bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Parkinson’s Disease Model: PINK1/PARKIN Double‐Knockout Rats
Authors: Almikhlafi Mohannad A. , Lamberty Benjamin G. , Totusek Steven A. , Fox Howard S. , Stauch Kelly L.
Published: April 2020

Introduction

Animals are important tools to study pathological changes, behavioral abnormalities, and therapeutics in Parkinson’s disease (PD), which is characterized by loss of dopaminergic neurons in the substantia nigra (SN). Loss‐of‐function mutations in PTEN‐induced putative kinase 1 (PINK1) and Parkin, mitophagy pathway genes are linked to autosomal recessive PD in humans; however, deficiency does not lead to PD symptoms or affect dopaminergic neurons in the SN in mice. In rats, loss of PINK1, but not Parkin, leads to progressive neurodegeneration in the SN and motor symptoms in a subset of animals. To try to obtain a more robust model of PD, we generated a PINK1/Parkin double‐knockout (PP‐DKO) rat model.

Hypothesis

We hypothesized that PP‐DKO have mitochondrial dysfunction leading to a loss in dopaminergic neurons in the SN causing motor deficits.

Methods

Using PP‐DKO rat model, mitochondrial bioenergetics were measured by seahorse‐XF96, neuronal quantification by immunohistochemistry and stereology, and behavioral assessment examining motor coordination, bradykinesia/agility, hindlimb strength, and gait.

Results

Striatal mitochondrial bioenergetics displayed dysfunction at 3‐month old rats. Maximal respiration and spare respiratory capacity were decreased. PP‐DKO at 6 and 8 months showed a 23% and 45% reduction in dopaminergic neurons in the SN; respectively. Also, a 30% reduction in the number of neurons was found in the ventral tegmental area at the 8 months. Motor coordination was assessed by rotarod, which showed a reduction in the latency to fall at 6‐ and 9‐months old rats. Bradykinesia and agility were tested by pole test that showed an increase in the time the rat required to turn and to walk down the pole at 6‐ and 9‐months old rats. Cylinder test showed a significant reduction in hindlimb strength when rearing frequency was measured at 6, and 8 months of age. Finally, gait abnormalities were also present at 8 months including reduction in hindlimb step length, reduction in fore‐ and hindlimb step pressure alongside with an increase in step angle.

Conclusion

PP‐DKO is a novel genetic model with reproducible neuropathology and motor abnormalities of PD that will help researchers to study the mechanisms by which recessive genes produce PD as well as testing preventative and potential therapeutic paradigms.

Support or Funding Information

This research was funded by Michael J. Fox Foundation for Parkinson’s research grant #15288
The Plasma Membrane Translocation of HspA1A, a Stress Inducible 70‐kDa Heat Shock Protein, Depends on its Interaction with Membrane Lipids
Authors: Smulders Larissa , Nikolaidis Nikolas
Published: April 2020

HspA1A is a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70). This protein is a molecular chaperone that plays important roles in cell survival. Normally, HSPA1A functions within the cytoplasm. Yet, in stressed and cancer cells HspA1A also localizes at the plasma membrane (PM). In cancer cells, in particular, the presence of HspA1A at the cell surface allows the tumor to increase invasiveness and develop resistance to radiation therapy. Therefore, blocking this protein from being at the surface of tumors is a promising therapeutic. However, the translocation pathway of HspA1A to the PM remains puzzling, because this protein lacks membrane localization signals. Based on the fact that HspA1A interacts with lipids, including phosphatidylserine (PS) and several mono‐phosphorylated phosphoinositides (PIPs), we hypothesized that the interaction of HspA1A with these lipids allows the chaperone to localize at the PM. To test this hypothesis, we subjected different cell lines to mild heat‐shock and the PM‐localized HspA1A was quantified using confocal microscopy and cell surface biotinylation. These experiments revealed that HspA1A’s membrane localization and embedding increased during recovery from non‐apoptotic heat‐shock. Next, we selectively reduced PS, PI(4)P, and PI(3)P targets by co‐transfecting HspA1A with known PS‐, PI(4)P‐, and PI(3)P‐ biosensors, and determined that HspA1A’s membrane localization was greatly reduced in all three cases. In contrast, the reduction of PI(4,5)P2 availability by overexpression of the PLCδ‐PH biosensor had minimal effects on HspA1A’s PM‐localization. Additionally, we manipulated the cellular lipid content using ionomycin, a calcium ionophore that activates PLC, and phenylarsine oxide (PAO), a PI4‐Kinase inhibitor, using several lipid‐biosensors as positive and negative controls. These experiments revealed that the PM localization of HspA1A was unaffected by ionomycin, but was greatly reduced in the presence of PAO, corroborating the findings obtained by the lipid‐biosensors on the importance of specific lipids in the PM translocation of HspA1A. Collectively, these findings strongly support the notion that HspA1A’s PM localization and anchorage is a complex lipid‐driven phenomenon controlled by the binding of the chaperone to endosomal and plasma membrane PS, PI(4)P, and PI(3)P. Our findings provide the basis for interventions that block HSPA1A’s translocation to the tumor cell’s PM allowing the cell to remain sensitive to radiation therapy and decrease its invasiveness.

