bluuebunny/crossref_metadata_2025 huggingface dataset:
A history of low dose ethanol modulates ventral hippocampus activity during motivated behavior
Authors: Bryant Kathleen G. , Nothem Mitch , Singh Binay , Barker Jacqueline M.
Published: May 2022

More than half of the 130 million people in the U.S. who consumed alcohol within the last month did so at a level that did not meet diagnostic criteria for an alcohol binge. However, even these sub diagnostic drinking levels can have neurobehavioral outcomes. Clinical studies have shown that low levels of acute ethanol drinking soon after learning can enhance task consolidation and memory recall. Our preclinical data in C57BL6J mice show that a history of repeated, low dose ethanol (0.5g/kg) exposure increases sucrose reward motivation as measured by progressive ratio. The ventral hippocampus (vHPC) is increasingly considered a critical contributor to reward seeking and has been shown to be sensitive to chronic ethanol exposure at higher doses. The present study sought to determine whether daily, post‐training, low dose ethanol exposure can modulate vHPC activity during motivated behavior. To investigate this, we implanted mice with an electrode array to record vHPC neural activity during performance of a progressive ratio task. We also investigated vHPC activity around important behavioral events in the task, including lever pressing (when the animal is seeking the reward) and magazine entries (when the animal is checking for reward delivery). Our results demonstrate that saline control mice show suppression of vHPC activity following a lever press, while mice with a history of low dose ethanol exposure exhibit this suppression prior to a lever press. In contrast, both saline and ethanol‐exposed mice exhibit similar vHPC activity patterns surrounding magazine entry. These results suggest that a history of post‐training low dose ethanol exposure modulates vHPC activity during reward seeking. Further, this shift in vHPC suppression around a lever press may be associated with the enhanced motivation for sucrose observed in ethanol‐exposed mice. Future studies will use optogenetics to determine whether this shift in activity contributes to heightened reward motivation in ethanol exposed mice. A greater understanding of the neurobiological substrates impacted by ethanol exposure can help identify factors placing casual drinkers at risk for transition to alcohol use disorder.
Effects of Vitamin D Supplementation on Overload‐Induced Muscle Growth and Function in Mice
Authors: Huey Kimberly , Conroy Zachary , Temperly McKenzie
Published: May 2022

Muscle hypertrophy induced by functional overload (FO) provides an in vivo model to study muscle growth. Research suggests vitamin D supplementation may stimulate muscle growth and may be associated with increased growth factor levels, such as insulin‐like growth factor‐1 (IGF‐1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). However, it is unclear if vitamin D impacts both muscle hypertrophy and contractile function. We hypothesized that vitamin D supplementation would positively impact the muscle response to a growth stimulus as evidenced by greater hypertrophy, increased IGF‐1, FGF, and VEGF levels, and improved contractile function compared to placebo. Adult mice underwent functional overload (FO) of the plantaris or sham surgery. In vivo plantaris force and fatigue resistance (% of maximal force after 10 contractions) were measured 14d post‐FO or sham in mice receiving daily vitamin D or placebo (n= 8‐10/group). IGF‐1, FGF, and VEGF were measured in the plantaris by ELISA. FO increased plantaris mass, independent of treatment (placebo: 22.8±1.7 vs. 13.4±1.3 and vitamin D: 20.3±3.3 vs. 13.2±0.7 mg, for FO and sham, respectively, p<0.05). Maximal force relative to body mass was unchanged with FO or vitamin D. Significant main effects of FO and vitamin D were found when maximal force was normalized to plantaris mass. With placebo, FO was associated with a significant decrease in force normalized to plantaris mass while vitamin D attenuated this decrease (placebo: 401±48 vs. 871±157 and vitamin D: 679±125 vs. 1062±120 g/g muscle, for FO and sham, respectively, p<0.05). Fatigue resistance was increased with FO, independent of treatment (placebo: 54±3% vs. 40±4% and vitamin D: 63±5% vs. 33±6%, for FO and sham, respectively, p<0.05). IGF‐1 levels were increased in the overloaded plantaris, independent of treatment (placebo: 842±284 vs. 99±9 and vitamin D: 576±138 vs. 115±10 mg/mg, for FO and sham, respectively, p<0.05), but were unchanged in the unloaded tibialis anterior. FGF levels were increased in the overloaded plantaris, independent of treatment (placebo: 378±56 vs. 105±37 and vitamin D: 414±58 vs. 153±82 mg/mg, for FO and sham, respectively, p<0.05). VEGF levels were unchanged with FO and/or Vitamin D. Vitamin D treatment in combination with FO did not significantly improve the ability of skeletal muscle to hypertrophy or increase growth factor expression in response to a growth stimulus. However, vitamin D supplementation may positively impact muscle strength during periods of growth.
Hybrid Rat Diversity Program (HRDP): A rat resource for mapping complex traits
Authors: Dwinell Melinda , Takizawa Akiko , Lazcares Lynn , Tutaj Monika , Schilling Rebecca , Geurts Aron , Kwitek Anne
Published: May 2022

