RNA-seq analysis of single bovine blastocysts

Background: Use of RNA-Seq presents unique benefits in terms of gene expression analysis because of its wide dynamic range and ability to identify functional sequence variants. This technology provides the opportunity to assay the developing embryo, but the paucity of biological material available from individual embryos has made this a challenging prospect. Results: We report here the first application of RNA-Seq for the analysis of individual blastocyst gene expression, SNP detection, and characterization of allele specific expression (ASE). RNA was extracted from single bovine blastocysts (n = 5), amplified, and analyzed using high-throughput sequencing. Approximately 38 million sequencing reads were generated per embryo and 9,489 known bovine genes were found to be expressed, with a high correlation of expression levels between samples (r > 0.97). Transcriptomic data was analyzed to identify SNP in expressed genes, and individual SNP were examined to characterize allele specific expression. Expressed biallelic SNP variants with allelic imbalances were observed in 473 SNP, where one allele represented between 65-95% of a variant’s transcripts. Conclusions: This study represents the first application of RNA-seq technology in single bovine embryos allowing a representation of the embryonic transcriptome and the analysis of transcript sequence variation to describe specific allele expression.EEA BalcarceFil: Chitwood, James L. University of California Davis. Department of Animal Science; Estados UnidosFil: Rincon, Gonzalo. University of California Davis. Department of Animal Science; Estados UnidosFil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Medrano, Juan F. University of California Davis. Department of Animal Science; Estados UnidosFil: Ross, Pablo J. University of California Davis. Department of Animal Science; Estados Unido

Molecular taxonomy. Bioinformatics and practical evaluation

Summary Molecular taxonomy is a field that studies the diversity of organisms based on molecular markers. This work is devoted to develop a methodology of molecular taxonomy of small organisms. The ribosomal RNA (rRNA) is used as a molecular marker since its nucleotide sequence includes stretches of various levels of conservation, which can be used as species, genus and taxa specific regions. The organisms live in complex communities. To discover the composition of these communities, a hybridization assay employing oligonucleotide microarrays is developed to indicate the presence of a certain rRNA, in a sample under investigation. An additional method based on the pyrosequencing process is proposed here. In this case the mixture of rRNA genes is directly sequenced and the proportion of individual sequences is then calculated from the obtained pyrogram. The work comprises two parts: theoretical bioinformatics and practical evaluation. The first part tackles the problem of DNA-RNA duplex stability prediction. As a result, an ad hoc stability function is proposed. An algorithm and a program are developed for the design of oligonucleotides employed in the microarray approach. The kinetics of DNA-RNA duplex dissociation is considered as well. In addition, the formalism of the pyrosequencing approach is elaborated theoretically. The experimental part deals with the issues of oligonucleotide microarray establishment, including fabrication, immobilization, hybridization and scanning. A real-time kinetic setup for observing the RNA-DNA duplex dissociation was developed. The theoretical findings and quality of the oligonucleotide design are practically evaluated. The theory is found to be in a good accordance with experiment. The pyrosequencing approach is tested as well and is demonstrated to have enough power to discover the composition of a complex mixture of rRNA genes

Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion

ABSTRACT The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture. Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture

Analysis of genome activation in early bovine embryos by bioinformatic evaluation of RNA-Seq data

During maternal-to-embryonic transition, control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation. Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts and to date there is no systematic study addressing the activation of specific genes during several stages of early bovine embryo development. Using the bovine model system, comparative analyses of RNA-seq data set were performed. The sequencing libraries had been constructed starting with germinal vesicle (GV) and metaphase II (MII) oocytes and embryos at the four-cell, eight-cell, 16-cell and blastocyst stage. The embryos had been generated in vitro by fertilization of Bos taurus taurus oocytes with sperm of a Bos taurus indicus sire. In total, approximately 13,000 RNA species could be identified in oocytes and each embryonic stages. The number of identified differential abundant transcripts increased in the course of development from roughly 100 to several thousands, with a sharp rise at the eight-cell stage. A bioinformatic approach could be developed to capture maternally delivered and de novo synthesized RNA species separately. It sensitively identified actively transcribed genes despite the fact that comparative analyses failed due to presence of the huge amount of RNA provided by the oocyte. Actively transcribed RNA species could be identified for approximately 8,000 genes, the majority of them at the eight-cell stage. This finding indicated, that the majority of all RNA species provided by oocytes was de novo transcribed during early embryonic development. Furthermore, it could be shown that the de novo transcription of larger genes was initiated later in embryonic development than smaller ones. A procedure was established to identify Bos t. indicus specific SNPs in RNA-Seq datasets which identified more than 60,000 SNPs occurring in 20% of all annotated genes. A major part of these SNPs could be detected at the eight-cell stage. This procedure enables a way to capture and study allele-specific transcription during early embryonic development. The described bioinformatic approaches were used to study major genome activation, an important step in the maternal-to-embryonic transition. More than 4,000 genes were de novo transcribed during major genome activation, which was found to occur at the eight-cell stage. These genes were functionally related to transcription, translation and their regulation. In summary, this thesis created and applied a powerful tool set for bioinformatic dissection of processes occurring during development of early bovine embryos and provided unprecedented insights in major genome activation

