bluuebunny/crossref_metadata_2025 huggingface dataset:
Alternately Translated Product of Cx37 Fails to Arrest Growth in Rin Cells but Alters Cx37 Hemichannel Function when Co‐Expressed
Authors: Alamban Andrew , Fye Margret , Pontifex Tasha , Burt Janis
Published: April 2020

Connexin proteins facilitate transmembrane and intercellular communication via channel formation (hemichannel) and intercellular docking of apposed hemichannels to form gap junction channels, respectively. Additionally, they play a role in determining growth phenotype (e.g., arrest, proliferation, death). Connexin 37 (Cx37), expressed in connexin‐deficient rat insulinoma (Rin) cells induces growth arrest or cell death in a manner dependent on the phosphorylation of specific carboxyl terminus (CT) residues. Notably, phosphorylation at serine 319 (S319) is both necessary and sufficient for growth arrest, and mimicking phosphorylation with aspartate substitution at either S275 or S321 is sufficient to induce cell death. 13 of the 21 members of the connexin gene family have a conserved methionine at position 213; in Cx43 mRNA, this methionine codon serves as an alternate translation start site that produces a CT‐spanning translation product. This 20kDa (20k) product interacts with Cx43, chaperoning its trafficking to the membrane. Here we test the hypotheses that 1) this conserved codon in Cx37 mRNA also serves as an alternate translation start site to produce a 13kDa product (13k) that, when expressed alone, regulates growth phenotype of Rin cells and 2) when co‐expressed with Cx37, regulates its channel function. To test these hypotheses, we expressed V5‐tagged 13k or one of two phosphomimetic mutants, 13k‐S321D and 13k‐S275D, in Rin cells or in Rin cells expressing Cx37. Despite a similar phosphorylation profile to Cx37, as determined by mass spectrometry, 13k did not arrest Rin cell growth and neither phosphomimetic mutant induced cell death nor growth arrest. In co‐expressing cells, immunoprecipitation (IP) of 13k failed to co‐IP full‐length Cx37, suggesting no direct interaction between these isoforms; however, 13k and the phosphomimetic mutants altered Cx37 hemichannel conductance and open probability properties, suggesting an indirect interaction of 13k isoforms with Cx37. In conclusion, these data suggest that while 13k and phosphomimetic mutants alone are insufficient to alter the growth phenotype of Rin cells toward growth arrest or death, they nevertheless lead to altered channel properties of full‐length Cx37. The findings herein will inform further research in understanding how connexins are regulated and, in turn, the role they play in regulating cell growth phenotypes.

Support or Funding Information

R01 HL131712 | Undergraduate Biology Research Program | University Funds
Obesity in Vascular and Alveolar Morphogenesis after Pneumonectomy
Authors: Mammoto Tadanori , Hendee Kathryn , Mammoto Akiko
Published: April 2020

Obese population is rapidly growing worldwide. Obesity is associated with chronic lung diseases including chronic obstructive pulmonary disease (COPD). Impairment of lung regeneration and repair contributes to the pathogenesis of COPD. Angiogenesis – the formation of new blood capillaries ‐ plays a key role in organ regeneration. Inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and obesity is accompanied by endothelial cell (EC) dysfunction. However, the effects of obesity on lung vascular and alveolar regeneration remain unclear. Compensatory lung growth and vascular and alveolar morphogenesis after PNX are suppressed in leptin‐deficient ob/ob mice treated with high‐fat diet compared to that in control lean mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the brown adipose tissue collected from post‐PNX mice compared to those from sham‐operated control mice, while these effects are attenuated in post‐PNX leptin‐deficient ob/ob mice with high‐fat diet. The serum collected from post‐PNX control mice stimulates EC sprouting, while EC sprouting is inhibited when treated with serum collected from leptin‐deficient ob/ob mice after PNX. These results suggest that obesity may inhibit post‐PNX regenerative lung growth through VEGF signaling. Modulation of VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese population.

Support or Funding Information

NIH R01HL142578
The Neuroprotective Effects of Estrogen in an Alzheimer’s Disease Hypothalamic Cell Line
Authors: Hindieh Jennifer Grace , Persaud Nikita , Cooperstein Deborah , Chakraborty Tandra R. , Chakraborty Sanjoy
Published: April 2020

Alzheimer’s Disease (AD), is a neurodegenerative disease affecting memory and cognition, eventually leading to dementia. Two of the most notable hallmarks of AD are the aggregation and depositing of beta‐amyloid fibrils as extracellular plaques as well as the intracellular accumulation of Tau as neurofibrillary tangles in the brain. Research suggests a possible neuroprotective role of the female sex hormone estrogen in neurodegenerative diseases, including AD. Using established AD cell line SH‐SY5Y as a control and N38 hypothalamic cells as an experimental cell line, AD will be modeled in vitro by incubating with synthetic, oligomerized beta‐amyloid, and subsequently, treating the culture with estrogen to observe its efficacy as a neuroprotective reagent. A western blot assay will be used to confirm the presence of total tau, before and after treatment. We hypothesize that cells treated with Aβ42 will express larger amounts of total Tau in contrast to cells treated with both estrogen and amyloid. Preliminary results confirm the presence of Tau in SH‐SY5Y and N38 cell line. Tau‐46 was used as the primary antibody, GAM‐HRP as a secondary antibody, with GADPH used as a control. Supplemental bioassays, MTT and LDH, were performed to determine cell viability, cytotoxicity, and proliferation. Preliminary results in MTT with N38 cells showed that Aβ42 reduced mitochondrial activity within 24hrs. These results also indicated estrogens’ neuroprotective capabilities within hypothalamic cells by counteracting the negative effect of Aβ42. Through analysis of both quantitative and qualitative data, there will be further clarification of the possible neuroprotective nature of estrogen with regards to beta amyloid plaque toxicity.

