bluuebunny/crossref_metadata_2025_split huggingface dataset:
CpG Methylation Differences Between Neurons and Glia are Highly Conserved from Mouse to Human
Authors: Kessler Noah J. , Van Baak Timothy E. , Baker Maria S. , Laritsky Eleonora , Coarfa Cristian , Waterland Robert A. 
Published: June 2021

Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study (Lister & Mukamel, et al., Science 2013) used genome‐wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That study minimized the importance of cell type‐specific differences in CpG methylation, claiming these are restricted to localized genomic regions, and instead emphasized that widespread and highly conserved differences in non‐CpG methylation distinguish neurons and glia. We reanalyzed the data from that study and came to markedly different conclusions. In particular, we found widespread cell type‐specific differences in CpG methylation, with a genome‐wide tendency for neuronal CpG‐hypermethylation punctuated by regions of glia‐specific hypermethylation. Alarmingly, our analysis indicated that the majority of genes identified by the primary study as exhibiting cell type‐specific CpG methylation differences were misclassified. To verify the accuracy of our analysis, we isolated neuronal and glial DNA from mouse cortex and performed quantitative bisulfite pyrosequencing at nine loci. The pyrosequencing results corroborated our analysis, without exception. Most interestingly, we found that gene‐associated neuron vs. glia CpG methylation differences are highly conserved across human and mouse, and are very likely to be functional. In addition to underscoring the importance of independent verification to confirm the conclusions of genome‐wide epigenetic analyses, our data indicate that CpG methylation plays a major role in neuroepigenetics, and that the mouse is likely an excellent model in which to study the role of DNA methylation in human neurodevelopment and disease, and effects of nutrition on these processes.Support or Funding InformationThis work was supported by grants from the National Institutes of Health Roadmap Epigenomics Program [grant number U01DA025956] and the U.S. Department of Agriculture (USDA) [grant numbers CRIS 6250‐51000‐055 and CRIS 3092‐5‐001‐059].
Aquaporin 9 in the Freeze Tolerance Mechanism of Cope's Gray Treefrogs
Authors: Stogsdill Brian , Frisbie James , Goldstein David 
Published: June 2021

Cope's Gray Treefrogs Hyla chrysoscelis accumulate glycerol in response to cold as part of their freeze tolerance mechanism. This glycerol may derive from glucose stores in the liver that are broken down and released through glycerol transporters. We therefore hypothesized that these treefrogs would express aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates rapid glycerol transport, predominately in the liver. We also hypothesized that this protein would be up‐regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we sequenced the AQP9 homologue HC‐9, analyzed RNA and protein expression, and characterized water and glycerol permeability through a Xenopus oocyte expression system.HC‐9 has 315 amino acids which include 2 conserved NPA motifs unique to aquaporins, 5 conserved residues typical of glyceroporins, a high homology (69%) to human AQP9, and a nearly identical homology (97%) to the hyla japonica. The mRNA strand also possesses a long untranslated tail unique to amphibians that appears to be necessary for translation. Furthermore, characterization using the Xenopus expression system has shown that HC‐9 is permeable to glycerol but has a poor water permeability compared to human AQP1.In warm‐acclimated animals, HC‐9 mRNA (RT‐PCR) and protein (western blotting) were expressed in many tissues, with some of the highest levels in liver, stomach, ventral skin, and muscle. Across most tissues, realtime mRNA expression (qRT‐PCR) decreased in cold‐acclimated (5ºC) and frozen (−2.5 ºC) treefrogs, with recovery in treefrogs returned to 5ºC for 24 hours. Meanwhile, protein expression increased in the liver of cold and frozen treefrogs, but remained unchanged or decreased in all other tissues.HC‐9 expression is clearly regulated as a part of the Cope's Gray Treefrogs freeze tolerance mechanism. Contrary to our hypothesis, most tissues have decreased expression, which may reduce glycerol permeability to protect cells during the freeze process. Liver is a notable exception by increasing protein expression, which may facilitate the re‐uptake of glycerol during ice formation. Liver appears to play a critical role in the distribution of the cryoprotectant glycerol, and this study suggests HC‐9 facilitates in this role.Support or Funding InformationSupported by NSF IOS‐1121457.
Effects of High‐Fat Diets on Mortality and Feeding in the Tobacco Hornworm Caterpillar, Manduca sexta
Authors: Greenlee Kendra J , Cambron Lizzette D , Thapa Gita , Baldwin Maxwell 
Published: June 2021

