Background: Extracellular vesicles (EVs) are recognized as potent vehicles for intercellular communication. To date, there is little information available regarding the role of EVs during the early stages of human embryonic development. The aim of this study was to develop techniques for the recovery of EVs secreted by a single human embryo in an in vitro culture system. The EVs were characterized according to size, concentration and electrical surface properties (zeta potential), in order to understand the role of EVs production in human embryos for determination of their quality at early stages of development. Methods: Human embryos were produced by in vitro fertilization (IVF) for 24 h in fertilization medium, cultured individually for 48 h (3 days) in cleavage medium and additionally 48 h in blastocyst medium (day 5). Conditioned media, at days 3 and 5 post-IVF, was collected and EVs were isolated using a series of centrifugations and size-exclusion chromatography. The size, concentration and zeta potential of EVs were characterized using a nanoparticle tracking analysis. Results: Using this method of isolation, we were able to collect and characterize EVs produced by a single human embryo. Analysis confirmed the presence of EVs at early stages of development, with the concentration of EVs being higher in early blastocysts (day 5), as compared to 4-8 cell-stage embryos (day 3). Moreover, already at day 3, we were able to discriminate between embryos that were properly developing and those that were later visually determined as degrading at day 5. The data indicates that embryos following normal development at day 3 but degrading at later stages (day 5) were producing significantly higher number of EVs (with size range of 100-160 nm) compared with those developing properly at day 3 and later progressing to early blastocysts at day 5. Summary/conclusion: In conclusion, we have developed a sensitive protocol for the isolation of EVs from human embryos cultured individually. We have demonstrated that human embryos secrete EVs in varying amounts and sizes during the early stages of their development. Further investigations are needed to establish EV characteristics of early human embryo as a quality marker for human clinical embryology.
Background and aims: Next to characteristic motor triad of Parkinson’s Disease (PD) due to loss of nigrostriatal neurons, more symptoms associated with non-neuronal tissues are emerging. Little is known about the molecular alterations underlying dermatologic issues or epidemiologic associations like increased incidence of melanoma in PD. The aim is to give an overview of the altered gene expression profiles of PD skin and blood using the novel method of RNA Sequencing. Networks of different genes are analyzed to map affected pathways that contribute to pathomolecular mechanism of PD in the periphery.
Methods: Whole transcriptomic profiling of 12+12 idiopathic PD patients’ and matched controls’ skin biopsies and venous whole blood was performed with highthroughput RNA-sequencing analysis. Followingly, pathway analysis of differentially changed gene expressions was performed. The results were validated using RT-qPCR.
Results: PD skin RNA-Seq resulted in a large collection of over 1000 differentially expressed genes, among which a clear pattern of global downregulation appeared. In blood, the differential changes were more subtle, blood being a heterogenous tissue. Pathways associated with mitochondrial metabolism and protein degradation by the ubiquitin-proteasome system were dysregulated in both.
Conclusion: The concordance of these results with previous gene expression profiling studies demonstrate that the molecular alterations in PD leading to neurodegeneration in the CNS are systemic and manifest also in peripheral tissues. Major affected pathways include dysfunction in protein metabolism, mitochondrial dysfunction and impaired immune system. Homeostatic imbalance in the skin can lead to increased susceptibility to mutagenic hazards and provide a possible molecular link between melanoma and PD.
Abstract Osteophytes are a prominent feature of osteoarthritis (OA) pathology. RNA-seq of osteophytes revealed patterns corresponding to active ECM re-modulation and participation of mast cells. The cells recruitment and their activity status were confirmed by anti-TPSAB1 and anti-FC epsilon RI antibodies in immunohistochemistry. Besides subchondral bone, which is a logical yet unproven route for the cells deployment into osteophytes, the authors propose that OA synovial fluid (SF) is necessary and sufficient for maturation of mast cell precursors (MCPs) in this channel. The authors present evidences to support their claim in the form of IHC, proteomics analysis of SF samples and in vitro cell differentiation assay, wherein human monocytes (ThP1) and hematopoietic stem cells (HSCs) showed differentiation in HLA-DR+/CD206+ and FCERI+ phenotype respectively after 9 days of SF treatment. These observations expound osteophytes and resident mast cells as yet unexplained functional epicentre in OA pathology.
