Notes

Age-Related Adjustments to the particular Structure from the Pie involving Koch: Implications regarding Catheter Ablation associated with Atrioventricular Nodal Re-entry Tachycardia.
13C-BTs are an indirect, cost-effective, and easy method to evaluate dynamic liver function and gastric kinetics in health and disease, with ongoing studies focusing on further applications in clinical medicine.Mitochondria play a key role in cell death and its regulation. The permeabilization of the outer mitochondrial membrane, which is mainly controlled by proteins of the BCL-2 family, is a key event that can be directly induced by different signaling pathways, including p53-mediated, and results in the release of proapoptotic factors to the cytosol, such as cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor-of-apoptosis (IAP) binding protein with low pI (SMAC/Diablo), Omi serine protease (Omi/HtrA2), apoptosis-inducing factor (AIF), or endonuclease G (Endo-G). Hence, the determination of subcellular localization of these proteins is extremely important to predict cell fate and elucidate the specific mechanism of apoptosis. Here we describe experimental protocols that can be used to study the subcellular location of different proapoptotic proteins to be used in basic cell biology and toxicology studies.Mitochondria are dynamic organelles that participate in a broad array of molecular functions within the cell. They are responsible for maintaining the appropriate energetic levels and control the cellular homeostasis throughout the generation of intermediary metabolites. Preserving a healthy and functional mitochondrial population is of fundamental importance throughout the life of the cells under pathophysiological conditions. Hence, cells have evolved fine-tuned mechanisms of quality control that help to preserve the right amount of functional mitochondria to meet the demand of the cell. The specific recycling of mitochondria by autophagy, termed mitophagy, represents the primary contributor to mitochondrial quality control. During this process, damaged or unnecessary mitochondria are recognized and selectively degraded. In the past few years, the knowledge in mitophagy has seen rapid progress, and a growing body of evidence confirms that mitophagy holds a central role in controlling cellular functions and the progression of various human diseases.In this chapter, we will discuss the pathophysiological roles of mitophagy and provide a general overview of the current methods used to monitor and quantify mitophagy. We will also outline the main established approaches to investigate the mitochondrial function, metabolism, morphology, and protein damage.Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter, we describe methods for simultaneous isolation of mtDNA and nuclear DNA (nucDNA), and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material, and availability of specific PCR reagents. We also briefly describe factors that affect mtDNA copy number and discuss caveats to its use as a biomarker.Mitochondria are the organelles where the most fundamental processes of energy transformation within the cell are located. They are also involved in several processes like apoptosis and autophagy, reactive oxygen species formation, and calcium signaling, which are crucial for proper cell functioning. In addition, mitochondrial genome hosts genes encoding important proteins incorporated in respiratory chain complexes and indispensable for the oxidative phosphorylation. Studying isolated mitochondria is, therefore, crucial for better understanding of cell physiology. The presented protocol describes a relatively simple and handy method for crude mitochondrial fraction isolation from different mammalian cell lines. It includes mechanical cells disruption (homogenization) and differential centrifugation. In addition, this chapter presents two basic ways to assess mitochondrial functionality by measuring mitochondrial inner membrane potential and coupled respiration.Investigation of mitochondrial metabolism perturbations and successful diagnosis of patients with mitochondrial abnormalities often requires assessment of human samples like muscle or liver biopsy as well as autopsy material. Immunohistochemical and histochemical examination is an important technique to investigate mitochondrial dysfunction that combined with spectrophotometric and Blue Native electrophoresis techniques can be an important tool to provide diagnosis of mitochondrial disorders. In this chapter, we focus on technical description of the methods that are suitable to detect the activity of complex I, II, and IV of mitochondrial respiratory chain in frozen sections of brain, heart, muscle, and liver biopsies/autopsy. The protocols provided can be useful not only for general assessment of mitochondrial activity in studied material, but they are also successfully used in the diagnostic procedures in case of suspicion of mitochondrial disorders. In the age of high-performance NGS sequencing, these methods can be used to confirm whether mutations are pathogenic by proving their impact on the activity of individual respiratory chain complexes.A positive relationship between mitochondrial functionality and gamete quality, ultimately contributing to fertilization success and normal embryo development has been established for some years now. Both mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production are major indicators of mitochondrial function, and the need for accurate biomarkers mirroring gamete quality highlights the importance of a precise assessment of mitochondrial quality and function. In this chapter, we discuss the use of some mitochondrial fluorescent probes coupled to flow cytometry and/or fluorescence microscopy to specifically assess mitochondrial ROS production and MMP in both sperm and oocytes. Furthermore, as the distribution/aggregation of mitochondria in the oocyte is of interest to determine its quality, a detailed protocol is also given. These methodologies are easy, accurate and can be safely applied in research- and/or clinical-based contexts.Mouse embryonic