Synergistically Bifunctional Paramagnetic Separation Enables Efficient Isolation of Urine Extracellular Vesicles and Downstream Phosphoproteomic Analysis.

Extracellular vesicles (EVs) have emerged as important carriers for intercellular communication and biological sources for diagnosis and therapeutics. Low efficiency in EV isolation from biofluids, however, severely restricts their downstream characterization and analysis. Here, we introduced a novel strategy for EV isolation from urine for prostate cancer diagnosis using bifunctionalized magnetic beads through high affinity Ti(IV) ions and the insertion of a phospholipid derivative, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, into the EV membrane synergistically. We demonstrated its efficient isolation of EVs from urine samples with low contamination, high recovery (>80%), and short separation time (within 1 h), resulting in the identification of 36,262 unique EV peptides corresponding to 3302 unique proteins and 3233 unique phosphopeptides representing 1098 unique phosphoproteins using only 100 µL and 5 mL urine samples, respectively. Coupled with trapped ion mobility spectrometry and parallel accumulation-serial fragmentation for phosphosite-specific resolution, quantitative phosphoproteomics of urine samples from prostate cancer patients and healthy individuals revealed 121 upregulated phosphoproteins in cancer patients in contrast to the healthy group. These particular advantages indicate that the novel bifunctional material enables sensitive EV phosphoproteomic analysis for noninvasive biomarker screening and early cancer diagnosis.

Glass Fiber-Supported Hybrid Monolithic Spin Tip for Enrichment of Phosphopeptides from Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) are attracting increasing interest with their intriguing role in intercellular communications. Protein phosphorylation in EVs is of great importance for understanding intercellular signaling processes. However, the study of EV phosphoproteomics is impeded by their relatively low amount in limited clinical sample volumes, and it is necessary to have a sensitive and efficient enrichment method for EV phosphopeptides. Herein, a novel Ti(IV)-functionalized and glass fiber-supported hybrid monolithic spin tip, termed PhosTip, was prepared for enriching phosphopeptides from urinary EVs. Glass fiber as the stationary phase positions the hybrid monolith in a standard pipet tip and prevents the monolith from distortion during experiments. The preparation procedure for the new PhosTip is simple and time-saving. The hybrid monolithic PhosTip provides excellent enrichment efficiency of low-abundance phosphopeptides from cell digests and urinary EVs with minimum contamination and sample loss. Using the PhosTip, we demonstrate that 5373 and 336 unique phosphopeptides were identified from 100 and 1 µg of cell lysates, while 3919 and 217 unique phosphopeptides were successfully identified from 10 and 1 mL of urinary samples, respectively. The PhosTip was finally applied to enrich phosphopeptides in urine EVs from prostate cancer patients and healthy controls and quantify 118 up-regulated proteins with phosphosites in prostate cancer samples. These results demonstrated that the PhosTip could be a simple and convenient tool for enriching phosphopeptides from clinical samples and for broader applications in biomarker discovery.

Deconstruction of Heterogeneity of Size-Dependent Exosome Subpopulations from Human Urine by Profiling N-Glycoproteomics and Phosphoproteomics Simultaneously.

The heterogeneous populations of exosomes with distinct nanosize have impeded our understanding of their corresponding function as intercellular communication agents. Profiling signaling proteins packaged in each size-dependent subtype can disclose this heterogeneity of exosomes. Herein, new strategy was developed for deconstructing heterogeneity of distinct-size urine exosome subpopulations by profiling N-glycoproteomics and phosphoproteomics simultaneously. Two-dimension size exclusion liquid chromatography (SEC) was utilized to isolate large exosomes (L-Exo), medium exosomes (M-Exo), and small exosomes (S-Exo) from human urine samples. Then, hydrophilic carbonyl-functionalized magnetic zirconium-organic framework (CFMZOF) was developed as probe for capturing the two kinds of post-translational modification (PTM) peptides simultaneously. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with database search was used to characterize PTM protein contents. We identified 144 glycoproteins and 44 phosphoproteins from L-Exo, 156 glycoproteins, and 46 phosphoproteins from M-Exo and 134 glycoproteins and 10 phosphoproteins from S-Exo. The ratio of the proteins with simultaneous glycosylation and phosphorylation is 11%, 9%, and 3% in L-Exo, M-Exo, and S-Exo, respectively. Based on label-free quantification intensity results, both principal component analysis and Pearson's correlation coefficients indicate that distinct-size exosome subpopulations exist significant differences in PTM protein contents. Analysis of high abundance PTM proteins in each exosome subset reveals that the preferentially packaged PTM proteins in L-Exo, M-Exo, and S-Exo are associated with immune response, biological metabolism, and molecule transport processes, respectively. Our PTM proteomics study based on size-dependent exosome subtypes opens a new avenue for deconstructing the heterogeneity of exosomes.