Support or Funding Information

This project was supported by funds from NIH
Sex Differences in Response to Acute Sympathetic Activation During Steady State Hypoxia
Authors: Jacob Dain W. , Harper Jennifer L. , Ott Elizabeth P. , Ivie Clayton L. , Limberg Jacqueline K.
Published: April 2020

Background

Activation of the sympathetic nervous system causes vasoconstriction and a reduction in peripheral blood flow. This sympathetically‐mediated vasoconstriction may be attenuated during hypoxia to maintain oxygen delivery – termed sympatholysis. However, hypoxic sympatholysis was previously shown to be absent in humans. A limitation of prior work was the failure to directly examine this response in women. Given young women exhibit an increase in blood flow during hypoxia, we hypothesized hypoxic sympatholysis would be present in young women but absent in young men.

Methods

Blood pressure (BP, finger photoplethysmography) and forearm blood flow (FBF, venous occlusion plethysmography) were assessed in healthy young men (n=9, 26±1 yrs) and women (n=10, 24±1 yrs). Subjects completed two trials consisting of a 2‐min cold pressor test (CPT) during baseline normoxia (0.21 FiO2, 98±0% SpO2) and following 3 min of steady‐state hypoxia (0.11±0.01 FiO2, 82±1% SpO2). FBF was normalized for BP (forearm vascular conductance, FVC). A change in FVC from steady‐state during the last 1‐min of CPT was calculated (ΔFVC). The difference in ΔFVC between normoxia and hypoxia trials was used as an index of hypoxic sympatholysis.

Results

In men, ΔFVC did not differ between normoxia and hypoxia (ΔFVC Normoxia: −0.30±0.45, Hypoxia: −0.77±0.41 mL/dL/100mmHg/min; p=0.46). In contrast, in women ΔFVC during normoxia (−0.70±0.27 mL/dL/100mmHg/min) was attenuated during hypoxia (+0.48±0.46 mL/dL/100mmHg/min; p=0.04) – indicating sympatholysis. The magnitude of hypoxic sympatholysis was greater in women than in men (Men: −0.46±0.38, Women: +1.18±0.39 mL/dL/100mmHg/min; p<0.01).

Conclusion

Young women attenuate sympathetically‐mediated vasoconstriction during steadystate hypoxia, whereas men do not. In other words, hypoxic sympatholysis is present in young women, but absent in young men. These data advance our understanding of sex‐related differences in hypoxic vascular control.

Support or Funding Information

Funding

NIH HL130339
The Effect of Preterm Birth and Recurrent Laryngeal Nerve Lesion on Palatopharyngeal Arch Function During Feeding
Authors: Edmonds Chloe Elise , Catchpole Emily A. , Mayerl Christopher J. , German Rebecca Z.
Published: April 2020

Feeding in infant mammals requires the precise coordination of multiple oropharyngeal structures, including the palatopharyngeal arch. During a typical swallow, the activity of the palatopharyngeus muscle produces constriction of the arches and lowers the soft palate, which may be responsible for flipping of the epiglottis to cover the airway. Most research on the role of the palatopharyngeal arches in swallowing has been limited to pharyngeal manometry, which provides insight into the relative pressures in the oropharynx during swallowing. Studies using manometry in preterm infants, or in those with iatrogenic sequelae such as recurrent laryngeal nerve (RLN) damage, suggest differences in function, but not on how the structures in the oropharynx move to contribute to those changes in function. In this study, we assessed palatopharyngeal arch kinematics during feeding by using our validated infant pig model to determine the impact of birth status (preterm versus term), lesion status (lesioned RLN versus intact) and their interaction on feeding performance. We placed tantalum radio‐opaque beads into relevant anatomical structures of 16 infant pigs, including the palatopharyngeal arches, the tongue, the hard and soft palate, and the nose. We recorded feeding at both seven and 17 days postnatal via biplanar high‐speed videofluoroscopy (100fps) across a total of 584 swallows. Using XMALab, we tracked the three‐dimensional movement of the arches relative to a rigid body comprised of the markers in the hard palate and the nose. We then used custom MATLAB code to calculate the three‐dimensional excursion of the arches during a swallow, as well as the timing of constriction initiation, and the point of maximal constriction during a swallow. We found that preterm infants had more variable arch excursion than term infants, and RLN lesion increased excursion for both term and preterm infants although there were minimal differences in arch excursion within an age between term and preterm infants. Term infants also reached maximal arch constriction later than preterm infants, most likely due to their larger arch excursion and larger bolus volume. Lesion infants, both term and preterm, also had delayed maximal arch constriction during swallows, although day 17 term lesion infants reached maximal constriction earlier than term day 17 infants. Lesion resulted in delayed initiation of arch constriction, which did not change with age, although older term infants exhibited later arch constriction initiation than older preterm infants. Our results show that preterm birth and RLN lesion have distinct yet pervasive effects on infant feeding. Altered feeding performance in these neurologically compromised infants may be due in part to changes in the activity of the palatopharyngeal arches.

Support or Funding Information

NIH R01HD088561
Effect of Histamine‐Receptor Antagonism on the Acute Inflammatory Response to Aerobic Cycling Exercise
Authors: Mangum Joshua , Needham Karen , Sieck Dylan , Minson Christopher , Halliwill John
Published: April 2020

