bluuebunny/crossref_metadata_2025 huggingface dataset:
A Fluorescent Based System to Monitor 2‐micron Copy Number in Saccharomyces cerevisiae
Authors: Howe Max , Butler Scott , Callahan Kevin
Published: April 2020

The 2‐micron plasmid is a small genetic element that persists in Saccharomyces cerevisiae with chromosome like stability. It encodes for four proteins (Rep1, Rep2, Raf1, and Flp) that appear to provide no advantage for the host cell, but instead have a role in replicating and maintaining a steady state concentration of the plasmid‐‐
1. Flp recombinase is known to promote amplification of the 2‐micron; disruption of the FLP gene is a valid way of preventing the necessary amplification of the plasmid to pass itself on. The 2‐micron is a selfish DNA element that is able to maintain a copy number of 40–60, however its ability to maintain these levels is not completely understood. To study this regulation we have established a quantitative and efficient means to monitor copy number. We used CRISPR‐Cas9 to replace ADE2 (encoded on 2‐micron/ADE22) with mCherry, enabling copy number to be monitored using flow cytometry. Preparation of the strains required no additional staining, allowing for quick processing and analysis. To establish the sensitivity of the system, mCherry expression was monitored in strains that had ectopic expression of the flp‐H305L mutant, which destabilizes the 2‐micron plasmid
2. We observed a decrease in mCherry expression in these strains, consistent with the role Flp plays in 2‐micron amplification. This reduction in copy number was confirmed using quantitative PCR to examine expression of mCherry from the 2‐micron. Current studies are employing this system to monitor how copy number is affected by two long non‐coding RNAs (lncRNA), which are expressed from the plasmid and regulated by Rrp6p and the TRAMP complex, components of the nuclear RNA surveillance pathway.

Support or Funding Information

SJFC internal funds
Progesterone prevents oxidative stress and respiratory dysfunctions in female rats exposed to intermittent hypoxia.
Authors: Bairam Aida , Laouafa Sofien , Marcouiller François , Pialoux Vincent , Joseph Vincent
Published: April 2020

The occurrence of sleep apnea (SA) increases after menopause in women, which is partly related to the decreased levels of ovarian hormones. Because progesterone (Prog) is a neuroprotective hormone and a potent respiratory stimulant, we tested the hypothesis that Prog reduces the apnea frequency and the oxidative stress induced by IH. We used OVX female rats implanted with an osmotic pump delivering vehicle (OVX‐Veh) or prog (4mg/kg/day). Two weeks following the surgery, rats were exposed to room air (RA), or IH for 7 days (nadir 10% O2, 10 cycles/hour, 8 hours/day). We studied three groups: OVX‐Veh‐RA; OVX‐Veh‐IH; and OVX‐Prog‐IH. At the end of exposure period, we measured the mean arterial pressure (tail cuff) and assessed the frequency of apneas during sleep and the ventilatory responses to hypoxia using whole body plethysmography. The brain was quickly harvested to measure the activity of the pro‐oxidant enzyme NADPH oxidase (NOX), and the antioxidant enzymes (mitochondrial and cytosolic superoxide dismutase ‐ SOD) in the cerebral cortex and brainstem. IH exposure increased the mean arterial blood pressure, the frequency of apneas during sleep, and the hypoxic ventilatory response. Prog did not prevent the elevation of blood pressure, but effectively reduced the frequency of apneas during sleep and prevented the elevation of the hypoxic ventilatory response. In the cerebral cortex, IH increased NOX activity, and decreased the activity of the cytosolic and mitochondrial SOD, these effects were prevented by Prog. None of these enzyme activities were altered by IH or Prog supplementation in the brainstem. We conclude that Prog prevents the respiratory alterations induced by IH, and reduces oxidative stress in the brain cortex. These studies are useful to better understand the responses to IH and sleep apnea in women particularly after menopause.

Support or Funding Information

Supported by CIHR.
Oxygen availability and mitochondrial respiration: Impact on permeabilized and isolated in vitro preparations
Authors: Park Soung Hun , Broxterman Ryan M. , Craig Jesse C. , Richardson Russell S.
Published: April 2020

Skeletal muscle mitochondrial respiration assessed in vitro is typically performed in hyperoxia utilizing either permeabilized fibers or isolated mitochondria preparations. However, although it remains to be comprehensively assessed, growing evidence supports that mitochondrial respiration is likely influenced by oxygen availability and this may have a different impact in these two preparations. Therefore, we tested the hypotheses that hyperoxia would 1) increase peak mitochondrial respiration (Vmax) in permeabilized fibers, but not in isolated mitochondria, without altering submaximal respiration in either preparation, and 2) not alter the mitochondrial critical partial pressure of O2 (PO2) in either preparation.

Methods

Fiber permeablization and mitochondrial isolation techniques were performed on muscle samples, and mitochondrial respiration was measured by high‐resolution respirometery (O2k) in both moderate (MOD, 100–125 mmHg) and high (HIGH, 250–300 mmHg) initial PO2 conditions, for both preparations. Respiration and chamber PO2 were continuously measured until the cessation of respiration, allowing for the assessment of Vmax, submaximal respiration, and critical PO2.

Results

Vmax was significantly greater in HIGH than MOD with permeabilized fibers (~57%, p<0.05, effect size (d) =2.8), but was not significantly different between conditions with isolated mitochondria (~1%, p>0.05, d=0.07). Submaximal respiration was not significantly different between HIGH and MOD conditions with permeabilized fibers (~4%, p>0.05, d=0.1), but was significantly lower in HIGH with isolated mitochondria (~33%, p<0.05, d=2.4). The critical PO2 with isolated mitochondria was lower in HIGH (0.48±0.25 mmHg) compared to MOD (0.97±0.08 mmHg, p=0.1, d=1.5). In contrast, in the permeabilized fibers, in both MOD and HIGH, there was no precipitous fall in respiration with falling oxygen availability, and respiration ceased despite 3–10 mmHg PO2 remaining in the chamber.