Towards A Unified Model Of Sperm Chromatin Structure

Sperm possess several layers of information that are delivered to the oocyte alongside the paternal DNA. Examples of potential sperm borne molecular cues of probable use to the embryo include RNAs and local and global chromatin structure. To identify candidate sperm RNAs that likely reach the oocyte cytoplasm following fertilization patterns of transcript compartmentalization in the mature gamete were identified. Though all sperm RNAs exhibited a preferential peripheral enrichment, a subset of RNAs were identified in which this trend was reduced. These RNAs are thought to be embedded with perinuclear theca and are correlated with late spermatogenic transcription. Malat1, a well-known nuclear non-coding RNAs, was relatively abundant within the intra-nuclear compartment of the gamete although enriched within the sperm extra-nuclear compartment. If these transcript are localized to the condensed sperm nucleus Maltat1 may contribute to the retention of somatic-like chromatin structures following nuclear remodeling. Histone-bound regions which persist in mature sperm are of interest as they may be informative of past spermatogenic and future embryonic genomic regulation. Genome wide nucleosome mapping in mature mouse sperm was complimented with a nuclease foot printing approach to identify sites of factor retention throughout the paternal gamete. Applying this analysis to a transgenic mouse model harboring the human protamine locus, highlighted the potential regulatory impact of the chromatin environment at the local and domain level. Consideration of these results within the context of the endogenous mouse protamine locus identified a candidate transcriptional regulatory mechanism. Factors predicted to be bound in mature sperm were correlated with genomic elements utilized in the testis and the very early embryo. This included Ctcf, which was significantly enriched within the boundary regions of topologically associated domains and the promoter regions of genes expressed in the zygote. These patterns were not observed in human or bull sperm. It is suggested that delivery of the paternal genome in association with regulatory factors may reflect the accelerated preimplantation development of the murine embryo. Sites of Ctcf retention in mature sperm were considered within a novel model of spermatogenic nuclear remodeling

Cytosolic nucleic acids as new markers of senescence and hyperglycemia-induced inflammation