Support or Funding Information

Adelphi University & New York City College of Technology
Exploring the Structural Stability and Assembly Mechanism of Hydrophobin Proteins
Authors: Vergunst Kathleen , Langelaan David N.
Published: April 2020

Hydrophobins are small, globular proteins with amphiphilic character that are produced and secreted by filamentous fungi. At hydrophobic‐hydrophilic interfaces they self‐assemble into durable amyloid‐containing structures, called rodlets, which create protective, water repellent coatings for fungal spores. Current models of hydrophobin self‐assembly predict that hydrophobin monomers undergo a conformational change at a hydrophobic‐hydrophilic interface and integrate into a growing rodlet, however the mechanistic details of rodlet assembly are unknown. To investigate the assembly mechanism of hydrophobins, we carried out stability studies with SC16, a hydrophobin isolated from Schizophyllum commune. SC16 was recombinantly expressed using E. coli and purified by immobilized Ni2+ affinity chromatography. NMR spectroscopy was used to determine that the structure of SC16 was minimally perturbed by denaturing (8 M urea) or reducing (2 mM DTT) conditions. Gel filtration chromatography indicated that in solution SC16 exists as a tetramer, suggesting that rodlet assembly does not initiate from hydrophobin monomers. Mutant forms of SC16 are being employed to determine the sequences and conformational changes required for rodlet assembly, with gel filtration chromatography used to determine the multimeric state, thioflavin T assays to quantify amyloid formation, and electron microscopy to visualize rodlet formation. X‐ray crystallography is being used to further characterize the tetrameric structure of SC16. Determining which hydrophobin sequences are responsible for self‐assembly will allow the rational modification of hydrophobins to add new functionalities or influence their self‐assembly.

Support or Funding Information

NSERC, Dalhousie University Faculty of Medicine, Nova Scotia Research Foundation, BioActives CREATE
Sympathetic Preganglionic Neurons (SPNs): a Promising Target to Regain Cardiorespiratory Control in Spinal Cord Injured (SCI) Patients
Authors: Lopes Luana Tenorio , Barioni Nicole Orsi , Yang Hannah , Wilson Richard
Published: April 2020

Background

The consequences of spinal cord injuries (SCI) are massive and become more severe with higher levels of injury. Lesions located on cervical portions, specifically between C1‐C3 cause an interruption of the descending bulbospinal pathways. These pathways include a key component of the neural control of respiration, linking the brainstem breathing rhythm generator with phrenic motor neurons that activate the diaphragm. Disruption to these pathways result in respiratory muscle paresis and/or paralysis, requiring mechanical ventilation. Respiratory complications, such as pneumonia and atelectasis complicate the process of weaning patients from ventilators, contributing to patient mortality. Previous data collected in the Wilson lab demonstrated that sympathetic preganglionic neurons (SPNs) located at the thoracic spinal cord have remarkable oxygen and pressure‐sensitive capabilities. Here, we hypothesized that SPNs can stimulate phrenic motor neurons independent of the brainstem.

Aim

To study the novel direct influence of SPNs on the phrenic motor nucleus.

Methods

An in situ artificially‐perfused cervico‐thoracic rat spinal cord preparation was used to record phrenic and splanchnic nerve activity while having the cervical and thoracic spinal cord separately perfused. This approach allowed us to selectively expose the thoracic segment to acute hypoxia (30 Torr; 10 min) in order to observe if cardiorespiratory responses elicited by the SPN’s could stimulate phrenic motor neurons located at the cervical spinal cord. Additionally, we recorded the electromyography activity from the diaphragm during hypoxia and ischemia (0 mmHg; 10 min).

Results

The results demonstrated that stimulation of the SPNs with hypoxia evoked phrenic nerve and diaphragm activity. This indicates that even without inputs from the brainstem, SPNs are responsive to hypoxia and ischemia and might have capability to stimulate phrenic motor neurons.

Conclusion

The implications of these findings point to the thoracic SPNs as a unique and promising target to regain cardiorespiratory control in SCI patients. Further pharmacological investigations may reveal effective treatment strategies to improve life quality and reduce mortality of SCI patients.

Support or Funding Information

Alberta Innovates Health Solutions (LL), Eyes High (NB), NSERC (HY), Canadian Institute of Health Research (RW).
Cannabidiol Induces Apoptosis and Perturbs Mitochondrial Function in both Human and Canine Glioma Cells
Authors: Gross Chase , Ramirez Dominique , Dickinson Peter , Gustafson Daniel , McGrath Stephanie
Published: April 2020