Conclusion

These findings reveal differing effects of oxygen availability on mitochondrial respiration, dependent upon the mitochondrial preparation, which need to be considered when making such measurements.

Support or Funding Information

National Heart, Lung, and Blood Institute at the National Institute of Health (PO1 HL1091830) and Veterans Administration Rehabilitation Research and Development Service (E6910‐R, E1697‐R, E1433‐P, E9275‐L and E1572‐P)
A meta‐analysis of sex inclusiveness within textbooks in the anatomic sciences
Authors: Barry Kelly , Rae Guenevere
Published: April 2020

Gender and sex minorities have been declared a health disparity by the NIH and discussions pertaining to inclusiveness of LGBTQI+ in medical curricula have soared. This study aimed at analyzing the current presentation of sex, reproductive anatomy and sexual function within gross anatomy, histology, embryology and neuroanatomy textbooks. Content analyses were performed using grounded theory approaches to measure the ideas and concepts presented by the authors about male, female and intersex anatomy, and also male, female and intersex sexual function. The analyses included breadth of topics covered, for example, whether or not the clitoris was included in the female reproduction chapter or whether or not female sexual function was described. Also, inclusive language pertaining to the anatomic variation in sexual anatomy and sexual function was noted as well as the devotion of words spent to each topic within the textbook chapter. In text that presented intersex and anatomic variation topics, a second content analysis was performed to describe the “inclusiveness” of the language used and presentation style when discussing individuals with atypical sex and/or gender anatomy and functioning. Overall, 6 gross anatomy, 9 histology, 5 embryology and 3 neuroanatomy textbooks were analyzed, some in a retrospective fashion to highlight changes in editions. Results indicate that there are inconsistent patterns of addressing sexual anatomy and functioning within textbooks of the anatomic sciences, with different fields (i.e. embryology versus gross anatomy) employing a different subset of approaches.
Concomitant Improvement of Synaptic Decline and NASH using Fermentable Fiber Inulin
Authors: Wan Yu-Jui Yvonne , Jena Prasant , Lucente Jacopo , Jin Lee-Way
Published: April 2020

Introduction

A Western diet with enriched sugar and fat is implicated in cognitive impairment and the development of non‐alcoholic steatohepatitis (NASH). This study examines the impact of fermentable fiber inulin in shifting gut microbiota and metabolites to affect liver and brain health in Western diet‐fed mice.

Methods

C57BL/6 mice were fed a healthy rodent control diet (CD) or a fructose, palmitate, and cholesterol (FPC)‐enriched diet after weaning until they were 3‐months old. Then, FPC diet‐fed mice were supplemented with or without 6% inulin for 5 months. All the mice were euthanized when they were 8 months of age.

Results

Long‐term consumption of the FPC diet induced NASH with elevated IL‐17A signaling. In addition, FPC diet reduced hippocampal long‐term potentiation (LTP) and induced microglia activation. Similarly, FPC diet induced Th17 pathway driven hepatic IL17A production along with increased IL17A inducers including Il1β, Il6, and Tgfβ. In contrast, inulin supplementation reduced these changes. Furthermore, FPC diet intake induced gut dysbiosis by increasing Proteobacteria whereas inulin supplementation prevented such changes. In the microbiota families Coriobacteriaceae and Erysipelotrichaceae, which are implicated in cholesterol metabolism, and the genus of Erysipelotrichaceae and Allobaculum also shifted in the same direction in response to FPC intake and inulin supplementation. Accordingly, cecum cholesterol and zymosterol, a precursor of cholesterol, were increased in FPC diet‐fed mice but reduced in inulin‐supplemented mice. In addition, FPC diet‐fed mice had reduced bile acid receptors TGR5 and FXR signaling, and inulin supplementation reversed such changes.

Conclusion

The data suggest that inulin via gut fermentation shifts gut microbes, alters IL‐17A signaling, as well as activates bile acid receptor‐regulated pathways to coordinately ameliorate impaired neuroplasticity and neuroinflammation as well as NASH found in Western diet‐fed mice. Together, diet simultaneously affects brain and liver health.

Support or Funding Information

This study is supported by grants funded by National Institutes of Health U01CA179582 and R01CA222490 and Mouse Metabolic Phenotyping Center (MMPC) Microbiome Program (30835‐60), Subaward A19‐2654.
The protective effect of vasopressin deficiency in liver fibrosis in rats with chronic portocaval anastomosis
Authors: Gonzalez Yesenia Danyeli Navarro , Quintanar-Stephano Andrés , Ventura-Juarez Javier , Muñoz-Ortega Martín Humberto , González-Blas Daniel , Tinajero-Ruelas Manuel
Published: April 2020

