Single-Cell Analysis Uncovers a Vast Diversity in Intracellular Viral Defective Interfering RNA Content Affecting the Large Cell-to-Cell Heterogeneity in Influenza A Virus Replication

Virus replication displays a large cell-to-cell heterogeneity; yet, not all sources of this variability are known. Here, we study the effect of defective interfering (DI) particle (DIP) co-infection on cell-to-cell variability in influenza A virus (IAV) replication. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNA) and are, thus, defective in virus replication. Moreover, they interfere with virus replication. Using single-cell isolation and reverse transcription polymerase chain reaction, we uncovered a large between-cell heterogeneity in the DI vRNA content of infected cells, which was confirmed for DI mRNAs by single-cell RNA sequencing. A high load of intracellular DI vRNAs and DI mRNAs was found in low-productive cells, indicating their contribution to the large cell-to-cell variability in virus release. Furthermore, we show that the magnitude of host cell mRNA expression (some factors may inhibit virus replication), but not the ribosome content, may further affect the strength of single-cell virus replication. Finally, we show that the load of viral mRNAs (facilitating viral protein production) and the DI mRNA content are, independently from one another, connected with single-cell virus production. Together, these insights advance single-cell virology research toward the elucidation of the complex multi-parametric origin of the large cell-to-cell heterogeneity in virus infections

Cell autonomous requirement of neurofibromin (Nf1) for postnatal muscle hypertrophic growth and metabolic homeostasis

Background Neurofibromatosis type 1 (NF1) is a multi-organ disease caused by mutations in neurofibromin 1 (NF1). Amongst other features, NF1 patients frequently show reduced muscle mass and strength, impairing patients' mobility and increasing the risk of fall. The role of Nf1 in muscle and the cause for the NF1-associated myopathy are mostly unknown. Methods To dissect the function of Nf1in muscle, we created muscle-specific knockout mouse models for NF1, inactivatingNf1in the prenatal myogenic lineage either under the Lbx1 promoter or under the Myf5 promoter. Mice were analysed during prenatal and postnatal myogenesis and muscle growth. Results Nf1(Lbx1)and Nf1(Myf5)animals showed only mild defects in prenatal myogenesis. Nf1(Lbx1)animals were perinatally lethal, while Nf1(Myf5)animals survived only up to approximately 25 weeks. A comprehensive phenotypic characterization of Nf1(Myf5)animals showed decreased postnatal growth, reduced muscle size, and fast fibre atrophy. Proteome and transcriptome analyses of muscle tissue indicated decreased protein synthesis and increased proteasomal degradation, and decreased glycolytic and increased oxidative activity in muscle tissue. High-resolution respirometry confirmed enhanced oxidative metabolism in Nf1(Myf5)muscles, which was concomitant to a fibre type shift from type 2B to type 2A and type 1. Moreover, Nf1(Myf5)muscles showed hallmarks of decreased activation of mTORC1 and increased expression of atrogenes. Remarkably, loss of Nf1 promoted a robust activation of AMPK with a gene expression profile indicative of increased fatty acid catabolism. Additionally, we observed a strong induction of genes encoding catabolic cytokines in muscle Nf1(Myf5)animals, in line with a drastic reduction of white, but not brown adipose tissue. Conclusions Our results demonstrate a cell autonomous role for Nf1 in myogenic cells during postnatal muscle growth required for metabolic and proteostatic homeostasis. Furthermore, Nf1 deficiency in muscle drives cross-tissue communication and mobilization of lipid reserves

ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer

Background Overexpression of ERG transcription factor due to genomic ERG- rearrangements defines a separate molecular subtype of prostate tumors. One of the consequences of ERG accumulation is modulation of the cell’s gene expression profile. Tudor domain-containing protein 1 gene (TDRD1) was reported to be differentially expressed between TMPRSS2:ERG-negative and TMPRSS2:ERG-positive prostate cancer. The aim of our study was to provide a mechanistic explanation for the transcriptional activation of TDRD1 in ERG rearrangement-positive prostate tumors. Methodology/Principal Findings Gene expression measurements by real-time quantitative PCR revealed a remarkable co-expression of TDRD1 and ERG (r2 = 0.77) but not ETV1 (r2<0.01) in human prostate cancer in vivo. DNA methylation analysis by MeDIP-Seq and bisulfite sequencing showed that TDRD1 expression is inversely correlated with DNA methylation at the TDRD1 promoter in vitro and in vivo (ρ = −0.57). Accordingly, demethylation of the TDRD1 promoter in TMPRSS2:ERG-negative prostate cancer cells by DNA methyltransferase inhibitors resulted in TDRD1 induction. By manipulation of ERG dosage through gene silencing and forced expression we show that ERG governs loss of DNA methylation at the TDRD1 promoter-associated CpG island, leading to TDRD1 overexpression. Conclusions/Significance We demonstrate that ERG is capable of disrupting a tissue-specific DNA methylation pattern at the TDRD1 promoter. As a result, TDRD1 becomes transcriptionally activated in TMPRSS2:ERG-positive prostate cancer. Given the prevalence of ERG fusions, TDRD1 overexpression is a common alteration in human prostate cancer which may be exploited for diagnostic or therapeutic procedures

High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal, Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options

Iranome: A catalogue of genomic variations in the Iranian population

Considering the application of human genome variation databases in precision medicine, population-specific genome projects are continuously being developed. However, the Middle Eastern population is underrepresented in current databases. Accordingly, we established Iranome database (www.iranome.com) by performing whole exome sequencing on 800 individuals from eight major Iranian ethnic groups representing the second largest population of Middle East. We identified 1,575,702 variants of which 308,311 were novel (19.6%). Also, by presenting higher frequency for 37,384 novel or known rare variants, Iranome database can improve the power of molecular diagnosis. Moreover, attainable clinical information makes this database a good resource for classifying pathogenicity of rare variants. Principal components analysis indicated that, apart from Iranian-Baluchs, Iranian-Turkmen, and Iranian-Persian Gulf Islanders, who form their own clusters, rest of the population were genetically linked, forming a super-population. Furthermore, only 0.6% of novel variants showed counterparts in "Greater Middle East Variome Project", emphasizing the value of Iranome at national level by releasing a comprehensive catalog of Iranian genomic variations and also filling another gap in the catalog of human genome variations at international level. We introduce Iranome as a resource which may also be applicable in other countries located in neighboring regions historically called Greater Iran (Persia)

Occasional paternal inheritance of the germline-restricted chromosome in songbirds

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby out-competing other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness

Ice-Age Climate Adaptations Trap the Alpine Marmot in a State of Low Genetic Diversity.

Some species responded successfully to prehistoric changes in climate [1, 2], while others failed to adapt and became extinct [3]. The factors that determine successful climate adaptation remain poorly understood. We constructed a reference genome and studied physiological adaptations in the Alpine marmot (Marmota marmota), a large ground-dwelling squirrel exquisitely adapted to the "ice-age" climate of the Pleistocene steppe [4, 5]. Since the disappearance of this habitat, the rodent persists in large numbers in the high-altitude Alpine meadow [6, 7]. Genome and metabolome showed evidence of adaptation consistent with cold climate, affecting white adipose tissue. Conversely, however, we found that the Alpine marmot has levels of genetic variation that are among the lowest for mammals, such that deleterious mutations are less effectively purged. Our data rule out typical explanations for low diversity, such as high levels of consanguineous mating, or a very recent bottleneck. Instead, ancient demographic reconstruction revealed that genetic diversity was lost during the climate shifts of the Pleistocene and has not recovered, despite the current high population size. We attribute this slow recovery to the marmot's adaptive life history. The case of the Alpine marmot reveals a complicated relationship between climatic changes, genetic diversity, and conservation status. It shows that species of extremely low genetic diversity can be very successful and persist over thousands of years, but also that climate-adapted life history can trap a species in a persistent state of low genetic diversity.This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001134), the UK Medical Research Council (FC001134), and the Wellcome Trust (FC001134). CB and AC are supported by the Agence Nationale de la Recherche (project ANR-13-JSV7-0005) and the Centre National de la Recherche Scientifique (CNRS), CB is supported by the Rhône-Alpes region (Grant 15.005146.01). LD is supported by Agence Nationale de la Recherche (project ANR-12-ADAP-0009). TIG is supported by a Leverhulme Early Career Fellowship (Grant ECF-2015-453) and a NERC grant (NE/N013832/1). JMG is supported by a Hertha Finberg Fellowship (FWF T703). LDR is supported by the Diabetes UK RD Lawrence Fellowship (16/0005382)