Evolution of the nitric oxide synthase family in vertebrates and novel insights in gill development

Nitric oxide (NO) is an ancestral key signaling molecule essential for life and has enormous versatility in biological systems, including cardiovascular homeostasis, neurotransmission, and immunity. Although our knowledge of nitric oxide synthases (Nos), the enzymes that synthesize NO in vivo, is substantial, the origin of a large and diversified repertoire of nos gene orthologs in fish with respect to tetrapods remains a puzzle. The recent identification of nos3 in the ray-finned fish spotted gar, which was considered lost in the ray-finned fish lineage, changed this perspective. This prompted us to explore nos gene evolution and expression in depth, surveying vertebrate species representing key evolutionary nodes. This study provides noteworthy findings: first, nos2 experienced several lineage-specific gene duplications and losses. Second, nos3 was found to be lost independently in two different teleost lineages, Elopomorpha and Clupeocephala. Third, the expression of at least one nos paralog in the gills of developing shark, bichir, sturgeon, and gar but not in arctic lamprey, suggest that nos expression in this organ likely arose in the last common ancestor of gnathostomes. These results provide a framework for continuing research on nos genes roles, highlighting subfunctionalization and reciprocal loss of function that occurred in different lineages during vertebrate genome duplications.

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Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

PLoS Pathogens ◽

10.1371/journal.ppat.1009291 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009291

Author(s):

Yuli Talyansky ◽

Travis B. Nielsen ◽

Jun Yan ◽

Ulrike Carlino-Macdonald ◽

Gisela Di Venanzio ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Bacterial Pathogen ◽

Specific Gene ◽

Dependent Manner ◽

Bacterial Burden ◽

Carbohydrate Structure ◽

Loss Of Function ◽

Capsule Assembly

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

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Oxygen tension limits nitric oxide synthesis by activated macrophages

Biochemical Journal ◽

10.1042/bj3500709 ◽

2000 ◽

Vol 350 (3) ◽

pp. 709-716 ◽

Cited By ~ 33

Author(s):

Charles C. MCCORMICK ◽

Wai Ping LI ◽

Monica CALERO

Keyword(s):

Nitric Oxide ◽

Oxygen Tension ◽

Interferon Γ ◽

Activated Macrophages ◽

Calcium Dependent ◽

Oxide Synthase ◽

Nos Gene ◽

High Output

Previous studies have established that constitutive calcium-dependent (‘low-output’) nitric oxide synthase (NOS) is regulated by oxygen tension. We have investigated the role of oxygen tension in the synthesis of NO by the ‘high-output’ calcium-independent NOS in activated macrophages. Hypoxia increased macrophage NOS gene expression in the presence of one additional activator, such as lipopolysaccharide or interferon-γ, but not in the presence of both. Hypoxia markedly reduced the synthesis of NO by activated macrophages (as measured by accumulation of nitrite and citrulline), such that, at 1% oxygen tension, NO accumulation was reduced by 80–90%. The apparent Km for oxygen calculated from cells exposed to a range of oxygen tensions was found to be 10.8%, or 137µM, O2 This value is considerably higher than the oxygen tension in tissues, and is virtually identical to that reported recently for purified recombinant macrophage NOS. The decrease in NO synthesis did not appear to be due to diminished arginine or cofactor availability, since arginine transport and NO synthesis during recovery in normoxia were normal. Analysis of NO synthesis during hypoxia as a function of extracellular arginine indicated that an altered Vmax, but not KmArg, accounted for the observed decrease in NO synthesis. We conclude that oxygen tension regulates the synthesis of NO in macrophages by a mechanism similar to that described previously for the calcium-dependent low-output NOS. Our data suggest that oxygen tension may be an important physiological regulator of macrophage NO synthesis in vivo.

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Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology

Non-Coding RNA ◽

10.3390/ncrna5010012 ◽

2019 ◽

Vol 5 (1) ◽

pp. 12 ◽

Cited By ~ 7

Author(s):

Nils R. Hansmeier ◽

Pia J. M. Widdershooven ◽

Sajjad Khani ◽

Jan-Wilhelm Kornfeld

Keyword(s):

Gene Targeting ◽

Mouse Models ◽

Noncoding Rna ◽

Genome Engineering ◽

Model Organisms ◽

Specific Gene ◽

Loss Of Function ◽

Mouse Lines

In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which <10 days hands-on working time is required.

