Obtaining miRNA from Saliva-Comparison of Sampling and Purification Methods.

The use of saliva as a biomarker source has advantages over other biofluids and imaging techniques, and miRNAs are ideal biomarker candidates. They are involved in numerous cellular processes, and their altered expression suggests that miRNAs play a crucial regulatory role in disease development. We wanted to find an easily reproducible and executable miRNA-obtaining methodology suitable for quantification. Three commercial miRNA extraction kits (mirVana, Nucleospin and miRNeasy) and three saliva collectors (50 mL tubes, Salimetrics and Oragene) were tested. Several features, including RNA quality and technical parameters, were evaluated. The expression of five synthetic spike-in controls and seven saliva-miRNAs was analyzed independently and grouped by the collectors and the extraction kits. The combination of Oragene and miRNeasy assured the most sensitive detection of all seven saliva miRNAs. Testing different combinations of saliva collectors and RNA purification kits permitted the establishment of combinations for different uses. The results of our study highlight that optimization of resources for biomarker studies is possible after careful planning of each study.

Alternative platelet activation pathways and their role in neurodegenerative diseases.

PURPOSE OF THE REVIEW: The study of platelets in the context of neurodegenerative diseases is intensifying, and increasing evidence suggests that platelets may play an important role in the pathogenesis of neurodegenerative disorders. Therefore, we aim to provide a comprehensive overview of the role of platelets and their diverse activation pathways in the development of these diseases. RECENT FINDINGS: Platelets participate in synaptic plasticity, learning, memory, and platelets activated by exercise promote neuronal differentiation in several brain regions. Platelets also contribute to the immune response by modulating their surface protein profile and releasing pro- and anti-inflammatory mediators. In Alzheimer's disease, increased levels of platelet amyloid precursor protein raise the production of amyloid-beta peptides promoting platelet activation, triggering at the same time amyloid-beta fibrillation. In Parkinson's disease, increased platelet α-synuclein is associated with elevated ROS production and mitochondrial dysfunction. SUMMARY: In this review, we revise different platelet activation pathways, those classically involved in hemostasis and wound healing, and alternative activation pathways recently described in the context of neurodegenerative diseases, especially in Alzheimer's disease.

Expression Levels of an Alpha-Synuclein Transcript in Blood May Distinguish between Early Dementia with Lewy Bodies and Parkinson's Disease.

Lewy body diseases (LBD) including dementia with Lewy bodies (DLB) and Parkinson disease (PD) are characterized by alpha-synuclein pathology. DLB is difficult to diagnose and peripheral biomarkers are urgently needed. Therefore, we analyzed the expression of five alpha-synuclein gene (SNCA) transcripts, SNCAtv1, SNCAtv2, SNCAtv3, SNCA126, and SNCA112, in 45 LBD and control temporal cortex samples and in the blood of 72 DLB, 59 PD, and 54 control subjects. The results revealed overexpression of SNCAtv1 and SNCA112 in DLB, and SNCAtv2 in PD temporal cortices. In DLB blood, diminution of all SNCA transcripts was observed. SNCAtv1 and SNCAtv2 were diminished in PD with disease onset before 70 years. SNCAtv3, driven by its own promoter, showed opposite expression in early DLB and PD, suggesting that its amount may be an early, DLB specific biomarker. Correlation between blood transcript levels and disease duration was positive in DLB and negative in PD, possibly reflecting differences in brain alpha-synuclein aggregation rates associated with differences in disease courses. In conclusion, SNCA transcripts showed a disease-specific increase in the brain and were diminished in blood of LBD patients. SNCAtv3 expression was decreased in early DLB and increased in early PD and could be a biomarker for early DLB diagnosis.

Epigenetics in Lewy Body Diseases: Impact on Gene Expression, Utility as a Biomarker, and Possibilities for Therapy.

