bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining the molecular determinants required for RNA binding functions of the Bicaudal‐C (Bicc1) translational repressor protein
Authors: Dolma Sonam , Tonelli Tomasso , Dowdle Megan , Sheets Michael
Published: April 2020

Bicaudal‐C (Bicc1) is an RNA binding protein that functions as translational repressor in all animal species. Its repression activities are required for a multitude of biological processes such as guiding the earliest steps of embryonic development and organ homeostasis. For example, defects in Bicc1 expression or its activities can give rise to polycystic kidney disease in humans, a disorder that is characterized by an abnormal collection of cysts in the kidney. In addition, Bicc1 functions in vertebrate embryos to control the translation of specific maternal mRNAs that encode key developmental regulatory proteins. During the early stages of vertebrate development defects in Bicc1 trigger cell fate changes that lead to embryonic malformations similar to some human birth defects. A general feature is that in all of its biological contexts Bicc1 binds to specific mRNAs and it is through this binding that it selects specific mRNAs for translational repression. My project is focused on defining the molecular determinants of the Bicc1 protein required for that specific mRNA binding. Recent results from the Sheets lab identified a 70 amino acid region of the Bicc1 protein, the KH2 domain that is required for mRNA binding. However, the particular molecular features within the KH2 domain important for RNA binding activity are unknown. To address this issue I am generating mutations of the KH2 domain that change evolutionarily conserved amino acid residues in all Bicc1 proteins. These mutant proteins will be analyzed with gel shift RNA binding assays in vitro as well as in vivo RNA binding experiments performed in Xenopus embryos. My goal is to define the amino acid residues in the KH2 domain that are responsible for providing specificity for Bicc1 binding to specific mRNAs, guide its repression activities and mediate its biological functions.

Support or Funding Information

This work is supported by a Hilldale Undergraduate Research Fellowship to SD and funding from NICHD (R01HD091921) to MDS. Megan Dowdle is supported by a SciMed GRS Advanced Opportunity Fellowship and Biotechnology Training Program of the National Institutes of Health (NIGMS T32GM008349).
Structural and Functional Analysis of RPE65 Folding by the Cytosolic Chaperonin CCT
Authors: Smith Theresa , Stewart Michael , Read Kristen , Shen Peter , Willardson Barry
Published: April 2020

A key step in the classical visual cycle is the trans‐to‐cis isomerization of retinoid intermediates by RPE65. Mutations in RPE65 disrupt this process and result in retinal dystrophies such as Leber congenital amaurosis (LCA). Many of these are single‐point missense mutations that occur far from the active site and appear to act by destabilizing the protein structure. Little is known, however, about how RPE65 is folded or how folding is disrupted by mutations. We have evidence that RPE65 folding is assisted by the cytosolic chaperonin CCT. CCT is a large double‐ring complex that is estimated to fold approximately 10% of the proteome and specializes in proteins with complex β‐propeller folds like RPE65. CCT interacts with both WT RPE65 and several LCA‐associated mutants (R91W, L408P, Y368H, R515W). Notably, the interactions between CCT and the mutants are approximately twice as strong as the interaction with the WT despite drastically reduced expression levels, consistent with their decreased stability. In addition, radiolabel pulse‐chase analysis shows that nascent RPE65 associates with CCT and dissociates over time, as expected of a substrate. Taken together, these results suggested that RPE65 is a true substrate of CCT and prompted us to seek a high‐resolution cryo‐EM structure of the complex in order to better understand how CCT folds RPE65. We isolated the complex using a tandem‐affinity approach and have obtained a preliminary 4.2 Å structure which shows the two rings of CCT and a mass attributable to RPE65 sitting between them. In contrast, our previously solved structure of Gβ1 bound to CCT showed Gβ1 interacting the with the apical domains of CCT at the top of the folding cavity. Further improvement of the RPE65‐CCT structure will shed light on mechanistic differences between RPE65 and Gβ1 folding and will provide a basis for understanding how CCT handles the folding defects caused by RPE65 mutations.
The Impact of Aging on Bone Regeneration in Mice
Authors: Stewart Kennon , Watt James , Busse Emily , Simkin Jennifer , Hoffseth Kevin , Sammarco Mimi
Published: April 2020

Older people are projected to outnumber children for the first time in U.S. history by 2030 (U.S. Census Bureau, 2017), creating a tsunami of people with reduced bone strength who are more susceptible to bone injury and fracture. Decreased bone quality has a multivariate causal path including loss of progenitor cells, reduced anabolic and catabolic capabilities of cells, and increased senescence. The age‐related cellular decline of bone begins prior to the onset of bone diseases such as osteoporosis. Thus, aging models exclusive of disease can be immensely beneficial in finding novel angles of approach for better bone healing during aging. With this in mind, our study uses the well‐characterized mouse digit amputation model. After amputation of the digit tip young mice are able to regrow patterned bone via direct ossification and soft tissue. However, regeneration in aged mice is delayed, and the regenerated bone is architecturally inferior showing decreased trabecular thickness, increased trabecular spacing, and an attenuated distal growth of the bone. Here we explore the impact of aging on regeneration and bone architecture, introduce a new method of analysis for bone architecture and discuss the advantages to alternative analysis for bone regeneration in the digit model.

