The Next Frontier: Translational Development of Ubiquitination, SUMOylation, and NEDDylation in Cancer.

Post-translational modifications of proteins ensure optimized cellular processes, including proteostasis, regulated signaling, cell survival, and stress adaptation to maintain a balanced homeostatic state. Abnormal post-translational modifications are associated with cellular dysfunction and the occurrence of life-threatening diseases, such as cancer and neurodegenerative diseases. Therefore, some of the frequently seen protein modifications have been used as disease markers, while others are targeted for developing specific therapies. The ubiquitin and ubiquitin-like post-translational modifiers, namely, small ubiquitin-like modifier (SUMO) and neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8), share several features, such as protein structures, enzymatic cascades mediating the conjugation process, and targeted amino acid residues. Alterations in the regulatory mechanisms lead to aberrations in biological processes during tumorigenesis, including the regulation of tumor metabolism, immunological modulation of the tumor microenvironment, and cancer stem cell stemness, besides many more. Novel insights into ubiquitin and ubiquitin-like pathways involved in cancer biology reveal a potential interplay between ubiquitination, SUMOylation, and NEDDylation. This review outlines the current understandings of the regulatory mechanisms and assay capabilities of ubiquitination, SUMOylation, and NEDDylation. It will further highlight the role of ubiquitination, SUMOylation, and NEDDylation in tumorigenesis.

Multiplexed LC-MS/MS analysis of methylsuccinic acid, ethylmalonic acid, and glutaric acid in plasma and urine.

Low molecular-mass aliphatic carboxylic acids are critically important for intermediate metabolism and may serve as important biomarkers for metabolic homeostasis. Here in, we focused on multiplexed method development of aliphatic carboxylic analytes, including methylsuccinic acid (MSA), ethylmalonic acid (EMA), and glutaric acid (GA). Also assessed was their utility in a population's health as well as metabolic disease screening in both plasma and urine matrices. MSA, EMA, and GA are constitutional isomers of dicarboxylic acid with high polarity and poor ionization efficiency, resulting in such challenges as poor signal intensity and retention, particularly in reversed-phase liquid chromatography with electrospray mass spectrometry (RP-LC-ESI-MS/MS). Derivatization using n-butanol was performed in the sample preparation to enhance the signal intensity accompanied with a positive charge from ionization in complicated biomatrices as well as to improve the separation of these isomers with optimal retention. Fit-for-purpose method validation results demonstrated quantitative ranges for MSA/EMA/GA from 5/10/20 ng/mL to 400 ng/mL in plasma analysis, and 100/200/100 ng/mL to 5000/10000/5000 ng/mL in urine analysis. This validated method demonstrates future utility when exploring population health analysis and biomarker development in metabolic diseases.

Development and validation of an immunoassay for quantification of NCAM-1 in human plasma.

BACKGROUND: Neural Cell Adhesion Molecule 1 (NCAM-1), a multifunctional member of the immunoglobulin superfamily, is expressed on the surface of neurons, glia, skeletal muscle, and natural killer cells. NCAM-1 has been implicated as having a role in cell-cell adhesion, involved in development of the nervous system, and for cells involved in the expansion of T cells and dendritic cells which play an important role in immune surveillance. Sensitive and specific methods to quantify non-surface bound NCAM-1 are not available. METHOD: A sandwich ligand binding assay was developed for quantification of NCAM-1 in plasma and validated using an electro-chemiluminescent (ECL) technology. RESULTS: The data presented here demonstrated that the validated method met all prespecified criteria for precision, linearity, and accuracy in the range of 62.5 ng/mL to 4000.0 ng/mL, the range believed to be most relevant for plasma. The bioanalytical validation of the assay established the inter-assay coefficient of variation <8 % for calibration points, <2 % for high quality control (HQC), <8 % for medium quality control (MQC) and <19 % for low quality control (LQC) samples. Purified NCAM-1 spike-recovery experiment in plasma was used to determine assay accuracy; nominal concentrations (%) of NCAM-1 ranged from 91 % to 112 % for high and low spike level, respectively. Assay performance was subsequently evaluated for parallelism, selectivity, interference, and stability. CONCLUSION: NCAM-1 assay has been developed and validated in human plasma and met all assay validation parameters pre-determined during development. Clinical testing of human plasma samples indicated that NCAM-1 does not seem to be influenced by age and was slightly influenced by gender. NCAM-1 assay has potential to be used as a biomarker assay once the assay is subjected to appropriate clinical assessment and diagnostic thresholds are established.

'Real-life experience': recurrence rate at 3 years with Hexvix® photodynamic diagnosis-assisted TURBT compared with good quality white light TURBT in new NMIBC-a prospective controlled study.

PURPOSE: To compare the recurrence rate at 3 years (RR-3y) for non-muscle invasive bladder cancer (NMIBC) between good quality (GQ) PDD-TURBT and GQWL-TURBT where PDD is used in routine practice for all new tumours. METHODS: All new, consecutive, NMIBC that received "good quality" criteria first TURBT across a university hospital service were prospectively recruited to this study over a 4-year period. Data were prospectively collected on all WL-TURBTs performed in 2007/8 and compared with PDD-TURBT from 2009/10. Only resection meeting strict "good quality criteria" were included from each cohort to control for resection quality, then cases were further matched 1:1 based on demographic and pathological criteria. The primary outcome was overall and risk group-specific recurrence rate at 3 years. RESULTS: Of 808 patients recruited, 345 had GQ-TURBT for NMIBC and were included. RR-3y was significantly less for GQ-PDD overall [RR-3y: GQ-PDD: 57/146 (39.0%), GQ-WL: 72/135 (53.3%) OR = 0.56 (0.35-0.90) p = 0.02] and on a 1:1 matched pair basis [RR GQ-PDD: 29/118 (24.6) vs. 59/118 (50.0) OR 0.33 (0.19-0.57) p < 0.001)]. Benefit was most marked in high-risk patients: RR-3y in high-risk patients treated with GQ-PDD was 25/48 (52.1%) vs. 28/35 (80%) for GQ-WL [OR 0.27 (0.10-0.74) p = 0.01]. CONCLUSION: When adopted for all new bladder tumour resections in routine practice, PDD appears to be associated with significantly reduced recurrence rates at 3 years in our "real life" experience, particularly in high-risk patients.