Identification and validation of N-acetylputrescine in combination with non-canonical clinical features as a Parkinson's disease biomarker panel.

Parkinson's disease is a progressive neurodegenerative disorder in which loss of dopaminergic neurons in the substantia nigra results in a clinically heterogeneous group with variable motor and non-motor symptoms with a degree of misdiagnosis. Only 3-25% of sporadic Parkinson's patients present with genetic abnormalities that could represent a risk factor, thus environmental, metabolic, and other unknown causes contribute to the pathogenesis of Parkinson's disease, which highlights the critical need for biomarkers. In the present study, we prospectively collected and analyzed plasma samples from 194 Parkinson's disease patients and 197 age-matched non-diseased controls. N-acetyl putrescine (NAP) in combination with sense of smell (B-SIT), depression/anxiety (HADS), and acting out dreams (RBD1Q) clinical measurements demonstrated combined diagnostic utility. NAP was increased by 28% in Parkinsons disease patients and exhibited an AUC of 0.72 as well as an OR of 4.79. The clinical and NAP panel demonstrated an area under the curve, AUC = 0.9 and an OR of 20.4. The assessed diagnostic panel demonstrates combinatorial utility in diagnosing Parkinson's disease, allowing for an integrated interpretation of disease pathophysiology and highlighting the use of multi-tiered panels in neurological disease diagnosis.

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.

Diablo ubiquitination analysis by sandwich immunoassay.

Ubiquitin plays an essential role in modulating protein function, and deregulation of the ubiquitin system leads to the development of a variety of human diseases. E3 Ubiquitin ligases that mediate ubiquitination and degradation of caspases prevent apoptosis, and as such belong to the family of inhibitors of apoptosis proteins (IAPs). Diablo is a substrate of IAPs but also a negative regulator of IAPs in apoptotic pathway as it blocks the interaction between IAPs and caspases. In efforts to identify IAP inhibitors, we developed sandwich immunoassays in conjunction with an electrochemical luminescence (ECL) platform for quantitation of total Diablo, ubiquitinated Diablo, and ubiquitinated Diablo with K48-specific linkage. The assay panel detects Diablo ubiquitination level changes in the presence of IAP inhibitor or proteasome inhibitor, demonstrating its potential as a cost-efficient high-throughput method for drug discovery involving IAP ubiquitination cascade. The ECL based sandwich assay panel performance was subsequently evaluated for precision, linearity, and limit of quantification.

Molecular understanding of sterically controlled compound release through an engineered channel protein (FhuA).

BACKGROUND: Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Delta1-160; Onaca et al., 2008). Compound fluxes within the FhuA Delta1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems. RESULTS: In total, 12 FhuA Delta1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A) six FhuA Delta1-160 variants in which one of the six lysines in the interior of FhuA Delta1-160 was substituted to alanine and Subset B) six FhuA Delta1-160 variants in which only one lysine inside the barrel was not changed to alanine. Translocation efficiencies were quantified with the colorimetric TMB (3,3',5,5'-tetramethylbenzidine) detection system employing horseradish peroxidase (HRP). Investigation of the six subset A variants identified position K556A as sterically important. The K556A substitution increases TMB diffusion from 15 to 97 [nM]/s and reaches nearly the TMB diffusion value of the unlabeled FhuA Delta1-160 (102 [nM]/s). The prominent role of position K556 is confirmed by the corresponding subset B variant which contains only the K556 lysine in the interior of the barrel. Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s). A first B-factor analysis based on MD simulations confirmed that position K556 is the least fluctuating lysine among the six in the channel interior of FhuA Delta1-160 and therefore well suited for controlling compound fluxes through steric hindrance. CONCLUSIONS: A FhuA Delta1-160 based reduction triggered release system has been shown to control the compound flux by the presence of only one inner channel sterical hindrance based on 3-(2-pyridyldithio)propionic-acid labeling (amino acid position K556). As a consequence, the release kinetic can be modulated by introducing an opportune number of hindrances. The FhuA Delta1-160 channel embedded in liposomes can be advanced to a universal and compound independent release system which allows a size selective compound release through rationally re-engineered channels.

Rapid and efficient method for the size separation of homogeneous fluorescein-encapsulating liposomes.

Liposomes are colloidal structures formed by the self-assembly of lipid molecules in solution into spherical, self-closed structures through their amphiphilic properties. All liposome preparation protocols reported consist of several steps of preparation, homogenization, and purification, which are labor-intensive, arduous, and lengthy to execute. In this work, a new procedure has been developed to reduce the time of the postrehydration sizing of liposomes from multilamellar vesicles, while improving the uniformity of the resulting liposomes produced and achieving high encapsulation efficiencies. For the homogenization step, the typically used method of filter extrusion was substituted by centrifugation. Purification of liposomes to eliminate nonencapsulated molecules and lipids is routinely carried out via gel permeation chromatography, an extremely lengthy procedure, and in the method we report, this lengthy step was replaced by the use of molecular-weight cut-off filters. Using this novel method, large unilamellar vesicles were produced and the time required, postrehydration, was dramatically reduced from almost 48 to less than 2 hours, with a highly uniformly sized population of liposomes being produced-the homogeneity of the liposome population achieved using our method was 99%, as compared to 88% attained by using the traditional method of production. We have used this approach to encapsulate fluorescein isothiocyanate (FITC), and 160,000 FITC molecules were encapsulated and the liposomes were demonstrated to be stable for at least 10 weeks at 4 degrees C.