bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating CRISPR Spacer Sequences of Escherichia coli in NJ/NY Metropolitan Area
Authors: Shah Rameen , Hassan Sherouk , Wei Yufeng 
Published: April 2020

Clustered regularly interspaced short palindromic repeats (CRISPR) is a DNA sequence, found in bacteria, that functions as protective system against bacteriophages. CRISPR DNA sequence is characterized by the presence of short, identical nucleotide repeats that are interspaced by spacers. The spacer sequences are unique in that they are records of viral infections experienced by the bacterium and act as an “immunization card”. If the same virus attempts to invade again, the spacer sequence recognizes it via the help of CRISPR‐RNA (crRNA) and CRISPR‐associated (Cas) proteins. Subsequently, Cas proteins cut the viral DNA to eliminate the threat. Thus, CRISPR/Cas system provides an effective mechanism for bacteria, such as Escherichia coli (E. coli), to resist infections from bacteriophage, but it could also pose challenges to phage therapy, which utilizes bacteriophages to kill bacteria. The focus of this research is to examine spacer sequences from soil samples collected around NY/NJ metropolitan area at various timepoints, locations, and weather conditions. To accomplish this goal, the genomic DNA was extracted from E. coli bacteria from the soil and purified. The CRISPR locus was amplified and purified using polymerase chain reaction (PCR) and gel electrophoresis, respectively. The sequences were run through the CRISPR Finder program to identify spacer sequences, and NCBI’s Blast alignment was carried out to determine the specific E. coli strain associated with the sequence. We have identified several new spacer sequences, which could putatively link the history of bacteriophage infections of the E. coli bacteria to the unique environments the bacteria are located.Support or Funding InformationWe would like to thank the Chemistry Department at NJCU. This research was funded by National Institutes of Health (NIH), grant number R21DA046223.
Membrane Substrate Transporters as Target to Re‐balance Cardiac Energy Metabolism to Mend the Failing Heart
Authors: Glatz Jan F.C. , Luiken Joost J.F.P. , Nabben Miranda 
Published: April 2020

BackgroundThere is growing recognition of the importance of energy metabolism in the cardiovascular system, and that disturbances therein are at the basis of causing cardiac disease. Specifically, chronic fuel shifts towards a single type of substrate are known to induce cardiac dysfunction. For instance, lipid overload due to Western life style or obesity eventually elicits diabetic cardiomyopathy, while excess glucose utilization is associated with hypertrophic heart failure (and vice versa). Therefore, interventions aimed at re‐balancing a tilted substrate preference toward an appropriate mix of substrates (in particular fatty acids and glucose) may result in restoration of cardiac contractile performance. Because the key‐governing kinetic step of substrate utilization is their sarcolemmal transport, we hypothesize that manipulating their recruitment to the sarcolemma is an effective approach to mend the failing heart.ResultsInhibition of the vesicular translocation of the fatty acid transporter CD36 to the sarcolemma by targeting endosomal H+‐ATPase in rat cardiomyocytes subjected to lipid overload completely restored both the fatty acid–glucose fuel balance and contractile function. Likewise, decreasing GLUT4‐mediated glucose uptake in experimental cardiac hypertrophy restored both the fuel balance and contractile function.ConclusionRe‐balancing cellular fuel supply, in particular with respect to fatty acids and glucose, appears an effective strategy to treat the failing heart. For such metabolic modulation therapy membrane substrate transporters, especially their subcellular recycling, are a promising target. Because vesicular trafficking involves >50 proteins and is highly (tissue‐)specific, manipulating transporter recycling would allow a heart‐specific approach.Support or Funding InformationMN is supported by the Dutch Heart Foundation, grant 2019T041.
Role of AQP1 and RhAG proteins in the deoxygenation of mouse red blood cells during aging
Authors: Zhao Pan , Taki Sara , Popple Jacob , Radford Thomas S. , Meyerson Howard J. , Occhipinti Rossana , Moss Fraser J. , Boron Walter F. 
