus, a SCFA‐producing genus, was decreased 3‐fold in mice with an aged microbiome (P=0.009).

Conclusion

We conclude that the gut microbiome has a major role in aged‐related cognitive impairment and inflammation. Furthermore, cognitive decline and an enhanced inflammatory state associated with aging can be transferred via the gut microbiome. Thus, manipulating the gut microbiome can protect from the detrimental effects associated with aging.

Support or Funding Information

RO1 AG058463 (RMB and LDM)

RO1 NS103592 (LDM and RMB)
Effect Of Sodium Azide And DDVP On Liver And Kidney Parameters Of Albino Rats
Authors: Agbasi Patrick Ugochukwu , Chukwu Leo Clinton , Ugorji Toochukwu Nze , Anyiam Chioma Kesiah , Daniel Jovita Ada
Published: April 2020

Background

In the Sub Sahara Africa, organophosphorous insecticides are extensively and intensively used in the living houses, offices and even against pests to preserve foods and agricultural products to preserve farm produce. Sodium azide (a herbicide) is also extensively used in the farms to control weeds. These conditions have necessitated this study.

Purpose

This research is designed to investigate the effect of low dose DDVP (dichlorvos) and sodium Azide co‐administration on some kidney and liver biochemical parameters of Albino rats bearing in mind the rising cases of liver diseases and kidney failures.

Method

Seven groups of five rats each were used for the study. Group A was used as control and received no chemical treatment, groups B and C were injected with 1% and 3% LD50 of Sodium Azide respectively, while groups D and E received 1% and 3% LD50 of DDVP respectively. Equally, group F received combined doses of 1% LD50 of DDVP and 1% LD50 of Sodium Azide while group G received 3% LD50 of DDVP and 3% of LD50 Sodium Azide. The chemicals were administered at alternate days for twenty one days. At the end of twenty one days study period, the animals were anesthetized with chloroform and blood were aspirated through cardiac puncture into empty clean 5ml containers and then allowed to clot. The serum were then separated and frozen before kidney and liver parameters were carried out.

Results

Sodium Azide caused significant reduction in liver transaminases, alkaline phosphotase and elecyrolytes (Na and K) while the creatinine level was not affected. DDVP, caused significant increases in the transaminases, Alkaline phosphotases while also causing causing significant reductions in urea, creatinine and the electrolytes when compared to control. Sodium Azide and DDVP caused significant reduction of the transaminases and urea while the creatinine level is unaffected.

Conclusion

Sodium Azide caused significant reduction in liver transaminases, alkaline phosphotase and elecyrolytes (Na and K) while DDVP caused significant increases in the enzymes and reductions in the urea and creatinine. Co‐administration of DDVP and Sodium Azide produced significant reduction of the transaminases, alkaline phosphotase and urea.
The inhibitory effects of Ganoderma Lucidum on NFκB related inflammatory cytokines in activated BV‐2 microglial cells
Authors: Hilliard Aaron , Mendonca Patricia , Soliman Karam F.A.
Published: April 2020

Neurodegenerative diseases have increased significantly over the last five years, and the increase in the elderly with Alzheimer’s and dementia requires increased attention and research design to combat this critical problem. Ganoderma Lucidum extract (GLE) is a potent ancient Asian remedy for extending the life and treatment of a variety of disease states. In the current study, we investigate the neuroprotective potential effects of Ganoderma Lucidum on LPS‐activated BV‐2 microglial cells. The LPS activated BV‐2 cells have been largely used as a model to illustrate the pro‐inflammatory process involved in the activation of signaling transduction pathways associated with neuronal injury. The methodology used in this work included cell viability, nitric oxide assay, screening of pro‐inflammatory cytokines using mouse cytokine arrays, individual ELISAs to validate data from the arrays, and RT‐PCR to investigate a possible signaling pathway for GLE neuroprotective effect in activated BV‐2 microglial cells. Cell viability results showed that increasing concentrations of GLE, ranging from 0.5 to 1.3 mg/ml, induced BV‐2 cytotoxicity in a dose‐response manner (IC50: 0.79 mg/ml). Also, the results obtained indicate that lower concentrations of GLE (< 0.5mg/ml) showed to be potent in reducing the release of nitric oxide in activated BV‐2 cells. The results from the cytokine arrays showed that GLE decreased the expression of several pro‐inflammatory cytokines including Granulocyte Colony Stimulating Factor (G‐CSF), Interleukin 1‐alpha (IL1‐α), Monocyte chemotactic protein‐5 (MCP‐5), Macrophage Inflammatory Protein 3 Alpha (MIP3‐α), and Regulated on Activation Normal T Expressed and Secreted (RANTES). The data obtained were further validated using individual ELISA, and the results showed that GLE was able to decrease the expression of each one of the downregulated proteins in the cytokine arrays. RT‐PCR was performed using different genes that are involved in the NFκB signaling activation. The results also showed that GLE decreases the mRNA expression of CHUK, NFκB 1/p150, and IKBKE, which are genes that participate in the NFκB translocation to the nucleus, and subsequent release of pro‐inflammatory cytokines. The findings suggest that GLE may be a new potential therapeutic option for decreasing inflammatory responses seen in Alzheimer’s and dementia patients.

