bluuebunny/crossref_metadata_2025 huggingface dataset:
Perforasomes of the facial artery
Authors: Hirtler Lena , Berger Vinzenz
Published: April 2020

Background

The facial artery perforator flap is a valuable alternative for reconstruction of small to medium defects in the central midface. Not only does it provide pleasing cosmetic results, it also allows reconstruction in a one‐stage procedure. Even though facial artery perforators have been examined by anatomical studies during the past years, there is still little knowledge of the vascular territories provided by these vessels. Aiming to make facial artery perforator flaps a plannable and safer procedure, this study examines the location and size of the vascular territories provided by facial artery perforators.

Material and methods

20 facial arteries of fresh specimen have been dissected. All perforators with an external diameter of at least 0,50 mm have been identified and injected with Methylene blue‐ or Eosin Y‐solutions to color their appendant vascular territories. All colored skin territories have been photographed, digitalized and measured. Their location, as well as the location of the perforating vessels, has been shown. All results were statistically analyzed.

Results

95 perforators with an external diameter of at least 0,50 mm have been identified. The average number of perforators per facial artery was 4,75. The mean external diameter at the origin of the facial artery was 0,64 ± 0,14 mm. The mean size of the colored skin territories was 5,62 ± 4,83 cm2. There was a statistical correlation between perforator diameter and the size of its vascular territory.

Conclusion

Two areas have been shown in which facial artery perforators originate frequently. Their location has been described in relation to a bone structure. Size and shape of the vascular territories show a high variability with regions of increased density. The graphical illustration of the location of the facial artery perforators and their vascular territories can help making facial artery perforator flaps a better plannable procedure.
Ppargc1a Regulates Prostaglandin Signaling to Control Ciliogenesis and Renal Multiciliated Cell Fate Choice During Development
Authors: Chambers Joseph M. , Addiego Amanda , Wingert Rebecca A.
Published: April 2020

Cilia are microtubule‐based organelles that function in a multitude of physiological contexts to perform chemosensing, mechanosensing, or fluid propulsion. The process of ciliogenesis is highly regulated and disruptions result in disease states termed ciliopathies. The genetic and molecular events that lead to dysfunctional ciliated cells are still not fully understood. Here, we show novel roles for peroxisome proliferator‐activated receptor gamma 1 alpha (ppargc1a) during ciliogenesis in mono‐ and multiciliated cells (MCCs) as well as discernment of renal tubule MCC fate choice during embryogenesis. We discovered that ppargc1a performs both roles by affecting prostaglandin levels, where cilia formation and renal MCC fate were restored with prostaglandin E2 (PGE2) treatment. Genetic disruption of ppargc1a specifically reduced expression of the prostanoid biosynthesis gene prostaglandin‐endoperoxide synthase 1 (ptgs1), and suboptimal knockdown of both genes revealed a synergistic effect. Further, ptgs1 overexpression rescued ciliogenesis and renal MCCs in ppargc1a deficient embryos. These findings position Ppargc1a as an essential genetic regulator of prostaglandin synthesis during ciliated cell ontogeny.

Support or Funding Information

National Institutes of Health (R01DK100237 to R.A.W.), a 2019 University of Notre Dame Advanced Diagnostics and Therapeutics Graduate Fellowship Award (to J.M.C.), and a 2019 University of Notre Dame College of Science Summer Undergraduate Research Fellowship Award (to A.A.)
Investigating the role of a V‐ATPase in the Development of the Air Sac Primordia
Authors: Salim Usman , Salim Hasan , Gondal Anas , Srivastava Ajay
Published: April 2020

Drosophila melanogaster is a commonly used organism in biological research. Their Air Sac Primordium (ASP) is analogous to the human lungs in that it supplies oxygen to the flight muscles. Furthermore, the ASP has emerged as a good structure to study cellular invasive processes with the potential to aid in our understanding of tumor metastasis. The ASP originates on the outside of the wing imaginal disc and then burrows into the wing imaginal disc as it develops. What genes are involved in this process is not well understood. A genetic screen for genes that are expressed in the ASP identified a proton pump gene encoding a V‐ATPase. The V‐ATPase is a vacuolar proton pump that plays a large array of roles from regulating the pH of cells internal space, to regulating endocytosis, to processing proteins and degradation. In insects, the V‐ATPase are important in establishing a membrane potential to drive K+ secretion. It has also been found to influence tracheal branching. Using RNA interference, the gene for the V‐ATPase was knocked down resulting in a defect in ASP development. Data from these and other experiments will be presented and their significance will be discussed.

Support or Funding Information

Kentucky Biomedical Research Infrastructure Network (KBRIN) Grant

Gatton Academy Research Internship Grant (RIG)

Faculty Undergraduate Student Engagement (FUSE) Grant
Transient ischemic stroke induces substantial leukocyte recruitment to both the ischemic and non‐ischemic cerebrovasculature as visualized through live animal imaging.
Authors: Batra Ayush , Nadkarni Neil A. , Muller William A. , Sullivan David P.
Published: April 2020

Ischemic stroke is a leading cause of a death and disability worldwide. Current treatment strategies rely on early restoration of blood flow; however, injury persists despite successful reperfusion. The innate immune response plays a pivotal role in reperfusion injury, but the mechanisms and time‐course of neutrophil and monocyte recruitment to the cerebrovasculature (CBV) during ischemic stroke remain unclear. We sought to characterize the real‐time leukocyte response at the CBV in a mouse stroke model with reperfusion.