Nutritionally‐balanced diets are important for fitness, but for insects the high energy value of fat storage is crucial for reproduction and development. Although studies have shown that insects fed high carbohydrate diets have increased fat storage, and excess fat storage in insects may have a fitness cost, little is known about how insects regulate fat consumption when presented with diets containing varying lipid content. To determine how insects respond to high fat diets, we fed caterpillars of different ages high (5.6%), medium (3.4%), or low (0.4%) fat diets. Young Manduca sexta larvae reared on high fat diet had 80% mortality and 43% lower body mass compared to those reared on medium or low fat diets. Older larvae showed no difference in mortality, but those fed a high fat diet had a lower average body mass by the third day of the fifth instar. Growth rates and development time also differed. High fat‐fed caterpillars wandered later than those fed low and medium fat diets. To test the hypothesis that the increased survival of older larvae was due to increased lipid transport, we measured mRNA expression of Apolipoprotein I and II (APO1 and 2), proteins responsible for transporting lipids to the fat body and to various tissues. APO1 and 2 expression did not differ with dietary fat content. In addition, we tested the hypothesis that caterpillars preferentially metabolize fat during the fifth instar. We measured oxygen consumption and carbon dioxide emission of caterpillars fed a high or low fat diet and calculated the respiratory quotient. To determine the mechanism underlying the decrease in body size, we tested the hypothesis that the high fat diet altered feeding. Caterpillars fed a high fat diet indeed ate less, as indicated by a decrease in food consumption and the number and mass of fecal pellets produced. These results suggest that increased dietary fat results in cessation of feeding in these insects. Due to conserved fat metabolism pathways in vertebrates and invertebrates, better understanding of the role of lipids in insects could contribute to knowledge of human metabolic diseases and provide support for using insect models for these studies.Support or Funding InformationThis research was funded in part by NSF EPS‐0447679 and IOS‐0953297 to K.J.G. and NIH National Center for Research Resources 2P20RR0l5566. The contents of this study are solely the responsibility of the authors and do not necessarily reflect the views of the NIH.
Endothelial Cell DNA Integrity following Anoxia/Reoxygenation Injury
Authors: Barr Johnny L , Gibson Ruby S , Dubick Michael A , Bowman Phillip D 
Published: June 2021

Ischemia/reperfusion injury (I/RI) is associated with trauma and various diseases including stroke, heart attack and hemorrhagic shock. This study investigated the extent of DNA damage in an in vitro system of hypoxia/reoxygenation that may be useful for screening for drugs to reduce this form of injury. The usual method of subjecting cells to reduced oxygen (O2) by purging with nitrogen is a slow process and does not reproduce the rapid local reduction in O2 that occurs in vivo during ischemia. Glucose oxidase (GOX), however, rapidly oxidizes extracellular glucose producing a very low O2 level (< 0.4 μM) within 5 min with only minor reductions in glucose levels. We previously showed 10 min of anoxia followed by reoxygenation of human umbilical vein endothelial cells (HUVEC) damaged various biomolecules, such as lipids, proteins, and DNA. In the present study, human umbilical vein endothelial cells (HUVEC) were subjected to GOX‐induced hypoxia for 10 min and returned to medium and normoxia for up to 6 hrs. The most commonly used biomarker of DNA oxidation, in epidemiological as well as experimental studies, is 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo), however this methods requires large amounts of DNA. Here we used a high‐throughput assessment of DNA damage utilizing a high‐sensitivity long‐run real‐time PCR technique for DNA‐damage quantification in both the mitochondrial and nuclear genome. Following hypoxia cells were provided normoxia (3% oxygen) for up to 6 hours, fixed in 50% isopropanol and DNA extracted with DNAzol (MRC, Cincinnati, OH). The isolated DNA was subjected to real‐time polymerase chain reaction (RT‐PCR) with primers designed to amplify large and small regions of mitochondrial and nuclear DNA where the extent of template amplification is inversely proportional to the lesion frequency within a given DNA sequence. Detected lesions per 10kb DNA were determined. Compared to hydrogen peroxide induced DNA damage, the test conditions produced extensive damage to both mitochondrial and nuclear DNA that could readily be detected by 4 and 6 hours. These results were also correlated with HPLC analysis of 8‐oxoguanine that indicated that this biomarker to oxidative stress was enhanced about 50%. This in vitro system of hypoxia/reoxygenation can be used to further understand responses of cells to I/RI and identify drugs and potential therapeutic windows in vitro that may be translated to ameliorate ischemia/reperfusion injury in vivo.Support or Funding InformationFunded by US Army Medical Research Materiel Command
Influence of social isolation on serotonergic fiber density in the auditory midbrain
Authors: Sandlain Rebecca L , Keesom Sarah M , Wise Bradley M , Hurley Laura M 
Published: June 2021

