bluuebunny/crossref_metadata_2025 huggingface dataset:
Vitamin D mitigates intestinal inflammation through modulation of oxidative stress, autophagy and intestinal permeability in Cftr‐null mice
Authors: Mailhot Genevieve , Morin Genevieve , Laffont Alisson
Published: April 2020

Background

The gut is the earliest organ affected by cystic fibrosis (CF), often in utero. Besides maldigestion and malabsorption, the other common intestinal manifestation is chronic inflammation, which exerts a negative impact on the nutrition, lung health and quality of life of these individuals. To date, few treatments exist to alleviate CF intestinal inflammation. Vitamin D plays anti‐inflammatory roles in the intestine through autocrine and/or paracrine actions, but its potential has been poorly investigated in the context of CF disease. Due to its anatomic location, the gut is the tissue exposed to the largest amount of vitamin D from external sources, making this vitamin an attractive therapeutic option. We previously showed that intestinal mucosa from CF mice exhibited increased eosinophilic infiltration compared to non‐CF mice, an effect that was reversed by the administration of a high‐vitamin D3 diet. We thus postulate that vitamin D mitigates CF intestinal inflammation through regulation of critical processes involved in inflammatory responses, such as permeability, oxidative stress and autophagy.

Methods

Twelve‐week old BALB/c CF (Cftr−/−) and non‐CF (Cftr+/+) mice were fed ad libitum a normal vitamin D3 diet (1,500 IU/kg diet) or a high‐vitamin D3 diet (5,000 IU/kg diet) for 8 weeks. Intestinal sections were harvested and assessed for the expression of markers of oxidative stress (e.g. deoxyguanidine), intestinal permeability (e.g. zonulin‐1) and autophagy (e.g.LC3, p62) by means of quantitative PCR, immunofluorescence and Western blotting.

Results

Deoxyguanidine (a marker of oxidative DNA damage) and zonulin‐1 duodenal immunostaining was increased in Cftr−/− mice fed with the normal vitamin D3 diet compared to diet‐matched Cftr+/+ mice. Protein ratio of LC3‐II to LC3‐I was higher whereas p62 expression was lower in the duodena of Cftr−/− mice, suggesting enhanced baseline autophagy. Cftr−/− mice fed the high vitamin D3 diet showed a decreased expression of deoxyguanidine, zonulin‐1 and LC3‐II to LC3‐I ratio and an increased p62 expression to levels of Cftr+/+ mice. Of note, the high‐vitamin D3 diet did not have any measurable impact on the intestinal mucosa of Cftr+/+ mice, which was comparable to that of Cftr+/+ mice fed the normal vitamin D3 diet.

Conclusions

Together, our findings show that the Cftr−/− duodenal mucosa exhibits increased permeability, autophagy and oxidative damage that may all contribute to the chronic inflammatory state. A high vitamin D3 diet given for 8 weeks reverses those alterations, making this vitamin a potential therapeutic option for CF intestinal inflammation.

Support or Funding Information

This work was supported by a grant from Cystic Fibrosis Canada
Sex‐Specific Alterations on Muscle Plasticity and Myogenesis in Offspring Rats from Vitamin D Deficient Mothers
Authors: Navegantes Luiz , Reis Natany , Turatti João , Gonçalves Dawit , Silveira Willian , Lautherbach Natália , Almeida Lucas , de Assis Ana Paula , Zanon Neusa , Heck Lilian , Kettelhut Isis
Published: April 2020

Objective

To analyze the impact of maternal Vitamin D deficiency (VDD) on offspring muscle development in adulthood of male and female rats.

Methods

Twelve 5‐week‐old female Wistar Hannover rats were fed either a Vit. D3+ diet (AIN93G with 1000 IU vitamin D3/kg diet) or Vit. D3− diet (AIN93G without vitamin D3) for six weeks and then bred to male rats. Mothers were maintained on the diets throughout gestation and lactation. At weaning, male and female offsprings were separated in four groups: male and female offspring control (M‐CTRL and F‐CTRL, respectively) pups of dams with Vit. D3+ diet; male and female offspring VDD (M‐VDD and F‐VDD, respectively) pups of dams with Vit. D3− diet. Offspring received a standard diet (Nuvilab) until 180 days of age, at which point tissues were harvested for analysis. *P≤0,05 (CEUA 052/2018).

Results

Both male and female VDD groups showed a reduction in the calcidiol serum concentration (23±1 vs 40±2 ng/ml in M‐CTRL and 23±2.2 ng/ml vs 36.6±0.9 ng/ml in F‐CTRL) without affecting Ca+2 serum. At weaning, M‐VDD weighed less than M‐CTRL rats but recovered weight in the 60 days post weaning and acquired a greater body weight accompanied by a higher mass of white and brown adipose tissue at 180 days. Extensor digitorum longus (EDL) muscles from the M‐VDD showed a decrease (20 %; p<0.05) in the number of total fibers and an increase in the cross sectional area of IIB (17 %), IIA (19 %) and IIAX (21%; p<0.05) fibers. VDD also deregulated myogenesis as indicated by the higher levels of MyoD (43%; p<0.05) and Myogenin (160%; p<0.05) and in the number of satellite cells (128.8±14 vs 91±7.6 nuclei Pax7+ in the MO‐CTRL). To further investigate the signaling pathways involved in the muscle hypertrophy effect in the M‐VDD group, p‐IGF‐1/insulin receptor levels, and different insulin downstream targets related to protein synthesis including Ser 473p‐Akt, Ser 21/9 p‐GSK‐3β and Ser 209p‐eIF4e were measured. VDD increased all these parameters and in parallel increased plasma insulin and muscle IGF‐1 mRNA levels, but did not affect mTOR signaling. These changes were not found in Soleus from the M‐VDD group and in both EDL and Soleus from the female offspring.

