Unraveling targetable systemic and cell-type-specific molecular phenotypes of Alzheimer’s and Parkinson’s brains with digital cytometry

Copyright © 2020 Bordone and Barbosa-Morais. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).The use, distribution or reproduction in other forums is permitted, provided theoriginal author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative disorders worldwide, with age being their major risk factor. The increasing worldwide life expectancy, together with the scarcity of available treatment choices, makes it thus pressing to find the molecular basis of AD and PD so that the causing mechanisms can be targeted. To study these mechanisms, gene expression profiles have been compared between diseased and control brain tissues. However, this approach is limited by mRNA expression profiles derived for brain tissues highly reflecting their degeneration in cellular composition but not necessarily disease-related molecular states. We therefore propose to account for cell type composition when comparing transcriptomes of healthy and diseased brain samples, so that the loss of neurons can be decoupled from pathology-associated molecular effects. This approach allowed us to identify genes and pathways putatively altered systemically and in a cell-type-dependent manner in AD and PD brains. Moreover, using chemical perturbagen data, we computationally identified candidate small molecules for specifically targeting the profiled AD/PD-associated molecular alterations. Our approach therefore not only brings new insights into the disease-specific and common molecular etiologies of AD and PD but also, in these realms, foster the discovery of more specific targets for functional and therapeutic exploration.This work was supported by European Molecular Biology Organization (EMBO Installation Grant 3057 to NB-M), Fundação para a Ciência e a Tecnologia (FCT Investigator Starting Grant IF/00595/2014 and CEEC Individual Assistant Researcher contract CEECIND/00436/2018 to NB-M, Ph.D.Studentship PD/BD/105854/2014 to MB), and Genome PT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 – Operational Programme for Competitiveness and Internationalization (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020),under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

The changes in classical and nonlinear parameters after a maximal bout to elicit fatigue in competitive swimming

The aim was to assess the effect of fatigue on linear and nonlinear parameters in swimming. Twenty-four fitness-oriented swimmers performed a maximal bout of 100m at front-crawl to elicit fatigue. Before (pre-) and immediately after (post-test) the bout, participants swam an allout 25m to derive the speed fluctuation (dv), approximate entropy (ApEn) and fractal dimension (FD) from the speed-time series collected by a speedo-meter. Swim speed was 10.85% slower in the post-test than in the pre-test (p < .001, η2=0.72). There was an effect of the fatigue on the dv with a moderate effect size. The dv increased shifting the 95CI band from 0.116–0.134 to 0.140–0.161. The ApEn showed non-significant variations between the pre- and post-test having the 95CI of pre- and post-test overlapped (pre: 0.659–0.700; post: 0.641–0.682). The FD showed as well a significant variation (the 95CI moved from 1.954–1.965 to 1.933–1.951). It can be concluded that in swimming there are changes in classical and nonlinear parameters under fatigue.This research was funded by the NIE AcRF grant (RI 11/13 TB)info:eu-repo/semantics/acceptedVersio

A Comparison of Experimental and Analytical Procedures to Measure Passive Drag in Human Swimming

The aim of this study was to compare the swimming hydrodynamics assessed with experimental and analytical procedures, as well as, to learn about the relative contributions of the friction drag and pressure drag to total passive drag. Sixty young talented swimmers (30 boys and 30 girls with 13.59±0.77 and 12.61±0.07 years-old, respectively) were assessed. Passive drag was assessed with inverse dynamics of the gliding decay speed. The theoretical modeling included a set of analytical procedures based on naval architecture adapted to human swimming. Linear regression models between experimental and analytical procedures showed a high correlation for both passive drag (Dp = 0.777*Df+pr; R2 = 0.90; R2a = 0.90; SEE = 8.528; P<0.001) and passive drag coefficient (CDp = 1.918*CDf+pr; R2 = 0.96; R2a = 0.96; SEE = 0.029; P<0.001). On average the difference between methods was -7.002N (95%CI: -40.480; 26.475) for the passive drag and 0.127 (95%CI: 0.007; 0.247) for the passive drag coefficient. The partial contribution of friction drag and pressure drag to total passive drag was 14.12±9.33% and 85.88±9.33%, respectively. As a conclusion, there is a strong relationship between the passive drag and passive drag coefficient assessed with experimental and analytical procedures. The analytical method is a novel, feasible and valid way to gather insight about one's passive drag during training and competition. Analytical methods can be selected not only to perform race analysis during official competitions but also to monitor the swimmer's status on regular basis during training sessions without disrupting or time-consuming procedures.info:eu-repo/semantics/publishedVersio

Alternative splicing : the pledge, the turn, and the prestige : the key role of alternative splicing in human biological systems