Lewy body disorders (LBD) include Parkinson's disease (PD) and dementia with Lewy bodies (DLB). They are synucleinopathies with a heterogeneous clinical manifestation. As a cause of neuropathological overlap with other neurodegenerative diseases, the establishment of a correct clinical diagnosis is still challenging, and clinical management may be difficult. The combination of genetic variation and epigenetic changes comprising gene expression-modulating DNA methylation and histone alterations modifies the phenotype, disease course, and susceptibility to disease. In this review, we summarize the results achieved in the deciphering of the LBD epigenome. To provide an appropriate context, first LBD genetics is briefly outlined. Afterwards, a detailed review of epigenetic modifications identified for LBD in human cells, postmortem, and peripheral tissues is provided. We also focus on the difficulty of identifying epigenome-related biomarker candidates and discuss the results obtained so far. Additionally, epigenetic changes as therapeutic targets, as well as different epigenome-based treatments, are revised. The number of studies focusing on PD is relatively limited and practically inexistent for DLB. There is a lack of replication studies, and some results are even contradictory, probably due to differences in sample collection and analytical techniques. In summary, we show the current achievements and directions for future research.

The ratio of shear to elastic modulus of in-plane loaded masonry.

When designing unreinforced masonry buildings, the wall stiffness and, consequently, the masonry elastic and shear modulus E and G are essential parameters. Current codes provide empirical estimates of the masonry elastic modulus and a ratio between the shear and elastic modulus, G/E. This ratio, commonly taken as 0.4, is not based on scientific evidence and there appears to be no consensus concerning its value and influencing parameters, meaning that current code standards might not accurately portray the shear deformations of masonry elements. To give the choice of the G/E ratio a theoretical foundation, this paper presents closed-form expressions for the G/E ratio of running-bond masonry that capture the effects of finite joint thickness, finite wall thickness and orthotropic block properties. Based on the geometry of blocks and joints as well as their elastic parameters, a validation of the developed expression using 3D finite element analyses shows good performance. For modern masonry typologies with hollow clay bricks, a G/E ratio of 0.20-0.25 is obtained. For historical masonry typologies, such as dry stacked or mortared stone masonry, as well as solid clay brick masonry, ratios between 0.30 and 0.40 are computed.

GBA Mutations Are Associated With Earlier Onset and Male Sex in Dementia With Lewy Bodies.

BACKGROUND: Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are Lewy body diseases characterized by similar pathological features. Several studies have shown a relation between alterations in the glucocerebrosidase gene (GBA) and the development of LB diseases. Here, we explored the role of GBA mutations in Spanish DLB patients. METHODS: GBA mRNA sequences were analyzed in a neuropathological (50 DLB, 43 PD, and 34 control brains) and in a clinical cohort (47 DLB patients and 131 unaffected individuals). RESULTS: Sixteen GBA mutation carriers were identified, 5 of which were brains with pure DLB. The most common mutation, E326K, was strongly associated with pure DLB and PD with dementia. GBA mutations were overrepresented in men and associated with earlier DLB onset. CONCLUSIONS: GBA mutations are also an important risk factor for DLB development in the Spanish population, are associated with earlier disease onset, and are more prevalent in men. © 2015 International Parkinson and Movement Disorder Society.

Cystatin C is differentially involved in multiple system atrophy phenotypes.

AIMS: As cystatin C (CysC) is involved in some forms of neurodegeneration, we investigated the possible relationship between CysC and multiple system atrophy (MSA), including its parkinsonian (MSAp) and cerebellar (MSAc) phenotypes. METHODS: Cystatin C gene (CST3) haplotypes were determined by PCR followed by KspI digestion in 50 MSA patients and 108 controls. CST3 and cathepsins B, D and L1 mRNA levels were studied in frozen post-mortem caudate nucleus and cerebellar samples of eight MSAp, four MSAc and 18 control brains and analysed by the ΔΔCt method. CysC immunohistochemistry was performed on three MSAp, three MSAc and three control cerebella. Additionally, determination of CST3 and cathepsins B, D and L1 mRNA levels and immunohistochemistry for CysC were carried out in cerebella from three patients with paraneoplastic cerebellar degeneration, three with spinocerebellar ataxia (type 3, SCA3) and three with cerebellar ischaemia (CI). RESULTS: In the set of blood samples, the CST3 B-haplotype was associated with MSAp (OR 4.86, confidence interval 1.84-13.3). High CST3 mRNA levels were found in MSAp caudate nuclei [expression change: 3.08 (2.98-3.18)] and MSAc cerebella [expression change: 2.44 (2.14-2.88)]. In the latter there was CysC over-expression in Purkinje cells, Bergmann glia and dentate nucleus neurones. No cathepsin increase was detected in MSA cerebella. High mRNA levels of CST3 and cathepsins B and L1 were observed in SCA3 and CI brains. CONCLUSIONS: CysC changes are differentially present in the parkinsonian and cerebellar forms of MSA and may play an important role in the pathogenesis of this neurodegenerative condition.

