bluuebunny/crossref_metadata_2025 huggingface dataset:
The Wright Variation of the Darrow‐Yannet Diagram: A New Tool to Teach Water Homeostasis
Authors: Wright Bruce E.
Published: May 2022

Purpose

To introduce an expanded version of the Darrow‐Yannet (D‐Y) diagram that can both improve basic understanding of water balance homeostasis and be more specifically applied to specific organ system topics (e.g., gastrointestinal secretion) than can the basic diagram.

Introduction and hypothesis

Since 1935, the Darrow‐Yannet diagram has been used to teach both water balance and homeostasis. Six models of ECF volume expansion or contraction of hypertonic, isotonic, or hypotonic fluid with subsequent water transfer to or from ICF show how ECF and ICF remain in balance. Yet when osmosis is used later in both gastrointestinal and renal physiology to describe movement of water from spaces that are technically outside the body into or out of part of the extracellular fluid compartment, students sometimes struggle with the concept of osmosis (water diffusion between compartments) not from ECF to ICF but from ECF to OB and back. They must also consider that through both structural characteristics of the epithelium and the autonomic and hormonal control of epithelium function, this water transfer can be considerably modified or even prevented. Because the classic D‐Y has no OB in its illustrations, it cannot be used to show either of these aspects of water balance. The question therefore arose, is there some way to expand the D‐Y diagram to show both, and emphasize other aspects of homeostasis too? The author hypothesized that it could be done.

Result

In the Wright variation of the D‐Y diagram, two changes are made to expand the D‐Y diagram and increase its usefulness. First, an OB compartment of open volume is introduced on the one side of the ECF while keeping ICF on the other side as usual. Second, the epithelial layer between OB and ECF is artificially widened so that it is both more easily visualized and potentially drawn with two cell populations (e.g., secretory vs. absorptive cells lining the duodenal lumen). Though volume is undefined in the OB compartment, osmolarity is defined, ranging from zero (pure water) to immeasurably high (dry), so osmotic OB‐to‐ECF gradients can be seen. Using control of the epithelium to determine whether fluid flows through or not regardless of gradient, nervous and hormonal control of water homeostasis can be shown. Finally, not just water movement but hormonal‐ or autonomic‐controlled ion movement across the epithelium can be graphically shown, showing off an important aspect of homeostasis. Loss of control of these ion movements (e.g., in GI secretory cells in cholera) can also be demonstrated, along with its pathophysiological effect.

Conclusion

An expanded D‐Y diagram with an artificially emphasized epithelial layer between an outside‐the‐body (OB) compartment and the extracellular fluid (ECF) has the potential to better illustrate the epithelium’s possible control in expanding or contracting ECF volume prior to ICF change than can a D‐Y diagram alone, both in health and disease.
Increase in venous [K+] During Hyperbaric Exposure Independent of Changes in pH or O2 Concentration
Authors: Lamers Jacob , MacLaughlin Kent , Marcou Matthew , Braun Rudolf , Eldridge Marlow
Published: May 2022

Introduction

Plasma potassium regulation is important for function of numerous cells in the body. Changes in potassium levels during exposure to an increased O2 concentration is thought to be the result of the changes in pH and increasing reactive oxygen species. However, the effects of hyperbaria on plasma potassium concentration are not well understood.

Methods

Eight subjects were exposed to 1.3 atmospheres absolute (ATA) of hyperbaric air for 90 minutes, 10‐times (M‐F) over 12‐days. Another eight subjects were exposed to 100% oxygen at 1 ATA over the same interval. Four venous blood draws were taken. On day 1 the first draw was taken immediately preceding treatment and the second was taken immediately following treatment. The third draw was taken prior to the 10th treatment and the 4th draw was taken 72 hours post final treatment. We analyzed samples on a blood gas analyzer and performed statistical analysis using a paired Wilcoxon signed‐rank test.

Results

The concentration group saw strong trend towards an increase in the potassium concentration from 4.09 ± 0.12 (mmol/L) to 4.28 ± 0.28 (mmol/L) (p = 0.065). In the hyperbaric group we see a significant increase in potassium concentration from 4.19 ± 0.26 (mmol/L) to 4.55 ± 0.27 (mmol/L) (p = 0.0068). In the concentration group we also see a significant increase in pH concentration from 7.37 ± 0.03 to 7.39 ± 0.01 (p= 0.021). A similar significant increase is not seen in the hyperbaric group.

These finding suggest that changes in potassium concentration in response to hyperbaria are not the result of oxygen concentration nor pH. Possible explanations include increased nitrogen levels due to hyperbaric air, increased CO2 concentration in hyperbaric chamber or changes in the activity of Na+,K+ ATPase pumps at the cellular level which may be a homeostatic response to combat pulmonary edema
Characterizing Hepatic Inflammation upon SGLT2 Inhibition in Diabetic TallyHo Mice
Authors: Choos Elijah N. D. , Rodriguez Olga , Albanese Chris , Ecelbarger Carolyn M. , Shepard Blythe D.
Published: May 2022