Blood flow to previously exercised muscle remains elevated for up to two hours following exercise, in response to activation of histamine receptors. Histamine also plays a significant role in the inflammatory response, increasing capillary permeability and allowing circulating cells to move in and out of tissues. Following a novel bout of exercise, there is typically an acute inflammatory response that resolves within 24–48 hours. Therefore, the purpose of this experiment was to determine if histamine receptors contribute to the acute inflammatory response following aerobic exercise in untrained individuals. We hypothesized that histamine‐receptor antagonism would decrease the inflammatory response to novel aerobic exercise. Six untrained volunteers (2F, 4M) completed 60 min of stationary cycling at 60% of their VO2peak. Volunteers completed the exercise twice, in random order, after placebo or blockade (combined oral H1/H2 histamine‐receptor antagonism with 540 mg fexofenadine and 300 mg ranitidine). Venous blood samples were collected prior to exercise and at 0, 6, 12, 24, and 48 hours after exercise. Flow cytometry (Beckman, Gallios) was utilized to quantify cell populations. A multiplex cytometric bead assay (LEGENDplex, Biolegend) was used for cytokine analysis and quantification. Following exercise, total circulating leukocytes increased from 1.70×106 ±0.29 to 3.08×106±0.58 in placebo (p<0.05) and 1.92×106±0.28 to 3.44×106±0.43% in blockade (p<0.01). Relative values of neutrophils increased from 33±1.5% to 46±1.6% in placebo (p=.13) and 36±5% to 56±2% in blockade (p<0.05). On average, total circulating leukocytes and neutrophils remained more elevated following exercise with blockade vs placebo (p<0.01). There was no effect of blockade on monocyte sub‐populations vs placebo. Following exercise, pro‐inflammatory cytokine MCP‐1 increased from 150±18pg/ml to 172±20pg/ml in placebo (p=0.18) and 151±20pg/ml to 192±13pg/ml in blockade (p<0.05). Similarly, pro‐inflammatory cytokine IL‐18 increased following exercise from 34±12pg/ml to 39±14pg/ml in placebo (p=0.21) and 35±20pg/ml to 58±13pg/ml in blockade (p<0.05). On average, MCP‐1 and IL‐18 remained more elevated following exercise with blockade vs placebo (p<0.05). Contrary to our hypothesis, the pattern of the acute inflammatory response to aerobic exercise appears to be augmented, not blunted, with histamine receptor antagonism. These data suggest that histamine receptor activation may play a complicated role in the acute inflammatory response to aerobic exercise.

Support or Funding Information

Support provided by NIH grant HL115027, AHA 17GRNT33660656, and The Eugene & Clarissa Evonuk Memorial Graduate Fellowship
Inhibiting Heptosyltransferase to Combat Gram‐Negative Bacterial Infections
Authors: Wang Alvin , Castro Colleen , Taylor Erika
Published: April 2020

Lipopolysaccharides (LPS) embedded in the outer membrane of Gram‐negative bacteria are known to enable antibiotic resistance by providing protection from hydrophobic antibiotics and assisting biofilm development. One of the key enzymes for synthesizing LPS is heptosyltransferase I (HepI), which catalyzes the reaction of ADP‐L‐glycero‐D‐manno‐heptose (ADP‐heptose) with Kdo2‐lipid A, a precursor of LPS. Previous studies have shown that HepI deficiency in bacteria results in increased susceptibility to antibiotics and host immune response. Here, we use molecular docking analysis to obtain binding affinity calculations of 419 natural product ligands provided by the National Cancer Institute (NCI) with HepI as the receptor. Docking calculations were performed with AutoDock Vina with 200 simulations per ligand. 118 ligands were found to have an average binding affinity calculation higher than that of ADP‐heptose. Selected ligands are being investigated for in vitro inhibition of HepI through kinetic analyses using a coupled assay system where ADP, a product of the heptose transfer reaction, is detected using NADH enzyme‐linked photometric assay. Potent HepI inhibitors will be further investigated as novel candidates for antimicrobial development.

Support or Funding Information

This project is supported by from Wesleyan University and the National Institutes of Health (1R15AI119907‐01).
SLC25A34 regulates bioenergetic metabolism in the murine liver
Authors: Roy Nairita , May Meredith , Delgado Evan Ryan , Alencastro Frances , Wilkinson Patrick David , Smyers Mei , Reynolds Michael John , Shiva Sruti , Duncan Andrew Wayne
Published: April 2020

Background and Rationale

Liver regeneration and diseases associated with altered mitochondrial metabolism, including NAFLD, are associated with alterations in ploidy. Many genes that regulate mitochondrial metabolism are also reported to alter hepatic polyploidy. SLC25A34 encodes an uncharacterized inner mitochondrial membrane protein, with a novel role in liver polyploidy. Interestingly, dysregulated Slc25a34 expression has been reported in numerous studies, which are all characterized by metabolic dysfunction, particularly altered glucose metabolism and fatty acid metabolism. We now hypothesize that SLC25A34 regulates various aspects of liver metabolism.

Results

In the current study, SLC25A34 was over expressed or knocked down in primary hepatocytes harvested from adult C57BL/6 mice and subsequent effects on different metabolic phenotypes were studied. Downregulation of SLC25A34 significantly increased the lipid droplets with a concurrent increase in expression of key players of fatty acid metabolism (Cpt1A, Srebf1, Acly). Maximal mitochondrial respiration and spare respiratory capacity were significantly elevated, possibly attributing to the increased levels of lipid droplets. Also, basal mitochondrial respiration and ATP‐linked mitochondrial respiration were significantly increased, accompanied by increased expression of regulators of mitochondrial biogenesis (PGC‐1 alpha, PGC‐1 beta, Prc and Tfam). In contrast, overexpression of SLC25A34 decreased expression of Cpt1A, Srebf1, Acly, but had no effect on lipid droplets. Unexpectedly, SLC25A34 overexpression also increased maximal mitochondrial respiration and spare respiratory capacity, as well as non‐mitochondrial respiration. Further, effect on energy intermediates (ADP, ATP and their ratio) and hepatocyte proliferation were studied. SLC25A34 depletion raised the ADP/ATP ratio and decreased hepatocyte proliferation, whereas overexpression of SLC25A34 decreased the ADP/ATP ratio and increased proliferation. Furthermore, SLC25A34 manipulation altered fatty acid oxidation and glycolysis functionally in primary hepatocytes as determined using the seahorse extracellular flux analyzer. Preliminary in‐vivo studies utilizing both whole‐body and liver‐specific SLC25A34 knockout mice indicate similar bioenergetic changes upon SLC25A34 deletion.

