Der Einfluss molekularer Mechanismen auf die Expression des NO-sensitiven Rezeptors lösliche Guanylatzyklase (sGC)

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO) which leads to the activation of GMP dependent protein kinases and to vasodilation. NO signaling may be affected by altered expression of sGC subunits, as has been shown in different pathological and physiological conditions and developmental stages. The molecular mechanisms underlying altered sGC expression in these and other conditions have not yet been revealed. Gene expression can also be regulated at the level of mRNA through alterations in translational efficiency and in mRNA stability. HuR (Human R) is a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins. Among other RNAs, there has been recent evidence that the expression of sGC is subject to post-transcriptional regulation by HuR. It has been shown that chronic hypertension induces changes in HuR expression and activity, which account for decreased sGC expression and activity in the aorta of hypertensive rats. This thesis should study was performed in an effort to provide some insight to the transcriptional and post-transcriptional regulation of sGC expression in a mammal, the rat. We investigated rat sGC alpha-1 transcriptional regulation in rat lung fibroblast (RLF-6) cells. The 3000bp 5' upstream region of the alpha-1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs- Alpha3000 (with -2794 bp), Alpha1100 (-1092 bp), Alpha350 (-346 bp) and Alpha200 (-200 bp). The promoter activity was the highest in the 200bp construct (about 6-fold higher than Alpha3000) suggesting that this fragment contains all the crucial elements necessary to support basal transcription of the alpha-1 sGC gene. Analysis of the 200 bp of the 5’ UTR of the alpha-1 gene was performed using the MATINSPECTOR V2.2 software for putative transcription factors. The constructs containing the deleted sites for NFY and Sp1 showed a significant decrease in constitutive promoter activity by almost 80% and 60% respectively, implying that these transcription factors are crucial elements in the basal expression of the of sGC alpha-1 subunit. Treatment of RLF-6 cells with genistein 50 microM and mithramycinA 100 nM, known to inhibit the NFY and Sp1 binding to DNA respectively, reflected the same effects. Furthermore the cGMP content of the cells was significantly reduced by both inhibitors, almost completely by genistein, and by about 40 % by mithramycinA. Electrophoretic mobility-shift assay (EMSA) clearly showed the formation of multiple complexes with the biotinylated ODN (decoy oligodeoxynucleotide) probes for NFY and Sp1 when incubated with RLF-6 nuclear extract. A “supershift” observed in the presence of antibodies to the individual transcription factors confirmed that these factors were present in the shifted band, indeed. NFY and Sp1 are instrumental in several physiological and pathophysiological effects mediated by several growth factors in smooth muscle cells. Thus the regulation of the promoter, in response to serum, was also analysed. 10% foetal calf serum led to decreased alpha-1 sGC level as shown by western blots performed with rat aorta. Decreased sGC alpha-1 mRNA expression was observed in RLF-6 cells and cultured rat aortic smooth muscle cells incubated with FCS for 24 hours. This decrease was reflected in the promoter activity in RLF-6 cells using both Alpha3000 and Alpha200 constructs confirming that the regulation took place at promoter level. EMSA performed with nuclear extracts from FCS treated RLF-6 cells led to diminished binding to NFY, but to an enhanced binding to Sp1 site. We concluded that the factors Sp1 and NFY (the sites overlapping) compete for binding, and in the presence of FCS, it is Sp1 that binds stronger, and hence results in diminishing promoter activity. In order to delineate the post-transcriptional regulation of sGC alpha-1 subunit, studies were performed to demonstrate the regulation of expression of the mRNA stabilizing protein HuR. It has been observed that exposure of isolated rat aortic segments to the activator of adenylyl cyclase, forskolin, strongly reduced