Liver fibrosis occur in liver diseases and its complications is cause of the increased rate of human mortality. The disease is characterized by the gradual accumulation of scar tissue in the liver parenchyma with decrease in metabolic capacity and liver failure. It is known that arginine vasopressin (AVP) participate in the regulation of the immune responses, including the scarring. However, the effect of how AVP regulates this process is not clear. Here we study the role of the AVP deficiency induced by the neurointermediate pituitary lobectomy (NIL) in rats with chronic anastomosis portocaval (APC) model (sixteen weeks). Groups of young male Wistar rats of 8–10 each were prepared: Sham operated (SHAM), APC, LNI and APC+NIL. Blood, plasma and serum biochemical variables were assessed: prothrombin time (TP), blood NH3, transaminases (TGO and TGP), bilirubins and γ‐Glutamyl Transpeptidase (GGT). Liver histological slides were stained with H‐E, Masson, Sirius Red and PAS. Results showed that in the APC group: the TP, NH3, TGO and TGP, bilirubins and GGT were significantly increased, whereas serum glucose levels, cholesterol, triglycerides, albumin, total proteins and the liver glycogen content where significantly augmented. Comparisons between SHAM and NIL groups did not shown significant differences in all these variables. As compared with the APC, the APC+NIL group developed a significant improvement in all these variables. The liver histopathology showed that the APC group developed a significant increase in fibrosis septa, inflammatory infiltrates and diminution of the glycogen content. The NIL group we do not have available data yet. In the NIL+APC group histology data showed a significant liver regeneration; less fibrosis, disappearance of inflammatory infiltrates and liver glycogen restoration.

Conclusions

Results indicate the AVP play a role in liver fibrosis (demonstrated by the AVP deficiency). Results also suggest that AVP antagonists may be used for liver fibrosis treatment.

Support or Funding Information

Universidad Autónoma de Aguascalientes PIFF14‐1 andCONACYT 221262. México
White Mountain Expedition 2019: Peaks and Valleys ‐ Oscillatory cerebral blood flow at high altitude
Authors: Anderson Garen K. , Rosenberg Alexander J. , Barnes Haley J. , Bird Jordan , Pentz Brandon , Byman Britta R.M. , Jendzjowsky Nicholas , Wilson Richard J. , Day Trevor A. , Rickards Caroline A.
Published: April 2020

Introduction

Cerebral tissue oxygenation can be impaired by decreases in oxygen delivery as a result of reduced cerebral blood flow, and environmental conditions such as ascent to high altitude. Recent evidence suggests that an oscillatory pattern in cerebral blood flow (at ~0.1 Hz) may protect cerebral oxygenation under conditions of cerebral hypoperfusion. In this study, we hypothesized that inducing oscillations in cerebral blood flow at 0.1 Hz would protect cerebral blood flow and cerebral tissue oxygen saturation during exposure to combined simulated hemorrhage and sustained hypobaric hypoxia (ascent and partial acclimatization to high altitude).

Methods

8 healthy human subjects (4 M, 24.7 ± 4.1 y; 4 F, 34.3 ± 8.3 y) participated in two experiments at high altitude (White Mountain, California, USA; altitude, 3800 m): 1) a control condition (CTRL) where lower body negative pressure (LBNP) was used to induce central hypovolemia by reducing chamber pressure to −60 mmHg for 10‐min, and 2) oscillatory LBNP (OLBNP) where chamber pressure was reduced to −60 mmHg, then oscillated every 5‐s between −30 mmHg and −90 mmHg for 10‐min (0.1 Hz). Measurements included internal carotid artery (ICA) blood flow via duplex Doppler ultrasound, middle cerebral artery velocity (MCAv) via transcranial Doppler ultrasound, and cerebral tissue oxygen saturation via near‐infrared spectroscopy. Frequency analysis (via fast Fourier transform) was performed to verify that oscillations in mean MCAv were generated at ~0.1 Hz. Data were analyzed with a linear mixed‐model. All data are represented as mean ± SE.

Results

Low frequency power (0.07–0.15 Hz) in mean MCAv increased during OLBNP vs. CTRL (P = 0.02). OLBNP did not protect ICA flow (OLBNP: −32.5 ± 4.5 Δ%; CTRL: −19.9 ± 8.9 Δ%; P = 0.18) or mean MCAv (OLBNP: −18.5 ± 3.4 Δ%; CTRL: −15.3 ± 5.4 Δ%; P = 0.58), but cerebral tissue oxygenation was protected (OLBNP: −0.67 ± 1.0 Δ%; CTRL: −4.07 ± 2.0 Δ%; P = 0.004).

Conclusions

These results support our hypothesis that inducing oscillatory blood flow leads to protection of cerebral tissue oxygenation, despite no differences in ICA blood flow or mean MCAv. Overall, these data suggest that therapies using oscillatory perfusion may help preserve cerebral tissue oxygen saturation under conditions of reduced oxygen delivery.

Support or Funding Information

AHA 17GRNT33671110
Potential Involvement of Peripheral Opioid Receptors in the Development of Tolerance to Mu‐Opioid Receptor Agonists
Authors: Sears Bryan , Voight Brett , Jutkiewicz Emily
Published: April 2020

Clinically used opioids are effective analgesics but are highly associated with many adverse effects including physical dependence, tolerance development, rewarding properties and respiratory depression, the cause of opioid‐related overdoses and deaths. The goal of this study is to evaluate tolerance development to centrally and peripherally‐acting mu‐opioid receptor (MOR) agonists. In this study, we used a model of acute, peripherally‐mediated visceral pain, the acetic acid stretch assay (AASA) and an inflammatory model of pain measuring mechanical allodynia using von Frey monofilaments. In the AASA, loperamide and morphine displayed acute antinociceptive effects with ED50 values of 0.62 and 0.15 mg/kg, respectively. Naloxone pretreatment attenuated the acute antinociceptive effects of both loperamide and morphine. Following 5 days of 2x daily drug administration, the ED50 for the loperamide and morphine dose effect curves were shifted to the right. In the model of inflammatory pain, acute administration of loperamide was ineffective at doses up to 3.2mg/kg. Morphine produced dose‐dependent acute antinociceptive effects in this assay with an ED50 of 4.2mg/kg. This effect was attenuated by naloxone but was not altered by naloxone‐methiodide. Following 5 days of 3x daily administration of morphine, the ED50 of the morphine dose effect curve was shifted 3.5 fold to the right. Interestingly, pretreatments of naloxone‐methiodide prior to each chronic morphine injection prevented the rightward shift in the morphine dose effect curve following chronic morphine treatment. These results suggest that repeated activation of peripheral opioid receptors produce tolerance and that peripheral opioid receptors may be involved in the development of tolerance to morphine.
The Functional Expression of the Lamin A/C Mutant Q517X in HL1 Cardiomyocytes Induces Electrophysiological Impairments Through Impaired Ca2+‐Homeostasis at the Endoplasmic Reticulum.
Authors: Gerbino Andrea , De Zio Roberta , Forleo Cinzia , Milano Serena , Procino Giuseppe , Favale Stefano , Svelto Maria , Carmosino Monica
Published: April 2020

