Exome Sequence Analysis of 14 Families With High Myopia

PURPOSE: To identify causal gene mutations in 14 families with autosomal dominant (AD) high myopia using exome sequencing. METHODS: Select individuals from 14 large Caucasian families with high myopia were exome sequenced. Gene variants were filtered to identify potential pathogenic changes. Sanger sequencing was used to confirm variants in original DNA, and to test for disease cosegregation in additional family members. Candidate genes and chromosomal loci previously associated with myopic refractive error and its endophenotypes were comprehensively screened. RESULTS: In 14 high myopia families, we identified 73 rare and 31 novel gene variants as candidates for pathogenicity. In seven of these families, two of the novel and eight of the rare variants were within known myopia loci. A total of 104 heterozygous nonsynonymous rare variants in 104 genes were identified in 10 out of 14 probands. Each variant cosegregated with affection status. No rare variants were identified in genes known to cause myopia or in genes closest to published genome-wide association study association signals for refractive error or its endophenotypes. CONCLUSIONS: Whole exome sequencing was performed to determine gene variants implicated in the pathogenesis of AD high myopia. This study provides new genes for consideration in the pathogenesis of high myopia, and may aid in the development of genetic profiling of those at greatest risk for attendant ocular morbidities of this disorder

The Integrative Conjugative Element clc (ICEclc) of Pseudomonas aeruginosa JB2

Integrative conjugative elements (ICE) are a diverse group of chromosomally integrated, self-transmissible mobile genetic elements (MGE) that are active in shaping the functions of bacteria and bacterial communities. Each type of ICE carries a characteristic set of core genes encoding functions essential for maintenance and self-transmission, and cargo genes that endow on hosts phenotypes beneficial for niche adaptation. An important area to which ICE can contribute beneficial functions is the biodegradation of xenobiotic compounds. In the biodegradation realm, the best-characterized ICE is ICEclc, which carries cargo genes encoding for ortho-cleavage of chlorocatechols (clc genes) and aminophenol metabolism (amn genes). The element was originally identified in the 3-chlorobenzoate-degrader Pseudomonas knackmussii B13, and the closest relative is a nearly identical element in Burkholderia xenovorans LB400 (designated ICEclc-B13 and ICEclc-LB400, respectively). In the present report, genome sequencing of the o-chlorobenzoate degrader Pseudomonas aeruginosa JB2 was used to identify a new member of the ICEclc family, ICEclc-JB2. The cargo of ICEclc-JB2 differs from that of ICEclc-B13 and ICEclc-LB400 in consisting of a unique combination of genes that encode for the utilization of o-halobenzoates and o-hydroxybenzoate as growth substrates (ohb genes and hyb genes, respectively) and which are duplicated in a tandem repeat. Also, ICEclc-JB2 lacks an operon of regulatory genes (tciR-marR-mfsR) that is present in the other two ICEclc, and which controls excision from the host. Thus, the mechanisms regulating intracellular behavior of ICEclc-JB2 may differ from that of its close relatives. The entire tandem repeat in ICEclc-JB2 can excise independently from the element in a process apparently involving transposases/insertion sequence associated with the repeats. Excision of the repeats removes important niche adaptation genes from ICEclc-JB2, rendering it less beneficial to the host. However, the reduced version of ICEclc-JB2 could now acquire new genes that might be beneficial to a future host and, consequently, to the survival of ICEclc-JB2. Collectively, the present identification and characterization of ICEclc-JB2 provides insights into roles of MGE in bacterial niche adaptation and the evolution of catabolic pathways for biodegradation of xenobiotic compounds

Characterization of ozone production in San Antonio, Texas, using measurements of total peroxy radicals

Observations of total peroxy radical concentrations ([XO2]&thinsp;≡&thinsp;[RO2]&thinsp;+&thinsp;[HO2]) made by the Ethane CHemical AMPlifier (ECHAMP) and concomitant observations of additional trace gases made on board the Aerodyne Mobile Laboratory (AML) during May 2017 were used to characterize ozone production at three sites in the San Antonio, Texas, region. Median daytime [O3] was 48&thinsp;ppbv at the site downwind of central San Antonio. Higher concentrations of NO and XO2 at the downwind site also led to median daytime ozone production rates (P(O3)) of 4.2&thinsp;ppbv&thinsp;h−1, a factor of 2 higher than at the two upwind sites. The 95th percentile of P(O3) at the upwind site was 15.1&thinsp;ppbv&thinsp;h−1, significantly lower than values observed in Houston. In situ observations, as well as satellite retrievals of HCHO and NO2, suggest that the region was predominantly NOx-limited. Only approximately 20&thinsp;% of observations were in the VOC-limited regime, predominantly before 11:00&thinsp;EST, when ozone production was low. Biogenic volatile organic compounds (VOCs) comprised 55&thinsp;% of total OH reactivity at the downwind site, with alkanes and non-biogenic alkenes responsible for less than 10&thinsp;% of total OH reactivity in the afternoon, when ozone production was highest. To control ozone formation rates at the three study sites effectively, policy efforts should be directed at reducing NOx emissions. Observations in the urban center of San Antonio are needed to determine whether this policy is true for the entire region.</p

A Pre-mRNA–Associating Factor Links Endogenous siRNAs to Chromatin Regulation

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA–driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans

RNAi Effector Diversity in Nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes

A Conserved PHD Finger Protein and Endogenous RNAi Modulate Insulin Signaling in Caenorhabditis elegans

Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16–dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.Leukemia & Lymphoma Society of America (3260-07 Special Fellow Award)Arnold and Mabel Beckman Foundation (Young Investigator Award)United States. National Institutes of Health (Director's New Innovator Award (1 DP2 OD006412-01))United States. National Institutes of Health (grant GM66269)modENCODE (grant U01 HG004270)United States. National Institutes of Health (training grant 5T32 GM07088-34

The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7–dependent siRNAs require Dicer. We identify that the eri-6/7–dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3–4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7–dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA–associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation

Single-cell RNA sequencing of neurofibromas reveals a tumor microenvironment favorable for neural regeneration and immune suppression in a neurofibromatosis type 1 porcine model

Neurofibromatosis Type 1 (NF1) is one of the most common genetically inherited disorders that affects 1 in 3000 children annually. Clinical manifestations vary widely but nearly always include the development of cutaneous, plexiform and diffuse neurofibromas that are managed over many years. Recent single-cell transcriptomics profiling efforts of neurofibromas have begun to reveal cell signaling processes. However, the cell signaling networks in mature, non-cutaneous neurofibromas remain unexplored. Here, we present insights into the cellular composition and signaling within mature neurofibromas, contrasting with normal adjacent tissue, in a porcine model of NF1 using single-cell RNA sequencing (scRNA-seq) analysis and histopathological characterization. These neurofibromas exhibited classic diffuse-type histologic morphology and expected patterns of S100, SOX10, GFAP, and CD34 immunohistochemistry. The porcine mature neurofibromas closely resemble human neurofibromas histologically and contain all known cellular components of their human counterparts. The scRNA-seq confirmed the presence of all expected cell types within these neurofibromas and identified novel populations of fibroblasts and immune cells, which may contribute to the tumor microenvironment by suppressing inflammation, promoting M2 macrophage polarization, increasing fibrosis, and driving the proliferation of Schwann cells. Notably, we identified tumor-associated IDO1+/CD274+ (PD-L1)+ dendritic cells, which represent the first such observation in any NF1 animal model and suggest the role of the upregulation of immune checkpoints in mature neurofibromas. Finally, we observed that cell types in the tumor microenvironment are poised to promote immune evasion, extracellular matrix reconstruction, and nerve regeneration