Conclusion

Maternal VDD selectively affect the development of glycolytic muscle in male offspring characterized by hypoplasia, hypertrophy and deregulated myogenesis. The muscle hypertrophy is a compensatory mechanism probably due to the greater insulin sensitivity and activation of the Akt pathway. Muscles from female offspring seem to be protected from this metabolic disturbance showing a clear sex‐specific effect induced by maternal VDD.

Support or Funding Information

FAPESP (19/06517‐1; 18/10089‐2)
Amniotic fluid mesenchymal stem cells ameliorate lung alveolar epithelium regeneration and reverse lung fibrosis of chronic obstructive pulmonary disease
Authors: Chen Chuan-Mu , Yang Jing-Chan , Yen Chih-Ching , Chen Hsiao-Ling
Published: April 2020

Pulmonary emphysema progression is attributed not only to alveolar structure loss and pulmonary regeneration impairment but also to excessive inflammatory response, proteolytic and anti‐proteolytic activity imbalance, lung epithelial cell apoptosis and abnormal lung remodeling. To ameliorate lung damage with higher efficiency through lung tissue engineering and cell therapy, predifferentiating graft cells into more restricted cell types before transplantation could enhance their ability to anatomically and functionally integrate into damaged lung. An optimal lung epithelial progenitor‐like cells (LEPLCs) predifferentiation condition has been established in eGFP‐expressing amniotic fluid mesenchymal stem cells (AFMSCs). Transplantation of LEPLCs significantly improved regeneration of lung tissue as they integrated into the lung alveolar structure, relieved airway inflammation, alleviated amount of DNA damage in alveolar cells, increased expression of growth factors such as vascular endothelial growth factor, and reduced expression of matrix metalloproteinases and lung remodeling factors when compared with the elastase‐induced pulmonary emphysema mice injected with AFMSCs. In conclusion, transplantation of predifferentiated AFMSCs through intratracheal injection showed better alveolar regeneration and reverse lung fibrosis in PPE‐induced pulmonary emphysema mice.

Support or Funding Information

This research was supported by grant MOE‐108‐S‐0023‐A from the Ministry of Education of Taiwan.
Investigation of Wnt Pathway Genes in a Highly Regenerative Marine Invertebrate
Authors: Hughes Jun-Wei Brendan , Ward Vivienne , Jamison Cameron Ann , Johnson Christine , Sumner Jack Thomas , Keeling Elena
Published: April 2020

Colonial ascidians are the closest known relatives to vertebrates that retain whole body regeneration. Understanding molecular pathways involved in regeneration in a diverse range of organisms will provide insights into evolutionary changes in regeneration. Specifically, our research explores Wnt signaling in the colonial ascidian Botrylloides violaceus; this highly conserved pathway is universally involved in studied animals with any regenerative ability. A recent rough draft genome for B. violaceus reveals multiple putative ligands, receptors, and downstream effectors from the Wnt pathway. Primers designed from the draft genome are being used in PCR to validate these predicted genes. Future studies will use qPCR to track gene expression of different Wnt pathway members through regeneration.

Support or Funding Information

The Frost Fund, College of Science and Mathematics at California Polytechnic State University, San Luis Obispo
Effects of Elevated CO2 on Yellowfin tuna (Thunnus albacares) Early Life Stage Respiration and Ammonia Excretion
Authors: Heuer Rachael M. , Wang Yadong M. , Pasparakis Christina , Scholey Vernon , Margulies Daniel , Grosell Martin
Published: April 2020

Although the impacts of near‐future projected CO2 levels on coastal fishes are well‐documented, few studies have addressed impacts to broad‐ranging pelagic fish species which generally live in a more stable environment and therefore may be more impacted by change. The early life stages of pelagic fish may be especially sensitive to CO2, since acid‐base regulatory mechanisms are not fully developed. Furthermore, few studies have examined how CO2 exposure could impact nitrogenous waste excretion in early life stage fish. In the present study, we examined the impacts of near‐future CO2 on early life stages of the Yellowfin tuna (Thunnus albacares). Tuna embryos were obtained from a captive spawning broodstock population of Yellowfin tuna at the IATTC Achotines Laboratory. Tuna were exposed to either control or 1900 matm CO2 (prediction for year 2300) directly after spawn and through the first 3 days of development as embryos hatched into yolk sac larvae. Metabolic rate, ammonia tissue accumulation, and ammonia excretion were quantified at various timepoints throughout development. Since Rh proteins putatively transport CO2 in addition to ammonia, samples to assess mRNA expression of Rh proteins were also taken at each developmental timepoint. Exposure to elevated CO2 did not significantly impact the metabolic rate of developing tuna, however, at the final larval stage, a 22% decrease in metabolic rate was noted. This decline was immediately preceded by a significant increase in ammonia excretion in CO2‐exposed larvae, despite no difference in ammonia tissue accumulation when comparing control and CO2‐exposed larvae. Interestingly, CO2‐exposed larvae exhibited a significant increase in urea accumulation at this timepoint. These data suggest that larvae are producing more nitrogenous waste, but are able to avoid the build‐up of toxic ammonia by maintaining higher levels of ammonia excretion and/or by converting ammonia to urea. Analysis of mRNA expression of Rh proteins is ongoing and may provide a mechanism for this increased nitrogenous waste excretion. These results suggest egg and yolk sac larvae life stages of yellowfin tuna may be equipped with mechanisms to counteract increases in CO2. This research was made possible by a grant from The Gulf of Mexico Research Initiative. Grant No: SA‐1520.