openAging is characterized by a gradual functional decline resulting from a complex interaction between genetic, epigenetic and stochastic factors and the rate of aging is recognized as the most important risk factor for the development of the most common age-related diseases (ARDs). Research on aging process is currently focused on understanding the molecular mechanisms underlying age-related features accompanying on one hand, the onset of ARDs and on the other hand, the chances to reach a successful aging. A chronic, systemic, low-grade, age-related pro-inflammatory status, called “Inflammaging” is correlated with ARDs development. The two main culprits of inflammaging are the repeated stimulation of immune system over time and the increased burden of senescent cells. Senescent cells are able to modify the microenvironment acquiring a senescence-associated secretory phenotype (SASP). In patients affected by ADRs, the rate of aging process in increased, as well as the burden of senescent cells with SASP. In this scenario, a better understanding of the molecular mechanisms that promote cellular senescence is of basic and clinical relevance. A number of mechanisms promoting senescence have been well characterized. Increasing evidence suggest that nuclear DNA fragility and a sub-functional DNA damage response (DDR) is associated with an increase release of nucleic acids in cytoplasm, including RNA:DNA hybrids. However, only few studies have tempted to assess the pro- or anti-inflammatory effects of this misplaced nucleic acid pool in different cellular models. In this framework, we have analysed the cytoplasmic pool of misplaced nucleic acids in a model of human endothelial cells (HUVECs), in two prototypical stress conditions related to inflammaging, such as “replicative cellular senescence” and “hyperglycemic condition”. A significant increased amount of misplaced nucleic acids was observed in the cytoplasm of senescent cells compared to the younger ones, and in young cells cultured for 1-week in hyperglycemic condition compared to young cells cultured in normoglycemic medium. The cytosolic pool of misplaced nucleic acids is composed of dsDNA, including micronuclei, bubbles and telomere sequences, dsRNA and hybrids. A reduced expression of RNaseH2, the enzyme involved in the degradation of RNA moiety of RNA:DNA hybrids was observed in senescent cells compared to the younger ones and in young cells treated with hyperglycemic medium. The amounts of mis incorporated ribonucleotides, biomarkers od genome instability, were inversely related to RNaseH2 expression level, suggesting that both “senescence” and “hyperglycemic condition” are associated with increased genomic instability. Since the presence of nucleic acids in cytoplasm can activate a number of cytosolic receptors inducing the antiviral response, characterized by increased release of type 1 Interferon (IFN-1), we analysed cGAS/STING/IRF3 axis and IFN-1 expression in “senescence” and “hyperglycemic condition”. Surprisingly, a strong reduction of cGAS expression was observed in senescent cells in normo and hyperglycemic conditions, in association with the absence of IFN-1 modulation, whereas the expression of proinflammatory cytokines, such as IL-1beta, IL-6 and IL-8 was significantly increased. Overall, our results suggest an imbalance between antiviral and proinflammatory response in senescent cells and in young cells under hyperglycemic conditions. Increasing evidence suggest that senescent cells have an enhanced susceptibility to viral infections. We hypothesized that such imbalance between antiviral and proinflammatory responses could be, almost in part, a direct consequence of their ability “to tolerate” a high cytosolic nucleic acids load. Overall, our results pave the way to explain why old subjects, especially the elderly patients affected by diabetes, are more susceptible to adverse outcomes of infectious diseases.L'invecchiamento è caratterizzato da un graduale declino funzionale derivante da una complessa interazione tra fattori genetici e epigenetici insieme ad un'aumentata velocità di invecchiamento, riconosciuta come il fattore di rischio più importante per lo sviluppo delle malattie associate all'età più comuni (ARD). La ricerca sul processo di invecchiamento è attualmente focalizzata sulla comprensione dei meccanismi molecolari alla base delle caratteristiche legate all'età che accompagnano, da un lato, l'insorgenza di ARD e dall'altro, le possibilità di raggiungere un invecchiamento di successo. Uno stato pro-infiammatorio cronico, sistemico, di basso grado e legato all'età, chiamato "Inflammaging" è correlato allo sviluppo degli ARD. I due principali responsabili dell'inflammaging sono la stimolazione ripetuta del sistema immunitario nel tempo e l'aumento del numero di cellule senescenti. Le cellule senescenti sono in grado di modificare il microambiente acquisendo un fenotipo secretorio associato alla senescenza (SASP). Nei pazienti affetti da ADR, la velocità del processo d’invecchiamento è aumentato, così come il numero delle cellule senescenti con SASP. In questo scenario, una migliore comprensione dei meccanismi molecolari che promuovono la senescenza cellulare è di rilevanza di base e clinica. Prove crescenti suggeriscono che la fragilità del DNA nucleare e una risposta sub-funzionale al danno al DNA (DDR) si associano a un aumento del rilascio di acidi nucleici nel citoplasma, inclusi gli ibridi RNA:DNA. In questa cornice, abbiamo analizzato il pool di acidi nucleici rilasciati nel citoplasma in un modello di cellule endoteliali umane (HUVEC), in due condizioni di stress legate all'inflammaging, come la "senescenza replicativa cellulare " e la "condizione iperglicemica". È stata osservata una quantità significativa di acidi nucleici nel citoplasma delle cellule senescenti rispetto a quelle più giovani e nelle cellule giovani coltivate per 1 settimana in condizioni iperglicemiche rispetto alle giovani cellule coltivate in mezzo normoglicemico. Il pool citosolico di acidi nucleici è composto da dsDNA, tra cui micronuclei, bubbles e sequenze telomeriche, dsRNA e ibridi. Una ridotta espressione di RNasi H2, l'enzima coinvolto nella degradazione della componente a RNA degli ibridi RNA:DNA è stato osservato nelle cellule senescenti rispetto a quelle più giovani e nelle cellule giovani trattate con mezzo iperglicemico. Le quantità di ribonucleotidi mal incorporati, biomarcatori dell’instabilità genomica, sono inversamente correlate al livello di espressione di RNasi H2, suggerendo che sia la "senescenza" che la "condizione iperglicemica" sono associate a una maggiore instabilità genomica. Poiché la presenza di acidi nucleici nel citoplasma può attivare un certo numero di recettori citosolici che inducono la risposta antivirale, caratterizzata da un maggiore rilascio di interferone di tipo 1 (IFN-1), abbiamo analizzato l'asse cGAS/STING/IRF3 e l'espressione IFN-1 in "senescenza" e "condizione iperglicemica". Sorprendentemente, una forte riduzione dell'espressione di cGAS è stata osservata nelle cellule senescenti in condizioni normo e iperglicemiche, in associazione con l'assenza di modulazione di IFN-1, mentre l'espressione di citochine pro-infiammatorie, come IL-1 beta, IL-6 e IL-8 è significativamente aumentata. Nel complesso, i nostri risultati suggeriscono uno squilibrio tra la risposta antivirale e pro-infiammatoria nelle cellule senescenti e nelle cellule giovani in condizioni iperglicemiche. Crescenti evidenze suggeriscono che le cellule senescenti hanno una maggiore suscettibilità alle infezioni virali. Abbiamo ipotizzato che tale squilibrio tra risposte antivirali e pro-infiammatorie potrebbe essere, almeno in parte, una conseguenza diretta della loro capacità di "tollerare" un elevato carico di acidi nucleici citosolici.SALUTE DELL'UOMOopenRamini, Debora