Published: April 2020

The traditional view had been that all O2 moves through red blood cell (RBC) membranes by dissolving in and diffusing through the membrane lipid. In a previous abstract, we reported data obtained using stopped‐flow analysis of hemoglobin (Hb) absorbance spectra on RBCs from wild‐type (WT) vs. double‐knockout (dKO) mice genetically deficient in both aquaporin‐1 (AQP1) and the rhesus (Rh) protein RhAG. We found that the rate constant of Hb deoxygenation (kHbO2)—which includes intra/extracellular diffusion and membrane permeability—was ~31% lower for RBCs from dKO than for WT mice. From these data, we computed that, together, AQP1 and the Rh complex account for ~60% of membrane O2 permeability. We now examine the effect of aging on kHbO2 of RBCs from WT vs. dKO mice, studying mice at six ages (2–3 months, 6 months, 12 months, 18 months, 24 months, and 29 months). The kHbO2 of RBCs from WT mice are indistinguishable from age 2–3 months through 12 months. However, compared to 2–3 months, kHbO2 is 9% lower at 18 months (p<0.05, N = 6), 12% lower at 24 months (p<0.05, N = 5), and 13% lower at 29 months (p<0.05, N = 3). For RBCs from dKO mice, kHbO2 starts off ~30% lower than that of WT mice at 2–3 months (see above), and remains at this same depressed level through 29 months (N = 5 to 6 at 6–24 months, 4 at 29 months). In the case of WT mice at 24 months, our hematology data—which show decreases for both mean corpuscular volume and mean corpuscular hemoglobin—cannot explain the observed decreases in kHbO2. DIC microscopy of living RBCs and microscopic observation of stained blood smears with RBCs from 3 months and 29 months with both WT and dKO groups confirm that RBCs are normal biconcave disks. Preliminary data from mass spectroscopy of RBC ghosts from WT mice (N = 1 each for 2–3 months and 29 months) suggests that, compared to 2–3 months, membrane protein levels at 29 months are lower by 11% for AQP1, 19% for RhAG, 27% for mRh, and 22% for AE1. We conclude from our dKO data that the non‐AQP1/RhAG component of RBC membrane O2 permeability is independent of aging. We hypothesize that the 12–13% decrease of kHbO2 at 24–29 months for WT mice is due in part to decreased expression of AQP1 and the Rh complex (i.e., the AQP1 and RhAG components of O2 membrane permeability decline with aging).Support or Funding InformationN00014‐15‐1‐2060 Gas Channels, ONRand N00014‐16‐1‐2535 ONR‐MURI
Carotid Artery Stiffness and Elasticity in Angiotensin II Treated Mice
Authors: Pardo Paulina V. , Chambers Laura C. , Yen Ting-Chieh , Jackson William F. , Dorrance Anne M. 