Abatacept might increase bone mineral density at femoral neck for patients with rheumatoid arthritis in clinical practice: AIRTIGHT study.

We investigated the influence of abatacept (ABT) on bone mineral density (BMD) and bone metabolic markers (BMMs) in patients with rheumatoid arthritis (RA) compared to other biologic disease-modifying anti-rheumatic drugs (bDMARDs). This prospective, comparative, non-randomized study (the AIRTIGHT study; UMIN000005570) investigated the effects of ABT and other bDMARDs on bone metabolism. A total of 165 RA patients were divided into ABT (n = 50) and non-ABT (n = 115). We evaluated percentage changes in BMD (%ΔBMD) at the lumbar spine and femoral neck using dual-energy X-ray absorptiometry. Urinary levels of cross-linked N-telopeptide of type I collagen (uNTx) and bone-specific alkaline phosphatase (BAP) were used as markers of bone resorption and formation, respectively. No significant differences in 1-year completion rates were seen between ABT (64%) and non-ABT (72%; p = 0.387). The %ΔBMD at the femoral neck was significantly higher in the ABT group (0.97%) than in the non-ABT group (- 2.19%; p = 0.026). Whereas, no significant difference in %ΔBMD at the lumbar spine was observed between groups (ABT, - 0.40%; Non-ABT, - 1.67%; p = 0.524). No significant differences were observed in changes to uNTx or BAP. ABT treatment was significantly associated with increased BMD at the femoral neck (odds ratio (OR) 8.84; 95% CI 1.08-72.4; p = 0.04), and baseline lumbar osteoarthritis was significantly associated with BMD at the lumbar spine (OR 2.97; 95% CI 1.23-7.13; p = 0.02). The efficacy of ABT for increasing BMD at the femoral neck was superior to that of other bDMARDs. ABT may offer good efficacy for improving BMD at the femoral neck in patients with RA.

Can exercise suppress tumour growth in advanced prostate cancer patients with sclerotic bone metastases? A randomised, controlled study protocol examining feasibility, safety and efficacy.

INTRODUCTION: Exercise may positively alter tumour biology through numerous modulatory and regulatory mechanisms in response to a variety of modes and dosages, evidenced in preclinical models to date. Specifically, localised and systemic biochemical alterations produced during and following exercise may suppress tumour formation, growth and distribution by virtue of altered epigenetics and endocrine-paracrine activity. Given the impressive ability of targeted mechanical loading to interfere with metastasis-driven tumour formation in human osteolytic tumour cells, it is of equal interest to determine whether a similar effect is observed in sclerotic tumour cells. The study aims to (1) establish the feasibility and safety of a combined modular multimodal exercise programme with spinal isometric training in advanced prostate cancer patients with sclerotic bone metastases and (2) examine whether targeted and supervised exercise can suppress sclerotic tumour growth and activity in spinal metastases in humans. METHODS AND ANALYSIS: A single-blinded, two-armed, randomised, controlled and explorative phase I clinical trial combining spinal isometric training with a modular multimodal exercise programme in 40 men with advanced prostate cancer and stable sclerotic spinal metastases. Participants will be randomly assigned to (1) the exercise intervention or (2) usual medical care. The intervention arm will receive a 3-month, supervised and individually tailored modular multimodal exercise programme with spinal isometric training. Primary endpoints (feasibility and safety) and secondary endpoints (tumour morphology; biomarker activity; anthropometry; musculoskeletal health; adiposity; physical function; quality of life; anxiety; distress; fatigue; insomnia; physical activity levels) will be measured at baseline and following the intervention. Statistical analyses will include descriptive characteristics, t-tests, effect sizes and two-way (group × time) repeated-measures analysis of variance (or analysis of covariance) to examine differences between groups over time. The data-set will be primarily examined using an intention-to-treat approach with multiple imputations, followed by a secondary sensitivity analysis to ensure data robustness using a complete cases approach. ETHICS AND DISSEMINATION: Ethics approval was obtained from the Human Research Ethics Committee (HREC) of Edith Cowan University and the Sir Charles Gairdner and Osborne Park Health Care Group. If proven to be feasible and safe, this study will form the basis of future phase II and III trials in human patients with advanced cancer. To reach a maximum number of clinicians, practitioners, patients and scientists, outcomes will be disseminated through national and international clinical, conference and patient presentations, as well as publication in high-impact, peer-reviewed academic journals. TRIAL REGISTRATION NUMBER: ACTRN 12616000179437.

