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1.
J Biol Chem ; 299(7): 104871, 2023 07.
Article in English | MEDLINE | ID: mdl-37247760

ABSTRACT

Malaria causes >600 thousand fatalities each year, with most cases attributed to the human-infectious Plasmodium falciparum species. Many rodent-infectious Plasmodium species, like Plasmodium berghei and Plasmodium yoelii, have been used as model species that can expedite studies of this pathogen. P. yoelii is an especially good model for investigating the mosquito and liver stages of development because key attributes closely resemble those of P. falciparum. Because of its importance, in 2002 the 17XNL strain of P. yoelii was the first rodent malaria parasite to be sequenced. Although this was a breakthrough effort, the assembly consisted of >5000 contiguous sequences that adversely impacted the annotated gene models. While other rodent malaria parasite genomes have been sequenced and annotated since then, including the related P. yoelii 17X strain, the 17XNL strain has not. As a result, genomic data for 17X has become the de facto reference genome for the 17XNL strain while leaving open questions surrounding possible differences between the 17XNL and 17X genomes. In this work, we present a high-quality genome assembly for P. yoelii 17XNL using PacBio DNA sequencing. In addition, we use Nanopore and Illumina RNA sequencing of mixed blood stages to create complete gene models that include coding sequences, alternate isoforms, and UTR designations. A comparison of the 17X and this new 17XNL assembly revealed biologically meaningful differences between the strains due to the presence of coding sequence variants. Taken together, our work provides a new genomic framework for studies with this commonly used rodent malaria model species.


Subject(s)
Malaria , Parasites , Plasmodium yoelii , Animals , Humans , Plasmodium yoelii/genetics , Rodentia , Malaria/parasitology , Liver
2.
PLoS Pathog ; 18(10): e1010926, 2022 10.
Article in English | MEDLINE | ID: mdl-36306287

ABSTRACT

The emergence of Plasmodium falciparum parasite resistance to dihydroartemisinin + piperaquine (PPQ) in Southeast Asia threatens plans to increase the global use of this first-line antimalarial combination. High-level PPQ resistance appears to be mediated primarily by novel mutations in the P. falciparum chloroquine resistance transporter (PfCRT), which enhance parasite survival at high PPQ concentrations in vitro and increase the risk of dihydroartemisinin + PPQ treatment failure in patients. Using isogenic Dd2 parasites expressing contemporary pfcrt alleles with differential in vitro PPQ susceptibilities, we herein characterize the molecular and physiological adaptations that define PPQ resistance in vitro. Using drug uptake and cellular heme fractionation assays we report that the F145I, M343L, and G353V PfCRT mutations differentially impact PPQ and chloroquine efflux. These mutations also modulate proteolytic degradation of host hemoglobin and the chemical inactivation of reactive heme species. Peptidomic analyses reveal significantly higher accumulation of putative hemoglobin-derived peptides in the PPQ-resistant mutant PfCRT isoforms compared to parental PPQ-sensitive Dd2. Joint transcriptomic and metabolomic profiling of late trophozoites from PPQ-resistant or -sensitive isogenic lines reveals differential expression of genes involved in protein translation and cellular metabolism. PPQ-resistant parasites also show increased susceptibility to an inhibitor of the P. falciparum M17 aminopeptidase that operates on short globin-derived peptides. These results reveal unique physiological changes caused by the gain of PPQ resistance and highlight the potential therapeutic value of targeting peptide metabolism in P. falciparum.


Subject(s)
Antimalarials , Artemisinins , Malaria, Falciparum , Parasites , Animals , Humans , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Chloroquine/pharmacology , Chloroquine/metabolism , Parasites/metabolism , Protozoan Proteins/metabolism , Drug Resistance/genetics , Malaria, Falciparum/drug therapy , Malaria, Falciparum/genetics , Malaria, Falciparum/parasitology , Antimalarials/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Artemisinins/pharmacology , Mutation , Hemoglobins/metabolism , Heme/metabolism
3.
Proc Natl Acad Sci U S A ; 117(2): 895-901, 2020 01 14.
Article in English | MEDLINE | ID: mdl-31882450