By 2030, Diabetes is estimated to become the 7th leading cause of death worldwide with roughly 90‐95% of all cases attributed to Type 2 diabetes (T2D). A common comorbidity of T2D is Non‐Alcoholic Fatty Liver Disease (NAFLD), characterized by the presence of lipid accumulation with or without hepatic steatosis (NASH). While the pathogenesis of NAFLD and NASH is not entirely understood, it is associated with insulin resistance, inflammation, and hyperlipidemia. Recently, sodium‐glucose co‐transporter 2 (SGLT2) inhibitors have emerged as a treatment option for patients with T2D. This drug effectively blocks renal glucose reabsorption and in turn, improves glycemic control. It is also associated with positive renal and cardiovascular outcomes. Given the attenuation of hyperglycemia, we sought to determine if SGLT2 inhibitors also offer protection from the development of NAFLD and NASH. To test this, spontaneously diabetic male and female TallyHo mice were maintained on either a control or high milk fat diet (60%) for 24‐weeks in the continued presence or absence of Empagliflozin, one of the most well‐characterized SGLT2 inhibitors on the market. As expected, at the conclusion of 24 weeks, blood glucose values were significantly lowered in the male mice treated with Empagliflozin. We also detected a reduction in hepatic lipid accumulation by both MRI and biochemical analysis. To determine if these protective effects correlated with a reduction in inflammation, we performed a large‐scale screen of more than 60 inflammatory markers on both plasma and livers collected from the high‐fat diet‐fed male mice. The data revealed that Empagliflozin attenuates systemic inflammation; we detected a downregulation in circulating pro‐inflammatory cytokines (CD30 ligand, IL‐1α, IL‐4, IL‐9, TIMP1, TPO) and an upregulation in MIP‐1γ, an anti‐inflammatory cytokine (p<0.05 for all cytokines). In contrast, we observed indications of increased inflammation in the Empagliflozin‐treated livers. Protein expression of cleaved caspase, the active form of the inflammatory‐driven protein, was elevated when compared to non‐treated milk fat diet‐fed mice (which was already elevated compared to normal chow controls). In addition, we noted heightened levels of five pro‐inflammatory cytokines (IL‐3, IL‐6, CCL2, M‐CSF, CCL1) while there was downregulation of another three (p40/p70, TNFSF8, CCL3; p<0.05). Efforts are currently underway to confirm the expression of these common inflammatory markers and to elucidate the mechanism behind the dysregulated hepatic inflammatory pathway upon SGLT2 inhibition.
Exercise‐Induced Shear Stress Upregulates eNOS Protein in vitro
Authors: Garcia Mario , Cabral Shelsea , Rodriguez Carmen , Conde Daniel , Gurovich Alvaro N.
Published: May 2022

Background

Endothelial cell dysfunction has been shown to be the first step in the pathogenesis of cardiovascular disease. A risk factor in the development of cardiovascular disease is physical inactivity. Endothelial shear stress (ESS) involves mechanoreceptors on the surface of endothelial cells that are capable of sensing different forces, triggering a biochemical response leading to cellular adaptations. Exercise‐induced ESS might act as a protective mechanism in preventing endothelial cell dysfunction. Endothelial nitric oxide synthase (eNOS), a key enzyme involved maintaining vascular homeostasis has been shown to be upregulated in response to higher shear stress levels. Although exercise has been shown to improve endothelial cell function, the role of varying exercise intensities on endothelial cell homeostasis has not been explored. The purpose of this study was to determine the effects of in vitro exercise‐induced ESS during 3 different exercise intensities on eNOS expression.

Methods

Commercially available human umbilical vein endothelial cells (HUVEC; Sigma‐Aldrich, St Louis, MO) were cultured until 90‐100% confluence. Exercise‐induced ESS in vivo was obtained from our previous reports. Cultured cells were exposed to pulsatile resting ESS (18 dynes/cm2) for 2 hours, followed by 1 hour at resting ESS, low‐intensity exercise‐induced ESS (35 dynes/cm2), moderate‐intensity exercise‐induced ESS (50 dynes/cm2), or high‐intensity exercise‐induced ESS (75 dynes/cm2) on a closed circuit pump and channeled slide (Ibidi pump system (PumpControl Software 1.5.4) and slides, Ibidi Inc., Fitchburg, WI). A set of cells were exposed to physiologically low shear stress levels (10 dynes/cm2) for 3 hours as a control. Following ESS conditions, total protein was extracted, and western immunoblotting was performed to measure eNOS protein expression with GAPDH used as the normalizing protein. Statistical analysis was performed using graphpad prism 8 software (Graphpad Software.). Significance was considered at p<0.05.

Results

Low and moderate intensity ESS increased eNOS protein expression compared to physiologically low conditions (P<0.05). There was a trend showing high intensity ESS increasing eNOS expression compared to physiologically low conditions (p<0.10). Additionally, moderate intensity ESS showed a significant increase in eNOS compared to resting conditions (P<0.05).

Conclusions

Our data shows that exercise‐induced ESS upregulates eNOS expression. Moderate intensity exercise was shown to produce the highest levels of eNOS expression. Our results from previous studies also show that moderate intensity exercise is capable of activating eNOS, which may act as a protective mechanism to prevent atherosclerosis development.
Regulation of PITX2 and ABCB1 in oncogenic multidrug resistance by tumorigenic HIF2 in VHL‐deficient renal cancer cells
Authors: Lee Wing‐Kee , Ow Calista J. , Schreiber Timm , Thevenod Frank
Published: May 2022

The tumour suppressor von Hippel‐Lindau (VHL) is involved in ubiquitination and subsequent proteasomal degradation of hypoxia‐inducible factors (HIFs). Under hypoxic conditions, HIFα‐subunits are stabilized, leading to dimerization with HIFβ and transactivation of genes for metabolism, survival and angiogenesis. Human renal cancer cells A498 are VHL‐deficient and harbor stabilized HIF‐2, whereas Caki‐1 cells are VHL‐intact and HIF‐1‐dominant. Previous studies demonstrated increased paired‐like homeodomain transcription factor 2 (PITX2) and its target gene, the ABCB1 drug transporter, in A498 cells. The relationship between VHL, HIF, PITX2 and ABCB1 remains unclear.