sGC alpha-1/beta-1 and HuR protein and mRNA expression in a time-dependent and actinomycin D-sensitive fashion. Transcription factor decoy approach proved that the cAMP-induced down-regulation of HuR is mediated by the activation of AP-1. It has been established that HuR stabilises the sGC alpha-1 and beta-1 mRNA. However the pathway underlying this regulation remains unknown. In order to identify the mechanism of this regulation, we looked for HuR interacting proteins employing the yeast two hybrid assay. The enzyme of the polyamine catabolic pathway spermidine/spermine N1-acetyltransferase (SSAT) was found to interact with the hinge region of HuR. This interaction was confirmed by performing immunoprecipitation and GST-pulldown experiments. A direct effect of these proteins on each other’s biological activity was not visible as tested through the SSAT activity assay and HuR gel shift. It might be possible that SSAT-mediated modulation of local polyamine concentrations enhances/reduces HuR activity and sGC expression to affect cell proliferation. In summary, this study represents an analysis of the rat sGC alpha-1 promoter regulation in rat fibroblast cells and identifies NFY and Sp1 as important factors in sGC alpha-1 expression. It also gives first evidence of sGC regulation at the transcriptional level in response to an external stimulus, and proposes the possible mechanism. It also identifies SSAT as a HuR interacting protein. These might have implications in the various pathophysiological conditions where sGC plays an important role.Die lösliche Guanylatzyklase (soluble guanylyl cyclase, sGC) ist ein Schlüsselenzym der NO/cGMP Signaltransduktion im kardiovaskulären System. Nach Aktivierung der sGC durch Stickstoffmonoxid (NO) kommt es in glatten Muskelzellen zur Produktion von zyklischem 3’,5’-Guanosinmonophosphat (cGMP), das als intrazelluläres Signaltransduktionsmolekül die Aktivierung von cGMP-abhängigen Proteinkinasen vermittelt und zur Vasodilatation führt. Die NO/cGMP-Signaltransduktion kann auch auf der Ebene der Gen-Expression beeinflusst werden. So konnte in verschiedenen physiologischen bzw. pathophysiologischen Modellen gezeigt werden, dass die unterschiedliche Expression von sGC-Untereinheiten die NO/cGMP-Signaltransduktion beeinflusst. Die molekularen Mechanismen, die auf der unterschiedlichen Expression der sGC-Untereinheiten basieren, wurden bisher noch nicht ausreichend aufgeklärt. Die Gen-Expression kann auf mRNA-Ebene durch Veränderung der Translation und der mRNA- (messenger ribonucleic acid, Boten-Ribonnukleinsäure) Stabilität reguliert werden. HuR (Human R) ist ist ein ubiquitär vorkommendes Mitglied der embryonic lethal abnormal vision (ELAV-) Familie der RNA-bindenden Proteine. Neben der Regulierung anderer Gene gibt es Hinweise darauf, dass auch die Expression der sGC einer post-translationalen Regulation durch HuR unterliegt. So wurde gezeigt, dass chronischer Bluthochdruck Veränderungen in der HuR-Expression und -Aktivität verursacht, was zu einer verminderten sGC-Expression und -Aktivität in der Aorta von hypertensiven Ratten führt. Diese Arbeit soll einen Einblick in die transkriptionale und post-transkriptionale Regulation der sGC-Expression im Rattenmodell liefern. Im Rahmen dieser Arbeit wurde die translationale Regulation der alpha-1 sGC-Untereinheit der Ratte in Rattenlungen-Fibroblastenzellen (rat lung fibroblast cells, RLA-6) untersucht. Es wurden die 3000 bp der 5´ upstream Region des Gens der alpha-1 sGC-Untereinheit isoliert und mit Hilfe von Luziferase-Reporter-Konstrukten - Alpha3000 (mit -2794 bp), Alpha1100 (-1092 bp), Alpha350 (-346 bp) und Alpha200 (-200 bp) - auf Promotor-Aktivität untersucht. Dabei war die Promotor-Aktivität des 200 bp Konstrukts am höchsten (ca. 6-fach im Vergleich zu Alpha3000). Dies deutete daraufhin, dass dieses Fragment entscheidende Elemente enthält, die für die basale Transkription des alpha-1 sGC Gens nötig sind. Um nach vermeintlichen Transkriptionsfaktoren zu suchen, wurde die Analyse der 200 bp am 5´ UTR des alpha-1 Gens mit der MATINSPECTOR V2.2 Software durchgeführt. Die Konstrukte mit den entfernten Stellen für NFY und Sp1 