ABSTRACT

Emerging and reemerging viruses are responsible for a number of recent epidemic outbreaks. A crucial step in predicting and controlling outbreaks is the timely and accurate characterization of emerging virus strains. We present a portable microfluidic platform containing carbon nanotube arrays with differential filtration porosity for the rapid enrichment and optical identification of viruses. Different emerging strains (or unknown viruses) can be enriched and identified in real time through a multivirus capture component in conjunction with surface-enhanced Raman spectroscopy. More importantly, after viral capture and detection on a chip, viruses remain viable and get purified in a microdevice that permits subsequent in-depth characterizations by various conventional methods. We validated this platform using different subtypes of avian influenza A viruses and human samples with respiratory infections. This technology successfully enriched rhinovirus, influenza virus, and parainfluenza viruses, and maintained the stoichiometric viral proportions when the samples contained more than one type of virus, thus emulating coinfection. Viral capture and detection took only a few minutes with a 70-fold enrichment enhancement; detection could be achieved with as little as 102 EID50/mL (50% egg infective dose per microliter), with a virus specificity of 90%. After enrichment using the device, we demonstrated by sequencing that the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for influenza virus. This enrichment method coupled to Raman virus identification constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real time.


Subject(s)
Microbiological Techniques/methods , Virology/methods , Virus Diseases/diagnosis , Viruses/isolation & purification , Humans , Influenza A virus/isolation & purification , Microbiological Techniques/instrumentation , Microtechnology/methods , Nanotubes, Carbon , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/virology , Respirovirus/isolation & purification , Rhinovirus/isolation & purification , Sensitivity and Specificity , Silicon Dioxide , Spectrum Analysis, Raman/methods , Staining and Labeling , Virion , Virology/instrumentation , Virus Diseases/virology , Viruses/genetics
4.
N Engl J Med ; 381(21): 2020-2031, 2019 11 21.
Article in English | MEDLINE | ID: mdl-31562796

ABSTRACT

BACKGROUND: In an early-phase study involving patients with advanced non-small-cell lung cancer (NSCLC), the response rate was better with nivolumab plus ipilimumab than with nivolumab monotherapy, particularly among patients with tumors that expressed programmed death ligand 1 (PD-L1). Data are needed to assess the long-term benefit of nivolumab plus ipilimumab in patients with NSCLC. METHODS: In this open-label, phase 3 trial, we randomly assigned patients with stage IV or recurrent NSCLC and a PD-L1 expression level of 1% or more in a 1:1:1 ratio to receive nivolumab plus ipilimumab, nivolumab alone, or chemotherapy. The patients who had a PD-L1 expression level of less than 1% were randomly assigned in a 1:1:1 ratio to receive nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy alone. All the patients had received no previous chemotherapy. The primary end point reported here was overall survival with nivolumab plus ipilimumab as compared with chemotherapy in patients with a PD-L1 expression level of 1% or more. RESULTS: Among the patients with a PD-L1 expression level of 1% or more, the median duration of overall survival was 17.1 months (95% confidence interval [CI], 15.0 to 20.1) with nivolumab plus ipilimumab and 14.9 months (95% CI, 12.7 to 16.7) with chemotherapy (P = 0.007), with 2-year overall survival rates of 40.0% and 32.8%, respectively. The median duration of response was 23.2 months with nivolumab plus ipilimumab and 6.2 months with chemotherapy. The overall survival benefit was also observed in patients with a PD-L1 expression level of less than 1%, with a median duration of 17.2 months (95% CI, 12.8 to 22.0) with nivolumab plus ipilimumab and 12.2 months (95% CI, 9.2 to 14.3) with chemotherapy. Among all the patients in the trial, the median duration of overall survival was 17.1 months (95% CI, 15.2 to 19.9) with nivolumab plus ipilimumab and 13.9 months (95% CI, 12.2 to 15.1) with chemotherapy. The percentage of patients with grade 3 or 4 treatment-related adverse events in the overall population was 32.8% with nivolumab plus ipilimumab and 36.0% with chemotherapy. CONCLUSIONS: First-line treatment with nivolumab plus ipilimumab resulted in a longer duration of overall survival than did chemotherapy in patients with NSCLC, independent of the PD-L1 expression level. No new safety concerns emerged with longer follow-up. (Funded by Bristol-Myers Squibb and Ono Pharmaceutical; CheckMate 227 ClinicalTrials.gov number, NCT02477826.).