Transient transfection of A498 cells with HIF‐1α or HIF‐2α siRNA (25 nM, 72 h, >85% protein knockdown) attenuated PITX2 protein by up to 75% by immunoblotting and PITX2 promoter activity by ~40% in a luciferase reporter gene assay. Immunoprecipitation studies did not propose direct HIF‐PITX2 protein‐protein interaction. HIF‐regulated PITX2 expression was proportional to ABCB1 expression, which was decreased at both mRNA (~20%) and protein (~70%) levels after HIF‐1α or HIF‐2α knockdown. Furthermore, HIF/PITX2 target gene CCND1 was reduced by >60%, but PITX2 targets LCN2 and WNT4 were increased >3‐fold following transient HIF downregulation. HIF‐2α siRNA was generally more effective than HIF‐1α siRNA in altering PITX2 and its target genes. Conversely, in Caki‐1 cells, HIF siRNA augmented PITX2 and ABCB1 mRNA and protein by ~1.5‐fold and ~2‐fold, respectively, with no significant changes in CCND1, LCN2 and WNT4. Polysome profiling show HIF‐2α siRNA negatively impacts active translation of PITX2 and ABCB1 mRNA. Finally, HIF2 knockdown sensitizes A498 cells to cell death by the chemotherapeutic drug and ABCB1 substrate, doxorubicin (1 µM, 24 h), as determined by trypan blue staining.

In summary, these findings suggest PITX2 and ABCB1 are positively regulated by tumorigenic HIF2 in drug‐resistant A498 cells whereas they are suppressed by HIF‐1 in drug‐sensitive Caki‐1 cells. These data may provide better understanding of membrane transporters and possible upstream causes of multidrug resistance in different renal cancers.
Activation of dorsal motor nucleus cholinergic neurons ameliorates the severity of acute pancreatitis
Authors: Thompson Dane A. , Tsaava Tea , Tracey Kevin , Sangeeta Chavan
Published: May 2022

Clusters of neuron cell bodies, termed nuclei, residing in the brainstem are the origin of cranial nerves which transmit action potentials controlling organ function. Neurons residing in the brainstem dorsal motor nucleus (DMN) project in the vagus nerve to communicate with the pancreas, liver, gastrointestinal tract and other organs. Cholinergic signaling in the vagus nerve‐mediated inflammatory reflex also controls immune system responses by inhibiting the production of cytokines in the spleen, although the function of DMN neurons in regulating pancreatic inflammation is not known. Here, we demonstrate that selective activation of cholinergic neurons in the DMN attenuates pancreatitis severity. Pancreatitis was induced with two intraperitoneal injections of the cholecystokinin analogue caerulein (50 mcg/kg), given one hour apart. A fiber‐optic cannula was inserted under stereotactic guidance into the DMN in transgenic mice expressing light‐responsive ion channel, channelrhodopsin, in cholinergic neurons under the choline acetyltransferase promoter. Animals were subjected to either optogenetic stimulation (473nm, 20 Hz, 25% duty cycle, 8‐12 mW total power) or sham stimulation (no light) for five minutes. Optogenetic stimulation but not sham stimulation of the cholinergic neurons in the DMN significantly attenuates serum amylase (4043mU/mL ± 264.4 vs. 2940mU/mL ± 170.9, p=0.0074; n=9‐11 mice/group) and pancreatic IL‐6 levels (1907pg/mg ± 879.3 vs. 1046pg/mg± 288.1, p=0.0330; n=9‐11 mice/group). The decreased severity of pancreatitis in optogenetically stimulated mice is further demonstrated by a significant improvement in pancreatic histologic severity score (6.86 ± 1.67 vs. 5.35 ± 1.44, p = 0.0462; n = 9‐11). Pharmacological blockade and surgical ablation of the vagus nerve signaling inhibit protective effects of DMN cholinergic neurons activation. Pre‐treatment with mecamylamine, a nicotinic receptor antagonist, attenuates the reduction in serum amylase following light stimulation (Sham stimulation vs. DMN vs DMN + Mecamylamine: 5047 ± 315.4 vs. 3769 ± 241.5 vs. 5620 ± 385.9, p = 0037; n = 10 mice/group). Additionally, mice with subdiaphragmatic vagotomy has no difference in serum amylase levels following optogenetic stimulation (2761mU/mL ± 787.3 vs. 2517mU/mL, p= 0.300; n=3), whereas sham surgery controls show a significant reduction in serum amylase levels (3224mU/mL ± 593.1 vs. 2087 mU/mL ± 951.2, p=0.0303; n=6 mice/group). These studies identify DMN as an important locus that regulates the severity of acute pancreatitis in vagus‐nerve‐dependent fashion, and provide new insights into the identity and central origin of the efferent vagus nerve fibers regulating acute pancreatitis.
The Role of Vts1/Smaug in Adaptive Heritable Gene Expression
Authors: Link Tina , Kona Abhishek , Kumar Navya , Malladi Pranav , Murugesan Vikram , Venkatesan Priya
Published: May 2022

