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Genome-wide DNA polymerase usage maps determined in fission yeast, using a new sequencing strategy based on ribonucleotide misincorporation, track the division of labor between replicative polymerases and reveal locations and efficiencies of replication origins. Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) α–primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polɛ replicates the leading strand, whereas Polδ performs lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδ followed by exchange for Polɛ.

Due to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body. IMPORTANCE Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome. Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome.

Background. An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy. Methods. RNA-Seq was performed on an Illumina Miseq, generating 20 million paired-end reads. Nonhost data were checked for similarity to known organisms using BLASTx. The full viral genome was sequenced by primer walking. Results. We identified an astrovirus, HAstV-VA1/HMO-C-UK1(a), which was highly divergent from human astrovirus (HAstV 1–8) genotypes, but closely related to VA1/HMO-C astroviruses, including one recovered from a case of fatal encephalitis in an immunosuppressed child. The virus was detected in stool and serum, with highest levels in brain and cerebrospinal fluid (CSF). Immunohistochemistry of the brain biopsy showed positive neuronal staining. A survey of 680 stool and 349 CSF samples identified a related virus in the stool of another immunosuppressed child. Conclusions. The discovery of HAstV-VA1/HMO-C-UK1(a) as the cause of encephalitis in this case provides further evidence that VA1/HMO-C viruses, unlike HAstV 1–8, are neuropathic, particularly in immunocompromised patients, and should be considered in the differential diagnosis of encephalopathy. With a turnaround from sample receipt to result of <1 week, we confirm that RNA-Seq presents a valuable diagnostic tool in unexplained encephalitis.

Background Clinical interpretation of the large number of rare variants identified by high throughput sequencing (HTS) technologies is challenging. The aim of this study was to explore the clinical implications of a HTS strategy for patients with hypertrophic cardiomyopathy (HCM) using a targeted HTS methodology and workflow developed for patients with a range of inherited cardiovascular diseases. By comparing the sequencing results with published findings and with sequence data from a large-scale exome sequencing screen of UK individuals, we sought to quantify the strength of the evidence supporting causality for detected candidate variants. Methods and results 223 unrelated patients with HCM (46±15 years at diagnosis, 74% males) were studied. In order to analyse coding, intronic and regulatory regions of 41 cardiovascular genes, we used solution-based sequence capture followed by massive parallel resequencing on Illumina GAIIx. Average read-depth in the 2.1 Mb target region was 120. Rare (frequency<0.5%) non-synonymous, loss-of-function and splice-site variants were defined as candidates. Excluding titin, we identified 152 distinct candidate variants in sarcomeric or associated genes (89 novel) in 143 patients (64%). Four sarcomeric genes (MYH7, MYBPC3, TNNI3, TNNT2) showed an excess of rare single non-synonymous single-nucleotide polymorphisms (nsSNPs) in cases compared to controls. The estimated probability that a nsSNP in these genes is pathogenic varied between 57% and near certainty depending on the location. We detected an additional 94 candidate variants (73 novel) in desmosomal, and ion-channel genes in 96 patients (43%). Conclusions This study provides the first large-scale quantitative analysis of the prevalence of sarcomere protein gene variants in patients with HCM using HTS technology. Inclusion of other genes implicated in inherited cardiac disease identifies a large number of non-synonymous rare variants of unknown clinical significance.

Optic cup-like structures generated from mouse ES cells produce photoreceptor precursors that integrate and mature in vivo in three models of retinal degeneration. Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair 1 and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice 2 , 3 . Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages 4 , 5 , 6 . However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs 6 . We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment–bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4–8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.

Background Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. Methods Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK -mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. Results Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRAS Q61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. Conclusions Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Background Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. Methods and results Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. Conclusions Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis.