To visualize leukocyte recruitment induced by ischemia, Ly6G‐tdTomato mice (which have fluorescent red neutrophils) were subjected to 90 minutes of transient middle cerebral artery occlusion (tMCAO) followed by reperfusion. Real‐time spinning‐disc confocal intravital microscopy of the CBV was performed at 24 and 72 hours following reperfusion. Intracranial windows were placed in both ischemic and non‐ischemic hemispheres; and tMCAO treated mice were compared to mice that underwent sham surgery. Neutrophil recruitment (adhesion, rolling) and extravasation were quantified in vivo on both the ischemic and contralateral non‐ischemic hemispheres. Postmortem staining with 2,3,5‐triphenyltetrazolium chloride was used to confirm induction of the stroke and to quantify infarct volume. In addition, wide‐field microscopy was used to examine neutrophil extravasation across the parenchyma.

Animals subjected to sham surgery showed negligible leukocyte recruitment to the CBV. However, marked increases in leukocyte rolling, adhesion, and extravasation were observed at 24 and 72 hours following the tMCAO procedure. Interestingly, non‐ischemic hemispheres also demonstrated increased leukocyte rolling and adhesion, but not extravasation tMCAO group. Also, substantially more neutrophil extravasation was observed in the core of the infarct at 72 hours. These findings are consistent with an early recruitment of neutrophils to the cortical surface at early time points and a delayed infiltration into the core, as reported by others.

These results show that a significant leukocyte response persists after reperfusion following tMCAO. This response is present both on the ischemic and, to a lesser extent, the non‐ischemic cortical CBV. Acute leukocyte recruitment observed on the non‐ischemic hemisphere suggests that ischemic stroke triggers a global inflammatory response within the CBV. Further reducing inflammation through immunomodulatory therapy in conjunction with reperfusion therapy may be beneficial in not only reducing stroke volume but potentially mitigating inflammation mediated secondary neuronal damage.

Support or Funding Information

NIH R01 HL046849 and R37 HL064774 to W.A.M.
Characterization and Quantification of Dopaminergic Phenotypes via Computer‐aided Video Analysis
Authors: Garriga Gustavo Adolfo , Nelms Brian
Published: April 2020

Dopamine is an important neuromodulator known to regulate a myriad of brain functions ranging from motor control to memory and learning. Dopamine dysregulation has been associated with multiple neurological disorders including schizophrenia, attention deficit hyperactivity disorder, addiction, Parkinson’s disease among many others. To better understand dopamine function at the molecular level, we are investigating the roles and regulation of dopaminergic genes using Caenorhabditis elegans (C. elegans) as a model system. We are conducting swimming induced paralysis (SWIP) assays using a computer‐aided video analysis system, allowing us to collect more nuanced data to uncover new phenotypes and discover roles for genes involved in dopaminergic signaling. We have selected several genes to focus on in this study. Each of these genes is highly enriched in dopamine neurons, based on RNASeq data from our laboratory. Mutant strains for these genes display accelerated swimming‐induced paralysis, a phenotype characteristic of hyperdopaminergic signaling, when compared to the wild‐type strain, N2. Via this analysis, we propose to gain more insight into specific rates and patterns of paralysis of these mutant strains in order to identify novel genes critical for dopaminergic functioning.

Support or Funding Information

Funding support from NSF CREST Grant #HRD1547757, NIH BD2K R25 Grant #1R25MD010396‐01, and DOE Title VII Grant MD‐HBCU #P382G090004.
Influence of RGS6 on Inhibitory G Protein Signaling in Mouse Sinoatrial Nodal Cells
Authors: Anderson Allison , Masuho Ikuo , Marron Ezequiel , Martemyanov Kirill , Wickman Kevin
Published: April 2020

Introduction

M2 muscarinic (M2R) activation of G protein‐gated inwardly rectifying K+ (GIRK) channels in sinoatrial nodal (SAN) cells is a key mediator of vagal/parasympathetic slowing of heart rate. The strength of M2R‐GIRK signaling is negatively regulated by Regulator of G protein Signaling 6 (RGS6). Adenosine, used clinically to diagnose and treat certain arrhythmias, exerts these actions through A1 adenosine receptor (A1R) activation of GIRK channels. The extent to which RGS6 regulates A1R‐GIRK signaling, however, is unclear. Here, we investigated the impact of RGS6 on A1R‐GIRK and M2R‐GIRK signaling in mouse SAN cells.

Methods and Results

We measured the efficacy and potency of M2R‐ and A1R‐GIRK signaling in adult SAN cells from wild‐type and RGS6−/− mice using whole‐cell patch‐clamp electrophysiology. As previously reported, SAN cells from RGS6−/− mice displayed prolonged channel deactivation kinetics and increased channel sensitivity to the non‐selective cholinergic agonist carbachol (CCh), relative to wild‐type counterparts. In contrast, we observed a striking increase in the amplitude of the GIRK response evoked by adenosine (Ado) in RGS6−/− SAN cells compared to wild‐type controls, with no impact on channel deactivation kinetics or sensitivity. Further recordings in wild‐type SAN cells revealed that M2R activation completely occludes the Ado‐induced response, whereas Ado application activates only a smaller fraction of GIRK channels. Both CCh‐ and Ado‐ induced currents were abolished in SAN cells treated by pertussis toxin (PTX), indicating these responses are mediated by Gi/o G proteins. Utilizing a bioluminescence resonance energy transfer (BRET) assay in transfected HEK cells, we found that RGS6 prefers Gαo as a substrate for over Gαi. Additionally, we found that while A1R does not discriminate between Gαo and Gαi, M2R displays a clear preference for Gαo. We next employed a Cre/flox strategy to remove Gαo from atrial tissue, including SAN cells. Loss of Gαo yielded significantly blunted CCh‐induced responses, which also displayed prolonged activation and deactivation kinetics. While Ado‐induced responses had prolonged deactivation kinetics, activation kinetics and response amplitude were unaffected.

