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Bladder cancer is common and has one of the highest recurrence rates. Cystoscopy, the current gold standard diagnosis approach, has recently benefited from the introduction of blue light assisted photodynamic diagnostic (PDD). While blue light cystoscopy improves diagnostic sensitivity, it remains a costly and invasive approach. Here, we present a microfluidic-based platform for non-invasive diagnosis which combines the principle of PDD with whole cell immunocapture technology to detect bladder cancer cells shed in patient urine ex vivo. Initially, we demonstrate with model cell lines that our non-invasive approach achieves highly specific capture rates of bladder cancer cells based on their Epithelial Cell Adhesion Molecule expression (>90%) and detection by the intensity levels of Hexaminolevulinic Acid-induced Protoporphyrin IX fluorescence. Then, we show in a pilot study that the biosensor platform successfully discriminates histopathologically diagnosed cancer patients (n = 10) from non-cancer controls (n = 25). Our platform can support the development of a novel non-invasive diagnostic device for post treatment surveillance in patients with bladder cancer and cancer detection in patients with suspected bladder cancer Refereed/Peer-reviewed

Prostate cancer is the second most common cancer in men and the second leading cause of male cancer deaths. The current blood test for detecting prostate cancers measures prostate-specific antigen. It has many limitations including a very high rate of false positives. Herein, prostate-specific membrane antigen (PSMA) based immunocapture and hexaminolevulinate (HAL) based photodetection are integrated into a new diagnostic device designed to selectively identify whole prostate cancer cells from voided urine with the aim of providing an accurate noninvasive alternative to current diagnosis methods. Prestained, prostate cancer cells spiked in urine samples at concentrations ranging from 1500 to 2000 cells/ml were captured with 89% sensitivity and 95% specificity. HAL, a cancer specific photosensitizer, was then used to circumvent the need for prestaining. Optimum HAL incubation conditions were identified (50 μM at 37 °C for 2 h) where the mean HAL-induced fluorescence intensity of LNCaP cells was three times that of healthy PNT2 cells, thus providing an independent way to discriminate captured cancer cells from background metabolites. Combining anti-PSMA immunocapture with HAL-induced fluorescent detection, 86% sensitivity and 88% selectivity were achieved, thereby proving the validity of the dual-method for the selective photospecific detection of prostate cancer cells Refereed/Peer-reviewed

Aim Extracellular vesicles, such as exosomes, are present in urine with reports of roles in intercellular signalling and diagnostic utility. However, the extent to which the concentration and characteristics of urinary vesicles are altered in albuminuric renal disease has not been well characterized. In this study, we examined the number and characteristics of extracellular vesicles in albuminuric urine. Methods Vesicles were isolated from the urine of 32 patients with varying levels of albuminuria using ultracentrifugation and density gradient purification and were examined using nanoparticle tracking analysis, immunoblotting and transmission electron microscopy. The size profile of particles in these urine preparations was compared with albumin-containing solutions. Results Overall, there were no substantial differences in the number, or characteristics, of vesicles released into proteinuric urine. Analysis of albumin-containing solutions showed particles of exosome-like size, suggesting that such particles can mimic exosomes in standard nanoparticle tracking analysis. Albumin and IgG depletion of proteinuric urine resulted in a substantial reduction in the concentration of particles detected by nanoparticle tracking analysis. Conclusion There was no increase in urinary vesicle concentration in patients with albuminuria. Furthermore, these results demonstrate the need for cautious interpretation of nanoparticle tracking analysis of vesicle concentration in biological fluids containing protein and for sophisticated preparative methods in vesicle purification from urine.

Photodynamic diagnosis and therapy have emerged as a promising tool in oncology. Using the visible fluorescence from photosensitisers excited by light, clinicians can both identify and treat tumour cells in situ. Protoporphyrin IX, produced in the penultimate step of the haem synthesis pathway, is a naturally occurring photosensitiser that visibly fluoresces when exposed to light. This fluorescence is enhanced considerably by the exogenous administration of the substrate 5-aminolaevulinic acid (5-ALA). Significantly, 5-ALA-induced protoporphyrin IX accumulates preferentially in cancer cells, and this enhanced fluorescence has been harnessed for the detection andphotodynamic treatment of brain, skin and bladder tumours. However, surprisingly little is known about the mechanistic basis for this phenomenon. This review focuses on alterations in the haem pathway in cancer and considers the unique features of the cancer environment, such as altered glucose metabolism, oncogenic mutations and hypoxia, and their potential effects on the protoporphyrin IXphenomenon. A better understanding of why cancer cells fluoresce with 5-ALA would improve its use in cancer diagnostics and therapies. Refereed/Peer-reviewed