Support or Funding Information

This research was made possible by a grant from The Gulf of Mexico Research Initiative. Grant No: SA‐1520.
Physical Inactivity‐induced Endothelial Dysfunction : The Role of Toll‐like Receptor 4
Authors: Guerrero Maria , Sun Chang , Kwon Oh Sung
Published: April 2020

Background

A hallmark characteristic of physical inactivity is not only the rapid deterioration of cardiovascular function and whole body insulin sensitivity, but also the accumulation of free fatty acids (FFAs) which contribute to vascular dysfunction, specifically endothelial cell dysfunction as a consequence of a mismatch between the synthesis and degradation of nitric oxide (NO). Although a mechanism that may contribute to physical inactivity‐induced endothelial dysfunction is unclear, the accumulation of the sphingolipid ceramide within endothelial cells may activate Toll‐like receptor (TLR) 4, which finally leads to vascular dysfunction. Therefore, this study sought to investigate the role of TLR4 signaling in physical‐inactivity (hindlimb unloading) induced endothelial dysfunction.

Methods

Adult female wild type (WT) mice were separated in three groups ; control (CON) (n=8), hindlimb unloading for 14 days (HU) (n=8), and hindlimb unloading with toll‐like receptor 4 inhibitor, TAK‐242 (4mg/kg) (HUT) (n=8). Following 14 days, using an in vitro preparation, endothelium‐dependent and ‐independent vasodilation were assessed in arteries in response to three stimuli 1) flow‐induced shear stress, 2) acetylcholine (ACh), and 3) sodium nitroprusside (SNP). Additionally, L‐NG‐monomethyl arginine citrate (L‐NMMA) was utilized to identify the impact of blocking NO synthase on these responses. Endothelial NOS (eNOS), TLR4, NFkB protein expression were analyzed via Western blot analysis.

Results

After 14 days of physical inactivity, HU group showed that progressive physical inactivy‐induced reductions in endothelial‐dependent vasodilatory function were present in response to the intraluminal flow (CON: 81 ± 3; HU: 40 ± 2%, P < 0.05) and ACh (CON: 93 ± 4, HU: 44 ± 3 %, P < 0.05). However, TLR4 inhibition using TAK‐242, significantly restored endothelial function following physical inactivity; intraluminal flow (HUT: 73 ± 2%, P < 0.05) and ACh (HU: 85 ± 3 %, P < 0.05). All groups had no significant effect on endothelium‐independent vasodilation (SNP). P‐eNOS/eNOS protein expression was significantly decreased with physical inactivity but TAK‐242 prevented HU induced eNOS reduction. In addition, HU significantly activated an inflammation marker, NFkB protein expression, but TAK‐242 prevented physical inactivity‐induced NFkB protein expression.

Conclusions

Overall, these findings suggest that physical inactivity induces endothelial dysfunction and endothelial TLR4 signaling may play a critical role in physical inactivity‐associated endothelial dysfunction. Therefore, TLR4 signaling could be a novel therapeutic target to restore endothelial function and NO bioavailability.
Comparing Lipid and Detergent Environments as Models for Studying Renal Transmembrane Proteins
Authors: Pavkov Isabela , Strickland Kerry , Cottrill Kirsten , Imhoff Barry R. , McCarty Nael A.
Published: April 2020

Background

Mutations in transmembrane proteins cause kidney diseases such as Dent’s disease, Bartter syndrome, and polycystic kidney disease.
1 Understanding the structure and function of renal membrane proteins reveals how they contribute to kidney function as well as kidney disease. Recent studies have shown the importance of generating a native lipid environment when studying membrane proteins.
2 However, membrane proteins are often studied in the presence of detergent, which may modify their natural structure and lead to decreased function. Nanodiscs, a novel lipid bilayer system, provide a more native environment for protein reconstitution.

Methods

In this study we used two proteins found in the kidney, the Na+/K+‐ATPase pump and CFTR ion channel, to determine whether nanodiscs or detergent micelles serve as the better model for studying renal transmembrane proteins. To do this, we expressed and purified our own Membrane Scaffold Protein (MSP), a base protein for nanodiscs, in Escherichia coli. We generated empty nanodiscs with three different lipid types: 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC), 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC), and 1‐Palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE).

Results

We have and are continuing to reconstitute CFTR and Na+/K+‐ATPase into nanodiscs with different lipid compositions. We evaluate membrane protein stability using ATPase activity in detergent and different lipid nanodisc environments. Preliminary data show improved ATPase activity for CFTR in nanodiscs versus detergent. Higher ATPase activity of proteins reconstituted in nanodiscs would indicate that detergent structurally modifies these proteins and consequently decreases their function. Nanodiscs provide a more stable environment in which to study these and other proteins found in the kidneys, allowing for more accurate assessment of structure and function.
Role of miR‐130b/301b cluster in macrophage polarization and obesity
Authors: Jiang Shaoning , McBride Aubrey , Luo Wenyi , Chernausek Steven
Published: April 2020

Macrophage activation status, such as M1 classic and M2 alternative activation, plays an important role in the development of obesity and other metabolic diseases. The molecular mechanisms regulating macrophage M1/M2 polarization are not fully understood. MicroRNAs are regulatory RNAs that suppress the translation and stability of the mRNA targets. In the present studies, we show that compared to wild type mice, knockout mice of miR‐130b/301b cluster exhibited decreased adiposity, increased whole body energy expenditure, and increased oxygen consumption in response to high fat diet. miR‐130b/301b knockout attenuated the LPS‐induced pro‐inflammatory activation of bone marrow macrophages, while promoted IL‐4 induced alternative macrophage activation. In addition, miR‐130b/301b knockout mice had increased AMP‐activated protein kinase (AMPK) activation in white adipose tissue, which can explain the increased energy expenditure. Together, the present study identified miR‐130b/301b cluster as a novel regulator of macrophage polarization and energy metabolism, which contributes to the development of obesity.