Rats are a major model for studying complex disease mechanisms, behavioral phenotypes, environmental factors, and for drug development and discovery. Inbred rat strains control for genetic background and allow for repeated, reproducible molecular, cellular, and whole animal phenotyping. Genetic susceptibility to disease, sensitivity to environmental elements, and pharmacogenomics are critical components of the concepts of precision medicine. The Hybrid Rat Diversity Program (HRDP) is a resource that combines the power of genetic stability within strains, power for genomic mapping strategies, and strength in an animal model that mirrors many human disease traits. The HRDP was designed to include 96 inbred rat strains with genomic and physiological diversity to maximize power to detect specific genetic loci associated with complex traits while maximizing the genetic diversity among strains. The 96 strain panel consists of 33 genetically diverse inbred strains and two panels of recombinant inbred panels: FEXL/LEXF (33 strains, Japan) and HXB/BXH (30 strains, Czech Republic). Embryo resuscitation and breeding is well underway at the Medical College of Wisconsin (MCW) with 55 strains available. Whole genome sequencing for 54 of the 96 strains is complete, having been generated on am Illumina NovaSeq S4 to achieve 30X coverage. An additional 14 related substrains have been sequenced. Liver and brain transcriptome analysis is underway through PhenoGen (http://phenogen.ucdenver.edu) at the University of Colorado. Genomic, phenotype, and strain information will be made available through the Hybrid Rat Diversity Panel portal at the Rat Genome Database (http://rgd.mcw.edu). The HRDP will provide a genetically stable population of rat strains with fully sequenced genomes, transcriptomes for brain and liver, and general phenotypic characterization to be used for systems genetic studies and fine mapping of complex traits.
A study of RNA polymerase‐Template interaction utilizing CRISPR/dCas9 protein
Authors: Hogaboom Jeremy R. , Enkhbaatar Khulan , King Maxwell J. M. , Rohlman Christopher E.
Published: May 2022

In vitro transcription is now a routine biochemical technique that allows for the synthesis of RNA molecules from a DNA template. This is done utilizing a variety of bacterial and phage RNA polymerases. Transcriptional pausing is a common biological phenomenon, yet the molecular mechanism of pausing is not yet well understood. Bacterial and phage RNA polymerases have been demonstrated to behave differently when they encounter a protein blockade during transcription. The goal of this project is to characterize the transcription complex as it encounters the protein blockade. Characterization of this complex will enable us to better understand the structural and chemical interactions between the polymerase, the DNA template, and the blockade protein. The protein blockade was engineered using the dCas9 protein from the CRISPR system. By using a nuclease deficient Cas9 protein (dCas9), we can create a protein blockade that will not cleave the template The CRISPR system is an adaptive immune system found in Archaea and many Bacteria. dCas9 protein binds guide RNA that is complementary to a target DNA sequence within the transcription template sequence creating a reversible blockade at specific locations in order to study the RNA polymerase interaction. In vitro conditions for T7 RNA polymerase have been optimized, and initial characterization of the complex has been completed. This work will allow us to better understand the mechanism behind a novel, flexible, and reversible transcriptional control system.
Neonatal and adult heterotypic inflammation sex‐dependently prime adult microglia in respiratory control regions without impairing adult breathing
Authors: Naidoo Robyn , Beyeler Sarah A. , Huxtable Adrianne G.
Published: May 2022

Adults often encounter diverse inflammatory stimuli, yet only some adults are prone to breathing deficits after adult inflammation. The determinants of adult susceptibility to breathing deficits during inflammation is understudied and likely involves microglia, the resident immune cells of the central nervous system. In non‐respiratory control regions, neonatal inflammation primes adult microglia for exaggerated inflammatory responses to otherwise harmless inflammatory stimuli, such as adult subthreshold heterotypic inflammation (differing neonatal and adult inflammatory stimuli). Since male and female microglia have sexually dimorphic developmental trajectories, we hypothesized that neonatal inflammation would sex‐dependently prime adult microglia in respiratory control regions to subthreshold heterotypic inflammation, contributing to impaired adult breathing. Using flow cytometry to assess microglia number (%CD11bhighCD45low / homogenates) and priming, adult male medullary microglia after neonatal inflammation (LPS 1mg/kg, i.p.) and adult subthreshold heterotypic inflammation (polyIC 478ug/kg, i.p.) were primed (neonatal LPS + adult polyIC: 25±7% microglia n=9; neonatal LPS + adult saline: 18±2% microglia, n=9; neonatal saline + adult saline: 10±2% microglia, n=9, p<0.05; neonatal saline + adult polyIC: 11±2% microglia, n=8, p<0.0001). In females, neonatal inflammation increased, but did not prime, medullary microglia to adult heterotypic inflammation (neonatal LPS + adult polyIC: 17±3% microglia, n=6; neonatal saline + adult saline: 11±1% microglia, n=6; neonatal saline + adult polyIC: 11±2% microglia, n=6, p<0.005; neonatal LPS + saline: 14±2% microglia, n=8, p>0.9). To test whether these primed microglia in respiratory control regions contribute to adult breathing deficits, breathing was assessed using plethysmography in adults after neonatal and adult heterotypic inflammation. Contrary to our hypothesis, neonatal and adult heterotypic inflammation did not impair adult eupneic breathing, the hypercapnic ventilatory response, or the hypoxic ventilatory response in adult males (p>0.9) or females (p>0.9). Therefore, adults after neonatal and adult heterotypic inflammation are able maintain breathing in response to low levels of inflammation despite primed medullary microglia. Primed microglia, however, are prone to augmented inflammatory responses and may induce significant deficits to breathing when faced with more severe inflammatory challenges.
The Role of Selective Adenosine Receptor Ligands on Inflammatory Pain
Authors: Alsalem Mohammad , Haddad Mansour
Published: May 2022