Conclusion

The data suggest that SLC25A34 is a key regulator of bioenergetic metabolism where it is involved in metabolic homeostasis and that modulation of SLC25A34 creates distinct metabolic phenotypes in the primary hepatocytes.

Support or Funding Information

This study is supported by NIDDKR01DK103645 and UPP Academic Foundation awarded to Dr. Andrew Wayne Duncan.
Inactive 5‐HT7 Receptors Associate with Inactive and Active Gs Heterotrimers
Authors: Jang Wonjo , Lambert Nevin A.
Published: April 2020

G protein‐coupled receptors (GPCRs) interact cooperatively with agonists and heterotrimeric G proteins to form ternary complexes, leading to activation of G proteins. Following exchange of GDP for GTP the complex is thought to rapidly disassemble as nucleotide‐bound G proteins adopt conformations that have low affinity for GPCRs, and heterotrimers dissociate into Gα and Gβγ subunits. Unlike most GPCRs, serotonin 5‐HT7 receptors are known to preassemble with GDP‐bound Gs heterotrimers prior to agonist binding. Here we show that these receptors also interact with GTP‐bound Gs heterotrimers. We used bioluminescence resonance energy transfer (BRET) and luciferase complementation assays to assess the effects of ligands and nucleotides on 5‐HT7‐Gs coupling. We find that stimulation with agonist (5‐HT) in intact cells promotes net dissociation of 5‐HT7‐Gs complexes, whereas inverse agonists (e.g. methiothepin) produce net association. To determine how nucleotide binding by Gs affects interactions with receptors, we replaced endogenous nucleotides with the non‐hydrolyzable analogs GTPγS or GDPβS in permeabilized, apyrase‐treated HEK293 cells. Unexpectedly, ligand‐mediated effects on 5‐HT7‐Gs complexes were largely maintained in the various nucleotide conditions. Agonist‐induced 5‐HT7‐Gs dissociation still occurred when only GDPβS was present, indicating that dissociation does not require Gs activation. Similarly, inverse agonist‐induced 5‐HT7‐Gs association still occurred when only GTPγS was present, suggesting that activated Gs heterotrimers retain affinity for inactive 5‐HT7 receptors. Moreover, we found that inverse agonist‐bound 5‐HT7 sequestered free Gβγ in the presence of GTPγS, suggesting that inactive 5‐HT7 receptors stabilized active Gs heterotrimers. These results reveal unconventional coupling of inactive 5‐HT7 receptors to both inactive and active Gs heterotrimers. This represents a significant departure from the standard allosteric model of GPCR‐G protein coupling, wherein G proteins interact primarily with active GPCRs, and GTP‐bound Gα subunits dissociate from both receptors and Gβγ subunits.

Support or Funding Information

Supported by GM130142 (to N.A.L.)
Mutation of Eftud2 in Mouse Neural Crest Cells Models MFDM and Reveals Contribution of P53 in Abnormal Craniofacial Development
Authors: Beauchamp Marie-Claude , Djedid Anissa , Bareke Eric , Aber Rachel , Majewski Jacek , Majewska Loydie Jerome
Published: April 2020

Haploinsufficiency of EFTUD2 is associated with MFDM (mandibulofacial dysostosis with microcephaly), but the etiology of this syndrome remains unknown. Our goal is to determine the tissue and temporal requirement for Eftud2 during craniofacial development. We generated a mouse with a conditional mutation of exon 2 of Eftud2 (Eftud2 flox) using the CRISPR/Cas9 editing system. We used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Embryos carrying one mutated allele of Eftud2 were normal. However, when both alleles were mutated, all embryos displayed hypoplasia of the midbrain and pharyngeal arches at E9.5. By E11.5, most of these embryos showed an open neural tube and all live embryos exhibited exencephaly at E14.5. At this stage, only 3 out of 12 embryos were still alive. Cartilage preparations revealed an absence of cartilage in the head, reduced/or absence of Meckel’s cartilage, and abnormal inner ear development. Crosses with the Rosa26RlacZ mice reporter line revealed a reduced number of neural crest cells in the pharyngeal region at E9.5 and E10.5, but not at E9.0. We next performed RNA sequencing analysis from the head of Eftud2 neural crest cells mutant embryos at E9.0, before any phenotype was observed. The P53 pathway was the most significantly upregulated in the mutant embryos, compared to their wild‐type littermates. Overexpression of P53 target genes identified in the RNAseq (Ccng1, Phlad3 and Trp53inp1) was validated by qRT‐PCR. Additionally, 373 genes showed increased skipped exons in the mutant embryos. Among them, only 2 genes were also overexpressed, including Mdm2, a major regulator of P53. We found that mutant embryos had significantly increased levels of the Mdm2 transcript lacking exon 3, which was previously reported to produce a truncated MDM2 protein, allowing stabilization of P53. Finally, we evaluated the contribution of P53 in craniofacial malformation of Eftud2 mutants by treating pregnant females with daily i.p. injection of pifithrinα, a P53 inhibitor, from E6.5 to E8.5. 37% of pifithrinα‐treated mutant embryos had normal midbrain development at E9.5. Also, the area perimeter of the first pharyngeal arch of pifithrinα‐treated mutant embryos was significantly higher than the vehicle‐treated ones. Altogether, these data suggest that Eftud2 specifically regulates Mdm2 splicing and leads to neural crest cell death due to overactivation of the P53 pathway. We propose that cranial neural crest cell death is responsible for craniofacial malformations in MFDM patients.