Support or Funding Information

COBRE Grant ‐ 2P20GM103629 in addition to CELT LS‐LAMP Grant
Beta‐lactam Antibiotic Potentiation in MRSA Using Small Molecule Adjuvants
Authors: Miller Heather , Blackledge Meghan
Published: April 2020

Methicillin‐resistant Staphylococcus aureus (MRSA) is a major public health concern, as it has developed resistance to our existing arsenal of antibiotics. These bacteria also form robust biofilms that are evident in chronic infections. MRSA expresses a master regulator of both antibiotic resistance and biofilm formation. This protein, Stk1, is a eukaryotic‐like serine/threonine kinase. We have identified novel small molecule inhibitors of Stk1 which combat both antibiotic resistance and biofilm formation. We hypothesized that if cultures were treated with an inhibitor of Stk1 as an adjuvant, then changes in resistance genes would be observed. In order to determine if the adjuvant, the antibiotic oxacillin, and/or cotreatment changed mRNA levels in MRSA, total RNA was purified and RT‐qPCR was used to measure changes in gene expression in key genes such as blaI, blaZ, mecI, mecA and pbp2. We observed differential gene expression that helps explain at the transcript level why treatment of MRSA with an adjuvant is effective at potentiating beta‐lactam antibiotics.

Support or Funding Information

Research reported in this presentation was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM134503. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Assessing Vascularization of the Heart of Young Pigs After Cardiac Injury by Ischemia/ Reperfusion
Authors: Matos-Ortiz Gabriela Nicole , Agnew Emma , Velayutham Nivedhitha , Moore Victoria , Riggs Kyle W. , Baker Richard Scott , Alsaied Tarek , Zafar Farhan , Yutzey Katherine E.
Published: April 2020

Existing studies in axolotls, river fish, zebrafish and newts demonstrate that the heart has the capacity to self‐repair following injury, with an increase in existing cardiomyocyte proliferation (CM). Studies on neonatal mice show that the heart can repair during the first week of life following injury, suggesting mammals have a transient window of cardiac regenerative capacity. However, how these findings extend to large mammals remains poorly understood. Recent findings from our laboratory show CM cell‐cycling activity occurring up to 2‐months of age in pigs. This suggests large mammals may have an elongated capacity of heart regeneration compared with previously studied organisms. To assess regeneration after injury, we generated a large mammalian heart injury model. Eight postnatal 30‐day (P30) old pigs underwent ischemia (1hr)/ reperfusion (IR) injury via occlusion of the second diagonal branch of the left anterior descending artery (LAD) and controls underwent sham surgery. We hypothesized that blood vessel density would increase as an effort to normalize blood perfusion to the heart after injury. Tissue harvest occurred 4 weeks after injury and subsequent histochemical analyses were performed within 3 specific zones (scar, border, remote) to assess cell cycling, scar formation, and vessel density. Similar patterns of cell‐cycling activity, measured via pHH3 immunohistochemistry, were found between sham and IR models. Vessel density, as measured via lectin‐DAB staining, was also similar between sham and IR pigs; suggesting no evidence of new blood vessel formation after injury. Collagen deposition, suggestive of scar formation, was evident following injury in IR pigs. This novel approach in assessing cardiac repair following injury in young large mammals highlights that scar formation, rather than an alteration in cardiomyocyte proliferation, might be hindering cardiac regeneration in P30 pigs. Hence, targeting scar formation to understand heart repair mechanisms in young pigs might provide a better understanding of the self‐repair capability of the heart, with potential translation to human systems.

Support or Funding Information

SURF‐American Heart Association Research Funding, Cincinnati Children’s Hospital Medical Center Research Foundation, Schmidlapp Young Women Scholarship
Relationship of Essential Oil Exposure to Anxiety in Zebrafish
Authors: Suits Danielle , Kasper Sherry
Published: April 2020

This study focused on the relationship between differing essential oils and their impact on zebrafish anxiety. Five test groups were used to observe the behavioral responses and measure the quantitative cortisol levels of the fish after being exposed to three different essential oils, a carrier oil, and a control group (no oil). Two separate tests were designed to study 20 zebrafish over the course of 7 weeks. First, the swimming behaviors of the fish were observed using a basic novel tank test (NTT). The swimming patterns of the fish were recorded and the percent time that each fish swam in the top half of the tank was compared to the time each fish swam in the bottom half of the tank. Second, the zebrafish blood cortisol concentrations were measured immediately following their exposure to the differing test treatments using a cortisol extraction ELISA kit. The two data sets supported a strong trend regarding essential oil types in relation to swimming patterns and cortisol concentrations. Both data sets agreed that the test groups exposed to lavender, coriander, and fractionated coconut oil exhibited less anxious swimming behavior and lower levels of cortisol, while the zebrafish exposed to lemongrass oil and only water showed increased anxious behavior and higher levels of cortisol. Both tests showed strong trends when analyzed with a one‐way ANOVA test. The novel tank test data set had a p‐value of 0.07 and the cortisol extraction test had a p‐value of 0.06. The results of this study show that many questions can still be asked and answered regarding essential oil effects on cortisol levels and stress responses in zebrafish.
Effect of Live‐Firefighting on Ventricular‐Vascular Coupling in Middle‐Aged Firefighters
Authors: Hibner Brooks Aaron , Schroeder Elizabeth C. , Lefferts Wesley K. , Lane-Cordova Abbi D. , Horn Gavin P. , Smith Denise L. , Fernhall Bo
Published: April 2020

Introduction

Cardiovascular events are a leading cause of line of duty‐related death among firefighters. Increased risk potentially manifests from detrimental changes in ventricular function, vascular load, or their interaction defined as ventricular‐vascular coupling (VVC). Young firefighters exhibit ventricular‐vascular uncoupling after live firefighting, however it is unknown whether higher‐risk, middle‐aged firefighters respond similarly.

Purpose

Determine the effect of live fire‐firefighting on ventricular function, vascular load, and VVC in middle‐aged firefighters (40–60 yr).

Methods

Eighteen middle‐aged male firefighters (48±5 years, 28±4.9 kg·m−2) underwent hemodynamic and cardiac measures before and 10 minutes after an 18‐minute bout of intermittent, high intensity live‐firefighting activities in personal protective equipment. Blood pressure (BP), left ventricular function (ejection fraction [EF]), ventricular elastance (Elv: end systolic pressure [ESP]/end systolic volume [ESV]), and vascular load, defined as arterial elastance (Ea: [ESP]/stroke volume [SV]) were measured. VVC quantified as the ratio of Ea to Elv.