The ancient RNA Binding Protein (RBP) Vts1 has been identified to self‐assemble along with the Smaug regulator to produce enhancing non‐amyloid prions in mRNA decay. Seen in Drosophila, the sterile‐alpha‐motif (SAM) domain of Vts and the Smaug regulator acts as an embryonic regulator for maternal transcript degradation through cytoplasmic deadenylase in progeny development. Specifically, of Saccharomyces cerevisiae, the Vts/Smaug regulator forms condensates that self‐template to create prions functioning towards epigenetic heritable inheritance. The RBP Vts1 SAM domain recognizes in RNA hairpins Smaug recognition elements, SREs, initiating the process of degradation and self‐assembly. The structure of RBPs are composed of an ordered RNA binding domain, and an unventured intrinsically disordered region (IDR). At the quaternary level structure, the Vts1 protein matches as a hexamer of 489 kDa dominant peak with an oligomer of more than eight monomers, and the IDR is around 490 kDa as well while the RBD‐Vts1 is around 75 kDa. The condensed version of Vts1 fuse into structures of 10 micrometers due to the IDR driving self‐association to create oligomers. The RNA binding SAM domain and IDR have induced transcription degradation and post‐transcriptional regulation in a new mechanism, prompting adaptive gene expression for developmental decisions in organisms over evolutionary time periods. Though many RBP prion condensates are not heritable and inactivate protein function, the SMAUG+ prions of the condensed Vts1/Smaug protein in S. Cerevisiae instead respond to environmental stress and produce new emerging phenotypes that are actually heritable over a large biological time‐scale. Furthermore, SMAUG+ conversion is reversible and an enhancer of RBP Vts1 function, producing adaptive gene expression systems in fluctuating environments. In various yeast models, the SMAUG+ allows yeast to decide nutrient replenishment after starvation which creates a new adaptive advantage for organisms. The new cellular decision making regulated by the prion is in two forms of rapid mitosis or meiosis creating a non‐stress state; SMAUG+ has created an adaptive memory that can influence developmental and cellular decisions in addition to plant growth in limited nutrients. Such advancements in heritable prion inheritance can uncover the function of intrinsically disordered sequences. The Walton SMART team has designed a 3D model with MSOE Center for Biomolecular Modeling to investigate the SAM domain of Vts1 further and examine the role of SMAUG+ prions in adaptive heritable gene expression.
SPARC: Development of a TRPA1 Reporter Mouse Model
Authors: Bahia Parmvir K. , Patil Mayur , Kim Seol‐Hee , Harsányiová Jana , Kollarik Marian , Taylor‐Clark Thomas
Published: May 2022

Transient receptor potential ankyrin 1 (TRPA1) is a cation channel activated by electrophilic irritants and oxidative stress. It is expressed in nociceptive sensory neurons and plays a role in pain and reflex signaling. Its expression in other cell types is controversial.

We generated a TRPA1‐Flp knock‐in mouse by incorporating a 2A‐FlpO after the last endogenous TRPA1 exon. This TRPA1‐Flp mouse was crossed with the Ai65f reporter strain which expresses the fluorescent protein tdTomato in a flp recombinase‐dependent manner. From these animals, we collected tissue to visualize tdTomato, as an indicator of TRPA1 expression.

37% of vagal neurons expressed tdTomato. Of these 74% expressed TRPV1 (capsaicin receptor), and 88% expressed the neuropeptide CGRP. In fura 2AM studies of dissociated neurons, the TRPA1 agonist AITC activated >90% of tdTomato+ neurons and only 24% of tdTomato‐ neurons. tdTomato expression was limited in the dorsal root ganglion, with only 10% of neurons expressing tdTomato. Of these 61% expressed TRPV1, and 81% expressed CGRP. AITC activated 80% of tdTomato+ neurons and 26% of tdTomato‐ neurons.

We found tdTomato in fibers projecting to the nucleus of the solitary tract and to superficial lamina of the spinal cord dorsal horn. Very little tdTomato expression was found elsewhere in the CNS. In the periphery, we found tdTomato+ fibers innervating the lungs and GI tract. We also found tdTomato expression in some stomach and colonic cells, although none were enterochromaffin cells.

In summary, we have created a knock‐in FlpO mouse model that can be used to identify TRPA1 expression.
A Mammoth Task: Identifying Mammoth Ivory Using Raman Spectroscopy
Authors: Shepherd Rebecca F. , Lister Adrian M. , Roberts Alice , Taylor Adam , Kerns Jemma G.
Published: May 2022

A mammoth tusk contains an inner mineralized protein matrix of dentine and an outer layer of cementum. Enamel is only present on the tips of the tusks of young mammoths, and is worn away in older mammoths. Dentine is a mineralized connective tissue containing the inorganic component of dahlite [Ca10(PO4)6(CO3)H2O]. To determine the species from which ivory originated, often destructive methods are used. Raman spectroscopy is a non‐invasive laser‐based technique that has proven applications in the chemistry of mineralized tissue. Ivory and bone have similar biochemical properties. The aim of this study is to test the hypothesis that mammoth ivory is identifiable using Raman spectroscopy.

Mammoth tusks were kindly loaned from the Natural History Museum, London, UK. All tusks were from the species Mammuthus primigenius discovered either in Lyakhov Islands or next to the Yenisei river, Krasnoyarsk (Siberia, Russia) and span the Pleistocene epoch, Cenerzoic era. The ivory was scanned with an inVia Raman micro spectrometer (Renishaw Ltd) equipped with a x50 objective lens and a 785nm laser. Spectra were acquired using line maps on cross sections of two samples, and individual spectral points were acquired independently at random or at points of interest on all samples. Data was analysed with principal component analysis (PCA) using an in‐house Matlab script.

To date, the results of this study establishes that well preserved mammoth ivory can be characterized through the comparison of peak intensity ratios between organic v(CH) collagen peaks and inorganic v(PO) hydroxyapatite peaks. Differences were observed in the hydroxyapatite peak from spectra acquired near the medulla of the tusk compared to the cortex. This suggests that the tusk is more mineralized towards the cortex compared to dentine found closer to the medulla. A comparison of the average data from each mammoth tusk demonstrated that the mammoth spectral ‘fingerprint’ remains similar for all samples, though there was some inter‐variation in the mineralization of the tusks from mammoths of the same species.

