bluuebunny/crossref_metadata_2025 huggingface dataset:
When Students Choose E‐learning Resources—the Importance of Ease and Convenience
Authors: Hortsch Michael , Rodenbarger Lauren
Published: June 2021

Electronic learning resources are popular with today's students. How students choose their favorite e‐learning tools has not been investigated in detail and therefore is not well understood. The popular SecondLook™ (SL) Histology e‐learning self‐review tool was offered in three different formats to students participating in two different histology courses at the University of Michigan (CDB450/550 and DENT510). The SL formats included downloadable PowerPoint files, an interactive online website, and a mobile smartphone and computer tablet application (app). Identical in content, each format had specific advantages and disadvantages with respect to the types of compatible devices, user features, and access limitations. Upon the conclusion of their courses, students were surveyed about their format choices and reasons for their selection, as well as usage patterns. Only 3 out of 213 participating students (91.4% participation rate) reported not having used the SL resource. Many students (46% CDB450/550, 64% DENT510) tried only one resource format, with PowerPoint being the most popular final choice (56% CBD450/550, 64% DENT510). Although the interactive website and mobile app offered several user‐friendly features not available within the PowerPoint files, they each only garnered approximately 20% final popularity in both courses. “Convenience,” “larger screen,” and “easy to use” were given most often as reasons for students' PowerPoint software format choice. The flexibility of time and place to use the resource and availability of features were also named as factors in the format selection. The access to a mobile learning tool enticed some students to use this resource in distractive environments. It also encouraged some students to forgo other learning resources, specifically textbooks, and the course website. These results suggest that today's students are in fact less motivated to actively seek out novel, high‐tech learning resources than commonly believed and instead often select easy to use and convenient review tools with which they are familiar. The study received an exempt status from the University of Michigan IRB.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Human Evolution Teaching Materials Project
Authors: Selba Molly Chisholm
Published: June 2021

The Human Evolution Teaching Materials Project (HETMP) was created to facilitate the inclusion of human evolution into existing middle school and high school curricula. HETMP utilizes open‐access 3D surface files of hominin crania, along with a variety of teaching materials, to reinforce a student's understanding of evolutionary processes through the analysis of hominin cranial morphology. It has been piloted in five middle school and high school classrooms during the period 2017–2018. Through a free and easy‐to‐use website, teachers have access to all of these materials as well as constant support in order to help facilitate the implementation of these materials into existing course content. The available teaching modules reference a variety of open source 3D models including: Pan troglodytes, Australopithecus africanus, A. afarensis, A. sediba, Paranthropus aethiopicus, P. boisei, Homo naledi, H. erectus, H. rudolfensis, H. habilis, H. heidelbergensis, H. sapiens, and H. neanderthalensis. These models can be printed by the school or by using a local 3D printer, or can be borrowed from a previously established set of crania. This program not only highlights the importance of keeping academic research open‐source, but also has a direct impact on evolution awareness in hundreds of middle and high school students. Teachers at the pilot schools noted increased student engagement and students have enjoyed learning about human evolution in a way that was previously not made available to them. Undoubtedly, HETMP has the potential to impact thousands of pre‐college students by demonstrating the tangible evidence of evolution and allowing middle school and high school students to learn about cranial morphology through hands‐on experiential learning.

Support or Funding Information

This project is supported by the Society for the Study of Evolution.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomical Exploration of the Tympanic Cavity Using Virtual and Printed 3D Models of the Human Petrous Bone
Authors: Zdilla Matthew J , Skrzat Janusz , Walecki Piotr , Tarasiuk Jacek
Published: June 2021

The tympanic cavity is, from a gross anatomical perspective, both small and complex. Scaling the anatomy of the tympanic cavity to a larger size, while maintaining accuracy with regard to detail, could improve the understanding of the anatomy. Therefore, the purpose of this study was to create an accurate 3D computer model of the tympanic cavity and materialize it, in a larger scale, via 3D printing. The 3D virtual model of the tympanic cavity was created from serial micro‐CT scans of a normal temporal bone and converted into stereolithography files dedicated for 3D printing. Micro‐CT scanning of the petrous bone was performed with aid of the Nanotom 180N device with a slice thickness of 15 μm. To obtain the 3D virtual model of the tympanic cavity, automatic image segmentation was performed with CT‐An software, which generated the mesh of triangles that approximated the geometry of the tympanic cavity. To create a polymer mass from the digital model, the Da Vinci 1.0 printer (nozzle diameter = 0.4 mm), which utilized prototyping technology based on fused filament fabrication, was employed in order to build the model with the acrylonitrile butadiene styrene at the layer height = 0.3 mm. The study demonstrated the possibility of building accurate models of the tympanic cavity that expose anatomical details including the oval window, round window, promontory, pyramidal evidence, subiculum, ponticulus, and sinus tympani— structures with dimensions in the range of only a few millimeters. Both 3D virtual and large‐scale printed models may improve the understanding of the complex anatomy of the tympanic cavity and spatial relationships between the ear components. Enlarged 3D virtual and printed models of the tympanic cavity facilitate demonstration and observation of the tympanic cavity anatomy in comparative studies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methocinnamox (MCAM) is an Effective Long‐Term Antagonist of Peripheral Mu, but not Kappa or Delta Opioid Receptors In Vivo
Authors: Jennings Elaine M , Disney Alex , Husbands Stephen , Winger Gail , Berg Kelly A. , Clarke William P. , Woods James H.
Published: June 2021