Support or Funding Information

The study is supported by OCAST (PI: Shaoning Jiang) and OK‐INBRE (RPI: Shaoning Jiang) grants.
Proteomic and Functional Characterization of STARD9
Authors: Clutario Kevin
Published: April 2020

The centrosome is a non‐membrane bound structural organelle that is crucial in spindle assembly, as the centrosomes act as the microtuble organizing centers at the spindle poles during mitosis. Deviation from normal spindle assembly can lead to activation of the spindle assembly checkpoint (SAC). Some of the most successful antimitotic drugs activate the SAC in order to arrest cancer cells in mitosis to induce subsequent apoptosis. Recently, kinesins and kinases implicated in spindle assembly have gained attention as possible therapeutic targets, as their functions rely on their ability to hydrolyze ATP. Our lab’s previous proteomic and genetic studies identified steroidogenic acute regulatory protein‐related lipid transfer domain containing 9 (STARD9) as a kinesin involved in spindle assembly. Its depletion invokes multipolar spindles in dividing cancer cells and most surprisingly, STARD9‐depleted cancer cells display increased Taxol sensitivity. STARD9 plays a functional role in the stabilization of pericentriolar material (PCM) during centriole maturation, but its mechanism of action and its regulation are not yet fully understood. To elucidate these mechanisms we have used proteomic and chemical‐genetic approaches to study both STARD9’s major domains and endogenous STARD9 protein. These approaches have identified putative interacting proteins that may play regulatory roles in PCM stabilization. We are currently focused on determining the importance of these STARD9 interactions for PCM stabilization. Our work looks to further the knowledge of STARD9’s function within centrosome assembly and more broadly cancer cell division.

Support or Funding Information

USHHS Ruth L. Kirschstein Institutional National Research Service Award #T32 CA009056
Molecular pathological mechanism of blue ray induces murine photoreceptor cell 661W apoptosis
Authors: Chang Wei-Min , Chen Chun-Yen , Li Li-Jie , Chan Ming-Hsien , Hsiao Michael
Published: April 2020

Modern life is full of artificial light in day and night. Smart phones, laptops, televisions, and high definition monitors provide unnatural human light source into human eye and cause ocular defects in modern human being. Understanding the molecular mechanism of the interplay between artificial light and ocular cells may help researcher society understanding the methods that preventing artificial light‐induced visual impairment. Here, we reported a short‐term exposure at short wavelength light source from blue and green light‐emitting diode (LED) could cause the murine photoreceptor cells 661W cells death, but not in red LED. However, the molecular mechanism remaining unclear. Using the molecular probe of caspase 3 reporter, we found the blue and green LED caused caspase 3 activation even as short as 10 minutes exposure and subsequently cell death at 16 hours later. We further confirmed 661W cells apoptosis by cleavage form caspase 3 and PARP formation. Moreover, we found the decreasing of mitochondria membrane potential and increasing of cellular relative‐oxygen species in 661W cells both indicate early cell apoptosis under short‐term exposure. Up to our knowledge, there was no transcriptome analysis which revealed immediately early gene expression in light‐induced cell death. Thus, we analyzed the 661W transcriptome at 2 hours after 10 minutes’ short wavelength light exposure and validated the candidate genes expression by RT‐Q‐PCR. Using the ingenuity pathway analysis, we found valuable up‐stream regulators, pathways, and potential mechanisms in short wavelength induced photoreceptor cell death and may provide novel therapy niches. In conclusion, our preliminary results prove that short wavelength light exposure can cause the photoreceptor cells death and the potential molecular clues. Further pathohistological analysis of key protein expression in the animal models may help us to prove the mechanism inside the eye damage from short wavelength light in future.

Support or Funding Information

This study was support by Shu Zen College of Medicine and Management with grant number (SZL10800012) and Academia Sinica
FoxO1: a potential target of cinnamon extract on the PI3K cascade
Authors: Borgman Rachael , Pearce Kathryn , Aulthouse Amy , Stockert Amy
Published: April 2020

Cinnamon has long been used medicinally for digestive discomforts. Literature highlights anti‐diabetic properties of cinnamon in cell culture and human clinical studies, suggesting a potential role for type 2 diabetes therapy. Despite these studies and patients’ use of cinnamon, the therapeutic mechanism is still poorly understood. Transcription factor PPAR‐gamma has been identified as one potential target of the cinnamon extract components given the effects of cinnamon on morphological changes in the 3T3‐L1 cells during treatment. We confirmed the presence of PPAR‐gamma in 3T3‐L1 cells using immunohistochemistry providing evidence for the spontaneous differentiation of cells when grown in three‐dimensional culture. Furthermore, we have characterized morphological changes in cinnamon‐treated cells while grown in monolayer and sought to determine what expression changes occur under these growth conditions. Due to the changes in expression that we observed, we identified FoxO1 as a potential target of the cinnamon extract. FoxO1 is a metabolic regulator that has been implemented in lipid localization, aging, and cell death. In addition to PPAR‐gamma, expression and localization of GLUT4 transporters is up‐regulated with cinnamon treatment in both muscle and adipose. Interestingly, FoxO1 is involved with both PPAR‐gamma and GLUT4 expression and activities, making it a reasonable target of the extract. Together, these data suggest that cinnamon not only affects multiple targets, but is also likely involved at several stages in the PI3K cascade. In addition to examining the expression levels of FoxO1 in treated and untreated cells, we sought to explore the localization of the FoxO1 transcription factor as it is proposed to shuttle between the nucleus and cytoplasm depending on conditions. We have initiated immunocytochemistry studies to determine the relative localization of the transcription factor when treated with cinnamon extract as compared to no treatment in order to correlate the location to the expression data.

