TransGeno - The ERA Chair for Translational Genomics and Personalized Medicine

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Adaptation of striated muscles to Wolframin deficiency in mice: Alterations in cellular bioenergetics
(Elsevier, 2020-01-11) Tepp, Kersti; Puurand, Marju; Timohhina, Natalja; Aid-Vanakova, Jekaterina; Reile, Indrek; Shevchuk, Igor; Chekulayev, Vladimir; Eimre, Margus; Peet, Nadežda; Kadaja, Lumme; Paju, Kalju; Käämbre, Tuuli
Background: Wolfram syndrome (WS), caused by mutations in WFS1 gene, is a multi-targeting disease affecting multiple organ systems. Wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. In this study we aimed to characterize alterations in energy metabolism in the cardiac and in the oxidative and glycolytic skeletal muscles in Wfs1-deficiency. Methods: Alterations in the bioenergetic profiles in the cardiac and skeletal muscles of Wfs1-knock-out (KO) male mice and their wild type male littermates were determined using high resolution respirometry, quantitative RT-PCR, NMR spectroscopy, and immunofluorescence confocal microscopy. Results: Oxygen consumption without ATP synthase activation (leak) was significantly higher in the glycolytic muscles of Wfs1 KO mice compared to wild types. ADP-stimulated respiration with glutamate and malate was reduced in the Wfs1-deficient cardiac as well as oxidative and glycolytic skeletal muscles. Conclusions: Wfs1-deficiency in both cardiac and skeletal muscles results in functional alterations of energy transport from mitochondria to ATP-ases. There was a substrate-dependent decrease in the maximal Complex I –linked respiratory capacity of the electron transport system in muscles of Wfs1 KO mice. Moreover, in cardiac and gastrocnemius white muscles a decrease in the function of one pathway were balanced by the increase in the activity of the parallel pathway. General significance: This work provides new insights to the muscle involvement at early stages of metabolic syndrome like WS as well as developing glucose intolerance
Analysis of repetitive element expression in the blood and skin of patients with Parkinson’s disease identifies differential expression of satellite elements
(Nature, 2019-03-13) Billingsley, Kimberley J.; Lättekivi, Freddy; Planken, Anu; Reimann, Ene; Kurvits, Lille; Kadastik-Eerme, Liis; Kasterpalu, Kristjan M.; Bubb, Vivien J.; Quinn, John P.; Kõks, Sulev; Taba, Pille
Repetitive elements (RE) constitute the majority of the human genome and have a range of functions both structural and regulatory on genomic function and gene expression. RE overexpression has been observed in several neurodegenerative diseases, consistent with the observation of aberrant expression of RE posing a mutagenic threat. Despite reports that associate RE expression with PD no study has comprehensively analyzed the role of these elements in the disease. This study presents the frst genome-wide analysis of RE expression in PD to date. Analysis of RNA-sequencing data of 12 PD patients and 12 healthy controls identified tissue-specific expression differences and more significantly, differential expression of four satellite elements; two simple satellite III (repName=CATTC_n and _GAATG_n) a high-copy satellite II (HSATII) and a centromeric satellite (ALR_Alpha) in the blood of PD patients. In support of the growing body of recent evidence associating REs with neurodegenerative disease, this study highlights the potential importance of characterization of RE expression in such diseases
Bovine follicular fluid and extracellular vesicles derived from follicular fluid alter the bovine oviductal epithelial cells transcriptome."
(MPDI, 2020-07-28) Hasan, Mohammad Mehedi; Viil, Janeli; Lättekivi, Freddy; Ord, James; Ul Ain Reshi, Qurat; Jääger, Kersti; Velthut-Meikas, Agne
While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.
Cellular, Extracellular and Extracellular Vesicular miRNA Profiles of Pre-Ovulatory Follicles Indicate Signaling Disturbances in Polycystic Ovaries
(MPDI, 2020-12-15) Rooda, Ilmatar; Hasan, Mohammad Mehedi; Roos, Kristine; Viil, Janeli; Andronowska, Aneta; Smolander, Olli-Pekka; Jaakma, Ülle; Salumets, Andres; Fazeli, Alireza; Velthut-Meikas, Agne
Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (p = 0.04) containing eight differentially expressed microRNAs (p < 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.
