Fluorescent Phenotyping of Blood Cells Using a Differential Sensing Strategy: Differentiating Physiological Aging Stages and Neuro-Degenerative Disease Drugs.

Blood continually contributes to the maintenance of homeostasis of the body and contains information regarding the health state of an individual. However, current hematological analyses predominantly rely on a limited number of CD markers and morphological analysis. In this work, differentially sensitive fluorescent compounds based on TCF scaffolds are introduced that are designed for fluorescent phenotyping of blood. Depending on their structures, TCF compounds displayed varied responses to reactive oxygen species, biothiols, redox-related biomolecules, and hemoglobin, which are the primary influential factors within blood. Contrary to conventional CD marker-based analysis, this unbiased fluorescent phenotyping method produces diverse fingerprints of the health state. Precise discrimination of blood samples from 37 mice was demonstrated based on their developmental stages, ranging from 10 to 19 weeks of age. Additionally, this fluorescent phenotyping method enabled the differentiation between drugs with distinct targets, serving as a simple yet potent tool for pharmacological analysis to understand the mode of action of various drugs.

Single-cell DNA amplicon sequencing reveals clonal heterogeneity and evolution in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia that is most frequent in children and is characterized by the presence of few chromosomal rearrangements and 10 to 20 somatic mutations in protein-coding regions at diagnosis. The majority of T-ALL cases harbor activating mutations in NOTCH1 together with mutations in genes implicated in kinase signaling, transcriptional regulation, or protein translation. To obtain more insight in the level of clonal heterogeneity at diagnosis and during treatment, we used single-cell targeted DNA sequencing with the Tapestri platform. We designed a custom ALL panel and obtained accurate single-nucleotide variant and small insertion-deletion mutation calling for 305 amplicons covering 110 genes in about 4400 cells per sample and time point. A total of 108 188 cells were analyzed for 25 samples of 8 T-ALL patients. We typically observed a major clone at diagnosis (>35% of the cells) accompanied by several minor clones of which some were less than 1% of the total number of cells. Four patients had >2 NOTCH1 mutations, some of which present in minor clones, indicating a strong pressure to acquire NOTCH1 mutations in developing T-ALL cells. By analyzing longitudinal samples, we detected the presence and clonal nature of residual leukemic cells and clones with a minor presence at diagnosis that evolved to clinically relevant major clones at later disease stages. Therefore, single-cell DNA amplicon sequencing is a sensitive assay to detect clonal architecture and evolution in T-ALL.

Blood transcriptome analysis revealed the immune changes and immunological adaptation of wildness training giant pandas.

The giant panda (Ailuropoda melanoleuca) is a global flagship species for biodiversity conservation. As the time for captive giant pandas to be released into the wild matures, wildness training is provided to allow adaptation to their natural environment. It is assumed that changes in the immune system would be integral in this adaptation from captive to wild, where many more pathogens would be encountered in their natural habitats. Therefore, this study aims to determine the expression changes of immune-related genes and their potential as immunoassay markers for adaptation monitoring in wildness training giant pandas, and then to understand the adaptation strategy of wildness training giant pandas to the wild environment, thereby improving the success rate of panda reintroduction. We obtained 300 differentially expressed genes (DEGs) by RNA-seq, with 239 up-regulated and 61 down-regulated DEGs in wildness training giant pandas compared to captive pandas. Functional enrichment analysis indicated that up-regulated DEGs were enriched in several immune-related terms and pathways. There were 21 immune-related DEGs, in which most of them were up-regulated in wildness training giant pandas, including several critical innate and cellular immune genes. IL1R2 was the most significantly up-regulated gene and is a signature of homeostasis within the immune system. In the protein-protein interaction (PPI) analysis, CXCL8, CXCL10, and CCL5 were identified as the hub immune genes. Our results suggested that wildness training giant pandas have stronger innate and cellular immunity than captive giant pandas, and we proposed that a gene set of CXCL8, CXCL10, CCL5, CD3D, NFKBIA, TBX21, IL12RB2, and IL1R2 may serve as potential immunoassay markers to monitor and assess the immune status of wildness training giant pandas. Our study offers the first insight into immune alterations of wildness training giant pandas, paving the way for monitoring and evaluating the immune status of giant pandas when reintroducing them into the wild.

Fluoride exposure and blood cell markers of inflammation in children and adolescents in the United States: NHANES, 2013-2016.