Conclusions

These results suggest that RGS6 negatively regulates A1R‐GIRK signaling distinct from its influence on M2R‐GIRK signaling in mouse SAN cells likely due to differing utilization of Gao by M2R and A1R.

Support or Funding Information

This work was supported by NIH grants R01 HL105550 and F31 HL139090.
Exploring The Relationship Between Optimized Bioavailable Curcumin And Performance After an Intensified Training Program
Authors: Kisiolek Jacob N. , Flores Victoria , Ramani Arjun , Johnston Nora , Taylor Sophia , Kheradia Nikeeta , Stewart Laura K.
Published: April 2020

Objective

The purpose of this double blind, randomized, study was to determine the effects of Longvida® Optimized Curcumin on time trial performance time as well as the inflammatory mediator C‐reactive protein (CRP) after 2 weeks of an intensified training program.

Methods

Healthy men and women (aged 18–40 y) were randomly assigned to placebo (PLA; n=11), Curcumin 1 (LV1; n=12) or Curcumin 2 (LV2; n=11) groups. Participants consumed capsules of rice flour placebo (1000mg/day) or curcumin (1000mg/day; Longvida 1 (LV1) or 1000mg/day Longvida 2 (LV2)) for 14 days, during a 2‐week intensified, cycle training period, which consisted of 3 training sessions per week. Each training session consisted of 6, 90 second stationary cycling sprints at 80–90% heart rate max (HRmax) followed by 180 seconds of low intensity cycling at 50–60% HRmax. Age, body mass index (BMI), percent body fat (BF%; BodPod, Cosmed, USA), and peak oxygen uptake (VO2peak) were assessed at the pre intervention time point (PRE). Time trial performance (16.1 km; TT) was obtained on a stationary cycle and resting and fasted serum was evaluated for CRP using an enzyme‐linked immunosorbent assay at both PRE and post intervention (POST) time points. Differences among groups were assessed using a one‐way ANOVA, and PRE and POST measurements among groups were assessed using a two‐way mixed model ANOVA. Data are presented as mean ± standard deviation with significance set at α=0.05.

Results

At PRE, there were no significant differences between groups with respect to mean participant characteristics (age: 24.8±4.2 y; BMI: 24.0±3.5; BF%: 20.55±8.90; VO2peak 42.07±8.90 ml/kg/min). TT performance significantly improved an average of 1.41±5.01% (p=0.05) when PRE times were compared to POST. Additionally, although not statistically significant, the average TT time change from PRE to POST was −205.67 sec for PLA, −2.17 sec for LV1, −105.32 sec for LV2. There were no significant group effects or group by time interactions for CRP (PLA: 3.2 ±3.4 mg/L; LV1: 0.7 ±0.9 mg/L; LV2: 1.2±01.3 mg/L).

Conclusions

The intensified training period elicited a significant improvement in TT performance. When taken daily, the dose of curcumin used in this study did not appear to provide additional performance related benefits. However, when compared to the existing literature, the dose and the timing of curcumin intake as well as the mode of the activity may be related to curcumin‐mediated improvements in performance.

Support or Funding Information

Verdure Sciences, Noblesville, IN
The E3 Ubiquitin Ligase Adaptor FBXL13 Mediates the cGMP‐dependent NKCC2 Ubiquitination in Thick Ascending Limbs
Authors: Ares Gustavo
Published: April 2020

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter (NKCC2). Nitric Oxide and atrial natriuretic peptide decrease NaCl absorption in the TAL by increasing the second messenger cyclic guanosine monophosphate (cGMP). NKCC2 is constitutively ubiquitinated, and cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification that regulates protein activity or fate. We recently showed that the cGMP‐dependent NKCC2 ubiquitination is mediated by the SCF (Skp1‐Cullin1‐FBox) complex, which is the largest family of E3 ubiquitin ligase in mammals. To study new proteins that interact and regulate NKCC2, we used a targeted proteomics approach. TALs were incubated with GST constructs of the NH2‐ or COOH‐terminal portion of NKCC2 followed by pull‐down and electrophoresis. We identified the ubiquitin‐ligase adaptor F‐Box leucine‐rich repeat 13 (FBXL13) that bind the NH2‐terminus portion of NKCC2. A band at the expected molecular weight for FBXL13 confirmed FBXL13 expression in rats TALs by Western blot. We studied the interaction of full length NKCC2 with FBXL13 and found that FBXL13 co‐immunoprecipitated with full length NKCC2 in TAL lysates Therefore, we hypothesized that the cGMP‐dependent NKCC2 ubiquitination is mediated by FBXL13. To test our hypothesis, we generated a global FBXL13 knockout mice by crossing FBXL13‐Flox with a global Cre mice. We confirmed the deletion of FBXL13 in tail by PCR and in TALs by Western blot. First, we studied whether deletion of FBXL13 affects total NKCC2 expression. For this, TALs from C57BL6J and FBXL13‐KO mice were lysed, and equal amount of proteins resolved by SDS‐PAGE and detected by Western blot. We found that total NKCC2 expression in FBXL13‐KO mice is increased by 73% (C57BL6J: 100% vs. FBXL13‐KO mice: 173 ± 37%, n = 4, p<0.05). These data suggest that in FBXL13‐KO mice, NKCC2 is been accumulated and not degraded. We tested whether the cGMP‐induced NKCC2 ubiquitination is blunted in FBXL13‐KO mice. TALs from C57BL6J control mice and FBXL13‐KO mice were incubated at 37°C during proteasome inhibition with MG132 and treated with vehicle or db‐cGMP 500μM. Ubiquitinated proteins were pulled down and NKCC2 measured by Western blot. We found that cGMP increased ubiquitinated‐NKCC2 by 24 ± 10% in C57BL6J mice, whereas this effect was completely absent in FBXL13‐KO mice (−14 ± 12%, n=3, p<0.05). These data indicate that FBXL13 mediates the cGMP‐dependent NKCC2 ubiquitination in native TALs. We conclude that the cGMP‐dependent NKCC2 ubiquitination is mediated by the novel E3 ubiquitin ligase adaptor FBXL13. To our knowledge, this is the first evidence showing an E3 ubiquitin ligase that interacts with the NH2‐terminus portion of NKCC2 in native TALs.