Norovirus Evolution: Understanding and characterising the emergence of novel strains in the population.

Human noroviruses (HuNoVs) are distributed globally, affect all age groups and place a significant burden upon health services. The diversity of this RNA virus is thought to play a significant role in the persistence of HuNoVs as the main cause of non-bacterial gastroenteritis globally. Molecular diagnostics have been critical for understanding the epidemiology of outbreaks and sporadic cases, and to design and implement effective intervention strategies and disease control measures. Immunocompromised individuals are widely considered to be a reservoir for epidemic variants of HuNoV and whilst there are studies investigating the emergence of novel strains in an immunocompetent general population, reports at the the individual level are scarce. Three separate methodologies were developed to characterise HuNoV persistence in acute convalescent and chronic infection. First, a standardised quantification method to accurately quantify the most prevalent HuNoV genogroup. Second, a PGM-MB capture method to select HuNoV prior to massively parallel sequencing (MPS). Third, an assay to measure host specific coproantibody responses to three epidemic variants from different epochs. Quantification of longitudinal samples from individuals with acute or chronic HuNoV infection showed the virus distribution was homogenous in stool and an RNA external standard, in contrast to DNA, did not underestimate virus titre. HuNoV PGM-MB capture meant near complete viral genomes could be recovered at variable mean coverage. A bioinformatics pipeline demonstrated over the course of chronic infection allele frequencies were much more variable. In acute infection, minor alleles were present at a much lower frequency, but potential immune escape mutants were present. Immune escape mutants existed as minority variants or conserved mutations in the consensus sequence, and were in the presence of HuNoV specific-coproantibody, which were mapped to the protein surface. In HuNoV chronic infection, immune pressure is variable or non-existent, and therefore epidemic variants could emerge over long periods of infection by random chance. However, under immune pressure exerted by coproantibodies, escape variants may be seen. In three individuals, acute HuNoV symptomatic infection occurred despite the presence of specific secretory Ab responses to the VLP classed as the closest phylogenetic relative. The closest relative (Sydney 2012), differed at two amino acids, one of which has been previously described (A340T) as belonging to an epitope, and another which can be classed as having a potential role in immune escape (A323T). A single individual with acute HuNoV infection established a more prominent response to an earlier strain of HuNoV, rather than two contemporary strains, which proposes a role for Original Antigenic Sin (OAS) or Antigenic Seniority in the secretory Ab immunity. Finally, the use of MPS in outbreak tracking was assessed and compared to the currently used amplicon and Sanger based method. Overall both methods showed significant correlation. However, MPS provided greater depth and the ability to identify variants among samples within an outbreak that represented consensus changes in one or more samples from the same outbreak. This meant that the MPS data would have been able to link all the samples into a single outbreak or transmission network, where the current Sanger sequencing may not have been able to link them all