BACKGROUND: Ingestion of fluoride in drinking water has been shown to result in increased cellular markers of inflammation in rodent models. However, the approximately 5-10 × increase in water fluoride concentrations required in rat and mouse models to obtain plasma fluoride concentrations similar to those found in humans has made relevant comparisons of animal to human studies difficult to assess. As an increased white blood cell count (WBC) is a marker of inflammation in humans, we used available NHANES survey data to assess the associations between plasma fluoride levels in the U.S. and blood cell counts children and adolescents.   METHODS: Multiple linear regressions were done to determine the association of blood cell counts and plasma fluoride in publicly available NHANES survey data from the 2013-2014 and 2015-2016 cycles. Plasma fluoride concentration measurements were available only for children aged 6 to 19, inclusive, and therefore this subpopulation was used for all analyses. Covariate predictors along with plasma fluoride were age, ethnicity, gender, and Body Mass Index (BMI).  RESULTS: Plasma fluoride was significantly positively associated with water fluoride, total WBC count, segmented neutrophils, and monocytes, and negatively associated with red blood cell count when adjusted for age, gender and BMI. CONCLUSION: Our finding that neutrophils and monocytes are associated with higher plasma fluoride in U.S. children and adolescents is consistent with animal data showing fluoride related effects of increased inflammation. These findings suggest the importance of further studies to assess potential mechanisms that are involved in absorption and filtration of ingested fluoride, particularly in tissues and organs such as the small intestine, liver and kidney.

Neutrophil-Selective Fluorescent Probe Development through Metabolism-Oriented Live-Cell Distinction.

Human neutrophils are the most abundant leukocytes and have been considered as the first line of defence in the innate immune system. Selective imaging of live neutrophils will facilitate the in situ study of neutrophils in infection or inflammation events as well as clinical diagnosis. However, small-molecule-based probes for the discrimination of live neutrophils among different granulocytes in human blood have yet to be reported. Herein, we report the first fluorescent probe NeutropG for the specific distinction and imaging of active neutrophils. The selective staining mechanism of NeutropG is elucidated as metabolism-oriented live-cell distinction (MOLD) through lipid droplet biogenesis with the help of ACSL and DGAT. Finally, NeutropG is applied to accurately quantify neutrophil levels in fresh blood samples by showing a high correlation with the current clinical method.

Smoking, blood cells and myeloproliferative neoplasms: meta-analysis and Mendelian randomization of 2·3 million people.

Meta-analyses and Mendelian randomization (MR) may clarify the associations of smoking, blood cells and myeloproliferative neoplasms (MPN). We investigated the association of smoking with blood cells in the Danish General Suburban Population Study (GESUS, n = 11 083), by meta-analyses (including GESUS) of 92 studies (n = 531 741) and MR of smoking variant CHRNA3 (rs1051730[A]) in UK Biobank, and with MPN in a meta-analysis of six studies (n (total/cases):1 425 529/2187), totalling 2 307 745 participants. In the meta-analysis the random-effects standardized mean difference (SMD) in current smokers versus non-smokers was 0·82 (0·75-0·89, P = 2·0 * 10-108 ) for leukocytes, 0·09 (-0·02 to 0·21, P = 0·12) for erythrocytes, 0·53 (0·42-0·64, P = 8·0 * 10-22 ) for haematocrit, 0·42 (0·34-0·51, P = 7·1 * 10-21 ) for haemoglobin, 0·19 (0·08-0·31, P = 1·2 * 10-3 ) for mean corpuscular haemoglobin (MCH), 0·29 (0·19-0·39, P = 1·6 * 10-8 ) for mean corpuscular volume (MCV), and 0·04 (-0·04 to 0·13, P = 0·34) for platelets with trends for ever/ex-/current smokers, light/heavy smokers and female/male smokers. Analyses presented high heterogeneity but low publication bias. Per allele in CHRNA3, cigarettes per day in current smokers was associated with increased blood cell counts (leukocytes, neutrophils), MCH, red cell distribution width (RDW) and MCV. The pooled fixed-effects odds ratio for MPN was 1·44 [95% confidence interval (CI): 1·33-1·56; P = 1·8 * 10-19 ; I2  = 0%] in current smokers, 1·29 (1·15-1·44; P = 8·0 * 10-6 ; I2  = 0%) in ex-smokers, 1·49 (1·26-1·77; P = 4·4 * 10-6 ; I2  = 0%) in light smokers and 2·04 (1·74-2·39, P = 2·3 * 10-18 ; I2  = 51%) in heavy smokers compared with non-smokers. Smoking is observationally and genetically associated with increased leukocyte counts and red blood cell indices (MCH, MCV, RDW) and observationally with risk of MPN in current and ex-smokers versus non/never-smokers.