Subject(s)
Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Ipilimumab/administration & dosage , Lung Neoplasms/drug therapy , Nivolumab/administration & dosage , Adult , Aged , Aged, 80 and over , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , B7-H1 Antigen/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/mortality , Female , Humans , Ipilimumab/adverse effects , Lung Neoplasms/metabolism , Lung Neoplasms/mortality , Male , Middle Aged , Nivolumab/adverse effects , Survival Analysis
5.
Addict Biol ; 26(1): e12859, 2021 01.
Article in English | MEDLINE | ID: mdl-31782218

ABSTRACT

Nicotine use remains highly prevalent with tobacco and e-cigarette products consumed worldwide. However, increasing evidence of transgenerational epigenetic inheritance suggests that nicotine use may alter behavior and neurobiology in subsequent generations. We tested the effects of chronic paternal nicotine exposure in C57BL6/J mice on fear conditioning in F1 and F2 offspring, as well as conditioned fear extinction and spontaneous recovery, nicotine self-administration, hippocampal cholinergic functioning, RNA expression, and DNA methylation in F1 offspring. Paternal nicotine exposure was associated with enhanced contextual and cued fear conditioning and spontaneous recovery of extinguished fear memories. Further, nicotine reinforcement was reduced in nicotine-sired mice, as assessed in a self-administration paradigm. These behavioral phenotypes were coupled with altered response to nicotine, upregulated hippocampal nicotinic acetylcholine receptor binding, reduced evoked hippocampal cholinergic currents, and altered methylation and expression of hippocampal genes related to neural development and plasticity. Gene expression analysis suggests multigenerational effects on broader gene networks potentially involved in neuroplasticity and mental disorders. The changes in fear conditioning similarly suggest phenotypes analogous to anxiety disorders similar to post-traumatic stress.


Subject(s)
Fear/drug effects , Hippocampus/metabolism , Memory/drug effects , Nicotine/pharmacology , Paternal Exposure/adverse effects , Animals , Conditioning, Psychological/drug effects , Cues , Extinction, Psychological , Female , Male , Mice , Mice, Inbred C57BL , Up-Regulation/drug effects
6.
BMC Bioinformatics ; 21(1): 292, 2020 Jul 08.
Article in English | MEDLINE | ID: mdl-32640986

ABSTRACT

BACKGROUND: Bioinformaticians collaborating with life scientists need software that allows them to involve their collaborators in the process of data analysis. RESULTS: We have developed a web application that allows researchers to publish and execute data analysis scripts. Within the platform bioinformaticians are able to deploy data analysis workflows (recipes) that their collaborators can execute via point and click interfaces. The results generated by the recipes are viewable via the web interface and consist of a snapshot of all the commands, printed messages and files that have been generated during the recipe run. A demonstration version of our software is available at https://www.bioinformatics.recipes/ . Detailed documentation for the software is available at: https://bioinformatics-recipes.readthedocs.io . The source code for the software is distributed through GitHub at https://github.com/ialbert/biostar-central . CONCLUSIONS: Our software platform supports collaborative interactions between bioinformaticians and life scientists. The software is presented via a web application that provides a high utility and user-friendly approach for conducting reproducible research. The recipes developed and shared through the web application are generic, with broad applicability and may be downloaded and executed on other computing platforms.


Subject(s)
Computational Biology/methods , Software , Data Analysis , Reproducibility of Results , User-Computer Interface , Workflow
7.
J Cell Sci ; 131(6)2018 03 26.
Article in English | MEDLINE | ID: mdl-29487181

ABSTRACT

In this study, we characterized the Puf family gene member Puf3 in the malaria parasites Plasmodium falciparum and Plasmodium yoelii Secondary structure prediction suggested that the RNA-binding domains of the Puf3 proteins consisted of 11 pumilio repeats that were similar to those in the human Puf-A (also known as PUM3) and Saccharomyces cerevisiae Puf6 proteins, which are involved in ribosome biogenesis. Neither P. falciparum (Pf)Puf3 nor P. yoelii (Py)Puf3 could be genetically disrupted, suggesting they may be essential for the intraerythrocytic developmental cycle. Cellular fractionation of PfPuf3 in the asexual stages revealed preferential partitioning to the nuclear fraction, consistent with nuclear localization of PfPuf3::GFP and PyPuf3::GFP as detected by immunofluorescence. Furthermore, PfPuf3 colocalized with the nucleolar marker PfNop1, demonstrating that PfPuf3 is a nucleolar protein in the asexual stages. We found, however, that PyPuf3 changed its localization from being nucleolar to being present in cytosolic puncta in the mosquito and liver stages, which may reflect alternative functions in these stages. Affinity purification of molecules that associated with a PTP-tagged variant of PfPuf3 revealed 31 proteins associated with the 60S ribosome, and an enrichment of 28S rRNA and internal transcribed spacer 2 sequences. Taken together, these results suggest an essential function for PfPuf3 in ribosomal biogenesis.