Published: April 2020

Hypertension, a risk factor for dementia, can impair the structure and function of cerebral arteries. Carotid arteries supply approximately 80% of the of blood required by the brain; increased stiffness and reduced distensibility in these arteries contribute to cerebrovascular disease. Angiotensin II (AngII)‐induced hypertension negatively affects the cerebral vasculature; it is possible that this is in part a response to increased carotid artery stiffness because stiffer arteries allow more of pulse pressure to be transmitted to the downstream vasculature. We hypothesized that AngII treatment in mice would lead to an increase in carotid artery wall stiffness, a loss of distensibility and impaired cognitive function. Sixteen‐week‐old C57BL/6 female mice were treated with AngII (1200 ng/kg/min via osmotic mini pump) for four weeks. Shams served as controls. Blood pressure was measured using tail‐cuff plethysmography. Barnes Maze was used to assess spatial memory and a nesting test was used to assess executive function. At euthanasia, arteries were isolated and mounted in a pressure myograph. To assess passive structure carotid arteries were incubated in Ca2+ free physiological salt solution (PSS) containing EGTA (2μM) and sodium nitroprusside (100μM). A pressure‐response curve was constructed; the intraluminal pressure was increased from 3 to 140 mmHg in 20‐mmHg increments and lumen diameter (μm), outer diameter (μm), and wall thickness (μm) were measured at each pressure. These data were used to calculate wall‐to‐lumen ratio, distensibility, circumferential wall stress (dynes/cm2), and wall strain. b‐coefficient, a measure of artery stiffness was calculated from stress/strain curves. Data are presented as means ± SEM (n=5–12). AngII infusion resulted in elevated systolic blood pressure (Sham: 142 ± 7; AngII 179 ± 7 mmHg; p<0.05). Barnes Maze tests showed that spatial memory was not impaired by AngII treatment (Sham: 25.2 ± 3.2; AngII 30.1 ± 3.4 seconds spent in target quadrant; p>0.05). Nesting test showed that executive function was impaired by Ang II treatment (Sham: 41.6 ± 7.7; AngII 10.5 ± 1.3 % nestlet shredded; p<0.05). There was no change in b‐coefficient in the AngII treated group (Sham: 4.91± 0.84; AngII 5.73 ± 1.20 p>0.05). Distensibility was also unaffected by Ang II treatment (Sham: 0.39 ± 0.01; AngII 0.39 ± 0.01; p>0.05). The change in executive function does not appear to be related to changes in carotid artery stiffness. Future studies will assess cerebral artery function. These data support the hypothesis that young female hypertensive mice are protected from changes in spatial memory, however, the impaired executive function is suggestive of initial negative changes in cognition.Support or Funding InformationRO1‐HL‐137694‐01, AM Dorrance and WFJackson
AMPK is Activated During Sleep Deprivation in Caenorhabditis elegans Ryan Schuck, Edwin Li, Matthew D. Nelson Saint Joseph’s University, Department of Biology, Philadelphia, PA 19131
Authors: Schuck Ryan James , Li Edwin , Nelson Matthew D. 
Published: April 2020

Sleep conserves energy and allows for its reallocation towards biosynthetic pathways, suggesting that one of sleep’s functions is cellular restoration. During times of sickness, cells are damaged and sleep is heightened, supporting an additional immune function for sleep. In some cases, sleep is directly induced by signaling mechanisms of the immune response. Work in model systems, such as Caenorhabditis elegans and Drosophila melanogaster, have identified a protective sleep state called stress‐induced sleep (SIS), which resembles sleep during sickness. SIS occurs following exposure to environmental stressors such as noxious temperatures, osmotic imbalances, physical damage and UV irradiation. While the signaling molecules regulating SIS have begun to be elucidated, the mechanisms linking sleep to cellular restoration are still unknown. A master regulator of nutrient/energy sensing, adenosine monophosphate‐activated protein kinase (AMPK), which is highly conserved from invertebrates to mammals, is a candidate for such a link. In mammalian cells, AMPK is activated by low levels of ATP and oxidative stressors, the latter occurring through the overproduction of reactive oxygen species. In both cases, activation of AMPK requires phosphorylation of a threonine residue (T172). We used CRISPR to make an AMPK mutant in C. elegans in which the predicted, homologous T172 site is changed to an alanine, to determine if this amino acid is conserved functionally. We find that these mutants live significantly shorter lives than wild type animals following exposure to paraquat, a known inducer of oxidative stress. Moreover, these animals provide us with an important control for testing our broader hypothesis that AMPK links sleep to cellular repair during SIS. To test this, we conducted a temporal western blot analysis, assessing AMPK’s phosphorylation levels during SIS in both wild type animals and those that are genetically sleep deprived. We find that AMPK is not significantly activated during times of normal SIS, however, mutant animals that lack sleep‐promoting neuropeptides, display increased AMPK activation 24 hours after stress exposure. These mutants also display reduced longevity in response to oxidative stress. We hypothesize that sleep deprivation promotes an increase in AMPK phosphorylation in an attempt to provide cellular protection/repair during SIS.Support or Funding InformationNSF Award #1845020 (PI Nelson)
Exercise Pressor Reflex in UC Davis Type‐2 Diabetes Mellitus (UCD‐T2DM) Rats Prior to the Onset of Diabetes
Authors: Huo Yu , Grotle Ann-Katrin , Lee Junghoon , Ybarbo Kai M. , Graham James , Stanhope Kimner L. , Havel Peter J. , Harison Michelle L. , Stone Audrey J. 