Isolation of exosomes from whole blood by integrating acoustics and microfluidics.

Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.

Highly Bioavailable Forms of Curcumin and Promising Avenues for Curcumin-Based Research and Application: A Review.

Curcumin exerts a wide range of beneficial physiological and pharmacological activities, including antioxidant, anti-amyloid, anti-inflammatory, anti-microbial, anti-neoplastic, immune-modulating, metabolism regulating, anti-depressant, neuroprotective and tissue protective effects. However, its poor solubility and poor absorption in the free form in the gastrointestinal tract and its rapid biotransformation to inactive metabolites greatly limit its utility as a health-promoting agent and dietary supplement. Recent advances in micro- and nano-formulations of curcumin with greatly enhanced absorption resulting in desirable blood levels of the active forms of curcumin now make it possible to address a wide range of potential applications, including pain management, and as tissue protective. Using these forms of highly bioavailable curcumin now enable a broad spectrum of appropriate studies to be conducted. This review discusses the formulations designed to enhance bioavailability, metabolism of curcumin, relationships between solubility and particle size relative to bioavailability, human pharmacokinetic studies involving formulated curcumin products, the widely used but inappropriate practice of hydrolyzing plasma samples for quantification of blood curcumin, current applications of curcumin and its metabolites and promising directions for health maintenance and applications.

Genome-wide association study of peripheral blood DNA methylation and conventional mammographic density measures.

Age- and body mass index (BMI)-adjusted mammographic density is one of the strongest breast cancer risk factors. DNA methylation is a molecular mechanism that could underlie inter-individual variation in mammographic density. We aimed to investigate the association between breast cancer risk-predicting mammographic density measures and blood DNA methylation. For 436 women from the Australian Mammographic Density Twins and Sisters Study and 591 women from the Melbourne Collaborative Cohort Study, mammographic density (dense area, nondense area and percentage dense area) defined by the conventional brightness threshold was measured using the CUMULUS software, and peripheral blood DNA methylation was measured using the HumanMethylation450 (HM450) BeadChip assay. Associations between DNA methylation at >400,000 sites and mammographic density measures adjusted for age and BMI were assessed within each cohort and pooled using fixed-effect meta-analysis. Associations with methylation at genetic loci known to be associated with mammographic density were also examined. We found no genome-wide significant (p < 10-7 ) association for any mammographic density measure from the meta-analysis, or from the cohort-specific analyses. None of the 299 methylation sites located at genetic loci associated with mammographic density was associated with any mammographic density measure after adjusting for multiple testing (all p > 0.05/299 = 1.7 × 10-4 ). In summary, our study did not find evidence for associations between blood DNA methylation, as measured by the HM450 assay, and conventional mammographic density measures that predict breast cancer risk.

Ultrasensitive Detection of Exosomes by Target-Triggered Three-Dimensional DNA Walking Machine and Exonuclease III-Assisted Electrochemical Ratiometric Biosensing.

Exosomes are membrane-enclosed phospholipid extracellular vesicles. In spite of their great promise as noninvasive biomarkers for cancer diagnosis, sensitive detection of exosomes is still challenging. Herein, the detection of exosomes was changed to the detection of DNA after recognition of exosomes with its aptamers. CD63 aptamer and EpCAM aptamer were used for the detection of MCF-7 cell-secreted exosome. The recognition process was amplified through the movements of a three-dimensional DNA walker. And then, Exonuclease III- assisted electrochemical ratiometric sensor was applied for further signal amplification. Under optimal conditions, the detection limit of 1.3 × 104 particles/mL was obtained with excellent selectivity. Furthermore, clinical application test for the detection of exosomes in human serum was also verified.

On the design of deterministic dielectrophoresis for continuous separation of circulating tumor cells from peripheral blood cells.