Further work in this study aims to compare the Raman spectra between mammoth and elephant ivory. This will have direct applications in archaeology, as the species from which an ivory sample is found could be identified without the need for more traditional, destructive techniques of valuable artefacts. Additionally, international trade regulations require proof of the species from which ivory is obtained. The information obtained in this study will be valuable in developing quick and non‐destructive methods for the identification of ivory from an unknown origin.
Application of a Novel Pancreas Perfusion Technique to Characterize Exocrine Pancreas Metabolism
Authors: Rushin Anna , McLeod Marc , Ragavan Mukundan , Merritt Matthew
Published: May 2022

The pancreas functions as both an endocrine and exocrine gland. The endocrine component consists of the islets of Langerhans, which contain several pancreatic cell types that produce hormones. In the islets, α‐cells produce glucagon and β‐cells produce insulin, both of which serve to regulate glucose metabolism. The exocrine tissue produces digestive enzymes such as chymotrypsin and is connected through the ductal system. Type I Diabetes (T1D) is an autoimmune disease in which immune cells attack the pancreatic β‐cells, resulting in the pancreas producing little to no insulin. Although the vast majority of research has focused on the islets, the exocrine tissue may be implicated in T1D as well. This research introduces a novel method to investigate exocrine pancreas metabolism specifically using a pancreas perfusion system through the common bile duct. I hypothesized that the exocrine pancreas would demonstrate metabolic activity through uptake and utilization of [2H7]glucose during a perfusion.

Using C57BL/6 male mice, the surgical procedure consisted of clamping the ampulla of Vater with a John Hopkins bulldog clamp and cannulation of the common bile duct. The pancreas was then perfused for 20 minutes with perfusate containing Krebs electrolytes, free fatty acids, and 16.7mM of either unlabeled or [2H7]glucose. The tissue was flash frozen in liquid nitrogen and subjected to an acetonitrile isopropanol water extraction. The extracts were derivatized using methoxyamine and MTBSTFA before gas chromatography‐mass spectrometry (GC‐MS) analysis.

GC‐MS measurements confirmed the pancreas exocrine tissue metabolized [2H7]glucose, as shown by an increased enrichment in lactate mass isotopologues in the tissue perfused with isotopically labeled glucose compared to the unlabeled control. Additional central carbon metabolites also demonstrated increased enrichment, namely malate and fumarate. The observed isotopologue labeling patterns are indicative of anaplerosis. This research demonstrates metabolism specific to the exocrine pancreas can be measured using a pancreas perfusion system and isotopic labeling. Future work will examine real‐time central carbon metabolic flux using dissolution dynamic nuclear polarization (dDNP) combined with the pancreas perfusion system. This research will use the T1D mouse model NOD. Rag1‐/‐.AI4α/β and compare the results to the NOD.Rag1‐/‐ control mouse model to examine how exocrine pancreas central carbon metabolism is altered in T1D models in a more physiologically relevant manner than previously possible.
Dopamine D2‐Like Receptor Signaling and Downregulation of Filamin‐A May Drive the Association Between Neighborhood Socioeconomic Status and CCR2 Expression on Monocytes
Authors: Pita Mario A. , Baumer Yvonne , Turner Briana S. , Gutierrez‐Huerta Cristhian A. , Neally Sam J. , Baez Andrew S. , Farmer Nicole , Mitchell Valerie M. , Collins Billy S. , Powell‐Wiley Tiffany M.
Published: May 2022

Social determinants of health (SDoH) include socioeconomic, environmental, and psychological factors that impact health. Low neighborhood socioeconomic status (nSES) is a SDoH that associates with cardiovascular mortality in longitudinal studies. While nSES as a chronic stress contributes to adverse health outcomes, the molecular mechanisms and pathogenesis are poorly understood. Previously, we found that residing in lower nSES areas may alter monocyte expression of C‐C chemokine receptor type 2 (CCR2), a regulator of monocyte recruitment during atherogenesis. To explore the effects of nSES as a chronic stress on monocyte CCR2 expression, we treated monocytes in vitro for 4 hours with catecholamines (epinephrine [Epi], norepinephrine [NE], or dopamine [DA]) used as stress biomarkers. Only DA increased CCR2 expression in a dose‐dependent manner (p<0.01), especially on non‐classical monocytes (NCM). In our current study, we investigated DA receptor signaling to learn which pathways may contribute to increases in CCR2 expression. Dopamine acts on five known receptors, the D1‐like receptors (D1 and D5) and D2‐like receptors (D2, D3, and D4). First, we performed flow cytometry on untreated monocytes and found D2 receptors were most abundant on the surface of all monocyte subsets (ie., classical monocytes [CM], intermediate monocytes [IM], and NCM as determined by CD14/CD16 expression). Furthermore, spearman correlation between D2‐like receptor surface expression and surface CCR2 expression in NCM suggested D2‐like receptor signaling (R2=0.79, p=0.04). Because the D2‐like receptor response is known to decrease cAMP levels compared to a D1‐like receptor response, a cAMP assay was performed on monocytes with DA treatment. Indicative of D2‐signaling, cAMP levels were found to be lower in DA‐treated monocytes compared to untreated controls (ctr 29.78pmol/ml vs DA 22.97 pmol/ml; p=0.038). We then investigated downstream mechanisms following DA signaling that could result in increased CCR2 expression. Filamin A (FlnA), a prominent actin‐cross‐linking protein, is known to regulate CCR2 recycling. We examined the expression of FlnA by flow cytometry and RT‐qPCR on flow sorted monocytes with and without DA treatment. There was a significant decrease in FlnA expression in NCM (p < 0.05), indicating a slowing of CCR2 recycling. Expression of an associated scaffold protein mRNA, b‐arrestin 1, was decreased by 66% in NCM (p= 0.008). Overall, we provide a novel immunological mechanism, driven by DA signaling and CCR2, for how nSES may contribute to atherogenesis. Future studies should investigate the importance of DA in CVD development and progression in populations disproportionately experiencing chronic stress due to SDoH.
Expression of ALMS1 in podocytes: possible role in filtration function
Authors: Maskey Dipak , Jayakumar Ankita , Monu Sumit R. , Ortiz Pablo A.
Published: May 2022