The United States is currently in the midst of an opioid abuse epidemic; deaths from synthetic opioids like fentanyl and its derivatives are increasing. Currently naloxone (Narcan), a competitive mu opioid receptor antagonist is the only pharmacological treatment for opioid overdose; reversing both respiratory depression and profound sedation. However, naloxone has a relatively short duration of action and thus its effects are limited. A long lasting mu opioid receptor antagonist called MCAM has been shown to attenuate the antinociceptive effects of morphine in mice for more than 48 hours (Broadbear et al., J Pharmacol Exp Ther, 294:933–940, 2000) and due to this long duration of action, may make a better treatment for opioid overdose and perhaps for opioid abuse. Because MCAM reduces mu receptor function for a longer period relative to kappa or delta opioid receptors, we have begun to examine if pain relief from either kappa or delta receptor agonists will be active while mu receptors remain occupied by MCAM. Using the Hargreaves method for thermal heat nociception, we determined the effect of intraplantar (i.pl.) pretreatment of MCAM or vehicle on peripherally mediated reduction in PGE2‐evoked thermal allodynia by DAMGO (mu agonist), U50488 (kappa agonist) or DPDPE (delta agonist). Injection of DAMGO (20μg/50 μl i.pl.), U50488 (0.1μg/50μl i.pl.) or DPDPE (20μg/50 μl i.pl.) produced a significant reduction in PGE2‐evoked thermal allodynia. Pretreatment with MCAM 15 min prior to PGE2 injection at 10nM (0.25μg/50μl i.pl), 50nM (1.25μg/50μl i.pl) or 20nM (0.5μg/50μl i.pl), had no effect on PGE2‐induced thermal hypersensitivity; however, it significantly inhibited the antinociceptive response to DAMGO, U50488 and DPDPE. The DAMGO‐mediated antinociception remained blocked 24 and 48h after a single administration of MCAM. By contrast, when tested 24h after administration, MCAM had no effect on U50488‐ or DPDPE‐mediated antinociceptive responses. Thus, MCAM displayed long‐term antagonism at mu opioid receptors but only a short antagonist effect at either kappa or delta opioid receptors. These data suggest that MCAM's long duration of antagonist action is selective for mu opioid receptors and that peripherally restricted delta and kappa receptor agonists may be a viable option for pain relief.

Support or Funding Information

The Addiction Research, Treatment & Training (ARTT) Center of Excellence at UT Health San Antonio.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Brominated Flame Retardants on Cytokine Production by Mast Cells
Authors: King Ethan D , Mahoney Jonathan , Krajewski Dylan , Smith Warren , Mathias Clinton , Bose Diptiman D
Published: June 2021

Polybrominated diphenyl ethers (PBDEs), extensively used as flame‐retardants, have been shown to cause neurotoxicity, immunotoxicity and endocrine disruption; however, the exact mechanisms are largely unknown. In this study, we propose to investigate PBDE‐induced immunotoxicity in mast cells. We examined the effects of two of the most commonly occurring brominated diphenyl ether (BDE) congeners, BDE‐47 and ‐99, and a metabolite of BDE‐47, 6‐OH‐BDE‐47 on MC/9 mast cells. MC/9 cells treated with BDE‐47, ‐99 and 6‐OH‐BDE‐47 (0.25, 0.5, 5, 20 and 40μM, 24h) were stimulated with IgE/antigen (1μg/ml), and levels of IL‐6 were measured by ELISA. Our results indicate that PBDEs inhibit the IgE‐mediated cytokine release in MC/9 cells in a concentration‐dependent manner. Interestingly, exposure to BDE‐47 and 6‐OH‐BDE47 decreased IL‐6 release in a concentration‐dependent manner; however, BDE‐99 decreased IL‐6 release only at concentrations above 20 μM. These results demonstrate that tetra‐brominated BDE‐47 and its 6‐OH metabolite can suppress cytokine release with increasing concentrations, while penta brominated BDE‐99 congener inhibits IL‐6 release only at higher concentrations. Mast cell degranulation measured by β‐hexoaminidase also showed that BDE congeners decreased degranulation in a concentration‐dependent manner in IgE stimulated mast cells. In order to test effects on cell viability, we exposed the MC/9 cells to increasing concentrations of the BDE congeners and measured cytotoxicity at 24 and 48h by MTS assay. We found that tetra‐brominated BDE‐47 exhibited more potent cytotoxicity (EC50 μM =42.38 at 24h and 32.33 at 48h) in comparison to the penta‐brominated BDE‐99 (EC50 μM =48.41 and 35.04 at 48h) in the MC/9 cells. However, the 6‐OH metabolite of BDE‐47 was more cytotoxic in comparison to its parent compound BDE‐47 as well as penta‐brominated BDE‐99 (EC50 μM =25.82 at 24h and 20.16 at 48h). Our results suggest that PBDE congeners can modulate cytokine release in mast cells and the extent of inhibition is dependent on the bromination of PBDEs. These results address the need to better understand the molecular and cellular mechanisms by which PBDE exposure can mediate immunotoxicity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of 16‐hour Nighttime Fasting on Markers of Oxidative Stress
Authors: Dirks‐Naylor Amie J , Wilson Jennifer A. , Waghel Rashi , Bush Mark , Cook Corbin
Published: June 2021