While pain is frequent manisfestation of several disease processes, chronic pain has been described as an established disorder with debilitating ramifications on health and lifestyle. The currently available analgesics for the treatment of chronic pain are often ineffective and accompanied by undesirable adverse effects, which invites for safer and more efficacious alternatives. Adenosine is a naturally‐occurring purinergic nucleoside that is involved in cell signaling in multiple tissue types. Although the activation of adenosine receptors can affect nociceptive, inflammatory, and neuropathic pain states, the specific regulatory functions of its subtypes (A1, A2A, A2B and A3 receptors) are not fully understood. The aim of this work was to investigate the role played by different adenosine receptor ligands on inflammatory pain conditions. Inflammatory pain was induced by intra‐plantar injection of Complete Freund’s Adjuvant (CFA) into the left hindpaw of adult male Sprague Dawley rats. Von Frey filaments were applied to the mid‐plantar aspect of the left hind paw using the “up‐down” method to determine the CFA‐induced mechanical allodynia (expressed as paw withdrawal threshold, PWT). Neither the A2A selective agonist CGS 21680 hydrochloride (0.1, 0.32 and 1 mg/kg) nor the A2B selective agonist BAY 60‐6583 (0.1, 0.32 and 1 mg/kg) produced any significant reversal of the PWT. However, both the A1 selective agonist (±)‐5′‐Chloro‐5′‐deoxy‐ENBA, and the A3 selective agonist 2‐Cl‐IB‐MECA produced a significant reversal of the PWT at the highest dose of 1 mg/kg, suggesting antinociceptive effects of the A1 and A3 adenosine receptors. Co‐administration of the selective antagonists of A1 and A3 receptors PSB36 (1 mg/ml) and MRS‐3777 (1 mg/ml); respectively, significantly reduced the anti‐nociceptive effects of both (±)‐5′‐Chloro‐5′‐deoxy‐ENBA, and 2‐Cl‐IB‐MECA (1 mg/kg) on PWT one hour post‐drug administration. Both the A2A selective antagonist ZM 241385 and the A2B selective antagonist PSB 603 produced a significant reversal of the PWT at the highest dose of 1 mg/kg. In conclusion, A1, A2A, A2B and A3 adenosine receptors are involved in mediating inflammatory pain states, and represent promising targets for the treatment of chronic pain conditions.
Exercise increases the expression of glucose transport and lipid metabolism genes at optimum level time point 6 hours post exercise in rat skeletal muscle
Authors: Fagbohun Oladapo F. , Joseph Jitcy C.
Published: May 2022

Aim

Type 2 diabetes constitutes more than 90% of all diabetic cases and is characterized by persistent increase in glucose level in the blood (hyperglycemia) as well as lipid and protein metabolic disorders that may induce insulin resistance. Increased lipid levels induce type 2 diabetes by interfering with the insulin‐signaling pathway responsible for glucose transport. Individuals who suffer from type 2 diabetes are usually characterized in part by down‐regulation of glucose transport genes such as glut4 and down‐regulation of mitochondrial lipid oxidizing genes such as carnitine palmitoyltransferase‐1 (Cpt‐1). Nuclear respiratory factor (NRF)‐1 is a mitochondrial transcriptional factor which is shown to be involved in glucose transport and is set as a potential therapeutic modality in the treatment and management of type 2 diabetes. In this study, we sought to access NRF‐1 and its target genes expression during exercise which is crucial in glucose transport and lipid oxidation.

Method

Five to six weeks old male Wistar rats were exercised to identify the time point for an optimum increase in the levels of NRF‐1 and its target genes. Gastrocnemius muscles were harvested after 0, 2, 4, 6, 8, 10, 12, and 15 h post‐exercise and non‐exercise rats (control). Primers were used to amplify the region of following genes; Nrf‐1, glut 4, carnitine palmitoyl transferase (Cpt‐1 & Cpt‐2), peroxisome proliferator‐activated receptor gamma co‐activator 1 (Pgc‐1), mef2a, and acetyl‐CoA carboxylase‐1 (Acc‐1). Relative mRNA expression was normalized to the Actin reference gene.

Results

From the result of this study, cpt‐1, Nrf‐1, mef2a, glut4, cpt2, and Pgc‐1 showed 2.5, 8, 1.2, 4.1, 4.6, 3.5‐folds increases respectively after 8h post‐exercise compared with control whereas Acc‐1 showed a 3.1‐fold decrease in gene expression ratio after 6 h post‐exercise compared with control. Nrf‐1 binding to cpt‐1 and mef2a increased with 3 and 3.5‐folds respectively 6h post‐exercise compared with control.

Conclusion

Conclusively, nrf‐1 was increased by exercise and also, it's binding to target genes which has huge implications in ameliorating type 2 diabetes and insulin resistance.
Implication of New identified Nicotine regulated genes in diabetic cardiomyopathy
Authors: Wang Yajing , Zhao Jing , Zhao Jianli , Xie Yaoli , Liu Caihong , Lau Wayne Bond , Lopez Bernard , Christopher Theodore , Ma Xinliang
Published: May 2022

Objectives

There is a paradox of nicotine and diabetes in patients with cardiomyopathy. Although great progress has been made in understanding the pathogenesis, it is urgent to clarify the in‐depth mechanism and identify new therapeutic targets for preventing diabetic cardiomyopathy (DCM) in smoker.

Methods

Human gene expression profiles in DCM (GSE4745 and GSE99203) corrected by SVA algorithm were analyzed and then “limma” packet gene screening was performed. By constructing the related co‐expression WGCNA network of DCM, combined with the analysis of Metascape pathway, the correlation of module and genes with DCM was determined. Furthermore, the Drug bank and STRING databases were used to screen nicotine targets and expand the gene network. The hub gene was identified by crossing the targets with DCM, which was obtained by WGCNA analysis. r‐packet “clusterprofiler” was used for GO analysis and KEGG pathway analysis. Then, the network of hub genes affecting DCM and the interaction diagram of core target related pathways that influence DCM is constructed by using the Cytoscape software. The relationship between hub genes was analyzed by using “corrplot”, “vioplot” packages and cibersort algorithm, and the potential mechanism of hub genes affecting DCM progress was further explored. To explore the relationship between the targets in the process of DCM, ROC curve of diagnostic efficacy was drawn with “pROC” package. Finally, a miRNA‐mRNA regulatory network including hub genes was further constructed by Targetscan. The regulatory network of transcription factors of hub genes was constructed from TRRUST database.