stem cells (mESCs) can be grown in culture, recapitulating the different states of pluripotency of their in vivo counterparts, with notably different metabolic profiles. mESCs in a naïve pluripotent state present an ambivalent metabolism, using both glycolysis and oxidative phosphorylation as energy sources. Here, we describe a method to evaluate the oxidative function of naïve mESCs using the Seahorse Extracellular Flux Analyzer coupled to flow cytometry analysis of mitochondrial transmembrane potential using the TMRM fluorescence probe, thus assessing both oxygen consumption and mitochondrial membrane potential. This may be a useful protocol for understanding how mitochondrial oxidative function and potential of mESCs change in certain circumstances, and how is it related with various pluripotency/differentiation phenotypes.In recent years, a number of advancements have been made in the study of entire mitochondrial proteomes in both physiological and pathological conditions. Naturally occurring iodothyronines (i.e., T3 and T2) greatly influence mitochondrial oxidative capacity, directly or indirectly affecting the structure and function of the respiratory chain components. Blue native PAGE (BN-PAGE) can be used to isolate enzymatically active oxidative phosphorylation (OXPHOS) complexes in one step, allowing the clinical diagnosis of mitochondrial metabolism by monitoring OXPHOS catalytic and/or structural features. Protocols for isolating mammalian liver mitochondria and subsequent one-dimensional (1D) BN-PAGE will be described in relation to the impact of thyroid hormones on mitochondrial bioenergetics.Mitochondria possess a genome that codes for proteins, in the same fashion as the nuclear genome. However, the small, circular mitochondrial DNA (mtDNA) molecule has a reduced base pair content, for it can only code for 2 rRNA, 22 tRNA molecules, and 13 proteins, all of them part of the mitochondrial respiratory chain. As such, all of the other mitochondrial components derive from nuclear genome. This separation leads to a requirement for a well-tuned coordination between both genomes, in order to produce fully functional mitochondria. A vast number of pathologies have been demonstrated to involve, to some extent, alterations in mitochondrial function that, no doubt, can be caused by alterations to the respiratory chain activity. As such, several methods and techniques have been developed to assess both content and function of mitochondrial proteins, in order to help understand mitochondrial involvement on the pathogenesis of disease. In this chapter, we will address some of these methods, with the main focus being on isolated mitochondria.Several studies have indicated the presence of microRNAs (miRNAs) within mitochondria although the origin, as well as the biological function, of these mitochondrially located miRNAs is largely unknown. The identification and significance of this subcellular localization is gaining increasing relevance to the pathogenesis of certain disease states. Here, we describe the isolation of highly purified mitochondria from rat liver by differential centrifugation, followed by RNAse A treatment to eliminate contaminating RNA. The coupled extraction of total RNA and protein is a more efficient design for allowing the downstream evaluation of miRNA and protein expression in mitochondria.Ectopic adrenal rests are a rare condition which can be found in various sites, generally in the retroperitoneum or pelvis along the path of gonadal descent. Their real prevalence is unknown. Males are more commonly affected, at least in the pediatric age. Adrenal rests are usually clinically silent and incidentally found in surgical samples, mostly in the pediatric population, and rarely in adults. With the aim of increasing knowledge and estimating the prevalence of ectopic adrenocortical tissue in the adult population, 44 adrenal rests in the urogenital tract of 40 adults are described. These represent approximately 0.07% of the total number of urogenital and gynecological surgeries performed in the 22 considered years. Adrenal rests were identified in the spermatic cord (10 males) and in paraovarian, parasalpingeal, or infundibulopelvic ligament locations (30 females). All but one was incidental findings. One case regarded an adrenocortical carcinoma arisen in adrenal rests. A literature review of adrenal ectopia in the urogenital tract of adults identified 57 reported cases from 53 patients, with similar clinicopathological features as those of our series, with the exception of a lower incidence of parasalpingeal locations. Despite their limited clinical implications, awareness of ectopic adrenal rests is essential also in adults for at least two reasons (a) to correctly identify sources of adrenocortical hormone production in case of adrenal insufficiency or hormonal imbalance and (b) to avoid misinterpretations in the diagnostic workup of renal cell carcinoma, adrenocortical tumors, and rare gonadal neoplasms, including Sertoli/Leydig cell tumors.
Arteriovenous fistula(AVF) is preferred vascular access for hemodialysis but has primary failure in 20-60%. Studying predictors of AVF failure would help plan appropriate management.We studied AVF outcomes, clinical and vascular factors predicting their failure in patients requiring hemodialysis.

Retrospective study of patients with AVF creation from January 2017 to December 2018. PF05221304 Outcomes studied were immediate (< 72h), primary (3months) AVF failure, six-month/one-year patency, analyzed for predictive clinical, vascular factors as assessed using Pre-operative Doppler Ultrasound(DUS).

Of 530 AVFs in 460 patients, DUS was done in 426/530 (80.4%), 349/460 (75.8%) were males, mean age was 53.10 ± 14.54 (18-91), 215/460(46.7%) had Diabetes mellitus(DM), 423/460(92%) hypertension. AVFs were radiocephalic in 79/530 (14.9%), brachiocephalic 418/530 (78.9%), brachiobasilic 33/530 (6.2%). AVF Immediate/Primary failure was seen in 64/530 (12.1%), 90/352 (25.6%); Patency at six months/one year in 253/352(71.8%),191/305 (62.
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