Caloric restriction and intermittent fasting have shown to confer a plethora of health benefits. However, traditional regimens of caloric restriction (restricting caloric intake by 30–40% every day) and intermittent fasting (fasting every other day), have proven difficult to comply. Prolonged nighttime fasting is a type of intermittent fasting which may be an easier regimen to maintain, but has been scarcely studied. Thus, the aim of this study was to determine the effects of this regimen on biomarkers of oxidative stress. Eleven healthy men ranging in age from 21–39 years fasted for 16‐hrs (~6pm–10am) each night for 4‐weeks. Subjects were instructed to eat their normal diet outside of the suggested fasting hours. Pre‐ and post‐fasting blood samples were collected. 24‐hr food and drink intake was self‐recorded periodically before and during the fasting period using the National Cancer Institute Automated Self‐Administered 24‐hour Recall (ASA24) website. Total antioxidant capacity, DNA oxidation, thiobarbituric acid reactive substances (TBARS), and glutathione reductase (GR) activity were measured in pre‐ and post‐plasma samples. Prolonged nighttime fasting increased plasma total antioxidant capacity (p=0.001), but did not affect DNA oxidation, lipid peroxidation, or GR activity. There was no difference in the 24‐hr recall caloric intake before and after starting the fasting regimen. The fasting regimen also did not affect the percent intake of proteins, carbohydrates, fats, sugar, or fiber. Thus, the results suggest that prolonged nighttime fasting, without altering daily caloric or dietary intake, may improve antioxidant capacity.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of GPCR6A and CaSR in Atlantic salmon (Salmo salar)
Authors: Gomes Ana Silva , Lai Floriana , Jordal Ann‐Elise O. , Rønnestad Ivar
Published: June 2021

The G protein‐coupled receptor family C, group 6, member A (GPCR6A) and the calcium‐sensing receptor (CaSR) belong to the GPCRs family C and play a crucial role in sensing amino acids in the mammalian gastrointestinal (GI)‐tract. These receptors, located in the enteroendocrine cells facing the GI‐tract lumen, are activated by dietary amino acids, which stimulate the release of gut hormones leading to the regulation of diverse physiological functions, including digestion, absorption, allocation of substrates for growth, feed intake, etc. While it is established that these receptors play a key role in amino acid sensing in mammals, there is little information in teleosts, including Atlantic salmon (Salmo salar L.).

The Atlantic salmon is a key commercial species in global aquaculture and provides an excellent model to study the fate of duplicated genes due to the salmonid‐specific 4th round of whole genome duplication. In the Atlantic salmon genome database we have identified at least three gpcr6a genes, named here gpcr6a‐like 1, ‐like 2 and –like 3. While GPCR6A‐like 1 and ‐like 2 share 93% of similarity at the amino acid level, both only share 46% of similarity with GPCR6A‐like 3. The Atlantic salmon gpcr6a exon‐intron diverges significantly from the mammalian homologue gene, however the 2D topology is well‐conserved. Regarding the Atlantic salmon CaSR, one casr gene in the chromosome (chr) ssa11 was identified and it shared a high similarity (78 %) with the human homologue. In addition, several other casr genes were found in Atlantic salmon, located in unplaced scaffolds and in the chr ssa9 and chr ssa20, which suggests that tandem duplication of casr have occurred in these chromosomes.

Further, we have started to investigate the spatial mRNA expression profile for all the salmon gpcr6a transcripts and the casr transcript in chr ssa11 in several tissues, including different regions of the GI‐tract. Preliminary results indicate that these receptors are present in the oropharyngeal area, and, even though in lower expression levels, present along the different regions of the GI‐tract. Our data collected this far support the hypothesis of a conserved system for amino acid sensing in the GI‐tract across the vertebrate lineage.

Support or Funding Information

Funded by Regional Research Fund West (RFFV 247978 SalmoFeedPlus), Cargill and Research Council of Norway (NFR 267626 LeuSENSE; 262096 GUTASTE).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prebiotics and Probiotics Maintain the Intestinal Barrier Function
Authors: Ducray Henri Alexandre Giblot , Globa Ludmila , Pustovyy Oleg , Vodyanoy Vitaly , Sorokulova Iryna
Published: June 2021

The intestinal barrier function is crucial in maintaining the host's homeostasis, as it acts as the first protective barrier between the lumenal content and circulation. Disruptions to this protective barrier have been linked to various pathological conditions. Excessive permeability can lead to tissue damage, systemic inflammation, and even organ failure. Studies have shown that the gut microbiota and its metabolites have an essential role in the regulation of the intestinal barrier function. Negative alterations to the gut microbiota, known as dysbiosis, have been shown to be a factor involved for the increased barrier permeability. Thus, restoration of the gut microbiota can provide a novel approach for the prevention of adverse effects caused by the loss of this protective barrier. The main goal of this study was to evaluate the efficacy of a prebiotic and probiotic in maintaining the intestinal barrier integrity during heat stress.

