EXS

By studying literature data and having performed an in silico analysis, the circulating microRNA expression profiles of healthy individuals appear to show an abundance of microRNAs with predominant tumor suppressor activity. We hypothesize that circulating tumor suppressor microRNAs might constitute a sort of continuous tumor surveillance, whereby circulating microRNAs delivering gene expression modulating epigenetic information might halt cell transformation and tumorigenesis. This mechanism might complement the well-known cancer immune surveillance...

Beside blood-borne circulating miRNAs, miRNAs have been identified in other body fluid and excrements including stool, bile, saliva, and urine. Given the direct link of these body fluids to certain organs, their analysis for potential diagnostic miRNA markers is plausible. Several independent findings underline the potential utility of stool-derived miRNAs in the diagnosis of colorectal and pancreatic cancer. Given the difficulties in the diagnosis of cholangiocellular cancer, biliary miRNAs might be envisaged as useful markers...

Diabetes, in all of its forms, is a disease state that demonstrates wide ranging pathological effects throughout the body. Until now, the only method of diagnosing and monitoring the progression of diabetes was through the measurement of blood glucose. Unfortunately, beta cell dysfunction initiates well before the clinical onset of diabetes, and so the development of an effective biomarker signature is of paramount importance to predict and monitor the progression of this disease. MicroRNAs (miRNAs/miRs) are small (18-22 nucleotide) noncoding (nc)RNAs that post-transcriptionally regulate endogenous gene expression by targeted inhibition or degradation of messenger (m)RNA...

Inflammatory bowel diseases (IBD) are chronic, idiopathic, polygenic diseases with significant genetic heterogeneity. The two major types of IBD are Crohn's disease (CD) and ulcerative colitis (UlC). It is well known that chronic intestinal inflammation results from the interplay of genetic, immunologic, and environmental factors, so the failure to properly downregulate nonspecific inflammation started by an environmental trigger may lead to the development of IBD. Recent studies indicate several microRNAs (miRNAs) as regulators of important pathways of the immune response and immune cell development, which are crucial to the pathogenesis of a variety of inflammatory diseases, including IBD...

MicroRNAs (miRNAs) are differentially regulated in healthy, activated, inflamed, neoplastic, or otherwise pathological cells and tissues. While their main functions are executed intracellularly, many miRNAs can reproducibly be detected extracellularly in plasma and serum. This circulating, extracellular miRNA is protected against degradation by complexation with carrier proteins and/or by being enclosed in subcellular membrane vesicles. This, together with their tissue- and disease-specific expression, has fuelled the interest in using circulating microRNA profiles as harbingers of disease, i...

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), are caused by a combination of events that impair normal neuronal function. Although they are considered different disorders, there are overlapping features among them from the clinical, pathological, and genetic points of view. Synaptic dysfunction and loss, neurite retraction, and the appearance of other abnormalities such as axonal transport defects normally precede the neuronal loss that is a relatively late event...

MicroRNAs, key regulators of biological processes, are involved in the pathophysiological mechanisms underlying human diseases, including cardiovascular diseases. Their recent discovery revealed a previously unknown layer of pathophysiologic regulators, which also play a key role in the regulation of several aspects of cardiovascular diseases. More recently, it was demonstrated that circulating microRNAs can be measured in the blood. Hence, the potential use of microRNAs as disease biomarkers attracted many research groups...

One of the major challenges in cancer research is the identification of stable biomarkers that could be routinely measured in easily accessible samples. Human blood and other body fluids represent rich sources for the identification of novel biomarkers. It is apparent that the availability of these biomarkers would improve an early detection of asymptomatic disease and the clinical management of cancer. MicroRNAs have been described to be present in various types of body fluids including cell-free serum and plasma...

Specific and sensitive noninvasive biofluid-based biomarkers are always needed in the laboratory diagnosis of diseases. Biomarkers are applied not only for diagnostic purposes but for stratifying a disease and for assessing the therapy response or disease progression. MicroRNAs (miRNAs) are short noncoding RNA molecules regulating gene expression posttranscriptionally. They are frequently dysregulated in many physiological and pathophysiological conditions. miRNAs are present in the circulation and in other biofluids that are common matrices for clinical laboratory testing that has raised the possibility that miRNAs may serve as novel biomarkers...

Great excitement has surrounded the finding that small RNAs are stable in various biofluids and carry specific signatures reflecting physiological and pathological states. In this chapter, we briefly describe the impact of this revolutionary discovery and introduce different subclasses of circulating microRNAs based on their mode of transport. Subsequently, we review the current state-of-the art knowledge on microRNA selection for export, secretion and possible uptake mechanisms and their potential function in circulation...