Support or Funding Information

Henry Ford Health System, Institutional Support, Grant #A20042.
Salinomycin and its derivatives as potent RET transcriptional inhibitors for the treatment of medullary thyroid carcinoma (MTC).
Authors: Alqahtani Tariq M. , Sun Daekyu M.
Published: April 2020

Rearranged during transfection kinase (RET) is a validated molecular target in medullary thyroid cancer (MTC), as activating mutations in RET are often associated with the development of MTC. The present study reports the first preclinical characterization of salinomycin and selected analogs as potent RET transcriptional inhibitors. Reverse transcription‐PCR and immunoblotting revealed that salinomycin profoundly decreased RET expression in the TT human MTC cell line by inhibiting RET transcription. Moreover, salinomycin resulted in remarkable anti‐proliferative activity against MTC that is driven by RET (gain of function mutation) by selectively inhibiting the intracellular PI3K/Akt/mTOR signaling pathway. Also, flow cytometry and fluorescence‐activated cell sorting showed that salinomycin induces G1 phase arrest and apoptosis by reducing the expression of retinoblastoma protein, E2F1, cyclin D and CDK4. The structure‐activity relationship of salinomycin was investigated in this study. Some of the salinomycin derivatives showed the ability to reduce RET expression where others fail to alter RET expression. These results suggest that the RET‐suppressing effect of salinomycin may be largely attributed to disruption of the Wnt pathway, presumably through interference with the ternary LRP6‐Frizzled‐Wnt complex. Furthermore, these findings support the further preclinical evaluation of salinomycin and its analogs as a promising new class of therapeutic agents for the improved treatment of MTC

Support or Funding Information

American Cancer Society for their support of this study (grant no. RSGM‐12‐046‐01‐CDD).
3D Virtual Models to Visualize Pelvic Floor & Perineum in Medical Gross Anatomy
Authors: Ismat Karim Hussain , Afifi Linda , Zumwalt Ann
Published: April 2020

Introduction

The pelvic floor and perineum are difficult regions to visualize with most educational tools. Cadaveric dissection of this region is cumbersome due to limited accessibility and ambiguity of structures, and presents challenges with teaching spatial orientation. Supplemental learning material such as two‐dimensional anatomical illustrations often lack the ability to demonstrate spatial depth and complex relationships of the pelvis and perineum. We developed 3D virtual models to teach this spatially complex region in a didactic session on pelvic and perineal anatomy.

Aim

This study explored the efficacy of 3D virtual models as a supplementary teaching tool for pelvic floor and perineum.

Methods

Two review sessions were provided to a total of 37 first year medical students. The content of the lesson was initially presented in a traditional lecture format alongside 2D illustrations and schematics. Immediately following the lecture portion, 3D models of each region of interest were presented to students using 3D4Medical Complete Anatomy application.

Prior to the session, students (n=31) completed a modified Likert scale (1–5) survey on their familiarity with pelvic musculature, perineal fasciae and spaces, and the course of the pudendal nerve. After the session, students (n=37) completed a similar survey reviewing their confidence in pelvic musculature, perineal fasciae and spaces, and the course of the pudendal nerve. Relative frequencies for each question have been reported as a percentage, with a score of 3 considered neutral.

Results

Of the students who responded to the pre‐session survey (n=31), 6.5% reported being familiar (a score of 4 or 5) with the muscles of the pelvic floor, 9.7% reported being familiar with the perineal fasciae and spaces, and 12.9% reported being familiar with the course of the pudendal nerve.

Responses to the post‐session survey (n=37), revealed that 91.9% of students reported improved confidence (a score of 4 or 5) in their understanding of pelvic floor musculature and perineal fasciae and spaces. While, 83.8% reported improved confidence in their understanding of the pudendal nerve course.