Support or Funding Information

Supported by a NIMHD Grant U54 007582
Mechanistic role of Gαo in striatal cAMP signal transduction
Authors: Muntean Brian S. , Sutton Laurie P. , Zucca Stefano , Dao Maria , Patil Dipak N. , Birnbaumer Lutz , Martemyanov Kirill A.
Published: April 2020

The G protein alpha subunit o (Gαo) is abundantly expressed in the nervous system and pathogenic mutations in its gene (GNAO1) cause movement disorder. In contrast to the other Gα subunits, Gαo has been relatively understudied and its function remains mechanistically ill defined. Using biochemical assays, optical imaging in primary neurons, and genetic mouse models we define mechanisms of Gαo action on the cAMP generating enzyme – adenylyl cyclase (AC) in a neuron subtype selective manner. Here, we show that Gαo transduces striatal signals to enable motor learning by opening an alternate signaling channel regulating dynamics of neuromodulatory interrogation of dopamine to the second messenger cAMP. We find that Gαo is a critical modifier of striatal AC activity through horizontal interplay with other G protein subunits. Gαo modulates Gβγ availability to impact processing of signaling by AC that sensitizes stimulatory inputs (Gαs/olf) while diminishing efficacy of inhibitory inputs (Gαi). These processes accompany adjustments in electrophysiological properties of striatal neurons that compromise motor learning in a circuit‐selective fashion. Based on our findings we propose a model for Gαo in programming motor command processing of GPCR signals to cAMP.

Support or Funding Information

This work was supported by funding from the following NIH grants: DA041207 (to BSM), DA048579 (to SZ), and DA036596, DA026405 (to KAM).
Tryptophan‐75 is a Potential Gating Residue of Cytochrome P450 2D6
Authors: McCarty Kevin D. , Furge Kyle A. , Furge Laura
Published: April 2020

Cytochrome P450s (CYPs) are responsible for the metabolism of an estimated 70 – 80% of pharmaceutical drugs in humans. Structurally, they have deeply buried active sites. Evidence suggests that access to the active site, and the resulting metabolism of substrates, is influenced by amino acid side chains ‐ a phenomenon referred to as tunnel‐gating. Our investigation hypothesized that tunnel‐gating in CYP2D6 exists and is mediated by a tryptophan‐75 (Trp‐75) residue. Analysis in silico and in vitro was conducted to understand gating in CYP2D6 and the role of plasticity in enzyme mechanisms. Molecular Dynamics simulations measured energy landscapes of Trp‐75 and simulated substrate passage within channel 2b. It was determined that Trp‐75 was able to obstruct both entry and exit of substrates from the enzyme within channel 2b, and that the residue alternates between three stable states. A Trp‐75 to alanine (*W75A) mutant of CYP2D6 was prepared along with a reference *1 enzyme and both were tested for their Michaelis‐Menten kinetics and ligand binding properties in vitro with the substrates bufuralol, dextromethorphan, and debrisoquine. Trp‐75 was not found to impact ligand binding by the CYP2D6 enzymes. The residue was observed to significantly impact Km and vmax, but no predictable kinetic trends were found. Furthermore, Trp‐75 was observed to impact enzymatic efficiency, where the absence of the residue doubled overall efficiency. Thus, we propose that the range of conformations available to Trp‐75 function in mediating channel‐gating that is substrate specific and that can serve to trap substrates into channel 2b for shuttling into the active site.