Results

Following a single bout of high intensity, intermittent live‐firefighting activities, mean arterial pressure and ESP decreased while heart rate increased (see table
). EF, Elv and Ea were maintained, and VVC was preserved.

Conclusion

Apparently healthy middle‐aged firefighters maintained the balance between ventricular function and vascular load following a single bout of live‐firefighting. Therefore, impaired VVC may not be related to the increased cardiovascular risk during a single bout of live‐firefighting in apparently healthy middle‐aged firefighters. Further investigations should examine the effect of an extended firefighting bout on ventricular function, vascular load, and VVC in individuals with cardiovascular disease risk factors.

Changes in cardiovascular parameters pre/post firefighting activity.

Pre

Post

p

SBP (mmHg)

132 ± 11

123 ± 10

<0.01

DBP (mmHg)

81 ± 6

79 ± 6

0.06

MAP (mmHg)

98 ± 7

94 ± 6

<0.01

HR (bpm)

65 ± 10

85 ± 11

<0.01

SV (ml)

85 ± 18

76 ± 15

0.11

EDV (ml)

126 ± 29

122 ± 27

0.40

ESV (ml)

43 ± 16

46 ± 17

0.40

ESP (mmHg)

102.6 ± 8.9

97.7 ± 6.7

<0.01

EF (%)

66.1 ± 6.9

63.0 ± 7.1

0.12

Ea (mmHg/ml)

1.3 ± 0.3

1.4 ± 0.2

0.24

Ely (mmHg/ml)

2.8 ± 1.0

2.5 ± 0.8

0.33

VVC

0.5 ± 0.1

0.6 ± 0.2

0.08

SBP: systolic blood pressure, DBP: diastolic blood pressure, MAP: mean arterial pressure, HR: heart rate, SV: stroke volume, EDV: end diastolic volume, ESV: end systolic volume, ESP: end systolic pressure, EF: ejection fraction, Ea: arterial elastance, Elv: ventricular elastance, VVC: ventricular‐vascular coupling
Translational Regulation of Cyclooxygenase‐2 (COX2) by Cyclin‐Dependent Kinase 7 (CDK7) in Endothelial Cells
Authors: Shetty Shruthi , Spaulding Amelia M. , Baker Cassadie , Baine Stephen H. , Hoyt Dale G.
Published: April 2020

Endothelial cell prostaglandins modulate the response to inflammatory stimuli. Combined treatment with E. coli endotoxin and interferon‐γ (LPS/IFN) induces cyclooxygenase‐2 (COX2) mRNA and protein. Modulation of COX2 expression may be therapeutically useful in different inflammatory states. Given the induction of COX2 mRNA by LPS/IFN, it was hypothesized that depletion of the transcription‐stimulating enzyme, cyclin‐dependent kinase 7 (CDK7), would antagonize induction of COX2. Here, mouse aortic endothelial cells were treated with CDK7 or non‐targeted siRNA, followed by LPS/IFN. Contrary to our expectations, knocking down CDK7 super‐induced the expression of COX2, without affecting mRNA or turnover of the protein. This suggested that CDK7 depletion increased protein synthesis. Here, signaling mechanisms known to regulate translation were investigated. CDK7 knockdown increased phosphorylation at Serine 209 of the mRNA cap‐binding translation initiation factor, eIF4E. Knockdown also increased cap‐binding activity of eIF4E. CDK7 depletion increased phosphorylation of the eIF4E repressor, 4EBP1, and decreased its association with eIF4E. Finally, increased phosphorylation of ribosomal protein S6 (RPS6), a component of the 40S ribosomal subunit, was also seen in the absence of CDK7, indicating increased translation. The results suggest that CDK7 represses COX2 expression by limiting translation through effects on RPS6, eIF4E and/or 4EBP1. CDK7 may serve as a target for the manipulation of COX2 expression in endothelial cells.

Support or Funding Information

Support was received from the Popat Patil Fellowship (SS).
APC Controls Wnt‐Induced β‐catenin Destruction Complex Recruitment in Human Colonocytes
Authors: Parker Taybor William , Neufeld Kristi Lynn
Published: April 2020

Wnt signaling is essential for intestinal homeostasis and is aberrantly activated in the majority of colorectal cancers (CRC). In over 80% of CRC, the tumor suppressor Adenomatous Polyposis Coli (APC) is mutated, resulting in expression of a truncated protein product. APC is a key component of the β‐catenin destruction complex, which maintains low cellular levels of β‐catenin but is inhibited following Wnt ligand presentation. The precise mechanism underlying β‐catenin destruction complex inhibition is not clear, nor is the exact role of APC in the complex. APC is primarily considered a core component of the destruction complex but has been shown to have other roles involving β‐catenin nuclear import/export and cytoskeletal functions. Here, we use Wnt3a beads to study the response of endogenous Wnt components to a local Wnt cue. Using three CRC cell lines, each with a different Wnt pathway status, we demonstrate that localized Wnt redistributes pathway components toward the Wnt source in the presence of full‐length, but not truncated APC. Further, use of the Wnt3a‐beads to perform protein pull‐down demonstrates that APC and β‐catenin both associate with the Wnt‐beads. APC depletion in nontransformed human colon epithelial cells diminishes this Wnt‐induced redistribution. Our results suggest revision of the current model as follows. In response to Wnt, the β‐catenin destruction complex: 1) maintains composition and binding to β‐catenin, 2) translocates to the plasma membrane, and 3) requires full‐length APC for this membrane trafficking. Currently, work is being performed to uncover mechanistic insights into the role of APC in destruction complex reorientation to a Wnt3a signal.