Support or Funding Information

Canadian Institutes of Health Research
Techniques for Successfully Flipping a Gross Anatomy Classroom for Graduate and Undergraduate Students
Authors: Garner Sarah
Published: April 2020

Many institutions are moving away from lecture‐based courses and are moving towards more active learning techniques. Re‐designing a course that is entirely lecture‐based to be focused on active learning can be a daunting task. One way to accomplish this is flipping a classroom by using technology to provide information outside of class time, and thus having more class time for active learning activities. An anatomy course at Tulane University for graduate and undergraduate students was completely redesigned as a flipped classroom course. Students were provided with pre‐recorded lectures and corresponding lecture documents prior to class. Students were expected to complete a homework assignment associated with the pre‐recorded lecture video before class to reinforce the information. During class, students participated in a variety of active learning activities including both individual work and group work. Video analytics were examined to see how often the students viewed the pre‐recorded videos and how early the students watched the video prior to coming to class. This presentation will discuss the steps involved in re‐designing an anatomy course to be a flipped classroom, including the challenges, time involved, strengths, and outcomes. Instructions and demonstration for creating quality pre‐recorded videos will be presented.
CTRP3 Prevents Macrophage Activation
Authors: Musick Adam
Published: April 2020

Sepsis is a life‐threatening medical emergency that is caused by the body’s extreme response to an infection. The initial infection results in an overproduction of cytokines which recruits additional immune cells. Adipose tissue is an active endocrine organ which secrets several anti‐inflammatory mediators, collectively called adipokines which could contribute to the control of the body’s response to a septic event. Our previous work has identified that the circulating levels of the anti‐inflammatory adipokine called C1q TNF Related Protein 3 (CTRP3) is significantly reduced in response to sepsis. We have also demonstrated that CTRP3 binds directly to circulating macrophages. Therefore, the purpose of this project was to examine the potential anti‐inflammatory effects of CTRP3 on macrophages.

METHODS

Thioglycolate‐induced peritoneal macrophages were isolated from CTRP3 overexpressing transgenic (Tg) and wild‐type (WT) mice. The isolated macrophages were then treated with lipopolysaccharides (LPS). Morphological changes were analyzed via bright‐field microscopy and the transcription levels of the inflammatory cytokines tumor necrosis factor alpha (TNF) and interleukin‐6 (IL6) were measured by Real‐Time PCR.

RESULTS

Morphological qualitative analysis indicated that LPS reduced macrophage adhesion and the effect was more pronounced in the Tg macrophages. Gene expression analysis demonstrated that LPS stimulation significantly increased IL6 and TNF levels in all samples (~4000‐fold and 300‐fold respectively). However, LPS‐induced increase in IL6 expression was significantly attenuated in Tg macrophages (fold elevation compared to vehicle treated: WT 273±92; Tg 78±68). There was no difference in LPS‐induced TNF expression in Tg compared to the WT macrophages.

CONCLUSION

Combined these data indicated that CTRP3 attenuates the ability of LPS to activate the pro‐inflammatory functions of the macrophages. Future studies are needed to confirm these findings and to determine the unique mechanism for the CTRP3‐induced alterations to macrophages. Regardless, CTRP3 is a unique contributor to macrophage’s response to inflammatory signals.

Support or Funding Information

1R15DK114740
Characterization Of Clinically Identified Mutations In Peripheral Arm Subunits NDUFV1, And NDUFS1 Of Respiratory Complex I, Using an E. coli Model System
Authors: Alkhaldi Hind , Vik Steven
Published: April 2020

Complex I (NADH: ubiquinone oxidoreductase) is the largest enzyme of the mammalian mitochondrial and bacteria respiratory chain. During the oxidation of NADH, hydrogen ions are translocated across the mitochondrial membrane, leading to the synthesis of ATP, the chemical energy currency of cells. Complex I in E. coli contains 13 different subunits, encoded by the nuo operon, which form an L‐shaped structure: a peripheral arm, containing 6 subunits involved in electron transport, and a membrane arm containing 7 subunits involved in pumping hydrogen ions. Peripheral arm subunits E, F, and G, form an NADH binding domain, and B, CD, and I interact with membrane subunits. Mutations in Complex I genes in humans lead to loss of vision, brain or muscle dysfunction, or other disorders. Here, we have modeled clinical mutations that have been reported to cause Complex I dysfunction in humans, using our bacterial system. Mutations have been constructed in subunit F (NDUFV1), the Flavin‐binding subunit: R358A, R358H, T173A, T173P, R102A, R102L, E349A, E349K, and in subunit G (NDUFV1), an Fe‐S protein: I154A, I154T, R219A, R219W. Analysis of additional mutations is underway. Expression levels of the mutant proteins have been determined by western blotting, and the NADH oxidase activities of the mutants have been measured. In conclusion, we have identified several mutations that are highly deleterious to the function or assembly of the enzyme. These results can be rationalized by consideration of the 3D structure of the Complex I.
Women In STEM‐ Intrinsic and Extrinsic Factors That Impact Retention and Yield
Authors: Benore Marilee Ann
Published: April 2020

Statistics gleaned from international sources paint a dismal picture of the number of women choosing, then leaving, STEM. The lack of women in the STEM workforce has become of significant concern as studies document the inequity, negative economic impact and loss of research when a disturbingly large number of capable individuals are discouraged or lost. The situation is not unique to the US; the percentage of women in STEM varies globally by field, nation and culture.

