Themed project case study: Quadruple tanks control with PLCs^†

A themed project based on the control of a quadruple tank rig using PLCs has been successfully carried out as part of the MSc in Advanced Control and Systems Engineering at the University of Manchester. The themed project involves ten students who address a single multivariable control challenge under the supervision of two academics and four PhD students. As every student is required to write their own MSc dissertation, the key point is the possibility of using different control techniques to be implemented on different hardware platforms. </jats:p

Human Genotyping and An Experimental Model Reveal NPR-C as A Possible Contributor to Morbidity In Coarctation Of The Aorta

Coarctation of the aorta (CoA) is a common congenital cardiovascular (CV) defect characterized by a stenosis of the descending thoracic aorta. Treatment exists, but many patients develop hypertension (HTN). Identifying the cause of HTN is challenging because of patient variability (e.g., age, follow-up duration, severity) and concurrent CV abnormalities. Our objective was to conduct RNA sequencing of aortic tissue from humans with CoA to identify a candidate gene for mechanistic studies of arterial dysfunction in a rabbit model of CoA devoid of the variability seen with humans. We present the first known evidence of natriuretic peptide receptor C (NPR-C; aka NPR3) downregulation in human aortic sections subjected to high blood pressure (BP) from CoA versus normal BP regions (validated to PCR). These changes in NPR-C, a gene associated with BP and proliferation, were replicated in the rabbit model of CoA. Artery segments from this model were used with human aortic endothelial cells to reveal the functional relevance of altered NPR-C activity. Results showed decreased intracellular calcium ([Ca2+]i) activity to C-type natriuretic peptide (CNP). Normal relaxation induced by CNP and atrial natriuretic peptide was impaired for aortic segments exposed to elevated BP from CoA. Inhibition of NPR-C (M372049) also impaired aortic relaxation and [Ca2+]i activity. Genotyping of NPR-Cvariants predicted to be damaging revealed that rs146301345 was enriched in our CoA patients, but sample size limited association with HTN. These results may ultimately be used to tailor treatment for CoA based on mechanical stimuli, genotyping, and/or changes in arterial function

Cathepsin B increases ENaC activity leading to hypertension early in nephrotic syndrome

The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria‐dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full‐length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+/K+‐ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified

Characterization of NPRC and its binding partners

The C type natriuretic peptide receptor (NPRC) also known as NPR3 is a widely expressed single transmembrane-spanning protein. NPRC functions as a homodimer at the cell surface for the metabolic clearance of a broad range of natriuretic peptides from circulation. The intracellular domain of NPRC is coupled to inhibitory G proteins and is involved in mediating signal transduction. In order to further elucidate the role of NPRC in signal transduction a proteomic approach was taken to identify putative protein binding partners for NPRC in different cell-types. An interrogation of the molecular association between NPRC and its identified protein binding partner(s) was carried out in different cell types to identify the specific interacting domains. The physiological role of the association between NPRC and its protein binding partner(s) were investigated in situ. Furthermore NPRC is subject to post translation modifications including glycosylation and phosphorylation. Although evidence suggests NPRC is phosphory ated on serine residues the specific amino acid residues that are phosphorylated and the kinases responsible for their phosphorylation has yet to be determined. A recombinant GST-NPRC fusion protein polyclonal NPRC antibody kinase prediction algorithm and several phosphospecific and substrate motif antibodies were utilized to characterize the phosphorylation state of NPRC in vitro