Support or Funding Information

This study was supported by the National Science Foundation [grant number IOS‐1456538] and by the National Institutes of Health [P30CA168524].
Essential Anatomy for Neurology and Psychiatry in the Undergraduate Medical Curriculum
Authors: Harmon Derek , Harrell Kelly , Keim Sarah , Topping Daniel , Sanky Charles , Laitman Jeffrey , Reidenberg Joy S. , Farias Anna , Lopez Lisa , Zumwalt Ann , Mork Amy , Wineski Lawrence , Cotter Megan , Brooks William , Davies David , Royer Danielle , Marzban Hassan , Notebaert Andrew , Martindale Jim , Hankin Mark
Published: April 2020

Introduction. To prepare medical students for clinical training and practice, it is critical to understand the anatomical knowledge considered most important for different clinical specialties.

Aim. To address this issue, a consortium of anatomists in the US and Canada is collecting data from clinical educators in Neurology and Psychiatry clerkships and electives to identify the anatomy they consider essential.

Methods. An IRB‐approved, online survey (Qualtrics, Seattle, WA) was used to assess the importance of 98 anatomical topics in seven body regions. The study first examined the percentage of Neurology and Psychiatry clinical educators (clerkship/elective directors and attending physicians) that considered each anatomical region important to their specialty. Second, the study examined the rank assigned to each anatomical topic using an ordinal scale from 1 (not important) to 4 (essential).

Results. At the time of abstract submission, data had been collected from 39 Neurology clinical educators at 11 medical schools and 19 Psychiatry clinical educators at 9 medical schools. The percentage of clinical educators that considered each anatomical region important to their specialty were (highest‐to‐lowest): Neurology – Back (97%), Upper Limb (97%), Lower Limb (97%), Head & Neck (97%), Thorax (54%), Pelvis & Perineum (46%), and Abdomen (31%); Psychiatry – Head & Neck (89%), Back (47%), Abdomen (37%), Thorax (32%), Upper Limb (26%), Lower Limb (26%), Pelvis & Perineum (16%). Further data analysis has identified the highest ranked anatomical topics in each body region for the Neurology and Psychiatry clerkships/electives.

Discussion and Conclusion. This database provides detailed information regarding the most clinically relevant anatomical topics as identified by Neurology and Psychiatry clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.
Connections between the RNAPII degradation, DNA damage response, and mRNA processing
Authors: Hofler Alex , Skidders Morgan , Moore Claire , Nazeer Fathima
Published: April 2020

Eukaryotic cells have evolved multiple mechanisms to deal with DNA damage in order to prevent disease. In the presence of helix distorting DNA damage, RNA polymerase II (RNAPII) blockage at damage sites can lead to RNAPII degradation and activation of the DNA damage response (DDR), if the damage is not repaired promptly. Our hypothesis is that proteins involved in RNAPII degradation, such as Elc1, Def1 and Rpb9 in yeast, are also involved in regulating the DDR and other related cellular processes like mRNA maturation in response to DNA damage. Our data suggest that Def1 and Rpb9 could be involved in activating the DNA damage response through Rad53 (a protein involved in DNA damage signaling) while Elc1 is involved in blocking mRNA maturation after UV‐type DNA damage. We are currently exploring the role of Rpb9 and Def1 in regulating mRNA maturation following helix distorting DNA damage, using a PCR based assay. We will present these unexpected interconnections between RNAPII degradation, the DNA damage response, and mRNA processing.

Support or Funding Information

Kilmer Grant for AH. Presidential Scholars Funding for AH. Kilmer Grant for MS. Research Grant and Sponsorship for FN.
Indoxyl Sulfate Exposure inhibits Vascular Smooth Muscle Cell migration and Potentiates Vascular Injury
Authors: Carr Sean , Okon Imoh , Zou Ming-hui
Published: April 2020

Introduction

Chronic Kidney Disease (CKD) has been implicated in cardiovascular complications. CKD results in decreased filtration and a concomitant increase in uremic toxins. Uremic toxins are a by‐product of organic molecules, including two prominent amino acid derivatives, namely Indoxyl sulfate (IS) obtained from tryptophan and p‐Cresyl sulfate from tyrosine. Of these uremic toxins, IS has been shown to be highly potent, and implicated in exacerbation of multiple cardiovascular diseases. However, the effect of IS during vascular injury and specific contributions of Vascular Smooth Muscle Cells (VSMCs) remain unclear.

Methods

Transwell migration and cell attachment assays were employed to examine the phenotypic effects of IS on VSMCs. Western blot analysis, gene silencing, and Immunofluorescence microscopy were utilized to examine the effects of IS on focal adhesion and actin related proteins.

Results

Exposure of VSMCs to IS resulted in the formation of stress fibers and cytoskeletal disorganization. In the presence or absence of chemoattractants, we observed decreased migration of IS‐treated cells. Additionally, a noticeable reduction in cell area and cell attachment was evident. This led to investigation of focal adhesion, and actin‐related proteins due to their established roles in cell migration. It was determined that Focal Adhesion Kinase (FAK) and Paxillin were significantly decreased after prolonged exposure to IS. Immunofluorescence microscopy confirmed decreased FAK and Paxillin intensity at the cell periphery of IS‐treated cells. To determine whether FAK or Paxillin were the main effectors of decreased cell migration instigated by IS, gene silencing was performed. Relative to Paxillin, loss of FAK expression correlated with a reduction VSMC migration.

Conclusion

These data suggest that IS contributes to alterations to focal adhesion related proteins, which in turn attenuates cell migration. IS induced loss of FAK potentiates reduced migration. Thus, IS‐induced inhibition of vascular repair may contribute to secondary complications in a subset of CVD patients.

