Elevated serum and urine levels of progranulin (PGRN) as a predictor of microglia activation in the early phase of traumatic brain injury: a further link with the development of neurodegenerative diseases.

Traumatic brain injury (TBI) is a frequent finding during forensic autopsies and neuropathological examinations in medico-legal practices. Despite the unprecedented attention currently focused on TBI pathogenesis, there is a need to improve its diagnostics through the use of novel biomarkers to facilitate detection, treatment, and prognosis. Recently, growth factor progranulin (PGRN) has attracted significant attention because of its neurotrophic and anti-inflammatory activities. The role of PGRN in TBI has not been widely discussed, although PGRN-related neuroinflammatory and neurodegenerative phenomena have been described. The aim of this study was to identify PGRN concentration levels in biofluids and examine PGRN and CD68 protein expression in brain tissue using immunohistochemical staining in individuals with fatal TBI in its early phase. The study was performed using cases (n = 30) of fatal head injury and control cases (n = 30) of sudden death. The serum and urine were collected within ~24 h after death and compared using the ELISA test, where brain specimens were stained with anti-PGRN and anti-CD68 antibodies. In our study, we observed elevated concentration levels of PGRN in the serum and urine of TBI individuals in the early phase of TBI. These changes were accompanied by increased expression of PGRN in the frontal cortex (1st-3rd layers), in which anti-CD68 immunostaining revealed disseminated cortical microglia activation. The possible implementation of performing such assays offers a novel and interesting tool for investigation and research regarding TBI diagnosis and pathogenesis. Furthermore, the above-mentioned surrogate biofluid assays may be useful in clinical prognosis and risk calculation of non-fatal cases of TBI, considering the development of neurodegenerative conditions of TBI individuals.

Competing Endogenous RNA Networks as Biomarkers in Neurodegenerative Diseases.

Protein aggregation is classically considered the main cause of neuronal death in neurodegenerative diseases (NDDs). However, increasing evidence suggests that alteration of RNA metabolism is a key factor in the etiopathogenesis of these complex disorders. Non-coding RNAs are the major contributor to the human transcriptome and are particularly abundant in the central nervous system, where they have been proposed to be involved in the onset and development of NDDs. Interestingly, some ncRNAs (such as lncRNAs, circRNAs and pseudogenes) share a common functionality in their ability to regulate gene expression by modulating miRNAs in a phenomenon known as the competing endogenous RNA mechanism. Moreover, ncRNAs are found in body fluids where their presence and concentration could serve as potential non-invasive biomarkers of NDDs. In this review, we summarize the ceRNA networks described in Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis and spinocerebellar ataxia type 7, and discuss their potential as biomarkers of these NDDs. Although numerous studies have been carried out, further research is needed to validate these complex interactions between RNAs and the alterations in RNA editing that could provide specific ceRNET profiles for neurodegenerative disorders, paving the way to a better understanding of these diseases.

A fully validated bioanalytical method using an UHPLC-MS/MS system for quantification of DNA and RNA oxidative stress biomarkers.

A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.

Developments in the synthesis of a water compatible molecularly imprinted polymer as artificial receptor for detection of 3-nitro-L-tyrosine in neurological diseases.

A highly selective water compatible molecularly imprinted polymer (MIP) for 3-nitro-L-tyrosine (3NT), an oxidative stress marker associated with neurodegenerative disorders, was prepared and its use as solid-phase extraction (SPE) sorbent material was demonstrated. The MIP was prepared by bulk polymerization using methacrylic acid as functional monomer and acetonitrile as porogen with traces of acetic acid and trifluoroacetic acid. In order to evaluate its binding properties, the MIP was analyzed by batch rebinding experiments and subsequently used as SPE sorbent for the selective clean-up and pre-concentration of 3NT from standard solutions and spiked human urine samples. The results obtained from batch rebinding experiments showed the presence of two association constants corresponding to high-affinity (Ka 4.20×10(3) M(-1)) and low-affinity (Ka 0.79×10(3) M(-1)) binding sites. Standard mixture solution loaded on MIP-SPE cartridge gave a recovery of 95% for 3NT, while the other compounds were totally eluted during washing step. Percentage of recovery higher than 90%, with relative standard deviation of 2%, was also obtained when a maximum of 55 µg of 3NT is used in spiked urine sample and loaded into the cartridge. Validation of the analytical method for 3NT quantification in human urine gave 0.7 µg mL(-1) of limit of detection, a linear range of 2.5-55 µg mL(-1) with a relative standard deviation of 2%.

Evaluation of a prototype point-of-care instrument based on monochromatic x-ray fluorescence spectrometry: potential for monitoring trace element status of subjects with neurodegenerative disease.