Mutations in the LMNA gene, which encodes A‐type nuclear Lamins, are among the most frequent genetic cause of dilated cardiomyopathy (DCM). We identified a novel LMNA nonsense mutation (Q517X) co‐segregating with DCM and permanent atrial fibrillation. Aim of this study is to gain insights into the unknown pathogenic mechanism induced by the expression of Q517X at cellular level.

HEK293 cells and HL‐1 cardiomyocytes were transiently transfected with either Lamin A or Q517X both tagged with mCherry. Q517X expression, localization and its effects on diverse cellular mechanisms were evaluated by western blotting, laser‐scanning confocal microscopy and real time FRET/imaging analysis in single cell. Also, patch clamp experiments were used to evaluate the effect of Q517X expression on HL‐1 spontaneous action potentials (APs).

When expressed in both cell types, Q517X mislocalized within the nucleoplasm where it clustered in aggregates of different sizes. Emerin was also irregularly distributed along the nuclear envelope probably indicating a general impairment of the inner nuclear membrane organization upon Q517X expression. HL‐1 cardiomyocytes expressing the mutant showed a significant reduction in APs frequency induced by an increased duration of both single APDs (APD100) and cycle length (i.e. the distance between two sequential APDs’ thresholds). In addition, after depolarizations events were constantly recorded in cells expressing the mutant. When we measured intracellular Ca2+ level in cardiomyocytes expressing either LMNA wild type or Q517X we found similar patterns of oscillation. However, in the absence of electrical activity at the plasma membrane, cardiomyocytes expressing the mutant showed spontaneous Ca2+ spikes, in free‐extracellular Ca2+, likely induced by abnormal release by the endoplasmic reticulum (ER) as directly confirmed by steady state FRET experiment with the ER Ca2+ probe ERD1.

So far, these results depict a pathogenic mechanism in which the exogenous expression of Q517X changed the geometry of emerin within the nucleus and altered the electrical activity at the plasma membrane by probably depleting ER Ca2+ content.

Support or Funding Information

This research was supported by funding from the ‘CLUSTER TECNOLOGICO REGIONALE DICLIMAX’ (project # MTJU9H8)
The Effects of Exercise Regimens on Perceived Stress in First‐Year Medical Students
Authors: Khamisani Azmeer , Tung Shouli , Chirico Erica N.
Published: April 2020

Stress in medical students is higher than the general population and burnout among those in the healthcare field is becoming a rising concern. Exercise has been shown to decrease anxiety and stress via a role in the oxidative stress and inflammatory pathways and could contribute to combating the increase in burnout and stress among medical students. We hypothesized that there would be an inverse correlation between activity level and perceived stress among medical students. Exercise activity of first‐year medical students at Cooper Medical School of Rowan University were monitored using FitBits throughout the course of the academic year and the data were uploaded to Fitabase, a comprehensive data management platform. Data such as heart rate, steps, activity level, calories and distance were collected throughout the year. During baseline and exam weeks, participants filled out a standard psychometric questionnaire to assess perceived stress levels. Students were divided into two groups based off their resting heart rates: high activity (HA) level (resting HR less than and including 60 beats per minute) and low activity (LA) level (resting heart rate greater than 60 beats per minute). Students were also categorized into high, moderate, and low stress groups based off their perceived stress scores. Those in the HA group had lower self‐reported stress scores throughout the entire school year during the week of the exam than those in the LA category (p=.014). There was a significant correlation between steps taken during the block (pre‐exam) and steps taken the week of the exam in each of the system blocks (p < 0.001). Furthermore, the HA groups consistently increased steps taken the week of the exam while the LA group decreased steps taken. Our data also indicated that the HA group performed more “very active” activity per day than the LA group, while “fairly active” and “lightly activity” did not differ between groups. Our results suggest a strong relationship between higher activity levels and a reduced perceived stress levels throughout the first year of medical school.
Screening Phytochemicals for Alternative Diabetes Treatments
Authors: Krueger Emily , Austin Mathew , Ekpo Idongesit , Beales Joseph , Yang Haokun , Nielson Andrew , Tessem Jeffery Sivert
Published: April 2020

One in 10 people suffer from diabetes mellitus, where Type 2 (T2D) makes up 90% of cases worldwide. T2D is characterized by hyperglycemia and insulin resistance stemming from dysregulation of insulin secreting pancreatic ß‐cells. The increased demand for insulin posed by insulin resistance leads to further exhaustion and apoptosis of the ß‐cells. Therefore, increasing functional ß‐cell mass by enhancing ß‐cell survival, proliferation and function, may be leveraged to help treat individuals with T2D diabetes. Despite spending 12% of the global health expenditure on diabetes, treatment limitations call for more research and superior therapeutics to protect and recover ß‐cells. Phytochemical flavanols, including naturally occurring polymers of epicatechin, are known to exert antioxidant bioactivity and improve cell health. We have previously demonstrated monomeric epicatechin enhances ß‐cell glucose‐stimulated insulin secretion. We hypothesize that other dietary flavanols may similarly be sufficient to enhance ß‐cell survival, proliferation and function. The objective of this study was to screen 13 dietary flavanols including epicatechin polymers, cinnamtannins, and procynidins, to define their in vitro ability to modulate ß‐cell survival, proliferation and function. Here we present our findings for these compounds, emphasizing their potential beneficial effects as alternative and complementary treatments for T2D.
3D Muscle Fiber Architecture of Avian Jaw Muscles and their Significance for Avian cranial functional morphology evolution
Authors: Verhulst Conner Dalen
Published: April 2020