Mechanisms of Extracellular Vesicle Biogenesis, Cargo Loading, and Release

Extracellular vesicles (EVs) are carriers of various biomolecules including bioactive enzymes, lipids, proteins, nucleic acids, and metabolites. EVs are classified into three main types based on their size, biogenesis, and cargo. Exosomes originate from endosomal membranes and are the smallest type of EV. Microvesicles (MVs) or microparticles are larger in size, and like apoptotic bodies which represent the largest type of EVs, both of these vesicles originate from outward budding of the plasma membrane. As discussed in this chapter, cargo loading of EVs and their release into the extracellular space where they can be taken up by neighboring or distant cells plays an important role in physiology and pathophysiology. This chapter will outline specific mechanisms involved in the loading and enrichment of miRNAs, proteins, and lipids within EVs. As explained here, various external and biological stimuli play a role in EV release. Finally, recent studies have shown that the biogenesis, cargo loading, and release of EVs are governed by circadian rhythms. Although EVs were once thought to serve as garbage disposals of cells, the numerous roles they serve in physiology and pathophysiology are now being appreciated

The C Type Natriuretic Peptide Receptor Tethers AHNAK1 at the Plasma Membrane to Potentiate Arachidonic Acid-Induced Calcium Mobilization

Arachidonic acid (AA) liberated from membrane phospholipids is known to activate phospholipase C γ1 (PLCγ1) concurrently with AHNAK in nonneuronal cells. The recruitment of AHNAK from the nucleus is required for it to activate PLCγ1 at the plasma membrane. Here, we identify the C-type natriuretic peptide receptor (NPR-C), an atypical G protein-coupled receptor, as a protein binding partner for AHNAK1 in various cell types. Mass spectrometry and MASCOT analysis of excised bands from NPR-C immunoprecipitation studies revealed multiple signature peptides corresponding to AHNAK1. Glutathione S-transferase (GST) pulldown assays using GST- AHNAK1 fusion proteins corresponding to each of the distinct domains of AHNAK1 showed the C1 domain of AHNAK1 associates with NPR-C. The role of NPR-C in mediating AA-dependent AHNAK1 calcium signaling was explored in various cell types, including 3T3-L1 preadipocytes during the early stages of differentiation. Sucrose density gradient centrifugation studies showed AHNAK1 resides in the nucleus, cytoplasm, and at the plasma membrane, but small interfering RNA (siRNA)-mediated knockdown of NPR-C resulted in AHNAK1 accumulation in the nucleus. Overexpression of a portion of AHNAK1 resulted in augmentation of intracellular calcium mobilization, whereas siRNA-mediated knockdown of NPR-C or AHNAK1 protein resulted in attenuation of intracellular calcium mobilization in response to phorbol 12-myristate 13-acetate. We characterize the novel association between AHNAK1 and NPR-C and provide evidence that this association potentiates the AA-induced mobilization of intracellular calcium. We address the role of intracellular calcium in the various cell types that AHNAK1 and NPR-C were found to associate

Cigarette smoke extract is a Nox agonist and regulates ENaC in alveolar type 2 cells

There is considerable evidence that cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD), and that oxidative stress occurs in COPD with the family of tissue nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes playing a significant role in lung pathogenesis. The purpose of this study was to determine the effects of cigarette smoke extract (CSE) on Nox signaling to epithelial sodium channels (ENaCs). Pre-treatment with diphenyleneiodonium (DPI), a pan-Nox inhibitor, prevented stimulatory effects of CSE on ENaC activity; open probability (Po) changed from 0.36 +/- 0.09 to 0.11 +/- 0.02; n=10, p=0.01 following CSE and DPI exposure. Likewise, Fulvene-5 (which inhibits Nox2 and Nox4 isoforms) decreased the number of ENaC per patch (from 2.75 +/- 0.25 to 1 +/- 0.5, n=9, p=0.002) and open probability (0.18 +/- 0.08 to 0.02 +/- 0.08, p=0.04). Cycloheximide chase assays show that CSE exposure prevented alpha-ENaC subunit degradation, whereas concurrent CSE exposure in the presence of Nox inhibitor, Fulvene 5, resulted in normal proteolytic degradation of alpha-ENaC protein in primary isolated lung cells. In vivo, co-instillation of CSE and Nox inhibitor promoted alveolar flooding in C57Bl6 mice compared to accelerated rates of fluid clearance observed in CSE alone instilled lungs. Real-time PCR indicates that mRNA levels of Nox2 were unaffected by CSE treatment while Nox4 transcript levels significantly increased 3.5 fold in response to CSE. Data indicate that CSE is an agonist of Nox4 enzymatic activity, and that CSE-mediated Nox4 plays an important role in altering lung ENaC activity.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Augmentation of Cathepsin Isoforms in Diabetic db/db Mouse Kidneys Is Associated with an Increase in Renal MARCKS Expression and Proteolysis.