The use of cannabidiol (CBD), the major non‐psychoactive compound derived from cannabis, as a nutraceutical in both human and veterinary medicine has increased steadily within the past ten years despite a lack of scientific evidence for its effectiveness. Of the cornucopia of ailments that CBD supposedly treats, epilepsy, pain, and cancer remain prominent areas of interest to scientists and the general public. In the world of cancer research, several publications have demonstrated that CBD is cytotoxic against breast, endometrial, glial, and blood cancers, and in all cases it appears that CBD triggers apoptosis through a cannabinoid receptor independent process. A recent publication suggests CBD causes cell death in leukemia cells via calcium dependent mitochondrial membrane disruption, leading to activation of intrinsic apoptosis. Other receptors, such as TRPV1, and pathways, such as autophagy, have been implicated in CBD‐induced cell death but the molecular details remain elusive. Because of their remarkable similarities, from a comparative oncology perspective, canine and human gliomas are of particular interest. Regardless of species, gliomas are aggressive tumors that are notoriously challenging to treat, given their resistance to multimodal treatment, thus creating a need for more effective therapeutics. We sought to characterize the mechanism of CBD‐induced cell death in both human and canine glioma cells. We show that CBD by itself is cytotoxic, anti‐proliferative, and anti‐migratory but at concentrations which may not be pharmacologically attainable. Using resazurin and the IncuCyte live cell imaging platform, we show that metabolic‐based assays artificially increase cell line sensitivity to CBD compared to the IncuCyte, suggesting that non‐cytotoxic concentrations of CBD perturb mitochondrial function. Furthermore, we show that CBD treatment results in the formation of large cellular structures whose existence are RIPK3‐dependent. After treatment with a variety of cell death inhibitors, we demonstrate that CBD‐induced cell death occurs via apoptosis; a classically necroptotic protein, RIPK3, also appears to be involved. Lastly, by combining CBD with the autophagy inhibitor hydroxychloroquine (HCQ) we show that sensitivity to CBD increases with HCQ treatment in canine cells, which suggests involvement of the autophagy pathway for mediating CBD‐induced cell death. These data demonstrate that human and canine glioma cells respond similarly to CBD, indicate a non‐canonical function of RIPK3, and support the involvement of autophagy in CBD‐induced cell death.

Support or Funding Information

Support provided by Extract Labs in Boulder, Colorado.
The Role of Sulfide in Reticulophagy through the Regulation of ATG18a by Persulfidation
Authors: Aroca Angeles , Bassham Diane C.
Published: April 2020

Autophagy is a major catabolic process in eukaryotic cells to degrade dysfunctional or unnecessary cellular components. It has conserved functions in development, cellular homeostasis, and stress responses from yeast to plants and mammals. Autophagy is implicated in cancer, liver disease, neurodegeneration, cardiac disease, pathogen infection, among other pathological symptoms in mammals, but in plants, it is critically important in the plant life, including seedling establishment, plant development, stress resistance, metabolism and reproduction. Autophagy also contributes to intracellular homeostasis in cells by selectively degrading aggregated proteins, damaged mitochondria, ribosomes, toxic macromolecules, excess peroxisomes, and pathogens to prevent toxicity. This selective autophagy is mediated by the binding of adaptor proteins, which link a cargo targeted for degradation to the autophagosome machinery. An increasing number of targets for selective autophagy under different stress conditions have emerged in recent years, but the underlying mechanisms of their regulation are still so far unknown. Hydrogen sulfide (H2S) is an endogenously generated gaseous signaling molecule, which has been recently implicated in autophagy regulation in both plants and mammals through persulfidation of specific targets. In previous works, it has been suggested that persulfidation may be the molecular mechanism through which sulfide regulates autophagy in plant cells. ATG18a is a core autophagy component that is required for bulk autophagy and is required for reticulophagy during ER stress and RNA interference (RNAi)‐ATG18a transgenic plants showed an autophagy‐defective phenotype. Thus, persulfidation of ATG18a may regulate ER stress–induced autophagy in Arabidopsis, in a similar way as other posttranslational modifications (PTMs) regulate the ability of the ATG proteins to interact with a number of autophagic regulators. In this research, we have revealed the role of sulfide in ER stress as a negative regulator of autophagy in plants. We demonstrate that persulfidation in ATG18a is sulfide‐concentration‐dependent using a dimedone switch method with Cy5 as reporting molecule for persulfide groups. We also show that sulfide regulates ATG18a phospholipid‐binding activity by reversible persulfidation in a specific residue. Our findings demonstrate that sulfide significantly activates AtATG18a binding capacity to specific phospholipids in a reversible manner.

Support or Funding Information

This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska‐Curie Individual Fellowship grant agreement No 834120.
Ca2+ Sensitivity of Limb Muscle Fibers in Young and Older Adults
Authors: Teigen Laura E. , Sundberg Christopher W. , Kelly Lauren J. , Hunter Sandra K. , Fitts Robert H.
Published: April 2020

Age‐induced declines in skeletal muscle contractile function have been attributed to multiple cellular factors, including a loss of peak force (PO), decreased Ca2+ sensitivity, and reduced shortening velocity (VO). However, changes in these cellular properties with aging remain unresolved, especially in older women, and the effect of submaximal Ca2+ on contractile function is unknown. Thus, we compared contractile properties of muscle fibers from 19 young (24±3 years; 11 women) and 21 older adults (77±7 years; 7 women) under maximal and submaximal Ca2+ and assessed the abundance of three proteins thought to influence Ca2+ sensitivity. Fast fibers showed marked atrophy with aging in both men (young=8,599 μm2; old=5,404 μm2) and women (young=4,909 μm2; old=3,174 μm2) that corresponded with a decreased absolute PO in men (young=1.48 mN; old=0.92 mN) and women (young=0.85 mN; old=0.60 mN). There were no differences in fast fiber size‐specific PO, indicating the age‐related decline in force was explained by differences in fiber size. Except for fiber size and absolute PO, no age or sex differences were observed in Ca2+ sensitivity, rate of force development (ktr), or VO. Submaximal Ca2+ depressed ktr and VO, but the effects were not altered by age in either sex. Contrary to rodent studies, regulatory light chain (RLC) and myosin binding protein‐C abundance and RLC phosphorylation were unaltered by age or sex. These data suggest the age‐induced decline in whole‐muscle contractile function is primarily due to the atrophy of fast fibers and that caution is warranted when extending results from rodent studies to humans.