Animal communication involves both a “sender”, which emits a signal, and a “receiver”, which processes the signal. Signal processing can be shaped by past social experience. In the auditory midbrain, serotonin is a potential mechanism for linking social experience with auditory processing for three reasons: 1) the serotonergic system is sensitive to social experience in other brain regions; 2) changes in serotonin concentration reflect contextual features of social encounters; and 3) serotonin influences single auditory neurons’ response to sounds, such as conspecific vocalizations. Laboratory mice (Mus musculus) provide an excellent model for addressing this potential mechanism, because mice demonstrate sensitivity to social experience via a simple manipulation of social housing conditions, and because laboratory mice are being increasingly studied as a model of auditory communication. In this study, we investigated how social isolation affects serotonergic fiber density in the inferior colliculus (IC), an auditory midbrain region that demonstrates selectivity for species‐specific vocalizations. Male CBA/J laboratory mice were given different social experiences by housing mice for four weeks either individually or in a social group of three mice. Subsequently, immunohistochemistry with florescence was used to label the serotonin transporter (SERT) as a marker for serotonergic fibers. Coronal sections of brain tissue were imaged, and ImageJ was used to quantify fiber density as the percent area of the IC covered by fibers. Mice from both social treatments demonstrated dense coverage (around 30%) of the IC by serotonergic fibers, corroborating with previous studies that demonstrate innervation of the IC by the serotonergic system. Individually housed mice had qualitatively lower percent coverage of the IC by serotonergic fibers than socially housed mice, particularly in sections of the IC that were more caudal; however, there were no statistically significant differences between social versus isolated treatment groups. Whether the smaller qualitative differences over the entire IC could be driven by disparities in the effects of social isolation among subregions of the IC has not yet been explored. These preliminary results suggest that social experience does not affect serotonergic fiber density in the IC. However, this does not rule out influences of social experience on other aspects of serotonergic signaling in the auditory midbrain, including socially induced serotonin release and profiles of serotonin receptors.Support or Funding InformationNSF grants: IOS 1456298 and DBI 1460949
Inhibition of Endocannabinoid Degradation Improves Recovery of Neurobehavioral Function and Resolution of Neuroinflammation and Synaptic Hyperexcitability in a Rodent Model of Traumatic Brain Injury
Authors: Mayeux Jacques P , Katz Paige , Edwards Scott , Middleton Jason , Molina Patricia 
Published: June 2021

Traumatic brain injury (TBI) is an increasingly prevalent condition affecting soldiers, athletes, and motor vehicle accident victims. It is the most common cause of death for people under 30 and currently lacks effective therapeutic interventions. TBI is defined as a primary mechanical insult followed by a secondary cascade involving inflammation, apoptosis, release of reactive oxygen species, and excitotoxicity, all of which can cause neuronal death and poor recovery. Previously we showed that inhibition of monoacylglycerol lipase (MAGL), the enzyme involved in 2‐acylglycerol (2‐AG) degradation, with JZL184 following mild TBI attenuated neuroinflammation and improved recovery of neurobehavioral function during the early 72 h post‐TBI period. The aim of this study was to examine if common pathologies resulting from the injury, including inflammation, neurobehavioral disruption, and synaptic excitability, could be reversed during the acute recovery period up to 10 days post‐injury. Adult male Wistar rats underwent a 5‐mm left lateral craniotomy, and TBI was induced three days later by lateral fluid percussion. Thirty minutes post‐TBI, rats received intraperitoneal injections of vehicle (alcohol, emulphor, and saline; 1:1:18) or JZL184 (16 mg/kg). Neurobehavioral function, neuroinflammation (immunohistochemistry) and synaptic excitability (brain slice electrophysiological recordings) were assessed up to 10 days post‐TBI. JZL184 administered‐TBI animals had improved neurobehavioral recovery compared to vehicle‐injected TBI animals beginning 24 hours post‐injury and persisting for two weeks. JZL184 administration significantly improved both ipsilateral cortex (gray matter) and corpus callosum (white matter) neuroinflammation seven days post‐TBI. JZL184 administration significantly attenuated TBI‐induced increases in mEPSC frequency and amplitude at 10 days post‐TBI. Taken together these results show that EC degradation inhibition 30 minutes post‐TBI has potential therapeutic benefits that persist throughout the acute recovery period up to 10 days post‐injury. Current studies are investigating the mechanisms of EC degradation inhibition‐mediated improvements from TBI.Support or Funding InformationSupport T‐32 AA007577, DOD W81XWH‐11‐2‐0011
Identification of inhibitors of Cytochrome P450 (CYP) 1B1
Authors: Roach Tyler , Kennedy Larry 
Published: June 2021