Results

3 gene modules were detected by WGCNA analysis, blue (202), grey (31) and turquoise (397), among which turquoise module had the highest correlation with DCM (cor =0.87, P = (3e −10)). The genes rich in monocarboxylic acid and sulfur compound metabolic processes in response to extracellular stimulus were identified. The top 13 nicotine‐related are targeted. The intersection of 113 target genes and turquoise module gene were screened out. There are the two hub genes involved Iron (III) ion response and retinoic acid biosynthetic process, involving Tryptophan Metabolism and Ovarian steroidogenesis pathways. The AUC values of the two hub genes are Cyp1a1(AUC: 0.769 (0.580 ‐ 0.958)) and Chrnb4(AUC: 0.707 (0.511 ‐ 0.902)) and both genes are strongly correlated with immune cell response. A total of 40 miRNA‐mRNA pairs, including 35 miRNAs and 2 mRNAs, were predicted by Targetscan. Three Cyp1a1 transcription factors were predicted by TRRUST database.

Conclusion

We demonstrated that two genes, Chrnb4 and Ccyp1a11, are the potential genes involved in nicotine modulating DCM, and these two genes may regulate DCM’s immune response through miRNA‐mRNA mode. It provides new potential targets for preventing DCM in smoking population.
Burrowing as an Index of Inflammatory Pain in Male vs. Female Rats
Authors: Craft Rebecca M.
Published: May 2022

The objective of this study was to determine whether burrowing behavior may be useful as a functional index of pain in both female and male rats, and whether a “no‐training” protocol can be used, to increase efficiency of testing. Adult Sprague‐Dawley rats were injected in one hindpaw with oil vehicle or complete Freund’s adjuvant (CFA), and then placed individually into cages containing a burrowing tube filled with aquarium gravel; the amount of gravel burrowed out of the tube after 1 h was measured each day for up to 7 days. To test the predictive validity of the burrowing test as a screen for analgesic effect, rats were pretreated with the non‐steroidal anti‐inflammatory drug (NSAID) ketoprofen (0.001‐3.2 mg/kg) or the opioid morphine (0.1‐3.2 mg/kg) as standard analgesics (true positives). Chlordiazepoxide (1.25‐10 mg/kg) was tested as a true negative, and delta‐9‐tetrahydrocannabinol (THC, 0.01‐2.0 mg/kg) was tested as a purported analgesic. CFA reliably suppressed burrowing in both sexes for 2‐3 days, using a “no‐training” protocol. In regard to predictive validity, ketoprofen restored CFA‐suppressed burrowing in both sexes by days 2‐3, and was more potent in females than males. In contrast, morphine only restored CFA‐suppressed burrowing in males. Chlordiazepoxide further decreased CFA‐suppressed burrowing in males, and did not alter burrowing behavior in females. THC did not significantly alter CFA‐suppressed burrowing in either sex, up to doses that decreased burrowing in oil‐treated controls. These results suggest that in male rats (as others have shown), burrowing is a useful, functional index of inflammatory pain. Furthermore, training/habituation to the burrowing procedure is not necessary. In contrast, although female rats’ burrowing behavior is suppressed by CFA to approximately the same extent as males’, only the NSAID restored CFA‐suppressed burrowing in females. Additional studies are underway to determine the extent to which CFA dose (i.e., pain intensity) and analgesic pretreatment time contribute to the limited efficacy of analgesics in the burrowing test, particularly on the first day after CFA injection.
Identification of Aloe‐derived natural products as lead scaffolds for SARS‐CoV‐2 main protease (Mpro) inhibitors
Authors: Hicks Emily G. , Kandel Sylvie E. , Lampe Jed N.
Published: May 2022

In the past two years, the COVID‐19 pandemic has caused over 5 million deaths and 250 million infections worldwide. Despite successful vaccination efforts and emergency approval of small molecule therapies, diverse antivirals are still needed to combat the inevitable viral resistance that will arise. The main protease of SARS‐CoV‐2 (Mpro) is an attractive drug target due to the clinical success of protease inhibitors against other viruses, such as HIV and HCV. However, in order to combat resistance, diverse chemical scaffolds need to be identified that have the potential to be developed into potent inhibitors. To this end, we screened a high‐content protease inhibitor library against Mpro in vitro, in order to identify structurally diverse compounds that may be further developed into antiviral leads. Our high‐content screening efforts retrieved 27 hits each with >50% inhibition in our Mpro FRET assay. Of these, three natural product compounds, anacardic acid, aloesin, and aloeresin D, were chosen for follow up IC50 screening due to their strong druglike properties (high bioavailability, low toxicity). Further analysis via dose response curves revealed IC50 values of 6.8 μM, 38.9 μM, and 125.3 μM respectively. Molecular docking studies demonstrated that the three inhibitors bound at the catalytic active site of Mpro with varying binding energies (‐7.8 to ‐5.6 kcal/mol). Furthermore, we determined that our kinetic data from the Mpro FRET assay fit better to a sigmoidal Hill model than the standard Michaelis‐Menten hyperbola, indicating substantial cooperativity between enzyme monomers, suggesting that the dimerization interface could be an attractive target for allosteric inhibitors. In conclusion, we identified two natural product compounds from the Aloe plant (aloesin and aloeresin D) as novel scaffolds for Mpro inhibitor design and confirmed the cooperative kinetics of Mpro. These results advance our knowledge of structure‐function relationships in Mpro and offer new molecular scaffolds for inhibitor design.
Development of humanized ACE2 mouse and rat models for COVID‐19 research
Authors: Jiang Hongmei , Hyddmark Emma M. V. , Gordon Sara , Bartels Angela , Wu Yumei , Warren Joe , Brown Andrew , Garrett Michael , Ehall Helmut , Zhao Guojun
Published: May 2022