Birds evolved a diversity of head shapes, feeding behaviors and jaw muscles which drive the feeding apparatus. However, capturing the anatomical and functional diversity of these muscles remains challenging and we know little about how jaw muscles develop, function and evolve among lineages of the clade and this hampers our ability to understand skeletal tissue biology, joint function, and patterns of evolution in bird skulls. Here we use contrast imaging, 3D muscle fiber tracking and new data visualization methods to explore the morphology, architecture and functional significance of the jaw muscles of a sample of birds to highlight the potential of these approaches. First, we show differences in functional morphology of jaw muscles between a hatchling and adult Mallard duck (Anas platyrhynchos). Ducks show a marked change in adductor chamber shape during ontogeny including an elongated postorbital process, quadrate orbital process and retroarticular process along with a broad temporal fossa which likely track with changes in muscle function and cranial kinesis. Indeed, we found m. depressor mandibulae in baby and adult ducks shifts from a rostrocadual orientation to a more dorsoventral orientation that offers additional mechanical advantage to jaw opening and propalinal mandible movement as well as concomitant changes in the 3D resultant and pennation of other jaw muscle bellies. Second, we show differences between a long‐ and short‐faced, dabbling birds (mallard, green‐wing teal), long faced orthally‐biting bird (belted kingfisher) and a short‐faced, hard‐biting bird, the grey parrot (Psittacus erithacus). We expected position, architecture and biomechanics of homologous muscles to differ substantially between these avian species given their different behaviors and overall cranial morphology. Although some muscles showed marked differences in resultant and pennation, others, including protractor pterygoideus, the primary actuator of kinesis, were remarkably similar in morphology suggesting a mosaic of changes happening during avian evolution. Together, these new, 3D high fidelity anatomical data on jaw muscle functional morphology in birds will not only better illuminate ecomorphological evolution but also illustrate how other clades of birds and vertebrates adapt their muscle architecture to meet functional demands.
Hyperglycemia‐Induced Inhibition of the Angiotensin II Type 1 Receptor Pathway in the Vascular Endothelium
Authors: Hoffmann Brian R. , Haspula Dhanush
Published: April 2020

Hyperglycemia, associated with diabetes, is known to damage blood vessels and contribute to negative cardiovascular health outcomes. However, the etiology behind the negative influence of the exposure to prolonged elevated glucose on endothelial cells is not yet fully understood. Our laboratories’ previous study identified changes in endothelial cell surface glycosylation resulting from hyperglycemia. One such modified protein was the Angiotensin II type 1 receptor (AT1R), which was N‐glycosylated adjacent to the active site. Activation of AT1R by angiotensin II (AngII) at subpressor doses is associated with positive endothelial cell growth, proliferation, and tube formation. However, AngII binding to the Angiotensin II type 2 receptor (AT2R), has been shown to elicit opposing effects. The objective of this study was to further investigate the effects of this hyperglycemia‐induced glycosylation of AT1R. A two‐week high glucose treatment (25 mM) of rat cardiac microvascular endothelial cells (RCMVECs) displayed impaired function by tube formation analysis (TFA) in comparison to a normal glucose treatment (4.5 mM). The decrease tube formation was further exacerbated by the addition of a subpressor dose of AngII. To further test the influence of hyperglycemia‐induced AT1R N‐glycosylation on signaling we treated RCMVECs with the PNGaseF enzyme for N‐glycosylation removal at the cell surface in combination with functional assays and AngII treatment under varying glycemic conditions. The efficacy of this treatment was tested through TFA and measurements of ERK1/2 activation through phosphorylation (known contributor to AT1R signaling). We observed a decrease in tube length, tube area, and number of branch points in a high glucose environment plus Ang‐II and were able to recover functionality with the addition of PNGaseF. While normal glucose treated cells had shown ERK1/2 activation, no observable increases in ERK1/2 were observed in response to AngII in the high glucose conditions. In the high glucose plus AngII treatment group we also observed a significant decrease in Vegfr2 gene expression compared to the normal glucose plus AngII treatment group. Further analysis is currently underway to further validate this mechanism of AT1R signaling impairment, but the data we have so far lead us to believe that hyperglycemia‐induced glycosylation blocks the AT1R active site to decrease functionality. It is possible that the hyperglycemia‐induced glycosylation of AT1R inhibits AngII binding, allowing for a higher rate of binding in the AT2R to further impair endothelial cell functionality.

Support or Funding Information

Support for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University.
High Fat Diet versus Disturbed Blood Flow Conditions: Implications for Endothelial Glycocalyx Integrity and Pre‐Atherosclerotic Inflammation
Authors: Mitra Ronodeep , Qiao Ju , Madhavan Sudharsan , O’Neil Gerard , Ritchie Bailey , Kulkarni Praveen , Sridhar Srinivas , van de Ven Anne , Cherry Erica , Ferris Craig , Hamilton James , Ebong Eno
Published: April 2020

Introduction

Endothelial glycocalyx (GCX) shedding plays a role in endothelial dysfunction and increases vessel wall permeability to inflammatory cells contributing to atherogenesis. We sought to determine whether a high fat diet (HFD) or disturbed blood flow conditions, both well‐known atherogenic risk factors, would more detrimentally contribute to pre‐atherosclerotic loss of GCX integrity and vascular inflammation.