Published: April 2020

Both the exercise pressor reflex and mechanoreflex are exaggerated after the development of T2DM in UCD‐T2DM rats. Whether the pressor reflex is also exaggerated before UCD‐T2DM rats developed diabetes is not known. Therefore, the purpose of this study was to determine if both the exercise pressor reflex and mechanoreflex are exaggerated in the UCD‐T2DM rats before the onset of diabetes.MethodsFor this study, we used young (8–12 wk old), male (n=12) and female (n=10), non‐diabetic UCD‐T2DM rats and age‐matched, male (n=11) and female (n=7), healthy Sprague Dawley rats. The exercise pressor reflex was evoked by statically and intermittently contracting the hindlimb muscle for 30s, and the mechanoreflex was evoked by passively stretching the Achilles tendon for 30s. Changes in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were continuously measured during both contraction and stretch.ResultsWe found that fasting blood glucose for both groups was below the threshold for diagnosis of diabetes (200mg/dl). Statically contracting the muscle did not significantly increase peak MAP (CTL: 11 ± 1 mmHg, n=12; UCD‐T2DM: 14 ± 2 mmHg, n=13, p>0.05), HR (CTL: 12 ± 4 bpm, n=12; UCD‐T2DM: 14 ± 5 bpm, n=13, p>0.05), or RSNA (CTL: 40 ± 20 %, n=4; UCD‐T2DM: 39 ± 23 %, n=3, p>0.05) in UCD‐T2DM rats compared to control rats with similar developed tensions. Likewise, intermittently contracting the muscle did not significantly increase peak MAP (CTL: 14 ± 2 mmHg, n=13; UCD‐T2DM: 14 ± 2 mmHg, n=6, p>0.05), HR (CTL: 16 ± 3 bpm, n=13; UCD‐T2DM: 14 ± 5 bpm, n=6, p>0.05), or RSNA (CTL: 73 ± 42 %, n=5; UCD‐T2DM: 34 ± 14 %, n=2, p>0.05) in UCD‐T2DM rats compared to control rats with similar developed tensions. Moreover, stretching the Achilles tendon did not significantly increase peak MAP (CTL: 15 ± 3 mmHg, n=11; UCD‐T2DM: 13 ± 1 mmHg, n=14, p>0.05), HR (CTL: 11 ± 4 bpm, n=11; UCD‐T2DM: 14 ± 2 bpm, n=14, p>0.05), or RSNA (CTL: 60 ± 12 %, n=3; UCD‐T2DM: 24 ± 19 %, n=4, p>0.05) in UCD‐T2DM rats compared to control rats with similar developed tensions.ConclusionWe conclude that both the exercise pressor reflex and the mechanoreflex are not exaggerated in UCD‐T2DM rats before the onset of diabetes. This study suggests that young UCD‐T2DM rats have similar MAP, HR, and RSNA responses to both muscle contraction and tendon stretch as seen in age‐matched Sprague Dawley rats prior to the onset of diabetes.Support or Funding InformationThis project was supported by NIH R01 HL144723.
Hypoxia and Hypercapnia Diminish Intestinal Barrier Integrity in a Human Enteroid Model System
Authors: Weber Gregory J. , van Sambeek Dana M. , Pearce Sarah C. 
