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Advances in mass spectrometry instrumentation have revolutionized analytical capability in clinical proteomics. In parallel, various sample preparation methods have been developed to try to address the inherent complexity and dynamic range of clinical samples, typically involving a combination of depletion of abundant proteins followed by extensive prefractionation. However, the depth of coverage routinely achieved in discovery proteomics experiments on peripheral fluids such as serum, still leaves something to be desired, especially if no depletion or prefractionation is done in order to increase the throughput of clinical samples. Remarkably, despite being an easily accessible, typically sterile and diagnostically rich clinical sample, urine is often overlooked and as such has received less development effort. As an ultrafiltrate of blood, urine contains proteins and protein fragments originating from all parts of the body which may have diagnostic or prognostic potential if accurately and reproducibly quantified. Acetohydroxamic Here, we describe an efficient and simple method for the concentration of urine samples by methanol-chloroform precipitation and subsequent in-solution tryptic digestion prior to discovery or targeted mass spectrometry analysis. We exemplify this method by reference to the discovery of novel candidate urinary biomarkers of schistosomiasis. Importantly, the methods described here have been used to identify >1900 protein groups in human urine by label-free discovery proteomics, without requiring any prior depletion or prefractionation, making this approach amenable to high throughput clinical biomarker studies in many diseases.Bladder cancer with an incidence of 15 cases per 100,000 persons in the global population is the most common tumor of the urinary tract. Imaging techniques, cytoscopy, and cytology are not sufficiently accurate to detect early stage tumors, and the need for new diagnostic markers is still an urgency. Among the biomarkers most recently proposed to improve diagnostic accuracy and especially sensitivity, increasing attention has been focused on the role of the ribonucleoprotein, telomerase. Previous studies have shown that the quantitative telomerase repeat amplification protocol (TRAP) assay performed in voided urine is an important noninvasive tool for the diagnosis of bladder tumors since it has very high sensitivity and specificity, even for early stage and low-grade tumors. Telomerase activity in urine determined by TRAP seems to be marker of great potential, even more advantageous in cost-benefit terms when used in selected symptomatic patients or professionally high-risk subgroups. Here we report the real-time PCR protocol to detect telomerase activity in urine sediment for bladder cancer.Bladder cancer has a very high frequency of recurrence and therefore requires close clinical surveillance throughout its life, with cystoscopies and serial cytological examinations. These tests are both invasive and expensive, with considerable interpersonal and inter-institutional variability. Moreover, cytological examination used for the diagnosis of low-grade tumors has a low sensitivity; thus, there is an increasing focus on the research for new, accurate, urinary markers. Herein, the biological basis, methodologies, and diagnostic performance of biomarkers are discussed.The analysis of liquid biopsy as a source of diagnostic, prognostic, and predictive biomarkers is still object of the main research in the prostate cancer field. Many advantages, such as less invasiveness compared to plasma or serum analysis and the rich content, confer to urine a role as an interesting fluid to be analysed especially in urological diseases. Here we report a workflow focused on profile, concentration, and protein surface characterization of EVs from urinary supernatant.Prostate cancer antigen 3 (PCA3) is a urinary biomarker for prostate cancer and has demonstrated a good specificity and sensitivity representing a minimally invasive test.PCA3 assay could be useful in combination with PSA to suggest an eventual rebiopsy in men who have had one or more previous negative prostate biopsies.Combination of multiple tumor biomarkers will be the trend in the near future to achieve the goal of evaluate the aggressiveness of cancer and at the same time reducing the number of unnecessary biopsies.Application of next generation sequencing techniques in the field of liquid biopsy, in particular urine, requires specific bioinformatics methods in order to deal with its peculiarity. Many aspects of cancer can be explored starting from nucleic acids, especially from cell-free DNA and circulating tumor DNA in order to characterize cancer. It is possible to detect small mutations, as single nucleotide variants, small insertions and deletions, copy-number alterations, and epigenetic profiles. Due to the low fraction of circulating tumor DNA over the whole cell-free DNA, some methods have been exploited. One of them is the application of unique barcodes to each DNA fragment in order to lower the limit of detection of cancer-related variants. Some bioinformatics workflows and tools are the same of a classic analysis of tumor tissue, but there are some steps in which specific algorithms have to be introduced.The characterization of circulating tumor cells (CTCs) is now widely studied as a promising source of cancer-derived biomarkers because of their role in tumor formation and progression. However, CTCs analysis presents some limitations and no standardized method for CTCs isolation from urine has been defined so far. In fact, besides blood, urine represents an ideal source of noninvasive biomarkers, especially for the early detection of genitourinary tumors. Besides CTCs, long noncoding RNAs (lncRNAs) have also been proposed as potential noninvasive biomarkers, and the evaluation of the diagnostic accuracy of urinary lncRNAs has dramatically increased over the last years, with many studies being published. Therefore, this review provides an update on the clinical utility of urinary lncRNAs as novel biomarkers for the diagnosis of bladder and prostate cancers.
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