Support or Funding Information

NIH 2R15GM086767‐03 and the Sherman Fairchild Foundation
Unbiased Identification of Key Residues in Splicing Protein Dib1
Authors: Turner Caitlyn E. , Maeder Corina
Published: April 2020

Pre‐messenger RNA (pre‐mRNA) is transcribed from a DNA segment, and then post‐transcriptionally modified before being translated into protein. To prepare the pre‐mRNA for proper protein production, the spliceosome facilitates the removal of non‐coding introns from the pre‐mRNA and the ligation of exons together, forming the mature messenger RNA (mRNA) in a process known as pre‐messenger RNA splicing. The spliceosome is a large ribonucleoprotein complex composed of 5‐snRNA and over 90 proteins that work in concert to facilitate pre‐mRNA splicing and ensure its accuracy. Due to its complexity and evolutionary importance to survival, mutations of the spliceosome have been linked to diseases. For instance, Burn‐McKeown syndrome is a craniofacial disorder caused by abnormalities in the gene for the U5‐15k protein, a small protein centered within human spliceosomes and an ortholog of Dib1 in yeast.

Dib1 is a 17‐kDa thioredoxin‐like protein that is associated with the U5 small nuclear ribonucleoprotein (snRNP) in the U5/U4.U6 complex. Located in the catalytic groove of the pre‐B complex of the spliceosome, Dib1 must vacate the complex so the spliceosome can complete its assembly. However, this “gate‐keeping” mechanism controlling this departure is still unknown. This project intends to discover residues that are crucial to the function of Dib1 by an unbiased method. To identify key residues in Dib1, a randomly mutagenized dib1 plasmid library was constructed. A temperature sensitive (ts) yeast screen was then performed with this library in order to identify Dib1 residues important for cell growth. From this screen, we have identified a new dib1 mutant and will discuss the significance of its location in Dib1 and its relevance to splicing.

Support or Funding Information

Trinity University, ASBMB Distinguished Undergraduate Scholarship, NIH‐NIGMS R15GM120720, and The Welch Foundation
Antimicrobial Activity of Extracts from the Leaves of Marrubium vulgare
Authors: Cravens Ashley , Krzysiak Amanda
Published: April 2020

Natural products have served as powerful therapeutics against pathogenic bacteria since the golden age of antibiotics of the mid‐20th century. In the United States, antibiotics are a key component of modern medicine and are one of the top written prescriptions every year. However, overuse and misuse of antibiotics has led to an increasing frequency of antibiotic‐resistant infections. These infections are difficult and expensive to treat, often resulting in increased length of illness, hospital stays, and mortality rates. This clearly demonstrates that new antibiotics are critical for modern medicine. Murrubium vulgare (White Horehound) is a shrub found in the Appalachia region that has been used in herbal medicine to treat many pulmonary aliments as an expectorant, tonic, and emmenagogue. Previous studies of this plant and its commercial products have indicated that it may contain antioxidant and antibacterial properties. The aim of this study is to determine if whole leaf extracts of M. vulgare contain antimicrobial properties. Five different plants were sampled for this project. The leaves were dried and then underwent extract in 95% ethanol in a 1:10 (m/v) ratio. The solvent was removed using a rotavapor at 60°C and hot plate evaporation. The crude product was left in a desiccator until constant weight was achieved. The crude samples were analyzed to characterize the different classes of natural products present. The crude extracts were dissolved in 5% DMSO at different ratios. The disk diffusion method was applied to determine the antimicrobial activity.

Support or Funding Information

Bellarmine University, Student Government Association.
A Cellular Based Assay to Monitor the Cleavage of the Extracellular Matrix by Matrix Metalloproteinases as a Tool for Drug Discovery Against Metastasis
Authors: Doyle Ryan , Canter Ben , Nader Joy , Wolkowicz Roland , Tandon Ruby
Published: April 2020

One of the most important genes up‐regulated in cancer is known as Matrix Metalloproteinase (MMPs). MMP’s are enzymes critical for remodeling the protein matrix, called extracellular matrix (ECM), that surrounds and supports cells. This protein can be secreted or membrane‐bound as it remodels the extracellular environments in healthy individuals. However, in cancerous cells, its overexpression leads to the metastasis of cancer cells.