Published: April 2020

Warfighters are commonly exposed to unique physiologic stressors that affect their physical and mental performance, as well as their overall health. The adverse impact of these stressors may be in part due to effects on the microorganisms residing in the human gut, collectively known as the gut microbiome. Numerous studies have revealed the gut microbiome can influence multiple physiological process, including immunity, nutrient metabolism, and cognition. Hypoxia refers to a lack of oxygen in a given system and exists as a gradient in intestinal tissue, however, the intestine is sensitive to changes in oxygenation and can alter normal cellular process, such as inflammation and barrier function. Similarly, an excess of carbon dioxide (CO2), or hypercapnia, also affects intestinal tissue, as levels of CO2 are inversely coupled with the level of oxygen and are found to be elevated in hypoxic tissue. The GI tract is thought to have a low‐grade, albeit constant, level of inflammation and is home to multiple immune cell types. In addition, both epithelial and immune cells in the intestine can sense and respond to various stimuli including pathogens, and compromises to intestinal barrier integrity due to injury or stress can result. In this study, we sought to determine the extent to which hypoxia and hypercapnia degrade intestinal cellular integrity through inflammation‐mediated mechanisms. Intestinal epithelial organoids (enteroids) derived from the duodenum were exposed to hypoxic (0.5% O2) or hypercapnic (10%, 20% CO2) conditions for a period of 72 hours. Initial results indicated intestinal barrier integrity was compromised in hypoxia and 20% CO2 conditions as evidenced by transepithelial electrical resistance (TEER), with hypoxia (0.7 Ω) and 20% CO2 (0.85 Ω) being significantly lower than control (1.19 Ω; P < 0.05). The Quantigene Plex array system was used to interrogate a panel of intestinal and inflammatory markers. Preliminary results suggest alterations in tight‐junction markers as well as in genes involved in inflammatory response pathways. In addition, IL‐8, a macrophage‐mediated chemokine produced in intestinal epithelial cells and involved in the innate immune response, was found to be significantly downregulated in hypoxia (58.7 pg/mL) and hypercapnia (42.5 pg in 10% CO2, 49.7 pg/mL in 20% CO2) compared to control (190.7 pg/mL; P < 0.05). In sum, our data indicate hypoxic and hypercapnic conditions lead to alterations in molecules involved in the innate immune response pathway, tight‐junction markers, as well as compromised epithelial barrier integrity. Future experiments will be aimed at identifying the mechanism(s) responsible for diminished intestinal barrier function as well as identify potential preventative strategies.Support or Funding InformationFunding support provided by the Office of Naval Research award #N0001419MP00144 to SP FY18‐FY20.
MDMA and TAAR1‐mediated RhoA Activation in Serotonin Neurons
Authors: Underhill Suzanne , Amara Susan 
Published: April 2020

We have recently described the biochemical cascade through which the psychostimulant amphetamine leads to internalization of the dopamine and norepinephrine transporters, (DAT and NET). Amphetamine is a relatively high affinity substrate for DAT and NET and they both serve as efficient conduits for amphetamine entry into the cell. Once inside the cell, amphetamine activates the trace amine associated receptor 1 (TAAR1), a GPCR that we have shown couples to both GS and G13 signaling pathways. TAAR1 signaling through G13 increases the activity of RhoA, a small GTPase that regulates trafficking of the catecholamine carriers and increases their internalization. We wanted to investigate if similar G‐protein coupled signaling events also occur in serotonergic neurons. Although amphetamine does not have a particularly high affinity for the serotonin transporter (SERT), other TAAR1 ligands do, including 3,4‐methylenedioxymethamphetamine (MDMA).We developed primary cultures from the raphe nuclei of E15 mice. 3H‐serotonin uptake mediated by these cultures was largely citalopram sensitive, indicating SERT‐mediated serotonin transport. Pre‐treatment of these cultures with MDMA resulted in a decrease of SERT activity that could be blocked by an inhibitor of RhoA. Similarly, by total internal reflection fluorescence microscopy in HEK293 cells we found internalization of GFP‐tagged SERT in response to MDMA was blocked by a RhoA inhibitor. In HEK293 cells in which the TAAR1 gene has been deleted, MDMA did not trigger SERT internalization. In noradrenergic neurons cultured from the locus coeruleus or dopamine neurons from the midbrain, only higher doses of MDMA pretreatment could decrease transporter activity at the cell surface, likely reflecting the differences in affinity of the drug for the different monoamine transporters.These data indicate that pharmacological agents can regulate TAAR1 signaling in serotonergic, as well as catecholaminergic neurons and that the selectivity of TAAR1 agonists depends not only on affinity for the GPCR but also on their transport efficiency. Moreover, these findings suggest that the activation of TAAR1 in serotonin neurons and the subsequent internalization of surface SERT likely contribute to the modulation of serotonin signaling observed in response to MDMA.Support or Funding InformationThis work was supported by Intramural Research Program at the NIH, NIMH (ZIAMH002946).