Assessment of trace elements such as Cu, Zn, and Se in patients with neurodegenerative disease, such as Alzheimer's (AD) and Parkinson's disease (PD), may be useful in etiologic studies and in assessing the risk of developing these conditions. A prototype point-of-care (POC) instrument based on monochromatic x-ray fluorescence (M-XRF) was assembled and evaluated for the determination of Cu, Zn, and Se in whole blood, plasma, and urine. The prototype instrument was validated using certified reference materials for Cu and Zn in serum/plasma, and the reported bias and relative imprecision were <10%. The M-XRF prototype performance was further assessed using human specimens collected from AD and PD subjects, and was found to be satisfactory (<20% bias) for monitoring Cu and Zn levels in plasma and whole blood. However, the prototype M-XRF sensitivity was not sufficient for quantifying Cu, Zn, or Se in urine. Nonetheless, while validating the prototype instrument, body fluids (whole blood, plasma, and urine) were collected from 19 AD patients, 23 PD patients, and 24 controls specifically for trace element analysis using well-validated methods based on inductively coupled plasma mass spectrometry (ICP-MS). This limited biomonitoring study provided robust data for up to 16 elements including Sb, As, Ba, Cd, Cs, Co, Cr, Cu, Hg, Pb, Mo, Se, Tl, Sn, Zn, and U in plasma, whole blood, and urine. The results did not indicate any significant differences in most trace elements studied between AD or PD patients compared to controls, although the sample size is limited. A statistically significant increase in plasma Se was identified for PD patients relative to AD patients, but this could be due to age differences.

Comparison of 24-h volume and creatinine-corrected total urinary polyphenol as a biomarker of total dietary polyphenols in the Invecchiare InCHIANTI study.

Polyphenols have beneficial effects on several chronic diseases but assessing polyphenols intake from self-reported dietary questionnaires tends to be inaccurate and not very reliable. A promising alternative is to use urinary excretion of polyphenols as a proxy measure of intake. The best method to assess urinary excretion is to collect 24-h urine. However, since collecting 24-h urine method is expensive, time consuming and may be difficult to implement in large population-based studies, measures obtained from spot urine normalized by creatinine are commonly used. The purpose of the study was to evaluate the correlation between polyphenols dietary intake and total urinary polyphenol excretion (TPE), expressed by both 24-h volume and urinary creatinine normalization in 928 participants from the InCHIANTI study. Dietary intake data were collected using a validated food frequency questionnaire. Urinary TPE was analyzed by Folin-Ciocalteau assay. Both urinary TPE expression models were statistically correlated (r=0.580), and the partial correlation coefficient improved (pr=0.722) after adjusting for the variables that modify the urinary creatinine excretion (i.e. gender, age, BMI, physical activity and renal function). In crude models, polyphenol intake was associated with TPE corrected by 24-h volume (r=0.211; P<0.001), but not with creatinine normalization (r=0.014; P=0.692). However, urinary TPE expressed by creatinine correction was significantly correlated with dietary polyphenols after adjusting for covariates (pr=0.113; P=0.002). We conclude that urinary TPE expressed by 24-h volume is a better biomarker of polyphenol dietary intake than by urinary creatinine normalization. After covariate adjustment, both can be used for studying the relationships between polyphenol intake and health in large-scale epidemiological studies.

Detection of proteinase K resistant proteins in the urine of patients with Creutzfeldt-Jakob and other neurodegenerative diseases.

Recent concern about the possible secondary spread of vCJD through blood transfusion and blood products has highlighted the need for a sensitive test for the identification of PrP(TSE/res) in clinical specimens collected in a non-invasive way. In addition, a more accurate estimate of the prevalence of pre-clinical vCJD in the population may be possible if there were a test that could be applied to easily available material such as urine. As a step towards this goal,the detection of putative PrP(TSE/res) in the urine of CJD patients has been improved, based on Proteinase K digestion of samples and western blotting. The modified western blot uses concentrated urine as a starting material. After proteolytic treatment followed by electrophoresis and western blotting, membranes are incubated with an anti-PrP antibody conjugated directly with horseradish peroxidase. This study was conducted on urine samples of CJD and other neurodegenerative disease affected individuals. Proteinase K resistant high molecular weight proteins were detected, which are suggested to be a complex of urinary PrP and immunoglobulin proteins. Whether urine can be used as a diagnostic tool for the detection of PrP could not be answered in this study.

Clinical and brain 18fluoro-2-deoxyglucose positron emission tomographic findings in ethylmalonic aciduria, a progressive neurometabolic disease.

We report a 2-year-old boy with ethylmalonic aciduria and vasculopathy syndrome evaluated by 18fluoro-2-deoxyglucose positron emission tomographic (18FDG PET) brain scan, with intense uptake of 18FDG in the caudate nucleus and putamen bilaterally but with no morphological changes on magnetic resonance imaging (MRI). A repeat 18FDG PET brain scan 1 year later showed a significant bilateral decreased uptake of glucose in the putamen and the head of the caudate nucleus as well as a decreased uptake in the frontal lobes. On MRI, there was atrophy and watershed infarcts in the basal ganglia, explaining the loss of glucose uptake. These results reflect a selective vulnerability of the basal ganglia, their functional derangement, and ultimate degeneration.