Support or Funding Information

National Institute on Aging grant (R01AG048262) to Robert Fitts and Sandra Hunter and an American Heart Association postdoctoral fellowship (19POST34380411) to Christopher Sundberg
What makes a good learning community?
Authors: Shaw Hannah , Richardson Kirsty
Published: April 2020

National Higher Education satisfaction surveys have recently introduced questions related to whether students feel part of a ‘learning community’. However, there are many ways to define a ‘learning community’ and limited understanding regarding the students’ perception of this term. Questionnaires assessing student satisfaction, their perception of a learning community, what factors may affect their learning experience, the sense of belonging and perceived competence for
learning scales were distributed to first, second and final year Bioscience students (n = 1500). Through qualitative and quantitative analysis, the results will highlight the breadth of what students think a learning community is, and whether there is a difference between cohort satisfaction when studying different subjects, and why this difference may exist. It will also demonstrate whether students with a greater sense of belonging and perceived competence for learning have a higher satisfaction rating and the factors affecting these scores. The results may be used to further understand the student perspective, manage their expectations and drive curriculum change to improve their sense of belonging and satisfaction in the learning community.

Ethical approval has been granted by the Cardiff School of Biosciences Ethics Review Committee
A Computational Model of Upper Thoracic High‐Frequency Spinal Cord Stimulation to Optimize Inspiratory Muscle Activation
Authors: Zander Hans , Kowalski Krzysztof E. , DiMarco Anthony F. , Lempka Scott F.
Published: April 2020

Background

High‐frequency spinal cord stimulation (HFSCS) has been previously shown to elicit physiological activation of the diaphragm and inspiratory intercostal muscles via spinal cord pathways in canines. It was previously hypothesized that during upper thoracic HF‐SCS, the phrenic motoneuron pools are activated via spinal pathways located in the ventro‐lateral funiculus (VLF).

Objective

The aim of the present study was to develop a computational model of ventral HFSCS to optimize stimulation parameters and electrode configurations for activation of the inspiratory muscles.

Methods

We created a computer model of an anatomically‐accurate finite element model (FEM) of the canine spinal cord and surrounding anatomy, along with an implanted HFSCS electrode array. From this FEM, we calculated extracellular voltages generated by SCS and applied them to multicompartment cable models of axons within the VLF. Model analysis compared activation thresholds for several unique electrode designs, configurations, and electrode placements in order to optimize activation of these VLF fibers to maximize inspiratory muscle activation.

Results

For initial model parameterization, we performed experimental measurements testing various electrode configurations and designs. We then used our computer model to evaluate several additional stimulation configurations (e.g. monopolar, bipolar) on VLF fiber selectivity. We then evaluated the effect of wider or narrow lateral lead spacing, contact spacing, and contact size. Finally, we considered several SCS waveform settings, such as rectangular waveforms and ramped waveforms, to potentially generate muscle activation more similar to natural inspiration.

Conclusions

We used our model to optimize electrode configurations, placements and novel stimulation waveforms of HFSCS to target fibers within the VLF. In particular, we found that wider lateral spacing between percutaneous electrodes better targets VLF fibers as opposed to the narrower spacing initially tested. We also show that ramped stimulation slowly recruits VLF fibers over time, which could improve naturalistic inspiratory muscle activation. In future work, we will perform experimental measurements in an animal model of HFSCS to confirm the ability of these optimized electrode designs and waveform parameters to better recreate normal inspiration.

Disclosure

Dr. DiMarco is a founder of and has a significant financial interest in Synapse BioMedical, a manufacturer of diaphragm pacing systems and holds patents for spinal cord stimulation to restore cough and respiration (5,678,535; 5,911,218; 5,999,855; 8,751,004). Drs. DiMarco and Kowalski hold the U.S. patents for technology related to the content of this manuscript: Respiratory Muscle Activation by Spinal Cord Stimulation (8,352,036).

Support or Funding Information

This work is supported by NIH‐NINDS (R01NS064157).
Morphological and molecular changes at dendritic spines orchestrate neuronal shifts in functional identity during monocular deprivation
Authors: Zepeda Jose , Ip Jacque , Tsimring Katya , Yun Dae Hee , Ku Taeyun , Chung Kwanghun , Sur Mriganka
Published: April 2020