To date (17 November 2021), there has been more than 254 million confirmed cases of COVID‐19, and more than 5 million death globally (World Health Organization. https://covid19.who.int/). The virus causing COVID‐19 is called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). SARS‐CoV‐2 infects host cells by the binding of its spike protein to the cellular surface protein angiotensin‐converting enzyme 2 (ACE2). The predicted 29 amino acid residues of ACE2 that interact with SARS‐CoV‐2 spike protein receptor binding domain (RBD) vary between human ACE2 and mouse or rat ACE2. Therefore, wildtype mice and rats show lower SARS‐CoV‐2 infection rate and mild symptoms compared to what is seen in humans. Small animal models that recapitulate human COVID‐19 disease are urgently needed for better understanding the transmission and therapeutic measurement. Currently, scientists use either mouse‐adapted SAS‐CoV‐2 (SAS‐CoV‐2 MA) models or random transgenic mouse models that artificially express human ACE2 under the control of cytokeratin 18 promoter or a constitutive promoter. SAS‐CoV‐2 MA may not completely reflect all aspects of the original human‐tropic SAS‐CoV‐2 and the current transgenic human ACE2 mouse models typically have high mortality rate caused by neuroinvasion and encephalitis due to very high human ACE2 expression. To overcome these limitations, we have developed humanized ACE2 mouse and rat models using CRISPR‐Cas9. Specifically, we inserted a ~3kb human ACE2 cDNA cassette into the mouse and rat Ace2 gene loci to ensure that human ACE2 expression is under the control of rodent Ace2 promoter and regulatory elements, while simultaneously disabling the rodent Ace2 gene. To accomplish this, CRISPR gRNAs targeting close to the translation initiation site of Ace2 were screened in cultured mouse and rat cells. Then CRISPR/Cas9 complex and donor DNA were subsequently microinjected into one‐cell stage embryos which were subsequently implanted into pseudo pregnant females. Resulting pups were screened for correct knockin by junction PCR and insert PCR, and the PCR products were Sanger sequenced. Targeted Locus Amplification (TLA) further confirmed the integration sites and transgene sequence. RT‐qPCR and Western blot analysis data showed that, in our models, human ACE2 is expressed in tissues expressing endogenous Ace2 (such as lung, kidney, and GI tract), while rodent endogenous Ace2 is absent from these tissues. Further breeding data indicated that both hemizygous and homozygous humanized ACE2 animals appear to be normal and fertile. Most importantly, animals displayed symptoms after infection with SARS‐CoV‐2. In summary, these data suggest that our humanized ACE2 models can be valuable for COVID‐19 research.
Enhanced Cerebral Blood Flow and Hemodynamic Status with an Alternative Left Ventricle Chest Compression Position during Cardiopulmonary Resuscitation in Swine
Authors: Marshall Rory A. , Morton Jude S. , Luchkanych Adam M. S. , El Karsh Yehia , El Karsh Zeyad , Morse Cameron , Turnbull Katie Y. , Schaefer Nici N. , Tomczak Corey R. , Grunau Brian E. , Olver T. D.
Published: May 2022

Introduction

The standard chest compression (SCC) position for cardiopulmonary resuscitation (CPR) is the centre of the chest and typically central to the aortic root. An alternative left ventricle chest compression (LVCC) position may facilitate superior global blood flow. We tested the hypothesis that, consistent with improved ETCO2 (indicative of cardiac output) and blood pressure (BP), LVCC would promote greater cerebral blood flow (CBF) than SCC.

Methods

Female pigs (N=32; 35±2kg) were rotated systematically to receive either SCC (n=14) or LVCC (n=18). Transthoracic echocardiography was used to identify and externally mark: (1) the SCC position, the midline at the level of the aortic route, or (2) the LVCC position, the midpoint of the left ventricle. After 2 minutes of untreated asphyxiated cardiac arrest (CA), swine were treated with three 2‐minute cycles of mechanical chest compressions (LUCAS III). ETCO2 (in‐line sampling), BP (arterial catheter line), and CBF velocity (CBFv, transcranial Doppler, primary outcome) were measured during the pre‐CA, untreated‐CA, and CPR‐treated phases. Between‐group outcomes were compared using a mixed model ANOVA with significant differences considered at P<0.05 and data are presented as mean±standard deviation.

Results

Pre‐ and untreated‐CA ETCO2, BP and CBFv were similar between the SCC and LVCC groups (P>0.100). During CPR, ETCO2 (36±6 mmHg versus 24±10 mmHg; P<0.001), Mean Arterial Pressure (MAP; 49±9 mmHg versus 37±9 mmHg; P=0.002), and Mean CBFv (11±5 cm/s versus 5±2 cm/s; P<0.001) were significantly higher in the LVCC, versus SCC, group.

Conclusion

In comparison to SCC,LVCC resulted in higher ETCO2, MAP, and Mean CBFv values throughout CPR in a swine model of CA.