Yet, in spite of the problems, many women persist and thrive. Understanding factors that impact persistence, resilience and self‐efficacy of successful women in STEM is necessary to determine how to encourage and retain women. Examining factors and experiences, especially those that are subtle or often unrealized, can be determined by utilizing techniques more commonly used by anthropologists and historians. Interviews and oral histories have emerged as excellent methods to identify personal experiences, as the narrative data can be more thoroughly analyzed, provides the opportunity to probe a topic deeply, and can be archived as videos for mentoring. By interviewing and surveying successful STEM women in Europe and the USA, specific factors were observed that may be valuable in retaining women and encouraging girls in STEM.

Support or Funding Information

University of Michigan
Pre‐hypertensive molecular alterations in the paraventricular nucleus of the hypothalamus during angiotensin‐II hypertension
Authors: Jeong Jin Kwon , Simonyan Hayk , Young Colin N.
Published: April 2020

The paraventricular nucleus of the hypothalamus (PVN) plays a critical role in the development of hypertension in response to elevated levels of angiotensin‐II (Ang‐II). Upstream circumventricular organs, regions lacking a blood‐brain‐barrier, sense circulating Ang‐II and in turn influence endocrine and autonomic control via efferent projections to the PVN. Despite progress in unraveling the function of the PVN in hypertension development, the underlying signaling mechanisms remain unclear. Notably, the majority of investigations have examined alterations in the PVN during established hypertension, which limits our understanding of temporal changes that may occur prior to elevations in arterial blood pressure. Based on this, we investigated key molecular targets in the PVN during the pre‐hypertensive phase of Ang‐II hypertension. C57Bl/6J male mice were implanted with subcutaneous osmotic minipumps for chronic delivery of low dose Ang‐II (600 ng/kg/min). Brains were harvested for immunohistochemistry analysis of target molecules (n=3–4/group) at baseline (day 0) and pre‐hypertensive (days 3, 5, 7) timepoints. Histological analysis of synaptophysin, a marker for synaptic terminal innervation, indicated a progressive increase in immunoreactivity that peaked by ~10% within the first week of Ang‐II infusion (day 7 fold change: 1.1± 0.03 density/area, p<0.05); indicative of increased synaptic input to the PVN prior to elevations in arterial blood pressure. Previous findings have demonstrated that circulating arginine vasopressin protein (AVP) is elevated during chronic Ang‐II hypertension. Consistent with this, AVP immunoreactivity in the PVN was elevated as early as day 3 of Ang‐II infusion (day 3 fold change: 1.7 ± 0.09 density/area, p<0.05), and this response was sustained for up to 7 days (day 5 fold change: 1.4 ± 0.02; day 7 fold change: 1.3 ± 0.03 density/area, both p<0.05). Interestingly, we also observed synaptic end AVP immunoreactivity in the circumventricular subfornical organ very early during Ang‐II infusion (e.g. day 3 fold change: 1.2 ± 0.02 density/area, p<0.05), suggesting a possible feedback loop between the PVN and subfornical organ in AVP regulation. Unlike AVP, PVN oxytocin immunoreactivity was not changed during Ang‐II hypertension (e.g. day 7 fold change: 1.01 ± 0.08 density/area, p>0.05). Collectively, these findings indicate early molecular changes within the PVN prior to the development of Ang‐II hypertension including: 1) Gradual synaptic innervation of the PVN during the first week of Ang‐II infusion; 2) Very early Ang‐II associated elevations in PVN AVP expression, but not oxytocin; and 3) Potential alterations in the innervation of hypothalamic AVP fibers to the circumventricular subfornical organ in response to Ang‐II.

Support or Funding Information

19CDA34630010 (to Jin Kwon Jeong) and 1R01HL141393 (to Colin N. Young)
Efficacy of Student‐Directed Learning in a Gross Anatomy Course for Undergraduate and Graduate Students
Authors: Garner Sarah
Published: April 2020

Introduction

With a decrease in the number of contact hours available to teach gross anatomy and no corresponding decrease in the amount of course material, instructors must come up with innovative ways to teach this information in less time. Contact hours involving active learning promote increased retention rates. Thus, many institutions are moving away from lecture‐based courses and towards active learning classroom sessions. Providing pre‐recorded lecture videos facilitates student‐directed learning outside of the classroom so that students can utilize class time and interaction with instructors in a productive, active manner. While the use of flipped classrooms has been widely studied in anatomy, students’ opinions of the efficacy in‐class activities has not been closely examined. The goal of this project is to determine if the implementation of pre‐recorded lecture videos increases conceptualization rather than rote memorization and which in‐class active learning techniques are well‐received by gross anatomy students.

Methods

Graduate and undergraduate students enrolled in a gross anatomy course will be provided with pre‐recorded lectures and corresponding lecture documents prior to entering the class. Once in class, students will participate in a variety of active in‐class activities including individual and group activities such as quizzes, clinically oriented questions and case studies, and osteology activities. Students will be invited to participate in surveys and interviews regarding course content, efficacy of pre‐recorded lecture videos and in‐class activities, methods of studying, and methods of assessment. Surveys and interviews will be utilized throughout the semester including the beginning of the semester, the end of each block of course material, and at the end of the course.