Simpson-Golabi-Behmel syndrome type 1 and hepatoblastoma in a patient with a novel exon 2-4 duplication of the GPC3 gene.

Mutations in the gene encoding glypican (GPC) 3 appear to be responsible for most cases of Simpson-Golabi-Behmel syndrome type 1. Duplication of the GPC4 gene has also been associated to this syndrome; however, no duplications involving GPC3 have been related. We describe a family that harbors a novel exon 2-4 duplication event leading to a truncating germline mutation of the GPC3 gene that, to our knowledge, has not been previously reported. GPC3 transcripts that carry this duplication bear non-functional proteins making its pathogenic role highly probable. The absence of a functional GPC3 may alter the normal differentiation of embryonal mesodermal tissues predisposing to the development of embryonal tumors, as the index case studied who developed a hepatoblastoma at age 9 months.

Geometrical digital twins of the as-built microstructure of three-leaf stone masonry walls with laser scanning.

Research on irregular stone masonry walls is hampered by the lack of detailed geometrical models of their internal micro-structure, i.e. the shape and size of each stone and its position within the wall. Without such a geometric digital twin of walls tested in the laboratory, it is difficult to evaluate the accuracy of existing numerical simulation techniques. Here, we describe the generation of geometrical digital twins of three irregular stone masonry walls built in the laboratory. We labelled each stone manually and then obtained the geometry of the individual stones using a portable laser scanning device. With the same device we scanned the wall after the construction of each layer. We then registered the position of each stone in the layer. This paper outlines the methodology for the data acquisition and digital reconstruction and presents the datasets for the walls. The developed geometrical digital twins provide unique information regarding the micro-structure of constructed walls that is key for the development and validation of numerical simulation techniques for stone masonry.

Peripheral α-synuclein isoforms are potential biomarkers for diagnosis and prognosis of isolated REM sleep behavior disorder.

INTRODUCTION: Isolated REM sleep behavior disorder (IRBD) represents an early manifestation of the synucleinopathies Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Aggregation of abnormal α-synuclein and its increased expression in the brain is crucial in the development of the synucleinopathies. Whereas α-synuclein gene (SNCA) transcripts are overexpressed in brain, a concomitant reduction occurs in blood of DLB patients. We assessed whether this decrease is also detectable in IRBD. METHODS: 108 IRBD patients and 149 controls were included of which 29 IRBD and 32 control cases were available for expression studies. Expression of SNCAtv1, SNCAtv2, SNCAtv3 and SNCA126 isoforms, and GBA were determined by real-time PCR. Genotype distribution of SNCA SNPs, rs356219 and rs2736990, and correlation with SNCA expression was analyzed. RESULTS: Expression of all SNCA transcripts was reduced in IRBD blood whereas GBA expression did not change. SNCAtv3 expression correlated inversely with IRBD duration, being lower in patients with longer follow-up. Rs356219-AA genotype frequency was increased in IRBD patients who later developed PD and DLB. Rs2736990-CC frequency was increased among IRBD cases who remained disease-free. No correlation was observed between rs356219 and rs2736990 genotypes and SNCA transcript levels. CONCLUSION: SNCA transcript expression is decreased in blood in IRBD, and levels decrease with IRBD duration. Our findings indicate that changes in SNCA expression occur in the earliest stages of the synucleinopathies before motor and cognitive symptoms become apparent.

Complete mitochondrial DNA profile in stroke: A geographical matched case-control study in Spanish population.