Previously, we identified Alstrom Syndrome 1 (ALMS1) as an interacting partner of NKCC2 in the Thick Ascending limb of the loop of Henle (TAL). Mutations in the ALMS1 gene in humans cause Alström syndrome, characterized by progressive metabolic alterations that include obesity, hypertension, and chronic kidney disease (CDK). SNPs in ALMS1 are associated with lower GFR in GWAS. However, role of ALMS1 in glomerular filtration has not been studied. We hypothesized that ALMS1 is expressed in glomerular podocytes where it may play a role in podocyte filtration function. To begin studying this, we localized ALMS1 expression to podocytes by immunofluorescence labeling of Actinin‐4, abundantly expressed in podocytes, and ALMS1. Confocal imaging showed that ALMS1 is co‐localized with Actinin‐4 in rat podocytes. In addition, ALMS1 was also expressed in an immortalized mouse podocyte cell line, as was Actinin‐4 and nephrin, as measured by Western blot and immunofluorescence microscopy. To determine if ALMS1 plays a role in podocyte pathology we measured nephrin expression in ALMS1 KO rats. These rats were generated in the Dahl SS genetic background and are hypertensive on a normal salt diet. We found that nephrin expression in renal cortical lysates was decreased by 61±08% (p<0.05, n=7) in ALMS1 KO rats. To study if ALMS1 deletion could play a role in filtration barrier function we measured albumin/creatinine ratio in urine samples from 14‐16 weeks old wild type (WT) and ALMS1 KO rats. In ALMS1 KO rats, urinary albumin excretion was higher than control rats (WT: 4±2; ALMS1 KO: 26±6, n=6, p<0.05). Taken together, our data show that ALMS1 is expressed in rat and mouse podocytes where it may play a role in filtration barrier function by maintaining podocyte biology.
Development and Characterization of Novel LRH‐1 Antagonist Using Structural and Computational Techniques
Authors: Cato Michael L. , Spurlin Racheal , Cornelison Jeffery , Jui Nathan , Ortlund Eric
Published: May 2022

Nuclear receptors are transcription factors that respond to external lipophilic signaling molecules by recruiting coactivators that enhance target gene expression. Liver receptor homolog‐1 (LRH‐1) is a nuclear receptor that drives ERα signaling and expression of cell cycle regulatory genes. LRH‐1 antagonists are therefore attractive potential therapeutics for the treatment of ERα positive and negative breast cancer. However, mechanistic studies exploring LRH‐1 antagonism are incredibly limited due to the lack of structural insight on the few existing compounds that decrease receptor activity. Using structure‐guided compound design, we have made modifications to the chemical scaffold of a highly potent LRH‐1 agonist to target and disrupt the region of the receptor responsible for coactivator binding. Our strategy effectively decreases LRH‐1 thermal stability, coactivator association, and transcriptional activity. Interestingly, molecular dynamics simulations reveal that antagonism is achieved through disruption of allosteric paths of communication unique to LRH‐1. Our work therefore characterizes a highly efficacious small molecule LRH‐1 antagonist and provides mechanistic insight into how signaling for this receptor can be effectively disrupted without large structural rearrangements.
Kidney and Gut Microbiome Crosstalk in Response to High Dietary Potassium
Authors: Cheema Muhammad U. , Subramanya Arohan R.
Published: May 2022

In this study, we tested the hypothesis that the gut microbiome influences the homeostatic response to a dietary K+ load. Male Specific Pathogen Free (SPF) and Germ‐Free (GF) mice (n = 5 per group) received 5% potassium citrate or K+alimentary matched control diets for 7 days. At the time of sacrifice, blood and urine parameters were compared, and the expression of relevant transporters and signaling molecules was assessed in the kidney by western blotting. No significant differences in whole blood K+ were observed between SPF and GF mice on control or high K+ diets. However, differences in other parameters were observed. Compared to control diet treated SPF mice, K+ loaded SPF mice exhibited a significant decrease in hematocrit (37 vs 39; p ≤ 0.0152) and hemoglobin (12.58 vs 13.26; p ≤ 0.0150), whereas GF mice exhibited no differences in these parameters regardless of dietary maneuver. Blood urea nitrogen (BUN) levels were significantly decreased in SPF mice (23.4mg/dL vs 27; p ≤ 0.0180) on High K+compared to SPF controls, while this was not the case for GF mice. These findings suggest that K+ loaded GF mice were less volume expanded than K+ loaded SPF mice. Consistent with this, plasma aldosterone measurements were higher in K+ loaded GF mice compared to K+ loaded SPF mice (966pg/mL vs 614; p ≤ 0.0155). In whole kidney homogenates, dietary K+ loading markedly increased the cleaved (active) form of the epithelial sodium channel gamma subunit (γENaC) relative to control diet treated mice in both the SPF and GF groups. However, cleaved γENaC was one‐fold higher in the K+ loaded GF mice compared to K+ loaded SPF mice. K+ citrate loading significantly upregulated pendrin in both GF and SPF groups (p ≤ 5.7146E‐05 and p ≤ 5.6654E‐06), likely an effect of the alkaline content of the diet. Interestingly, we also observed a significant 91% increase in Aquaporin 2 (AQP2) levels in the GF high K+ group (p ≤ 0.0168) relative to GF mice treated with control diet, an effect that was not observed in SPF mice (p= 0.165). These findings indicate that male germ‐free mice maintain plasma [K+] in response to a dietary alkaline potassium load, but develop reduced volume status compared to K+ loaded SPF controls. To compensate, germ free mice exhibit plasma elevated aldosterone concentrations, and upregulate cleaved γENaC and AQP2 abundance in the kidney. This suggests that an intact gut microbiome helps to defend against excessive volume depletion during the homeostatic response to alkaline K+ loading.
Response of Osteosarcoma Cell Lines to a Novel CHD1L Inhibitor
Authors: Gustafson Daniel L. , Viola Lindsey , LaBarbera Daniel V.
Published: May 2022