zeigten eine signifikante Verminderung in der konstitutiven Promotor-Aktivität zu fast 80 und 60%. Dies deutete daraufhin, dass diese Transkriptionsfaktoren entscheidend sind für die basale Expression der alpha-1 sGC-Untereinheit. Die Behandlung der RLF-6 Zellen mit Genistein (50 mikroM) und Mithramycin A (100 nM), die die Anbindung von NFY und Sp1 an die DNA inhibieren, führte ebenfalls zu einer Reduktion der Promotor-Aktivität. Außerdem war der Gehalt von cGMP in den Zellen durch die beiden Inhibitoren signifikant reduziert - mit Genistein wurde eine fast komplette Inhibition erreicht und mit Mithramycin A eine Reduktion um ca. 40%. Der EMSA (electrophoretic mobility-shift assay) zeigte deutlich die Bildung von multiplen Komplexen mit biotinylierten ODN (decoy oligodeoxynucleotide) Proben für NFY und Sp1, wenn diese mit RLF-6 Zellkern-Extrakt inkubiert wurden. In einem „supershift“ konnten die einzelnen Transkriptionsfaktoren mit Antikörpern nachgewiesen werden. NFY und Sp1 sind an zahlreichen physiologischen und pathophysiologischen Prozessen in glatten Muskelzellen beteiligt, die durch Wachstumsfaktoren gesteuert werden. Aus diesem Grund wurde die Regulation des Promotors in Gegenwart von Serum analysiert. Mit Western-Blot Analysen konnte gezeigt werden, dass 10 % fötales Kälber-Serum (foetal calf serum, FCS) zu einer verminderten Expression von alpha-1 sGC in Rattenaortenringen führte. Eine verminderte sGC alpha-1 mRNA-Expression wurde in RLF-6 Zellen und in kultivierten glatten Muskelzellen der Rattenaorta, die mit FCS über 24 Stunden inkubiert wurden, demonstriert. Diese Verminderung ging einher mit einer verminderten Promotor-Aktivität der Konstrukte Alpha3000 und Alpha200 in RLF-6 Zellen, was eine Regulation auf Promotor-Ebene aufzeigte. EMSA Studien mit Zellkern-Extrakt aus FCS-vorbehandelten RLF-6 Zellen zeigten eine verminderte NFY-, aber eine gesteigerte Sp1-Bindung. Daraus wurde gefolgert, dass die Faktoren Sp1 und NFY um eine Bindungsstelle am Promotor konkurrieren und dass die Gegenwart von FCS zu einer verstärkten Sp1-Bindung und damit zu einer verminderten Promotor-Aktivität führt. Um die post-transkriptionale Regulation der alpha-1 sGC-Untereinheit zu demonstrieren, wurden Versuche zur Regulation der Expression des mRNA-stablisierenden Proteins HuR durchgeführt. Es konnte gezeigt werden, dass die Inkubation von isolierten Rattengefäßringen mit Forskolin, einem Aktivator der Adenylatzyklase, die sGC alpha-1/beta-1 und die HuR Protein- und mRNA-Expression zeitabhängig und Actinomycin D-sensitiv reduziert. Der Transkriptionsfaktor-Abbau bewies, dass die cAMP-induzierte Runterregulation von HuR durch die Aktivierung von AP-1 vermittelt wird. Es konnte festgestellt werden, dass HuR die alpha-1 und beta-1 mRNA der sGC stabilisiert. Aber die Regulation dieser Signalwege war bisher unbekannt. Um den Mechanismus der Regulation zu identifizieren, haben wir nach HuR-interagierenden Proteinen durch Anwendung des Yeast-two-hybrid assays gesucht. Die Enzyme des Polyamin-katabolischen Signalwegs Spermidin/ Spermin N1-Acetyltransferase (SSAT) wurden als eine hinge Region von HuR entdeckt. Diese Interaktion wurde bestätigt durch Immunopräzipitation und GST-pulldown Experimente. Einen direkten gegenseitigen Effekt auf die Aktivität dieser Proteine war nicht sichtbar, was durch SSAT activity assays und HuR gel shifts getested wurde. Es könnte möglich sein, dass die SSAT-vermittelte Modulation der lokalen Polyamin-Konzentration die HuR-Aktivität und die sGC-Expression fördert bzw. vermindert bei der Zellproliferation. Diese Arbeit zeigt eine Analyse der Promotor-Regulation der alpha-1 sGC-Untereinheit der Ratte in Fibroblastenzellen der Ratte und identifiziert NFY und Sp1 als wichtige Faktoren in der alpha-1 sGC-Expression. Die sGC-Regulation auf transkriptionaler Ebene unter Beteiligung der Transkriptionsfaktoren NFY und Sp1, ausgelöst durch Serum als externen Stimulus, weist auf einen möglichen Mechanismus hin. Außerdem wurde SSAT als HuR-Interaktionsprotein identifiziert. Die in dieser Arbeit dargestellte Regulation der sGC-Expression könnte Einfluss haben auf verschiedene pathophysiologische Zustände