INTRODUCTION: Stroke, the second leading cause of death worldwide, is a complex disease influenced by many risk factors among which we can find reactive oxygen species (ROS). Since mitochondria are the main producers of cellular ROS, nowadays studies are trying to elucidate the role of these organelles and its DNA (mtDNA) variation in stroke risk. The aim of the present study was to perform a comprehensive evaluation of the association between mtDNA mutations and mtDNA content and stroke risk. MATERIAL AND METHODS: Homoplasmic and heteroplasmic mutations of the mtDNA were analysed in a case-controls study using 110 S cases and their corresponding control individuals. Mitochondrial DNA copy number (mtDNA-CN) was analysed in 73 of those case-control pairs. RESULTS: Our results suggest that haplogroup V, specifically variants m.72C > T, m.4580G > A, m.15904C > T and m.16298 T > C have a protective role in relation to stroke risk. On the contrary, variants m.73A > G, m.11719G > A and m.14766C > T appear to be genetic risk factors for stroke. In this study, we found no statistically significant association between stroke risk and mitochondrial DNA copy number. CONCLUSIONS: These results demonstrate the possible role of mtDNA genetics on the pathogenesis of stroke, probably through alterations in mitochondrial ROS production.

Cognitive and Motor Decline in Dementia with Lewy Bodies and Parkinson's Disease Dementia.

Background: There is a need to better understand the rate of cognitive and motor decline of Dementia with Lewy bodies (DLB) and Parkinson's disease Dementia (PDD). Objectives: To compare the rate of cognitive and motor decline in patients with DLB and PDD from the E-DLB Consortium and the Parkinson's Incidence Cohorts Collaboration (PICC) Cohorts. Methods: The annual change in MMSE and MDS-UPDRS part III was estimated using linear mixed regression models in patients with at least one follow-up (DLB n = 837 and PDD n = 157). Results: When adjusting for confounders, we found no difference in the annual change in MMSE between DLB and PDD (-1.8 [95% CI -2.3, -1.3] vs. -1.9 [95% CI -2.6, -1.2] [P = 0.74]). MDS-UPDRS part III showed nearly identical annual changes (DLB 4.8 [95% CI 2.1, 7.5]) (PDD 4.8 [95% CI 2.7, 6.9], [P = 0.98]). Conclusions: DLB and PDD showed similar rates of cognitive and motor decline. This is relevant for future clinical trial designs.

New brain-specific beta-synuclein isoforms show expression ratio changes in Lewy body diseases.

Lewy body diseases (LBDs) include dementia with Lewy bodies (DLB) and Parkinson disease (PD). Alpha-synuclein (AS) aggregation is a key event in the pathogenesis of LBDs and beta-synuclein (BS) inhibits AS aggregation in vitro and in vivo. Recently, BS has been shown to interact directly with AS regulating its functionality and preventing its oligomerization, and a molecular subgroup of pure DLB lacks BS in cortical regions. In this study, we characterized four new BS transcript variants and analyzed their expression in neuronal and non-neuronal tissue, and their differential expression in frozen samples of three areas from brains of patients with pure Lewy body pathology (LBP), common LBP, Alzheimer pathology, and of controls. Relative mRNA expression was determined by real-time PCR with neuron-specific enolase 2 and synaptophysin as housekeeping genes, and expression changes were evaluated by the ΔΔCt method. Two main findings are in concordance with earlier studies. First, all BS isoforms are drastically diminished in the cortex of patients with pure LBP that had presented clinically as DLB but not PD with dementia. Second, an important shift of the isoform expression ratio was observed in the temporal cortex of all LBD cases, and the minor isoforms, normally absent in the midbrain, were detected in the caudate nucleus of all DLB samples. Our results provide further evidence for the role of minor transcript variants in the development of complex diseases and provide new insights into the pathogenesis of LBDs that may be important for the understanding of molecular mechanisms involved in these complex diseases.

Genetic landscape of Segawa disease in Spain. Long-term treatment outcomes.