Support or Funding Information

National Institutes of Health
The Search Continues: Determination of a Lipid Raft Targeting Motif in Cytochromes P450
Authors: Fuchs Robert , Reed James , Backes Wayne
Published: April 2020

Cytochromes P450 play an essential role in metabolism of both xenobiotics and endogenous compounds. The liver P450s responsible for drug metabolism are predominantly expressed in hepatocytes, where they closely associate with the endoplasmic reticulum (ER). This membranous organelle is mostly composed of phospholipids, which can assemble into disordered regions (ld) or ordered regions (lo, “lipid rafts”).

Our group previously showed that different P450 species show strong preference for either ordered or disordered regions. When expressed in human embryonic kidney (HEK) cells, we demonstrated that CYP1A2 is found in ordered (raft) regions, and that CYP1A1 exhibits a strong preference for the disordered region. This pattern was striking, as the two P450 species have over 70% sequence homology. We then showed that a chimeric CYP1A2 construct containing the first 107 amino acids of CYP1A1 no longer localized in the ordered regions, but mobilized to the disordered region. These findings indicate that a P450 raft targeting sequence exists within the first 107 amino acids of CYP1A2.

The length and composition of this motif remain unclear. We hypothesize that a 5–9 amino acid sequence within the first 107 amino acids is responsible for the localization of CYP1A2 into the ordered domains, and that substitution of the corresponding region of CYP1A1 can mobilize full‐length CYP1A2 to the disordered region. To test this, we generated 5‐ to 30‐amino acid chimeras using overhang PCR cloning and expressed these constructs in HEK cells. The disordered membrane regions from these cells were solubilized using Brij98, with the ordered regions remaining in the membrane. The ordered membranes were isolated as a pellet after centrifugation at 100,000 x g for 1 h. We found that insertion of the first 28 amino acids of CYP1A1 into CYP1A2 led to its partial relocation into the disordered regions, and that the first 30 amino acids led to more complete relocation of the chimeric protein. We then generated smaller chimeras in an effort to identify the minimum amino acid segment responsible for membrane relocation. Substitution of amino acids 28–30 led to relocation of CYP1A2 from the ordered to disordered regions. These results demonstrate that a raft‐targeting sequence resides within the first 30 N‐terminal amino acids of CYP1A2 and, when eliminated, permits mobilization of the P450.

Support or Funding Information

Supported by NIH GM123253 and ES013648 and by a predoctoral fellowship from the PhRMA Foundation.
Endothelial‐Dependent, but not Endothelial‐Independent, Vasodilation Is Reduced in Non‐Hispanic Blacks versus Non‐Hispanic Whites
Authors: Hollowed Casey G. , Miller James T. , Wong Brett J.
Published: April 2020

Nitric oxide (NO)‐dependent vasodilation is reduced in non‐Hispanic Blacks (NHB) compared to non‐Hispanic Whites (NHW), but it is not well‐established if this reduction is due to reduced endothelial‐dependent or endothelial‐independent vasodilation, or both. The purpose of this study was to examine endothelium‐dependent and endothelium‐independent mechanisms of cutaneous microvascular vasodilation between young, healthy NHB and NHW. Six participants who self‐identified as either NHB or NHW were instrumented with two microdialysis fibers in the skin of the forearm. A dose‐response was then completed at each site: 1) acetylcholine (ACh; 10−10– 10−1M) to assess endothelial‐dependent vasodilation, and 2) sodium nitroprusside (SNP; 10−10– 10−1M) to assess endothelial‐independent vasodilation. Each dose was perfused for 5 minutes. Local heaters were kept at 33°C throughout baseline and the dose‐response protocol. Maximal vasodilation was elicited by heating the skin to 43°C and infusing 54 mM SNP. Laser‐Doppler flowmetry (LDF) was used to assess an index of skin blood flow. Cutaneous vascular conductance (CVC) was calculated (LDF/MAP) and normalized to maximal blood flow (%CVCmax). Data are presented as mean ± standard deviation. The %CVCmax response to ACh in NHB was significantly reduced compared to NHW at all concentrations >10−4 M (10−3: NHB 20 ± 13 vs. NHW 66 ± 8 %CVCmax; 10−2: NHB 25 ± 3 vs. NHW 76 ± 8 %CVCmax; 10−1: NHB 39 ± 15 vs. NHW 84 ± 8 %CVCmax;P< 0.05 for all). The ACh log EC50 was higher for NHB (−1 ± 0.6 M) compared to NHW (−3.5 ± 0.8 M; P< 0.05). The %CVCmax response to SNP was similar between NHB and NHW for all concentrations tested. Similarly, there was no difference in log EC50 between NHB (−1.5 ± 0.6 M) and NHW (−2.1 ± 0.4 M). Reduced NO‐dependent vasodilation in NHB compared NHW appears to result from decreased endothelial‐dependent mechanisms, and not endothelial‐independent mechanisms. This information supports suggestions from previous research in the field and may aid in understanding the discrepancy in disease risk between NHB and NHW.
“Smac”ing the Life out of XIAP
Authors: Greig James , Akteke Elfin , Doshi Sarah , He Eva , Fujikawa Kento , Lawrence Katya , Feberova Natalie , Hayashi Miwa , Jia Rizhe , Jin Maggie , Leha Emily , Powter Morgan , Shen Delphine Jiameng , Walker Samuel , Korneluk Robert
Published: April 2020

X‐linked inhibitor of apoptosis protein (XIAP) is a key prosurvival protein that blocks apoptosis by inhibiting both the intrinsic and extrinsic pathways. Released by the mitochondria when under stress, XIAP is a homodimer, with 2 sets of repeating type I and type II baculoviral IAP domains: BIR1, BIR2 and BIR3, and RING. XIAP has a capacity to block apoptosis by directly inhibiting caspases 3,7 and 9. Second mitochondria‐derived activator of caspases (Smac) is a 184 amino acid chain. Smac antagonizes XIAP by attaching to the BIR3 domain inhibiting the binding of caspase‐9. Synthetic Smac mimetics (SMs) have been designed to duplicate the N‐terminal tetrapeptide (AVPI) or the IAP‐binding motif (IBM). Our model aims to show two BIR3 domain of a homodimer XIAP, bound to the IAP‐binding motif (IBM) of a Smac dimer. Selective antagonism of XIAP, specifically the BIR3 by Smac/SMs or monotherapy is not enough to kill cancer cells, however, it could be combined with immunotherapy, specifically virotherapy. This tricks the immune system to think that the cancer cells have a virus, which stimulates the immune antiviral response and creates an overproduction of immune cells known as a “cytokine storm”, to kill cancer cells.

