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

RNA-seq analysis of glycosylation related gene expression in STZ-induced diabetic rat kidney inner medulla

The UT-A1 urea transporter is crucial to the kidney’s ability to generate concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, α-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ) - induced diabetic rat kidney. Differential gene expression analysis combining quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8 and -9. In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes. Conclusions: In diabetes, not only is UT-A1 protein abundance increased but the protein’s glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes

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

قراءة في رواية" الشمعة والدهاليز " للؕاهر وؕار

تبدأ أحداث الرواية باستيقاظ الشاعر/ البؕل على أصوات متفاوتة القوة والتقارب، غامضة مبهمة الكلمات، ولكنها واضحة اللحن. لم تكن هذه الأصوات لمدافع ، ولا حتى لدبابات وجنازر، بل كانت� لهدير بشري قوي. وقد ظلت هذه الأصوات تنبعث من كل ساحات الجزائر العاصمة، من آلاف الحناجر، في حالة ودر صوفي متناه. لقد قرر الشاعر النزول إلى المدينة أو بالأحرى أن يتبع مصدر الأصوات، ليعرف ماذا يجري هناك، لأن المسألة تعنيه باعتباره جزائري أولا ، وباحث اجتماع ثانيا.وأثناء ذلك كانت الأصوات تتعالى آتية من بعيد، وبدأت تنتشر في أماكن كثيرة ، وليس في موضع واحد، حاول الشاعر استحضار ؕبيعة الأصوات أو عبارات اللحن كما كانت تردد لأول مرة " لا إله إلا الله محمد رسول الله� عليها نحيا وعليها نموت عليها نلقى الله

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