New and Noteworthy

We provide novel evidence that compared with mechanical compressions performed over the centre of the chest, compressions performed over the left ventricle promote greater CBF during BLS CPR. Clinical validation of these results may improve survival rates and attenuate neurological deficits following CA.
Effect of Intermittent Hypoxia on Plasma Glucose Levels during an Oral Glucose Tolerance Test
Authors: Massoudian Sahar D. , Wojan Frank , Zhao Jiahui , Lalande Sophie
Published: May 2022

Hypoxia triggers glucose uptake independently from the action of insulin. The purpose of this study was to determine the acute effect of intermittent hypoxia, defined as alternating short bouts of breathing hypoxic and room air, on plasma glucose levels during an oral glucose tolerance test in healthy individuals. We hypothesized that exposure to intermittent hypoxia would attenuate the increase in glucose levels in response to an oral glucose tolerance test. Nine individuals (5 men, age: 24 ± 4 years, height: 175 ± 9 cm, weight: 71.0 ± 13.5 kg, HbA1c: 5.4 ± 0.1%) participated in the study. Participants visited the laboratory on two occasions. On both visits, a 2‐hour oral glucose tolerance test was performed, with venous blood samples collected 0, 30, 60, 90 and 120 minutes following the ingestion of a 75 g glucose drink. On visit 1, an intermittent hypoxia (IH) protocol, consisting of eight 4‐minute hypoxic cycles at a targeted arterial oxygen saturation of 80% interspersed with breathing room air to resaturation, was performed following ingestion of the glucose drink. On visit 2, an intermittent normoxia protocol consisting of eight 4‐minute normoxic cycles interspersed with breathing room air was performed following ingestion of the glucose drink. Visit order was randomized and participants were blinded to the condition. As expected, intermittent hypoxia resulted in a lower arterial oxygen saturation than intermittent normoxia (IH: 83 ± 3, IN: 98 ± 1%, p < 0.01) which corresponded to lower levels of inspired oxygen (IH: 10.9 ± 0.7, IN: 20.9 ± 0.3%, p < 0.01). Plasma glucose responses to the oral glucose tolerance test were not different between conditions (IH vs. IN: 0: 90 ± 7 vs. 89 ± 6; 30: 135 ± 21 vs. 137 ± 24; 60: 110 ± 28 vs. 108 ± 25; 90: 96 ± 18 vs. 88 ± 14; and 120: 101 ± 19 vs. 83 ± 14 mg/dl, p = 0.29). Intermittent hypoxia triggered an increase in cardiac output (6.1 ± 0.9 to 6.8 ± 1.3 L/min, p < 0.01) caused by an increase in heart rate (67 ± 10 to 79 ± 12 bpm, p < 0.01). Contrary to our hypothesis, intermittent exposure to hypoxia did not attenuate the increase in plasma glucose levels during an oral glucose tolerance test in individuals with normal glycemic control. It remains to be determined whether intermittent hypoxia can attenuate the increase in plasma glucose levels in response to an oral glucose tolerance test in individuals with impaired glucose tolerance.
Identifying the vagal bronchopulmonary afferents responsible for eliciting the cough response to inhaled sulfur dioxide in mice
Authors: Lin Ruei‐Lung‐ , Lin An‐Hsuan , Lee Lu‐Yuan
Published: May 2022

Among the three major types of vagal sensory nerves innervating the lung and airways, C‐fibers and rapidly adapting receptors (RARs or irritant receptors) are known to possess the properties and functions of “cough receptors”, but their relative roles in regulating cough reflex responses to inhaled irritants are not yet clearly defined. To address this question, we have recently carried out a series of studies in a mouse cough model, in which the intra‐pleural pressure was continuously recorded by a surgically implanted telemetry sensor. Sulfur dioxide (SO2), a common irritant gas, was chosen for the study because its widespread use as a chemical agent in various industrial operations has led to severe lung injuries in workers in numerous incidences resulting from accidental release of high concentrations of SO2 in industrial facilities. Inhalation of SO2 (300 and 600 ppm) consistently and reproducibly elicited coughs in a concentration‐dependent manner in awake mice moving freely in a recording chamber. The cough frequency increased rapidly after the onset of the SO2 inhalation challenge, which continued for the remaining duration of 8‐min exposure. In the current study, single‐fiber electrophysiological recording of vagal afferent activity in anesthetized mice showed that the high concentration of SO2 (600 ppm) evoked a sustained stimulatory effect on C‐fibers for the entire duration (8 min) of the inhalation challenge. However, in sharp contrast, inhalation of SO2 at the same concentration and duration generated a distinct and consistent inhibitory effect on both phasic RARs and slowly adapting receptors. Their respiratory cycle‐related phasic discharge declined progressively and/or ceased completely within 1‐3 min after the onset of the SO2 inhalation challenge, which gradually recovered after >15 min following the termination of the SO2 inhalation. In a small percentage of RARs that displayed no phasic activity during eupneic breathing and often exhibited sensitivity to certain inhaled irritants, the same SO2 inhalation challenge did not evoke any significant stimulatory effect. Taken together, these observations suggest that stimulation of bronchopulmonary C‐fibers is primarily responsible for triggering airway irritation and coughing in response to the SO2 inhalation challenge.
T18/S19 diphosphorylation of myosin regulatory Light Chain is Responsible for the impaired relaxation of pulmonary arteries in pulmonary hypertensive rats
Authors: Cho Suhan , Oh Seung Beom , Kim Sung Joon
Published: May 2022