Subject(s)
Plasmodium falciparum/metabolism , Plasmodium yoelii/metabolism , Protozoan Proteins/chemistry , Ribosomes/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Cytosol/metabolism , Life Cycle Stages , Plasmodium falciparum/chemistry , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Plasmodium yoelii/chemistry , Plasmodium yoelii/genetics , Plasmodium yoelii/growth & development , Protein Transport , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomes/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
8.
Int J Syst Evol Microbiol ; 70(11): 5701-5710, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32931408

ABSTRACT

A novel thermophilic phototrophic purple sulphur bacterium was isolated from microbial mats (56 °C) at Nakabusa hot springs, Nagano prefecture, Japan. Cells were motile, rod-shaped, stain Gram-negative and stored sulphur globules intracellularly. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were the major pigments. Dense liquid cultures were red in colour. Strain No.7T was able to grow photoautotrophically using sulfide, thiosulfate, sulfite and hydrogen (in the presence of sulfide) as electron donors and bicarbonate as the sole carbon source. Optimum growth occurred under anaerobic conditions in the light at 50 °C (range, 40-56 °C) and pH 7.2 (range, pH 7-8). Major fatty acids were C16 : 0 (46.8 %), C16 : 1 ω7c (19.9 %), C18 : 1 ω7c (21.1 %), C14 : 0 (4.6 %) and C18 : 0 (2.4 %). The polar lipid profile showed phosphatidylglycerol and unidentified aminophospholipids to be the major lipids. The only quinone detected was ubiquinone-8. 16S rRNA gene sequence comparisons indicated that the novel bacterium is only distantly related to Thermochromatium tepidum with a nucleotide identity of 90.4 %. The phylogenetic analysis supported the high novelty of strain No.7T with a long-branching phylogenetic position within the Chromatiaceae next to Thermochromatium tepidum. The genome comprised a circular chromosome of 2.99 Mbp (2 989 870 bp), included no plasmids and had a DNA G+C content of 61.2 mol%. Polyphasic taxonomic analyses of the isolate suggested strain No.7T is a novel genus within the Chromatiaceae. The proposed genus name of the second truly thermophilic purple sulphur bacterium is Caldichromatium gen. nov. with the type species Caldichromatium japonicum sp. nov. (DSM 110881=JCM 39101).


Subject(s)
Chromatiaceae/classification , Hot Springs/microbiology , Phylogeny , Bacterial Typing Techniques , Base Composition , Chromatiaceae/isolation & purification , DNA, Bacterial/genetics , Fatty Acids/chemistry , Japan , Phospholipids/chemistry , Pigmentation , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sulfides , Sulfur , Thiosulfates , Ubiquinone/chemistry
9.
Nucleic Acids Res ; 46(16): 8385-8403, 2018 09 19.
Article in English | MEDLINE | ID: mdl-30102401

ABSTRACT

The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor orchestrating complex roles in cell and systems biology. Species differences in CAR's effector pathways remain poorly understood, including its role in regulating liver tumor promotion. We developed transgenic mouse models to assess genome-wide binding of mouse and human CAR, following receptor activation in liver with direct ligands and with phenobarbital, an indirect CAR activator. Genomic interaction profiles were integrated with transcriptional and biological pathway analyses. Newly identified CAR target genes included Gdf15 and Foxo3, important regulators of the carcinogenic process. Approximately 1000 genes exhibited differential binding interactions between mouse and human CAR, including the proto-oncogenes, Myc and Ikbke, which demonstrated preferential binding by mouse CAR as well as mouse CAR-selective transcriptional enhancement. The ChIP-exo analyses also identified distinct binding motifs for the respective mouse and human receptors. Together, the results provide new insights into the important roles that CAR contributes as a key modulator of numerous signaling pathways in mammalian organisms, presenting a genomic context that specifies species variation in biological processes under CAR's control, including liver cell proliferation and tumor promotion.