An Intelligent Framework for Estimating Software Development Projects using Machine Learning

The IT industry has faced many challenges related to software effort and cost estimation. A cost assessment is conducted after software effort estimation, which benefits customers as well as developers. The purpose of this paper is to discuss various methods for the estimation of software effort and cost in the context of software engineering, such as algorithmic methods, expert judgment methods, analogy-based estimation methods, and machine learning methods, as well as their different aspects. In spite of this, estimation of the effort involved in software development are subject to uncertainty. Several methods have been developed in the literature for improving estimation accuracy, many of which involve the use of machine learning techniques. A machine learning framework is proposed in this paper to address this challenging problem. In addition to being completely independent of algorithmic models and estimation problems, this framework also features a modular architecture. It has high interpretability, learning capability, and robustness to imprecise and uncertain inputs

Silybin B and Cianidanol Inhibit M pro and Spike Protein of SARS-CoV-2: Evidence from in Silico Molecular Docking Studies

BACKGROUND: The main proteases (Mpro) and Spike Proteins (SP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV-2) play a major role in viral infection development by producing several non-structural proteins (nsPs) and penetrating the host cells respectively. In this study, the potential of in silico molecular docking-based drug repositioning approach was exploited for identifying the inhibitors of Mpro and SP of SARS-CoV-2. METHODS: A total of 196 compounds including various US-FDA-approved drugs, vitamins and their analogs were docked with Mpro (PDB IDs: 6YB7 and 6Y84), and the top six ligands were further tested for ADME properties followed by docking with SP (PDB IDs: 6LXT and 6W41). RESULTS: Out of 196 compounds, binding energy (DE) of Silybin B (6YB7: DE: -11.20 kcal/mol; 6Y84: DE: -10.18 kcal/mol; 6LXT:DE: -10.47 kcal/mol; 6W41:DE: -10.96 kcal/mol) and Cianidanol (6YB7:DE: -8.85 kcal/mol; 6Y84:DE:-10.02 kcal/mol; 6LXT:DE:-9.36 kcal/mol; 6W41:DE: -9.52 kcal/mol) demonstrated better binding and ADME properties compared with the currently endeavored drugs like Hydroxychloroquine and Lopinavir. Additionally, Elliptinone, Diospyirin, SCHEMBL94263 and Fiboflavin have shown encouraging results. Fiboflavin, an immunity booster, was found to inhibit both the Mpro and spike protein of SARS-CoV-2. It was observed that amino acid residues MET6, ALA7, PHE8, PRO9, ASP295, GLY302, VAL303 and THR304 play significant roles in protein-ligand interactions through hydrogen bonds and Vander Waals forces. CONCLUSION: Silybin B and Cianidanol showed excellent binding and ADME properties compared with the currently endeavored drugs and can be exploited as therapeutic options against SARS-CoV-2 infection after experimental validation and clinical trials

Advances in pulmonary drug delivery targeting microbial biofilms in respiratory diseases

The increasing burden of respiratory diseases caused by microbial infections poses an immense threat to global health. This review focuses on the various types of biofilms that affect the respiratory system and cause pulmonary infections, specifically bacterial biofilms. The article also sheds light on the current strategies employed for the treatment of such pulmonary infection-causing biofilms. The potential of nanocarriers as an effective treatment modality for pulmonary infections is discussed, along with the challenges faced during treatment and the measures that may be implemented to overcome these. Understanding the primary approaches of treatment against biofilm infection and applications of drug-delivery systems that employ nanoparticle-based approaches in the disruption of biofilms are of utmost interest which may guide scientists to explore the vistas of biofilm research while determining suitable treatment modalities for pulmonary respiratory infections. </jats:p

Exploring state-of-the-art advances in targeted nanomedicines for managing acute and chronic inflammatory lung diseases

