bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polycystin 1 ciliary localization is regulated by its aGPCR activity
Authors: Gresko Nikolay , Caplan Michael 
Published: May 2022

Autosomal dominant polycystic kidney disease (ADPKD) develops due to loss of function mutations in the PKD1 and PKD2 genes, which encode PC1 and PC2, respectively. PC1 is a 460kD multi‐spanning membrane protein that undergoes multiple proteolytic cleavages, at least two of which release C‐terminal fragments. Polycystin 1 and Polycystin 2 both prominently localize to the primary cilium where they contribute to cellular mechano‐sensation and also form a cation‐permeable heterotetrameric channel that may contribute to Ca2+ flux into the cilia. Recent studies demonstrate that PC1 can function as an atypical adhesion GPCR that is activated by Wnt ligands. Wnt ligand binding leads to dissociation of the PC1 extracellular N terminal fragment and allows the PC1 receptor function to be activated by a tethered agonist peptide that resides at the N terminus of the PC1 transmembrane domains.Here we report that Polycystin 1 cilia and cell surface localization is regulated by its activity status. We demonstrate that a constitutively active form of the Polycystin 1 protein is absent from the primary cilia whereas a constitutively inactive version of the PC1 protein that lacks its tethered agonist peptide resides in the primary cilia. Moreover, we show that interruption of the cellular receptor desensitization process by β‐arrestin inhibition permits the constitutively active PC1 construct to accumulate in the cilium. We also studied the surface localization of the full length PC1 and found that extended Wnt9b ligand treatment or mechanical stimulation both lead to reduced quantity of the full length PC1 at the cell surface and in the primary cilium, a process that can be disrupted by inhibition of β‐arrestin. In vivo, we find that reduction of the luminal flow in renal tubules by ureter obstruction leads to accumulation of the PC1 in the primary cilia of the tubule cells.Taken together, our data suggest that PC1 surface trafficking is regulated by receptor‐activating stimuli including ligand binding and mechanical stress and, similar to the regulation of aGPCR receptors, is subject to an activity‐dependent receptor desensitization process. Our findings indicate that the aGPCR receptor‐like properties of Polycystin 1 offer new insights into this protein’s physiological function and the regulation of its trafficking. Understanding these processes may suggest targets for the development of new therapeutic interventions for ADPKD.
Acute Hypernatremia Increases Functional Connectivity of Sodium Sensing Regions in the Brain
Authors: Stock Joseph M. , Burciu Roxana G. , Chung Jae Woo , Romberger Nathan T. , McMillan Ronald K. , Patel Darshil , Salhab Ibrahim , Stocker Sean D. , Farquhar William B. 
Published: May 2022

Rodent studies have identified specialized sodium chloride (NaCl)‐sensing neurons in the circumventricular organs (CVOs), which mediate NaCl‐induced changes in sympathetic nerve activity, arginine vasopressin (AVP), thirst, and blood pressure (BP). In humans, acute hypernatremia has been shown to increase sympathetic nerve activity, AVP, thirst and BP. However, few human studies have investigated the network of NaCl sensing regions of the brain using functional magnetic resonance imaging (fMRI).ObjectiveTo determine how the functional connectivity of sodium sensing regions of the brain change during an acute hypernatremic perturbation.MethodsResting‐state fMRI was performed on 9 healthy young adults (29±4 yrs, 5M) at baseline and during a 30 min 3% NaCl intravenous infusion at a rate of 0.15 ml/kg/min. Venous blood samples and perceived thirst (Likert scale, cm) were obtained before and immediately following the infusion. Serum electrolytes, plasma osmolality, hematocrit, and hemoglobin were assessed. Beat‐to‐beat blood pressure was measured continuously. Seed‐based voxel‐wise functional connectivity analysis was performed in AFNI using spherical seeds placed bilaterally in the subfornical organ (SFO), the organum vasculosum lamina terminalis (OVLT) as well as cortical sensory regions such as the insula. Functional connectivity was compared at baseline vs. early infusion (min 0‐15) and baseline vs. late infusion (min 16‐30) at p < 0.01, corrected for multiple comparisons with 3dClustSim.ResultsThirst (∆1.9±2.0 cm), serum sodium (∆3.2±2.1 mmol/L), and plasma osmolality (∆6.9±4.7 mOsm/kg H2O) all increased from baseline to post‐infusion (p<0.05). Head motion was minimal and did not change throughout the stages of the infusion (mean motion = 0.11±0.10 mm, p>0.80). Hypertonic saline infusion increased local functional connectivity between the SFO and OVLT (Z score baseline=0.07±0.01, Z score late phase=0.17+0.01, p<0.01). A similar increase in functional connectivity was seen between the insula and somatosensory cortex.ConclusionAcute hypernatremia increases local functional connectivity within the CVOs and between cortical sensory regions of the brain.
The Wright Variation of the Darrow‐Yannet Diagram: A New Tool to Teach Water Homeostasis
Authors: Wright Bruce E. 
Published: May 2022

PurposeTo 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 hypothesisSince 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.ResultIn 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.ConclusionAn 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

IntroductionPlasma 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.MethodsEight 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.ResultsThe 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.SummaryThese 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
Exercise‐Induced Shear Stress Upregulates eNOS Protein in vitro
Authors: Garcia Mario , Cabral Shelsea , Rodriguez Carmen , Conde Daniel , Gurovich Alvaro N. 
Published: May 2022

BackgroundEndothelial 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.MethodsCommercially 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.ResultsLow 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).ConclusionsOur 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.
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.