Pulmonary arteries (PA) are exposed to hemodynamic environment and contractile stimuli different from those of systemic arteries. Previous studies reported that in situapplication of sodium nitroprusside (SNP, NO donor) lowered the PA pressure, which was less prominent than the response of systemic arteries. Here, we investigated the relaxation speed and the in vitro effects of NO‐dependent signaling modulators to compare between isolated PA and mesenteric arteries (MA) of rats. In the dual‐wire myography study, PA showed significantly slower relaxation after the response to 80 mM KCl (80K) or to pharmacological agonists (U46619 and PGF2a). Surprisingly, addition of 30 mM SNP could not prevent the 80K contraction in PA while significantly attenuated the contraction of MA. The pretreatment with soluble guanylate cyclase (sGC) inhibitor (ODQ, 10 mM) markedly further slowed the relaxation of PA after 80K or agonist‐induced contraction. Perplexingly, immunoblot assay revealed higher expression of sGC‐a with similar level of sGC‐b1 in PA than MA. The expression of RhoA‐kinase (ROCK) was higher while myosin light chain phosphatase (MYPT) appeared lower in PA than MA. The treatment with ROCK inhibitor (Y27632, 10 mM) could abolish the different of relaxation response between PA and MA. Taken together, we speculate that despite the higher expression and the functional importance of NO/sGC pathway in PA, the higher level of ROCK and relatively low MYPT in PA are responsible for the characteristic slow relaxation after removal of stimuli.
Examining the Interaction Between Cas9 and Ku, and its Implications for Gene Editing
Authors: Hayes Catherine M. , Bharti Aditya , Hanakahi Les A.
Published: May 2022

Gene editing via CRISPR/Cas9 is an innovative new tool that allows scientists and clinicians to alter DNA. This can be done by inserting new DNA or by knocking out the function of preexisting DNA. Both processes begin with Cas9, an enzyme that acts as molecular scissors to introduce a DNA double‐strand break (DSB) at a specific location. The way this DSB is repaired determines whether new DNA is inserted or preexisting DNA is knocked out.

Insertion of the new DNA sequence relies on a DNA‐repair process called homologous recombination. Knocking out the function of a gene is achieved through the DNA‐repair process non‐homologous end joining (NHEJ). Unfortunately, the majority of Cas9‐induced DSBs are repaired by NHEJ, thereby posing a barrier to inserting new genes. So why is NHEJ so prominent at Cas9‐induced DSBs?

We have evidence that Cas9 interacts with the NHEJ initiating factor Ku. We, and others, used lysates from Cas9‐expressing human cells and found that Cas9 co‐immunoprecipitated with Ku. These observations may reflect either a direct Ku‐Cas9 protein‐protein interaction, or an indirect interaction mediated by other human cellular proteins, DNA, or both.

I hypothesize that Cas9 and Ku have a direct physical interaction. To test my hypothesis, I carried out affinity‐capture assays using purified recombinant proteins. I found that Cas9 captured Ku, indicating that the interaction is not dependent on other human cellular proteins. This interaction also occurred when Ku‐DNA interactions are disrupted by adding ethidium bromide or benzonase. This indicates that the interaction is not due to the DNA‐binding capabilities of Ku.

These experiments indicate that Ku and Cas9 have a direct protein‐protein interaction. Because Ku initiates NHEJ, our results suggest the possibility that the Cas9‐Ku interaction may recruit NHEJ factors to Cas9‐induced DSBs and provides a plausible explanation of the bias towards NHEJ repair of Cas9‐induced DSBs.
REDD1 deletion prevents the development of renal dysfunction in diabetic mice
Authors: Sunilkumar Siddharth , Miller William P. , Toro Allyson , Yerlikaya Esma , Dennis Michael D.
Published: May 2022

Diabetic kidney disease (DKD) results in chronic loss of kidney function and is the leading cause of end‐stage renal disease. Hyperglycemia is an important mediator of diabetic complications, including DKD. Hyperglycemic conditions promote renal redox imbalance, resulting in glomerular/tubular damage and compromised renal function. Presently, much remains unknown regarding the molecular events that contribute to development of oxidative stress in the context of DKD. In the present study, we evaluated the hypothesis that hyperglycemic conditions promote expression of the stress response protein REDD1 in the kidney in a manner that contributes to the development of renal pathology and compromised function. After 16 weeks of streptozotocin (STZ)‐induced diabetes, urinary albumin to creatinine ratio and kidney to body weight ratios were elevated in wild‐type mice. These markers of renal dysfunction were associated with increased renal REDD1 expression. In contrast to wild‐type mice, REDD1 knockout (KO) mice failed to exhibit a diabetes‐induced deficit in kidney function. Histopathologic examination of the kidney found glomerular damage in diabetic wild‐type mice, but not in diabetic REDD1 KO mice. In cultured human podocytes, exposure to hyperglycemic conditions elevated REDD1 expression concomitant with increased oxidative stress and cell death. Oxidative stress and cellular apoptosis were not observed in podocyte cultures upon REDD1 deletion. Similarly, REDD1 deletion prevented diabetes‐induced oxidative stress in the kidney in coordination with upregulation of the Nrf2 antioxidant response. These findings provide new insights into how diabetes and hyperglycemia contribute to development of renal pathology and support the possibility that therapeutics targeting REDD1 could be beneficial in the context of DKD.
High‐fat/high‐carbohydrate diet increases glycogen accumulation in lung tissue in vivo
Authors: James Alexis N. , Conroy Lindsey R. , Hawkinson Tara R. , Chang Josephine E. , Manauis Elena C. , Lamb Jessica F. , Buoncristiani Michael D. , Young Lyndsay E. A. , Filmore Brainson Christine , Sun Ramon C.
Published: May 2022