Experience‐dependent plasticity refers to the brain’s ability to sculpt and remodel its circuits in an experience‐dependent manner. In the binocular visual cortex, where inputs from both eyes converge onto single neurons, the relative responsiveness of a neuron to input from one eye can shift when that eye is deprived of input. Ocular dominance plasticity (ODP) is a well‐established model for experience‐dependent plasticity, and it is triggered by monocular deprivation (MD) by suturing an eyelid. This shift is classically measured by calculating the ocular dominance index (ODI), a measure of responsiveness to either eye. The cellular and molecular mechanisms for this reorganization during ODP are not well understood. We hypothesize that morphological and molecular changes at dendritic spines (synapses) allow for functional reprogramming of neurons. To test this, we introduced a plasmid into binocular neurons via single‐cell electroporation that encodes for the calcium sensor GCaMP6s, together with a structural marker mRuby2. Using two‐photon microscopy, spines were imaged in vivo in awake mice to determine their ODIs. We then imaged at later time points during MD to measure how spine size and their responsiveness was affected. Finally, the same neurons were visualized using magnified analysis of proteome (MAP), which evenly expanded tissue by 4X to closely examine scaffolding and transmission‐related proteins at the postsynaptic density. Preliminary data suggests that a combination of extensive spine loss and potentiation of few specific spines allows for shifts in responsiveness. In addition to synaptic plasticity being important for learning and memory in adulthood, many autism spectrum disorders (ASDs) have been associated with mutations in synaptic proteins. Investigation of the molecular mechanisms behind changes in synaptic morphology are therefore important to better understand the mechanisms of learning and memory, as well as these disorders.

Support or Funding Information

This research was supported by:IMSD R25GM076321 (JZ)NIH grants EY007023 and EY028219 (MS).
Differentiation of 3T3‐L1 adipocytes with arachidonic acid increases inflammatory markers
Authors: Beck Jordan , Caldari Cristina
Published: April 2020

Adipose tissue is responsible for insulating internal organs, storing energy for times of negative energy balance, and secreting adipokines into circulation. The major cell types found in adipose tissue include undifferentiated adipocytes (pre‐adipocytes), adipocytes, and adipose tissue macrophages macrophages (ATMs). Adipocytes and ATMs are the major sources of adipokines, adipose tissue cytokines with functions ranging from inflammatory mediators to metabolic regulators. Polyunsaturated fatty acids (PUFAs) have been shown to affect the differentiation and lipid droplet formation in 3T3‐L1 adipocytes, but few studies have examined their effects on inflammatory adipokine production from these cells. Adipocyte differentiation, which is marked by lipid droplet (triacylglycerol; TAG) accumulation, is regulated by various transcription activators, including a splice variant of the nuclear receptor PPAR□. PUFAs are major regulators of PPAR□. For example, the n‐3 PUFA EPA has been shown to have an effect on lipid droplet size through effects on lipid catabolism, as well as mediate reductions in pro‐inflammatory adipokine secretion in favor of anti‐inflammatory secretions. The objectives of this experiment were to determine the effects of n‐3 and ‐6 PUFAs on TAG lipolysis, as measured by free fatty acid (FFA) release, and inflammatory cytokine production, as measured by interleukin (IL)‐6 and macrophage inflammatory protein (MIP)‐1α production. In order to examine these effects, the 3T3‐L1 cells were differentiated in media with 100μM of PUFA treatment (α‐linoleic acid [LA], arachidonic acid [ARA], and EPA), followed by a 6 h, 1μg/mL of lipopolysaccharide (LPS) challenge. Fully differentiated 3T3‐L1 adipocytes under these treatments did not release FFA in quantifiable amounts. The LPS+ARA treatment resulted in significantly higher levels of MIP‐1□ (26.3±2.5 pg/ml) and IL‐6 (146.13±17.0 pg/ml) in conditioned media, compared to control (MIP‐1□:0.55±2.5; IL‐6:0.23±17.0), LPS (MIP‐1□:0.06±2.5; IL‐6:16.5±17.0), and LPS+LA (MIP‐1□:7.5±2.5; IL‐6:27.0±17.0). Cells treated with LPS+EPA released similar amounts of MIP‐1□ (14.9±2.5) but significantly lower amounts of IL‐6 (20.3±17.0) compared to LPS+ARA‐treated cells. These data suggest that differentiating 3T3‐L1 adipocytes in the presence of the n‐6 PUFA ARA results in increased release of LPS‐stimulated IL‐6 and MIP‐1□. These results indicate a role of this n‐6 fatty acid on adipocyte‐derived inflammation.

Support or Funding Information

Laurie Bukovac Hodgson & David Hodgson Endowed Fund, as well as the Denison Summer Science Research Supply Fund
Hormesis ultraviolet radiation and its effects on Drosophila melanogaster performance
Authors: Berry Raymond , Lopez-Martinez Giancarlo
Published: April 2020

Hormesis can be protective as a multifarious response that results in upregulation of genes and organismal performance. The protective effects of hormesis in animal models includes improved survival, mating, extended longevity, and improved performance at old age (i.e. mating); amongst others. The sun is a source of energy used by terrestrial life in many trophic levels allowing for exposure to irradiation (UVR), a natural environmental stressor that increases production of reactive oxygen species (ROS) .This type of environmental stress is damaging by attacking lipids, proteins and DNA. Exposure to prolonged UVR conditions early in life (pupae) leads to changes in performance later in life. This response can be classified as preconditioning, pretreatment, conditioning, or cross tolerance. Previous studies scantily provide appropriate levels of exposure needed for UVR hormesis that would increase production of ROS and antioxidant enzymes. The age of the individual at the time of treatment can have dramatic effects on hormesis with low dose UVR effects that induce protection, an extension of normal life span and improved organismal performance shown in other insects. The vinegar fly, Drosophila melanogaster, was exposed to UVA at different intensities. Vials containing pupae, separated from food and kept in same vial were exposed to 0, 30, 45 or 60 minutes of ultraviolet radiation A (nm = 365). Different rates of UVA exposure (mW/m2), produced by the distance from the source, show slight differences in treatment survival/adult emergence and flight ability. Low doses of UVR lead to protective and beneficial responses, while exposures to high doses are damaging and detrimental. Ultimately, data such as these can be refined to develop a model of dose response that can be used to estimate hormesis, or the biphasic response.