Osteosarcoma (OSA) is a primary tumor of bone that occurs in both humans (hOSA) and dogs (cOSA) and has been recognized as being very similar across these species. In fact, OSA is probably the best example where surgical, radiological and drug trials have been translated from testing in pet dogs with OSA to trials in human patients. In both humans and dogs treated for OSA, the primary treatment modalities are surgery and cytotoxic chemotherapy with doxorubicin and platinum‐based drugs standard for both and methotrexate also commonly added in humans. The treatment of OSA in both dogs and humans has been virtually unchanged in the last 40 years leading to stagnation in outcome improvement with lung metastasis and drug resistance being the primary mechanism of disease progression and patient morbidity and mortality. Thus, drug treatments that can effectively treat OSA and inhibit disease progression at metastatic sites are badly needed. Chromodomain Helicase DNA‐binding protein 1‐Like (CHD1L), a.k.a. Amplified in Liver Cancer (ALC1), is an oncogene that was originally identified in hepatocellular carcinoma. It has recently gained more attention with regards to resistance to PARP inhibitors in BRCA‐mutant cancers as well as a potential target for anticancer drug therapy. Studies have also suggested that CHD1L plays a role in resistance to platinum‐based chemotherapy agents through the modulation of DNA repair processes. This has led to a great deal of interest into drugs that can inhibit CHD1L. The first and only known small molecule CHD1L inhibitors (CHD1Li) have recently been reported by Dr. LaBarbera’s lab in the CU School of Pharmacy (Mol Cancer Ther 19:1598, 2020) who has an active medicinal chemistry program developing these molecules. Preliminary analysis of CHD1L gene expression in cOSA cell lines and tumors shows that expression levels are significantly higher in cOSA cell lines as opposed to other canine cell lines and that CHD1L expression is higher in cOSA tumors as opposed to normal bone. Treatment of cOSA and hOSA cell lines with a CHD1Li show a steep dose‐response of inhibition of cell proliferation and induction of cell death with IC50 values ranging between 2 and 3 μM and LD50values between 3 and 4 μM. These μM drug concentrations are safely achievable in mice dosed daily with this drug based on in vivo studies. The results suggest that CHD1L inhibition should be further pursued for therapeutic development in OSA and that cOSA and hOSA cell lines have a similar response to these agents.
Immune‐modulating Effects of Low Carbohydrate Ketogenic Foods in Healthy Canines
Authors: Tavener Selena K. , Jackson Matthew I. , Panickar Kiran S.
Published: May 2022

Ketogenic foods (KF) limit digestible carbohydrate but contain high fat, and have antioxidant and anti‐inflammatory effects as well as improving mitochondrial function. Beta hydroxybutyrate (BHB), one of the ketone bodies, has been reported to reduce pro‐inflammatory NLRP3 inflammasome, as well chemokines including CXCL2 and CCL5 in cultures. We assessed the immune‐modulating effects of two low carbohydrate (LoCHO) foods varying in protein and fat, and compared their effects to a food replete with carbohydrate (CON) in healthy canines. This study was approved by IACUC and the Animal Welfare Committee, Hills PNC. Macronutrients (P/F/C; % energy) for the foods were: CON (23/34/35), LoCHO1 (53/39/8) and LoCHO2 (27/67/5). We previously reported that LoCHO2 significantly increased circulating BHB compared to both CON and LoCHO1 although LoCHO1 did not increase BHB above that found when dogs consumed CON food. In the present study, dogs were fed control food (CON; ketogenic ratio (KR 0.46) followed by LoCHO1 (KR: 0.97), then LoCHO2 (KR: 1.63) or LoCHO2 followed by LoCHO1. Each food was fed for 5 weeks, with collections in the 5th week; 15 weeks feeding total. Gene expression for inflammatory cytokines and receptors from whole blood collected in PAXgene RNA tubes from 10 dogs was assessed using Canine RT2 Profiler PCR array. When compared to CON there was a significant decrease in several pro‐inflammatory cytokines/chemokines in both LoCHO1 and LoCHO2 groups including CCL1, CCL8, CCL13, CCL17,CCL24, CX3CL1, CXCR1, IL‐10RA, IL‐17A, IL‐1RN, IL‐5, IL‐9, and SPP1 (all p<0.05). Interestingly, a subset of inflammatory proteins that decreased in LoCHO1, but not in LoCHO2, included IL‐33, IL‐6R, Il‐7, IL‐8, NAMPT, and TNFRSF11B. In contrast, the decrease in inflammatory markers in LoCHO2, but not in LoCHO1, included C5, CSF3, IFN‐γ, IL‐3, IL‐10RB, IL‐17C, TNFSF13, TNFSF13B, and TNFSF14. Decreased levels of selected cytokines indicate the ability of both low‐carbohydrate foods to exert an anti‐inflammatory effect and provide a strong rationale for testing its efficacy in dogs with inflammatory conditions. For instance, cytokines including IL‐7 and IL‐8 are increased in chronic inflammatory conditions such as colitis, dermatitis, as well as in autoimmune conditions. Increased IL‐33 is also associated with chronic lesional skin of atopic dermatitis in canines and these conditions might benefit from LoCHO1 food. LoCHO2 food decreased complement component 5 (C5) and blockade of C5 attenuates renal dysfunction including glomerulopathy. LoCHO2 also decreased TNFSF13, a member of the tumor necrosis factor superfamily, highly expressed in tumors, and associated with poor prognosis, indicating its potential as a nutritional intervention strategy for cancer. Taken together, our results indicate that both LoCHO1 and LoCHO2 foods might be important as part of immune‐modulating therapeutic nutritional strategies to reduce inflammation or inflammatory pathways to maintain health in canines.
Applying high‐throughput analysis to understand cell proliferation under different adhesion conditions
Authors: Nuñez Clarissa , Ashraf Naghmana , Shuster Charles B.
Published: May 2022