Support or Funding Information

The Ashbury College MSOE Center for BioMolecular Modeling SMART Team used 3D modeling and printing technology to examine structure‐function relationships between Smac and BIR3 of XIAP.
The Effects of XIAP on Alzheimer's Disease
Authors: MacGillivray Brendan Cameron , Cobo Nicolas Alonso , Bai Kathleen , Efraim Margaret , Fu Celine , Fu Henry , Fujikawa Ayaka , Goodall Lauchlann , Han Brian , He Lucy , Hernandez-Oviedo Isabela , Ilianu Richard , Muscant Rebecca , Niu Catherina , Ramirez Victoria , Samad Hana , Ticala Iza , Wang Oscar , Wong Andrew , Korneluk Robert
Published: April 2020

Inhibitor of apoptosis proteins (IAPs) prevents programmed cell death by interfering with the activation of caspases that cleave cytoskeleton fibres. X‐chromosome‐linked inhibitor of apoptosis protein (XIAP) is believed to be the most potent natural apoptosis inhibitor in the IAP family, as it effectively suppresses both intrinsic and extrinsic pathways of apoptosis. XIAP is characterized by three Baculoviral IAP Repeat (BIR) domains, a ubiquitin‐associated (UBA) domain, and one Really Interesting New Gene (RING) domain. The BIR‐2 domain binds with caspases 3 and 7, whereas the BIR‐3 domain binds with caspase 9.

XIAP is closely associated with several neurodegenerative disorders, including Alzheimer’s Disease (AD), which affects over 44 million individuals worldwide. AD is characterized by the formation of ß‐amyloid plaques and tau tangles, which obstruct nutrients from reaching the nerve cells and result in cell death. Caspases cleave tau proteins that consequently collapse into tangles. Since XIAP prevents the activation of caspases, a lower level of XIAP leads to the acceleration of nerve cell death and cognitive impairment. While XIAP is unable to arrest tangle formation, an increase of XIAP will lessen cognitive decline.

Support or Funding Information

The Ashbury MSOE Center for BioMolecular Modeling SMART Team used 3D modelling and printing technology to examine the effects of the XIAP BIR‐3 and BIR‐2 domains on the caspases that interact with the Alzheimer’s pathway.
Does lysine acetylation affect water permeability of the collecting duct?
Authors: Hyndman Kelly Anne , Davis Parker , Mendoza Luciano Deven
Published: April 2020

We recently reported that there are > 400 proteins in the inner medullary collecting duct that are post‐translationally lysine acetylated (ac‐K). Although historically, ac‐K proteins were thought to be predominantly histone proteins in the nucleus, we now know that all cellular compartments are enriched with ac‐K proteins. Pathway analyses determined that ac‐K proteins are enriched in various physiological processes such as glycolysis and vasopressin‐mediated water reabsorption. We recently identified the basolateral water channel, aquaporin‐3 (AQP3), as undergoing lysine acetylation (AQP3 K282). Thus, the purpose of this study was to determine if ac‐K AQP3 affects water permeability. We developed antibodies to specifically detected ac‐K AQP3 or total AQP3. In both male and female mice, ac‐K AQP3 was expressed in the basolateral membrane of the cortical and outer medullary collecting duct. However, following 24 h of water deprivation, ac‐K AQP3 was also found in the inner medullary collecting duct. Next, we developed AQP3 K point mutation plasmids, with K282Q representing an acetylated mimic, and K282 representing a deacetylated mimic. These constructs were stably expressed in vasopressin‐responsive mouse cortical collecting duct cells (AQP3mpkCCDs). Using the cell volume sensitive dye, calcein, water permeability of these mutant cells was determined following an osmotic stimulus. We found the acetylated mimic (K282Q) had the highest water permeability followed by the AQP3 wild type K282 cells and the deacetylated mimic (K282R). Finally, using CRISPR/CAS we engineered whole body point mutation mice. The K282Q mutant mice and littermate controls (K282) all developed normally. As adults, K282Q mice presented with elevated plasma osmolality (307 ± 2 mOsm/Kg H2O vs 298 ± 4 mOsm/kg H2O, P = 0.04) at baseline. Following 3 days of hydration with 5% sucrose water, K282Q produced twice as much urine as controls during their active period (2.16 ± 0.5 ml/12h vs 0.88 ± 0.2, P = 0.03). Moreover, when challenged with a 2 ml sterile water i.p., K282Q mice excreted majority of the water load within the first 3 h, while controls reached peak urine flow after 3 h. Finally, we quantified AQP2 (the apical water channel) and AQP3 mRNA expression and protein abundance. AQP2, AQP3 and AQP4 mRNA expression was similar between control and mutant mice. Surprisingly, K282Q mutant mice presented with significantly less AQP2 and AQP3 protein abundance compared to controls. Thus, mutating K282 in vivo resulted in a loss of AQP3 (a potentially AQP2) stability, and highlights the importance of this lysine residue. To conclude, lysine acetylation of AQP3 is a novel posttranslational modification that may regulate AQP function and water permeability in the collecting duct.