Lung adenocarcinoma (LUAD) is the major histological subgroup of non‐small cell lung cancer (NSCLC) with an extremely high mortality rate even when diagnosed at an early stage. LUAD makes up around 40% of lung cancer cases and can quickly metastasize to other areas of the body. Glycogen is the short‐term storage of carbohydrates and can be easily mobilized in the body. Recently, other and our own data suggests glycogen can drive lung cancer progression, however, the mechanism of which glycogen accumulates in LUAD is poorly understood and potentially arises from a number of genetic and environmental factors. Although specific genetic variants driving aberrant LUAD glycogen accumulation have been identified, several studies have shown that caloric excess as well as specific nutrients in the diet can promote tumor growth. Further, LUAD patients with type II diabetes or high BMI have significantly poorer survival. Therefore, we hypothesized that a diet rich in fats and carbohydrates that mimics a Western diet increases glycogen accumulation in lung tissue and potentially drives LUAD tumor progression. To test our hypothesis that a Western diet alters glycogen metabolism independent of genetic drivers and tumor formation, we administered via oral gavage a Western diet comprised of corn oil and high fructose corn syrup (150 μl corn oil: 25% high‐fructose corn syrup solution) to wild‐type C57BL/6 mice at acute (1‐, 3‐, and 6‐hours) and chronic (two weeks) time points. At the end of each time point, mice were sacrificed and lung tissue was harvested. Using a combination of gas chromatography mass spectrometry (GCMS) and matrix‐assisted laser desorption ionization mass spectrometry imaging, we quantified central carbon metabolites and glycogen content in lung tissue resected from these mice. Mice administered H2O were used as a control. We found that the mice administered a Western diet for two weeks exhibited elevated levels of glycogen as well as metabolite pools within glycolysis and the TCA cycle in lung tissue compared to the control group. However, our acute studies revealed no change in lung glycogen up to 6‐hours after receiving the Western diet. Overall, these data highlight a link between diet and glycogen metabolism in the lung and suggest that intake of different dietary nutrients may play a role in glycogen accumulation in LUAD tumors and disease progression. Our results demonstrate that a high fat/high carbohydrate diet in wild‐type mice increases central carbon metabolism and glycogen accumulation in lung tissue after chronic exposure. Moving forward, we will examine the contribution of diet to glycogen metabolism in a mouse of LUAD and assess the effect of increased glycogen accumulation on tumor growth in vivo.
Stretching the Face: Mimetic Muscles Have Muscle Spindles
Authors: Omstead K. M. , Marazita Mary L. , Weinberg Seth , Williams Jade , Burrows Anne M.
Published: May 2022

Most skeletal muscles contain muscle spindles (MS) – sensory receptors which inform the CNS about changes in muscle length and rate of change in lengths. Muscle spindles are the most frequently found sense organ in skeletal muscles. While the face is the primary communication tool for mammals, it remains unclear if MS are present in the facial mimetic muscles. In order to investigate the potential presence of MS in mimetic musculature, we used hematoxylin and eosin and sirius red collagen staining to examine select mimetic muscles among a wide phylogenetic range of mammals (N=23 individuals). Bright light microscopy was used to identify and quantify MS. Contrary to previous studies, we found MS throughout mimetic muscles, including in humans. Preliminary results identified 2 MS in human (Homo sapiens) sections, while only a single muscle spindle was identified across chimpanzee (Pan troglodytes) samples – fewer than other primate and non‐primates investigated. Muscle spindles have been shown to atrophy with increasing age, which may explain this discrepancy. Focusing on canids, we were able to identify 23 MS across various dog breeds. There was a significantly greater number of MS in wolf (Canis lupus) and husky breed of dog samples (an “ancient” dog breed) when compared with more derived domestic dog breeds (beagle/chihuahua) and with other mammals. An increased presence of muscle spindles identified in dog and wolf species in both the zygomatic major and orbicularis oris muscles suggests that the mobility of facial muscles in canids may be influenced by proprioceptive mechanisms that are required for finer motor movements corresponding to vocal communication.
The Utility of Anatomical Diagrams Shared on Social Media
Authors: Motzko Micaela , Dennis Jennifer F.
Published: May 2022

Introduction/Objective

Medical schools have made an increasing effort to provide students with online anatomical resources. Yet, studies report that most students who encounter a question during their anatomy study will consult the internet or social media to find supplemental resources before using course materials. A quick internet search by a novel anatomist may lead to inaccurate information and overwhelming results. We created sets of accurate and unique anatomical diagrams and shared these via a social media account to assess their perceived utility and accessibility to students learning anatomy.

Materials/Methods

Anatomical diagrams were created and shared on a public Instagram account, Anatomy Adventures, during the 2020‐21 academic year. The systems‐oriented graphics covered musculoskeletal, nervous, gastrointestinal, and reproductive systems, including the osteology, myology, and neurovasculature of each system. An IRB‐approved survey (10.6% response rate) was distributed from March‐April 2021, assessing: 1) the demographics of the surveyed population, 2) how the diagrams were utilized, and 3) how they could be improved. A free‐form space was included for optional feedback. Responses were tabulated and converted to frequencies and percentages; descriptive codes were applied to the data according to published methods to summarize verbatim responses. Chi square test assessed differences among the responses when applicable. Alpha<5%.

Results

Of the 111 survey responses, 95% participants fell within the 18‐30 age range, with 78% identifying as female. The majority of participants were medical students (69%), but the sample also included undergraduate/graduate students (16%), as well as fully employed participants (14%). Geographically, most participants were located in the North and Central (69%) or other (27%) regions of the United States (US), with 4% located outside of the US. As for utility, 54% of participants reported using the diagrams to study for medical school coursework or board examinations, and 42% of responses indicated non‐medical use of the diagrams, such as leisure viewing or reviewing for their occupation. Free‐form responses (n=65) were coded into categories characterizing the utility of the diagrams: 1) color‐coding (12.3%), 2) simplicity (43%), and 3) style (24.6%) (P=0.0025).

Significance

We demonstrate that supplemental resources shared via social media have the potential to be widely used by students, specifically by those 18‐30 years. The inherent flexibility of social media is how consumers preferred to utilize information. Qualitative data indicated presentation style largely impacts how resources are utilized by students, as an overwhelming majority report gaining unique information from this resource.

Conclusion

Social media platforms can be used by educators to disseminate accurate supplemental materials to students. Anatomy educators should consider utilizing social media to fill gaps in anatomy education to provide accurate and accessible resources.