Materials and Methods

C57BL/6‐background apolipoprotein E knockout (ApoE‐KO) mice were fed either a HFD or chow diet, and/or underwent a ligation of the left carotid artery (LCA), inducing disturbed flow conditions. After one week, mice were sacrificed and LCAs and right carotid arteries (RCAs) were preserved to compare GCX coverage and thickness and inflammatory macrophage accumulation in carotid arterial walls amongst and between cohorts.

Results and Discussion

Endothelial GCX damage occurred in LCAs of HFD fed mice when compared to the control. More significant GCX damage occurred in the LCAs of mice exposed to disturbed flow by partial LCA ligation, also compared to control. Flow simulations conducted through SimVascular adequately characterized flow patterns, pressure, and wall shear stresses in non‐ligated vs. ligated mice. They confirmed the notion that the LCA partial ligation surgery does indeed reduce wall shear stress and increase pressure, characteristics of disturbed flow. No difference in macrophage accumulation in carotid arterial walls was observed when comparing the LCAs of control mice to the LCAs of HFD fed mice. However, macrophage infiltration in vessel walls showed a 20‐fold increase in LCAs exposed to disturbed flow following ligation, when compared to control LCAs.

Conclusions

This study was the first to demonstrate that disturbed flow contributes more detrimentally to pre‐atherosclerotic loss of GCX integrity and vascular inflammation. The study concluded that disturbed flow conditions induced by partial ligation, compared to HFD conditions, compromised the integrity of the endothelial GCX to a greater extent. We anticipate more rapid atherogenesis as a consequence of the severity of this flow induced GCX damage.

Support or Funding Information

We are pleased to acknowledge that this work was funded by the National Institute of Health (NIH) K01‐ HL125499 awarded to E Ebong, NIH R21‐DA042583 awarded to S Sridhar, and Air Force Office of Scientific Research (AFOSR) FA2386‐17‐1‐4042 awarded to S Sridhar.
Does Hypoxia Affect Pulse Transit Time in Healthy Controls?
Authors: Brubaker Justin M , Carter Katrina J , Ward Aaron T , Kellawan J. Mikhail , Harrell John W , Eldridge Marlowe W , Roldan Alejandro , Wieben Oliver , Schrage William G
Published: April 2020

Hypoxia is known to increase cerebral blood flow and increase sympathetic nervous activity (SNA). Given the strong link between elevated vascular stiffness and cardiovascular diseases exhibiting elevated SNA, we tested whether a hypoxic stressor in healthy subjects would acutely change cerebral vascular stiffness. We hypothesized hypoxia would lead to increased cerebrovascular stiffness as measured by a reduced pulse wave transit time. Eight healthy controls (31±8 yrs, M=3, F=5), with a BMI of 18.5 – 25 kg/m2, laid supine in a MRI, breathing room air (Normoxia) for ~10 minutes followed by a hypoxia condition, in which subjects breathed a gas mixture of 11% O2 and 89% nitrogen through a mouthpiece. Inspired CO2 between 1 – 5% of inspired air was bled in to maintain a constant end‐tidal CO2. Heart rate, end‐tidal CO2, arterial oxygen saturation, and blood pressure were continuously monitored. A 3 Tesla MRI using 4‐dimensional flow phase contrast vastly under‐sampled isotropic projection reconstruction (PC VIPR; ~6 minutes) was used to capture cerebral blood flow (CBF = blood velocity • vessel cross sectional area) in the internal carotid arteries (ICA) and superior sagittal sinus (SSS). Cerebral blood flow across 20 equal segments of the average cardiac cycle was used to determine the time to peak flow in the ICAs and SSS during normoxia and hypoxia. Pulse transit time (PTT) was calculated as the difference in time to peak in the SSS from the time to peak in the ICAs. Data were analyzed via paired t‐tests and are presented as mean ± SD. Significance was taken as p≤0.05. Per design, hypoxia decreased SpO2 from 98%±1 to 80%±5 and increased CBF from 642±122 to 747±150 ml/min (p<0.001), while end‐tidal CO2 did not change 40±2 and 39±2 mmHg. During normoxia, pulse transit time from ICA to SSS was 98±50 ms, whereas pulse transit time during hypoxia was 167±82 ms (one‐tailed P‐value=0.06). Contrary to our hypothesis, acutely increasing CBF with hypoxia led to increased pulse transit time between the ICA and SSS. These findings suggest conditions that increase cerebral blood flow may positively impact hemodynamics in a manner associated with decreased vascular stiffness.

Support or Funding Information

ADA 1‐12‐IN‐39
Are We Teaching to the Test? Medical Student Perceptions of the USMLE Step 1 Exam
Authors: Loder Danielle
Published: April 2020

With many medical schools having undergone significant curricular reform, including those who have transitioned to a pass/fail curriculum model, residency programs have little quantitative data with which to compare individual medical students who apply to their programs. For these programs, the primary source of quantitative data is a student’s USMLE Step 1 exam score, because it provides a common measurement across medical schools with different curricula. This circumstance puts an immense amount of pressure on this one exam, sparking discussions at medical schools across the nation about the “Step 1 Climate” (
Chen et. al., 2019). In 2016, Indiana University School of Medicine – Bloomington (IUSM‐BL) underwent curricular reform. The new curriculum uses a pass/fail system for all courses. Given the emphasis placed on the Step 1 exam, I am interested in learning more about student perceptions of the Step 1 exam, specifically how it impacts their relationship with the IUSM‐BL curriculum.