Diagnosis and treatment of lung diseases pose serious challenges. Currently, diagnostic as well as therapeutic methods show poor efficacy toward drug-resistant bacterial infections, while chemotherapy causes toxicity and nonspecific delivery of drugs. Advanced treatment methods that cure lung-related diseases, by enabling drug bioavailability via nasal passages during mucosal formation, which interferes with drug penetration to targeted sites, are in demand. Nanotechnology confers several advantages. Currently, different nanoparticles, or their combinations, are being used to enhance targeted drug delivery. Nanomedicine, a combination of nanoparticles and therapeutic agents, that delivers drugs to targeted sites increases the bioavailability of drugs at these sites. Thus, nanotechnology is superior to conventional chemotherapeutic strategies. Here, the authors review the latest advancements in nanomedicine-based drug-delivery methods for managing acute and chronic inflammatory lung diseases

Pharmacological Potentials of Royal Jelly in Dentistry: A Review

“Royal Jelly” is a viscous material which is secreted by the cephalic gland of the “worker bee.” This is the key component of the “queen bee’s” diet, rendering it with longer life span compared to worker bees.“Royal Jelly” possess various pharmacological properties. These includes antioxidant, neurotropic, anti-inflammatory, immunomodulatory and antibiotic, anti-aging, antihypertensive properties. This article summarizes biological properties, composition, criteria of quality evaluation of “Royal Jelly”and published studies on the efficacy of “Royal Jelly” as a “wound healing agent” , as a component of mouth wash used in treatment of oral mucositis, periodontal diseases, as a storage media and as a pulp capping agent.“Royal Jelly” can emerge as a viable alternative to conventional synthetic agents, which may avoid usual drawbacks of synthetic medicaments

Antioxidants in Alzheimer&rsquo;s Disease: Current Therapeutic Significance and Future Prospects

Alzheimer&rsquo;s disease (AD) rate is accelerating with the increasing aging of the world&rsquo;s population. The World Health Organization (WHO) stated AD as a global health priority. According to the WHO report, around 82 million people in 2030 and 152 million in 2050 will develop dementia (AD contributes 60% to 70% of cases), considering the current scenario. AD is the most common neurodegenerative disease, intensifying impairments in cognition, behavior, and memory. Histopathological AD variations include extracellular senile plaques&rsquo; formation, tangling of intracellular neurofibrils, and synaptic and neuronal loss in the brain. Multiple evidence directly indicates that oxidative stress participates in an early phase of AD before cytopathology. Moreover, oxidative stress is induced by almost all misfolded protein lumps like &alpha;-synuclein, amyloid-&beta;, and others. Oxidative stress plays a crucial role in activating and causing various cell signaling pathways that result in lesion formations of toxic substances, which foster the development of the disease. Antioxidants are widely preferred to combat oxidative stress, and those derived from natural sources, which are often incorporated into dietary habits, can play an important role in delaying the onset as well as reducing the progression of AD. However, this approach has not been extensively explored yet. Moreover, there has been growing evidence that a combination of antioxidants in conjugation with a nutrient-rich diet might be more effective in tackling AD pathogenesis. Thus, considering the above-stated fact, this comprehensive review aims to elaborate the basics of AD and antioxidants, including the vitality of antioxidants in AD. Moreover, this review may help researchers to develop effectively and potentially improved antioxidant therapeutic strategies for this disease as it also deals with the clinical trials in the stated field

Exploring state-of-the-art advances in targeted nanomedicines for managing acute and chronic inflammatory lung diseases

Diagnosis and treatment of lung diseases pose serious challenges. Currently, diagnostic as well as therapeutic methods show poor efficacy toward drug-resistant bacterial infections, while chemotherapy causes toxicity and nonspecific delivery of drugs. Advanced treatment methods that cure lung-related diseases, by enabling drug bioavailability via nasal passages during mucosal formation, which interferes with drug penetration to targeted sites, are in demand. Nanotechnology confers several advantages. Currently, different nanoparticles, or their combinations, are being used to enhance targeted drug delivery. Nanomedicine, a combination of nanoparticles and therapeutic agents, that delivers drugs to targeted sites increases the bioavailability of drugs at these sites. Thus, nanotechnology is superior to conventional chemotherapeutic strategies. Here, the authors review the latest advancements in nanomedicine-based drug-delivery methods for managing acute and chronic inflammatory lung diseases