Due to their size, small extracellular vesicles such as exosomes have been difficult to identify and to quantify. As the roles that exosomes play in intercellular signalling become clearer, so does their potential utility as both diagnostic biomarkers for disease and as therapeutic vectors. Accurate assessment of exosomes, both their number and their cargo, is important for continued advancement in the field of vesicle research. To that end, several technologies, including nanoparticle tracking analysis, have been developed to define the physical characteristics of vesicle preparations and determine their concentration. This chapter describes a method for identifying the size and concentration of a subpopulation of vesicles in biological samples, using nanoparticle tracking analysis. Characterization of distinct exosomes is enabled by specific marker antibodies, coupled to fluorescent quantum dots.

Due to their size, small extracellular vesicles such as exosomes have been difficult to identify and to quantify. As the roles that exosomes play in intercellular signalling become clearer, so does their potential utility as both diagnostic biomarkers for disease and as therapeutic vectors. Accurate assessment of exosomes, both their number and their cargo, is important for continued advancement in the field of vesicle research. To that end, several technologies, including nanoparticle tracking analysis, have been developed to define the physical characteristics of vesicle preparations and determine their concentration. This chapter describes a method for identifying the size and concentration of a subpopulation of vesicles in biological samples, using nanoparticle tracking analysis. Characterization of distinct exosomes is enabled by specific marker antibodies, coupled to fluorescent quantum dots.

Background: Biological roles for intracellular dipeptidyl peptidases 8 and 9 are unknown. Results: By degradomics, 29 new in vivo substrates were identified (nine validated) for DP8/DP9, including adenylate kinase 2 and calreticulin. Conclusion: These substrates indicate roles for DP8 and DP9 in metabolism and energy homeostasis. Significance: Being the first proteomics screen for DP8/DP9 substrates, unexpected new cellular roles were revealed., Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.

Extracellular vesicles, such as exosomes, are present in urine with reports of roles in intercellular signalling and diagnostic utility. However, the extent to which the concentration and characteristics of urinary vesicles are altered in albuminuric renal disease has not been well characterized. In this study, we examined the number and characteristics of extracellular vesicles in albuminuric urine.Vesicles were isolated from the urine of 32 patients with varying levels of albuminuria using ultracentrifugation and density gradient purification and were examined using nanoparticle tracking analysis, immunoblotting and transmission electron microscopy. The size profile of particles in these urine preparations was compared with albumin-containing solutions.Overall, there were no substantial differences in the number, or characteristics, of vesicles released into proteinuric urine. Analysis of albumin-containing solutions showed particles of exosome-like size, suggesting that such particles can mimic exosomes in standard nanoparticle tracking analysis. Albumin and IgG depletion of proteinuric urine resulted in a substantial reduction in the concentration of particles detected by nanoparticle tracking analysis.There was no increase in urinary vesicle concentration in patients with albuminuria. Furthermore, these results demonstrate the need for cautious interpretation of nanoparticle tracking analysis of vesicle concentration in biological fluids containing protein and for sophisticated preparative methods in vesicle purification from urine.

In the article by Lv et al. [1][1] published in the Journal of the American Society of Nephrology , the authors describe enhanced urinary exosomal release in experimental models of acute and chronic renal injury. They also describe increased exosomal release in patients with IgA nephropathy and a

BK viral infection is an important cause of renal transplant dysfunction and failure. Current strategies utilize surveillance for infection with DNA polymerase chain reaction assays and modulation of immunosuppression. Many viruses including polyomaviruses encode microRNAs (miRNAs). We have detected BK virus (BKV) encoded miRNAs in the blood of infected renal transplant recipients, and see a strong correlation between BKV encoded miRNA and BKV DNA in blood and a relationship between levels of bkv-miR-B1-5p and the presence of biopsy-proven BK viral nephropathy. Further research is needed to determine whether the detection of this and other virally encoded miRNAs may be useful in the diagnosis of active viral replication.

Dipeptidyl peptidase (DP) 6 and DP10 are non-enzyme members of the dipeptidyl peptidase IV family, which includes fibroblast activation protein, DP8, and DP9. DP6 and DP10 proteins have been shown to be critical components of voltage-gated potassium (Kv) channels important in determining cellular excitability. The aim of this paper was to review the research to date on DP6 and DP10 structure, expression, and functions. To date, the protein region responsible for modulating Kv4 channels has not been conclusively identified and the significance of the splice variants has not been resolved. Resolution of these issues will improve our overall knowledge of DP6 and DP10 and lead to a better understanding of their role in diseases, such as asthma and Alzheimer's disease.Clin Chem Lab Med 2009;47:262–7.