Support or Funding Information

Ohio Northern University Raabe College of Pharmacy Bower, Bennett and Bennett Research Award
Analysis of Antidepressant Agonist Activity at LPA1, LPA2 and LPA3 lysophosphatidic acid receptors
Authors: Olianas Maria C. , Dedoni Simona , Marras Luisa , Ingianni Angela , Onali Pierluigi
Published: April 2020

Besides inhibiting monoamine reuptake, antidepressants have been shown to affect a number of neurotransmitter receptor systems, at which they most often act as antagonists. We have previously shown that in neuronal and non‐neuronal cells different antidepressants, predominantly belonging to the tricyclic and tetracyclic classes, activate the LPA1 lysophosphatidic acid (LPA) receptor to induce proliferative and prosurvival signaling. In the present study we further characterize this unique agonist activity of antidepressants by examining their ability to activate two additional members of the LPA receptor family, LPA2 and LPA3, which display high sequence similarity to LPA1. The human LPA receptors were stably transfected in HEK293 cells (HEK293‐LPA1–3 cells), and their activation was monitored by measuring the phosphorylation/activation of ERK1/2 by Western blot. Exposure to LPA caused a concentration‐dependent increase of phospho‐ERK1/2 levels with EC50 values of 145, 210 and 45 nM in HEK293‐LPA1, ‐LPA2 and ‐LPA3 cells, respectively. As previously observed in other cell types endogenously expressing LPA receptors, amitriptyline, imipramine, clomipramine, desipramine, mianserin and mirtazapine effectively increased ERK1/2 phosphorylation in HEK293‐LPA1 cells. In HEK293 cells stably transfected with a LPA1 mutant (Q3.29A), which was unresponsive to LPA, the stimulatory effects of amitriptyline, clomipramine and mianserin were greatly suppressed, indicating the dependence on receptor structural integrity. Tricyclic antidepressants failed to stimulate ERK1/2 phosphorylation in HEK293‐LPA2 cells, whereas the tetracyclic compounds mianserin and mirtazapine displayed agonist activity, although with relative efficacies lower than those showed in HEK293‐LPA1 cells. Conversely, both tricyclic and tetracyclic antidepressants induced ERK1/2 activation in HEK293‐LPA3 cells. These data demonstrate that some classes of antidepressants can exert agonist activity at more than one LPA receptor, thus extending their spectrum of pharmacological activity. In particular, the present finding that antidepressants can activate LPA3, which is expressed in human hippocampus, amygdala, frontal cortex and in different peripheral tissues, suggests that this receptor can be a potential target for the therapeutic and adverse effects of these drugs.

Support or Funding Information

University of Cagliari FIR 2019
Understanding the Cell‐Cycle Dependence of Mus81‐Mms4 Recruitment and its Reliance on Slx4
Authors: Siavoshi Mehrnaz Ashley , Le Lindsey , Buo Sorinna , Fischhaber Paula
Published: April 2020

DNA damage repair pathways are a key element in maintaining genomic stability and preventing cancer and other diseases, particularly given the prevalence of DNA damaging agents. One form of DNA damage is double‐strand breaks (DSBs), the repair of which utilizes numerous repair proteins regardless of which subpathway is implemented. One such repair protein in S. cerevisiae is Mus81, which forms a complex with Mms4, that functions as a structure‐specific endonuclease that cleaves flapped DNA structures present in intertwined chromosomes that have undergone DSB repair in late S and G2 phases.

Here, we seek to understand the cell cycle dependence of Mus81‐ recruitment to DSBs and determine if this recruitment is Slx4‐dependent. This exploration requires complete cell cycle synchronization with □‐factor followed by release back into cell cycle following □‐factor removal. As they progress through the cell cycle, cultures are monitored by fluorescence microscopy and flow cytometry to properly correlate fluorescence data with discrete stages of the cell cycle. The yeast strain under study contains a Mus81 protein tagged with yEmRFP which enables its tracking during the fluorescence microscopy portion of the experiment. Preliminary microscopy experiments on a Mus81‐yEmRFP labeled strain have demonstrated that Mus81 is present in low abundance, making it difficult to track the RFP signal throughout the cell cycle. This creates the need for increased expression of Mus81 if it is to be investigated via fluorescence microscopy. Therefore, a plasmid has been constructed that incorporates an ADH1 promoter in front of the Mus81‐yEmRFP gene to enable overexpression. The use of this modified strain will allow for experimental correlation of Mus81‐Mms4 activity with a specific stage of the cell cycle.

Support or Funding Information

National Institutes of Health (NIH) (Grant SC1GM127204)NIH BUILD Program (NIH Grant 5TL4GM118977)
Teaching venous return without throwing students a curve
Authors: Jawad Rasha , McCabe Richard D.
Published: April 2020

Here we develop a comprehensive presentation of venous return.

Guyton’s venous return curve describes venous return in terms of right atrial pressure, mean circulatory filling pressure, and sympathetic activity.
1 This work demonstrated the key role of venous return in cardiac function, and it has been part of undergraduate medical education since that time. However, experience shows that venous return curves are difficult to present or understand, and have limited direct clinical application.
2,
3

Venous return remains of central importance to physiology and the pathophysiology underlying many clinical disorders.
4,
5 We propose a simple, intuitive model to explain venous return that can be progressively presented to explain both venous filling and circulatory mobilization of blood. The latter concept encompasses venoconstriction as well as other mobilizing factors.

The presentation centers around venous return for students familiar with the role of central venous pressure (CVP) in cardiac preload. The model is presented in the following progression:

Start with a simple circuit consisting of the heart, arteries, a set of capillaries, and veins at stop‐flow condition with a specified filling pressure.

Illustrate the development of normal CVP and mean arterial pressure (MAP), starting from stop‐flow to normal cardiac output.