INTRODUCTION: In 2009, we described a possible founder effect of autosomal dominant Segawa disease in Córdoba (Spain) due to mutation c.265C>T (p. Q89*) in the GCH1 gene. We present a retrospective multicentre study aimed at improving our knowledge of Segawa disease in Spain and providing a detailed phenotypic-genotypic description of patients. METHODS: Clinical-genetic information were obtained from standardized questionnaires that were completed by the neurologists attending children and/or adults from 16 Spanish hospitals. RESULTS: Eighty subjects belonging to 24 pedigrees had heterozygous mutations in GCH1. Seven genetic variants have been described only in our cohort of patients, 5 of which are novel mutations. Five families not previously described with p. Q89* were detected in Andalusia due to a possible founder effect. The median latency to diagnosis was 5 years (IQR 0-16). The most frequent signs and/or symptoms were lower limb dystonia (38/56, 67.8%, p = 0.008) and diurnal fluctuations (38/56, 67.8%, p = 0.008). Diurnal fluctuations were not present in the phenotypes other than dystonia. Fifty-three of 56 symptomatic patients were treated with a levodopa/decarboxylase inhibitor for (mean ± SD) 12.4 ± 8.12 years, with 81% at doses lower than 350 mg/day (≤5 mg/kg/d in children). Eleven of 53 (20%) patients had nonresponsive symptoms that affected daily life activities. Dyskinesias (4 subjects) were the most prominent adverse effects. CONCLUSION: This study identifies 5 novel mutations and supports the hypothesis of a founder effect of p. Q89* in Andalusia. New insights are provided for the phenotypes and long-term treatment responses, which may improve early recognition and therapeutic management.

The decrease of ß-synuclein in cortical brain areas defines a molecular subgroup of dementia with Lewy bodies.

Lewy body diseases include dementia with Lewy bodies and Parkinson's disease. Whereas dementia with Lewy bodies and Parkinson's disease can be distinguished as separate clinical entities, the pathological picture is very often identical. α-synuclein aggregation is a key event in the pathogenesis of Lewy body diseases and ß-synuclein inhibits α-synuclein aggregation in vitro and in vivo. Recently, ß-synuclein has been shown to interact directly with α-synuclein, regulating its functionality and preventing its oligomerization. In this study, we analysed the expression of two ß-synuclein transcript variants and the main α-synuclein transcript SNCA140, in frozen samples of three areas from brains of patients with (i) pure diffuse Lewy body pathology; (ii) pure Alzheimer's disease pathology; (iii) diffuse Lewy body pathology and concomitant Alzheimer's disease pathology and (iv) controls. Relative messenger RNA expression was determined by real-time polymerase chain reaction, expression changes were evaluated by the ΔΔC(t) method and messenger RNA expression data were confirmed at the protein level. A drastic diminution of ß-synuclein expression was observed in cortical areas of all samples that presented neuropathological features corresponding to pure diffuse Lewy body pathology and the clinical phenotype of dementia with Lewy bodies, but not in those with neuropathological features corresponding to diffuse Lewy body pathology and concomitant Alzheimer's disease pathology or the clinical phenotype of Parkinson's disease with dementia. The correlation of expression data with the clinical phenotype and neuropathological diagnosis of the patients suggested the existence of a specific molecular subtype of dementia with Lewy bodies, characterized by a strong decrease of ß-synuclein in the frontal and temporal cortices. Furthermore, our findings provide new insights into the pathogenesis of Lewy body diseases that may be important for the understanding of molecular mechanisms involved in these complex diseases.

Pareto-like sequential sampling heuristic for global optimisation.

In this paper, we propose a simple global optimisation algorithm inspired by Pareto's principle. This algorithm samples most of its solutions within prominent search domains and is equipped with a self-adaptive mechanism to control the dynamic tightening of the prominent domains while the greediness of the algorithm increases over time (iterations). Unlike traditional metaheuristics, the proposed method has no direct mutation- or crossover-like operations. It depends solely on the sequential random sampling that can be used in diversification and intensification processes while keeping the information-flow between generations and the structural bias at a minimum. By using a simple topology, the algorithm avoids premature convergence by sampling new solutions every generation. A simple theoretical derivation revealed that the exploration of this approach is unbiased and the rate of the diversification is constant during the runtime. The trade-off balance between the diversification and the intensification is explained theoretically and experimentally. This proposed approach has been benchmarked against standard optimisation problems as well as a selected set of simple and complex engineering applications. We used 26 standard benchmarks with different properties that cover most of the optimisation problems' nature, three traditional engineering problems, and one real complex engineering problem from the state-of-the-art literature. The algorithm performs well in finding global minima for nonconvex and multimodal functions, especially with high dimensional problems and it was found very competitive in comparison with the recent algorithmic proposals. Moreover, the algorithm outperforms and scales better than recent algorithms when it is benchmarked under a limited number of iterations for the composite CEC2017 problems. The design of this algorithm is kept simple so it can be easily coupled or hybridised with other search paradigms. The code of the algorithm is provided in C++14, Python3.7, and Octave (Matlab).