Conclusion

The use of 3D models, as a supplement to existing course content, is an effective method for improving student confidence in pelvic floor and perineum anatomy. Our results suggest that the combination of 3D virtual models and didactic knowledge transmission, facilitates the learning of pelvic floor and perineal regions.
Exploring Protein‐Protein Interactions for MS2‐L Lysis Activity
Authors: Bart Olivia M. , Piefer Andrew J.
Published: April 2020

Bacteriophage like MS2 are viruses that infect and replicate in host cell bacteria. MS2 is a small single‐stranded RNA bacteriophage that infects Escherichia coli. MS2 has a lysis protein L that hypothetically requires a host protein for cell lysis. Expression of L alone in E. coli is sufficient for cell lysis, although the mechanism remains unknown. Interestingly, our data suggests that N‐terminally tagged L is functional for lysis, although with slower kinetics than an un‐tagged version. As a control experiment for exploring protein‐protein interactions, we are overexpressing the chaperone protein DnaJ to confirm its known interaction with L.
1 To accomplish this we are using the bi‐cistronic expression vector pET‐Duet‐1 to co‐express DnaJ and L. pET‐Duet provides epitope tags to permit confirmation of expression and interaction via immunoblotting and pull‐down experiments, respectively. It is known that DnaJ and L interact, however, L does not require DnaJ for lysis.
1 Using Photo‐leucine‐crosslinking, a process that incorporates a leucine analog reactive to UV light into expressed proteins, we hope to covalently capture other E. coli proteins that are necessary for lysis. Overexpressed DnaJ will serve as a positive control to confirm covalent cross‐linking with a known interaction partner.
Procalcitonin and Bacterial Infections in the Human Body
Authors: Link Tina , Jacobson Reagan , Escorcia Isabella , Fisher Joseph , Nguyen Lan , Reddy Megan
Published: April 2020

Procalcitonin (PCT), a member of the calcitonin (CT) superfamily, is a 116 amino acid, 13 kilodalton precursor protein produced in humans in the parafollicular cells of the thyroid as well as the neuroendocrine cells of the lungs and intestines of healthy individuals. PCT is coded for in the Calc‐I gene, located on chromosome 11. PCT is the precursor to the regulatory protein calcitonin but may have its own, separate role in the human body’s response to bacterial infection. There is a positive correlation between the concentration of PCT in the blood and the severity of a bacterial infection, making PCT an acute phase reactant. Under normal conditions, PCT undergoes proteolytic cleavage in the thyroid and is converted into CT, which then leaves the thyroid and enters the bloodstream; however, under inflammatory conditions, PCT is not cleaved and enters the bloodstream in a three‐section state comprising an amino terminus, immature calcitonin, and calcitonin carboxyl‐terminus peptide. Additionally, under inflammatory conditions, PCT is produced in other locations besides the parafollicular cells of the thyroid and the neuroendocrine cells of the lungs and intestines due to an increase in the expression of the Calc‐I gene. The reason for this increase may be related to potential anti‐inflammatory characteristics of PCT, including an ability to signal a reduction in the production of pro‐inflammatory proteins such as tumor necrosis factor‐alpha and interleukin 1‐beta. Furthermore, PCT in a specific concentration has been shown to reduce the reactivity of lipopolysaccharide (LPS) in gram‐negative bacteria. These properties of PCT have many potential applications in the medical field that are currently being studied or used. For example, detection of PCT in the blood can be used to determine if an infection is viral or bacterial or to evaluate the severity of infection in patients with sepsis. The determination of the pathogen allows for the correct course of appropriate treatment, potentially preventing the overprescription and overuse of antibiotics, a critical concern with the continued rise of bacterial antibiotic resistance. Additionally, as patients recover from a systemic bacterial infection, monitoring the level of PCT allows physicians to determine when the course of antibiotics may be stopped, once again minimizing antibiotic overuse. The Walton High School SMART Team has designed a 3D model of procalcitonin to investigate the relationship between procalcitonin’s structure and its function.

Support or Funding Information

MSOE Center for Biomolecular Modeling
Activating Resident Neural Precursor Cells in the Spinal Cord to Promote Neural Repair
Authors: Gilbert Emily , Livingston Jessica , Khan Monoleena , Kandavel Harini , Kehtari Tarlan , Morshead Cindi
Published: April 2020

Within the mammalian spinal cord there are two distinct populations of neural precursor cells: neural stem and progenitor cells (NSPCs) and oligodendrocyte progenitor cells (OPCs). NSPCs are located within the periventricular zone of the spinal cord and are relatively quiescent and non‐neurogenic under homeostatic conditions. OPCs are located throughout the grey and white matter of the spinal cord and give rise to new oligodendrocytes throughout life. Both of these populations are activated following injury, however, their response is not sufficient for functional recovery. We propose that enhancing resident precursor activation is a promising approach to improve structural and functional outcomes following SCI. The FDA‐approved drug metformin (MET) has demonstrated efficacy in promoting improved outcomes in the injured brain through pleiotropic effects including: decreased inflammation, enhanced precursor cell activation and increased neurogenesis and oligogenesis. Here, we seek to establish the influence of MET treatment on NSPC and OPC populations and explore whether MET can improve functional recovery following injury in the spinal cord. Our cellular data reveal that in uninjured animals, MET treatment significantly expands the NSPC pool in females, but not males; increases neurogenesis in males only and enhances oligogenesis across both sexes. Conversely, we observed no effect of MET treatment on the number of OPCs in uninjured mice, irrespective of sex. To test the efficacy of MET treatment following injury, we used two distinct models: (1) a dorsal column injury in the thoracic spinal cord (SCI), and (2), a model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE). In both models, MET was delivered to mice immediately following injury and for 14 days. Following SCI, mice were tested on a skilled walking test on post‐injury days (PID) 7 and 14. While all mice showed a significant deficit on PID7, by PID14, MET treated animals were not significantly impaired relative to pre‐injury performance and naive controls. In the EAE model, we evaluated the efficacy of MET treatment through both clinical scoring (based on degree of paralysis) and gait analysis. EAE resulted in significant clinical deficits 14 days following induction, which were mitigated in mice that received MET. Additionally, our gait analysis revealed improved metrics in MET treated mice. Across both injury models we observed significantly reduced inflammation. Taken together, our results support MET as a viable therapeutic strategy to expand and enhance NSPCs, improve the response of OPCs and support functional recovery following spinal cord injury.