Transcriptomic studies usually focus on either gene or exon-based annotations, and only limited experiments have reported changes in reads mapping to introns. The analysis of intronic reads allows the detection of nascent transcription that is not influenced by steady-state RNA levels and provides information on actively transcribed genes. Here, we describe substantial intronic transcriptional changes in Parkinson's disease (PD) patients compared to healthy controls (CO) at two different timepoints; at the time of diagnosis (BL) and three years later (V08). We used blood RNA-Seq data from the Parkinson's Progression Markers Initiative (PPMI) cohort and identified significantly changed transcription of intronic reads only in PD patients during this follow-up period. In CO subjects, only nine transcripts demonstrated differentially expressed introns between visits. However, in PD patients, 4873 transcripts had differentially expressed introns at visit V08 compared to BL, many of them in genes previously associated with neurodegenerative diseases, such as LRRK2, C9orf72, LGALS3, KANSL1AS1, and ALS2. In addition, at the time of diagnosis (BL visit), we identified 836 transcripts (e.g. SNCA, DNAJC19, PRRG4) and at visit V08, 2184 transcripts (e.g. PINK1, GBA, ALS2, PLEKHM1) with differential intronic expression specific to PD patients. In contrast, reads mapping to exonic regions demonstrated little variation indicating highly specific changes only in intronic transcription. Our study demonstrated that PD is characterized by substantial changes in the nascent transcription, and description of these changes could help to understand the molecular pathology underpinning this disease.
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder leading to upper and lower motoneurons degeneration. Although several mechanisms potentially involved in disease development have been identified, its pathogenesis is not fully understood. From the patient side, ALS diagnosis, still based on clinical criteria, can be difficult and may take up to 1 year. More than 30 genes have been associated to genetically inherited ALS, among which four (C9ORF72, SOD1, TARDBP and FUS) would explain around 60–70% of cases. However, familial ALS represents only 5–10% of ALS cases while the remaining are sporadic, with genetics explaining 6–10% of such cases only. In this context, short tandem repeats (STRs) expansions, have recently been found in clinically diagnosed ALS patients. In this review, we discuss the recent discoveries on ALS associated STRs and their potential as biomarkers as well as prognosis and therapy targets.
The human genome contains human endogenous retroviruses (HERVs) and they occupy around 8% of our genome. These viruses are derived from ancient infections of retroviruses that were reverse transcribed and integrated into the genome of ancestral host animals. These viral genomes were fixed and inherited for tens of millions of years ago. Almost all of the HERVs have lost their infectivity and no known HERV is presently. The role of HERVs has been shown in the different inflammatory diseases and cancers. The goal of present study was to analyse the genome‐wide transcriptional activity of HERVs in osteosarcoma. We performed RNA‐seq analysis of 36 tumor‐normal paired samples, raw sequencing data were used to identify HERV sequences and counts data as expressional activity. DESeq2 package was used for statistical analysis. We found massive and activation of HERVs in the osteosarcoma sample. More precisely, 58 different repeat element were differently described between normal and tumor bone with the FDR levele below 0.05. The most significantly upregulated elements were satellite elements ALR, ALR/ALPHA, ALR2, ALR6 and ALRA SAT elements. In addition, over‐expression of the pericentromeric human satellite II (HSATII) and the ancient mariner transposon (HSMAR1) was found. The most down‐regulated elements were HERVK22I, LTR40A, LTR40B, LTR40C and MER87B. Up‐regulation of the satellite elements during cancer has been described in some previous studies. However, genome‐wide analysis of all repetitive elements in the human genome is novel approach and could give additional information about the oncogenesis. Support or Funding Information This study was supported by institutional research funding (IUT20‐46) from the Estonian Research Agency and by the European Union's Seventh Framework Programme (FP7/2007‐2013) under grant agreement no. 602398 (Hyporth).
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