Highly selective manganese-doped zinc sulfide quantum dots based label free phosphorescent sensor for phosphopeptides in presence of zirconium (IV).

We report a room-temperature phosphorescence (RTP) sensor for phosphopeptides based on zirconium (IV)-modulated mercaptopropionic acid (MPA)-capped Mn-doped ZnS quantum dots (QDs). This sensor incorporates the advantages of the well-known Zr(4+)-phosphopeptide affinity pair and the RTP properties of doped QDs. The RTP of Mn-doped ZnS QDs capped with MPA can be effectively quenched by Zr(4+). The high affinity of phosphopeptides to Zr(4+) enables the dissociation of the ion from the surface of MPA-capped ZnS QDs, thereby forming a stable complex with phosphopeptides in the solution, and recovering the RTP of the QDs. The Zr(4+)-induced RTP quenching and subsequent phosphopeptide-induced RTP recovery for MPA-capped ZnS QDs provide a solid basis for the present RTP sensor based on QDs for the detection of phosphopeptides. The detection limit for phosphopeptides is 0.9ngmL(-1), the relative standard deviations is 2.5%, and the recovery of urine and serum samples with phosphopeptides addition rangs from 96% to 105% at optimal conditions. The proposed method was successfully applied to biological fluids and obtained satisfactory results.

Relationships between age-related biochemical markers of bone turnover and OPG, TGF-ß1 and TGF-ß2 in native Chinese women.

Osteoprotegerin (OPG), transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 are cytokines closely associated with bone metabolism. However, their association with bone turnover markers in native Chinese women remains unknown. The study aims to investigate the relationship between bone metabolism related cytokines including OPG, TGF-ß1, TGF-ß2 and bone turnover markers in native Chinese women. The cross-sectional study was conducted on 691 healthy Chinese women (20-80 years old). Levels of OPG, TGF-ß1, TGF-ß2, serum bone-specific alkaline phosphatase (BAP), osteocalcin (OC), cross-linked N-terminal telopeptides of type I collagen (sNTX), cross-linked C-terminal telopeptides of type I collagen (sCTX), urinary NTX (uNTX), urinary CTX (uCTX) and total urinary deoxypyridinoline (uDPD) were determined. The present study showed that OPG and TGF-ß2 had positive correlation with BAP, OC, uNTX, uCTX and uDPD, while TGF-ß1 showed negative correlation with BAP, OC, sCTX, uNTX and uCTX, and most of the coefficients of partial correlation remained significant after adjustments for age and body mass index (BMI). Multiple linear regression stepwise analysis showed that OPG and TGF-ß2 were positive determinative factors for BAP, sCTX, uNTX and uCTX, which could explain 0.6-16.6% of the variation in these markers. TGF-ß1 was a negative determinative factor for BAP, OC, sCTX and uCTX, which could explain 0.7-7.3% of the variation in these markers. This study suggested that measuring bone turnover indicators and serum cytokines simultaneously might help evaluating changes in bone turnover rate caused by aging or menopause in women.

Identification of free phosphopeptides in different biological fluids by a mass spectrometry approach.

Human body fluids have been rediscovered in the post-genomic era as a great source of biological markers and perhaps as source of potential biomarkers of disease. Recently, it has been found that not only proteins but also peptides and their modifications can be indicators of early pathogenic processes. This paper reports the identification of free phosphopeptides in human fluids using an improved IMAC strategy coupled to iterative mass spectrometry-based scanning techniques (neutral loss, precursor ion, multiple reaction monitoring). Many peptides were detected in the enriched extract samples when submitted to the MS-integrated strategy, whereas they were not detected in the initial extract samples. The combination of the IMAC-modified protocol with selective "precursor ion" and constant "neutral loss" triple quadrupole scan modes confers a high sensitivity on the analysis, allowing rapid phosphopeptide identification and characterization, even at low concentrations. To the best of our knowledge this work represents the first report exclusively focused on the detection of free phosphorylated peptides in biological fluids.