Support or Funding Information

Canadian Institute of Health Research

Medicine by Design
Protein Binding to Lipid Droplets with Altered Ratios of Surface Phospholipids
Authors: Paetz Olivia , Renier Timothy , Vuong Sunny , Listenberger Laura
Published: April 2020

Alcoholic Fatty Liver Disease (AFLD) is characterized by an increase in lipid droplets in the liver after acute ingestion of alcohol. Previous experiments with a rat model of AFLD show hepatic lipid droplets contain altered levels of the predominant classes of phospholipids. Specifically, hepatic lipid droplets from AFLD rats were found with lower amounts of phosphatidylcholine (PC) relative to phosphatidylethanolamine (PE). Our experiments aim to determine if the altered phospholipid composition of AFLD lipid droplets causes differences in the numbers or types of lipid droplet‐associated proteins. We selected proteins based on their membrane binding motifs and roles in lipid droplet homeostasis. AML12 cells cultured in choline‐deficient media simulate the altered phospholipid composition of AFLD lipid droplets. We isolated lipid droplets from AML12 cells cultured with and without choline and examined proteins of interest by SDS‐PAGE and western blotting. We found equivalent levels of rab18 and lanosterol synthase in lipid droplet fractions from cells grown with and without choline. Alternatively, perilipin 1, perilipin 2, and CIDE‐C are predominantly found in lipid droplet fractions from cells grown without choline (with lower ratios of PC:PE). The proteins that are most influenced by lipid droplet surface composition are all predicted to associate with lipid droplets through amphipathic alpha helices. By evaluating how altered phospholipid monolayer composition affects the binding of lipid droplet proteins, we seek to further elucidate the biochemical pathways that contribute to AFLD.

Support or Funding Information

This research was supported by St. Olaf College and the TRIO McNair Scholars Program.
Perfectionistic Cognitions and Interleukin‐6: The Moderating Role of Child Maltreatment
Authors: Molnar Danielle S. , MacNeil Adam J. , Moore Jessy , Maguire Aindriu R. R. , O’Leary Deborah D. , Wade Terrance J.
Published: April 2020

Research is beginning to establish that perfectionism (i.e., the need to be absolutely perfect accompanied by harsh self‐criticism) contributes to poorer health and early mortality. Although few studies have addressed any personality/immune system links, there is growing evidence that personality can influence immune system functioning by altering cytokine production. Yet, to our knowledge, no study has examined the effect of perfectionism on immunological functioning. According to the Perfectionism Diathesis‐Stress Model, the deleterious effects of perfectionism on health should be further exacerbated by adverse childhood experiences (ACEs) such as maltreatment. However, this contention has yet to be tested empirically. The aim of this multidisciplinary research was to test how perfectionism is related to inflammatory analytes and whether these associations were moderated by child maltreatment. This study included a sample of young adults from the Niagara Longitudinal Heart Study (NLHS). Blood samples were collected, and serum was used to analyze levels of various immune biomarkers including Interleukin‐6 (IL‐6). Participants completed a self‐reported questionnaire that included the Childhood Trust Events Survey to identify exposure to sexual, physical, and emotional abuse before 18 years of age. Positive responses were then summed to create a count from 0 to 3 types of exposure. The Perfectionistic Cognitions Inventory assessed perfectionism. Results from multiple regression using Andrew Haye’s Process in which log‐transformed IL‐6 was regressed on perfectionistic cognitions, child maltreatment, and the interaction between perfectionistic cognitions and child maltreatment indicated that the relationship between perfectionistic cognitions and IL‐6 was moderated by child maltreatment. Specifically, higher perfectionistic cognitions were related to higher levels of IL‐6 under conditions of higher maltreatment. In sum, findings indicate that perfectionistic cognitions are related to immune system functioning and specifically to higher levels of IL‐6 when participants have experienced high levels of child maltreatment. These results support the Perfectionism Diathesis‐Stress Model and may provide key insights into both early prevention and intervention efforts aimed at reducing the toll of perfectionism on health.

Support or Funding Information

The NLHS is funded by the Canadian Institutes of Health Research (CIHR #s 363774, 399332). DSM is supported by an Early Researcher Award (Ministry of Research and Innovation, Government of Ontario). ARRM is supported by the Ontario Graduate Scholarship (OGS) Program.
Met‐β‐catenin induced hepatocellular cancer requires Ras but not Akt or Stat3 signaling
Authors: Tao Junyan , Liu Silvia , Bell Aaron , Chen Xin , Monga Satdarshan P.
Published: April 2020