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Evolution of gene expression after whole-genome duplication: new insights from the spotted gar genome

10.1101/151944 ◽

2017 ◽

Author(s):

Jeremy Pasquier ◽

Ingo Braasch ◽

Peter Batzel ◽

Cedric Cabau ◽

Jérome Montfort ◽

...

Keyword(s):

Gene Expression ◽

Genome Duplication ◽

Expression Patterns ◽

Duplicate Gene ◽

Current Status ◽

Specific Gene ◽

Whole Genome ◽

Spotted Gar ◽

Genome Duplications ◽

Underlying Mechanisms

AbstractWhole genome duplications (WGD) are important evolutionary events. Our understanding of underlying mechanisms, including the evolution of duplicated genes after WGD, however remains incomplete. Teleost fish experienced a common WGD (teleost-specific genome duplication, or TGD) followed by a dramatic adaptive radiation leading to more than half of all vertebrate species. The analysis of gene expression patterns following TGD at the genome level has been limited by the lack of suitable genomic resources. The recent concomitant release of the genome sequence of spotted gar (a representative of holosteans, the closest lineage of teleosts that lacks the TGD) and the tissue-specific gene expression repertoires of over 20 holostean and teleostean fish species, including spotted gar, zebrafish and medaka (the PhyloFish project), offered a unique opportunity to study the evolution of gene expression following TGD in teleosts. We show that most TGD duplicates gained their current status (loss of one duplicate gene or retention of both duplicates) relatively rapidly after TGD (i.e. prior to the divergence of medaka and zebrafish lineages). The loss of one duplicate is the most common fate after TGD with a probability of approximately 80%. In addition, the fate of duplicate genes after TGD, including subfunctionalization, neofunctionalization, or retention of two ‘similar’ copies occurred not only before, but also after the radiation of species tested, in consistency with a role of the TGD in speciation and/or evolution of gene function. Finally, we report novel cases of TGD ohnolog subfunctionalization and neofunctionalization that further illustrate the importance of these processes.

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Synthetic Lethality Screening Highlights Colorectal Cancer Vulnerability to Concomitant Blockade of NEDD8 and EGFR Pathways

Cancers ◽

10.3390/cancers13153805 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3805

Author(s):

Federica Invrea ◽

Simona Punzi ◽

Consalvo Petti ◽

Rosalba Minelli ◽

Michael D. Peoples ◽

...

Keyword(s):

Colorectal Cancer ◽

Target Genes ◽

Enzyme Inhibitor ◽

Synthetic Lethality ◽

Drop Out ◽

Specific Gene ◽

Loss Of Function ◽

Egfr Signaling Pathway

Colorectal cancer (CRC) is a heterogeneous disease showing significant variability in clinical aggressiveness. Primary and acquired resistance limits the efficacy of available treatments, and identification of effective drug combinations is needed to further improve patients’ outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a “drop-out” loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. These results unveil possible therapeutic opportunities in specific CRC clinical settings.

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The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish

Blood ◽

10.1182/blood-2008-12-189910 ◽

2009 ◽

Vol 114 (21) ◽

pp. 4654-4663 ◽

Cited By ~ 32

Author(s):

Julio D. Amigo ◽

Gabriele E. Ackermann ◽

John J. Cope ◽

Ming Yu ◽

Jeffrey D. Cooney ◽

...

Keyword(s):

Binding Sites ◽

Regulatory Elements ◽

Specific Gene ◽

Loss Of Function ◽

Gata Factors ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Regulatory Loop

Abstract The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.

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In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with Autism risk genes

10.1101/791525 ◽

2019 ◽

Cited By ~ 7

Author(s):

Xin Jin ◽

Sean K. Simmons ◽

Amy X. Guo ◽

Ashwin S. Shetty ◽

Michelle Ko ◽

...