Detection and analysis of circulating tumor cells (CTCs) have emerged as a promising way to diagnose cancer, study its cellular mechanism, and test or develop potential treatments. However, the rarity of CTCs among peripheral blood cells is a big challenge toward CTC detection. In addition, in cases where there is similar size range between certain types of CTCs (e.g. breast cancer cells) and white blood cells (WBCs), high-resolution techniques are needed. In the present work, we propose a deterministic dielectrophoresis (DEP) method that combines the concept of deterministic lateral displacement (DLD) and insulator-based dielectrophoresis (iDEP) techniques that rely on physical markers such as size and dielectric properties to differentiate different type of cells. The proposed deterministic DEP technology takes advantage of frequency-controlled AC electric field for continuous separation of CTCs from peripheral blood cells. Utilizing numerical modeling, different aspects of coupled DLD-DEP design such as the required applied voltages, velocities, and geometrical parameters of DLD arrays of microposts are investigated. Regarding the inevitable difference and uncertainty ranges for the reported crossover frequencies of cells, a comprehensive analysis is conducted on applied electric field frequency as design's determinant factor. Deterministic DEP design provides continuous sorting of CTCs from WBCs even with similar size and has the future potential for high throughput and efficiency.

Increasing DNA Isolation Performance from Blood Cells Using a Modified Commercial Low-Cost Reagent.

BACKGROUND: Genomic studies facilitate comprehension of the pathophysiology, diagnosis, and treatment of chronic diseases. Such studies require sufficient and good quality DNA isolated from a large number of blood samples. This study attempts to obtain a high-quality genomic DNA isolated from a large number of blood samples using a simple and cheap method. METHODS: The EasyPure® Genomic DNA Kit (Transgen Biotech) was modified to increase the amount of DNA recovery: a few steps and two additional column elutions were added to the original manufacturer´s procedure. RESULTS: The amount of DNA isolated from frozen blood samples increased by an average of 56%. Its 260/280 ratio and electrophoretic mobility properties make it suitable for genomic studies. CONCLUSIONS: A relatively low-cost commercial column and a simple modification of the manufacturer´s protocol, provided a simple and cheap procedure to isolate high-quality DNA from a large number of blood samples suitable for genomic studies.

Transcriptome of blood cells as a reservoir of cardiovascular biomarkers.

Biomarkers have the potential to move personalized medicine a step forward for the patients' benefit. Blood is a non-invasive source of biomarkers. The cell-free compartment of the blood has traditionally been used to search for biomarkers and currently used cardiovascular biomarkers are mainly measured in plasma or serum fractions. While most of these biomarkers are proteins, very few belong to the RNA family due to methodological constraints inherent to the assessment of RNAs. Over the past decade, technological developments have overcome these constraints and have allowed the assessment of RNAs in blood cells. Thus, the search for novel cardiovascular biomarkers among RNAs expressed in blood cells has emerged. This review highlights the potential of the transcriptome of blood cells to be used as a reservoir of novel cardiovascular biomarkers. First, the benefit of using biomarkers for personalized medicine is introduced. The focus is on the RNA family of biomarkers, both coding and non-coding. The value of systems-based approaches for biomarker discovery is discussed. Then, the current knowledge of the use of blood cells' transcriptome for biomarker discovery is reviewed. Recent technological developments that have facilitated the study of the transcriptome of blood cells are presented. Further axes of developments are proposed to bring RNA-based biomarkers to clinical application. Lastly, some directions for future work are presented.

Design of a Single-Layer Microchannel for Continuous Sheathless Single-Stream Particle Inertial Focusing.

High-throughput, high-precision single-stream focusing of microparticles has a potentially wide range of applications in biochemical analysis and clinical diagnosis. In this work, we develop a sheathless three-dimensional (3D) particle-focusing method in a single-layer microchannel. This novel microchannel consists of periodic high-aspect-ratio curved channels and straight channels. The proposed method takes advantage of both the curved channels, which induce Dean flow to promote particle migration, and straight channels, which suppress the remaining stirring effects of Dean flow to stabilize the achieved particle focusing. The 3D particle focusing is demonstrated experimentally, and the mechanism is analyzed theoretically. The effects of flow rate, particle size, and cycle number on the focusing performance were also investigated. The experimental results demonstrate that polystyrene particles with diameters of 5-20 µm can be focused into a 3D single file within seven channel cycles, with the focusing accuracy up to 98.5% and focusing rate up to 98.97%. The focusing throughput could reach up to ∼105 counts/min. Furthermore, its applicability to biological cells is also demonstrated by 3D focusing of HeLa and melanoma cells and bovine blood cells in the proposed microchannel. The proposed sheathless passive focusing scheme, featuring a simple channel structure, small footprint (9 mm × 1.2 mm), compact layout, and uncomplicated fabrication procedure, holds great promise as an efficient 3D focusing unit for the development of next-generation on-chip flow cytometry.