Cytochrome P450(CYP)1B1 is known to be overexpressed in several types of human malignancies. The role of CYP1B1 in estradiol metabolism to catalyze the hydroxylation at the C‐4 position is of specific importance in estrogen‐related tumorigenesis. Finding a safe, potent inhibitor to this critical step could lead to chemoprevention of several forms of human cancer. We initially used the Chembridge online database (www.hit2lead.com) to perform similarity searches, using 2,3′,4,5′‐tetramethoxystilbene (TMS), a compound known to inhibit CYP1B1 activity, as the search target. Using A 50% similarity threshold, 64 compounds were ultimately tested at a single concentration for inhibition of CYP1B1 catalyzed ethoxyresorufin‐o‐deethylase (EROD) activity. Five compounds inhibited EROD activity to an extent equal to or greater than TMS, and were considered positive hits. CYP1‐selectivity studies were performed on these five compounds, establishing selectivity of CYP1B1 over both CYP1A1 and CYP1A2. Kinetic studies were used to determine the Ki and the mechanism of enzyme inhibition. Cell viability studies were performed using the MTT assay on breast cancer cell lines. Additionally, follow on studies have been performed using analogs of the original five compounds to continue the search for safe, potent inhibitors of CYP1B1.Support or Funding InformationONR
miRNA profiling in blood and tissue samples in non‐human primates in response to Yersinia pestis infection using next generation sequencing approach
Authors: Dimitrov George , Kumar Raina , Moyler Candace , Gautam Aarti , Miller Stacy‐Ann , Hammamieh Rasha , Jett Marti 
Published: June 2021

MicroRNAs (miRNAs) are a class of small non‐coding RNA molecules (20–30 nucleotides long) that regulate gene expression negatively by transcriptional inhibition or post‐translational degradation. It is increasingly clear that miRNAs are an important part of the host response to microbes. In the current study, African green monkeys (Cercopithecus aethiops) were challenged intranasally with aerosolized Yersinia pestis strain CO92. Blood and tissue samples were collected post‐exposure from 6 hr through 42 hr time points. We used a next generation sequencing approach to profile miRNA expression patterns in different tissues and blood samples. Y. pestis is a gram‐negative facultative anaerobe and the causative agent of plague, which throughout history has had devastating effects on human populations. Our data enabled us to examine miRNA expression profiles during infection and identify tissue‐specific expression patterns during progression of the disease. We observed 180 upregulated miRNAs and 272 downregulated miRNAs across all time points in blood samples. At the 24 hr time point there were 52 differentially expressed (DE) miRNAs in blood samples that targeted 5381 genes. Functional analysis of DE miRNAs and their targets showed activation of TGF‐β and STAT3 pathways. TGF‐ β activated kinase 1 (TAK1, also called MAP3K7) is a known key regulator that inhibits innate immune signaling. In the kidney tissues there were a total of 32 DE miRNAs across all time points. At the 24 hr time point there were 11 unique mature DE miRNAs in exposed animal tissues as compared with non‐exposed animal tissues. In the lung tissues there were 67 DE miRNAs across all time points. At the 24 hr time point there were seven mature DE miRNAs when compared with non‐exposed animal tissues. These seven miRNAs were also DE in the blood samples. In the mediastinal lymph node tissues we had 52 DE miRNAs, eight of which were in common with blood samples. In the submandibular lymph nodes there were 25 DE miRNAs, three of which were in common with blood. We found that blood, lung, submandibular lymph and mediastinal lymph tissues shares two common miRNAs: mml‐miR‐590‐5p, which has 301 gene targets, and mml‐miR‐451, which has 17 gene targets. Both of these miRNAs are known to contribute to cytokine and grown factor activity, and miR‐590‐5p is a known circulating biomarker miRNA in cardiovascular and inflammatory diseases. Follow‐up comprehensive investigations are currently underway to further elucidate disease progression.Support or Funding InformationThis work was supported by the Defense Threat Reduction Agency, contractTMTI0029_09_WR_T
Translocation of Gold Nanoparticles in Model Epithelial Cells (Caco‐2 Monolayers)
Authors: Yao Mingfei , He Lili , McClements David Julian , Xiao Hang 
Published: June 2021

At present, there is limited knowledge about the gastrointestinal fate and potential cytotoxicity of ingested inorganic nanoparticles. In this study, we determined the influence of particle size and concentration of gold nanoparticles (AuNPs) on their intestinal absorption, accumulation and cytotoxicity. AuNPs with different sizes (15, 50, and 100 nm) were subjected to an intestinal epithelium monolayer model to establish their patterns of absorption, accumulation and cytotoxicity. Results from inductively coupled plasma mass spectrometry (ICP‐MS) analysis revealed that as the mean particle diameter of the AuNPs decreased, their rate of absorption by the intestinal epithelium cells increased, but their cellular accumulation in the epithelial cells decreased. Moreover, fluoresce microscopy analysis indicated that accumulation of AuNPs caused cytotoxicity in the intestinal epithelial cells, which was evidenced by depolarization of mitochondria membranes. Our results demonstrated that particle size play an important role in dictating intestinal absorption, accumulation and cytotoxicity of AuNPs.Support or Funding InformationUSDA (2011‐67021, 2014‐67021)