First‐ and second‐year medical students at IUSM‐BL were interviewed using a 30–60‐minute semi‐structured interview format. The questions were designed to obtain student perceptions of the USMLE Step 1 exam and its relationship to the current medical school curriculum at IUSM‐BL. Interview questions included the following: 1) Having students explain the Step 1 exam and its importance in their future education, 2) Explanations of how students are studying for the Step 1 exam, 3) Perceptions of how well the IUSM‐BL curriculum is preparing students for the Step 1 exam. A thematic analysis of the interview transcripts was performed to determine commonalities in responses. Resultant themes indicate that the USMLE Step 1 exam is ever‐present in the minds of our medical students. They stress the overemphasis on this exam for their future residency applications, and suggest some ideas for ways to alleviate the emphasis placed on the Step 1 exam. This study will contribute to answering the larger questions surrounding the effect of the Step 1 exam on students and medical education.
Full body loss of the nuclear hormone receptor Nr4a3 results induces obesity and glucose intolerance.
Authors: Yang Haokun , Herring Jacob , Wynn Adam , Elison Weston , Walton Chase , Good Lance , Marchant Erik , Marchant Nathan , Bikman Benjamin , Tessem Jeffery Sivert
Published: April 2020

Type 2 diabetes (T2D) is increasing at an alarming rate worldwide. T2D is characterized by obesity, glucose intolerance, and elevated blood glucose level. The orphan nuclear receptor Nr4a3 has been shown to play critical roles in controlling expression of genes associated with proliferation, cell survival and fuel metabolism. However, Nr4a3’s role in glucose homeostasis has yet to be explored. Here we present data demonstrating that male and female Nr4a3 full‐body knock out (KO) mice have a ~22% increase in body mass when compared to control animals and fed a standard diet, and a ~30% increase when fed a high fat diet. In addition, these mice present with significantly elevated blood glucose and impaired glucose tolerance. Given Nr4a3’s role in mitochondrial function and fuel utilization, we measured respiration of liver, adipose and muscle. Nr4a3 KO mice fed a standard diet presented no difference in liver or muscle respiration, however adipose respiration was significantly impaired regardless of sex. Animals fed a high fat diet presented with impaired adipose, liver and muscle respiration. These data begin to demonstrate that Nr4a3 is necessary for whole‐body glucose homeostasis, that loss of Nr4a3 predisposes animals to obesity and glucose intolerance, and that high fat feeding worsens the observed phenotypes. These data serve as a step toward understanding the pathway of T2D progression and the role that Nr4a3 plays in this progression.
High‐resolution Epigenome Mapping Reveals Distinct and Divergent Roles for UHRF1 in the Maintenance of DNA Methylation
Authors: Tiedemann Rochelle L. , Du Qian , Dickson Bradley M. , Kong Xiangqian , Chomiak Alison A. , Zhou Wanding , Johnson Benjamin K. , Baylin Stephen B. , Jones Peter A. , Clark Susan J. , Rothbart Scott B.
Published: April 2020

The E3 ubiquitin ligase UHRF1 is an established cofactor for replication‐coupled DNA methylation inheritance. Our group and others have made recent progress dissecting biochemical mechanisms linking UHRF1 to DNA methylation control. Our current model posits a step‐wise pathway in which UHRF1 engages nucleosomes through histone and DNA binding. This multivalent nucleosome readout directs UHRF1 ubiquitin ligase activity toward the N‐terminal tail of histone H3, a binding site for DNMT1. While this elegant chromatin regulatory process is now taking shape, it remains unclear whether the reliance on UHRF1 for DNA methylation maintenance is localized or genome‐wide. Here we present comparative, high‐resolution, genome‐scale analysis of DNA methylation maintenance following acute depletion of UHRF1 and DNMT1 in a human colorectal cancer cell line. As expected, DNMT1 depletion resulted in global reduction of DNA methylation genome‐wide. However, depletion of UHRF1 revealed distinct attributes of the genome that rely on UHRF1 for DNA methylation maintenance. Significantly, these distinct genomic attributes are divergent on their dependency of ubiquitin deposition on the histone H3 tail by UHRF1 to recruit DNMT1 and maintain DNA methylation. Notably, these newly identified UHRF1‐ubiquitin dependent DNA methylation signatures are consistent with the observed loss of DNA methylation that occurs during aging and cancer progression. We propose that UHRF1 misregulation is a key contributor to DNA methylation erosion that occurs through human lifespan and cancer.

Support or Funding Information

This work was supported by the Van Andel Institute, and grants from the National Institutes of Health to S.B.R. (R35GM124736) and the American Cancer Society ‐ Michigan Cancer Research Fund to R.L.T. (PF‐16‐245‐01‐DMC).
Jurkat T‐cells chronically growing at acidic pH 7.2 show increased CO2 hydration activity
Authors: Snitsarev Vladislav , Vega Quinn C. , Petroff Elena
Published: April 2020

In our previous study (Vega et al. Faseb J. 32, S787.14) we reported that a clone of Jurkat T‐cell line that we have been chronically propagating at pH 7.2 (10% CO2) showed a significantly different growth pattern than the control clone at pH 7.4 (5% CO2).

Since carbonic anhydrase (CA) is a key contributor to cellular pH regulation, here we tested if pH7.2 and pH7.4 clones have a different CA activity that was measured with the Wilbur‐Anderson assay; briefly, one unit is defined as 2*(T0‐T)/T, where T0 and T are the times in seconds in the absence and presence of enzyme, respectively, required for a saturated CO2 solution (4 mL) to lower pH of 20 mM TRIS buffer (6 mL) from 8.3 to 6.3 at 0°C.