Neuropeptide Y and Pial Artery Vasomotor Control: An Ordered Affair
Authors: Morse Cameron , Boerman Erika M. , Thorne Pamela K. , Emter Craig A. , Padilla Jaume , Olver T. Dylan 
Published: April 2020

Evidence indicates sympathetic neurotransmitters influence cerebral vasomotor tone in a branch order‐dependent manner. Further, in addition to eliciting vasoconstriction, sympathetic co‐transmitter neuropeptide Y (NPY) may also elicit dilation in the cererbrovasculature depending on whether it acts on vascular smooth muscle or the endothelium. Herein, we investigated vasomotor responses to NPY in pial arteries (intracranial arteries on the surface of the brain) along the cerebrovascular tree. Female pigs (n=15, total arteries=88; age=2±0 months) were euthanized and their brains were harvested. First order (1A; n=7–8 per experiment), second order (2A; n=7–8 per experiment), and third order (3A; n=7–8 per experiment) pial arterial branches from the middle cerebral artery were dissected for pressure myography experiments. Following stabilization, NPY was administered either extra‐luminally (1e‐10 ‐‐to 1e‐6 M; half log doses, n=8 pigs) or intra‐luminally (1e‐9 M, n=7 pigs). To evaluate the contribution of nitric oxide (NO) on vasomotor responses to NPY, a subset of arteries were pre‐treated with a NO synthase (NOS) inhibitor (L‐NAME 3e‐4 M) 30 minutes prior to NPY administration. Whereas extra‐luminal NPY elicited vasoconstriction (24/24 arteries), intra‐luminal NPY tended to elicit vasodilation (16/21 arteries). Maximal vasoconstriction (% of baseline diameter) in response to extra‐luminal NPY was greater in 3a vs. 2a and 1a pial arteries (1a=13±1%; 2a=15±2%; *3a=23±4%; p≤0.01). Differences between 1a and 2a pial arteries were not significant (p=0.17). Pre‐treatment with a NOS inhibitor enhanced the maximal vasoconstriction in 1a, 2a and 3a pial arteries (grouped data; untreated=17±1%; *following NOS inhibition=29±1%; p<0.01). Dilation (% of baseline diameter) in response to intra‐luminal NPY was greater in 1a and 3a vs. 2a pial arteries (*1a=4±4%; 2a=−3±6%; *3a=9±4%; p≤0.01). Of note, whereas 86% of 1a and 3a pial arteries dilated, only 57% of 2a pial arteries dilated following transient exposure to intra‐luminal NPY. NOS inhibition abolished NPY‐induced dilation and promoted an NPY‐mediated vasoconstriction (grouped data; untreated=4±3%; *following NOS inhibition=−15±3%; p<0.01). In summary, NPY regulates cerebral vasomotor control through both vasoconstriction and dilation. These results indicate vasomotor responses to NPY are branch order‐dependent and most apparent in 3a pial arteries. NOS inhibition exacerbated NPY‐induced constriction and ablated NPY‐induced dilation, implicating NO signaling as a regulator of NPY‐dependent effects on cerebrovascular tone.Support or Funding InformationThis work was supported by a MU CVM COR grant and a SHRF Establishment grant (#4522).