Animals were pre‐treated by oral gavage with Bacillus subtilis probiotic strain, a yeast fermentate product having prebiotic properties, or phosphate buffered saline (PBS) prior to exposure to heat stress. Control rats received identical treatments, but without the exposure to stressor. The results demonstrated that heat stress lead to significant changes to the intestinal barrier in the stressed animals pre‐treated with PBS, causing significant decreases in villi height and total mucosal thickness, and reduced expression of tight junction proteins. The level of lipopolysaccharides in serum also significantly increased in those animals. All registered adverse effects were prevented in stressed animals pre‐treated with the probiotic and prebiotic. Analysis of the gut microbiota revealed significant disruption of microbial composition only in the stressed rats pre‐treated with PBS. These results demonstrate the high efficacy of the prebiotic and probiotic in preventing the loss of the intestinal barrier function. We can speculate that the beneficial modulation of the gut microbiota could be a valuable approach for prevention of stress‐related adverse effects.

Support or Funding Information

This work was supported by Auburn University funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Clinical implications of the cleido‐occipitalis cervicalis muscle: A muscular variant of the trapezius muscle
Authors: Maslowski Doug J. , Snyder Sean B. , Pellis Zachary , Petrone Ashley B. , Zdilla Matthew J. , Lambert H. Wayne
Published: June 2021

Anatomical variation in the supraclavicular region is unusual, aside from the scalene muscles. This report presents a left‐sided cleido‐occipitalis cervicalis muscle found during routine dissection of an 87 year‐old female body donor. The variant muscle traveled from the occipital bone as part of the trapezius muscle, before deviating from the trapezius to descend inferiorly in the neck to insert into the superior and posterior aspect of the middle of the clavicle. As it traveled inferiorly towards the clavicle, the variant muscle passed immediately superficial to the transverse cervical artery. The muscle was innervated by the accessory nerve (or CN XI). With regard to clinical relevancy during imaging and physical examination, the cleido‐occipitalis cervicalis muscle may appear to be an arterial aneurysm, thrombosed vein, lymphovenous malformation, neck cyst, tumor, enlarged lymph node, or other soft tissue pathology. Within the literature, it has been reported that it may be difficult to distinguish the cleido‐occipitalis cervicalis muscle from the levator claviculae muscle, which is a more common muscular variant within the lateral cervical triangle. Furthermore, the presence of the cleido‐occipitalis cervicalis may present difficulty in the management of clavicular fractures— the most common type of bone fracture.

Support or Funding Information

WVU Initiation to Research Opportunities (INTRO) Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alternative mechanisms of ribosome stalling rescue in the gram‐negative bacterium Francisella tularensis
Authors: Srinivas Pooja , Goralski Tyler D. P. , Keiler Kenneth C. , Dunham Christine M.
Published: June 2021

The number of new antibiotics being developed has plateaued, particularly compared to the sharp increase in antibiotic‐resistant bacteria plaguing humanity. Many current antibiotics target the bacterial ribosome, but have cross‐reactivity with eukaryotic ribosomes, causing host toxicity. One approach to prevent this is the identification and development of antibiotics that target bacterial‐specific pathways, such as ribosome stalling rescue. At any given moment, 5–10% of E. coli ribosomes are stalled. Stalled ribosome complexes arise when the ribosome halts during translation, which may arise due to the absence of an in‐frame stop codon, depletion of aminoacyl‐tRNAs, or proline‐rich motifs. Trans‐translation, found in 99.9% of bacterial species, is the main rescue pathway for stalled complexes that arise due to no in‐frame stop codon. Most bacteria remain viable without trans‐translation because they encode additional rescue systems, such as Alternative Rescue Factor A and B (ArfA and ArfB). The pathogenic bacterium F. tularensis appears to only encode trans‐translation factors, but trans‐translation deletion strains remain viable. How does F. tularensis survive without known ribosome rescue mechanisms present? Transposon mutagenesis data from the Keiler lab identifies a novel release factor, ArfT, which allows F. tularensis survival in the absence of trans‐translation. In vitro translation assays find that ArfT facilitates peptidyl‐tRNA hydrolysis with both RF1 and RF2, in contrast to the model of ArfA‐RF2‐mediated rescue in other well‐studied systems. These data suggest that ArfT‐mediated ribosome rescue occurs via a novel mechanism. Our results indicate that F. tularensis 70S‐ArfT stalled complexes have an increase of RF1 binding, suggesting a novel mechanism of recognition by RF1. Future studies include structural and biochemical characterization of the molecular mechanism of ArfT and release factor binding to stalled F. tularensis 70S ribosome complexes. These studies will provide new insights on the fundamental mechanism and role of ribosome rescue in bacteria.