Support or Funding Information

NIH RISE 5R25GM061222‐19
Effects of Cannabidiol on RAW 264.7 Macrophage Viability and Inflammatory Markers
Authors: Danchine Veronika Denise , Caldari Cristina
Published: April 2020

Cannabidiol (CBD) is a non‐psychoactive derivative from the marijuana plant. There has been an explosion of readily available commercial products infused with and marketing CBD contents, and yet there is a paucity of information regarding the specific effects of CBD on immunity. Our laboratory is interested in examining the effects of CBD on innate immunity, using the immortalized murine macrophage cell line, RAW 264.7. The objective of this research was to determine the effects of CBD on the proliferation, activation, and inflammatory cytokine production in lipopolysaccharide (LPS)‐activated murine macrophages. Additionally, we wanted to determine if maintaining the murine macrophages in serum‐free media during CBD incubation would have an effect on cell viability. RAW 264.7 cells were incubated with either 0.2 or 20 μM of CBD for 24 hours in serum‐free media (SFM) or complete media (CM), followed by a 6 hour, 0.01 μg/ml LPS challenge in SFM. Cell viability was determined via cell counts with Trypan blue exclusion and MTS assay. Macrophages cultured in CM tended to have higher percentages of live cells (Trypan blue exclusion method) compared to those cultured in SFM (54.7+5.4% vs 31.8+5.4% live; ANOVA, p=0.07). Additionally, culturing macrophages in CM also resulted in significantly higher viability (MTS assay) compared to macrophages in SFM (0.58+0.06 vs 0.29+0.06 490nm absorbance; ANOVA, p=0.03). There was no significant effect of CBD concentration (ANOVA; p=0.4) or CBD concentration*media (ANOVA; p=0.2) type on cell viability. These results suggest that concentrations of 0.2 and 20 μM CBD have no detrimental effects on murine macrophage viability and proliferation and that CBD pre‐incubation of macrophages should be performed in complete media. Additionally, data will be presented on the effects of CBD on macrophage activation, including nitric oxide (NO) and interleukin (IL)‐6 production.

Support or Funding Information

Denison University Biology Department and Denison Lisska Center for Scholarly Engagement
Creation of a Novel Absorbent Bandage for Pressure Ulcer Treatment
Authors: Dunlap Jared N. , Fischer Kristin , Wolyniak Michael J.
Published: April 2020

Research was undertaken to explore the possibility of using solid, microporous materials in a bandage that when applied to a pressure ulcer that achieves the following criteria:

‐contains a microporous material as the secondary absorbent that withholds moisture under bodily conditions.

‐sustains an ideal moisture level on the wound bed to create an ideal healing environment.

‐expect an absorbance capacity for water from the microporous material under body‐like conditions over 25%.

‐accounts for multiple different secretion types in and around wounds.

‐integrates this adsorbent into a comfortable gauze bandage for easy for use by doctors and nurses.

‐prevents infection and bacterial growth in this bandage by integrating a heat‐stable antibiotic into the bandage.

The goal of this research was to understand if these goals were feasible, then design a bandage containing a microporous material that could prevent pressure ulcer progression and infection, and further test the used components for effectiveness and unintended reactions. Comparative literature research was conducted on several heat stable antibiotics and on multiple microporous materials including kaolin clays, silica gel, and zeolite. It was determined that synthetic zeolite (4 Angstrom Molecular Sieve) was the ideal candidate to use in the bandage. A highly effective, heat stable antibiotic was also chosen to integrate into the bandage: Trimethoprim‐Sulfamethoxazole. The combination of the two materials yields the potential for the long‐term application of this bandage. However, when acting as a Hemostatic Agent, zeolite has been found to cause an exothermic reaction when it contacts blood. In order to continue my construction and study of the bandage, this interaction needs to be further understood in relation to pressure ulcers. Bovine serum, whole blood, and ratios of fractionated blood was tested in tissue wells with zeolite at physiological temp (37□) for any significant exothermic reaction amounting to a temperature change over 5□ from physiological temp. No significant temperature change was noted with Fetal Bovine Serum and will be used as a control. Another goal includes testing upon the zeolite mechanism of action if such a temperature change occurs with blood ratios found in pressure ulcers, in order to potentially mitigate the effects when implemented in a bandage. Another goal is testing to study effectiveness of antibiotics when integrated and subjected to heat and vacuum with zeolite. The end goal is finally designing and constructing a prototype bandage.