Support or Funding Information

K01DK105038, R03DK120503, the University of Alabama at Birmingham Pittman Scholarship, NIH P30 DK074038
Aligned Nanofibrillar Scaffolds as a Platform for the Treatment of Volumetric Muscle Loss Injury
Authors: Miles Lindsey M. , Raymond-Pope Christiana J. , Corona Benjamin T. , Paukshto Michael V. , Zaitseva Tatiana S. , Greising Sarah M.
Published: April 2020

Volumetric muscle loss (VML) injury is the traumatic loss of skeletal muscle fibers, along with associated vascular and neural structures, resulting in long‐term functional disability. To date, several strategies have been developed to target skeletal muscle and vascular regeneration and recovery of muscle function, with only limited improvements reported. Our objective, using a standardized unilateral VML injury to the rat tibialis anterior (TA) muscle, was two‐fold: (1) examine the rate of cell population and distribution of cell phenotypes in a bioengineered aligned collagen scaffold; and (2) evaluate the in vivo transfection efficiency of aligned collagen scaffolds loaded with firefly luciferase (Fluc) using bioluminescence imaging. We hypothesize that a modified mRNA (mmRNA)‐incorporated aligned collagen scaffold could be an ideal platform to stimulate vascular and muscle fiber regeneration following VML injury. In total, 25 adult male Lewis rats were used across studies. In Study 1, following unilateral VML injury to the TA muscle (~82mg removed), rats received implantation of an aligned collagen scaffold (6mm in diameter, 0.5–3.0mm thickness), consisting of two interconnected bundles of BioBridge threads that were cross‐linked, lyophilized, and sterilized. The TA muscle was evaluated histologically at 1, 2, and 4 weeks post‐VML to examine cell population and distribution, fibrotic content, and extent of scaffold degradation within the remaining TA muscle and VML defect area. In Study 2, in vivo bioluminescence imaging quantified localized RNA‐transfected cell expression in rats up to 2 weeks post‐Fluc injection within the TA muscle, with or without VML injury, or implantation of an aligned collagen scaffold loaded with Fluc‐RNA following VML injury to evaluate transfection efficiency for potential mmRNA cargo. Scaffolds exhibited minimal degradation through 4 weeks following implantation. Quantification of cellular distributions within the VML defect area indicated significant macrophage infiltration and inflammation up to 4 weeks post‐VML. Intramuscular Fluc injections indicated peak bioluminescence signal (3.0x105 p/s/cm2/sr) occurs at 7 days post‐injection. These results serve as a reference for our ongoing development of a mmRNA‐loaded scaffold delivery system. Our long‐term goal is to evaluate candidate growth factors to co‐deliver in the mmRNA‐loaded aligned collagen scaffolds to facilitate organized, functional skeletal muscle and vascular regeneration following VML injury.

Support or Funding Information

This work is supported by: US Army Medical Research and Materiel Command under Contract Number W81XWH19C0021; T32AR050938; P217A170051.
An Increase in Thermotolerance Is Associated with Elevated Levels of Heat Shock Protein 70 (HSP70) in a Mouse Exertional Heat Stroke Model
Authors: Dineen Shauna M. , Ward Jermaine A. , Plamper Mark L. , Mayer Thomas A. , Leon Lisa R.
Published: April 2020

Recent data from our laboratory showedthat one exertional heat stroke (EHS) exposure improved thermal tolerance inmice who were exposed to the same conditions again within 24 h. This was evident by longer average runningtime, higher thermal load, higher maximum core temperature, and less severehypothermia during the second EHS exposure and recovery compared to the first. Theaim of this study was to determine if traditional biomarkers of EHS severitywere reflective of the improved thermoregulatory performance observed during the second EHS. We collected blood and liver samples from male C57BL/6J mice at30 min and 3 h into recovery from a second EHS event that occurred 24 h after the initial EHS exposure. Mice displayed significantly lower circulating cytokines and chemokines 30 min (MIP‐1β, MIP‐2; P<0.05) and 3 h (G‐CSF,IL‐10, IP‐10, KC, MCP‐1; P<0.05) following the second vs. first EHS exposure. Plasma corticosterone was also significantly lower in mice 3 h after the second vs. first EHS (36458 vs. 262914 pg/ml, respectively; P=0.0005). Liver heat shock protein (HSP)70 was significantly elevated in mice 24 h after the first EHS and was further increased after the second EHS at 30 min (5‐fold) and 3 h(2‐fold) of recovery. The elevated levels of liver HSP70 protein correlated with a reduction in HSP70 gene expression at these time points, suggestive of a negative feedback loop. Although HSP70 is thought to protect against heat‐induced organ damage, aspartate aminotransferase (AST; a clinical biomarker of liver damage) was significantly higher 30 min after the second vs. first EHS exposure (994 U/L vs. 374 U/L, respectively; P<0.05). Taken together, our results suggest that one EHS event induces partial thermal tolerance in mice that improves performance in the heat 24 h later and may protect against systemic inflammation during the subsequent heat exposure, as evidenced by changes in the expression of pro‐ and anti‐inflammatory factors, such as cytokines, chemokines and corticosterone. Elevated liver HSP70 suggests protection against organ damage, but this interpretation needs to be validated with histological analysis of the liver since elevated AST levels suggest increased stress to this organ following the second EHS. Perhaps the adaptive responses of improved run time and thermal tolerance in the heat 24 h after the initial EHS come at the cost of additional organ damage. Author views not official US Army or DOD policy.
Ferric Reductases CG1275 and Nemy in Iron Uptake in Drosophila S2 cells
Authors: Lewis Keyata , Holst Jessica , Kane Gregory , Castaneda Mirella , Gorman Maureen J. , Ragan Emily
Published: April 2020