Introduce positive or negative external filling sources that control filling pressure (i.e. gastrointestinal [GI], urinary, respiratory and integumentary systems).

Add transcapillary forces as the factors controlling internal filling, via shifting volume between interstitial fluid (ISF) and plasma.

Model venules ‐ added in parallel in the diagram ‐ as mobilizers of blood under the control of sympathetic nerves.

Add other mobilizing factors (i.e. GI/splenic blood, muscular pumping, muscular filling) as additional parallel elements under appropriate controls.

The model is qualitative, while the venous return curve is quantitative. However, we anticipate it being more easily understood. It is also more comprehensive and more easily applied to clinical medicine. Additional regulatory and pathophysiological factors are easily incorporated into this model. We plan to compare students’ understanding of venous return following presentation of this model versus their understanding following presentation of Guyton’s venous return curve.
Novel Binge Drinking Model Causes Perturbations in Muscle Metabolism with Refeeding
Authors: Steiner Jennifer , Tice Abigail , Laudato Joseph , Johnson Bonde
Published: April 2020

Introduction

It is well known that acute alcohol intoxication impairs metabolic signaling within skeletal muscle including suppression of protein synthesis which is at least partially due to inhibition of mTOR activity as assessed by phosphorylation of its downstream substrates p70 ribosomal protein S6 Kinase 1 (S6K1), ribosomal protein S6 (rpS6) and eukaryotic initiation factor 4E (4E‐BP1). The current mouse model used to mimic binge drinking employs either oral gavage or intraperitoneal (IP) injection, both of which are stressful to the animal and in the case of the injection, lack physiological relevance as it bathes all organs in a high dose of alcohol while bypassing normal intestinal metabolism. Therefore, we wanted to determine if an acute ‘dose’ of alcohol incorporated into the food would be sufficient to mimic the binge drinking effects previously reported.

Methods

C57BL6 male (n=9) and female mice (n=10) were randomized to receive either control liquid diet food (# 710027, DYETS, Bethlehem, PA), or a calorie (kcal) matched liquid diet containing 32% kcal from alcohol (# 710260, DYETS, Bethlehem, PA). Following a 1 week acclimation period to the control liquid diet, mice were fasted for 16 hours during their light cycle and then, at the onset of the dark cycle, were provided with liquid diet (Con or EtOH) for 30 minutes. The gastrocnemius (GAS) and blood were collected 1.5 hour after the beginning of the refeeding period. Western blots were performed on GAS for protein expression. T‐tests (2 tailed, unpaired) for males (Con vs. EtOH), and females (Con vs. EtOH), or between sexes of the same dietary treatment group, were used for analysis (p<0.05).

Results

Despite the high concentration of alcohol in the liquid diet and its novel introduction to the EtOH group, male mice consumed the same volume of food regardless of treatment group. Control females though, consumed significantly more food than EtOH females (4.5 mL vs. 3.6 mL). Alcohol intake per kg body weight was higher in females than males; however, total alcohol intake (g), and blood alcohol levels (213 vs. 187 mg/dL), were not different between males and females. Alcohol intake decreased AKT S473 phosphorylation in GAS, along with phosphorylation of mTOR S2448 in males and females. Downstream substrates of mTOR were also suppressed by alcohol, including phosphorylation of S6K1 T389 and rpS6 S240/244.

Conclusion

Despite lower blood alcohol levels (compared to injection), alcohol reversed or prevented the increase in mTOR signaling typically induced by an anabolic stimulus (i.e. nutrient intake). Further, novel findings included the consumption of a high percentage of alcohol in liquid diet form without any prior exposure to a lower dose or acclimation period to alcohol. Future work will continue to characterize this model and lead to optimization of models of alcohol intoxication that best mimic human practice.
Renal Deafferentation Improves Renal Function and Blood Pressure, but not Cardiac Parameters in an experimental model chronic kidney disease.
Authors: Bergamaschi Cássia Toledo , Sanders-Silveira Sarah , Veiga Amanda Costa , Aires Regina Sousa , Milanez Maycon Igor , Campos Ruy Ribeiro , Nishi Erika Emy
Published: April 2020

Chronic kidney disease is highly prevalent and its mortality is associated with cardiovascular disease. Experimental evidence shows that the renal nerves contribute to arterial hypertension and renal sympathetic hyperactivity worsening the disease; renal denervation has shown renoprotective and cardioprotective effects. However, renal sympathetic innervation plays a fundamental role in renal homeostasis and its removal is associated with impaired compensatory mechanisms during hemodynamic changes. Thus, the aim of this work was to investigate the effects of selective renal denervation of afferent fibers on renal and cardiovascular repercussions in chronic kidney disease (CKD) induced by 5/6 nephrectomy in Wistar rats. The use of animals was approved by the Federal University of São Paulo Animal Care and Use Committee and performed according to the National Institutes of Health guidelines for the conduct of experiments in animals. After five weeks of nephrectomy, denervation of renal afferent fibers with a periaxonal application of 33 mM capsaicin was performed. Cardiovascular and renal function parameters were evaluated three weeks after deafferentation.

Results

CKD induced increase in blood pressure (CTL 113±7.2 × CKD 185±14 mmHg), creatinine plasma levels (CTL 0.5±0.1 × CKD 1.2±0.5 mg/dl) and proteinuria (CTL 14±10 × 300±129 mg/24h). It was also observed an increase in cardiac hypertrophy, reduction in baroreflex sensitivity and in parasympathetic tone after autonomic double blockade. Afferent renal denervation reduced blood pressure (165±21 mmHg), improved creatinine plasma levels (0.8 ±0.2 mg/dl) and proteinuria (178±102 mg/24h). No alterations were observed after deafferentation over baroreflex sensitivity, cardiac hypertrophy and autonomic control to the heart. Together these results suggest that renal deafferentation improves blood pressure and renal function, without, however, improving the cardiac changes found in this CKD model. It is possible that the time expected after deafferentation or the stage of the disease in which it was performed was not sufficient to induce cardiac improvements. Further studies are necessary to elucidate this question.