We have previously shown that co‐expression of hMet and mutant‐β‐catenin (S33Y or S45Y) using sleeping beauty transposon/transposase via hydrodynamic tail vein injection (SB‐HTVI) leads to HCC in mice referred to as Met‐b‐catenin model, which represented 11% of human HCC. Since Met signaling can activate downstream signaling including Ras, AKT/PI3K and STAT3, we next wanted to address if all these downstream components are equally or differentially required to lead to Met‐b‐catenin HCC. Recently, we replaced Met with G12D‐Kras to activate Ras/MAPK/Erk signaling and co‐expressed with mutant b‐catenin, which showed >90% gene expression similarity to Met‐b‐catenin HCC. Here, in leiu of Met, we co‐delivered mutant‐β‐catenin (S45Y/S33Y) with either myr‐AKT or H1047R‐PIK3CA or STAT3 via SB‐HTVI. Tumorigenesis was monitored at different stages after injection and molecular analysis was performed. Co‐injection of H1047R‐PIK3CA and S45Y/S33Y‐β‐catenin or myr‐ AKT and S45Y‐β‐catenin showed a notably slower tumor development as compared to Met‐b‐catenin or Kras‐b‐catenin model, taking >15 weeks post injection to develop appreciable tumor burden seen in the other 2 models at around 8 weeks. In fact, it took about 16 weeks and 22 weeks to induce comparable tumors for AKT‐S33Y‐β‐catenin group, and H1047R‐PIK3CA‐S33Y‐β‐catenin group, respectively. Co‐expression of active AKT/PI3K with mutant‐b‐catenin led to HCC displaying lipid accumulation in tumor cells. Co‐delivery of STAT3 and mutant‐catenin induced none or few small tumor nodules even around 34‐weeks post injection. There was evidence of increased p‐ERK, p‐AKT, and mTORC1/mTORC2 and downstream signaling including p‐EIF4E, p‐4E‐BP1 and p‐S6 Ribosomal protein as well as β‐catenin targets GS and CCND1 in AKT/PI3K‐b‐catenin. In conclusion, Ras signaling appears to be the most important downstream effector of Met signaling which cooperates with mutant β‐catenin for HCC development, while AKT/PI3K are of lesser relevance, and STAT3 of least relevance. These findings will have implications in biology as well as therapeutics for these subsets of human HCC as use of inhibitors of appropriate downstream effectors of Met may be more efficacious and with lesser off‐target effects than Met inhibition by itself in the Met‐b‐catenin HCC.

Support or Funding Information

This work was supported by NIH grants R01CA204586 to S.P.M
Role of the Candida albicans glycerophosphocholine acyltransferase, Gpc1, in phosphatidylcholine biosynthesis and cell physiology
Authors: King Will Ray , Patton-Vogt Jana
Published: April 2020

Phosphatidylcholine (PC) is an abundant phospholipid in eukaryotic membranes. Our understanding of PC biosynthesis and acyl chain remodeling in Saccharomyces cerevisiae has been expanded with the identification of a glycerophosphosphocholine acyltransferase (GPCAT), Gpc1. Gpc1 acylates glycerophosphocholine (GPC), a product of complete deacylation of PC, to form lysophosphotidylcholine (LysoPC). LysoPC can subsequently be converted to PC by Ale1. This PC deacylation reacylation pathway (PC‐DRP) defines a novel route for PC biosynthesis and provides a potential mechanism to alter the acyl chain composition of a PC molecule. Changes in acyl chain composition can, in turn, affect physical properties of a membrane, such as fluidity, width, thickness and shape. Gpc1 displays no homology to known acyltransferases and defines a new gene family with members in plants, animals and fungi. The pathogenic fungus Candida albicans displays robust transport of GPC into the cell. We have previously shown that the inability of C. albicans to transport GPC results in decreased virulence in a mouse model of disseminated candidiasis, implying this metabolite plays a role during pathogenicity. We have created a C. albicans diploid knockout of Gpc1 homolog (orf19.998) using a transient CRISPR Cas‐9 system. Preliminary lipid analyses indicate that Gpc1 impacts PC biosynthesis in vivo. The physiological roles of CaGpc1 are being examined through growth and expression studies. Conditions to be examined include those associated with the human host and virulence, including elevated temperature, serum exposure, and hyphae inducing conditions.

Support or Funding Information

NIH R15 GM104876
Inhibition of Oxidative DNA‐protein Crosslinking Via an Aqueous Extract of Kale
Authors: Gallano Alyssa Daniele , Nguyen Tanya , Lezo Daisy , Ramirez Ashley , Stemp Eric
Published: April 2020

Oxidative damage is involved in the formation of free radicals, and if deregulated, can ultimately facilitate a variety of diseases ranging from Alzheimer’s disease to cancer. DNA‐protein crosslinking is a recognized form of oxidative stress that involves proteins interacting with radical‐induced lesions in the DNA molecule. In DNA, this damage is observed primarily at guanine (G) since it is the most easily oxidized base. Studies on oxidative stress show that substances – the most familiar being kale and spinach – high on antioxidants aid in protection from the excessive oxidizing effects of free radical species. The exact mechanism of inhibition has been hypothesized as a redox mechanism between the antioxidant and the radical. Therefore, the primary counteraction used to observe this mechanism and to reverse oxidative DNA‐protein crosslinking in this experiment was
kale.

The flash‐quench technique is a method used for guanine oxidation and can induce DNA‐protein crosslinking. The intercalator, Ru(phen)2dppz2+[phen = phenanthroline, dppz = dipyridophenazine], is excited with a laser and gives an electron to the quencher, Co(NH3)5Cl2+. The intercalator then takes an electron from guanine, creating the guanine radical, which then reacts with protein. Samples containing Ru(phen)2dppz2+, Co(NH3)5Cl2+, histone protein, PUC19 DNA and kale were irradiated with blue laser light for 0 to 140 seconds to induce guanine damage. The extent of crosslinking was determined by the gel shift assay where SDS was added to the samples to disrupt the noncovalent interactions between the DNA and protein. Furthermore, the samples were compared with the control which substituted water for the kale extract. The inhibition of oxidative DNA damage by kale was studied further by focusing on kaempferol, a component of kale. Likewise, the gel shift assay was performed on samples containing water and two different concentrations – 50 μM and 200 μM – of the antioxidant. The interaction between kaempferol and the guanine radical was then displayed through transient absorption spectroscopy. Since kaempferol holds known antioxidative properties, the inhibition of DNA oxidation at the guanine site was also expected. Indeed, upon comparison, minimal DNA protein crosslinking was observed when kale or kaempferol were present in the samples. Specifically, as the irradiation time increased, the absorption of free DNA was low, assuming that a redox mechanism between the antioxidant and the guanine radical occurred.