Iron plays key roles in humans and insects, including in the electron transport chain and other electron transport processes. There is still uncertainty about how iron enters the cell in insects. Currently our group is investigating two cytochrome b561 family members, CG1275 and nemy, as possible ferric reductase components in a Drosophila S2 cell iron uptake pathway. Nemy is known to have ferric reductase properties in Drosophila cells, but its main function occurs in the insect’s brain where it is expressed in neuroendocrine neurons. Nemy has not yet been directly linked to how flies intake iron, but we do hypothesize that a ferric reductase such as nemy is needed in order for the cell to uptake iron. We hypothesize that Fe3+ is reduced to Fe2+ using a ferric reductase (CG1275 or nemy), which will then pass through the malvolio protein for entry into the cytosol. Our experiment involves RNA interference (RNAi) gene silencing of CG1275 and nemy. By adding dsRNA with a sequence matching CG1275 or nemy mRNA, we cause selective degradation of mRNA. After RNAi we extract total RNA and use quantitative PCR to analyze mRNA levels to see if degradation of targeted mRNA has been achieved. We will also measure cellular iron content using a ferrozine‐based assay. If we detect a decrease in iron content in the cells then we will have support for our hypothesis.

Support or Funding Information

NSF IOS‐1656407
Impact of Multiple Exertional Heat Strokes on Thermoregulation and Performance
Authors: Mayer Thomas , Dineen Shauna , Ward Jermaine , Plamper Mark , Leon Lisa
Published: April 2020

Heat stress can result in beneficial acclimation and/or harmful injury depending on the severity of exposure. The impact of multiple heat exposures on thermoregulatory and physical performance has not been well‐studied, but is an important consideration for warfighters, athletes, and others who routinely encounter hot environments. The aim of this study was to examine the effects of multiple exertional heat strokes (EHS) on thermoregulatory and performance variables. C57BL/6J male mice were implanted with an intra‐abdominal radiotelemetry device to measure core temperature (Tc; ±0.1°C). Four exercise training sessions were conducted at thermoneutral conditions (25°C) prior to heat exposure to familiarize mice with the forced running protocol. For the EHS protocol, mice rested inside a running wheel for approximately 35 minutes until the environmental chamber reached 37.5°C and then began running at 2.5m/min, accelerating 0.3m/min every 10 minutes until reaching their max speed when Tc reached 41.0°C; mice were removed from the heat when they collapsed. After recovering for either one day (1D), three days (3D), or seven days (7D), mice repeated the EHS protocol. Regardless of the number of recovery days, mice spent more time in the heat prior to collapse during the second heat exposure compared to the first (1D: 335±9 minutes vs 275±8 minutes, p<0.001; 3D: 331±9 minutes vs 275±7 minutes, p<0.001; 7D: 320±9 minutes vs 274±7 minutes, p<0.001). Maximum Tc (Tcmax) was higher during the second heat exposure after both 1D and 3D recovery compared to the first heat exposure (1D: 42.50±0.03°C vs 42.15±0.04°C, p<0.001; 3D: 42.40±0.04°C vs 42.13±0.04°C, p<0.001; 7D: 42.17±0.03°C vs 42.12±0.03°C, not significant) and during the second heat exposure Tcmax was higher in 1D compared to 3D (p<0.05) and 7D (p<0.001) and higher in 3D than 7D (p<0.001). Hypothermia depth, a measure of injury severity, was less pronounced after the second heat exposure compared to the first heat (1D: 34.57±0.13°C vs 31.31±0.34°C, p < 0.001; 3D: 33.33±0.19°C vs 31.86±0.22°C, p < 0.001; 7D: 33.59±0.30°C vs 31.77±0.46°C, p < 0.001). Hypothermia depth after the second heat was also less severe in 1D mice compared to 3D mice (p < 0.05) and trended less severe compared to 7D mice (p = 0.07). Taken together, these results demonstrate that a single exertional heat exposure can induce acclimation‐like measures that enhance performance and may protect against injury during subsequent heat exposures. Author views are not official US Army or DoD policy.
Neuraminidase 1 and 2 Active Site Comparison for Viral Drug Resistance Analysis
Authors: Sieradzki Guinevere , Mierswa Sabrina , Sabin Amber , DeBruine Anna , Koprek Jessica , Perez Victoria , Minx Connor , Persaud Krishna , Selvik Josephine , Smith Behrgen , Zieman Joshua , Kanungo Muskan , Berkowicz Alexandra , Cabrera-Brito Bella , Pung Benjamin , Alexander Anne
Published: April 2020

Neuraminidase is an important protein on the surface of influenza and is an antiviral target. There are several types of neuraminidase, but this project focuses on neuraminidase 1 (N1) and 2 (N2) because of their prevalence in human influenza A infections. The amino acid residues in the active sites of N1 and N2 are 94% identical. However, there are several key differences that affect how N1 and N2 interact with the same antiviral drug. The purpose of this project is to gain a deeper understanding of viral drug resistance by modeling the active sites of N1 and N2 and their interactions with oseltamivir (Tamiflu). Two PDB files have been selected for N1 (2HU4) and N2 (4GZP). These PDB files will initially be overlaid in Jmol to identify the level of structural similarity, then active site boxes will be 3D printed for N1 and N2. Active site boxes model only the active site of a molecule, giving a detailed view and understanding of this specific location. Within the models for N1 and N2, Jmol will be used to highlight amino acids E276, I222, and H274Y to investigate their effects on inhibitor interactions. Additionally, a model of the inhibitor oseltamivir (Tamiflu) will be 3D printed to compare its interactions with N1 and N2. These active site boxes will enhance the scientific education and study of these active sites.

Support or Funding Information

This work is part of the CREST program, supported by National Science Foundation Grants I022793, I323414, and 172s940.