The expression of the myristoylated alanine-rich C-kinase substrate (MARCKS) family of proteins in the kidneys plays an important role in the regulation of the renal epithelial sodium channel (ENaC) and hence overall blood pressure regulation. The function of MARCKS is regulated by post-translational modifications including myristoylation, phosphorylation, and proteolysis. Proteases known to cleave both ENaC and MARCKS have been shown to contribute to the development of high blood pressure, or hypertension. Here, we investigated protein expression and proteolysis of MARCKS, protein expression of multiple protein kinase C (PKC) isoforms, and protein expression and activity of several different proteases in the kidneys of diabetic db/db mice compared to wild-type littermate mice. In addition, MARCKS protein expression was assessed in cultured mouse cortical collecting duct (mpkCCD) cells treated with normal glucose and high glucose concentrations. Western blot and densitometric analysis showed less abundance of the unprocessed form of MARCKS and increased expression of a proteolytically cleaved form of MARCKS in the kidneys of diabetic db/db mice compared to wild-type mice. The protein expression levels of PKC delta and PKC epsilon were increased, while cathepsin B, cathepsin S, and cathepsin D were augmented in diabetic db/db kidneys compared to those of wild-type mice. An increase in the cleaved form of MARCKS was observed in mpkCCD cells cultured in high glucose compared to normal glucose concentrations. Taken together, these results suggest that high glucose may contribute to an increase in the proteolysis of renal MARCKS, while the upregulation of the cathepsin proteolytic pathway positively correlates with increased proteolysis of MARCKS in diabetic kidneys, where PKC expression is augmented

Dapagliflozin Treatment Augments Bioactive Phosphatidylethanolamine Concentrations in Kidney Cortex Membrane Fractions of Hypertensive Diabetic db/db Mice and Alters the Density of Lipid Rafts in Mouse Proximal Tubule Cells

In addition to inhibiting renal glucose reabsorption and allowing for glucose excretion, the sodium/glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin may be efficacious in treating various comorbidities associated with type 2 diabetes mellitus (T2DM). The molecular mechanisms by which dapagliflozin exerts its beneficial effects are largely unknown. We hypothesized dapagliflozin treatment in the diabetic kidney alters plasma membrane lipid composition, suppresses extracellular vesicle (EV) release from kidney cells, and disrupts lipid rafts in proximal tubule cells. In order to test this hypothesis, we treated diabetic db/db mice with dapagliflozin (N = 8) or vehicle (N = 8) and performed mass spectrometry-based lipidomics to investigate changes in the concentrations of membrane lipids in the kidney cortex. In addition, we isolated urinary EVs (uEVs) from urine samples collected during the active phase and the inactive phase of the mice and then probed for changes in membrane proteins enriched in the EVs. Multiple triacylglycerols (TAGs) were enriched in the kidney cortex membrane fractions of vehicle-treated diabetic db/db mice, while the levels of multiple phosphatidylethanolamines were significantly higher in similar mice treated with dapagliflozin. EV concentration and size were lesser in the urine samples collected during the inactive phase of dapagliflozin-treated diabetic mice. In cultured mouse proximal tubule cells treated with dapagliflozin, the lipid raft protein caveolin-1 shifted from less dense fractions to more dense sucrose density gradient fractions. Taken together, these results suggest dapagliflozin may regulate lipid-mediated signal transduction in the diabetic kidney