Future directions include determining the lowest concentration at which kaempferol is active, assessing the effects of additional antioxidants from kale, and examining a pure sample of kaempferol via high performance liquid chromatography (HPLC).

Support or Funding Information

Supported and Funded by:Loring‐Denault Endowed Chair in Chemistry, Dr. Eleanor Siebert, and Professor Emeritus
HPV: The Power of Vaccination
Authors: Gite Jasmine , Ramanathan Siddharth , Hussein Inaya Hajj
Published: April 2020

INTRODUCTION

Genital human papillomavirus (HPV) is a sexually transmitted infection (STI) that can lead to oropharyngeal, anal, cervical, vaginal, and penile cancers. It is the initiating event for malignant transformation and cervical dysplasia classified by the thickness of squamous intraepithelial lesions. Histologic evidence shows disorganized architecture and koilocytic changes in the epithelium, with a predilection for the squamocolumnar junction or transformation zone. Compared to heterosexual population, LGBTQIA+ subgroups may be at higher risk to HPV infections. This study aims to determine how knowledgeable Oakland University (OU) students are about HPV infection and vaccination, their perception and attitude towards it, and willingness to receive the vaccine, with a focus on gender and sexual orientation.

METHODS

A cross‐sectional survey collecting data on various parameters with measurable indicators such as: general non‐identifying information, knowledge of HPV vaccination, attitude towards HPV vaccination, and intent for receiving HPV vaccine was conducted. An information section regarding HPV and its vaccine was provided to assess change in knowledge of and attitude towards vaccination. Comparisons were analyzed with ANOVA and Chi‐Square tests.

RESULTS

On average, heterosexual respondents agreed less than LGBTQIA+ respondents that the current vaccine is capable of preventing cervical, penile, or anal cancer (P = 0.0091). Vaccinated respondents agreed more than unvaccinated respondents that all gynecologists and personal physicians should recommend the vaccine to patients whether or not they come from conservative families (P = 0.0002), and that they would recommend this vaccine for their college friends (P = < 0.0001). Vaccinated respondents were significantly younger than unvaccinated respondents (P = 0.0005), but respondents who have heard of the HPV vaccine were significantly older than respondents who have not heard of the HPV vaccine (P = 0.0188). Adjusting for all factors, smokers have a significantly lower knowledge score than non‐smokers (P = 0.0290). A lower proportion of alcohol drinking respondents received the HPV vaccine as compared to non‐alcohol drinking respondents (P = 0.0358). On average, respondents who were unvaccinated were significantly more likely to undergo the HPV vaccine after taking the survey as compared to before taking the survey (P = 0.0016).

CONCLUSION

Despite increasing social acceptance of the LGBTQIA+ community, few studies have addressed HPV infection and vaccination awareness among sexual minorities. Our findings show discrepancies in vaccine knowledge and vaccination status among different at‐risk groups. The findings also show increased likelihood of undergoing vaccination after reading an informational section within the survey, indicating the significant impact of and need to have widespread educational campaigns to influence behavioral perceptions and increase vaccine uptake.
CDK1‐dependent Phosphorylation of the Tumor Suppressor Phosphatase, PHLPP1, Regulates the Mitotic PHLPP1 Interactome
Authors: Kawashima Agnieszka Grzechnik , Wong Cassandra , King Charles C. , Gingras Anne-Claude , Newton Alexandra
Published: April 2020

PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor originally discovered for its ability to directly dephosphorylate and inactivate the pro‐survival kinase Akt, a key transducer of growth factor signaling. A number of other PHLPP1 targets have been identified, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP1 itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis in a CDK1‐dependent manner, and that this hyperphosphorylation regulates its interaction with mitotic proteins. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost with lambda phosphatase treatment, and is prevented by CDK1 inhibition. This mobility shift can be recreated in vitro using recombinant CDK1‐Cyclin B. Mass spectrometry and biochemical analysis reveals that these phosphorylations modify the N‐terminus of PHLPP1, a functionally uncharacterized region. A proximity dependent biotin identification (BioID) interaction screen revealed that mitotic PHLPP1 interacts with components of the mitotic spindle apparatus and the kinetochore. Additionally, the data suggest that the N‐terminus is required for the dissociation of PHLPP1 from interphase scaffolds, such as Scribble, during mitosis. During mitotic exit, PHLPP1 protein levels decrease, suggesting that PHLPP1 is degraded during mitotic exit. This correlates with an increase in Akt Ser473 phosphorylation, a validated cellular target of PHLPP1. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows proper passage through mitosis. Reversible protein phosphorylation, orchestrated by kinases and phosphatases, plays a role in controlling proper progression through mitosis, which is essential as errors in mitosis can result in aneuploidy, a hallmark of cancer. The finding that PHLPP1 binds mitotic proteins in a cell cycle and phosphorylation‐dependent manner may have relevance to its tumor suppressive function.

Support or Funding Information

This work was supported by NIH R35 GM122523, T32 GM007752, & The Frontiers of Innovation Scholars Program.