Results

It is anticipated that the students will feel uncomfortable with self‐directed learning at the beginning of the semester, due to unfamiliarity and a feeling of increased work. However, it is also anticipated that the subsequent surveys and interviews will show an increased appreciation for the pre‐recorded lecture videos and an increased focus on conceptualization, with a decreased focus on memorization.

Conclusion

While pre‐recorded lecture videos may be more time consuming for instructors to create and increase the amount of work outside of class expected from students, the videos will improve conceptual knowledge of material and the quality of in‐class contact hours. Students will find group work most effective and prefer group work and clinically oriented questions to individual quizzes. Furthermore, students will feel more prepared for examinations and enjoy classroom time more with the interactive in‐class activities as opposed to traditional lectures. Therefore, the utilization of pre‐recorded lecture videos as an adjunct tool in anatomy education should be considered.
Chronic Binge Alcohol‐induced Hyperalgesia in SIV‐infected Rhesus Macaques
Authors: Foret Brittany , Coleman Larry , McGarrah Heather , Simon Liz , Molina Patricia
Published: April 2020

Chronic pain affects more than half of people living with HIV (PLWH), and the current pain management strategies, such as opioids, capsaicin, and gabapentin, remain inefficient in PLWH. Moreover, the pathogenesis of HIV‐associated pain remains widely unknown, hindering implementation of evidence‐based treatments. Alcohol and pain have a bidirectional relationship, where people consume alcohol to relieve pain, and unhealthy alcohol uses leads to chronic pain. Approximately 30% of PLWH develop alcohol use disorder; therefore, it is important to understand the effects of alcohol on pain in this population. Using a relevant preclinical model of HIV‐infection, the aim of the study was to determine if chronic binge alcohol (CBA)‐administration causes hyperalgesia in SIV‐infected macaques. We hypothesized that CBA‐administration causes hyperalgesia in rhesus macaques prior to infection, and these findings will be exacerbated during peak viremia in SIV infection. Rhesus macaques, four to six years old were administered CBA or isovolumetric water (VEH) through an intragastric catheter for 30 minutes, five days a week, with blood alcohol concentrations reaching 50–60 mM. Three months after the start of CBA administration, macaques were inoculated with SIVMac251 and 2.5 months later initiated on antiretroviral therapy. To assess thermal hyperalgesia, tail withdrawal test was used. Conscious macaques were restrained in chairs and their tails are placed in water baths at 40, 50, and 55C, and the latency to remove their tail (in seconds) determined. The test was performed three months after the initiation of CBA administration (24 hours after a CBA administration session), and repeated 2.5 months after SIV‐infection. Molecular analysis for proteins involved in pain circuitry will be determined in the spinal cord and periaqueductal gray. Our results indicate that three months of CBA administration causes hyperalgesia in rhesus macaques. Whether SIV infection exacerbates hyperalgesia, and if the molecular measures correlates to behavioral measures warrants further investigation. These data provide mechanistic insight into how unhealthy alcohol use affects hyperalgesia in HIV infection, and will provide a scientific basis for pain management strategies in this population.

Support or Funding Information

NIH/NIAAA P60 AA009803 and 5T32AA007577‐19
Knockout of Per1 Exacerbates the Hypertensive Phenotype of the Dahl Salt Sensitive Rat
Authors: Spires Denisha R. , Zietara Adrian , Levchenko Vladislav , Gumz Michelle L. , Staruschenko Alexander
Published: April 2020

The dynamic between blood pressure, its circadian rhythm, and the circadian clock genes has been considered as a factor in the subsequent renal injury that accompanies hypertension. Various circadian clock proteins such as BMAL1 and PERIOD (PER) function in a marriage of positive and negative feedback loops that control transcriptional regulation. Of particular interest is PER1 which naturally acts in the negative loop of the cycle as a repressor, but recent findings show that it may have additional roles as a transcription activator. PER1 has a role in renal sodium handling as a target of aldosterone, and it has been linked to the expression of the α‐subunit of the epithelial sodium channel (α‐ENaC). Investigators have found that knockout of PER1 in 129/sv mice causes a decrease in mean arterial pressure (MAP); however, when the gene is knocked out in the C57BL/6J mouse on a 4% NaCl diet (high salt, HS) with deoxycorticosterone pivalate (DOCP) treatment, there is a significant increase in MAP with animals displaying a “non‐dipping” pattern of MAP. The wildtype mice that were on HS with DOCP treatment had a 11.4 ± 1.9% decrease in MAP from night to day whereas the knockout mice with the same conditions had only a 5.7 ± 1.4% decrease. To test the contribution of PER1 in the development of salt‐induced hypertension, we created the Per1 knockout rat on the Dahl Salt‐Sensitive rat background (SS; knock out SSPer1−/−). Eight to 9‐week old SS and SSPer1−/− male rats were implanted with DSI telemetry units to measure blood pressure on a 4% NaCl diet (high salt, HS) for 3 weeks. MAP in SSPer1−/− rats was significantly higher compared to wild type SS rats (191 ± 7 vs. 168 ± 4 mmHg, P = 0.031; respectively) after 3 weeks of HS. We also revealed a significant increase in albuminuria in SSPer1−/− rats on HS compared to SS rats on HS (0.10 ± 0.01 vs. 0.05 ± 0.01 Alb/Cre Clearance ratio, P = 0.027; respectively). Knockout of Per1 in SS rats on HS also resulted in a significant increase in cholesterol (200.0 ± 11.8 vs. 135.0 ± 9.7 mg/Dl, P = 0.003; respectively) and alkaline phosphatase (37.5 ± 3.6 vs. 77.6 ± 3.3 U/L, P = <0.001; respectively). Furthermore, SSPer1−/− rats after 3 weeks of HS diet had significant decreases in creatinine clearance compared to SS rats (65 ± 6 vs. 127 ± 24 mL/hour, P = 0.036; respectively). Here, we show that lack of PER1 causes a dramatization of the salt‐sensitive phenotype of the SS rat with a decline in renal function. These data propose that the role PER1 plays in sodium homeostasis in the kidney is pivotal for proper blood pressure regulation.