Subject(s)
Cell Proliferation/genetics , DNA-Binding Proteins/genetics , Liver Neoplasms/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Androstanes/chemistry , Androstanes/metabolism , Animals , Constitutive Androstane Receptor , Forkhead Box Protein O3/genetics , Genes, myc/genetics , Genome/genetics , Growth Differentiation Factor 15/genetics , Hepatocytes/metabolism , Humans , I-kappa B Kinase/genetics , Ligands , Liver/chemistry , Liver/metabolism , Liver Neoplasms/pathology , Mice , Mice, Transgenic , Protein Binding/genetics
10.
Proc Natl Acad Sci U S A ; 113(23): 6478-83, 2016 Jun 07.
Article in English | MEDLINE | ID: mdl-27222581

ABSTRACT

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.


Subject(s)
Decapodiformes/genetics , Peptides , Proteins , Tandem Repeat Sequences , Animals , Mechanical Phenomena , Peptides/chemistry , Peptides/genetics , Proteins/chemistry , Proteins/genetics
11.
Proc Natl Acad Sci U S A ; 113(4): 1020-5, 2016 Jan 26.
Article in English | MEDLINE | ID: mdl-26755583

ABSTRACT

Sexual reproduction brings genes from two parents (matrigenes and patrigenes) together into one individual. These genes, despite being unrelated, should show nearly perfect cooperation because each gains equally through the production of offspring. However, an individual's matrigenes and patrigenes can have different probabilities of being present in other relatives, so kin selection could act on them differently. Such intragenomic conflict could be implemented by partial or complete silencing (imprinting) of an allele by one of the parents. Evidence supporting this theory is seen in offspring-mother interactions, with patrigenes favoring acquisition of more of the mother's resources if some of the costs fall on half-siblings who do not share the patrigene. The kinship theory of intragenomic conflict is little tested in other contexts, but it predicts that matrigene-patrigene conflict may be rife in social insects. We tested the hypothesis that honey bee worker reproduction is promoted more by patrigenes than matrigenes by comparing across nine reciprocal crosses of two distinct genetic stocks. As predicted, hybrid workers show reproductive trait characteristics of their paternal stock, (indicating enhanced activity of the patrigenes on these traits), greater patrigenic than matrigenic expression, and significantly increased patrigenic-biased expression in reproductive workers. These results support both the general prediction that matrigene-patrigene conflict occurs in social insects and the specific prediction that honey bee worker reproduction is driven more by patrigenes. The success of these predictions suggests that intragenomic conflict may occur in many contexts where matrigenes and patrigenes have different relatednesses to affected kin.


Subject(s)
Bees/genetics , Animals , Bees/physiology , Crosses, Genetic , DNA Methylation , Family , Female , Male , Polymorphism, Single Nucleotide , Reproduction
12.
Biochim Biophys Acta ; 1859(9): 1228-1237, 2016 09.
Article in English | MEDLINE | ID: mdl-27080131

ABSTRACT

The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily that functions as a xenosensor, serving to regulate xenobiotic detoxification, lipid homeostasis and energy metabolism. CAR activation is also a key contributor to the development of chemical hepatocarcinogenesis in mice. The underlying pathways affected by CAR in these processes are complex and not fully elucidated. MicroRNAs (miRNAs) have emerged as critical modulators of gene expression and appear to impact many cellular pathways, including those involved in chemical detoxification and liver tumor development. In this study, we used deep sequencing approaches with an Illumina HiSeq platform to differentially profile microRNA expression patterns in livers from wild type C57BL/6J mice following CAR activation with the mouse CAR-specific ligand activator, 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzene (TCPOBOP). Bioinformatic analyses and pathway evaluations were performed leading to the identification of 51 miRNAs whose expression levels were significantly altered by TCPOBOP treatment, including mmu-miR-802-5p and miR-485-3p. Ingenuity Pathway Analysis of the differentially expressed microRNAs revealed altered effector pathways, including those involved in liver cell growth and proliferation. A functional network among CAR targeted genes and the affected microRNAs was constructed to illustrate how CAR modulation of microRNA expression may potentially mediate its biological role in mouse hepatocyte proliferation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.