Support or Funding Information

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001; by the São Paulo Research Foundation (FAPESP 18/02671‐3) and by the Brazilian National Research Council (CNPq)
Flow Cytometry Analysis of Anastasis using CellTrace Far Red (CTFR) cell tracker in Cultured Mammalian T Lymphocytes
Authors: Smith Perianne , Lauzon Robert
Published: April 2020

Anastasis is a cellular recovery process that rescues apoptotic cells from the brink of death. Our lab has developed a model for studying anastasis using cultured mouse HT2 lymphocytes dependent on the cytokine interleukin 2 (IL2). Within 24 hours of IL2 deprivation, HT2 cells display characteristic changes underlying apoptosis, including cellular condensation, membrane blebbing and fragmentation into apoptotic bodies, phosphatidylserine externalization, caspase 3 activation as well as G1 cell cycle arrest. HT2 cells can subsequently be rescued by adding IL2 back into the culture medium. In this study, we have used quantitative flow cytometry in conjunction with CellTrace Far Red (CTFR) and SYTOX Green (live/dead indicator) to track the kinetics of anastasis and re‐entry of viable apoptotic cells into the cell cycle. HT2 lymphocytes continuously maintained in IL2 comprised a homogeneous, viable population of CTFR+ cells which decreased in fluorescence intensity with each subsequent cell cycle for up to 96 hours post‐CTFR labelling. To investigate apoptosis reversal, HT2 cells were labeled with CTFR, deprived of IL2 for 24 hours and then rescued with IL2 re‐addition for periods of 24, 48, and 72 hours. Our findings indicate that viable, CTFR+ HT2 cells gradually resumed cell cycle entry over the 72‐hour rescue period. As caspase activation represents a cellular marker of early‐mid stage apoptotic cells, we also investigated caspase 3/7 activity in viable CTFR+ cells using the Nucview‐488 fluorescent caspase 3 substrate. Three distinct populations of caspase‐high, medium and low, viable CTFR+ HT2 cells were observed at 24 hours following IL2 deprivation. IL2‐rescued cells subsequently segregated into caspase‐high and low populations, ultimately merging into a single, caspase‐low population by 72 hours post‐rescue. Collectively, our findings suggest that CTFR tracking can be used successfully as a tool to follow the kinetics of apoptosis reversal in IL2‐dependent mouse HT2 lymphocytes, and is a promising model for the study of the mechanistic basis of anastasis.

Support or Funding Information

Union College Faculty and Student Research Grants
Cardiovascular Responses During Graded Lower‐Body Negative Pressure in Heart Failure with Preserved Ejection Fraction
Authors: Alpenglow Jeremy , Bunsawat Kanokwan , Craig Jesse , Ryan John , Richardson Russel , Wray Walter
Published: April 2020

Heart failure with preserved ejection fraction (HFpEF) represents approximately half of the known cases of heart failure in the United States, with equally poor outcomes and rising prevalence compared to heart failure with reduced ejection fraction (HFrEF). While changes in autonomic nervous system function is a well‐known feature of HFrEF pathophysiology, whether patients with HFpEF experience a similar decrement remains unknown. Therefore, we sought to evaluate the reflex cardiovascular responses to baroreflex unloading achieved via graded levels of lower‐body negative pressure (LBNP) in patients with HFpEF compared to healthy, age‐matched controls (CON). In six participants (three HFpEF (1 male, 70±3 yrs) and three CON (2 male, 74±2 yrs)), we assessed forearm blood flow (FBF; Doppler ultrasound), mean arterial blood pressure (MAP; Finapres), heart rate (HR; electrocardiogram) and forearm vascular conductance (FVC) at rest and throughout graded levels of LBNP (−10, −20, −30 and −40 mmHg). At rest, there were no significant differences between HFpEF and CON in FBF (109±32 vs. 85±29 ml/min), MAP (90±4 vs. 96±3 mmHg), HR (60±5 vs. 61±5 bpm), or FVC (1.19±0.33 vs. 0.89±0.28 ml/min/mmHg). The reflex tachycardic response to baroreflex unloading induced by LBNP was similar between patients with HFpEF and CON. Vasoconstriction in response to LBNP decreased FBF to a similar level in both HFpEF and CON (−10: 102±31 vs. 88±28 ml/min; −20: 95±30 vs. 76±25 ml/min; −30: 91±30 vs. 78±28 ml/min; −40: 91±33 vs. 71±21 ml/min, HFpEF vs. CON), with similar changes in FVC. Together, these preliminary data suggest that baroreflex‐mediated cardiac and vascular responses are not altered in patients with HFpEF, suggesting an absence of overt autonomic dysfunction is this patient group.