Cross-Sectional Study to Characterise Nicotine Dependence in Central Vietnamese Men
(SAGE, 2019) Kõks, Gea; Tran, Ha Diep Thi; Ngoc Bich Thi, Ngoc Bich Thi; Nhat Nguyen Hoang, Linh; Tran, Hue Minh Thi; Ngoc, Thanh Cao; Phuoc, Thuoc Doan; Ho, Xuan Dung; Duy, Binh Ho; Lättekivi, Freddy
Tobacco is legally permitted for adults, easily available, and the prevalence of smoking is high. Tobacco use is the largest preventable risk factor for human disease. To reduce smoking, many countries have introduced public policy to restrict the distribution of tobacco. The aim of this study was to analyse tobacco smoking and nicotine dependence in Central Vietnamese men around Hue and Da Nang cities. Nicotine dependence was measured using the Fagerström Test for Nicotine Dependence (FTND) score. The cohort contained total of 1822 Central Vietnamese men from Hue and Da Nang: 1453 smokers and 369 non-smokers. Individuals completed a questionnaire and factors such as smoking initiation, quitting behaviour, and success in quitting were also recorded. In the smoking group, the average amount of time in which the individual had smoked was 26.4years. Average FTND value was 4.02, median was 4, the first quartile was 2, and the third quartile was 6. In all, 431 smokers (30%) had an FTND score of 6 or higher; an FTND score of this value is considered to equate to an individual having high nicotine dependence. Therefore, it could be noted that high nicotine dependence is very common in Central Vietnam. High nicotine dependence was significantly correlated with years of smoking. The longer the smoking period, the higher the FTND score. A high FTND score correlated with the individual being less likely to successfully quit smoking. The results of the questionnaire demonstrate that even when there is no restriction in public policy concerning the distribution of tobacco, individuals still wish to quit smoking. This study identified a high prevalence of severe nicotine dependence in Central Vietnamese men and the majority of smokers wished to quit smoking. Consequently, the results of this study highlight the acute need for a specific program to aid smokers in Central Vietnam to quit smoking.
De novo and inherited pathogenic variants in collagen-related osteogenesis imperfecta
(Wiley Periodicals, Inc., 2019) Zhytnik, Lidiia; Maasalu, Katre; Duy, Ho Binh; Pashenko, Andrey; Khmyzov, Sergey; Reimann, Ene; Prans, Ele; Kõks, Sulev; Märtson, Aare
Background: Osteogenesis imperfecta (OI) is a rare genetic bone fragility disorder. In the current study, differences between the genotypes and phenotypes of de novo and inherited collagen‐related OI were investigated. Methods: A comparative analysis was performed of the genotypes and phenotypes of 146 unrelated inherited and de novo collagen I OI cases from Estonia, Ukraine, and Vietnam. Mutational analysis of the subjects and all available parents were per formed with Sanger sequencing. Results: Results showed that 56.16% of the OI cases were caused by de novo patho genic variants. The proportion of OI types OI1, OI4, and OI3 among subjects with inherited OI was 45.31%, 46.88%, and 7.81%, respectively. Among subjects with de novo OI, the proportions of OI types (OI1, OI4, and OI3) were almost equal. Both inherited and de novo OI pathogenic variants occurred more often in the COL1A1 gene than in the COL1A2. The majority of de novo cases were missense pathogenic variants, whereas inherited OI was mostly caused by loss of function pathogenic variants. Conclusion: In summary, there were significant differences between the phenotypes and genotypes of subjects with de novo and inherited OI. These findings may pro mote the further understanding of OI etiology, and assist with diagnostics proce dures, as well as with family plannin
Identification of an optimal method for extracting RNA from human skin biopsy, using domestic pig as a model system.
(Nature, 2019-12-27) Reimann, Ene; Kristi, Abram; Kõks, Sulev; Kingo, Külli; Fazeli, Alireza
To evaluate skin tissue gene expression patterns correctly, extracting sufficient quantities of good quality RNA is essential. However, RNA extraction from skin tissue is challenging, as the hyaluronic acid-collagen matrix is extremely difficult to homogenize. Although there are multiple ways to extract RNA from skin, there are no comparative studies that identify the most critical steps, e.g. sample collection, storage and homogenization. We analysed the various steps involved in RNA extraction (i.e. biopsy collection as dry biopsy or into nucleotide stabilizing reagents, different storage conditions, enzymatic digestion, stator-rotor and bead motion-based homogenizing combined with column-based RNA purification). We hypothesised that domestic pig skin is applicable as a model for human skin studies. Altogether twenty different workflows were tested on pig skin and the four most promising workflows were tested on human skin samples. The optimal strategy for extracting human skin RNA was to collect, store and homogenize the sample in RLT lysis buffer from the RNeasy Fibrous Tissue Kit combined with beta-mercaptoethanol. Both stator-rotor and bead motion-based homogenizing were found to result in high quality and quantity of extracted RNA. Our results confirmed that domestic pig skin can be successfully used as a model for human skin RNA studies.
Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers
(2020-06) Dissanayake, Keerthie; Nõmm, Monika; Lättekivi, Freddy; Ressaissi, Yosra; Godakumara, Kasun; Lavrits, Arina; Midekessa, Getnet; Viil, Janeli; Bæk, Rikke; Møller Jørgensen, Malene; Bhattacharjee, Sourav; Aneta, Andronowska; Salumets, Andres; Jaakma, Ülle; Fazeli, Alireza
Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.
Multicomponent biomarker approach improves the accuracy of diagnostic biomarkers for psoriasis vulgaris
(2019-10-14) Reimann, Ene; Lättekivi, Freddy; Keermann, Maris; Abram, Kristi; Kõks, Sulev; Kingo, Külli; Fazeli, Alireza
Accurate biomarker-based diagnosis of psoriasis vulgaris has remained a challenge; no reliable disease-specific biomarkers have yet been identified. There are several different chronic inflammatory skin diseases that can present similar clinical and dermoscopy features to psoriasis vulgaris, making accurate diagnosis more difficult. Both literature-based and data-driven selection of biomarker was conducted to select candidates for a multicomponent biomarker for psoriasis vulgaris. Support vector machine-based classification models were trained using gene expression data from locally recruited patients and validated on 7 public datasets, which included gene expression data of other inflammatory skin diseases in addition to psoriasis vulgaris. The resulting accuracy of the best classification model based on the expression levels of 4 genes (IL36G, CCL27, NOS2 and C10orf99) was 96.4%, outperforming classification based on other marker gene combinations, which were more affected by variability in gene expression profiles between different datasets and patient groups. This approach has the potential to fill the void of clinically applicable diagnostic biomarkers for psoriasis vulgaris and other inflammatory skin diseases.
Paternal effects in a wild-type zebrafish implicate a role of sperm-derived small RNAs
(Wiley Online Library, 2020-06-11) Ord, James; Heath, Paul R.; Fazeli, Alireza; Watt, Penelope J.
While the importance of maternal effects has long been appreciated, a growing body of evidence now points to the paternal environment having an important influence on offspring phenotype. Indeed, research on rodent models suggests that paternal stress leaves an imprint on the behaviour and physiology of offspring via nonge netic information carried in the spermatozoa; however, fish have been understudied with regard to these sperm-mediated effects. Here, we investigated whether the ze brafish was subjected to heritable influences of paternal stress by exposing males to stressors (conspecific-derived alarm cue, chasing and bright light) before mating and assessing the behavioural and endocrine responses of their offspring, including their behavioural response to conspecific-derived alarm cue. We found that after males are exposed to stress, their larval offspring show weakened responses to stressors. Small RNA sequencing subsequently revealed that the levels of several small noncoding RNAs, including microRNAs, PIWI-interacting RNAs and tRNA-derived small RNAs, were altered in the spermatozoa of stressed fathers, suggesting that stress-induced alterations to the spermatozoal RNA landscape may contribute to shaping offspring phenotype. The work demonstrates that paternal stress should not be overlooked as a source of phenotypic variation and that spermatozoal small RNAs may be important intergenerational messengers in fish.
Specific trophoblast transcripts transferred by extracellular vesicles affect gene expression in endometrial epithelial cells and may have a role in embryo-maternal crosstalk
(BioMed Central Ltd, 2019-11-14) Es-Haghi, Masoumeh; Godakumara, Kasun; Häling, Annika; Lättekivi, Freddy; Lavrits, Arina; Viil, Janeli; Andronowska, Aneta; Nafee, Tamer; James, Victoria; Jaakma, Ülle; Salumets, Andres; Fazeli, Alireza
Background. Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA. Methods. We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR. Results. We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication. Conclusion. Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.