Subject(s)
Gene Regulatory Networks , Hepatocytes/drug effects , Liver Neoplasms/genetics , MicroRNAs/genetics , Pyridines/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Animals , Cell Proliferation/drug effects , Computational Biology , Constitutive Androstane Receptor , Gene Expression Profiling , Gene Expression Regulation , Gene Library , Gene Ontology , Hepatocytes/cytology , Hepatocytes/metabolism , High-Throughput Nucleotide Sequencing , Injections, Intraperitoneal , Ligands , Liver/cytology , Liver/drug effects , Liver/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Metabolic Networks and Pathways/genetics , Mice , Mice, Inbred C57BL , MicroRNAs/classification , MicroRNAs/metabolism , Molecular Sequence Annotation , Primary Cell Culture , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction
13.
BMC Genomics ; 16: 620, 2015 Aug 19.
Article in English | MEDLINE | ID: mdl-26285697

ABSTRACT

BACKGROUND: Adaptive manipulation of animal behavior by parasites functions to increase parasite transmission through changes in host behavior. These changes can range from slight alterations in existing behaviors of the host to the establishment of wholly novel behaviors. The biting behavior observed in Carpenter ants infected by the specialized fungus Ophiocordyceps unilateralis s.l. is an example of the latter. Though parasitic manipulation of host behavior is generally assumed to be due to the parasite's gene expression, few studies have set out to test this. RESULTS: We experimentally infected Carpenter ants to collect tissue from both parasite and host during the time period when manipulated biting behavior is experienced. Upon observation of synchronized biting, samples were collected and subjected to mixed RNA-Seq analysis. We also sequenced and annotated the O. unilateralis s.l. genome as a reference for the fungal sequencing reads. CONCLUSIONS: Our mixed transcriptomics approach, together with a comparative genomics study, shows that the majority of the fungal genes that are up-regulated during manipulated biting behavior are unique to the O. unilateralis s.l. genome. This study furthermore reveals that the fungal parasite might be regulating immune- and neuronal stress responses in the host during manipulated biting, as well as impairing its chemosensory communication and causing apoptosis. Moreover, we found genes up-regulated during manipulation that putatively encode for proteins with reported effects on behavioral outputs, proteins involved in various neuropathologies and proteins involved in the biosynthesis of secondary metabolites such as alkaloids.


Subject(s)
Ants/genetics , Ants/microbiology , Behavior, Animal/physiology , Saccharomycetales/physiology , Sequence Analysis, RNA/methods , Animals , Gene Expression Profiling/methods , Gene Expression Regulation , Genome, Fungal , Genome, Insect , Host-Pathogen Interactions , Phylogeny , Saccharomycetales/genetics
14.
Nat Genet ; 38(10): 1210-5, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16964265

ABSTRACT

DNA sequence has long been recognized as an important contributor to nucleosome positioning, which has the potential to regulate access to genes. The extent to which the nucleosomal architecture at promoters is delineated by the underlying sequence is now being worked out. Here we use comparative genomics to report a genome-wide map of nucleosome positioning sequences (NPSs) located in the vicinity of all Saccharomyces cerevisiae genes. We find that the underlying DNA sequence provides a very good predictor of nucleosome locations that have been experimentally mapped to a small fraction of the genome. Notably, distinct classes of genes possess characteristic arrangements of NPSs that may be important for their regulation. In particular, genes that have a relatively compact NPS arrangement over the promoter region tend to have a TATA box buried in an NPS and tend to be highly regulated by chromatin modifying and remodeling factors.


Subject(s)
DNA, Fungal/genetics , Genomics/methods , Nucleosomes/chemistry , Saccharomyces cerevisiae/genetics , DNA, Fungal/chemistry , Gene Expression Regulation, Fungal , Genome, Fungal , Models, Genetic , Nucleosomes/genetics , Promoter Regions, Genetic , TATA Box
15.
Nature ; 453(7193): 358-62, 2008 May 15.
Article in English | MEDLINE | ID: mdl-18408708

ABSTRACT

Comparative genomics of nucleosome positions provides a powerful means for understanding how the organization of chromatin and the transcription machinery co-evolve. Here we produce a high-resolution reference map of H2A.Z and bulk nucleosome locations across the genome of the fly Drosophila melanogaster and compare it to that from the yeast Saccharomyces cerevisiae. Like Saccharomyces, Drosophila nucleosomes are organized around active transcription start sites in a canonical -1, nucleosome-free region, +1 arrangement. However, Drosophila does not incorporate H2A.Z into the -1 nucleosome and does not bury its transcriptional start site in the +1 nucleosome. At thousands of genes, RNA polymerase II engages the +1 nucleosome and pauses. How the transcription initiation machinery contends with the +1 nucleosome seems to be fundamentally different across major eukaryotic lines.