Support or Funding Information

T32‐GM008602 R01‐GM093278‐09S1 R01‐GM121650‐02

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methylation of Ded1 Affects Its Role in Translation
Authors: Murvin Mickey , Yang Banruo , D'Alessandro Alec , Freniere Christian , Hilliker Angie
Published: June 2021

Ded1p is an essential ATP‐dependent RNA helicase involved in translation initiation through association with translation factors such as eIF4A and eIF4G (1, 2), nuclear export and accumulation of stress granules. We are interested in how a post translational modification (PTM) of Ded1, methylation, modulates these roles of Ded1. The methyltransferase Hmt1 methylates Ded1 at four arginine residues in the N and C terminus (3). Methylation strengthens the interaction between Ded1 and Npl3, a protein involved in nuclear export (3). However, it is unclear how methylation impacts Ded1 in translation.

To test Ded1 methylation in vivo, we mutated the four methylated arginines to either alanine or lysine. Methylation of Ded1p is not required for normal cell growth, making it unlikely that Ded1 methylation is required for global translation. However, Ded1 is important for the translation of many mRNAs, especially those with highly structured 5′ UTRs (8). We find a reporter with the highly structured CLG1 5′ UTR is not efficiently translated by a Ded1 methylation mutant, suggesting Ded1 methylation may be important for the translation of specific mRNAs.

However, as the arginines are mutated, we cannot rule out another role for these arginines aside from methylation. In order to directly test the effect of Ded1 methylation on translation, we will purify recombinant wild type Ded1 protein with or without co‐expression with Hmt1. Translation efficiency of recombinant methylated versus unmethylated Ded1 protein will be tested in vitro using both simple and highly structured 5′ UTRs, such as CLG1, before a luciferase reporter. We will also perform helicase assays to test the impact of Ded1 methylation on its biochemical function.

Since Ded1 interacts with other proteins through the N or C terminus, it is possible that methylation influences Ded1‐protein interactions. We use a conditional two‐hybrid assay in E. coli (3) to test the effect of methylation on Ded1's binding partners. We show methylation strengthens the known interaction between Ded1 and eIF4A. A novel interaction between Ded1 and Xrn1 and a reported oligomerization of Ded1 (4) has been validated through immunoprecipitation. Furthermore, we will test the effects of methylation on known Ded1 interactions with other translation factors such as eIF4G and eIF4E (2, 5) and RNA exporters such as Gle1 and XpoI (6, 7). Besides testing Ded1 interactions with a single binding partner, we will also test interactions between Ded1 and a combination of two different initiation factors. Ded1 is required for translation of >600 mRNAs in yeast (8). Our investigation further elucidates how methylation regulates Ded1 and its effect on mRNA translation.

Support or Funding Information

NIH R15 GM128068‐01; HHMI; Arts & Sciences (UR); Smart Fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ensemble Docking of Potential BACE1 Inhibitors for Alzheimer's Disease
Authors: Guevara Johnny , Zahran Mai
Published: June 2021

A wealth of evidence suggests that inhibiting β‐secretase 1 (BACE1) can delay or halt the progression of Alzheimer's disease (AD). Studies have shown that small molecules able to interact with the asp dyad and the flap in the active site of BACE1 can promote inhibition. In our previous published work, six potential BACE1 inhibitors (1–6) were designed, synthesized, and evaluated by pIC50 measures obtained from in vitro FRET inhibition assays. The aim of this work was to implement an in silico ensemble docking approach to study the binding mechanism between 1–6 and flexible conformations of BACE1. We performed a 50 ns molecular dynamics (MD) simulation of a free BACE1 structure to generate an ensemble of flexible BACE1 conformations and docked 1–6 to each conformation in the ensemble. Docking results revealed 3, 5, and 1 exhibiting the most favorable binding affinities across all dockings, while retaining binding orientations relative to the catalytic asp dyad sustained by hydrogen bonds, as well as exhibiting stabilizing hydrophobic and pi‐pi interactions to flap residues. MMGBSA residue decomposition analysis further revealed residues ASP32, ASP228, and TYR71 as the main contributors for binding of 3, 5, and 1 to BACE1. Conversely, 2, 4 and 6 exhibited less favorable binding affinities and as a result formed fewer interactions to critical active site residues responsible for BACE1 inhibition. In addition, the Pearson correlation coefficient (R) values between computational binding affinity and experimental pIC50 values across all dockings, R < −0.75, showed a strong correlation between computational and experimental results. Overall, our computational results support our experimental results, provide crucial information about the binding mechanism behind our potential six BACE1 inhibitors, and highlight the importance of addressing receptor flexibility in docking simulations.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Azadirachtin on Bacterial Biofilm Formation
Authors: Kaverimanian Vishnu , Heuertz Rita
Published: June 2021