Spermatozoa induce transcriptomic alterations in bovine oviductal epithelial cells prior to initial contact
(2020-09-03) Qurat Ul Ain, Qurat; Viil, Janeli; Ord, James; Lättekivi, Freddy; Godakumara, Kasun; Hasan, Mohammad Mehedi; Nõmm, Monika; Jääger, Kersti; Velthut-Meikas, Agne; Jaakma, Ülle; Salumets, Andres; Fazeli, Alireza
The capability of spermatozoa to directly influence maternal gene expression is already established. Indeed, some of the changes induced by spermatozoa may have a direct functional importance in the pre-conceptional period. Although the mechanisms underlying these sperm-maternal interactions are not well characterized, it is possible that they could involve ligands that are released from the spermatozoa. This study therefore aimed to test whether physical contact between bovine spermatozoa and bovine oviductal epithelial cells (BOECs) is a prerequisite for spermatozoa-induced gene expression changes. We used two co-culture models: a contact co-culture model in which spermatozoa interact directly with BOECs, and a non-contact co-culture model in which an insert with the pore size of 0.4 μm was placed between spermatozoa and BOECs. Messenger RNA sequencing analysis of BOECs by RNA-seq revealed ten differentially expressed genes in contact system and 108 differentially expressed genes in the non-contact system after 10 h of co-culture. Retinol metabolism pathway and ovarian steroidogenesis pathway were significantly enriched in the non-contact co-culture system. Q-PCR analysis revealed that transcriptional responses can be rapid, with increased expression of four genes (DHRS3, CYP1B1, PTGS2, and ATF3) detectable within just 90 min of co-incubation, but with expression levels highly dependent on the type of co-culture system. The findings from our study demonstrate that direct contact with spermatozoa is not necessary to induce changes in gene expression of oviductal epithelial cells, suggesting that spermatozoa may be able to signal to maternal tissues in advance of their arrival.
Time-critical influences of gestational diet in a seahorse model of male pregnancy
(The Company of Biologists, 2020-02-07) Otero-Ferrer, Francisco; Lättekivi, Freddy; Ord, James; Reimann, Ene; Kõks, Sulev; Izquierdo, Marisol; Holt, William V.; Fazeli, Alireza
Sex role reversal is not uncommon in the animal kingdom but is taken to the extreme by the Syngnathidae, in which male pregnancy is one of the most astonishing idiosyncrasies. However, critical and time-dependent environmental effects on developing embryos, such as those extensively studied in mammalian pregnancy, have not been investigated in the male pregnancy context. Here, we tested the hypothesis that seahorse pregnancy is subject to ‘critical windows’ of environmental sensitivity by feeding male long-snouted seahorses (Hippocampus reidi) a diet deficient in polyunsaturated fatty acids during specific periods before and during pregnancy. Despite embryos being nourished principally by maternally supplied yolk, we found that offspring morphology, fatty acid composition and gene expression profiles were influenced by paternal diet in a manner that depended critically on the timing of manipulation. Specifically, reception of a diet deficient in polyunsaturated fatty acids in the days preceding pregnancy resulted in smaller newborn offspring, while the same diet administered towards the end of pregnancy resulted in substantial alterations to newborn gene expression and elongation of the snout at 10 days old. Although paternal diet did not affect 10 day survival, the observed morphological alterations in some cases could have important fitness consequences in the face of natural selective pressures such as predation and food availability. Our results demonstrate that, under male pregnancy, fine-scale temporal variation in parental diet quality and subsequent critical window effects should not be overlooked as determinants of developing offspring fitness.
Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability
(ACS Publications, 2020-06-30) Midekessa, Getnet; Godakumara, Kasun; Ord, James; Viil, Janeli; Lättekivi, Freddy; Dissanayake, Keerthie; Kopanchuk, Sergei; Rinken, Ago; Aneta, Andronowska; Bhattacharjee, Sourav; Rinken, Toonika; Fazeli, Alireza
Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl2, and AlCl3) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant (p < 0.05) lowering of the ZP. Moreover, the ZP was significantly (p < 0.05) less negative in the presence of ions with higher valency (Al3+/Ca2+) than in the presence of monovalent ones (Na+/K+). Besides, the ZP of EVs became less negative at acidic pH, and vice versa. The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs.

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