Bioconversion of duck blood cell: process optimization of hydrolytic conditions and peptide hydrolysate characterization.

BACKGROUND: As the protein-laden by-product, red blood cells (RBCs) from poultry blood is a potential source of protein used as food and feed ingredient. However, RBC was currently underutilized. Therefore, it is an urgent need to develop feasible and cost-effective methods for converting poultry waste into nutritional and functional products. RESULTS: To take full advantage of this poultry waste, peptide hydrolysate was produced by deep controllable bioconversion of RBC, by means of synergistic combination of neutrase and flavourzyme. In this work, the functional properties and antioxidant activity of peptide hydrolysate were also characterized. The degree of hydrolysis (DH) was optimized using response surface methodology, and optimal hydrolysis conditions were found to be: temperature 51 °C, substrate concentration 14% (w/v), initial pH 7.0, and time 7.5 h. The red blood cell hydrolysate (RBCH) obtained not only possessed plentiful small peptides (< 3 kDa, 68.14%), but also was abundant in essential amino acids, accounting for over 50% of total amino acids. In addition to its excellent solubility (> 80%), emulsifying and foaming properties, RBCH also exhibited notable antioxidant activities, such as DPPH (2,2-diphenyl- 1-picrylhydrazyl) radical-scavenging activity (IC50, 4.16 mg/mL), reducing power, metal chelating ability and inhibiting lipid peroxidation. CONCLUSIONS: RBCH enriched in small peptides has the potential to be a new food additive with outstanding functional and antioxidant properties, and a process was established for converting poultry waste into peptide hydrolysate using neutrase and flavourzyme.

Social defeat stress exacerbates the blood abnormalities in Opisthorchis felineus-infected mice.

A model of chronic opisthorchiasis combined with social stress is examined; this situation is more likely for humans and animals than a separate impact of the infectious factor. For this purpose, we evaluated the effects of Opisthorchis felineus ("OP" group) and 30-day social stress (confrontations between males, "SS" group) alone and in combination ("OP + SS" group) in inbred C57BL/6 male mice and compared these effects according to the parameters listed below. The animals exposed to neither factor formed the control group ("CON"). All animals were assayed for blood biochemical parameters, changes in blood cell composition, and pattern of bone marrow hematopoiesis. By the end of the experiment, we have observed crucial effects of the two factors on the blood and liver of "OP" and "OP + SS". Eosinophil and basophil counts increased and relative segmented neutrophil and monocyte counts decreased in "OP + SS" mice on the background of activated myelopoiesis, mainly determined by social stress. Despite depressed erythropoiesis, "OP" mice displayed no changes in the relative peripheral erythrocyte counts. On the contrary, social stress, which stimulated erythropoiesis in "SS" and "OP + SS" mice, was accompanied by a decrease in the relative erythrocyte counts and hematocrit. Hepatosplenomegaly was observed on the background of these two impacts. Changes in transaminase (ALT and AST) and alkaline phosphatase activities as well as an increase in cholesterol and product of lipid peroxidation suggest a pronounced destruction of the liver. Altogether, social stress exacerbates many of the assayed blood parameters in the mice infected with the liver fluke.

Prioritizing and selecting likely novel miRNAs from NGS data.

Small non-coding RNAs play a key role in many physiological and pathological processes. Since 2004, miRNA sequences have been catalogued in miRBase, which is currently in its 21st version. We investigated sequence and structural features of miRNAs annotated in the miRBase and compared them between different versions of this reference database. We have identified that the two most recent releases (v20 and v21) are influenced by next-generation sequencing based miRNA predictions and show significant deviation from miRNAs discovered prior to the high-throughput profiling period. From the analysis of miRBase, we derived a set of key characteristics to predict new miRNAs and applied the implemented algorithm to evaluate novel blood-borne miRNA candidates. We carried out 705 individual whole miRNA sequencings of blood cells and collected a total of 9.7 billion reads. Using miRDeep2 we initially predicted 1452 potentially novel miRNAs. After excluding false positives, 518 candidates remained. These novel candidates were ranked according to their distance to the features in the early miRBase versions allowing for an easier selection of a subset of putative miRNAs for validation. Selected candidates were successfully validated by qRT-PCR and northern blotting. In addition, we implemented a web-server for ranking potential miRNA candidates, which is available at:www.ccb.uni-saarland.de/novomirank.