Due to the overuse of currently available antibiotics, bacteria are rapidly evolving resistance to modern medical treatments. Because of this fact, it is important to find alternative antimicrobic agents for which bacteria have not yet developed resistance. To compound this problem, it has been identified that bacteria living in biofilms (as aggregates protected by extracellular polymeric matrix) can be up to 1000 times more antibiotic‐resistant than their planktonic counterparts. The NIH has reported that up to 80% of chronic and 65% of all human infections are biofilm‐associated. One major therapeutic alternative to modern antibiotics is phytochemicals (plant‐derived compounds). A phytochemical of interest, azadirachtin, is derived from the neem tree (Azadirachta indica) which has been historically used in the Indian subcontinent as an herbal remedy for many ailments including those caused by parasites and bacteria. A significant source of infection in hospitals is Staphylococcus aureus which is known for emerging antimicrobic resistance and tendency to form biofilms. The purpose of my project was to identify if azadirachtin inhibits biofilm formation. In order to determine the effects of azadirachtin on biofilm, a microplate biofilm quantitative assay was performed. S. aureus that were methicillin resistant (MRSA, 4 different strains) or susceptible (MSSA, 3 different strains) were assessed. Bacteria were first inoculated into tryptic soy broth (TSB) and incubated (37°C, overnight). Afterwards, bacteria were standardized to a spectrophotometric A590 of 0.1–0.2 growth. Once standardized, the bacteria were transferred to 96‐well microplates with/without azadirachtin (0–0.8 mM) and incubated (37°C, 20 h). Following incubation, bacteria were killed with glutaraldehyde and microplates were washed (three times with dH2O). Subsequently, crystal violet (0.1% aqueous) was added to stain the biofilm. Excess dye was removed by washing (three times with dH2O) and stained biofilm was solubilized (8% acetic acid). Solubilized biofilm was quantitated by A590 readings. Results indicated that azadirachtin had no significant (p<0.05) effect on mean biofilm amounts for MRSA and MSSA biofilm formation. As azadirachtin appears to elicit no anti‐biofilm effect on the Gram positive S. aureus, our next step will be to ascertain its effect on Gram negative Pseudomonas aeruginosa biofilm.

Support or Funding Information

Financial support for VK was received from the DeNardo Education and Research Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Amygdalin improves burn wound healing in diabetic rats
Authors: Haidar Hanine , Lauricella Marianna , Jurjus Rosalyn , Daouk Hisham , Leone Angelo , Capello Francesco , Eid Assaad , Jurjus Abdo
Published: June 2021

Introduction

The skin has a unique plasticity and regenerative abilities which are partly impaired in diabetics. On the other hand, amygdalin or vitamin B17 has an array of functions affecting inflammation and cell regeneration.

Aim

This study aimed to assess the effect of amygdalin on burn wound healing in diabetic rats

Materials and methods

Sixty‐four male Sprague‐Dawley rats were subjected to burn according to standard published models. Diabetes was induced by sterptozocin and treatment by vitamin B17 followed every other day for 28 days, the duration of the experiment. Biopsies were taken on days 1, 3, 7, 14, and 28 for light microscopy evaluation and cytokines modulation.

Results

Histological assessment of the burn wounds showed a remarkable improvement in earlier healing, smaller wound area and a better scar in the amygdalin treated group. In addition, a significant decrease in the inflammatory cytokines (IL‐1α, Il‐6 and IL‐12) was noted.

Conclusion

Amygdalin improved burn wound healing in diabetic rats, in quality and time.

Support or Funding Information

Ph D Program (University of Palermo and American University of Beirut)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin‐like Growth Factor‐1 Receptor Signaling Regulates Adult Tendon Growth Through ERK1/2‐dependent Tenocyte Proliferation and Initiation of Protein Synthesis
Authors: Disser Nathaniel P , Sugg Kristoffer B , Kharaz Yalda Ashraf , Talarek Jeffrey R , Comerford Eithne , Mendias Christopher L
Published: June 2021

Tendon fibroblasts, or tenocytes, are the main cell type in tendon and serve to synthesize and maintain collagen fibrils and other extracellular matrix proteins within the tissue. Despite the high prevalence of tendon injury, the underlying biological mechanisms of postnatal tendon growth and repair are not well understood. Insulin‐like growth factor (IGF‐1) signaling is stimulated by growth hormone (GH) and promotes the growth and development of many different tissue types. While IGF‐1 is a potent hypertrophic signaling molecule in skeletal muscle, less is known about the role of IGF‐1 in tendon biology. Therefore, the purpose of this study was to determine the importance of IGF‐1 signaling in postnatal tendon growth. Our hypothesis was that IGF‐1 signaling is required for the response of tendon to a mechanical load stimulus. To test this hypothesis, we used both in vitro studies of cultured tenocytes, and in vivo studies using genetically modified mice that allowed us to delete the IGF‐1 receptor (IGF1R) specifically in tenocytes. Treatment of tenocytes in vitro with IGF‐1 increased the expression of Mki67 transcript levels after 24 hours, and resulted in activation of the Akt and ERK1/2 pathways. When tenocyte proliferative activity was measured, cells treated with IGF‐1 displayed increased proliferation compared to controls, but inhibition of ERK prevented the IGF‐1‐mediated increase in cell proliferation and Mki67 (Ki67) expression. Inhibition of Akt signaling did not impact tenocyte proliferation or Mki67 expression. In addition, treatment of tenocytes with IGF1 increased the rate of protein synthesis and ribosomal RNA expression. Analysis of mechanically overloaded plantaris tendons in vivo revealed significantly reduced size of the neotendon matrix which forms around the original tendon in response to mechanical loads, and total tendon cross‐sectional area (CSA) at 14 days in the IGF‐1 knockdown mice compared to the controls. This was accompanied by decreased number of proliferating cells and proteins involved in cell proliferation and initiation of translation, as measured by proteomic analysis. The results of this work support IGF‐1 signaling as a fundamental component of postnatal tendon growth in response to mechanical loading, specifically through the regulation of ERK1/2‐dependent tenocyte proliferation and initiation of protein synthesis. Further studies of the growth factors and proteins involved, and the potential to evaluate IGF‐1 to promote tendon regeneration, are warranted.