Support or Funding Information

National Institutes of Health Grants R35 HL135749 (AS) and R01 DK109570 (MLG) and by the American Heart Association 18PRE34030127 (DS)
Age‐Related Loss of Phrenic Motor Neurons: Reduced Myogenic Influence?
Authors: Wilfred Ann M. , Fogarty Matthew J. , Mantilla Carlos B. , Sieck Gary C.
Published: April 2020

There is an age‐associated loss of phrenic motor neurons (PhMNs) and withdrawal of pre‐synaptic terminals at neuromuscular junctions (NMJs) in 24‐month old Fischer 344 (F344) rats. Previously, we showed that age‐related PhMN loss is concomitant with neuromuscular transmission failure and diaphragm muscle (DIAm) sarcopenia (an age‐related atrophy of muscle fibers and a decrease in specific force). It is unknown if pre‐synaptic terminal withdrawal precedes PhMN loss, which would implicate a reduction in a trophic influence emanating from muscle fibers (myogenic). We hypothesized that age‐related PhMN loss results from pre‐synaptic terminal withdrawal and reduced myogenic influence, which occurs predominantly at type IIx and/or IIb DIAm fibers (innervated by larger PhMNs). F344 rats were obtained from the National Institutes of Aging. Experimental ages were chosen to represent 100% and 50% survival at 6 months and 24 months‐old, respectively. PhMNs were first retrogradely labeled by intrapleural injection of cholera toxin‐subunit B (CTB), which is dependent on binding to pre‐synaptic terminal gangliosides. Thus, with pre‐synaptic terminal withdrawal, PhMNs would not be labeled. Subsequently (3 days after CTB), PhMNs were retrogradely labeled by sectioning the phrenic nerve and dipping the proximal end into a Rhodamine dextran (Rho) solution – this labeling is not dependent on pre‐synaptic terminals. CTB and Rho labeled PhMNs were visualized by confocal microscopy. Only optical slices containing the nucleus of Rho‐ and/or CTB‐labeled PhMNs were counted. We analyzed the differences in labeling of PhMNs by CTB and Rho in both age groups. In labeled PhMNs, short and long axis measurements were used to determine somal surface areas by assuming a prolate spheroid shape. In 6‐month old rats, there was no significant difference in the number of PhMNs labeled by CTB or Rho. In 24‐month old rats, the total number of labeled PhMNs was reduced compared to the 6‐month old rats, which primarily affected larger PhMNs. Furthermore, at 24‐months, fewer PhMNs were labeled with CTB compared to Rho and those labeled with Rho had larger somal surface areas. These results support our hypothesis that the age‐related loss of larger PhMNs results from pre‐synaptic terminal withdrawal and reduced myogenic influence.

Support or Funding Information

Support: NIH R01‐AG044615

American Physiological Society
Sex‐Specific Estrogen Regulation of Hypothalamic Astrocyte Estrogen Receptor and Glycogen Metabolic Enzyme Protein Expression, and Glycogen Content in Rats
Authors: Ibrahim Mostafa M.H. , Bheemanapally Khaggeswar , Sylvester Paul W. , Briski Karen P.
Published: April 2020

Brain astrocytes are implicated in estrogenic neuroprotection against bio‐energetic insults, which may involve their glycogen energy reserve. Forebrain estrogen receptor (ER)‐alpha (ERα) and ‐beta (ERβ) exert differential control of glycogen metabolic enzyme [glycogen synthase (GS); phosphorylase (GP)] expression in hypoglycemic male versus female rats. Studies were conducted using a rat hypothalamic astrocyte primary culture model along with selective ER agonists to investigate the premise that estradiol (E2) exerts sex‐dimorphic control over astrocyte glycogen mass and metabolism. Female astrocyte GS and GP profiles are more sensitive to E2 stimulation than the male. E2 did not regulate expression of phospho‐GS (inactive enzyme form) in either sex. Data also show that transmembrane G protein‐coupled ER‐1 (GPER) signaling is implicated in E2 control of GS profiles in each sex, and regulates GP expression in females. E2 increases total 5′‐AMK‐activated protein kinase (AMPK) protein in female astrocytes, but stimulated pAMPK (active form) expression with equivalent potency via GPER in females and ERα in males. In female astrocytes, ERα protein was up‐regulated at a lower E2 concentration and over a broader dosage range compared to males, whereas ERβ was increased after exposure to 1–10 nM versus 100 pM E2 levels in females and males, respectively. GPER profiles were stimulated by E2 in female, but not male astrocytes. E2 increased astrocyte glycogen content in female, but not male astrocytes; selective ERβ or ERα stimulation elevated glycogen levels in the female and male, respectively. Outcomes imply that dimorphic astrocyte protein and glycogen responses to E2 may reflect, in part, differential steroid induction of ER variant expression and/or regulation of post‐receptor signaling in each sex.

Support or Funding Information

This research was supported by NIH DK 109382.