© The Author(s) 2017. This article is an open access publication. Open Access. This article is distributed under the terms of the Crea-tive Commons Attribution 4.0 International License (http://crea-tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Alternative pre-mRNA splicing is a tightly controlled process conducted by the spliceosome, with the assistance of several regulators, resulting in the expression of different transcript isoforms from the same gene and increasing both transcriptome and proteome complexity. The differences between alternative isoforms may be subtle but enough to change the function or localization of the translated proteins. A fine control of the isoform balance is, therefore, needed throughout developmental stages and adult tissues or physiological conditions and it does not come as a surprise that several diseases are caused by its deregulation. In this review, we aim to bring the splicing machinery on stage and raise the curtain on its mechanisms and regulation throughout several systems and tissues of the human body, from neurodevelopment to the interactions with the human microbiome. We discuss, on one hand, the essential role of alternative splicing in assuring tissue function, diversity, and swiftness of response in these systems or tissues, and on the other hand, what goes wrong when its regulatory mechanisms fail. We also focus on the possibilities that splicing modulation therapies open for the future of personalized medicine, along with the leading techniques in this field. The final act of the spliceosome, however, is yet to be fully revealed, as more knowledge is needed regarding the complex regulatory network that coordinates alternative splicing and how its dysfunction leads to disease.The authors are supported by: EMBO Installation Grant (3057), Investigador FCT Starting Grant (IF/00595/2014) and iMM Lisboa start-up funds to NLB-M; Postdoctoral fellowships by UNESCO-L’Oreal For Women in Science Program (ERI/NCS/FLP/CDC.13.94) and iMM/FCT/MEC/FEDER (IMM/BPD/45-2016, LISBOA-01-0145-FEDER-007391) to LG-P; Fundação para a Ciência e Tecnologia (FCT) PhD fellowships to MCB (PD/BD/105854/2014) and MA-F (PD/BD/128283/2017); Fundação AstraZeneca Innovate Competition Award to MA-F.info:eu-repo/semantics/publishedVersio

Spike-in validation of an Illumina-specific variance-stabilizing transformation

BACKGROUND: Variance-stabilizing techniques have been used for some time in the analysis of gene expression microarray data. A new adaptation, the variance-stabilizing transformation (VST), has recently been developed to take advantage of the unique features of Illumina BeadArrays. VST has been shown to perform well in comparison with the widely-used approach of taking a log2 transformation, but has not been validated on a spike-in experiment. We apply VST to the data from a recently published spike-in experiment and compare it both to a regular log2 analysis and a recently recommended analysis that can be applied if all raw data are available. FINDINGS: VST provides more power to detect differentially expressed genes than a log2 transformation. However, the gain in power is roughly the same as utilizing the raw data from an experiment and weighting observations accordingly. VST is still advantageous when large changes in expression are anticipated, while a weighted log2 approach performs better for smaller changes. CONCLUSION: VST can be recommended for summarized Illumina data regardless of which Illumina pre-processing options have been used. However, using the raw data is still encouraged whenever possible

Genome-Wide Identification of Functionally Distinct Subsets of Cellular mRNAs Associated with Two Nucleocytoplasmic-Shuttling Mammalian Splicing Factors

Background: Pre-mRNA splicing is an essential step in gene expression that occurs co-transcriptionally in the cell nucleus, involving a large number of RNA binding protein splicing factors, in addition to core spliceosome components. Several of these proteins are required for the recognition of intronic sequence elements, transiently associating with the primary transcript during splicing. Some protein splicing factors, such as the U2 small nuclear RNP auxiliary factor (U2AF), are known to be exported to the cytoplasm, despite being implicated solely in nuclear functions. This observation raises the question of whether U2AF associates with mature mRNA-ribonucleoprotein particles in transit to the cytoplasm, participating in additional cellular functions. Results: Here we report the identification of RNAs immunoprecipitated by a monoclonal antibody specific for the U2AF 65 kDa subunit (U2AF$^{65}$) and demonstrate its association with spliced mRNAs. For comparison, we analyzed mRNAs associated with the polypyrimidine tract binding protein (PTB), a splicing factor that also binds to intronic pyrimidine-rich sequences but additionally participates in mRNA localization, stability, and translation. Our results show that 10% of cellular mRNAs expressed in HeLa cells associate differentially with U2AF$^{65}$ and PTB. Among U2AF$^{65}$ -associated mRNAs there is a predominance of transcription factors and cell cycle regulators, whereas PTB-associated transcripts are enriched in mRNA species that encode proteins implicated in intracellular transport, vesicle trafficking, and apoptosis. Conclusion: Our results show that U2AF$^{65}$ associates with specific subsets of spliced mRNAs, strongly suggesting that it is involved in novel cellular functions in addition to splicing

Expression microarray reproducibility is improved by optimising purification steps in RNA amplification and labelling.