Support or Funding Information

NIH R01‐AR063649 and F32‐AR067086.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Norepinephrine on Gut Bacterial Community Structure and Function
Authors: Menon Rani , Fitzsimmons Ben , Vanajakumari Manoj U , Lee Kyongbum , Jayaraman Arul
Published: June 2021

Metabolites produced by both the microbial community and the host are present in the gastrointestinal tract environment. Several metabolites have been identified as important mediators of different physiological functions in the host. Small molecules and metabolites produced by commensal bacteria resident in the gastrointestinal tract can both promote and attenuate colonization of pathogens and inflammation. However, interactions between different metabolites in the GI tract and their net effect on the host are poorly understood. We hypothesized that NE promotes EHEC virulence by altering the microbial community structure and function to facilitate colonization. An in vitro anaerobic batch culture system was used to investigate the effect of NE on the microbial community structure and function. Cecal contents from 6–8 week‐old mice (n = 7) were both collected and cultured anaerobically in Gut Microbiota Medium (GMM) with or without NE (500μM) at 37°C for seven days. Exposure to NE increased the diversity of the bacterial community (p < 0.05, Mann‐Whitney test), as determined from 16S rRNA sequencing. Genera containing facultative pathogens and reported with pro‐inflammatory roles like Hathewaya, Clostridium and Streptococcus were highly abundant in the NE group while beneficial bacterial genera like Lactobacillus were decreased in abundance with NE treatment. Untargeted LC‐MS metabolomics showed significant changes in the levels of metabolites derived from aromatic amino acids. Targeted LC‐MS metabolomics analysis confirmed that the tryptophan metabolites indole and 5‐hydroxyindole were significantly decreased and tyramine was increased with NE treatment (p < 0.05, Wilcoxon test). Integration of metabolomic and metagenomic data using partial least squares regression (PLS‐R) showed correlation between the downregulation of indole and indole producing bacteria in the presence of NE. NE also down‐regulated the expression of the enzyme Tryptophanase A (TnaA) that is required for the conversion of dietary tryptophan to indole, in both nonpathogenic E. coli and EHEC. These results suggest that NE can modulate the microbial community composition and metabolite output to potentially increase pathogen colonization and infection.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CAIDE Dementia Risk Score Indicates Cortical Thinning in Low‐Risk, Middle‐Aged Adults
Authors: Gourley Drew , Pasha Evan P. , Kaur Sonya S. , Haley Andreana P. , Tanaka Hirofumi
Published: June 2021

Increased risk for the future development of Alzheimer's disease begins as early as midlife and can be predicted using modifiable cardiovascular and lifestyle‐related risk factors. The Cardiovascular Risk Factors, Aging and Incidence of Dementia (CAIDE) dementia risk score has been used to predict late life risk of dementia based on midlife vascular risk factors. Additionally, in older adults with mild or subjective cognitive impairment, higher CAIDE scores were associated with greater atrophy in the middle temporal and frontal regions of the brain.

Purpose

We aimed to determine if CAIDE score is associated with cortical thickness in middle‐aged cognitively intact adults at risk for cardiovascular disease. We hypothesized individuals with higher CAIDE scores would have reduced cortical thickness in a priori regions associated with memory and attention‐executive function.

Methods

A total of 155 middle‐age adults (49±6 years, 52.4% female) classified as cognitively normal were studied. CAIDE score was calculated using the original model that excludes ApoE e4 genotype. This includes age, sex, education level, systolic blood pressure, body mass index, total cholesterol, and physical inactivity. Each factor is given a specific weight and the total score ranges from 0 to 14. Cortical thickness was obtained from high resolution magnetization‐prepared rapid acquisition gradient‐echo images. Thickness values for 24 a priori regions of interest associated with memory and executive function were extracted and analyzed.

Results

A majority of the participants (95.5%) in this sample were categorized as low‐risk, having a CAIDE score of 8 or lower. After using a corrected p‐value (p=0.006) for multiple correlations, there were several significant negative associations between CAIDE score and cortical thickness. These include the right parahippocampal (r=−0.251, p=0.002), left pars opercularis (r=−0.244, p=0.002), right superior frontal (r=−0.229, p=0.004) and left superior frontal (r=−0.247, p=0.002) regions.