Subject(s)
Drosophila melanogaster/genetics , Genome, Insect/genetics , Nucleosomes/genetics , Nucleosomes/metabolism , Animals , Conserved Sequence/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/enzymology , Gene Expression Regulation/genetics , Genes, Insect/genetics , Histones/metabolism , Promoter Regions, Genetic/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/genetics , Transcription Initiation Site , Transcription, Genetic/genetics
16.
bioRxiv ; 2024 Jul 11.
Article in English | MEDLINE | ID: mdl-39026743

ABSTRACT

Twister ribozymes are an extensively studied class of nucleolytic RNAs. Thousands of natural twisters have been proposed using sequence homology and structural descriptors. Yet, most of these candidates have not been validated experimentally. To address this gap, we developed CHiTA (Cleavage High-Throughput Assay), a high-throughput pipeline utilizing massively parallel oligonucleotide synthesis and next-generation sequencing to test putative ribozymes en masse in a scarless fashion. As proof of principle, we applied CHiTA to a small set of known active and mutant ribozymes. We then used CHiTA to test two large sets of naturally occurring twister ribozymes: over 1, 600 previously reported putative twisters and ∼1, 000 new candidate twisters. The new candidates were identified computationally in ∼1, 000 organisms, representing a massive increase in the number of ribozyme-harboring organisms. Approximately 94% of the twisters we tested were active and cleaved site-specifically. Analysis of their structural features revealed that many substitutions and helical imperfections can be tolerated. We repeated our computational search with structural descriptors updated from this analysis, whereupon we identified and confirmed the first intrinsically active twister ribozyme in mammals. CHiTA broadly expands the number of active twister ribozymes found in nature and provides a powerful method for functional analyses of other RNAs.

17.
J Nutr ; 143(4): 526-32, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23343678

ABSTRACT

The effect of feeding C57BL/6 mice white button (WB) mushrooms or control (CTRL) diets for 6 wk was determined on the bacterial microflora, urinary metabolome, and resistance to a gastrointestinal (GI) pathogen. Feeding mice a diet containing 1 g WB mushrooms/100 g diet resulted in changes in the microflora that were evident at 2 wk and stabilized after 4 wk of WB feeding. Compared with CTRL-fed mice, WB feeding (1 g/100 g diet) increased the diversity of the microflora and reduced potentially pathogenic (e.g., Clostridia) bacteria in the GI tract. Bacteria from the Bacteroidetes phylum increased and the Firmicutes phylum decreased in mushroom-fed mice compared with CTRL. The changes in the microflora were also reflected in the urinary metabolome that showed a metabolic shift in the WB-fed compared with the CTRL-fed mice. The WB feeding and changes in the microbiome were associated with fewer inflammatory cells and decreased colitis severity in the GI mucosa following Citrobacter rodentium infection compared with CTRL. Paradoxically, the clearance of C. rodentium infection did not differ even though Ifn-γ and Il-17 were higher in the colons of the WB-fed mice compared with CTRL. Adding modest amounts of WB mushrooms (1 g/100 g diet) to the diet changed the composition of the normal flora and the urinary metabolome of mice and these changes resulted in better control of inflammation and resolution of infection with C. rodentium.


Subject(s)
Agaricales , Citrobacter rodentium , Diet , Enterobacteriaceae Infections/veterinary , Gastrointestinal Tract/microbiology , Rodent Diseases/microbiology , Animals , Bacteria/classification , Bacteria/genetics , Colitis/microbiology , Colon/chemistry , Colon/microbiology , Cytokines/genetics , Enterobacteriaceae Infections/diet therapy , Enterobacteriaceae Infections/microbiology , Feces/microbiology , Female , Male , Metagenome , Mice , Mice, Inbred C57BL , RNA, Messenger/analysis , Rodent Diseases/diet therapy
18.
Biotechnol Bioeng ; 110(11): 3059-62, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23616357