Support or Funding Information

This project is funded in part by the National Institutes of Health (HL118313, R56AG057584, T32HL139451) and the U.S. Department of Veterans Affairs (I01RX001311, I01RX001697, I01RX002323, I01RX000182).
Development of GPR183 Antagonists to Treat Neuropathic Pain
Authors: Latzo Nicholas George , Braden Kathryn , Giancotti Luigi , Chen Zhoumou , DeLeon Chelsea , Boehm Terri , D’Cunha Napoleon , Luongo Livio , Doyle Timothy , Kolar Grant , Walker John , Arnatt Christopher , Salvemini Daniela
Published: April 2020

Chronic neuropathic pain affects millions of people in the United States today. Opioids are currently the frontline agent for the treatment of neuropathic pain; however, as their use continues to grow, serious side effects can arise including addiction and death. The need for a safer alternative to opioids is becoming increasingly more urgent. GPR183 (Ebstein‐Barr induced gene 2, EBI2) is a G‐protein coupled receptor (GPCR) that binds to 7a,25‐dihydroxycholesterol (7a,25‐OHC). A homology model for GPR183 was constructed and a pharmacophore hypothesis was built around the binding of other known ligands to the receptor. Over 5 million compounds were screened in silico for their similarity to our pharmacophore hypothesis and the top 20k hits were funneled into a high‐throughput docking protocol. Several compounds showed strong inhibition of 7a,25‐OHC calcium mobilization with nanomolar potency. Intrathecal injections of these GPR183 antagonists reversed CCI‐induced mechano‐allodynia in mice when given at peak neuropathic pain. These results identify a prominent and functional role for GPR183 in the spinal cord in the maintenance of neuropathic pain. A preliminary structure‐activity relationship study of one of these compounds is being performed to study the pharmacology and medicinal properties of this new class of compounds.

Support or Funding Information

We would like to acknowledge Dr. Dale Dorsett and the Saint Louis University Genomics Core Facility for their assistance with the RNA‐Sequencing. This research was supported by funds from Dr. Salvemini and Dr. Arnatt and T32 GM008306‐01 training grant (KB). Dr. Salvemini and Dr. Arnatt have filed a patent describing the intellectual property generated at SLU. All other authors claim no conflict of interest.
Isolation of the exon‐switching variant, CYP4F2b, from human hepatic carcinoma (HCC) and its role in modulating epithelial‐mesenchymal transition (EMT) through Twist2.
Authors: Kang Puneet , Rote Paul , Saleem Abraham , Danso Prince , Shirota Leticia , Hardwick James
Published: April 2020

Cytochrome P450 IV (CYP4) are a family of omega‐hydroxylase enzymes. We have previously shown that these fatty acid omega‐hydroxylases function in the hydroxylation of various chain‐length saturated and unsaturated fatty acids in the endoplasmic reticulum. These dicarboxylate products then undergo peroxisomal β‐oxidation, yielding shorter chain fatty acids. In humans, the CYP4F family members hydroxylate long‐chain fatty acid (>C16) and the bioactive eicosanoids, arachidonic acid (HETE) and pro‐inflammatory leukotrienes B4 (LTB4). In the human liver, CYP4F2a metabolizes long‐chain fatty acids and arachidonic acid to the vasoactive 20‐HETE. We have identified a new variant, CYP4F2b from human hepatic carcinoma that is produced from the CYP4F2 gene by exon switching. We have compared the expression of CYP4 members in human HCC and matched healthy human liver. We observed increased expression of CYP4F2b in HCC compared to controls, as well as increased fatty acid omega‐hydroxylation, with associated elevations of acetyl‐CoA/acetate in HCC, and decreased activity and expression of the acetyl‐CoA thioesterase 12 (ACOT12). Increased acetyl‐CoA levels activate the basic helix‐loop‐helix (bHLH) transcription factor Twist2, which initiates an epithelial‐mesenchymal cell transition (EMT). We propose that increased omega‐hydroxylation of fatty acids by CYP4F2b and subsequent peroxisomal β‐oxidation increases cellular acetyl‐CoA levels in HCC, leading to activation of Twist2 and progression of EMT.

Support or Funding Information

NEOMED foundation
Development of Craniofacial Biomineralization in Mice with Osteogenesis Imperfecta (OI)
Authors: Wright Tommy R. , McBride Alexandra H. , Organ Jason M. , Menegaz Rachel A.
Published: April 2020

Osteogenesis imperfecta (OI), or “brittle bone disease,” is a rare autosomal dominant disorder characterized by mutations in the genes that encode the alpha 1 (COL1A1) and alpha 2 (COL1A2) chains of type I collagen, a significant component of bone. Misfolding of the mutated collagen polypeptides interfere with proper assembly of collagen chains, or the triple helical structures, leading to extreme skeletal fragility. OI manifests in a variety of types, with type I being the most mild and most common, and type III, the subject of this study, being more severe and more rare. Clinical manifestations of OI type III generally include a short stature, low bone mineral density (BMD), dental problems, hearing loss, and blue sclerae. In this study, we use a mouse model of OI type III to examine how mutations in type I collagen synthesis impact craniofacial biomineralization during postnatal growth.

The homozygous recessive osteogenesis imperfecta murine (OIM) is a model of OI type III and is characterized by increased skeletal fractures, low postcranial BMD, osteopenia, progressive skeletal deformities, and small body size. Wild type mice (WT) and OIM littermates (n≥3/genotype) were micro‐CT scanned at weaning (4 weeks) and adulthood (16 weeks). BMD at three regions of interest (ROI) within the mouse skull was measured using Bruker CTAnalyzer software. ROIs were chosen based on the amount of load during mastication, and included the temporomandibular joint (TMJ), the parietal bone (1mm from the sagittal suture), and the anterior maxilla at the incisal alveolus. Mann‐Whitney U‐tests (α=0.05) were used to compare BMD values between the genotypes for each ROI.

At weaning, OIM mice had significantly lower BMD values (g.cm−3 CaHA) than their wild type littermates at the TMJ (0.39 vs 0.58), parietal bone (0.35 vs 0.64), and anterior maxilla (0.53 vs 0.85). These statistical differences persist into adulthood. This decrease in BMD reflects decreased deposition of CaHA due to an abnormal collagen framework in the craniofacial skeleton of OIM mice. Ongoing efforts will expand this analysis to quantify the development of BMD at additional regions within the craniofacial skeleton.

Support or Funding Information

This research was supported by an Indiana University Collaborative Research Grant and the Ralph W. and Grace Showalter Trust.