Sepsis causes extensive morbidity and mortality in children worldwide. Prompt recognition and timely treatment of sepsis is critical in reducing morbidity and mortality. Genomic approaches are used to discover novel pathways, therapeutic targets and biomarkers. These may facilitate diagnosis and risk stratification to tailor treatment strategies. To investigate the temporal gene expression during the evolution of sepsis induced multi-organ failure in response to a single organism, Neisseria meningitidis, in previously healthy children. RNA was extracted from serial blood samples (6 time points over 48 hours from presentation) from five critically ill children with meningococcal sepsis. Extracted RNA was hybridized to Affymetrix arrays. The RNA underwent strict quality control and standardized quantitation. Gene expression results were analyzed using GeneSpring software and Ingenuity Pathway Analysis. A marked variability in differential gene expression was observed between time points and between patients revealing dynamic expression changes during the evolution of sepsis. While there was evidence of time-dependent changes in expected gene networks including those involving immune responses and inflammatory pathways, temporal variation was also evident in specific \"biomarkers\" that have been proposed for diagnostic and risk stratification functions. The extent and nature of this variability was not readily explained by clinical phenotype. This is the first study of its kind detailing extensive expression changes in children during the evolution of sepsis. This highlights a limitation of static or single time point biomarker estimation. Serial estimations or more comprehensive network approaches may be required to optimize risk stratification in complex, time-critical conditions such as evolving sepsis.

Background Developing sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal. This process requires de novo gene expression but only a small number of genes induced by NGF deprivation have been identified so far, either by a candidate gene approach or in mRNA differential display experiments. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. Here, we describe for the first time, how advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons cultured in the presence and absence of NGF. Results We have used Affymetrix Exon arrays to study the pattern of expression of all known genes in NGF-deprived sympathetic neurons. We identified 415 up- and 813 down-regulated genes, including most of the genes previously known to be regulated in this system. NGF withdrawal activates the mixed lineage kinase (MLK)-c-Jun N-terminal kinase (JNK)-c-Jun pathway which is required for NGF deprivation-induced death. By including a mixed lineage kinase (MLK) inhibitor, CEP-11004, in our experimental design we identified which of the genes induced after NGF withdrawal are potential targets of the MLK-JNK-c-Jun pathway. A detailed Gene Ontology and functional enrichment analysis also identified genetic pathways that are highly enriched and overrepresented amongst the genes expressed after NGF withdrawal. Five genes not previously studied in sympathetic neurons - trib3, ddit3, txnip, ndrg1 and mxi1 - were validated by real time-PCR. The proteins encoded by these genes also increased in level after NGF withdrawal and this increase was prevented by CEP-11004, suggesting that these genes are potential targets of the MLK-JNK-c-Jun pathway. Conclusions The sympathetic neuron model is one of the best studied models of neuronal apoptosis. Overall, our microarray data gives a comprehensive overview of, and provides new information about, signalling pathways and transcription factors that are regulated by NGF withdrawal.

Background: In humans, the adrenal glands and gonads undergo distinct biological events between 6-10 weeks post conception (wpc), such as testis determination, the onset of steroidogenesis and primordial germ cell development. However, relatively little is currently known about the genetic mechanisms underlying these processes. We therefore aimed to generate a detailed genomic atlas of adrenal and gonad development across these critical stages of human embryonic and fetal development. Methods: RNA was extracted from 53 tissue samples between 6-10 wpc (adrenal, testis, ovary and control). Affymetrix array analysis was performed and differential gene expression was analysed using Bioconductor. A mathematical model was constructed to investigate time-series changes across the dataset. Pathway analysis was performed using ClueGo and cellular localisation of novel factors confirmed using immunohistochemistry. Results: Using this approach, we have identified novel components of adrenal development (e.g. ASB4 , NPR3 ) and confirmed the role of SRY as the main human testis-determining gene. By mathematical modelling time-series data we have found new genes up-regulated with SOX9 in the testis (e.g. CITED1 ), which may represent components of the testis development pathway. We have shown that testicular steroidogenesis has a distinct onset at around 8 wpc and identified potential novel components in adrenal and testicular steroidogenesis (e.g. MGARP , FOXO4 , MAP3K15 , GRAMD1B , RMND2 ), as well as testis biomarkers (e.g. SCUBE1 ). We have also shown that the developing human ovary expresses distinct subsets of genes (e.g. OR10G9 , OR4D5 ), but enrichment for established biological pathways is limited. Conclusion: This genomic atlas is revealing important novel aspects of human development and new candidate genes for adrenal and reproductive disorders.