To investigate novel ways to block metastasis, our lab engineered a new cellular Two‐Tag Assay as a tool for drug discovery, enabling the search for inhibitors. The assay, based on a system we developed for HIV and Dengue Virus, was adapted to MMP‐14. It relies on a scaffold composed of two antibody epitopes, HA and FLAG, flanking a protein substrate. Because the FLAG epitope is encoded upstream of the substrate and HA, it is cleaved while HA remains attached to the cell membrane. We then use flow cytometry to monitor the presence of HA and FLAG. Based on the presence of one or two tags, we can determine the robustness of cleavage. Previously, we inserted viral proteases between the tags and showed autocatalytic cleavage or lack of while the protein travels through the classical secretory pathway. The MMP‐14 assay has a consensus substrate of MMP‐14 instead of a viral protease, as well as a cell line obtained through retroviral technology. In addition, we expressed MMP‐14 enzyme in cells expressing substrate, as well as naïve cells. Comparing substrate‐expressing cells with substrate and enzyme expressing cells, we showed that MMP‐14 cleaves the substrate. We performed mixing experiments with substrate‐expressing and enzyme‐expressing cells to specifically corroborate cleavage at the cell surface. Results were corroborated with confocal microscopy and western blotting. Decrease in FLAG expression in the presence of MMP‐14 proved the assay’s ability to discover new inhibitors of MMP‐14.

Support or Funding Information

This research is supported by fellow researchers in the Wolkowicz Lab and the National Cancer Institute of the National Institutes of Health under award numbers: U54CA132384 & U54CA132379
Immunohistochemical analysis of macrophage polarization in aged kidneys and spleen
Authors: Moyer Eric , Bloomer Steven
Published: April 2020

Aging is accompanied by a pro‐inflammatory state that adversely affects physiological function in many organs. Macrophages, innate immune cells of the myeloid lineage, exist on a phenotypic spectrum ranging from pro‐inflammatory (M1) to anti‐inflammatory (M2), and play a major role in tissue homeostasis throughout life. Previously, we have shown that aging increases the number of macrophages of both phenotypes (M1 and M2) in the liver; however, in other organs, there are conflicting findings as to the effects of aging on macrophage number and polarization. Therefore, the purpose of this study was to determine the effects of aging on macrophage abundance and phenotype in the kidneys and spleen. We hypothesized that aging would increase the number of M1 macrophages, and decrease the number of M2 macrophages in each organ. To investigate this, we utilized immunohistochemistry to detect the M1 marker, inducible nitric oxide synthase (iNOS), and the M2 marker, cluster of differentiation 206 (CD206), in kidney and spleen sections from young (6 months) and old (24 months) male Fischer 344 rats. The number of cells were counted in 6–7 visual fields, the area of the field was determined, and the results are expressed as the number of cells per square millimeter. In the kidney, we detected diffuse iNOS staining in tubular cells, however there was no visible staining in cells with morphologic characteristics of macrophages in either age group. Cells positive for CD206 were detected most frequently in the medulla, with no significant differences in number between young (3.3 cells/mm2) and old (4.1 cells/mm2) animals. In the spleen, macrophages resided in the marginal zone, and in the red pulp. We observed a trend (p=0.07) for fewer splenic iNOS+ macrophages in young animals, compared to the old (0.015 cells/mm2 vs. 0.072 cells/mm2). However, there was no difference in splenic CD206+ macrophages between young and old groups (59 vs 57 cells/mm2). Unlike the liver, we have observed similar numbers of M2 macrophages between young and old animals in the kidneys and spleen, which reflects the heterogeneous nature of aging. Our results have also identified a unique M2 macrophage population in the kidney, which resides in the renal medulla.

Support or Funding Information

Faculty Development Grant, Penn State Abington. Erickson Discovery Grant, Penn State University.
Transforming Growth Factor‐α Acts via Epidermal Growth Factor Receptor to Increase p44/42 Mitogen‐Activated Protein Kinase Signaling and Expression of Excitatory Mediators in the Hypothalamic Paraventricular Nucleus in Rats
Authors: Yu Yang , Wei Shun-Guang , Weiss Robert M. , Felder Robert B.
Published: April 2020