Platelet miRNA Biosignature Discriminates between Dementia with Lewy Bodies and Alzheimer's Disease.

Dementia with Lewy bodies (DLB) is one of the most common causes of degenerative dementia, after Alzheimer's disease (AD), and presents pathological and clinical overlap with both AD and Parkinson's disease (PD). Consequently, only one in three DLB cases is diagnosed correctly. Platelets, previously related to neurodegeneration, contain microRNAs (miRNAs) whose analysis may provide disease biomarkers. Here, we profiled the whole platelet miRNA transcriptome from DLB patients and healthy controls. Differentially expressed miRNAs were further validated in three consecutive studies from 2017 to 2019 enrolling 162 individuals, including DLB, AD, and PD patients, and healthy controls. Results comprised a seven-miRNA biosignature, showing the highest diagnostic potential for the differentiation between DLB and AD. Additionally, compared to controls, two miRNAs were down-regulated in DLB, four miRNAs were up-regulated in AD, and two miRNAs were down-regulated in PD. Predictive target analysis identified three disease-specific clusters of pathways as a result of platelet-miRNA deregulation. Our cross-sectional study assesses the identification of a novel, highly specific and sensitive platelet-associated miRNA-based biosignature, which distinguishes DLB from AD.

Molecular pathology of Lewy body diseases.

Lewy body diseases are characterized by the presence of Lewy bodies, alpha-synuclein(AS)-positive inclusions in the brain. Since their main component is conformationally modified AS, aggregation of the latter is thought to be a key pathogenic event in these diseases. The analysis of inclusion body constituents gives additional information about pathways also involved in the pathology of synucleinopathies. Widespread mitochondrial dysfunction is very closely related to disease development. The impairment of protein degradation pathways, including both the ubiquitin-proteasome system and the autophagy-lysosome pathway also play an important role during the development of Lewy body diseases. Finally, differential expression changes of isoforms corresponding to genes primarily involved in Lewy body formation point to alternative splicing as another important mechanism in the development of Parkinson's disease, as well as dementia with Lewy bodies. The present paper attempts to give an overview of recent molecular findings related to the pathogenesis of Lewy body diseases.

Extracellular vesicles, new actors in the search for biomarkers of dementias.

Increased life expectancy impacts directly on the number of older people worldwide with the associated increase in neurodegenerative diseases. Besides their social implications, the different forms of dementia, including Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies or frontotemporal dementia, show clinical and pathological overlaps; this hinders their specific and differential diagnosis. To date, biomarkers for each of these types of dementia have been investigated in the cerebrospinal fluid or blood. More recently, the field of biomarker search found a new opportunity to improve diagnosis in extracellular vesicles (EVs). EVs are released by cells including those of the central nervous system and these can be isolated from cerebrospinal fluid and blood. This review summarizes the current knowledge related to the search for dementia biomarkers in the field of EVs including studies of specific EV content, mainly proteins such as α-synuclein or tau and RNA species.

Comprehensive proteomic profiling of plasma-derived Extracellular Vesicles from dementia with Lewy Bodies patients.

Proteins and nucleic acids contained in extracellular vesicles (EVs) are considered a feasible source of putative biomarkers for physiological and pathological conditions. Within the nervous system, not only neurons but also other brain cells are able to produce EVs, which have been involved in their physiological processes and also in the development and course of several neurodegenerative diseases. Among these, dementia with Lewy bodies (DLB) is the second cause of dementia worldwide, though most cases are missed or misdiagnosed as Alzheimer's disease (AD) due to the important clinical and pathological overlap between both diseases. In an attempt to find reliable biomarkers for DLB diagnosis, our group characterized the proteome of plasma-derived EVs from DLB patients compared to aged-matched healthy controls (HCs) using two different proteomic LC-MS/MS approaches. Remarkably, we found that gelsolin and butyrylcholinesterase were differentially identified between DLB and HCs. Further validation of these results using conventional ELISA techniques, and including an additional group of AD patients, pointed to decreased levels of gelsolin in plasma-EVs from DLB compared to HCs and to AD samples. Thus, gelsolin may be considered a possible biomarker for the differentiation between DLB and AD.