Support or Funding Information

‐Hampden‐Sydney College Undergraduate Research Council.
How does GDP‐fucose get into the Endoplasmic Reticulum for O‐fucosylation of EGF Repeats and TSRs?
Authors: Yuan Youxi , Haltiwanger Robert S.
Published: April 2020

Fucose is a common monosaccharide found in glycan chains of all mammalian cells. Fucose is transferred from GDP‐fucose to glycoproteins by fucosyltransferases, resulting in terminal fucosylation of N‐linked glycans or mucin‐type O‐glycans, or direct modification of serine or threonine residues by Protein O‐fucosyltransferases (POFUT) 1 or 2. Defects in fucosylation can lead to developmental disorders or cancer. In particular, POFUT1 adds O‐fucose to Epidermal Growth Factor‐like (EGF) repeats of Notch receptors, while POFUT2 adds O‐fucose to Thrombospondin type 1 Repeats (TSRs) found in multiple cell‐surface and secreted proteins, including the A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) family of extracellular proteases. Elimination of Pofut1 or Pofut2 results in early embryonic lethality in mice, indicating these two fucosylation pathways play essential roles in mouse development. Both POFUT1 and POFUT2 are located in the endoplasmic reticulum (ER), while fucosyltransferases involved in terminal fucosyaltion are localized in the trans‐Golgi. GDP‐fucose is synthesized in the cytoplasm either through a de novo or salvage pathway, and the GDP‐fucose is then transported into the lumen of the ER or Golgi for use by the fucosyltransferases. Currently two GDP‐fucose transporters, SLC35C1 and SLC35C2, have been identified, both of which are localized to the Golgi. It is unknown how GDP‐fucose is transported into the ER where Notch receptors and ADAMTS proteins are fucosylated. In order to uncover where the GDP‐fucose for POFUT1 and POFUT2 comes from, we have generated CRISPR‐Cas9 mutant cell lines in which fucosylation is impaired in two ways: 1. Inactivating GDP‐fucose biosynthetic enzymes from the salvage or de novo pathways; 2. Inactivating the GDP‐fucose transporters. HEK293T cells have proven to be an excellent model for fucosylation studies. All the fucose biosynthesis genes are normally expressed, and impaired fucosylation can be expected in knockout mutant cell lines. Using CRISPR‐Cas9 technology, the following genes have been knocked out: GMDS and TSTA3 of the de novo pathway, FUK and GFPP of the salvage pathway, and the GDP‐fucose transporters SLC35C1 and SLC35C2. O‐Fucosylation of EGF repeats and TSRs and terminal fucosylation of N‐linked glycans is being analyzed using Mass Spectrometry and lectin binding. Notch function is also being evaluated. These results should reveal whether a preferred biosynthetic pathway or transporter for different forms of fucosylation exists.

Support or Funding Information

Supported by NIH grant GM061126.
Elucidating the Mechanism of DNA Repair in Bdelloid Rotifers by Using RNAi Knockdown and CRISPR/Cas9 Editing
Authors: Gardner Storm Devin , Schurko Andrew Michael
Published: April 2020

Genomic DNA regularly becomes damaged by numerous ways, including desiccation, radiation exposure, and during recombination in cell division. Fortunately, cells have DNA repair pathways that seek out and correct damage. The bdelloid rotifer Adineta vaga is an excellent model organism to study DNA repair because it has a highly efficient DNA repair system. Bdelloids can recover from desiccation and return to life by repairing DNA damage endured in the desiccated state. Bdelloids have also been shown to recover from DNA damage caused by massive doses of ionizing radiation. The mechanisms of this miraculous DNA repair in these aquatic invertebrates are unknown. This project explores using RNA interference (RNAi) and CRISPR/Cas9 genome editing as tools to investigate the function of DNA repair genes, MSH6 and MLH1, in bdelloid rotifers. When target genes are knocked down or inactivated, DNA repair in bdelloids should be compromised. For RNAi, two methods were explored. For the “feeding method,” bdelloids were fed strains of E. coli that express double stranded RNA (dsRNA) copies of MLH1 and MSH6. For the “soaking method,” dsRNA copies of MLH1 and MSH6 were synthesized, and bdelloids were transfected with these molecules. The expectation was that ingested or transfected dsRNA would induce RNAi in bdelloid rotifers. Real‐time PCR followed to quantify expression of the target gene and determine if the RNAi knockdown was successful. For the feeding method, an MLH1 knockdown was observed 6–12 hours after feeding, but the effect was absent 24 hours after feeding. For the soaking method, an MSH6 knockdown was only present when rotifers were transfected with 0.2 μg dsRNA and incubated for 48 hours. As an alternative tool for studying gene function, CRISPR/Cas9 genome editing was done to mutate DNA repair genes. For utilizing CRISPR/Cas9, a single guide RNA that targets MLH1 was synthesized and used in a Cas9 in vitro cleavage assay to determine its ability to cut the target sequence. The guide RNA was then combined with Cas9, and these ribonucleoprotein complexes were electroporated into bdelloids. Mutation detection using T7 endonuclease I was then done to identify alterations to the gene sequence. Gene knockdowns using RNAi and gene mutations using CRISPR/Cas9 will provide a reverse genetics approach to identify genes that have a role in bdelloid DNA repair. This could enhance our understanding of similar mechanisms in traditional model systems, which could have implications for biomedical research.