Myocardial infarction (MI)‐induced heart failure (HF) in rats leads to activation of brain p44/42 mitogen‐activated protein kinase (MAPK) signaling, which may increase proinflammatory cytokine expression and upregulate renin‐angiotensin system (RAS) activity in the brain, contributing to sympathetic excitation. We recently reported (Hypertension, 2019; 74: AP2010) that activation of the epidermal growth factor receptor (EGFR) in the hypothalamic paraventricular nucleus (PVN), a critical cardiovascular and autonomic center, increases p44/42 MAPK signaling in rats with HF. Both transforming growth factor (TGF)‐α and TGF‐β1 can activate EGFR and p44/42 MAPK signaling in peripheral tissues. Both are expressed in the PVN. In the present study, we first examined whether HF alters TGF‐α and TGF‐β1 expression in the PVN. Male rats underwent coronary artery ligation (CL) to induce HF or sham surgery, and brains were collected 4 weeks after CL. Compared with sham rats, HF rats had significantly (*P<0.05) increased TGF‐α mRNA expression (1.93±0.83* vs 1.03±0.29, fold change) in the PVN, but TGF‐β1 mRNA expression in the PVN was comparable between HF and sham rats (1.42±0.63 vs 1.01±0.20). We next examined whether TGF‐α increases p44/42 MAPK activity in the PVN via EGFR. Male rats received bilateral PVN microinjections of a lentiviral vector encoding a small interfering RNA (siRNA) for EGFR or a scrambled siRNA. One week later, these rats received an intracerebroventricular (ICV) injection of TGF‐α (10 μl, 0.1 μg/μl over 5 min) and the brains were collected 4 hours after the TGF‐α injection. Normal rats without any treatments served as control. Compared with control rats, scrambled siRNA+TGF‐α rats had significantly increases in protein levels of phosphorylated p44/42 MAPK (0.70±0.15* vs 0.23±0.11, corrected by total p44/42 MAPK) and in mRNA expression of the proinflammatory cytokines tumor necrosis factor‐α (5.45±2.10* vs 1.24±0.77, fold change) and interleukin‐1β (2.58±0.33* vs 1.00±0.09, fold change), with a tendency toward an increase in mRNA expression of the RAS component angiotensin II type 1a receptor (1.40±0.29 vs 1.02±0.24, P = 0.08, fold change) in the PVN. All of these findings were ameliorated in the EGFR siRNA+TGF‐α rats (by 21–48%), in which EGFR mRNA expression was reduced by 57%. These results indicate that TGF‐α, an endogenous ligand for EGFR, increases in the PVN in HF and may increase p44/42 MAPK signaling, thereby contributing to the augmented inflammation and RAS activity that drives sympathetic excitation in HF.

Support or Funding Information

Supported by NIH grant R01HL136149 and the Department of Veterans Affairs.
Alzheimer’s Disease‐Related Pathology and Impaired Cognitive Function Associate with Reduced O‐GlcNAc Transferase in Aged Metabolic Syndrome KKAy+/− Mice
Authors: Jinka Sanjay , Lallo Jason , Gupta Shreya , Mathias Amy , Khanal Saugat , Al-Rhayyel Yasmine , Herman Danielle , Fleming Sheila , Raman Priya
Published: April 2020

Metabolic syndrome (MetS) refers to a cluster of anomalies including type 2 diabetes, obesity, insulin resistance and dyslipidemia. Patients with MetS are 1.5 times more likely to develop late‐onset Alzheimer’s Disease (AD), with impaired glucose metabolism, defective insulin signaling, increased oxidative stress and inflammation being shared between the comorbid diseases. Notably, risk of AD is profoundly enhanced in the aging MetS population. Recent studies in AD patients and AD mouse models suggest a putative link between hyperphosphorylated tau neurofibrillary tangles, a commonly accepted AD pathology, and cerebral glucose hypometabolism. Impaired glucose metabolism is characterized by reduced glucose flux through the hexosamine metabolic pathway triggering attenuated signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), a major regulator of intracellular protein O‐GlcNAcylation. However, the role of OGT in the etiology of MetS‐induced AD remains incompletely understood. The goal of the present study was to examine the link between cognitive function, AD pathology and OGT signaling in a mouse model of MetS (KKAy+/−) that develops increased body weight, hyperglycemia, elevated total cholesterol and total triglyceride levels. Briefly, male and female obese agouti KKAy+/−, lean non‐agouti KKAy−/− and normal C57BL/6 control mice weaned at 4 wks of age on regular chow diet were subjected to periodic body weight and random blood glucose monitoring followed by a battery of behavioral tests at 12+ months of age. Plasma and brain (frontal cortex and hippocampus) tissues were then harvested from each genotype for biochemical and molecular studies. In an object recognition test of attention and memory, obese KKAy+/− mice showed a more severe impairment in discrimination between a novel and familiar object compared to lean KKAy−/− and normal C57BL/6 mice. Additionally, in a test of spontaneous activity obese KKAy+/− mice made significantly fewer rears vs lean KKAy−/− mice and wild‐type C57BL/6 controls; these results suggest diminished cognitive function in the obese KKAy+/− mice. Furthermore, immunoblotting of brain tissue lysates derived from the agouti KKAy+/− mice revealed increased ptau expression coupled with reduced pERK (signaling mediator of neuronal function) and pGSK (inactive form of tau kinase GSK3β) expression compared to non‐agouti KKAy−/−. Importantly, augmented tau phosphorylation was concomitant to attenuated OGT expression in brain lysates of obese KKAy+/− mice. Together, these data demonstrate a direct association between cognitive dysfunction, AD‐related pathology and reduced OGT expression in aged MetS KKAy+/− mice. Overall, our study implicates a role of OGT in MetS‐induced cognitive decline and AD pathogenesis.