BACKGROUND: Expression microarrays have evolved into a powerful tool with great potential for clinical application and therefore reliability of data is essential. RNA amplification is used when the amount of starting material is scarce, as is frequently the case with clinical samples. Purification steps are critical in RNA amplification and labelling protocols, and there is a lack of sufficient data to validate and optimise the process. RESULTS: Here the purification steps involved in the protocol for indirect labelling of amplified RNA are evaluated and the experimentally determined best method for each step with respect to yield, purity, size distribution of the transcripts, and dye coupling is used to generate targets tested in replicate hybridisations. DNase treatment of diluted total RNA samples followed by phenol extraction is the optimal way to remove genomic DNA contamination. Purification of double-stranded cDNA is best achieved by phenol extraction followed by isopropanol precipitation at room temperature. Extraction with guanidinium-phenol and Lithium Chloride precipitation are the optimal methods for purification of amplified RNA and labelled aRNA respectively. CONCLUSION: This protocol provides targets that generate highly reproducible microarray data with good representation of transcripts across the size spectrum and a coefficient of repeatability significantly better than that reported previously.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Agreement between different methods to measure the active drag coefficient in front-crawl swimming

The aim of this study was to analyze the agreement of the active drag coefficient measured through drag and propulsion methods. The sample was composed of 18 swimmers (nine boys: 15.9 ± 0.9 years; nine girls: 15.3 ± 1.2 years) recruited from a national swimming team. The velocity perturbation method was used as the drag measurement system and the Aquanex system as the propulsion system. For both sexes combined, the frontal surface area was 0.1128 ± 0.016 m2, swim velocity 1.54 ± 0.13 m∙s-1, active drag 62.81 ± 11.37 N, propulsion 68.81 ± 12.41 N. The level of the active drag coefficient agreement was calculated based on the mean values comparison, simple linear regression, and Bland Altman plots. The mean data comparison revealed non-significant differences (p > 0.05) between methods to measure the active drag coefficient. Both the linear regression (R2 = 0.82, p < 0.001) and Bland Altman plots revealed a very high agreement. The active drag coefficient should be the main outcome used in the interpretation of the swimmers’ hydrodynamic profile, because it is less sensitive to swimming velocity. Coaches and researchers should be aware that the active drag coefficient can also be calculated based on propulsion methods and not just based on drag methods. Thus, the swimming community can now use different equipment to measure the hydrodynamics of their swimmersThis work was supported by national funds (FCT - Portuguese Foundation for Science and Technology) under the project UIDB/DTP/04045/2020info:eu-repo/semantics/publishedVersio

Aerodynamic analysis of human walking, running and sprinting by numerical simulations

The drag in walking, running, and sprinting locomotion can be assessed by analytical procedures and experimental techniques. However, assessing the drag variations by these three main locomotion’s (i.e., walking, running, and sprinting) were not found using computational fluid dynamics. (CFD). Thus, the aim of this study was two-fold: (1) to assess the aerodynamics of human walking, running, and sprinting by CFD technique; 2) compare such aerodynamic characteristics between walking and running. Three 3D models were produced depicting the walking, running, and sprinting locomotion techniques, converted to computer aided design models and meshed. The drag varied with 4 locomotion type. Walking had the lowest drag, followed-up by running and then sprinting. At the same velocities, the drag was larger in walking than in running and increased with velocity. In conclusion, drag varied with locomotion type. Walking had the lowest drag, followed-up by running and then sprinting. At the same velocities, the drag was larger in walking than in running and increased with velocity.This project was founded by the Portuguese Foundation for Science and Technology, I.P. (project UIDB04045/2020)info:eu-repo/semantics/publishedVersio

A MECHANICAL SPEEDO-METER TO ASSESS SWIMMER’S HORIZONTAL INTRA-CYCLIC VELOCITY: VALIDATION FOR BREASTSTROKE AND BUTTERFLY STROKE

The aim was to validate a system to assess speed-fluctuation at breaststroke and butterfly stroke. 12 boys and 11 undertook a set of maximal 2x25 m swims (Breaststroke and Butterfly stroke). The speedo-meter cable was attached to the subject’s hip and data was acquired on-line. At the same time, subjects were recorded in the sagital plane with an underwater video camera enabling the hip to be digitized. The following were analysed: (i) maximal velocity; (ii) minimal velocity; (iii) difference between maximal and minimal velocity; (iv) coefficient of variation. There were no significant differences for the mean values between methods, except for the coefficient of variation. Linear regression models were highly related. More than 80% of the Bland-Altman plots were within the 1.96 standard-deviation criteria used as a rule of thumb for technique validation