Support or Funding Information

This work was supported by grants from the Hendrix College Odyssey Program and the Hugh Stevens Moseley Undergraduate Research Awards.
Renomedullary Interstitial Cell Endothelin A Receptors Regulated Blood Pressure and Renal Function
Authors: Hu Chunyan , Lakshmipathi Jayalakshmi , Stuart Deborah , Peti-Peterdi Janos , Kohan Donald E.
Published: April 2020

Renomedullary interstitial cells (RMIC) may modulate blood pressure (BP) and renal function through release of natriuretic factors and through direct effects on medullary blood flow (RMIC encircle vasa recta). Endothelin‐1 (ET‐1) modulates RMIC vasoactive factor release and contraction via ETA receptors (ETAR) in vitro. Consequently, we tested the hypothesis that knockout of ETAR in RMIC regulates renal function and BP in vivo. We bred ETARf/f mice containing loxP‐flanked exons 6–8 of the EDNRA gene with tamoxifen‐inducible tenascin‐C‐CreER2 knockin mice. Tenascin‐C‐CreER2:ETARf/f mice were injected with tamoxifen (160 mg/kg IP) for 5 consecutive days at 3 months of age to obtain RMIC ETAR KO. Littermates of the same genotype without injection served as controls. EDNRA gene recombination occurred in inner medulla, but not cortex. Inner medulla ETAR mRNA content fell by 80%, while ETBR mRNA levels were unaffected. ETRA KO did not affect BP in mice on normal (0.3% Na+) or low (0.03% Na+) salt diets. In contrast, ETAR KO reduced salt‐sensitive systolic BP elevation in mice on a high (3.2% Na+) salt diet (ETAR KO 127±3 vs. control 134±2 mmHg, n=13, 15, p<0.05). Urine volume and urinary Na+ and K+ excretion were higher in high‐salt diet‐treated ETAR KO mice compared with controls during the first 3 days of high salt intake. Urinary ET‐1, nitrite/nitrate, and prostaglandin E2 excretion were increased in high salt diet‐treated ETAR KO mice vs. controls. No significant differences were found in GFR, vasopressin, aldosterone, or renin levels between high salt fed ETAR KO and control mice. Inner medullary protein and mRNA levels of total NKCC2 and ENaC‐α and ‐β were decreased, and inner medullary cyclooxygenase‐2 (COX‐2), but not COX‐1, protein levels were increased, in high salt fed ETAR KO vs. control mice (n=8). Exogenous ET‐1 did not detectably alter RMIC intracellular Ca2+ concentration or cause RMIC contraction as assessed by ex vivo inner medulla confocal imaging. We conclude that disruption of RMIC ETAR reduces salt‐sensitive hypertension and this is associated with reductions in NKCC2 and ENaC together with elevations in several autocrine and paracrine natriuretic factors. These effects of RMIC ETAR, at least based on ex vivo studies, may not involve RMIC contraction. While further study is required, our data suggest a novel regulatory pathway for maintaining water/electrolyte homeostasis.

Support or Funding Information

NHLBI P01 HL136267
Coronary Vascular Cell Stiffness Influences Coronary Blood Flow
Authors: Montgomery Kathlyene R. , McCallinhart Patricia E. , Trask Aaron J.
Published: April 2020

Our laboratory has previously shown that type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) undergo inward hypertrophic remodeling associated with reduced stiffness. Reduced T2DM CRM tissue stiffness is associated with decreased elastic modulus of coronary vascular smooth muscle cells (VSMCs). The goal of this study was to test the hypothesis that reducing or augmenting coronary vascular cell stiffness will increase or decrease coronary blood flow, respectively. Coronary blood flow was measured from hearts isolated from 16‐week‐old control Db/db and diabetic db/db mice and perfused on a Langendorff system at a constant pressure of 80 mmHg. Latrunculin B (1 μM final concentration; n=2 per group) was infused to depolymerize actin and reduce vascular cell stiffness and jasplakinolide (0.2 μM final concentration; n=4 per group) was infused to increase actin polymerization and increase vascular cell stiffness. After drug infusions, hearts were immediately transferred to an adjacent Langendorff apparatus and were perfusion fixed with 4% paraformaldehyde for 2 mins at 1 mL/min. Hearts were then placed in 4% PFA for 24–48 hours and were stored in 70% EtOH until embedding in paraffin and sectioning. Sections were evaluated for F‐ and G‐actin by immunofluorescence. In pooled Db/db and db/db analyses, Latrunculin B caused an increase in coronary blood flow over baseline (34.1 ± 15.0% above baseline, n=7), and jasplakinolide caused a 26.5 ± 4.1% reduction in coronary blood flow from baseline (n=8). Immunohistochemical analysis confirmed a decreased trend in F/G actin in the coronary microvessels of latrunculin B‐perfused hearts (1.00 vs. 0.82 ± 0.08, n=1–4 per group), while F/G actin tended to be increased in the CRMs of jasplakinolide‐perfused hearts (1.00 ± 0.00 vs. 1.50 ± 0.16, p=0.12, n=2–6 per group). Importantly, the F/G actin ratio in jasplakinolide‐treated hearts was not different measured in the myocardium 50–100 μm away from a CRM (1.00 ± 0.06 in vehicle‐perfused hearts vs. 1.01 ± 0.10 in jasplakinolide‐perfused hearts, p=0.96, n=2–6 per group), demonstrating that the drug was efficacious only in the peri‐vascular region. These data support the hypothesis that coronary blood flow increases as vascular cell stiffness is decreased and coronary blood flow decreases as vascular cell stiffness is increased. Collectively, these data suggest that coronary vascular cell stiffness can modulate coronary blood flow.

Support or Funding Information

KRM was supported by an American Heart Association Summer Undergraduate Research Fellowship (17UFEL33420025). NIH R00HL116769, R21EB026518, and S10OD023438 to AJT.