Conclusions

Higher CAIDE scores in midlife are associated with lower cortical thickness in middle‐age adults at risk for cardiovascular disease. This finding supports the notion that cardiovascular risks may contribute to early alteration of brain structure. We propose that the CAIDE dementia risk score may be useful in predicting future risk of dementia even in low‐risk populations who have no indication of clinically significant cognitive impairment.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Free Radical Scavenging Activities of Extracts and Bioactive Constituents from the Roots of Plumbago zeylanica (Linn.)
Authors: Ajayi Gabriel Olukayode , Ademuyiwa Oladipo , Olagunju Joseph Abayomi , Lasisi Aliyu
Published: June 2021

Bioactive constituents from the methanolic extract (ME) and ethylacetate extract (EA) of Plumbago zeylanica were characterized by gas chromatography‐mass spectrometry. The free radical scavenging activities of ME and EA was determined using 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH). Major bioactive compounds found in the ME includes oxalic acid allyltridecyl ester, decane, 2‐piperidinone‐N‐[4‐bromo‐n‐butyl] and tetradecane, compounds obtained from EA were 1‐(ethenyloxyl) octadecane and cis‐13‐octadecenoic acid. Cis‐13‐octadecenoic acid, though known, was isolated for the first time from the root of P. zeylanica. The highest percentage antioxidant activities of ME and EA were 98.5% and 45.5% respectively at 350 μg/ml concentration of extracts. Results obtained from this study justify the use of P. zeylanica in traditional medicine for treatment of different ailments and it could be a potential source for novel drug compounds.

Support or Funding Information

Contributions from authors

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Wound healing property of aerial parts of Coleus vettiveroides
Authors: Ponnan Jeyasekhar M , Edward Jaslin J , Johnson Rajasingh K
Published: June 2021

Background

Coleus vettiveroides is a Labiatae species native to India. In Kenya, the leaves of some species within this genus having diterpenes, triterpenes and flavones compounds were used traditionally for treating the cough. However, to our knowledge, there is no experimental study in supporting its pharmacological activities.

Hypothesis

We hypothesize that topical administration of ethanolic extracts from the leaves of C. vettiveroides enhances wound healing in rat model.

Methods and Results

Four groups of male albino rats were selected (n=6) in this study. Full thickness of excisional circular wounds approximately 10 mm diameter were inflicted on the clear skin cutting under anesthesia. A diagrammatic representation of the murine wound‐healing model is presented in the figure. The ointment developed namely ethanolic extract of C. vettiveroides (EEVS) 2.5 mg/g and 5 mg/g was applied twice a day topically to 2nd and 3rd group respectively for 15 days. Whereas, the 1st group applied with simple ointment serve as negative control and the 4th group applied with silver sulfadiazine (10 mg/gm) topically serves as positive control. Wound contraction was measured on day 2, 4, 6, 8,12,14 and 15. The measurements were compared to the control groups. On day 15, the wounds treated with the EEVS show a significantly reduced wound size and a better wound healing effect both physiologically and histologically when comparted to the control groups. These results suggest that EEVS ointment has the wound healing potential.

Conclusion

Overall, these data suggest that the ethanolic extract from leaves of C. vettiveroides has the wound healing activity. Thus, these plant extracts have a therapeutic application.

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ENaC Activity and Regulation in Renal Distal Convoluted Tubule Cells
Authors: Song Chang , Le Janet , Yue Qiang , Mistry Monisha , Wynne Brandi , Ma Heping , Eaton Douglas C.
Published: June 2021

Our previous work has shown that epithelial sodium channel (ENaC) proteins are present in distal convoluted tubule (DCT) cells of the distal nephron. The purpose of these experiments was to show whether these proteins were functional. We also wish to show whether the channels were stimulated by aldosterone, inhibited by amiloride, and regulated by the ENaC chaperone protein, MLP‐1, as ENaC is regulated in collecting duct principal cells. We used DCT‐15 cells in monolayer culture on permeable polyester supports to measure transepithelial voltage, resistance, and current. The properties of DCT‐15 cells are quite similar to those of in situ cells of the DCT2 segment of the nephron. Our results showed that DCT‐15 cells had relatively low resistances (206 ± 5.71 Ω‐cm2, n=36) and small, but measurable amiloride‐sensitive currents (2.87 ± 0.237 μA, n=36), showing that these cells contain functional ENaC. 100 nM aldosterone significantly increased mean DCT‐15 cell current (from 2.87 ± 0.237, n=36, to 5.33 ± 0.395μA, n=8). Amiloride significantly lowered the current across DCT cells to very low levels (from 2.74 ± 0.219, n=42, to 0.825 ± 0.186μA, n=15). MLP‐1 is a myristoylated protein that when associated with the membrane promotes ENaC activity. When three critical serines are phosphorylated by PKC, MLP‐1 dissociates from the membrane and ENaC activity decreases. We transfected the cells with four MLP FLAG‐tagged mutants: wild type (WT), constitutively active (S3A), constitutively inactive (S3D), and myristoylation negative (GA) and measured DCT‐15 current. For comparison, we examined a principal cell line, mpkCCD cells. Transfection with MLP‐1 modified current in both DCT and CCD cells: median current was highest in S3A mutant and lowest in S3D mutant; the current after transfection with either construct was significantly different from WT. ENaC and MLP construct expression was also studied in lysates of both cell types by Western blotting with FLAG and anti‐ENaC subunit antibodies. In DCT‐15 cells with the highest current, expression of ENaC was greatest. Lastly, we calculated based on an average current of 2 μA/cm2 and ENaC single channel current of 0.4 pA that DCT‐15 cells must have approximately 5 million functional channels per square centimeter.

Support or Funding Information

Supported by R01 DK‐110409 to DCE and K01 DK‐115660 and ASN Gottschalk AWARD to BMW

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.