Keyword(s):

De Novo ◽

Disease Risk ◽

Autism Spectrum ◽

Specific Gene ◽

Functional Evaluation ◽

Loss Of Function ◽

Risk Genes ◽

Biologically Relevant ◽

Relevant Context

AbstractThe thousands of disease risk genes and loci identified through human genetic studies far outstrip our current capacity to systematically study their functions. New experimental approaches are needed for functional investigations of large panels of genes in a biologically relevant context. Here, we developed a scalable genetic screen approach, in vivo Perturb-Seq, and applied this method to the functional evaluation of 35 autism spectrum disorder (ASD) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing brain in utero, and then performed single-cell RNA-Seq in the postnatal brain. We identified cell type-specific gene signatures from both neuronal and glial cell classes that are affected by genetic perturbations, and pointed at elements of both convergent and divergent cellular effects across this cohort of ASD risk genes. In vivo Perturb-Seq pioneers a systems genetics approach to investigate at scale how diverse mutations affect cell types and states in the biologically relevant context of the developing organism.

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Impact of RNA-Guided Technologies for Target Identification and Deconvolution

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057114548414 ◽

2014 ◽

Vol 19 (10) ◽

pp. 1327-1337 ◽

Cited By ~ 16

Author(s):

Myles Fennell ◽

Qing Xiang ◽

Alexia Hwang ◽

Chong Chen ◽

Chun-Hao Huang ◽

...

Keyword(s):

High Efficiency ◽

Virus Production ◽

Expression Vectors ◽

Specific Gene ◽

Loss Of Function ◽

Shrna Expression ◽

Rnai Technology ◽

Target Effects

For well over a decade, RNA interference (RNAi) has provided a powerful tool for investigators to query specific gene targets in an easily modulated loss-of-function setting, both in vitro and in vivo. Hundreds of publications have demonstrated the utility of RNAi in arrayed and pooled-based formats, in a wide variety of cell-based systems, including clonal, stem, transformed, and primary cells. Over the years, there have been significant improvements in the design of target-specific small-interfering RNA (siRNA) and short-hairpin RNA (shRNA), expression vectors, methods for mitigating off-target effects, and accurately interpreting screening results. Recent developments in RNAi technology include the Sensor assay, high-efficiency miR-E shRNAs, improved shRNA virus production with Pasha (DRGC8) knockdown, and assessment of RNAi off-target effects by using the C9-11 method. An exciting addition to the arsenal of RNA-mediated gene modulation is the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas) system for genomic editing, allowing for gene functional knockout rather than knockdown.

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A Long Lost Key Opens an Ancient Lock: Drosophila Myb Causes a Synthetic Multivulval Phenotype in Nematodes

10.1101/788265 ◽

2019 ◽

Author(s):

Paul J. Vorster ◽

Paul Goetsch ◽

Tilini U. Wijeratne ◽

Keelan Z. Guiley ◽

Laura Andrejka ◽

...

Keyword(s):

Cell Fate ◽

Cell Cycle Progression ◽

Target Genes ◽

Suppressor Gene ◽

Last Common Ancestor ◽

Loss Of Function ◽

Core Complex ◽

C Elegans

ABSTRACTThe five-protein MuvB core complex (LIN9/Mip130, LIN37/Mip40, LIN52, LIN54/Mip120, and LIN53/p55CAF1/RBBP4) has been highly conserved during the evolution of animals. This nuclear complex interacts with proteins encoded by the RB tumor suppressor gene family and its associated E2F-DP transcription factors to form DREAM complexes that repress the expression of genes that regulate cell cycle progression and cell fate. The MuvB core complex also interacts with proteins encoded by the Myb oncogene family to form the Myb-MuvB complexes that activate many of the same target genes. We show that animal-type Myb genes and proteins are present in Bilateria, Cnidaria, and Placozoa, the latter including some of the simplest known animal species. However, bilaterian nematode worms appear to have lost their animal-type Myb genes hundreds of millions of years ago. Nevertheless, the amino acids in the LIN9 and LIN52 proteins that directly interact with the MuvB-binding domains of human B-Myb and Drosophila Myb are conserved in C. elegans. Here we show that, despite greater than 500 million years since their last common ancestor, the Drosophila melanogaster Myb protein can bind to the nematode LIN9 and LIN52 family proteins in vitro and can cause a synthetic multivulval (synMuv) phenotype in vivo. This phenotype is similar to that caused by loss-of-function mutations in C. elegans synMuvB class genes including those that encode homologs of the MuvB core, RB, E2F, and DP. Furthermore, amino acid substitutions in the MuvB-binding domain of Drosophila Myb that disrupt its functions in vitro and in vivo also disrupt its activity in C. elegans. We speculate that nematodes and other animals may contain another protein that can bind to LIN9 and LIN52 in order to activate transcription of genes repressed by DREAM complexes.

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