Circular RNAs in Blood.

Recent advances in RNA sequencing and bioinformatic analysis have allowed the development of a new research field: circular RNAs (circRNAs). These members of the non-coding transcriptome are generated by backsplicing, which results in a covalently closed, single-stranded RNA molecule. To date, thousands of circRNAs have been identified in different human cell types. CircRNAs are evolutionarily conserved, highly stable, cell-/developmental stage-specific and have longer half-lives compared with linear RNAs. Interestingly, different studies have demonstrated that circRNAs are abundantly expressed in the bloodstream. In this chapter, we review the current knowledge of circRNA biology in blood cells and the cell-free compartment, including extracellular vesicles. The potential clinical application of blood circRNAs in the biomarker and therapy fields is also discussed. Finally, perspectives for future studies are proposed.

Comparison of different cell type correction methods for genome-scale epigenetics studies.

BACKGROUND: Whole blood is frequently utilized in genome-wide association studies of DNA methylation patterns in relation to environmental exposures or clinical outcomes. These associations can be confounded by cellular heterogeneity. Algorithms have been developed to measure or adjust for this heterogeneity, and some have been compared in the literature. However, with new methods available, it is unknown whether the findings will be consistent, if not which method(s) perform better. RESULTS: Methods: We compared eight cell-type correction methods including the method in the minfi R package, the method by Houseman et al., the Removing unwanted variation (RUV) approach, the methods in FaST-LMM-EWASher, ReFACTor, RefFreeEWAS, and RefFreeCellMix R programs, along with one approach utilizing surrogate variables (SVAs). We first evaluated the association of DNA methylation at each CpG across the whole genome with prenatal arsenic exposure levels and with cancer status, adjusted for estimated cell-type information obtained from different methods. We then compared CpGs showing statistical significance from different approaches. For the methods implemented in minfi and proposed by Houseman et al., we utilized homogeneous data with composition of some blood cells available and compared them with the estimated cell compositions. Finally, for methods not explicitly estimating cell compositions, we evaluated their performance using simulated DNA methylation data with a set of latent variables representing "cell types". RESULTS: Results from the SVA-based method overall showed the highest agreement with all other methods except for FaST-LMM-EWASher. Using homogeneous data, minfi provided better estimations on cell types compared to the originally proposed method by Houseman et al. Further simulation studies on methods free of reference data revealed that SVA provided good sensitivities and specificities, RefFreeCellMix in general produced high sensitivities but specificities tended to be low when confounding is present, and FaST-LMM-EWASher gave the lowest sensitivity but highest specificity. CONCLUSIONS: Results from real data and simulations indicated that SVA is recommended when the focus is on the identification of informative CpGs. When appropriate reference data are available, the method implemented in the minfi package is recommended. However, if no such reference data are available or if the focus is not on estimating cell proportions, the SVA method is suggested.

Total α-synuclein levels in human blood cells, CSF, and saliva determined by a lipid-ELISA.

The validity of α-synuclein (α-Syn) as a biomarker for Parkinson's disease (PD) is still under investigation. Conventional methods for capture and quantitation of α-Syn protein in human samples are primarily based on anti-α-Syn antibodies. Specific and competent antibodies were raised against α-Syn. However, capture by anti-α-Syn antibodies may be limited to specific epitope recognition, attributed to protein structure or post-translational modifications. Hence, antibody-based methods for α-Syn capture raise a concern regarding their efficacy to detect the intracellular, unfolded α-Syn pool. An alternative is α-Syn capture by membrane lipids, i.e., to utilize the biochemical property of α-Syn to specifically bind membrane lipids and acquire a characteristic structure following binding. We determined α-Syn levels in human samples using immobilized lipids for α-Syn capture. The lipids used for α-Syn capture consist of phosphatidyl inositol (PI), phosphatidyl serine (PS), and phosphatidyl ethanolamine (PE). Addition of mono-sialoganglioside, GM1 ganglioside, to the immobilized lipids significantly improved α-Syn detection. Following capture, the lipid-bound α-Syn was detected using an anti-α-Syn antibody. Total α-Syn levels in whole blood cells (WBC), cerebrospinal fluid (CSF), and saliva were determined by the lipid-ELISA method.