In a control experiment, CA with the reported activity (≥ 3,000 units/mg dry weight) showed in duplicate experiments 3,048 (T0=50.5 sec, T=10.5 sec, 0.0025 mg, determined with Hanna HI10832) and 3,111 (T0=66 sec, T=13.5 sec, 0.0025 mg, determined with a timer).

To avoid the osmotic shock to the cells, we added 0.85% NaCl to all the assay’s solutions and tested if the presence of 0.85% NaCl affects CA activity.

In the presence of 0.85% NaCl of NaCl, the values were 1,869±95 (T0=72.3±0.9 sec, T=21.8±0.8 sec, n=5), both blank T0 and enzyme T times being slower most likely because CO2 content in the saturated aqueous solution was smaller due to the presence of another solute, 0.85% NaCl.

For the experiment, cell suspensions were span twice in the TRIS (20 mM)+NaCl 0.85% buffer at 0°C to achieve 1 mL of 10 million cells/mL density that was added to 5 mL of the TRIS buffer (pH 8.3), then immediately 4 mL of 0.85% NaCl saturated with CO2 was added, and time for pH to drop from 8.3 to 6.3 was measured; the final cell count being 10 million/mL in 10 mL of the reaction volume.

For the pH7.4 clone, T= 82.3±1.1 sec (n=6) was slower than blank determination in 0.85% NaCl resulting in the apparent negative CA activity of −0.24 formally calculated as 2*(T0‐T)/T; while for the pH7.2 clone, the T= 77.3±1.4 sec (n=6) resulted in −0.13 (p<0.05).

If the increased CO2 hydration in the pH7.2 clone is caused by the increase in CA molecules alone, we would expect that these cells would have an additional 0.11/(10*106 cells)/(3000 units/mg)/(1000 mg/g)/(30,000 g/mole) * 6.022*1023 = 7.4*104 CA molecules per cell.

We conclude that 1) isotonic NaCl significantly inhibits apparent CA activity in the context of Wilbur‐Anderson assay units, 2) both pH7.2 and 7.4 clones at 1 million/mL cell density inhibited CO2 hydration compared to the blank CO2 hydration in the Wilbur‐Anderson assay supplemented with 0.85% NaCl; 3) in relation to each other, pH7.2 clone has a higher CO2 hydration activity than pH7.4 clone speculatively due to estimated additional 7.4*104 CA molecules.

Differential CA activity and expression in the pH7.2 and 7.4 clones will be further compared using physiology and molecular biology tools.
Differential effects of n‐3 and n‐6 fatty acids on inflammatory cytokine production in 3T3‐L1 adipocytes and RAW264.7 macrophages.
Authors: Caldari Cristina , Beck Jordan , Danchine Veronika
Published: April 2020

There is a current focus on obese adipose tissue as an organ involved in the development of meta‐inflammation—chronic low‐grade inflammation that can predispose the individual to chronic diseases. As an immunomodulating and endocrine organ, it is important to study the relationship between cells found in adipose tissue. Looking into the interplay between two of these adipose tissue cells, adipocytes and adipose tissue macrophages (ATMs), can give scientists information on how to reduce adipose tissue‐derived meta‐inflammation. Hypertrophic adipocytes attract circulating monocytes, which encounter inflammatory molecules when they infiltrate the adipose tissue, causing them to differentiate into macrophages with an inflammatory phenotype. Inflammatory cytokines produced by the ATMs can in turn exacerbate the localized inflammatory condition in the adipose tissue, contributing to the chronic release of inflammatory cytokines from this organ. Certain fatty acids (FAs), like omega‐3 (n‐3) FAs, have long been known to possess anti‐inflammatory properties. The objective of this study was to determine the effects of both n‐3 and n‐6 FAs on inflammatory molecule release by 3T3‐L1 murine adipocytes and RAW264.7 murine macrophages. Macrophages were pre‐incubated with 100 μM of linoleic acid (LA, n‐6), arachidonic acid (ARA, n‐6), or eicosapentaenoic acid (EPA, n‐3) for 24 h, followed by a 6 h, 0.01 μg/ml lipopolysaccharide (LPS) challenge. Fibroblasts were differentiated into adipocytes with the addition of 100 μM of one of the n‐6 or n‐3 FAs throughout the differentiation period, followed by a 6 h, 1 μg/ml LPS challenge once they were fully differentiated. All FA treatments resulted in a significant decrease in macrophage activation (as measured by nitric oxide [NO] production; p<0.001, two‐way ANOVA) and interleukin (IL)‐6 production (p<0.001, two‐way ANOVA) compared to control and LPS‐challenged macrophages. Fatty acid treatment did not affect the release of transforming growth factor (TGF)‐β or monocyte inflammatory protein (MIP)‐1α production by LPS‐challenged macrophages. Adipocytes differentiated with ARA had significantly higher levels of IL‐6 (p<0.05, Tukey’s HSD), compared to control and adipocytes differentiated with LA or EPA. Additionally, ARA resulted in the highest production of MIP‐1α in differentiated adipocytes, followed by EPA (p<0.05, Tukey’s HSD). These results suggest that n‐3 and n‐6 fatty acids have differential effects on inflammatory and chemotactic molecule production in cultured murine macrophages and adipocytes. The n‐6 fatty acid ARA exhibited the strongest immunomodulating effect on both cell types, suppressing activation and IL‐6 production in macrophages, while significantly increasing IL‐6 and MIP‐1α production in differentiated adipocytes. The effects of these fatty acids on co‐incubation of these cell types and total inflammatory molecule production warrants investigation.