Support or Funding Information

NEOMED Start‐Up Funds
Targeting primary cilia with polymeric nanoparticles
Authors: Pala Rajasekharreddy , Nauli Surya M.
Published: April 2020

The field of nanomedicine is a promising future in providing huge clinical impacts on advanced disease management and personalized medicine. Nanoparticles (NPs) have been used for targeted delivery at a desired site and a sustained release of a drug, which decreases the overall toxicity by delivering a smaller drug dosage. An extensively investigated NPs is poly lactic‐co‐glycolic acid (PLGA) which is an FDA approved biodegradable polymer and they are currently used in clinical setting. Hence in our current study, we used PLGA nanoparticles labeled with dopamine receptor 5 antibody to target primary cilia. Further these NPs were loaded with fenoldopam to improve cilia structure and function. The conjugation and loading was confirmed by UV‐visible spectroscopy, transmission electron microscopy (TEM), X‐ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), zeta potential measurements and Fourier transform infrared spectroscopy (FTIR). TEM confirmed the synthesized nanoparticles were around 120 nm and spherical in shape. FTIR confirms the drug loading and antibody interactions with the PLGA. When we injected these NPs to ciliopathic hypertensive mice, significantly improved cardiac function in the ciliopathic hypertensive models, in which the hearts also exhibited arrhythmia, which was corrected with the PLGA‐NPs. Intraciliary and cytosolic Ca2+ were increased when cilia were induced with fluid flow, and this served as a cilia‐dependent mechanism of the PLGA‐NPs. Fenoldopam‐alone caused an immediate decrease in blood pressure, followed by hypertrophy with compromised functions in left ventricle pressure, stroke volume, ejection fraction. Our drug delivery profiles confirmed that the PLGA‐NPs were a potential delivery system for targeting cilia more specifically, efficiently, and effectively than fenoldopam‐alone. In conclusion, our results indicate that nanomedicine provides a possibility for targeted ciliotherapy.

Support or Funding Information

This work was supported by the National Institute of Health (HL131577), Congressionally Directed Medical Research Program (PR130153) and American Heart association (19IPLOI34730020).
Gene Expression in LEW.1WR1 Rats Indicate Insulin Resistance in Muscle and Liver Tissue
Authors: Wilkerson Vidal Quiana C. , Collins Genoah , Mercado Luis , Martin Sidney , Wright Laura Catherine , Gibson Helen , Love-Rutledge Sharifa
Published: April 2020

FAT10 is a ubiquitin‐like protein and type 1 diabetes (T1D) susceptibility gene that may play a role in age‐related inflammation, adiposity, cancer, and kidney disease. The young T1D susceptible, LEW.1WR1 rat overexpresses FAT10 and has higher fasting concentrations of blood insulin. It is unclear how the overexpression of FAT10 directly affects insulin sensitivity. It is also unclear if the initial insulin sensitivity of this animal model plays a role in disease susceptibility. We measured the relative expression of lipid and glucose metabolism genes in skeletal muscle and liver tissue in LEW.1WR1 and LEW/ssNHsd rats to determine if 12‐week old LEW.1WR1 rats were insulin resistant. We also tested the gene expression in pancreatic islets to determine if there were changes in endocrine pancreas cell health and hormone production. The results indicate that gene expression increased in the islets for glucagon, insulin, and PLIN5 (Perilipin‐5), and cyclin D expression decreased in LEW.1WR1 rats, suggesting reduced compensation and a potentially lipotoxic environment for beta cells. The increase in glucagon and insulin also supports peripheral insulin resistance. In liver tissue, UBD (Ubiquitin‐D), a gene suspected to regulate beta‐oxidation, was increased. INSIG (Insulin‐induced gene 1) and GLUT2 (Glucose transporter 2) gene expression decreased, indicating decreased expression of insulin‐regulated gene and glucose transporters, respectively. In muscle tissues, ATGL (Adipose triglyceride lipase), GAPDH (Glyceraldehyde‐3‐Phosphate Dehydrogenase), and PNPLA2 (Patatin Like Phospholipase Domain Containing 2) gene expression decreased, indicating a reduced ability to burn fat as energy and glucose intolerance in muscle tissue. These results, in tandem with previous work, suggest that fat storage in organs may account for the difference in weight previously observed between LEW.1WR1 and LEW/ssNHsd rats, and that LEW.1WR1 rats were developing insulin resistance which further suggests these rats are a novel animal model for pre‐diabetes.