ABSTRACT

Pyrosequencing was used to characterize bacterial communities in air-cathode microbial fuel cells across a volumetric (graphite fiber brush) and a planar (carbon cloth) anode, where different physical and chemical gradients would be expected associated with the distance between anode location and the air cathode. As expected, the stable operational voltage and the coulombic efficiency (CE) were higher for the volumetric anode than the planar anode (0.57 V and CE = 22% vs. 0.51 V and CE = 12%). The genus Geobacter was the only known exoelectrogen among the observed dominant groups, comprising 57 ± 4% of recovered sequences for the brush and 27 ± 5% for the carbon-cloth anode. While the bacterial communities differed between the two anode materials, results showed that Geobacter spp. and other dominant bacterial groups were homogenously distributed across both planar and volumetric anodes. This lends support to previous community analysis interpretations based on a single biofilm sampling location in these systems.


Subject(s)
Bacteria/classification , Bacteria/genetics , Bioelectric Energy Sources , Biota , Electrodes/microbiology , Air , Carbon , Sequence Analysis, DNA
19.
Nature ; 446(7135): 572-6, 2007 Mar 29.
Article in English | MEDLINE | ID: mdl-17392789

ABSTRACT

The nucleosome is the fundamental building block of eukaryotic chromosomes. Access to genetic information encoded in chromosomes is dependent on the position of nucleosomes along the DNA. Alternative locations just a few nucleotides apart can have profound effects on gene expression. Yet the nucleosomal context in which chromosomal and gene regulatory elements reside remains ill-defined on a genomic scale. Here we sequence the DNA of 322,000 individual Saccharomyces cerevisiae nucleosomes, containing the histone variant H2A.Z, to provide a comprehensive map of H2A.Z nucleosomes in functionally important regions. With a median 4-base-pair resolution, we identify new and established signatures of nucleosome positioning. A single predominant rotational setting and multiple translational settings are evident. Chromosomal elements, ranging from telomeres to centromeres and transcriptional units, are found to possess characteristic nucleosomal architecture that may be important for their function. Promoter regulatory elements, including transcription factor binding sites and transcriptional start sites, show topological relationships with nucleosomes, such that transcription factor binding sites tend to be rotationally exposed on the nucleosome surface near its border. Transcriptional start sites tended to reside about one helical turn inside the nucleosome border. These findings reveal an intimate relationship between chromatin architecture and the underlying DNA sequence it regulates.


Subject(s)
Chromatin Assembly and Disassembly , Genome, Fungal/genetics , Histones/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Saccharomyces cerevisiae/genetics , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , Gene Expression Regulation/genetics , Nucleosomes/chemistry , Promoter Regions, Genetic/genetics , Rotation , Transcription, Genetic/genetics
20.
bioRxiv ; 2023 Jan 07.
Article in English | MEDLINE | ID: mdl-36711553

ABSTRACT

Malaria causes over 200 million infections and over 600 thousand fatalities each year, with most cases attributed to a human-infectious Plasmodium species, Plasmodium falciparum . Many rodent-infectious Plasmodium species, like Plasmodium berghei, Plasmodium chabaudi , and Plasmodium yoelii , have been used as genetically tractable model species that can expedite studies of this pathogen. In particular, P. yoelii is an especially good model for investigating the mosquito and liver stages of parasite development because key attributes closely resemble those of P. falciparum . Because of its importance to malaria research, in 2002 the 17XNL strain of P. yoelii was the first rodent malaria parasite to be sequenced. While sequencing and assembling this genome was a breakthrough effort, the final assembly consisted of >5000 contiguous sequences that impacted the creation of annotated gene models. While other important rodent malaria parasite genomes have been sequenced and annotated since then, including the related P. yoelii 17X strain, the 17XNL strain has not. As a result, genomic data for 17X has become the de facto reference genome for the 17XNL strain while leaving open questions surrounding possible differences between the 17XNL and 17X genomes. In this work, we present a high-quality genome assembly for P. yoelii 17XNL using HiFi PacBio long-read DNA sequencing. In addition, we use Nanopore long-read direct RNA-seq and Illumina short-read sequencing of mixed blood stages to create complete gene models that include not only coding sequences but also alternate transcript isoforms, and 5' and 3' UTR designations. A comparison of the 17X and this new 17XNL assembly revealed biologically meaningful differences between the strains due to the presence of coding sequence variants. Taken together, our work provides a new genomic and gene expression framework for studies with this commonly used rodent malaria model species.

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