Mammalian cell culture is a staple of biomedical research, and yet it has long been recognized that the conditions in which cells are cultured do not reflect the cellular environment in vivo. Adherent cells are cultured on a plastic surface with an elastic modulus over 10,000‐fold stiffer than what is experienced by neuronal, epithelial and muscle cells in living tissues. These altered conditions have a variety of effects on cellular behaviors, especially in regard to cell shape, motility and division. Previous work in the lab suggest that cell adhesion and motility play a role in the mechanics of cytokinesis in adherent cells. To test this hypothesis, we are conducting a study of the cell cycle and cell division dynamics of human Retinal Pigmented Epithelial (RPE1) cells cultured on substrates of differing mechanical properties. We have genetically engineered lines of RPE1 cells that express either a nuclear Red Fluorescent Protein (RFP) marker or a two‐color fluorescent cell cycle biosensor that respectively quantify cell number and cell cycle timing using a high‐throughput imaging system. The doubling time as well as the lengths of each phase of the cell cycle will be measured in cells grown in matrices with decreasing stiffness, and these experiments will also allow us to define a minimal matrix stiffness that will still afford successful cytokinesis. These data will then form the foundation for future studies where we perturb different components of the cytokinetic machinery and test the ability of cells to compensate through increased cell adhesion and motility.
A novel cell‐based Screening System Identifies Adefovir‐dipivoxil as Suppressor of RET expression in medullary thyroid carcinoma
Authors: Alqahtani Tariq , Alsuhebany Nada , Aldhaeefi Mohammed , Sun Daekyu
Published: May 2022

The aberrant gene expression is often associated with the progression of several cancers and hence targeting the transcriptional activation of oncogenes using small‐molecules could be a potential strategy for controlling the tumor growth and development. The gain‐of‐function mutation in the RET proto‐oncogene has been identified as the major cause for the development of medullary thyroid carcinoma (MTC), which is a part of multiple endocrine neoplasia type 2 (MEN2) syndrome. In this study we utilized a cell‐based bioluminescence reporter system in which the luciferase gene expression is driven by the RET promoter to screen for drug‐like molecules that potentially suppress the transcription of this gene. Using this system, we identified adefovir‐ dipivoxil as a transcriptional inhibitor for the RET gene, which further suppressed the endogenous RET protein expression in MTC TT cells. Consistent with this effect, adefovir‐dipivoxil also inhibited RET dependent TT cell proliferation and increased apoptosis in these cells. These results highlight the potential of the cell‐based screening assay to identify transcriptional inhibitors for other oncogenes.
Sleep Disturbance and Sympathetic Reactivity in Menopausal Women
Authors: Tahsin Chowdhury Tasnova , Panigrahy Nisha , Stokes William , Anselmo Miguel , Glazos Aline , Lee Emma , Reilly Cavan , Vanden Noven Marnie L. , Carter Jason R. , Keller‐Ross Manda L.
Published: May 2022

Introduction

Cardiovascular disease (CVD) is the leading cause of death in women. Sleep disturbances (DS), such as insomnia or frequent nocturnal awakenings, may increase the risk of CVD. Prior work suggests that autonomic blood pressure (BP) dysregulation in postmenopausal women may exaggerate risk of hypertension (HTN) and CVD. In addition, individuals with greater BP and sympathetic neural responses to stressors, such as a cold pressor test (CPT), demonstrate future risk for HTN development. Further, when exposed to 24‐hour sleep deprivation, older women, but not older men, demonstrate greater sympathetic activity. It is unknown, however, if women who experience DS demonstrate a greater sympathetic reactivity to a CPT. The purpose of this study was to determine if postmenopausal women with DS exhibit sympathetic reactivity to a CPT compared to women without DS.

Methods

Eighteen women (62±1yrs old; mean±SE) participated in two study visits. Visit 1: Informed consent and medical and health questionnaires. Visit 2: DS was assessed by the Menopause‐Specific Quality of Life questionnaire. Participants were classified as having disturbed sleep (DS; n=10) or non‐disturbed sleep (NDS; n=8) based on a scale of 1 indicating ‘none’ and 2‐8 being how bothersome it is. The study was conducted in the morning after overnight fast and abstinence of alcohol, caffeine, and exercise. Muscle sympathetic nerve activity (MSNA) was measured from the peroneal nerve using microneurography. Heart rate (HR) via 3‐lead electrocardiography, MSNA, and beat‐to‐beat BP using a non‐invasive finger cuff were measured during a 10‐min quiet rest and a two‐min CPT of the left hand.

Results

Menopause onset ranged from 40‐57yrs (50±1yrs). Body mass index was similar between groups (DS=25±1kg/m2, NDS=24±2kg/m2, p=0.53). MSNA burst incidence (bursts/100 heart beats, hb) increased throughout the CPT (time effect, p<0.01), but increases were not significantly different between DS (Δ19±6b/100hb; 95% CI [7,31]) and NDS (Δ11±6b/100hb; 95% CI [‐2,24]) (time*group, p=0.36) groups. Systolic BP, Diastolic BP and HR increased similarly (p>0.05) during the CPT for both groups. Sleep difficulty score was correlated with resting diastolic BP (r=0.43, p=0.03) and trended to correlate with the age of menopause (r= ‐0.37, p=0.05).

Conclusion

These findings suggest that women with DS do not demonstrate greater sympathetic reactivity to a CPT, however, we did observe that DS was linked to greater diastolic BP at rest and appeared to be more bothersome for women who completed menopause at an earlier age. These findings suggest a pathway by which disrupted sleep may contribute to the future development of HTN and CVD in postmenopausal women.