Support or Funding Information

The authors would like to thank the UAH Office of the Vice President for Research and Economic Development, The Alabama Space Grant Consortium, ALSAMP, Adriel D. Johnson Sr. Fellowship, College of Science, Department of Chemistry, Department of Biology, Honors College, and Office of the Provost for the financial support for the resources, supplies, and student salaries.
Atheroprotective Effects Induced by Glyceraldehyde‐3′‐phosphate Dehydrogenase (GAPDH) in Murine Model of Atherosclerosis
Authors: Sukhanov Sergiy , Hou Xuwei , Snarski Patricia , Yoshida Tadashi , Delafontaine Patrice
Published: April 2020

GAPDH was shown to protect smooth muscle cells (SMC) against oxidant‐induced apoptosis via stimulation of DNA repair. GAPDH levels were reduced in atherosclerotic plaque SMC and therefore we hypothesized that SMC‐specific GAPDH overexpression will reduce atherosclerosis. GAPDH protein levels were increased >2‐fold (P<0.05) in aortas isolated from Apoe‐null mice with SM22α promoter‐driven GAPDH overexpression (SM‐GAPDH mice) compared to Apoe‐null controls. SM‐GAPDH mice have BW and BP similar to control. Aortic circumference, lumen area and media area were not changed by GAPDH overexpression. SMC GAPDH did not change pulse wave velocity (SM‐GAPDH, 1.1±0.1 m/s, control, 1.2±0.3 m/s) (ultrasound imaging) as well as endothelium‐dependent and ‐independent vascular responses to PE, Ach or SNP (organ chamber assay with thoracic aortic rings). High‐cholesterol fed SM‐GAPDH had reduced atherosclerotic burden (H&E‐stained aortic valve cross‐sections: 28±3% decrease, P<0.05), elevated plaque SMC levels (IHC for a‐SM actin, 3.1‐fold increase, P<0.01; IHC for calponin, 3.8‐fold increase, P<0.05), increased plaque collagen (Trichrome, 2.1‐fold increase, P<0.05), reduced plaque SMC apoptosis (TUNEL assay with IHC for calponin, 3.2‐fold decrease, IHC for cleaved caspase‐3, 2.6‐fold decrease both are P<0.05) without changes in macrophages (IHC with Mac3, P=NS). Aortas isolated from SM‐GAPDH have increased expression of Ape1 endonuclease (major DNA repair enzyme, 31±5% increase, P<0.05), reduced cleaved PARP (apoptotic marker, 74±12% decrease, P<0.001) and decreased oxidative DNA damage (AP sites assay, 2.5‐fold decrease, P<0.05; pSer139 histone H2AX, 33±3% decrease, P<0.05) compared to controls. Plaques in SM‐GAPDH mice had thicker SMC‐rich fibrous plaque cap (3.5‐fold increase, P<0.05) and decreased area of necrotic core (1.8‐fold reduction, P<0.05) suggesting enhanced plaque stability. These data taken together with our in vitro findings indicate that Ape1 upregulation mediates GAPDH effect on DNA and apoptosis. Our results suggest that stimulation of GAPDH/Ape1 axis is a novel potential anti‐atherosclerotic therapy.

Support or Funding Information

Study was supported by NIH/NHLBI 1R01HL142796‐01A1 (SS), AHA Grant‐in‐aid (SS) and NIH/NHLBI 7R01HL070241 (PD).