MicroRNAs as the endogenous mediators of RNA interference have principal roles in gene expression regulation. Since their discovery in the early 1990s, their number has steadily grown to approximately 2500 known microRNAs at present in humans. MicroRNAs encoded by distinct genes regulate the expression of about 30-60 % of human protein coding genes by targeting their messenger RNAs (mRNAs) and induce mostly posttranscriptional inhibition, or in some cases enhancement. MicroRNAs, as fine regulators of the gene expression, have important roles in development, the physiological functioning of the organism, e...

This year marks the 30th anniversary of the first description of the cellular distribution of actin within a yeast cell. Since then advances in both molecular genetics and imaging technologies have ensured research within these simple model organisms has blazed a trail in the field of actomyosin research. Many yeast proteins and their functions are functionally conserved in human cells. This, combined with experimental speed, minimal cost and ease of use make the yeasts extremely attractive model organisms for researching diverse cellular processes, including those involving actomyosin...

Molecular machines are the workhorses of the cell that efficiently convert chemical energy into mechanical motion through conformational changes. They can be considered powerful machines, exerting forces and torque on the molecular level of several piconewtons and piconewton-nanometer, respectively. For studying translocation and conformational changes of these machines, fluorescence methods, like FRET, as well as "mechanical" methods, like optical and magnetic tweezers, have proven well suited over the past decades...

In this chapter, we describe experimental techniques used in vitro to illuminate how small teams of motors can work to translocate cargos. We will focus on experiments utilizing in vitro reconstitution, artificial or ex vivo purified cargos, and fluorescence imaging. A number of studies have been able to recapitulate the activities of cargo transport driven by small teams of motors elucidating how multiple motors can work together to transport cargos within the cell. Here, we describe some of the methods employed and highlight important experimental details needed to perform these experiments...

Motor proteins convert the chemical energy of adenosine triphosphate (ATP) hydrolysis into directed movement along filamentous tracks, such as DNA, microtubule, and actin. The motile properties of motors are essential to their wide variety of cellular functions, including cargo transport, mitosis, cell motility, nuclear positioning, and ciliogenesis. Detailed understanding of the biophysical mechanisms of motor motility is therefore essential to understanding the physical basis of these processes. In which direction is the motor going? How fast and how far can a single motor walk down its track? How is ATP hydrolysis coupled to directed motion? How do multiple subunits of a motor coordinate with each other during motility? These questions can be addressed directly by tracking motors at a single-molecule level...

Myosins are a large superfamily of actin-dependent molecule motors that carry out many functions in cells. Some myosins are cargo carriers that move processively along actin which means that a single molecule of myosin can take many ATP-dependent steps on actin per initial encounter. Other myosins are designed to work in large ensembles such as myosin thick filaments. In vitro motility assays are a powerful method for studying the function of myosins. These assays in general use small amounts of protein, are simple to implement, and can be done on microscopes commonly found in many laboratories...

DEAD-box proteins catalyze the ATP-dependent unwinding of RNA duplexes and accompany RNA molecules throughout their cellular life. Conformational changes in the helicase core of DEAD-box proteins are intimately linked to duplex unwinding. In the absence of ligands, the two RecA domains of the helicase core are separated. ATP and RNA binding induces a closure of the cleft between the RecA domains that is coupled to the distortion of bound RNA, leading to duplex destabilization and dissociation of one RNA strand...

The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples...

Studying the dynamics of the interaction between actin and myosin and how this is modulated by ATP and other nucleotides is fundamental to any understanding of myosin motor protein activity. The fluorescent label pyrene, covalently attached to actin (at Cys 374), has been one of the most useful optical probes to report myosin binding to actin. The unique spectral features of pyrene make it sensitive to changes in the microenvironment of the probe and allow to monitor processes such as conformational changes and protein-protein interactions...

This chapter provides an overview of different methodologies to dissect the ATPase mechanism of motor proteins. The use of ATP is fundamental to how these molecular engines work and how they can use the energy to perform various cellular roles. Rapid reaction and single-molecule techniques will be discussed to monitor reactions in real time through the application of fluorescence intensity, anisotropy and FRET. These approaches utilise fluorescent nucleotides and biosensors. While not every technique may be suitable for your motor protein, the different ways to determine the ATPase mechanism should allow a good evaluation of the kinetic parameters...